Numerous intestinal diseases are characterized by immune cell activation and compromised epithelial barrier function. We have shown that cytokine treatment of epithelial monolayers increases myosin II regulatory light chain (MLC) phosphorylation and decreases barrier function and that these are both reversed by MLC kinase (MLCK) inhibition. The aim of this study was to determine the mechanisms by which interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha regulate MLC phosphorylation and disrupt epithelial barrier function. We developed a model in which both cytokines were required for barrier dysfunction. Barrier dysfunction was also induced by TNF-alpha addition to IFN-gamma-primed, but not control, Caco-2 monolayers. TNF-alpha treatment of IFN-gamma-primed monolayers caused increases in both MLCK expression and MLC phosphorylation, suggesting that MLCK is a TNF-alpha-inducible protein. These effects of TNF-alpha were not mediated by nuclear factor-kappaB. However, at doses below those needed for nuclear factor-kappaB inhibition, sulfasalazine was able to prevent TNF-alpha-induced barrier dysfunction, MLCK up-regulation, and MLC phosphorylation. Low-dose sulfasalazine also prevented morphologically evident tight junction disruption induced by TNF-alpha. These data show that IFN-gamma can prime intestinal epithelial monolayers to respond to TNF-alpha by disrupting tight junction morphology and barrier function via MLCK up-regulation and MLC phosphorylation. These TNF-alpha-induced events can be prevented by the clinically relevant drug sulfasalazine.

Although tight junction morphology is not obviously affected by TNF, this proinflammatory cytokine promotes internalization of occludin, resulting in disrupted barrier function within the intestine.

IFN-gamma primes intestinal epithelia to respond to TNF by inducing TNFR2 expression, which in turn mediates TNF-induced MLCK-dependent barrier dysfunction. The data further suggest that epithelial TNFR2 blockade may be a novel approach to restore barrier function in intestinal disease.

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BACKGROUND & AIMS-LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpes virus entry on T cells) is a TNF core family member that regulates T cell activation and causes experimental inflammatory bowel disease. Additional data suggest that LIGHT may be involved in the pathogenesis of human inflammatory bowel disease. The aim of this study was to determine if LIGHT was capable of signaling directly to intestinal epithelia and to define the mechanisms and consequences of such signaling.

Two myosin light chain kinase (MLCK) isoforms, MLCK1 and MLCK2, which differ only by a short insertion in MLCK1 are expressed in intestinal epithelia. MLCK1 localizes to the perijunctional actomyosin ring and tight junction while MLCK2 is distributed more diffusely. We have previously shown that intestinal epithelial MLCK is transcriptionally and enzymatically activated by TNF, both in vitro and in vivo, and that this upregulation is critical to TNF‐induced barrier dysfunction. Since MLCK1 regulates epithelial barrier function, we hypothesized that TNF might specifically upregulate MLCK1 transcription or targeting to the perijunctional actomyosin ring. MLCK1 and total MLCK were detected using PCR primers or antibodies directed against the unique MLCK1 insertion or regions shared by both isoforms, respectively. Real‐time RT PCR analysis of Caco‐2 monolayers showed that MLCK1 and total MLCK mRNA transcripts increased 4.1‐ and 3.5‐fold, respectively, 4 hours after TNF treatment. Immunofluorescence microscopy demonstrated concomitant recruitment of MLCK1 to the perijunctional actomyosin ring. TNF caused similar MLCK1 recruitment to the perijunctional actomyosin ring of murine enterocytes in vivo. These data demonstrate that TNF upregulates MLCK1 expression and, perhaps more importantly, that TNF enhances MLCK1 targeting to the perijunctional actomyosin ring in vitro and in vivo. Supported by NIDDK.

TNF causes epithelial barrier dysfunction in vitro and in vivo and is accompanied by redistribution of the tight junction protein occludin. Mechanistic characterization of this vesicular trafficking process and associated tight junction regulation has been limited by the inability to image occludin internalization in real time. Our aim was to characterize the route and mechanisms of TNF‐induced occludin endocytosis in vivo.We developed mice expressing EGFP‐occludin and mRFP1‐ZO‐1 in intestinal epithelium, and both fusion proteins colocalized with their endogenous counterparts. Jejunal mucosa was imaged by confocal microscopy of live anaesthetized mice.TNF (5μg i.p.) triggered focal intra‐junctional occludin aggregation followed by occludin endocytosis. The small endocytic vesicles contained caveolin‐1 but not clathrin heavy chain. Occludin endocytosis was prevented by dynasore, a dynamin II inhibitor; L‐t‐LacCer, a synthetic lipid inhibitor of caveolar endocytosis; and cholesterol chelation with MβCD; while neither macropinocytosis nor clathrin‐mediated endocytosis inhibitors blocked internalization. Occludin endocytosis was also defective in caveolin‐1 knockout mice, though TNF activated myosin normally.These data are the first to image occludin protein trafficking in vivo and demonstrate the pivotal role of caveolin‐1‐dependent caveolar endocytosis in this process.Supported by NIH.