Tumour necrosis factor-alpha (TNF-alpha) increases nuclear factor kappaB (NFkappaB) 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 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.

Corticotropin-releasing hormone signaling in synovial tissue from patients with early inflammatory arthritis is mediated by the type 1 alpha corticotropin-releasing hormone receptor.

OBJECTIVE: Elevated levels of corticotropin-releasing hormone (CRH) are produced locally in inflamed human synovial tissue, and previous observations indicate a role for CRH in the pathogenesis of inflammatory joint disease. To further elucidate the biologic role of CRH at peripheral sites, we examined the expression of known CRH receptor subtypes in inflamed human synovium and compared the expression patterns in normal synovium. METHODS: Immunohistochemical analysis was used to confirm enhanced expression of specific CRH receptor subtypes in rheumatoid arthritis (RA) and psoriatic arthritis (PsA) synovium. Immunofluorescence double-labeling was used to further characterize CRH receptor-expressing cells. Reverse transcriptase-polymerase chain reaction analysis was performed to examine CRH receptor subtype messenger RNA (mRNA) expression in RA, PsA, and normal synovial tissue. In addition, CRH receptor expression was examined in isolated synovial endothelial cells and synoviocytes. RESULTS: Selective up-regulation of CRH receptors in inflamed synovial tissue indicated that CRH functions locally, in an autocrine/paracrine receptor-mediated manner. Immunoreactive CRH receptor type 1 (CRH-R1) was expressed abundantly on vascular endothelial cells and discrete perivascular cell populations, identified as mast cells. In all samples of inflamed synovium studied, CRH-R1alpha mRNA was detected; however, we were unable to identify CRH-R1beta or any CRH-R2 isoforms in samples from the same cohort of, patients. CRH receptor subtype expression in separated synovial cell populations revealed a pattern of staining similar to that seen in vivo. In contrast, neither CRH receptor subtype was expressed in normal synovial tissue. CONCLUSION: Our findings suggest that CRH signaling, via CRH-R1alpha, may play a role in both the vascular changes and the pathologic mechanisms associated with joint inflammation in human arthritis.