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Am J Physiol Cell Physiol 276: C1139-C1147, 1999;
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Vol. 276, Issue 5, C1139-C1147, May 1999

P2Z/P2X7 receptor-dependent apoptosis of dendritic cells

Robson Coutinho-Silva1, Pedro M. Persechini2, Rodrigo Da Cunha Bisaggio2, Jean-Luc Perfettini1, Ana Cristina Torres De Sa Neto2, Jean M. Kanellopoulos3, Iris Motta-Ly3, Alice Dautry-Varsat1, and David M. Ojcius1

1 Unité de Biologie des Interactions Cellulaires, Centre National de la Recherche Scientifique 1960, and 3 Unité de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale 277, Institut Pasteur, Paris, France; and 2 Laboratorio de Imunobiofisica, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Macrophages and thymocytes express P2Z/P2X7 nucleotide receptors that bind extracellular ATP. These receptors play a role in immune development and control of microbial infections, but their presence on dendritic cells has not been reported. We investigated whether extracellular ATP could trigger P2Z/P2X7 receptor-dependent apoptosis of dendritic cells. Apoptosis could be selectively triggered by tetrabasic ATP, since other purine/pyrimidine nucleotides were ineffective, and it was mimicked by the P2Z receptor agonist, benzoylbenzoyl ATP, and blocked by magnesium and the irreversible antagonist, oxidized ATP. RT-PCR analysis confirmed the mRNA expression of the P2Z/P2X7 receptor and the absence of P2X1. Caspase inhibitors and cycloheximide had only a partial effect on the apoptosis, suggesting that a caspase-independent mechanism may also be operative. Brief treatment with ATP led to an increase in the intracellular calcium concentration and permeabilization of the plasma membrane to Lucifer yellow, which diffused throughout the dendritic cell cytosol. Other small extracellular molecules may thus attain a similar intracellular distribution, perhaps activating endogenous proteases that contribute to initiation of apoptosis.

adenosine 5'-triphosphate; inflammatory mediators


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DENDRITIC CELLS are efficient antigen-presenting cells and play a leading role in activating T cell-dependent immune responses (3). In nonlymphoid tissues, dendritic cells are present as "immature dendritic cells," which are capable of internalizing and processing antigen and expressing high levels of myosin heavy chain (MHC) molecules (2). Pathogens and inflammatory stimuli [tumor necrosis factor-alpha (TNF-alpha ), interleukin-1 (IL-1), and lipopolysaccharide (LPS)] can induce the differentiation of immature dendritic cells into "mature dendritic cells," which have decreased capacity for antigen processing but stable presentation of previously acquired antigens by the MHC molecules. The mature dendritic cells migrate into secondary lymphoid tissues, where they initiate T cell-dependent immune responses (3). At the same time, LPS and gram-negative bacteria are potent inducers of cytokine release by dendritic cells. Proinflammatory cytokine secretion by dendritic cells after encounters with pathogens may thus lead to autocrine activation of dendritic cells.

The signals that maintain dendritic cells in an immature state or that activate differentiation in mature dendritic cells in vivo are largely unknown. LPS stimulates dendritic cell depletion and movement from nonlymphoid tissues via a TNF-alpha -dependent mechanism (36), and TNF-alpha and IL-1 cause Langerhans cell migration from the epidermis to the dermis and then to the lymph nodes (3, 36). Hence bacterial products and cytokines most likely play an important role in directing and activating dendritic cells.

Other factors that may be involved in regulating the function of dendritic cells have yet to be considered. Prominent among these is extracellular ATP (ATPo), which is thought to influence macrophage function during inflammation and to play an important role in controlling infections by intracellular pathogens (10, 22).

ATPo interacts with P2 receptors, which are widely present on different types of tissues. Based on pharmacological, functional, and cloning data, two classes of P2 receptors for extracellular nucleotides have been identified: P2X ligand-gated ion channels and P2Y G protein-coupled receptors. Several members of both classes have been cloned, expressed, and characterized (10, 12, 13, 31, 44). The P2Z/P2X7 receptors are expressed on the surface of macrophages and other cells of immune and hematopoetic origin (10, 12, 13, 31). Activation of the P2Z/P2X7 receptor by ATPo, most likely in its fully dissociated tetra-anionic ATP4- form, results in the permeabilization of cells by opening cation-specific ion channels and nonspecific pores that conduct a range of low-molecular-mass solutes of molecular mass <900 Da (8, 9, 41). In addition, incubation with ATPo increases cytoplasmic calcium concentrations and causes apoptosis of thymocytes, T cells, and macrophages (23, 48, 49), and these effects are thought to be mediated via P2Z receptors (10). Further interest in the receptor was generated by the finding that IL-1beta is processed proteolytically and secreted during ATPo-mediated apoptosis of monocytes and macrophages (19, 21). IL-1beta is a proinflammatory cytokine produced by monocytes and macrophages after LPS stimulation, which causes cleavage of the inactive pro-IL-1beta precursor by caspase-1 into the active mature IL-1beta . However, a second stimulus is required for IL-1beta to be released into the medium, and ATPo engagement of the P2Z receptor is thought to provide this signal (21). Conversely, exposure to LPS enhances P2Z receptor activity, and pro- and anti-inflammatory stimuli modulate receptor expression (10, 20).

Despite the fact that macrophages and dendritic cells share a common myeloid lineage (32) and are both involved in warding off microbial infections, the presence of the P2Z receptor on dendritic cells has not been previously reported. Moreover, the phagocytic cell of the thymus reticulum, which displays several characteristics of dendritic cells, has been shown to display P2Z receptors, as assayed by permeabilization assays and patch-clamp experiments (7, 8). We have therefore characterized the effects of ATPo on dendritic cells, using dendritic cell permeabilization and apoptosis as readouts for P2Z receptor engagement. Most of these studies were conducted with a fully functional murine dendritic cell line with the properties of immature dendritic cells (14, 16, 29), and ATPo-mediated apoptosis was also observed in primary immature dendritic cells derived from human peripheral blood cells. Known antagonists of the P2Z receptor were unable to induce apoptosis, whereas agonists promoted apoptosis. The identity of the P2 receptor responsible for these effects was confirmed by RT-PCR analysis, which revealed the presence of the P2X7 receptor. The mechanisms possibly involved in mediating apoptosis were investigated by confocal microscopy and the use of specific inhibitors.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells and materials. The immortalized dendritic cell line, D2SC/1, was derived from Balb/c mouse spleen (16) and was generously provided by Dr. P. Ricciardi-Castagnoli. The cells were maintained at 37°C in an atmosphere of 5% CO2 in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (GIBCO BRL) and 2 mM L-glutamine. Primary human dendritic cells were prepared from peripheral blood precursors of normal donors, as described elsewhere (37). Murine dendritic cells were used for experiments after 5 days of incubation in granulocyte/macrophage colony-stimulating factor (GM-CSF), and human dendritic cells were recovered after 7 days of incubation in GM-CSF and IL-4.

ATP, ADP, AMP, UTP, benzoylbenzoyl ATP (BzATP), oxidized ATP, and adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S) were purchased from Sigma (St. Louis, MO). The reagents were prepared as stock 100 mM solutions in PBS and stored at -20°C until use. The ICE (caspase-1) inhibitor II (Ac-YVAD-CMK), and the CPP32/Apopain (caspase-3) inhibitor II (Z-DEVD-FMK) were from Calbiochem (La Jolla, CA). Lucifer yellow (LY) was from Molecular Probes (Eugene, OR).

Measurement of apoptosis. Dendritic cells were first grown on 75-cm2 tissue culture flasks (Costar) until 60-70% confluence and then were incubated with ATP, ATP analogs, or nucleotides with inhibitors in PBS for 30 min at 37°C in 5% CO2, and the medium was then removed and replaced by cell culture medium. The cells were then incubated for the indicated time at 37°C in 5% CO2. For caspase inhibition experiments, 50 µM of the caspase-1 or caspase-3 inhibitor was added 15 min before addition of ATP and was maintained with the dendritic cells during the duration of the incubation. Dendritic cells were incubated with the P2Z blocker, oxidized ATP, for 2 h, and oxidized ATP was then removed from the medium before addition of ATP.

Quantitative measurements of apoptosis were performed by cytofluorometry of detergent-permeabilized propidium iodide (PI)-stained cells as described previously (11, 27). Viability was measured using the standard PI-exclusion assay with unpermeabilized cells. Unless noted otherwise, both adherent cells and cells in the supernatant were collected for analysis.

The cells were transferred into 12 × 75 mm FALCON 2052 FACS tubes (Becton Dickinson, San Jose, CA). Data from 10,000 dendritic cells were collected on a FACScan flow cytometer (Becton Dickinson) with an argon laser tuned to 488 nm.

DNA fragmentation was measured as previously described (30).

Intracellular calcium measurements. Cells were loaded with 6 µM fura 2-AM (Molecular Probes) for 1 h at room temperature in culture medium. The cells were then washed and perfused with PBS supplemented with 1 mM CaCl2, with the use of a three-compartment superfusion chamber whose bottom was formed by the coverslip containing the cells (18). Intracellular calcium concentrations in groups of 20-40 cells were monitored continuously at 37°C with the use of a fluorescence photometer (Photon Technology, Princeton, NJ). Fura 2 was excited alternatively at 340 and 380 nm, and the emission at 510 nm was measured. The ratio measurement, which is proportional to the intracellular calcium concentration, was determined every 100 ms.

RT-PCR detection of nucleotide receptors. Total RNA was isolated from dendritic cell cultures using TRIzol (GIBCO). RNA was reverse transcribed using the First Strand Kit (Pharmacia Biotechnology). The reaction was conducted for 1 h at 37°C. The cDNA obtained was then amplified using primers designed from the rat P2X7 sequence (sense: 5'-GGC AGT TCA GGG AGG AAT CAT GG-3'; antisense: 5'-AAA GCG CCA GGT GGC ATA GCT C-3') (9) or from the mouse P2X1 sequence (sense: 5'-CAT TGT GCA GAG AAC CCA GAA-3'; antisense: 5'-ATG TCC TCC GCA TAC TTG AAC-3') and gave rise to 939- and 776-bp products, respectively. The P2X7 and P2X1 PCR reactions contained 1.0 µM of each primer, 62.5 µM of each dNTP, 20 mM Tris · HCl (pH 8.4), 50 mM KCl, 3.5 mM MgCl2, and 0.5 unit of Taq polymerase (GIBCO). The PCR cycling protocol was 1 min at 94°C, 2 min at 55°C, and 2 min at 72°C. The protocol was conducted for 35 cycles and included an initial 5-min denaturation and a final 10-min extension at 72°C. Mock RT-PCR reactions, using RNA from the different cells, were carried out to test for genomic DNA contamination (data shown only for the dendritic cell line). PCR amplification of a constitutively expressed mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sense: 5'-ATC ACC ATC TTC CAG GAG CG-3'; antisense: 5'-CCT GCT TCA CCA CCT TCT TG-3') was used as a control of the presence of cDNA in the reactions. Products were subjected to electrophoresis on 1% agarose gels containing ethidium bromide and were photographed.

Demonstration of permeabilization by confocal microscopy. Cell permeabilization was assessed by observing the differential uptake of LY in cells that had been treated with 5 mg/ml LY with or without 5 mM ATP at 37°C for 10 min. Culture dishes were then rapidly washed four times with PBS, the coverslips were transferred to a confocal microscope at room temperature, and cells were examined immediately. Images were acquired with a Zeiss LSM 510 confocal microscope equipped with an Ar/HeNe laser. Serial optical sections were typically recorded at 0.5-µm intervals with ×63 and ×100 lenses.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Apoptosis of the dendritic cell line induced by extracellular ATP. We investigated the effects of extracellular nucleotides by incubating the dendritic cell line for 30 min with the nucleotide in PBS at 37°C, and then removing the supernatant and incubating for an additional 6 h with cell culture medium at 37°C. The cells were then collected and analyzed for apoptosis by cytofluorometry, based on the differential PI staining of viable and apoptotic cells (11, 27). In the presence of ATPo, the dendritic cells underwent rounding and swelling and detached from the cell culture medium (not shown). As preliminary cytofluorometry experiments indicated that most of the apoptotic cells were in the supernatant, the cells in suspension and adherent cells were pooled and analyzed together. Five millimolar ATP induced apoptosis of one third of the cells, compared with <10% of the cells dying spontaneously (Fig.1).


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  		Fig. 1.   Effect of extracellular ATP (ATPo) and other extracellular nucleotides on apoptosis of dendritic cells. Dentritic cell line was incubated with control buffer or 5 mM of the indicated nucleotides for 30 min. Supernatant was then removed and replaced by cell culture medium, and cells were incubated in CO2 incubator at 37°C for an additional 6 h. Apoptosis was quantified by cytofluorometry with propidium iodide (PI)-stained cells, as described in MATERIALS AND METHODS. All apoptosis values were normalized with respect to value obtained with ATP, which was 31%. Values represent average and SD of 3 separate experiments.

To determine whether a P2Z receptor may be responsible for the apoptosis, we evaluated whether UTP, AMP, and ADP could promote apoptosis, since these nucleotides have been previously shown to stimulate other P2 receptors but not P2Z receptors (10, 12, 13). After incubation of the dendritic cells with 5 mM UTP, AMP, ADP, or ATP, only the cells incubated in the presence of ATP underwent apoptosis to a significant extent (Fig. 1). Given the variability in total apoptosis due to ATP that we observed in different experiments, the values obtained with AMP, ADP, UTP, and buffer control were normalized with respect to the ATP levels, which caused death of 31% of the cells (n = 3). However, in each separate experiment, the hierarchy of the effects for each nucleotide was the same.

A salient feature of apoptosis is fragmentation of DNA into a ladder of 200-bp units (46). The identification of PI-stained cells by cytofluorometry as live or apoptotic was therefore confirmed by analysis of dendritic cell DNA by gel electrophoresis. Figure 2 shows that 5 mM ATPo induced high levels of DNA fragmentation in treated dendritic cells. There was a low level of spontaneous DNA fragmentation in the untreated dendritic cell sample, which was not affected by UTP, AMP, or ADP.


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  		Fig. 2.   Effect of ATPo on DNA fragmentation of dendritic cells. Dendritic cell line was incubated with nucleotides or control buffer as described in legend to Fig. 1. Low level of spontaneous DNA fragmentation is seen in dendritic cell line incubated in presence of control buffer or 5 mM AMP, ADP, or UTP, but a high level of fragmentation is evident in cells that had been incubated with 5 mM ATP. Same number of starting cells (before incubation with nucleotides) was loaded on each lane. DNA fragmentation was visualized on a 1.5% agarose gel stained with ethidium bromide, and arrows on left correspond to 200, 400, and 600 bp.

Because it has previously been reported that millimolar concentrations of ATPo are required to activate the P2Z receptor (10, 12, 13), we characterized the concentration dependence of ATPo-mediated apoptosis in dendritic cells. No measurable effects were observed at 1 mM ATPo, but 5 mM ATPo caused a high level of dendritic cells to die after 6 h (Fig. 3). Thus the concentration dependence is similar to that observed for the P2Z-associated permeabilization phenomenon in macrophages and other cells.


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  		Fig. 3.   Concentration dependence of ATPo-mediated apoptosis of dendritic cells. Dendritic cell line was incubated with indicated concentration of ATP for 30 min. Supernatant was then removed and replaced by cell culture medium and cells were incubated in CO2 incubator at 37°C for additional 6 h. Apoptosis was quantified by cytofluorometry with PI-stained cells, as described in MATERIALS AND METHODS. Apoptosis obtained with different ATP concentrations was normalized with respect to value obtained with 10 mM ATP. Values represent average and SD of 3 separate experiments.

Effect of P2Z receptor agonists and antagonists on dendritic cell apoptosis. The identity of the receptor responsible for dendritic cell apoptosis was further confirmed by characterizing the effects of selective agonists and antagonists on ATPo-mediated apoptosis.

BzATP and the poorly hydrolyzable ATPgamma S are selective agonists for the P2Z receptor, and oxidized ATP behaves as an irreversible inhibitor (26, 28, 45). Figure 4A shows that both of the ATP agonists, BzATP and ATPgamma S, induced apoptosis of dendritic cells. Although BzATP is more effective than ATP in inducing permeabilization of cells, BzATP and ATP have a comparable potency in eliciting apoptosis, consistent with previous studies (50).


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  		Fig. 4.   Effect of P2Z receptor agonists and antagonists on apoptosis of dendritic cells. A: effect of agonists adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S) and benzoylbenzoyl ATP (BzATP) on apoptosis. Dendritic cell line was incubated with 5 mM BzATP, ATP, or ATPgamma S, as described for ATP in legend to Fig. 1. B: effect of antagonist Mg2+ and irreversible antagonist oxidized ATP (oxATP) on apoptosis of dendritic cells. Dendritic cell line was incubated with (in mM) 10 MgCl2 alone, 5 ATP and 10 MgCl2, 0.3 oxidized ATP alone, 5 ATP alone, or 0.3 oxidized ATP for 2 h followed by 5 ATP. Apoptosis was quantified by cytofluorometry, as described in MATERIALS AND METHODS, and apoptosis values were normalized with respect to value for ATP alone. Values represent average and SD of 3 separate experiments.

Low concentrations of oxidized ATP (100 µM) have been reported to block ATPo-dependent lysis of macrophages, as well as the permeabilization of the membrane to extracellular hydrophilic markers in J774 macrophages (26). We similarly observed that oxidized ATP does not induce apoptosis of dendritic cells (Fig. 4B). Moreover, because the effect in macrophages is irreversible (26), suggesting covalent modification of the receptor, we also preincubated the dendritic cells with oxidized ATP for 2 h and removed the oxidized ATP before addition of 5 mM ATP; under these conditions there is inhibition of most of the ATPo-induced apoptosis (Fig. 4B). The P2Z receptor is also antagonized by Mg2+, which chelates ATP4- (10, 12, 13), and we find that it inhibits ATPo-mediated apoptosis of dendritic cells (Fig. 4B), implying that the active substance is the tetrabasic form of ATP.

Microscopic characterization of ATP-induced permeabilization of dendritic cells. In macrophages and other cells expressing the P2Z receptor, the ATPo-gated pore has a molecular cut-off of ~900 Da (34, 41). Hence otherwise impermeant markers such as LY and PI traverse this pore easily. We therefore evaluated the functional expression of the receptor by measuring the uptake of extracellular LY after brief (10-min) exposure of the cells to ATPo, visualizing the LY distribution by confocal microscopy. In the absence of ATPo, the dye is internalized into large intracellular vacuoles reminiscent of macropinosomes (Fig. 5A), as previously reported for uptake of fluorescent dextran by the same cell line (29). In contrast, in the presence of ATPo, the dye is quickly detected throughout the dendritic cell cytoplasm and in the nuclear region (Fig. 5C). Contrast phase analysis of the same cell sample was used to identify the nuclei (Fig. 5D).


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  		Fig. 5.   Effect of ATPo on uptake of Lucifer yellow (LY) by dendritic cells. Dendritic cell line was incubated with control buffer and LY for 10 min, and cells were visualized by fluorescence microscopy (A) and phase-contrast microscopy (B) with a confocal microscope, as described in MATERIALS AND METHODS. Arrow points out a typical large LY-laden vacuole. Same cells were incubated with 5 mM ATP and LY for 10 min, and cells were visualized by fluorescence microscopy (C) and phase-contrast microscopy (D). Arrows point out same dendritic cell nucleus.

Because ATPo has been shown to induce calcium fluxes in thymocytes and macrophages (12, 31, 35, 42, 43), we investigated whether ATPo may also trigger calcium changes in dendritic cells. Cells in the spectrofluorometer maintained a basal intracellular calcium concentration, but sustained exposure of dendritic cells to 50 µM ATPo resulted in a rapid rise in the calcium concentration, which subsided to basal levels within a minute (Fig. 6A). Because this concentration of ATPo does not cause apoptosis of dendritic cells, the same experiment was repeated with 5 mM ATPo, which showed that a calcium peak is followed by a sustained plateau (Fig. 6B). Similarly, continuous exposure to 5 mM extracellular UTP, which does not induce apoptosis, can trigger the transient calcium peak but not the sustained increase in calcium levels (Fig. 6C). These results suggest that only the exposure to ATPo at the same conditions required to induce apoptosis can cause sustained levels of calcium increase.


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  		Fig. 6.   Effect of extracellular nucleotides on intracellular calcium concentration of dendritic cells. Dendritic cell line was loaded with calcium-sensitive indicator fura 2, and a final concentration of 50 µM ATP (A), 5 mM ATP (B), or 5 mM UTP (C) was continuously added to cells starting at onset of calcium peak. D: final concentration of 200 µM ATP was added rapidly (first arrow); after addition of 10 mM Mg2+ to perfusion medium (2nd arrow), cells were mixed with 200 µM ATP (3rd arrow) and then 200 µM UTP (4th arrow). Reagent concentration in chamber decreased to zero within 1 min due to continuous perfusion of extracellular medium. Fluorescence is proportional to intracellular calcium concentration and was measured as described in MATERIALS AND METHODS.

To further characterize the nature of the receptor responsible for dendritic cell apoptosis, intracellular calcium concentrations were measured in the absence and presence of 10 mM Mg2+ in the perfusion medium, which abrogated the calcium peaks due to 200 µM ATPo (Fig. 6D). However, the extracellular Mg2+ had no effect on the calcium flux due to 200 µM extracellular UTP (Fig. 6D), consistent with the proposal that a P2U (P2Y subtype) receptor is present in macrophages (1). This receptor is thus probably present in dendritic cells, although it does not induce apoptosis.

Finally, no apoptosis was measured within 10 min after incubation with ATP (not shown), whereas permeabilization, as assayed by LY internalization or calcium fluxes, begins much earlier, suggesting that permeabilization precedes the earliest steps of apoptosis.

Detection of P2X7 receptor mRNA in dendritic cells. The results on apoptosis induced by ATP and its analogs, and on the permeabilization of the plasma membrane due to ATP, suggest the functional activity of the P2Z-type receptor on dendritic cells. Because this activity has been assigned to the cloned P2X7 receptor (44), we have determined whether the latter is present in dendritic cells. J774 macrophages were used as positive control for P2Z/P2X7 expression (5). RT-PCR analysis revealed the presence of P2X7 mRNA in dendritic cells (Fig. 7), thereby corroborating the identity of the P2Z receptor based on functional studies.


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  		Fig. 7.   Demonstration of P2X7 receptor mRNA expression on dendritic cells (DC). Expression of the P2X7 receptor was analyzed by RT-PCR, as described in MATERIALS AND METHODS. Expected size of P2X7 mRNA is 939 bp (arrow on right), and J774 cells were used as a positive control. Mock reaction was used to control for possible genomic contamination in dendritic cell preparation, while cDNA was omitted from the reaction in the negative lane.

Because the P2X1 receptor has been shown to play a role in ATP-mediated apoptosis of thymocytes (6), we evaluated whether the receptor mRNA may be present in dendritic cells. HL-60 cells (differentiated promyelocytes) were used as a positive control for P2X1 (4), but no message was detected in dendritic cells (Fig. 8). The absence of measurable P2X1 mRNA in dendritic cells thus argues against a role for this receptor in ATP-induced apoptosis of dendritic cells.


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  		Fig. 8.   Absence of P2X1 receptor mRNA expression in dendritic cells. Top: cDNA was synthesized and amplified by RT-PCR P2X1 primers from HL-60 cells and dendritic cells. Bottom: PCR amplification of a constitutively expressed control mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a positive control. PCR product was separated on a 1.2% agarose gel and was ethidium bromide stained. cDNAs were omitted from reactions in negative lane.

Effect of inhibitors on dendritic cell apoptosis. Most types of apoptosis characterized to date are executed by specific cysteine proteases known as caspases (40). Caspase-1 (ICE) is involved in proinflammatory reactions, whereas caspase-3 has a role executing final stages of apoptosis. After pretreatment of dendritic cells with 50 µM of a caspase-1 inhibitor or 50 µM of a caspase-3 inhibitor, ATPo induced 63 ± 7 and 73 ± 2%, respectively, of the apoptosis due to ATPo in the absence of inhibitor. In separate experiments, we verified that the caspase-1 inhibitor prevented IL-1beta secretion from monocytes and that the caspase-3 inhibitor blocked Fas-mediated apoptosis (30). These results reveal a partial contribution of caspase-1 and caspase-3 activity in ATPo-induced apoptosis but suggest that other proapoptotic mechanisms may also be operative. Because neosynthesis of proteins is required for certain types of apoptosis (15), we also measured ATPo-mediated apoptosis in the presence of 50 µM cycloheximide and found that it reached 79 ± 2% of the apoptosis observed in the absence of cycloheximide. Hence the dendritic cells appear to synthesize constitutively the proteins employed for executing the ATPo-induced apoptotic program.

ATP-mediated apoptosis of primary dendritic cells. We investigated whether the effects observed by us are limited to the dendritic cell line or whether other dendritic cells may also be sensitive to ATPo-induced apoptosis. We therefore isolated primary dendritic cells from human blood and observed that they are essentially as sensitive as the cell line studied to the apoptotic effects of ATPo (Fig. 9).


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  		Fig. 9.   Apoptosis of primary dendritic cells induced by incubation with ATPo. Primary dendritic cells from human blood and dendritic cell line were incubated with 5 mM ATP for 30 min. Supernatant was then removed and replaced by cell culture medium, and cells were incubated in CO2 incubator at 37°C for additional 6 h. Apoptosis was quantified by cytofluorometry with PI-stained cells, and specific apoptosis was obtained by subtracting control values for each separate cell type from values obtained in presence of ATP.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We show here that dendritic cells express a functional P2Z/P2X7 receptor that is capable of initiating apoptosis after its engagement by ATPo but not by other extracellular purine and pyrimidine nucleotides. Functional apoptosis studies with agonists (BzATP, ATPgamma S) and antagonists (oxidized ATP) specific for the P2Z receptor distinguished this receptor from other P2 receptors, and RT-PCR analysis confirmed the presence of P2X7 mRNA in dendritic cells.

Two types of dendritic cells were used for these studies, a murine cell line derived from spleen and primary dendritic cells prepared from human peripheral blood. Both have the characteristics of immature dendritic cells that are capable of internalizing antigen or microbes and processing them for presentation via MHC molecules (14, 29, 33, 37). Maturation of dendritic cells is necessary for initiation of T cell responses, and it can be induced by several factors, especially microbial and inflammatory products (3).

Immature dendritic cells use macropinocytosis to concentrate extracellular macromolecules with high efficiency into large intracellular compartments (39). We have previously shown that the dendritic cell line internalizes fluorescent dextran into large vacuoles consistent with macropinosomes (29), and we now observe similar vacuoles after a short incubation of the dendritic cell line with LY. However, addition of ATP to the same medium caused LY to distribute rapidly throughout the dendritic cell cytoplasm, implying that ATPo promotes opening of the large pores typically associated with the P2Z receptor. Interestingly, LY was also observed in the dendritic cell nucleus. Given the size of the LY dye (mol wt = 457), these results suggest that ATPo may also permit entry into the cytosol and nucleus of other small molecules.

ATP-induced apoptosis of dendritic cells was not affected by cycloheximide, indicating that the proteins required for ATP-mediated apoptosis are synthesized constitutively by the dendritic cells. In addition, specific inhibitors for caspase-1 or caspase-3, which execute many of the apoptotic programs characterized to date (40), blocked ATPo-induced apoptosis only partially, suggesting that this cell death pathway is, for the most part, independent of known caspases. ATPo-mediated apoptosis of dendritic cells may therefore resemble other pathways of caspase-independent apoptosis that have been recently reported, such as those induced by deregulated oncogenes, DNA damage, infection by Chlamydia, or overexpression of the proapoptotic protein Bax (24, 30, 38, 47). Unlike apoptosis pathways initiated by various surface ligands, the signal for Bax-mediated apoptosis is integrated within the cell and may be mediated by enzymes other than caspases, such as nucleases and protein kinases (40). The observation that ATPo causes LY to diffuse throughout the dendritic cell cytosol and nucleus raises the possibility that other extracellular compounds may also acquire the same distribution, perhaps activating endogenous proteases and/or endonucleases.

High doses of ATPo are required for activation of the P2Z receptor, raising the obvious question of how such high-nucleotide concentrations could be achieved in the extracellular space in vivo. Although a number of cell types, including platelets, are known to secrete ATP (12), ATP can also be released from the cytosol of injured cells, such as may be found during inflammatory reactions. Transient exposure to ATPo is sufficient to initiate subsequent events leading to apoptosis.

Dendritic cells are distributed in tissues such as skin and mucous membranes, which are strategically located to encounter microbes from the external environment, and dendritic cells have been shown to internalize and process antigens from a number of microbes, including Staphylococcus, Leishmania, mycobacteria (2), Chlamydia (29), and Listeria (17). Although the effects of ATPo on this process have yet to be investigated, it is worthwhile noting that ATPo, but not other apoptotic ligands or stimuli that induce necrosis, inhibits growth of mycobacteria in macrophages (22, 25). Because the P2Z nucleotide receptor may be used to lyse macrophages infected with intracellular pathogens in such a way that the pathogens are not released in viable form, it becomes imperative to determine the effects of ATPo on the survival of microbes in dendritic cells.


    ACKNOWLEDGEMENTS

We are grateful to Philippe Souque and Vandir da Costa for technical assistance, to Raymond Hellio for help with the confocal microscope, and to Dr. Mauro Eduardo Weine da Costa for generously providing the GAPDH primers and for technical advice on RT-PCR.


    FOOTNOTES

This work was supported by funds from the Institut Pasteur and from Conselho Nacional de Desenvolvimento Cientifico e Technológico do Brasil, Financiadora de Estudos e Projetos, Fundacão de Amparo à Pesquisa do Estado do Rio de Janeiro, Programa de Apoio a Nucleos de Excelencia, and Fundacão Universitaria Jose Bonifacio FUJB. The confocal microscope was purchased with a donation from Marcel and Liliane Pollack.

Permanent address of R. Coutinho-Silva: Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

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

Address for reprint requests and other correspondence: D. M. Ojcius, Institut Pasteur, Unité de Biologie des Interactions Cellulaires, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France (E-mail: ojcius{at}pasteur.fr).

Received 28 August 1998; accepted in final form 17 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 276(5):C1139-C1147
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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