Activation of A2A Adenosine Receptor Attenuates Intestinal Inflammation in Animal Models of Inflammatory Bowel Disease
2005; Elsevier BV; Volume: 129; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2005.05.032
ISSN1528-0012
AutoresMasaru Odashima, Giorgos Bamias, Jesús Rivera–Nieves, Joel Linden, Cynthia C. Nast, Christopher A. Moskaluk, Marco Marini, Kazuhiko Sugawara, Kosuke Kozaiwa, Michiro Otaka, Sumio Watanabe, Fabio Cominelli,
Tópico(s)Pregnancy and Medication Impact
ResumoBackground & Aims: Adenosine has been implicated as an important regulator of the inflammatory response. Four subtypes of adenosine receptors (A1, A2A, A2B, and A3) have been described, of which A2A potentially inhibits inflammation. The aim of this study was to investigate the role of A2A in mucosal inflammation by administering a selective A2A agonist (ATL-146e) to experimental models of inflammatory bowel disease. Methods: The anti-inflammatory effects of ATL-146e were studied in the acute and chronic rabbit formalin-immune complex models of colitis and the SAMP1/YitFc mouse model of spontaneous ileitis. Results: ATL-146e significantly reduced the acute inflammatory index and tissue necrosis compared with vehicle (P < .01) in the acute model of rabbit immune colitis. In the chronic rabbit immune colitis model, ATL-146e significantly suppressed inflammatory cell infiltration into the colonic mucosa (P < .05) and prevented mortality. The administration of ATL-146e significantly decreased the chronic inflammatory index (P < .01) and villus distortion index (P < .01) in the ileum of SAMP1/YitFc mice, and ameliorated adoptively transferred ileitis in severe combined immunodeficient mice injected with CD4+ T cells from SAMP1/Yit mice (P < .05). Tumor necrosis factor, interferon γ, and interleukin 4 concentrations were significantly suppressed by ATL-146e treatment in supernatants from cultures of mesenteric lymph node cells of SAMP1/YitFc mice (P < .05 vs vehicle-treated mice). Conclusions: A2A adenosine receptor activation by ATL-146e significantly reduced inflammation in the intestinal mucosa. This effect was associated with decreased leukocyte infiltration and inhibition of proinflammatory cytokines. Activation of A2A by selective agonism may therefore serve as a novel therapy for the treatment of inflammatory bowel disease. Background & Aims: Adenosine has been implicated as an important regulator of the inflammatory response. Four subtypes of adenosine receptors (A1, A2A, A2B, and A3) have been described, of which A2A potentially inhibits inflammation. The aim of this study was to investigate the role of A2A in mucosal inflammation by administering a selective A2A agonist (ATL-146e) to experimental models of inflammatory bowel disease. Methods: The anti-inflammatory effects of ATL-146e were studied in the acute and chronic rabbit formalin-immune complex models of colitis and the SAMP1/YitFc mouse model of spontaneous ileitis. Results: ATL-146e significantly reduced the acute inflammatory index and tissue necrosis compared with vehicle (P < .01) in the acute model of rabbit immune colitis. In the chronic rabbit immune colitis model, ATL-146e significantly suppressed inflammatory cell infiltration into the colonic mucosa (P < .05) and prevented mortality. The administration of ATL-146e significantly decreased the chronic inflammatory index (P < .01) and villus distortion index (P < .01) in the ileum of SAMP1/YitFc mice, and ameliorated adoptively transferred ileitis in severe combined immunodeficient mice injected with CD4+ T cells from SAMP1/Yit mice (P < .05). Tumor necrosis factor, interferon γ, and interleukin 4 concentrations were significantly suppressed by ATL-146e treatment in supernatants from cultures of mesenteric lymph node cells of SAMP1/YitFc mice (P < .05 vs vehicle-treated mice). Conclusions: A2A adenosine receptor activation by ATL-146e significantly reduced inflammation in the intestinal mucosa. This effect was associated with decreased leukocyte infiltration and inhibition of proinflammatory cytokines. Activation of A2A by selective agonism may therefore serve as a novel therapy for the treatment of inflammatory bowel disease. Recent advances in inflammatory bowel disease (IBD) research have led to the hypothesis that Crohn's disease (CD) and ulcerative colitis (UC) are dysregulated immune responses that occur within the intestinal mucosa and are directed against intraluminal antigens of bacterial origin.1Bouma G. Strober W. The immunological and genetic basis of inflammatory bowel disease.Nat Rev Immunol. 2003; 3: 521-533Crossref PubMed Scopus (1538) Google Scholar During this process, cytokines play central and multifaceted roles through the induction of intestinal recruitment of inflammatory cells, stimulation of inflammatory mediator release, and generation of imbalanced immunologic effector pathways (T-helper [Th]1, Th2, or both).2Fiocchi C. Inflammatory bowel disease etiology and pathogenesis.Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1892) Google Scholar Consequently, recent strategies for the treatment of IBD have primarily targeted the immunopathogenic processes that mediate chronic intestinal inflammation at the cytokine level.3Sandborn W.J. Targan S.R. Biologic therapy of inflammatory bowel disease.Gastroenterology. 2002; 122: 1592-1608Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 4Bamias G. Sugawara K. Pagnini C. Cominelli F. The Th1 immune pathway as a therapeutic target in Crohn's disease.Curr Opin Investig Drugs. 2003; 4: 1279-1286PubMed Google Scholar Adenosine is a primordial signaling molecule that plays an important role in many physiological responses in mammalian tissues. It acts through 4 G protein-coupled receptors (A1, A2A, A2B, and A3).5Linden J. Molecular approach to adenosine receptors receptor-mediated mechanisms of tissue protection.Annu Rev Pharmacol Toxicol. 2001; 41: 775-787Crossref PubMed Scopus (583) Google Scholar, 6Sullivan G.W. Linden J. Role of adenosine receptors in inflammation.Drug Dev Res. 1998; 45: 103-112Crossref Scopus (86) Google Scholar, 7Cronstein B.N. Adenosine, an endogenous anti-inflammatory agent.J Appl Physiol. 1994; 76: 5-13Crossref PubMed Scopus (592) Google Scholar These receptors are expressed on cells of the immune system.5Linden J. Molecular approach to adenosine receptors receptor-mediated mechanisms of tissue protection.Annu Rev Pharmacol Toxicol. 2001; 41: 775-787Crossref PubMed Scopus (583) Google Scholar Among them, A2A receptors are unique in that they induce anti-inflammatory signals upon activation.6Sullivan G.W. Linden J. Role of adenosine receptors in inflammation.Drug Dev Res. 1998; 45: 103-112Crossref Scopus (86) Google Scholar, 7Cronstein B.N. Adenosine, an endogenous anti-inflammatory agent.J Appl Physiol. 1994; 76: 5-13Crossref PubMed Scopus (592) Google Scholar Accordingly, A2A receptor activation effectively attenuates ischemia/reperfusion injury of the heart, lung, liver, and kidney in vivo.8Ross S.D. Tribble C.G. Linden J. Gangemi J.J. Lanpher B. Wang A.Y. Kron I.L. Selective adenosine-A2A activation reduces lung reperfusion injury following transplantation.J Heart Lung Transplant. 1999; 18: 381-402Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 9Okusa M.D. Linden J. Huang L. Rieger J.M. Macdonald T.L. Huynh L.P. A2A adenosine receptor-mediated inhibition of renal injury and neutrophil adhesion.Am J Physiol Renal Physiol. 2000; 279: F809-F818PubMed Google Scholar, 10Jordan J.E. Zhao Z.Q. Sato H. Taft S. Vinten-Johansen J. Adenosine A2 receptor activation attenuates reperfusion injury by inhibiting neutrophil accumulation, superoxide generation, and coronary endothelial adherence.J Pharmacol Exp Ther. 1997; 280: 301-309PubMed Google Scholar, 11Harada N. Okajima K. Murakami K. Usune S. Sato C. Ohshima K. Katsuragi T. Adenosine and selective A2A receptor agonists reduce ischemia/reperfusion injury of rat liver mainly by inhibiting leukocyte activation.J Pharmacol Exp Ther. 2000; 294: 1034-1042PubMed Google Scholar These effects were primarily mediated through decreased expression of adhesion molecules, compromised endothelial adherence, reduced neutrophilic infiltration, and diminished superoxide generation.8Ross S.D. Tribble C.G. Linden J. Gangemi J.J. Lanpher B. Wang A.Y. Kron I.L. Selective adenosine-A2A activation reduces lung reperfusion injury following transplantation.J Heart Lung Transplant. 1999; 18: 381-402Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 9Okusa M.D. Linden J. Huang L. Rieger J.M. Macdonald T.L. Huynh L.P. A2A adenosine receptor-mediated inhibition of renal injury and neutrophil adhesion.Am J Physiol Renal Physiol. 2000; 279: F809-F818PubMed Google Scholar, 10Jordan J.E. Zhao Z.Q. Sato H. Taft S. Vinten-Johansen J. Adenosine A2 receptor activation attenuates reperfusion injury by inhibiting neutrophil accumulation, superoxide generation, and coronary endothelial adherence.J Pharmacol Exp Ther. 1997; 280: 301-309PubMed Google Scholar, 11Harada N. Okajima K. Murakami K. Usune S. Sato C. Ohshima K. Katsuragi T. Adenosine and selective A2A receptor agonists reduce ischemia/reperfusion injury of rat liver mainly by inhibiting leukocyte activation.J Pharmacol Exp Ther. 2000; 294: 1034-1042PubMed Google Scholar At the cellular level, activation of A2A receptors down-regulated the secretion of proinflammatory cytokines in vitro.12Link A.A. Kino T. Worth J.A. McGuire J.L. Crane M.L. Chrousos G.P. Wilder R.L. Elenkov I.J. Ligand-activation of the adenosine A2A receptor inhibits IL-12 production by human monocytes.J Immunol. 2000; 164: 436-442PubMed Google Scholar, 13Hasko G. Kuhel D.G. Chen J.F. Schwarzschild M.A. Deithch E.A. Mabley J.G. Marton A. Szabo C. Adenosine inhibits IL-12 and TNF production via adenosine A2A receptor-dependent and independent mechanisms.FASEB J. 2000; 14: 2065-2074Crossref PubMed Scopus (442) Google Scholar, 14Lappas C.M. Rieger J.M. Linden J. A2A adenosine receptor induction inhibits IFN-γ production in murine CD4+ T cells.J Immunol. 2005; 174: 1073-1080Crossref PubMed Scopus (330) Google Scholar Taken together, these data raise the possibility that A2A receptor activation may be of therapeutic benefit in chronic inflammatory conditions, including IBD. In this study, we hypothesized that activation of A2A receptors would reduce the severity of experimental IBD and modify the profile of inflammatory mediators during mucosal inflammation. To test this hypothesis, we used the newly developed, highly selective A2A adenosine receptor agonist ATL-146e.15Rieger J.M. Brown M.L. Sullivan G.W. Linden J. MacDonald T.L. Design, synthesis, and evaluation of novel A2A adenosine receptor agonists.J Med Chem. 2001; 44: 531-539Crossref PubMed Scopus (90) Google Scholar Administration of ATL-146e prevented acute and relapsing formalin-immune complex-induced colitis in rabbits, a disease that resembles UC, and is primarily dependent on infiltration with neutrophils and secretion of inflammatory mediators.16Cominelli F. Nast C.C. Clark B.D. Schindler R. Eysselein V.E. Thompson R.C. Dinarello C.A. Interleukin-1 (IL-1) gene expression, synthesis, and effects of specific IL-1 receptor blockade in rabbit immune colitis.J Clin Invest. 1990; 86: 972-980Crossref PubMed Scopus (314) Google Scholar, 17Cominelli F. Nast C.C. Duchini A. Lee M. Recombinant interleukin 1 receptor antagonist blocks the proinflammatory activity of endogenous interleukin-1 in rabbit immune colitis.Gastroenterology. 1992; 103: 65-71Abstract PubMed Google Scholar In addition, administration of ATL-146e effectively ameliorated spontaneous chronic ileitis in SAMP1/YitFc mice, a model that bears clinical, histological, and immunologic similarity to CD.18Kosiewicz M.M. Nast C.C. Krishnan A. Rivera-Nieves J. Moskaluk C.A. Matsumoto S. Kozaiwa K. Cominelli F. Th1-type responses mediate spontaneous ileitis in a novel murine model of Crohn's disease.J Clin Invest. 2001; 107: 695-702Crossref PubMed Scopus (220) Google Scholar, 19Rivera-Nieves J. Bamias G. Vidrich A. Marini M. Pizarro T.T. McDuffie M.J. Moskaluk C.A. Cohn S.M. Cominelli F. Emergence of perianal fistulizing disease in the SAMP1/YitFc mouse, a spontaneous model of chronic ileitis.Gastroenterology. 2003; 124: 972-982Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar Our study provides evidence that, during chronic small-intestinal inflammation, A2A receptor activation may down-regulate lymphocyte function. This was demonstrated by the suppression of proinflammatory cytokine secretion and the diminished ability of CD4+ lymphocytes to transfer ileitis into recipient severe combined immunodeficient (SCID) mice after administration of ATL-146e. The selective A2A adenosine receptor agonist ATL-146e was synthesized in the Department of Chemistry at the University of Virginia by introducing substitutions at the C-2 and 5′ positions of adenosine.15Rieger J.M. Brown M.L. Sullivan G.W. Linden J. MacDonald T.L. Design, synthesis, and evaluation of novel A2A adenosine receptor agonists.J Med Chem. 2001; 44: 531-539Crossref PubMed Scopus (90) Google Scholar For in vivo administration, ATL-146e was dissolved in phosphate-buffered saline (PBS) or 0.9% saline with <0.01% dimethyl sulfoxide. All animal protocols were approved by the Animal Care and Use Committee of the University of Virginia. Formalin-immune complex colitis was induced in the distal colon of New Zealand White male rabbits (2.0–2.5 kg) by using a modification of the method by Hogdson et al, as previously described by us.16Cominelli F. Nast C.C. Clark B.D. Schindler R. Eysselein V.E. Thompson R.C. Dinarello C.A. Interleukin-1 (IL-1) gene expression, synthesis, and effects of specific IL-1 receptor blockade in rabbit immune colitis.J Clin Invest. 1990; 86: 972-980Crossref PubMed Scopus (314) Google Scholar After sedation, rabbits (5 per group on each study day) were administered 4 mL of unbuffered formaldehyde (0.45% vol/vol; Electron Microscopy Sciences, Fort Washington, PA) intrarectally, followed 2 hours later by intravenous injection of 1 mL of immune complexes in antigen excess (Sigma Immunochemicals, St Louis, MO). ATL-146e or vehicle was administered intraperitoneally 1 hour before and 5, 11, 17, 23, 29, 35, and 41 hours after the induction of colitis. Animals were killed 48 hours after induction. Longitudinal sections of distal colon were obtained for histological examination and assay of myeloperoxidase (MPO) activity. Colitis was initially induced as described previously. After 48 hours, Alzet osmotic minipumps (Model 1002; Alza, Palo Alto, CA) were implanted into the rabbits. Minipumps contained ATL-146e, dexamethasone, or PBS (vehicle-treated controls). After a 7-day recovery period, colitis was again induced, and then animals were killed 48 hours later. Representative colonic sections were studied by a single pathologist (C.C.N.) in a blinded manner. The mucosa and submucosa of each sample were examined separately. The acute and chronic inflammatory indices, as well as edema and percentage necrosis, were evaluated as previously described.20Ferretti M. Casini-Raggi V. Pizarro T.T. Eisenberg S.P. Nast C.C. Cominelli F. Neutralization of endogenous IL-1 receptor antagonist exacerbates and prolongs inflammation in rabbit immune colitis.J Clin Invest. 1994; 94: 449-453Crossref PubMed Scopus (148) Google Scholar SAMP1/YitFc mice were maintained under specific pathogen-free conditions in a barrier facility at the University of Virginia. Alzet osmotic minipumps containing vehicle or ATL-146e were inserted subcutaneously into 40-week-old mice; animals were killed 3 days later. SAMP1/YitFc mice administered dexamethasone (100 μg daily intraperitoneally) served as positive controls. For adoptive transfer, CD4+ T cells were isolated from mesenteric lymph nodes (MLNs) of 40-week-old SAMP1/YitFc mice by using anti-CD4-bound magnetic beads in a positive-selection immunomagnetic protocol (Miltenyi Biotec, Auburn, CA). Six- to eight-week-old SCID mice (C3HSmn.C-Prkdcscid/J; Jackson Laboratories, Bar Harbor, ME) were injected intraperitoneally with 105 to 106 CD4+ T cells. ATL-146e, PBS, or dexamethasone was administered intraperitoneally every 6 hours during the 3 days before death. Recipients were killed 6 weeks after the transfer of donor CD4+ cells. In both models, the severity of ileitis was evaluated histologically in a blinded manner by a single pathologist (C.A.M.), as previously described.19Rivera-Nieves J. Bamias G. Vidrich A. Marini M. Pizarro T.T. McDuffie M.J. Moskaluk C.A. Cohn S.M. Cominelli F. Emergence of perianal fistulizing disease in the SAMP1/YitFc mouse, a spontaneous model of chronic ileitis.Gastroenterology. 2003; 124: 972-982Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar A modification of the method described by Krawisz et al21Krawisz J.E. Sharon P. Stenson W.F. Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity assessment of inflammation in rat and hamster models.Gastroenterology. 1984; 87: 1344-1350Abstract Full Text PDF PubMed Scopus (1626) Google Scholar was used to assay for MPO activity. Briefly, 100 mg of colon tissue was placed in a polypropylene tube in 2 mL of 0.5% (vol/vol) hexadecyltrimethylammonium bromide buffer (Eastman Kodak, Rochester, NY) in 50 mmol/L phosphate buffer (pH 6.0). Samples were homogenized for 2 seconds (Brinkmann Polytron; Brinkmann Instruments, Westbury, NY), and homogenates were then centrifuged at 10,000g for 10 minutes. A total of 0.1 mL of supernatant was combined with 2 mL of 50 mmol/L phosphate buffer (pH 6.0). The change in absorbance was measured by spectrophotometry at 460 nm for up to 2 minutes. One unit of MPO was defined as the amount of enzyme that will degrade 1 mol of hydrogen peroxide per minute at 25°C (pH 6.0). MLN cells (1 × 106/mL) in complete media (RPMI 1640, 10% fetal calf serum, 2 mmol/L l-glutamine, 1% penicillin/streptomycin, 10 mmol/L HEPES, 1 mmol/L sodium pyruvate, nonessential amino acids, and 1 × 10−5 mol/L 2-mercaptoethanol) were cultured with immobilized anti-CD3 antibody (10 μg/mL; BD Biosciences PharMingen, San Diego, CA). Supernatants were harvested after 24 hours. The concentrations of interferon (IFN)-γ, tumor necrosis factor (TNF), interleukin (IL)-4, and IL-10 were measured in cell culture supernatants by using commercially available enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN). All data were expressed as mean ± SEM. Statistical significance was determined with the Mann-Whitney U test by using the StatView-J 4.11 statistical program (Abacus Concepts, Berkeley, CA) for Macintosh. P values of <.05 were considered significant. Single induction of formalin-immune complex colitis results in an acute inflammatory response.16Cominelli F. Nast C.C. Clark B.D. Schindler R. Eysselein V.E. Thompson R.C. Dinarello C.A. Interleukin-1 (IL-1) gene expression, synthesis, and effects of specific IL-1 receptor blockade in rabbit immune colitis.J Clin Invest. 1990; 86: 972-980Crossref PubMed Scopus (314) Google Scholar Administration of ATL-146e before the induction of colitis significantly attenuated tissue damage in a dose-dependent manner, because both 20 and 40 μg/kg ATL-146e significantly reduced the acute inflammatory index (vehicle, 2.8 ± 0.2; ATL-146e 40 μg/kg, 1.3 ± 0.2; P < .01), edema (vehicle, 2.2 ± 0.2; ATL-146e 40 μg/kg, 1.3 ± 0.2; P < .05), and the percentage of necrosis (vehicle, 24.3% ± 4.9%; ATL-146e 40 μg/kg, 2.0% ± 1.5%; P < .05; Figure 1A). Mucosal erosions and necrosis were seen in control rabbits, with extensive inflammatory infiltration of the mucosa and submucosa. However, treatment with ATL-146e resulted in preservation of the mucosa, intact glands, and few patchy infiltrates (Figure 1B). In line with the histological study, rabbits administered ATL-146e had significantly lower colonic MPO activity (vehicle, 3.2 ± 0.4 U/g of tissue per minute; ATL-146e 40 μg/kg, 0.07 ± 0.01 U/g of tissue per minute; P < .001), which is indicative of decreased recruitment and activation of neutrophils (Figure 1C). The relapsing form of formalin-immune complex colitis consists of reinduction of colitis, which increases mononuclear infiltration of the distal colon (chronic-type inflammation). Indeed, when compared with single induction, reinduced colitis showed a significantly higher chronic inflammatory index (1.3 ± 0.3 vs 0.8 ± 0.1; P < .05), although acute inflammation, necrosis, and edema were comparable. Reinduction of colitis in vehicle-treated rabbits resulted in high mortality (5/12 rabbits; 41.7%), an effect that was completely prevented by ATL-146e (0.1 μg · kg−1 · min−1) or dexamethasone (1 mg/day). In addition, the vehicle-treated animals that did survive had marked acute and chronic mucosal and submucosal inflammatory infiltrates and erosions (Figure 2A). In contrast, ATL-146e-treated animals displayed mild mucosal inflammation with normal glands and intact surfaces (Figure 2B). The effect of ATL-146e administration was comparable to that of dexamethasone (Figure 2C and D). Continuous infusion of ATL-146e significantly reduced the acute inflammatory index (0.8 ± 0.3 vs 2.8 ± 0.7 in vehicle; P < .05) and edema (1.1 ± 0.3 vs 2.2 ± 0.3; P < .05). The percentage of necrosis and the chronic inflammatory index were reduced in both the ATL-146e and dexamethasone groups, but not to a significant level, probably because of the high variability of percentage necrosis in this model. The anti-inflammatory effects of ATL-146e treatment were confirmed by the dramatic decrease of colonic MPO activity, shown in Figure 2E (vehicle, 3.6 ± 0.1 U/g of tissue per minute; ATL-146e, 0.2 ± 0.1 U/g of tissue per minute [P < .01]; dexamethasone, 0.4 ± 0.1 U/g of tissue per minute [P < .05]). In contrast to formalin-immune complex colitis, disease in SAMP1/YitFc mice develops spontaneously and persists throughout life. It is localized to the terminal ileum and is characterized by chronic inflammatory cell infiltration in the lamina propria. The effects of continuous administration of ATL-146e on the severity of ileitis in SAMP1/YitFc mice are shown in Figure 3. Compared with vehicle treatment, ATL-146e administration resulted in significantly reduced indices for active inflammation (2.7 ± 0.4 vs 3.8 ± 0.3; P < .05), chronic inflammation (1.6 ± 0.2 vs 2.5 ± 0.1; P < .01), and villus distortion (2.4 ± 0.4 vs 4.7 ± 0.2; P < .01), whereas the total inflammatory score was reduced by 41% (6.8 ± 0.9 vs 11.5 ± 0.5; P < .001). These effects were comparable to those obtained with dexamethasone administration (total inflammatory score, 6.7 ± 0.9 [42% reduction]; P < .01 vs vehicle). Representative sections of terminal ilea in Figure 3B clearly show that, compared with vehicle-treated mice (left), ATL-146e-treated mice (right) showed preserved mucosal architecture with nearly normal-appearing villi, minimal inflammatory infiltration of the lamina propria, and normal thickness of the muscularis propria. Chronic inflammation in SAMP1/YitFc mice is associated with increased secretion of both Th1 (IFN-γ and TNF) and Th2 (IL-4 and IL-5) cytokines by mucosal lymphocytes.22Bamias G. Martin III, C. Mishina M. Ross W.G. Rivera-Nieves J. Marini M. Cominelli F. Pro-inflammatory effects of TH2 cytokines in a murine model of chronic small intestinal inflammation.Gastroenterology. 2005; 128: 654-666Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar We therefore hypothesized that the therapeutic effects of ATL-146e on SAMP1/YitFc mice may be mediated through the suppression of Th1 and/or Th2 cytokine production. We found that freshly isolated SAMP1/YitFc MLN cells from ATL-146e-treated mice secreted significantly less IFN-γ and TNF when stimulated compared with vehicle-treated controls (Figure 4A). In addition, ATL-146e significantly reduced IL-4 secretion, whereas IL-10 was not affected (Figure 4B). To further test the ability of ATL-146e to interfere with lymphocytic function, we tested its effects on an ileitis model that is well established in our laboratory and consists of the adoptive transfer of SAMP1/YitFc MLN CD4+ cells into SCID recipients.18Kosiewicz M.M. Nast C.C. Krishnan A. Rivera-Nieves J. Moskaluk C.A. Matsumoto S. Kozaiwa K. Cominelli F. Th1-type responses mediate spontaneous ileitis in a novel murine model of Crohn's disease.J Clin Invest. 2001; 107: 695-702Crossref PubMed Scopus (220) Google Scholar Administration of ATL-146e resulted in significant amelioration of disease in SCID recipients (total inflammatory score: ATL-146e 50 μg/kg, 7.3 ± 0.6; vehicle, 10.1 ± 0.9; P < .05; Figure 4C and D). It is interesting to note that the therapeutic effect on the chronic inflammatory index was the most pronounced (ATL-146e 50 μg/kg, 1.5 ± 0.3; vehicle, 2.8 ± 0.4; P < .05) compared with villus distortion and active inflammatory indices (Figure 4D). This result indicates that ATL-146e primarily exerted its beneficial effects by targeting the transferred lymphocytic population. By comparison, dexamethasone treatment resulted in an active inflammatory index of 0.9 ± 0.2 (not significant), a chronic inflammatory index of 2.0 ± 0.5 (not significant), villus distortion of 3.8 ± 0.6 (P < .05 vs vehicle), and a total inflammatory score of 6.7 ± 0.9 (P < .05). In this study, we report that a selective A2A receptor agonist, ATL-146e, abrogates experimental intestinal inflammation. By using animal models that represent both acute and chronic forms of gut inflammation, we show that the pharmacological properties of ATL-146e modulate a variety of inflammatory pathways in vivo. Our results suggest that A2A activation interferes with lymphocytic function during chronic ileitis and may exert anti-inflammatory effects, in part by suppressing cytokine secretion by mucosal lymphocytes. Formalin-immune complex distal colitis shares several histological features with UC.16Cominelli F. Nast C.C. Clark B.D. Schindler R. Eysselein V.E. Thompson R.C. Dinarello C.A. Interleukin-1 (IL-1) gene expression, synthesis, and effects of specific IL-1 receptor blockade in rabbit immune colitis.J Clin Invest. 1990; 86: 972-980Crossref PubMed Scopus (314) Google Scholar, 17Cominelli F. Nast C.C. Duchini A. Lee M. Recombinant interleukin 1 receptor antagonist blocks the proinflammatory activity of endogenous interleukin-1 in rabbit immune colitis.Gastroenterology. 1992; 103: 65-71Abstract PubMed Google Scholar In our study, ATL-146e effectively prevented the induction of acute colitis and reversed chronic/relapsing disease. Both forms depend on neutrophilic infiltration and local secretion of mediators, including proinflammatory cytokines.16Cominelli F. Nast C.C. Clark B.D. Schindler R. Eysselein V.E. Thompson R.C. Dinarello C.A. Interleukin-1 (IL-1) gene expression, synthesis, and effects of specific IL-1 receptor blockade in rabbit immune colitis.J Clin Invest. 1990; 86: 972-980Crossref PubMed Scopus (314) Google Scholar, 17Cominelli F. Nast C.C. Duchini A. Lee M. Recombinant interleukin 1 receptor antagonist blocks the proinflammatory activity of endogenous interleukin-1 in rabbit immune colitis.Gastroenterology. 1992; 103: 65-71Abstract PubMed Google Scholar Our data clearly show that activation of A2A receptor signaling effectively suppressed neutrophil-mediated inflammatory responses. First, histological parameters of active inflammation and edema were dramatically decreased in ATL-146e-treated rabbits compared with controls. Second, severe necrosis was almost absent in the treated group, thus indicating reduced tissue damage. Third, colonic MPO activity, an accurate biochemical marker of neutrophilic infiltration, was significantly lower in animals administered the receptor agonist. Finally, no mortality occurred in the chronic/relapsing model after administration of ATL-146e, in striking contrast to the 42% baseline mortality in control animals. This signifies a beneficial effect of ATL-146e on accelerating tissue healing once the initial induction of colitis is imposed. Indeed, recent studies have shown that ATL-146e enhances wound healing through increased fibroblast migration and angiogenesis.23Montesinos M.C. Gadangi P. Longaker M. Sung J. Levine J. Nilsen D. Reibman J. Li M. Jiang C.K. Hirschhorn R. Recht P.A. Ostad E. Levin R. Cronstein B.N. Wound healing is accelerated by agonists of adenosine A2A (Gα s-linked) receptors.J Exp Med. 1997; 186: 1615-1620Crossref PubMed Scopus (181) Google Scholar Taken together, these data strongly suggest that selective A2A receptor agonism may have preventative and therapeutic efficacy during gut inflammation mediated by neutrophils and their products. Another recent study reported that the colonic epithelium expresses A2B but not other types of adenosine receptors and that the A2B receptor shows proinflammatory properties, including induction of IL-6 and activation of neutrophils.24Feoktistov I. Biaggion I. Adenosine A2B receptors.Pharmacol Rev. 1997; 49: 381-402PubMed Google Scholar, 25Sullivan G.W. Rieger J.M. Scheld W.M. MacDonald T.L. Linden J. Cyclic AMP-dependent inhibition of human neutrophil oxidative activity by substituted 2-propynylcyclohexyl adenosine A(2A) receptor agonists.Br J Pharmacol. 2001; 32: 1017-1026Crossref Scopus (146) Google Scholar Our results suggest that, during colitis, activation of the A2A receptor may offer a counterregulatory signal and suppress these proinflammatory effects, thereby ameliorating tissue damage. It is interesting to note that Morabito et al26Morabito L. Montesinos M.C. Schreibman D.M. Balter L. Thompson L.F. Resta R. Carlin G. Huie M.A. Cronstein B.N. Methotrexate and sulfasalazine promote adenosine release by a mechanism that requires ecto-5′-nucleotidase-mediated conversion of adenine nucleotides.J Clin Invest. 1998; 101: 295-300Crossref PubMed Scopus (233) Google Scholar have shown that sulfasalazine and methotrexate, 2 drugs that are commonly used in the treatment of IBD, may induce their anti-inflammatory effects by promoting adenosine release. A2A receptors are present on T cells and regulate many lymphocytic functions.27Koshiba M. Kojima H. Huang S. Apsov S. Sitkovsky M.V. Memory of extracellular adenosine A2A purinergic receptor-mediated signaling in murine T cells.J Biol Chem. 1997; 272: 25881-25889Crossref PubMed Scopus (164) Google Scholar, 28Huang S. Apsov S. Koshiba M. Sitkovsky M. Role of A2A extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T cell activation and expansion.Blood. 1997; 90: 1600-1610Crossref PubMed Google Scholar An important finding in our study was that A2A activation significantly ameliorated lymphocyte-mediated immune responses. This was clearly shown by the dramatic improvement of chronic ileitis in SAMP1/YitFc mice, a model that bears remarkable clinical, pathologic, and immunologic similarity to CD. Although the exact mechanisms of action for ATL-146e remain unknown, our study provides evidence that they include suppression of effector lymphocyte functions. First, the chronic inflammatory index was significantly decreased after ATL-146e administration, thus indicating decreased recruitment of lymphocytes to the inflamed ileum. This effect could be mediated by down-regulation of adhesion molecule expression and their ligands. In fact, a recent study reported that ATL-146e administration decreased endothelial expression of P selectin and intercellular adhesion molecule 1 in an ischemia/reperfusion injury model.10Jordan J.E. Zhao Z.Q. Sato H. Taft S. Vinten-Johansen J. Adenosine A2 receptor activation attenuates reperfusion injury by inhibiting neutrophil accumulation, superoxide generation, and coronary endothelial adherence.J Pharmacol Exp Ther. 1997; 280: 301-309PubMed Google Scholar Second, ileitis in SCID mice induced by SAMP1/YitFc CD4+ cells was significantly ameliorated by ATL-146e treatment, thus indicating compromised pathogenic potential of donor lymphocytes in recipient mice in the presence of A2A activation. Finally, MLN lymphocytes isolated from mice administered ATL-146e produced significantly lower amounts of IFN-γ and TNF, 2 proinflammatory cytokines that are central to the pathogenesis of CD.1Bouma G. Strober W. The immunological and genetic basis of inflammatory bowel disease.Nat Rev Immunol. 2003; 3: 521-533Crossref PubMed Scopus (1538) Google Scholar, 2Fiocchi C. Inflammatory bowel disease etiology and pathogenesis.Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1892) Google Scholar Similarly to our studies, it was shown that adenosine acts via the A2A receptor to exert anti-inflammatory effects, suppressing the production of IL-12, TNF, and IFN-γ by monocytes and lymphocytes.14Lappas C.M. Rieger J.M. Linden J. A2A adenosine receptor induction inhibits IFN-γ production in murine CD4+ T cells.J Immunol. 2005; 174: 1073-1080Crossref PubMed Scopus (330) Google Scholar, 29Lukashev D. Ohta A. Apasov S. Chen J.F. Sitkovsky M. Physiologic attenuation of proinflammatory transcription by the Gs protein-coupled A2A adenosine receptor in vivo.J Immunol. 2004; 173: 21-24PubMed Google Scholar In addition, Koshiba et al30Koshiba M. Rosin D.L. Hayashi N. Linden J. 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Adenosine and selective A2A receptor agonists reduce ischemia/reperfusion injury of rat liver mainly by inhibiting leukocyte activation.J Pharmacol Exp Ther. 2000; 294: 1034-1042PubMed Google Scholar Our study suggests that acute and chronic intestinal inflammation represent additional targets for therapeutic administration of selective A2A receptor agonists. It is impressive that systemic administration of ATL-146e reversed established ileitis in both the spontaneous and adoptive transfer models of ileitis, both of which show virtually 100% disease penetrance.18Kosiewicz M.M. Nast C.C. Krishnan A. Rivera-Nieves J. Moskaluk C.A. Matsumoto S. Kozaiwa K. Cominelli F. Th1-type responses mediate spontaneous ileitis in a novel murine model of Crohn's disease.J Clin Invest. 2001; 107: 695-702Crossref PubMed Scopus (220) Google Scholar These effects were comparable to those of dexamethasone, which is one of the most potent therapeutics currently available for the treatment of IBD. In fact, the data for ATL-146e are in line with a previous study which reported that the adenosine kinase inhibitor GP515 had similar beneficial effects in mice with dextran sulfate sodium-induced colitis.32Siegmund B. Rieder F. Albrich S. Wolf K. Bidlingmaier C. Firestein G.S. Boyle D. Lehr H.A. Loher F. Hartmann G. Endres S. Eigler A. Adenosine kinase inhibitor GP515 improves experimental colitis in mice.J Pharmacol Exp Ther. 2001; 296: 99-105PubMed Google Scholar Given the great similarities between the SAMP1/YitFc model and the human condition, our results raise the possibility that interference with adenosine pathways may offer a therapeutic alternative in CD. Although high doses (>100 ng · kg−1 · min−1) of A2A agonists can cause vasodilatation, hypotension, and reflex tachycardia, no cardiovascular effects are usually observed in the dose range used in our study (10 ng · kg−1 · min−1). In conclusion, data presented herein support the existence of important anti-inflammatory actions of ATL-146e in the intestine through the inhibition of neutrophil activation and the suppression of lymphocyte-derived cytokine-mediated proinflammatory responses. Activation of A2A receptor-mediated signaling through selective agonism may be a novel therapeutic approach for patients with IBD. The authors thank the Histology/Imaging Core (Dr Theresa Pizarro, Sharon Hoang, Greg Harp, and Michelle Wartan) of the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases UVA Digestive Health Research Center, and Dr Sarah A. De La Rue for critical revision of the manuscript.
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