NF-kappa B-dependent synergistic regulation of CXCL10 gene expression by IL-1 beta and IFN-gamma in human intestinal epithelial cell lines

Abstract

Background and aims Little is known about the intestinal epithelial expression and secretion of CXCL10 (IP-10), a chemokine involved in recruiting T cells and monocytes. We aimed to study CXCL10 gene expression and regulation by the pro-inflammatory cytokines interleukin (IL)-1 beta, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha in intestinal epithelial cell lines. Materials and methods CXCL10 expression and secretion kinetics were assessed in Caco-2, HT-29 and DLD1 human colon epithelial cells, treated with IL-1 beta, TNF-alpha, IFN-gamma alone or in combination with each other by real-time polymerase chain reaction (PCR), Northern blotting and enzyme-linked immunoabsorbent assay (ELISA). Transient transfections with TGL-IP10 (CXCL10 promoter) and TGL-IP10-kappa B2 mutant promoter and gelshifts and supershifts for nuclear factor (NF)-kappa B were also performed. Results Real-time PCRs and ELISA experiments revealed that IL-1 beta was the strongest and earliest inducer of CXCL10 messenger ribonucleic acid (mRNA) expression and protein secretion in Caco-2 cell line, whereas INF-gamma had a delayed kinetics. There was a strong synergistic effect of either TNF-alpha or IL-1 beta with IFN-gamma both on CXCL10 mRNA expression and protein secretion in all three cell lines. Real-time PCR and ELISA experiments using a specific NF-kappa B inhibitor and transfection experiments with a NF-kappa B-binding defective CXCL10 promoter construct revealed that the induction of CXCL10 by IL-1 beta and its synergism with IFN-gamma is NF-kappa B dependent. Conclusion These data demonstrate that in colonic epithelial cells, depending on the cellular context and utilizing the NF-kappa B pathway, IL-1 beta alone and/or in synergism with IFN-gamma may play a major role in the induction of CXCL10.