Adenosine augments IL-10 production by macrophages through an A2B receptor-mediated posttranscriptional mechanism

Adenosine receptor ligands have anti-inflammatory effects and modulate immune responses by up-regulating IL-10 production by immunostimulated macrophages. The adenosine receptor family comprises G protein-coupled heptahelical transmembrane receptors classified into four types: A(1), A(2A), A(2B), and A(3). Our understanding of the signaling mechanisms leading to enhanced IL-10 production following adenosine receptor occupancy on macrophages is limited. In this study, we demonstrate that adenosine receptor occupancy increases IL-10 production by LPS-stimulated macrophages without affecting IL-10 promoter activity and IL-10 mRNA levels, indicating a posttranscriptional mechanism. Transfection experiments with reporter constructs containing sequences corresponding to the AU-rich Y-untranslated region (UTR) of IL-10 mRNA confirmed that adenosine receptor activation acts by relieving the translational repressive effect of the IL-10 3'-UTR. By contrast, adenosine receptor activation failed to liberate the translational arrest conferred by the 3'-UTR of TNF-alpha mRNA. The IL-10 3'-UTR formed specific complexes with proteins present in cytoplasmic extracts of RAW 264.7 cells. Adenosine enhanced binding of proteins to a region of the IL-10 3'-UTR containing the GUAUUUAUU nonamer. The stimulatory effect of adenosine on IL-10 production was mediated through the A(2B) receptor, because the order of potency of selective agonists was 5'-N-ethylcarboxamidoadenosine (NECA) > N-6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 2-chloro-N-6-cyclopentyladenosine (CCPA) = 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS-21680). Also, the selective A(2B) antagonist, alloxazine, prevented the effect of adenosine. Collectively, these studies identify a novel pathway in which activation of a G protein-coupled receptor augments translation of an anti-inflammatory gene.