1. Ligands of the various adenosine receptor subtypes modulate the production of pro-and anti-inflammatory cytokines. Here we evaluated the effect of adenosine and various ligands of the adenosine receptor subtypes (A1, A2, A3) on the chemokine macrophage inflammatory protein (MIP) 1α production in immunostimulated RAW macrophages in vitro. Furthermore, we studied whether a selected A3 adenosine receptor agonist inhibits MIP-1α production and affects the course of inflammation in collagen-induced arthritis. 2. In the cultured macrophages, the A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA), and, less potently, the A2 receptor agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS; 1-200 μM) dose-dependently suppressed the production of MIP-1α. The selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 1-200 μM) was ineffective, and adenosine was a weak inhibitor. The inhibition of MIP-1α production by the A3 and A2 agonist was associated with suppression of its steady-state mRNA levels. 3. Based on the in vitro data, we concluded that activation of A3, and to a lesser extent A2 adenosine receptors suppresses MIP-1α expression. Since IB-MECA was the most potent inhibitor of MIP-1α expression, we next investigated whether it affects the production of other pro-inflammatory mediators. We observed that IB-MECA (1-300 μM) inhibited, in a dose-dependent manner, the production of IL-12, IL-6, and, to a lesser extent, nitric oxide in the immunostimulated cultured macrophages. 4. Since MIP-α is a chemokine which enhances neutrophil recruitment into inflammatory sites, we investigated whether the A3 agonist IB-MECA affects the course of inflammation, MIP-α production and the degree of neutrophil recruitment in arthritis. In a model of collagen-induced arthritis in mice, IB-MECA (0.5 mg/kg/day) reduced the severity of joint inflammation. IB-MECA inhibited the formation of MIP-1α, IL-12 and nitrotyrosine (an indicator of reactive nitrogen species) in the paws, and suppressed neutrophil infiltration. 5. We conclude that adenosine receptor agonists, most notably the A3 agonist IB-MECA suppress the production of MIP-α, and exert anti-inflammatory effects. Therefore, stimulation of adenosine receptor subtypes A3 and A2 may be a strategy worthy of further evaluation for the abrogation of acute or chronic inflammatory disorders.
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