Release of soluble immune complexes from immune adherence receptors on human erythrocytes is mediated by C3b inactivator independently of β1H and is accompanied by generation of C3c

Abstract

Antigen.cntdot.antibody complexes (Ag.cntdot.Ab) prepared from 125I-labeled bovine serum albumin and guinea pig anti-albumin were incubated at 37.degree. C for 30 min with normal human serum diluted optimally for binding (1:16) and then with autologous erythrocytes (RBC). After washing, RBC-bearing antigen.cntdot.antibody.cntdot.complement complexes (Ag.cntdot.Ab.cntdot.C) were resuspended in serum reagents or solutions of purified C components, and the kinetics of dissociation were analyzed. Ag.cntdot.Ab-C dissociated in serum heated at 56.degree. C for 30 min (S.DELTA.30) but not in serum heated for 120 min (S.DELTA.120). Dissociation in S.DELTA.30 markedly decreased after adsorption with anti-C3b inactivator but not anti-.beta.1H or anti-C4 binding protein (C4bp), and dissociation in S.DELTA.120 markedly increased after addition of C3b inactivator. Hemolytic assays revealed that S.DELTA.30 retained inactivator activity; S.DELTA.120 lacked significant activity. Ag.cntdot.Ab.cntdot.C dissociated in the presence of purified inactivator or C3b but not .beta.1H or C3. Dissociation was more rapid with inactivator than with C3b and occurred at 0.degree. C and at 37.degree. C. Treatment with inactivator inhibitor abolished dissociation in S.DELTA.30; dissociation in inactivator deficient serum was markedly reduced. Addition of .beta.1H did not enhance inactivator-mediated dissociation at limiting dilutions of inactivator, and adsorption of Ag.cntdot.Ab.cntdot.C with anti-.beta.1H or preparation of Ag.cntdot.Ab.cntdot.C with serum adsorbed with anti-.beta.1H did not diminish dissociation. After dissociation with inactivator, Ag.cntdot.Ab.cntdot.C were unchanged in size but were no longer able to bind to fresh RBC and gave enhanced binding to Raji and Daudi human lymphoblastoid cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Ag.cntdot.Ab.cntdot.C, prepared with 125I labeled C3 revealed that, after binding to RBC, dissociation with inactivator was accompanied by generation of a C3 fragment the size of C3c. Preincubation of Ag.cntdot.Ab.cntdot.C with excess inactivator did not prevent subsequent binding of Ag.cntdot.Ab.cntdot.C to RBC but, immediately after binding, Ag.cntdot.Ab.cntdot.C dissociated rapidly. Thus, C3b inactivator can release immune complexes from immmune adherence receptors on human RBC and release occurs independently of .beta.1H, alters cell binding properties of immune complexes and involves multiple cleavages of the C3b .alpha.'' chain; receptors in human RBC memebrane are required for this C3b inactivator-mediated breakdown.