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Mechanism of androgen-induced thymic atrophy in the wall lizard, Hemidactylus flaviviridis: An in vitro study
General and Comparative Endocrinology 144 (2005) 10–19 www.elsevier.com/locate/ygcen
Mechanism of androgen-induced thymic atrophy in the wall lizard, Hemidactylus Xaviviridis: An in vitro study B. Hareramadas, U. Rai ¤ Comparative Endocrinology Laboratory, Department of Zoology, University of Delhi (North Campus), Delhi 110 007, India Received 14 November 2004; revised 9 April 2005; accepted 13 April 2005 Available online 7 July 2005
Abstract The present in vitro study demonstrates the eVect of androgen on thymocyte apoptosis leading to thymic atrophy in the wall lizard, Hemidactylus Xaviviridis. Thymocytes collected from castrated lizards were incubated with varying concentrations of dihydrotestosterone (DHT) to observe its eVect on proliferation and apoptosis. DHT treatment reduced the tritiated thymidine incorporation in thymocytes, suggesting that androgen directly inhibits thymocyte proliferation. It also caused apoptosis of thymocytes eVectively at 10¡7 M. However, the increased apoptotic action of DHT was indirectly mediated through thymic epithelial cell-rich stromal cell components (TEC). This observation was reaYrmed by in vitro incubation of thymocytes with DHT-pretreated TEC-conditioned medium. However, the DHT-induced TEC-secreted apoptotic factors could induce thymocyte DNA fragmentation only when DHT was added to the conditioned medium. It implies that DHT priming of thymocytes is required for the apoptotic eVect of DHTinduced TEC-secreted factor. DHT-induced thymocyte apoptosis was found to be caspase-dependent since it activated the initiator (caspase-9) and eVector caspases (caspases-3 and -7) as well as cleaved the enzyme substrate poly(ADP-ribose) polymerase (PARP). Further, the apoptotic eVect of DHT was routed through its classical receptors, as non-steroidal antiandrogen Xutamide blocked the DHT-induced thymocyte apoptosis. The inhibition of apoptosis by transcription/translation inhibitors further substantiates the genomic pathway of DHT action. It can be concluded that DHT, in addition to inhibiting thymocyte proliferation directly, accelerates caspase-dependent apoptotic process in thymocytes indirectly through TEC via a genomic pathway. Nevertheless, the priming of thymocytes with DHT is required for the apoptotic eVect of TEC-secreted factor. 2005 Elsevier Inc. All rights reserved. Keywords: Androgen; Thymocyte proliferation; Apoptosis; Caspases; Lizard
1. Introduction The thymic–testicular axis has been extensively studied in mammals. Castration results in thymic enlargement, whereas androgen replacement causes reversal of this eVect (Grossman, 1984; Olsen and Kovacs, 2001; Olsen et al., 1998). The androgen-induced thymic atrophy might be due to alterations in various mechanisms including cell proliferation, cell traYcking, and/or cell death. Androgen has been reported to inhibit T cell line
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proliferation and increase apoptosis in a concentrationdependent manner (McMurray et al., 2001). This suggests that thymocytes are the potential targets for direct action of androgen. The localization of androgen receptors in thymocytes further supports this possibility (Kumar et al., 1995; Viselli et al., 1995). So far apoptosis is concerned, Nieto and Lopez-Rivas (1989) has reported that androgen has no eVect on murine T cell line apoptosis; while other in vivo and in vitro thymic organ culture experimental studies demonstrate acceleration in T cell apoptosis after androgen treatment (Dulos and Bagchus, 2001; Kumar et al., 1995; Olsen and Kovacs, 2001; Olsen et al., 1998). These results raised the possibility of the involvement of cells other
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than thymocytes in androgen-induced apoptosis. This line of thinking is reinforced by the Wndings that thymic epithelial cells possess androgen receptors (Grossman, 1984; Iwata et al., 1996; Kumar et al., 1995; Seiki and Sakabe, 1997). Further, the thymic epithelial cells are reported to be required for the apoptotic action of androgen on thymocytes (Dulos and Bagchus, 2001; Kumar et al., 1995; Olsen and Kovacs, 2001). In reptiles, the seasonal changes in thymic–testicular axis and thymic regression noted following androgen replacement therapy demonstrate a reciprocal relationship between androgen level and thymic growth/development (el Masri et al., 1995; Hareramadas and Rai, 2001; Muñoz and De la Fuente, 2001; Varas et al., 1991; Zapata et al., 1992). However, the immune cellular targets for androgen action have not been studied in reptiles, despite their phylogenic importance as ancestors to both birds and mammals. The present study, therefore, was undertaken to determine the direct and indirect eVects of androgen on thymocyte proliferation and apoptosis, and to delineate its pathway of action in thymic cells in the wall lizard. Also, the role of caspases in androgen-induced thymocyte apoptosis in wall lizards was studied since both caspase-dependent (Mann et al., 2000; Viselli and Barrett, 1999) and -independent (Kawahara et al., 1998) pathways of apoptosis are reported in thymocytes or T lymphoma cell line of mammals.
2. Materials and methods 2.1. Animals Adult male wall lizards (Hemidactylus Xaviviridis) weighing » 8 to 10 g were procured locally (Delhi:latitude: 28°12⬘–28°53⬘N, longitude: 76°50⬘–77°23⬘E) during regressed (May–August) and recrudescence (September– November) phases of the reproductive cycle. They were housed (12L:12D) in wooden cages with mesh wire at the top and fed live house Xies ad libitum. After 1 week of acclimation, lizards were castrated and kept for 7 days. Guidelines of Animal Ethics Committee, University of Delhi and Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Statistics, and Programme Implementation, Government of India, were followed in handling, maintenance, and sacriWce of animals. 2.2. Reagents and culture media Tissue culture medium RPMI-1640, dihydrotestosterone (DHT), Xutamide, acridine orange (AO), propidium iodide (PI), and DiBAC4(3) [bis-(1,3-dibarbituric acid)trimethine oxanol], RNase I-A, and concanavalin A (ConA) were obtained from Sigma Chemicals (St. Louis, MO, USA). Proteinase K and FITC-conjugated secondary
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antibody (FITC-anti IgG antibody) were procured from Bangalore Genei (Bangalore, India). All other chemicals were procured from SRL (New Delhi, India). RPMI-1640 was supplemented with 40 g/ml antibiotic gentamycin, 100 g/ml streptomycin, 100 IU/ml penicillin (Ranbaxy Ind., New Delhi, India), 50 M -mercaptoethanol, 25 mM Hepes buVer, pH 7.2 (Merck India, New Delhi), and 10% heat-inactivated fetal calf serum (FCS; Biological Industries, Beth Haemek, Israel) prior to use. Tritiated thymidine ([methyl-3H]TdR; 2.0 Ci/mmol) was purchased from Board of Radiation and Isotope Technology (BRIT:Hyderabad, India). Primary antibodies for active caspases (3, 7, and 9), and PARP and HRP-conjugated secondary antibody (HRP-anti-IgG antibody) were obtained from Cell Signaling Technology (Beverly, MA, USA). Stock solutions (1 M) of DHT and DiBAC4(3) were prepared in dimethylsulphoxide (DMSO) and further dilutions were made in RPMI-1640 prior to use. 2.3. Isolation of thymic cells For each set of experiments, 25–30 castrated lizards were sacriWced. The thymic lobes were dissected out under aseptic conditions and transferred into cold phosphate-buVered saline (PBS, pH 7.2) containing 2% heatinactivated FCS (PBS–FCS). Tissues were pooled and washed thoroughly with PBS to remove the bloodstains. 2.3.1. Thymocytes Thymic lobes were gently minced and passed through a nylon mesh of pore size 40 M into cold PBS–FCS. Thymocytes were isolated following the procedure of Hareramadas et al. (2004). The single cell suspension of thymocytes was washed with PBS–FCS and the cell pellet was resuspended in tissue culture medium RPMI-1640 supplemented with 10% heat-inactivated FCS, 50 M -mercaptoethanol, 2 mM glutamine, and 25 mM Hepes buVer. Cell viability was checked by trypan blue exclusion, and the Wnal cell density was adjusted to 5 £ 106 cells/ml. 2.3.2. Thymic epithelial cell-rich stromal cell suspension A crude preparation of thymic epithelial cell-rich stromal cell suspension was made following the method of Rosen et al. (1976). In brief, the minced tissue of thymic lobes left-over on nylon mesh during thymocyte isolation was collected and treated with 0.1% trypsin/ EDTA solution for 15 min on ice. After washing, the crude preparation of thymic epithelial cell-rich stromal cells (TEC) was used for further experiments. 2.4. EVect of DHT on thymocyte proliferation and apoptosis 2.4.1. Thymocyte proliferation Thymocytes were incubated with either 5 g/ml concanavalin A (Con-A) alone or Con-A with diVerent
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concentrations of DHT for 72 h. Cells incubated in medium alone served as control. The cultures were pulsed with 1 Ci/ml [3H]TdR 18 h prior to the completion of incubation. After 72 h, thymocytes were washed with PBS and lysed with 1% SDS. The radioactivity in the lysate was counted using a liquid scintillation counter (Beckman, USA) and proliferation was expressed in terms of counts per min (§SEM)/106 thymocytes. 2.4.2. Thymocyte apoptosis 2.4.2.1. Experiment I. Thymocytes, TEC, and thymocytes along with TEC were cultured separately either in the medium alone (controls) or with varying concentrations of DHT (10¡5–10¡13 M) for 24 h in a humidiWed chamber of 5% CO2 incubator. Similar experimental groups were also undertaken for immunocytochemistry. In case of thymocyte–TEC co-culture experimental groups, thymocytes were separated out from TEC mass following centrifugation (100 rpm) at 4 °C for 5 min. The supernatant containing thymocytes was then used for Xow cytometric analysis, Xuorescence microscopy, immunocytochemistry, and agarose gel electrophoresis. 2.4.2.1.1. DNA extraction and gel electrophoretic analysis. DNA was extracted following the method of Nicoletti et al. (1991) modiWed by Hareramadas et al. (2004). BrieXy, cells from various experimental groups were harvested, washed, and resuspended in 500 l of hypotonic lysis buVer containing 200 g/ml proteinase K. DNA was extracted with Tris-equilibrated phenol–chloroform and then with chloroform alone. The aqueous phase containing nucleic acids was treated with RNase I-A, extracted with phenol–chloroform, chloroform–isoamyl alcohol, and Wnally with chloroform. DNA present in aqueous phase was then precipitated with 0.5 M ammonium acetate and absolute ethanol at ¡20 °C for overnight. The precipitated DNA was recovered (20,000g at 4 °C for 30 min) and dissolved in Tris–EDTA buVer (10 mM Tris, pH 8.0, and 1 mM EDTA). DNA was quantiWed (260 nm) and resolved on 1.5% agarose gel. DNA molecular weight marker ( DNA/EcoRI + HindIII double digest; Bangalore Genei, Bangalore, India) was also run along with the samples and DNA was visualized under UV transilluminator after staining with ethidium bromide. 2.4.2.1.2. Flow cytometric analysis. DNA content. Cells harvested from various experimental groups were processed for Xow cytometric analysis of DNA content following the method of Nicoletti et al. (1997). BrieXy, cells were harvested and washed twice with PBS. The cell pellet was then Wxed in 70% (v/v) chilled ethanol (¡20 °C) for 24 h. After Wxation, cells were washed with PBS and resuspended in hypotonic Xuorochrome, propidium iodide solution (50 g/ml PI in 0.1% sodium citrate plus 0.1% Triton X-100), and incubated in dark for 30 min at room temperature. The PI Xuorescence of
individual nuclei was analyzed by Becton Dickinson‘FACSVantage SE’ Xow cytometer. DNA integrity was then determined by generating histograms of cell number versus DNA content (PI Xuorescence). Plasma membrane potential. It was analyzed with the anionic bis-oxanol Xuorescent dye, DiBAC4(3) [bis(1,3-dibarbituric acid)-trimethine oxanol]. It has been demonstrated that DiBAC4(3) serves as an indicator for plasma membrane potential in lymphoid cells (Mann and Cidlowski, 2001). During resting state, the dye is excluded from the lymphocytes, whereas it enters the cell upon membrane depolarization and can be detected with Xuorescence activated cell sorting (FACS) analysis by measuring the increase in DiBAC4(3) Xuorescence. For membrane potential analysis, cells of various experimental groups were harvested, washed Wrst with PBS, and then with staining buVer (0.06 M sodium phosphate buVer, pH 7.0, supplemented with 5 mM KCl, 130 mM NaCl, 1.3 mM CaCl2, 0.6 mM MgCl2, and 10 mM glucose) . The same buVer was used to dissolve the dye. Cells were then incubated with 150 nM DiBAC4(3) for 30 min at 37 °C. To exclude the cells that had lost membrane integrity, PI was added to a concentration of 10 g/ml. Then the cells were analyzed by FACS. FACS analysis. All Xuorescence measurements of individual cells were made with a Becton Dickenson (BD) ‘FACSVantage SE’ Xow cytometer (Becton Dickenson, Mountain View, CA, USA) equipped with an air-cooled argon ion laser. PI Xuorescence was measured on FL-3 (excitation: 488 nm, emission: 620 § 11 nm) to exclude non-viable cells. Cell size was monitored by alterations in the forward light scattering properties of the cells as described by Mann et al. (2000). DiBAC4(3) Xuorescence of viable cells was measured on FL-1 (excitation: 488 nm, emission: 530 nm) to determine the membrane potential. An increase in DiBAC4(3) indicated a decrease in plasma membrane potential. Data were recorded on an Apple’s Macintosh Power PC running on Mac OS 9 and interpreted using ‘Cell Quest’ software of BD. Cell debris was excluded from analysis by appropriately raising the forward scatter (FSC) threshold. A total of 10,000 cells were analyzed for each sample. 2.4.2.1.3. Fluorescence microscopy. Based on gel electrophoretic and Xow cytotometric analyses of the present study, and the range of physiological testosterone concentrations (10¡7–10¡5 M) reported in diVerent lacertilian species (Ando et al., 1990; Huf et al., 1987; Radder et al., 2001; Smith and John-Alder, 1999), 10¡7 M DHT was used for Xuorescence microscopic study. Propidium iodide staining. Thymocytes and TEC were cultured either separately or co-cultured with DHT for 24 h. For controls, thymocytes, TEC, and thymocytes + TEC were cultured in the medium
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alone. After incubation, cells were washed with PBS; pellet was resuspended in a solution of 90% methanol and 10% propidium iodide, and kept for 10 min in the dark at room temperature. The cells were washed with PBS and resuspended in PBS-glycerine (1:1) solution. Then they were examined under ‘Nikon TE2000-U’ Xuorescence inverted microscope. Apoptotic cells were identiWed by their smaller, condensed, and intensely stained nuclei as compared to normal cells. Acridine orange staining for DNA strand breaks. Acridine orange (AO) is a metachromatic dye which diVerentially stains double-stranded (ds) and single-stranded (ss) nucleic acids. AO after intercalating into dsDNA emits green Xuorescence upon excitation at 480–490 nm. On the contrary, it emits red Xuorescence while interacting with ssDNA or RNA. Chromatin condensation is an early event of apoptosis and the condensed chromatin is much more sensitive to DNA denaturation than normal chromatin. Therefore, if RNA is removed by treating with RNase I-A and DNA is denatured in situ by exposure to HCl shortly before AO staining, apoptotic cells display an intense red Xuorescence, and a reduced green emission when compared to non-apoptotic interphase cells. AO staining was done following the method of Nicoletti and Mannucci (1997). In brief, cells at diVerent time intervals of incubation (6, 12, and 24 h) were taken out, washed, and Wxed with 1% paraformaldehyde in PBS on ice for 15 min. Fixed cells were washed and permeated with cold 70% ethanol for 4 h on ice. After permeation, cells were washed and incubated in RNAse I-A solution for 30 min at 37 °C. Cells were then resuspended in PBS and 0.1 M HCl was added to denature DNA. After 30–45 s, AO staining solution (6 g AO/ml in 0.1 M citric acid + 0.2 M Na2HPO4, pH 2.6) was added and kept for 10 min. Cells were observed under ‘Nikon E-400’ Xuorescence microscope. 2.4.2.1.4. Caspases-3, -7, and -9 (Immunocytochemistry). To understand whether the DHT-induced apoptotic pathway is caspase-dependent or -independent, active caspases-3, -7, and -9, and cleaved poly(ADP-ribose) polymerase (PARP) were immunocytochemically identiWed using antibodies. Thymocytes, TEC, and thymocytes + TEC incubated in medium alone (control) or with DHT were washed and Wxed with 3% paraformaldehyde. After Wxation, cells were washed thrice with Tris-buVered saline (TBS) containing 0.1% Triton X-100 (TBS/T), non-speciWc binding was blocked with 5% normal goat serum in TBS/T, and then incubated with primary antibodies for 24 h at 4 °C. Unbound antibodies were removed by washing with TBS/T, and cells were then incubated either with Xuorescein isothiocyanate (FITC)-conjugated or horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit IgG-FITC; and goat antirabbit IgG-HRP) for 2 h at room temperature. In case of HRP-conjugated secondary antibody incubation,
13
cells were pretreated with H2O2 to block endogenous peroxidase activity. Subsequently, cells were stained with 3-3⬘-diaminobenzidine tetrahydrochloride (DAB) solution, washed, and observed under ‘Nikon TE2000-U’ Xuorescence inverted microscope. Negative (without primary and/or secondary antibody) as well as positive (thymocytes treated with 10¡14 M corticosterone known to induce apoptosis, Hareramadas et al., 2004) controls were also maintained. 2.4.2.1.5. Microscopy. The Xuorescence observations were accomplished using a blue Wlter set (excitation: 450–490 nm, emission: 505 nm, and dichroic barrier Wlter: 520 nm). Images of PI stained cells, and caspases-3, -7, and -9 as well as cleaved PARP-positive (FITC Xuorescence) cells were captured with ‘Nikon Digital Sight DS-5M-L1’ digital camera equipped with 1⬙ relay lens and a 2/3⬙ CCD chip, whereas ‘Nikon Coolpix 4500’ digital camera equipped with 1⬙ relay lens and a 1/2⬙ CCD chip was used for the photography of AO stained cells. 2.4.2.2. Experiment II 2.4.2.2.1. EVect of DHT-pretreated TEC-conditioned medium on thymocyte apoptosis. TEC were pretreated with 10¡7 M DHT for 24 h, washed, and then incubated in medium alone for 24 h. The conditioned medium was collected and its diVerent dilutions were made (conditioned medium:fresh medium, 3:1, 1:1, and 1:3) to see its concentration-dependent apoptotic eVect on thymocytes following DNA gel electrophoresis. Untreated TEC-conditioned medium served as control. Further, thymocytes were incubated in the pre-treated TECconditioned medium with or without DHT to examine whether the DHT-induced TEC-secreted factor alone is capable to cause thymocyte apoptosis or the presence of DHT is also required. 2.4.2.3. Experiment III 2.4.2.3.1. EVect of Xutamide, an antiandrogen. To delineate the action of DHT on thymic cells of wall lizards, thymocytes, TEC, and thymocytes + TEC were Wrst incubated with anti-androgen Xutamide (10¡9 M) for 24 h, washed, and then incubated with varying concentrations of DHT for 24 h. After incubation, cells were processed for DNA gel electrophoretic and Xow cytometric analyses. 2.4.2.3.2. EVect of actinomycin D and cycloheximide. To understand the genomic action of DHT leading to de novo synthesis of proteins responsible for thymocyte apoptosis, thymocytes, and TEC were incubated separately as well as in combination with 10¡7 M DHT and 3 g/ml of actinomycin D (transcription inhibitor) or 10¡5 M cycloheximide (translation inhibitor) for 24 h in a humidiWed chamber of 5% CO2 incubator. Controls were incubated in medium alone or with actinomycin D/cycloheximide. After incubations, cells were processed for DNA gel electrophoresis.
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2.5. Statistical analysis All experiments were carried out in triplicate. In vitro dose-related eVect of DHT on thymocyte proliferation and apoptosis of thymic cells by Xow cytometric study was analyzed by one-way analysis of variance (ANOVA) followed by Newman–Keuls’ multiple range test. The results are based on the data of one of the repeated experiments.
3. Results 3.1. EVect of DHT on thymocyte proliferation and apoptosis 3.1.1. Thymocyte proliferation Con-A signiWcantly enhanced the uptake of [3H]TdR by thymocytes as compared to the control (Fig. 1, error bars with superscripts a vs. b, p < 0.01). However, DHT when supplemented with Con-A, signiWcantly reduced the mitogen-induced thymocyte proliferation, maximum inhibition occurred at 10¡5 M (error bars with superscripts b vs. c and d, p < 0.01). 3.1.2. Thymocyte apoptosis 3.1.2.1. Experiment I. DNA fragmentation and FACS analysis. Thymocytes incubated with DHT for 24 h showed no DNA fragmentation at any of the concentrations ranging from 10¡13 to 10¡5 M (Fig. 2, lanes 2–6)
Fig. 1. Histogram showing in vitro eVect of varying concentrations of dihydrotestosterone (DHT) on Con-A-induced thymocyte proliferation. Tritiated thymidine ([3H]TdR) incorporation is expressed as ‘mean counts per minute (CPM) § SEM’ (one-way analysis of variance (ANOVA) followed by Newman–Keuls’ multiple range test, p < 0.01). Data (means § SEM) represent one of the repeated experiments (N D 3). The error bars with diVerent superscripts (a–d) diVer signiWcantly.
Fig. 2. Agarose gel electrophoresis of thymocyte DNA. Thymocytes either alone or along with TEC were treated with diVerent concentrations of DHT (10¡5–10¡13 M) for 24 h. Lanes M, Marker ( DNA double digest); 1, thymocytes in medium alone (control), 2–6, thymocytes incubated with varying concentrations of DHT (10¡5, 10¡7, 10¡9, 10¡11, and 10¡13 M, respectively); 7, TEC in medium alone; 8, thymocytes + TEC in medium alone; 9–13, thymocytes + TEC incubated with 10¡5, 10¡7, 10¡9, 10¡11, and 10¡13 M of DHT, respectively. DHT-induced DNA laddering can be seen in thymocytes when incubated in the presence of TEC (lanes 9 and 10), though DNA fragmentation was more pronounced at 10¡7 M (lane 10). Results shown is one of the representative experiment of the experiments performed Wve times (N D 5).
and was found comparable to the control (lane 1), suggesting that DHT did not have any direct eVect on thymocyte apoptosis. Similarly, no apoptotic eVect of DHT was seen on TEC. Interestingly, DHT-induced apoptosis in thymocytes when they were co-cultured with TEC, and greater amount of fragmented DNA was seen at 10¡7 M concentration (lane 10), though TEC did not show any DNA laddering (lane 7). This indicates that DHT indirectly, through TEC, could induce thymocyte apoptosis. This indirect apoptotic eVect of DHT on thymocytes via TEC was also conWrmed by FACS analysis of ethanol-Wxed PI-stained cells. Like the control, the representative PI histograms of DHT treated thymocytes (without TEC) for 24 h showed a normal cell cycle distribution with a few hypodiploid cells (Fig. 3A). However, the hypodiploid DNA peak in response to DHT treatment was greatly enhanced when thymocytes were co-cultured with TEC for 24 h. DHT-induced hypodiploid cell percentage increased with orderly increase in concentration and reached its maximum at 10¡7 M (Fig. 3A); though DNA laddering was not seen following gel electrophoresis at low concentrations of DHT (Fig. 2), possibly due to DNA fragmentation appearing at later stages of apoptosis. 3.1.2.1.1. Membrane potential. After DHT treatment of thymocytes, the population of unWxed cells exhibiting DiBAC4(3) Xuorescence was comparable to that of the control, indicating that a large population of cells excluded the dye, since DHT failed to aVect the membrane potential of thymocytes. In contrast, in the
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3.1.2.1.3. Caspases-3,-7, and -9 (Immunocytochemistry). Immunocytochemical examination of in vitro eVect of DHT on caspases in thymocytes cultured alone or with TEC showed that DHT-treated thymocytes were positive to caspases-3, -7, and -9 and PARP (caspase substrate) only when they were co-cultured with TEC (Fig. 6). The maximum number of caspase-positive cells were seen at 10¡7 M DHT. It appears that DHT through TEC activates the cascade of caspases in thymocytes which lead to their apoptosis.
Fig. 3. (A) DNA Xuorescence Xow cytometric proWle of ethanolWxed PI-stained thymocytes treated with diVerent doses of DHT 10 ¡5–10 ¡13 M in the absence or presence of TEC for 24 h. For controls, thymocytes alone or with TEC were incubated in medium alone. Data suggest the indirect apoptotic eVect of DHT through TEC on thymocytes. Maximum percentage hypodiploid thymocytes were noted at 10¡7 M DHT. Result presented here is one of the representative experiments repeated Wve times (N D 5). (B) Plasma membrane depolarization of thymocytes treated with varying concentrations of DHT in the absence or presence of TEC. Percentage DiBAC4(3)-positive apoptotic thymocytes showing their loss of membrane potential was assessed by Xow cytometry. Experiment was repeated thrice (N D 3). Data represent one of the repeated experiments.
presence of TEC, DHT increased the population of DiBAC4(3) Xuorescence-positive thymocytes (Fig. 3B). This further substantiates that androgen accelerates thymocytes apoptosis indirectly through TEC. 3.1.2.1.2. Fluorescence microscopy. The PI-stained nuclei of thymocytes treated with 10¡7 M DHT were normal and comparable to the control (Fig. 4A); whereas smaller, condensed, and intensely stained nuclei of DHT-treated thymocytes were seen when they were co-cultured with TEC (Fig. 4B). Similar observations were noted following AO staining after DNA denaturation. Emission of green Xuorescence from nuclei of DHT-treated thymocytes was comparable to those incubated in medium alone (control, Fig. 5A). But, TEC when co-cultured with thymocytes in medium containing DHT for diVerent time intervals, i.e., 6, 12, and 24 h, resulted in decrease of green Xuorescence emission from thymocytes and increased red Xuorescence showing signs of apoptosis gradually with an increase of time duration from 6 to 24 h (Figs. 5B–D). This suggests that DHT through TEC induces lizard thymocyte cell death.
3.1.2.2. Experiment II. EVect of TEC-conditioned medium on thymocyte apoptosis. Thymocytes incubated either in medium alone or with diVerent dilutions of DHT-pretreated TEC-conditioned medium (conditioned medium:fresh medium::3:1, 1:1, and 1:3) did not exhibit DNA fragmentation as evident by gel electrophoretic analysis (Fig. 7). However, thymocytes DNA fragmentation indicating apoptosis was noted when DHT was supplemented to the DHT-pretreated TEC-conditioned medium, and the eVect decreased with the increase in dilution of DHT-pretreated TEC-conditioned medium (Fig. 7). This suggests that priming of thymocytes with DHT is required for the apoptotic action of DHTinduced TEC-secreted factor. 3.1.2.3. Experiment III. EVect of antiandrogen. Gel electrophoresis for DNA fragmentation and Xow cytometric analysis of diploid/hypodiploid cell distribution showed that antiandrogen Xutamide (10¡9 M) did not have apoptotic eVect on thymocytes/TEC alone or co-culture of thymocytes and TEC. However, pre-treatment of thymocytes+ TEC with Xutamide blocked the apoptotic eVect of DHT as no DNA laddering was seen in Xutamide pretreated group, and Xow cytometric data were comparable to that of control (thymocytes + TEC in medium alone) (Fig. 8). This shows that the androgen apoptotic action is mediated through its classical receptors in thymic cells. 3.1.2.3.1. EVect of transcription and translation inhibitors. Actinomycin D and cycloheximide, transcription and translation inhibitors, respectively, blocked the DHTinduced apoptosis in thymocytes co-cultured with TEC. This suggests the genomic action of DHT in inducing thymocyte apoptosis, indirectly through TEC (data not shown).
4. Discussion In the present in vitro study, DHT profoundly inhibited the mitogen-induced uptake of [3H]TdR by thymocytes, suggesting that androgen directly attenuates thymocyte proliferation in wall lizards. The inhibitory eVect of androgen on thymocyte proliferation has also been reported in turtles, Mauremys caspica (Muñoz and De la Fuente, 2001), and Chelonia mydas (Work et al., 2000). In mammals, androgen also induces thymocyte cell death
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Fig. 4. Fluorescence photomicrographs of propidium iodide (PI) stained thymocytes treated with 10¡7 M of DHT for 24 h in the presence of TEC. (A) Thymocytes in medium alone (control); (B) DHTinduced apoptotic thymocytes showing condensation (long arrow), fragmentation (arrow head), and fragmented DNA (short arrow). The experiment was repeated three times (N D 3) to verify the consistency of results (original magniWcation:1560£).
(Olsen et al., 1998), besides inhibiting proliferation (Bilbo and Nelson, 2001; McMurray et al., 2001). Using Jurkat T cell line (human acute T cell leukemia), testosterone was shown to increase thymocyte apoptosis in a concentrationand time-dependent manner (McMurray et al., 2001), thereby indicating the direct eVect of androgen on thymocyte apoptosis. On the contrary, androgen has no apoptotic eVect on murine T cell line, CTLL-2 (Nieto and LopezRivas, 1989). Moreover, administration of androgen in castrated mice or in vitro thymic organ culture with androgen has been reported to cause thymocyte apoptosis (Olsen et al., 1998). This indicates that cells other than thymocytes are involved in mediating the apoptotic eVect of androgen on murine thymocytes.
Fig. 5. Acridine orange (AO)-stained thymocytes treated with DHT (10¡7 M) for diVerent time intervals (0, 6, 12, and 24 h) in the presence of TEC. (A) Emission of green Xuorescence by non-apoptotic thymocytes incubated in medium alone for 24 h (control); (B–D) exhibit decrease in green Xuorescence and increase in red Xuorescence, indicating their transformation from non-apoptotic to apoptotic cells following 6, 12, and 24 h DHT treatment, respectively. The experiment was repeated three times (N D 3) to verify the consistency of results (original magniWcation: 1080£).
The presence of androgen receptors in thymic epithelial cells (Kumar et al., 1995; Olsen and Kovacs, 2001; Olsen et al., 2001) suggests their role in thymocyte apoptosis as thymic epithelial cell network is known to provide microenvironment for thymocyte maturation. Further, androgen-induced TGF- secretion from thymic epithelial cells has been implicated crucial for the activation of CPP32/caspase-3 which, in turn, causes thymocyte apoptosis (Hadden, 1998; Olsen et al., 1993; Viselli and Barrett, 1999). In view of these Wndings, it has been proposed that androgen also lead to indirect eVect on thymocyte apoptosis, through TEC (Olsen and Kovacs, 2001).
Fig. 6. Immunocytochemical demonstration of active caspases and cleaved enzyme substrate PARP in apoptotic thymocytes following DHT (10¡7 M) treatment in the presence of TEC. For caspase-3 and PARP, cells after challenging with primary antibodies were incubated with FITC-conjugated secondary antibody and counter stained with PI (A) i–iv. Control nuclei were stained bright red (caspase-3: (A) i; PARP: (A) iii, whereas condensed apoptotic cells showed yellowish-green Xuorescence due to the overlapping of FITC and PI signals (A) ii and iv for active caspase-3 and cleaved PARP, respectively). For caspases-7 and -9, after counter challenging with HRP-conjugated secondary antibody, thymocytes were stained with DAB. (B) vi and viii showed active caspases-7 (control, (B) v) and -9 (control, (B) vii), respectively. Figures represent one of the repeated experiments (N D 5) (original magniWcation:1560£).
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Fig. 7. EVect of DHT-pretreated TEC-conditioned medium on DNA fragmentation of thymocytes incubated with or without DHT. M, marker; lane 1, thymocytes + TEC in medium alone; 2, thymocytes + TEC + DHT 10¡7 M; 3–5, thymocytes + untreated TECconditioned medium (dilutions: 25:75, 50:50, and 75:25%); 6–8, thymocytes + DHT 10¡7 M + diVerent dilutions of untreated TEC-conditioned medium; 9–11, thymocytes + diVerent dilutions of DHT-pretreated TEC-conditioned medium (25:75, 50:50, and 75:25%); 12–14, thymocytes + DHT 10¡7 M + diVerent dilutions of DHT-pretreated TEC-conditioned medium. DNA laddering in response to DHTinduced TEC-secreted factor can be seen in thymocytes only when the conditioned medium was supplemented with DHT (lanes 12–14). Data presented are one of the representative experiments (N D 3).
Fig. 8. EVect of antiandrogen, Xutamide on DHT-induced thymocyte apoptosis. Lane M, marker; 1, thymocytes + TEC in medium; 2, thymocytes + TEC + Xutamide; 3, thymocytes + TEC + DHT 10¡5 M; 4, thymocytes + TEC + DHT 10¡7 M; 5, thymocytes + TEC + Xutamide + DHT 10¡5 M, and 6, thymocytes + TEC + Xutamide + DHT 10¡7 M. Data presented are one of the representative experiments (N D 3).
In wall lizards, unlike proliferation, DHT did not have any direct eVect on thymocyte cell death at any concentration ranging from 10¡13 to 10¡5, as evident by DNA gel electrophoresis, Xow cytometry as well as Xuorescence microscopic analyses. However, when lizard thymocytes were treated with DHT in the presence of TEC, membrane depolarization and loss of membrane potential, cell shrinkage, DNA condensation, and internucleosomal DNA fragmentation were observed. These
17
results suggest that in wall lizards DHT regulates thymocyte apoptosis, indirectly, through TEC-secreted paracrine factor. This conclusion is further substantiated by the Wndings that DHT-pretreated TEC-conditioned medium accelerated the thymocyte apoptosis. However, it may be noted that the DHT-pretreated TEC-conditioned medium was capable of inducing thymocyte apoptosis only when thymocytes were exposed to DHT. Several studies using mammalian models have shown caspase-dependent pathway of thymocyte apoptosis (Mann et al., 2000; Opferman and Korsmeyer, 2003; Villa et al., 1997; Viselli and Barrett, 1999; Vugmeyster et al., 2002), though caspase-independent pathway of apoptosis has also been reported in embryonic stem cells (Joza et al., 2001) and other cell lines (Kawahara et al., 1998; Perfettini et al., 2002; Wu et al., 2002). Following caspasedependent pathway, cytochrome c released from mitochondria in response to death stimuli, activates caspase-9 (a key initiator caspase) which, in turn, activates downstream eVector caspases, caspases-3, -6, and -7, which are evolutionarily conserved (Samuilov et al., 2000). These caspases then cleave their preferential cellular substrates, PARP (a DNA repair enzyme cleaved by caspases-3 and 7) and lamin A (a component of nuclear lamina cleaved by caspase-6). Also, caspase cleavage of ICAD/DFF (inhibitor of caspase activated DNase/DNA fragmentation factor) releases the nuclease CAD, presumably allowing it to enter the nucleus and degrade genomic DNA (Villa et al., 1997). In this study, DHT was found able to activate caspases-9, -7, and -3 and subsequently cleave PARP in thymocytes only when they were co-cultured with TEC. This suggests that androgen-induced TECsecreted paracrine factor might activate thymocyte apoptosis following caspase-dependent pathway in wall lizards, as reported in mammals (Viselli and Barrett, 1999). Flutamide is a potent nonsteroidal antiandrogen that competitively binds with intracellular nuclear/cytosolic androgen receptors to block the androgen action (Kumar et al., 1995; Wright and Cacale, 2004). In the present study, DHT-induced internucleosomal DNA fragmentation of thymocytes was blocked when TEC alone or thymocyte–TEC co-culture were treated with Xutamide. It seems that androgen exerts its apoptotic eVect on thymocytes through TEC following the classical genomic pathway. Further, treatment of thymocyte–TEC co-culture with transcription or translation inhibitor also blocked the DHT-induced DNA laddering of thymocytes. This reaYrms that DHT via its genomic action induced the de novo synthesis of apoptotic factors by TEC that in turn acted on thymocytes. In conclusion, we suggest that besides inhibiting thymocyte proliferation directly, androgen also induces thymocyte cell death indirectly through thymic stromal cell components, possibly TEC, via genomic pathway. The DHT-induced apoptotic factor secreted by TEC may acts through caspase-dependent pathway leading to
18
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thymocyte apoptosis and eventually produces atrophy of the thymus of wall lizard. The androgen-induced thymic atrophy in wall lizards may lead to suppression of T cell-mediated immune response, increase in disease susceptibility and death, in turn, during winter, as experienced in our laboratory.
Acknowledgment The Wrst author sincerely acknowledges the Wnancial support from Council of ScientiWc and Industrial Research (CSIR), New Delhi-110 007, India.
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Mechanism of androgen-induced thymic atrophy in the wall lizard, Hemidactylus Xaviviridis: An in vitro study B. Hareramadas, U. Rai ¤ Comparative Endocrinology Laboratory, Department of Zoology, University of Delhi (North Campus), Delhi 110 007, India Received 14 November 2004; revised 9 April 2005; accepted 13 April 2005 Available online 7 July 2005
Abstract The present in vitro study demonstrates the eVect of androgen on thymocyte apoptosis leading to thymic atrophy in the wall lizard, Hemidactylus Xaviviridis. Thymocytes collected from castrated lizards were incubated with varying concentrations of dihydrotestosterone (DHT) to observe its eVect on proliferation and apoptosis. DHT treatment reduced the tritiated thymidine incorporation in thymocytes, suggesting that androgen directly inhibits thymocyte proliferation. It also caused apoptosis of thymocytes eVectively at 10¡7 M. However, the increased apoptotic action of DHT was indirectly mediated through thymic epithelial cell-rich stromal cell components (TEC). This observation was reaYrmed by in vitro incubation of thymocytes with DHT-pretreated TEC-conditioned medium. However, the DHT-induced TEC-secreted apoptotic factors could induce thymocyte DNA fragmentation only when DHT was added to the conditioned medium. It implies that DHT priming of thymocytes is required for the apoptotic eVect of DHTinduced TEC-secreted factor. DHT-induced thymocyte apoptosis was found to be caspase-dependent since it activated the initiator (caspase-9) and eVector caspases (caspases-3 and -7) as well as cleaved the enzyme substrate poly(ADP-ribose) polymerase (PARP). Further, the apoptotic eVect of DHT was routed through its classical receptors, as non-steroidal antiandrogen Xutamide blocked the DHT-induced thymocyte apoptosis. The inhibition of apoptosis by transcription/translation inhibitors further substantiates the genomic pathway of DHT action. It can be concluded that DHT, in addition to inhibiting thymocyte proliferation directly, accelerates caspase-dependent apoptotic process in thymocytes indirectly through TEC via a genomic pathway. Nevertheless, the priming of thymocytes with DHT is required for the apoptotic eVect of TEC-secreted factor. 2005 Elsevier Inc. All rights reserved. Keywords: Androgen; Thymocyte proliferation; Apoptosis; Caspases; Lizard
1. Introduction The thymic–testicular axis has been extensively studied in mammals. Castration results in thymic enlargement, whereas androgen replacement causes reversal of this eVect (Grossman, 1984; Olsen and Kovacs, 2001; Olsen et al., 1998). The androgen-induced thymic atrophy might be due to alterations in various mechanisms including cell proliferation, cell traYcking, and/or cell death. Androgen has been reported to inhibit T cell line
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proliferation and increase apoptosis in a concentrationdependent manner (McMurray et al., 2001). This suggests that thymocytes are the potential targets for direct action of androgen. The localization of androgen receptors in thymocytes further supports this possibility (Kumar et al., 1995; Viselli et al., 1995). So far apoptosis is concerned, Nieto and Lopez-Rivas (1989) has reported that androgen has no eVect on murine T cell line apoptosis; while other in vivo and in vitro thymic organ culture experimental studies demonstrate acceleration in T cell apoptosis after androgen treatment (Dulos and Bagchus, 2001; Kumar et al., 1995; Olsen and Kovacs, 2001; Olsen et al., 1998). These results raised the possibility of the involvement of cells other
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than thymocytes in androgen-induced apoptosis. This line of thinking is reinforced by the Wndings that thymic epithelial cells possess androgen receptors (Grossman, 1984; Iwata et al., 1996; Kumar et al., 1995; Seiki and Sakabe, 1997). Further, the thymic epithelial cells are reported to be required for the apoptotic action of androgen on thymocytes (Dulos and Bagchus, 2001; Kumar et al., 1995; Olsen and Kovacs, 2001). In reptiles, the seasonal changes in thymic–testicular axis and thymic regression noted following androgen replacement therapy demonstrate a reciprocal relationship between androgen level and thymic growth/development (el Masri et al., 1995; Hareramadas and Rai, 2001; Muñoz and De la Fuente, 2001; Varas et al., 1991; Zapata et al., 1992). However, the immune cellular targets for androgen action have not been studied in reptiles, despite their phylogenic importance as ancestors to both birds and mammals. The present study, therefore, was undertaken to determine the direct and indirect eVects of androgen on thymocyte proliferation and apoptosis, and to delineate its pathway of action in thymic cells in the wall lizard. Also, the role of caspases in androgen-induced thymocyte apoptosis in wall lizards was studied since both caspase-dependent (Mann et al., 2000; Viselli and Barrett, 1999) and -independent (Kawahara et al., 1998) pathways of apoptosis are reported in thymocytes or T lymphoma cell line of mammals.
2. Materials and methods 2.1. Animals Adult male wall lizards (Hemidactylus Xaviviridis) weighing » 8 to 10 g were procured locally (Delhi:latitude: 28°12⬘–28°53⬘N, longitude: 76°50⬘–77°23⬘E) during regressed (May–August) and recrudescence (September– November) phases of the reproductive cycle. They were housed (12L:12D) in wooden cages with mesh wire at the top and fed live house Xies ad libitum. After 1 week of acclimation, lizards were castrated and kept for 7 days. Guidelines of Animal Ethics Committee, University of Delhi and Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Statistics, and Programme Implementation, Government of India, were followed in handling, maintenance, and sacriWce of animals. 2.2. Reagents and culture media Tissue culture medium RPMI-1640, dihydrotestosterone (DHT), Xutamide, acridine orange (AO), propidium iodide (PI), and DiBAC4(3) [bis-(1,3-dibarbituric acid)trimethine oxanol], RNase I-A, and concanavalin A (ConA) were obtained from Sigma Chemicals (St. Louis, MO, USA). Proteinase K and FITC-conjugated secondary
11
antibody (FITC-anti IgG antibody) were procured from Bangalore Genei (Bangalore, India). All other chemicals were procured from SRL (New Delhi, India). RPMI-1640 was supplemented with 40 g/ml antibiotic gentamycin, 100 g/ml streptomycin, 100 IU/ml penicillin (Ranbaxy Ind., New Delhi, India), 50 M -mercaptoethanol, 25 mM Hepes buVer, pH 7.2 (Merck India, New Delhi), and 10% heat-inactivated fetal calf serum (FCS; Biological Industries, Beth Haemek, Israel) prior to use. Tritiated thymidine ([methyl-3H]TdR; 2.0 Ci/mmol) was purchased from Board of Radiation and Isotope Technology (BRIT:Hyderabad, India). Primary antibodies for active caspases (3, 7, and 9), and PARP and HRP-conjugated secondary antibody (HRP-anti-IgG antibody) were obtained from Cell Signaling Technology (Beverly, MA, USA). Stock solutions (1 M) of DHT and DiBAC4(3) were prepared in dimethylsulphoxide (DMSO) and further dilutions were made in RPMI-1640 prior to use. 2.3. Isolation of thymic cells For each set of experiments, 25–30 castrated lizards were sacriWced. The thymic lobes were dissected out under aseptic conditions and transferred into cold phosphate-buVered saline (PBS, pH 7.2) containing 2% heatinactivated FCS (PBS–FCS). Tissues were pooled and washed thoroughly with PBS to remove the bloodstains. 2.3.1. Thymocytes Thymic lobes were gently minced and passed through a nylon mesh of pore size 40 M into cold PBS–FCS. Thymocytes were isolated following the procedure of Hareramadas et al. (2004). The single cell suspension of thymocytes was washed with PBS–FCS and the cell pellet was resuspended in tissue culture medium RPMI-1640 supplemented with 10% heat-inactivated FCS, 50 M -mercaptoethanol, 2 mM glutamine, and 25 mM Hepes buVer. Cell viability was checked by trypan blue exclusion, and the Wnal cell density was adjusted to 5 £ 106 cells/ml. 2.3.2. Thymic epithelial cell-rich stromal cell suspension A crude preparation of thymic epithelial cell-rich stromal cell suspension was made following the method of Rosen et al. (1976). In brief, the minced tissue of thymic lobes left-over on nylon mesh during thymocyte isolation was collected and treated with 0.1% trypsin/ EDTA solution for 15 min on ice. After washing, the crude preparation of thymic epithelial cell-rich stromal cells (TEC) was used for further experiments. 2.4. EVect of DHT on thymocyte proliferation and apoptosis 2.4.1. Thymocyte proliferation Thymocytes were incubated with either 5 g/ml concanavalin A (Con-A) alone or Con-A with diVerent
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concentrations of DHT for 72 h. Cells incubated in medium alone served as control. The cultures were pulsed with 1 Ci/ml [3H]TdR 18 h prior to the completion of incubation. After 72 h, thymocytes were washed with PBS and lysed with 1% SDS. The radioactivity in the lysate was counted using a liquid scintillation counter (Beckman, USA) and proliferation was expressed in terms of counts per min (§SEM)/106 thymocytes. 2.4.2. Thymocyte apoptosis 2.4.2.1. Experiment I. Thymocytes, TEC, and thymocytes along with TEC were cultured separately either in the medium alone (controls) or with varying concentrations of DHT (10¡5–10¡13 M) for 24 h in a humidiWed chamber of 5% CO2 incubator. Similar experimental groups were also undertaken for immunocytochemistry. In case of thymocyte–TEC co-culture experimental groups, thymocytes were separated out from TEC mass following centrifugation (100 rpm) at 4 °C for 5 min. The supernatant containing thymocytes was then used for Xow cytometric analysis, Xuorescence microscopy, immunocytochemistry, and agarose gel electrophoresis. 2.4.2.1.1. DNA extraction and gel electrophoretic analysis. DNA was extracted following the method of Nicoletti et al. (1991) modiWed by Hareramadas et al. (2004). BrieXy, cells from various experimental groups were harvested, washed, and resuspended in 500 l of hypotonic lysis buVer containing 200 g/ml proteinase K. DNA was extracted with Tris-equilibrated phenol–chloroform and then with chloroform alone. The aqueous phase containing nucleic acids was treated with RNase I-A, extracted with phenol–chloroform, chloroform–isoamyl alcohol, and Wnally with chloroform. DNA present in aqueous phase was then precipitated with 0.5 M ammonium acetate and absolute ethanol at ¡20 °C for overnight. The precipitated DNA was recovered (20,000g at 4 °C for 30 min) and dissolved in Tris–EDTA buVer (10 mM Tris, pH 8.0, and 1 mM EDTA). DNA was quantiWed (260 nm) and resolved on 1.5% agarose gel. DNA molecular weight marker ( DNA/EcoRI + HindIII double digest; Bangalore Genei, Bangalore, India) was also run along with the samples and DNA was visualized under UV transilluminator after staining with ethidium bromide. 2.4.2.1.2. Flow cytometric analysis. DNA content. Cells harvested from various experimental groups were processed for Xow cytometric analysis of DNA content following the method of Nicoletti et al. (1997). BrieXy, cells were harvested and washed twice with PBS. The cell pellet was then Wxed in 70% (v/v) chilled ethanol (¡20 °C) for 24 h. After Wxation, cells were washed with PBS and resuspended in hypotonic Xuorochrome, propidium iodide solution (50 g/ml PI in 0.1% sodium citrate plus 0.1% Triton X-100), and incubated in dark for 30 min at room temperature. The PI Xuorescence of
individual nuclei was analyzed by Becton Dickinson‘FACSVantage SE’ Xow cytometer. DNA integrity was then determined by generating histograms of cell number versus DNA content (PI Xuorescence). Plasma membrane potential. It was analyzed with the anionic bis-oxanol Xuorescent dye, DiBAC4(3) [bis(1,3-dibarbituric acid)-trimethine oxanol]. It has been demonstrated that DiBAC4(3) serves as an indicator for plasma membrane potential in lymphoid cells (Mann and Cidlowski, 2001). During resting state, the dye is excluded from the lymphocytes, whereas it enters the cell upon membrane depolarization and can be detected with Xuorescence activated cell sorting (FACS) analysis by measuring the increase in DiBAC4(3) Xuorescence. For membrane potential analysis, cells of various experimental groups were harvested, washed Wrst with PBS, and then with staining buVer (0.06 M sodium phosphate buVer, pH 7.0, supplemented with 5 mM KCl, 130 mM NaCl, 1.3 mM CaCl2, 0.6 mM MgCl2, and 10 mM glucose) . The same buVer was used to dissolve the dye. Cells were then incubated with 150 nM DiBAC4(3) for 30 min at 37 °C. To exclude the cells that had lost membrane integrity, PI was added to a concentration of 10 g/ml. Then the cells were analyzed by FACS. FACS analysis. All Xuorescence measurements of individual cells were made with a Becton Dickenson (BD) ‘FACSVantage SE’ Xow cytometer (Becton Dickenson, Mountain View, CA, USA) equipped with an air-cooled argon ion laser. PI Xuorescence was measured on FL-3 (excitation: 488 nm, emission: 620 § 11 nm) to exclude non-viable cells. Cell size was monitored by alterations in the forward light scattering properties of the cells as described by Mann et al. (2000). DiBAC4(3) Xuorescence of viable cells was measured on FL-1 (excitation: 488 nm, emission: 530 nm) to determine the membrane potential. An increase in DiBAC4(3) indicated a decrease in plasma membrane potential. Data were recorded on an Apple’s Macintosh Power PC running on Mac OS 9 and interpreted using ‘Cell Quest’ software of BD. Cell debris was excluded from analysis by appropriately raising the forward scatter (FSC) threshold. A total of 10,000 cells were analyzed for each sample. 2.4.2.1.3. Fluorescence microscopy. Based on gel electrophoretic and Xow cytotometric analyses of the present study, and the range of physiological testosterone concentrations (10¡7–10¡5 M) reported in diVerent lacertilian species (Ando et al., 1990; Huf et al., 1987; Radder et al., 2001; Smith and John-Alder, 1999), 10¡7 M DHT was used for Xuorescence microscopic study. Propidium iodide staining. Thymocytes and TEC were cultured either separately or co-cultured with DHT for 24 h. For controls, thymocytes, TEC, and thymocytes + TEC were cultured in the medium
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alone. After incubation, cells were washed with PBS; pellet was resuspended in a solution of 90% methanol and 10% propidium iodide, and kept for 10 min in the dark at room temperature. The cells were washed with PBS and resuspended in PBS-glycerine (1:1) solution. Then they were examined under ‘Nikon TE2000-U’ Xuorescence inverted microscope. Apoptotic cells were identiWed by their smaller, condensed, and intensely stained nuclei as compared to normal cells. Acridine orange staining for DNA strand breaks. Acridine orange (AO) is a metachromatic dye which diVerentially stains double-stranded (ds) and single-stranded (ss) nucleic acids. AO after intercalating into dsDNA emits green Xuorescence upon excitation at 480–490 nm. On the contrary, it emits red Xuorescence while interacting with ssDNA or RNA. Chromatin condensation is an early event of apoptosis and the condensed chromatin is much more sensitive to DNA denaturation than normal chromatin. Therefore, if RNA is removed by treating with RNase I-A and DNA is denatured in situ by exposure to HCl shortly before AO staining, apoptotic cells display an intense red Xuorescence, and a reduced green emission when compared to non-apoptotic interphase cells. AO staining was done following the method of Nicoletti and Mannucci (1997). In brief, cells at diVerent time intervals of incubation (6, 12, and 24 h) were taken out, washed, and Wxed with 1% paraformaldehyde in PBS on ice for 15 min. Fixed cells were washed and permeated with cold 70% ethanol for 4 h on ice. After permeation, cells were washed and incubated in RNAse I-A solution for 30 min at 37 °C. Cells were then resuspended in PBS and 0.1 M HCl was added to denature DNA. After 30–45 s, AO staining solution (6 g AO/ml in 0.1 M citric acid + 0.2 M Na2HPO4, pH 2.6) was added and kept for 10 min. Cells were observed under ‘Nikon E-400’ Xuorescence microscope. 2.4.2.1.4. Caspases-3, -7, and -9 (Immunocytochemistry). To understand whether the DHT-induced apoptotic pathway is caspase-dependent or -independent, active caspases-3, -7, and -9, and cleaved poly(ADP-ribose) polymerase (PARP) were immunocytochemically identiWed using antibodies. Thymocytes, TEC, and thymocytes + TEC incubated in medium alone (control) or with DHT were washed and Wxed with 3% paraformaldehyde. After Wxation, cells were washed thrice with Tris-buVered saline (TBS) containing 0.1% Triton X-100 (TBS/T), non-speciWc binding was blocked with 5% normal goat serum in TBS/T, and then incubated with primary antibodies for 24 h at 4 °C. Unbound antibodies were removed by washing with TBS/T, and cells were then incubated either with Xuorescein isothiocyanate (FITC)-conjugated or horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit IgG-FITC; and goat antirabbit IgG-HRP) for 2 h at room temperature. In case of HRP-conjugated secondary antibody incubation,
13
cells were pretreated with H2O2 to block endogenous peroxidase activity. Subsequently, cells were stained with 3-3⬘-diaminobenzidine tetrahydrochloride (DAB) solution, washed, and observed under ‘Nikon TE2000-U’ Xuorescence inverted microscope. Negative (without primary and/or secondary antibody) as well as positive (thymocytes treated with 10¡14 M corticosterone known to induce apoptosis, Hareramadas et al., 2004) controls were also maintained. 2.4.2.1.5. Microscopy. The Xuorescence observations were accomplished using a blue Wlter set (excitation: 450–490 nm, emission: 505 nm, and dichroic barrier Wlter: 520 nm). Images of PI stained cells, and caspases-3, -7, and -9 as well as cleaved PARP-positive (FITC Xuorescence) cells were captured with ‘Nikon Digital Sight DS-5M-L1’ digital camera equipped with 1⬙ relay lens and a 2/3⬙ CCD chip, whereas ‘Nikon Coolpix 4500’ digital camera equipped with 1⬙ relay lens and a 1/2⬙ CCD chip was used for the photography of AO stained cells. 2.4.2.2. Experiment II 2.4.2.2.1. EVect of DHT-pretreated TEC-conditioned medium on thymocyte apoptosis. TEC were pretreated with 10¡7 M DHT for 24 h, washed, and then incubated in medium alone for 24 h. The conditioned medium was collected and its diVerent dilutions were made (conditioned medium:fresh medium, 3:1, 1:1, and 1:3) to see its concentration-dependent apoptotic eVect on thymocytes following DNA gel electrophoresis. Untreated TEC-conditioned medium served as control. Further, thymocytes were incubated in the pre-treated TECconditioned medium with or without DHT to examine whether the DHT-induced TEC-secreted factor alone is capable to cause thymocyte apoptosis or the presence of DHT is also required. 2.4.2.3. Experiment III 2.4.2.3.1. EVect of Xutamide, an antiandrogen. To delineate the action of DHT on thymic cells of wall lizards, thymocytes, TEC, and thymocytes + TEC were Wrst incubated with anti-androgen Xutamide (10¡9 M) for 24 h, washed, and then incubated with varying concentrations of DHT for 24 h. After incubation, cells were processed for DNA gel electrophoretic and Xow cytometric analyses. 2.4.2.3.2. EVect of actinomycin D and cycloheximide. To understand the genomic action of DHT leading to de novo synthesis of proteins responsible for thymocyte apoptosis, thymocytes, and TEC were incubated separately as well as in combination with 10¡7 M DHT and 3 g/ml of actinomycin D (transcription inhibitor) or 10¡5 M cycloheximide (translation inhibitor) for 24 h in a humidiWed chamber of 5% CO2 incubator. Controls were incubated in medium alone or with actinomycin D/cycloheximide. After incubations, cells were processed for DNA gel electrophoresis.
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2.5. Statistical analysis All experiments were carried out in triplicate. In vitro dose-related eVect of DHT on thymocyte proliferation and apoptosis of thymic cells by Xow cytometric study was analyzed by one-way analysis of variance (ANOVA) followed by Newman–Keuls’ multiple range test. The results are based on the data of one of the repeated experiments.
3. Results 3.1. EVect of DHT on thymocyte proliferation and apoptosis 3.1.1. Thymocyte proliferation Con-A signiWcantly enhanced the uptake of [3H]TdR by thymocytes as compared to the control (Fig. 1, error bars with superscripts a vs. b, p < 0.01). However, DHT when supplemented with Con-A, signiWcantly reduced the mitogen-induced thymocyte proliferation, maximum inhibition occurred at 10¡5 M (error bars with superscripts b vs. c and d, p < 0.01). 3.1.2. Thymocyte apoptosis 3.1.2.1. Experiment I. DNA fragmentation and FACS analysis. Thymocytes incubated with DHT for 24 h showed no DNA fragmentation at any of the concentrations ranging from 10¡13 to 10¡5 M (Fig. 2, lanes 2–6)
Fig. 1. Histogram showing in vitro eVect of varying concentrations of dihydrotestosterone (DHT) on Con-A-induced thymocyte proliferation. Tritiated thymidine ([3H]TdR) incorporation is expressed as ‘mean counts per minute (CPM) § SEM’ (one-way analysis of variance (ANOVA) followed by Newman–Keuls’ multiple range test, p < 0.01). Data (means § SEM) represent one of the repeated experiments (N D 3). The error bars with diVerent superscripts (a–d) diVer signiWcantly.
Fig. 2. Agarose gel electrophoresis of thymocyte DNA. Thymocytes either alone or along with TEC were treated with diVerent concentrations of DHT (10¡5–10¡13 M) for 24 h. Lanes M, Marker ( DNA double digest); 1, thymocytes in medium alone (control), 2–6, thymocytes incubated with varying concentrations of DHT (10¡5, 10¡7, 10¡9, 10¡11, and 10¡13 M, respectively); 7, TEC in medium alone; 8, thymocytes + TEC in medium alone; 9–13, thymocytes + TEC incubated with 10¡5, 10¡7, 10¡9, 10¡11, and 10¡13 M of DHT, respectively. DHT-induced DNA laddering can be seen in thymocytes when incubated in the presence of TEC (lanes 9 and 10), though DNA fragmentation was more pronounced at 10¡7 M (lane 10). Results shown is one of the representative experiment of the experiments performed Wve times (N D 5).
and was found comparable to the control (lane 1), suggesting that DHT did not have any direct eVect on thymocyte apoptosis. Similarly, no apoptotic eVect of DHT was seen on TEC. Interestingly, DHT-induced apoptosis in thymocytes when they were co-cultured with TEC, and greater amount of fragmented DNA was seen at 10¡7 M concentration (lane 10), though TEC did not show any DNA laddering (lane 7). This indicates that DHT indirectly, through TEC, could induce thymocyte apoptosis. This indirect apoptotic eVect of DHT on thymocytes via TEC was also conWrmed by FACS analysis of ethanol-Wxed PI-stained cells. Like the control, the representative PI histograms of DHT treated thymocytes (without TEC) for 24 h showed a normal cell cycle distribution with a few hypodiploid cells (Fig. 3A). However, the hypodiploid DNA peak in response to DHT treatment was greatly enhanced when thymocytes were co-cultured with TEC for 24 h. DHT-induced hypodiploid cell percentage increased with orderly increase in concentration and reached its maximum at 10¡7 M (Fig. 3A); though DNA laddering was not seen following gel electrophoresis at low concentrations of DHT (Fig. 2), possibly due to DNA fragmentation appearing at later stages of apoptosis. 3.1.2.1.1. Membrane potential. After DHT treatment of thymocytes, the population of unWxed cells exhibiting DiBAC4(3) Xuorescence was comparable to that of the control, indicating that a large population of cells excluded the dye, since DHT failed to aVect the membrane potential of thymocytes. In contrast, in the
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3.1.2.1.3. Caspases-3,-7, and -9 (Immunocytochemistry). Immunocytochemical examination of in vitro eVect of DHT on caspases in thymocytes cultured alone or with TEC showed that DHT-treated thymocytes were positive to caspases-3, -7, and -9 and PARP (caspase substrate) only when they were co-cultured with TEC (Fig. 6). The maximum number of caspase-positive cells were seen at 10¡7 M DHT. It appears that DHT through TEC activates the cascade of caspases in thymocytes which lead to their apoptosis.
Fig. 3. (A) DNA Xuorescence Xow cytometric proWle of ethanolWxed PI-stained thymocytes treated with diVerent doses of DHT 10 ¡5–10 ¡13 M in the absence or presence of TEC for 24 h. For controls, thymocytes alone or with TEC were incubated in medium alone. Data suggest the indirect apoptotic eVect of DHT through TEC on thymocytes. Maximum percentage hypodiploid thymocytes were noted at 10¡7 M DHT. Result presented here is one of the representative experiments repeated Wve times (N D 5). (B) Plasma membrane depolarization of thymocytes treated with varying concentrations of DHT in the absence or presence of TEC. Percentage DiBAC4(3)-positive apoptotic thymocytes showing their loss of membrane potential was assessed by Xow cytometry. Experiment was repeated thrice (N D 3). Data represent one of the repeated experiments.
presence of TEC, DHT increased the population of DiBAC4(3) Xuorescence-positive thymocytes (Fig. 3B). This further substantiates that androgen accelerates thymocytes apoptosis indirectly through TEC. 3.1.2.1.2. Fluorescence microscopy. The PI-stained nuclei of thymocytes treated with 10¡7 M DHT were normal and comparable to the control (Fig. 4A); whereas smaller, condensed, and intensely stained nuclei of DHT-treated thymocytes were seen when they were co-cultured with TEC (Fig. 4B). Similar observations were noted following AO staining after DNA denaturation. Emission of green Xuorescence from nuclei of DHT-treated thymocytes was comparable to those incubated in medium alone (control, Fig. 5A). But, TEC when co-cultured with thymocytes in medium containing DHT for diVerent time intervals, i.e., 6, 12, and 24 h, resulted in decrease of green Xuorescence emission from thymocytes and increased red Xuorescence showing signs of apoptosis gradually with an increase of time duration from 6 to 24 h (Figs. 5B–D). This suggests that DHT through TEC induces lizard thymocyte cell death.
3.1.2.2. Experiment II. EVect of TEC-conditioned medium on thymocyte apoptosis. Thymocytes incubated either in medium alone or with diVerent dilutions of DHT-pretreated TEC-conditioned medium (conditioned medium:fresh medium::3:1, 1:1, and 1:3) did not exhibit DNA fragmentation as evident by gel electrophoretic analysis (Fig. 7). However, thymocytes DNA fragmentation indicating apoptosis was noted when DHT was supplemented to the DHT-pretreated TEC-conditioned medium, and the eVect decreased with the increase in dilution of DHT-pretreated TEC-conditioned medium (Fig. 7). This suggests that priming of thymocytes with DHT is required for the apoptotic action of DHTinduced TEC-secreted factor. 3.1.2.3. Experiment III. EVect of antiandrogen. Gel electrophoresis for DNA fragmentation and Xow cytometric analysis of diploid/hypodiploid cell distribution showed that antiandrogen Xutamide (10¡9 M) did not have apoptotic eVect on thymocytes/TEC alone or co-culture of thymocytes and TEC. However, pre-treatment of thymocytes+ TEC with Xutamide blocked the apoptotic eVect of DHT as no DNA laddering was seen in Xutamide pretreated group, and Xow cytometric data were comparable to that of control (thymocytes + TEC in medium alone) (Fig. 8). This shows that the androgen apoptotic action is mediated through its classical receptors in thymic cells. 3.1.2.3.1. EVect of transcription and translation inhibitors. Actinomycin D and cycloheximide, transcription and translation inhibitors, respectively, blocked the DHTinduced apoptosis in thymocytes co-cultured with TEC. This suggests the genomic action of DHT in inducing thymocyte apoptosis, indirectly through TEC (data not shown).
4. Discussion In the present in vitro study, DHT profoundly inhibited the mitogen-induced uptake of [3H]TdR by thymocytes, suggesting that androgen directly attenuates thymocyte proliferation in wall lizards. The inhibitory eVect of androgen on thymocyte proliferation has also been reported in turtles, Mauremys caspica (Muñoz and De la Fuente, 2001), and Chelonia mydas (Work et al., 2000). In mammals, androgen also induces thymocyte cell death
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Fig. 4. Fluorescence photomicrographs of propidium iodide (PI) stained thymocytes treated with 10¡7 M of DHT for 24 h in the presence of TEC. (A) Thymocytes in medium alone (control); (B) DHTinduced apoptotic thymocytes showing condensation (long arrow), fragmentation (arrow head), and fragmented DNA (short arrow). The experiment was repeated three times (N D 3) to verify the consistency of results (original magniWcation:1560£).
(Olsen et al., 1998), besides inhibiting proliferation (Bilbo and Nelson, 2001; McMurray et al., 2001). Using Jurkat T cell line (human acute T cell leukemia), testosterone was shown to increase thymocyte apoptosis in a concentrationand time-dependent manner (McMurray et al., 2001), thereby indicating the direct eVect of androgen on thymocyte apoptosis. On the contrary, androgen has no apoptotic eVect on murine T cell line, CTLL-2 (Nieto and LopezRivas, 1989). Moreover, administration of androgen in castrated mice or in vitro thymic organ culture with androgen has been reported to cause thymocyte apoptosis (Olsen et al., 1998). This indicates that cells other than thymocytes are involved in mediating the apoptotic eVect of androgen on murine thymocytes.
Fig. 5. Acridine orange (AO)-stained thymocytes treated with DHT (10¡7 M) for diVerent time intervals (0, 6, 12, and 24 h) in the presence of TEC. (A) Emission of green Xuorescence by non-apoptotic thymocytes incubated in medium alone for 24 h (control); (B–D) exhibit decrease in green Xuorescence and increase in red Xuorescence, indicating their transformation from non-apoptotic to apoptotic cells following 6, 12, and 24 h DHT treatment, respectively. The experiment was repeated three times (N D 3) to verify the consistency of results (original magniWcation: 1080£).
The presence of androgen receptors in thymic epithelial cells (Kumar et al., 1995; Olsen and Kovacs, 2001; Olsen et al., 2001) suggests their role in thymocyte apoptosis as thymic epithelial cell network is known to provide microenvironment for thymocyte maturation. Further, androgen-induced TGF- secretion from thymic epithelial cells has been implicated crucial for the activation of CPP32/caspase-3 which, in turn, causes thymocyte apoptosis (Hadden, 1998; Olsen et al., 1993; Viselli and Barrett, 1999). In view of these Wndings, it has been proposed that androgen also lead to indirect eVect on thymocyte apoptosis, through TEC (Olsen and Kovacs, 2001).
Fig. 6. Immunocytochemical demonstration of active caspases and cleaved enzyme substrate PARP in apoptotic thymocytes following DHT (10¡7 M) treatment in the presence of TEC. For caspase-3 and PARP, cells after challenging with primary antibodies were incubated with FITC-conjugated secondary antibody and counter stained with PI (A) i–iv. Control nuclei were stained bright red (caspase-3: (A) i; PARP: (A) iii, whereas condensed apoptotic cells showed yellowish-green Xuorescence due to the overlapping of FITC and PI signals (A) ii and iv for active caspase-3 and cleaved PARP, respectively). For caspases-7 and -9, after counter challenging with HRP-conjugated secondary antibody, thymocytes were stained with DAB. (B) vi and viii showed active caspases-7 (control, (B) v) and -9 (control, (B) vii), respectively. Figures represent one of the repeated experiments (N D 5) (original magniWcation:1560£).
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Fig. 7. EVect of DHT-pretreated TEC-conditioned medium on DNA fragmentation of thymocytes incubated with or without DHT. M, marker; lane 1, thymocytes + TEC in medium alone; 2, thymocytes + TEC + DHT 10¡7 M; 3–5, thymocytes + untreated TECconditioned medium (dilutions: 25:75, 50:50, and 75:25%); 6–8, thymocytes + DHT 10¡7 M + diVerent dilutions of untreated TEC-conditioned medium; 9–11, thymocytes + diVerent dilutions of DHT-pretreated TEC-conditioned medium (25:75, 50:50, and 75:25%); 12–14, thymocytes + DHT 10¡7 M + diVerent dilutions of DHT-pretreated TEC-conditioned medium. DNA laddering in response to DHTinduced TEC-secreted factor can be seen in thymocytes only when the conditioned medium was supplemented with DHT (lanes 12–14). Data presented are one of the representative experiments (N D 3).
Fig. 8. EVect of antiandrogen, Xutamide on DHT-induced thymocyte apoptosis. Lane M, marker; 1, thymocytes + TEC in medium; 2, thymocytes + TEC + Xutamide; 3, thymocytes + TEC + DHT 10¡5 M; 4, thymocytes + TEC + DHT 10¡7 M; 5, thymocytes + TEC + Xutamide + DHT 10¡5 M, and 6, thymocytes + TEC + Xutamide + DHT 10¡7 M. Data presented are one of the representative experiments (N D 3).
In wall lizards, unlike proliferation, DHT did not have any direct eVect on thymocyte cell death at any concentration ranging from 10¡13 to 10¡5, as evident by DNA gel electrophoresis, Xow cytometry as well as Xuorescence microscopic analyses. However, when lizard thymocytes were treated with DHT in the presence of TEC, membrane depolarization and loss of membrane potential, cell shrinkage, DNA condensation, and internucleosomal DNA fragmentation were observed. These
17
results suggest that in wall lizards DHT regulates thymocyte apoptosis, indirectly, through TEC-secreted paracrine factor. This conclusion is further substantiated by the Wndings that DHT-pretreated TEC-conditioned medium accelerated the thymocyte apoptosis. However, it may be noted that the DHT-pretreated TEC-conditioned medium was capable of inducing thymocyte apoptosis only when thymocytes were exposed to DHT. Several studies using mammalian models have shown caspase-dependent pathway of thymocyte apoptosis (Mann et al., 2000; Opferman and Korsmeyer, 2003; Villa et al., 1997; Viselli and Barrett, 1999; Vugmeyster et al., 2002), though caspase-independent pathway of apoptosis has also been reported in embryonic stem cells (Joza et al., 2001) and other cell lines (Kawahara et al., 1998; Perfettini et al., 2002; Wu et al., 2002). Following caspasedependent pathway, cytochrome c released from mitochondria in response to death stimuli, activates caspase-9 (a key initiator caspase) which, in turn, activates downstream eVector caspases, caspases-3, -6, and -7, which are evolutionarily conserved (Samuilov et al., 2000). These caspases then cleave their preferential cellular substrates, PARP (a DNA repair enzyme cleaved by caspases-3 and 7) and lamin A (a component of nuclear lamina cleaved by caspase-6). Also, caspase cleavage of ICAD/DFF (inhibitor of caspase activated DNase/DNA fragmentation factor) releases the nuclease CAD, presumably allowing it to enter the nucleus and degrade genomic DNA (Villa et al., 1997). In this study, DHT was found able to activate caspases-9, -7, and -3 and subsequently cleave PARP in thymocytes only when they were co-cultured with TEC. This suggests that androgen-induced TECsecreted paracrine factor might activate thymocyte apoptosis following caspase-dependent pathway in wall lizards, as reported in mammals (Viselli and Barrett, 1999). Flutamide is a potent nonsteroidal antiandrogen that competitively binds with intracellular nuclear/cytosolic androgen receptors to block the androgen action (Kumar et al., 1995; Wright and Cacale, 2004). In the present study, DHT-induced internucleosomal DNA fragmentation of thymocytes was blocked when TEC alone or thymocyte–TEC co-culture were treated with Xutamide. It seems that androgen exerts its apoptotic eVect on thymocytes through TEC following the classical genomic pathway. Further, treatment of thymocyte–TEC co-culture with transcription or translation inhibitor also blocked the DHT-induced DNA laddering of thymocytes. This reaYrms that DHT via its genomic action induced the de novo synthesis of apoptotic factors by TEC that in turn acted on thymocytes. In conclusion, we suggest that besides inhibiting thymocyte proliferation directly, androgen also induces thymocyte cell death indirectly through thymic stromal cell components, possibly TEC, via genomic pathway. The DHT-induced apoptotic factor secreted by TEC may acts through caspase-dependent pathway leading to
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thymocyte apoptosis and eventually produces atrophy of the thymus of wall lizard. The androgen-induced thymic atrophy in wall lizards may lead to suppression of T cell-mediated immune response, increase in disease susceptibility and death, in turn, during winter, as experienced in our laboratory.
Acknowledgment The Wrst author sincerely acknowledges the Wnancial support from Council of ScientiWc and Industrial Research (CSIR), New Delhi-110 007, India.
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