1 Unité de Biologie des Interactions Cellulaires, Centre National de la Recherche Scientifique, Unité de Recherche Associée 1960, Institut Pasteur, 75724 Paris Cedex 15, France; and 2 Laboratorio de Imunobiofisica, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil
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ABSTRACT |
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Given the role that extracellular ATP (ATPo)-mediated apoptosis may play in inflammatory responses and in controlling mycobacterial growth in macrophages, we investigated whether ATPo has any effect on the viability of chlamydiae in macrophages and, conversely, whether the infection has any effect on susceptibility to ATPo-induced killing via P2Z/P2X7 purinergic receptors. Apoptosis of J774 macrophages could be selectively triggered by ATPo, because other purine/pyrimidine nucleotides were ineffective, and it was inhibited by oxidized ATP, which irreversibly inhibits P2Z/P2X7 purinergic receptors. Incubation with ATPo but not other extracellular nucleotides inhibits the growth of intracellular chlamydiae, consistent with previous observations on ATPo effects on growth of intracellular mycobacteria. However, chlamydial infection for 1 day also inhibits ATPo-mediated apoptosis, which may be a mechanism to partially protect infected cells against the immune response. Infection by Chlamydia appears to protect cells by decreasing the ability of ATPo to permeabilize macrophages to small molecules and by abrogating a sustained Ca2+ influx previously associated with ATPo-induced apoptosis.
apoptosis; bacteria; immunity; purinergic receptors
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INTRODUCTION |
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THE CHLAMYDIA SPECIES are the causative agents of several significant diseases in humans and a wide variety of animals. Over 600 million humans are estimated to be infected with C. trachomatis strains, which invade primarily epithelial cells of the eyes and genital tract, leading to conjunctivitis and trachoma and causing sexually transmitted diseases that result in sterility (4, 14, 30, 59). The chlamydiae are obligate intracellular bacteria that exist in two developmental forms. The metabolically inert elementary bodies (EB) are internalized by epithelial cells into vacuoles that avoid fusion with host-cell lysosomes and that differentiate a few hours after infection into the metabolically active reticulate bodies (RB). The RB proliferate within a membrane-bound inclusion and, after 1 day of infection, begin differentiating back into EB. The EB are released from the infected cell after about 2 days of infection, allowing a new infection cycle to begin (4, 39). In addition to infecting epithelial cells, C. trachomatis and C. psittaci also infect macrophages in vitro and in vivo (31).
Macrophages and other eukaryotic cells die mainly through one of two mechanisms, necrosis or apoptosis (8, 15, 55, 64). Necrosis is often referred to as accidental cell death and is caused through irreversible damage of the plasma membrane. Perforin, produced by cytotoxic lymphocytes, and complement kill cells by destroying the integrity of the plasma membrane, leading to osmotic swelling and necrosis (7, 35, 36). A perforin-dependent mechanism, however, does not appear to be necessary for controlling Chlamydia infections (51, 56). Apoptosis, or programmed cell death, is distinguished from necrosis by morphological and biochemical criteria. Apoptosis is associated with nuclear and cytosolic condensation and with fragmentation of chromatin and DNA. Until recently, caspase activation was often used as a criterion for defining apoptosis, although caspase-independent pathways have also been identified (8, 15, 55). Apoptosis is induced by, among other signals, the Fas ligand or antibodies against Fas (41), glucocorticoids (63), beauvericin and valinomycin (48, 65), staurosporine (37), and extracellular ATP (ATPo) (1, 2).
The Fas receptor is constitutively expressed on many cells, including peripheral blood monocytes. Treatment with monoclonal antibodies directed against Fas in vitro and in vivo induces caspase-dependent apoptosis of cells expressing the Fas receptor (41). Chlamydia protects infected cells against apoptosis induced by external ligands such as anti-Fas antibodies by preventing cytochrome release from mitochondria and caspase activation (20), but this protection cannot prevent apoptosis caused by the Chlamydia infection itself, which does not rely on known caspases (47). Consistent with these results, infection by C. trachomatis causes apoptosis in the genital tract in vivo (49), and Fas-induced apoptosis is not critical for clearance of the bacteria from the same tissues (51).
Caspases are also activated during ATPo-mediated apoptosis but are not required for this cell death pathway (17). ATPo is thought to be involved in inflammatory responses (2, 16, 19), and the ability of ATPo to trigger apoptosis has been demonstrated in a number of cell types, including macrophages, thymocytes, and dendritic cells (1, 9, 13, 16, 68). ATPo has its effect primarily via nucleotide receptors, which are distributed through the body and belong to several families: P2X receptors, which are ligand-gated ion channels; P2Y and P2U receptors, which are G protein-coupled receptors; and P2Z receptors, which are ligand-gated ion channels that can be activated by ATPo (1, 19, 23). The different receptors can be distinguished on the basis of the behavior of different agonist and antagonist nucleotides (19, 23), and the corresponding genes have been cloned (23, 62). The P2Z/P2X7 receptor, which represents the P2Z receptor previously characterized on macrophages (62), generates a pore that is reversibly permeable to hydrophilic molecules smaller than 900 Da (16). Macrophages and dendritic cells have been shown to express the genes for both the P2Y and P2Z/P2X7 receptors (10, 13, 44).
Interestingly, induction of apoptosis in Mycobacterium tuberculosis-infected macrophages with ATPo is associated with killing of the intracellular mycobacteria (33, 38). In studies with monocytes infected with bacillus Calmette-Guerin, both H2O2 and ATPo killed the monocytes, but only ATPo treatment killed the mycobacteria (38). In a comparison with other ligands that can trigger lysis of macrophages, including complement-mediated cytolysis, Fas ligation, and CD69 activation, only ATPo treatment led to death of both host cells and intracellular mycobacteria (33).
We therefore evaluated the effects of ATPo treatment on the viability of chlamydiae in infected macrophages. Consistent with the results on mycobacteria-infected cells, ATPo inhibited the infectious activity of the chlamydiae. Moreover, we found that Chlamydia infection also partially inhibits ATPo-induced macrophage death. Because this inhibition would presumably operate on a level different from that previously reported for inhibition of caspase-dependent pathways, we investigated the effects of the infection on the activity of the P2Z/P2X7 purinergic receptor. We found that the permeabilizing activity of the receptor decreases partially after infection. Thus the immune response may use an ATPo-dependent mechanism to control Chlamydia infections, but the bacteria themselves may attempt to limit the effectiveness of the immune response by protecting the infected cells against ATPo-mediated apoptosis.
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METHODS |
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Cells and materials. The human cervical adenocarcinoma cell line HeLa 229 and the mouse macrophage cell line J774 were from American Type Culture Collection. The cells were maintained at 37°C in an atmosphere of 5% CO2 in Dulbecco's modified minimal essential medium (GIBCO BRL) (for HeLa) or RPMI 1640 (for J774) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO BRL) and 2 mM L-glutamine. The J774 cells were purchased in 1988 and maintained in culture almost continuously since then. The Chlamydia strain used here, the guinea pig inclusion conjunctivitis serovar of C. psittaci (4), was obtained from Dr. Roger Rank (University of Arkansas). FITC-labeled anti-Chlamydia monoclonal antibodies (MAb) were from Argene (Varilhes, France).
ATP, ADP, AMP, UDP, UTP, benzoylbenzoyl ATP (BzATP), oxidized ATP (oxATP), and adenosine 5'-O-(3-thiotriphosphate) were purchased from Sigma (St. Louis, MO). The reagents were prepared as stock 100 mM solutions in PBS and stored atPreparation of chlamydiae and infection of macrophages. The chlamydiae were grown in infected HeLa cell monolayer cultures as described (46). For infections, adherent J774 cells were typically grown on coverslips or on 75-cm2 tissue culture flasks (Costar) until 60-70% confluence and then incubated with chlamydiae in cell culture medium for the indicated times at 37°C in 5% CO2. The Chlamydia preparation was used at a multiplicity of infection (MOI) of 4.0. For chloramphenicol inhibition experiments, the cells were incubated with 68 µg/ml chloramphenicol for 30 min at 37°C before bacteria were added, and the antibiotic was maintained with the cells during the duration of the infection.
Measurement of nucleotide-induced apoptosis by cytofluorometry. Macrophages were first grown on 75-cm2 tissue culture flasks (Costar) until 60-70% confluence and then 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 with cell culture medium. The cells were then incubated for the indicated times (usually 6 h) at 37°C in 5% CO2. Cells were incubated with the P2Z/P2X7 blocker oxATP for 2 h, and oxATP was then removed from the medium before ATP was added. For experiments with infected cells, macrophages were first infected with C. psittaci for 24 h, as indicated, and then incubated with nucleotides at 37°C for an additional 6 h.
Quantitative measurements of apoptosis were performed by cytofluorometry of detergent-permeabilized propidium iodide (PI)-stained cells, as described previously (18, 43). 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 J774 cells were collected on a FACScan flow cytometer (Becton Dickinson) with an argon laser tuned to 488 nm.DNA fragmentation assay. After J774 cells were incubated with 5 mM ATP, ADP, AMP, UDP, or UTP as described above and then incubated for an additional 6 h at 37°C in a CO2 incubator, both adherent cells and cells in suspension (1-3 × 106) were collected and centrifuged (270 g for 5 min). The pellet was then lysed with 0.6% SDS, 10 mM EDTA, 10 mM Tris, and 20 µg/ml RNase A, pH 7.5, for 1 h at 37°C in 3-ml aliquots. Three hundred microliters of 5 M NaCl were added, and the preparation was incubated for 1 h on ice and finally centrifuged for 30 min at 13,000 g. The supernatant, containing the DNA, was extracted with phenol-chloroform-isoamyl alcohol (25:24:1), and low-molecular-weight DNA was precipitated with ethanol. The same quantity of DNA sample was loaded per gel well. Samples were separated by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining.
Measurement of infectious activity of chlamydiae. Macrophages that had been infected with C. psittaci at an MOI of 4.0 for 24 h were incubated with 5 mM ATP, UTP, ADP, or AMP 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 incubated for an additional 6 h at 37°C in 5% CO2. The cells and supernatant were then combined and centrifuged for 60 min at 12,000 rpm in a Sorvall type GSA rotor. The pellet was resuspended in ice-cold culture medium with a 21-gauge 2-ml syringe to dissociate aggregates, giving the final suspension of Chlamydia used to infect HeLa cells. Serial dilutions of the chlamydial preparation were used to infect HeLa cells on coverslips for 72 h, and the chlamydial vacuoles were revealed by fixing the cells with paraformaldehyde, permeabilizing with saponin, and incubating with FITC-conjugated anti-Chlamydia MAb, as previously described (45). Samples were examined with a Zeiss microscope (Axiophot Zeiss) attached to a cooled charge-coupled device camera (Photometrics), and images were acquired and analyzed with the IPLab spectrum program (Signal Analytics, Vienna, VA). The bacteria recovered from macrophages did not give rise to large inclusions. Thus, to define the relative size (in square pixels) of noninfectious bacteria, HeLa cells were also incubated with ultraviolet-inactivated chlamydiae, prepared as previously described (47); all fluorescent particles with a size larger than the noninfectious bacteria were then counted as infectious vacuoles. At least 10 separate fields containing an average of 200-300 HeLa cells were counted per sample, and the experiment was repeated on three separate occasions.
Demonstration of permeabilization by fluorescence microscopy. Cell permeabilization was assessed by observing the differential uptake of LY in infected or uninfected macrophages growing on coverslips that had been treated with 5 mg/ml LY with or without 5 mM ATP at 37°C for 10 min. Coverslips were then rapidly rinsed with PBS at room temperature, and samples were examined immediately with the Zeiss microscope. Acquired images were then analyzed with the IPLab spectrum program.
To distinguish fluorescently positive (permeabilized) cells from nonfluorescent (nonpermeabilized) cells, a threshold of fluorescence intensity was defined with the use of a sample that had been exposed to LY in the absence of ATP. To identify permeabilized cells in images, the IPLab spectrum program was used to quantify the number of cells per field that had fluorescence intensities higher than the threshold level. At least 50 cells per sample were analyzed, and the percentage of permeabilized (LY positive) cells in the different samples was calculated.Intracellular Ca2+ measurements. Cells were loaded with 6 µM fura 2-AM (Molecular Probes) for 1 h at room temperature in RMPI culture medium. The cells were then washed and perfused with PBS supplemented with 1 mM CaCl2 by using a three-compartment superfusion chamber whose bottom is formed by the coverslip containing the cells (26). Intracellular Ca2+ concentrations in groups of 20-40 cells were monitored continuously at 37°C with 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 logarithm of the intracellular Ca2+ concentration, was determined every 100 ms.
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RESULTS |
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Apoptosis of macrophages due to treatment with extracellular
nucleotides.
The effects of ATPo on the viability of J774 macrophages
were characterized after a 30-min incubation of the macrophages with ATPo, followed by a 6-h incubation in the absence of
ATPo. Apoptosis was then assayed by measuring nuclear
condensation in PI-stained macrophages, as described in MATERIALS
AND METHODS. There was very little effect at
ATPo concentrations below 2 mM, but over one-third of the
cells were apoptotic after exposure to 5 mM ATPo (Fig.
1). The concentration dependence of
ATPo-induced apoptosis in J774 cells is thus consistent
with previous reports on the ability of ATPo to trigger
apoptosis in macrophages, microglial, mesangial, and dendritic cells
(13, 16, 22, 42, 57). Previous studies have also confirmed
the apoptotic nature of macrophage cell death by showing DNA
fragmentation and morphological features of apoptosis due to
ATPo treatment (33, 38, 42).
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Effect of P2Z/P2X7 receptor agonists and antagonists on
macrophage apoptosis.
The specificity of the receptor was confirmed by evaluating the effect
of various agonists and antagonists of the P2Z/P2X7 receptor. BzATP is a selective agonist of the receptor
(19), and Fig. 4A
shows that it induces apoptosis of J774 cells. BzATP is believed to
induce permeabilization of cells better than ATP, but both nucleotides
trigger apoptosis (13, 69).
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Effect of ATPo on viability of intracellular
chlamydiae.
It has previously been shown that intracellular mycobacteria are killed
if the host macrophages undergo apoptosis induced by ATPo,
but not if induced by ligation of surface CD95, while treatment of
extracellular mycobacteria with ATPo has no toxic effect
(33, 34). To determine if ATPo might have any
effect on the viability of intracellular chlamydiae, we infected J774 macrophages with C. psittaci for 24 h, incubated the
infected cells with 5 mM extracellular nucleotides (ATP, UTP, ADP, AMP) for 30 min and, after incubating the infected cells for an additional 6 h at 37°C in the absence of exogenous nucleotide, harvested the bacteria from both the supernatant and adherent infected cells. Infectious activity was then determined by infecting HeLa cells with
the bacteria recovered from macrophages and then quantifying infectious
units as described in MATERIALS AND METHODS. Of the four
nucleotides tested, only ATPo had a significant effect on the ability of the chlamydiae to reinfect new host cells (Fig. 5).
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Effect of Chlamydia infection on ATPo-induced
apoptosis.
It has recently been shown that infection with Chlamydia
protects infected cells against apoptosis triggered by external
ligands, such as anti-Fas antibodies or staurosporine
(20). To evaluate whether Chlamydia infection
may also protect against apoptosis induced by ATPo, we
infected J774 cells with C. psittaci and then measured the
ability of ATPo to induce apoptosis by using the standard
6-h assay described above. Because Chlamydia infection can
also lead to apoptosis after 1 day of infection, becoming especially
significant after 2 days of infection (47), we infected J774 cells for 24 h before incubating with ATPo. At
this length of infection, very little if any apoptosis was measured as
a result of the infection itself, compared with apoptosis induced by
ATPo (Fig. 6A).
However, preinfecting macrophages for 1 day inhibited almost all the
apoptosis due to subsequent incubation with ATPo (Fig.
6A). To exclude the possibility that a toxic component of Chlamydia may have inhibited ATPo-mediated
apoptosis, in the absence of infection, the chlamydiae and macrophages
were incubated in the presence of chloramphenicol, which inhibits
protein synthesis of chlamydiae but not the eukaryotic host cell
(54). Under these conditions, ATPo induced as
much apoptosis as in the complete absence of bacteria (Fig.
6B).
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Effect of Chlamydia infection on ATPo-induced permeabilization and Ca2+ fluxes. Chlamydia infection was reported to protect cells against apoptosis due to inhibition of caspase activation in infected host cells (20). To investigate whether the infection may also inhibit apoptosis at some level other than the caspases, we have studied the effect of the infection on the activity of the P2Z/P2X7 receptor.
One of the early events following P2Z/P2X7 receptor engagement is permeabilization of the plasma membrane via the opening of cation-specific ion channels and nonspecific pores that conduct a range of small solutes of molecular mass <900 (11, 12, 60). We have therefore measured permeabilization of J774 cells to the fluorescent dye LY in the presence and absence of infection. In uninfected macrophages, ~70% of cells were loaded with LY after a 10-min incubation with 5 mM ATPo (Fig. 7), measured by fluorescence microscopy as described in MATERIALS AND METHODS. ATPo could induce permeabilization to LY in less than half of these cells after a 24-h infection with C. psittaci (Fig. 7), suggesting that the infection inhibits some of the pore-forming activity of the receptor. The infection apparently affected the permeability of the plasma membrane to LY, because there was no difference in the level of micropinocytosis of LY in infected or uninfected cells (not shown).
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DISCUSSION |
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A number of macrophage cell lines and primary monocytes and macrophages are known to undergo apoptosis after treatment with ATPo (33, 38, 42), and the J774 macrophage cell line has been extensively used to show that ATPo can permeabilize the plasma membrane through engagement of the P2Z/P2X7 purinergic receptor. Using agonists and antagonists specific for the P2Z/P2X7 receptor, we have shown here that ATPo also induces apoptosis of J774 macrophages through engagement of the same receptor. When Chlamydia-infected J774 cells are incubated with ATPo for 30 min, the bacteria recovered 6 h later have significantly reduced infectious activity. Because other extracellular nucleotides have no effect on Chlamydia viability, we have concluded that ATPo displays its bacteriocytic effect via the P2Z/P2X7 receptor. However, at the present we cannot determine whether the chlamydiae die as an indirect consequence of macrophage apoptosis or whether the P2Z/P2X7 receptor may have an effect directly on the bacteria, independently of host cell apoptosis. In previous studies with macrophages infected with mycobacteria (33, 34, 38), it was shown that apoptosis induced by ATPo, but not by Fas ligation, resulted in loss in viability of most of the intracellular mycobacteria through a process involving active killing, rather than bacteriostatis, of the mycobacteria.
Different species and strains of Chlamydia can infect
monocytes and macrophages, although as a rule the infection is less efficient than in epithelial cells (31). It has been
reported that the infection of C. trachomatis in human
monocytes and macrophages in vitro is not productive but that the
bacteria are transcriptionally active and resemble bacteria present in
vivo during persistent infections (24). These chlamydiae
could therefore serve as a source of bacteria for subsequent
infections. Activation of Chlamydia-infected epithelial
cells with interferon- (IFN-
) has also been used as an in vitro
model for persistence of infection (5, 6).
Most of the damage resulting from Chlamydia infection is due
to the inflammatory response of the host, rather than to the infection
itself (53). ATPo is also thought to be
involved in initiating proinflammatory reactions (16), and
it has been shown that the P2Z/P2X7 receptor regulates
processing of the inflammatory cytokine interleukin-1 by
lipopolysaccharide-treated macrophages (21, 27, 32).
During an inflammatory response following infection with
Chlamydia, IFN-
is produced (53), which
activates macrophages and increases expression of the
P2Z/P2X7 receptor (28, 29). A primary role of
P2Z/P2X7-dependent apoptosis may thus be used to lyse
macrophages infected with intracellular pathogens such as chlamydiae or
mycobacteria during inflammatory responses in such a way that the
pathogens are not released from the host cells in viable form.
While the immune system may use inflammatory mediators such as ATPo to eliminate intracellular pathogens, Chlamydia also attempts to minimize the damage to itself by partially inhibiting ATPo-mediated apoptosis. It has recently been reported that chlamydiae can inhibit apoptosis due to external ligands such as Fas by blocking caspase activation (20), and we find that the bacteria can also interfere with apoptosis by decreasing the activity of the P2Z/P2X7 receptor. Because Chlamydia can also directly induce apoptosis (25, 47, 49), the bacteria appear to modulate host-cell apoptosis at several levels simultaneously. Most of these phenomena have been observed in vitro, and a role for the respective activities in vivo now awaits elucidation.
Chlamydia infection inhibits ATPo-induced permeabilization to LY only partially, consistent with the effects of the infection on ATPo-induced apoptosis. In both infected and uninfected macrophages, ATPo was able to trigger a rapid, transient rise in the intracellular Ca2+ concentration. We have recently shown that treatment of dendritic cells with 50 µM ATPo, a concentration that does not induce apoptosis, also causes the short-lived spike in intracellular Ca2+, but without the large, sustained increase observed afterward following treatment with 5 mM ATPo, which does cause apoptosis (13). In infected macrophages, 5 mM ATPo did cause a small, sustained increase in the long-lived component, compared with the basal level in untreated cells, but the sustained increase was significantly larger in uninfected cells. Thus the Ca2+ measurements are also in line with the partial inhibition of ATPo-apoptosis observed in infected macrophages.
The effects of infection on P2Z/P2X7 receptor activity have been previously characterized for two pathogens, mycobacteria and Pseudomonas aeruginosa. Mycobacteria inhabit macrophages as their preferential host cell, and ATPo-induced apoptosis of infected cells results in mycobacterial death (33, 38). Because host cell death is detrimental to the survival of the intracellular pathogens, the mycobacteria secrete ATP-scavenging enzymes such as ATPase to minimize the cytotoxic effect of extracellular ATP (66). Conversely, the extracellular pathogen P. aeruginosa secretes as yet uncharacterized factors that increase the sensitivity of macrophages to ATPo-induced cytolysis, presumably by modulating the activity of the P2Z/P2X7 receptor (67). The factors are produced by a mucoid strain of P. aeruginosa isolated from the lungs of a cystic fibrosis patient, but not in a nonmucoid laboratory strain, suggesting that the secreted enzymes may represent virulence factors used in vivo. It is thus interesting that chlamydiae protect the infected macrophage, which may be used as a vehicle for chlamydial persistence, while P. aeruginosa, which does not need to grow within macrophages, secretes factors that kill the same cells.
Finally, the cytotoxic activity of the P2Z/P2X7 receptor requires high doses of ATPo (100 µM-1 mM), raising the obvious question of how these nucleotide concentrations could be achieved in the extracellular space. A number of cell types, including platelets, are known to secrete ATP (19), and ATP is thought to be released from cytotoxic lymphocytes as part of their cytolytic arsenal (3). High local concentrations of ATPo might therefore be attained in the contact zone formed between a cytotoxic lymphocyte and its target cell. ATP can also be released from the cytosol of damaged cells, and cells infected with Chlamydia contain ATP concentrations that are two- to threefold higher than in uninfected cells (46). Transient release of ATP from dying infected cells could therefore engage the P2Z/P2X7 receptor of neighboring macrophages. It has been shown, in fact, that transient exposure to extracellular ATP is sufficient to trigger cytoplasmic Ca2+ fluxes (13, 52) and that cells need to be exposed to ATPo for only short periods of time (<15 min) for the cells to die several hours later (27, 38, 50). Because the intracellular ATP concentration in uninfected cells is 5-10 mM, it is thus conceivable that sufficiently high concentrations of ATPo to activate the P2Z/P2X7 receptor could be produced over short periods locally, such as from infected cells during inflammatory reactions.
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ACKNOWLEDGEMENTS |
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We are grateful to Philippe Souque and Vandir da Costa for excellent technical assistance.
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FOOTNOTES |
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These studies were supported by funds from the Institut Pasteur, Centre National de la Recherche Scientifique, Conselho Nacional de Desenvolvimento Cientifico e Tecnológico, Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro, Pronex, and Fundacão Universitaria Jose Bonifacio.
Address for reprint requests and other correspondence: R. Coutinho-Silva, Instituto de Biofisica, Universidade Federal do Rio de Janeiro, Bloco G, CCS, Cidade Universitaria, 21941-590 Rio de Janeiro, Brazil (E-mail: rcsilva{at}ibccf.biof.ufrj.br) or D. M. Ojcius, Institut Pasteur, Unité de Biologie Moléculaire du Gène, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France (E-mail address: ojcius{at}pasteur.fr).
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. Section 1734 solely to indicate this fact.
Received 17 May 2000; accepted in final form 8 August 2000.
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