COMPLEMENT REGULATION IN THE RAT GLOMERULUS - CRRY AND CD59 REGULATE COMPLEMENT IN GLOMERULAR MESANGIAL AND ENDOTHELIAL-CELLS

The complement regulators, decay accelerating factor, membrane cofacto r protein, and CD59 are present in human glomeruli. Crry is the rodent analogue to the former two proteins. In this study, we examined compl ement regulation in cultured rat glomerular endothelial cells (GEnC) a nd mesangial cells (MES). Immunoprecipitation of I-125-labeled membran e proteins and Western blotting studies were performed with anti-Crry and anti-CD59. In both GEnC and MES, Crry was present as 53, 65, and 7 8 kD proteins. The 20 kD CD59 was apparent in GEnC. CD59 was, also pre sent in MES, but in relatively smaller quantities. By Northern analyse s, 1.8 kb CD59 mRNA was present in GEnC as well as in RNA from isolate d rat glomeruli. mRNA for Crry was present in both GEnC and MES as 2.2 kb species. The functional significance of these proteins was evaluat ed next. Anti-Thy 1.1 IgG was used to activate the complement classica l pathway in MES. To inhibit the function of the complement regulators , anti-CD59 and/or anti-Crry F(ab')(2) antibodies were added with anti -Thy 1.1. Inhibition of Crry function led to enhanced cytotoxicity, wh ile there was no effect when CD59 function was inhibited. The compleme nt alternative pathway was studied by adding complement in Mg-EGTA buf fer. Inhibition of Crry led to productive alternative pathway activati on, which was accentuated by anti-CD59 when Crry was incompletely inhi bited. Alternative pathway regulation was also evaluated in GEnC. Inhi bition of CD59 function alone had no effect in GEnC, while inhibition of Crry led to significant cytotoxicity from alternative pathway activ ation. Under conditions in which Crry was inactive, inhibition of CD59 further enhanced cytotoxicity. Therefore, Crry is present in both GEn C and MES and restricts the complement alternative pathway in both cel l types. Crry also regulates the classical pathway in MES. CD59 is pre sent and functionally active in GEnC, while it appears to have a minor role in MES.