Tumour necrosis factor-α (TNF-α) increases nuclear factor κB (NFκB) activity in and interleukin-8 (IL-8) release from bovine mammary epithelial cells

Epithelia play important immunological roles at a variety of mucosal sites. We examined NF kappa B 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 KB consensus sequence was identified bioinformatically. We used this sequence to analyse nuclear extracts for IL-8 specific NF kappa B activity. As a surrogate marker of NF kappa B 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 NF kappa B activity in bovine mammary epithelial monolayers. p65 NF kappa B homodimer was identified in both control and TNF-alpha treated cells. Novel sequencing of the bovine IL-8 promoter identified a putative kappa B 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-1 beta (IL-1 beta) 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-1 beta but inhibited by both dexamethasone and anti-TNE These data support a role for TNF-alpha in activation of NF kappa B and release of IL-8 from bovine mammary epithelial cells. (c) 2007 Elsevier B.V. All rights reserved.