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 8

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210-kDa MLCK–/– mice are protected from anti-CD3–induced diarrhea. (A) I...
210-kDa MLCK–/– mice are protected from anti-CD3–induced diarrhea. (A) Intestinal weight-to-length ratio did not increase in MLCK–/– mice 3 hours after anti-CD3 treatment, while wild-type mice showed a large increase in weight-to-length ratio (P = 0.52; n = 4) (B) Consistent with the lack of an increase in weight-to-length ratio, perfused segments of small intestine in MLCK–/– mice treated with anti-CD3 did not show significant changes in water movement relative to untreated MLCK–/– mice (P = 0.35; n = 4). (C) Paracellular permeability, indicated by BSA efflux, did not increase in MLCK–/– mice treated with anti-CD3 relative to untreated MLCK–/– mice (P = 0.12; n = 4). (D) Immunofluorescent detection of occludin in intestinal epithelia of MLCK–/– mice demonstrated that occludin remained confined to the tight junction 3 hours after anti-CD3 treatment, without any evidence of internalization (scale bar, 5 μm).