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 6

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Systemic T cell activation increases intestinal epithelial MLC phosphory...
Systemic T cell activation increases intestinal epithelial MLC phosphorylation. (A) Electron micrographs of villous enterocytes from jejunum of control and anti-CD3–treated mice demonstrate disruption of the tight junction and marked perijunctional cytoskeletal condensation (arrow) after T cell activation (scale bar, 250 nm). (B) Phosphorylated MLC (red) in control intestinal villi was localized to the perijunctional actomyosin ring and demonstrates enhancement at cell junctions. Three hours after anti-CD3 treatment, MLC phosphorylation was markedly increased. Matched exposures are shown (scale bar, 5 μm). (C) Intestinal epithelial MLC phosphorylation, determined by immunoblot for phosphorylated and total MLC in isolated intestinal epithelial cells, revealed an increase in MLC phosphorylation 3 hours after anti-CD3 injection. This increase in MLC phosphorylation was largely prevented by the TNF-neutralizing antibody. (D) Immunoblots of phosphorylated MLC at several time points after anti-CD3 injection showed that epithelial MLC phosphorylation peaked 2–3 hours after anti-CD3 injection and returned to baseline levels within 5 hours after anti-CD3 injection. (E) Quantitative analysis of the blots showed that MLC phosphorylation (red line) increased and decreased in parallel with changes in intestinal weight-to-length ratio (blue line) induced by T cell activation (n = 3).