| Adenosine, acting at its receptors, particularly A2A receptors (A2AR), is a potent endogenous anti-inflammatory agent that modulates functions of inflammatory and immune cells. To understand how adenosine receptors are regulated during inflammation in which the inflammatory milieu abounds in proinflammatory cytokines, we investigated the effects of IL-1, TNF-alpha and IFN-gamma on expression, function and desensitization of A2AR in human monocytic THP-1 cells and human microvascular endothelial cells (HMVEC). We observed that, in both cultured cell lines, expression of A2AR message and protein was upregulated by IL-1 and TNF-alpha but down-regulated by IFN-gamma. The changes in A2AR expression appeared physiologically functional as selective agonists of A2AR (CGS-21680 and MRE0094) significantly stimulated increased cAMP accumulation in IL-1- and TNF-alpha-treated cells but not in either resting or IFNgamma-treated cells. Consistently, IL-1 and TNF-alpha enhanced the effects of A2AR occupancy on IL-10 and IL-12 production in LPS-activated THP-1 cells and expression of vascular endothelial growth factor (VEGF) in HMVEC, whereas IFN-gamma attenuated these effects. Since the increased A2AR function by IL-1 and TNF-alpha was disproportionate to the increment in expression, the effect of TNF-alpha on desensitization of A2AR was further studied. We found that desensitization of A2AR, determined by cAMP responses to repeated agonist stimulation, was greatly inhibited following TNF-alpha treatment. Consistent with its effect on receptor desensitization, TNF-alpha blocked agonist-induced translocation of G protein-coupled receptor (GPCR) kinase 2 (GRK2) and beta-arrestins, which mediate the phosphorylation and desensitization of A2AR and other GPCRs, to the plasma membrane. The effects of TNF-alpha on GRK2 translocation and subsequent A2AR desensitization are most likely mediated via sphingomyelinase (SMase)-dependent pathways as these effects of TNF-alpha were mimicked by C2-ceramide, a second messenger in the SMase pathways, and reversed by inhibitors of SMases and c-Jun N-terminal kinase (JNK). Together, these results demonstrate signaling crosstalk between TNF-alpha and GPCRs. TNF-alpha enhances GPCR activity by not only increasing the receptor number but, more importantly, by preventing agonist-induced desensitization of GPCRs by diminishing agonist-dependent recruitment of GRK2 and beta-arrestin to the plasma membrane by a sphingolipid/JNK-mediated mechanism. |
