Adenosine inhibits tumor necrosis factor-α release from mouse peritoneal macrophages via A_2A and A_2B but not the A ₃ adenosine receptor

Adenosine is elaborated in injured tissues where it suppresses inflammatory responses of essentially all immune cells, including production of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α). Most of the anti-inflammatory actions of adenosine have been attributed to signaling through the A_2A adenosine receptor (A_2AAR). Previously, however, it has been shown that the A₃AR agonist N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (IB-MECA) potently inhibited TNF-α release from macrophages obtained from A _2AAR "knockout" (A_2AKO) mice, suggesting that the A₃AR may also regulate cytokine expression. Here, we confirmed that the A_2AAR is the primary AR subtype that suppresses TNF-α release from thioglycollate-elicited mouse peritoneal macrophages induced by both Toll-like receptor-dependent (TLR) and TLR-independent stimuli, but we determined that the A_2BAR rather than the A₃AR mediates the non-A_2AAR actions of adenosine since 1) the ability of IB-MECA to inhibit TNF-α release was not altered in macrophages isolated from A ₃KO mice, and 2) the A_2BAR antagonist 1,3-dipropyl-8-[4- [((4-cyanophenyl) carbamoylmethyl)oxy]phenyl]xanthine (MRS 1754) blocked the ability of the nonselective AR agonist adenosine-5′-N-ethylcarboxamide (NECA) to inhibit TNF-α release from macrophages isolated from A _2AKO mice. Although A_2BARs seem capable of inhibiting TNF-α release, the A_2AAR plays a dominant suppressive role since MRS 1754 did not block the ability of NECA to inhibit TNF-α release from macrophages isolated from wild-type (WT) mice. Furthermore, the potency and efficacy of adenosine to inhibit TNF-α release from WT macrophages were not influenced by blocking A_2BARs with MRS 1754. The data indicate that adenosine suppresses TNF-α release from macrophages primarily via A_2AARs, although the A_2BAR seems to play an underlying inhibitory role that may contribute to the anti-inflammatory actions of adenosine under select circumstances.