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Endocrinology Vol. 138, No. 2 810-818
Copyright © 1997 by The Endocrine Society

Articles

In Vivo Resistance to Glucocorticoid-Induced Apoptosis in Rat Thymocytes with Normal Steroid Receptor Function in Vitro

Nicklas B. E. Oldenburg1,2, Rosemary B. Evans-Storms1 and John A. Cidlowski

Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709

Address all correspondence and request for reprints to: John A. Cidlowski, National Institute of Environmental Health Sciences, NIH, P.O. Box 12233, MDE2-02 Research Triangle Park, North Carolina 27709. E-mail: Cidlowski{at}NIEHS.NIH.GOV


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Discussion
 References
 
Previous studies have shown that although the majority of rat thymic lymphocytes are sensitive to glucocorticoid-induced apoptosis in vivo, a small population of mature thymic lymphocytes remains even after high dose steroid administration. Here, we describe experiments that were performed to understand the molecular basis of the resistance of these cells to glucocorticoid-induced apoptosis. Adrenalectomized rats were treated for 72 h with a bolus dose (5 mg/kg body weight) of dexamethasone to produce a population of thymocytes that survived glucocorticoid administration. Reinjection of these animals with equivalent doses of dexamethasone failed to induce further thymic regression or apoptosis in these cells. Glucocorticoid receptor number and receptor binding affinity for dexamethasone were similar in control and resistant thymocytes. Western blot analysis using epitope-purified antiglucocorticoid receptor antibodies confirmed this observation. To delineate the mechanism of resistance, we evaluated whether cells resistant to dexamethasone in vivo showed any response to this glucocorticoid in vitro. The ability of glucocorticoid to inhibit [3H]lysine incorporation into protein in cells treated with dexamethasone in vitro was equivalent to control cells, indicating that glucocorticoid receptor function was normal in both populations. To evaluate whether in vivo glucocorticoid-resistant thymocytes retain any capacity to undergo apoptosis, in vitro studies were performed on these cells using the calcium ionophore A23187 to induce programmed cell death. Cleavage of chromatin into 30- to 50-kilobase fragments or oligonucleosomal fragments characteristic of apoptosis was observed in both sensitive and resistant thymocytes treated in vitro with A23187. Cells resistant to glucocorticoid in vivo unexpectedly exhibited internucleosomal cleavage of chromatin and apoptosis in response to dexamethasone in vitro. We examined the levels of the apoptosis suppressor Bcl-2 in thymocytes isolated from control and 72 h dexamethasone-treated rats to determine whether increased expression of this protein could explain the resistance to glucocorticoid-induced apoptosis that we observed. Both glucocorticoid-sensitive and -resistant thymocytes expressed similar levels of Bcl-2. Together, these data indicate that resistance to glucocorticoid in vivo is not due to alteration of the glucocorticoid receptor or to expression of Bcl-2, but rather to some endogenous thymic factor and/or cell-to-cell contact that probably alters glucocorticoid receptor signaling.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Discussion
 References
 
OF THE WIDELY recognized effects of glucocorticoids on the immune system, the killing of immature T cells is the most profound. Glucocorticoids were identified as the inducers of lymphocytolysis (1, 2) decades before a mechanism to explain this process was described. It is now clear that glucocorticoid-induced lymphoid death represents the activation of a physiological program of cell death known as apoptosis (3, 4, 5). Morphologically and biochemically, apoptotic forms of cell death can be distinguished from degenerative processes such as necrosis. Apoptosis occurs by an orderly series of events that includes rapid reduction in cell volume, chromatin condensation, increased nuclear fragility, and ordered cleavage of DNA into nucleosome-sized fragments (4, 5, 6).

Programmed cell death in rat and mouse thymocytes has been extensively studied because these predominantly immature cells can be stimulated to undergo apoptosis both in vivo and in vitro (3, 4). Internucleosomal cleavage of thymocyte DNA is a time-dependent process beginning approximately 2 h after glucocorticoid administration (3). Approximately 90% of thymocytes (predominantly immature cortical cells) die by apoptosis within 48–72 h after steroid treatment (2). After 72 h of in vivo glucocorticoid administration, however, DNA degradation products are no longer detectable in the remaining thymocytes (3). This remaining population is predominantly composed of mature thymocytes (CD4 or CD8 single-positive) that are physically restricted to the thymic medulla (7, 8, 9). These data suggest that the population of cells remaining in the thymus gland after dexamethasone administration is resistant to glucocorticoid-induced apoptosis. Here, we describe experiments performed to explore the molecular basis of this resistance. We demonstrate that these thymocytes are resistant to glucocorticoid-induced apoptosis in vivo, but protection is lost when cells are removed from the natural milieu of the thymus gland.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Discussion
 References
 
Thymocyte preparation
Thymocytes were prepared essentially as previously described (3). All protocols were approved by the National Institute of Environmental Health Sciences animal care and use committee and were performed in accordance with the regulations in the NIH Guide for the Care and Use of Laboratory Animals published by the U.S. Public Health Service. In brief, male Sprague-Dawley rats (85–100 g BW) were bilaterally adrenalectomized 5–14 days before use and were maintained on 0.85% NaCl and rat chow ad libitum. Rats were administered dexamethasone (5 mg/kg BW) (Steraloids, Wilton, NH) by ip injection of the steroid suspended by sonication in PBS. Control animals received PBS alone. Animals were killed by rapid decapitation, and the thymus glands were removed and placed in ice-cold PBS. Thymus tissue was cut with scissors, and aliquots were homogenized in a loose-fitting Kontes No. 22 glass/glass homogenizer (Kontes Co., Vineland, NJ). The homogenized suspension was filtered through 202-µm Nitex mesh (Tetko, New York, NY) and centrifuged at 2600 x g for 5 min at 4 C. The supernatant was discarded, and the pellet was resuspended in PBS, refiltered, and centrifuged as described above. Cells were resuspended in cold PBS, and the cell concentration was determined by counting on a hemocytometer or by using a Coulter counter (Coulter Electronic, Hialeah, FL).

Cell culture and in vitro experiments
Thymocytes were isolated as described above, and then cells were centrifuged over Lymphocyte Separation Medium (Organon Teknika, Durham, NC) at 18 C for 20 min at 400 x g to remove dead cells. Thymocytes were resuspended in cold RPMI-1640 and centrifuged at 100 x g at 4 C for 10 min. Cell number and viability were determined by trypan blue dye exclusion and counting using a hemocytometer. Thymocytes (5 x 106 cells/ml) were placed in 6-well flat-bottom plates (Costar, Cambridge, MA) and incubated in RPMI-1640 medium with 10% FCS and either 1 µM dexamethasone, 0.1 µM A23187 (Calbiochem, San Diego, CA), or vehicle. A23187 was prepared as a 100 mM stock in tissue culture grade dimethylsulfoxide and stored at 4 C. Dimethylsulfoxide was used as a control when appropriate. Dexamethasone was prepared as a saturated solution in H2O and stored at 4 C. The concentration was determined using the molar extinction coefficient method. Thymocytes were harvested, and cell viability was determined by the trypan blue dye exclusion method. DNA was isolated and analyzed as described below. The HTC rat hepatoma cell line was a generous gift of Dr. Stoney Simons (NIDDK), and was maintained in DMEM containing 5% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, and 75 U/ml streptomycin in 5% CO2 at 37 C.

Histology
For histological analysis, thymocytes were isolated as described above, pelleted, and fixed in ethanol/acetic acid (3:1) for 10 min (10). Cells were then dried on slides, stained with Wright’s stain, and subsequently photographed with standard light microscopic techniques using a Nikon camera (Nikon Corp., Tokyo, Japan) and Kodak Gold 200 ASA film (Eastman Kodak, Rochester, NY) (10).

DNA fragmentation analysis
DNA was isolated by solubilizing the cells in lysis buffer (0.2 M Tris, pH 8.5, 0.1 M Na2EDTA, 1% SDS). Lysate was treated with RNase A (0.2 mg/ml) for 1 h at 37 C, followed by treatment with proteinase K (0.5 mg/ml) for 1 h at 55 C. The samples were extracted three times with phenol/chloroform (1:1 vol/vol; plus 2% isoamyl alcohol) followed by one chloroform extraction and precipitated by addition of 100 mM NaCl and 2.5 vol ethanol at -70 C. The DNA was resuspended in TE buffer (10 mM Tris, pH 7.5, 1 mM Na2 EDTA), the concentration was determined by absorbance at 260 nm, and 15 µg/lane of DNA was electrophoresed on 1.8% agarose gels. The gels were stained with ethidium bromide, visualized by UV transillumination, and photographed as previously described (3).

Pulsed field gel electrophoretic analysis of rat thymocyte DNA
Thymocytes from appropriately treated adrenalectomized rats were isolated and resuspended in 0.5% InCert agarose (FMC Bioproducts, Rockland, ME) at a concentration of 2 x 107 cells/ml and solidified in a 6.5-mm square mold. Agarose plugs were placed in 5 times their volume of lysis buffer (100 mM EDTA, 1% Sarcosyl) (Sigma Chemical Co., St. Louis, MO) for 24 h at 37 C. Plugs were then placed in fresh lysis buffer containing 100 µg/ml proteinase K for 24 h at 50 C. A 2-mm slice of the plug was washed twice in 50 ml running buffer (22 mM Tris-borate, 1 mM EDTA), and then the DNA was electrophoretically separated on a 1.0% agarose gel by pulsed field gel electrophoresis for 20 h with an initial switch time of 0.5 sec and a final switch time of 45 sec and a forward to backward ratio of 1 (11).

Whole cell glucocorticoid receptor binding
Thymocytes were washed 1 h at 37 C in a 5% CO2 incubator in RPMI-1640 containing 10% FCS (JRH Bioscience, Lenexa, KS), and then the hormone binding capacity of these cells was determined by incubating 5 x 107 cells in 0.5 ml RPMI with concentrations of [3]H-dexamethasone (New England Nuclear, Boston, MA) (specific activity: 49.9 Ci/mmol) ranging from 1 x 10-7 to 8 x 10-10 M. Cells were incubated for 1 h at 37 C with gentle shaking, and then cells were lysed by placing them in ice-cold 1.5 mM MgCl2 for 30 min at 4 C. Nuclei were obtained by centrifugation for 30 min at 4000 x g, and radioactivity present in the nuclei was determined using a scintillation counter (12). The radians method of Rosenthal (14) was used to determine nonspecific hormone binding, and binding capacity expressed as receptors/cell, as well as receptor dissociation constants, were determined according to the method of Scatchard (13). Regression analysis was also performed on this data to determine whether only one class of receptors was present.

Electrophoresis and Western analysis of glucocorticoid receptor and Bcl-2
For analysis of glucocorticoid receptor, cells were washed once in cold PBS and then resuspended in buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.3) containing the protease inhibitors phenylmethylsulfonyl fluoride (0.1 mM) and aprotinin (1 µg/ml), and the phosphatase inhibitors NaF (20 mM), Na3VO4 (1 mM), and Na4P2O7 (20 mM). Cells were homogenized by several 10-sec bursts from a Tekmar Ultra Turrax homogenizer (Tekmar Co., Cincinnati, OH). Cytosol was prepared by centrifugation of this homogenate at 100,000 x g for 1 h at 4 C. Protein concentrations were determined by the method of Bradford (15) using the Bio-Rad (Richmond, CA) protein microassay, and samples were diluted with Fairbank’s sample buffer to give a final concentration of 1% sodium lauryl sulfate, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5% sucrose, 10 µg/ml pyronin Y, and 12 mg/ml dithiothreitol. Samples were heated to 100 C for 10 min, and then 150 µg total protein from each cytosol was separated by Fairbanks PAGE using a mixture of 30% acrylamide and 0.8% bis-acrylamide to compose a 7.5% resolving gel and a 3% stacking gel (16). Proteins were subsequently transferred electrophoretically to 0.1 µm nitrocellulose (Schleicher and Schuell, Keene, NH) in Tris-glycine buffer (25 mM Tris-HCl, pH 8.3, 150 mM glycine, 15% methanol) overnight at 35 V with cooling. Loading equivalency and transfer efficiency were verified by staining with Ponceau S (Sigma) (0.5% in 1% acetic acid) (17), and then membranes were treated with blocking buffer composed of 10% nonfat dry milk in Tris-buffered saline (TBS) (10 mM Tris-HCl, 154 mM NaCl, 0.05% Tween-20, pH 7.4) for 1 h at room temperature with mixing. Membranes were then reacted with antisera generated against the human glucocorticoid receptor (no. 57–1:1000 in TBS) (18) for 1 h at room temperature with mixing. After washing with TBS, the membranes were reacted with horseradish peroxidase-linked donkey antirabbit immunoglobulin diluted 1:15,000 in TBS for 1 h at room temperature. Proteins were visualized after washing by reaction with luminescence detection reagents (Amersham Corp., Arlington Heights, IL) followed by autoradiography at room temperature using hyperfilm-ECL (Amersham Corp.).

For analysis of Bcl-2, all protocols were as described above except that whole cell lysates were prepared by homogenization of cells in buffer composed of 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 150 mM NaCl, and 0.5% Triton X-100 (containing protease and phosphatase inhibitors), and 100 µg of total protein was separated by Laemmli SDS-PAGE (5% stacking gel with a 12% resolving gel) (19) after samples were denatured in sample buffer (final concentration: 50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol). Western analysis was performed as described using polyclonal rabbit antirat/mouse Bcl-2 antiserum (cat no. 13456E, Pharmingen, San Diego, CA) diluted 1:2000.

[3H]Lysine incorporation into protein
In vitro sensitivity to glucocorticoids was measured by studying the effects of dexamethasone on the incorporation of radiolabeled lysine. Thymocytes (5 x 106 cells/well) were placed in 96-well microtiter plates (Costar, Cambridge, MA) and treated with either vehicle or 1 µM dexamethasone for 3 h. Before harvesting, cells were pulsed with 2 µCi/ml L-lysine[4, 5-3H] (specific activity = 70 Ci/mmol) (ICN Radiochemicals, Irvine, CA) for 1 h. Cells were harvested onto glass fiber filters (Titertek, Rockville, MD) and rinsed for 60 sec with distilled water. The filters were then washed with 15 ml 10% trichloroacetic acid, air dried, and radioactivity determined using a scintillation counter. Counts per minute were normalized for cell number, and percent [3H]lysine incorporation was calculated as cpm/105 treated cells divided by cpm/105 control cells x 100.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Discussion
 References
 
Morphological characteristics of thymocytes
The profound lympholytic effect of glucocorticoids on developing thymocytes has been described in depth (2), however, little is known about the cells that survive long-term glucocorticoid treatment. Figure 1Go shows the effect of an in vivo dexamethasone dose on thymus gland size and cell number. The thymus gland of a rat treated for 72 h with dexamethasone (5 mg/kg BW) was dramatically smaller than that of a vehicle-treated animal, and only approximately 10% of the cells remained in the gland after this treatment. To control for the possibility that metabolism of the dexamethasone was decreasing the concentration of the apoptotic stimulus and thereby allowing survival of some cells, we readministered an equivalent dose (5 mg/kg) of dexamethasone to 72-h steroid-treated animals and then determined cell number and thymus weight after 6 h (3). This time point was chosen because it has previously been shown that in vivo administration of dexamethasone induces apoptosis of thymocytes within 6 h (3). Neither cell number nor thymus weight changed significantly after this second administration of hormone (Fig. 1Go). Apoptosis of thymocytes after in vivo treatment with dexamethasone peaks after 18–24 h (3). Therefore, it was possible that there was a delay in onset of apoptosis after readministration of hormone. To ensure that apoptosis was not being overlooked, thymus weight and thymocyte number were also evaluated 18 h after readministration of dexamethasone. No change was observed when compared with thymus glands from animals 6 h after readministration of hormone (data not shown). Together, these data demonstrate that the thymocytes remaining in the thymus of rats treated with steroid are not sensitive to glucocorticoid administration in vivo and thus appear to be glucocorticoid resistant.



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  		Figure 1. Effect of glucocorticoids on thymus gland. Photographs of representative thymus glands from adrenalectomized rats 72 h after treatment with vehicle (con) (left), 72 h after a single ip injection of dexamethasone (DEX) (5 mg/kg BW) (center), or after reinjection of an equivalent dose of dexamethasone for 6 h to rats previously treated with dexamethasone for 72 h (right) are shown and their weights given. In addition, average number of cells per thymus is reported ± SD (control n = 16, dexamethasone treated n = 12).

 
Cells undergoing apoptosis exhibit unique morphological features that characterize this form of cell death. Initially, cells undergo a reduction in cell volume, followed by nuclear condensation, and the eventual compartmentalization of structurally intact organelles into membrane-bound vesicles. These vesicles, termed apoptotic bodies, detach from the cell surface and are phagocytosed and degraded by surrounding cells (6). Thymocytes from rats injected with dexamethasone for varying times were isolated, and their morphology assessed after staining with Wright’s stain. In addition to control thymocytes and thymocytes from rats treated with dexamethasone for 72 h, cells from rats treated for 6 and 18 h with dexamethasone were also stained to evaluate the presence of immature vs. mature thymocytes. Figure 2Go shows the results of these studies. The thymus of control rats contained a mixed population of mature (dense nuclei with scant cytoplasm) and immature (larger nuclei with more cytoplasm) thymocytes. Based on morphology, less than 1% of these cells were apoptotic. Thymocytes isolated from animals after treatment with dexamethasone for 6 h showed similar morphology (Fig. 2BGo). Failure to detect a large number of apoptotic thymocytes after 6 h most likely reflects efficient clearing by phagocytic cells. After 18 h of hormone treatment, there was clear morphological evidence of apoptosis (approximately 17%) with many shrunken cells with condensed chromatin, abundant cytoplasmic blebbing, and apoptotic bodies (Fig. 2CGo). After 72 h of dexamethasone administration, the population of cells remaining in the thymus gland appeared to be composed of predominantly mature thymocytes (Fig. 2DGo). These data support previous observations that demonstrated no internucleosomal cleavage of the cellular chromatin in these 72-h dexamethasone-treated thymocytes (3). Administration of an additional dose of dexamethasone (5 mg/kg BW) for 6 or 18 h to animals that had already been treated for 72 h did not cause any morphological changes in these thymocytes that would be indicative of apoptosis (Fig. 2Go, E and F). These morphological data demonstrate that the population of thymocytes remaining in the thymus gland after chronic glucocorticoid treatment is mature and appears to be resistant to glucocorticoid-induced apoptosis, and that resistance of these cells to glucocorticoid is not due to unavailablity of hormone.



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  		Figure 2. Morphology of thymocytes following in vivo dexamethasone administration. Adrenalectomized rats were treated with an ip injection of dexamethasone (5 mg/kg BW) and killed 6, 18, and 72 h later. In addition, 72-h-treated animals were reinjected with identical doses of dexamethasone for an additional 6 and 18 h. Thymocytes were isolated, fixed in 3:1 ethanol/acetic acid, dried onto microscope slides, and then stained with Wright’s stain. Cells were photographed on a Nikon camera using Kodak Gold 200 ASA film. A, Control; B, 6 h dexamethasone; C, 18 h dexamethasone; D, 72 h dexamethasone; E, 72 + 6 h dexamethasone; F, 72 + 18 h dexamethasone.

 
Biochemical characteristics of thymocytes after glucocorticoid treatment
To further evaluate the resistance of these cells to glucocorticoid-induced apoptosis, we analyzed internucleosomal cleavage of chromatin, which is a widely accepted reflection of apoptosis (4). Previous observations have shown that there is no internucleosomal DNA degradation in long-term in vivo dexamethasone-treated thymocytes (3), however it has not been determined whether the absence of DNA degradation was an effect of reduced steroid levels in vivo 72 h after dexamethasone administration. Initially, DNA degradation was examined in animals that had been treated for 6 h with dexamethasone. The 6-h time point was selected because 50% of the thymocyte DNA appears as degradation products, while viability remains greater that 90% (3). Electrophoretic analysis of the DNA revealed characteristic fragments composed of multiples of approximately 180 bp in the dexamethasone-treated animals 6 h after steroid administration (Fig. 3AGo) that were not present in the control cells. After 72 h of steroid administration, all evidence of DNA degradation disappeared, and DNA from treated animals was identical to controls (Fig. 3AGo). Reinjection of 72-h dexamethasone-treated rats with an equivalent dose of steroid for an additional 6 h did not cause additional internucleosomal degradation to occur in the remaining cells (Fig. 3AGo).



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  		Figure 3. Detection of rat thymocyte DNA fragmentation after in vivo administration of dexamethasone. A, Detection of internucleosomal DNA cleavage. Adrenalectomized rats were treated with vehicle (CON) or dexamethasone (DEX) (5 mg/kg BW) and killed 6 h or 72 h later. In addition, some animals that had been treated with dexamethasone for 72 h were reinjected with either vehicle or dexamethasone (5 mg/kg) and killed 6 h later. DNA was extracted from thymocytes and 15 µg/lane was electrophoretically separated on 1.8% agarose gels, stained with ethidium bromide, and visualized by UV transillumination. Data are representative of three experiments. B, Detection of 30- to 50-Kb DNA fragmentation. Rats were treated as above, but DNA was prepared for pulsed field gel electrophoresis as described in Materials and Methods. After electrophoresis, DNA was visualized as described above. Data are representative of three experiments. Control lanes in both panels are from animals treated with vehicle for 6 h. Controls from all other time points were similar to these (none showed evidence of internucleosomal or 30- to 50-Kb DNA cleavage, data not shown).

 
Recently, it has been reported that dexamethasone-induced apoptosis involves the cleavage of DNA into 30- to 50-kilobase (Kb) fragments before, and perhaps in the absence of, internucleosomal fragmentation (20, 21). This form of DNA degradation may result from the same nuclease(s) responsible for internucleosomal DNA cleavage, or it may be independently regulated, and represent an additional pathway that cells may use for programmed cell death. We therefore evaluated the integrity of thymocyte DNA by pulsed field gel electrophoresis for evidence of high mol wt fragments under the same experimental conditions in which they were previously tested for internucleosomal DNA degradation. This technique is more sensitive for detection of DNA cleavage during apoptosis, because lesser amounts of large DNA fragments can be detected by ethidium bromide binding compared with the smaller DNA fragments generated by internucleosomal cleavage, and would therefore be expected to show evidence of apoptosis earlier than would internucleosomal DNA degradation. Figure 3BGo demonstrates that a 30- to 50-Kb band of DNA was detectable after 6 h of dexamethasone administration that was not present in the control cells. No high mol wt chromatin cleavage is evident in the thymocytes that survive the initial 72 h of dexamethasone administration (Fig. 3BGo). Thymocyte DNA from 72-h dexamethasone-treated animals reinjected with dexamethasone for an additional 6 h also did not undergo any such DNA fragmentation (Fig. 3BGo). The lack of cleavage of DNA into 30- to 50-Kb fragments corroborates the previous morphological and biochemical analyses indicating that in vivo dexamethasone-treated thymocytes are truly resistant to glucocorticoid-induced apoptosis, and that this is not a function of decreasing levels of hormone due to metabolic loss. In addition, this data demonstrates that this possible alternate pathway for DNA degradation during apoptosis has not been activated in these dexamethasone-resistant cells.

Steroid binding and Western analysis of glucocorticoid receptor
Glucocorticoid resistance may occur by down-regulation of transcription of the glucocorticoid receptor after chronic exposure to hormone, or by mutations that arise in the receptor (22, 23, 24, 25). Experiments were therefore designed to determine whether in vivo dexamethasone-resistant thymocytes had a decreased number of glucocorticoid receptors or an altered binding affinity of receptor that might explain why they do not undergo apoptosis. Thymocytes from both control and 72-h dexamethasone-treated adrenalectomized rats were isolated and analyzed for glucocorticoid receptor binding to [3H]dexamethasone via the method of Scatchard (13). Regression analysis of representative data from one experiment (Fig. 4Go) demonstrates that there was a single class of glucocorticoid receptors in both control thymocytes and the subpopulation of thymocytes resistant to glucocorticoid-induced apoptosis. Table 1Go combines results from several experiments that show that the average receptor number of the control thymocytes was not significantly different from that of the 72-h dexamethasone-treated cells. The binding affinity of glucocorticoid receptor in the control and dexamethasone-resistant thymocytes was comparable. Thus, the observed resistance to glucocorticoids apparently does not reflect altered receptor number or hormone binding affinity, nor the emergence of a glucocorticoid receptor containing hormone binding sites of differing affinity in the dexamethasone-resistant cells.



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  		Figure 4. Scatchard plot analysis of glucocorticoid receptor binding in rat thymocytes. Rats were treated with vehicle ({blacksquare}) or dexamethasone, (•) (5 mg/kg BW) and killed 72 h later. Hormone binding capacity of thymocytes was determined as described in Materials and Methods. r = 0.99 for thymocytes from vehicle-treated animals and r = 0.98 for thymocytes from dexamethasone-treated animals.

 

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  		Table 1. Number of binding sites (Bmax) and binding affinity (Kd) for total control thymocytes and apoptosis-resistant thymocytes

 
We evaluated the integrity of the glucocorticoid receptor by Western analysis to assess whether receptor from in vivo glucocorticoid-resistant cells was modified in some manner (such as proteolysis or phosphorylation) that could account for steroid resistance with normal hormone binding. This assay was performed using epitope-purified antisera generated against the human glucocorticoid receptor, which has previously been shown to cross-react with rat glucocorticoid receptor (18, 26). The cells from which protein was isolated were treated identically to those used for the binding assays (cells were incubated for 2 h in complete media to mimic conditions under which steroid binding assays were performed). Figure 5Go demonstrates that the molecular mass of glucocorticoid receptor was the same in cells isolated from dexamethasone-treated rats and from control animals, indicating that alteration in the glucocorticoid receptor had not occurred in thymocytes resistant to dexamethasone-induced apoptosis in vivo. This data also confirms the whole cell steroid binding studies that demonstrated that thymocytes from control and dexamethasone treated rats contained equivalent amounts of glucocorticoid receptor.



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  		Figure 5. Western analysis of glucocorticoid receptor in rat thymocytes treated for 72 h with dexamethasone. Adrenalectomized rats were treated with vehicle or dexamethasone (5 mg/kg BW) and killed after 72 h. Whole cell lysates were prepared from HTC cells (HTC), thymocytes isolated from control rats (O h), and thymocytes isolated from rats after 72 h dexamethasone treatment (72 h). Cytosolic protein (150 µg/lane) was separated electrophoretically and transferred to nitrocellulose as described in Materials and Methods. Glucocorticoid receptor was detected by reaction with rabbit antisera generated against human glucocorticoid receptor, followed by treatment with antiserum specific for rabbit immunoglobulin and detection as described in Materials and Methods.

 
Effect of dexamethasone on protein synthesis
In an effort to explain the absence of induction of apoptosis by dexamethasone administration in vivo in the resistant thymocyte population, we sought to determine whether the glucocorticoid receptors functioned properly in these cells by measuring the inhibition of protein synthesis in lymphocytes exposed to dexamethasone in vitro (which requires a functional glucocorticoid receptor) (5, 27). Total thymocytes from control animals or glucocorticoid-resistant thymocytes from animals treated for 72 h with dexamethasone were placed in unsupplemented media, or media containing 1 µM of dexamethasone and incubated for 3 h. Tritiated lysine was then administered for 1 h, and radioactive amino acid incorporation into protein was compared between control and dexamethasone-treated groups. Data presented in Fig. 6Go demonstrates that the response of these two populations of cells to the same dose of dexamethasone was the same, because protein synthesis was inhibited equally in both apoptosis-resistant thymocytes (32%) and control thymocytes (31%). These data indicate that both populations had properly functioning glucocorticoid receptors that were capable of eliciting a typical glucocorticoid response. When taken in conjunction with the previous binding studies and Western analysis, these data suggest that the resistance to dexamethasone-induced apoptosis observed in the 72-h dexamethasone-treated thymocytes is not due to any malfunction of glucocorticoid receptor binding or signaling in vitro.



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  		Figure 6. Effect of dexamethasone on [3H]lysine incorporation in rat thymocytes. Total thymocytes from control rats and apoptosis-resistant thymocytes were cultured with vehicle (CON) or 1 µM dexamethasone (DEX) for 3 h and then pulse labeled for 1 h with [3H]lysine. Cells were harvested and radioactive incorporation assayed as described in Materials and Methods. Counts per minutes were normalized for cell number, and percent [3H]lysine incorporation was calculated as cpm/105 treated cells divided by cpm/105 control cells x 100. Each data point represents mean of three samples ± SD.

 
Induction of apoptosis in glucocorticoid resistant thymocytes in vitro
We next sought to determine whether cells resistant to induction of apoptosis by administration of dexamethasone in vivo retained any capacity to undergo apoptosis induced by compounds other than dexamethasone. Lymphoid cell lines previously have been described that are resistant to glucocorticoid-induced apoptosis, but that can undergo programmed cell death when exposed to the calcium ionophore A23187, demonstrating that other initiators of apoptosis exist that operate by nonglucocorticoid-mediated mechanisms to activate the nuclease(s) responsible for programmed cell death (28, 29).

To determine whether the population of thymocytes resistant to dexamethasone-induced apoptosis could undergo calcium ionophore-induced apoptosis, thymocytes were isolated from 72-h dexamethasone-treated animals and placed in media with 0.1 µM A23187 or vehicle. As an additional control, in vivo dexamethasone-resistant thymocytes were also treated with 1 µM dexamethasone. DNA was isolated from all cells, and its integrity analyzed by agarose gel electrophoresis at 0, 3, and 6 h after treatment. As shown in Fig. 7Go, apoptosis-resistant thymocytes in the control group did not undergo internucleosomal DNA degradation at any time point. In cells treated with A23187, an internucleosomal DNA cleavage ladder was evident at 3 h after treatment and increased in intensity at 6 h, demonstrating that thymocytes resistant to glucocorticoid in vivo do have the capacity to undergo apoptosis by nonglucocorticoid-mediated mechanisms. Surprisingly, these cells also underwent apoptosis in response to dexamethasone treatment in vitro. This effect was detected after 6 h of dexamethasone treatment. Furthermore, pulsed field gel electrophoresis analysis of in vitro treated thymocytes detected a 30- to 50-Kb DNA band in the treatment groups in which internucleosomal DNA degradation was observed (data not shown). Therefore, although these cells are resistant to glucocorticoid-induced apoptosis in vivo, they become sensitive when removed from the natural environment of the animal.



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  		Figure 7. Effect of in vitro administration of dexamethasone and calcium ionophore A23187 on thymocytes resistant to dexamethasone-induced apoptosis in vivo. Adrenalectomized rats were treated with dexamethasone (5 mg/kg BW) and killed 72 h later. Thymocytes were isolated, placed in culture, and treated with vehicle (CONTROL), dexamethasone (DEX) (1.0 µM), or calcium ionophore (A23187) (0.1 µM). DNA was isolated from these cells 3 or 6 h later, and 15 µg of each sample was electrophoretically separated on 1.8% agarose gels, stained with ethidium bromide, and visualized by UV transillumination.

 
Western analysis of Bcl-2 expression in glucocorticoid-sensitive and -resistant thymocytes
Thymocytes that are resistant to induction of apoptosis by glucocorticoids in vivo could be influenced by an inhibitor of programmed cell death. The product of the bcl-2 proto-oncogene is one such inhibitor that is widely known to suppress apoptosis induced by glucocorticoids in many lymphoid cell lines, and is expressed in many cells in the thymus (30, 31, 32). Surprisingly, a majority of apoptotic thymic cells appear to express Bcl-2. Dexamethasone has been recently demonstrated to down-regulate expression of bcl-2 in myeloid leukemic cells (33, 34), however, its effect on thymocytes has not been documented. Therefore, it became important to determine whether the resistance to glucocorticoids that we observed reflected an increase in Bcl-2 expression. We therefore analyzed the expression of this protein in thymocytes isolated from vehicle-treated control rats, and rats that had been treated for 72 h with dexamethasone (Fig. 8Go). There was no appreciable difference in the level of Bcl-2 in the glucocorticoid-resistant thymocyte population when compared with control thymocytes, suggesting that Bcl-2 expression is not responsible for the suppression of apoptosis in the resistant population.



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  		Figure 8. Western analysis of Bcl-2 expression in rat thymocytes. Thymocytes were isolated from vehicle-treated rats (CON) and rats treated with 1 µM dexamethasone for 72 h (DEX). Total protein (100 µg/lane) from these thymocytes was separated by SDS-PAGE and electrophoretically transferred to nitrocellulose as described in Materials and Methods. Western analysis was performed as described in Materials and Methods using antiserum generated against rat/mouse Bcl-2.

 

   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
References
 
Glucocorticoid hormone induces programmed cell death in the majority of cells in the rat thymus. Immature cortical thymocytes are extremely sensitive to glucocorticoid-induced death, whereas the more mature medullary thymocytes persist after steroid treatment (7). This observation has been confirmed by the absence of internucleosomal DNA degradation in surviving thymocytes obtained from rats treated with dexamethasone for 72 h (3). Furthermore, nuclease activity detected in thymocyte extracts between 2 and 48 h after dexamethasone administration is no longer observed after 72 h treatment with hormone (35). We have demonstrated that a population of thymocytes exists that is resistant to dexamethasone-induced apoptosis in vivo, and that this resistance is probably not due to a decreased level of dexamethasone in the animal or to increased expression of Bcl-2.

Resistance to glucocorticoids has previously been described in lymphoid cell lines (22, 23, 24, 25, 35). These studies have revealed that down-regulation of receptor or gene mutations that affect receptor function can convey steroid resistance. We therefore examined a thymocyte population that is resistant to induction of apoptosis by administration of dexamethasone in vivo for possible alterations in receptor level or hormone-receptor binding properties in an attempt to explain their decreased sensitivity to glucocorticoid-induced apoptosis. We observed that the apoptosis-resistant thymocytes have the same number of glucocorticoid receptors with equivalent ligand binding affinity as control cells. In similar studies performed in mice, a decreased binding affinity for glucocorticoids was observed in apoptosis-resistant thymocytes after long-term steroid treatment (36, 37). This apparent reduction in affinity probably reflects occupation of receptors by hormone added in vivo, because these cells were evidently not washed before receptor measurement. During our analysis of binding affinity of glucocorticoid receptor, cells were washed at 37 C under conditions previously shown to promote ligand dissociation and minimal receptor occupancy by hormone administered in vivo (38). Western analysis of glucocorticoid receptor in thymocytes from control and dexamethasone-treated rats confirmed that glucocorticoid receptor level and mol wt does not change after dexamethasone treatment. We have also demonstrated inhibition of protein synthesis in surviving cells after treatment with dexamethasone in vitro, indicating that the glucocorticoid receptor is functional. Studies in mice found that variations between corticosterone-resistant and -sensitive thymocytes obtained under other experimental paradigms were not due to impaired receptor function (37, 39). Resistance to steroid-induced cell death was attributed to the stage of cell differentiation (37), as well as to some mechanism in the thymic medulla responsible for protecting some cells from cytolysis (39). Our data indicate that resistance to apoptosis in the population of cells we examined is not due to diminished numbers or altered function of glucocorticoid receptors (when evaluated in vitro).

In an attempt to determine whether in vivo dexamethasone-resistant thymocytes retain any capacity to undergo apoptosis, we treated them with the calcium ionophore A23187 in vitro. This ionophore initiates apoptosis by nonglucocorticoid-mediated mechanisms by activating the calcium-magnesium dependent nuclease(s) responsible for internucleosomal degradation (29). Evidence of chromatin cleavage after 3 h in vitro indicates that these resistant cells are still able to undergo apoptosis. Most surprising was the observation that thymocytes resistant to glucocorticoid-induced apoptosis, when treated with a maximal dose of dexamethasone in vivo, undergo programmed cell death in vitro when treated with a concentration of dexamethasone (1 µM) less than that achieved in tissues during in vivo dexamethasone administration (3). This novel finding suggests a protective mechanism exists in vivo that is not present or functioning in vitro.

We wished to examine whether overexpression of the apoptosis inhibitor Bcl-2 might explain why surviving thymocytes are resistant to induction of apoptosis by glucocorticoids. Bcl-2 is expressed at equivalent levels in both normal rat thymocytes and thymocytes that survive 72 h treatment with dexamethasone, and therefore does not appear to be responsible for the resistant phenotype. Although it is possible that expression of other proteins that modulate the susceptibility of thymocytes to induction of apoptosis by glucocorticoids is altered in these resistant cells, their rapid susceptibility to dexamethasone in vitro suggests that other mechanisms are responsible for their resistance to apoptosis.

A major role of the thymus gland is to orchestrate the maturation of T cells. T cell differentiation is a complex process involving both positive and negative selection (40), and experimental evidence indicates that apoptosis plays an important role in the latter (41, 42). Recent studies have attempted to elucidate the intricate role that the thymus plays in this maturation process (43, 44). T cell maturation occurs by both direct contact of developing thymocytes with thymic stromal cells as well as by secretion of cytokines, including interleukin-6, granulocyte colony-stimulating factor, and macrophage colony-stimulating factor by these stromal cells (45, 46, 47, 48). A variety of studies have demonstrated that interleukins modulate glucocorticoid-induced apoptosis (36, 49, 50), as well as T cell receptor-mediated (51) and anti-CDs-induced cell death (52). The inhibitory effect of interleukins on programmed cell death therefore offers a plausible explanation for in vivo resistance of cells to apoptosis that is lost when cells are removed from the thymus. Interleukin-4 was unable to protect rat thymocytes resistant to induction of apoptosis by in vivo dexamethasone administration from apoptosis induced in vitro (data not shown). Cell-to-cell interaction with thymic stromal cells is necessary for proper differentiation of T cells, and such interplay within the natural environment of the thymus gland may confer protection from apoptosis to the glucocorticoid-resistant cells (44). Indeed, a thymic stromal cell line has been shown to be able to partially protect thymocytes from induction of apoptosis (53). This protection was not conferred solely by soluble factors or by cell-cell contact. Although the exact mechanism of resistance of a subpopulation of thymic lymphocytes to glucocorticoid-induced apoptosis has yet to be determined, there appears to be a complex array of regulatory path-ways that both induce and block glucocorticoid-mediated apoptosis.


   Footnotes
 
1 The first two authors contributed equally to this manuscript. Back

2 Howard Hughes Medical Institute Medical Student Research Training Fellow during completion of this research. Back

Received July 29, 1996.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 References
 

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