title: Tumour necrosis factor-alpha (TNF-alpha) increases nuclear factor kappaB (NFkappaB) activity in and interleukin-8 (IL-8) release from bovine mammary epithelial cells.
abstract: Epithelia play important immunological roles at a variety of mucosal sites. We examined NFkappaB activity in control and TNF-alpha treated bovine mammary epithelial monolayers (BME-UV cells). A region of the bovine IL-8 (bIL-8) promoter was sequenced and a putative kappaB consensus sequence was identified bioinformatically. We used this sequence to analyse nuclear extracts for IL-8 specific NFkappaB activity. As a surrogate marker of NFkappaB activation, we investigated IL-8 release in two models. Firstly in BME-UV monolayers, IL-8 release in the presence of pro- and anti-inflammatory agents was determined by enzyme-linked immunosorbent assay (ELISA). Secondly, we measured IL-8 secretion from a novel model of intact mucosal sheets of bovine teat sinus. IL-8 release into bathing solutions was assessed following treatment with pro- and anti-inflammatory agents. TNF-alpha enhanced NFkappaB activity in bovine mammary epithelial monolayers. p65 NFkappaB homodimer was identified in both control and TNF-alpha treated cells. Novel sequencing of the bovine IL-8 promoter identified a putative kappaB consensus sequence, which specifically bound TNF-alpha inducible p50/p65 heterodimer. TNF-alpha induced primarily serosal IL-8 release in the cell culture model. Pre-treatment with anti-TNF or dexamethasone inhibited TNF-alpha induced IL-8 release. High dose interleukin-1beta (IL-1beta) induced IL-8 release, however significantly less potently than TNF-alpha. Bovine mammary mucosal tissue released high basal levels of IL-8 which were unaffected by TNF-alpha or IL-1beta but inhibited by both dexamethasone and anti-TNF. These data support a role for TNF-alpha in activation of NFkappaB and release of IL-8 from bovine mammary epithelial cells.
authors.affiliation: UCD School of Agriculture, Food Science & Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: D C
authors.initials: DC
authors.lastname: Fitzgerald
authors.forename: K G
authors.initials: KG
authors.lastname: Meade
authors.forename: A N
authors.initials: AN
authors.lastname: McEvoy
authors.forename: L
authors.initials: L
authors.lastname: Lillis
authors.forename: E P
authors.initials: EP
authors.lastname: Murphy
authors.forename: D E
authors.initials: DE
authors.lastname: MacHugh
authors.forename: A W
authors.initials: AW
authors.lastname: Baird
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001483:Base Sequence; D002417:Cattle; D003907:Dexamethasone / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay / Q000662:veterinary; D004847:Epithelial Cells / Q000276:immunology / Q000378:metabolism; D005260:Female; D066298:In Vitro Techniques; D000069285:Infliximab; D016209:Interleukin-8 / Q000276:immunology* / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000276:immunology / Q000378:metabolism; D008414:Mastitis, Bovine / Q000276:immunology*; D008969:Molecular Sequence Data; D009092:Mucous Membrane / Q000276:immunology; D016328:NF-kappa B / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000276:immunology*
chemical_list: D000893:Anti-Inflammatory Agents; D000911:Antibodies, Monoclonal; D016209:Interleukin-8; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D003907:Dexamethasone; D000069285:Infliximab