Oxidized glucocorticoids counteract glucocorticoid-induced apoptosis in murine thymocytes in vitro

Life Sciences 68 (2001) 2905–2916

Oxidized glucocorticoids counteract glucocorticoid-induced apoptosis in murine thymocytes in vitro Toshihiko Hirano*, Hiroshi Horigome, Hiroyuki Ishishita, Sayaka Uda, Kitaro Oka Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan Received 27 July 2000; accepted 13 October 2000

Abstract 11b-hydroxyglucocorticoids (HGCs) are known to induce apoptosis in immature T cells. Here we show that 11-oxoglucocorticoids (OGCs), which are oxidized metabolites of HGCs, counteract the apoptosis-inducing effects of HGC in murine thymocytes in vitro. Corticosterone at concentrations ranging from 0.1–100 mM induced apoptosis in thymocytes obtained from C57BL/6J mice aged 4 weeks, as demonstrated by cell staining with anti-phosphatidylserine antibody, a decrease in mitochondrial membrane potential, and DNA fragmentation. Co-culture of the cells with 10–100 mM of OGCs, dehydrocorticosterone, cortisone, and prednisone significantly inhibited thymocyte apoptosis induced by 1 mM corticosterone, (p,0.006). Among the other 6 physiological metabolites of the HGCs we tested, 20a-dehydrocortisol also showed considerable inhibitory effect on corticosteroneinduced thymocyte apoptosis. Corticosterone-treatment of thymocytes in vitro decreased the number of CD4 and CD8 double positive cells, while co-culturing the cells with dehydrocorticosterone significantly attenuated this corticosterone effect (p,0.0001). Numbers of double-negative cells and singlepositive cells were not significantly affected by corticosterone, dehydrocorticosterone, or both together. These results raised the possibility that OGCs and probably other HGC metabolites can regulate apoptotic cell death of immature double-positive thymocytes induced by HGC. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Glucocorticoid; Oxidized glucocorticoid; Attenuation of apoptosis; Negative selection; Thymocytes

Introduction HGCs have been considered to play an important role in the induction of apoptosis in immature T cells in thymus [1,2]. Accordingly, HGC levels in thymus microenvironments might be critical for regulation of negative selection in thymus. Tissue local-levels of HGCs * Corresponding author. Tel.: 181-426-76-5796; fax: 181-426-76-5798. E-mail address: [email protected] (T. Hirano) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 6 9 -4

2906

T. Hirano et al. / Life Sciences 68 (2001) 2905–2916

are extensively influenced by their metabolizing enzyme 11 b-hydroxysteroid dehydrogenase (11 b-HSD), which oxidizes HGCs to their corresponding metabolite OGCs and protects specific cells or tissues from glucocorticoid (GC)- and/or mineralocorticoid-actions of HGCs [3,4]. We and other researchers have shown that murine thymocytes include HGC oxidizing enzyme activity, suggesting that this enzyme may regulate thymocyte apoptosis induced by HGCs in thymus [5,6]. However, the physiological roles of HGC metabolites, OGCs, produced by the actions of 11 b-HSD on the apoptosis-inducing effects of HGCs in thymus have not yet been examined, for these OGCs themselves have been believed so far to possess little or negligible hormonal actions. One interesting observation was found in a report by Ohyama et al., in which they showed that one of the OGCs cortisone suppress apoptotic cell death in chorion laeve tissues of human fetal membranes [7]. As an approach to determine physiological roles of OGCs in regulating apoptotic cell death of immature T cells in thymus, we examined in our present study the possible effects of OGCs and other physiological metabolites of HGCs on the apoptosisinducing actions of HGCs in murine thymocytes in vitro. Our results showed for the first time that some of these metabolites significantly counteracted the apoptosis-inducing effects of one of the physiological HGCs, corticosterone, in mouse thymocytes. Materials and methods Mice and reagents Male C57BL/6 mice (4 weeks of age) obtained from Charles River Co., Japan, were used throughout the study. Corticosterone, dehydrocorticosterone, cortisone, 5 b-dehydrocortisol, 5 b-dehydrocortisone, 6b-hydroxycortisone, 20a-dehydrocortisol, 20b-dehydrocortisol, tetrahydro-cortisol and prednisone were purchased from Sigma Chemical Co., USA. Thymocyte culture Thymuses were obtained from ether-anesthetized mice. The mice thymuses were minced, gently pressed to release thymocytes in ice-cold PBS, and passed through a nylon mesh (35 mm) and washed. The cells were cultured using a modification of the method in our previous report [5]. Briefly, the thymocytes were suspended in ice-cold RPMI 1640 medium supplemented with 10 % fetal calf serum (Gibco, USA), 100 IU/ml of penicillin, and 100 mg/ ml of streptomycin. Viable cells were then counted using a trypan blue dye exclusion test, and were adjusted to 13106 cells/ml of culture. The thymocytes were then placed in 24-well, flat-bottom plates (Iwaki Glass Co., Japan) and incubated with 131029 z131024 M of GC for 6h in 5 % CO2 /air at 37 8C. GC was dissolved in ethanol, and the final culture ethanol concentration was adjusted to 1 %. Ethanol was also used in the control wells. Flow cytometric analysis Apoptotic cell detection and phenotype analysis were performed in the following ways using flow cytometry. (i) Cell cycle analysis: Cells were fixed for more than 30 min at 220 8C in 70 % ethanol at a density of 23106 cells/ml. After washing with PBS, 100 ml of cell suspension was treated with 2 ml of 10 mg/ml RNase A in PBS at 37 8C for 30 min. To assess

T. Hirano et al. / Life Sciences 68 (2001) 2905–2916

2907

DNA fragmentation, the cells were then stained with 0.2 mg/ml of propidium iodide (PI) in PBS for 30 min. The percentage of apoptotic cells corresponding to the amount of fragmented DNA in the hypoploid sub-G0/G1 cell cycle peak was then calculated. (ii) Detection of mitochondria specific dye and T cell subtype: Disruption of mitochondrial transmembrane is known to occur in apoptotic cells [8], and such cells are not stained with chloromethyl Xarosamine (CMXRos). One million cells were stained with CMXRos (Molecular Probes, Eugene, USA) at 37 8C for 30 min. CMXRos (1 mM) was prepared as a stock solution in dimethylsulfoxide and stocked at 220 8C. The stock solution was then diluted in a 100 nM working solution with PBS. After CMXRos staining, the cells were washed in an ice-cold PBS solution supplemented with 2% heat-inactivated FBS, and incubated for 20 min at 4 8C with a phycoerythrin-conjugated anti-CD4 monoclonal antibody (0.1 ng/106 cells in 100 ml; PharMingen, Co., USA) and with FITC-conjugated anti-CD8 monoclonal antibody (0.1 ng/ 106 cells in 100 ml; PharMingen). (iii) Detection of cell surface phosphatidylserine: Increase in phosphatidylserine exposure to the outer leaflet of the plasma membrane in apoptotic cells has been reported [9]. One million cells were stained with an FITC-conjugated antiphosphatidylserine monoclonal antibody, Annexin V (Molecular Probes, Eugene, USA) at room temperature for 15 min. After staining, a total of 20,000 non-gated cells were analyzed using a FACSCalibur analyzer and CellQuest software (Becton Dickinson, Mountain View, USA). Agarose gel electrophoresis of fragmented DNA The extraction of thymocyte DNA and gel electrophoresis were performed with the techniques similar to those of previous report [10]. In brief, approximately 1 3 106 cells were solubilized with 100 ml of lysis buffer (10 mM Tris-hydrochloride buffer, pH 8.0, containing 0.1 mM EDTA and 150 mM sodium chloride) and placed on ice for 10 min. The sample was then centrifuged at 20,0003g for 20 min. The supernatant was placed in an 1.5 ml microtube, and was treated with 2 ml of 10 mg/ml RNase A at 37 8C for 30 min, followed by treatment with 2 ml of 50 mg/ml proteinase K at 37 8C for 60 min. The sample was then extracted with an equal volume of phenol-chloroform using DNA isolating-gel (Iwaki Glass, Japan) and was precipitated by adding of 10 times the volume of 3 M sodium acetate and an equal volume of ethanol at 220 8C. After the ethanol evaporation, the DNA was resuspended in a TE buffer (10 mM Tris-hydrochloride, pH 8.0, containing 1 mM EDTA). The DNA samples (10 ml/lane) were electrophoretically separated on 2.0 % agarose gels containing 0.5 mg/ml ethidium bromide (Sigma, USA) using a Mupid-2 (Advance Co., Japan). The DNA was then visualized with an UV transilluminator. Quantification of fragmented DNA Fragmented DNA was measured with an ELISA cell death detection kit, as performed in our previous study [11]. In brief, thymocytes cultured as described above were washed with culture medium and incubated with an incubation buffer to be lysed at 48C for 30 min. The lysates were centrifuged at 20,0003g for 10 min and supernatants were collected. The supernatants were diluted to 104 cell equivalents/ml with incubation buffer and stored at 270 8C until the assay. Specific enrichment of mono- and oligo-nucleosomes released into the cytoplasm were determined by ELISA (Boeringer-Manheim, Germany). Diluted supernatant was

2908

T. Hirano et al. / Life Sciences 68 (2001) 2905–2916

then transferred to an anti-histone-precorted microtiter plate and incubated for 90 min at room temperature. After washing with incubation buffer, anti-DNA conjugated with peroxidase solution was added and incubated for 90 min at room temperature. After washing with incubation buffer, a solution of ABTS (2,29-amino-di-[3-ethyl]benzthiazoline sulfonate) as the substrate was added and the mixture was incubated for 20 min at room temperature, then absorbance was measured at 405 nm. Specific enrichment of DNA fragmentation was expressed as the enrichment factor which was obtained using the following equation: absorbance of test culture/absorbance of control culture. Statistical analysis We used Bonferoni/Dunn multiple analysis to compare means. Values are reported as mean (SD) unless otherwise mentioned. These analysis were performed with Statview [12] on a Macintosh Power PC computer (Apple Computer, USA). In each case, two-sided p values less than 0.05 were considered to be significant. Results Cellular and biochemical apoptotic markers in thymocytes Physiological mouse glucocorticoid, corticosterone, induced features of apoptosis in thymocytes at concentrations ranging from 0.1 to 100 mM after 6 h of treatment in vitro. Fig.1 shows dose-dependent inductions of Annexin V positive cells (Fig.1A), CMXRos negative cells (Fig.1B), and DNA fragmentation (Fig.1C) by corticosterone in thymocytes of C57BL/ 6 mice. These results showed that maximal apoptosis induction with corticosterone occurred at concentrations higher than 1 mM. Accordingly, we examined the effects of OGCs against the apoptosis-inducing action of this concentration (1 mM) of corticosterone. Fig.2 shows the attenuating effects of physiological OGCs, dehydrocorticosterone and cortisone, and the synthetic OGC prednisone on corticosterone-induced increases in percentages of Annexin V positive thymocytes (Fig.2A) and CMXRos negative thymocytes (Fig.2B). These OGCs by themselves alone did not affect expression of cell marker of apoptosis, whereas they significantly counteracted the action of 1 mM corticosterone at a concentration of 1 00 mM (p,0.006). Inhibitory effects of these OGCs were also detectable at 10 mM, but the effects became apparent at 100 mM. Thymocytes treated with 1 mM corticosterone for 6 h showed a typical DNA ladder formation as detected by agarose-gel electrophoresis, whereas the formation of the DNA ladder was attenuated by combination of 0.1–10 mM dehydrocorticosterone as shown in Fig.3. Fragmented DNAs in corticosterone-treated thymocytes were also quantified with a cell death ELISA (Fig.4). Corticosterone induced DNA-fragmentation dose-dependently as shown in Fig.1C, whereas this effect of corticosterone at 1 mM was significantly attenuated by co-culturing the cells with dehydrocorticosterone at 10 mM (Fig.4; p,0.05). Effects of glucocorticoid treatment on CD4/CD8 positive thymocytes To characterize thymocytes, which underwent apoptosis from corticosterone, we stained them with anti-CD4 and anti-CD8 monoclonal antibodies, and the numbers of CD4/CD8 dou-

T. Hirano et al. / Life Sciences 68 (2001) 2905–2916

2909

Fig. 1. Induction of cellular and biochemical features of apoptosis by corticosterone in murine thymocytes. (A) phosphatidylserine positive/PI negative cells; (B) CMXRos negative cells; (C) relative amount of fragmented DNA (enrichment factor). Each error bar indicates mean (SD) of 6 experiments.

ble positive-, double negative-, and single positive-cells were counted with flow cytometry as shown in Fig.5. Corticosterone (1 mM) treatment of thymocytes in vitro significantly decreased the total number of viable thymocytes and the number of CD41/CD81 (double positive) cells. Corticosterone-treatment of the cells in the presence of 100 mM dehydrocorticos-

2910

T. Hirano et al. / Life Sciences 68 (2001) 2905–2916

Fig. 2. Attenuation of corticosterone-induced apoptosis of thymocytes by OGCs. Thymocytes were treated with vehicle (control), 100 mM OGC (DB; dehydrocorticosterone, E; cortisone, PSN; prednisone) alone, 1 mM corticosterone (B) alone, or 1 mM corticosterone (B) plus 100 mM OGC. A; % of annexin V positive/PI negative cells, B; % of CMXRos negative cells. Error bars are mean (SD) of 6 experiments. * p,0.006, ** p,0.0004, *** p,0.0001.

terone resulted in significant attenuation of the effects of corticosterone on total number of thymocytes and the number of CD4/CD8 double positive cells (p,0.0001). Whereas, dehydrocorticosterone alone did not significantly affect total number of thymocytes and the number of CD4/CD8 double positive cells (Figs 5a,b). In addition, the numbers of double negative cells and single positive cells were not significantly affected by either corticosterone, dehydrocorticosterone, or both (Figs 5c–e). Other OGSs, cortisone and prednisolone, also exhibited significant effects on corticosterone-induced decreases in total number of thymocytes, while the effects of these OGCs on numbers of thymocyte subsets were not statistically significant. Effects of other HGC-metabolites In addition to OGC effects, we also examined the counteracting effects of some of the other physiological metabolites of cortisol: 6b-hydroxycortisone, 20a-dehydrocortisol, 20b-

T. Hirano et al. / Life Sciences 68 (2001) 2905–2916

2911

Fig. 3. Agarose-gel electrophoresis of DNA extracted from thymocytes treated with 1 mM corticosterone alone (lane 2), 1 mM corticosterone plus 0.1 mM dehydrocorticosterone (lane 3), 1 mM corticosterone plus 1 mM dehydrocorticosterone (lane 4), and 1 mM corticosterone plus 10 mM dehydrocorticosterone (lane 5). Lane 1; DNA from control (untreated) thymocytes, Lane 6; DNA size marker.

dehydrocortisol, tetrahydrocortisol, 5 b-dehydrocortisol, and 5 b-dehydrocortisone. The inhibitory effects of these metabolites at a concentration of 100 mM on corticosterone (1 mM)induced apoptosis as detected by staining the cells with anti-phosphatidylserine antibody (Annexin V) are indicated in Fig. 6. 20a-dehydrocortisol inhibited corticosterone-induced thymocyte apoptosis by 55 %. 5 b-Dehydrocortisone also showed moderate effect (inhibition

Fig. 4. Attenuation of corticosterone-induced DNA fragmentation of thymocytes by dehydrocorticosterone. Thymocytes were incubated in the presence of 1 mM corticosterone alone or 1 mM corticosterone plus dehydrocorticosterone, and the fragmented DNA were analyzed with a cell death ELISA. Error bars indicate means (SD) of 6 experiments.

2912

T. Hirano et al. / Life Sciences 68 (2001) 2905–2916

Fig. 5. Effects of corticosterone (B), OGCs, and their combinations on numbers of total viable thymocytes (a), CD41/CD81 cells (b), CD42/CD82 cells (c), CD41/CD82 cells (d), and CD42/CD81 cells (e). DB; dehydrocorticosterone, E; cortisone, PSN; prednisone * p,0.002, ** p,0.001, *** p,0.0001.

T. Hirano et al. / Life Sciences 68 (2001) 2905–2916

2913

Fig. 6. Inhibition of corticosterone-induced thymocyte apoptosis by physiological cortisol metabolites. Thymocytes were incubated in the presence of 1 mM corticosterone plus 100 mM of each metabolite, and the Annexin V positive/PI negative cells were analyzed. Data were expressed as % inhibition against corticosterone-induced apoptosis. 6b-OHE; 6b-hydroxycortisone, 20a-DHF; 20a-dehydrocortisol, 20b-DHF; 20b-dehydrocortisol, THF; tetrahydrocortisol, 5 b-DHF; 5 b-dehydrocortisol, 5 b-DHE; 5 b-dehydrocortisone.

of 27 %) against corticosterone-induced apoptosis, while the other four metabolites examined in this study did not significantly affect corticosterone-induced apoptosis of thymocytes. Discussion The results described above raised the possibility that OGCs can regulate apoptotic cell death of immature double-positive thymocytes induced by HGCs. In addition, our data suggested that other physiological metabolites of cortisol, for instance 20a-dehydrocortisol, may also possess protective effects on thymocytes against HGC-induced apoptosis. Thymocyte apoptosis was demonstrated by several cellular and biochemical observations: (i) an increase in phospholipid phosphatidylserine exposure to the outer leaflet of the plasma membrane [9], (ii) disruption of mitochondrial trans-membrane potential [8], and (iii) an increase in DNA fragmentation as detected by agarose gel electrophoresis and cell death ELISA [11,13]. Corticosterone-treatment of thymocytes in vitro gave all of these cellular and biochemical features of apoptosis, while dehydrocorticosterone significantly attenuated these signs of apoptosis induced by corticosterone. Immature thymocytes which undergo apoptosis induced by HGCs are considered to be CD4/CD8 double positive cells, as has been previously demonstrated by several reports [14,15]. Our present data of thymocyte-subset analysis are consistent with these previous observations, and show that corticosterone decreased thymocytes mainly through induction of apoptosis in CD4/CD8 double positive cells (Fig. 5). Dehydrocorticosterone protected this effect of corticosterone resulting in a recovery in the number of CD4/CD8 double positive cells, while the steroid did not affect the numbers of any other subsets of thymocytes. Thus, our

2914

T. Hirano et al. / Life Sciences 68 (2001) 2905–2916

results suggest that OGCs may have a role in regulating apoptotic cell death of immature T cells by counteracting the apoptosis-inducing effects of HGCs on double positive thymocytes. Cortisone and prednisone also showed significant apoptosis inhibitory activity, though prednisone appeared to be less effective as compared to the effects of other OGCs (Fig.2B). Prednisone has been reported to show apoptosis-inducing, rather than inhibiting, effects on mitogen-stimulated human peripheral-blood mononuclear cells [16]. Thus, prednisone’s effects on immune cell apoptosis appears to be complex, which may result from enzymatic conversion of prednisone to its active HGC form prednisolone in certain tissues by a reductive action of 11b-HSD. The backgrounds of the assays presently used are sometimes high in certain experimental conditions, especially when the apoptotic cells were estimated with CMXRos-staining procedures. We could not clearly explain the reason for these high backgrounds, but it could be possible to consider that in vitro incubation of thymocytes in the culture medium, even in the absence of GCs, caused spontaneous apoptosis in these cells. Moreover, CMXRos-negative cells include both apoptotic and necrotic cells, but CMXRos stains only living cells. Thus the necrotic cells might increase the backgrounds in this assay. Some previous reports [1,17] showed that concentration of dexamethasone which cause maximum apoptosis in murine thymocytes in vitro was 0.1–1 mM, which was 10–100 times lower than that of GC we used to obtain maximum apoptosis in the present study. However, generally used GC in these kinds of studies is dexamethasone, which is known to have GC potency of .20 times higher than the potency of corticosterone. Moreover, the time to reach maximum apoptosis in vitro was 12h in their study [1], which was 2 times more than the incubation time we used in our experiment to examine thymocyte apoptosis induced by corticosterone in vitro. Thus, in reference to these previous reports, 10 mM of corticosterone might be a reasonable concentration of the GC to obtain maximum apoptosis in mouse thymocytes in vitro in our experimental conditions. In vitro concentrations of the OGCs we used to obtain significant counteractive effects are 10–100 times higher than the concentration of corticosterone needed to induce apoptosis in thymocytes, and thus these OGC concentrations appear to be supraphysiological. However, it could be possible to consider that OGC levels might increase and the products may accumulate in local tissues or cells harboring 11b-HSD which converts HGCs to their corresponding oxidized metabolites OGCs. Indeed, we and other researchers have reported that thymus contains considerable activity of this type of enzyme [5,6]. High activity of the enzymes which convert HGCs to OGCs is also demonstrated in human fetus thymus [18]. We have shown in our previous report [5] that the thymocytes remaining after induction of apoptosis by systemically elevated levels of HGC expressed a relatively high level of 11b-HSD activity. This means that the higher the cellular 11b-HSD activity, the stronger the resistance of the cells to the apoptosis-inducing action of HGC. Moreover, pharmacological intervention of 11b-HSD has been reported to result in an increase of HGC levels and subsequent induction of apoptosis in thymocytes [5]. 11b-HSD may regulate thymocyte apoptosis by acting not only as an inactivator of HGCs, but also as a producer of the counteracting hormone OGCs. Suppression of enzymatic activity of 11b-HSD by inhibitors such as glycyyrhetinic acid, as we described previously [5], may result in promotion of thymocyte apoptosis by not only accumulating HGCs but also suppressing production of the counteracting hormone OGCs. Chronic

T. Hirano et al. / Life Sciences 68 (2001) 2905–2916

2915

stress has been reported to induce elevation in serum corticosterone level and reduction of rat thymus weight, while it did not have an influence on 11b-HSD activity in thymus [19]. Since 11b-HSD activity did not change after chronic stress [19], the relative increase in HGC level as compared to the OGC level in the thymus microenvironment after stress might efficiently cause apoptosis of thymus under these experimental conditions. The mechanism for the counteracting action of OGCs against the apoptosis-inducing effect of HGCs is unknown, but it is likely that OGCs intervene in the binding of HGCs to their cellular GC-receptors (GC-Rs) in thymocytes. In a general sense, oxidized metabolites of HGCs are inactive as GCs, and are believed to have little capacity to bind and activate or inhibit GC-Rs. However, a replacement assay of [3H]dexamethasone binding to a cytosol fraction prepared from human thymus hyperplasia suggested that cortisone, an 11-oxo metabolite of cortisol, possess considerable binding capacity to GC-R [20]. Another GC-binding study using carp blood leukocytes also suggested that, although the binding capacity of cortisone to GC-R was several times lower than that of cortisol, cortisone possesses the activity as a ligand for GC-R [21]. On the other hand, expression of phospholipid phosphatidylserine to the outer leaflet of the plasma membrane and disruption of the mitochondria trans-membrane potential are considered to be relatively early events in cell apoptosis [8,22,23]. We found in the present study that OGCs attenuated these signs of apoptosis induced by HGC, suggesting that OGCs might prevent relatively early apoptosis events. All of these observations support the counteracting mechanism of OGC against HGC-induced apoptosis of thymocytes by intervening with GC-R in thymocytes. The results, in summary, showed that OGCs have the pharmacological effect of attenuating the action of corticosterone on CD4 and CD8 double positive thymocytes to induce apoptosis. From these observations, it could be possible to consider that some HGC metabolites may implicate in the thymocyte selection process in thymus by intervening in HGC action.

References 1. Cohen JJ, Duke RC. Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. Journal of Immunology 1984;132(1):38–42. 2. Wyllie AH. Glucocorticoid –induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 1980;284:555–6. 3. Walker BR. Organ-specific action of 11b-hydroxysteroid dehydrogenase in humans: implications for the pathophysiology of hypertension. Steroids 1994;59:84–9. 4. Funder JW, Pearce PT, Smith R, Smith L. Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science 1988;242: 583–5. 5. Horigome H, Horigome A, Homma M, Hirano T, Oka K. Glycyrrhetinic acid induced apoptosis in thymocytes: impact of 11b-hydroxysteroid dehydrogenase inhibition. American Journal of Physiology 1999;277:E624–30. 6. Dougherty TF, Berliner ML, Berliner DL. 11b-Hydroxy dehydrogenase system activity in thymi of mice following prolonged cortisol treatment. Endocrinology 1960:66:550–8. 7. Ohyama K, Oka K, Emura A, Tamura H, Suga T, Bessho T, Hirakawa S, Yamakawa T. Suppression of apoptotic cell death progressed in vitro with incubation of the chorion laeve tissues of human fetal membrane by glucocorticoid. Biological and Pharmaceutical Bulletin 1998:21(10); 1024–9. 8. Castedo M, Hirsh T, Susin SA, Zamzami N, Marchetti P, Macho A, Kromer G. Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis. Journal of Immunology 1996;157:512–21.

2916

T. Hirano et al. / Life Sciences 68 (2001) 2905–2916

9. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C, A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V. Journal of Immunological Method 1995;184:39–51. 10. Horigome A, Hirano T, and Oka K. Tacrolimus-induced apoptosis and its prevention by interleukins in mitogenactivated human peripheral mononuclear cells. Immunopharmacology 1998;39:21–30. 11. Horigome A, Hirano T, Oka K, Takeuchi H, Sakurai E, Kozaki K, Matsuno N, Nagao T, Kozaki M. Glucocorticoids and cyclosporine induce apoptosis in mitogen-activated human peripheral mononuclear cells. Immunopharmacology 1997;37:87–94. 12. Haycock KA, Roth R, Gagnon J. Statview. Berkeley, Calif. Abacus Concepts 1994. 13. Ito M, Watanabe M, Ihara T, Kamiya H, Sakurai M. Fas antigen and bcl-2 expression on lymphocytes cultured with cytomegalovirus and varicella-zoster virus antigen. Cellular Immunology 1995;160:173–7. 14. Homo F, Duval D, Hatzfeld J, Evrard C. Glucocorticoid sensitive and resistant cell populations in the mouse thymocytes. Journal of Steroid Biochemistry 1980;13:135–43. 15. Jondal M, Okret S, McConkey D. Killing of immature CD41CD81 thymocytes in vivo by anti-CD3 or 59-(N-ethyl)-carboxamide adenosine is blocked by glucocorticoid receptor antagonist RU-486. European Journal of Immunology 1993;23:1246–50. 16. Lanza L, Schudeletti M, Puppo F, Bosco O, Peirano L, Filaci G, Fecararotta E, Vidali G, Indiveri F. Prednisone increases apoptosis in in vitro activated human peripheral blood T lymphocytes. Clinical and Experimental Immunology 1996;103:482–90. 17. Oldenburg NBE, Evans-Storms RB, Cidlowski JA. In vivo resistance to glucocorticoid-induced apoptosis in rat thymocytes with normal steroid receptor function in vitro. Endocrinology 1997;138(2):810–8. 18. Murphy BEP. Ontogeny of cortisol-cortisone interconversion in human tissues: a role for cortisone in human fetal development. Journal of Steroid Biochemistry 1981;14:811–7. 19. Jellinck PH, Dhabhar FS, Sakai RR, McEwen BS. Long-term corticosteroid treatment but not chronic stress affects 11b-hydroxysteroid dehydrogenase type I activity in rat brain and peripheral tissues. Journal of Steroid Biochemistry 1997;60(5):319–23. 20. Ranelletti FO, Carmignani M, Iacobelli S, Tonali P. Glucocorticoid-binding components in human thymus hyperplasia. Cancer Research 1978;38(3):516–20. 21. Weyts FA, Verburg-van-Kemenade BM, Flik G. Characterization of glucocorticoid receptors in peripheral blood leukocytes of Carp, Cyprinus carpio L. Gen Comp Endocrinology 1998;111(1):1–8. 22. Van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsperger CP. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 1998;31:1–9. 23. Zamzami N, Marchetti P, Castedo M, Zanin C, Vayssiere JL, Petit PX, Kroemer G. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. Journal of Experimental Medicine 1995;181:1661–72.