In vitro synthesis of factor B of the alternative pathway of complement activation by mouse peritoneal macrophages

Abstract

Factor B of the alternative pathway of complement activation was shown to be synthesized and secreted by unstimulated mouse peritoneal macrophages. The activity of B in the culture supernatants from macrophage monolayers was detected by consumption of C3 in reaction mixtures containing supernatant and guinea pig factors C3, D and insoluble C3b. Using a monospecific antiserum, factor B in concentrated culture supernatants was shown by immunodiffusion and immunoelectrophoresis to be identical to factor B in mouse plasma and to form a characteristic complex with cobra venom factor in the presence of D. A steady rate of factor B secretion was observed for 4 days providing the medium was changed every 24 h. Cycloheximide (0.5 μg/ml), an inhibitor of protein synthesis, caused inhibition (90%) of factor B production. Incubation of culture medium containing ¹⁴C-labeled amino acids with the macrophage monolayer resulted in incorporation of radioactivity into factor B as detected by autoradiography of precipitation lines formed with anti-B antiserum. This indicated that synthesis of factor B had occurred. In the same culture supernatants the presence of newly synthesized C3 was also demonstrated.