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

Am J Pathol. 1997 Jun;150(6):2019-31.

Abstract

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)-kappa B DNA binding activity within 60 minutes as determined by serial electrophoretic mobility shift assay. Cytosolic to nuclear translocation of NF-kappa B was confirmed by Western immunoblot and immunocytochemical analyses. Formation of the C5b-8 complex also promoted NF-kappa B translocation but to a lesser degree than observed in HUVECs containing complete MAC. No cytosolic to nuclear translocation of the p65 NF-kappa B 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-kappa B 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-kappa B 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-kappa B is necessary for this response.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Blotting, Northern
  • Blotting, Western
  • Cell Nucleus / metabolism
  • Cells, Cultured
  • Chemokine CCL2 / metabolism*
  • Complement Membrane Attack Complex / physiology*
  • Cytosol / metabolism
  • DNA / metabolism
  • Endothelium, Vascular / drug effects
  • Endothelium, Vascular / metabolism*
  • Humans
  • Immunohistochemistry
  • Interleukin-8 / metabolism*
  • Lipopolysaccharides / pharmacology
  • NF-kappa B / metabolism*
  • Pyrrolidines / pharmacology
  • Thiocarbamates / pharmacology
  • Time Factors
  • Umbilical Veins / drug effects
  • Umbilical Veins / metabolism

Substances

  • Chemokine CCL2
  • Complement Membrane Attack Complex
  • Interleukin-8
  • Lipopolysaccharides
  • NF-kappa B
  • Pyrrolidines
  • Thiocarbamates
  • pyrrolidine dithiocarbamic acid
  • DNA