Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8 and monocyte chemoattractant protein-1 through nuclear factor-κB activation

Activation of the complement cascade and subsequent assembly of the membrane attack complex (MAC) occur in a number of pathophysiological settings. When formed on the surface of endothelial cells in sublytic concentrations, the MAC can induce a number of proinflammatory activities, 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 transduction pathways, but little is known about the intranuclear mechanisms by which complement- derived products promote the up-regulation of inflammatory mediators. Using purified distal complement proteins (C5-9)) to assemble functional MAC on early-passage human umbilical vein endothelial cells (HUVECs), we examined mechanisms of MCP-1 and IL-8 induction. Formation of sublytic concentrations of MAC promoted an increase in nuclear factor (NF)-κB DNA binding activity within 60 minutes as determined by serial electrophoretic mobility shift assay. Cytosolic to nuclear translocation of NF-κB was confirmed by Western immunoblot and immunocytochemical analyses. Formation of the C5b-8 complex also promoted NF-κB translocation but to a lesser degree than observed in HUVECs containing complete MAC. No cytosolic to nuclear translocation of the p65 NF-κB subunit was observed in unstimulated HUVECs or in cells incubated with the MAC components devoid of C7. Preincubation of HUVECs with pyrrolidine dithiocarbamate prevented MAC-induced increases in IL-8 and MCP- 1 mRNA concentrations and protein secretion. A direct cause and effect linkage between MAC assembly and NF-κB activation was established through examination of the pharmacological effect of the peptide SN50 on IL-8 and MCP-1 expression. SN50 is a recently engineered 26-amino-acid peptide that contains a lipophilic cell-membrane-permeable motif and a nuclear localization sequence that specifically competes with the nuclear localization sequence of the NF-κB p50 subunit. This study provides direct in vitro evidence 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 nuclear translocation of NF-κB is necessary for this response.