Studies on the mechanism of C3b inhibition of immune complex induced C1 activation.
We had previously demonstrated that in normal human serum (NHS) nascent C3b inhibited C1 activation by immune complexes (IC). We have now investigated the mechanism of this feedback inhibition. For these studies, EA-IgG were added to solutions containing physiological concns of purified C1, C1-In, C2, C3 and C4. Mixtures were then incubated at 37 degrees C for 30 min. Western blot and autoradiographic analyses revealed that almost half of the IgG molecules had become covalently linked to C3b in a 1:1 complex with the C3 alpha' chain of C3b being bound to the heavy chain of IgG. IgG-C3b and free IgG were separated by ion exchange chromatography and immune complexes were formed with each. The consumption of complement in NHS by EA-IgG and EA-(IgG-C3b) were then compared. The results indicate that binding of C3b to IgG did not significantly inhibit the C1 activating potential of the IgG. Thus feedback inhibition is not due to the binding of C3b to IgG. An alternative mechanism was next explored. After incubation of EA-IgG with C1 through C3, EA were separated from supernatant fluid by centrifugation. It was determined that one-third to one-half of the IgG had been released from the erythrocytes. Release appears not to have been due to C3b binding to IgG, since the released IgG-C3b readily bind to fresh sheep erythrocyte (E), and since IgG that was free of C3b was also released from EA by complement, it is more likely that C3b binding to the E caused the dissociation of antibody. These results indicate that under physiological conditions, the C1 activating potential of an immune complex is greatly reduced as the result of the binding of nascent C3b to the antigen moiety of the IC, thereby causing the displacement of complement activating antibody. In addition to IgG, IgG-C3b is also released from the IC.
Ziccardi
R J
RJ
Department of Immunology, IMM-11, Research Institute of Scripps Clinic, La Jolla, CA 92037.
D000818:Animals; D000936:Antigen-Antibody Complex / Q000276:immunology*; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003167:Complement Activation; D003174:Complement C1 Inactivator Proteins / Q000276:immunology*; D003179:Complement C3b / Q000276:immunology* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000276:immunology; D007074:Immunoglobulin G / Q000378:metabolism
D000936:Antigen-Antibody Complex; D003174:Complement C1 Inactivator Proteins; D007074:Immunoglobulin G; D003179:Complement C3b