CELLULAR
IMMUNOLOGY
1452 10-2 I7 (1992)
Glutathione Influences the Proliferation as Well as the Extent of Mitochondrial Activation in Rat Splenocytes C. PIERI, F. MORONI,
AND R. RECCHIONI
Cytology Center, Gerontology Research Department of I.N.R.C.A., Via Birarelli No. 8, 60121 Ancona, Italy Received June 1, 1992; accepted July I2, 1992 The time-dependent changes of mitochondrial membrane potential and mass have been investigated on rat splenic lymphocytes stimulated with Con A in the presence and absence of reduced glutathione (GSH). Rhodamine(Rh-123) and nonyl acridine orange (NAO) were used as specific dyes to monitor the membrane potential and mass of mitochondria, respectively. The percentage of cells showing blast transformation ‘and the level of Rh-123 or NAO uptake were analyzed by flow cytometry. Present results demonstrate that a large number of cells showed activated mitochondria already at 24 hr after Con A stimulation and the activation of these organelles was not related to blast transformation. The addition of GSH into the culture medium increased the number of cells responding to mitogenic stimulation. In parallel it augumented the percentage of lymphocytes with activated mitochondria and also prevented their depolarization. 0 1992 Academx
Press, Inc.
INTRODUCTION
Glutathione (GSH) is a tripeptide thiol which is widely distributed in cells. GSH is supposed to play an important role as an intracellular free radical scavenger and also protects against radiation damages (l-3). Some other cellular functions have also been shown to be connected to intracellular GSH concentration, such as DNA repair (4) cellular redox balance (5), and amino acid transport (6). From the vast array of different effects influenced by GSH, it appears obvious that cell proliferation cannot be independent of the intracellular GSH concentration. Studies have shown that depletion of the GSH level impaired cell proliferation (7-9). Recently, it has been demonstrated that an increase in the intracellular GSH content significantly enhanced the proliferative response to mitogenic stimuli ( lo- 13). Despite several attempts to identify the actual mechanisms which participate in the up- and down-regulation of cell activation in the presence and absence of GSH, respectively, no definitive conclusions could be drawn thus far. Since a beneficial effect of the increased redox potential on the energetic background of cell activation processes seems to be a reasonable generalized conclusion, we paid special attention to mitochondrial parameters in the presence and absence of GSH during mitogenic stimulation of rat splenic lymphocytes. In order to keep the system as physiological as possible, live cells were studied. Changes in the mitochondrial membrane potential, monitoring its energetic state, were followed by the mitochondrium-specific fluorescent dye rhodamine- 123 (Rh- 123) ( 14). In addition, the uptake of another fluorescent dye, the nonyl acridine orange (NAO), was reported on the 210 0008-8749192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
COMMUNICATIONS
211
mass of mitochondria during cell proliferation (15). Obviously the effect of GSH on the proliferative capability of splenic rat lymphocytes was also investigated. Our data, presented here, support the view that cell proliferation and mitochondrial metabolism are significantly up-regulated by the presence of GSH. MATERIALS
AND
METHODS
Cell preparation and culture. Splenic lymphocytes were prepared from 5-monthold female Wistar rats from our own breed. After cervical dislocation, spleens were removed and teased. The lymphocytes were prepared by Ficoll-Hypaque gradient centrifugation according to Boyum ( 16) and after repeated washes in Hanks’ solution were resuspended (2 X lo6 cells/ml) in RPM1 1640 supplemented with 2 mA4 glutamine, 10% fetal calf serum, 100 units/ml penicillin, and 100 pg/ml streptomicin. Cell stimulation. The cells were stimulated in microwells, containing 3 X lo5 cell in each, with different concentrations of concanavalin A ( 1- 10 pg/ml) in the presence or absence of 5 mA4 GSH (Sigma). [3H]Thymidine incorporation was measured after 24, 48, and 72 hr of incubation at 37°C in 5% CO2 atmosphere. The 2-&i doses of [3H]thymidine (6.7 Ci/mmol, Amersham) were added to each well 5 hr before collecting the cells on glass fiber filters (Labtek) by means of a cell harvester (Skatron AS). Filters were dried and their radioactivity was measured in a liquid scintillation counter (Packard-Tricarb). The results were the means of the analysis of samples from five animals. Flow cytometric cell analysis. The viability of the stimulated and unstimulated lymphocytes was determined by the ability of the viable cells to exclude ethidium bromide (EB). The percentage of the lymphocytes undergoing blast transformations was calculated from the forward angle light scatter signals of those cells which were EB negative. The splenocytes were stained for mitochondrial potential determination by rhodamine- 123 (Molec. Probes), as described by Darzynkiewicz et al. ( 17). Samples of cell (2 X 106/ml) treated with Con A (5 pug/ml) in the presence and absence of 5 mM GSH were incubated for various durations. At different time points the cells were stained with 25 puM Rh-123 at room temperature for 20 min in the dark. The cells were washed twice with phosphate-buffered saline (pH 7.4) and were suspended in RPM1 1640. The same procedure was followed for the staining with 5 PM NAO for 15 min according to Retinaud et al. (18). Before the analyses, 10 pug/ml EB was added to each sample to monitor the dead cells, which were excluded from the analysis of the Rh-123 and NAO fluorescence by gating for red fluorescence. Measurements of the fluorescence and light scatter parameters on a cell-by-cell basis were carried out in a Coulter Epics V flow cytometer (Coulter, Hialeah, FL). The argon ion laser was tuned to 488 nm. The green fluorescence of the Rh-123- or NAO-stained cells was detected between 500 and 540 nm, whereas the red fluorescence emitted by the EB that penetrated only dead cells was measured at wavelengths higher than 6 15 nm. Data were analyzed by program packages provided by the manufacturer. RESULTS Measurements on the prohjzration. Figure IA shows data on the viability of lymphocytes cultured for 24, 48, and 72 hr. The control cells without mitogen preserved their viability within 95% at all three time points. The percentage of dead cells in the stimulated population reached 16% after 48 hr and was slightly further increased at 72 hr. GSH, added together with the Con A, provided a 4-5% protection against cell
212
SHORT COMMUNICATIONS
0:
24
48
72
hours
24 48 72 hours FIG. 1. (A) Viability of the lymphocytes cultured for 3 days. (B) Percentage of cells undergoing blast transformation. The cells were cultured as described under Materials and Methods without Con A (a), with 5 pg/ml Con A (O), and with the mitogen and 5 mM GSH (0). SEM did not exceed 2 and 4% of the means in A and B, respectively.
mortality practically at all time points. Figure 1B presents data on the number of lymphocytes undergoing blast transformation. Few cells moved from the resting state in the control population, but their number could reach lo-12% after 72 hr of incubation. Con A stimulation induced blast formation in 32 and 55% of the cells at 24 and 48 hr, respectively, practically without further significant increase by 72 hr. Addition of GSH significantly influenced this parameter. The percentage of the blasts went up to 50% at 24 hr and 74% at 48 hr; however, at 72 hr it declined by approximately 10% on an average. Figure 2 indicates the [3H]thymidine incorporation into newly synthethized DNA, a key parameter for cell proliferation, at different doses of Con A in three different time points and presence or absence of GSH. The [3H]thymidine incorporation was low and concentration independent at 24 hr and showed a positive dependence on the lectin concentrations of up to 5 pg/ml and above that dropped to nearly half of the maximal values reached at the optimal 5 pg/ml concentration after 72 hr of incubation. GSH-treated samples showed a very different pattern for all three different durations of incubations. The cycling of the cells was obvious at 24 hr. The optimum was also at the same lectin concentration, but the [3H]thymidine incorporation reached 75% of the incorporated isotope level of those
213
SHORT COMMUNICATIONS 300
1
0 ~ 0
E 2
4
6
8
10
,
12
Con A (rig/ml) FIG. 2. Time-dependent [‘Hlthymidine incorporation in Con A-stimulated lymphocytes in the presence (closed symbols) and absence (open symbols) 5 mkf GSH. Determination was performed at 24 (O,O), 48 (0,m) and 72 @,A) hr. SEM of each point was comprised between 5 and 10% of the means.
of the 48- and 72-hr samples without GSH. The optimal lectin concentration shifted to 2.5 pg/ml after 48 or 72 hr and the nucleotide incorporation level was more than doubled. Maximal incorporation was reached earlier, at 48 hr, and was by and large concentration independent of the lectin above the optimal 2.5 pg/ml level. After 72 hr incubation doses above this optimal lectin concentration caused a 10% decrease in the incorporation. Mitochondrial parameters. An example of histograms showing the distribution of Rh- 123 fluorescence and mitochondrial membrane potential over the cell population are seen in Fig. 3, obtained with nonstimulated control, Con A-stimulated, and Con
Log of fluorescence
intensity
(channel
number).
FIG. 3. Histograms of Rh-123 fluorescence distribution of control (Cl), Con A (0) and the mitogen plus ). The intervals delimited by dashed lines contain cell populations with depolarized (I), normally polarized (II), and hyperpolarized (III) mitochondria.
214
SHORT
COMMUNICATIONS
A- and GSH-stimulated cells at 72 hr. In control cultures three distinctly different fluorescent populations can be recognized after Rh- 123 staining. The first population (between channels O-25) binds low amounts of dye, representing partially or totally depolarized cells. The second cell population (channels 25-90) is likely to include the lymphocytes which had normally polarized mitochondria and the third one, highly fluorescent (greater than channel 90) population can arise from the hyperpolarized organelles. Mitogenic stimulation increased the first and the third population at the expense of the second one. Addition of GSH increased the number of the hyperpolarized (highly fluorescence) population. A similar picture was seen in case of the NAO fluorescence, reporting on the overall mass of mitochondria present in the investigated populations. The populations based on NAO fluorescence, i.e., mitochondrial mass, sometimes showed an inhomogeneity in unstimulated lymphocytes. Two or even three populations could be identified in some samples. However, after Con A stimulation the mass homogeneity significantly increased allowing us to focus our interest on three populations, as in the case of Rh123 fluorescence. Figure 4 summarizes the results, i.e., the changes in cell populations showing low (I), normal (II), and high (III) Rh-123, and NAO fluorescence. Small changes in the percentage of cells with depolarized mitochondria occurred in the population of unstimulated lymphocytes (approximately 12% at 72 hr). After Con A stimulation 55 and 70% of lymphocytes showed highly fluorescent mitochondria at 24 and 48 hr, respectively. An additional day did not increase this percentage. The hyperpolarization of mitochondria was strongly dependent on the presence of GSH. Indeed, 75% of cells showed an increased uptake of Rh-123 already after 24 hr incubation. At 48 hr this percentage reached 85% and thereafter began declining. In the case of the NAO fluorescence the first histogram was practically constant within the 3-day cultures. The general trend of the changes was the same as shown for Rh- 123. However, after 24 hr of incubation, fewer cells were able to modify the mass of their mitochondria than those increasing their membrane potential even in the absence of GSH. Not less than 75 and 85% of the viable cells increased their mitochondrial mass after 48 and 72 hr, respectively. DISCUSSION The principal aim of the present work was to investigate the time-dependent changes in the pattern of mitochondrial parameters as mitochondrial membrane potential and mass during cell proliferation in the presence and absence of glutathione. Earlier attempts studied the uptake of Rh-123 in normal and malignantly transformed cells ( 14, 19) and also during cell proliferation ( 17,20,2 1); however, none of them analyzed their values for changes in the mitochondrial mass. A recent and also the first combined Rh- 123 and NAO analysis by Leprat et al. (22), studying mitochondrial changes during aging of mouse splenic lymphocyte, neglected the possibility of looking for the time kinetics and analyzed their data only after 72 hr. Thus, our data seem to be the first presenting a combined analysis of mitochondrial potential changes, normalized for mass alterations during cell activation, and expanding the focus of interest over the whole period of activation process. Thus, the effect of GSH could be compared to such a set of control data which had not been available yet. The effect of GSH upon cell proliferation. In accordance with earlier investigators, an increase in the [3H]thymidine incorporation was observed in the presence of GSH
215
SHORT COMMUNICATIONS
Rh-123
NAO
100 -
24h
24h III
II -* 100 -
48h
III
80 -
20 0L
100 _
J
80 -
i? 2 6CG 2 . a 40 10 0F
11 I
x,
x72h
III
72h
80 60 40 20
II I
Con A 0
Con A 5
GSH
0
Con A 0
Con A 5
GSH
4. Changes of cell populations showing low (I), normal (II), and high (III) Rh-123 and NAO fluorescence during lymphocyte proliferation in the presence and absence of GSH. SEM of each point was comprised between 4 and 7% of the means. FIG.
when the cells were stimulated by mitogens (lo- 13). The highest values of the DNA synthesis were moved to the 48-hr samples, in contrast to the 72-hr peaks when lectin alone was applied. The blast transformation was also increased in the GSH-treated cell populations by 20% at 24 and 48 hr. This finding supports the earlier view that cells are apt to respond to mitogenic stimulation according to their GSH content (23). This enhancement of blast populations might be due to the effect of GSH on the
216
SHORT COMMUNICATIONS
transmembrane signaling, namely the increase in the free intracellular calcium concentration and the tyrosine kinase activity (24). The shortening of the cell cycle, found by us, can be explained by the recently published effect of GSH on the IL-2 activation. This second phase of well-controlled self-activation cycle can be increased twofold by GSH, which is supposed to induce a rapid internalization of IL-2 and IL-2 receptor complexes (25). On the other hand, it is interesting that the GSH had no effect on the expression of IL-2-coding mRNAs or the IL-2 synthesis at other levels (26). These data may refer to the effects of GSH at the plasma membrane level. Neither normal nor transformed lymphocytes are supposed to take up GSH from extracellular sources (27-29). Nevertheless, intracellular GSH level increased in stimulated lymphocytes when incubated together (11). The extracellular breakdown of GSH and a transport of the products for new GSH synthesis (11) was one suggestion that was also supported by others observing an increased cystein transport activity of the alanine-serine-cysteine (ASC) transport system (30). A nonbiochemical approach can be that the redox potential represented by the GSH even at the outer surface of the cells may save internal redox capacity for other processes. Thus, the observed increase of internal GSH level may partially be due to the lower “spending” of redox material. Transmembrane potential and mass of mitochondria. The analysis of the time kinetics of mitochondrial parameters enable us to draw some quantitative conclusions. First of all the increase in the membrane potential and mass of mitochondria is a precocious phenomenon, already evident at 24 hr. In addition, another interesting feature emerges comparing the number of cells undergoing blast transformation with that showing activated mitochondria. Indeed, considering the curve III in Fig. 4, at 24 hr of incubation the number of the cells displaying hyperpolarized mitochondria increased from 12% (resting cells) to 55% upon lectin stimulation. The percentage of cells showing a mitochondrial mass change was about the same. The percentage of changes in blast transformation was lower (32%). Thus, changes in mass and membrane potential of mitochondria must proceed and do not seem to be dependent on blast transformation. A similar conclusion can be drawn from analyzing the data obtained in the presence of GSH. Another interesting point of information arises from the analysis of the kinetic of change of Rh- 123 and NAO fluorescence. Indeed, a comparison of fluorescence changes of these two probes suggests that the membrane potential changes proceed the mass changes and return earlier. As shown for the cell cycle progression, the presence of GSH in the culture medium strongly influences the mitochondrial parameters. An increase of the number of cells showing activated mitochondria was found in GSH-treated cultures as compared to the GSH-untreated ones, at any time taken into account. In addition, it is of importance to note that GSH was able to decrease the number of cells showing depolarized mitochondria (Fig. 4). This suggests that GSH exerts its action either at the level of the mechanism(s) which induce(s) the activation of mitochondria and on the mitochondrium in itself, probably protecting these organelles against peroxidative damages. ACKNOWLEDGMENTS The authors are grateful to Mr. G. Mazzarini and R. Pierandrei for their technical assistance.
SHORT COMMUNICATIONS
217
REFERENCES 1. Andreoli, S. P., Mallett, C. P., and Bergstein, J. M., J. Lab. Clin. Med. 108, 190, 1986. 2. Biaglow, J. E., Varness, M. E., Clark, E. P., and Epp, E. R., Radiat. Rex 95, 437, 1983. 3. Harlan, J. M., Levine, J. D., Callahan, K. S., Schwartz, B. R., and Harker, L. A., J. Clin. Invest. 73, 706, 1984. 4. Edgren, M., and Revesz, L., Int. J. Radiat. Biol. 48, 207, 1985. 5. Reed. D. J., Biochem. Pharmacol. 35, 7, 1986. 6. Meister, A., Methods Enzymol. 113, 571, 1985. 7. Chaplin, D. D., and Wedner, H. J., Cell. Immunol. 36, 303, 1978. 8. Messina, J. P., and Lawrence, D. A., J. Immunol. 143, 1974, 1989. 9. Fidelus, R. K., Ginouves, P., Lawrence, D., and Tsan, M. F., Exp. Cell Res. 170, 269, 1987. 10. Fidelus, R. K., and Tsan, M. F., J. Immunol. 97, 155, 1986. 11. Suthanthiran, M., Anderson, M. E., Sharma, V. K., and Meister, A., Proc. Natl. Acad. Sci. USA 87, 3343, 1990. 12. Franklin, R. A., Arkins, S., and Keller, K. W., Cell. Immunol. 130, 416, 1990. 13. Smyth, M. J., J. Immunol. 146, 1921, 1991. 14. Johnson, L. V., Walsh, M. L., and Chen, L. B., Proc. Natl. Acad. Sci. USA 77, 990, 1980. 15. Erbrich, V., Septinus, N., Naujak, A., Zimmerman, H. W., Histochemistry 80, 385, 1984. 16. Boyum, A., J. Clin. Invest. (Suppl.) 21, 197, 1968. 17. Darzynkiewicz, Z., Staiano-Coico, L., Melamed, M. R., Proc. Natl. Acad. Sci. USA 78, 2383, 1981. 18. Retinaud, M. E., Leprat, P., and Julien, R., Cytometry 9, 206, 1988. 19. Nadakuvakaren, K. K., Nadakuvakaren, J. J., and Chen, L. B., Cancer Res. 45, 6093, 1985. 20. Benel, L., Ronot, X., Koruprobst, M., Adolphe, M., and Mounolou, J. C., Cytometry 7, 281, 1986. 21. James, T. W., and Bahman, R., J. Cell. Biol. 89, 256, 1981. 22. Leprat, P., Retinaud, M. H., and Julien, R., Mech. Age. Dev. 52, 149, 1990. 23. Kavanagh, T. J., Grossmann, A., Jaecks,E. P., Jinneman, J. C., Eaton, D. L., Martin, G., and Rabinovich, P. S., J. Cell. Phvsiol. 145,472, 1990. 24. Grossmann, A., Kavanagh, T. J., Ledbetter, J. A., and Rabinovich, P. S., Cytometry (Suppl.) 5, 60, 199 1 (Abstract). 25. Liang, C. M., Lee, H., Cattel, D., and Liang, S. M., J. Biol. Chem. 264, 135 19, 1989. 26. Gmiinder, H., Roth, S., Eck, H. P., Gallas, H., Mihm, S., and Driige, W., Cell. Immunol. 130, 520, 1990. 21. Martensson, J., Jain, A., Frayer, W., and Meister, A., Proc. Natl. Acad. Sci. USA 86, 5296, 1989. 28. Dethmers, J. K., and Meister, A., Proc. Natl. Acad. Sci. USA 78, 7792, 1981. 29. Hamilos, D. L., Zelamey, P., and Mascali, J. J., Immunopharmacology 18, 223, 1989. 30. Ishii, T., Sugita, Y., and Bannai, S., J. Cell. Physiol. 133, 330, 1987.
IMMUNOLOGY
1452 10-2 I7 (1992)
Glutathione Influences the Proliferation as Well as the Extent of Mitochondrial Activation in Rat Splenocytes C. PIERI, F. MORONI,
AND R. RECCHIONI
Cytology Center, Gerontology Research Department of I.N.R.C.A., Via Birarelli No. 8, 60121 Ancona, Italy Received June 1, 1992; accepted July I2, 1992 The time-dependent changes of mitochondrial membrane potential and mass have been investigated on rat splenic lymphocytes stimulated with Con A in the presence and absence of reduced glutathione (GSH). Rhodamine(Rh-123) and nonyl acridine orange (NAO) were used as specific dyes to monitor the membrane potential and mass of mitochondria, respectively. The percentage of cells showing blast transformation ‘and the level of Rh-123 or NAO uptake were analyzed by flow cytometry. Present results demonstrate that a large number of cells showed activated mitochondria already at 24 hr after Con A stimulation and the activation of these organelles was not related to blast transformation. The addition of GSH into the culture medium increased the number of cells responding to mitogenic stimulation. In parallel it augumented the percentage of lymphocytes with activated mitochondria and also prevented their depolarization. 0 1992 Academx
Press, Inc.
INTRODUCTION
Glutathione (GSH) is a tripeptide thiol which is widely distributed in cells. GSH is supposed to play an important role as an intracellular free radical scavenger and also protects against radiation damages (l-3). Some other cellular functions have also been shown to be connected to intracellular GSH concentration, such as DNA repair (4) cellular redox balance (5), and amino acid transport (6). From the vast array of different effects influenced by GSH, it appears obvious that cell proliferation cannot be independent of the intracellular GSH concentration. Studies have shown that depletion of the GSH level impaired cell proliferation (7-9). Recently, it has been demonstrated that an increase in the intracellular GSH content significantly enhanced the proliferative response to mitogenic stimuli ( lo- 13). Despite several attempts to identify the actual mechanisms which participate in the up- and down-regulation of cell activation in the presence and absence of GSH, respectively, no definitive conclusions could be drawn thus far. Since a beneficial effect of the increased redox potential on the energetic background of cell activation processes seems to be a reasonable generalized conclusion, we paid special attention to mitochondrial parameters in the presence and absence of GSH during mitogenic stimulation of rat splenic lymphocytes. In order to keep the system as physiological as possible, live cells were studied. Changes in the mitochondrial membrane potential, monitoring its energetic state, were followed by the mitochondrium-specific fluorescent dye rhodamine- 123 (Rh- 123) ( 14). In addition, the uptake of another fluorescent dye, the nonyl acridine orange (NAO), was reported on the 210 0008-8749192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
COMMUNICATIONS
211
mass of mitochondria during cell proliferation (15). Obviously the effect of GSH on the proliferative capability of splenic rat lymphocytes was also investigated. Our data, presented here, support the view that cell proliferation and mitochondrial metabolism are significantly up-regulated by the presence of GSH. MATERIALS
AND
METHODS
Cell preparation and culture. Splenic lymphocytes were prepared from 5-monthold female Wistar rats from our own breed. After cervical dislocation, spleens were removed and teased. The lymphocytes were prepared by Ficoll-Hypaque gradient centrifugation according to Boyum ( 16) and after repeated washes in Hanks’ solution were resuspended (2 X lo6 cells/ml) in RPM1 1640 supplemented with 2 mA4 glutamine, 10% fetal calf serum, 100 units/ml penicillin, and 100 pg/ml streptomicin. Cell stimulation. The cells were stimulated in microwells, containing 3 X lo5 cell in each, with different concentrations of concanavalin A ( 1- 10 pg/ml) in the presence or absence of 5 mA4 GSH (Sigma). [3H]Thymidine incorporation was measured after 24, 48, and 72 hr of incubation at 37°C in 5% CO2 atmosphere. The 2-&i doses of [3H]thymidine (6.7 Ci/mmol, Amersham) were added to each well 5 hr before collecting the cells on glass fiber filters (Labtek) by means of a cell harvester (Skatron AS). Filters were dried and their radioactivity was measured in a liquid scintillation counter (Packard-Tricarb). The results were the means of the analysis of samples from five animals. Flow cytometric cell analysis. The viability of the stimulated and unstimulated lymphocytes was determined by the ability of the viable cells to exclude ethidium bromide (EB). The percentage of the lymphocytes undergoing blast transformations was calculated from the forward angle light scatter signals of those cells which were EB negative. The splenocytes were stained for mitochondrial potential determination by rhodamine- 123 (Molec. Probes), as described by Darzynkiewicz et al. ( 17). Samples of cell (2 X 106/ml) treated with Con A (5 pug/ml) in the presence and absence of 5 mM GSH were incubated for various durations. At different time points the cells were stained with 25 puM Rh-123 at room temperature for 20 min in the dark. The cells were washed twice with phosphate-buffered saline (pH 7.4) and were suspended in RPM1 1640. The same procedure was followed for the staining with 5 PM NAO for 15 min according to Retinaud et al. (18). Before the analyses, 10 pug/ml EB was added to each sample to monitor the dead cells, which were excluded from the analysis of the Rh-123 and NAO fluorescence by gating for red fluorescence. Measurements of the fluorescence and light scatter parameters on a cell-by-cell basis were carried out in a Coulter Epics V flow cytometer (Coulter, Hialeah, FL). The argon ion laser was tuned to 488 nm. The green fluorescence of the Rh-123- or NAO-stained cells was detected between 500 and 540 nm, whereas the red fluorescence emitted by the EB that penetrated only dead cells was measured at wavelengths higher than 6 15 nm. Data were analyzed by program packages provided by the manufacturer. RESULTS Measurements on the prohjzration. Figure IA shows data on the viability of lymphocytes cultured for 24, 48, and 72 hr. The control cells without mitogen preserved their viability within 95% at all three time points. The percentage of dead cells in the stimulated population reached 16% after 48 hr and was slightly further increased at 72 hr. GSH, added together with the Con A, provided a 4-5% protection against cell
212
SHORT COMMUNICATIONS
0:
24
48
72
hours
24 48 72 hours FIG. 1. (A) Viability of the lymphocytes cultured for 3 days. (B) Percentage of cells undergoing blast transformation. The cells were cultured as described under Materials and Methods without Con A (a), with 5 pg/ml Con A (O), and with the mitogen and 5 mM GSH (0). SEM did not exceed 2 and 4% of the means in A and B, respectively.
mortality practically at all time points. Figure 1B presents data on the number of lymphocytes undergoing blast transformation. Few cells moved from the resting state in the control population, but their number could reach lo-12% after 72 hr of incubation. Con A stimulation induced blast formation in 32 and 55% of the cells at 24 and 48 hr, respectively, practically without further significant increase by 72 hr. Addition of GSH significantly influenced this parameter. The percentage of the blasts went up to 50% at 24 hr and 74% at 48 hr; however, at 72 hr it declined by approximately 10% on an average. Figure 2 indicates the [3H]thymidine incorporation into newly synthethized DNA, a key parameter for cell proliferation, at different doses of Con A in three different time points and presence or absence of GSH. The [3H]thymidine incorporation was low and concentration independent at 24 hr and showed a positive dependence on the lectin concentrations of up to 5 pg/ml and above that dropped to nearly half of the maximal values reached at the optimal 5 pg/ml concentration after 72 hr of incubation. GSH-treated samples showed a very different pattern for all three different durations of incubations. The cycling of the cells was obvious at 24 hr. The optimum was also at the same lectin concentration, but the [3H]thymidine incorporation reached 75% of the incorporated isotope level of those
213
SHORT COMMUNICATIONS 300
1
0 ~ 0
E 2
4
6
8
10
,
12
Con A (rig/ml) FIG. 2. Time-dependent [‘Hlthymidine incorporation in Con A-stimulated lymphocytes in the presence (closed symbols) and absence (open symbols) 5 mkf GSH. Determination was performed at 24 (O,O), 48 (0,m) and 72 @,A) hr. SEM of each point was comprised between 5 and 10% of the means.
of the 48- and 72-hr samples without GSH. The optimal lectin concentration shifted to 2.5 pg/ml after 48 or 72 hr and the nucleotide incorporation level was more than doubled. Maximal incorporation was reached earlier, at 48 hr, and was by and large concentration independent of the lectin above the optimal 2.5 pg/ml level. After 72 hr incubation doses above this optimal lectin concentration caused a 10% decrease in the incorporation. Mitochondrial parameters. An example of histograms showing the distribution of Rh- 123 fluorescence and mitochondrial membrane potential over the cell population are seen in Fig. 3, obtained with nonstimulated control, Con A-stimulated, and Con
Log of fluorescence
intensity
(channel
number).
FIG. 3. Histograms of Rh-123 fluorescence distribution of control (Cl), Con A (0) and the mitogen plus ). The intervals delimited by dashed lines contain cell populations with depolarized (I), normally polarized (II), and hyperpolarized (III) mitochondria.
214
SHORT
COMMUNICATIONS
A- and GSH-stimulated cells at 72 hr. In control cultures three distinctly different fluorescent populations can be recognized after Rh- 123 staining. The first population (between channels O-25) binds low amounts of dye, representing partially or totally depolarized cells. The second cell population (channels 25-90) is likely to include the lymphocytes which had normally polarized mitochondria and the third one, highly fluorescent (greater than channel 90) population can arise from the hyperpolarized organelles. Mitogenic stimulation increased the first and the third population at the expense of the second one. Addition of GSH increased the number of the hyperpolarized (highly fluorescence) population. A similar picture was seen in case of the NAO fluorescence, reporting on the overall mass of mitochondria present in the investigated populations. The populations based on NAO fluorescence, i.e., mitochondrial mass, sometimes showed an inhomogeneity in unstimulated lymphocytes. Two or even three populations could be identified in some samples. However, after Con A stimulation the mass homogeneity significantly increased allowing us to focus our interest on three populations, as in the case of Rh123 fluorescence. Figure 4 summarizes the results, i.e., the changes in cell populations showing low (I), normal (II), and high (III) Rh-123, and NAO fluorescence. Small changes in the percentage of cells with depolarized mitochondria occurred in the population of unstimulated lymphocytes (approximately 12% at 72 hr). After Con A stimulation 55 and 70% of lymphocytes showed highly fluorescent mitochondria at 24 and 48 hr, respectively. An additional day did not increase this percentage. The hyperpolarization of mitochondria was strongly dependent on the presence of GSH. Indeed, 75% of cells showed an increased uptake of Rh-123 already after 24 hr incubation. At 48 hr this percentage reached 85% and thereafter began declining. In the case of the NAO fluorescence the first histogram was practically constant within the 3-day cultures. The general trend of the changes was the same as shown for Rh- 123. However, after 24 hr of incubation, fewer cells were able to modify the mass of their mitochondria than those increasing their membrane potential even in the absence of GSH. Not less than 75 and 85% of the viable cells increased their mitochondrial mass after 48 and 72 hr, respectively. DISCUSSION The principal aim of the present work was to investigate the time-dependent changes in the pattern of mitochondrial parameters as mitochondrial membrane potential and mass during cell proliferation in the presence and absence of glutathione. Earlier attempts studied the uptake of Rh-123 in normal and malignantly transformed cells ( 14, 19) and also during cell proliferation ( 17,20,2 1); however, none of them analyzed their values for changes in the mitochondrial mass. A recent and also the first combined Rh- 123 and NAO analysis by Leprat et al. (22), studying mitochondrial changes during aging of mouse splenic lymphocyte, neglected the possibility of looking for the time kinetics and analyzed their data only after 72 hr. Thus, our data seem to be the first presenting a combined analysis of mitochondrial potential changes, normalized for mass alterations during cell activation, and expanding the focus of interest over the whole period of activation process. Thus, the effect of GSH could be compared to such a set of control data which had not been available yet. The effect of GSH upon cell proliferation. In accordance with earlier investigators, an increase in the [3H]thymidine incorporation was observed in the presence of GSH
215
SHORT COMMUNICATIONS
Rh-123
NAO
100 -
24h
24h III
II -* 100 -
48h
III
80 -
20 0L
100 _
J
80 -
i? 2 6CG 2 . a 40 10 0F
11 I
x,
x72h
III
72h
80 60 40 20
II I
Con A 0
Con A 5
GSH
0
Con A 0
Con A 5
GSH
4. Changes of cell populations showing low (I), normal (II), and high (III) Rh-123 and NAO fluorescence during lymphocyte proliferation in the presence and absence of GSH. SEM of each point was comprised between 4 and 7% of the means. FIG.
when the cells were stimulated by mitogens (lo- 13). The highest values of the DNA synthesis were moved to the 48-hr samples, in contrast to the 72-hr peaks when lectin alone was applied. The blast transformation was also increased in the GSH-treated cell populations by 20% at 24 and 48 hr. This finding supports the earlier view that cells are apt to respond to mitogenic stimulation according to their GSH content (23). This enhancement of blast populations might be due to the effect of GSH on the
216
SHORT COMMUNICATIONS
transmembrane signaling, namely the increase in the free intracellular calcium concentration and the tyrosine kinase activity (24). The shortening of the cell cycle, found by us, can be explained by the recently published effect of GSH on the IL-2 activation. This second phase of well-controlled self-activation cycle can be increased twofold by GSH, which is supposed to induce a rapid internalization of IL-2 and IL-2 receptor complexes (25). On the other hand, it is interesting that the GSH had no effect on the expression of IL-2-coding mRNAs or the IL-2 synthesis at other levels (26). These data may refer to the effects of GSH at the plasma membrane level. Neither normal nor transformed lymphocytes are supposed to take up GSH from extracellular sources (27-29). Nevertheless, intracellular GSH level increased in stimulated lymphocytes when incubated together (11). The extracellular breakdown of GSH and a transport of the products for new GSH synthesis (11) was one suggestion that was also supported by others observing an increased cystein transport activity of the alanine-serine-cysteine (ASC) transport system (30). A nonbiochemical approach can be that the redox potential represented by the GSH even at the outer surface of the cells may save internal redox capacity for other processes. Thus, the observed increase of internal GSH level may partially be due to the lower “spending” of redox material. Transmembrane potential and mass of mitochondria. The analysis of the time kinetics of mitochondrial parameters enable us to draw some quantitative conclusions. First of all the increase in the membrane potential and mass of mitochondria is a precocious phenomenon, already evident at 24 hr. In addition, another interesting feature emerges comparing the number of cells undergoing blast transformation with that showing activated mitochondria. Indeed, considering the curve III in Fig. 4, at 24 hr of incubation the number of the cells displaying hyperpolarized mitochondria increased from 12% (resting cells) to 55% upon lectin stimulation. The percentage of cells showing a mitochondrial mass change was about the same. The percentage of changes in blast transformation was lower (32%). Thus, changes in mass and membrane potential of mitochondria must proceed and do not seem to be dependent on blast transformation. A similar conclusion can be drawn from analyzing the data obtained in the presence of GSH. Another interesting point of information arises from the analysis of the kinetic of change of Rh- 123 and NAO fluorescence. Indeed, a comparison of fluorescence changes of these two probes suggests that the membrane potential changes proceed the mass changes and return earlier. As shown for the cell cycle progression, the presence of GSH in the culture medium strongly influences the mitochondrial parameters. An increase of the number of cells showing activated mitochondria was found in GSH-treated cultures as compared to the GSH-untreated ones, at any time taken into account. In addition, it is of importance to note that GSH was able to decrease the number of cells showing depolarized mitochondria (Fig. 4). This suggests that GSH exerts its action either at the level of the mechanism(s) which induce(s) the activation of mitochondria and on the mitochondrium in itself, probably protecting these organelles against peroxidative damages. ACKNOWLEDGMENTS The authors are grateful to Mr. G. Mazzarini and R. Pierandrei for their technical assistance.
SHORT COMMUNICATIONS
217
REFERENCES 1. Andreoli, S. P., Mallett, C. P., and Bergstein, J. M., J. Lab. Clin. Med. 108, 190, 1986. 2. Biaglow, J. E., Varness, M. E., Clark, E. P., and Epp, E. R., Radiat. Rex 95, 437, 1983. 3. Harlan, J. M., Levine, J. D., Callahan, K. S., Schwartz, B. R., and Harker, L. A., J. Clin. Invest. 73, 706, 1984. 4. Edgren, M., and Revesz, L., Int. J. Radiat. Biol. 48, 207, 1985. 5. Reed. D. J., Biochem. Pharmacol. 35, 7, 1986. 6. Meister, A., Methods Enzymol. 113, 571, 1985. 7. Chaplin, D. D., and Wedner, H. J., Cell. Immunol. 36, 303, 1978. 8. Messina, J. P., and Lawrence, D. A., J. Immunol. 143, 1974, 1989. 9. Fidelus, R. K., Ginouves, P., Lawrence, D., and Tsan, M. F., Exp. Cell Res. 170, 269, 1987. 10. Fidelus, R. K., and Tsan, M. F., J. Immunol. 97, 155, 1986. 11. Suthanthiran, M., Anderson, M. E., Sharma, V. K., and Meister, A., Proc. Natl. Acad. Sci. USA 87, 3343, 1990. 12. Franklin, R. A., Arkins, S., and Keller, K. W., Cell. Immunol. 130, 416, 1990. 13. Smyth, M. J., J. Immunol. 146, 1921, 1991. 14. Johnson, L. V., Walsh, M. L., and Chen, L. B., Proc. Natl. Acad. Sci. USA 77, 990, 1980. 15. Erbrich, V., Septinus, N., Naujak, A., Zimmerman, H. W., Histochemistry 80, 385, 1984. 16. Boyum, A., J. Clin. Invest. (Suppl.) 21, 197, 1968. 17. Darzynkiewicz, Z., Staiano-Coico, L., Melamed, M. R., Proc. Natl. Acad. Sci. USA 78, 2383, 1981. 18. Retinaud, M. E., Leprat, P., and Julien, R., Cytometry 9, 206, 1988. 19. Nadakuvakaren, K. K., Nadakuvakaren, J. J., and Chen, L. B., Cancer Res. 45, 6093, 1985. 20. Benel, L., Ronot, X., Koruprobst, M., Adolphe, M., and Mounolou, J. C., Cytometry 7, 281, 1986. 21. James, T. W., and Bahman, R., J. Cell. Biol. 89, 256, 1981. 22. Leprat, P., Retinaud, M. H., and Julien, R., Mech. Age. Dev. 52, 149, 1990. 23. Kavanagh, T. J., Grossmann, A., Jaecks,E. P., Jinneman, J. C., Eaton, D. L., Martin, G., and Rabinovich, P. S., J. Cell. Phvsiol. 145,472, 1990. 24. Grossmann, A., Kavanagh, T. J., Ledbetter, J. A., and Rabinovich, P. S., Cytometry (Suppl.) 5, 60, 199 1 (Abstract). 25. Liang, C. M., Lee, H., Cattel, D., and Liang, S. M., J. Biol. Chem. 264, 135 19, 1989. 26. Gmiinder, H., Roth, S., Eck, H. P., Gallas, H., Mihm, S., and Driige, W., Cell. Immunol. 130, 520, 1990. 21. Martensson, J., Jain, A., Frayer, W., and Meister, A., Proc. Natl. Acad. Sci. USA 86, 5296, 1989. 28. Dethmers, J. K., and Meister, A., Proc. Natl. Acad. Sci. USA 78, 7792, 1981. 29. Hamilos, D. L., Zelamey, P., and Mascali, J. J., Immunopharmacology 18, 223, 1989. 30. Ishii, T., Sugita, Y., and Bannai, S., J. Cell. Physiol. 133, 330, 1987.
