Tumour necrosis factor-α (TNF-α) increases nuclear factor κB (NFκB) 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 NFκB activity in control and TNF-α treated bovine mammary epithelial monolayers (BME-UV cells). A region of the bovine IL-8 (bIL-8) promoter was sequenced and a putative κB consensus sequence was identified bioinformatically. We used this sequence to analyse nuclear extracts for IL-8 specific NFκB activity. As a surrogate marker of NFκB 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-α enhanced NFκB activity in bovine mammary epithelial monolayers. p65 NFκB homodimer was identified in both control and TNF-α treated cells. Novel sequencing of the bovine IL-8 promoter identified a putative κB consensus sequence, which specifically bound TNF-α inducible p50/p65 heterodimer. TNF-α induced primarily serosal IL-8 release in the cell culture model. Pre-treatment with anti-TNF or dexamethasone inhibited TNF-α induced IL-8 release. High dose interleukin-1β (IL-1β) induced IL-8 release, however significantly less potently than TNF-α. Bovine mammary mucosal tissue released high basal levels of IL-8 which were unaffected by TNF-α or IL-1β but inhibited by both dexamethasone and anti-TNF. These data support a role for TNF-α in activation of NFκB and release of IL-8 from bovine mammary epithelial cells.