Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0104032 on July 7, 2004

Published online before print July 7, 2004
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(Journal of Leukocyte Biology. 2004;76:796-803.)
© 2004 by Society for Leukocyte Biology

Involvement of multiple P2Y receptors and signaling pathways in the action of adenine nucleotides diphosphates on human monocyte-derived dendritic cells

Frédéric Marteau*,1, Didier Communi*, Jean-Marie Boeynaems*,{dagger} and Nathalie Suarez Gonzalez*

* Institute of Interdisciplinary Research, IRIBHM, and
{dagger} Departement of Medical Chemistry, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium

1Correspondence: Institute of Interdisciplinary Research, IRIBHM, Building C5-110, 808, route de Lennik, 1070 Brussels, Belgium. E-mail: fmarteau{at}ulb.ac.be


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 ADPßS 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. ADPßS alone was less potent for up-regulation of CD83 than ATP{gamma}S and did not increase the CD83 expression by DC stimulated with lipopolysaccharide (LPS). Similar to ATP{gamma}S, ADPßS inhibited the release of IL-12p40, IL-12p70, and TNF-{alpha} stimulated by LPS (1–100 ng/ml). The inhibitory effect of ADPßS 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 ADPßS and potentiation by ATP{gamma}S. 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.

Key Words: IL-12 • IL-10 • ATP • ADP


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immature dendritic cells (DC) in peripheral tissues are resting sentinels characterized by their capacity for endocytosis. Maturation of DC is induced by different pathogen-related molecules [lipopolysaccharide (LPS), bacterial DNA, double-stranded RNA] and endogeneous signals, such as CD40 ligand. It is associated with the loss of endocytosis, the surface expression of antigen-presenting molecules (major histocompatibility complex class II) and costimulatory molecules (CD80, CD86), a migratory behavior, and the release of proinflammatory cytokines, such as interleukin (IL)-12. It became clear recently that DC can exist in a third developmental stage, semimaturation, which is induced by several agents [tumor necrosis factor {alpha} (TNF-{alpha}) and prostaglandin E2 (PGE2)] and involves the surface expression of antigen-presenting and costimulatory molecules but no release of proinflammatory cytokines and might result in tolerance [1 ].

The modulation of DC function by adenosine 5'-triphosphate (ATP) and other extracellular adenine nucleotides has been demonstrated recently. Schnurr et al. [2 ] and la Sala et al. [3 ] showed that ATP (or its slowly hydrolyzed derivative ATP{gamma}S) and TNF-{alpha} have a synergistic effect on the expression of CD83 and T cell stimulation by monocyte-derived human DC. However, they reported opposite effects on IL-12 secretion, potentiation [2], or inhibition [3 ]. This contradiction was resolved by Wilkin et al. [4 ], who showed that ATP{gamma}S potentiated the IL-12p40 release induced by different maturation agents (TNF-{alpha}, LPS, and CD40L) as long as the IL-12p40 concentration is inferior to a threshold value, whereas inhibition was observed above this value, and that LPS-induced production of IL-12p70 was always inhibited by ATP{gamma}S. Furthermore Wilkin et al. [5 ] provided evidence that the action of ATP{gamma}S involves the P2Y11 receptor and is mediated by cyclic adenosine monophosphate (cAMP). Indeed, the rank order of potency of various adenine nucleotides was consistent with the pharmacological profile of recombinant human (rh)P2Y11 receptor, their action was associated with an increase in cAMP, which could not be explained by degradation into adenosine and activation of A2 receptors, and the expression of P2Y11 in human DC was documented by quantitative reverse transcriptase-polymerase chain reaction. Further studies showed that ATP modulates the expression of chemokines and chemokine receptors in a way similar to known cAMP-elevating agents [6 ] and confirmed the role of the P2Y11 receptor [7 ]. Taken together, these data indicate that similar to the action of PGE2 [1 , 8 ], ATP acting via P2Y11 and cAMP favors a semimaturation state of DC associated with a T helper cell type 2 (Th2) response or a T regulatory (Treg) response.

Until now, almost no data are available concerning the effect of other adenine nucleotides on DC maturation, especially adenosine 5'-diphosphate (ADP) and other diphosphates. la Sala et al. [3 ] showed that ADP also inhibits IL-12 production but apparently to a lower extent than ATP. Calcium transients were observed in human monocyte-derived DC stimulated with ADP, but the identity of the P2Y receptor(s) involved was not characterized [9 , 10 ]. The recent discovery of the P2Y13 receptor, an ADP-selective receptor preferentially expressed in the immune system [11 ], in particular, in DC [12 ], was an incentive to further characterize the actions of ADP on DC. The observation in mice that Langerhans and DC require CD39, an enzyme that degrades ATP into ADP, for optimal stimulation of T cells [13 ] is another indication that ATP and ADP might have a distinct profile of action on DC. We therefore decided to study in detail the effects of ADP and other adenine nucleotides diphosphates on the maturation of monocyte-derived DC. We compared the effect of slowly hydrolyzed derivatives of ADP and ATP, ADPßS and ATP{gamma}S, respectively, on the production of cytokines (IL-12, IL-10, TNF-{alpha}) and the surface expression of CD83. We also investigated the signaling pathway of ADP and its analogs using intracellular calcium ([Ca2+]i) measurements, cAMP assay, and Western blotting measurements of extracellular signal-regulated kinase (ERK)1/2 phosphorylation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
rh Granulocyte macrophage-colony stimulating factor (GM-CSF) or Leucomax was supplied by Novartis (Basel, Switzerland) and rhIL-4, by R&D Systems (Abingdon, GB). ADP, ADPßS, ATP, ATP{gamma}S, Ap3A, 2-methylthio-ADP (2MeSADP), pertussis toxin (PTX), and 1,2-bis(O-aminophenyl-ethane-ethane)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) were purchased from Sigma Chemical Co. (St. Louis, MO). AR-C69931MX was a generous gift from Drs. Jonathan D. Turner and Darshan Shah (AstraZeneca, Wilmington, DE). MRS-2179 was obtained from Tocris Chemicals (Ellisville, MO). Forskolin and PD98059 were purchased from Calbiochem (La Jolla, CA). Rolipram was a gift from the Laboratories Jacques Logeais (Trappes, France). Methanol Spectranal was purchased from Riedel-de-Haen (Seelze, Germany). The culture medium used in this study was RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 25 mM HEPES buffer, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 mg/ml gentamicin, all purchase from Gibco-BRL (Ghent, Belgium), and 5 x 10–5 M ß-mercaptoethanol (ß-ME; Merck, Darmstadt, Germany).

DC generation and maturation
Immature human DC were derived from adherent peripheral blood monocytes of normal donors as described previously [14 ]. In brief, peripheral blood mononuclear cells (PBMC) were isolated from leukocyte-enriched buffy coats by standard density gradient centrifugation using Lymphoprep solution from Nycomed (Oslo, Norway), resuspended in complete medium and 2.5 x 108 PBMC, and were allowed to adhere during 2 h at 37°C at 5% CO2 in air in 75 cm2 cell-culture flasks. Nonadherent cells were removed and rinsed; adherent cells were cultured in 15 ml complete medium supplemented with 800 U/ml GM-CSF and 500 U/ml IL-4. GM-CSF and IL-4 were added twice a week. After 5 or 6 days of culture, cells were replated at a density of 1 x 106 cells/ml in 24 multiwells in complete medium with GM-CSF and IL-4. Nucleotides or other tested agents were added for 24 h. The purity of each cell preparation was evaluated by looking at the expression of CD1a, human leukocyte antigen (HLA)-DR, CD14, CD83, and CD3: 90–95% of cells were CD1a- and HLA-DR-positive.

Flow cytometric analysis
Cell staining was performed using fluorescein isothiocyanate-conjugated anti-human CD83 [immunoglobulin G (IgG)1,k, HB15e] supplied by PharMingen (San Diego, CA) on 2 x 105 cells in 100 µl phosphate-buffered saline (PBS) with 0.1% sodium azide of 30 min in the dark at 4°C. After washing with 2 ml PBS, samples were analyzed on a FACSort® (Becton Dickinson, Franklin Lakes, NJ). Data were analyzed and presented using Cellquest software (Becton Dickinson); the number of gated events was at least 10,000.

Cytokine assay
Human IL-12p40, IL-12p70, IL-10, and TNF-{alpha} were measured in DC supernatants by enzyme-linked immunosorbent assay (ELISA) using commercially available kits. IL-12p40, IL-10, and TNF-{alpha} detection kits were purchased from Biosource (Camarillo, CA). IL-12p70 detection kits were purchased from R&D Systems. IL-12p40 kits recognize p40 monomer, p40 homodimer, and p70 heterodimer, whereas IL-12p70 kits only recognize bioactive p70 heterodimer.

[Ca2+]i measurements
Levels of [Ca2+]i were measured at room temperature with the fura-2 or fluo-4 fluorescent Ca2+-binding dyes. After 5–6 days of incubation with IL-4 and GM-CSF at 37°C in a 5% CO2 and humidified atmosphere, DC were washed twice in Ca2+- and Mg2+-free Hanks’ balanced saline solution (HBSS; Gibco-BRL) and loaded at a concentration of 107 cells/ml with 0.25 mM sulfinpyrazone (Sigma Chemical Co.), 5 µM cell-permeant fura-2/AM or fluo-4/AM (Molecular Probes, Leiden, The Netherlands), and 100 µg/ml pluronic acid F-127 (Molecular Probes) used to facilitate cell loading for 30 min at 37°C. Cells were then washed twice HBSS with 0.25 mM sulfinpyrazone, resuspended at a final concentration of 106 cells/ml, and placed in a 37°C water bath. For fura-2 assays, fluorescence in a 2-ml suspension was determined in an LS-50B fluorometer (Perkin Elmer, Überlingen, Germany) equipped with a computer. Excitation wavelengths were 340 and 380 nm, and the fluorescence emission was 510 nm. All measurements were carried out at room temperature. For fluo-4 assays, we used a 96-well black plate with a transparent bottom (Packard-Bell, The Netherlands). Fluorescence of 50 µl suspension per well was determined using the functional drug-screening system (Hamamatsu, Japan); the excitation wavelength was 488 nm, and the fluorescence emission was 520 nm.

cAMP measurements
Cells were preincubated for 30 min in complete culture medium with 25 µM rolipram and then incubated in the same medium for 12 min in the presence of the agonists with or without forskolin (3 µM). The incubation was stopped by the addition of 1 ml HCl 0.1 M. The incubation medium was dried up, and the samples were resuspended in water and diluted as required. cAMP was quantified by radioimmunoassay (RIA) after acetylation as described previously [15 ]. Each experimental condition was tested in triplicate.

Western blot analysis of phosphorylated ERK1 and ERK2 proteins
Immature DC were washed with RPMI 1640 and serum-starved with RPMI 1640 during 2 h in 24-well plates, each well containing 500 µl cell suspensionat a density of 106 cell/ml. Cells were washed with HBSS and lysed on ice in 120 µl Laemmli buffer [10% (w/v)] glycerol, 5% (v/v) ß-ME, 2.3% (w/v) sodium dodecyl sulfate (SDS), 62.5 mM Tris-HCl, pH 6.8, with proteinase inhibitors (Roche, Belgium): 1 µg ml–1 leupeptin, 60 µg ml–1 pefabloc, 1 µg ml–1 aprotinin; and phosphatase inhibitors (Sigma, Belgium): 1 mM sodium orthovanadate and 10 mM sodium fluoride. The protein concentration was determined using the method of Minamide and Bamburg [16 ]. The same amount of protein (25 µg) for each condition was electrophoresed on a 12% SDS-polyacrylamide gel. Proteins were then transferred overnight at 30 V and 4°C onto a nitrocellulose membrane using 20 mM Tris, 154 mM glycine, 20% (v/v) methanol as a transfer buffer. Immunodetection was achieved using the enhanced chemiluminescence (ECL) Western blotting detection system (ECL, Amersham Pharmacia Biotech, Bergrand, The Netherlands) with a biotinylated secondary mouse antibody (1:25,000, Amersham Pharmacia Biotech). The monoclonal antibody (mAb), specific for the dually phosphorylated forms of ERK1 and ERK2 (at Th202 and Tyr204), was used at a 1:1000 dilution (New England Biolabs, Beverly, MA).

Evaluation of nucleotide degradation
After 5–6 days with IL-4 and GM-CSF at 37°C in a 5% CO2 and humidified atmosphere, DC were washed twice and resuspended in Krebs-Ringer-HEPES buffer (124 mM NaCl, 5 mM KCl, 1.25 mM MgSO4, 1.45 mM CaCl2, 1.25 mM KH2PO4, 25 mM HEPES, pH 7.4, and 8 mM D-glucose) at 106 cells/ml. Cells were then incubated in the presence of 100 µM ADP, ATP, ADPßS, or ATP{gamma}S for various times. Cell supernatants were filtered through a 0.4-µm filter (Millipore, Brussels, Belgium) and stored at –20°C. Nucleotides were separated by high-pressure liquid chromatography (HPLC) on a µBondapack C18 reverse-phase 3.9 x 300 mm column (Millipore) and eluted at 1.5 ml/min with a linear methanol gradient between solution A [5 mM tetrabutylammonium dihydrogen phosphate (TBAP), 60 mM KH2PO4, and 5% methanol, pH=6] and solution B (5 mM TBAP, 60 mM KH2PO4, and 35% methanol, pH=6) at 2% per min. The column was calibrated using a panel of nucleotides (AMP, ADP, ADPßS, ATP, and ATP{gamma}S).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Nucleotide degradation at the DC surface
Rapid degradation of extracellular nucleotides by ecto-nucleotidases, in particular the conversion of ATP into ADP, complicates the interpretation of the pharmacological effects of nucleotides. The presence of ecto-nucleotidases on the surface of human monocyte-derived DC has been reported previously [17 ]. As shown in Figure 1 , we confirmed that ATP and ADP undergo rapid degradation with similar kinetics. However, no degradation of ATP{gamma}S and ADPßS was observed after 10 min. The degradation over a 10-min period was 37 ± 7.48% for ADP and 55.3 ± 14.6% for ATP (mean±SD of at least two independent experiments).



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  		Figure 1. Degradation rate of di- and triphosphate nucleotides in the presence of immature DC. ATP, ADP, ATP{gamma}S, and ADPßS at 100 µM were incubated in the presence of immature DC (106 cells/ml). The concentrations of nucleotides were assayed at different times by HPLC as described in experimental procedures. These data are representative of at least two independent experiments.

 
ADP induces calcium increase
We tested the ability of ADP, ADPßS, and 2MeSADP to increase [Ca2+]i in immature DC. In these assays, ATP{gamma}S and regulated on activation, normal T expressed and secreted (RANTES), a ligand of CC chemokine receptor 5, were used as positive controls. As shown in Figure 2A , ADP (10 µM) and RANTES (100 nM) induced a fast and transient increase of calcium, whereas ATP{gamma}S (100 µM) exhibits early and delayed phase (data not shown). The pretreatment of DC with PTX (5 µg/ml) for 5 h totally abolished the ADP and RANTES response (Fig. 2A) . The insensitivity of ATP{gamma}S response to PTX treatment was already reported [9] and confirmed in our experiments. To exclude any effect mediated by a calcium channel, we used EGTA (7.5 mM) as a calcium chelator agent. As shown in Figure 2A , the response to ADP was not affected, whereas the response to RANTES and the late phase of ATP{gamma}S response (data not shown) were abolished. The same pattern of response was observed using ADPßS (100 µM) instead of ADP (data not shown). To investigate the P2Y subtype involved in the ADP response, we tested different antagonists of ADP-sensitive P2Y receptors: MRS-2179 (P2Y1) and AR-C69931MX, a known P2Y12 antagonist that was recently shown to also inhibit the P2Y13 receptor [18 ]. Immature DC were pretreated for 5 min with the antagonists and stimulated with ADP (10 µM). ATP{gamma}S and RANTES were used as controls. None of the two antagonists had an effect on the ADP response (Fig. 2B) . The response to ADPßS (100 µM) was also unaffected by these inhibitors (data not shown). Furthermore, we compared concentration-action curves for ADP, ADPßS, 2MeSADP, and Ap3A. As shown in Figure 3 , the rank order of potency was ADP = 2MeSADP > ADPßS > Ap3A.



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  		Figure 2. Effect of ADP on [Ca2+]i mobilization in immature DC loaded with fura-2 (A) ADP (10 µM) induces a transient increase in [Ca2+]i, which is blocked by PTX (5 µg/ml) pretreatment (5 h) and unaffected by EGTA (7.5 mM). RANTES (100 nM) and ATP{gamma}S (100 µM) were used as controls. (B) Immature DC were pretreated 5 min with MRS-2179 (10 µM), a P2Y1 antagonist, or with AR-C69931MX (10 µM), a P2Y12 and P2Y13 antagonist, before being challenged with ADP, ATP{gamma}S, and RANTES. The ratio of emission represents the ratio between 340 nm and 380 nm. These data (A, B) are representative out of four independent experiments.

 


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  		Figure 3. Immature DC loaded with fluo-4 (as reported in experimental procedures) were challenged with various concentrations of ADP, ADPßS, 2MeSADP, and Ap3A. The ratio of emission represents the ratio between the maximum value of the peak and the basal value before stimulation mesured at 520 nm. Data are given as the mean ± SD of triplicate points and are representative of three independent experiments.

 
ADP does not act through the cAMP pathway like ATP{gamma}S
The ATP-induced maturation of monocyte-derived DC is mediated through the elevation of cAMP [5 ]. We tested different diphosphate adenine nucleotides, and we compared their effects to the ATP{gamma}S-induced elevation of cAMP. As shown in Figure 4 , ADP, ADPßS, and 2MeSADP (100 µM) did not increase intracellular cAMP in contrast to ATP{gamma}S (10 µM). We also assessed the capacity of ADP to inhibit the cAMP accumulation induced by forskolin (3 µM), but no effect was observed (data not shown).



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  		Figure 4. Effect of triphosphate and diphosphate adenine nucleotides on cAMP accumulation. DC were preincubated for 20 min in RPMI-1640/HEPES buffer with rolipram and incubated in the same medium for 10 min in the absence (CTL) or presence of ATP{gamma}S (10 µM), ADP (100 µM), ADPßS (100 µM), or 2MeSADP (100 µM). cAMP was quantified by RIA after acetylation. Data are given as the mean ± SD of triplicate points and are representative of three independent experiments.

 
Adenine nucleotides diphosphates induce ERK1 phosphorylation
As described previously [19 ], ERK1/2 signaling plays a key role in the regulation of cytokine production by DC following their activation with LPS or TNF-{alpha}. Moreover, our group previously showed that ADP can induce the phosphorylation of ERK1/2 through the stimulation of human P2Y13 [11 ]. Considering these data, we stimulated immature DC with ADP, ADPßS, and 2MeSADP for different times and concentrations. As shown in Figure 5A , the phosphorylation of ERK1 was already detectable after 5 min of stimulation and still present after 20 min. The time-courses of ADP and 2MeSADP actions were similar. As shown in Figure 5B , the phosphorylation was already detectable at 1 µM for all the compounds. AR-C69931MX (10 µM) inhibited ERK1 phosphorylation, whereas MRS-2179 (10 µM) had no effect (Fig. 5C) . AR-C69931MX also inhibited ERK1 phosphorylation at 1 µM (data not shown). As reported in the previous section, ADP, 2MeSADP, and ADPßS induce a release of calcium from intracellular stores. We used an [Ca2+]i chelator BAPTA-AM to investigate a possible calcium dependency of ERK1 phosphorylation. As shown in Figure 5D 1 h pretreatment with BAPTA-AM (50 µM) totally abolished the phosphorylation of ERK1. Finally, we pretreated the cells overnight with 100 ng/ml PTX, and we observed that the response to ADPßS (10 µM) was totally abolished by this treatment (Fig. 5E) .



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  		Figure 5. Effect of adenosine diphosphate nucleotides on ERK1 phosphorylation in human immature DC. (A) Immature DC were not stimulated (Ctrl) or stimulated with ADP (100 µM) or 2MeSADP (100 µM) for various times. (B) Immature DC were not challenged (Ctrl) or challenged with different concentrations of ADP, 2MeSADP, or ADPßS for 10 min. (C) Immature DC were preincubated with MRS-2179 (10 µM) or with AR-C69931MX (10 µM). After 5 min of incubation, cells were not challenged (Ctrl) or challenged with ADP (10 µM) or 2MeSADP (10 µM) and ADPßS (10 µM). (D) DC were preincubated with BAPTA-AM (50 µM) for 1 h before a 10-min stimulation by ADP (10 µM) or ADPßS (10 µM). (E) Immature DC were treated with PTX for 18 h before being challenged with 10 µM ADPßS for 10 min. The phosphorylation of ERK1 in response to ADP, 2MeSADP, and ADPßS was observed for six donors, and these data are representative of two (E) or at least three (A–D) independent experiments.

 
Differential effects of ADPßS and ATP{gamma}S on cytokine production and cell-surface markers
The previously reported effect of ATP on CD83 up-regulation in the absence or presence of LPS [6] was compared with the effect of ADPßS. As shown in Figure 6 , ADPßS (250 µM) alone is less efficient, 33% ± 2.3% CD83-positive cells, in up-regulating the CD83 marker in comparison with ATP{gamma}S (250 µM), 59.6% ± 1.0% CD83-positive cells (mean±SD of four independent experiments). No potentiating effect of ADPßS was observed in the presence of LPS (10 µg/ml), even when PD98059, a selective inhibitor of MEK, the mitogen-activated protein kinase (MAPK) kinase that phosphorylates ERK1/2, was present (data not shown).



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  		Figure 6. ADPßS and ATP{gamma}S effect on CD83 up-regulation. Immature DC were untreated (Ctrl), stimulated with 250 µM ADPßS or ATP{gamma}S, or induced to mature with 10 µg/ml LPS in the absence or presence of nucleotides. After 24 h, DC were stained with mAb against CD83 (thin-lined histograms) or with isotype-matched control Ig (bold-lined histograms). Numbers indicate the percentage of CD83-positive cells. These data are representative of four independent experiments.

 
ADPßS (10 µM) inhibited the production of IL-12p40 (Fig. 7A ) and IL-12p70 (Fig. 7B) at all LPS concentrations used (1–100 ng/mL). The same inhibitory effect of ADPßS was also observed for TNF-{alpha} secretion (Fig. 7C) . These effects of ADPßS were similar to those of ATP{gamma}S, tested in the same experiments. The main difference between ADPßS and ATP{gamma}S resided in their capacity of modulating the production of IL-10. In fact, ADPßS totally suppressed IL-10, whereas ATP{gamma}S amplified the production of this cytokine (Fig. 7D) .



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  		Figure 7. Effect of ADPßS and ATP{gamma}S alone or in combination with LPS (1, 10, 100 ng/ml) on IL-12p40 (A), IL-12p70 (B), TNF-{alpha} (C), and IL-10 (D) secretion by DC, which were incubated with medium (CTRL) or ADPßS (10 µM) or ATP{gamma}S (100 µM) alone or with different concentrations of LPS. After 24 h, supernatants were harvested for ELISA measurement of cytokines. Results are expressed as ng/106 cells/ml (IL-12p40 and TNF-{alpha}) and as pg/106 cells/ml (IL-12p70 and IL-10) and represent the mean ± range of duplicate points in one representative out of four independent experiments.

 
The rank order of potency to inhibit LPS-induced IL-12p70 release was ADPßS > ADP > 2MeSADP = Ap3A (Fig. 8A ). To identify which P2Y receptor could mediate the observed effects of adenine nucleotides diphosphates on cytokine production, we tested the antagonists AR-C69931MX and MRS-2179. As shown in Figure 8B , none of these antagonists reversed the inhibitory effect of ADPßS (10 µM) on LPS-induced IL-12p70 production. The inhibition of IL-10 and TNF-{alpha} production by ADPßS were also resistant to those antagonists (data not shown).



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  		Figure 8. Pharmacological characterization of the effect of adenine diphosphate nucleotides. (A) Concentration action curves of different adenine diphosphate nucelotides. Immature DC were challenged with LPS (100 ng/ml) in the presence of ADP, ADPßS, 2MeSADP, or Ap3A at different concentrations. After 24 h, supernatants were harvested for ELISA detection of IL-12p70. Results are expressed as pg/106 cells/ml and represent the mean ± range of duplicate points in one representative experiment out of three independent experiments. (B) Effect of ADP-sensitive P2Y receptor antagonists on IL-12p70 production. DC were preincubated or not (w/o antagonist) with MRS-2179 (10 µM) or AR-C69931MX (10 µM) for 10 min. DC were then challenged with LPS (100 ng/ml) alone or in the presence of ADPßS (10 µM). After 24 h, supernatants were harvested for ELISA detection of IL-12p70. Results are expressed as pg/106 cells/ml and represent the mean ± range of duplicate points in one representative experiment out of four independent experiments. N.D., Not detected.

 
To address the possible role of ADPßS-induced ERK1 phosphorylation in the cytokine production, we preincubated the cells for 1 h in the presence of PD98059 at 25 µM. As shown in Figure 9 , PD98059, which potentiated the LPS-induced production of IL-12p40, as previously reported [19 ], did not reverse the inhibition of IL-12p40 production induced by ADPßS (10 µM). Similary, PD98059 did not reverse the inhibitory effect of ADPßS on LPS-induced stimulation of TNF-{alpha} and IL-10 (data not shown). We also tested that inhibition of cytokine production by ADPßS was not a result of cytotoxicity: Propidium iodide incorporation was not modified by ADPßS (data not shown).



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  		Figure 9. Effect PD98059, a selective inhibitor of MEK, the MAPK that phosphorylates ERK1/2, on IL-12p40 production. DC were preincubated for 1 h in the presence of PD98059 (25 µM) before stimulation with medium (CTRL) or with ADPßS (10 µM) alone or in addition, with LPS (100 ng/ml). After 24 h, supernatants were harvested for ELISA detection of IL12p40. Results are expressed as ng/106 cells/ml and represent the mean ± range of duplicate points in one representative experiment of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several groups have reported that extracellular ATP modulates the function of human monocyte-derived DC [2 3 4 5 6 7 , 20 ]. These cells express functional P2X receptors and also P2Y1,2,4,6 and P2Y11 receptors [17 ]. ATP induces the semimaturation of DC, characterized by the up-regulation of costimulatory molecules (such as CD83) [3 ] and various chemokines [6 ] but by an inhibition of the production of proinflammatory cytokines such as IL-12 [2 , 4 ]. This profile of action is similar to that of cAMP-elevating agents, i.e., PGE2 [8 ], and is indeed associated with an increase in cAMP, presumably mediated by the P2Y11 receptor [5 , 7 ]. ATP, via inhibition of IL-12 and potentiation of IL-10, will impair the initiation of a Th1 response and favor a Th2 response or a Treg cell response [1 ]. As DC express ecto-nucleotidases such as CD39 [13 , 17 ], ADP is likely to be generated following release of ATP from necrotic cells or from CD4+ T lymphocytes [13 ]. An important role of CD39 in the immune system was revealed by its inactivation in mice, which caused exacerbated skin inflammation but attenuated T cell-mediated, allergic contact hypersensitivity as a result of a decreased capacity of CD39–/– DC to activate T cells [13 ]. As CD39 converts ATP into ADP, this suggests that these two nucleotides can have distinct effects on DC. The effects of ADP on DC have been little studied so far. la Sala et al. [3] showed that ADP also inhibited IL-12 production but apparently, to a lesser extent than ATP. Idzko et al. [10] showed that ADP, like ATP, was chemotactic for immature but not LPS-matured DC, an action associated with a rise in cytoplasmic calcium and actin polymerization. In the present study, we have investigated the signaling mechanisms of ADP and related nucleotides in DC and compared their effects on DC function with those of nucleotide triphosphates.

There were two major differences between the effects of ATP{gamma}S and those of ADP, ADPßS, or 2MeSADP on DC. First, unlike ATP{gamma}S, the diphosphates had no stimulatory effect on cAMP accumulation. Second, the effect of the diphosphates on [Ca2+]i increase was sensitive to PTX, whereas the action of ATP{gamma}S was not. This suggests the involvement of a G-protein-coupled receptor (GPCR) coupled to Go/Gi protein. However, the [Ca2+]i rise was neither inhibited by AR-C69931MX, which is an antagonist of the two Gi-coupled ADP receptors, P2Y12 and P2Y13 [18 ], or by MRS-2179, an antagonist of the third known ADP receptor, P2Y1. As we checked that degradation of ADP was minimal during the time-course of the calcium transient and that no degradation of ADPßS occurred over 10 min, these data suggest that a previously uncharacterized ADP receptor is involved this action on DC. Several orphan GPCRs are structurally related to P2Y receptors. In particular, H963 and GPR87, the genes that are colocalized with those of P2Y1, P2Y12, and P2Y13 on chromosome 3q24-3q25 [21 ], are potential candidates, as their mRNA is expressed in organs such as spleen and thymus. It is interesting that ADP, 2MeSADP, and ADPßS also induced ERK1 phosphorylation and that the effect was partially inhibited by AR-C69931MX. This suggests an involvement of the P2Y13 receptor, as its mRNA is preferentially expressed in organs such as the spleen and in DC [11, 12], but a role of the P2Y12 receptor cannot be excluded. Taken together, our results suggest the involvement of two distinct Go/i-coupled receptors in the action of ADP on DC: the P2Y13 (or P2Y12) receptor on one hand and an uncharacterized receptor on the other hand. That last receptor would be solely responsible for the Ca2+ rise, whereas both would be required for ERK phosphorylation that was indeed sensitive to inhibition by PTX and [Ca2+]i chelation. There are numerous examples of the simultaneous involvement of Gi and Ca2+ in ERK phosphorylation [22 23 24 25 ].

The action of adenine nucleotides diphosphates on DC function, especially that of ADPßS, which is particularly relevant in view of its slow degradation (Fig. 1) , is globally similar to that of ATP and ATP{gamma}S, previously reported: up-regulation of CD83, inhibition of IL-12, and TNF-{alpha} production. There was, however, one major difference: IL-10 production was potentiated by ATP{gamma}S and inhibited by ADPßS. It is known that inhibition of IL-12 production can result from the activation of Gs- and Go/i-coupled receptors [26 , 27 ]. Inhibition of IL-12 can be a result of an increase in [Ca2+]i [28 ] or to ERK activation [19 , 29 , 30 ]. In the case of ADPßS, it seems that calcium plays a dominant role over ERK, as AR-C69931MX, which blocks the effect on ERK but not on [Ca2+]i, did not prevent the inhibition of IL-12 production. This is also consistent with the inability of PD98059, an inhibitor of ERK1/2 phosphorylation, to reverse the inhibitory effect of ADPßS on IL-12, TNF-{alpha}, and IL-10 production.

In conclusion, it now appears that ATP released from dying cells or from activated CD4+ T lymphocytes [13 ] can modulate DC function by several mechanisms. It can act directly via a cAMP increase, presumably mediated by the P2Y11 receptor, and indirectly via its degradation into ADP, which seems to act via 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.


   ACKNOWLEDGEMENTS
 
This work was supported by an Action de Recherche Concertée of the Communauté Française de Belgique, by the Belgian Program on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Federal Service for Science, Technology and Culture, and by grants of the Fonds de la Recherche Scientifique Médicale and the Fonds Emile DEFAY. F. M. is supported by the Fonds National de la Recherche Scientifique/FRIA, Belgium. D. C. is an associated researcher of the Fonds National de la Recherche Scientifique. N. S. G. is supported by Euroscreen. We thank F. Wilkin for her help and Drs. J. D. Turner and D. Shah (AstraZeneca, Wilmington, DE) for their generous gift of AR-C69931MX.

Received January 21, 2004; revised May 18, 2004; accepted June 7, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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