Recently, it has been shown that ATP and TNF-α synergize in the activation and maturation of human dendritic cells (DC); the effect of ATP was reproduced by hydrolysis-resistant derivatives of ATP and was blocked by suramin, suggesting the involvement of a P2 receptor, but the particular subtype involved was not identified. In this report we confirm that ATP and various derivatives synergize with TNF-α and LPS to induce the maturation of human monocyte-derived DC, as revealed by up-regulation of the CD83 marker and the secretion of IL-12. The rank order of potency of various analogs (AR-C67085 > adenosine 5′-O-(3-thiotriphosphate) = 2′- and 3′-O-(4-benzoyl-benzoyl) ATP > ATP > 2-methylthio-ATP) was close to that of the recombinant human P2Y11 receptor. Furthermore, these compounds activated cAMP production in DC, in a xanthine-insensitive way, consistent with the involvement of the P2Y11 receptor, which among P2Y subtypes has the unique feature of being dually coupled to phospholipase C and adenylyl cyclase activation. The involvement of the P2Y11/cAMP/protein kinase A signaling pathway in the nucleotide-induced maturation of DC is supported by the inhibitory effect of H89, a protein kinase A inhibitor. Taken together, our results demonstrate that ATP activates DC through stimulation of the P2Y11 receptor and subsequent increase in intracellular cAMP.

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ATP has been reported to inhibit or stimulate lymphoid cell proliferation, depending on the origin of the cells. Agents that increase cAMP, such as PGE2, inhibit human CD4+ T cell activation. We demonstrate that several ATP derivatives increase cAMP in both freshly purified and activated human peripheral blood CD4+ T cells. The rank order of potency of the various nucleotides was: adenosine 5′-O-(3-thiotriphosphate) (ATPγS) ≈ 2′- and 3′-O-(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP ≫ dATP, 2-propylthio-β,γ-dichloromethylene-d-ATP, UDP, UTP. This effect did not involve the activation of A2Rs by adenosine or the synthesis of prostaglandins. ATPγS had no effect on cytosolic calcium, whereas BzATP induced an influx of extracellular calcium. ATPγS and BzATP inhibited secretion of IL-2, IL-5, IL-10, and IFN-γ; expression of CD25; and proliferation after activation of CD4+ T cells by immobilized anti-CD3 and soluble anti-CD28 Abs, without increasing cell death. Taken together, our results suggest that extracellular adenine nucleotides inhibit CD4+ T cell activation via an increase in cAMP mediated by an unidentified P2YR, which might thus constitute a new therapeutic target in immunosuppressive treatments.

The orphan receptor GPR86 was recently identified as a new ADP receptor coupled to G i and named P2Y 13 . This brings to eight the number of genuine P2Y receptors. On the basis of sequence homology, they can be subdivided into two subgroups: P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 ; and P2Y 12 , P2Y 13 , and the UDP-glucose receptor that should be renamed P2Y 14 . Some pharmacological properties of the P2Y 13 receptor are significantly different from those of the P2Y 12 receptor:ATP and 2MeSATP behaved as weak partial agonists. Potency of 2MeSADP was greater than or equal to that of ADP, depending on the expressing cell line and signaling pathway studied.AR-C67085MX behaved as an antagonist, but only with a mM potency as compared to nM at the P2Y 12 receptor.The active metabolite of clopidogrel had no effect. Expression of P2Y 13 mRNA was highest in brain, spleen, and various types of leukocytes, in particular dendritic cells. Recent results underscore the role of other P2Y receptors, especially P2Y 11 , in dendritic cell biology. Indeed, ATP and other nucleotides have multiple effects on human dendritic cells: potentiation of maturation, potentiation, or inhibition of IL-12p40 secretion (depending on the level of stimulation), inhibition of IL-12p70 production, potentiation of IL-10 secretion, and modulation of the repertoire of expressed chemokines and chemokine receptors. These actions are mediated by a rise in cAMP and the rank order of potency of various nucleotides is consistent with the involvement of the P2Y 11 receptor. On the other hand, several nucleotides inhibit the proliferation of human CD4 + T-cells and their secretion of cytokines: these actions also involve a rise in cAMP, apparently mediated by a receptor other than P2Y 11 . These actions are reminiscent of those of PGE 2 and might represent a protective mechanism operating DDR Abbreviations: ATPgS, adenosine 5'-O-(3-thiotriphosphate); BzATP, 2'-and 3'-O-(4-benzoyl-benzoyl) ATP; 2Me-

We have previously reported that ATPgS, a slowly hydrolyzed analog of ATP, inhibits the activation of human CD4 + T lymphocytes by anti-CD3 and anti-CD28 mAb. In this report we have partially characterized the signaling mechanisms involved in this immunosuppressive effect. ATPgS had no inhibitory effect on CD4 + T-cell activation induced by PMA and anti-CD28, indicating that it acts proximally to the TCR. It had no effect on the calcium rise induced by CD3/CD28 stimulation, but inhibited the phosphorylation of three kinases, ERK2, p38 MAPK and PKB, that play a key role in the activation of T cells. The receptor involved in these actions remains unidentified.Abbreviations: 5 0 -FSBA -5 0 -p-(fluorosulfonyl)benzoyl adenosine; ATPgS -adenosine 5 0 -O-(3-thiotriphosphate); E-NTPDase -ecto-nucleoside triphosphate diphosphohydrolase; ERK -extracellular signal-regulated kinase; mAb -monoclonal antibody; MAPK -mitogen activated protein kinase; PKA -protein kinase A; PKB -protein kinase B; PMA -phorbol 12-myristate 13-acetate; TCR -T-cell receptor