title: Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression.
abstract: 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.
authors.affiliation: Department of Pathology, The University of Chicago, 5841 South Maryland Ave., MC 1089, Chicago, IL 60637, USA.
authors.forename: Fengjun
authors.initials: F
authors.lastname: Wang
authors.forename: W Vallen
authors.initials: WV
authors.lastname: Graham
authors.forename: Yingmin
authors.initials: Y
authors.lastname: Wang
authors.forename: Edwina D
authors.initials: ED
authors.lastname: Witkowski
authors.forename: Brad T
authors.initials: BT
authors.lastname: Schwarz
authors.forename: Jerrold R
authors.initials: JR
authors.lastname: Turner
mesh_terms: D018938:Caco-2 Cells; D049109:Cell Proliferation; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007371:Interferon-gamma / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D009219:Myosin-Light-Chain Kinase / Q000096:biosynthesis*; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D012460:Sulfasalazine / Q000494:pharmacology; D019108:Tight Junctions; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000502:physiology*; D015854:Up-Regulation
chemical_list: D016207:Cytokines; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D012460:Sulfasalazine; D007371:Interferon-gamma; D009219:Myosin-Light-Chain Kinase