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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 996-1005
IMMUNOBIOLOGY
Pharmacologic properties of P2Z/P2X7
receptor characterized in murine dendritic cells: role on
the induction of apoptosis
Oscar Kenji Nihei,
Antonio Carlos Campos de Carvalho,
Wilson Savino, and
Luiz Anastacio Alves
From the Laboratory on Thymus Research, Department of Immunology,
Oswaldo Cruz Institute, FIOCRUZ; and the Laboratory of Excitable
Membranes, "Carlos Chagas Filho" Institute of Biophysics, Federal
University of Rio de Janeiro, Rio de Janeiro, Brazil.
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Abstract |
In the immune system, extracellular adenosine 5'-triphosphate
(ATP) mediates a variety of effects mainly through activation of a
particular receptor subtype, the pore-forming
P2Z/P2X7 purinoceptor. This purinergic receptor
has been described chiefly in cells of hemopoietic origin such as T
cells, thymocytes, monocytes, macrophages, and phagocytic cells of
thymic reticulum. In this study, we characterized the
P2Z/P2X7 purinoceptor and the ATP-mediated
apoptosis in murine spleen-derived dendritic cells (DCs). Dye uptake
and apoptosis were evaluated by flow cytometry. ATP-treated DCs were
permeable to different low-molecular-weight fluorescent probes such as
ethidium bromide, YO-PRO 1, and lucifer yellow. Such an effect was
dose-dependent (EC50: 721 µmol/L); mediated by the fully
anionic agonist (ATP4 ); and specifically stimulated by
ATP, BzATP, and ATP S. Additionally, an ATP-induced increase in
intracellular calcium was detected by microfluorometry. Furthermore,
ATP treatment induced a significant increase in apoptotic DCs
(64.46% ± 3.8%) when compared with untreated control cells
(34% ± 5.8%), as ascertained by the TdT-mediated dUTP nick end
labeling technique. Both ATP-induced DC permeabilization and apoptosis
were inhibited by oxidized ATP, a
P2Z/P2X7-specific antagonist. In conclusion, we
characterized the expression of the P2Z/P2X7
purinoceptor in murine spleen-derived DCs and described its role on
the induction of apoptosis.
(Blood. 2000;96:996-1005)
© 2000 by The American Society of Hematology.
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Introduction |
Adenosine 5'-triphosphate (ATP) has been
described as an important molecule in both the intracellular and
extracellular microenvironments of the cell. Despite the regulatory
control exerted by ectonucleotidases, which maintain its low
physiologic concentrations, extracellular ATP1 may reach
high concentrations when released exocytotically from various cell
types such as neurons, platelets, basophils, and mast cells, or when
released nonexocytotically from damaged cells.2 Since the
pioneering work of Drury and Szent-Györgyi3 showing
the effects of extracellular adenine compounds in mammalian hearts,
modulatory effects of extracellular ATP have been described in the
majority of cells and tissues studied so far.2,4,5 Such
effects are mediated by the activation of P2 receptors, which were
recently classified into 2 major families, P2X and P2Y.6 The receptors that represent the P2X and P2Y families are linked to
ligand-gated ion channels and G proteins, respectively.6,7 The P2X family-related P2Z/P2X7 purinoceptor
has been described in various cell types including those of hemopoietic
origin, such as thymocytes, peripheral T lymphocytes, mast cells,
monocytes, macrophages, and phagocytic cells of the thymic
reticulum.8-10 The activation of the
P2Z/P2X7 receptor induces progressive pore opening and cell membrane permeabilization, allowing the exchange of
ions and molecules of up to 900 d and an increase in intracellular calcium.11,12 Recently, the activation of NFAT and
NF-  transcriptional factors, as well as
phospholipase D, was also associated with the intracellular signaling
triggered by the P2Z/P2X7
receptor.13-15
Yet, the physiologic importance of the P2Z/P2X7
purinoceptor described in different cells of the immune system remains
unclear and has become a major focus of investigation. In macrophages, the activation of P2Z/P2X7 has been related to
the formation of multinucleated giant cells, enhancement of
intracellular bactericide mechanisms, increase of posttranscriptional
processing and release of interleukin-1 and cell
death.16-18 In peripheral T lymphocytes, loss of L-selectin
expression has been described after P2Z/P2X7 activation.19 Recently, we demonstrated the expression of
the P2Z/P2X7 purinoceptor in phagocytic cells
of the thymic reticulum.20 These cells, first characterized
by Papiernik et al,21 share several features with
interdigitating dendritic cells (DCs) and macrophages,
such as few lysosomes, DC shape, high expression of major
histocompatibility complex (MHC) class II molecules, CR3 complement
receptor, and phagocytosis of IgG-opsonized sheep red blood cells. Such
similarities prompted us to investigate the presence of P2 receptors in
peripheral DCs, described as the major professional antigen-presenting
cells. These cells express high levels of MHC class II and
costimulatory molecules, enabling the activation of naive T lymphocytes
in vivo.22-24 In addition, DCs are widely
distributed in lymphoid and nonlymphoid organs, initiating
migration to T-cell areas of draining lymph nodes upon activation.25 Such a feature theoretically increases the
possibility to interact with extracellular adenine nucleotides released
by different sources.2
In this study, we characterize the P2Z/P2X7
receptor in murine spleen-derived DCs by pharmacologic and functional
criteria. Moreover, we show that apoptosis in these cells is enhanced
following P2Z/P2X7 activation by extracellular ATP.
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Materials and methods |
Reagents
Acridine orange, adenosine, cyclic adenosine 5'-monophosphate
(cAMP), adenosine 5'-diphosphate (ADP), adenosine
5'-triphosphate (ATP), 3'-0-(4-benzoylbenzoyl)-adenosine
5'-triphosphate (BzATP), N,N-dimethyl formamide (DMF), ethidium
bromide, ethylenediaminetetraacetic acid (EDTA), ethylene
glycol-bis(-aminoethyl ether) N,N,N',N'-tetraacetic acid
(EGTA), 5'-p-fluorosulfonyl-benzoyladenosine (5'FSBA),
L-glutamine, lucifer yellow, periodate oxidized ATP (oATP),
probenecid, propidium iodide, RPMI 1640 medium, uridine
5'-triphosphate (UTP), and trypan blue were purchased from Sigma
Chemical Co (St Louis, MO). FURA-2AM and YO-PRO 1 were
obtained from Molecular Probes (Eugene, OR), and adenosine
5'-O-(3-thiotriphosphate) (ATPS) was from Boehringer Mannheim
Biochemicals (Indianapolis, IN). Fetal calf serum was obtained from
GIBCO/BRL (Gaithersburg, MD).
Animals
Swiss-Webster female mice, 8 to 10 weeks old, were obtained from the
animal facilities of the Oswaldo Cruz Foundation and the Institute of
Microbiology and Immunology of the Federal University of Rio de
Janeiro. The animals were maintained in a light-controlled environment
and fed ad libitum.
Antibodies
Spleen-derived DCs were stained with fluorescein isothiocyanate
(FITC)- or phycoerythrin (PE)-labeled monoclonal antibodies (mAbs). The
following mAbs were also used: anti-CD3 (clone 29B; purchased from
GIBCO/BRL), anti-CD45R (clone RA3-6B2), biotinylated anti-CD80 (clone
1G10), and anti-CD86 (clone GL-1), which were obtained from Pharmingen
(San Diego, CA). The anti-F4/80 (macrophage marker) was from Caltag
(Burlingame, CA), and the anti-Ia (clone Ox 6) was from Crawley Down
(Sussex, England). The unlabeled hamster anti-mouse CD11c (clone N418)
was a gift from Dr Mireille Dardenne (Hospital Necker, Paris, France),
whereas the anti-FcRII/FcRIII mAb (clone 2.4G2) was kindly
provided by Dr Tania C. de Araújo Jorge (Oswaldo Cruz Foundation,
Rio de Janeiro, Brazil). The biotin-labeled anti-hamster IgG was
obtained from Vector Laboratories (Burlingame, CA), and both FITC- and
PE-streptavidin conjugates were from Amersham Pharmacia
Biotech (Buckinghamshire, UK).
Isolation and phenotypic characterization of DCs
DCs were obtained as described.26 Briefly, spleens were
disrupted and the cells were centrifuged at 1300 rpm for 5 minutes, resuspended in supplemented RPMI 1640 medium (10% heat-inactivated fetal calf serum, 2 mmol/L L-glutamine, 1 mmol/L pyruvate,
50 µmol/L mercaptoethanol, 100 U/mL penicillin, and 100 µg/mL
streptomycin), and incubated for 2 hours at 37°C in a 5%
CO2 atmosphere in plastic cell culture plates. Culture
plates were then washed vigorously 3 times with supplemented RPMI 1640 medium, and the nonadherent cells were discarded. The adherent cells
were maintained in the culture medium and incubated overnight at
37°C in a 5% CO2 atmosphere. After incubation, DCs
(which exhibit adherence capacity in the first hours of culture) become
nonadherent and float in the medium. The DCs were collected and
immediately used in our assays. To verify the degree of contaminating
cells in DC preparations, we stained floating cells with the following
mAbs: anti-F4/80 (macrophage), anti-CD45R (B cells), anti-Ia (MHC class
II molecules), anti-CD3 (T cells), and anti-CD11c (all DC
subpopulations); 80% to 95% of the cells were
Ia/CD11cHigh (Figure 1).
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| Fig 1.
Cytofluorometric profiles of DCs labeled with different
antibodies.
The specificity or type of each antibody is displayed above the
respective panel. The anti-F4/80, anti-CD3, anti-CD45R (B220), and
anti-MHC II (Ia) antibodies were analyzed taking into account the
negative control Ct-1 (rat IgG), and the anti- CD11c antibody was
analyzed based on the negative control Ct-2 (hamster IgG). The M1
marker defined the positive cells (%). The profiles are representative
of 3 separate experiments.
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Immunofluorescence
Freshly isolated DCs were centrifuged for 5 minutes at 1300 rpm and
suspended in phosphate-buffered saline (PBS) with 5% normal mouse
serum (vol/vol) and 2% of the 2.4G2 hybridoma supernatant. The cell
suspensions were distributed in microplate wells and incubated at
4°C for 20 minutes. After incubation, the cells were centrifuged as
described earlier and incubated separately with anti-CD3-FITC,
anti-CD45R-PE, anti-Ia-FITC, or anti-F4/80-PE at 4°C for 20 minutes. Isotype control was performed with unrelated PE- or
FITC-labeled rat IgG (Pharmingen). When anti-CD11c was used as the
primary antibody, the cell suspension was subsequently labeled with
biotinylated goat anti-hamster mAb, followed by FITC-streptavidin or
PE-streptavidin; both incubations were performed at 4°C for 20 minutes. In this case, a hamster anti-mouse  T cell receptor (TCR) was used as the isotype control (Pharmingen). The cells were
further washed with PBS and ultimately fixed with 1% formaldehyde/PBS. The data were analyzed by flow cytometry using an Epics Elite (Beckman
Coulter, Fullerton, CA) with WinMDI software.
Permeabilization assay
Freshly isolated DCs were suspended in RPMI 1640 medium. In some
experiments, RPMI 1640 medium with 5 mmol/L EDTA was used to deplete
calcium and magnesium divalent cations. DCs were incubated with
ethidium bromide (10 µmol/L), lucifer yellow (0.5 mg/mL), or YO-PRO 1 (10 µg/mL) for 15 minutes at 37°C in the presence or absence of
ATP, or another selected nucleotide analog. The cell suspensions were
washed 3 times with medium before the fluorescence analysis using a
Zeiss Axiovert 100 microscope (Carl Zeiss, Oberkochen, Germany). The
fluorescence pattern was also analyzed by flow cytometry, as described
previously. In this case, at least 104 cells were analyzed
in each group. Dead cells and cellular debris were excluded based on
low forward and side scatters and the very high fluorescence profile.
The dose-response curves obtained from permeabilization assays were
fitted with the logistic equation (Microcal Origin 4.1 software,
Microcal, Northampton, MA) depicted as follows:
y = [A1  A2] / [1 + (X /Xo)p] + A2,
where A1 and A2 correspond to minimal and maximal percentages of
permeabilized cells, respectively; y corresponds to permeabilized cells
(%) at each treatment point; and X corresponds to the agonist concentration. XO represents the half-maximal effective
concentration (EC50), and P represents the Hill
coefficient,27,28 which indicates the possible number of
ligand-binding sites in the receptor.
In our evaluations, we substituted saline solutions for RPMI 1640 medium to ascertain optimal culture conditions in DC permeabilization assays. We did this because, for us and our colleagues, these cells
presented sensitivity to great variations in culture conditions (ie,
temperature, medium, cytokines, serum, and pH). The RPMI 1640 medium is
a complex solution that contains amino acids and some vitamins.
However, the known binding constants between amino acids or vitamins
and the ions of interest are much lower when compared with those of
EDTA and ATP. Assuming this, we calculated the ATP4
concentration based on the total concentration of K+,
Mg2+, Ca2+, ATP, and EDTA. The pH
and the Fabiato binding constants were also considered.29
The calculation was done with the aid of a computer program originally
developed by Mark Kurzmack.30
Inhibition of ectonucleotidase activity
To ascertain the interference of ectonucleotidase activity in
ATP-dependent DC permeabilization, we pretreated the cells with 5'FSBA, a known irreversible ectonucleotidase
inhibitor.31,32 Briefly, the DCs were suspended in RPMI
1640 medium and treated with 1 mmol/L of 5'FSBA for 1 hour at
37°C (5% CO2). The cells were washed twice, suspended
in RPMI 1640 medium, and incubated with ATP (5 mmol/L) and ethidium
bromide (10 µmol/L) for 15 minutes at 37°C (5% CO2).
DC permeabilization was analyzed by flow cytometry. To evaluate the
specificity of the 5'FSBA effect, we also treated DCs with DMF
only (1%), the 5'FSBA diluent. In these experiments, DMF
application did not induce any effect (data not shown).
Intracellular calcium measurement
Intracellular calcium measurement in DCs was performed as
described.33 Briefly, DC suspensions (106
cells/mL in RPMI 1640 medium) were plated in 31-mm-diameter round glass
coverslips (Biophysica Technologies, Sparks, MD), treated with 2.5 mmol/L probenecid, and loaded with 6 µmol/L Fura2-AM in RPMI
1640 medium for 45 minutes at room temperature. After incubation, the
coverslips were rinsed with standard saline (composition in mmol/L: 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.4) and the experiments were performed using a photomultiplier-based calcium detection system (Photon Technology, Princeton, NJ) in a
modified 3-compartment superfusion chamber with the bottom formed by
the coverslip.34 Microscopic fields containing 20 to 40 DCs were selected and excited at 340- and 380-nm wavelengths; the intracellular calcium was monitored based on the ratio of emission at
510 nm (340 nm/380 nm). In all of the experiments, cells were perfused
with standard saline at room temperature and ATP was applied in the
initial mixing compartment, reaching the cells at a final concentration
of 5 mmol/L. In some cases, saline without calcium was perfused
(composition in mmol/L: 140 NaCl, 5 KCl, 1 MgCl2, 10 HEPES,
1 EGTA, pH 7.4).
Morphologic analysis of apoptosis
DCs were suspended in RPMI 1640 medium and plated in a V-bottom
96-well microtiter plate (105 cells per well). The cells
were treated with ATP (5 mmol/L) for 30 minutes, washed
twice, and further incubated for 6 hours with supplemented
medium alone at 37°C in a 5% CO2 incubator. The DC suspension was then centrifuged and suspended in PBS containing the
fluorescent dyes acridine orange (5 µg/mL) and propidium iodide (10 µg/mL). Both of these dyes intercalate DNA; however,
acridine orange (green labeling) is lipophilic whereas propidium iodide (red labeling) is hydrophilic. Thus, acridine orange allows
the analysis of nuclear morphology of viable cells if normal (intact) or apoptotic (fragmented), whereas propidium iodide allows cell viability determination. Additionally, the nuclear morphology of dead
cells labeled with propidium iodide can suggest whether it is
just necrotic or in advanced apoptosis (secondary necrosis).
The DCs were analyzed by confocal laser scanning microscopy (LSM 410;
Zeiss, Jena, Germany). Acridine orange was excited at 488 nm (argon
laser) and propidium iodide at 543 nm (helium/neon laser); the filter
settings were BP 510-525 nm and LP 570 nm, respectively. The
correspondent transmitted light images were obtained at 543 nm
(helium/neon laser) using a differential interference contrast device.
Quantification of apoptosis
The DCs were suspended in RPMI 1640 medium and treated with ATP,
BzATP, or UTP (all at 5 mmol/L) for 30 minutes; later, the cells were
washed twice, resuspended in supplemented RPMI 1640 medium, and further
incubated for 6 hours at 37°C (5% CO2). Apoptosis was
quantified by cytofluorometry using the terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) technique, as
described elsewhere.35 Briefly, cells were fixed in 2%
paraformaldehyde/PBS for 30 minutes, washed twice with PBS (1% bovine
serum albumin [BSA]), and incubated with permeabilization solution
(0.1% Triton X-100, 0.1% sodium citrate) for 2 minutes at 4°C.
Cells were further washed twice with PBS and resuspended in TUNEL
reaction mixture (in situ cell death detection kit; Boehringer
Mannheim, Mannheim, Germany) composed of buffer solution with TdT and
FITC-conjugated dUTP. After 50 minutes of incubation, the cells were
washed with PBS and analyzed by flow cytometry. In some cases, the DCs
were labeled with anti-CD11c mAb (followed by biotinylated anti-hamster IgG and PE-streptavidin) or PE-conjugated anti-CD80 or anti-CD86 mAb
before the TUNEL assay. As a negative control, DCs were incubated with
FITC-conjugated dUTP only. A positive control was performed by treating
the cells with DNAse (10 µg/mL) immediately before incubation with
TUNEL reaction mixture.
Statistical analysis
Statistical comparisons were done by the Student t test.
Differences were considered significant at P < .05.
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Results |
Permeabilization effect of ATP
The expression of the P2Z/P2X7 purinoceptor
can be detected by low-molecular-weight dye-uptake
assays19,36,37 based on the feature of this receptor, which
opens a poorly selective pore when activated. Figure
2 shows the fluorescence microscopy of DCs
left untreated or treated with ATP (5 mmol/L) in the presence of the
fluorescent dye lucifer yellow. The fluorescent dye could be seen
diffusely throughout the cytoplasm of treated DCs, indicating membrane
permeabilization (Figure 2A). This same pattern was not observed in
control cells (Figure 2B). The degree of permeabilization was also
quantified by cytofluorometry. Figure 3
shows that DCs treated with ATP became permeabilized to 2 other
fluorescent dyes, ethidium bromide (Figure 3B) and YO-PRO 1 (Figure
3C). Analysis by phase contrast microscopy demonstrated that DCs
treated with ATP did not become permeabilized to trypan blue (MW 961)
(data not shown), indicating a molecular-weight limitation of the
permeabilization phenomenon.
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| Fig 2.
Extracellular ATP induces permeabilization in DCs.
Phase contrast microscopy (left panels) and the respective fluorescence
microscopy (right panels) of DCs incubated for 15 minutes at 37°C
with lucifer yellow (0.5 mg/mL) either with (A) or without (B) 5 mmol/L
ATP. It is clear that ATP treatment enhanced the entrance of the
fluorescence dye into the cells. (Magnification, 320×.)
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| Fig 3.
Cytofluorometric profiles of DC permeabilization induced
by ATP.
(A) Forward-scatter (FSC) and side-scatter (SSC) dot plot. R1
represents the DC population that was gated for analysis, thus
excluding cellular debris and dead cells. DCs were incubated with 10 µmol/L ethidium bromide (B) or 10 µg/mL YO-PRO 1 (C) and treated
(ATP) or not (Ct) with 5 mmol/L ATP for 15 minutes at 37°C.
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Cell suspensions prelabeled with the anti-CD11c mAb were also subject
to the permeabilization assay, confirming the phenotype of the cells
sensitive to extracellular ATP (Figure 4).
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| Fig 4.
Permeabilization assay of DCs previously labeled with the
anti-CD11c mAb.
(A) Dual DC labeling with ethidium bromide (EB) and anti-CD11c antibody
(FITC). The upper panel represents the DCs labeled with just the
unrelated control antibody (Ct-Ab); the central panel represents the
DCs labeled with anti-CD11c mAb and incubated with ethidium bromide
alone (Ct-CD11c); the lower panel represents the DCs labeled with
anti-CD11c mAb and incubated with ethidium bromide plus 5 mmol/L ATP
(ATP-CD11c) for 15 minutes at 37°C. (B) The top histogram
represents the fluorescence profile of CD11c+ cells
incubated with 10 µmol/L ethidium bromide, either with (ATP) or
without (Ct) 5 mmol/L ATP. The M1 marker defines the threshold for
defining cell permeabilization. The bottom histogram represents the
fluorescence profile of the DC population labeled with anti-CD11c mAb
(CD11c) or with unrelated control antibody (Ct-Ab).
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ATP4 as the active ligand that triggers
permeabilization
ATP4 normally binds to divalent cations, such as
calcium and magnesium, to form a complex.38 It is well
established that the tetra-anionic
ATP4 is the active ligand of the
P2Z/P2X7 purinoceptor.8,36
Figure 5 shows the fluorescence profile of
DCs incubated with ethidium bromide (10 µmol/L) plus MgCl2
(5 mmol/L) before the application of ATP (5 mmol/L). The
ATP-induced permeabilization was completely inhibited by
MgCl2 treatment, indicating that the active form of the
ligand is actually the fully ionized ATP4.
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| Fig 5.
Treatment of DCs with MgCl2 completely blocks
the ATP-induced permeabilization.
DCs were incubated in the presence of 10 µmol/L ethidium bromide and
5 mmol/L MgCl2 before the application of 5 mmol/L ATP. The
histogram represents the fluorescence intensity of control DCs (Ct)
incubated with ethidium bromide alone, those treated with ATP in the
presence of ethidium bromide (ATP), and those subjected to 5 mmol/L ATP
in the presence of ethidium bromide plus 5 mmol/L MgCl2
(MgCl2-ATP).
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Dose dependence
To verify the dose dependence of ATP-triggered permeabilization, we
treated DCs with different concentrations of ATP (Figure 6). In RPMI 1640 medium, the effective
permeabilization began at 0.1 mmol/L and the peak, at which 44.5% of
the DC population became permeabilized, was reached at 5 mmol/L. The
EC50 was 0.721 mmol/L (Figure 6A, circle), and the
calculated Hill coefficient was 2.46.
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| Fig 6.
Dose-response curve of DC permeabilization
following ATP treatment: effect of Ca2+ and
Mg2+ chelation.
(A) DCs were treated with different concentrations of ATP (0.01 to 20 mmol/L) and exposed to ethidium bromide (10 µmol/L) for 15 minutes at 37°C. The experiments were performed in RPMI 1640 medium
alone (circle) or in the presence of EDTA (5 mmol/L) (triangle). The
dose-response curves were also plotted based on the
P2Z/P2X7 active ligand ATP4
concentration calculated from the experiments performed in RPMI
1640 medium alone (B) or in the presence of EDTA (C). Plotted data
represent the mean ± SD of 3 distinct experiments.
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The fact that just half of the DC population responded to the assay
prompted us to investigate the possible effect of ectonucleotidases in
this process. The same assay was carried out in RPMI 1640 medium with 5 mmol/L of EDTA to decrease manyfold the Ca2+ and Mg2+
concentrations, which have been described as being essential to the activity of ectonucleotidases.1,39 Under such
conditions, the EC50 shifted to 0.094 mmol/L
(Figure 6A, triangle). As shown in Figure 5, it was the anionic
form of the agonist (ATP4) that induced
the permeabilization of DCs. Hence, to investigate whether the shift in
EC50, observed during permeabilization performed in
medium containing EDTA, was due to an increase of noncomplexed active
ligand (ATP4) or to an inhibition of
ectonucleotidases, we normalized the dose-response curves based on the
concentration of ATP4 calculated in each condition.
Even after normalization, we observed that differences in
EC50 were still present (Figure 6B,C), indicating that part
of this effect might well be the result of ectonucleotidase inhibition.
Our assays using 5'FSBA, an irreversible ectonucleotidase inhibitor, further indicated that this was the case. As seen in Figure 7, when the DCs were previously
treated with 5'FSBA (1 mmol/L), we observed an increased number
of permeabilized cells (71.8% ± 6.2%) when compared
with the untreated control (41.2% ± 7.9%).
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| Fig 7.
The ectonucleotidase inhibitor, 5'FSBA, enhances
ATP-mediated DC permeabilization.
The DC suspension was previously treated with 1 mmol/L of 5'FSBA
for 1 hour at 37°C. These cells were then washed twice and
incubated with ATP (5 mmol/L) and ethidium bromide (10 µmol/L) for 15 minutes at 37°C. The degree of permeabilization was analyzed by
flow cytometry. Data shown were normalized and compared by Student
t test. *Results from DCs treated with 5'FSBA were
significantly different from those of untreated cells
(P < .001). Data are the average of 3 experiments (mean ± SD).
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Pharmacologic profile
Different nucleotide analogs were tested for their capacity to
permeabilize DCs. Adenosine, cAMP, ADP, UTP, BzATP, and ATPS were
tested at 5 mmol/L in all assays, and their effects were compared with
that of 5 mmol/L ATP. As depicted in Figure
8, ATP, BzATP, and ATPS selectively
permeabilized DCs above the background level, whereas the other analogs
did not cause any effect. In additional experiments, DCs were treated
with increasing concentrations of oATP, previously described as a
selective P2Z/P2X7 receptor antagonist that binds covalently and irreversibly to the receptor, inhibiting its effects.40 DCs were incubated with oATP for
2 hours at 37°C. The cells were then incubated with or without ATP in the presence of ethidium bromide for 15 minutes. As shown in Figure
9, pretreatment with oATP at concentrations
greater than 300 µmol/L completely blocked the ATP-mediated
permeabilization.
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| Fig 8.
DC permeabilization was induced selectively by adenine
triphosphate nucleotide analogs.
Adenosine, cAMP, ADP, UTP, ATPS, and ATP were analyzed for their
ability to permeabilize DCs. DC suspensions containing 10 µmol/L
ethidium bromide were incubated for 15 minutes at 37°C with
different nucleotide analogs (all at 5 mmol/L). Data shown were not
normalized. Results express the average of 3 experiments (mean ± SD).
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| Fig 9.
Oxidized ATP treatment blocks the ATP-mediated DC
permeabilization.
DC suspensions were pretreated with different concentrations of
oxidized ATP (oATP) for 2 hours at 37°C. These cells were
subsequently treated with ATP (5 mmol/L) plus ethidium bromide (10 µmol/L) and incubated for 15 minutes more. The permeabilization
inhibition was analyzed by flow cytometry.
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ATP treatment induces intracellular calcium increase in DCs
An increase in intracellular calcium ([Ca2+]i) has
been described as a hallmark of the activation of all subtypes of P2
receptors studied so far.41 Thus, we measured by
microfluorometry with FURA2-AM DC
[Ca2+]i before and after ATP treatment. Figure
10A depicts the 510-nm emission
wavelength ratio showing the [Ca2+]i
increase in DCs after a 5-mmol/L ATP application in standard saline;
this increase was not observed when saline alone was applied as a
negative control. When the same microscopic field was maintained and
perfused with saline without calcium (see "Materials and
methods"), ATP application was not able to induce any intracellular
calcium alteration (Figure 10B). In contrast, when the same cells were subsequently perfused with standard saline, the intracellular calcium
increase was again observed after ATP application (Figure 10C),
indicating that the extracellular environment is the source for the
calcium increase detected in the intracellular milieu after the ATP
treatment.
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| Fig 10.
Intracellular calcium increase induced by ATP treatment.
DCs previously loaded with 6 µmol/L FURA2-AM and
treated with 2.5 mmol/L probenecid were tested in perfusion with either
standard saline (including 1 mmol/L CaCl2) (A,C) or
calcium-free saline (including 1 mmol/L EGTA) (B). The arrows indicate
the application of control saline alone (Ct) or 5 mmol/L ATP (ATP).
Intracellular calcium changes were detected by monitoring the variation
of the ratio obtained from emission at 510 nm elicited under 340-nm and
380-nm excitation wavelengths. These measurements are representative of
3 separate experiments and were obtained from the same cellular
microscopic field.
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ATP-induced apoptosis of DCs
After establishing the presence of the
P2Z/P2X7 purinoceptor in murine spleen-derived
DCs, we investigated one possible function associated with its
activation. One of the effects associated with
P2Z/P2X7 activation in other cell types is the
induction of apoptosis.8,42,43 To investigate this
possibility, we incubated DCs with ATP for 30 minutes and subsequently
with medium alone for 6 hours more. First, ATP-treated DCs were
evaluated morphologically. As ascertained by double staining with
acridine orange (green labeling) and propidium iodide (red labeling),
the DCs presented morphologic characteristics of apoptotic cells upon ATP treatment (Figure 11B): (1) chromatin
condensation, evidenced by the increased fluorescence intensity of the
nucleus; (2) fragmented nuclei; and (3) apoptotic bodies.
Interestingly, even the nonviable cells, labeled with propidium iodide,
presented fragmented nuclei (Figure 11B, right panel), an
indication of secondary necrosis that follows the initial apoptosis.
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| Fig 11.
ATP treatment induces morphologic changes in DCs
compatible with apoptosis.
DC suspension was incubated with ATP (5 mmol/L) for 30 minutes at
37°C, washed twice with PBS, and incubated additionally for 6 hours
in complete RPMI 1640 medium. DCs were incubated immediately before the
analyses with acridine orange (5 µg/mL) and propidium iodide (10 µmol/L) to determine nuclear morphology and cell integrity,
respectively. The DC apoptotic phenotype was ascertained by laser
scanning confocal microscopy. The right panels represent the images of
fluorescence data and the left ones represent the corresponding
transmitted images. (A) Control DCs were viable, presenting
green-labeled round nuclei. (B) The ATP-treated DC suspension presented
cells with features associated with different stages of apoptosis:
chromatin condensation (increased nuclei fluorescence), fragmented
nuclei and apoptotic bodies (arrows), and secondary necrosis
(fragmented nuclei labeled red with propidium iodide). Bar: 10 µm.
|
|
In subsequent experiments, the executive phase of apoptosis
characterized by genomic DNA cleavage was ascertained by the TUNEL technique. The incorporation of FITC-conjugated dUTP could be evidenced
as an increase in fluorescence profile of the cells when analyzed by
flow cytometry (Figure 12). A high
concentration of ATP as that applied to permeabilize the cells (5 mmol/L) induced a significant increase in apoptotic DCs
(65% ± 4.4%) when compared with controls (35.4% ± 7.6%)
(Figure 13A). This effect was dose dependent (Figure 13B), and the EC50 was 0.989 mmol/L. DC
phenotype was ascertained by labeling the cells with the anti-CD11c mAb before the TUNEL assay. Thus, we confirmed that all cells analyzed, independent of being resistant or susceptible to ATP-induced apoptosis, presented the DC CD11chigh phenotype (Figure
14).
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| Fig 12.
ATP-induced apoptosis detected by the TdT-mediated dUTP
nick end labeling (TUNEL) technique.
DCs were incubated with ATP (5 mmol/L) for 30 minutes at 37°C,
washed twice with PBS/BSA, and incubated additionally for 6 hours in
complete RPMI medium. Apoptosis was ascertained by the TUNEL technique
and flow cytometry. The resulting fluorescence intensity profiles of
control DCs (lower left histogram; Ct2) and those treated with 5 mmol/L
ATP (lower right histogram; ATP) are shown. As a negative control, DCs
were incubated with FITC-conjugated dUTP only (upper left histogram;
Ct1) without TdT. Positive control was performed by treating the DCs
with DNAse (10 µg/mL) immediately before incubation with TUNEL
reaction mixture (upper right histogram; DNAse).
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| Fig 13.
P2ZP2X7 activation-dependent
apoptosis of DCs.
(A) DCs were incubated with UTP, BzATP, or ATP (all at 5 mmol/L) for 30 minutes at 37°C; washed twice with PBS/BSA; and incubated
additionally for 6 hours in complete RPMI 1640 medium. Alternatively,
DCs were incubated with 500 µmol/L of the
P2Z/P2X7 antagonist, oATP, for 2 hours before
ATP (5 mmol/L) treatment. The DCs treated with oATP plus ATP
(oATP + ATP) were compared with the control untreated DCs
(Control) and with those treated with ATP alone (5 mmol/L). Data
are based on at least 3 independent experiments. The results
(mean ± SD) were compared with Student t test.
*Data from DCs treated with ATP or BzATP were significantly different
from control (P < .001); **data from ATP-treated DCs
submitted to oATP inhibition were significantly different from
ATP-treated DCs (P < .001). (B) Dose-response curve of
ATP-induced apoptosis. Apoptosis was ascertained by the TUNEL
technique and flow cytometry. Data are based on 3 independent
experiments (mean ± SD).
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| Fig 14.
Anti-CD11c antibody labeling of apoptotic and
nonapoptotic DCs.
The DCs were treated with 5 mmol/L ATP for 30 minutes, washed, and
incubated for an additional 6 hours. These cells were then labeled with
the anti-CD11c mAb before the detection of apoptosis by TUNEL. The dot
plots show the double labeling for CD11c (PE) and TUNEL (FITC) of
either control cells (Ct) (upper panel) or those treated with ATP (ATP)
(lower panel).
|
|
To analyze whether the ATP-induced apoptosis of DCs is mediated
specifically by P2Z/P2X7 activation, we
incubated the DCs with 500 µmol/L of the antagonist oATP for 2 hours
before the treatment with ATP. As shown, this antagonist significantly
inhibited the apoptotic effect triggered by ATP (P < .001)
(Figure 13A). Additionally, the
P2Z/P2X7-specific receptor agonist BzATP also induced DC apoptosis, in contrast to UTP, which did not induce this
effect (Figure 13A).
Evaluation of costimulatory molecules in ATP-treated DCs
After normalizing ATP-induced apoptosis, we observed that only
39.6% of the DCs entered apoptosis after treatment with 10 mmol/L ATP
(Figure 13B). Thus, not all DCs are susceptible to ATP-induced apoptosis. To verify whether the apoptosis-resistant DCs had been activated by a purinergic stimulus, we evaluated the expression of
costimulatory molecules CD80 (B7.1) and CD86 (B7.2) because up-regulation of these molecules has been related to DC activation by
different inflammatory stimuli.44,45 For this purpose, we labeled the cells with anti-CD80 and anti-CD86 mAbs before the TUNEL
assay. Thus, we could analyze the expression of these costimulatory molecules in apoptotic and nonapoptotic cells after ATP treatment. As
expected, DCs presented a high expression of CD80 and CD86 molecules
(Figure 15A,B). Such expression was not
modified in apoptosis-resistant cells after ATP treatment. Conversely,
the expression of both molecules was decreased in cells susceptible to
ATP-induced apoptosis.
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| Fig 15.
Evaluation of CD80 and CD86 expression on apoptotic and
nonapoptotic DCs following ATP treatment.
DCs were exposed to ATP for 30 minutes, washed, and incubated for an
additional 6 hours. They were then labeled with the PE-conjugated
anti-CD80 (B7.1) or anti-CD86 (B7.2) mAb before the detection of
apoptosis by TUNEL (FITC). The dot plots show the CD80 (A) or CD86 (B)
and TUNEL double labeling of control cells (Ct) (left panel) and those
treated with 5 mmol/L ATP (ATP) (right panel).
|
|
| |
Discussion |
Studies concerning P2 receptors and their importance in cell and
systemic physiology have increased substantially46 since Drury and Szent-Györgyi's pioneering work.3 The P2
receptors have been described in many cell types, either excitable or
not, and the effects observed upon their activation are quite
variable.2,5 In the present work, we demonstrate the
expression of the P2Z/P2X7 purinoceptor in
murine spleen-derived DCs and the relation of this receptor to
ATP-induced apoptosis in these cells.
The basic approach used to characterize the
P2Z/P2X7 purinoceptor was the dye-uptake assay,
ascertained by fluorescence microscopy and cytofluorometry. Different
molecular-weight fluorescent probes were tested, and most of them
(ethidium bromide, a 314-d cation; YO-PRO 1, a 374-d
cation; and lucifer yellow, a 443-d anion) entered into ATP-treated
DCs, except for the nonfluorescent probe trypan blue (Mr 961), which
did not cross the cell membrane. These results are consistent with
those described in other cell types such as macrophages, microglia, and
lymphocytes, whose P2Z/P2X7-associated pores
are permeable to low-molecular-weight dyes.36,47,48
In DCs, the permeabilization phenomenon was effectively seen with ATP
concentrations higher than 100 µmol/L, a result that is consistent
with the high concentrations necessary to activate the
P2Z/P2X7 receptor, as compared with the
requirements of all other P2X and P2Y purinoceptor subtypes, which can
be activated with ATP concentrations as low as 1 µmol/L.4,5,8 When the permeabilization was performed in
divalent cation-depleted medium, a shift in ATP EC50 was
observed from 0.721 mmol/L in standard medium to 0.094 mmol/L. This
finding can be explained in 2 ways: (1) In standard medium,
Mg2+ and Ca2+ can bind to fully ionized
ATP4 forming MgATP2 and
CaATP2 complexes,38 which have not been
described as active ligands of the P2Z/P2X7
receptor,49,50 thus affecting the available active
ATP4 concentration; or (2) DCs express
Ca2+/Mg2+-dependent ectonucleotidases that
hydrolyze ATP4, reducing its
concentration.39 We observed that both factors are involved
because application of extra MgCl2 before ATP treatment blocked the permeabilization effect. In addition, the previous treatment of DC suspension with 5'FSBA, an irreversible
ectonucleotidase inhibitor, significantly increased the ATP
permeabilization effect. Moreover, when the dose-response curves were
plotted as a function of ATP4 concentration, the
EC50 and the curves obtained under these different conditions (using standard or divalent cation-depleted media) did not
overlap, thus also supporting the action of
Ca2+/Mg2+-dependent ectonucleotidases on DCs,
as previously described.39
Additionally, based on the dose-response curve obtained under standard
conditions (medium without EDTA), a Hill coefficient of 2.46 was found,
which is very close to that attributed to the P2Z/P2X7 purinoceptor present in macrophages
(2.4-3),51 lymphocytes (2.3),47 and mast cells
(1.6-2.1),52 suggesting that this receptor presents at
least 2 binding sites to the ligand.
In our permeabilization assays, even under optimized conditions, no
more than 70% of the DCs became permeabilized when treated with ATP.
Such a permeabilization resistance cannot be merely attributed to
possible ATP-resistant contaminant cells because they were all
CD11c+. Therefore, mechanisms other than
Ca2+/Mg2+-dependent ectonucleotidases may
be affecting P2Z/P2X7 activation. Even a
differential expression of the receptor cannot be discarded because 3 phenotypically distinct subpopulations of DCs were characterized recently in the spleen53,54:
CD8 +DEC205+CD11b,
CD8DEC205CD11b+,
and CD8+- DEC205+CD11bdull,
with all of them presenting the CD11chigh MHC
IIhigh phenotype.
Among the various nucleotides tested, only ATP, BzATP, and the
nonhydrolyzable ATPS were able to induce membrane permeabilization in DCs. Such an agonist selectivity matches that described for the
P2Z/P2X7 receptor.11,12
Accordingly, ATP-mediated permeabilization was completely blocked by
long-term treatment with 300 µmol/L oATP, a well-established
P2Z/P2X7 inhibitor.40 Moreover, an increase in intracellular calcium was detected after ATP application, which was blocked when calcium-free saline was perfused, demonstrating its extracellular origin. All of these features strongly suggest the
presence of P2Z/P2X7 receptors in murine
spleen-derived DCs.
Interestingly, recent studies have also suggested the expression of P2Y
receptors on DCs.55,56 In our calcium microfluorometric assays, we also tested the effect of UTP on DCs. However, the calcium
responses to UTP were not consistent. We obtained calcium responses in 3 experiments and no response in 4 experiments. In this case, we need to consider the interference of
ectonucleotidases, receptor desensitization, and even the DC mature phenotype.
In a second vein, we investigated a putative role of the
P2Z/P2X7 purinergic receptor in inducing
apoptosis in DCs, as described previously for
macrophages8,16,17 and recently for D2SC/1 cells, an
immortalized DC line.57 Using double staining with acridine
orange and propidium iodide, we ascertained that ATP-treated DCs
presented morphologic alterations associated with different stages of
apoptosis, such as chromatin condensation, fragmented nuclei, apoptotic
bodies, and secondary necrosis.58 Additionally, DNA
cleavage, an important apoptosis signal, was evidenced and quantified
by the TUNEL technique. The flow cytometric analysis of DCs assayed by
TUNEL showed that the ATP treatment induced a significant increase of
apoptotic cells from the basal level of 34.08% ± 5.8% in
control cultures to 64.46% ± 3.8% after 5 mmol/L ATP treatment.
Spontaneous apoptosis of cultured DCs was described by Ludewig et
al.59 To see whether such basal apoptosis was also due to
ATP spontaneously released in the culture medium, we incubated the
cells with the P2Z/P2X7 antagonist oATP during
the last phase of DC isolation, which consists of an overnight
incubation at 37°C. Nevertheless, under this condition the basal
value of cells undergoing apoptosis was not significantly modified
(data not shown), suggesting that it is not mediated by the
P2Z/P2X7 receptor.
When the data were normalized, we observed that the apoptotic effect
induced by ATP reached a maximum of 39.6% of the DC population. The DC
phenotype of the ATP-induced apoptosis-resistant population was
confirmed by anti-CD11c antibody labeling. Additionally, these cells
already presented an activated phenotype, expressing high levels of
CD80 and CD86 costimulatory molecules. Such an apparently mature
phenotype is likely due to the isolation procedure
adopted,60 which was based on differential adherence
properties of DCs. Interestingly, the apoptotic DCs presented a
diminished expression of these molecules when compared with the
nonapoptotic ones. Such a loss of surface molecules has been described
and was related to the apoptotic process.61 In contrast,
Berchtold et al62 have demonstrated an increased expression
of CD80 and CD86 molecules in maturing DCs treated with ATP, suggesting
the involvement of P2 receptors in their activation. Thus, further
investigation that takes into account both immature and mature DC
phenotypes is necessary to clarify the relative importance of the
P2Z/P2X7 receptor in determining the DC
activation status.
To verify the receptor specificity of such ATP-induced apoptosis, we
treated DCs with the P2Z/P2X7 antagonist oATP
(500 µmol/L), which significantly inhibited the ATP-induced
apoptosis. Such an inhibition was partial because the apoptotic
response did not return completely to control levels. Thus, the
concomitant participation of other P2 receptor subtypes in apoptosis
induction cannot be discarded. However, in our experiments when we
treated DCs with UTP (5 mmol/L), a P2Y agonist, we were unable to
induce apoptosis in DCs, in contrast to BzATP, which also resulted in
apoptosis induction. Hence, the possible co-participation of other P2
receptors may be limited to those of the P2X subtypes.
Specific cytotoxicity induced by the P2Z/P2X7
purinoceptor has been widely described. The ATP-sensitive J774
macrophage cell line, expressing a high level of
P2Z/P2X7 receptors, presents an increased rate
of spontaneous cell death in culture.42 Similarly, embryonic kidney cells (HEK293) transfected with P2X7 cDNA
underwent apoptosis when exposed to ATP.43 Interestingly,
in macrophages, P2Z/P2X7 activation has been
associated with differentiation63 and with an improvement
of mycobactericidal activity that is followed by cell death by
apoptosis.17 Therefore, it is conceivable that apoptosis
induction contributes to the elimination of intracellular parasites and
infected cells.
In the case of DCs, an enhancement of their effector functions by
P2Z/P2X7 activation also needs to be
investigated because fetal skin-derived DCs showed reduced
antigen-presenting capacity when treated with oATP, the
P2Z/P2X7 antagonist.64 In addition, ATP has acted synergistically with tumor necrosis factor-, inducing human monocyte-derived DC maturation.62 This possible
activation function may have many implications because DCs exert a
major role as antigen-presenting cells and have been involved in the initiation of diverse autoreactive responses, such as thyroiditis and
experimental allergic encephalomyelitis.65,66 In this
context, we cannot discard the possibility that the apoptosis induced
by P2Z/P2X7 activation in mature DCs may
consist of a mechanism that self-limits and negatively controls their
function, avoiding the possible induction of undesired autoimmune
responses. We hope that further investigation will clarify this
putative dual function of the P2Z/P2X7
purinoceptor in DCs, namely to induce cell activation while
simultaneously self-limiting the initiated response.
| |
Acknowledgments |
We thank Dr Mireille Dardenne (Hospital Necker, Paris, France) for
providing the anti-CD11c mAb (N418 clone), Dr Tania C. de Araújo
Jorge (Oswaldo Cruz Foundation, Rio de Janeiro, Brazil) for providing
the anti-FcRII/FcRIII mAb (clone 2.4G2), and Dr Adriana Bonomo
(National Cancer Institute of Rio de Janeiro, Brazil) for helping us to
isolate dendritic cells in the beginning of this work.
| |
Footnotes |
Submitted August 3, 1999; accepted March 20, 2000.
Supported in part by grants from CNPq, PRONEX/CNPq, PADCT/CNPq, and
FAPERJ (Brazil).
Reprints: Luiz Anastácio Alves, Laboratório de
Pesquisas sobre o Timo, Departamento de Imunologia, Instituto Oswaldo Cruz-Fundação Oswaldo Cruz, Av. Brasil, 4365, Manguinhos,
21045-900, Rio de Janeiro, Brazil.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction