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Intestinal epithelial barrier function is impaired after the exposure of enterocytes to proinflammatory cytokines. The mechanism(s) responsible for this phenomenon remain incompletely understood. We used cultured monolayers of Caco-2 enterocyte-like cells to characterize the effect of cytomix, a mixture of interferon-γ, tumor necrosis factor-α, and interleukin-1β, on the expression and localization of several tight junctions proteins. Cells were stimulated with cytomix in the presence or absence of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), an NO· scavenger. Some cells were treated with (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate] (DETA-NONOate), an NO· donor. Tight junction protein expression was measured in cellular extracts by Western blotting and localized in cells using immunofluorescence. Steady-state mRNA levels were determined using semi-quantitative reverse-transcription polymerase chain reaction. Incubation of cells with DETA-NONOate or cytomix decreased epithelial barrier function, decreased expression of ZO-1 mRNA, decreased expression of ZO-1, ZO-3, and occludin protein, and increased expression of claudin-1 protein. The effects of cytomix on barrier function and tight junction protein expression were modulated by cPTIO. Cytomix caused incorrect subcellular localization of ZO-1, occludin, and claudin-1, and this was modulated by co-incubation with cPTIO. DETA-NONOate caused similar protein mislocalization as observed with cytomix. The effectiveness of cPTIO in maintaining tight junction protein expression and correct subcellular localization was less apparent at early time points (12 h) compared with later points, suggesting an NO·-independent effect of cytokines on barrier function. Thus, cytomix appears to increase the permeability of Caco-2 monolayers through NO·-dependent and -independent mechanisms that are associated with changes in the expression and/or targeting of proteins involved in tight junction function.