Gastroenterology

Gastroenterology

Volume 117, Issue 1, July 1999, Pages 115-122
Gastroenterology

Alimentary Tract
Heat-shock protein 72 protects against oxidant-induced injury of barrier function of human colonic epithelial Caco2/bbe cells,☆☆,

https://doi.org/10.1016/S0016-5085(99)70557-3Get rights and content

Abstract

Background & Aims: Barrier function of the inflamed intestinal mucosa can be compromised by reactive oxygen metabolites that increase mucosal permeability and disrupt the actin cytoskeleton, the integrity of which is important for maintaining tight epithelial junctions. Because heat-shock protein 72 (hsp72) protects intestinal epithelial cells against injury, we determined whether resistance of Caco2/bbe (C2) intestinal monolayer barrier function was related to their high endogenous hsp72 expression. Methods: hsp72 anti-sense (C2/AS) and vector-only transfected C2 (C2/CEP4) clones, lines that exhibit low and high hsp72 expression, respectively, were studied. Permeability was assessed by measuring electrical resistance and mannitol fluxes and actin organization by confocal fluorescein isothiocyanate–phalloidin analysis. Results: Basal transepithelial electrical resistance (TER) and mannitol fluxes were not significantly different between groups. However, the oxidant monochloramine rapidly decreased TER and increased mannitol permeability of C2/AS monolayers compared with C2/CEP4 (50% effective doses at 30 minutes were 0.53 ± 0.11 and 2.06 ± 0.34 mmol/L, respectively). Associated with these changes, decreased cell viability, dissociation and aggregation of perijunctional and stress actin filaments, loss of cell height, and increased intercellular separation were observed only in C2/AS cells treated with monochloramine. Conclusions: hsp72 protects intestinal epithelial barrier function against oxidant-induced stress, in part, by protecting the integrity of the actin cytoskeleton.

GASTROENTEROLOGY 1999;117:115-122

Section snippets

Cell culture and cell protection

The Caco2/bbe subclone (C2)13 was grown in Dulbecco's modified Eagle medium (DMEM; high glucose, 4.5 g/L) with 10% (vol/vol) fetal bovine serum, 10 μg/mL transferrin, 50 μg/mL streptomycin, and 50 U/mL penicillin. Cells were used between passages 50 and 80. IEC-18 cells were grown in DMEM containing 5% (vol/vol) fetal bovine serum, 0.1 U/mL insulin, and penicillin/streptomycin as above. Cells were used between passages 20 and 27.

The ability of the C2 and IEC-18 cell lines to withstand injury

Monochloramine injury in C2 and IEC-18 cells

Normal diploid intestinal epithelial cells such as the IEC-18 cell line are easily injured by exposure to oxidants, as previously shown.11, 12 In contrast, we had observed that C2 cells were far more resistant to the injurious effects of monochloramine treatment. The difference in cell response to monochloramine induced injury is shown in Figure 1.

. Ability of the oxidant monochloramine (NH2Cl) to injure IEC-18 and C2 cells. Cellular injury was assessed by loading cells with 51CrCl, injuring the

Discussion

One of the most important functions of the intestinal mucosa is the maintenance of barrier function, which is not only essential for preventing systemic entry of foreign antigens, bacteria, and bacterial products, but also for other gut functions such as vectorial transport of nutrients, water, and electrolytes. However, mucosal barrier function can be significantly compromised in the setting of inflammatory bowel diseases, particularly by immune and inflammatory mediators such as interferon

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    Supported by National Institutes of Health grant DK-47722 (to E.B.C.), the Digestive Disease Center at the University of Chicago DK-42086, National Cancer Institute grant CA-14599 to the University of Chicago Cancer Research Center, and the Gastrointestinal Research Foundation of Chicago.

    ☆☆

    Address requests for reprints to: Eugene B. Chang, M.D., Department of Medicine, University of Chicago, MC 6084, 5841 South Maryland Avenue, Chicago, Illinois 60637. e-mail: [email protected]; fax: (773) 702-2281.

    Confocal microscopy was performed in the Al Robin Confocal Core Facility.

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