The P2Y 13 receptor has recently been identified as a new P2Y receptor sharing a high sequence homology with the P2Y 12 receptor as well as similar functional properties: coupling to G i and responsiveness to ADP (Communi et al., 2001). In the present study, the pharmacology of the P2Y 13 receptor and its differences with that of the P2Y 12 Similarly, 2MeSADP was more potent than ADP in stimulating IP 3 accumulation after 10 min in AG32 cells and increasing cAMP in pertussis toxin-treated CHO-K1 cells stimulated by forskolin. On the other hand, ADP and 2MeSADP were equipotent at stimulating IP 3 formation in AG32 cells after 30 s and inhibiting forskolininduced cAMP accumulation in CHO-K1 cells. These differences in potency cannot be explained by differences in degradation rate, which in AG32 cells was similar for the two nucleotides. When contaminating diphosphates were enzymatically removed and assay of IP 3 was performed after 30 s, ATP and 2MeSATP seemed to be weak partial agonists of the P2Y 13 receptor expressed in AG32 cells. The stimulatory effect of ADP on the P2Y 13 receptor in AG32 cells was antagonized by reactive blue 2, suramin, pyridoxal-phosphate-6-azophenyl-2Ј,4Јdisulfonic acid, diadenosine tetraphosphate, and 2-(propylthio)-5Ј-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (AR-C67085MX), but not by N 6

Extracellular adenosine triphosphate affects the maturation of human monocytederived dendritic cells (DCs), mainly by inhibiting T-helper 1 (Th1) cytokines, promoting Th2 cytokines, and modulating the expression of costimulatory molecules. In this study, we report that adenosine triphosphate (ATP) can induce immunosuppression through its action on DCs, defining a new role for extracellular nucleotides. Microarray analysis of ATPstimulated human DCs revealed inter alia a drastic up-regulation of 2 genes encoding mediators involved in immunosuppression: thrombospondin-1 (TSP-1) and indoleamine 2,3-dioxygenase (IDO). The release of TSP-1 by DCs in response to ATP was confirmed at the protein level by enzyme-linked immunosorbent assay (ELISA), immunodetection, and mass spectrometry analysis, and has an antiproliferative effect on T CD4 ؉ lymphocytes through TSP-1/CD47 interaction. Our pharmacologic data support the involvement of purinergic receptor P2Y 11 in this ATP-mediated TSP-1 secretion. We demonstrate also that ATP significantly potentiates the up-regulation of IDO-a negative regulator of T lymphocyte proliferation-and kynurenine production initiated by interferon-␥ (IFN-␥) in human DCs.Thus, extracellular ATP released from damaged cells and previously considered as a danger signal is also a potent regulator of mediators playing key roles in immune tolerance. Consequently, nucleotides' derivatives may be considered as useful tools for DC-based immunotherapies. ( IntroductionDendritic cells (DCs) are antigen-presenting cells (APCs) playing a crucial role in the induction and regulation of immune responses. In response to danger signals like proinflammatory cytokines such as tumor necrosis factor-␣ (TNF-␣) and interleukin-1␤ (IL-1␤); pathogen-related molecules (lipopolysaccharide [LPS], doublestranded RNA, bacterial DNA); and T cell-derived signals such as CD40 ligand, immature dendritic cells undergo maturation. Maturation of DCs induces the loss of endocytosis, the surface expression of stable major histocompatibility complex (MHC)-peptide complexes and costimulatory molecules (CD80, CD86) and the production of cytokines such as IL-12. 1 Maturation is also accompanied by a shift in the expression of chemokines and their receptors, allowing DC migration to lymphoid organs. High secretion of IL-12 by mature DCs induces differentiation of CD4 ϩ T cells into T-helper 1 (Th1) cells secreting interferon-␥ (IFN-␥), whereas low IL-12 release combined with IL-10 production induces a Th2 response or a T regulatory response associated with induced tolerance. 1,2 In the past several years, our concepts about the role of DCs as the most potent initiators of the immune response against foreign antigens have evolved and the existence of an intermediate state between immaturity and maturation, called semimaturation, has been proposed. 3 An increasing number of reports provide strong evidence that APCs, and particularly DCs, are also involved in central and peripheral tolerance. 4,5 This tolerogenicity seems to be mediated b...

Adenosine 5'-triphosphate (ATP), which is released from necrotic cells, induces a semimaturation state of dendritic cells (DC), characterized by the up-regulation of costimulatory molecules and the inhibition of proinflammatory cytokines. This action is mediated by cyclic adenosine monophosphate (cAMP) and involves the P2Y11 receptor. As DC express the ecto-enzyme CD39, which converts ATP into adenosine 5'-diphosphate (ADP), the effects of adenine nucleotides diphosphates on molecular signaling [intracellular calcium ([Ca2+]i), cAMP, extracellular signal-regulated kinase 1 (ERK1)], costimulatory molecule expression (CD83), and cytokine production [interleukin (IL)-12, tumor necrosis factor alpha (TNF-alpha), IL-10] were investigated in human monocyte-derived DC. ADP, 2-methylthio-ADP, and ADPbetaS had no effect on cAMP, increased [Ca2+]i, and stimulated the phosphorylation of ERK1. The effect on ERK1 was inhibited by AR-C69931MX, a P2Y12 and P2Y13 antagonist. On the contrary the effect on [Ca2+]i was neither inhibited by AR-C69931MX or by the P2Y1 antagonist MRS-2179. Both effects were inhibited by pertussis toxin. ADPbetaS alone was less potent for up-regulation of CD83 than ATPgammaS and did not increase the CD83 expression by DC stimulated with lipopolysaccharide (LPS). Similar to ATPgammaS, ADPbetaS inhibited the release of IL-12p40, IL-12p70, and TNF-alpha stimulated by LPS (1-100 ng/ml). The inhibitory effect of ADPbetaS on IL-12 release was neither reversed by AR-C69931MX or by MRS-2179. The two nucleotides had opposite effects on IL-10 production: inhibition by ADPbetaS and potentiation by ATPgammaS. In conclusion, ATP can modulate the function of DC, directly via a cAMP increase mediated by the P2Y11 receptor and indirectly via its degradation into ADP, which acts via Gi-coupled receptors coupled to ERK activation and calcium mobilization. These distinct mechanisms converge on the inhibition of inflammatory cytokine production, particularly IL-12, but have a differential effect on IL-10.

We have synthesized a series of derivatives of the known P2 receptor antagonist PPADS (pyridoxal-5'-phosphate-6-azo-phenyl-2,4-disulfonate) and examined their ability to inhibit functional activity of the recombinant human P2Y13 nucleotide receptor expressed in 1321N1 human astrocytoma cells co-expressing G(alpha)16 protein (AG32). Analogues of PPADS modified through substitution of the phenylazo ring, including halo and nitro substitution, and 5'-alkyl phosphonate analogues were synthesized and tested. A 6-benzyl-5'-methyl phosphonate analogue was prepared to examine the effect of stable replacement of the azo linkage. The highest antagonistic potency was observed for 6-(3-nitrophenylazo) derivatives of pyridoxal-5'-phosphate. The 2-chloro-5-nitro analogue (MRS 2211) and 4-chloro-3-nitro analogue (MRS 2603) inhibited ADP (100 nM)-induced inositol trisphosphate (IP3) formation with pIC50 values of 5.97 and 6.18, respectively, being 45- and 74-fold more potent than PPADS. The antagonism of MRS 2211 was competitive with a pA2 value of 6.3. MRS2211 and MRS2603 inhibited phospholipase C (PLC) responses to 30 nM 2-methylthio-ADP in human P2Y1 receptor-mediated 1321N1 astrocytoma cells with IC50 values of >10 and 0.245 microM, respectively. Both analogues were inactive (IC50>10 microM) as antagonists of human P2Y12 receptor-mediated PLC responses in 1321N1 astrocytoma cells. Thus, MRS2211 displayed >20-fold selectivity as antagonist of the P2Y13 receptor in comparison to P2Y1 and P2Y12 receptors, while MRS2603 antagonized both P2Y1 and P2Y13 receptors.

Extracellular ATP and PGE2 are two cAMP-elevating agents inducing semimaturation of human monocyte-derived dendritic cells (MoDCs). We have extensively compared the gene expression profiles induced by adenosine 5′-O-(3-thiotriphosphate) (ATPγS) and PGE2 in human MoDCs using microarray technology. At 6 h of stimulation, ATPγS initiated an impressive expression profile compared with that of PGE2 (1125 genes compared with 133 genes, respectively) but after 24 h the number of genes regulated by ATPγS or PGE2 was more comparable. Many target genes involved in inflammation have been identified and validated by quantitative RT-PCR experiments. We have then focused on novel ATPγS and PGE2 target genes in MoDCs including CSF-1, MCP-4/CCL13 chemokine, vascular endothelial growth factor-A, and neuropilin-1. ATPγS strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion, which are involved in monocyte and dendritic cell (DC) differentiation. Additionally, ATPγS down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL8/MCP-2, and CCL13/MCP-4. Interestingly, vascular endothelial growth factor A, a major angiogenic factor displaying immunosuppressive properties, was secreted by MoDCs in response to ATPγS, ATP, or PGE2, alone or in synergy with LPS. Finally, flow cytometry experiments have demonstrated that ATPγS, ATP, and PGE2 down-regulate neuropilin-1, a receptor playing inter alia an important role in the activation of T lymphocytes by DCs. Our data give an extensive overview of the genes regulated by ATPγS and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.

Extracellular ATP is known to affect the maturation of monocyte-derived dendritic cells mainly by regulation of cytokines and costimulatory molecules. The present study describes the inhibition of MCP-1 (CCL2) and MIP-1a (CCL3) release by human monocyte-derived dendritic cells in response to adenine nucleotides. Our pharmacological data support the involvement of P2Y 11 and P2Y 1 purinergic receptors in the downregulation of these major monocyte recruiters. Migration assays have demonstrated that supernatants of dendritic cells treated with adenine nucleotides or anti-MCP-1/MIP-1a blocking antibodies display a strongly reduced capacity to attract monocytes and immature dendritic cells.

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-

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