A monoclonal antibody to the rat Crry/p65 antigen, a complement regulatory membrane protein, stimulates adhesion and proliferation of thymocytes.
A murine monoclonal antibody (mAb), 3F10, was produced by fusion of spleen cells obtained from mice immunized with a rat cortical thymic epithelial cell line (R-TNC.1) stimulated with interferon-gamma and P3X myeloma cells. 3F10 recognized an antigen expressed both on thymocytes and non-lymphoid cells in the thymus. Flow cytometry showed that 3F10 stained more than 98% thymocytes and 90% R-TNC.1 cells. Immunoprecipitation and Western blot studies demonstrated that 3F10 reacted with molecules of 55000 and 65000 MW from both thymocyte and R-TNC.1 cell lysates. 3F10 recognized the same antigen on Chinese hamster ovary cells transfected with rat Crry as did 5I2 mAb, confirming the specificity of 3F10 mAb for the rat homologue of mouse Crry/p65, a membrane-bound complement regulatory protein. 3F10 mAb induced homotypic aggregation of thymocytes and exhibited an additive effect on the aggregation evoked by phorbol myristate acetate. The aggregation was dependent on active cell metabolism, intact cytoskeleton, divalent cations and activation of protein phosphatases 1 and 2A (as assessed by use of okadaic acid). In contrast, H-7, HA1004 and genistein partially inhibited, whereas staurosporine potentiated the aggregation of thymocytes triggered by 3F10. 3F10 mAb also stimulated binding of thymocytes to the R-TNC.1 line. Both homotypic and heterotypic adhesive interactions are mediated by leucocyte function-associated antigen-1 (LFA-1). In addition, 3F10 stimulated proliferation of thymocytes induced by suboptimal concentrations of concanavalin A. These data suggest that rat Crry/p65 might be involved in the regulation of both cell adhesion and activation of thymocytes. This is a novel, non-complement-dependent function of Crry/p65.
University School of Pharmacy, Belgrade, Yugoslavia.
N
N
Arsenović-Ranin
D
D
Vucević
N
N
Okada
M
M
Dimitrijević
M
M
Colić
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface; D002448:Cell Adhesion / Q000276:immunology; D002455:Cell Division / Q000276:immunology; D002460:Cell Line; D003208:Concanavalin A / Q000276:immunology; D006224:Cricetinae; D003412:Cricetulus; D007124:Immunoenzyme Techniques; D018799:Intercellular Adhesion Molecule-1 / Q000276:immunology; D016169:Lymphocyte Function-Associated Antigen-1 / Q000276:immunology; D008297:Male; D051379:Mice; D051381:Rats; D011956:Receptors, Cell Surface; D011951:Receptors, Complement / Q000276:immunology*; D017463:Receptors, Complement 3b; D013601:T-Lymphocytes / Q000276:immunology*; D013950:Thymus Gland / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; C081470:Cr1l protein, mouse; C513105:Cr1l protein, rat; D016169:Lymphocyte Function-Associated Antigen-1; D011956:Receptors, Cell Surface; D011951:Receptors, Complement; D017463:Receptors, Complement 3b; D003208:Concanavalin A; D018799:Intercellular Adhesion Molecule-1