Regulation of P2X7 nucleotide receptor function in human monocytes by extracellular ions and receptor density

Lalitha Gudipaty1, Benjamin D. Humphreys1, Gary Buell2, and George R. Dubyak1

1 Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106; and 2 Serono Pharmaceutical Research Institute, Geneva, Switzerland


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

P2X receptors function as ATP-gated cation channels. The P2X7 receptor subtype is distinguished from other P2X family members by a very low affinity for extracellular ATP (millimolar EC50) and its ability to trigger induction of nonselective pores on repeated or prolonged stimulation. Previous studies have indicated that certain P2X7 receptor-positive cell types, such as human blood monocytes and murine thymocytes, lack this pore-forming response. In the present study we compared pore formation in response to P2X7 receptor activation in human blood monocytes with that in macrophages derived from these monocytes by in vitro tissue culture. ATP induced nonselective pores in macrophages but not in freshly isolated monocytes when both cell types were identically stimulated in standard NaCl-based salines. However, ion substitution studies revealed that replacement of extracellular Na+ and Cl- with K+ and nonhalide anions strongly facilitated ATP-dependent pore formation in monocytes. These ionic conditions also resulted in increased agonist affinity, such that 30-100 µM ATP was sufficient for activation of nonselective pores by P2X7 receptors. Comparison of P2X7 receptor expression in blood monocytes with that in macrophages indicated no differences in steady-state receptor mRNA levels but significant increases (up to 10-fold) in the amount of immunoreactive P2X7 receptor protein at the cell surface of macrophages. Thus ability of ATP to activate nonselective pores in cells that natively express P2X7 receptors can be modulated by receptor subunit density at the cell surface and ambient levels of extracellular Na+ and Cl-. These mechanisms may prevent adventitious P2X7 receptor activation in monocytes until these proinflammatory leukocytes migrate to extravascular sites of tissue damage.

macrophage; extracellular ATP; inflammation; ligand-gated ion channel


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE P2X FAMILY OF NUCLEOTIDE receptors comprises seven genetically distinct subtypes, all of which function as ATP-gated cation channels (6, 23). The P2X7 receptor subtype, which corresponds to the functionally defined P2Z receptor (33), is distinguished by an extraordinarily low affinity for extracellular ATP, the presumed physiological agonist. Activation of P2X7 receptor channels triggers depolarization, Ca2+ influx, and rapid equilibration of Na+ and K+ gradients. However, on repeated or prolonged stimulation by ATP, P2X7 receptors additionally facilitate the induction of a nonselective pore that is permeable to inorganic ions and organic molecules (anionic and cationic) with masses (Mr) as large as 800 Da (29). The structural and functional nature of this nonselective pore is a subject of interest given the ability of extracellular ATP to rapidly induce necrotic or apoptotic death in cells that natively express the P2X7 receptor (4, 32). Recent studies indicate that other P2X receptor subtypes, including P2X2 and P2X4, can also activate a nonselective permeability when overexpressed in heterologous cell types (18, 19, 36).

Because individual P2X7 receptor subunits possess only two transmembrane-spanning segments, it is assumed that functional channels are oligomers of several individual subunits. This assumption is supported by studies showing that recombinant P2X7 receptor subunits can self-assemble during translation and processing into stable, detergent-resistant complexes (34). Although the mechanisms underlying nonselective pore formation by P2X7 receptors are unknown, induction of the pore may involve further multimerization of the P2X7 receptor subunits or a dynamic change in the selectivity filter of existing P2X7 channel complexes (18, 36). However, recent studies by Schilling and colleagues (30, 31) suggest that nonselective pore formation may involve intrinsic membrane proteins that are regulated by, but distinct from, the P2X7 receptor per se.

Functional analyses of natively expressed human P2X7 receptors have largely utilized monocyte-derived macrophages (8, 13), monocytic leukemia cell lines (16), or blood lymphocytes from patients with chronic lymphocytic leukemia (21, 38). Multiple studies have described the ATP-dependent induction of nonselective pores [permeable to ethidium+ (Mr = 314 Da) or YO-PRO2+ (Mr = 375 Da) ion] in human macrophages (3, 8, 14, 32). In contrast, such pores were not observed when human blood monocytes, which are the precursors of macrophages, were similarly stimulated with high concentrations of extracellular ATP under physiological ionic conditions (8, 14). Using a monoclonal antibody suitable for fluorescence-activated cell sorter (FACS) analysis, Buell et al. (2) demonstrated that immunoreactive P2X7 receptors are expressed in >80% of human monocytes. Similar studies have been recently reported by Gu et al. (13). Thus the inability of ATP to efficiently activate the nonselective pore in blood monocytes, despite the expression of plasma membrane P2X7 receptor subunits, indicates that posttranslational mechanisms limit P2X7 receptor-dependent pore formation until monocytes leave the blood, enter proinflammatory tissue spaces, and differentiate into macrophages. A similar lack of nonselective pore formation has been described in murine thymocytes that express P2X7 receptor mRNA and exhibit ATP-gated cation channel activity (16, 31).

Pore formation may require a threshold number of agonist-bound P2X7 receptor subunits, and the surface density of P2X7 receptors may be insufficient on circulating monocytes. Environmental factors such as pH, ionic strength, and ionic composition may additionally limit the ability of P2X7 receptor subunits to undergo the conformational changes required for induction/activation of the nonselective pores. Recent studies have shown that activation of recombinant or native P2X7 receptors by ATP and other nucleotide agonists is exquisitely sensitive to the ionic composition of the extracellular medium (13, 22, 24, 37). In this study, we have used human blood monocytes and monocyte-derived macrophages as a model system to demonstrate critical roles for receptor surface density and allosteric modulation by extracellular ionic composition in the induction of nonselective pores by natively expressed human P2X7 receptors.


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

Isolation of blood monocytes. Venous blood (20-30 ml) from healthy volunteers was collected into 4-5 ml of sterile acid-citrate-dextrose containing 100 mM disodium citrate and 128 mM D-glucose, pH 5. The citrated whole blood was diluted 1:1 with PBS, and 30-ml aliquots were layered over 13 ml of Histopaque-1077 (Sigma Chemical, St. Louis, MO) and centrifuged at 400 g for 30 min. Mononuclear leukocytes on top of the Histopaque layer were collected into PBS-citrate and centrifuged at 200 g. The supernatant containing >95% platelets was discarded, while the pellet comprising the peripheral blood leukocytes (PBML) was subjected to two more washes in PBS-citrate and finally resuspended in Iscove's modified DMEM plus 10% iron-supplemented newborn calf serum (Hyclone Laboratories, Logan, UT).

Experimental salt solutions. PBML, isolated as described above, were resuspended in one of the basic salt solutions (BSS) described in Table 1. For the experiments involving reduced concentrations of extracellular divalent cations, each BSS was adjusted to contain 0.5 mM CaCl2 and no added MgCl2.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Ionic composition of BSS

Antibodies. R-phycoerythrin-conjugated anti-CD14 (a monocyte marker) and FITC-conjugated anti-CD14, anti-mouse IgG (FITC conjugated), and anti-mouse IgG (R-phycoerythrin conjugated) were purchased from Sigma Chemical. The monoclonal antibody to the human P2X7 receptor was generated as previously described by Buell et al. (2) and provided by Serono Pharmaceuticals. The exact region of antibody recognition has not yet been determined.

Analysis of P2X7 receptor mRNA by RT-PCR. RT-PCR was carried out exactly as previously described (15). Primers specific for the human P2X7 receptor cDNA sequence [5'-GGCAGTTCAGGGAGGAATCATGG-3' (sense) and 5'-AAAGCGCCAGGTGGCATAGCTC-3' (antisense)] generated a 939-bp product. Commercial primers to human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Stratagene, La Jolla, CA) generated a 600-bp product. Products of PCR were electrophoresed on 1% agarose gels containing ethidium bromide and photographed. In control experiments, standard curves were generated using serial dilutions of the RT reactions as templates for PCR with each primer set. The linear range of the assay was thus determined for each primer set, and products from the original 20-µl RT were diluted (1:100 for P2X7 and 1:500 for GAPDH) into the final PCR volume to ensure nonsaturation of the PCR amplification.

Flow cytometric analysis of P2X7 receptor expression. PBML were isolated from whole blood as described above but were pelleted and resuspended (107 cells/ml) in PBS containing 4% BSA (FACS buffer). For experiments involving mature macrophages, the PBML were first plated and allowed to adhere for 3 h. After removal of the nonadherent lymphocytes, the monocytes were cultured for 1-9 days in Iscove's DMEM supplemented with 10% calf serum and 1% penicillin-streptomycin. In some experiments (specified below), recombinant human interferon-gamma (1,000 U/ml; Genentech) was included in the tissue culture medium. The adherent monocyte-derived macrophages were nonenzymatically detached by incubation at 37°C for 20 min using cell dissociation solution (Sigma Chemical). Monocytes or macrophages were then incubated in FACS buffer supplemented with human IgG (50 µg/ml) for 60 min on ice to block nonspecific binding of specific antibodies to Fc receptors. After addition of the monoclonal anti-human P2X7 antibody (1.7 µg/ml), the intact cell samples were rotated for 2 h at 4°C. The cells were centrifuged and washed three times in FACS buffer, resuspended in FACS buffer containing FITC-conjugated anti-mouse IgG (0.17 µg/ml), and incubated for 30 min at 4°C. They were then rinsed twice in FACS buffer and once in 0.02% sodium azide in PBS and fixed with 2% paraformaldehyde in PBS for 30 min. The fixed cells were then centrifuged and resuspended in FACS buffer for analysis. An EPICS XL-mCL flow cytometer (Coulter, Miami, FL) with an air-cooled argon ion laser operated at 15 mW was used to analyze fluorescence. The forward scatter was linear, but the side scatter, as well as the FITC and PE, were acquired by logarithmic amplification with excitation wavelength of 488 nm and emission band-pass filters at 530 and 575 nm, respectively. Before the data were acquired, live gates were set to obtain well-shaped cells and to exclude cellular debris. A sample of unstained PBML was used to set the gates for negative values. PBML were identified and gated on the basis of forward and side scatter and by the CD14-positive marker for monocytes and CD3/CD19-positive markers for lymphocytes. Acquisition of data from 10,000 cells was performed in list mode and processed using XL System 3.0 software with an attached Dell computer. The fluorescence distribution was displayed as a single histogram. Further analysis was done using WinList 4.0 software (Verity Software, Topsham, ME).

Analysis of P2X7-dependent pore formation by fluorescence microscopy. For microscopic analysis of dye uptake by human monocytes or macrophages, PBML were plated onto Labtek chambered slides (Nalge-Nunc) and allowed to adhere for 2-3 h at 37°C. The nonadherent cells (mostly lymphocytes) were washed off, and the adherent monocytes were assayed immediately or were tissue cultured for 9 days. For dye uptake assays, the parallel wells containing similar numbers of adherent cells were incubated in complete NaCl BSS containing 2 µM YO-PRO-1 (Molecular Probes, Eugene, OR) for 15 min at 37°C. The YO-PRO-1 medium was variously supplemented with no additions (as a control for basal dye uptake), 3 mM ATP (to test for P2X7-dependent pore formation), or 50 µg/ml digitonin (to nonselectively permeabilize all cells within each sample well). After the 15-min test incubation, the YO-PRO-1-containing BSS was removed and replaced with dye-free NaCl BSS (after 2 washes). The number of YO-PRO-1-positive cells within equivalent optical fields of each separate well was immediately assayed by fluorescence microscopy (Nikon Eclipse E600) using a standard fluorescein filter set.

Analysis of P2X7-dependent pore formation by flow cytometry. For flow cytometric analyses, freshly isolated PBML were resuspended in one of the BSS media containing 2 µM YO-PRO-1. Parallel samples of each cell suspension were stimulated with various concentrations of ATP or no additions (as controls) for 15 min at 37°C. The cells were then centrifuged, washed twice, resuspended in complete NaCl BSS, and immediately subjected to FACS analysis. Monocytes were gated as described for analysis of CD14 and P2X7 receptor expression. YO-PRO-1 uptake was measured using 488-nm excitation and 530-nm emission filters. The distribution and single cell fluorescence intensities were calculated as described above.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ATP-dependent induction of nonselective pores in blood monocytes vs. monocyte-derived macrophages. When stimulated with high concentrations of ATP (>1 mM), cells that express functional P2X7 receptors can become permeable to large organic dyes, such as YO-PRO2+ (Mr = 375 Da) and ethidium+ (Mr = 314 Da), which form highly fluorescent complexes on binding to intracellular nucleic acids. We compared the ATP-dependent accumulation of YO-PRO-1-nucleic acid complexes in freshly isolated human blood monocytes with that in monocytes that had differentiated into macrophages after 9 days of in vitro culture (Fig. 1). The plating densities were adjusted to contain similar numbers of monocytes or macrophages per optical field (~350 cells at ×20 magnification). This was verified by permeabilizing all cells within each well with digitonin in the presence of YO-PRO-1 and counting the number of fluorescent nuclei. The labeled nuclei were evenly dispersed in freshly isolated monocyte cultures. In contrast, macrophage nuclei were generally clustered within islands of aggregated single cells, while some nuclei were colocalized within multinucleate giant cells that are formed by the fusion of single macrophages. Such cell clusters and multinucleate giant cells typify in vitro cultures of monocyte-derived macrophages. ATP-dependent pore formation was assayed in parallel wells of monocytes or macrophages bathed in medium containing physiological concentrations of extracellular Na+, K+, Cl-, Ca2+, and Mg2+. When incubated for 15 min at 37°C in the absence of extracellular ATP, few monocytes (~2%) and only modest numbers of macrophages (~10%) accumulated YO-PRO-1. When 3 mM ATP was included in the YO-PRO-1 assay medium, >95% of the macrophages within each optical field exhibited strong cytoplasmic and nuclear fluorescence after the 15-min test incubation. In contrast, the same ATP challenge produced no significant change in the numbers of YO-PRO-1-labeled monocytes. Prolonging the incubation period (up to 30 min) did not increase YO-PRO-1 labeling of the monocyte samples (data not shown). These observations, which are similar to those previously reported by Falzoni et al. (8) and Hickman et al. (14), underscore the significant quantitative and qualitative differences between the ATP-dependent induction of nonselective pores in human monocytes and that in the macrophages derived from these monocytes.


View larger version (68K):
[in this window]
[in a new window]
 
Fig. 1.   ATP induced YO-PRO-1 dye uptake in macrophages but not monocytes. A: YO-PRO-1 dye uptake by monocytes/macrophages was measured as described in MATERIALS AND METHODS. Cells were adhered onto a multiwell chambered slide. Each experiment was performed in a separate well. Cells were stimulated for 15 min with 3 mM ATP or 50 µg/ml digitonin or remained unstimulated in the presence of 2 µM YO-PRO-1 in NaCl-containing HEPES-buffered saline. Data were obtained from a single donor preparation of monocytes that were assayed on the day of isolation (day 0, top) or after 9 days of in vitro culture (bottom). Results are representative of 6 similar experiments using cells from 3 different blood donors. B: averaged values from 6 experiments in A.

Expression of P2X7 receptor mRNA and protein in blood monocytes vs. monocyte-derived macrophages. Using P2X7 receptor-specific monoclonal antibodies, Buell et al. (2) and Gu et al. (13) demonstrated expression of cell surface P2X7 receptors in >80% of human blood monocytes (2). Given the absence of ATP-dependent pore formation in freshly isolated human monocytes (Fig. 1) (8, 14), we tested the possibility that P2X7 receptor gene expression is low in circulating monocytes but is significantly increased when monocytes are incubated under in vitro conditions that promote their differentiation into macrophages. RT-PCR analysis indicated that the steady-state levels of P2X7 receptor mRNA were similar in freshly isolated blood monocytes and in monocyte-derived macrophages generated by incubating monocytes for 3 days in the presence of interferon-gamma (Fig. 2). This suggests that differentiation of blood monocytes into macrophages does not trigger significant changes in P2X7 receptor gene transcription or stability of P2X7 receptor mRNA.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 2.   P2X7 receptor mRNA levels are constant in monocytes and macrophages. Total mRNA was isolated from monocytes (day 0) and interferon-gamma (1,000 U/ml)-induced macrophages (day 3). mRNA was reverse transcribed to cDNA, and PCR were performed using P2X7-specific or human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers. Mock RT samples were subjected to PCR with GAPDH primers.

We also compared the expression of P2X7 receptor protein in monocytes with that in macrophages using a monoclonal anti-human P2X7 receptor antibody and FACS analysis. Figure 3, C and D, illustrates the FACS histograms of P2X7 receptor surface expression in the monocytes vs. monocyte-derived macrophages obtained from a representative blood donor, and Fig. 4 summarizes the averaged FACS analyses of monocyte/macrophages from multiple donors. Parallel immunocytochemical analyses with the CD14 monocyte marker (Fig. 3B) and the CD3 T lymphocyte marker (data not shown) verified that cells within the region 2 subpopulation of the scatter profile were predominantly monocytes (Fig. 3A), while the region 1 subpopulation was enriched in lymphocytes. FACS analysis of intact leukocyte samples revealed P2X7 receptor protein expression in 68 ± 6.0% of freshly isolated monocytes (n = 8 experiments with 6 donors) and in 88 ± 4.4% of 9-day-old monocyte-derived macrophages (n = 3). Thus ~70% of blood monocytes stain positive for cell surface P2X7 receptor expression, and this percentage only marginally changes during in vitro differentiation into macrophages. In contrast, the mean fluorescence intensity (MFI, an index of receptor number per cell) of surface labeled P2X7 receptors was only 27.3 ± 2.4 (n = 8) in fresh monocytes but increased 10-fold to 304 ± 40.7 (n = 3) in 9-day-old macrophages. Thus, whereas the majority of blood monocytes express detectable numbers of P2X7 receptors, the number of cell surface receptors increases significantly as these monocytes develop into fully differentiated macrophages.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 3.   P2X7 receptor protein expression in monocytes and macrophages. Monocytes and macrophages were stained with fluorescein-labeled monoclonal antibody to P2X7 receptor or with its isotype-matched control antibody (IgG2a). Cells were maintained in suspension at 4°C during the experimental procedure. A parallel sample of cells was labeled with fluorescein-conjugated CD14. All experimental samples were analyzed by fluorescence-activated cell sorting (FACS). On the basis of the characteristic forward (A, x-axis) and side (A, y-axis) scatter profiles, cells in region R2 were gated to represent the monocytic population (A). These cells were confirmed to be CD14 positive (B). C and D: monocyte and macrophage data, respectively. Traces are obtained from a single experiment but are representative of 12 experiments from 9 additional donors for monocytes and 2 experiments each from 3 donors for macrophages.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Change in P2X7 receptor protein expression during monocyte-to-macrophage differentiation. Monocytes were differentiated with 1,000 U/ml interferon-gamma . P2X7 receptor protein expression was monitored during various stages of in vitro differentiation from day 0 (blood monocytes) to day 9 (macrophages). Values are means ± SD from 3 independent experiments with separate donors. Cell surface P2X7 receptor protein was measured as described in MATERIALS AND METHODS and Fig. 3 legend. Fluorescence due to nonspecific binding was assessed by the isotype-matched IgG2a levels. Cell surface expression of the monocytic marker CD14 was also measured. Cells having higher fluorescence than a parallel sample of unstained cells were gated positive. A: plot of the mean fluorescence intensity vs. time in culture. B: percentage of cells (of 10,000) with positive fluorescence plotted vs. time in culture.

The time course characterizing this quantitative change in P2X7 receptor expression was characterized by culturing blood monocytes in the presence of interferon-gamma for various times before FACS analysis of P2X7 receptor and CD14 surface expression (Fig. 4). The percentage of P2X7-positive cells was relatively stable (68-85%) throughout the 9-day test period, while the percentage of CD14-positive cells declined modestly from 85 to 65%. Consistent with previous reports (1, 25, 26), the average density of CD14 sites on individual monocytes sharply decreased (a 10-fold reduction in MFI) during the first 3 days of in vitro differentiation due to shedding of this glycosylphosphatidylinositol-anchored surface protein. In contrast, the average number of P2X7 receptor sites per cell increased twofold (MFI = 55) over this same interval. By day 6, P2X7 receptor staining intensity increased fourfold (MFI = 95). Thus the development of blood monocytes into macrophages is characterized by a progressive increase in the number of P2X7 receptors that are targeted to the cell surface. To verify the specificity of this increased labeling with anti-P2X7 antibody, the cells were also stained with identical concentrations of isotype-matched, nonimmune IgG2a. The nonspecific IgG2a binding remained low (MFI = 6) and constant during the initial 3 days of in vitro differentiation but increased modestly to MFI of 15 and 25 in the 6- and 9-day-old differentiated macrophages, respectively. This is most likely due to increased expression of IgG-binding Fc receptors (5).

Inhibitory effects of extracellular Na+ and Cl- on nonselective pore formation by the P2X7 receptors of blood monocytes. The results described above suggest that limited expression of cell surface P2X7 receptor subunits may be one mechanism for the lack of nonselective pore formation in freshly isolated blood monocytes stimulated with high concentrations of extracellular ATP. However, pore formation by the P2X7 receptors natively expressed in freshly isolated normal or leukemic blood lymphocytes can only be observed when these cells are stimulated in medium lacking extracellular Na+ and Mg2+ (13, 37, 38). We hypothesized that the absence of ATP-induced pore formation in human blood monocytes may be similarly repressed by extracellular ions. This hypothesis was tested by assaying the ability of 3 mM ATP to stimulate YO-PRO-1 accumulation in freshly isolated blood mononuclear leukocytes that were suspended in HEPES-buffered solutions of different ionic composition and analyzed by FACS (Fig. 5). Consistent with the studies using adherent blood monocytes (Fig. 1), no significant number of monocytes suspended in standard NaCl-based saline accumulated YO-PRO-1 in response to ATP (Fig. 5A). Although Mg2+ is known to strongly modulate P2X7 receptor function (6, 23, 35), removal of extracellular Mg2+ from the NaCl-based saline did not facilitate ATP-induced YO-PRO-1 uptake (Fig. 5C). In contrast, when NaCl was isosmotically replaced by potassium glutamate, 3 mM ATP triggered a robust uptake of YO-PRO-1 in the majority of blood monocytes (~90%) regardless of the presence or absence of extracellular Mg2+ (Fig. 5, B and D, respectively). Although extracellular Mg2+ did not alter the percentage of ATP-responsive monocytes, it did affect the absolute amount of YO-PRO-1 that was accumulated during the 15-min test incubation. Thus monocytes suspended in Mg2+-free, potassium glutamate BSS accumulated 4- to 10-fold more YO-PRO-1 dye (MFI = 2,800 for the experiment in Fig. 5D) than monocytes activated in medium containing 1 mM Mg2+ (MFI = 270 for the experiment in Fig. 5B). Although the FACS profiles in Fig. 5 illustrate the responses of monocytes from a single donor, they are representative of similar experiments performed with different blood donors (n = 9). The averaged values from these experiments are presented in Fig. 5E. The results indicate that nonselective pores can be induced in blood monocytes, despite the relatively low number of P2X7 receptor subunits that are targeted to the surface of these cells.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 5.   Inhibitory effects of NaCl on ATP-induced YO-PRO-1 uptake in monocytes. Peripheral blood leukocytes were suspended at 107 cells/ml in NaCl basic salt solution (BSS; A and C) or potassium glutamate BSS (B and D) in the presence of 2 µM YO-PRO-1. The cells were then unstimulated or stimulated with 3 mM ATP for 15 min at 37°C and washed twice in NaCl BSS. Flow cytometric analysis of YO-PRO-1 uptake was performed as described in MATERIALS AND METHODS. Additional effects of Mg2+ were compared in media containing 1.0 mM Mg2+ (A and B) or no added Mg2+ (C and D). E: mean fluorescence intensity values from similar experiments using 9 different monocyte preparations.

Enhanced potency of ATP as an agonist for monocyte P2X7 receptors in the absence of extracellular NaCl. The experiments in Fig. 5 demonstrate that physiological concentrations of Na+ and Cl- strongly attenuate ATP-mediated pore formation in human monocytes. Previous studies have shown that millimolar concentrations of ATP are required for the activation of the P2X7/P2Z receptor (29, 33). However, Michel et al. (22) reported that removal of extracellular NaCl can increase the potency of BzATP as an inducer of pore formation in HEK-293 cells that express recombinant human P2X7 receptors. We observed that replacement of extracellular Na+ and Cl- similarly affects the potency of ATP as an agonist for the natively expressed P2X7 receptors of human blood monocytes (Fig. 6). Thus, although significant YO-PRO-1 accumulation (as assayed by MFI values from FACS analyses) in response to 100 µM ATP was observed when monocytes were stimulated in potassium glutamate BSS, even 3 mM ATP triggered no dye uptake when the same cells were suspended in NaCl BSS. The same ATP sensitivity was observed in potassium glutamate BSS in the presence or absence of millimolar Mg2+. The ATP dose-response relationship in the potassium glutamate media was biphasic, with an initial rise in the MFI per cell at 30-100 µM and an apparent maximum between 500 and 1 mM ATP. However, a further increase in the MFI was observed at >1 mM ATP. This is most likely due to a reduction in intracellular Mg2+ under these conditions, resulting in enhanced fluorescence of the YO-PRO-1-nucleic acid complexes. Mg2+ will permeate the nonselective pore, and ATP is a strong chelator of Mg2+ (and Ca2+). Thus, when 3 mM extracellular ATP is in excess over the concentrations of extracellular Mg2+ (1 mM) and Ca2+ (1.5 mM), the free concentration of Mg2+ will be reduced to micromolar levels, leading to a transmembrane gradient that will favor efflux of intracellular Mg2+ (given the 0.3-0.5 mM free Mg2+ within the cytosol). Thus reduction of extracellular Na+ and Cl- increases the potency of ATP as an agonist for P2X7 receptor-dependent pore formation, while removal of extracellular Mg2+ primarily facilitates an increased rate of dye influx through these nonselective pores.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6.   Dose-response relationships describing effects of ATP on YO-PRO-1 uptake by blood monocytes. YO-PRO-1 uptake per cell was measured by FACS analysis of peripheral blood leukocytes stimulated for 15 min at 37°C with ATP. Peripheral blood leukocytes were suspended in NaCl BSS containing no added MgCl2 (), NaCl BSS containing 1 mM MgCl2 (open circle ), potassium glutamate BSS containing no added MgCl2 (), or potassium glutamate BSS containing 1 mM MgCl2 (). Data represent average ± range from 2 independent experiments.

Effects of Na+ vs. Cl- on nonselective pore formation by monocyte P2X7 receptors. Figure 7 illustrates the independent effects of Na+ vs. Cl- replacement on ATP-dependent YO-PRO-1 uptake by blood monocytes using medium containing physiological concentrations of Mg2+ (1 mM) and Ca2+ (1.5 mM). Individual substitution of Na+ with K+ (KCl BSS) facilitated significant YO-PRO-1 accumulation (MFI = 40) in 70% of the monocytes (Fig. 7C). Substitution of Cl- with glutamate (sodium glutamate BSS; Fig. 7B) produced similar changes in ATP-induced YO-PRO-1 uptake (MFI = 30) in the same percentage of cells. Replacement of Cl- with gluconate also potentiated ATP-activated dye accumulation (data not shown). Consistent with previous studies (Fig. 5), simultaneous replacement of Na+ and Cl- with K+ and glutamate (Fig. 7D) resulted in a synergistic potentiation of ATP-dependent YO-PRO-1 influx in the same monocytes (MFI = 270). The averaged data from three independent experiments of this type are illustrated in Fig. 7E.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of Na+ vs. Cl- on YO-PRO-1 uptake in monocytes. Peripheral blood leukocytes were suspended at 107 cells/ml in NaCl (A), sodium glutamate (B), KCl (C), or potassium glutamate (D) in the presence of 2 µM YO-PRO-1. The cells were then unstimulated or stimulated with 3 mM ATP for 15 min at 37°C and washed twice in NaCl BSS. YO-PRO-1 uptake was measured by FACS analysis. E: mean fluorescence intensity values from 3 independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These studies demonstrate that the P2X7 receptor gene is transcriptionally active in human blood monocytes and that P2X7 receptor protein traffics to the plasma membrane of these cells. However, ATP can induce the nonselective pore that typifies activated P2X7 receptors only when these blood leukocytes are assayed in media containing reduced extracellular Na+ or Cl-. This contrasts with the robust induction of the nonselective pore in macrophages, which are derived from these human monocytes, when assayed in normal NaCl-containing medium. These findings suggest that multiple mechanisms have evolved to minimize the adventitious activation to P2X7 receptors in circulating monocytes while permitting these receptors to be rapidly upregulated and activated when monocytes leave the blood and enter peripheral sites of tissue inflammation.

All members of the P2X receptor family, including P2X7, function primarily as ATP-gated ion channels that can conduct small, inorganic cations (e.g., Na+, K+, and Ca2+). Analysis of most P2X family receptors indicates that the assembled, cation-permeable channels minimally conduct larger organic molecules such as Tris+ (121 Da), N-methylglucamine+ (195 Da), or the YO-PRO-1 dye (375 Da) used in our experiments (24). In contrast, the sustained activation of P2X7 receptors by ATP results in a time- and temperature-dependent acquisition of permeability to such large molecules (7, 24, 29). This delayed change in permeability has been termed "pore formation" to distinguish it from the rapidly gated change in conductance to small inorganic cations. The molecular nature of these nonselective pores remains to be established but might be consistent with several mechanisms. 1) P2X7 homooligomeric channels with fixed subunit stoichiometry may have multiple permeability states. 2) P2X7 homooligomers might physically interact with other P2X7 channels or other membrane proteins to form higher-order pore complexes. 3) Intracellular signaling cascades activated by P2X7 receptor channels may trigger covalent or allosteric changes in other proteins that comprise the nonselective pore (30, 31). Regardless of mechanism, extracellular Na+ and Cl- clearly exert strong inhibitory effects on the ability of ATP to activate pore formation via the cell surface P2X7 receptors expressed in circulating monocytes (Figs. 5-7). Isosmotic replacement of these ions with K+ and nonhalide anions results in an apparent "unmasking" of the pore-inducing function. One likely effect of Na+ and Cl- is to inhibit the binding of ATP to existing P2X7 receptor oligomers and thereby attenuate rapid gating of the cation channel and subsequent induction of the nonselective pore. Michel et al. (22) demonstrated that the repressive actions of Na+ and Cl- on the function of recombinant P2X7 receptors primarily reflect effects on agonist potency, rather than agonist efficacy, regardless of whether cation channel activity or YO-PRO-1 accumulation was measured as an index of receptor activation. We similarly observed that replacement of extracellular NaCl facilitated pore formation in blood monocytes stimulated with ATP concentrations as low as 30 µM, even in the presence of millimolar Mg2+ (Fig. 6).

In addition to reducing the ATP affinity of the P2X7 receptor, Na+ and Cl- may directly repress conformational changes that permit assembly or activation of the nonselective pore. Pore formation may require a threshold number of ATP-occupied P2X7 receptor complexes to drive efficient interaction with other putative pore-forming proteins or to activate intracellular signaling cascades that regulate such proteins. It should be stressed that millimolar ATP does activate pore formation in many P2X7-expressing cell types (8, 15, 29, 32), including human monocyte-derived macrophages (Fig. 1), bathed in physiological saline solutions. The inability of millimolar ATP to increase YO-PRO-1 uptake by human blood monocytes under identical assay conditions suggests that the number of ATP-occupied P2X7 receptors that can be formed is insufficient to drive formation of the pore. If a threshold number of ATP-occupied receptors is required for pore formation, the requisite number of receptors occupied at a given concentration of ATP may be achieved by increasing ATP affinity (as acutely induced by reducing extracellular NaCl) or by increasing the number of cell surface receptors. Consistent with this latter possibility, our FACS analyses indicated that monocyte-derived human macrophages express significantly higher numbers (by 3- to 10-fold) of cell surface P2X7 receptors than circulating monocytes. Presumably, the number of ATP-occupied P2X7 receptors required for threshold pore formation can accumulate when such macrophages are stimulated with millimolar ATP, even in the presence of high extracellular NaCl.

It is noteworthy that, with some blood donors, 500 µM BzATP [which is more potent and efficacious than ATP as a P2X7 agonist (33, 39)] did stimulate significant YO-PRO-1 accumulation by blood monocytes assayed in NaCl medium (data not shown). This further supports the possibility that pore formation requires a threshold number of agonist-occupied P2X7 receptors. On the basis of similar studies of the P2X7 receptors expressed in human leukemic lymphocytes, Wiley et al. (39) suggested that the rate and extent of pore formation are dependent on the fraction of P2X7 channels held in the open state due to agonist occupancy.

FACS analyses of digitonin-permeabilized blood monocytes using the monoclonal anti-P2X7 receptor indicated additional pools of intracellular P2X7 receptors (data not shown). Gu et al. (13) also described significant intracellular levels of immunoreactive P2X7 receptor proteins in human lymphocytes, monocytes, and neutrophils. Such findings suggest that trafficking of intracellular P2X7 receptors to the cell surface is a potential mechanism for regulating the pore-forming function. We considered the possibility that incubation of monocytes in low-Na+ or low-Cl- saline promoted a rapid redistribution of P2X7 receptors. An acutely induced increase in the number of cell surface P2X7 receptor channels could underlie the enhanced pore formation observed under such experimental conditions. This was tested by stimulating monocytes with 3 mM ATP in potassium glutamate BSS for 15 min (optimal conditions for pore formation) before analysis of cell surface P2X7 receptor by the same FACS protocols used in the experiments illustrated in Figs. 4 and 5. No changes were observed in the anti-P2X7 FACS profiles of these monocytes (data not shown). This suggests that rapid insertion of additional P2X7 receptors into the plasma membrane is an unlikely mechanism for the potentiation of ATP-induced pore formation observed when monocyte P2X7 receptors are activated in salines containing low Na+ or low Cl-.

The physiological roles for these nonselective pores also remain speculative. However, many biological responses associated with P2X7 receptor stimulation involve the activation of protease-based signaling cascades. These include caspase-1-mediated processing of interleukin-1beta in monocytes and macrophages, caspase-3-dependent apoptosis of macrophages and dendritic cells (4, 11, 27, 28), and matrix metalloprotease-mediated shedding of L-selectin and CD23 in lymphocytes (12, 17). Initiation of these protease-based cascades by ATP-activated P2X7 receptors appears to involve the sustained collapse of normal transmembrane gradients for K+, Na+, and Ca2+. Induction of the nonselective pores may provide a unique mechanism for ensuring the rapid and complete loss of intracellular K+ and accumulation of Na+ and Ca2+. Unlike kinase-based signaling, protease-dependent signal transduction is not readily reversible. Thus it is not surprising that activation of the P2X7 receptor and nonselective pore formation appear to be stringently regulated at multiple levels.

It is striking that the ATP affinity of the P2X7 receptor is maximal when the extracellular ionic composition approximates the cytoplasmic ionic milieu, i.e., a high-K+, low-Na+ solution wherein organic anions substitute for Cl-. When cells lyse within the immediate vicinity of a P2X7 receptor-expressing cell, they will not only release ATP but will also modify the local extracellular ionic composition. The extracellular NaCl solution will be "diluted" by the intracellular K+-organic anion solution. Obviously, this sort of ionic perturbation will be maximal in physiological environments wherein the ratio of cellular volume to extracellular volume is large (20). Such conditions may also be operative when monocytes or macrophages populate inflammatory tissue sites that are characterized by high cell density and significant numbers of damaged or dying cells. Thus the ability of extracellular monovalent ions to allosterically modulate ATP affinity may be a heretofore unrecognized mechanism for the physiological regulation of the pore-forming P2X7 receptor.


    ACKNOWLEDGEMENTS

We are grateful to Sylvia Kertesy for excellent technical support, R. Michael Sramkoski for assistance with FACS analysis, and Reza Beigi and Karen Parker for helpful discussions.


    FOOTNOTES

This study was supported by National Institute of General Medical Sciences Grant GM-36387 (G. R. Dubyak).

Address for reprint requests and other correspondence: G. R. Dubyak, Dept. of Physiology and Biophysics, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4970 (E-mail: gxd3{at}po.cwru.edu).

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 31 August 2000; accepted in final form 26 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bazil, V, and Strominger JL. Shedding as a mechanism of down-modulation of CD14 on stimulated human monocytes. J Immunol 147: 1567-1574, 1991[Abstract/Free Full Text].

2.   Buell, G, Chessell IP, Michel AD, Collo G, Salazzo M, Herren S, Gretener D, Grahames C, Kaur R, Kosco-Vilbois MH, and Humphrey PP. Blockade of human P2X7 receptor function with a monoclonal antibody. Blood 92: 3521-3528, 1998[Abstract/Free Full Text].

3.   Chiozzi, P, Sanz JM, Ferrari D, Falzoni S, Aleotti A, Buell GN, Collo G, and Di Virgilio F. Spontaneous cell fusion in macrophage cultures expressing high levels of the P2X/P2Z receptor. J Cell Biol 138: 697-706, 1997[Free Full Text].

4.   Coutinho-Silva, R, Persechini PM, Bisaggio RD, Perfettini JL, Neto AC, Kanellopoulos JM, Motta-Ly I, Dautry-Varsat A, and Ojcius DM. P2Z/P2X7 receptor-dependent apoptosis of dendritic cells. Am J Physiol Cell Physiol 276: C1139-C1147, 1999[Abstract/Free Full Text].

5.   Dorrington, KJ. Properties of the Fc receptor on macrophages. Immunol Commun 5: 263-280, 1976[ISI][Medline].

6.   Dubyak, GR, Clifford EE, Humphreys BD, Kertesy SB, and Martin KA. Expression of multiple ATP receptor subtypes during the differentiation and inflammatory activation of myeloid leukocytes. Drug Dev Res 38: 1-10, 1996[ISI].

7.   Evans, RJ, Surprenant A, and North RA. P2X receptors: cloned and expressed. In: The P2 Nucleotide Receptors, edited by Turner JT, Weisman GA, and Fedan JS.. Totawa, NJ: Humana, 1998, p. 43-62.

8.   Falzoni, S, Munerati M, Ferrari D, Spisani S, Moretti S, and Di Virgilio F. The purinergic P2Z receptor of human macrophage cells: characterization and possible physiological role. J Clin Invest 95: 1207-1216, 1995[ISI][Medline].

9.   Gantner, F, Kupferschmidt R, Schudt C, Wendel A, and Hatzelmann A. In vitro differentiation of human monocytes to macrophages: change in PDE profile and its relationship to suppression of tumour necrosis factor-alpha release by PDE inhibitors. Br J Pharmacol 121: 221-231, 1997[Abstract].

10.   Gargett, CE, Cornish JE, and Wiley JS. ATP, a partial agonist for the P2Z receptor of human lymphocytes. Br J Pharmacol 122: 911-917, 1997[Abstract].

11.   Griffiths, RJ, Stam EJ, Downs JT, and Otterness IG. ATP induces the release of IL-1 from LPS-primed cells in vivo. J Immunol 154: 2821-2828, 1995[Abstract/Free Full Text].

12.   Gu, B, Bendall LJ, and Wiley JS. Adenosine triphosphate-induced shedding of CD23 and L-selectin (CD62L) from lymphocytes is mediated by the same receptor but different metalloproteases. Blood 92: 946-951, 1998[Abstract/Free Full Text].

13.   Gu, B, Zhang WY, Bendall LJ, Chessel IP, Buell GN, and Wiley JS. Expression of P2X7 purinoceptors on human lymphocytes and monocytes: evidence for nonfunctional P2X7 receptors. Am J Physiol Cell Physiol 279: C1189-C1197, 2000[Abstract/Free Full Text].

14.   Hickman, SE, Khoury JE, Greenberg S, Schieren I, and Silverstein SC. P2Z adenosine triphosphate receptor activity in cultured human monocyte-derived macrophages. Blood 84: 2452-2456, 1994[Abstract/Free Full Text].

15.   Humphreys, BD, and Dubyak GR. Induction of the P2Z/P2X7 nucleotide receptor and associated phospholipase D by lipopolysaccharide and IFN-gamma in the human THP-1 monocytic cell line. J Immunol 157: 5627-5637, 1996[Abstract].

16.   Humphreys, BD, Virginio C, Surprenant A, Rice J, and Dubyak GR. Isoquinolines as antagonists of the P2X7 nucleotide receptor: high selectivity for the human versus rat receptor homologues. Mol Pharmacol 54: 22-32, 1998[Abstract/Free Full Text].

17.   Jamieson, GP, Snook MB, Thurlow PJ, and Wiley JS. Extracellular ATP causes loss of L-selectin from human lymphocytes via occupancy of P2Z purinoceptors. J Cell Physiol 166: 637-642, 1996[ISI][Medline].

18.   Khakh, BSS, Bao XR, Labarca C, and Lester HA. Neuronal P2X transmitter-gated channels change their ion selectivity in seconds. Nat Neurosci 2: 322-330, 1999[ISI][Medline].

19.   Khakh, BS, and Lester HA. Dynamic selectivity filters in ion channels. Rev Neurol 23: 653-658, 1999.

20.   Ma, W, Komgreen A, Uzlaner N, Priel Z, and Silberberg SD. Extracellular sodium regulates airway ciliary motility by inhibiting a P2X receptor. Nature 400: 894-897, 1999[ISI][Medline].

21.   Markwardt, F, Lohn M, Bohm T, and Klapperstuck M. Purinoceptor-operated cationic channels in human B lymphocytes. J Physiol (Lond) 498: 143-154, 1997[Abstract].

22.   Michel, AD, Chessell IP, and Humphrey PPA Ionic effects on human recombinant P2X7 receptor function. Naunyn Schmiedebergs Arch Pharmacol 359: 102-109, 1999[ISI][Medline].

23.   North, RA, and Barnard EA. Nucleotide receptors. Curr Opin Neurobiol 7: 346-357, 1997[ISI][Medline].

24.   Nuttle, LC, and Dubyak GR. Differential activation of cation channels and non-selective pores by macrophage P2Z purinergic receptors expressed in Xenopus oocytes. J Biol Chem 269: 13988-13996, 1994[Abstract/Free Full Text].

25.   Payne, JB, Nichols FC, and Peluso JF. The effects of interferon-gamma and bacterial lipopolysaccharide on CD14 expression in human monocytes. J Interferon Res 12: 307-310, 1992[ISI][Medline].

26.   Pedron, T, Girard R, and Chaby R. Variation of LPS-binding capacity, epitope expression and shedding of membrane-bound CD14 during differentiation of human monocytes. J Immunol 155: 1460-1471, 1995[Abstract].

27.   Perregaux, D, and Gabel CA. Interleukin-1beta maturation and release in response to ATP and nigericin. J Biol Chem 269: 15195-15203, 1994[Abstract/Free Full Text].

28.   Perregaux, DG, Laliberte RE, and Gabel CA. Human monocyte interleukin-1beta posttranslational processing. J Biol Chem 271: 29830-29838, 1996[Abstract/Free Full Text].

29.   Rassendren, F, Buell GN, Virginio C, Collo G, North A, and Surprenant A. The permeabilizing ATP receptor, P2X7. J Biol Chem 272: 5482-5486, 1997[Abstract/Free Full Text].

30.   Schilling, WP, Sinkins WG, and Estacion M. Maitotoxin activates a nonselective cation channel and P2Z/P2X7-like cytolytic pore in human skin fibroblasts. Am J Physiol Cell Physiol 277: C755-C765, 1999[Abstract/Free Full Text].

31.   Schilling, WP, Wasylyna T, Dubyak GR, Humphreys BD, and Sinkins WG. Maitotoxin and P2Z/P2X7 purinergic receptor stimulation activate a common cytolytic pore. Am J Physiol Cell Physiol 277: C766-C776, 1999[Abstract/Free Full Text].

32.   Steinberg, TH, Newman AS, Swanson JA, and Silverstein SC. ATP4- permeabilizes the plasma membrane of mouse macrophages to fluorescent dyes. J Biol Chem 262: 8884-8888, 1987[Abstract/Free Full Text].

33.   Surprenant Rassendren, AF, Kawashima E, North RA, and Buell G. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science 272: 735-738, 1996[Abstract].

34.   Torres, GE, Egan TM, and Voigt MM. Hetero-oligomeric assembly of P2X receptor subunits. J Biol Chem 274: 6653-6659, 1999[Abstract/Free Full Text].

35.   Virginio, C, Church D, North RA, and Surprenant A. Effects of divalent cations, protons and calmidazolium at the rat P2X7 receptor. Neuropharmacology 36: 1285-1294, 1997[ISI][Medline].

36.   Virginio, C, MacKenzie A, Rassendren FA, North RA, and Surprenant A. Pore dilation of neuronal P2X receptor channels. Nat Neurosci 2: 315-321, 1999[ISI][Medline].

37.   Wiley, JS, Chen R, Wiley MJ, and Jamieson GP. The ATP4- receptor-operated ion channel of human lymphocytes: inhibition by ion fluxes by amiloride analogs and by extracellular sodium ions. Arch Biochem Biophys 292: 411-418, 1992[ISI][Medline].

38.   Wiley, JS, and Dubyak GR. Extracellular adenosine triphosphate increases cation permeability of chronic lymphocytic leukemic lymphocytes. Blood 73: 1316-1323, 1989[Abstract].

39.   Wiley, JS, Gargett CE, Zhang W, Snook MB, and Jamieson GP. Partial agonists and antagonists reveal a second permeability state of human lymphocyte P2Z/P2X7 channel. Am J Physiol Cell Physiol 275: C1224-C1231, 1998[Abstract/Free Full Text].


Am J Physiol Cell Physiol 280(4):C943-C953
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society