NO-DEPENDENT AND NO-INDEPENDENT IL-1 PRODUCTION BY A HUMAN COLONIC EPITHELIAL-CELL LINE UNDER INFLAMMATORY STRESS

1 The present study was designed to investigate, in an in vitro model of the human intestinal barrier, the ability of epithelial cells to pr oduce interleukin-1 (IL-I), the cellular mechanisms involved in IL-1 r elease, and the intracellular signalling pathways involved in IL-1 up- regulation during inflammatory stress. 2 This study was based on the h uman colonic epithelial cell line HT29-C1.16E, maintained as polarized monolayers on filters mounted in culture chambers and treated with va rious proinflammatory cytokines (interferon gamma (IFN gamma), tumour necrosis factor alpha (TNF alpha), IL-1 beta) alone or in combination. 3 IL-1 production, restricted to IL-1 alpha, was induced by the combi nation of IFN gamma/TNF alpha. When IL-1 beta was added to IFN gamma/T NF alpha, it led to an additional production of IL-1 alpha. IL-1 alpha release was associated with cell damage, as shown by the correlation between lactate dehydrogenase (LDH) release and extracellular IL-1 pro duction, and was not accounted for by a secretory mechanism. 4 Both IF N gamma/TNF alpha and IFN gamma/TNF alpha/IL-1 beta induced inducible nitric oxide synthase (iNOS) expression as shown by quantitation of NO 2-/NO3- by use of the Griess reagent, quantitation of cells scoring po sitive with an anti-iNOS antibody and detection of mRNAs coding for iN OS by RT-PCR. The use of NG-monomethyl-L-arginine (L-NMMA), an inhibit or of NOS, led to the demonstration of two distinct signalling pathway s in IL-1 production by HT29-Cl.16E cells, one dependent on NO (r-NMMA -sensitive) under treatment with IFN gamma/TNF alpha/IL-1 beta, and th e other independent of NO (L-NMMA-insensitive) under treatment with IF N gamma/TNF alpha. 5 Moreover, we examined whether a redox-based mecha nism could be responsible for the apparent discrepancy between NO prod uction and NO implication in IL-I production under IFN gamma/TNF alpha and IFN gamma/TNF alpha/IL-1 beta treatments. Experiments with cystei ne, which acts as a powerful reductant, suggest that the nitrosonium c haracter of NO is involved. in the NO-dependent pathway in IL-1 produc tion.