Restriction of Complement-Mediated Membrane Damage by the Eighth Component of Complement: A Dual Role for C8 in the Complement Attack Sequence

Abstract

Abstract Inhibitors of the fluid phase complex were examined in serum from which the lipoproteins had been removed. A limited number of these nonlipoprotein inhibitors were observed, and one was shown to be C8 by several criteria. The inhibitory activity exhibited numerous physiochemical similarities to C8 including identical chromatographic profiles on DEAE-cellulose, CM-cellulose, and QAE-Sephadex ion exchange columns as well as an indistinguishable m.w. as determined by molecular-sieve chromatography and sucrose gradient ultracentrifugation. In addition, serum from a patient genetically deficient in C8 was shown to lack the inhibitory activity that co-purified with C8 in normal serum. Subsequent purification of C8 by established methods verified that these activities were indeed due to the same molecule and furthermore that the inhibitory activity was not due to inactive C8, thus establishing a new function for this C component. A comparison of the inhibitory activity of C8 with its hemolytic effeciency revealed that very small amounts were required for both activities, although higher amounts of C8 were required for inhibition of formation than for lysis of preformed EAC1-7 in the presence of excess C9. Experiments employing purified and radiolabeled C7 indicated that C8 limited the formation of intermediates by preventing the attachment of the fluid phase complexes to target membranes. In addition, the inhibitory activity of C8 could be counteracted by the simultaneous addition of C56̅ and C7 but not by the addition of either component alone, verifying that C8 restricts formation by interacting with the nascent complex in the fluid phase. The ability of C8 to limit the attachment of fluid phase , while being required for the lysis of cells already bearing , suggests a dual role for C8 in complement-mediated cytolysis and thus represents a point of internal regulation in the complement attack sequence.