REGULATION OF THE MEMBRANE ATTACK MECHANISM OF COMPLEMENT BY SERUM-DERIVED C5B67-INHIBITOR

The regulation and assembly of the C attack mechanism by C8 and its constituent polypeptide chains was investigated. Of 5 functional C8 deficient (C8D) sera that were available for study, 3 were characterized as C8(beta)-chain deficient and 2 as C8(alpha)-(gamma) subunit deficient by functional C8 reconstitution. Treatment of membrane-associated C5b67 with functional C8D sera demonstrated the sequential interaction of C8(beta)-chain followed by C8(alpha)-(gamma) subunit. C8(beta), present in C8(alpha)-(gamma) deficient serum, was identified as having a dual role in C attack. EAC1-7((beta)) were lysed upon the addition of C8(beta) deficient serum, whereas, in contrast, C8(beta) blocked lysis mediated by whole C8 and C9. Similarly, EC5b67 prepared by reactive lysis were spared from C-EDTA induced lysis by prior treatment with C8(alpha)-(gamma) deficient serum. C8(beta)-chain regulatory and hemolytic activities were inhibited by 10mM EDTA suggesting a requirement for metal cations in C8(beta)-chain function.;Serum lipoproteins and C8 each have been shown to inhibit the binding of C5b67 to erythrocytes. The relative contribution of these proteins to total serum C5b67-INH activity was determined. Inhibitor levels were assessed by titration of C-deficient human sera in a C5b6-initiated lysis assay system employing guinea pig E. C5b67-INH activity in 3 C9 deficient sera (C9D) was 2550 (+OR-) 407 U/ml ((+OR-) 1 SD) compared to only 428 (+OR-) 136 U/ml for 5 functional C8D sera, representing a decrease of approximately 80%. C5b67-INH activity was identical for all 5 C8D sera irrespective of which polypeptide subunit of C8 was missing. C8D or C9D sera incubated with preformed EC5b67 did not prevent lysis indicating that inhibition occurred in the fluid phase. The relative deficiency of C5b67-INH in C8D serum was due to the absence of C8 as demonstrated by antibody neutralization experiments. Incubation of C9D serum with IgG antibody to C8 reduced the amount of C5b67-INH to levels found in C8D serum. Removal of lipoproteins from C8D serum by ultracentrifugal flotation further reduced the residual C5b67-INH to only 10% of the activity seen in C9D serum. Support for these findings come from studies demonstrating the functional reconstitution of C5b67-INH to levels found in C9D serum upon combining purified C8 and lipoproteins at normal serum concentrations. Additionally, purified lipoproteins assayed alone provided the amount of C5b67-INH present in C8D serum. These results suggest that C8 contributes the substantial majority of serum-derived C5b67-INH activity. Together, C8 and lipoproteins provide approximately 90% of the C5b67-INH present in serum.