Conditional Expression of a Glucocorticoid Receptor Transgene in Thymocytes Reveals a Role for Thymic-Derived Glucocorticoids in Thymopoiesi
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内分泌学杂志 2005年第6期
Department of Medical Nutrition (A.P., S.O.), Karolinska Institutet, Karolinska University Hospital Huddinge, Novum, SE-141 86 Huddinge, Sweden; and Microbiology and Tumour Biology Center (M.J.), Karolinska Institutet, SE-171 77 Stockholm, Sweden
Address all correspondence and requests for reprints to: Prof. Sam Okret, Department of Medical Nutrition, Karolinska Institutet, Karolinska University Hospital Huddinge, Novum, SE-141 86 Huddinge, Sweden. E-mail: Sam.Okret@mednut.ki.se.
Abstract
We and others have previously reported that thymic epithelial cells produce glucocorticoids (GCs). In vitro studies have also suggested that thymic-derived GCs play a role in the development of thymocytes. However, until now it has not yet been established whether thymic-derived GCs play a role in thymopoiesis in vivo. To investigate this, we conditionally overexpressed the GC receptor (GR) in thymocytes using transgenic mice with a tetracycline-inducible expression system. The influence of systemic GCs was excluded by adrenalectomizing the transgenic mice before the GR induction. Conditional expression of transgenic GR in the thymocytes of adrenalectomized transgenic mice led to a decrease in the thymocyte number. This was associated with increased thymocyte apoptosis. The effect of thymic-derived GCs on the thymocytes was confirmed after transgenic GR induction in a thymic organ culture system. Finally, the GR antagonist RU486 increased thymocyte number in adrenalectomized mice in vivo and prevented a reduction in thymocyte number in thymic organ culture after transgenic GR induction. These observations further confirmed a role for the thymic-derived GCs in regulating thymocyte homeostasis in vivo.
Introduction
GLUCOCORTICOIDS (GCs) ARE A GROUP of steroid hormones that, in addition to their metabolic and developmental effects, have immunosuppressive and antiinflammatory properties and are essential in regulation of immune responses (1). GCs are also very potent inducers of apoptosis in thymocytes (2, 3). GCs act through interaction with GC receptor (GR), which is expressed in most cell types including thymocytes (4). Notably, the GR concentration is a major factor determining cellular sensitivity toward GCs both in vitro and in vivo (5, 6, 7, 8, 9).
Thymus plays a crucial role in the immune system by generating T cells. In the thymus, highly immature CD4–CD8– double-negative thymocytes proliferate and undergo extensive genetic and phenotypic alteration to yield the CD4+CD8+ double-positive (DP) population. Most of the DP thymocytes undergo apoptosis, and less than 5% of the DP thymocytes survive and differentiate into either CD4+ or CD8+ single-positive (SP) thymocytes, which then will seed the peripheral lymphoid tissues (1). Thus, apoptosis and cell proliferation are major regulators of thymocyte homeostasis. It has been shown that endogenous GCs play a role in the development, differentiation, and homeostasis of thymocytes (1, 9, 10), although the direction of the response is a matter of debate (10, 11, 12).
It has also been reported that the thymus is cable of producing GCs (13, 14, 15, 16). Vacchio et al. (13) showed that cultured thymic non-T cells produced soluble pregnenolone and deoxycorticosterone after addition of the precursor 22R- hydroxycholesterol. Furthermore, we and others using various techniques have demonstrated the presence of steroidogenic enzymes such as cytochrome P450 hydroxylases Cyp11A1, Cyp21, and Cyp11B1 in the thymus (13, 14, 15, 16). These enzymes, in combination with the enzyme 3?-hydroxysteroid dehydrogenase, convert cholesterol into corticosterone, the major GC in rodents. Most results suggest thymic epithelial cells (TEC) as the main source of the thymic derived GCs. We have previously reported that when TEC were cocultured with reporter cells containing the GR and a GR-dependent reporter gene, a specific induction of reporter gene activity was observed, showing that TEC can produce active GCs (14).
Previous studies have shown that thymic-derived GCs affect the development of thymocytes in vitro (1). However, until now it has not been clear whether thymic-derived GCs play a role in regulating thymopoiesis in vivo. The aim of the present work was to address this question. This was investigated by creating a tetracycline-inducible expression system to conditionally express a GR transgene in thymocytes after treatment of the transgenic animals with the tetracycline analog doxycycline (DOX). Transgenic mice were adrenalectomized before transgene induction to exclude the effects of systemic GCs. To investigate the effects of thymic-derived GCs, the number of thymocytes and the percentage of thymocyte populations were examined. Because apoptosis and cell proliferation play a crucial role in the regulation of thymopoiesis, we also analyzed the percentage of apoptotic and cycling thymocytes. Our results demonstrated that overexpression of the GR in thymocytes in the absence of systemic GCs led to an increase in the percentage of apoptotic cells and to a decrease in the number of thymocytes. We also examined the effect of the GR antagonist RU486 on thymopoiesis in adrenalectomized mice and on thymic organ culture after transgenic GR induction. Results from the above-described in vivo experiments, in combination with results obtained from thymic organ culture experiments, demonstrated that thymic-derived GCs play a regulatory role in thymopoiesis in vivo.
Materials and Methods
Antibodies and reagents
Fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (01064A) and phycoerythrin (PE)-conjugated anti-CD8 (01045A) were purchased from BD Pharmingen (San Diego, CA). Mouse monoclonal antibody recognizing only the rat GR made in our lab has previously been described (9). Horseradish peroxidase-linked sheep antimouse Ig was purchased from Amersham Biosciences (Buckinghamshire, UK). BSA, mifepristone (RU486), DOX, and propidium iodide (PI) were purchased from Sigma (St. Louis, MO). Deoxyribonuclease-free ribonuclease A was purchased from Roche Molecular Biochemicals (Mannheim, Germany). RPMI 1640 medium, penicillin, streptomycin, and L-glutamine were purchased from Life Technologies, Inc.-BRL (Grand Island, NY).
Generation of hCD2-rtTA/TRE/CMVmin-rGR double-transgenic mice
Mice with a conditional tetracycline-regulated expression of a transgenic rat GR in the thymocytes (hCD2-GR) were generated by crossing TRE/CMVmin-rGR transgenic mice (generated in our laboratory) with hCD2-rtTA transgenic mice. The transgene for generation of TRE/CMVmin-rGR transgenic mice was generated by cloning the cDNA encoding the full coding region of the rat GR cDNA [BamH1 fragment from SVGR1 (17) downstream of the TRE/CMVmin promoter of the pTRE2 vector (Clontech Laboratories, BD Biosciences, Palo Alto, CA)]. The TRE/CMVmin-rGR transgenic mice were generated by microinjection of the construct into the (CBA-C57BL/6) fertilized eggs by standard procedure. Offspring from two tested founder TRE/CMVmin-rGR transgenic mice gave the same phenotype when crossed with the hCD2-rtTA transgenic mice. The hCD2-rtTA transgenic mice were kindly provided by Dr. Rose Zamoyska (18) These were on a C57BL/6 background. The hCD2-rtTA transgenic mice express a reverse transactivator (rtTA) under the control of the hCD2 gene regulatory sequence, directing expression of this chimeric transactivator to the T cell lineage. Generation of these mice has been described previously (Division of Molecular Immunology, National Institute for Medical Research, London, UK) (18). Transmission of transgenes was monitored by PCR analysis of tail DNA, using the following primers. Sense and antisense primers used for the hCD2-rtTA mice were 5'-ATA AAA AGC ACT GTG GAT TTC TGC-3' and 5'-AGA GGG GAC AGG AAA TCT CTA AGA-3', respectively. The sense and antisense primers for TRE/CMVmin-rGR mice were 5'-AAT GAG GTC GGA ATC GAA GG-3' and 5'-TAG CTT GTC GTA ATA ATG GCG G-3', respectively. To conditionally express the transgenic rat GR in the thymocytes, double-transgenic mice (hCD2-GR) were treated with 1 mg/ml DOX in 2% sucrose in their drinking water, which was changed every second day. DOX/sucrose-treated single-transgenic or just sucrose-treated double-transgenic mice served as controls. Four- to 8-wk-old male mice were used for all experiments. The number of thymocytes presented in the figures refers to thymocyte number per thymus. For controls, thymuses from littermates, nontreated double-transgenic hCD2-GR or single-transgenic TRE/CMVmin-rGR or hCD2-rtTA transgenic mice, were used as indicated in figure legends. All animal experimentation was conducted in accord with accepted standards of humane animal care, as approved by the local animal ethical committee.
Western blot analysis
Western blot analysis of GR in whole-cell protein extracts from purified thymocytes was performed as explained before (9). Briefly, protein concentrations in the cell extracts were quantified with the Bio-Rad kit (Bio-Rad Laboratories, Inc., Hercules, CA). Extracted proteins were separated on an 8% sodium dodecyl sulfate polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were incubated with antibodies recognizing only transgenic rat GR (9). After incubation membranes were washed and incubated with horseradish peroxidase-linked sheep antimouse Ig. To check for efficiency of transfer and equal loading, membranes were stained with Ponceau S (Sigma) solution.
Isolation and flow cytometric analysis of thymocytes
Thymus was gently passed through a steel net. Cells were washed with and resuspended in ice-cold RPMI 1640 containing penicillin-streptomycin, L-glutamine, and 0.5% BSA. For flow cytometric analysis of thymocyte populations, cells were stained with anti-CD4-FITC and anti-CD8-PE monoclonal antibodies in ice-cold complete RPMI 1640 medium for 1 h on ice. Cells were washed and resuspended in PBS containing 1% BSA. A total of 10,000 gated cells were analyzed by flow cytometry (FACScan) and the CellQuest program (both from BD Biosciences, Immunocytometry Systems, San Jose, CA).
Adrenalectomy
Adrenalectomy was performed by standard procedure under Avertin (tribromoethanol) anesthesia. Adrenalectomized mice were given 0.7% NaCl in their drinking water to maintain mineral balance. Animals were allowed to recover for 3 d before experiments were performed.
Treatment of mice with RU486
Mice were daily injected ip with 100 μg RU486 (dissolved in sesame oil) for 1 wk. Control mice were injected with an equal volume of sesame oil.
Measuring serum corticosterone level
A solid-phase 125I-RIA system (Diagnostic Products Corp., Los Angeles, CA) was used to determine serum corticosterone levels according to the manufacturer’s recommendations. Mice were retroorbitally bled under methoxyflurane (Metofane) anesthesia before killing. The sensitivity of the assay is 5 ng/ml.
Cell cycle analysis of thymocytes
For flow cytometric analysis of cell cycle, 1 x 106 thymocytes were washed with sample buffer (PBS supplemented with 1% BSA and 1 g/liter glucose), fixed in cold (–20 C) 70% ethanol, and stored at –20 C overnight. The samples were centrifuged and the pellets were resuspended in 1 ml sample buffer containing PI (50 μg/ml) and deoxyribonuclease-free ribonuclease A (50 μg/ml) for 1 h at room temperature while the tubes were vigorously agitated on a shaking platform. A total of 10,000 gated cells (to exclude debris) were analyzed by flow cytometry in a FACScan.
In vitro studies on the effect of induced GR expression on thymocyte survival
Thymus lobes were cut into small pieces and transferred into the Eppendorf tubes containing 0.5% BSA in serum-free RPMI 1640 medium in the absence or presence of 2 μg/ml DOX and 1 μM RU486 for 3 d. Thymus pieces were gently passed through a steel net. Cells were washed with and resuspended in ice-cold PBS containing 1% BSA. The percentage of living cells was determined by flow cytometric analysis of the morphological characteristics of living cells using the forward vs. side scatter, representing the size and density of the cells respectively.
Statistical methods
All results are expressed as mean ± SD, unless otherwise stated. For data analysis, a two-tailed homoscedastic Student’s t test was used with P 0.05 considered significant. When indicated, one-way ANOVA using the GraphPad Prism software (Graph Pad Software Inc., San Diego, CA) was performed.
Results
Conditional expression of a transgenic GR in the thymocytes of the hCD2-GR transgenic mice
The generation of transgenic mice that conditionally express transgenic GR in the thymocytes was based on the tetracycline-inducible expression system (19). To produce these mice, mice containing the GR cDNA under the control of tetracycline-controlled promoter (TRE/CMVmin-rGR) (Fig. 1A, 2) were crossed with mice constitutively expressing the chimeric transactivator rtTA under the control of the hCD2 gene regulatory sequence, which directs expression of this chimeric transactivator to the thymocytes (18) (Fig. 1A, 1). This will allow the transgenic rat GR to be conditionally expressed in the thymocytes of double-transgenic mice (hCD2-GR) after treatment of mice with DOX. To demonstrate the expression of transgenic GR, protein extracts from the thymocytes of hCD2-GR mice were analyzed by Western blotting using a monoclonal antibody that recognizes the transgenic rat GR but not the endogenous mouse GR. As demonstrated in Fig. 1B, the transgenic rat GR was expressed in the thymocytes only after treatment of mice with DOX. Expression after DOX administration was not detected in other tissues such as the liver and lung (data not shown).
FIG. 1. Transgene constructs and conditional expression of the GR transgene in the thymocytes of hCD2-GR transgenic mice. A, Schematic representation of the two transgene constructs (1 and 2) used for generation of hCD2-GR double-transgenic mice. The hCD2-GR double-transgenic mice were produced by crossing the transgenic mice containing a rat GR transgene downstream of a tetracycline-controlled TRE/CMVmin promoter (2) with transgenic mice constitutively expressing a chimeric transactivator rtTA under the control of the hCD2 gene regulatory sequence (1). B, Conditional expression of the transgenic GR in the thymocytes. Transgenic mice (4 wk of age) were treated with DOX (1 mg/ml in drinking water supplemented with 2% sucrose) for 1 wk. Control transgenic mice (–DOX) were given drinking water supplemented with 2% sucrose alone. After treatment, thymocytes were isolated and protein extracts from thymocytes were analyzed by Western immunoblotting. Transgenic rat GR was detected with an anti-GR antibody that recognizes the rat but not the endogenous mouse GR.
Conditional expression of transgenic GR in the thymocytes decreased the number thymocytes
To investigate the in vivo effects of conditional expression of transgenic GR in the thymocytes on thymocyte homeostasis, double-transgenic mice were treated with DOX for 1 wk. Thymocytes were then isolated and counted. As can be seen in Fig. 2A, expression of the transgenic GR resulted in a reduction in the total number of thymocytes (P < 0.001). This demonstrated that transgenic GR expression affects thymopoiesis.
FIG. 2. Effects of conditional transgenic GR expression on thymocyte number. The hCD2-GR double-transgenic mice and the hCD2-rtTA single-transgenic mice were treated with DOX (1 mg/ml in drinking water supplemented with 2% sucrose) for 1 wk. Control transgenic mice (–DOX) were given drinking water supplemented with 2% sucrose alone. After treatment thymocytes were isolated and counted. A, Shown is absolute number of thymocytes from 6-wk-old control (–DOX) and DOX-treated (+DOX) hCD2-GR double-transgenic mice. B, Shown is absolute number of thymocyte populations from 4 wk control (–DOX) and DOX-treated (+DOX) hCD2-rtTA single-transgenic mice. Data are shown as mean ± SD from six mice in each group. Note that the difference in the total cell number of thymocytes between the control hCD2-GR double-transgenic mice (A) and the control hCD2-rtTA single-transgenic mice (B) is due to analyzing these mice at slightly different ages.
To exclude a nonspecific effect of rtTA expression and DOX treatment on thymopoiesis, we treated the single-transgenic hCD2-rtTA mice, in which thymocytes constitutively express the rtTA, with DOX in the same way as the hCD2-GR double-transgenic mice were treated. DOX treatment of these mice did not affect the number of thymocytes (Fig. 2B). Also flow cytometric analysis of thymocytes from DOX-treated single-transgenic hCD2-rtTA, tetracycline-responsive element-GR, or wild-type mice did not show any nonspecific effects of these factors on the number of different populations of thymocytes (data not shown). These control experiments confirmed that the effects observed after DOX treatment of the double-transgenic hCD2-GR mice were mediated only by the expression of the transgenic GR in the thymocytes.
Conditional expression of transgenic GR in thymocytes in the absence of systemic GCs decreased the number of thymocytes
To investigate possible in vivo effect of the thymic-derived GCs on thymopoiesis, we first adrenalectomized hCD2-GR transgenic mice to exclude the influence of systemic GCs. Measuring serum corticosterone levels after adrenalectomy confirmed the absence of circulating GCs (Fig. 3A). Adrenalectomy resulted in an approximately 2-fold increase in the number of thymocytes compared with the number of thymocytes in intact (nonadrenalectomized) hCD2-GR transgenic mice (Fig. 3B, P < 0.001, n = 6). A group of adrenalectomized hCD2-GR transgenic mice were given DOX in the drinking water for 1 wk, after which thymocytes were counted and analyzed by flow cytometry. The results demonstrated a reduction in the total number of thymocytes (Fig. 3B, P < 0.01, n = 6). However, no significant change was observed in the percentage of different thymocyte populations, demonstrating that all four populations were similarly affected by the thymic-derived GCs (Fig. 3C). The values for the percentage of different thymocyte populations in treated vs. controls were as follows. CD8+ SP: 2.7 ± 0.2 vs. 2.6 ± 0.3, CD4+ SP: 9.7 ± 0.3 vs. 9.6 ± 0.3, DP: 82 ± 0.9 vs. 81 ± 1.7, double negative: 4.5 ± 0.2 vs. 4.9 ± 0.3.
FIG. 3. Effects of conditional transgenic GR expression in the thymocytes on thymocyte number and populations in adrenalectomized mice. The hCD2-GR double-transgenic mice (8 wk of age) were adrenalectomized. After recovery, mice were treated with DOX (1 mg/ml in drinking water supplemented with 0.7% NaCl and 2% sucrose) for 1 wk. Control transgenic mice (Ctrl) were given drinking water supplemented with just 0.7% NaCl and 2% sucrose. A, Serum corticosterone concentration in adrenalectomized (ADX) and control (Ctrl) mice as mean ± SD from six mice in each group. The corticosterone concentrations in ADX mice were below detection level for the assay (5 ng/ml). B, The absolute number of thymocyte from nonadrenalectomized (Non-Adx), adrenalectomized (Adx), or from DOX-treated adrenalectomized (Adx/Dox) hCD2-GR double-transgenic mice as mean ± SD from six mice in each group. C, The flow cytometric analysis of thymocyte populations. Thymocytes from control (Ctrl) or DOX-treated (DOX) adrenalectomized hCD2-GR double-transgenic mice were stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 monoclonal antibodies, and analyzed by flow cytometry. Single cells are displayed on a dot plot of CD4 vs. CD8. The numbers indicate the percentage of cells in each quadrant.
Conditional expression of a transgenic GR in thymocytes in the absence of the systemic GCs increased the percentage of apoptotic cells but not the cycling thymocytes
Thymocyte homeostasis is mainly regulated by apoptosis and cell proliferation. Furthermore, thymocytes are highly sensitive to apoptotic effects of GCs. Thus we measured the percentage of apoptotic cells in isolated thymocytes from control and DOX-treated hCD2-GR transgenic mice by flow cytometry. As can be seen in Fig. 4A, DOX treatment of adrenalectomized hCD2-GR transgenic mice led to an increase in the percentage of apoptotic thymocytes (10.1 ± 1.7% in treated mice vs. 5.2 ± 1.2% in nontreated mice, P < 0.01, n = 6). DOX did not have any effect on the percentage of apoptotic cells in nontransgenic mice (data not shown, see also Fig. 6, A and B). Because the number of thymocytes also is regulated by cell proliferation, we investigated the effects of thymic-derived GCs on the percentage of cycling cells. Flow cytometric analysis of the cell cycle in thymocytes from control and DOX-treated adrenalectomized hCD2-GR transgenic mice did not show any effect of thymic-derived GCs on the percentage of cycling thymocytes (S/G2/M phase of the cell cycle) (10.5 ± 0.9% in treated mice vs. 10.3 ± 0.8% in nontreated mice, n = 6) (Fig. 4B).
FIG. 4. Effects of conditional transgenic GR expression in the thymocytes of adrenalectomized mice on thymocyte apoptosis and cell cycle. The hCD2-GR double-transgenic mice were adrenalectomized. After recovery, mice were treated with DOX (1 mg/ml in drinking water supplemented with 0.7% NaCl and 2% sucrose) for 1 wk. Control transgenic mice (Ctrl) were given drinking water supplemented with just 0.7% NaCl and 2% sucrose. Thymocytes were then isolated, counted, and analyzed by flow cytometry. A, Determining the percentage of apoptosis in isolated thymocytes from control (Ctrl) and DOX-treated (DOX) adrenalectomized hCD2-GR transgenic mice. Shown are representative flow cytometric analysis of thymocytes based on the morphological characteristics of living and apoptotic cells using the forward scatter vs. side scatter, representing the size and density of the cells, respectively. The apoptotic cells give lower forward scatter and higher side scatter values. The percentages of apoptotic cells are given in the figures. The results are representative of one of six mice in each group. P < 0.01. B, Flow cytometric analysis of the cell cycle in thymocytes from control (Ctrl) and DOX-treated (DOX) adrenalectomized hCD2-GR transgenic mice. Thymocytes were fixed, premeabilized, stained with PI, and analyzed by flow cytometry. Shown are DNA histograms of thymocytes. The percentages of cycling cells (S/G2/M phase of the cell cycle) are given in the figures (P = not significant). The results are representative of one of six mice in each group.
FIG. 6. Effect of thymic-derived GCs on the survival of thymocytes in vitro. A, Thymuses obtained from 6-wk double-transgenic (hCD2-GR) and control (hCD2-rtTA) mice were cultured in serum-free RPMI 1640 medium containing 0.5% BSA in the absence (–DOX) or presence (+DOX) of 2 μg/ml DOX for 3 d. Thymocytes were then isolated and the percentage of living cells was determined by flow cytometric analysis. In the figures, single cells are displayed on a dot plot of forward scatter vs. side scatter. The percentages of living cells are given in the figures. The results are representative of one of six samples in each group. B, The bars show the percentage of living cells as mean ± SD from six samples in each group. P < 0.001 for DOX-treated vs. nontreated thymuses from hCD2-GR double-transgenic mice. C, Thymuses obtained from double-transgenic hCD2-GR mice were treated with 2 μg/ml DOX in the absence or presence of 1 μM RU486. The bars show the percentage of living cells as mean ± SD from six samples in each group. A one-way ANOVA showed that all the means are significant different (P < 0.05). The Bonferroni’s multiple comparison test showed that P < 0.05 between control (Ctrl) vs. Dox/RU. The other P values are shown in the figure.
Treatment of adrenalectomized and intact hCD2-GR transgenic mice with the GR antagonist RU486 increased the number of thymocytes
To find further support for a role for the thymic-derived GCs on thymopoiesis in vivo, we treated adrenalectomized hCD2-GR transgenic mice with the GR antagonist RU486. If thymic-derived GCs participate in the regulation of thymocyte homeostasis, it is expected that the GR antagonist RU486 will antagonize this effect. As demonstrated in Fig. 5A, treatment of adrenalectomized transgenic mice with RU486 resulted in increased number of thymocytes, as compared with the control mice (adrenalectomized transgenic mice treated with just the solvent alone, P < 0.001, n = 5). This observation further supported that thymic-derived GCs affects thymopoiesis. In addition, we treated intact nonadrenalectomized hCD2-GR transgenic mice with RU486. The result showed an increase in the number of thymocytes, confirming a suppressive effect of endogenous GCs on thymocyte homeostasis (Fig. 5B).
FIG. 5. Effect of RU486 treatment of adrenalectomized and intact hCD2-GR transgenic mice on the number of thymocytes. A, hCD2-GR transgenic mice (8 wk of age) were adrenalectomized and given 0.7% NaCl in their drinking water to maintain mineral balance. After recovery, the mice were treated by daily ip injections with the GR antagonist RU486 (100 μg/d dissolved in sesame oil) for 1 wk. Control adrenalectomized hCD2-GR transgenic mice were injected with the solvent (sesame oil) alone. Thymuses were removed. Thymocytes were isolated and counted. Shown is the absolute number of total thymocytes from adrenalectomized hCD2-GR transgenic mice (Ctrl) or from RU486-treated adrenalectomized hCD2-GR transgenic mice as mean ± SD from five mice in each group. B, Nonadrenalectomized hCD2-GR transgenic mice (8 wk of age) were treated with RU486 or vehicle alone as explained above. Shown is the absolute number of total thymocytes from nonadrenalectomized vehicle-treated mice (Ctrl) or from nonadrenalectomized RU486-treated mice as mean ± SD from five mice in each group.
Expression of the transgenic GR reduced the percentage of living thymocytes in vitro
We also examined the effects of thymic-derived GCs on the thymocyte survival in vitro. This was performed by treating isolated thymuses obtained from hCD2-GR transgenic mice in a thymic organ culture system with DOX. Thymocytes were then isolated and analyzed for the percentage of living cells. Similar treatment was performed in parallel for thymuses obtained from control (hCD2-rtTA) mice. As can be seen in Fig. 6, A and B, DOX treatment of isolated thymuses from hCD2-GR mice reduced the percentage of living thymocytes by approximately 50% compared with the nontreated hCD2-GR transgenic thymocytes (P < 0.001, n = 6). DOX treatment of the thymuses obtained from control (hCD2-rtTA) mice did not affect the survival of thymocytes (Fig. 6, A and B). In an additional experiment, thymuses obtained from transgenic hCD2-GR mice were treated with DOX in the absence or presence of RU486 (Fig. 6C). The results showed that RU486 could partly prevent the effects of GR overexpression on thymocyte death. These in vitro results derived using isolated thymuses indeed demonstrated that the survival of thymocytes was influenced by the thymic-derived GCs.
Discussion
Endogenous GCs are known to play an important regulatory role in the function of the immune system. This includes a role in the development, homeostasis, and function of T cells (1, 9). It is also well established that thymocytes are highly sensitive to the apoptotic effects of GCs (2, 3). Of interest to this work, we and others have previously reported that the thymus is capable of producing GCs (13, 14, 15, 16). Later studies suggested that the thymic-derived GCs regulate the development of the thymocytes (1). This was mainly based on in vitro studies in a thymic organ culture system in which corticosteroid biosynthesis was blocked by steroidogenic inhibitors. However, whether thymic-derived GCs exert an effect on thymopoiesis in vivo has not been established. In this report we addressed this question by using a system in which a DOX-inducible transgenic GR was expressed in the thymocytes of transgenic mice in vivo. The transgenic mice were depleted of systemic GCs by adrenalectomy to exclude the influence of systemic GCs on thymopoiesis. The absence of systemic GCs was verified by serum measurements using a sensitive RIA. In such a condition, paracrine effects of thymus-derived GCs are most likely responsible for the observed effects. The results obtained from these in vivo studies showed that thymic-derived GCs suppress thymopoiesis. This could be explained by decreased survival of thymocytes. This is in concord with our previous observation showing that constitutive overexpression of GR in the thymocytes in transgenic mice led to an increased apoptosis and a decrease in the number of thymocytes (9). Thus, thymocyte homeostasis is controlled by both adrenal (systemic) and thymus-derived GCs. The observation that indeed thymic-derived GCs affect the thymocytes was confirmed by our complementary in vitro experiments, in which thymuses were isolated and the transgenic GR was induced in vitro in a thymic organ culture system. An additional advantage of the inducible expression system is that it excludes "metabolic programming" effects of a transgene during embryonic or neonatal stages that would possibly affect the phenotype later in life. The effect of thymic-derived GCs on thymopoiesis in vivo was further supported by treating adrenalectomized mice with the GR antagonist RU486, as this resulted in increased thymocyte number. RU486 also partially prevented the DOX-induced reduction in thymocyte number in thymic organ cultures of hCD2-GR transgenic mice. Although precautions have to be taken when it comes to conclusions drawn from RU486 experi-ments alone, because RU486 also may act via the androgen or progestin receptor, these experiments support the results from the GR-inducible transgenic mice that the decrease in thymocyte number after transgenic GR expression is a GC-GR-mediated effect.
In our in vivo and in vitro studies we observed a decreased survival of thymocytes after the GR induction in the thymocytes, whereas the cell cycle of thymocytes were not affected. The observed decreased survival suggests a mechanism by which thymic-derived GCs could reduce the number of thymocytes in vivo. This result was in line with our previous report demonstrating that coculturing of TEC with thymocytes decreased survival of thymocytes (14). In our in vivo and in vitro experiments we also found no indication that DOX treatment by itself, especially in combination with expression of the rtTA transcription factor, had any nonspecific effect on thymopoiesis. A similar observation has previously been reported (20). These experiments excluded a nonspecific effect of the tetracycline-inducible expression system on thymopoiesis. Furthermore, expression of the transgenic rat GR does not affect the endogenous GR level (4). In addition, we found no evidence that expression in the thymocytes of the transgenic GR itself results in a ligand-independent activation of GR. This conclusion was based on the observation that the percentage of apoptosis in purified hCD2-GR thymocytes was identical in the absence or presence of DOX (data not shown). Thus, in the absence of GC-producing TEC, no activation of the transgenic GR occurs.
In summary, the results from the present work using transgenic mice with a tetracycline-regulated inducible expression system further demonstrate a role for endogenous GCs in regulating the homeostasis of thymocytes. The results from studies in adrenalectomized mice shed new light on the in vivo role that thymic-derived GCs play in thymopoiesis. In the thymus of hCD2-GR transgenic mice, the expression of the transgenic GR was restricted to the thymocytes. Hence, the results provided clear evidence that direct GC effects on these cells primed the above effects of the thymic-derived GCs on the thymocytes.
Acknowledgments
We thank Dr. Rose Zamoyska for kindly providing us with the hCD2-rtTA transgenic mice. The Embryo and Genome Research Core Facility and the animal care unit at the Karolinska University Hospital Huddinge are acknowledged for the pronuclear injections and animal care.
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Address all correspondence and requests for reprints to: Prof. Sam Okret, Department of Medical Nutrition, Karolinska Institutet, Karolinska University Hospital Huddinge, Novum, SE-141 86 Huddinge, Sweden. E-mail: Sam.Okret@mednut.ki.se.
Abstract
We and others have previously reported that thymic epithelial cells produce glucocorticoids (GCs). In vitro studies have also suggested that thymic-derived GCs play a role in the development of thymocytes. However, until now it has not yet been established whether thymic-derived GCs play a role in thymopoiesis in vivo. To investigate this, we conditionally overexpressed the GC receptor (GR) in thymocytes using transgenic mice with a tetracycline-inducible expression system. The influence of systemic GCs was excluded by adrenalectomizing the transgenic mice before the GR induction. Conditional expression of transgenic GR in the thymocytes of adrenalectomized transgenic mice led to a decrease in the thymocyte number. This was associated with increased thymocyte apoptosis. The effect of thymic-derived GCs on the thymocytes was confirmed after transgenic GR induction in a thymic organ culture system. Finally, the GR antagonist RU486 increased thymocyte number in adrenalectomized mice in vivo and prevented a reduction in thymocyte number in thymic organ culture after transgenic GR induction. These observations further confirmed a role for the thymic-derived GCs in regulating thymocyte homeostasis in vivo.
Introduction
GLUCOCORTICOIDS (GCs) ARE A GROUP of steroid hormones that, in addition to their metabolic and developmental effects, have immunosuppressive and antiinflammatory properties and are essential in regulation of immune responses (1). GCs are also very potent inducers of apoptosis in thymocytes (2, 3). GCs act through interaction with GC receptor (GR), which is expressed in most cell types including thymocytes (4). Notably, the GR concentration is a major factor determining cellular sensitivity toward GCs both in vitro and in vivo (5, 6, 7, 8, 9).
Thymus plays a crucial role in the immune system by generating T cells. In the thymus, highly immature CD4–CD8– double-negative thymocytes proliferate and undergo extensive genetic and phenotypic alteration to yield the CD4+CD8+ double-positive (DP) population. Most of the DP thymocytes undergo apoptosis, and less than 5% of the DP thymocytes survive and differentiate into either CD4+ or CD8+ single-positive (SP) thymocytes, which then will seed the peripheral lymphoid tissues (1). Thus, apoptosis and cell proliferation are major regulators of thymocyte homeostasis. It has been shown that endogenous GCs play a role in the development, differentiation, and homeostasis of thymocytes (1, 9, 10), although the direction of the response is a matter of debate (10, 11, 12).
It has also been reported that the thymus is cable of producing GCs (13, 14, 15, 16). Vacchio et al. (13) showed that cultured thymic non-T cells produced soluble pregnenolone and deoxycorticosterone after addition of the precursor 22R- hydroxycholesterol. Furthermore, we and others using various techniques have demonstrated the presence of steroidogenic enzymes such as cytochrome P450 hydroxylases Cyp11A1, Cyp21, and Cyp11B1 in the thymus (13, 14, 15, 16). These enzymes, in combination with the enzyme 3?-hydroxysteroid dehydrogenase, convert cholesterol into corticosterone, the major GC in rodents. Most results suggest thymic epithelial cells (TEC) as the main source of the thymic derived GCs. We have previously reported that when TEC were cocultured with reporter cells containing the GR and a GR-dependent reporter gene, a specific induction of reporter gene activity was observed, showing that TEC can produce active GCs (14).
Previous studies have shown that thymic-derived GCs affect the development of thymocytes in vitro (1). However, until now it has not been clear whether thymic-derived GCs play a role in regulating thymopoiesis in vivo. The aim of the present work was to address this question. This was investigated by creating a tetracycline-inducible expression system to conditionally express a GR transgene in thymocytes after treatment of the transgenic animals with the tetracycline analog doxycycline (DOX). Transgenic mice were adrenalectomized before transgene induction to exclude the effects of systemic GCs. To investigate the effects of thymic-derived GCs, the number of thymocytes and the percentage of thymocyte populations were examined. Because apoptosis and cell proliferation play a crucial role in the regulation of thymopoiesis, we also analyzed the percentage of apoptotic and cycling thymocytes. Our results demonstrated that overexpression of the GR in thymocytes in the absence of systemic GCs led to an increase in the percentage of apoptotic cells and to a decrease in the number of thymocytes. We also examined the effect of the GR antagonist RU486 on thymopoiesis in adrenalectomized mice and on thymic organ culture after transgenic GR induction. Results from the above-described in vivo experiments, in combination with results obtained from thymic organ culture experiments, demonstrated that thymic-derived GCs play a regulatory role in thymopoiesis in vivo.
Materials and Methods
Antibodies and reagents
Fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (01064A) and phycoerythrin (PE)-conjugated anti-CD8 (01045A) were purchased from BD Pharmingen (San Diego, CA). Mouse monoclonal antibody recognizing only the rat GR made in our lab has previously been described (9). Horseradish peroxidase-linked sheep antimouse Ig was purchased from Amersham Biosciences (Buckinghamshire, UK). BSA, mifepristone (RU486), DOX, and propidium iodide (PI) were purchased from Sigma (St. Louis, MO). Deoxyribonuclease-free ribonuclease A was purchased from Roche Molecular Biochemicals (Mannheim, Germany). RPMI 1640 medium, penicillin, streptomycin, and L-glutamine were purchased from Life Technologies, Inc.-BRL (Grand Island, NY).
Generation of hCD2-rtTA/TRE/CMVmin-rGR double-transgenic mice
Mice with a conditional tetracycline-regulated expression of a transgenic rat GR in the thymocytes (hCD2-GR) were generated by crossing TRE/CMVmin-rGR transgenic mice (generated in our laboratory) with hCD2-rtTA transgenic mice. The transgene for generation of TRE/CMVmin-rGR transgenic mice was generated by cloning the cDNA encoding the full coding region of the rat GR cDNA [BamH1 fragment from SVGR1 (17) downstream of the TRE/CMVmin promoter of the pTRE2 vector (Clontech Laboratories, BD Biosciences, Palo Alto, CA)]. The TRE/CMVmin-rGR transgenic mice were generated by microinjection of the construct into the (CBA-C57BL/6) fertilized eggs by standard procedure. Offspring from two tested founder TRE/CMVmin-rGR transgenic mice gave the same phenotype when crossed with the hCD2-rtTA transgenic mice. The hCD2-rtTA transgenic mice were kindly provided by Dr. Rose Zamoyska (18) These were on a C57BL/6 background. The hCD2-rtTA transgenic mice express a reverse transactivator (rtTA) under the control of the hCD2 gene regulatory sequence, directing expression of this chimeric transactivator to the T cell lineage. Generation of these mice has been described previously (Division of Molecular Immunology, National Institute for Medical Research, London, UK) (18). Transmission of transgenes was monitored by PCR analysis of tail DNA, using the following primers. Sense and antisense primers used for the hCD2-rtTA mice were 5'-ATA AAA AGC ACT GTG GAT TTC TGC-3' and 5'-AGA GGG GAC AGG AAA TCT CTA AGA-3', respectively. The sense and antisense primers for TRE/CMVmin-rGR mice were 5'-AAT GAG GTC GGA ATC GAA GG-3' and 5'-TAG CTT GTC GTA ATA ATG GCG G-3', respectively. To conditionally express the transgenic rat GR in the thymocytes, double-transgenic mice (hCD2-GR) were treated with 1 mg/ml DOX in 2% sucrose in their drinking water, which was changed every second day. DOX/sucrose-treated single-transgenic or just sucrose-treated double-transgenic mice served as controls. Four- to 8-wk-old male mice were used for all experiments. The number of thymocytes presented in the figures refers to thymocyte number per thymus. For controls, thymuses from littermates, nontreated double-transgenic hCD2-GR or single-transgenic TRE/CMVmin-rGR or hCD2-rtTA transgenic mice, were used as indicated in figure legends. All animal experimentation was conducted in accord with accepted standards of humane animal care, as approved by the local animal ethical committee.
Western blot analysis
Western blot analysis of GR in whole-cell protein extracts from purified thymocytes was performed as explained before (9). Briefly, protein concentrations in the cell extracts were quantified with the Bio-Rad kit (Bio-Rad Laboratories, Inc., Hercules, CA). Extracted proteins were separated on an 8% sodium dodecyl sulfate polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were incubated with antibodies recognizing only transgenic rat GR (9). After incubation membranes were washed and incubated with horseradish peroxidase-linked sheep antimouse Ig. To check for efficiency of transfer and equal loading, membranes were stained with Ponceau S (Sigma) solution.
Isolation and flow cytometric analysis of thymocytes
Thymus was gently passed through a steel net. Cells were washed with and resuspended in ice-cold RPMI 1640 containing penicillin-streptomycin, L-glutamine, and 0.5% BSA. For flow cytometric analysis of thymocyte populations, cells were stained with anti-CD4-FITC and anti-CD8-PE monoclonal antibodies in ice-cold complete RPMI 1640 medium for 1 h on ice. Cells were washed and resuspended in PBS containing 1% BSA. A total of 10,000 gated cells were analyzed by flow cytometry (FACScan) and the CellQuest program (both from BD Biosciences, Immunocytometry Systems, San Jose, CA).
Adrenalectomy
Adrenalectomy was performed by standard procedure under Avertin (tribromoethanol) anesthesia. Adrenalectomized mice were given 0.7% NaCl in their drinking water to maintain mineral balance. Animals were allowed to recover for 3 d before experiments were performed.
Treatment of mice with RU486
Mice were daily injected ip with 100 μg RU486 (dissolved in sesame oil) for 1 wk. Control mice were injected with an equal volume of sesame oil.
Measuring serum corticosterone level
A solid-phase 125I-RIA system (Diagnostic Products Corp., Los Angeles, CA) was used to determine serum corticosterone levels according to the manufacturer’s recommendations. Mice were retroorbitally bled under methoxyflurane (Metofane) anesthesia before killing. The sensitivity of the assay is 5 ng/ml.
Cell cycle analysis of thymocytes
For flow cytometric analysis of cell cycle, 1 x 106 thymocytes were washed with sample buffer (PBS supplemented with 1% BSA and 1 g/liter glucose), fixed in cold (–20 C) 70% ethanol, and stored at –20 C overnight. The samples were centrifuged and the pellets were resuspended in 1 ml sample buffer containing PI (50 μg/ml) and deoxyribonuclease-free ribonuclease A (50 μg/ml) for 1 h at room temperature while the tubes were vigorously agitated on a shaking platform. A total of 10,000 gated cells (to exclude debris) were analyzed by flow cytometry in a FACScan.
In vitro studies on the effect of induced GR expression on thymocyte survival
Thymus lobes were cut into small pieces and transferred into the Eppendorf tubes containing 0.5% BSA in serum-free RPMI 1640 medium in the absence or presence of 2 μg/ml DOX and 1 μM RU486 for 3 d. Thymus pieces were gently passed through a steel net. Cells were washed with and resuspended in ice-cold PBS containing 1% BSA. The percentage of living cells was determined by flow cytometric analysis of the morphological characteristics of living cells using the forward vs. side scatter, representing the size and density of the cells respectively.
Statistical methods
All results are expressed as mean ± SD, unless otherwise stated. For data analysis, a two-tailed homoscedastic Student’s t test was used with P 0.05 considered significant. When indicated, one-way ANOVA using the GraphPad Prism software (Graph Pad Software Inc., San Diego, CA) was performed.
Results
Conditional expression of a transgenic GR in the thymocytes of the hCD2-GR transgenic mice
The generation of transgenic mice that conditionally express transgenic GR in the thymocytes was based on the tetracycline-inducible expression system (19). To produce these mice, mice containing the GR cDNA under the control of tetracycline-controlled promoter (TRE/CMVmin-rGR) (Fig. 1A, 2) were crossed with mice constitutively expressing the chimeric transactivator rtTA under the control of the hCD2 gene regulatory sequence, which directs expression of this chimeric transactivator to the thymocytes (18) (Fig. 1A, 1). This will allow the transgenic rat GR to be conditionally expressed in the thymocytes of double-transgenic mice (hCD2-GR) after treatment of mice with DOX. To demonstrate the expression of transgenic GR, protein extracts from the thymocytes of hCD2-GR mice were analyzed by Western blotting using a monoclonal antibody that recognizes the transgenic rat GR but not the endogenous mouse GR. As demonstrated in Fig. 1B, the transgenic rat GR was expressed in the thymocytes only after treatment of mice with DOX. Expression after DOX administration was not detected in other tissues such as the liver and lung (data not shown).
FIG. 1. Transgene constructs and conditional expression of the GR transgene in the thymocytes of hCD2-GR transgenic mice. A, Schematic representation of the two transgene constructs (1 and 2) used for generation of hCD2-GR double-transgenic mice. The hCD2-GR double-transgenic mice were produced by crossing the transgenic mice containing a rat GR transgene downstream of a tetracycline-controlled TRE/CMVmin promoter (2) with transgenic mice constitutively expressing a chimeric transactivator rtTA under the control of the hCD2 gene regulatory sequence (1). B, Conditional expression of the transgenic GR in the thymocytes. Transgenic mice (4 wk of age) were treated with DOX (1 mg/ml in drinking water supplemented with 2% sucrose) for 1 wk. Control transgenic mice (–DOX) were given drinking water supplemented with 2% sucrose alone. After treatment, thymocytes were isolated and protein extracts from thymocytes were analyzed by Western immunoblotting. Transgenic rat GR was detected with an anti-GR antibody that recognizes the rat but not the endogenous mouse GR.
Conditional expression of transgenic GR in the thymocytes decreased the number thymocytes
To investigate the in vivo effects of conditional expression of transgenic GR in the thymocytes on thymocyte homeostasis, double-transgenic mice were treated with DOX for 1 wk. Thymocytes were then isolated and counted. As can be seen in Fig. 2A, expression of the transgenic GR resulted in a reduction in the total number of thymocytes (P < 0.001). This demonstrated that transgenic GR expression affects thymopoiesis.
FIG. 2. Effects of conditional transgenic GR expression on thymocyte number. The hCD2-GR double-transgenic mice and the hCD2-rtTA single-transgenic mice were treated with DOX (1 mg/ml in drinking water supplemented with 2% sucrose) for 1 wk. Control transgenic mice (–DOX) were given drinking water supplemented with 2% sucrose alone. After treatment thymocytes were isolated and counted. A, Shown is absolute number of thymocytes from 6-wk-old control (–DOX) and DOX-treated (+DOX) hCD2-GR double-transgenic mice. B, Shown is absolute number of thymocyte populations from 4 wk control (–DOX) and DOX-treated (+DOX) hCD2-rtTA single-transgenic mice. Data are shown as mean ± SD from six mice in each group. Note that the difference in the total cell number of thymocytes between the control hCD2-GR double-transgenic mice (A) and the control hCD2-rtTA single-transgenic mice (B) is due to analyzing these mice at slightly different ages.
To exclude a nonspecific effect of rtTA expression and DOX treatment on thymopoiesis, we treated the single-transgenic hCD2-rtTA mice, in which thymocytes constitutively express the rtTA, with DOX in the same way as the hCD2-GR double-transgenic mice were treated. DOX treatment of these mice did not affect the number of thymocytes (Fig. 2B). Also flow cytometric analysis of thymocytes from DOX-treated single-transgenic hCD2-rtTA, tetracycline-responsive element-GR, or wild-type mice did not show any nonspecific effects of these factors on the number of different populations of thymocytes (data not shown). These control experiments confirmed that the effects observed after DOX treatment of the double-transgenic hCD2-GR mice were mediated only by the expression of the transgenic GR in the thymocytes.
Conditional expression of transgenic GR in thymocytes in the absence of systemic GCs decreased the number of thymocytes
To investigate possible in vivo effect of the thymic-derived GCs on thymopoiesis, we first adrenalectomized hCD2-GR transgenic mice to exclude the influence of systemic GCs. Measuring serum corticosterone levels after adrenalectomy confirmed the absence of circulating GCs (Fig. 3A). Adrenalectomy resulted in an approximately 2-fold increase in the number of thymocytes compared with the number of thymocytes in intact (nonadrenalectomized) hCD2-GR transgenic mice (Fig. 3B, P < 0.001, n = 6). A group of adrenalectomized hCD2-GR transgenic mice were given DOX in the drinking water for 1 wk, after which thymocytes were counted and analyzed by flow cytometry. The results demonstrated a reduction in the total number of thymocytes (Fig. 3B, P < 0.01, n = 6). However, no significant change was observed in the percentage of different thymocyte populations, demonstrating that all four populations were similarly affected by the thymic-derived GCs (Fig. 3C). The values for the percentage of different thymocyte populations in treated vs. controls were as follows. CD8+ SP: 2.7 ± 0.2 vs. 2.6 ± 0.3, CD4+ SP: 9.7 ± 0.3 vs. 9.6 ± 0.3, DP: 82 ± 0.9 vs. 81 ± 1.7, double negative: 4.5 ± 0.2 vs. 4.9 ± 0.3.
FIG. 3. Effects of conditional transgenic GR expression in the thymocytes on thymocyte number and populations in adrenalectomized mice. The hCD2-GR double-transgenic mice (8 wk of age) were adrenalectomized. After recovery, mice were treated with DOX (1 mg/ml in drinking water supplemented with 0.7% NaCl and 2% sucrose) for 1 wk. Control transgenic mice (Ctrl) were given drinking water supplemented with just 0.7% NaCl and 2% sucrose. A, Serum corticosterone concentration in adrenalectomized (ADX) and control (Ctrl) mice as mean ± SD from six mice in each group. The corticosterone concentrations in ADX mice were below detection level for the assay (5 ng/ml). B, The absolute number of thymocyte from nonadrenalectomized (Non-Adx), adrenalectomized (Adx), or from DOX-treated adrenalectomized (Adx/Dox) hCD2-GR double-transgenic mice as mean ± SD from six mice in each group. C, The flow cytometric analysis of thymocyte populations. Thymocytes from control (Ctrl) or DOX-treated (DOX) adrenalectomized hCD2-GR double-transgenic mice were stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 monoclonal antibodies, and analyzed by flow cytometry. Single cells are displayed on a dot plot of CD4 vs. CD8. The numbers indicate the percentage of cells in each quadrant.
Conditional expression of a transgenic GR in thymocytes in the absence of the systemic GCs increased the percentage of apoptotic cells but not the cycling thymocytes
Thymocyte homeostasis is mainly regulated by apoptosis and cell proliferation. Furthermore, thymocytes are highly sensitive to apoptotic effects of GCs. Thus we measured the percentage of apoptotic cells in isolated thymocytes from control and DOX-treated hCD2-GR transgenic mice by flow cytometry. As can be seen in Fig. 4A, DOX treatment of adrenalectomized hCD2-GR transgenic mice led to an increase in the percentage of apoptotic thymocytes (10.1 ± 1.7% in treated mice vs. 5.2 ± 1.2% in nontreated mice, P < 0.01, n = 6). DOX did not have any effect on the percentage of apoptotic cells in nontransgenic mice (data not shown, see also Fig. 6, A and B). Because the number of thymocytes also is regulated by cell proliferation, we investigated the effects of thymic-derived GCs on the percentage of cycling cells. Flow cytometric analysis of the cell cycle in thymocytes from control and DOX-treated adrenalectomized hCD2-GR transgenic mice did not show any effect of thymic-derived GCs on the percentage of cycling thymocytes (S/G2/M phase of the cell cycle) (10.5 ± 0.9% in treated mice vs. 10.3 ± 0.8% in nontreated mice, n = 6) (Fig. 4B).
FIG. 4. Effects of conditional transgenic GR expression in the thymocytes of adrenalectomized mice on thymocyte apoptosis and cell cycle. The hCD2-GR double-transgenic mice were adrenalectomized. After recovery, mice were treated with DOX (1 mg/ml in drinking water supplemented with 0.7% NaCl and 2% sucrose) for 1 wk. Control transgenic mice (Ctrl) were given drinking water supplemented with just 0.7% NaCl and 2% sucrose. Thymocytes were then isolated, counted, and analyzed by flow cytometry. A, Determining the percentage of apoptosis in isolated thymocytes from control (Ctrl) and DOX-treated (DOX) adrenalectomized hCD2-GR transgenic mice. Shown are representative flow cytometric analysis of thymocytes based on the morphological characteristics of living and apoptotic cells using the forward scatter vs. side scatter, representing the size and density of the cells, respectively. The apoptotic cells give lower forward scatter and higher side scatter values. The percentages of apoptotic cells are given in the figures. The results are representative of one of six mice in each group. P < 0.01. B, Flow cytometric analysis of the cell cycle in thymocytes from control (Ctrl) and DOX-treated (DOX) adrenalectomized hCD2-GR transgenic mice. Thymocytes were fixed, premeabilized, stained with PI, and analyzed by flow cytometry. Shown are DNA histograms of thymocytes. The percentages of cycling cells (S/G2/M phase of the cell cycle) are given in the figures (P = not significant). The results are representative of one of six mice in each group.
FIG. 6. Effect of thymic-derived GCs on the survival of thymocytes in vitro. A, Thymuses obtained from 6-wk double-transgenic (hCD2-GR) and control (hCD2-rtTA) mice were cultured in serum-free RPMI 1640 medium containing 0.5% BSA in the absence (–DOX) or presence (+DOX) of 2 μg/ml DOX for 3 d. Thymocytes were then isolated and the percentage of living cells was determined by flow cytometric analysis. In the figures, single cells are displayed on a dot plot of forward scatter vs. side scatter. The percentages of living cells are given in the figures. The results are representative of one of six samples in each group. B, The bars show the percentage of living cells as mean ± SD from six samples in each group. P < 0.001 for DOX-treated vs. nontreated thymuses from hCD2-GR double-transgenic mice. C, Thymuses obtained from double-transgenic hCD2-GR mice were treated with 2 μg/ml DOX in the absence or presence of 1 μM RU486. The bars show the percentage of living cells as mean ± SD from six samples in each group. A one-way ANOVA showed that all the means are significant different (P < 0.05). The Bonferroni’s multiple comparison test showed that P < 0.05 between control (Ctrl) vs. Dox/RU. The other P values are shown in the figure.
Treatment of adrenalectomized and intact hCD2-GR transgenic mice with the GR antagonist RU486 increased the number of thymocytes
To find further support for a role for the thymic-derived GCs on thymopoiesis in vivo, we treated adrenalectomized hCD2-GR transgenic mice with the GR antagonist RU486. If thymic-derived GCs participate in the regulation of thymocyte homeostasis, it is expected that the GR antagonist RU486 will antagonize this effect. As demonstrated in Fig. 5A, treatment of adrenalectomized transgenic mice with RU486 resulted in increased number of thymocytes, as compared with the control mice (adrenalectomized transgenic mice treated with just the solvent alone, P < 0.001, n = 5). This observation further supported that thymic-derived GCs affects thymopoiesis. In addition, we treated intact nonadrenalectomized hCD2-GR transgenic mice with RU486. The result showed an increase in the number of thymocytes, confirming a suppressive effect of endogenous GCs on thymocyte homeostasis (Fig. 5B).
FIG. 5. Effect of RU486 treatment of adrenalectomized and intact hCD2-GR transgenic mice on the number of thymocytes. A, hCD2-GR transgenic mice (8 wk of age) were adrenalectomized and given 0.7% NaCl in their drinking water to maintain mineral balance. After recovery, the mice were treated by daily ip injections with the GR antagonist RU486 (100 μg/d dissolved in sesame oil) for 1 wk. Control adrenalectomized hCD2-GR transgenic mice were injected with the solvent (sesame oil) alone. Thymuses were removed. Thymocytes were isolated and counted. Shown is the absolute number of total thymocytes from adrenalectomized hCD2-GR transgenic mice (Ctrl) or from RU486-treated adrenalectomized hCD2-GR transgenic mice as mean ± SD from five mice in each group. B, Nonadrenalectomized hCD2-GR transgenic mice (8 wk of age) were treated with RU486 or vehicle alone as explained above. Shown is the absolute number of total thymocytes from nonadrenalectomized vehicle-treated mice (Ctrl) or from nonadrenalectomized RU486-treated mice as mean ± SD from five mice in each group.
Expression of the transgenic GR reduced the percentage of living thymocytes in vitro
We also examined the effects of thymic-derived GCs on the thymocyte survival in vitro. This was performed by treating isolated thymuses obtained from hCD2-GR transgenic mice in a thymic organ culture system with DOX. Thymocytes were then isolated and analyzed for the percentage of living cells. Similar treatment was performed in parallel for thymuses obtained from control (hCD2-rtTA) mice. As can be seen in Fig. 6, A and B, DOX treatment of isolated thymuses from hCD2-GR mice reduced the percentage of living thymocytes by approximately 50% compared with the nontreated hCD2-GR transgenic thymocytes (P < 0.001, n = 6). DOX treatment of the thymuses obtained from control (hCD2-rtTA) mice did not affect the survival of thymocytes (Fig. 6, A and B). In an additional experiment, thymuses obtained from transgenic hCD2-GR mice were treated with DOX in the absence or presence of RU486 (Fig. 6C). The results showed that RU486 could partly prevent the effects of GR overexpression on thymocyte death. These in vitro results derived using isolated thymuses indeed demonstrated that the survival of thymocytes was influenced by the thymic-derived GCs.
Discussion
Endogenous GCs are known to play an important regulatory role in the function of the immune system. This includes a role in the development, homeostasis, and function of T cells (1, 9). It is also well established that thymocytes are highly sensitive to the apoptotic effects of GCs (2, 3). Of interest to this work, we and others have previously reported that the thymus is capable of producing GCs (13, 14, 15, 16). Later studies suggested that the thymic-derived GCs regulate the development of the thymocytes (1). This was mainly based on in vitro studies in a thymic organ culture system in which corticosteroid biosynthesis was blocked by steroidogenic inhibitors. However, whether thymic-derived GCs exert an effect on thymopoiesis in vivo has not been established. In this report we addressed this question by using a system in which a DOX-inducible transgenic GR was expressed in the thymocytes of transgenic mice in vivo. The transgenic mice were depleted of systemic GCs by adrenalectomy to exclude the influence of systemic GCs on thymopoiesis. The absence of systemic GCs was verified by serum measurements using a sensitive RIA. In such a condition, paracrine effects of thymus-derived GCs are most likely responsible for the observed effects. The results obtained from these in vivo studies showed that thymic-derived GCs suppress thymopoiesis. This could be explained by decreased survival of thymocytes. This is in concord with our previous observation showing that constitutive overexpression of GR in the thymocytes in transgenic mice led to an increased apoptosis and a decrease in the number of thymocytes (9). Thus, thymocyte homeostasis is controlled by both adrenal (systemic) and thymus-derived GCs. The observation that indeed thymic-derived GCs affect the thymocytes was confirmed by our complementary in vitro experiments, in which thymuses were isolated and the transgenic GR was induced in vitro in a thymic organ culture system. An additional advantage of the inducible expression system is that it excludes "metabolic programming" effects of a transgene during embryonic or neonatal stages that would possibly affect the phenotype later in life. The effect of thymic-derived GCs on thymopoiesis in vivo was further supported by treating adrenalectomized mice with the GR antagonist RU486, as this resulted in increased thymocyte number. RU486 also partially prevented the DOX-induced reduction in thymocyte number in thymic organ cultures of hCD2-GR transgenic mice. Although precautions have to be taken when it comes to conclusions drawn from RU486 experi-ments alone, because RU486 also may act via the androgen or progestin receptor, these experiments support the results from the GR-inducible transgenic mice that the decrease in thymocyte number after transgenic GR expression is a GC-GR-mediated effect.
In our in vivo and in vitro studies we observed a decreased survival of thymocytes after the GR induction in the thymocytes, whereas the cell cycle of thymocytes were not affected. The observed decreased survival suggests a mechanism by which thymic-derived GCs could reduce the number of thymocytes in vivo. This result was in line with our previous report demonstrating that coculturing of TEC with thymocytes decreased survival of thymocytes (14). In our in vivo and in vitro experiments we also found no indication that DOX treatment by itself, especially in combination with expression of the rtTA transcription factor, had any nonspecific effect on thymopoiesis. A similar observation has previously been reported (20). These experiments excluded a nonspecific effect of the tetracycline-inducible expression system on thymopoiesis. Furthermore, expression of the transgenic rat GR does not affect the endogenous GR level (4). In addition, we found no evidence that expression in the thymocytes of the transgenic GR itself results in a ligand-independent activation of GR. This conclusion was based on the observation that the percentage of apoptosis in purified hCD2-GR thymocytes was identical in the absence or presence of DOX (data not shown). Thus, in the absence of GC-producing TEC, no activation of the transgenic GR occurs.
In summary, the results from the present work using transgenic mice with a tetracycline-regulated inducible expression system further demonstrate a role for endogenous GCs in regulating the homeostasis of thymocytes. The results from studies in adrenalectomized mice shed new light on the in vivo role that thymic-derived GCs play in thymopoiesis. In the thymus of hCD2-GR transgenic mice, the expression of the transgenic GR was restricted to the thymocytes. Hence, the results provided clear evidence that direct GC effects on these cells primed the above effects of the thymic-derived GCs on the thymocytes.
Acknowledgments
We thank Dr. Rose Zamoyska for kindly providing us with the hCD2-rtTA transgenic mice. The Embryo and Genome Research Core Facility and the animal care unit at the Karolinska University Hospital Huddinge are acknowledged for the pronuclear injections and animal care.
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