T Lymphocytes + Activation of Human CD4 Extracellular Adenine ...

The Journal of Immunology

Extracellular Adenine Nucleotides Inhibit the Activation of Human CD4ⴙ T Lymphocytes1 Xavier Duhant,2* Liliane Schandene´,† Catherine Bruyns,* Nathalie Suarez Gonzalez,* Michel Goldman,† Jean-Marie Boeynaems,*‡ and Didier Communi* 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 CD4ⴙ T cell activation. We demonstrate that several ATP derivatives increase cAMP in both freshly purified and activated human peripheral blood CD4ⴙ T cells. The rank order of potency of the various nucleotides was: adenosine 5ⴕ-O-(3-thiotriphosphate) (ATP␥S) ⬇ 2ⴕ- and 3ⴕ-O-(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP ⬎⬎ dATP, 2-propylthio-␤,␥-dichloromethylene-D-ATP, UDP, UTP. This effect did not involve the activation of A2Rs by adenosine or the synthesis of prostaglandins. ATP␥S had no effect on cytosolic calcium, whereas BzATP induced an influx of extracellular calcium. ATP␥S and BzATP inhibited secretion of IL-2, IL-5, IL-10, and IFN-␥; expression of CD25; and proliferation after activation of CD4ⴙ 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 CD4ⴙ 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 Journal of Immunology, 2002, 169: 15–21.

E

xtracellular nucleotides constitute an ubiquitous family of messengers that exert autocrine or paracrine actions. Their release in fluids results from cell lysis, exocytosis of nucleotide-concentrating granules (synaptic vesicles, plateletdense bodies), or efflux from cytoplasm through membrane transport proteins (1–3). They induce a wide spectrum of biological effects that are mediated by P2 purinergic receptors. P2Rs can be divided in two families: the ionotropic P2X, which are ligandgated cation channels; and the metabotropic P2Y, which are part of the superfamily of G-protein-coupled receptors. At this stage, eight genuine human P2YRs have been identified and characterized: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 (4), P2Y12 (5, 6), and P2Y13 (7) as well as the uridine 5⬘-diphosphoglucose receptor which is structurally related to P2Y12 (8). In particular, extracellular nucleotides exert numerous actions, mediated by several P2Y and P2XRs, on monocytes and macrophages, dendritic cells, lymphocytes, and granulocytes (reviewed in Ref. 3). Among the P2YRs, the P2Y11 subtype has the unique property of being dually coupled to phospholipase C and adenylyl cyclase activation. Northern blotting analysis revealed a restricted expression of this receptor in spleen and HL-60 cells, supporting a role

*Institute of Interdisciplinary Research, School of Medicine, Departments of †Immunology and ‡Medical Chemistry, Erasme Hospital, Universite´ Libre de Brussels, Brussels, Belgium Received for publication August 29, 2001. Accepted for publication April 22, 2002. 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 Concerte´e of the Communaute´ Franc¸aise 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 Me´dicale, the Bekales Foundation, the Fonds Me´dical Reine Elisabeth and Boehringer Ingelheim, the Fonds Emile DEFAY, and the Fonds de la Fondation de la Chirurgie Cardiaque, the Fonds National de la Recherche Scientifique/Te´le´vie, Belgium (to X.D.), and Euroscreen (to N.S.G.).

2 Address correspondence and reprint requests to Dr. Xavier Duhant, Institute of Interdisciplinary Research, Building C5-110, 808 route de Lennik, 1070 Brussels, Belgium. E-mail address: [email protected]

Copyright © 2002 by The American Association of Immunologists, Inc.

of ATP in the regulation of immune responses and hemopoiesis (9). Recently, Wilkin et al. (10) showed that ATP and hydrolysisresistant derivatives of ATP, such as adenosine 5⬘-O-(3-thiotriphosphate) (ATP␥S),3 which are agonists of the P2Y11R, induce the maturation of human monocyte-derived dendritic cells via an increase in cAMP. A coherent picture of lymphoid cells control by extracellular nucleotides has not yet emerged, and it is generally believed that human peripheral T lymphocytes express P2XRs but lack P2YRs (3). In this study, we have investigated the effects of P2Y11R agonists on the activation of human blood-derived CD4⫹ T lymphocytes.

Materials and Methods Reagents ATP, ATP␥S, 2⬘- and 3⬘-O-(4-benzoylbenzoyl)-ATP (BzATP), dATP, UDP, UTP, PGE2, 8-bromo-cAMP, and indomethacin (Indo) were obtained from Sigma (St. Louis, MO). 2-Methylthio-ATP, CGS-21680 (a potent agonist of A2Rs), and 8-( p-sulfophenyl)theophylline (8-p-SPT) were purchased from Research Biochemicals International (Natick, MA). 2-Propylthio-␤,␥-dichloromethylene-D-ATP (AR-C67085) was a gift of Drs. J. D. Turner and P. Leff (Astra Zeneca R&D, Loughborough, U.K.). H-89 was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). The anti-CD3 mAb OKT3 (Orthoclone OKT3) was provided by Janssen-Cilag (Berchem, Belgium), and the anti-CD28 mAb (clone CD28.2) was supplied by BD PharMingen (San Diego, CA). [3H]Thymidine (25 Ci/mmol) was from Moravek Biochemicals (Brea, CA). Rolipram was a gift from the Laboratoires Jacques Logeais (Trappes, France).

Isolation of resting CD4⫹ T cells from peripheral blood PBMC were isolated from buffy coats of healthy blood donors by density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). After three washes in HBSS (Invitrogen, Merelbeke, Belgium). CD4⫹ T cells were isolated from PBMC using the MACS negative depletion system (Miltenyi Biotec, Auburn, CA). No contaminating CD8⫹ T cells, B cells, monocytes, or NK cells were detected. 3 Abbreviations used in this paper: ATP␥S, adenosine 5⬘-O-(3-thiotriphosphate); BzATP, 2⬘- and 3⬘-O-(4-benzoylbenzoyl)adenosine 5⬘-triphosphate; AR-C67085 (2propylthio-␤,␥-dichloromethylene-D-ATP); PKA, protein kinase; 8-p-SPT, 8-(psulfophenyl)theophylline; Indo, indomethacin; [Ca2⫹]i, intracellular calcium concentration.

0022-1767/02/$02.00

INHIBITION OF CD4⫹ T CELL ACTIVATION BY ADENINE NUCLEOTIDES

16 CD4⫹ T cell activation and proliferation. ⫹

Purified CD4 T cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated FCS from HyClone (Logan, UT), 25 mM HEPES buffer, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 ␮g/ml gentamicin, and 50 ␮M 2-ME at 37°C in 5% CO2. The CD4⫹ T cells (2 ⫻ 105/well) were activated in flat-bottom 96-well plates precoated with the anti-CD3 mAb (10 ␮g/ml) in the presence of soluble anti-CD28 mAb (1 ␮g/ml) and the presence or absence of different concentrations of nucleotides. Culture supernatants were harvested after 24, 72, or 96 h for measurement of cytokine concentration and the remaining cells were resuspended in PBS to determine CD25 surface expression as well as apoptosis and necrosis by flow cytometry. After 56 h of culture, proliferation was assessed by [3H]thymidine (0.5 ␮Ci/well) uptake during the next 16 h. Each experimental condition was tested in triplicate.

Flow cytometry Flow cytometric analysis of surface phenotype of the CD4⫹ T cells was performed by two- or three-color staining using FITC-, PE-, and PerCPconjugated mouse anti-human mAb. Purified CD4⫹ T cells were stained with mAbs against CD3, CD4, CD8, CD14, CD16, CD19, CD25, CD45, CD56, and CD69 (all from BD PharMingen). The percentage of apoptotic and necrotic CD4⫹ T cells was determined using FITC-conjugated annexin V and propidium iodide, both from BD PharMingen (San Diego, CA). Samples were assayed in duplicate and analyzed using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ) and CellQuest software (BD Biosciences).

Cytokine measurements Commercially available kits were used for quantification of various cytokine levels: IL-2 (R&D Systems, Oxon, U.K.); IL-10 and IFN-␥ from Biosource International (Camarillo, CA). IL-5 levels were measured by two-site sandwich ELISA using Abs from BD PharMingen (San Diego, CA). Each experimental condition was tested in triplicate.

cAMP measurements Cells were preincubated for 30 min in complete culture medium with 25 ␮M rolipram and then incubated in the same medium for 12 min in the presence of the agonists. The incubation was stopped by the addition of 1 ml 0.1 M HCl. The incubation medium was dried up, and the samples were resuspended in water and diluted as required. cAMP was quantified by radioimmunoassay after acetylation as previously described (11). Each experimental condition was tested in triplicate.

FIGURE 1. Effect of adenine and uridine nucleotides on cAMP accumulation in freshly purified (FP) or activated (Act) human CD4⫹ T cells. CD4⫹ T cell activation was performed by incubating during 72 h with precoated anti-CD3 and soluble anti-CD28 mAb. Freshly purified (A–D) or activated (A and D) cells were preincubated for 30 min with rolipram (25 ␮M) and then incubated in the same medium in the presence of various concentrations of nucleotides for 12 min. cAMP was quantified by RIA after acetylation. In the experiments shown in D, freshly purified cells were preincubated with 8-p-SPT or Indo during 15 min and then stimulated with the agonists. Data represent the mean ⫾ SD of triplicate experimental points obtained in one representative experiment of six (A and D) or three (B and C). Ctrl, Control, 2MeSATP, 2-methylthio-ATP; CGS, CGS-21680.

Calcium measurements Levels of intracellular calcium were measured with the calcium-binding dye fluo-3 (12). Directly after the isolation or after 24 h incubation at 37°C in a 5% CO2, humidified atmosphere, purified human CD4⫹ T cells were washed twice in calcium- and magnesium-free HBSS and incubated at a concentration of 5 ⫻ 106 cells/ml with 0.25 mM sulfinpyrazone (Sigma), 100 ␮g/ml pluronic acid F-127 (Molecular Probes, Leiden, The Netherlands), and 5 ␮M fluo-3 (Molecular Probes), for 30 min at 37°C. Cells were then washed twice in complete medium or HBSS with 0.25 mM sulfinpyrazone, resuspended at a final concentration of 5 ⫻ 105/ml, and placed in a 37°C water bath. After 10 s acquisition on a FACScan flow cytometer, the cells were stimulated with either 100 nM RANTES or 100 ␮M nucleotide (ATP␥S or BzATP). The FL1 signal for fluo-3-bound calcium was calibrated by transporting in saturating calcium with 100 ng/ml A23187 to obtain the maximum signal (Fmax) and then adding 2 mM MgCl2 to obtain the minimum signal (Fmin). The intracellular calcium concentration ([Ca2⫹]i) was calculated using the formula [Ca2⫹]i ⫽ Kd (F ⫺ Fmin)/ (Fmax ⫺ F), where Kd ⫽ 400 nM for the fluo-3 intracellular dye (13).

Quantitative RT-PCR assay of P2Y11 mRNA Total RNA was isolated using the Tripur Isolation Reagent (Roche Diagnostics, Basel, Switzerland). To avoid DNA contamination, 1 ␮g total RNA was treated with DNA-free from Ambion (Austin, TX). Reverse transcription was performed with 500 ng RNA using the Superscript II preamplification system with random hexamers (Life Technologies). The primers and TaqMan fluorogenic probe for P2Y11 (GenBank accession number AF030335) were from Eurogentec (Seraing, Belgium): forward (19 –39), 5⬘-CTG CCC TGC CAA CTT CTT G-3⬘; reverse P2Y11 (76 –96), 5⬘-CAG TAT GGG CCA CAG GAA GTC-3⬘; probe (45– 69), 5⬘-(FAM)TGC CGA CGA CAA ACT CAG TGG GTT-(TAMRA)-3⬘. TaqMan PCR assays were performed in duplicate on cDNA samples or RNA in 96-well optical plates on an ABI Prism 7700 sequence detection system (PE Applied Biosystems, Foster City, CA). For each 25-␮l reaction, 1/20 of reverse transcription was mixed with 12.5 ␮l 2⫻ qPCR Mastermix kit (Eurogentec), primers (300 nM each), and fluorogenic probe (200 nM). PCR parameters were 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were analyzed with Sequence Detector Software (SDS version 1.6; PE Applied Biosystems).

Western blot analysis of phosphorylated CREB Purified CD4⫹ T cells were preincubated with 3 ␮M H-89 for 4 h and then stimulated for 30 min in flat-bottom plates precoated with 10 ␮g/ml antiCD3 mAb in the presence of 1 ␮g/ml soluble anti-CD28 mAb, and in the

The Journal of Immunology

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FIGURE 2. Effect of ATP␥S and BzATP on the concentration of cytosolic calcium in human CD4⫹ T cells. Freshly purified cells were loaded with the fluorescent dye fluo-3, and calcium mobilization was followed by flow cytometry after gating on living cells. As described in Materials and Methods, A23187 (100 ng/ml) and MgCl2 (2 mM) were used to obtain respectively maximum and minimum signal (Fmin and Fmax). Nucleotides were added at a final concentration of 100 ␮M (arrows) and the cells were in medium with (A) or without (B) extracellular calcium. Data are from one of five experiments. [Ca2⫹]i was computed as described previously (12), and the baseline value was subtracted.

presence or absence of 100 ␮M ATP␥S. Cells were washed with HBSS and lysed on ice in 120 ␮l of Laemmli buffer (10% (w/v) glycerol, 5% (v/v) 2-ME, 2.3% (w/v) SDS, 62.5 mM Tris-HCl, pH 6.8) with proteinase inhibitors: 1 ␮g/ml leupeptin, 60 ␮g/ml Pefabloc, 1 ␮g/ml aprotinin, and phosphatase inhibitors (Roche); 1 mM sodium orthovanadate and 10 mM sodium fluoride (Sigma). The protein concentration was determined using the method of Minamide and Bamburg (14). The same amount of protein (10 ␮g) for each condition was electrophoresed on a 12% SDS-polyacrylamide gel. Proteins were then transferred overnight at 28 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 an Ab specific for phospho-CREB at a 1/1,000 dilution and the ECL Western blotting detection system (Amersham Pharmacia Biotech) with a biotinylated secondary rabbit Ab (1/50,000). The anti-phospho-CREB Ab was a gift from Dr. M. R. Montminy (The Clayton Foundation Laboratories for Peptide Biology, La Jolla, CA).

preparations tested. At 100 ␮M, suramin reduced the cAMP accumulation in ATP␥S-stimulated CD4⫹ T cells by 51% (mean of four experiments; data not shown). In three experiments of six, a stimulation of cAMP accumulation could also be detected in response to adenosine (100 ␮M) or the A2AR agonist CGS-21680 (100 nM). That response was observed only in activated cells and not in freshly isolated ones (Fig. 1D). That cAMP response was sensitive to inhibition by 8-p-SPT (300 ␮M), an A2R antagonist, whereas the effect of ATP␥S was not (Fig. 1D). In contrast, Indo, an inhibitor of PG release, had a minimal effect on the cAMP response to ATP␥S of 25 ⫾ 14% inhibition (mean of three experiments ⫾ SD; range, 11–38%).

Statistical method

To test whether human CD4⫹ T cells exhibit a phospholipase C/inositol 1,4,5-triphosphate/Ca2⫹ response to adenine nucleotides, we performed calcium-binding dye fluo-3 assays. To verify the functionality of the calcium mobilization, we used the chemokine RANTES (100 nM), known to induce an increase of [Ca2⫹]i (data not shown) (16). In the presence of extracellular calcium, BzATP (100 ␮M) increased [Ca2⫹]i in freshly isolated CD4⫹ T cells, whereas ATP␥S (100 ␮M) had no effect (Fig. 2, A and 2B). The effect of BzATP was abolished in a Ca2⫹-free medium (Fig. 2B).

Triplicates were obtained for each measurements (i.e., for each agonist, each donor, and each concentration). ANOVA for repeated measures (concentration at three levels) with two intersubject factors (donor and agonist) was performed. Adjustment for multiple comparisons between the three concentrations was performed using the Sidak correction (SPSS 10.0; SPSS, Chicago, IL).

Results Effects of nucleotides on cAMP To evaluate the functional expression of P2Y11Rs, we determined the intracellular cAMP accumulation in response to nucleotides. A comparison was made between freshly purified CD4⫹ T cells and CD4⫹ T cells activated during 72 h with the association of immobilized anti-CD3 mAb and soluble anti-CD28 mAb used as an APC-independent and polyclonal T cell stimulus. At 100 ␮M, ATP␥S and BzATP, both agonists of P2Y11R (15), stimulated significantly the cAMP production in both populations (Fig. 1A). This stimulation was observed in all 16 preparations of CD4⫹ T cells that were tested (6 blood donors for Fig. 1, A and D, 3 for Fig. 1B, 3 for Fig. 1C, and 4 for the suramin experiments; data not shown). 2MeSATP was clearly less potent, and uridine nucleotides were inactive (Fig. 1A). ATP␥S (100 ␮M) produced a 20- ⫾ 11-fold cAMP increase in freshly isolated cells and a 28- ⫾ 15-fold cAMP increase 72 h after activation (mean ⫾ SD of six independent experiments). Concentration-action curves showed that the rank order of agonist potency was ATP␥S ⬵ BzATP ⬎ ATP (Fig. 1B). Although the profile observed thus far was consistent with the P2Y11R (15), two other P2Y11 agonists, dATP and AR-C67085, had no effect on cAMP accumulation (Fig. 1C) in all the three

Effect of nucleotides on calcium

Quantitative RT-PCR assay of P2Y11 mRNA P2Y11 mRNA was quantified by RT-PCR using TaqMan fluorigenic detection. In human peripheral CD4⫹ T cells, these experiments revealed a number of copies smaller than in dendritic cells Table I. Quantification of P2Y11 mRNA in CD4⫹ T cells and other human cellsa

Cell Type

P2Y11 Copy ⫾ SD

P2Y11 Copy/ng Total RNA

1321-N1 Monocytes DC HL-60 CD4⫹

3,435 ⫾ 177 1,495 ⫾ 157 6,282 ⫾ 352 14,320 ⫾ 559 1,445 ⫾ 615

69 30 126 286 29

a The P2Y11 copy number is calculated using the standard curve method and normalized to the equivalent total RNA quantity engaged in the PCR (50 ng). Data were obtained from duplicate points and are given as the mean ⫾ SD of three independent experiments, except for CD4⫹ T cells, of which five preparations were studied.


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FIGURE 3. Concentration-inhibition curves for ATP␥S (F) and BzATP (E) on IL-2 (A), IFN-␥ (B), IL-5 (C), and IL-10 (D) secretion by human CD4⫹ T cells. The freshly purified cells were incubated with mAb-precoated antiCD3 and soluble anti-CD28 and various concentrations of nucleotides. After 24 h (IL-2 (A)), 72 h (IFN-␥ (B) and IL-10 (C)), or 96 h (IL-5 (D)) of incubation, the supernatants were harvested for ELISA.

and HL-60 cells and comparable with those in 1321N1 astrocytoma cells that do not express functional P2Y11Rs (Table I). Effects of nucleotides on cytokine production by CD4⫹ T cells IFN-␥, IL-2, IL-5, and IL-10 production by CD4⫹ T cells obtained from three donors and activated with immobilized anti-CD3 and soluble anti-CD28 mAb (106 cells/ml) was measured in the absence or presence of nucleotide derivatives added at the beginning of the culture. As shown in Fig. 3 and Tables II and III, both ATP␥S and BzATP significantly inhibited in a concentration-dependent manner the secretion of the four cytokines tested. The inhibition increased significantly with the concentration ( p ⬍ 0.001 for each cytokine), but no significant difference could be detected between the agonists, except for IL-10. These effects were insensitive to 8-p-SPT (data not shown), indicating that they are

not mediated by adenosine and by activation of A2Rs. Moreover, the inhibition of IL-2 and IL-10 production by CD4⫹ T cells was not modified by the addition of Indo (5 ␮g/ml; data not shown), whereas, as previously described (17), the cAMP-elevating agent forskolin (10 ␮M) suppressed the secretion of the four cytokines (data not shown). No significant amounts of cytokines were detectable in culture supernatants of nonactivated CD4⫹ T cell (⬍10 pg/ml). To study the involvement of the protein kinase A (PKA) in the cAMP-dependent responses in CD4⫹ T cells, we tested H-89, which is an inhibitor of PKA that acts by competitive inhibition of the ATP binding site (18, 19). At 3 ␮M, H-89 was able to prevent the phosphorylation of CREB induced by ATP␥S (Fig. 4B). H-89 per se had a small inhibitory effect on IFN-␥ secretion during a 24-h stimulation (31 ⫾ 14% inhibition; mean ⫾ SD of three

Table II. Effect of ATP␥S and BzATP on cytokine secretion by activated CD4⫹ T cellsa IFN-␥

IL-2

IL-5

IL-10

Donor

Mean of triplicate (pg/ ml) ⫾ SD

Control

1 2 3

97 ⫾ 6 255 ⫾ 27 273 ⫾ 13

ATP␥S (100 ␮M)

1 2 3

41 ⫾ 8 155 ⫾ 3 236 ⫾ 6

36 ⫾ 22

1228 ⫾ 160 871 ⫾ 6 104 ⫾ 14

83 ⫾ 16

1820 ⫾ 735 952 ⫾ 82 513 ⫾ 49

61 ⫾ 10

262 ⫾ 13 78 ⫾ 88 150 ⫾ 32

84 ⫾ 6

BzATP (100 ␮M)

1 2 3

18 ⫾ 5 92 ⫾ 6 104 ⫾ 14

69 ⫾ 11

827 ⫾ 120 1243 ⫾ 124 265 ⫾ 53

83 ⫾ 9

1120 ⫾ 135 737 ⫾ 88 150 ⫾ 32

79 ⫾ 8

188 ⫾ 11 126 ⫾ 15 192 ⫾ 36

81 ⫾ 6

Mean % inhibition


Mean % inhibition

3580 ⫾ 113 5946 ⫾ 558 4536 ⫾ 465


Mean % inhibition

3946 ⫾ 379 3448 ⫾ 153 1211 ⫾ 253


Mean % inhibition

1605 ⫾ 156 596 ⫾ 51 798 ⫾ 58

a Data represent the mean (picograms per milliliter) ⫾ SD of each triplicate for each donor and the mean percentage of inhibition of each cytokine secretion from activated CD4⫹ T cells in presence of ATP␥S or BzATP (100 ␮M). Control represents the normal level of secretion of activated cells (no nucleotide).

The Journal of Immunology Table III.

19 Values of p after statistical analysis of inhibition of cytokine secretion dataa Concentration

Cytokine

Agonist

Donor

Concentration (global effect)

IL-2 IFN-␥ IL-5 IL-10

0.242 0.738 0.748 0.038

0.001 0.414 0.002 ⬍0.001

⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001

1 vs 10 (␮M)

1 vs 100 (␮M)

10 vs 100 (␮M)

0.05 ⬍0.001 ⬍0.001 ⬍0.001

⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001

⬍0.001 ⬍0.001 ⬍0.001 0.002

a ANOVA for repeated measures (concentration at three levels) with two intersubject factors (donor and agonist) was performed. Adjustment for multiple comparisons between the three concentrations was performed using the Sidak correction (SPSS 10.0 from SPSS).

experiments) and did not reverse the inhibitory effect of ATP␥S, BzATP, 8-bromo-cAMP, or PGE2 (Fig. 4A). Effects of ATP analogs on CD4⫹ T cell proliferation As shown in Fig. 5, ATP␥S and BzATP, added at concentrations ranging from 1 to 100 ␮M, inhibited the proliferation of CD4⫹ T cells activated by immobilized anti-CD3 and soluble anti-CD28 mAb as assessed by [3H]thymidine incorporation. An average of 41 ⫾ 13 and 46 ⫾ 7% inhibition (mean ⫾ SD of three experiments) of CD4⫹ T cell proliferation were observed with ATP␥S and BzATP (both 100 ␮M), respectively. Moreover, the decrease in proliferation was not reversed by the simultaneous addition of 8-p-SPT (data not shown). Labeling of the CD4⫹ T cells with annexin-FITC and propidium iodide showed that nucleotides did not increase the number of apoptotic and necrotic cells (data not shown). ATP analogs down-regulate the expression of CD25 during CD4⫹ T cell activation For further insight into the mechanism of action of the ATP derivatives on CD4⫹ T cell proliferation, CD25 expression induced on these cells during activation by immobilized anti-CD3 and soluble anti-CD28 mAb was evaluated by flow cytometry in the presence or absence of the nucleotides. As shown in Fig. 6, the addition of ATP␥S (100 ␮M) or BzATP (100 ␮M) at the beginning of the culture inhibited CD25 expression on the CD4⫹ T cells after 72 h of activation by ⬎40%.

Discussion

In both activated and nonactivated CD4⫹ T cell, cAMP accumulation was observed in response to ATP and ATP derivatives, ATP␥S and BzATP, known as agonists of the P2Y11R. The rank order of potency was ATP␥S ⬇ BzATP ⬎ ATP ⬎ 2-methylthioATP ⬎⬎ UTP, UDP. This pharmacological profile is similar to that FIGURE 4. Effect of H-89 on the nucleotidemediated inhibition of CD4⫹ T cell activation (A and B). A, Cells were preincubated for 4 h with 3 ␮M H-89 and then stimulated with the two activator Abs, and the supernatants were harvested, in this case after 24 h, for ELISA of IFN-␥. Data represent the mean ⫾ SD of triplicate experimental points obtained in one representative experiment of three. B, Cells were preincubated for 4 h with 3 ␮M H-89 and then stimulated for 30 min with precoated antiCD3 and soluble anti-CD28 or ATP␥S, with or without H-89. Total proteins were harvested for Western blot analysis. A representative blot for Ser133-phosphorylated CREB (p-CREB) is shown (one of five independent experiments).

previously observed in HL-60 cells (20, 21), human dendritic cells (10), or CHO-K1 cells stably expressing the recombinant human P2Y11R (15). However, several arguments do not support the hypothesis that these responses were mediated by the P2Y11R. First, dATP and AR-C67085, two potent agonists of the human recombinant P2Y11R previously found to be active in HL-60 (21) and dendritic cells (10), had no effect on cAMP accumulation in human CD4⫹ T cells. Furthermore, whereas the P2Y11R is dually coupled to adenylyl cyclase and phospholipase C stimulation, neither ATP␥S nor BzATP induced a significant mobilization of intracellular calcium in CD4⫹ T cells. As previously shown for total blood T lymphocytes, BzATP, but not ATP␥S, induced an influx of extracellular calcium in CD4⫹ T cells, presumably via the activation of a P2XR (22). Finally, quantitative RT-PCR revealed that the number of P2Y11 mRNA copies is lower in CD4⫹ T cells than in HL-60 and dendritic cells and comparable with that found in 1321N1 human astrocytoma cells, a cell line devoid of functional P2YRs. These results suggest that the cAMP response to ATP␥S and BzATP is mediated by another receptor than the P2Y11R. These cAMP responses were not due to the degradation of adenine nucleotides into adenosine and the activation of A2Rs. Indeed, a stimulation of cAMP accumulation in response to adenosine or the A2AR agonist CGS-21680 was detected in only a few experiments and exclusively in activated cells. That cAMP response was sensitive to inhibition by 8-p-SPT, an A2R antagonist, whereas the effect of ATP␥S was insensitive to 8-p-SPT but partially inhibited by suramin, a nonselective antagonist of P2Rs. The identity of the receptor involved in the effect of ATP␥S and BzATP remains thus unknown. Thus far, the P2Y11R is the only P2R directly coupled to adenylyl cyclase stimulation, but several orphan G protein-coupled receptors are structurally close to the P2YRs and constitute potential candidates, some of which are expressed in thymus (23, 24).

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FIGURE 5. Inhibition of cell proliferation by ATP derivatives. Human CD4⫹ T cells were purified and activated with precoated anti-CD3 and soluble anti-CD28 mAb. Nucleotides were added as the same time as the two Abs. The cells were incubated during 56 h and then 16 h in presence of [3H]thymidine (0.5 ␮Ci/well). Data are the mean percentage of control [3H]thymidine incorporation. Data represent the mean ⫾ SD of triplicate experimental points obtained in one representative experiment of three.

According to these data, we have studied the effects of adenine nucleotides on cytokine release by CD4⫹ T cells. Our results demonstrate that ATP␥S and BzATP induce an inhibition of the CD4⫹ T cell activation. The release of four cytokines (IL-2, IL-5, IL-10, and IFN-␥) involved in both Th1 and Th2 responses was significantly inhibited in the presence of adenine nucleotides. Simultaneously with inhibition of IL-2 secretion, the level of the IL-2R (CD25) expression was down-regulated; these two effects contribute to the inhibition of proliferation, assessed with the classical [3H]thymidine uptake test. We checked that the inhibition of [3H]thymidine uptake in our experiments was not due to apoptosis or necrosis. The immunosuppressive effects of ATP␥S and BzATP on both Th1 and Th2 responses are reminiscent of the action of PGE2 on human CD4⫹ T cells, which has been described by many authors (25–28) and is mediated by cAMP. It is now well established that an increase in cAMP inhibits T cell activation, via several mechanisms more or less proximal to TCR signaling. These mechanisms include inter alia: activation of C-terminal Src kinase (29); inhibition of c-Jun kinase (30); and phosphorylation of NFAT, preventing its nuclear translocation (31). The up-regulation of sev-

eral phosphodiesterases during T cell activation supports the concept that cAMP exerts a physiological role of T cell response moderator (32–34). The immunomodulatory effects of cAMP on T lymphocytes are mediated partially by PKA activation and partially via other mechanisms. It has indeed been shown that the inhibitory effect of cAMP-elevating agents on IL-5 release from activated T lymphocytes was not relieved by the PKA inhibitor H-89, although it was confirmed that this agent inhibited the activation of PKA and the resulting phosphorylation of CREB (18, 19, 35). Likewise we have shown that although H-89 inhibited the ATP␥S-induced CREB phosphorylation, the inhibitory effect of ATP␥S on IFN-␥ secretion was not suppressed by this PKA antagonist. Discrepant effects of adenine nucleotides on lymphocyte proliferation have been reported in the past. Gregory and Kern (36) showed that extracellular ATP stimulated the proliferation of murine thymocytes, whereas Fishman et al. (37) observed that ATP suppressed the proliferation of human peripheral lymphocytes, presumably as a result of its degradation into adenosine. Ikehara et al. (38) observed both inhibitory and stimulatory effects of ATP on murine lymphocyte proliferation, depending on the origin of the cells. ATP had a synergistic effect on the proliferation of human peripheral blood T cells stimulated by PHA or anti-CD3 Ab, an effect apparently mediated by a P2XR (22). Inhibitory effects of ATP on T cell proliferation might be mediated by its degradation into adenosine and activation of A2ARs which are indeed expressed on some T cell populations (39, 40). It is well known that T cell responses may greatly differ according to the subpopulation studied and the nature of the stimulus used to induce their activation. In our study, in which purified human CD4⫹ T cells were activated by immobilized anti-CD3 and soluble anti-CD28 Abs that closely mimic the physiological activation by the APC, we have observed an inhibitory effect of adenine nucleotides on cytokine secretion and cell proliferation, which is mediated by cAMP independently from adenosine receptors. Interestingly, unlike the P2Y2 (41) or P2Y6 (42) receptors that are up-regulated after T cell activation, the cAMP response to adenine nucleotides is present on nonactivated CD4⫹ T cells. The receptor involved in this action might constitute a new interesting target for topical immunosuppression in eye, skin, or airway inflammatory diseases.

Acknowledgments We thank Martine Ducarme, Christine Servais and Alain Crusiaux for excellent technical assistance and Dr. Viviane De Maertelaer for the biostatistical analysis.

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FIGURE 6. Effect of ATP derivatives on CD25 expression in human CD4⫹ T cells after 72 h of activation with precoated anti-CD3 and soluble anti-CD28 mAb. The nucleotides were added as the same time as the two Abs. CD25 expression was analyzed by flow cytometry after 72 h of incubation. Data are the mean ⫾ range of duplicate points obtained in one representative experiment of three.

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