Tumour necrosis factor-alpha (TNF-alpha) increases nuclear factor kappaB (NFkappaB) activity in and interleukin-8 (IL-8) release from bovine mammary epithelial cells

D C Fitzgerald, K G Meade, A N McEvoy, L Lillis, E P Murphy, D E MacHugh, A W Baird

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62 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)59-68
Number of pages10
JournalVeterinary Immunology and Immunopathology
Volume116
Issue number1-2
DOIs
Publication statusPublished - 15 Mar 2007

Keywords

  • Animals
  • Anti-Inflammatory Agents
  • Antibodies, Monoclonal
  • Base Sequence
  • Cattle
  • Dexamethasone
  • Electrophoretic Mobility Shift Assay
  • Epithelial Cells
  • Female
  • Interleukin-8
  • Mammary Glands, Animal
  • Mastitis, Bovine
  • Molecular Sequence Data
  • Mucous Membrane
  • NF-kappa B
  • Tumor Necrosis Factor-alpha

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