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Glu⁴⁹⁶ to Ala Polymorphism in the P2X₇ Receptor Impairs ATP-Induced IL-1 Release from Human Monocytes¹

Department of Medicine, University of Sydney at Nepean Hospital, Penrith, New South Wales, Australia

	Abstract

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Priming of monocytes with LPS produces large quantities of intracellular, biologically inactive IL-1 that can be processed and released by subsequent activation of the P2X₇ receptor by extracellular ATP. We examined whether a loss-of-function polymorphism of the human P2X₇ receptor (Glu⁴⁹⁶Ala) impairs this process. Both ATP-induced ethidium⁺ uptake and ATP-induced shedding of L-selectin (CD62L) were nearly absent in monocytes from four subjects homozygous for Glu⁴⁹⁶Ala confirming that this polymorphism impairs P2X₇ function. The level of ATP-induced IL-1 released in 2 h from LPS-activated whole blood from homozygous subjects was 50% of that from wild-type samples. A more marked defect in IL-1 release was observed from LPS-activated monocytes of homozygous subjects which was only 22% of that released from wild-type monocytes after a 30-min incubation with ATP. However, after a 60-min incubation with ATP, the amount of IL-1 released from homozygous monocytes was 70% of that released from wild-type monocytes. Incubation of monocytes of either genotype with nigericin resulted in a similar release of IL-1. Western blotting demonstrated that ATP induced the release of mature 17-kDa IL-1 from monocytes, and confirmed that this process was impaired in homozygous monocytes. Finally, ATP-induced ⁸⁶Rb⁺ efflux was 9-fold lower from homozygous monocytes than from wild-type monocytes. The results indicate that ATP-induced release of IL-1 is slower in monocytes from subjects homozygous for the Glu⁴⁹⁶Ala polymorphism in the P2X₇ receptor and that this reduced rate of IL-1 release is associated with a lower ATP-induced K⁺ efflux.

	Introduction

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Interleukin-1 is a pleiotropic, inflammatory cytokine released from a variety of cells including activated monocytes, macrophages, and microglia (1). This cytokine is synthesized as a biologically inactive, immature 33-kDa form which is cleaved to a biologically active, mature 17-kDa form by caspase-1 (alternatively termed IL-1-converting enzyme) (2, 3). However, in some inflammatory conditions other proteases may also contribute to IL-1 maturation (4). Inflammatory stimuli, such as LPS, induce synthesis of immature IL-1, but an additional stimulus, such as extracellular ATP, K⁺ ionophores, hypotonic stress, or bacterial toxins, is necessary to cause the release and maturation of IL-1 (5, 6, 7, 8). These secondary stimuli contribute to this process by causing changes in the intracellular ionic environment including the depletion of intracellular K⁺ (5, 6, 7, 8).

The release of IL-1 from monocytes occurs by a pathway other than the classical endoplasmic reticulum-Golgi route (9). Extracellular ATP can induce the release of IL-1 from monocytes via the exocytosis of endolysosome-related vesicles (10) or shedding of microvesicles (11); however, other mechanisms may also exist (12, 13, 14). ATP-induced release of IL-1 is independent of caspase-1 maturation (15), but activation of this protease is necessary for ATP-induced maturation of IL-1 (16). Conversely, ATP-induced release but not maturation of IL-1 is dependent on an influx of extracellular Ca²⁺ (17). Studies with P2X₇ receptor-deficient mice indicate that extracellular ATP mediates the release and maturation of IL-1 via activation of the P2X₇ receptor (18, 19, 20, 21), while the P2X₇ antagonists, KN-62, oxidized ATP, and anti-P2X₇ mAb, inhibit ATP-induced IL-1 release from human monocytes or monocytic cell lines (15, 22, 23).

The P2X₇ receptor is a ligand-gated cation channel which has a wide distribution including cells of the immune and hemopoietic system (24, 25, 26). Activation of this receptor by brief exposure to extracellular ATP opens a cation channel, which allows Ca²⁺ influx, as well as K⁺ efflux (27, 28). Longer exposure to ATP (10–40 s) leads to dilatation of the P2X₇ channel to a pore, which allows uptake of permeants up to the size of ethidium⁺ (29). In addition to the release of mature IL-1 from cells of monocytic lineage (6, 23, 30, 31, 32), P2X₇ activation initiates a number of downstream events including the stimulation of a membrane metalloproteinase which causes shedding of L-selectin (CD62L) from lymphocytes and monocytes (19, 33, 34, 35). Although chemoattractants such as IL-8 can induce the rapid shedding of L-selectin from neutrophils (36), extracellular ATP is the only natural mediator so far described, which can cause L-selectin shedding from monocytes and lymphocytes. Recently, we have identified a single nucleotide polymorphism at nucleotide position 1513 of the human P2X₇ gene (1513AC) that changes a glutamic acid to alanine at aa 496 (Glu⁴⁹⁶Ala) and which leads to a loss of receptor function as assessed by ATP-induced Ca²⁺ and ethidium⁺ influx (37). In this study, we investigated whether the ATP-induced release of IL-1 is impaired in monocytes from subjects homozygous for the Glu⁴⁹⁶Ala polymorphism.

	Materials and Methods

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Materials

LPS (Escherichia coli serotype 055:B5), ATP, nigericin, PMA, RPMI 1640 medium, BSA (Fraction V), DMSO, 7-aminoactinomycin D (7-AAD)³, and ethidium bromide were from Sigma-Aldrich (St. Louis, MO). HEPES, FCS, nonessential amino acids, 2-ME, and PCR reagents were from Invitrogen (Auckland, New Zealand). Ficoll-Paque PLUS was from Amersham Pharmacia Biotech (Uppsala, Sweden). Acetyl-Tyr-Val-Ala-Asp chloromethylketone (YVAD-CMK) was from Bachem (Bubendorf, Switzerland). Protease inhibitor mixture tablets (Complete, Mini, EDTA-free) were from Roche (Penzberg, Germany) and used according to the manufacturer’s instructions. Goat anti-human IL-1 Ab was from R&D Systems (Minneapolis, MN). Peroxidase-conjugated ImmunoPure mouse anti-goat IgG Ab and SuperSignal West Pico Chemiluminescent Substrate were from Pierce Endogen (Rockford, IL). PerkinElmer Life Sciences (Boston, MA) supplied [⁸⁶Rb⁺]Cl (specific activity 5.7 mCi/mg). Di-n-butyl phthalate and di-isooctyl phthalate (BDH Chemicals, Poole, England) were blended 80:20 to give a mixture of density 1.030 g/ml. FITC- and PE-conjugated anti-CD14 mAb and isotype control mAb were from DAKO (Carpinteria, CA). FITC-conjugated L-selectin mAb was from Bender MedSystems (Vienna, Austria), and FITC-conjugated anti-human P2X₇ mAb (clone L4) (22) was prepared as described (35).

Normal subjects and genotyping

Peripheral blood was collected in heparin-containing vacutainer tubes from normal volunteers previously identified as either wild-type or homozygous for the Glu⁴⁹⁶Ala polymorphism (37, 38, 39). Additional genotyping of the P2X₇ gene was performed by sequencing of PCR products as described (40). All subjects were wild type at nucleotide position 1729 of the P2X₇ gene which codes a second loss-of-function polymorphism (Ile⁵⁶⁸Asn) (40). Blood was collected with informed consent and in most instances, blood from each homozygous subject was studied in parallel with blood from a wild-type subject. The experimental protocol was approved by the Wentworth Area Health Service (Penrith, Australia) and University of Sydney Human Ethics Committees (Sydney, Australia).

Measurement of ethidium⁺ influx into monocytes

PBMC were isolated by gradient centrifugation using Ficoll-Paque PLUS, and ATP-induced ethidium⁺ influx into monocytes was measured by time-resolved flow cytometry as described (35). Briefly, 25 µM ethidium⁺ was added to 1 x 10⁶ PBMC, prelabeled with FITC-conjugated anti-CD14 mAb in 1 ml KCl medium (150 mM KCl, 5 mM D-glucose, 0.1% BSA, 10 mM HEPES, pH 7.5) at 37°C, followed 40 s later by the addition of 1 mM ATP. Data was acquired at 1 x 10³ events per second on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). The linear mean channel of ethidium⁺ fluorescence intensity for the gated monocyte population (based on forward and side scatter, and CD14 expression) over successive 5-s intervals was analyzed by WinMDI 2.7 software developed by J. Trotter (The Scripps Research Institute, La Jolla CA; www.scripps.edu) and plotted against time. P2X₇ function was quantitated as the difference in arbitrary units of area under the uptake curves in the presence and absence of ATP in the first 5 min of incubation.

Measurement of ATP-induced shedding of L-selectin

PBMC (2 x 10⁶/ml) were incubated for 5 min in KCl medium at 37°C in the presence or absence of either 0.5 mM ATP or 20 nM PMA. The incubations were terminated by adding 2 volumes of cold isotonic MgCl₂ buffer (10 mM MgCl₂, 145 mM NaCl, 5 mM KCl, 10 mM HEPES, pH 7.5) and centrifugation. Cells were washed once with PBS containing 0.1% NaN₃, and the mean fluorescence intensity of L-selectin expression determined using immunofluorescence labeling and flow cytometry. Results are presented as the percentage of L-selectin expression in the presence of ATP compared with L-selectin expression in the absence of ATP.

Immunolabeling and flow cytometry

PBMC (1 x 10⁶) were labeled with FITC- and/or PE-conjugated mAb, and 7-AAD for 20 min, washed, and analyzed using a FACSCalibur flow cytometer and CellQuest Software (BD Biosciences).

Blood-based IL-1 assay

The release of IL-1 from cells in whole blood was performed as described (41) with some minor changes. Briefly, 100 µl of whole blood and 100 µl of RPMI 1640 medium containing 10 mM HEPES (incomplete medium) were dispensed into flat-bottom 96-well plates. The diluted samples were incubated in the presence of 100 ng/ml LPS for 2 h at 37°C/5% CO₂ and then in the presence or absence of 6 mM ATP for a further 2 h. The samples were centrifuged at 700 x g for 10 min, and the resulting supernatants stored at -30°C until the levels of IL-1 were measured using a human IL-1 ELISA kit (Pierce Endogen).

Mononuclear cell-based IL-1 assay

PBMC were resuspended at 5 x 10⁶ PBMC/ml in RPMI 1640 medium containing 10% heat-inactivated FCS, 100 µM nonessential amino acids, 50 µM 2-ME, 5 µg/ml gentamicin, and 10 mM HEPES (complete medium), and cultured in 24-well plates (0.5 ml/well) at 37°C/5% CO₂. At 2 h the nonadherent cells were removed by gently washing the wells twice with warm incomplete medium. The plastic-adherent cells were cultured for a further 4 h in complete medium containing 100 ng/ml LPS (0.5 ml/well). The medium was then removed and the plastic-adherent cells cultured in the presence or absence of 3 mM ATP or 20 µM nigericin in incomplete medium containing 0.1% BSA (0.5 ml/well) for 30 or 60 min. The samples were centrifuged at 11,000 x g for 10 s, and the resulting supernatants stored at -30°C until the levels of IL-1 were measured by ELISA. Cell pellets and cells remaining in the wells were lysed in a total of 0.5 ml incomplete medium containing 0.2% Triton X-100, and the cell lysates stored at -30°C. Supernatants and cell lysates were assayed for lactate dehydrogenase (LDH) using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI).

Measurements of IL-1 release by Western blotting

PBMC at 3 x 10⁶ PBMC/ml in complete medium were cultured in 6-well plates (5 ml/well) at 37°C/5% CO₂. At 2 h the nonadherent cells were removed by gently washing the wells twice with warm incomplete medium. The plastic-adherent cells were cultured for a further 4 h in complete medium containing 100 ng/ml LPS (5 ml/well). The medium was then removed and the plastic-adherent cells cultured in the presence or absence of 3 mM ATP or 20 µM nigericin in incomplete medium (5 ml/well) for 30 or 60 min. Media were then collected into tubes containing protease inhibitor mixture, and centrifuged at 2500 x g for 5 min at 4°C, and the supernatants were stored at -30°C until required. In some experiments, plastic-adherent cells were preincubated for 15 min in the presence of 60 µM YVAD-CMK or an equal volume of DMSO vehicle before the addition of ATP. Supernatants were concentrated with Ultrafree-15 Centrifugal Filter Devices (5-kDa molecular mass cut-off; Millipore, Bedford, MA), and the proteins separated by SDS-PAGE using 12% polyacrylamide gels (Bio-Rad, Hercules, CA) under reducing conditions and transferred to nitrocellulose (Bio-Rad) as described (42). Immunoblotting was performed as described (23). Briefly, blots were blocked overnight with TBST (50 mM Tris, 250 mM NaCl, 0.1% Tween 20, pH 7.5) containing 5% skim milk powder, washed three times with TBST, and incubated with anti-IL-1 Ab diluted in TBST for 1 h. After washing five times with TBST, blots were incubated with peroxidase-conjugated anti-goat IgG Ab diluted in TBST for 1 h, washed, and incubated with chemiluminescent substrate for 5 min. Images were captured on Biomax MS Imaging Film (Kodak, Rochester, NY).

Measurement of ⁸⁶Rb⁺ efflux from cultured monocytes

Plastic-adherent PBMC in 24-well plates were prepared as above (for the IL-1 ELISA) and incubated for 4 h at 37°C in complete medium containing 100 ng/ml LPS and 5 µCi/ml [⁸⁶Rb⁺]Cl (1 ml/well). The medium was removed and the plastic-adherent cells washed twice with incomplete medium containing 0.1% BSA. The cells were then incubated at 37°C in the presence or absence of 3 mM ATP or 20 µM nigericin in incomplete medium containing 0.1% BSA (1 ml/well) for up to 4 min. At specific time points sample media were overlaid onto 0.3 ml of phthalate oil mixture and centrifuged at 8000 x g for 30 s. Cells underlying the oil layer, which were collected as described (43), and cells remaining in the wells were solubilized in incomplete medium containing 1% Triton X-100. The level of radioactivity in the supernatants overlying the oil layer and in the cell lysates was determined by Cerenkov counting, and the percentage release of ⁸⁶Rb⁺ calculated.

Statistics

Results are expressed as mean ± SEM. Differences in P2X₇ expression, ethidium⁺ uptake, L-selectin loss, and LDH release were compared using the two-tailed Student’s unpaired t test. Differences in IL-1 release and ⁸⁶Rb⁺ release were compared using ANOVA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
ATP-induced ethidium⁺ uptake and L-selectin shedding is impaired in monocytes homozygous for the Glu⁴⁹⁶Ala polymorphism

We have previously shown near complete loss of ATP-induced ethidium⁺ uptake into lymphocytes and monocytes from a normal subject homozygous for the Glu⁴⁹⁶Ala polymorphism (37), while ATP-induced shedding of L-selectin is greatly slowed from leukemic B-lymphocytes expressing nonfunctional P2X₇ (35). Study of an additional three homozygote subjects confirmed that the ATP-induced uptake of ethidium⁺ in monocytes homozygous for the Glu⁴⁹⁶Ala polymorphism was nearly absent when compared with monocytes from wild-type subjects (mean arbitrary units of uptake of 118 ± 72, n = 4 vs 18750 ± 1801, n = 7 respectively; p < 0.001; Fig. 1). The surface expression of P2X₇ on monocytes from homozygous subjects was lower than P2X₇ expression on monocytes from wild-type subjects (mean fluorescence intensity of 24.8 ± 7.6, n = 4 vs 39.5 ± 5.6, n = 7 respectively; Fig. 2), but this difference did not reach significance (p = 0.18).

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Impaired ATP-induced ethidium+ uptake into monocytes homozygous for the Glu496Ala polymorphism. PBMC from a subject either wild type at 496 (•) or homozygous for the Glu496Ala polymorphism () were labeled with FITC-CD14 mAb and suspended in KCl medium at 37°C. Ethidium+ (25 µM) was added, followed 40 s later by the addition of 1 mM ATP (arrow). Mean channel of cell-associated fluorescence was measured by time-resolved flow cytometry for monocytes incubated in the presence (•, ) or absence of extracellular ATP ().

View larger version (13K): [in this window] [in a new window]   FIGURE 2. P2X7 receptor expression on monocytes. PBMC from a subject either wild type at 496 (top panel) or homozygous for the Glu496Ala polymorphism (bottom panel) were stained with FITC-P2X7 (solid line) or FITC-isotype control (shaded line) mAb, PE-CD14 mAb, and 7-AAD, and analyzed by flow cytometry.

Impaired ATP-induced release of IL-1 in subjects homozygous for the Glu⁴⁹⁶Ala polymorphism

The release of IL-1 from leukocytes in the presence or absence of ATP was assayed by a blood-based method in which whole blood is pretreated with LPS (41). Incubation of blood from wild-type subjects with ATP produced a release of IL-1 of 5806 ± 875 pg/ml (Fig. 3). Incubation of blood from homozygous subjects with ATP produced a release of IL-1 of 3034 ± 531 pg/ml which was 50% of that observed for wild-type subjects (p < 0.01; Fig. 3). Release of IL-1 from blood in the absence of ATP was <450 pg/ml (Fig. 3).

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Impaired ATP-induced release of IL-1 from peripheral blood cells homozygous for the Glu496Ala polymorphism. Diluted blood samples from subjects either wild type at 496 () or homozygous for the Glu496Ala polymorphism () were incubated at 37°C in the presence of 100 ng/ml LPS for 2 h and then for a further 2 h in the absence (control) or presence of 6 mM ATP. Supernatants were collected and the amount of IL-1 released was measured by ELISA. Results are expressed as the mean ± SEM (n = 6 wild-type values and 3 homozygote values); *, p < 0.02 or **, p < 0.001 to corresponding control sample; , p < 0.01, ATP-treated wild-type vs ATP-treated homozygote.

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Impaired ATP-induced release of IL-1 from monocytes homozygous for the Glu496Ala polymorphism. Plastic-adherent monocytes from subjects either wild type at 496 () or homozygous for the Glu496Ala polymorphism () were incubated at 37°C in the presence of 100 ng/ml LPS for 4 h and then for a further 30 min (top panel) or 60 min (bottom panel) in the absence (control) or presence of 3 mM ATP or 20 µM nigericin. Supernatants were collected and the amount of IL-1 released was measured by ELISA. Results are expressed as the mean ± SEM (n = 6–7 wild-type values and 3–4 homozygote values); *, p < 0.001 to corresponding control sample; , p < 0.01, ATP-treated wild-type vs ATP-treated homozygote.

ATP induces the release of mature IL-1

Western blotting of cell supernatants was performed to determine the maturation status of the IL-1 released. Plastic-adherent monocytes from wild-type and homozygote subjects were activated for 4 h with LPS and then incubated a further 30 min with or without ATP. In the absence of ATP no detectable IL-1 was released from monocytes of either genotype (Fig. 5). In the presence of ATP, IL-1 was present in supernatants from monocytes but only in the mature 17-kDa form (Fig. 5). Lesser amounts of 17-kDa IL-1 were detectable in supernatants from homozygote monocytes than from wild-type monocytes (Fig. 5). A similar amount of mature 17-kDa IL-1 was present in supernatants from monocytes of either genotype incubated with nigericin (Fig. 5).

View larger version (12K): [in this window] [in a new window]   FIGURE 5. Impaired ATP-induced release of mature IL-1 from monocytes homozygous for the Glu496Ala polymorphism. Plastic-adherent monocytes from subjects either wild type at 496 or homozygous for the Glu496Ala polymorphism were incubated at 37°C in the presence of 100 ng/ml LPS for 4 h and then for a further 30 min in the absence (control) or presence of 3 mM ATP or 20 µM nigericin. IL-1 released into the medium was visualized using Western blotting. Results are representative of two separate experiments using different wild-type and homozygote subjects.

View larger version (17K): [in this window] [in a new window]   FIGURE 6. ATP-induced maturation of IL-1 from monocytes is impaired by the caspase-1 inhibitor, YVAD-CMK. Plastic-adherent monocytes from subjects either wild type at 496 or homozygous for the Glu496Ala polymorphism were incubated at 37°C in the presence of 100 ng/ml LPS for 4 h, then preincubated for 15 min with DMSO vehicle or 60 µM YVAD-CMK before incubation for a further 30 or 60 min with 3 mM ATP. IL-1 released into the medium was visualized using Western blotting. Results are representative of two separate experiments using different wild-type and homozygote subjects.

Supernatants and cell lysates from LPS-treated monocytes were assayed for LDH release as a measure of cell lysis. Monocytes from homozygous subjects after 30 and 60 min with ATP released a similar but minimal amount of extracellular LDH (5.8 ± 3.9% vs 6.6 ± 2.9% respectively, n = 4, p = 0.86). The release of LDH from wild-type monocytes after 30 and 60 min with ATP was also similar (5.1 ± 1.9% vs 9.6 ± 4.4% respectively, n = 7, p = 0.32). There was no measurable extracellular LDH in supernatants from monocytes of either genotype after incubation with nigericin (results not shown).

Impaired ATP-induced ⁸⁶Rb⁺ efflux in subjects homozygous for the Glu⁴⁹⁶Ala polymorphism

K⁺ efflux from monocytes is necessary for the release and maturation of IL-1 in response to either ATP or nigericin (6, 31). Therefore, to investigate whether ATP-induced K⁺ efflux is impaired from monocytes homozygous for the Glu⁴⁹⁶Ala polymorphism, LPS-primed monocytes were loaded with ⁸⁶Rb⁺, a surrogate for K⁺, and its release was measured. Preliminary results indicated that ⁸⁶Rb⁺ release reached a plateau at 2 min or longer after addition of ATP (results not shown). Incubation of wild-type monocytes with ATP for 2 min produced a release of ⁸⁶Rb⁺ of 63.0 ± 2.1% (Fig. 7), while incubation of homozygous monocytes with ATP produced a 35.9 ± 1.5% release which was significantly lower than that observed for wild-type monocytes (p < 0.001; Fig. 7). In contrast, incubation of wild-type and homozygous monocytes with nigericin resulted in a similar release of ⁸⁶Rb⁺ at 2 min (59.5 ± 3.1% vs 62.6 ± 4.2% respectively, p = 0.35; Fig. 7). Release of ⁸⁶Rb⁺ from monocytes in the absence of agonist was 24.3 ± 1.0% for wild-type monocytes and 31.7 ± 3.0% for homozygous monocytes (Fig. 7). Subtraction of these control values from the total ⁸⁶Rb⁺ release gives values for ATP-induced ⁸⁶Rb⁺ release of 38.7 ± 1.1% for wild type and 4.2 ± 2.0% for homozygous monocytes. Thus the ATP-induced ⁸⁶Rb⁺ efflux was 9-fold greater for wild type than homozygous monocytes. Similar results were also observed when ⁸⁶Rb⁺ release was measured from monocytes after 1 min of incubation (results not shown).

View larger version (14K): [in this window] [in a new window]   FIGURE 7. Impaired ATP-induced 86Rb+ efflux from monocytes homozygous for the Glu496Ala polymorphism. Plastic-adherent monocytes from subjects either wild type at 496 () or homozygous for the Glu496Ala polymorphism () were incubated at 37°C in the presence of 100 ng/ml LPS and 5 µCi/ml [86Rb+]Cl for 4 h, washed, and then incubated in the absence (control) or presence of 3 mM ATP or 20 µM nigericin for 2 min. Radioactivity in supernatants and cell lysates was counted, and the percentage release of 86Rb+ determined. Results are expressed as the mean ± SEM (n = 3 wild-type values and 3 homozygote values); *, p < 0.001 to corresponding control sample; , p < 0.001, ATP-treated wild-type vs ATP-treated homozygote.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It is generally accepted that ATP-induced release of IL-1 occurs before cell lysis (49) and is independent of caspase-1 maturation (15). The low but similar levels of LDH released from monocytes of either genotype and at either time point indicates that the ATP-induced release of IL-1 from homozygote monocytes after 60 min incubation with ATP was not due to cell lysis. Cleavage of caspase-1 is necessary for ATP-induced maturation of IL-1 (16). Both ELISA, which preferentially detects mature IL-1 (14, 17), and Western blotting demonstrated that monocytes from Glu⁴⁹⁶Ala homozygotes release much lower amounts of 17-kDa IL-1 than wild-type monocytes after a 30-min incubation with ATP. After a 60-min incubation with ATP, monocytes of either genotype released increased levels of IL-1 which was predominately in the 17-kDa form. Pretreatment of monocytes with the caspase-1 inhibitor, YVAD-CMK, caused a reduction in the levels of 17-kDa IL-1 released and an increase in the quantities of 33-kDa IL-1 released following ATP treatment, which indicates that activation of caspase-1 is necessary for ATP-induced maturation of IL-1 in both wild-type and homozygote monocytes.

K⁺ efflux is thought to be essential for IL-1 release as increasing the extracellular K⁺ concentration is known to impair IL-1 release from both murine and human macrophages stimulated with either ATP or nigericin (6, 31). Whether this ATP-induced K⁺ efflux from monocytes activates the Ca²⁺-independent phospholipase A₂ to cause the subsequent processing of IL-1 as observed for nigericin (50) is unknown. However, our data confirms previous observations with murine macrophages (6, 51) and human monocytes (52) that ATP causes a substantial efflux of ⁸⁶Rb⁺ from monocytic cells, which is an order of magnitude more rapid than that from lymphocytes (40, 43). Moreover, our data shows that ATP- but not nigericin-induced ⁸⁶Rb⁺ (K⁺) efflux was reduced 9-fold from homozygous monocytes, supporting a role for K⁺ efflux in promoting IL-1 release. The flow cytometric measurements of ATP-induced ethidium⁺ uptake on three Glu⁴⁹⁶Ala homozygous subjects, in addition to the one previously published (37), confirm that ATP-induced pore formation is significantly impaired in monocytes homozygous for this polymorphism. However, the Glu⁴⁹⁶Ala polymorphism may not result in a total loss of P2X₇ channel function. Significant ATP-induced currents can be seen in either Xenopus oocytes or HEK293 cells transfected with Glu⁴⁹⁶Ala mutant human P2X₇ constructs (53). Recent work from our group has shown that truncation of the rat P2X₇ construct at several sites in the C-terminal tail, including aa 460, abolishes ATP-induced ethidium⁺ uptake but not channel activity (54) suggesting that amino acids in the C-terminal tail, such as Glu⁴⁹⁶, may be essential for dilatation of the channel to a larger pore. Therefore, although ATP-induced ⁸⁶Rb⁺ (K⁺) efflux from homozygous monocytes is greatly reduced compared with wild-type monocytes, the smaller residual K⁺ efflux through the P2X₇ channel may be sufficient to cause the delayed release of IL-1 from homozygous monocytes after a 60-min incubation with ATP.

The availability of biologically active IL-1 is known to be affected by other polymorphisms in the genes coding IL-1, the IL-1 receptor, and IL-1 receptor antagonist, and such polymorphisms are associated with a number of diseases including rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis, and tuberculosis (see Ref. 55 and references therein). Recent studies with knockout mice indicate that extracellular ATP released at sites of inflammation can activate P2X₇ receptors in vivo. In a murine model of mAb anti-collagen-induced arthritis the incidence and severity of disease was impaired in P2X₇-deficient animals, which corresponded to a reduction in cellular infiltrate within the cartilage (19). These results suggest that P2X₇ activation by ATP released at sites of injury or infection may be important in inflammation and immunity, and that impairment of IL-1 release due to loss-of-function polymorphisms of the P2X₇ receptor may be of significance in certain diseases. Whether a second loss-of-function polymorphism recently identified in the P2X₇ receptor (40) affects IL-1 release is currently unknown, but examination of loss-of-function polymorphisms within the P2X₇ receptor is warranted in certain inflammatory and infectious diseases.

	Acknowledgments

 
We thank Dr. George Dubyak and Dr. Christopher Gabel for helpful advice regarding the IL-1 assays, as well as Kristin Skarratt for additional genotyping, Dr. Ben Gu for preparing the L4 mAb, and Dr. Stephen Fuller for coordinating the collection of some blood samples.

	Footnotes

 
¹ This work was supported by the National Health and Medical Research Council of Australia, the Leukaemia Foundation of New South Wales, and the Clive and Vera Ramaciotti Foundation. A.N.S. was a recipient of a Faculty of Medicine Postgraduate Award from the University of Sydney.

² Address correspondence and reprint requests to Dr. Ronald Sluyter, Department of Medicine, University of Sydney, Level 5 South Block, Nepean Hospital, Penrith, New South Wales 2750, Australia. E-mail address: rons{at}med.usyd.edu.au

³ Abbreviations used in this paper: YVAD-CMK, acetyl-Tyr-Val-Ala-Asp chloromethylketone; LDH, lactate dehydrogenase; 7-AAD, 7-aminoactinomycin D.

Received for publication June 16, 2003. Accepted for publication December 18, 2003.

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