Characterization of ICAM-2 and evidence for a third counter-receptor for LFA-1.
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.
de Fougerolles
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Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115.
Stacker
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Schwarting
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Springer
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D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D002449:Cell Aggregation / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology; D005434:Flow Cytometry; D006801:Humans; D018799:Intercellular Adhesion Molecule-1; D016169:Lymphocyte Function-Associated Antigen-1 / Q000032:analysis* / Q000494:pharmacology; D051379:Mice; D009000:Monocytes / Q000166:cytology; D009928:Organ Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015815:Cell Adhesion Molecules; C065289:ICAM2 protein, human; D016169:Lymphocyte Function-Associated Antigen-1; D018799:Intercellular Adhesion Molecule-1; D013755:Tetradecanoylphorbol Acetate