SUBLYTIC CONCENTRATIONS OF THE MEMBRANE ATTACK COMPLEX OF COMPLEMENT INDUCE ENDOTHELIAL INTERLEUKIN-8 AND MONOCYTE CHEMOATTRACTANT PROTEIN-1 THROUGH NUCLEAR FACTOR-KAPPA-B ACTIVATION

Activation of the complement cascade and subsequent assembly of the me mbrane attack, complex (MAC) occur in a number of pathophysiological s ettings, when formed on the surface of endothelial cells in sublytic c oncentrations, the MAC can induce a number of proinflammatory activiti es, including the secretion of soluble mediators (eg, interleukin (IL) -8 and monocyte chemoattractant protein (MCP)-1) and the up-regulation of cell surface adhesion molecules, Available data indicate that MAC- induced cell activation may occur through several complex signal trans duction pathways, but little is known about the intranuclear mechanism s by which complement-derived products promote the up-regulation of in flammatory mediators. Using purified distal complement proteins (C5-9) to assemble functional MAC on early-passage human umbilical vein endo thelial cells (HUVECs), we examined mechanisms of IMCP-1 and IL-8 indu ction. Formation of sublytic concentrations of MAC promoted an increas e in nuclear factor (NF)-kappa B DNA binding activity within 60 minute s as determined by serial electrophoretic mobility shift assay, Cytoso lic to nuclear translocation of NF-kappa B was confirmed by Western im munoblot and immunocytochemical analyses. Formation of the C5b-8 compl ex also promoted NF-kappa B translocation but to a lesser degree than observed in HUVECs containing complete MAC. No cytosolic to nuclear tr anslocation of the p65 NF-kappa B subunit was observed in unstimulated HUVECs or in cells incubated with the MAC components devoid of C7. Pr eincubation of HUVECs with pyrrolidine dithiocarbamate prevented MAC-i nduced increases in IL-8 and MCP-I mRNA concentrations and protein sec retion, A direct cause and effect linkage between MAC assembly and NF- kappa B activation was established through examination of the pharmaco logical effect of the peptide SN50 on IL-8 and MCP-1 expression. SN50 Is a recently engineered 26-amino-acid peptide that contains a lipophi lic cell-membrane-permeable motif and a nuclear localization sequence that specifically competes with the nuclear localization sequence of t he NF-kappa B p50 subunit, This study provides direct in vitro evidenc e that the distal complement system (MAC) can promote proinflammatory endothelial cell activation, specifically, increases in IL-8 and MCP-1 mRNA concentrations and protein secretion, and that cytosolic to nucl ear translocation of NF-kappa B is necessary for this response.