Isolation and characterization of macrophage-derived C1q and its similarities to serum C1q.

Recently, we have shown that the collagen-like, Fc-recognizing subcomponent C1q of the first complement component is synthesized by human, guinea pig and mouse peritoneal macrophages. To test whether macrophages may contribute to the serum pool of C1q, C1q was purified from guinea pig serum and from guinea pig peritoneal macrophage supernatants and compared for similarities. Both molecules had a similar sedimentation rate (macrophage C1q: 11.3 S, serum C1q: 11.2 S) and showed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions three identical bands with molecular weights of Mr, 29 000, Mr, 27 000 and Mr 23 000 for the A, B and C chains, respectively. Both C1q molecules migrated by immunoelectrophoresis in the gamma region and, in Ouchterlony analysis, showed complete antigenic identity with rabbit anti-serum C1q. These experiments demonstrate the antigenic and protein chemical similarities between serum C1q and C1q secreted by macrophages supporting the idea that macrophages have to be considered as one potential source of serum C1q. Furthermore, macrophage-derived C1q may be of importance in the local microenvironment at an inflammatory site involving macrophages.

Activation of the first component of complement, C1, by a monoclonal antibody recognizing the C chain of C1q.

The effect of a purified monoclonal anti-C1q anti-body (Ab 242 G3) on the function of C1q, a subcomponent of the first component of complement C1, was studied. No inhibition of purified activated C1 was observed, whereas binding of the Ab to fluid phase C1q, to C1q bound to immune complexes (EAC1q), or to serum C1 in fluid phase resulted in a dose-dependent inhibition of the hemolytic activity of C1. In contrast, when the effect of the Ab on serum C1 bound to immune complexes (EAC1) was measured, no inhibition, but a dose-dependent enhancement, of the hemolytic activity was obtained. The dose-response curve of the Ab-treated cell-bound serum C1 was indistinguishable from that of activated C1. Isolated Fab fragments of this Ab did not cause an increase in C1 activity. After separation of the A, B, and C chains of C1q by SDS-PAGE, Ab 242 G3 reacted in immunoblotting selectively with the C chain. These data indicate that cross-linking of C1q via the C chain of C1q might lead to an internal activation of C1.

Purification and physicochemical properties of C1q from guinea-pig serum.

An efficient method is described for the isolation of highly purified, IgG-free and stable guinea-pig serum C1q. The procedure includes the chromatography of EDTA-treated serum (25 mM EDTA) on CM- and DEAE-cellulose followed by gel filtration on ACA 34-Ultrogel whereby ammonium sulfate precipitation was used for concentration. The final product stored in a glycerol containing buffer was purified 700-fold with a yield of approximately 50%. It was judged to be homogeneous by several criteria including SDS-PAGE, analytical ultracentrifugation, gel filtration and immunoprecipitation. The protein has a sedimentation rate of 11.3 S and consists of three distinct polypeptide chains A, B and C with mol. wts of 30,200, 28,200 and 24,000. Amino acid analysis revealed a content of 4.42% hydroxyproline, 1.81% hydroxylysine and 18.7% glycine. In contrast to human serum C1q a very low content of cysteine residues was detected. SDS-PAGE analysis performed in the absence of 2-mercaptoethanol but in the presence of 5-10% SDS revealed clearly that gps-C1q is dissociated in a time-dependent manner into the individual chains.

Conformational changes in C1q after binding to immune complexes: detection of neoantigens with monoclonal antibodies.

The formation of neoantigens within the C1q molecule after the binding of C1r and C1s to C1q and the binding of C1q to immune complexes is described. The neoantigens were detected by different monoclonal anti-C1q antibodies. This immunochemical study supports the hypothesis drawn from functional studies that the activation of the classical C pathway results from conformational changes within the C1q molecule leading to the activation of C1r and subsequently C1s.