@article {1761746, title = {De novo design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8}, journal = {Nat Commun}, volume = {14}, number = {1}, year = {2023}, month = {2023 Sep 13}, pages = {5660}, abstract = {The RGD (Arg-Gly-Asp)-binding integrins αvβ6 and αvβ8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvβ6 and αvβ8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence\ new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvβ6 and αvβ8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very\ close to the computational design models,\ and\ show\ that the inhibitors stabilize specific conformational states of the\ αvβ6 and the αvβ8 integrins. In a lung fibrosis mouse model, the αvβ6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.}, keywords = {Animals, Cell Membrane, Cryoelectron Microscopy, Disease Models, Animal, Integrins, Mice, Pulmonary Fibrosis}, issn = {2041-1723}, doi = {10.1038/s41467-023-41272-z}, author = {Roy, Anindya and Shi, Lei and Chang, Ashley and Dong, Xianchi and Fernandez, Andres and Kraft, John C and Li, Jing and Le, Viet Q and Winegar, Rebecca Viazzo and Cherf, Gerald Maxwell and Slocum, Dean and Poulson, P Daniel and Casper, Garrett E and Vallecillo-Z{\'u}niga, Mary L and Valdoz, Jonard Corpuz and Miranda, Marcos C and Bai, Hua and Kipnis, Yakov and Olshefsky, Audrey and Priya, Tanu and Carter, Lauren and Ravichandran, Rashmi and Chow, Cameron M and Johnson, Max R and Cheng, Suna and Smith, McKaela and Overed-Sayer, Catherine and Finch, Donna K and Lowe, David and Bera, Asim K and Matute-Bello, Gustavo and Birkland, Timothy P and Frank DiMaio and Raghu, Ganesh and Cochran, Jennifer R and Stewart, Lance J and Campbell, Melody G and Van Ry, Pam M and Springer, Timothy and Baker, David} } @article {1748851, title = {Structural insights into MIC2 recognition by MIC2-associated protein in Toxoplasma gondii}, journal = {Commun Biol}, volume = {6}, number = {1}, year = {2023}, month = {2023 Aug 31}, pages = {895}, abstract = {Microneme protein 2 (MIC2) and MIC2-associated protein (M2AP) play crucial roles in the gliding motility and host cell invasion of Toxoplasma gondii. Complex formation between MIC2 and M2AP is required for maturation and transport from the microneme to the parasite surface. Previous studies showed that M2AP associates with the 6th TSR domain of MIC2 (TSR6), but the detailed interaction remains unclear. In this study, we report crystal structures of M2AP alone and in complex with TSR6. TSR domains have an unusually thin, long structure with a layer of intercalated residues on one side. The non-layered side of TSR6 with hotspot residue His-620 at the center binds to M2AP. Remarkably, we show that TSR6 residue Y602 is dynamic; it equilibrates between being part of the layer (the layered state) and in a flipped-out state in the absence of M2AP. However, when bound to M2AP, Y602 shifts to the flipped-out state. Our findings provide insights into the association and stabilization of MIC2-M2AP complex, and may be used to develop new therapies to prevent infections caused by this parasite.}, keywords = {Humans, Problem Solving, Recognition, Psychology, Research Personnel, Toxoplasma}, issn = {2399-3642}, doi = {10.1038/s42003-023-05277-0}, author = {Zhang, Su and Wang, Fangfang and Zhang, Dujuan and Liu, Dongsheng and Ding, Wei and Springer, Timothy A. and Song, Gaojie} } @article {1710131, title = {A recombinant technique for mapping functional sites of hetero-trimeric collagen helices: collagen IV CB3 fragment as a prototype for integrin binding}, journal = {J Biol Chem}, year = {2023}, month = {2023 Jun 09}, pages = {104901}, abstract = {Collagen superfamily of proteins is a major component of the extracellular matrix. Defects in collagens underlie the cause of nearly 40 human genetic diseases in millions of people worldwide. Pathogenesis typically involves genetic alterations of the triple helix, a hallmark structural feature that bestows exceptional mechanical resistance to tensile forces and a capacity to bind a plethora of macromolecules. Yet, there is a paramount knowledge gap in understanding the functionality of distinct sites along the triple helix. Here, we present a recombinant technique to produce triple helical fragments for functional studies. The experimental strategy utilizes the unique capacity of the NC2 hetero-trimerization domain of collagen IX to drive three α-chain selection and registering the triple helix stagger. For proof of principle, we produced and characterized long triple helical fragments of collagen IV that were expressed in a mammalian system. The hetero-trimeric fragments encompassed the CB3 trimeric peptide of collagen IV which harbors the binding motifs for α1β1 and α2β1 integrins. Fragments were characterized and shown to have a stable triple helix, post-translational modifications, and high affinity and specific binding of integrins. The NC2 technique is a universal tool for the high yield production of hetero-trimeric fragments of collagens. Fragments are suitable for mapping functional sites, determining coding sequences of binding sites, elucidating pathogenicity and pathogenic mechanisms of genetic mutations, and production of fragments for protein replacement therapy.}, issn = {1083-351X}, doi = {10.1016/j.jbc.2023.104901}, author = {Boudko, Sergei P and Konopka, Elizabeth H and Kim, Woo Jin and Taga, Yuki and Mizuno, Kazunori and Springer, Timothy A. and Hudson, Billy G and Moy, Terence I and Lin, Fu-Yang} } @article {1703891, title = {Conformational change of Plasmodium TRAP is essential for sporozoite migration and transmission}, journal = {EMBO Rep}, year = {2023}, month = {2023 Jun 12}, pages = {e57064}, abstract = {Eukaryotic cell adhesion and migration rely on surface adhesins connecting extracellular ligands to the intracellular actin cytoskeleton. Plasmodium sporozoites are transmitted by mosquitoes and rely on adhesion and gliding motility to colonize the salivary glands and to reach the liver after transmission. During gliding, the essential sporozoite adhesin TRAP engages actin filaments in the cytoplasm of the parasite, while binding ligands on the substrate through its inserted (I) domain. Crystal structures of TRAP from different Plasmodium species reveal the I domain in closed and open conformations. Here, we probe the importance of these two conformational states by generating parasites expressing versions of TRAP with the I domain stabilized in either the open or closed state with disulfide bonds. Strikingly, both mutations impact sporozoite gliding, mosquito salivary gland entry, and transmission. Absence of gliding in sporozoites expressing the open TRAP I domain can be partially rescued by adding a reducing agent. This suggests that dynamic conformational change is required for ligand binding, gliding motility, and organ invasion and hence sporozoite transmission from mosquito to mammal.}, issn = {1469-3178}, doi = {10.15252/embr.202357064}, author = {Braumann, Friedrich and Klug, Dennis and Kehrer, Jessica and Song, Gaojie and Feng, Juan and Springer, Timothy A. and Frischknecht, Friedrich} } @article {1703896, title = {A specialized integrin-binding motif enables proTGF-β2 activation by integrin αVβ6 but not αVβ8}, journal = {Proc Natl Acad Sci U S A}, volume = {120}, number = {24}, year = {2023}, month = {2023 Jun 13}, pages = {e2304874120}, abstract = {Activation of latent transforming growth factor (TGF)-β2 is incompletely understood. Unlike TGF-β1 and β3, the TGF-β2 prodomain lacks a seven-residue RGDLXX (L/I) integrin-recognition motif and is thought not to be activated by integrins. Here, we report the surprising finding that TGF-β2 contains a related but divergent 13-residue integrin-recognition motif (YTSGDQKTIKSTR) that specializes it for activation by integrin αVβ6 but not αVβ8. Both classes of motifs compete for the same binding site in αVβ6. Multiple changes in the longer motif underlie its specificity. ProTGF-β2 structures define interesting differences from proTGF-β1 and the structural context for activation by αVβ6. Some integrin-independent activation is also seen for proTGF-β2 and even more so for proTGF-β3. Our findings have important implications for therapeutics to αVβ6 in clinical trials for fibrosis, in which inhibition of TGF-β2 activation has not been anticipated.}, keywords = {Antigens, Neoplasm, Fibrosis, Humans, Integrins, Transforming Growth Factor beta, Transforming Growth Factor beta1, Transforming Growth Factor beta2}, issn = {1091-6490}, doi = {10.1073/pnas.2304874120}, author = {Le, Viet Q and Zhao, Bo and Ramesh, Siddanth and Toohey, Cameron and Adam DeCosta and Mintseris, Julian and Liu, Xinyue and Gygi, Steven and Springer, Timothy A.} } @article {1659169, title = {Reflections on Integrins-Past, Present, and Future: The Albert Lasker Basic Medical Research Award}, journal = {JAMA}, volume = {328}, number = {13}, year = {2022}, month = {2022 Oct 04}, pages = {1291-1292}, keywords = {Awards and Prizes, Biomedical Research, Cell Biology, History, 21st Century, Integrins, United States}, issn = {1538-3598}, doi = {10.1001/jama.2022.17005}, author = {Hynes, Richard O and Ruoslahti, Erkki and Springer, Timothy A.} } @article {1659168, title = {Single-molecule characterization of subtype-specific β1 integrin mechanics}, journal = {Nat Commun}, volume = {13}, number = {1}, year = {2022}, month = {2022 Dec 03}, pages = {7471}, abstract = {Although integrins are known to be mechanosensitive and to possess many subtypes that have distinct physiological roles, single molecule studies of force exertion have thus far been limited to RGD-binding integrins. Here, we show that integrin α4β1 and RGD-binding integrins (αVβ1 and α5β1) require markedly different tension thresholds to support cell spreading. Furthermore, actin assembled downstream of α4β1 forms cross-linked networks in circularly spread cells, is in rapid retrograde flow, and exerts low forces from actin polymerization. In contrast, actin assembled downstream of αVβ1 forms stress fibers linking focal adhesions in elongated cells, is in slow retrograde flow, and matures to exert high forces (\>54-pN) via myosin II. Conformational activation of both integrins occurs below 12-pN, suggesting that post-activation subtype-specific cytoskeletal remodeling imposes the higher threshold for spreading on RGD substrates. Multiple layers of single integrin mechanics for activation, mechanotransduction and cytoskeleton remodeling revealed here may underlie subtype-dependence of diverse processes such as somite formation and durotaxis.}, issn = {2041-1723}, doi = {10.1038/s41467-022-35173-w}, author = {Jo, Myung Hyun and Li, Jing and Jaumouill{\'e}, Valentin and Hao, Yuxin and Coppola, Jessica and Yan, Jiabin and Waterman, Clare M and Springer, Timothy A. and Ha, Taekjip} } @article {1652765, title = {A general chemical principle for creating closure-stabilizing integrin inhibitors}, journal = {Cell}, volume = {185}, number = {19}, year = {2022}, month = {2022 Sep 15}, pages = {3533-3550.e27}, abstract = {Integrins are validated drug targets with six approved therapeutics. However, small-molecule inhibitors to three integrins failed in late-stage clinical trials for chronic indications. Such unfavorable outcomes may in part be caused by partial agonism, i.e., the stabilization of the high-affinity, extended-open integrin conformation. Here, we show that the failed, small-molecule inhibitors of integrins αIIbβ3 and α4β1 stabilize the high-affinity conformation. Furthermore, we discovered a simple chemical feature present in multiple αIIbβ3 antagonists that stabilizes integrins in their bent-closed conformation. Closing inhibitors contain a polar nitrogen atom that stabilizes, via hydrogen bonds, a water molecule that intervenes between a serine residue and the metal in the metal-ion-dependent adhesion site (MIDAS). Expulsion of this water is a requisite for transition to the open conformation. This change in metal coordination is general to integrins, suggesting broad applicability of the drug-design principle to the integrin family, as validated with a distantly related integrin, α4β1.}, keywords = {Drug Design, Integrin alpha4beta1, Protein Conformation, Serine, Water}, issn = {1097-4172}, doi = {10.1016/j.cell.2022.08.008}, author = {Lin, Fu-Yang and Li, Jing and Xie, Yonghua and Zhu, Jianghai and Huong Nguyen, Thi Thu and Zhang, Yonghui and Zhu, Jieqing and Springer, Timothy A.} } @article {1651435, title = {Conformation of von Willebrand factor in shear flow revealed with stroboscopic single-molecule imaging}, journal = {Blood}, year = {2022}, month = {2022 Aug 30}, abstract = {von Willebrand factor (VWF) is a multimeric blood protein that acts as a mechanical probe, responding to changes in flow to initiate platelet plug formation. Previously, our labs had shown using single-molecule imaging that shear stress can extend surface-tethered VWF, but paradoxically we found that the required shear stress was higher than reported for free-in-flow VWF-an observation inconsistent with basic physical principles. To resolve this inconsistency critical to VWF{\textquoteright}s molecular mechanism, we measured free VWF extension in shear flow using PULSIS-Pulsed Laser Stroboscopic Imaging of Single molecules. Here, laser pulses of different durations are used to capture multiple images of the same molecule within each frame, enabling accurate length measurements in the presence of motion blur. At high shear stresses, we observed a mean shift in VWF extension of less than 200 nm, much shorter than the multiple-micron extensions previously reported with no evidence for the predicted sharp globule-stretch conformational transition. Modeling VWF with a Brownian dynamics simulation, our results are consistent with VWF behaving as an uncollapsed polymer rather than the theorized compact ball. The muted response of free VWF to high shear rates implies that 1) the tension experienced by free VWF in physiological shear flow is lower than indicated by previous reports and 2) that tethering to platelets or the vessel wall is required to mechanically activate VWF adhesive function for primary hemostasis.}, issn = {1528-0020}, doi = {10.1182/blood.2022016969}, author = {Bergal, Hans T and Jiang, Yan and Yang, Darren and Springer, Timothy A. and Wong, Wesley P} } @article {1651434, title = {Dynamics of integrin α5β1, fibronectin, and their complex reveal sites of interaction and conformational change}, journal = {J Biol Chem}, year = {2022}, month = {2022 Aug 02}, pages = {102323}, abstract = {Integrin α5β1 mediates cell adhesion to the extracellular matrix (ECM) by binding fibronectin (Fn). Selectivity for Fn by α5β1 is achieved through recognition of an RGD motif in the 10th type-III Fn domain (Fn10) and the synergy site in the 9th type-III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5β1-binding affinities and stabilities. We also interrogated binding of the α5β1 ectodomain headpiece fragment to Fn using hydrogen deuterium exchange mass spectrometry (HDX MS) to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two β-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit β-propeller domain for the Fn9 synergy site and in the β1-subunit βI domain for Fn10 based on decreases in α5β1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5β1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5β1 binding to Fn and dynamics associated with this interaction.}, issn = {1083-351X}, doi = {10.1016/j.jbc.2022.102323}, author = {Su, Yang and Iacob, Roxana E and Li, Jing and Engen, John R and Springer, Timothy A.} } @article {1651437, title = {Monomeric prefusion structure of an extremophile gamete fusogen and stepwise formation of the postfusion trimeric state}, journal = {Nat Commun}, volume = {13}, number = {1}, year = {2022}, month = {2022 07 13}, pages = {4064}, abstract = {Here, we study the gamete fusogen HAP2 from Cyanidioschyzon merolae (Cyani), an extremophile red algae that grows at acidic pH at 45 {\textdegree}C. HAP2 has a trimeric postfusion structure with similarity to viral class II fusion proteins, but its prefusion structure has been elusive. The crystal structure of a monomeric prefusion state of Cyani HAP2 shows it is highly extended with three domains in the order D2, D1, and D3. Three hydrophobic fusion loops at the tip of D2 are each required for postfusion state formation. We followed by negative stain electron microscopy steps in the process of detergent micelle-stimulated postfusion state formation. In an intermediate state, two or three linear HAP2 monomers associate at the end of D2 bearing its fusion loops. Subsequently, D2 and D1 line the core of a trimer and D3 folds back over the exterior of D1 and D2. D3 is not required for formation of intermediate or postfusion-like states.}, keywords = {Extremophiles, Germ Cells, Protein Conformation, Viral Envelope Proteins, Viral Fusion Proteins}, issn = {2041-1723}, doi = {10.1038/s41467-022-31744-z}, author = {Feng, Juan and Dong, Xianchi and Su, Yang and Lu, Chafen and Springer, Timothy A.} } @article {1651436, title = {Structures of VWF tubules before and after concatemerization reveal a mechanism of disulfide bond exchange}, journal = {Blood}, year = {2022}, month = {2022 Jul 01}, abstract = {von Willebrand Factor (VWF) is an adhesive glycoprotein that circulates in the blood as disulfide-linked concatemers and functions in primary hemostasis. The loss of long VWF concatemers is associated with the excess bleeding of type 2A von Willebrand (VW) disease. Formation of the disulfide bonds that concatemerize VWF requires VWF to self-associate into helical tubules, yet how the helical tubules template intermolecular disulfide bonds is not known. Here, we report cryo-EM structures of complete VWF tubules before and after intermolecular disulfide-bond formation. The structures provide evidence that VWF tubulates through a charge-neutralization mechanism and that the A1 domain enhances tubule length by crosslinking successive helical turns. In addition, the structures reveal disulfide states prior to and after disulfide bond-mediated concatemerization. The structures and proposed assembly mechanism provide a foundation to rationalize VW disease-causing mutations.}, issn = {1528-0020}, doi = {10.1182/blood.2022016467}, author = {Anderson, Jacob Ronald and Li, Jing and Springer, Timothy A. and Brown, Alan} } @article {1639542, title = {Loss of LRRC33-dependent TGFβ1 activation enhances antitumor immunity and checkpoint blockade therapy}, journal = {Cancer Immunol Res}, volume = {10}, number = {4}, year = {2022}, month = {2022 Apr 01}, pages = {453-467}, abstract = {TGFβ has multiple roles and gene products (TGFβ1, -β2, and -β3), which make global targeting of TGFβ undesirable. Expression of TGFβ requires association with milieu molecules, which localize TGFβ to the surface of specific cells or extracellular matrices. Here, we found that LRRC33 was specifically associated with TGFβ1, not TGFβ2 and TGFβ3, and was required for surface display and activation of TGFβ1 on tumor-infiltrating myeloid cells. Loss of LRRC33-dependent TGFβ1 activation slowed tumor growth and metastasis by enhancing innate and adaptive antitumor immunity in multiple mouse syngeneic tumor models. LRRC33 loss resulted in a more immunogenic microenvironment, with decreased myeloid-derived suppressor cells, more active CD8+ T and NK cells, and more skewing toward tumor-suppressive M1 macrophages. LRRC33 loss and PD-1 blockade synergized in controlling B16.F10 tumor growth. Our results demonstrate the importance of LRRC33 in tumor biology and highlight the therapeutic potential of dual blockade of the LRRC33/TGFβ1 axis and PD-1/PD-L1 in cancer immunotherapy.}, keywords = {Animals, Cell Line, Tumor, Disease Models, Animal, Immunotherapy, Macrophages, Mice, Neoplasms, Transforming Growth Factor beta, Tumor Microenvironment}, issn = {2326-6074}, doi = {10.1158/2326-6066.CIR-21-0593}, author = {Jiang, Aiping and Qin, Yan and Springer, Timothy A.} } @article {1639541, title = {Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker}, journal = {Elife}, volume = {11}, year = {2022}, month = {2022 May 09}, abstract = {Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N- and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIbα, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIbα, which is induced by tensile force physiologically and regulated allosterically by the N-linker.}, keywords = {Blood Platelets, Polysaccharides, Protein Binding, Urea, von Willebrand Factor}, issn = {2050-084X}, doi = {10.7554/eLife.75760}, author = {Bonazza, Klaus and Iacob, Roxana E and Hudson, Nathan E and Li, Jing and Lu, Chafen and Engen, John R and Springer, Timothy A.} } @article {1630950, title = {Regulation by metal ions and the ADMIDAS of integrin α5β1 conformational states and intrinsic affinities}, journal = {Mol Biol Cell}, year = {2022}, month = {2022 Feb 02}, pages = {mbcE21110536}, abstract = {Activation of integrins by Mn2+ is a benchmark in the integrin field, but how it works and whether it reproduces physiologic activation is unknown. We show that Mn2+ and high Mg2+ concentrations compete with Ca2+ at the ADMIDAS and shift the conformational equilibrium toward the open state, but the shift is far from complete. Additionally, replacement of Mg2+ by Mn2+ at the MIDAS increases the intrinsic affinities of both the high affinity open and low affinity closed states of integrins, in agreement with stronger binding of Mn2+ than Mg2+ to oxygen atoms. Mutation of the ADMIDAS increases the affinity of closed states and decreases the affinity of the open state and thus reduces the difference in affinity between the open and closed states. An important biological function of the ADMIDAS may be to stabilize integrins in highly discrete states, so that when integrins support cell adhesion and migration, their high and low affinity correspond to discrete on- and off-states, respectively.}, issn = {1939-4586}, doi = {10.1091/mbc.E21-11-0536}, author = {Anderson, Jordan M and Li, Jing and Springer, Timothy A.} } @article {1629400, title = {Protection of the Prodomain α1-Helix Correlates with Latency in the Transforming Growth Factor-β Family}, journal = {J Mol Biol}, volume = {434}, number = {5}, year = {2022}, month = {2022 Jan 04}, pages = {167439}, abstract = {The 33 members of the transforming growth factor beta (TGF-β) family are fundamentally important for organismal development and homeostasis. Family members are synthesized and secreted as pro-complexes of non-covalently associated prodomains and growth factors (GF). Pro-complexes from a subset of family members are latent and require activation steps to release the GF for signaling. Why some members are latent while others are non-latent is incompletely understood, particularly because of large family diversity. Here, we have examined representative family members in negative stain electron microscopy (nsEM) and hydrogen deuterium exchange (HDX) to identify features that differentiate latent from non-latent members. nsEM showed three overall pro-complex conformations that differed in prodomain arm domain orientation relative to the bound growth factor. Two cross-armed members, TGF-β1 and TGF-β2, were each latent. However, among V-armed members, GDF8 was latent whereas ActA was not. All open-armed members, BMP7, BMP9, and BMP10, were non-latent. Family members exhibited remarkably varying HDX patterns, consistent with large prodomain sequence divergence. A strong correlation emerged between latency and protection of the prodomain α1-helix from exchange. Furthermore, latency and protection from exchange correlated structurally with increased α1-helix buried surface area, hydrogen bonds, and cation-pi bonds. Moreover, a specific pattern of conserved basic and hydrophobic residues in the α1-helix and aromatic residues in the interacting fastener were found only in latent members. Thus, this first comparative survey of TGF-β family members reveals not only diversity in conformation and dynamics but also unique features that distinguish latent members.}, issn = {1089-8638}, doi = {10.1016/j.jmb.2021.167439}, author = {Le, Viet Q and Iacob, Roxana E and Zhao, Bo and Su, Yang and Tian, Yuan and Toohey, Cameron and Engen, John R and Springer, Timothy A.} } @article {1629401, title = {Structural basis of malaria transmission blockade by a monoclonal antibody to gamete fusogen HAP2}, journal = {Elife}, volume = {10}, year = {2021}, month = {2021 Dec 23}, abstract = {HAP2 is a transmembrane gamete fusogen found in multiple eukaryotic kingdoms and is structurally homologous to viral class II fusogens. Studies in Plasmodium have suggested that HAP2 is an attractive target for vaccines that block transmission of malaria. HAP2 has three extracellular domains, arranged in the order D2, D1, and D3. Here, we report monoclonal antibodies against the D3 fragment of Plasmodium berghei HAP2 and crystal structures of D3 in complex with Fab fragments of two of these antibodies, one of which blocks fertilization of Plasmodium berghei in vitro and transmission of malaria in mosquitoes. We also show how this Fab binds the complete HAP2 ectodomain with electron microscopy. The two antibodies cross-react with HAP2 among multiple plasmodial species. Our characterization of the Plasmodium D3 structure, HAP2 ectodomain architecture, and mechanism of inhibition provide insights for the development of a vaccine to block malaria transmission.}, issn = {2050-084X}, doi = {10.7554/eLife.74707}, author = {Feng, Juan and Dong, Xianchi and Adam DeCosta and Su, Yang and Fiona Angrisano and Sala, Katarzyna A and Blagborough, Andrew M and Lu, Chafen and Springer, Timothy A.} } @article {1623546, title = {Low affinity integrin states have faster ligand binding kinetics than the high affinity state}, journal = {eLife}, volume = {10}, number = {e73359}, year = {2021}, abstract = {Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins α4β1 and α5β1 and show that the low-affinity states bind substantially faster than the high-affinity state. On and off-rates are similar for integrins on cell surfaces and as ectodomain fragments. Although the extended-open conformation{\textquoteright}s on-rate is ~20-fold slower, its off-rate is ~25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. Low affinity integrin states not only bind ligand more rapidly, but are also more populous on the cell surface than high affinity states. Thus, our results suggest that integrin binding to ligand may precede, rather than follow, activation by "inside-out signaling".}, url = {https://pubmed.ncbi.nlm.nih.gov/34854380/}, author = {Li, Jing and Yan, Jiabin and Tim Springer} } @article {1620029, title = {Congenital X-linked neutropenia with myelodysplasia and somatic tetraploidy due to a germline mutation in SEPT6}, journal = {Am J Hematol}, year = {2021}, month = {2021 Oct 22}, abstract = {Septins play key roles in mammalian cell division and cytokinesis but have not previously been implicated in a germline human disorder. A male infant with severe neutropenia and progressive dysmyelopoiesis with tetraploid myeloid precursors was identified. No known genetic etiologies for neutropenia or bone marrow failure were found. However, next-generation sequencing of germline samples from the patient revealed a novel, de novo germline stop-loss mutation in the X-linked gene SEPT6 that resulted in reduced SEPT6 staining in bone marrow granulocyte precursors and megakaryocytes. Patient skin fibroblast-derived induced pluripotent stem cells (iPSCs) produced reduced myeloid colonies, particularly of the granulocyte lineage. CRISPR/Cas9 knock-in of the patient{\textquoteright}s mutation or complete knock-out of SEPT6 was not tolerated in non-patient-derived iPSCs or human myeloid cell lines, but SEPT6 knock-out was successful in an erythroid cell line and resulting clones revealed a propensity to multinucleation. In silico analysis predicts that the mutated protein hinders the dimerization of SEPT6 coiled-coils in both parallel and antiparallel arrangements, which could in turn impair filament formation. These data demonstrate a critical role for SEPT6 in chromosomal segregation in myeloid progenitors that can account for the unusual predisposition to aneuploidy and dysmyelopoiesis.}, issn = {1096-8652}, doi = {10.1002/ajh.26382}, author = {Renella, Raffaele and Gagne, Katelyn and Beauchamp, Ellen and Fogel, Jonathan and Perlov, Aleksej and Sola, Mireia and Schlaeger, Thorsten and Hofmann, Inga and Shimamura, Akiko and Ebert, Benjamin L and Schmitz-Abe, Klaus and Markianos, Kyriacos and Murphy, Kristi and Sun, Liang and Rockowitz, Shira and Sliz, Piotr and Campagna, Dean R and Springer, Timothy A. and Bahl, Christopher and Agarwal, Suneet and Fleming, Mark D and Williams, David A} } @article {1623545, title = {Single-molecule imaging of von Willebrand factor reveals tension-dependent self-association}, journal = {Blood}, volume = {138}, number = {23}, year = {2021}, pages = {2425-2434}, abstract = {Von Willebrand factor (VWF) is an ultra-long concatemeric protein important in hemostasis and thrombosis. VWF molecules can associate with other VWF molecules, but little is known about the mechanism. Hydrodynamic drag exerts tensile force on surface-tethered VWF that extends it and is maximal at the tether point and declines linearly to zero at the downstream, free end. Using single-molecule fluorescence microscopy, we directly visualize the kinetics of binding of free VWF in flow to surface-tethered single VWF molecules and show that self-association requires elongation of tethered VWF and that association increases with tension in tethered VWF, reaches half maximum at a characteristic tension of ~10 pN, and plateaus above ~ 25 pN. Association is reversible and hence noncovalent; a sharp decrease in shear flow results in rapid dissociation of bound VWF. Tethered, primary VWF molecules can recruit more than their own mass of secondary VWF from the flow stream. Kinetics show that instead of accelerating, the rate of accumulation decreases with time, revealing an inherently self-limiting self-association mechanism. We propose that this may be because multiple tether points between secondary and primary VWF result in lower tension on the secondary VWF, which shields more highly tensioned primary VWF from further association. GPIbα binding and VWF self-association occur in the same region of high tension in tethered VWF concatemers; however, the half-maximal tension required for activation of GPIbα is higher, suggesting differences in molecular mechanisms. These results have important implications for the mechanism of platelet plug formation in hemostasis and thrombosis.}, url = {https://pubmed.ncbi.nlm.nih.gov/34882208/}, author = {Fu, Hongxia and Jiang, Yan and Wesley Wong and Springer, Timothy A.} } @article {1618480, title = {Complement Receptor 3 Forms a Compact High-Affinity Complex with iC3b}, journal = {J Immunol}, year = {2021}, month = {2021 Jun 11}, abstract = {Complement receptor 3 (CR3, also known as Mac-1, integrin αMβ2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment iC3b and, to a lesser extent, C3dg. Although the interaction between the iC3b thioester domain and the ligand binding CR3 αM I-domain is structurally and functionally well characterized, the nature of additional CR3-iC3b interactions required for phagocytosis of complement-opsonized objects remains obscure. In this study, we analyzed the interaction between iC3b and the 150-kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of the CR3 headpiece for iC3b compared with 515 nM for the iC3b thioester domain, whereas experiments monitoring binding of iC3b to CR3-expressing cells suggested an affinity of 50 nM for the CR3-iC3b interaction. Small angle x-ray scattering analysis revealed that iC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, iC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the iC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the iC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by iC3b than by C3dg and pave the way for the development of specific therapeutics for the treatment of inflammatory and neurodegenerative diseases that do not interfere with the recognition of noncomplement CR3 ligands.}, issn = {1550-6606}, doi = {10.4049/jimmunol.2001208}, author = {Jensen, Rasmus K and Bajic, Goran and Sen, Mehmet and Springer, Timothy A. and Vorup-Jensen, Thomas and Andersen, Gregers R} } @article {1618481, title = {CD11c regulates hematopoietic stem and progenitor cells under stress}, journal = {Blood Adv}, volume = {4}, number = {24}, year = {2020}, month = {2020 12 22}, pages = {6086-6097}, abstract = {β2 integrins are well-known leukocyte adhesion molecules consisting of 4 members: CD11a-d. Their known biological functions range widely from leukocyte recruitment, phagocytosis, to immunological synapse formation, but the studies have been primarily focused on CD11a and CD11b. CD11c is 1 of the 4 members and is extremely homologous to CD11b. It has been well known as a dendritic cell marker, but the characterization of its function has been limited. We found that CD11c was expressed on the short-term hematopoietic stem cells and multipotent progenitor cells. The lack of CD11c did not affect the number of hematopoietic stem and progenitor cells (HSPCs) in healthy CD11c knockout mice. Different from other β2 integrin members, however, CD11c deficiency was associated with increased apoptosis and significant loss of HSPCs in sepsis and bone marrow transplantation. Although integrins are generally known for their overlapping and redundant roles, we showed that CD11c had a distinct role of regulating the expansion of HSPCs under stress. This study shows that CD11c, a well-known dendritic cell marker, is expressed on HSPCs and serves as their functional regulator. CD11c deficiency leads to the loss of HSPCs via apoptosis in sepsis and bone marrow transplantation.}, keywords = {Animals, CD11 Antigens, CD11c Antigen, CD18 Antigens, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells, Mice, Mice, Knockout}, issn = {2473-9537}, doi = {10.1182/bloodadvances.2020002504}, author = {Hou, Lifei and Voit, Richard A and Sankaran, Vijay G and Springer, Timothy A. and Yuki, Koichi} } @article {1532243, title = {Evolutionarily distant I domains can functionally replace the essential ligand-binding domain of TRAP}, journal = {Elife}, volume = {9}, year = {2020}, month = {2020 07 10}, abstract = {Inserted (I) domains function as ligand-binding domains in adhesins that support cell adhesion and migration in many eukaryotic phyla. These adhesins include integrin αβ heterodimers in metazoans and single subunit transmembrane proteins in apicomplexans such as TRAP in and MIC2 in . Here we show that the I domain of TRAP is essential for sporozoite gliding motility, mosquito salivary gland invasion and mouse infection. Its replacement with the I domain from Toxoplasma MIC2 fully restores tissue invasion and parasite transmission, while replacement with the aX I domain from human integrins still partially restores liver infection. Mutations around the ligand binding site allowed salivary gland invasion but led to inefficient transmission to the rodent host. These results suggest that apicomplexan parasites appropriated polyspecific I domains in part for their ability to engage with multiple ligands and to provide traction for emigration into diverse organs in distant phyla.}, issn = {2050-084X}, doi = {10.7554/eLife.57572}, author = {Klug, Dennis and Goellner, Sarah and Kehrer, Jessica and Sattler, Julia and Strauss, L{\'e}anne and Singer, Mirko and Lu, Chafen and Springer, Timothy A. and Frischknecht, Friedrich} } @article {1532242, title = {Disulfide Exchange in von Willebrand factor Dimerization in the Golgi}, journal = {Blood}, year = {2020}, month = {2020 Sep 22}, issn = {1528-0020}, doi = {10.1182/blood.2020005989}, url = {https://pubmed.ncbi.nlm.nih.gov/32961556/}, author = {Dong, Xianchi and Springer, Timothy A.} } @article {1487026, title = {Design and assessment of TRAP-CSP fusion antigens as effective malaria vaccines}, journal = {PLoS One}, volume = {15}, number = {1}, year = {2020}, month = {2020}, pages = {e0216260}, abstract = {The circumsporozoite protein (CSP) and thrombospondin-related adhesion protein (TRAP) are major targets for pre-erythrocytic malaria vaccine development. However, the CSP-based vaccine RTS,S provides only marginal protection, highlighting the need for innovative vaccine design and development. Here we design and characterize expression and folding of P. berghei (Pb) and P. falciparum (Pf) TRAP-CSP fusion proteins, and evaluate immunogenicity and sterilizing immunity in mice. TRAP N-terminal domains were fused to the CSP C-terminal αTSR domain with or without the CSP repeat region, expressed in mammalian cells, and evaluated with or without N-glycan shaving. Pb and Pf fusions were each expressed substantially better than the TRAP or CSP components alone; furthermore, the fusions but not the CSP component could be purified to homogeneity and were well folded and monomeric. As yields of TRAP and CSP fragments were insufficient, we immunized BALB/c mice with Pb TRAP-CSP fusions in AddaVax adjuvant and tested the effects of absence or presence of the CSP repeats and absence or presence of high mannose N-glycans on total antibody titer and protection from infection by mosquito bite both 2.5 months and 6 months after the last immunization. Fusions containing the repeats were completely protective against challenge and re-challenge, while those lacking repeats were significantly less effective. These results correlated with higher total antibody titers when repeats were present. Our results show that TRAP-CSP fusions increase protein antigen production, have the potential to yield effective vaccines, and also guide design of effective proteins that can be encoded by nucleic acid-based and virally vectored vaccines.}, keywords = {Animals, Antibodies, Antigens, Disease Models, Animal, Gene Expression Regulation, Humans, Immunization, Malaria, Malaria Vaccines, Mice, Plasmodium berghei, Plasmodium falciparum, Polysaccharides, Protein Folding, Protozoan Proteins, Recombinant Fusion Proteins}, issn = {1932-6203}, doi = {10.1371/journal.pone.0216260}, author = {Lu, Chafen and Song, Gaojie and Beale, Kristin and Yan, Jiabin and Garst, Emma and Feng, Juan and Lund, Emily and Catteruccia, Flaminia and Springer, Timothy A.} } @article {1481807, title = {General structural features that regulate integrin affinity revealed by atypical αVβ8}, journal = {Nat Commun}, volume = {10}, number = {1}, year = {2019}, month = {2019 Dec 02}, pages = {5481}, abstract = {Integrin αVβ8, which like αVβ6 functions to activate TGF-βs, is atypical. Its β8 subunit binds to a distinctive cytoskeleton adaptor and does not exhibit large changes in conformation upon binding to ligand. Here, crystal structures, hydrogen-deuterium exchange dynamics, and affinity measurements on mutants are used to compare αVβ8 and αVβ6. Lack of a binding site for one of three βI domain divalent cations and a unique β6-α7 loop conformation in β8 facilitate movements of the α1 and α1{\textquoteright} helices at the ligand binding pocket toward the high affinity state, without coupling to β6-α7 loop reshaping and α7-helix pistoning that drive large changes in βI domain-hybrid domain orientation seen in other integrins. Reciprocal swaps between β6 and β8 βI domains increase affinity of αVβ6 and decrease affinity of αVβ8 and define features that regulate affinity of the βI domain and its coupling to the hybrid domain.}, issn = {2041-1723}, doi = {10.1038/s41467-019-13248-5}, author = {Wang, Jianchuan and Su, Yang and Iacob, Roxana E and Engen, John R and Springer, Timothy A.} } @article {1445478, title = {Specific high affinity interaction of Helicobacter~pylori CagL with integrin α β promotes type IV secretion of CagA into human cells}, journal = {FEBS J}, volume = {286}, number = {20}, year = {2019}, month = {2019 Oct}, pages = {3980-3997}, abstract = {CagL is an essential pilus surface component of the virulence-associated type IV secretion system (T4SS) employed by Helicobacter~pylori to translocate the oncogenic effector protein CagA into human gastric epithelial cells. CagL contains an RGD motif and integrin α β is widely accepted as its host cell receptor. Here, we show that CagL binds integrin α β with substantially higher affinity and that this interaction is functionally important. Cell surface expression of α β on various cell lines correlated perfectly with cell adhesion to immobilized CagL and with binding of soluble CagL to cells. We found no such correlation for α β . The purified α β ectodomain bound CagL with high affinity. This interaction was highly specific, as the affinity of CagL for other RGD-binding integrins was two to three orders of magnitude weaker. Mutation of either conserved leucine in the CagL RGDLXXL motif, a motif that generally confers specificity for integrin α β and α β , lowered the affinity of CagL for α β . Stable expression of α β in α β -negative but α β -expressing human cells promoted two hallmarks of the functional H.~pylori T4SS, namely translocation of CagA into host cells and induction of interleukin-8 secretion by host cells. These findings suggest that integrin α β , although not essential for T4SS function, represents an important host cell receptor for CagL.}, issn = {1742-4658}, doi = {10.1111/febs.14962}, author = {Bu{\ss}, Maren and Tegtmeyer, Nicole and Schnieder, Jennifer and Dong, Xianchi and Li, Jing and Springer, Timothy A. and Backert, Steffen and Niemann, Hartmut H} } @article {1430782, title = {Electrostatic Steering Enables Flow-Activated Von Willebrand Factor to Bind Platelet Glycoprotein, Revealed by Single-Molecule Stretching and Imaging}, journal = {J Mol Biol}, volume = {431}, number = {7}, year = {2019}, month = {2019 Mar 29}, pages = {1380-1396}, abstract = {Von Willebrand factor (VWF), a large multimeric blood protein, senses changes in shear stress during bleeding and responds by binding platelets to plug ruptures in the vessel wall. Molecular mechanisms underlying this dynamic process are difficult to uncover using standard approaches due to the challenge of applying mechanical forces while monitoring structure and activity. By combining single-molecule fluorescence imaging with high-pressure, rapidly switching microfluidics, we reveal the key role of electrostatic steering in accelerating the binding between flow-activated VWF and GPIbα, and in rapidly immobilizing platelets under flow. We measure the elongation and tension-dependent activation of individual VWF multimers under a range of ionic strengths and pH levels, and find that the association rate is enhanced by 4 orders of magnitude by electrostatic steering. Under supraphysiologic salt concentrations, strong electrostatic screening dramatically decreases platelet binding to VWF in flow, revealing the critical role of electrostatic attraction in VWF-platelet binding during bleeding.}, issn = {1089-8638}, doi = {10.1016/j.jmb.2019.02.014}, author = {Jiang, Yan and Fu, Hongxia and Springer, Timothy A. and Wong, Wesley P} } @article {1430781, title = {The von Willebrand factor D{\textquoteright}D3 assembly and structural principles for factor VIII binding and concatemer biogenesis}, journal = {Blood}, year = {2019}, month = {2019 Jan 14}, abstract = {D assemblies comprise half of von Willebrand factor yet are of unknown structure. D1 and D2 in the prodomain and D{\textquoteright}D3 in mature VWF at Golgi pH form helical VWF tubules in Weibel Palade bodies and template dimerization of D3 through disulfides to form ultralong VWF concatemers. D{\textquoteright}D3 forms the binding site for factor VIII. The crystal structure of monomeric D{\textquoteright}D3 with cysteine residues required for dimerization mutated to alanine was determined at endoplasmic reticulum (ER)-like pH. The smaller C8-3, TIL3 and E3 modules pack through specific interfaces as they wind around the larger, N-terminal, Ca-binding VWD3 module to form a wedge shape. D{\textquoteright} with its TIL{\textquoteright} and E{\textquoteright} modules projects away from D3. The two mutated cysteines implicated in D3 dimerization are buried, providing a mechanism for protecting them against premature disulfide linkage in the ER where intrachain disulfide linkages are formed. D3 dimerization requires co-association with D1 and D2, Ca, and Golgi-like acidic pH. Associated structural rearrangements in the C8-3 and TIL3 modules are required to expose cysteine residues for disulfide linkage. Our structure provides insight into many von Willebrand disease mutations including those that diminish FVIII binding, which suggest that factor VIII binds not only to the N-terminal TIL{\textquoteright} domain of D{\textquoteright} distal from D3 but also extends across one side of D3. The organizing principle for the D3 assembly has implications for other D assemblies and the construction of higher order, disulfide-linked assemblies in the Golgi in both VWF and mucins.}, issn = {1528-0020}, doi = {10.1182/blood-2018-10-876300}, author = {Dong, Xianchi and Leksa, Nina C and Chhabra, Ekta Seth and Arndt, Joseph W and Lu, Qi and Knockenhauer, Kevin E and Peters, Robert T and Springer, Timothy A.} } @article {1404801, title = {A Tandem Mass Spectrometry Sequence Database Search Method for Identification of O-Fucosylated Proteins by Mass Spectrometry}, journal = {J Proteome Res}, volume = {18}, number = {2}, year = {2019}, month = {2019 Feb 01}, pages = {652-663}, abstract = {Thrombospondin type 1 repeats (TSRs), small adhesive protein domains with a wide range of functions, are usually modified with O-linked fucose, which may be extended to O-fucose-β1,3-glucose. Collision-induced dissociation (CID) spectra of O-fucosylated peptides cannot be sequenced by standard tandem mass spectrometry (MS/MS) sequence database search engines because O-linked glycans are highly labile in the gas phase and are effectively absent from the CID peptide fragment spectra, resulting in a large mass error. Electron transfer dissociation (ETD) preserves O-linked glycans on peptide fragments, but only a subset of tryptic peptides with low m/ z can be reliably sequenced from ETD spectra compared to CID. Accordingly, studies to date that have used MS to identify O-fucosylated TSRs have required manual interpretation of CID mass spectra even when ETD was also employed. In order to facilitate high-throughput, automatic identification of O-fucosylated peptides from CID spectra, we re-engineered the MS/MS sequence database search engine Comet and the MS data analysis suite Trans-Proteomic Pipeline to enable automated sequencing of peptides exhibiting the neutral losses characteristic of labile O-linked glycans. We used our approach to reanalyze published proteomics data from Plasmodium parasites and identified multiple glycoforms of TSR-containing proteins.}, issn = {1535-3907}, doi = {10.1021/acs.jproteome.8b00638}, author = {Swearingen, Kristian E and Eng, Jimmy K and Shteynberg, David and Vigdorovich, Vladimir and Springer, Timothy A. and Mendoza, Luis and Sather, Noah D and Deutsch, Eric W and Kappe, Stefan H I and Moritz, Robert L} } @article {1404800, title = {Dendritic cell-expressed common gamma-chain recruits IL-15 for trans-presentation at the murine immunological synapse}, journal = {Wellcome Open Res}, volume = {3}, year = {2018}, month = {2018}, pages = {84}, abstract = {Mutations of the common cytokine receptor gamma chain (γc) cause Severe Combined Immunodeficiency characterized by absent T and NK cell development. Although stem cell therapy restores these lineages, residual immune defects are observed that may result from selective persistence of γc-deficiency in myeloid lineages. However, little is known about the contribution of myeloid-expressed γc to protective immune responses.\  Here we examine the importance of γc for myeloid dendritic cell (DC) function. We utilize a combination of DC/T-cell co-culture assays and a novel lipid bilayer system mimicking the T cell surface to delineate the role of DC-expressed γc during DC/T-cell interaction. We observed that γc in DC was recruited to the contact interface following MHCII ligation, and promoted IL-15Rα colocalization with engaged MHCII. Unexpectedly, trans-presentation of IL-15 was required for optimal CD4+T cell activation by DC and depended on DC γc expression. Neither recruitment of IL-15Rα nor IL-15 trans-signaling at the DC immune synapse (IS), required γc signaling in DC, suggesting that γc facilitates IL-15 transpresentation through induced intermolecular associations or cytoskeletal reorganization following MHCII ligation. These findings show that DC-expressed γc is required for effective antigen-induced CD4+ T cell activation. We reveal a novel mechanism for recruitment of DC IL-15/IL-15Rα complexes to the IS, leading to CD4+ T cell costimulation through localized IL-15 transpresentation that is coordinated with antigen-recognition.}, issn = {2398-502X}, doi = {10.12688/wellcomeopenres.14493.2}, author = {Beilin, Chiara and Choudhuri, Kaushik and Bouma, Gerben and Malinova, Dessislava and Llodra, Jaime and Stokes, David L and Shimaoka, Motumu and Springer, Timothy A. and Dustin, Michael L and Thrasher, Adrian J and Burns, Siobhan O} } @article {1344641, title = {Fusion surface structure, function, and dynamics of gamete fusogen HAP2}, journal = {Elife}, volume = {7}, year = {2018}, month = {2018 10 03}, abstract = {HAP2 is a class II gamete fusogen in many eukaryotic kingdoms. A crystal structure of HAP2 shows a trimeric fusion state. Domains D1, D2.1 and D2.2 line the 3-fold axis; D3 and a stem pack against the outer surface. Surprisingly, hydrogen-deuterium exchange shows that surfaces of D1, D2.2 and D3 closest to the 3-fold axis are more dynamic than exposed surfaces. Three fusion helices in the fusion loops of each monomer expose hydrophobic residues at the trimer apex that are splayed from the 3-fold axis, leaving a solvent-filled cavity between the fusion loops in each monomer. At the base of the two fusion loops, Arg185 docks in a carbonyl cage. Comparisons to other structures, dynamics, and the greater effect on gamete fusion of mutation of axis-proximal than axis-distal fusion helices suggest that the apical portion of each monomer could tilt toward the 3-fold axis with merger of the fusion helices into a common fusion surface.}, issn = {2050-084X}, doi = {10.7554/eLife.39772}, author = {Feng, Juan and Dong, Xianchi and Pinello, Jennifer and Zhang, Jun and Lu, Chafen and Iacob, Roxana E and Engen, John R and Snell, William J and Springer, Timothy A.} } @article {1317302, title = {A Milieu Molecule for TGF-β Required for Microglia Function in the Nervous System}, journal = {Cell}, year = {2018}, month = {2018 Jun 12}, abstract = {Extracellular proTGF-β is covalently linked to "milieu" molecules in the matrix or on cell surfaces and is latent until TGF-β is released by integrins. Here, we show that LRRC33 on the surface of microglia functions as a milieu molecule and enables highly localized, integrin-αVβ8-dependent TGF-β activation. Lrrc33 mice lack CNS vascular abnormalities associated with deficiency in TGF-β-activating integrins but have microglia with a reactive phenotype and after 2\ months develop ascending paraparesis with loss of myelinated axons and death by 5\ months. Whole bone marrow transplantation results in selective repopulation of Lrrc33 brains with WT microglia and halts disease progression. The phenotypes of WT and Lrrc33 microglia in the same brain suggest that there is little spreading of TGF-β activated from one microglial cell to neighboring microglia. Our results suggest that interactions between integrin-bearing cells and cells bearing milieu molecule-associated TGF-β provide localized and selective activation of TGF-β.}, issn = {1097-4172}, doi = {10.1016/j.cell.2018.05.027}, author = {Qin, Yan and Garrison, Brian S and Ma, Wenjiang and Wang, Rui and Jiang, Aiping and Li, Jing and Mistry, Meeta and Bronson, Roderick T. and Santoro, Daria and Franco, Charlotte and Robinton, Daisy A and Beth Stevens and Rossi, Derrick J and Lu, Chafen and Springer, Timothy A.} } @article {1309106, title = {Ligand and cation-induced structural alterations of the leukocyte integrin LFA-1}, journal = {J Biol Chem}, year = {2018}, month = {2018 Mar 05}, abstract = {In aI integrins including leukocyte function-associated antigen-1 (LFA-1), ligand-binding function is delegated to the aI domain, requiring extra steps in the relay of signals that activate ligand binding and coordinate it with cytoplasmic signals. Crystal structures reveal great variation in orientation between the aI domain and the remainder of the integrin head. Here, we investigated the mechanisms involved in signal relay to the aI domain, including whether binding of the ligand intercellular adhesion molecule-1 (ICAM-1) to the aI domain is linked to headpiece opening and engenders a preferred aI domain orientation. Using small-angle Xray scattering (SAXS) and negative-stain EM we define structures of ICAM-1, LFA-1, and their complex, and the effect of activation by Mn2+. Headpiece opening was substantially stabilized by substitution of Mg2+ with Mn2+ and became complete upon ICAM-1 addition. These agents stabilized aI-headpiece orientation, resulting in a well-defined orientation of ICAM-1 such that its tandem Iglike domains pointed in the opposite direction from the β-subunit leg of LFA-1. Mutations in the integrin βI domain α1/α1{\textquoteleft} helix stabilizing either the open or the closed βI-domain conformation indicated that α1/α1{\textquoteleft} helix movements are linked to ICAM-1 binding by the aI domain and to the extended-open conformation of the ectodomain. The LFA-1--ICAM-1 orientation described here with ICAM-1 pointing anti-parallel to the LFA-1 β-subunit leg is the same orientation that would be stabilized by tensile force transmitted between the ligand and the actin cytoskeleton, and is consistent with the cytoskeletal force model of integrin activation.}, issn = {1083-351X}, doi = {10.1074/jbc.RA117.000710}, author = {Sen, Mehmet and Koksal, Adem C and Yuki, Koichi and Wang, Jianchuan and Springer, Timothy A.} } @article {1300615, title = {Measuring integrin conformational change on the cell surface with super-resolution microscopy}, journal = {Cell Reports}, volume = {In Press}, year = {2018}, abstract = {We use super-resolution interferometric photoactivation and localization microscopy (iPALM) and a constrained photoactivatable fluorescent protein integrin fusion to measure the displacement of the head of integrin lymphocyte function-associated 1 (LFA-1) resulting from integrin conformational change on the cell surface. We demonstrate that the distance of the LFA-1 head increases substantially between basal and ligand-engaged conformations, which can only be explained at the molecular level by integrin extension. We further demonstrate that one class of integrin antagonist maintains the bent conformation, while another antagonist class induces extension. Our molecular scale measurements on cell-surface LFA-1 are in excellent agreement with distances derived from crystallographic and electron microscopy structures of bent and extended integrins. Our distance measurements are also in excellent agreement with a previous model of LFA-1 bound to ICAM-1 derived from the orientation of LFA-1 on the cell surface measured using fluorescence polarization microscopy.}, url = {http://www.cell.com/cell-reports/abstract/S2211-1247(18)30111-6}, author = {Travis I. Moore and Jesse Aaron and Teng-Leong Chew and Springer, Timothy A.} } @article {1297825, title = {High integrin αVβ6 affinity reached by hybrid domain deletion slows ligand-binding on-rate}, journal = {Proc Natl Acad Sci U S A}, year = {2018}, month = {2018 Jan 29}, abstract = {The role of the hybrid domain in integrin affinity regulation is unknown, as is whether the kinetics of ligand binding is modulated by integrin affinity state. Here, we compare cell surface and soluble integrin αVβ6 truncation mutants for ligand-binding affinity, kinetics, and thermodynamics. Removal of the integrin transmembrane/cytoplasmic domains or lower legs has little effect on αVβ6 affinity, in contrast to β1 integrins. In integrin opening, rearrangement at the interface between the βI and hybrid domains is linked to remodeling at the ligand-binding site at the opposite end of the βI domain, which greatly increases in affinity in the open conformation. The larger size of the βI-hybrid interface in the closed state suggests that the hybrid domain stabilizes closing. In agreement, deletion of the hybrid domain raised affinity by 50-fold. Surface plasmon resonance and isothermal titration calorimetry gave similar results and the latter revealed tradeoffs between enthalpy and entropy not apparent from affinity. At extremely high affinity reached in Mn2+ with hybrid domain truncation, αVβ6 on-rate for both pro-TGF-β1 and fibronectin declined. The results suggest that the open conformation of αVβ6 has lower on-rate than the closed conformation, correlate with constriction of the ligand-binding pocket in open αVβ6 structures, and suggest that the extended-closed conformation is kinetically selected for ligand binding. Subsequent transition to the extended-open conformation is stabilized by its much higher affinity for ligand and would also be stabilized by force exerted across ligand-bound integrins by the actin cytoskeleton.}, issn = {1091-6490}, doi = {10.1073/pnas.1718662115}, author = {Dong, Xianchi and Zhao, Bo and Lin, Fu-Yang and Lu, Chafen and Rogers, Bruce N and Springer, Timothy A.} } @article {1297567, title = {Tolloid cleavage activates latent GDF8 by priming the pro-complex for dissociation}, journal = {EMBO J}, year = {2018}, month = {2018 Jan 17}, abstract = {Growth differentiation factor 8 (GDF8)/myostatin is a latent TGF-β family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid-cleaved GDF8 pro-complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro-complexes reveals a V-shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring-like, cross-armed conformation of latent TGF-β1. Surprisingly, Tolloid-cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-β1 to the α1-helix, latency lasso, and β1-strand in the prodomain and to the β6{\textquoteright}- and β7{\textquoteright}-strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain.}, issn = {1460-2075}, doi = {10.15252/embj.201797931}, author = {Le, Viet Q and Iacob, Roxana E and Tian, Yuan and McConaughy, William and Jackson, Justin and Su, Yang and Zhao, Bo and Engen, John R and Pirruccello-Straub, Michelle and Springer, Timothy A.} } @article {1281966, title = {Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration}, journal = {Nat Commun}, volume = {8}, number = {1}, year = {2017}, month = {2017 Dec 11}, pages = {2047}, abstract = {Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, {\r A}-scale structures of cell surface receptors in the context of functional and cellular, μm-scale measurements.}, issn = {2041-1723}, doi = {10.1038/s41467-017-01848-y}, author = {Nordenfelt, Pontus and Moore, Travis I and Mehta, Shalin B and Kalappurakkal, Joseph Mathew and Swaminathan, Vinay and Koga, Nobuyasu and Lambert, Talley J and Baker, David and Waters, Jennifer C and Oldenbourg, Rudolf and Tani, Tomomi and Mayor, Satyajit and Waterman, Clare M and Springer, Timothy A.} } @article {1281971, title = {Energy landscape differences among integrins establish the framework for understanding activation}, journal = {J Cell Biol}, year = {2017}, month = {2017 Nov 09}, abstract = {Why do integrins differ in basal activity, and how does affinity for soluble ligand correlate with cellular adhesiveness? We show that basal conformational equilibrium set points for integrin α4β1 are cell type specific and differ from integrin α5β1 when the two integrins are coexpressed on the same cell. Although α4β1 is easier to activate, its high-affinity state binds vascular cell adhesion molecule and fibronectin 100- to 1,000-fold more weakly than α5β1 binds fibronectin. Furthermore, the difference in affinity between the high- and low-affinity states is more compressed in α4β1 (600- to 800-fold) than in α5β1 (4,000- to 6,000-fold). α4β1 basal conformational equilibria differ among three cell types, define affinity for soluble ligand and readiness for priming, and may reflect differences in interactions with intracellular adaptors but do not predict cellular adhesiveness for immobilized ligand. The measurements here provide a necessary framework for understanding integrin activation in intact cells, including activation of integrin adhesiveness by application of tensile force by the cytoskeleton, across ligand-integrin-adaptor complexes.}, issn = {1540-8140}, doi = {10.1083/jcb.201701169}, author = {Li, Jing and Springer, Timothy A.} } @article {1246711, title = {Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions}, journal = {Proc Natl Acad Sci U S A}, volume = {114}, number = {40}, year = {2017}, month = {2017 Oct 03}, pages = {10648-10653}, abstract = {Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven "retrograde flow" of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues.}, issn = {1091-6490}, doi = {10.1073/pnas.1701136114}, author = {Swaminathan, Vinay and Kalappurakkal, Joseph Mathew and Mehta, Shalin B and Nordenfelt, Pontus and Moore, Travis I and Koga, Nobuyasu and Baker, David A and Oldenbourg, Rudolf and Tani, Tomomi and Mayor, Satyajit and Springer, Timothy A. and Waterman, Clare M} } @article {1246696, title = {Prodomain-Growth Factor Swapping in the Structure of pro-TGF-β1}, journal = {J Biol Chem}, year = {2017}, month = {2017 Nov 05}, abstract = {Transforming growth factor (TGF)-β is synthesized as a proprotein that dimerizes in the endoplasmic reticulum. After processing in the Golgi to cleave the N-terminal prodomain from the C-terminal growth factor (GF) domain in each monomer, pro-TGF-β is secreted and stored in latent complexes. It is unclear which prodomain and GF monomer are linked prior to proprotein convertase (PC) cleavage, and how much conformational change occurs following cleavage. We have determined a structure of pro-TGF-β1 with the PC cleavage site mutated, to mimic the structure of the TGF-β1 proprotein. Structure, mutation, and model building demonstrate that the prodomain arm domain in one monomer is linked to the GF that interacts with the arm domain in the other monomer in the dimeric structure, i.e., the prodomain arm domain and GF domain in each monomer are swapped. Swapping has important implications for the mechanism of biosynthesis in the TGF-β family and is relevant to the mechanism for preferential formation of heterodimers over homodimers for some members of the TGF-β family. Our structure, together with two previous ones, also provides insights into which regions of the prodomain-GF complex are highly structurally conserved, and which are perturbed by crystal lattice contacts.}, issn = {1083-351X}, doi = {10.1074/jbc.M117.809657}, author = {Zhao, Bo and Xu, Shutong and Dong, Xianchi and Lu, Chafen and Springer, Timothy A.} } @article {1170526, title = {Flow-induced elongation of von Willebrand factor precedes tension-dependent activation}, journal = {Nat Commun}, volume = {8}, number = {1}, year = {2017}, month = {2017 Aug 23}, pages = {324}, abstract = {Von Willebrand factor, an ultralarge concatemeric blood protein, must bind to platelet GPIbα during bleeding to mediate hemostasis, but not in the normal circulation to avoid thrombosis. Von Willebrand factor is proposed to be mechanically activated by flow, but the mechanism remains unclear. Using microfluidics with single-molecule imaging, we simultaneously monitored reversible Von Willebrand factor extension and binding to GPIbα under flow. We show that Von Willebrand factor is activated through a two-step conformational transition: first, elongation from compact to linear form, and subsequently, a tension-dependent local transition to a state with high affinity for GPIbα. High-affinity sites develop only in upstream regions of VWF where tension exceeds ~21 pN and depend upon electrostatic interactions. Re-compaction of Von Willebrand factor is accelerated by intramolecular interactions and increases GPIbα dissociation rate. This mechanism enables VWF to be locally activated by hydrodynamic force in hemorrhage and rapidly deactivated downstream, providing a paradigm for hierarchical mechano-regulation of receptor-ligand binding.Von Willebrand factor (VWF) is a blood protein involved in clotting and is proposed to be activated by flow, but the mechanism is unknown. Here the authors show that VWF is first converted from a compact to linear form by flow, and is subsequently activated to bind GPIbα in a tension-dependent manner.}, issn = {2041-1723}, doi = {10.1038/s41467-017-00230-2}, author = {Fu, Hongxia and Jiang, Yan and Yang, Darren and Scheiflinger, Friedrich and Wong, Wesley P and Springer, Timothy A.} } @article {1077996, title = {Rules of engagement between αvβ6 integrin and foot-and-mouth disease virus}, journal = {Nat Commun}, volume = {8}, year = {2017}, month = {2017 May 23}, pages = {15408}, abstract = {Foot-and-mouth disease virus (FMDV) mediates cell entry by attachment to an integrin receptor, generally αvβ6, via a conserved arginine-glycine-aspartic acid (RGD) motif in the exposed, antigenic, GH loop of capsid protein VP1. Infection can also occur in tissue culture adapted virus in the absence of integrin via acquired basic mutations interacting with heparin sulphate (HS); this virus is attenuated in natural infections. HS interaction has been visualized at a conserved site in two serotypes suggesting a propensity for sulfated-sugar binding. Here we determined the interaction between αvβ6 and two tissue culture adapted FMDV strains by cryo-electron microscopy. In the preferred mode of engagement, the fully open form of the integrin, hitherto unseen at high resolution, attaches to an extended GH loop via interactions with the RGD motif plus downstream hydrophobic residues. In addition, an N-linked sugar of the integrin attaches to the previously identified HS binding site, suggesting a functional role.}, issn = {2041-1723}, doi = {10.1038/ncomms15408}, author = {Kotecha, Abhay and Wang, Quan and Dong, Xianchi and Ilca, Serban L and Ondiviela, Marina and Zihe, Rao and Seago, Julian and Charleston, Bryan and Fry, Elizabeth E and Abrescia, Nicola G A and Springer, Timothy A. and Huiskonen, Juha T and Stuart, David I} } @article {1067526, title = {Atypical interactions of integrin αVβ8 with pro-TGF-β1}, journal = {Proc Natl Acad Sci U S A}, year = {2017}, month = {2017 May 08}, abstract = { Integrins αVβ6 and αVβ8 are specialized for recognizing pro-TGF-β and activating its growth factor by releasing it from the latency imposed by its surrounding prodomain. The integrin αVβ8 is atypical among integrins in lacking sites in its cytoplasmic domain for binding to actin cytoskeleton adaptors. Here, we examine αVβ8 for atypical binding to pro-TGF-β1. In contrast to αVβ6, αVβ8 has a constitutive extended-closed conformation, and binding to pro-TGF-β1 does not stabilize the open conformation of its headpiece. Although Mn(2+) potently activates other integrins and increases affinity of αVβ6 for pro-TGF-β1 25- to 55-fold, it increases αVβ8 affinity only 2- to 3-fold. This minimal effect correlates with the inability of Mn(2+) and pro-TGF-β1 to stabilize the open conformation of the αVβ8 headpiece. Moreover, αVβ8 was inhibited by high concentrations of Mn(2+) and was stimulated and inhibited at markedly different Ca(2+) concentrations than αVβ6 These unusual characteristics are likely to be important in the still incompletely understood physiologic mechanisms that regulate αVβ8 binding to and activation of pro-TGF-β. }, issn = {1091-6490}, doi = {10.1073/pnas.1705129114}, author = {Wang, Jianchuan and Dong, Xianchi and Zhao, Bo and Li, Jing and Lu, Chafen and Springer, Timothy A.} } @article {1062021, title = {Integrin extension enables ultrasensitive regulation by cytoskeletal force}, journal = {Proc Natl Acad Sci U S A}, year = {2017}, month = {2017 Apr 17}, abstract = { Integrins undergo large-scale conformational changes upon activation. Signaling events driving integrin activation have previously been discussed conceptually, but not quantitatively. Here, recent measurements of the intrinsic ligand-binding affinity and free energy of each integrin conformational state on the cell surface, together with the length scales of conformational change, are used to quantitatively compare models of activation. We examine whether binding of cytoskeletal adaptors to integrin cytoplasmic domains is sufficient for activation or whether exertion of tensile force by the actin cytoskeleton across the integrin-ligand complex is also required. We find that only the combination of adaptor binding and cytoskeletal force provides ultrasensitive regulation. Moreover, switch-like activation by force depends on the large, \>130 {\r A} length-scale change in integrin extension, which is well tailored to match the free-energy difference between the inactive (bent-closed) and active (extended-open) conformations. The length scale and energy cost in integrin extension enable activation by force in the low pN range and appear to be the key specializations that enable cell adhesion through integrins to be coordinated with cytoskeletal dynamics. }, issn = {1091-6490}, doi = {10.1073/pnas.1704171114}, author = {Li, Jing and Springer, Timothy A.} } @article {1039511, title = {Distinct recognition of complement iC3b by integrins αXβ2 and αMβ2}, journal = {Proc Natl Acad Sci U S A}, year = {2017}, month = {2017 Mar 14}, abstract = { Recognition by the leukocyte integrins αXβ2 and αMβ2 of complement iC3b-opsonized targets is essential for effector functions including phagocytosis. The integrin-binding sites on iC3b remain incompletely characterized. Here, we describe negative-stain electron microscopy and biochemical studies of αXβ2 and αMβ2 in complex with iC3b. Despite high homology, the two integrins bind iC3b at multiple distinct sites. αXβ2 uses the αX αI domain to bind iC3b on its C3c moiety at one of two sites: a major site at the interface between macroglobulin (MG) 3 and MG4 domains, and a less frequently used site near the C345C domain. In contrast, αMβ2 uses its αI domain to bind iC3b at the thioester domain and simultaneously interacts through a region near the αM β-propeller and β2 βI domain with a region of the C3c moiety near the C345C domain. Remarkably, there is no overlap between the primary binding site of αXβ2 and the binding site of αMβ2 on iC3b. Distinctive binding sites on iC3b by integrins αXβ2 and αMβ2 may be biologically beneficial for leukocytes to more efficiently capture opsonized pathogens and to avoid subversion by pathogen factors. [[{"fid":3464186,"view_mode":"default","type":"media","attributes":{"height":"550","width":"980","class":"media-element file-default"}}]] }, issn = {1091-6490}, doi = {10.1073/pnas.1620881114}, author = {Xu, Shutong and Wang, Jianchuan and Wang, Jia-Huai and Springer, Timothy A.} } @article {990116, title = {Sorting zebrafish thrombocyte lineage cells with a Cd41 monoclonal antibody enriches hematopoietic stem cell activity}, journal = {Blood}, year = {2017}, month = {2017 Jan 26}, issn = {1528-0020}, doi = {10.1182/blood-2016-12-759993}, author = {Gansner, John M and Leung, Alexander D and Superdock, Michael and Blair, Megan C and Ammerman, Michelle B and Durand, Ellen M and Barut, Bruce and Handin, Robert I and Stachura, David L and Lu, Chafen and Springer, Timothy A. and Zon, Leonard I} } @article {988991, title = {Conformational equilibria and intrinsic affinities define integrin activation}, journal = {EMBO J}, year = {2017}, month = {2017 Jan 25}, abstract = {We show that the three conformational states of integrin α5β1 have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000-fold higher affinity of the extended-open state than the bent-closed and extended-closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α5β1 On the surface of K562 cells, α5β1 is 99.8\% bent-closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N-glycans and leg domains in each subunit that connect the ligand-binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single-span transmembrane domains.}, issn = {1460-2075}, doi = {10.15252/embj.201695803}, author = {Li, Jing and Su, Yang and Xia, Wei and Qin, Yan and Humphries, Martin J and Vestweber, Dietmar and Caba{\~n}as, Carlos and Lu, Chafen and Springer, Timothy A.} } @article {988986, title = {Force interacts with macromolecular structure in activation of TGF-β}, journal = {Nature}, volume = {542}, number = {7639}, year = {2017}, month = {2017 Feb 02}, pages = {55-59}, abstract = {Integrins are adhesion receptors that transmit force across the plasma membrane between extracellular ligands and the actin cytoskeleton. In activation of the transforming growth factor-β1 precursor (pro-TGF-β1), integrins bind to the prodomain, apply force, and release the TGF-β growth factor. However, we know little about how integrins bind macromolecular ligands in the extracellular matrix or transmit force to them. Here we show how integrin αVβ6 binds pro-TGF-β1 in an orientation biologically relevant for force-dependent release of TGF-β from latency. The conformation of the prodomain integrin-binding motif differs in the presence and absence of integrin binding; differences extend well outside the interface and illustrate how integrins can remodel extracellular matrix. Remodelled residues outside the interface stabilize the integrin-bound conformation, adopt a conformation similar to earlier-evolving family members, and show how macromolecular components outside the binding motif contribute to integrin recognition. Regions in and outside the highly interdigitated interface stabilize a specific integrin/pro-TGF-β orientation that defines the pathway through these macromolecules which actin-cytoskeleton-generated tensile force takes when applied through the integrin β-subunit. Simulations of force-dependent activation of TGF-β demonstrate evolutionary specializations for force application through the TGF-β prodomain and through the β- and not α-subunit of the integrin.}, issn = {1476-4687}, doi = {10.1038/nature21035}, author = {Dong, Xianchi and Zhao, Bo and Iacob, Roxana E and Zhu, Jianghai and Koksal, Adem C and Lu, Chafen and Engen, John R and Springer, Timothy A.} } @article {989016, title = {Selective Targeting of High-Affinity LFA-1 Does Not Augment Costimulation Blockade in a Nonhuman Primate Renal Transplantation Model}, journal = {Am J Transplant}, year = {2016}, month = {2016 Nov 26}, abstract = {Costimulation blockade (CoB) via belatacept is a lower-morbidity alternative to calcineurin inhibitor (CNI)-based immunosuppression. However, it has higher rates of early acute rejection. These early rejections are mediated in part by memory T cells, which have reduced dependence on the pathway targeted by belatacept and increased adhesion molecule expression. One such molecule is leukocyte function antigen (LFA)-1. LFA-1 exists in two forms: a commonly expressed, low-affinity form and a transient, high-affinity form, expressed only during activation. We have shown that antibodies reactive with LFA-1 regardless of its configuration are effective in eliminating memory T cells but at the cost of impaired protective immunity. Here we test two novel agents, leukotoxin A and AL-579, each of which targets the high-affinity form of LFA-1, to determine whether this more precise targeting prevents belatacept-resistant rejection. Despite evidence of ex vivo and in vivo ligand-specific activity, neither agent when combined with belatacept proved superior to belatacept monotherapy. Leukotoxin A approached a ceiling of toxicity before efficacy, while AL-579 failed to significantly alter the peripheral immune response. These data, and prior studies, suggest that LFA-1 blockade may not be a suitable adjuvant agent for CoB-resistant rejection.}, issn = {1600-6143}, doi = {10.1111/ajt.14141}, author = {Samy, K P and Anderson, D J and Lo, D J and Mulvihill, M S and Song, M. and Farris, A B and Parker, B S and MacDonald, A L and Lu, C. and Springer, T.A. and Kachlany, S C and Reimann, K A and How, T and Leopardi, F V and Franke, K S and Williams, K D and Collins, B H and Kirk, A D} } @article {938306, title = {Coordinated integrin activation by actin-dependent force during T-cell migration.}, journal = {Nat Commun}, volume = {7}, year = {2016}, month = {2016 Oct 10}, pages = {13119}, abstract = {For a cell to move forward it must convert chemical energy into mechanical propulsion. Force produced by actin polymerization can generate traction across the plasma membrane by transmission through integrins to their ligands. However, the role this force plays in integrin activation is unknown. Here we show that integrin activity and cytoskeletal dynamics are reciprocally linked, where actin-dependent force itself appears to regulate integrin activity. We generated fluorescent tension-sensing constructs of integrin αLβ2 (LFA-1) to visualize intramolecular tension during cell migration. Using quantitative imaging of migrating T cells, we correlate tension in the αL or β2 subunit with cell and actin dynamics. We find that actin engagement produces tension within the β2 subunit to induce and stabilize an active integrin conformational state and that this requires intact talin and kindlin motifs. This supports a general mechanism where localized actin polymerization can coordinate activation of the complex machinery required for cell migration.}, issn = {2041-1723}, doi = {10.1038/ncomms13119}, author = {Nordenfelt, Pontus and Elliott, Hunter L and Springer, Timothy A.} } @article {938321, title = {Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia.}, journal = {Haematologica}, volume = {101}, number = {1}, year = {2016}, month = {2016 Jan}, pages = {46-56}, abstract = {Several patients have been reported to have variant dominant forms of Glanzmann thrombasthenia, associated with macrothrombocytopenia and caused by gain-of-function mutations of ITGB3 or ITGA2B leading to reduced surface expression and constitutive activation of integrin αIIbβ3. The mechanisms leading to a bleeding phenotype of these patients have never been addressed. The aim of this study was to unravel the mechanism by which ITGB3 mutations causing activation of αIIbβ3 lead to platelet dysfunction and macrothrombocytopenia. Using platelets from two patients carrying the β3 del647-686 mutation and Chinese hamster ovary cells expressing different αIIbβ3-activating mutations, we showed that reduced surface expression of αIIbβ3 is due to receptor internalization. Moreover, we demonstrated that permanent triggering of αIIbβ3-mediated outside-in signaling causes an impairment of cytoskeletal reorganization arresting actin turnover at the stage of polymerization. The induction of actin polymerization by jasplakinolide, a natural toxin that promotes actin nucleation and prevents depolymerization of stress fibers, in control platelets produced an impairment of platelet function similar to that of patients with variant forms of dominant Glanzmann thrombasthenia. del647-686β3-transduced murine megakaryocytes generated proplatelets with a reduced number of large tips and asymmetric barbell-proplatelets, suggesting that impaired cytoskeletal rearrangement is the cause of macrothrombocytopenia. These data show that impaired cytoskeletal remodeling caused by a constitutively activated αIIbβ3 is the main effector of platelet dysfunction and macrothrombocytopenia, and thus of bleeding, in variant forms of dominant Glanzmann thrombasthenia.}, keywords = {Animals, Blood Platelets, CHO Cells, Cricetinae, Cricetulus, Cytoskeleton, Female, Humans, Integrin alpha2, Integrin beta3, Male, Mutation, Thrombasthenia, Thrombocytopenia}, issn = {1592-8721}, doi = {10.3324/haematol.2015.130849}, author = {Bury, Loredana and Falcinelli, Emanuela and Chiasserini, Davide and Springer, Timothy A. and Italiano, Joseph E and Gresele, Paolo} } @article {938316, title = {Interrogating the Plasmodium Sporozoite Surface: Identification of Surface-Exposed Proteins and Demonstration of Glycosylation on CSP and TRAP by Mass Spectrometry-Based Proteomics.}, journal = {PLoS Pathog}, volume = {12}, number = {4}, year = {2016}, month = {2016 Apr}, pages = {e1005606}, abstract = {Malaria parasite infection is initiated by the mosquito-transmitted sporozoite stage, a highly motile invasive cell that targets hepatocytes in the liver for infection. A promising approach to developing a malaria vaccine is the use of proteins located on the sporozoite surface as antigens to elicit humoral immune responses that prevent the establishment of infection. Very little of the P. falciparum genome has been considered as potential vaccine targets, and candidate vaccines have been almost exclusively based on single antigens, generating the need for novel target identification. The most advanced malaria vaccine to date, RTS,S, a subunit vaccine consisting of a portion of the major surface protein circumsporozoite protein (CSP), conferred limited protection in Phase III trials, falling short of community-established vaccine efficacy goals. In striking contrast to the limited protection seen in current vaccine trials, sterilizing immunity can be achieved by immunization with radiation-attenuated sporozoites, suggesting that more potent protection may be achievable with a multivalent protein vaccine. Here, we provide the most comprehensive analysis to date of proteins located on the surface of or secreted by Plasmodium falciparum salivary gland sporozoites. We used chemical labeling to isolate surface-exposed proteins on sporozoites and identified these proteins by mass spectrometry. We validated several of these targets and also provide evidence that components of the inner membrane complex are in fact surface-exposed and accessible to antibodies in live sporozoites. Finally, our mass spectrometry data provide the first direct evidence that the Plasmodium surface proteins CSP and TRAP are glycosylated in sporozoites, a finding that could impact the selection of vaccine antigens.}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1005606}, author = {Swearingen, Kristian E and Lindner, Scott E and Shi, Lirong and Shears, Melanie J and Harupa, Anke and Hopp, Christine S and Vaughan, Ashley M and Springer, Timothy A. and Moritz, Robert L and Kappe, Stefan H I and Sinnis, Photini} } @article {938311, title = {Structural Biology and Evolution of the TGF-β Family.}, journal = {Cold Spring Harb Perspect Biol}, year = {2016}, month = {2016 Sep 16}, abstract = {We review the evolution and structure of members of the transforming growth factor β (TGF-β) family, antagonistic or agonistic modulators, and receptors that regulate TGF-β signaling in extracellular environments. The growth factor (GF) domain common to all family members and many of their antagonists evolved from a common cystine knot growth factor (CKGF) domain. The CKGF superfamily comprises six distinct families in primitive metazoans, including the TGF-β and Dan families. Compared with Wnt/Frizzled and Notch/Delta families that also specify body axes, cell fate, tissues, and other families that contain CKGF domains that evolved in parallel, the TGF-β family was the most fruitful in evolution. Complexes between the prodomains and GFs of the TGF-β family suggest a new paradigm for regulating GF release by conversion from closed- to open-arm procomplex conformations. Ternary complexes of the final step in extracellular signaling show how TGF-β GF dimers bind type I and type II receptors on the cell surface, and enable understanding of much of the specificity and promiscuity in extracellular signaling. However, structures suggest that when GFs bind repulsive guidance molecule (RGM) family coreceptors, type I receptors do not bind until reaching an intracellular, membrane-enveloped compartment, blurring the line between extra- and intracellular signaling. Modulator protein structures show how structurally diverse antagonists including follistatins, noggin, and members of the chordin family bind GFs to regulate signaling; complexes with the Dan family remain elusive. Much work is needed to understand how these molecular components assemble to form signaling hubs in extracellular environments in vivo.}, issn = {1943-0264}, doi = {10.1101/cshperspect.a022103}, author = {Hinck, Andrew P and Mueller, Thomas D and Springer, Timothy A.} } @article {746696, title = {Relating conformation to function in integrin α5β1}, journal = {Proc Natl Acad Sci U S A.}, year = {2016}, month = {17 Jun 2016}, abstract = { Whether β1\ integrin ectodomains visit conformational states similarly to β2\ and β3\ integrins has not been characterized. Furthermore, despite a wealth of activating and inhibitory antibodies to β1\ integrins, the conformational states that these antibodies stabilize, and the relation of these conformations to function, remain incompletely characterized. Using negative-stain electron microscopy, we show that the integrin α5β1\ ectodomain adopts extended-closed and extended-open conformations as well as a bent conformation. Antibodies SNAKA51, 8E3, N29, and 9EG7 bind to different domains in the α5\ or β1\ legs, activate, and stabilize extended ectodomain conformations. Antibodies 12G10 and HUTS-4 bind to the β1\ βI domain and hybrid domains, respectively, activate, and stabilize the open headpiece conformation. Antibody TS2/16 binds a similar epitope as 12G10, activates, and appears to stabilize an open βI domain conformation without requiring extension or hybrid domain swing-out. mAb13 and SG/19 bind to the βI domain and βI-hybrid domain interface, respectively, inhibit, and stabilize the closed conformation of the headpiece. The effects of the antibodies on cell adhesion to fibronectin substrates suggest that the extended-open conformation of α5β1\ is adhesive and that the extended-closed and bent-closed conformations are nonadhesive. The functional effects and binding sites of antibodies and fibronectin were consistent with their ability in binding to α5β1\ on cell surfaces to cross-enhance or inhibit one another by competitive or noncompetitive (allosteric) mechanisms.}, url = {http://www.pnas.org.ezp-prod1.hul.harvard.edu/content/early/2016/06/16/1605074113.long}, author = {Su, Yang and Xia, Wei and Li. Jing and Walz, Thomas and Humphries, Martin J. and Vestweber, Dietmar and Caba{\~n}as, Carlos and Lu, Chafen and Springer, T.A.} } @article {689086, title = {Leukocyte integrin αLβ2 headpiece structures: The αI domain, the pocket for the internal ligand, and concerted movements of its loops}, journal = {Proc Natl Acad Sci U S A.}, year = {2016}, month = {2 March, 2016}, abstract = {High-resolution crystal structures of the headpiece of lymphocyte function-associated antigen-1 (integrin αLβ2) reveal how the αI domain interacts with its platform formed by the α-subunit β-propeller and β-subunit βI domains. The αLβ2\ structures compared with αXβ2\ structures show that the αI domain, tethered through its N-linker and a disulfide to a stable β-ribbon pillar near the center of the platform, can undergo remarkable pivoting and tilting motions that appear buffered by N-glycan decorations that differ between αL\ and αX\ subunits. Rerefined β2\ integrin structures reveal details including pyroglutamic acid at the β2\ N terminus and bending within the EGF1 domain. Allostery is relayed to the αI domain by an internal ligand that binds to a pocket at the interface between the β-propeller and βI domains. Marked differences between the αL\ and αX\ subunit β-propeller domains concentrate near the binding pocket and αI domain interfaces. Remarkably, movement in allostery in the βI domain of specificity determining loop 1 (SDL1) causes concerted movement of SDL2 and thereby tightens the binding pocket for the internal ligand.}, author = {Sen, Mehmet and Springer, T.A.} } @article {624496, title = {β-subunit Binding is Sufficient for Ligands to open the Integrin αIIbβ3 Headpiece.}, journal = {J Biol Chem.}, volume = {291}, number = {9}, year = {2016}, month = {16 Feb 2016}, pages = {4537-56}, abstract = {The platelet integrin αIIbβ3 binds to a KQAGDV motif at the fibrinogen γ-chain C-terminus and to RGD motifs present in loops in many extracellular matrix proteins. These ligands bind in a groove between the integrin α and β subunits; the basic Lys or Arg sidechain hydrogen bonds to the αIIb-subunit and the acidic Asp sidechain coordinates to a metal ion held by the β3-subunit. Ligand binding induces headpiece opening, with conformational change in the β-subunit. During this opening, RGD slides in the ligand-binding pocket towards αIIb, with movement of the βI-domain β1-α1 loop toward αIIb, enabling formation of direct, charged hydrogen bonds between the Arg sidechain and αIIb. Here we test whether ligand interactions with β3 suffice for stable ligand binding and headpiece opening. We find that the AGDV tetrapeptide from KQAGDV binds to the αIIbβ3 headpiece with affinity comparable to the RGDSP peptide from fibronectin. AGDV induced complete headpiece opening in solution as shown by increase in hydrodynamic radius. Soaking of AGDV into closed αIIbβ3 headpiece crystals induced intermediate states similarly to RGDSP. AGDV has very little contact with the α subunit. Furthermore, as measured by epitope exposure, AGDV, like the fibrinogen γ C-terminal peptide and RGD, caused integrin extension on the cell surface. Thus, pushing by the β3 subunit on Asp is sufficient for headpiece opening and ligand sliding, and no pulling by the αIIb subunit on Arg is required. Copyright {\textcopyright} 2015, The American Society for Biochemistry and Molecular Biology.}, url = {http://www.ncbi.nlm.nih.gov/pubmed/26631735}, author = {Lin, Fu-Yang and Zhu, Jianghai and Eng, Edward T. and Hudson, Nathan E. and Springer, Timothy A.} } @article {938301, title = {Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13.}, journal = {J Thromb Haemost}, volume = {13}, number = {11}, year = {2015}, month = {2015 Nov}, pages = {2063-75}, abstract = {BACKGROUND: Recently, conformational activation of ADAMTS-13 was identified. This mechanism showed the evolution from a condensed conformation, in which the proximal MDTCS and distal T2-CUB2 domains are in close contact with each other, to an activated, open structure due to binding with von Willebrand factor (VWF). OBJECTIVES: Identification of cryptic epitope/exosite exposure after conformational activation and of sites of flexibility in ADAMTS-13. METHODS: The activating effect of 25 anti-T2-CUB2 antibodies was studied in the FRETS-VWF73 and the vortex assay. Cryptic epitope/exosite exposure was determined with ELISA and VWF binding assay. The molecular basis for flexibility was hypothesized through rapid automatic detection and alignment of repeats (RADAR) analysis, tested with ELISA using deletion variants and visualized using electron microscopy. RESULTS: Eleven activating anti-ADAMTS-13 antibodies, directed against the T5-CUB2 domains, were identified in the FRETS-VWF73 assay. RADAR analysis identified three linker regions in the distal domains. Interestingly, identification of an antibody recognizing a cryptic epitope in the metalloprotease domain confirmed the contribution of these linker regions to conformational activation of the enzyme. The proof of flexibility around both the T2 and metalloprotease domains, as shown by by electron microscopy, further supported this contribution. In addition, cryptic epitope exposure was identified in the distal domains, because activating anti-T2-CUB2 antibodies increased the binding to folded VWF up to ~3-fold. CONCLUSION: Conformational activation of ADAMTS-13 leads to cryptic epitope/exosite exposure in both proximal and distal domains, subsequently inducing increased activity. Furthermore, three linker regions in the distal domains are responsible for flexibility and enable the interaction between the proximal and the T8-CUB2 domains.}, keywords = {ADAM Proteins, Allosteric Regulation, Allosteric Site, Amino Acid Sequence, Antibodies, Monoclonal, Antigen-Antibody Reactions, Catalysis, Consensus Sequence, Enzyme Activation, Epitopes, Humans, Microscopy, Electron, Molecular Sequence Data, Protein Binding, Protein Conformation, Protein Folding, Protein Processing, Post-Translational, Protein Structure, Tertiary, Sequence Alignment, Sequence Homology, Amino Acid, Thrombospondin 1, von Willebrand Factor}, issn = {1538-7933}, doi = {10.1111/jth.13149}, author = {Deforche, L and Roose, E and Vandenbulcke, A and Vandeputte, N and Feys, H B and Springer, T.A. and Mi, L.Z. and Muia, J and Sadler, J E and Soejima, K and Rottensteiner, H and Deckmyn, H. and De Meyer, S. F. and Vanhoorelbeke, K.} } @article {624501, title = {Structural basis for quinine-dependent antibody binding to platelet integrin αIIbβ3.}, journal = {Blood}, volume = {126}, number = {18}, year = {2015}, pages = {2138-45}, abstract = {Drug-induced immune thrombocytopenia (DITP) is caused by antibodies that react with specific platelet-membrane glycoproteins when the provoking drug is present. More than 100 drugs have been implicated as triggers for this condition, quinine being one of the most common. The cause of DITP in most cases appears to be a drug-induced antibody that binds to a platelet membrane glycoprotein only when the drug is present. How a soluble drug promotes binding of an otherwise nonreactive immunoglobulin to its target, leading to platelet destruction, is uncertain, in part because of the difficulties of working with polyclonal human antibodies usually available only in small quantities. Recently, quinine-dependent murine monoclonal antibodies were developed that recognize a defined epitope on the β-propeller domain of the platelet integrin αIIb subunit (GPIIb) only when the drug is present and closely mimic the behavior of antibodies found in human patients with quinine-induced thrombocytopenia in vitro and in vivo. Here, we demonstrate specific, high-affinity binding of quinine to the complementarity-determining regions (CDRs) of these antibodies and define in crystal structures the changes induced in the CDR by this interaction. Because no detectable binding of quinine to the target integrin could be demonstrated in previous studies, the findings indicate that a hybrid paratope consisting of quinine and reconfigured antibody CDR plays a critical role in recognition of its target epitope by an antibody and suggest that, in this type of drug-induced immunologic injury, the primary reaction involves binding of the drug to antibody CDRs, causing it to acquire specificity for a site on a platelet integrin. {\textcopyright} 2015 by The American Society of Hematology.}, url = {http://www.ncbi.nlm.nih.gov/pubmed/26282540}, author = {Zhu, Jianghai and Zhu, Jieqing and Bougie, Daniel W. and Aster, Richard H. and Springer, Timothy A.} } @article {546711, title = {Carbon nanotube-assisted optical activation of TGF-β signalling by near-infrared light.}, journal = {Nat Nanotechnol.}, volume = {10}, number = {5}, year = {2015}, pages = {465-71}, abstract = {Receptor-mediated signal transduction modulates complex cellular behaviours such as cell growth, migration and differentiation. Although photoactivatable proteins have emerged as a powerful tool for controlling molecular interactions and\ signalling\ cascades at precise times and spaces using\ light, many of these\ light-sensitive proteins are activated by ultraviolent or visible\ light, which has limited tissue penetration. Here, we report a single-walled\ carbon\ nanotube (SWCNT)-assisted approach that enables\ near-infraredlight-triggered\ activation\ of transforming growth factor β (TGF-β) signal transduction, an important\ signalling\ pathway in embryonic development and cancer progression. The protein complex of TGF-β and its latency-associated peptide is conjugated onto SWCNTs, where TGF-β is inactive. Upon\ near-infrared\ irradiation, TGF-β is released through the photothermal effect of SWCNTs and becomes active. The released TGF-β activates downstream signal transduction in live cells and modulates cellular behaviours. Furthermore, preliminary studies show that the method can be used to mediate TGF-β\ signalling\ in living mice.}, author = {Lin L. and Liu L. and Zhao B. and Xie R. and Lin W. and Li H. and Li Y. and Shi M. and Chen Y.G. and Springer T.A. and Chen X.} } @article {546716, title = {Force-induced on-rate switching and modulation by mutations in gain-of-function von Willebrand diseases.}, journal = {Proc Natl Acad Sci USA}, volume = {112}, number = {15}, year = {2015}, pages = {4648-53}, abstract = {Mutations\ in the ultralong vascular protein\ von\ Willebrand\ factor (VWF) cause the common human bleeding disorder,\ von\ Willebranddisease (VWD). The A1 domain in VWF binds to glycoprotein Ibα (GPIbα) on platelets, in a reaction triggered, in part, by alterations in flow during bleeding.\ Gain-of-function\ mutations\ in A1 and GPIbα in VWD suggest conformational regulation. We report that force application switches A1 and/or GPIbα to a second state with faster\ on-rate, providing a mechanism for activating VWF binding to platelets.\ Switching\ occurs near 10 pN, a force that also induces a state of the receptor-ligand complex with slower off-rate. Force greatly increases the effects of VWD\ mutations, explaining pathophysiology. Conversion of single molecule kon (s(-1)) to bulk phase kon (s(-1)M(-1)) and the kon and koff values extrapolated to zero force for the low-force pathways show remarkably good agreement with bulk-phase measurements.\ }, author = {Kim, J. and Hudson, N.E. and Springer, T.A.} } @article {505526, title = {Structure of bone morphogenetic protein 9 procomplex}, journal = {Proc Natl Acad Sci USA}, number = {112}, year = {2015}, pages = {3710-5}, abstract = {Bone morphogenetic proteins (BMPs) belong to the TGF-β family, whose 33 members regulate multiple aspects of morphogenesis. TGF-β family members are secreted as procomplexes containing a small growth factor dimer associated with two larger prodomains. As isolated procomplexes, some members are latent, whereas most are active; what determines these differences is unknown. Here, studies on pro-BMP structures and binding to receptors lead to insights into mechanisms that regulate latency in the TGF-β family and into the functions of their highly divergent prodomains. The observed open-armed, nonlatent conformation of pro-BMP9 and pro-BMP7 contrasts with the cross-armed, latent conformation of pro-TGF-β1. Despite markedly different arm orientations in pro-BMP and pro-TGF-β, the arm domain of the prodomain can similarly associate with the growth factor, whereas prodomain elements N- and C-terminal to the arm associate differently with the growth factor and may compete with one another to regulate latency and stepwise displacement by type I and II receptors. Sequence conservation suggests that pro-BMP9 can adopt both cross-armed and open-armed conformations. We propose that interactors in the matrix stabilize a cross-armed pro-BMP conformation and regulate transition between cross-armed, latent and open-armed, nonlatent pro-BMP conformations.}, author = {Mi, L-Z. and Brown, C.T. and Gao, Y. and Tian, Y. and Le, V. Q. and Walz, T. and Springer, T.A.} } @article {546736, title = {Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists.}, journal = {Bioorg Med Chem. }, volume = {22}, number = {7}, year = {2014}, pages = {2353-65}, abstract = { The inhibition of\ protein-protein\ interactions remains a challenge for traditional small molecule drug\ discovery. Here we describe the use of DNA-encoded\ library\ technology\ for the\ discovery\ of small molecules that are\ potent\ inhibitors of the\ interaction\ betweenlymphocyte\ function-associated\ antigen\ 1\ and its ligand intercellular adhesion molecule\ 1. A DNA-encoded\ library\ with a potential complexity of 4.1\ billion compounds was exposed to the I-domain of the\ target\ protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the\ lymphocyte\ function-associated\ antigen\ 1/intercellular adhesion molecule-1\ interaction\ with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the\ target\ protein. Copyright {\textcopyright} 2014 Elsevier Ltd. All rights reserved. }, author = {Kollman, CS and Bai, X and Tsai, CH and Yang, H and Lind, KE and Skinner, SR and Zhu, Z. and Israel, DI and Cuozzo, JW and Morgan, BA and Yuki, K and Xie, C. and Springer, T.A. and Shimaoka, M. and Evindar, G} } @article {546731, title = {Highly reinforced structure of a C-terminal dimerization domain in von Willebrand factor.}, journal = {Blood}, volume = {123}, number = {12}, year = {2014}, pages = {1785-93}, abstract = {The\ C-terminal\ cystine knot (CK) (CTCK)\ domain\ in\ von\ Willebrand\ factor\ (VWF) mediates\ dimerization\ of proVWF in the endoplasmic reticulum and is essential for long multimers required for hemostatic function. The CTCK dimer crystal\ structure\ revealshighly\ elongated monomers with 2 β-ribbons and 4 intra-chain disulfides, including 3 in the CK.\ Dimerization\ buries an extensive interface of 1500 {\r A}(2) corresponding to 32\% of the surface of each monomer and forms a super β-sheet and 3 inter-chain disulfides. The shape, dimensions, and N-terminal connections of the crystal\ structure\ agree perfectly with previous electron microscopic images of VWF dimeric bouquets with the CTCK dimer forming a down-curved base. The dimer interface is suited to resist hydrodynamic force and disulfide reduction. CKs in each monomer flank the 3 inter-chain disulfides, and their presence in β-structures with dense backbone hydrogen bonds creates a rigid,\ highly\ crosslinked interface. The\ structure\ reveals the basis for\ vonWillebrand\ disease phenotypes and the fold and disulfide linkages for CTCK domains in diverse protein families involved in barrier function, eye and inner ear development, insect coagulation and innate immunity, axon guidance, and signaling in extracellular matrices.}, author = {Zhou, Y.F. and Springer, T.A.} } @article {546746, title = {Metal ion and ligand binding of integrin α5β1.}, journal = {Proc Natl Acad Sci USA}, volume = {111}, number = {50}, year = {2014}, pages = {17863-8}, abstract = {Integrin\ α5β1 binds to an Arg-Gly-Asp (RGD) motif in its\ ligand\ fibronectin. We report high-resolution crystal structures of a four-domain α5β1 headpiece fragment, alone or with RGD peptides soaked into crystals, and RGD peptide affinity measurements. The headpiece crystallizes in a closed conformation essentially identical to that seen previously for α5β1 complexed with a Fab that allosterically inhibits\ ligand\ binding\ by stabilizing the closed conformation. Soaking experiments show that\ binding\ of cyclic RGD peptide with 20-fold higher affinity than a linear RGD peptide induces conformational change in the β1-subunit βI domain to a state that is intermediate between closed (low affinity) and open (high affinity). In contrast,\ binding\ of a linear RGD peptide induces no shape shifting. However, linear peptide\ binding\ induces shape shifting when Ca(2+) is depleted during soaking. Ca(2+) bound to the adjacent to\ metal\ ion-dependent adhesion site (ADMIDAS), at the locus of shape shifting, moves and decreases in occupancy, correlating with an increase in affinity for RGD measured when Ca(2+) is depleted. The results directly demonstrate that Ca(2+)binding\ to the ADMIDAS stabilizes integrins in the low-affinity, closed conformation. Comparisons in affinity between four-domain and six-domain headpiece constructs suggest that flexible\ integrin\ leg domains contribute to conformational equilibria. High-resolution views of the hybrid domain interface with the plexin-semaphorin-integrin\ (PSI) domain in different orientations show a ball-and-socket joint with a hybrid domain Arg side chain that rocks in a PSI domain socket lined with carbonyl oxygens.}, author = {Xia W and Springer TA} } @article {546721, title = {Structural basis of regulation of von Willebrand factor binding to glycoprotein Ib.}, journal = {J Biol Chem.}, volume = {289}, number = {9}, year = {2014}, pages = {5565-79}, abstract = {Activation by elongational flow of\ von Willebrand factor\ (VWF) is critical for primary hemostasis. Mutations causing type 2B\ vonWillebrand\ disease (VWD),\ platelet-type VWD (PT-VWD), and tensile force each increase affinity of the VWF A1 domain and\ plateletglycoprotein Ibα (GPIbα) for one another; however, the\ structural\ basis\ for these observations remains elusive. Directed evolution was used to discover a further gain-of-function mutation in A1 that shifts the long range disulfide bond by one residue. We solved multiple crystal structures of this mutant A1 and A1 containing two VWD mutations complexed with GPIbα containing two PT-VWD mutations. We observed a gained interaction between A1 and the central leucine-rich repeats (LRRs) of GPIbα, previously shown to be important at high shear stress, and verified its importance mutationally. These findings suggest that\ structural\ changes, including central GPIbα LRR-A1 contact, contribute to VWF affinity\ regulation. Among the mutant complexes, variation in contacts and poor complementarity between the GPIbα β-finger and the region of A1 harboring VWD mutations lead us to hypothesize that the structures are on a pathway to, but have not yet reached, a force-induced super high affinity state.}, author = {Blenner, M.A. and Dong, X. and Springer, T.A.} } @article {546751, title = {Structural determinants of integrin β-subunit specificity for latent TGF-β.}, journal = {Nat Struct Mol Biol.}, volume = {21}, number = {12}, year = {2014}, pages = {1091-6}, abstract = {Eight\ integrin\ α-β heterodimers recognize ligands with an Arg-Gly-Asp (RGD) motif. However, the\ structural\ mechanism by which integrins differentiate among extracellular proteins with RGD motifs is not understood. Here, crystal structures, mutations and peptide-affinity measurements show that αVβ6 binds with high affinity to a RGDLXXL/I motif within the prodomains of TGF-β1 and TGF-β3. The LXXL/I motif forms an amphipathic α-helix that binds in a hydrophobic pocket in the β6 subunit. Elucidation of the basis for ligand binding\ specificity\ by the\ integrin\ β subunit reveals contributions by three different βI-domain loops, which we designatespecificity-determining loops (SDLs) 1, 2 and 3. Variation in a pair of single key residues in SDL1 and SDL3 correlates with the variation of the entire β subunit in\ integrin\ evolution, thus suggesting a paradigmatic role in overall β-subunit function.}, author = {Dong, X. and Hudson, N.E. and Lu, C. and Springer, T.A.} } @article {546726, title = {Structures of the Toxoplasma gliding motility adhesin}, journal = {Proc Natl Acad Sci USA}, volume = {111}, number = {13}, year = {2014}, pages = {4862-7}, abstract = {Micronemal protein 2 (MIC2) is the key\ adhesin\ that supports\ gliding\ motility\ and host cell invasion by\ Toxoplasma\ gondii. With a von Willebrand factor A (VWA) domain and six thrombospondin repeat domains (TSR1-6) in its ectodomain, MIC2 connects to the parasite actomyosin system through its cytoplasmic tail. MIC2-associated protein (M2AP) binds noncovalently to the MIC2 ectodomain. MIC2 and M2AP are stored in micronemes as proforms. We find that the MIC2-M2AP ectodomain complex is a highly elongated 1:1 monomer with M2AP bound to the TSR6 domain. Crystal\ structures\ of N-terminal fragments containing the VWA and TSR1 domains for proMIC2 and MIC2 reveal a closed conformation of the VWA domain and how it associates with the TSR1 domain. A long, proline-rich, disulfide-bonded pigtail loop in TSR1 overlaps the VWA domain. Mannose α-C-linked to Trp-276 in TSR1 has an unusual (1)C4 chair conformation. The MIC2 VWA domain includes a mobile α5-helix and a 22-residue disordered region containing two disulfide bonds in place of an α6-helix. A hydrophobic residue in the prodomain binds to a pocket adjacent to the α7-helix that pistons in opening of the VWA domain to a putative high-affinity state.}, author = {Song, G. and Springer, TA.} } @article {546741, title = {von Willebrand Factor, Jedi Knight of the Bloodstream.}, journal = {Blood}, volume = {124}, number = {9}, year = {2014}, pages = {1412-25}, abstract = { When blood vessels are cut, the forces in the\ bloodstream\ increase and change character. The dark side of these forces causes hemorrhage and death. However,\ von\ Willebrand\ factor\ (VWF), with help from our circulatory system and platelets, harnesses the same forces to form a hemostatic plug. Force and VWF function are so closely intertwined that, like members of the\ Jedi\ Order in the movie Star Wars who learn to use "the Force" to do good, VWF may be considered the\ Jedi\ knight\ of the\ bloodstream. The long length of VWF enables responsiveness to flow. The shape of VWF is predicted to alter from irregularly coiled to extended thread-like in the transition from shear to elongational flow at sites of hemostasis and thrombosis. Elongational force propagated through the length of VWF in its thread-like shape exposes its monomers for multimeric binding to platelets and subendothelium and likely also increases affinity of the A1 domain for platelets. Specialized domains concatenate and compact VWF during biosynthesis. A2 domain unfolding by hydrodynamic force enables postsecretion regulation of VWF length. Mutations in VWF in\ von\ Willebranddisease contribute to and are illuminated by VWF biology. I attempt to integrate classic studies on the physiology of hemostatic plug formation into modern molecular understanding, and point out what remains to be learned. {\textcopyright} 2014 by The American Society of Hematology. }, author = {Springer, T.A.} } @article {546756, title = {Complete integrin headpiece opening in eight steps.}, journal = {J Cell Biol}, volume = {201}, number = {7}, year = {2013}, pages = {1053-68}, abstract = { Carefully soaking crystals with Arg-Gly-Asp (RGD) peptides, we captured\ eight\ distinct RGD-bound conformations of the αIIbβ3integrin\ headpiece. Starting from the closed βI domain conformation, we saw six intermediate βI conformations and finally the fully open βI with the hybrid domain swung out in the crystal lattice. The β1-α1 backbone that hydrogen bonds to the Asp side chain of RGD was the first element to move followed by adjacent to metal ion-dependent adhesion site Ca(2+), α1 helix, α1{\textquoteright} helix, β6-α7 loop, α7 helix, and hybrid domain. We define in atomic detail how conformational change was transmitted over long distances in integrins, 40 {\r A} from the ligand binding site to the opposite end of the βI domain and 80 {\r A} to the far end of the hybrid domain. During these movements, RGD slid in its binding groove toward αIIb, and its Arg side chain became ordered. RGD concentration requirements in soaking suggested a \>200-fold higher affinity after\ opening. The thermodynamic cycle shows how higher affinity pays the energetic cost of\ opening. }, author = {Zhu, J. and Zhu, J. and Springer, T.A.} } @article {546771, title = {Domain 1 of mucosal addressin cell adhesion molecule has an I1-set fold and a flexible integrin-binding loop.}, journal = {J Biol Chem.}, volume = {288}, number = {9}, year = {2013}, pages = {6284-94}, abstract = {Mucosal\ addressin\ cell\ adhesion\ molecule\ (MAdCAM) binds integrin α4β7. Their interaction directs lymphocyte homing to mucosa-associated lymphoid tissues. The interaction between the two immunoglobulin superfamily (IgSF) domains of MAdCAM and integrin α4β7 is unusual in its ability to mediate either rolling\ adhesion\ or firm\ adhesion\ of lymphocytes on vascular surfaces. We determined four crystal structures of the IgSF domains of MAdCAM to test for unusual structural features that might correlate with this functional diversity. Higher resolution\ 1.7- and\ 1.4-A structures of the IgSF domains of MAdCAM in a previously described crystal lattice revealed two alternative conformations of the\ integrin-binding\ loop, which were deformed by large lattice contacts. New crystal forms in the presence of two different Fabs to MAdCAM demonstrate a shift in IgSF\ domain\ topology from the I2- to\ I1-set, with a switch ofintegrin-binding\ loop\ from CC{\textquoteright} to CD. The\ I1-set\ fold\ and CD\ loop\ appear biologically relevant. The different conformations seen in crystal structures suggest that the\ integrin-binding\ loop\ of MAdCAM is inherently\ flexible. This contrasts with rigidity of the corresponding loops in vascular\ cell\ adhesion\ molecule, intercellular\ adhesion\ molecule\ (ICAM)-1, ICAM-2, ICAM-3, and ICAM-5 and may reflect a specialization of MAdCAM to mediate both rolling and firm\ adhesion\ by binding to different α4β7 integrin conformations.}, author = {Yu, Y. and Zhu, J. and Huang, PS and Wang, J H and Pullen, N and Springer, T.A.} } @article {546781, title = {How Natalizumab Binds and Antagonizes α4 Integrins.}, journal = {J Biol Chem.}, volume = {288}, number = {45}, year = {2013}, pages = {32314-25}, abstract = {Natalizumab\ antibody to α4-integrins\ is used in therapy of multiple sclerosis and Crohn{\textquoteright}s disease. A crystal structure of the Fab bound to an α4 integrin β-propeller and thigh domain fragment shows that\ natalizumab\ recognizes human-mouse differences on the circumference of the β-propeller domain. The epitope is adjacent to but outside of a ligand-binding groove formed at the interface with the β-subunit βI domain and shows no difference in structure when bound to Fab. Competition between Fab and the ligand vascular cell adhesion molecule (VCAM) for binding to cell surface α4β1 shows noncompetitive antagonism. In agreement, VCAM docking models suggest that binding of domain 1 of VCAM to α4-integrins\ is unimpeded by the Fab, and that bound Fab requires a change in orientation between domains 1 and 2 of VCAM for binding to α4β1. Mapping of species-specific differences onto α4β1 and α4β7 shows that their ligand-binding sites are highly conserved. Skewing away from these conserved regions of the epitopes recognized by current therapeutic function-blocking antibodies has resulted in previously unanticipated mechanisms of action.}, author = {Yu, Y. and Sch{\"u}rpf, T. and Springer, T.A.} } @article {546786, title = {An internal ligand-bound, metastable state of a leukocyte integrin, αXβ2.}, journal = {J Cell Biol}, volume = {203}, number = {4}, year = {2013}, pages = {629-42}, abstract = {How is massive conformational change in integrins achieved on a rapid timescale? We report crystal structures of a\ metastable, putative transition\ state\ of\ integrin\ αXβ2. The αXβ2 ectodomain is bent; however, a lattice contact stabilizes its ligand-binding αI domain in a high affinity, open conformation. Much of the αI α7 helix unwinds, loses contact with the αI domain, and reshapes to form an\ internal\ ligand that binds to the interface between the β propeller and βI domains. Lift-off of the αI domain above this platform enables a range of extensional and rotational motions without precedent in allosteric machines. Movements of secondary structure elements in the β2 βI domain occur in an order different than in β3 integrins, showing that\ integrin\ β subunits can be specialized to assume different intermediate states between closed and open. Mutations demonstrate that the structure trapped here is\ metastable\ and can enable rapid equilibration between bent and extended-open\ integrin\ conformations and up-regulation of\ leukocyte\ adhesiveness.}, author = {Sen, M and Yuki, K and Springer, T.A.} } @article {546761, title = {Mechanisms by which von Willebrand disease mutations destabilize the A2 domain.}, journal = {J Biol Chem.}, volume = {288}, number = {9}, year = {2013}, pages = {6317-24}, abstract = {von\ Willebrand\ Factor (VWF) is an ultralong, concatameric, and adhesive glycoprotein. On short time scales, adhesiveness for platelets is activated by elongation of VWF by altered hydrodynamics at sites of hemostasis. Over longer time scales, the length of VWF is regulated by ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13), cleavage by which in the VWF\ A2\ domain\ is dependent on elongational force. Patients with\ von\ Willebrand\ disease\ type 2A present with increased bleeding due to\ mutations\ within the VWF\ A2\ domain\ that enhance cleavage. We tested using temperature and force the hypothesis that\ von\ Willebrand\ disease\ mutations\ disrupt\ A2\ force sensing by destabilizing the folded state.\ Mutations\ R1597W, M1528V, and E1638K reduced\ A2\ thermal stability by 10-18 {\textdegree}C. M1528V and E1638K showed a marked further decrease in stability upon calcium removal. In contrast, R1597W, which resides within the\ A2\ calcium-binding loop, exhibited similar stability in the presence and absence of calcium. Using single molecule optical tweezers and R1597W, we measured the force dependence of unfolding and refolding kinetics. In the presence of calcium, the R1597W mutation slowed the rate of refolding but had no effect on unfolding. The three\ mutations\ highlight the calcium-binding loop (R1597W), the hydrophobic core around the vicinal disulfide (M1528V), and hydrogen bonds to the α4-less loop (E1638K), as structural features critically important to the function of\ A2\ as a force sensor in regulating thrombogenic activity in the vasculature.}, author = {Xu, AJ and Springer, T.A.} } @article {546791, title = {Release of cellular tension signals self-restorative ventral lamellipodia to heal barrier micro-wounds.}, journal = {J Cell Biol}, volume = {201}, number = {3}, year = {2013}, pages = {449-65}, abstract = {Basic mechanisms by which\ cellular\ barriers sense and respond to integrity disruptions remain poorly understood. Despite its tenuous structure and constitutive exposure to disruptive strains, the vascular endothelium exhibits robust\ barrier\ function. We show that in response to micrometer-scale disruptions induced by transmigrating leukocytes, endothelial cells generate unique\ ventrallamellipodia\ that propagate via integrins toward and across these "micro-wounds" to close them. This novel actin remodeling activity progressively healed multiple\ micro-wounds\ in succession and changed direction during this process. Mechanical probe-induced micro-wounding of both endothelia and epithelia suggests that\ ventral\ lamellipodia\ formed as a response to force imbalance and specifically loss of isometric\ tension.\ Ventral\ lamellipodia\ were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of hydrogen peroxide. Together with Apr2/3, these were functionally required for effective micro-wound healing. We propose that\ barrier\ disruptions are detected as local\ release\ of isometric\ tension/force unloading, which is directly coupled to reactive oxygen species-dependent\ self-restorative\ actin remodeling dynamics.}, author = {Martinelli R and Sage PT and Massol R and Varghese L and Sciuto T and Toporsian M and Dvorak AM and Kirchhausen T and Springer TA and Carman CV} } @article {548731, title = {Calcium Stabilizes the von Willebrand Factor A2 Domain by Promoting Folding.}, journal = {Proc Natl Acad Sci USA}, volume = {109}, number = {10}, year = {2012}, pages = {3742-7}, abstract = {Von Willebrand factor (VWF) is a large, multimeric plasma glycoprotein that critically mediates hemostasis at sites of vascular injury. Very large VWF multimers have the greatest thrombogenic activity, which is attenuated by cleavage in the A2 domain by the metalloproteinase ADAMTS13. ADAMTS13 proteolysis requires mechanical force to expose the scissile bond and is regulated by a calcium-binding site within A2. In this study, we characterized the interaction between VWF A2 and calcium by examining the effect of calcium on VWF A2 stability and mechanical unfolding and refolding. Isothermal calorimetry yielded a calcium binding K(d) = 3.8 {\textpm} 1.0 μM and reversible thermal denaturation showed that 5 mM calcium stabilized the unfolding transition from 56.7 {\textpm} 0.1 to 69.1 {\textpm} 0.1 {\textdegree}C. Using optical tweezers to apply tensile force to single domains, we found that calcium did not affect VWF A2 unfolding, but rather enhanced refolding kinetics fivefold, resulting in a 0.9 kcal/mol stabilization in the folding activation energy in the presence of calcium. Taken together, our data demonstrate that VWF binds calcium at physiologic calcium concentrations and that calcium stabilizes VWF A2 by accelerating refolding.}, author = {Xu, A and Springer, T.A.} } @article {548736, title = {GARP regulates the bioavailability and activation of TGF-β.}, journal = {Mol Biol Cell.}, volume = {23}, number = {6}, year = {2012}, pages = {1129-39}, abstract = {Glycoprotein-A repetitions predominant protein (GARP) associates with latent transforming growth factor-β (proTGFβ) on the surface of T regulatory cells and platelets; however, whether GARP functions in latent TGFβ activation and the structural basis of coassociation remain unknown. We find that Cys-192 and Cys-331 of GARP disulfide link to the TGFβ1 prodomain and that GARP with C192A and C331A mutations can also noncovalently associate with proTGFβ1. Noncovalent association is sufficiently strong for GARP to outcompete latent TGFβ-binding protein for binding to proTGFβ1. Association between GARP and proTGFβ1 prevents the secretion of TGFβ1. Integrin α(V)β(6) and to a lesser extent α(V)β(8) are able to activate TGFβ from the GARP-proTGFβ1 complex. Activation requires the RGD motif of latent TGFβ, disulfide linkage between GARP and latent TGFβ, and membrane association of GARP. Our results show that GARP is a latent TGFβ-binding protein that functions in regulating the bioavailability and activation of TGFβ.}, author = {Wang, R. and Zhu, J. and Dong, X. and Shi, M. and Lu, C. and Springer, T.A.} } @article {548741, title = {Integrin Inside-Out Signaling and the Immunological Synapse.}, journal = {Curr Opin Cell Biol.}, volume = {24}, number = {1}, year = {2012}, pages = {107-15}, abstract = {Integrins dynamically equilibrate between three conformational states on cell surfaces. A bent conformation has a closed headpiece. Two extended conformations contain either a closed or an open headpiece. Headpiece opening involves hybrid domain swing-out and a 70 {\r A} separation at the integrin knees, which is conveyed by allostery from the hybrid-proximal end of the βI domain to a 3 {\r A} rearrangement of the ligand-binding site at the opposite end of the βI domain. Both bent-closed and extended-closed integrins have low affinity, whereas extended-open integrin affinity is 10(3) to 10(4) higher. Integrin-mediated adhesion requires the extended-open conformation, which in physiological contexts is stabilized by post-ligand binding events. Integrins thus discriminate between substrate-bound and soluble ligands. Analysis of LFA-1-ICAM-1 interactions in the immunological synapse suggests that bond lifetimes are on the order of seconds, which is consistent with high affinity interactions subjected to cytoskeletal forces that increase the dissociation rate. LFA-1 βI domain antagonists abrogate function in the immunological synapse, further supporting a critical role for high affinity LFA-1.Copyright {\textcopyright} 2011 Elsevier Ltd. All rights reserved.}, author = {Springer, T.A. and Dustin,M.L.} } @article {548746, title = {Mechanisms for kinase-mediated dimerization of the EGF receptor.}, journal = {J Biol Chem.}, volume = {287}, number = {45}, year = {2012}, pages = {38244-53}, abstract = {We study a mechanism by which dimerization of the EGF receptor (EGFR) cytoplasmic domain is transmitted to the ectodomain. Therapeutic and other small molecule antagonists to the kinase domain that stabilize its active conformation, but not those that stabilize an inactive conformation, stabilize ectodomain dimerization. Inhibitor-induced dimerization requires an asymmetric kinase domain interface associated with activation. EGF and kinase inhibitors stimulate formation of identical dimer interfaces in the EGFR transmembrane domain, as shown by disulfide cross-linking. Disulfide cross-linking at an interface in domain IV in the ectodomain was also stimulated similarly; however, EGF but not inhibitors stimulated cross-linking in domain II. Inhibitors similarly induced noncovalent dimerization in nearly full-length, detergent-solubilized EGFR as shown by gel filtration. EGFR ectodomain deletion resulted in spontaneous dimerization, whereas deletion of exons 2-7, in which extracellular domains III and IV are retained, did not. In EM, kinase inhibitor-induced dimers lacked any well defined orientation between the ectodomain monomers. Fab of the therapeutic antibody cetuximab to domain III confirmed a variable position and orientation of this domain in inhibitor-induced dimers but suggested that the C termini of domain IV of the two monomers were in close proximity, consistent with dimerization in the transmembrane domains. The results provide insights into the relative energetics of intracellular and extracellular dimerization in EGFR and have significance for physiologic dimerization through the asymmetric kinase interface, bidirectional signal transmission in EGFR, and mechanism of action of therapeutics.}, author = {Lu, C. and Mi, L-Z. and Schurpf, T. and Walz, T. and Springer, T.A.} } @article {548771, title = {Structural specializations of α4β7, an Integrin that Mediates Rolling Adhesion.}, journal = {J Cell Biol.}, volume = {196}, number = {1}, year = {2012}, pages = {131-46}, abstract = {The lymphocyte homing receptor integrin α(4)β(7) is unusual for its ability to mediate both rolling and firm adhesion. α(4)β(1) and α(4)β(7) are targeted by therapeutics approved for multiple sclerosis and Crohn{\textquoteright}s disease. Here, we show by electron microscopy and crystallography how two therapeutic Fabs, a small molecule (RO0505376), and mucosal adhesion molecule-1 (MAdCAM-1) bind α(4)β(7). A long binding groove at the α(4)-β(7) interface for immunoglobulin superfamily domains differs in shape from integrin pockets that bind Arg-Gly-Asp motifs. RO0505376 mimics an Ile/Leu-Asp motif in α(4) ligands, and orients differently from Arg-Gly-Asp mimics. A novel auxiliary residue at the metal ion-dependent adhesion site in α(4)β(7) is essential for binding to MAdCAM-1 in Mg(2+) yet swings away when RO0505376 binds. A novel intermediate conformation of the α(4)β(7) headpiece binds MAdCAM-1 and supports rolling adhesion. Lack of induction of the open headpiece conformation by ligand binding enables rolling adhesion to persist until integrin activation is signaled.}, author = {Yu Y. and Zhu J. and Mi L-Z. and Walz T. and Sun H. and Chen JF. and Springer TA.} } @article {547201, title = {Shape Change in the Receptor for Gliding Motility in Plasmodium Sporozoites.}, journal = {Proc Natl Acad Sci USA}, volume = {109}, number = {52}, year = {2012}, pages = {21420-21425}, abstract = {Sporozoite gliding motility and invasion of mosquito and vertebrate host cells in malaria is mediated by thrombospondin repeat anonymous protein (TRAP). Tandem von Willebrand factor A (VWA) and thrombospondin type I repeat (TSR) domains in TRAP connect through proline-rich stalk, transmembrane, and cytoplasmic domains to the parasite actin-dependent motility apparatus. We crystallized fragments containing the VWA and TSR domains fromPlasmodium vivax\ and\ Plasmodium falciparum\ in different crystal lattices. TRAP VWA domains adopt closed and open conformations, and bind a Mg2+\ ion at a metal ion{\textendash}dependent adhesion site implicated in ligand binding. Metal ion coordination in the open state is identical to that seen in the open high-affinity state of integrin I domains. The closed VWA conformation associates with a disordered TSR domain. In contrast, the open VWA conformation crystallizes with an extensible β ribbon and ordered TSR domain. The extensible β ribbon is composed of disulfide-bonded segments N- and C-terminal to the VWA domain that are largely drawn out of the closed VWA domain in a 15 {\r A} movement to the open conformation. The extensible β ribbon and TSR domain overlap at a conserved interface. The VWA, extensible β ribbon, and TSR domains adopt a highly elongated overall orientation that would be stabilized by tensile force exerted across a ligand-receptor complex by the actin motility apparatus of the sporozoite. Our results provide insights into regulation of {\textquotedblleft}stick-and-slip{\textquotedblright} parasite motility and for development of sporozoite subunit vaccines.}, author = {Song, G. and Koksal, A.C. and Lu, C. and Springer, T.A.} } @article {530011, title = {Complex structure of engineered modular domains defining molecular interaction between ICAM-1 and integrin LFA-1}, journal = {PLoS OnePloS OnePloS One}, volume = {7}, year = {2012}, note = {Kang, SungkwonKim, Chae UnGu, XiaolingOwens, Roisin Mvan Rijn, Sarah JBoonyaleepun, VanissraMao, YuxinSpringer, Timothy AJin, Moonsoo MGM103485/GM/NIGMS NIH HHS/R21 AI079532/AI/NIAID NIH HHS/PLoS One. 2012;7(8):e44124. doi: 10.1371/journal.pone.0044124. Epub 2012 Aug 30.}, pages = {e44124}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2012/09/08}, abstract = {Intermolecular contacts between integrin LFA-1 (alpha(L)beta(2)) and ICAM-1 derive solely from the integrin alpha(L) I domain and the first domain (D1) of ICAM-1. This study presents a crystal structure of the engineered complex of the alpha(L) I domain and ICAM-1 D1. Previously, we engineered the I domain for high affinity by point mutations that were identified by a directed evolution approach. In order to examine alpha(L) I domain allostery between the C-terminal alpha7-helix (allosteric site) and the metal-ion dependent adhesion site (active site), we have chosen a high affinity variant without mutations directly influencing either the position of the alpha7-helix or the active sites. In our crystal, the alpha(L) I domain was found to have a high affinity conformation to D1 with its alpha7-helix displaced downward away from the binding interface, recapitulating a current understanding of the allostery in the I domain and its linkage to neighboring domains of integrins in signaling. To enable soluble D1 of ICAM-1 to fold on its own, we also engineered D1 to be functional by mutations, which were found to be those that would convert hydrogen bond networks in the solvent-excluded core into vdW contacts. The backbone structure of the beta-sandwich fold and the epitope for I domain binding of the engineered D1 were essentially identical to those of wild-type D1. Most deviations in engineered D1 were found in the loops at the N-terminal region that interacts with human rhinovirus (HRV). Structural deviation found in engineered D1 was overall in agreement with the function of engineered D1 observed previously, i.e., full capacity binding to alpha(L) I domain but reduced interaction with HRV.}, isbn = {1932-6203 (Electronic)1932-6203 (Linking)}, author = {Kang, S. and Kim, C. U. and Gu, X. and Owens, R. M. and van Rijn, S. J. and Boonyaleepun, V. and Mao, Y. and Springer, T.A. and Jin, M. M.} } @article {529011, title = {Antigen recognition is facilitated by invadosome-like protrusions formed by memory/effector T cells}, journal = {J. Immunol}, volume = {188}, year = {2012}, note = {Journal articleJournal of immunology (Baltimore, Md. : 1950)J Immunol. 2012 Mar 21.}, month = {Mar 21}, pages = {3686-99}, edition = {2012/03/24}, abstract = {Adaptive immunity requires that T cells efficiently scan diverse cell surfaces to identify cognate Ag. However, the basic cellular mechanisms remain unclear. In this study, we investigated this process using vascular endothelial cells, APCs that possess a unique and extremely advantageous, planar morphology. High-resolution imaging revealed that CD4 memory/effector T cells dynamically probe the endothelium by extending submicron-scale, actin-rich "invadosome/podosome-like protrusions" (ILPs). The intimate intercellular contacts enforced by ILPs consistently preceded and supported T cell activation in response to endothelial MHC class II/Ag. The resulting calcium flux stabilized dense arrays of ILPs (each enriched in TCR, protein kinase C-theta, ZAP70, phosphotyrosine, and HS1), forming what we term a podo-synapse. Similar findings were made using CD8 CTLs on endothelium. Furthermore, careful re-examination of both traditional APC models and professional APCs suggests broad relevance for ILPs in facilitating Ag recognition. Together, our results indicate that ILPs function as sensory organelles that serve as actuators of immune surveillance.}, isbn = {1550-6606 (Electronic)0022-1767 (Linking)}, author = {Sage, P. T. and Varghese, L. M. and Martinelli, R. and Sciuto, T. E. and Kamei, M. and Dvorak, A. M. and Springer, T.A. and Sharpe, A. H. and Carman, C. V.} } @article {530066, title = {Molecular basis for complement recognition by integrin αXβ2}, journal = {Proc Natl Acad Sci USA}, volume = {109}, year = {2012}, pages = {4586-4591}, edition = {March 5}, author = {Chen, X. and Yu, Y. and Mi, L.Z. and Walz, T. and Springer, T.A.} } @article {528231, title = {The {\textquoteleft}RGD finger{\textquoteright} of Del-1 is a unique structural feature critical for integrin binding}, journal = {FASEB J.}, volume = {26}, year = {2012}, pages = {3412-20}, abstract = {Developmental endothelial cell locus-1 (Del-1) glycoprotein is secreted by endothelial cells and a subset of macrophages.\ Del-1plays a regulatory role in vascular remodeling and functions in innate immunity through interaction with\ integrin\ α(V)β(3).\ Del-1contains 3 epidermal growth factor (EGF)-like repeats and 2 discoidin-like domains. An Arg-Gly-Asp (RGD) motif in the second EGF domain (EGF2) mediates adhesion by endothelial cells and phagocytes. We report the crystal structure of its 3 EGF domains. TheRGD\ motif of EGF2 forms a type II{\textquoteright} β turn at the tip of a long protruding loop, dubbed the\ RGD\ finger. Whereas EGF2 and EGF3 constitute a rigid rod via an interdomain calcium ion\ binding\ site, the long linker between EGF1 and EGF2 lends considerable flexibility to EGF1. Two\ unique\ O-linked glycans and 1 N-linked glycan locate to the opposite side of EGF2 from the\ RGD\ motif. These\ structural\ features favor\ integrin\ binding\ of the\ RGD\ finger. Mutagenesis data confirm the importance of having the\ RGD\ motif at the tip of the\ RGD\ finger. A database search for EGF domain sequences shows that this\ RGD\ finger\ is likely an evolutionary insertion and\ unique\ to the EGF domain of\ Del-1\ and its homologue milk fat globule-EGF 8.}, author = {Sch{\"u}rpf, T. and Chen, Q. and Liu, J-H. and Wang, R. and Springer, T. and J.-H. Wang} } @article {527921, title = {Sequence and structure relationships within von Willebrand factor}, journal = {Blood}, volume = {120}, year = {2012}, pages = {449-58}, abstract = {In the present study, we re-annotated von Willebrand factor (VWF), assigned its entire sequence to specific modules, and related these modules to structure using electron microscopy (EM). The D domains are assemblies of smaller modules visible as lobes in EM. Modules in the D-domain assemblies include von Willebrand D, 8-cysteine, trypsin inhibitor-like, E or fibronectin type 1-like domains, and a unique D4N module in D4. The D1-D2 prodomain shows 2 large connected assemblies, each containing smaller lobes. The previous B and C regions of VWF are re-annotated as 6 tandem von Willebrand C (VWC) and VWC-like domains. These 6 VWC domains correspond to 6 elongated domains that associate in pairs at acidic pH in the stem region of VWF dimeric bouquets. This correspondence is demonstrated by binding of integrin αIIbβ3\ to the fourth module seen in EM, VWC4, which bears the VWF Arg-Gly-Asp motif. The C-terminal cystine knot domain dimerizes end-to-end in a manner predicted by homology to TGF-β and orients approximately perpendicular to the VWC domains in dimeric bouquets. Homologies of domains in VWF to domains in other proteins allow many disulfide bonds to be tentatively assigned, which may have functional implications.}, author = {Zhou, Y.F. and Eng, E. and Zhu, J. and Lu, C. and Walz, T. and Springer, T.A.} } @article {530271, title = {Structure-guided design of a high affinity platelet integrin αIIbβ3 receptor antagonist that disrupts Mg2+ binding to the MIDAS}, journal = {Sci. Transl. Med.}, volume = {4}, year = {2012}, pages = {125ra32}, abstract = {An integrin found on platelets, α(IIb)β(3) mediates platelet aggregation, and α(IIb)β(3) antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg(2+)) located in the β subunit metal ion-dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the α(IIb) subunit and a carboxyl group that coordinates the MIDAS Mg(2+) in the β(3) subunits. They induce conformational changes in the β(3) subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of α(IIb)β(3) (RUC-1) that binds exclusively to the α(IIb) subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β(3) as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2-α(IIb)β(3) headpiece complex in 1 mM calcium ion (Ca(2+))/5 mM Mg(2+) at 2.6 {\r A} revealed that RUC-2 binds to α(IIb) the way RUC-1 does, but in addition, it binds to the β(3) MIDAS residue glutamic acid 220, thus displacing Mg(2+) from the MIDAS. When the Mg(2+) concentration was increased to 20 mM, however, Mg(2+) was identified in the MIDAS and RUC-2 was absent. RUC-2{\textquoteright}s ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg(2+) concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other α(IIb)β(3) antagonists and may offer advantages as a therapeutic agent.}, author = {Zhu, J. and Choi, W-S. and McCoy, J.G. and Negri,A. and Zhu, J. and Naini, S. and Li, J. and Shen, M. and Huang, W and Bougie, D. and M Rasmussen and Aster, R. and Thomas, C.J. and Filizola, M. and Springer, T.A. and Coller,B.S.} } @article {530081, title = {An unexpected fold in the circumsporozoite protein target of malaria vaccines}, journal = {Proc Natl Acad Sci USA}, volume = {109}, year = {2012}, pages = {7817-22}, abstract = {Circumsporozoite\ (CS)\ protein\ is the major surface component of Plasmodium falciparum sporozoites and is essential for host cell invasion. A vaccine containing tandem repeats, region III, and thrombospondin type-I repeat (TSR) of CS is efficacious in phase III trials but gives only a 35\% reduction in severe\ malaria\ in the first year postimmunization. We solved crystal structures showing that region III and TSR\ fold\ into a single unit, an "αTSR" domain. The αTSR domain possesses a hydrophobic pocket and core, missing in TSR domains. CS binds heparin, but αTSR does not. Interestingly, polymorphic T-cell epitopes map to specialized αTSR regions. The N and C termini are unexpectedly close, providing clues for sporozoite sheath organization. Elucidation of a unique structure of a domain within CS enables rational design of next-generation subunit\ vaccines\ and functional and medicinal chemical investigation of the conserved hydrophobic pocket.}, author = {Doud, M.B. and Koksal, A.C. and Mi, L.Z. and Song, G. and Lu, C. and Springer, T.A.} } @article {527856, title = {αVβ3 integrin crystal structures and their functional implications}, journal = {Biochemistry}, volume = {51}, year = {2012}, pages = {8814-28}, abstract = {Many questions about the significance of structural features of integrin α(V)β(3) with respect to its mechanism of activation remain. We have determined and re-refined crystal structures of the α(V)β(3) ectodomain linked to C-terminal coiled coils (α(V)β(3)-AB) and four transmembrane (TM) residues in each subunit (α(V)β(3)-1TM), respectively. The α(V) and β(3) subunits with four and eight extracellular domains, respectively, are bent at knees between the integrin headpiece and lower legs, and the headpiece has the closed, low-affinity conformation. The structures differ in the occupancy of three metal-binding sites in the βI domain. Occupancy appears to be related to the pH of crystallization, rather than to the physiologic regulation of ligand binding at the central, metal ion-dependent adhesion site. No electron density was observed for TM residues and much of theα(V) linker. α(V)β(3)-AB and α(V)β(3)-1TM demonstrate flexibility in the linker between their extracellular and TM domains, rather than the previously proposed rigid linkage. A previously postulated interface between the α(V) and β(3) subunits at their knees was also not supported, because it lacks high-quality density, required rebuilding in α(V)β(3)-1TM, and differed markedly between α(V)β(3)-1TM and α(V)β(3)-AB. Together with the variation in domain-domain orientation within their bent ectodomains between α(V)β(3)-AB and α(V)β(3)-1TM, these findings are compatible with the requirement for large structural changes, such as extension at the knees and headpiece opening, in conveying activation signals between the extracellular ligand-binding site and the cytoplasm.}, author = {Dong, X. and Mi, L.Z., and Zhu, J. and Wang, W. and Hu, P. and Luo, B. H. and Springer, T.A.} } @article {548781, title = {Latent TGF-β structure and activation.}, journal = {Nature}, volume = {474}, number = {7351}, year = {2011}, pages = {343-9}, abstract = {Transforming growth factor (TGF)-β is stored in the extracellular matrix as a latent complex with its prodomain. Activation of TGF-β1 requires the binding of α(v) integrin to an RGD sequence in the prodomain and exertion of force on this domain, which is held in the extracellular matrix by latent TGF-β binding proteins. Crystals of dimeric porcine proTGF-β1 reveal a ring-shaped complex, a novel fold for the prodomain, and show how the prodomain shields the growth factor from recognition by receptors and alters its conformation. Complex formation between α(v)β(6) integrin and the prodomain is insufficient for TGF-β1 release. Force-dependent activation requires unfastening of a {\textquoteright}straitjacket{\textquoteright} that encircles each growth-factor monomer at a position that can be locked by a disulphide bond. Sequences of all 33 TGF-β family members indicate a similar prodomain fold. The structure provides insights into the regulation of a family of growth and differentiation factors of fundamental importance in morphogenesis and homeostasis.}, author = {Shi, M. and Zhu, J. and Wang, R. and Chen, X. and Mi, L-Z. and Walz, T. and Springer, TA.} } @article {548791, title = {A pH-regulated dimeric bouquet in the structure of von Willebrand factor.}, journal = {EMBO J.}, volume = {30}, number = {19}, year = {2011}, pages = {4098-111}, abstract = {At the acidic pH of the trans-Golgi and Weibel-Palade bodies (WPBs), but not at the alkaline pH of secretion, the C-terminal \~{}1350 residues of von Willebrand factor (VWF) zip up into an elongated, dimeric bouquet. Six small domains visualized here for the first time between the D4 and cystine-knot domains form a stem. The A2, A3, and D4 domains form a raceme with three pairs of opposed, large, flower-like domains. N-terminal VWF domains mediate helical tubule formation in WPBs and template N-terminal disulphide linkage between VWF dimers, to form ultralong VWF concatamers. The dimensions we measure in VWF at pH 6.2 and 7.4, and the distance between tubules in nascent WPB, suggest that dimeric bouquets are essential for correct VWF dimer incorporation into growing tubules and to prevent crosslinking between neighbouring tubules. Further insights into the structure of the domains and flexible segments in VWF provide an overall view of VWF structure important for understanding both the biogenesis of ultralong concatamers at acidic pH and flow-regulated changes in concatamer conformation in plasma at alkaline pH that trigger hemostasis.}, author = {Zhou, YF. and Eng, E. and Nishida, N. and Lu, C. and Walz, T. and Springer, TA.} } @article {548796, title = {Regulation of integrin affinity on cell surfaces.}, journal = {EMBO J.}, volume = {30}, number = {23}, year = {2011}, pages = {4712-27}, abstract = {Lymphocyte activation triggers adhesiveness of lymphocyte function-associated antigen-1 (LFA-1; integrin α(L)β(2)) for intercellular adhesion molecules (ICAMs) on endothelia or antigen-presenting cells. Whether the activation signal, after transmission through multiple domains to the ligand-binding αI domain, results in affinity changes for ligand has been hotly debated. Here, we present the first comprehensive measurements of LFA-1 affinities on T lymphocytes for ICAM-1 under a broad array of activating conditions. Only a modest increase in affinity for soluble ligand was detected after activation by chemokine or T-cell receptor ligation, conditions that primed LFA-1 and robustly induced lymphocyte adhesion to ICAM-1 substrates. By stabilizing well-defined LFA-1 conformations by Fab, we demonstrate the absolute requirement of the open LFA-1 headpiece for adhesiveness and high affinity. Interaction of primed LFA-1 with immobilized but not soluble ICAM-1 triggers energy-dependent affinity maturation of LFA-1 to an adhesive, high affinity state. Our results lend support to the traction or translational motion dependence of integrin activation.}, author = {Sch{\"u}rpf, T. and Springer, TA.} } @article {548786, title = {Simultaneous visualization of the extracellular and cytoplasmic domains of the epidermal growth factor receptor.}, journal = {Nat Struct Mol Biol.}, volume = {18}, number = {9}, year = {2011}, pages = {984-9}, abstract = {To our knowledge, no structural study to date has characterized, in an intact receptor, the coupling of conformational change in extracellular domains through a single-pass transmembrane domain to conformational change in cytoplasmic domains. Here we examine such coupling, and its unexpected complexity, using nearly full-length epidermal growth factor receptor (EGFR) and negative-stain EM. The liganded, dimeric EGFR ectodomain can couple both to putatively active, asymmetrically associated kinase dimers and to putatively inactive, symmetrically associated kinase dimers and monomers. Inhibitors that stabilize the active or inactive conformation of the kinase active site, as well as mutations in the kinase dimer interface and a juxtamembrane phosphorylation site, shift the equilibrium among the three kinase association states. This coupling of one conformation of an activated receptor ectodomain to multiple kinase-domain arrangements reveals previously unanticipated complexity in transmembrane signaling and facilitates regulation of receptor function in the juxtamembrane and cytoplasmic environments.}, author = {Mi, L-Z. and Lu, C. and Nishida, N. and Walz, T. and Springer, TA.} } @article {529456, title = {Biology and physics of von Willebrand factor concatamers}, journal = {J Thromb Haemost.}, volume = {9}, number = {Suppl 1}, year = {2011}, pages = {130-143}, abstract = { Structural specialisations enable\ von\ Willebrand\ factor\ (VWF) to assemble during biosynthesis into helical tubules in Weibel-Palade bodies (WPB). Specialisations include a pH-regulated dimeric bouquet formed by the C-terminal half of VWF and helical assembly guided by the N-terminal half that templates inter-dimer disulphide bridges. Orderly assembly and storage of ultra-long\ concatamersin helical tubules, without crosslinking of neighboring tubules, enables unfurling during secretion without entanglement. Length regulation occurs post-secretion, by hydrodynamic force-regulated unfolding of the VWF A2 domain, and its cleavage by the plasma protease ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13). VWF is longest at its site of secretion, where its haemostatic function is most important. Moreover, elongational hydrodynamic forces on VWF are strongest just where needed, when bound to the vessel wall, or in elongational flow in the circulation at sites of vessel rupture or vasoconstriction in haemostasis. Elongational forces regulate haemostasis by activating binding of the A1 domain to platelet GPIbα, and over longer time periods, regulate VWF length by unfolding of the A2 domain for cleavage by ADAMTS13. Recent structures of A2 and single molecule measurements of A2 unfolding and cleavage by ADAMTS13 illuminate the mechanisms of VWF length regulation. Single molecule studies on the A1-GPIb receptor-ligand bond demonstrate a specialised flex-bond that enhances resistance to the strong hydrodynamic forces experienced at sites of haemorrhage. {\textcopyright} 2011 International Society on Thrombosis and Haemostasis. }, author = {Springer, T.A.} } @article {528621, title = {The C-terminal αI domain linker as a critical structural element in the conformational activation of αI integrins}, journal = {J Biol Chem.}, volume = {286}, number = {49}, year = {2011}, pages = {42115-22}, abstract = {The activation of α/β heterodimeric integrins is the result of highly coordinated rearrangements within both subunits. The molecular interactions between the two subunits, however, remain to be characterized. In this study we use the integrin α(L)β(2) to investigate the functional role of the C-linker polypeptide, which connects the C-terminal end of the inserted (I) domain with the β-propeller domain on the α subunit and is located at the interface with the βI domain of the β chain. We demonstrate that shortening of the C-linker by eight or more amino acids results in constitutively active α(L)β(2), in which the αI domain is no longer responsive to the regulation by the βI domain. Despite this inter-subunit uncoupling, both I domains individually remain sensitive to intra-subunit conformational changes induced by allosteric modulators. Interestingly, the length and not the sequence of the C-linker appears to be critical for its functionality in the α/β inter-subunit communication. Using two monoclonal antibodies (R7.1 and CBR LFA-1/1) we further demonstrate that shortening of the C-linker results in the gradual loss of combinational epitopes that require both the αI and β-propeller domains for full reactivity. Taken together, our findings highlight the role of the C-linker as a spring-like element which allows relaxation of the αI domain in the resting state and controlled tension of the αI domain during activation, exerted by the β chain.}, author = {Weitz-Schmidt, G. and Schurpf, T. and Springer, T.A.} } @article {528476, title = {Intact αIIbβ3 extends after activation measured by solution X-ray scattering and electron microscopy}, journal = {J Biol Chem}, volume = {286}, number = {40}, year = {2011}, pages = {35218-35226}, abstract = {Integrins are bidirectional signaling molecules on the cell surface that have fundamental roles in regulating cell behavior and contribute to cell migration and adhesion. Understanding of the mechanism of integrin signaling and activation has been advanced with truncated ectodomain preparations; however, the nature of conformational change in the full-length intact integrin molecule remains an active area of research. Here we used small angle x-ray scattering and electron microscopy to study detergent-solubilized, intact platelet integrin α(IIb)β(3). In the resting state, the intact α(IIb)β(3) adopted a compact, bent conformation. Upon activation with Mn(2+), the average integrin extension increased. Further activation by addition of ligand led to stabilization of the extended state and opening of the headpiece. The observed extension and conformational rearrangement upon activation are consistent with the extension and headpiece opening model of integrin activation}, author = {Eng, E. and Smagghe, B. and Walz, T. and Springer, T.A.} } @article {527916, title = {A novel calcium-binding site of von Willebrand factor A2 domain regulates its cleavage by ADAMTS13}, journal = {Blood}, volume = {117}, year = {2011}, note = {Zhou, MinyunDong, XianchiBaldauf, CarstenChen, HuaZhou, YanfengSpringer, Timothy ALuo, XinpingZhong, ChenGrater, FraukeDing, JianpingUnited StatesBloodBlood. 2011 Apr 28;117(17):4623-31. Epub 2011 Mar 8.}, month = {Apr 28}, pages = {4623-31}, edition = {2011/03/10}, abstract = {The proteolysis of VWF by ADAMTS13 is an essential step in the regulation of its hemostatic and thrombogenic potential. The cleavage occurs at strand beta4 in the structural core of the A2 domain of VWF, so unfolding of the A2 domain is a prerequisite for cleavage. In the present study, we present the crystal structure of an engineered A2 domain that exhibits a significant difference in the alpha3-beta4 loop compared with the previously reported structure of wild-type A2. Intriguingly, a metal ion was detected at a site formed mainly by the C-terminal region of the alpha3-beta4 loop that was later identified as Ca(2+) after various biophysical and biochemical studies. Force-probe molecular dynamic simulations of a modeled structure of the wild-type A2 featuring the discovered Ca(2+)-binding site revealed that an increase in force was needed to unfold strand beta4 when Ca(2+) was bound. Cleavage assays consistently demonstrated that Ca(2+) binding stabilized the A2 domain and impeded its unfolding, and consequently protected it from cleavage by ADAMTS13. We have revealed a novel Ca(2+)-binding site at the A2 domain of VWF and demonstrated a relationship between Ca(2+) and force in the regulation of VWF and primary hemostasis.}, isbn = {1528-0020 (Electronic)0006-4971 (Linking)}, author = {Zhou, M and Dong, X. and Baldauf, C and Chen, H and Zhou, Y. and Springer, T.A. and Lu, X. and Zhong, C and Grater, F and Ding, J} } @article {528616, title = {Tests of integrin transmembrane domain homo-oligomerization during integrin ligand binding and signaling}, journal = {J Biol Chem}, volume = {286}, year = {2011}, note = {Wang, WeiZhu, JieqingSpringer, Timothy ALuo, Bing-HaoResearch Support, Non-U.S. Gov{\textquoteright}tUnited StatesThe Journal of biological chemistryJ Biol Chem. 2011 Jan 21;286(3):1860-7. Epub 2010 Nov 16.}, month = {Jan 21}, pages = {1860-7}, edition = {2010/11/18}, abstract = {Integrin transmembrane (TM) and/or cytoplasmic domains play a critical role in integrin bidirectional signaling. Although it has been shown that TM and/or cytoplasmic alpha and beta domains associate in the resting state and separation of these domains is required for both inside-out and outside-in signaling, the role of TM homomeric association remains elusive. Formation of TM homo-oligomers was observed in micelles and bacterial membranes previously, and it has been proposed that homomeric association is important for integrin activation and clustering. This study addresses whether integrin TM domains form homo-oligomers in mammalian cell membranes using cysteine scanning mutagenesis. Our results show that TM homomeric interaction does not occur before or after soluble ligand binding or during inside-out activation. In addition, even though the cysteine mutants and the heterodimeric disulfide-bounded mutant could form clusters after adhering to immobilized ligand, the integrin TM domains do not form homo-oligomers, suggesting that integrin TM homomeric association is not critical for integrin clustering or outside-in signaling. Therefore, integrin TM homo-oligomerization is not required for integrin activation, ligand binding, or signaling.}, keywords = {Amino Acid Substitution, Cell Adhesion, Cell Line, Humans, Integrins/genetics/*metabolism, Ligands, Mutagenesis, Protein Binding, Protein Multimerization/*physiology, Protein Structure, Tertiary, Signal Transduction/*physiology}, isbn = {1083-351X (Electronic)0021-9258 (Linking)}, author = {Wang, W. and Zhu, J. and Springer, T.A. and Luo, B. H.} } @article {548801, title = {A mechanically stabilized receptor-ligand flex-bond important in the vasculature.}, journal = {Nature}, volume = {466}, number = {7309}, year = {2010}, pages = {992-5}, abstract = {Haemostasis in the arteriolar circulation mediated by von Willebrand factor (VWF) binding to platelets is an example of an adhesive interaction that must withstand strong hydrodynamic forces acting on cells. VWF is a concatenated, multifunctional protein that has binding sites for platelets as well as subendothelial collagen. Binding of the A1 domain in VWF to the glycoprotein Ib alpha subunit (GPIbalpha) on the surface of platelets mediates crosslinking of platelets to one another and the formation of a platelet plug for arterioles. The importance of VWF is illustrated by its mutation in von Willebrand disease, a bleeding diathesis. Here, we describe a novel mechanochemical specialization of the A1-GPIbalpha bond for force-resistance. We have developed a method that enables, for the first time, repeated measurements of the binding and unbinding of a receptor and ligand in a single molecule (ReaLiSM). We demonstrate two states of the receptor-ligand bond, that is, a flex-bond. One state is seen at low force; a second state begins to engage at 10 pN with a approximately 20-fold longer lifetime and greater force resistance. The lifetimes of the two states, how force exponentiates lifetime, and the kinetics of switching between the two states are all measured. For the first time, single-molecule measurements on this system are in agreement with bulk phase measurements. The results have important implications not only for how platelets bound to VWF are able to resist force to plug arterioles, but also how increased flow activates platelet plug formation.}, author = {Kim, J. and Zhang, C. and Zhang, X. and Springer, TA.} } @article {548811, title = {Modulation of integrin activation by an entropic spring in the β-knee.}, journal = {J Biol Chem.}, volume = {285}, number = {43}, year = {2010}, pages = {32954-66}, abstract = {We show that the length of a loop in the β-knee, between the first and second cysteines (C1-C2) in integrin EGF-like (I-EGF) domain 2, modulates integrin activation. Three independent sets of mutants, including swaps among different integrin β-subunits, show that C1-C2 loop lengths of 12 and longer favor the low affinity state and masking of ligand-induced binding site (LIBS) epitopes. Shortening length from 12 to 4 residues progressively increases ligand binding and LIBS epitope exposure. Compared with length, the loop sequence had a smaller effect, which was ascribable to stabilizing loop conformation, and not interactions with the α-subunit. The data together with structural calculations support the concept that the C1-C2 loop is an entropic spring and an emerging theme that disordered regions can regulate allostery. Diversity in the length of this loop may have evolved among integrin β-subunits to adjust the equilibrium between the bent and extended conformations at different set points.}, author = {Smagghe, B. and Huang, P. and Ban, Y-E. and Baker, D. and Springer, TA.} } @article {548816, title = {Structural Evidence for Loose Linkage between Ligand Binding and Kinase Activation in the Epidermal Growth Factor Receptor.}, journal = {Mol Cell Biol.}, volume = {30}, number = {22}, year = {2010}, pages = {5432-43}, abstract = {The mechanisms by which signals are transmitted across the plasma membrane to regulate signaling are largely unknown for receptors with single-pass transmembrane domains such as the epidermal growth factor receptor (EGFR). A crystal structure of the extracellular domain of EGFR dimerized by epidermal growth factor (EGF) reveals the extended, rod-like domain IV and a small, hydrophobic domain IV interface compatible with flexibility. The crystal structure and disulfide cross-linking suggest that the 7-residue linker between the extracellular and transmembrane domains is flexible. Disulfide cross-linking of the transmembrane domain shows that EGF stimulates only moderate association in the first two α-helical turns, in contrast to association throughout the membrane over five α-helical turns in glycophorin A and integrin. Furthermore, systematic mutagenesis to leucine and phenylalanine suggests that no specific transmembrane interfaces are required for EGFR kinase activation. These results suggest that linkage between ligand-induced dimerization and tyrosine kinase activation is much looser than was previously envisioned.}, author = {Lu, C. and Mi, L-Z. and Grey, MJ. and Zhu, J. and Graef, E. and Yokoyama, S. and Springer, TA.} } @article {548821, title = {Structure of an integrin with an αI domain, complement receptor type 4.}, journal = {Proc Natl Acad Sci USA}, volume = {109}, number = {12}, year = {2010}, pages = {4586-91}, abstract = {Integrin α(X)β(2) functions as complement receptor for iC3b and mediates recognition and phagocytosis of pathogens. We used negative-stain EM to examine the α(X)β(2) interaction with iC3b. EM class averages of α(X)β(2) in complex with iC3b define the binding sites on both the integrin and iC3b. iC3b contains C3c and thioester domain moieties linked by a long flexible linker. The binding site is on the key ring of the C3c moiety, at the interface between the MG3 and MG4 domains. Similar complexes are seen between α(X)β(2) and the C3c fragment. α(X)β(2) binds through the α(X) αI domain, on the face known to bear the metal ion-dependent adhesion site, at the opposite end of the αI domain from its site of insertion in the β-propeller domain.}, author = {Xie, C. and Zhu, J. and Chen, X. and Mi, L-Z. and Nishida, N. and Springer, T.A.} } @article {530161, title = {A cation-π interaction regulates ligand binding affinity and signaling of integrin α4β7}, journal = {Proc Natl Acad Sci USA}, volume = {107}, year = {2010}, pages = {21388-21393}, abstract = {Integrin α(4)β(7) mediates rolling and firm adhesion of leucocytes, two of the critical steps in leukocyte migration and tissue specific homing. Affinity of α(4)β(7) for ligand is dynamically regulated by three interlinked metal ion-binding sites in β(7)-subunit I domain. In this study, we found that Phe185 (F185), a highly conserved aromatic residue in β(7)-subunit, links the specificity-determining loop and the synergistic metal ion-binding site (SyMBS) through cation-π interaction. Mutations of F185 that disrupted the SyMBS cation-F185 interaction led to deficient firm cell adhesion mediated by high affinity α(4)β(7), and only slightly affected rolling adhesion mediated by low affinity α(4)β(7). Disruption of SyMBS cation-F185 interaction induced partial extension of integrin ectodomain and separation of cytoplasmic tails, and impaired α(4)β(7)-mediated bidirectional signaling. In addition, loss of SyMBS cation-F185 interaction increased paxillin expression and promoted paxillin-integrin binding, leading to deficient cell spreading. Furthermore, integrin α(4)β(7)-mediated cell migration was decreased by the abolishment of SyMBS cation-F185 interaction. Thus, these findings reveal a cation-π interaction playing vital roles in the regulation of integrin affinity, signaling, and biological functions.}, author = {Pan, Youdong and Zhang, Kun and Qi, Junpeng and Yue, Jiao and Springer, Timothy A. and Chen, Jianfeng} } @article {527926, title = {The closed headpiece of integrin αIIbβ3 and its complex with an αIIbβ3-specific antagonist that does not induce opening}, journal = {Blood}, volume = {116}, year = {2010}, pages = {5050-9}, edition = {Aug. 2}, abstract = {The platelet integrin α(IIb)β(3) is essential for hemostasis and thrombosis through its binding of adhesive plasma proteins. We have determined crystal structures of the α(IIb)β(3) headpiece in the absence of ligand and after soaking in RUC-1, a novel small molecule antagonist. In the absence of ligand, the α(IIb)β(3) headpiece is in a closed conformation, distinct from the open conformation visualized in presence of Arg-Gly-Asp (RGD) antagonists. In contrast to RGD antagonists, RUC-1 binds only to the α(IIb) subunit. Molecular dynamics revealed nearly identical binding. Two species-specific residues, α(IIb) Y190 and α(IIb) D232, in the RUC-1 binding site were confirmed as important by mutagenesis. In sharp contrast to RGD-based antagonists, RUC-1 did not induce α(IIb)β(3) to adopt an open conformation, as determined by gel filtration and dynamic light scattering. These studies provide insights into the factors that regulate integrin headpiece opening, and demonstrate the molecular basis for a novel mechanism of integrin antagonism.}, author = {Zhu, J. and Negri,A. and Provasi, D. and Filizola, M. and Coller,B.S. and Springer, T.A.} } @article {528566, title = {Engineering of single Ig superfamily domain of intercellular adhesion molecule 1 (ICAM-1) for native fold and function}, journal = {J Biol Chem.}, volume = {285}, year = {2010}, note = {Owens, Roisin MGu, XiaolingShin, MiranSpringer, Timothy AJin, Moonsoo MAI079532/AI/NIAID NIH HHS/United StatesCA31798/CA/NCI NIH HHS/United StatesResearch Support, N.I.H., ExtramuralUnited StatesThe Journal of biological chemistryJ Biol Chem. 2010 May 21;285(21):15906-15. Epub 2010 Mar 19.}, month = {May 21}, pages = {15906-15}, edition = {2010/03/23}, abstract = {The immunoglobulin (Ig) superfamily is one of the largest families in the vertebrate genome, found most frequently in cell surface molecules. Intercellular adhesion molecule-1 (ICAM-1) contains five extracellular Ig superfamily domains (D1-D5) of which the first domain, D1, is the binding site for the integrin lymphocyte function-associated antigen-1 (LFA-1) and human rhinovirus. Despite the modular nature of many Ig superfamily domains with respect to domain folding and ligand recognition, D1 does not fold on its own due to the loss of its interaction with the second domain. The goal of this study was to engineer ICAM-1 D1 by introducing mutations that would stabilize the Ig superfamily domain fold while retaining its ability to bind to LFA-1 and rhinovirus. First, with a directed evolution approach, we isolated mutations in D1 that showed binding to conformation-specific antibodies and the ligand binding domain of LFA-1 called the inserted, or I, domain. Then, with a rational design approach we introduced mutations that contributed to the stability of ICAM-1 D1 in solution. The mutations that restored native folding of D1 in isolation were those that would convert hydrogen bond networks in buried regions into hydrophobic contacts. Notably, for most mutations, identical or similar types of substitutions were found in ICAM-1 molecules of different species and other ICAM family members. The systematic approach demonstrated in this study to engineer a single Ig superfamily fold in ICAM-1 can be broadly applicable to the engineering of modular Ig superfamily domains in other cell surface molecules.}, keywords = {525}, isbn = {1083-351X (Electronic)0021-9258 (Linking)}, author = {Owens, R. M. and Gu, X. and Shin, M. and Springer, T.A. and Jin, M. M.} } @article {530061, title = {Requirement of open headpiece conformation for activation of leukocyte integrin αXβ2}, journal = {Proc Natl Acad Sci USA}, volume = {107}, year = {2010}, pages = {14727-32}, abstract = {Negative stain electron microscopy (EM) and adhesion assays show that alpha(X)beta(2) integrin activation requires headpiece opening as well as extension. An extension-inducing Fab to the beta(2) leg, in combination with representative activating and inhibitory Fabs, were examined for effect on the equilibrium between the open and closed headpiece conformations. The two activating Fabs stabilized the open headpiece conformation. Conversely, two different inhibitory Fabs stabilized the closed headpiece conformation. Adhesion assays revealed that alpha(X)beta(2) in the extended-open headpiece conformation had high affinity for ligand, whereas both the bent conformation and the extended-closed headpiece conformation represented the low affinity state. Intermediate integrin affinity appears to result not from a single conformational state, but from a mixture of equilibrating conformational states.}, author = {Chen, X. and Xie, C. and Nishida, N. and Li, Z. and Walz, T. and Springer, T.A.} } @article {548831, title = {Mechanoenzymatic cleavage of the ultralarge vascular protein, von Willebrand Factor.}, journal = {Science}, volume = {324}, number = {5932}, year = {2009}, pages = {1330-4}, abstract = {Von Willebrand factor (VWF) is secreted as ultralarge multimers that are cleaved in the A2 domain by the metalloprotease ADAMTS13 to give smaller multimers. Cleaved VWF is activated by hydrodynamic forces found in arteriolar bleeding to promote hemostasis, whereas uncleaved VWF is activated at lower, physiologic shear stresses and causes thrombosis. Single-molecule experiments demonstrate that elongational forces in the range experienced by VWF in the vasculature unfold the A2 domain, and only the unfolded A2 domain is cleaved by ADAMTS13. In shear flow, tensile force on a VWF multimer increases with the square of multimer length and is highest at the middle, providing an efficient mechanism for homeostatic regulation of VWF size distribution by force-induced A2 unfolding and cleavage by ADAMTS13, as well as providing a counterbalance for VWF-mediated platelet aggregation.}, author = {Zhang, X. and Halvorsen, K. and Zhang, CZ. and Wong, WP. and Springer, TA.} } @article {548841, title = {Rationally designed integrin beta3 mutants stabilized in the high affinity conformation.}, journal = {J Biol Chem.}, volume = {284}, number = {6}, year = {2009}, pages = {3917-24}, abstract = {Integrins are important cell surface receptors that transmit bidirectional signals across the membrane. It has been shown that a conformational change of the integrin beta-subunit headpiece (i.e. the beta I domain and the hybrid domain) plays a critical role in regulating integrin ligand binding affinity and function. Previous studies have used coarse methods (a glycan wedge, mutations in transmembrane contacts) to force the beta-subunit into either the open or closed conformation. Here, we demonstrate a detailed understanding of this conformational change by applying computational design techniques to select five amino acid side chains that play an important role in the energetic balance between the open and closed conformations of alphaIIbbeta3. Eight single-point mutants were designed at these sites, of which five bound ligands much better than wild type. Further, these mutants were found to be in a more extended conformation than wild type, suggesting that the conformational change at the ligand binding headpiece was propagated to the legs of the integrin. This detailed understanding of the conformational change will assist in the development of allosteric drugs that either stabilize or destabilize specific integrin conformations without occluding the ligand-binding site.}, author = {Luo, BH. and Karanicolas, J. and Harmacek, LD. and Baker, D. and Springer, TA.} } @article {548846, title = {Structural basis for selectin mechanochemistry.}, journal = {Proc Natl Acad Sci USA}, volume = {106}, number = {1}, year = {2009}, pages = {91-6}, abstract = {Selectins are adhesion molecules that resist large tensile forces applied by hydrodynamic forces to leukocytes binding to vessel walls. In crystals, the liganded (high-affinity) and unliganded (low-affinity) conformations differ in orientation between their tandem lectin and EGF domains. I examine how tensile force exerted on a selectin-ligand complex in vivo could favor the more extended, high-affinity conformation. Allostery is transmitted from the EGF-lectin domain interface to the ligand-binding interface on the lectin domain, 30 A away. Trp-1 of the lectin domain and the long axis of the EGF domain form an L-shaped prybar that is welded together by hydrogen bonds to the Trp-1 alpha-amino group. Pivoting of the prybar induced by force demolishes an interface between the Trp-1 side chain and the lectin domain at a switch1 region. These changes are transmitted by rigid body movement of the switch2 region to rearrangements in the switch3 region at the ligand binding site. Another switch region corresponds to a single residue in the EGF domain with large effects on ligand binding and rolling adhesion. Allostery in selectins, and the alignment of tensile force on a selectin-ligand complex with the transition pathway for conformational change, explain much of the structural basis for selectin mechanochemistry.}, author = {Springer, TA.} } @article {548851, title = {Structural specializations of A2, a force-sensing domain in the ultralarge vascular protein von Willebrand factor.}, journal = {Proc Natl Acad Sci USA}, volume = {106}, number = {23}, year = {2009}, pages = {9226-31}, abstract = {The lengths of von Willebrand factor (VWF) concatamers correlate with hemostatic potency. After secretion in plasma, length is regulated by hydrodynamic shear force-dependent unfolding of the A2 domain, which is then cleaved by a specific protease. The 1.9-A crystal structure of the A2 domain demonstrates evolutionary adaptations to this shear sensor function. Unique among VWF A (VWA) domains, A2 contains a loop in place of the alpha4 helix, and a cis-proline. The central beta4-strand is poorly packed, with multiple side-chain rotamers. The Tyr-Met cleavage site is buried in the beta4-strand in the central hydrophobic core, and the Tyr structurally links to the C-terminal alpha6-helix. The alpha6-helix ends in 2 Cys residues that are linked by an unusual vicinal disulfide bond that is buried in a hydrophobic pocket. These features may narrow the force range over which unfolding occurs and may also slow refolding. Von Willebrand disease mutations, which presumably lower the force at which A2 unfolds, are illuminated by the structure.}, author = {Zhang, Q. and Zhou, YF. and Zhang, CZ. and Springer, TA.} } @article {548856, title = {Transmission of Allostery through the Lectin Domain in Selectin-Mediated Cell Adhesion.}, journal = {Proc Natl Acad Sci USA}, volume = {106}, number = {1}, year = {2009}, pages = {85-90}, abstract = {The selectins are cell adhesion proteins that must resist applied forces to mediate leukocyte tethering and rolling along the endothelium and have 2 conformational states. Selectin-ligand bond dissociation increases only modestly with applied force, and exhibits catch bond behavior in a low-force regime where bond lifetimes counterintuitively increase with increasing force. Both allosteric and sliding-rebinding models have emerged to explain catch bonds. Here, we introduce a large residue into a cleft that opens within the lectin domain to stabilize the more extended, high-affinity selectin conformation. This mutation stabilizes the high-affinity state, but surprisingly makes rolling less stable. The position of the mutation in the lectin domain provides evidence for an allosteric pathway through the lectin domain, connecting changes at the lectin-EGF interface to the distal binding interface.}, author = {Waldron, TT. and Springer, TA.} } @article {528611, title = {The novel S527F mutation in the integrin β3 chain induces a high affinity αIIbβ3 receptor by hindering adoption of the bent conformation}, journal = {J Biol Chem.}, volume = {284}, number = {22}, year = {2009}, pages = {14917-14918}, abstract = {Three heterozygous mutations were identified in the genes encoding platelet integrin receptor alphaIIbbeta3 in a patient with an ill defined platelet disorder: one in the beta3 gene (S527F) and two in the alphaIIb gene (R512W and L841M). Five stable Chinese hamster ovary cell lines were constructed expressing recombinant alphaIIbbeta3 receptors bearing the individual R512W, L841M, or S527F mutation; both the R512W and L841M mutations; or all three mutations. All receptors were expressed on the cell surface, and mutations R512W and L841M had no effect on integrin function. Interestingly, the beta3 S527F mutation produced a constitutively active receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound to non-activated alphaIIbbeta3 receptors carrying the S527F mutation, indicating that the conformation of this receptor was altered and corresponded to the high affinity ligand binding state. In addition, the conformational change induced by S527F was evident from basal anti-ligand-induced binding site antibody binding to the receptor. A molecular model bearing this mutation was constructed based on the crystal structure of alphaIIbbeta3 and revealed that the S527F mutation, situated in the third integrin epidermal growth factor-like (I-EGF3) domain, hindered the alphaIIbbeta3 receptor from adopting a wild type-like bent conformation. Movement of I-EGF3 into a cleft in the bent conformation may be hampered both by steric hindrance between Phe(527) in beta3 and the calf-1 domain in alphaIIb and by decreased flexibility between I-EGF2 and I-EGF3.}, author = {Vanhoorelbeke, K. and De Meyer, S. F. and Pareyn, I. and Melchior, C. and Plancon, S. and Margue, C. and Pradier, O. and Fondu, P. and Kieffer, N. and Springer, T.A. and Deckmyn, H.} } @article {530246, title = {Structural basis of activation-dependent binding of ligand-mimetic antibody AL-57 to integrin LFA-1}, journal = {Proc Natl Acad Sci USA}, volume = {106}, number = {43}, year = {2009}, note = {Zhang, HongminLiu, Jin-HuanYang, WeiSpringer, TimothyShimaoka, MotomuWang, Jia-HuaiAI063421/AI/NIAID NIH HHS/United StatesCA31798/CA/NCI NIH HHS/United StatesHL48675/HL/NHLBI NIH HHS/United StatesResearch Support, N.I.H., ExtramuralUnited StatesProceedings of the National Academy of Sciences of the United States of AmericaProc Natl Acad Sci U S A. 2009 Oct 27;106(43):18345-50. Epub 2009 Sep 23.}, month = {Oct 27}, pages = {18345-50}, edition = {2009/10/07}, abstract = {The activity of integrin LFA-1 (alpha(L)beta(2)) to its ligand ICAM-1 is regulated through the conformational changes of its ligand-binding domain, the I domain of alpha(L) chain, from an inactive, low-affinity closed form (LA), to an intermediate-affinity form (IA), and then finally, to a high-affinity open form (HA). A ligand-mimetic human monoclonal antibody AL-57 (activated LFA-1 clone 57) was identified by phage display to specifically recognize the affinity-upregulated I domain. Here, we describe the crystal structures of the Fab fragment of AL-57 in complex with IA, as well as in its unligated form. We discuss the structural features conferring AL-57{\textquoteright}s strong selectivity for the high affinity, open conformation of the I domain. The AL-57-binding site overlaps the ICAM-1 binding site on the I domain. Furthermore, an antibody Asp mimics an ICAM Glu by forming a coordination to the metal-ion dependent adhesion site (MIDAS). The structure also reveals better shape complementarity and a more hydrophobic interacting interface in AL-57 binding than in ICAM-1 binding. The results explain AL-57{\textquoteright}s antagonistic mimicry of LFA-1{\textquoteright}s natural ligands, the ICAM molecules.}, isbn = {1091-6490 (Electronic)}, author = {Zhang, H and Liu, J-H. and Yang, W and Springer, T. and Shimaoka, M. and J.-H. Wang} } @article {529801, title = {The structure of a receptor with two associating transmembrane domains on the cell surface: integrin αIIbβ3}, journal = {Mol Cell.}, volume = {34}, number = {2}, year = {2009}, pages = {234-249}, abstract = {Structures of intact receptors with single-pass transmembrane domains are essential to understand how extracellular and cytoplasmic domains regulate association and signaling through transmembrane domains. A chemical and computational method to determine structures of the membrane regions of such receptors on the cell surface is developed here and validated with glycophorin A. An integrin heterodimer structure reveals association over most of the lengths of the alpha and beta transmembrane domains and shows that the principles governing association of hetero and homo transmembrane dimers differ. A turn at the Gly of the juxtamembrane GFFKR motif caps the alpha TM helix and brings the two Phe of GFFKR into the alpha/beta interface. A juxtamembrane Lys residue in beta also has an important role in the interface. The structure shows how transmembrane association/dissociation regulates integrin signaling. A joint ectodomain and membrane structure shows that substantial flexibility between the extracellular and TM domains is compatible with TM signaling.}, author = {Zhu, J. and Luo, B. H. and Barth, P. and Schonbrun, J. and Baker, D. and Springer, T.A.} } @article {548866, title = {Functional and Structural Stability of the Epidermal Growth Factor Receptor in Detergent Micelles and Phospholipid Nanodiscs.}, journal = {Biochemistry}, volume = {47}, number = {39}, year = {2008}, pages = {10314-23}, abstract = {Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro.}, author = {Mi, LZ. and Grey, MJ. and Nishida, N. and Walz, T. and Lu, C. and Springer, TA.} } @article {548876, title = {Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.}, journal = {J Exp Med.}, volume = {205}, number = {1}, year = {2008}, pages = {195-205}, abstract = {Precise spatial and temporal regulation of cell adhesion and de-adhesion is critical for dynamic lymphocyte migration. Although a great deal of information has been learned about integrin lymphocyte function-associated antigen (LFA)-1 adhesion, the mechanism that regulates efficient LFA-1 de-adhesion from intercellular adhesion molecule (ICAM)-1 during T lymphocyte migration is unknown. Here, we show that nonmuscle myosin heavy chain IIA (MyH9) is recruited to LFA-1 at the uropod of migrating T lymphocytes, and inhibition of the association of MyH9 with LFA-1 results in extreme uropod elongation, defective tail detachment, and decreased lymphocyte migration on ICAM-1, without affecting LFA-1 activation by chemokine CXCL-12. This defect was reversed by a small molecule antagonist that inhibits both LFA-1 affinity and avidity regulation, but not by an antagonist that inhibits only affinity regulation. Total internal reflection fluorescence microscopy of the contact zone between migrating T lymphocytes and ICAM-1 substrate revealed that inactive LFA-1 is selectively localized to the posterior of polarized T lymphocytes, whereas active LFA-1 is localized to their anterior. Thus, during T lymphocyte migration, uropodal adhesion depends on LFA-1 avidity, where MyH9 serves as a key mechanical link between LFA-1 and the cytoskeleton that is critical for LFA-1 de-adhesion.}, author = {Morin, NA. and Oakes, PW. and Hyun, YM. and Lee, D. and Chin, EY. and King, MR. and Springer, TA. and Shimaoka, M. and Tang, JX. and Reichner, JS. and Kim, M.} } @article {548881, title = {Structural basis for distinctive recognition of fibrinogen by the platelet integrin αIIbβ3.}, journal = {J Cell Biol.}, volume = {182}, number = {4}, year = {2008}, pages = {791-800}, abstract = {Hemostasis and thrombosis (blood clotting) involve fibrinogen binding to integrin alpha(IIb)beta(3) on platelets, resulting in platelet aggregation. alpha(v)beta(3) binds fibrinogen via an Arg-Asp-Gly (RGD) motif in fibrinogen{\textquoteright}s alpha subunit. alpha(IIb)beta(3) also binds to fibrinogen; however, it does so via an unstructured RGD-lacking C-terminal region of the gamma subunit (gammaC peptide). These distinct modes of fibrinogen binding enable alpha(IIb)beta(3) and alpha(v)beta(3) to function cooperatively in hemostasis. In this study, crystal structures reveal the integrin alpha(IIb)beta(3)-gammaC peptide interface, and, for comparison, integrin alpha(IIb)beta(3) bound to a lamprey gammaC primordial RGD motif. Compared with RGD, the GAKQAGDV motif in gammaC adopts a different backbone configuration and binds over a more extended region. The integrin metal ion-dependent adhesion site (MIDAS) Mg(2+) ion binds the gammaC Asp side chain. The adjacent to MIDAS (ADMIDAS) Ca(2+) ion binds the gammaC C terminus, revealing a contribution for ADMIDAS in ligand binding. Structural data from this natively disordered gammaC peptide enhances our understanding of the involvement of gammaC peptide and integrin alpha(IIb)beta(3) in hemostasis and thrombosis.}, author = {Springer, TA. and Zhu, J. and Xiao, T.} } @article {548886, title = {Structure of a Complete Integrin Ectodomain in a Physiologic Resting State and Activation and Deactivation by Applied Forces.}, journal = {Mol Cell.}, volume = {32}, number = {6}, year = {2008}, pages = {849-61}, abstract = {The complete ectodomain of integrin alpha(IIb)beta(3) reveals a bent, closed, low-affinity conformation, the beta knee, and a mechanism for linking cytoskeleton attachment to high affinity for ligand. Ca and Mg ions in the recognition site, including the synergistic metal ion binding site (SyMBS), are loaded prior to ligand binding. Electrophilicity of the ligand-binding Mg ion is increased in the open conformation. The beta(3) knee passes between the beta(3)-PSI and alpha(IIb)-knob to bury the lower beta leg in a cleft, from which it is released for extension. Different integrin molecules in crystals and EM reveal breathing that appears on pathway to extension. Tensile force applied to the extended ligand-receptor complex stabilizes the closed, low-affinity conformation. By contrast, an additional lateral force applied to the beta subunit to mimic attachment to moving actin filaments stabilizes the open, high-affinity conformation. This mechanism propagates allostery over long distances and couples cytoskeleton attachment of integrins to their high-affinity state.}, author = {Zhu, J. and Luo, BH. and Xiao, T. and Zhang, C. and Nishida, N. and Springer, TA.} } @article {548891, title = {An unusual allosteric mobility of the C-terminal helix of a high-affinity alphaL integrin I domain variant bound to ICAM-5.}, journal = {Mol Cell.}, volume = {31}, number = {3}, year = {2008}, pages = {432-7}, abstract = {Integrins are cell surface receptors that transduce signals bidirectionally across the plasma membrane. The key event of integrin signaling is the allosteric regulation between its ligand-binding site and the C-terminal helix (alpha7) of integrin{\textquoteright}s inserted (I) domain. A significant axial movement of the alpha7 helix is associated with the open, active conformation of integrins. We describe the crystal structure of an engineered high-affinity I domain from the integrin alpha(L)beta(2) (LFA-1) alpha subunit in complex with the N-terminal two domains of ICAM-5, an adhesion molecule expressed in telencephalic neurons. The finding that the alpha7 helix swings out and inserts into a neighboring I domain in an upside-down orientation in the crystals implies an intrinsically unusual mobility of this helix. This remarkable feature allows the alpha7 helix to trigger integrin{\textquoteright}s large-scale conformational changes with little energy penalty. It serves as a mechanistic example of how a weakly bound adhesion molecule works in signaling.}, author = {Zhang, H. and Casanovas, JM. and Jin, M. and Liu JH. and Gahmberg, CG. and Springer, TA. and Wang, JH.} } @article {528091, title = {Trans-cellular migration: cell-cell contacts get intimate}, journal = {Curr. Opin. Cell Biol.}, volume = {20}, year = {2008}, note = {Journal ArticleUnited States}, pages = {533-40}, abstract = {Trans-cellular migration, the movement of one cell directly through another, seems an unlikely, counterintuitive, and even bizarre process. Trans-cellular migration has been reported for nearly half a century in leukocyte transendothelial migration in vivo, but is not well enough accepted to widely feature in textbook accounts of diapedesis. Recently, the first in vitro and additional in vivo observations of trans-cellular diapedesis have been reported. Mechanisms by which this occurs are just beginning to be elucidated and point to podosome-like protrusive activities in leukocytes and specific fusogenic functions in endothelial cells. Emerging evidence for a quantitatively significant contribution of trans-cellular migration to leukocyte trafficking in increasingly diverse settings suggests that this phenomenon represents an important and physiologic cell biological process.}, isbn = {0955-0674 (Print)}, author = {Carman, C. V. and Springer, T.A.} } @article {548901, title = {Binding between the integrin αXβ2 (CD11c/CD18) and heparin.}, journal = {J Biol Chem.}, volume = {282}, number = {42}, year = {2007}, pages = {30869-77}, abstract = {The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor alphaXbeta2 (p150,95, CD11c/CD18). Here, we analyze the structural motifs within heparin that constitute high affinity binding sites for the I domain of integrin alphaXbeta2. Heparin oligomers with chain lengths of 10 saccharide residues or higher provide strong inhibition of the binding by the alphaX I domain to the complement fragment iC3b. By contrast, smaller oligomers or the synthetic heparinoid fondaparinux were not able to block the binding. Semipurified heparin oligomers with 12 saccharide residues identified the fully sulfated species as the most potent antagonist of iC3b, with a 1.3 microM affinity for the alphaX I domain. In studies of direct binding by the alphaX I domain to immobilized heparin, we found that the interaction is conformationally regulated and requires Mg2+. Furthermore, the fully sulfated heparin fragment induced conformational change in the ectodomain of the alphaXbeta2 receptor, also demonstrating allosteric linkage between heparin binding and integrin conformation.}, author = {Vorup-Jensen, T. and Chi, L and Gjelstrup, L. C and Jensen, U. B and Jewett, C. A and Xie, C. and Shimaoka, M. and Linhardt, R. J and Springer, T.A.} } @article {548941, title = {The connection between metal ion affinity and ligand affinity in integrin I domains.}, journal = {Biochim Biophys Acta.}, volume = {1774}, number = {9}, year = {2007}, pages = {1148-55}, abstract = {Integrins are cell-surface heterodimeric proteins that mediate cell-cell, cell-matrix, and cell-pathogen interactions. Half of the known integrin alpha subunits contain inserted domains (I domains) that coordinate ligand through a metal ion. Although the importance of conformational changes within isolated I domains in regulating ligand binding has been reported, the relationship between metal ion binding affinity and ligand binding affinity has not been elucidated. Metal and ligand binding by several I domain mutants that are stabilized in different conformations are investigated using isothermal titration calorimetry and surface plasmon resonance studies. This work suggests an inverse relationship between metal ion affinity and ligand binding affinity (i.e. constructs with a high affinity for ligand exhibit a low affinity for metal). This trend is discussed in the context of structural studies to provide an understanding of interplay between metal ion binding and ligand affinities and conformational changes.}, author = {Vorup-Jensen, T. and Waldron, T. T and Astrof, N. and Shimaoka, M. and Springer, T.A.} } @article {548906, title = {Requirement of α and β subunit transmembrane helix separation for integrin outside-in signaling.}, journal = {Blood}, volume = {110}, number = {7}, year = {2007}, pages = {2475-83}, abstract = {Adhesion to extracellular ligands through integrins regulates cell shape, migration, growth, and survival. How integrins transmit signals in the outside-to-in direction remains unknown. Whereas in resting integrins the alpha and beta subunit transmembrane domains are associated, ligand binding promotes dissociation and separation of these domains. Here we address whether such separation is required for outside-in signaling. By introduction of an intersubunit disulfide bond, we generated mutant integrin alphaIIbbeta3 with blocked transmembrane separation that binds ligand, mediates adhesion, adopts an extended conformation after ligand binding, and forms antibody-induced macroclusters on the cell surface similarly to wild type. However, the mutant integrin exhibits a profound defect in adhesion-induced outside-in signaling as measured by cell spreading, actin stress-fiber and focal adhesion formation, and focal adhesion kinase activation. This defect was rescued by reduction of the disulfide bond. Our results demonstrate that the separation of transmembrane domains is required for integrin outside-in signal transduction.}, author = {Zhu, J. and Carman, C. V and Kim, M. and Shimaoka, M. and Springer, T. A and Luo, B-H.} } @article {548911, title = {Specific and covalent labeling of a membrane protein with organic fluorochromes and quantume dots.}, journal = {Proc Natl Acad Sci USA}, volume = {104}, number = {37}, year = {2007}, pages = {14753-8}, abstract = {The real-time observation of protein dynamics in living cells and organisms is of fundamental importance for understanding biological processes. Most approaches to labeling proteins exploit noncovalent interactions, unsuitable to long-term studies, or genetic fusion to naturally occurring fluorescent proteins that often have unsatisfactory optical properties. Here we used the fungal enzyme cutinase and its suicide substrate p-nitrophenyl phosphonate to covalently attach a variety of labels to the integrin lymphocyte function-associated antigen-1 (LFA-1) on the surface of living cells. Cutinase was embedded in the extracellular domain of LFA-1 with no appreciable influence on integrin function and conformational regulation. p-nitrophenyl phosphonate-conjugated fluorochromes, including the very bright and stable quantum dots, bound efficiently and specifically to LFA-1/cutinase. The availability of a genetically encoded tag that binds covalently to quantum dots could foster the development of new experimental strategies for the study of protein dynamics in vivo.}, author = {Bonasio, R and Carman, C. V and Kim, E and Sage, P. T and Love, K. R and Mempel, TR and Springer, T. A and von Andrian, U. H} } @article {548916, title = {Structural basis of integrin regulation and signaling.}, journal = {Annu Rev Immunol.}, volume = {25}, year = {2007}, pages = {619-47}, abstract = {Integrins are cell adhesion molecules that mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions. They play critical roles for the immune system in leukocyte trafficking and migration, immunological synapse formation, costimulation, and phagocytosis. Integrin adhesiveness can be dynamically regulated through a process termed inside-out signaling. In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm in the classical outside-in direction. Recent structural, biochemical, and biophysical studies have greatly advanced our understanding of the mechanisms of integrin bidirectional signaling across the plasma membrane. Large-scale reorientations of the ectodomain of up to 200 A couple to conformational change in ligand-binding sites and are linked to changes in alpha and beta subunit transmembrane domain association. In this review, we focus on integrin structure as it relates to affinity modulation, ligand binding, outside-in signaling, and cell surface distribution dynamics.}, author = {Luo, B-H. and Carman, C. V and Springer, T.A.} } @article {548931, title = {Structural plasticity in IgSF domain 4 of ICAM-1 mediates cell surface dimerization.}, journal = {Proc Natl Acad Sci USA}, volume = {104}, number = {39}, year = {2007}, pages = {15358-63}, abstract = {The Ig superfamily (IgSF) intercellular adhesion molecule-1 (ICAM-1) equilibrates between monomeric and dimeric forms on the cell surface, and dimerization enhances cell adhesion. A crystal structure of ICAM-1 IgSF domains (D) 3-5 revealed a unique dimerization interface in which D4s of two protomers fuse through edge beta-strands to form a single super beta-sandwich domain. Here, we describe a crystal structure at 2.7-A resolution of monomeric ICAM-1 D3-D5, stabilized by the monomer-specific Fab CA7. CA7 binds to D5 in a region that is buried in the dimeric interface and is distal from the dimerization site in D4. In monomeric ICAM-1 D3-D5, a 16-residue loop in D4 that is disordered in the dimeric structure could clearly be traced as a BC loop, a short C strand, and a CE meander with a cis-Pro followed by a solvent-exposed, flexible four-residue region. Deletions of 6 or 10 residues showed that the C-strand is essential for monomer stability, whereas a distinct six-residue deletion showed little contribution of the CE meander. Mutation of two inward-pointing Leu residues in edge beta-strand E to Lys increased monomer stability, confirming the hypothesis that inward-pointing charged side chains on edge beta-strands are an important design feature to prevent beta-supersheet formation. Overall, the studies reveal that monomer-dimer transition is associated with a surprisingly large, physiologically relevant, IgSF domain rearrangement.}, author = {Chen, X. and Kim, T. D and Carman, C. V and Mi, L and Song, G. and Springer, T. A} } @article {548936, title = {Tests of the Extension and Deadbolt Models of Integrin Activation.}, journal = {J Biol Chem.}, volume = {282}, number = {16}, year = {2007}, pages = {11914-20}, abstract = {Despite extensive evidence that integrin conformational changes between bent and extended conformations regulate affinity for ligands, an alternative hypothesis has been proposed in which a "deadbolt" can regulate affinity for ligand in the absence of extension. Here, we tested both the deadbolt and the extension models. According to the deadbolt model, a hairpin loop in the beta3 tail domain could act as a deadbolt to restrain the displacement of the beta3 I domain beta6-alpha7 loop and maintain integrin in the low affinity state. We found that mutating or deleting the beta3 tail domain loop has no effect on ligand binding by either alphaIIbbeta 3 or alphaVbeta3 integrins. In contrast, we found that mutations that lock integrins in the bent conformation with disulfide bonds resist inside-out activation induced by cytoplasmic domain mutation. Furthermore, we demonstrated that extension is required for accessibility to fibronectin but not smaller fragments. The data demonstrate that integrin extension is required for ligand binding during integrin inside-out signaling and that the deadbolt does not regulate integrin activation.}, author = {Zhu, J. and Boylan, B and Luo, B-H. and Newman, P. J and Springer, T. A} } @article {548946, title = {Transcellular diapedesis is initiated by invasive podosomes.}, journal = {Immunity}, volume = {26}, number = {6}, year = {2007}, pages = {784-97}, abstract = {Diapedesis is critical for immune system function and inflammatory responses. This occurs by migration of blood leukocytes either directly through individual microvascular endothelial cells (the "transcellular" route) or between them (the "paracellular" route). Mechanisms for transcellular pore formation in endothelium remain unknown. Here we demonstrate that lymphocytes used podosomes and extended "invasive podosomes" to palpate the surface of, and ultimately form transcellular pores through, the endothelium. In lymphocytes, these structures were dependent on Src kinase and the actin regulatory protein WASP; inhibition of podosome formation selectively blocked the transcellular route of diapedesis. In endothelium, membrane fusion events dependent on the SNARE-containing membrane fusion complex and intracellular calcium were required for efficient transcellular pore formation in response to podosomes. These findings provide insights into basic mechanisms for leukocyte trafficking and the functions of podosomes.}, author = {Carman, C. V and Sage, P. T. and Sciuto, T. E. and de la Fuente, M. A. and Geha, R. S. and Ochs, H. D. and Dvorak, H. F. and Dvorak, A. M. and Springer, T.A.} } @article {548956, title = {Complement and the multifaceted functions of VWA and integrin I domains.}, journal = {Structure}, volume = {14}, number = {11}, year = {2006}, pages = {1611-6}, abstract = {The recent crystal structure of complement protein component C2a reveals an interface between its VWA and serine protease domains that could not exist in the zymogen C2. The implied change in VWA domain conformation between C2 and C2a differs from that described for other VWA domains, including the I domains in integrins. Here, the remarkable diversity in both conformational regulation and ligand binding among VWA domains that function in complement, hemostasis, cell adhesion, anthrax toxin binding, vesicle transport, DNA break repair, and RNA quality control is reviewed. Finally, implications for metastability of complement convertases are discussed.}, author = {Springer, T.A.} } @article {548961, title = {Importance of force linkage in mechanochemistry of adhesion receptors.}, journal = {Biochemistry}, volume = {45}, number = {50}, year = {2006}, pages = {15020-8}, abstract = {The alpha subunit-inserted (I) domain of integrin alphaLbeta2 [lymphocyte function-associated antigen-1 (LFA-1)] binds to intercellular adhesion molecule-1 (ICAM-1). The C- and N-termini of the alpha I domain are near one another on the "lower" face, opposite the metal ion-dependent adhesion site (MIDAS) on the "upper face". In conversion to the open alpha I domain conformation, a 7 A downward, axial displacement of C-terminal helix alpha7 is allosterically linked to rearrangement of the MIDAS into its high-affinity conformation. Here, we test the hypothesis that when an applied force is appropriately linked to conformational change, the conformational change can stabilize adhesive interactions that resist the applied force. Integrin alpha I domains were anchored to the cell surface through their C- or N-termini using type I or II transmembrane domains, respectively. C-terminal but not N-terminal anchorage robustly supported cell rolling on ICAM-1 substrates in shear flow. In contrast, when the alphaL I domain was mutationally stabilized in the open conformation with a disulfide bond, it mediated comparable levels of firm adhesion with type I and type II membrane anchors. To exclude other effects as the source of differential adhesion, these results were replicated using alpha I domains conjugated through the N- or C-terminus to polystyrene microspheres. Our results demonstrate a mechanical feedback system for regulating the strength of an adhesive bond. A review of crystal structures of integrin alpha and beta subunit I domains and selectins in high- and low-affinity conformations demonstrates a common mechanochemical design in which biologically applied tensile force stabilizes the more extended, high-affinity conformation.}, author = {Astrof, N. S. and Salas, A. and Shimaoka, M. and Chen, J. F and Springer, T. A} } @article {548951, title = {A small molecule agonist of an integrin, aLb2.}, journal = {J Biol Chem.}, volume = {281}, number = {49}, year = {2006}, pages = {37904-12}, abstract = {The binding of integrin alpha(L)beta(2) to its ligand intercellular adhesion molecule-1 is required for immune responses and leukocyte trafficking. Small molecule antagonists of alpha(L)beta(2) are under intense investigation as potential anti-inflammatory drugs. We describe for the first time a small molecule integrin agonist. A previously described alpha/beta I allosteric inhibitor, compound 4, functions as an agonist of alpha(L)beta(2) in Ca(2+) and Mg(2+)and as an antagonist in Mn(2+). We have characterized the mechanism of activation and its competitive and noncompetitive inhibition by different compounds. Although it stimulates ligand binding, compound 4 nonetheless inhibits lymphocyte transendothelial migration. Agonism by compound 4 results in accumulation of alpha(L)beta(2) in the uropod, extreme uropod elongation, and defective de-adhesion. Small molecule integrin agonists open up novel therapeutic possibilities.}, author = {Yang, W and Carman, C. V and Kim, M. and Salas, A. and Shimaoka, M. and Springer, T.A.} } @article {548966, title = {Structural transitions of complement component C3 and its activation products.}, journal = {Proc Natl Acad Sci USA }, volume = {103}, number = {52}, year = {2006}, pages = {19737-42}, abstract = {Complement sensitizes pathogens for phagocytosis and lysis. We use electron microscopy to examine the structural transitions in the activation of the pivotal protein in the complement pathway, C3. In the cleavage product C3b, the position of the thioester domain moves approximately 100 Angstrom, which becomes covalently coupled to antigenic surfaces. In the iC3b fragment, cleavage in an intervening domain creates a long flexible linker between the thioester domain and the macroglobulin domain ring of C3. Studies on two products of nucleophile addition to C3 reveal a structural intermediate in activation, and a final product, in which the anaphylatoxin domain has undergone a remarkable movement through the macroglobulin ring.}, author = {Nishida, N. and Walz, T. and Springer, T. A} } @article {528351, title = {Activation of leukocyte β2 integrins by conversion from bent to extended conformations}, journal = {Immunity}, volume = {25}, number = {4}, year = {2006}, note = {in file}, pages = {583-594}, abstract = {We used negative stain electron microscopy (EM) to examine the conformational changes in the ectodomains required for activation of the leukocyte integrins alpha(X)beta(2) and alpha(L)beta(2). They transitioned between a bent conformation and two extended conformations in which the headpiece was in either a closed or an open state. Extended integrins exhibited marked flexibility at the alpha subunit genu and between integrin epidermal growth factor-like (I-EGF) domains 1 and 2. A clasp to mimic juxtamembrane association between the integrin alpha and beta subunits stabilized the bent conformation strongly for alpha(X)beta(2) and less so for alpha(L)beta(2). A small molecule allosteric antagonist induced the extended, open headpiece conformation. A Fab known to activate beta(2) integrins on leukocytes induced extension, and a Fab reporter of activation bound only after extension had been induced. The results establish an intimate relationship between extension of beta(2) integrins and their activation in immune responses and leukocyte trafficking.}, author = {Nishida, N. and Xie, C. and Shimaoka, M. and Cheng, Y. and Walz, T. and Springer, T.A.} } @article {530176, title = {AL-57, a ligand-mimetic antibody to integrin LFA-1, reveals chemokine-induced affinity upregulation in lymphocytes}, journal = {Proc Natl Acad Sci USA}, volume = {103}, number = {38}, year = {2006}, note = {in file}, pages = {13991-13996}, abstract = {Affinity of integrin lymphocyte function-associated antigen 1 (LFA-1) is enhanced by conformational changes from the low-affinity closed form to the high-affinity (HA) open form of the ligand-binding inserted (I) domain as shown by work with purified I domains. However, affinity up-regulation of LFA-1 on the cell surface by physiological agonists such as chemokines has yet to be demonstrated by monovalent reagents. We characterize a mAb, AL-57 (activated LFA-1 clone 57), that has been developed by phage display that selectively targets the HA open conformation of the LFA-1 I domain. AL-57 discriminates among low-affinity, intermediate-affinity, and HA states of LFA-1. Furthermore, AL-57 functions as a ligand mimetic that binds only upon activation and requires Mg2+ for binding. Compared with the natural ligand intercellular adhesion molecule-1, AL-57 shows a tighter binding to the open I domain and a 250-fold slower off rate. Monovalent Fab AL-57 demonstrates affinity increases on a subset (approximately 10\%) of lymphocyte cell surface LFA-1 molecules upon stimulation with CXCL-12 (CXC chemokine ligand 12). Affinity up-regulation correlates with global conformational changes of LFA-1 to the extended form. Affinity increase stimulated by CXCL-12 is transient and peaks 2 to 5 min after stimulation.}, author = {Shimaoka, M. and Kim, M. and Cohen, E. H. and Yang, W and Astrof, N. and Peer, D. and Salas, A. and Ferrand, A. and Springer, T.A.} } @article {530111, title = {Directed evolution to probe protein allostery and integrin I domains of 200,000-fold higher affinity}, journal = {Proc Natl Acad Sci USA}, volume = {103}, number = {15}, year = {2006}, note = {in file}, pages = {5758-5763}, abstract = {Understanding allostery may serve to both elucidate mechanisms of protein regulation and provide a basis for engineering active mutants. Herein we describe directed evolution applied to the integrin alpha(L) inserted domain for studying allostery by using a yeast surface display system. Many hot spots for activation are identified, and some single mutants exhibit remarkable increases of 10,000-fold in affinity for a physiological ligand, intercellular adhesion molecule-1. The location of activating mutations traces out an allosteric interface in the interior of the inserted domain that connects the ligand binding site to the alpha7-helix, which communicates allostery to neighboring domains in intact integrins. The combination of two activating mutations (F265S/F292G) leads to an increase of 200,000-fold in affinity to intercellular adhesion molecule-1. The F265S/F292G mutant is potent in antagonizing lymphocyte function-associated antigen 1-dependent lymphocyte adhesion, aggregation, and transmigration.}, keywords = {CA31799}, author = {Jin, M. and Song, G. and Kim, Y-S. and Astrof, N. and Shimaoka, M. and Wittrup, D. and Springer, T.A.} } @article {527841, title = {A high affinity human antibody antagonist of P-selectin mediated rolling}, journal = {Biochem. Biophys. Res. Commun.}, volume = {350}, number = {3}, year = {2006}, note = {in file}, pages = {508-513}, abstract = {We have characterized the IgG form of a previously isolated and engineered single-chain Fv (scFv), named RR2r3s4-1, that binds to human PSGL-1. This fully human IgG was determined to have a Kd of 1.8+/-0.7 nM by fluorescence quenching titration. It better inhibits P-selectin-PSGL-1 interactions than a commercially available murine monoclonal antibody KPL1 and better inhibits neutrophil rolling than KPL1. Thus, RR2r3s4-1 is the most effective antibody at inhibiting P-selectin-PSGL-1 interactions known. Specificity analysis reveals that RR2r3s4-1 does not cross react with murine PSGL-1 and thus requires more than tyrosine sulfate for binding to human PSGL-1. This evidence demonstrates the therapeutic potential of this antibody as a potent anti-inflammatory therapeutic.}, author = {Swers, J. S. and Widom, A. and Phan, U. and Springer, T.A. and Wittrup, K. D.} } @article {529431, title = {Identification and characterization of a human monoclonal antagonistic antibody AL-57 that preferentially binds the high-affinity form of lymphocyte function-associated antigen-1}, journal = {J Leukoc Biol.}, volume = {80}, number = {4}, year = {2006}, pages = {905-914}, abstract = {LFA-1 (alpha(L)beta(2)) mediates cell-cell and cell-extracellular matrix adhesions essential for immune and inflammatory responses. One critical mechanism regulating LFA-1 activity is the conformational change of the ligand-binding alpha(L) I domain from low-affinity (LA), closed form, to the high-affinity (HA), open form. Most known integrin antagonists bind both forms. Antagonists specific for the HA alpha(L) I domain have not been described. Here, we report the identification and characterization of a human antibody AL-57, which binds to the alpha(L) I domain in a HA but not LA conformation. AL-57 was discovered by selection from a human Fab-displaying library using a locked-open HA I domain as target. AL-57 Fab-phage bound HA I domain-expressing K562 cells (HA cells) in a Mg(2+)-dependent manner. AL-57 IgG also bound HA cells and PBMCs, activated by Mg(2+)/EGTA, PMA, or DTT. The binding profile of AL-57 IgG on PBMCs was the same as that of ICAM-1, the main ligand of LFA-1. In contrast, an anti-alpha(L) murine mAb MHM24 did not distinguish between the HA and LA forms. Moreover, AL-57 IgG blocked ICAM-1 binding to HA cells with a potency greater than MHM24. It also inhibited ICAM-1 binding to PBMCs, blocked adhesion of HA cells to keratinocytes, and inhibited PHA-induced lymphocyte proliferation with potencies comparable with MHM24. These results indicate that specifically targeting the HA I domain is sufficient to inhibit LFA-1-mediated, adhesive functions. AL-57 represents a therapeutic candidate for treatment of inflammatory and autoimmune diseases.}, author = {Huang, L. and Shimaoka, M. and Rondon, I. and Roy, I. and Chang, Q. and Po, M. and Dransfield, D. T. and Ladner, R. C. and Edge, A. S. and Salas, A. and Wood, C. R. and Springer, T.A. and Cohen, E. H.} } @article {528096, title = {Integrin structures and conformational signaling}, journal = {Curr Opin Cell Biol.}, volume = {18}, number = {5}, year = {2006}, note = {in file}, pages = {579-586}, abstract = {Integrins are cell adhesion molecules that play critical roles in development, wound healing, hemostasis, immunity and cancer. Advances in the past two years have shed light on the structural basis for integrin regulation and signaling, especially on how global conformational changes between bent and extended conformations relate to the inter-domain and intra-domain shape shifting that regulates affinity for ligand. The downward movements of the C-terminal helices of the alpha I and beta I domains and the swing-out of the hybrid domain play pivotal roles in integrin conformational signaling. Experiments have also shown that integrins transmit bidirectional signals across the plasma membrane by coupling extracellular conformational change with an unclasping and separation of the alpha and beta transmembrane and cytoplasmic domains.}, author = {Luo, B-H. and Springer, T.A.} } @article {528586, title = {Rational design of ICAM-1 variants for antagonizing integrin LFA-1-dependent adhesion}, journal = {J Biol Chem.}, volume = {281}, number = {8}, year = {2006}, note = {in file}, pages = {5042-5049}, abstract = {The interaction between integrin lymphocyte function-associated antigen-1 (LFA-1) and its ligand intercellular adhesion molecule-1 (ICAM-1) is critical in immunological and inflammatory reactions but, like other adhesive interactions, is of low affinity. Here, multiple rational design methods were used to engineer ICAM-1 mutants with enhanced affinity for LFA-1. Five amino acid substitutions 1) enhance the hydrophobicity and packing of residues surrounding Glu-34 of ICAM-1, which coordinates to a Mg2+ in the LFA-1 I domain, and 2) alter associations at the edges of the binding interface. The affinity of the most improved ICAM-1 mutant for intermediate- and high-affinity LFA-1 I domains was increased by 19-fold and 22-fold, respectively, relative to wild type. Moreover, potency was similarly enhanced for inhibition of LFA-1-dependent ligand binding and cell adhesion. Thus, rational design can be used to engineer novel adhesion molecules with high monomeric affinity; furthermore, the ICAM-1 mutant holds promise for targeting LFA-1-ICAM-1 interaction for biological studies and therapeutic purposes.}, author = {Song, G. and Lazar, G. A. and Kortemme, T. and Shimaoka, M. and Desjarlais, J. R. and Baker, D. and Springer, T.A.} } @article {530046, title = {Regulation of outside-in signaling by the β2 I domain of integrin αLβ2}, journal = {Proc Natl Acad Sci USA}, volume = {103}, number = {35}, year = {2006}, pages = {13062-13067}, abstract = {The adhesiveness of integrin alpha(L)beta(2) is modulated by divalent cations. We mutated three metal ion-binding sites in the beta(2) I domain. The metal ion-dependent adhesion site (MIDAS) and the ligand-induced metal-binding site are required for ligand binding and sufficient for synergism between Ca(2+) and Mg(2+). Adjacent to MIDAS (ADMIDAS) mutants are constitutively active but remain bent, with poor exposure of a beta(2) stalk region epitope. Fluorescence resonance energy transfer between fluorescent protein-fused alpha(L) and beta(2) cytoplasmic domains showed that ADMIDAS mutation abrogated ligand binding-induced spatial separation of cytoplasmic domains. Furthermore, ADMIDAS mutation abolished spreading on ligand-bearing substrates. Thus, beta(2) I domain metal ion-binding sites regulate alpha(L) I domain affinity, and the ADMIDAS is required for outside-in signaling.}, keywords = {in file}, author = {Chen, J. F. and Yang, W and Kim, M. and Carman, C. V. and Springer, T.A.} } @article {529846, title = {Remodeling of the lectin/EGF-like interface in P- and L-selectin increases adhesiveness and shear resistance under hydrodynamic force}, journal = {Nat. Immunol.}, volume = {7}, number = {8}, year = {2006}, note = {in file}, pages = {883-889}, abstract = {Crystal structures of the lectin and epidermal growth factor (EGF)-like domains of P-selectin show {\textquoteright}bent{\textquoteright} and {\textquoteright}extended{\textquoteright} conformations. An extended conformation would be {\textquoteright}favored{\textquoteright} by forces exerted on a selectin bound at one end to a ligand and at the other end to a cell experiencing hydrodynamic drag forces. To determine whether the extended conformation has higher affinity for ligand, we introduced an N-glycosylation site to {\textquoteright}wedge open{\textquoteright} the interface between the lectin and EGF-like domains of P-selectin. This alteration increased the affinity of P-selectin for its ligand P-selectin glycoprotein 1 (PSGL-1) and thereby the strength of P-selectin-mediated rolling adhesion. Similarly, an asparagine-to-glycine substitution in the lectin-EGF-like domain interface of L-selectin enhanced rolling adhesion under shear flow. Our results demonstrate that force, by {\textquoteright}favoring{\textquoteright} an extended selectin conformation, can strengthen selectin-ligand bonds.}, author = {Phan, U. T. and Waldron, T. T. and Springer, T.A.} } @article {528581, title = {Transition from rolling to firm adhesion can be mimicked by extension of integrin αLβ2 in an intermediate-affinity state}, journal = {J Biol Chem.}, volume = {281}, number = {16}, year = {2006}, note = {in file}, pages = {10876-10882}, abstract = {AlphaLbeta2 affinity for intercellular adhesion molecule-1 (ICAM-1) is regulated by the conformation of the alphaL I domain, which is in turn controlled by the conformation and orientation of other adjacent domains. Additionally, overall integrin conformation (bent versus straightened) influences the orientation of the I domain and access to its ligands, influencing adhesive efficiency. The open or high affinity I domain conformation supports strong adhesion, whereas the closed, low affinity conformation mediates weak interactions or rolling. We have previously suggested that alphaLbeta2 can also exist on the cell surface in an intermediate affinity state. Here we have studied the adhesive properties of integrin alphaLbeta2 containing mutant I domains with intermediate affinities for ICAM-1. In an overall bent conformation, the intermediate affinity state of alphaLbeta2 is hardly detected by conventional adhesion assays, but robust adhesion is seen when an extended conformation is induced by a small molecule alpha/beta I allosteric antagonist. Intermediate affinity alphaLbeta2 supports more stable rolling than wild-type alphaLbeta2 under shear conditions. Moreover, antagonist-induced extension transforms rolling adhesion into firm adhesion in a manner reminiscent of chemokine activation of integrin alphaLbeta2. These findings suggest the relevance of intermediate affinity states of alphaLbeta2 to the transition between inactive and active states and demonstrate the importance of both I domain affinity and overall integrin conformation for cell adhesion.}, author = {Salas, A. and Shimaoka, M. and Phan, U. and Kim, M. and Springer, T.A.} } @article {530191, title = {An atomic resolution view of ICAM recognition in a complex between the binding domains of ICAM-3 and integrin αLβ2}, journal = {Proc Natl Acad Sci USA }, volume = {102}, number = {9}, year = {2005}, note = {in file}, pages = {3366-3371}, abstract = {Within the Ig superfamily (IgSF), intercellular adhesion molecules (ICAMs) form a subfamily that binds the leukocyte integrin alphaLbeta2. We report a 1.65-A-resolution crystal structure of the ICAM-3 N-terminal domain (D1) in complex with the inserted domain, the ligand-binding domain of alphaLbeta2. This high-resolution structure and comparisons among ICAM subfamily members establish that the binding of ICAM-3 D1 onto the inserted domain represents a common docking mode for ICAM subfamily members. The markedly different off-rates of ICAM-1, -2, and -3 appear to be determined by the hydrophobicity of residues that surround a metal coordination bond in the alphaLbeta2-binding interfaces. Variation in composition of glycans on the periphery of the interfaces influences on-rate.}, author = {Song, G. and Yang, Y. and Liu, J-H. and Casasnovas,J. and Shimaoka, M. and Springer, T.A. and J.-H. Wang} } @article {528506, title = {Contribution of N-linked glycans to the conformation and function of intercellular adhesion molecules (ICAMs)}, journal = {J Biol Chem.}, volume = {280}, number = {7}, year = {2005}, note = {in file}, month = {Feb 18}, pages = {5854-5861}, abstract = {The crystal structures of the glycosylated N-terminal two domains of ICAM-1 and ICAM-2 provided a framework for understanding the role of glycosylation in the structure and function of intercellular adhesion molecules (ICAMs). The most conserved glycans were less flexible in the structures, interacting with protein residues and contributing to receptor folding and expression. The first N-linked glycan in ICAM-2 contacts an exposed tryptophan residue, defining a conserved glycan-W motif critical for the conformation of the integrin binding domain. The absence of this motif in human ICAM-1 exposes regions used in receptor dimerization and rhinovirus recognition. Experiments with soluble molecules having the N-terminal two domains of human ICAMs identified glycans of the high mannose type N-linked to the second domain of the dendritic cell-specific ICAM-grabbing nonintegrin lectin-ligands ICAM-2 and ICAM-3. About 40\% of those receptor molecules bear endoglycosidase H sensitive glycans responsible of the lectin binding activity. High mannose glycans were absent in ICAM-1, which did not bind to the lectin, but they appeared in ICAM-1 mutants with additional N-linked glycosylation and lectin binding activity. N-Linked glycosylation regulate both conformation and immune related functions of ICAM receptors.}, author = {Jimenez, D. and Roda-Navarro, P. and Springer, T.A. and Casasnovas,J.M.} } @article {530131, title = {Disrupting integrin transmembrane domain heterodimerization increases ligand binding affinity, not valency or clustering}, journal = {Proc Natl Acad Sci USA }, volume = {102}, year = {2005}, note = {in fileerased duplicate data $\#$16899}, pages = {3679-3684}, abstract = {Residues important in the interaction between the 23-residue transmembrane (TM) domains of the integrin alpha(IIb)- and beta(3)-subunits were identified by mutating each non-Leu residue to Leu. Leu substitutions of alpha(IIb) at G972, G976, and T981, and of beta(3) at I693 and G708, increased ligand binding. Substitutions with other amino acids at alpha(IIb)G972 and beta(3)G708 could also increase ligand binding. The results are consistent with and extend the helical interface between the integrin alpha- and beta-subunit TM domains previously defined by cysteine scanning and disulfide bond formation. We differentiated between affinity- and valency-based modes of activation by TM domain mutations. The mutant alpha(IIb) W967C forms disulfide-linked alpha(IIb)-subunits within an (alpha(IIb)beta(3))(2) tetramer. This tetramer behaved as an ideal model for the valency mode of regulation, because it exhibited significantly increased binding to multivalent but not monovalent ligands and basally retained the bent conformation. By contrast, the activating Leu mutants showed increased binding to the monovalent, ligand-mimetic PAC-1 Fab and increased exposure of ligand-induced binding site (LIBS) epitopes, suggesting that they partially adopt an extended conformation. Furthermore, the previously described beta(3)G708N mutation in Chinese hamster ovary cells enhanced ligand binding affinity, not valency, and did not alter cell-surface clustering as defined by confocal microscopy. Our studies provide evidence that disrupting the integrin heterodimeric TM helix-helix interface activates ligand binding mainly by increasing the monomeric affinity for ligand, but not the receptor valency, i.e., clustering.}, author = {Luo, B-H. and Carman, C. V. and Takagi, J. and Springer, T.A.} } @article {530216, title = {Exposure of acidic residues as a danger signal for recognition of fibrinogen and other macromolecules by integrin αXβ2}, journal = {Proc Natl Acad Sci USA }, volume = {102}, number = {5}, year = {2005}, pages = {1614-1619}, abstract = {The structural integrity of tissue proteins is damaged in processes ranging from remodeling of the extracellular matrix to destruction by microbial pathogens. Leukocytes play a prominent role in tissue surveillance and repair. However, it remains enigmatic what features of structurally decayed proteins prompt recognition by leukocyte cell-surface receptors. Here, we report that adhesion of human neutrophil granulocytes to fibrinogen is greatly increased by plasmin digestion in a mode where alphaXbeta2 dominates the integrin-dependent binding. The bacterial protease subtilisin also enhances binding by alphaXbeta2. The alphaX ligand binding domain has an unusually high affinity for carboxyl groups, with KD at approximately 100 microM. Our findings implicate enhanced accessibility of negatively charged residues in structurally decayed proteins as a pattern recognition motif for alphaXbeta2 integrin. Comparisons among integrins show relevance of these findings to the large number of ligands recognized by alphaMbeta2 and alphaXbeta2 but not alphaLbeta2. The observations suggest that the pericellular proteolysis at the leading edge of neutrophils not only facilitates passage through the extracellular matrix but also manufactures binding sites for alphaXbeta2.}, author = {Vorup-Jensen, T. and Carman, C. V. and Shimaoka, M. and Schuck, P. and Svitel, J. and Springer, T.A.} } @article {528641, title = {Two-dimensional kinetics regulation of αLβ2-ICAM-1 interaction by conformational changes of the αL inserted domain}, journal = {J Biol Chem.}, volume = {280}, number = {51}, year = {2005}, note = {in file}, month = {Oct 18}, pages = {42207-42218}, abstract = {The leukocyte integrin alphaLbeta2 mediates cell adhesion and migration during inflammatory and immune responses. Ligand binding of alphaLbeta2 is regulated by or induces conformational changes in the inserted (I) domain. By using a micropipette, we measured the conformational regulation of two-dimensional (2D) binding affinity and the kinetics of cell-bound intercellular adhesion molecule-1 interacting with alphaLbeta2 or isolated I domain expressed on K562 cells. Locking the I domain into open and intermediate conformations with a disulfide bond increased the affinities by approximately 8000- and approximately 30-fold, respectively, from the locked closed conformation, which has similar affinity as the wild-type I domain. Most surprisingly, the 2D affinity increases were due mostly to the 2D on-rate increases, as the 2D off-rates only decreased by severalfold. The wild-type alphaLbeta2, but not its I domain in isolation, could be up-regulated by Mn2+ or Mg2+ to have high affinities and on-rates. Locking the I domain in any of the three conformations abolished the ability of divalent cations to regulate 2D affinity. These results indicate that a downward displacement of the I domain C-terminal helix, induced by conformational changes of other domains of the alphaLbeta2, is required for affinity and on-rate up-regulation.}, author = {Zhang, F. and Marcus, W. D. and Goyal, N. H. and Selvaraj, P. and Springer, T.A. and Zhu, C.} } @article {530236, title = {Activation of integrin β subunit I-like domains by one-turn C-terminal α-helix deletions}, journal = {Proc Natl Acad Sci USA}, volume = {101}, number = {8}, year = {2004}, note = {in file}, pages = {2333-2338}, abstract = {Integrins contain two structurally homologous but distantly related domains: an I-like domain that is present in all beta-subunits and an I domain that is present in some alpha-subunits. Atomic resolution and mutagenesis studies of alpha I domains demonstrate a C-terminal, axial displacement of the alpha7-helix that allosterically regulates the shape and affinity of the ligand-binding site. Atomic resolution studies of beta I-like domains have thus far demonstrated no similar alpha7-helix displacement; however, other studies are consistent with the idea that alpha I and beta I-like domains undergo structurally analogous rearrangements. To test the hypothesis that C-terminal, axial displacement of the alpha7-helix, coupled with beta6-alpha7 loop reshaping, activates beta I-like domains, we have mimicked the effect of alpha7-helix displacement on the beta6-alpha7 loop by shortening the alpha7-helix by two independent, four-residue deletions of about one turn of alpha-helix. In the case of integrin alphaLbeta2, each mutant exhibits constitutively high affinity for the physiological ligand intercellular adhesion molecule 1 and full exposure of a beta I-like domain activation-dependent antibody epitope. In the case of analogous mutants in integrin alpha4beta7, each mutant shows the activated phenotype of firm adhesion, rather than rolling adhesion, in shear flow. The results show that integrins that contain or lack alpha I domains share a common pathway of beta I-like domain activation, and they suggest that beta I-like and alpha I domain activation involves structurally analogous alpha7-helix axial displacements.}, author = {Yang, W and Shimaoka, M. and Chen, J. F. and Springer, T.A.} } @article {528551, title = {Allosteric β1 integrin antibodies that stabilize the low affinity state by preventing the swing-out of the hybrid domain}, journal = {J. Biol. Chem.}, volume = {279}, number = {26}, year = {2004}, note = {in file}, pages = {27466-27471}, abstract = {The ligand binding function of integrins can be modulated by various monoclonal antibodies by both direct and indirect mechanisms. We have characterized an anti-beta(1) antibody, SG/19, that had been reported to inhibit the function of the beta(1) integrin on the cell surface. SG/19 recognized the wild type beta(1) subunit that exists in a conformational equilibrium between the high and low affinity states but bound poorly to a mutant beta(1) integrin that had been locked in a high affinity state. Epitope mapping of SG/19 revealed that Thr(82) in the beta(1) subunit, located at the outer face of the boundary between the I-like and hybrid domains, was the key binding determinant for this antibody. Direct visualization of the alpha (5)beta(1) headpiece fragment in complex with SG/19 Fab with electron microscopy confirmed the location of the binding surface and showed that the ligand binding site is not occluded by the bound Fab. Surface plasmon resonance showed that alpha (5)beta(1) integrin bound by SG/19 maintained a low affinity toward its physiological ligand fibronectin (Fn) whereas binding by function-blocking anti-alpha(5) antibodies resulted in a complete loss of fibronectin binding. Thus a class of the anti-beta antibodies represented by SG/19 attenuate the ligand binding function by restricting the conformational shift to the high affinity state involving the swing-out of the hybrid domain without directly interfering with ligand docking.}, author = {Luo, B-H. and Strokovich, K. and Walz, T. and Springer, T.A. and Takagi, J.} } @article {529206, title = {The binding sites for competitive antagonistic, allosteric antagonistic, and agonistic antibodies to the I domain of integrin LFA-1}, journal = {J. Immunol.}, volume = {173}, number = {6}, year = {2004}, pages = {3972-3978}, abstract = {We explore the binding sites for mAbs to the alpha I domain of the integrin alphaLbeta2 that can competitively inhibit, allosterically inhibit, or activate binding to the ligand ICAM-1. Ten mAbs, some of them clinically important, were mapped to species-specific residues. The results are interpreted with independent structures of the alphaL I domain determined in seven different crystal lattices and in solution, and which are present in three conformational states that differ in affinity for ligand. Six mAbs bind to adjacent regions of the beta1-alpha1 and alpha3-alpha4 loops, which show only small (mean, 0.8 angstroms; maximum, 1.8 angstroms) displacements among the eight I domain structures. Proximity to the ligand binding site and to noncontacting portions of the ICAM-1 molecule explains competitive inhibition by these mAbs. Three mAbs bind to a segment of seven residues in the beta5-alpha6 loop and alpha6 helix, in similar proximity to the ligand binding site, but on the side opposite from the beta1-alpha1/alpha3-alpha4 epitopes, and far from noncontacting portions of ICAM-1. These residues show large displacements among the eight structures in response to lattice contacts (mean, 3.6 angstroms; maximum, 9.4 angstroms), and movement of a buried Phe in the beta5-alpha6 loop is partially correlated with affinity change at the ligand binding site. Together with a lack of proximity to noncontacting portions of ICAM-1, these observations explain variation among this group of mAbs, which can either act as competitive or allosteric antagonists. One agonistic mAb binds distant from the ligand binding site of the I domain, to residues that show little movement (mean, 0.5 angstroms; maximum, 1.0 angstroms). Agonism by this mAb is thus likely to result from altering the orientation of the I domain with respect to other domains within an intact integrin alphaLbeta2 heterodimer.Copyright 2004 The American Association of Immunologists, Inc.}, author = {Lu, C. and Shimaoka, M. and Salas, A. and Springer, T.A.} } @article {528156, title = {Boca-dependent assembly of β-propeller/EGF modules in low-density lipoprotein receptor proteins}, journal = {EMBO J.}, volume = {23}, number = {6}, year = {2004}, note = {in file}, pages = {1372-1380}, abstract = {The extracellular portions of cell surface receptor proteins are often comprised of independently folding protein domains. As they are translated into the endoplasmic reticulum (ER), some of these domains require protein chaperones to assist in their folding. Members of the low-density lipoprotein receptor (LDLR) family require the chaperone called Boca in Drosophila or its ortholog, Mesoderm development, in the mouse. All LDLRs have at least one six-bladed beta-propeller domain, which is immediately followed by an epidermal growth factor (EGF) repeat. We show here that Boca is specifically required for the maturation of these beta-propeller/EGF modules through the secretory pathway, but is not required for other LDLR domains. Protein interaction data suggest that as LDLRs are translated into the ER, Boca binds to the beta-propeller. Subsequently, once the EGF repeat is translated, the beta-propeller/EGF module achieves a more mature state that has lower affinity for Boca. We also show that Boca-dependent beta-propeller/EGF modules are found not only throughout the LDLR family but also in the precursor to the mammalian EGF ligand.}, author = {Culi, J. and Springer, T.A. and Mann, R. S.} } @article {530311, title = {Conversion between three conformational states of integrin I domains with a C-terminal pull spring studied with molecular dynamics}, journal = {Structure}, volume = {12}, number = {12}, year = {2004}, pages = {2137-2147}, abstract = {We test with molecular dynamics the hypothesis that interdomain forces in integrins, simulated with a spring attached to the C-terminal alpha 7-helix of an integrin I domain, can allosterically stabilize alternative I domain conformations. Depending on the force applied and timecourse, in alpha(L) and alpha(M) I domains the beta 6-alpha 7 loop moves successively between three ratchet positions; i.e. from closed to intermediate, and then to open. More distal, linked alterations in MIDAS loops and metal coordination closely resemble those seen when the MIDAS becomes ligated. Simulations show that the intermediate state is populated over a wider range of forces for alpha(L) than alpha(M) I domains. Simulations with mutant I domains suggest that specific ratchet residues regulate conformational equilibria. Simulations with alpha(1) and alpha(2) I domains reveal a lack of the intermediate conformation, owing to Phe to Glu substitution at the second ratchet residue. The findings have important implications for biological regulation of integrin adhesiveness.}, author = {Jin, M. and Andricioaei, I. and Springer, T.A.} } @article {530231, title = {The integrin α subunit leg extends at a Ca2+-dependent epitope in the thigh/genu interface upon activation}, journal = {Proc Natl Acad Sci USA }, volume = {101}, number = {43}, year = {2004}, note = {in file}, pages = {15422-15427}, abstract = {Two activation-dependent Abs to the integrin alphaL-subunit were used to study conformational rearrangement of alphaLbeta2 on the cell surface. Activation lowered the concentration of Ca2+ required for maximal expression of each epitope. Each Ab requires the Ca2+-binding loop in the integrin genu and nearby species-specific residues in the thigh domain. Key thigh residues are shielded from Ab in the bent integrin conformation by the alpha-subunit calf-1 domain and the nearby bent beta leg, suggesting that extension at the genu is required for epitope exposure. Activating stimuli and alpha/beta I-like small molecule antagonists demonstrate that exposure of epitopes in the integrin alpha- and beta-subunit legs is coordinate during integrin activation. A coordinating residue donated by the calf-1 domain is as important as Ca2+ for mAb binding. Together with inspection of the alphaV structure, this result suggests that the genu/calf-1 interface is maintained in integrin activation, and that extension occurs by a rearrangement at the thigh/genu interface.}, author = {Xie, C. and Shimaoka, M. and Xiao, T. and Schwab, P. and Klickstein,L.B. and Springer, T.A.} } @article {530021, title = {Integrin β3 regions controlling binding of murine mAb 7E3: Implications for the mechanism of integrin αIIbβ3 activation}, journal = {Proc Natl Acad Sci USA}, volume = {101}, number = {36}, year = {2004}, note = {in file}, pages = {13114-13120}, abstract = {Abciximab, a derivative of the murine mAb 7E3, protects against ischemic complications of percutaneous coronary interventions by inhibiting ligand binding to the alphaIIbbeta3 receptor. In this study we identified regions on integrin beta3 that control 7E3 binding. Murine/human amino acid substitutions were created in two regions of the betaA domain that previous studies found to influence 7E3 binding: the C177-C184 loop and K125-N133. The T182N substitution and a K125Q mutation reduced 7E3 binding to human beta3 in complex with alphaIIb. The introduction of both the human C177-C184 region and human W129 into murine beta3 was necessary and sufficient to permit 7E3 binding to the human alphaIIb/murine beta3 complex. Although we cannot exclude allosteric effects, we propose that 7E3 binds between C177-C184 and W129, which are within 15 A of each other in the crystal structure and close to the beta3 metal ion-dependent adhesion site. We previously demonstrated that 7E3 binds more rapidly to activated than unactivated platelets. Because it has been proposed that alphaIIbbeta3 changes from a bent to an extended conformation upon activation, we hypothesized that 7E3 binds less well to the bent than the extended conformation. In support of this hypothesis we found that 7E3 bound less well to an alphaIIbbeta3 construct locked in a bent conformation, and unlocking the conformation restored 7E3 binding. Thus, our data are consistent with alphaIIbbeta3 existing in variably bent conformations in equilibrium with each other on unactivated platelets, and activation resulting in alphaIIbbeta3 adopting a more extended conformation.}, keywords = {cd61}, author = {Artoni, A. and Li, J. and Mitchell, B. and Ruan, J. and Takagi, J. and Springer, T.A. and Coller,B.S. and French, D. L.} } @article {530241, title = {Inter-subunit signal transmission in integrins by a receptor-like interaction with a pull spring}, journal = {Proc Natl Acad Sci USA}, volume = {101}, number = {9}, year = {2004}, note = {in file}, pages = {2906-2911}, abstract = {The function of some multidomain proteins is regulated by interdomain communication. We use second-site suppressor cysteine mutations to test a hypothesis on how the inserted (I)-like domain in the integrin beta-subunit regulates ligand binding by the neighboring I domain in the integrin alpha-subunit [Huth, J. R., Olejniczak, E. T., Mendoza, R., Liang, H., Harris, E. A., et al. (2000) Proc. Natl. Acad. Sci. USA 97, 5231-5236; and Alonso, J. L., Essafi, M., Xiong, J. P., Stehle, T. \& Arnaout, M. A. (2002) Curr. Biol. 12, R340-R342]. The hypothesis is that an interaction between the beta I-like metal ion-dependent adhesion site (MIDAS) and an intrinsic ligand in the linker following the alpha I domain, Glu-310, exerts a pull that activates the alpha I domain. Individual mutation of alpha(L) linker residue Glu-310 or beta(2) MIDAS residues Ala-210 or Tyr-115 to cysteine abolishes I domain activation, whereas the double mutation of alpha(L)-E310C with either beta(2)-A210C or beta(2)-Y115C forms a disulfide bond that constitutively activates ligand binding. The disulfide-bonded mutant is resistant to small molecule antagonists that bind to the beta I-like domain near its interface with the alpha I domain and inhibit communication between these domains but remains susceptible to small molecule antagonists that bind underneath the I domain alpha 7-helix and certain allosteric antagonistic antibodies. Thus, the alpha 7-helix and its linker are better modeled as a pull spring than a bell rope. The results suggest that alpha(L) residue Glu-310, which is universally conserved in all I domain-containing integrins, functions as an intrinsic ligand for the beta I-like domain, and that when integrins are activated, the beta I-like MIDAS binds to Glu-310, pulls the spring, and thereby activates the alpha I domain.}, author = {Yang, W and Shimaoka, M. and Salas, A. and Takagi, J. and Springer, T.A.} } @article {528556, title = {Locking the β3 integrin I-like domain into high and low affinity conformations with disulfides}, journal = {J. Biol. Chem.}, volume = {279}, number = {11}, year = {2004}, note = {in file}, pages = {10215-10221}, abstract = {Although integrin alpha subunit I domains exist in multiple conformations, it is controversial whether integrin beta subunit I-like domains undergo structurally analogous movements of the alpha7-helix that are linked to affinity for ligand. Disulfide bonds were introduced into the beta(3) integrin I-like domain to lock its beta6-alpha7 loop and alpha7-helix in two distinct conformations. Soluble ligand binding, ligand mimetic mAb binding and cell adhesion studies showed that disulfide-bonded receptor alpha(IIb)beta(3)(T329C/A347C) was locked in a low affinity state, and dithiothreitol treatment restored the capability of being activated to high affinity binding; by contrast, disulfide-bonded alpha(IIb)beta(3)(V332C/M335C) was locked in a high affinity state. The results suggest that activation of the beta subunit I-like domain is analogous to that of the alpha subunit I domain, i.e. that axial movement in the C-terminal direction of the alpha7-helix is linked to rearrangement of the I-like domain metal ion-dependent adhesion site into a high affinity conformation.}, author = {Luo, B-H. and Takagi, J. and Springer, T.A.} } @article {528741, title = {The primacy of affinity over clustering in regulation of adhesiveness of the integrin αLβ2}, journal = {J. Cell Biol.}, volume = {167}, number = {6}, year = {2004}, pages = {1241-1253}, abstract = {Dynamic regulation of integrin adhesiveness is required for immune cell-cell interactions and leukocyte migration. Here, we investigate the relationship between cell adhesion and integrin microclustering as measured by fluorescence resonance energy transfer, and macroclustering as measured by high resolution fluorescence microscopy. Stimuli that activate adhesion through leukocyte function-associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand. Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering. Increased diffusivity in the membrane by cytoskeleton-disrupting agents was sufficient to drive adhesion in the absence of affinity modulation and was associated with a greater accumulation of LFA-1 to the zone of adhesion, but redistribution did not precede cell adhesion. Disruption of conformational communication within the extracellular domain of LFA-1 blocked adhesion stimulated by affinity-modulating agents, but not adhesion stimulated by cytoskeleton-disrupting agents. Thus, LFA-1 clustering does not precede ligand binding, and instead functions in adhesion strengthening after binding to multivalent ligands.}, author = {Kim, M. and Carman, C. V. and Yang, W and Salas, A. and Springer, T.A.} } @article {528461, title = {The relative influence of metal ion binding sites in the I-like domain and the interface with the hybrid domain on rolling and firm adhesion by integrin α4β7}, journal = {J. Biol. Chem.}, volume = {279}, number = {53}, year = {2004}, note = {erased duplicate data $\#$16614}, pages = {55556-61}, abstract = {We examined the effect of conformational change at the beta(7) I-like/hybrid domain interface on regulating the transition between rolling and firm adhesion by integrin alpha(4)beta(7). An N-glycosylation site was introduced into the I-like/hybrid domain interface to act as a wedge and to stabilize the open conformation of this interface and hence the open conformation of the alpha(4) beta(7) headpiece. Wild-type alpha(4)beta(7) mediates rolling adhesion in Ca(2+) and Ca(2+)/Mg(2+) but firm adhesion in Mg(2+) and Mn(2+). Stabilizing the open headpiece resulted in firm adhesion in all divalent cations. The interaction between metal binding sites in the I-like domain and the interface with the hybrid domain was examined in double mutants. Changes at these two sites can either counterbalance one another or be additive, emphasizing mutuality and the importance of multiple interfaces in integrin regulation. A double mutant with counterbalancing deactivating ligand-induced metal ion binding site (LIMBS) and activating wedge mutations could still be activated by Mn(2+), confirming the importance of the adjacent to metal ion-dependent adhesion site (ADMIDAS) in integrin activation by Mn(2+). Overall, the results demonstrate the importance of headpiece allostery in the conversion of rolling to firm adhesion.}, author = {Chen, J. F. and Takagi, J. and Xie, C. and Xiao, T. and Luo, B-H. and Springer, T.A.} } @article {528141, title = {RIAM, an Ena/VASP and profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion}, journal = {Dev. Cell}, volume = {7}, number = {4}, year = {2004}, pages = {585-595}, abstract = {The small GTPase Rap1 induces integrin-mediated adhesion and changes in the actin cytoskeleton. The mechanisms that mediate these effects of Rap1 are poorly understood. We have identified RIAM as a Rap1-GTP-interacting adaptor molecule. RIAM defines a family of adaptor molecules that contain a RA-like (Ras association) domain, a PH (pleckstrin homology) domain, and various proline-rich motifs. RIAM also interacts with Profilin and Ena/VASP proteins, molecules that regulate actin dynamics. Overexpression of RIAM induced cell spreading and lamellipodia formation, changes that require actin polymerization. In contrast, RIAM knockdown cells had reduced content of polymerized actin. RIAM overexpression also induced integrin activation and cell adhesion. RIAM knockdown displaced Rap1-GTP from the plasma membrane and abrogated Rap1-induced adhesion. Thus, RIAM links Rap1 to integrin activation and plays a role in regulating actin dynamics.}, author = {Lafuente, E. M. and van Puijenbroek, A. A. and Krause, M. and Carman, C. V. and Freeman, G. J. and Berezovskaya, A. and Constantine, E. and Springer, T.A. and Gertler, F. B. and Boussiotis, V. A.} } @article {528356, title = {Rolling adhesion through an extended conformation of integrin αLβ2 and relation to αI and βI-like domain interaction}, journal = {Immunity}, volume = {20}, number = {4}, year = {2004}, pages = {393-406}, abstract = {In vivo, beta(2) integrins and particularly alpha(L)beta(2) (LFA-1) robustly support firm adhesion of leukocytes, but can also cooperate with other molecules in supporting rolling adhesion. Strikingly, a small molecule alpha/beta I-like allosteric antagonist, XVA143, inhibits LFA-1-dependent firm adhesion, while at the same time it enhances adhesion in shear flow and rolling both in vitro and in vivo. XVA143 appears to induce the extended conformation of integrins as shown by increased activation epitope exposure. Fab to the beta(2) I-like domain converts firm adhesion to rolling adhesion, but does not enhance adhesion. Residue alpha(L)-Glu-310 in the linker following the I domain is critical for communication to the beta(2) I-like domain, rolling, integrin extension, and activation by Mn(2+) of firm adhesion. The results demonstrate the importance of integrin extension in rolling, and suggest that rolling and firm adhesion are mediated by extended conformations of alpha(L)beta(2) that differ in the affinity of the alpha(L) I domain for ICAM-1.}, author = {Salas, A. and Shimaoka, M. and Kogan, A. N. and Harwood, C. and von Andrian, U. H. and Springer, T.A.} } @article {530006, title = {A specific interface between integrin transmembrane helices and affinity for ligand}, journal = {PLoS Biol.}, volume = {2}, number = {6}, year = {2004}, note = {in file}, pages = {776-786}, abstract = {Conformational communication across the plasma membrane between the extracellular and intracellular domains of integrins is beginning to be defined by structural work on both domains. However, the role of the alpha and beta subunit transmembrane domains and the nature of signal transmission through these domains have been elusive. Disulfide bond scanning of the exofacial portions of the integrin alpha(IIbeta) and beta(3) transmembrane domains reveals a specific heterodimerization interface in the resting receptor. This interface is lost rather than rearranged upon activation of the receptor by cytoplasmic mutations of the alpha subunit that mimic physiologic inside-out activation, demonstrating a link between activation of the extracellular domain and lateral separation of transmembrane helices. Introduction of disulfide bridges to prevent or reverse separation abolishes the activating effect of cytoplasmic mutations, confirming transmembrane domain separation but not hinging or piston-like motions as the mechanism of transmembrane signaling by integrins.}, author = {Luo, B-H. and Springer, T.A. and Takagi, J.} } @article {530001, title = {Structural basis for allostery in integrins and binding of fibrinogen-mimetic therapeutics}, journal = {Nature}, volume = {432}, number = {7013}, year = {2004}, pages = {59-67}, abstract = {Integrins are important adhesion receptors in all Metazoa that transmit conformational change bidirectionally across the membrane. Integrin alpha and beta subunits form a head and two long legs in the ectodomain and span the membrane. Here, we define with crystal structures the atomic basis for allosteric regulation of the conformation and affinity for ligand of the integrin ectodomain, and how fibrinogen-mimetic therapeutics bind to platelet integrin alpha(IIb)beta3. Allostery in the beta3 I domain alters three metal binding sites, associated loops and alpha1- and alpha7-helices. Piston-like displacement of the alpha7-helix causes a 62 degrees reorientation between the beta3 I and hybrid domains. Transmission through the rigidly connected plexin/semaphorin/integrin (PSI) domain in the upper beta3 leg causes a 70 A separation between the knees of the alpha and beta legs. Allostery in the head thus disrupts interaction between the legs in a previously described low-affinity bent integrin conformation, and leg extension positions the high-affinity head far above the cell surface.}, author = {Xiao, T. and Takagi, J. and J.-H. Wang and Coller,B.S. and Springer, T.A.} } @article {529796, title = {Structural basis for dimerization of ICAM-1 on the cell surface}, journal = {Mol. Cell}, volume = {14}, number = {2}, year = {2004}, note = {in file}, pages = {269-276}, abstract = {We have determined the 3.0 A crystal structure of the three C-terminal domains 3-5 (D3-D5) of ICAM-1. Combined with the previously known N-terminal two-domain structure (D1D2), a model of an entire ICAM-1 extracellular fragment has been constructed. This model should represent a general architecture of other ICAM family members, particularly ICAM-3 and ICAM-5. The observed intimate dimerization interaction at D4 and a stiff D4-D5 stem-like architecture provide a good structural explanation for the existence of preformed ICAM-1 cis dimers on the cell membrane. Together with another dimerization interface at D1, a band-like one-dimensional linear cluster of ICAM-1 on an antigen-presenting cell (APC) surface can be envisioned, which might explain the formation of an immunological synapse between an activated T cell and APC which is critical for T cell receptor signaling.}, author = {Yang, Y. and Jun, C-D. and Liu, J-H. and Zhang, R-G. and Jochimiak, A. and Springer, T.A. and J.-H. Wang} } @article {528106, title = {Therapeutic antagonists and the conformational regulation of the β2 integrins}, journal = {Current Topics in Medicinal Chemistry}, volume = {4}, year = {2004}, pages = {1485-1495}, abstract = {Integrins are a structurally elaborate family of adhesion molecules that transmit signals bi-directionally across the plasma membrane by undergoing large-scale structural rearrangements. By regulating cell-cell and cell-matrix contacts, integrins participate in a wide range of biological processes, including development, tissue repair, angiogenesis, inflammation and haemostasis. From a therapeutic standpoint, integrins are probably the most important class of cell-adhesion receptors. Recent progress in the development of integrin antagonists has resulted in their clinical application and has shed new light on integrin biology. On the basis of their mechanism of action, small-molecule integrin antagonists fall into three different classes. Each of these classes affect the equilibria that relate integrin conformational states, but in different ways.}, author = {Shimaoka, M. and Springer, T.A.} } @article {527766, title = {The three-dimensional structure of integrins and their ligands, and conformational regulation of cell adhesion}, journal = {Adv. Prot. Chem.}, volume = {68}, year = {2004}, pages = {29-63}, publisher = {Elsevier}, address = {San Diego, CA}, abstract = {Integrins are a structurally elaborate family of adhesion molecules that transmit signals bidirectionally across the plasma membrane by undergoing large-scale structural rearrangements. By regulating cell-cell and cell-matrix contacts, integrins participate in a wide-range of biological interactions including development, tissue repair, angiogenesis, inflammation and hemostasis. From a therapeutic standpoint, integrins are probably the most important class of cell adhesion receptors. Structural investigations on integrin-ligand interactions reveal remarkable features in molecular detail. These details include the atomic basis for divalent cation-dependent ligand binding and how conformational signals are propagated long distances from one domain to another between the cytoplasm and the extracellular ligand binding site that regulate affinity for ligand, and conversely, cytosolic signaling pathways.}, author = {Springer, T.A. and J.-H. Wang}, editor = {Garcia, K Christopher} } @article {528666, title = {A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them}, journal = {J. Cell Biol.}, volume = {167}, number = {2}, year = {2004}, note = {in file}, pages = {377-388}, abstract = {The basic route and mechanisms for leukocyte migration across the endothelium remain poorly defined. We provide definitive evidence for transcellular (i.e., through individual endothelial cells) diapedesis in vitro and demonstrate that virtually all, both para- and transcellular, diapedesis occurs in the context of a novel "cuplike" transmigratory structure. This endothelial structure was comprised of highly intercellular adhesion molecule-1- and vascular cell adhesion molecule-1-enriched vertical microvilli-like projections that surrounded transmigrating leukocytes and drove redistribution of their integrins into linear tracks oriented parallel to the direction of diapedesis. Disruption of projections was highly correlated with inhibition of transmigration. These findings suggest a novel mechanism, the "transmigratory cup", by which the endothelium provides directional guidance to leukocytes for extravasation.}, author = {Carman, C. V. and Springer, T.A.} } @article {530301, title = {Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins}, journal = {Science}, volume = {301}, number = {5640}, year = {2003}, month = {Sep 19}, pages = {1720-1725}, abstract = {Although critical for development, immunity, wound healing, and metastasis, integrins represent one of the few classes of plasma membrane receptors for which the basic signaling mechanism remains a mystery. We investigated cytoplasmic conformational changes in the integrin LFA-1 (alphaLbeta2) in living cells by measuring fluorescence resonance energy transfer between cyan fluorescent protein-fused and yellow fluorescent protein-fused alphaL and beta2 cytoplasmic domains. In the resting state these domains were close to each other, but underwent significant spatial separation upon either intracellular activation of integrin adhesiveness (inside-out signaling) or ligand binding (outside-in signaling). Thus, bidirectional integrin signaling is accomplished by coupling extracellular conformational changes to an unclasping and separation of the alpha and beta cytoplasmic domains, a distinctive mechanism for transmitting information across the plasma membrane.}, author = {Kim, M. and Carman, C. V. and Springer, T.A.} } @article {529866, title = {Bistable regulation of integrin adhesiveness by a bipolar metal ion cluster}, journal = {Nat. Struct. Biol.}, volume = {10}, number = {12}, year = {2003}, note = {in filein lab goodies}, pages = {995-1001}, abstract = {Integrin alpha(4)beta(7) mediates rolling adhesion in Ca(2+) and Ca(2+) + Mg(2+), and firm adhesion in Mg(2+) and Mn(2+), mimicking the two key steps in leukocyte accumulation in inflamed vasculature. We mutated an interlinked linear array of three divalent cation-binding sites present in integrin beta-subunit I-like domains. The middle, metal ion-dependent adhesion site (MIDAS) is required for both rolling and firm adhesion. One polar site, that adjacent to MIDAS (ADMIDAS), is required for rolling because its mutation results in firm adhesion. The other polar site, the ligand-induced metal binding site (LIMBS), is required for firm adhesion because its mutation results in rolling. The LIMBS mediates the positive regulatory effects of low Ca(2+) concentrations, whereas the ADMIDAS mediates the negative regulatory effects of higher Ca(2+) concentrations, which are competed by Mn(2+). The bipolar sites thus stabilize two alternative phases of adhesion.}, author = {Chen, J. F. and Salas, A. and Springer, T.A.} } @article {529996, title = {Complex between nidogen and laminin fragments reveals a paradigmatic β-propeller interface}, journal = {Nature}, volume = {424}, number = {6951}, year = {2003}, note = {in file}, pages = {969-974}, abstract = {Basement membranes are fundamental to tissue organization and physiology in all metazoans. The interaction between laminin and nidogen is crucial to the assembly of basement membranes. The structure of the interacting domains reveals a six-bladed Tyr-Trp-Thr-Asp (YWTD) beta-propeller domain in nidogen bound to laminin epidermal-growth-factor-like (LE) modules III3-5 in laminin (LE3-5). Laminin LE module 4 binds to an amphitheatre-shaped surface on the pseudo-6-fold axis of the beta-propeller, and LE module 3 binds over its rim. A Phe residue that shutters the water-filled central aperture of the beta-propeller, the rigidity of the amphitheatre, and high shape complementarity enable the construction of an evolutionarily conserved binding surface for LE4 of unprecedentedly high affinity for its small size. Hypermorphic mutations in the Wnt co-receptor LRP5 (refs 6-9) suggest that a similar YWTD beta-propeller interface is used to bind ligands that function in developmental pathways. A related interface, but shifted off-centre from the pseudo-6-fold axis and lacking the shutter over the central aperture, is used in the low-density lipoprotein receptor for an intramolecular interaction that is regulated by pH in receptor recycling.}, author = {Takagi, J. and Yang, Y. and Liu, J-H. and J.-H. Wang and Springer, T.A.} } @article {529791, title = {The critical cytoplasmic regions of the αL/β2 integrin in Rap1-induced adhesion and migration}, journal = {Mol. Biol. Cell}, volume = {14}, number = {6}, year = {2003}, note = {in lab goodiesin file}, pages = {2570-2582}, abstract = {Rap1 is a potent inside-out signal that increases LFA-1 adhesive activity. In this study, we have defined the cytoplasmic region of the alphaL and beta2 integrin that are required for Rap1-stimulated adhesion and subsequent migration on ICAM-1. Human LFA-1 bearing truncated and point-mutated alphaL and beta2 cytoplasmic regions were reconstituted in mouse IL-3-dependent proB cells, BAF/3. Truncation of the alphaL, but not beta2 subunit cytoplasmic region, abolished Rap1V12-dependent adhesion to ICAM-1. The alanine substitution of two lysine residues (K1097/K1099) in the alphaL subunit was found to be critical in adhesion induced by Rap1V12, but not PMA. This mutation suppressed Rap1V12-induced LFA-1 conformation changes and ligand-binding affinity. The K1097/K1099 mutation also impaired binding to ICAM-1 induced by TCR cross-linking or SDF-1. In contrast, the alanine substitution for tyrosine in the beta2 subunit endocytosis motif inhibited internalization of LFA-1, and severely impaired detachment at the cell rear, which resulted in long-elongated cell shapes. This result demonstrates that internalization of LFA-1 is a critical step in the deadhesion process. Our study revealed novel requirements of amino acid residues of the LFA-1 cytoplasmic region in the response to the inside-out signaling and the subsequent deadhesion process.}, author = {Tohyama, Y. and Katagiri, K. and Pardi, R. and Lu, C. and Springer, T.A. and Kinashi,T.} } @article {529051, title = {Endothelial cells proactively form microvilli-like membrane projections upon ICAM-1 engagement of leukocyte LFA-1}, journal = {J. Immunol.}, volume = {171}, number = {11}, year = {2003}, note = {in file}, pages = {6135-6144}, abstract = {Specific leukocyte/endothelial interactions are critical for immunity and inflammation, yet the molecular details of this interaction interface remain poorly understood. Thus, we investigated, with confocal microscopy, the distribution dynamics of the central adhesion molecules ICAM-1 and LFA-1 in this context. Monolayers of activated HUVECs stained with fluorescent anti-ICAM-1 Fabs or Chinese hamster ovary-K1 cells expressing ICAM-1-green fluorescent protein were allowed to bind LFA-1-bearing monocytes, neutrophils, or K562 LFA-1 transfectants. ICAM-1 was rapidly relocalized to newly formed microvilli-like membrane projections in response to binding LFA-1 on leukocytes. These ICAM-1-enriched projections encircled the leukocytes extending up their sides and clustered LFA-1 underneath into linear tracks. Projections formed independently of VCAM-1/very late Ag 4 interactions, shear, and proactive contributions from the LFA-1-bearing cells. In the ICAM-1-bearing endothelial cells, projections were enriched in actin but not microtubules, required intracellular calcium, and intact microfilament and microtubule cytoskeletons and were independent of Rho/Rho kinase signaling. Disruption of these projections with cytochalasin D, colchicine, or BAPTA-AM had no affect on firm adhesion. These data show that in response to LFA-1 engagement the endothelium proactively forms an ICAM-1-enriched cup-like structure that surrounds adherent leukocytes but is not important for firm adhesion. This finding leaves open a possible role in leukocyte transendothelial migration, which would be consistent with the geometry and kinetics of formation of the cup-like structure.}, author = {Carman, C. V. and Jun, C-D. and Salas, A. and Springer, T.A.} } @article {528546, title = {High affinity ligand binding by integrins does not involve head separation}, journal = {J. Biol. Chem.}, volume = {278}, number = {19}, year = {2003}, note = {in filein lab goodies}, pages = {17185-17189}, abstract = {Conformational change in the integrin extracellular domain is required for high affinity ligand binding and is also involved in post-ligand binding cellular signaling. Although there is evidence to the contrary, electron microscopic studies showing that ligand binding triggers alpha- and beta-subunit dissociation in the integrin headpiece have gained popularity and support the hypothesis that head separation activates integrins. To test directly the head separation hypothesis, we enforced head association by introducing disulfide bonds across the interface between the alpha-subunit beta-propeller domain and the beta-subunit I-like domain. Basal and activation-dependent ligand binding by alpha(IIb)beta(3) and alpha(V)beta(3) was unaffected. The covalent linkage prevented dissociation of alpha(IIb)beta(3) into its subunits on EDTA-treated cells. Whereas EDTA dissociated wild type alpha(IIb)beta(3) on the cell surface, a ligand-mimetic Arg-Gly-Asp peptide did not, as judged by binding of complex-specific antibodies. Finally, a high affinity ligand-mimetic compound stabilized noncovalent association between alpha(IIb) and beta(3) headpiece fragments in the presence of SDS, indicating that ligand binding actually stabilized subunit association at the head, as opposed to the suggested subunit separation. The mechanisms of conformational regulation of integrin function should therefore be considered in the context of the associated alphabeta headpiece.}, author = {Luo, B-H. and Springer, T.A. and Takagi, J.} } @article {528086, title = {Integrin avidity regulation: Are changes in affinity and conformation underemphasized?}, journal = {Curr. Opin. Cell Biol.}, volume = {15}, number = {5}, year = {2003}, note = {in file}, pages = {547-556}, abstract = {Integrins play critical roles in development, wound healing, immunity and cancer. Central to their function is their unique ability to modulate dynamically their adhesiveness through both affinity- and valency-based mechanisms. Recent advances have shed light on the structural basis for affinity regulation and on the signaling mechanisms responsible for both affinity and valency modes of regulation.}, author = {Carman, C. V. and Springer, T.A.} } @article {529196, title = {Overlapping and selective roles of endothelial ICAM-1 and ICAM-2 in lymphocyte trafficking}, journal = {J. Immunol.}, volume = {171}, number = {5}, year = {2003}, note = {in file}, pages = {2588-2593}, abstract = {The integrin LFA-1 interacts with a variety of ligands termed ICAMs. ICAM-1 and ICAM-2 are both expressed on endothelium and serve as counterreceptors during lymphocyte trafficking. In this study, we analyzed their relative contribution to lymphocyte recirculation through lymph nodes and to recruitment into lung and inflamed skin by blocking mAbs against ICAM-1 and ICAM-2 and mice deficient for ICAM-1. The entry of lymphocytes into peripheral and mesenteric lymph nodes was found to be unaffected by the functional deletion of either ICAM-1 or ICAM-2. However, when both pathways were blocked, recirculation through lymph nodes was strongly reduced. Trapping of lymphocytes in the lung after i.v. injection is partly mediated by LFA-1/ICAM interactions; the data presented in this study show an exclusive role of ICAM-1 in LFA-1-dependent lung trapping. Similarly, ICAM-1, but not ICAM-2, was required for the migration of T effector cells into the inflamed skin. These results indicate that ICAM-1 and ICAM-2 have redundant functions in lymphocyte recirculation through lymph nodes, but ICAM-1 is unique in supporting migration into inflamed sites and trapping within the lung.}, author = {Lehmann, J. C. U. and Jablonski-Westrich, D. and Haubold, U. and Gutierrez-Ramos,J-C. and Springer, T.A. and Hamann, A.} } @article {528361, title = {Small molecule integrin antagonists that bind to the β2 subunit I-like domain and activate signals in one direction and block them in another}, journal = {Immunity}, volume = {19}, number = {3}, year = {2003}, note = {in file}, pages = {391-402}, abstract = {Leukocyte integrins contain an inserted (I) domain in their alpha subunits and an I-like domain in their beta(2) subunit, which directly bind ligand and regulate ligand binding, respectively. We describe a novel mechanistic class of integrin inhibitors that bind to the metal ion-dependent adhesion site of the beta(2) I-like domain and prevent its interaction with and activation of the alpha(L) I domain. The inhibitors do not bind to the alpha(L) I domain but stabilize alpha/beta subunit association and can show selectivity for alpha(L)beta(2) compared to alpha(M)beta(2). The inhibitors reveal a crucial intersection for relaying conformational signals within integrin extracellular domains. While blocking signals in one direction to the I domain, the antagonists induce the active conformation of the I-like domain and stalk domains, and thus transmit conformational signals in the other direction toward the transmembrane domains.}, author = {Shimaoka, M. and Salas, A. and Yang, W and Weitz-Schmidt, G. and Springer, T.A.} } @article {530136, title = {Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand}, journal = {Proc Natl Acad Sci USA}, volume = {100}, number = {5}, year = {2003}, note = {in file}, pages = {2403-2408}, abstract = {The affinity of the extracellular domain of integrins for ligand is regulated by conformational changes signaled from the cytoplasm. Alternative types of conformational movement in the ligand-binding headpiece have been proposed. In one study, electron micrograph image averages of the headpiece of integrin aV beta 3 show two different conformations. The open conformation of the headpiece is present when a ligand mimetic peptide is bound and differs from the closed conformation in the presence of an obtuse angle between the beta 3 subunit hybrid and I-like domains. We tested the hypothesis that opening of the hybrid-I-like domain interface increases ligand-binding affinity by mutationally introducing an N-glycosylation site into it. Both beta 3 and beta1 integrin glycan wedge mutants exhibit constitutively high affinity for physiological ligands. The data uniquely support one model of integrin activation and suggest that movement at the interface with the hybrid domain pulls down the C-terminal helix of the I-like domain and activates its metal ion-dependent adhesion site, analogously to activation of the integrin I domain.}, author = {Luo, B-H. and Springer, T.A. and Takagi, J.} } @article {530221, title = {Structure and allosteric regulation of the αXβ2 integrin I domain}, journal = {Proc Natl Acad Sci USA}, volume = {100}, number = {4}, year = {2003}, note = {in lab goodies}, pages = {1873-1878}, abstract = {The integrin alpha X beta 2 (CD11c/CD18, p150,95) binds ligands through the I domain of the alpha X subunit. Ligands include the complement factor fragment iC3b, a key component in the innate immune defense, which, together with the expression of alpha X beta 2 on dendritic cells and on other leukocytes, suggests a role in the immune response. We now report the structure of the alpha X I domain resolved at 1.65 A by x-ray crystallography. To analyze structural requirements for ligand binding we made a mutation in the alpha X I domain C-terminal helix, which increased the affinity for iC3b approximately 200-fold to 2.4 microM compared with the wild-type domain affinity of approximately 400 microM. Gel permeation chromatography supported a conformational change between the wild-type and mutated domains. Conservation of allosteric regulation in the alpha X I domain points to the functional importance of this phenomenon.}, author = {Vorup-Jensen, T. and Ostermeier, C. and Shimaoka, M. and Hommel, U. and Springer, T.A.} } @article {528161, title = {Structure of integrin α5β1 in complex with fibronectin}, journal = {EMBO J.}, volume = {22}, number = {18}, year = {2003}, note = {in file}, pages = {4607-4615}, abstract = {The membrane-distal headpiece of integrins has evolved to specifically bind large extracellular protein ligands, but the molecular architecture of the resulting complexes has not been determined. We used molecular electron microscopy to determine the three-dimensional structure of the ligand-binding headpiece of integrin alpha5beta1 complexed with fragments of its physiological ligand fibronectin. The density map for the unliganded alpha5beta1 headpiece shows a {\textquoteright}closed{\textquoteright} conformation similar to that seen in the alphaVbeta3 crystal structure. By contrast, binding to fibronectin induces an {\textquoteright}open{\textquoteright} conformation with a dramatic, approximately 80 degrees change in the angle of the hybrid domain of the beta subunit relative to its I-like domain. The fibronectin fragment binds to the interface between the beta-propeller and I-like domains in the integrin headpiece through the RGD-containing module 10, but direct contact of the synergy-region-containing module 9 to integrin is not evident. This finding is corroborated by kinetic analysis of real-time binding data, which shows that the synergy site greatly enhances k(on) but has little effect on the stability or k(off) of the complex.}, author = {Takagi, J. and Strokovich, K. and Springer, T.A. and Walz, T.} } @article {527966, title = {Structures of the αL I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation}, journal = {Cell}, volume = {112}, number = {1}, year = {2003}, note = {in tearoom in lab goodies}, pages = {99-111}, abstract = {The structure of the I domain of integrin alpha L beta 2 bound to the Ig superfamily ligand ICAM-1 reveals the open ligand binding conformation and the first example of an integrin-IgSF interface. The I domain Mg2+ directly coordinates Glu-34 of ICAM-1, and a dramatic swing of I domain residue Glu-241 enables a critical salt bridge. Liganded and unliganded structures for both high- and intermediate-affinity mutant I domains reveal that ligand binding can induce conformational change in the alpha L I domain and that allosteric signals can convert the closed conformation to intermediate or open conformations without ligand binding. Pulling down on the C-terminal alpha 7 helix with introduced disulfide bonds ratchets the beta 6-alpha 7 loop into three different positions in the closed, intermediate, and open conformations, with a progressive increase in affinity.}, author = {Shimaoka, M. and Xiao, T. and Liu, J-H. and Yang, Y. and Dong, Y. and Jun, C-D. and McCormack, A. and Zhang, R and Joachimiak, A. and Takagi, J. and J.-H. Wang and Springer, T.A.} } @article {529856, title = {Therapeutic antagonists and the conformational regulation of integrin structure and function}, journal = {Nat. Rev. Drug Disc.}, volume = {2}, number = {9}, year = {2003}, note = {in file}, pages = {703-716}, abstract = {Integrins are a structurally elaborate family of adhesion molecules that transmit signals bi-directionally across the plasma membrane by undergoing large-scale structural rearrangements. By regulating cell-cell and cell-matrix contacts, integrins participate in a wide range of biological processes, including development, tissue repair, angiogenesis, inflammation and haemostasis. From a therapeutic standpoint, integrins are probably the most important class of cell-adhesion receptors. Recent progress in the development of integrin antagonists has resulted in their clinical application and has shed new light on integrin biology. On the basis of their mechanism of action, small-molecule integrin antagonists fall into three different classes. Each of these classes affect the equilibria that relate integrin conformational states, but in different ways.}, author = {Shimaoka, M. and Springer, T.A.} } @article {528561, title = {Activation induced conformational changes in the I domain region of LFA-1}, journal = {J. Biol. Chem.}, volume = {277}, number = {12}, year = {2002}, note = {In press.in lab goodies.}, pages = {10638-10641}, abstract = {Conformational changes in integrins are important for efficient ligand binding during activation. We proposed that the I domain of the integrin lymphocyte function-associated antigen 1 (LFA-1) could exist in both open and closed conformations and generated constitutively activated LFA-1 by locking the I domain in the open conformation. Here we provide structural and biochemical evidence to validate conformational change in the I domain of LFA-1 upon activation. Two monoclonal antibodies to alpha(L), HI111 and CBR LFA-1/1, bind wild-type LFA-1 well, but their binding is significantly reduced when LFA-1 is locked in the open conformation. Furthermore, this reduction in monoclonal antibody binding also occurs when LFA-1 is activated by divalent cations. HI111 maps to the top region of the I domain that is close to the putative ligand-binding site surrounding the MIDAS (metal ion-dependent adhesion site). The epitope of CBR LFA-1/1 is at the C-terminal segment of the I domain that links to the beta-propeller, and undergoes a large movement between the open and closed conformations. Our data demonstrate that these two regions undergo significant conformational changes during LFA-1 activation and that the I domain of activated LFA-1 adopts a similar tertiary structure as the predicted locked open form.}, author = {Ma,Q. and Shimaoka, M. and Lu, C. and Jing, H. and Carman, C. V. and Springer, T.A.} } @article {530316, title = {Archaeal surface layer proteins contain β-propeller, polycystic kidney disease, and β-helix domains, and are related to metazoan cell surface proteins}, journal = {Structure}, volume = {10}, number = {10}, year = {2002}, note = {in file}, pages = {1453-1464}, abstract = {The surface layer of archaeobacteria protects cells from extreme environments and, in Methanosarcina, may regulate cell adhesion. We identify three domain types that account for the complete architecture of numerous Methanosarcina surface layer proteins (SLPs). We solve the crystal structure for two of these domains, which correspond to the two N-terminal domains of an M. mazei SLP. One domain displays a unique, highly symmetrical, seven-bladed beta propeller fold, and the other belongs to the polycystic kidney disease (PKD) superfamily fold. The third domain is predicted to adopt a beta helix fold. These domains have homologs in metazoan cell surface proteins, suggesting remarkable relationships between domains in archaeal SLPs and metazoan cell surface proteins.}, author = {Jing, H. and Takagi, J. and Liu, J-H. and Lindgren, S. and Zhang, R-G. and Joachimiak, A. and J.-H. Wang and Springer, T.A.} } @article {530306, title = {Cesar Milstein (1927-2002)}, journal = {Science}, volume = {296}, number = {5571}, year = {2002}, note = {in tea roomin lab goodies}, pages = {1253}, author = {Springer, T.A.} } @article {529851, title = {Cesar Milstein, the father of modern immunology}, journal = {Nat. Immunol.}, volume = {3}, number = {6}, year = {2002}, note = {in file}, pages = {501-503}, author = {Springer, T.A.} } @article {527871, title = {Chondroitin sulfate B exerts its inhibitory effect on secondary lymphoid tissue chemokine (SLC) by binding to the C-terminus of SLC}, journal = {Biochim. Biophys. Acta}, volume = {1571}, number = {3}, year = {2002}, note = {in lab goodies}, pages = {219-224}, abstract = {We previously reported that certain glycosaminoglycans (GAGs) bind secondary lymphoid tissue chemokine (SLC, CCL21) and that the SLC-binding GAGs, including chondroitin sulfate B (CS B), negatively modulate the function of SLC, although the mechanism remains unknown [J. Biol. Chem. 276 (2001) 5228]. To gain insight into the mechanism of inhibition, we used a C-terminally truncated SLC (SLC-T) that lacked clusters of basic amino acid residues that have been implicated in GAG binding. While SLC-T failed to bind any GAGs, it induced prominent intracellular Ca(2+) mobilization in CC chemokine receptor (CCR) 7-expressing cells, as did wild-type SLC. However, the SLC-T-induced Ca(2+) influx was not inhibited by CS B, unlike the SLC-induced Ca(2+) influx. These results demonstrate the requirement of the C-terminus of SLC for the inhibition of chemokine responses by CS B; that is, CS B exerts its inhibitory effect by binding to the C-terminus of SLC, thus defining the mode of action of CS B on certain chemokines.}, author = {Hirose, J. and Kawashima, H. and Willis, M. S. and Springer, T.A. and Hasegawa, H. and Yoshie, O. and Miyasaka, M.} } @article {527796, title = {Conformational regulation of integrin structure and function}, journal = {Annu. Rev. Biophys. Biomol. Struct.}, volume = {31}, year = {2002}, note = {In file.}, pages = {485-516}, abstract = {Integrins are a structurally elaborate family of heterodimers that mediate divalent cation-dependent cell adhesion in a wide range of biological contexts. The inserted (I) domain binds ligand in the subset of integrins in which it is present. Its structure has been determined in two alternative conformations, termed open and closed. In striking similarity to signaling G proteins, rearrangement of a Mg(2+)-binding site is linked to large conformational movements in distant backbone regions. Mutations have been used to stabilize either the closed or open structures. These show that the snapshots of the open conformation seen only in the presence of a ligand or a ligand mimetic represent a high-affinity, ligand-binding conformation, whereas those of the closed conformation correspond to a low-affinity conformation. The C-terminal alpha-helix moves 10 A down the side of the domain in the open conformation. Locking in the conformation of the preceding loop is sufficient to increase affinity for ligand 9000-fold. This C-terminal "bell-rope" provides a mechanism for linkage to conformational movements in other domains. The transition from the closed to open conformation has been implicated in fast (\<1 s) regulation of integrin affinity in response to activation signals from inside the cell. Recent integrin structures and functional studies reveal interactions between beta-propeller, I, and I-like domains in the headpiece, and a critical role for integrin EGF domains in the stalk region. These studies suggest that the headpiece of the integrin faces down toward the membrane in the inactive conformation and extends upward in a "switchblade"-like opening motion upon activation. These long-range structural rearrangements of the entire integrin molecule involving multiple interdomain contacts appear closely linked to conformational changes in the I domain, which result in increased affinity and competence for ligand binding.}, author = {Shimaoka, M. and Takagi, J. and Springer, T.A.} } @article {529861, title = {Cysteine-rich module structure reveals a fulcrum for integrin rearrangement upon activation}, journal = {Nat. Struct. Biol.}, volume = {9}, number = {4}, year = {2002}, note = {in filein lab goodies}, pages = {282-287}, abstract = {Cysteine-rich repeats in the integrin beta subunit stalk region relay activation signals to the ligand-binding headpiece. The NMR solution structure and disulfide bond connectivity of Cys-rich module-3 of the integrin beta2 subunit reveal a nosecone-shaped variant of the EGF fold, termed an integrin-EGF (I-EGF) domain. Interdomain contacts between I-EGF domains 2 and 3 observed by NMR support a model in which the modules are related by an approximate two-fold screw axis in an extended arrangement. Our findings complement a 3.1 A crystal structure of the extracellular portion of integrin alphaVbeta3, which lacks an atomic model for I-EGF2 and a portion of I-EGF3. The disulfide connectivity of I-EGF3 chemically assigned here differs from the pairings suggested in the alphaVbeta3 structure. Epitopes that become exposed upon integrin activation and residues that restrain activation are defined in beta2 I-EGF domains 2 and 3. Superposition on the alphaVbeta3 structure reveals that they are buried. This observation suggests that the highly bent alphaVbeta3 structure represents the inactive conformation and that release of contacts with I-EGF modules 2 and 3 triggers a switchblade-like opening motion extending the integrin into its active conformation.}, author = {Beglova, N. and Blacklow, S. C. and Takagi, J. and Springer, T.A.} } @article {528251, title = {The genome of Methanosarcina acetivorans reveals extensive metabolic and physiological diversity}, journal = {Gen. Res.}, volume = {12}, number = {4}, year = {2002}, note = {in lab goodies}, pages = {532-542}, abstract = {Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon cycle and contributes significantly to global warming. The majority of methane in nature is derived from acetate. Here we report the complete genome sequence of an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are the most metabolically diverse methanogens, thrive in a broad range of environments, and are unique among the Archaea in forming complex multicellular structures. This diversity is reflected in the genome of M. acetivorans. At 5,751,492 base pairs it is by far the largest known archaeal genome. The 4524 open reading frames code for a strikingly wide and unanticipated variety of metabolic and cellular capabilities. The presence of novel methyltransferases indicates the likelihood of undiscovered natural energy sources for methanogenesis, whereas the presence of single-subunit carbon monoxide dehydrogenases raises the possibility of nonmethanogenic growth. Although motility has not been observed in any Methanosarcineae, a flagellin gene cluster and two complete chemotaxis gene clusters were identified. The availability of genetic methods, coupled with its physiological and metabolic diversity, makes M. acetivorans a powerful model organism for the study of archaeal biology. [Sequence, data, annotations and analyses are available at http://www-genome.wi.mit.edu/.]}, author = {Galagan, J.E. and Nusbaum, C. and Roy, A. and Endrizzi, M. G. and Macdonald, P. and FitzHugh, W. and Calvo, S. and Engels, R. and Smirnov, S. and Atnoor, D. and Brown, A. and Allen, N. and Naylor, J. and Stange-Thomann, N. and DeArellano, K. and Johnson, R. and Linton, L. and McEwan, P. and McKernan, K. and Talamas, J. and Tirrell, A. and Ye, W. and Zimmer, A. and Barber, R. D. and Cann, I. and Graham, D. E. and Grahame, D. A. and Guss, A. and Hedderich, R. and Ingram-Smith, C. and Kuettner, H. C. and Krzycki, J. A. and Leigh, J. A. and W Li and Liu, J. and Mukhopadhyay, B. and Reeve, J. N. and K. Smith and Springer, T.A. and Umayam, L. A. and White, O. and White, R. H. and Conway de Macario, E. and Ferry, J. G. and Jarrell, K. F. and Jing, H. and Macario, A. J. and Paulsen, I. and Pritchett, M. and Sowers, K. R. and Swanson, R. V. and Zinder, S. H. and Lander, E. and Metcalf, W. W. and Birren, B.} } @article {528001, title = {Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling}, journal = {Cell}, volume = {110}, number = {5}, year = {2002}, note = {in file.}, pages = {599-611}, abstract = {How ligand binding alters integrin conformation in outside-in signaling, and how inside-out signals alter integrin affinity for ligand, have been mysterious. We address this with electron microscopy, physicochemical measurements, mutational introduction of disulfides, and ligand binding to alphaVbeta3 and alphaIIbbeta3 integrins. We show that a highly bent integrin conformation is physiological and has low affinity for biological ligands. Addition of a high affinity ligand mimetic peptide or Mn(2+) results in a switchblade-like opening to an extended structure. An outward swing of the hybrid domain at its junction with the I-like domain shows conformational change within the headpiece that is linked to ligand binding. Breakage of a C-terminal clasp between the alpha and beta subunits enhances Mn(2+)-induced unbending and ligand binding.}, author = {Takagi, J. and Petre, B. M. and Walz, T. and Springer, T.A.} } @article {528396, title = {Integrin activation and structural rearrangement}, journal = {Immunol. Rev.}, volume = {186}, year = {2002}, note = {in lab goodies}, pages = {141-163}, abstract = {Among adhesion receptor families, integrins are particularly important in biological processes that require rapid modulation of adhesion and de-adhesion. Activation on a timescale of \< 1 s of beta2 integrins on leukocytes and beta3 integrins on platelets enables deposition of these cells at sites of inflammation or vessel wall injury. Recent crystal, nuclear magnetic resonance (NMR), and electron microscope (EM) structures of integrins and their domains lead to a unifying mechanism of activation for both integrins that contain and those that lack an inserted (I) domain. The I domain adopts two alternative conformations, termed open and closed. In striking similarity to signaling G-proteins, rearrangement of a Mg2+-binding site is linked to large conformational movements in distant backbone regions. Mutations that stabilize a particular conformation show that the open conformation has high affinity for ligand, whereas the closed conformation has low affinity. Movement of the C-terminal alpha-helix 10 A down the side of the domain in the open conformation is sufficient to increase affinity at the distal ligand-binding site 9,000-fold. This C-terminal "bell-rope" provides a mechanism for linkage to conformational movements in other domains. Recent structures and functional studies reveal interactions between beta-propeller, I, and I-like domains in the integrin headpiece, and a critical role for integrin epidermal growth factor (EGF) domains in the stalk region. The headpiece of the integrin faces down towards the membrane in the inactive conformation, and extends upward in a "switchblade"-like opening upon activation. These long-range structural rearrangements of the entire integrin molecule involving interdomain contacts appear closely linked to conformational changes within the I and I-like domains, which result in increased affinity and competence for ligand binding.}, author = {Takagi, J. and Springer, T.A.} } @article {527876, title = {Measurement of selectin tether bond lifetimes}, journal = {Biophys. J.}, volume = {83}, number = {4}, year = {2002}, note = {in file}, pages = {2318-2320}, abstract = {A direct comparison of selectin-mediated transient, adhesive events using high temporal resolution. [Biophys J. 1999]}, author = {Springer, T.A. and Chen, S. and Alon, R.} } @article {528456, title = {Molecular basis for interaction between Icap1α PTB domain and β1 integrin}, journal = {J. Biol. Chem.}, volume = {277}, number = {10}, year = {2002}, note = {IN FILE}, pages = {8140-8145}, abstract = {Icap1 alpha is a 200-amino acid protein that binds to the COOH-terminal 13 amino acids ((786)AVTTVVNPKYEGK(798)) of the integrin beta(1) subunit. Alanine scanning mutagenesis of this region revealed that Val(787), Val(790), and (792)NPKY(795) are critical for Icap1 alpha binding. The NPXY motif is a known binding substrate for phosphotyrosine binding (PTB) domain proteins. The sequences of Icap1 alpha, residues 58--200, and the beta(1) integrin, residues 786-797, were aligned to the available PTB-peptide structures to generate a high quality structural model. Site-directed mutagenesis showed that Leu(135), Ile(138), and Ile(139) of Icap1 alpha, residues predicted by the model to be in close proximity to (792)NPKY(795), and Leu(82) and Tyr(144), residues expected to form a hydrophobic pocket near Val(787), are required for the Icap1 alpha-beta(1) integrin interaction. These findings indicate that Icap1 alpha is a PTB domain protein, which recognizes the NPXY motif of beta(1) integrin. Furthermore, our date suggest that an interaction between Val(787) and the hydrophobic pocket created by Leu(82) and Tyr(144) of Icap1 alpha forms the basis for the specificity of Icap1 alpha for the beta(1) integrin subunit.}, author = {Chang, D. D. and Hoang, B. Q. and Liu, J. and Springer, T.A.} } @article {528101, title = {Predicted and experimental structures of integrins and β-propellers}, journal = {Curr. Opin. Struct. Biol.}, volume = {12}, number = {6}, year = {2002}, note = {in lab goodies}, pages = {802-813}, abstract = {Integrins and other cell surface receptors have been fertile grounds for structure prediction experiments. Recently determined structures show remarkable successes, especially with beta-propeller domain predictions, and also reveal how ligand binding by integrins is conformationally regulated.}, author = {Springer, T.A.} } @article {528366, title = {Role for CCR7 ligands in the emigration of newly generated T lymphocytes from the neonatal thymus}, journal = {Immunity}, volume = {16}, number = {2}, year = {2002}, note = {In file.}, pages = {205-218}, abstract = {Most T lymphocytes are generated within the thymus. It is unclear, however, how newly generated T cells relocate out of the thymus to the circulation. The present study shows that a CC chemokine CCL19 attracts mature T cells out of the fetal thymus organ culture. Another CC chemokine CCL21, which shares CCR7 with CCL19 but has a unique C-terminal extension containing positively charged amino acids, failed to show involvement in thymic emigration. Neonatal appearance of circulating T cells was defective in CCL19-neutralized mice as well as in CCR7-deficient mice but not in CCL21-neutralized mice. In the thymus, CCL19 is predominantly localized in the medulla including endothelial venules. These results indicate a CCL19- and CCR7-dependent pathway of thymic emigration, which represents a major pathway of neonatal T cell export.}, author = {Ueno, T. and Hara, K. and Swope Willis, M. and Malin, M. and Hoepken, U. and Gray, D. H. D. and Matsushima, K. and Lipp, M. and Springer, T.A. and Boyd, R. L. and Yoshie, O. and Takahama, Y.} } @article {527861, title = {The role of specificity-determining loop of the integrin β-subunit I-like domain in folding, association with the α subunit, and ligand binding}, journal = {Biochemistry}, volume = {41}, number = {13}, year = {2002}, note = {IN FILE}, pages = {4339-4347}, abstract = {Integrin beta subunits contain a highly conserved I-like domain that is known to be important for ligand binding. Unlike integrin I domains, the I-like domain requires integrin alpha and beta subunit association for optimal folding. Pactolus is a novel gene product that is highly homologous to integrin beta subunits but lacks associating alpha subunits [Chen, Y., Garrison, S., Weis, J. J., and Weis, J. H. (1998) J. Biol. Chem. 273, 8711-8718] and a approximately 30 amino acid segment corresponding to the specificity-determining loop (SDL) in the I-like domain. We find that the SDL is responsible for the defects in integrin beta subunit expression and folding in the absence of alpha subunits. When transfected in the absence of alpha subunits into cells, extracellular domains of mutant beta subunits lacking SDL, but not wild-type beta subunits, were well secreted and contained immunoreactive I-like domains. The purified recombinant soluble beta1 subunit with the SDL deletion showed an elongated shape in electron microscopy, consistent with its structure in alphabeta complexes. The SDL segment is not required for formation of alpha5beta1, alpha4beta1, alphaVbeta3, and alpha6beta4 heterodimers, but is essential for fomation of alpha6beta1, alphaVbeta1, and alphaLbeta2 heterodimers, suggesting that usage of subunit interface residues is variable among integrins. The beta1 SDL is required for ligand binding and for the formation of the epitope for the alpha5 monoclonal antibody 16 that maps to loop segments connecting blades 2 and 3 of beta-propeller domain of alpha5, but is not essential for nearby beta-propeller epitopes.}, author = {Takagi, J. and Debottis, D.P. and Erickson, H. P. and Springer, T.A.} } @article {530186, title = {Stabilizing the integrin αM inserted domain in alternative conformations with a range of engineered disulfide bonds}, journal = {Proc Natl Acad Sci USA}, volume = {99}, number = {26}, year = {2002}, note = {in lab goodiesin tearoom}, pages = {16737-16741}, abstract = {Conformational movement of the C-terminal alpha7 helix in the integrin inserted (I) domain, a major ligand-binding domain that adopts an alpha/beta Rossmann fold, has been proposed to allosterically regulate ligand-binding activity. Disulfide bonds were engineered here to reversibly lock the position of the alpha7 helix in one of two alternative conformations seen in crystal structures, termed open and closed. Our results show that pairs of residues with Cbeta atoms farther apart than optimal for disulfide bond stereochemistry can be successfully replaced by cysteine, suggesting that backbone movement accommodates disulfide formation. We also find more success with substituting partially exposed than buried residues. Disulfides stabilizing the open conformation resulted in constitutively active alphaMbeta2 heterodimers and isolated alphaM inserted domains, which were reverted to an inactive form by dithiothreitol reduction. By contrast, a disulfide stabilizing the closed conformation resulted in inactive alphaMbeta2 that was resistant to activation but became activatable after dithiothreitol treatment.}, author = {Shimaoka, M. and Lu, C. and Salas, A. and Xiao, T. and Takagi, J. and Springer, T.A.} } @article {528576, title = {Transition from rolling to firm adhesion is regulated by the conformation of the I domain of the integrin LFA-1}, journal = {J. Biol. Chem.}, volume = {277}, number = {52}, year = {2002}, note = {in file}, pages = {50255-50262}, abstract = {The integrin lymphocyte function-associated antigen-1 (alpha(L)beta(2)), which is known for its ability to mediate firm adhesion and migration, can also contribute to tethering and rolling in shear flow. The alpha(L) I domain can be mutationally locked with disulfide bonds into two distinct conformations, open and closed, which have high and low affinity for the ligand intercellular adhesion molecule 1 (ICAM-1), respectively. The wild type I domain exists primarily in the lower energy closed conformation. We have measured for the first time the effect of conformational change on adhesive behavior in shear flow. We show that wild type and locked open I domains, expressed in alpha(L)beta(2) heterodimers or as isolated domains on the cell surface, mediate rolling adhesion and firm adhesion, respectively. alpha(L)beta(2) is thus poised for the conversion of rolling to firm adhesion upon integrin activation in vivo. Isolated I domains are surprisingly more effective than alpha(L)beta(2) in interactions in shear flow, which may in part be a consequence of the presence of alpha(L)beta(2) in a bent conformation. Furthermore, the force exerted on the C-terminal alpha-helix appears to stabilize the open conformation of the wild type isolated I domain and contribute to its robustness in supporting rolling. An allosteric small molecule antagonist of alpha(L)beta(2) inhibits both rolling adhesion and firm adhesion, which has important implications for its mode of action in vivo.}, author = {Salas, A. and Shimaoka, M. and Chen, S. and Carman, C. V. and Springer, T.A.} } @article {528636, title = {Amino acid residues in the PSI domain and cysteine-rich repeats of the integrin β2 subunit that restrain activation of the integrin αXβ2}, journal = {J. Biol. Chem.}, volume = {276}, number = {10}, year = {2001}, pages = {6922-6929}, abstract = {The leukocyte integrin alpha(X)beta(2) (p150,95) recognizes the iC3b complement fragment and functions as the complement receptor type 4. alpha(X)beta(2) is more resistant to activation than other beta(2) integrins and is inactive in transfected cells. However, when human alpha(X) is paired with chicken or mouse beta(2), alpha(X)beta(2) is activated for binding to iC3b. Activating substitutions were mapped to individual residues or groups of residues in the N-terminal plexin/semaphorin/integrin (PSI) domain and C-terminal cysteine-rich repeats 2 and 3. These regions are linked by a long range disulfide bond. Substitutions in the PSI domain synergized with substitutions in the cysteine-rich repeats. Substitutions T4P, T22A, Q525S, and V526L gave full activation. Activation of binding to iC3b correlated with exposure of the CBR LFA-1/2 epitope in cysteine-rich repeat 3. The data suggest that the activating substitutions are present in an interface that restrains the human alpha(X)/human beta(2) integrin in the inactive state. The opening of this interface is linked to structural rearrangements in other domains that activate ligand binding.}, author = {Zang, Q. and Springer, T.A.} } @article {528516, title = {Amino acid residues in the αIIb subunit that are critical for ligand binding to integrin αIIbβ3 are clustered in the β-propeller model}, journal = {J. Biol. Chem.Journal of Biological Chemistry}, volume = {276}, number = {47}, year = {2001}, note = {Reprint in fileUsing Smart Source ParsingSep}, pages = {44275-44283}, abstract = {Several distinct regions of the integrin alpha(IIb) subunit have been implicated in ligand binding. To localize the ligand binding sites in alpha(IIb), we swapped all 27 predicted loops with the corresponding sequences of alpha(4) or alpha(5). 19 of the 27 swapping mutations had no effect on binding to both fibrinogen and ligand-mimetic antibodies (e.g. LJ-CP3), suggesting that these regions do not contain major ligand binding sites. In contrast, swapping the remaining 8 predicted loops completely blocked ligand binding. Ala scanning mutagenesis of these critical predicted loops identified more than 30 discontinuous residues in repeats 2-4 and at the boundary between repeats 4 and 5 as critical for ligand binding. Interestingly, these residues are clustered in the predicted beta-propeller model, consistent with this model. Most of the critical residues are located at the edge of the upper face of the propeller, and several critical residues are located on the side of the propeller domain. None of the predicted loops in repeats 1, 6, and 7, and none of the four putative Ca(2+)-binding predicted loops on the lower surface of the beta-propeller were important for ligand binding. The results map an important ligand binding interface at the edge of the top and on the side of the beta-propeller toroid, centering on repeat 3.}, keywords = {374}, author = {Kamata, T. and Tieu, K. K. and Springer, T.A. and Takada, Y} } @article {528541, title = {Association of the membrane-proximal regions of the α and β subunit cytoplasmic domains constrains an integrin in the inactive state}, journal = {J. Biol. Chem.}, volume = {276}, number = {18}, year = {2001}, pages = {14642-14648.}, abstract = {The adhesiveness of integrins is regulated through a process termed "inside-out" signaling. To understand the molecular mechanism of integrin inside-out signaling, we generated K562 stable cell lines that expressed LFA-1 (alpha(L)beta(2)) or Mac-1 (alpha(M)beta(2)) with mutations in the cytoplasmic domain. Complete truncation of the beta(2) cytoplasmic domain, but not a truncation that retained the membrane proximal eight residues, resulted in constitutive activation of alpha(L)beta(2) and alpha(M)beta(2), demonstrating the importance of this membrane proximal region in the regulation of integrin adhesive function. Furthermore, replacement of the alpha(L) and beta(2) cytoplasmic domains with acidic and basic peptides that form an alpha-helical coiled coil caused inactivation of alpha(L)beta(2). Association of these artificial cytoplasmic domains was directly demonstrated. By contrast, replacement of the alpha(L) and beta(2) cytoplasmic domains with two basic peptides that do not form an alpha-helical coiled coil activated alpha(L)beta(2). Induction of ligand binding by the activating cytoplasmic domain mutations correlated with the induction of activation epitopes in the extracellular domain. Our data demonstrate that cytoplasmic, membrane proximal association between integrin alpha and beta subunits, constrains an integrin in the inactive conformation.}, author = {Lu, C. and Takagi, J. and Springer, T.A.} } @article {529881, title = {C-terminal opening mimics "inside-out" activation of integrin α5β1}, journal = {Nat. Struct. Biol.}, volume = {8}, number = {5}, year = {2001}, pages = {412-416}, abstract = {Integrins are adhesion molecules that convey signals both to and from the cytoplasm across the plasma membrane. In resting cells, integrins in a low affinity state can be activated by {\textquoteright}inside-out signaling{\textquoteright}, in which signals affecting integrin heterodimer cytoplasmic domains cause a conformational change in the integrin ligand-binding headpiece connected to the membrane by two long, approximately 16 nm stalks. Here we demonstrate a mechanism for conveying a conformational change over the long distance from the plasma membrane to the headpiece. We prepared soluble, alpha5beta1 integrin heterodimer extracellular fragments in which interactions between alpha- and beta-subunit cytoplasmic domains were replaced with an artificial clasp. Release of this C-terminal clasp by specific protease cleavage resulted in an approximately 14 nm separation of the stalks coupled to increased binding to fibronectin. This activation did not require any associated molecules or clustering and was observed with physiological concentrations of divalent cations. These findings suggest that the overall mechanism for integrin inside-out activation involves the spatial separation of the cytoplasmic and/or transmembrane domains.}, keywords = {CD49e}, author = {Takagi, J. and Erickson, H. P. and Springer, T.A.} } @article {530211, title = {Definition of EGF-like, closely interacting modules that bear activation epitopes in integrin β subunits}, journal = {Proc Natl Acad Sci USA}, volume = {98}, number = {20}, year = {2001}, pages = {11175-11180.}, abstract = {Integrin beta subunits contain four cysteine-rich repeats in a long extracellular stalk that connects the headpiece to the membrane. Most mAbs to integrin activation epitopes map to these repeats, and they are important in propagating conformational signals from the membrane/cytosol to the ligand-binding headpiece. Sequence analysis of a protein containing only 10 integrin-like, cysteine-rich repeats suggests that these repeats start one cysteine earlier than previously reported. By using the new repeat boundaries, statistically significant sequence homology to epidermal growth factor-like domains is found, and a disulfide bond connectivity of the eight cysteines is predicted that differs in three of four disulfides from a previous prediction of epidermal growth factor-like modules [Berg, R. W., Leung, E., Gough, S., Morris, C., Yao, W.-P., Wang, S.-x., Ni, J. \& Krissansen, G. W. (1999) Genomics 56, 169-178]. N-terminally truncated beta2 integrin stalk fragments were well expressed and secreted from 293 T cells when they began at repeat boundaries but not when they began one cysteine earlier or later. Furthermore, peptides that correspond to module 3 or modules 2 + 3 were expressed in bacteria and refolded. The module 2 + 3 fragment was as reactive with three mAbs to activation epitopes as a beta2 fragment expressed in eukaryotic cells, indicating a native fold. Only one residue intervenes between the last cysteine of one module and the first cysteine of the next. This arrangement is consistent with a tight intermodule connection, a prerequisite for signal propagation from the membrane to the ligand binding headpiece.}, author = {Takagi, J. and Beglova, N. and Yalamanchili, P. and Blacklow, S. C. and Springer, T.A.} } @article {530116, title = {Dimerization and the effectiveness of ICAM-1 in mediating LFA-1 dependent adhesion}, journal = {Proc Natl Acad Sci USA}, volume = {98}, number = {12}, year = {2001}, pages = {6830-6835}, abstract = {Dimeric intercellular adhesion molecule-1 (ICAM-1) binds more efficiently to lymphocyte function-associated antigen-1 (LFA-1) than monomeric ICAM-1. However, it is unknown whether dimerization enhances binding simply by providing two ligand-binding sites and thereby increasing avidity, or whether it serves to generate a single "fully competent" LFA-1-binding surface. Domain 1 of ICAM-1 contains both the binding site for LFA-1, centered on residue E34, and a homodimerization interface. Whether the LFA-1-binding site extends across the homodimerization interface has not been tested. To address this question, we constructed four different heterodimeric soluble forms of ICAM-1 joined at the C terminus via an alpha-helical coiled coil (ACID-BASE). These heterodimeric ICAM-1 constructs include, (i) E34/E34 (two intact LFA-1-binding sites), (ii) E34/K34 (one disrupted LFA-1-binding site), (iii) E34/DeltaD1-2 (one deleted LFA-1-binding site), and (iv) K34/K34 (two disrupted LFA-1-binding sites). Cells bearing activated LFA-1 bound similarly to surfaces coated with either E34/K34 or E34/DeltaD1-2 and with an approximately 2-fold reduction in efficiency compared with E34/E34, suggesting that D1 dimerization, which is precluded in E34/DeltaD1-D2, is not necessary for optimal LFA-1 binding. Furthermore, BIAcore (BIAcore, Piscataway, NJ) affinity measurements revealed that soluble open LFA-1 I domain bound to immobilized soluble ICAM-1, E34/E34, E34/K34, and E34/DeltaD1-D2 with nearly identical affinities. These studies demonstrate that a single ICAM-1 monomer, not dimeric ICAM-1, represents the complete, "fully competent" LFA-1-binding surface.}, author = {Jun, C-D. and Shimaoka, M. and Carman, C. V. and Takagi, J. and Springer, T.A.} } @article {528821, title = {Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes}, journal = {J. Clin. Invest.}, volume = {108}, year = {2001}, pages = {871-878}, abstract = {The lineage relationship between short-lived effector T cells and long-lived memory cells is not fully understood. We have described T-GFP mice previously, in which naive and early activated T cells express GFP uniformly, whereas cells that have differentiated into effector cytotoxic T cells selectively lose GFP expression. Here we studied antigen-specific CD8 T cell differentiation using T-GFP mice crossed to the TCR transgenic (Tg) mice P14 (specific for the lymphocytic choriomeningitis virus glycoprotein peptide, gp33-41). After activation with antigenic peptide, P14XT-GFP CD8(+) T cells cultured in high-dose IL-2 developed into cells with effector phenotype and function: they were blastoid, lost GFP expression, expressed high levels of activation and effector markers, and were capable of immediate cytotoxic function. In contrast, cells cultured in IL-15 or low-dose IL-2 never developed into full-fledged effector cells. Rather, they resembled memory cells: they were smaller, were GFP(+), did not express effector markers, and were incapable of immediate cytotoxicity. However, they mediated rapid-recall responses in vitro. After adoptive transfer, they survived in vivo for at least 10 weeks and mounted a secondary immune response after antigen rechallenge that was as potent as endogenously generated memory cells. In addition to providing a simple means to generate memory cells in virtually unlimited numbers, our results suggest that effector differentiation is not a prerequisite for memory cell generation.}, author = {Manjunath, N. and Shankar, P. and Wan, J. and Weninger, W. and Crowley, M. A. and Hieshima, K. and Springer, T.A. and Fan, X. and Shen, H. and Lieberman, J. and von Andrian, U. H.} } @article {529201, title = {Epitope mapping of antibodies to the C-terminal region of the integrin β2 subunit reveals regions that become exposed upon receptor activation}, journal = {J. Immunol.}, volume = {166}, number = {9}, year = {2001}, pages = {5629-5637.}, abstract = {The cysteine-rich repeats in the stalk region of integrin beta subunits appear to convey signals impinging on the cytoplasmic domains to the ligand-binding headpiece of integrins. We have examined the functional properties of mAbs to the stalk region and mapped their epitopes, providing a structure-function map. Among a panel of 14 mAbs to the beta(2) subunit, one, KIM127, preferentially bound to alpha(L)beta(2) that was activated by mutations in the cytoplasmic domains, and by Mn(2+). KIM127 also bound preferentially to the free beta(2) subunit compared with resting alpha(L)beta(2). Activating beta(2) mutations also greatly enhanced binding of KIM127 to integrins alpha(M)beta(2) and alpha(X)beta(2). Thus, the KIM127 epitope is shielded by the alpha subunit, and becomes reexposed upon receptor activation. Three other mAbs, CBR LFA-1/2, MEM48, and KIM185, activated alpha(L)beta(2) and bound equally well to resting and activated alpha(L)beta(2), differentially recognized resting alpha(M)beta(2) and alpha(X)beta(2), and bound fully to activated alpha(M)beta(2) and alpha(X)beta(2). The KIM127 epitope localizes within cysteine-rich repeat 2, to residues 504, 506, and 508. By contrast, the two activating mAbs CBR LFA-1/2 and MEM48 bind to overlapping epitopes involving residues 534, 536, 541, 543, and 546 in cysteine-rich repeat 3, and the activating mAb KIM185 maps near the end of cysteine-rich repeat 4. The nonactivating mAbs, 6.7 and CBR LFA-1/7, map more N-terminal, to subregions 344-432 and 432-487, respectively. We thus define five different beta(2) stalk subregions, mAb binding to which correlates with effect on activation, and define regions in an interface that becomes exposed upon integrin activation.}, keywords = {CD18}, author = {Lu, C. and Ferzly, M. and Takagi, J. and Springer, T.A.} } @article {529086, title = {Expression of stromal-derived factor-1 is decreased by IL-1 and TNF and regulates dermal wound healing}, journal = {J. Immunol.}, volume = {166}, number = {9}, year = {2001}, pages = {5749-5754.}, abstract = {Stromal-derived factor-1 (SDF-1) is a CXC chemokine that is believed to be constitutively expressed by stromal cells of numerous tissues. In this report, we demonstrate that dermal fibroblasts and vessels of noninflamed tissues express SDF-1. Unexpectedly, we found that expression of SDF-1 is regulated by inflammation. Expression of SDF-1 by primary cultures of human gingival fibroblasts is potently inhibited by activated macrophages via secretion of IL-1alpha and TNF-alpha. Levels of SDF-1 mRNA also decrease in acutely inflamed mouse dermal wounds. We propose that SDF-1 functions as a homeostatic regulator of tissue remodeling, whose expression stabilizes existing dermal architecture.}, keywords = {121.24}, author = {Fedyk, E.R. and Jones,D. and Critchley, H.O.D. and Phipps, R.P. and Blieden, T.A. and Springer, T.A.} } @article {529316, title = {IL-8 production in human lung fibroblasts and epithelial cells activated by the Pseudomonas autoinducer N-3-oxododecanoyl homoserine lactone is transcriptionally regulated by NF-κB and activator protein-2}, journal = {J. Immunol.}, volume = {167}, number = {1}, year = {2001}, note = {Reprint: not in file}, pages = {366-374.}, abstract = {The destructive pulmonary inflammation associated with Pseudomonas aeruginosa colonization is caused, in part, by the production of the chemokine IL-8, which recruits neutrophils into the lung. The Pseudomonas autoinducer, N-3-oxododecanoyl homoserine lactone (3-O-C12-HSL), is a small lipid-soluble molecule that is essential in the regulation of many P. aeruginosa virulence factors, but little is known about how it affects eukaryotic cells. In this report we demonstrate that 3-O-C12-HSL is a potent stimulator of both IL-8 mRNA and protein from human fibroblasts and epithelial cells in vitro. The IL-8 produced from these 3-O-C12-HSL-stimulated cells was found to be functionally active by inducing the chemotaxis of neutrophils. To determine a mechanism for this IL-8 induction, deletion constructs of the IL-8 promoter were examined. It was found that the DNA region between nucleotides -1481 and -546 and the transcription factor NF-kappaB were essential for the maximal induction of IL-8 by 3-O-C12-HSL. This was confirmed by EMSAs, where 3-O-C12-HSL induced a shift with both AP-2 and NF-kappaB consensus DNA. The activation of NF-kappaB and subsequent production of IL-8 were found to be regulated by a mitogen-activated protein kinase pathway. These findings support the concept that the severe lung damage that accompanies P. aeruginosa infections is caused by an exuberant neutrophil response stimulated by 3-O-C12-HSL-induced IL-8. Understanding the mechanisms of 3-O-C12-HSL activation of lung structural cells may provide a means to help control lung damage during infections with P. aeruginosa.}, author = {Smith, R. S. and Fedyk, E.R. and Springer, T.A. and Mukaida, N. and Iglewski, B. H. and Phipps, R.P.} } @article {529871, title = {Implications for familial hypercholesterolemia from structure of the LDL receptor YWTD-EGF domain pair}, journal = {Nat. Struct. Biol.}, volume = {8}, number = {6}, year = {2001}, pages = {499-504}, abstract = {The low-density lipoprotein receptor (LDLR) is the primary mechanism for uptake of cholesterol-carrying particles into cells. The region of the LDLR implicated in receptor recycling and lipoprotein release at low pH contains a pair of calcium-binding EGF-like modules, followed by a series of six YWTD repeats and a third EGF-like module. The crystal structure at 1.5 A resolution of a receptor fragment spanning the YWTD repeats and its two flanking EGF modules reveals that the YWTD repeats form a six-bladed beta-propeller that packs tightly against the C-terminal EGF module, whereas the EGF module that precedes the propeller is disordered in the crystal. Numerous point mutations of the LDLR that result in the genetic disease familial hypercholesterolemia (FH) alter side chains that form conserved packing and hydrogen bonding interactions in the interior and between propeller blades. A second subset of FH mutations are located at the interface between the propeller and the C-terminal EGF module, suggesting a structural requirement for maintaining the integrity of the interdomain interface.}, author = {Jeon, H and Meng, W. and Takagi, J. and Eck, M. J. and Springer, T.A. and Blacklow, S. C.} } @article {530121, title = {An isolated, surface-expressed I domain of the integrin αLβ2 is sufficient for strong adhesive function when locked in the open conformation with a disulfide}, journal = {Proc Natl Acad Sci USA}, volume = {98}, number = {5}, year = {2001}, pages = {2387-2392}, abstract = {We introduced disulfide bonds to lock the integrin alphaLbeta2 I domain in predicted open, ligand binding or closed, nonbinding conformations. Transfectants expressing alphaLbeta2 heterodimers containing locked-open but not locked-closed or wild-type I domains constitutively adhered to intercellular adhesion molecule-1 (ICAM-1) substrates. Locking the I domain closed abolished constitutive and activatable adhesion. The isolated locked-open I domain bound as well as the activated alphaLbeta2 heterodimer, and binding was abolished by reduction of the disulfide. Lovastatin, which binds under the conformationally mobile C-terminal alpha-helix of the I domain, inhibited binding to ICAM-1 by alphaLbeta2 with wild-type, but not locked-open I domains. These data establish the importance of conformational change in the alphaL I domain for adhesive function and show that this domain is sufficient for full adhesive activity.}, author = {Lu, C. and Shimaoka, M. and Ferzly, M. and Oxvig,C. and Takagi, J. and Springer, T.A.} } @article {527851, title = {Kinetic and mechanical basis of rolling through an integrin and novel Ca2+-dependent rolling and Mg2+-dependent firm adhesion modalities for the α4β7-MAdCAM-1 interaction}, journal = {Biochemistry}, volume = {40}, number = {46}, year = {2001}, pages = {13972-13979}, abstract = {We studied interactions in shear flow of cells bearing integrins alpha4beta1 or alpha4beta7 with VCAM-1 and MAdCAM-1 substrates in different divalent cations. Interestingly, Ca(2+) was essential for tethering in flow and rolling interactions through both alpha4 integrins. Mg(2+) promoted firm adhesion of alpha4beta7-expressing cells on MAdCAM-1 but with much lower tethering efficiency in shear flow. The k(off) degrees of 1.28 s(-1) and resistance of the receptor-ligand bond to force (estimated as a bond interaction distance or sigma) for transient tethers on MAdCAM-1 were similar to values for E- and P-selectins. By contrast to results in Ca(2+) or Ca(2+) + Mg(2+), in Mg(2+) the alpha4beta7-MAdCAM-1 k(off) degrees decreased 20-fold to 0.046 s(-1), and the bond was weaker, providing an explanation for the finding of firm adhesion under these conditions. Shear enhanced tethering to MAdCAM-1, thereby contributing to the stability of rolling. Comparisons to selectins demonstrate that the kinetic and mechanical properties of the alpha4beta7 integrin are well suited to its intermediate position in adhesion cascades, in which it bridges rapid rolling through selectins to firm adhesion through beta2 integrins.}, keywords = {451}, author = {de Chateau, M. and Chen, S. and Salas, A. and Springer, T.A.} } @article {529476, title = {LFA-1 expression on target cells promotes human immunodeficiency virus type 1 infection and transmission}, journal = {J. Virol.}, volume = {75}, number = {2}, year = {2001}, note = {In file}, pages = {1077-1082}, abstract = {While CD4 and the chemokine receptors are the principal receptors for human immunodeficiency virus (HIV), other cellular proteins, such as LFA-1, are also involved in HIV infection. LFA-1 and its ligands, ICAM-1, ICAM-2, and ICAM-3, can be expressed on the cells infected by HIV, as well as on the HIV virions themselves. To examine the role of LFA-1 expressed on target cells in HIV infection, Jurkat-derived Jbeta2.7 T-cell lines that express either wild-type LFA-1, a constitutively active mutant LFA-1, or no LFA-1 were used. The presence of wild-type LFA-1 enhanced the initial processes of HIV infection, as well as the subsequent replication and transmission from cell to cell. In contrast, the constitutively active LFA-1 mutant failed to promote virus replication and spread, even though this mutant could help HIV enter cells and establish the initial infection. This study clearly demonstrates the contribution of LFA-1 in the different stages of HIV infection. Moreover, not only is LFA-1 expression important for initial HIV-cell interaction, subsequent replication, and transmission, but its activity must also be properly regulated.}, author = {Hioe, C. E. and Chien, P. C. and Lu, C. and Springer, T.A. and Wang, X. H. and Bandres, J. and Tuen, M.} } @article {530126, title = {Locking in alternate conformations of the integrin αLβ2 I domain with disulfide bonds reveals functional relationships among integrin domains}, journal = {Proc Natl Acad Sci USA}, volume = {98}, number = {5}, year = {2001}, pages = {2393-2398}, abstract = {We used integrin alphaLbeta2 heterodimers containing I domains locked open (active) or closed (inactive) with disulfide bonds to investigate regulatory interactions among domains in integrins. mAbs to the alphaL I domain and beta2 I-like domain inhibit adhesion of wild-type alphaLbeta2 to intercellular adhesion molecule-1. However, with alphaLbeta2 containing a locked open I domain, mAbs to the I domain were subdivided into subsets (i) that did not inhibit, and thus appear to inhibit by favoring the closed conformation, and (ii) that did inhibit, and thus appear to bind to the ligand binding site. Furthermore, alphaLbeta2 containing a locked open I domain was completely resistant to inhibition by mAbs to the beta2 I-like domain, but became fully susceptible to inhibition after disulfide reduction with DTT. This finding suggests that the I-like domain indirectly contributes to ligand binding by regulating opening of the I domain in wild-type alphaLbeta2. Conversely, locking the I domain closed partially restrained conformational change of the I-like domain by Mn(2+), as measured with mAb m24, which we map here to the beta2 I-like domain. By contrast, locking the I domain closed or open did not affect constitutive or Mn(2+)-induced exposure of the KIM127 epitope in the beta2 stalk region. Furthermore, locked open I domains, in alphaLbeta2 complexes or expressed in isolation on the cell surface, bound to intercellular adhesion molecule-1 equivalently in Mg(2+) and Mn(2+). These results suggest that Mn(2+) activates alphaLbeta2 by binding to a site other than the I domain, most likely the I-like domain of beta2.}, author = {Lu, C. and Shimaoka, M. and Zang, Q. and Takagi, J. and Springer, T.A.} } @article {530181, title = {Reversibly locking a protein fold in an active conformation with a disulfide bond: integrin αL I domains with high affinity and antagonist activity in vivo}, journal = {Proc Natl Acad Sci USA}, volume = {98}, number = {11}, year = {2001}, pages = {6009-6014}, abstract = {The integrin alphaLbeta2 has three different domains in its headpiece that have been suggested to either bind ligand or to regulate ligand binding. One of these, the inserted or I domain, has a fold similar to that of small G proteins. The I domain of the alphaM and alpha2 subunits has been crystallized in both open and closed conformations; however, the alphaL I domain has been crystallized in only the closed conformation. We hypothesized that the alphaL domain also would have an open conformation, and that this would be the ligand binding conformation. Therefore, we introduced pairs of cysteine residues to form disulfides that would lock the alphaL I domain in either the open or closed conformation. Locking the I domain open resulted in a 9,000-fold increase in affinity to intercellular adhesion molecule-1 (ICAM-1), which was reversed by disulfide reduction. By contrast, the affinity of the locked closed conformer was similar to wild type. Binding completely depended on Mg(2+). Orders of affinity were ICAM-1 \> ICAM-2 \> ICAM-3. The k(on), k(off), and K(D) values for the locked open I domain were within 1.5-fold of values previously determined for the alphaLbeta2 complex, showing that the I domain is sufficient for full affinity binding to ICAM-1. The locked open I domain antagonized alphaLbeta2-dependent adhesion in vitro, lymphocyte homing in vivo, and firm adhesion but not rolling on high endothelial venules. The ability to reversibly lock a protein fold in an active conformation with dramatically increased affinity opens vistas in therapeutics and proteomics.}, author = {Shimaoka, M. and Lu, C. and R. Palframan and von Andrian, U. H. and Takagi, J. and Springer, T.A.} } @article {530056, title = {Selectin receptor-ligand bonds: Formation limited by shear rate and dissociation governed by the Bell model}, journal = {Proc Natl Acad Sci USA}, volume = {98}, number = {3}, year = {2001}, pages = {950-955}, abstract = {We have studied the principles that govern the formation and dissociation of an adhesive bond between a cell moving in shear flow and a substrate and tested different theories of how force affects bond dissociation. Viscosity relates the kinematics of fluid movement (shear rate, units of time(-1)) to shear stress (units of force/area, the product of shear rate and viscosity). At different medium viscosities, the formation of receptor-ligand bonds between a cell in the flowstream and P-selectin on the vessel wall showed a similar efficiency as a function of shear rate but not of shear stress. Therefore, bond formation was a function of shear rate and hence of the kinematics of receptor and ligand movement. By contrast, the kinetics of bond dissociation was a function of shear stress and hence of force on the bond. The different requirements for bond formation and dissociation allowed dissociation kinetics to be measured at higher forces on the bond by increasing medium viscosity. Data over an extended range of forces on the bond therefore could be collected that enabled five different proposed equations, relating force to bond dissociation, to be compared for fit to experimental data. The relationship proposed by Bell [Bell, G. I. (1978) Science 200, 618-627] fit the data significantly the best and also predicted an off-rate in the absence of force that best matched an independent measurement [Mehta, P., Cummings, R. D. \& McEver, R. P. (1998) J. Biol. Chem. 273, 32506-32513].}, author = {Chen, S. and Springer, T.A.} } @article {528511, title = {Ultrastructure and function of dimeric, soluble intercellular adhesion molecule-1 (ICAM-1)}, journal = {J. Biol. Chem.}, volume = {276}, number = {31}, year = {2001}, pages = {29019-29027}, abstract = {Previous studies have demonstrated dimerization of intercellular adhesion molecule-1 (ICAM-1) on the cell surface and suggested a role for immunoglobulin superfamily domain 5 and/or the transmembrane domain in mediating such dimerization. Crystallization studies suggest that domain 1 may also mediate dimerization. ICAM-1 binds through domain 1 to the I domain of the integrin alpha(L)beta(2) (lymphocyte function-associated antigen 1). Soluble C-terminally dimerized ICAM-1 was made by replacing the transmembrane and cytoplasmic domains with an alpha-helical coiled coil. Electron microscopy revealed C-terminal dimers that were straight, slightly bent, and sometimes U-shaped. A small number of apparently closed ring-like dimers and W-shaped tetramers were found. To capture ICAM-1 dimerized at the crystallographically defined dimer interface in domain 1, cysteines were introduced into this interface. Several of these mutations resulted in the formation of soluble disulfide-bonded ICAM-1 dimers (domain 1 dimers). Combining a domain 1 cysteine mutation with the C-terminal dimers (domain 1/C-terminal dimers) resulted in significant amounts of both closed ring-like dimers and W-shaped tetramers. Surface plasmon resonance studies showed that all of the dimeric forms of ICAM-1 (domain 1, C-terminal, and domain 1/C-terminal dimers) bound similarly to the integrin alpha(L)beta(2) I domain, with affinities approximately 1.5--3-fold greater than that of monomeric ICAM-1. These studies demonstrate that ICAM-1 can form at least three different topologies and that dimerization at domain 1 does not interfere with binding in domain 1 to alpha(L)beta(2).}, author = {Jun, C-D. and Carman, C. V. and Redick, S. D. and Shimaoka, M. and Erickson, H. P. and Springer, T.A.} } @article {529876, title = {Computational design of an integrin I domain stabilized in the open, high affinity conformation}, journal = {Nat. Struct. Biol.}, volume = {7}, number = {8}, year = {2000}, note = {Reprint in file}, pages = {674-678}, abstract = {We have taken a computational approach to design mutations that stabilize a large protein domain of approximately 200 residues in two alternative conformations. Mutations in the hydrophobic core of the alphaMbeta2 integrin I domain were designed to stabilize the crystallographically defined open or closed conformers. When expressed on the cell surface as part of the intact heterodimeric receptor, binding of the designed open and closed I domains to the ligand iC3b, a form of the complement component C3, was either increased or decreased, respectively, compared to wild type. Moreover, when expressed in isolation from other integrin domains using an artificial transmembrane domain, designed open I domains were active in ligand binding, whereas designed closed and wild type I domains were inactive. Comparison to a human expert designed open mutant showed that the computationally designed mutants are far more active. Thus, computational design can be used to stabilize a molecule in a desired conformation, and conformational change in the I domain is physiologically relevant to regulation of ligand binding.}, author = {Shimaoka, M. and Shifman, J. M. and Jing, H. and Takagi, J. and Mayo, S. L. and Springer, T.A.} } @article {528626, title = {Folding and function of I-domain deleted Mac-1 and LFA-1}, journal = {J. Biol. Chem.}, volume = {275}, number = {29}, year = {2000}, note = {Reprint is not in file.}, pages = {21877-21882}, abstract = {In those integrins that contain it, the I domain is a major ligand recognition site. The I domain is inserted between beta-sheets 2 and 3 of the predicted beta-propeller domain of the integrin alpha subunit. We deleted the I domain from the integrin alpha(M) and alpha(L) subunits to give I-less Mac-1 and lymphocyte function-associated antigen-1 (LFA-1), respectively. The I-less alpha(M) and alpha(L) subunits were expressed in association with the wild-type beta(2) subunit on the surface of transfected cells and bound to all the monoclonal antibodies mapped to the putative beta-propeller and C-terminal regions of the alpha(M) and alpha(L) subunits, suggesting that the folding of these domains is independent of the I domain. I-less Mac-1 bound to the ligands iC3b and factor X, but this binding was reduced compared with wild-type Mac-1. In contrast, I-less Mac-1 did not bind to fibrinogen or denatured bovine serum albumin. Binding to iC3b and factor X by I-less Mac-1 was inhibited by the function-blocking antibody CBRM1/32, which binds to the beta-propeller domain of the alpha(M) subunit. I-less LFA-1 did not bind its ligands intercellular adhesion molecule-1 and -3. Thus, the I domain is not essential for the folding, heterodimer formation, and surface expression of Mac-1 and LFA-1 and is required for binding to some ligands, but not others.}, author = {Yalamanchili, P. and Lu, C. and Oxvig,C. and Springer, T.A.} } @article {527976, title = {A novel Ca2+-binding β-hairpin loop better resembles integrin sequence motifs than the EF-hand}, journal = {CellCell}, volume = {102}, year = {2000}, pages = {275-277}, author = {Springer, T.A. and Jing, H. and Takagi, J.} } @article {528501, title = {Structural and functional studies with antibodies to the integrin β2 subunit: a model for the I-like domain}, journal = {J. Biol. Chem.}, volume = {275}, number = {28}, year = {2000}, note = {Reprint Status: NOT In File}, pages = {21514-21524}, abstract = {To establish a structure and function map of the beta2 integrin subunit, we mapped the epitopes of a panel of beta2 monoclonal antibodies including function-blocking, nonblocking, and activating antibodies using human/mouse beta2 subunit chimeras. Activating antibodies recognize the C-terminal half of the cysteine-rich region, residues 522-612. Antibodies that do not affect ligand binding map to residues 1-98 and residues 344-521. Monoclonal antibodies to epitopes within a predicted I-like domain (residues 104-341) strongly inhibit LFA-1-dependent adhesion. These function-blocking monoclonal antibodies were mapped to specific residues with human --\> mouse knock-out or mouse --\> human knock-in mutations. Combinatorial epitopes involving residues distant in the sequence provide support for a specific alignment between the beta-subunit and I domains that was used to construct a three-dimensional model. Antigenic residues 133, 332, and 339 are on the first and last predicted alpha-helices of the I-like domain, which are adjacent on its "front." Other antigenic residues in beta2 and in other integrin beta subunits are present on the front. No antigenic residues are present on the "back" of the domain, which is predicted to be in an interface with other domains, such as the alpha subunit beta-propeller domain. Most mutations in the beta2 subunit in leukocyte adhesion deficiency are predicted to be buried in the beta2 subunit I-like domain. Two long insertions are present relative to alpha-subunit I-domains. One is tied down to the back of the I-like domain by a disulfide bond. The other corresponds to the "specificity-determining loop" defined in beta1 and beta3 integrins and contains the antigenic residue Glu(175) in a disulfide-bonded loop located near the "top" of the domain.}, author = {Huang, C and Zang, Q. and Takagi, J. and Springer, T.A.} } @article {528631, title = {The top of the I-like domain of the integrin LFA-1 β subunit contacts the α subunit β-propeller domain near β-sheet 3}, journal = {J. Biol. Chem.}, volume = {275}, number = {29}, year = {2000}, note = {Reprint is not in file}, pages = {22202-22212}, abstract = {We find that monoclonal antibody YTA-1 recognizes an epitope formed by a combination of the integrin alpha(L) and beta(2) subunits of LFA-1. Using human/mouse chimeras of the alpha(L) and beta(2) subunits, we determined that YTA-1 binds to the predicted inserted (I)-like domain of the beta(2) subunit and the predicted beta-propeller domain of the alpha(L) subunit. Substitution into mouse LFA-1 of human residues Ser(302) and Arg(303) of the beta(2) subunit and Pro(78), Thr(79), Asp(80), Ile(365), and Asn(367) of the alpha(L) subunit is sufficient to completely reconstitute YTA-1 reactivity. Antibodies that bind to epitopes that are nearby in models of the I-like and beta-propeller domains compete with YTA-1 monoclonal antibody for binding. The predicted beta-propeller domain of integrin alpha subunits contains seven beta-sheets arranged like blades of a propeller around a pseudosymmetry axis. The antigenic residues cluster on the bottom of this domain in the 1-2 loop of blade 2, and on the side of the domain in beta-strand 4 of blade 3. The I domain is inserted between these blades on the top of the beta-propeller domain. The antigenic residues in the beta subunit localize to the top of the I-like domain near the putative Mg(2+) ion binding site. Thus, the I-like domain contacts the bottom or side of the beta-propeller domain near beta-sheets 2 and 3. YTA-1 preferentially reacts with activated LFA-1 and is a function-blocking antibody, suggesting that conformational movements occur near the interface it defines between the LFA-1 alpha and beta subunits.}, author = {Zang, Q. and Lu, C. and Huang, C and Takagi, J. and Springer, T.A.} } @article {528686, title = {An automatic braking system that stabilizes leukocyte rolling by an increase in selectin bond number with shear}, journal = {J. Cell Biol.}, volume = {144}, number = {1}, year = {1999}, note = {Reprint Status: NOT In File}, pages = {185-200}, abstract = {Wall shear stress in postcapillary venules varies widely within and between tissues and in response to inflammation and exercise. However, the speed at which leukocytes roll in vivo has been shown to be almost constant within a wide range of wall shear stress, i.e., force on the cell. Similarly, rolling velocities on purified selectins and their ligands in vitro tend to plateau. This may be important to enable rolling leukocytes to be exposed uniformly to activating stimuli on endothelium, independent of local hemodynamic conditions. Wall shear stress increases the rate of dissociation of individual selectin-ligand tether bonds exponentially (, ) thereby destabilizing rolling. We find that this is compensated by a shear-dependent increase in the number of bonds per rolling step. We also find an increase in the number of microvillous tethers to the substrate. This explains (a) the lack of firm adhesion through selectins at low shear stress or high ligand density, and (b) the stability of rolling on selectins to wide variation in wall shear stress and ligand density, in contrast to rolling on antibodies (). Furthermore, our data successfully predict the threshold wall shear stress below which rolling does not occur. This is a special case of the more general regulation by shear of the number of bonds, in which the number of bonds falls below one.}, author = {Chen, S. and Springer, T.A.} } @inbook {528271, title = {CD2/LFA-3 (CD2: T11, E rosette receptor; LFA-3: CD58)}, booktitle = {Guidebook to the Extracellular Matrix and Adhesion Proteins}, year = {1999}, note = {Reprint Status: In File}, pages = {154-157}, publisher = {Sambrook and Tooze}, organization = {Sambrook and Tooze}, edition = {2}, address = {New York}, author = {Dustin,M.L. and Springer, T.A.}, editor = {Kreis,T. and Vale,R.} } @article {528346, title = {The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment}, journal = {Immunity}, volume = {10}, number = {4}, year = {1999}, note = {Reprint Status: NOT In File}, pages = {463-471}, abstract = {We report that the chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within fetal liver and bone marrow microenvironment. In CXCR4-deficient embryos, pro-B cells are present in blood but hardly detectable in liver; myeloid cells are elevated in blood and reduced in liver compared to wild-type embryos. Mice reconstituted with CXCR4-deficient fetal liver cells have reduced donor-derived mature B lymphocytes in blood and lymphoid organs. The numbers of pro-B and pre-B cells are reduced in bone marrow and abnormally high in blood. Granulocytic cells are reduced in bone marrow but elevated and less mature in the blood. B lineage and granulocytic precursors are released into the periphery in absence of CXCR4.}, author = {Ma,Q. and Jones,D. and Springer, T.A.} } @article {530151, title = {Conformational changes in tertiary structure near the ligand binding site of an integrin I domain}, journal = {Proc Natl Acad Sci USA}, volume = {96}, number = {5}, year = {1999}, note = {Reprint Status: In File}, pages = {2215-2220}, abstract = {For efficient ligand binding, integrins must be activated. Specifically, a conformational change has been proposed in a ligand binding domain present within some integrins, the inserted (I) domain [Lee, J., Bankston, L., Arnaout, M. \& Liddington, R. C. (1995) Structure (London) 3, 1333-1340]. This proposal remains controversial, however, despite extensive crystal structure studies on the I domain [Lee, J., Bankston, L., Arnaout, M. \& Liddington, R. C. (1995) Structure (London) 3, 1333-1340; Liddington, R. \& Bankston, L. (1998) Structure (London) 6, 937-938; Qu, A. \& Leahy, D. J. (1996) Structure (London) 4, 931-942; and Baldwin, E. T., Sarver, R. W., Bryant, G. L., Jr., Curry, K. A., Fairbanks, M. B., Finzel, B. C. , Garlick, R. L., Heinrikson, R. L., Horton, N. C. \& Kelly, L. L. (1998) Structure (London) 6, 923-935]. By defining the residues present in the epitope of a mAb against the human Mac-1 integrin (alphaMbeta2, CD11b/CD18) that binds only the active receptor, we provide biochemical evidence that the I domain itself undergoes a conformational change with activation. This mAb, CBRM1/5, binds the I domain very close to the ligand binding site in a region that is widely exposed regardless of activation as judged by reactivity with other antibodies. The conformation of the epitope differs in two crystal forms of the I domain, previously suggested to represent active and inactive receptor. Our data suggests that conformational differences in the I domain are physiologically relevant and not merely a consequence of different crystal lattice interactions. We also demonstrate that the transition between the two conformational states depends on species-specific residues at the bottom of the I domain, which are proposed to be in an interface with another integrin domain, and that this transition correlates with functional activity.}, author = {Oxvig,C. and Lu, C. and Springer, T.A.} } @article {530346, title = {Domains in plexins: Links to integrins and transcription factors}, journal = {Trends Biochem. Sci.}, volume = {24}, number = {7}, year = {1999}, note = {Reprint Status: In File}, pages = {261-263}, keywords = {CD18}, author = {Bork, P. and Doerks, T. and Springer, T.A. and Snel, B.} } @inbook {528136, title = {Immunoaffinity chromatography}, booktitle = {Current Protocols in Protein Science}, year = {1999}, pages = {9.5.1-9.5.11}, publisher = {John Wiley and Sons, Inc.}, organization = {John Wiley and Sons, Inc.}, address = {New York}, author = {Springer, T.A.}, editor = {Coligan,J.E. and Dunn, B.M. and Ploegh, H.L. and Speicher, D.W. and Wingfield, P.T.} } @inbook {528276, title = {Intercellular adhesion molecules (ICAMs)}, booktitle = {Guidebook to the Extracellular Matrix and Adhesion Proteins}, year = {1999}, note = {Reprint Status: In File}, pages = {216-220}, publisher = {Sambrook and Tooze}, organization = {Sambrook and Tooze}, edition = {2}, address = {New York}, author = {Dustin,M.L. and Springer, T.A.}, editor = {Kreis,T. and Vale,R.} } @inbook {528281, title = {Lymphocyte function associated-1 (LFA-1, CD11a/CD18)}, booktitle = {Guidebook to the Extracellular Matrix and Adhesion Proteins,}, year = {1999}, note = {Reprint Status: In File}, pages = {228-232}, publisher = {Sambrook and Tooze}, organization = {Sambrook and Tooze}, edition = {2}, address = {New York}, author = {Dustin,M.L. and Springer, T.A.}, editor = {Kreis,T. and Vale,R.} } @article {530036, title = {Lymphocyte function-associated antigen-1 binding residues in intercellular adhesion molecule-2 (ICAM-2) and the integrin binding surface in the ICAM subfamily}, journal = {Proc Natl Acad Sci USA}, volume = {96}, number = {6}, year = {1999}, note = {Reprint Status: In File}, pages = {3017-3022}, abstract = {The crystal structure of intercellular adhesion molecule-2 (ICAM-2) revealed significant differences in the presentation of the critical acidic residue important for integrin binding between I and non-I-domain integrin ligands. Based on this crystal structure, we mutagenized ICAM-2 to localize the binding site for the integrin lymphocyte function-associated antigen-1 (LFA-1). The integrin binding site runs diagonally across the GFC beta-sheet and includes residues on the CD edge of the beta-sandwich. The site is oblong and runs along a flat ridge on the upper half of domain 1, which is proposed to dock to a groove in the I domain of LFA-1, with the critical Glu-37 residue ligating the Mg2+ in the I domain. Previous mutagenesis of ICAM-1 and ICAM-3, interpreted in light of the recently determined ICAM-1 and ICAM-2 structures, suggests similar binding sites. By contrast, major differences are seen with vascular cell adhesion molecule-1 (VCAM-1), which binds alpha4 integrins that lack an I domain. The binding site on VCAM-1 includes the lower portion of domain 1 and the upper part of domain 2, whereas the LFA-1 binding site on ICAM is confined to the upper part of domain 1.}, author = {Casasnovas,J. and Pieroni,C. and Springer, T.A.} } @article {529426, title = {Maturation decreases responsiveness of human bone marrow B lineage cells to stromal-derived factor 1 (SDF-1)}, journal = {J. Leukoc. Biol.}, volume = {66}, number = {4}, year = {1999}, pages = {667-673}, abstract = {We compared the chemotactic responsiveness of different subsets of human B lineage cells to stromal derived factor-1 (SDF-1). High percentages (30-40\% of input) of purified bone marrow progenitors including non-B lineage progenitors, pro-B cells, and pre-B cells migrated to SDF-1alpha, demonstrating that SDF-1 is an efficacious chemoattractant of these cells. Pro-B cells responded optimally to a lower concentration of SDF-1 than other subsets, demonstrating that SDF-1 is a more potent chemoattractant of this subset. A lower percentage (10-15\% of input) of mature B lymphocytes migrated to SDF-1alpha than pro-B cells, demonstrating that responsiveness of B lineage cells to SDF-1 decreases during differentiation. Inhibition by anti-CXCR4 mAb demonstrated that migration of B lineage cells to SDF-1 was completely dependent on CXC chemokine receptor-4 (CXCR4). Mature B cells expressed higher levels of CXCR4 receptors than uncommitted progenitors and pro-B cells, despite differences in responsiveness to SDF-1. CXCR4 receptors expressed by unresponsive and SDF-1-responsive B cells bound SDF-1alpha with similar affinities (K(D) = 1.7-3.3 x 10(-9) M). Therefore, elements downstream from CXCR4 appear to regulate responsiveness of B cells to SDF-1. We speculate that SDF-1 and CXCR4 direct migration of progenitor cells in microenvironments that promote B lymphopoiesis.}, author = {Fedyk, E.R. and Ryan, D.H. and Ritterman, I. and Springer, T.A.} } @article {528006, title = {Mutational analysis of MAdCAM-1/α4β7 interactions reveals significant binding determinants in both the first and second immunoglobulin domains}, journal = {Cell Adhes. Comm.}, volume = {7}, year = {1999}, note = {Reprints in file.}, pages = {167-181}, abstract = {The selective emigration of blood born leukocytes into tissues is mediated, in part by interactions of Ig-like cell adhesion molecules (IgCAMs) expressed on vascular endothelium and their cognate ligands, the leukocyte integrins. Within mucosal lymphoid tissues and gastrointestinal sites the mucosal vascular addressin. MAdCAM-1 is the predominant IgCAM, mediating specific lymphocyte homing via interactions with its ligand on lymphocytes, the integrin alpha4beta7. Previous studies have shown that an essential binding motif resides in the first Ig domain of all IgCAMs, containing an acidic residue (D or E) preceded by an aliphatic residue (L or I) that resides in strand C or the CD loop. However, domain swap experiments with MAdCAM-1 and VCAM-1 have shown a requirement for both Ig domains 1 and 2 for efficient integrin binding. We describe the use of chimeric MAdCAM-1/VCAM-1 receptors and point mutations in MAdCAM-1 to define other sites that are required for binding to the integrin alpha4beta7. We find that, in addition to critical CD loop residues, other regions in both domain one and two contribute to MAdCAM-1/alpha4beta7 interactions, including a buried arginine residue in the F strand of domain one and several acidic residues in a highly extended DE ribbon in domain 2. These mutations, when placed in the recently solved crystal structure of human MAdCAM-1 give insight into the integrin binding preference of this unique receptor.}, keywords = {CD MAdCAM}, author = {Green, N. and Rosebrook, J. and Cochran, N. and Tan, K. and J.-H. Wang and Springer, T.A. and Briskin, M.J.} } @article {528331, title = {Prolonged eosinophil accumulation in allergic lung interstitium of ICAM-2 deficient mice results in extended hyperresponsiveness}, journal = {Immunity}, volume = {10}, number = {1}, year = {1999}, note = {Reprint Status: NOT In File}, pages = {9-19}, abstract = {ICAM-2-deficient mice exhibit prolonged accumulation of eosinophils in lung interstitium concomitant with a delayed increase in eosinophil numbers in the airway lumen during the development of allergic lung inflammation. The ICAM-2-dependent increased and prolonged accumulation of eosinophils in lung interstitium results in prolonged, heightened airway hyperresponsiveness. These findings reveal an essential role for ICAM-2 in the development of the inflammatory and respiratory components of allergic lung disease. This phenotype is caused by the lack of ICAM-2 expression on non-hematopoietic cells. ICAM-2 deficiency on endothelial cells causes reduced eosinophil transmigration in vitro. ICAM-2 is not essential for lymphocyte homing or the development of leukocytes, with the exception of megakaryocyte progenitors, which are significantly reduced.}, author = {Gerwin,N. and Gonzalo,J.A. and Lloyd,C. and Coyle,A.J. and Reiss,Y. and Banu,N. and Wang, B. and Xu, H and Avraham,H. and Engelhardt,B. and Springer, T.A. and Gutierrez-Ramos,J.C.} } @article {529436, title = {Role of ICAM-1 and ICAM-2 and alternate CD11/CD18 ligands in neutrophil transendothelial migration}, journal = {J. Leukoc. Biol.}, volume = {65}, year = {1999}, note = {Reprint Status: In File}, pages = {117-126}, abstract = {We evaluated the relative contribution of ICAM-1 and ICAM-2, known ligands on endothelium for LFA-1 and Mac-1, in spontaneous neutrophil (PMN) transendothelial migration (TEM) across IL-1-activated HUVEC monolayers or TEM induced by C5a or IL-8 across unstimulated HUVEC grown on polycarbonate filters. Adhesion blocking mAb to ICAM-1 [R6.5 F(ab)2] or ICAM-2 [CBR IC2/2 F(ab)2] tended to inhibit TEM under each condition but, in general, inhibition was significant only with both ICAM-1 and ICAM-2 blockade. mAb to LFA-1 partially inhibited migration to C5a or IL-8 across unstimulated HUVEC and inhibition was not altered by additional treatment of HUVEC with mAbs to ICAM-1 and -2. In contrast, with IL-1 HUVEC, mAb to ICAM-1 significantly inhibited this LFA-1-independent TEM. mAb to Mac-1 alone partially inhibited TEM and, when combined with mAb to LFA-1, migration was almost completely blocked with all TEM conditions tested. The contribution of alternate ligands for Mac-1 in mediating Mac-1-dependent but ICAM-1/-2-independent C5a-induced TEM was examined using anti-LFA-1-treated PMN and anti-ICAM-treated resting HUVEC. Addition of RGD peptides, fibronectin, fibrinogen, heparins, collagens alone or in combination, even to heparinase-treated HUVEC, did not inhibit this Mac-1-mediated PMN TEM. The results indicate that: (1) LFA-1 mediates PMN TEM primarily by interaction with ICAM-1 and ICAM-2; (2) ICAM-2 may function in concert with ICAM-1 in this role, especially on unstimulated endothelium, and (3) Mac-1 on PMN also plays a major role in TEM and can utilize yet to be identified ligands distinct from ICAM-1 or -2, especially on unstimulated endothelium.}, keywords = {276.2}, author = {Issekutz, A. C. and Rowter, D. and Springer, T.A.} } @article {528876, title = {B lymphocyte chemotaxis regulated in association with microanatomic localization, differentiation state, and B cell receptor engagement}, journal = {J. Exp. Med.}, volume = {187}, number = {5}, year = {1998}, note = {Reprint Status: In File}, pages = {753-762}, abstract = {Migration of mature B lymphocytes within secondary lymphoid organs and recirculation between these sites are thought to allow B cells to obtain T cell help, to undergo somatic hypermutation, to differentiate into effector cells, and to home to sites of antibody production. The mechanisms that direct migration of B lymphocytes are unknown, but there is evidence that G protein-coupled receptors, and possibly chemokine receptors, may be involved. Stromal cell- derived factor (SDF)-1alpha is a CXC chemokine previously characterized as an efficacious chemoattractant for T lymphocytes and monocytes in peripheral blood. Here we show with purified tonsillar B cells that SDF-1alpha also attracts naive and memory, but not germinal center (GC) B lymphocytes. Furthermore, GC B cells could be converted to respond to SDF-1alpha by in vitro differentiation into memory B lymphocytes. Conversely, the migratory response in naive and memory B cells was significantly reduced after B cell receptor engagement and CD40 signaling. The receptor for SDF-1, CXC chemokine receptor 4 (CXCR4), was found to be expressed on responsive as well as unresponsive B cell subsets, but was more rapidly downregulated on responsive cells by ligand. Finally, messenger RNA for SDF-1 was detected by in situ hybridization in a layer of cells surrounding the GC. These findings show that responsiveness to the chemoattractant SDF-1alpha is regulated during B lymphocyte activation, and correlates with positioning of B lymphocytes within a secondary lymphoid organ.}, author = {Bleul,C.C. and Schultze,J.L. and Springer, T.A.} } @article {528041, title = {Cardiac graft intercellular adhesion molecule-1 (ICAM-1) and interleukin-1 expression mediate primary isograft failure and induction of ICAM-1 in organs remote from the site of transplantation}, journal = {Circ. Res.}, volume = {82}, number = {7}, year = {1998}, note = {Reprint Status: In File}, pages = {762-772}, abstract = {During the first few hours after heart transplantation, the occurrence of graft failure is unpredictable and devastating. An explosive cascade of inflammatory events within the reperfused graft vasculature is likely to be mediated, at least in part, by the local expression of the leukocyte adhesion receptor intercellular adhesion molecule-1 (ICAM-1, CD54). Furthermore, although proinflammatory cytokines such as interleukin-1 (IL-1) are known to autoinduce their own (and ICAM-1) expression in vitro, there are no data to identify their functional in vivo cross talk in the setting of isograft transplantation. To determine the role of ICAM-1 in primary graft failure, we used an isogeneic vascularized model of heterotopic cardiac transplantation. ICAM-1 mRNA and protein increased in grafts during the early posttransplant period and were predominantly localized in the endothelium. The functional significance of this was established using donor hearts obtained from either ICAM-1-deficient (ICAM-1 -/-) or control (ICAM-1 +/+) mice. ICAM-1 +/+ grafts exhibited increased neutrophil infiltration, reduced left ventricular compliance, and poorer survival than did ICAM-1 -/- grafts. Increased ICAM-1 expression was not limited to ICAM-1 +/+ grafts but also occurred in unmanipulated recipient organs located remote from the site of surgery (but only after transplantation of ICAM-1 +/+, not ICAM-1 -/-, cardiac grafts). This expression of ICAM-1 in remote organs appeared to be triggered by IL-1alpha released from the graft, because (1) in situ hybridization revealed increased IL-1 mRNA within cells of the reperfused graft, including myocytes and endothelial cells; (2) ICAM-1 expression in remote organs coincided with a significant increase in serum levels of IL-1alpha after transplantation of ICAM-1 +/+ grafts; both remote organ ICAM-1 expression and IL-1alpha levels were blunted by implantation of ICAM-1 -/- grafts; and (3) remote organ ICAM-1 expression and neutrophil infiltration and IL-1 levels could be blocked by the administration of an IL-1 receptor antagonist. These data demonstrate an apparent positive-feedback loop in which local ICAM-1 and IL-1 expression leads to a mutual amplification of each other{\textquoteright}s expression within the reperfused graft, promulgating inflammatory events that are likely to be an important cause of primary cardiac graft failure. Because IL-1 receptor blockade reduces the IL-1-mediated autoinduction of IL-1, reduces the expression of ICAM-1 in both the graft and remote organs, and improves graft survival, it may provide a new and effective strategy to prevent the occurrence of primary cardiac graft failure.}, author = {Wang,C.Y. and Naka,Y. and Liao,H. and Oz,M.C. and Springer, T.A. and Gutierrez-Ramos,J-C. and Pinsky,D.J.} } @article {528816, title = {The C-C chemokine receptor CCR3 participates in stimulation of eosinophil arrest on inflammatory endothelium in shear flow}, journal = {J. Clin. Invest.}, volume = {101}, number = {9}, year = {1998}, note = {Grant HL-48675 (NIH)Reprint Status: In FileNov-4th}, pages = {2017-2024}, abstract = {Chemokines are widely hypothesized to stimulate firm adhesion of leukocytes on endothelium in shear flow. Thus far, this has been demonstrated experimentally for exogenously added chemoattractants, but not for those released by endothelium. We found that human umbilical cord endothelial cells (HUVEC) stimulated with TNF-alpha and IFN-gamma secreted eosinophil chemoattractants into the culture supernatant. This material induced transendothelial chemotaxis, stimulated eosinophil binding to purified intercellular adhesion molecule 1, and augmented binding to purified vascular cell adhesion molecule 1 in a 3-min static assay. Chemotaxis and stimulation of adhesion were abrogated completely by the pretreatment of eosinophils with an mAb to the C-C chemokine receptor 3 (CCR3). Eosinophils accumulated efficiently on HUVEC stimulated with TNF-alpha and IFN-gamma in shear flow at 1.5 dyn/cm2. CCR3 mAb slightly but significantly reduced eosinophil arrest and accumulation, by preventing development of firm adhesion by some of the tethered eosinophils, so that they detached within 30 s after the initial tethering. In the presence of mAb to the alpha4 integrin subunit, the effect of CCR3 mAb was more prominent, and approximately half of eosinophil arrest and accumulation was abolished. Inhibition by CCR3 mAb in the presence of beta2 integrin mAb was similar to that in control eosinophils. This is the first evidence that endothelial cell-derived chemokines can activate firm adhesion through alpha4 and beta2 integrins even in the presence of shear flow.}, keywords = {None}, author = {Kitayama,J. and Mackay,C.R. and Ponath,P.D. and Springer, T.A.} } @article {528191, title = {The chemokine receptor CXCR3 mediates rapid and shear-resistant adhesion-induction of effector T lymphocytes by the chemokines IP10 and Mig}, journal = {Eur. J. Immunol.}, volume = {28}, number = {3}, year = {1998}, note = {No TAS grant listedReprint Status: In File}, pages = {961-972}, abstract = {Integrin-mediated adhesion to the vascular endothelium is an essential step in leukocyte diapedesis. We show that the chemokines 10-kDa inflammatory protein (IP10) and monokine induced by IFN (Mig) induce rapid and transient adhesion of human IL-2-stimulated T lymphocytes (IL-2 T cells) to immobilized integrin ligands through their receptor CXCR3, which is selectively expressed on activated T cells. Induction of adhesion by IP10 and Mig was already observed at subnanomolar concentrations and was maximal at 5-10 nM, resulting in three- to sixfold increase in adhesion of IL-2 T cells over background. No effect was seen with resting naive/memory T cells which lack CXCR3 and migration responses to IP10 and Mig. Both chemokines are produced in human umbilical vein endothelial cells (HUVEC) upon stimulation with IFN-gamma and TNF-alpha. These chemokines induce IL-2 T cell adhesion also when captured on the surface of endothelial cells. Under conditions of flow, IL-2 T cells roll and rapidly adhere to IP10/Mig-expressing HUVEC, and anti-CXCR3 mAb treatment reduces arrest and firm adhesion. This is the first study that shows chemokine-induced adhesion in activated memory/effector T cells which represent the fraction of T cells that are selectively mobilized in inflammation. The critical role of IFN-gamma as inducer of IP10/Mig production in HUVEC indicates that these chemokines are essential mediators of effector T cell recruitment to IFN-gamma-dependent pathologies.}, author = {Piali,L. and Weber,C. and LaRosa,G. and Mackay,C.R. and Springer, T.A. and Clark-Lewis,I. and Moser,B.} } @article {529241, title = {Differential requirements for LFA-1 binding to ICAM-1 and LFA-1-mediated cell aggregation}, journal = {J. Immunol.}, volume = {160}, number = {9}, year = {1998}, note = {This work was supported by NIH grant CA31798Reprint Status: In File}, pages = {4208-4216}, abstract = {Cellular adhesion through the beta2 integrin lymphocyte function-associated Ag (LFA)-1 is a complex event involving activation, ligand binding, and cell shape changes that ultimately result in enhanced adhesion. In this report we define requirements for ligand binding and post receptor signaling by comparing two mechanisms of activation of LFA-1: 1) inside-out signaling and 2) direct activation by the beta2 Ab, CBR LFA-1/2. Our results demonstrate that activation of LFA-1 binding to ICAM-1 by CBR LFA-1/2, in contrast to inside-out signaling mechanisms, does not require protein kinase C activation or protein phosphatase 2A activity nor is it affected by agents that interfere with reorganization of the cytoskeleton. Inhibition of protein tyrosine kinase activity does not affect ICAM- binding by either mechanism of activation. However, activation by either mode does require the presence of the beta cytoplasmic domain; deletion of the C-terminal phenylalanine or the five amino acid stretch between 756-762 abolished activation of LFA-1. This, combined with the observation that intracellular energy pools must be preserved, implicates the beta cytoplasmic domain in a key energy-dependent conformational change in LFA-1 that is required to achieve enhanced ligand binding. Post ligand binding events induced by both PMA and Ab stimulation, as measured by homotypic aggregation, require protein tyrosine kinase, phosphatase, and RhoA activities. By examining both ligand binding and aggregation, we have been able to dissect the signaling components critical in the multistep process of LFA-1-mediated cellular adhesion.}, author = {Petruzzelli,L. and Maduzia,L. and Springer, T.A.} } @article {530041, title = {A dimeric crystal structure for the N-terminal two domains of ICAM-1}, journal = {Proc Natl Acad Sci USA}, volume = {95}, number = {8}, year = {1998}, note = {Reprint Status: In File}, pages = {4134-4139}, abstract = {The 3.0-A structure of a 190-residue fragment of intercellular adhesion molecule-1 (ICAM-1, CD54) reveals two tandem Ig-superfamily (IgSF) domains. Each of two independent molecules dimerizes identically with a symmetry-related molecule over a hydrophobic interface on the BED sheet of domain 1, in agreement with dimerization of ICAM-1 on the cell surface. The residues that bind to the integrin LFA-1 are well oriented for bivalent binding in the dimer, with the critical Glu-34 residues pointing away from each other on the periphery. Residues that bind to rhinovirus are in the flexible BC and FG loops at the tip of domain 1, and these and the upper half of domain 1 are well exposed in the dimer for docking to virus. By contrast, a residue important for binding to Plasmodium falciparum-infected erythrocytes is in the dimer interface. The presence of A{\textquoteright} strands in both domains 1 and 2, conserved hydrogen bonds at domain junctions, and elaborate hydrogen bond networks around the key integrin binding residues in domain 1 make these domains suited to resist tensile forces during adhesive interactions. A subdivision of the intermediate (I) set of IgSF domains is proposed in which domain 1 of ICAM-1 and previously described I set domains belong to the I1 set and domain 2 of ICAM-1, ICAM-2, and vascular cell adhesion molecule-1 belong to the I2 set.}, author = {Casasnovas,J.M. and Stehle,T. and Liu, J-H. and J.-H. Wang and Springer, T.A.} } @article {529466, title = {The domain structure of ICAM-1 and the kinetics of binding to rhinovirus}, journal = {J. Virol.}, volume = {72}, number = {7}, year = {1998}, note = {NIH grant $\#$AI31921Reprint Status: In File}, pages = {6244-6246}, abstract = {Fragments of intercellular adhesion molecule 1 (ICAM- 1) containing only the two most N terminal of its five immunoglobulin SF domains bind to rhinovirus 3 with the same affinity and kinetics as a fragment with the entire extracellular domain. The fully active two-domain fragments contain 5 or 14 more residues than a previously described fragment that is only partially active. Comparison of X-ray crystal structures show differences at the bottom of domain 2. Four different glycoforms of ICAM- 1 bind with identical kinetics.}, author = {Casasnovas,J.M. and Bickford,J.K. and Springer, T.A.} } @article {528046, title = {Effects of a monoclonal antibody to P-selectin on recovery of neonatal lamb hearts after cold cardioplegic ischemia}, journal = {Circulation}, volume = {98}, year = {1998}, note = {No TAS grant listedReprint Status: In File}, pages = {II391-II398}, abstract = {BACKGROUND: The interaction between endothelium and leukocytes plays a crucial role in ischemia-reperfusion injury. P-selectin, which is expressed on activated endothelium, mediates the first step in leukocyte adherence to the endothelium. This study examined the effects of a monoclonal antibody (mAb) against P-selectin on the recovery of cardiac function and myocardial neutrophil infiltration after ischemia.METHODS AND RESULTS: Thirteen blood-perfused, isolated neonatal lamb hearts underwent 2 hours of hypothermic cardioplegic arrest and 2 hours of reperfusion. Immediately before reperfusion, mAb to P-selectin was administered to the perfusate (15 micrograms/mL) in 6 hearts (group P-sel). In control (n = 7), the same volume of saline was added. Isovolumic left ventricular function and coronary blood flow were measured. At 2 hours after reperfusion, myocardial myeloperoxidase activity, an index of neutrophil accumulation, was assayed. At 30 minutes of reperfusion, hearts treated with mAb to P-selectin achieved significantly greater recovery of maximum developed pressure (70 +/- 4\% in control versus 77 +/- 2\% in group P-sel, P \< 0.01), maximum positive first derivative of pressure (dP/dt) (64 +/- 7\% in control versus 73 +/- 5\% in group P-sel, P \< 0.05), and maximum negative dP/dt (61 +/- 6\% in control versus 70 +/- 6\% in group P-sel, P \< 0.05) compared with control. Percent baseline of coronary blood flow was also significantly increased in group P-sel (135 +/- 40\% in control versus 205 +/- 43\% in group P-sel, P \< 0.05). Myocardial myeloperoxidase activity was significantly lower (P \< 0.05) in group P-sel (4.7 +/- 3.2) versus control (16.0 +/- 10.1). (Units are change in absorbance/min/g tissue.)CONCLUSIONS: The functional blockade of P-selectin resulted in better recovery of cardiac function and attenuated neutrophil accumulation during early reperfusion. Strategies to block P-selectin mediated neutrophil adherence may have clinical application in improving myocardial function at early reperfusion.}, author = {Nagashima,M. and Shin{\textquoteright}oka, T and Nollert,G. and Shum-Tim,D. and Hickey,P.R. and Kirchhoff,A. and Springer, T.A. and Burke,P.R. and Mayer,J.E.,Jr.} } @article {530156, title = {Experimental support for a β-propeller domain in integrin α-subunits and a calcium binding site on its lower surface}, journal = {Proc Natl Acad Sci USA}, volume = {95}, number = {9}, year = {1998}, note = {Reprint Status: In File}, pages = {4870-4875}, abstract = {Integrins are large, heterodimeric surface molecules of wide importance in cell adhesion. The N-terminal half of all integrin alpha-subunits contains seven weak sequence repeats of approximately 60 amino acids that are important in ligand binding and have been predicted to fold cooperatively into a single beta-propeller domain with seven beta-sheets. We provide evidence supporting this model with a mouse mAb to human Mac-1 (alphaM beta2, CD11b/CD18). This antibody, CBRM1/20, binds to amino acid residues that are in different repeats and are 94 residues apart in the primary structure in the loop between strands 1 and 2 of beta-sheet 5 and in the loop between strands 3 and 4 of beta-sheet 6. The 1-2 loops of beta-sheets 5-7 in integrins have EF hand-like Ca2+-binding motifs. CBRM1/20 binds to Mac-1 in the presence of Ca2+ or Sr2+ with an EC50 of 0.2 mM. Mg2+ or Mn2+ cannot substitute. Antibodies to other epitopes on the Mac-1 beta-propeller domain bind in the absence of calcium. mAb CBRM1/20 does not block ligand binding. Thus, the region on the lower surface of the beta-propeller domain to which mAb CBRM1/20 binds does not bind ligand and, furthermore, cannot bind other integrin domains, such as those of the beta-subunit.}, author = {Oxvig,C. and Springer, T.A.} } @article {529451, title = {An extracellular β-propeller module predicted in lipoprotein and scavenger receptors, tyrosine kinases, epidermal growth factor precursor, and extracellular matrix components}, journal = {J. Mol. Biol.}, volume = {283}, number = {4}, year = {1998}, note = {Supported by NIH grant HL48675Reprint Status: In File}, pages = {837-862}, abstract = {An abundant, widely dispersed, extracellular sequence repeat that contains a consensus YWTD motif is shown here to occur in groups of six contiguous repeats. Thirteen lines of evidence, including experimental and computational data, predict with p\<3x10(-9) that the repeats do not form tandem domains, but rather each group of six repeats folds into a compact beta-propeller structure. The six beta-sheets are arranged about a 6-fold pseudosymmetry axis, and each repeat contributes loops to the faces surrounding the pseudosymmetry axis. Seven different endocytic receptors that contain from one to eight YWTD beta-propeller domains act as lipoprotein, vitellogenin, and scavenger receptors. In the low density lipoprotein receptor (LDLR), the many mutations in familial hypercholesterolaemia that map to the YWTD domain can now be interpreted. In the extracellular matrix component nidogen, the YWTD domain functions to bind laminin. Three YWTD domains and interspersed fibronectin type III (FN3) domains constitute almost the entire extracellular domain of the sevenless and c-ros receptor tyrosine kinases. YWTD domains often are bounded by epidermal growth factor (EGF) modules, including in the EGF precursor itself. YWTD beta-propellers have a circular folding pattern that brings neighboring modules into close proximity, and may have important consequences for the architecture of multi-domain proteins.}, author = {Springer, T.A.} } @article {530141, title = {Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice}, journal = {Proc Natl Acad Sci USA}, volume = {95}, number = {16}, year = {1998}, note = {This work was supported by NIH grant HL-48675Reprint Status: In File}, pages = {9448-9453}, abstract = {The chemokine stromal cell-derived factor 1, SDF-1, is an important regulator of leukocyte and hematopoietic precursor migration and pre-B cell proliferation. The receptor for SDF-1, CXCR4, also functions as a coreceptor for T-tropic HIV-1 entry. We find that mice deficient for CXCR4 die perinatally and display profound defects in the hematopoietic and nervous systems. CXCR4-deficient mice have severely reduced B-lymphopoiesis, reduced myelopoiesis in fetal liver, and a virtual absence of myelopoiesis in bone marrow. However, T-lymphopoiesis is unaffected. Furthermore, the cerebellum develops abnormally with an irregular external granule cell layer, ectopically located Purkinje cells, and numerous chromophilic cell clumps of abnormally migrated granule cells within the cerebellar anlage. Identical defects are observed in mice lacking SDF-1, suggesting a monogamous relationship between CXCR4 and SDF-1. This receptor-ligand selectivity is unusual among chemokines and their receptors, as is the function in migration of nonhematopoietic cells.}, author = {Ma,Q. and Jones,D. and Borghesan,P.R. and Segal,R.A. and Nagasawa,T. and Kishimoto,T. and Bronson,R.T. and Springer, T.A.} } @article {529376, title = {Interaction of very late antigen-4 with VCAM-1 supports transendothelial chemotaxis of monocytes by facilitating lateral migration}, journal = {J. Immunol.}, volume = {161}, number = {12}, year = {1998}, note = {Supported by Deutsche Forschungsgemeinschaft grant $\#$ We-1913/2 and NIH grant $\#$CA31798Reprint Status: In File}, pages = {6825-6834}, abstract = {The transient regulation of very late antigen (VLA)-4 avidity by CC chemokines may promote chemotaxis of monocytes across VCAM-1-bearing barriers, whereas late and prolonged activation of VLA-5 may mediate subsequent localization in the extracellular matrix. We demonstrate that interactions of VLA-4 with VCAM-1, fibronectin, or a 40-kDa fragment but not a 120-kDa fragment of fibronectin supported the lateral random migration of isolated blood monocytes induced by CC chemokines, termed chemokinesis. This effect was optimal at intermediate substrate concentrations. Moreover, coimmobilization of VCAM-1 with ICAM-1 allowed better migration than ICAM-1 alone. Chemokinesis on VCAM-1 appeared to be associated with transient regulation of VLA-4 avidity by CC chemokines, given that locking VLA-4 in a high avidity state markedly inhibited migration and the locomotion rate was inversely correlated with the adhesive strength of VLA-4 to VCAM-1 following stimulation with monocyte chemoattractant protein-1. Induction of VCAM-1 expression by endothelial activation with IL-4 improved chemokinesis and lateral migration toward a monocyte chemoattractant protein-1 or a monocyte inflammatory protein-1alpha gradient on endothelium and increased transendothelial chemotaxis of monocytes by a VLA-4-dependent mechanism. In contrast, endothelial activation with IL-4 did not affect the time required for diapedesis of monocytes itself. Hence, VCAM-1 may facilitate transendothelial chemotaxis by supporting lateral migration of attached monocytes along endothelium.}, author = {Weber,C. and Springer, T.A.} } @article {530016, title = {The kinetics and shear threshold of transient and rolling interactions of L-selectin with its ligand on leukocytes}, journal = {Proc Natl Acad Sci USA}, volume = {95}, number = {20}, year = {1998}, note = {Reprint Status: NOT In File}, pages = {11631-11636}, abstract = {The kinetics of rolling and transient adhesions through selectins may depend on the kinetics and mechanical properties of the selectin:ligand bond, as well as on cellular properties including receptor-anchoring to the cell membrane and cytoskeleton. Kinetics are known to depend on the selectin and may also be ligand dependent. Here, we study the kinetics of transient and rolling interactions of leukocytes with L-selectin immobilized on a substrate. Remarkably, all properties examined are similar to those seen when the sidedness is opposite, i.e., when the L-selectin ligand is on the substrate and when the ligand is isolated from HEV rather than present on leukocytes. The similar properties include rolling velocity, a threshold shear stress above 0.4 dyn/cm2 required to support rolling, a k degreesoff of 7.0 to 6.8 s-1 for the L-selectin tether bond, and a mechanical bond length of 0.24 to 0.20 A. Our results argue against a model in which L-selectin shedding mediates rolling. Furthermore, the fast and force-resistant kinetic properties suggest that L-selectin is specialized dynamically for tethering leukocytes to vessel walls and adherent leukocytes.}, author = {Alon, R. and Chen, S. and Fuhlbrigge,R. and Puri,K.D. and Springer, T.A.} } @article {528701, title = {L-selectin ligands that are O-glycoprotease-resistant and distinct from MECA-79 antigen are sufficient for tethering and rolling of lymphocytes on human high endothelial venules}, journal = {J. Cell Biol.}, volume = {140}, number = {3}, year = {1998}, note = {Reprint Status: In File}, pages = {721-731}, abstract = {During the process of lymphocyte recirculation, lymphocytes bind via L-selectin to sulfated sialyl-Lewisx (sLex)-containing carbohydrate ligands expressed on the surface of high endothelial venules (HEV). We have examined the expression of sLex on HEV using a panel of mAbs specific for sLex and sLex-related structures, and have examined the function of different sLex-bearing structures using an in vitro assay of lymphocyte rolling on HEV. We report that three sLex mAbs, 2F3, 2H5, and CSLEX-1, previously noted to bind with high affinity to glycolipid-linked sLex, vary in their ability to stain HEV in different lymphoid tissues and bind differentially to O-linked versus N-linked sLex on glycoproteins. Treatment of tissue sections with neuraminidase abolished staining with all three mAbs but slightly increased staining with MECA-79, a mAb to a sulfation-dependent HEV-associated carbohydrate determinant. Treatment of tissue sections with O-sialoglycoprotease under conditions that removed the vast majority of MECA-79 staining, only partially reduced staining with the 2F3 and 2H5 mAbs. Using a novel rolling assay in which cells bind under flow to HEV of frozen tissue sections, we demonstrate that a pool of O-sialoglycoprotease-resistant molecules is present on HEV that is sufficient for attachment and rolling of lymphocytes via L-selectin. This interaction is not inhibited by the mAb MECA-79. Furthermore, MECA-79 mAb blocks binding to untreated sections by only 30\%, whereas the sLex mAb 2H5 blocks binding by approximately 60\% and a combination of MECA-79 and 2H5 mAb blocks binding by 75\%. We conclude that a pool of O-glycoprotease-resistant sLex-like L-selectin ligands exist on human HEV that is distinct from the mucin-associated moieties recognized by MECA-79 mAb. We postulate that these ligands may participate in lymphocyte binding to HEV.}, author = {Clark,R.A. and Fuhlbrigge,R.C. and Springer, T.A.} } @article {529941, title = {Modifying the mechanical property and shear threshold of L-selectin adhesion independently of equilibrium properties}, journal = {Nature}, volume = {392}, number = {6697}, year = {1998}, note = {no grant listedReprint Status: In File}, pages = {930-933}, abstract = {Interactions between adhesion molecules on two different cells differ from interactions between receptors and soluble ligands in that the adhesion molecule interaction (bond) is often subjected to force. It is widely assumed by cell biologists that the {\textquoteright}strength{\textquoteright} of a bond is a simple function of the affinity of one adhesion molecule for the other, whereas biophysicists suggest that bonds have {\textquoteright}mechanical properties{\textquoteright} that affect their strength. Mechanical properties are a function of the shape of the energy landscape related to bond formation and dissociation, whereas affinity is related only to the net energy change. Mechanical properties determine the amount by which the kinetics and affinity of bonds are altered by applied force. To date there has been no experimental manipulation of an adhesion molecule that has been shown to affect mechanical properties. L-selectin is an adhesion molecule that mediates lymphocyte binding to, and rolling on, high endothelial venules; these are prerequisites for the emigration of lymphocytes from the bloodstream into lymph nodes. Here we report a selective and reversible chemical modification of a mucin-like ligand that alters the mechanical properties of its bond with L-selectin. The effect of force on the rate of bond dissociation, that is, on a mechanical property, is altered, whereas there is little or no effect of the modification on the rate of bond dissociation in the absence of force. Moreover, the puzzling requirement for hydrodynamic shear flow above a threshold level for L-selectin interactions is dramatically altered.}, author = {Puri,K.D. and Chen, S. and Springer, T.A.} } @article {529026, title = {Protection from lymphoma cell metastasis in ICAM-1 mutant mice: A posthoming event}, journal = {J. Immunol.}, volume = {161}, number = {5}, year = {1998}, note = {Reprint Status: NOT In File}, pages = {2333-2338}, abstract = {It has been hypothesized that the intercellular adhesion receptors used by normal cells could also be operative in the spreading of circulating malignant cells to target organs. In the present work, we show that genetic ablation of the ICAM-1 gene confers resistance to T cell lymphoma metastasis. Following i.v. inoculation of LFA-1-expressing malignant T lymphoma cells, we found that ICAM-1-deficient mice were almost completely resistant to the development of lymphoid malignancy compared with wild-type control mice that developed lymphoid tumors in the kidneys, spleen, and liver. Histologic examinations confirmed that ICAM-1-deficient mice, in contrast to wild-type mice, had no evidence of lymphoid infiltration in these organs. The effect of ICAM-1 on T cell lymphoma metastasis was observed in two distinct strains of ICAM-1-deficient animals. Nonetheless, lymphoma cells migrated with the same efficiency to target organs in both normal and ICAM-1-deficient mice, indicating not only that ICAM-1 expression by the host is essential in lymphoma metastasis, but also that this is so at stages subsequent to homing and extravasation into target organs. These results point to posthoming events as a focus of future investigation on the control of metastasis mediated by ICAM-1.}, author = {Aoudjit,F. and Potworowski,E.F. and Springer, T.A. and St-Pierre,Y.} } @article {528401, title = {Structural specializations of immunoglobulin superfamily members for adhesion to integrins and viruses}, journal = {Immunol. Rev.}, volume = {163}, year = {1998}, note = {This work was supported by NIH grants HL48675, AI31921, and CA31798.Reprint Status: In File}, pages = {197-215}, abstract = {The circulation and migration of leukocytes are critical for immune surveillance and immune response to infection or injury. The key step of leukocyte recruitment involves the adhesion between immunoglobulin superfamily (IgSF) proteins on endothelium and integrin molecules on leukocyte surfaces. Some of the IgSF members are subverted as virus receptors. Four crystal structures of N-terminal two-domain fragments of these IgSF proteins have been determined: intercellular adhesion molecule-1 (ICAM-1), ICAM-2, vascular adhesion molecule-1 (VCAM-1), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). An acidic residue near the bottom of domain 1 plays a key role in integrin binding. For ICAM-1 and ICAM-2, this glutamic acid residue is located on a flat surface, complementary to the flat surface of the I domain of the integrin to which they bind, lymphocyte function-associated antigen-1 (LFA-1). For VCAM-1 and MAdCAM-1, the acidic residue is aspartic acid, and it resides on a protruded CD loop which may be complementary to a more pocket-like structure in the alpha 4 integrins to which they bind, which lack I domains. A number of unique structural features of this subclass of IgSF have been identified which are proposed to consolidate the domain structure to resist force during adhesion to integrins. Different mechanisms are proposed for the different CAMs to present the integrin-binding surface toward the opposing cell for adhesion, and prevent cis interaction with integrins on the same cell. Finally, CD4 and ICAM-1 are compared in the context of ligand binding and virus binding, which shows how human immunodeficiency virus and rhinovirus fit well with the distinct structural feature of their cognate receptors.}, author = {J.-H. Wang and Springer, T.A.} } @article {530321, title = {The structure of immunoglobulin superfamily domains 1 and 2 of MAdCAM-1 reveals novel features important for integrin recognition}, journal = {Structure}, volume = {6}, number = {6}, year = {1998}, note = {No specific grants mentioned in paperReprint Status: In File}, pages = {793-801}, abstract = {BACKGROUND: Mucosal addressin cell adhesion molecule 1 (MAdCAM-1) is a cell adhesion molecule that is expressed on the endothelium in mucosa, and guides the specific homing of lymphocytes into mucosal tissues. MAdCAM-1 belongs to a subclass of the immunoglobulin superfamily (IgSF), the members of which are ligands for integrins. Human MAdCAM-1 has a unique dual function compared to other members in the same subclass in that it binds both the integrin alpha4beta7, through its two IgSF domains, and a selectin expressed on leukocytes, via carbohydrate sidechains. The structure determination of the two IgSF domains and comparison to the N-terminal two-domain structures of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecules (ICAM-1 and ICAM-2) allow us to assess the molecular basis of the interactions between integrins and their preferred ligands.RESULTS: The crystal structure of a fragment containing the two IgSF domains of human MAdCAM-1 has been determined to 2.2 A resolution. The structure of MAdCAM-1 reveals two separate integrin-recognition motifs. The key integrin-binding residue, Asp42, resides in the CD loop of domain 1; a buried arginine residue (Arg70) plays a critical role in maintaining the conformation of this loop. The second binding site is associated with an unusual long D strand in domain 2. The D and E strands extend beyond the main body of the domain, forming a negatively charged beta ribbon unique to MAdCAM-1. This ribbon is located on the same face as the key aspartate residue in domain 1, consistent with evidence that it is involved in integrin binding.CONCLUSIONS: The structural comparison of MAdCAM-1 to other members of the same IgSF subclass reveals some interesting features. Firstly, MAdCAM-1, like VCAM-1, has the key integrin-binding residue located on the protruding CD loop of domain 1 and binds to an integrin that lacks an I domain. This is in contrast to ICAM-1 and ICAM-2 where the key residue is located at the end of the C strand on a flat surface and which bind to integrins that contain I domains. Secondly, architectural differences in the CD loops of MAdCAM-1 and VCAM-1 cause an 8 A shift in position of the critical aspartate residue, and may partly determine their binding preference for different integrins. Finally, the unusual charge distribution of the two-domain fragment of MAdCAM-1 is predicted to orient the molecule optimally for integrin binding on the top of its long mucin-like stalk.}, keywords = {CD MAdCAM}, author = {Tan, K. and Casasnovas,J.M. and Liu, J-H. and Briskin, M.J. and Springer, T.A. and J.-H. Wang} } @article {528536, title = {The structure of the β-propeller domain and C-terminal region of the integrin αM subunit}, journal = {J. Biol. Chem.}, volume = {273}, number = {24}, year = {1998}, note = {This work was supported by NIH grant CA31799Reprint Status: In File}, pages = {15138-15147}, abstract = {The alphaM subunit of integrin Mac-1 contains several distinct regions in its extracellular segment. The N-terminal region has been predicted to fold into a beta-propeller domain composed of seven beta-sheets each about 60 amino acid residues long, with the I-domain inserted between beta-sheets 2 and 3. The structure of the C-terminal region is unknown. We have used monoclonal antibodies (mAbs) as probes to study the dependence of the structure of different regions of the alphaM subunit on association with the beta2 subunit in the alphaM/beta2 heterodimer. All of the mAbs to the I-domain immunoprecipitated the unassociated alphaM precursor and reacted with the alphaM subunit expressed alone on the surface of COS cells. By contrast, four mAbs to the beta-propeller domain did not react with the unassociated alphaM precursor nor with the uncomplexed alphaM subunit expressed on COS cell surface. The four mAbs were mapped to three subregions in three different beta-sheets, making it unlikely that each recognized an interface between the alpha and beta subunits. These results suggest that folding of different beta-propeller subregions is coordinate and is dependent on association with the beta2 subunit. The segment C-terminal to the beta-propeller domain, residues 599-1092, was studied with nine mAbs. A subset of four mAbs that reacted with the alphaM/beta2 complex but not with the unassociated alphaM subunit were mapped to one subregion, residues 718-759, and five other mAbs that recognized both the unassociated and the complexed alphaM subunit were localized to three other subregions, residues 599-679, 820-882, and 943-1047. This suggests that much of the region C-terminal to the beta-propeller domain folds independently of association with the beta2 subunit. Our data provide new insights into how different domains in the integrin alpha and beta subunits may interact.}, author = {Lu, C. and Oxvig,C. and Springer, T.A.} } @article {528206, title = {Transendothelial chemotaxis of human α/β and γ/δ T lymphocytes to chemokines}, journal = {Eur. J. Immunol.}, volume = {28}, number = {1}, year = {1998}, note = {Reprint Status: In File}, pages = {104-113}, abstract = {Two subpopulations of human T lymphocytes expressing different antigen receptors, alpha/beta and gamma/delta, emigrate into inflamed tissues in distinctive patterns. We compared the transmigration of alpha/beta and gamma/delta T cells to C-C and C-X-C chemokines using an in vitro transendothelial chemotaxis assay. The C-C chemokines monocyte chemoattractant protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta stimulated similar, dose-dependent chemotaxis of purified gamma/delta T cells, whereas MCP-1, RANTES, and MIP-1alpha produced greater chemotaxis of purified alpha/beta T cells than MIP-1beta. In contrast, the C-X-C chemokines interleukin (IL)-8 and interferon-gamma inducible protein-10 (IP-10) did not promote chemotaxis of either alpha/beta or gamma/delta T cells. Three gamma/delta T cell clones with differing CD4 and CD8 phenotypes also migrated exclusively to C-C chemokines. Phenotypic analysis of mononuclear cells that transmigrated from an input population of unfractionated peripheral blood mononuclear cells confirmed the results with purified gamma/delta T cells. Our data demonstrate that human peripheral blood alpha/beta and gamma/delta T cells can transmigrate to MCP-1, RANTES, MIP-1alpha, and MIP-1beta, and suggest that both T lymphocyte subpopulations share the capacity to emigrate in response to C-C chemokines during inflammation.}, author = {Roth,S.J. and Diacovo,T.G. and Brenner,M.B. and Rosat,J-P. and Buccola,J. and Morita,C.T. and Springer, T.A.} } @article {529486, title = {Use of murine CXCR-4 as a second receptor by some T-cell-tropic human immunodeficiency viruses}, journal = {J. Virol.}, volume = {72}, number = {2}, year = {1998}, note = {No TAS grant listedReprint Status: NOT In File}, pages = {1652-1656}, abstract = {The human CXCR-4 molecule serves as a second receptor for primary, T-cell-tropic, and laboratory-adapted human immunodeficiency virus type 1 (HIV-1) isolates. Here we show that murine CXCR-4 can support the entry of some of these HIV-1 isolates. Differences between mouse and human CXCR-4 in the ability to function as an HIV-1 receptor are determined by sequences in the second extracellular loop of the CXCR-4 protein.}, author = {Parolin, C. and Borsetti, A. and Choe, H. and Farzan, M. and Kolchinsky, P. and Heesen,M. and Ma,Q. and Gerard, C. and Palu, G. and Dorf,M.E. and Springer, T.A. and Sodroski, J.} } @inbook {529676, title = {CDw145 workshop panel report}, booktitle = {Leucocyte Typing VI: White Cell Differentiation Antigens}, year = {1997}, note = {Reprint Status: In File}, pages = {754-755}, publisher = {Garland Publishing}, organization = {Garland Publishing}, address = {New York}, author = {Kitayama,J. and Hancock,W.W. and Springer, T.A.}, editor = {Kishimoto,T. and Kikutani,H. and Borne,A.E.G.Kr.v.d. and Goyert,S.M. and Mason,D. and Miyasaka, M. and Moretta,L. and Okumura, K. and Shaw,S. and Springer, T. and Sugamura,K. and Zola,H.} } @article {529381, title = {Characterization of lymphocyte function-associated antigen 1 (LFA-1)-deficient T cell lines: The αL and β2 subunits are interdependent for cell surface expression}, journal = {J. Immunol.}, volume = {158}, number = {1}, year = {1997}, note = {no springer grant number listedReprint Status: In File}, pages = {273-279}, abstract = {The leukocyte, or beta2, integrins are heterodimeric cell surface molecules that share a common beta subunit, and have unique alpha subunits. LFA-1 is the predominant beta2 integrin on lymphocytes, and plays an important role in many lymphocyte functions; however, most studies of the cytoplasmic regions of LFA-1 have been performed in transfected epithelial cells, such as COS, in part because no lymphoid cell lines deficient in the LFA-1 alpha subunit have been described. To address structure-function studies of beta2 integrins in relevant cell types, two T lymphoblastoid cell clones that completely lack cell surface LFA-1, J-(beta2).7 and SK-(beta2).7, derived from Jurkat and SKW3, respectively, were prepared by chemical mutagenesis and selection. Biosynthetic labeling and immunoprecipitation showed that the J-(beta2).7 clone did not translate any LFA-1 alpha subunit protein, while the SK-(beta2).7 cells did not synthesize any beta2 subunit protein. Northern blot analysis of poly(A+) RNA from these cells revealed an absence of the corresponding mRNA in each case. By transfection analysis, only the alpha subunit reconstituted LFA-1 expression in the J-(beta2).7 cells, while only the beta subunit restored cell surface LFA-1 expression in the SK-(beta2).7 cells. Functional studies with the parental cell lines, the J-(beta2).7 and SK-(beta2).7 cells, and the transfectants showed that all binding of Jurkat and SKW3 cells to purified ICAM-1 is mediated by LFA-1, and the reconstituted LFA-1 expressed by the J-(beta2).7 and SK-(beta2).7 transfected cells is regulated normally.}, keywords = {T cells}, author = {Weber,K.S.C. and York,M.R. and Springer, T.A. and Klickstein,L.B.} } @article {528856, title = {The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood}, journal = {J. Exp. Med.}, volume = {185}, number = {1}, year = {1997}, note = {HL48675Reprint Status: In File}, pages = {111-120}, abstract = {Hematopoietic progenitor cells migrate in vitro and in vivo towards a gradient of the chemotactic factor stromal cell-derived factor-1 (SDF-1) produced by stromal cells. This is the first chemoattractant reported for human CD34+ progenitor cells. Concentrations of SDF-1 that elicit chemotaxis also induce a transient elevation of cytoplasmic calcium in CD34+ cells. SDF-1-induced chemotaxis is inhibited by pertussis toxin, suggesting that its signaling in CD34+ cells is mediated by seven transmembrane receptors coupled to Gi proteins. CD34+ cells migrating to SDF-1 include cells with a more primitive (CD34+/CD38- or CD34+/DR-) phenotype as well as CD34+ cells phenotypically committed to the erythroid, lymphoid and myeloid lineages, including functional BFU-E, CFU-GM, and CFU-MIX progenitors. Chemotaxis of CD34+ cells in response to SDF-1 is increased by IL-3 in vitro and is lower in CD34+ progenitors from peripheral blood than in CD34+ progenitors from bone marrow, suggesting that an altered response to SDF-1 may be associated with CD34 progenitor mobilization.}, author = {Aiuti,A. and Webb,I.J. and Bleul,C. and Springer, T.A. and Gutierrez-Ramos,J.C.} } @article {529151, title = {Contrasting responses to multiple chemotactic stimuli in transendothelial migration: heterologous desensitization in neutrophils and augmentation of migration in eosinophils}, journal = {J. Immunol.}, volume = {158}, number = {5}, year = {1997}, note = {HL48675Reprint Status: In File}, pages = {2340-2349}, abstract = {At inflammatory sites in vivo, leukocytes may confront multiple, competing chemoattractive signals. We found significant differences between eosinophils and neutrophils in transendothelial chemotaxis to a chemoattractant diffusing from the lower chamber, when a chemoattractant that binds to another receptor is present at uniform concentration. The transendothelial migration of eosinophils to FMLP, C5a, RANTES, or MCP-3 was totally inhibited by the presence of the homologous chemoattractant, and only RANTES and MCP-3 showed mutual inhibition. C5a and to a lesser extent FMLP chemokinetically stimulated migration to RANTES and MCP-3, without stimulating random migration. Results with neutrophils contrasted. The presence of FMLP not only abrogated neutrophil transmigration to FMLP but also strongly decreased chemotaxis to C5a, IL-8, and Gro-alpha. Similarly, C5a inhibited neutrophil chemotaxis to IL-8 and Gro-alpha. IL-8 almost totally abrogated chemotaxis to Gro-alpha, but Gro-alpha only moderately inhibited chemotaxis to IL-8. Neither IL-8 nor Gro-alpha significantly inhibited transmigration to FMLP or C5a. Actin polymerization in eosinophils and neutrophils was desensitized by the same combinations of chemoattractants that desensitized chemotaxis. We conclude that eosinophils have at least three noninterfering receptor-signal transduction pathways for chemotaxis and actin polymerization. In contrast, the signaling pathways for FMLP, C5a, and IL-8/Gro-alpha in neutrophils are heterologously cross-desensitized, with a hierarchy of resistance to competing signals of FMLP \> C5a \> IL-8 \> Gro-alpha, in agreement with previous results in neutrophils on the Ca2+-mobilizing response. These results may have important implications for the behavior of these cell types in inflammatory sites.}, author = {Kitayama,J. and Carr,M.W. and Roth,S.J. and Buccola,J. and Springer, T.A.} } @article {529901, title = {The crystal structure of ICAM-2 reveals a distinctive integrin recognition surface}, journal = {Nature}, volume = {387}, number = {6630}, year = {1997}, note = {NIH grantReprint Status: NOT In File}, pages = {312-315}, abstract = {Recognition by integrin proteins on the cell surface regulates the adhesive interactions between cells and their surroundings. The structure of the {\textquoteright}I{\textquoteright} domain that is found in some but not all integrins, has been determined. However, the only integrin ligands for which structures are known, namely fibronectin and VCAM-1, are recognized by integrins that lack I domains. The intercellular adhesion molecules ICAM-1, 2 and 3 are, like VCAM-1, members of the immunoglobulin superfamily (IgSF), but they are recognized by an I domain-containing integrin, lymphocyte-function-associated antigen 1 (LFA-1, or CD11a/CD18). Here we present the crystal structure of the extracellular region of ICAM-2. The glutamic acid residue at position 37 is critical for LFA-1 binding and is proposed to coordinate the Mg2+ ion in the I domain; this Glu 37 is surrounded by a relatively flat recognition surface and lies in a beta-strand, whereas the critical aspartic acid residue in VCAM-1 and fibronectin lie in protruding loops. This finding suggests that there are differences in the architecture of recognition sites between integrins that contain or lack I domains. A bend between domains 1 and 2 of ICAM-2 and a tripod-like arrangement of N-linked glycans in the membrane-proximal region of domain 2 may be important for presenting the recognition surface to LFA-1. A model of ICAM-1 based on the ICAM-2 structure provides a framework for understanding its recognition by pathogens.}, keywords = {CD102}, author = {Casasnovas,J.M. and Springer, T.A. and Liu, J-H. and Harrison,S.C. and J.-H. Wang} } @article {529786, title = {Defining extracellular integrin α chain sites that affect cell adhesion and adhesion strengthening without altering soluble ligand binding}, journal = {Mol. Biol. Cell}, volume = {8}, number = {12}, year = {1997}, note = {This work was supported by TAS grants CA31798 and HL48675Reprint Status: In File}, pages = {2647-2657}, abstract = {It was previously shown that mutations of integrin alpha4 chain sites, within putative EF-hand-type divalent cation-binding domains, each caused a marked reduction in alpha4beta1-dependent cell adhesion. Some reports have suggested that alpha-chain "EF-hand" sites may interact directly with ligands. However, we show here that mutations of three different alpha4 "EF-hand" sites each had no effect on binding of soluble monovalent or bivalent vascular cell adhesion molecule 1 whether measured indirectly or directly. Furthermore, these mutations had minimal effect on alpha4beta1-dependent cell tethering to vascular cell adhesion molecule 1 under shear. However, EF-hand mutants did show severe impairments in cellular resistance to detachment under shear flow. Thus, mutation of integrin alpha4 "EF-hand-like" sites may impair 1) static cell adhesion and 2) adhesion strengthening under shear flow by a mechanism that does not involve alterations of initial ligand binding.}, keywords = {CD49d}, author = {Pujades,C. and Yauch,R. and Alon, R. and Masumoto,A. and Burkly,L. and Springer, T.A. and Chen, C and Lobb,R.R. and Hemler,M.E.} } @inbook {529691, title = {Endothelial cell antigens: Section report}, booktitle = {Leucocyte Typing VI: White Cell Differentiation Antigens}, year = {1997}, note = {Reprint Status: In File}, pages = {693-702}, publisher = {Garland Publishing, Inc.}, organization = {Garland Publishing, Inc.}, address = {New York and London}, author = {Springer, T.A. and Kitayama,J.}, editor = {Kishimoto,T. and Kikutani,H. and Borne,A.E.G.Kr.v.d. and Goyert,S.M. and Mason,D. and Miyasaka, M. and Moretta,L. and Okumura, K. and Shaw,S. and Springer, T. and Sugamura,K. and Zola,H.} } @inbook {529686, title = {Endothelial cell blind panel: Biochemical analysis report}, booktitle = {Leucocyte Typing VI: White Cell Differentiation Antigens}, year = {1997}, note = {Reprint Status: In File}, pages = {721-724}, publisher = {Garland Publishing, Inc.}, organization = {Garland Publishing, Inc.}, address = {New York}, author = {Kitayama,J. and Springer, T.A.}, editor = {Kishimoto,T. and Kikutani,H. and Borne,A.E.G.Kr.v.d. and Goyert,S.M. and Mason,D. and Miyasaka, M. and Moretta,L. and Okumura, K. and Shaw,S. and Springer, T.} } @inbook {529681, title = {Endothelial cell blind panel: Flow cytometric analysis report}, booktitle = {Leucocyte Typing VI: White Cell Differentiation Antigens}, year = {1997}, note = {Reprint Status: In File}, pages = {725-733}, publisher = {Garland Publishing, Inc.}, organization = {Garland Publishing, Inc.}, address = {New York}, author = {Kitayama,J. and Ryan,M. and Clark,R. and Springer, T.A.}, editor = {Kishimoto,T. and Kikutani,H. and Borne,A.E.G.Kr.v.d. and Goyert,S.M. and Mason,D. and Miyasaka, M. and Moretta,L. and Okumura, K. and Shaw,S. and Springer, T. and Sugamura,K. and Zola,H.} } @article {529261, title = {The faster kinetics of L-selectin than of E-selectin and P-selectin rolling at comparable binding strength}, journal = {J. Immunol.}, volume = {158}, number = {1}, year = {1997}, note = {no grant number listedReprint Status: In File}, pages = {405-413}, abstract = {Selectins are a family of lectins that mediate tethering and rolling of leukocytes on endothelium in vascular shear flow. To test the hypothesis that the kinetics and the strength of rolling interactions can be independently varied for different selectin:ligand pairs, we have directly compared all three selectins with regard to distinct measures of selectin-mediated interactions in shear flow: tethering, rolling velocity, and strength of rolling adhesions. At comparable site densities of E-selectin, P-selectin, and the L-selectin counter-receptor CD34, neutrophils tethered with similar efficiency and developed rolling adhesions of similar strength as measured by resistance to detachment. Under the same conditions, neutrophils rolled 7.5- to 10.5-fold faster on CD34 than on E-selectin and P-selectin. These findings suggest that the kinetics of bond dissociation and bond formation are faster for L-selectin than for E- and P-selectin. We also compared the behavior of neutrophils and lymphocytes on the same selectin. Both cell types showed comparable strength of binding to CD34; however, neutrophils rolled with faster velocities than lymphocytes.}, author = {Puri,K.D. and Finger,E.B. and Springer, T.A.} } @article {530101, title = {Folding of the conserved domain but not of flanking regions in the integrin β2 subunit requires association with the α subunit}, journal = {Proc Natl Acad Sci USA}, volume = {94}, number = {7}, year = {1997}, note = {CA31798Reprint Status: In File}, pages = {3156-3161}, abstract = {We have used immunoprecipitation with mAbs to probe folding during biosynthesis of the beta2 integrin subunit of lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) before and after association with the alphaL subunit. An evolutionarily conserved region is present in the beta2 subunit between amino acid residues 102 and 344. mAbs to one subregion before the conserved region, and two subregions after the conserved domain, immunoprecipitated both the unassociated beta{\textquoteright}2 precursor and mature alphaL/beta2 complex, suggesting portions of these subregions are folded before association with alphaL. An activating mAb to the C-terminal cysteine-rich region, KIM127, preferentially bound to the unassociated beta subunit, suggesting that it may bind to an epitope that is in an alphabeta interface in unactivated LFA-1. By contrast, mAbs to five different epitopes in the conserved region did not react with unassociated beta{\textquoteright}2 precursor, suggesting that this region folds after alphaL association and is intimately associated with the alphaL subunit in the alphaL/beta2 complex. mAbs to two different epitopes that involve the border between the conserved region and the C-terminal segment, were fully or partially reactive with the beta{\textquoteright}2 precursor, suggesting that this region is partially folded before association with alphaL. The findings suggest that the conserved region is a distinct folding and hence structural unit, and is intimately associated with the alpha subunit.}, author = {Huang, C and Lu, C. and Springer, T.A.} } @article {530196, title = {Folding of the N-terminal, ligand-binding region of integrin α-subunits into a β-propeller domain}, journal = {Proc Natl Acad Sci USA}, volume = {94}, number = {1}, year = {1997}, note = {CA31798, CA31799Reprint Status: In File}, pages = {65-72}, abstract = {The N-terminal approximately 440 aa of integrin alpha subunits contain seven sequence repeats. These are predicted here to fold into a beta-propeller domain. A homologous domain from the enzyme phosphatidylinositol phospholipase D is predicted to have the same fold. The domains contain seven four-stranded beta-sheets arranged in a torus around a pseudosymmetry axis. The trimeric G-protein beta subunit (G beta) appears to be the most closely related beta-propeller. Integrin ligands and a putative Mg2+ ion are predicted to bind to the upper face of the beta-propeller. This face binds substrates in beta-propeller enzymes and is used by the G protein beta subunit to bind the G protein alpha subunit. The integrin alpha subunit I domain, which is structurally homologous to the G protein alpha subunit, is tethered to the top of the beta-propeller domain by a hinge that may allow movement of the domains relative to one another. The Ca2+-binding motifs in integrin alpha subunits are on the lower face of the beta-propeller.}, author = {Springer, T.A.} } @article {530106, title = {Folding of the β-propeller domain of the integrin αL subunit is independent of the I domain and dependent on the β2 subunit}, journal = {Proc Natl Acad Sci USA}, volume = {94}, number = {7}, year = {1997}, note = {CA31798Reprint Status: In File}, pages = {3162-3167}, abstract = {We have studied the folding during biosynthesis of the lymphocyte function-associated antigen 1 (LFA-1) alphaL subunit using mAb to epitopes that map to seven different regions within the amino acid sequence. The N-terminal portion of alphaL is predicted to contain a beta-propeller domain, consisting of seven beta-sheets, and an I domain that is predicted to be inserted between beta-sheet 2 and beta-sheet 3 of the beta-propeller. The I domain of alphaL folds before association with the beta2 subunit, as shown by immunoprecipitation of the unassociated alphaL subunit by mAbs specific for four different sequence elements within the I domain. By contrast, the beta-propeller domain is not folded in unassociated alphaL after a chase of as long as 12 h after synthesis, but does fold upon association with beta2. This is shown with mAbs to regions of alphaL, that precede and follow the I domain in the primary structure. A mAb that maps near the junction of the C terminus of the I domain with the beta-propeller domain suggests that this region is partially folded before subunit association. The results show that the I domain and beta-propeller domains fold independently of one another, and suggest that the beta-propeller domain bears an interface for association with the beta subunit.}, author = {Huang, C and Springer, T.A.} } @article {529351, title = {Functional expression of the CXC-chemokine receptor-4/fusin on mouse microglial cells and astrocytes}, journal = {J. Immunol.}, volume = {159}, number = {2}, year = {1997}, note = {Reprint Status: In File}, pages = {905-911}, abstract = {The mRNA for the seven-transmembrane-spanning G protein-coupled receptor fusin/CXCR-4 is expressed in primary mouse astrocyte cultures and the transformed mouse microglial cell line, N9. Cell surface expression of fusin in these cells was confirmed by staining with a polyclonal anti-fusin Ab. The functional capacity of this chemokine receptor was examined by evaluating the calcium responses following stimulation of glial cells with the CXC-chemokine, stromal-derived cell factor-1alpha (SDF-1alpha). Both astrocytes and microglial cells mobilized calcium following stimulation with chemically synthesized SDF-1alpha. SDF-1alpha- and carbachol-mediated calcium responses of astrocytes were partially inhibited by treatment with pertussis toxin (PTx), suggesting receptor coupling to a combination of G alpha(i) and other G proteins. In contrast, the calcium responses of microglial cells to SDF-1alpha were completely PTx sensitive, while responses to carbachol stimulation were PTx resistant. The ability of SDF-1alpha to induce glial cell migration was also examined. Synthetic SDF-1alpha was a potent chemoattractant for mouse microglial cells at ligand concentrations of 10 to 500 ng/ml; peak responses were noted at 100 ng/ml. In contrast, astrocytes did not migrate toward a gradient of SDF-1alpha. The failure of SDF-1alpha to induce astrocyte migration was specific, as another chemokine, macrophage inflammatory protein-1alpha, triggered astrocyte chemotaxis.}, author = {Tanabe,S. and Heesen,M. and Yoshizawa,I. and Berman,M.A. and Luo,Y. and Bleul,C.C. and Springer, T.A. and Okuda,K. and Gerard,N. and Dorf,M.E.} } @article {530026, title = {The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes}, journal = {Proc Natl Acad Sci USA}, volume = {94}, number = {5}, year = {1997}, note = {HL48675Reprint Status: In File}, pages = {1925-1930}, abstract = {The chemokine receptors CXCR4 and CCR5 function as coreceptors for HIV-1 entry into CD4+ cells. During the early stages of HIV infection, viral isolates tend to use CCR5 for viral entry, while later isolates tend to use CXCR4. The pattern of expression of these chemokine receptors on T cell subsets and their regulation has important implications for AIDS pathogenesis and lymphocyte recirculation. A mAb to CXCR4, 12G5, showed partial inhibition of chemotaxis and calcium influx induced by SDF-1, the natural ligand of CXCR4. 12G5 stained predominantly the naive, unactivated CD26(low) CD45RA+ CD45R0- T lymphocyte subset of peripheral blood lymphocytes. In contrast, a mAb specific for CCR5, 5C7, stained CD26(high) CD45RA(low) CD45R0+ T lymphocytes, a subset thought to represent previously activated/memory cells. CXCR4 expression was rapidly up-regulated on peripheral blood mononuclear cells during phytohemagglutinin stimulation and interleukin 2 priming, and responsiveness to SDF-1 increased simultaneously. CCR5 expression, however, showed only a gradual increase over 12 days of culture with interleukin 2, while T cell activation with phytohemagglutinin was ineffective. Taken together, the data suggest distinct functions for the two receptors and their ligands in the migration of lymphocyte subsets through lymphoid and nonlymphoid tissues. Furthermore, the largely reciprocal expression of CXCR4 and CCR5 among peripheral blood T cells implies distinct susceptibility of T cell subsets to viral entry by T cell line-tropic versus macrophage-tropic strains during the course of HIV infection.}, author = {Bleul,C.C. and L. Wu and Hoxie,J.A. and Springer, T.A. and Mackay,C.R.} } @article {528646, title = {The kinetics of L-selectin tethers and the mechanics of selectin-mediated rolling}, journal = {J. Cell Biol.}, volume = {138}, number = {5}, year = {1997}, note = {Supported by grant $\#$ HL48675Reprint Status: In File}, pages = {1169-1180}, abstract = {Two mechanisms have been proposed for regulating rolling velocities on selectins. These are (a) the intrinsic kinetics of bond dissociation, and (b) the reactive compliance, i.e., the susceptibility of the bond dissociation reaction to applied force. To determine which of these mechanisms explains the 7.5-11.5-fold faster rolling of leukocytes on L-selectin than on E- and P-selectins, we have compared the three selectins by examining the dissociation of transient tethers. We find that the intrinsic kinetics for tether bond dissociation are 7-10-fold more rapid for L-selectin than for E- and P-selectins, and are proportional to the rolling velocities through these selectins. The durations of pauses during rolling correspond to the duration of transient tethers on low density substrates. Moreover, applied force increases dissociation kinetics less for L-selectin than for E- and P-selectins, demonstrating that reactive compliance is not responsible for the faster rolling through L-selectin. Further measurements provide a biochemical and biophysical framework for understanding the molecular basis of rolling. Displacements of tethered cells during flow reversal, and measurements of the distance between successive pauses during rolling provide estimates of the length of a tether and the length of the adhesive contact zone, and suggest that rolling occurs with as few as two tethers per contact zone. Tether bond lifetime is an exponential function of the force on the bond, and the upper limit for the tether bond spring constant is of the same order of magnitude as the estimated elastic spring constant of the lectin-EGF unit. Shear uniquely enhances the rate of L-selectin transient tether formation, and conversion of tethers to rolling adhesions, providing further understanding of the shear threshold requirement for rolling through L-selectin.}, keywords = {CD62L}, author = {Alon, R. and Chen, S. and Puri,K.D. and Finger,E.B. and Springer, T.A.} } @article {528851, title = {Neutrophil accumulation on activated, surface-adherent platelets in flow is mediated by interaction of Mac-1 with fibrinogen bound to αIIbβ3 and stimulated by platelet-activating factor}, journal = {J. Clin. Invest.}, volume = {100}, number = {8}, year = {1997}, note = {Supported by grant $\#$HL48675Reprint Status: In File}, pages = {2085-2093}, abstract = {We have studied the pathways that lead to arrest and firm adhesion of rolling PMN on activated, surface-adherent platelets. Stable arrest and adhesion strengthening of PMN on thrombin-stimulated, surface-adherent platelets in flow required distinct Ca2+- and Mg2+-dependent regions of Mac-1 (alphaMbeta2), and involved interactions of Mac-1 with fibrinogen, which was bound to platelets via alphaIIbbeta3. Mac-1 also bound to other unidentified ligands on platelets, which were not intracellular adhesion molecule-2 (ICAM-2), heparin, or heparan-sulfate proteoglycans. This was shown by inhibition with mAbs or peptides, by treatment of platelets with heparitinase, and by using platelets with defective alphaIIbbeta3 from a patient with Glanzmann thrombasthenia. Tethering of PMN on platelet ICAM-2 via LFA-1 (alphaLbeta2) was observed, which may facilitate the transition between rolling on selectins and Mac-1-dependent arrest. Arrest and adhesion strengthening was pertussis toxin sensitive and in flow was mainly induced by platelet-activating factor but not through activation of the chemokine receptor CXCR2. In stasis, spreading occurred and the CXCR2 contributed to firm adhesion.}, author = {Weber,C. and Springer, T.A.} } @article {529156, title = {P-selectin, L-selectin, and α4 integrin have distinct roles in eosinophil tethering and arrest on vascular endothelial cells under physiological flow conditions}, journal = {J. Immunol.}, volume = {159}, number = {8}, year = {1997}, note = {Supported by grant $\#$HL48675Reprint Status: In File}, pages = {3929-3939}, abstract = {The adhesive interactions of eosinophils with purified E-, P-, and L-selectins; vascular cell adhesion molecule-1 molecule; and HUVEC were examined in shear flow. Compared with neutrophils, eosinophils showed markedly less binding to E-selectin, but significantly stronger avidity for P-selectin. Both cell types showed a similar level of tethering and rolling on L-selectin. Eosinophils tethered and arrested abruptly on vascular cell adhesion molecule-1. However, some of the tethers were detached within several seconds; this was prevented by stimulation with eotaxin. Eosinophils also showed immediate arrest on HUVEC stimulated with 100 U/ml TNF-alpha for 6 h. Treatment with L-selectin mAb decreased eosinophil accumulation on the HUVEC by abrogating secondary tethers through interactions between flowing and attached eosinophils. mAb to P-selectin but not to E-selectin strongly inhibited primary tethers and accumulation of eosinophils. mAb to the integrin alpha 4 subunit inhibited arrest, induced rolling or detachment of tethered eosinophils, and resulted in partial reduction of eosinophil accumulation. mAb to the integrin beta 2 subunit had only a slight effect, whereas treatment with mAb to the integrin alpha 4 and beta 2 subunits together abolished rolling interactions as well as arrest, and thus almost totally inhibited eosinophil accumulation. Our data indicate that P-selectin, but not E-selectin, is directly involved in eosinophil tethering on inflammatory endothelium while L-selectin mainly mediates intereosinophil interaction. VLA-4 has a crucial role in eosinophil arrest, and arrest is enhanced by exposure to chemoattractants.}, author = {Kitayama,J. and Fuhlbrigge,R.C. and Puri,K.D. and Springer, T.A.} } @article {529371, title = {Role of αLβ2 integrin avidity in transendothelial chemotaxis of mononuclear cells}, journal = {J. Immunol.}, volume = {159}, number = {8}, year = {1997}, note = {Supported by grant$\#$ CA31798Reprint Status: In File}, pages = {3968-3975}, abstract = {The leukocyte integrin alpha L beta 2 (LFA-1) is important in transendothelial migration. Since it is not fully understood how LFA-1 mediates transmigration, we studied the effects of alpha L and beta 2 cytoplasmic domain mutants that alter LFA-1 adhesiveness for intercellular adhesion molecule-1. Monocyte chemotactic protein-1 (MCP-1) induced LFA-1-dependent transendothelial migration of Jurkat and J-beta 2.7 transfectants coexpressing the MCP-1 receptor CCR2B and wild-type alpha L. No transendothelial chemotaxis was observed with truncation mutants of the alpha L cytoplasmic tail, which rendered LFA-1 constitutively active or locked LFA-1 in a low avidity state unresponsive to cellular activation. Moreover, transendothelial chemotaxis of lymphoblastoid SLA transfectants was abolished by truncation of the beta 2 cytoplasmic domain, but not by mutation of its TTT motif, which is important in phorbol ester-induced adhesion. These data indicate that transmigration may require both alpha L and beta 2 cytoplasmic domains. We further show that MCP-1-induced transendothelial chemotaxis of PBMC was inhibited by sustained activation of LFA-1 with Mn2+ or a stimulatory mAb to beta 2. Dimeric soluble intercellular adhesion molecule-1 also reduced transendothelial chemotaxis of PBMC. Taken together, our data suggest that transendothelial chemotaxis of mononuclear cells may involve dynamic changes in LFA-1 avidity.}, author = {Weber,C. and Lu,C-F. and Casasnovas,J.M. and Springer, T.A.} } @article {530051, title = {Rolling and transient tethering of leukocytes on antibodies reveal specializations of selectins}, journal = {Proc Natl Acad Sci USA}, volume = {94}, number = {7}, year = {1997}, note = {HL48675Reprint Status: In File}, pages = {3172-3177}, abstract = {Antibodies immobilized on the wall of a flow chamber can support leukocyte rolling in shear flow. IgM mAb to Lewis(x) (CD15) and sialyl Lewis(x) (CD15s), which are carbohydrate antigens related to selectin ligands, plus mAb to CD48 and CD59, could mediate rolling. IgM and IgG mAb to L-selectin (CD62L), lymphocyte function-associated antigen 1 (CD11a), CD43, intercellular adhesion molecule 3 (CD50), and CD45 mediated only firm adhesion. In contrast to selectins, antibodies supported rolling only within a restricted range of site densities and wall shear stresses, outside of which firm adhesion or detachment occurred. When wall shear stress was increased, rolling velocity increased rapidly for antibodies but not for selectins. The kinetics of dissociation from the substrate of transiently tethered cells also increased more rapidly as a function of shear stress for antibodies than for selectins. These comparisons emphasize a number of remarkable features of selectins, including the lack of development of firm adhesion, and suggest that specialized molecular or cellular mechanisms must be required to explain their ability to support rolling over a wide range of environmental variables.}, author = {Chen, S. and Alon, R. and Fuhlbrigge,R.C. and Springer, T.A.} } @article {528696, title = {Tenascin supports lymphocyte rolling}, journal = {J. Cell Biol.}, volume = {137}, number = {3}, year = {1997}, note = {CA31798Reprint Status: In File}, pages = {755-765}, abstract = {Tenascin is a large extracellular matrix molecule expressed at specific sites in the adult, including immune system tissues such as the bone marrow, thymus, spleen, and T cell areas of lymph nodes. Tenascin has been reported to have both adhesive and anti-adhesive effects in static assays. We report here that tenascin supports the tethering and rolling of lymphocytes and lymphoblastic cell lines under flow conditions. Binding was calcium dependent and was not inhibited by treatment of lymphocytes with O-glycoprotease or a panel of glycosidases including neuraminidase and heparitinase but was inhibited by treatment of cells with proteinase K. Binding was to the fibrinogen-like terminal domain of tenascin as determined by antibody blocking studies and binding to recombinant tenascin proteins. When compared to rolling of the same cell type on E-selectin, rolling on tenascin was found to be smoother at all shear stresses tested, suggesting that cells formed a larger number of bonds on the tenascin substrate than on the E-selectin substrate. When protein plating densities were adjusted to give similar profiles of cell detachment under increasing shears, the density of tenascin was 8.5-fold greater than that of E-selectin. Binding to tenascin was not dependent on any molecules previously identified as tenascin receptors and is likely to involve a novel tenascin receptor on lymphocytes. We postulate that the ability of tenascin to support lymphocyte rolling may reflect its ability to support cell migration and that this interaction may be used by lymphocytes migrating through secondary lymphoid organs.}, author = {Clark,R.A. and Erickson, H. P. and Springer, T.A.} } @article {529211, title = {The α subunit cytoplasmic domain regulates the assembly and adhesiveness of integrin lymphocyte function-associated antigen-1 (LFA-1)}, journal = {J. Immunol.}, volume = {159}, number = {1}, year = {1997}, note = {Supported by grant $\#$ CA31798Reprint Status: In File}, pages = {268-278}, abstract = {The integrin LFA-1 mediates activation-dependent leukocyte adhesion. The beta subunit cytoplasmic domain has been demonstrated previously to modulate the adhesiveness of LFA-1. To investigate whether the alpha subunit cytoplasmic domain is also involved in the regulation of LFA-1-adhesive function, we stably expressed cytoplasmic domain truncated forms of the alpha subunit in a Jurkat mutant (Jurkat-beta2.7) deficient in the endogenous LFA-1 alpha subunit and in K562 cells. Clones expressing similar levels of cell surface LFA-1 were tested for their ability to bind to immobilized ICAM-1. Truncation of the alpha subunit cytoplasmic domain before, but not after, the conserved GFFKR sequence motif resulted in constitutive ICAM-1 binding of both Jurkat-beta2.7 and K562 transfectants. However, truncation after the GFFKR motif reduced sensitivity to stimulation by PMA or stimulatory Abs. Internal deletion of the GFFKR motif, or point mutations of the Gly (G), the two Phe (F), or the Arg (R) in the GFFKR motif to Ala (A) rendered LFA-1 constitutively active. Mutation of the Lys (K) did not affect LFA-1 adhesion to ICAM-1. These findings indicate that the GFFKR motif maintains the low adhesive state of LFA-1, possibly by restraining the receptor conformation. We further demonstrate that the alpha subunit cytoplasmic domain and the conserved GFFKR motif are also required for efficient formation of LFA-1 alphabeta heterodimers.}, keywords = {LFA-1}, author = {Lu, C. and Springer, T.A.} } @article {529916, title = {Adhesion through L-selectin requires a threshold hydrodynamic shear}, journal = {Nature}, volume = {379}, number = {6562}, year = {1996}, note = {Reprint Status: In File}, pages = {266-269}, abstract = {Selectins are cell adhesion molecules that bind carbohydrate ligands and promote interaction between leukocytes and the vessel wall in vascular shear flow. Selectin-ligand bonds have high mechanical strength, allowing initial tethering to the vessel wall through one or few bonds, and have fast on and off rates, permitting rolling in response to hydrodynamic drag. The L-selectin molecule on leukocytes binds to peripheral node addressin on high endothelial venules of lymph nodes to mediate leukocyte rolling and binds to a ligand on neutrophils to mediate rolling of leukocytes over one another. Here we describe a surprising mechanism for regulation of these interactions, both in vitro and in vivo. Shear above a critical threshold is required to promote and maintain rolling interactions through L-selectin, but not through E-selectin, P-selectin or VCAM-1. The shear threshold requirement for L-selectin may be physiologically important in low shear to prevent inappropriate aggregation of leukocytes and interaction with the vessel wall.}, keywords = {CD62L}, author = {Finger,E.B. and Puri,K.D. and Alon, R. and Lawrence,M.B. and von Andrian, U. H. and Springer, T.A.} } @article {528326, title = {The C-C chemokine MCP-1 modulates the avidity of β1 but not β2 integrins on T lymphocytes}, journal = {Immunity}, volume = {4}, number = {2}, year = {1996}, note = {Supported by grants HL48675 \& CA31798Reprint Status: In File}, pages = {179-187}, abstract = {The ability of chemokines, particularly MCP-1, to induce integrin-dependent binding of T lymphocytes to endothelial adhesion molecules or extracellular matrix (ECM) components was examined. MCP-1 induced significant adhesion to fibronectin (FN) and to endothelial-secreted ECM but not to purified ICAM-1 or VCAM-1, or to activated endothelium. The MCP-1-induced binding of T lymphocytes to FN was rapid, dose dependent, and resulted from activation of both VLA-4 and VLA-5. Like MCP-1, the chemokines RANTES and MIP-1 beta induced T lymphocyte binding to FN, but not to ICAM-1. We suggest therefore, that these T lymphocyte chemokines may be most important, not in initiating integrin-dependent firm adhesion of T lymphocytes to the vascular wall, but rather, in subsequent adhesive interactions during migration into tissue.}, author = {Carr,M.W. and Alon, R. and Springer, T.A.} } @article {528691, title = {CD44 and hyaluronan-dependent rolling interactions of lymphocytes on tonsillar stroma}, journal = {J. Cell Biol.}, volume = {134}, number = {4}, year = {1996}, note = {CA31798Reprint Status: NOT In File}, pages = {1075-1087}, abstract = {Little is known about how lymphocytes migrate within secondary lymphoid organs. Stromal cells and their associated reticular fibers form a network of fibers that radiate from high endothelial venules to all areas of the lymph node and may provide a scaffold for lymphocyte migration. We studied interactions of lymphocytes with cultured human tonsillar stromal cells and their extracellular matrix using shear stress to distinguish transient interactions from firm adhesion. Tonsillar lymphocytes and SKW3 T lymphoma cells tethered and rolled on monolayers of cultured tonsillar stromal cells and their matrix. A significant proportion of these rolling interactions were independent of divalent cations and were mediated by CD44 binding to hyaluronan, as shown by inhibition with mAb to CD44, soluble hyaluronan, as hyaluronidase treatment of the substrate, and O-glycoprotease treatment of the rolling cells. O-glycoprotease treatment of the substrate also blocked binding completely to stromal matrix and partially to stromal monolayers. SKW3 cells tethered and rolled on plastic-immobilized hyaluronan, confirming the specificity of this interaction. By contrast, monolayers of resting or stimulated human umbilical vein endothelial cells failed to support CD44- and hyaluronan-dependent rolling. SKW3 cells added under flow conditions to frozen sections of human tonsil bound and rolled along reticular fibers in the presence of EDTA. Rolling was blocked by either CD44 mAb or hyaluronan. We propose that lymphocytes migrating through secondary lymphoid organs may use CD44 to bind to hyaluronan immobilized on stromal cells and reticular fibers.}, keywords = {CD44}, author = {Clark,R.A. and Alon, R. and Springer, T.A.} } @article {528786, title = {Cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion: Role of neutrophil adhesion in the pathogenesis of stroke}, journal = {J. Clin. Invest.}, volume = {97}, number = {1}, year = {1996}, note = {Reprint Status: NOT In File}, pages = {209-216}, abstract = {Acute neutrophil (PMN) recruitment to postischemic cardiac or pulmonary tissue has deleterious effects in the early reperfusion period, but the mechanisms and effects of neutrophil influx in the pathogenesis of evolving stroke remain controversial. To investigate whether PMNs contribute to adverse neurologic sequelae and mortality after stroke, and to study the potential role of the leukocyte adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of stroke, we used a murine model of transient focal cerebral ischemia consisting of intraluminal middle cerebral artery occlusion for 45 min followed by 22 h of reperfusion. PMN accumulation, monitored by deposition of 111In-labeled PMNs in postischemic cerebral tissue, was increased 2.5-fold in the ipsilateral (infarcted) hemisphere compared with the contralateral (noninfarcted) hemisphere (P \< 0.01). Mice immunodepleted of neutrophils before surgery demonstrated a 3.0-fold reduction in infarct volumes (P \< 0.001), based on triphenyltetrazolium chloride staining of serial cerebral sections, improved ipsilateral cortical cerebral blood flow (measured by laser Doppler), and reduced neurological deficit compared with controls. In wild-type mice subjected to 45 min of ischemia followed by 22 h of reperfusion, ICAM-1 mRNA was increased in the ipsilateral hemisphere, with immunohistochemistry localizing increased ICAM-1 expression on cerebral microvascular endothelium. The role of ICAM-1 expression in stroke was investigated in homozygous null ICAM-1 mice (ICAM-1 -/-) in comparison with wild-type controls (ICAM-1 +/+). ICAM-1 -/- mice demonstrated a 3.7-fold reduction in infarct volume (P \< 0.005), a 35\% increase in survival (P \< 0.05), and reduced neurologic deficit compared with ICAM-1 +/+ controls. Cerebral blood flow to the infarcted hemisphere was 3.1-fold greater in ICAM-1 -/- mice compared with ICAM-1 +/+ controls (P \< 0.01), suggesting an important role for ICAM-1 in the genesis of postischemic cerebral no-reflow. Because PMN-depleted and ICAM-1-deficient mice are relatively resistant to cerebral ischemia-reperfusion injury, these studies suggest an important role for ICAM-1-mediated PMN adhesion in the pathophysiology of evolving stroke.}, keywords = {ICAM1}, author = {Connolly,E.S.,Jr. and Winfree,C.J. and Springer, T.A. and Naka,Y. and Liao,H. and Yan,S.D. and Stern,D.M. and Solomon,R.A. and Gutierrez-Ramos,J-C. and Pinsky,D.J.} } @article {529391, title = {Characterization of murine intercellular adhesion molecule-2}, journal = {J. Immunol.}, volume = {156}, number = {12}, year = {1996}, note = {CA31798Reprint Status: In File}, pages = {4909-4914}, abstract = {Rat mAbs were raised against murine intercellular adhesion molecule-2 (ICAM-2). Immune precipitation and purification reveal that the murine ICAM-2 glycoprotein is 55 kDa and is similar in size to human ICAM-2. ICAM-2 is expressed on a variety of leukocyte cell lines, including T and B lymphoma, mastocytoma, and macrophage lines. ICAM-2 is well expressed on endothelioma cell lines, and in contrast to ICAM-1, expression is not increased by inflammatory cytokines. One of the mAb to ICAM-2 partially or completely inhibits binding of cells expressing LFA-1 to purified ICAM-2, and binding of cells expressing ICAM-2 to purified LFA-1. The findings in the mouse are congruent with those in the human, suggesting functional conservation of ICAM-2 across species.}, author = {Xu, H and Bickford,J.K. and Luther,E. and Carpenito,C. and Takei,F. and Springer, T.A.} } @article {528256, title = {Cloning and chromosomal localization of a novel gene encoding a human β2-integrin α subunit}, journal = {Gene}, volume = {171}, number = {2}, year = {1996}, note = {Supported by grant $\#$CA31799Reprint Status: In File}, pages = {291-294}, abstract = {We isolated a partial genomic clone encoding ITGAD, a novel beta 2-integrin alpha subunit. The ITGAD gene is highly homologous to the three previously known alpha subunit-encoding genes, that compose the beta 2 integrin family, in deduced amino acid sequence, intron/exon structure and mapping location (chromosome 16p11).}, author = {Wong,D.A. and Davis,E.M. and LeBeau,M. and Springer, T.A.} } @article {530226, title = {Differential regulation of β1- and β2-integrin avidity by chemoattractants in eosinophils}, journal = {Proc Natl Acad Sci USA}, volume = {93}, number = {20}, year = {1996}, note = {CA31799Reprint Status: In File}, pages = {10939-10944}, abstract = {The CC chemokines regulated on activation normal T expressed and secreted (RANTES) and monocyte chemotactic protein 3 (MCP-3), and the anaphylatoxin C5a, induce activation, degranulation, chemotaxis, and transendothelial migration of eosinophils. Adhesion assays on purified ligands showed differential regulation of beta 1 and beta 2 integrin avidity in eosinophils. Adhesiveness of VLA-4 (alpha 4 beta 1, CD29/CD49d) for vascular cell adhesion molecule 1 or fibronectin was rapidly increased but subsequently reduced by RANTES, MCP-3, or C5a. The deactivation of VLA-4 lead to cell detachment, whereas phorbol 12-myristate 13-acetate induced sustained activation of VLA-4. In contrast, chemoattractants stimulated a prolonged increase in the adhesiveness of Mac-1 (alpha M beta 2, CD11b/CD18) for intercellular adhesion molecule 1. Inhibition by pertussis toxin confirmed signaling via G protein-coupled receptors. Chemoattractants induced transient, while phorbol 12-myristate 13-acetate induced sustained actin polymerization. Disruption of actin filaments by cytochalasins inhibited increases in avidity of VLA-4 but not of Mac-1. Chemoattractants did not upregulate a Mn2+-inducible beta 1 neoepitope defined by the mAb 9EG7, but induced prolonged expression of a Mac-1 activation epitope recognized by the mAb CBRM1/5. This mAb inhibited chemoattractant-stimulated adhesion of eosinophils to intercellular adhesion molecule 1. Thus, regulation of VLA-4 was dependent on the actin cytoskeleton, whereas conformational changes appeared to be crucial for activation of Mac-1. To our knowledge, this is the first demonstration that physiological agonists, such as chemoattractants, can differentially regulate the avidity of a beta 1 and a beta 2 integrin expressed on the same leukocyte.}, author = {Weber,C. and Kitayama,J. and Springer, T.A.} } @article {529091, title = {A differential role for cell shape in neutrophil tethering and rolling on endothelial selectins under flow}, journal = {J. Immunol.}, volume = {157}, number = {11}, year = {1996}, note = {CA31799Reprint Status: In File}, pages = {5085-5096}, abstract = {We investigated the role of neutrophil microvilli in interactions with E-selectin and P-selectin in hydrodynamic shear flow by disruption with cytochalasin B, hypotonic swelling, and chilling. Cytochalasin B only marginally reduced microvilli numbers (from 30 +/- 6 to 16 +/- 6 per cell perimeter, p \< 0.005) as shown by electron microscopy, completely disrupted tethering in shear flow to E-selectin and P-selectin, increased the strength of rolling adhesions on E-selectin and P-selectin, and increased cell deformability in shear flow with a likely increase in the area of cell:substrate contact. Hypoosmotic swelling markedly reduced microvilli number (to 6 +/- 5 per perimeter, p \< 0.005), almost completely inhibited tethering on E- and P-selectin, and increased the strength of rolling adhesions on P-selectin but not on E-selectin. Chilling almost completely abolished microvilli (to 3 +/- 3 per perimeter, p \< 0.005), but pseudopod-like structures were present, and had little effect on tethering in flow. Immunogold labeling of L-selectin, which is normally clustered on tips of microvilli, showed that in the absence of microvilli it remained in small clusters. Our studies show that alterations in cell morphology and viscoelasticity can have opposing effects on tethering and rolling, showing that they are independently regulatable. Furthermore, our results suggest that the association of molecules that mediate rolling with microvilli tips may be important not just to enhance presentation, but for other functions such as to promote resistance to extraction from the membrane or cooperative interactions among clustered receptors.}, author = {Finger,E.B. and Bruehl,R.E. and Bainton,D.F. and Springer, T.A.} } @article {529806, title = {Expression and polarization of intercellular adhesion molecule-1 on intestinal epithelia: Consequences for CD11b/CD18-mediated interactions with neutrophils}, journal = {Mol. Med.}, volume = {2}, number = {4}, year = {1996}, note = {CA31798,CA31799Reprint Status: In File}, pages = {489-505}, abstract = {BACKGROUND:Epithelial dysfunction and patient symptoms in inflammatory intestinal diseases such as ulcerative colitis and Crohn{\textquoteright}s disease correlate with migration of neutrophils (PMN) across the intestinal epithelium. In vitro modeling of PMN transepithelial migration has revealed distinct differences from transendothelial migration. By using polarized monolayers of human intestinal epithelia (T84), PMN transepithelial migration has been shown to be dependent on the leukocyte integrin CD11b/CD18 (Mac-1), but not on CD11a/CD18 (LFA-1). Since intercellular adhesion molecule-I (ICAM-1) is an important endothelial counterreceptor for these integrins, its expression in intestinal epithelia and role in PMN-intestinal epithelial interactions was investigated.MATERIALS AND METHODS: A panel of antibodies against different domains of ICAM-1, polarized monolayers of human intestinal epithelia (T84), and natural human colonic epithelia were used to examine the polarity of epithelial ICAM-1 surface expression and the functional role of ICAM-1 in neutrophil-intestinal epithelial adhesive interactions.RESULTS: While no surface expression of ICAM-1 was detected on unstimulated T84 cells, interferon-gamma (IFN gamma) elicited a marked expression of ICAM-1 that selectively polarized to the apical epithelial membrane. Similarly, apically restricted surface expression of ICAM-1 was detected in natural human colonic epithelium only in association with active inflammation. With or without IFN gamma pre-exposure, physiologically directed (basolateral-to-apical) transepithelial migration of PMN was unaffected by blocking monoclonal antibodies (mAbs) to ICAM-1. In contrast, PMN migration across IFN gamma-stimulated monolayers in the reverse (apical-to-basolateral) direction was inhibited by anti-ICAM-1 antibodies. Adhesion studies revealed that T84 cells adhered selectively to purified CD11b/CD18 and such adherence, with or without IFN gamma pre-exposure, was unaffected by ICAM-1 mAb. Similarly, freshly isolated epithelial cells from inflamed human intestine bound to CD11b/CD18 in an ICAM-1-independent fashion.CONCLUSIONS: These data indicate that ICAM-1 is strictly polarized in intestinal epithelia and does not represent a counterreceptor for neutrophil CD11b/CD18 during physiologically directed transmigration, but may facilitate apical membrane-PMN interactions after the arrival of PMN in the intestinal lumen.}, keywords = {ICAM1}, author = {Parkos,C.A. and Colgan,S.P. and Diamond,M.S. and Nusrat,A. and Liang,T.W. and Springer, T.A. and Madara,J.L.} } @article {528871, title = {A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1)}, journal = {Journal of Experimental Medicine}, volume = {184}, number = {3}, year = {1996}, note = {DK45104Reprint Status: NOT In File}, pages = {1101-1110}, abstract = {Chemotactic factors are postulated to direct emigration of lymphocytes from the blood stream into sites of inflammation. Members of a family of chemotactic cytokines, termed chemokines, have been shown to attract lymphocytes but efficacy, i.e., the maximal percentage of attracted cells, has been low. We have identified a highly efficacious lymphocyte chemotactic activity in the supernatants of the murine bone marrow stroma cell line MS-5 which attracts 10-fold more lymphocytes in vitro than currently described lymphocyte chemoattractants. Purification of this chemotactic activity revealed identity to stromal cell-derived factor 1 (SDF-1). SDF-1 acts on lymphocytes and monocytes but not neutrophils in vitro and is both a highly efficacious and highly potent mononuclear cell attractant in vivo. In addition, SDF-1 induces intracellular actin polymerization in lymphocytes, a process that is thought to be a prerequisite for cell motility. Since SDF-1 is expressed constitutively in a broad range of tissues it may have a role in immune surveillance and in basal extravasation of lymphocytes and monocytes rather than in inflammation.}, author = {Bleul,C.C. and Fuhlbrigge,R.C. and Casasnovas,J.M. and Aiuti,A. and Springer, T.A.} } @article {528906, title = {Interactions of human α/β and γ/δ T lymphocyte subsets in shear flow with E-selectin and P-selectin}, journal = {J. Exp. Med.}, volume = {183}, number = {3}, year = {1996}, note = {Supported by grant $\#$HL48675Reprint Status: In File}, pages = {1193-1203}, abstract = {We have compared the ability of human alpha/beta and gamma/delta T lymphocytes to adhere to selectin-bearing substrates, an interaction thought to be essential for homing and localization at sites of inflammation. Both T cell populations form rolling adhesions on E- and P-selectin substrates under physiologic flow conditions. Although equivalent to alpha/beta T cells in binding to E-selectin, gamma/delta T cells demonstrated greater ability to adhere to P-selectin that was purified or expressed on the surface of activated, adherent platelets. Under static conditions, 80\% of gamma/delta T cells and 53\% of alpha/beta T cells formed shear-resistant adhesions to P-selectin, whereas only 30\% of gamma/delta and alpha/beta T cells adhered to E-selectin. The enhance ability of gamma/delta T cells to adhere to P-selectin cannot be attributed to differences in expression of the P-selectin glycoprotein ligand (PSGL-1), as all alpha/beta T cells versus approximately 75\% of gamma/delta T cells expressed PSGL-1. Both cell populations expressed a similar percentage of the carbohydrate antigens sialyl LewisX and cutaneous lymphocyte-associated antigen. Depletion of lymphocyte populations or T cell clones bearing these oligosaccharides with the monoclonal antibody CSLEX-1 and HECA-452, respectively, resulted in a substantial reduction in adhesion to E-selectin and slight reduction in adhesion to P-selectin under flow conditions. Treatment of cells with an endopeptidase that selectively degrades O-sialomucins such as PSGL-1, abolished P-selectin but not E-selectin adhesion. Removal of terminal sialic acids with neuraminidase or protease treatment of cells abrogated cell adhesion to both selectin substrates. These results provide direct evidence for the presence of distinct E- and P-selectin ligands on T lymphocytes and suggest that gamma/delta T cells may be preferentially recruited to inflammatory sites during the early stages of an immune response when P-selectin is upregulated.}, author = {Diacovo,T. and Roth,S.J. and Morita,C.T. and Rosat,J-P. and Brenner,M.B. and Springer, T.A.} } @article {528651, title = {Interactions through L-selectin between leukocytes and adherent leukocytes nucleate rolling adhesions on selectins and VCAM-1 in shear flow}, journal = {J. Cell Biol.}, volume = {135}, number = {3}, year = {1996}, note = {HL48675Reprint Status: In File}, pages = {849-865}, abstract = {We demonstrate an additional step and a positive feedback loop in leukocyte accumulation on inflamed endothelium. Leukocytes in shear flow bind to adherent leukocytes through L-selectin/ligand interactions and subsequently bind downstream and roll on inflamed endothelium, purified E-selectin, P-selectin, L-selectin, VCAM-1, or peripheral node addressin. Thus adherent leukocytes nucleate formation of strings of rolling cells and synergistically enhance leukocyte accumulation. Neutrophils, monocytes, and activated T cell lines, but not peripheral blood T lymphocytes, tether to each other through L-selectin. L-selectin is not involved in direct binding to either E- or P-selectin and is not a major counterreceptor of endothelial selectins. Leukocyte-leukocyte tethers are more tolerant to high shear than direct tethers to endothelial selectins and, like other L-selectin-mediated interactions, require a shear threshold. Synergism between leukocyte-leukocyte and leukocyte-endothelial interactions introduces novel regulatory mechanisms in recruitment of leukocytes in inflammation.}, keywords = {VCAM-1}, author = {Alon, R. and Fuhlbrigge,R.C. and Finger,E.B. and Springer, T.A.} } @article {527786, title = {Intercellular adhesion molecule (ICAM-1) deficient mice are resistant to cerebral ischemia-reperfusion injury}, journal = {Ann. Neurol.}, volume = {39}, number = {1}, year = {1996}, note = {Supported by grant $\#$HL48675Reprint Status: In File}, pages = {618-624}, abstract = {Acute neutrophil (PMN) recruitment to postischemic cardiac or pulmonary tissue has deleterious effects in the early reperfusion period, but the mechanisms and effects of neutrophil influx in the pathogenesis of evolving stroke remain controversial. To investigate whether PMNs contribute to adverse neurologic sequelae and mortality after stroke, and to study the potential role of the leukocyte adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of stroke, we used a murine model of transient focal cerebral ischemia consisting of intraluminal middle cerebral artery occlusion for 45 min followed by 22 h of reperfusion. PMN accumulation, monitored by deposition of 111In-labeled PMNs in postischemic cerebral tissue, was increased 2.5-fold in the ipsilateral (infarcted) hemisphere compared with the contralateral (noninfarcted) hemisphere (P \< 0.01). Mice immunodepleted of neutrophils before surgery demonstrated a 3.0-fold reduction in infarct volumes (P \< 0.001), based on triphenyltetrazolium chloride staining of serial cerebral sections, improved ipsilateral cortical cerebral blood flow (measured by laser Doppler), and reduced neurological deficit compared with controls. In wild-type mice subjected to 45 min of ischemia followed by 22 h of reperfusion, ICAM-1 mRNA was increased in the ipsilateral hemisphere, with immunohistochemistry localizing increased ICAM-1 expression on cerebral microvascular endothelium. The role of ICAM-1 expression in stroke was investigated in homozygous null ICAM-1 mice (ICAM-1 -/-) in comparison with wild-type controls (ICAM-1 +/+). ICAM-1 -/- mice demonstrated a 3.7-fold reduction in infarct volume (P \< 0.005), a 35\% increase in survival (P \< 0.05), and reduced neurologic deficit compared with ICAM-1 +/+ controls. Cerebral blood flow to the infarcted hemisphere was 3.1-fold greater in ICAM-1 -/- mice compared with ICAM-1 +/+ controls (P \< 0.01), suggesting an important role for ICAM-1 in the genesis of postischemic cerebral no-reflow. Because PMN-depleted and ICAM-1-deficient mice are relatively resistant to cerebral ischemia-reperfusion injury, these studies suggest an important role for ICAM-1-mediated PMN adhesion in the pathophysiology of evolving stroke.}, author = {Soriano,S.G. and Wang,Y.F. and Lipton,S.A. and Springer, T.A. and Gutierrez-Ramos,J-C. and Hickey,P.R.} } @article {528811, title = {Intercellular adhesion molecule-1 deficient mice are protected against ischemic renal injury}, journal = {J. Clin. Invest.}, volume = {97}, number = {1}, year = {1996}, note = {Reprint Status: In File}, pages = {1056-1063}, abstract = {Studies in the rat have pointed to a role for intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of acute tubular necrosis. These studies used antibodies, which may have nonspecific effects. We report that renal ICAM-1 mRNA levels and systemic levels of the cytokines IL-1 and TNF-alpha increase 1 h after ischemia/ reperfusion in the mouse. We sought direct proof for a critical role for ICAM-1 in the pathophysiology of ischemic renal failure using mutant mice genetically deficient in ICAM-1. ICAM-1 is undetectable in mutant mice in contrast with normal mice, in which ICAM-1 is prominent in the endothelium of the vasa recta. Mutant mice are protected from acute renal ischemic injury as judged by serum creatinine, renal histology, and animal survival . Renal leukocyte infiltration, quantitated morphologically and by measuring tissue myeloperoxidase, was markedly less in ICAM-1-deficient than control mice. To evaluate whether prevention of neutrophil infiltration could be responsible for the protection observed in the mutant mice, we treated normal mice with antineutrophil serum to reduce absolute neutrophil counts to \< 100 cells/mm3. These neutrophil-depleted animals were protected against ischemic renal failure. Anti-1CAm-1 antibody protected normal mice against renal ischemic injury but did not provide additional protection to neutrophil-depleted animals. Thus, ICAM-1 is a key mediator of ischemic acute renal failure likely acting via potentiation of neutrophilendothelial interactions.}, author = {Kelly,K.J. and Williams,W.W.,Jr. and Colvin,R.B. and Meehan,S.M. and Springer, T.A. and Guti{\'e}rrez-Ramos,J-C. and Bonventre, J V} } @article {528526, title = {Localization of the binding site on intercellular adhesion molecule-3 (ICAM-3) for lymphocyte function-associated antigen-1 (LFA-1)}, journal = {J. Biol. Chem.}, volume = {271}, number = {39}, year = {1996}, note = {CA31798Reprint Status: In File}, pages = {23920-23927}, abstract = {Intercellular adhesion molecule 3 (ICAM-3; CD50) is the predominant counter-receptor on resting T cells and monocytes for the leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18), and may play an important role in the initial stages of the T cell-dependent immune response. Deletion of individual immunoglobulin superfamily (IgSF) domains of ICAM-3 and ICAM-3 IgSF domain chimeras with CD21 showed there is a single LFA-1 binding site in ICAM-3 and that IgSF domain 1 is necessary and sufficient for LFA-1 binding. Epitope mapping and functional studies performed with 17 anti-ICAM-3 monoclonal antibodies demonstrated that only some monoclonal antibodies, with epitopes wholly within domain 1 of ICAM-3, were able to block binding of ICAM-3 bearing cells to purified LFA-1, in agreement with the data obtained from the domain deletion mutants and CD21 chimeras. Analysis of a panel of 45 point mutants of domain 1 of ICAM-3 identified five residues that may contact LFA-1 as part of the binding site, Asn23, Ser25, Glu37, Phe54, and Gln75. These five residues are predicted by molecular modeling, based on the structure of vascular cell adhesion molecule 1 (VCAM-1), to cluster in two distinct locations on domain 1 of ICAM-3 on the BED face (Asn23 and Ser25) and on the C strand or CD loop (E37), the E strand (F54), and the FG loop (Q75). The residues, Asn23 and Ser25, comprise a consensus N-linked glycosylation site.}, keywords = {LFA-1}, author = {Klickstein,L.B. and York, M.B. and de Fougerolles, A.R. and Springer, T.A.} } @article {529896, title = {The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry}, journal = {Nature}, volume = {382}, number = {6594}, year = {1996}, note = {"grants from NIH- no number listed"Reprint Status: In File}, pages = {829-833}, abstract = {Chemokines are chemotactic cytokines that activate and direct the migration of leukocytes. There are two subfamilies, the CXC and the CC chemokines. We recently found that the CXC-chemokine stromal cell-derived factor-1 (SDF-1) is a highly efficacious lymphocyte chemoattractant. Chemokines act on responsive leukocyte subsets through G-protein-coupled seven-transmembrane receptors, which are also used by distinct strains of HIV-1 as cofactors for viral entry. Laboratory-adapted and some T-cell-line-tropic (T-tropic) primary viruses use the orphan chemokine receptor LESTR/fusin (also known as fusin), whereas macrophage-tropic primary HIV-1 isolates use CCR-5 and CCR-3 (refs 7-11), which are receptors for known CC chemokines. Testing of potential receptors demonstrated that SDF-1 signalled through, and hence {\textquoteright}adopted{\textquoteright}, the orphan receptor LESTR, which we therefore designate CXC-chemokine receptor-4 (CXCR-4). SDF-1 induced an increase in intracellular free Ca2+ and chemotaxis in CXCR-4-transfected cells. Because SDF-1 is a biological ligand for the HIV-1 entry cofactor LESTR, we tested whether it inhibited HIV-1. SDF-1 inhibited infection by T-tropic HIV-1 of HeLa-CD4 cells, CXCR-4 transfectants, and peripheral blood mononuclear cells (PBMCs), but did not affect CCR-5-mediated infection by macrophage-tropic (M-tropic) and dual-tropic primary HIV-1.}, keywords = {122.11}, author = {Bleul,C.C. and Farzan, M. and Choe, H. and Parolin, C. and Clark-Lewis,I. and Sodroski, J. and Springer, T.A.} } @article {528481, title = {Modulation of endothelial cell adhesion by hevin, an acidic protein associated with high endothelial venules}, journal = {J. Biol. Chem.}, volume = {271}, number = {8}, year = {1996}, note = {Supported by grant $\#$CA31798Reprint Status: NOT In File}, pages = {4511-4517}, abstract = {High endothelial venules (HEV) are specialized plump postcapillary venules in lymphoid tissues that support high levels of lymphocyte extravasation from the blood. We have recently identified a novel human transcript, expressed to high levels in HEV, that encodes a secreted, acidic protein closely related to the anti-adhesive extracellular matrix protein known as BM-40, osteonectin, and SPARC (secreted protein acidic and rich in cysteine). Here, we show that this protein, designated hevin, is associated with basal, lateral, and apical surfaces of HEV cells, and unlike MECA-79 antigen, is not expressed on the underlying basement membrane. In contrast to fibronectin or other adhesive extracellular matrix proteins, purified hevin does not support endothelial cell adhesion in vitro. Moreover, addition of soluble exogenous hevin inhibits attachment and spreading of endothelial cells on fibronectin substrates. Hevin-treated cells do not form focal adhesions and exhibit a rounded morphology. Together, these results suggest that hevin is an abundant extracellular protein that modulates high endothelial cell adhesion to the basement membrane.}, author = {Girard, J-P. and Springer, T.A.} } @article {530146, title = {Molecular cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin}, journal = {Proc Natl Acad Sci USA}, volume = {93}, number = {25}, year = {1996}, note = {no springer grant number listedReprint Status: In File}, pages = {14726-14729}, abstract = {Pre-B-cell growth-stimulating factor/ stromal cell-derived factor 1 (PBSF/SDF-1) is a member of the CXC group of chemokines that is initially identified as a bone marrow stromal cell-derived factor and as a pre-B-cell stimulatory factor. Although most chemokines are thought to be inducible inflammatory mediators, PBSF/SDF-1 is essential for perinatal viability,. B lymphopoiesis, bone marrow myelopoiesis, and cardiac ventricular septal formation, and it has chemotactic activities on resting lymphocytes and monocytes. In this paper, we have isolated a cDNA that encodes a seven transmembrane-spanning-domain receptor, designated pre-B-cell-derived chemokine receptor (PB-CKR) from a murine pre-B-cell clone, DW34. The deduced amino acid sequence has 90\% identity with that of a HUMSTSR/fusin, a human immunodeficiency virus 1 (HIV-1) entry coreceptor. However, the second extracellular region has lower identity (67\%) compared with HUMSTSR/fusin. PB-CKR is expressed during embryo genesis and in many organs and T cells of adult mice. Murine PBSF/SDF-1 induced an increase in intracellular free Ca2+ in DW34 cells and PB-CKR-transfected Chinese hamster ovary (CHO) cells, suggesting that PB-CKR is a functional receptor for murine PBSF/SDF-1. Murine PBSF/ SDF-1 also induced Ca2+ influx in fusin-transfected CHO cells. On the other hand, considering previous results that HIV-1 does not enter murine T cells that expressed human CD4, PB-CKR may not support HIV-1 infection. Thus, PB-CKR will be an important tool for functional mapping of HIV-1 entry coreceptor fusin and for understanding the function of PBSF/SDF-1 further.}, keywords = {122.11, 124.24}, author = {Nagasawa,T. and Nakajima,T. and Tachibana,K. and Iizasa,H. and Bleul,C.C. and Yoshie, O. and Matsushima, K. and Yoshida,N. and Springer, T.A. and Kishimoto,T.} } @article {527891, title = {Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the β2-integrin CD11b/CD18}, journal = {Blood}, volume = {88}, number = {1}, year = {1996}, note = {CA31799Reprint Status: NOT In File}, pages = {146-157}, abstract = {Platelets bound to thrombogenic surfaces have been shown to support activation-dependent firm adhesion of neutrophils in flow following selectin-mediated tethering and rolling. The specific receptor(s) responsible for mediating adhesion-strengthening interactions between neutrophils and platelets has not previously been identified. Furthermore, the ability of adherent platelets to support the migration of bound neutrophils has not been tested. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte binding in vascular shear flow and emigration at thrombogenic sites. Our results demonstrate that the beta 2-integrin Mac-1 (CD11b/CD18) is required for both firm attachment to and transmigration of neutrophils across surface-adherent platelets. In flow assays, neutrophils from patients with leukocyte adhesion deficiency-1 (LAD-I), which lack beta 2-integrin receptors, formed P-selectin-mediated rolling interactions, but were unable to develop firm adhesion to activated platelets, in contrast to healthy neutrophils, which developed firm adhesion within 5 to 30 seconds after initiation of rolling. Furthermore, the adhesion-strengthening interaction observed for healthy neutrophils could be specifically inhibited by monoclonal antibodies (mAbs) to Mac-1, but not to lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) or intercellular adhesion molecule-2 (ICAM-2; CD102). Further evidence for a beta 2-integrin-dependent neutrophil/platelet interaction is demonstrated by the complete inhibition of interleukin (IL)-8-induced neutrophil transmigration across platelets bound to fibronectin-coated polycarbonate filters by mAbs to Mac-1. Thus, Mac-1 is required for firm adhesion of neutrophils to activated, adherent platelets and may play an important role in promoting neutrophil accumulation on and migration across platelets deposited at sites of vascular injury.}, author = {Diacovo,T.G. and Roth,S.J. and Buccola,J.M. and Bainton,D.F. and Springer, T.A.} } @article {530286, title = {Platelet-mediated lymphocyte delivery to high endothelial venules}, journal = {Science}, volume = {273}, number = {5272}, year = {1996}, note = {HL48675Reprint Status: In File}, pages = {252-255}, abstract = {Circulating lymphocytes gain access to lymph nodes owing to their ability to initiate rolling along specialized high endothelial venules (HEVs). One mechanism of rolling involves L-selectin binding to peripheral node addressin (PNAd) on HEVs. Activated platelets are shown to bind to circulating lymphocytes and to mediate rolling in HEVs, in vivo, through another molecule, P-selectin, which also interacts with PNAd. In vitro, activated platelets enhanced tethering of lymphocytes to PNAd and sustained lymphocyte rolling, even in the absence of functional L-selectin. Thus, a platelet pathway operating through P-selectin provides a second mechanism for lymphocyte delivery to HEVs.}, author = {Diacovo,T.G. and Puri,K.D. and Warnock,R.A. and Springer, T.A. and von Andrian, U. H.} } @article {529006, title = {Quantitation of L-selectin distribution on human leukocyte microvilli by immunogold labeling and electron microscopy}, journal = {J. Histochem. Cytochem.}, volume = {44}, number = {8}, year = {1996}, note = {Supported by grant CA31799Reprint Status: In File}, pages = {835-844}, abstract = {L-Selectin is a leukocyte cell adhesion receptor that contributes to neutrophil (PMN) rolling on activated endothelium at sites of inflammation and mediates lymphocyte attachment to high endothelial venules in peripheral lymph nodes. Localization of this receptor to the tips of PMN and lymphocyte microvilli has been demonstrated. However, its distribution on these cells has not been quantified, and its localization on other leukocytes and the morphometry of microvilli on different leukocyte subpopulations have not been previously examined. In this study, PMN and mononuclear leukocytes were isolated from anticoagulated blood by dextran sedimentation and density centrifugation, fixed in 2\% paraformaldehyde and 0.05\% glutaraldehyde, immunogold-labeled for L-selectin, and embedded in Epon resin. The distribution of L-selectin was determined by counting gold particles on the plasma membrane of sectioned cells, and the surface microstructure of these cells was surveyed on two-dimensional transmission electron micrographs. On average, 78\% of PMN, 72\% of monocyte, and 71\% of lymphocyte L-selectin was observed on the microvilli, with more variance on lymphocytes than the other cell types. Typical PMN and monocyte sections had 26 microvilli, whereas typical lymphocyte sections had 23. Quantitation of the distribution of L-selectin and leukocyte surface topology offers a foundation from which to study the requirement of microvilli or microvillus-localized L-selectin for leukocyte tethering and rolling in model systems that mimic microvascular environments.}, author = {Bruehl,R.E. and Springer, T.A. and Bainton,D.F.} } @article {528426, title = {Role of intercellular adhesion molecule 1 in pathogenesis of staphylococcal arthritis and in host defense against staphylococcal bacteremia}, journal = {Infect. Immun.}, volume = {64}, number = {7}, year = {1996}, note = {Reprint Status: In File}, pages = {2804-2807}, abstract = {Intercellular adhesion molecule 1 (ICAM-1) is a member of the immunoglobulin superfamily that interacts with two integrins, LFA-1 and Mac-1. These interactions are critical for leukocyte extravasation into inflamed tissue. To assess the role of ICAM-1 expression in the pathogenesis of bacterial infection, homozygously mutant mice lacking the ICAM-1 gene were exposed to Staphylococcus aureus. Within 6 days after inoculation 50\% of the animals in the ICAM-1(-/-) group, but none of the controls, had died. Despite the high level of mortality, ICAM-1(-/-) mice developed less frequent and less severe arthritis than their wild-type littermates. In agreement, normal mice inoculated with staphylococci and administered anti-ICAM-1 antibodies exhibited a higher frequency of mortality but less severe arthritis than the controls. Our results indicate that ICAM-1 on the one hand provides protection against systemic disease but on the other hand aggravates the local disease manifestation.}, author = {Verdrengh, M. and Springer, T.A. and Gutierrez-Ramos,J-C. and Tarkowski, A.} } @article {528571, title = {A Schiff base with mildly oxidized carbohydrate ligands stabilizes L-selectin and not P-selectin or E-selectin rolling adhesions in shear flow}, journal = {J. Biol. Chem.}, volume = {271}, number = {10}, year = {1996}, note = {Supported by grants CA31799 \& HL48675Reprint Status: In File}, pages = {5404-5413}, abstract = {Selectins are a family of lectins, that mediate tethering and rolling of leukocytes on endothelium in vascular shear flow. Mild periodate oxidation of the L-selectin ligand CD34, or L-selectin ligands on leukocytes, enhanced resistance to detachment in shear and decreased rolling velocity equivalent to an 8-fold increase in ligand density, yet had little effect on the rate of tethering. Enhanced interactions were also seen with mildly oxidized sialyl Lewisa and sialyl Lewisx glycolipids. Enhancement was completely reversed by borohydride reduction, yielding a strength of interaction equivalent to that with the native ligands. No effect on the strength of P-selectin and E-selectin interactions was seen after mild oxidation of their ligands. Completeness of modification of sialic acid by mild periodate was verified with monoclonal antibody to sialyl Lewisx-related structures and resistance to neuraminidase. The addition of cyanoborohydride to leukocytes rolling through L-selectin on mildly oxidized but not native CD34 caused arrest of rolling cells and formation of EDTA-resistant bonds to the substrate, suggesting that a Schiff base was reduced. Cyanoborohydride reduction of mildly oxidized cells rolling on P-selectin and E-selectin also caused arrest and formation of EDTA-resistant bonds but with slower kinetics. These data suggest that interactions with a sialic acid aldehyde group on mildly oxidized ligands that include interconversion to a Schiff base can occur with three selectins yet only stabilize binding through the selectin with the fastest koff, L-selectin.}, author = {Puri,K.D. and Springer, T.A.} } @article {528771, title = {Sequential regulation of α4β1 and α5β1 integrin avidity by CC chemokines in monocytes: Implications for transendothelial chemotaxis}, journal = {J. Cell Biol.}, volume = {134}, year = {1996}, note = {CA31798Reprint Status: NOT In File}, pages = {1063-1073}, abstract = {Leukocyte emigration possibly requires dynamic regulation of integrin adhesiveness for endothelial and extracellular matrix ligands. Adhesion assays on purified vascular cell adhension molecule (VCAM)-1, fibronectin, and fibronectin fragments revealed distinct kinetic patterns for the regulation of very late antigen (VLA)-4 (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) avidity by the CC chemokines monocyte inflammatory protein (MIP)-1 alpha, RANTES (regulated on activation, normal T expressed and secreted), or monocyte chemoattractant protein (MCP)-1 in monocytes. CC chemokines induced early activation and subsequent deactivation of VLA-4, whereas upregulation of VLA-5 avidity occurred later and persisted. Controlled detachment assays in shear flow suggested that adhesive strength of VLA-4 for VCAM-1 or the 40-kD fragment of fibronectin (FN40) is more rapidly increased and subsequently reduced by MCP-1 than by MIP-1 alpha, and confirmed late and sustained activation of the adhesive strength of VLA-5 for the 120-kD fragment of fibronectin (FN120). Mn2+ or the stimulating beta 1 mAb TS2/16 strongly and stably enhanced monocyte binding to VCAM-1 or fibronectin, and locked beta 1 integrins in a high avidity state, which was not further modulated by CC chemokines. Mn2+ and mAb TS2/16 inhibited CC chemokine-induced transendothelial migration, particularly chemotaxis across stimulated endothelium that involved VLA-4 and VCAM-1. VLA-4 on Jurkat cells is of constitutively high avidity and interfered with migration across barriers expressing VCAM-1. Low but not high site densities of VCAM-1 or FN40 promoted, while FN120 impaired, beta 1 integrin-dependent monocyte chemotaxis to MCP-1 across filters coated with these substrates. Thus, we show that CC chemokines can differentially and selectively regulate avidity of integrins sharing common beta subunits. Transient activation and deactivation of VLA-4 may serve to facilitate transendothelial diapedesis, whereas late and prolonged activation of VLA-5 may mediate subsequent interactions with the basement membrane and extracellular matrix.}, author = {Weber,C. and Alon, R. and Moser,B. and Springer, T.A.} } @article {528736, title = {Sialylated, fucosylated ligands for L-selectin expressed on leukocytes mediate tethering and rolling adhesions in physiologic flow conditions}, journal = {J. Cell Biol.}, volume = {135}, number = {3}, year = {1996}, note = {HL48675Reprint Status: In File}, pages = {837-848}, abstract = {Interaction of leukocytes in flow with adherent leukocytes may contribute to their accumulation at sites of inflammation. Using L-selectin immobilized in a flow chamber, a model system that mimics presentation of L-selectin by adherent leukocytes, we characterize ligands for L-selectin on leukocytes and show that they mediate tethering and rolling in shear flow. We demonstrate the presence of L-selectin ligands on granulocytes, monocytes, and myeloid and lymphoid cell lines, and not on peripheral blood T lymphocytes. These ligands are calcium dependent, sensitive to protease and neuraminidase, and structurally distinct from previously described ligands for L-selectin on high endothelial venules (HEV). Differential sensitivity to O-sialo-glycoprotease provides evidence for ligand activity on both mucin-like and nonmucin-like structures. Transfection with fucosyltransferase induces expression of functional L-selectin ligands on both a lymphoid cell line and a nonhematopoietic cell line. L-selectin presented on adherent cells is also capable of supporting tethering and rolling interactions in physiologic shear flow. L-selectin ligands on leukocytes may be important in promoting leukocyte-leukocyte and subsequent leukocyte endothelial interactions in vivo, thereby enhancing leukocyte localization at sites of inflammation.}, author = {Fuhlbrigge,R.C. and Alon, R. and Puri,K.D. and Lowe, J.B. and Springer, T.A.} } @article {528726, title = {Visualization of CD2 interaction with LFA-3 and determination of the two-dimensional dissociation constant for adhesion receptors in a contact area}, journal = {J. Cell Biol.}, volume = {132}, number = {3}, year = {1996}, note = {Supported by grant $\#$CA31798Reprint Status: NOT In File}, pages = {465-474}, abstract = {Many adhesion receptors have high three-dimensional dissociation constants (Kd) for counter-receptors compared to the KdS of receptors for soluble extracellular ligands such as cytokines and hormones. Interaction of the T lymphocyte adhesion receptor CD2 with its counter-receptor, LFA-3, has a high solution-phase Kd (16 microM at 37 degrees C), yet the CD2/LFA-3 interaction serves as an effective adhesion mechanism. We have studied the interaction of CD2 with LFA-3 in the contact area between Jurkat T lymphoblasts and planar phospholipid bilayers containing purified, fluorescently labeled LFA-3. Redistribution and lateral mobility of LFA-3 were measured in contact areas as functions of the initial LFA-3 surface density and of time after contact of the cells with the bilayers. LFA-3 accumulated at sites of contact with a half-time of approximately 15 min, consistent with the previously determined kinetics of adhesion strengthening. The two-dimensional Kd for the CD2/LFA-3 interaction was 21 molecules/microns 2, which is lower than the surface densities of CD2 on T cells and LFA-3 on most target or stimulator cells. Thus, formation of CD2/LFA-3 complexes should be highly favored in physiological interactions. Comparison of the two-dimensional (membrane-bound) and three-dimensional (solution-phase) KdS suggest that cell-cell contact favors CD2/LFA-3 interaction to a greater extent than that predicted by the three-dimensional Kd and the intermembrane distance at the site of contact. LFA-3 molecules in the contact site were capable of lateral diffusion in the plane of the phospholipid bilayer and did not appear to be irreversibly trapped in the contact area, consistent with a rapid off-rate. These data provide insights into the function of low affinity interactions in adhesion.}, keywords = {CD2, LFA-3}, author = {Dustin,M.L. and Ferguson, L.M. and Chan, P.-Y. and Springer, T.A. and Golan, D.E.} } @article {553451, title = {Heterogenous glycosylation of ICAM-3 and lack of interaction with Mac-1 and p150,95.}, journal = {Eur J Immunol.}, volume = {25}, number = {4}, year = {1995}, pages = {1008-12}, abstract = {Intercellular adhesion molecule (ICAM)-1, ICAM-2, and ICAM-3 have been identified as counter-receptors for the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1). The other leukocyte integrins, Mac-1 and p150,95, also interact with ICAM-1. ICAM-1 and ICAM-3 are highly homologous, and an undefined ligand for Mac-1 is present on neutrophils where ICAM-3 is well expressed. In addition, glycosylation has been shown to affect the interaction of ICAM-1 with Mac-1. We therefore sought to characterize ICAM-3 heterogeneity and determine whether ICAM-3 was a ligand for either Mac-1 or p150,95. Despite extensive differences in N-linked glycosylation, ICAM-3 purified from lymphoid cells and from neutrophils supports adhesion of LFA-1-bearing cells equally well; however, neither supports adhesion of Mac-1 or p150,95-expressing chinese hamster ovary cell transfectants. Similarly, purified Mac-1 does not support adhesion of ICAM-2 or ICAM-3-expressing L cell transfectants. ICAM-3 on neutrophils does not participate in Mac-1-dependent homotypic aggregation. Thus, ICAM-3 is not a counter-receptor for either Mac-1 or p150,95.}, author = {de Fougerolles, A.R. and Diamond,M.S. and Springer, T.A.} } @inbook {529641, title = {Stimulation of binding of LFA-1 bearing cells to ICAM-1 and ICAM-3}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1594-1595}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Petruzzelli,L. and Maduzia,L. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529551, title = {Localization of the LFA-1 binding site on ICAM-3 (CD50) by epitope mapping and deletion mutagenesis}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1563-1566}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Klickstein,L.B. and de Fougerolles,A.R. and York,M.R. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @article {529236, title = {Activation of LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) mimicked by an antibody directed against CD18}, journal = {J. Immunol.}, volume = {155}, number = {2}, year = {1995}, note = {Reprint Status: In File}, pages = {854-866}, abstract = {The beta 2-integrin (CD18) family members bind to their ligands subsequent to activation of a number of well defined and diverse signal transduction pathways. The precise molecular changes associated with activation of the integrin family members have remained elusive. Here, we characterize a monoclonal, CBR LFA-1/2, that binds to the beta 2-subunit and is able to mimic activation induced upon stimulation by phorbol esters. The Ab induces binding of the LFA-1-expressing cell line, JY, to ICAM-1 (CD54) and ICAM-3 (CD50). Activation of binding by this Ab is independent of Fc interactions and does not occur through cross-linking at the cell surface, because the Fab fragment of the Ab is able to modulate the same effect. Stimulation of neutrophils with CBR LFA-1/2 induces binding to ICAM-1 through activation of both LFA-1 and Mac-1. Activation of Mac-1 by CBR LFA-1/2 was further confirmed by stimulation of neutrophil binding to fibrinogen, a ligand for Mac-1. CBR LFA-1/2 lowers by 10-fold the concentration of Mg2+ required to achieve maximal binding of LFA-1 to ICAM-1. It therefore appears that CBR LFA-1/2 induces a conformational change that directly increases the avidity of beta 2-integrins for ligands.}, author = {Petruzzelli,L. and Maduzia,L. and Springer, T.A.} } @article {528441, title = {Activation of natural killer cells by the mAb YTA-1 that recognizes leukocyte function-associated antigen-1}, journal = {Int. Immunol.}, volume = {7}, number = {5}, year = {1995}, note = {Reprint Status: In File}, pages = {763-769}, abstract = {The mAb YTA-1, which brightly stains CD3-CD16+ large granular lymphocytes (LGL)/natural killer (NK) cells and CD8+ T cells by immunofluorescence, is specific for leukocyte function-associated antigen (LFA)-1. Some mAbs recognizing the LFA-1 alpha chain (CD11a) or LFA-1 beta chain (CD18) inhibited the binding of YTA-1 to peripheral blood mononuclear cells. YTA-1 mAb could be chemically cross-linked to 170 and 96 kDa molecules, whose molecular weights correspond to those of LFA-1 alpha and beta respectively. YTA-1 bound to COS-7 cells co-transfected with CD11a and CD18 cDNAs, but not to untransfected cells. Reactivities of YTA-1 to K562 cells transfected with LFA-1 alpha and beta (CD11a/CD18) cDNAs and to CHO cells transfected with Mac-1 (CD11b/CD18) or p150, 95 (CD11c/CD18) cDNAs strongly suggest that YTA-1 recognizes either LFA-1 alpha or an epitope formed by a combination of LFA-1 alpha and beta. Treatment of fresh CD3-CD16+ LGL with YTA-1 augmented cytolytic activity and induced proliferation. F(ab{\textquoteright})2 fragments of YTA-1 augmented NK cytotoxicity, indicating that the NK activating signal was transmitted through LFA-1 without involvement of Fc gamma receptor III. In contrast, the other mAbs against LFA-1 could not activate NK cells. These results collectively indicate that YTA-1 recognizes a unique epitope of LFA-1, which is involved in activation of fresh NK cells.}, keywords = {ACTIVATION}, author = {Sugie, K. and Nakamura, K. and Teshigawara, K. and Diamond,M.S. and Springer, T.A. and Nakamura, Y. and Leonard, W.J. and Uchida, A. and Yodoi, J.} } @inbook {529556, title = {Adhesion structure subpanel 1, E rosetting/GPI anchor: CD2, CD48, CD55, CD58, CD99 and CDw108}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1468-1471}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Klickstein,L.B. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,C. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529516, title = {Adhesion structure subpanel 2, selectins: CD62E, CD62L, and CD62P}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1498-1499}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Diacovo,T. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529586, title = {Adhesion structure subpanel 4: CD50 (ICAM-3), CD54 (ICAM-1), and CD102 (ICAM-2)}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1542-1545}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Klickstein,L.B. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529636, title = {Adhesion structure subpanel 5, leukocyte integrins: CD11a, CD11b, CD11c, CD18}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1581-1585}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Petruzzelli,L. and Luk,J. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529651, title = {Adhesion structure subpanels 7 and 8, β3, β4, β7 integrins and novel functional antigens: CD51, CD61, CD103, and CD104}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1655-1659}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Wong,D.A. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529731, title = {Adhesion Structures: Section Report}, booktitle = {Leukocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1443-1467}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Springer, T.A. and Luther,E. and Klickstein,L.B.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @article {528496, title = {A binding interface on the I domain of lymphocyte function associated antigen-1 (LFA-1) required for specific interaction with intercellular adhesion molecule 1 (ICAM-1)}, journal = {J. Biol. Chem.}, volume = {270}, number = {32}, year = {1995}, note = {Reprint Status: In File}, pages = {19008-19016}, abstract = {Previous studies have shown that lymphocyte function-associated antigen-1 (LFA-1) molecules containing the human alpha (CD11a) and human beta (CD18) subunits but not the murine alpha and human beta subunits can bind to human intercellular adhesion molecule 1 (ICAM-1). Using human/mouse LFA-1 alpha subunit chimeras, we mapped regions required for binding to ICAM-1 N-terminal to amino acid (aa) residue 350. Ligand binding sites were mapped in greater detail by scanning this region with murine sequences from 56 down to 17 aa in length and finally by introducing single or few murine aa residue replacements into the human sequence. Replacement of two non-contiguous regions of aa residues 119-153 and 218-248 in the me domain with the corresponding mouse sequences abolished most binding to human ICAM-1, without affecting alpha beta subunit association or expression on the surface of transfected COS cells. Specific residues within the I domain found to be important were Met-140, Glu-146, Thr-243, and Ser-245. Using the recently solved structure of the Mac-1 (CD11b) I domain as a model (Lee, J.-O., Rieu, P., Arnaout, M.A., and Liddington, R. (1995) Cell 80, 631-638), these residues are shown to be located on the surface of the I domain surrounding the site to which Mg2+ is chelated, and fine a ligand binding interface. Mapping of the epitopes of a panel of mouse anti-human and rat anti-mouse monoclonal antibodies gave concordant results. Epitopes were mapped to two different regions in the N-terminal domain, four regions within the I domain, and two regions between the I domain and the EF hand-like repeats. Monoclonal antibodies to epitopes within the mid- to C-terminal portion of the I domain and the N-terminal portion of the region between the I domain and the EF hand-like repeats gave good inhibition of LFA-1-dependent homotypic aggregation with cells that express either ICAM-1 or ICAM-3 as the major LFA-1 ligand.}, keywords = {ICAM1}, author = {Huang, C and Springer, T.A.} } @article {528201, title = {C-C chemokines, but not the C-X-C chemokines Il-8 and IP-10, stimulate transendothelial chemotaxis of T lymphocytes}, journal = {Eur. J. Immunol.}, volume = {25}, number = {12}, year = {1995}, note = {Reprint Status: On Request 000000}, pages = {3482-3488}, abstract = {Eight chemokines were tested for ability to elicit transendothelial chemotaxis of unstimulated peripheral blood T lymphocytes. The C-C chemokines monocyte chemotactic protein (MCP)-2, MCP-3, RANTES, macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and, as previously described, MCP-1 induced significant, dose-dependent transendothelial chemotaxis of CD3+ T lymphocytes. In contrast, the C-X-C chemokines interleukin-8 (IL-8) and interferon-gamma inducible protein-10 (IP-10) failed to induce transendothelial chemotaxis of CD3+ T lymphocytes or T lymphocyte subsets. RANTES, MIP-1 alpha, and MIP-1 beta induced significant transendothelial chemotaxis of CD4+, CD8+, and CD45R0+ T lymphocyte subsets. Phenotyping of mononuclear cells that underwent transendothelial migration to MCP-2, MCP-3, RANTES, or MIP-1 alpha showed both monocytes and activated (CD26 high), memory-type (CD45R0+) T cells. Both CD4+ and CD8+ T lymphocytes were recruited, but not natural killer cells or significant numbers of B cells. MCP-2 was the only C-C chemokine tested that attracted a significant number of naive-type (CD45RA+) T lymphocytes. In the absence of endothelium, IL-8 but not IP-10 promoted modest but significant chemotoxis of CD3+ T lymphocytes. Our data support the hypothesis that C-C, not the C-X-C chemokines IL-8 or IP-10, promote transendothelial chemotaxis of T lymphocytes.}, author = {Roth,S.J. and Carr,M.W. and Springer, T.A.} } @inbook {529601, title = {CD102 (ICAM-2) cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1550-1552}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Klickstein,L.B. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529506, title = {CD103 (αE) cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1666-1667}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, keywords = {CD103}, author = {Cepek,K.L. and Wong,D.A. and Brenner,M.B. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529666, title = {CD104 (β4) cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1667-1668}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Wong,D.A. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L.Gilks,W. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529631, title = {CD11a cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {manuscript in fileReprint Status: In File}, pages = {1586-1587}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Petruzzelli,L. and Huang, C and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,C. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529621, title = {CD11b cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1588-1590}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Luk,J. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529626, title = {CD11c cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1590-1592}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Luk,J. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529646, title = {CD18 cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1592-1593}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Petruzzelli,L. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529561, title = {CD48 cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1472-1473}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Klickstein,L.B. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529591, title = {CD50 (ICAM-3) cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1546-1547}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Klickstein,L.B. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529656, title = {CD51 (αV) and CD51/CD61 complex cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1663-1664}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Wong,D.A. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529596, title = {CD54 (ICAM-1) cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1548-1550}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Klickstein,L.B. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529566, title = {CD55 cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: NOT In File}, pages = {1473-1474}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Klickstein,L.B. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529571, title = {CD58 cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1475-1476}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Klickstein,L.B. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529576, title = {CD59 cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1476-1477}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Klickstein,L.B. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529661, title = {CD61 (β3) cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1664-1665}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Wong,D.A. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529526, title = {CD62E (E-selectin) cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1501-1503}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Diacovo,T. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529531, title = {CD62L (L-selectin) cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1503-1504}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Diacovo,T. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529521, title = {CD62P (P-selectin) cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1500-1501}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Diacovo,T. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529581, title = {CDw108 cluster report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1477-1478}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Klickstein,L.B. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529511, title = {Characterization of the ICAM-3/LFA-1 interaction and its role in immune responses}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1557-1559}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {de Fougerolles, A.R. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @article {529401, title = {Characterization of transendothelial chemotaxis of T lymphocytes}, journal = {J. Immunol.}, volume = {188}, number = {1}, year = {1995}, note = {Reprint Status: NOT In File}, pages = {97-116}, abstract = {We have adapted a chemotaxis assay using human umbilical vein endothelial cell (HUVEC) monolayers on microporous membranes for studying lymphocyte transendothelial chemotaxis in vitro. Supernatants of peripheral blood mononuclear cells stimulated with phytohemagglutinin (PHA) were identified as an excellent source of lymphocyte chemoattractant activity. The activity in PHA supernatant typically caused 2-6\% of peripheral blood lymphocytes (PBL) to transmigrate compared to 0.1-0.3\% to media control. Checkerboard analysis demonstrated that transmigration was directional and not attributable to random locomotion. Purified T lymphocytes also underwent transendothelial chemotaxis to PHA supernatant. Using monoclonal antibodies to several human adhesion receptors, we found that the interaction between LFA-1 and ICAM-1/ICAM-2 was more important for transendothelial lymphocyte chemotaxis than the interaction between VLA-4 and VCAM-1. A monoclonal antibody to the beta 1 integrin subunit inhibited chemotaxis more than antibodies to the VLA alpha 2, alpha 3, alpha 4, or alpha 5 subunits. The transendothelial assay was used to guide purification of the lymphocyte chemoattractant activity, which we reported previously to be monocyte chemoattractant protein-1 (MCP-1) (Carr et al., Proc. Natl. Acad. Sci. USA (1994) 91, 3652). The adhesion molecules required for chemotaxis to MCP-1 were similar to those with PHA supernatant. The use of HUVEC in the assay enhances the signal-to-background ratio of chemotaxis and provides a model that is physiologically relevant to lymphocyte emigration from the bloodstream into sites of inflammation.}, author = {Roth,S.J. and Carr,M.W. and Rose,S.S. and Springer, T.A.} } @article {528336, title = {Cloning from purified high endothelial venule cells of hevin, a close relative of the antiadhesive extracellular matrix protein SPARC}, journal = {Immunity}, volume = {2}, number = {1}, year = {1995}, note = {Reprint Status: NOT In File}, pages = {113-122}, abstract = {High endothelial venules (HEV) in lymphoid tissues support high levels of lymphocyte extravasion from the blood. We purified high endothelial cells from human tonsils by immunomagnetic selection with MECA-79 MAb to construct an HEV cDNA library. Differential screening of this library using cDNA probes from HEV (plus) or flat-walled vessel (minus) endothelial cells allowed us to characterize a novel human cDNA expressed to high levels in HEV. The cDNA encodes a secreted acidic calcium-binding glycoprotein of 664 aa residues, designated hevin, exhibiting 62\% identity with the antiadhesive extracellular matrix protein SPARC, over a region of 232 aa spanning more than four fifths of the SPARC coding sequence. The primary structure and sequence of hevin and similar to SPARC-like proteins from rat and quail, called SC1 or QR1. Hevin could contribute to the induction or maintenance of features of the HEV endothelium that facilitate lymphocyte migration.}, author = {Girard, J-P. and Springer, T.A.} } @inbook {529546, title = {Domain localization and correlation with inhibition of function of workshop CD11a mAb}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1595-1597}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Huang, C and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @article {529441, title = {Expression of glycophosphatidylinositol (GPI)-anchored and non-anchored isoforms of vascular cell adhesion molecule 1 in murine stromal and endothelial cells}, journal = {J. Leukoc. Biol.}, volume = {57}, number = {1}, year = {1995}, note = {Reprint Status: In File}, pages = {168-173}, abstract = {Monoclonal antibodies to murine vascular cell adhesion molecule-1 (VCAM-1, CD106) revealed not only the expected VCAM-1 molecule with an apparent molecular weight of 100 kDa, but also a molecule with a smaller size of 46 kDa in stromal cells and stimulated endothelial cells. Peptide mapping suggested the 46 kDa and 100 kDa proteins were closely related. The 46 kDa, but not 100 kDa protein, was cleaved from the cell surface with phosphatidylinositol-specific phospholipase C (PI-PLC), showing that the 46 kDa protein was a GPI-linked molecule. The 46 kDa and 100 kDa isoforms of VCAM-1 were shown to be N-glycosylated, have similar kinetics of biosynthesis, and to be partially shed from the cell surface with a slight reduction of size. TNF-alpha induced both isoforms of VCAM-1 with a similar time course of appearance on the surface of endothelial cells. The relative amounts of the 46 kDa and 100 kDa isoforms depended on the cell type examined. The GPI-anchored isoform is functionally important, because on a cell on which it was expressed almost as well as the 100 kDa isoform, treatment with PI-PLC reduced VLA-4-dependent conjugate formation.}, keywords = {CD106, CD29, CD49d}, author = {Kinashi,T. and St.Pierre,Y. and Springer, T.A.} } @inbook {529536, title = {Expression of sialomucin CD34 by high endothelial venules (HEV) in human tonsils}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: NOT In File}, pages = {1801-1803}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Girard, J-P. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J.M. and Kishimoto,T. and Morimoto,C. and Ritz,J. and Shaw,S. and Silverstein,R.L. and Springer, T.A. and Tedder,T.F. and Todd,R.F.} } @article {529016, title = {Glycolipid ligands for selectins support leukocyte tethering and rolling under physiologic flow conditions}, journal = {J. Immunol.}, volume = {154}, number = {10}, year = {1995}, note = {Reprint Status: NOT In File}, pages = {5356-5366}, abstract = {Selectin interactions with glycolipids have been examined previously under static conditions, whereas physiologic interactions mediated by selectins take place under flow. We find that under physiologic flow conditions, sialyl Lewis(x) (sLe(x)) glycolipid and sialyl Lewisa (sLe(a)) neoglycolipid support tethering and rolling adhesions of Chinese hamster ovary (CHO) cells expressing E-selectin and lymphoid and myeloid cells expressing L-selectin. These selectin-mediated adhesions persist at the highest shear stresses that occur in postcapillary venules in vivo and occur at lower site densities than found for sLe(x) on neutrophils. The interactions are Ca(2+)-dependent and can be specifically and completely blocked with anti-selectin mAbs. Asialo nonfucosylated glycolipids are inactive, and sulfatide supports weak tethering, but not rolling, of L-selectin-expressing cells. Rolling velocities and resistance to detachment are related to the glycolipid site density and fall within the range measured for neutrophil and myeloid cell rolling on substrates containing purified selectins. These observations are the first indication that glycolipids can interact with selectins in physiologic flow conditions, and can contribute to rolling adhesions.}, author = {Alon, R. and Feizi,T. and Yuen,C-T. and Fuhlbrigge,R.C. and Springer, T.A.} } @article {528706, title = {Heparin is an adhesive ligand for the leukocyte integrin Mac-1 (CD11b/CD18)}, journal = {J. Cell Biol.}, volume = {130}, number = {6}, year = {1995}, note = {Reprint Status: In File}, pages = {1473-1482}, abstract = {Previous studies have demonstrated that the leukocyte integrin Mac-1 adheres to several cell surface and soluble ligands including intercellular adhesion molecule-1, fibrinogen, iC3b, and factor X. However, experiments with Mac-1-expressing transfectants, purified Mac-1, and mAbs to Mac-1 indicate the existence of additional ligands. In this paper, we demonstrate a direct interaction between Mac-1 and heparan sulfate glycans. Heparin affinity resins immunoprecipitate Mac-1, and neutrophils and transfectant cells that express Mac-1 bind to heparin and heparan sulfate, but not to other sulfated glycosaminoglycans. Inhibition studies with mAbs and chemically modified forms of heparin suggest the I domain as a recognition site on Mac-1 for heparin, and suggest that either N- or O-sulfation is sufficient for heparin to bind efficiently to Mac-1. Under conditions of continuous flow in which heparins and E-selectin are cosubstrates, neutrophils tether to E-selectin and form firm adhesions through a Mac-1-heparin interaction.}, author = {Diamond,M.S. and Alon, R. and Parkos,C.A. and Quinn,M.T. and Springer, T.A.} } @article {528411, title = {High endothelial venules (HEVs): Specialized endothelium for lymphocyte migration}, journal = {Immunol. Today}, volume = {16}, number = {9}, year = {1995}, note = {Reprint Status: NOT In File}, pages = {449-457}, abstract = {High endothelial venules (HEVs) are specialized postcapillary venules found in lymphoid tissues that support high levels of lymphocyte extravasation from the blood. Here, Jean-Philippe Girard and Timothy Springer highlight the unique properties of HEV endothelium, discuss the molecular mechanisms controlling HEV specialization and review evidence suggesting that HEVs could play an important role in the pathogenesis of chronic inflammatory diseases.}, author = {Girard, J-P. and Springer, T.A.} } @article {528436, title = {ICAM-1 is required for T cell proliferation but not for anergy or apoptosis induced by Staphylococcus aureus enterotoxin B in vivo}, journal = {Int. Immunol.}, volume = {7}, number = {10}, year = {1995}, note = {Reprint Status: In File}, pages = {1691-1698}, abstract = {The response of T lymphocytes to superantigens requires expression of the appropriate TCR V beta gene products as well as the establishment of cellular interactions mediated by adhesion molecules. To study the role of intercellular adhesion molecule (ICAM)-1 in the response in vivo to superantigens, we have analyzed the effects induced by the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) in mice which have been made genetically deficient in ICAM-1. SEB treatment of wild-type mice causes proliferation, deletion and anergy of the SEB-reactive V beta 8+ T cell population. Here we show that cellular interactions mediated by ICAM-1 are not essential for the induction of anergy or for the deletion of CD4+ V beta 8+ or CD8+ V beta 8+ T cells, but are required for the proliferation of these peripheral T lymphocytes. This is the first demonstration in vivo that the absence of the co-stimulatory signals provided by the interaction of ICAM-1 with its specific ligands impairs the proliferation of SEB-reactive T cells. Interestingly, our study showed that SEB-induced proliferation of CD8+ V beta 8+ T cells from lymph nodes (not from spleen) is independent of the interactions mediated by ICAM-1.}, author = {Gonzalo,J.A. and Martinez-A,C. and Springer, T.A. and Gutierrez-Ramos,J-C.} } @article {528036, title = {Ideas crystallized on immunoglobulin superfamily-integrin interactions}, journal = {Chem. Biol.}, volume = {2}, number = {10}, year = {1995}, note = {Expected publication date: October 1995Reprint Status: In File}, pages = {639-43}, abstract = {Interactions between immunoglobulin superfamily (IgSF) members and integrins are central to lymphocyte homing, leukocyte emigration into tissues at inflammatory sites, and in cell-cell interactions that lead to immune responses. Recent X-ray crystal structures reveal that the interaction of a divalent cation found in the integrin structure with an acidic residue from the IgSF partner may be important in binding.}, author = {de Fougerolles,A. and Springer, T.A.} } @inbook {529606, title = {Identification of novel GPI-anchored antigens by analysis of GPI-anchor-deficient cells with mAb in the blind panel}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1478-1481}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Klickstein,L.B. and York,M.R. and Luther,E. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {529611, title = {Identification of workshop endothelial section mAb that recognize novel glycosylphosphatidylinositol-anchored antigens}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: NOT In File}, pages = {1850-1852}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Klickstein,L.B. and York,M.R. and Luther,E. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @article {528656, title = {The integrin VLA-4 supports tethering and rolling in flow on VCAM-1}, journal = {J. Cell Biol.}, volume = {128}, number = {6}, year = {1995}, note = {Reprint Status: In File}, pages = {1243-1253}, abstract = {Selectins have previously been shown to tether a flowing leukocyte to a vessel wall and mediate rolling. Here, we report that an intergrin, VLA-4, can also support tethering and rolling. Blood T lymphocytes and alpha 4 integrin-transfected cells can tether in shear flow, and then roll, through binding of the intergrin VLA-4 to purified VCAM-1 on the wall of a flow chamber. VLA-4 transfectants showed similar tethering and rolling on TNF-stimulated endothelium. Tethering efficiency, rolling velocity, and resistance to detachment are related to VCAM-1 density. Tethering and rolling did not occur on ICAM-1, fibronectin, or fibronectin fragments, and tethering did not require integrin activation or the presence of an alpha 4 cytoplasmic domain. Arrest of rolling cells on VCAM-1 occurred spontaneously, and/or was triggered by integrin activating agents Mn2+, phorbol ester, and mAb TS2/16. These agents, and the alpha 4 cytoplasmic domain, promoted increased resistance to detachment. Together the results show that VLA-4 is a versatile integrin that can mediate tethering, rolling, and firm arrest on VCAM-1.}, keywords = {451}, author = {Alon, R. and Kassner,P.D. and Carr,M.W. and Finger,E.B. and Hemler,M.E. and Springer, T.A.} } @inbook {529671, title = {Integrin β7 pre-CD report}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1669-1670}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Wong,D.A. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @article {528451, title = {Kinetics and thermodynamics of virus binding to receptor: Studies with rhinovirus, intercellular adhesion molecule-1 (ICAM-1), and surface plasmon resonance}, journal = {J. Biol. Chem.}, volume = {270}, number = {22}, year = {1995}, note = {Reprint Status: In File}, pages = {13216-13224}, abstract = {We have studied the kinetics and thermodynamics of a virus interacting with its receptor using human rhinovirus serotype 3 (HRV3), soluble intercellular adhesion molecule-1 (ICAM-1, CD54) containing Ig superfamily domains 1-5 (sICAM-1), and surface plasmon resonance. There were two classes of binding sites for sICAM-1 on HRV3, each comprising about 50\% of the total sites, with association rate constants of 2450 +/- 300 and 134 +/- 11 M-1 s-1. These rates are low, consistent with binding to a relatively inaccessible site in the rhinovirus canyon. By contrast, three monoclonal antibodies bound to sICAM-1 with a single rate constant of 17,000-48,000 M-1 s-1. The dissociation rate constant for HRV3 was 1.7 +/- 0.1 x 10(-3) s-1, giving calculated dissociation constants of 0.7 +/- 0.1 and 12.5 +/- 1.2 microM. Agreement was good with saturation binding in solution, which showed two sites of similar abundance with KD of 0.55 +/- 0.2 and 5.7 +/- 2.0 microM. A bivalent chimera of ICAM-1 with the IgA1 Fc region bound with KD = 50 and 410 nM, showing 17-fold enhanced affinity. Lowering pH from 8.0 to 6.0 reduced affinity by approximately 50-fold, primarily by reducing the on rate. Thermodynamic measurements showed that binding of ICAM-1 to HRV3 is endothermic, by contrast to binding to monoclonal antibody. The heat that is absorbed of 3.5 and 6.3 kcal/mol for the two classes of ICAM-1 binding sites may contribute to receptor-mediated disruption of virions, which has an activation energy of about 42 kcal/mol.}, keywords = {ICAM1}, author = {Casasnovas,J.M. and Springer, T.A.} } @article {529891, title = {Lifetime of the P-selectin: carbohydrate bond and its response to tensile force in hydrodynamic flow}, journal = {Nature}, volume = {374}, number = {6522}, year = {1995}, note = {Reprint Status: In File}, pages = {539}, abstract = {Selectins tether to the blood vessel wall leukocytes that are flowing in the bloodstream and support subsequent labile rolling interactions as the leukocytes are subjected to hydrodynamic drag forces. To support this rolling, selectins have been proposed to have rapid bond association and dissociation rate constants, and special mechanical properties linking tensile forces and bond dissociation. We have visualized transient tethering and release of neutrophils in hydrodynamic flow on lipid bilayers containing densities of P-selectin below those required to support rolling. We report here that transient tethers had first-order kinetics and other characteristics suggesting a unimolecular interaction between P-selectin and its glycoprotein ligand (PSGL-1). The unstressed dissociation constant (off rate) was 1 s-1. Hydrodynamic shear stresses of up to 1.1 dyn cm-2, corresponding to a force on the bond of up to 110 pN, increased the off rate only modestly, to 3.5 s-1. The data was adequately matched by a proposed equation relating off rate to the exponential of tensile force on the bond and the bond interaction distance, and gave a bond interaction distance of 0.5 A. This distance is compatible with hydrogen and metal coordination bonds between P-selectin and PSGL-1. Fast on and off rates, together with the high tensile strength of the selectin bond, appear necessary to support rolling at physiological shear stresses.}, author = {Alon, R. and Hammer, D. A. and Springer, T.A.} } @article {527896, title = {Receptor tyrosine kinase stimulates cell-matrix adhesion by phosphatidylinositol 3 kinase and phospholipase C-γ1 pathways}, journal = {Blood}, volume = {86}, number = {6}, year = {1995}, note = {Reprint Status: In File}, pages = {2086-2090}, abstract = {Receptor tyrosine kinases are known to be important in growth and differentiation. We have recently found that c-kit, the tyrosine kinase receptor for steel factor, also regulates cell-matrix adhesion. Because Steel factor helps regulate cell migration and localization, this may be an important biologic function. Integrin adhesiveness is regulated within minutes by c-kit. The signaling pathways for tyrosine kinase stimulation of integrin adhesiveness and their relation to pathways that regulate growth and differentiation over much longer time periods remain uncharacterized. We have studied the effector pathways by which receptor tyrosine kinases regulate cell-matrix adhesion using wild-type and mutant forms of the platelet-derived growth factor (PDGF) receptor, which is closely related to c-kit. The PDGF receptor expressed in mast cells is as potent as c-kit in stimulating adhesion to fibronectin. We show that induction of adhesion is regulated through two independent pathways of phosphatidylinositol 3 kinase (PI3K) and phospholipase C-gamma 1 (PLC gamma)-protein kinase C by elimination of autophosphorylation sites required for activation of PI3K and PLC gamma or in combination with downregulation of protein kinase C or wortmannin. By contrast, a receptor mutated in both the PI3K and PLC gamma association sites can still stimulate mast cell growth, indicating a crucial role of these effector molecules in regulating adhesion rather than cell growth.}, author = {Kinashi,T. and Escobedo,J.A. and Williams,L.T. and Takatsu,K. and Springer, T.A.} } @article {529701, title = {Regulation of cell-matrix adhesion by receptor tyrosine kinases}, journal = {Leuk. Lymphoma}, volume = {18}, number = {3-4}, year = {1995}, note = {Reprint Status: In File}, pages = {203-208}, abstract = {Cell-cell and cell-matrix adhesive interactions mediated by integrins play crucial roles in leukocyte migration to inflamed tissues, and also in cell migration during embryogenesis. Much remains to be learned about the molecular mechanisms of regulation of adhesion mediated by integrins. Recently we found that steel factor and c-kit induce adhesion to fibronectin by VLA-5 in mast cells. Activation of adhesiveness is transient, and occurs at concentrations of steel factor 100-fold lower than required for growth stimulation. This suggests that regulation of adhesion is an important biological function of steel factor and c-kit. Other receptor tyrosine kinases such as the PDGF receptor can substitute for c-kit. Signaling through receptor tyrosine kinases may offer a general mechanism for the regulation of integrin avidity.}, author = {Kinashi,T. and Springer, T.A.} } @inbook {529541, title = {Report on the CD34 cluster workshop}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {840-846}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, keywords = {CD34}, author = {Greaves,M.F. and Titley,I. and Colman,S.M. and Buhring,H.-J. and Campos,L. and Castoldi,G.L. and Garrido,F. and Gaudernack,G. and Girard, J-P. and Ingles-Esteve,J. and Invernizzi,R. and Knapp,W. and Lansdorp,P.M. and Lanza,F. and Merle-Beral,H. and Parravicini,C. and Razak,K. and Ruiz-Cabello,F. and Springer, T.A. and Van Der Schoot,C.E. and Sutherland,D.R.}, editor = {Schlossman,S. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,C. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @article {528176, title = {Rolling of lymphocytes and neutrophils on peripheral node addressin and subsequent arrest on ICAM-1 in shear flow}, journal = {Eur. J. Immunol.}, volume = {25}, number = {4}, year = {1995}, note = {Reprint Status: In File}, pages = {1025-1031}, abstract = {We studied leukocyte interactions in shear flow with peripheral lymph node addressin (PNAd), a mixture of glycoproteins expressed on high endothelial venules (HEV) that is required for lymphocyte homing and has been shown to contain a ligand for L-selectin. T lymphocytes and neutrophils tether and roll on plastic-immobilized PNAd and E-selectin at 1.8 dyn/cm2 wall shear stress, but fail to interact with immobilized ICAM-1, a ligand for LFA-1 and Mac-1, at the same flow rate. Cells roll faster on PNAd than on P-selectin or E-selectin. L-selectin mAb inhibit T lymphocyte and neutrophil tethering to PNAd, but do not inhibit T lymphocyte tethering to purified E-selectin. If allowed to interact with ICAM-1 under static conditions, phorbol ester-treated T lymphocytes, but not resting T lymphocytes, are able to form stationary adhesions that withstand the detachment force generated by 36 dyn/cm2 wall shear stress. In contrast, a wall shear stress of 7.3 dyn/cm2 detaches 50\% of resting T lymphocytes bound to PNAd. Incubating T lymphocytes on PNAd and ICAM-1 does not result in adhesion strengthening, suggesting that adhesion through PNAd by L-selectin does not stimulate lymphocyte LFA-1 avidity for ICAM-1. Chemoattractant stimulation of neutrophils or phorbol ester stimulation of lymphoblasts rolling on coimmobilized PNAd and ICAM-1 results in rapid arrest and firm sticking, extending the model of sequential selectin-mediated rolling and subsequent integrin-mediated firm arrest to lymphocytes and ligands expressed on HEV.}, author = {Lawrence,M.B. and Berg,E.L. and Butcher,E.C. and Springer, T.A.} } @article {528756, title = {Sialomucin CD34 is the major L-selectin ligand in human tonsil high endothelial venules}, journal = {J. Cell Biol.}, volume = {131}, number = {1}, year = {1995}, note = {Reprint Status: In File}, pages = {261-270}, abstract = {Peripheral node addressin (PNAd) is a complex mixture of glycoproteins with L-selectin ligand activity that functions in lymphocyte homing. We have investigated the contribution of the sialomucin CD34 relative to other components of PNAd in lymphocyte tethering and rolling in in vitro laminar flow assays. PNAd was isolated with MECA-79 mAb-Sepharose from tonsillar stroma, and the CD34 component (PNAd,CD34+) and CD34-negative component (PNAd,CD34-) separated on CD34 mAb-Sepharose. Lymphocytes on the PNAd,CD34- fraction tether less efficiently, roll faster and are less resistant to shear detachment than on PNAd. The PNAd,CD34+ fraction constitutes about half the total functional activity. These studies show that CD34 is a major functional component of PNAd. Ligand activity in both the PNAd,CD34+ and PNAd,CD34- fractions is expressed on mucin-like domains, as shown with O-sialoglycoprotease. The CD34 component of PNAd has about four times higher tethering efficiency than total tonsillar CD34. CD34 from spleen shows no lymphocyte tethering. Although less efficient than the PNAd,CD34+ fraction from tonsil, CD34 from the KG1a hematopoietic cell line is functionally active as an L-selectin ligand despite lack of reactivity with MECA-79 mAb, which binds to a sulfation-dependent epitope. All four forms of CD34 are active in binding to E-selectin. KG1a CD34 but not spleen CD34 are active as L-selectin ligands, yet both lack MECA-79 reactivity and possess E-selectin ligand activity. This suggests that L-selectin ligands and E-selectin ligands differ in more respects than presence of the MECA-79 epitope.}, author = {Puri,K.D. and Finger,E.B. and Gaudernack,G. and Springer, T.A.} } @inbook {528291, title = {Signals on endothelium for lymphocyte recirculation and leukocyte emigration: The area code paradigm}, booktitle = {The Harvey Lectures, Series 89}, year = {1995}, pages = {53-103}, publisher = {Wiley-Liss, Inc.}, organization = {Wiley-Liss, Inc.}, address = {New York}, author = {Springer, T.A.} } @article {529781, title = {Specialized functional properties of the integrin α4 cytoplasmic domain}, journal = {Mol. Biol. Cell}, volume = {6}, number = {6}, year = {1995}, note = {Reprint Status: In File}, pages = {661-74}, abstract = {For functional studies of the integrin alpha 4 cytoplasmic domain, we have expressed the following in K562 and Chinese hamster ovary (CHO) cells: 1) wild-type alpha 4 (called X4C4), 2) two chimeric forms of alpha 4 (called X4C2 and X4C5) that contain the cytoplasmic domains of alpha 2 and alpha 5, respectively, and 3) alpha 4 with no cytoplasmic domain (X4C0). Cytoplasmic domain exchange had no effect on VLA-4-dependent static cell adhesion or tethering to VCAM-1 in conditions of shear flow. However, the presence of the alpha 2 or alpha 5 tails markedly enhanced VLA-4-dependent K562 cells spreading (X4C2 \> X4C5 \> X4C4 \> X4C0), increased localization of VLA-4 into focal adhesion-like complexes in CHO cells (X4C2 \> X4C5 \> X4C4), and strengthened CHO and K562 cell resistance to detachment from VCAM-1 in conditions of shear flow (X4C2 \> X4C5 \> X4C4 \> X4C0). Conversely, the alpha 4 tail supported greater VLA-4-dependent haptotactic and chemotactic cell migration. In the absence of any alpha tail (i.e., X4C0), robust focal adhesions were observed, even though cell spreading and adhesion strengthening were minimal. Thus, such focal adhesions may have relatively little functional importance, and should not be compared with focal adhesions formed when alpha tails are present. Together, these results indicate that all three alpha-chain tails exert defined positive effects (compared with no tail at all), but suggest that the alpha 4 cytoplasmic domain may be specialized to engage in weaker cytoskeletal interactions, leading to diminished focal adhesion formation, cell spreading, and adhesion strengthening, while augmenting cell migration and facilitating rolling under shear flow. These properties of the alpha 4 tail are consistent with the role of alpha 4 integrins on highly motile lymphocytes, monocytes, and eosinophils.}, keywords = {CD49d}, author = {Kassner,P.D. and Alon, R. and Springer, T.A. and Hemler,M.E.} } @inbook {529616, title = {Subunit specificity and epitope mapping of Mac-1 and p150,95 mAb using chimeric CD11b x CD11c transfectants}, booktitle = {Leucocyte Typing V: White Cell Differentiation Antigens}, year = {1995}, note = {Reprint Status: In File}, pages = {1599-1601}, publisher = {Oxford University Press}, organization = {Oxford University Press}, address = {New York}, author = {Luk,J. and Luther,E. and Diamond,M.S. and Springer, T.A.}, editor = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J. and Kishimoto,T. and Morimoto,T. and Ritz,J. and Shaw,S. and Silverstein,R. and Springer, T. and Tedder,T. and Todd,R.} } @inbook {527836, title = {Traffic signals on endothelium for leukocytes in health, inflammation, and atherosclerosis}, booktitle = {Atherosclerosis and coronary artery disease}, year = {1995}, note = {Reprint Status: NOT In File}, pages = {511-537}, publisher = {Lippincott-Raven Publishers}, organization = {Lippincott-Raven Publishers}, address = {Philadelphia}, author = {Springer, T.A. and Cybulsky,M.I.}, editor = {Fuster,V. and Ross,R. and Topol,E.J.} } @article {527816, title = {Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration}, journal = {Annu. Rev. Physiol.}, volume = {57}, year = {1995}, note = {Reprint Status: In File}, pages = {827-872}, author = {Springer, T.A.} } @article {527931, title = {Adhesion molecules in hematopoietic cells}, journal = {Blood Cells}, volume = {20}, number = {1}, year = {1994}, note = {Reprint Status: In File}, pages = {25-44}, abstract = {Interaction with stromal cells is known to be crucial for growth and differentiation of hematopoietic cells. To characterize adhesion molecules involved in this interaction, we examined adhesion of a panel of lymphoid, myeloid, and mast cell lines with stromal cells. We found that very late antigen-4 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1) were major adhesion molecules in lymphoid and myeloid cells, whereas myeloma cells adhered to stromal cells through hyaluronate. We investigated regulation of VLA-4 during differentiation of myeloid cells using a neutrophil precursor cell line, L-G3. Differentiation of neutrophils induced by granulocyte colony-stimulating factor was accompanied with down-regulation of VLA-4. Induced L-G3 cells adhered to stromal cells in proportion to the expression of VLA-4. Mast cells used two mechanisms to adhere to fibroblasts and stromal cells. They adhered to fibronectin through VLA-5 when stimulated with steel factor and also directly to membrane-anchored steel factor through c-kit.}, author = {Kinashi,T. and Springer, T.A.} } @article {529296, title = {CD antigens 1993}, journal = {J. Immunol.}, volume = {152}, number = {1}, year = {1994}, pages = {98-9}, author = {Schlossman,S.F. and Boumsell,L. and Gilks,W. and Harlan,J.M. and Kishimoto,T. and Morimoto,C. and Ritz,J. and Shaw,S. and Silverstein,R.L. and Springer, T.A. and Tedder,T.F. and Todd,R.F.} } @article {529421, title = {Changes in subcellular localization and surface expression of L-selectin, alkaline phosphatase, and Mac-1 in human neutrophils during stimulation with inflammatory mediators}, journal = {J. Leukoc. Biol.}, volume = {56}, number = {1}, year = {1994}, note = {Reprint Status: In File}, pages = {80-87}, abstract = {The localization of the adhesion protein L-selectin in human neutrophils was determined by subcellular fractionation and immunoelectron microscopy and compared with the localization of Mac-1 (alpha m beta 2) and alkaline phosphatase, the marker for secretory vesicles. L-selectin was found to be localized exclusively on the plasma membrane of unstimulated cells and also of stimulated cells, although markedly diminished. This was in contrast to Mac-1, which was also localized in secretory vesicles and in specific/gelatinase granules as shown previously [Sengel{\o}v, H., et al. J. Clin. Invest. (1993) 92, 1467-1476]. Stimulation of neutrophils with inflammatory mediators such as tumor necrosis factor (TNF), platelet-activating factor (PAF), or f-Met-Leu-Phe (fMLP), induced parallel up-regulation of the surface membrane content of alkaline phosphatase and Mac-1 and down-regulation of L-selectin, as evidenced by flow cytometry. Preimbedding immunoelectron microscopy confirmed that L-selectin was present mainly on tips of microvilli in unstimulated cells and showed that alkaline phosphatase and Mac-1 were randomly distributed on the surface membrane of fMLP-stimulated cells. These studies indicate that the transition of neutrophils from L-selectin-presenting cells to Mac-1-presenting cells induced by inflammatory mediators is mediated by incorporation of secretory vesicle membrane, rich in Mac-1 and devoid of L-selectin, into the plasma membrane.}, keywords = {CD62L}, author = {Borregaard,N. and Kjeldsen,L. and Sengelov,H. and Diamond,M.S. and Springer, T.A. and Anderson,H.C. and Kishimoto,T.K. and Bainton,D.F.} } @article {528891, title = {Characterization of the function of ICAM-3 and comparison to ICAM-1 and ICAM-2 in immune responses}, journal = {J. Exp. Med.}, volume = {179}, number = {2}, year = {1994}, note = {Reprint Status: NOT In File}, pages = {619-629}, abstract = {We have characterized the immunobiology of the interaction of intercellular adhesion molecule 3 (ICAM-3; CD50) with its counter-receptor, leukocyte function-associated antigen 1 (LFA-1; CD11a/CD18). Purified ICAM-3 supported LFA-1-dependent adhesion in a temperature- and cation-dependent manner. Activation of cells bearing LFA-1 increased adhesiveness for ICAM-3 in parallel to adhesiveness for ICAM-1. Although CBR-IC3/1 monoclonal antibody (mAb) blocked adhesion of cells to purified LFA-1, when tested alone, neither CBR-IC3/1 nor five novel ICAM-3 mAbs characterized here blocked adhesion of cells to purified ICAM-3 or homotypic adhesion. Two ICAM-3 mAbs, CBR-IC3/1 and CBR-IC3/2, were required to block LFA-1-dependent adhesion to purified ICAM-3- or LFA-1-dependent, ICAM-1-, ICAM-2-independent homotypic adhesion of lymphoid cell lines. Two ICAM-3 mAbs, CBR-IC3/1 and CBR-IC3/6, induced LFA-1-independent aggregation that was temperature and divalent cation dependent and was completely inhibited by ICAM-3 mAb, CBR-IC3/2, recognizing a distinct epitope. Purified ICAM-3 provided a costimulatory signal for proliferation of resting T lymphocytes. mAb to ICAM-3, together with mAbs to ICAM-1 and ICAM-2, inhibited peripheral blood lymphocyte proliferation in response to phytohemagglutinin, allogeneic stimulator cells, and specific antigen. Inhibition was almost complete and to the same level as with mAb to LFA-1, suggesting the most functionally important, and possibly all, of the ligands for LFA-1 have been defined.}, keywords = {CD50}, author = {de Fougerolles, A.R. and Qin,X. and Springer, T.A.} } @article {529416, title = {Cloning and expression of the chicken CD18 cDNA}, journal = {J. Leukoc. Biol.}, volume = {55}, number = {4}, year = {1994}, note = {Reprint Status: In File}, pages = {501-506}, abstract = {The leukocyte integrins play a critical role in a number of cellular adhesive interactions during the immune response. We describe here the isolation and characterization of the chicken beta 2 (CD18) subunit, common to the leukocyte integrin family. The deduced 748-amino-acid sequence reveals a transmembrane protein with 65\% and 64\% identity with its human and murine homologues, respectively. The chicken beta 2 can associate on the cell surface with the human alpha subunit of LFA-1 and yields a hybrid molecule capable of binding to purified ICAM-1 and ICAM-3.}, keywords = {CD18}, author = {Bilsland,C.A.G. and Springer, T.A.} } @article {528661, title = {Distinct cell surface ligands mediate T lymphocyte attachment and rolling on P- and E-selectin under physiological flow}, journal = {J. Cell Biol.}, volume = {127}, number = {5}, year = {1994}, note = {Reprint Status: In File}, pages = {1485-1495}, abstract = {Memory T lymphocytes extravasate at sites of inflammation, but the mechanisms employed by these cells to initiate contact and tethering with endothelium are incompletely understood. An important part of leukocyte extravasation is the initiation of rolling adhesions on endothelial selectins; such events have been studied in monocytes and neutrophils but not lymphocytes. In this study, the potential of T lymphocytes to adhere and roll on endothelial selectins in vitro was investigated. We demonstrate that T cells can form tethers and rolling adhesions on P selectin and E selectin under physiologic flow conditions. Tethering and rolling on P selectin was independent of cell-surface cutaneous lymphocyte antigen (CLA) expression, which correlated strictly with the capacity of T cells to form rolling adhesions under flow on E selectin. T cell tethering to P selectin was abolished by selective removal of cell surface sialomucins by a P. haemolytica O-glycoprotease, while cutaneous lymphocyte antigen expression was unaffected. A sialomucin molecule identical or closely related to P selectin glycoprotein ligand-1 (PSGL-1), the major P selectin ligand on neutrophils and HL-60 cells, appears to be a major T cell ligand for P selectin. P selectin glycoprotein ligand-1 does not appear to support T cell rolling on E selectin. In turn, E selectin ligands do not appear to be associated with sialomucins. These data demonstrate the presence of structurally distinct ligands for P or E selectins on T cells, provide evidence that both ligands can be coexpressed on a single T cell, and mediate tethering and rolling on the respective selectins in a mutually exclusive fashion.}, author = {Alon, R. and Rossiter,H. and Wang, X and Springer, T.A. and Kupper,T.S.} } @article {528081, title = {The dynamic regulation of integrin adhesiveness}, journal = {Curr. Biol.}, volume = {4}, number = {6}, year = {1994}, note = {Reprint Status: In File}, pages = {506-517}, abstract = {The integrins are a family of transmembrane heterodimeric adhesion molecules that play important roles in wound healing, immune system function and organ development. Recent studies indicate that adhesion of integrins to their ligands is not constitutive but is dynamically regulated by intracellular signal transduction pathways.}, author = {Diamond,M.S. and Springer, T.A.} } @article {528796, title = {A functional integrin ligand on the surface of platelets: Intercellular adhesion molecule-2}, journal = {J. Clin. Invest.}, volume = {94}, number = {3}, year = {1994}, note = {Reprint Status: In File}, pages = {1243-1251}, abstract = {Activated platelets express P-selectin and release leukocyte chemoattractants; however, they have not been known to express integrin ligands important in the stabilization of leukocyte interactions with the vasculature. We now demonstrate the presence of intercellular adhesion molecular-2 (ICAM-2) (CD102), and lack of expression of other beta 2-integrin ligands, ICAM-1 (CD54) and ICAM-3 (CD50), on the surface of resting and stimulated platelets. ICAM-2 isolated from platelets migrates as a band of 59,000 M(r) in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staining of bone marrow aspirates with anti-ICAM-2 mAb demonstrates strong reactivity to megakaryocytes. Using frozen thin sections and immunogold labeling, the antigen was shown to be present on the plasma membrane and surface-connected canalicular system of resting platelets. The average number of ICAM-2 molecules per platelet is 3,000 +/- 230 and does not change after activation. In adhesion assays, resting and stimulated platelets were capable of binding through ICAM-2 to purified leukocyte function-associated antigen-1. Activation of T lymphocytes with PMA stimulated binding to platelets that was Mg2+ dependent and could be specifically inhibited by mAbs to either ICAM-2 or leukocyte function-associated antigen-1. ICAM-2 is the only known beta 2-integrin ligand present on platelets, suggesting that it may play an important role in leukocyte-platelet interactions in inflammation and thrombosis.}, author = {Diacovo,T.G. and de Fougerolles, A.R. and Bainton,D.F. and Springer, T.A.} } @article {528166, title = {Intercellular adhesion molecules (ICAM)-1 ICAM-2 and ICAM-3 function as counter-receptors for lymphocyte function-associated molecule 1 in human immunodeficiency virus-mediated syncytia formation}, journal = {Eur. J. Immunol.}, volume = {24}, number = {9}, year = {1994}, note = {Reprint Status: In File}, pages = {2191-2195}, abstract = {It has been previously demonstrated that lymphocyte function-associated molecule 1 (LFA-1) plays a major role in human immunodeficiency virus (HIV)-mediated syncytia formation. In the present study we investigated the involvement of intercellular adhesion molecule-1 (ICAM-1), ICAM-2 and ICAM-3 in the process. The ability of monoclonal antibodies (mAb) directed against ICAM-1, ICAM-2 and ICAM-3 to block syncytia was analyzed either in phytohemagglutinin (PHA)-activated lymphocytes infected in vitro with primary or laboratory strains of HIV or by coculturing a T cell line stably expressing HIV envelope with PHA-activated lymphocytes. Complete inhibition of syncytia formation was observed only by the simultaneous addition to the cell cultures of all (i.e. anti-ICAM-1, anti-ICAM-2 and anti-ICAM-3) mAb. These results indicate that the interaction between LFA-1 and ICAM is a critical step in HIV-mediated syncytia formation, and that ICAM-1, ICAM-2 and ICAM-3 are the receptor molecules for the LFA-1-dependent syncytia formation.}, author = {Butini,L. and de Fougerolles, A.R. and Vaccarezza,M. and Graziosi,C. and Cohen,D.I. and Montroni,M. and Springer, T.A. and Pantaleo,G. and Fauci,A.S.} } @inbook {529751, title = {Kinetics of receptor and virus interaction and receptor-induced virus disruption: Methods for study with surface plasmon resonance}, booktitle = {Methods Enzymol.}, volume = {6}, year = {1994}, pages = {157-167}, publisher = {Academic Press}, organization = {Academic Press}, edition = {2}, address = {New York}, abstract = {We have used BIAcore to analyze the kinetics of the interaction between rhinovirus and soluble intercellular adhesion molecule-1 (sICAM-1). Human rhinovirus serotype 3 (HRV3) was immobilized in the carboxymethylated dextran of the sensor chip, and sICAM-1 expressed with baculovirus was injected through the rhinovirus surface. sICAM-1 bound specifically to HRV3. The virus remained intact in the surface after 12 successive cycles of association and dissociation at 20{\textdegree}C. The association rate was slow (720 M-1 s-1) and the dissociation rate was moderate (1.8 {\texttimes} 10-3 s-1) for protein interaction. A dissociation constant (KD) of 2.5 ({\textpm}0.18) μM was obtained from the kinetic constants. A slightly higher KD of 7.2 μM was obtained when equilibrium between virus and soluble receptor was reached in solution. At 30{\textdegree}C, binding of sICAM-1 disrupted HRV3, as monitored by a loss in resonance units.}, author = {Casasnovas,J.M. and Reed, R. R. and Springer, T.A.} } @article {529041, title = {The leukocyte integrin p150,95 (CD11c/CD18) as a receptor for iC3b: Activation by a heterologous β subunit and localization of a ligand recognition site to the I domain}, journal = {J. Immunol.}, volume = {152}, number = {9}, year = {1994}, note = {Reprint Status: NOT In File}, pages = {4582-4589}, abstract = {p150,95 is a member of the leukocyte integrin family of adhesion proteins. Compared with LFA-1 and Mac-1, p150,95 is less well functionally characterized. Although p150,95 has complement receptor activity for iC3b and has been designated complement receptor type 4, transfected cells expressing p150,95 do not bind iC3b-sensitized cells. We report that cells cotransfected with a human p150,95 alpha subunit and a chicken, but not human, beta subunit bind IgM-iC3b-coated erythrocytes, suggesting that interactions between the alpha and beta subunits can regulate p150,95 adhesiveness. Furthermore, purified human p150,95 binds to cell-bound iC3b-coated erythrocytes. Because binding to iC3b by cellular and purified p150,95 is specifically abolished by mAbs that localize to the I domain of p150,95, we suggest that the I domain of the p150,95 alpha subunit is an important ligand recognition site for iC3b.}, keywords = {CD11c}, author = {Bilsland,C.A.G. and Diamond,M.S. and Springer, T.A.} } @article {529001, title = {Leukocytosis and resistance to septic shock in intercellular adhesion molecule 1-deficient mice}, journal = {J. Exp. Med.}, volume = {180}, number = {1}, year = {1994}, note = {Reprint Status: NOT In Fileerased duplicate data $\#$10227.}, pages = {95-109}, abstract = {Intercellular adhesion molecule 1 (ICAM-1) is one of three immunoglobulin superfamily members that bind to the integrins lymphocyte function associated 1 (LFA-1) and Mac-1 on leukocytes. We have generated mice that are genetically and functionally deficient in ICAM-1. These mice have elevated numbers of circulating neutrophils and lymphocytes, as well as diminished allogeneic T cell responses and delayed type hypersensitivity. Mutant mice are resistant to lethal effects of high doses of endotoxin (lipopolysaccharide [LPS]), and this correlates with a significant decrease in neutrophil infiltration in the liver. Production of inflammatory cytokines such as tumor necrosis factor alpha or interleukin 1 is normal in ICAM-1-deficient mice, and thus protection appears to be related to a diminution in critical leukocyte-endothelial interactions. After sensitization with D-galactosamine (D-Gal), ICAM-1-deficient mice are resistant to the lethal effect of low doses of exotoxin (Staphylococcus aureus enterotoxin B [SEB]), which has been shown to mediate its toxic effects via the activation of specific T cells. In this model, ICAM-1-mediated protection against SEB lethality correlates with a decrease in the systemic release of inflammatory cytokines, as well as with prevention of extensive hepatocyte necrosis and hemorrhage. ICAM-1-deficient mice sensitized with D-Gal, however, are not protected from lethality when challenged with low doses of endotoxin (LPS). These studies show that the different contribution of ICAM-1 in the activation of either T cells or macrophages is decisive for the fatal outcome of the shock in these two models. This work suggests that anti-ICAM-1 therapy may be beneficial in both gram-positive and -negative septic shock, either by reducing T cell activation or by diminishing neutrophil infiltration.}, author = {Xu, H and Gonzalo,J.A. and St.Pierre,Y. and Williams,I.R. and Kupper,T.S. and Cotran,R.S. and Springer, T.A. and Gutierrez-Ramos,J-C.} } @article {530031, title = {Monocyte chemoattractant protein-1 is a major T lymphocyte chemoattractant}, journal = {Proc Natl Acad Sci USA}, volume = {91}, year = {1994}, note = {Reprint Status: In File}, pages = {3652-3656}, abstract = {We have utilized a transendothelial lymphocyte chemotaxis assay to identify and purify a lymphocyte chemoattractant in supernatants of mitogen-stimulated peripheral blood mononuclear cells. Amino acid sequence analysis revealed identity with monocyte chemoattractant protein 1 (MCP-1), a chemoattractant previously thought to be specific for monocytes. Recombinant MCP-1 is chemoattractive for purified T lymphocytes and for CD3+ lymphocytes in peripheral blood lymphocyte preparations. The T-cell response to MCP-1 is dose-dependent and chemotactic, rather than chemokinetic. Phenotyping of chemoattracted T lymphocytes shows they are an activated memory subset. The response to MCP-1 by T lymphocytes can be duplicated in the absence of an endothelial monolayer and the majority of T-lymphocyte chemotactic activity in mitogen-stimulated peripheral blood mononuclear cell supernatants can be neutralized by antibody to MCP-1. Thus, MCP-1 is the major lymphocyte chemoattractant secreted by mitogen-stimulated peripheral blood mononuclear cells and is capable of acting as a potent T-lymphocyte, as well as monocyte, chemoattractant. This may help explain why monocytes and T lymphocytes of the memory subset are always found together at sites of antigen-induced inflammation.}, author = {Carr,M.W. and Roth,S.J. and Luther,E. and Rose,S.S. and Springer, T.A.} } @article {528341, title = {Neutrophil tethering to and rolling on E-selectin are separable by requirement for L-selectin}, journal = {Immunity}, volume = {1}, number = {2}, year = {1994}, note = {Reprint Status: In File}, pages = {137-145}, abstract = {Neutrophil tethering and rolling in shear flow are mediated by selectins and have been thought to be two indistinguishable manifestations of a single molecular interaction between selectin and ligand. However, we report that under physiologic flow conditions, tethering to E-selectin requires a ligand distinct from the one that supports neutrophil rolling. Tethering under shear to E-selectin requires a carbohydrate ligand that is closely associated with the lectin domain of L-selectin on the neutrophil surface, as enzymatic removal of L-selectin, chemotactic factor-induced shedding of L-selectin, and L-selectin MAbs effectively block tethering. In contrast, this ligand is dispensable for the ability to roll on E-selectin, since rolling adhesions formed after static incubations were not affected by the presence or absence of L-selectin. Thus, E-selectin interactions with ligands on neutrophils persist after L-selectin shedding. These findings add an additional step for regulation of leukocyte localization in inflammatory sites.}, author = {Lawrence,M.B. and Bainton,D.F. and Springer, T.A.} } @article {529471, title = {The pathway of rhinovirus disruption by soluble intercellular adhesion molecule 1 (ICAM-1): An intermediate in which ICAM-1 is bound and RNA is released}, journal = {J. Virol.}, volume = {68}, number = {9}, year = {1994}, note = {Reprint Status: In File}, pages = {5882-5889}, abstract = {We have examined the pathway of rhinovirus interaction with soluble intercellular adhesion molecule 1 (sICAM-1). Binding of sICAM-1 to rhinovirus serotypes 3 and 14 gives particles with sedimentation coefficients from 145 to 120S, depending on the amount of sICAM-1 bound. The formation of 120S particles is faster and more extensive at a neutral pH than at an acidic pH. A large number of receptors (\> 30) can bind to human rhinovirus 3 without disruption. Disruption by sICAM-1 of rhinovirus that yields 80S particles is strongly temperature dependent and is antagonized by a low pH. Interestingly, sICAM-1 remains bound to the viral capsid after RNA is released, although in smaller amounts than those observed for the native virus. We have found heterogeneity both between and within 80S particle preparations in the VP4 content and number of bound receptors. The ability of the virus to remain bound to its receptor during the uncoating process may facilitate the transport of the viral genome into the cytoplasm in vivo.}, author = {Casasnovas,J.M. and Springer, T.A.} } @article {528766, title = {Residues within a conserved amino acid motif of domains 1 and 4 of VCAM-1 are required for binding to VLA-4}, journal = {J. Cell Biol.}, volume = {125}, number = {1}, year = {1994}, note = {Reprint Status: NOT In File}, pages = {215-222}, abstract = {Vascular cell adhesion molecule 1 (VCAM-1), a member of the Ig superfamily originally identified on activated endothelium, binds to the integrin very late antigen-4 (VLA-4), also known as alpha 4 beta 1 or CD49d/CD29, to support cell-cell adhesion. Studies based on cell adhesion to two alternatively spliced forms of VCAM-1 or to chimeric molecules generated from them and intercellular adhesion molecule-1 (ICAM-1) have demonstrated two VLA-4 binding sites on the predominate form of VCAM-1. Here, we studied VLA-4-dependent adhesion of the lymphoid tumor cell line Ramos to cells expressing wild type and mutant forms of VCAM-1. Results based on domain deletion mutants demonstrated the existence and independence of two VLA-4-binding sites located in the first and fourth domains of VCAM-1. Results based on amino acid substitution mutants demonstrated that residues within a linear sequence of six amino acids found in both domain 1 and 4 were required for VLA-4 binding to either domain. Five of these amino acids represent a conserved motif also found in ICAM domains. We propose that integrin binding to these Ig-like domains depends on residues within this conserved motif. Specificity of integrin binding to Ig-like domains may be regulated by a set of nonconserved residues distinct from the conserved motif.}, author = {Vonderheide,R.H. and Tedder,T.F. and Springer, T.A. and Staunton,D.E.} } @article {527901, title = {Steel factor and c-kit regulate cell-matrix adhesion}, journal = {Blood}, volume = {83}, number = {4}, year = {1994}, note = {Reprint Status: NOT In File}, pages = {1033-1038}, abstract = {Steel (SI) and white spotting (W) loci encode steel factor (c-kit ligand) and the c-kit tyrosine kinase receptor, respectively. Mutations at these loci affect migration and differentiation of primordial germ cells, neural crest-derived melanoblasts, and hematopoietic cells. In these processes, cell adhesion molecules are hypothesized to be crucial. We have examined the role of steel factor and c-kit in cell-extracellular matrix adhesion using bone marrow-derived mast cells as a model system. Steel factor stimulates mast cells to bind to fibronectin and, to a lesser extent, to vitronectin, whereas interleukin-3 and interleukin-4, which are also mast cell growth factors, do not. Activation of adhesiveness is transient, occurs at concentrations of steel factor 100-fold lower than required for growth stimulation, and requires the integrin VLA-5. Mast cells from c-kit mutant mice adhere to fibronectin on stimulation with phorbol 12-myristate 13-acetate (PMA), but not on stimulation with steel factor, indicating that stimulation of integrin adhesiveness requires activation of the c-kit protein tyrosine kinase. By contrast, c-kit mutant and wild-type mast cells adhere equally well to COS cells expressing membrane-anchored steel factor, showing that the kinase activity of c-kit is not required for adhesion directly mediated by c-kit. Our findings suggest that regulation of adhesion is an important biologic function of steel factor.}, author = {Kinashi,T. and Springer, T.A.} } @article {527971, title = {Traffic signals for lymphocyte recirculation and leukocyte emigration: the multi-step paradigm}, journal = {Cell}, volume = {76}, number = {2}, year = {1994}, note = {Reprint Status: In File}, pages = {301-314}, author = {Springer, T.A.} } @inbook {528056, title = {Adhesion molecules in immunity and inflammation}, booktitle = {Clinical Aspects of immunology}, year = {1993}, note = {Reprint Status: In Fileerased duplicate data $\#$6839}, pages = {199-219}, publisher = {Blackwell Scientific Publications}, organization = {Blackwell Scientific Publications}, edition = {5}, address = {Boston}, author = {Springer, T.A.}, editor = {Lachmann,P.J. and Peters,K. and Rosen,F.S. and Walport,M.J.} } @inbook {527826, title = {Adhesion receptors in inflammation: a pr{\'e}cis}, booktitle = {Application of Basic Science to Hematopoiesis and Treatment of Disease}, year = {1993}, note = {MS $\#$235Reprint Status: NOT In File}, pages = {231-239}, publisher = {Raven Press}, organization = {Raven Press}, edition = {1}, address = {New York}, author = {Springer, T.A.}, editor = {Thomas,E.D. and Carter,S.K.} } @article {528171, title = {CDw50 and ICAM-3: Two names for the same molecule}, journal = {Eur. J. Immunol.}, volume = {23}, number = {7}, year = {1993}, note = {Reprint Status: NOT In File}, pages = {1508-1512}, abstract = {CDw50 differentiation antigen is a molecule broadly expressed on hematopoetic cells but not on other cells. Previous experiments showed that CDw50 monoclonal antibodies (mAb) inhibited primary mixed lymphocyte culture (MLC). To understand the function of CDw50 better, we purified it and obtained peptide sequence. At the same time, intercellular adhesion molecule (ICAM)-3, the third ligand of lymphocyte function-associated molecule 1, was described by mAb and subsequent cDNA cloning. Immunochemical, functional, and protein sequencing studies show that ICAM-3 and CDw50 are the same glycoprotein, a 120-kDa surface molecule with presumably an important role in the immune responses.}, author = {Juan,M. and Vilella,R. and Mila,J. and Yag{\"u}e,J. and Miralles,A. and Campbell,K.S. and Friedrich,R.J. and Cambier,J. and Vives,J. and de Fougerolles, A.R. and Springer, T.A.} } @article {528196, title = {Characterization of two new CD18 alleles causing severe leukocyte adhesion deficiency}, journal = {Eur. J. Immunol.}, volume = {23}, number = {11}, year = {1993}, note = {Reprint Status: In File}, pages = {2792-2798}, abstract = {Leukocyte adhesion deficiency (LAD) is an autosomal recessive disease caused by heterogeneous mutations within the gene encoding the common beta subunit (CD18) of the three leukocyte integrins LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and p150,95 (CD11c/CD18). Based on the level of expression of CD18 on patient leukocytes, two phenotypes of LAD have been defined (severe and moderate) which correlate with the severity of the disease. We have investigated the molecular basis of the disease in two unrelated severe patients (HS and ZJO). Both patients share a complete absence of CD18 protein precursor and cell surface expression, but they differ in the level of CD18 mRNA, which is normal in HS and undetectable by Northern blot in ZJO. Determination of the primary structure of the patient HS CD18 mRNA revealed a 10-base pair deletion between nucleotides 190-200 (CD18 exon 3), which eliminates residues 41-43 and causes a frameshift into a premature termination codon 17 base pairs downstream from the deleted region. The 10-base pair frameshift deletion maps to a region of the CD18 gene where aberrant mRNA processing has been detected in HS and two other unrelated LAD patients. In the ZJO patient, amplification of lymphoblast CD18 mRNA demonstrated the presence of a non-sense mutation in the third nucleotide of the triplet encoding Cys534 (TGC--\>TGA), within exon 12. Both genetic abnormalities were also detected at the genomic level, and affect the restriction pattern of their corresponding genes, thus enabling the detection of the mutant alleles among healthy heterozygous alleles in family studies. The identification of two new LAD CD18 alleles, either carrying a non-sense mutation (ZJO) or a partial gene deletion (HS), further illustrates the heterogeneity of the genetic alterations in LAD.}, author = {Rodr{\'\i}guez,C.L. and Nueda,A. and Grospierre,B. and S{\'a}nchez-Madrid,F. and Fischer,A. and Springer, T.A. and Corb{\'\i},A.L.} } @article {528886, title = {Cloning and expression of intercellular adhesion molecule 3 reveals strong homology to other immunoglobulin family counter-receptors for lymphocyte function-associated antigen-1}, journal = {J. Exp. Med.}, volume = {177}, number = {4}, year = {1993}, note = {Reprint Status: In File}, pages = {1187-1192}, abstract = {Based on protein sequence, we have isolated a cDNA for intercellular adhesion molecule 3 (ICAM-3), the most recently defined counter-receptor for lymphocyte function-associated antigen 1 (LFA-1). Expression of the cDNA yields a product that reacts with monoclonal antibody to ICAM-3 and functions as a ligand for LFA-1. The deduced 518-amino acid sequence of the predicted mature protein defines a highly glycosylated type I integral membrane protein with five immunoglobulin (Ig)-like domains. The five Ig-like domains of ICAM-3 are highly homologous with those of human ICAM-1 (52\% identity) and human ICAM-2 (37\% identity).}, keywords = {CD50}, author = {de Fougerolles, A.R. and Klickstein,L.B. and Springer, T.A.} } @article {529481, title = {Efficient neutralization and disruption of rhinovirus by chimeric ICAM-1/immunoglobulin molecules}, journal = {J. Virol.}, volume = {67}, number = {6}, year = {1993}, note = {Reprint Status: In File}, pages = {3561-3568}, abstract = {The intercellular adhesion molecule 1 (ICAM-1) is used as a cellular receptor by 90\% of human rhinoviruses (HRVs). Chimeric immunoadhesin molecules containing extracellular domains of ICAM-1 and constant regions of immunoglobulins (Igs) were designed in order to determine the effect of increased valency, Ig isotype, and number of ICAM-1 domains on neutralization and disruption of rhinovirus structure. These immunoadhesins include ICAM-1 amino-terminal domains 1 and 2 fused to the hinge and constant domains of the heavy chains of IgA1, IgM, and IgG1 (IC1-2D/IgA, -/IgM, and -/IgG). In addition, all five extracellular domains were fused to IgA1 (IC1-5D/IgA). Immunoadhesins were compared with soluble forms of ICAM-1 containing five and two domains (sICAM-1 and ICI-2D, respectively) in assays of HRV binding, infectivity, and conformation. In prevention of HRV plaque formation, IC1-5D/IgA was 200 times and IC1-2D/IgM and IC1-2D/IgA were 25 and 10 times more effective, respectively, than ICAM-1. The same chimeras were highly effective in inhibiting binding of rhinovirus to cells and disrupting the conformation of the virus capsid, as demonstrated by generation of approximately 65S particles. The results show that the number of ICAM-1 domains and a flexible Ig hinge are important factors contributing to the efficacy of neutralization. The higher efficiency of chimeras that bound bivalently in disrupting HRV was attributed to higher binding avidity. The IC1-5D/IgA immunoadhesin was effective at nanomolar concentrations, making it feasible therapy for rhinovirus infection.}, author = {Martin,S. and Casasnovas,J.M. and Staunton,D.E. and Springer, T.A.} } @article {527821, title = {Functional studies of truncated soluble ICAM-1 expressed in E. coli}, journal = {Antimicrob. Agents Chemother.}, volume = {37}, number = {6}, year = {1993}, note = {MS $\#$242Reprint Status: In File}, pages = {1278-1284}, abstract = {We have expressed in Escherichia coli the two N-terminal immunoglobulin (Ig)-like domains of the intercellular adhesion molecule 1 (ICAM-1). The first 188 residues of ICAM-1 were expressed with an N-terminal methionine (MP188) or as a maltose-binding fusion protein which was cleaved with factor Xa (XP188). After refolding, both MP188 and XP188 were active in binding to the leukocyte integrin lymphocyte function-associated antigen 1, which has previously been shown to bind to the N-terminal Ig domain of ICAM-1. The major group of rhinoviruses and malaria-infected erythrocytes bind to distinct sites within the first Ig-like domain of ICAM-1. Both MP188 and XP188 bound to malaria-infected erythrocytes; however, only XP188 inhibited human rhinovirus plaque formation. A product (MdQ1P188) with the initiation methionine fused to residue 2, i.e., with glutamine 1 deleted, inhibited plaque formation. MdQ1P188 was able to induce a conformational change of the virus capsid as shown by conversion of 149S particles to 85S particles, whereas MP188 had no effect. These results show that functionally active fragments of ICAM-1 can be produced in E. coli, that glycosylation is not required for ligand binding, and that the N-terminal residue of ICAM-1 is proximal to or part of the human rhinovirus-binding site.}, author = {Martin,S. and Martin,A. and Staunton,D.E. and Springer, T.A.} } @article {528711, title = {The I domain is a major recognition site on the leukocyte integrin Mac-1 (CD11b/CD18) for four distinct adhesion ligands}, journal = {J. Cell Biol.}, volume = {120}, number = {4}, year = {1993}, note = {Reprint Status: In File}, pages = {1031-1043}, abstract = {Despite the identification and characterization of several distinct ligands for the leukocyte integrin (CD11/CD18) family of adhesion receptors, little is known about the structural regions on these molecules that mediate ligand recognition. In this report, we use alpha subunit chimeras of Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18), and an extended panel of newly generated and previously characterized mAbs specific to the alpha chain of Mac-1 to map the binding sites for four distinct ligands for Mac-1: iC3b, fibrinogen, ICAM-1, and the as-yet uncharacterized counter-receptor responsible for neutrophil homotypic adhesion. Epitopes of mAbs that blocked ligand binding were mapped with the chimeras and used to localize the ligand recognition sites because the data obtained from functional assays with the Mac-1/p150,95 chimeras were not easily interpreted. Results show that the I domain on the alpha chain of Mac-1 is an important recognition site for all four ligands, and that the NH2-terminal and perhaps divalent cation binding regions but not the COOH-terminal segment may contribute. The recognition sites in the I domain appear overlapping but not identical as individual Mac-1-ligand interactions are distinguished by the discrete patterns of inhibitory mAbs. Additionally, we find that the alpha subunit NH2-terminal region and divalent cation binding region, despite being separated by over 200 amino acids of the I domain, appear structurally apposed because three mAbs require the presence of both of these regions for antigenic reactivity, and chimeras that contain the NH2 terminus of p150,95 require the divalent cation binding region of p150,95 to associate firmly with the beta subunit.}, keywords = {CD11b}, author = {Diamond,M.S. and Garcia-Aguilar,J. and Bickford,J.K. and Corbi,A.L. and Springer, T.A.} } @article {528421, title = {The intracellular bacterium rhodococcus equi uses complement receptors to bind to mammalian cells}, journal = {Infect. Immun.}, volume = {61}, year = {1993}, note = {MS $\#$238Reprint Status: In File}, pages = {2919-2929}, author = {Hondalus,M.K. and Diamond,M.S. and Springer, T.A. and Mosser,D.M.} } @article {529446, title = {Location of the domains of ICAM-1 by immunolabeling and single-molecule electron microscopy}, journal = {J. Leukoc. Biol.}, volume = {53}, number = {3}, year = {1993}, note = {MS $\#$236, manuscriptReprint Status: In File}, pages = {342-346}, abstract = {Intercellular adhesion molecule 1 (ICAM-1), a member of the immunoglobulin gene superfamily, is a cell surface glycoprotein with an extracellular domain comprising five immunoglobulin-like domains. Soluble ICAM-1, a recombinant protein truncated at the transmembrane domain, has a rod-like shape, 19 nm long overall, with a characteristic bend 7.6 nm from one end of the molecule. Because the link between domain D2 and domain D3 is proline rich, it has been proposed that the short arm contains domains D1 and D2 and the long arm contains domains D3-D5. We used single-molecule electron microscopy of soluble ICAM-1 decorated with monoclonal antibodies specific for domains D1 and D4 to show that the bend instead lies between domains D3 and D4. Therefore, the short arm lies closer to the plasma membrane, whereas the long arm, containing all the known ligand binding sites on ICAM-1, is positioned toward the target cell surface.}, keywords = {CD54}, author = {Kirchhausen,T. and Staunton,D.E. and Springer, T.A.} } @article {529191, title = {Neutrophils roll on E-selectin}, journal = {J. Immunol.}, volume = {151}, number = {11}, year = {1993}, note = {Reprint Status: NOT In File}, pages = {6338-6346}, abstract = {Using flow conditions that simulate those in post capillary venules, we have found that neutrophils attach and roll on a substrate bearing purified E-selectin. E-selectin resembles P-selectin (CD62) with regard to the dependence of attachment efficiency on wall shear stress and selectin density. In contrast, once attached, neutrophils form rolling adhesions on E-selectin that are much stronger than those on P-selectin. Rolling velocities on E-selectin are slower and have less variance than on P-selectin. With increasing shear stress, rolling velocities reach a plateau level that is dependent on E-selectin density, suggesting that the number of receptor-ligand bonds and the bond dissociation rate limit rolling velocity, and that the bonds are not broken by the applied force.}, author = {Lawrence,M.B. and Springer, T.A.} } @article {528841, title = {Subcellular localization and dynamics of Mac-1 (αMβ2) in human neutrophils}, journal = {J. Clin. Invest.}, volume = {92}, number = {3}, year = {1993}, note = {Reprint Status: In File}, pages = {1467-1476}, abstract = {The subcellular localization of Mac-1 was determined in resting and stimulated human neutrophils after disruption by nitrogen cavitation and fractionation on two-layer Percoll density gradients. Light membranes were further separated by high voltage free flow electrophoresis. Mac-1 was determined by an ELISA with monoclonal antibodies that were specific for the alpha-chain (CD11b). In unstimulated neutrophils, 75\% of Mac-1 colocalized with specific granules including gelatinase granules, 20\% with secretory vesicles and the rest with plasma membranes. Stimulation with nanomolar concentrations of FMLP resulted in the translocation of Mac-1 from secretory vesicles to the plasma membrane, and only minimal translocation from specific granules and gelatinase granules. Stimulation with PMA or Ionomycin resulted in full translocation of Mac-1 from secretory vesicles and gelatinase granules to the plasma membrane, and partial translocation of Mac-1 from specific granules. These findings were corroborated by flow cytometry, which demonstrated a 6-10-fold increase in the surface membrane content of Mac-1 in response to stimulation with FMLP, granulocyte-macrophage colony stimulating factor, IL-8, leukotriene B4, platelet-activating factor, TNF-alpha, and zymosan-activated serum, and a 25-fold increase in response to Ionomycin. Thus, secretory vesicles constitute the most important reservoir of Mac-1 that is incorporated into the plasma membrane during stimulation with inflammatory mediators.}, author = {Sengelov,H. and Kjeldsen,L. and Diamond,M.S. and Springer, T.A. and Borregaard,N.} } @article {528716, title = {A subpopulation of Mac-1 (CD11b/CD18) molecules mediates neutrophil adhesion to ICAM-1 and fibrinogen}, journal = {J. Cell Biol.}, volume = {120}, number = {2}, year = {1993}, note = {Reprint Status: In File}, pages = {545-556}, abstract = {We report that a subpopulation (10\%) of the Mac-1 (CD1 1b/CD18) molecules on activated neutrophils mediates adhesion to ICAM-1 and fibrinogen. We describe a novel mAb (CBRM1/5) that binds to an activation-specific neoepitope on a subset of Mac-1 molecules on neutrophils and monocytes after stimulation with chemoattractants or phorobol esters but does not recognize Mac-1 on resting myeloid cells. CBRM1/5 immunoprecipitates a subpopulation of Mac-1 molecules from detergent lysates of neutrophils, binds to immunoaffinity-purified Mac-1, and localizes to the I domain on the alpha chain of Mac-1. Because CBRM1/5 recognizes a fraction of Mac-1 on activated neutrophils, but still blocks Mac-1-dependent adhesion to fibrinogen and ICAM-1, we suggest that only a small subset of Mac-1 molecules is competent to mediate adhesion.}, keywords = {CD 11b}, author = {Diamond,M.S. and Springer, T.A.} } @article {528676, title = {Association of intercellular adhesion molecule-1 (ICAM-1) with actin-containing cytoskeleton and α-actinin}, journal = {J. Cell Biol.}, volume = {118}, number = {5}, year = {1992}, note = {Reprint Status: In File}, pages = {1223-1234}, abstract = {We have studied the cytoskeletal association of intercellular adhesion molecule-1 (ICAM-1, CD54), an integral membrane protein that functions as a counterreceptor for leukocyte integrins (CD11/CD18). A linkage between ICAM-1 and cytoskeletal elements was suggested by studies showing a different ICAM-1 staining pattern for COS cells transfected with wild-type ICAM-1 or with an ICAM-1 construct that replaces the cytoplasmic and transmembrane domains of ICAM-1 with a glycophosphatidylinositol (GPI) anchor. Wild-type ICAM-1 appeared to localize most prominently in microvilli whereas GPI-ICAM-1 demonstrated a uniform cell surface distribution. Disruption of microfilaments with cytochalasin B (CCB) changed the localization of wild-type ICAM-1 but had no effect on GPI-ICAM-1. Some B-cell lines demonstrated a prominent accumulation of ICAM-1 into the uropod region whereas other cell surface proteins examined were not preferentially localized. CCB also induced redistribution of ICAM-1 in these cells. For characterization of cytoskeletal proteins interacting with ICAM-1, a 28-residue peptide that encompasses the entire predicted cytoplasmic domain (ICAM-1,478-505) was synthesized, coupled to Sepharose-4B, and used as an affinity matrix. One of the most predominant proteins eluted either with soluble ICAM-1,478-505-peptide or EDTA, was 100 kD, had a pI of 5.5, and in Western blots reacted with alpha-actinin antibodies. A direct association between alpha-actinin and ICAM-1 was demonstrated by binding of purified alpha-actinin to ICAM-1,478-505-peptide and to immunoaffinity purified ICAM-1 and by a strict colocalization of ICAM-1 with alpha-actinin, but not with the cytoskeletal proteins talin, tensin, and vinculin. The region of ICAM-1,478-505 interacting with alpha-actinin was mapped to the area close to the membrane spanning region. This region contains several positively charged residues and appears to mediate a charged interaction with alpha-actinin which is not highly dependent on the order of the residues.}, author = {Carpen,O. and Pallai,P. and Staunton,D.E. and Springer, T.A.} } @article {529776, title = {Effect of lengthening lymphocyte function-associated antigen 3 on adhesion to CD2}, journal = {Mol. Biol. Cell}, volume = {3}, number = {2}, year = {1992}, note = {MS$\#$227Reprint Status: In File}, pages = {157-166}, abstract = {The effect of lengthening the distance in an adhesion molecule between the receptor binding site and the membrane anchor was studied by inserting four Ig-like domains into the two Ig domain lymphocyte function-associated antigen 3 (LFA-3) molecule. The extended molecule expressed in Chinese hamster ovary (CHO) cells bound to CD2 on T lymphocytes 4- to 20-fold more efficiently than the wild-type molecule at 4 degrees C. Treatment of the CHO clones with neuraminidase to remove sialic acid, or with deoxymannojirimycin to reduce the bulk of N-linked glycosylation, showed that adhesion to both the wild-type and the chimeric LFA-3 molecules was under the influence of cell-cell repulsive forces to a similar extent and that these treatments had less effect than lengthening LFA-3. At higher temperatures, such as 22 and 37 degrees C, the efficiency of binding to the wild-type LFA-3 increased to levels comparable with binding to extended LFA-3. Our results suggest that more distal locations of the adhesive binding site from the cell membrane anchor increase the efficiency of cell-cell adhesion by enhancing the frequency of receptor encounter with ligand and that more proximal locations of the adhesive binding site can provide efficient cell-cell adhesion at physiological temperatures.}, author = {Chan, P.-Y. and Springer, T.A.} } @article {528896, title = {Intercellular adhesion molecule 3, a third adhesion counter-receptor for lymphocyte function-associated molecule 1 on resting lymphocytes}, journal = {J. Exp. Med.}, volume = {175}, number = {1}, year = {1992}, note = {MS$\#$225Reprint Status: NOT In File}, pages = {185-190}, abstract = {Recent studies suggest that some T and B lymphocyte cell lines bind to the integrin lymphocyte function-associated molecule 1 (LFA-1) chiefly through a pathway independent of its two known counter-receptors, intercellular adhesion molecules (ICAMs)-1 and -2. A monoclonal antibody (mAb) was raised that, in combination with blocking mAb to ICAM-1 and ICAM-2, can completely inhibit binding of these cell lines to purified LFA-1. This third ligand, designated ICAM-3 based on its functional relatedness to ICAM-1 and -2, is a highly glycosylated protein of 124,000 Mr. It is well expressed on all leukocytes and absent from endothelial cells. In assays of adhesion of resting lymphocytes to purified LFA-1, ICAM-3 is by far the most functionally important ICAM, implying an important role for ICAM-3 in the generation of immune responses.}, author = {de Fougerolles, A.R. and Springer, T.A.} } @article {529336, title = {Internalization of a major group human rhinovirus does not require cytoplasmic or transmembrane domains of ICAM-1}, journal = {J. Immunol.}, volume = {148}, number = {10}, year = {1992}, note = {MS$\#$227Reprint Status: In File}, pages = {3271-3274}, abstract = {Intercellular adhesion molecule-1 (CD54), a cell adhesion molecule and the receptor for the major group of rhinoviruses, is a class 1 membrane protein with five Ig-like domains in its extracellular region, a transmembrane domain, and a short cytoplasmic domain. The amino-terminal domains (D1 and D2) are sufficient for virus binding and the first is most important (1). We have investigated whether other extracellular domains, transmembrane or cytoplasmic domains are required for virus entry as determined by postinfection virion protein biosynthesis. We demonstrate that cytoplasmic, transmembrane, and Ig-like domains 3, 4, and 5 are not essential for rhinovirus entry into transfected COS cells. The efficiency of rhinovirus infection directly correlates with the efficiency of rhinovirus binding and a form of intercellular adhesion molecule-1 that is glycophosphatidyl-inositol anchored, and thus does not extend into the inner leaflet of the membrane bilayer or the cytoplasm efficiently supports virus entry.}, keywords = {970}, author = {Staunton,D.E. and Gaur,A. and Chan, P.-Y. and Springer, T.A.} } @article {529396, title = {Isolation, characterization, and expression of mouse ICAM-2 complementary and genomic DNA}, journal = {J. Immunol.}, volume = {149}, number = {8}, year = {1992}, note = {Reprint Status: In File}, pages = {2650-2655}, abstract = {Intercellular adhesion molecule-2 (ICAM-2), a cell surface glycoprotein, is a second counter-receptor for lymphocyte function-associated Ag-1 (LFA-1). We report here the isolation and characterization of the cDNA and the gene that encode murine ICAM-2 (Accession numbers X65493 and X65490, respectively). The deduced sequence of the cDNA has 60\% amino acid identity with its human counterpart and has the same expression pattern in cells and tissues. Furthermore, COS cells transfected with mouse ICAM-2 complementary and genomic DNA bind to purified human LFA-1, demonstrating the conservation of the function of ICAM-2 as a ligand for LFA-1 and conservation across species of sequences that are critical for binding to human LFA-1. COS cells transfected with the ICAM-2 cDNA do not react with mAb PA3, previously suggested to define ICAM-2 in the mouse. The mouse ICAM-2 gene was isolated and its structural organization determined. The gene is present in a single copy in the mouse genome and contains four exons spanning about 5.0 kb of DNA. The exon/intron architecture correlates to the structural domains of the protein and resembles that of other Ig superfamily members. The gene for ICAM-2, which is constitutively expressed in endothelial cells, has several conserved sequence motifs in its promoter region, including a direct repeat, and lacks transcription factor-binding sites present in the ICAM-1 gene, which is inducible in endothelial cells.}, author = {Xu, H and Tong,I.L. and de Fougerolles, A.R. and Springer, T.A.} } @article {528751, title = {Leishmania promastigotes require opsonic complement to bind to the human leukocyte integrin Mac-1 (CD11b/CD18)}, journal = {J. Cell Biol.}, volume = {116}, number = {2}, year = {1992}, note = {Reprint Status: NOT In File}, pages = {511-520}, abstract = {Previous reports have suggested that Leishmania spp. interact with macrophages by binding to Mac-1 (CD1 1b/CD18), a member of the leukocyte integrin family. To better define this interaction, we tested the ability of leishmania promastigotes to bind to purified leukocyte integrins and to cloned integrins expressed in COS cells. We show that leishmania promastigotes bind to cellular or purified Mac-1 but not lymphocyte function-associated antigen-1 in a specific, dose-dependent manner that requires the presence of serum. Binding is inhibited with specific monoclonal antibodies to Mac-1. In the absence of complement opsonization, three different species of leishmania tested fail to bind directly to any of the three leukocyte integrins. We show that binding to Mac-1 requires the third component of complement (C3). Organisms incubated in heat-inactivated serum or serum that has been immunologically depleted of C3 fail to bind to Mac-1. Because the addition of purified C3 to C3-depleted serum restores leishmania binding to Mac-1, we suggest that parasites gain entry into macrophages by fixing complement and subverting a well-characterized adhesive interaction in the immune system between Mac-1 and iC3b.}, author = {Mosser,D.M. and Springer, T.A. and Diamond,M.S.} } @article {528986, title = {Lymphocyte adhesion through VLA-4: Evidence for a novel binding site in the alternatively spliced domain of VCAM-1 and an additional α4 integrin counter-receptor on stimulated endothelium}, journal = {J. Exp. Med.}, volume = {175}, number = {6}, year = {1992}, note = {MS$\#$231Reprint Status: NOT In File}, pages = {1433-1442}, abstract = {Recent studies demonstrate that alternative splicing of mRNA from a single gene can produce two forms of vascular cell adhesion molecule 1 (VCAM-1): a six-immunoglobulin (Ig) domain form (VCAM-6D) and a seven-Ig domain form (VCAM-7D). Using a COS cell transient expression assay, we investigated whether VCAM-6D and VCAM-7D differ functionally in adhesion to the integrin VLA-4 (CD49d/CD29) on lymphoid cells. Binding of lymphoid cell lines and peripheral blood lymphocytes was completely blocked by VLA-4 monoclonal antibody (mAb) and one VCAM-1 mAb (4B9) to both VCAM-6D and VCAM-7D, whereas one VCAM-1 mAb (E1/6) completely blocked binding to VCAM-6D but only partially inhibited binding to VCAM-7D. We conclude that there is one VLA-4 binding site in the six Ig domains shared between VCAM-6D and VCAM-7D, and that the alternatively spliced domain 4 present in VCAM-7D provides a second VLA-4 binding site that is blocked by 4B9 but not the E1/6 mAb. We compared the inhibitory effects of anti-VCAM-1 and anti-VLA-4 mAbs on lymphoid cell adhesion to cultured human umbilical vein endothelial cells (HUVEC). The anti-VCAM-1 mAb 4B9 blocked the binding of PBL and lymphoid tumor cells to stimulated HUVEC better than the anti-VCAM-1 mAb E1/6. Because VCAM-7D is the predominant form of VCAM-1 expressed by stimulated endothelial cells, this difference in VCAM-1 mAb inhibition is attributed to lymphoid cell binding to VCAM-7D on stimulated HUVEC. Although the anti-VLA-4 mAb and anti-VCAM-1 mAb 4B9 equally inhibited PBL binding to stimulated HUVEC, mAb 4B9 inhibited the binding of two lymphoid cell lines significantly less than anti-VLA-4 mAb. Combination of 4B9 mAb with function-blocking antiserum to human fibronectin, a second known ligand for VLA-4, also failed to inhibit as much as anti-VLA-4 mAb. These findings suggest that adhesion of lymphoid cell lines through VLA-4 or other alpha 4 integrins may involve inducible counter-receptor(s) on endothelium distinct from either VCAM-1 or fibronectin. Time course experiments indicate that the fraction of alpha 4 integrin-dependent binding that can be blocked by anti-VCAM-1 mAb E1/6 rises and peaks within 2 h of tumor necrosis factor (TNF) stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)}, keywords = {CD49d}, author = {Vonderheide,R.H. and Springer, T.A.} } @article {528761, title = {Micromanipulation of adhesion of a Jurkat cell to a planar bilayer membrane containing lymphocyte function-associated antigen 3 molecules}, journal = {J. Cell Biol.}, volume = {116}, number = {4}, year = {1992}, note = {MS$\#$227Reprint Status: In File}, pages = {997-1006}, abstract = {Cell adhesion plays a fundamental role in the organization of cells in differentiated organs, cell motility, and immune response. A novel micromanipulation method is employed to quantify the direct contribution of surface adhesion receptors to the physical strength of cell adhesion. In this technique, a cell is brought into contact with a glass-supported planar membrane reconstituted with a known concentration of a given type of adhesion molecules. After a period of incubation (5-10 min), the cell is detached from the planar bilayer by pulling away the pipette holding the cell in the direction perpendicular to the glass-supported planar bilayer. In particular, we investigated the adhesion between a Jurkat cell expressing CD2 and a glass-supported planar bilayer containing either the glycosyl-phosphatidylinositol (GPI) or the transmembrane (TM) isoform of the counter-receptor lymphocyte function-associated antigen 3 (LFA-3) at a concentration of 1,000 molecules/microns 2. In response to the pipette force the Jurkat cells that adhered to the planar bilayer containing the GPI isoform of LFA-3 underwent extensive elongation. When the contact radius was reduced by approximately 50\%, the cell then detached quickly from its substrate. The aspiration pressure required to detach a Jurkat cell from its substrate was comparable to that required to detach a cytotoxic T cell from its target cell. Jurkat cells that had been separated from the substrate again adhered strongly to the planar bilayer when brought to proximity by micromanipulation. In experiments using the planar bilayer containing the TM isoform of LFA-3, Jurkat cells detached with little resistance to micromanipulation and without changing their round shape.}, author = {T{\"o}zeren,A. and Sung,K.-L.P. and Sung,L.A. and Dustin,M.L. and Chan, P.-Y. and Springer, T.A. and Chien,S.} } @article {527881, title = {Micromanipulation of adhesion of phorbol 12-myristate-13-acetate-stimulated T lymphocytes to planar membranes containing intercellular adhesion molecule-1}, journal = {Biophys. J.}, volume = {63}, number = {1}, year = {1992}, note = {MS$\#$226Reprint Status: In File}, pages = {247-258}, abstract = {This paper presents an analytical and experimental methodology to determine the physical strength of cell adhesion to a planar membrane containing one set of adhesion molecules. In particular, the T lymphocyte adhesion due to the interaction of the lymphocyte function associated molecule 1 on the surface of the cell, with its counter-receptor, intercellular adhesion molecule-1 (ICAM-1), on the planar membrane, was investigated. A micromanipulation method and mathematical analysis of cell deformation were used to determine (a) the area of conjugation between the cell and the substrate and (b) the energy that must be supplied to detach a unit area of the cell membrane from its substrate. T lymphocytes stimulated with phorbol 12-myristate-13-acetate (PMA) conjugated strongly with the planar membrane containing purified ICAM-1. The T lymphocytes attached to the planar membrane deviated occasionally from their round configuration by extending pseudopods but without changing the size of the contact area. These adherent cells were dramatically deformed and then detached when pulled away from the planar membrane by a micropipette. Detachment occurred by a gradual decrease in the radius of the contact area. The physical strength of adhesion between a PMA-stimulated T lymphocyte and a planar membrane containing 1,000 ICAM-1 molecules/micron 2 was comparable to the strength of adhesion between a cytotoxic T cell and its target cell. The comparison of the adhesive energy density, measured at constant cell shape, with the model predictions suggests that the physical strength of cell adhesion may increase significantly when the adhesion bonds in the contact area are immobilized by the actin cytoskeleton.}, author = {T{\"o}zeren,A. and Mackie,L.H. and Lawrence,M.B. and Chan, P.-Y. and Dustin,M.L. and Springer, T.A.} } @article {527961, title = {Plasmodium falciparum-infected erythrocytes bind ICAM-1 at a site distinct from LFA-1, Mac-1, and human rhinovirus}, journal = {Cell}, volume = {68}, number = {1}, year = {1992}, note = {MS$\#$229Reprint Status: In File}, pages = {63-69}, abstract = {The attachment of erythrocytes infected with P. falciparum to human venular endothelium is the primary step leading to complications from severe and cerebral malaria. Intercellular adhesion molecule-1 (ICAM-1, CD54) has been implicated as a cytoadhesion receptor for P. falciparum-infected erythrocytes. Characterization of domain deletion, human/murine chimeric ICAM-1 molecules, and amino acid substitution mutants localized the primary binding site for parasitized erythrocytes to the first amino-terminal immunoglobulin-like domain of ICAM-1. The ICAM-1 binding site is distinct from those recognized by LFA-1, Mac-1, and the human major-type rhinoviruses. Synthetic peptides encompassing the binding site on ICAM-1 inhibited malaria-infected erythrocyte adhesion to ICAM-1-coated surfaces with a Ki of 0.1-0.3 mM, whereas the Ki for soluble ICAM-1 is 0.15 microM. These findings have implications for the therapeutic reversal of malaria-infected erythrocyte sequestration in the host microvasculature.}, author = {Ockenhouse,C.F. and Betageri,R. and Springer, T.A. and Staunton,D.E.} } @article {529076, title = {Regulation of locomotion and cell-cell contact area by the LFA-1 and ICAM-1 adhesion receptors}, journal = {J. Immunol.}, volume = {148}, number = {9}, year = {1992}, note = {MS$\#$222Reprint Status: NOT In File}, pages = {2654-2663}, abstract = {We demonstrate complementary differences in the behavior of B lymphoblastoid cells adhering to LFA-1 or its counter-receptor ICAM-1. The interaction of B lymphoblastoid cells with glass-supported planar bilayers bearing LFA-1 or ICAM-1 was observed by time-lapse video microscopy, and the distribution of adhesion receptors on cells interacting with the planar bilayers was studied by immunofluorescence microscopy. B lymphoblasts formed a large contact area and crawled rapidly (up to 25 microns/min) on planar bilayers bearing ICAM-1. In contrast, these cells attached to planar bilayers bearing LFA-1 through a fixed point about which the cells actively pivoted, using a single stalk-like projection. Phorbol ester-stimulated lymphoblasts, which adhere more strongly to ICAM-1-bearing substrates than unstimulated lymphoblasts, were still capable of locomotion on ICAM-1. Phorbol ester stimulation of B lymphoblasts on planar bilayers bearing LFA-1 promoted a rapid conversion from "stalk" attachment to symmetrical spreading of the cell on the substrate. Cellular LFA-1 remained uniformly distributed on the cell surface during interaction with bilayers bearing purified ICAM-1 as determined by immunofluorescence. In contrast, ICAM-1 was concentrated in the stalk-like structure through which the unstimulated B lymphoblasts adhered to LFA-1 in planar bilayers, but ICAM-1 immunofluorescence became more uniformly distributed over the cell surface within minutes of phorbol ester addition. Neither LFA-1 or ICAM-1 colocalized with the prominent staining of filamentous actin in the ruffling membrane regions. Interaction through cell surface LFA-1 and ICAM-1, 2, or 3 promotes different cellular morphologies and behaviors, the correlation of which with previously observed patterns of lymphocyte interaction with different cell types is discussed.}, author = {Dustin,M.L. and Carpen,O. and Springer, T.A.} } @article {528976, title = {Soluble intercellular adhesion molecule-1 immunoglobulin G1 immunoadhesion mediates phagocytosis of malaria-infected erythrocytes}, journal = {J. Exp. Med.}, volume = {176}, year = {1992}, note = {Old $\#$8764Reprint Status: NOT In File}, pages = {1471-1476}, abstract = {We describe an immunoadhesin molecule containing intercellular adhesion molecule 1 (ICAM-1) molecularly fused to hinge and CH2 and CH3 domains of the human immunoglobulin G1 H chain that binds Plasmodium falciparum-infected erythrocytes. This receptor-based immunoadhesin is an effective and specific inhibitor of P. falciparum-infected erythrocyte adhesion to ICAM-1-bearing surfaces, but does not inhibit leukocyte function antigen 1 (LFA-1) interaction with ICAM-1. Furthermore, the immunoadhesin promotes phagocytosis and destruction of parasitized erythrocytes by human monocytes. Each of these modes of action has potential for the therapy of malaria.}, keywords = {765.1}, author = {Staunton,D.E. and Ockenhouse,C.F. and Springer, T.A.} } @article {527936, title = {Binding of the integrin Mac-1 (CD11b/CD18) to the third Ig-like domain of ICAM-1 (CD54) and its regulation by glycosylation}, journal = {Cell}, volume = {65}, number = {6}, year = {1991}, note = {MS$\#$218Reprint Status: In File}, pages = {961-971}, abstract = {Both the integrins LFA-1 and Mac-1 bind to ICAM-1, an immunoglobulin superfamily member. Previously, we localized the binding sites of LFA-1 and the major group of human rhinoviruses to the first NH2-terminal immunoglobulin-like domain of ICAM-1. Here, we show that the binding site on ICAM-1 for Mac-1 is unexpectedly distinct from that for LFA-1 and maps to the third NH2-terminal immunoglobulin-like domain. These findings provide a function for the tandem duplication of immunoglobulin-like domains in ICAM-1 and have implications for other immunoglobulin superfamily members. Mutations at two sites in the third domain that destroy consensus sequences for N-linked glycosylation enhance binding to purified Mac-1. Agents that interfere with carbohydrate processing provide evidence that the size of the N-linked oligosaccharide side chains on ICAM-1 affects binding to Mac-1 but not to LFA-1. Thus, we suggest that the extent of glycosylation on ICAM-1 may regulate adhesion to LFA-1 or Mac-1 in vivo.}, author = {Diamond,M.S. and Staunton,D.E. and Marlin,S.D. and Springer, T.A.} } @article {529966, title = {Cell adhesion: A birth certificate for CD2}, journal = {Nature}, volume = {353}, number = {6346}, year = {1991}, note = {Review, MS$\#$223Reprint Status: In File}, pages = {704-705}, keywords = {CD2}, author = {Springer, T.A.} } @article {528901, title = {Characterization of ICAM-2 and evidence for a third counter-receptor for LFA-1}, journal = {J. Exp. Med.}, volume = {174}, number = {1}, year = {1991}, note = {MS$\#$216Reprint Status: In File}, pages = {253-267}, abstract = {In an endeavor to further characterize human intercellular adhesion molecule-2 (ICAM-2), two murine monoclonal antibodies (mAb) were generated to ICAM-2 transfected COS cells, and designated CBR-IC2/1 and CBR-IC2/2. Immunoprecipitated, reduced ICAM-2 migrated as a broad band of Mr 60,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with N-glycanase revealed a peptide backbone of Mr 31,000, consistent with the size predicted from the cDNA. ICAM-2 had a broad distribution on hematopoietic cell lines and little expression on other cell lines, the sole exception being cultured endothelial cells which possess high levels of ICAM-2. Resting lymphocytes and monocytes expressed ICAM-2, while neutrophils did not. Staining of tissue sections with anti-ICAM-2 mAb confirmed their strong reactivity to vascular endothelium, but demonstrated a lack of ICAM-2 expression on other tissues. Small clusters of ICAM-2 positive cells were, however, seen in germinal centers. In contrast to ICAM-1 there was little or no induction of ICAM-2 expression on lymphocytes or cultured endothelium upon stimulation with inflammatory mediators. One of the two mAb, CBR-IC2/2, was found to totally inhibit binding of ICAM-2+ COS cells to purified lymphocyte function-associated antigen-1 (LFA-1). Using this mAb, LFA-1-dependent binding to both stimulated and unstimulated endothelium was found to be totally accounted for by ICAM-1 and ICAM-2. Homotypic aggregation of an Epstein-Barr virus-transformed B cell line, JY, was found to be solely ICAM-1 and ICAM-2-dependent, while in the case of the T cell lymphoma cell line, SKW3, anti- ICAM-2 mAb in conjunction with anti-ICAM-1 mAb could not inhibit the LFA-1-dependent aggregation. This suggests an additional LFA-1 ligand exists. Using a cell binding assay to purified LFA-1 in conjunction with anti-ICAM-1 and anti-ICAM-2 mAb, we have demonstrated that this putative third ligand for LFA-1 exists on SKW3 and other cell lines.}, keywords = {CD 102}, author = {de Fougerolles, A.R. and Stacker,S.A. and Schwarting,R. and Springer, T.A.} } @article {529146, title = {Cloning of the murine lymphocyte function-associated molecule-1 (LFA-1) α subunit and its expression in COS cells}, journal = {J. Immunol.}, volume = {147}, number = {1}, year = {1991}, note = {MS$\#$219Reprint Status: In File}, pages = {369-371}, abstract = {The lymphocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is an integrin that mediates adhesion of immune cells by interaction with two members of the Ig superfamily, ICAM-1 and ICAM-2. LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report here the isolation and expression of the murine alpha subunit cDNA (GenBank accession no. M60778). The deduced sequence comprises a 1061 amino acid extracellular domain, a 29 amino acid transmembrane region, and a 50 amino acid cytoplasmic domain. It has a 72\% amino acid identity with its human counterpart and 34\% identity with the murine Mac-1 alpha subunit. The murine LFA-1 alpha subunit could be expressed on the cell surface of a fibroblastoid cell line, COS, by cotransfection with either the human or murine beta subunit cDNA.}, keywords = {CD11a}, author = {Kaufman,Y. and Tseng,E. and Springer, T.A.} } @article {528931, title = {The cytoplasmic domain of the integrin lymphocyte function-associated antigen 1 β subunit: sites required for binding to intercellular adhesion molecule 1 and the phorbol ester-stimulated phosphorylation site}, journal = {J. Exp. Med.}, volume = {174}, number = {5}, year = {1991}, note = {MS$\#$223Reprint Status: In File}, pages = {1227-1238}, abstract = {We have defined the regions of the cytoplasmic domain of the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) that are required for active binding of its extracellular domain to intercellular adhesion molecule 1 (ICAM-1). The NH2-terminal 28 amino acids in the cytoplasmic domain are dispensable, but a segment of 5 amino acids including three contiguous threonines (758-760) and Phe 766 in the COOH-terminal third of the cytoplasmic domain are required for binding to ICAM-1. Mutation and phosphoamino acid analysis show that Ser 756 is the major residue phosphorylated in response to phorbol ester. Furthermore, multiple mutations demonstrate that serine phosphorylation can be dissociated from phorbol ester-stimulated binding of LFA-1 to ICAM-1. The sites we have defined are previously unremarked, are well conserved in the beta 1, beta 3, and beta 7 integrin subunits, and may be of broad importance in regulating adhesiveness of integrins.}, keywords = {CD18}, author = {Hibbs,M.L. and Jakes,S. and Stacker,S.A. and Wallace,R.W. and Springer, T.A.} } @article {528681, title = {Influence of receptor lateral mobility on adhesion strengthening between membranes containing LFA-3 and CD2}, journal = {J. Cell Biol.}, volume = {115}, year = {1991}, note = {MS$\#$217Reprint Status: In File}, pages = {245-255}, abstract = {We have used an in vitro model system of glass-supported planar membranes to study the effects of lateral mobility of membrane-bound receptors on cell adhesion. Egg phosphatidylcholine (PC) bilayers were reconstituted with two anchorage isoforms of the adhesion molecule lymphocyte function-associated antigen 3 (LFA-3). The diffusion coefficient of glycosyl phosphatidylinositol (GPI)-anchored LFA-3 approached that of phospholipids in the bilayers, whereas the transmembrane (TM)-anchored isoform of LFA-3 was immobile. Both static and laminar flow assays were used to quantify the strength of adherence to the lipid bilayers of the T lymphoma cell line Jurkat that expresses the counter-receptor CD2. Cell adhesion was dependent on LFA-3 density and was more efficient on membranes containing the GPI isoform than the TM isoform. Kinetic measurements demonstrated an influence of contact time on the strength of adhesion to the GPI isoform at lower site densities (25-50 sites/microns2), showing that the mobility of LFA-3 is important in adhesion strengthening. At higher site densities (1,500 sites/microns2) and longer contact times (20 min), Jurkat cell binding to the TM and GPI isoforms of LFA-3 showed equivalent adhesion strengths, although adhesion strength of the GPI isoform developed twofold more rapidly than the TM isoform. Reduction of CD2 mobility on Jurkat cells at 5 degrees C greatly decreased the rate of adhesion strengthening with the TM isoform of LFA-3, resulting in a 30-fold difference between the two LFA-3 isoforms. Our results demonstrate that the ability of a membrane receptor and its membrane-bound counter-receptor to diffuse laterally enhances cell adhesion both by allowing accumulation of ligands in the cell contact area and by increasing the rate of receptor-ligand bond formation.}, keywords = {CD2}, author = {Chan, P.-Y. and Lawrence,M.B. and Dustin,M.L. and Ferguson, L.M. and Golan, D.E. and Springer, T.A.} } @inbook {528121, title = {Isolation of proteins using antibodies: Immunoaffinity chromatography and immunoprecipitation}, booktitle = {Current Protocols in Immunology}, year = {1991}, note = {MS$\#$214Reprint Status: In File}, pages = {8.2.1-8.3.11}, publisher = {Greene Publishing Associates and Wiley Interscience}, organization = {Greene Publishing Associates and Wiley Interscience}, address = {New York}, author = {Springer, T.A.}, editor = {Coligan,J.E. and Kruisbeek,A.M. and Margulies,D.H. and Shevach,E.M. and Strober,W.} } @article {529326, title = {Leukocyte integrin p150,95 (CD11c/CD18) functions as an adhesion molecule binding to a counter-receptor on stimulated endothelium}, journal = {J. Immunol.}, volume = {146}, number = {2}, year = {1991}, note = {MS$\#$213; endothelial cellsReprint Status: In File}, pages = {648-655}, abstract = {p150,95 is a member of the beta 2 family of integrins, which includes both LFA-1 and Mac-1. These molecules are known to play a role in the adhesion of lymphocytes, granulocytes, and monocytes to various cell types including vascular endothelium. p150,95 is presumed to have an adhesive function because of its structural relationship to the other beta 2 integrins and the ability of anti-p150,95 mAb to inhibit some myeloid cell interactions with tumor cells, endothelial cells, and other substrates. In an endeavor to demonstrate directly that p150,95 can act as an adhesion molecule, we raised a mAb (CBRp150/4G1) to the alpha subunit of p150,95, which allows for the purification of functional intact p150,95 heterodimers. The antibody was selected by using a high pH elution ELISA. The assay was designed to select for antibodies directed to the alpha-chain of p150,95, which could be readily dissociated from p150,95 under conditions of high pH and 2 mM MgCl2. p150,95 purified under these conditions with CBRp150/4G1-Sepharose could be immunoprecipitated by using antibodies to the alpha- and beta-chains of p150,95 indicating that the structural integrity of the heterodimer was preserved during purification and elution. Elution in the absence of divalent cations yielded primarily dissociated alpha and beta subunits. Other antibodies previously made to p150,95 alpha-chain such as SHCL3 were greatly reduced in their efficiency of yielding intact heterodimer under these conditions. Mapping of the epitopes by using chimeric molecules of p150,95/Mac-1 revealed that antibodies that react with the divalent cation sites of p150,95 are inferior for the purification of intact p150,95. The adhesive capacity of p150,95 was demonstrated by the specific binding of 18-h rIL-1 beta or LPS-stimulated endothelial cells to purified p150,95 absorbed to plastic microtiter plates. These results indicate that p150,95 can function independently as an adhesion molecule and that it can interact with a counter-receptor on stimulated endothelium.}, keywords = {CD11c}, author = {Stacker,S.A. and Springer, T.A.} } @article {527951, title = {Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins}, journal = {Cell}, volume = {65}, number = {5}, year = {1991}, note = {MS$\#$221Reprint Status: In File}, pages = {859-873}, abstract = {Rolling of leukocytes on vascular endothelial cells, an early event in inflammation, can be reproduced in vitro on artificial lipid bilayers containing purified CD62, a selectin also named PADGEM and GMP-140 that is inducible on endothelial cells. Neutrophils roll on this selectin under flow conditions similar to those found in postcapillary venules. Adhesion of resting or activated neutrophils through the integrins LFA-1 and Mac-1 to ICAM-1 in a lipid bilayer does not occur at physiologic shear stresses; however, static incubation of activated neutrophils allows development of adhesion that is greater than 100-fold more shear resistant than found on CD62. Addition of a chemoattractant to activate LFA-1 and Mac-1 results in the arrest of neutrophils rolling on bilayers containing both CD62 and ICAM-1. Thus, at physiologic shear stress, rolling on a selectin is a prerequisite for activation-induced adhesion strengthening through integrins.}, author = {Lawrence,M.B. and Springer, T.A.} } @article {528671, title = {Motility and ultrastructure of large granular lymphocytes on lipid bilayers reconstituted with adhesion receptors LFA-1, ICAM-1 and two isoforms of LFA-3}, journal = {J. Cell Biol.}, volume = {115}, number = {3}, year = {1991}, note = {MS$\#$224Reprint Status: NOT In File}, pages = {861-871}, abstract = {Large granular lymphocytes, mediators of NK activity, bind to other cells using both the LFA (lymphocyte function-associated)-1-ICAM and the CD2-LFA-3 adhesion pathways. Here we have studied the motility and ultrastructure of large granule lymphocyte (LGL) on lipid bilayers containing purified LFA-1, ICAM-1, and the transmembrane and glycophosphatidylinositol isoforms of LFA-3. LGLs moved at 8 microns/min on ICAM-1 but poorly (less than 1 microns/min) on its receptor pair LFA-1. TM-LFA-3 promoted locomotion at a rate close to ICAM-1, whereas the cells were less motile on GPI-LFA-3. The difference in the rates of locomotion on the two isoforms of LFA-3 is presumably attributable to their difference in anchoring and lateral mobility in the bilayer. In spite of the variation in motility the ultrastructure of the adhering cells was similar on all four ligands. LGLs contacted the membrane variably, i.e., cells adhering only in a few small areas or in larger areas were detected on each ligand. The relative percentage of the plasma membrane facing the lipid bilayer was greatest on ICAM-1 and least on the transmembrane isoform of LFA-3, demonstrating no correlation with motility. The ratio of adjacent plasma membrane to lipid bilayer was virtually constant for all four ligands. Activation of the LGLs with a combination of CD2 mAb T11(2) and T11(3) (T11(2/3) mAb) reduced the movement on ICAM-1 and virtually immobilized the cells on the other bilayers. In the presence of T11(2/3) mAb, the area of cell membrane attaching to bilayers containing ICAM-1 and GPI-LFA-3 was decreased and the percentage of plasma membrane facing other cells was increased. No preferential orientation of the Golgi apparatus or degranulation was detected in the absence or presence of T11(2/3) mAb, but a significantly lower percentage of LGLs on ICAM-1 contained a profile of the Golgi apparatus after exposure to T11(2/3) mAb. The results demonstrate that the motility of LGLs depends on the type of receptor in the opposing bilayer, the receptor mobility in the bilayer, and the activation of the cells. The ultrastructure of LGLs binding to any of the adhesion molecules does not have the characteristics of LGLs in cytolytic contact with target cells, suggesting that the mediation of an attack on a target requires more complex stimulus than any one of the single adhesion proteins tested here.}, author = {Carpen,O. and Dustin,M.L. and Springer, T.A. and Swafford,J.A. and Smith,L.A. and Caulfield,J.P.} } @article {528226, title = {Multiple integrins share the ability to induce elevation of intracellular pH}, journal = {Exp. Cell Res.}, volume = {195}, number = {2}, year = {1991}, note = {MS$\#$222Reprint Status: In File}, pages = {533-535}, abstract = {Previous work has shown that adhesion of anchorage-dependent cells to fibronectin via integrin alpha 5 beta 1 leads to activation of the Na-H antiporter and a rise in intracellular pH (pHi). We now show that adhesion of bovine capillary endothelial cells (BCE) to fibrinogen; collagens type III, IV, and V; laminin; and vitronectin; ligands that bind other members of the integrin family, resulted in significant elevations in pHi. Other ligands (basic fibroblast growth factor, concanavalin A, and thrombin), which bind cells when immobilized on plastic, but that do not bind integrins and do not support cell growth, do not elevate pHi. Adhesion to an antibody against integrin alpha v beta 3 also elevates pHi. Adhesion of peripheral human T lymphocytes to an antibody against the integrin LFA-1 induced a rise in pHi. Antibodies to CD2 or ICAM-2 had only slight effects on pHi, whereas an antibody to the T cell receptor complex that strongly activates T cells induced a large increase in pHi. We conclude that elevation of pHi by integrins is specific and is a property shared by many members of the integrin family.}, keywords = {CD18}, author = {Schwartz,M.A. and Ingber, D. E. and Lawrence,M. and Springer, T.A. and Lechene, C.} } @article {529971, title = {The next cluster of differentiation (CD) workshop}, journal = {Nature}, volume = {354}, number = {6352}, year = {1991}, note = {MS$\#$224, reviewReprint Status: In File}, pages = {415-416}, author = {Springer, T.A.} } @article {530296, title = {Regulation of adhesion to ICAM-1 by the cytoplasmic domain of LFA-1 integrin β subunit}, journal = {Science}, volume = {251}, number = {5001}, year = {1991}, note = {MS$\#$208Reprint Status: In File}, pages = {1611-1613}, abstract = {Interactions between cytotoxic lymphocytes and their targets require the T cell antigen receptor (TCR) and the integrin lymphocyte function-associated molecule-1 (LFA-1, CD11a/CD18). LFA-1 is not constitutively avid for its counter-receptors, intercellular adhesion molecules (ICAMs)-1 and -2. Cross-linking of the TCR transiently converts LFA-1 to a high avidity state and thus provides a mechanism for regulating cellular adhesion and de-adhesion in an antigen-specific manner. Truncation of the cytoplasmic domain of the beta, but not the alpha, subunit of LFA-1 eliminated binding to ICAM-1 and sensitivity to phorbol esters. Thus, LFA-1 binding to ICAM-1 was found to be regulated by the cytoplasmic domain of the beta subunit of LFA-1.}, keywords = {CD18}, author = {Hibbs,M.L. and Xu, H and Stacker,S.A. and Springer, T.A.} } @article {527806, title = {Role of lymphocyte adhesion receptors in transient interactions and cell locomotion}, journal = {Annu. Rev. Immunol.}, volume = {9}, year = {1991}, note = {MS$\#$209Reprint Status: In File}, pages = {27-66}, abstract = {Lymphocytes adhere to other cells and extracellular matrix in the process of immunological recognition and lymphocyte recirculation. This review focuses on regulation of lymphocyte adhesion and the use of adhesion mechanisms by lymphocytes to obtain information about their immediate environment. The CD2 and LFA-1 adhesion receptors appear to have distinct roles in the regulation of adhesion and modulation of T lymphocyte activation. Adhesion mediated by interaction of CD2 with LFA-3 is dramatically altered by surface charge and adhesion receptor density in such a way that this pathway is latent in resting T lymphocytes but becomes active over a period of hours following T-cell activation. CD2 ligation can mediate or enhance T-cell activation, suggesting that signals from CD2/LFA-3 adhesive interactions are integrated with signals from the T-cell antigen receptor during immunological recognition. A model for the role of LFA-3 lateral diffusion in adhesion is presented, based on the lateral diffusion of different LFA-3 forms in glass supported planar membranes. Interaction of LFA-1 with ICAMs is also regulated by cell activation but in a different way than in interaction of CD2 with LFA-3. LFA-1 avidity for ICAMs is transiently increased by T-cell activation over a period of minutes. Cycles of avidity change are also observed for other T lymphocyte integrins which bind to extracellular matrix components. We propose that integrin avidity cycles may have an important role in the interconnected phenomena of locomotion, initial cell-cell adhesion, and cell-cell deadhesion. Recent observations on recirculation of T lymphocyte subpopulations are discussed in the context of general lessons learned from study of the CD2/LFA-3 and LFA-1/ICAM adhesion mechanisms.}, author = {Dustin,M.L. and Springer, T.A.} } @article {529981, title = {Sticky sugars for selectins}, journal = {Nature}, volume = {349}, number = {6306}, year = {1991}, note = {MS$\#$220, reviewReprint Status: In File}, pages = {196-197}, author = {Springer, T.A. and Lasky,L.A.} } @inbook {529736, title = {VLA-4-dependent adhesion of lymphocytic cell lines to cultured endothelium}, booktitle = {Lymphatic Tissues and In Vivo Immune Responses}, year = {1991}, note = {MS$\#$211Reprint Status: In File}, pages = {873-876}, publisher = {Marcel Dekker, Inc.}, organization = {Marcel Dekker, Inc.}, address = {New York}, author = {Vonderheide,R.H. and Springer, T.A.}, editor = {Imhof,B. and et al.} } @inbook {529886, title = {The ability of NK cell and granulocyte forms of CD16 to trigger cytolytic function}, booktitle = {Natural Killer Cells: Biology and Clinical Application. 6th International Natural Killer Cell Workshop}, year = {1990}, note = {MS$\#$196Reprint Status: In File}, pages = {50-54}, publisher = {S. Karger AG}, organization = {S. Karger AG}, address = {Basel}, author = {Carpen,O. and Selvaraj, P. and Hibbs,M.L. and Springer, T.A.} } @article {529961, title = {Adhesion receptors of the immune system}, journal = {Nature}, volume = {346}, number = {6283}, year = {1990}, note = {MS $\#$195, review articleReprint Status: In File}, pages = {425-433}, abstract = {The adhesive interactions of cells with other cells and with the extracellular matrix are crucial to all developmental processes, but have a central role in the functions of the immune system throughout life. Three families of cell-surface molecules regulate the migration of lymphocytes and the interactions of activated cells during immune responses.}, keywords = {015.1}, author = {Springer, T.A.} } @inbook {528016, title = {Area code molecules of lymphocytes}, booktitle = {Cell to Cell Interaction: a Karger Symposium}, year = {1990}, note = {MS$\#$215Reprint Status: In File}, pages = {16-39}, publisher = {S. Karger AG}, organization = {S. Karger AG}, address = {Basel}, author = {Springer, T.A.}, editor = {Burger,M.M. and Sordat,B. and Zinkernagel,R.M.} } @article {527981, title = {The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinovirus}, journal = {Cell}, volume = {61}, number = {2}, year = {1990}, note = {MS$\#$199Reprint Status: In File}, pages = {243-254}, abstract = {Intercellular adhesion molecule 1 (ICAM-1, CD54) binds to the integrin LFA-1 (CD11a/CD18), promoting cell adhesion in immune and inflammatory reactions. ICAM-1 is also subverted as a receptor by the major group of rhinoviruses. Electron micrographs show that ICAM-1 is a bent rod, 18.7 nm long, suggesting a model in which the five immunoglobulin-like domains are oriented head to tail at a small angle to the rod axis. ICAM-1 sequences important to binding LFA-1, rhinovirus, and four monoclonal antibodies were identified through the characterization of chimeric ICAM-1 molecules and mutants. The amino-terminal two immunoglobulin-like domains of ICAM-1 appear to interact conformationally. Domain 1 of ICAM-1 contains the primary site of contact for both LFA-1 and rhinovirus; the presence of domains 3-5 markedly affects the accessibility of the binding site for rhinovirus and less so for LFA-1. The binding sites appear to be distinct but overlapping; rhinovirus binding also differs from LFA-1 binding in its lack of divalent cation dependence. Our analysis suggests that rhinoviruses mimic LFA-1 in binding to the most membrane-distal, and thus most accessible, site of ICAM-1.}, keywords = {CD54}, author = {Staunton,D.E. and Dustin,M.L. and Erickson, H. P. and Springer, T.A.} } @article {528996, title = {Distinct mutations in two patients with leukocyte adhesion deficiency and their functional correlates}, journal = {J. Exp. Med.}, volume = {172}, number = {1}, year = {1990}, note = {MS$\#$199Reprint Status: In File}, pages = {335-345}, abstract = {Two patients with leukocyte adhesion deficiency (LAD), one with a moderate phenotype (patient 14) and one with a severe phenotype (patient 2) who had been shown to have a normal sized beta subunit protein precursor, were analyzed in an attempt to determine the molecular basis for their disease. RNase mapping located possible mutations to two distinct but adjacent regions of the beta subunit cDNA. Sequencing of patient-derived cDNA clones in this region revealed a C for T difference at amino acid 149 in patient 14 which resulted in the substitution of a leucine for a proline, and an A for G substitution at amino acid 169 in patient 2 which mutated a glycine to an arginine. The mutated amino acids are in a region of the cDNA that is highly conserved between the beta subunits of the integrin family and are identical in all known integrin beta subunits. Co-transfection of the beta subunit cDNA containing the patient 2 mutation with the wild-type alpha subunit of LFA-1 in a mammalian expression system resulted in no expression of LFA-1. In the case of the mutation in patient 14 there was markedly diminished expression of LFA-1 with loss of function and loss of the epitope for a number of anti-beta mAbs. Normal half-life of the mutant beta subunits, and previous demonstration of a lack of alpha/beta complex formation during biosynthesis in patient cells, suggest a defect in association with the alpha subunit. Association with beta is required for expression of the alpha subunit of LFA-1. Loss of functional expression with both of these beta subunit mutations suggests that they lie in a site critical for association with the alpha subunit.}, keywords = {CD18}, author = {Wardlaw,A.J. and Hibbs,M.L. and Stacker,S.A. and Springer, T.A.} } @article {528466, title = {Genomic structure of an integrin α subunit, the leukocyte p150,95 molecule}, journal = {J. Biol. Chem.}, volume = {265}, number = {5}, year = {1990}, note = {MS$\#$201Reprint Status: In File}, pages = {2782-2788}, abstract = {The genomic structure of integrins is important to our understanding of the evolution of this complex family. The alpha subunit of the leukocyte integrin p150,95 (CD11c) is a transmembrane polypeptide of 1144 residues whose long extracellular region contains three putative divalent cation binding repeats and a 200- amino acid inserted or "I" domain. The p150,95 alpha subunit gene extends over 25 kilobases and is comprised of at least 31 exons grouped in five clusters. The I domain, which is only present in some integrins and is homologous to domains in von Willebrand factor, cartilage matrix protein, complement factor B and the alpha 1 and alpha 2 chains of collagen type VI, is distributed in four exons. Each one of the three divalent cation binding repeats is encoded by a separate exon. Surprisingly, a sequence homologous to the first two putative divalent cation binding repeats is present in an inverted orientation in the intron following the last exon of the I domain. Both the signal peptide and the transmembrane domain are split in two exons. Putative proteolytic cleavage sequences in other integrin alpha subunits align as inserts within the p150,95 alpha subunit gene falling at exon boundaries. The organization of the p150,95 alpha subunit gene provides further insights into the structure and evolution of the integrins.}, author = {Corbi,A.L. and Garcia-Aguilar,J. and Springer, T.A.} } @article {528721, title = {ICAM-1 (CD54): A counter-receptor for Mac-1 (CD11b/CD18)}, journal = {J. Cell Biol.}, volume = {111}, number = {6 Pt 2}, year = {1990}, note = {MS$\#$210Reprint Status: In File}, pages = {3129-3139}, abstract = {While the leukocyte integrin lymphocyte function-associated antigen (LFA)-1 has been demonstrated to bind intercellular adhesion molecule (ICAM)-1, results with the related Mac-1 molecule have been controversial. We have used multiple cell binding assays, purified Mac-1 and ICAM-1, and cell lines transfected with Mac-1 and ICAM-1 cDNAs to examine the interaction of ICAM-1 with Mac-1. Stimulated human umbilical vein endothelial cells (HUVECs), which express a high surface density of ICAM-1, bind to immunoaffinity-purified Mac-1 adsorbed to artificial substrates in a manner that is inhibited by mAbs to Mac-1 and ICAM-1. Transfected murine L cells or monkey COS cells expressing human ICAM-1 bind to purified Mac-1 in a specific and dose-dependent manner; the attachment to Mac-1 is more temperature sensitive, lower in avidity, and blocked by a different series of ICAM-1 mAbs when compared to LFA-1. In a reciprocal assay, COS cells cotransfected with the alpha and beta chain cDNAs of Mac-1 or LFA-1 attach to immunoaffinity-purified ICAM-1 substrates; this adhesion is blocked by mAbs to ICAM-1 and Mac-1 or LFA-1. Two color fluorescence cell conjugate experiments show that neutrophils stimulated with fMLP bind to HUVEC stimulated with lipopolysaccharide for 24 h in an ICAM-1-, Mac-1-, and LFA-1-dependent fashion. Because cellular and purified Mac-1 interact with cellular and purified ICAM-1, we conclude that ICAM-1 is a counter receptor for Mac-1 and that this receptor pair is responsible, in part, for the adhesion between stimulated neutrophils and stimulated endothelial cells.}, author = {Diamond,M.S. and Staunton,D.E. and de Fougerolles, A.R. and Stacker,S.A. and Garcia-Aguilar,J. and Hibbs,M.L. and Springer, T.A.} } @article {528011, title = {The leukocyte integrin LFA-1 reconstituted by cDNA transfection in a nonhematopoietic cell line is functionally active and not transiently regulated}, journal = {Cell Regul.}, volume = {1}, number = {4}, year = {1990}, note = {MS$\#$202Reprint Status: In File}, pages = {359-367}, abstract = {The functional activity of lymphocyte function-associated antigen 1 (LFA-1) on leukocytes can be regulated by T-cell receptor (TCR) stimulation and pharmacologic agents. It was of interest to determine if functionally active LFA-1 could be reconstituted on a nonhematopoietic, LFA-1-negative cell line. We report the expression of LFA-1 and diethylaminoethyl (DEAE) Mac-1 alpha beta heterodimers on the cell surface of a fibroblastoid cell line, COS, by DEAE dextran cotransfection of the alpha and beta subunit cDNAs. Immunoprecipitation studies demonstrated that the alpha and beta subunit was expressed in heterodimers. The alpha or beta subunit was expressed at lower levels after transfection with the alpha or beta subunit cDNA alone. Cotransfection of the alpha and beta subunit cDNAs, but not transfection of alpha or beta alone, was sufficient to reconstitute intercellular adhesion molecule-1 (ICAM-1) binding activity. Consistent with this observation, LFA-1 on the fibroblastoid cells possesses the activation epitope defined by the L16 monoclonal antibody (mAb). This epitope marks the conversion of LFA-1 from the low to high avidity state on peripheral blood T lymphocytes (PBLs) and is constitutively present on activated cell lines. In contrast to LFA-1 on leukocytes, the functional activity of LFA-1 on fibroblastoid cells was not influenced by phorbol ester treatment. Furthermore, the use of agents that interfere with intracellular signaling, a protein kinase C inhibitor, cAMP analogue, or the combination of a phosphodiesterase inhibitor and adenyl cyclase activator, did not affect the binding of COS cells expressing LFA-1 to purified ICAM-1.}, author = {Larson,R.S. and Hibbs,M.L. and Springer, T.A.} } @article {527801, title = {The sensation and regulation of interactions with the extracellular environment: The cell biology of lymphocyte adhesion receptors}, journal = {Annu. Rev. Cell Biol.Annual Review of Cell Biology}, volume = {6}, year = {1990}, note = {MS$\#$207Reprint Status: In File}, pages = {359-402}, author = {Springer, T.A.} } @article {529936, title = {A soluble form of intercellular adhesion molecule-1 inhibits rhinovirus infection}, journal = {Nature}, volume = {344}, number = {6261}, year = {1990}, note = {MS$\#$204Reprint Status: In File}, pages = {70-72}, abstract = {Rhinoviruses belong to the picornavirus family and cause about 50\% of common colds. Most rhinoviruses and some coxsackie viruses share a common receptor on human cells. The glycoprotein intercellular adhesion molecule-1 (ICAM-1) has recently been identified as the cellular receptor for the subgroup of rhinoviruses known as the major groups. ICAM-1 is a member of the immunoglobulin supergene family and is a ligand for lymphocyte function-associated antigen-1 (LFA-1); these ICAM-1/LFA-1 interactions are critical to many cell adhesion processes involved in the immunological response. Because anti-ICAM-1 antibodies can block binding of major-group rhinoviruses to cells, we considered that antagonism of virus-receptor interaction might be a way of preventing rhinovirus infection. We have constructed and purified a soluble form of the ICAM-1 molecule, which is normally membrane-bound, and demonstrated that it is a potent and specific inhibitor of rhinovirus infection.}, keywords = {970, CD54}, author = {Marlin,S.D. and Staunton,D.E. and Springer, T.A. and Stratowa,C. and Sommergruber,W. and Merluzzi,V.} } @article {529141, title = {On the species specificity of the interaction of LFA-1 with intercellular adhesion molecules}, journal = {J. Immunol.}, volume = {145}, number = {4}, year = {1990}, note = {MS$\#$206Reprint Status: In File}, pages = {1181-1187}, abstract = {Species restrictions in immune cell interactions have been demonstrated both in Ag-specific responses of T lymphocytes and the phenomenon of natural attachment. To determine the possible contribution of adhesion receptors to these restrictions, we have studied binding between the murine and human homologues of LFA-1 (CD11a/CD18) and ICAM employing purified human LFA-1 and ICAM-1 (CD54) bound to solid substrates. Murine cell lines bind to purified human LFA-1 through ICAM-1 and at least one other counter-receptor. This provides evidence for multiple counter-receptors for LFA-1 in the mouse as well as in the human. In contrast to binding of murine ICAM-1 to human LFA-1, murine LFA-1 does not bind to human ICAM-1. The species specificity maps to the LFA-1 alpha subunit, because mouse x human hybrid cells expressing the human alpha subunit associated with a mouse beta subunit bind to human ICAM-1, whereas those with a human beta subunit associated with a murine alpha subunit do not. Increased adhesiveness for ICAM-1 stimulated by phorbol esters could be demonstrated for hybrid LFA-1 molecules with human alpha and murine beta subunits.}, keywords = {CD54}, author = {Johnston,S.C. and Dustin,M.L. and Hibbs,M.L. and Springer, T.A.} } @article {528386, title = {Structure and function of leukocyte integrins}, journal = {Immunol. Rev.}, volume = {114}, year = {1990}, note = {Review, MS$\#$203Reprint Status: In File}, pages = {181-217}, keywords = {REVIEW}, author = {Larson,R.S. and Springer, T.A.} } @article {528806, title = {Transfection of cells from patients with leukocyte adhesion deficiency with an integrin β subunit (CD18) restores LFA-1 expression and function}, journal = {J. Clin. Invest.}, volume = {85}, number = {3}, year = {1990}, note = {MS$\#$198Reprint Status: In File}, pages = {674-681}, abstract = {Leukocyte adhesion deficiency (LAD) is an inherited immunodeficiency disease that is characterized by the deficient expression of the leukocyte adhesion glycoproteins lymphocyte function-associated antigen-1 (LFA-1), Mac-1, and p150,95. This loss of expression is attributed to heterogeneous defects in the common beta subunit shared by these glycoproteins. Here we demonstrate that expression of the LFA-1 alpha beta heterodimer in EBV-transformed B lymphoblastoid cells from LAD patients can be recovered after transfection with the beta subunit cDNA contained in an EBV-based vector. Four patients with differing severities of LAD comprising three distinct classes of mutations were studied. Flow cytometry analysis of stably transfected patient cells revealed near normal levels of expression of both the alpha and beta chains of LFA-1, and immunoprecipitation studies confirmed that fully processed alpha and beta chains were being expressed at the cell surface. In addition, Northern analysis of mRNA expression also demonstrated that the transfected LAD patient cells were expressing high quantities of exogenous beta subunit mRNA. Functional studies such as homotypic adhesion and adhesion to a purified counterreceptor for LFA-1, intracellular adhesion molecule-1, demonstrated that LFA-1 function had been restored in the stably transfected LAD patient cell lines. These studies unequivocally show that the defect in cells from patients with LAD is in the leukocyte integrin beta subunit.}, author = {Hibbs,M.L. and Wardlaw,A.J. and Stacker,S.A. and Anderson,D.C. and Lee,A. and Roberts,T.M. and Springer, T.A.} } @article {528846, title = {Adherence of neutrophils to cultured human microvascular endothelial cells: Dependence upon the Mac-1, LFA-1, p150,95 glycoprotein family}, journal = {J. Clin. Invest.}, volume = {83}, number = {2}, year = {1989}, note = {MS $\#$171Reprint Status: In File}, pages = {637-646}, abstract = {The process of neutrophil adhesion to and migration through the microvascular endothelium, an early event in the induction of the acute inflammatory response, has been attributed to the generation of extravascular chemoattractants. Although both chemotactic peptides and lipid mediators enhance neutrophil adherence in vitro and in vivo, the mechanism(s) involved in the interaction between circulating neutrophils and microvascular endothelial cells is still not completely understood. In a microtiter well adherence assay, the chemotactic peptides, FMLP and C5a, and the lipid mediators, leukotriene B4 (LTB4) and platelet activating factor (PAF), enhanced human neutrophil adherence to cultured human microvascular endothelial cells as well as to human umbilical vein endothelial cells in a dose-dependent manner with a rapid time course. This stimulated adhesive interaction between neutrophils and cultured human endothelial cells was dependent on the expression of the Mac-1, LFA-1, p150,95 glycoprotein family on the neutrophil surface since neutrophils from patients with leukocyte adhesion deficiency, lacking surface expression of the adhesive glycoproteins, exhibited markedly diminished adherence to human endothelial cells in response to stimulation with chemotactic factors compared to normal control neutrophils. All four mediators enhanced expression of the glycoprotein family on the surface of normal neutrophils as determined by flow cytofluorimetry using a monoclonal antibody (TS1/18) to the glycoprotein common beta subunit. In addition, TS1/18 inhibited up to 100\% the adherence of normal neutrophils to endothelial cells stimulated by maximal concentrations of FMLP, C5a, LTB4, or PAF. Moreover, HL-60 cells, human promyelocytic leukemia cells, neither increased glycoprotein surface expression nor adherence in response to stimulation. Thus, peptide and lipid mediators of the acute inflammatory response appear to enhance adherence of circulating neutrophils to the microvascular endothelium by a mechanism dependent on expression of the Mac-1, LFA-1, p150,95 glycoprotein family on the neutrophil surface.}, author = {Tonnesen,M.G. and Anderson,D.C. and Springer, T.A. and Knedler,A. and Avdi,N. and Henson,P.M.} } @inbook {530256, title = {Adhesion receptors regulate antigen-specific interactions, localization, and differentiation in the immune system}, booktitle = {Progress in Immunology}, volume = {7}, year = {1989}, pages = {121-130}, publisher = {Springer-Verlag Berlin Heidelberg}, organization = {Springer-Verlag Berlin Heidelberg}, author = {Springer, T.A.}, editor = {Melchers,F. and Albert, E. D. and Boehmer, H. V. and Dierich, M. P. and Du Pasquier, L. and Eichmann, K. and Gemsa, D. and G{\"o}tze, O. and Kalden, J. R. and Kaufmann, S. H. E. and Kirchner, H. and Resch, K. and Riethm{\"u}ller, G. and Schimpl, A. and Sorg, C. and Steinmetz, M. and Wagner, H. and Zachau, H.G. and Nicklin, L.} } @inbook {529711, title = {B cell and activation workshop monoclonal antibodies to phosphatidylinositol-anchored proteins}, booktitle = {Leukocyte Typing IV: White Cell Differentiation Antigens}, year = {1989}, note = {$\#$188Reprint Status: In File}, pages = {182-183}, publisher = {Oxford University}, organization = {Oxford University}, edition = {4}, address = {London}, author = {Hollander,N. and Low,M.G. and Springer, T.A.}, editor = {Knapp,W. and Dorken,B. and Gilks,W.R. and Rieber,E.P. and Schmidt,R.E. and Stein, H. and von dem Borne,A.E.G.Kr.} } @article {527991, title = {A cell adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses}, journal = {Cell}, volume = {56}, number = {5}, year = {1989}, note = {$\#$180Reprint Status: In File}, pages = {849-853}, abstract = {Rhinoviruses, which cause common colds, possess over 100 serotypes, 90\% of which (the major group) share a single receptor. Lymphocyte function associated molecule 1 (LFA-1) mediates leukocyte adhesion to a wide variety of cell types by binding to intercellular adhesion molecule 1 (ICAM-1). We demonstrate identity between the receptor for the major group of rhinoviruses and ICAM-1. A major group rhinovirus binds specifically to purified ICAM-1 and to ICAM-1 expressed on transfected COS cells, and binding is blocked by three ICAM-1 monoclonal antibodies (MAb) that block ICAM-1-LFA-1 interaction, but not by an ICAM-1 MAb that does not block ICAM-1-LFA-1 interaction. This suggests that the ICAM-1 contact site(s) for LFA-1 and rhinoviruses is proximal or identical. In addition, ICAM-1 MAb block the cytopathic effect in HeLa cells mediated by representative major but not minor group rhinoviruses. ICAM-1 is induced by soluble mediators of inflammation, suggesting that the host immune response to rhinovirus may facilitate spread to uninfected cells.}, keywords = {970}, author = {Staunton,D.E. and Merluzzi,V.J. and Rothlein,R. and Barton,R. and Marlin,S.D. and Springer, T.A.} } @inbook {528371, title = {A cluster of antibodies (RR1/1, LB-2 and 84H10) that inhibit LFA-1-dependent lymphoid and myeloid cell adhesion bind intercellular adhesion molecule-1 (ICAM-1)}, booktitle = {Immunobiology of HLA Volume II: Immunogenetics and Histocompatibility}, year = {1989}, note = {MS$\#$191Reprint Status: In File}, pages = {577-580}, publisher = {Springer-Verlag}, organization = {Springer-Verlag}, address = {New York}, author = {Makgoba,M.W. and Sanders,M.E. and Ginther Luce,G.E. and Dustin,M.L. and Mannoni,P. and Clark,E.A. and Springer, T.A. and Shaw,S.}, editor = {Dupont,B.} } @inbook {528376, title = {Coordinate-enhanced expression of three adhesion molecules (LFA-3, CD2, and LFA-1) and three other molecules (4B4, UCHL1, and Pgp-1) defines a human T-cell subset containing memory cells and characterized by enhanced γ interferon production}, booktitle = {Immunobiology of HLA Volume II: Immunogenetics and Histocompatibility}, year = {1989}, note = {MS$\#$190Reprint Status: In File}, pages = {560-562}, publisher = {Springer-Verlag}, organization = {Springer-Verlag}, address = {New York}, author = {Sanders,M.E. and Makgoba,M.W. and Sharrow,S.O. and Stephany,D. and Springer, T.A. and Young,H.A. and Shaw,S.}, editor = {Dupont,B.} } @article {528911, title = {Correlation of CD2 binding and functional properties of multimeric and monomeric lymphocyte function associated antigen-3}, journal = {J. Exp. Med.}, volume = {169}, number = {2}, year = {1989}, pages = {503-517}, abstract = {LFA-3 was purified with an intact (mLFA-3) or an enzymatically removed membrane-anchoring domain (sLFA-3). Gel filtration and sucrose gradient sedimentation showed sLFA-3 to be a single highly glycosylated polypeptide chain in solution, while mLFA-3 formed micelles of 8 LFA-3 monomers. 125I-mLFA-3 bound to Jurkat T leukemic cell surface CD2 with much higher avidity than sLFA-3. mLFA-3 binding had characteristics of a multivalent interaction with cell surface CD2 and had an avidity of 1.5 nM for Jurkat cells and 12 nM for resting T cells. Two CD2 mAbs tested did not block mLFA-3 binding: 9-1 and CD2.1. These mAbs were tested in combination with LFA-3 for their ability to activate T cells. The combination of mLFA-3 and CD2.1 mAbs induced a rapid increase in cytosolic free Ca2+ in Jurkat cells which was proportional to mLFA-3 occupation of CD2 sites. sLFA-3 showed no activity in the Ca2+ flux assay. The combination of mLFA-3 and the CD2.1 mAbs significantly stimulated proliferation of PBMC. The combination of mLFA-3 and 9-1 mAbs was weakly or not mitogenic for the same cells. The combination of CD2.1 and sLFA-3 at concentrations up to 480 nM was not consistently mitogenic. Therefore, monomeric LFA-3/CD2 interaction appears to have a relatively low affinity, while multimeric LFA-3 binds with high avidity. T cell activation by binding of LFA-3 to CD2 appears to require occupation of 10(4) to 10(5) CD2 sites, which is likely to occur during adhesion, but is rare in receptor systems with soluble ligands.}, keywords = {CD16}, author = {Dustin,M.L. and Olive,D. and Springer, T.A.} } @inbook {529501, title = {Differential effects on leukocyte functions of CD11a, CD11b, and CD18 monoclonal antibodies}, booktitle = {Leucocyte Typing IV: White Cell Differentiation Antigens}, year = {1989}, note = {$\#$185Reprint Status: In Fileerased duplicate data $\#$6206.}, pages = {570-574}, publisher = {Oxford University Press}, organization = {Oxford University Press}, edition = {4}, address = {Oxford}, author = {Diamond,M.S. and Johnston,S.C. and Dustin,M.L. and McCaffery,P. and Springer, T.A.}, editor = {Knapp,W. and Dorken,B. and Gilks,W.R. and Rieber,E.P. and Schmidt,R.E. and Stein, H. and von dem Borne,A.E.G.Kr.} } @article {529131, title = {Distinct restriction of complement- and cell-mediated lysis}, journal = {J. Immunol.}, volume = {142}, number = {11}, year = {1989}, note = {$\#$179Reprint Status: In File}, pages = {3913-3916}, abstract = {Complement- and cell-mediated killing utilize related effector proteins (C8/C9 and perforin, respectively), suggesting that proteins which protect cells against complement- and cell-mediated attack may also be similar. In homologous complement-mediated killing two protective proteins, which are anchored to the cell membrane by phosphatidylinositol glycan (PIG) tails, are known. To study whether similar PIG-tailed proteins protect against lymphocyte-mediated killing, nucleated cell lines with a mutation in the biosynthesis of the PIG anchor were used. It was found that PIG-tailed membrane proteins restrict homologous complement-mediated lysis but not three different types of cell-mediated killing or lysis by purified perforin. Furthermore, E from patients with an acquired defect in PIG tail biosynthesis did not differ from normal E in sensitivity to antibody-dependent cell-mediated cytotoxicity, in spite of their increased sensitivity to human C8 and C9.}, keywords = {770.3}, author = {Hollander,N. and Shin,M.L. and Rosse,W. and Springer, T.A.} } @article {529991, title = {Functional cloning of ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1}, journal = {Nature}, volume = {339}, number = {6219}, year = {1989}, note = {$\#$181Reprint Status: In File}, pages = {61-64}, abstract = {The leukocyte adhesion molecule LFA-1 mediates a wide range of lymphocyte, monocyte, natural killer cell, and granulocyte interactions with other cells in immunity and inflammation. LFA-1 (CD11a/CD18) is a receptor for intercellular adhesion molecule 1 (ICAM-1, CD54), a surface molecule which is constitutively expressed on some tissues and induced on other in inflammation. Induction of ICAM-1 on epithelial cells, endothelial cells and fibroblasts mediates LFA-1-dependent adhesion of lymphocytes. Several lines of evidence have suggested the existence of a second LFA-1 ligand: homotypic adhesion of one cell line was inhibited by a monoclonal antibody to LFA-1, but not by one to ICAM-1; there exists an LFA-1-dependent, ICAM-1-independent pathway of adhesion to endothelial cells; and also, there are some types of target cells in which LFA-1-dependent T-lymphocyte adhesion and lysis are independent of ICAM-1. We have cloned this second ligand, designated ICAM-2, using a novel method for identifying ligands of adhesion molecules. ICAM-2 is an integral membrane protein with two immunoglobulin-like domains, whereas ICAM-1 has five. Remarkably, ICAM-2 is much more closely related to the two most N-terminal domains of ICAM-1 (34\% identity) than either ICAM-1 or ICAM-2 is to other members of the immunoglobulin superfamily, demonstrating the existence of a subfamily of immunoglobulin-like ligands that bind the same integrin receptor.}, author = {Staunton,D.E. and Dustin,M.L. and Springer, T.A.} } @inbook {528116, title = {Human leukocyte adhesion deficiency: Molecular basis for a defective immune response to infections of the skin}, booktitle = {Current Problems in Dermatology}, year = {1989}, note = {Proceedings of the Immunology and Skin Congress, European Dermatology Society, Innsbruck, Austria, MS $\#$172Reprint Status: In File}, pages = {106-115}, publisher = {Karger}, organization = {Karger}, edition = {18}, address = {Basel}, author = {Kishimoto,T.K. and Springer, T.A.}, editor = {Fritsch,P. and Schuler,G. and Hintner,H.} } @article {527831, title = {Immunohistologic analysis of the distribution of cell adhesion molecules within the inflammatory synovial microenvironment}, journal = {Arthritis Rheu.}, volume = {32}, number = {1}, year = {1989}, note = {MS $\#$170Reprint Status: In File}, pages = {22-30}, abstract = {Antigen-independent binding of T lymphocytes to a variety of cell types has been shown to be mediated by receptor-ligand pairs of adhesion molecules. In forms of inflammatory synovitis (including rheumatoid arthritis), T cells home to synovium, become activated, and participate in the generation of chronic synovitis. Using indirect immunofluorescence assays on synovial frozen tissue sections and on synovial fibroblast cell lines, we studied the distribution of cell adhesion molecules on components of the synovial microenvironment in inflammatory synovitis. We reasoned that analysis of the cell types within synovium that express adhesion molecules might provide clues to lymphocyte-stromal interactions that occur in inflammatory synovitis. We found that antibodies against the lymphocyte function-associated antigen 3 (LFA-3) molecule and the intercellular adhesion molecule 1 (ICAM-1) both reacted with macrophage-like type A synovial cells and synovial fibroblasts, as well as with tissue macrophages and vessel endothelium. Using flow cytometry, we found that anti-LFA-3 and anti-ICAM-1 (but not antibodies against their ligands CD2 and LFA-1) reacted with synovial fibroblast cells cultured in vitro. Thus, these data demonstrate that the ligands for lymphocyte LFA-1 molecules (ICAM-1) and for T cell CD2 molecules (LFA-3) are widely distributed among cell types of the synovial microenvironment and provide numerous cell types with which lymphocytes can interact via these 2 adhesion pathways during the course of inflammatory synovitis.}, author = {Hale,L.P. and Martin,M.E. and McCollum,D.E. and Nunley,J.A. and Springer, T.A. and Singer,K.H. and Haynes,B.F.} } @article {527776, title = {Intercellular adhesion molecule-1 (ICAM-1) monoclonal antibody inhibits cytotoxic T lymphocyte recognition}, journal = {Ann. N. Y. Acad. Sci.Annals of the New York Academy of Sciences}, volume = {532}, year = {1989}, note = {MS$\#$193Reprint Status: In File}, pages = {427-428}, author = {Makgoba,M.W. and Sanders,M.E. and Luce,G.E.G. and Gugel,E.A. and Dustin,M.L. and Springer, T.A. and Shaw,S.} } @article {528521, title = {Leukocyte adhesion deficiency: Aberrant splicing of a conserved integrin sequence causes a moderate deficiency phenotype}, journal = {J. Biol. Chem.}, volume = {264}, number = {6}, year = {1989}, note = {MS $\#$177}, pages = {3588-3595}, abstract = {Leukocyte adhesion deficiency (LAD) is a heritable deficiency of the LFA-1, Mac-1, p150,95 family of leukocyte alpha beta heterodimers (the leukocyte integrins). We have studied the defect in patients who synthesize an aberrantly small form of the beta subunit common to all three proteins. S1 nuclease protection showed the presence of a 90-nucleotide mismatch in RNA from patients and relatives, correlating with inheritance of the disease. Use of the Taq polymerase chain reaction to amplify this region of RNA after first strand cDNA synthesis and sequencing showed an in-frame deletion of 90 nucleotides in the extracellular domain. Thus, this highly conserved region, 63\% and 53\% identical in amino acid sequence to two other beta subunits of the integrin family, is required for association of the beta subunit with alpha subunits. The 90-nucleotide region corresponds to a single exon present in both the normal and patient genome. The patient DNA has a single G to C substitution in the 5{\textquoteright} splice site. This results in the direct joining of nonconsecutive exons in an unusual type of abnormal RNA splicing. A small amount of normally spliced message, detected by S1 nuclease protection and Taq polymerase chain reaction, encodes a normal sized beta subunit which is surface-expressed and accounts for the low levels of leukocyte integrin expression observed in these patients, and hence the moderate phenotype.}, author = {Kishimoto,T.K. and O{\textquoteright}Connor,K. and Springer, T.A.} } @inbook {529746, title = {Leukocyte adhesion deficiency and other disorders of leukocyte motility}, booktitle = {Metabolic Basis of Inherited Disease}, year = {1989}, note = {$\#$158Reprint Status: In File}, pages = {2751-2777}, publisher = {McGraw-Hill}, organization = {McGraw-Hill}, edition = {6}, address = {New York}, author = {Anderson,D.C. and Smith,C.W. and Springer, T.A.}, editor = {Stanbury,J.B. and Wyngaarden,J.B. and Frederickson,D.S.} } @article {527761, title = {The leukocyte integrins: LFA-1, Mac-1, and p150,95}, journal = {Adv. Immunol.}, volume = {46}, year = {1989}, note = {$\#$178Reprint Status: In File}, pages = {149-182}, author = {Kishimoto,T.K. and Larson,R.S. and Corbi,A.L. and Dustin,M.L. and Staunton,D.E. and Springer, T.A.} } @article {530291, title = {Mechanisms for regulating expression of membrane isoforms of FcγRIII (CD16)}, journal = {Science}, volume = {246}, number = {4937}, year = {1989}, note = {MS$\#$197}, pages = {1608-1611}, abstract = {Granulocyte and natural killer (NK) cell Fc receptors for immunoglobulin G (CD16) differ in only a few amino acids, yet have phosphatidylinositol glycan (PIG) or polypeptide membrane anchors, respectively. Mutagenesis shows that anchoring is regulated by a serine residue near the PIG anchor attachment site in the extracellular domain. The NK cell isoform was not expressed on the surface of COS cells unless cotransfected with a subunit that was expressed in NK cells and that was identical to the gamma subunit of the high affinity IgE Fc receptor (Fc epsilon RI). However, the CD16 sequence and not expression of the gamma subunit is dominant in regulating PIG reanchoring.}, keywords = {CD16}, author = {Hibbs,M.L. and Selvaraj, P. and Carpen,O. and Springer, T.A. and Kuster,H. and Jouvin,M.H. and Kinet,J.P.} } @article {529301, title = {Natural killer cell and granulocyte Fcγ receptor III (CD16) differ in membrane anchor and signal transduction}, journal = {J. Immunol.}, volume = {143}, number = {10}, year = {1989}, note = {$\#$181Reprint Status: In File}, pages = {3283-3288}, abstract = {CD16 is a low affinity Fc gamma R III expressed on granulocytes, macrophages and large granular lymphocytes, the mediators of antibody-dependent cellular cytotoxicity and NK. The occupancy of CD16 by aggregated IgG on large granular lymphocytes induces expression of activation markers, release of inflammatory mediators and triggering of effector functions such as antibody-dependent cellular cytotoxicity. Recently we and others described that CD16 is anchored to the membrane of granulocytes via a phosphatidylinositol glycan moiety. Here we show that the CD16 molecule expressed on NK cells, cultured monocytes, and lung macrophages is not phosphatidylinositol glycan moiety anchored. It is not released with phosphatidylinositol-specific phospholipase C, and after removal of N-linked carbohydrate is 5 to 7 kDa larger than the granulocyte CD16 molecule, strongly suggesting the presence of transmembrane and cytoplasmic protein domains. Redirected killing of hybridoma targets expressing anti-CD16 surface Ig shows that NK cell CD16 is unable to do so. These findings demonstrate that NK cell and granulocyte CD16 have different membrane anchors and indicate that the type of membrane anchor is an important biologic mechanism for regulating the functional capacity of surface receptors.}, author = {Selvaraj, P. and Carpen,O. and Hibbs,M.L. and Springer, T.A.} } @inbook {529726, title = {Phosphatidylinositol-anchored antigens defined by non-lineage monoclonal antibodies}, booktitle = {Leukocyte Typing IV: White Cell Differentiation Antigens}, year = {1989}, note = {$\#$186Reprint Status: In File}, pages = {743-744}, publisher = {Oxford University}, organization = {Oxford University}, edition = {4}, address = {London}, author = {Selvaraj, P. and Low,M.G. and Lopez,P. and Springer, T.A.}, editor = {Dorken,B. and Gilks,W.R. and Rieber,E.P. and Schmidt,R.E. and Stein, H. and von dem Borne,A.E.G.Kr.} } @article {528746, title = {Primary structure of the LFA-1 α subunit: An integrin with an embedded domain defining a protein superfamily}, journal = {J. Cell Biol.}, volume = {108}, number = {2}, year = {1989}, note = {$\#$180Reprint Status: In File}, pages = {703-712}, abstract = {The leukocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is a membrane glycoprotein which functions in cell-cell adhesion by heterophilic interaction with intercellular adhesion molecule 1 (ICAM-1). LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report the molecular biology and protein sequence of the alpha subunit. Overlapping cDNAs containing 5,139 nucleotides were isolated using an oligonucleotide specified by tryptic peptide sequence. The mRNA of 5.5 kb is expressed in lymphoid and myeloid cells but not in a bladder carcinoma cell line. The protein has a 1,063-amino acid extracellular domain, a 29-amino acid transmembrane region, and a 53-amino acid cytoplasmic tail. The extracellular domain contains seven repeats. Repeats V-VII are in tandem and contain putative divalent cation binding sites. LFA-1 has significant homology to the members of the integrin superfamily, having 36\% identity with the Mac-1 and p150,95 alpha subunits and 28\% identity with other integrin alpha subunits. An insertion of approximately 200 amino acids is present in the NH2-terminal region of LFA-1. This "inserted/interactive" or I domain is also present in the p150,95 and Mac-1 alpha subunits but is absent from other integrin alpha subunits sequenced to date. The I domain has striking homology to three repeats in human von Willebrand factor, two repeats in chicken cartilage matrix protein, and a region of complement factor B. These structural features indicate a bipartite evolution from the integrin family and from an I domain family. These features may also correspond to relevant functional domains.}, author = {Larson,R.S. and Corbi,A.L. and Berman,L. and Springer, T.A.} } @inbook {529721, title = {Reactivity of workshop CD16 panel monoclonal antibodies with distinct membrane anchored forms of CD16}, booktitle = {Leukocyte Typing IV: White Cell Differentiation Antigens}, year = {1989}, note = {$\#$187Reprint Status: In File}, pages = {595-597}, publisher = {Oxford University}, organization = {Oxford University}, edition = {4}, address = {London}, author = {Selvaraj, P. and Hibbs,M.L. and Carpen,O. and Springer, T.A.}, editor = {Dorken,B. and Gilks,W.R. and Rieber,E.P. and Schmidt,R.E. and Stein, H. and von dem Borne,A.E.G.Kr.} } @article {527781, title = {Spontaneous rosetting of T lymphocytes to Reed-Sternberg cells is mediated by the CD2/LFA-3 and LFA-1/ICAM-1 pathways of antigen-independent adhesion}, journal = {Ann. N. Y. Acad. Sci.}, volume = {532}, year = {1989}, note = {MS$\#$192Reprint Status: In File}, pages = {436-438}, author = {Sanders,M.E. and Makgoba,M.W. and Sussman,E.H. and Luce,G.E.G. and Springer, T.A. and Cossman,J. and Shaw,S.} } @inbook {528061, title = {Structure and regulation of the leukocyte adhesion receptor LFA-1 and its counter-receptors, ICAM-1 and ICAM-2}, booktitle = {Cold Spring Harbor Symp. Quant. Biol.}, volume = {54}, year = {1989}, pages = {753-765}, author = {Dustin,M.L. and Aguilar,J.G. and Hibbs,M.L. and Larson,R.S. and Stacker,S.A. and Staunton,D.E. and Wardlaw,A.J. and Springer, T.A.} } @inbook {529716, title = {The subunit specificity of the CD11a/CD18, CD11b, and CD11c panels of antibodies}, booktitle = {Leukocyte Typing IV: White Cell Differentiation Antigens}, year = {1989}, note = {$\#$184Reprint Status: In File}, pages = {566-570}, publisher = {Oxford University}, organization = {Oxford University}, edition = {4}, address = {London}, author = {Larson,R.S. and Hibbs,M.L. and Corbi,A.L. and Luther,E. and Garcia-Aguilar,J. and Springer, T.A.}, editor = {Knapp,W. and Dorken,B. and Gilks,W.R. and Rieber,E.P. and Schmidt,R.E. and Stein, H. and von dem Borne,A.E.G.Kr.} } @article {529911, title = {T cell receptor cross-linking transiently stimulates adhesiveness through LFA-1}, journal = {Nature}, volume = {341}, number = {6243}, year = {1989}, note = {$\#$189Reprint Status: In File}, pages = {619-624}, abstract = {Effective interaction between T cells and their targets requires that recognition of specific antigen be coordinated with increased cell-cell adhesion. We show that antigen-receptor cross-linking increases the strength of the adhesion mechanism between lymphocyte function-associated molecule-1 (LFA-1) and intercellular adhesion molecules (ICAMs), with intracellular signals transmitted from the T-cell antigen receptor to the LFA-1 adhesion molecule. The increase in avidity is rapid and transient, providing a dynamic mechanism for antigen-specific regulation of lymphocyte adhesion and de-adhesion.}, keywords = {189, CD11a}, author = {Dustin,M.L. and Springer, T.A.} } @article {528921, title = {Adhesion of T lymphoblasts to epidermal keratinocytes is regulated by interferon γ and is mediated by intercellular adhesion molecule-1 (ICAM-1)}, journal = {J. Exp. Med.}, volume = {167}, number = {4}, year = {1988}, note = {MS$\#$160, CA31798, Tobacco researchReprint Status: In File}, pages = {1323-1340}, abstract = {The cell surface expression and function of the LFA-1 ligand, intercellular adhesion molecule 1 (ICAM-1), on epidermal keratinocytes (EK) was studied. ICAM-1 expression on the surface of cultured EK was either absent or weak, but was induced by treating EK with rIFN-gamma or TNF for 4-48 h. IFN-gamma and TNF were synergistic. IFN-gamma treatment increased T lymphoblast adhesion from less than 2\% to 20-40\%, with a concentration dependence similar to that seen for ICAM-1 induction. All of the adhesion to EK was inhibited by LFA-1 and ICAM-1 mAbs, but not by HLA-DR, CD2, or LFA-3 mAbs. There was no difference in the level of T lymphoblast adhesion to IFN-gamma-treated allogeneic or autologous EK. ICAM-1 purified from the HeLa epithelioid cell line and reconstituted into planar membranes also supported efficient adhesion of T lymphoblasts that was blocked by LFA-1 mAb bound to the T lymphoblasts or ICAM-1 mAb bound to the planar membranes. T lymphoblasts adherent to EK or ICAM-1 planar membranes were isolated by panning, and surface markers were analyzed by immunofluorescence flow cytometry. The adherent T cells were a phenotypically skewed subpopulation. They were enriched for CD8+ cells and expressed 1.5-2.5-fold higher LFA-1 and CD2 compared with the unseparated population.}, author = {Dustin,M.L. and Singer,K.H. and Tuck,D.T. and Springer, T.A.} } @article {529126, title = {Biosynthesis and function of LFA-3 in human mutant cells deficient in phosphatidylinositol anchored proteins}, journal = {J. Immunol.}, volume = {141}, number = {12}, year = {1988}, note = {MS $\#$176Reprint Status: In File}, pages = {4283-4290}, abstract = {Mutants that lack expression of phosphatidylinositol (PI)-anchored proteins were derived from the human B lymphoblastoid JY cell line. It was demonstrated that unlike wild-type cells, which normally express both a transmembrane and a PI-linked form of LFA-3 glycoprotein, the mutant cells expressed only the transmembrane form of LFA-3. [3H]Ethanolamine was not incorporated into LFA-3 of mutant cells, indicating that the anchor moiety was entirely missing. Blockade of normal biosynthesis of the PI-anchored form led to accumulation of two intermediates that may have intact and truncated polypeptide chains. The truncated LFA-3, which was not attached to the cell membrane, was secreted by mutant cells into culture supernatants. A possible division of adhesion function between the two forms of LFA-3 was studied by using the JY cell lines as targets for CTL. Wild-type and mutant JY cells formed conjugates with CTL and were subsequently lysed to a similar extent. In addition, wild-type and mutant JY cells stimulated CTL proliferation to the same extent. Antibody-blocking experiments demonstrated a predominant role for the CD2/LFA-3 pathway in interaction of both wild-type and mutant cells with CTL. Because E exclusively express only the PI-linked LFA-3 form, and this form is known to mediate cell adhesion, the present results indicate that the two distinct membrane-anchored LFA-3 forms are each capable of mediating adhesion. A possible division of signaling functions between the two forms of LFA-3 is under investigation.}, keywords = {618}, author = {Hollander,N. and Selvaraj, P. and Springer, T.A.} } @article {529046, title = {The CD2 ligand LFA-3 activates T cells but depends on the expression and function of the antigen receptor}, journal = {J. Immunol.}, volume = {141}, number = {6}, year = {1988}, note = {MS$\#$163Reprint Status: NOT In File}, pages = {1904-1911}, abstract = {The T cell Ag receptor (CD3/Ti) and the sheep E receptor (CD2) expressed on the surface of human T cells are both capable of initiating intracellular signals necessary for T cell activation. CD3/Ti interacts with Ag to initiate cellular immune responses. Although the exact function of CD2 is unknown, lymphocyte function-associated Ag 3 (LFA-3), a 55- to 70-kDa receptor expressed on a broad spectrum of hemopoietic and nonhemopoietic cells, has recently been shown to be its natural ligand. We show here that although purified multimeric LFA-3 is not capable of initiating transmembrane signaling events on its own, the combination of LFA-3 and the anti-CD2 mAb CD2.1 induces intracellular calcium increases, phosphatidylinositol second messenger generation and lymphokine secretion in the T cell leukemic line Jurkat. In order to study the signaling requirements of CD2, we compared the ability of CD2 mAb and LFA-3 to initiate activation signals in Jurkat and in three Jurkat-derived mutants. A CD3-CD2+ mutant failed to increase calcium or exhibit phosphatidylinositol hydrolysis to either the combination of agonist CD2 mAb 9-1 and 9.6 or LFA-3 and CD2.1. Reconstitution of the Ag receptor by transfection of the Ti-beta-chain restored the expression of the CD3/Ti complex and the ability to respond to either combination of CD2 ligands. However, no response to CD2 ligands was detected in a CD3+CD2+ mutant selected for signaling defects to CD3/Ti ligands. Complementation of the CD3/Ti signaling defect by cell fusion also restored competency to respond to CD2 agonists. These results demonstrate that LFA-3 under appropriate conditions can activate T cells via the CD2 complex and that this activation requires not only the cell surface expression of the CD3/Ti complex but also a functional Ag receptor pathway.}, author = {Bockenstedt,L.K. and Goldsmith,M.A. and Dustin,M.L. and Olive,D. and Springer, T.A. and Weiss,A.} } @article {528881, title = {Chromosomal location of the genes encoding the leukocyte adhesion receptors LFA-1, Mac-1 and p150,95. Identification of a gene cluster involved in cell adhesion}, journal = {J. Exp. Med.}, volume = {167}, number = {5}, year = {1988}, note = {MS$\#$165Reprint Status: In File}, pages = {1597-1607}, abstract = {The adhesion receptors Mac-1, LFA-1, and p150,95 are cell surface alpha/beta heterodimers that play a key role in leukocyte adhesion processes. The genes for Mac-1, LFA-1, and p150,95 alpha subunits have been located to chromosome 16 by means of Southern blot analysis using a series of somatic cell hybrids. Chromosomal in situ hybridization has demonstrated that the genes for the three alpha subunits map to the short arm of chromosome 16, between bands p11 and p13.1, defining a cluster of genes involved in leukocyte adhesion. The gene encoding the LFA-1/Mac-1/p150,95 beta subunit, and defective in leukocyte adhesion deficiency, has been located on chromosome 21, band q22. The leukocyte adhesion receptor alpha and beta subunits are mapped to chromosomal regions that have been shown to be involved in cytogenetic rearrangements in certain patients with acute myelomonocytic leukemia and the blast phase of chronic myelogenous leukemia, respectively.}, author = {Corbi,A.L. and Larson,R.S. and Kishimoto,T.K. and Springer, T.A. and Morton,C.C.} } @article {529056, title = {Endothelial activation during interleukin 2 (IL 2) immunotherapy: A possible mechanism for the vascular leak syndrome}, journal = {J. Immunol.}, volume = {140}, year = {1988}, note = {MS $\#$152Reprint Status: In File}, pages = {1883-1888}, abstract = {A major sequela of immunotherapy with interleukin 2 (IL-2) is development of a vascular leak syndrome. The pathogenesis of this toxic effect is not known. We have examined pre- and post-treatment skin biopsies from 14 patients undergoing systemic administration of IL-2 for evidence of endothelial cell activation. Specifically, we have used the immunoperoxidase technique to detect the expression of three different activation antigens: endothelial-leukocyte adhesion molecule 1, detected with monoclonal antibody H4/18; intercellular adhesion molecule 1, detected with antibody RR1/1; and histocompatibility leukocyte antigen-DQ, detected with antibody Leu 10. Each of these antigens may be induced on cultured endothelial cells by various cytokines (although not by IL-2) and is expressed during endothelial cell activation in vivo at sites of delayed hypersensitivity and other immune responses. Pretreatment biopsies from each patient showed no endothelial expression of endothelial-leukocyte adhesion molecule 1 and only weak to moderate expression of intercellular adhesion molecule 1 and histocompatibility leukocyte antigen-DQ (except for one specimen unreactive with Leu 10). After 5 days of treatment, every patient showed marked endothelial expression of all three antigens (except for the same patient who remained unreactive with Leu 10). Endothelial-leukocyte adhesion molecule-1 expression was confined to postcapillary venular endothelium whereas intercellular adhesion molecule-1 and Leu 10 also were expressed on stromal cells and mononuclear cells. Thus, we conclude that i.v. administration of IL-2 leads to endothelial cell activation. Because IL-2 fails to induce the same antigens on cultured endothelial cells, we infer that IL-2 acts in vivo by inducing the production of other cytokines (e.g., interleukin 1, tumor necrosis factor, lymphotoxin, and interferon-gamma). Finally, since endothelial cell activation at sites of cell-mediated immune responses is well known to result in vascular leakiness to macromolecules, we propose that the vascular leak syndrome accompanying IL-2 therapy may arise from widespread inappropriate endothelial cell activation.}, author = {Cotran,R.S. and Pober,J.S. and Gimbrone,M.A.,Jr. and Springer, T.A. and Wiebke,E.A. and Gaspari,A.A. and Rosenberg,S.A. and Lotze,M.T.} } @article {529491, title = {Epstein-Barr virus latent infection membrane protein alters the human B lymphocyte phenotype: Deletion of the amino terminus abolishes activity}, journal = {J. Virol.}, volume = {62}, number = {11}, year = {1988}, note = {MS $\#$173Reprint Status: In File}, pages = {4173-4184}, abstract = {A latent infection membrane protein (LMP) encoded by the Epstein-Barr virus (EBV) genome in latently infected, growth-transformed lymphocytes alters the phenotype of a human EBV-negative B-lymphoma cell line (Louckes) when introduced by gene transfer. These LMP-expressing cells exhibit increased homotypic adhesion due to increased expression of the adhesion molecules LFA-1 and ICAM-1. Increased homotypic adhesion could foster B-cell growth by facilitating autocrine growth factor effects. LFA-3 expression is also induced. The induction of LFA-3 and ICAM-1 results in increased heterotypic adhesion to T lymphocytes. This could result in more effective T-cell immune surveillance. Since LMP is expressed in EBV-transformed lymphocytes and has been demonstrated to transform rodent fibroblasts in vitro, a wide range of possible effects on B-lymphoma cell growth were assayed. In the Louckes B-lymphoma cell line, EBV LMP causes increased cell size, acid production, plasma membrane ruffling, and villous projections. Although cell proliferation rate was not greatly affected, the steady-state intracellular free calcium level, transforming growth factor beta responsiveness, and expression of the lymphocyte activation markers (CD23 and transferrin receptor) were increased. Thus, LMP appears to be a mediator of EBV effects on B-cell transformation. In transfected lymphoma cells, LMP localizes to patches at the cell periphery and associates with the cytoskeleton as it does in EBV-transformed B lymphocytes or in rodent fibroblasts. A partially deleted form of LMP (D1LMP) does not aggregate in patches or associate with the cytoskeleton and had little effect on B-cell growth. Thus, cytoskeletal association may be integral to LMP activity.}, author = {Wang, D and Liebowitz, D. and Wang, F and Gregory, C. and Rickinson, A. and Larson,R.S. and Springer, T.A. and Kieff, E.} } @article {528186, title = {Functional evidence that intercellular adhesion molecule-1 (ICAM-1) is a ligand for LFA-1 in cytotoxic T cell recognition}, journal = {Eur. J. Immunol.}, volume = {18}, number = {4}, year = {1988}, note = {MS $\#$144Reprint Status: In File}, pages = {637-640}, abstract = {Although intercellular adhesion molecule-1 (ICAM-1) has been implicated as a ligand in some LFA-1-dependent adhesion, its importance to T cell function has not been established. The present studies investigate the importance of ICAM-1 for human cytotoxic T lymphocytes (CTL), both in their formation of antigen-independent conjugates (AIC) and in their lysis of targets. Analysis of monoclonal antibody (mAb) inhibition of AIC formation indicate that ICAM-1 mAb 1 blocks (a) AIC formation with some but not all targets; (b) the LFA-1 pathway but not the CD2/LFA-3 pathway of adhesion; (c) by binding to the target cell, not the T cell. In studies of cell-mediated lysis (CML) ICAM-1 mAb inhibited lysis of some targets, such as U-937, that use ICAM-1 predominantly in AIC formation; CML on some other targets is not inhibited by ICAM-1 mAb. These data indicate that ICAM-1 is a ligand for AIC formation, antigen-specific CTL recognition and cytolysis of particular target cells. The data also indicate that ICAM-1 is not used in LFA-1-dependent CTL interactions with all kinds of target cells, suggesting the existence of alternative ligands for LFA-1.}, author = {Makgoba,M.W. and Sanders,M.E. and Ginther Luce,G.E. and Gugel,E.A. and Dustin,M.L. and Springer, T.A. and Shaw,S.} } @article {528471, title = {The human leukocyte adhesion glycoprotein Mac-1 (complement receptor type 3, CD11b) α subunit}, journal = {J. Biol. Chem.}, volume = {263}, number = {25}, year = {1988}, pages = {12403-12411}, abstract = {Mac-1 (CD 11b/CD18) is a leukocyte adhesion heterodimeric glycoprotein which functions both as a receptor for iC3b (CR3) and in several cell-cell and cell-substrate adhesive interactions. We describe full-length cDNA clones for the alpha subunit of Mac-1. Mac-1 alpha subunit message was detected in blood monocytes and phorbol-12-myristate acetate-induced myeloid cell lines, but not in cells of the T or B lineages, correlating with Mac-1 protein surface expression. The alpha subunit of Mac-1 is a transmembrane protein of 1137 residues with a long extracellular domain (1092 residues) and a 19-amino acid cytoplasmic tail. The extracellular domain contains three putative divalent cation-binding sequences and 19 potential N-glycosylation sites. The amino acid sequence of Mac-1 alpha shows that it is a member of the integrin superfamily; Mac-1 alpha shows 63\% identity to the alpha subunit of the leukocyte adhesion glycoprotein p150.95 and 25\% to the alpha subunits of the extracellular matrix receptors platelet glycoprotein IIb/IIIa, the fibronectin receptor, and the vitronectin receptor. The Mac-1 alpha subunit putative divalent cation-binding sites and the flanking regions exhibit a high degree of identity both to the p150.95 alpha subunit (87\% identity at the amino acid level) and to the rest of the integrin alpha subunits (38\%). The alpha subunit of Mac-1, like the p150.95 alpha subunit, contains a domain of 187 amino acids in the extracellular region which is absent in other integrins. This leukocyte or "L" domain is homologous to the A domains of von Willebrand factor, which in turn are homologous to regions of the C3-binding proteins factor B and C2. These findings draw attention to this region of Mac-1 as a potential binding site for iC3b.}, keywords = {551}, author = {Corbi,A.L. and Kishimoto,T.K. and Miller,L.J. and Springer, T.A.} } @article {529286, title = {Human memory T lymphocytes express increased levels of three cell adhesion molecules (LFA-3, CD3, LFA-1) and three other molecules (UCHL1, CDw29, and Pgp-1) and have enhanced IFN-γ production}, journal = {J. Immunol.}, volume = {140}, number = {5}, year = {1988}, note = {MS $\#$153Reprint Status: In File}, pages = {1401-1407}, abstract = {Studies of cell-surface molecules involved in human T cell interaction reveal that differential expression of each of three adhesion molecules (LFA-3, CD2, and LFA-1) subdivides human peripheral blood T cells into major subpopulations. Systematic analysis of the relationship between expression of these and other markers of T cell subsets demonstrates a single major subset of human peripheral blood T lymphocytes distinguished by enhanced expression of LFA-3, CD2, LFA-1, and three other markers (CDw29 [4B4], UCHL1, and Pgp-1). Large differences in relative expression are observed for UCHL1 (29-fold) and LFA-3 (greater than 8-fold), and smaller differences (2- to 4-fold) are seen for CDw29, CD2, LFA-1, and Pgp-1. Bimodal distribution of LFA-3 is found on both CD4+ cells and on CD8+ cells as well as on B lymphocytes (CD19+). Neonatal T cells (CD3+) are comprised almost exclusively of the subset expressing low LFA-3, CD2, LFA-1, CDw29, and UCHL1. Activation of cord peripheral blood mononuclear leukocytes with PHA leads to uniform enhanced expression of each of these molecules on CD3+ cells. Functional analyses of these T cell subsets were performed after sorting of adult T cells based on differential LFA-3 expression. Only the LFA-3+ subset proliferated in response to the Ag tetanus toxoid, even though the LFA-3- subset proliferated more strongly to PHA. Furthermore, the LFA-3+ subset made greater than fivefold more IFN-gamma than the LFA-3- subset in response to PHA, despite the fact that both subsets made equivalent amounts of IL-2. This phenotypic and functional analysis of resting and activated newborn and adult T cells indicates that human memory T cells express enhanced levels of LFA-3, CD2, LFA-1, UCHL1, CDw29, and Pgp-1; we speculate that the increase in expression of T cell adhesion molecules LFA-3, CD2, and LFA-1 on memory cells is functionally important in their enhanced responsiveness.}, keywords = {615, 616, 618}, author = {Sanders,M.E. and Makgoba,M.W. and Sharrow,S.O. and Stephany,D. and Springer, T.A. and Young,H.A. and Shaw,S.} } @article {529931, title = {ICAM-1 a ligand for LFA-1 dependent adhesion of B, T and myeloid cells}, journal = {Nature}, volume = {331}, number = {6151}, year = {1988}, note = {MS $\#$155Reprint Status: In File}, pages = {86-88}, abstract = {Cell-cell adhesion is essential for many immunological functions. The LFA-1 molecule, a member of a superfamily of adhesion molecules, participates in adhesion which is critical to the function of each of the three major subsets of leukocytes: lymphocytes, monocytes and granulocytes. Putative LFA-1 ligands have been identified functionally in different laboratories using three different monoclonal antibodies that inhibit LFA-1-mediated leukocyte adhesion in particular model systems; however, there may be more than one LFA-1 ligand. We have directly compared the three relevant monoclonal antibodies, and show that each binds to the same molecule, intercellular-adhesion molecule-1 (ICAM-1). Most important, B, T and myeloid cells adhere specifically to purified ICAM-1-coated surfaces; such adhesion has distinctive requirements for Mg2+ and Ca2+. This constitutes biochemical evidence that ICAM-1 functions as a ligand for LFA-1-dependent adhesion by a variety of leukocytes.}, author = {Makgoba,M.W. and Sanders,M.E. and Luce,G.E.G. and Dustin,M.L. and Springer, T.A. and Clark,E.A. and Mannoni,P. and Shaw,S.} } @article {528776, title = {Intercellular adhesion molecule-1 (ICAM-1) is involved in the cytolytic T lymphocyte interaction with human synovial cells}, journal = {J. Cell. Physiol.}, volume = {137}, number = {1}, year = {1988}, note = {MS$\#$159Reprint Status: In File}, pages = {173-178}, abstract = {The cell surface molecules involved in the human cytolytic T lymphocyte (CTL)-synovial cell interaction may play an important role in T cell interactions with connective tissue mesenchymal cells. To examine the molecular basis for the CTL-synovial cell interaction, we immortalized synovial cell explants to establish the cell line SYN.SPP. The SYN.SPP cell line was compared to the established B lymphoblastoid cell line JY. Cell surface immunofluorescence demonstrated significantly different levels of the immunologically relevant cell surface molecules ICAM-1 and LFA-3. Both cell lines were used to stimulate CTL precursors. After several months in culture, CTL lines stimulated by the SYN.SPP and JY cell lines demonstrated HLA class I-directed cytolytic activity. The cell surface molecules utilized by the anti-SYN.SPP and anti-JY CTL lines were identified by monoclonal antibody (MAb) inhibition. MAb recognizing the CTL cell surface molecules CD3, CD8 and LFA-1 (CD11a) significantly inhibited CTL-mediated lysis of both target cells. An interesting observation was that the anti-SYN.SPP CTL line appeared to utilize the ICAM-1 and not the LFA-3 target cell molecule. In contrast, the anti-JY CTL line utilized the LFA-3 and not the ICAM-1 membrane molecule. These results indicate that CTL interactions with connective tissue mesenchymal cells may be regulated by a unique pattern of antigen nonspecific cell-cell interaction molecules.}, author = {Mentzer,S.J. and Rothlein,R. and Springer, T.A. and Faller,D.V.} } @article {528381, title = {The leukocyte adhesion deficiency: Molecular basis and functional consequences}, journal = {Immunodefic. Rev.}, volume = {1}, year = {1988}, note = {Leukocyte adhesion deficience, LFA-1, Mac-1, P150,95, bone marrow, MS $\#$168Reprint Status: In File}, pages = {39-54}, author = {Fischer,A. and Lisowska-Grospierre,B. and Anderson,D.C. and Springer, T.A.} } @article {528731, title = {Lymphocyte function associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells}, journal = {J. Cell Biol.}, volume = {107}, number = {1}, year = {1988}, note = {MS$\#$162, CA31798, Tobacco researchReprint Status: NOT In File}, pages = {321-331}, abstract = {Intercellular adhesion molecule-1 (ICAM-1) on the surface of cultured umbilical vein and saphenous vein endothelial cells was upregulated between 2.5- and 40-fold by rIL-1, rTNF, LPS and rIFN gamma corresponding to up to 5 X 10(6) sites/cell. Endothelial cell ICAM-1 was a single band of 90 kD in SDS-PAGE. Purified endothelial cell ICAM-1 reconstituted into liposomes and bound to plastic was an excellent substrate for both JY B lymphoblastoid cell and T lymphoblast adhesion. Adhesion to endothelial cell ICAM-1 in planar membranes was blocked completely by monoclonal antibodies to lymphocyte function associated antigen-1 (LFA-1) or ICAM-1. Adhesion to artificial membranes was most sensitive to ICAM-1 density within the physiological range found on resting and stimulated endothelial cells. Adhesion of JY B lymphoblastoid cells, normal and genetically LFA-1 deficient T lymphoblasts and resting peripheral blood lymphocytes to endothelial cell monolayers was also assayed. In summary, LFA-1 dependent (60-90\% of total adhesion) and LFA-1-independent basal adhesion was observed and the use of both adhesion pathways by different interacting cell pairs was increased by monokine or lipopolysaccharide stimulation of endothelial cells. The LFA-1-dependent adhesion could be further subdivided into an LFA-1/ICAM-1-dependent component which was increased by cytokines and a basal LFA-1-dependent, ICAM-1-independent component which did not appear to be affected by cytokines. We conclude that ICAM-1 is a regulated ligand for lymphocyte-endothelial cell adhesion, but at least two other major adhesion pathways exist.}, keywords = {480}, author = {Dustin,M.L. and Springer, T.A.} } @article {529951, title = {The major Fc receptor in blood has a phosphatidylinositol anchor and is deficient in paroxysmal noctural hemoglobinuria}, journal = {Nature}, volume = {333}, number = {6173}, year = {1988}, note = {MS $\#$169Reprint Status: In File}, pages = {565-567}, abstract = {Fc receptors on phagocytic cells in the blood mediate binding and clearance of immune complexes, phagocytosis of antibody-opsonized microorganisms, and potently trigger effector functions, including superoxide anion production and antibody-dependent cellular cytotoxicity. The Fc receptor type III (Fc gamma R III, CD 16), present in 135,000 sites per cell 1 on neutrophils and accounting for most of FcR in blood, unexpectedly has a phosphatidylinositol glycan (PIG) membrane anchor. Deficiency of Fc gamma R III is observed in paroxysmal nocturnal haemoglobinuria (PNH), an acquired abnormality of haematopoietic cells affecting PIG tail biosynthesis or attachment, and is probably responsible for circulating immune complexes and susceptibility to bacterial infections associated with this disease. Although a growing number of eukaryotic cell-surface proteins with PIG-tails are being described, none has thus far been implicated in receptor-mediated endocytosis or in triggering of cell-mediated killing. Our findings on the Fc gamma R III raise the question of how a PIG-tailed protein important in immune complex clearance in vivo and in antibody-dependent killing mediates ligand internalization and cytotoxicity. Together with our results, previous functional studies on Fc gamma R III and Fc gamma R II suggest that these two receptors may cooperate and that the type of membrane anchor is an important mechanism whereby the functional capacity of surface receptors can be regulated.}, keywords = {285}, author = {Selvaraj, P. and Rosse,W.F. and Silber,R. and Springer, T.A.} } @article {527986, title = {Primary structure of intercellular adhesion molecule 1 (ICAM-1) demonstrates interaction between members of the immunoglobulin and integrin supergene families}, journal = {Cell}, volume = {52}, number = {6}, year = {1988}, note = {MS$\#$166; CTR1307BReprint Status: In File}, pages = {925-933}, abstract = {Intercellular adhesion molecule 1 (ICAM-1) is a 90 kd inducible surface glycoprotein that promotes adhesion in immunological and inflammatory reactions. ICAM-1 is a ligand of lymphocyte function-associated antigen-1 (LFA-1), an alpha beta complex that is a member of the integrin family of cell-cell and cell-matrix receptors. ICAM-1 is encoded by an inducible 3.3 kb mRNA. The amino acid sequence specifies an integral membrane protein with an extracellular domain of 453 residues containing five immunoglobulin-like domains. Highest homology is found with neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG), which also contain five Ig-like domains. NCAM and MAG are nervous system adhesion molecules, but unlike ICAM-1, NCAM is homophilic. The ICAM-1 and LFA-1 interaction is heterophilic and unusual in that it is between members of the immunoglobulin and intergrin families. Unlike other integrin ligands, ICAM-1 does not contain an RGD sequence.}, author = {Staunton,D.E. and Marlin,S.D. and Stratowa,C. and Dustin,M.L. and Springer, T.A.} } @article {529066, title = {Purified lymphocyte function-associated antigen-3 (LFA-3) activates human thymocytes via the CD2 pathway}, journal = {J. Immunol.}, volume = {141}, number = {9}, year = {1988}, note = {MS$\#$164Reprint Status: In File}, pages = {2980-2985}, abstract = {Defining the cellular and molecular mechanisms of interaction of developing thymocytes with nonlymphoid cells of the thymic microenvironment is critical for understanding normal thymus function. We have previously shown that the CD2/LFA-3 adhesion pathway is important in the interaction of thymocytes with a variety of LFA-3+ nonlymphoid thymic microenvironment cell types. Moreover, T cell activation via the CD2 (alternative, Ag independent) pathway is considered an important mechanism for intrathymic T cell proliferation. To study the relevance of CD2/LFA-3 interactions to human thymocyte activation, we have used purified LFA-3 Ag in several in vitro assays of thymocyte proliferation. Whereas LFA-3 Ag alone did not induce thymocyte proliferation, LFA-3 Ag in combination with the anti-CD2 antibody, CD2.1, and rIL-2 induced marked thymocyte proliferation. Additionally, the anti-CD28 antibody, Kolt2, could substitute for rIL-2, resulting in thymocyte activation induced by LFA-3 Ag in combination with antibodies CD2.1 and Kolt2. In both triggering systems, LFA-3 induced thymocyte activation was dependent upon the concentration of LFA-3 Ag. LFA-3 Ag-dependent thymocyte activation was directed primarily toward CD1-, mature thymocytes. Finally, intact SRBC that express the sheep homolog of LFA-3, T11TS, in combination with antibody CD2.1 and rIL-2 could also induce thymocyte activation. These data suggest that interaction of LFA-3 molecules with thymocyte CD2 molecules may provide a component of the stimulus for normal intrathymic thymocyte activation leading to thymocyte proliferation.}, keywords = {616}, author = {Denning,S.M. and Dustin,M.L. and Springer, T.A. and Singer,K.H. and Haynes,B.F.} } @article {528406, title = {Supergene families meet in the immune system}, journal = {Immunol. Today}, volume = {9}, number = {7-8}, year = {1988}, note = {MS $\#$175, Commentary for Immunology TodayReprint Status: In File}, pages = {213-215}, author = {Dustin,M.L. and Staunton,D.E. and Springer, T.A.} } @article {529256, title = {Activation of cultured human endothelial cells by recombinant lymphotoxin: Comparison with tumor necrosis factor and interleukin 1 species}, journal = {J. Immunol.}, volume = {138}, number = {10}, year = {1987}, note = {MS $\#$141Reprint Status: In File}, pages = {3319-3324}, abstract = {Recombinant human lymphotoxin (LT) was compared with recombinant human tumor necrosis factor (TNF) for direct actions on cultured human endothelial cells (HEC). At equivalent half-maximal concentrations (based on L929 cytotoxicity units) LT and TNF each caused rapid and transient induction (peak 4 to 6 hr) of an antigen associated with leukocyte adhesion (detected by monoclonal antibody H4/18), a rapid but sustained increased expression (plateau 24 hr) of a lymphocyte adhesion structure (ICAM-1), a gradual (plateau 4 to 6 days) increase in expression of HLA-A,B antigens, and gradual (4 to 6 days) conversion of HEC culture morphology from epithelioid to fibroblastoid, an effect enhanced by immune interferon (IFN-gamma). Induction of H4/18 binding by maximal concentrations of LT or TNF could not be augmented by addition of the other cytokine, and 24 hr pretreatment with LT or TNF produced hyporesponsiveness to both mediators for reinduction. H4/18 binding can be transiently induced by tumor-promoting phorbol esters. Pretreatment with either LT or TNF also fully inhibited induction of H4/18 binding by phorbol ester, whereas phorbol ester pretreatment only variably and partially inhibited reinduction by LT or TNF. These actions of LT on endothelium shared with TNF may serve in vivo to promote lymphocyte and inflammatory leukocyte adhesion and transendothelial migration. Recombinant human interleukin 1 species (IL 1 alpha and IL 1 beta) shared many of the actions of LT and TNF and were indistinguishable from each other. However, IL 1 species could be distinguished from LT/TNF by their relative inability to enhance HLA-A,B expression, by their ability to augment H4/18 binding caused by maximally effective concentrations of LT or TNF, and by their inability to inhibit reinduction of H4/18 binding by LT or TNF. In contrast to the actions of LT or TNF, pretreatment with IL 1 alpha or IL 1 beta only partially inhibited induction of H4/18 binding by phorbol ester, and phorbol ester pretreatment consistently, albeit partially, inhibited induction by IL 1 species. These studies suggest that activated T cells through the secretion of LT can in turn activate the local endothelial lining so as to promote homing and extravasation of inflammatory cells. Furthermore, these LT actions can be augmented or complemented by other locally produced mediators such as IFN-gamma or IL 1.}, author = {Pober,J.S. and Lapierre,L.A. and Stolpen,A.H. and Brock,T.A. and Springer, T.A. and Fiers,W. and Bevilacqua,M.P. and Mendrick,D.L. and Gimbrone,M.A.,Jr.} } @article {529906, title = {Anchoring mechanisms for LFA-3 cell adhesion glycoprotein at membrane surface}, journal = {Nature}, volume = {329}, number = {6142}, year = {1987}, note = {MS $\#$156, CA31798Reprint Status: In File}, pages = {846-848}, abstract = {The manner in which a membrane protein is anchored to the lipid bilayer may have a profound influence on its function. Most cell surface membrane proteins are anchored by a membrane-spanning segment(s) of the polypeptide chain, but another type of anchor has been described for several proteins: a phosphatidyl inositol glycan moiety, attached to the protein C terminus. This type of linkage has been identified on membrane proteins involved in adhesion and transmembrane signalling and could be important in the execution of these functions. We report here that an immunologically important adhesion glycoprotein, lymphocyte function-associated antigen 3 (LFA-3), can be anchored to the membrane by both types of mechanism. These two distinct cell-surface forms of LFA-3 are derived from different biosynthetic precursors. The existence of a phosphatidyl-inositol-linked and a transmembrane anchored form of LFA-3 has important implications for adhesion and transmembrane signalling by LFA-3.}, keywords = {618}, author = {Dustin,M.L. and Selvaraj, P. and Mattaliano,R.J. and Springer, T.A.} } @article {529226, title = {Biosynthesis and glycosylation of p150,95 and related leukocyte adhesion proteins}, journal = {J. Immunol.}, volume = {139}, number = {3}, year = {1987}, note = {MS $\#$142Reprint Status: NOT In File}, pages = {842-847}, abstract = {The p150,95 cell surface protein is a member of a family of heterodimeric leukocyte adhesion proteins that have homologous alpha subunits, each noncovalently associated with a common beta subunit. In this report we have metabolically labeled the U937 cell line at various timepoints during its phorbol myristic acetate-induced maturation to examine the kinetics of synthesis of these proteins during monocytic differentiation, and their maturation and glycosylation. The p150,95 alpha subunit was immunoprecipitated with p150,95-specific monoclonal antibody (MAb), or an antiserum to the denatured, purified alpha X subunit. The glycosylation and polypeptide chain length of the p150,95, Mac-1, and lymphocyte function associated antigen (LFA-1) alpha and beta subunits were compared by immunoprecipitation with subunit specific MAb and antisera, and by digestion with Endo H and N-glycanase. The p150,95 alpha subunit is synthesized as a precursor of 146,000 Mr, has five to six N-linked oligosaccharides, and has a polypeptide chain backbone of 132,000 Mr. Over 50\% of the carbohydrate on the mature alpha subunit of 150,000 Mr was sensitive to Endo H digestion. The p150,95 alpha and beta precursors can associate before maturation into the mature form. Conversion to the mature form was accompanied by loss of reactivity with the antiserum to the denatured alpha X subunit, suggesting a change in conformation. Mac-1 and LFA-1 alpha subunits have precursors of 160,000 Mr and 165,000 Mr, respectively, and contain N-linked carbohydrates. The polypeptide chain length for the Mac-1 alpha subunit is 137,000 Mr, and for LFA-1 is 149,000 Mr. Only 14\% of the oligosaccharide on the mature LFA-1 alpha subunit was sensitive to Endo H, suggesting that unlike p150,95, most is converted to the complex type. The differences noted in the Mr of the three homologous alpha subunits are therefore due to differences in both polypeptide chain length and carbohydrate processing during biosynthesis.}, author = {Miller,L.J. and Springer, T.A.} } @article {528151, title = {cDNA cloning and complete primary structure of the α subunit of a leukocyte adhesion glycoprotein, p150,95}, journal = {EMBO J.}, volume = {6}, number = {13}, year = {1987}, note = {MS $\#$146, CA31799, ACSA Fundacion Juan March fellowshipReprint Status: In File}, pages = {4023-4028}, abstract = {The leukocyte adhesion receptors, p150,95, Mac-1 and LFA-1 are integral membrane glycoproteins which contain distinct alpha subunits of 180,000-150,000 Mr associated with identical beta subunits of 95,000 Mr in alpha beta complexes. p150,95 alpha subunit tryptic peptides were used to specify oligonucleotide probes and a cDNA clone of 4.7 kb containing the entire coding sequence was isolated from a size-selected myeloid cell cDNA library. The 4.7-kb cDNA clone encodes a signal sequence, an extracellular domain of 1081 amino acids containing 10 potential glycosylation sites, a transmembrane domain of 26 amino acids, and a C-terminal cytoplasmic tail of 29 residues. The extracellular domain contains three tandem homologous repeats of approximately 60 amino acids with putative divalent cation-binding sites, and four weaker repeats which lack such binding sites. The cDNA clone hybridizes with a mRNA of 4.7 kb which is induced during in vitro differentiation of myeloid cell lines. The p150,95 alpha subunit is homologous to the alpha subunits of receptors which recognize the RGD sequence in extracellular matrix components, as has previously been shown for the beta subunits, supporting the concept that receptors involved in both cell-cell and cell-matrix interactions belong to a single gene superfamily termed the integrins. Distinctive features of the p150,95 alpha subunit include an insertion of 126 residues N-terminal to the putative metal binding region and a deletion of the region in which the matrix receptors are proteolytically cleaved during processing.}, author = {Corbi,A.L. and Miller,L.J. and O{\textquoteright}Connor,K. and Larson,R.S. and Springer, T.A.} } @article {530276, title = {Chemoattractant-regulated fusion of a novel, mobilizable intracellular compartment with the plasma membrane in human neutrophils}, journal = {Science}, volume = {237}, number = {4819}, year = {1987}, note = {MS $\#$139, CA31799Reprint Status: In File}, pages = {1204-1206}, abstract = {A novel mobilizable intracellular compartment was identified in human neutrophils by latent alkaline phosphatase activity. This compartment is mobilized to the plasma membrane much more readily than any identified granule subset and has kinetics of up-regulation in the membrane similar to those reported for a variety of receptor proteins. Triton X-100 permeabilization of both intact human neutrophils and subcellular fractions obtained by density-gradient centrifugation revealed that 70 percent of the alkaline phosphatase is located in an intracellular compartment distinct from primary, secondary, and gelatinase granules and from the plasma membrane. This compartment fully translocates to the plasma membrane after stimulation with nanomolar concentrations of the chemotactic peptide N-formylmethionylleucylphenylalanine.}, keywords = {137, 310}, author = {Borregaard,N. and Miller,L.J. and Springer, T.A.} } @article {527946, title = {Cloning of the β subunit of the leukocyte adhesion proteins: Homology to an extracellular matrix receptor defines a novel supergene family}, journal = {Cell}, volume = {48}, number = {4}, year = {1987}, note = {MS $\#$134, CA31798Reprint Status: In File}, pages = {681-690}, abstract = {We have isolated cDNA clones encoding the beta subunit of the human LFA-1, Mac-1, and p150,95 family of leukocyte adhesion proteins. The deduced 769-amino-acid sequence defines a cysteine-rich polypeptide with the characteristic features of an integral membrane protein. Peptide sequence data, Northern blot analysis, and Southern blot analysis suggest that a single gene encodes the beta subunit of all three leukocyte adhesion proteins. There is 45\% homology between the beta subunit sequence and band III of integrin, a chick fibronectin and laminin receptor. This homology defines a new supergene family of cellular adhesion proteins.}, author = {Kishimoto,T.K. and O{\textquoteright}Connor,K. and Lee,A. and Roberts,T.M. and Springer, T.A.} } @article {528966, title = {Deficiency of lymphocyte function-associated antigen-3 (LFA-3) in paroxysmal nocturnal hemoglobinuria: Functional correlates and evidence for a phosphatidylinositol membrane anchor}, journal = {J. Exp. Med.}, volume = {166}, number = {4}, year = {1987}, note = {MS $\#$149, CA31798, ACSReprint Status: NOT In File}, pages = {1011-1025}, abstract = {Lymphocyte function-associated antigen 3 (LFA-3) is a widely distributed cell surface glycoprotein that binds to the T lymphocyte CD2 surface glycoprotein. This interaction mediates CTL-target cell conjugate formation and adhesion of thymocytes to thymic epithelial cells. CD2 is also the E rosette receptor of T lymphocytes and mediates rosetting with autologous E by binding to LFA-3. We describe deficient expression of LFA-3 on E from paroxysmal nocturnal hemoglobinuria (PNH) patients. PNH is an acquired defect affecting phosphatidylinositol-anchored membrane proteins, of which decay-accelerating factor (DAF) is most important in the clinical symptoms of PNH. LFA-3-negative, weakly positive, and positive populations were found among PNH E. There was a good correlation with DAF deficiency. PNH E exhibited decreased binding of 125I-CD2 and rosetting with a human T lymphoma cell line. PNH E readily incorporated purified LFA-3, restoring LFA-3 expression and the CD2 binding and rosetting activity to normal levels. The expression of DAF was not restored after the incorporation of purified LFA-3 into PNH E, showing that LFA-3 and DAF are different molecules. Phosphatidylinositol-specific phospholipase C (PIPLC) treatment of a B lymphoma cell line released 35\% of the cell surface LFA-3 and 62\% of DAF. LFA-3 on E was resistant to PIPLC. However, when LFA-3 purified from human E was reconstituted in sheep E or human E and subjected to PIPLC treatment, 40-50\% of LFA-3 was released from the cell membrane. The results show that LFA-3 is attached to the cell membrane by a phosphatidylinositol glycolipid moiety, and confirm previous findings (37-41) that LFA-3 is a cell adhesion molecule that mediates adhesion by interacting with CD2 antigen.}, author = {Selvaraj, P. and Dustin,M.L. and Silber,R. and Low,M.G. and Springer, T.A.} } @article {527941, title = {Heterogenous mutations in the β subunit common to the LFA-1, Mac-1, and p150,95 glycoproteins cause leukocyte adhesion deficiency}, journal = {Cell}, volume = {50}, number = {2}, year = {1987}, note = {MS $\#$145, CA31798Reprint Status: In File}, pages = {193-202}, abstract = {Leukocyte adhesion deficiency (LAD) is a heritable disease involving deficient expression of three related leukocyte adhesion glycoproteins: LFA-1, Mac-1, and p150,95. These proteins are alpha beta heterodimers containing identical 95,000 dalton beta subunits. Here we demonstrate that the primary defect in LAD is in the beta subunit gene. We identified five distinct beta subunit phenotypes in LAD patients: undetectable beta subunit mRNA and protein precursor; low levels of beta subunit mRNA and precursor; an aberrantly large beta subunit precursor, probably due to an extra glycosylation site; an aberrantly small precursor; and a grossly normal precursor. Mutant beta subunit precursors from LAD patients failed to associate with the LFA-1 alpha subunit. In family studies, inheritance of the aberrant precursors correlates with the known inheritance of the LAD defect.}, keywords = {LFA-1}, author = {Kishimoto,T.K. and Hollander,N. and Roberts,T.M. and Anderson,D.C. and Springer, T.A.} } @inbook {529706, title = {Human cytotoxic T cells adhere to potential targets by two antigen-independent pathways: CD2 binding to LFA-3 or LFA-1 binding to an undefined ligand}, booktitle = {Leukocyte Typing III: White Cell Differentiation Antigens}, year = {1987}, note = {MS $\#$132Reprint Status: In File}, pages = {137-139}, publisher = {Springer-Verlag}, organization = {Springer-Verlag}, address = {New York}, author = {Shaw,S. and Luce,G.G. and Springer, T.A. and Plunkett,M.L. and Quinones,R. and Gress,R.E. and Sanders,M.E.}, editor = {McMichael,A.} } @inbook {528126, title = {Immunoaffinity chromatography}, booktitle = {Current Protocols in Molecular BiologyCurrent Protocols in Molecular Biology}, year = {1987}, note = {1992 ed. 10.11. - 10.11.7. Reprint Status: In Fileerased duplicate data $\#$5838.}, pages = {10.11.1-10.11.7}, publisher = {Greene Publishing Associates}, organization = {Greene Publishing Associates}, address = {New York}, author = {Springer, T.A.}, editor = {Ansubel,R.M. and Brent,R. and Kingston,R.E. and Moore,D.D. and Smith,J.A. and Seidman, J G and Struhl,K.} } @article {527791, title = {Leukocyte adhesion deficiency: An inherited defect in the Mac-1, LFA-1, and p150,95 glycoproteins}, journal = {Ann. Rev. Med.}, volume = {38}, year = {1987}, note = {MS$\#$129Reprint Status: NOT In File}, pages = {175-194}, abstract = {Leukocyte adhesion deficiency (LAD) is a recently recognized autosomal-recessive trait characterized by recurrent bacterial infections, impaired pus formation and wound healing, and abnormalities in a wide spectrum of adherence-dependent functions of granulocytes, monocytes, and lymphoid cells. Features of this disease are attributable to deficiency (or absence) of cell surface expression of a family of functionally and structurally related glycoproteins. These include Mac-1 (complement receptor type 3), lymphocyte function-associated antigen-1 (LFA-1), and p150,95. Defective biosynthesis of the beta chain shared by each molecule (comprised of alpha 1 beta 1 complexes) represents the fundamental molecular basis of this disease. Recognition of the molecular pathogenesis of this disorder has allowed rich insights into the role of cellular adherence reactions in inflammation and host defense.}, author = {Anderson,D.C. and Springer, T.A.} } @article {528861, title = {Leukocyte adhesion receptors are stored in peroxidase-negative granules of human neutrophils}, journal = {J. Exp. Med.}, volume = {166}, number = {6}, year = {1987}, note = {CA31799Reprint Status: NOT In File}, pages = {1641-1653}, abstract = {Previous studies have suggested that the leukocyte adhesion proteins Mac-1 and p150,95 are stored in a latent intracellular pool in neutrophils, and cellular fractionation studies have shown that Mac-1 is localized primarily in the peroxidase-negative specific granules. To determine the subcellular location of leukocyte adhesion receptors (LAR), we used immunocytochemical techniques on frozen thin sections of human blood leukocytes that had been incubated for peroxidase to mark the peroxidase-positive azurophil granules. To enhance the sensitivity of detection, polyclonal antibodies against immunoaffinity-purified p150,95 were raised in rabbits and absorbed with leukocytes from a patient deficient in this protein. The antiserum reacted with p150,95 and two other antigens with the same beta subunit, Mac-1 and lymphocyte function-associated antigen 1 (LFA-1). In neutrophils, we observed immunogold label for LAR predominantly on the membranes of peroxidase-negative granules, and in smaller amounts on the plasma and perinuclear membranes. In double-label experiments, there was colocalization of LAR with lactoferrin in some of the peroxidase-negative granules. We conclude that the latent pool of LAR resides in the membranes of peroxidase-negative granules. A significant increase in label on the plasma membrane of neutrophils stimulated with PMA is consistent with secretion of LAR to the exterior of the cell during degranulation. While LFA-1 appears very early in neutrophil maturation, it is becoming clear that Mac-1 and p150,95 are upregulated from an intracellular storage pool of peroxidase-negative granules that appear during the myelocyte stage of differentiation. Further studies are indicated to determine the significance of these proteins on the plasma membrane of two other granulocytes, eosinophils and basophils.}, keywords = {150}, author = {Bainton,D.F. and Miller,L.J. and Kishimoto,T.K. and Springer, T.A.} } @article {527811, title = {The lymphocyte function-associated LFA-1, CD2, and LFA-3 molecules: cell adhesion receptors of the immune system}, journal = {Annu. Rev. Immunol.}, volume = {5}, year = {1987}, note = {MS $\#$130Reprint Status: NOT In File}, pages = {223-252}, author = {Springer, T.A. and Dustin,M.L. and Kishimoto,T.K. and Marlin,S.D.} } @article {529071, title = {Monoclonal antibodies to CD2 and lymphocyte function-associated antigen 3 inhibit human thymic epithelial cell-dependent mature thymocyte activation}, journal = {J. Immunol.}, volume = {139}, number = {8}, year = {1987}, note = {MS $\#$127Reprint Status: In File}, pages = {2573-2578}, abstract = {Recent study of human thymocyte-thymic epithelial (TE) cell interactions has demonstrated that thymocytes bind to TE cells, and a consequence of this binding is the provision of accessory cell signals by TE cells for phytohemagglutinin (PHA)-induced mature thymocyte activation. In this paper we report on studies of the molecules involved in TE cell-dependent mature thymocyte activation. TE-thymocyte interactions necessary for PHA-induced thymocyte activation were inhibited by monoclonal antibodies against the cluster of differentiation (CD)2 antigen on thymocytes and lymphocyte function-associated (LFA)-3 antigen on TE cells. Inhibition of TE accessory cell signals by antibodies against CD2 (alpha CD2) and LFA-3 (alpha LFA-3) antigens occurred early on during thymocyte activation and prevented thymocyte interleukin 2 receptor expression. Further, alpha CD2 and alpha LFA-3 inhibited PHA-induced thymocyte activation in whole thymic explant cultures suggesting a significant role of the CD2 and LFA-3 antigens in thymocyte activation when accessory cell signals for PHA-induced thymocyte triggering were delivered by cells within an intact thymic microenvironment.}, keywords = {616, 618}, author = {Denning,S.M. and Tuck,D.T. and Vollger,L.W. and Springer, T.A. and Singer,K.H. and Haynes,B.F.} } @article {528836, title = {Polymorphism of lymphocyte function associated antigen-1, LFA-1, demonstrated by a Lupus patient{\textquoteright}s alloantiserum}, journal = {J. Clin. Invest.}, volume = {79}, number = {6}, year = {1987}, note = {MS $\#$137Reprint Status: In File}, pages = {1607-1614}, abstract = {We have found a human serum, E27, obtained from a multiply transfused patient with systemic lupus erythematosus, which immunoprecipitates the lymphocyte function associated antigen-1 (LFA-1). The immunoprecipitated molecules were identified as the LFA-1 alpha and beta chains by their comigration on SDS-PAGE, two-dimensional SDS-PAGE, and by sequential clearance experiments. Serum E27 did not immunoprecipitate LFA-1 from autologous cells, though LFA-1 molecules were present. In contrast, serum E27 immunoprecipitated LFA-1 from most but not all normal donor lymphocytes. Thus, serum E27 defines two serological phenotypes of LFA-1. 95\% of normal individuals tested exhibited the LFA-1 phenotype precipitated by serum E27. Serum E27 appears to be directed at determinants of the LFA-1 alpha-chain and not the beta-chain since it immunoprecipitated LFA-1 molecules but not the Mac-1 molecules. Additional evidence for the alpha chain specificity was provided by immunoprecipitation of mouse-human heterohybridoma cells. LFA-1 was immunoprecipitated by serum E27 from mouse-human heterohybridoma cells expressing the human alpha-chain, not from a hybrid cell line expressing the human beta-chain. Together these findings demonstrate an antigenic polymorphism of the human LFA-1 alpha-chain molecule.}, author = {Pischel,K.D. and Marlin,S.D. and Springer, T.A. and Woods,V.L.,Jr. and Bluestein,H.G.} } @article {528991, title = {Primary structure of lymphocyte function associated antigen-3 (LFA-3): The ligand of the T-lymphocyte CD2 glycoprotein}, journal = {J. Exp. Med.}, volume = {166}, number = {4}, year = {1987}, note = {MS $\#$151, CA31798, Biogen GrantReprint Status: NOT In File}, pages = {923-934}, abstract = {We have isolated the cDNA for human lymphocyte function-associated antigen 3 (LFA-3), the ligand of the T lymphocyte CD2 molecule. The identity of the clones was established by comparison of the deduced amino acid sequence to the LFA-3 NH2-terminal and tryptic peptide sequences. The cDNA defines a mature protein of 222 amino acids that structurally resembles typical membrane-anchored proteins. An extracellular domain with six N-linked glycosylation sites is followed by a hydrophobic putative transmembrane region and a short cytoplasmic domain. The mature glycoprotein is estimated to be 44-68\% carbohydrate. Southern blots of human genomic DNA indicate that only one gene codes for human LFA-3. Northern blot analysis demonstrates that the LFA-3 mRNA of 1.3 kb is widely distributed in human tissues and cell lines.}, author = {Wallner,B.P. and Frey,A.Z. and Tizard,R. and Mattaliano,R.J. and Hession,C. and Sanders,M.E. and Dustin,M.L. and Springer, T.A.} } @article {529231, title = {Purification and α subunit N-terminal sequences of human Mac-1 and p150,95 leukocyte adhesion proteins}, journal = {J. Immunol.}, volume = {138}, number = {8}, year = {1987}, note = {MS $\#$133, CA31798, CA31799Reprint Status: In File}, pages = {2381-2383}, keywords = {290, 332}, author = {Miller,L.J. and Wiebe,M. and Springer, T.A.} } @inbook {528131, title = {Purification of proteins by precipitation}, booktitle = {Current Protocols in Molecular BiologyCurrent Protocols in Molecular Biology}, year = {1987}, pages = {10.16.01-10.16.11}, publisher = {Greene Publishing Associates}, organization = {Greene Publishing Associates}, address = {New York}, author = {Springer, T.A.}, editor = {Ausubel, F M and Brent,R. and Kingston,R.E. and Moore,D.D. and Smith,J.A. and Seidman, J G and Struhl,K.} } @article {527956, title = {Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1)}, journal = {Cell}, volume = {51}, number = {5}, year = {1987}, note = {MS $\#$157, CA31798, Tobacco researchReprint Status: In File}, pages = {813-819}, abstract = {Lymphocyte function-associated antigen 1 (LFA-1) is a leukocyte cell surface glycoprotein that promotes intercellular adhesion in immunological and inflammatory reactions. It is an alpha beta complex that is structurally related to receptors for extracellular matrix components, and thus belongs to the integrin family. ICAM-1 (intercellular adhesion molecule-1) is a distinct cell surface glycoprotein. Its broad distribution, regulated expression in inflammation, and involvement in LFA-1-dependent cell-cell adhesion have suggested that ICAM-1 may be a ligand for LFA-1. We have purified ICAM-1 and incorporated it into artificial supported lipid membranes. LFA-1+ but not LFA-1- cells bound to ICAM-1 in the artificial membranes, and the binding could be specifically inhibited by anti-ICAM-1 treatment of the membranes or by anti-LFA-1 treatment of the cells. The cell binding to ICAM-1 required metabolic energy production, an intact cytoskeleton, and the presence of Mg2+ and was temperature dependent, characteristics of LFA-1- and ICAM-1-dependent cell-cell adhesion.}, keywords = {615}, author = {Marlin,S.D. and Springer, T.A.} } @article {528221, title = {Purified lymphocyte function-associated antigen-3 and T11 target structure are active in CD2-mediated T cell stimulation}, journal = {Eur. J. Immunol.}, volume = {17}, number = {12}, year = {1987}, note = {MS$\#$154; CA31798Reprint Status: In File}, pages = {1847-1850}, abstract = {In this study we have used cells expressing LFA-3 or T11TS, the human and sheep forms of the ligand of CD2, as well as the purified LFA-3 and T11TS molecules themselves to study their effects on T cell activation via the CD2-mediated "alternative pathway". Sheep red blood cells, which bind to CD2 via T11TS in E-rosette formation, and human autologous monocytes, which express the LFA-3 molecule, both induce proliferation of resting T cells in the presence of per se submitogenic concentrations of anti-T11(2) plus anti-T11(3) monoclonal antibodies (mAb). This effect is blocked by mAb to LFA-3, T11TS and CD2 known to inhibit CD2-ligand interaction. In addition, purified LFA-3 and T11TS, when added at ng amounts to cultures containing submitogenic concentrations of anti-T11(2 + 3) mAb, are also strongly mitogenic for resting human T cells. Thus, both LFA-3 and T11TS are potent co-stimulators of the alternative pathway of T cell activation but by themselves do not provide a mitogenic signal. This finding is discussed with regard to a physiological role of CD2-LFA-3 interaction in T cell activation.}, author = {Tiefenthaler,G. and H{\"u}nig,T. and Dustin,M.L. and Springer, T.A. and Meuer,S.C.} } @article {528916, title = {Purified lymphocyte function-associated antigen-3 (LFA-3) binds to CD2 and mediates T lymphocyte adhesion}, journal = {J. Exp. Med.}, volume = {165}, number = {3}, year = {1987}, note = {MS $\#$139, CA31798, ACSFAReprint Status: In File}, pages = {677-692}, abstract = {CD2 is a T lymphocyte glycoprotein that functions in adhesion of T lymphocytes and also as a putative receptor for activation signals. Functional data suggest that LFA-3, a widely distributed cell surface glycoprotein, may be the biological ligand of CD2. We have purified LFA-3 from human erythrocytes and characterized the purified protein functionally. LFA-3 bound specifically to CD2+ cells, and this binding was inhibited by CD2 mAb. Conversely, purified LFA-3 inhibited binding of CD2 mAb to cells, and the concentration required for this effect suggests that LFA-3 half-saturated CD2 at 1-5 nM LFA-3. Purified LFA-3 inhibited rosetting of human and sheep erythrocytes with CD2+ T lymphoma cells and T lymphocytes, and mediated aggregation of a CD2+ T lymphoma cell line. Purified LFA-3 reconstituted into planar membranes mediated efficient CD2-dependent adhesion of T lymphoblasts. These data demonstrate that LFA-3 is a ligand for CD2 and that LFA-3 can mediate T lymphocyte adhesion.}, keywords = {618, LFA-3}, author = {Dustin,M.L. and Sanders,M.E. and Shaw,S. and Springer, T.A.} } @article {528946, title = {Rosetting of activated human T lymphocytes with autologous erythrocytes: Definition of the receptor and ligand molecules as CD2 and lymphocyte function-associated antigen 3 (LFA-3)}, journal = {J. Exp. Med.}, volume = {165}, number = {3}, year = {1987}, note = {MS $\#$128Reprint Status: In File}, pages = {664-676}, abstract = {CD2, also known as LFA-2, T11, and the E rosette receptor, is a T lymphocyte surface protein functionally important in adhesion to target cells and T cell triggering. LFA-3 is a widely distributed cell surface protein that functions in adhesion on target cells. We find that LFA-3 is expressed on human E, and that CD2 is a receptor for LFA-3 that mediates T cell adhesion to human E. Pretreatment of T lymphocytes with CD2 mAb or of E with LFA-3 mAb inhibits rosetting. Purified CD2 molecules bind to human E and inhibit rosetting. 125I-CD2 binding to E is inhibited by LFA-3 mAb; reciprocally, binding of LFA-3 mAb to human E is inhibited by pretreatment with purified CD2. Higher concentrations of CD2 aggregate human E; aggregation is inhibited by mAb to LFA-3.}, keywords = {616}, author = {Plunkett,M.L. and Sanders,M.E. and Selvaraj, P. and Dustin,M.L. and Springer, T.A.} } @article {529306, title = {Rosetting of human T-lymphocytes with sheep and human erythrocytes: Comparison of human and sheep ligand binding using purified E receptor}, journal = {J. Immunol.}, volume = {139}, number = {8}, year = {1987}, note = {MS $\#$140, CA31798, ACSFAReprint Status: In File}, pages = {2690-2695}, abstract = {Previous studies have shown that the purified T lymphocyte glycoprotein, cluster differentiation 2 (CD2) (also known as T11, lymphocyte function-associated antigen (LFA)-2, and the erythrocyte (E) rosette receptor) interacts with the LFA-3 molecule on human E. We have examined the interaction of the purified CD2 molecule with the T11 target structure (T11TS) molecule on sheep E, and compared the two interactions. Purified, 125I-labeled CD2 bound to sheep E and the binding was inhibited by anti-T11TS monoclonal antibody (mAb). Reciprocally, the binding of T11TS mAb to sheep E was inhibited by pretreatment of sheep E with purified CD2. High concentrations of purified CD2 aggregated sheep E, possibly by inserting into the membrane, and the aggregation was inhibited by T11TS mAb. The affinity and number of binding sites for purified CD2 on sheep and human E was found to be similar, with Ka of 9 X 10(7)/M and 6 X 10(7)/M and 9800 and 8300 CD2 binding sites/E, respectively. Thus, the human T lymphocyte CD2 molecule is a receptor that cross-reacts between LFA-3 on human E and T11TS on sheep E, suggesting that LFA-3 and T11TS are functionally homologous ligands. As measured by saturation mAb binding, there are 8100 and 3900 ligand molecules/sheep and human E, respectively. Human and sheep E have surface areas of 145 and 54 micron 2, respectively. The 3.2- to 5.6-fold higher ligand density on sheep E appears to account for the ability of sheep but not human E to rosette with certain types of human T lymphocytes.}, keywords = {616, 618}, author = {Selvaraj, P. and Dustin,M.L. and Mitnacht,R. and H{\"u}nig,T. and Springer, T.A. and Plunkett,M.L.} } @article {529356, title = {Serological crossreactivity of T11 target structure (T11TS) and lymphocyte function-associated antigen 3 (LFA-3): evidence for structural homology of the sheep and human ligands of CD2}, journal = {J. Immunol.}, volume = {139}, number = {8}, year = {1987}, note = {MS $\#$143, CA31798, ACSFAReprint Status: In File}, pages = {2696-2701}, abstract = {T11 target structure (T11TS) and lymphocyte function-associated antigen (LFA) 3 are the cell-surface glycoproteins on sheep and human erythrocytes (E) binding to cluster differentiation 2 (the E-receptor) on T cells in E rosette formation. Here we show that this functional cross-reactivity is most likely due to a structural homology of these molecules. A rabbit antiserum to sheep T11TS is shown to cross-react with LFA-3 in several independent assays: (a) rabbit anti-T11TS antiserum blocks the formation of E rosettes by human T cells with both autologous and xenogeneic (sheep) E by binding to the respective E; (b) the antiserum blocks the binding of anti-LFA-3 monoclonal antibody to human E; and (c) it reacts with purified LFA-3 in Western blotting. Together, these findings demonstrate that T11TS on sheep E and LFA-3 on human E are serologically related, providing further support for the notion that T11TS and LFA-3 are the sheep and human forms of the same cell interaction molecule.}, keywords = {616, 618}, author = {Tiefenthaler,G. and Dustin,M.L. and Springer, T.A. and H{\"u}nig,T.} } @article {528831, title = {Stimulated mobilization of monocyte Mac-1 and p150,95 adhesion proteins from an intracellular vesicular compartment to the cell surface}, journal = {J. Clin. Invest.}, volume = {80}, number = {2}, year = {1987}, note = {MS $\#$131Reprint Status: In File}, pages = {535-544}, abstract = {Monocytes were stimulated to increase their cell surface quantity of leukocyte adhesion proteins p150,95 and Mac-1 by the chemoattractant formyl-methionyl-leucyl-phenylalanine, or other mediators such as platelet-derived growth factor, tumor necrosis factor, C5a, and leukotriene B4. Dose-response curves indicated variations in the sensitivity of monocytes and granulocytes to these mediators. These increases were independent of protein synthesis and half-maximal at 2 min. Human alveolar and murine peritoneal macrophages, cells that had previously diapedised, could not be induced to upregulate Mac-1 or p150,95. Detergent permeabilization studies in monocytes indicated that these proteins were stored in internal latent pools, which were reduced upon stimulation. Electron microscopy utilizing rabbit antiserum against p150,95 revealed these proteins on the plasma membrane, and in intracellular vesicles and peroxidase negative granules. Together with other functional studies, these findings suggest that the mobilization of Mac-1 and p150,95 from an intracellular compartment to the plasma membrane regulates the monocyte{\textquoteright}s ability to adhere and diapedese.}, author = {Miller,L.J. and Bainton,D.F. and Borregaard,N. and Springer, T.A.} } @article {529946, title = {The T lymphocyte glycoprotein CD2 (LFA-2/T11/E-rosette receptor) binds the cell surface ligand LFA-3.}, journal = {Nature}, volume = {326}, number = {6111}, year = {1987}, note = {MS$\#$136; 31798; ACS faculty awardReprint Status: In File}, pages = {400-403}, abstract = {CD2 (known also as T11 (ref. 1), LFA-2 (ref. 2) and the erythrocyte rosette receptor (ref. 3] is a functionally important T lymphocyte surface glycoprotein of relative molecular mass 50,000 to 58,000 (Mr 50-58 K) which appears early in thymocyte ontogeny and is present on all mature T cells. Monoclonal antibodies to CD2 inhibit cytotoxic T-lymphocyte (CTL)-mediated killing by binding to the T lymphocyte and blocking adhesion to the target cell. Such antibodies also inhibit T helper cell responses including antigen-stimulated proliferation, interleukin-2 (IL-2) secretion, and IL-2 receptor expression. Certain combinations of monoclonal antibodies to CD2 epitopes trigger proliferation of peripheral blood T lymphocytes, cytotoxic effector function and expression of IL-2 receptors by thymocytes, resulting in thymocyte proliferation in the presence of exogenous IL-2 (ref. 11). These findings suggest that CD2 can function in signalling as well as being an adhesion molecule. To understand the role of CD2 in T-cell adhesion and activation, it is essential to define its natural ligand. Our previous observation that purified CD2 inhibits rosetting of T lymphocytes with sheep erythrocytes and can be absorbed by sheep erythrocytes suggested it also might bind with detectable affinity to human cells. We now report that CD2 binds to a cell-surface antigen known as lymphocyte function-associated antigen-3 (LFA-3) with high affinity, and can mediate adhesion of lymphoid cells via interaction with LFA-3.}, author = {Selvaraj, P. and Plunkett,M.L. and Dustin,M.L. and Sanders,M.E. and Shaw,S. and Springer, T.A.} } @article {529361, title = {Thymocyte binding to human thymic epithelial cells is inhibited by monoclonal antibodies to CD-2 and LFA-3 antigens}, journal = {J. Immunol.}, volume = {138}, number = {2}, year = {1987}, note = {MS $\#$124Reprint Status: In File}, pages = {358-363}, abstract = {With the use of cultured human thymic epithelial (TE) cells, we have previously shown that thymocytes bind to TE cells in suspension in a rosette-forming assay. To identify cell surface molecules involved in human TE-thymocyte rosette formation, we assayed a large panel of monoclonal antibodies for their ability to inhibit rosette formation. We found anti-CD-2 (LFA-2, T11), and anti-LFA-3 antibodies all inhibited binding of TE cells to thymocytes. By using indirect immunofluorescence assays, we determined that cultured TE cells were 90\% LFA-3 positive and CD-2 negative, whereas thymocytes were 10\% LFA-3 positive and 98\% CD-2 positive. Pretreatment of TE cells with anti-LFA-3 but not anti-LFA-2 inhibited TE-thymocyte binding. In contrast, pretreatment of thymocytes with anti-CD-2 but not anti-LFA-3 antibodies inhibited TE-thymocyte binding. Thus TE cell-thymocyte binding is blocked by antibodies to the CD-2 (T11) antigen on thymocytes and by an antibody to the LFA-3 antigen on TE cells. Because the CD-2 antigen has been implicated in T cell activation, these data suggest that a natural ligand for T cell activation via the CD-2 molecule is present on human thymic epithelial cells.}, keywords = {616}, author = {Vollger,L.W. and Tuck,D.T. and Springer, T.A. and Haynes,B.F. and Singer,K.H.} } @article {528826, title = {Abnormal cytolytic activity of lymphocyte function-associated antigen-1-deficient human cytolytic T lymphocyte clones}, journal = {J. Clin. Invest.}, volume = {78}, number = {5}, year = {1986}, note = {Reprint Status: In File}, pages = {1387-1391}, abstract = {The involvement of the lymphocyte function-associated antigen-1 (LFA-1) membrane molecule in cytolytic T lymphocyte (CTL) interactions with lymphoid target cells was investigated using CTL clones derived from two patients with a heritable deficiency of LFA-1. LFA-1 surface expression on the CTL clones was 1\% of the normal level of LFA-1, unchanged with prolonged culture, and identical on 14 different CTL clones. The function of the LFA-1 molecule was addressed using the LFA-1-deficient CTL clones and LFA-1-deficient lymphoid target cells. The lytic activity of the LFA-1-deficient CTL clones was 43\% of control when tested against a target cell line expressing normal levels of LFA-1 and less than 10\% of control when tested against an LFA-1-deficient target cell line. These results demonstrate a direct involvement of LFA-1 in CTL-mediated cytolysis and suggest a more general dependence on LFA-1 in lymphoid cell-cell interactions.}, keywords = {016, 615}, author = {Mentzer,S.J. and Bierer,B.E. and Anderson,D.C. and Springer, T.A. and Burakoff,S.J.} } @inbook {528301, title = {Antibodies specific for the Mac-1, LFA-1, p150,95 glycoproteins or their family, or for other granulocyte proteins, in the 2nd International Workshop on Human Leukocyte Differentiation Antigens}, booktitle = {Human Myeloid and Hematopoietic Cells}, year = {1986}, note = {Reprint Status: In File}, pages = {55-68}, publisher = {Springer-Verlag}, organization = {Springer-Verlag}, address = {New York}, author = {Springer, T.A. and Anderson,D.C.}, editor = {Reinherz,E.L. and Haynes,B.F. and Nadler,L.M. and Bernstein,I.D.} } @article {528026, title = {Biochemical models of interferon-γ-mediated macrophage activation. Independent regulation of lymphocyte function associated antigen (LFA)-1 and I-A antigen on murine peritoneal macrophages}, journal = {Cell. Immunol.}, volume = {97}, number = {1}, year = {1986}, note = {Reprint Status: NOT In File}, pages = {110-120}, abstract = {IFN-gamma can induce the expression of both class II histocompatibility antigens (Ia) and the lymphocyte function associated (LFA)-1 antigen on murine peritoneal macrophages. We have examined the molecular changes which lead to altered expression of these two cell surface proteins to determine whether they are regulated by similar or independent mechanisms. While I-A antigen expression can be induced or enhanced by treatment of macrophages with either phorbol diesters and/or the Ca2+ ionophore A23187, these agents had no effect upon expression of LFA-1 under similar conditions. Macrophages from the A/J strain mouse exhibit a deficiency in their sensitivity to IFN-gamma which is seen in our studies as an inability of IFN-gamma to elevate I-A antigen expression. However, expression of I-A could be modulated in these cells by treatment with either phorbol diesters or A23187. In contrast, IFN-gamma could induce LFA-1 antigen on A/J derived macrophages and this was not affected by either phorbol or A23187. Thus these two antigens, despite coordinate expression in response to IFN-gamma in normal mouse strains, are clearly regulated independently. These results suggest that IFN-gamma generates at least two independent molecular events in macrophages which ultimately modulate the expression of cell surface proteins important to the performance of activated functions.}, keywords = {615}, author = {Strassmann,G. and Somers,S.D. and Springer, T.A. and Adams,D.O. and Hamilton,T.A.} } @article {529021, title = {Contributions of the Mac-1 glycoprotein family to adherence-dependent granulocyte functions: Structure-function assessments employing subunit-specific monoclonal antibodies}, journal = {J. Immunol.}, volume = {137}, number = {1}, year = {1986}, note = {Reprint Status: In File}, pages = {15-27}, abstract = {MAb directed at the alpha-subunits of Mac-1 (alpha M), LFA-1 (alpha L), p150,95 (alpha X), or their common beta-subunit were used to characterize the contributions of the Mac-1 glycoprotein family to granulocyte adherence reactions. Inhibitory effects of these MAb in incubation experiments with normal granulocytes indicated distinct adhesive contributions of each subunit. Significantly greater adherence, and inhibition of adherence by anti alpha M, alpha X, and beta MAb, was observed under chemotactic conditions designed to "up-regulate" the surface expression of the alpha M beta and alpha X beta complexes. Adherence to protein-coated glass and binding of albumin-coated latex beads were significantly inhibited by anti-beta greater than anti-alpha M (OKM-10, M1/70, LM2/1.6 and OKM-1) greater than anti-alpha X greater than anti-alpha L MAb, but no effects of anti-HLA, AB, or anti-CR-1 MAb were evident. A similar rank order of inhibition was observed in granulocyte aggregation assays in response to C5a, PMA, or f-Met-Leu-Phe. Significant inhibition of directed migration by anti-beta or anti-alpha M (OKM-1 or OKM-10) MAb was observed in subagarose but not Boyden chemotaxis assays; inhibition was dependent on a continuous cell exposure to anti-Mac-1 alpha or beta during the assay, suggesting that a continuum of new Mac-1 expression is required for directed translocation. Phagocytosis of Oil-Red-O paraffin or zymosan selectively opsonized with C3-derived ligands was significantly inhibited by anti-alpha M MAb (OKM-10 greater than LM2/1.6 greater than M1/70 greater than OKM-1) or by combinations of anti-alpha M + anti-CR-1 MAb, but only minimal inhibitory effects of anti-beta MAb and no effects of anti-alpha L or anti-alpha X MAb were seen. Similarly, complement-dependent phagocytosis-associated lactoferrin release, ingestion, and intracellular killing of Staphylococcus aureus 502A, and binding of iC3b-opsonized SRBC, were significantly inhibited by anti-alpha M (OKM-10, M1/70) or combinations of anti-alpha M + anti-CR-1 MAb, but not by anti-beta, alpha L, or alpha X MAb. Notably, none of the anti-Mac-1 MAb demonstrated inhibitory effects in assays of adherence-independent functions including shape change, specific f-Met-Leu-3H-Phe binding, O-2 generation, chemiluminescence evolution, or lactoferrin release in response to PMA.(ABSTRACT TRUNCATED AT 400 WORDS)}, keywords = {015, CD11b}, author = {Anderson,D.C. and Miller,L.J. and Schmalstieg,F.C. and Rothlein,R. and Springer, T.A.} } @article {528181, title = {Defective membrane expression of the LFA-1 complex may be secondary to the absence of the β chain in a child with recurrent bacterial infection}, journal = {Eur. J. Immunol.}, volume = {16}, number = {2}, year = {1986}, note = {publication $\#$103Reprint Status: In File}, pages = {205-208}, abstract = {Membrane and intracellular processing of the LFA-1 macromolecular complex, known to be involved in cytolytic function of T lymphocytes, was investigated in a child with recurrent bacterial infections, impaired natural killer activity, T cell-mediated lymphocytolysis and absent adhesion and migration of phagocytic cells. Monoclonal antibodies to the LFA-1 alpha and beta subunits, able to precipitate the LFA-1 alpha, 180-kDa chain, the p151 chain and beta 94-kDa chain (shared by both alpha chains), were used in immunoprecipitation studies of patient and control phytohemagglutinin-blasts. Neither of the alpha chains nor the beta chain were found in precipitates obtained from 125I-surface-labeled patient cells in contrast to controls. However, the precursor of the LFA-1 alpha chain, a 170-kDa polypeptide, was identified in lysates of biosynthetically labeled patients{\textquoteright} cells. These results suggest that the defective membrane expression of the LFA-1 complex may be secondary to the absence of the mature beta chain.}, author = {Lisowska-Grospierre,B. and Bohler,M.C. and Fischer,A. and Mawas,C. and Springer, T.A. and Griscelli,C.} } @article {530086, title = {Differentiation of myeloid cells is accompanied by increased levels of pp60c-src protein and kinase activity}, journal = {Proc Natl Acad Sci USA}, volume = {83}, number = {14}, year = {1986}, note = {Reprint Status: NOT In File}, pages = {5131-5135}, abstract = {We have detected a significant increase in the levels of pp60c-src kinase activity associated with the differentiation of myeloid cell lines HL-60 and U-937. The induction of pp60c-src kinase activity becomes apparent approximately 14 hr after the addition of phorbol 12-myristate 13-acetate and increases 20-fold by 72 hr. The enhanced kinase activity can be accounted for by elevated levels of c-src protein in the differentiated cells. When nonleukemic bone marrow cells were examined, myeloid progenitor cells exhibited a low level of pp60c-src kinase activity. As these cells are allowed to differentiate in culture, the resulting adherent monocytes are as high in pp60c-src kinase activity as HL-60 cells induced to differentiate into monocytes. A strong correlation is found between the levels of pp60c-src kinase activity and the degree of monocytic differentiation of the cells from patients with acute myeloid leukemia. Our findings suggest that the activation of pp60c-src kinase activity is a normal physiological event associated with myeloid differentiation.}, keywords = {550}, author = {Gee,C.E. and Griffin,J. and Sastre,L. and Miller,L.J. and Springer, T.A. and Piwnica-Worms,H. and Roberts,T.M.} } @article {529271, title = {A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1}, journal = {J. Immunol.}, volume = {137}, number = {4}, year = {1986}, note = {Reprint Status: In File}, pages = {1270-1274}, abstract = {Homotypic adhesion by phorbol ester-stimulated lymphocytes requires LFA-1 and Mg+2 and does not involve like-like interactions between LFA-1 molecules on adjacent cells. The latter finding suggested that a second molecule, distinct from LFA-1, also participates in LFA-1-dependent adhesion. The identification of such a molecule was the object of this investigation. After immunization with LFA-1-deficient EBV-transformed lymphoblastoid cells, a MAb was obtained that inhibits phorbol ester-stimulated aggregation of LFA-1+ EBV lines. This MAb defines a novel cell surface molecule, which is designated intercellular adhesion molecule 1 (ICAM-1). ICAM-1 is distinct from LFA-1 in both cell distribution and structure. In SDS-PAGE, ICAM-1 isolated from JY cells is a single chain of Mr = 90,000. As shown by MAb inhibition, ICAM-1 participates in phorbol ester-stimulated adhesion reactions of B lymphocyte and myeloid cell lines and T lymphocyte blasts. However, aggregation of one T lymphocyte cell line (SKW-3) was inhibited by LFA-1 but not ICAM-1 MAb. It is proposed that ICAM-1 may be a ligand in many, but not all, LFA-1-dependent adhesion reactions.}, keywords = {615}, author = {Rothlein,R. and Dustin,M.L. and Marlin,S.D. and Springer, T.A.} } @inbook {527866, title = {The importance of the Mac-1, LFA-1 glycoprotein family in monocyte and granulocyte adherence, chemotaxis, and migration into inflammatory sites: Insights from an experiment of nature}, booktitle = {Biochemistry of Macrophages (Ciba Symposium 118)}, year = {1986}, note = {Reprint Status: NOT In File}, pages = {102-126}, publisher = {Pitman}, organization = {Pitman}, address = {London}, abstract = {The Mac-1, LFA-1 (lymphocyte function-associated 1), p150,95 family of glycoproteins, which share a common beta subunit of Mr 95 000, are of widespread importance in leucocyte adhesion reactions. This paper focuses on the role of this glycoprotein family in granulocyte and monocyte adhesion and chemotaxis in vitro, and in migration into inflammatory sites in vivo. Most findings have been made with granulocytes, but results with monocytes are similar. Some studies have used leucocytes from patients exhibiting a severe or moderate deficiency in expression of this glycoprotein family, which is secondary to a defect in the common beta subunit. Patients are susceptible to bacterial infections and have defective pus formation and Rebuck skin-window tests, despite chronic granulocytosis. Granulocytes from such patients exhibit defective adherence to serum albumin and fibronectin-coated glass or plastic, defective orientation and directed migration in response to chemoattractants, and are defective in chemoattractant-stimulated aggregation and hyperadherence. Antibodies to the common beta subunit, to the Mac-1 alpha subunit, and to a lesser extent to the LFA-1 and p150,95 alpha subunits, inhibit many of the same functional responses by normal cells. In normal granulocytes and monocytes chemoattractants stimulate a five-fold increase in Mac-1 and p150,95 surface expression, by mobilization of a latent, presumably intracellular, pool. Cells from patients are deficient in up-regulation of these molecules but show normal up-regulation of other surface receptors, degranulation and oxidative burst. The hypothesis is presented that Mac-1 and p150,95 regulate or directly mediate the increase in granulocyte and monocyte adhesivity, which is essential for diapedesis, chemotaxis and migration into inflammatory sites.}, author = {Springer, T.A. and Anderson,D.C.} } @article {529081, title = {Induction by IL-1 and interferon-γ, tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1)}, journal = {J. Immunol.}, volume = {137}, number = {86}, year = {1986}, note = {Reprint Status: In File}, pages = {245-254}, keywords = {615}, author = {Dustin,M.L. and Rothlein,R. and Bhan,A.K. and Dinarello,C.A. and Springer, T.A.} } @article {529461, title = {Interaction of West Nile virus with primary murine macrophages: Role of cell activation and receptors for antibody and complement}, journal = {J. Virol.}, volume = {57}, number = {3}, year = {1986}, note = {Reprint Status: In File}, pages = {952-959}, abstract = {We have measured growth of West Nile virus in mouse primary peritoneal macrophages (resident, thioglycolate elicited, and Mycobacterium bovis BCG activated) and in macrophagelike (P338D1) and nonmacrophage (L929, PS clone D) cell lines infected in the absence or presence of specific antibodies (immunoglobulin G ([IgG], IgM), and complement. Monoclonal antibodies directed against Fc receptors (IgG1/2b, 2.4G2) and type 3 complement receptors (Mac-1) were used to define the role of each receptor. Virus yield depended on a balance between enhancement and neutralization and was influenced by the physiologic state of the macrophage, the receptor pathway of viral entry, the mouse strain and age of donor. BCG-activated macrophages displayed a greater ability to restrict West Nile virus than nonactivated cells only in the presence of antiviral IgM, with or without complement; the Fc receptors for various classes of IgG mediated striking enhancement. These studies identify some of the complex innate and acquired factors that determine the interaction between West Nile virus and primary macrophages in vitro.}, author = {Cardosa, M. J. and Gordon, S. and Hirsch, S. and Springer, T.A. and Porterfield, J. S.} } @inbook {528261, title = {Leukocyte complement receptors and adhesion proteins in the inflammatory response: Insights from an experiment of nature}, booktitle = {Genes and Proteins in Immunity, Biochem. Soc. Symp. 51}, year = {1986}, note = {Reprint Status: In File}, pages = {47-57}, publisher = {A. R. Liss}, organization = {A. R. Liss}, address = {London}, abstract = {The complement receptor type 3 (CR3) mediates phagocytosis and degradation of iC3b-opsonized particles by macrophages and granulocytes. The CR3 is identical to the Mac-1 molecule, which is composed of two non-covalently associated glycoprotein subunits, alpha M of Mr 170,000 and beta of Mr 95,000. Patients with recurring, life-threatening bacterial infections have been identified who have moderate (95\%) or severe (greater than 99\%) deficiency of Mac-1 and of the related LFA-1 and p150,95 molecules. The primary defect is in the shared beta subunit of these molecules. Patient leukocytes are not only deficient in CR3 but in a wide variety of adhesion-dependent functions, including granulocyte chemotaxis, adherence to surfaces, and aggregation. Monoclonal antibodies to the Mac-1 alpha subunit and to the beta subunit block these functions. The hypothesis will be advanced that Mac-1 functions dually as the CR3 and in {\textquoteright}nonspecific{\textquoteright} adherence reactions. Adherence functions are stimulated in normal granulocytes by chemoattractants, which also induce a rapid 5-fold increase in Mac-1 and p150,95 on the cell surface. It is proposed that absence of Mac-1 and p150,95 expression and upregulation by patient granulocytes is causally related to their inability to extravasate and migrate into inflammatory sites.}, author = {Springer, T.A. and Anderson,D.C.}, editor = {Kay, J and Kerr,M.A. and Williams,A.F. and Reid,K.B.M.} } @article {528936, title = {LFA-1 immunodeficiency disease: Definition of the genetic defect and chromosomal mapping of α and β subunits of the lymphocyte function-associated antigen 1 (LFA-1) by complementation in hybrid cells}, journal = {J. Exp. Med.}, volume = {164}, number = {3}, year = {1986}, note = {Reprint Status: In File}, pages = {855-867}, abstract = {Lymphocyte function associated antigen 1 (LFA-1) is a leukocyte cell adhesion protein. We have studied a novel human immunodeficiency disease in which LFA-1 and two other proteins which share the same beta subunit are lacking from the surface of leukocytes. The basis of the inherited defect in cell surface expression of both the alpha and beta subunits of LFA-1 was determined by somatic cell fusion of patient or normal human cells with an LFA-1+ mouse T cell line. Human LFA-1 alpha and beta subunits from normal cells could associate with mouse LFA-1 subunits to form interspecies hybrid alpha beta complexes. Surface expression of the alpha but not the beta subunit of patient cells was rescued by the formation of interspecies complexes. The findings show that the LFA-1 alpha subunit in genetically deficient cells is competent for surface expression in the presence of an appropriate beta subunit, and suggest that the genetic lesion affects the beta subunit. The human LFA-1 alpha and beta subunits were mapped to chromosomes 16 and 21, respectively. The genetic defect is inferred to be on chromosome 21.}, keywords = {615}, author = {Marlin,S.D. and Morton,C.C. and Anderson,D.C. and Springer, T.A.} } @inbook {528286, title = {Macrophages}, booktitle = {Handbook of experimental immunology}, volume = {Vol.IV: Applications of immunological methods in biomedical sciences}, year = {1986}, note = {Reprint Status: In File}, pages = {118}, publisher = {Blackwell}, organization = {Blackwell}, edition = {4}, address = {Oxford}, author = {Unkeless,J.C. and Springer, T.A.}, editor = {Weir,D.M. and Herzenberg,L.A. and Blackwell,C.C.} } @article {529346, title = {Mechanisms of tumor cell capture by activated macrophages: Evidence for involvement of lymphocyte function associated (LFA)-1 antigen}, journal = {J. Immunol.}, volume = {136}, number = {11}, year = {1986}, note = {Reprint Status: NOT In File}, pages = {4328-4333}, abstract = {The lymphocyte function-associated (LFA)-1 molecule is expressed on certain populations of macrophages that have an augmented capacity to capture tumor cells. Accordingly, we analyzed the role of LFA-1 in the establishment of such cell-cell interactions. F(ab{\textquoteright})2 fragments of the M17/4, anti-LFA-1 monoclonal antibody (MAb) inhibited the interaction between activated macrophages and tumor cells by up to 80\% in a dose-dependent manner. The anti-LFA-1 MAb reduced (between 55 to 79\%) the number of P815, LSTRA, or EL-4 tumor cells bound to trypsin-sensitive structures on bacillus Calmette Guerin activated macrophages. The inhibition appeared selective, because a F(ab{\textquoteright})2 fragment of anti-Mac-1 did not inhibit such binding. Inhibition of tumor cell capture could be observed as soon as 15 min after the onset of the cell-cell interaction between activated macrophages and tumor cells. Optimal inhibition occurred when both tumor targets and macrophages were precoated with the MAb. Although P815, LSTRA, EL-4, and BW5147 tumor cells all expressed LFA-1, only the first three but not BW5147 cells were bound by activated macrophages. Furthermore, endotoxin-pulsed macrophages elicited by thioglycollate broth expressed the LFA-1 antigen but did not exhibit selective tumor cell capture. Finally, anti-LFA-1 inhibited the development of weak into strong binding. Taken together, the results suggest that LFA-1 molecules can participate in the interaction between activated macrophages and neoplastic cells.}, author = {Strassmann,G. and Springer, T.A. and Somers,S.D. and Adams,D.O.} } @article {529291, title = {The mouse leukocyte adhesion proteins Mac-1 and LFA-1: Studies on mRNA translation and protein glycosylation with emphasis on Mac-1}, journal = {J. Immunol.}, volume = {137}, number = {3}, year = {1986}, note = {Reprint Status: In File}, pages = {1060-1065}, abstract = {Translation in vitro of mRNA and immunoprecipitation with specific rabbit antisera showed that the unglycosylated precursor polypeptides of the mouse Mac-1 and lymphocyte function associated antigen (LFA-1) alpha subunits are 130,000 Mr and 140,000 Mr, respectively. Furthermore, polysomes purified by using anti-Mac-1 IgG yielded a similar major product of translation in vitro of Mr = 130,000. The Mac-1 and LFA-1 alpha subunit translation products are immunologically noncross-reactive, showing that differences between these related proteins are not due to post-translational processing. Mac-1 and LFA-1 alpha subunits could only be in vitro translated from mRNA from cell lines the surfaces of which express the corresponding Mac-1 and LFA-1 alpha-beta complexes, showing tissue-specific expression is regulated at the mRNA level. The glycosylation of Mac-1 was examined by both translation in vitro in the presence of dog pancreas microsomes and by biosynthesis in vivo and treatment with tunicamycin, endoglycosidase H, and the deglycosylating agent trifluoromethane sulfonic acid. High mannose oligosaccharides are added to the Mac-1 alpha and beta polypeptide backbones of Mr = 130,000 and 72,000, respectively, to yield precursors of Mr = 164,000 and 91,000, respectively. The alpha and beta subunit precursors are then processed with partial conversion of high mannose to complex type carbohydrate to yield the mature subunits of Mr = 170,000 and 95,000, respectively.}, keywords = {550}, author = {Sastre,L. and Kishimoto,T.K. and Gee,C. and Roberts,T. and Springer, T.A.} } @article {529251, title = {Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor and immune interferon}, journal = {J. Immunol.}, volume = {137}, number = {6}, year = {1986}, note = {Reprint Status: In File}, pages = {1893-1896}, abstract = {We have used the quantitative binding of murine monoclonal antibodies to the surface of cultured human umbilical vein endothelial (HUVE) cells to study the responses of HUVE cells to three different immune mediators: interleukin 1 (IL 1), tumor necrosis factor (TNF), and immune interferon (IFN-gamma). Antibody H4/18, reactive with an endothelial cell-specific activation antigen, does not bind to unstimulated HUVE cells but shows rapidly and transiently inducible binding (peak 4 to 6 hr) to cells stimulated by IL 1 or TNF that declines to basal levels by 24 hr, even in the continued presence of mediator. Binding of H4/18 is unaffected by IFN-gamma. Antibody RR1/1, reactive with intercellular adhesion molecule 1, binds to unstimulated HUVE cells, but binding is rapidly increased (plateau 24 hr) after stimulation by IL 1 or TNF and slowly increased (over several days) by IFN-gamma. In contrast to H4/18 binding, the increase in RR1/1 binding is sustained in the continued presence of mediator. Antibody W6/32, reactive with HLA-A,B antigens, binds to unstimulated HUVE cells and shows gradually progressive increases (over several days) in binding upon treatment with IFN-gamma or TNF. These observations demonstrate that HUVE cells show distinct but overlapping patterns of antigenic modulation in response to three different lymphokines, and suggest that the "activation" of endothelial cells observed in situ may represent a complex integration of several lymphokine-mediated signals.}, keywords = {615, CD ICAM-1}, author = {Pober,J.S. and Gimbrone,M.A.,Jr. and Lapierre,L.A. and Mendrick,D.L. and Fiers,W. and Rothlein,R. and Springer, T.A.} } @article {529321, title = {p150,95, the third member of the Mac-1, LFA-1 human leukocyte adhesion glycoprotein family}, journal = {J. Immunol.}, volume = {136}, number = {1}, year = {1986}, note = {Reprint Status: In File}, pages = {240-245}, abstract = {Monoclonal antibodies specific for p150,95, a third member of the Mac-1 and lymphocyte function-associated antigen-1 (LFA-1) leukocyte adhesion protein family, have been identified and used to study the biochemistry and cellular expression of p150,95. p150,95 is a noncovalently associated heterodimer containing alpha X and beta subunits of Mr = 150,000 and 95,000 respectively. Findings suggest that the p150,95 alpha X beta complex shares a common beta subunit with the alpha L beta LFA-1 and alpha M beta Mac-1 complexes. Co-precipitation experiments demonstrated identity between the p150,95 molecule precipitated by anti-beta MAb and by p150,95-specific MAb. Patients with a previously demonstrated genetic deficiency in Mac-1 and LFA-1 fail to express p150,95. Deficiency of the Mac-1, LFA-1, and p150,95 alpha beta complexes on the surface of patient cells appears due to a defect in the common beta subunit. The lack of cross-reaction of p150,95-specific MAb with LFA-1 and Mac-1, which appear to utilize identical beta subunits, suggests that the determinant is specified by the alpha X rather than the beta subunit of p150,95. The data suggest that alpha X is yet a third member of a family of alpha subunit proteins that associate with a common beta subunit, are differentially regulated in leukocyte differentiation, and function in adhesion reactions. p150,95 is normally expressed on blood monocytes and granulocytes. Chemoattractants such as f-Met-Leu-Phe stimulate a rapid, fivefold increase in surface expression that is not dependent on protein synthesis and appears to reflect mobilization of an intracellular latent pool. The intimate relation between the lack of chemoattractant-stimulated upregulation of p150,95 and Mac-1 by patient granulocytes and their failure to upregulate adhesiveness to these same stimuli in vitro, or to diapedese and migrate into inflammatory sites in vivo, is discussed.}, author = {Springer, T.A. and Miller,L.J. and Anderson,D.C.} } @article {530171, title = {A partial genomic DNA clone for the α subunit of the mouse complement receptor type 3 and cellular adhesion molecule Mac-1}, journal = {Proc Natl Acad Sci USA}, volume = {83}, number = {15}, year = {1986}, note = {Reprint Status: NOT In File}, pages = {5644-5648}, abstract = {A genomic clone coding for the alpha subunit of the mouse complement receptor type 3 and the cellular adhesion molecule Mac-1 has been isolated directly from a genomic library using synthetic oligonucleotide probes based on the amino-terminal amino acid sequence of the protein. The identity of the clone has been established by DNA sequencing and in vitro translation of hybrid-selected mRNA. The gene is present in a single copy in the murine genome. The region containing the amino-terminal exon has been sequenced. RNA gel blotting shows that the Mac-1 alpha-subunit mRNA is 6 kilobases in length. Mac-1 alpha-subunit mRNA is present in macrophages but not T lymphoma or L cells. During gamma interferon-stimulated maturation of the mouse premyelocytic cell line M1, Mac-1 alpha-subunit mRNA is induced. This corresponds with the tissue distribution of the Mac-1 alpha subunit, showing expression is regulated at least partially at the message level.}, keywords = {136, 550, CD ICAM-1}, author = {Sastre,L. and Roman,J.M. and Teplow,D.B. and Dreyer,W.J. and Gee,C.E. and Larson,R.S. and Roberts,T.M. and Springer, T.A.} } @inbook {529761, title = {Production of syrian and armenian hamster monoclonal antibodies of defined specificity}, booktitle = {Methods Enzymol.}, volume = {121}, year = {1986}, pages = {239-244}, publisher = {Academic Press}, organization = {Academic Press}, address = {New York}, author = {Sanchez-Madrid,F. and Springer, T.A.} } @article {529246, title = {Purification and characterization of the lymphocyte function-associated-2 (LFA-2) molecule}, journal = {J. Immunol.}, volume = {136}, number = {11}, year = {1986}, note = {Reprint Status: In File}, pages = {4181-4187}, abstract = {The lymphocyte function-associated-2 (LFA-2) molecule, equivalent to CD2 and the E rosette receptor, was purified by MAb affinity chromatography from the Jurkat T lymphoma cell line. Jurkat was selected for its high level of expression of 1.0 X 10(5) sites/cell. A two-site radioimmunometric assay was developed to monitor purification. From 50 g of packed cells, 230 micrograms of LFA-2 was obtained with 65\% yield of antigenic activity with a purification factor of 13,000. A major component of 58,000 and 54,000 was obtained that corresponded to LFA-2 antigenic activity as shown by immunoblotting and immunoprecipitation. The doublet was resolved by 2D IEF-SDS-PAGE into components of pI = 5.5 and 5.6. Smaller amounts of lower Mr components were also seen. All these components appeared related by processing or proteolytic breakdown, as shown by Cleveland peptide mapping. The LFA-2 deoxycholate complex had an apparent Mr of 68,000 by gel filtration, suggesting it was monomeric. Purified LFA-2 inhibited rosetting of T lymphocytes with sheep E, and addition to preformed rosettes caused their disruption. Inhibitory activity was absorbed by sheep E. This is the first evidence that the CD2/LFA-2 molecule can directly bind to sheep E. Purified LFA-2 should be useful for the further biochemical and functional characterization of this molecule.}, author = {Plunkett,M.L. and Springer, T.A.} } @article {529221, title = {Regulated expression of the Mac-1, LFA-1, p150,95 glycoprotein family during leukocyte differentiation}, journal = {J. Immunol.}, volume = {137}, number = {9}, year = {1986}, note = {Reprint Status: In File}, pages = {2891-2900}, abstract = {The regulation of Mac-1, LFA-1, and p150,95 expression during leukocyte differentiation was examined. LFA-1 was present on almost all cell types studied. Both Mac-1 and p150,95 were present on the more mature cells of the myelomonocytic series, but only p150,95 was detected on some B cell lines and cloned cytotoxic T lymphocytes. Phorbol myristate acetate (PMA) stimulation of B chronic lymphocytic leukemia cells dramatically increased p150,95 expression. The resultant Mac-1, LFA-1, p150,95 phenotype resembled hairy cell leukemia, a B cell plasmacytoid leukemia. The promonocytic cell line U937 and the promyeloblastic cell line HL-60 expressed only LFA-1. Monocytic differentiation of U937 cells was stimulated by PMA, and induced the concomitant expression of Mac-1 and p150,95, with more p150,95 induced than Mac-1. Granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulation of U937 cells gave similar results. PMA-stimulated monocytic differentiation of the HL-60 cell line also induced expression of both Mac-1 and p150,95. The number of p150,95 molecules on PMA-stimulated U937 and HL-60 cells were 5 X 10(5) and 3 X 10(5), respectively. Retinoic acid stimulated myeloid differentiation of HL-60 cells and induced expression of both Mac-1 and p150,95. These cells acquired a Mac-1, LFA-1, p150,95 profile that resembled that of granulocytes, with more Mac-1 than p150,95 induced. GM-CSF stimulation of HL-60 cells induced a similar Mac-1 and p150,95 phenotype. The contributions of Mac-1, LFA-1, and p150,95 to aggregation of PMA-differentiated U937 cells were assessed. Monoclonal antibodies to the beta subunit and the LFA-1 alpha subunit, but not those to p150,95 or Mac-1 alpha subunit, inhibited this homotypic adherence.}, keywords = {550}, author = {Miller,L.J. and Schwarting,R. and Springer, T.A.} } @article {528951, title = {The requirement for lymphocyte function-associated antigen 1 in homotypic leukocyte adhesion stimulated by phorbol ester}, journal = {J. Exp. Med.}, volume = {163}, number = {5}, year = {1986}, note = {Reprint Status: In File}, pages = {1132-1149}, abstract = {Lymphocytes become adherent and aggregate after stimulation with phorbol esters such as PMA. Time-lapse video showed that aggregating cells were motile and exhibited vigorous pseudopodial movements. Adhesion sites were initiated between pseudopodia of neighboring cells, and then moved to the uropod. PMA-stimulated aggregation by EBV-transformed B cell lines, SKW-3 (a T cell line), differentiated U937 (a monocytic line), and blood lymphocytes was inhibited by mAbs to LFA-1. A number of different mAb to the LFA-1 alpha and beta subunits and F(ab{\textquoteright})2 and Fab{\textquoteright} fragments inhibited aggregation. Furthermore, lymphoblasts from normal individuals, but not from LFA-1-deficient patients, aggregated in response to PMA. These findings suggest LFA-1 is critically involved in stimulated lymphocyte adhesion. LFA-1 expression was not increased by PMA stimulation, showing that other mechanisms regulate LFA-1-dependent adherence. LFA-1-deficient patient cells were able to coaggregate with LFA-1+ cells, showing that aggregation is not mediated by like-like interactions between LFA-1 molecules on opposite cells. Aggregation was Mg+2-dependent, inhibited by cytochalasin B, and was reversed when LFA-1 mAb was added to preformed aggregates. Previous findings suggesting that LFA-1 is important in a wide variety of leukocyte functions are elucidated by this work, which shows that LFA-1 is a general leukocyte cell adhesion molecule, the activity of which is regulated by cell activation.}, author = {Rothlein,R. and Springer, T.A.} } @article {529106, title = {T lymphocyte adhesion to endothelial cells: Mechanisms demonstrated by anti-LFA-1 monoclonal antibodies}, journal = {J. Immunol.}, volume = {137}, number = {9}, year = {1986}, note = {Old $\#$4775Reprint Status: NOT In File}, pages = {2901-2906}, abstract = {Adhesion of lymphocytes to vascular endothelium is the first event in the passage of lymphocytes into a chronic inflammatory reaction. To investigate molecular mechanisms of T-EC adhesion, monoclonal antibodies (Mab) against T cell surface antigens have been tested for inhibition of binding. Baseline and phorbol ester-stimulated adhesion were strongly inhibited by either Mab 60.3 (reactive with the beta-chain of the LFA-1, OKM1, and p150,95 molecules) or by Mab TS 1/22 (specific for the alpha-chain of LFA-1). Although the increased binding of phorbol ester-stimulated lymphocytes was inhibited by anti-LFA-1 antibody, there was no increased expression of LFA-1 on phorbol ester-stimulated T cells, as determined by FACS analysis. Maximal inhibition of unstimulated and phorbol ester-stimulated T-EC adhesion was seen at Mab concentrations of 1 microgram/ml. In contrast, LPS- and IL 1-enhanced T-EC adhesion were only weakly inhibited by these antibodies. Mab 60.3 and TS 1/22 did not stain either unstimulated EC or LPS- or IL 1-stimulated EC, as measured by FACS analysis; moreover, preincubation of EC alone with these antibodies did not lead to inhibition of T-EC binding. Adhesion was not affected by Mab against the sheep erythrocyte receptor (LFA-2), a nonpolymorphic HLA class 1 framework antigen, or against LFA-3, the alpha-chain of OKM1, or the alpha-chain of p150,95. These results suggest that the mechanism of binding of lymphocytes to unstimulated endothelium differs from that to stimulated endothelium. LFA-1 appears to be an important adhesion-related molecule for binding to unstimulated endothelium. However, the increased lymphocyte adhesion to IL 1- or LPS-stimulated EC observed in these experiments appears to be relatively independent of LFA-1. The increased adhesion to stimulated EC could be due either to an increase in the avidity or the density of the EC receptor molecules ordinarily involved in unstimulated T-EC binding or to the formation of alternative receptors on the stimulated EC that are not present on unstimulated cells.}, keywords = {276}, author = {Haskard,D. and Cavender,D. and Beatty,P. and Springer, T.A. and Ziff,M.} } @article {529956, title = {Two antigen-independent adhesion pathways used by human cytotoxic T cell clones}, journal = {Nature}, volume = {323}, number = {6085}, year = {1986}, note = {MS $\#$125Reprint Status: In File}, pages = {262-264}, abstract = {Cell-cell adhesion is essential for many immunological functions, including interaction of cytotoxic T lymphocytes (CTLs) with their targets. We have explored CTL-target interactions using well-characterized cloned human CTLs. Conjugate formation between these CTLs and many antigen-negative targets is almost as efficient as with specific target cells, but does not lead to target-cell lysis. Thus, on specific target cells, adhesion by antigen-independent pathways may occur concurrently with or precede antigen recognition. The molecules LFA-1, CD2 (T11, LFA-2) and LFA-3 have been shown to be involved in human CTL conjugation with and lysis of specific target cells. Here we describe monoclonal antibody inhibition studies using individual monoclonal antibodies and mixes which demonstrate (1) that LFA-1, CD2 and LFA-3 are involved in antigen-independent conjugate formation; and (2) suggest that CD2 and LFA-3 are involved in one pathway and LFA-1 in another. We confirmed the existence of distinct pathways by the demonstration that LFA-1-dependent adhesion requires divalent cations and is temperature-sensitive whereas CD2- and LFA-3-dependent adhesion does not require divalent cations and is temperature-insensitive. Together with previous data, our studies suggest that CD2 on the effector interacts with LFA-3 as its ligand on targets.}, author = {Shaw,S. and Luce,G.E.G. and Quinones,R. and Gress,R.E. and Springer, T.A. and Sanders,M.E.} } @article {528031, title = {Antigens associated with the activation of murine mononuclear phagocytes in vivo: Differential expression of lymphocyte function-associated antigen in the several stages of development}, journal = {Cell. Immunol.}, volume = {94}, number = {1}, year = {1985}, note = {Reprint Status: In File}, pages = {265-275}, abstract = {Two well-characterized antigens [Mac-1 and lymphocyte-function-associated antigen (LFA-1)], expressed on a variety of leukocytes, are members of a family of surface proteins associated with multiple recognition functions. To analyze expression of these proteins during macrophage development, we utilized both radioimmunoassay and flow cytometry. As previously reported, Mac-1 is expressed on murine macrophages in all stages of development. We found LFA-1 to be present on murine mononuclear phagocytes but only in certain stages of their development. Specifically, we found LFA-1 was expressed on murine tissue macrophages but only on those activated in vivo by bacillus Calmette Guerin (BCG) or, to a lesser extent, primed by pyran copolymer. Although LFA-1 was absent on inflammatory (responsive) and resident tissue macrophages it was also present on blood-borne monocytes. Activated macrophages also selectively expressed the H-11 and Ly-6 antigens. Thus, these data indicate that LFA-1 is selectively expressed on mononuclear phagocytes of the tissues but only on those in the primed and activated stages of development.}, author = {Strassmann,G. and Springer, T.A. and Haskill,S.J. and Miraglia,C.C. and Lanier,L.L. and Adams,D.O.} } @article {528051, title = {Characterization of a monoclonal rat anti-mouse interleukin 2 (IL-2) receptor antibody and its use in the biochemical characterization of the murine IL-2 receptor}, journal = {Clin. Immunol. Immunopathol.}, volume = {36}, number = {1}, year = {1985}, note = {Reprint Status: NOT In File}, pages = {18-29}, abstract = {Anti-murine interleukin 2 (IL-2) receptor monoclonal antibodies (mAb) were made from rats immunized with murine cytotoxic lymphocytes. One mAb, designated M7/20, strongly inhibited the proliferation of both IL-2 dependent CTLL-2 cells and concanavalin A (Con A)-induced T-cell blasts. Inhibition was linearly dependent on the concentrations of both M7/20 and IL-2. Utilizing FACS analysis, M7/20 was shown to bind selectively to mitogen-activated T lymphocytes and, to a lesser degree, to activated B lymphocytes. 125I-Labeled M7/20 binding assays indicated that 48-hr Con A-induced T-cell blasts possessed 89,000 binding sites/cell with a Kd of 1.2 X 10(-9) M. Competitive binding analyses indicated that M7/20 and IL-2 occupy the same or overlapping cell surface sites. Preliminary biochemical characterization of M7/20 immunoprecipitates of detergent extracts from both surface-iodinated and internally D-[3H]glucosamine-labeled T lymphoblasts indicated that the murine IL-2 receptor is an N-glycosylated 58,000-Da glycoprotein. Together these results suggest that mAb M7/20 binds at or near the IL-2-binding epitope on the murine IL-2 receptor and, thus, upon manipulation may act as an IL-2 agonist.}, author = {Gaulton,G.N. and Bangs,J. and Maddock,S. and Springer, T.A. and Eardley,D.D. and Strom,T.B.} } @article {527911, title = {Characterization of patients with an increased susceptibility to bacterial infections and a genetic deficiency of leukocyte membrane complement receptor type 3 and the related membrane antigen LFA-1}, journal = {Blood}, volume = {66}, number = {4}, year = {1985}, note = {Reprint Status: NOT In File}, pages = {882-890}, abstract = {Three children from two unrelated families had a history of recurrent bacterial infections, and their neutrophils were shown to have deficient phagocytic and respiratory responses and possible deficiencies in chemotaxis or adherence. Their neutrophils were strikingly deficient in the ability to ingest or give a respiratory burst in response to unopsonized bakers{\textquoteright} yeast or zymosan (Z). Tests for neutrophil and monocyte CR1 (C3b/iC3b receptor) and CR3 (iC3b receptor) demonstrated rosettes with both EC3b and EC3bi. However, EC3bi were bound only to CR1, and not to CR3, because EC3bi rosettes were inhibited completely by anti-CR1. Neutrophils, monocytes, and natural killer (NK) cells also did not fluorescence stain with monoclonal antibodies specific for the alpha-chain of CR3 (anti-Mac-1, anti-Mol, OKM1, and MN-41). Quantitation of C receptors with 125I monoclonal anti-CR1 and anti-CR3 indicated that neutrophils from each patient expressed normal amounts of CR1 per cell but less than 10\% of the normal amount of CR3. Examination of neutrophils by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that a normal glycoprotein of approximately 165,000 daltons was missing. Immunoblotting of these gels indicated that the missing band was the alpha-chain of CR3. Subsequent analysis of all three patients{\textquoteright} cells also demonstrated a deficiency of LFA-1 alpha-chain and the common beta-chain that is shared by the CR3/LFA-1/p150,95 membrane antigen family. The deficiency of LFA-1 probably explained the absent NK cell function, as normal NK cell activity is inhibited by anti-LFA-1 but not by anti-CR3. The reduced phagocytic and respiratory responses to Z were probably due to CR3 deficiency, because treatment of normal neutrophils with anti-CR3, but not anti-FLA-1, inhibits responses to Z by 80\% to 90\%. Ingestion of Staphylococcus epidermidis by normal neutrophils was shown to be partially inhibited by monoclonal antibodies to the alpha-chain of either CR3 or LFA-1, and monoclonal antibody to the common beta-chain inhibited ingestion by 75\%. Thus, both CR3 and LFA-1 may have previously unrecognized functions as phagocyte receptors for bacteria. The absence of this type of nonimmune recognition of bacteria by these children{\textquoteright}s neutrophils may be one of the reasons for their increased susceptibility to bacterial infections.}, author = {Ross,G.D. and Thompson,R.A. and Walport,M.J. and Springer, T.A. and Watson,J.V. and Ward,R.H.R. and Lida,J. and Newman,S.L. and Harrison,R.A. and Lachmann,P.J.} } @article {529276, title = {Complement receptor type three-dependent degradation of opsonized erythrocytes by mouse macrophages}, journal = {J. Immunol.}, volume = {135}, number = {4}, year = {1985}, note = {Reprint Status: NOT In File}, pages = {2668-2672}, abstract = {The role of the complement receptor type 3 (CR3) on thioglycollate-elicited peritoneal macrophages (TG-PM) in the destruction of opsonized particles was studied. We found that sheep red blood cells (E) that were opsonized with an IgM monoclonal anti-Forssman antibody and complement (E-IgM-C) were lysed by TG-PM, whereas there was little lysis of E pretreated with either the antibody or the complement source alone. Furthermore, this lysis could be inhibited by anti-CR3 monoclonal antibodies that had previously been shown to inhibit binding of E-IgM-C to the CR3. Kinetic studies of phagocytosis and lysis indicated that lysis of E-IgM-C occurs after phagocytosis, suggesting that lysis is an intracellular event. Further findings suggested that intra-cellular lysis was promoted by CR3 bound to the phagocytosed target, because a monoclonal anti-CR3 antibody decreased the rate of phagocytosis of E-IgM-C but not its magnitude, whereas the rate and extent of lysis were strikingly inhibited. Furthermore, TG-PM that had already internalized unopsonized E selectively lysed E-IgM-C that were added later. These data confirm that the interaction of the CR3 with its ligand on E-IgM-C promotes rapid phagocytosis, and further suggest that the CR3 facilitates degradation of the target particle once internalization has occurred.}, author = {Rothlein,R. and Springer, T.A.} } @article {528801, title = {Deficiency of the adhesive protein complex lymphocyte function antigen 1, complement receptor type 3, glycoprotein p150,95 in a girl with recurrent bacterial infections}, journal = {J. Clin. Invest.}, volume = {76}, number = {6}, year = {1985}, note = {Reprint Status: NOT In File}, pages = {2385-2392}, abstract = {A patient presenting delayed umbilical cord detachment, severe recurrent bacterial infections, and inability to form pus exhibited a profound defect in the expression of alpha- and beta-chains of the receptor for the C3bi fragment of C3 (CR3), lymphocyte function antigen 1 (LFA-1) molecule, and the p150,95 molecule found on neutrophils, monocytes, and lymphocyte membranes. This was shown by immunofluorescence studies using specific monoclonal antibodies, rosette formation with C3bi-coated erythrocytes, and immunoprecipitation for the LFA-1 complex. These membrane defects were responsible for abnormal phagocytic cell functions including adherence to nylon wool, cell movement, phagocytosis, and opsonized particle-induced oxidative response and for defective natural killer cell activity. In addition, lymphocyte function deficiencies previously unobserved in this disease were found. Cytolytic T lymphocyte activity was profoundly reduced; alpha- and gamma-interferon production were impaired. Finally, there was no antibody production to vaccinal antigens whereas the antibody responses to polysaccharides and to cytomegalovirus were found to be normal. The cytotoxic T cell deficiency could be expected from previous blocking experiments of this function with monoclonal antibodies to LFA-1 and is probably related to an extremely severe deficiency in LFA-1 expression in this patient. Anomalies in interferon and in antibody production suggest additional role(s) of the LFA-1 complex in monocyte/T lymphocyte/B lymphocyte cell interactions that have not yet been envisaged.}, author = {Fischer,A. and Seger,R. and Durandy,A. and Grospierre,B. and Virelizier,J.L. and Le Deist,F. and Griscelli,C. and Fischer,E. and Kazatchkine,M. and Bohler,M.-C. and Descamps-Latscha,B. and Trung,P.H. and Springer, T.A. and Olive,D. and Mawas,C.} } @inbook {529741, title = {The function of LFA-1 in cell-mediated killing and adhesion: Studies on heritable LFA-1, Mac-1 deficiency and on lymphoid cell self-aggregation}, booktitle = {Mechanisms of Cell-Mediated Cytotoxicity II}, year = {1985}, note = {Reprint Status: NOT In File}, pages = {311-322}, publisher = {Plenum Press}, organization = {Plenum Press}, address = {New York, London}, author = {Springer, T.A. and Rothlein,R. and Anderson,D.C. and Burakoff,S.J. and Krensky,A.M.}, editor = {Henkart,P. and Martz,E.} } @inbook {528316, title = {Functional and structural interrelationships among the Mac-1, LFA-1 family of leukocyte adhesion glycoproteins, and their deficiency in a novel, heritable disease}, booktitle = {Hybridoma Technology in the Biosciences and Medicine}, year = {1985}, note = {Reprint Status: In File}, pages = {191-206}, publisher = {Plenum Press}, organization = {Plenum Press}, address = {New York and London}, author = {Springer, T.A. and Anderson,D.C.}, editor = {Springer, T.A.} } @article {529171, title = {Heritable lymphocyte function-associated antigen-1 deficiency: Abnormalities of cytotoxicity and proliferation associated with abnormal expression of LFA-1}, journal = {J. Immunol.}, volume = {135}, number = {5}, year = {1985}, pages = {3102-3108}, abstract = {The effect of heritable LFA-1 deficiency on T lymphocyte function was measured. After primary mixed lymphocyte stimulation, all six patients studied showed diminished allospecific T lymphocyte cytolytic and NK activity as compared with kindred and normal controls. MLR and mitogen-induced proliferative responses were consistently depressed. LFA-1-deficient, EBV-transformed B cell lines were poor stimulators of T cell responses. Primary cytolytic responses by lymphocytes from severely LFA-1-deficient patients (less than 0.2\% of normal surface expression) were consistently more profoundly depressed than those by lymphocytes from moderately deficient patients (about 5\% of normal surface expression). These results demonstrate the importance of LFA-1 in lymphocyte function. After repeated MLR restimulation, proliferative and cytolytic capacity improved and CTL lines could be established from all patients. Cytolysis by lines from one but not a second severe patient, and by four of four moderate patients, was inhibited by anti-LFA-1 MAb, and at 10-fold lower concentrations than required for inhibition of killing by control CTL lines. The locus of inhibition was on the target cell for the severely deficient CTL line, and on both the target and effector cells for moderately deficient CTL lines. In contrast, the locus of inhibition for normal CTL is on the effector cell. These findings show that LFA-1 can participate bidirectionally in cell interactions. The in vitro results are discussed in terms of the clinical findings in patients.}, author = {Krensky,A.M. and Mentzer,S.J. and Clayberger,C. and Anderson,D.C. and Schmalstieg,F.C. and Burakoff,S.J. and Springer, T.A.} } @inbook {528311, title = {Human cytolytic T-lymphocyte clones and their function-associated cell surface molecules}, booktitle = {Hybridoma Technology in the Biosciences and Medicine}, year = {1985}, note = {Reprint Status: In File}, pages = {559-573}, publisher = {Plenum Press}, organization = {Plenum Press}, address = {New York and London}, author = {Krensky,A.M. and Mentzer,S.J. and Greenstein,J.L. and Crimmins,M. and Clayberger,C. and Springer, T.A. and Burakoff,S.J.}, editor = {Springer, T.A.} } @article {529216, title = {Identification of cell surface antigens present on murine hematopoietic stem cells}, journal = {J. Immunol.}, volume = {134}, number = {5}, year = {1985}, note = {Reprint Status: In File}, pages = {3286-3290}, abstract = {Nine antigens found on murine bone marrow cells were examined to define their pattern of expression in murine hematopoietic differentiation. Lymphocyte function antigen (LFA-1), heat stable antigen (recognized by M1/69), common leukocyte antigen (CLA, T200, Ly-5) and Lgp100a (recognized by 30-C7) were present on early hematopoietic progenitors, BFU-E, CFU-E, CFU-GM, and CFU-M. All antigens found on progenitors were found on some immature precursor cells, myeloblasts, erythroblasts, or monoblasts, but their pattern of expression on identifiable hematopoietic cells varied. Three of these antigens, LFA-1, heat stable antigen recognized by M1/69, and CLA, were expressed on leukocytes of all stages of maturity but were lost from the erythroid lineage during differentiation. MAC-1, Forssman antigen, heat stable antigen (recognized by M1/75), anti-P-95 (recognized by M5/113), and Ia (recognized by M5/114) were found only on differentiated hematopoietic precursors or mature cells. The expression of these antigens was more lineage-specific. MAC-1 and heat stable antigen (recognized by M1/75) were restricted to either mature myeloid or erythroid cells, respectively. The marked differences in distribution of these antigens suggest that they may be useful in negative or positive selection experiments to enrich progenitors, and that some of them may have a functional role in differentiation.}, keywords = {CD24}, author = {Miller,B.A. and Antognetti,G. and Springer, T.A.} } @inbook {528431, title = {The importance of the Mac-1, LFA-1 glycoprotein family in adherence-dependent inflammatory functions: Insights from an experiment of nature}, booktitle = {Infection and Malignancy (RES Symposium)}, year = {1985}, note = {Reprint Status: In File}, publisher = {Alan Liss}, organization = {Alan Liss}, address = {New York}, abstract = {The Mac-1, LFA-1 (lymphocyte function-associated 1), p150,95 family of glycoproteins, which share a common beta subunit of Mr 95 000, are of widespread importance in leucocyte adhesion reactions. This paper focuses on the role of this glycoprotein family in granulocyte and monocyte adhesion and chemotaxis in vitro, and in migration into inflammatory sites in vivo. Most findings have been made with granulocytes, but results with monocytes are similar. Some studies have used leucocytes from patients exhibiting a severe or moderate deficiency in expression of this glycoprotein family, which is secondary to a defect in the common beta subunit. Patients are susceptible to bacterial infections and have defective pus formation and Rebuck skin-window tests, despite chronic granulocytosis. Granulocytes from such patients exhibit defective adherence to serum albumin and fibronectin-coated glass or plastic, defective orientation and directed migration in response to chemoattractants, and are defective in chemoattractant-stimulated aggregation and hyperadherence. Antibodies to the common beta subunit, to the Mac-1 alpha subunit, and to a lesser extent to the LFA-1 and p150,95 alpha subunits, inhibit many of the same functional responses by normal cells. In normal granulocytes and monocytes chemoattractants stimulate a five-fold increase in Mac-1 and p150,95 surface expression, by mobilization of a latent, presumably intracellular, pool. Cells from patients are deficient in up-regulation of these molecules but show normal up-regulation of other surface receptors, degranulation and oxidative burst. The hypothesis is presented that Mac-1 and p150,95 regulate or directly mediate the increase in granulocyte and monocyte adhesivity, which is essential for diapedesis, chemotaxis and migration into inflammatory sites.}, author = {Anderson,D.C. and Springer, T.A.} } @inbook {529841, title = {Inherited LFA-1, Mac-1 deficiency and its molecular biology}, booktitle = {Mononuclear Phagocytes: Physiology and Function}, year = {1985}, note = {Reprint Status: In File}, pages = {115-123}, publisher = {Martinus Nijhoff}, organization = {Martinus Nijhoff}, address = {Dordrecht, Boston, Lancaster}, author = {Springer, T.A. and Sastre,L. and Schmalstieg,F. and Anderson,D.}, editor = {van Furth,R.} } @article {528236, title = {Leukocyte LFA-1, OKM1, p150,95 deficiency syndrome: functional and biosynthetic studies in three kindreds}, journal = {Fed. Proc.}, volume = {44}, number = {10}, year = {1985}, note = {Reprint Status: In File}, pages = {2671-2677}, abstract = {Three patients (2 female, 1 male) with recurrent infection, granulocytosis, impaired pus formation, and/or delayed umbilical cord separation were identified. Assessments of polymorphonuclear leukocytes (PMN)/monocyte function in each patient revealed profound abnormalities of adherence and adherence-dependent functions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of their PMN lysates demonstrated a deficient or absent protein(s) of 138 kilodaltons (gp 138). Na3HB4 labeling demonstrated the absence of a major cell surface glycoprotein complex in each patient. Among parental and sibling PMN suspensions, functional assessments revealed no consistent abnormalities, although variably diminished gp138 was identified by SDS-PAGE and Na3HB4 labeling. Analysis by fluorescence-activated cell sorting and monoclonal antibodies (MAb) to LFA-1 alpha, OKM1 alpha, and their common beta subunit demonstrated a severe or total deficiency of PMN/monocyte surface expression of each protein among all patients; intermediate values were observed for parental and affected sibling suspensions, findings consistent with an autosomal recessive mode of inheritance for this disorder. Cell surface labeling (125I) and immunoprecipitation with the same MAb demonstrated the absence of these glycoproteins in addition to a 150-kilodalton protein (p150,95). Identical abnormalities of surface expression of patient lymphocytes blast-transformed with phytohemagglutinin (PHA) or Epstein-Barr virus were demonstrated. Further, significantly diminished natural killer cell cytotoxicity was observed for each patient tested. PHA blast-transformed patient lymphocytes labeled with [35S]methionine demonstrated a total absence of the beta molecule but indicated the presence of an LFA-1 alpha precursor. These findings indicate that LFA-1 alpha synthesis and surface expression require beta association. It is concluded that impaired inflammatory function in this disorder is casually related to a heritable deficiency of critical "adhesive" leukocyte glycoproteins.}, author = {Anderson,D.C. and Schmalstieg,F.C. and Shearer,W. and Becker-Freeman,K. and Kohl,S. and Smith,C.W. and Tosi,M.F. and Springer, T.A.} } @article {528241, title = {The LFA-1, Mac-1 glycoprotein family and its deficiency in an inherited disease}, journal = {Fed. Proc.}, volume = {44}, number = {10}, year = {1985}, note = {MS $\#$78Reprint Status: In File}, pages = {2660-2663}, abstract = {A family of functionally important, high-molecular-weight glycoproteins with identical beta subunits has recently been defined on leukocyte cell surfaces. Soon after these molecules and at least some of their functions had been defined with monoclonal antibodies, an inherited disease, LFA-1, Mac-1 deficiency, was discovered in humans. This deficiency has confirmed that this glycoprotein family is of central importance in leukocyte cell surface adhesion reactions.}, keywords = {016}, author = {Springer, T.A.} } @article {527846, title = {The LFA-1, Mac-1 leukocyte adhesion glycoprotein family and its deficiency in a heritable human disease}, journal = {Biochem. Soc. Trans.}, volume = {13}, year = {1985}, note = {Reprint Status: In File}, pages = {3-6}, author = {Springer, T.A. and Sastre,L. and Anderson,D.C.} } @article {530096, title = {The Ly-15 alloantigenic system: A genetically determined polymorphism of the murine lymphocyte function-associated antigen-1 molecule}, journal = {Proc Natl Acad Sci USA}, volume = {82}, number = {2}, year = {1985}, note = {Reprint Status: NOT In File}, pages = {526-530}, abstract = {Serological and biochemical studies using monoclonal antibodies have demonstrated that the Ly-15 cell membrane alloantigens are polymorphic sites on the lymphocyte function-associated antigen-1 (LFA-1) molecule. Ly-15.2 and LFA-1 show identical tissue distributions, being present on all thymocytes, lymphocytes, and neutrophils, and flow cytofluorometric analysis indicated identical cell surface expression of these molecules. Identity of Ly-15.2 and LFA-1 was confirmed by immunochemical analysis. The Ly-15.2 and LFA-1 molecules have an identical heterodimeric structure of Mr 180,000 alpha chain and Mr 94,000 beta chain, which coelectrophorese on two-dimensional NaDodSO4/PAGE. Furthermore, anti-Ly-15.2 and anti-LFA-1 antibodies coprecipitate the same molecule from thymocyte lysates, and peptide mapping studies show that the Ly-15.2 and LFA-1 alpha chains are identical, as are the beta chains.}, author = {Hogarth,P.M. and Walker,I.D. and McKenzie,I.F.C. and Springer, T.A.} } @article {529986, title = {Sequence homology of the LFA-1 and Mac-1 leukocyte adhesion glycoproteins and unexpected relation to leukocyte interferon}, journal = {Nature}, volume = {314}, number = {6011}, year = {1985}, note = {Reprint Status: In File}, pages = {540-542}, abstract = {Cell-surface adherence reactions are fundamental to the biology of lymphocytes, monocytes and granulocytes. The lymphocyte function-associated 1 (LFA-1) and macrophage 1 (Mac-1) glycoproteins mediate differing types of adhesion reactions on these cells. LFA-1 participates in T-lymphocyte and natural killer-cell adhesion to target cells, whereas the Mac-1 antigen is identical to the complement receptor type 3, which mediates adhesion of monocytes and granulocytes to C3bi-sensitized particles. Deficiency of these proteins, in a heritable disease, results in multiple adhesion-related leukocyte defects. LFA-1 and Mac-1 resemble one another in overall structure, having alpha-subunits of relative molecular mass (Mr) 180,000 and 170,000, respectively, which are non-covalently associated with beta-subunits of Mr 95,000 in alpha 1 beta 1 complexes. Peptide mapping and immunological cross-reactivity have shown that the beta-subunits are highly related if not identical, but have revealed no similarities between the alpha-subunits. Nonetheless, the shared beta-subunit suggested that LFA-1 and Mac-1 might be members of a protein family containing diversified but evolutionarily related alpha-subunits. Therefore, we examine here the structure of the alpha-subunits by N-terminal amino-acid sequencing. Sequence homology shows that the alpha-subunits are members of a novel leukocyte adhesion protein family, and suggests that their evolution occurred by gene duplication. A search for similarities to previously sequenced proteins reveals a further unexpected homology between LFA-1 and leukocyte (alpha) interferons.}, keywords = {551}, author = {Springer, T.A. and Teplow,D.B. and Dreyer,W.J.} } @article {529411, title = {The severe and moderate phenotypes of heritable Mac-1, LFA-1 deficiency: Their quantitative definition and relation to leukocyte dysfunction and clinical features}, journal = {J. Infect. Dis.}, volume = {152}, number = {4}, year = {1985}, note = {MS $\#$89Reprint Status: In File}, pages = {668-689}, abstract = {An inherited syndrome characterized by recurrent or progressive necrotic soft-tissue infections, diminished pus formation, impaired wound healing, granulocytosis, and/or delayed umbilical cord severance was recognized in four male and four female patients. As shown with subunit-specific monoclonal antibodies in immunofluorescence flow cytometry and 125I immunoprecipitation techniques, in addition to a NaB3H4-galactose oxidase labeling assay, granulocytes, monocytes, or lymphocytes from these individuals had a "moderate" or "severe" deficiency of Mac-1, LFA-1, or p150,95 (or a combination)--three structurally related "adhesive" surface glycoproteins. Two distinct phenotypes were defined on the basis of the quantity of antigen expressed. Three patients with severe deficiency and four patients with moderate deficiency expressed less than 0.3\% and 2.5\%-31\% of normal amounts of these molecules on granulocyte surfaces, respectively. The severity of clinical infectious complications among these patients was directly related to the degree of glycoprotein deficiency. More profound abnormalities of tissue leukocyte mobilization, granulocyte-directed migration, hyperadherence, phagocytosis of iC3b-opsonized particles, and complement- or antibody-dependent cytotoxicity were found in individuals with severe, as compared with moderate, deficiency. It is proposed that in vivo abnormalities of leukocyte mobilization reflect the critical roles of Mac-1 glycoproteins in adhesive events required for endothelial margination and tissue exudation. The recognition of phenotypic variation among patients with Mac-1, LFA-1 deficiency may be important with respect to therapeutic strategies.}, author = {Anderson,D.C. and Schmalsteig,F.C. and Finegold,M.J. and Hughes,B.J. and Rothlein,R. and Miller,L.J. and Kohl,S. and Tosi,M.F. and Jacobs,R.L. and Waldrop,T.C. and Goldman,A.S. and Shearer,W.T. and Springer, T.A.} } @article {529341, title = {Studies on antigens associated with the activation of murine mononuclear phagocytes: Kinetics of and requirements for induction of lymphocyte function-associated (LFA)-1 antigen in vitro}, journal = {J. Immunol.}, volume = {135}, number = {1}, year = {1985}, note = {Reprint Status: In File}, pages = {147-151}, abstract = {Macrophages activated and primed in vivo, although not resident or responsive macrophages, express the lymphocyte function associated (LFA)-1 antigen. By contrast, the biochemically related Mac-1 antigen is expressed on all populations of macrophages. In the present paper, we studied regulation of the LFA-1 antigen in vitro. LFA-1 could be induced in vitro on thioglycollate (TG)-elicited but not on proteose peptone (PP)-elicited or resident macrophages. Specifically, macrophage-activating factor (MAF), interferon-gamma (IFN-gamma), or picogram amounts of endotoxin (LPS) induced LFA-1 on TG-elicited macrophages following overnight incubation. Interferon, -alpha or -beta, fucoidin, and colony-stimulating factor were not effective. While some levels of LFA-1 could be detected as soon as 10 hr, peak expression was observed after 16 to 32 hr of incubation. The induction could be completely abrogated by cycloheximide, suggesting that protein synthesis was required. These results indicate that the induction of LFA-1 on mononuclear phagocytes is closely regulated and that the requirements for such induction are distinct from but share certain similarities with induction of cytotoxic functions and expression of Ia antigen.}, keywords = {645}, author = {Strassmann,G. and Springer, T.A. and Adams,D.O.} } @article {529311, title = {Susceptibility of cytotoxic T lymphocyte (CTL) clones to inhibition by anti-T3 and anti-T4 (but not anti-LFA-1) monoclonal antibodies varies with the "avidity" of CTL-target interaction}, journal = {J. Immunol.}, volume = {134}, number = {5}, year = {1985}, note = {Reprint Status: In File}, pages = {3019-3026}, abstract = {To explore the role of the T3, T4, and LFA-1 molecules in high and low "avidity" interactions between SB2-specific cytotoxic T lymphocyte (CTL) clones and their targets, monoclonal antibody-mediated inhibition of cytotoxicity has been studied in experiments that vary the "avidity" of interaction in three different ways. 1) Previous results have been extended with respect to different CTL clones assayed on the same SB2-positive target cells. Differences between clones in susceptibility to anti-T3 inhibition paralleled variations in anti-T4 inhibition, and both correlated inversely with the "avidity" of the effector-target interaction (inferred previously from studies of conjugate dissociation). 2) A high "avidity" clone, 8.4, was identified that lysed not only SB2-positive cells but also cross-reacted on a few SB2-negative cells. Cold target inhibition studies confirmed the cross-reaction, and together with conjugate dissociation studies, indicated that cross-reaction to be of lower "avidity" than the specific recognition of SB2. Cross-reactive lysis was much more susceptible to inhibition by anti-T3 and anti-T4 than was specific lysis. 3) Anti-T3 and anti-T4 blocking was analyzed in the presence of anti-Ia antibody to reduce the amount of Ia antigen available on the target. Anti-T3 and anti-T4 antibody blocking was more efficient after the addition of anti-Ia antibody concentrations that (by themselves) produced minimal inhibition of lysis. As a control, anti-LFA-1 antibody blocking was analyzed in each of these three experimental systems that compare interactions of different "avidity"; minimal variation was observed in the efficiency of inhibition by anti-LFA-1. Thus, anti-T3 and anti-T4 inhibition correlates inversely with the "avidity" of that CTL-target interaction, but anti-LFA-1 inhibition does not.}, keywords = {615}, author = {Shaw,S. and Goldstein,G. and Springer, T.A. and Biddison,W.E.} } @article {528781, title = {Abnormalities of polymorphonuclear leukocyte function associated with a heritable deficiency of high molecular weight surface glycoproteins (GP138): Common relationship to diminished cell adherence}, journal = {J. Clin. Invest.}, volume = {74}, number = {2}, year = {1984}, note = {Reprint Status: NOT In File}, pages = {536-551}, abstract = {Investigations of polymorphonuclear leukocyte (PMN) function were performed in a 5-yr-old white female with delayed umbilical cord separation, impaired pus formation, and a severe defect of PMN chemotaxis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated an almost total deficiency of a high molecular weight glycoprotein(s) (GP138) in the granule and membrane fractions of the patient{\textquoteright}s cells, and NaB3H4-galactose oxidase labeling demonstrated the absence of a major glycoprotein complex on the surface of her PMNs. Monoclonal antibodies (MAb) were employed in flow cytometry experiments to demonstrate that two previously characterized glycoproteins (Mo1 and LFA1) were undetectable on the surface of the patient{\textquoteright}s PMNs and monocytes. Immunoprecipitation of 125I-labeled patient cells with subunit specific MAbs confirmed that the alpha-subunits of Mo1 (155 kD) and LFA1 (177 kD) and their common beta-subunit (94 kD) were totally deficient. Functional analyses of patient PMNs demonstrated severe impairment of adherence- and adhesion-dependent cell functions including spreading, aggregation, orientation in chemotactic gradients, antibody-dependent cellular cytotoxicity, and phagocytosis of particles (Oil-Red-0-paraffin, zymosan) selectively opsonized with C3-derived ligands. Patient PMNs demonstrated a normal capacity to rosette with IgG or C3b-coated sheep erythrocytes, but rosette formation with C3bi-coated erythrocytes was profoundly diminished. Adhesion-independent functions including shape change, N-formyl-methionyl-leucyl-3H-phenylalanine binding, and O-2 generation or secretion elicited by soluble stimuli were normal. Membrane fluidity, surface charge, and microtubule assembly were also normal. These findings provide new evidence that critical PMN surface glycoproteins are required to facilitate multiple adhesion-dependent cellular functions of the inflammatory response.}, author = {Anderson,D.C. and Schmalstieg,F.C. and Arnaout,M.A. and Kohl,S. and Tosi,M.F. and Dana,N. and Buffone,G.J. and Hughes,B.J. and Brinkley,B.R. and Dickey,W.D. and Abramson,J.S. and Springer, T.A. and Boxer,L.A. and Hollers,J.M. and Smith,C.W.} } @inbook {528076, title = {Analysis of macrophage differentiation and function with monoclonal antibodies}, booktitle = {Contemporary Topics in Immunobiology}, volume = {14}, year = {1984}, pages = {1-31}, publisher = {Plenum Press}, organization = {Plenum Press}, address = {New York}, author = {Springer, T.A. and Unkeless,J.C.}, editor = {Adams,D.O. and Hanna,M.G.,Jr.} } @inbook {530261, title = {Cell surface structures involved in the human cytolytic T lymphocyte response}, booktitle = {Regulation of the Immune System}, year = {1984}, note = {Reprint Status: NOT In File}, pages = {209-219}, publisher = {Alan R. Liss}, organization = {Alan R. Liss}, address = {New York}, abstract = {Two long-term cytolytic T lymphocyte (CTL) lines derived from the peripheral blood lymphocytes (PBL) of a single donor were analyzed for target specificity and involvement of cell surface molecules in CTL-target interactions. One line, AH2, was generated after stimulation with B lymphoblastoid cells. Cytolysis by these cells was restricted to targets expressing the appropriate HLA-A2 specificity and was blocked by mAb recognizing CD2, CD3, CD8, LFA-1, and LFA-3. The second line, AE1, was generated after stimulation with cultured endothelial cells derived from human newborn preputial microvessels. These CTL lysed all human target cells tested, except autologous cells and the Class I negative cell line Daudi. In addition, mAb specific for CD2, CD3, and CD8 did not affect cytolysis. Anti-LFA-1 and -LFA-3 mAb blocked cytolysis of B lymphoblastoid targets but not endothelial targets. These results indicate that some CTL utilize as yet uncharacterized cell surface structures for CTL-target interactions.}, keywords = {85}, author = {Krensky,A.M. and Clayberger,C. and Greenstein,J.L. and Collins,T. and Pober,J.S. and Robbins,E. and Anderson,D. and Springer, T.A. and Burakoff,S.J.}, editor = {Sercarz,E. and Cantor,H. and Chess, L.} } @article {529161, title = {Defective natural killer cytotoxicity and polymorphonuclear leukocyte antibody-dependent cellular cytotoxicity in patients with LFA-1/OKM-1 deficiency}, journal = {J. Immunol.}, volume = {133}, number = {6}, year = {1984}, note = {Reprint Status: In File}, pages = {2972-2978}, abstract = {Four children with an immunodeficiency involving the absence of leukocyte membrane glycoproteins reacting with anti-LFA-1 and OKM-1 monoclonal antibodies were unable to mediate adherence-dependent leukocyte functions. Even with normal Fc receptor function, their PMN-ADCC and MC-NKC were markedly deficient. Single cell analysis demonstrated deficient antibody-mediated PMN-target cell adherence. Monoclonal antibodies against LFA-1 and OKM-1 reproduced this immunodeficiency in leukocytes from normal adults. LFA-1/OKM-1 mediates a PMN-target cell adhesive step.}, author = {Kohl,S. and Springer, T.A. and Schmalstieg,F.C. and Loo,L.S. and Anderson,D.C.} } @article {528791, title = {Deficiency of a surface membrane glycoprotein (Mo1) in man}, journal = {J. Clin. Invest.}, volume = {73}, number = {1}, year = {1984}, note = {Reprint Status: NOT In File}, pages = {153-159}, abstract = {Deficiency of a granulocyte surface glycoprotein of 150,000-D had been associated with defective C3- and IgG-dependent phagocytosis in a patient with recurrent bacterial infections. By using monoclonal antibodies, we found that this patient{\textquoteright}s granulocytes, monocytes, and null cells were deficient in Mo1 (equivalent to OKM1 and Mac-1), a cell surface molecule consisting of two noncovalently linked glycoproteins of 155,000 and 94,000 D. The 155,000-D subunit is closely associated with the human complement receptor that recognizes C3bi and/or a further degradation product termed C3dg (C3bi receptor); the 94,000-D subunit has been shown to be shared, on normal cells, by two other surface membrane glycoproteins: lymphocyte function-associated antigen-1 (LFA-1) and P-150, 95. Both subunits of Mo1 were deficient on the patient{\textquoteright}s granulocytes as determined by immunoprecipitation with subunit-specific monoclonal antibodies as well as fluorescence analysis. Mol-deficient monocytes, like granulocytes, had defective C3-and IgG-dependent phagocytosis. Natural killing activity by the patient{\textquoteright}s peripheral blood leukocytes was normal. Mo1-deficient granulocytes and monocytes rosetted normally with sheep erythrocytes coated with C3bi. This rosetting was totally inhibited by a mixture of anti-Mo1 and anti-C3b (the major fragment of C3) receptor antibodies but not by either antibody alone. Since monoclonal antibodies to the 155,000-D subunit of Mo1 can inhibit C3bi receptor binding, immune phagocytosis, opsonized zymosan-induced degranulation, and superoxide generation by normal phagocytes (functions which are defective in Mo1-deficient cells), it appears likely that Mo1 deficiency may in part underlie the functional aberrations leading to recurrent bacterial infections in man.}, author = {Dana,N. and Todd,R.F.,III and Pitt,J. and Springer, T.A. and Arnaout,M.A.} } @article {529111, title = {Glycoproteins of 210,000 and 130,000 M.W. on activated T cells: Cell distribution and antigenic relation to components on resting cells and T cell lines}, journal = {J. Immunol.}, volume = {132}, number = {6}, year = {1984}, note = {Reprint Status: NOT In File}, pages = {3011-3018}, abstract = {A glycoprotein complex of 210,000 and 130,000 m.w., found on mitogen or alloantigen-stimulated human T cells and not on other hematopoietic cells, has been defined by a monoclonal antibody (Mab). The components of this complex are a subset of a larger family of proteins (210,000, 165,000 and 130,000 m.w.) defined by a second Mab. In a panel of hematopoietic cell lines and cell types, only activated T cells (including the cell line HUT-102) express the 210,000/130,000 complex and these cells also express the IL 2 receptor, a characteristic marker for activated T cells. The 210,000/130,000 m.w. complex (reactive with the Mab TS2/7) is present on all long-term activated T cells, including both the OKT4 and OKT8 subsets. The 210,000 m.w. subunit is expressed only on activated T cells. Other lymphoid cells express either the 130,000 m.w. subunit alone (unactivated lymphocytes, thymocytes, HUT-78) or the 130,000 subunit together with a 165,000 subunit (MOLT-4, HSB, and other leukemic T cell lines). The 210,000/130,000 m.w., 165,000/130,000 m.w. and 130,000 m.w. complexes are antigenically related in that all share reactivity with the Mab A- 1A5 . Among non-lymphoid hematopoietic cells and cell lines, none express the 210,000 m.w. chain; adherent cells (monocytes) and myeloid cell lines each express single proteins of 130,000 to 155,000 m.w. Granulocytes and red blood cells are negative and platelets express multiple bands (165,000 and 140,000 m.w.). Immunoperoxidase staining of tissue sections showed that a broad range of tissues and cell types had material cross-reactive with the lymphoid 130,000 m.w. protein. However, only a discrete subset of those tissues and cells including blood vessel walls, connective tissue, smooth muscle, kidney mesangial cells, and some non-cellular matrix tissue, had material cross-reactive with the 210,000 m.w. protein on activated T lymphocytes.}, author = {Hemler,M.E. and Sanchez-Madrid,F. and Flotte,T.J. and Krensky,A.M. and Burakoff,S.J. and Bhan,A.K. and Springer, T.A. and Strominger, J. L.} } @article {530326, title = {Human lymphocyte function associated antigens}, journal = {Surv. Immunol. Res.}, volume = {3}, number = {1}, year = {1984}, note = {Reprint Status: NOT In File}, pages = {39-44}, abstract = {Three cell surface molecules, designated LFA-1, LFA-2, and LFA-3 were identified by mAbs selected for their ability to block cytolysis by an OKT4+, HLA-DR-specific CTL line. The LFA mAbs block all CTL and proliferative functions studied. In addition, anti-LFA-1 mAbs inhibit NK-mediated cytolysis. By analogy with murine LFA-1, human LFA-1 may be involved in the adhesion stage of cellular interactions. LFA-2, the SRBC receptor molecule, appears to be a T cell function-specific molecule. We have not yet established whether LFA-2 participates in antigen recognition or whether it is involved in antigen-non-specific interactions. The anti-LFA-3 mAb specifically blocks function by binding to the target cells, implying that LFA-3 may be a target ligand for an effector-specific receptor. The CTL-target interaction involves a number of steps, including antigen recognition, cell adhesion, and delivery of the lethal hit [22]. The LFA antigens show the complexity of this process at the molecular level. The anti-LFA monoclonal antibodies will be useful probes into the T cell immune response and may prove clinically relevant, both diagnostically and therapeutically.}, author = {Krensky,A.M. and Sanchez-Madrid,F. and Springer, T.A. and Burakoff,S.J.} } @article {528971, title = {Inherited deficiency of the Mac-1, LFA-1, p150,95 glycoprotein family and its molecular basis}, journal = {J. Exp. Med.}, volume = {160}, number = {6}, year = {1984}, note = {Reprint Status: NOT In File}, pages = {1901-1918}, abstract = {Leukocyte surface glycoproteins that share a common beta subunit have been found to be congenitally deficient in three unrelated patients with recurring bacterial infection. The glycoproteins, Mac-1, LFA-1, and p150,95, have the subunit compositions alpha M beta, alpha L beta, and alpha X beta, respectively. Using subunit-specific monoclonal antibodies, both the alpha M and beta subunits of Mac-1, the alpha L and beta subunits of LFA-1, and at the least the beta subunit of p150,95, were found to be deficient at the cell surface by the techniques of immunofluorescence flow cytometry, radioimmunoassay, and immunoprecipitation. A latent pool of Mac-1 that can be expressed on granulocyte surfaces in response to secretory stimuli, such as f-Met-Leu-Phe, was also lacking in patients. Deficiency was found on all leukocytes tested, including granulocytes, monocytes, and T and B lymphocytes. Quantitation by immunofluorescence cytometry of subunits on granulocytes from parents of these patients and of a fourth deceased patient showed approximately half-normal surface expression, and, together with data on other siblings and a family with an affected father and children, demonstrate autosomal recessive inheritance. Deficiency appears to be quantitative rather than qualitative, with two patients expressing approximately 0.5\% and one patient approximately 5\% of normal amounts. The latter patient had alpha beta complexes on the cell surface detectable by immunoprecipitation. Biosynthesis experiments showed the presence of normal amounts of alpha{\textquoteright}L intracellular precursor in lymphoid lines of all three patients. Together with surface deficiency of three molecules that share a common beta subunit but have differing alpha subunits, this suggests the primary deficiency is of the beta subunit. The lack of maturation of alpha{\textquoteright}L to alpha L and the deficiency of the alpha subunits at the cell surface and in latent pools suggests that association with the beta subunit is required for alpha subunit processing and transport to the cell surface or to latent pools. The molecular basis of this disease is discussed in light of adhesion-related functional abnormalities in patients{\textquoteright} leukocytes and the blockade of similar functions in healthy cells by monoclonal antibodies.}, author = {Springer, T.A. and Thompson,W.S. and Miller,L.J. and Schmalstieg,F.C. and Anderson,D.C.} } @article {529176, title = {LFA-1, LFA-2 and LFA-3 antigens are involved in CTL-target conjugation}, journal = {J. Immunol.}, volume = {132}, number = {5}, year = {1984}, note = {Reprint Status: In File}, pages = {2180-2182}, abstract = {Three cell surface antigens associated with the CTL-target cell interaction were previously identified by generation of mAb against OKT4+, HLA-DR-specific CTL, and selection for inhibition of cytolysis in a 51Cr-release assay. In this report, we showed that these mAb inhibit cytolysis by blocking CTL-target cell conjugate formation. It appears that LFA-1, LFA-2, and LFA-3 are cell surface structures involved in strengthening effector-target adhesion that accompanies antigen-specific recognition.}, keywords = {160}, author = {Krensky,A.M. and Robbins,E. and Springer, T.A. and Burakoff,S.J.} } @inbook {529836, title = {Macrophage and T lymphocyte-mediated immunity: Similarities at the level of the Mac-1 and LFA-1 molecules}, booktitle = {Mononuclear Phagocyte Biology}, year = {1984}, note = {Reprint Status: In File}, pages = {109-128}, publisher = {Marcel Dekker}, organization = {Marcel Dekker}, address = {New York}, author = {Springer, T.A.}, editor = {Volkman,A.} } @inbook {529756, title = {Preparation and use of monoclonal antimacrophage antibodies}, booktitle = {Methods Enzymol.}, volume = {108}, year = {1984}, pages = {313-324}, publisher = {Academic Press}, organization = {Academic Press}, address = {New York}, author = {Ho,M. and Springer, T.A.}, editor = {DiSabato,G.} } @inbook {529831, title = {The relation of Langerhans cells to other dendritic cells and macrophages}, booktitle = {Mononuclear Phagocyte Biology}, year = {1984}, note = {Reprint Status: In File}, pages = {51-66}, publisher = {Marcel Dekker, New York}, organization = {Marcel Dekker, New York}, address = {New York}, keywords = {235, 550, 702, 710}, author = {Flotte,T.J. and Haines,K.A. and Pechman,K.M. and Springer, T.A. and Gigli,I. and Thorbecke,G.J.}, editor = {Volkman,A.} } @article {530331, title = {All monocyte antigens are not expressed on renal endothelium}, journal = {Tissue Antigens}, volume = {21}, number = {3}, year = {1983}, note = {Reprint Status: In File}, pages = {254-259}, abstract = {Recently a tissue restricted antigen system, which is expressed on endothelial cells and monocytes (E-M antigens) but not lymphocytes, has been associated with kidney graft rejection. In screening sera from recipients of kidney, bone marrow or skin grafts for possible reactivity with endothelial cell antigens, we have found that all (13 of 13) endothelial reactive sera also reacted with monocytes, but that many (21 of 34) monocyte reactive sera did not react with endothelial cells. Additionally, one well-defined monoclonal antibody (M1/70), which was cytotoxic for human monocytes, neither stained renal endothelium nor was absorbed by renal endothelium when perfused through a human kidney. Thus, not all monocyte antigens appear to be expressed in high concentrations on renal vascular endothelium. This may explain why monocyte reactive antibodies do not always correlate with kidney graft rejection.}, author = {Baldwin,W.M.,III and Claas,F.H.J. and Paul,L.C. and Springer, T.A. and Hendriks,G.F.J. and van Es,L.A. and Van Rood,J.J.} } @article {528491, title = {Biosynthesis and assembly of the α and β subunits of Mac-1, a macrophage glycoprotein associated with complement receptor function}, journal = {J. Biol. Chem.}, volume = {258}, number = {5}, year = {1983}, note = {Reprint Status: NOT In File}, pages = {2766-2769}, abstract = {Mac-1 is a macrophage surface antigen containing noncovalently associated alpha and beta subunits of Mr = 170,000 and 95,000, respectively (K{\"u}rzinger, K., and Springer, T.A. (1982) J. Biol. Chem. 257, 12412-12418). To determine whether the subunits are derived from a common or separate precursor, the biosynthesis of Mac-1 was studied. [35S]Methionine pulse-chase-labeled material was immunoprecipitated with either a monoclonal antibody recognizing an alpha chain determinant present in the associated alpha 1 beta 1 complex or a polyclonal antiserum recognizing the alpha 1 beta 1 complex as well as the free beta subunit. In peritoneal exudate macrophages, the alpha subunit was derived from a precursor of Mr = 161,000 which was converted to the mature Mr = 170,000 chain with a t1/2 of 30 to 45 min. The beta subunit was derived from a Mr = 87,000 precursor which became associated with the alpha subunit and was converted to Mr = 95,000 with a t1/2 of 2 h. Labeled beta chain took longer than alpha to become associated with the alpha 1 beta 1 complex in a number of different types of peritoneal macrophage populations, correlating with synthesis of an excess of beta. In the P388D1 macrophage-like tumor line, alpha and beta were processed with t1/2s of about 2 and 1 h. Both alpha and beta precursors were present in the complex, suggesting that complex formation preceded processing.}, author = {Ho,M-K. and Springer, T.A.} } @article {529101, title = {Blocking of CTL-mediated killing by monoclonal antibodies to LFA-1 and Lyt-2,3. I: Increased susceptibility to blocking after papain treatment of target cells}, journal = {J. Immunol.}, volume = {130}, number = {6}, year = {1983}, note = {Reprint Status: NOT In File}, pages = {2546-2551}, abstract = {It is now established that monoclonal antibodies (MAb) against LFA-1 and Lyt-2,3 antigens on cytolytic T lymphocytes (CTL) block killing function in the absence of C. It has been suggested that the blocking is inversely related to CTL-target affinity. In this report, we studied the effect of papain pretreatment of target cells, because papain is known to remove H-2 and to render target cells more resistant to allospecific CTL. CTL-target conjugate formation was weaker with papain-treated target cells (based on reduced post-dispersion lysis in dextran-containing medium). The concentration of MAb required to produce 40 to 60\% inhibition of 51Cr release (2-hr assay) was reduced four to 29-fold for alpha LFA-1 and 64 to 114-fold for alpha Lyt-2,3. Papain, however, did not induce blocking by MAb to other CTL antigens such as Thy-1, H-2, and T200. Flow cytometric analysis confirmed that papain selectively removed more than 95\% of H-2. In kinetic studies of removal and recovery, H-2 density and conjugate formation correlated well with each other. Sensitivity to blocking was not as well correlated, raising the possibility that an unidentified papain-sensitive target cell molecule other than H-2 plays an important role in CTL-target interaction.}, author = {Gromkowski,S.H. and Heagy,W.E. and Sanchez-Madrid,F. and Springer, T.A. and Martz,E.} } @article {528146, title = {Cytotoxic T cells directed against HLA-DR antigens and their surface proteins}, journal = {Diagn. Immunol.}, volume = {1}, number = {3}, year = {1983}, note = {Reprint Status: NOT In File}, pages = {116-119}, abstract = {The authors review their recent research involving the generation of cytotoxic T lymphocytes (CTL) directed against HLA-DR antigens. A mouse anti-human xenogeneic system first suggested that HLA-DR antigens could be recognized by CTL. Human allogeneic CTL specific for HLA-DR6 were generated and found to be OKT4+. The fact that these CTL were OKT4+ while anti HLA-A,B CTL were OKT8+ suggested that these T cell surface antigens may be involved in MHC antigen recognition; ie, they may be part of the T cell receptor. These OKT4+, HLA-DR specific CTL were further used to generate monoclonal antibodies (1) which block cytolysis and define novel antigens involved in the CTL-target interaction and (2) which define an antigenic complex on alloantigen activated T cells.}, author = {Burakoff,S.J. and Clayberger,C. and Hemler,M. and Krensky,A.M. and Reiss,C.S. and Robbins,E. and Sanchez-Madrid,F. and Springer, T.A. and Strominger, J. L. and Ware,C.F.} } @article {527771, title = {Dendritic cell and macrophage staining by monoclonal antibodies in tissue sections and epidermal sheets}, journal = {Am. J. Pathol.}, volume = {111}, number = {1}, year = {1983}, note = {Reprint Status: In File}, pages = {112-124}, abstract = {Mouse tissue sections were stained by monoclonal antibodies to macrophage antigens (Mac-1 (M1/70), Mac-2 (M3/38), Mac-3 (M3/84) with the use of immunoperoxidase. Mac-1 was located diffusely in the cytoplasm of round cells in a high percentage of alveolar macrophages, resident peritoneal and bone marrow cells, in splenic red pulp, and in rare perivascular cells in the thymus. Mac-1 was absent in epithelial cells and Langerhans cells. Mac-2 was strongly positive in many dendritic cells in the thymic medulla, more than the cortex, in paracortex and medulla of lymph nodes, sparing the follicles, and in the marginal zone of spleen. There were a few positive cells in germinal centers. Mac-2 was located in a low percentage of bone marrow and a high percentage of resident peritoneal cells. When positive in sections Mac-3 always showed granular cytoplasmic staining. Bone marrow showed a high percentage of cytoplasmic staining (greater than 50\%), as compared with low surface staining (less than 1\%). It was found in hematopoietic cells, and in all endothelium, including postcapillary venules and lining of sinuses. It was probable that the resulting dendritic staining pattern for Mac-3 in paracortex of lymph node, white and red pulp, thymic cortex, and medulla included dendritic cells other than endothelial cells. Alveolar macrophages and Kupffer cells were positive for Mac-2 and Mac-3. Mac-3 also stained bile canaliculi. Clearly different staining patterns were found in epithelial cells for Mac-2 and Mac-3 in kidney tubules, intestinal mucosal lining, bronchi, choroid plexus, and epidermis.}, keywords = {G/2,51}, author = {Flotte,T. and Springer, T.A. and Thorbecke,G.J.} } @article {529266, title = {Expression and induction in vitro of macrophage differentiation antigens on murine cell lines}, journal = {J. Immunol.}, volume = {130}, number = {1}, year = {1983}, note = {Reprint Status: In File}, pages = {108-114}, keywords = {550}, author = {Ralph,P. and Ho,M-K. and Litcofsky,P.B. and Springer, T.A.} } @article {529181, title = {The functional significance, distribution, and structure of LFA-1, LFA-2, and LFA-3: cell surface antigens associated with CTL-target interactions}, journal = {J. Immunol.}, volume = {131}, number = {2}, year = {1983}, note = {Reprint Status: NOT In File}, pages = {611-616}, abstract = {Three cell surface antigens associated with the cytolytic T lymphocyte(CTL)-target cell interaction were identified by generation of monoclonal antibodies (MAb) against OKT4+, HLA-DR-specific CTL and selection for inhibition of cytolysis in a 51Cr-release assay. These MAb block cytolysis by both OKT4+ and OKT8+ CTL and the proliferative responses to PHA and the mixed lymphocyte response (MLR). LFA-1 is an antigen widely distributed on lymphoid tissues and is composed of two polypeptides of 177,000 and 95,000 Mr on all cell types studied. Anti-LFA-1 MAb block NK cell-mediated cytolysis in addition to T lymphocyte-mediated cytotoxicity and proliferation. LFA-2 (Mr = 55,000 to 47,000), a determinant on the sheep red blood cell receptor, is expressed by T cells but not B cells and appears specific for T cell functions. LFA-3 (Mr = 60,000) is a widely distributed antigen present on both hematopoietic and nonhematopoietic tissues and appears to only be involved in T cell functions. MAb to LFA-1 and LFA-2 inhibit function by binding to effector cell surface molecules, whereas anti-LFA-3 MAb appear to block by binding to the target cells. Together with previously described molecules, LFA-1, LFA-2, and LFA-3 demonstrate the complexity of CTL-mediated cytotoxicity at the molecular level.}, author = {Krensky,A.M. and Sanchez-Madrid,F. and Robbins,E. and Nagy,J. and Springer, T.A. and Burakoff,S.J.} } @article {528956, title = {A human leukocyte differentiation antigen family with distinct α subunits and a common β subunit: The lymphocyte function-associated antigen (LFA-1), the C3bi complement receptor (OKM1/Mac-1), and the p150,95 molecule}, journal = {J. Exp. Med.}, volume = {158}, number = {6}, year = {1983}, pages = {1785-1803}, abstract = {The human lymphocyte function-associated antigen-1 (LFA-1), the complement receptor-associated OKM1 molecule, and a previously undescribed molecule termed p150,95, have been found to be structurally and antigenically related. Each antigen contains an alpha- and beta-subunit noncovalently associated in an alpha 1 beta 1-structure as shown by cross-linking experiments. LFA-1, OKM1, and p150,95 alpha-subunit designations and their molecular weights are alpha L = 177,000 Mr, alpha M = 165,000 Mr, and alpha X = 150,000 Mr, respectively. The beta-subunits are all = 95,000 Mr. Some MAb precipitated only LFA-1, others only OKM1, and another precipitates all three antigens. The specificity of these MAb for particular subunits was examined after subunit dissociation by high pH. MAb specific for LFA-1 or OKM1 bind to the alpha L- or alpha M-subunits, respectively, while the cross-reactive MAb binds to the beta-subunits. Coprecipitation experiments with intact alpha 1 beta 1-complexes showed anti-alpha and anti-beta MAb can precipitate the same molecules. In two-dimensional (2D) isoelectric focusing-SDS-PAGE, the alpha subunits of the three antigens are distinct, while the beta-subunits are identical. Biosynthesis experiments showed alpha L, alpha M, and alpha X are synthesized from distinct precursors, as is beta. The three antigens differ in expression on lymphocytes, granulocytes, and monocytes. During maturation of the monoblast-like U937 line, alpha M and alpha X are upregulated and alpha L is downregulated. Some MAb to the alpha subunit of OKM1 inhibited the complement receptor type three. LFA-1, OKM1, and p150,95 constitute a novel family of functionally important human leukocyte antigens that share a common beta-subunit.}, author = {Sanchez-Madrid,F. and Nagy,J. and Robbins,E. and Simon,P. and Springer, T.A.} } @article {529366, title = {Human lymphocyte function associated antigen-1 (LFA-1): identification of multiple antigenic epitopes and their relationship to CTL-mediated cytotoxicity}, journal = {J. Immunol.}, volume = {131}, number = {3}, year = {1983}, note = {Reprint Status: NOT In File}, pages = {1182-1187}, abstract = {Human lymphocyte function-associated antigen (LFA)-1, a heterodimeric lymphocyte surface glycoprotein of 177,000 and 95,000 relative molecular weight has been implicated to function in the cytotoxic T lymphocyte (CTL) effector mechanism. Seven mouse hybridoma lines producing monoclonal antibodies (MAb) reactive with this structure were studied. Three unique and 3 partially over-lapping epitopes on human LFA-1 were defined by competitive cross inhibition binding assays using biosynthetically labeled anti-LFA-1 MAb. In contrast, of five rat antimouse LFA-1 MAb, all five recognized a common or shared epitope. An HLA-B7 specific human CTL line expressed 1.1 X 10(5) LFA-1 sites per cell with a direct saturation binding assay. Human CTL expressed two to four times more LFA-1 than peripheral blood lymphocytes or B and T lymphoblastoid cell lines. Titration of each of the anti-LFA-1 MAb in a 51chromium release cytolytic assay revealed quantitative differences in the ability of the different anti-LFA-1 MAb to block cytolysis indicating distinct functional and antigenic epitopes exist on the human LFA-1 molecule. Anti-LFA-1 MAb reversibly inhibited the CTL reaction by slowing the initial rate of cytolysis. These results suggest anti-LFA-1 MAb inhibit CTL function by specific blockade of a functionally relevant molecule.}, author = {Ware,C.F. and Sanchez-Madrid,F. and Krensky,A.M. and Burakoff,S.J. and Strominger, J. L. and Springer, T.A.} } @article {527906, title = {Lymphokine inducing "terminal differentiation" of the human monoblast leukemia line U937: a role for γ interferon}, journal = {Blood}, volume = {62}, number = {6}, year = {1983}, note = {Reprint Status: in File}, pages = {1169-1175}, abstract = {The human monoblast leukemia line, U937, is growth-inhibited and induced to develop markers of mature monocytes by lymphokine preparations. Lymphokine is cytostatic and induces expression of Fc receptors in U937 and in myelomonocytic leukemic lines RC-2A and KG-1, but does not have these effects on T- and B-lymphocytic lines. In addition to previously described properties, including complement receptors, phagocytosis, and antibody-dependent cellular cytotoxicity (ADCC), Mac-1 and Mac-3 surface antigens defined by monoclonal antibodies are induced on U937 cells by lymphokine and phorbol ester. The Mac-1 surface component appears to have a regulatory role in differentiation of the monocyte lineage line, since antibodies to this antigen block the induction of Mac-3 antigen. The lymphokine activity was concentrated by salt precipitation and characterized by ion-exchange and size chromatography. Fractions of about 40,000 daltons were responsible for growth inhibition and induction of Fc receptors and Mac-1 antigen in U937 cells. However, ADCC was not induced in U937 by individual fractions of lymphokine, suggesting that this cytotoxic capacity may be regulated by a lymphokine of a different size, which is only effective after initial maturation steps. Since gamma-interferon is present on the 40K size range of lymphokine, the possibility that interferon is a differentiation modulator for the monoblast cells was investigated. Highly purified gamma-interferon (10(7) U/mg protein) at 10-300 U/ml inhibited growth and induced Fc receptors in U937 similar to the effect of lymphokine. The Fc-receptor-inducing activity of lymphokine was inhibited by a neutralizing monoclonal antibody to gamma-interferon, suggesting that this differentiation factor in lymphokine is gamma-interferon.}, author = {Ralph,P. and Harris, P and Punjabi,C.J. and Welte,K. and Litcofsky,P.B. and Ho,M-K. and Rubin,B. and Moore,M.A.S. and Springer, T.A.} } @article {528961, title = {Mapping of antigenic and functional epitopes on the α and β subunits of two related glycoproteins involved in cell interactions, LFA-1 and Mac-1}, journal = {J. Exp. Med.}, volume = {158}, number = {2}, year = {1983}, pages = {586-602}, abstract = {Mouse Mac-1, a complement receptor-associated surface structure on macrophages, and LFA-1, a function-associated structure on lymphocytes, comprise a novel family of leukocyte differentiation antigens participating in adhesive cell interactions. Mac-1 and LFA-1 contain alpha-subunits of 170,000 and 180,000 Mr, respectively, and beta-subunits of 95,000 Mr noncovalently associated in alpha 1 beta 1 complexes. The structural relation between the alpha- and between the beta-subunits, and the location of functionally important sites on the molecules, have been probed with antibodies. Both non-cross-reactive and cross-reactive monoclonal antibodies (MAb) and antisera prepared to the purified molecules or the LFA-1 alpha-subunits were used. Reactivity with individual subunits was studied by immunoprecipitation after dissociation induced by high pH treatment, or by immunoblotting after SDS-PAGE. Cross-reactive epitopes on Mac-1 and LFA-1 were found to be present on the beta-subunits, which were immunologically identical. Non-cross-reactive epitopes that are distinctive for Mac-1 or LFA-1 were localized to the alpha-subunits. MAb to LFA-1 alpha-subunit epitopes inhibited CTL-mediated killing. Two MAb to Mac-1 alpha-subunit epitopes but not a third MAb to a spatially distinct alpha-epitope inhibited complement receptor function. Neither function was inhibited by a MAb binding to a common beta-subunit epitope. Therefore, sites of Mac-1 and LFA-1 involved in their respective adhesion-related functions, as well as distinctive structural features, have been localized to the alpha-subunits.}, author = {Sanchez-Madrid,F. and Simon,P. and Thompson,S. and Springer, T.A.} } @article {529496, title = {Monoclonal antibodies against rat glomerular antigens: production and specificity}, journal = {Lab. Invest.}, volume = {49}, number = {1}, year = {1983}, note = {Reprint Status: In File}, pages = {107-117}, abstract = {To define the characteristics and target antigens (Ags) of nephrotoxic antibodies (Abs) and to analyze the factors that govern the evolution of Ab-mediated glomerular injury, we have prepared monoclonal Abs against rat glomerular Ags. BALB/c mice were immunized with Lewis rat cortex or glomeruli, and their spleens were removed and fused with hypoxanthine-aminopterin-thymidine supplement-sensitive myelomas. Hybrids were selected for production of Abs against Lewis rat kidney by indirect immunofluorescence. To date, more than 50 positive hybrids have been selected and their tissue reactivity defined by indirect immunofluorescence and immunoelectron microscopy. Of these, 14 are presented here in detail. One of these monoclonal Abs, K9/4, recognizes a unique Ag present exclusively on the cell surface of rat glomerular visceral epithelial cells. Four Abs (K12/2, K17/4, K12/5, and K12/8) recognize sites within the glomerular basement membrane; K12/2 and K17/4 also bind to vascular basement membranes of the rat, whereas K12/5 and K12/8 bind to glomerular basement membrane, tubular basement membranes, and vascular and epithelial basement membranes in all tissues of the rat. Two hybridomas (K6/1 and K6/3) recognize determinant(s) present on cell surfaces of endothelial and epithelial cells as well as within the glomerular basement membrane. All of these previously mentioned Abs are species restricted (i.e., they bind only to rat tissue) and, with the exception of K9/4, bind upon in vivo administration. Several others, however, recognize ubiquitous Ags that are present on intracellular structures in every species tested. The tissue distribution of these Ags suggests that they are present in contractile or cytoskeletal elements and, as expected from their intracellular location, monoclonal Abs directed against these components do not bind upon in vivo administration. Future studies will be directed at defining the antigenic composition of the glomerular capillary wall and the relevance of such Ags in immune-mediated glomerular injury.}, author = {Mendrick,D.L. and Rennke,H.G. and Cotran,R.S. and Springer, T.A. and Abbas,A.K.} } @inbook {529766, title = {Quantitation of hybridoma immunoglobulins and selection of myeloma or specific light chain loss variants}, booktitle = {Methods Enzymol.}, volume = {92}, year = {1983}, pages = {147-160}, publisher = {Academic Press}, organization = {Academic Press}, address = {New York}, keywords = {G/2,45}, author = {Springer, T.A.} } @article {529281, title = {Stable hamster-mouse hybridomas producing IgG and IgM hamster monoclonal antibodies of defined specificity}, journal = {J. Immunol.}, volume = {130}, number = {1}, year = {1983}, note = {Reprint Status: NOT In File}, pages = {309-312}, abstract = {Specific antibody-secreting hybridomas have been obtained by fusing Syrian or Armenian hamster (Mesocricetus auratus or Cricetulus migratorius) spleen cells with mouse myeloma cells. The hamsters were immunized to mouse cytolytic T lymphocytes. Hybrids were selected either by an indirect binding assay using an 125I-monoclonal antibody (MAb) reactive with hamster kappa-chains or by their ability to block T cell-mediated cytolysis. Three hybridoma clones were obtained that secreted intact IgM-like and IgG-like hamster MAb as shown by SDS-PAGE. The clones were stable as shown by subcloning. Two MAb recognized antigens of wide tissue distribution; the third bound specifically to T lymphocytes, gave strong inhibition of T cell-mediated cytolysis, and immunoprecipitated the Lyt-2,3 molecule.}, author = {Sanchez-Madrid,F. and Szklut,P. and Springer, T.A.} } @article {530091, title = {Staining of Langerhans cells with monoclonal antibodies to macrophages and lymphoid cells}, journal = {Proc Natl Acad Sci USA}, volume = {80}, number = {11}, year = {1983}, note = {Reprint Status: In File}, pages = {3448-3451}, abstract = {Langerhans cells are Ia-bearing antigen-presenting cells in the epidermis that share many functions with macrophages. We have used monoclonal antibodies to the macrophage antigens, Mac-2 and-3, Ia antigen, Fc fragment receptor and the common leukocyte antigen CLA to compare the cell surface antigens of these cells with those of interdigitating and follicular dendritic cells and of macrophages in lymphoid tissues. Immunoperoxidase staining was carried out with epidermal sheets from BALB/c mice and epidermal cell suspensions enriched for Langerhans cells by Fc rosetting. Langerhans cells stained for all of these antigens. Comparison with the staining properties of other dendritic cells and macrophages, in combination with previous observations, indicates a close relationship of Langerhans cells to the interdigitating cells of lymphoid tissues.}, keywords = {715}, author = {Haines,K.A. and Flotte,T.J. and Springer, T.A. and Gigli,I. and Thorbecke,G.J.} } @article {528486, title = {Tissue distribution, structural characterization and biosynthesis of Mac-3, a macrophage surface glycoprotein exhibiting molecular weight heterogeneity}, journal = {J. Biol. Chem.}, volume = {258}, number = {1}, year = {1983}, note = {Reprint Status: In File}, pages = {636-642}, abstract = {Mac-3 is a mouse macrophage differentiation antigen defined by a rat anti-mouse monoclonal antibody (MAb),M3/84. The structure, biosynthesis, quantitative surface expression, and distribution of Mac-3 have been studied by radiolabeling and isolation with MAb-Sepharose, saturation binding, absorption, and immunofluorescence flow cytometry. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Mac-3 migrates as a diffuse band with average Mr = 110,000. Labeling of intact cells with 125I and accessibility to MAb show it is present at least in part on the cell surface. Saturation labeling with 125I-MAb shows 4.2 X 10(4) cell surface sites on thioglycollate medium-elicited peritoneal macrophages. [35S]Methionine and [3H]glucosamine incorporation into Mac-3 by purified macrophages show it is a glycoprotein synthesized by these cells. Absorption shows Mac-3 is strongest in macrophages, present in lower quantities in lung, liver, bone marrow, and spleen, and undetectable in thymus, lymph node, brain, and heart. Immunofluorescent flow cytometry shows surface expression on thioglycollate-elicited macrophages but not bone marrow, spleen, lymph node, or thymus cell suspensions. Similar amounts of Mac-3 are immunoprecipitated from resident macrophages or macrophages elicited by sterile inflammatory agents, intracellular parasites, or immunomodulators, but the average Mr of Mac-3 varies from 92,000 to 110,000. Mac-3 is synthesized from precursor(s) of Mr = 74,000 and 79,000, identical in the different macrophages. Processing into the mature molecule, which migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a more diffuse band and varies in Mr among macrophage elicited by different agents and to a lesser degree between different preparations of the same type of macrophage, occurs in 15 to 30 min.}, author = {Ho,M-K. and Springer, T.A.} } @article {528021, title = {Antigens involved in mouse cytolytic T-lymphocyte (CTL)-mediated killing: functional screening and topographic relationship}, journal = {Cell. Immunol.}, volume = {73}, number = {1}, year = {1982}, note = {Reprint Status: In File}, pages = {1-11}, keywords = {G/2,48}, author = {Sanchez-Madrid,F. and Davignon,D. and Martz,E. and Springer, T.A.} } @article {528866, title = {Anti-Mac-1 selectively inhibits the mouse and human type three complement receptor}, journal = {J. Exp. Med.}, volume = {156}, number = {4}, year = {1982}, note = {Reprint Status: In File}, pages = {1000-1009}, abstract = {Anti-Mac-1 (M1/70), a rat monoclonal antibody that reacts with mouse and human macrophages, polymorphonuclear leukocytes (PMNL), and natural killer cells, selectively inhibited complement receptor-mediated rosetting by murine macrophages and human PMNL. Preincubation of macrophages with anti-Mac-1 inhibited formation of rosettes with sheep erythrocytes bearing IgM antibody and murine C3 fragments. No inhibition was observed when other monoclonal antibodies that react with macrophages (such as anti-Ly5, anti-H-2, or anti-pan-leukocyte) were tested at 10-fold higher concentrations. Anti-Mac-1 did not affect macrophage Fc receptor-mediated rosetting. Erythrocytes bearing homogeneous human C3 fragments C3b (EC3b) or C3bi (EC3bi) were used to test the specificity of the murine macrophage and human PMNL complement receptor inhibited by anti-Mac-1. In both cases, anti-Mac-1 inhibited CR3-mediated rosetting of EC3bi but not CR1-dependent rosetting of EC3b. The results show that Mac-1 is either identical to CR3 or closely associated with CR3 function. This is one of the first cases in which a monoclonal antibody-defined differentiation antigen has been associated with a specific cell surface function.}, keywords = {G/2,49}, author = {Beller,D.I. and Springer, T.A. and Schreiber,R.D.} } @article {528391, title = {LFA-1 and Lyt-2,3, molecules associated with T lymphocyte-mediated killing; and Mac-1, an LFA-1 homologue associated with complement receptor function}, journal = {Immunol. Rev.}, volume = {68}, year = {1982}, note = {Reprint Status: In File}, pages = {171-195}, keywords = {G/2,50}, author = {Springer, T.A. and Davignon,D. and Ho,M.K. and K{\"u}rzinger,K. and Martz,E. and Sanchez-Madrid,F.} } @article {529921, title = {LFA-1 but not Lyt-2 is associated with killing activity of cytotoxic T lymphocyte hybridomas}, journal = {Nature}, volume = {300}, number = {5890}, year = {1982}, note = {Reprint Status: In File}, pages = {357-360}, keywords = {160}, author = {Kaufman,Y. and Golstein,P. and Pierres,M. and Springer, T.A. and Eshhar,Z.} } @article {529121, title = {Mac-1 antigen: Quantitative expression in macrophage populations and tissues, and immunofluorescent localization in spleen}, journal = {J. Immunol.}, volume = {128}, year = {1982}, note = {Reprint Status: In File}, pages = {2281-2286}, keywords = {G/2,44, GD/3}, author = {Ho,M.K. and Springer, T.A.} } @article {529116, title = {Mac-2, a novel 32,000 Mr macrophage subpopulation-specific antigen defined by monoclonal antibody}, journal = {J. Immunol.}, volume = {128}, number = {3}, year = {1982}, note = {Reprint Status: NOT In File}, pages = {1221-1228}, abstract = {Two monoclonal antibodies, M3/31 and M3/38, were obtained by fusion of mouse myeloma cells with rat spleen cells immunized to immunoadsorbent-purified macrophage glycoproteins. Co-precipitation experiments show that antigenic determinants recognized by these two antibodies reside on the same molecular species, termed Mac-2, Mac-2, an antigen of 32,000 Mr, is synthesized by and expressed on the surface of thioglycollate-elicited macrophages as shown by [35S]-methionine and 125I labeling. Saturation binding experiments show that thioglycollate-elicited macrophages express 1.7 X 10(5) Mac-2 sites/cell. Thioglycollate-elicited macrophages are strongly absorptive for 125I-labeled M3/38 MAb. Kidneys are also absorptive; however, evidence is presented pointing to the nonspecificity of this absorption. Lymph node and thymus are negative, whereas spleen and bone marrow are weakly absorptive, probably due to stromal cells. Nonlymphoid tissues, such as lung, liver, heart, and brain, exhibit slight or no absorbing capacity. Cell suspensions from spleen, bone marrow, thymus, and peripheral lymph node are greater than 99\% Mac-2- by immunofluorescent flow cytometry. In contrast, thioglycollate-elicited macrophages are greater than 96\% strongly positive for Mac-2. Only 20\% of peptone-elicited cells are weakly positive, whereas resident peritoneal macrophages and other macrophage elicited by Listeria monocytogenes, Con A, or LPS are greater than 98\% negative. SDS-PAGE of [35S]-methionine-labeled Mac-2 shows that thioglycollate-elicited macrophages synthesize 10- to 30-fold more Mac-2 than other peritoneal macrophage subpopulations, whereas all types of peritoneal macrophages synthesize and express on their surfaces similar amounts of the Mac-1 antigen. Mac-2 antigen is therefore induced in macrophages only in response to specific differentiative signals.}, keywords = {G/2,35, G/3,5, I/HHY}, author = {Ho,M.K. and Springer, T.A.} } @inbook {528321, title = {Macrophage differentiation antigens: markers for macrophage subpopulations and tissue localization}, booktitle = {Hybridomas in cancer diagnosis and treatment}, year = {1982}, note = {Reprint Status: In File}, pages = {53-61}, publisher = {Raven Press}, organization = {Raven Press}, address = {New York}, keywords = {G/2,40, G/3,16}, author = {Springer, T.A. and Ho,M-K.}, editor = {Mitchell,M.S. and Oettgen,H.F.} } @article {527756, title = {The molecular basis for cytolytic T lymphocyte function: Analysis with blocking monoclonal antibodies}, journal = {Adv. Exper. Med. Biol.}, volume = {146}, year = {1982}, note = {Reprint Status: In File}, pages = {447-465}, abstract = {During the past decade the mechanism of CTL-mediated killing has been resolved into 3 steps, and its cation requirements, and general nature have been well defined. However, biochemical understanding of the CTL-target interaction has made little progress. Recently, we have developed a monoclonal antibody (MAb) which blocks killing by binding to a previously undescribed molecule on the CTL membrane, a molecule which we therefore have termed lymphocyte function-associated antigen one (LFA-1). LFA-1 and Lyt-2,3 are the only presently identified sites for such blocking; antibodies to over a dozen other molecules expressed on the CTL do not block killing. Present evidence suggests that LFA-1 is crucial in the adhesive interaction of T cells with other cells (e.g., targets, macrophages, perhaps B cells) The continuing search for blocking MAbs provides a systematic way to link specific molecules with CTL function.}, keywords = {G/2,43}, author = {Martz,E. and Davignon,D. and K{\"u}rzinger,K. and Springer, T.A.} } @article {528306, title = {Monoclonal antibodies specific for rat IgG1, IgG2a, and IgG2b subclasses, and kappa chain monotypic and allotypic determinants: Reagents for use with rat monoclonal antibodies}, journal = {Hybridoma}, volume = {l}, number = {3}, year = {1982}, note = {Reprint Status: In File}, pages = {257-273}, abstract = {Mouse monoclonal antibodies to rat IgG were obtained by fusion of immune SJL mouse spleen cells to NSI myeloma cells. Seven monoclonal antibodies have been labeled with 125I and studied as to specificity and avidity by using a panel of rat monoclonal antibodies both as inhibitors and target antigens in soft well plate and indirect cell binding assays. All MAb were selected for high avidity of 4 X 10(7) to greater than or equal to 2 X 10(9) M-1. Four MAb were subclass-specific. RG11/39, RG7/1, and RG7/11 were absolutely specific for the Fc{\textquoteright} region of IgG1, IgG2a, and IgG2b, respectively. RG9/6 showed specificity for the Fab{\textquoteright} region of IgG2a but crossreacted with lower avidity with IgG2c. Three MAb reacted with rat kappa chains. RG7/9 defined a monotypic (common) kappa chain determinant. RG11/15 and RG7/7 were specific for allelic kappa 1a and kappa 1b determinants, respectively. The monotypic and kappa 1a allotypic determinants are topographically separated. The antibodies can be used as screening reagents in indirect cell binding assays. They have sensitivity similar to affinity-purified rabbit anti-rat IgG and more defined specificity. They do not crossreact with mouse or human IgG, making them particularly suitable companion reagents for rat anti-mouse or anti-human MAb. One Mab, RG7/7, strongly crossreacts with Syrian hamster IgG.}, keywords = {G/2,46}, author = {Springer, T.A. and Bhattacharya,A. and Cardoza,J.T. and Sanchez-Madrid,F.} } @inbook {529826, title = {Murine macrophage differentiation antigens defined by monoclonal antibodies}, booktitle = {Monoclonal hybridoma antibodies: techniques and applications}, year = {1982}, note = {Reprint Status: In File}, pages = {169-175}, publisher = {CRC Press.}, organization = {CRC Press.}, address = {New York.}, keywords = {G/2,37, NKW}, author = {Springer, T.A.}, editor = {Hurrel,J.G.R.} } @article {528531, title = {Purification and structural characterization of LFA-1, a lymphocyte function-associated antigen, and Mac-1, a related macrophage differentiation antigen}, journal = {J. Biol. Chem.}, volume = {257}, number = {20}, year = {1982}, note = {Reprint Status: In File}, pages = {12412-12418}, abstract = {LFA-1, an antigen associated with antigen-specific T lymphocyte-mediated killing, and Mac-1, a macrophage differentiation antigen associated with type three complement receptor function, contain alpha chains of Mr = 180,000 and 170,000, respectively, and beta chains of Mr = 95,000. The monoclonal antibodies defining these antigens do not cross-react. The LFA-1 and Mac-1 beta chains are highly homologous or identical, whereas the alpha chains are highly different by tyrosyl tryptic peptide mapping (K{\"u}rzinger, K., Ho, M. K., and Springer, T. A. (1982) Nature (Lond.) 296, 668-670). T lymphoma cell lines express LFA-1 but not Mac-1 as shown by immunofluorescence and immunoprecipitation. Conversely, some macrophage-like lines express Mac-1 but not LFA-1. Other macrophage-like lines co-express Mac-1 and small amounts of LFA-1. Mac-1 and LFA-1 are present as separate molecules in these cells. [35S]Methionine and [[3H]glucosamine are incorporated into both alpha and beta chains of Mac-1 and LFA-1, showing both chains are endogenously synthesized and are glycoproteins. Cross-linking and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments show that in both Mac-1 and LFA-1 the alpha and beta chains are noncovalently associated in alpha 1 beta 1 quaternary structures. By quantitative immunofluorescence flow cytometry, the EL-4 T lymphoma and P388D1 macrophage-like lines were estimated to express 10(5) LFA-1 and 1.6 x 10(5) Mac-1 molecules/cell, respectively. From these sources the antigens have been purified to homogeneity in 200-400-micrograms quantities by monoclonal antibody affinity chromatography. The purified antigens contain only the alpha and beta subunits.}, keywords = {G/2,47}, author = {K{\"u}rzinger,K. and Springer, T.A.} } @article {529386, title = {Role of syngeneic Ia+ accessory cells in the generation of allospecific CTL responses}, journal = {J. Immunol.}, volume = {129}, number = {2}, year = {1982}, note = {Reprint Status: NOT In File}, pages = {694-697}, abstract = {Under conditions in which antigen dose is suboptimal, the recognition of Ia determinants is a necessary component of the generation of allospecific CTL responses. With monoclonal anti-Ia antibodies used as blocking reagents, it is demonstrated that the recognition of alloantigens may proceed via two pathways. Alloantigens can be recognized in the context of syngeneic Ia determinants in a similar fashion to conventional antigens. If, however, the Ia+ cells are removed from the responder population, the generation of such responses involves the recognition of allogeneic Ia determinants directly. A non-T, non-B, adherent accessory cell was identified as the critical syngeneic Ia+ cell required for CTL responses in these cultures.}, author = {Weinberger,O. and Germain,R.N. and Springer, T.A. and Burakoff,S.J.} } @article {529096, title = {A single monoclonal anti-Ia antibody inhibits antigen-specific T cell proliferation controlled by distinct Ir genes mapping in different H-2 I subregions}, journal = {J. Immunol.}, volume = {128}, number = {3}, year = {1982}, note = {Reprint Status: In File}, pages = {1409-1413}, abstract = {A xenogeneic rat anti-mouse Ia monoclonal antibody, M5/114 (gamma 2b, kappa), was studied for its effects in vitro on T cell proliferative responses. Strain distribution studies revealed that M5/114 could inhibit I-A subregion-restricted T cell responses of the H-2b,d,q,u but not the H-2f,k,s haplotypes, indicating that this xenoantibody recognizes a polymorphic determinant on mouse Ia molecules. This same monoclonal antibody was found to inhibit BALB/c (H-2d) T cell proliferation to both G60A30T10 and G58L38 phi 4. The Ir genes regulating responses to these antigens map to either the I-A subregion (GAT), or the I-A and I-E subregions (GL phi), raising the possibility that M5/114 recognizes both I-A and I-E subregion-encoded Ia glycoproteins. It could be shown, using appropriate F1 responding cells, that M5/114 does in fact affect GAT and GL phi responses by interaction with both the I-A and the I-E subregion products, and not by any nonspecific effect resulting from binding to the I-A subregion product alone. These results are consistent with genetic and biochemical studies directly demonstrating that M5/114 recognizes A alpha A beta and E alpha E beta molecular complexes. The existence of a shared epitope on I-A and I-E subregion products suggests the possibility that these molecules arose by gene duplication. Finally, the precise correlation between the Ia molecules recognized by M5/114 and the ability of this antibody to block T cell responses under Ir gene control strengthens the hypothesis that Ia antigens are Ir gene products.}, keywords = {G/2,42}, author = {Germain,R.N. and Bhattacharya,A. and Dorf,M.E. and Springer, T.A.} } @article {529926, title = {Structural homology of a macrophage differentiation antigen and an antigen involved in T-cell-mediated killing}, journal = {Nature}, volume = {296}, number = {5858}, year = {1982}, note = {Reprint Status: In File}, pages = {668-670}, keywords = {G/2,39, NKW}, author = {K{\"u}rzinger,K. and Ho,M.K. and Springer, T.A.} } @article {530166, title = {Three distinct antigens associated with human T lymphocyte-mediated cytolysis: LFA-1, LFA-2, and LFA-3}, journal = {Proc Natl Acad Sci USA}, volume = {79}, number = {23}, year = {1982}, note = {MS $\#$57Reprint Status: NOT In File}, pages = {7489-7493}, abstract = {Monoclonal antibodies were prepared to anti-HLA-DR cytolytic T lymphocytes (CTLs) and screened for inhibition of CTL-mediated killing. Binding of monoclonal antibodies to four types of molecules, LFA-1, LFA-2, LFA-3, and HLA-DR, inhibited killing, suggesting that these molecules participate in the CTL-target cell interaction. The antigens were characterized by immunoprecipitation, crosslinking, NaDodSO4/polyacrylamide gel electrophoresis, and immunofluorescence flow cytometry. The LFA-1 antigen contains alpha and beta polypeptide chains of Mr 177,000 and 95,000 that are noncovalently associated in an alpha 1 beta 1 structure. It is present on both B and T lymphocytes and marks subpopulations that differ in quantitative expression. Human LFA-1 appears to be the homologue of mouse LFA-1. Human LFA-2 is of Mr 49,000 with a minor component of Mr 36,000. It is expressed on CTL lines but not on a B-cell line and in peripheral blood preferentially on T lymphocytes. Human LFA-3 is of Mr 60,000 and is expressed on both B and T lymphocytes.}, author = {Sanchez-Madrid,F. and Krensky,A.M. and Ware,C.F. and Robbins,E. and Strominger, J. L. and Burakoff,S.J. and Springer, T.A.} } @article {529331, title = {Characterization of an anti-H-2 monoclonal antibody and its use in large-scale antigen purification}, journal = {J. Immunol.}, volume = {127}, number = {3}, year = {1981}, note = {Reprint Status: In File}, pages = {923-930}, abstract = {A rat anti-mouse monoclonal antibody (MAb), M1/42, has been found to react with H-2 antigens from cells of the a, b, d, j, k, s, and u haplotypes (all haplotypes tested). This antibody, when bound to cells and reacted with FITC-conjugated anti-rat Ig, could be used to quantitate H-2 expression on several cell types. The antibody was also useful in comparing the H-2 products precipitated from a variety of haplotypes. M1/42-coupled Sepharose-4B beads were used to purify H-2d antigens by affinity chromatography. Pure H-2 molecules eluted from the column in 0.5\% DOC, 0.65 M NaCl, 20 mM Tris, pH 8.0, yielding 110 to 180 micrograms H-2d/10(10) P815 tumor cells. This antibody, when used in series with H-2Kk-specific MAb 11-4.1, allowed purification of Dk and Dd from RDM-4 and YAC cells, respectively. H-2d purified by column chromatography on M1/42 was found to be serologically and biologically active, as determined by MAb rebinding, inhibition of cell lysis by alloantisera plus complement and ability to stimulate alloreactive CTL. This antibody and the described protocols should be useful in the preparation of relatively large quantities of a number of H-2 antigens.}, keywords = {340, 570, 630, G/2,36}, author = {Stallcup,K.C. and Springer, T.A. and Mescher,M.F.} } @article {529031, title = {Cross reaction of a rat-anti-mouse phagocyte-specific monoclonal antibody (anti-Mac-1) with human monocytes and natural killer cells}, journal = {J. Immunol.}, volume = {126}, number = {1}, year = {1981}, note = {Reprint Status: NOT In File}, pages = {359-364}, abstract = {A monoclonal antibody produced by a hybridoma cell line that has previously been shown to recognize an antigen present on murine macrophages and granulocytes (Mac-1) has now been shown to bind to human monocytes and polymorphonuclear leukocytes. Human monocytes bind about 40,000 M1/70 (anti-Mac-1) F(ab{\textquoteright})2 or IgG molecules per cell in saturating conditions. In addition, M1/70 antibody recognizes a small population (less than 10\%) of human blood lymphocytes. These cells express approximately 3-fold fewer Mac-1 antigen determinants than monocytes. Separation of this lymphocyte subset on a fluorescence-activated cell sorter has shown that all the natural killing activity in human blood can be found among these cells. Similarly, separation of the natural killer cells by an independent method based on their surface Fc receptor has shown that nearly all of them can be labeled by the hybridoma antibody. The same results are obtained when an F(ab{\textquoteright})2 fragment of the M1/70 hybridoma antibody is used. The anti-Mac-1 antibody does not interfere with binding to the Fc receptor, nor does it interfere with either natural killing or antibody-dependent cellular cytotoxicity mediated by these cells. We conclude that there is a similar antigenic structure on the surface of murine and human monocytes and granulocytes and that this structure is also found on human natural killer cells.}, keywords = {G/2,27, G/3,4}, author = {Ault,K.A. and Springer, T.A.} } @article {530076, title = {Lymphocyte function-associated antigen 1 (LFA-1): A surface antigen distinct from Lyt-2,3 that participates in T lymphocyte-mediated killing}, journal = {Proc Natl Acad Sci USA}, volume = {78}, number = {7}, year = {1981}, note = {Reprint Status: In File}, pages = {4535-4539}, abstract = {Monoclonal antibodies (MAb) have been used to probe the relationship of cytolytic T lymphocyte (CTL) surface molecules to CTL function. Rat MAb to mouse CTL were generated. Twelve MAb so obtained gave preferential binding to T cells as compared to B cells, and three of these recognized previously undescribed surface polypeptides. These Mab and more broadly reactive and previously obtained MAb were tested for their ability to block CTL-mediated killing in the absence of complement. To ensure that any observed blocking was due to binding of MAb to the effector cell rather than the target cell, a xenogeneic mouse CTL anti-rat BN lymphoma target cell system was utilized (MAb and target cells both of rat origin). Of 24 MAb tested here, 21 had little or no effect on CTL function, including those to H-2, Thy-1, Lyt-1, Ly 5, Ly 6, Lgp 100, and at least six other defined antigens. We confirmed inhibition of killing with two MAb to Lyt-2,3. Another MAb, M7/14, gave profound and consistent blockade of CTL function. It was confirmed that M7/14 MAb blocks killing by binding to the mouse CTL and does not bind to the rat lymphoma target cells used for the CTL assay. The findings suggest that the antigen defined by M7/14, termed a lymphocyte function-associated antigen, LFA-1, participates in or is closely associated with the mechanism of CTL-mediated killing. LFA-1 contains two polypeptide chains of 180,000 and 95,000 Mr and is distinct from other described lymphocyte glycoproteins. LFA-1 thus represents both a previously undescribed lymphocyte surface antigen and molecular site for blockade of CTL-mediated killing.}, keywords = {160, 645, 665, G/2,29}, author = {Davignon,D. and Martz,E. and Reynolds,T. and K{\"u}rzinger,K. and Springer, T.A.} } @inbook {529821, title = {Monoclonal antibodies as probes of surface structures participating in T-lymphocyte function}, booktitle = {Monoclonal antibodies and T cell hybridomas}, year = {1981}, note = {Reprint Status: In File}, pages = {45-52}, publisher = {Elsevier}, organization = {Elsevier}, address = {New York}, keywords = {G/2,30}, author = {Springer, T.A. and K{\"u}rzinger,K. and Reynolds,T. and Germain,R.N. and Davignon,D. and Martz,E.}, editor = {Hammerling,U. and Hammerling,G. and Kearney,J.} } @inbook {529771, title = {Monoclonal antibodies as tools for the study of mononuclear phagocytes}, booktitle = {Methods for studying mononuclear phagocytes}, year = {1981}, note = {Reprint Status: In File}, pages = {305-313}, publisher = {Academic Press}, organization = {Academic Press}, address = {New York}, keywords = {G/2,31}, author = {Springer, T.A.}, editor = {Adams,D.O. and Edelson,P.J. and Koren,H.} } @article {528591, title = {Monoclonal antibody analysis of complex biological systems: Combination of cell hybridization and immunoadsorbents in a novel cascade procedure and its application to the macrophage cell surface}, journal = {J. Biol. Chem.}, volume = {256}, number = {8}, year = {1981}, note = {Reprint Status: NOT In File}, pages = {3833-3839}, keywords = {G/1,2, G/2,28, I/HHY}, author = {Springer, T.A.} } @article {529061, title = {Monoclonal antibody to a novel lymphocyte function-associated antigen (LFA-1): Mechanism of blocking of T lymphocyte-mediated killing and effects on other T and B lymphocyte functions}, journal = {J. Immunol.}, volume = {127}, number = {2}, year = {1981}, note = {Reprint Status: NOT In File}, pages = {590-595}, keywords = {G/2,32}, author = {Davignon,D. and Martz,E. and Reynolds,T. and K{\"u}rzinger,K. and Springer, T.A.} } @article {529136, title = {Natural killer activity in the peritoneal exudates of mice infected with Listeria monocytogenes: Characterization of the natural killer cells by using a monoclonal rat anti-murine macrophage antibody (M1/70)}, journal = {J. Immunol.}, volume = {127}, number = {5}, year = {1981}, note = {Reprint Status: In File}, pages = {1792-1799}, abstract = {Exudates induced by i.p. injection of five listeria monocytogenes (LM) constituted a rich source of CBA/J murine natural killer (NK) cells. Maximum expression of NK activity was seen from day 2 through day 6 after initial exposure to LM. When nylon wool nonadherent peritoneal exudate cells were examined by a single-cell cytotoxicity assay, the number of cells binding to YAC-1 target cells increased after infection as did their individual lytic capacity. A monoclonal rat anti-murine macrophage antibody (M1/70), previously shown by our group to recognize human NK cells, can also be used as a marker for murine NK cells. Utilizing M1/70 and the fluorescence-activated cell sorter, selection of M1/70-labeled mononuclear cells led to the enrichment of both NK and antibody-dependent cellular cytotoxicity. These M1/70-positive cells had a distinctive morphology and contained granules on Wright-Giemsa staining. They were not phagocytic, did not contain nonspecific esterase, and lacked surface I-Ak, IgM determinants, complement receptors, and high levels of Thy 1.2.}, keywords = {550, 740, G/2,38, G/3,2}, author = {Holmberg,L.A. and Springer, T.A. and Ault,K.A.} } @article {529186, title = {A novel lymphocyte function-associated antigen (LFA-1): cellular distribution, quantitative expression, and structure}, journal = {J. Immunol.}, volume = {127}, number = {2}, year = {1981}, note = {Reprint Status: NOT In File}, pages = {596-602}, abstract = {We have previously described a monoclonal antibody (MAb), M7/14, which blocks a variety of T cell functions, including CTL-mediated killing, the mixed lymphocyte response, and antigen-specific proliferation. The antigen defined by M7/14 has been designated lymphocyte function-associated antigen one (LFA-1). In this report, LFA-1 has been studied as to cell distribution, surface abundance, structure, and in comparison to other CTL surface antigens, LFA-1 is expressed on lymphoid cells of both the T and the B lineages and on a large fraction of bone marrow cells, but not on exudate macrophages or nonlymphoid tissues. T cells express more LFA-1 than B cells, both in the unstimulated and stimulated states. Compared with unstimulated spleen cells, cytolytic T lymphocyte cell preparations (CTLP) and Con A blasts, but not LPS blasts, show increased LFA-1 expression relative to H-2, and for T cell-containing populations, Lyt-2. M7/14 MAb binds to about 1.5 X 10(4) and 7 X 10(4) LFA-1 sites per average spleen cell or CTLP cell, respectively. M7/14 Mab binds to cTLP in quantitites of 2.5-fold and ies 10.4-fold less than H-2 and Thy-1 Mab, respectively; since the latter have little or no effect on CTL function, inhibition of killing by M7/14 MAb is specific for the LFA-1 surface site. M7/14 MAb and a blocking Lyt-2 MAb are bound in similar quantities of CTLP. LFA-1 is a glycoprotein and consists of 2 noncovalently linked polypeptide chains of 180,000 and 95,000 Mr. The same molecular species as on CTL is present on other T cells and on B cells. The molecular structure and cell distribution of LFA-1 clearly distinguishes it from Lyt-2,3, Ly-5, T145, and T11, which were previously suggested to be either associated with the function of and/or present on the surface of CTL.}, keywords = {G/2,33, G/3,10}, author = {K{\"u}rzinger,K. and Reynolds,T. and Germain,R.N. and Davignon,D. and Martz,E. and Springer, T.A.} } @inbook {529816, title = {Rat anti-mouse macrophage monoclonal antibodies and their use in immunofluorescent studies of macrophages in tissue sections}, booktitle = {Monoclonal antibodies and T cell hybridomas}, year = {1981}, note = {Reprint Status: In File}, pages = {53-61}, publisher = {Elsevier}, organization = {Elsevier}, address = {New York}, keywords = {G/1,5, G/2,34, I/HHY}, author = {Ho,M.K. and Springer, T.A.}, editor = {Hammerling,U. and Hammerling,G. and Kearney,J.} } @article {529036, title = {A shared alloantigenic determinant on Ia antigens encoded by the I-A and I-E subregions: evidence for I region gene duplication}, journal = {J. Immunol.}, volume = {127}, number = {6}, year = {1981}, note = {Reprint Status: In File}, pages = {2488-2495}, abstract = {Two monoclonal antibodies to mouse Ia antigens were produced by fusion of xenoimmune rat spleen cells with the NSI myeloma. These monoclonal antibodies detect polymorphic determinants present on B cells and activated T lymphocytes from mice carrying the H-2b, H-2d, H-2k, H-2r, and H-2q haplotypes but not from mice carrying the H-2s or H-2r haplotypes. Antigenic site number determinations showed the positive haplotypes can be divided into 2 groups. Mice bearing the H-2b, H-2d, and H-2q haplotypes express a high number--40,000 to 80,000--of antigenic sites per B lymphocyte, and monoclonal antibody plus complement can lyse B cells from these mice. In contrast, mice bearing the H-2k and H-2r haplotypes express a low number of antigenic sites--about 5000 per cell. Spleen cells from mice carrying the latter haplotypes are not lysed with monoclonal antibody and complement. Genetic mapping demonstrated that high and low expression map to the I-A and I-E subregions, respectively. The monoclonal antibodies detect an Ia specificity on I-Ab, I-Ad, I-Ed, and I-Ek molecules. These observations were confirmed using several different experimental approaches, i.e., cytotoxicity, fluorescent staining, competitive inhibition of monoclonal antibody binding, and 2-dimensional gel electrophoresis of immunoprecipitates. The avidity for A alpha b A beta b and E alpha k E beta k is 5 to 7 x 10(-9) M-1. The antigenic determinant is heat labile, which suggests that it is not carbohydrate. The results imply that Ia antigens encoded by distinct subregions share sequence homology, which may be a consequence of ancestral gene duplication.}, keywords = {G/2,41}, author = {Bhattacharya,A. and Dorf,M.E. and Springer, T.A.} } @article {528416, title = {Use of a monoclonal antibody specifically non-reactive with T cells to delineate lymphocyte subpopulations}, journal = {Immunology}, volume = {42}, number = {3}, year = {1981}, note = {Reprint Status: In File}, pages = {371-378}, abstract = {Rat monoclonal antibody M1/69.16 reacts with a heat stable antigen of mouse commonly expressed in the majority of cell types in blood, spleen, bone marrow and thymus, including cells of erythroid, myeloid and lymphoid series. However, subpopulations of cells in lymphoid tissues can be identified which are non-reactive with this antibody using the fluorescence-activated cell sorter. All surface Ig positive cells seem to react with M1/69.16 while more than 96\% of Ig negative cells in spleen and lymph nodes are M1/69.16 negative. Most cells (80\%-90\%) in the M1/69.16 negative populations in spleen lymph nodes and bone marrow express Thy-l. Thus, peripheral T cells are specifically non-reactive with this antibody. In contrast, approximately 95\% of thymocytes react with M1/69.16, leaving a minor population which is negative. The negative population (5\%) is enriched in cells expressing high amounts of H-2 antigen and those bearing H9/25 antigen which is specific for lymphocyte subsets, indicating that M1/69.16 negative thymocytes represent a specific subpopulation, possibly "mature{\textquoteright} thymocytes.}, keywords = {CD24, G/2,26}, author = {Takei,F. and Secher,D.S. and Milstein,C. and Springer, T.A.} } @inbook {529811, title = {Cell-surface differentiation in the mouse. Characterization of "jumping" and "lineage" antigens using xenogeneic rat monoclonal antibodies}, booktitle = {Monoclonal antibodies}, year = {1980}, note = {Reprint Status: NOT In File}, pages = {185-217}, publisher = {Plenum Press}, organization = {Plenum Press}, address = {New York}, keywords = {G/1,3, G/2,23, G/3,8}, author = {Springer, T.A.}, editor = {Kennett,R.H. and McKearn,T.J. and Bechtol,K.B.} } @article {529406, title = {Quantitation of light chain synthesis in myeloma x spleen cell hybrids and identification of myeloma chain loss variants using radioimmunoassay}, journal = {J. Immunol. Methods}, volume = {37}, number = {2}, year = {1980}, note = {Reprint Status: In File}, pages = {139-152}, abstract = {A radioimmunoassay specific for the MOPC 21 kappa (K) myeloma chain of NSI and X63 myeloma x spleen cell hybrids was used to study light chain secretion in myeloma-hybrid lines. The M1 series of rat spleen cell x NSI mouse myeloma hybrid lines was chosen to illustrate the application of the radioimmunoassay for K chain quantitation and identification of K chain loss variants. Most of these lines secrete H (specific heavy), L (specific light), and K (myeloma kappa) chains, i.e., are HLK lines. Assays specific for rat L chain and mouse K chain showed that the ratio of L/K chain secreted by 6 different hybrid HLK lines ranged from 1.1 to 12.4. Using the rapid radioimmunoassay screening procedure, HL clonal variants which had lost K chain secretion were isolated at a frequency of approximately 10(-2) and characterized. K chain loss was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of radiolabelled secreted products. Stability of one HL line and its HLK parent was examined during 9 months of growth in vitro. The HL line remained stable, while antibody secreted by the HLK line became inactive, apparently due to overgrowth by clonally dominant HK cells which no longer secreted specific L chains. The radioimmunoassay appears to detect MOPC 21 kappa chain variable region determinants. Therefore, although used here with rat-mouse hybrids, it should also be possible to use the assay to obtain mouse-mouse variant hybrid lines secreting antibody of improved homogeneity.}, keywords = {G/2,24}, author = {Springer, T.A.} } @article {528216, title = {Mac-1: a macrophage differentiation antigen identified by monoclonal antibody}, journal = {Eur. J. Immunol.}, volume = {9}, number = {4}, year = {1979}, note = {Reprint Status: In File}, pages = {301-306}, keywords = {G/1,1, G/2,22, G/3,1}, author = {Springer, T.A. and Galfre,G. and Secher,D.S. and Milstein,C.} } @inbook {528266, title = {Monoclonal antibodies and cell surface antigens}, booktitle = {Genetics and human biology: possibilities and realities. Ciba Foundation Symp. No. 66}, volume = {3}, year = {1979}, note = {Reprint Status: In File}, pages = {1-16}, publisher = {Excerpta Medica}, organization = {Excerpta Medica}, edition = {1}, address = {Amsterdam, pp. 251-266, 1979, and reprinted in Cell. Biol. Internatl. Rep.}, keywords = {G/2,21, I/HHY}, author = {Milstein,C. and Galfre,G. and Secher,D.S. and Springer, T.A.} } @article {527996, title = {Monoclonal antibodies as probes for differentiation and tumor-associated antigens: A Forssman specificity on teratocarcinoma stem cells}, journal = {Cell}, volume = {14}, number = {4}, year = {1978}, note = {Reprint Status: In File}, pages = {775-783}, abstract = {A set of monoclonal antibodies derived by fusing P3-NS1/1-Ag4-1 myeloma cells with spleen cells from a rat immunized with mouse spleen were screened for activity against a tumor cell panel. One of these antibodies was found to react only with mouse embryonal carcinoma cells and no other tumor cell type tested, including differentiated derivatives of teratocarcinomas. In the adult mouse, this antigen is expressed by subpopulations of cells in the spleen, bone marrow, lymph node, brain, kidney and testes, although not in liver and thymus. This antigen has a species and tissue distribution consistent with that of Forssman antigen. The molecules which carry this specificity on the embryonal carcinoma cells appear to be glycolipids.}, keywords = {G/2,19}, author = {Stern,P.L. and Willison,K.R. and Lennox,E. and Galfre,G. and Milstein,C. and Secher,D. and Ziegler,A. and Springer, T.A.} } @article {528111, title = {Monoclonal xenogeneic antibodies to mouse leukocyte antigens: Identification of macrophage-specific and other differentiation antigens}, journal = {Curr. Topics Microbiol. Immunol.}, volume = {81}, year = {1978}, note = {BOXReprint Status: In File}, pages = {45-50}, keywords = {G/2,18}, author = {Springer, T.A. and Galfre,G. and Secher,D. and Milstein,C.} } @article {528211, title = {Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens}, journal = {Eur. J. Immunol.}, volume = {8}, number = {8}, year = {1978}, note = {Reprint Status: In File}, pages = {539-551}, abstract = {Hybrid myeloma cell lines secreting monoclonal antibodies to mouse cell surface antigens have been prepared. Spleen cells from a DA rat immunized with B10 mouse spleen cells that had been enriched for T cells were fused to cells from a nonsecreting mouse myeloma line (NSI). The presence in the culture supernatants of antibodies binding to mouse spleen cells was tested by a binding assay with 125I-labeled anti-rat IgG. From a large number of positive cultures, ten independent hybrid clones were purified, each secreting a different antibody. Each antigenic target was analyzed by (a) gel electrophoresis of immunoprecipitated 125 I-labeled cell surface molecules, (b) heat stability, (c) strain and species distribution and (d) cross-inhibition of binding of different monoclonal antibodies. It was concluded that the ten monoclonal antibodies regognized four types of antigen. One was the heterophile, heat-stable, Forssman antigen. The second (mol.wt. 210 000) appears to be a major 125I-labeled lymphoid cell surface protein. The third, a minor component of spleen cells, was precipitated as two polypeptides of mol.wt. 190 000 and 105 000. Five IgG-secreting clones identify the fourth antigen, a heat-stable, possibly glycolipid component expressed on mouse red blood cells and also on thymocytes. Cross-inhibition studies suggest that these last monoclonal antibodies bind to overlapping, but not identical, determinants. The class and chain composition of the monoclonal antibodies were studied by gel electrophoresis, isoelectric focusing and ability to lyse red blood cells and thymocytes.}, keywords = {G/2,20, G/3,11}, author = {Springer, T.A. and Galfre,G. and Secher,D.S. and Milstein,C.} } @inbook {528446, title = {Studies of the structure of the human Ia-like antigen}, booktitle = {Ir genes and Ia antigens}, year = {1978}, note = {Reprint Status: In File}, pages = {229-234}, publisher = {Academic Press}, organization = {Academic Press}, address = {New York.}, keywords = {G/2,17}, author = {Springer, T.A. and Kaufman,J. and Terhorst, C. and Strominger, J. L.}, editor = {McDevit,H.O.} } @article {528066, title = {Chemical and immunological characterization of HLA-linked B-lymphocyte alloantigens}, journal = {Cold Spring Harbor Symp. Quant. Biol.}, volume = {41}, year = {1977}, note = {Reprint Status: NOT In File}, pages = {387-395}, keywords = {G/2,12}, author = {Springer, T.A. and Kaufman, J. F. and Siddoway,L.A. and Giphart,M. and Mann, D. L. and Terhorst, C. and Strominger, J. L.} } @article {528601, title = {Detergent solubilization, purification, and separation of specificities of HLA antigens from a cultured human lymphoblastoid line, RPMI 4265}, journal = {J. Biol. Chem.}, volume = {252}, number = {13}, year = {1977}, note = {Reprint Status: NOT In File}, pages = {4682-4693}, abstract = {HLA antigens have been purified to homogeneity after detergent solubilization from RPMI 4265, a human lymphoblastoid line. The inhibition of cytotoxicity assay for HLA antigen was modified, using preincubation with bovine serum albumin of antigen samples containing detergent to prevent lysis of target cells by detergent. Solubilization was tested with many types of detergents. A polyethyleneglycol oleyl ether nonionic detergent mixture, Brij 99:Brij 97 (2:1) was selected for solubilization, since it selectively solubilized HLA antigens, had a low absorbance at 280 nm and was uncharded. HLA antigens were then purified by Lens culinaris lectin affinity chromatography and Bio-Gel A-5m filtration. The antigen specifity HLA-A2 was separated from specificities HLA-B7,12 by isoelectric focusing. Purified HLA antigens contained a subunit of Mr=44,000 with NH2-terminal glycine, and a subunit of Mr=12,000, beta2-microglobulin, with NH2-terminal isoleucine.}, keywords = {G/2,13}, author = {Springer, T.A. and Mann, D. L. and DeFranco,A.L. and Strominger, J. L.} } @article {530336, title = {Detergent-soluble products of the HLA region}, journal = {Transplant. Proc.}, volume = {9}, number = {(Suppl. 1)}, year = {1977}, note = {Reprint Status: In File}, pages = {21-28}, keywords = {G/2,10}, author = {Springer, T.A. and Strominger, J. L.} } @article {529976, title = {Purification and structural characterisation of human HLA-linked B-cell antigens}, journal = {Nature}, volume = {268}, number = {5617}, year = {1977}, note = {Reprint Status: In File}, pages = {213-218}, abstract = {The human B cell-specific alloantigen which is closely linked genetically to HLA contains two non-covalently associated, sialogycoprotein subunits of molecular weight (MW) 29,000 (p29), and 34,000 (p34). Although p29 and p34 have different amino-terminal sequences, their tyrosine peptide maps indicate considerable similarity in other portions of their polypeptide chains. Thus the genes for their proteins may have evolved by duplication of a common ancestral gene. Another lymphocyte cell surface protein of MW 16,000 (p16) has also been characterised. Both p16 and p44 (the heavy chain of HLA-A,B antigens) have been compared with p29 and p34.}, keywords = {G/2,15}, author = {Springer, T.A. and Kaufman, J. F. and Terhorst, C. and Strominger, J. L.} } @article {528596, title = {Purification of HLA-linked B lymphocyte alloantigens in immunologically active form by preparative sodium dodecyl sulfate-gel electrophoresis and studies on their subunit association}, journal = {J. Biol. Chem.}, volume = {252}, year = {1977}, note = {Reprint Status: NOT In File}, pages = {6201-6207}, abstract = {The\ HLA-linked\ B\ cell alloantigen (p29,34) is composed of two subunits of 29,000 (p29) and 34,000 (p34) molecular weight. The partially purified\ HLA-linked\ B\ cell alloantigen was purified by a final step of\ preparative\ sodium\ dodecyl\ sulfate-gel\ electrophoresis. An antiserum was prepared against p29,34 which specifically lysed\ B\ lymphocytes. In\ sodium\ dodecyl\ sulfate at 21 degrees, p29 and p34 remained noncovalently associated and retained immunologic activity;\ subunit\ dissociation at higher temperatures correlated with loss of immunologic activity. Although the pI values of p29 and p34 are 6.1 and 5.2, respectively, the subunits co-electrofocus under nondenaturing conditions. In addition, cross-linking\ studies\ showed the\ B\ cell antigen has a (p29)1(p34)1\ subunit\ structure.}, keywords = {G/2,16}, author = {Springer, T.A. and Kaufman, J. F. and Siddoway,L.A. and Mann, D. L. and Strominger, J. L.} } @article {528071, title = {Structure of HLA-A and HLA-B antigens isolated from cultured human lymphocytes}, journal = {Cold Spring Harbor Symp. Quant. Biol.}, volume = {41}, year = {1977}, note = {Reprint Status: NOT In File}, pages = {323-329}, abstract = {From these data, a model was prepared which summarizes schematically our present knowledge of the structure and orientation of the HL-A antigenic molecule in the lymphocyte membrane (Fig. 3). It seems likely that the heavy chain spans the membrane, with the hydrophobic region inserted in the membrane and the hydrophilic C-terminus inside the cell. This C-terminal region bears one (possible two) SH residue which has the potential for forming interchain disulfides. Whether or not these are actually formed physiologically remains an interesting question. There is the attractive possibility that whatever the physiological functions of HL-A antigens are, structurally these molecules provide the potential for signaling from outside the cell to inside the cell because they span the membrane. It is even conceivable that this function might be expressed via the opening and closing of disulfide bridges.}, keywords = {G/2,11}, author = {Strominger, J. L. and Mann, D. L. and Parham, P. and Robb, R. and Springer, T.A. and Terhorst, C.} } @article {528606, title = {Subunit and disulfide structure of monomeric and dimeric forms of detergent-soluble HLA antigens}, journal = {J. Biol. Chem.}, volume = {252}, year = {1977}, note = {Reprint Status: NOT In File}, pages = {4694-4700}, abstract = {The\ structure\ of\ monomeric\ and\ disulfide-bonded\ dimeric\ forms\ of\ HLA antigens\ has been studied.\ Detergent-soluble\ HLA\ antigen heavy chains contain one or two easily reduced sulfhydryl groups not found in papain-solubilized\ HLA antigens, as demonstrated by amino acid analysis (Springer, T. A., and Strominger, J.L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2481-2485, and Terhorst, C., Parham, P., Mann, D.L., and Strominger, J.L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 910-914) and by labeling with iodo[3H]acetate. Dimer formation occurred during purification, since it was prevented by pretreatment of membranes containing\ HLA\ antigen with iodoacetamide. Cross-linking studies showed that the non-disulfide-bonded form of\ HLA antigens\ contains one\ subunit\ each of the Mr = 44,000 heavy chain and the Mr = 12,000 light chain (beta2-microglobulin).}, keywords = {G/2,14}, author = {Springer, T.A. and Robb,R.J. and Terhorst, C. and Strominger, J. L.} } @article {530201, title = {Detergent-soluble HLA antigens contain a hydrophilic region at the COOH-terminus and a penultimate hydrophobic region}, journal = {Proc. Natl. Acad. Sci. U. S. A.}, volume = {73}, year = {1976}, note = {Reprint Status: In File}, pages = {2481-2485}, abstract = {Purified,\ detergent-soluble\ HLA\ antigens\ (p44,12) are composed of a glycoprotein of molecular weight 44,000 (p44) and a peptide of molecular weight 12,000 (p12), beta2-microglobulin. Upon digestion with papain, p44,12 is converted to p39,12, then to p34,12, which retains antigenic activity. The NH2-terminal amino acid sequences of p34 and p44 are identical. p44, p39, and p34 were purified, and comparison of their amino acid compositions showed that the COOH-terminal peptide removed by the first papain cleavage is\ hydrophilic\ and contains cysteine that can be alkylated after mild reduction. The\ penultimate\ COOH-terminal peptide removed by the second papain cleavage is\ hydrophobic, and presumably anchors\ HLA\ antigens\ to the membrane. This correlates with the observation that p44,12 and p39,12 bind detergent, while p34,12 does not. The orientation and integration of\ HLA\ antigensin the lymphocyte membrane were thus defined, and the structure suggests that\ HLA\ antigens\ span the plasma membrane.}, keywords = {610, 775, 822, G/2,8}, author = {Springer, T.A. and Strominger, J. L.} } @article {528246, title = {The immunoglobulin-like structure of human histocompatibility antigens}, journal = {Federation Proceedings}, volume = {35}, year = {1976}, note = {Reprint Status: In File}, pages = {1177-1182}, keywords = {610, G/2,6}, author = {Strominger, J. L. and Humphreys, R. E. and McCune, J. M. and Parham, P. and Robb, R. and Springer, T.A. and Terhorst, C.} } @inbook {530266, title = {Isolation and structure of products of the human histocompatibility gene complex}, booktitle = {Role of histocompatibility gene complex in immune responses. Proc. Int. Conf. at Brook Lodge, Mich.}, volume = {35}, year = {1976}, note = {Reprint Status: In File}, pages = {621-643}, publisher = {Academic Press}, organization = {Academic Press}, address = {New York}, keywords = {G/2,7}, author = {Strominger, J. L. and Chess, L. and Humphreys, R. E. and Mann, D. and Parham, P. and Robb, R. and Schlossman,S. and Springer, T.A. and Terhorst, C.}, editor = {Katz, D. and Benacerraf,B.} } @inbook {527751, title = {The structure of products of the major histocompatibility complex in man}, booktitle = {27. Mosbach Colloquium: The immune system}, year = {1976}, pages = {202-219}, publisher = {Springer-Verlag}, organization = {Springer-Verlag}, address = {Berlin}, keywords = {G/2,9}, author = {Strominger, J. L. and Humphreys, R. E. and Kaufman, J. F. and Mann, D. L. and Parham, P. and Robb, R. and Springer, T.A. and Terhorst, C.}, editor = {Melchers,F. and Rajewsky,K.} } @inbook {528296, title = {Isolation of histocompatibility antigens and of several B-specific proteins from cultured human lymphocytes}, booktitle = {Histocompatibility Testing 1975}, year = {1975}, note = {Reprint Status: In File}, pages = {719-730}, publisher = {Munksgaard}, organization = {Munksgaard}, address = {Copenhagen}, keywords = {605, G/2,5}, author = {Strominger, J. L. and Chess, L. and Herrmann, H. C. and Humphreys, R. E. and Malenka, D. and Mann, D. and McCune, J. M. and Parham, P. and Robb, R. and Springer, T.A. and Terhorst, C.}, editor = {Kissmeyer-Nielsen} } @article {530341, title = {The immunoglobulin-like structure of human histocompatibility antigens}, journal = {Transplantation Reviews}, volume = {21}, year = {1974}, note = {Reprint Status: In File}, pages = {126-143}, keywords = {610, G/2,4}, author = {Strominger, J. L. and Cresswell, P. and Grey, H. and Humphreys, R. E. and Mann, D. and McCune, J. and Parham, P. and Robb, R. and Sanderson, A. R. and Springer, T.A. and Terhorst, C. and Turner, M. J.} } @article {530071, title = {Immunological identity of the small subunit of HL-A antigens and β2-microglobulin and its turnover on the cell membrane}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {71}, year = {1974}, pages = {2123-2127}, abstract = {A number of\ immunological\ methods have been employed to show that the\ small\ subunit\ of\ HL-A\ antigens, isolated either after papain treatment or after solubilization with detergent, is identical to beta(2)-microglobulin, a protein previously isolated from human urine and shown to be homologous in structure to constant region domains of immunoglobulins. Moreover, quantitative data indicate virtually total\ identity\ between the\ small\ subunit\ of\ HL-A\ antigens\ and beta(2)-microglobulin. Studies of the\ turnover\ of labeled\ HL-Aantigens\ from the lymphocyte surface indicate that the two subunits turn over at similar rates, although only the\ small\ subunit\ could be detected in the culture medium. The significance of these observations is discussed.}, keywords = {050, G/2,3}, author = {Cresswell, P. and Springer, T.A. and Strominger, J. L. and Turner, M. J. and Grey, H. M. and Kubo, R. T.} } @article {530206, title = {Partial purification of detergent solubilized HL-A antigen and its cleavage by papain}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {71}, year = {1974}, pages = {1539-1543}, abstract = {HL-A\ antigen\ solubilized\ with the non-ionic\ detergent, Brij 99, has been purified to about 50\% of homogeneity from a cultured human lymphoblast line. It consists of two nonidentical subunits of 44,000 and 12,000 molecular weight (MW). Upon\ papain\ proteolysis the 44,000 MW peptide is converted by at least two cleavages to a 34,000 MW peptide, but the 12,000 MW peptide appears to be unchanged. Concomitantly, the apparent molecular weight in gel filtration chromatography under nondenaturing conditions in the presence of Brij 99 is reduced from 460,000 to 45,000.\ HL-A\ molecules produced by direct\ papain\ proteolysis of membranes and bypapain\ treatment of purified\ detergent-soluble\ HL-A\ are identical.}, keywords = {610, G/2,2}, author = {Springer, T.A. and Strominger, J. L. and Mann, D.} } @article {528926, title = {The small subunit of HL-A antigens is β2-microglobulin}, journal = {Journal of Experimental Medicine}, volume = {138}, year = {1973}, pages = {1608-1612}, author = {Grey, H. M. and Kubo, R. T. and Colon, S. M. and Poulik, M. D. and Cresswell, P. and Springer, T.A. and Turner, M. and Strominger, J. L.} }