title: Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8 and monocyte chemoattractant protein-1 through nuclear factor-kappa B activation.
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.
authors.affiliation: Department of Pathology, University of Michigan Medical School, Ann Arbor 48109-0602, USA.
authors.forename: K S
authors.initials: KS
authors.lastname: Kilgore
authors.forename: E
authors.initials: E
authors.lastname: Schmid
authors.forename: T P
authors.initials: TP
authors.lastname: Shanley
authors.forename: C M
authors.initials: CM
authors.lastname: Flory
authors.forename: V
authors.initials: V
authors.lastname: Maheswari
authors.forename: N L
authors.initials: NL
authors.lastname: Tramontini
authors.forename: H
authors.initials: H
authors.lastname: Cohen
authors.forename: P A
authors.initials: PA
authors.lastname: Ward
authors.forename: H P
authors.initials: HP
authors.lastname: Friedl
authors.forename: J S
authors.initials: JS
authors.lastname: Warren
mesh_terms: D015152:Blotting, Northern; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism*; D015938:Complement Membrane Attack Complex / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D016209:Interleukin-8 / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D011759:Pyrrolidines / Q000494:pharmacology; D013859:Thiocarbamates / Q000494:pharmacology; D013997:Time Factors; D014471:Umbilical Veins / Q000187:drug effects / Q000378:metabolism
chemical_list: D018932:Chemokine CCL2; D015938:Complement Membrane Attack Complex; D016209:Interleukin-8; D008070:Lipopolysaccharides; D016328:NF-kappa B; D011759:Pyrrolidines; D013859:Thiocarbamates; C020972:pyrrolidine dithiocarbamic acid; D004247:DNA