Figure 5.

Tumour necrosis factor (TNF) induces interleukin (IL)−33 expression in a hypoxia inducible factor‐1α (HIF‐1α)‐dependent manner. (a) HT‐29 cells were treated with TNF (10 ng/ml), TNF (10 ng/ml) and YC‐1 (10 μM) or vehicle for 24 h, and the levels of IL‐33 mRNA were determined by quantitative real‐time–polymerase chain reaction (qRT–PCR). ^* P < 0·05. (b) HT‐29 cells transfected with si‐HIF‐1α or si‐control (50 nM) for 48 h were then stimulated with TNF (10 ng/ml) for 24 h, and IL‐33 mRNA was determined by qRT–PCR and normalized to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) expression. ^** P < 0·01. Representative results from three independent experiments are shown. (c) HT‐29 cells were first treated with or without YC‐1 (10 μM) for 24 h, and then stimulated with TNF (10 ng/ml) for 0, 5, 15, 30 and 60 min, respectively, the activation of extracellular‐regulated kinase (ERK) and p‐38 pathway was determined by Western blot. (d) Relative expression of IL‐33 mRNA in colonic tissues from the same Crohn's disease (CD) patients (n = 10) before and after anti‐TNF monoclonal antibody (mAb) treatment [infliximab (IFX), 5 mg/kg]. GAPDH was used as a housekeeping gene. ^* P < 0·05. (e,f) Freshly isolated colonic biopsies from patients with CD (n = 10) (e) or ulcerative colitis (UC) (n = 10) (f) were incubated in the presence of IFX (50 μg/ml) or control human IgG (HIg, 50 μg/ml) for 24 h. The levels of IL‐33 mRNA were analysed by qRT–PCR and normalized to GAPDH. ^** P < 0·01; ^*** P < 0·001. Data are compiled from three independent experiments.