Introduction 1 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 12 22 23 24 25 Materials and methods Biological reagents The recombinant cytokines IL-1β, TNF-α and IFN-γ were purchased from Roche applied science (Mannheim, Germany). An enzyme-linked immunosorbent assay (ELISA) kit for CXCL10 was purchased from RnD systems (Weisbaden, Germany). The nuclear factor (NF)-κB inhibitor BAY11-7082 was purchased from Calbiochem (San Diego, CA). Cell culture and stimulation protocols 2 2 5 RNA isolation, real-time PCR Ribonucleic acid (RNA) was isolated by using Qiagen RNeasy mini kit according to the manufacturer’s protocol. The RNA concentrations were determined photometrically using a Gene Quant RNA/deoxyribonucleic acid (DNA) calculator (Pharmacia, Freiburg, Germany). RNA was subsequently used for real-time PCR and Northern blot analysis. Reverse transcription of messenger RNA (mRNA) was performed using 1 μg of total cellular RNA. To determine the mRNA expression of CXCL10, real-time PCR was carried out using gene-specific primers for human CXCL10 (Invitrogen GmbH, Karlsruhe) forward 5′-CCA GAA TCG AAG GCC ATC AA-3′, reverse 5′-CAT TTC CTT GCT AAC TGC TTT CAG-3′ and β-actin (Invitrogen GmbH) forward 5′-CTG GCA CCC AGC ACA ATG-3′, reverse 5′-CCG ATC CAC ACG GAG TAC TTG-3′ in an ABI Prism 7000 sequence detection system. PCR reaction was set up with Sybr® Green PCR Master mix (Invitrogen) containing 0.3 μmol/l primers each and 1 μl of RT-product in 25 μl volume. A two-step amplification protocol was chosen consisting of initial denaturation at 95°C for 10 min followed by 45 cycles with 15 s denaturation at 95°C and 30 s annealing/extension at 60°C. Finally, a dissociation protocol was performed to control specificity of amplification products. \documentclass[12pt]{minimal}
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\begin{document}$${{{\rm{CXCL10}}} \mathord{\left/ {\vphantom {{{\rm{CXCL10}}} {{\rm{\beta  - actin}}}}} \right. \kern-\nulldelimiterspace} {{\rm{\beta  - actin}}}}\;{\rm{ratio}} = 2^{\Delta {\rm{CT}}} $$\end{document} Transient transfections 26 Luciferase assays Twenty-four hours after transfection, cells were stimulated with IL-1β (0.1,1, 2, 10 and 50 ng/ml), TNF-α (2, 10 and 50 ng/ml), IFN-γ (2, 10 and 50 ng/ml), IL-1β (1 ng/ml) + TNFα (50 ng/ml), IL-1β (1 ng/ml) + IFN-γ (50 ng/ml) and TNFα (50 ng/ml) + IFN-γ (50 ng/ml) for 24 h, harvested and lysed, and luciferase was assayed by using a luciferase kit (Promega). Values were normalised to renilla luciferase. Enzyme-linked immunosorbent assay To study the synergistic effects of cytokines on CXCL10 secretion, supernatants were collected at 0, 3, 6, 12 and 24 h in Caco-2 and HT29 cells stimulated with IL-1β (1 ng/ml), TNF-α (50 ng/ml), IFN-γ (50 ng/ml), IL-1β (0.5 ng/ml) + TNFα (50 ng/ml), IL-1β (0.5 ng/ml) + IFN-γ (50 ng/ml) and TNFα (50 ng/ml) + IFN-γ (50 ng/ml) and stored at −70°C until measured. CXCL10 protein secretion was measured by ELISA according to the manufacturer’s protocol. Preparation of nuclear extract g Electrophoretic mobility shift assay and super-shift assay 32 4 Statistical analysis t p Results Differential expression of CXCL10 mRNA in intestinal epithelial cell lines under basal conditions 1 Fig. 1 5 bars Asterisk p  IL-1β, TNF-α and IFN-γ dose-dependent influence on CXCL10 gene expression in IECs 2 Fig. 2 5 p Asterisk p  IL-1β, TNF-α and IFN-γ time-dependent influence on CXCL10 gene expression in IECs 3 3 3 3 3 Fig. 3 a b c 5 p N.S. Number sign p  IL-1β, TNF-α and IFN-γ time-dependent influence on CXCL10 protein secretion in IECs 4 4 4 Fig. 4 5 a b Asterisk p  CXCL10 promoter activation by cytokines 5 5 5 Fig. 5 a b c Asterisk p C bars number sign p N.S.  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. 6 6 Fig. 6 5 a b c asterisk p N.S.  7 7 Fig. 7 a b c C asterisk p number sign p N.S.  8 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. 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  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 local inflammation and immune responses. The intestinal epithelium participates in inflammatory responses in part through the generation of numerous cytokines and chemokines that mediate recruitment and activation of inflammatory 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. 27 12 25 28 26 29 12 16 30 31 32 33 34 17 35 38 35 36 26 39 39 26 40 43 26 26 44 45 46 Taken together, our data suggest that while IFN-γ, TNF-α and IL-1β may work individually through disparate signalling pathways in CXCL10 gene regulation, a combination of these cytokines enhances transcription of the CXCL10 gene in an NF-κB-dependent manner. The fact that IL-1β via activation of NF-κB is able to mimic the effect of TNF-α in augmenting CXCL10 expression might be of importance in patients with IBD, who do not respond to anti TNF-α antibody therapy. In those cases, an additional inhibition of IL-1β might be desirable as a future therapy option in treating IBD.