The epithelial barrier is maintained by in vivo tight junction expansion during pathologic intestinal epithelial shedding

Abstract

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.

Figure 1. High dose TNF increases cell shedding but does not compromise barrier function

A. in vivo imaging of cell shedding in wild type mice. Low magnification time-lapse images at indicated times after 7.5 µg TNF. Bar = 20 µm. Time after TNF in minutes is indicated on images. Nuclei were labeled with Hoechst 33342. Alexa-633 (1µg/ml) was used as a luminal permeability marker (red). Zoomed views show a time lapse of shedding events, bar = 10µm. B. High magnification time-lapse images of a single shedding event. Bar = 10 µm. C. High magnification time-lapse images of a single shedding event after EDTA. Bar = 10 µm. D. Fixed tissue sections of jejunum from wild type mice 120 min after 7.5 µg TNF injection; F-actin (red) and nuclei (blue). Bar = 10 µm.

Figure 2. Tight junction proteins are remodeled during cell shedding

A. in vivo imaging of EGFP-occludin and mRFP1-ZO-1 during cell shedding in transgenic mice after 7.5 µg TNF. Bar=20 µm. Zoomed views bar = 10 µm. B & C. High magnification time-lapse images of (B) mRFP1-ZO-1 (red) and (C) EGFP-occludin (green) redistribution during a single shedding event. The luminal dye is 1µg/ml Alexa 633. Bar = 10 µm.

Figure 3. Proteins are remodeled in shedding cells

A. Jejunum was harvested from wild type mice 120 min after i.p. injection of 7.5 µg TNF and labeled for claudin-15, claudin-7, E-cadherin, ROCK, MLCK, pMLC, myosin IIA, myosin IIB, myosin IIC, cleaved caspase 3, tubulin (green), F-actin (red) and nuclei (blue). Arrow indicates nucleus of shedding cell. Bar = 10 µm. B. Wild-type mice were injected with 7.5 µg TNF, and a segment of jejunum perfused with 50 µm dynasore, 40 µm cytochalasin D, 500 µm blebbistatin, 400 µm Y27632, 400 µm colcemid, 50 µm Q-VD-OPH, or isotonic solution. MLCK^−/− mice were injected with 7.5 µg TNF and a segment of jejunum perfused with isotonic solution. Jejunum was harvested 120 min after TNF and labeled for ZO-1 (green), F-actin (red), and nuclei (blue). The number of shedding events was counted in at least 60,000 µm of basement membrane per condition.

Figure 4. Cell shedding can be defined by early, middle, and late stages

A. Electron micrographs of aldehyde-fixed, plastic-embedded jejunum harvested from wild type mice 120 min after TNF injection. As shedding progresses organelles break down, arrow in a and b; cells assumed a funnel shape, arrow indicates lateral membrane of shedding cell in b; and microvilli vesiculated, arrow in c. Nuclei of shedding cells condensed and fragmented, arrow in e; and the nuclear envelope became indistinct, arrow in d. Neighboring cells filled the space left by the shedding cell from beneath, arrows in f. B–D. Jejunum was harvested from wild type, mRFP1-ZO-1, or EGFP-occludin mice 120 min after 7.5 µg TNF i.p. and labeled for ZO-1 (red) and F-actin (green) or occludin (green) and F-actin (red). Nuclei are blue. B. Early-stage cell shedding. C. Middle-stage cell shedding. D. Late stage cell shedding. Bar=10 µm.

Figure 5. Extrusion of apoptotic shedding cells requires cytoskeletal elements and membrane traffic

A. Wild-type mice were injected with 7.5 µg TNF, and a segment of jejunum perfused with 50 µm Q-VDOPH, 400 µm colcemid, 500 µm blebbistatin, 40 µm cytochalasin D, 400 µm Y27632, 50 µm dynasore, or isotonic solution. MLCK^−/− mice were injected with 7.5 µg TNF and a segment of jejunum perfused with isotonic solution. Jejunum was harvested 120 min after TNF and labeled for ZO-1 (green), F-actin (red), and nuclei (blue). At least 60,000 µm of basement membrane was examined per condition. The stages of cell shedding were determined by F-actin and ZO-1 staining as in figure 4B–D. The percentage of events observed in each stage of shedding varied between the inhibitors used. B. Remodeling of ZO-1 (green) and F-actin (red) along lateral membranes of shedding cells in wild type mice after perfusion with Q-VD-OPH, colcemid, blebbistatin, cytochalasin D, Y27632, and dynasore, and in MLCK^−/− mice. Arrow indicates nucleus of shedding cell. C. A segment of jejunum was perfused with 50µm dynasore for 60 min prior to injection of 7.5 µg TNF. Tissue was harvested 120 min after TNF injection and labeled for F-actin (red) and nuclei (blue). Low magnification images show an accumulation of partially extruded cells (arrows). Bar = 10 µm.

Figure 6. A model for the mechanism of TNF-induced intestinal epithelial extrusion

Caspase cleavage is required to initiate the process, with additional contributions from MLCK and myosin ATPase activity as well as microtubule-dependent events. This is followed by redistribution of cytoskeletal, tight junction, and adherens junction proteins along lateral membranes of cell that will be shed. Myosin ATPase activity and both microtubule- and dynamin-dependent events are required for development of tension along lateral membranes and progression of extrusion. Finally, adjacent cells move into, and beneath, the space left by the shedding cell. This resolution requires actin reorganization, ROCK, MLCK, and dynamin.