Dependence of Complement Rosettes on a Nonenzymatic and an Enzymatic Step

Abstract Rosette formation between typical EAC14oxy23 (human complement components; 1.2 hemolytically active C2 sites and 1 × 104 C3 molecules) and Raji cells can be reduced by 20 to 30%, of 0.5 mM diisopropylfluorophosphate (DFP) are present during the rosette assay. Reduction of rosette formation is only slightly less pronounced, if EAC14oxy23 are pretreated for 10 min at 23°C with 0.5 mM DFP. To elucidate the possible contribution of the C42Ѕ enzyme to the formation of rosettes, EAC1423 were prepared with nonoxidized C2. These EAC1423 were kept at 37°C for 30 min to decay the C2 and to obtain EAC143. Such cells formed about 10 to 15% rosettes. By treating the EAC143 with increasing amounts of oxidized C2 to form EAC14oxy23, rosette formation was increased up to 40%. This C2 dependent increase in rosette formation could be suppressed by DFP or by performing the rosette assay at 4°C. For comparison SRBC were treated with tannic acid and coated with purified C3 (Etan-C3) to exlcude any enzymatic activity. These Etan-C3 formed about 20% rosettes with Raji cells (Etan-BSA were negative), which could not be inhibited by DFP and which reacted equally well at 4°C as at 37°C. Treatment of Raji cells with glutaraldehyde (0.01 to 0.1%) left their interaction with Etan-C3 unaltered but blocked their rosette formation with EAC14oxy23. The same reaction pattern resulted from pretreat-ment of the lymphoid cells with IgG anti-C3. These results suggest the participation of an enzymatic action in complement dependent rosette formation. In conjunction with the report about C4 in Raji cell membranes (Ferrone, S. Pellegrino, M. A. and N. R. Cooper, Science 193, 53, 1976) and our own data, indicating the presence of C3 in the lymphoid cell plasma membrane, it is tempting to assume the "bridge formation mechanism" as a possible explanation for this interaction: proteolytic cleavage of a Raji cell membrane component (C4 or C3?) by the C42 enzyme → liberation of new binding sites (from C4 or C3?) → interlinkage of the Raji cell and the EAC14oxy23.