Int J Colorectal Dis (2008) 23:305–317 Fig. 3 Time kinetics of CXCL10 mRNA expression in IECs stimulated with cytokines. a Caco-2, b HT29 and c DLD1 cells were plated at a density of 5×105 cells in six-well plates and grown for 24 h and then stimulated with respective cytokines and their combinations. Cells were harvested at different time points as indicated, and total RNA from the cells was isolated and first-strand cDNA was prepared from 1 μg of total RNA. Real-time PCR was performed for CXCL10, and β-actin was used as an internal control. Graphs represent the mean±SEM of three to five experiments. IFN-γ led to a significant increase in CXCL10 expression from 2 h onwards. A significant increase (p<0.05) from control was observed in all expect in those marked as N.S. (non-significant). Number sign indicates time points with significant differences (p<0.05) of cytokine combinations or IL-1β compared to IFN-γ alone IL-1β + TNF-α had no synergistic effect on CXCL10 promoter activity. Evaluating the role of NF-κB in CXCL10 gene expression To evaluate the possible role of NF-κB in cytokine-induced CXCL10 gene expression, experiments with a specific NF- κB inhibitor were performed. The effect of BAY 11-7082 on NF-κB inhibition was evaluated by reporter assays. BAY11-7082 was able to dose-dependently inhibit IL-1β- induced pNF-κB-SEAP gene reporter activity in Caco-2 cells (data not shown). Based on this experiment, we could determine that 10 μM of BAY11-7082 was sufficient to inhibit the NF-κB activation. The induction of CXCL10 gene expression by cytokines was inhibited by addition of the NF-κB-specific inhibitor BAY11-7082 in Caco-2, HT29 and DLD1 cells, with a varying degree of inhibition in the individual cell lines. Inhibition of NF-κB led to almost complete inhibition (approximately 90–95% inhibition) of CXCL10 mRNA and protein secretion induced by cytokines in Caco-2 and 311 DLD1 cells (Fig. 6a and c). In DLD1 cells, IL-1β (1 ng/ml) did not have any inducing effect on CXCL10 secretion. In HT29, inhibition of NF-κB had approximately 60–75% inhibitory effect on cytokine-induced CXCL10 mRNA expression and secretion (Fig. 6b). To further evaluate the role of NF-κB, transient trans- fections were done with a CXCL10 promoter construct bearing a mutated NFkB-binding site (TGL-IP10 κB2 Mut). In Caco-2 cells, TGL-IP10 κB2 Mut was not responsive to any of the cytokines and their combinations, suggesting that CXCL10 gene induction is strongly dependent on NF-κB in this cell line (Fig. 7a). In HT29 and DLD1 cells, the combination of TNF-α + IFN-γ led to 6.49 ± 2.13 and 4.87 ± 1.43-fold increase, and the combination of IL-1β + IFN-γ led to 2.26 ± 0.63 and 4.75 ± 0.53-fold increase of the TGL- IP10 κB2 Mut promoter activity (Fig. 7b and c), revealing that a smaller portion of CXCL10 gene expression might be regulated independently from NF-κB. To confirm further our results from promoter and inhibitor studies, we did gel-shift and super shift assays to show that NF-κB is activated by cytokines and that the activated NF-κB complex contains the NF-κB functional sub-unit p65. In the nuclear extracts of Caco-2 cells, we could clearly see a shift in the samples treated with IL-1β, TNF-α alone or the combinations of IL-1β + IFN-γ and TNF-α + IFN-γ. The protein-DNA complex was efficiently competed by unlabelled κB oligonucleotides. There was no shift in the samples treated with the NF-κB inhibitor BAY11-7082, stating that the shift was indeed because of NF-κB binding. Furthermore, antibody super shift assay using anti-p65 antibody had clearly shown that the complex contains NF-κB functional sub-unit, p65 (Fig. 8). Fig. 4 Time kinetics of CXCL10 protein secretion in Caco-2 and HT29 cells stimulated with cytokines. 5 ×105 cells were plated into six-well plates and grown for 24 h and then stimulated with IL-1β, TNF-α and IFN-γ as indicated. Supernatants were collected at respective time points from a Caco-2 and b HT29 cells and ELISA for CXCL10 protein was performed. Data represent means of three to five experiments±SEM done in duplicates for each sample. Asterisk indicates time points with significant differences (p <0.05) of cytokine combinations compared to IFN-γ alone