Formation of EAC{ie} and EAC{ie} with Macrophage Culture Supernatant Containing the Secreted Complement Components C1 to C3

Abstract

Culture supernatants of thioglycollate-elicited guinea pig peritoneal macrophages containedhemolytic C1, C4, C2 and C3, whereas hemolytic C5, C6, C7, C8 or C9 were not detected. Activity of C1, C2 and C3 increased up to a 48 h culture period, whereas C4 activity already declined in 2 day old cultures. After secretion, the hemolytic activity of C1 was least stable in culture supernatant. Sensitized sheep erythrocytes (EA) when incubated with culture super-natant initiated activation and functional cooperation of secreted C1 to C3 as indicated by formation of EAC{ie} and EA{ie} intermediates. Decay and regeneration with purified C2 was shown for EAC{ie} and deposition of C3 fragments on EAC{ie} was demonstrated with anti-C3. On an average, supernatants of 2 day old macrophage cultures were most suitable for formation of EAC{ie} and EAC{ie}. The rate of EAC{ie} and EAC{ie} formation, and also of C2 and C3 inactivation, during incubation of EA with culture supernatant was slow; addition of purified C1 to culture supernatant, however, greatly enhanced the same reactions of EA with supernatant which indicated that C1 was the rate limiting factor. Local secretion of hemolytic C1, C4, C2 and C3 by macrophages may have an important role in antimicrobial defense mechanisms due to the well-known functional cooperation between macrophages and activated C3.