Gastroenterology

Gastroenterology

Volume 140, Issue 4, April 2011, Pages 1208-1218.e2
Gastroenterology

Basic—Alimentary Tract
The Epithelial Barrier Is Maintained by In Vivo Tight Junction Expansion During Pathologic Intestinal Epithelial Shedding

https://doi.org/10.1053/j.gastro.2011.01.004Get rights and content

Background & Aims

Tumor necrosis factor (TNF) increases intestinal epithelial cell shedding and apoptosis, potentially challenging the barrier between the gastrointestinal lumen and internal tissues. We investigated the mechanism of tight junction remodeling and barrier maintenance as well as the roles of cytoskeletal regulatory molecules during TNF-induced shedding.

Methods

We studied wild-type and transgenic mice that express the fluorescent-tagged proteins enhanced green fluorescent protein–occludin or monomeric red fluorescent protein 1–ZO-1. After injection of high doses of TNF (7.5 μg intraperitoneally), laparotomies were performed and segments of small intestine were opened to visualize the mucosa by video confocal microscopy. Pharmacologic inhibitors and knockout mice were used to determine the roles of caspase activation, actomyosin, and microtubule remodeling and membrane trafficking in epithelial shedding.

Results

Changes detected included redistribution of the tight junction proteins ZO-1 and occludin to lateral membranes of shedding cells. These proteins ultimately formed a funnel around the shedding cell that defined the site of barrier preservation. Claudins, E-cadherin, F-actin, myosin II, Rho-associated kinase (ROCK), and myosin light chain kinase (MLCK) were also recruited to lateral membranes. Caspase activity, myosin motor activity, and microtubules were required to initiate shedding, whereas completion of the process required microfilament remodeling and ROCK, MLCK, and dynamin II activities.

Conclusions

Maintenance of the epithelial barrier during TNF-induced cell shedding is a complex process that involves integration of microtubules, microfilaments, and membrane traffic to remove apoptotic cells. This process is accompanied by redistribution of apical junctional complex proteins to form intercellular barriers between lateral membranes and maintain mucosal function.

Section snippets

Animals

Seven- to 10-week old C57BL/6 wild-type, transgenic, and 210-kilodalton MLCK−/− mice were housed in an Association for Assessment and Accreditation of Laboratory Animal Care–accredited facility using Institutional Animal Care and Use Committee–approved procedures, as described.4, 9

Live Animal Imaging

Imaging of anesthetized animals was performed as previously described (Supplementary Materials and Methods).4, 10 Inhibitors were perfused luminally for 1 hour before imaging.

Immunofluorescence

Images of immunostained sections were

Barrier Function Is Maintained During TNF-Induced Cell Shedding

To visualize shedding in intact epithelia, in vivo imaging of wild-type mice was performed as described previously.4, 11 Only rare shedding events followed administration of low-dose (5 μg) TNF, which causes tight junction barrier loss and water secretion.4, 9 In contrast, high-dose (7.5 μg) TNF induced abundant epithelial shedding beginning 90 minutes after administration (6.2 × 10−3 ± 4.7 × 10−3 events/mm basement membrane at 120 minutes). Neighboring cells shed in rapid succession in some

Discussion

Epithelial extrusion occurs millions of times each day in the mammalian intestine. The frequency of such events is further amplified in inflammatory disease, where epithelial turnover occurs at significantly increased rates. Barrier defects associated with epithelial extrusion have been implicated as a cause of the barrier dysfunction associated with enterocolitis in experimental models and patients with inflammatory bowel disease6, 25 and may even be sites of pathogen entry.26 Despite these

Acknowledgments

L.S. and W.V.G. contributed equally to this work as second authors. J.R.T. and A.J.M.W. contributed equally to this work.

The authors thank V. Bindokas and Yimei Chen for confocal and electron microscopy support, respectively, and Danielle S. Smith for her outstanding efforts in cataloging and preparing data for analysis.

References (34)

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Conflicts of interest The authors disclose no conflicts.

Funding Supported by the National Institutes of Health (grants R01DK61931, R01DK68271, and P01DK67887 to J.R.T.), the University of Chicago Cancer Center (P30CA14599), T32HL007237 (to A.M.M.), the University of Chicago Institute for Translational Medicine (UL1RR024999), a Crohn's and Colitis Foundation of America research fellowship supported by Ms Laura McAteer Hoffman (to L.S.), and Wellcome Trust grant WT087768AIA (to A.J.M.W.).

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