Epithelial myosin light chain kinase–dependent barrier dysfunction mediates T cell activation–induced diarrhea in vivo
Daniel R. Clayburgh, … , Randall J. Mrsny, Jerrold R. Turner
Daniel R. Clayburgh, … , Randall J. Mrsny, Jerrold R. Turner
Published October 3, 2005
Citation Information: J Clin Invest. 2005;115(10):2702-2715. https://doi.org/10.1172/JCI24970.
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Research Article

Epithelial myosin light chain kinase–dependent barrier dysfunction mediates T cell activation–induced diarrhea in vivo

Abstract

Disruption of the intestinal epithelial barrier occurs in many intestinal diseases, but neither the mechanisms nor the contribution of barrier dysfunction to disease pathogenesis have been defined. We utilized a murine model of T cell–mediated acute diarrhea to investigate the role of the epithelial barrier in diarrheal disease. We show that epithelial barrier dysfunction is required for the development of diarrhea. This diarrhea is characterized by reversal of net water flux, from absorption to secretion; increased leak of serum protein into the intestinal lumen; and altered tight junction structure. Phosphorylation of epithelial myosin II regulatory light chain (MLC), which has been correlated with tight junction regulation in vitro, increased abruptly after T cell activation and coincided with the development of diarrhea. Genetic knockout of long myosin light chain kinase (MLCK) or treatment of wild-type mice with a highly specific peptide MLCK inhibitor prevented epithelial MLC phosphorylation, tight junction disruption, protein leak, and diarrhea following T cell activation. These data show that epithelial MLCK is essential for intestinal barrier dysfunction and that this barrier dysfunction is critical to pathogenesis of diarrheal disease. The data also indicate that inhibition of epithelial MLCK may be an effective non-immunosuppressive therapy for treatment of immune-mediated intestinal disease.

Authors

Daniel R. Clayburgh, Terrence A. Barrett, Yueming Tang, Jon B. Meddings, Linda J. Van Eldik, D. Martin Watterson, Lane L. Clarke, Randall J. Mrsny, Jerrold R. Turner

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Figure 4

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Changes in tight junction morphology after anti-CD3 treatment. (A) Immun...
Changes in tight junction morphology after anti-CD3 treatment. (A) Immunofluorescent localization of the tight junction proteins claudin-1, claudin-4, claudin-5, and JAM-A in the small intestinal epithelium of wild-type mice demonstrates the localization of these proteins to the tight junction before (left panels) and 3 hours after (right panels) anti-CD3 treatment. Claudin-1, claudin-4, and claudin-5 distributions were not affected by anti-CD3 treatment. However, JAM-A demonstrated limited redistribution to intracellular sites after anti-CD3 treatment. (B) ZO-1 distribution appeared unchanged in transverse sections, but when viewed in en face sections, ZO-1 adopted a thinner, more sinuous distribution in anti-CD3–treated tissue. (C) Occludin was localized to the tight junction in control tissue, but after anti-CD3 treatment, occludin was internalized into intracellular vesicles (arrows), visible in both transverse and en face sections. Scale bars in A–C, 5 μm. (D) Detergent-insoluble subcellular fractions of jejunal epithelial cells were isolated on isopycnic sucrose gradients (black line). Protein concentration was determined for each fraction of gradients prepared from control (blue line) and anti-CD3–treated (red line) mice. A protein peak associated with the low-density, detergent-insoluble membrane fraction was present (bracket). The arrow designates the insoluble high-density pellet (fraction 24). These fractions were assessed by immunoblot. (E) Immunoblots for occludin in lysate (diluted), pellet, and low-density, detergent-insoluble membrane fractions showed loss of occludin from the detergent-insoluble membrane fraction after anti-CD3 treatment.