Regulation of mucosal structure and barrier function in rat colon exposed to tumor necrosis factor alpha and interferon gamma in vitro : A novel model for studying the pathomechanisms of inflammatory bowel disease cytokines

• Objective. In Inflammatory bowel disease (IBD), elevated cytokines are responsible for disturbed intestinal transport and barrier function. The mechanisms of cytokine action have usually been studied in cell culture models only; therefore the aim of this study was to establish an in vitro model based on native intestine to analyze distinct cytokine effects on barrier function, mucosal structure, and inherent regulatory mechanisms. Material and methods. Rat colon was exposed to tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) in Ussing chambers. Transepithelial resistance (R-t) and H-3-mannitol fluxes were measured for characterization of the paracellular pathway. Transcellular transport was analyzed by horseradish peroxidase (HRP) flux measurements. Expression and distribution of tight junction proteins were characterized in immunoblots and by means of confocal laser-scanning microscopy (LSM). Results. Colonic viability could be preserved for 20 h in a specialized in vitro set-up. This was sufficient to alter mucosal architecture with crypt surface reduction. R-t was decreased (101 +/- 10 versus 189 +/- 10 Omega . cm(2)) with a parallel increase in mannitol permeability after cytokine exposure. Tight junction proteins claudin-1, -5, -7, and occludin decreased (45 +/- 10%, 16 +/- 7%, 42 +/- 8%, and 42 +/- 13% of controls, respectively), while claudin- 2 increased to 208 +/- 32%. Occludin and claudin- 1 translocated from the plasma membrane to the cytoplasm. HRP flux increased from 0.73 +/- 0.09 to 8.55 +/- 2.92 pmol . h(-1) . cm(-2). Conclusions. A new experimental IBD model with native colon in vitro is presented. One-day exposure to TNFa and IFNg alters mucosal morphology and impairs epithelial barrier function by up-regulation of the paracellular pore-former claudin-2 and down-regulation of the barrier-builders claudin-1, -5, and -7. These alterations resemble changes seen in IBD and thus underline their prominent role in IBD pathogenicity.

Nyckelord

Barrier function

claudins

colon

crypts

cytokines

interferon-gamma

large intestine

mucosal structure

occludin

permeability

tight junctions

transepithelial resistance

tumor necrosis factor-alpha

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MEDICIN

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