Volume 129, Issue 1, Pages 26-33 (July 2005) Activation of A2A Adenosine Receptor Attenuates Intestinal Inflammation in Animal Models of Inflammatory Bowel Disease  Masaru Odashima, Giorgos Bamias, Jesus Rivera-Nieves, Joel Linden, Cynthia C. Nast, Christopher A. Moskaluk, Marco Marini, Kazuhiko Sugawara, Kosuke Kozaiwa, Michiro Otaka, Sumio Watanabe, Fabio Cominelli  Gastroenterology  Volume 129, Issue 1, Pages 26-33 (July 2005) DOI: 10.1053/j.gastro.2005.05.032 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Effects of preventive administration of the A2A agonist ATL-146e on the severity of acute formalin-immune complex colitis. Rabbits were treated intraperitoneally with either ATL-146e (n = 10–12 per dose group) or vehicle (PBS; n = 38) 1 hour before and 5, 11, 17, 23, 29, 35, and 41 hours after the induction of colitis. Distal colonic specimens were obtained 48 hours after induction for histological assessment of colitis. (A) Acute inflammatory index, edema, and percentage of necrosis were assessed as described in Materials and Methods. Each bar represents the mean ± SEM. (B) Representative microphotographs from ATL-146e-treated or vehicle-treated animals. Compared with vehicle, ATL-146e-treated animals had significantly less acute inflammatory infiltrate in the mucosa, had normal glands, and had intact surfaces (stain, H&E; original magnification, 80×). (C) MPO activity was measured as described in Materials and Methods. Each bar represents the mean ± SEM. Gastroenterology 2005 129, 26-33DOI: (10.1053/j.gastro.2005.05.032) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Effects of ATL-146e administration on the severity of relapsing formalin-immune complex colitis. Rabbits were treated with ATL-146e (0.1 μg·kg−1·min−1; n = 5), dexamethasone (1 mg/day; n = 5), or vehicle (n = 7) by using miniosmotic pumps 48 hours after the initial formalin enema. (A) Vehicle-treated animals displayed marked acute and chronic inflammatory infiltration of the mucosa and, to a lesser degree, the submucosa. An area of surface mucosal erosion can be seen. (B) ATL-146e administration resulted in mild mucosal inflammation, normal glands, and an intact surface. (C) Dexamethasone treatment resulted in minimal inflammation with no necrosis or erosions. (D) Acute inflammatory index, edema, percentage of necrosis, and chronic inflammatory index were assessed 48 hours after reinduction of colitis. Dex, dexamethasone. (E) MPO activity was measured as described in Materials and Methods. Data are presented as mean ± SEM. Gastroenterology 2005 129, 26-33DOI: (10.1053/j.gastro.2005.05.032) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Effects of ATL-146e treatment on the severity of SAMP1/YitFc ileitis. Forty-week-old SAMP1/YitFc mice were administered ATL-146e 0.1 μg·kg−1·min−1 (n = 20), dexamethasone 100 μg/day (n = 10), or vehicle (n = 30). (A) Villus distortion, active and chronic inflammatory indices, and the total inflammatory score were assessed histologically in samples of ileum obtained after 3 days of treatment. Data are presented as mean ± SEM. (B) Vehicle-treated mouse showing typical characteristics of SAMP1/YitFc ileitis (acute and chronic inflammatory cells, distorted villi, and muscular hypertrophy). ATL-146e administration resulted in reconstitution of the mucosal architecture, amelioration of inflammatory infiltration, and normal muscular thickness. Gastroenterology 2005 129, 26-33DOI: (10.1053/j.gastro.2005.05.032) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Effects of ATL-146e treatment on the function of mucosal lymphocytes. (A and B) Forty-week-old SAMP1/YitFc mice were treated with ATL-146e or vehicle. Isolated MLN cells (106/mL) were cultured with immobilized anti-CD3 (10 μg/mL) for 24 hours. Secreted IFN-γ, TNF, IL-4, and IL-10 were measured by enzyme-linked immunosorbent assay. Each bar represents the mean ± SEM. (C) CD4+ T cells from MLNs of inflamed SAMP1/YitFc mice were injected into SCID mice. Recipients were treated with ATL-146e, dexamethasone, or vehicle (n = 10 per group) for 3 days before death (6 weeks after transfer). Vehicle-treated mice showed severe mucosal damage, with heavy infiltration by neutrophils and mononuclear cells (left). In contrast, ATL-146e treatment resulted in almost normal intestinal mucosa and scarce inflammatory infiltrates (right). (D) Villus distortion, active and chronic inflammatory indices, and total inflammatory scores were assessed as described in Materials and Methods. Data are presented as mean ± SEM. Gastroenterology 2005 129, 26-33DOI: (10.1053/j.gastro.2005.05.032) Copyright © 2005 American Gastroenterological Association Terms and Conditions