Opposite effect of oxidative stress on inducible nitric oxide synthase and haem oxygenase-1 expression in intestinal inflammation: anti-inflammatory effect of carbon monoxide

Inducible nitric oxide synthase (iNOS) is expressed in intestinal epithelial cells (IEC) of patients with active inflammatory bowel disease (IBD) and in IEC of endotoxaemic rats. The induction of iNOS in IEC is an element of the NF-kappaB-mediated survival pathway. Haem oxygenase-1 (HO-1) is an AP-1-regulated gene that is induced by oxidative stress. The enzyme produces carbon monoxide (CO), which may attenuate the inflammatory response. The aim of this study was to investigate the regulation and interaction of iNOS and HO-1 in response to inflammation and oxidative stress. Male Wistar rats were treated with the thiol-modifying agent diethylmaleate (DEM) to induce oxidative stress and rendered endotoxaemic by LPS injection. Human colonic biopsies and the human colon carcinoma cell line DLD-1 were treated with DEM and the lipid peroxidation end-product 4-hydroxynonenal to induce oxidative stress and exposed to cytokine mix (CM) to mimic inflammation. In some experiments, cells were incubated with 250-400 ppm CO prior to and during stimulation with CM. HO-1 and iNOS expression was evaluated by RT-PCR, western blotting, and immunohistology. NF-kappaB activation was evaluated by EMSA. LPS induced iNOS but not HO-1 in epithelial cells of the ileum and colon. Oxidative stress strongly induced HO-1 in epithelial and inflammatory cells. Combined oxidative stress and endotoxaemia decreased iNOS expression but strongly induced HO-1 expression. Similarly, CM induced iNOS but not HO-1 in colonic biopsies and DLD-1 cells. Oxidative stress prevented iNOS induction in an NF-kappaB-dependent manner but increased HO-1 expression in CM-exposed DLD-1 cells. CO inhibited iNOS mRNA induction in CM-stimulated DLD-1 cells. These data demonstrate opposite regulation of iNOS and HO-1 in intestinal epithelial cells in response to cytokine exposure and oxidative stress. These findings suggest that iNOS (NF-kappaB driven) and HO-1 (AP-1 driven) represent mutually exclusive survival mechanisms in intestinal epithelial cells.