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

Background & Aims: Barrier function of the inflamed intestinal mucosa can b e compromised by reactive oxygen metabolites that increase mucosal permeabi lity and disrupt the actin cytoskeleton, the integrity of which is importan t 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. Permeabi lity was assessed by measuring electrical resistance and mannitol fluxes an d actin organization by confocal fluorescein isothiocyanate-phalloidin anal ysis. Results: Basal transepithelial electrical resistance (TER) and mannit ol fluxes were not significantly different between groups. However, the oxi dant monochloramine rapidly decreased TER and increased mannitol permeabili ty of C2/AS monolayers compared with C2/ CEP4 (50% effective doses at 30 mi nutes were 0.53 +/- 0.11 and 2.06 +/- 0.34 mmol/L, respectively). Associate d with these changes, decreased cell viability, dissociation and aggregatio n of perijunctional;and stress actin filaments, loss of cell height, and in creased intercellular separation were observed only in C2/AS cells treated with monochloramine. Conclusions: hsp72 protects intestinal epithelial barr ier function against oxidant-induced stress, in part, by protecting the int egrity of the actin cytoskeleton.