Activation of the Alternative Pathway of Human Complement by Apoptotic Human Umbilical Vein Endothelial Cells1

Abstract

Complement activation generally does not occur on homologous cells. We observed C3 deposition on cultured human umbilical vein endothelial cells (HUVEC) when those which had died of apoptosis were treated with human serum. The C3 deposition on apoptotic HUVEC required Mg2+ but not Ca2+. In addition, the incubation of apoptotic HUVEC with purified C3, B, and D in the presence of Mg2+ resulted in C3 deposition. These results indicated that the C3 deposition on apoptotic HUVEC is mediated by the activation of the alternative complement pathway. C3 contains an intrachain thioester bond in the alpha chain (110 kDa) and upon activation to C3b, binds with membrane molecules by forming an ester or amide bond. Western blotting of reduced C3b-membrane molecule complexes, isolated from serum-treated apoptotic HUVEC by immunoprecipitation with anti-C3, revealed the covalent binding of C3b to several membrane molecules. Most of the C3b-membrane molecule complexes were cleaved by hydroxylamine, suggesting covalent binding via an ester bond. The molecular mass of the major alpha chain fragment released by hydroxylamine treatment was not 105 kDa but 68 kDa, which corresponds to the alpha 1 fragment of iC3b. These results indicate that most of the C3b on HUVEC was cleaved at its alpha' chain to yield iC3b, which consists of three disulfide-linked polypeptide chains and is a ligand of the complement receptor type 3 (CR3) of phagocytes. These results suggest that apoptotic HUVEC can activate the alternative pathway of the homologous complement and that the complement is related to the clearance of apoptotic cells by phagocytes.