Although regulatory lymphocytes play an important role in the immune system, the regulation of their functions is poorly understood and remains to be elucidated. In this study we demonstrate that micromolar concentrations of the common cell metabolite NAD induce death in murine forkhead/winged helix transcription factor gene-expressing CD4+CD25+ regulatory T cells with high efficiency and within minutes. Similar, but less dramatic, effects are demonstrable with ATP and its nonhydrolysable derivative, benzoylbenzoyl-ATP. Other T cell subsets are more resistant, with CD8 cells being the least sensitive and CD4 cells expressing intermediate sensitivity. The higher sensitivity of CD4+CD25+ cells is demonstrable in vivo. Injection of NAD or benzoylbenzoyl-ATP causes preferential induction of a cell death signal in CD4+CD25+ cells. Transmission of the death signal requires functional P2X7 receptors, pointing to a role for these receptors in regulation and homeostasis of CD4+CD25+ regulatory T cells. Consistent with this, P2X7R gene-deleted mice possess increased levels of forkhead/winged helix transcription factor gene-expressing CD4+CD25+ cells.

A fundamental question in immunology is how nonresponsiveness to self-Ags is maintained while enabling an efficient response to infections. Tolerance is established by a central mechanism in the thymus and is maintained in the periphery by regulatory T (Treg)4 cells, which control autoreactive T cells. The importance of peripheral tolerance is well documented by experiments in which elimination of Treg cells results in severe autoimmune pathology (1, 2). Given the pivotal role of Treg cells, the question of how these cells are regulated remains to be elucidated. It is well documented that immune responses to microbial infections often trigger autoimmunity (3, 4), which raises the question of why Treg cells fail to protect against breakage of tolerance in the course of an infection. An explanation could be that the function of Treg cells is curtailed in favor of a more potent response to the infectious pathogen. It is postulated that there are mechanisms that inactivate Treg cells at sites of an infection. A common feature of infections is tissue inflammation and necrosis, associated with the release of cellular components into the extracellular space. Some of these components could provide a danger signal that modulates Treg cell functions. We tested this by examining the effects of two common cell metabolites that are released from necrotic cells and undergo rapid degradation outside cells.

We report in this study that CD4+CD25+ Treg cells undergo death by necrotic lysis within seconds when brought into contact with low concentrations of the common metabolites, NAD and ATP. Conventional T cells are more resistant and undergo slower death, associated with annexin V staining. We also show that the two metabolites act by providing ligands for the purinergic receptor P2X7 (P2X7R) and that cells lacking the receptor are resistant to rapid death induction. Moreover, by demonstrating that mice with deleted P2X7Rs possess increased numbers of CD4+CD25+ cells expressing forkhead/winged helix transcription factor gene (Foxp3), we suggest a role for this receptor in the homeostasis of CD4+CD25+ cells. It is proposed that P2X7R provides a signaling structure by which intracellular components NAD and ATP regulate CD4+CD25+ Treg cells.

Pathogen-free female C57BL/6 mice, 6–8 wk of age, were obtained from The Jackson Laboratory. C57BL/6 P2X7R gene-deleted mice (P2X7R−/−) were provided by Dr. C. Gabel (Pfizer, Inc., Ann Arbor, MI) (5) and were bred at the University of Southern California animal facility. Mice were injected i.v. with 1 or 10 mg of NAD or benzoylbenzoyl-ATP (Bz-ATP; Sigma-Aldrich) dissolved in 300 μl of PBS either once or three times at 30-min intervals. All mice were killed 2 h after the first injection.

Spleen, cervical, and inguinal lymph nodes or peripheral blood cells were used in all experiments as indicated. Erythrocytes were removed before analysis or culture by treatment for 5 min on ice with 155 mM NH4Cl, 10 mM KHCO3, and 1 mM EDTA, pH 7.3.

To purify CD4+CD25+ and CD4+CD25 cells, spleen cells were incubated with IMag anti-mouse CD4 magnetic particles (BD Biosciences) in 1× IMag Buffer (BD Biosciences) for 30 min at 4°C and then separated by IMagnet (BD Biosciences). The enriched CD4+ population was then incubated with FITC-conjugated anti-mouse CD4 Ab and PE-conjugated anti-mouse CD25 Ab for 30 min at 4°C. CD4+CD25+ and CD4+CD25 cells were separated by FACSVantage SE (BD Biosciences). Purity was verified by fluorometry to be >95%. The CD4 cell population was used to provide APCs to cultures.

To assay the function of CD4+CD25+ T cells, purified CD4+CD25 T cells (5 × 104/well) were cultured with 10 μg/ml anti-CD3 mAb (eBioscience) in the presence of CD4, APC-containing cells and varying numbers of CD4+CD25+ T cells for 3 days in complete RPMI 1640 medium containing 10% FBS (6). The APC-containing CD4 population (2 × 105 cells/well) was irradiated with 3000 rad. [3H]thymidine (Amersham Biosciences; 0.5 μCi/well) was added during the last 16 h of culture.

To assay induction of cell death, spleen cells were incubated with or without NAD, ATP (Sigma-Aldrich), or Bz-ATP in RPMI 1640 for various times and assayed for annexin V staining or by microscopic counting in the presence of trypan blue. To assay the short-term effects of NAD (≤5 min), a 10-fold excess of ice-cold PBS was added to the cell suspension to dilute out NAD (7). After centrifugation, cells were washed twice in ice-cold PBS before culture in complete RPMI 1640 medium. To inhibit P2X7 receptors, 20 μM KN-62 (Sigma-Aldrich) dissolved in 0.01% DMSO was added 10 min before addition of NAD or ATP (7, 8).

For FACS analysis, cells were preincubated with anti-mouse CD16/CD32 (2.4G2) mAb (BD Biosciences) to block FcγRs, followed by incubation with mAbs for 30 min at 4°C. The following mAbs were used: PerCP-conjugated anti-mouse CD4 (L3T4), PE-conjugated anti-mouse CD25 (PC61), and allophycocyanin-conjugated anti-mouse CD8 (Ly-2; BD Biosciences). To monitor induction of death, cells were stained with the Annexin VFITC Apoptosis Detection Kit I (BD Biosciences). To detect Foxp3 protein expression, cells stained with anti-CD4 and anti-CD25 Abs were fixed using Fix/Perm Buffer (eBioscience) for 24 h, then incubated with FITC-conjugated anti-mouse Foxp3 Ab (FJK-16; eBioscience) for 30 min at 4°C. For quantification of cell surface ADP-ribosylation, cells (4 × 106 cells/ml in RPMI 1640) were incubated with 300 μM etheno-NAD (Sigma-Aldrich) for 30 min at 37°C, followed by incubation with etheno-ADP-ribose-specific Ab 1G4, provided by Dr. R. Santella (Mailman School of Public Health of Columbia University, New York, NY) (9) and FITC-conjugated goat anti-mouse Ig (BD Biosciences). FACS analysis was performed on a FACSCalibur (BD Biosciences).

For assay of Foxp3 expression by RT-PCR, spleen cells were incubated in normal medium or in the presence of NAD for 15 min at 37°C. CD4+ cells were then purified using CD4 magnetic particles (BD Biosciences) as described above. Total RNA from CD4+ cells was extracted using TRIzol reagent (Invitrogen Life Technologies). RNA was reverse transcribed into cDNA using the Omniscript RT Kit (Qiagen). PCR was conducted using the Taq PCR MasterMix Kit (Qiagen) with the following primers synthesized at the University of Southern California Microchemical Core Facility: Foxp3, 5′-CAG CTG CCT ACA GTG CCC CTA-3′ and 5′-CAT TTG CCA GCA GTG GGT AG-3′; and GAPDH, 5′-TGA AGG TCG GTG TGA ACG GAT-3′ and 5′-CAG GGG GGC TAA GCA GTT GGT-3′. PCRs consisted of an initial 5-min 94°C denaturing step, followed by 35 cycles of 45 s at 94°C, 45 s at 57°C, and 60 s at 72°C (10). PCR was performed using a DNA Thermal Cycler 480 (PerkinElmer).

Results are expressed as the mean ± SD. Statistical significance of differences between experimental groups was calculated by Student’s t test.

We had previously shown that NAD, when added to cultures of B6 spleen cells, induces death in a small proportion of T cells (11), raising the question of whether a defined T cell subset expresses preferential sensitivity to the death-inducing signal. Subsequent experiments demonstrated that rapid death induction requires the expression of ADP-ribosyltransferase 2 (ART-2), an NAD-consuming cell surface enzyme that attaches ADP-ribosyl groups to arginines of cell surface proteins (7, 12, 13), prompting the hypothesis that it is this reaction that induces the death signal. Therefore, to assess effects of NAD, we assayed ART-2 activity on T cell subsets.

B6 spleen cells were incubated with the ART-2 substrate etheno-NAD (7, 9) and assayed for cell surface etheno-APP-ribosylation by labeling with etheno-ADP-ribose-specific Ab 1G4. Fig. 1 shows that CD4+CD25 and CD4+CD25+ cells undergo comparable labeling, whereas CD8+CD25 cells show somewhat lower labeling. An expectation, therefore, is that all three T cell subsets should be sensitive to NAD-induced death. To test this, spleen cells were incubated with NAD and assayed for the recovery of CD4+CD25+ T cells. Fig. 2,A shows the percentages of CD4+ CD25+, CD4+CD25, and CD8+CD25 T cells in spleen cells incubated with 500 μM NAD for 30 min; Fig. 2,B shows the complete time kinetics. As early as 30 s after addition of NAD, a 40% decrease in CD4+CD25+ cells was seen, followed by only a minor decrease over the next 30 min. In contrast, CD4+CD25 and CD8+CD25 cells decreased only slightly during this period. To determine the NAD concentrations required for this effect, spleen cells were incubated with increasing NAD concentrations for 30 min. Fig. 2 C shows that addition of 1 μM NAD induced a substantial decrease in CD4+CD25+ cells. In contrast, there was almost no effect on CD4+CD25 and CD8+CD25 cells.

FIGURE 1.

CD4+CD25+ cells express ART-2 enzyme activity. B6 spleen cells were incubated with 300 μM etheno-NAD for 30 min at 37°C. Samples were stained for CD25, CD4, CD8, and etheno-ADP-ribose-specific 1G4 Ab and analyzed by FACS. To generate the histograms, FACS data were gated for CD4+CD25, CD4+CD25+, or CD8+ cells. □, Samples not incubated with etheno-NAD; ▪, samples treated with etheno-NAD. This experiment was repeated at least three times with similar results.

FIGURE 1.

CD4+CD25+ cells express ART-2 enzyme activity. B6 spleen cells were incubated with 300 μM etheno-NAD for 30 min at 37°C. Samples were stained for CD25, CD4, CD8, and etheno-ADP-ribose-specific 1G4 Ab and analyzed by FACS. To generate the histograms, FACS data were gated for CD4+CD25, CD4+CD25+, or CD8+ cells. □, Samples not incubated with etheno-NAD; ▪, samples treated with etheno-NAD. This experiment was repeated at least three times with similar results.

Close modal
FIGURE 2.

CD4+CD25+ Treg cells disappear from spleen cell cultures incubated with NAD. A and B, B6 spleen cells were incubated with 500 μM NAD for the indicated periods of time; stained for CD25, CD4, and CD8; and analyzed by FACS. Scattergrams of untreated controls and the 30 min end point are shown. Numbers in the scattergrams represent percentages specified by the gates. The plot on the right shows all data, of which only the controls and respective end points are shown on the left. C, B6 spleen cells were incubated with the indicated concentrations of NAD for 30 min and analyzed as described above (A and B). D, B6 spleen cells were incubated with or without 100 μM NAD for 15 min. CD4+ cells were isolated by magnetic bead adsorption. In the isolated cell population, FOXP3 mRNA was assayed by RT-PCR as described in Materials and Methods. All experiments were repeated at least three times.

FIGURE 2.

CD4+CD25+ Treg cells disappear from spleen cell cultures incubated with NAD. A and B, B6 spleen cells were incubated with 500 μM NAD for the indicated periods of time; stained for CD25, CD4, and CD8; and analyzed by FACS. Scattergrams of untreated controls and the 30 min end point are shown. Numbers in the scattergrams represent percentages specified by the gates. The plot on the right shows all data, of which only the controls and respective end points are shown on the left. C, B6 spleen cells were incubated with the indicated concentrations of NAD for 30 min and analyzed as described above (A and B). D, B6 spleen cells were incubated with or without 100 μM NAD for 15 min. CD4+ cells were isolated by magnetic bead adsorption. In the isolated cell population, FOXP3 mRNA was assayed by RT-PCR as described in Materials and Methods. All experiments were repeated at least three times.

Close modal

These results suggest that NAD contact causes preferential elimination of Treg cells in spleen cell cultures. However, although CD4 and CD25 are markers for Treg cells, the expression of Foxp3 is a more definitive marker of regulatory cells (14, 15). Therefore, spleen cells were incubated for 15 min with 100 μM NAD, and Foxp3 mRNA expression in CD4 cells was assayed by RT-PCR. Fig. 2,D shows that the Foxp3 signal disappeared after incubation with NAD. This effect was higher than expected, because only ∼50% of CD4+CD25+ cells disappeared from NAD-incubated cultures (Fig. 2, B and C). Therefore, it is likely that the remaining CD4+CD25+ cells were also undergoing cell death.

To further examine this, cells from NAD-incubated cultures were stained for annexin V. Fig. 3, A and B, shows that contact with 500 μM NAD for only 30 s led to ∼80–90% annexin V staining in the CD4+CD25+ T cell population. Moreover, the addition of only 10 μM NAD sufficed to induce annexin V staining to a similar degree after a 30-min incubation (Fig. 3 C). In contrast, induction of annexin V staining in CD4+CD25 and CD8+CD25 cells was much lower and increased gradually with time and NAD concentrations. These results show that NAD induces cell death in CD4+CD25+ Treg cells at a much faster rate and at lower NAD concentrations than in conventional T cells.

FIGURE 3.

NAD induces rapid and preferential annexin V staining in CD4+CD25+ cells. A and B, B6 spleen cells were incubated with 500 μM NAD for the indicated periods of time and then stained for CD25, CD4, CD8, and annexin V and analyzed by FACS. To generate the histograms, FACS data were gated for CD4+CD25, CD4+CD25+, or CD8+ cells as shown in Fig. 2. Histograms of untreated controls and the 30 min end point are shown, and numbers in histograms indicate the percentage of annexin V-staining cells. The plot in B shows all data, of which only the controls and respective end points are shown in A. C, B6 spleen cells were incubated with the indicated concentrations of NAD for 30 min, then stained for CD25, CD4, CD8, and annexin V and analyzed as described in A and B. These experiments were repeated at least three times with similar results.

FIGURE 3.

NAD induces rapid and preferential annexin V staining in CD4+CD25+ cells. A and B, B6 spleen cells were incubated with 500 μM NAD for the indicated periods of time and then stained for CD25, CD4, CD8, and annexin V and analyzed by FACS. To generate the histograms, FACS data were gated for CD4+CD25, CD4+CD25+, or CD8+ cells as shown in Fig. 2. Histograms of untreated controls and the 30 min end point are shown, and numbers in histograms indicate the percentage of annexin V-staining cells. The plot in B shows all data, of which only the controls and respective end points are shown in A. C, B6 spleen cells were incubated with the indicated concentrations of NAD for 30 min, then stained for CD25, CD4, CD8, and annexin V and analyzed as described in A and B. These experiments were repeated at least three times with similar results.

Close modal

Recent experiments had shown that P2X7Rs play a pivotal role in NAD-induced death of conventional T cells (7, 16), which suggests that these receptors play a similar role in CD4+CD25+ cells. To examine this, the ability of P2X7R inhibitor KN-62 (8, 17) to interfere with cell death induction was assayed at a maximal NAD concentration of 500 μM. Fig. 4shows that in the presence of NAD, KN-62 inhibited the induction of annexin V staining in CD4+CD25+ T cells. Moreover, KN-62 increased cell recoveries of CD4+CD25+ T cells cultured without addition of NAD. The effects of KN-62 on conventional CD4 and CD8 cells were similar. Addition of KN-62 to cultures of B6 cells incubated with lower concentrations of NAD showed concordant results (data not shown).

FIGURE 4.

KN-62 inhibits NAD- and ATP-induced cell death in CD4+CD25+ cells. B6 spleen cells were incubated with 500 μM NAD or ATP for 30 min, then stained for CD25, CD4, CD8, and annexin V and analyzed by FACS. To generate the histograms, FACS data were gated for CD4+CD25, CD4+CD25+, or CD8+ cells. Where indicated, cells were preincubated for 10 min with 20 μM KN-62 before addition of NAD or ATP, and incubation in the presence of KN-62 was continued for 30 min. This experiment was repeated at least three times with similar results.

FIGURE 4.

KN-62 inhibits NAD- and ATP-induced cell death in CD4+CD25+ cells. B6 spleen cells were incubated with 500 μM NAD or ATP for 30 min, then stained for CD25, CD4, CD8, and annexin V and analyzed by FACS. To generate the histograms, FACS data were gated for CD4+CD25, CD4+CD25+, or CD8+ cells. Where indicated, cells were preincubated for 10 min with 20 μM KN-62 before addition of NAD or ATP, and incubation in the presence of KN-62 was continued for 30 min. This experiment was repeated at least three times with similar results.

Close modal

To provide additional evidence for the involvement of P2X7Rs, spleen cells from P2X7R−/− mice (5) were incubated with NAD and assayed for annexin V staining. Fig. 5shows that although B6 CD4+CD25+ T cells underwent a significant increase in annexin V staining under normal culture conditions, cells from P2X7R−/− mice showed no increase, even at 500 μM NAD. Conventional CD4 and CD8 cells from P2X7R−/− mice were also resistant to induction of annexin V staining by NAD. These results demonstrate that functional P2X7Rs are required for induction of rapid cell death in CD4+CD25+ cells. Moreover, the finding that death of CD4+CD25+ cells lacking functional P2X7Rs and cultured under normal culture conditions was very low, suggested that components in the medium or released by cells, one of which is most likely NAD, induce death via signaling through P2X7Rs.

FIGURE 5.

CD4+CD25+ cells from P2X7R−/− mice are resistant to NAD- and ATP-induced cell death. Spleen cells from B6 or P2X7R−/− mice were incubated with 500 μM NAD or ATP for 30 min, then stained for CD25, CD4, CD8, and annexin V and analyzed by FACS. To generate the histograms, FACS data were gated for CD4+CD25, CD4+CD25+, or CD8+ cells. This experiment was repeated at least three times with similar results.

FIGURE 5.

CD4+CD25+ cells from P2X7R−/− mice are resistant to NAD- and ATP-induced cell death. Spleen cells from B6 or P2X7R−/− mice were incubated with 500 μM NAD or ATP for 30 min, then stained for CD25, CD4, CD8, and annexin V and analyzed by FACS. To generate the histograms, FACS data were gated for CD4+CD25, CD4+CD25+, or CD8+ cells. This experiment was repeated at least three times with similar results.

Close modal

The finding that NAD-induced death in CD4+CD25+ T cells involved P2X7Rs, predicts that ATP, the well-established ligand of this receptor (18, 19), should exert effects similar to those of NAD. To examine this, spleen cells were incubated with increasing concentrations of ATP and assayed for cell recovery and annexin V staining. Fig. 6 A shows that 300 μM ATP caused a significant decrease in CD4+CD25+ cells and an increase in annexin V staining in the remaining cells, effects that further increase at 1000 μM ATP. Effects on conventional CD4 and CD8 cells were concordant, but much less pronounced.

FIGURE 6.

CD4+CD25+ cells are more sensitive to ATP-induced cell death than conventional CD4+ and CD8+ cells. A, B6 spleen cells were incubated with the indicated concentrations of ATP for 120 min, then stained for CD25, CD4, CD8, and annexin V and analyzed by FACS. To generate the histograms, FACS data were gated for CD4+CD25, CD4+CD25+, or CD8+ cells. Scattergrams and histograms of untreated controls and the highest ATP concentrations are shown. The plots show all data from scattergrams and histograms, of which only the controls and respective end points are shown on the left. This experiment was repeated at least three times with similar results. B, B6 spleen cells were incubated with 500 μM ATP for the times indicated, then stained for CD25, CD4, CD8, and annexin V and analyzed by FACS as described in A. This experiment was repeated at least three times with similar results.

FIGURE 6.

CD4+CD25+ cells are more sensitive to ATP-induced cell death than conventional CD4+ and CD8+ cells. A, B6 spleen cells were incubated with the indicated concentrations of ATP for 120 min, then stained for CD25, CD4, CD8, and annexin V and analyzed by FACS. To generate the histograms, FACS data were gated for CD4+CD25, CD4+CD25+, or CD8+ cells. Scattergrams and histograms of untreated controls and the highest ATP concentrations are shown. The plots show all data from scattergrams and histograms, of which only the controls and respective end points are shown on the left. This experiment was repeated at least three times with similar results. B, B6 spleen cells were incubated with 500 μM ATP for the times indicated, then stained for CD25, CD4, CD8, and annexin V and analyzed by FACS as described in A. This experiment was repeated at least three times with similar results.

Close modal

To determine the kinetics of death induction, cells were incubated with 500 μM ATP for various times. Fig. 6,B shows that at 2 min there was a significant decrease in CD4+CD25+ cells as well as an increase in the percentage of annexin V-staining cells, effects that were even more pronounced at 15 min. Concordant, but less dramatic, effects were seen with conventional CD4 and CD8 cells. To confirm that ATP-induced death of CD4+CD25+ cells was mediated by P2X7Rs, effects in the presence of KN-62 and in spleen cells from P2X7R−/− mice were assayed. At the high concentration of 500 μM ATP, there was no increase in annexin V staining of B6 cells in the presence of KN-62, or in cells from P2X7R−/− mice (Figs. 4 and 5). These results show that CD4+CD25+ cells are highly sensitive to ATP-induced death, and this response requires functional P2X7Rs.

Bz-ATP is a high affinity, nonhydrolysable ligand, of the P2X7R (18). It was therefore interesting to test the effects of this ligand on CD4+CD25+ cells. Fig. 7,A shows that Bz-ATP concentrations one-third or less than those of ATP induced a decrease in CD4+CD25+ cells and an increase in annexin V staining. As expected, much smaller effects were elicited in conventional CD4 and CD8 cells (Fig. 7,A). Importantly, no effects of Bz-ATP were demonstrable in CD4+CD25+ cells from P2X7R−/− mice (Fig. 7 B). These results demonstrate that a nonhydrolysable derivative of ATP induces effects in CD4+CD25+ cells similar to those of ATP and that these effects are mediated by P2X7Rs.

FIGURE 7.

Bz-ATP induces more efficient cell death than ATP in CD4+CD25+ cells and requires functional P2X7Rs. A, B6 spleen cells were incubated with the indicated concentrations of Bz-ATP for 120 min, then stained for CD25, CD4, CD8, and annexin V and analyzed by FACS. To generate histograms, FACS data were gated for CD4+CD25, CD4+CD25+, or CD8+ cells. Scattergrams and histograms of untreated controls and the highest Bz-ATP concentrations are shown. The plots show all data from scattergrams and histograms, of which only the controls and respective end points are shown on the left. B, Spleen cells from P2X7R−/− mice were incubated with 300 μM Bz-ATP for 120 min and processed and analyzed as described in A. These experiments were repeated twice with similar results.

FIGURE 7.

Bz-ATP induces more efficient cell death than ATP in CD4+CD25+ cells and requires functional P2X7Rs. A, B6 spleen cells were incubated with the indicated concentrations of Bz-ATP for 120 min, then stained for CD25, CD4, CD8, and annexin V and analyzed by FACS. To generate histograms, FACS data were gated for CD4+CD25, CD4+CD25+, or CD8+ cells. Scattergrams and histograms of untreated controls and the highest Bz-ATP concentrations are shown. The plots show all data from scattergrams and histograms, of which only the controls and respective end points are shown on the left. B, Spleen cells from P2X7R−/− mice were incubated with 300 μM Bz-ATP for 120 min and processed and analyzed as described in A. These experiments were repeated twice with similar results.

Close modal

The results presented in Figs. 2 and 6 show that a large percentage of CD4+CD25+ cells disappear within 30 s of NAD or ATP contact. To test whether this rapid disappearance is due to cell death, rather than a loss of cell surface CD4 or CD25 expression, CD4+CD25+ cells were isolated by magnetic bead adsorption and cell sorting. FACS analysis revealed >95% homogeneity (Fig. 8,A), but no expression of ART-2 activity (data not shown), consistent with our previous observation that stimulation of T cells causes the release of cell surface ART-2 (20). Therefore, the effects of P2X7R stimulation were tested with ATP. The results presented in Fig. 8 B show that addition of 500 μM ATP for 15 min led to 80% lysis of purified CD4+CD25+ cells. We conclude, the observed disappearance of CD4+CD25+ cells incubated with NAD or ATP is due to cell lysis and not loss of cell surface CD4 or CD25 Ags.

FIGURE 8.

CD4+CD25+ cells rapidly lyse upon engagement of the P2X7R. A, CD4+CD25+ and CD4+CD25 cells were isolated, and purity was analyzed by FACS. B, The isolated CD4+CD25+ and CD4+CD25 cells were treated with 500 μM ATP for 15 min, and cell viability was determined by trypan blue exclusion. ∗, p < 0.02; ∗∗, p < 0.01 (control vs ATP-treated cells). Error bars indicate SD values from triplicate samples. This experiment was repeated three times with similar results.

FIGURE 8.

CD4+CD25+ cells rapidly lyse upon engagement of the P2X7R. A, CD4+CD25+ and CD4+CD25 cells were isolated, and purity was analyzed by FACS. B, The isolated CD4+CD25+ and CD4+CD25 cells were treated with 500 μM ATP for 15 min, and cell viability was determined by trypan blue exclusion. ∗, p < 0.02; ∗∗, p < 0.01 (control vs ATP-treated cells). Error bars indicate SD values from triplicate samples. This experiment was repeated three times with similar results.

Close modal

The finding that efficient death is induced within seconds of NAD contact in CD4+CD25+ cells raises the possibility that this is also demonstrable in intact animals. To examine this, B6 mice were injected with increasing doses of NAD, and spleen cells were assayed for annexin V staining 2 h later. The results revealed that a single injection of 1 mg of NAD had no demonstrable effect (data not shown); however, a dose 10 times higher, injected once or three times, induced a 50% decrease in recovered CD4+CD25+ cells and a significant increase in annexin V staining of the remaining CD4+CD25+ cell population (Fig. 9,A). Much smaller, if any, effects were seen in conventional CD4 and CD8 cells (Fig. 9,A and data not shown). Because NAD action requires functional P2X7Rs, it is predicted that there should be no effects of NAD in P2X7R−/− mice. Fig. 9 A shows that at the highest NAD dose there was neither a decrease nor annexin V staining of CD4+CD25+ cells in P2X7R−/− mice.

FIGURE 9.

Effects of NAD and Bz-ATP on CD4+CD25+ cells in B6 and P2X7R−/− mice. A, Groups of three B6 and P2X7R−/− mice received 10 mg of NAD in 300 μl of PBS i.v. once or additional injections 30 and 60 min later. Spleen cells were harvested 2 h later; stained for CD25, CD4, CD8, and annexin V; and analyzed by FACS. To generate histograms, FACS data were gated for CD4+CD25+ or CD4+CD25 cells. B, Groups of three B6 and P2X7R−/− mice received one injection of 1 mg of Bz-ATP/mouse. Spleen cells were harvested 2 h later and processed as described in A. These experiments were repeated twice with similar results.

FIGURE 9.

Effects of NAD and Bz-ATP on CD4+CD25+ cells in B6 and P2X7R−/− mice. A, Groups of three B6 and P2X7R−/− mice received 10 mg of NAD in 300 μl of PBS i.v. once or additional injections 30 and 60 min later. Spleen cells were harvested 2 h later; stained for CD25, CD4, CD8, and annexin V; and analyzed by FACS. To generate histograms, FACS data were gated for CD4+CD25+ or CD4+CD25 cells. B, Groups of three B6 and P2X7R−/− mice received one injection of 1 mg of Bz-ATP/mouse. Spleen cells were harvested 2 h later and processed as described in A. These experiments were repeated twice with similar results.

Close modal

Although these results show that NAD can exert effects on CD4+CD25+ cells in vivo and by action on P2X7Rs, they raise the question of why such high doses are required. A likely reason is that effects are limited by rapid, CD38-mediated, NAD hydrolysis (21). We therefore tested whether injection of a lower dose of a nonhydrolysable P2X7 ligand, i.e., Bz-ATP, has effects on CD4+CD25+ cells. Fig. 9 B shows that this is indeed the case. Injection of 1 mg of Bz-ATP induced annexin V staining in CD4+CD25+ cells of B6, but not P2X7R−/−, mice. Therefore, the action of NAD is most likely local and in the direct vicinity of lysing cells.

The demonstration that NAD induces a death signal in CD4+CD25+ T cells of normal, but not P2X7R−/− mice, raises the possibility that P2X7R regulates the homeostasis and/or function of CD4+CD25+ Treg cells in vivo. To examine this, the distributions of CD4+CD25+ cells in normal and P2X7R−/− mice were compared. Table I shows that in peripheral blood, lymph node, and spleen of P2X7R−/− mice, there were significantly more CD4+CD25+ T cells than in normal B6 mice. To test whether this increase in CD4+CD25+ cells reflected an increase in Treg cells, CD4+CD25+ cells were assayed for the expression of Foxp3. The results presented in Fig. 10 A demonstrate that spleen, lymph nodes, and PBL from P2X7R−/− mice contained significantly more Foxp3-expressing CD4+CD25+ cells than respective organs from B6 mice.

Table I.

Counts of CD4+ CD25+ or CD3+ cells in spleen, lymph nodes, or blood

CompartmentCell TypeMice
WT B6aB6 P2X7R−/−a
No. of cells% cellsNo. of cells% cells
Spleenb CD4+CD25+ 1.27 × 106 1.59 ± 0.10 2.42 × 106 3.03 ± 0.12e 
 CD3+ 2.25 × 107 28.13 ± 3.62 2.76 × 107 34.51 ± 1.25 
Lymph nodesc CD4+CD25+ 3.05 × 104 3.11 ± 0.27 4.69 × 104 4.72 ± 0.19e 
 CD3+ 5.20 × 105 52.03 ± 6.17 6.20 × 105 61.13 ± 3.18 
Bloodd CD4+CD25+ 1.88 × 103 0.14 ± 0.04 2.80 × 104 2.09 ± 0.23e 
 CD3+ 3.09 × 105 23.07 ± 4.38 5.32 × 105 39.71 ± 3.45 
CompartmentCell TypeMice
WT B6aB6 P2X7R−/−a
No. of cells% cellsNo. of cells% cells
Spleenb CD4+CD25+ 1.27 × 106 1.59 ± 0.10 2.42 × 106 3.03 ± 0.12e 
 CD3+ 2.25 × 107 28.13 ± 3.62 2.76 × 107 34.51 ± 1.25 
Lymph nodesc CD4+CD25+ 3.05 × 104 3.11 ± 0.27 4.69 × 104 4.72 ± 0.19e 
 CD3+ 5.20 × 105 52.03 ± 6.17 6.20 × 105 61.13 ± 3.18 
Bloodd CD4+CD25+ 1.88 × 103 0.14 ± 0.04 2.80 × 104 2.09 ± 0.23e 
 CD3+ 3.09 × 105 23.07 ± 4.38 5.32 × 105 39.71 ± 3.45 
a

No. of mice = 3.

b

No. of cells per spleen.

c

No. of cells per lymph node of three lymph nodes extracted.

d

No. of cells per 1 ml of blood.

e

, p < 0.01 (between B6 P2X7R−/− and WT B6).

FIGURE 10.

P2X7R−/− mice possess more Foxp3-expressing CD4+CD25+ cells with normal regulatory function. A, Spleen cells, lymph node cells, and PBL from P2X7R−/− and wt B6 mice were first stained for CD25 and CD4, then fixed and stained for Foxp3 and analyzed by FACS. The scattergrams show data from CD4+ gated cells analyzed for the expression of CD25 and Foxp3. Numbers in the scattergrams represent the relative percentage of each respective cell population. These experiments were repeated twice with similar results. B, CD4+CD25+ cells were isolated from normal B6 and P2X7R−/− mice and cultured at the ratios indicated with B6 CD4+CD25 cells as well as an APC-containing population and anti-CD3 Ab. Cell proliferation was assayed by incorporation of [3H]TdR on day 3, as indicated. Error bars indicate SDs from triplicate cultures.

FIGURE 10.

P2X7R−/− mice possess more Foxp3-expressing CD4+CD25+ cells with normal regulatory function. A, Spleen cells, lymph node cells, and PBL from P2X7R−/− and wt B6 mice were first stained for CD25 and CD4, then fixed and stained for Foxp3 and analyzed by FACS. The scattergrams show data from CD4+ gated cells analyzed for the expression of CD25 and Foxp3. Numbers in the scattergrams represent the relative percentage of each respective cell population. These experiments were repeated twice with similar results. B, CD4+CD25+ cells were isolated from normal B6 and P2X7R−/− mice and cultured at the ratios indicated with B6 CD4+CD25 cells as well as an APC-containing population and anti-CD3 Ab. Cell proliferation was assayed by incorporation of [3H]TdR on day 3, as indicated. Error bars indicate SDs from triplicate cultures.

Close modal

To examine whether CD4+CD25+ cells from P2X7R−/− mice are functional, the regulatory activity of these cells was assayed. CD4+CD25+ cells were isolated by magnetic bead adsorption and cell sorting from B6 and P2X7R−/− mice. Cells of 95% homogeneity by FACS analysis were mixed at ratios between 1:1 and 1:8 with purified B6 CD4+CD25 cells. APCs and anti-CD3 Ab were added to induce proliferation of CD4+CD25 cells (6). Fig. 10 B reveals that on a per cell basis, CD4+CD25+ cells from P2X7R−/− mice were as effective in their suppressive activity as cells from B6 mice. Therefore, the absence of P2X7Rs appears to cause an increase in functional CD4+CD25+ Treg cells in vivo.

Experiments are presented in this report showing that B6 CD4+CD25+ T cells express high sensitivity to the common cell metabolites, NAD and ATP. A concentration of 1 μM NAD, i.e., 1/1000 that inside cells and only ∼10 times that in the extracellular fluid (22, 23, 24, 25), induces almost instantaneous death. Effects of ATP are concordant, but not as impressive, because concentrations of 300 μM, i.e., 1/10th those inside cells, are required to induce effects comparable to those of NAD. Although these results show that very low concentrations of NAD suffice to induce cell death in Treg cells, our experiments also suggest that this probably only occurs at specific sites at which NAD is released by cell necrosis or other means. Indeed, very high doses of NAD had to be injected i.v. to induce systemic cell death in CD4+CD25+ T cells, and such high doses are not likely to ever be reached by cell lysis.

Our data show that P2X7Rs play an obligatory role in rapid NAD- and ATP-induced death of CD4+CD25+ cells. Therefore, we suggest that free ATP as well as ADP-ribosyl groups attached close to the binding site of the P2X7R provide ligands for its activation. Free ADP-ribose, the breakdown product of NAD by action of CD38 (21), induces only minimal, if any, effects in CD4+CD25+ cells (data not shown), which is consistent with our previous demonstration that rapid death of conventional T cells is induced by NAD, but not by ADP-ribose and requires the expression of functional ART-2 (7). Apparently, the affinity of P2X7Rs for free ADP-ribose molecules is too low to induce efficient signaling, a conclusion consistent with previous results in the P2X7R-oocyte expression system (26). It is interesting that even the canonical ligand for P2X7Rs, i.e., ATP, has to be added in substantial concentrations to induce a death signal. Therefore, free ligands appear to be less efficient in receptor signaling than ADP-ribosyl groups attached to proteins. This enables regulation of the P2X7R function on several levels, i.e., release of NAD by dying cells and presence of ART-2 on CD4+CD25+ cells. Shedding of ART-2 induced by cell activation would increase resistance to NAD-induced cell death (20). Consequently, high concentrations of ATP would be required to trigger cell death. In this respect, it is interesting to note that human T cells lack expression of ART-2 (Ref.27 and our unpublished observations), making regulation of human T cells by cell surface ADP-ribosylation unlikely. However, the signaling sensitivity of the human P2X7R appears to be higher than that of the mouse receptor (28), perhaps enabling more efficient regulation of human T cells via soluble ligands, such as ATP. It is also interesting that mouse strains differ in their P2X7Rs. Indeed, the P2X7Rs in B6 and BALB/c mice differ by one amino acid, resulting in a difference in efficiency of receptor signaling in the two mouse strains (29). Despite this difference, Treg cells from BALB/c mice are also more sensitive to death induction by P2X7R signaling than conventional T cells (data not shown).

The P2X7R is a 595-aa polypeptide with two membrane-spanning domains and N- and C-terminal cytoplasmic domains (18). It is predominantly expressed on monocytes, macrophages, and DC’s (30, 31, 32), but, as shown in this study and elsewhere, it is also expressed on other cell types of the hemopoietic cell lineage, including T cells (7, 16, 33, 34). Engagement of the receptor with ATP (18, 19) opens nonselective ion channels, followed by formation of pores that allow passage of 900-Da molecules (35, 36, 37, 38). Although shorter permeabilization periods may be tolerated as cells reseal their membranes, longer periods often induce death (39, 40, 41). We show that engagement of the P2X7R leads to rapid cell death in a large proportion of the CD4+CD25+ population, associated with complete cell disintegration, consistent with the earlier demonstration that P2X7Rs can induce cell death by a necrotic, osmoid colloidal, lysis mechanism (42). In addition, CD4+CD25+ cells as well as conventional CD4 and CD8 cells can undergo much slower death, indicated by annexin V staining and consistent with apoptosis inducible by the receptor (7, 42, 43). The reason why there exist these dramatic differences in sensitivity among T cell populations remains to be investigated. Reasons could be differences in P2X7R structure, densities, or proteins associated with the receptor. Recent gene array analyses have indicated higher expression of P2X7Rs on Treg cells compared with conventional T effector cells (44). It is therefore quite possible that the observed differences in sensitivity are due to differential expression of the P2X7R on T cell subsets.

The finding that CD4+CD25+ cells undergo much faster death than conventional CD4 and CD8 T cells may point to an important role of this pathway in immune regulation. Both NAD and ATP have exceedingly short half-lives outside cells, due to an abundance of extracellular NAD- and ATP-degrading enzymes. Therefore, a rapid response of CD4+CD25+ cells to NAD and ATP compared with conventional T cells may cause a shift toward conventional T cells, enabling a more efficient immune response.

We demonstrate that CD4+CD25+ cells from P2X7R−/− mice exert normal suppression of T cell proliferation in vitro, which shows that P2X7Rs are not required for their function. The finding that P2X7R−/− mice have increased numbers of Foxp3-expressing CD4+CD25+ cells in the circulation and lymphoid organs points to the possibility that P2X7Rs play a role in Treg cell homeostasis. Low levels of Treg cells correlate with the development of autoimmunity, such as type I diabetes in NOD mice, and a role of P2X7Rs in this disease model has been suggested (45). Consistent with this, P2X7R−/− mice are relatively resistant to Ab-induced collagen arthritis (33).

In conclusion, our data show that NAD and ATP, at concentrations well below those inside cells, induce rapid and efficient death in CD4+CD25+ Treg cells via action on P2X7Rs. Conventional T cells are relatively resistant, resulting in preferential elimination of CD4+CD25+ cells. Our data prompt the hypothesis that metabolites NAD and ATP, released during cell necrosis, serve to limit the action of CD4+CD25+ Treg cells, thereby promoting increased responses of conventional T cells. This newly uncovered mechanism of cell regulation may lead to novel approaches to specifically eliminate Treg cells for therapeutic purposes.

We thank Dr. C Gabel for the generous gift of P2X7R−/− mouse breeding pairs, and Drs. William Stohl and Dixon Gray (University of Southern California) for critical reading of this manuscript.

The authors have no financial conflict of interest.

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 U.S. Public Health Service Grants AI40038 and AI43954.

4

Abbreviations used in this paper: Treg cell, regulatory T cell; Bz-ATP, benzoylbenzoyl-ATP; Foxp3, forkhead/winged helix transcription factor gene.

1
Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda.
1995
. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor α-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.
J. Immunol.
155
:
1151
.-1164.
2
Suri-Payer, E., A. Z. Amar, A. M. Thornton, E. M. Shevach.
1998
. CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells.
J. Immunol.
160
:
1212
.-1218.
3
Bach, J. F..
2002
. The effect of infections on susceptibility to autoimmune and allergic diseases.
N. Engl. J. Med.
347
:
911
.-920.
4
Wucherpfennig, K. W..
2001
. Mechanisms for the induction of autoimmunity by infectious agents.
J. Clin. Invest.
108
:
1097
.-1104.
5
Solle, M., J. Labasi, D. G. Perregaux, E. Stam, N. Petrushova, B. H. Koller, R. J. Griffiths, C. A. Gabel.
2001
. Altered cytokine production in mice lacking P2X7 receptors.
J. Biol. Chem.
276
:
125
.-132.
6
Takeda, I., S. Ine, N. Killeen, L. C. Ndhlovu, K. Murata, S. Satomi, K. Sugamura, N. Ishii.
2004
. Distinct roles for the OX40-OX40 ligand interaction in regulatory and nonregulatory T cells.
J. Immunol.
172
:
3580
.-3589.
7
Kawamura, H., F. Aswad, M. Minagawa, K. Malone, H. Kaslow, F. Koch-Nolte, W. H. Schott, E. H. Leiter, G. Dennert.
2005
. P2X7 receptor dependent and independent T cell death is induced by nicotinamide adenine dinucleotide.
J. Immunol.
174
:
1971
.-1979.
8
Humphreys, B. D., C. Virginio, A. Surprenant, J. Rice, G. R. Dubyak.
1998
. Isoquinolines as antagonists of the P2X7 nucleotide receptor: high selectivity for the human versus rat receptor homologues.
Mol. Pharmacol.
54
:
22
.-32.
9
Krebs, C., W. Koestner, M. Nissen, V. Welge, I. Parusel, F. Malavasi, E. H. Leiter, R. M. Santella, F. Haag, F. Koch-Nolte.
2003
. Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine.
Anal. Biochem.
314
:
108
.-115.
10
Fu, A., S. , N. Zhang, A. C. Yopp, D. Chen, M. Mao, D. Chen, H. Zhang, J. S. Ding, J. S. Bromberg.
2004
. TGF-β induces Foxp3+ T-regulatory cells from CD4+ CD25 precursors.
Am. J. Transplant.
4
:
1614
.-1627.
11
Liu, Z. X., O. Azhipa, S. Okamoto, S. Govindarajan, G. Dennert.
2001
. Extracellular nicotinamide adenine dinucleotide induces T cell apoptosis in vivo and in vitro.
J. Immunol.
167
:
4942
.-4947.
12
Wang, J., E. Nemoto, A. Y. Kots, H. R. Kaslow, G. Dennert.
1994
. Regulation of cytotoxic T cells by ecto-nicotinamide adenine dinucleotide (NAD) correlates with cell surface GPI-anchored/arginine ADP-ribosyltransferase.
J. Immunol.
153
:
4048
.-4058.
13
Adriouch, S., W. Ohlrogge, F. Haag, F. Koch-Nolte, M. Seman.
2001
. Rapid induction of naive T cell apoptosis by ecto-nicotinamide adenine dinucleotide: requirement for mono (ADP-ribosyl)transferase 2 and a downstream effector.
J. Immunol.
167
:
196
.-203.
14
Khattri, R., T. Cox, S. A. Yasayko, F. Ramsdell.
2003
. An essential role for scurfin in CD4+CD25+ T regulatory cells.
Nat. Immunol.
4
:
337
.-342.
15
Fontenot, J. D., M. A. Gavin, A. Y. Rudensky.
2003
. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells.
Nat. Immunol.
4
:
330
.-336.
16
Seman, M., S. Adriouch, F. Scheuplein, C. Krebs, D. Freese, G. Glowacki, P. Deterre, F. Haag, F. Koch-Nolte.
2003
. NAD-induced T cell death: ADP-ribosylation of cell surface proteins by ART2 activates the cytolytic P2X7 purinoceptor.
Immunity
19
:
571
.-582.
17
Gargett, C. E., J. S. Wiley.
1997
. The isoquinoline derivative KN-62 a potent antagonist of the P2Z-receptor of human lymphocytes.
Br. J. Pharmacol.
120
:
1483
.-1490.
18
North, R. A., A. Surprenant.
2000
. Pharmacology of cloned P2X receptors.
Annu. Rev. Pharmacol. Toxicol.
40
:
563
.-580.
19
Di Virgilio, F., P. Chiozzi, D. Ferrari, S. Falzoni, J. M. Sanz, A. Morelli, M. Torboli, G. Bolognesi, O. R. Baricordi.
2001
. Nucleotide receptors: an emerging family of regulatory molecules in blood cells.
Blood
97
:
587
.-600.
20
Nemoto, E., S. Stohlman, G. Dennert.
1996
. Release of a glycosylphosphatidylinositol-anchored ADP-ribosyltransferase from cytotoxic T cells upon activation.
J. Immunol.
156
:
85
.-92.
21
Howard, M., J. C. Grimaldi, J. F. Bazan, F. E. Lund, L. Santos-Argumedo, R. M. Parkhouse, T. F. Walseth, H. C. Lee.
1993
. Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen CD38.
Science
262
:
1056
.-1059.
22
Jacobson, E. L., M. K. Jacobson.
1997
. Tissue NAD as a biochemical measure of niacin status in humans.
Methods Enzymol.
280
:
221
.-230.
23
Kim, U. H., M. K. Kim, J. S. Kim, M. K. Han, B. H. Park, H. R. Kim.
1993
. Purification and characterization of NAD glycohydrolase from rabbit erythrocytes.
Arch. Biochem. Biophys.
305
:
147
.-152.
24
Aleo, M. F., M. L. Giudici, S. Sestini, P. Danesi, G. Pompucci, A. Preti.
2001
. Metabolic fate of extracellular NAD in human skin fibroblasts.
J. Cell Biochem.
80
:
360
.-366.
25
Traut, T. W..
1994
. Physiological concentrations of purines and pyrimidines.
Mol. Cell. Biochem.
140
:
1
.-22.
26
Chakfe, Y., R. Seguin, J. P. Antel, C. Morissette, D. Malo, D. Henderson, P. Seguela.
2002
. ADP and AMP induce interleukin-1β release from microglial cells through activation of ATP-primed P2X7 receptor channels.
J. Neurosci.
22
:
3061
.-3069.
27
Haag, F., F. Koch-Nolte, M. Kuhl, S. Lorenzen, H. G. Thiele.
1994
. Premature stop codons inactivate the RT6 genes of the human and chimpanzee species.
J. Mol. Biol.
243
:
537
.-546.
28
Hibell, A. D., E. J. Kidd, I. P. Chessell, I. P. , P. P. Humphrey, A. D. Michel.
2000
. Apparent species differences in the kinetic properties of P2X7 receptors.
Br. J. Pharmacol.
130
:
167
.-173.
29
Adriouch, S., C. Dox, V. Welge, M. Seman, F. Koch-Nolte, F. Haag.
2002
. Cutting edge: a natural P451L mutation in the cytoplasmic domain impairs the function of the mouse P2X7 receptor.
J. Immunol.
169
:
4108
.-4112.
30
Mutini, C., S. Falzoni, D. Ferrari, P. Chiozzi, A. Morelli, O. R. Baricordi, G. Collo, P. Ricciardi-Castagnoli, F. Di Virgilio.
1999
. Mouse dendritic cells express the P2X7 purinergic receptor: characterization and possible participation in antigen presentation.
J. Immunol.
163
:
1958
.-1965.
31
Collo, G., S. Neidhart, E. Kawashima, M. Kosco-Vilbois, R. A. North, G. Buell.
1997
. Tissue distribution of the P2X7 receptor.
Neuropharmacology
36
:
1277
.-1283.
32
Mehta, V. B., J. Hart, M. D. Wewers.
2001
. ATP-stimulated release of interleukin (IL)-1β and IL-18 requires priming by lipopolysaccharide and is independent of caspase-1 cleavage.
J. Biol. Chem.
276
:
3820
.-3826.
33
Labasi, J. M., N. Petrushova, C. Donovan, S. McCurdy, P. Lira, M. M. Payette, W. Brissette, J. R. Wicks, L. Audoly, C. A. Gabel.
2002
. Absence of the P2X7 receptor alters leukocyte function and attenuates an inflammatory response.
J. Immunol.
168
:
6436
.-6445.
34
Baricordi, O. R., D. Ferrari, L. Melchiorri, P. Chiozzi, S. Hanau, E. Chiari, M. Rubini, F. Di Virgilio.
1996
. An ATP-activated channel is involved in mitogenic stimulation of human T lymphocytes.
Blood
87
:
682
.-690.
35
Schilling, W. P., T. Wasylyna, G. R. Dubyak, B. D. Humphreys, W. G. Sinkins.
1999
. Maitotoxin and P2Z/P2X7 purinergic receptor stimulation activate a common cytolytic pore.
Am. J. Physiol.
277
:
C766
.-C776.
36
Steinberg, T. H., A. S. Newman, J. A. Swanson, S. C. Silverstein.
1987
. ATP4- permeabilizes the plasma membrane of mouse macrophages to fluorescent dyes.
J. Biol. Chem.
262
:
8884
.-8888.
37
Hickman, S. E., J. el Khoury, S. Greenberg, I. Schieren, S. C. Silverstein.
1994
. P2Z adenosine triphosphate receptor activity in cultured human monocyte-derived macrophages.
Blood
84
:
2452
.-2456.
38
Virginio, C., A. MacKenzie, R. A. North, A. Surprenant.
1999
. Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor.
J. Physiol.
519
:
335
.-346.
39
MacKenzie, A., H. L. Wilson, E. Kiss-Toth, S. K. Dower, R. A. North, A. Surprenant.
2001
. Rapid secretion of interleukin-1β by microvesicle shedding.
Immunity
5
:
825
.-835.
40
Buisman, H. P., T. H. Steinberg, J. Fischbarg, S. C. Silverstein, S. A. Vogelzang, C. Ince, D. L. Ypey, P. C. Leijh.
1988
. Extracellular ATP induces a large nonselective conductance in macrophage plasma membranes.
Proc. Natl. Acad. Sci. USA
85
:
7988
.-7992.
41
Ferrari, D., P. Chiozzi, S. Falzoni, M. Dal Susino, G. Collo, G. Buell, F. Di Virgilio.
1997
. ATP-mediated cytotoxicity in microglial cells.
Neuropharmacology
36
:
1295
.-1301.
42
Ferrari, D., M. Los, M. K. Bauer, P. Vandenabeele, S. Wesselborg, K. Schulze-Osthoff.
1999
. P2Z purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death.
FEBS Lett.
447
:
71
.-75.
43
Hogquist, K. A., M. A. Nett, E. R. Unanue, D. D. Chaplin.
1991
. Interleukin 1 is processed and released during apoptosis.
Proc. Natl. Acad. Sci. USA
88
:
8485
.-8489.
44
Herman, A. E., G. J. Freeman, D. Mathis, C. Benoist.
2004
. CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion.
J. Exp. Med.
199
:
1479
.-1489.
45
Elliott, J. I., C. F. Higgins.
2004
. Major histocompatibility complex class I shedding and programmed cell death stimulated through the proinflammatory P2X7 receptor: a candidate susceptibility gene for NOD diabetes.
Diabetes
53
:
2012
.-2017.