Cytokine-stimulated nitric oxide production by Caco-2 human colonic epithelial cells is not inhibited by the corticosteroid, dexamethasone

Abstract

NO production by inducible NOS (iNOS) may be involved in the pathogenesis of inflammatory bowel disease (IBD). In many cell types, iNOS expression is regulated transcriptionally and post-transcriptionally by corticosteroids, one of the main treatments for IBD. Aim: To study iNOS expression and iNOS sensitivity to corticosteroids in the human enterocytic differentiated colonic cell line, Caco-2. Methods: Caco-2 cells were grown under standard culture conditions (DMEM, FCS 20%) and used 10 days after cell confluence to permit differentiation. NOS activity was assessed by using nitrate reduction and Griess reaction, which measures the NO stable oxidative products nitrite and nitrate (NOx). Cytokine stimulation was IFN'/ (200U/ml), IL1-13 (5 ng/ml), and TNFa (100 ng/ml) (cytomix). All results are for a 24 hours incubation. Results: Combined cytokine stimulation induced NOx production in Caco-2 cells (unstimulated ceils: 1.2 ± 0.4 pM; cytomix-treated ceils: 8.5 -+ 0.7 pM, P<0.0001, Student t test). The cytokine-induced NOx production was dependent on de novo transcription as shown by its complete inhibition with actinomycin D (5 pg/ml). This production was calcium-independent since EGTA (0.1-1 mM) had no effect on cytokine-induced NOx release. This production was inhibited by the NOS inhibitor L-nitro-arginine and by the novel selective iNOS inhibitor 1400W (EC50 12±4 pM). Moreover, the cytokine-induced NOx production was inhibited by pre-treatment with pyrrolidine dithiocarbamate and dichloroisocoumarine, specific inhibitors of NF-lcB (EC50 150±38 pM and 50 pM, respectively). Conversely, dexamethasone (0.1-10 pM) had no effect on NOx production even with 6 hours pre-treatment before induction. Under the same conditions dexamethasone inhibited cytokine-induced nitrite production in the wellcharacterised murine macrophage cell line J774 (53 ±4 % with dexamethasone 10 }aM) and the murine intestinal epithelial cell line IEC-6 (60 ± 2% with 0.1 }aM dexamethasone). Conclusions: Thus, the differentiated human intestinal cell line Caco-2 produced NO after cytokine stimulation via the transcriptional induction of a calcium-independent NOS, and NF4cB was involved in this pathway. In contrast to many other cell lines, the corticosteroid, dexamethasone did not inhibit NOS induction. This could be due to a defect at the steroid receptor level or in the transduction pathway involving this receptor. M.Cavicchi received a research grant from Institut de Recherche des Maladies de rAppareil Digestif, France.