title: Regulation of bovine E-selectin expression by recombinant tumor necrosis factor alpha and lipopolysaccharide.
abstract: Induction of adhesion molecules by cytokines and LPS is an important mechanism of regulating leukocyte migration into tissue. Expression and regulation of E-selectin may be differentially influenced by the stimuli involved with effects on mRNA or surface protein kinetics. Surface protein and mRNA expression kinetics of bovine E-selectin were measured and compared in primary cultures of bovine aortic endothelial cells (BAEC) stimulated for various periods of time with recombinant bovine tumor necrosis factor alpha (rbTNF-alpha) or Escherichia coli lipopolysaccharide (LPS). E-selectin mRNA expression was measured via quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) using a construct that contained multiple synthetic oligonucleotides for several bovine adhesion molecules and cytokines. Surface expression of E-selectin was measured by flow cytometry. Unstimulated BAECs expressed minimum or no E-selectin on the surface. A low number of endothelial cells expressed surface E-selectin as early as 1h post-stimulation and surface expression was sustained after both stimuli for 24-72h. Mean fluorescence intensity (MFI) indicated peak surface concentration of E-selectin at 6 h post-stimulation after LPS followed by a gradual decrease to 72h without returning to baseline values. Mean fluorescence intensity following stimulation with TNF-alpha increased slightly between 0 and 72h. The pattern of mRNA expression differed between stimuli. LPS-stimulated BAECs expressed peak amounts of E-selectin mRNA at 6 h, followed by a decline to baseline by 24 h. Conversely, BAECs stimulated with rbTNF-alpha expressed significantly (p pound 0.05) higher amounts of mRNA at 1h than compared to unstimulated controls (0 h), but this decreased to below baseline levels by 6h; followed by a gradual increase and eventually a sharp increase between 18 and 72 h. To account for the lack of correlation between mRNA and protein expression, it was hypothesized that shedding of surface E-selectin accounted at least in part, for the large increase in mRNA expression seen at 18-72h. Culture supernatants from rbTNF-alpha-treated BAECs were harvested, and tested for the presence of shed E-selectin using ELISA. Unstimulated culture supernatants contained little or no E-selectin. Between 6 and 48 h, the concentration of E-selectin in culture supernatants from rbTNF-alpha-stimulated BAECs increased approximately two-fold, suggesting that the sharp increase in E-selectin mRNA expression around 18 h may be related to significant loss of surface E-selectin during this period.
authors.affiliation: Department of Pathobiology, University of Guelph, Ont., N1G 2W1, Guelph, Canada.
authors.forename: C
authors.initials: C
authors.lastname: Van Kampen
authors.forename: B A
authors.initials: BA
authors.lastname: Mallard
mesh_terms: D000818:Animals; D002417:Cattle / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000737:chemistry; D019040:E-Selectin / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects; D018950:Lipopolysaccharide Receptors / Q000276:immunology / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000276:immunology / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000494:pharmacology*; D001613:beta 2-Microglobulin / Q000096:biosynthesis / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D019040:E-Selectin; D018950:Lipopolysaccharide Receptors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D001613:beta 2-Microglobulin