title: Activation of human endothelial cells by viable or heat-killed gram-negative bacteria requires soluble CD14.
abstract: In response to bacterial lipopolysaccharides (LPS; endotoxin), endothelial cells are converted to an activation phenotype expressing both proinflammatory and procoagulant properties that include the induction of leukocyte adhesion molecules and tissue factor expression. LPS-induced endothelial cell activation requires a soluble form of the monocyte LPS receptor, sCD14. We evaluated the capacity of multiple strains of gram-negative and gram-positive bacteria to induce endothelial E-selectin and tissue factor expression through sCD14-dependent pathways with cultured human umbilical vein endothelial cells (HUVE). Both viable and heat-killed gram-negative bacteria (Bacteroides fragilis, Enterobacter cloacae, Haemophilus influenzae, and Klebsiella pneumoniae) but not viable or heat-killed gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae) induced prominent E-selectin surface expression detected by enzyme-linked immunosorbent assay. Tissue factor activity on HUVE, indicated by factor X activation, was induced in response to gram-negative bacteria but not in response to gram-positive bacteria. Gram-negative bacteria induced transcriptional activation in HUVE, indicated by the appearance of E-selectin-specific mRNA and by the demonstration of activation of NF-kappa B, a trans-activating factor necessary for E-selectin and tissue factor gene transcription. In contrast, neither E-selectin mRNA nor activation of NF-kappa B was detected in HUVE treated with gram-positive bacteria. Endothelial cell activation by gram-negative bacteria in each of these assays was inhibited with a monoclonal antibody (60bd) against CD14. Furthermore, CHO-K1 cells, transfected with human recombinant CD14, responded to all strains of gram-negative bacteria (viable or heat killed), indicated by CHO-K1 NF-kappa B activation. We conclude that gram-negative bacteria induce endothelial cell activation through a common sCD14-dependent pathway.
authors.affiliation: Department of Surgery, University of Washington School of Medicine, Seattle 98104, USA.
authors.forename: R F
authors.initials: RF
authors.lastname: Noel
authors.forename: T T
authors.initials: TT
authors.lastname: Sato
authors.forename: C
authors.initials: C
authors.lastname: Mendez
authors.forename: M C
authors.initials: MC
authors.lastname: Johnson
authors.forename: T H
authors.initials: TH
authors.lastname: Pohlman
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D019040:E-Selectin / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D006090:Gram-Negative Bacteria / Q000502:physiology*; D006358:Hot Temperature; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000502:physiology*; D008070:Lipopolysaccharides / Q000633:toxicity; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D013925:Thromboplastin / Q000096:biosynthesis; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D019040:E-Selectin; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D013925:Thromboplastin