NO-dependent and NO-independent IL-1 production by a human colonic epithelial cell line under inflammatory stress

Abstract

1. The present study was designed to investigate, in an in vitro model of the human intestinal barrier, the ability of epithelial cells to produce interleukin-1 (IL-1), the cellular mechanisms involved in IL-1 release, and the intracellular signalling pathways involved in IL-1 up-regulation during inflammatory stress.

2. This study was based on the human colonic epithelial cell line HT29-Cl.16E, maintained as polarized monolayers on filters mounted in culture chambers and treated with various proinflammatory cytokines (interferon γ (IFNγ), tumour necrosis factor α (TNFα), IL-1β) alone or in combination.

3. IL-1 production, restricted to IL-1α, was induced by the combination of IFNγ/TNFα. When IL-1β was added to IFNγ/TNFα, it led to an additional production of IL-1α. IL-1α release was associated with cell damage, as shown by the correlation between lactate dehydrogenase (LDH) release and extracellular IL-1 production, and was not accounted for by a secretory mechanism.

4. Both IFNγ/TNFα and IFNγ/TNFα/IL-1β induced inducible nitric oxide synthase (iNOS) expression as shown by quantitation of NO₂⁻/NO₃⁻ by use of the Griess reagent, quantitation of cells scoring positive with an anti-iNOS antibody and detection of mRNAs coding for iNOS by RT–PCR. The use of N^G-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS, led to the demonstration of two distinct signalling pathways in IL-1 production by HT29-Cl.16E cells, one dependent on NO (L-NMMA-sensitive) under treatment with IFNγ/TNFα/IL-1β, and the other independent of NO (L-NMMA-insensitive) under treatment with IFNγ/TNFα.

5. Moreover, we examined whether a redox-based mechanism could be responsible for the apparent discrepancy between NO production and NO implication in IL-1 production under IFNγ/TNFα and IFNγ/TNFα/IL-1β treatments. Experiments with cysteine, which acts as a powerful reductant, suggest that the nitrosonium character of NO is involved in the NO-dependent pathway in IL-1 production.

Full Text

The Full Text of this article is available as a PDF (387.7 KB).