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    Rothlein, R. & Springer, T.A. The requirement for lymphocyte function-associated antigen 1 in homotypic leukocyte adhesion stimulated by phorbol ester. J. Exp. Med. 163, 5, 1132-1149 (1986).Abstract
    Rothlein_1986_4513.pdf

    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')2 and Fab' 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.

    Lisowska-Grospierre, B., et al. 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. Eur. J. Immunol. 16, 2, 205-208 (1986).Abstract
    Lisowska_Grospierre_1986_44110pdf.pdf

    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' 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.

    Sastre, L., et al. A partial genomic DNA clone for the α subunit of the mouse complement receptor type 3 and cellular adhesion molecule Mac-1. Proc Natl Acad Sci USA 83, 15, 5644-5648 (1986).Abstract
    Sastre_1986_4703.pdf

    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.

    Marlin, S.D., Morton, C.C., Anderson, D.C. & Springer, T.A. 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. J. Exp. Med. 164, 3, 855-867 (1986).Abstract
    Marlin_1986_4748.pdf

    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.

    Cardosa, M.J., Gordon, S., Hirsch, S., Springer, T.A. & Porterfield, J.S. Interaction of West Nile virus with primary murine macrophages: Role of cell activation and receptors for antibody and complement. J. Virol. 57, 3, 952-959 (1986).Abstract
    Cardosa_1986_4349.pdf

    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.

    Unkeless, J.C. & Springer, T.A. Macrophages. Handbook of experimental immunology Vol.IV: Applications of immunological methods in biomedical sciences, 118 (1986).
    Unkeless_1986_3821.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
    Hogarth_1985_4221.pdf

    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.

    Anderson, D.C., et al. Leukocyte LFA-1, OKM1, p150,95 deficiency syndrome: functional and biosynthetic studies in three kindreds. Fed. Proc. 44, 10, 2671-2677 (1985).Abstract
    Anderson_1985_4150.pdf

    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.

    Krensky, A.M., et al. Heritable lymphocyte function-associated antigen-1 deficiency: Abnormalities of cytotoxicity and proliferation associated with abnormal expression of LFA-1. J. Immunol. 135, 5, 3102-3108 (1985).Abstract
    Krensky_1985_4424.pdf

    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.

    Strassmann, G., et al. Antigens associated with the activation of murine mononuclear phagocytes in vivo: Differential expression of lymphocyte function-associated antigen in the several stages of development. Cell. Immunol. 94, 1, 265-275 (1985).Abstract
    Strassmann_1985_3873.pdf

    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.

    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
    Strassman_1985_4350.pdf

    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.

    Anderson, D.C. & Springer, T.A. The importance of the Mac-1, LFA-1 glycoprotein family in adherence-dependent inflammatory functions: Insights from an experiment of nature. Infection and Malignancy (RES Symposium) (1985).Abstract
    Anderson_1995_4423.pdf

    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.

    Fischer, A., et al. Deficiency of the adhesive protein complex lymphocyte function antigen 1, complement receptor type 3, glycoprotein p150,95 in a girl with recurrent bacterial infections. J. Clin. Invest. 76, 6, 2385-2392 (1985).Abstract
    Fischer_1985_4153.pdf

    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.

    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
    Springer_1985_3313.pdf

    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.

    Ross, G.D., et al. 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. Blood 66, 4, 882-890 (1985).Abstract
    Ross_1985_4151.pdf

    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' 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' 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's neutrophils may be one of the reasons for their increased susceptibility to bacterial infections.

    Springer, T.A. The LFA-1, Mac-1 glycoprotein family and its deficiency in an inherited disease. Fed. Proc. 44, 10, 2660-2663 (1985).Abstract
    Springer_1985_4147.pdf

    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.

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