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
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ABSTRACT |
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
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INTRODUCTION |
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-
(TNF-
), 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-
-dependent mechanism (36), and TNF-
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-1
is
processed proteolytically and secreted during
ATPo-mediated apoptosis of
monocytes and macrophages (19, 21). IL-1
is a proinflammatory
cytokine produced by monocytes and macrophages after LPS stimulation,
which causes cleavage of the inactive pro-IL-1
precursor by
caspase-1 into the active mature IL-1
. However, a second stimulus is
required for IL-1
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.
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MATERIALS AND METHODS |
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)
(ATP
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.
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RESULTS |
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.
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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.
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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.
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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 ATP
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 ATP
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)
(ATP S) and benzoylbenzoyl ATP (BzATP) on apoptosis. Dendritic cell
line was incubated with 5 mM BzATP, ATP, or ATP 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.
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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.
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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.
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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.
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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.
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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-1
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.
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DISCUSSION |
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, ATP
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.
 |
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