Cross-Talk between Gs- and Gq-Coupled Pathways in Regulation of Interleukin-4 by A2BAdenosine Receptors in Human Mast Cells

Abstract

Human mast cells express functional A_2A and A_2B adenosine receptors. However, only stimulation of A_2B, not A_2A, leads to secretion of interleukin (IL)-4, an important step in adenosine receptor-mediated induction of IgE synthesis by B-cells. In this study, we investigate intracellular pathways that link stimulation of A_2B receptors to IL-4 up-regulation in HMC-1 mast cells. Both A_2A and A_2B receptors couple to G_s proteins and stimulate adenylate cyclase, but only A_2B stimulates phospholipase Cβ through coupling to G_q proteins leading to activation of protein kinase C and calcium mobilization. Inhibition of phospholipase Cβ completely blocked A_2B receptor-dependent IL-4 secretion. The protein kinase C inhibitor 2-{8-[(dimethylamino)-methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-(1-methyl-1H-indol-3-yl)maleimide (Ro-32-0432) had no effect on A_2B receptor-mediated IL-4 secretion but inhibited phorbol 12-myristate 13-acetate-stimulated IL-4 secretion. In contrast, chelation of intracellular Ca²⁺ inhibited both A_2B receptor- and ionomycin-dependent IL-4 secretion. This Ca²⁺-sensitive pathway probably includes calcineurin and nuclear factor of activated T cells, because A_2B receptor-dependent IL-4 secretion was blocked with cyclosporin A or 11R-VIVIT peptide. G_s-linked pathways also play a role in the A_2B receptor-dependent stimulation of IL-4 secretion; inhibition of adenylate cyclase or protein kinase A attenuated A_2B receptor-dependent IL-4 secretion. Although stimulation of adenylate cyclase with forskolin did not increase IL-4 secretion on its own, it potentiated the effect of Pasteurella multocida toxin by 2-fold and ionomycin by 3-fold. Both forskolin and stimulation of A_2B receptors up-regulated NFATc1 protein levels. We conclude that A_2B receptors up-regulate IL-4 through G_q signaling that is potentiated via cross-talk with G_s-coupled pathways.