Volume 121, Issue 4, Pages 875-888 (October 2001) Macrophage-derived IL-18–mediated intestinal inflammation in the murine model of Crohn's disease  Takanori Kanai, Mamoru Watanabe, Akira Okazawa, Toshiro Sato, Motomi Yamazaki, Susumu Okamoto, Hiromasa Ishii, Teruji Totsuka, Ryoichi Iiyama, Ryuichi Okamoto, Masao Ikeda, Masashi Kurimoto, Kiyoshi Takeda, Shizuo Akira, Toshifumi Hibi  Gastroenterology  Volume 121, Issue 4, Pages 875-888 (October 2001) DOI: 10.1053/gast.2001.28021 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Induction of TNBS colitis. Systemic immunization of TNBS conjugated with BSA followed by intrarectal administration of TNBS/EtOH reproducibly developed pancolitis in almost all treated C57BL/6J mice. (A) Survival rate after intrarectal TNBS administration. Time on the X-axis corresponds to the number of days after intrarectal administration of EtOH alone or TNBS/EtOH as described in Materials and Methods. Each group contained 20 mice. (B) Weight changes of mice with TNBS immune colitis. The weights of mice treated with EtOH only were significantly higher than mice treated with TNBS/EtOH on days 1, 3, and 5 (*P < 0.05). Gastroenterology 2001 121, 875-888DOI: (10.1053/gast.2001.28021) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Mac-1 and IL-18 at colitis lesion in TNBS-treated mice. (A) Flow cytometric analysis of the Mac-1–positive splenocyte population after EtOH (upper) or TNBS/EtOH (lower) 8 days after intrarectal administration. Splenocytes were stained with either anti–Mac-1-PE (thick line) or isotype-matched mouse IgG-PE (thin line). (B) 2-color flow cytometric analysis of the Mac-1/Gr-1 or Mac-1/CD11c in splenocytes after EtOH (upper) or TNBS/EtOH (lower) 8 days after intrarectal administration. Splenocytes were stained with anti–Gr1-FITC/anti–Mac-1-PE or anti–CD11c-FITC/anti–Mac-1-PE. (C) Flow cytometric analysis of PMA+Ca ionophore-stimulated splenocytes for the expression of intracytoplasmic IFN-γ in mice with EtOH (upper) or TNBS/EtOH (lower) challenged mice. Histograms show reactivity with anti–IFN-γ-PE (thick line) or a negative control (rat-IgG-PE; thin line). (D) IL-18 protein level assayed by ELISA in cell lysates of splenocyte population after EtOH (left) or TNBS/EtOH (right). The data are expressed as the IL-18 protein level in cell lysates after adjusting protein concentrations of cell lysates to 1 mg/mL. Each bar represents the mean ± SD from 5 mice (*P < 0.05). (E) Flow cytometric analysis of PMA+Ca ionophore-stimulated LPMCs for the expression of intracytoplasmic IFN-γ in mice with EtOH (upper) or TNBS/EtOH (lower) challenged mice 8 days after intrarectal administration. Histograms show reactivity with anti–IFN-γ-PE (thick line) or a negative control (rat-IgG-PE; thin line). (F) IFN-γ production of LPMCs from EtOH (left) or TNBS/EtOH (right) challenged mice on day 8. Each bar represents the mean ± SD from 5 mice (*P < 0.05). (G) Intracytoplasmic IL-18 staining. Flow cytometric analysis of lipopolysaccharide-stimulated LPMCs obtained from EtOH-treated mice (upper), and TNBS/EtOH-treated mice received control IgG (lower). IL-18 production for TNBS/EtOH-treated mice was significantly higher than that of control EtOH-treated mice at 7 days after intrarectal administration. (H) Western blot analysis of murine IL-18 expression in colonic tissue homogenates derived from freshly obtained colonic tissue samples. Mature form of murine IL-18 (18.3 kilodalton) is more abundant in colonic tissues from TNBS colitis mice compared with those from EtOH-given mice. Gastroenterology 2001 121, 875-888DOI: (10.1053/gast.2001.28021) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Administration of anti–Mac-1-saporin represses colitis in TNBS-treated mice. (A) Weight changes of mice with TNBS immune colitis. Each group contained 8 mice. The weights of TNBS/EtOH-treated mice that received anti–Mac-1-saporin were significantly higher than those of mice treated with TNBS/EtOH without anti–Mac-1-saporin on days 3, 5, and 7 (*P < 0.05). (B) Macroscopic appearance of colons of (a) EtOH-treated mice, (b) TNBS/EtOH-treated mice, and (c) TNBS/EtOH-treated mice that received anti–Mac-1-saporin antibody 7 days after intrarectal administration. (C) Immunohistochemical staining for Mac-1 in colonic sections from EtOH-treated mice, TNBS/EtOH-treated mice, and TNBS/EtOH-treated mice that received anti–Mac-1 mAb (15.6 μg/body); or TNBS/EtOH-treated mice that received anti–Mac-1-saporin (20 μg/body) 7 days after intrarectal administration. (Upper panels) Photomicrograph of H&E-stained cross section. (Lower panels) Photomicrograph of immunohistochemical staining for Mac-1. (D) Flow cytometric analysis of PMA+Ca ionophore-stimulated splenocytes obtained from EtOH-treated mice, TNBS/EtOH-treated mice, or TNBS/EtOH-treated mice that received anti–Mac-1-saporin stained for intracytoplasmic IFN-γ 7 days after intrarectal administration. Histograms show reactivity with anti–IFN-γ-PE (thick line) or a negative control (rat-IgG-PE; thin line). (E) IFN-γ production of anti-CD3/anti-CD28 stimulated LPMCs obtained from EtOH-treated mice (left), TNBS/EtOH-treated mice (middle), or TNBS/EtOH-treated mice (right) that received anti–Mac-1-saporin. Each bar represents the mean ± SD from 5 mice 7 days after intrarectal administration (*P < 0.05). Gastroenterology 2001 121, 875-888DOI: (10.1053/gast.2001.28021) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Administration of anti–IL-18 antibody represses colitis in TNBS-treated mice. The inhibition studies with the anti–IL-18 antibody used 1 mg of either neutralizing rabbit anti–IL-18 antibodies or control rabbit IgG given intraperitoneally to mice given at the indicated time points (days 0, 1, 3, and 5) with day 0 corresponding to the day of intrarectal challenge with TNBS/EtOH. Each group contained 6 mice. (A) Weight changes of EtOH-treated C57BL/6J mice and TNBS/EtOH-treated C57BL/6J mice, which received control IgG or anti–IL-18 antibodies (*P < 0.05). The weights of TNBS/EtOH-treated mice that received anti–IL-18 antibodies were significantly higher than TNBS/EtOH-treated C57BL/6J mice, which received control IgG on days 1, 3, and 5 (*P < 0.05). (B) Macroscopic appearance of colons and spleens of (a) EtOH-treated mice, (b) TNBS/EtOH-treated mice that received control IgG, and (c) TNBS/EtOH-treated mice that received anti–IL-18 antibodies at 7 days after intrarectal administration. (C) Histologic examination of the colon in EtOH-treated mice, TNBS/EtOH-treated mice that received control IgG, and TNBS/EtOH-treated mice that received anti–IL-18 antibodies at 7 days after intrarectal administration. Original magnification 100×. (D) Histologic scoring of colitis in treated and untreated mice. The average score for TNBS/EtOH-treated mice that received anti–IL-18 antibodies was significantly less than that of TNBS colitis control mice that received control IgG (*P < 0.05) 7 days after intrarectal administration. (E) Quantitation of LPMC in treated and untreated mice. The number of LPMC in anti–IL-18–treated mice was significantly less than that of TNBS colitis control mice (*P < 0.05) 7 days after intrarectal administration. (F) Intracytoplasmic IFN-γ staining. Flow cytometric analysis of PMA+Ca ionophore-stimulated LPMCs obtained from EtOH-treated mice, TNBS/EtOH-treated mice that received control IgG, and TNBS/EtOH-treated mice that received anti–IL-18 antibodies. IFN-γ production for TNBS/EtOH-treated mice that received anti–IL-18 antibodies was significantly less than that of TNBS/EtOH-treated mice that received control IgG 7 days after intrarectal administration. Gastroenterology 2001 121, 875-888DOI: (10.1053/gast.2001.28021) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Lack of TNBS colitis in IL-18 mice. The clinical course of TNBS immune colitis in IL-18+/+ and IL-18−/− mice. Each group contained 15 mice. (A) Growth curves for mice receiving standard TNBS immunization/intrarectal challenge protocol. The weights of IL-18−/− mice treated with TNBS/EtOH enema were significantly higher than those of IL-18+/+ mice on days 1 and 3 (*P < 0.05). (B) Macroscopic appearance of colons and spleens of IL-18+/+ mice (a) treated with EtOH/TNBS and (b) those of IL-18−/− mice treated with TNBS/EtOH at 7 days after intrarectal administration. (C) Histologic analysis of colonic specimens 7 days after intrarectal administration. Histopathology of colon specimens of (left) IL-18+/+ mice with TNBS/EtOH-treated mice and (right) IL-18−/− mice treated with TNBS/EtOH. Original magnification 100×. (D) Quantitation of Mac-1–positive cells was significantly increased in (left) spleens and (right) colons in TNBS-treated IL-18+/+ mice, compared with TNBS-treated IL-18−/− mice 7 days after intrarectal administration (*P < 0.05). (E) IFN-γ production of LPMCs obtained from TNBS/EtOH-treated IL-18+/+ mice and TNBS/EtOH-treated IL-18−/− mice 7 days after intrarectal administration. (F) Conventional TNBS-enema colitis model without TNBS immunization in IL-18−/− mice. Some IL-18+/+ wild-type mice developed colitis and, in contrast, IL-18−/− mice never developed colitis at all. Gastroenterology 2001 121, 875-888DOI: (10.1053/gast.2001.28021) Copyright © 2001 American Gastroenterological Association Terms and Conditions