Volume 121, Issue 3, Pages 619-630 (September 2001) Macrophage migration inhibitory factor is an important mediator in the pathogenesis of gastric inflammation in rats  Xiao Ru Huang, Connie Wun Chun Hui, Yong-Xiong Chen, Benjamin Chun, Yu Wong, Peter C.W. Fung, Christine Metz, Chi Hin Cho, Wai Mo Hui, Richard Bucala, Shiu-Kum Lam, Hui Y. Lan  Gastroenterology  Volume 121, Issue 3, Pages 619-630 (September 2001) DOI: 10.1053/gast.2001.27205 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 In situ hybridization shows MIF mRNA expression in normal stomach and in gastric ulcer. (A) Normal rat stomach showing weak MIF expression within mucosa. (B) Day 1 and (C) day 3 gastric ulcer tissues. Many MIF-positive mononuclear cells are visible within the necrotic mucosa (NM) and diffusely infiltrate the submucosa and muscular wall (arrows), peaking at day 3 with massive necrosis and deep ulcer (C, enlargement in the insert). (D) Day 3 of anti-MIF treatment. MIF-positive mononuclear cells are substantially reduced, and the severity of ulcer is attenuated, compared with day 3 untreated rats (C). (E) Day 5. Most MIF-positive cells are capillary endothelial cells (enlargement in insert). (F) Day 8 and (G) day 14. MIF mRNA expression in the ulcer base is largely decreased, and ulcer healing occurs. (H) Day 3 gastric ulcer tissue with the MIF sense probe showing no MIF mRNA–positive cells detectable. Original magnification 100×. Gastroenterology 2001 121, 619-630DOI: (10.1053/gast.2001.27205) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Double immunostaining of MIF and iNOS expression in normal stomach and in gastric ulcer. (A–F) Double staining of MIF (blue) with ED1+ macrophages (brown), and (G–I) double staining of MIF (blue) and iNOS expression (brown). (A–B) Serial sections of a normal rat stomach. Weak and constitutive expression of MIF by gastric epithelial cells with few ED1+ macrophages was detected by the anti-MIF and macrophage mAb (A), but these were completely absent in the negative control section (B). (C) Day 1 and (D) day 3 ulcer tissues. Strong MIF expression is associated with ED1+ macrophage infiltration within the necrotic mucosa (C) and throughout the gastric wall, leading to the development of severe ulceration (D). Note that many ED1+ cells (brown) strongly express MIF (D, insert). (E) Day 3 anti-MIF treatment. Up-regulation of MIF expression and macrophage accumulation are virtually abrogated and ulceration is attenuated, compared with the untreated animal (D). (F) Day 8. Up-regulation of MIF expression and macrophage accumulation in the ulcer base are largely reduced when ulcer healing occurs. (G) Day 1 and (H) day 3. Strong MIF (blue) and iNOS (brown) expression is associated with the development of severe gastric ulcer. Note that many MIF+ iNOS+ cells are in the macrophage-rich area in serial sections (C, D). (I) Day 3 anti-MIF treatment. Up-regulation and MIF and iNOS expression is completely abrogated by anti-MIF treatment. Magnifications: 100× (A, B, D–F, H, I); 125× (C, G). Gastroenterology 2001 121, 619-630DOI: (10.1053/gast.2001.27205) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Semiquantitation of MIF mRNA and protein expression, macrophage and neutrophil accumulation, and iNOS expression in gastric ulcer. Each bar represents the mean value ± SEM for each group of 6 animals. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the normal (day 0). Gastroenterology 2001 121, 619-630DOI: (10.1053/gast.2001.27205) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 In situ hybridization and RT-PCR show that anti-MIF treatment abrogates up-regulation of TNF-α, iNOS, and ICAM-1 mRNA expression in gastric ulcer at day 3. (A–F) In situ hybridization and (G, H) RT-PCR. Results show that up-regulation of TNF-α (A), iNOS (B), and ICAM-1 (C) mRNA expression in the ulcer base is completely abrogated by anti-MIF treatment (D, TNF-α; E, iNOS; F, ICAM-1). Similar results are found by RT-PCR (G, H). Ratio represents the mean ± SEM for groups of 5 animals (ICAM-1 or iNOS/GAPDH). *P < 0.05, **P < 0.01, compared with the normal and anti-MIF treatment. Original magnification 100×. Gastroenterology 2001 121, 619-630DOI: (10.1053/gast.2001.27205) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Immunostaining shows that anti-MIF treatment suppresses up-regulation of TNF-α and ICAM-1 expression in gastric ulcer at day 3. (A, B) Day 3 control and (C, D) anti-MIF treatment. Results show that up-regulation of TNF-α (A) and ICAM-1 (B) in the ulcer base is completely abrogated by anti-MIF treatment (C, TNF-α; D, ICAM-1). Original magnification 100×. Gastroenterology 2001 121, 619-630DOI: (10.1053/gast.2001.27205) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Comparison at day 3 gastric ulcer of MIF, TNF-α, ICAM-1, and iNOS mRNA with protein expression and macrophage and neutrophil infiltration after anti-MIF treatment. Each bar represents the mean value ± SD for groups of 6 animals in control (solid bars) and in anti-MIF (open bars) mAb treatment animals. MΦ, macrophages; PMN, neutrophils. ***P < 0.001 compared with control animals. Gastroenterology 2001 121, 619-630DOI: (10.1053/gast.2001.27205) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 RT-PCR or ELISA shows that MIF induces ICAM-1 and iNOS mRNA expression, or TNF-α production by peripheral blood leukocytes. (A) MIF induces ICAM-1 mRNA expression in a dose-dependent fashion (4 hours) by RT-PCR. (B) MIF induces iNOS mRNA expression (4 hours) in a dose-dependent manner by RT-PCR. (C) MIF induces TNF-α production in a dose- and time-dependent manner by ELISA. Note that the ability of MIF (100 ng/mL) induced up-regulation of ICAM-1 and iNOS mRNA expression at 4 hours, as well as TNF-α production at 4 and 24 hours are abrogated by a neutralizing anti-MIF mAb. Ratio represents the mean ± SD for 3 experiments. *P < 0.05, compared with MIF (0); #P < 0.05, ##P < 0.001, compared with the control. Gastroenterology 2001 121, 619-630DOI: (10.1053/gast.2001.27205) Copyright © 2001 American Gastroenterological Association Terms and Conditions