SYNTHESIS OF FACTOR-D, FACTOR-B AND FACTOR-P OF ALTERNATIVE PATHWAY OF COMPLEMENT ACTIVATION, AS WELL AS OF C3, BY GUINEA-PIG PERITONEAL MACROPHAGES INVITRO

Abstract

Culture supernatants from monolayers prepared with guinea-pig peritoneal macrophages contained functional factor D and C3 [complement component 3] activity. Factor D was detected by consumption of C3 in the presence of culture supernatant, factor B and insoluble C3b. Preincubation of culture supernatant with anti-D Ig[immunoglobulin]G totally inhibited C3 consumption in the D assay which identified factor D as the B activating enzyme. The synthesis of D and C3 by macrophages was proven by the fact that cycloheximide in the culture medium strongly reduced the amount of detectable D and C3 and by incorporation experiments with 14C-labeled amino acids which resulted in the production by macrophages of radiolabeled D and C3. Radiolabeled B and P were also detected. The majority of the B protein appeared in its cleaved (Bb) form and, therefore, factor B escaped detection in the functional assay. For unknown reasons functional activity of P was not detectable. The enzyme responsible for B cleavage in the culture supernatants was identified as factor D. Activation of B by the D enzyme in culture supernatants probably occurred through the C3b-dependent feedback cycle of the alternative pathway. This is concluded from the observation that C3 was consumed on incubation of the culture supernatant at 37.degree. C, although at a low rate.