The central serine esterase of the alternative pathway of complement (APC) activation, activated factor B (Bb), has been shown recently to induce murine macrophages and human monocytes to become spread on a glass substrata. It has also been established that to induce the spreading reaction, the catalytic site of the Bb enzyme must be structurally intact since treatment of Bb with heat (56 degrees C for 30 min) or diisopropylfluorophosphate (10(-3) M) destroyed both enzymatic and spreading activities. In the C3b,Bb complex, Bb exhibits restricted substrate specificity for C3 and C5. With this in mind, the role of C3 and C5 in the monocyte spreading reaction was explored in the present study. Expression of C3 and C5 on the surface of human peripheral blood monocytes was investigated by the direct fluorescent antibody technique employing fluorescein isothiocyanate-conjugated anti-C3 or C5 F(ab')2 antibody fragments. It was found that C3 and C5 were present on 6 +/- 7% of freshly prepared monocytes and that expression of C5, but not C3, increased to 70 +/- 6% when monocytes were incubated for 3 d 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 mol wt 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. It was found that 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 less than 10%. That the inhibitory effect of anti-C5 Fab' was exerted directly on the monocyte was established 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: greater than 4 microgram of C5 absorbed by 100% the inhibitory activity of 10-20 microgram of anti-C5 Fab'. That factor Bb exerted its effect on monocytes by interacting directly with cell surface C5 was indicated when it was found that purified C5 inhibited the monocyte spreading reaction induced by Bb; greater than 25 microgram of C5 inhibited by 100% the spreading reaction induced by 3 microgram factor Bb.

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