Protective Effects of Different Combinations of Human MCP, DAF, and CD59 on Complement-Dependent Cytolysis in NIH 3T3 Cells

Abstract

Objectives: To analyze the protective effects against complement-mediated cytolysis of the MCP, DAF, and CD59 human complement regulatory proteins, alone and in combination, on NIH 3T3 mouse fibroblast cells.

Materials and Methods: We constructed 3 double- and 3 single-human complement regulatory protein plasmids (pIRES-hMCP-hDAF, pIRES-hMCP-hCD59, pIRES-hDAF-hCD59, pIRES-A-hMCP, pIRES-B-hDAF, and pIRES-B-hCD59). The plasmids were transfected into NIH 3T3 cells, and stable transfectants were obtained by treatment with 200 kg/m3 G418 for 2 weeks. Normal human serum (50%) as a source of complement was added to the culture medium of stable transfectants. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to analyze the protective ability of different human complement regulatory protein plasmids on complement-dependent cytolysis.

Results: The viability of double-human complement regulatory protein stable transfectants was significantly higher than that of single-human complement regulatory protein stable transfectants (P < .05). Among the double-transfectants, cells expressing pIRES-hMCP-hDAF and pIRES-hMCP-hCD59 survived better than cells expressing pIRES-hDAF-hCD59 (91.75% ± 3.30% and 84.88% ± 2.36% vs 66.19% ± 6.52%; P < .05). Among the single-transfectants, cells expressing pIRES-A-hMCP or pIRES-B-hDAF survived better than cells expressing pIRES-B-hCD59 or pIRES empty vector (53.76% ± 3.84% and 56.32% ± 2.83% vs 43.28% ± 0.96% and 40.27% ± 1.11%; P < .05).

Conclusions: These results suggest that the MCP+DAF and MCP+CD59 combinations could be more effective than DAF+CD59 in protecting the NIH 3T3 cells from injury caused by complement-dependent cytolysis, whereas MCP or DAF alone is stronger than CD59 alone in inhibiting membrane attack complex formation.