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The Journal of Immunology, 2008, 180, 300 -308
Copyright © 2008 by The American Association of Immunologists, Inc.
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Sequential Shrinkage and Swelling Underlie P2X7-Stimulated Lymphocyte Phosphatidylserine Exposure and Death1

Simon R. J. Taylor*, Mireya Gonzalez-Begne{dagger}, Stephen Dewhurst{dagger}, Giovanna Chimini{ddagger}, Christopher F. Higgins*, James E. Melvin{dagger} and James I. Elliott2,*

* Medical Research Council, Clinical Sciences Centre, Faculty of Medicine, Imperial College, London, United Kingdom; {dagger} Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642; and {ddagger} Centre d’Immunologie de Marseille-Luminy, Parc Scientifique et Technologique de Luminy, Marseille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Patterns of change in cell volume and plasma membrane phospholipid distribution during cell death are regarded as diagnostic means of distinguishing apoptosis from necrosis, the former being associated with cell shrinkage and early phosphatidylserine (PS) exposure, whereas necrosis is associated with cell swelling and consequent lysis. We demonstrate that cell volume regulation during lymphocyte death stimulated via the purinergic receptor P2X7 is distinct from both. Within seconds of stimulation, murine lymphocytes undergo rapid shrinkage concomitant with, but also required for, PS exposure. However, within 2 min shrinkage is reversed and swelling ensues ending in cell rupture. P2X7-induced shrinkage and PS translocation depend upon K+ efflux via KCa3.1, but use a pathway of Cl– efflux distinct from that previously implicated in apoptosis. Thus, P2X7 stimulation activates a novel pathway of cell death that does not conform to those conventionally associated with apoptosis and necrosis. The mixed apoptotic/necrotic phenotype of P2X7-stimulated cells is consistent with a potential role for this death pathway in lupus disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cell volume regulation is essential to cell survival and its dysregulation results in cell death. The pattern of cell volume change is a principal difference between apoptosis and necrosis, the two major forms of cell death. Thus, apoptosis is associated with cell shrinkage following efflux of K+ and Cl– ions and the consequent loss of water, whereas necrosis is associated with swelling due to uptake of osmolytes (1). We have suggested that one consequence of cell shrinkage is to promote the movement of the phospholipid phosphatidylserine (PS)3 from the inner to the outer leaflet of the plasma membrane early in apoptosis (but not necrosis), before membrane disruption (2, 3). Thus, regulation of cell volume and PS translocation in apoptosis appear to be mechanistically related and distinguish this cell death pathway from necrosis.

The purinergic receptor P2X7 is a ligand-gated, relatively nonspecific cation channel principally, though not exclusively, expressed by cells of the immune system. Its stimulation by ATP or its analog BzATP (2'-3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate; Sigma-Aldrich) induces rapid exposure of PS at the cell surface and, if stimulation is longer than a few minutes (4), cell death. P2X7-stimulated cell death has been variably reported to be either apoptotic (5, 6, 7, 8) or necrotic, based on observations of low cleavage of poly(ADP-ribose) polymerase, and release of lactate dehydrogenase and the nuclear protein high mobility group box 1 (HMGB1) (9, 10, 11). Some of these differences in interpretation are likely to be due to the fact that apoptosis and necrosis are not "either/or" alternative death mechanisms, but appear to be the opposite ends of a spectrum of cell death pathways (12). For example, lactate dehydrogenase release has been used to identify necrosis by some researchers, but apoptosis by another (13), whereas unengulfed apoptotic cells may also release lactate dehydrogenase following secondary necrosis (14), suggesting this marker may not efficiently discriminate between the two pathways. Similarly, release of HMGB1 has also been reported to occur during both necrosis and apoptosis (15). Clearly such results are consistent with the suggestion that most indicators detect death pathways intermediate between classical apoptosis and necrosis. By contrast, the nature of cell volume changes is purported to distinguish fundamentally between pathways that are principally apoptotic and necrotic (16). The volume changes occurring during P2X7-stimulated death are unclear, having been associated with cell swelling (10), a swelling-associated "oncotic" pore (17, 18), or shrinkage (5, 6, 7). Consequently whether P2X7-induced death is essentially apoptotic or necrotic remains unresolved.

We previously reported that cell shrinkage is required for surface exposure of PS following lymphocyte stimulation with calcium ionophore (2, 3). As PS is translocated from the inner leaflet of the plasma membrane to the outer leaflet within seconds of P2X7 activation (19), this model predicts that cell shrinkage should be equally rapid and not, as has been reported, a late phenotype occurring at around 30 min (7). However, as noted on the issue of volume change following P2X7 activation, the literature is inconsistent, with some reporting cell swelling not shrinkage (10, 17, 18).

To resolve these apparent discrepancies, we undertook a real-time study of cell volume changes following P2X7 stimulation. We show that P2X7-stimulated lymphocytes undergo waves of cell shrinkage and swelling. Initially, within seconds of activation, stimulated lymphocytes shrink, which is concomitant with, and also required for, PS translocation to the outer leaflet of the plasma membrane. PS translocation appears to occur by an alternative mechanism to that reported recently (2) as indicated by differences in chloride channel involvement and lack of macroscopic bleb formation. Secondly, cell shrinkage stops within 2 min of P2X7 activation and swelling ensues, which ultimately results in catastrophic cell lysis. Thirdly, cell lysis is associated with the collapse of the remnant body, which we suggest has previously been mistaken for apoptotic shrinkage (6, 7). Thus, P2X7 activation appears to induce a novel physiological cell death pathway associated with sequential cell shrinkage, swelling, and collapse. This blurs the distinction between volume changes hitherto believed to discriminate between apoptosis and necrosis. Furthermore, the data support a general role for cell shrinkage in promoting PS translocation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

BALB/c, C57BL/10, and FVB/n mice were from Harlan Olac and bred at the Biological Services Unit (Imperial College, London, United Kingdom). Mice with a mutation in Abca1 have been described elsewhere (20) and were maintained on a DBA/1J background in a pathogen free facility at Charles River Breeding Laboratories. Where studied, Abca1-deficient mice were compared with wild-type mice from the same breeding colony. Mice with mutations in KCa3.1 (also known as IK1, IKCa1, KCNN4), or in P2X7 were bred onto a C57BL/6 background have been previously described (21, 22) and were maintained at the Center for Oral Biology in the Aab Institute of Biomedical Sciences (University of Rochester Medical Center, Rochester, NY). P2X7-deficient mice were a gift from C. Gabel (Pfizer, Groton, CT). Where studied, KCa3.1-and P2X7-deficient mice were compared with wild-type mice from the same breeding colony. All Home Office and local ethical guidelines for the care of laboratory animals were followed.

Real-time flow cytometry

Mesenteric lymph node cells from adult mice were prepared by teasing with needles into phenol red-free DMEM (Sigma-Aldrich) supplemented with 5% FSC. Cell suspensions were stained with CD4-allophycocyanin, CD4-PerCP, CD8-allophycocyanin, CD45RB-PE, or CD45RB-FITC (BD Biosciences) Abs, as indicated. The use of differently labeled Abs enabled T cells from two mouse strains to be detected differentially, and hence studied simultaneously in the same tube. Cells were washed and resuspended in DMEM and, where indicated, equilibrated with annexin V-FITC or annexin V-allophycocyanin (annexin V; BD Biosciences) for 3 min, in some cases together with 0.15 µM merocyanine 540 (MC540; Sigma-Aldrich). Where cells were stimulated, baseline fluorescence was established from 30 s to 1 min before addition of 150 µM BzATP (Sigma-Aldrich). Cells were monitored for PS exposure and MC540 binding continuously in real-time by flow cytometry on a FACSCalibur machine and analyzed using CellQuest (BD Biosciences) or FlowJo software (Tree Star). Forward light scatter (FSC) was used as a measure of the volume of spherical cells, its sensitivity being greatest when light is collected over an angle of <10 degrees as in the FACSCalibur. Macrophages were excluded on the basis of their high FSC and side light scatter.

To measure calcium uptake, Ab-labeled cells were incubated for 10 min with 0.25 µM fluo-4-AM (Invitrogen Life Technologies), washed in phenol red-free DMEM, and incubated with half the level annexin V-allophycocyanin recommended by the manufacturer before flow cytometry. Use of limiting quantities of annexin V enables cells with constitutively exposed PS to be distinguished from cells translocating PS following stimulation as, under these conditions, the former bind higher levels of annexin V.

Data are presented as the percentage of responder cells only in conditions in which a clear, stable negative population could be gated in baseline conditions. Thresholds were set by eye, typically between 10 and 17 fluorescence units for annexin V and propidium iodide (PI) binding. For calcium uptake experiments, the negative threshold is more variable and dependent on fluo-4 loading.

Carbenoxolone (Sigma-Aldrich) was dissolved in water. Tamoxifen and quinine (Sigma-Aldrich) were dissolved in ethanol. Clotrimazole (Sigma-Aldrich) and fluo-4-AM were dissolved in DMSO. Charybdotoxin (Sigma-Aldrich) was dissolved in DMEM supplemented with 0.1% BSA. Concentrations for ion channel inhibitors are as used previously (3) and references therein. Diluents alone had no effect on any parameter studied. All results are representative of a minimum of three independent experiments.

Real-time microscopy

Murine mesenteric lymph node cells were freshly dissected and adhered to glass-bottomed petri dishes coated with poly-L-lysine (Sigma-Aldrich). Cells were maintained in DMEM at 37°C in a humidified environmental chamber and exposed to ~150 µM BzATP in the presence of annexin V-Alexa Fluor 568 (Molecular Probes). Cells were imaged with a DeltaVision inverted fluorescence widefield microscope (Applied Precision) equipped with a x100 1.35NA UPlanApo oil immersion objective lens (Leica). Alexa Fluor 568 was stimulated by with a HQ545/30x excitation filter and the emitted fluorescence collected through a dichroic mirror (660DCLP) and an HQ620/60m emission filter (Chroma Technology). Sequential brightfield and fluorescence images were taken every 20 s for ~20 min. Images were analyzed with SoftWoRx v3.3.5 (Applied Precision) and figures prepared with Adobe Illustrator 11.0 software (Adobe Systems).

Western blotting

Mice were rendered unconscious by exposure to CO2 and killed by exsanguination before isolation of cervical, mediastinal, axillary, inguinal, lumbar, sciatic, and sacral lymph nodes. Isolated lymph nodes were minced in 2 ml of homogenization buffer solution containing 250 mM sucrose (J.T. Baker), 10 mM triethanolamine, leupeptin, 0.125 M phenylmethyl sulfonyl fluoride (all from Sigma-Aldrich) and Complete Protease Inhibitor Cocktail (1 tablet/50 ml working solution; Roche Diagnostics). Nodes were homogenized using a glass-teflon tissue grinder (20 passes; Wheaton Science Products; Millville, NJ). Protein (100 µg) was heated at 55°C for 20 min before separation in a 10% SDS-PAGE Tris-glycine minigel (Bio-Rad). Proteins were transferred onto polyvinylidene difluoride membrane (Invitrogen Life Technologies) overnight at 4°C using a transfer buffer (containing 10 mM CAPS (3-(cyclohexylamino)-1-propanesulphonic) acid (pH 11)) in 10% methanol. Membrane was blocked overnight at 4°C with 5% nonfat dry milk in TBS (25 mM Tris (pH 7.5), 150 mM NaCl) and then incubated with primary Ab anti-P2X7 receptor (Chemicon/Millipore) at a dilution of 1/300 in 2.5% nonfat dry milk solution at 4°C overnight. After washing with TBST/0.1%, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG secondary Ab (Pierce) at a dilution of 1/2500 in TBST/2.5% nonfat dry milk for 1 h at room temperature. Labeled proteins were visualized using ECL detection kit (GE-Amersham Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
P2X7-stimulated lymphocytes undergo sequential shrinkage, swelling, and collapse

Whether P2X7-stimulated cells undergo apoptosis or necrosis (associated with persistent cell shrinkage or swelling, respectively) is unclear, as is the temporal relationship between any volume change and PS exposure. We therefore assessed the kinetics of P2X7-stimulated volume change and PS exposure by lymphocytes from BALB/c mice by real-time flow cytometry in the continuous presence of fluorescently conjugated annexin V. Annexin V binds to extracellular PS and thus an increase in cell-bound fluorescence indicates increased PS exposure. After basal binding of annexin V-FITC was established, cells were treated with BzATP to stimulate P2X7 receptors. BzATP is an analog of ATP with ~3-fold greater potency for the murine receptor (23) and evokes equivalent cellular effects. PS exposure, changes in cell volume as indicated by FSC, and membrane breakdown indicated by uptake of PI were measured simultaneously in the same population of cells. P2X7-stimulated cells shrank within seconds of activation but after ~90 s began to swell beyond their original size (Fig. 1). Swelling continued until cells finally collapsed concomitant with membrane rupture as indicated by the increase in small dead cell remnants taking up PI. Though BzATP also activates other P2X receptors, rapid PS translocation and cell death are characteristic of the P2X7 stimulation (19, 24). Furthermore, lymphocytes from mice lacking P2X7 (22) were not stimulated by BzATP in our assays (Fig. 1) indicating the complete dependence of this response on the P2X7 receptor.


Figure 1
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  		FIGURE 1. P2X7-stimulated cells undergo sequential shrinkage, swelling, and lysis. Mesenteric lymph node cells from BALB/c mice were labeled with anti-CD4-allophycocyanin, maintained at 37°C (until commencement of flow cytometry), and incubated with PI for 2 min. Only cells staining positively for CD4 were analyzed thereafter. Cells were stimulated with the P2X7 agonist BzATP (150 µM) at the time shown by an arrow and monitored in real time by flow cytometry. All parameters were measured simultaneously in the same population of cells. Change in mean cell size (i) of live cells (those excluding PI) as a function of time is shown. Cell size is measured in mean FSC units. Cells shrink abruptly within seconds of activation, but begin to recover volume after about 1 min. The rate of death (cells taking up PI) (ii) following P2X7 stimulation is shown. Density plot of cell size (iii) in FSC units as a function of time, including both live and dead cells, is shown. Two major populations are apparent. The peak density trace in the live cell population and the trough between peaks are indicated. An alternative to this density plot (iii) is shown of the data (iv) and shows the bimodal distribution of cell size over the course of the experiment. The population of small, "collapsed" PI+ cells is indicated (iii and iv). Note that the final collapse as cells take up PI is associated with a much greater decrease in cell size than that which occurs during the initial cell shrinkage immediately following P2X7 stimulation. The initial shrinkage is therefore not apparent (iv) in density plot. Data are representative of six independent experiments and in the plots shown reflect the analysis of 500,000 CD4+ cells. Lymphocytes from mice lacking P2X7 (22 ) fail to respond to BzATP (v and vi) as evidenced by a lack of change in cell size (mean FSC units, (v)) and PS exposure (annexin V-FITC binding, (vi)), which precedes PI uptake. Mesenteric lymph node cells from P2X7-deficient mice were labeled with anti-CD4-PerCP and incubated with annexin V-FITC for 2 min. Only cells staining positively for CD4 were analyzed thereafter. Cells were stimulated with the P2X7 agonist BzATP (150 µM) at the time indicated by an arrow and monitored in real time by flow cytometry. Data show two representative experiments of six performed and each trace reflects the analysis of ~13,000 cells.

 
Cell collapse was associated with a much greater decrease in cell size than that occurring during the initial shrinkage phase and was sudden and catastrophic as evidenced by real-time microscopy (Fig. 2i) and shown in supplemental video.4 Consistent with flow cytometry data, although PS translocation occurs rapidly and relatively synchronously, the timing of lysis is somewhat more variable. Notably, in contrast to the sequence of events following treatment with calcium ionophore (2), P2X7-stimulated PS translocation and cell death appears to occur in the absence of macroscopic bleb formation (Fig. 2i and supplemental video).4


Figure 2
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  		FIGURE 2. P2X7-stimulated swelling and lysis are temperature-sensitive. BALB/c mesenteric lymph node cells were incubated with annexin V-Alexa Fluor 568 (to measure exposed PS) for 2 min before stimulation with 150 µM BzATP and monitored by real-time microscopy. Cells were maintained at 37°C. Fluorescence at 4 min reflects binding of annexin V-Alexa Fluor 568. Scale bar represents size in micrometers. Mesenteric lymph node cells from BALB/c mice were labeled with anti-CD4-allophycocyanin, maintained at 37°C (red line) or 20°C (blue line) until commencement of flow cytometry and incubated with PI for 2 min (ii). Cells were stimulated with the P2X7 agonist BzATP (150 µM) at the time indicated by an arrow and monitored in real time by flow cytometry. The effect of temperature on the rate of change (left) in cell size of live CD4+ cells (those excluding PI) as indicated by mean FSC units. The effect of temperature on the rate of cell death (right) (uptake of PI) among CD4+ cells is also shown. Data are representative results of sequential experiments performed on the same day and reflect the response of between 600,000 and 900,000 CD4+ T cells. Data are representative of five independent experiments.

 
The downstream effects of P2X7 activation, including the uptake of a variety of fluorochromes, have been reported to be temperature-sensitive (18, 25) and biphasic (26, 27). Consistent with these observations, though P2X7-stimulated cell shrinkage was largely unaffected by temperature, the rate of subsequent cell volume recovery was reduced at 20°C and cell lysis was eliminated (Fig. 2ii). Hence, P2X7-stimulated cell lysis does not occur in cells that fail to swell following initial shrinkage.

Thus, changes associated with P2X7-stimulated cell death are characteristic neither of apoptosis nor necrosis, but exhibit elements of both. As in apoptosis, cells initially shrink and expose PS before membrane breakdown yet, as in necrosis, cell death occurs after a period of swelling up to and beyond the initial cell size. Notably, membrane rupture was associated with a sudden collapse in cell size, the decrease in volume being much greater than the initial, transient shrinkage. This terminal reduction in cell size is similar in kinetics and extent to that previously reported as apoptotic shrinkage (6, 7), but we suggest, both studies may reflect a marked loss of cytoplasmic material after cell lysis and not early stage apoptotic volume decrease (AVD). Presumed apoptosis in the latter studies was linked to dye uptake. Nevertheless, how permeability to fluorochromes such as ethidium and propidium develops remains controversial. indeed, as has been suggested by other reports, the literature has been confused by the fact that dye uptake is sometimes used to measure cell death but can also be stimulated in live cells by P2X7 stimulation (28). Our data emphasize the need for a careful kinetic separation of such rapid, P2X7-stimulated effects.

P2X7-stimulated decrease in lipid packing occurs before PS translocation

We have recently reported that PS translocation following lymphocyte stimulation with calcium ionophore is dependent on a decrease in lipid packing, as evidenced by uptake of the dye MC540 concomitant with cell shrinkage, and occur initially on macroscopic blebs protruding from the cell (2). However, as PS translocation following P2X7 activation appears kinetically distinct from that in conventional apoptosis, and to occur in the absence of overt bleb formation (Fig. 2 and supplemental video), we assessed the kinetics of MC540 uptake after P2X7 stimulation. Consistent with a role for decreased lipid packing in PS translocation, binding of MC540 precedes that of annexin V following P2X7 stimulation (Fig. 3). For this experiment we used lymphocytes from mice (FVB/n) bearing the low-sensitivity allele of P2X7 (29) (allele assessed using conditions outlined previously (30)) as these T cells exhibit a dose-dependent, bimodal response that allowed us to follow responder and nonresponder populations in the same tube. Those cells that do not exhibit increased MC540 binding also failed to translocate PS (Fig. 3, A and B, iii). In such experiments, binding of annexin V appears to be slightly inhibited by prior insertion of MC540, (data not shown) consistent with the suggestion that MC540 binding is not dependent on PS translocation. Thus, as in the response to calcium ionophore, decreased lipid packing precedes and appears to be required for PS exposure following P2X7 stimulation.


Figure 3
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  		FIGURE 3. Decreased lipid packing precedes PS translocation in response to P2X7 stimulation. Mesenteric lymph node cells from FVB/n mice were labeled with anti-CD4-PerCP, and anti-CD45RB-FITC (to exclude CD45RBlow cells with constitutively exposed PS (35 )) and incubated with annexin V-allophycocyanin and 0.15 µM MC540 for 3 min. Cells were stimulated with the P2X7 agonist BzATP (150 µM) at the time indicated by an arrow and were monitored in real time by flow cytometry. Two equivalent experiments (A and B) are shown. Density plots for CD4+ cells as a function of time of lipid packing (decreased lipid packing measured as increased MC540 binding, FL-2 units) (i), PS exposure (AV binding, FL-4 units) (ii), and PS exposure (AV binding, FL-4 units) by cells failing to exhibit decreased lipid packing following P2X7 stimulation (iii) (unboxed cells shown in (i)). Additionally in A, PS exposure by cells exhibiting increased MC540 binding following P2X7 stimulation (iv) is shown (boxed cells shown in (i)). Data shown reflect the response of 60,000–70,000 CD4+ T cells and are representative of eight independent experiments.

 
P2X7-stimulated shrinkage is KCa3.1-dependent and is required for PS translocation

Calcium ionophore-stimulated lymphocyte cell shrinkage precedes and is required for PS translocation, with PS initially translocated at the site of macroscopic blebs (2, 3). However, P2X7 activation does not lead to bleb formation on lymphocytes, whereas the rapid kinetics of cell shrinkage and its reversibility indicate that the mechanisms underlying loss of lipid asymmetry may differ from that following ionophore treatment. We therefore assessed whether or not PS translocation following P2X7 activation depends on cell shrinkage, and whether the same pathways mediate ion efflux as are involved following calcium ionophore treatment.

Volume decrease and therefore PS translocation following calcium ionophore stimulation of T lymphocytes is dependent on the efflux on K+ via the KCa3.1 channel, Cl– ions by undefined channels, and the osmotically obliged loss of water (2, 3, 21). Similarly, P2X7-stimulated cell shrinkage and PS translocation were significantly reduced, though not entirely blocked, by inhibitors of KCa3.1, and were diminished in mice bearing a targeted mutation in KCa3.1 (Fig. 4), despite P2X7 expression being unaffected in these animals (Fig. 5A). Though activation of large conductance Ca2+-activated K+ (BKCa) has been reported following P2X7 stimulation (31), its inhibitor iberiotoxin (32) was without effect (Fig. 4), as was agitoxin, an inhibitor of Kv1.3 (33) used as a potassium channel implicated in the activation of naive T cells (34).


Figure 4
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  		FIGURE 4. P2X7-stimulated cell shrinkage and PS translocation require the activity of the KCa3.1 channel. Mesenteric lymph node cells from C57BL/10 mice were labeled with anti-CD4-allophycocyanin and anti-CD45RB-PE and incubated with annexin V-FITC for 2 min in the presence or absence of 10 µM clotrimazole (i), 0.5 mM quinine (ii), or for 10 min in the presence of 250 nM charybdotoxin (iii). Cells were stimulated with the P2X7 agonist BzATP (150 µM) as indicated by an arrow and were monitored in real time by flow cytometry. Cells with low levels of CD45RB were excluded from the analysis as this population contains a high frequency of cells with constitutively exposed PS (35 ). The percentage of cells with exposed PS are shown as indicated by annexin V binding (left). Change in cell size is indicated by mean FSC units (right) as a function of time. BzATP alone (red) and BzATP in the presence of inhibitor (blue) are shown. Mesenteric lymph node cells from mice lacking KCa3.1 (iv). Parental controls were labeled with anti-CD4-allophycocyanin and anti-CD4-PerCP, respectively, and mixed, stained with anti-CD45RB-PE, and incubated with annexin V-FITC for 2 min. Thus, labeled cells from KCa3.1-deficient and parental mice could subsequently be distinguished by flow cytometric gating. Cells were stimulated and responses were monitored as in previous three experiments (i-iii). Parental controls (red) and KCa3.1-deficient (KCa3.1 –/–) (blue) are indicated. Mesenteric lymph node cells from C57BL/10 mice were labeled with anti-CD4-allophycocyanin and anti-CD45RB-PE and incubated with annexin V-FITC for 10 min in the presence or absence of 150 nM iberiotoxin (v), an inhibitor of the potassium channel BKCa, and 50 nM agitoxin (vi), an inhibitor of Kv1.3. Cells were stimulated with the P2X7 agonist BzATP (150 µM) at the time indicated by an arrow and monitored in real time by flow cytometry. In plots shown the data reflect the response of between 25,000 and 70,000 CD4+ cells. In all experiments, data (red or blue) represent the results of a single experiment. Experiments are only compared when conducted on the same day and are representative of four (i-iv) or three (v and vi) independent experiments.

 

Figure 5
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  		FIGURE 5. KCa3.1 inhibitors do not act directly on P2X7. A, Western blot with P2X7 Ab in lymph node cells. Protein derived from lymph node cells from wild-type and KCa3.1-deficient mice was subjected to Western blotting as described in Materials and Methods. Equivalent levels of 70 kDa P2X7 monomer and 140 kDa dimer (as reported elsewhere (47 )) were apparent. A lower band is presumed to be a nonglycosylated form of P2X7. B, Mesenteric lymph node cells from C57BL/10 mice were labeled with anti-CD4-PerCP and anti-CD45RB-PE, the Ca2+ indicator fluo-4-AM and incubated with annexin V-allophycocyanin for 2 min. Cells were defined as high annexin V (AVhigh) or low (AVlow) before stimulation. A representative density plot (i) of Ca2+ uptake (increase in FL-1 units) following P2X7 stimulation by 150 µM BzATP added at time indicated by arrow with naive (CD45RBhighAVlow) (left) and activated/memory (CD45RBlowAVhigh) (right) CD4+ cells in the same tube. Ca2+ uptake shown (ii) as the percentage of naive phenotype cells (left) exhibiting an increase in Ca2+ uptake or the change in mean FL-1 fluorescence (right) by activated/memory phenotype (AVhigh) cells in the same tube following P2X7 stimulation in the presence (blue) or absence (red) of 250 µM quinine. An equivalent experiment (iii) was conducted but in the presence or absence of 250 nM charybdotoxin. The different measures of increased Ca2+ uptake in left and right hand panels were required because almost all AVhigh cells respond in this assay. Data are representative of four independent experiments.

 
We have previously shown that most P2X7-stimulated Ca2+ uptake by T cells is dependent on PS exposure at the cell surface (35). To exclude the possibility that KCa3.1 inhibitors were blocking P2X7 itself, we therefore took advantage of a population of activated/mature phenotype T cells on which PS is constitutively exposed (35). Thus, we reasoned that if inhibitors were acting, as intended, on KCa3.1 they would, by inhibiting PS translocation, block P2X7-stimulated Ca2+ uptake by naive T cells, but would not inhibit Ca2+ uptake by activated/memory cells on which PS is already exposed. By contrast if presumed KCa3.1 inhibitors were in fact acting on P2X7 they would inhibit Ca2+ uptake by both populations. Consistent with the former, quinine and charybdotoxin did not inhibit BzATP-activated Ca2+ uptake by those T cells that bound annexin V-FITC before stimulation (Fig. 5B) and do not, therefore, inhibit P2X7 directly, but reduce Ca2+ uptake by naive cells through blocking KCa3.1-dependent PS translocation.

Thus, as for calcium ionophore-mediated stimulation, P2X7-stimulated PS translocation is strongly facilitated by cell shrinkage, this being dependent on efflux of K+ ions via KCa3.1.

P2X7-stimulated shrinkage and PS translocation require chloride efflux but are not blocked by tamoxifen

To maintain electroneutrality, K+ and Cl– ions are effluxed concomitantly during cell shrinkage. Due to the relatively nonspecific nature of anion channel inhibitors, the Cl– channels and/or transporters involved are difficult to define. AVD is associated with efflux of chloride via an undefined channel/transporter with a characteristic inhibitor profile, with one of the most potent blockers being the anti-estrogen, tamoxifen (36). This channel is known variously as the volume-regulated anion channel (VRAC), volume-sensitive outwardly rectifying (VSOR) Cl– channel, volume-sensitive organic osmolyte anion channel (VSOAC), or swelling activated Cl– channel ICl,swell (16, 37). Consistent with a role for VRAC in AVD, we have previously shown that cell shrinkage and PS translocation by B cells (but also by T cells; J.I. Elliott, unpublished data) during calcium ionophore-stimulated apoptosis is markedly inhibited by tamoxifen (2). By contrast, tamoxifen has no effect on P2X7-induced cell shrinkage and, surprisingly, appears to slightly increase the level of PS exposed per cell (Fig. 6). The broad chloride channel blockers niflumic acid and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) did, however, inhibit both P2X7-induced (Fig. 6) and calcium ionophore-induced (data not shown) shrinkage and PS translocation. Niflumic acid efficiently blocked PS translocation even though its effects on cell shrinkage were modest. Thus, P2X7-stimulated PS translocation is dependent on cell shrinkage achieved through efflux of K+ and Cl– ions, though loss of chloride is mediated by different channels/transporters to those required for PS exposure following stimulation with calcium ionophore. The molecular identities of the tamoxifen-inhibitable VRAC and P2X7-activated chloride channels are unknown.


Figure 6
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  		FIGURE 6. P2X7-stimulated cell shrinkage and PS translocation require chloride efflux, but not through the VRAC. Mesenteric lymph node cells from BALB/c mice were labeled with anti-CD4-allophycocyanin and anti-CD45RB-PE and incubated with annexin V-FITC for 2 min in the presence or absence of 5 µM tamoxifen (i), an inhibitor of the VRAC (36 ), 100 µM NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) (ii), and 0.5 mM niflumic acid (NFA) (iii). NPPB and niflumic acid are broad inhibitors of chloride channels. Cells were stimulated with the P2X7 agonist BzATP (150 µM) at the time indicated by an arrow and monitored in real time by flow cytometry. Cells with low levels of CD45RB were excluded from the analysis as this population contains a high frequency of cells with constitutively exposed PS (35 ). Data shown are the percentage of cells (left) with exposed PS as indicated by annexin V-binding or change in cell size (right) in mean FSC units as a function of time. BzATP alone (red) and BzATP in the presence of inhibitor (blue) represent the results of a single experiment indicated. Data reflect the response between 11,000 and 50,000 CD4+ cells and are representative of four (ii and iii) or six (i) independent experiments. Experiments are only compared when conducted on the same day.

 
P2X7-stimulated shrinkage and dye uptake by T cells are pannexin-independent

It has recently been reported that early, P2X7-stimulated dye uptake, though not ion conductance, PS translocation, membrane blebbing, or cell death, is dependent on the hemichannel, pannexin-1 (26, 27). In contrast with such studies in macrophages, we found the pannexin inhibitor carbenoxolone had no effect on any parameter studied in T cells, including early fluorochrome uptake and cell shrinkage (Fig. 7). These data suggest that pannexin-1 plays a tissue-specific, but not obligate role in P2X7-stimulated dye uptake.


Figure 7
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  		FIGURE 7. P2X7-stimulated cell shrinkage and dye uptake in T cells are pannexin-independent. Mesenteric lymph node cells were stained with anti-CD8-allophycocyanin Ab and monitored by flow cytometry. BODIPY-taxol (BT, 0.5 µM) (i) or ethidium bromide (EBr, 0.5 µM) (ii) were added at 25 s followed by 150 mM BzATP at 90 s (shown by arrow). Panels show dye uptake (as mean FL-1 units) in the presence (blue) or absence (red) of 20 µM pannexin inhibitor, carbenoxolone. The presence of 20 µM carbenoxolone (blue) also had no effect on the rate of P2X7-stimulated cell shrinkage (FSC units) (iii). Diluent controls for the experiments in this study. Mesenteric lymph node cells from BALB/c mice were labeled with anti-CD4-allophycocyanin and anti-CD45RB-PE and incubated in 300 µl of DMEM with annexin V-FITC for 2 min. The rate of PS exposure (AV binding) (iv and v) and cell shrinkage (vi and vii) stimulated by 150 µM BzATP alone (red) or in the presence or absence of 0.8% ethanol (blue) (iv and vi) or 0.5% DMSO (blue) (v and vii). Data shown reflect the response of 23,000–40,000 CD4+ cells and are representative of four (i-iii) or 12 (iv-vii) independent experiments.

 
Although several proteins have been postulated to mediate specific or relatively nonspecific transport of membrane phospholipids, none has been clearly identified. Decreased translocation of PS has, for example, been reported in cells from mice lacking Abca1 (20), though not in Abca1-deficient B lymphocytes following calcium ionophore stimulation (2). The role of Abca1 in PS translocation therefore remains unclear but it may function as a regulator rather than a direct transporter. Given that PS exposure appears differently regulated in response to different stimuli, as described, we assessed the role of Abca1 in P2X7-stimulated loss of lipid asymmetry. Rates of P2X7-stimulated PS exposure in Abca1-deficient and parental mice could not be distinguished (Fig. 8) and the process therefore appears Abca1-independent.


Figure 8
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  		FIGURE 8. P2X7-stimulated PS translocation does not depend on the activity of Abca1. Mesenteric lymph node cells from mice lacking Abca1 and parental controls were labeled with anti-CD4-allophycocyanin and anti-CD4-PerCP, respectively, and mixed, stained with anti-CD45RB-PE, and incubated with annexin V-FITC for 2 min. Thus, labeled cells from Abca1-deficient and parental mice could subsequently be distinguished in the same tube by flow cytometric gating. Cells were stimulated with the P2X7 agonist BzATP (150 µM) at the time indicated by an arrow and monitored in real time by flow cytometry. Parental controls (red) and Abca1-deficient (blue) are indicated. Data reflect the response of 50,000 CD4+ cells and are representative of four independent experiments.

 

   Discussion
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 Abstract
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 Materials and Methods
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 Discussion
Disclosures
 References
 
Cell death pathways are broadly classified as either apoptotic or necrotic, albeit with autophagy as a recognized although less extensively studied pathway that may have evolved to enable cells to withstand starvation (38, 39). However, the distinction between apoptosis and necrosis is not absolute and it has been argued that a spectrum of death pathways exists between the two (12). At one end, classical apoptosis is characterized by distinct chromatin condensation, DNA fragmentation, caspase-dependence, cell shrinkage, PS exposure, blebbing, and the formation of apoptotic bodies. At the other end, classical necrosis is caspase-independent, lacking chromatin condensation, accompanied by cell swelling and lacking PS exposure before membrane rupture. In between, several intermediate pathways have been identified with varying degrees of association with features such as caspase involvement and chromatin condensation. However, despite this association, cell volume changes in the two fundamental pathways appear distinct, with apoptosis and apoptosis-like forms of cell death associated with persistent cell shrinkage, whereas necrosis is characterized by persistent cell swelling (16). Though apoptosis-like changes associated with cell swelling have been reported occasionally, these associations appear to require nonphysiologic conditions such as the use of sodium-substituted medium (40).

Where P2X7-stimulated cell death fits into the apoptotic-necrotic spectrum has been unclear, presumably in part reflecting attempts to categorize the death pathway itself as either/or apoptotic or necrotic, based on assays that are rarely, if ever, diagnostic between the two. Another problem has been that although cell volume change is perhaps the most definitive means of classifying cell death as essentially apoptotic or necrotic, reports as to whether P2X7 induces shrinkage or swelling have been mixed (5, 6, 7, 8, 9, 10, 11, 18). Indeed, problematic to our hypothesis that cell shrinkage is required for PS translocation (3, 35), P2X7-induced shrinkage has been reported to be a relatively late (30 min) phenotype (7) when compared with PS translocation, which commences within seconds (though PS translocation was not directly measured in that study). Data we present in this study resolve this issue and describe for the first time a cell death pathway that is associated neither with persistent shrinkage nor persistent swelling, but waves of the two. We have previously used the term "aponecrosis" to describe the apparent mixed apoptotic-necrotic process induced by P2X7 stimulation (30). We show that P2X7 stimulation induces, within seconds, transient cell shrinkage that is required for PS translocation. This response is rapidly followed by cell swelling, ultimately ending in cell lysis. Consequently, the P2X7 death pathway possesses "apoptotic-like" features, such as cell shrinkage and early PS translocation, and features of necrosis such as swelling and lysis.

Despite providing further evidence of a link between cell shrinkage and PS exposure, our data also show that mechanisms underlying PS translocation are stimulus-dependent. More precisely, while stimulation of lymphocytes with calcium ionophore or via P2X7 both result in cell shrinkage and PS translocation, the two use distinct pathways of chloride ion efflux to bring about cell volume decrease. This distinction appears to be associated with differences in the mechanics and distribution of PS exposure. P2X7-induced cell shrinkage appears to depend on a pathway of chloride ion efflux distinct from that described in all other forms of AVD to date (1, 16, 41). Hence, in response to cell swelling induced by hypo-osmotic shock, all cells are able to efflux Cl– ions via an unidentified chloride channel and thereby restore normal volume. The various Cl– channels (e.g. VRAC and others) appear identical with that used in AVD (16, 41) and are sensitive to inhibition by the anti-estrogen tamoxifen (36, 37). Consistent with this appearance, we have recently shown that tamoxifen inhibits lymphocyte shrinkage and PS exposure stimulated by calcium ionophore (2). By contrast, though broad-spectrum chloride channel inhibitors such as niflumic acid and NPPB inhibit P2X7-induced shrinkage and PS translocation, these phenotypes were either insensitive to or increased in the presence of tamoxifen. The use of a pathway of Cl– efflux distinct to that in more conventional forms of apoptosis is also associated with a difference in the macroscopic behavior of cell membranes and sites of PS exposure following stimulation. Thus, in response to calcium ionophore, large blebs form that are the site of both decreased lipid packing and subsequent PS exposure (2). By contrast, P2X7-stimulated lymphocytes do not appear to bleb and, consistent with this, PS translocation occurs rapidly across the cell surface without an overt site of initial exposure.

Why then does P2X7-activated cell death use pathways of Cl– efflux and PS translocation that differ from those in conventional apoptosis? We suggest that there may be two fundamental reasons. Both may reflect that although prolonged stimulation (beyond a few minutes (4)) results in cell death, shorter periods of activation do not have similar results, such that case phenotypes such as PS exposure are reversible. First, therefore, brief P2X7 stimulation has a role in immune cell activation in which PS exposure plays an important part. We have recently shown, for example, that P2X7-stimulated PS exposure is associated with the altered activity of membrane associated proteins and suggested that loss of lipid asymmetry is a rapid means of coordinating changes in signaling across a cell (35). Furthermore, we suggested that PS exposure may be required to promote membrane deformation required for lymphocyte extravasation. Clearly, an association of PS exposure with localized membrane blebs that may be pinched off the cell is compatible neither with coordinating changes in the activity of membrane proteins across a cell, nor with promoting lymphocyte extravasation. For both of these functions, a widespread distribution of PS would be a likely prerequisite. It should be noted that the function of P2X7 may be partly cell type-dependent, and this finding may influence the behavior of stimulated cells and the distribution of PS. For example, P2X7 has been shown to stimulate the release of IL-1β from macrophage/monocyte lineage cells within PS-exposed shed microvesicles (19). However, we have not observed microvesicle (or bleb) shedding from stimulated lymphocytes. Such cell type differences in the behavior of membranes subsequent to P2X7 stimulation may explain the finding that whereas zeiotic blebbing occurs on stimulated adherent cells (4), this blebbing does not appear to occur on lymphocytes.

Secondly, activation of the (unidentified) Cl– channel conventionally associated with AVD may have sequelae independent of PS translocation that nonetheless promote apoptosis. If so, it would clearly be undesirable for this channel to be activated during brief, nonapoptotic P2X7 stimulation.

Although the identity of the Cl– channel is uncertain, our data indicate a role for K+ efflux via KCa3.1 in P2X7-stimulated cell shrinkage and PS translocation. Consistent with this observation, it has been shown that, at least in some cell types, BzATP stimulates membrane hyperpolarization that is prevented by the KCa3.1 inhibitor, charybdotoxin (42). However, our data do not imply that the majority of P2X7-stimulated K+ efflux occurs via KCa3.1, nor does it exclude K+ efflux occurring via P2X7 itself. Indeed, as with dye uptake, the route of K+ efflux following P2X7 stimulation has been elusive, and it has been argued that both occur through a dilated P2X7"pore" (43). Although in macrophages the pore may, in fact, be pannexin-1 (26, 27), we have found no role for this hemichannel in dye uptake by T cells. Rather, we have argued that alterations in dye and ion uptake and efflux may reflect changes in the activity of multiple channels or the permeability of the lipid bilayer itself upon shrinkage or PS translocation (35). Thus we envisage that initial P2X7-stimulated, KCa3.1-dependent cell shrinkage and PS exposure may promote K+ movement through other channels and/or P2X7 itself as downstream events, but not that KCa3.1 is the major conduit of K+ efflux per se.

Our results once more raise the question as to whether specific transporters of PS exist. In particular, if the mechanism of PS translocation is stimulus-specific, it is possible that several PS transporters exist, the functions of which may be partly redundant. The study of cells from Abca1-deficient mice indicates that this protein, at least in certain cells/conditions, regulates the rate of PS exposure (20), perhaps acting as an anion transporter (or regulator thereof) (44). However, we showed in this study that whatever the role of Abca1 in PS exposure in some conditions (20), Abca1 does not appear to be involved in P2X7-stimulated loss of lipid asymmetry. A case could be made that P2X7 itself directly transports PS upon appropriate stimulation and, indeed, transfection of P2X7 confers sensitivity to BzATP-stimulated PS exposure to a variety of cell types (4). Nevertheless, given the requirement for cell shrinkage in P2X7-stimulated PS exposure and in the absence of evidence for a direct association between PS and P2X7, we prefer the hypothesis that PS transport depends on the formation of energetically favorable sites during cell shrinkage (2), not on specific phospholipid transporters. We have suggested that following calcium ionophore treatment, such favored sites of PS translocation are formed as a consequence of shrinkage-dependent bleb formation (2). Macroscopic blebs do not form in response to P2X7 activation, implying a mechanistic difference. Indeed, it appears very unlikely that lymphocytes, which contain little cytoplasm, would be capable of forming a sufficient number of macroscopic blebs in response to P2X7 activation to achieve the rapid, synchronous exposure of PS across the cell that we observe. However, it also seems unlikely that the P2X7-stimulated shrinkage we see could occur without significant ruffling of the membrane. We therefore speculate that, concomitant with cell shrinkage, P2X7 stimulation induces transient distortions of the membrane sufficiently small to be beyond observation by light microscopy yet, as following calcium ionophore, associated with locally decreased lipid packing (MC540 uptake). As suggested previously, such membrane distortions would serve as energetically favorable sites for PS translocation.

Finally, our data are consistent with the hypothesis that excess P2X7 stimulation predisposes development of the autoimmune disease systemic lupus erythematosus (30). The disease is widely believed to be an autoimmune response to apoptotic debris (45), including PS and associated proteins found at the surface of apoptotic debris. Although removal of apoptotic debris is generally considered to be immunologically silent, excess apoptosis, or failure to remove such material, may result in secondary necrosis and an ensuing autoimmune response. We have argued that although P2X7 stimulation is itself proinflammatory through the release of mediators such as IL-1β (19, 46), its prolonged stimulation may have an additional role in systemic lupus erythematosus pathogenesis through the generation of debris that is phenotypically apoptotic yet released into a proinflammatory milieu formed as a consequence of cell lysis. In support of this suggestion, the P2X7 gene is associated with both mouse and human lupus susceptibility loci (30). Data presented in this study showing the catastrophic (presumably proinflammatory) lysis of stimulated, PS-exposed cells following cell swelling are therefore consistent with a potential role for P2X7 stimulation in lupus susceptibility.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
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 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants R01 DE009692 (to J.E.M. and M.G.-B.) and R37 DE008921 (to J.E.M. and S.D.) from the National Institutes of Health. S.T., J.I.E., and C.F.H. were also supported by the Medical Research Council of the United Kingdom. G.C. was supported by funding from the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and the European Union. Back

2 Address correspondence and reprint requests to Dr. James I. Elliott, Medical Research Council, Clinical Sciences Centre, Faculty of Medicine, Imperial College Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, United Kingdom. E-mail address: james.elliott{at}imperial.ac.uk Back

3 Abbreviations used in this paper: PS, phosphatidylserine; HMGB1, high mobility group box 1; PI, propidium iodide; MC540, merocyanine 540; AVD, apoptotic volume decrease; FSC, forward light scatter; VRAC, volume-regulated anion channel. Back

4 The online version of this article contains supplemental material. Back

Received for publication October 19, 2007. Accepted for publication October 19, 2007.


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