Publications

1985
Springer, T.A., Sastre, L. & Anderson, D.C. The LFA-1, Mac-1 leukocyte adhesion glycoprotein family and its deficiency in a heritable human disease. Biochem. Soc. Trans. 13, 3-6 (1985). Springer_1985_4173.pdf
Hogarth, P.M., Walker, I.D., McKenzie, I.F.C. & Springer, T.A. The Ly-15 alloantigenic system: A genetically determined polymorphism of the murine lymphocyte function-associated antigen-1 molecule. Proc Natl Acad Sci USA 82, 2, 526-530 (1985).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.

Hogarth_1985_4221.pdf
Springer, T.A., Teplow, D.B. & Dreyer, W.J. Sequence homology of the LFA-1 and Mac-1 leukocyte adhesion glycoproteins and unexpected relation to leukocyte interferon. Nature 314, 6011, 540-542 (1985).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.

Springer_1985_3313.pdf
Anderson, D.C., et al. The severe and moderate phenotypes of heritable Mac-1, LFA-1 deficiency: Their quantitative definition and relation to leukocyte dysfunction and clinical features. J. Infect. Dis. 152, 4, 668-689 (1985).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.

Anderson_1985_4205.pdf
Strassmann, G., Springer, T.A. & Adams, D.O. 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. J. Immunol. 135, 1, 147-151 (1985).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.

Strassman_1985_4350.pdf
Shaw, S., Goldstein, G., Springer, T.A. & Biddison, W.E. 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. J. Immunol. 134, 5, 3019-3026 (1985).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.

Shaw_1985_4328.pdf
1984
Anderson, D.C., et al. Abnormalities of polymorphonuclear leukocyte function associated with a heritable deficiency of high molecular weight surface glycoproteins (GP138): Common relationship to diminished cell adherence. J. Clin. Invest. 74, 2, 536-551 (1984).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'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'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.

Anderson_1984_4055.pdf
Springer, T.A. & Unkeless, J.C. Analysis of macrophage differentiation and function with monoclonal antibodies. Contemporary Topics in Immunobiology 14, 1-31 (1984). Springer_1984_3544.pdf
Krensky, A.M., et al. Cell surface structures involved in the human cytolytic T lymphocyte response. Regulation of the Immune System 209-219 (1984).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.

Krensky_1984_2972.pdf
Kohl, S., Springer, T.A., Schmalstieg, F.C., Loo, L.S. & Anderson, D.C. Defective natural killer cytotoxicity and polymorphonuclear leukocyte antibody-dependent cellular cytotoxicity in patients with LFA-1/OKM-1 deficiency. J. Immunol. 133, 6, 2972-2978 (1984).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.

Kohl_1984_3377.pdf
Dana, N., Todd, R.F., I.I.I., Pitt, J., Springer, T.A. & Arnaout, M.A. Deficiency of a surface membrane glycoprotein (Mo1) in man. J. Clin. Invest. 73, 1, 153-159 (1984).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'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'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'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.

Dana_1984_3874.pdf
Hemler, M.E., et al. 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. J. Immunol. 132, 6, 3011-3018 (1984).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.

Hemler_1984_4003.pdf
Krensky, A.M., Sanchez-Madrid, F., Springer, T.A. & Burakoff, S.J. Human lymphocyte function associated antigens. Surv. Immunol. Res. 3, 1, 39-44 (1984).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.

Krensky_1984_4081.pdf
Springer, T.A., Thompson, W.S., Miller, L.J., Schmalstieg, F.C. & Anderson, D.C. Inherited deficiency of the Mac-1, LFA-1, p150,95 glycoprotein family and its molecular basis. J. Exp. Med. 160, 6, 1901-1918 (1984).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'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'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' leukocytes and the blockade of similar functions in healthy cells by monoclonal antibodies.

Springer_1984_4163.pdf
Krensky, A.M., Robbins, E., Springer, T.A. & Burakoff, S.J. LFA-1, LFA-2 and LFA-3 antigens are involved in CTL-target conjugation. J. Immunol. 132, 5, 2180-2182 (1984).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.

Krensky_1984_4143.pdf
Springer, T.A. Macrophage and T lymphocyte-mediated immunity: Similarities at the level of the Mac-1 and LFA-1 molecules. Mononuclear Phagocyte Biology 109-128 (1984). Springer_1984_3384.pdf
Ho, M. & Springer, T.A. Preparation and use of monoclonal antimacrophage antibodies. Methods Enzymol. 108, 313-324 (1984). Ho_1984_3546.pdf
Flotte, T.J., et al. The relation of Langerhans cells to other dendritic cells and macrophages. Mononuclear Phagocyte Biology 51-66 (1984). Flotte_1984_3223.pdf
1983
Baldwin, W.M., I.I.I., et al. All monocyte antigens are not expressed on renal endothelium. Tissue Antigens 21, 3, 254-259 (1983).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.

Baldwin_1983_3464.pdf
Ho, M.-K. & Springer, T.A. Biosynthesis and assembly of the α and β subunits of Mac-1, a macrophage glycoprotein associated with complement receptor function. J. Biol. Chem. 258, 5, 2766-2769 (1983).Abstract

Mac-1 is a macrophage surface antigen containing noncovalently associated alpha and beta subunits of Mr = 170,000 and 95,000, respectively (Kürzinger, 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.

Ho_1983_3312.pdf

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