Induction of IL-13 Triggers TGF-β₁-Dependent Tissue Fibrosis in Chronic 2,4,6-Trinitrobenzene Sulfonic Acid Colitis

Cytokine production of colonic LPMC at weekly time points during chronic TNBS colitis in BALB/c mice. LPMC were extracted from the lamina propria and cultured for 48 h in the presence of T cell or APC stimulants (see Materials and Methods). Cytokine concentrations in the culture supernatants were determined by cytokine-specific ELISA. Data shown are mean values ± SD from individual cultures of cells derived from mice in two separate experiments, each containing at least five mice per group; ∗, p < 0.05 and ∗∗, p < 0.01.

In situ cytokine production in the colonic mucosa of mice with chronic TNBS colitis measured at weekly time points. Total colonic protein was extracted from mice in the various groups and subjected to cytokine-specific ELISAs. Data shown are representative of two independent experiments each containing at least five mice per group. Data shown are mean values ± SD derived from at least five mice per group; ∗, p < 0.05 and ∗∗, p < 0.01.

IL-13 (a) and IL-4 (b) production by isolated colonic LPMC stimulated in vitro in the presence of anti-cytokine Abs. Cells were extracted from the lamina propria on day 42 of chronic TNBS colitis and cultured for 48 h in the presence of plate-bound anti-CD3 and soluble anti-CD28 as well as specific blocking Abs or control IgG. Cytokine concentrations in the supernatants were determined by ELISA. ∗, p < 0.05 and ∗∗, p < 0.01.

IL-13Rα₁ and IL-13Rα₂ expression and function in BALB/c mice during the course of chronic TNBS colitis. a, IL-13Rα₁ expression is constitutive whereas IL-13Rα₂ expression is induced on day 35 during chronic TNBS colitis. IL-13Rα₁ mRNA expression was determined by RT-PCR of RNA extracted from colonic LPMC, and IL-13Rα₂ protein expression was determined by Western blot analysis analyzed of colonic LPMC lysates. b, IL-13 production after administration of pCI-sIL-13Rα₂-Fc, IL-13Rα₂-specific siRNA, or anti-TGF-β₁-Ab. Cells were extracted from the lamina propria and cultured for 48 h in the presence of plate-bound anti-CD3 and soluble anti-CD28. Cytokine concentration was determined in culture supernatants by ELISA. Data shown are mean values ± SD for the pooled data derived from two separate experiments. Groups in each experiment contained at least five mice; ∗∗, p < 0.01. c, IL-13Rα₂ expression (but not IL-13Rα₁) is reduced in chronic TNBS after treatment with pCI-sIL-13Rα₂-Fc and IL-13Rα₂-specific siRNA. IL-13Rα₁ and IL-13Rα₂ expression were determined as described in a. d, DNA-binding activity of c-jun in nuclear extracts of cells derived from isolated lamina propria is reduced after administration of pCI-sIL-13Rα₂-Fc and IL-13Rα₂-specific siRNA but not anti-TGF-β₁-Ab. DNA-binding activity was determined in nuclear extracts from colonic lamina propria cells by the TransAM assay (see Materials and Methods). Data shown are mean values ± SD for the pooled data derived from three separate experiments. Groups in each experiment contained at least five mice; ∗∗, p < 0.01.

Body weight, collagen deposition, and histological scores of mice on day 49 of chronic TNBS colitis after administration of pCI-sIL-13Rα₂-Fc, IL-13Rα₂-specific siRNA, or anti-TGF-β₁ beginning on day 35 of chronic TNBS colitis. a, Body weight as a percentage of starting weight after administration of pCI-sIL-13Rα₂-Fc, IL-13Rα₂-specific siRNA, or anti-TGF-β₁-Ab (see text). Data shown are mean values ± SD derived from at least five mice per group. b, Masson’s trichrome staining of representative colon cross-sections during chronic TNBS colitis after administration of pCI-sIL-13Rα₂-Fc, IL-13Rα₂-specific siRNA, or anti-TGF-β₁-Ab (magnification, ×5). c, Histological scores after administration of pCI-sIL-13Rα₂-Fc or anti-TGF-β₁-Ab. Scores shown are mean values ± SD from at least five mice per group.

LPMC cytokine production of mice on day 49 of chronic TNBS colitis after administration of pCI-sIL-13Rα₂-Fc, IL-13Rα₂-specific siRNA, or anti-TGF-β₁ beginning on day 35 of chronic TNBS colitis. Cells were extracted from the lamina propria and cultured for 48 h in the presence of plate-bound anti-CD3 and soluble anti-CD28. Cytokine concentration was determined in culture supernatants by ELISA. Data shown are mean values ± SD from individual cultures of cells derived from mice in two separate experiments, each containing at least five mice per group. Note that the scales used in these panels are frequently different from those used in Fig. 2, due to the lower cytokine production at this time point.

In situ cytokine production in the colonic mucosa of mice with chronic TNBS colitis measured after administration of pCI-sIL-13Rα₂-Fc, IL-13Rα₂-specific siRNA, or anti-TGF-β₁-Ab. Total colonic protein was extracted from mice in the various groups and subjected to cytokine-specific ELISAs. Data shown are mean values ± SD derived from at least five mice per group; ∗∗, p < 0.01.

TGF-β₁ secretion by colonic lamina propria cells on day 49 of chronic TNBS colitis in BALB/c mice. a, TGF-β₁ secretion after treatment with pCI-sIL-13Rα₂-Fc, IL-13Rα₂-specific siRNA, or anti-TGF-β₁-Ab. Cells were extracted from the lamina propria and cultured for 48 h in the presence of stimulants (see Materials and Methods). Cytokine concentration was determined in supernatants by ELISA. Data shown are mean values ± SD from two separate experiments. Groups in each experiment contained at least five mice; ∗∗, p < 0.01. b, Collagen content of the colon after administration of pCI-sIL-13Rα₂-Fc, IL-13Rα₂-specific siRNA, or anti-TGF-β₁-Ab. Collagen content was determined during chronic TNBS colitis by a Sircol assay. Data shown are mean values ± SD derived from at least five mice per group; ∗∗, p < 0.01.