Int J Colorectal Dis (2008) 23:305–317 309 Fig. 2 Dose-dependent expression of CXCL10 mRNA in IECs 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 at an increasing doses of 2, 10 and 50 ng/ml. The cells were harvested after 4 h, total RNA from the cells was isolated, and first- strand cDNA was prepared from 1 μg of total RNA. Real-time PCR IL-1β, TNF-α and IFN-γ time-dependent influence on CXCL10 protein secretion in IECs To test whether cytokine stimulation of IECs would lead not only to the induction of CXCL10 mRNA but also to the synthesis and secretion of the protein, Caco-2, HT29 and DLD1 cells were incubated with respective cytokines (from 3 to 24 h), and secretion of CXCL10 was detected by ELISA. Under basal conditions, Caco-2 cells secreted 0.012 ± 0.003 ng/ml CXCL10. Treatment with IL-1β, TNF-α and IFN-γ led to a time-dependent increase in CXCL10 protein, with IL-1β being the most potent stimulus during the first 6 h (Fig. 4a). Of importance, after 12 h, CXCL10 secretion was stimulated strongly by IFN-γ. Co-incubation of Caco-2 with respective cytokines revealed a synergistic effect of IL-1β +IFN-γ and TNF-α+ IFN-γ on CXCL10 secretion, reaching levels of 3.354 ±0.690 and 7.808.1 ±0.173 ng/ml after 24 h. The combination of IL-1β+ TNF-α had no synergistic effect on CXCL10 secretion when compared to the respective cytokines alone. These time kinetic studies revealed that in Caco-2, IL-1β alone or in combination with IFN-γ induced the secretion of CXCL10 very early compared to the combination of TNF-α+ IFN-γ, which induced a delayed but stronger synergistic secretion of CXCL10 protein, suggesting that in case of IL-1β+ IFN-γ, a synergism could take place very early, e.g., at the level of intracellular signal cascades. In HT29 cultures, CXCL10 secretion was not detectable under basal condition. TNF-α or IFN-γ alone led to an induction of CXCL10 protein secretion within 3 h (0.0172±0.005 and 0.011±0.001 ng/ml), whereas IL-1β could not induce the secretion of CXCL10 protein. IL-1β, TNF-α and IFN-γ led to a time-dependent increase in CXCL10 concentrations in HT29 cell supernatants reaching maximum levels of 0.039± 0.0057, 0.632±0.0222 and 19.929±0.838 ng/ml showing was performed for CXCL10, and β-actin was used as an internal control. Graph represents the mean±SEM of four individual experi- ments. All cytokine-stimulated expressions were significantly different from unstimulated controls (p<0.05). Asterisk, no significant differ- ence between different IL-1β concentrations (p<0.05) that IFN-γ is the strongest inducer of CXCL10 protein secretion when compared to other two cytokines in this cell line (Fig. 4b). The combinations of TNFα+IFN-γ and IL- 1β+IFN-γ induced CXCL10 protein secretion to 1.822± 0.109 and 0.410±0.027 ng/ml within 3 h and led to a maximum of 492.728±83.776 and 87.158±7.913 ng/ml after 24 h (Fig. 4b). CXCL10 promoter activation by cytokines TGL-IP10, a promoter-reporter containing 435 bp of human CXCL10 sequence upstream of the transcriptional start site, was used to evaluate the role of IL-1β, TNF-α and IFN-γ alone or in combination with each other in the transcription of CXCL10. When TGL-IP10-transfected Caco-2 cells were stimulated with single cytokines, IL-1β (1 ng/ml) led to a stronger induction (6.53±1.65-fold increase) than TNF-α (50 ng/ml) and IFN-γ (50 ng/ml) alone (2.76±0.53 and 3.14± 1.2-fold increase). The combination of IL-1β+IFN-γ showed a stronger synergistic induction (26.18 ±11.68) than the combination of TNF-α+IFN-γ (13.97±3.25-fold increase) (Fig. 5a). In HT29 cells, when stimulated with single cytokines, IFN-γ (50 ng/ml) led to a maximum induction (6.52±0.58-fold increase), followed by TNF-α (4.44±0.16- fold increase) and IL-1β (1.43±0.05-fold increase). The combination of IL-1β+IFN-γ synergistically led to 12.76± 3.21-fold induction, and TNF-α+IFN-γ led to 26.70±7.5- fold induction of promoter activity (Fig. 5b). In DLD1 cells (Fig. 5c), IFN-γ (50 ng/ml) led to a maximum induction (8.06±0.51-fold increase) of CXCL10 promoter activity followed by TNF-α (50 ng/ml) induction (3.59±0.08-fold increase) and IL-1β (1 ng/ml) induction (2.32±0.12-fold increase). The combination of IL-1β+IFN-γ led to a 40.72± 12.19-fold induction, and TNF-α+IFN-γ led to a 20.56± 6.27 fold induction. In all three cell lines, the combination of