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The Journal of Immunology, 2007, 179, 3550 -3558
Copyright © 2007 by The American Association of Immunologists, Inc.
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Gene Expression Profiling Defines ATP as a Key Regulator of Human Dendritic Cell Functions1

Nathalie Bles2,*, Michael Horckmans2,*, Anne Lefort*, Frédéric Libert*, Pascale Macours{dagger}, Hakim El Housni{ddagger}, Frédéric Marteau*, Jean-Marie Boeynaems*,{dagger} and Didier Communi3,*

* Institute of Interdisciplinary Research, Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, Brussels, Belgium; {dagger} Department of Medical Chemistry, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium; and {ddagger} Department of Genetics, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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) (ATP{gamma}S) and PGE2 in human MoDCs using microarray technology. At 6 h of stimulation, ATP{gamma}S 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 ATP{gamma}S 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 ATP{gamma}S and PGE2 target genes in MoDCs including CSF-1, MCP-4/CCL13 chemokine, vascular endothelial growth factor-A, and neuropilin-1. ATP{gamma}S strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion, which are involved in monocyte and dendritic cell (DC) differentiation. Additionally, ATP{gamma}S down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1{alpha}, CCL4/MIP-1beta, 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 ATP{gamma}S, ATP, or PGE2, alone or in synergy with LPS. Finally, flow cytometry experiments have demonstrated that ATP{gamma}S, 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 ATP{gamma}S and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Maturation of dendritic cells (DCs)4 in response to LPS, CD40 ligand, or proinflammatory cytokines is reflected by a loss of endocytosis, the surface expression of stable MHC-peptide complexes and costimulatory molecules (CD80, CD86), the production of cytokines like IL-12, and a shift in the expression of chemokines and their receptors allowing DC migration to lymphoid organs (1). In human monocyte-derived DCs (MoDCs), ATP, which is released inter alia from necrotic cells, induces the up-regulation of costimulatory molecules (2, 3, 4) and regulates various chemokines and chemokine receptors (5, 6). ATP also regulates the action of LPS and other maturating agents on human DCs by inhibiting the production of proinflammatory cytokines such as IL-12, IL-1beta, TNF-{alpha}, and IL-6 and by potentiating anti-inflammatory IL-10 (2, 7). These features of ATP-treated DCs are compatible with semimature DCs whose potential involvement in central and peripheral tolerance has been previously discussed (8, 9, 10). This profile of action of ATP is similar to that of cAMP-elevating agents such as PGE2 and is indeed associated with an increase in cAMP, presumably mediated by the P2Y11 receptor (4). ATP, via inhibition of IL-12 and potentiation of IL-10, will thus impair the initiation of a Th1 response and favor a Th2 response or tolerance (2, 7). Recently, we reported the critical role of ATP-mediated signal transduction in triggering two targets (thrombospondin-1 (TSP-1) and IDO) involved in T cell immunosuppression, suggesting a potential role of extracellular nucleotides in immune tolerance (11).

In the present study, we have extensively compared the target genes of adenosine 5'-O-(3-thiotriphosphate) (ATP{gamma}S) and PGE2 in human MoDCs using a combination of microarray technology, quantitative RT-PCR experiments, ELISAs, and flow cytometry analysis. This study is the first one to provide gene expression profiles of ATP{gamma}S and PGE2 in MoDCs and gives an overview of the potential actions of ATP and PGE2 on human DCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

ATP, ATP{gamma}S, PGE2, forskolin, and LPS were obtained from Sigma-Aldrich.

Preparation of MoDCs

PBMCs were isolated from leukocyte-enriched buffy coats of healthy volunteer donors by standard density gradient centrifugation using Lymphoprep solution from Nycomed. PBMCs (2.5 x 108) were allowed to adhere for 1 h and 30 min at 37°C at 5% CO2 in air in 75-cm2 cell culture flasks. Nonadherent cells were removed and adherent cells were cultured in 15 ml of RPMI 1640 medium supplemented with 800 U/ml GM-CSF and 500 U/ml IL-4. GM-CSF and IL-4 were also added a second time 2 days after the adhesion step. Five days after the adhesion step, the purity of each cell preparation was evaluated using flow cytometry by analyzing the expression of two markers of DCs, HLA-DR and CD1a. Moreover, the absence of monocytes, lymphocytes, and mature DCs was always checked by staining cell preparation using CD14, CD3, and CD83 markers, respectively. For our experiments, we have only used cell preparations of HLA-DR+CD1a+ immature DCs displaying at least 95% purity. Cells were then plated at 106 cells/ml in 24 multiwells in complete medium. Agents were then added for different periods of time.

Flow cytometry analysis

Cells were labeled with FITC-conjugated anti-human CD83 and PE-conjugated anti-human CD1a, HLA-DR, CD14, CD3, and neuropilin-1 (NRP-1) Abs (BD Pharmingen). Cells (2 x 105) were incubated in 100 µl of PBS with 0.1% sodium azide for 30 min in the dark at 4°C, washed with 1 ml of PBS, and analyzed on a Cytomics FC 500 flow cytometry system (Beckman Coulter). Data were analyzed using CXP cytometry software; the number of events was at least 10,000. We have checked the purity (>95%) and immaturity of our preparations of DCs by FACS analysis, identifying CD1a+HLA-DR+CD83–CD14–CD3– cells.

RNA isolation and microarray analysis

Immature DCs (106 cells/ml) were stimulated by ATP{gamma}S (100 µM) or PGE2 (500 nM) for 2, 6, 12, or 24 h in complete RPMI 1640 medium. RNA was isolated using TRIzol reagent (Invitrogen Life Technologies) and an RNeasy kit column (Qiagen). RNA was reverse transcribed using oligo(dT) primers and ArrayScript (Ambion; Applied Biosystems), labeled and hybridized as previously described (12) to Human Exonic Evidence Based Oligonucleotide (HEEBO) arrays containing on average 44,544 human 70-mer oligonucleotides (Stanford University, Palo Alto, CA). Data were obtained for four time points using RNA from two independent donors.

Quantitative RT-PCR (qRT-PCR) experiments

For each target gene, primers were selected using Primer Express 2.0 software (PCR product size: 100–150 bp; primer size: 20–25 bp; Tm: 58°C to 60°C). Several control genes were tested for their stability in our system (YWHAZ, B2M, RPL13A, and SDHA) (13). Two of these control genes (B2M and SDHA) were selected after analysis using the geNorm program. RT-PCR amplification mixtures (25 µl) contained 2 ng of template cDNA, Power SYBR Green PCR Master Mix (12.5 µl) (Applied Biosystems), and 200 nM forward and reverse primer. Reactions were run on a 7500 Fast Real-Time PCR System (Applied Biosystems). The cycling conditions were 10 min for polymerase activation at 95°C and 40 cycles at 95°C for 15 s and 60°C for 60 s. Mean ± SD values were obtained for each gene using qBase software. Each assay was performed in duplicate for two independent donors.

ELISA

Immature DCs were stimulated by different agents for 24 h at 106 cells/ml in 24 multiwells. DC supernatants were collected and CSF-1, vascular endothelial growth factor (VEGF)-A, and CCL13 were measured by ELISA using commercially available kits from R&D Systems.

Kynurenine measurements

DCs were stimulated with IFN-{gamma} at 100 U/ml alone or in combination with ATP{gamma}S at 100 µM or PGE2 at 5 µM for 24 h. Cells were then washed and resuspended in red phenol-free complete medium supplemented with 300 µM L-tryptophan. After 5 h, supernatants were collected and kynurenine concentration was quantified by HPLC. Culture supernatants (400 µl) were extracted with 80 µl of 10% trichloroacetic acid, the precipitate was removed by centrifugation, and the supernatant was diluted in the initial mobile phase composed of deionized water, 5% (v/v) methanol, 1% (v/v) acetic acid, and 5 mM hexane sulfonic acid. Samples were injected onto an Atlantis dC18 reverse phase column (4.6 x 300 mm, 3 µm; Waters) and eluted with a linear gradient of methanol (5–40% over 35 min) at 1 ml/min. Absorbance was measured at 370 nm and compared against a standard curve of L-kynurenine.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Comparison of ATP{gamma}S and PGE2 gene expression profiles in MoDCs

First, we obtained the gene expression profiles of ATP{gamma}S and PGE2 in MoDCs by using microarray technology. ATP{gamma}S, which is more resistant to degradation by ectonucleotidases, was used instead of ATP to avoid additional gene regulations due to its degradation products such as ADP and adenosine. PGE2 displays effects similar to those of ATP on DC maturation and is also able to activate the cAMP pathway in MoDCs (14, 15). Immature MoDCs (106/ml) were stimulated for 2, 6, 12, or 24 h with 100 µM ATP{gamma}S or 500 nM PGE2. Microarray experiments have been performed using total RNA extracted from the stimulated and unstimulated DCs obtained from two independent donors. After amplification, the RNAs were labeled to hybridize arrays containing ~40,000 human specific oligonucleotides. We first performed a selection of sequences regulated at least 2-fold for at least for one agent and one time point (see Table S1 in the supplementary data).5 Among these 3,512 regulated sequences, 1,896 oligonucleotides were regulated at least 2-fold by ATP{gamma}S and only 97 oligonucleotides were regulated at least 2-fold by PGE2. From these experiments, it thus appeared that ATP{gamma}S was able to induce a very wide and early gene expression profile in MoDCs. As shown in Table I, there was a significant disproportion between the number of genes regulated by ATP{gamma}S and by PGE2 at a short time of stimulation (2 h and 6 h). At 24 h of stimulation, the number of genes commonly regulated by ATP{gamma}S and PGE2 was the highest (Table I).


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  		Table I. Number of genes regulated by ATP{gamma}S and/or PGE2 at 2, 6, 12, or 24 h in human MoDCsa

 
We then identified 57 regulated genes playing a role in inflammation, including chemokines, interleukins, CD markers, receptors, and glycoproteins. Among these genes, 26 sequences were regulated at least 2-fold by ATP{gamma}S only (Table II) and 31 sequences were regulated at least 2-fold by both ATP{gamma}S and PGE2 (Table III).


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  		Table II. List of genes regulated by ATP{gamma}S only and involved in inflammationa

 

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  		Table III. List of genes regulated by both ATP{gamma}S and PGE2and involved in inflammationa

 
Common ATP{gamma}S and PGE2 target genes in human MoDCs

First, our microarray data were consistent with the effects of ATP{gamma}S and PGE2 on DC maturation (2, 3, 4, 6, 14, 15). ATP{gamma}S and PGE2 both regulated genes encoding chemokine receptors such as CXCR4, CCR5, and CCR1 (Table III). Moreover, ATP{gamma}S up-regulated CD83, which is a surface marker specifically up-regulated upon DC maturation (3, 16). Many genes encoding chemokines (e.g., CCL2, CCL3, CCL4, and CCL22), glycoproteins (e.g., TSP-1), IL-7 and IL-15 receptors (Table III), and many other known or unknown genes (see supplemental data) were regulated in a similar way by ATP{gamma}S and PGE2 and more particularly at 24 h of stimulation.

ATP{gamma}S regulates a large panel of genes not regulated by PGE2 in MoDCs

The large expression profile of ATP{gamma}S included genes encoding chemokines (CCL7 and CCL24), interleukins (IL-1A and IL-16), CD markers (e.g., CD55, CD69, and CD72) and receptors (erythropoietin and CSF-1 receptors) (Table II), and a large number of other known or unknown genes (Table II and Table S1 in the online supplemental material).

Validation of target genes using quantitative PCR experiments

We have validated the regulation of several promising target genes displaying a link with the immune system using quantitative PCR experiments. SYBR Green experiments have been performed for CCL2, CCL3, CCL4, CCL13, CCR5, CD36, NRP-1, THBS-1, VEGF-A, INDO, CSF-1, and CSF-1R. The primer sequences for these 12 genes are listed in Table IV. The data have been obtained on two independent preparations of DCs and there was a good correlation with our microarray data (Fig. 1). Quantitative PCR experiments confirmed that VEGF-A, CCL13, CSF-1 and NRP-1 were effectively regulated by both ATP{gamma}S and PGE2 at least at one stimulation time point.


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  		Table IV. List of the specific primers used for the SYBR Green experiments

 

Figure 1
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  		FIGURE 1. Quantitative RT-PCR data obtained for 12 genes regulated by ATP{gamma}S and/or PGE2 and involved in inflammation. Ratios were obtained for ATP{gamma}S (—) and PGE2 (- - -) at 2, 6, 12, and 24 h for two independent preparation of MoDCs (means ± SEM) using SYBR Green technology. mRNA expression in ATP{gamma}S- or PGE2-treated cells and untreated cells has been normalized for each gene and each time point using two housekeeping genes (B2M and SDHA). Ratios were calculated comparing normalized expression of each gene in ATP{gamma}S- or PGE2- treated DCs to its normalized expression in untreated DCs.

 
Even if ATP{gamma}S expression profile was very large, we decided to focus our attention on four genes commonly regulated by ATP{gamma}S and PGE2 such as CSF-1, VEGF-A, CCL13/MCP-4 chemokine, and NRP-1 because their regulation was not yet reported, unexpected, and promising. Some particular genes regulated by ATP{gamma}S such as FOXO3A transcription factor and SOD2 (Table II) have also retained our attention.

ATP{gamma}S is a potent negative signal for the secretion of several chemokines by MoDCs

We have previously shown that ATP{gamma}S down-regulated the secretion of chemokines such as CCL2 and CCL3 by MoDCs (5). These down-regulations were confirmed in our microarray experiments and were also observed in response to PGE2 (Table III). Additionally, we have observed down-regulation of genes encoding CCL4/MIP-1beta and CCL8/MCP-2 chemokines in response to ATP{gamma}S and PGE2 (Table III).

Our microarray and quantitative PCR data additionally revealed the significant down-regulation of CCL13 by ATP{gamma}S and to a lesser extent by PGE2 (Table III), which was confirmed by quantitative PCR (Fig. 1). CCL2, CCL3, CCL4, CCL8, and CCL13 are all recruiters of monocytes and immature DCs. Their concomitant down-regulation in response to ATP{gamma}S could be correlated with the reduced capacity of adenine nucleotide-treated MoDCs to recruit monocytes and DCs (5). We have observed by ELISA using DC supernatants that ATP, PGE2, and more strongly ATP{gamma}S inhibit CCL13 release by MoDCs both in basal and LPS-stimulated conditions (Fig. 2). We have also shown that forskolin inhibited CCL13 release both in the absence or the presence of LPS at 100 ng/ml (Fig. 2).


Figure 2
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  		FIGURE 2. Effects of ATP, ATP{gamma}S, PGE2, and forskolin on CCL13/MCP-4 release by human MoDCs. DCs were stimulated by ATP (300 µM), ATP{gamma}S (100 µM), PGE2 (500 nM), or forskolin (FK; 10 µM) in the absence or presence of LPS (100 ng/ml) for 24 h. Supernatants of treated DCs were collected for ELISA measurements of human CCL13/MCP-4. Results are expressed as picograms per 106 cells/ml and represent the mean ± SEM of three independent experiments. CONT, Control. (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Student’s t test were performed using GraphPad Prism.

 
ATP{gamma}S strongly down-regulates CSF-1 cytokine and CSF-1 receptor

An interesting observation was the down-regulation of CSF-1 and the gene encoding its receptor CSF-1R in response to ATP{gamma}S (Tables II and III). PGE2 was able to down-regulate CSF-1 cytokine but not its receptor (Table II). These regulations were first confirmed using quantitative PCR experiments (Fig. 1). To quantify CSF-1 release, we have performed ELISAs using supernatants of DCs treated for 24 h with ATP{gamma}S, ATP, or PGE2. We have observed that ATP{gamma}S, ATP, and PGE2 induce an inhibition of CSF-1 release in the absence or the presence of LPS (100 ng/ml) (Fig. 3).


Figure 3
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  		FIGURE 3. Effects of ATP, ATP{gamma}S, and PGE2 on CSF-1 release by human MoDCs. DCs were stimulated by ATP (300 µM), ATP{gamma}S (100 µM), or PGE2 (500 nM) in the absence or the presence of LPS (100 ng/ml) for 24 h. Supernatants of treated DCs were collected for ELISA measurements of human CSF-1. Results are expressed as picograms per 106 cells/ml and represent the mean ± SEM of five independent experiments. CONT, Control. (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Student’s t test were performed using GraphPad Prism.

 
MoDCs release VEGF-A in response to ATP{gamma}S, ATP and PGE2

In our microarray and qRT-PCR experiments, VEGF was a gene up-regulated by ATP{gamma}S and PGE2 (Table III and Fig. 1). We have used an ELISA kit specific for the most common form of VEGF called VEGF-A. VEGF-A protein levels have been quantified in supernatants of MoDCs treated during 24 h by ATP, ATP{gamma}S, and PGE2 in the presence or the absence of LPS (Fig. 4). ATP, ATP{gamma}S, and PGE2 were able to induce a moderate secretion of VEGF-A. Whereas LPS induced only a weak VEGF-A secretion, it was able to significantly potentiate the production of VEGF-A induced by ATP, ATP{gamma}S, and PGE2. VEGF-A concentration was >2 ng/ml in the supernatant of DCs treated with a combination of 100 ng/ml LPS and ATP{gamma}S (100 µM) or PGE2 (500 nM). No VEGF-C production was detected using the same DC supernatants (data not shown).


Figure 4
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  		FIGURE 4. Effects of ATP, ATP{gamma}S ,and PGE2 on VEGF-A release by human MoDCs. DCs were stimulated by ATP (300 µM), ATP{gamma}S (100 µM), or PGE2 (500 nM) in the absence or the presence of LPS (100 ng/ml) for 24 h. Supernatants of treated DCs were collected for ELISA measurements of human VEGF-A. Results are expressed as picograms per 106 cells/ml and represent the mean ± SEM of three independent experiments. CONT, Control. (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Student’s t test were performed using GraphPad Prism.

 
ATP{gamma}S and PGE2 down-regulate NRP-1 expression on MoDCs

One of the down-regulations observed in response to ATP{gamma}S was that of NRP-1 (Table III). In addition to its known functions in axon guidance (17) and angiogenesis (18) as a semaphorin and VEGF coreceptor, NRP-1 also plays a key role in the initiation of the primary immune response by regulating interactions between DCs and T cells (19). We have confirmed down-regulation of NRP-1 mRNA in response to ATP{gamma}S and more weakly in response to PGE2 by quantitative PCR experiments (Fig. 1). Flow cytometry experiments have been performed using PE-conjugated anti-human NRP-1 Ab. We have observed a strong down-regulation of NRP-1 expression at the membrane of MoDCs in response to ATP{gamma}S (Fig. 5). Weaker effects were observed in response to ATP and PGE2 (Fig. 5).


Figure 5
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  		FIGURE 5. Effects of ATP, ATP{gamma}S, and PGE2 on NRP-1 expression on human MoDC cells. DCs were stimulated by ATP (300 µM), ATP{gamma}S (100 µM), or PGE2 (500 nM) for 24 h. NRP-1 expression was analyzed by flow cytometry analysis using an anti-human PE-NRP-1. The flow cytometry data in A were obtained in an experiment representative of five independent experiments. In B is displayed the mean of fluorescence ± S.D. obtained from these five independent experiments. CONT, Control. (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Student’s t test were performed using GraphPad Prism.

 
ATP{gamma}S regulated expression of genes related to the tryptophan metabolism and IDO activation

We have previously reported that ATP and ATP{gamma}S significantly potentiate the activity of IDO, a negative regulator of T lymphocyte proliferation, and kynurenine production from tryptophan initiated by IFN-{gamma} in human DCs (11). PGE2 was reported to up-regulate IDO in DCs both in vitro (20) and in vivo (21). We have observed a stronger INDO up-regulation in MoDCs in response to ATP{gamma}S than in response to PGE2 in microarray (Table III). Additionally, several genes potentially linked to IDO up-regulation and tryptophan metabolism such as genes encoding the superoxide dismutase SOD2 and the FOXO3a transcription factor were up-regulated in response to ATP{gamma}S (Table II). Up-regulation of FOXO3a and SOD2 has been related to a potential alternative pathway that up-regulates IDO in response to CTLA4 independently of IFN-{gamma} increase (22). We observed that SOD2 and FOXO3A mRNAs were up-regulated at 6 h in response to ATP{gamma}S but not in response to PGE2 or forskolin by quantitative PCR analysis (Table V).


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  		Table V. Quantitative RT-PCR data obtained for FOXO3A and SOD2 in response to ATP{gamma}S, PGE2, or forskolina

 
We then compared the effect of ATP{gamma}S and PGE2 on the generation of kynurenine derivatives in the absence or the presence of IFN-{gamma} (100 U/ml). As shown in Fig. 6, ATP{gamma}S was able to potentiate IFN-{gamma} effect as previously described (11) whereas PGE2 displayed a weak effect alone but was not able to potentiate an IFN-{gamma} response. These data suggest that ATP{gamma}S could also stimulate IDO activity in MoDCs through an alternative IDO induction pathway as described for CTLA4 (22).


Figure 6
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  		FIGURE 6. ATP{gamma}S but not PGE2 potentiates IFN-{gamma} action on kynurenine production. DCs were either untreated (CONT, Control) or treated with ATP{gamma}S (100 µM) or PGE2 (5 µM) alone or in combination with 100 U/ml IFN-{gamma} for 24 h in complete medium. DCs were then washed and incubated for five additional hours in red phenol-free RPMI 1640 supplemented with 300 µM L-tryptophan. Kynurenine levels were determined in each supernatant by HPLC. Data (mean ± range) were obtained in duplicate and are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study combines gene profiling, qRT-PCR, ELISA, and flow cytometry experiments to identify and compare target genes of ATP{gamma}S and PGE2 in human MoDCs. Several papers have previously described the effects of ATP on the expression of DC maturation markers and some chemokines and chemokine receptors (2, 3, 4, 5, 6). ATP also regulates the action of LPS on human DCs by inhibiting the production of proinflammatory cytokines like IL-12, IL-1beta, IL-6, and TNF-{alpha} and by potentiating anti-inflammatory IL-10 (2, 4, 7). These effects of ATP are reproduced by other cAMP-elevating agents such as PGE2 (14, 15) and are most probably mediated by the P2Y11 receptor (4). PGE2 effects on DC maturation are likely to result from activation of the EP2/EP4 receptors (14). Many of the effects of ATP{gamma}S on MoDCs, such as its effect on CD83 expression and TSP-1 release, are reproduced by forskolin or dibutyryl-cAMP, demonstrating their action through cAMP elevation (4, 5, 11, 14).

It appeared necessary and useful to follow and compare the expression of all of the target genes of ATP{gamma}S and PGE2 simultaneously in MoDCs. Microarray technology was a good option for performing this study and revealed an impressive expression profile of ATP{gamma}S compared with that of PGE2, especially at a short time of stimulation. At 24 h of stimulation, ATP{gamma}S and PGE2 regulated a significant pool of common genes including regulations compatible with their effect on DC maturation. The comparison between the genes regulated by ATP{gamma}S and by PGE2 identified the regulation of many ATP target genes that could not depend on cAMP increase. The large and early expression profile of ATP{gamma}S results most likely from the activation of P2Y11 receptor and other purinergic receptors coupled to calcium-dependent pathways (e.g., P2Y2 and P2X7) (23, 24). The use of forskolin allows us to confirm the involvement of the cAMP pathway in the regulation of genes regulated by ATP{gamma}S and PGE2, such as CCL13, as we had previously shown for other target genes including CCL2 and CCL3 (5) as well as TSP-1 (11).

We then performed a validation of microarray analysis by using qRT-PCR experiments for 12 target genes that displayed a link with the immune system. The expression profile of ATP{gamma}S was very large and provided a large series of novel target genes that will be studied in the future. We decided to focus our attention on unexpected and promising target genes: four genes regulated by ATP{gamma}S and more weakly by PGE2 such as NRP-1, VEGF-A, CCL13, and CSF-1 and some particular genes regulated more specifically by ATP{gamma}S such as FOXO3A transcription factor and superoxide dismutase SOD2.

We observed CSF-1 and CSF-1R down-regulation in response to ATP{gamma}S in our microarray and qRT-PCR experiments. CSF-1 is known to modulate the development and immune function of DCs but also the survival, proliferation, and differentiation of mononuclear phagocytes (25). The transcription of the CSF-1 receptor CSF-1R is inactive in precursors of DCs and up-regulated in DCs during differentiation (26). Reduction of CSF-1 secretion and CSF-1R expression on DCs has been associated with a loss of proliferative response as well as a loss of their phagocytic and adhesive properties (27).

Besides the strong down-regulation of the CCL2, CCL3, CCL4, and CCL8 chemokines by ATP{gamma}S and PGE2 that was observed our in microoarray data, ATP{gamma}S, ATP, and PGE2 were able to inhibit CCL13 release from MoDCs. CCL13 and these other chemokines are all involved in the recruitment of monocytes, immature DCs, NKs, and activated lymphocytes (28, 29). The down-regulation of CCL13 release in response to ATP{gamma}S and PGE2 could be correlated with the reduced capacity of adenine nucleotide-treated DCs to attract monocytes and immature DCs (5). Inhibition of CSF-1 and CCL13 release in response to ATP could reduce monocyte and DC differentiation and recruitment at the site of inflammation.

We have also shown that ATP, ATP{gamma}S, and PGE2 induced a VEGF-A secretion by MoDCs that was strongly potentiated in combination with LPS. Released VEGF-A concentrations were sufficient to activate known VEGF receptors. DCs that matured in the presence of anti-inflammatory molecules such as PGE2, IL-10, and calcitriol have been previously reported to secrete VEGF-A (30). This regulation may provide a link between DCs and angiogenesis, but VEGF-A secreted by DCs could also be able to inhibit T cell development and may contribute to tumor-induced immune suppression (31). Interestingly, we have previously reported that the most up-regulated gene in the expression profile induced by ATP{gamma}S and PGE2 is the gene THBS1 encoding TSP-1 (11), a protein that displays antiangiogenic properties but is also able to down-regulate CD4+ T cell proliferation and behave as an autocrine inhibitor of IL-12 release by DCs (32). Finally, another ATP{gamma}S and PGE2 target gene, NRP-1, is expressed on human DCs and resting T cells. NRP-1 was described as a critical membrane protein in the interaction between DCs and T lymphocytes and in the initiation of DC-induced proliferation of resting T cells (19). These data support the notion that the down-regulation of NRP-1 expression on MoDCs in response to ATP, ATP{gamma}S, and PGE2 could also contribute to their immunosuppressive effects.

The tolerogenic properties of ATP-treated DCs was also supported inter alia by the up-regulation of TSP-1 and IDO in MoDCs as well as the secretion of kynurenine derivatives (11). IDO is an enzyme involved in tryptophan metabolism and considered as a negative regulator of T lymphocyte proliferation. PGE2 was shown to induce IDO activity in DCs by both in vitro (20) and in vivo (21) studies. We have shown that ATP{gamma}S up-regulated IDO mRNA more strongly than PGE2 by microarray and qRT-PCR experiments. It was interesting to observe the additional up-regulations of the FOXO3A transcription factor and superoxide dismutase SOD2 genes in response to ATP{gamma}S. Quantitative PCR experiments have confirmed their regulation in response to ATP{gamma}S, whereas PGE2 and forskolin had no significant effect. FOXO3A and SOD2 are associated with an alternative pathway of IDO activation described in response to CTLA4 (22). It has been proposed that CTLA4-Ig up-regulates IDO through an IFN-{gamma}-dependent pathway and an IFN-{gamma}-independent pathway involving FOXO3A and SOD2 up-regulations coupled with peroxynitrite down-regulation (22). FOXO3A and SOD2 up-regulation could explain why ATP{gamma}S but not PGE2 potentiated IFN-{gamma}-mediated up-regulation of IDO and kynurenine production. As there has been a proposal to explain the synergy between PGE2 and TNF-{alpha} on IDO activity (20), our experiments suggest a synergy between the signaling pathways induced by the activation of ATP and IFN-{gamma} receptors.

The identification and study of ATP{gamma}S and PGE2 target genes extend the scope and give a large overview of ATP and PGE2 effects on human MoDCs. Apart from its effect on DC maturation markers, ATP{gamma}S regulated a lot of interesting target genes in MoDCs. First, ATP might be considered as a strong negative signal for chemokine secretion by DCs. ATP is also able to down-regulate the expression of membrane receptors involved in primary immune response and DC functions such as NRP-1 and CSF-1R. Additionally, this is the first time that VEGF-A secretion by human DCs in response to extracellular nucleotides has been described. High concentrations of TSP-1 released by DCs in response to ATP suggest a negative global effect of ATP-treated DCs on angiogenesis, but TSP-1 and VEGF-A release by DCs could lead to immunosuppression through their negative paracrine effect on T cell proliferation (33).

The large expression profile of ATP in human DCs reflects the complexity of its action on human DCs and in the immune system in general. Several studies have described both proinflammatory and anti-inflammatory actions of ATP. Swennen et al. (34) recently reported that ATP inhibits the release of the proinflammatory cytokine TNF-{alpha} and stimulates the release of the anti-inflammatory cytokine IL-10. ATP also stimulates IL-1 release through the P2X7 receptor, a low affinity ATP receptor expressed on DCs (24). The balance between proinflammatory and anti-inflammatory actions of ATP depends on the immune cell types involved but could also be a matter of the concentration and/or the location of released ATP. At the level of DCs ATP could exert a proinflammatory action when it is released at a low concentration, and a massive release of ATP through cell lysis could lead to an anti-inflammatory or tolerogenic signal through the activation of its low affinity receptor P2Y11. It has been described, for example, that low concentrations of ATP and ADP induce DC migration through P2Y2 and P2Y1 activation, respectively, whereas high concentrations of ATP inhibit DC migration through the P2Y11 receptor (35). The synergy between LPS and ATP on VEGF release and between IFN-{gamma} and ATP on IDO activity highlights possible cross-talks between their distinct signaling pathways and defines ATP as a key cosignal on human DCs. The action of released ATP will thus also depend on the identity and the concentration of the different factors present at the site of inflammation.

We have previously reported that ATP could act as an immunosuppressive agent through a direct negative effect on cytokine release from T CD4+ (36). In this report we have shown that, at the level of DCs, besides its negative effect on chemokine release ATP down-regulates the expression of major receptors involved in DC differentiation and function (CSF-1R and NRP-1) and stimulates the expression of proteins displaying immunosuppressive properties. Even if proinflammatory actions of ATP have been previously described, it is important to consider ATP and also PGE2 as agents that might inhibit or reduce inflammation in specific conditions. Our data suggest that ATP derivatives specific of a cAMP-coupled P2Y11 receptor could be used as therapeutic agents to regulate major DC functions and leukocyte recruitment.


   Acknowledgments
 
We thank F. Bulté for technical assistance. We thank Dr. B. Dessars for helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by an Action de Recherche Concertée of the Communauté Française de Belgique, by the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Federal Service for Science, Technology and Culture, by grants of the Fonds de la Recherche Scientifique Médicale, the Fonds Emile DEFAY, and the LifeSciHealth programme of the European Community (Grant LSHB-2003-503337). N. B., M. H., and F. M. were supported by the Fonds National de la Recherche Scientifique/Fonds pour la Recherche dans l’Industrie et dans l’Agriculture, Belgium. D. C. and F. L. are Research Associate of the Fonds National de la Recherche Scientifique (FNRS). Back

2 N. B. and M. H. contributed equally to the work. Back

3 Address correspondence and reprint requests to Dr. Didier Communi, Institute of Interdisciplinary Research (IRIBHM), Université Libre de Bruxelles, Building C (5th floor), Campus Erasme, 808 Route de Lennik, Brussels, Belgium. E-mail address: communid{at}ulb.ac.be Back

4 Abbreviations used in this paper: DC, dendritic cell; ATP{gamma}S, adenosine 5'-O-(3-thiotriphosphate); MoDC, monocyte-derived DC; TSP-1, thrombospondin-1; NRP-1, neuropilin-1; qRT-PCR, quantitative RT-PCR; VEGF, vascular endothelial growth factor. Back

5 The online version of this article contains supplemental material. Back

Received for publication October 31, 2006. Accepted for publication July 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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