Transient P2X7 Receptor Activation Triggers Macrophage Death Independent of Toll-like Receptors 2 and 4, Caspase-1, and Pannexin-1 Proteins*

Background: P2X7 receptors are thought to be primarily involved in inflammatory signaling. Results: Transient (1–4 min) high ATP induced delayed (hours) cell death in macrophages from WT and TLR2/4, Casp1, or Panx1 knock-out mice. Conclusion: Transient P2X7 receptor activation triggers macrophage-selective apoptotic cell death independent of TLR signaling, Casp1, and Panx1. Significance: P2X7 receptors function foremost as death triggers in macrophages. The function of P2X7 receptors (ATP-gated ion channels) in innate immune cells is unclear. In the setting of Toll-like receptor (TLR) stimulation, secondary activation of P2X7 ion channels has been linked to pro-caspase-1 cleavage and cell death. Here we show that cell death is a surprisingly early triggered event. We show using live-cell imaging that transient (1–4 min) stimulation of mouse macrophages with high extracellular ATP ([ATP]e) triggers delayed (hours) cell death, indexed as DEVDase (caspase-3 and caspase-7) activity. Continuous or transient high [ATP]e did not induce cell death in P2X7-deficient (P2X7−/−) macrophages or neutrophils (in which P2X7 could not be detected). Blocking sustained Ca2+ influx, a signature of P2X7 ligation, was highly protective, whereas no protection was conferred in macrophages lacking caspase-1 or TLR2 and TLR4. Furthermore, pannexin-1 (Panx1) deficiency had no effect on transient ATP-induced delayed cell death or ATP-induced Yo-Pro-1 uptake (an index of large pore pathway formation). Thus, “transient” P2X7 receptor activation and Ca2+ overload act as a death trigger for native mouse macrophages independent of Panx1 and pro-inflammatory caspase-1 and TLR signaling.

Typically, pro-inflammatory IL-1␤ and IL-18 cytokine processing and release can be detected after about 20 -30 min of P2X 7 receptor stimulation, and cell death, commonly indexed as lactate dehydrogenase release, is moderately low under such conditions (10 -12, 15). In contrast, prolonged (Ͼ30 min) P2X 7 receptor stimulation is well known to be lethal (4,11,15). Hence, cell death is generally assumed to be a late event in relation to inflammatory cytokine processing. However, the extracellular [ATP] is tightly controlled by ectonucleotidases (21), and it is difficult to imagine a situation in which macro-* This work was supported in part by the Deutsche Forschungsgemeinschaft phage P2X 7 receptors are stimulated by high [ATP]e for a long duration (15-20 min) after sensing bacteria via TLR4. Brief (5 min) stimulation of LPS-primed macrophages with millimolar ATP concentrations is a more likely signaling scenario. Indeed, brief stimulation of P2X 7 receptors is sufficient to initiate the processing of IL-1␤ in macrophages (16,18). The aim of this study was to elucidate the role of P2X 7 receptors in innate immune cells. We used resident macrophages from mice deficient in various genes (Casp1, P2rx7, Tlr2, Tlr4, P2ry2, and Panx1) and real-time single-cell imaging to elucidate, in particular, a link between transient P2X 7 receptor activation and cell death.
Resident Peritoneal Macrophages-Resident macrophages were isolated and seeded into fibronectin-coated -Slide I chambers (Ibidi, Martinsried, Germany), as described previously (25,26). In brief, mice were killed by overdose with isoflurane. A 24-gauge catheter (BD Insyte-W, BD Infusion Therapy Systems) was inserted into the peritoneum, and resident peritoneal cells were harvested by lavage with 8 ml of ice-cold Hanks' buffered salt solution (Invitrogen). After centrifugation at 360 ϫ g for 5 min, cells were resuspended in RPMI 1640 medium (Biochrom AG, Berlin, Germany) containing 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin. -Slide I chambers (Ibidi), which have a channel volume of 100 l, were filled with the cell suspension and incubated at 37°C in air with 5% CO 2 . After 2 h, nonadherent cells were removed by washing the channel with 2 ml of fresh medium. Experiments were performed after 1-2 days of incubation, and the medium was switched to bicarbonate-free RPMI 1640 containing 20 mM Hepes (Biochrom AG). Hanks' buffered salt solutions were used for single-cell Ca 2ϩ -imaging experiments. EGTA (0.5 mM) was added to Ca 2ϩ -free Hanks' solution. Alternatively, nominally Ca 2ϩ -free RPMI 1640 medium was made by adding 5 mM EGTA and subsequently titrating the pH back to 7.4. In selected experiments, macrophages were primed (pretreated) with LPS by incubation in medium containing 1 g/ml LPS from Escherichia coli 0111:B4 (L3012, Sigma) for 4 h.
Bone Marrow-derived Neutrophils-Bone marrow cells were flushed from the hind leg femurs of mice using Hanks' buffered salt solution (Invitrogen) containing 10% FCS and Hepes (pH 7.4). After filtration via a cell strainer with 70-m pores (BD Falcon, BD Biosciences), the cell suspension was centrifuged at 1200 rpm for 10 min. During this time, a density gradient was prepared in a round bottom 14-ml tube (BD Falcon, BD Biosciences) by layering 4-ml Histopaque-1119 underneath a 4-ml Histopaque-1077 via a long syringe needle. The bone marrow cell pellet was resuspended in 1 ml of solution, layered onto the Histopaque density gradient, and centrifuged at 1800 rpm (without using brakes) for 30 min at room temperature. The granulocyte layer, sandwiched between the Histopaque-1077 and -1119, was removed using a pipette, washed once, and resuspended in 10 ml of conditioned medium containing RPMI 1640 medium (Biochrom AG), 20% heat-inactivated fetal calf serum, 10% culture supernatant from WEHI-3B cells (mouse myelomonocytic leukemia cell line; ATCC TIB-68), 100 units/ml penicillin, and 100 g/ml streptomycin. Cells were cultured overnight at 37°C (5% CO 2 ). The following morning, cells were centrifuged at 1200 rpm for 8 min and resuspended in 10 ml of Hepes-Ringer solution. Subsequently, cells were seeded into -Slide I chambers (Ibidi) freshly coated with fibronectin. After allowing 5-10 min for adherence, the medium was replaced with bicarbonate-free RPMI 1640 containing 20 mM Hepes (Biochrom AG), but no fetal calf serum.
Bone Marrow-derived Macrophages-For selected experiments, bone marrow-derived macrophages were used to produce glass bottom WillCo (WillCo Wells) dishes (40-mm glass diameter and 0.17-mm thickness) with highly confluent macrophages. The femurs of mice were cleared of tissue and completely fractured in the middle of the shaft (diaphysis) using a surgical scalpel blade (number 21). Bone marrow cells were flushed out of each bone fragment using ϳ5 ml of Dulbecco's modified Eagle's medium (DMEM), injected via a 90°bent 23-gauge needle. The cell suspension, collected in a 50-ml Falcon tube, was centrifuged for 8 min at 1100 rpm and 4°C. The supernatant was discarded, and the pellet was resuspended in 1 ml of lysis buffer for 5 min (before recentrifugation) to induce hemolysis. The lysis buffer contained: 155 mM NH 4 Cl, 10 mM KHCO 3 , and 0.1 mM EDTA (pH 7.4). The suspension was then centrifuged for 10 min at 300 ϫ g and room temperature. The supernatant was aspirated, and the cells were washed once using 10 ml of Dulbecco's PBS and centrifuged (300 ϫ g for 8 min at room temperature). Next, the pellet was resuspended in 30 ml of incubation medium, which consisted of DMEM, 2% glutamine, 1% kanamycin, 1% nonessential amino acids, 10% heat-inactivated FCS, and 20 ng/ml recombinant mouse colony-stimulating factor 1 (macrophage), which is also called macrophage colony-stimulating factor (R&D Systems). The resuspended cells were incubated (37°C, 5% CO 2 ) in 30-ml Teflon bags for 6 days. After this period, the cells were resuspended after placement on ice for 15 min and transferred to glass-bottomed WillCo dishes. After 2 h (to allow cell adhesion), the dishes were washed with RPMI 1640 medium (Biochrom) containing 10% heat-inactivated FCS, 100 units/ml penicillin, and 100 g/ml streptomycin. Before use, the cells were incubated overnight.
Time-lapse Video Microscopy-Macrophages were placed on the stage of an inverted Axio Observer microscope (Carl Zeiss MicroImaging, Göttingen, Germany) maintained at 37°C by a temperature-controlled XL incubator (Zeiss). Differential interference contrast (DIC) and fluorescence images were obtained via a 63ϫ/1.40 oil immersion objective lens and charge-coupled device camera (AxioCam MRm, Zeiss) controlled by AxioVision software (Zeiss). Typically, time-lapse images were captured every 15 s or 1-2 min (6 -12 h of recording).
Nuclear Staining and Cytochrome c Labeling-Macrophages were fixed with 4% paraformaldehyde in Dulbecco's PBS for 15 min at 37°C followed by permeabilization with 0.1% Triton X-100 in PBS, containing 5% normal goat serum, for 10 min. Cells were incubated with Alexa Fluor 555-conjugated anticytochrome c antibodies (BD Pharmingen) for 30 min. Subsequently, the nuclei were stained by incubating cells with 280 nM 4Ј,6-diamidino-2-phenylindole (DAPI), a nucleic acid stain, for 90 s. Note that NucView488, similar to DAPI, is a nucleic acid stain, which allows assessment of nuclear morphology (28).
Single-cell Cytosolic [Ca 2ϩ ] Measurements-A glass coverslip seeded with macrophages was sealed onto the bottom of a Perspex bath (volume, 100 l) using silicone lubricant (Fine Science Tools). Macrophages were imaged via a 40ϫ/1.40 oil objective lens and superfused at 1 ml/min. To monitor intracellular [Ca 2ϩ ], cells were incubated for 15 min with 10 M fluo-3/AM. To reduce the rate of fluo-3 loss, solutions contained 0.8 mM probenecid, and experiments were performed at room temperature (20 -23°C). In each experiment, a single macrophage (selected with a bilateral iris) was excited at 488 nm, whereas fluorescence was detected at 530 Ϯ 15 nm using a microscopebased spectrofluorometer system (Photon Technology International, Seefeld, Germany). Fluorescence signals were normalized with respect to the resting fluorescence intensity (F 0 ) and expressed as F/F 0 . Solutions were rapidly switched by means of miniature three-way valves (The Lee Co., Westbrook, CT).
Statistical Analysis-Normality and homoscedasticity were tested using the Kolmogorov-Smirnov and Levene tests, respectively. A one-way analysis of variance was performed using an ␣ value of 0.05. The post hoc Scheffé test was used to compare groups. In the case of two independent groups, an unpaired t test was used to test for statistical significance. Statistical analyses were performed using SPSS software, and data are presented as means Ϯ S.E.

RESULTS
Continuous ATP Application-We initially tested whether prolonged P2X 7 receptor stimulation activated caspase-3/7 and induced cell death in resident peritoneal macrophages using live-cell time-lapse imaging. After preloading with TMRE to monitor mitochondrial membrane potential, macrophages were continuously incubated in medium containing the cellpermeable and nonfluorescent probe DEVD-NucView488 to detect caspase-3/7 activity. Application of high [ATP]e (3 mM), required to activate P2X 7 receptors, induced microblebbing of wild-type (WT) and Casp1 Ϫ/Ϫ macrophages (Fig. 1A) within 60 s and dissipated the mitochondrial membrane potential in all cells within 2-3 min. Subsequently, between 10 and 60 min, one cell after the other became caspase-3/7-positive (Fig. 1B), indicated by the appearance of green fluorescence; the nonfluorescent probe DEVD-NucView488 is cleaved by caspase-3/7 (DEVDases), and the NucView488 moiety becomes fluorescent upon binding to RNA and DNA (28).
In contrast to macrophages, prolonged application of ATP (3 mM) did not induce microblebbing, activation of caspase-3/7, or cell death in bone marrow-derived neutrophils (Gr-1 ϩ cells) (Fig. 1C). This could be explained by a lack of P2X 7 receptors in this cell type. Indeed, we could not detect P2X 7 mRNA in Gr-1 high /CD11b high cells (neutrophils) purified by cell sorting.
manifest. However, it is unlikely that macrophages are confronted with high [ATP]e for prolonged periods. Other unidentified factors may increase the sensitivity of P2X 7 ion channels to ATP. Alternatively, transient increases of ATP above 500 M may be physiologically more relevant.
Transient ATP Application-Next, we tested whether transient (1-4 min) P2X 7 receptor stimulation is sufficient to induce cell death in macrophages. Macrophages were stimulated with 3 mM ATP for 1, 2, 3, or 4 min. Following washout of ATP, macrophages were incubated in medium containing DEVD-NucView488, and fluorescent and DIC images were taken every 1 min for 6 h. After application of ATP for 4 min and washout, the macrophages had "fuzzy" edges (probably due to cell swelling) and a paucity of lamellipodia ( Fig. 2A and supplemental Video 1). However, within 1-2 h, the cells resumed a normal morphology characterized by broad lamellipodial membrane extensions and peripheral ruffling ( Fig. 2A and supplemental Video 1). This pseudo-recovery period was interrupted by abrupt cell contraction and dynamic membrane blebbing, characteristic of apoptosis, followed by concurrent caspase-3/7 activity and massive membrane blebbing, indicating secondary necrosis (Fig. 2, A-C, and supplemental Video 1; for comparison, see the supplemental Video accompanying the review by Taylor et al. (29)). The kinetics of caspase-3/7 activation in individual cells is shown in Fig. 2B.
The sequence of events described above (and shown in Fig. 2, A-C) was also observed in bone marrow-derived macrophage plated at high (Ͼ80%) confluency (supplemental Fig. 1). The macrophage purity was high, as indicated by anti-F4/80 labeling of living macrophages (supplemental Fig. 1A). At such high confluency, individual macrophages are in close contact with neighboring cells. However, following transient stimulation with high [ATP]e, confluency is decreased due to the retraction of lamellipodia (supplemental Fig. 1, B and C).
Activation of DEVDase (caspase-3/7) activity and massive blebbing consistently coincided, as shown in the example in Fig.  2C. Note that the cell becomes diffusely green due to the binding of NucView488 to RNA, and then NucView488 redistributes to DNA (28). Thus, in addition to reporting DEVDase activity, NucView488 provides a "view" of nuclear morphology. The nucleus of macrophages appeared condensed or fragmented, indicative of apoptosis, during delayed cell death (supplemental Video 1). As in the case of prolonged P2X 7 receptor stimulation, delayed caspase-3/7 activation and massive blebbing following transient high [ATP]e were end-stage apoptotic events. To confirm that transient high [ATP]e leads to caspase-3 activation, we performed Western blot (Fig. 2D). Fully cleaved caspase-3 (17-kDa fragment) could be detected 4 h after transient high [ATP]e. Susceptibility to delayed caspase-3/7 activation and cell death increased steeply as a function of the duration of ATP application (Fig. 2E).
Loss of Cytochrome c and Nuclear Contraction-Cytochrome c was localized to the mitochondria in fixed and permeabilized macrophages labeled with Alexa Fluor 555-conjugated anti-cytochrome c antibodies (supplemental Fig. 2A). Apoptotic macrophages fixed 3 h after transient (4 min) ATP challenge were characterized by loss of mitochondrial cytochrome c and contracted nuclei (supplemental Fig. 2, B and C).

JOURNAL OF BIOLOGICAL CHEMISTRY 10655
Transient ATP-induced Cell Death Is Independent of Caspase-1 and TLR Signaling-P2X 7 receptor stimulation has been shown to activate caspase-1, and we speculated that this pro-inflammatory and pro-apoptotic enzyme could play a role in delayed ATP-induced cell death. However, Casp1 Ϫ/Ϫ macrophages were clearly not protected from delayed P2X 7 -dependent cell death (Fig. 4A and supplemental Video 3). In addition, we found that macrophages isolated from TLR2 and TLR4 double knock-out (TLR2/4 dKO) mice were not protected from ATP-induced cell death (Fig. 4B). The cumulative death rates of individual WT, Casp1 Ϫ/Ϫ , and TLR2/4 dKO macrophages (solid symbols) are plotted in Fig. 4C. It can be seen that one cell after the other becomes caspase-3/7-positive (cell death end point) starting at around 1 h after washout of ATP. For comparison, the data from Fig. 1B, showing the cumulative death rates during continuous ATP application, are superimposed (open symbols). Most cells die within 1 h of continuous high [ATP]e. As in the case of WT cells, the nucleus or Casp1 Ϫ/Ϫ and TLR2/4 dKO macrophages became fragmented during delayed cell death (for example, Fig. 4D), and by 12 h after washout, less than ϳ20% of WT, Casp1 Ϫ/Ϫ , and TLR2/4 dKO cells survived (Fig. 4E). Thus, the data in Fig. 4 indicate that TLR signaling and cleavage of pro-caspase-1 to active caspase-1 are not required in the P2X 7 -triggered signaling cascade culminating in caspase-3/7 activation and cell death.

cells.
Massive Ca 2ϩ Influx Is Critical Determinant of P2X 7 -mediated Delayed Cell Death-Superfusion of macrophages with ATP concentrations up to 300 M has previously been reported to induce a large Ca 2ϩ transient attributable to P2Y 2 receptor activation and a small Ca 2ϩ influx signal via P2X 1 and P2X 4 receptors (30). In single-cell Ca 2ϩ recordings, we found that high [ATP]e (3 mM) induced a sustained increase in cytosolic [Ca 2ϩ ] following the initial Ca 2ϩ transient (Fig. 6A). The elevated Ca 2ϩ plateau could be interrupted by superfusing the cell with Ca 2ϩ -free medium. We presumed that the sustained Ca 2ϩ component was solely due to P2X 7 receptor activation. Indeed, there was no Ca 2ϩ plateau in P2X 7 Ϫ/Ϫ macrophages superfused with high [ATP]e (Fig. 6B). Instead, ATP induced a single Ca 2ϩ spike in P2X 7 Ϫ/Ϫ macrophages, consistent with P2Y 2 receptor activation. The lack of sustained Ca 2ϩ influx in P2X 7 Ϫ/Ϫ macrophages is good evidence, together with the lack of microblebbing and cell death, that the functional P2X 7 splice variant found in some tissues (31) is not (functionally) expressed in macrophages. Using P2Y 2 Ϫ/Ϫ macrophages, we could "isolate" the P2X 7 -mediated sustained Ca 2ϩ signal (Fig.  6C). An overlay of the P2Y 2 -and P2X 7 -mediated Ca 2ϩ signaling components is shown in Fig. 6D.
Ca 2ϩ has been recognized as a death trigger for a long time (32), and we speculated that the striking Ca 2ϩ influx and Ca 2ϩ overload mediated by P2X 7 receptor activation may play a role in transient high [ATP]e-induced cell death. Consistent with  MARCH 23, 2012 • VOLUME 287 • NUMBER 13 this notion, application of millimolar ATP in Ca 2ϩ -free (0 Ca 2ϩ ) medium protected macrophages from delayed cell death (Fig. 6E). That is, the transient (4 min) application of ATP was bracketed with Ca 2ϩ -free medium, and under these conditions, a single Ca 2ϩ spike, similar to that seen in P2X 7

Transient P2X 7 Activation and Cell Death
Ϫ/Ϫ cells (Fig.  6B), is evoked. Hence, the duration of P2X 7 ligation and accompanying sustained Ca 2ϩ influx are important determinants of death signaling.
Pan-caspase and Calpain Inhibitors Attenuate Transient ATP-induced Delayed Cell Death-In the presence of the cellpermeable pan-caspase inhibitor z-VAD-fmk (40 M), macrophages were partially protected from delayed cell death induced by 4 min of exposure to 3 mM ATP (Fig. 7A). Cells were similarly protected by calpeptin (100 M), an inhibitor of the calpain family of Ca 2ϩ -activated proteases (Fig. 7B). These inhibitor experiments indicate that calpain and caspases contribute to transient ATP-induced cell death signaling cascade.
we compared the rates of ATP-induced dye (Yo-Pro-1; M r 375) uptake in single WT and Panx1 Ϫ/Ϫ macrophages using timelapse microscopy. Yo-Pro-1 is a green fluorescent DNA and RNA stain that is normally cell-impermeable. The mean rates of ATP-induced Yo-Pro-1 uptake by WT and Panx1 Ϫ/Ϫ macrophages are shown in Fig. 8B. These results indicate that Panx1 is not the large pore pathway in native macrophages.
The kinetics of Yo-Pro-1 uptake in individual cells is shown in Fig. 8 (C-F). During the initial 20-min incubation period with 2 M Yo-Pro-1, there was no constitutive uptake of Yo-Pro-1 in WT macrophages (Fig. 8, C and D). However, application of 3 mM ATP induced Yo-Pro-1 uptake, consistent with the opening of a large pore ( Fig. 8C and supplemental Video 4). Yo-Pro-1 uptake was not impaired in Panx1 Ϫ/Ϫ macrophages. The time course of Yo-Pro-1 uptake by Panx1 Ϫ/Ϫ macrophages is shown in Fig. 8, E and F (see also supplemental Video 5).
We tested whether stimulation of P2X 7 receptors with a lower concentration of ligand, 500 M instead of 3 mM ATP, was sufficient to induce Yo-Pro-1 uptake (supplemental Fig. 3). There was no obvious change in cell morphology after 20 min (supplemental Fig. 3A), or even 60 min, of continuous stimulation with 500 M ATP. However, a weak increase in Yo-Pro-1 uptake could be detected (supplemental Fig. 3, A and B). The magnitude of Yo-Pro-1 fluorescence induced by 500 M ATP was much less than that evoked by 3 mM (compare supplemental Fig. 3B with Fig. 8D). A similar weak increase in Yo-Pro-1 uptake was measured in Panx1 Ϫ/Ϫ macrophages challenged with 500 M ATP (supplemental Fig. 3, C and D). Thus, Panx1 is not responsible for the weak dye uptake response to 500 M ATP. As in the case for WT macrophages, continuous stimulation of Panx1-deficient cells with 500 M ATP also did not induce cell death.
Lack of ATP-induced Yo-Pro-1 Uptake in P2X 7 Ϫ/Ϫ Macrophages-We speculated that the stimulation of P2Y 2 receptors may cause weak (low capacity) Yo-Pro-1 uptake, which is otherwise masked by the dominant effect of P2X 7 . However, this was not the case because no Yo-Pro-1 uptake could be detected when P2X 7 Ϫ/Ϫ macrophages were challenged with ATP (Fig. 9, A and B, and supplemental Video 6). These findings indicate that P2X 7 receptors mediate the weak Yo-Pro-1 uptake induced by 500 M ATP, as well as the robust dye uptake evoked by 3 mM ATP.
Panx1 Deficiency Does Not Protect Macrophages from Transient ATP-induced Cell Death-Panx1 activation has been implicated in cell death in response to P2X 7 receptor ligands (33,35). Therefore, we investigated whether Panx1 Ϫ/Ϫ macrophages were protected from cell death (Fig. 9, C and D). Delayed transient ATP-induced cell death was clearly not reduced in Panx1 Ϫ/Ϫ macrophages (Fig. 9, C and D). Thus, the pro-death signaling triggered by P2X 7 receptor activation in native macrophages is not dependent on Panx1.
High [ATP]e (acting at P2X 7 receptors) can also induce macrophage-specific cell death, and our data indicate that activation of P2X 7 receptors for 1-4 min is sufficient to trigger apoptotic cell death. What was already known about the effects of transient P2X 7 receptor stimulation on cell fate? In 2002, Le Feuvre et al. (15) reported that "a brief pulse of ATP (5 mM for 5 min) had no effect on basal lactate dehydrogenase release (measured in supernatant samples taken 0.5 and 2 h after stimulation) from control WT (mouse peritoneal) macrophages, and the cells completely excluded trypan blue (data not shown)." In light of our findings, revealed by live-cell imaging, that transient high [ATP]e leads to caspase-3/7 activation and cell death delayed by hours (Figs. 2 and 4), we presume that Le Feuvre et al. (15) would have detected loss of cell integrity (secondary necrosis) if they had followed the fate of the cells for a longer period. The authors, however, found that a brief pulse (5 min) of ATP variably induced massive lactate dehydrogenase release (ϳ40 and ϳ100% of total release measured in two separate experiments, respectively) within 30 min from WT, but not caspase-1-deficient, macrophages that had been primed with LPS. In contrast, Pelegrin et al. (20) reported that application of 5 mM ATP for 20 min caused negligible lactate dehydrogenase release (2-4% of total) in mouse peritoneal macrophages primed with LPS for 4 h. Thus, the extent to which LPS priming sensitizes macrophages to ATP-induced cell death is unclear. In our study, we found that transient ATP similarly induced delayed cell death in WT, Casp1 Ϫ/Ϫ , TLR2/4 dKO, and Panx1 Ϫ/Ϫ macrophages, as well as in LPS-primed (4 h) WT and Casp1 Ϫ/Ϫ cells. Cell death, indexed as caspase-3/7 activity, was negligible in the first 60 min after transient ATP application, except in LPS-primed WT macrophages, ϳ20% of which became caspase-3/7-positive within 30 min. Thus, priming with LPS, and possibly other pathogen-associated molecular patterns or even macrophage infection, may shorten the delay between transient ATP stimulation and cell death in a caspase-1-dependent fashion.
Live-cell imaging revealed that the fate of macrophages after transient P2X 7 receptor stimulation follows a distinct sequence of events. After washout of ATP, the cells appear to recover from the initial microblebbing and loss of lamellipodial membrane protrusive activity, but in the following hours, suddenly one cell after the other tightly contracts and further dynamic microblebbing manifests, followed by the formation of large, expansive blebs. The nuclei become contracted or fragmented, features typical for apoptosis (29). A consistent feature of delayed death in individual cells is that massive bleb formation coincides with the emergence of caspase-3/7 activity. We assume that the effector caspases caspase-3 and caspase-7 contribute to the massive blebbing. These effector caspases target multiple proteins of the cytoskeleton, including key components of actin filaments, intermediate filaments, and microtubules (29). The pan-caspase inhibitor z-VAD-fmk did not completely abrogate transient ATP-induced delayed cell death, indicating that the mode of death signaling is only partially caspase-dependent.
What is the function of P2X 7 receptors expressed on macrophages? The major differences between P2X 7 receptors and other members of the P2X family are (i) activation by high [ATP]e (0.5-3 mM rather than 0.1-100 M range), (ii) lack of desensitization (P2X receptors typically desensitize within seconds), and (iii) the permeability transition to larger molecules upon stimulation. The requirement for high [ATP]e suggests that P2X 7 ion channels are activated under extreme conditions, such as massive tissue injury. Our data indicate that the Ca 2ϩ overload induced by several minutes of P2X 7 receptor stimulation serves as a death trigger. Identifying pathophysiological scenarios in which [ATP]e and cytosolic [Ca 2ϩ ] are increased, even for several minutes, remains a challenge in the fields of purinergic signaling and immunology. Unidentified endogenous or pathogen-associated factors may come into play to increase the affinity of P2X 7 receptors for ATP. The contribution of P2X 7 receptor-induced large pore formation to transient high [ATP]e-induced cell death remains unclear. Genetic deletion of pannexin-1, putative P2X 7 -mediated large pore pathway, did not affect the kinetics of ATP-induced Yo-Pro-1 uptake, and transient ATP-induced delayed cell death was unaffected. We speculate that instead of recruiting another transport pathway, the P2X 7 channel protein probably changes configuration to allow the passage of larger molecules, although to date single-channel current recordings have not been able to reveal such dramatic changes in size selectivity (41). In summary, there is considerable evidence linking P2X 7 receptor function with caspase-1 and TLR4 signaling pathways, and prolonged P2X 7 ion channel stimulation is known to induce cytolytic cell death. We now show, surprisingly, that death is an early triggered event, such that transient (1-4 min) high [ATP]e leads to delayed (hours) cell death. Moreover, our results clearly show that death signaling depends on the duration of trigger ATP and Ca 2ϩ overload, but it is independent of pro-inflammatory caspase-1 activation and TLR signaling. Finally, we also show that pannexin-1 is not involved in P2X 7dependent Yo-Pro-1 uptake or transient high [ATP]e-induced delayed cell death.