Suppression of inflammation by low-dose methotrexate is mediated by adenosine A2A receptor but not A3 receptor activation in thioglycollate-induced peritonitis

Abstract

Prior studies demonstrate that adenosine, acting at one or more of its receptors, mediates the anti-inflammatory effects of methotrexate in animal models of both acute and chronic inflammation. Both adenosine A2A and A3 receptors contribute to the anti-inflammatory effects of methotrexate treatment in the air pouch model of inflammation, and the regulation of inflammation by these two receptors differs at the cellular level. Because different factors may regulate inflammation at different sites we examined the effect of low-dose weekly methotrexate treatment (0.75 mg/kg/week) in a model of acute peritoneal inflammation in adenosine A2A receptor knockout mice and A3 receptor knockout mice and their wild-type littermates. Following intraperitoneal injection of thioglycollate there was no significant difference in the number or type of leukocytes, tumor necrosis factor alpha (TNF-alpha) and IL-10 levels that accumulated in the thioglycollate-induced peritoneal exudates in adenosine A2A knockout mice or wild-type control mice. In contrast, there were more leukocytes, TNF-alpha and IL-10 in the exudates of the adenosine A3 receptor-deficient mice. Low-dose, weekly methotrexate treatment increased the adenosine concentration in the peritoneal exudates of all mice studied, and reduced the leukocyte accumulation in the wild-type mice and A3 receptor knockout mice but not in the A2A receptor knockout mice. Methotrexate reduced exudate levels of TNF-alpha in the wild-type mice and A3 receptor knockout mice but not the A2A receptor knockout mice. More strikingly, IL-10, a critical regulator of peritoneal inflammation, was increased in the methotrexate-treated wild-type mice and A3 knockout mice but decreased in the A2A knockout mice. Dexamethasone, an agent that suppresses inflammation by a different mechanism, was similarly effective in wild-type mice, A2A mice and A3 knockout mice. These findings provide further evidence that adenosine is a potent regulator of inflammation that mediates the anti-inflammatory effects of methotrexate. Moreover, these data provide strong evidence that the anti-inflammatory effects of methotrexate and adenosine are mediated by different receptors in different inflammatory loci, an observation that may explain why inflammatory diseases of some organs but not of other organs respond to methotrexate therapy.

Figure 1

Effect of methotrexate and dexamethasone treatment on leukocyte accumulation in peritoneal exudates of mice. (a) A_2A wild-type mice and A_2A receptor knockout mice or (b) A₃ wild-type mice and A₃ receptor knockout mice either were treated with weekly injections of methotrexate (0.75 mg/kg) or saline control for 4 weeks prior to induction of inflammation or were treated with a single intraperitoneal injection of dexamethasone (1.5 mg/kg) or saline 1 hour before induction of inflammation and subsequent collection of inflammatory exudates, as described. Results are presented as the mean (± SEM) million cells per exudate. **P < 0.001 vs wild-type control mice, ⁺⁺P < 0.001 vs knockout control mice, ⁺P < 0.05 vs knockout control mice, all one-way analysis of variance (Bonferroni t test).