308 Diego, CA). Comparisons between two or more different treatment groups were made with the non-paired t test or analysis of variance (ANOVA) where appropriate. For time kinetics experiments, a two-way ANOVA test followed by the Bonferroni post-test was performed. In case of RNA expression, a log transformation was performed before. Statistical differences were regarded significant at a p level below 0.05. Data were expressed as means±standard error of the mean (SEM). Results Differential expression of CXCL10 mRNA in intestinal epithelial cell lines under basal conditions In the present study, the basal mRNA expression of CXCL10 in three different IECs, Caco-2, HT29 and DLD1 cells, were studied first. Real-time PCR experiments using gene-specific primers for CXCL10 revealed that the basal mRNA expression of CXCL10 was highest in Caco-2 followed by HT29 and DLD1 (Fig. 1). IL-1β, TNF-α and IFN-γ dose-dependent influence on CXCL10 gene expression in IECs Caco-2, HT29 and DLD1 cells were tested for their ability to respond to pro-inflammatory cytokines by the Fig. 1 Basal level expression of CXCL10 in IECs. Caco-2, HT29 and DLD1 cells were plated at a density of 5×105 cells in six well plates, grown for 24 h and total RNA from the cells was isolated. First-strand complementary DNA was prepared from 1 μg of total RNA. Real-time PCR was performed for CXCL10 and β-actin was used as an internal control. The bars indicate the mean of six individual experiments. Asterisk corresponds to p<0.01 Int J Colorectal Dis (2008) 23:305–317 induction of CXCL10 mRNA expression. Cells were stimulated for 4 h with various concentrations of cytokines, and CXCL10 expression was analysed by real-time PCR. In response to IFN-γ and TNF-α, Caco- 2, HT29 and DLD1 cells showed a dose-dependent induction of CXCL10 mRNA (Fig. 2). Increased expres- sion of CXCL10 mRNA was observed when 2 ng/ml IL- 1β was used, while a higher concentration did not further enhance expression. Further, we checked the response of IECs to lower doses of IL-1β showing a dose response in the range of 0.1 to 1 ng/ml in the induction of CXCL10 mRNA. IL-1β at a concentration of 0.5 ng/ml was able to induce maximum expression of CXCL10 mRNA at 4 h (data not shown). IL-1β, TNF-α and IFN-γ time-dependent influence on CXCL10 gene expression in IECs Once we confirmed the responsiveness of IECs to pro- inflammatory cytokines, we next examined the mRNA kinetics of CXCL10 induced by the cytokines alone or in combination with each other. Each cell line had a different time course of response when stimulated with respective cytokines and their combinations (Fig. 3a–c). In Caco-2, stimulated with IL-1β or TNF-α, CXCL10 mRNA was induced within the first 40 min, and the induction reached to a peak after 4 h, while IFN-γ had a later initial effect inducing CXCL10 mRNA after 2 h and reached to a maximum at 8 h. Combination of IL-1β + IFN-γ had a stronger synergistic effect in the induction of CXCL10 mRNA within 20 min and reaching to maximum after 4 h, whereas TNF-α + IFN-γ synergistically led to a later induction of CXCL10 mRNA starting at 40 min but had a strong synergistic effect after 8 h when compared to IL- 1β + IFN-γ (Fig. 3a). In HT29 (Fig. 3b) and DLD1 (Fig. 3c), CXCL10 mRNA time kinetics deviated from that in Caco-2 (Fig. 3a). CXCL10 mRNA was induced after 1 h of stimulation with the respective single cytokines in DLD1. In case of HT29, either TNF-α or IFN-γ alone led to delayed induction of CXCL10 mRNA after 1 h, whereas IL-1β alone led to an induction starting at 2 h. HT29 and DLD1 cells treated with combinations of IL-1β + IFN-γ showed a strong synergistic induction of CXCL10 mRNA within 40 min, whereas the TNF-α + IFN-γ combination led to an even stronger induction of CXCL10 mRNA within 20 min in HT29 and within 40 min in DLD1. IL-1β + TNFα in combination had no synergistic effect on CXCL10 mRNA induction in all the cell lines used for this study. The real-time PCR results could be confirmed by Northern blot analysis in Caco-2 and HT29 cells. However, the detection threshold was not low enough to display basal and single cytokine-induced CXCL10 mRNA (data not shown).