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Blood, 15 June 2006, Vol. 107, No. 12, pp. 4946-4953. Prepublished online as a Blood First Edition Paper on March 2, 2006; DOI 10.1182/blood-2005-07-2994.
PHAGOCYTES Rapid ATP-induced release of matrix metalloproteinase 9 is mediated by the P2X7 receptorFrom the Department of Medicine, University of Sydney at Nepean Hospital, Penrith, New South Wales, Australia.
Matrix metalloproteinase-9 (MMP-9) activity is required for inflammatory response, leukocyte recruitment, and tumor invasion. There is increasing evidence suggesting that the P2X7 receptor of mononuclear cells, which is activated by extracellular adenosine triphosphate (ATP), is involved in inflammatory responses. In this study, ATP caused a rapid release of MMP-9 and a moderate decrease in tissue inhibitor of metalloproteinase 1 (TIMP-1) release from human peripheral-blood mononuclear cells (PBMCs) over a 30-minute time course. The release was time- and dose-dependent and dissociated from ATP-induced cell death. BzATP, which is the most potent agonist for the P2X7 receptor, also caused a similar effect at a lower dosage. ATP-induced MMP-9 release was inhibited by the P2X7 receptor antagonists periodate oxidized ATP and KN-62, or by calcium chelators, as well as by a loss-of-function polymorphism in the P2X7 receptor, but not by brefeldin A, monensin, or cycloheximide, or by anti–tumor necrosis factor-  (TNF-) or anti–interleukin-1 (IL-1) monoclonal antibodies. Results from purified subsets of PBMCs showed monocytes were the major source for MMP-9 and TIMP-1 release, and ATP remained effective in purified monocyte and T-cell populations. These observations suggest a novel role for P2X7 as a pro-inflammatory receptor involved in rapid MMP-9 release and leukocyte recruitment.
Synthesis and secretion of matrix metalloproteinases (MMPs) have been demonstrated for all cells of the immune and hemopoietic lineages as well as fibroblasts and endothelial cells. A wide variety of neoplastic cells also have been shown to secrete MMPs, and these MMPs are considered to participate in the metastatic dissemination of tumors1 and cyclic changes in the endometrium.2 Other studies have shown that MMPs are involved in physiologic processes such as angiogenesis wound repair3 and the inflammatory response.4 Among the members of the MMP family, the gelatinases A and B (MMP-2 and 9) are important, since both can produce proteolytic degradation of type 4 collagen, which is the principal component of basement membrane. Many studies have shown that MMP-2 is secreted constitutively, while secretion of MMP-9 is regulated by inflammatory stimuli that differ according to cell type.5 Like the other MMPs, gelatinases are produced in a latent form containing a prodomain, which requires activation by a cysteine-switch mechanism.6 Both gelatinases form tight noncovalent and stable complexes with tissue inhibitor of metalloproteinases (TIMPs). Thus, pro–MMP-2 (72 kDa) specifically binds TIMP-2,7 whereas pro–MMP-9 (92 kDa) specifically binds TIMP-1, which plays a major role in regulating MMP-9 activity.8 Besides the inhibitory effect of TIMP-1, MMP-9 activity also is regulated at various levels from production to secretion, including transcription of the gene by cytokines such as tumor necrosis factor- (TNF-) and interleukin 1 (IL-1) or by cellular interactions such as cell-cell contact between monocytes and activated T lymphocytes, activation of pro-enzyme by other MMPs,9,10 or by variant proteinases such as lipopolysaccharide (LPS)–associated proteinases.11
The P2X7 receptor is a ligand-gated cation channel activated by extracellular adenosine triphosphate (ATP) and highly expressed on monocytes, macrophages, and lymphocytes. Activation of this receptor by exposure to extracellular ATP opens a cation selective channel that allows Ca2+ and Na+ influx and K+ efflux. The channel dilates within the first minute to a larger pore, followed by a cascade of downstream effects, which include the stimulation of phospholipase D,12,13 the activation of membrane metalloproteases,14,15 extensive membrane blebbing,16 and the stimulation of intracellular caspases, which eventually lead to the apoptotic death of the target cell.17 P2X7 activation also leads to release and maturation of both IL-1
Materials
ATP, 2',3'-O-(4-benzoyl)benzoyl ATP (BzATP), periodate oxidized ATP (OxATP), phorbol 12-myristate 13-acetate (PMA), brefeldin A (BFA), monensin, cycloheximide, barium chloride, magnesium chloride, D-glucose, bovine serum albumin (BSA), RPMI-1640 medium, gentamicin, bistris, ethylenediaminetetraacetic acid (EDTA), 4-aminophenylmercuric acetate (APMA), Source of human peripheral-blood mononuclear cells Peripheral blood was collected from nonsmoking donors and diluted with an equal volume of RPMI-1640 medium. Peripheral mononuclear cells were separated by density gradient centrifugation over Ficoll-Hypaque and washed once in RPMI-1640 medium and resuspended in RPMI-1640 medium containing 10% fetal calf serum and 5 µg/mL gentamicin (complete RPMI-1640 medium). Monocytes comprised between 9% and 18% of PBMCs from these healthy subjects. Before each experiment, cells were washed twice and resuspended in either HEPES-buffered NaCl medium (145 mM NaCl, 5 mM KCl, 10 mM HEPES, 5 mM D-glucose, and 1 mg/mL BSA, pH 7.5) or HEPES-buffered KCl medium (150 mM KCl, 10 mM HEPES, 5 mM D-glucose, and 1 mg/mL BSA, pH 7.5) in NoStick Hydrophobic 1.5 mL polypropylene tubes (Scientific Specialties Inc, Lodi, CA). In this study, healthy subjects with wild-type P2X7 receptor do not carry any of the known loss-of-function polymorphisms and have high P2X7 function measured by ATP-induced ethidium uptake. Subjects homozygous for the Glu496Ala polymorphism were identified by direct sequencing as previously described22-24 and showed no P2X7 function, as assayed by ethidium influx measurement, in either lymphocyte or monocyte populations. The study was approved by the Human Research Ethics Committee in Sydney West Area Health Service (05/066). Informed consent was provided according to the Declaration of Helsinki. Gelatin zymography
Mononuclear cells (2.0 x 107/mL) were incubated in NaCl medium or KCl medium with/without ATP or other reagents at 37°C. Both supernatants and cell pellets were collected by centrifugation at 1200g for 3 minutes and frozen at –30°C for 10 to 35 hours. Before assaying, cell pellets were lysed in lysate buffer containing mini-complete protease inhibitor cocktail (EDTA-free, 1 tablet/7 mL), 50 mM bistris, 750 mM Immunostaining of MMP-9 PBMCs (1 x 107/mL) from subjects with wild-type P2X7 function were incubated in Na medium containing 0.1 mM Ca2+ at 37°C with or without 1 µM KN-62 for 15 minutes, followed by addition of 1 mM ATP or 100 nM phorbol myristate acetate (PMA). Cells were washed twice and resuspended in phosphate-buffered saline (PBS). For surface staining, cells were incubated with 4 µg/mL anti–MMP-9 mAb for 20 minutes and washed twice, followed by staining with FITC-conjugated goat anti–mouse IgG + IgM Ab for 15 minutes. For intracellular staining, cells were fixed in 1% paraformaldehyde for 15 minutes at 4°C, washed twice, and resuspended in PBS containing 0.1% saponin. Cells were incubated with 4 µg/mL anti–MMP-9 mAb for 30 minutes and washed twice, followed by staining with FITC-conjugated goat anti–mouse IgG + IgM Ab for another 30 minutes. Fluorescence intensity was measured by BD FACSCalibur flow cytometry. Purification of monocytes, T lymphocytes, and NK cells by positive selection PBMCs (5-10 x 107) from randomly selected healthy subjects were resuspended in NaCl medium and incubated with MACS magnetic beads coated with anti-CD14 mAb for 15 minutes at 4°C. Cells were washed once and loaded onto a MACS LS separation column. CD14+ monocytes were collected, as well as the negative fraction. The same steps were repeated to extract CD3+ T lymphocytes and CD56+ natural killer (NK) cells from the negative fraction. The final negative fraction was collected as B lymphocytes. The CD14+ monocytes had fewer than 15% of CD3+, CD19+, or CD56+ cells; the CD3+ T lymphocytes had fewer than 5% of CD14+, CD19+, or CD56+ cells; CD56+ NK cells had fewer than 5% of CD3+, CD14+, or CD19+ cells, while the B lymphocytes had more than 90% of CD19+ cells. All cell suspensions were counted, washed, and resuspended in NaCl medium with 1 mM CaCl2 at a concentration of 2.0 x 107/mL. Supernatants were collected after 60 minutes, and MMP-9 and TIMP-1 concentrations were determined using ELISA kits. Purification of monocytes and T lymphocytes by negative selection PBMCs (5-10 x 107) from healthy subjects with fully functional wild-type P2X7 receptors were resuspended in NaCl medium. MACS magnetic beads coated with anti-CD14, CD19, and CD56 mAb were added. Cells were washed once after 15 minutes of incubation at 4°C. T cells were collected through a MACS LD separation column. For monocyte separation, anti-CD3 MACS beads were used instead of anti-CD14 beads. More than 95% purified T cells were CD3+, and more than 90% purified monocytes were CD14+. MMP-9 and TIMP-1 ELISA Human MMP-9 Biotrak assay kit (Amersham), which recognizes the 92-kDa MMP-9 and the complex of 92-kDa MMP-9 with TIMP-1, was used to determine MMP-9 concentration. Human TIMP-1 ELISA kit (Oncogene), which reacts with free TIMP-1 and TIMP-1 complexes with MMPs, was used to determine TIMP-1 concentration. Samples were diluted 1:10 for TIMP-1 assay. ELISAs were performed according to the manufacturer's manuals. All samples were stored at –80°C for 1 to 3 weeks before assay. Ethidium influx measurement by flow cytometry PBMCs (2 x 106) prelabeled with appropriate fluorophore-conjugated anti-CD19 (for B cell) or anti-CD14 (for monocyte) mAbs were washed once and resuspended in 1.0 mL HEPES-buffered KCl medium (10 mM HEPES, 150 mM KCl, 5 mM D-glucose, 0.1% BSA, pH 7.5) at 37°C. Cells were analyzed at 1000 events per second on a FACSCalibur flow cytometer (Becton Dickinson) and were gated by forward and side scatter and by cell-type–specific antibodies. All samples were stirred and temperature controlled at 37°C using a Time Zero module (Cytek, Fremont, CA). Ethidium (25 µM) was added, followed 40 seconds later by addition of 1.0 mM ATP. The linear mean channel of fluorescence intensity for each gated subpopulation over successive 5-second intervals was analyzed by WinMDI software (version 2.8; Joseph Trotter, The Scripps Institute, La Jolla, CA) and plotted against time. The area under ethidium uptake curve was calculated as the arbitrary unit of the P2X7 receptor function. LDH detection The CytoTox 96 nonradioactive cytotoxicity assay kit (Promega) was used to detect L-lactate dehydrogenase (LDH) in supernatant and cell-pellet lysate. Titration of 1:5000 diluted positive control provided in the kit was used as a standard curve. Assay was performed according to the manufacturer's manual. Statistics One-sided paired t test was used in this study.
Variability of ATP-induced MMP-9 release To study ATP effect on MMP-9 and TIMP-1 release, PBMCs from randomly selected healthy subjects (n = 21) were incubated with or without ATP for 30 minutes, and MMP-9 and TIMP-1 concentrations in supernatants were determined using ELISA. Another 3 subjects were studied with genetic absence of P2X7 function due to homozygosity for a loss-of-function polymorphism in the P2RX7 gene. Although the spontaneous releases of MMP-9 and TIMP-1 were quite variable between individuals, an increment in MMP-9 release, which was observed with ATP in 14 of 21 randomly selected subjects, was significant (P = .0002, n = 21). After incubation with ATP for 30 minutes, TIMP-1 release decreased in 9 of the 20 randomly selected subjects and remained at a similar level in the others (Figure 1), although TIMP-1 release increased at longer times of incubation (> 60 minutes) (Figure 2C). In each of the 3 subjects with absent P2X7 function, ATP had a negligible effect on MMP-9 and TIMP-1 release (Figure 1, closed circles). Zymography for subjects with good MMP-9 release showed 3 MMP bands at 220, 130, and 92 kDa in the gel (Figures 2A, 3A, 4A, and S1A, which is available on the Blood website; see the Supplemental Materials link at the top of the online article), with the 220-kDa bands not shown), corresponding to the homodimer of 92-kDa MMP-9, the complex of 92-kDa MMP-9 with 25-kDa lipocalin, and the monomeric MMP-9 in latent form, respectively, as previous described.29 An 84-kDa band showing MMP-9 in active form also was observed when the supernatant was incubated with 2 mM 4-aminophenylmercuric acetate (APMA), an in vitro activator for MMP-9 and MMP-2, for 30 minutes at 37°C before loaded on the gel; however, no MMP band was observed when the MMP inhibitor, 10 mM EDTA or 50 µg/mL Ro31-9790, was added in the developing buffer (data not shown). A 72-kDa MMP-2 band also was found in supernatants from some of the subjects by gelatin zymography; however, ATP failed to alter MMP-2 or TIMP-2 concentration as measured by ELISA after 30 and 60 minutes of incubation (data not shown). To normalize data and reduce variability between individuals, the use of PMA to induce the maximum release of MMP-9 and TIMP-1 was examined.30,31 PBMCs were incubated with various concentrations of PMA (0.1-500 nM) for 15 minutes at 37°C, and MMP-9 activities in supernatant and cell pellet were determined by gelatin zymography, whereas MMP-9 and TIMP-1 concentrations were detected using ELISA kits. PMA (at 5 nM) stimulated maximum MMP-9 and TIMP-1 secretion in 15 minutes (EC50 = 2 nM) when measured either by zymography or by ELISA (Figure S1). Control cells incubated with 10 nM PMA were routinely included in further experiments to obtain maximum MMP-9 release for each subject.
Time course of ATP-induced MMP-9 and TIMP-1 releases PBMCs from healthy subjects with fully functional wild-type P2X7 receptors were incubated with ATP for 5 to 120 minutes. Increased MMP-9 activity (Figure 2A) and MMP-9 protein level (Figure 2B) appeared in supernatants as soon as 5 minutes after addition of ATP. On average, ATP released 60% (range, 40% to 90%) of the MMP-9 released by PMA ("total") at 30 to 60 minutes, while only 20% (range, 10% to 60%) of total was spontaneously released over the same time. The increment in MMP-9 due to ATP was significant at 15 minutes and 30 minutes (P value is .003 and .016, respectively, n = 4; Figure 2B). Figure 2C shows that 10% to 45% of total TIMP-1 was released at 30 to 60 minutes, and there was a trend for ATP to inhibit TIMP-1 release. In contrast, quantitation of LDH in the supernatant revealed that ATP-induced cytolysis over the 2 hours of incubation was less than 3% of total, indicating that release of MMP-9 and TIMP-1 was not a result of cell lysis (Figure 2D). Agonist dose-response for MMP-9 and TIMP-1 release The release of MMP-9 and TIMP-1 from PBMCs was measured over a range of concentrations of ATP as well as BzATP, which is the most potent agonist for the P2X7 receptor. Over a 30-minute incubation, ATP increased MMP-9 release with an EC50 of 300 µM and decreased TIMP-1 release with an IC50 of 500 µM (Figure 3A,C,D), while the more potent BzATP had an EC50 of 50 µM for increasing MMP-9 release and an IC50 of 70 µM for the reduction in TIMP-1 release (Figure 3B-D).
ATP-induced MMP-9 release is inhibited by P2X7 antagonists KN-62, which is an isoquinolinesulfonamide derivative and a potent and specific inhibitor of the human P2X7 receptor,32 blocked most of the ATP-induced MMP-9 release with an IC50 of 50 nM (Figure 4A,B). Another P2X7 antagonist, OxATP, is widely used as an irreversible antagonist of the P2X7 receptor. Both ATP and BzATP failed to stimulate MMP-9 release in PBMCs pretreated with OxATP (Figure S2). Neither KN-62 nor OxATP affected the spontaneous release of MMP-9. ATP-induced MMP-9 release is Ca2+-dependent
Ca2+ is an important cofactor for a number of secretory events such as P2X7–mediated maturation and secretion of IL-1
MMP-9 and TIMP-1 release from PBMCs is mostly from monocytes To identify the cell of origin of MMP-9, the different cells of the mononuclear preparation were separated by using antibody-coated magnetic beads. Tables 1 and 2 shows that the basal release of MMP-9 was far greater from the monocytes of PBMCs than for T lymphocytes, whether these cells were separated by either positive or negative selection by magnetic beads. The ATP-stimulated components of MMP-9 release also were far greater for monocytes than for T lymphocytes, although an increment of MMP-9 release could be demonstrated only for negatively selected monocytes, as adhesion of these cells to beads (positive selection) largely abolished the ATP response (Tables 1,2). Table S1 also shows that monocytes released the largest amount of MMP-9 and TIMP-1, followed by B lymphocytes, T lymphocytes, and NK cells. Gelatin zymography also showed monocytes released the highest activity of MMP-9 (data not shown).
Rapid MMP-9 release is mainly via secretion from intracellular stores To investigate the source of MMP-9 released to the medium, PBMCs from subjects with wild-type P2X7 receptors were stained with anti–MMP-9 mAb. No MMP-9 was found on the surface of lymphocytes, while monocytes showed weak surface anti–MMP-9 mAb binding (Figure 6A). However, neither ATP nor PMA induced any change in the weak staining on the monocyte surface. In contrast, both lymphocytes and monocytes showed large amounts of intracellular MMP-9, and the amounts were decreased after treatment with either ATP or PMA. Pretreatment of KN-62 abolished this effect of ATP (Figure 6A).
Rapid MMP-9 release is via brefeldin A–insensitive pathway The antiviral antibiotic brefeldin A34 and the sodium ionophore monensin35 are widely used to block protein secretion. Figure 6B shows that the secretion of MMP-9 induced by both ATP and PMA was unaffected by pretreatment of PBMCs with brefeldin A or monensin. Cycloheximide, a widely used inhibitor of protein synthesis,36 also showed no inhibitory effect on ATP- or PMA-induced rapid MMP-9 release over a 30-minute incubation (Figure 6B).
ATP-induced MMP-9 release is independent of TNF-
The pro-inflammatory cytokines IL-1
There is increasing evidence that the P2X7 receptor on cells of monocytic or lymphoid origin participates in the inflammatory response. P2X7 knockout mice show reduced IL-1 release and absence of IL-1 maturation in response to ATP stimulation41 and less severe disease outcomes compared to wild-type mice in anticollagen antibody–induced arthritis, a model of inflammatory joint disease.27 Our data show that the P2X7 receptor also plays a role in rapid release of MMP-9 from monocytes, which is required in the early phase of the inflammatory response, as these cells migrate through basement membrane into the inflammatory focus. Most previous studies have measured the appearance of MMP-9 in supernatants over long time periods (12 to 48 hours) when both production and secretion have to be taken into consideration. However, the short time frame (< 60 minutes) used in the present study may be more relevant to the early events in inflammation, since it has been shown that monocytes can be recruited into inflammatory sites in just 2 to 3 hours, although the numbers steadily increase over 24 hours.42,43 In this short time period of 30 minutes, ATP increased MMP-9 release and decreased TIMP-1 release in a time- and dose-dependent manner (Figures 2,3). This ATP-induced MMP-9 release was inhibited by the P2X7 receptor antagonists KN62 and OxATP (Figures 4, S2), and ATP also failed to induce MMP-9 release from PBMCs of subjects who were homozygous for the P2X7 loss-of-function polymorphism (Figure 1), which abolishes ATP-induced entry of Ca2+ into PBMCs.22 Furthermore, Figure 7 and Tables 1 and 2 show that ATP-induced MMP-9 release is independent of TNF-– or IL-1–stimulated MMP-9 release and that the ATP-induced release does not require the interaction of T lymphocytes with monocytes. These observations suggest a novel role for P2X7 as a pro-inflammatory receptor mediating rapid MMP-9 release from monocytes as they migrate into an inflammatory focus.
Our previous studies have shown that there is wide variation of P2X7 function between individuals,44 largely due to allelic variations in the P2RX7 gene.22-26 In this study, the increments of MMP-9 release after ATP stimulation also varied in randomly selected subjects, and this genetic influence plays an important part. However, other factors, such as spontaneous MMP-9 release and intracellular MMP-9 protein level, also may affect ATP-induced MMP-9 release. Figure 2 shows that spontaneous MMP-9 release increases progressively at longer times of incubation (> 60 minutes). Figure 7 suggests that cytokines, such as TNF- , may play a major role in spontaneous MMP-9 release as anti–TNF- mAb significantly reduced spontaneous MMP-9 release. Since monocytes make the major contribution to MMP-9 release (Tables 1, 2, and S1), the percentage of monocytes in PBMCs may be another important factor influencing ATP-induced MMP-9 release. Thus, all of these factors contribute to the considerable variability in MMP-9 release shown in Figure 1.
The P2X7 receptor also has been shown to mediate release and maturation of the pro-inflammatory cytokines IL-1
Although MMP-9 is not a membrane protein, it has been shown that MMP-9 does bind to a number of surface adhesion molecules and receptors including M2 integrin (Mac-1),46 intercellular adhesion molecule-1 (ICAM-1),47 and Ku heterodimer (Ku70/Ku80),48 which are expressed on the surface of monocytes. It is possible that MMP-9 is rapidly released from the cell surface together with its docking molecules, which are shed by ATP or PMA. However, our results did not support this hypothesis, as ATP or PMA failed to shed MMP-9 on monocyte surface. Instead, large amounts of intracellular MMP-9 were found in monocytes as well as in lymphocytes. ATP reduced the MMP-9 intracellular pool, and this effect was abolished by pretreatment of KN62, suggesting that ATP-induced MMP-9 release is due to secretion of the intracellular MMP-9 store, via activation of the P2X7 receptor. MMP-9 (92 kDa) has a proposed leading single sequence in its first 19 residues, and it is well-known that long-term (> 7 hours) MMP-9 secretion requires protein synthesis36 and involves the regular protein transport pathway from the endoplasmic reticulum to the Golgi apparatus.49,50 However, in this study, both brefeldin A and monensin failed to block the rapid MMP-9 release induced by ATP or PMA over 30 minutes, suggesting that rapid MMP-9 release may occur via granule secretion, similar to membrane type 1 matrix metalloproteinase (MT1-MMP), which rapidly traffics to the plasma membrane via an unconventional brefeldin A–resistant pathway.51 In support of this concept, it has been shown that B lymphocytes contain MMP-9 in a granular distribution.52 Cycloheximide also failed to block rapid MMP-9 release in this study, in line with a previous report that protein synthesis is not required for rapid MMP-9 release from neutrophils.30 Among the 4 subsets of mononuclear cells, monocytes showed the highest spontaneous release of MMP-9, which may assist the emigration of this cell type from blood into the tissues of the reticulo-endothelial system. Previous studies have shown that monocytes are the first leukocyte type found in an inflammatory site, with cells appearing within 2 to 3 hours.42,43 Elevated concentrations of ATP and its metabolites are found within the inflammatory focus from the breakdown of cellular integrity, and this ATP may help the recruitment of monocytes to the lesion by secretion of MMP-9 and digestion of the extracellular matrix. However, the full biologic significance of the rapid MMP-9 secretion from monocytes remains to be established.
We thank Dr Steven Fuller for recruitment of healthy subjects, Ms Kristen Skarratt for identification of P2X7 genotype, Dr Ronald Sluyter and A/Prof Lois A. Salamonsen for critical review and discussion, Dr Jin Zhang for advice on gelatin zymography technique, and Ms Jennifer Georgiou for separation of peripheral-blood mononuclear cells from some healthy subjects.
Submitted July 26, 2005; accepted February 14, 2006.
Prepublished online as Blood First Edition Paper, March 2, 2006; DOI 10.1182/blood-2005-07-2994.
Supported by the National Health and Medical Research Council, the Cure Cancer Australia Foundation, the Leukemia Foundation of Australia, and a Sesqui Fellowship from the University of Sydney (B.J.G.).
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: James S. Wiley, Level 5, South Block, Nepean Hospital, Penrith, NSW 2750, Australia; e-mail: wileyj{at}medicine.usyd.edu.au.
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Abstract
Introduction
-amino-N-caproic acid, N-dodecyl 
) and subjects homozygous for the P2X7 Glu496Ala loss-of-function polymorphism (
). PBMCs (2 x 107/mL) were incubated with or without 1 mM ATP at 37°C in NaCl medium containing 1 mM CaCl2. Supernatants were collected after 30 minutes, and MMP-9 and TIMP-1 concentrations were measured by ELISA. The genotypes of the P2X7 receptor in those randomly selected healthy donors were unknown at the time of assay.
