Human monocyte spreading induced by factor Bb of the alternative pathway of complement activation. A possible role for C5 in monocyte spreading.

Abstract

The central serine esterase of the alternative pathway of complement (APC) activation, activated factor B (Bb), induces murine macrophages and human monocytes to become spread on a glass substrata. To induce the spreading reaction, the catalytic site of the Bb enzyme must be structurally intact since treatment of Bb with heat (56.degree. C for 30 min) or DFP (10-3 M) destroyed both enzymatic and spreading activities. In the C3b (fragment b of complement component 5), Bb complex, Bb exhibits restricted substrate specificity for C3 and C5. The role of C3 and C5 in the monocyte spreading reaction was studied. Expression of C3 and C5 on the surface of human peripheral blood monocytes was investigated by the direct fluorescent antibody technique using fluorescein isothiocyanate-conjugated anti-C3 or C5 F(ab'')-2 antibody fragments. C3 and C5 were present on 6 .+-. 7% of freshly prepared monocytes and expression of C5, but not C3, increased to 70 .+-. 6% when monocytes were incubated for 3 days in serum-free medium. Biosynthesis of C5 was indicated when it was found that under serum-free conditions, monocytes incorporated [3H]leucine into immunoprecipitable C5 with an apparent MW of 180,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The role of C3 and C5 in the monocyte spreading reaction induced by factor Bb was explored by testing for the ability of anti-C3 and anti-C5 Fab'' antibody fragments to block monocyte spreading. Anti-C5 Fab'' inhibited by up to 100% the 3 h human monocyte spreading reaction induced by Bb; in contrast, anti-C3 Fab'' or anti-C4 Fab'' inhibited by < 10%. It was established that the inhibitory effect of anti-C5 Fab'' was exerted directly on the monocyte when it was found that the 3 h monocyte spreading reaction was significantly inhibited by pretreating monocytes with anti-C5 Fab'' for 20 min and then washing before the addition of Bb. The specificity of the inhibitory effect of anti-C5 Fab'' was established by quantitatively absorbing the antibody fragments with polyacrylamide gel-purified C5 antigen. Factor Bb exerted its effect on monocytes by interacting directly with cell surface C5 since purified C5 inhibited the monocyte spreading reaction induced by Bb.