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The Journal of Immunology, 2001, 166: 6754-6763.
Copyright © 2001 by The American Association of Immunologists

P2X7 Nucleotide Receptor Mediation of Membrane Pore Formation and Superoxide Generation in Human Promyelocytes and Neutrophils1

Byung-Chang Suh*, Jong-So Kim*, Uk Namgung*, Hyunjung Ha{dagger} and Kyong-Tai Kim2,*

* Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Hyoja-Dong, Pohang, Republic of Korea; and {dagger} Division of Life Sciences, Chungbuk National University, Cheongju, Republic of Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The P2X7 receptor, which induces cation channel opening imparting significant permeability to Ca2+ and pore formation with changes in the plasma membrane potential, has been known to be rather restrictedly expressed in cells of the macrophage lineage including dendrites, mature macrophages, and microglial cells. However, we show here that the P2X7 receptor is also expressed in cells of granulocytic lineage such as HL-60 promyelocytes, granulocytic differentiated cells, and neutrophils. Exposure of these cells to 2',3'-O-(4-benzoyl)benzoyl-ATP (BzATP) triggered intracellular Ca2+ rise through the mediation of phospholipase C-independent and suramin-sensitive pathways. BzATP also induced depolarization of the plasma membrane in the absence of extracellular Ca2+, whereas it hyperpolarized the cells in the presence of external Ca2+, probably in part through the activation of Ca2+-activated K+ channels. However, the hyperpolarization phenomenon was markedly attenuated in differentiated HL-60 cells and neutrophils. RT-PCR and Northern blot analysis revealed the presence of P2X7 receptors on both HL-60 and neutrophil-like cells. This was further confirmed by pore formation through which the uptake of Lucifer yellow and YO-PRO1 occurred on BzATP treatment. BzATP stimulated in a concentration-dependent manner the production of superoxide in differentiated HL-60 cells via a pathway partially dependent on extracellular Ca2+. Moreover, in human neutrophils, BzATP was a more effective inducer of superoxide generation than PMA. Taken together, this is a first demonstration of the expression of P2X7 receptors on neutrophils, which shows that the receptor is functionally involved in the defense mechanism by activation of the respiratory burst pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Extracellular ATP evokes many physiological responses in various cell types. Among them are such different phenomena as platelet aggregation, neurotransmission, and muscle contraction (1). In the immune system, ATP plays important roles in leukocyte functions. They include DNA synthesis, blastogenesis, cell-mediated killing, apoptosis by priming superoxide release, and degranulation of mast cells (2, 3). These various effects of ATP are mediated by the interactions of the nucleotide with P2 receptors on the plasma membrane; however, the receptors involved in these actions are not fully characterized either pharmacologically or physiologically. The P2 purinoceptors can be broadly classified into metabotropic P2Y and ionotropic P2X receptors (4). Release of calcium from intracellular stores is generally thought to result from the activation of P2Y receptors which belong to the G protein-coupled receptor family. P2X receptors are ligand-gated ion channels, currently thought to be multisubunit proteins with two transmembrane domains per subunit (5, 6). There is biochemical evidence showing that P2X receptor subunits can form heterooligomeric channel assemblies, except for the P2X7 subtype which preferentially forms homooligomeric complexes (6). P2X channels are highly permeable to calcium and mediate rapid depolarization of the cells when activated by ATP (7).

The P2X7 receptors cloned from rat macrophages and brain proved the identity of cytolytic P2Z receptors previously described for mast cells, macrophages, lymphocytes, and erythrocytes (8, 9). A unique feature of cloned P2X7 and endogenous P2X7-like receptors is that, whereas under physiological conditions these receptors function like other P2X receptors in that they are selectively permeable to small cations only in the continued presence of ATP and when divalent cation levels are low, the cation channel can be converted to a pore permeable to small molecules with a molecular mass of up to 900 Da as well as to ions (10). This increase in membrane permeability results finally in the induction of apoptotic processes with a concomitant activation of the rapid release of proinflammatory cytokine IL-1{beta} (11, 12). The recombinant receptor of rat P2X7 responds to agonists eliciting inward currents with a potency order of 2',3'-O-(4-benzoyl)benzoyl-ATP (BzATP)3 > ATP > 2-methylthio-ATP (2MeSATP) > adenosine 5'-O-(3-thiotriphosphate) > ADP, although there are marked differences in the maximal effective concentrations between rat and human P2X7 receptors (2, 10).

The present work tested P2X receptors in HL-60 promyelocytes and human neutrophils. HL-60 cell is a human cell line derived from peripheral blood leukocytes of a patient with acute promyelocytic leukemia (13). Addition of DMSO to the growth medium induces differentiation of these progenitors into morphologically and functionally mature neutrophils (14, 15). Neutrophils play a crucial role in host defenses by migrating to the site of the infection and eliminating foreign bodies from the tissue. This complex process involves multiple steps including transmembrane and intracellular signaling which causes directed motility and cell activation and culminates in phagocytosis, degranulation, and superoxide production (16). The biological effect of ATP on neutrophils includes the stimulation of cell proliferation and differentiation as well as modulation of proinflammatory activities. Previous studies of P2X1, P2Y2, and P2Y11 receptors in HL-60 cells indicated that the expression levels of the purinergic receptors underlie the significant changes occurring during hemopoietic differentiation (17, 18, 19). However, the whole pattern of P2 purinoceptor expression and their functional effect in HL-60 promyelocytes and neutrophils have not yet been thoroughly analyzed (20).

In the present study, we found that HL-60 promyelocytes and human neutrophils express another subtype of purinergic receptor, P2X7, and that the activation of this receptor is coupled to the induction of membrane depolarization, pore formation, and reactive oxygen species production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
HL-60 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). To exclude the possibility of a HL-60 batch-specific effect of extracellular nucleotide on permeability, HL-60 cells obtained from Drs. P. G. Suh and S. H. Ryu (POSTECH, Pohang, Korea) were also used for comparison. KN-62, {alpha},{beta}-Methylene-ATP ({alpha},{beta}-MeATP) and {beta},{gamma}-methylene-ATP ({beta},{gamma}-MeATP) were obtained from Research Biochemicals (Natick, MA). Fura-2 pentaacetoxymethyl ester (fura-2-AM), 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), Lucifer yellow, and bisoxonol (DiSBAC2(3)) were purchased from Molecular Probes (Eugene, OR). FK-506 was obtained from Calbiochem-Novobiochem (La Jolla, CA). Other reagents were purchased from Sigma (St. Louis, MO).

Culture of HL-60 cells

HL-60 cells were cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD) buffered with HEPES and sodium bicarbonate (pH 7.4) and supplemented with 10% (v/v) heat-inactivated bovine calf serum (HyClone, Logan, UT) and 1% (v/v) penicillin-streptomycin (Life Technologies) under a humidified atmosphere of 5% CO2 at 37°C. Fresh medium was added to culture flasks every 2 days, and the cells were subcultured once a week.

Isolation of human neutrophils

Human neutrophils were isolated from the peripheral blood of healthy donors, using dextran sedimentation and Ficoll-Plaque gradient centrifugation (21). Isolated cells were washed and resuspended in PBS-glucose buffer containing 2.6 mM KCl, 1.5 mM KH2PO4, 0.5 mM MgCl2, 136 mM NaCl, 8 mM Na2PO4, and 5.5 mM glucose, pH 7.4, and used immediately.

Measurement of intracellular Ca2+ level

Intracellular free Ca2+ concentration ([Ca2+]i) was determined using the fluorescent Ca2+ indicator fura-2-AM as previously described (22). Fluorescence ratios were taken by dual excitation at 340 and 380 nm and emission at 500 nm by an alternative wavelength-time scanning method. Calibration of the fluorescence signal in term of [Ca2+]i was performed according to the method of Grynkiewicz et al. (23). The cells were suspended in Locke’s solution composed of 154 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl2, 1.2 mM MgCl2, 10 mM glucose, and 5 mM HEPES buffer adjusted to pH 7.4. Sulfinpyrazone (250 µM) was added to all solutions to prevent dye leakage (24). For extracellular Ca2+-free Locke’s solution, CaCl2 was omitted and 100 µM EGTA was included.

Measurement of inositol 1,4,5-trisphosphate (IP3)

To determine the IP3 production, the HL-60 cells were stimulated with agonist for the indicated periods of time, and the reaction was terminated by aspirating the medium followed by addition of 0.3 ml ice-cold 15% (w/v) TCA containing 10 mM EGTA. The extract was then transferred to an Eppendorf tube and the TCA was removed by extractions with diethyl ether four times. Finally, the extract was neutralized with 200 mM Trizma base, and its pH was adjusted to 7.4. As we previously described in detail, IP3 concentration in the cells was determined by [3H]IP3 competition assay in binding to IP3-binding protein (25). The IP3 concentration in the sample was determined based on a standard curve and expressed as picomole per milligram protein. The IP3 binding protein was prepared from bovine adrenal cortex according to the method of Challiss et al. (26).

Measurement of membrane potential with bisoxonol

Changes in the membrane potential were monitored using a fluorescent potential-sensitive anionic dye, bisoxonol (DiSBAC2(3)) as reported by Barry and Cheek (27) with minor modifications. Briefly, HL-60 cells, after preincubation for 1 h at 37°C in incubation solution (125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM Na2HPO4, 5.5 mM glucose, 5 mM NaHCO3, 20 mM HEPES, and 1 mM CaCl2, pH 7.4), were washed and resuspended at a density of 1.5 x 106 cells/ml. The cells were then incubated with 300 nM bisoxonol for 10 min at 37°C before the addition of stimulants. Fluorescence was measured at an excitation wavelength of 540 nm and an emission wavelength of 580 nm. In Ca2+-free solution, CaCl2 was omitted and 200 µM EGTA was included.

Changes in plasma membrane permeability

BzATP-dependent increases in plasma membrane permeability were measured with the help of the extracellular fluorescent tracer Lucifer yellow (Molecular Probes). Harvested cells were incubated for the indicated time periods at 37°C in Locke’s solution containing 250 µM sulfinpyrazone and 1 mg/ml Lucifer yellow and stimulated with 300 µM BzATP. At the end of the stimulation, the cells were washed twice with Ca2+-free Locke’s solution and once with Ca2+-containing Locke’s solution. A 125-W xenon lamp filtered to emit 450 nm light illuminated the cells. Fluorescence emission was filtered to >520 nm and monitored with a CCD camera (Hamamatsu Photonics, Hamamatsu, Japan). Images were captured and processed using an imaging processing system (Universal Imaging, West Chester, PA) equipped with a 20x objective. Total cellular uptake of Lucifer yellow per 1 x 106 cells was measured at the excitation wavelength of 450 nm and emission wavelength of 521 nm using a spectrofluorophotometer (28).

RT-PCR analysis

Total RNA was extracted from HL-60 cells or human neutrophils using acid guanidinium thiocyanate-phenol-chloroform (Tri-reagent; Molecular Research Center, Cincinnati, OH) (29). One microgram of total RNA was added to 0.5 µg oligo(dT) in diethyl pyrocarbonate-treated water, incubated at 70°C for 5 min, and then cooled at 4°C for 5 min. A total of 1 mM concentrations of each of the four dNTPs, 5 µl 5x reverse transcription buffer, and 200 U superscript II reverse transcriptase (Life Technologies) were added, and the reactions were incubated at 42°C for 1 h and at 75°C for 10 min and then stored at 4°C. For PCR amplification, an aliquot of the cDNA synthesis reaction was added to a reaction buffer containing 1 mM dNTPs, 1 mM oligonucleotide primers, and 2 U Taq DNA polymerase (Promega, Madison, WI). Forty temperature cycles were conducted as follows: denaturation at 95°C for 1 min; annealing at temperatures specific for each set of primers for 1 min; and extension at 72°C for 1 min in a Minicycler (MJ Research, Watertown, MA). The resultant amplification products were analyzed by agarose gel electrophoresis.

The following oligonucleotide primers were used for amplification of P2X7: sense primer, 5'-agatcgtggagaatggagtg-3' (bases 247–266); antisense primer, 5'-ttctcgtggtgtagttgtgg-3' (bases 626–645) (GenBank accession no. NM002562). Amplified PCR products were sequenced according to the manufacturer’s instructions (Amersham Life Science, Cleveland, OH).

Northern blotting

Total RNA (15 µg) was resolved by electrophoresis through 1% agarose gels containing 0.66 M formaldehyde and transferred onto nylon membranes (ICN, East Hills, NY). The blots were then probed with a cDNA fragment of P2X7 (bases 247–645) labeled with [{alpha}-32P]dCTP by the random primer extension method. The hybridization proceeded at 65°C in a solution containing 10% polyethylene glycol, 7% SDS, 10 mM EDTA, 250 mM NaCl, 85 mM Na2HPO4 (pH 7.2), denatured salmon sperm DNA, and the probe (5 x 105 cpm/ml). After hybridization, the blots were washed briefly twice in 1x SSC, 0.1% SDS at 65°C and twice in 0.1% SSC at room temperature. After having been washed extensively, the membranes were mounted for autoradiography with the use of a BAS 1000 film (Fuji Film, Minami-Ashigara-Shi, Japan). The filter was then reprobed with a cDNA to the human ribosomal large subunit protein 32 (RPL-32).

Measurement of intracellular reactive oxygen species production

The production of intracellular reactive oxygen species like superoxide and hydrogen peroxide was determined based on changes in fluorescence of DCFH-DA, an oxidation-sensitive fluorescence probe, using a slight modification of a previously published procedure (30, 31). Briefly, the cell suspension was incubated in fresh serum-free RPMI 1640 with 2 µM DCFH-DA at 37°C for 40 min under continuous stirring. The loaded cells were then washed twice with Locke’s solution. Then 2 x 106 cells were placed into a cuvette in a thermostatically controlled cell holder at 37°C and stirred continuously. Fluorescence was excited at 488 nm, and emission was recorded at 530 nm. The change in fluorescence intensity was monitored.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phospholipase C (PLC)-independent [Ca2+]i rise induced by extracellular nucleotides in HL-60 promyelocytes

Many previous studies have shown that application of ATP to HL-60 promyelocytes results in a prominent increase in [Ca2+]i through the activation of PLC-coupled P2Y2 and/or P2Y11 receptors (18, 19). However, we found that extracellular ATP could also elevate the [Ca2+]i without inositol phosphate generation, but instead in an extracellular Ca2+-dependent manner. Fig. 1GoA shows that in the presence of 2.2 mM extracellular Ca2+, ATP triggered an increase in the [Ca2+]i at a maximal effective concentration of ~3 µM. Higher concentrations of ATP, above 3 µM, raised the peak [Ca2+]i in an amount comparable with that of 3 µM ATP, but the peak rapidly decreased to the basal level as the concentration of ATP was increased, which is caused by the differential activation of protein kinases as we have previously reported (32). In contrast, in the absence of extracellular Ca2+, the ATP effect on the [Ca2+]i increase was detectable above at least ~1 µM ATP, continuously increasing in the presence of up to 300 µM ATP. The concentration dependency is shown in Fig. 1GoB, in which the ATP-mediated [Ca2+]i rise in the presence of external Ca2+ and the Ca2+ mobilization in the absence of external Ca2+ are evoked with an EC50 seen at 85 ± 17 nM and 10.2 ± 2.5 µM, respectively. ATP also elevated IP3 generation in a concentration-dependent manner (Fig. 1GoC). The IP3 generation was barely detected at 1 µM ATP, and a significant elevation was only seen at 10 µM followed by a continuous increase of up to 300 µM ATP, which is comparable with the data of the Ca2+ release in the absence of external Ca2+ but not with those of the total [Ca2+]i rise under 2.2 mM extracellular Ca2+. The data suggest that extracellular ATP may evoke a [Ca2+]i rise also, through a PLC-independent pathway by inducing Ca2+ influx from the extracellular space, although PLC-dependent [Ca2+]i rise remains prominently involved on stimulation of the cells with ATP concentrations higher than 1 µM.



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  		FIGURE 1. Effects of extracellular ATP on the [Ca2+]i rise and IP3 generation in HL-60 promyelocytes. A, Typical patterns of [Ca2+]i rise in cells treated with various concentrations of ATP in the presence or absence of extracellular Ca2+. Ca2+ profiles shown are from a typical experiment. B, Concentration-dependent effect of ATP on total [Ca2+]i rise and Ca2+ mobilization. Fura-2-AM-loaded cells were stimulated with various concentrations of ATP and the peak internal Ca2+ level was measured as described in Materials and Methods. C, Concentration-dependent IP3 generation evoked by ATP in HL-60 cells. The cells were treated with various concentrations of ATP for 15 s, and the reaction was stopped by addition of 15% (v/v) TCA containing 10 mM EGTA. The IP3 generation was measured as described in Materials and Methods. The data are means ± SEM of five independent experiments performed in triplicate.

 
BzATP-mediated Ca2+ influx through ionotropic P2X receptors

To investigate which type of purinoceptor was involved in the ATP-mediated and PLC-independent [Ca2+]i rise, we determined the effects of sequential treatments of the cells with P2 receptor selective ATP analogues. Fig. 2GoA shows that treatment with a maximal concentration of ATP (100 µM) almost completely prevented a subsequent BzATP- and UTP-mediated increase in [Ca2+]i indicating that all the P2 receptors had been desensitized by the ATP treatment. Furthermore, BzATP blocked a subsequent 2MeSATP response, whereas ATP still triggered another increase in [Ca2+]i. However, treatment of the cells with 2MeSATP partially decreased the BzATP-mediated [Ca2+]i rise, although it did not effect a subsequent UTP-stimulated Ca2+ response. These data suggested that a novel P2 receptor responsive to BzATP and less effectively to 2MeSATP was present on the cells.



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  		FIGURE 2. BzATP-mediated [Ca2+]i rise in HL-60 cells. A, Fura-2/AM-loaded cells were stimulated with 100 µM ATP, BzATP, UTP, 2 MeSATP, and {alpha},{beta}-MeATP in different combinational orders in the presence of 2.2 mM extracellular Ca2+. The experiments were conducted >10 times, and Ca2+ transients obtained from a typical experiment are presented. B, The HL-60 cells were stimulated with 100 µM BzATP initially in the absence of external Ca2+, and then 3 mM CaCl2 was added to the medium 2 min after agonist stimulation. C, Concentration-dependent effect of ATP on [Ca2+]i rise in cells treated with BzATP. Cells were treated with 100 µM BzATP for 5 min and then stimulated with various concentrations of ATP. Inset, The experiment was conducted more than three times, and the results are presented. D, Effect of suramin on the BzATP-stimulated [Ca2+]i rise. The cells were treated with various concentrations of suramin for 3 min and then stimulated with 100 µM BzATP. Each point is the mean ± SEM of three independent experiments in triplicate.

 
Previously, it was reported that the P2X1 receptor was present on HL-60 cells and that its expression was prominently increased in granulocytic differentiated cells (17). However, the P2X1 receptors were not involved in the ATP-induced Ca2+ response, because the selective P2X1 agonist {alpha},{beta}-MeATP slightly increased the [Ca2+]i and did not inhibit a subsequent BzATP- and ATP-stimulated increase in [Ca2+]i. Interestingly, the BzATP- and the 2MeSATP-stimulated [Ca2+]i rises were maintained along with the stimulation in the continuous presence of agonist. In addition, BzATP had no effect on Ca2+ mobilization in the absence of extracellular Ca2+, but it prominently triggered Ca2+ influxes (Fig. 2GoB). Table IGo shows that BzATP has little effect on IP3 generation, whereas ATP and UTP significantly increased the IP3 level. 2MeSATP and {alpha},{beta}-MeATP showed detectable IP3 generation only at higher concentrations (>100 µM). The concentration-dependent Ca2+ elevation on ATP treatment after BzATP treatment in Fig. 2GoC shows that the subsequent ATP treatment induced a rise in [Ca2+]i at a potency comparable with that of the Ca2+ release or IP3 generation (see Fig. 1Go). Fig. 2GoD shows that the BzATP-response was inhibited by suramin treatment in a concentration-dependent manner. Incubation with KN-62 (50 nM) also decreased the BzATP responses by ~35–40% (data not shown). Above the concentrations used, KN-62 (50 nM) or suramin (50 µM) had a nonspecific effect on the plasma membrane and increased the basal [Ca2+]i level. Thus, the results indicated that the BzATP-stimulated responses were the consequence of a suramin- or KN-62-sensitive P2X purinoceptor activation. Taken together, these results suggest that the ionotropic P2X7 receptor, which is specifically sensitive to BzATP and 2 MeSATP and prominently induces Ca2+ influx, is expressed and involved in maintaining the sustained [Ca2+]i level during the stimulation of HL-60 cells with ATP.


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  		Table I. Effect of ATP and its analogues on IP3 generation in HL-60 promyelocytes1

 
BzATP-mediated membrane depolarization of HL-60 promyelocytes

One of the earliest changes occurring at P2X7 receptor activation is plasma membrane depolarization due to a fast transmembrane cation influx (33). Fig. 3Go shows that this response is also evoked in BzATP-stimulated HL-60 cells. Surprisingly, however, although the larger depolarization in response to BzATP was anticipated, BzATP hyperpolarized the cells in normal Locke’s solution containing 2.2 mM CaCl2, although the addition of 30 mM K+ as control still triggered depolarization of the cell membrane (Fig. 3GoA). Thapsigargin and ionomycin also evoked membrane hyperpolarization.



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  		FIGURE 3. Effect of BzATP on HL-60 plasma membrane potential. A, HL-60 cells pretreated with vehicle or 50 nM charybdotoxin (ChTX) for 5 min were incubated at 37°C in 1 mM Ca2+-containing (normal) or Ca2+-free solution at a concentration of ~1 x 106 cells/ml in the presence of 200 nM bisoxonol. BzATP (100 µM ), 1 µM thapsigargin (Tg), or 1 µM ionomycin (Iono) was applied for 2 min, and 30 mM KCl was used as the control. B, BzATP concentration dependency of plasma membrane depolarization in Ca2+-free solution. Bisoxonol-loaded cells were treated with various concentrations of BzATP, and the net depolarization level is presented as percent of the 30 mM KCl-induced response. C, The cells were stimulated with various ATP analogues (100 µM), and the depolarization level is presented. ATP{gamma}S, adenosine 5'-O-(3-thiotriphosphate). The experiments were performed more than five times; each point is the mean ± SEM.

 
Previously, it has been reported that HL-60 promyelocytes express a K+-selective ion channel that is activated by a [Ca2+]i above 100 nM (35). We, therefore, examined the effect of the Ca2+-activated K+ channel inhibitor charybdotoxin on the hyperpolarization response. When the cells were treated with maximal effective concentrations of the toxin (50 nM) for 5 min, the subsequent BzATP-mediated hyperpolarization disappeared; instead, there occurred a slight depolarization. However, charybdotoxin partially decreased the thapsigargin- and ionomycin-stimulated hyperpolarization, suggesting that there might be another K+ channels insensitive to charybdotoxin. In the absence of extracellular Ca2+, BzATP substantially depolarized the cells to up to 40–50% of the 30 mM K+-mediated response, whereas the thapsigargin- and ionomycin-induced hyperpolarization remained, probably due to the intracellular Ca2+ mobilization. Fig. 3GoB shows the concentration-dependency of the BzATP effect on plasma membrane depolarization in a Ca2+-free solution. The EC50 was 62 ± 15 µM, which is comparable with values of the activation of hP2X7 receptors (10). In addition, the pharmacological efficacy for plasma membrane depolarization in Ca2+-free saline was BzATP >= ATP > 2 MeSATP, adenosine 5'-O-(3-thiotrisphosphate) >> {alpha},{beta}-MeATP, {beta},{gamma}-MeATP (Fig. 3GoC). This matches the potency order of the nucleotides on the P2X7 receptor (2, 10).

Expression of P2X7 receptors in HL-60 promyelocytes

The presence of P2X7 receptors in the neutrophil lineage was investigated by RT-PCR of mRNA prepared from control, DMSO-treated HL-60 cells and human neutrophils, using primers designed to specifically amplify a fragment of the human P2X7 receptor cDNA. As shown in Fig. 4GoA, amplified products of the expected sizes for P2X7 (399 bp) was detected in control and DMSO-treated cells. In addition, human neutrophils also revealed the expression of P2X7. As a control, a 320-bp DNA derived from {alpha}-tubulin mRNA was successfully amplified in each sample (data not shown). In addition, the nucleotide sequences of the amplified DNA products matched that of the human P2X7. Northern blot analysis using the RT-PCR product as a probe further demonstrated the expression of mRNA for P2X7 (Fig. 4GoB). A band of ~2 kb was detected in control and differentiated HL-60 cells. In contrast, it appears that the level of the P2X7 receptor mRNA transcription in DMSO-differentiated cells was much higher than that in the control cells, while the housekeeping gene RPL-32 was slightly decreased. Fig. 4GoC shows that BzATP stimulated the [Ca2+]i rise in both HL-60 cells and neutrophils. Interestingly, the BzATP-stimulated response increased during DMSO differentiation. A time course of the DMSO treatment shows that the BzATP-stimulated [Ca2+]i response increased with the length of time of the DMSO treatment for up to 4 days and was then followed by a decrease in the response (Fig. 4GoD). Taken together, our results indicate that P2X7 receptors are prominently expressed on HL-60 promyelocytes and neutrophils and that the expression level increased during granulocytic differentiation of the HL-60 cells.



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  		FIGURE 4. Expression of P2X7 mRNAs in the granulocytic lineage. Total RNA was extracted from HL-60 promyelocytes (CONT), granulocytic differentiated cells (DMSO), or human neutrophils for RT-PCR (A) and Northern blot (B) analysis as described in Materials and Methods. Human RPL-32 was used as a loading control. A typical result of three to five separate experiments is shown. C, Fura-2-AM-loaded cells were treated with 100 µM BzATP, and typical Ca2+ profiles are presented. D, BzATP-mediated [Ca2+]i rise in DMSO-differentiated HL-60 cells. The HL-60 cells were treated for the indicated times (days) with 1.25% (v/v) DMSO, and the net [Ca2+]i rise induced by 100 µM BzATP is presented. Data are means ± SEM of triple experiments

 
Membrane depolarization of neutrophils by BzATP

To study the effect of the P2X7 receptor stimulation on neutrophil membranes, we used the BzATP effect on membrane depolarization. Fig. 5GoA shows that the BzATP treatment slightly depolarized the cells regardless of the absence or presence of charybdotoxin in normal Locke’s solution, whereas it dramatically increased depolarization in the absence of extracellular Ca2+. The concentration dependence examined in Fig. 5GoB shows that BzATP evoked membrane depolarization with a similar potency as determined for the HL-60 cells (see Fig. 3GoB). Previously, it has been reported that granulocytic differentiation of HL-60 cells resulted in a decreased expression of Ca2+-activated K+ channels (34). BzATP had consistently little effect on hyperpolarization in normal Locke’s solution of differentiated HL-60 cells, whereas it substantially depolarized the membrane in Ca2+-free solution as in the control cells. Therefore, our results suggest that the phenomenon displayed by BzATP on neutrophils might be indeed due to a lower expression of Ca2+ activated-K+ channels.



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  		FIGURE 5. Effect of BzATP on membrane potential in granulocytes. A, Human neutrophils pretreated with vehicle or 50 nM charybdotoxin (ChTX) for 5 min were incubated at 37°C in 1 mM Ca2+-containing (normal) or Ca2+-free solution at a concentration of ~1 x 106 cells/ml in the presence of 200 nM bisoxonol. BzATP (100 µM) was applied for 2 min, and 30 mM KCl was added for the control. B, BzATP concentration dependency of the plasma membrane depolarization in Ca2+-free solution. Bisoxonol-loaded cells were treated with various concentrations of BzATP. The net depolarization level is presented as percent of the 30 mM KCl-induced response. Each point is the mean ± SEM of three independent experiments. C, HL-60 promyelocytes treated with vehicle (control) or 1.25% (v/v) DMSO for 5 days were stimulated with BzATP in Ca2+-containing (normal) or Ca2+-free solution. BzATP (100 µM) was applied for 2 min, and then 30 mM KCl was added for the control. The experiments were performed more than five times and a typical result is shown.

 
Microscopic characterization of the BzATP-mediated permeabilization of HL-60 cells

In various cell types, the P2X7 receptor was linked to the formation of membrane pores permeable to generally impermeable markers such as Lucifer yellow and YO-PRO1. We therefore evaluated the functional effect of the receptor by measuring the uptake of extracellular Lucifer yellow or YO-PRO1 after exposure of the cells to BzATP. In Fig. 6Go, we treated the cells with BzATP for 30 min in Ca2+-free Locke’s solution then measuring the Lucifer yellow entry into the cells. Most of the HL-60 promyelocytes were positive in terms of Lucifer yellow uptake, and this was even more intense for DMSO-differentiated cells (Fig. 6Go, F and H). Differentiated cells are smaller than the control cells and more irregular in cellular morphology (see Fig. 6Go, A–D). The time course of Lucifer yellow fluorescence uptake shows that a brief (15-min) treatment with BzATP already triggered a significant Lucifer yellow uptake which then increased for up to 1 h of incubation time. However, this does not seem to be the consequence of a nonspecific increase in plasma membrane permeability or due to a cytotoxic effect, because in up to 2 h of incubation no uptake of Lucifer yellow was detected in control cells (Fig. 6GoI). Fig. 7GoA shows that the BzATP treatment also induces the uptake of YO-PRO1, which was measured 30 min after adding BzATP and peaked at 1 h after the BzATP addition at ~2-fold the basal fluorescence. Differentiated HL-60 cells showed more uptake of YO-PRO1, ~1.5- to 2-fold over the undifferentiated cells (Fig. 7GoB), which is consistent with the up-regulation of the P2X7 mRNA expression along with the granulocytic differentiation.



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  		FIGURE 6. BzATP causes permeabilization of the plasma membrane in HL-60 cells. HL-60 cells treated with (A, B, E, and F) or without (C, D, G, and H) 1.25% (v/v) DMSO for 5 days were rinsed with Locke’s solution twice and incubated in Ca2+-free warm (37°C) solution containing 1 mg/ml Lucifer yellow and 250 µM sulfinpyrazone in either the absence (A, C, E, and G) or the presence (B, D, F, and H) of 100 µM BzATP. After 30 min, the cells were rinsed twice with Ca2+-containing Locke’s solution and photographed using a x40 objective. A–D, Bright-field photographs of HL-60 cells; E–H, fluorescence images of HL-60 cells. Bar = 25 µm. I, Time course of Lucifer yellow uptake in HL-60 promyelocytes. Cells (1 x 106) were treated with vehicle (control) or 100 µM BzATP for the designated times, and the fluorescence was measured using a spectrofluorophotometer as described in Materials and Methods.

 


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  		FIGURE 7. YO-PRO1 uptake in HL-60 promyelocytes. A, Time course of YO-PRO1 uptake and fluorescence (arbitrary units) in HL-60 promyelocytes in response to 100 µM BzATP. Aliquots of 1 x 106 HL-60 cells per Eppendorf tube were incubated with Ca2+-free Locke’s solution containing 5 µM YO-PRO1 and 250 µM sulfinpyrazone in the presence or absence of BzATP for the designated times at 37°C. B, Control and DMSO-differentiated cells were stimulated with vehicle or 100 µM BzATP for 60 min, and the fluorescence intensity was measured. Each time point is the mean ± SEM of experiments performed in triplicate.

 
P2X7 receptor-mediated production of reactive oxygen species

Neutrophils are considered functional participants in a number of inflammatory conditions such as parasite infections and atopic diseases (16). We therefore investigated the effects of the P2X7 receptor on the production of reactive oxygen metabolites in granulocytic differentiated HL-60 cells and neutrophils. Fig. 8GoA shows that BzATP treatment significantly evoked the generation of superoxides in a concentration-dependent manner in granulocytic-differentiated HL-60 cells. PMA treatment as a positive control also triggered prominently the generation of superoxides. Interestingly, BzATP was much more potent in generating superoxides than the general chemotactic factors fMLP and platelet-activating factor (PAF), which produced only a slight increase in fluorescence (Fig. 8GoB). Removal of extracellular Ca2+ significantly reduced the PMA- and BzATP-induced superoxide generation (Fig. 8GoC). Ca2+ readdition augmented the superoxide generation, although it failed to restore superoxide generation to the response induced in the presence of extracellular Ca2+ (Fig. 8DGo). In contrast, BzATP, fMLP, and PMA had no effect on the oxygen radical generation in undifferentiated HL-60 promyelocytes (data not shown). More interestingly, in neutrophils, BzATP exhibited a more potent capacity to generate superoxides than PMA (Fig. 9GoA). Under extracellular Ca2+-free conditions, PMA exhibited a similar potency to produce superoxides, whereas the response to BzATP was very much weaker (Fig. 9GoB). However, pretreatment of the cells with the inhibitor of Ca2+-calmodulin-activated phosphoprotein phosphatase FK-506 did not decrease but slightly enhanced the BzATP-mediated superoxide generation (Fig. 9GoC). The results therefore indicate that the activation of P2X7 receptors is linked to the production of superoxide in neutrophils through, at least in part, an extracellular Ca2+-dependent, and Ca2+-calmodulin-activated phosphoprotein phosphatase-independent pathway.



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  		FIGURE 8. BzATP-mediated production of reactive oxygen species in DMSO-differentiated HL-60 cells. DCFH/DA-loaded cells (2 x 106) were stimulated with 1 µM PMA (A), various concentrations of BzATP (A) 1 µM fMLP (B), or 300 nM PAF (B) in the presence of 2.2 mM extracellular Ca2+, and elevation of fluorescence was detected as described in Materials and Methods. C, The cells were treated with 1 µM PMA, 100 µM BzATP, and 1 µM fMLP in the absence of extracellular Ca2+. D, The cells were treated with 100 µM BzATP under Ca2+-free conditions followed by the addition of 3 mM CaCl2. The experiments were repeated at least four times and were reproducible. Typical fluorescence transients are presented.

 


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  		FIGURE 9. BzATP-mediated production of reactive oxygen species in human neutrophils. DCFH/DA-loaded cells (2 x 106) were stimulated with PMA (1 µM), BzATP (100 µM), or fMLP (1 µM) in the presence (A) or absence (B) of 2.2 mM extracellular Ca2+, and the elevation of fluorescence was detected as described in Materials and Methods. C, DCFH/DA-loaded cells pretreated with vehicle (0.2% DMSO) or FK-506 (1 µM) for 20 min were stimulated with BzATP (100 µM) in the presence of 2.2 mM extracellular Ca2+. The experiments were repeated three times, and typical fluorescence transients are presented.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Polymorphonuclear neutrophils are phagocytic cells specializing in host defenses against bacterial and fungal infections (35). They can emigrate from blood vessels to the site of infection, a process triggered by specific substances termed chemotactic factors, such as the PAF, arachidonate metabolite leukotriene B4, anaphylatoxin complement fragment C5a, IL-8, and a number of N-formylmethionyl peptides. These chemoattractants stimulate a variety of coordinated biochemical and cellular responses in neutrophils, including aggregation, phagocytosis of particles, production of cytotoxic and microbicidal superoxide radicals, and release of proteolytic enzymes from the granules. All this happens via specific surface receptors coupled to guanine nucleotide-regulatory proteins or ion channels (16, 36). In the present study, we demonstrated that extracellular nucleotides also modulate proinflammatory functions through the activation of P2X7 purinoceptors on human neutrophils and granulocytic differentiated HL-60 cells. However, although the P2X7 receptor is also expressed by undifferentiated HL-60 cells, reactive oxygen species were not detected on treatment of the undifferentiated cells with BzATP or PMA, indicating that differentiation to the neutrophil-like status is specifically required for the induction of the respiratory burst machinery.

It has been known that the P2X7 receptor is expressed primarily in mature macrophages of the myeloid lineage and a limited number of other cell types including parotid acinar cells, testis, and fibroblasts (8, 10, 33). However, this study suggests that P2X7 receptors are expressed on neutrophils as well as HL-60 promyelocytes and granulocytic differentiated cells. Our conclusion is based on several lines of evidence. First, BzATP triggers extracellular Ca2+ influx without IP3 generation and Ca2+ mobilization, and the currents are maintained for at least a few minutes during the continuous presence of the agonist, whereas P2X1 and P2X3 receptors activated by {alpha},{beta}-MeATP and {beta},{gamma}-MeATP are rapidly desensitized within 100 to 300 ms (6). Second, BzATP treatment induces depolarization of the plasma membrane, particularly in a Ca2+-free medium. In addition, the efficacy of various nucleotides able to induce depolarization correlates with the known P2X7 receptor responses. Third, BzATP stimulation causes a drastic increase in plasma membrane permeability to low molecular mass aqueous solutes such as Lucifer yellow and YO-PRO1. It has been determined that the P2X7 receptor activation induces nonselective pores to let molecules of up to 900 Da pass (10), a characteristic shared to a lesser degree with other P2X members; however, the pore structure has remained uncharacterized at present. Fourth, RT-PCR and Northern analysis showed that the mRNA for P2X7 receptors is expressed in human neutrophils as well as HL-60 cells. Therefore, we must conclude that BzATP-mediated responses in neutrophils and HL-60 cells are evoked through the mediation of P2X7 receptors and that the receptors are not limited to macrophage of the myeloid lineage but also expressed and functional in granulocytes.

Many studies have shown that multiple types of P2 nucleotide receptors are expressed by blood cells and that the expression levels of certain P2 receptor can be rapidly modulated during cell activation processes or during differentiation of hemopoietic progenitor cells (37). In HL-60 cells, three types of P2 receptors, P2Y2, P2Y11, and P2X1 were proved to be expressed, and among them the expressions of P2Y11 and P2X1 receptors are increased during monocytic and/or granulocytic differentiation, whereas P2Y2 expression remains unchanged (17, 18, 19). In the present report, we first demonstrated that P2X7 receptors are functionally expressed by HL-60 cells and that the expression level of the receptor increases on granulocytic differentiation. Several researchers have reported that extracellular nucleotides can activate inflammatory responses via the production of cytokines and superoxide radicals through the activation of purine and pyrimidine responsive receptors on granulocytic differentiated cells (38, 39). At present, although the physiological significance of the P2X7 receptor remains largely unknown, the changes in the expression levels of P2 receptors seem to be strictly regulated according to their functional capabilities.

Previous studies of human hemopoietic cell types have demonstrated the presence of P2X1 receptor mRNA in total human blood leukocytes and P2X1 receptor protein in PMA- and dibutyryl cAMP-differentiated HL-60 cells (17, 40). However, our data show that the selective agonist of the P2X1 receptor {alpha},{beta}-MeATP has little effect on the [Ca2+]i rise and membrane depolarization of undifferentiated HL-60 cells (Figs. 2Go and 3Go). This may be due to the lower expression level of the receptor in promyelocytes, although its level drastically increased on granulocytic differentiation (17). Recently, Clifford et al. (37) reported that the P2X1 receptor was not expressed in purified human neutrophils. Therefore, the P2X1 receptor may not have a critical role in the BzATP-mediated receptor signalings in HL-60 cells and mature human neutrophils.

In agreement with previous reports, we found that the activation of the P2X7 receptor triggers a potent plasma membrane depolarization (Figs. 3Go and 5Go). However, signals that produce elevation in [Ca2+]i hyperpolarized the membrane probably through the activation of Ca2+-activated K+ channels, because the selective blocker of the Ca2+-activated K+ channel, charybdotoxin, inhibits membrane hyperpolarization. This may be the result of the relative abundance of these ion channels on HL-60 promyelocytes (34). On the contrary, in granulocytic differentiated cells and neutrophils, the exposure to ligands produced depolarization of the membrane potential without significant hyperpolarization, which is consistent with previous results indicating that the expression of the K+-selective channels is suppressed in granulocytes (34, 41). However, mature macrophages largely do express K+ channels and reveal hyperpolarization on treatment with chemoattractants (42). Therefore, the results suggest that there may be differences in the Ca2+-based signalings between granulocytic and monocytic lineages. Additionally, there may be several types of Ca2+-activated K+ conductances in HL-60 cells, because charybdotoxin treatment does not completely remove the membrane hyperpolarization (Fig. 3Go) (43, 44).

Related to the P2X7 receptor activation is the ability of BzATP to induce superoxides in mature granulocytes. However, the mechanism by which BzATP triggers the activation of the signaling cascade that results in superoxide generation is still unclear. Previously, it has been proposed that the activation of PKC and/or of the small GTP-binding protein Rac is required to mediate the activation of the NADPH-oxidase complex (45). In addition, an excessive Ca2+ influx from the extracellular space, but not internal Ca2+ mobilization, seems also important for the generation of the oxygen radical cascade (46, 47). Thus, the BzATP-stimulated Ca2+ influx through nonselective cation channels might play an important role in oxygen radical generation. However, a simple rise in [Ca2+]i is unlikely to explain the BzATP-mediated superoxide generation, because the selective P2Y2 receptor agonist UTP does not trigger superoxide generation despite its ability to induce a transient increase in [Ca2+]i comparable with that of BzATP in HL-60 cells. Many studies showed that the stimulation of P2X7 receptors is associated with a marked increase in the activity of phospholipase D, an enzyme that has been linked to several leukocyte antimicrobial mechanisms, including phagocytosis, generation of reactive oxygen species, and granule secretion (48, 49). Recently, the activation of P2X7 receptors was also seen to trigger a strong activation of the c-Jun N-terminal kinase pathway, which primarily mediates inflammation signals and apoptosis (50). Therefore, although the BzATP-stimulated superoxide generation is not yet totally understood, several complex signaling systems may be involved in the activation of the respiratory burst assembly and production of reactive oxygen species.

Many cell types are readily killed by a sustained ATP stimulation in the millimolar range (9, 51). This cytotoxicity is one of the most striking consequences of the activation of the P2X7 receptor, although it has been suggested that P2X1 may also confer sensitivity to ATP (52). The biological significance of the P2X7 receptor to trigger a cytotoxic effect is not clear, but it may have a role in the elimination of unwanted cells during physiological or pathological cell and tissue turnover (9, 50). However, we did not see any unusual alterations in cell morphology of the HL-60 cells during up to 60 min of exposure to 100 µM BzATP, but prolonged incubation, longer than 1 h, or high BzATP concentrations, >1 mM, did evoke changes such as swelling and blebbing (data not shown).

In conclusion, BzATP induces Ca2+ influx, membrane depolarization, and pore formation in HL-60 cells by activating P2X7 purinoceptors. The present discoveries are particularly significant insofar as they show that the activation of the receptor is related to the generation of superoxide and that this response is also evoked in mature human neutrophils. The pharmacological and molecular data clearly show that the expression of P2X7 receptors increases during granulocytic differentiation of HL-60 cells.


   Acknowledgments
 
We thank G. Hoschek for editing the manuscript.


   Footnotes
 
1 This work was supported by the National Research Laboratory Program and Brain Science and Engineering Research Program supported by the Ministry of Science and Technology and Korea Science and Engineering Foundation and by The Korea Research Foundation and Brain Korea 21 Program sponsored by the Ministry of Education. Back

2 Address correspondence and reprint requests to Dr. Kyong-Tai Kim, Department of Life Science, POSTECH, San 31, Hyoja-Dong, Pohang 790-784, South Korea. E-mail address: ktk{at}postech.ac.kr Back

3 Abbreviations used in this paper: BzATP, 2',3'-O-(4-benzoyl)benzoyl-ATP; {alpha},{beta}-MeATP, {alpha},{beta}-methylene-ATP; {beta},{gamma}-MeATP, {beta},{gamma}-methylene-ATP; [Ca2+]i, intracellular free Ca2+ concentration; DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate; fura-2-AM, fura-2 pentaacetoxymethyl ester; IP3, inositol 1,4,5-trisphosphate; 2MeSATP, 2-methylthio-ATP triphosphate; PAF, platelet-activating factor; PLC, phospholipase C. Back

Received for publication November 27, 2000. Accepted for publication March 22, 2001.


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 Introduction
 Materials and Methods
 Results
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