Search

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.

CD59 is a 18-20-kDa membrane glycoprotein that inhibits formation of the membrane attack complex of complement (C) on homologous cells. In the present study we analyzed the expression and function of CD59 on human endothelial cells. Immunohistochemical analysis of renal cortex demonstrated a predominant expression of CD59 on peritubular capillary endothelial cells and glomerular endothelial cells. Flow cytometry analysis showed that human umbilical vein endothelial cells (HUVEC) expressed CD59 and the fluorescence intensity was approximately four times that of peripheral blood lymphocytes. CD59 is detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single 20-kDa molecule in 2% deoxycholate extracts of HUVEC. CD59 was released from the surface of HUVEC by phosphatidylinositol-specific phospholipase C, demonstrating that it is attached to the cell membrane by means of a glycolipid anchor. The functional activity of CD59 expressed on HUVEC was studied. Blocking of CD59 antigen with F(ab')2 fragments of polyclonal anti-CD59 enhanced markedly the susceptibility of HUVEC to C-mediated lysis. This effect was dependent on the amount of blocking antibodies added. Northern blot analysis revealed the presence of three species of mRNA expressed in HUVEC, which hybridized to a cDNA probe specific for CD59, with sizes of about 800, 1400 and 2000 bp. These findings suggest that CD59 may be important in protection of endothelial cells against C-mediated damage at local sites of inflammation, thereby maintaining the vascular integrity in vivo.