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The P2 purinergic receptors of human dendritic cells: identification and coupling to cytokine release

DAVIDE FERRARI^*1,2, ANDREA LA SALA^,2, PAOLA CHIOZZI^*, ANNA MORELLI^*, SIMONETTA FALZONI^*, GIAMPIERO GIROLOMONI, MARCO IDZKO^§, STEFAN DICHMANN^¶, JOHANNES NORGAUER^¶ and FRANCESCO DI VIRGILIO^*,

^* Department of Experimental and Diagnostic Medicine, Section of General Pathology, and
Center of Biotechnology, University of Ferrara, Ferrara, Italy;
Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, IRCCS, Rome, Italy; and
^§ Departments of Pneumology and
^¶ Dermatology, University of Freiburg, Freiburg, Germany

¹Correspondence: Department of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara, via L. Borsari 46, I-44100 Ferrara, Italy. E-mail: dfr{at}dns.unife.it

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We investigated the expression of purinoceptors in human dendritic cells, providing functional, pharmacological, and biochemical evidence that immature and mature cells express P2Y and P2X subtypes, coupled to increase in the intracellular Ca²⁺, membrane depolarization, and secretion of inflammatory cytokines. The ATP-activated Ca²⁺ change was biphasic, with a fast release from intracellular stores and a delayed influx across the plasma membrane. A prolonged exposure to ATP was toxic to dendritic cells that swelled, lost typical dendrites, became phase lucent, detached from the substrate, and eventually died. These changes were highly suggestive of expression of the cytotoxic receptor P2X₇, as confirmed by ability of dendritic cells to become permeant to membrane impermeant dyes such as Lucifer yellow or ethidium bromide. The P2X₇ receptor ligand 2',3'-(4-benzoylbenzoyl)-ATP was a better agonist then ATP for Ca²⁺ increase and plasma membrane depolarization. Oxidized ATP, a covalent blocker of P2X receptors, and the selective P2X₇ antagonist KN-62 inhibited both permeabilization and Ca²⁺ changes induced by ATP. The following purinoceptors were expressed by immature and mature dendritic cells: P2Y₁, P2Y₂, P2Y₅, P2Y₁₁ and P2X₁, P2X₄, P2X₇. Finally, stimulation of LPS-matured cells with ATP triggered release of IL-1ß and TNF-. Purinoceptors may provide a new avenue to modulation of dendritic cells function.—Ferrari, D., La Sala, A., Chiozzi, P., Morelli, A., Falzoni, S., Girolomoni, G., Idzko, M., Dichmann, S., Norgauer, J., Di Virgilio, F. The P2 purinergic receptors of human dendritic cells: identification and coupling to cytokine release.

Key Words: extracellular nucleotides • nucleotide receptors • IL-1ß • TNF- • inflammation

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
DENDRITIC CELLS (DCS) are antigen-presenting cells specialized to activate naive T lymphocytes and initiate primary immune responses (1 2 3) . DCs are widely distributed in tissues where they efficiently capture antigens. After exposure to inflammatory stimuli, DCs undergo maturation, a process that encompasses acquisition of a high level of surface MHC, costimulatory molecules, chemokine, and cytokine production as well as increased antigen-presenting activity, and migrate to lymphoid organs to start immune responses (1) . Although DCs are the focus of increasing attention, many aspects of their biology and specialized functions remain unknown. DCs are known to be activated by immune and nonimmune mediators, such as the poorly characterized factors released from virally infected, stressed, or necrotic cells (4) . ATP could be a good candidate to the role of a nonimmune activatory stimulus for DCs since it is released by infected or injured cells. Very recently, we reported the expression by mouse DCs of the purinergic P2X₇ receptor, a member of the family of plasma membrane receptors for extracellular nucleotides (5) .

Extracellular nucleotides have been recognized as important mediators in many systems, including the immune system, where they trigger different responses via activation of plasma membrane receptors known as P2 purinoceptors (6 7 8 9) . On the basis of pharmacological, functional, and cloning data, two P2 receptor subfamilies have been described: P2Y and P2X (10) . P2Y receptors are seven membrane-spanning, G-protein-coupled receptors. Their activation triggers generation of inositol 1,4,5-trisphosphate and release of Ca²⁺ from intracellular stores. P2Y receptors are ubiquitous, being expressed by monocytes, macrophages, neurons, smooth and striated muscle cells, as well as epithelial and endothelial cells (7) . Responses to extracellular nucleotides suggestive of P2Y expression have been reported in human DCs (11) . P2X receptors are identified with plasma membrane channels selective for monovalent and divalent cations, which are directly activated by extracellular ATP without requiring hydrolysis of the nucleotide or generation of intracellular second messengers (12) . These channels were originally identified in mammalian sensory neurons, and subsequently also found in smooth muscle cells, fibroblasts, and thymocytes (7 , 12) . An interesting member of the P2X subfamily is the recently cloned P2X₇ receptor, previously known as P2Z (13) . The P2X₇ receptor subunit is a 595 AA protein that assembles in the plasma membrane to form multimeric complexes of unknown stoichiometry (14) . P2X₇ differs from the other P2X receptors by its extended amino-terminal domain that endows this receptor with the ability to form large plasma membrane pores permeable to small hydrophilic molecules. An interesting property of the P2X₇ pore is its reversibility: removal of ATP triggers resealing of the plasma membrane and, provided the exposure to ATP is brief, cell recovery. The only known physiological ligand of P2X₇ is ATP, most probably in its fully dissociated form, but synthetic ligands based on the ATP structure, among which 2',3'-(4-benzoyl-benzoyl)-ATP (BzATP) is the most potent, are also active (13) . The P2X₇ receptor is absent from freshly isolated monocytes but appears during differentiation to macrophages, and is up regulated by interferon (IFN) or tumor necrosis factor (TNF-) plus lipopolysaccharide (LPS) (15 , 16) . The physiological role of P2X₇ is unknown, although it has been suggested that it might participate in the modulation of lymphocyte proliferation (17) and interleukin 1ß (IL-1ß) release from macrophage and microglial cells (18 19 20) . In addition, we have recently provided evidence that this receptor also modulates the interaction between mouse dendritic cells and T_H lymphocytes (5) .

Given the emerging and potentially important role of P2 receptors in immunomodulation, we carried out a thorough characterization of P2X and P2Y receptor expression and function in human DCs derived from peripheral blood monocytes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
ATP, UTP, CTP, GTP, and ADP were purchased from Boehringer Mannheim (Mannheim, Germany). 2',3'-(4-benzoyl-benzoyl)-ATP, digitonin, ionomycin, and lipopolysaccharide from Escherichia coli, serotype 055:B5, were purchased from Sigma (Sigma-Aldrich, Milan, Italy), KN-62 was from Calbiochem (Calbiochem-Novabiochem Corporation, La Jolla, Calif.); Percoll and Ficoll-Paque were obtained from Pharmacia (Pharmacia Biotech AB, Uppsala, Sweden). Periodate-oxidized ATP (oATP) was a kind gift of Dr. Stefania Hanau, University of Ferrara, (Ferrara, Italy).

Solutions
Fluorescence measurements were performed either in saline solution containing 125 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 1 mM Na₂HPO₄, 5.5 mM glucose, 5 mM NaHCO₃, 1 mM CaCl₂, and 20 mM HEPES (pH 7.4 with NaOH), hereafter also referred to as standard saline solution, or in a Na⁺-free saline solution containing 300 mM sucrose, 1 mM MgSO₄, 1 mM K₂HPO₄, 5.5 mM glucose, 1 mM CaCl₂, and 20 mM HEPES (pH 7.4 with KOH).

Purification of dendritic cells
Dendritic cells were obtained from peripheral blood of healthy donors, as described previously (21) . Briefly, monocytes were separated by a two step Ficoll and Percoll gradient, and then cultured in RPMI 1640 (Life Technologies, Gaithersburg, Md.) complemented with 10% heat-inactivated FBS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.05 mM 2-ME (all from Life Technologies) at 37°C with 5% CO₂, in the presence of 200 U/ml IL-4 and 100 ng/ml GM-CSF (Pepro Tech, Rocky Hill, N.J.), we will refer to this medium as complete culture medium. After 6 days, DCs were negatively selected by magnetic separation with anti-CD2 and anti-CD19 Ab-coniugated beads (Dynal, Oslo, Norway) to remove contaminating lymphocytes. The procedure resulted >98% pure CD1a⁺ and CD14^-, CD2^-, and CD19^- DC preparations. Maturation of DCs was obtained by overnight incubation with 50 µg/ml LPS. LPS-stimulated DCs were characterized by FACS for their expression of the differentiation markers CD40, CD54, CD83, and CD86.

Cytoplasmic free Ca²⁺ measurements
Changes in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) were measured with the fluorescent indicator Fura-2/AM, using an LS50 Perkin Elmer fluorometer (Perkin Elmer Ltd., Beaconsfield, U.K.), as described previously (15 , 17) . For Fura-2/AM loading, cells (1x10⁷/ml) were resuspended in standard saline solution, in the presence of 4 µM Fura-2/AM and 250 µM sulfinpyrazone (Sigma-Aldrich). Incubation was performed at 37°C for 15 min. Cells were then washed in the same solution and [Ca²⁺]_i changes were determined in a thermostated, magnetically stirred cuvette, with the 340/380 excitation ratio at an emission wavelength of 505 nm.

Plasma membrane potential measurement
Changes in plasma membrane potential were measured with the fluorescent dye bis[1,3-diethylthiobarbiturate)trimethineoxonal (bisoxonol) (Molecular Probes, Leiden, The Netherlands) at the wavelength pair 540/580 nm, as described previously (15) . Experiments were performed in a spectrofluorometer (model LS50, Perkin Elmer Ltd., Beaconsfield, U.K.) equipped with a thermostat-controlled (37°C) cuvette holder and magnetic stirrer.

Changes in plasma membrane permeability
ATP-dependent increases in plasma membrane permeability were measured by monitoring the uptake of the dyes ethidium bromide or Lucifer yellow (Molecular Probes) as reported previously (15 , 22) . For Lucifer yellow uptake, cells were incubated at 37°C for 15 min, in a Ca²⁺-free standard saline solution containing 1 mg/ml Lucifer yellow, 250 µM sulfinpyrazone to block fluorescent dye extrusion, and 1 mM ATP. After several rinsings with culture medium to remove the extracellular dye, cells were analyzed with an inverted fluorescence microscope (Olympus IMT-2, Olympus Optical Co. Ltd., Tokyo, Japan) equipped with a 40x objective.

Measurement of enzymatic activity
Lactate dehydrogenase activity was measured according to standard methods (23) , briefly, DCs (5x10⁵/ml) were plated overnight in 24-well plates in the presence of 50 µg/ml LPS. Cells were then rinsed and incubated in culture medium with the nucleotides. Supernatants were collected, cleared by centrifugation (10 min at 2500 g) and added to a solution containing, 0.63 mM pyruvate, 11.3 mM NADH, 44.4 mM K₂HPO₄, 16.8 mM KH₂PO₄ (pH 7.5). Absorbance was measured in a spectrofluorometer (Ultrospec 3000, Pharmacia Biotech, Milan, Italy) at a wavelength of 340 nm. DCs were also lysed with 0.1% Triton X-100 (J.T. Baker, Milan, Italy) and cleared by centrifugation. Absorbance of these samples was considered as 100% of lactic dehydrogenase release.

Western blot analysis
Cells were lysed in lysis buffer containing 300 mM sucrose, 1 mM MgSO₄, 1 mM K₂HPO₄, 5.5 mM glucose, 20 mM HEPES (pH 7.4), 1 mM benzamidine, 1 mM PMSF, 0.2 µM DNase, and 0.2 µg of RNase by repeated freeze/thawing (three cycles). Proteins were separated on 7.5% SDS-polyacrylamide gels according to Laemmli (1970) and blotted overnight on nitrocellulose paper (Schleicher and Schull Italia, Legnano, Italy). The rabbit polyclonal anti-P2X₇ serum was raised against the synthetic peptide corresponding to the last 20 amino acids of the P2X₇ protein (KIRKEFPKTQGQYSGFKYPY) and was kindly provided by Dr. Gary Buell (Ares-Serono Pharmaceutical Research Institute, Geneva, Switzerland) (14) . The primary antibody was used at a dilution of 1:100 in TBS buffer (10 mM Tris-Cl, 150 mM NaCl, pH 8.0). The secondary antibody was a goat anti-rabbit antibody conjugated to alkaline phosphatase.

Flow cytometry analysis of P2X₇ expression
Cells were recovered from culture plates, washed with 2% FBS, 0.01% NaN₃ PBS, incubated for 30 min at 4°C with anti-P2X₇ mAb (kind gift of Dr. Gary Buell) or irrelevant mouse Ig (clone 107.3, PharMingen, San Diego, Calif.) as negative control. After three washes, cells were incubated with FITC-conjugated goat anti-mouse Ig (Southern Biotechnology Associates, Birmingham, Ala.), for 30 min at 4°C, washed and immediately analyzed using a FACScan with Cell Quest software (Becton Dickinson, Mountain View, Calif.).

RT-PCR
Total RNA was isolated by using RNAzol-B (TEL-TEST, Friendswood, Tex.). 100 ng RNA was reversed transcribed using the ACCESS reverse transcription-polymerase chain reaction (RT-PCR) kit (Promega Corporation, Madison, Wis.), then amplified by polymerase chain reaction (35 cycles); 10 µl of the PCR products for P2 receptors and 5 µl for ß-actin were then loaded and separated in a 2% agarose, ethidium bromide-containing gel. Control reactions in the absence of reverse transcriptase were also carried out. The sequence specific primers used for P2 receptors were P2X₁ (248 bp), 5'-CGCCTTCCTCTTCGAGTATG-3' forward, and 5'-GGAAGACGTAGTCAGCCACA-3' reverse primers. P2X₄ (521 bp), 5'-TGCATTTATGATGCTAAAACAG-3' forward, and 5'-CAAGACCCTGCTCGTAATC-3' reverse primers. P2X₇ (399 bp), 5'-AGATCGTGGAGAATGGAGTG-3' forward, and 5'-TTCTCGTGGTGTAGTTGTGG-3' reverse primers. P2Y₁ (260), 5'-TGTGGTGTACCCCCTCAAGTCCC-3' forward, and 5'-ATCCGTAACAGCCCAGAATCAGCA-3' reverse. P2Y₂ (367), 5'-CCAGGCCCCCGTGCTCTACTTTG-3' forward, and 5'-CATGTTGATGGCGTTGAGGGTGTG-3' reverse. P2Y₅ (282), 5'-CTTCACAACACGGAATTGGC-3' forward, and 5'-TCAAAGCAGGCTTCTGAGGC-3' reverse. P2Y₁₁ (238), 5'-GTGGTTGAGTTCCTGGTGGC-3' forward, and 5'-CCAGCAGGTTGCAGGTGAAG-3' reverse. ß-actin (661 bp), forward 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' and 5'-AGTCATAGTCCGCCTAGAAGCATTTGCGGT-3' reverse primers.

Cytokine measurement
For measurement of ATP-dependent cytokine release, DCs were washed free of LPS after the overnight incubation, further incubated for 3 days, and then stimulated again with 50 µg/ml LPS for 3 h before addition of ATP. Cytokines present in the supernatants of DC cultures (1x10⁶ cells/ml) were measured by ELISA, using commercially available ELISA kits from R&D Systems (Minneapolis, Minn.) according to the manufacturer’s instructions.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Human DCs express functional P2X receptors
Figure 1 shows the effect on plasma membrane permeability of a brief (15 min) stimulation of human DCs with 1 mM ATP. The near totality of the cells took up the extracellular dye, irrespectively of whether they had been induced to maturation with LPS. The level of positivity to the Lucifer yellow uptake test was striking: no other blood cells have ever been reported to exhibit a similar level of ATP-dependent increase in plasma membrane permeability, as circulating monocytes are only 10–17% positive and normal peripheral lymphocytes are negative (15 , 24 , 25) . Even macrophages, the current paradigm for P2X receptor studies in the immune system, are at the most 50% positive to Lucifer yellow uptake, a percentage that can increase to 80% after IFN treatment (15) . A prediction of the high susceptibility to ATP-dependent permeabilization is that DCs should also be very sensitive to ATP-dependent cytotoxicity. This prediction was fulfilled, as shown in the experiments reported in Fig. 2 , where a prolonged (2 h) incubation with 1 mM ATP (Fig. 2C , D ) caused marked morphological changes (rounding, loss of filopodia, and lamellipodia), which were exacerbated at higher (3 mM, Fig. 2E , F ; 5 mM, Fig. 2G , H ) ATP concentrations. Membrane blebbing, swelling (but in some cases also shrinkage), and cell detachment from the substrate became clearly apparent. There were no major differences in the susceptibility to ATP-mediated cytotoxic effects between immature and mature DCs. Figure 3A , B reports the time course and dose dependency of ATP-induced release of lactic dehydrogenase. Leakage of this cytosolic marker started 15 min after ATP addition and increased rapidly until 60 min. While the time course was shifted to the left, dose dependency was not significantly changed compared to that measured in other cell types, suggesting that affinity of the receptor was the same.

View larger version (154K): [in this window] [in a new window]   Figure 1. ATP stimulation induces Lucifer yellow uptake in human DCs. Cells, 2.5 x 105/ml, were incubated for 15 min with the fluorescent dye Lucifer yellow in the absence (A, B, E, F) or in the presence of 1 mM ATP (C, D, G, H), as described in Materials and Methods. A—D) Immature DCs; E–H) mature DCs.

View larger version (154K): [in this window] [in a new window]   Figure 2. Changes in cell morphology induced by ATP in human DCs. Cells, 2.5 x 105/ml, were incubated in culture medium in the absence (A, B) or in the presence of 1 (C, D), 3 (E, F), or 5 mM ATP (G, H) for 2 h. Immature DCs, A, C, E, G; mature DCs, B, D, F, H.

View larger version (13K): [in this window] [in a new window]   Figure 3. Effect of ATP on release of lactic dehydrogenase (LDH). A) Time course. ATP concentration was 5 mM. B) Dose dependency. Cells were incubated for 90 min in the presence of increasing ATP concentrations. Experiments were performed in a standard saline at a concentration of 5 x 105 cells/ml. Data are means ± SD of triplicate determinations.

ATP induces a rise in [Ca²⁺]_i and plasma membrane depolarization
ATP and other nucleotides are known to induce [Ca²⁺]_i changes in many cell systems, and we and others have shown that this is also the case in mononuclear phagocytes (15 , 19) . Human DCs also responded to a nonlytic ATP dose with a [Ca²⁺]_i rise suggestive of the operation of two main ATP-stimulated pathways: release from intracellular stores (fast early transient) and influx across the plasma membrane (delayed plateau) (Fig. 4A , trace a). Removal of extracellular Ca²⁺ (Fig. 4A , trace b) abolished the late, but not the early phase, thus confirming the intracellular source of the Ca²⁺ responsible for the early increase. This experiment strongly suggests that human DCs express both P2Y and P2X receptors. To further investigate the role of P2X receptors in [Ca²⁺]_i increases, we tested the effect of replacement of external Na⁺ with iso-osmolar sucrose. It is known that Ca²⁺ permeability through the P2X receptors is decreased in the presence of extracellular sodium; thus, it is expected that ATP-mediated [Ca²⁺]_i changes should be higher in the absence of Na⁺ (26 , 27) . Figure 4B shows that in the absence of Na⁺, ATP triggered an increase in [Ca²⁺]_i much larger, especially in the delayed phase, although the fast transient was also 30% increased. The ATP pharmacological analogs BzATP and oATP have been described as a preferential agonist and a covalent blocker, respectively, for P2X receptors with very little activity at P2Y receptors (28 , 29) , and so are valuable tools to test the involvement of P2X receptors in [Ca²⁺]_i mobilization. Figure 4C shows that BzATP triggered a large [Ca²⁺]_i rise even in the presence of extracellular Na⁺ and that this increase was almost completely abolished by pretreatment with oATP, further confirming expression of P2X receptors by human DCs. More recently, the Ca²⁺/calmodulin-dependent protein kinase II inhibitor KN-62 has been proposed as a selective P2X₇ antagonist (30) . Figure 4D shows that this compound also reduced the [Ca²⁺]_i rise triggered by BzATP, albeit incompletely. Besides ATP, other nucleotides such as ADP, UTP, and GTP applied in the low µM range elicited a transient [Ca²⁺]_i rise (not shown). Since these nucleotides are potent and selective activators of P2Y receptors, especially when applied at low concentrations, their ability to raise [Ca²⁺]_i is a further indication that dendritic cells also express P2Y receptors in addition to P2X. In the absence of extracellular Ca²⁺, BzATP was unable to increase the [Ca²⁺]_i, thus confirming its preferential agonist activity at P2X rather than P2Y receptors (Fig. 4E ).

View larger version (26K): [in this window] [in a new window]   Figure 4. Kinetics of ATP- and BzATP-triggered [Ca2+]i changes. DCs were loaded with the Ca2+ indicator Fura-2/AM, as specified in Materials and Methods, and then stimulated with ATP (A) in a Ca2+-containing (trace a) or in a Ca2+-free, 0.5 mM EGTA-containing medium (trace b). B) Cells were stimulated with ATP in a Na+-containing saline solution (trace b) or in a medium where Na+ was replaced with isotonic sucrose (trace a). C, D) Inhibition of BzATP-induced Ca2+ rise by oATP and KN-62, respectively. Cells were pretreated with 300 µM oATP for 2 h at 37°C, rinsed, and challenged with BzATP (C, trace b). KN-62 (50 nM) was added to the cells 10 min prior to BzATP stimulation and left in the samples throughout the experiment (D, trace b); traces a in panels C, D, controls. E) Effect of chelation of extracellular Ca2+ on BzATP-triggered [Ca2+]i rise. Cells were incubated in Ca2+-containing (trace a) or Ca2+-free, EGTA-supplemented (0.5 mM) (trace b) medium. Nucleotides were added at the concentration of 1 mM. Ionomycin (iono) was 100 nM. Added Ca2+ was 1.5 mM. Traces are from a single experiment representative of five similar.

An early event that precedes the large increase in plasma membrane permeabilization triggered by P2X₇ is a long-lasting depolarization of the plasma membrane. Figure 5A shows that DCs, like other cells expressing this receptor, are depolarized by ATP (trace a) and BzATP (trace b). BzATP was a more potent depolarizing agent than ATP, as shown by the full and long-lasting collapse of the plasma membrane potential and by the comparison of dose-response curves for plasma membrane depolarization induced by ATP (Fig. 5B , open circles) and BzATP (Fig. 5B , filled circles). A decrease in plasma membrane potential was observed at an ATP concentration as low as 10 µM, and plateau was reached at 1 mM. BzATP was more potent then ATP as, although the shape of the curves was similar, an effect was detectable even at 3 µM and a plateau was reached at 100 µM.

View larger version (13K): [in this window] [in a new window]   Figure 5. ATP and BzATP induce plasma membrane depolarization. A) Cells were loaded with the fluorescent dye bisoxonol as reported in Materials and Methods, then challenged with 100 µM ATP (trace a) or BzATP (trace b). Traces are from a single experiment representative of five similar. B) Dose dependency curves for plasma membrane depolarization induced by ATP () or BzATP (•). Data are means ± SD of triplicate determinations.

oATP was previously shown to completely block permeabilization in macrophages (29) and microglial cells (19) . DCs were challenged with ATP in the presence of the extracellular fluorescent dye ethidium bromide. Activation of P2X₇ receptor allows the tracer to penetrate the cells, where it increases its fluorescence upon binding to nucleic acids. Figure 6 , trace a shows a kinetic of ATP-mediated permeabilization to ethidium bromide. oATP (trace b) fully blocked ethidium bromide uptake, further suggesting that ATP-triggered permeabilization was a P2X₇ receptor-mediated event. Digitonin was added at the end of the experiment to elicit maximal permeabilization.

View larger version (13K): [in this window] [in a new window]   Figure 6. Effects of oATP on the ATP-induced plasma membrane permeabilization. Trace a, control; trace b, pretreatment with oATP. ATP and ethidium bromide concentrations were 3 mM and 20 µM, respectively. Cells were incubated with oATP as detailed in Fig. 4 . Digitonin (D1G) was 100 nM. Traces are from a single experiment representative of five similar.

Although until recently it was thought that P2X₇ was the P2X receptor mainly expressed by immune cells, more recent reports suggest that P2X₁ and P2X₄ receptors are also expressed (13) . Figure 7A shows that P2X₁, P2X₄, and P2X₇ messages are present in human DCs and that maturation does not appreciably change the level of expression. Figure 7B shows that besides P2X, four P2Y receptor subtypes—P2Y₁, P2Y₂, P2Y₅, and P2Y₁₁—are also expressed to about the same level in mature and immature DCs. Unfortunately, there are as yet no Abs specific for P2Y receptors; however, we previously characterized a polyclonal Ab that recognizes the P2X₇ receptor in Western blotting (31) . Furthermore, a MoAb raised against the P2X₇ ectodomain has recently become available; this MoAb does not recognize the denatured P2X₇ protein but, on the contrary, is a good ligand for the native receptor (32) . Figure 8A shows that the polyclonal Ab stains a band of approximately 72 kDa in both immature and mature DCs. Accordingly, both immature and mature DCs showed a significant reactivity to the anti-P2X₇ MoAb by FACS analysis (Fig. 8B ).

View larger version (26K): [in this window] [in a new window]   Figure 7. P2X (A) and P2Y (B) receptor subtypes expressed by DCs. RT-PCR was performed as described in Materials and Methods. Immature (I) and mature (M) DCs. MWM, molecular weight markers.

View larger version (16K): [in this window] [in a new window]   Figure 8. Western blot (A) and FACS analysis (B) of P2X7 expression by human DCs. Western blot and FACS analysis were performed as described in Materials and Methods. I, immature, and M, mature DCs. Boldface line: DCs treated with the anti-P2X7 MoAb; dotted line, irrelevant mouse Ig control.

ATP induces cytokine release from mature DCs
Dendritic cells are very efficient producers of many key inflammatory cytokines, including IL-1ß and TNF-. We therefore tested whether cytokine secretion was affected by stimulation with extracellular ATP. It is known that secretion of IL-1ß from LPS-stimulated macrophage and microglial cells is a slow and inefficient process that leads to extracellular accumulation of small amounts of this cytokine and mainly of the biologically inactive 31–34 kDa procytokine form (33 , 34) . We and others have reported that extracellular ATP induces a fast and efficient processing and release of IL-1ß from macrophages (18 , 20 , 34) . Figure 9A shows a nucleotide dose dependency for IL-1ß release from mature DCs. A 30 min stimulation with ATP, but not with other nucleotides such as UTP, GTP, ADP, triggered a rapid secretion of IL-1ß that plateaued at 3 mM ATP, similarly to human macrophages. The time course, was biphasic, with the initial phase, reaching a maximum after 30 min, followed by a second steady, nonsaturating, release possibly due to the ensuing cell damage (Fig. 9C ). ATP was also a potent stimulus for TNF- secretion, albeit dose dependency and time course differed from those of IL-1ß. The ATP threshold was much lower, as stimulation of TNF- release could be observed at a concentration as low as 100 µM (Fig. 9B ); whereas on the one hand TNF- release was about as rapid as IL-1ß release reaching a maximum after 30 min, on the other hand it did not significantly increase further after longer incubations (Fig. 9D ). As expected for a P2X₇-dependent process, BzATP was a better agonist than ATP for IL-1ß secretion (Fig. 9E ). Finally, Fig. 9F shows that IL-1ß production due to stimulation with ATP was completely blocked by oATP, further supporting involvement of P2X₇. It is of interest that oATP reduced secretion of IL-1ß triggered by the mere stimulation with LPS.

View larger version (16K): [in this window] [in a new window]   Figure 9. ATP triggers secretion of IL-1ß and TNF- from mature DCs. A, C) Dose dependency and time course, respectively, of ATP-induced IL-1ß production. A) Cells were incubated with ATP (•) or other nucleotides such as UTP, GTP, CTP, ADP (, average ± SD of 10 determinations) for 30 min. C) ATP concentration was 5 mM. B, D) Dose dependency and time course, respectively, for ATP-induced TNF- release; for conditions, see panels A and C. E) Dose dependency of BzATP-stimulated IL-1ß secretion; cells were stimulated with BzATP for 30 min. F) Inhibition of ATP-stimulated IL-1ß secretion by oATP; LPS (L). Cells were pretreated with oATP, as detailed in Fig. 4 , then incubated for 30 min with 5 mM ATP. Cytokines released in the supernatants were measured by ELISA as described in Materials and Methods. Data are averages ± SD of triplicate determinations from an experiment representative of four others replicated with similar results with samples from four different donors.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
It is increasingly appreciated that receptors for extracellular nucleotides have a central role in inflammation and immunomodulation (8 , 9 , 35 36 37) . They are expressed by monocytes/macrophages (15 , 22 , 28) , microglial cells (19) , lymphocytes (38 , 39) , eosinophils (40) , and recently have also been demonstrated in mouse dendritic cells (5) . Two major proinflammatory cytokines, IFN and TNF-, and endotoxin were shown to selectively up-regulate some P2 receptors while decreasing expression of others (15 , 16) . Furthermore, activation of P2 receptors has been associated with stimulation of IL-1ß processing and release from macrophages and microglial cells (18 , 19) as well as induction of apoptosis in macrophages, microglial cells and lymphocytes (41 , 42) . Despite the increasing amount of observations accumulated over the last few years, no thorough characterization has ever been carried out in human DCs, a key element of the immune response.

A first and intriguing result of our investigation is that human DCs express the P2X₇ receptor to a very high level not usually found in other immune or nonimmune cells. More than 90% of DCs generated from peripheral blood monocytes were positive for P2X₇ expression, a percentage much higher compared to other mononuclear phagocytes so far investigated. Peripheral blood monocytes are weakly P2X₇ positive; in a typical Lucifer yellow uptake test, only 15–17% are stained. After 5 days of in vitro culture, 50% of the monocytes become positive, although there is some donor-dependent variability; only upon stimulation with IFN does the percentage of positive macrophages rise to 80% (15 , 16) . The high-level positivity of DCs is not totally unprecedented as mouse DCs also strongly express P2X₇ (5 , 43) , suggesting that high expression of this P2 receptor could be a phenotypic marker of dendritic cells.

The most striking functional correlate of high-level P2X₇ expression is the increased sensitivity to ATP-dependent cytotoxicity. Both immature and mature DCs undergo the typical alterations of all cells highly sensitive to ATP: rounding, loss of cell protrusions, swelling, and then lysis. All these changes indicate that under massive ATP stimulation, death of DCs occurs by colloido-osmotic lysis, i.e., necrosis. However, several shrunken cells were also clearly visible in the microscopic field, suggesting that death by apoptosis could also take place. A thorough investigation is needed to clarify in detail the death pathways activated via P2 receptors in human DCs.

Biochemical analysis confirmed expression of P2X₇ at the mRNA and protein level and showed that two other P2X receptors, P2X₁ and P2X₄, were also expressed. Although P2X₇ can unequivocally be assigned some well-defined functions (i.e., plasma membrane permeabilization, cytotoxicity, or stimulation of IL-1ß secretion), it is difficult to link either P2X₁ or P2X₄ receptor to a given cellular response. They also are ion channels, albeit with smaller conductance than P2X₇ (44) . A recent report (45) suggests that P2X₄ might, under certain conditions, also generate nonselective plasma membrane pores like P2X₇, but there is no evidence that this occurs in immune cells. On the other hand, it cannot be excluded that the P2X₁ and P2X₄ subunits participate with P2X₇ in the formation of the native ‘permeabilizing ATP receptor’ and modulate the intrinsic pore-forming activity of the P2X₇ subunit. There is no experimental proof that the ‘ATP-gated channel’ of immune cells is a heteromeric structure like, for example, that of sensory ganglia (46) , but it is a fact that the electrophysiological characteristics of cloned human P2X₇ do not faithfully match that of the native macrophage or lymphocyte receptor.

High-level expression of P2X₇ by human DCs raises the issue of the role of this receptor in dendritic cell physiology. These cells are the key elements in the stimulation of primary immune response and in immunomodulation, since they are the main antigen-presenting cells and a major source of cytokines (1 2 3) . It has long been known that DCs also express high plasma membrane ectonuclotidase activity, which has been widely exploited to specifically stain and identify epidermal Langerhans cells in skin sections (47 , 48) ; however, the physiological significance of this enzyme is obscure. In the light of high expression of the P2X₇ receptor, a high ectonucleotidase activity is not surprising as it is increasingly appreciated that ATP release, and therefore accumulation in the extracellular milieu, is a far more common event than once thought (49 50 51 52) . T and B lymphocytes, macrophages, microglial cells, epithelial and endothelial cells, not to mention platelets, are capable of releasing via nonlytic pathways up to 10–15% of their total ATP content. This leads to the accumulation of ATP that, while never exceeding tens of micromoles in the bulk phase, could easily reach much higher concentrations in protected compartments at the level of the plasma membrane, and thus be sufficient to activate a low-affinity ATP receptor such as P2X₇. Given the peculiar sensitivity of human DCs to this nucleotide, they are at risk of being severely damaged in all those occasions during which close interaction occurs with other cells (e.g., virus infected cells, tumor cells and lymphocytes) unless they also possess an efficient ATP-degrading mechanism. On the other hand, expression of an active P2X₇ receptor offers to the immune system a quick way to eliminate unwanted DCs simply by down-modulating their intrinsic ecto-ATPase activity, making them more susceptible to even low extracellular ATP concentrations. Down-modulation of endothelial cell ecto-ATPase activity by TNF- has been previously reported (53) .

P2X receptor stimulation does not necessarily lead to irreversible DC injury, but if it is pulsatile it can also transduce activatory signals for cytokine secretion. As in other mononuclear phagocytes, IL-1ß release is powerfully stimulated by ATP doses suggestive of a main involvement of the P2X₇ receptor, and with a time course indicating that, at least the initial rapid phase, is not due to ATP-dependent cell injury. In mononuclear phagocytes it has been shown that P2X₇-mediated IL-1ß release is due to activation of ICE/caspase-1 (54) . We have not dealt with caspase stimulation in the present work, but this is clearly an issue to be further investigated.

We show here that human DCs also express four P2Y subtypes: P2Y₁, P2Y₂, P2Y₅, and P2Y₁₁. P2Y receptors are G-protein coupled to Ca²⁺ release from intracellular stores and their role in immune cells is not well understood. The ATP dose dependency suggests that these receptors might also be involved in ATP-dependent TNF- release.

Expression of P2 receptors renders DCs sensitive to the concentration of nucleotides in the pericellular environment. Nucleotides can be released under many circumstances: injury of the plasma membrane, cell death, platelet activation, lymphocyte or macrophage stimulation by antigens or bacterial endotoxin. Furthermore, high expression of P2X₇, to a level unusual in other human mononuclear phagocytes makes DCs very sensitive to cytotoxic effects mediated by extracellular ATP and suggests a new pathway for down-modulating the immune response.

	ACKNOWLEDGMENTS

 
We wish to thank the blood bank of Arcispedale St. Anna, Ferrara, Italy, and particularly Mr. Paolo Gamberoni for his helpful collaboration. This work was supported by grants from the Italian Association for Cancer Research, the National Research Council of Italy (target project on Biotechnology), the Ministry of Scientific Research, Telethon of Italy, and the AIDS Project, Instituto Superiore di Sanitá.

	FOOTNOTES

 
² The first two authors contributed equally to the paper.

Received for publication January 27, 2000. Revision received June 2, 2000.

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