312 Discussion In addition to its many homeostatic functions such as barrier protection, mucus production and resorption, the intestinal epithelium plays an important role in regulating Int J Colorectal Dis (2008) 23:305–317 Fig. 5 Transient transfection analysis of TGL-IP10 in IECs. The CXCL10 promoter containing reporter construct, pTGL-IP10 and pRL-TK (renilla) plasmids, was transiently transfected into a Caco-2, b HT29 and c DLD1 cells. 24 h after transfection, cells were stimulated with cytokines as indicated for 24 h, harvested, and promoter activity was monitored by luciferase assay. Data were normalised with renilla luciferase values and included as fold increase vs control. Data represent the means ± SEM of three to five independent experiments done in duplicates for each sample. Asterisk indicates an induction significantly different (p<0.05) from control (C). Significant differences between combinations of cytokines and the respective single cytokines are indicated with bars with a number sign (p<0.05). N.S. is not significant local inflammation and immune responses. The intestinal epithelium participates in inflammatory responses in part through the generation of numerous cytokines and chemo- kines that mediate recruitment and activation of inflamma- tory cells. The epithelium is involved in local cytokine networks allowing first response to noxious agents as well as cross-talk with immune cells to help provide an effective inflammatory response. These bi-directional inflammatory communications are necessary for normal host defence, but they also play an important role in the pathogenesis of IBD and might be also of importance in modulating anti- tumoural immunity in colonic malignancies. In this report, we demonstrate that pro-inflammatory cytokines, which may be produced by sub-mucosal immune cells, can differentially and co-ordinately regulate CXCL10 gene expression in IECs. CXCL10 is one of several chemokines produced by IEC. Previous studies could show that IFN-γ in combination with TNF-α caused induction of RANTES (regulated upon activation, normal t-cell expressed, and secreted protein) and monocyte chemotactic protein-1 in colorectal tumour cell lines, while IFN-γ had no effect on CXCL8 (IL-8) secretion [27], the latter observation being confirmed in all three cell lines in the present study (data not shown). Dwinell et al. [12] had previously shown that stimulation of IECs with TNF-α or IL-1α strongly potentiated IFN-γ- induced CXCL10. It is well known that among IL-1α and IL-1β, the latter is secreted and has an active role in inflammation. Importantly, IL-1β was shown to be over- expressed in IBD patients [25, 28]. This makes IL-1β an Fig. 6 Role of NF-κB in cytokine-induced CXCL10 mRNA expression by IECs. 5×105 cells were plated and grown for 24 h. Then, the cells were pre-treated with 10 μM of BAY 11-7082 for 1 h and then stimulated with respective cytokines and their combinations as stated in the figure for 2 and 24 h. After 2 h, cells were harvested for RNA isolation to perform real-time PCRs, and at 24 h, cell supernatants were collected and stored at −70°C until ELISA was performed for CXCL10. Data presented are the means±SEM of three independent experiments done in duplicates for each sample in a Caco-2, b HT29 and c DLD1 cells. Significant CXCL10 inhibition by BAY 11-7082 is indicated by an asterisk (p< 0.05). N.S. is not significant