CD14-dependent activation of human endothelial cells by Bacteroides fragilis outer membrane.
We studied the capacity of isolated Bacteriodes fragilis outer membrane, B. fragilis NCTC9343 lipopolysaccharide (LPS; endotoxin), and B. fragilis NCTC9343 capsular polysaccharides to activate human umbilical vein endothelial cell (HUVEC) monolayers. To assess HUVEC activation, E-selectin expression was measured by enzyme-linked immunosorbent assay (ELISA), Northern blot analysis for E-selectin-specific mRNA, and electrophoretic gel mobility shift assay (EMSA) for NF-kappa B, a transcription factor necessary for E-selectin gene activation. Exposure of HUVECs to B. fragilis outer membrane fractions, separated from other components of the B. fragilis cell wall by isopycnic, sucrose gradient centrifugation, significantly increased surface expression of E-selectin and induced functional endothelial cell-dependent leukocyte adhesion. B. fragilis outer membranes induced translocation of NF-kappa B to HUVEC nuclei and accumulation of E-selectin mRNA in HUVEC cytoplasm. E-selectin expression induced by B. fragilis outer membranes was not blocked by polymixin B. In contrast, E-selectin expression induced by outer membrane fractions purified from E. coli was competitively inhibited by polymixin B. Neither purified B. fragilis LPS, a prominent constituent of the outer membrane, nor purified B. fragilis capsular polysaccharides induced HUVEC activation. Two different monoclonal antibodies directed against human CD14 completely inhibited B. fragilis outer membrane-induced NF-kappa B activation, E-selectin transcription, and E-selectin surface expression. We conclude that the outer membrane component of the B. fragilis cell wall contains a proinflammatory factor(s), that is not LPS, which induces human endothelial cell activation by a soluble CD14-dependent mechanism.
Sato
T T
TT
Department of Surgery, Harborview Medical Center, University of Washington School of Medicine, Seattle 98104-9796, USA.
Kovacich
J C
JC
Boyle
E M
EM
Haddix
T L
TL
Weintraub
A
A
Pohlman
T H
TH
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins / Q000494:pharmacology*; D001441:Bacteroides fragilis; D015152:Blotting, Northern; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D019040:E-Selectin / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D011112:Polymyxin B / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D015533:Transcriptional Activation
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D019040:E-Selectin; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D011112:Polymyxin B