Volume 123, Issue 1, Pages 256-270 (July 2002) Amelioration of dextran sulfate sodium–induced colitis by anti-macrophage migration inhibitory factor antibody in mice  Tatsuya Ohkawara, Jun Nishihira, Hiroshi Takeda, Shuhei Hige, Mototsugu Kato, Toshiro Sugiyama, Toshihiko Iwanaga, Hideki Nakamura, Yuka Mizue, Masahiro Asaka  Gastroenterology  Volume 123, Issue 1, Pages 256-270 (July 2002) DOI: 10.1053/gast.2002.34236 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Time course of plasma MIF level of DSS-induced colitis in BALB/c mice. Mice were killed before and at 1, 3, 5, and 7 days after administration of 4% DSS solution. Each point represents the mean ± standard error (n = 5). *Statistically significant compared with nontreated control mice (P < 0.05). Gastroenterology 2002 123, 256-270DOI: (10.1053/gast.2002.34236) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Northern blot analysis. Northern blot analysis was performed for MIF mRNA expression in the cecum (A) and the colon (B) of BALB/c mice as described in Materials and Methods. Samples were collected at days 0, 1, 3, 5, and 7 after DSS. The bands of GAPDH and relative intensities are shown at the bottom of each lane. The experiments were repeated at least 3 times, and representative results are shown. Gastroenterology 2002 123, 256-270DOI: (10.1053/gast.2002.34236) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Immunohistochemical analysis of MIF expression in the cecum and colon of BALB/c mice. The cecum and colon were surgically removed from control mice and from mice treated with DSS. The tissue specimens of the cecum (A, B, C) and the colon (D, E, F) were stained with a Vector ABC immunostaining kit. (A and D) Negative controls reacted with nonimmune rabbit IgG as the primary antibody; (B and E) before DSS treatment with the anti-MIF antibody; (C and F) reacted with the anti-MIF antibody 7 days after DSS. Gastroenterology 2002 123, 256-270DOI: (10.1053/gast.2002.34236) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Effects of anti-MIF antibody on rectal bleeding and diarrhea induced by DSS. The frequencies (%) of rectal bleeding and diarrhea in BALB/c mice (A) and C57BL/6 mice (B) are shown at day 7 post-DSS with (■) and without (□) anti-MIF antibody administration (n = 20). Statistical analysis was carried out comparing anti-MIF antibody and nonimmune rabbit IgG treatments using the Mann–Whitney U test. Gastroenterology 2002 123, 256-270DOI: (10.1053/gast.2002.34236) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of anti-MIF antibody on survival rate in TNBS colitis. The effect of the anti-MIF antibody on the survival rate of BALB/c mice with TNBS colitis was evaluated. Column 1, nontreated control; column 2, treated with 100 μL enema of 50% ethanol; column 3, treated with TNBS (270 mg/kg) in ethanol and nonimmune IgG; and column 4, treated with TNBS in ethanol and anti-MIF antibody. Gastroenterology 2002 123, 256-270DOI: (10.1053/gast.2002.34236) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Histopathologic examination for the effect of antibody on DSS-induced tissue damage in BALB/c mice. The cecum and colon tissues were opened longitudinally and the tissue specimens were prepared for H&E staining. Histopathologic changes at day 7 after DSS with and without the anti-MIF antibody are shown for the cecum (A, B, C) and the colon (D, E, F). (A and D) Normal tissues; (B and E) the tissues 7 days after DSS; (C and F) the tissues 7 days after DSS treated with the anti-MIF antibody. Gastroenterology 2002 123, 256-270DOI: (10.1053/gast.2002.34236) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Evaluation of the DSS-induced colocecal damage and the extent of inflammatory lesions in mice with and without anti-MIF antibody treatment. (A and B) The cecum and colon were obtained from BALB/c mice (n = 20) at day 7 after DSS, and histologic scores for the tissue damage (A) and the extent of lesions (B) in mice with (■) and without (□) anti-MIF antibody treatment are shown. (C and D) The cecum and colon were obtained from C57BL/6 mice (n = 20) at day 7 after DSS, and histologic scores for the tissue damage (C) and the extent of lesions (D) with (■) and without (□) anti-MIF antibody treatment are shown. The scores were assessed by H&E staining. NS, not significant. Gastroenterology 2002 123, 256-270DOI: (10.1053/gast.2002.34236) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Effects of anti-MIF antibody on TNF-α production in the cecum and colon induced by DSS in BALB/c mice. We measured TNF-α contents in the cecum and colon at day 7 post-DSS in mice with and without anti-MIF antibody treatment. TNF-α contents in the cecum and colon were up-regulated at day 7 after DSS in comparison with that at day 0. The up-regulation was significantly suppressed by anti-MIF antibody treatment. □, control without any treatment; ▨, treated with nonimmune rabbit IgG; and ■, treated with anti-MIF antibody (n = 20). NS, not significant. Gastroenterology 2002 123, 256-270DOI: (10.1053/gast.2002.34236) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 Effects of anti-MIF antibody on TNF-α, IFN-γ, and IL-4 production in the cecum and colon induced by DSS in C57BL/6 mice. We measured (A) TNF-α (B) IFN-γ, and (C) IL-4 in the cecum and colon at day 7 after DSS with and without anti-MIF antibody treatment (n = 10). □, control without treatment; ▨, treated with nonimmune rabbit IgG; and ■, treated with anti-MIF antibody (n = 20). Gastroenterology 2002 123, 256-270DOI: (10.1053/gast.2002.34236) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 10 Effect of anti-MIF antibody on the expression of MMP-13 mRNA in C57BL/6 mice. Northern blot analysis was carried out for (A) cecum and (B) colon. The tissues were obtained with and without administration of anti-MIF antibodies at the indicated times. The bands of GAPDH are shown at the bottom of each lane. We repeated the experiments at least 3 times and show representative results. Gastroenterology 2002 123, 256-270DOI: (10.1053/gast.2002.34236) Copyright © 2002 American Gastroenterological Association Terms and Conditions