Cytokine-induced intestinal epithelial hyperpermeability: Role of nitric oxide

Objective: Incubation of enterocytic monolayers with interferon (IFN)-gamma increases nitric oxide (NO) production and permeability, but NO synthesis inhibitors ameliorate the development of IFN-gamma-induced hyperpermeabilit y. Induction of inducible nitric oxide synthase (iNOS), an isoform of the e nzyme responsible for NO biosynthesis, is often enhanced by the synergistic effects of multiple cytokines. Moreover, many of the cytopathic effects of NO are mediated by peroxynitrite, which is produced by the reaction of NO with superoxide radical anion. In the present study, we sought to determine whether combinations of several proinflammatory cytokines, including IFN-g amma, interleukin-1 beta, and tumor necrosis factor-alpha, have synergistic effects on the induction of iNOS expression and/or hyperpermeability to hy drophilic solutes in cultured enterocytic monolayers. We also assessed the effects of aminoguanidine (a relatively selective iNOS inhibitor), L-N-G-mo nomethyl arginine (an isoform-nonselective NO synthase inhibitor), and Tiro n (a superoxide radical anion scavenger) on the development of cytokine-ind uced hyperpermeability. Design: Caco-2 monolayers were incubated under control conditions or with I FN-gamma, interleukin-1 beta, or tumor necrosis factor-alpha atone, pairwis e combinations of these cytokines, or all three cytokines together (cytomix ; CM). iNOS messenger RNA (mRNA) expression was assessed using Northern blo t analysis. The permeability of Caco-2 monolayers growing on permeable supp orts in bicameral chambers was assessed by measuring the apical-to-basolate ral flux of fluorescein disulfonic acid. The concentration of nitrate plus nitrite in culture supernatants, an indirect measure of NO production, was determined using the Griess reaction. Results: After 24 hrs of incubation, up-regulation of iNOS mRNA expression was evident only in cells exposed to IFN-gamma-containing formulations. Exp ression of iNOS mRNA was far greater in cells incubated with GM than in cel ls treated with IFN-gamma alone or either of the two-component IFN-gamma-co ntaining cytokine combinations. Compared with IFN-gamma, CM resulted in a h igher rate of NO production over 48 hrs of incubation. The permeability of Caco-2 monolayers increased by approximately six-fold and similar to 20-fol d after incubation for 48 hrs with IFN-gamma alone and CM, respectively. Th e increase in permeability induced by incubation with CM was significantly ameliorated by the addition of aminoguanidine, L-N-G-monomethyl arginine, o r Tiron. Conclusions: IFN-gamma-containing combinations of cytokines are potent indu cers of iNOS in cultured enterocytic monolayers. Peroxynitrite may be an im portant mediator of cytokine-induced gut epithelial hyperpermeability.