Acquired interferon gamma responsiveness during Caco-2 cell differentiation: effects on iNOS gene expression

Background-Impairment of intestinal barrier function occurs under a variety of inflammatory conditions and is mediated at least in part by interferon gamma (IFN-gamma) induced nitric oxide (NO) production. Previous in vivo st udies have shown that systemic lipopolysaccharide treatment caused an induc tion of the rat inducible nitric oxide synthase (iNOS) mRNA primarily in vi llus cells, rather than in undifferentiated crypt cells. Aims-To examine iNOS induction by IFN-gamma in vitro as a function of enter ocyte differentiation. Methods-Preconfluent and postconfluent Caco-2 cells were treated with IFN-g amma in the presence or absence of various inhibitors. Northern analyses we re performed to assess the magnitude of iNOS mRNA induction. IFN-gamma rece ptor mRNA and protein levels were determined. Results-iNOS mRNA induction by IFN-gamma occurred at two hours and was not blocked by cycloheximide, indicating that it is an immediate early response . iNOS induction and nitrite/nitrate increases were inhibited by dexamethas one and pyrrolidine dithiocarbamate, supporting an important role for the N F-kappa B transcription factor in this process. The stimulated iNOS inducti on was seen almost exclusively under conditions of cellular differentiation -that is, in postconfluent Caco-2 cells. This increased IFN-gamma responsiv eness seen in postconfluent Caco-2 cells correlated with an increased expre ssion of IFN-gamma receptor, whereas T84 and HT-29 cells did not show any s ignificant alterations in either iNOS induction or IFN-gamma receptor level s as a function of postconfluent growth. Conclusions-With regard to iNOS mRNA induction, IFN-gamma responsiveness is acquired during Caco-2 cell differentiation, perhaps related to an increas e in the numbers of IFN-gamma receptors.