Mouse complement components C4 and Slp act synergistically in a homologoushemolytic C4 assay

Goals of the present study were to compare the hemolytic activities of mous e C4 and Slp in a homologous system and to study a possible interaction bet ween these proteins during complement activation. As reagents for mouse C4 and Sip, we used serum of C4(-/-) knockout C57BL/6 (C4(-)/Slp(-)) mice and sensitized rabbit erythrocytes as target cells. Sera to be tested contained none, either of the two or both proteins. We found that C4(-)/Slp(+) serum has some hemolytic C4 activity, but less than C4(+)/Slp(-) serum. Comparin g C4 activities of C4(+)/Slp(-) and C4(+)/Slp(+) sera, we found a threefold enhanced activity in double-positive serum. Hemolytic C4 levels of mixture s of solely C4- and Slp-sufficient sera did not overlap with expected C4 le vels, but rather these sera showed synergy. This explains the enhanced acti vity of double-positive serum. Similar results were observed for total comp lement activation. In conclusion, Slp has measurable, but poor C4 activity as compared with mouse C4. Using our homologous system, we showed that the enhanced classical pathway activity of double-positive sera is most probabl y based on synergy between C4 and Slp. Our results answer an old question a s to why C4(+)/Slp(+) mice have higher complement levels than C4(+)/Slp(-) mice.