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CD69 expression induced by thapsigargin, phorbol ester and ouabain on thymocytes is dependent on external Ca2+ entry
Life Sciences 73 (2003) 1037 – 1051 www.elsevier.com/locate/lifescie
CD69 expression induced by thapsigargin, phorbol ester and ouabain on thymocytes is dependent on external Ca2+ entry Sandra Rodrigues Mascarenhas a,b, Juliana Echevarria-Lima a,b, Neusa Fernandes dos Santos a, Vivian M. Rumjanek a,* a
Laboratory of Tumoral Immunology, Department of Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil b Biophysics Institute Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Received 23 September 2002; accepted 10 February 2003
Abstract In the present work murine thymocytes exposed to Thapsigargin (TG 10, 20 and 50 nM), Phorbol-12,13,20triacetate (TPA16 nM) and Ouabain (OUA100 nM) exhibited an increased expression of CD69, a molecule related to cellular activation and associated to Ca+ + influx in other systems. The kinetics of CD69 appearance depended on the stimuli and dose used. TG 50 nM induced an increased expression by 6 h whereas with lower doses (10 and 20 nM) an increase was detected at 18 h. TPA maximal increase was evident at 6 h. OUA lead to an observable increase at 18 h. However, in the case of TPA or TG the presence of the stimuli was only necessary for the first 2 h of culture, whereas OUA needed to be present during the whole assay. It was also demonstrated that Ca+ + influx was an essential feature, as EGTA diminished or abolished CD69 increased expression. Nevertheless, EGTA was only capable of this effect when present at the time of the stimuli. No correlation of CD69 expression with thymocyte death was observed. Similarly, the agents under study did not promote the maturation from doublepositive into single-positive thymocytes. TPA and Thapsigargin were capable of decreasing the level of CD4 molecules on the cell surface, probably due to the loss of these molecules. OUA, on the other hand, did not modify CD4/CD8 expression on these cells. D 2003 Elsevier Science Inc. All rights reserved. Keywords: CD69; Thymocytes; Ouabain; Phorbol ester; Thapsigargin; Calcium
* Corresponding author. Laborato´rio de Imunologia Tumoral, Departamento de Bioquı´mica Me´dica, Instituto de Cieˆncias Biome´dicas, Centro de Cieˆncias da Sau´de, Bloco H, Sala 003, Universidade Federal do Rio de Janeiro, 21941-590, Rio de Janeiro, RJ, Brazil. Fax: +55-21-25612936. E-mail address: [email protected] (V.M. Rumjanek). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00377-1
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Introduction Cellular activation is regulated by intracellular calcium levels. Changes in these levels may lead to proliferation, differentiation or apoptotic cell death. However, not only the levels, but also the source of cytosolic calcium (intracellular calcium stores such as endoplasmic reticulum, mitochondria or extra cellular sources) may define the fate of the cell. The molecule CD69, has been associated to the process of activation in a number of cells (Testi et al., 1994), being the earliest molecule to appear de novo on the surface of lymphocytes under activation (Testi et al., 1989). This molecule, a disulphide-linked dimeric structure including a 33 kDa and a 27 kDa chain, may be constitutively expressed by some double-positive (DP) thymocytes undergoing positive selection and are maintained on all single-positive (SP) cells (Swat et al., 1993; Anderson et al., 1994; Marrack and Kappler, 1997). Additionally CD69 expression may be induced in thymocytes following activation by phorbol myristate acetate (TPA) or anti-CD3 antibodies (Lanier et al., 1986). In Jurkat cells the expression of CD69 following activation via CD3 or thapsigargin is dependent on extra cellular calcium entry (Aussel et al., 1996). Thapsigargin is a blocker of the endoplasmic reticulum calcium ATPase capable of raising intracellular calcium levels as a result of the inhibition of the pump activity associated with the constant leakage of calcium from the stores. The emptying of the endoplasmic reticulum generates a calcium influx through a calcium release activated channel (CRAC) that sustains the calcium rise (Putney, 1990; Hoth and Penner, 1992; Breittmayer et al., 1993). Thapsigargin has also been used on thymocytes to induce apoptosis in these cells with extra cellular calcium entry being necessary for cell death to take place (Jiang et al., 1994). However, there is no reference to CD69 expression on thymocytes treated with Thapsigargin. Ouabain, a specific inhibitor of NA+K+ ATPase, was also shown to increase CD69 expression in stimulated lymphocytes (Pires et al., 1997). Furthermore, it has been shown that Ouabain induces intracellular calcium accumulation in lymphocytes (Balasubramanyam et al., 1994) as well as interfering with a number of functions in stimulated lymphocytes (Stoeck et al., 1983; Lillehoj and Shevach, 1985; Dornand et al., 1986; Moraes et al., 1989; Olej et al., 1994 and Olej et al., 1998), but little is known on its effect on thymocytes. In the present study we associated the need of calcium influx and CD69 expression in thymocytes using drugs known to induce intracellular calcium rises via different mechanisms. It is shown that for all substances used: TPA, Thapsigargin and Ouabain the entry of extra cellular calcium was important for CD69 expression to take place.
Methods Preparation of thymocytes suspension Male inbred Balb/c mice (3–5 weeks old) were used in all experiments. Animals were housed in a temperature-controlled room and received water and food ad libitum. During all the experiments performed, animals were treated in accordance with published regulations for animal laboratorial use. Mice were sacrificed by cervical dislocation and their thymuses were removed. Cell suspensions were prepared, centrifuged at 1200 rpm for 7 minutes and ressuspended in RPMI 1640 (Sigma), supplemented with 5 10 5 M h-mercapethanol, Penicillin 60 mg/l, Streptomycin 100 mg/ml and
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10% Fetal Calf Serum (FCS; Highclone). The thymocyte number was adjusted to 1 106 cells/ml and aliquots of 5 105 cells were cultured in 96 well tissue culture plates for 6 and 18 hours. Treatments Thymocytes at 1 106 cells/ml were incubated for 2, 6 and 18 h at 37 jC in an atmosphere of 5% CO2 with the following drugs: Thapsigargin (TG) at different doses, kept as a stock solution dissolved in dimethylsulfoxide (DMSO) at a concentration of 100 AM; Phorbol Ester (TPA) 16 nM (10 ng/ml), kept as a stock solution dissolved in DMSO medium at a concentration of 1 mg/ml; OUA (100 nM), dissolved in RPMI medium just before use at concentration of 1 AM and EGTA(10 mM), dissolved in RPMI medium at concentration of 100 mM. After incubation cells were analyzed in a flow cytometer. All chemicals were obtained from Sigma. Flow cytometric analysis Cell viability was measured by propidium iodide (PI 10 Ag/ml) exclusion determined by flow cytometer, after 6 h and 18 h. In some experiments an Annexin Kit (R&D Systems) was used to verify the presence of apoptotic cells. Thymocytes were analyzed for CD69, CD4 and CD8 expression 6 h and 18 h after the various treatments. For this, 1 106 cells/ml were incubated with a saturating amount of anti-mouse CD69 PE conjugated (Pharmingen, USA) or anti-mouse CD4 PE conjugated (R&D Systems, USA) and antimouse CD8 FITC conjugated (R&D Systems, USA), for 30 min. at 4 jC. Cells were then washed once with chilled PBS and ressuspended in PBS. For those experiments data were acquired in a mode of 10 000 events. Cell viability and analysis of CD69, CD4 and CD8 expression were performed by flow cytometer FACScan (Beckton and Dickinson, USA). The data were analyzed by WinMid software. Calcium mobilization analysis Calcium concentration was monitored as previously described by Balasubramanyam et al. (1994). Thymocytes were incubated for 40 minutes at room temperature with the calcium sensitive fluorescent probe 1.5 AM acetoxymethyl ester form of FURA-2 (FURA-2 AM; Molecular Probes) in supplemented RPMI medium. Cells were washed twice and resuspended in PBS with 1 mM CaCl2 pH 7.4. The cell number was adjusted at 1 106 cells/ml and used in the specific assays. After 100 seconds cells were stimulated with the different reagents. Calcium measurement was performed in a F4500 fluorometer (Hitachi). Excitation and emission were set at 340 nm, slit width 5 nm, and 505 nm, slit width 5 nm wavelengths, respectively. Results are plotted as fluorescence intensity vs time (600 s). In some experiments cells were preincubated for 2 h with TG with or without EGTA and Ca+ + measurement made after this time. Statistical analysis Statistical analysis of the data was performed using one-way analysis of variance (ANOVA) followed by the student T-Test to compare drug-induced changes with respective controls. A value of p < 0.05 was selected as indicating statistical significance and data were expressed as the mean F S.D.
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Results Thymocytes survival after in vitro treatment with OUA, TG and TPA We examined the effect of drugs used in this work on thymocytes survival. Cells were exposed to TPA 16 nM, TG 10, 20 and 50 nM and OUA 100 nM for 6 and 18 hours. After these periods, cells were treated with PI as described in Materials and Methods to determine the percentage of necrotic cells. As it can observed in Fig. 1A, cell viability, as measured by PI exclusion, was unaffected by 6 h of treatment. However, TG 20 nM or 50 nM and TPA 16 nM produced 40 to 60% cell death after 18 h in culture. To verify if at earlier periods apoptosis, as opposed to necrosis, could be observed, an Annexin assay was performed at 6 h with all treatments and showed no difference between control and treated cells (data not shown). Similarly, the addition of EGTA did not modify the effect on cell viability
Fig. 1. Thymocytes survival after in vitro treatment with Ouabain (OUA), Thapsigargin (TG), Phorbol Ester (TPA) and EGTA. Thymocytes (5 105 cells) were incubated for 6 and 18 hours in RPMI medium and 10% FCS in the presence or absence of TPA, TG and OUA (A) and the same drugs in the presence of EGTA 10 mM (B). Viability was determined by propidium iodide (PI) exclusion as described under Materials and Methods. Results are expressed as means F S.D. of three independent experiments. * significant at the p < 0.05 compared to control, ** significant at the p < 0.05 compared to Figure A versus B; as well as compared to control.
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produced by the drugs alone (Fig. 1B), with the exception of cells treated with TG 50 nM where the addition of EGTA partially protected these cells from death. TPA, TG and OUA increase CD69 expression on thymocytes Previous observations have indicated that phorbol esters are capable of inducing CD69 expression on mature and immature T cells (Testi et al., 1989). Figs. 2 and 3 shows that TPA 16 nM is capable of increasing CD69 expression on thymocytes after incubation periods of 6 and 18 hours. Another substance known to induce CD69 expression is TG, as described before in Jurkat T cells (Aussel et al., 1996). Our results demonstrate that TG is also capable of increasing CD69 expression on thymocytes in a dose-dependent way (Fig. 3), albeit to lower levels than that of TPA. Thymocytes cultured for 6 hours in the presence of TG 50 nM presented increased levels of CD69 compared to control cells, however the doses of 10 and 20 nM had no effect. A longer incubation period, on the other hand, led to a small increase in the expression of this molecule with the lower doses. In a previous work our group has shown that the inhibitor of Na+K+-ATPase, OUA, causes an augmentation of CD69 expression on activated human lymphocytes (Pires et al., 1997), but there is no information available on the effects of this drug on thymocytes. An 18 h exposure of thymocytes to OUA 100 nM increases CD69 expression, as shown in Fig. 3. Conversely, cells treated for 6 hours with
Fig. 2. CD69 expression on thymocytes in the presence of Phorbol Ester (TPA). Thymocytes (5 105 cells) were incubated for 6 h and 18 h in RPMI medium and 10% FCS in the absence or presence of TPA (16 nM). The thin line represents control background staining levels, M1 represents auto fluorescence, M2 represents low fluorescence intensity and M3 represents high fluorescence intensity. The numbers represent the percentage of CD69high cells (M3). One representative of five experiments is shown.
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Fig. 3. CD69 expression on thymocytes in the presence of Phorbol Ester (TPA), Thapsigargin (TG) and Ouabain (OUA). Thymocytes (5 105 cells) were incubated for 6 h (black columns) and 18 h (gray columns) in the absence or presence of the drugs. At 18 h TG 50 nM was not used because of the high degree of mortality. The numbers represent the percentage of CD69high cells (M3). Results are expressed as means F S.D. of at least three independent experiments. * significant at the p < 0.05 compared to control.
OUA did not show an increase of this surface molecule (Fig. 3). Higher doses of OUA (1 mM–10 AM) did not induce CD69 expression at 6 h (data not shown). TPA, TG and OUA cause calcium mobilization on thymocytes It has been reported a link between Ca+ + mobilization and CD69 expression. Our results show that TPA16 nM, TG 50 nM and OUA 100 nM have the ability of inducing intracellular (Ca+ +) elevation on thymocytes (Fig. 4). EGTA is capable of reducing CD69 expression induced by TPA, TG and OUA Inhibition of Ca+ + influx in Jurkat T cells impaired TG-induced CD69 expression at the cell surface (Aussel et al., 1996). To analyze if CD69 expression on thymocytes depended on Ca+ + influx, cells were incubated in the presence or absence of the Ca+ + chelator EGTA (10 mM). For this, thymocytes were incubated with the optimal conditions of CD69 expression: TPA 16 nM, TG 50 nM; for 6 hours; and with OUA 100 nM; for 18 hours. EGTA per se did not modify CD69 staining on control thymocytes, however the results show that EGTA down-regulates TPA-, TG- and OUA-induced CD69 expression (Fig. 5). EGTA prevents the sustained Ca++ increase It has already been described that the maintance of increased Ca+ + levels produced by TG is dependent on external entry (Putney, 1990). Furthermore, because the presence of EGTA in the first
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Fig. 4. Calcium mobilization by Phorbol Ester (TPA), Thapsigargin (TG) and Ouabain (OUA). The phorbol ester TPA, the SERCA inhibitor TG and the Na+K+-ATPase inhibitor OUA were shown, using FURA-2, to induce intracellular calcium mobilization on thymocytes. Thymocytes (106 cells) were exposed to different drugs for 600 seconds. (A) Cells exposed or not to TPA 16 nM. (B) Cells exposed or not to TG 50 nM. (C) Cells exposed or not to OUA 100 nM. The thick black line represents the effects of the drugs and the thin gray line represents untreated cells. This figure is representative of five different experiments.
2 hours completely abolished CD69 increased expression, an experiment was performed to verify if there was external Ca+ + entry under this circumstance. Table 1 shows that cells pretreated with TG and EGTA for 2 hours washed and exposed to Ca+ + medium did not show a second bout of Ca+ +
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Fig. 5. EGTA is capable of reducing CD69 expression induced by Phorbol Ester (TPA), Thapsigargin (TG) and Ouabain (OUA). The Ca+ + chelator EGTA (10 mM) was added to thymocytes for 6 h in the absence or presence of various drugs. Cells were treated either with EGTA alone, with drugs and with EGTA plus drugs. The numbers represent the percentage of CD69high cells (M3). Results are expressed as means F S.D. of at least three independent experiments. * significant at the p < 0.05 compared to EGTA.
entry, reinforcing the idea that external Ca+ + is essential at the moment TG is present generating CD69 induction. Kinetics of the need of the presence of the stimulus and external calcium on CD69 expression A 2 h exposure to TPA 16 nM or TG 50 nM is enough to produce a similar increase in CD69 expression as that seen when the drugs were present for the whole culture period (Fig. 6 lane 5 compared to lane 2). Similarly, the presence of EGTA restricted to the same first 2 h (Fig. 6 lane 6) produce the same inhibition as that seen when it was present for the whole time of the assay (Fig. 6 lane 6 compared to lane 8 and 3). This inhibitory effect was only partial for TPA and complete for TG. EGTA present only in the last 4 h did not modify the effect produced by the drugs (Fig. 6 lane 7 compared to lane 5).
Table 1 Effect of EGTA on the Ca+ + increase maintained 2 h after TG treatment Treatment*
EXP 1
EXP 2
EXP 3
EXP 4
Mean
TG TG + EGTA
41% 4%
51% 0%
23% 0%
83% 16%
50% 3%
* Cells were treated for 2 h with TG or TG plus EGTA, washed and Ca+ + measurements performed in a medium containing Ca . ++
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Fig. 6. Kinetics of the need of external calcium on CD69 expression. The drugs TG 50 nM or TPA 16 nM were added to thymocytes culture during the whole period (6 h) or for the first 2 h of incubation in the presence or absence of EGTA 10 mM (E). Lanes 1, 2 and 3 represent the effect of drugs and /or EGTA present for 6 h; lanes 4, 5 and 6 represent the effect of drugs and/or EGTA present for 2 h in thymocyte culture, washed away and ressuspended in drug free medium (M) for a further 4 hours; lanes 7 and 8 represent the effect of EGTA given in the last 4 hours or present during the whole period of culture. One representative of three experiments is shown.
CD4/CD8 expression on thymocytes in the presence of Phorbol Ester (TPA), Thapsigargin (TG) and Ouabain (OUA) Previous observations have indicated that phorbol esters are capable to produce CD4 internalization on single-positive and double-positive murine thymocytes (Anderson and Coleclough, 1993 and Tagawa et al., 1991). Similarly, our results shows that TPA 16 nM is capable of diminishing
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the level of CD4 molecules on the surface of thymocytes after 6 h (data not shown) and after18 h (Fig. 7). Our results demonstrate for the first time that TG is also capable of reducing CD4 molecules on thymocytes in a dose-dependent way (Fig. 7), albeit to lower levels than those of TPA. OUA 100 nM did not modify the expression of CD4, but it may might be necessary a longer incubation period, for this phenomenon to take place with OUA. Neither TG (10,20 and 50 nM) nor OUA (100 nM) were capable of modifying CD4 expression during the 6 h culture (data not shown).
Fig. 7. CD4/CD8 expression on thymocytes in the presence of Phorbol Ester (TPA), Thapsigargin (TG) and Ouabain (OUA). Thymocytes (5 105 cells) were incubated for 18 hours in the absence or presence of TPA (16 nM), TG (10 and 20 nM) and OUA (100 nM). TG 50 nM was not used because of the high degree of mortality. The numbers represent the percentage of CD4, CD8 or DP cells. One representative of three experiments is shown.
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Discussion The present work demonstrated for the first time that agents such as Thapsigargin and Ouabain were capable of increasing CD69 expression on thymocytes, similar to what has been reported using TPA (Hara et al., 1986 and Lanier et al., 1986). All these three agents (TPA, Thapsigargin and Ouabain) increased cytosolic Ca+ + levels in thymocytes. The Ca+ + mobilized by these substances originates from both internal and external pools. This Ca+ + increase has been described in thymocytes using both Thapsigargin (Jiang et al., 1994) and TPA (Klip et al., 1984). The present results show a similar response in these cells and extend the capability of mobilizing Ca+ + to Ouabain. Furthermore, the present results suggest that the transient Ca+ + release from internal stores was not sufficient to induce CD69 expression on thymocytes. External calcium influx was essential for this process to take place, as it was possible to modulate CD69 increased expression when EGTA was used to buffer external Ca+ +. This is in agreement with what has been described in human T lymphocytes (Vandenberghe et al., 1992) and Jurkat (Aussel et al., 1996) cells exposed to different stimuli. We found that the percentage of CD69high positive cells in unstimulated thymocytes diminished to nearly half by 18 h of culture. A decrease in CD69 expression with time on cultured thymocyte has been described by Testi et al. (1989), who suggested the need of interactions with the thymic stroma to maintain the levels of CD69 on thymocytes. It is possible that, in our study, the substances added: TPA, Thapsigargin and Ouabain could, in some way, substitute for the signals produced by these interactions. Activation of protein kinase C (PKC) by stimulation of the TCR/CD3 or by phorbol esters induces the expression of CD69 on thymocytes (Hara et al., 1986 and Lanier et al., 1986). It is, however, unknown the signaling pathways involved when Thapsigargin and Ouabain are used. It is noteworthy that Thapsigargin has been shown before, by other authors, to induce the expression of this molecule in Jurkat T lymphocytes (Aussel et al., 1996) and Ouabain on activated peripheral blood lymphocytes (Pires et al., 1997). A possible pathway for CD69 induction by these substances could involve the expression of c-fos.There are evidences that the expression of CD69 is regulated by c-fos via the transcription factor AP-1 (Castellanos et al., 1997), and in different systems, all these three agents, TPA (Kontny et al., 1992), Thapsigargin (He et al., 2000) and Ouabain (Nakagawa et al., 1992; Peng et al., 1996), were capable of inducing c-fos expression. The kinectics of c-fos RNA expression vary being very rapid in the case of TPA (Kontny et al., 1992) and Thapsigargin (He et al., 2000) contrasting with the slow induction of the gene by Ouabain (Nakagawa et al., 1992). In agreement to what has been observed with c-fos the kinetics of CD69 appearance depended on the stimuli and dose used, being faster with TPA and Thapsigargin, and slower with Ouabain. However, TPA and Thapsigargin needed only to be present for the first two hours for the induction of expression to take place. Independent of the removal of the drug after 2 h, maximal increase with TPA was already evident by 6 h. When Thapsigargin was used, a dose of 50 nM induced an increased expression by 6 h whereas with lower doses an increase was detected at 18 h. In the case of Ouabain an observable increase was only seen at 18 h, but the drug needed to be present for the whole time (data not shown). The need for external Ca+ + is limited to the period necessary for the drugs to induce CD69 expression. When EGTA was only present for the first 2 h (together with the stimulus) the effect
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of Thapsigargin was completely abrogated whilst the effect of TPA was only partially affected. In this case EGTA present only in the last 4 hours of culture was of no avail. In the case of Ouabain, EGTA needs to be present the whole time of the culture period to inhibit CD69 increased expression. This might be due to different kinetics in Ca+ + entry produced by the various drugs. Despite being the best inducer of CD69 expression, TPA produced the lowest levels of cytosolic Ca+ +,either as a result of activating the plasma membrane Ca+ + pump (Rink and Sage, 1987) and/or as a direct effect on endoplasmatic reticulum calcium uptake (Cardoso et al., 1997). This suggests that Ca+ + influx is more important for CD69 expression then the final levels of intracellular Ca+ + achieved. It is unlikely that the decrease in CD69 expression produced by EGTA resulted from cell death. Thymocytes remained as viable in the presence of EGTA as cells kept in medium or in the presence of drugs alone. In the particular case of 50 nM Thapsigargin the addition of EGTA partially protected these cells from death. Similarly, Jiang et al.(Jiang et al., 1994) demonstrated that the presence of EGTA for 2 hours after exposure to Thapsigargin was capable of preventing rat thymocytes of undergoing apoptosis. Not only Jiang et al. (1994), but also McConkey et al. (1989) and Hampton et al. (1996) demonstrated a clear involvement of the increase in cytosolic Ca+ + levels during apoptosis. However, there is controversy regarding the role played by intracellular Ca+ + during the apoptotic process. Others workers (Alnemri and Litwack, 1990; Duke et al., 1994 and Beaver and Waring, 1994) suggested that Ca+ + increase was not sufficient to induce apoptosis. A correlation between CD69 expression and thymocyte death has also been suggested by Kishimoto et al. (1995) who demonstrated that thymocytes undergoing apoptosis were expressing this molecule. In the present work the percentage of necrotic thymocytes after 6 hour of culture was similar between control and cells treated with TPA, Thapsigargin or Ouabain (around 16%) despite the fact that TPA induced CD69high expression in 95% of the cells. It has also been suggested, in different systems, that CD69 expression may precede cell proliferation, maturation or differentiation (Testi et al., 1994). Furthermore, it has been reported that mice that overexpressed CD69 in the thymus displayed a dramatic increase in the number of singlepositive cells (Nakayama et al., 2002). In the present study no evidence of thymocyte maturation was obtained. Even if TPA and Thapsigargin led to a decrease in double positive cells, this seemed to reflect a loss of CD4 molecules, as previously described in thymocytes with TPA (Anderson and Coleclough, 1993 and Tagawa et al., 1991) as opposed to a maturation towards single positive cells. Ouabain on the other hand did not modify CD4/CD8 expression on these cells. This might result from the fact that OUA takes much longer to induce CD69 expression and it was not studied for longer periods. Negative effects of Ouabain on the proliferation of mature lymphocytes have been reported (Lillehoj and Shevach, 1985, Brodie et al., 1995 and Moraes et al., 1989) even in situations where CD69 expression was increased (Pires et al., 1997). CD69 expression may be stimulated via different pathways (Borrego et al., 1993) and it is possible that the one induced by Ouabain differs from the pathway that produces CD4 internalization. In this regard it has been suggested that both TPA and Thapsigargin are capable of activating cells via PKC (Lanier et al., 1986 and Huber et al., 2000) whereas it has been suggested that OUA may use a tyrosine kinase pathway leading to the expression of RAS (Haas et al., 2000) and that, at least for c-fos RNA expression, the activation of PKC is not the primary mechanism by which Ouabain exert its action (Nakagawa et al., 1992).
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Conclusion The present work demonstrated in thymocytes that TPA, Thapsigargin and Ouabain were capable of Ca+ + mobilization, that these three drugs induced increased CD69 expression, suggesting cellular activation, and that this expression depended on external Ca+ + entry. Finally, no correlation between CD69 and thymocyte death or thymocyte maturation could be established. Acknowledgements This work was supported by the Brazilian National Research Council (CNPq, PRONEX) and FAPERJ (Fundacßa˜o de Amparo a` Pesquisa do Rio de Janeiro). Sandra Rodrigues Mascarenhas was a recipient of a Master Degree Scholarship from CAPES (Coordenacßa˜o de Aperfeicßoamento de Pessoal de Nı´vel Superior).
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Hara, T., Jung, L.K.L., Bjorndahl, J.M., Fu, S.M., 1986. Human T cell activation. II- rapid induction of a phosphorilated 28KD/ 32KD disufide-linked early activation antigen (EA 1) by 12- o- tetradecanoyl phorbol - 13- acetate, mitogens and antigen. Journal of Experimental Medicine 164, 1988 – 2005. Haas, M., Askari, A., Xie, Z., 2000. Involvment of Src and epidermal growth factor receptor in the signal-transducing function of Na+/K+-ATPase. Journal of Biological Chemistry 275 (36), 27832 – 27837. He, H., McColl, K., Distelhorst, C.W., 2000. Involvement of c-Fos in signaling grp78 induction following ER calcium release. Oncogene 19 (51), 5936 – 5943. Hoth, M., Penner, R., 1992. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 355, 353 – 355. Huber, M., Hughes, M.R., Krystal, G., 2000. Thapsigargin-induced degranulation of mast cells is dependent is dependent on transient activation of phosphatidyl-3 Kinase. Journal of Immunology 165 (1), 124 – 133. Jiang, S., Chow, S.C., Nicotera, P., Orrenius, S., 1994. Intracellular Ca2 + signals activate apoptosis in thymocytes: studies using the Ca2+-ATPase inhibitor thapsigargin. Experimental Cellular Research 212 (1), 84 – 92. Kishimoto, H., Surh, C.D., Sprent, J., 1995. Upregulation of surface markers on dying thymocytes. Journal of Experimental Medicine 181 (2), 649 – 655. Klip, A., Rothstein, A., Mack, E., 1984. Stimulation of hexose uptake in rat thymic lymphocytes by phorbol ester. A role for Ca2+ and Na+/H+ exchange? Biochemical. Biophysical Research Communication 124 (1), 14 – 22. Kontny, E., Kvanta, A., Fredholm, B.B., 1992. Activation of protein kinase C and elevation of cAMP interact synergistically to raise c-fos and AP-1 activity in Jurkat cells. European Journal Pharmacology 227 (3), 333 – 338. Lanier, L.L., Allison, J.P., Phillips, J.H., 1986. Correlation of cell surface antigen expression on human thymocytes by multicolor flow cytometric analysis: implication for differentiation. Journal of Immunology 137, 2501 – 2507. Lillehoj, H., Shevach, E.M., 1985. A comparison of the effects of cyclosporin A, dexamethasone, and ouabain on the interleukin-2 cascate. Journal of Immunopharmacology 7 (3), 267 – 284. Marrack, P.C., Kappler, J.W., 1997. Positive selection of thymocytes bearing ah T cell receptors. Current Opinion Immunology 9, 250 – 255. McConkey, D.J., Hartzell, P., Amador-Perez, J.F., Orrenius, S., Jondal, M., 1989. Calcium-dependent killing of immature thymocytes by stimulation via the CD3/T cell receptor complex. Journal of Immunology 143 (6), 1801 – 1806. Moraes, V.L., Olej, B., De La roque, L., d Rumjanek, V.M., 1989. Lack of sensitivity to Ouabain in Natural killer activity. Faseb Journal 3, 2425 – 2429. Nakagawa, Y., Rivera, V., Larner, A.C., 1992. A role for the Na/K-ATPase in the control of Human c-fos and c-jun Transcription. Journal of Biological Chemistry 267 (13), 8785 – 8788. Nakayama, T., Kasprowicz, D.J., Yamashita, M., Schubert, L.A., Gillard, G., Kimura, M., Didierlaurent, A., Koseki, H., Ziegler, S.F., 2002. The generation of mature, single-positive thymocytes in vivo is dysregulated by CD69 blockade or overexpression. Journal of Immunology 168 (1), 87 – 94. Olej, B., De la Rocque, L., Castilho, F.P.D., Mediano, I.F., Campos, M.M., Rumjanek, V.M., 1994. Effect of Ouabain on lymphokine-activated killer cells. International Journal of Immunopharmacology 16, 769 – 774. Olej, B., dos Santos, N.F., Leal, L., Rumjanek, V.M., 1998. Ouabain induces apoptosis on PHA-activated lymphocytes. Bioscience Reports 18, 1 – 7. Pires, V., Harab, R., Olej, B., Rumjanek, V.M., 1997. Ouabain effects on activated lymphocytes: augmentation of CD25 expression on TPA - stimulated cells and of CD69 on PHA- and TPA- stimulated cells. International Journal of Immunopharmacology 9, 143 – 148. Peng, M., Huang, M., Xie, Z., Huang, W.H., Askari, A., 1996. Partial Inhibition of Na/K-ATPase by Ouabain Induces the Ca2+ - dependent expression of early-response gene in cardiac myocytes. Journal of Biological Chemistry 271 (17), 10372 – 10378. Putney, J.W.J., 1990. Capacitative calcium entry revisited. Cellular Calcium 11, 611 – 624. Rink, T.J., Sage, S.O., 1987. Stimulated calcium efflux from fura-2-loaded human platelets. Journal Physiology 393, 513 – 524. Swat, W., Dessing, M., von Boehmer, H., Kisielow, P., 1993. CD69 expression during selection and maturation of CD4 +8+ thymocytes. European Journal Immunology 23, 739 – 746. Stoeck, M., Northoff, H., Resch, K., 1983. Inhibition of mitogen-induced lymphocyte proliferation by ouabain: interference with interleukin 2 production and interleukin 2 action. Journal of Immunology 131 (3), 1433 – 1437. Tagawa, M., Koike, T., Griffith, L.C., 1991. Phosphorylation of mouse thymocytes CD4 and CD8: regulation of surface expression. Biochemical International 24, 739 – 747.
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Testi, R., Ambrosio, D., de Maria, R., Santoni, A., 1994. The CD69 receptor; a multipurpose cell-surface trigger for hematopoietic cells. Immunology Today 15, 479 – 483. Testi, R., Phillips, J.H., Lanier, L.L., 1989. T Cell activation via Leu-23 (CD69). Journal of Immunology 143, 1123 – 1128. Vandenberghe, P., Verwilghen, J., Van Vaeck, F., Ceuppens, J.L., 1992. Ligation of the CD5 or CD28 molecules on resting human T cells induces expression of the early activation antigen CD69 by a calcium- and tyrosine kinase-dependent mechanism. Immunology 78, 210 – 217.
CD69 expression induced by thapsigargin, phorbol ester and ouabain on thymocytes is dependent on external Ca2+ entry Sandra Rodrigues Mascarenhas a,b, Juliana Echevarria-Lima a,b, Neusa Fernandes dos Santos a, Vivian M. Rumjanek a,* a
Laboratory of Tumoral Immunology, Department of Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil b Biophysics Institute Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Received 23 September 2002; accepted 10 February 2003
Abstract In the present work murine thymocytes exposed to Thapsigargin (TG 10, 20 and 50 nM), Phorbol-12,13,20triacetate (TPA16 nM) and Ouabain (OUA100 nM) exhibited an increased expression of CD69, a molecule related to cellular activation and associated to Ca+ + influx in other systems. The kinetics of CD69 appearance depended on the stimuli and dose used. TG 50 nM induced an increased expression by 6 h whereas with lower doses (10 and 20 nM) an increase was detected at 18 h. TPA maximal increase was evident at 6 h. OUA lead to an observable increase at 18 h. However, in the case of TPA or TG the presence of the stimuli was only necessary for the first 2 h of culture, whereas OUA needed to be present during the whole assay. It was also demonstrated that Ca+ + influx was an essential feature, as EGTA diminished or abolished CD69 increased expression. Nevertheless, EGTA was only capable of this effect when present at the time of the stimuli. No correlation of CD69 expression with thymocyte death was observed. Similarly, the agents under study did not promote the maturation from doublepositive into single-positive thymocytes. TPA and Thapsigargin were capable of decreasing the level of CD4 molecules on the cell surface, probably due to the loss of these molecules. OUA, on the other hand, did not modify CD4/CD8 expression on these cells. D 2003 Elsevier Science Inc. All rights reserved. Keywords: CD69; Thymocytes; Ouabain; Phorbol ester; Thapsigargin; Calcium
* Corresponding author. Laborato´rio de Imunologia Tumoral, Departamento de Bioquı´mica Me´dica, Instituto de Cieˆncias Biome´dicas, Centro de Cieˆncias da Sau´de, Bloco H, Sala 003, Universidade Federal do Rio de Janeiro, 21941-590, Rio de Janeiro, RJ, Brazil. Fax: +55-21-25612936. E-mail address: [email protected] (V.M. Rumjanek). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00377-1
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Introduction Cellular activation is regulated by intracellular calcium levels. Changes in these levels may lead to proliferation, differentiation or apoptotic cell death. However, not only the levels, but also the source of cytosolic calcium (intracellular calcium stores such as endoplasmic reticulum, mitochondria or extra cellular sources) may define the fate of the cell. The molecule CD69, has been associated to the process of activation in a number of cells (Testi et al., 1994), being the earliest molecule to appear de novo on the surface of lymphocytes under activation (Testi et al., 1989). This molecule, a disulphide-linked dimeric structure including a 33 kDa and a 27 kDa chain, may be constitutively expressed by some double-positive (DP) thymocytes undergoing positive selection and are maintained on all single-positive (SP) cells (Swat et al., 1993; Anderson et al., 1994; Marrack and Kappler, 1997). Additionally CD69 expression may be induced in thymocytes following activation by phorbol myristate acetate (TPA) or anti-CD3 antibodies (Lanier et al., 1986). In Jurkat cells the expression of CD69 following activation via CD3 or thapsigargin is dependent on extra cellular calcium entry (Aussel et al., 1996). Thapsigargin is a blocker of the endoplasmic reticulum calcium ATPase capable of raising intracellular calcium levels as a result of the inhibition of the pump activity associated with the constant leakage of calcium from the stores. The emptying of the endoplasmic reticulum generates a calcium influx through a calcium release activated channel (CRAC) that sustains the calcium rise (Putney, 1990; Hoth and Penner, 1992; Breittmayer et al., 1993). Thapsigargin has also been used on thymocytes to induce apoptosis in these cells with extra cellular calcium entry being necessary for cell death to take place (Jiang et al., 1994). However, there is no reference to CD69 expression on thymocytes treated with Thapsigargin. Ouabain, a specific inhibitor of NA+K+ ATPase, was also shown to increase CD69 expression in stimulated lymphocytes (Pires et al., 1997). Furthermore, it has been shown that Ouabain induces intracellular calcium accumulation in lymphocytes (Balasubramanyam et al., 1994) as well as interfering with a number of functions in stimulated lymphocytes (Stoeck et al., 1983; Lillehoj and Shevach, 1985; Dornand et al., 1986; Moraes et al., 1989; Olej et al., 1994 and Olej et al., 1998), but little is known on its effect on thymocytes. In the present study we associated the need of calcium influx and CD69 expression in thymocytes using drugs known to induce intracellular calcium rises via different mechanisms. It is shown that for all substances used: TPA, Thapsigargin and Ouabain the entry of extra cellular calcium was important for CD69 expression to take place.
Methods Preparation of thymocytes suspension Male inbred Balb/c mice (3–5 weeks old) were used in all experiments. Animals were housed in a temperature-controlled room and received water and food ad libitum. During all the experiments performed, animals were treated in accordance with published regulations for animal laboratorial use. Mice were sacrificed by cervical dislocation and their thymuses were removed. Cell suspensions were prepared, centrifuged at 1200 rpm for 7 minutes and ressuspended in RPMI 1640 (Sigma), supplemented with 5 10 5 M h-mercapethanol, Penicillin 60 mg/l, Streptomycin 100 mg/ml and
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10% Fetal Calf Serum (FCS; Highclone). The thymocyte number was adjusted to 1 106 cells/ml and aliquots of 5 105 cells were cultured in 96 well tissue culture plates for 6 and 18 hours. Treatments Thymocytes at 1 106 cells/ml were incubated for 2, 6 and 18 h at 37 jC in an atmosphere of 5% CO2 with the following drugs: Thapsigargin (TG) at different doses, kept as a stock solution dissolved in dimethylsulfoxide (DMSO) at a concentration of 100 AM; Phorbol Ester (TPA) 16 nM (10 ng/ml), kept as a stock solution dissolved in DMSO medium at a concentration of 1 mg/ml; OUA (100 nM), dissolved in RPMI medium just before use at concentration of 1 AM and EGTA(10 mM), dissolved in RPMI medium at concentration of 100 mM. After incubation cells were analyzed in a flow cytometer. All chemicals were obtained from Sigma. Flow cytometric analysis Cell viability was measured by propidium iodide (PI 10 Ag/ml) exclusion determined by flow cytometer, after 6 h and 18 h. In some experiments an Annexin Kit (R&D Systems) was used to verify the presence of apoptotic cells. Thymocytes were analyzed for CD69, CD4 and CD8 expression 6 h and 18 h after the various treatments. For this, 1 106 cells/ml were incubated with a saturating amount of anti-mouse CD69 PE conjugated (Pharmingen, USA) or anti-mouse CD4 PE conjugated (R&D Systems, USA) and antimouse CD8 FITC conjugated (R&D Systems, USA), for 30 min. at 4 jC. Cells were then washed once with chilled PBS and ressuspended in PBS. For those experiments data were acquired in a mode of 10 000 events. Cell viability and analysis of CD69, CD4 and CD8 expression were performed by flow cytometer FACScan (Beckton and Dickinson, USA). The data were analyzed by WinMid software. Calcium mobilization analysis Calcium concentration was monitored as previously described by Balasubramanyam et al. (1994). Thymocytes were incubated for 40 minutes at room temperature with the calcium sensitive fluorescent probe 1.5 AM acetoxymethyl ester form of FURA-2 (FURA-2 AM; Molecular Probes) in supplemented RPMI medium. Cells were washed twice and resuspended in PBS with 1 mM CaCl2 pH 7.4. The cell number was adjusted at 1 106 cells/ml and used in the specific assays. After 100 seconds cells were stimulated with the different reagents. Calcium measurement was performed in a F4500 fluorometer (Hitachi). Excitation and emission were set at 340 nm, slit width 5 nm, and 505 nm, slit width 5 nm wavelengths, respectively. Results are plotted as fluorescence intensity vs time (600 s). In some experiments cells were preincubated for 2 h with TG with or without EGTA and Ca+ + measurement made after this time. Statistical analysis Statistical analysis of the data was performed using one-way analysis of variance (ANOVA) followed by the student T-Test to compare drug-induced changes with respective controls. A value of p < 0.05 was selected as indicating statistical significance and data were expressed as the mean F S.D.
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Results Thymocytes survival after in vitro treatment with OUA, TG and TPA We examined the effect of drugs used in this work on thymocytes survival. Cells were exposed to TPA 16 nM, TG 10, 20 and 50 nM and OUA 100 nM for 6 and 18 hours. After these periods, cells were treated with PI as described in Materials and Methods to determine the percentage of necrotic cells. As it can observed in Fig. 1A, cell viability, as measured by PI exclusion, was unaffected by 6 h of treatment. However, TG 20 nM or 50 nM and TPA 16 nM produced 40 to 60% cell death after 18 h in culture. To verify if at earlier periods apoptosis, as opposed to necrosis, could be observed, an Annexin assay was performed at 6 h with all treatments and showed no difference between control and treated cells (data not shown). Similarly, the addition of EGTA did not modify the effect on cell viability
Fig. 1. Thymocytes survival after in vitro treatment with Ouabain (OUA), Thapsigargin (TG), Phorbol Ester (TPA) and EGTA. Thymocytes (5 105 cells) were incubated for 6 and 18 hours in RPMI medium and 10% FCS in the presence or absence of TPA, TG and OUA (A) and the same drugs in the presence of EGTA 10 mM (B). Viability was determined by propidium iodide (PI) exclusion as described under Materials and Methods. Results are expressed as means F S.D. of three independent experiments. * significant at the p < 0.05 compared to control, ** significant at the p < 0.05 compared to Figure A versus B; as well as compared to control.
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produced by the drugs alone (Fig. 1B), with the exception of cells treated with TG 50 nM where the addition of EGTA partially protected these cells from death. TPA, TG and OUA increase CD69 expression on thymocytes Previous observations have indicated that phorbol esters are capable of inducing CD69 expression on mature and immature T cells (Testi et al., 1989). Figs. 2 and 3 shows that TPA 16 nM is capable of increasing CD69 expression on thymocytes after incubation periods of 6 and 18 hours. Another substance known to induce CD69 expression is TG, as described before in Jurkat T cells (Aussel et al., 1996). Our results demonstrate that TG is also capable of increasing CD69 expression on thymocytes in a dose-dependent way (Fig. 3), albeit to lower levels than that of TPA. Thymocytes cultured for 6 hours in the presence of TG 50 nM presented increased levels of CD69 compared to control cells, however the doses of 10 and 20 nM had no effect. A longer incubation period, on the other hand, led to a small increase in the expression of this molecule with the lower doses. In a previous work our group has shown that the inhibitor of Na+K+-ATPase, OUA, causes an augmentation of CD69 expression on activated human lymphocytes (Pires et al., 1997), but there is no information available on the effects of this drug on thymocytes. An 18 h exposure of thymocytes to OUA 100 nM increases CD69 expression, as shown in Fig. 3. Conversely, cells treated for 6 hours with
Fig. 2. CD69 expression on thymocytes in the presence of Phorbol Ester (TPA). Thymocytes (5 105 cells) were incubated for 6 h and 18 h in RPMI medium and 10% FCS in the absence or presence of TPA (16 nM). The thin line represents control background staining levels, M1 represents auto fluorescence, M2 represents low fluorescence intensity and M3 represents high fluorescence intensity. The numbers represent the percentage of CD69high cells (M3). One representative of five experiments is shown.
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Fig. 3. CD69 expression on thymocytes in the presence of Phorbol Ester (TPA), Thapsigargin (TG) and Ouabain (OUA). Thymocytes (5 105 cells) were incubated for 6 h (black columns) and 18 h (gray columns) in the absence or presence of the drugs. At 18 h TG 50 nM was not used because of the high degree of mortality. The numbers represent the percentage of CD69high cells (M3). Results are expressed as means F S.D. of at least three independent experiments. * significant at the p < 0.05 compared to control.
OUA did not show an increase of this surface molecule (Fig. 3). Higher doses of OUA (1 mM–10 AM) did not induce CD69 expression at 6 h (data not shown). TPA, TG and OUA cause calcium mobilization on thymocytes It has been reported a link between Ca+ + mobilization and CD69 expression. Our results show that TPA16 nM, TG 50 nM and OUA 100 nM have the ability of inducing intracellular (Ca+ +) elevation on thymocytes (Fig. 4). EGTA is capable of reducing CD69 expression induced by TPA, TG and OUA Inhibition of Ca+ + influx in Jurkat T cells impaired TG-induced CD69 expression at the cell surface (Aussel et al., 1996). To analyze if CD69 expression on thymocytes depended on Ca+ + influx, cells were incubated in the presence or absence of the Ca+ + chelator EGTA (10 mM). For this, thymocytes were incubated with the optimal conditions of CD69 expression: TPA 16 nM, TG 50 nM; for 6 hours; and with OUA 100 nM; for 18 hours. EGTA per se did not modify CD69 staining on control thymocytes, however the results show that EGTA down-regulates TPA-, TG- and OUA-induced CD69 expression (Fig. 5). EGTA prevents the sustained Ca++ increase It has already been described that the maintance of increased Ca+ + levels produced by TG is dependent on external entry (Putney, 1990). Furthermore, because the presence of EGTA in the first
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Fig. 4. Calcium mobilization by Phorbol Ester (TPA), Thapsigargin (TG) and Ouabain (OUA). The phorbol ester TPA, the SERCA inhibitor TG and the Na+K+-ATPase inhibitor OUA were shown, using FURA-2, to induce intracellular calcium mobilization on thymocytes. Thymocytes (106 cells) were exposed to different drugs for 600 seconds. (A) Cells exposed or not to TPA 16 nM. (B) Cells exposed or not to TG 50 nM. (C) Cells exposed or not to OUA 100 nM. The thick black line represents the effects of the drugs and the thin gray line represents untreated cells. This figure is representative of five different experiments.
2 hours completely abolished CD69 increased expression, an experiment was performed to verify if there was external Ca+ + entry under this circumstance. Table 1 shows that cells pretreated with TG and EGTA for 2 hours washed and exposed to Ca+ + medium did not show a second bout of Ca+ +
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Fig. 5. EGTA is capable of reducing CD69 expression induced by Phorbol Ester (TPA), Thapsigargin (TG) and Ouabain (OUA). The Ca+ + chelator EGTA (10 mM) was added to thymocytes for 6 h in the absence or presence of various drugs. Cells were treated either with EGTA alone, with drugs and with EGTA plus drugs. The numbers represent the percentage of CD69high cells (M3). Results are expressed as means F S.D. of at least three independent experiments. * significant at the p < 0.05 compared to EGTA.
entry, reinforcing the idea that external Ca+ + is essential at the moment TG is present generating CD69 induction. Kinetics of the need of the presence of the stimulus and external calcium on CD69 expression A 2 h exposure to TPA 16 nM or TG 50 nM is enough to produce a similar increase in CD69 expression as that seen when the drugs were present for the whole culture period (Fig. 6 lane 5 compared to lane 2). Similarly, the presence of EGTA restricted to the same first 2 h (Fig. 6 lane 6) produce the same inhibition as that seen when it was present for the whole time of the assay (Fig. 6 lane 6 compared to lane 8 and 3). This inhibitory effect was only partial for TPA and complete for TG. EGTA present only in the last 4 h did not modify the effect produced by the drugs (Fig. 6 lane 7 compared to lane 5).
Table 1 Effect of EGTA on the Ca+ + increase maintained 2 h after TG treatment Treatment*
EXP 1
EXP 2
EXP 3
EXP 4
Mean
TG TG + EGTA
41% 4%
51% 0%
23% 0%
83% 16%
50% 3%
* Cells were treated for 2 h with TG or TG plus EGTA, washed and Ca+ + measurements performed in a medium containing Ca . ++
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Fig. 6. Kinetics of the need of external calcium on CD69 expression. The drugs TG 50 nM or TPA 16 nM were added to thymocytes culture during the whole period (6 h) or for the first 2 h of incubation in the presence or absence of EGTA 10 mM (E). Lanes 1, 2 and 3 represent the effect of drugs and /or EGTA present for 6 h; lanes 4, 5 and 6 represent the effect of drugs and/or EGTA present for 2 h in thymocyte culture, washed away and ressuspended in drug free medium (M) for a further 4 hours; lanes 7 and 8 represent the effect of EGTA given in the last 4 hours or present during the whole period of culture. One representative of three experiments is shown.
CD4/CD8 expression on thymocytes in the presence of Phorbol Ester (TPA), Thapsigargin (TG) and Ouabain (OUA) Previous observations have indicated that phorbol esters are capable to produce CD4 internalization on single-positive and double-positive murine thymocytes (Anderson and Coleclough, 1993 and Tagawa et al., 1991). Similarly, our results shows that TPA 16 nM is capable of diminishing
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the level of CD4 molecules on the surface of thymocytes after 6 h (data not shown) and after18 h (Fig. 7). Our results demonstrate for the first time that TG is also capable of reducing CD4 molecules on thymocytes in a dose-dependent way (Fig. 7), albeit to lower levels than those of TPA. OUA 100 nM did not modify the expression of CD4, but it may might be necessary a longer incubation period, for this phenomenon to take place with OUA. Neither TG (10,20 and 50 nM) nor OUA (100 nM) were capable of modifying CD4 expression during the 6 h culture (data not shown).
Fig. 7. CD4/CD8 expression on thymocytes in the presence of Phorbol Ester (TPA), Thapsigargin (TG) and Ouabain (OUA). Thymocytes (5 105 cells) were incubated for 18 hours in the absence or presence of TPA (16 nM), TG (10 and 20 nM) and OUA (100 nM). TG 50 nM was not used because of the high degree of mortality. The numbers represent the percentage of CD4, CD8 or DP cells. One representative of three experiments is shown.
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Discussion The present work demonstrated for the first time that agents such as Thapsigargin and Ouabain were capable of increasing CD69 expression on thymocytes, similar to what has been reported using TPA (Hara et al., 1986 and Lanier et al., 1986). All these three agents (TPA, Thapsigargin and Ouabain) increased cytosolic Ca+ + levels in thymocytes. The Ca+ + mobilized by these substances originates from both internal and external pools. This Ca+ + increase has been described in thymocytes using both Thapsigargin (Jiang et al., 1994) and TPA (Klip et al., 1984). The present results show a similar response in these cells and extend the capability of mobilizing Ca+ + to Ouabain. Furthermore, the present results suggest that the transient Ca+ + release from internal stores was not sufficient to induce CD69 expression on thymocytes. External calcium influx was essential for this process to take place, as it was possible to modulate CD69 increased expression when EGTA was used to buffer external Ca+ +. This is in agreement with what has been described in human T lymphocytes (Vandenberghe et al., 1992) and Jurkat (Aussel et al., 1996) cells exposed to different stimuli. We found that the percentage of CD69high positive cells in unstimulated thymocytes diminished to nearly half by 18 h of culture. A decrease in CD69 expression with time on cultured thymocyte has been described by Testi et al. (1989), who suggested the need of interactions with the thymic stroma to maintain the levels of CD69 on thymocytes. It is possible that, in our study, the substances added: TPA, Thapsigargin and Ouabain could, in some way, substitute for the signals produced by these interactions. Activation of protein kinase C (PKC) by stimulation of the TCR/CD3 or by phorbol esters induces the expression of CD69 on thymocytes (Hara et al., 1986 and Lanier et al., 1986). It is, however, unknown the signaling pathways involved when Thapsigargin and Ouabain are used. It is noteworthy that Thapsigargin has been shown before, by other authors, to induce the expression of this molecule in Jurkat T lymphocytes (Aussel et al., 1996) and Ouabain on activated peripheral blood lymphocytes (Pires et al., 1997). A possible pathway for CD69 induction by these substances could involve the expression of c-fos.There are evidences that the expression of CD69 is regulated by c-fos via the transcription factor AP-1 (Castellanos et al., 1997), and in different systems, all these three agents, TPA (Kontny et al., 1992), Thapsigargin (He et al., 2000) and Ouabain (Nakagawa et al., 1992; Peng et al., 1996), were capable of inducing c-fos expression. The kinectics of c-fos RNA expression vary being very rapid in the case of TPA (Kontny et al., 1992) and Thapsigargin (He et al., 2000) contrasting with the slow induction of the gene by Ouabain (Nakagawa et al., 1992). In agreement to what has been observed with c-fos the kinetics of CD69 appearance depended on the stimuli and dose used, being faster with TPA and Thapsigargin, and slower with Ouabain. However, TPA and Thapsigargin needed only to be present for the first two hours for the induction of expression to take place. Independent of the removal of the drug after 2 h, maximal increase with TPA was already evident by 6 h. When Thapsigargin was used, a dose of 50 nM induced an increased expression by 6 h whereas with lower doses an increase was detected at 18 h. In the case of Ouabain an observable increase was only seen at 18 h, but the drug needed to be present for the whole time (data not shown). The need for external Ca+ + is limited to the period necessary for the drugs to induce CD69 expression. When EGTA was only present for the first 2 h (together with the stimulus) the effect
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of Thapsigargin was completely abrogated whilst the effect of TPA was only partially affected. In this case EGTA present only in the last 4 hours of culture was of no avail. In the case of Ouabain, EGTA needs to be present the whole time of the culture period to inhibit CD69 increased expression. This might be due to different kinetics in Ca+ + entry produced by the various drugs. Despite being the best inducer of CD69 expression, TPA produced the lowest levels of cytosolic Ca+ +,either as a result of activating the plasma membrane Ca+ + pump (Rink and Sage, 1987) and/or as a direct effect on endoplasmatic reticulum calcium uptake (Cardoso et al., 1997). This suggests that Ca+ + influx is more important for CD69 expression then the final levels of intracellular Ca+ + achieved. It is unlikely that the decrease in CD69 expression produced by EGTA resulted from cell death. Thymocytes remained as viable in the presence of EGTA as cells kept in medium or in the presence of drugs alone. In the particular case of 50 nM Thapsigargin the addition of EGTA partially protected these cells from death. Similarly, Jiang et al.(Jiang et al., 1994) demonstrated that the presence of EGTA for 2 hours after exposure to Thapsigargin was capable of preventing rat thymocytes of undergoing apoptosis. Not only Jiang et al. (1994), but also McConkey et al. (1989) and Hampton et al. (1996) demonstrated a clear involvement of the increase in cytosolic Ca+ + levels during apoptosis. However, there is controversy regarding the role played by intracellular Ca+ + during the apoptotic process. Others workers (Alnemri and Litwack, 1990; Duke et al., 1994 and Beaver and Waring, 1994) suggested that Ca+ + increase was not sufficient to induce apoptosis. A correlation between CD69 expression and thymocyte death has also been suggested by Kishimoto et al. (1995) who demonstrated that thymocytes undergoing apoptosis were expressing this molecule. In the present work the percentage of necrotic thymocytes after 6 hour of culture was similar between control and cells treated with TPA, Thapsigargin or Ouabain (around 16%) despite the fact that TPA induced CD69high expression in 95% of the cells. It has also been suggested, in different systems, that CD69 expression may precede cell proliferation, maturation or differentiation (Testi et al., 1994). Furthermore, it has been reported that mice that overexpressed CD69 in the thymus displayed a dramatic increase in the number of singlepositive cells (Nakayama et al., 2002). In the present study no evidence of thymocyte maturation was obtained. Even if TPA and Thapsigargin led to a decrease in double positive cells, this seemed to reflect a loss of CD4 molecules, as previously described in thymocytes with TPA (Anderson and Coleclough, 1993 and Tagawa et al., 1991) as opposed to a maturation towards single positive cells. Ouabain on the other hand did not modify CD4/CD8 expression on these cells. This might result from the fact that OUA takes much longer to induce CD69 expression and it was not studied for longer periods. Negative effects of Ouabain on the proliferation of mature lymphocytes have been reported (Lillehoj and Shevach, 1985, Brodie et al., 1995 and Moraes et al., 1989) even in situations where CD69 expression was increased (Pires et al., 1997). CD69 expression may be stimulated via different pathways (Borrego et al., 1993) and it is possible that the one induced by Ouabain differs from the pathway that produces CD4 internalization. In this regard it has been suggested that both TPA and Thapsigargin are capable of activating cells via PKC (Lanier et al., 1986 and Huber et al., 2000) whereas it has been suggested that OUA may use a tyrosine kinase pathway leading to the expression of RAS (Haas et al., 2000) and that, at least for c-fos RNA expression, the activation of PKC is not the primary mechanism by which Ouabain exert its action (Nakagawa et al., 1992).
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Conclusion The present work demonstrated in thymocytes that TPA, Thapsigargin and Ouabain were capable of Ca+ + mobilization, that these three drugs induced increased CD69 expression, suggesting cellular activation, and that this expression depended on external Ca+ + entry. Finally, no correlation between CD69 and thymocyte death or thymocyte maturation could be established. Acknowledgements This work was supported by the Brazilian National Research Council (CNPq, PRONEX) and FAPERJ (Fundacßa˜o de Amparo a` Pesquisa do Rio de Janeiro). Sandra Rodrigues Mascarenhas was a recipient of a Master Degree Scholarship from CAPES (Coordenacßa˜o de Aperfeicßoamento de Pessoal de Nı´vel Superior).
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