Protective effects of different combinations of human MCP, DAF, and CD59 on complement-dependent cytolysis in NIH 3T3 cells.
OBJECTIVE
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.


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-hMCPhCD59 survived better than cells expressing pIREShDAF- 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.
College of Animal Science and Technology, Guangxi University, Guangxi Academy of Sciences, Nanning 530003, China.
Xiurong
X
Yang
Jixian
J
Deng
Zhiliang
Z
Jiang
D Joshua
DJ
Liao
Hesheng
H
Jiang
D000818:Animals; D018958:CD55 Antigens / Q000235:genetics / Q000502:physiology*; D018957:CD59 Antigens / Q000235:genetics / Q000502:physiology*; D002470:Cell Survival; D015938:Complement Membrane Attack Complex / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D006801:Humans; D051916:Membrane Cofactor Protein / Q000235:genetics / Q000502:physiology*; D051379:Mice; D023421:Models, Animal; D041681:NIH 3T3 Cells; D010957:Plasmids; D014162:Transfection
C497857:CD46 protein, human; D018958:CD55 Antigens; D018957:CD59 Antigens; D015938:Complement Membrane Attack Complex; D051916:Membrane Cofactor Protein