Relative roles of decay‐accelerating factor, membrane cofactor protein, and CD59 in the protection of human endothelial cells against complement‐mediated lysis

Abstract

Human umbilical vein endothelial cells (HUVEC) were found by Western blot analysis to express three membrane-bound C regulatory proteins, decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. DAF was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 70-kDa molecule under nonreducing conditions in 2% deoxycholate extracts of HUVEC, MCP as a 63-kDa protein and CD59 as a 20-kDa molecule. Northern blot analysis revealed the presence of two species of mRNA expressed in HUVEC, which hybridized to a cDNA probe specific for DAF, with sizes of about 2.0 kb and 2.7 kb. MCP mRNA was detected at 4.2 kb and a CD59 cDNA probe hybridized with three mRNA species with sizes of about 800, 1400 and 2000 bp. DAF and CD59 were released from the surface of HUVEC by phosphatidylinositol-phospholipase C, demonstrating that both are attached to the cell membrane by means of a glycolipid anchor. The relative contribution of DAF, MCP and CD59 in regulating the sensitivity to lysis of HUVEC by autologous complement was determined by incubation of sensitized endothelial cells with F(ab′)₂ fragments of polyclonal antibodies raised against these proteins. The susceptibility of sensitized cells to lysis by homologous complement was markedly increased in the presence of F(ab′)₂ anti-CD59 and to a lesser, but significant, extent in the presence of F(ab′)₂ anti-DAF. F(ab′)₂ anti-MCP did not significantly alter the susceptibility of HUVEC to complement-mediated lysis.