Gastroenterology

Gastroenterology

Volume 131, Issue 4, October 2006, Pages 1153-1163
Gastroenterology

Basic–alimentary tract
IFN-γ-Induced TNFR2 Expression Is Required for TNF-Dependent Intestinal Epithelial Barrier Dysfunction

https://doi.org/10.1053/j.gastro.2006.08.022Get rights and content

Background & Aims: Tumor necrosis factor (TNF) plays a critical role in intestinal disease. In intestinal epithelia, TNF causes tight junction disruption and epithelial barrier loss by up-regulating myosin light chain kinase (MLCK) activity and expression. The aim of this study was to determine the signaling pathways by which TNF causes intestinal epithelial barrier loss. Methods: Caco-2 cells that were either nontransfected or stably transfected with human TNF receptor 1 (TNFR1) or TNFR2 and mouse colonocytes were used for physiologic, morphologic, and biochemical analyses. Results: Colitis induced in vivo by adoptive transfer of CD4+CD45RBhi T cells was associated with increased epithelial MLCK expression and myosin II regulatory light chain (MLC) phosphorylation as well as morphologic tight junction disruption. In vitro studies showed that TNF caused similar increases in MLCK expression and MLC phosphorylation, as well as barrier dysfunction, in Caco-2 monolayers only after interferon (IFN)-γ pretreatment. This reductionist model was therefore used to determine the molecular mechanism by which IFN-γ and TNF synergize to cause intestinal epithelial barrier loss. IFN-γ priming increased TNFR1 and TNFR2 expression, and blocking antibody studies showed that TNFR2, but not TNFR1, was required for TNF-induced barrier dysfunction. Transgenic TNFR2, but not TNFR1, expression allowed IFN-γ-independent TNF responses. Conclusions: IFN-γ 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.

Section snippets

CD4+CD45RBhi-Adoptive Transfer

C57BL/6 and RAG1−/− mice were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained in a specific pathogen-free facility. All experiments were carried out in accordance with National Institutes of Health guidelines under protocols approved by the Institutional Animal Care and Use Committee at the University of Chicago. CD4+CD45RBhi lymphocytes were isolated from C57BL/6 splenocytes using a MO-FLO sorter (Dako, Carpinteria, CA) after staining with allophycocyanin-conjugated anti-CD4

Experimental Immune-Mediated Inflammatory Bowel Disease Is Associated With Increased MLCK Expression, Increased MLC Phosphorylation, and Tight Junction Disruption In Vivo

Several studies using in vitro2, 5, 18 and acute in vivo4 models have shown that MLCK-mediated MLC phosphorylation is central to TNF-induced epithelial tight junction disruption and barrier dysfunction. In the setting of chronic disease, it has recently been reported that intestinal epithelial MLCK expression and MLC phosphorylation are increased in patients with active inflammatory bowel disease and that the magnitude of these changes correlates with the grade of disease activity.6 However,

Discussion

Compromised intestinal barrier function is a common feature of inflammatory, infectious, ischemic, and immune-mediated intestinal disease,35 and abundant data suggest that compromised intestinal barrier function may be a critical pathogenic event in these processes.36, 37, 38, 39, 40 For example, in patients with inactive Crohn’s disease, barrier loss predicts disease reactivation.41, 42 The degree of barrier dysfunction also parallels expression of activation markers on circulating lymphocytes.

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    Supported by the National Institutes of Health (DK61931 and DK68271), the Crohn’s Colitis Foundation of America, The University of Chicago Digestive Disease Center (DK42086), and The University of Chicago Cancer Center (CA14599).

    1

    F.W. and B.T.S. contributed equally to this work.

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