Biochimica et Biophysica Acta 1798 (2010) 1797–1804

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Biochimica et Biophysica Acta

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P2X7 receptor activation induces cell death and microparticle release in murineerythroleukemia cells

Patrick Constantinescu a, Bin Wang a, Kati Kovacevic a, Iman Jalilian a, Giel J.C.G.M. Bosman b,James S. Wiley c, Ronald Sluyter a,⁎a School of Biological Sciences, University of Wollongong, Wollongong, Australiab Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlandsc Florey Neurosciences Institutes, University of Melbourne, Australia

Abbreviations: BzATP, 2′- and 3′-0(4-benzoylbenzoyMP, microparticle; MEL, murine erythroleukemia; MTbromide; mAb, monoclonal antibody; 7AAD, 7-aminoactmean fluorescence intensity⁎ Corresponding author. Tel.: +61 2 4221 5508; fax:

E-mail address: rsluyter@uow.edu.au (R. Sluyter).

0005-2736/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.bbamem.2010.06.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 March 2010Received in revised form 17 May 2010Accepted 1 June 2010Available online 8 June 2010

Keywords:P2X receptorRed blood cellMacrophageApoptosisMicroparticle

Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in amanner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells isunknown. This study investigated whether MEL cells express functional P2X7. RT-PCR, immunoblotting andimmunofluorescence staining demonstrated the presence of P2X7 in MEL cells. Cytofluorometricmeasurements demonstrated that ATP induced ethidium+ uptake into MEL cells in a concentration-dependent fashion and with an EC50 of ∼154 μM. The most potent P2X7 agonist 2′- and 3′-0(4-benzoylbenzoyl) ATP, but not ADP or UTP, induced ethidium+ uptake. ATP-induced ethidium+ and YO-PRO-12+ uptake were impaired by the P2X7 antagonist, A-438079. A colourmetric assay demonstrated thatATP impaired MEL cell growth. A cytofluorometric assay showed that ATP induced MEL cell death and thatthis process was impaired by A-438079. Finally, cytofluorometric measurements of Annexin-V binding andbio-maleimide staining demonstrated that ATP could induce rapid phosphatidylserine exposure andmicroparticle release in MEL cells respectively, both of which were impaired by A-438079. These resultsdemonstrate that MEL cells express functional P2X7, and indicate that activation of this receptor may beimportant in the death and release of microparticles from red blood cells in vivo.

l) ATP; PS, phosphatidylserine;T, thiazolyl blue tetrazoliuminomycin D; Ab, antibody; MFI,

+61 2 4221 4135.

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

It is now firmly established that extracellular nucleotides andnucleosides operate through a complex network of purinergicsignalling cascades involving various integral membrane receptorsand ectoenzymes [1–4]. One suchmember of this network is the P2X7receptor, a trimeric ligand-gated cation channel belonging to thefamily of P2X receptors [2,5]. Activation of P2X7 by ATP causes themovement of Ca2+, Na+ and K+ across the plasma membrane, as wellas the uptake of organic cations including fluorescent dyes such asethidium+ and YO-PRO-12+ [5,6]. P2X7 is also activated by 2′- and 3′-0(4-benzoylbenzoyl) ATP (BzATP) but not other nucleotides such asADP and UTP, while its relative function is increased in the absence ofextracellular Ca2+, Na+ and Mg2+ ions [5,6]. P2X7 activation inducesan array of downstream events, in a cell specificmanner, including the

rapid exposure of phosphatidylserine (PS) and subsequent micropar-ticle (MP) release in the form of either microvesicles or exosomes [7],as well as cell death [8].

P2X7 is predominately expressed on mononuclear leukocytes [9]and epithelial cells [10], and osteoblasts and osteoclasts [11], where itplays a role in inflammatory and immune responses [12,13] and bonehomeostasis [14] respectively. P2X7 is also present in mature redblood cells (erythrocytes) but its physiological role is not welldefined. P2X7 has been observed in erythrocytes from several specieswhere it mediates ATP-induced cation fluxes, epoxyeicosatrienoicacid release, PS exposure and cell death [15–20]. However, with theexception of P2X7 mRNA in human reticulocytes [21,22], theexpression of P2X7 in immature red blood cells is limited. Over25 years ago, Chatawala and Cantley observed that extracellular ATPcould induce Ca2+, Na+ and K+

fluxes in and impair the growth ofmurine erythroleukemia (MEL) cells [23], a commonly usedmodel ofred blood cell biology [24]. Although not considered at the time, theATP-induced cation fluxes in MEL cells were characteristic of P2X7,being maximal at 1 mM ATP and with an EC50 value of ∼700 μM,andwere increased in the absence of extracellular Ca2+ andNa+ [23].Collectively this data suggested that MEL cells express P2X7,however the identity of the receptor in these cells has never beenconfirmed.

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Using molecular, immunochemical and pharmacological evidence,we report that MEL cells express P2X7, and that activation of thisreceptor in these cells by ATP induces organic cation uptake and celldeath, as well as rapid PS exposure and MP release. This cell line willprovide a useful model for future studies of P2X7 in red cell biology,and suggests that P2X7 may be important in the death and release ofmicroparticles from red blood cells in vivo.

2. Materials and methods

2.1. Materials

RPMI-1640 medium, fetal calf serum (heat-inactivated beforeuse), horse serum and L-glutamine were from Invitrogen (GrandIsland, NY). ATP, BzATP, ADP, UTP, ethidium bromide and thiazolylblue tetrazolium bromide (MTT) were from Sigma (St. Louis, MO). A-438079 was from Tocris Bioscience (Ellisville, MO). Rat anti-murineP2X7 monoclonal antibody (mAb) (clone HANO43) and 7-aminoacti-nomycin D (7AAD) were from Alexis Biochemicals (Lausen,Switzerland). Rat IgG2a isotype control mAb and FITC-conjugatedmurine anti-rat IgG polyclonal antibody (Ab) were from eBioscience(San Diego, CA). Rabbit anti-rat P2X7 (C-termini epitope) polyclonalAb and blocking peptide (used according to the manufacturer'sinstructions) were fromAlomone Labs (Jerusalem, Israel). Peroxidase-conjugated goat anti-rabbit IgG Ab was from Rockland (Gilbertsville,PA). Protease inhibitor cocktail tablets (complete, Mini, EDTA-free),phenyl-methyl-sulfonyl-fluoride and Annexin-V-FLUOS were fromRoche Diagnostics (Pensberg, Germany). SuperSignal® West PicoChemiluminescent Substrate was from Pierce (Rockford, IL). YO-PRO-12+ and bio-maleimide, alternatively known as BODIPY FL N-(2-aminoethyl)maleimide, were from Molecular Probes (Invitrogen,Eugene, OR). Fluoresbrite YG 0.5 μm microspheres were fromPolysciences Inc. (Warrington, PA). Flow-Count fluorospheres werefrom Beckman Coulter (Fullerton, CA). Other reagent grade chemicalswere from Sigma or Amresco (Solon, OH).

2.2. Cells

RAW264.7 cells were obtained from the American Type CultureCollection (Manassas, VA). MEL cells were kindly provided by Dr. SallyA. Eaton (University of Sydney, Sydney, Australia). Both cell lines weremaintained in complete culture medium (RPMI-1640 mediumcontaining 10% fetal calf serum and 5 mM L-glutamine) at 37 °C and95% air/5% CO2. RAW264.7 cells were harvested by cell scraping.

2.3. P2X7 expression by RT-PCR

Total RNA was isolated from cells using the RNeasy Mini Kit(Qiagen, Hilden, Germany) according to the manufacturer's instruc-tions. PCR amplification was performed using Superscript™ III One-Step RT-PCR System Platinum Taq DNA polymerase (Invitrogen), andforward primer 5′-ATATCCACTTCCCCGGCCAC-3′ and reverse primer5′-TCGGCAGTGATGGGACCAG-3′ (GeneWorks, Hindmarsh, Australia)[25] for 42 cycles (94 °C, 1 min; 54 °C, 1 min; 72 °C, 1 min) as des-cribed [26]. PCR products were separated on a 2% agarose gel andvisualised using ethidium bromide staining.

2.4. P2X7 expression by immunoblotting

Cells were washed three times in ice-cold PBS and then lysed(1×107 cells/ml) in ice-cold 50 mM BisTris (pH 7.0) containing750 mM 6-aminohexanoic acid, 1% n-dodecyl β-D-maltoside, 1 mMphenyl-methyl-sulfonyl-fluoride and a protease inhibitor cocktailover 60 min, sheared 10 times through a 21 G needle and centrifuged(16,000×g for 10 min at 4 °C). Cell supernatants (equivalent to1×105 cells/well) were then separated under reducing conditions

(5% 2-mercaptoethanol) using 4–20% gradient iGels (NuSep, Austell,GA) and transferred to nitrocellulosemembranes (Bio-Rad, Hercules,CA). Nitrocellulose membranes were blocked overnight at 4 °Cwith Tris-buffered saline (250 mM NaCl and 50 mM Tris, pH 7.5)containing 0.2% Tween-20 and 5% skim milk powder, and thenincubated for 2 h at room temperature with anti-P2X7 Ab diluted inTris-buffered saline containing 0.2% Tween-20 and 5% skim milkpowder. Membraneswerewashed three times over 30 minwith Tris-buffered saline containing 0.2% Tween-20 and then incubated for 1 hat room temperature with peroxidase-conjugated anti-rabbit IgGantibody diluted in Tris-buffered saline containing 0.2% Tween-20and 5% skim milk powder. Membranes were washed as above,incubated with chemiluminescent substrate and visualised usingAmersham Hyperfilm ECL (GE Healthcare, Little Chalfont, Buckin-ghamshire, UK).

2.5. P2X7 expression by immunofluorescence staining

Cells in PBS containing 10% horse serum and 0.01% NaN3 (1×106

cells/100 μl) were incubated with isotype control or anti-P2X7 mAbfor 30 min room temperature, washed twice with PBS containing0.01% NaN3, incubated with FITC-conjugated murine anti-rat IgGpolyclonal Ab and 7AAD (to exclude dead cells) for 30 min at roomtemperature, and washed as above. Data was collected using a LSR IIflow cytometer (BD Biosciences, San Diego, CA) and the mean fluores-cence intensity (MFI) determined using FlowJo software (Tree Star,Ashland, OR).

2.6. Fluorescent cation dye uptake assay

P2X7-induced pore formation was assessed using a fixed-timeassay as described [27]. Briefly, cells resuspended in NaCl medium(145 mM NaCl, 5 mM KCl, 5 mM glucose, 0.1% bovine serum albumin,and 10 mM HEPES, pH 7.5) (1×106 cells/ml) were incubated with25 μM ethidium+ (or 1 μM YO-PRO-12+ were indicated) in theabsence or presence of nucleotide for 5 to 15 min as indicated at37 °C. Incubationswere stopped by addition of an equal volume of ice-cold NaCl medium containing 20 mM MgCl2 (MgCl2 medium) andcentrifugation (300×g for 5 min). Cells were washed once with NaClmedium. In some experiments, cells were pre-incubated in theabsence or presence of 10 μMA-438079 for 15 min at 37 °C before theaddition of ethidium+ or YO-PRO-12+. Data was collected by flowcytometry and the MFI of ethidium+ uptake determined using FlowJosoftware.

2.7. Measurement of cell growth by the MTT assay

MEL cells in complete culture media in 96-well plates (5×104

cells/100 μl/well) were incubated in the absence or presence of ATP(as indicated) for 24 h at 37 °C and 95% air/5% CO2. During the final4 h of ATP incubation, 0.5 mg/ml MTT was added (10 μl/well). Cellswere then incubated with 10 mM HCl containing 10% SDS (solubili-zation solution) (100 μl/well) overnight at 37 °C. Absorbances wereread at a wavelength of 550 nm and a reference wavelength of690 nm using a SpectaMax Plus384 microplate reader (MolecularDevices, Sunnyvale, CA).

2.8. Measurement of cell death by a cytofluorometric assay

MEL cells in complete culture media in 24-well plates (5×105

cells/ml/well) were incubated in the absence or presence of 2 mMATP for 24 h at 37 °C and 95% air/5% CO2. In some experiments, cellswere pre-incubated in the absence or presence of 10 μMA-438079 for15 min at 37 °C before the addition of ATP. Following incubation withATP, cells were harvested and washed once with NaCl mediumcontaining 5 mM CaCl2. Cells in 100 μl NaCl medium containing 5 mM

Fig. 1. P2X7 is present on MEL cells. (A) RNA was isolated from RAW264.7 and MELcells, and analysed by RT-PCR using primers to P2X7. RNA substituted with H2O wasused as a negative control. PCR products were visualised using ethidium bromide.(B) Whole cell lysates of RAW264.7 and MEL cells were separated by electrophoresis,transferred to nitrocellulose membrane and probed with anti-P2X7 Ab, pre-incubatedin the absence (Ab) or presence of blocking peptide (Peptide/Ab). (C) RAW264.7 andMEL cells were labelled with anti-P2X7 (solid line) or isotype control (shaded) mAb, andwith FITC-conjugated anti-rat IgG Ab and 7AAD (to exclude dead cells), and analysed byflow cytometry. Results are representative of 2–3 experiments.

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CaCl2 were then incubated with Annexin-V-FLUOS and 7AAD for15 min at room temperature, before addition of 400 μl NaCl mediumcontaining 5 mMCaCl2. Data was collected by flow cytometry, and thecells gated to exclude dust particles and small cellular debris (eventsof low forward and side scatter), and the percentage of Annexin-V+/7AAD−, Annexin-V+/7AAD+ and Annexin-V−/7AAD+ cells withinthe gate determined using FlowJo software.

2.9. PS exposure assay

Cells were resuspended in PS exposure assay medium (147 mMNaCl, 2 mM KCl, 12 mM glucose, 2.5 mM CaCl2, 1 mM MgCl2, and10 mM HEPES, pH 7.5) (1×106 cells/500 μl) and pre-incubated in theabsence or presence of 10 μMA-438079 for 15 min at 37 °C. Cells werethen incubated with Annexin-V-FLUOS in the absence or presence of2 mM ATP for a further 5 min at 37 °C. Incubations were stopped byaddition of an equal volume of ice-cold MgCl2 medium andcentrifugation (300×g for 5 min). Cells were washed once withNaCl medium and incubated with 7AAD (to exclude dead cells) for5 min before flow cytometric analysis to determine the MFI ofAnnexin-V-FLUOS binding using FlowJo software.

2.10. MP release assay

ATP-induced MP release was conducted as previously described[28]. Adherent RAW264.7 cells or suspended MEL cells in MP releaseassay medium (147 mMNaCl, 2 mM KCl, 12 mM glucose, 2 mM CaCl2,1 mM MgCl2, and 10 mM HEPES, pH 7.5; 0.2 μM filtered) in 24-wellplates (0.5×106 cells/500 μl/well) were incubated in the absence orpresence of 2 mM ATP for 10 min at 37 °C and 95% air/5% CO2.Incubations were stopped by centrifugation (300×g for 5 min) and450 μl of each supernatant collected. In some experiments, cells werepre-incubated in the absence or presence of 10 μM A-438079 for15 min at 37 °C before the addition of ATP.

The amount of MPs in each supernatant was then quantified usingbio-maleimide as previously described [29,30]. Briefly, supernatants(450 μl) were incubated with 25 μl of 50 μMbio-maleimide for 15 minat room temperature. Then to each sample, 25 μl of Flow-Countfluorospheres was added, the samples vortexed and analysed by flowcytometry collecting 2000 events within the Flow-Count fluoro-spheres gate. Amounts of bio-maleimide+ MPs per ml were thenenumerated based on the number of Flow-Count fluorospheresaccording to the manufacturer's instructions. Fluoresbrite YG 0.5 μmmicrospheres, suspended in MP release assay medium, were alsoanalysed by flow cytometry and used to approximate the size of MPs.All data was analysed using FlowJo software.

2.11. Presentation of data, statistics and nomenclature

Results are represented as means±SD. Data was compared usingeither the unpaired Student's t-test for single comparisons or one-wayanalysis of variance (using Tukey's post test) for multiple comparisonsusing Prism 5 for Mac OS X Version 5.0a (GraphPad Software,San Diego, CA) with Pb0.05 considered significant. The nomenclaturefor P2X7, rather than P2X7, has been adopted according to recentguidelines [5,31,32].

3. Results

3.1. P2X7 is expressed in MEL cells

In order to determine if MEL cells express P2X7, mRNA was isolatedand amplified by RT-PCR. RAW264.7 cells, which are well known toexpress P2X7 [27,28,33,34], were included as a positive control. P2X7mRNAwasexpressed in both cell types (Fig. 1A).Next,whole cell lysateswere prepared and the presence of P2X7 protein examined by

immunoblotting. Immunoblotting with an anti-P2X7 polyclonal Abrevealed the presence of a major band at 75 kDa, the predicted size ofP2X7, in both RAW264.7 and MEL cells (Fig. 1B). Pre-incubation of theanti-P2X7 polyclonal Ab with blocking peptide abolished immunoreac-tivity (Fig. 1B). Finally, cells were incubated with an anti-P2X7 mAb[35] and examined by flow cytometry. Immunolabelling demonstratedcell-surface P2X7 expression on RAW264.7 and MEL cells with a MFIof 25.6±12.1 and 5.2±3.0 respectively (Fig. 1C). Collectively, theseresults indicate that MEL cells express P2X7.

3.2. P2X7 agonists induce ethidium+ uptake into MEL cells

Next, a fixed-time ethidium+ uptake assay [27] was used todetermine if P2X7 on MEL cells is functional. Again RAW264.7 cells

Fig. 2. ATP induces ethidium+ uptake into MEL cells. RAW264.7 and MEL cells in NaClmedium containing 25 μM ethidium+ were incubated in the absence (basal) orpresence of 1 mM ATP at 37 °C for up to 15 min. Incubations were stopped by additionof MgCl2 medium and centrifugation, and the cells analysed by flow cytometry. Resultsare mean±SD (n=3); where SDs are not visible, SDs are≤1.1; ⁎⁎Pb0.01 compared tocorresponding basal.

Fig. 3. P2X7 agonists induce ethidium+ uptake intoMEL cells. MEL cells in NaClmediumcontaining 25 μM ethidium+ were incubated at 37 °C for 15 min in the (A) absence orpresence of varying ATP concentrations (as indicated), or (B) in the absence (basal) or1 mM agonist (except BzATP, 200 μM). Incubations were stopped by addition of MgCl2medium and centrifugation, and the cells analysed by flow cytometry. Ethidium+

uptake is expressed as (A) percent maximum response compared to 2 mM ATP or(B) mean fluorescence intensity. Results are mean±SD (n=3); ⁎⁎Pb0.01 compared tobasal.

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were used as a positive control. Consistent with previous findings[28,34], ATP induced ethidium+ uptake into RAW264.7 cells in a time-dependent manner (Fig. 2). ATP also induced ethidium+ uptake intoMEL cells in a time-dependent manner (Fig. 2), although the level ofuptake was lower than that of RAW264.7 cells. Ethidium+ uptake inthe absence of ATP was similar in both cell lines (Fig. 2).

MEL cells were then incubated with increasing concentrations ofATP. ATP induced ethidium+ uptake in a concentration-dependentmanner, withmaximal uptake occurring at 1 to 2 mMATP andwith anEC50 of 154±13 μM (Fig. 3A). MEL cells were also incubated in thepresence of the most potent P2X7 agonist BzATP, as well as ADP andUTP, which do not activate P2X7 [36]. BzATP, but not ADP or UTP,induced ethidium+ uptake into MEL cells (Fig. 3B).

3.3. A P2X7 antagonist impairs ATP-induced ethidium+ uptake intoMEL cells

To confirm that the ATP-induced ethidium+ uptake into MEL cellswas mediated by P2X7, cells were pre-incubated in the absence orpresence of the P2X7 antagonist A-438079 [37]. Pre-incubation withA-438079 significantly inhibited ATP-induced ethidium+ uptake intoMEL cells by 80±2% (Fig. 4A). To determine if P2X7 activation couldinduce the uptake of a second cation, cells were pre-incubated in theabsence or presence of A-438079, and ATP-induced YO-PRO-12+

uptake examined. As for ethidium+, ATP induced YO-PRO-12+ uptakeinto MEL cells, and pre-incubation with A-438079 significantlyimpaired this uptake by 79±9% (Fig. 4B).

3.4. P2X7 induces cell death in MEL cells

A previous study has shown that extracellular ATP impairs thegrowth of MEL cells [23], however the mechanism was never deter-mined. Therefore, to confirm if extracellular ATP can impair thegrowth of MEL cells, cells were incubated with increasing concentra-tions of ATP and the relative cell number (expressed as absorbance)determined using the MTT assay. ATP reduced cell numbers in aconcentration-dependent manner, with a maximal reduction occur-ring at 2 mM ATP (Fig. 5).

The MTT assay measures metabolic activity rather than cellnumbers directly, thus the decrease in absorbances following ATP

incubation may be a result of decreased metabolic activity rather thandecreased cell numbers (as a result of either cell death or reducedproliferation). However, given the well-known cytolytic role of P2X7in various cell types [8], we reasoned that the reduced absorbancesfollowing ATP incubation were due to increased cell death. Therefore,MEL cells were incubated with 2 mM ATP for 24 h, and the amount ofcell death was determined by flow cytometry using Annexin-Vbinding (PS exposure) and 7AAD uptake (loss of plasma membraneintegrity) (Fig. 6A). Incubation with 2 mM ATP induced an approx-imately 3.1-fold increase in Annexin-V+ and 7AAD+ cells, whichrepresents either necrotic or late apoptotic cells or a combination ofboth [38], compared to cells incubated in the absence of ATP (basal)(Fig. 6B). Incubation with 2 mMATP resulted in an approximately 3.3-fold and 1.6-fold increase in Annexin-V+/7AAD− (early apoptotic)cells and Annexin-V−/7AAD+ (necrotic) cells compared to basal cells(Fig. 6B).

To determine if ATP induced MEL cell death via activation of P2X7,cells were pre-incubated in the absence or presence of A-438079,before overnight incubation in the absence or presence of 2 mM ATP.As above, incubation with 2 mM ATP induced a significant increase inAnnexin-V+/7AAD– (Fig. 7A) and Annexin-V+/7AAD+ cells (Fig. 7B)compared to basal cells. In contrast, 2 mM ATP induced a small in-crease in the percentage Annexin-V−/7AAD+cells compared to basalcells, but this failed to reach statistical significance (results not shown).

Fig. 4. The P2X7 antagonist, A-438079, impairs ATP-induced ethidium+ and YO-PRO-12+

uptake into MEL cells. MEL cells in NaCl medium were pre-incubated in the absence orpresence of 10 μM A-438079 at 37 °C for 15 min, and then with (A) 25 μM ethidium+ or(B) 1 μMYO-PRO-12+ in the absence (basal) or presence of 1 mMATP at 37 °C for 15 min.Incubations were stopped by addition of MgCl2 medium and centrifugation, and the cellsanalysed by flow cytometry. Results are mean±SD (n=3); ⁎Pb0.05 and ⁎⁎Pb0.01compared to corresponding basal; ††Pb0.01 compared to ATP alone.

Fig. 6. ATP induces cell death in MEL cells. MEL cells in complete culture media wereincubated in the absence (basal) or presence of 2 mM ATP at 37 °C for 24 h. Cells wereharvested, and incubated with Annexin-V-FLUOS and 7AAD, and analysed by flowcytometry. (A) Representative dot plots shown (B)with results expressed asmean±SD(n=3); ⁎⁎Pb0.01 compared to corresponding basal.

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Pre-incubation with A-438079 significantly impaired the ATP-in-duced increase of Annexin-V+/7AAD− and Annexin-V+/7AAD+ cellsby 88±6% and 78±15% respectively (Fig. 7).

Fig. 5. ATP impairs the growth of MEL cells. MEL cells in complete culture media wereincubated in the absence (basal) or presence of ATP (as indicated) at 37 °C for 24 h. MTTwas added during the final 4 h, before overnight incubation with solubilization solutionat 37 °C. Absorbances were then read at 550 and 690 nm. Results are mean absorbances(A550 nm−A690 nm)±SD (n=3); ⁎⁎Pb0.01 compared to basal.

3.5. P2X7 induces rapid PS exposure and MP release in MEL cells

P2X7 activation can induce MP release from various cell types,which is preceded by the rapid and reversible exposure of PS on thecell-surface which can be measured by Annexin-V binding [7].Therefore, the ability of P2X7 activation to induce MP release fromMEL cells was firstly studied indirectly by assessing P2X7-inducedrapid PS exposure. Cells were pre-incubated in the absence or presenceof A-438079, and then for 5 min in the presence of Annexin-V in theabsence or presence of ATP. In the absence of A-438079, ATP causedsignificantly higher amounts of Annexin-V binding on MEL cells,compared to cells incubated in the absence of ATP (Fig. 8). Pre-incubationwithA-438079 reduced the amountof Annexin-Vbindingby63±3% (Fig. 8).

The above results indicate that P2X7 activation can induce rapid PSexposure in MEL cells, and that P2X7 activation may also lead to MPfrom these cells. Therefore, using incubation conditions previouslyused for RAW264.7 cells [28], MEL cells, as well as positive controlRAW264.7 cells, were incubated in the absence or presence of ATP for10 min as described [28]. The amount of MP released into thesupernatants was then quantified using the MP stain, bio-maleimide[29,30] and flow cytometry (Fig. 9). Incubation of RAW264.7 cellswith ATP resulted in an approximately 1.7-fold increase in the releaseof MPs into the extracellular medium compared to RAW264.7 cells

Fig. 7. The P2X7 antagonist, A-438079, impairs ATP-induced cell death in MEL cells.MEL cells in complete culture media were pre-incubated in the absence or presence of10 μM A-438079 at 37 °C for 15 min, and then incubated in the absence (basal) orpresence of 2 mM ATP at 37 °C for 24 h. Cells were harvested, and incubated withAnnexin-V-FLUOS and 7AAD, and analysed by flow cytometry. Results are mean±SD(n=3); ⁎⁎Pb0.01 compared to corresponding basal; ††Pb0.01 compared to ATP alone.

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incubated in the absence of ATP (35718±7468 versus 21256±11803MPs/ml respectively, n=9, P=0.007; Fig. 9A). Similarly, incubationof MEL cells with ATP resulted in an approximately 1.9-fold increasein the release of MPs into the extracellular medium compared toMEL cells incubated in the absence of ATP (54321±23358 versus28027±14377 MPs/ml respectively, n=9, P=0.011; Fig. 9B). To

Fig. 8. P2X7 activation mediates ATP-induced rapid PS exposure in MEL cells. MEL cellsin PS exposure assay medium were pre-incubated in the absence or presence of 10 μMA-438079 at 37 °C for 15 min, and then with Annexin-V-FLUOS in the absence (basal)or 2 mMATP at 37 °C for 5 min. Incubations were stopped by addition of MgCl2 mediumand centrifugation. Cells were incubated for 5 min with 7AAD (to exclude dead cells),and then analysed by flow cytometry. Results are mean±SD (n=3); ⁎⁎Pb0.01compared to corresponding basal; ††Pb0.01 compared to ATP alone.

Fig. 9. ATP induces MP release from MEL cells. (A) Adherent RAW264.7 cells or(B) suspended MEL cells in MP release assay medium were incubated in the absence(basal; left hand plots) or presence of 2 mM ATP (right hand plots) at 37 °C for 10 min.Incubations were stopped by centrifugation and supernatants collected. Supernatantswere incubated with 2.5 μM bio-maleimide for 15 min. Then to each sample, Flow-Count fluorospheres were added, the samples vortexed and analysed by flowcytometry. MPs were gated by forward and side scatter, and then the amount of bio-maleimide+ MPs per ml enumerated. Representative dot plots of 9 experiments areshown.

determine if the ATP-induced MP release was mediated by P2X7, cellswere pre-incubated in the absence or presence of A-438079. Pre-incubation with A-438079 significantly inhibited MP release fromRAW264.7 andMEL cells by 83±2% and 71±11% respectively (Fig. 10).

4. Discussion

Over 25 years ago, Chatawala and Cantley reported that extracel-lular ATP could induce Ca2+, Na+ and K+

fluxes in and reduce thegrowth of MEL cells [23]. These ATP-induced effects were character-istic of P2X7, however this receptor was not considered at the time.Therefore, we hypothesised that this murine erythroleukemia cell lineexpresses functional P2X7 receptors. Our study supports thishypothesis. First, RT-PCR and immunoblotting demonstrated thatP2X7 mRNA and protein respectively were present in MEL cells.

Fig. 10. The P2X7 antagonist, A-438079, impairs ATP-induced MP release from MELcells. (A) Adherent RAW264.7 cells or (B) suspended MEL cells in MP release assaymedium were pre-incubated in the absence or presence of 10 μM A-438079 at 37 °C for15 min, and then in the absence (basal) or presence of 2 mM ATP at 37 °C for 10 min.Incubations were stopped by centrifugation and supernatants collected. Supernatantswere incubated with 2.5 μM bio-maleimide for 15 min. Then to each sample, Flow-Count fluorospheres were added, the samples vortexed and analysed by flow cyto-metry. Results are mean±SD (n=9); ⁎⁎Pb0.01 compared to corresponding control;†Pb0.05 compared to ATP alone.

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Second, immunofluorescence staining revealed the presence of P2X7on MEL cells. Third, ATP induced ethidium+ uptake into MEL cellswith an EC50 value typical of ATP-induced cation fluxes mediated byrecombinant P2X7 [36]. Fourth, BzATP, but not ADP or UTP inducedethidium+ uptake into MEL cells, again typical of recombinant P2X7[36]. Fifth, ATP-induced ethidium+ and YO-PRO-1+ uptake into MELcells was impaired by the specific P2X7 antagonist, A-438079 [37].Finally, ATP could induce cell death, rapid PS exposure and MP releasefrom MEL cells, and each of these events, which are commonlyassociated with P2X7 activation [7,8], could be impaired by A-438079.

P2X7 activation could induce cell death in MEL cells. Chatawalaand Cantley reported that extracellular ATP could inhibit the growthof MEL cells [23], however whether this was due to a block in celldivision or due to increased cell death was never established. Ourstudy confirmed that extracellular ATP could impair MEL cell growth,and further demonstrated that this effect was a result of P2X7-induced cell death. In the cytofluorometric assay, the Annexin-V−/7AAD+ cells most likely represented early apoptotic cells, howeverwhether the Annexin-V+/7AAD+ cells represented late apoptotic ornecrotic cells, or both cannot be exclusively determined using onlythese two markers of cell death [38]. We have previously shown thatP2X7 activation can induce PS exposure and haemolysis in canineerythrocytes [18], while others have shown that the bacterialvirulence factor, α-hemolysin, can induce haemolysis of murine,human and equine erythrocytes in a P2X7-dependent fashion [16].P2X7 activation is known to induce cell death in a variety of cell types[8], however the physiological role of P2X7-induced cell death

remains obscure. Studies of P2X7 deficient mice suggest that thereceptor is not involved in regulating cell numbers under normal,physiological conditions [39,40], except in bone homeostasis [11].Both human and murine studies indicate that P2X7-induced macro-phage death may play a role in controlling intracellular bacterialinfections [12], while impaired P2X7-induced cell death may promoteepithelial malignancies [41]. Thus, we hypothesise that P2X7activation may play a role in removing either wanted or unwantedcells, including red blood cells, during various disease states includinginfection and malignancy, but not during normal homeostasis, withthe exception of bone homeostasis.

P2X7 activation could also induce MP release from MEL cells. Usingincubation conditions previously used to study P2X7-induced MPrelease from RAW264.7 macrophages [28], coupled with a cytofluoro-metric assay utilising the MP stain, bio-maleimide [29,30], wedemonstrated that P2X7 activation could induce rapid PS exposureandMP release fromMEL cells. P2X7 activation can induceMP release inthe form ofmicrovesicles or exosomes from various cell types includingmacrophages,microglia, dendritic cells and endothelial cells [28,42–45].MPs released from these cell types can promote either coagulation orinflammation through the expression of tissue factor and PS, orinterleukin-1β and major histocompatibility class II complexes respec-tively. Althoughwe have not specifically characterised theMPs releasedfollowing P2X7 activation in our study, using microspheres weestimated the MPs to be approximately 0.5 μM in diameter (results notshown), which is within the range (0.1 to 1 μM in diameter) ofmicrovesicles [46]. It is possible, that P2X7 activation may have alsoinduced the release of smaller MPs, butwas beyond the detection limitsof the cytofluorometric technique used in our study.Moreover, whetherthe MPs detected in our study represent true microvesicles or simplymembrane fragments is unknown. Nevertheless, our study provides thefirst evidence thatP2X7activation can induceMP release fromredbloodcells. Microvesiculation or MP release is a well-described phenomenonin both reticulocytes and erythrocytes, where it is postulated to play animportant role during the terminal differentiation of reticulocytes intoerythrocytes to remove no longer required cellular molecules ororganelles [47,48], and in extending erythrocyte life-span through theremoval of non-functional proteins and potential auto-antigens whichmay cause the pre-mature removal of otherwise functional erythrocytes[49,50]. Themechanisms however that lead to red blood cell MP releasein vivo and during the cold-storage of red blood cells are unknown. Thus,our study suggests a possible role for P2X7 in these processes. It will beof interest to characterise the nature of theMPs released fromMEL cellsfollowing P2X7 activation, and whether P2X7 can induce MP releasefromprimary reticulocytes and erythrocytes in future in vitro and in vivostudies.

Acknowledgements

This work was supported by Cure Cancer Australia and theUniversity of Wollongong (Wollongong, Australia). R.S. was arecipient of a European Travel Award from the Australian Academyof Science. We gratefully acknowledge Dr. Jason D. McArthur(University of Wollongong) andMs. Yvonne Groenen-Döpp (RijnstateHospital, Arnhem, The Netherlands) for technical advice regardingRT-PCR and MP assays respectively, and Ms. Margaret Phillips(University of Wollongong) for technical assistance.

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