Extracellular ATP Induces a Distorted Maturation of Dendritic Cells and Inhibits Their Capacity to Initiate Th1 Responses1 Andrea la Sala,2,3* Davide Ferrari,2† Silvia Corinti,* Andrea Cavani,* Francesco Di Virgilio,† and Giampiero Girolomoni* Dendritic cells (DCs) express functional purinergic receptors, but the effects of purine nucleotides on DC functions have been marginally investigated. In this study, we report on the ability of micromolar concentrations of ATP to affect the maturation and Ag-presenting function of monocyte-derived DCs in vitro. Chronic stimulation (24 h) of DCs with low, noncytotoxic ATP doses increased membrane expression of CD54, CD80, CD86, and CD83, slightly reduced the endocytic activity of DCs, and augmented their capacity to promote proliferation of allogeneic naive T lymphocytes. Moreover, ATP enhanced LPS- and soluble CD40 ligand-induced CD54, CD86, and CD83 expression. On the other hand, ATP markedly and dose-dependently inhibited LPS- and soluble CD40 ligand-dependent production of IL-1␣, IL-1, TNF-␣, IL-6, and IL-12, whereas IL-1 receptor antagonist and IL-10 production was not affected. As a result, T cell lines generated from allogeneic naive CD45RAⴙ T cells primed with DCs matured in the presence of ATP produced lower amounts of IFN-␥ and higher levels of IL-4, IL-5, and IL-10 compared with T cell lines obtained with LPS-stimulated DCs. ATP inhibition of TNF-␣ and IL-12 production by mature DCs was not mediated by PGs or elevation of intracellular cAMP and did not require ATP degradation. The inability of UTP and the similar potency of ADP to reproduce ATP effects indicated that ATP could function through the P2X receptor family. These results suggest that extracellular ATP may serve as an important regulatory signal to dampen IL-12 production by DCs and thus prevent exaggerated and harmful immune responses. The Journal of Immunology, 2001, 166: 1611–1617.
D
endritic cells (DCs)4 subserve a fundamental task in the immune system, as they are the most efficient APCs for the activation of naive T cells and thus for the induction of primary immune responses (1, 2). DCs reside in unperturbed tissues in an immature form where they are very capable of taking up Ags but weak at stimulating T cells. Under the influence of a variety of danger signals, including pathogens, proinflammatory cytokines, and dying cells, DCs undergo a process of differentiation known as maturation and migrate to the T cell areas of secondary lymphoid organs. DC maturation is associated with a reduced phagocytic and endocytic capacity, increased membrane expression of MHC and costimulatory molecules, and acquisition of potent T cell-stimulating functions. Mature DCs also release abundant amounts of cytokines and chemokines. Maturation of
*Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy; and †Department of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara, Ferrara, Italy Received for publication September 25, 2000. Accepted for publication November 7, 2000. 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 the Associazione Italiana per la Ricerca sul Cancro, the Istituto Superiore di Sanita` (AIDS project, Grant 40B/1.18), the Consiglio Nazionale delle Ricerche Target Project on Biotechnology, the European Community (Biomed 2 Program, Grant BMH4-CT98 –3713), the Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (Cofin), and the University of Ferrara. 2
A.L.S. and D.F. contributed equally to this work.
3
Address correspondence and reprint requests to Dr. Andrea la Sala, Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, Via dei Monti di Creta, 104, 00167 Rome, Italy. E-mail address:
[email protected] 4 Abbreviations used in this paper: DC, dendritic cell; sCD40L, soluble CD40 ligand; IL-1ra, IL-1 receptor antagonist; DPCPX, 8-cyclopentyl-1–3-dipropylxanthine; CSC, 8-(3-chlorostyryl)caffeine; LDH, lactate dehydrogenase; Rp-cAMPS, Rp-adenosine 3⬘,5⬘-cyclic monophosphothioate triethylamine.
Copyright © 2001 by The American Association of Immunologists
DCs is reinforced during interactions with T cells by membrane and soluble molecules such as CD40 ligand (CD40L) and IFN-␥ provided by T cells themselves (1–5). At the initial stage of T cell-DC interaction, the cytokine microenvironment plays a key role in Th cell differentiation toward Th1 or Th2 cell types. In particular, mature DCs produce high levels of IL-12 and preferentially promote the development of Th1 responses (6). DC maturation is thus a fundamental checkpoint in the initiation and shaping of immune responses, and it is tightly regulated. In particular, IL-10, TGF-, corticosteroids, vitamin D3, and PGE2 inhibit DC maturation induced by different stimuli and can convert DCs from Th1- to Th2-skewing APCs (2). Extracellular nucleotides have emerged as important regulators of inflammatory and immune responses. In particular, ATP can affect the functions of B cells, T cells, macrophages, and eosinophils (7–9) via activation of plasma membrane receptors known as P2 purinoceptors (10 –12). Two P2 receptors subfamilies have been recognized: P2X and P2Y, the former identified as membrane channels and the latter as seven membrane-spanning G proteincoupled receptors (10, 13, 14). ATP can be released by regulated exocytosis, traumatic cell lysis, or passive leakage from damaged cells, and thus extracellular ATP concentration is likely raised during tissue injury or inflammation (10). Recent evidence from our and other laboratories showed that functional P2 receptors of the P2X and P2Y subtypes are expressed on both mouse and human DCs (15–18). Most studies however have addressed the effects of acute exposure and using relatively high doses of extracellular ATP. In this study, we report on the ability of chronic (24 h) stimulation with low doses of extracellular ATP to affect the maturation and Ag-presenting functions of DCs. The major finding of this study was that ATP at micromolar concentrations induced a distorted maturation of DCs, with blocked LPS- and CD40L-dependent production of IL-1, TNF-␣, IL-6, and IL-12 but unaffected release of IL-1 receptor antagonist (IL-1ra), and IL-10. As a result, 0022-1767/01/$02.00
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FIGURE 2. ATP up-regulates CD83, CD86, CD80, and CD54 in a dose-dependent manner. Immature DCs were incubated for 24 h with grading doses of ATP (E), ATP and 10 g/ml LPS (f), or ATP plus 1 g/ml sCD40L (Œ). DCs were then stained with mAbs against the indicated surface markers. ATP induced higher expression of CD83, CD86, and CD54 in both immature and LPS- or sCD40L-stimulated DCs, whereas CD80 was up-regulated only in immature DCs. Similar results were observed in three distinct experiments. FIGURE 1. Extracellular ATP induces up-regulation of CD83, CD86, and CD54 in both immature and mature DCs. DCs were generated from peripheral blood CD14⫹ cells cultured with GM-CSF and IL-4. On day 6, CD2⫹ and CD19⫹ cells were removed, and the resulting cells were ⬎95% CD1a⫹ and CD14⫺. Immature DCs were left untreated, stimulated with 250 M ATP, or induced to mature with 10 g/ml LPS or 1 g/ml sCD40L in the presence or absence of 250 M ATP. After a 24-h incubation, DCs were stained with mAbs against the indicated surface molecules (bold line) or with isotype control Abs (thin line). Numbers indicate the net mean fluorescence intensity. The results are representative of 10 independent experiments.
mature DCs exposed to ATP showed an impaired ability to initiate Th1 responses.
Materials and Methods Reagents and Abs LPS (from Escherichia coli 055:B5), ADP, and indomethacin were purchased from Sigma-Aldrich (Milan, Italy). ATP, ATP-␥-S, and UTP were obtained from Boehringer Mannheim (Mannheim, Germany). Soluble CD40L (sCD40L) was obtained from Alexis (San Diego, CA). Adenosine receptors antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 8-(3chlorostyryl)caffeine (CSC), MRS-1220, and the inhibitor of cAMP-mediated activation of protein kinase A, Rp-adenosine 3⬘,5⬘-cyclic monophosphothioate triethylamine (Rp-cAMPS), were purchased from Research Biochemicals International (Natick, MA). The mAbs FITC-conjugated and pure anti-HLA-DR, anti-CD45RO and anti-CD45RA, and FITC-conjugated anti-CD14, anti-CD3, and anti-CD4 were purchased from Becton Dickinson (San Jose, CA). FITC-conjugated anti-CD1a, anti-CD86, anti-CD40, and antiIFN-␥; pure anti-CD28; and PE-conjugated rat anti-IL-4 were obtained from PharMingen (San Diego, CA). FITC-conjugated anti-CD54, anti-CD80, and anti-CD83 and pure anti-CD3 were purchased from Immunotech (Marseille, France). Anti-MHC class I was obtained from Dako (Glostrup, Denmark). Control mouse or rat Ig were purchased from Becton Dickinson or PharMingen. The anti-human IL-10 mAb and human rIL-12 were provided by R&D Systems (Minneapolis, MN). Anti-IL-10 mAb, sCD40L, and ATP had undetectable endotoxin levels (⬍10 pg/mg) by the Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD).
DC preparation and stimulation DCs were prepared from purified peripheral blood monocytes of healthy individuals, as described previously (19). Briefly, ⬎90% pure CD14⫹cells were cultured at 1 ⫻ 106 cells/ml in RPMI 1640 supplemented with 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 25 mM HEPES, 0.05 mM 2-ME, 100 U/ml penicillin, and 100 g/ml streptomycin (all from Life Technologies, Chagrin Falls, OH; complete RPMI) supplemented with 10% FBS, 100 ng/ml human rGM-CSF, and 200 U/ml human rIL-4 (R&D Systems) at 37°C with 5% CO2. Medium was changed after 3 days, and, at day 6 of culture, cells were recovered and depleted of
CD2⫹ and CD19⫹ cells by means of immunomagnetic beads coated with specific mAbs (Dynal, Oslo, Norway). This procedure gave ⬎97% pure CD1a⫹ and CD14⫺ DC preparations. DCs were left untreated or incubated for 24 h at 37°C with nucleotides or induced to mature with LPS (10 g/ml) or sCD40L (1 g/ml) in the presence or absence of nucleotides. Where indicated, DCs cultures were added with 25 M DPCPX, 25 M indomethacin, 50 M MRS-1220, 1 M CSC, or 300 M Rp-cAMPS 30 min before the addition of ATP.
Flow cytometry analysis of DCs DCs either untreated or stimulated for 24 h with LPS or sCD40L in the presence or absence of nucleotides were washed and then incubated with FITC-conjugated mAbs in PBS containing 2% FBS and 0.01% NaN3 for 40 min at 4°C. When pure mAbs were used, a second incubation with a FITC-coupled goat (Fab⬘)2 anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) was performed. Matched isotype mouse Ig were used in control samples. To detect DC apoptosis and necrosis, cells were stained with FITC-conjugated annexin V and propidium iodide using the annexin V-FITC apoptosis detection kit from Genzyme (Cambridge, MA). Cells were analyzed using a FACScan (Becton Dickinson).
Quantitation of endocytosis DCs stimulated as reported above were washed, resuspended in complete medium, and then incubated with 1 mg/ml Texas Red-conjugated BSA (Molecular Probes, Eugene, OR) at 37°C or 4°C. At selected time points, uptake was stopped by adding cold PBS containing 2% FBS and 0.01% NaN3. Cells were then washed four times and analyzed in a FACScan. Surface binding values obtained by incubating cells at 4°C were subtracted from values measured at 37°C.
DC cytokine release IL-10 was measured in DC supernatants by ELISA using a matched pair of mAbs from PharMingen. IL-1␣, IL-1, IL-1ra, and IL-6 concentrations were determined using ELISA kits from R&D Systems. IL-12 (p70) and TNF-␣ were measured using OptiEIA kits from PharMingen. Samples were assayed in triplicate for each condition.
RNase protection assay Total RNA was extracted from purified DCs after a 24-h incubation with the indicated stimuli using TRIzol (Life Technologies) according to the manufacturer’s instructions. Two multiprobe template sets, hCK2 and hCK3 (RiboQuant; PharMingen), were used for in vitro transcription reactions in the presence of a GACU pool and a T7 RNA-polymerase to synthesize [32P]UTP-labeled antisense probes. RNase protection analysis of 3 g of total RNA was performed after overnight hybridization at 60°C with 2.5 ⫻ 106 cpm of hCK2 or hCK3, followed by digestion with RNase A and T1 according to standard protocols. Protected fragments were treated with proteinase K, extracted with phenol-chloroform plus isoamyl alcohol (50:1), and finally precipitated in ethanol in the presence of ammonium
The Journal of Immunology
1613 man serum. Cocultures were pulsed at day 3 with 1 Ci/well [3H]thymidine for about 16 h at 37°C and then harvested onto fiber-coated 96-well plates. Radioactivity was measured in a beta counter. Results are given as mean cpm ⫾ SD of triplicate cultures. Where indicated, allogeneic naive T cells were purified (⬎95% CD45RA⫹) by incubation of T cells with antiCD45RO mAb followed by a goat anti-mouse Ig coupled to immunomagnetic beads and then cocultured (106 cells/well) with DCs (5 ⫻ 104 cells/ well) in a 24-well plate in complete RPMI plus 5% human serum for 6 days. Thereafter, T cells were restimulated or not with plate-coated antiCD3 and soluble anti-CD28 mAbs (both at 1 g/ml), and examined for intracellular IFN-␥ and IL-4 after 6 h. For two-color intracellular staining, monensin (10 M; Sigma, St. Louis, MO) and brefeldin A (10 g/ml; Sigma) were added to the cultures before the staining to prevent cytokine secretion. T cells were then fixed with 2% paraformaldehyde, permeabilized with 0.5% saponin, stained with FITC-conjugated mouse anti-IFN-␥ and PE-conjugated rat anti-IL-4, and finally analyzed with a FACScan. In control samples, staining was performed using isotype-matched control Ab. Cytokines were also measured in the T cell supernatants 48 h after activation by ELISA using matched pairs of mAbs (for IL-4, IL-10, and IFN-␥) and OptEIA kit (for IL-5) from PharMingen. T cells that were not restimulated at day 6 did not show any lymphokine production (data not shown).
Statistical analysis The Mann-Whitney U test was used to compare differences in cytokine release, endocytosis, and T cell proliferation. The p values ⱕ 0.05 were considered significant. FIGURE 3. ATP induces functional maturation of DCs. Immature DCs were left untreated (E) or stimulated with 250 M ATP (䡺), 10 g/ml LPS (F), or LPS plus ATP (f) for 24 h. Cells were then extensively washed, and Ag-presenting capacity and endocytic activity were measured. A, DCs were incubated with 1 mg/ml Texas Red-BSA at 37°C or 4°C. At various time points, cells were washed and the accumulation of labeled BSA was measured by flow cytometry. Differences in BSA uptake between untreated and ATP-treated immature DCs were significant (p ⬍ 0.03) at time points for ⬎30 min. Results are given as the average (⫾SD of duplicate cultures) of the mean fluorescence subtracted from the fluorescence of cells incubated at 4°C. Three distinct experiments gave similar results. B, Graded numbers of DCs were cocultured with 105/well purified CD45RA⫹ naive allogeneic T lymphocytes. [3H]Thymidine incorporation was measured after 3 days. Differences in T cell proliferation induced by untreated immature DCs and ATP-stimulated DCs were significant (p ⬍ 0.02) at APC doses ⬎625/well. Results are given as mean cpm ⫾ SD of triplicate cultures and are representative of four different experiments.
acetate. The samples were electrophoresed on 5% denaturing sequencing gels and then exposed to Kodak films (Kodak, Rochester, NY).
DC-T cell cocultures For the primary MLR assay, T lymphocytes were purified (⬎95% CD3⫹) from the heavy density fraction (50 – 60%) of Percoll gradients followed by immunomagnetic depletion using a mixture of anti-HLA-DR and antiCD19 mAb-conjugated beads (Dynal). DCs were washed and then cultured in 96-well microculture plates in serial dilutions along with 1.5 ⫻ 105/well allogeneic T lymphocytes in complete RPMI complemented with 5% hu-
Results Extracellular ATP induces phenotypic and functional maturation of DCs DCs maturation is associated with higher expression of MHC and costimulatory molecules, as well as de novo expression of CD83. Immature DCs incubated for 24 h with 250 M ATP displayed enhanced levels of CD83, CD86, CD80, and CD54 (Fig. 1), as previously reported by Berchtold et al. (16). In addition, ATP further increased the expression of CD83, CD86, and CD54, but not CD80, induced by either LPS or sCD40L, whereas the expression of MHC molecules was not at all affected. The effects of ATP on CD83, CD86, CD80, and CD54 membrane levels were dose dependent (Fig. 2). As a consequence of the increased expression of CD86, but not CD80, the CD86:CD80 ratio was 30 – 40% higher in ATP/LPS- or ATP/sCD40L-stimulated DCs compared with DC stimulated solely with LPS or sCD40L. A reduced ability to capture exogenous Ags is an early event during DC maturation and has been demonstrated for fluid phase tracers, receptor-mediated endocytosis, as well as for the uptake of particulates and bacteria (1, 19). In keeping with these observations, ATP induced a significant decrease in the uptake of BSA in immature DCs (Fig. 3A). Since ATP affected DC phenotype and endocytic activity, we next studied the effect of ATP on the Ag-presenting function of DCs. Fig. 3B shows that ATP stimulation increased the capacity of immature DCs to induce proliferation of allogeneic naive CD45RA⫹
Table I. ATP inhibits release of IL-12 and proinflammatory cytokines, but not IL-10 and IL-1ra, from maturing DCs Treatmenta
TNF-␣
IL-12
IL-6
IL-10
IL-1␣
IL-1
IL-1ra
None ATP LPS LPS ⫹ ATP sCD40L sCD40L ⫹ ATP
⬍0.1 ⬍0.1 22.9 ⫾ 3.1 0.6 ⫾ 0.0† 15.5 ⫾ 1.8 4.1 ⫾ 0.8‡
⬍0.1 ⬍0.1 2.2 ⫾ 0.3 0.1 ⫾ 0.0† 2.7 ⫾ 0.4 0.3 ⫾ 0.1‡
⬍0.1 ⬍0.1 22.0 ⫾ 2.6 5.6 ⫾ 0.9† 23.7 ⫾ 2.9 11.0 ⫾ 1.8‡
⬍0.1 ⬍0.1 6.8 ⫾ 1.2 6.3 ⫾ 1.0 12.0 ⫾ 1.6 13.6 ⫾ 1.5
⬍0.1 ⬍0.1 0.4 ⫾ 0.1 ⬍0.1† 0.2 ⫾ 0.0 ⬍0.1‡
⬍0.1 ⬍0.1 11 ⫾ 0.9 2.5 ⫾ 0.1† 10 ⫾ 0.7 3.4 ⫾ 0.8‡
49 ⫾ 11 82 ⫾ 10* 134 ⫾ 15 122 ⫾ 14 121 ⫾ 9.5 115 ⫾ 13
a DCs were left untreated or stimulated with 250 M ATP, 10 g/ml LPS, LPS plus ATP, 1 g/ml sCD40L, or sCD40L plus ATP. After 24 h, cytokines were measured in the supernatants by ELISA. Results are expressed as ng/106 cells/ml and represent the mean ⫾ SD of triplicate cultures. * p ⬍ 0.04 vs untreated DCs; †, p ⬍ 0.03 vs LPS-stimulated DCs; ‡, p ⬍ 0.01 vs DCs treated with sCD40L.
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T lymphocytes. In contrast, ATP did not increase the alloantigenpresenting functions of LPS-stimulated DCs.
ATP inhibits the capacity of mature DCs to induce Th1 responses
ATP inhibits the production of IL-12 and proinflammatory cytokines, but not IL-10 or IL-1ra, by mature DCs
IL-12 released by mature DCs is the most important factor that drives the differentiation of naive T cells toward the IFN-␥-producing Th1 phenotype (6). Given the inhibitory effect of ATP on IL-12 production by DCs, we next examined the quality of primary T cell response induced by DCs matured in the presence of extracellular ATP. Naive allogeneic T cells primed with mature DCs differentiated into Th1 lymphocytes as they produced large amounts of IFN-␥ and low levels of IL-4, IL-5, and IL-10 (Fig. 5).
A major attribute of mature DCs is the synthesis and release of cytokines with important modulatory function on inflammation and T cell differentiation. ATP did not induce cytokine release by immature DCs, with the exception of IL-1ra, whereas strongly inhibited LPS or sCD40L induced production of TNF-␣, IL-1␣, IL1, IL-6, and IL-12. In contrast, IL-10 and IL-1ra synthesis and release by mature DCs were only slightly or not affected (Table I and Fig. 4). The changes in cytokine production were not due to a toxic effect of ATP since neither the trypan blue exclusion test nor lactate dehydrogenase release assay showed increased cell death after 250 M ATP stimulation for 24 h (data not shown). In addition, 250 M ATP did not induce DC apoptosis (see below). The cytokine profile of DCs matured in the presence of extracellular ATP was thus characterized by abundant IL-10 and IL-1ra, but absent production of IL-12 and proinflammatory cytokines. Because autocrine IL-10 inhibits IL-12 and TNF-␣ production by DCs,5 we stimulated DCs with LPS along with ATP in the presence of anti-IL-10-neutralizing mAb or isotype control mAb. Neutralization of autocrine IL-10 only partially restored IL-12 and TNF-␣ production, indicating that ATP suppression of IL-12 and TNF-␣ production by mature DCs was largely independent from autocrine IL-10 (data not shown). 5 S. Corinti, C. Albanesi, A. la Sala, S. Pastore, and G. Girolomoni. Regulatory activity of autocrine interleukin-10 on dendritic cell functions. Submitted for publication.
FIGURE 4. ATP inhibits mRNA transcription of TNF-␣, IL-12p40, IL1␣, IL-1 and IL-6 but not of IL-10 and IL-1ra by mature DCs. Immature DCs were left untreated (lane 1) or stimulated with 250 M ATP (lane 2), 10 g/ml LPS (lane 3), LPS plus ATP (lane 4), 1 g/ml sCD40L (lane 5), or sCD40L plus ATP (lane 6). After 24 h, total RNA was extracted and the RNase protection assay performed to visualize cytokine-specific mRNA transcripts using two multiprobe template sets.
FIGURE 5. DCs matured in the presence of extracellular ATP display impaired ability to initiate Th1 responses in vitro. Immature DCs were left untreated or stimulated with 250 M ATP, or induced to undergo maturation with LPS in the presence or absence of 250 M ATP. After 24 h, DCs were washed and used to prime purified allogeneic CD45RA⫹ naive T lymphocytes. T cell priming was also performed with LPS- or LPS/ATPtreated DCs, adding exogenous IL-12 (2 ng/ml) at the beginning of the cocultures. T cells were cultured in the presence of 30 U/ml IL-2 and after 10 days restimulated with plate-coated anti-CD3 and soluble anti-CD28 mAbs. Cells were then examined for intracellular IFN-␥ and IL-4 by flow cytometry after 6 h (A) and for cytokine release by ELISA after 48 h (B). In A, the numbers indicate the percentage of positive cells in each quadrant. In B, data are expressed as ng/ml/106 cells ⫾ SD of triplicate cultures. ⴱ, p ⬍ 0.03 vs T cells primed with immature DCs; ⴱⴱ, p ⬍ 0.05 vs T cells primed with LPS-matured DCs; ⴱⴱⴱ, p ⬍ 0.02 vs T cells primed with DCs treated with LPS and ATP. Shown is a representative experiment of four performed.
The Journal of Immunology In contrast, T lymphocytes primed with DCs matured in the presence of ATP displayed a reduced production of IFN-␥ and a higher release of IL-4, IL-5, and IL-10. Addition of rIL-12 at the beginning of the T cell culture restored IFN-␥ production and inhibited IL-4, IL-5, and IL-10 release, suggesting that most of the effects of ATP on the T cell-differentiating properties of DCs were consequent to inhibition of IL-12 production. Suppressed IL-12 and TNF-␣ release by ATP is not mediated by PGs or elevation in intracellular cAMP and does not require extracellular ATP degradation PGE2 and various cAMP-elevating agents such as cholera toxin, rolipram, and cicaprost have been reported to inhibit TNF-␣ and IL-12, but not IL-10 production by monocytes and DCs (20 –22). Fig. 6 shows that blocking PG synthesis by indomethacin or the activation of cAMP-dependent protein kinase A by Rp-cAMPS could not restore TNF-␣ and IL-12 production. Interestingly, indomethacin markedly increased IL-12 and TNF-␣ release by mature DCs, confirming previously published data (23). Extracellular nucleotides are subjected to rapid degradation by plasma membrane ectoenzymes, with ATP and ADP degraded to AMP by apyrase, and AMP metabolized to adenosine by 5⬘-nucleotidase (24). DCs express functional apyrase and 5⬘-nucleotidase (16, 25),
FIGURE 6. ATP-induced abrogation of TNF-␣ and IL-12 release by mature DCs is not mediated by endogenous PGE2, cAMP, or P1 adenosine receptors. Immature DCs were stimulated with LPS (䡺) or with LPS plus 250 M ATP (f) in the presence or absence of 25 M indomethacin, 300 M Rp-cAMPS, or 25 M DPCPX (added 30 min before stimulation). After a 24-h culture, cytokines were measured in the supernatants by ELISA. Results are given as mean ng/ml/106 cells ⫾ SD of triplicate cultures and are representative of two different experiments. Differences in TNF-␣ and IL-12 release between LPS- and LPS plus ATP-stimulated DCs were always statistically significant (p ⬍ 0.001).
1615 and adenosine and its metabolite inosine have been reported to inhibit TNF-␣ and IL-12 production by monocytes (26). Incubation of DCs with the adenosine receptors antagonist DPCPX did not prevent the inhibitory effect of ATP on TNF-␣ and IL-12 production by maturing DCs (Fig. 6). Selective inhibitors of adenosine A1 (8CSC) and A2 (MRS-1220) receptors also failed to counteract ATP effects (data not shown). To further asses whether ATP could act through its metabolites, we tested the nonhydrolyzable ATP analogue ATP-␥-S, which dose-dependently inhibited TNF-␣ and IL-12 release by LPS-stimulated DCs with an efficiency 100fold higher than ATP (Fig. 7). However, ATP-␥-S at concentrations ⱖ10 M reduced also IL-10 release, most likely as a result of a direct toxic effect. Indeed, ATP-␥-S at these concentrations increased significantly the number of apoptotic and necrotic DCs, a property not shared by ATP, UTP, or ADP (Fig. 8 and data not
FIGURE 7. Different potency of ATP, ADP, ATP-␥-S, and the inability of UTP to inhibit TNF-␣ and IL-12 release by mature DCs suggest P2X purinoceptor involvement. Immature DCs were stimulated with 10 g/ml LPS plus graded concentrations of ATP (F), ADP (⽧), the nonhydrolyzable ATP analogue ATP-␥-S (Œ), or UTP (f). After a 24-h incubation, ELISA was performed on culture supernatants. Results are given as mean ng/ml/106 cells ⫾ SD of triplicate cultures. Differences in TNF-␣ release between DCs treated with LPS and LPS plus nucleotides were significant (p ⬍ 0.03) when ATP was ⱖ25 M, ADP ⱖ 250 M, and ATP-␥-S ⱖ 2.5 M. Differences in IL-12 secretion between DCs stimulated with LPS and LPS plus nucleotides were significant (p ⬍ 0.03) when ATP was ⱖ 25 M, ADP ⱖ 25 M, and ATP-␥-S ⱖ 2.5 M. IL-10 release was significantly lower (p ⬍ 0.05) only when ATP-␥-S was ⱖ10 M. Shown is a representative experiment of three performed.
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FIGURE 8. ATP-␥-S, but not ATP or UTP, increases DC apoptosis and necrosis. A, Immature DCs were stimulated with 10 g/ml LPS plus 250 M ATP, UTP, or ATP-␥-S. After a 6-h incubation at 37°C with 5% CO2, DCs were analyzed for propidium iodide staining and annexin V binding by double-color flow cytometry. The numbers indicate the percentage of necrotic cells (upper right quadrant) and apoptotic cells (lower right quadrant). B, Dose-response effect of the nucleotides on the percentage of annexin V⫹ DCs. Two independent experiments gave similar results.
shown). Fig. 7 also shows that UTP, which activates P2Y but not P2X receptors, was unable to change the cytokine profile of mature DCs. In contrast, ADP inhibited TNF-␣ and IL-12 production by mature DCs, with a potency similar to ATP.
Discussion In this study, we found that chronic (24-h) stimulation of immature DCs with low, noncytotoxic ATP doses increased membrane expression of CD54, CD86, CD80, and CD83, slightly reduced the endocytic activity of DCs, and augmented their capacity to promote proliferation of naive allogeneic T cells. Moreover, ATP enhanced the expression of CD54, CD83, and CD86, but not CD80, on DCs stimulated with LPS or sCD40L. On the other hand, ATP markedly and dose-dependently inhibited LPS- and sCD40L-dependent production of IL-12 and proinflammatory cytokines, whereas production of IL-1ra and IL-10 was not altered. As a consequence of blocked IL-12 production, DCs matured in the presence of extracellular ATP showed an impaired capacity to initiate Th1 responses in vitro. In fact, naive T cells stimulated with mature DCs exposed to extracellular ATP produced lower levels of IFN-␥, but higher amounts of IL-4, IL-5, and IL-10. Addition of exogenous IL-12 at the time of T cell priming restored a potent Th1 polarization, suggesting that the immune deviation induced by ATP is mainly due to the suppression of IL-12 production in maturing DCs. Moreover, DCs matured in the presence of ATP expressed a higher CD86:CD80 ratio, and this can be an additional factor that may favor the induction of Th2 response, in line with the selective role of CD86 in Th2 polarization (27, 28). DC maturation is an important control point in the development of immune responses, as it converts Ag-capturing DCs into potent immunostimulatory APCs and can also determine whether periph-
ATP ALTERS DENDRITIC CELL MATURATION eral tolerance or immunity is induced (29). Maturation of DCs is stimulated by factors signaling tissue danger such as microorganisms, dying cells, and proinflammatory cytokines. Recently, a variety of factors has emerged that can limit DC maturation. For example, intracellular cAMP-elevating agents such as PGE2, cholera toxin, and 2-adrenoreceptor agonists inhibit IL-12 and TNF-␣ and enhance IL-10 expression by LPS-stimulated DCs (21, 23, 30). In contrast, IL-10, glucocorticoids, and vitamin D3 interfere with DC maturation as a whole by blocking the up-regulation of presenting and costimulatory molecules, the release of IL-12 and TNF-␣ but not IL-10, and maintain DCs in an Ag-capturing state (31–33). Extracellular ATP acted independently of the above-cited mechanisms since its activity was not affected by indomethacin, inhibition of cAMP-dependent protein kinase A, or neutralization of endogenous IL-10. We also excluded that ATP worked through products of its extracellular metabolism. DCs express on their surface both ecto-apyrase and ecto-5⬘-nucleotidase which rapidly hydrolyze ATP to AMP and adenosine (16), and adenosine receptors activation is reported to selectively inhibit IL-12 and TNF-␣ production by human monocytes (26, 34, 35). The nonhydrolyzable ATP analogue, ATP-␥-S, was more efficient than ATP at inhibiting IL-12 and TNF-␣ production, and antagonists of adenosine receptors could not prevent ATP activity. Thus, extracellular ATP and P2 purinoceptors appear to play a direct role in the modulation of DCs response to both noncognate and cognate maturation stimuli. Given the number of P2X and P2Y receptors subtypes expressed by DCs and the lack of specific agonists or inhibitors, it is difficult to link receptors subtypes to a given cellular response, with the exception of the P2X7 receptor which was shown previously to mediate membrane permeabilization, cell death, and IL-1 release at concentrations much higher than those used in this study (18). However, the inability of UTP and the similar potency of ADP to reproduce ATP effects indicated that ATP could function through the P2X receptor family. Exposure of DCs to necrotic cells induces full maturation of DCs, including high membrane expression of costimulatory and MHC-presenting molecules and acquisition of potent capacity to stimulate effector T cell responses (4, 5), but the nature of the activating stimuli provided by necrotic cells are not known. We have shown here that ATP increases DC expression of CD83, CD86, and CD54, but not MHC molecules, and inhibits selectively the production of proinflammatory cytokines and IL-12. The effects of ATP on cytokine production are rather reminiscent of those observed in macrophages that have ingested apoptotic cells (36, 37). The intracellular concentration of ATP ranges from 5 to 10 mM, and any agent that causes plasma membrane damage or rapid cell death can promote the release of intracellular ATP (14). Hence, micromolar concentrations of ATP in the extracellular milieu are likely to be present at sites of inflammation associated with tissue damage. In this context, extracellular ATP may act as an important signal which can limit the initiation of Th1 response and favor Th2 immune deviation and prevent detrimental proinflammatory cytokine release by DCs. Moreover, the unchanged IL-10 production by maturing DCs exposed to ATP may allow the emergence of immune regulatory cells, such as T regulatory cells 1 (38, 39), an hypothesis currently tested in our laboratory. In conclusion, extracellular ATP may serve as an important signal for an alternative DC maturation (40) and thus for the prevention of exaggerated and harmful immune responses. This phenomenon may be especially relevant in the context of chronic inflammatory disorders during which ATP can be easily released by damaged cells and DCs can differentiate from monocyte precursors (41).
The Journal of Immunology
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