Volume 129, Issue 3, Pages 969-984 (September 2005) Interleukin-22, a Member of the IL-10 Subfamily, Induces Inflammatory Responses in Colonic Subepithelial Myofibroblasts  Akira Andoh, Zhuobin Zhang, Osamu Inatomi, Sanae Fujino, Yasuyuki Deguchi, Yoshio Araki, Tomoyuki Tsujikawa, Katsuyuki Kitoh, Shokei Kim–Mitsuyama, Atsushi Takayanagi, Nobuyoshi Shimizu, Yoshihide Fujiyama  Gastroenterology  Volume 129, Issue 3, Pages 969-984 (September 2005) DOI: 10.1053/j.gastro.2005.06.071 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Immunohistochemical analyses of interleukin (IL)-22 expression in the colon. (A-a) Control normal mucosa (×200), (b) ulcerative colitis (×200), and (c) Crohn’s disease (×200). (B) Dual-colored immunofluorescence was used to determine CD3, CD4, and CD8 expression (FITC, green fluorescence; d, e, or f), IL-22 expression (rhodamine-red, red fluorescence; g, h, and i), and merged (CD3 plus IL-22 [j], CD4 plus IL-22 [k], CD8 plus IL-22 [l], yellow fluorescence). (C) Dual-colored immunofluorescence was used to determine α-smooth muscle actin (α-SMA; FITC, m) and IL-22R1 (rhodamine-red, red fluorescence, n) in normal human colonic mucosa. Double-positive staining can be seen as yellow fluorescence in merged panel (o). Specificity of IL-22R1 immunoreactivity was confirmed by using anti-IL-22R1 antibodies treated with blocking peptide (p). Gastroenterology 2005 129, 969-984DOI: (10.1053/j.gastro.2005.06.071) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Number of IL-22-positive cells in the mucosa. Results were expressed as number of positive cells per field (×400). The lower and upper margins of the box represent the 25th and 75th percentiles, with the extended arms representing the 10th and 90th percentiles, respectively. The median is shown as a horizontal line within the box. Gastroenterology 2005 129, 969-984DOI: (10.1053/j.gastro.2005.06.071) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Effects of IL-22 on [3H]-thymidine incorporation, collagen, and MMP-1 secretion. (A) SEMFs were stimulated for 24 hours, and the [3H]-thymidine incorporation was determined. Each factor was used at 200 ng/mL. Values are expressed as means ± SD (n = 5). **P < .01. (B) SEMFs were incubated with each factor for 24 hours, and collagen secretion was analyzed by SDS-PAGE and Western blotting. (C) SEMFs were stimulated with IL-22 (200 ng/mL) for 24 hours, and secreted MMP-1 levels were determined by ELISA. Values are expressed as mean ± SD (n = 5). **P < .01. (D) SEMFs were stimulated with IL-22 (200 ng/mL) for 24 hours, and secreted MMP-3 was analyzed by Western blotting. Gastroenterology 2005 129, 969-984DOI: (10.1053/j.gastro.2005.06.071) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 (A) Effects of IL-22 on the expression of IL-6, IL-8, IL-11, and LIF mRNA and protein in human colonic subepithelial myofibroblasts (SEMFs). Cells were incubated for 6 hours with 200 ng/mL of IL-22. Total RNA was extracted, and expression of IL-6, IL-8, IL-11, and LIF mRNA was determined by Northern blots. (B and C) Cells were incubated for 24 hours with increasing concentrations of IL-22. The secreted cytokine levels were determined by ELISA. Values are expressed as mean ± SD (n = 5). *P < .05, **P < .01, a significant difference from the values of medium alone. Gastroenterology 2005 129, 969-984DOI: (10.1053/j.gastro.2005.06.071) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 (A) RT-PCR analyses for mRNA expressions of IL-10R2 and IL-22R1 in colonic subepithelial myofibroblasts (SEMFs). HT-29 colon cancer cells were used as positive controls. (B) Effects on IL-22 on IL-6 and IL-8 promoter activities. Human IL-6 and IL-8 promoter DNA and β-galactosidase reporter vectors were cotransfected to colonic SEMFs and incubated for 24 hours. Next, cells were incubated with IL-22 (200 ng/mL) for 6 hours. Luciferase activites were measured by the Luciferase Assay System Kit (Promega, Madison, WI) and expressed as relative activities normalized to β-galactosidase activities. Values are expressed as mean ± SD (n = 5). **P < .01, a significant difference from the values of medium alone. (C) Electrophoretic gel mobility shift assays (EMSAs) for NF-κB and AP-1 DNA-binding activities. Cells were incubated with medium alone, IL-22 (200 ng/mL) for 1 hour, and then nuclear extracts were prepared. The dashed arrow indicates nonspecific DNA-protein bound complexes. Gastroenterology 2005 129, 969-984DOI: (10.1053/j.gastro.2005.06.071) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 (A) Western blots for IκBα and c-Jun expression. Colonic subepithelial myofibroblasts (SEMFs) were infected with an adenovirus expressing the IκBΔN or DN-c-Jun and then incubated for 48 hours. Expression of IκBα or c-Jun protein was assessed by Western blots. (B) Forty-eight hours after infection with the adenovirus, cells were stimulated with IL-22 (200 ng/mL) for 6 hours. Cytokine mRNA expression was then determined by Northern blots. (C and D) Forty-eight hours after infection with the adenovirus, cells were stimulated with IL-22 (200 ng/mL) for 24 hours, and secreted cytokine levels were determined by ELISA. Values are expressed as mean ± SD (n = 5). *P< .05, **P < .01, a significant difference from the values of IL-22 stimulation. Gastroenterology 2005 129, 969-984DOI: (10.1053/j.gastro.2005.06.071) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 MAP-kinase and JAK/STAT activation and the effect of inhibitors on cytokine secretion. (A) Colonic subepithelial myofibroblasts (SEMFs) were stimulated with IL-22 (200 ng/mL), and the activation of MAP kinases and JAK/STAT were evaluated by Western blots. Antibodies directed against phosphorylated (p)- and total-MAP kinase and JAK1/STAT3 were used. (B and C) Cells were pretreated with 20 μmol/L MAP kinase inhibitors (SB203580, PD098059, or U02016) and JAK2 inhibitor (AG490) for 15 minutes. Cells were stimulated with IL-22 (200 ng/mL) for 24 hours, and cytokine levels in supernatants were determined by ELISA. Values are expressed as mean ± SD (n=5). *P < .05, **P < .01, a significant difference from the values of IL-22 stimulation. Gastroenterology 2005 129, 969-984DOI: (10.1053/j.gastro.2005.06.071) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 The combined effects from IL-17 and IL-22. (A) EMSAs for NF-κB and AP-1 DNA-binding activities. Cells were incubated for 1 hour with medium alone, IL-22 (200 ng/mL), IL-17 (200 ng/mL), and IL-17 (200 ng/mL) plus IL-22 (200 ng/mL), and nuclear extracts were prepared. (B) Northern blots for cytokine mRNA expression. Cells were stimulated for 6 hours, and total RNA was extracted. (C and D) Cells were stimulated for 24 hours, and secreted cytokine levels were determined by ELISA. Values are expressed as mean ± SD (n = 5). *P < .05, **P < .01, a significant difference from IL-17 stimulation; and +P < .05, ++P < .01, a significant difference from IL-22 stimulation. Gastroenterology 2005 129, 969-984DOI: (10.1053/j.gastro.2005.06.071) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 Effects from members of the IL-10 subfamily. (A and B) Colonic subepithelial myofibroblasts (SEMFs) were stimulated for 24 hours with 200 ng/mL of each cytokine, and secreted IL-6, IL-8, IL-11, and LIF levels were determined by ELISA. Values are expressed as mean ± SD (n = 5). *P < .05, **P < .01, a significant difference from the values of medium alone. (C) The combined effects of IL-19 and IL-22. Cells were stimulated for 6 hours with IL-19 (200 ng/mL), IL-22 (200 ng/mL), or IL-19 plus IL-22, and mRNA expression for IL-6, IL-8, and LIF was evaluated by Northern blots. (D) The combined effects of IL-19 and IL-22 on cytokine secretion. Cells were stimulated for 24 hours, and secreted cytokine levels were determined by ELISA. Values are expressed as mean ± SD (n = 5). *P < .05, **P < .01, a significant difference from IL-17 stimulation; and +P < .05, ++P < .01, a significant difference from IL-22 stimulation. Gastroenterology 2005 129, 969-984DOI: (10.1053/j.gastro.2005.06.071) Copyright © 2005 American Gastroenterological Association Terms and Conditions