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Apoptosis of murine thymocytes induced by extracellular ATP is dose- and cytosolic pH-dependent
Immunology Letters 72 (2000) 23 – 30
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Apoptosis of murine thymocytes induced by extracellular ATP is dose- and cytosolic pH-dependent Pe´ter V. Nagy, Tama´s Fehe´r, Sabina Morga, Ja´nos Matko´ * Biophysics Research Group of the Hungarian Academy of Sciences, Department of Biophysics and Cell Biology, Uni6ersity Medical School of Debrecen, PO Box 39, 4012 Debrecen, Hungary Received 1 December 1999; received in revised form 24 January 2000; accepted 25 January 2000
Abstract Thymocytes from young Balb/C mice responded to low extracellular ATP (ATPec) doses (50.3 mM) with a rapid intracellular acidification (mean pH: ca. 0.3 pH unit) that was inhibited by the Ca2 + channel blocker verapamil, or by suramin (50 mM) and TNP-ATP (40 mM), potent P2x (and P2y) purinoreceptor antagonists. ATPec also triggered a remarkable DNA fragmentation and cell shrinkage detectable only at these low doses. DNA fragmentation gradually disappears with increasing [ATPec] above 0.5 mM, with a concomitant dominance of cytosolic alkalinization of the cells. Suramin and TNP-ATP also blocked the ATPec-triggered DNA fragmentation efficiently. oATP, inhibitor of P2z nonspecific ATP-gated membrane pores, and 2 mM extracellular Mg2 + did not influence either the cytosolic acidification or the DNA fragmentation, but almost completely abolished the intracellular alkalinization characteristic of P2z receptor activation at high ATPec doses. Antagonist-sensitivity of the ATPec-induced membrane potential responses indicates that hyperpolarization is associated with intracellular acidification, while rapid depolarization is linked to alkalinization. These data together indicate that the Ca2 + -dependent hyperpolarization and cytosolic acidification triggered by low ATPec doses are essential early signals in apoptosis of murine thymocytes and are likely mediated by P2x1 type ATP-gated ion channels. Subset specificity of the early purinergic signals suggests that the double positive thymocytes are most sensitive to ATPec showing both P2z and P2x receptor activation characteristics, the double negative thymocytes preferentially show P2z-type, while single positive (CD4−CD8+ or CD4+CD8−) thymocytes respond mostly by weaker P2x-type changes, indicating that ATPec, similarly to adenosine may serve as a potential regulator of cell death and differentiation in the thymus. © 2000 Elsevier Science B.V. All rights reserved. Keywords: P2 purinoreceptors; Cytosolic pH; Plasma membrane potential; DNA fragmentation; Thymocyte apoptosis
1. Introduction Extracellular ATP (ATPec) is known to induce various responses in thymocytes, such as necrotic cell death/lysis, apoptosis or blastogenesis [1 – 3]. These diverse responses are possibly due to differential expression/activity of several distinct ATP-specific P2 purinoreceptors and/or activation of different signaling pathways in these cells. P2z (P2x7) lytic purinoreceptors have been identified and characterized in thymocytes, based on functional and pharmacological properties [1,4,5]. Activation of this receptor by its specific agonist, ATP4 − , results in opening of a non-selective * Corresponding author. Tel./fax: +36-52-412623. E-mail address: [email protected] (J. Matko´)
cation channel/membrane pore permeable even for compounds with a molecular weight of 1 kDa [1,5], followed by lysis of the cells. Other P2 receptor subclasses, like the P2x1 ATP-gated ion channel [6] and the G protein-coupled P2y receptor [7] have been cloned recently and reported to appear also on thymocytes upon apoptotic or activation stimuli [8–10]. Recent studies proposed a potential regulatory role for ATPec in cell-mediated cytotoxicity or intrathymic development/selection [11], thereby making the mechanism of ATP-thymocyte interaction of central interest. The molecular details of the signal pathways involved in purinergically-induced apoptosis, however, are still not fully understood. Moreover, considerable controversy exists about the P2 receptor subclasses mediating apoptotic signals in murine thymocytes [4,8–10].
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Earlier works reported on a biphasic nature of membrane potential [12,13], cytosolic calcium ion level [14] and membrane fluidity responses [15] of murine thymocytes to low and high doses of ATPec. Therefore, in the present study, we analyzed several early and late cellular responses induced by low ATPec doses in murine thymocytes, with special attention to intracellular pH (pHic). Using specific ion channel blockers and P2 receptor antagonists we demonstrated a strong correlation between two ATPec-induced early cellular responses (transient hyperpolarization and cytosolic acidification). Moreover, the cytosolic acidification, hyperpolarization and the late apoptotic response characterized by DNA fragmentation were all inhibited by selective P2x1 (and P2y) purinoreceptor antagonists. These data imply that cytosolic acidification is an important early event in purinergically-induced thymocyte apoptosis and may be mediated by P2x type ATP-gated channel proteins. Maturation-dependency of the early ATP-induced responses suggests that ATPec may act as a potential regulator of different cell death mechanisms in the thymus.
2. Materials and methods
2.1. Chemicals and cells ATP, oxidized ATP, nigericin, Triton X-100 and propidium iodide were purchased from Sigma Chemical, (St Louis, MO), verapamil from Calbiochem-Novabiochem AG (Lucerne, Switzerland). Bis-oxonol (DiBaC4(3), bis-(1,3-dibutylbarbituric acid)trimethine oxonol), BCECF- AM, Pluronic F-127 and TNP-ATP were purchased from Molecular Probes (Eugene, OR). Suramin was from Research Biochemicals International (Natick, MA). All other reagents were of research grade. Thymocytes were prepared from freshly sacrificed, 3 – 4-week old Balb/C mice and their viability was always above 95%, as tested by propidium iodide exclusion [16]. Thymocytes were suspended before the experiments in a standard buffer containing 10 mM Hepes (Fluka AG), 143 mM NaCl, 1 mM Na2SO4, 1 mM NaH2PO4, 5 mM KCl, 1 mM CaCl2 (MgCl2 if indicated) and 5 mM glucose at pH 7.4. In DNA fragmentation experiments, cells were cultured in RPMI 1640 medium supplemented with the appropriate ion milieu, at 37oC, in 5% CO2 incubator.
2.2. Measurement of membrane potential Membrane potential changes were analyzed using a potential sensitive fluorescent probe, bis- oxonol, whose distribution between the extracellular and cellular compartments in potential sensitive. Depolarization of the
plasma membrane leads to an increased, while hyperpolarization results in a reduced concentration of negatively charged oxonol in cellular compartments compared to the resting membrane potential. Bis-oxonol was used at a final concentration of 200 nM [12]. Oxonol fluorescence was excited with the 488 nm line of an argon ion laser, emission was collected with a 520 nm long-pass filter in a modified Becton Dickinson FACStar Plus flow cytometer. Membrane potential changes were determined from 10 000 cells.
2.3. Intracellular pH measurements BCECF was used as an indicator of intracellular pH of cells [17]. The acetoxy-methylester (AM) derivative of this indicator readily permeates the cell membrane. Thereafter it is hydrolyzed by non-specific esterases to yield free, membrane impermeable indicator, whose fluorescence depends on the ambient concentration of free protons. BCECF-AM was dissolved in DMSO at a concentration of 1 mg ml − 1 and applied in a final concentration of : 2 mg/ml, at a cell density of ca. 3–4× 107 cells/cm3. The cells were incubated with the dye for 30 min at 37°C. Calibration of pHic was carried out with the modified method of Balkay et al. [18] using nigericin. Briefly, after increasing the concentration of extracellular K+ to the intracellular level, the pHic can be shifted to that of the extracellular space using nigericin. Using solutions of different pH values a calibration curve can be set up.
2.4. Digital imaging microscopy Signal transduction measurements were carried out in an inverted fluorescence microscope (Axiovert 135TV, Zeiss) equipped with two CCD cameras and an Attofluor digital imaging system 5.44 (Atto Inst, Rockville, MD). Cell density was set to 5–8×106 cm − 3 and the cells were attached onto the surface of poly-L-lysine treated coverslips. Excitation at wavelengths of 460 and 488 nm was used for BCECF. Emission was monitored through a 520 nm band pass filter. Exciting and emitted radiations were discriminated with a 510 nm dichroic mirror for pH measurements. Histograms presented in the figures demonstrate microscopic measurements comprising data of 80–100 individual cells.
2.5. DNA extraction and electrophoresis Cells exposed to ATPec for 6 hours at 37 oC in RPMI-1640 were lysed in the presence of 0.2% Triton X-100, 10 mM Tris, 2 mM EDTA, at pH 7.4. After centrifuging the sample at 13 000×g, the supernatant was digested with 50 mg/ml RNase A for 30 min, and with 0.5 mg/ml proteinase K, in the presence of 1%
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SDS overnight, at 37°C. Then DNA was precipitated with 0.5 M NaCl and 66% ethanol, at − 70°C. The precipitate was centrifuged at 13 000× g, washed in 70% ethanol, and centrifuged again at 13 000× g. The pellet was dried, resuspended and run on a 0.8% agarose gel.
2.6. Flow cytometric analysis of DNA fragmentation DNA fragmentation of thymocytes was followed by flow cytometry using a sensitive technique described in [19]. Briefly, the control and ATPec-treated thymocytes were centrifuged at 200 g and the pellet was gently resuspended in 1.5 ml hypotonic fluorochrome solution (50 mg/ml propidium iodide in 0.1% sodium citrate plus 0.1% Triton X-100) and stored overnight at 4°C before flow cytometry. This procedure allowed an easy discrimination of diploid and subdiploid peaks because of the very low C.V. value of the diploid peak and the large shift of the subdiploid relative to the diploid fluorescence. The samples were run on a Becton–Dickinson FACStar Plus flow cytometer using 514 nm excitation and a 600 nm longpass filter to collect PI fluorescence.
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2.7. Treatment of cells with P2 purinoreceptor antagonists Oxidized ATP (oATP) is known as a specific and irreversible inhibitor of P2z lytic receptors [20]. The cells were incubated in the presence of 0.3 mM oATP for 2 h at 37°C. Afterwards the sample was washed, centrifuged and resuspended in the appropriate buffer for the experiment. In some experiments the cells were pretreated with 50 mM suramin or 40 mM TNP-ATP, specific antagonists of P2x purinoreceptors [21,22] for 3 min, at 37°C, before addition of ATPec.
2.8. Separation of thymocyte subsets by immunomagnetic beads The four subsets of thymocytes were separated magnetically using Magnetic Cell Sorting kit with anti-CD4 or anti-CD8 antibodies coupled to superparamagnetic microbeads in a strong magnetic field (Milte´nyi Biotec GmbH, Germany). Briefly, first the CD8+ thymocytes were separated and then the CD8 antibody-coupled microbeads were removed from the cells by 20 ml/ml Multisort Release Enzyme. Next, both retained and flow-through cells of the first separation step were labeled with anti- CD4 antibody-microbead and separated again in a magnetic field. This three-step procedure yielded all four phenotypic subsets of thymocytes with high purity.
3. Results and discussion
3.1. Extracellular ATP induces rapid and dose-dependent changes in the cytosolic pH of thymocytes
Fig. 1. ATPec induces rapid, dose-dependent changes in the cytosolic pH of murine thymocytes. (A.) The pHic histograms of quiescent cells ( ), of thymocytes treated with 0.1 mM ATPec alone (2) or with 10 mM verapamil +0.1 mM ATPec ( ) were monitored by BCECF and analyzed with single cell imaging microscopy. The histograms represent the pHic values measured at 37°C, 3 min after ATP addition and contain data of 80 – 100 cells. Relative frequency of cells versus pHic is displayed on the figure. (B.) The pHic histograms of quiescent cells ( ), of cells treated with 0.5 mM ATPec alone () and with 100 mM amiloride+ 0.5 mM ATPec (), measured under the same conditions as in part A, are displayed here.
Thymocytes freshly taken from Balb/C mice underwent a rapid and remarkable cytosolic acidification after treatment with low doses of ATPec (5 0.3 mM, Fig. 1). Under these conditions transient membrane hyperpolarization [12,13], moderate rise of cytosolic free calcium level [14] and membrane fluidization [15] were also reported earlier for these cells. In addition, single cell microscopic imaging with the pHic indicator BCECF revealed an interesting biphasic response to ATPec. Low [ATPec] preferentially induced cytosolic acidification (Fig. 1A), while higher ATPec doses ( \ 0.5 mM) provoked alkalinization (Fig. 1B). Both responses were highly specific to ATP, since AMP, UTP or GTP were all found ineffective. Cytosolic acidification was inhibited by 10 mM verapamil, a Ca2 + -channel blocker (Fig. 1A), and by suramin, a selective P2x receptor antagonist (Fig. 3A). Pretreatment of cells
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Fig. 2. Flow cytometric DNA fluorescence histograms of PI-stained thymocytes recorded after 24 h incubation in RPMI medium alone (A) and in the presence of 0.3 mM ATP (+ 1mM Mg2 + ) (B). Data were acquisited in logarithmic mode giving a good resolution of diploid and subdiploid peaks. The proportion of subdiploid cells, as percentages of total cell number, are displayed on both panels. Panel C shows agarose DNA-electrophoretograms of untreated (control) thymocytes (1) and thymocytes treated with 0.1 mM (2), 0.2 mM (3), 0.3 mM (4), 0.5 mM (5) and 0.8 mM (6) ATPec. Lane M displays markers with the indicated number of DNA base pairs. Sample preparation is given in Section 2.
with oATP, 2 mM extracellular Mg2 + known to desensitize P2z lytic purinoreceptors, or with 100 mM amiloride, a blocker of Na+/H+ antiport, all abolished the intracellular alkalinization observed at high ATPec doses, while did not affect cytosolic acidification observed only at low ATPec doses (Table 1). This biphasic pHic response may reflect a possible receptor heterogeneity in mediating the early purinergic signals in murine thymocytes. The rapid cytosolic acidification could be a result of a stimulus-dependent inhibition of the Na+/H+ antiport with a yet unknown mechanism. Since our preliminary investigations have shown an inhibitory effect of certain Ser/Thr phosphatase inhibitors on DNA fragmentation (Fehe´r,T., unpublished data), dephosphorylation of the antiport and/or other apoptosis regulators by these enzyzmes [31] might be a possible mechanism. Further investigations are required, however, to elucidate the contribution of protein phoshatases to the regulation of thymocyte apoptosis. Activation of Ca2 + -dependent H+-flux (e.g. countertransport with Ca2 + -extrusion via plasma membrane Ca2 + - ATPase [28]) or metabolic H+-generation [29] could also be responsible for the rapid acidification. Cytosolic alkalinization observed at high ATPec doses is likely linked to activation of Na+/H+ antiport or other mechanisms sensitive to amiloride (Fig. 1B), consistent with earlier data showing requirement of extracellular Na+ for intracellular alkalinization as opposed to intracellular acidification [30]. Since activation of P2z ATP-gated membrane pores is followed by a large Na+ conductance and sustained membrane depolarization [1,5,12], the cytosolic alkalinization of murine thymocytes is most likely linked to activation of these receptors. This is further supported by the efficient blockade of alkalinization response with oATP and Mg2 + .
3.2. ATPec -triggered DNA fragmentation in thymocytes is dose-dependent and sensiti6e to P2x purinoreceptor antagonists Thymocytes taken from young Balb/C mice display a spontaneous basal DNA fragmentation (ca. 20–25% subdiploid cells in 24 h) in culture (Fig. 2A), that is significantly increased by low (5 0.3 mM) ATPec doses (up to 60% subdiploid cells in 24 h) (Fig. 2B). In addition, we have shown that the cells display typical morphological features of apoptosis with decreased cell volume several hours after ATP addition [13]. At higher concentrations (]0.5 mM) the DNA fragmentation decreased (Fig. 2C) possibly due to a dominating lytic effect of ATPec. This indicates that ATPec can, itself, initiate thymocyte apoptosis and the purinoreceptors mediating apoptotic signals can be saturated already by low doses insufficient to cause significant activation of P2z lytic purinoreceptors. Our data are consistent with the observed additive or antagonistic effects of ATPec at low and high doses, respectively, on TcR-engagement-induced rapid thymoTable 1 pHic- — response of thymocytes to ATPec under different conditionsa Cells, treatment
Intracellular pH
Control 0.1 mM 0.1 mM 0.5 mM 0.5 mM 0.5 mM 0.5 mM
7.21 90.03 6.94 9 0.04 7.3590.03 7.41 90.04 6.95 9 0.02 6.89 90.04 6.99 9 0.03
ATPec ATPec+10 mM verapamil ATPec ATPec+100 mM amiloride ATPec+0.3 mM oATP ATP+2 mM Mg2+
a Intracellular pH values of control and ATP-treated thymocytes are presented as mean 9SEM. pHic values were calculated from at least four independent image microscopic experiments ( : 600 cells) using the mean values of histograms.
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3.3. Correlation between the early purinergic signals and the late apoptotic response
Fig. 3. Effect of P2 purinoreceptor antagonists on the ATPec-induced cytosolic acidification and DNA fragmentation. (A.) Quiescent thymocytes were treated with 0.3 mM ATP and intracellular pH was monitored in time ( ). Thymocytes pretreated with 50 mM suramin were challenged with the same ATPec dose (). Curves display the mean pHic value of :50 cells from three independent experiments. Typical error bars representing SEM values are also shown. (B.) Percentages of subdiploid cells (determined from flow cytometric histograms) incubated for 24 h in RPMI-1640 are shown for untreated thymocytes (1), for cells treated with 0.3 mM ATPec alone (2), with 50 mM suramin+0.3 mM ATPec (3), with 40 mM TNP-ATP+ 0.3 mM ATPec (4), and with 0.3 mM oATP+ 0.3 mM ATPec (5).
Induction of apoptosis was often found associated with changes in pHic in various cell types under apoptotic stimuli, such as etoposide [25], somatostatin [26] or staurosporin [27]. In most cases the cytosolic acidification was shown to be Ca2 + -dependent. We also found a strong time-dependent relation between the rapid rise of cytosolic free calcium and the subsequent intracellular acidification, with a maximal effect appearing 2–3 min after addition of ATPec (Fig. 3A). The transient hyperpolarization observed at low ATPec doses was also reported to be calcium dependent [12,13], and possibly linked to calcium-dependent potassium channel (KCa) activation [23,24]. In addition, we show here that the Ca2 + -dependent acidification and hyperpolarization were both sensitive to suramin (Table 1 and Table 2), suggesting a cellular signaling mechanism mediated by suramin-sensitive P2 purinoreceptors. The relationship between cytosolic acidification and DNA fragmentation/apoptosis, in general, is still not well understood. Increasing number of recent reports demonstrated that intracellular acidification may stimulate the ICE/CED-3 protease family [31,32], acidic endonucleases [33] and the pH-sensitive DNAse g [34] associated with initiation of apoptotic DNA fragmentation. The observed relation between the ATPec-trigTable 2 Receptor- and thymocyte subset-specificity of the early membrane potential responses to low and high ATPec dosesa Thymocyte phenotype, pretreatment
Membrane potential response (percentage change of indicator fluorescence, %)b 0.1 mM ATP
cyte cell death [10]. Concentration dependence of DNA fragmentation observed here may resolve an apparent controversy reported earlier [9]. In this work P2x1 receptor mRNA (supposed to be involved in apoptosis [8]) was found in stimulated thymocytes, but no significant DNA fragmentation was detected. However, 0.5–5 mM ATPec concentrations were used, which, according to our data (Fig. 2C), falls into the range where lytic action of ATPec becomes dominant without detectable DNA fragmentation. Suramin and TNP-ATP, potent antagonists of P2x receptors [21,22], both inhibited DNA fragmentation, while oATP, an irreversible P2z receptor inhibitor did not (Fig. 3B). This suggests that the ATPec-induced DNA fragmentation is associated to P2x-type receptor activation that is likely responsible also for cytosolic acidification.
Whole thymocyte popu- −14 ( 9 4) lation Whole thymocyte popuND lation, 0.3 mM oATP Whole thymocyte popu- +9 (9 4) lation, 50 mM suramin Double negative subset +5 (98) Double positive subset −20 ( 9 6) Single positive subset −28 ( 9 5) a
1 mM ATP +37 ( 9 8) −6 (9 5) ND
+87 ( 9 12) +53 ( 9 7) +19 ( 9 6)
Thymocytes were treated with the indicated amount of ATP. Membrane potential changes were measured 5 min after ATP challenge, and are expressed as relative changes in oxonol indicator fluorescence intensity ( 9standard error of the mean). Fluorescence intensity was determined from the mean values of fluorescence histograms from flow cytometric experiments (10 000 cells/sample). Increased fluorescence intensity refers to depolarization, while a negative change represents hyperpolarization. Subset specifications refer to the expression of CD4 and CD8 differentiation antigens (ND, not determined). b
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Fig. 4. Staining of separated thymocytes with anti-CD4 and anti-CD8 antibodies. Thymocytes separated with magnetic beads were stained with fluoresceinated anti-CD8 and rhodaminated anti-CD4 primary antibodies, and 10 000 cells per sample were recorded on a flow cytometer. Fluorescence intensities were corrected for spectral overlap, and corrected values are presented as dot plots for the four subpopulations stained and analyzed separately.
gered cytosolic acidification and DNA fragmentation indicates that the decrease in pHic seen in thymocytes is essential for initiation of nucleosomal DNA degradation. The efficient inhibition of apoptosis by suramin and TNP-ATP, and not by oATP, suggests involvement of the P2x or P2y receptor subfamilies selectively sensitive to these antagonists [21,22]. Since the early cellular signals were not associated with phosphoinositide hydrolysis [35] and were insensitive to G protein inhibitors (pertussis toxin or GTP-g-S) (Matko´ J., unpublished data), involvement of G protein-linked P2y receptors seems less likely. The observed agonist and antagonist specificity of the investigated signals, consistently with recent immunocytochemical staining of Balb/C mouse thymocytes [8], all imply involvement of the P2x1 ATPgated cation channel [6]. Another possibility; that P2z (P2x7) purinoreceptors in thymocytes may function under different conditions as either lytic P2z membrane pores or as P2x1 ATP-gated cation channels, cannot be excluded either, although this hypothesis [4,10,36] needs further support. Apoptosis is thought to play a key role in the negative selection of thymocytes. The biphasic pH and membrane potential responses reported, respectively, in
this paper and previously [12] could be explained by at least two types of purinoreceptor activities (e.g. P2x and P2z, as discussed above) and/or by heterogeneity in purinoreceptor expression among thymocytes. The latter possibility would also be consistent with the notion of differentiation-dependent expression of thymocyte cell surface receptors in general, and with the maturation-dependent expression of purionoreceptors in particular [8,11,14]. In an attempt to correlate ATP-induced early signals with expression of CD4 and CD8 molecules we separated double negative (CD4− CD8−), double positive (CD4+CD8+) and single positive (CD4−CD8+ and CD4+CD8−) thymocytes using magnetic beads. The purity of the separated subsets was \95%, as shown in Fig. 4, using fluorescent anti-CD4 and -CD8 antibodies and flow cytometric analysis of CD4 and CD8 positivity, respectively. However, the number of isolated cells was low, especially in the double negative and single positive subsets. Therefore we used the plasma membrane potential of cells as an indicator of early cellular responses, since labeling of cells with a pHic indicator would have resulted in further cell loss. Characterizing the dose dependence of the membrane potential response ([12] and Table 2) we found that high ATP dose (\ 0.5 mM) preferentially
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induces depolarization, while low concentration of ATP ( B 0.3 mM) elicits hyperpolarization. Previously we have also shown that hyperpolarization is associated with cytosolic acidification. In order to assign distinct purinoreceptor activities to the membrane potential responses, oATP and suramin were used. The depolarization and alkalinization observed at high ATP-dose were blocked by oATP implying the importance of P2z receptor activity in this process. On the other hand, hyperpolarization and acidification were sensitive to the P2x receptor antagonist, suramin (Table 2). Having established a link between purinoreceptor activity and membrane potential changes, we characterized the subset specificity of the above responses. The most immature, double negative thymocytes did not respond by hyperpolarization to low ATPec dose (0.1 mM), while they displayed the highest sensitivity to high ATPec dose (1 mM) responding by a remarkable depolarization. The mature, single positive subsets displayed a much reduced depolarization response to high ATPec dose, while they showed a well detectable hyperpolarization response to low ATPec dose. The double positive cells responded to low ATPec dose by hyperpolarization with a remarkable sensitivity, and also demonstrated significant depolarization to high ATPec dose (Table 2). Characterization of purinoreceptor activities may have important implications in the maturation of thymocytes. Our data imply that P2x purinoreceptors, but not the P2z receptors, are important in ATPec-induced apoptosis of thymocytes. Assuming that hyper- and depolarization responses are characteristic markers of P2x and P2z receptor activation, respectively, our data imply that the most immature double negative cells lack P2x receptor activity, but produce functional P2z receptors. In contrast, in single positive cells the membrane potential responses indicated a low abundance of P2z receptors, while P2x receptor expression/activity was high in these cells. The CD4+CD8+ cells seem to be in transition between double negative and mature cells with respect to purinoreceptor activity: they show both P2x- and P2z-characteristic responses depending on the ATPec dose, therefore being most sensitive to this effector molecule. Expression of both types of P2 receptors on the most abundant thymocyte subset may explain the fact that in the case of inhibition of the P2x receptor pathway pHic changes were characteristic of the P2z-type response, and the other way around (Figs. 1 and 3A and Table 1). Our results are consistent with the work of Di Virgilio et al. [37] demonstrating that mature lymphocytes are much less sensitive to P2z receptor-mediated lysis, and also coincide with our previous results [38] showing that medullary thymocytes were more sensitive to the apoptosis inducing effect of ATPec. Our functional data coincide well with direct immunohistochemical staining of P2x receptor
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proteins, showing that P2x1 receptors are absent in the most mature, double negative cells, while they are more abundant in single or double positive cells [8].
3.4. Concluding remarks The functional role of P2 purinoreceptor-mediated responses is not fully understood. P2x receptors may take part in thymocyte apoptosis either on their own or in cooperation with other apoptosis inducing agents, e.g. steroids [8]. According to our results P2x-mediated signaling may be important in apoptosis of cells in the double positive subset, where most selection processes take place, but also in late stage selection of mature thymocytes. Since the P1 receptor agonist, adenosine is believed to play a role in the negative selection of double positive thymocytes [11,39], the concerted action of P1 and P2 purinoreceptors would allow a sensitive control of cell survival depending on purinoreceptor and ecto-ATPase expression of cells and local agonist concentration. Resolution of the receptor heterogeneity on thymocyte subsets, as well as identification of further potential intracellular signaling components, such as protein phosphatases and proteases, need further investigations. Receptor gene knock-out animals, recently published P2 receptor cDNA sequences and the availability of monoclonal antibodies and structural models for the receptors can further stimulate these studies.
Acknowledgements We thank Drs Sa´ndor Damjanovich, Ja´nos Szo¨llo¨si and Gyo¨rgy Panyi for their valuable criticism and discussions. The skillful technical assistance of Istvan Gal and Terez Lakatos in the experiments is gratefully acknowledged. This work was supported by research grants OTKA 23873 and 29947 from the Hungarian Academy of Sciences.
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Apoptosis of murine thymocytes induced by extracellular ATP is dose- and cytosolic pH-dependent Pe´ter V. Nagy, Tama´s Fehe´r, Sabina Morga, Ja´nos Matko´ * Biophysics Research Group of the Hungarian Academy of Sciences, Department of Biophysics and Cell Biology, Uni6ersity Medical School of Debrecen, PO Box 39, 4012 Debrecen, Hungary Received 1 December 1999; received in revised form 24 January 2000; accepted 25 January 2000
Abstract Thymocytes from young Balb/C mice responded to low extracellular ATP (ATPec) doses (50.3 mM) with a rapid intracellular acidification (mean pH: ca. 0.3 pH unit) that was inhibited by the Ca2 + channel blocker verapamil, or by suramin (50 mM) and TNP-ATP (40 mM), potent P2x (and P2y) purinoreceptor antagonists. ATPec also triggered a remarkable DNA fragmentation and cell shrinkage detectable only at these low doses. DNA fragmentation gradually disappears with increasing [ATPec] above 0.5 mM, with a concomitant dominance of cytosolic alkalinization of the cells. Suramin and TNP-ATP also blocked the ATPec-triggered DNA fragmentation efficiently. oATP, inhibitor of P2z nonspecific ATP-gated membrane pores, and 2 mM extracellular Mg2 + did not influence either the cytosolic acidification or the DNA fragmentation, but almost completely abolished the intracellular alkalinization characteristic of P2z receptor activation at high ATPec doses. Antagonist-sensitivity of the ATPec-induced membrane potential responses indicates that hyperpolarization is associated with intracellular acidification, while rapid depolarization is linked to alkalinization. These data together indicate that the Ca2 + -dependent hyperpolarization and cytosolic acidification triggered by low ATPec doses are essential early signals in apoptosis of murine thymocytes and are likely mediated by P2x1 type ATP-gated ion channels. Subset specificity of the early purinergic signals suggests that the double positive thymocytes are most sensitive to ATPec showing both P2z and P2x receptor activation characteristics, the double negative thymocytes preferentially show P2z-type, while single positive (CD4−CD8+ or CD4+CD8−) thymocytes respond mostly by weaker P2x-type changes, indicating that ATPec, similarly to adenosine may serve as a potential regulator of cell death and differentiation in the thymus. © 2000 Elsevier Science B.V. All rights reserved. Keywords: P2 purinoreceptors; Cytosolic pH; Plasma membrane potential; DNA fragmentation; Thymocyte apoptosis
1. Introduction Extracellular ATP (ATPec) is known to induce various responses in thymocytes, such as necrotic cell death/lysis, apoptosis or blastogenesis [1 – 3]. These diverse responses are possibly due to differential expression/activity of several distinct ATP-specific P2 purinoreceptors and/or activation of different signaling pathways in these cells. P2z (P2x7) lytic purinoreceptors have been identified and characterized in thymocytes, based on functional and pharmacological properties [1,4,5]. Activation of this receptor by its specific agonist, ATP4 − , results in opening of a non-selective * Corresponding author. Tel./fax: +36-52-412623. E-mail address: [email protected] (J. Matko´)
cation channel/membrane pore permeable even for compounds with a molecular weight of 1 kDa [1,5], followed by lysis of the cells. Other P2 receptor subclasses, like the P2x1 ATP-gated ion channel [6] and the G protein-coupled P2y receptor [7] have been cloned recently and reported to appear also on thymocytes upon apoptotic or activation stimuli [8–10]. Recent studies proposed a potential regulatory role for ATPec in cell-mediated cytotoxicity or intrathymic development/selection [11], thereby making the mechanism of ATP-thymocyte interaction of central interest. The molecular details of the signal pathways involved in purinergically-induced apoptosis, however, are still not fully understood. Moreover, considerable controversy exists about the P2 receptor subclasses mediating apoptotic signals in murine thymocytes [4,8–10].
0165-2478/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 0 ) 0 0 1 6 8 - 1
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Earlier works reported on a biphasic nature of membrane potential [12,13], cytosolic calcium ion level [14] and membrane fluidity responses [15] of murine thymocytes to low and high doses of ATPec. Therefore, in the present study, we analyzed several early and late cellular responses induced by low ATPec doses in murine thymocytes, with special attention to intracellular pH (pHic). Using specific ion channel blockers and P2 receptor antagonists we demonstrated a strong correlation between two ATPec-induced early cellular responses (transient hyperpolarization and cytosolic acidification). Moreover, the cytosolic acidification, hyperpolarization and the late apoptotic response characterized by DNA fragmentation were all inhibited by selective P2x1 (and P2y) purinoreceptor antagonists. These data imply that cytosolic acidification is an important early event in purinergically-induced thymocyte apoptosis and may be mediated by P2x type ATP-gated channel proteins. Maturation-dependency of the early ATP-induced responses suggests that ATPec may act as a potential regulator of different cell death mechanisms in the thymus.
2. Materials and methods
2.1. Chemicals and cells ATP, oxidized ATP, nigericin, Triton X-100 and propidium iodide were purchased from Sigma Chemical, (St Louis, MO), verapamil from Calbiochem-Novabiochem AG (Lucerne, Switzerland). Bis-oxonol (DiBaC4(3), bis-(1,3-dibutylbarbituric acid)trimethine oxonol), BCECF- AM, Pluronic F-127 and TNP-ATP were purchased from Molecular Probes (Eugene, OR). Suramin was from Research Biochemicals International (Natick, MA). All other reagents were of research grade. Thymocytes were prepared from freshly sacrificed, 3 – 4-week old Balb/C mice and their viability was always above 95%, as tested by propidium iodide exclusion [16]. Thymocytes were suspended before the experiments in a standard buffer containing 10 mM Hepes (Fluka AG), 143 mM NaCl, 1 mM Na2SO4, 1 mM NaH2PO4, 5 mM KCl, 1 mM CaCl2 (MgCl2 if indicated) and 5 mM glucose at pH 7.4. In DNA fragmentation experiments, cells were cultured in RPMI 1640 medium supplemented with the appropriate ion milieu, at 37oC, in 5% CO2 incubator.
2.2. Measurement of membrane potential Membrane potential changes were analyzed using a potential sensitive fluorescent probe, bis- oxonol, whose distribution between the extracellular and cellular compartments in potential sensitive. Depolarization of the
plasma membrane leads to an increased, while hyperpolarization results in a reduced concentration of negatively charged oxonol in cellular compartments compared to the resting membrane potential. Bis-oxonol was used at a final concentration of 200 nM [12]. Oxonol fluorescence was excited with the 488 nm line of an argon ion laser, emission was collected with a 520 nm long-pass filter in a modified Becton Dickinson FACStar Plus flow cytometer. Membrane potential changes were determined from 10 000 cells.
2.3. Intracellular pH measurements BCECF was used as an indicator of intracellular pH of cells [17]. The acetoxy-methylester (AM) derivative of this indicator readily permeates the cell membrane. Thereafter it is hydrolyzed by non-specific esterases to yield free, membrane impermeable indicator, whose fluorescence depends on the ambient concentration of free protons. BCECF-AM was dissolved in DMSO at a concentration of 1 mg ml − 1 and applied in a final concentration of : 2 mg/ml, at a cell density of ca. 3–4× 107 cells/cm3. The cells were incubated with the dye for 30 min at 37°C. Calibration of pHic was carried out with the modified method of Balkay et al. [18] using nigericin. Briefly, after increasing the concentration of extracellular K+ to the intracellular level, the pHic can be shifted to that of the extracellular space using nigericin. Using solutions of different pH values a calibration curve can be set up.
2.4. Digital imaging microscopy Signal transduction measurements were carried out in an inverted fluorescence microscope (Axiovert 135TV, Zeiss) equipped with two CCD cameras and an Attofluor digital imaging system 5.44 (Atto Inst, Rockville, MD). Cell density was set to 5–8×106 cm − 3 and the cells were attached onto the surface of poly-L-lysine treated coverslips. Excitation at wavelengths of 460 and 488 nm was used for BCECF. Emission was monitored through a 520 nm band pass filter. Exciting and emitted radiations were discriminated with a 510 nm dichroic mirror for pH measurements. Histograms presented in the figures demonstrate microscopic measurements comprising data of 80–100 individual cells.
2.5. DNA extraction and electrophoresis Cells exposed to ATPec for 6 hours at 37 oC in RPMI-1640 were lysed in the presence of 0.2% Triton X-100, 10 mM Tris, 2 mM EDTA, at pH 7.4. After centrifuging the sample at 13 000×g, the supernatant was digested with 50 mg/ml RNase A for 30 min, and with 0.5 mg/ml proteinase K, in the presence of 1%
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SDS overnight, at 37°C. Then DNA was precipitated with 0.5 M NaCl and 66% ethanol, at − 70°C. The precipitate was centrifuged at 13 000× g, washed in 70% ethanol, and centrifuged again at 13 000× g. The pellet was dried, resuspended and run on a 0.8% agarose gel.
2.6. Flow cytometric analysis of DNA fragmentation DNA fragmentation of thymocytes was followed by flow cytometry using a sensitive technique described in [19]. Briefly, the control and ATPec-treated thymocytes were centrifuged at 200 g and the pellet was gently resuspended in 1.5 ml hypotonic fluorochrome solution (50 mg/ml propidium iodide in 0.1% sodium citrate plus 0.1% Triton X-100) and stored overnight at 4°C before flow cytometry. This procedure allowed an easy discrimination of diploid and subdiploid peaks because of the very low C.V. value of the diploid peak and the large shift of the subdiploid relative to the diploid fluorescence. The samples were run on a Becton–Dickinson FACStar Plus flow cytometer using 514 nm excitation and a 600 nm longpass filter to collect PI fluorescence.
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2.7. Treatment of cells with P2 purinoreceptor antagonists Oxidized ATP (oATP) is known as a specific and irreversible inhibitor of P2z lytic receptors [20]. The cells were incubated in the presence of 0.3 mM oATP for 2 h at 37°C. Afterwards the sample was washed, centrifuged and resuspended in the appropriate buffer for the experiment. In some experiments the cells were pretreated with 50 mM suramin or 40 mM TNP-ATP, specific antagonists of P2x purinoreceptors [21,22] for 3 min, at 37°C, before addition of ATPec.
2.8. Separation of thymocyte subsets by immunomagnetic beads The four subsets of thymocytes were separated magnetically using Magnetic Cell Sorting kit with anti-CD4 or anti-CD8 antibodies coupled to superparamagnetic microbeads in a strong magnetic field (Milte´nyi Biotec GmbH, Germany). Briefly, first the CD8+ thymocytes were separated and then the CD8 antibody-coupled microbeads were removed from the cells by 20 ml/ml Multisort Release Enzyme. Next, both retained and flow-through cells of the first separation step were labeled with anti- CD4 antibody-microbead and separated again in a magnetic field. This three-step procedure yielded all four phenotypic subsets of thymocytes with high purity.
3. Results and discussion
3.1. Extracellular ATP induces rapid and dose-dependent changes in the cytosolic pH of thymocytes
Fig. 1. ATPec induces rapid, dose-dependent changes in the cytosolic pH of murine thymocytes. (A.) The pHic histograms of quiescent cells ( ), of thymocytes treated with 0.1 mM ATPec alone (2) or with 10 mM verapamil +0.1 mM ATPec ( ) were monitored by BCECF and analyzed with single cell imaging microscopy. The histograms represent the pHic values measured at 37°C, 3 min after ATP addition and contain data of 80 – 100 cells. Relative frequency of cells versus pHic is displayed on the figure. (B.) The pHic histograms of quiescent cells ( ), of cells treated with 0.5 mM ATPec alone () and with 100 mM amiloride+ 0.5 mM ATPec (), measured under the same conditions as in part A, are displayed here.
Thymocytes freshly taken from Balb/C mice underwent a rapid and remarkable cytosolic acidification after treatment with low doses of ATPec (5 0.3 mM, Fig. 1). Under these conditions transient membrane hyperpolarization [12,13], moderate rise of cytosolic free calcium level [14] and membrane fluidization [15] were also reported earlier for these cells. In addition, single cell microscopic imaging with the pHic indicator BCECF revealed an interesting biphasic response to ATPec. Low [ATPec] preferentially induced cytosolic acidification (Fig. 1A), while higher ATPec doses ( \ 0.5 mM) provoked alkalinization (Fig. 1B). Both responses were highly specific to ATP, since AMP, UTP or GTP were all found ineffective. Cytosolic acidification was inhibited by 10 mM verapamil, a Ca2 + -channel blocker (Fig. 1A), and by suramin, a selective P2x receptor antagonist (Fig. 3A). Pretreatment of cells
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Fig. 2. Flow cytometric DNA fluorescence histograms of PI-stained thymocytes recorded after 24 h incubation in RPMI medium alone (A) and in the presence of 0.3 mM ATP (+ 1mM Mg2 + ) (B). Data were acquisited in logarithmic mode giving a good resolution of diploid and subdiploid peaks. The proportion of subdiploid cells, as percentages of total cell number, are displayed on both panels. Panel C shows agarose DNA-electrophoretograms of untreated (control) thymocytes (1) and thymocytes treated with 0.1 mM (2), 0.2 mM (3), 0.3 mM (4), 0.5 mM (5) and 0.8 mM (6) ATPec. Lane M displays markers with the indicated number of DNA base pairs. Sample preparation is given in Section 2.
with oATP, 2 mM extracellular Mg2 + known to desensitize P2z lytic purinoreceptors, or with 100 mM amiloride, a blocker of Na+/H+ antiport, all abolished the intracellular alkalinization observed at high ATPec doses, while did not affect cytosolic acidification observed only at low ATPec doses (Table 1). This biphasic pHic response may reflect a possible receptor heterogeneity in mediating the early purinergic signals in murine thymocytes. The rapid cytosolic acidification could be a result of a stimulus-dependent inhibition of the Na+/H+ antiport with a yet unknown mechanism. Since our preliminary investigations have shown an inhibitory effect of certain Ser/Thr phosphatase inhibitors on DNA fragmentation (Fehe´r,T., unpublished data), dephosphorylation of the antiport and/or other apoptosis regulators by these enzyzmes [31] might be a possible mechanism. Further investigations are required, however, to elucidate the contribution of protein phoshatases to the regulation of thymocyte apoptosis. Activation of Ca2 + -dependent H+-flux (e.g. countertransport with Ca2 + -extrusion via plasma membrane Ca2 + - ATPase [28]) or metabolic H+-generation [29] could also be responsible for the rapid acidification. Cytosolic alkalinization observed at high ATPec doses is likely linked to activation of Na+/H+ antiport or other mechanisms sensitive to amiloride (Fig. 1B), consistent with earlier data showing requirement of extracellular Na+ for intracellular alkalinization as opposed to intracellular acidification [30]. Since activation of P2z ATP-gated membrane pores is followed by a large Na+ conductance and sustained membrane depolarization [1,5,12], the cytosolic alkalinization of murine thymocytes is most likely linked to activation of these receptors. This is further supported by the efficient blockade of alkalinization response with oATP and Mg2 + .
3.2. ATPec -triggered DNA fragmentation in thymocytes is dose-dependent and sensiti6e to P2x purinoreceptor antagonists Thymocytes taken from young Balb/C mice display a spontaneous basal DNA fragmentation (ca. 20–25% subdiploid cells in 24 h) in culture (Fig. 2A), that is significantly increased by low (5 0.3 mM) ATPec doses (up to 60% subdiploid cells in 24 h) (Fig. 2B). In addition, we have shown that the cells display typical morphological features of apoptosis with decreased cell volume several hours after ATP addition [13]. At higher concentrations (]0.5 mM) the DNA fragmentation decreased (Fig. 2C) possibly due to a dominating lytic effect of ATPec. This indicates that ATPec can, itself, initiate thymocyte apoptosis and the purinoreceptors mediating apoptotic signals can be saturated already by low doses insufficient to cause significant activation of P2z lytic purinoreceptors. Our data are consistent with the observed additive or antagonistic effects of ATPec at low and high doses, respectively, on TcR-engagement-induced rapid thymoTable 1 pHic- — response of thymocytes to ATPec under different conditionsa Cells, treatment
Intracellular pH
Control 0.1 mM 0.1 mM 0.5 mM 0.5 mM 0.5 mM 0.5 mM
7.21 90.03 6.94 9 0.04 7.3590.03 7.41 90.04 6.95 9 0.02 6.89 90.04 6.99 9 0.03
ATPec ATPec+10 mM verapamil ATPec ATPec+100 mM amiloride ATPec+0.3 mM oATP ATP+2 mM Mg2+
a Intracellular pH values of control and ATP-treated thymocytes are presented as mean 9SEM. pHic values were calculated from at least four independent image microscopic experiments ( : 600 cells) using the mean values of histograms.
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3.3. Correlation between the early purinergic signals and the late apoptotic response
Fig. 3. Effect of P2 purinoreceptor antagonists on the ATPec-induced cytosolic acidification and DNA fragmentation. (A.) Quiescent thymocytes were treated with 0.3 mM ATP and intracellular pH was monitored in time ( ). Thymocytes pretreated with 50 mM suramin were challenged with the same ATPec dose (). Curves display the mean pHic value of :50 cells from three independent experiments. Typical error bars representing SEM values are also shown. (B.) Percentages of subdiploid cells (determined from flow cytometric histograms) incubated for 24 h in RPMI-1640 are shown for untreated thymocytes (1), for cells treated with 0.3 mM ATPec alone (2), with 50 mM suramin+0.3 mM ATPec (3), with 40 mM TNP-ATP+ 0.3 mM ATPec (4), and with 0.3 mM oATP+ 0.3 mM ATPec (5).
Induction of apoptosis was often found associated with changes in pHic in various cell types under apoptotic stimuli, such as etoposide [25], somatostatin [26] or staurosporin [27]. In most cases the cytosolic acidification was shown to be Ca2 + -dependent. We also found a strong time-dependent relation between the rapid rise of cytosolic free calcium and the subsequent intracellular acidification, with a maximal effect appearing 2–3 min after addition of ATPec (Fig. 3A). The transient hyperpolarization observed at low ATPec doses was also reported to be calcium dependent [12,13], and possibly linked to calcium-dependent potassium channel (KCa) activation [23,24]. In addition, we show here that the Ca2 + -dependent acidification and hyperpolarization were both sensitive to suramin (Table 1 and Table 2), suggesting a cellular signaling mechanism mediated by suramin-sensitive P2 purinoreceptors. The relationship between cytosolic acidification and DNA fragmentation/apoptosis, in general, is still not well understood. Increasing number of recent reports demonstrated that intracellular acidification may stimulate the ICE/CED-3 protease family [31,32], acidic endonucleases [33] and the pH-sensitive DNAse g [34] associated with initiation of apoptotic DNA fragmentation. The observed relation between the ATPec-trigTable 2 Receptor- and thymocyte subset-specificity of the early membrane potential responses to low and high ATPec dosesa Thymocyte phenotype, pretreatment
Membrane potential response (percentage change of indicator fluorescence, %)b 0.1 mM ATP
cyte cell death [10]. Concentration dependence of DNA fragmentation observed here may resolve an apparent controversy reported earlier [9]. In this work P2x1 receptor mRNA (supposed to be involved in apoptosis [8]) was found in stimulated thymocytes, but no significant DNA fragmentation was detected. However, 0.5–5 mM ATPec concentrations were used, which, according to our data (Fig. 2C), falls into the range where lytic action of ATPec becomes dominant without detectable DNA fragmentation. Suramin and TNP-ATP, potent antagonists of P2x receptors [21,22], both inhibited DNA fragmentation, while oATP, an irreversible P2z receptor inhibitor did not (Fig. 3B). This suggests that the ATPec-induced DNA fragmentation is associated to P2x-type receptor activation that is likely responsible also for cytosolic acidification.
Whole thymocyte popu- −14 ( 9 4) lation Whole thymocyte popuND lation, 0.3 mM oATP Whole thymocyte popu- +9 (9 4) lation, 50 mM suramin Double negative subset +5 (98) Double positive subset −20 ( 9 6) Single positive subset −28 ( 9 5) a
1 mM ATP +37 ( 9 8) −6 (9 5) ND
+87 ( 9 12) +53 ( 9 7) +19 ( 9 6)
Thymocytes were treated with the indicated amount of ATP. Membrane potential changes were measured 5 min after ATP challenge, and are expressed as relative changes in oxonol indicator fluorescence intensity ( 9standard error of the mean). Fluorescence intensity was determined from the mean values of fluorescence histograms from flow cytometric experiments (10 000 cells/sample). Increased fluorescence intensity refers to depolarization, while a negative change represents hyperpolarization. Subset specifications refer to the expression of CD4 and CD8 differentiation antigens (ND, not determined). b
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Fig. 4. Staining of separated thymocytes with anti-CD4 and anti-CD8 antibodies. Thymocytes separated with magnetic beads were stained with fluoresceinated anti-CD8 and rhodaminated anti-CD4 primary antibodies, and 10 000 cells per sample were recorded on a flow cytometer. Fluorescence intensities were corrected for spectral overlap, and corrected values are presented as dot plots for the four subpopulations stained and analyzed separately.
gered cytosolic acidification and DNA fragmentation indicates that the decrease in pHic seen in thymocytes is essential for initiation of nucleosomal DNA degradation. The efficient inhibition of apoptosis by suramin and TNP-ATP, and not by oATP, suggests involvement of the P2x or P2y receptor subfamilies selectively sensitive to these antagonists [21,22]. Since the early cellular signals were not associated with phosphoinositide hydrolysis [35] and were insensitive to G protein inhibitors (pertussis toxin or GTP-g-S) (Matko´ J., unpublished data), involvement of G protein-linked P2y receptors seems less likely. The observed agonist and antagonist specificity of the investigated signals, consistently with recent immunocytochemical staining of Balb/C mouse thymocytes [8], all imply involvement of the P2x1 ATPgated cation channel [6]. Another possibility; that P2z (P2x7) purinoreceptors in thymocytes may function under different conditions as either lytic P2z membrane pores or as P2x1 ATP-gated cation channels, cannot be excluded either, although this hypothesis [4,10,36] needs further support. Apoptosis is thought to play a key role in the negative selection of thymocytes. The biphasic pH and membrane potential responses reported, respectively, in
this paper and previously [12] could be explained by at least two types of purinoreceptor activities (e.g. P2x and P2z, as discussed above) and/or by heterogeneity in purinoreceptor expression among thymocytes. The latter possibility would also be consistent with the notion of differentiation-dependent expression of thymocyte cell surface receptors in general, and with the maturation-dependent expression of purionoreceptors in particular [8,11,14]. In an attempt to correlate ATP-induced early signals with expression of CD4 and CD8 molecules we separated double negative (CD4− CD8−), double positive (CD4+CD8+) and single positive (CD4−CD8+ and CD4+CD8−) thymocytes using magnetic beads. The purity of the separated subsets was \95%, as shown in Fig. 4, using fluorescent anti-CD4 and -CD8 antibodies and flow cytometric analysis of CD4 and CD8 positivity, respectively. However, the number of isolated cells was low, especially in the double negative and single positive subsets. Therefore we used the plasma membrane potential of cells as an indicator of early cellular responses, since labeling of cells with a pHic indicator would have resulted in further cell loss. Characterizing the dose dependence of the membrane potential response ([12] and Table 2) we found that high ATP dose (\ 0.5 mM) preferentially
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induces depolarization, while low concentration of ATP ( B 0.3 mM) elicits hyperpolarization. Previously we have also shown that hyperpolarization is associated with cytosolic acidification. In order to assign distinct purinoreceptor activities to the membrane potential responses, oATP and suramin were used. The depolarization and alkalinization observed at high ATP-dose were blocked by oATP implying the importance of P2z receptor activity in this process. On the other hand, hyperpolarization and acidification were sensitive to the P2x receptor antagonist, suramin (Table 2). Having established a link between purinoreceptor activity and membrane potential changes, we characterized the subset specificity of the above responses. The most immature, double negative thymocytes did not respond by hyperpolarization to low ATPec dose (0.1 mM), while they displayed the highest sensitivity to high ATPec dose (1 mM) responding by a remarkable depolarization. The mature, single positive subsets displayed a much reduced depolarization response to high ATPec dose, while they showed a well detectable hyperpolarization response to low ATPec dose. The double positive cells responded to low ATPec dose by hyperpolarization with a remarkable sensitivity, and also demonstrated significant depolarization to high ATPec dose (Table 2). Characterization of purinoreceptor activities may have important implications in the maturation of thymocytes. Our data imply that P2x purinoreceptors, but not the P2z receptors, are important in ATPec-induced apoptosis of thymocytes. Assuming that hyper- and depolarization responses are characteristic markers of P2x and P2z receptor activation, respectively, our data imply that the most immature double negative cells lack P2x receptor activity, but produce functional P2z receptors. In contrast, in single positive cells the membrane potential responses indicated a low abundance of P2z receptors, while P2x receptor expression/activity was high in these cells. The CD4+CD8+ cells seem to be in transition between double negative and mature cells with respect to purinoreceptor activity: they show both P2x- and P2z-characteristic responses depending on the ATPec dose, therefore being most sensitive to this effector molecule. Expression of both types of P2 receptors on the most abundant thymocyte subset may explain the fact that in the case of inhibition of the P2x receptor pathway pHic changes were characteristic of the P2z-type response, and the other way around (Figs. 1 and 3A and Table 1). Our results are consistent with the work of Di Virgilio et al. [37] demonstrating that mature lymphocytes are much less sensitive to P2z receptor-mediated lysis, and also coincide with our previous results [38] showing that medullary thymocytes were more sensitive to the apoptosis inducing effect of ATPec. Our functional data coincide well with direct immunohistochemical staining of P2x receptor
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proteins, showing that P2x1 receptors are absent in the most mature, double negative cells, while they are more abundant in single or double positive cells [8].
3.4. Concluding remarks The functional role of P2 purinoreceptor-mediated responses is not fully understood. P2x receptors may take part in thymocyte apoptosis either on their own or in cooperation with other apoptosis inducing agents, e.g. steroids [8]. According to our results P2x-mediated signaling may be important in apoptosis of cells in the double positive subset, where most selection processes take place, but also in late stage selection of mature thymocytes. Since the P1 receptor agonist, adenosine is believed to play a role in the negative selection of double positive thymocytes [11,39], the concerted action of P1 and P2 purinoreceptors would allow a sensitive control of cell survival depending on purinoreceptor and ecto-ATPase expression of cells and local agonist concentration. Resolution of the receptor heterogeneity on thymocyte subsets, as well as identification of further potential intracellular signaling components, such as protein phosphatases and proteases, need further investigations. Receptor gene knock-out animals, recently published P2 receptor cDNA sequences and the availability of monoclonal antibodies and structural models for the receptors can further stimulate these studies.
Acknowledgements We thank Drs Sa´ndor Damjanovich, Ja´nos Szo¨llo¨si and Gyo¨rgy Panyi for their valuable criticism and discussions. The skillful technical assistance of Istvan Gal and Terez Lakatos in the experiments is gratefully acknowledged. This work was supported by research grants OTKA 23873 and 29947 from the Hungarian Academy of Sciences.
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