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

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 A(2A) and A(3) 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 A(2A) receptor knockout mice and A(3) 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 A(2A) knockout mice or wild-type control mice. In contrast, there were more leukocytes, TNF-alpha and IL-10 in the exudates of the adenosine A(3) 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 A(3) receptor knockout mice but not in the A(2A) receptor knockout mice. Methotrexate reduced exudate levels of TNF-alpha in the wild-type mice and A(3) receptor knockout mice but not the A(2A) receptor knockout mice. More strikingly, IL-10, a critical regulator of peritoneal inflammation, was increased in the methotrexate-treated wild-type mice and A(3) knockout mice but decreased in the A(2A) knockout mice. Dexamethasone, an agent that suppresses inflammation by a different mechanism, was similarly effective in wild-type mice, A(2A) mice and A(3) 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.