Int J Colorectal Dis (2008) 23:305–317 315 Fig. 8 Gel shift and super shift analysis for NF-κB. Caco-2 cells were seeded and grown for 24 h before pre-treating with BAY 11-7082 (10 μM) for 1 h and then stimulated with respective cytokines for 30 min; cells were then harvested, and nuclear extracts were prepared. in a variety of cell types and have demonstrated that this synergy depends on the co-existence of TNF-α-responsive NF-κB-binding sites and IFN-γ-responsive signal trans- ducers and activators of transcription (STAT) protein- binding elements within the promoters of the genes of interest [35, 36]. In our study, when IFN-γ and TNF-α were combined, the accumulation of CXCL10 mRNA and protein was elevated compared to unstimulated cells or cells stimulated with either cytokine alone, implying a synergistic effect on transcription of the CXCL10 gene. A synergistic effect by these two cytokines on CXCL10 expression was also demonstrated in astrocytes, and this synergy has been shown to be at least in part dependent on NF-κB activation and binding to a NF-κB-binding site within the CXCL10 promoter [26]. The synergistic action of IL-1β with IFN-γ in IECs has not been characterised so far. Recently, Takami et al. [39] showed the synergistic induction of hepatocyte growth factor in human skin fibroblasts by IL-1β+ IFN-γ+ IFN-γ [39]. We here report for the first time that in intestinal epithelial cell lines, IL-1β alone or in combination with IFN-γ induced CXCL10 gene expression. In Caco-2 cells, the combination led to an early synergistic induction of both CXCL10 mRNA and protein compared to the combination of IFN-γ+TNF-α. This early induction of CXCL10 mRNA and protein by IL-1β+IFN-γ in Caco-2 might be due to the early convergence of signalling pathways that activates transcription factors like NF-κB, IRF-1, STAT1 or other factors that mediate transcription factor interactions. Transient transfections with human CXCL10 promoter [26], in Caco-2, HT29 and DLD1 cells, showed that the synergistic effect of IL-1β+ IFN-γ and IFN-γ+ TNF-α on 5 μg of nuclear extracts were used for gel shifts with a wild-type κB oligonucleotide. Super shifts were performed with anti-p65 antibody. Similar results were obtained in two separate experiments CXCL10 gene expression in these cell lines was indeed because of the transcriptional synergy employed at the level of the CXCL10 promoter. A number of studies had revealed that cytokines like IL-1β, TNF-α or enteroinva- sive bacteria and the bacterial cell wall product lipopoly- saccharide induce the expression of several inflammatory genes through the transcription factor NF-κB in IECs [40– 43]. To find out the signalling mechanism involved in CXCL10 gene expression in IECs, we explored the role of transcription factor NF-κB. Inhibition of NF-κB with BAY11-7082 clearly inhibited both CXCL10 mRNA and protein induced by single cytokine alone or in combinations in Caco-2, HT29 and DLD1 cells. Gel shift in Caco-2 could confirm that NF-κB is activated and binds to consensus κB oligonucleotides in cells treated with either IL-1β or TNF- α alone and in combination with IFN-γ. It was shown previously that the binding complex of NF-κB to the κB2- binding site of the CXCL10 promoter contains the homodimer of p65 [26]. In our study, super shift assays in Caco-2 cells treated with IL-1β alone or in combination with IFN-γ confirmed the presence of p65 in the complexes bound to the consensus κB oligo nucleotides. In addition to these experiments, transient transfection experiments with a κB2 mutant of the CXCL10 promoter [26] clearly showed that in Caco-2 cells, none of the cytokines and their combinations were able to induce the CXCL10 promoter activity. In HT29 cells, the combination of IFN-γ+TNF-α led to a weaker induction of TGL-IP10κB-2 mutant activity, whereas in DLD1cells, the combination of IL-1β+IFN-γ and IFN-γ+TNF-α led to a weak induction of TGL-IP10 κB-2 mutant activity. Based on these data, we can conclude that NF-κB plays a critical role in CXCL10 gene expression