Quigg R J, Morgan B P, Holers V M, Adler S, Sneed A E, Lo C F
Department of Medicine, University of Chicago, Illinois, USA.
Kidney Int. 1995 Aug;48(2):412-21. doi: 10.1038/ki.1995.309.
The complement regulators, decay accelerating factor, membrane cofactor protein, and CD59 are present in human glomeruli. Crry is the rodent analogue to the former two proteins. In this study, we examined complement regulation in cultured rat glomerular endothelial cells (GEnC) and mesangial cells (MES). Immunoprecipitation of 125I-labeled membrane 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 78 kD proteins. The 20 kD CD59 was apparent in GEnC. CD59 was also present in MES, but in relatively smaller quantities. By Northern analyses, 1.8 kb CD59 mRNA was present in GEnC as well as in RNA from isolated rat glomeruli. mRNA for Crry was present in both GEnC and MES as 2.2 kb species. The functional significance of these proteins was evaluated next. Anti-Thy 1.1 IgG was used to activate the complement classical 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, while there was no effect when CD59 function was inhibited. The complement alternative pathway was studied by adding complement in Mg-EGTA buffer. Inhibition of Crry led to productive alternative pathway activation, which was accentuated by anti-CD59 when Crry was incompletely inhibited. Alternative pathway regulation was also evaluated in GEnC. Inhibition of CD59 function alone had no effect in GEnC, while inhibition of Crry led to significant cytotoxicity from alternative pathway activation. Under conditions in which Crry was inactive, inhibition of CD59 further enhanced cytotoxicity. Therefore, Crry is present in both GEnC and MES and restricts the complement alternative pathway in both cell types. Crry also regulates the classical pathway in MES. CD59 is present and functionally active in GEnC, while it appears to have a minor role in MES.
补体调节蛋白、衰变加速因子、膜辅因子蛋白和CD59存在于人类肾小球中。Crry是前两种蛋白在啮齿动物中的类似物。在本研究中，我们检测了培养的大鼠肾小球内皮细胞（GEnC）和系膜细胞（MES）中的补体调节。用抗Crry和抗CD59对125I标记的膜蛋白进行免疫沉淀及蛋白质印迹研究。在GEnC和MES中，Crry均以53、65和78kD的蛋白形式存在。20kD的CD59在GEnC中明显可见。CD59也存在于MES中，但含量相对较少。通过Northern分析，1.8kb的CD59 mRNA存在于GEnC以及分离的大鼠肾小球RNA中。Crry的mRNA以2.2kb的形式存在于GEnC和MES中。接下来评估了这些蛋白的功能意义。抗Thy 1.1 IgG用于激活MES中的补体经典途径。为抑制补体调节蛋白的功能，将抗CD59和/或抗Crry F(ab')2抗体与抗Thy 1.1一起加入。抑制Crry功能导致细胞毒性增强，而抑制CD59功能则无影响。通过在Mg-EGTA缓冲液中添加补体来研究补体替代途径。抑制Crry导致替代途径有效激活，当Crry未被完全抑制时，抗CD59会加剧这种激活。也在GEnC中评估了替代途径调节。单独抑制CD59功能对GEnC无影响，而抑制Crry则导致替代途径激活产生显著的细胞毒性。在Crry无活性的条件下，抑制CD59会进一步增强细胞毒性。因此，Crry存在于GEnC和MES中，并在两种细胞类型中限制补体替代途径。Crry也在MES中调节经典途径。CD59存在于GEnC中且功能活跃，而它在MES中似乎作用较小。
