Brooimans R A, Hiemstra P S, van der Ark A A, Sim R B, van Es L A, Daha M R
Department of Nephrology, University Hospital Leiden, The Netherlands.
J Immunol. 1989 Mar 15;142(6):2024-30.
The present studies were initiated to characterize a 150-kDa molecule with inhibitory activity for C3bBb formation, which is present in human umbilical vein endothelial cells (HUVEC). Therefore, human endothelial culture supernatants (HECS) were analyzed for the presence of human complement factor H by ELISA. It was found that H was present in HECS. An immunoblot analysis of affinity purified H from HECS showed that the size of HUVEC H was identical to that of plasma H. The mean production of H by HUVEC of first passage cultures was 40 ng/10(6) cells/day. The synthesis of HUVEC H was fully inhibitable by the addition of cycloheximide to the cultures, suggesting that H is de novo synthesized. Additional evidence for de novo synthesis was obtained by using biosynthetic labeling with [35S] methionine, immunoprecipitation, and SDS-PAGE. It was demonstrated that, indeed, HUVEC produce and secrete factor H. Two forms of the protein were identified, the 150-kDa form and also a 45-kDa form, both forms have been identified in plasma. The functional activity of HUVEC H is identical to that of plasma H. IFN-gamma induced enhanced synthesis of H by HUVEC, whereas it had no effect on C3 synthesis. Supernatant from stimulated PBMC, T cell growth factor, enhanced synthesis of both H and C3. The present studies indicate that H is produced by HU-VEC and that H may function as an inhibitor of complement activation at the endothelial cell level and, thereby, together with molecules like decay accelerating factor and membrane cofactor protein, may influence resistance of endothelial cells to complement mediated damage.
本研究旨在鉴定一种存在于人类脐静脉内皮细胞（HUVEC）中、对C3bBb形成具有抑制活性的150 kDa分子。因此，采用酶联免疫吸附测定法（ELISA）分析了人内皮细胞培养上清液（HECS）中是否存在人补体因子H。结果发现HECS中存在H。对从HECS中亲和纯化得到的H进行免疫印迹分析表明，HUVEC来源的H的大小与血浆来源的H相同。初代培养的HUVEC产生H的平均量为40 ng/10⁶细胞/天。向培养物中添加环己酰亚胺可完全抑制HUVEC产生H，这表明H是重新合成的。通过使用[³⁵S]甲硫氨酸进行生物合成标记、免疫沉淀和SDS-PAGE获得了重新合成的更多证据。结果表明，HUVEC确实能产生并分泌因子H。鉴定出了该蛋白的两种形式，即150 kDa形式和45 kDa形式，这两种形式在血浆中均已被鉴定出。HUVEC来源的H的功能活性与血浆来源的H相同。干扰素-γ可诱导HUVEC增强H的合成，而对C3合成无影响。刺激后的外周血单个核细胞（PBMC）的上清液、T细胞生长因子可增强H和C3的合成。本研究表明，H由HUVEC产生，并且H可能在内皮细胞水平上作为补体激活的抑制剂发挥作用，因此，与衰变加速因子和膜辅因子蛋白等分子一起，可能会影响内皮细胞对补体介导损伤的抵抗力。
