Gastrointestinal
Loss of the Tight Junction Protein ZO-1 in Dextran Sulfate Sodium Induced Colitis

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Background

Inflammatory bowel disease (IBD) is associated with increased intestinal permeability and decreased expression of tight junction (TJ) proteins in the inflamed mucosa. Whether this alteration in TJ expression is a prerequisite for the development of intestinal inflammation or a secondary result of that inflammation is unknown. This study looked at the expression of the TJ protein ZO-1 and the corresponding permeability changes in dextran sulfate sodium (DSS) induced colitis in a mouse model.

Materials and methods

BALB/c mice were fed 3% DSS or water for 1, 3, 5, or 7 days. The animals were weighed, stool was checked for blood, and the colon length measured. Segments of the colon were used for histology, immunohistochemistry for ZO-1, or Western blot for TJ proteins. Colonic permeability was measured using Evan’s Blue dye.

Results

DSS treated animals had heme positive stools, colitis by histology, significant weight loss, and colon shortening. There was an absence of ZO-1 by Western blot in the 7-day DSS treated animals, double the amount of claudin-1 and normal cytokeratin. The loss of ZO-1 started after 1 d of DSS treatment and was followed by a significant increase in permeability to Evan’s blue by day 3.

Conclusions

The loss of ZO-1 and increased permeability preceded the development of significant intestinal inflammation suggesting that in DSS colitis alterations in the TJ complex occur before the intestinal inflammation and not as a consequence of it. These changes in the TJ complex may facilitate the development of the inflammatory infiltrate seen in colitis.

Introduction

Inflammatory bowel disease (IBD), which consists of Crohn’s disease (CD) and ulcerative colitis (UC), is an immune-mediated illness characterized by chronic intestinal inflammation with or without other systemic manifestations. The etiology of IBD is unknown and there is no known medical cure. One theory of pathogenesis suggests that there is a compromise in the intestinal permeability barrier exposing the underlying immuno-regulatory mechanisms to normally excluded agents that results in an aberrant and self-perpetuating inflammatory process. Consistent with this theory is the finding of increased intestinal permeability in both patients with CD and at-risk relatives who remain asymptomatic [1].

The tight junction complex (TJ) is a cluster of proteins that forms a physiologically active barrier at the level of the intestinal epithelial cell that can change its permeability based on the cellular environment. The organization of the TJ is similar to other intercellular junctions and consists of transmembrane proteins that mediate adhesive function linked to the underlying plaque proteins that in turn associate with the cytoskeleton [2]. Three of the key proteins of the TJ are ZO-1, occludin, and the more recently identified family of claudins. ZO-1, one of the plaque proteins, was the first TJ protein characterized. It is a 225 kda membrane bound protein that localizes to the TJ. It binds the transmembrane proteins occludin and the claudins linking them to cytoskeletal actin [3, 4]. Contraction of cytoskeletal actin is thought to have a role in the regulation of paracellular permeability and ZO-1 may be the direct link between actin and the transmembrane proteins.

The expression of TJ proteins has been shown to be decreased in the intestinal inflammation of IBD [5, 6, 7]. This corresponds to an increase in intestinal permeability and decrease in transepithelial resistance (TER) [8, 9]. Increased intestinal permeability in IBD patients correlates with disease activity and has been demonstrated to be a predictor of relapse, after both medical and surgical remission [10, 11, 12, 13]. Whether this alteration in tight junction expression and increase in permeability is a prerequisite for the development of intestinal inflammation or a secondary result of that inflammation is unknown. The goal of this present study was to measure expression of the TJ protein ZO-1 and the corresponding permeability changes associated with dextran sulfate sodium (DSS) induced colitis in a mouse model.

Section snippets

Mice

Female BALB/c mice, 8 to 10 weeks (17–25 g) were purchased from Jackson Laboratory (Bar Harbor, ME) and handled in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).

Induction of Colitis

Colitis was induced in the mice by replacing their drinking water with 3% (wt/vol) DSS in tap water. Control mice received tap water. Animals were allowed water or 3% DSS and food ad lib. See Table 1 for animal groups. Weight change and

Weight Loss

Control animals had about a 5% weight gain over the 7 d study period. DSS treated animals, on the other hand, had significant weight loss compared to water treated control animals (Fig. 1). The DSS treated animals gained weight on day 1 but then lost weight progressively through day 7. The increased weight on day 1 may be explained by edema in the colon from the DSS induced injury.

Fecal Blood

None of the control animals developed fecal blood. In the DSS treated animals feces were Hemoccult positive in 19%

Discussion

The cause of IBD is unknown but is thought to be due to an uncontrolled inflammatory response of the intestinal tract to an environmental agent in the genetically predispositioned patient. What the environmental agent is and how it interacts with the intestine is also unknown. It has been demonstrated that there is an alteration in the intestinal TJ complex and a corresponding increase in intestinal permeability in the intestinal inflammation associated with IBD [5, 6, 7, 17]. One of the

Acknowledgments

We thank Rona Ellis of the Microscopy Imaging Core Facility of the Section of Research Resources, Penn State College of Medicine for preparing the immunofluorescence and for assisting with the confocal images.

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