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Endocrinology, doi:10.1210/en.2003-1660
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Endocrinology Vol. 145, No. 5 2392-2401
Copyright © 2004 by The Endocrine Society

Glucocorticoids Delay Age-Associated Thymic Involution through Directly Affecting the Thymocytes

Ahmad Pazirandeh, Mikael Jondal and Sam Okret

Department of Medical Nutrition (A.P., S.O.), Karolinska Institutet, Huddinge University Hospital, Novum, SE-141 86 Huddinge, Sweden; and Microbiology and Tumor Biology Center (A.P., M.J.), Karolinska Institutet, SE-171 77 Stockholm, Sweden

Address all correspondence and requests for reprints to: Professor Sam Okret, Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, Novum, SE-141 86 Huddinge, Sweden. E-mail: sam.okret{at}mednut.ki.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
After puberty, the thymus undergoes a dramatic loss in size, resulting in a reduction in the number of thymocytes, a phenomenon termed age-associated thymic involution. The factors regulating this process are poorly understood. We investigated the role of endogenous glucocorticoids (GCs) in this process by studying transgenic mice with increased GC sensitivity restricted to the T-cell lineage due to overexpression of a GC-receptor transgene under the control of the proximal lck promoter. Surprisingly, in these transgenic mice, the age-associated thymic involution did not start until after 6 months of age, demonstrating that endogenous GCs through directly affecting the thymocytes delay the age-associated thymic involution process. The delayed age-associated thymic involution resulted in a significantly higher number of thymocytes in transgenic mice, compared with wild-type mice at 6 months of age or older. The higher number of thymocytes was associated with increased percentage of cycling double-negative and single-positive thymocytes, whereas thymic apoptosis was unaffected. The above effects of GCs were restricted to the thymocytes and were not reflected on the peripheral T cells, in which GCs suppressed the number of peripheral T cells in aged transgenic mice, demonstrating that thymocytes and T cells are differentially regulated by GCs. Furthermore, CD4+ T cells were more affected than CD8+ T cells, resulting in a decrease in the CD4/CD8 T-cell ratio. In summary, our results reveal novel biological effects of endogenous GCs on thymic involution and T-cell homeostasis in aged mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE THYMUS IS the key organ of the immune system for the maturation of T cells. The development of the thymus continues until puberty (around 4–6 wk of age in mice) at which time it reaches its maximum size, containing the highest number of thymocytes. After puberty the thymus undergoes a dramatic loss in weight and volume coupled to an inefficient functioning of the thymus and reduction in thymopoiesis, a phenomenon termed age-associated thymic involution (1, 2, 3). Thymus exhibits the most profound involution during normal postnatal histogenesis, and aging. This has been proposed to contribute to immune senescence, characterized by defects in cellular and humoral immune responses to immunization, infectious agents, and cancer (4, 5, 6). Despite awareness of the age-associated thymic involution phenomenon for decades, the mechanisms responsible for this apparently normal developmental program have remained unknown. However, it has been demonstrated that the involution is an intrinsic effect of the thymus and not associated to the precursor cells derived from the bone marrow that seed into the thymus (1). Furthermore, it has been suggested that changes in the hormonal environment of gonadal hormones, growth factors, or cytokines participate in this process, although their contribution is still a matter of debate (7, 8, 9).

Intrathymic development and homeostasis of thymocytes are mainly regulated by cell proliferation and apoptosis. Highly immature CD4–CD8– double-negative (DN) thymocytes undergo extensive gene rearrangement, proliferation, 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 (10).

The immune system is very sensitive to the actions of glucocorticoids (GCs). GCs are key regulators in the immune response to antigenic or proinflammatory stimuli (11, 12). GCs are also very potent inducers of apoptosis in thymocytes and lymphocytes (13, 14, 15, 16). It has been shown that GCs play a role in the development, differentiation, homeostasis, and functions of T cells (17, 18), although the direction of the response is a matter of debate (19, 20, 21, 22, 23). The role of endogenous GCs in the age-associated thymic involution process is also unclear. In addition to the systemic GCs, thymic-derived GCs have been suggested to play a role in T-cell development (24, 25, 26). Some of the effects of GCs on thymocytes and T cells are suggested to involve cross-communication between the GC and T-cell receptor (TCR) signaling pathways (27, 28, 29, 30, 31). GCs can repress or activate gene transcription. This is initiated through interaction of the GCs with the intracellular GC receptor (GR), which is expressed in most cell types including thymocytes and lymphocytes (32, 33, 34). Notably, the GR expression level is a major factor in determining cellular sensitivity toward GCs both in vitro and in vivo (35, 36, 37). This concept has been used by us and others to investigate the in vivo role of GCs in different tissues by generating transgenic mice with an altered GC sensitivity due to modified GR expression (18, 35, 38, 39).

We recently reported the involvement of normal levels of endogenous GCs in negatively regulating the homeostasis of thymocytes and peripheral T cells in young mice (3–8 wk of age) (18). This conclusion was mainly based on studies in transgenic mice with increased GC sensitivity restricted to the T-cell lineage. These transgenic mice [Lck(Pr)-sGR] were generated by overexpression of the rat GR gene specifically in their T-cell lineage by using the lck proximal promoter (40). The aim of the present study was to investigate the effects of endogenous GCs on the age-associated thymic involution and homeostasis of T cells in aged mice (up to 18 months of age). Using the transgenic mice approach, we here, for the first time, demonstrate that endogenous GCs through direct effects on thymocytes delay the initiation of the age-associated thymic involution. In addition, endogenous GCs suppress the number of peripheral T cells in aged mice, providing evidence that thymocyte and peripheral T-cell homeostasis are differentially regulated by GCs in elderly mice. Finally, our results demonstrate that GCs also play a regulatory role in controlling the CD4+/CD8+ T-cell ratio.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of transgenic mice
The Lck(Pr)-sGR transgenic mice were generated and bred as explained previously (18). Briefly, a 2.8-kb BamH1 fragment containing the full-length coding region of the rat GR cDNA cloned in sense orientation into a expression vector containing the proximal p56lck promoter [Lck(Pr)] (40). The constructs were removed from bacterial plasmid DNA and introduced into male pronuclei of zygotes obtained from F1 (C57BL/6JxCBA/J) mice by standard microinjection techniques. Transgenic mice were identified by PCR and Southern blot analysis of DNA from tail as explained previously (18). The mice were bred on C57BL/6JxCBA/J background. The phenotypic analysis of Lck(Pr)-sGR transgenic mice was performed on two separate homozygotic transgenic lines with similar transgene expression levels. The two independent lines showed the same phenotype. For all experiments, male Lck(Pr)-sGR and control [wild-type (WT)] mice were used, which were obtained from parallel matings to ensure the same conditions and age.

Antibodies and reagents
Fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (01064A), 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 (18). Polyclonal antibody (H-300) recognizing both the rat and mouse GR was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-linked sheep antimouse immunoglobulin and HRP-linked donkey antirabbit immunoglobulin were purchased from Amersham Biosciences (Buckinghamshire, UK). Mouse monoclonal antibody against the ß-tubulin (N357) was from Amersham. Rhodamine 123, propidium iodide, and 7-aminoactinomycin D (7-AAD) were purchased from Sigma (St. Louis, MO).

Isolation of cells
Thymus and spleen were passaged through a steel net. Red blood cells were lysed with ACK lysing buffer [0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM Na2EDTA, (pH 7.3)]. Cells were washed with and resuspended in cold RPMI 1640 containing penicillin-streptomycin, L-glutamine, and heat-inactivated fetal calf serum (10%) all obtained from Life Technologies, Inc.-BRL (Grand Island, NY).

Western blot analysis
Western blot analysis of GR in whole-cell protein extracts from purified thymocytes was performed as explained before (18). 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 or both transgenic rat and endogenous mouse GR. Then incubation membranes were washed and incubated with HRP-linked sheep antimouse immunoglobulin or with HRP-linked donkey antirabbit immunoglobulin, depending on the source of the primary antibody. Intensity of the bands was also measured using a luminescent image analyzer (LAS-1000, Fujifilm, Tokyo, Japan). To check for efficiency of transfer and equal loading, membranes were stained with Ponceau S solution and also incubated with an antibody against the ß-tubulin protein.

Histology of the thymus
For histology, thymus was removed and fixed in paraformaldehyde, and sections were stained with hematoxylin-eosin by standard procedure.

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 Metofane anesthesia.

GC sensitivity assay
Thymocytes (2 x 106) were incubated in complete RPMI 1640 medium at 37 C and 5% CO2 for 20 h in the absence or presence of indicated concentrations of corticosterone. Viability of cells was determined by flow cytometric analysis after rhodamine-123 staining (18, 41, 42). Briefly, thymocytes in complete RPMI 1640 medium were stained with 2.5 mM rhodamine-123 for 1 h at 37 C and washed with cold PBS + 1% BSA. The results were confirmed by analyzing the morphological characteristics of living and apoptotic thymocytes using the forward vs. side scatter, representing the size and density of the cells, respectively. The apoptotic cells give lower forward scatter and higher side scatter values. Analysis was performed by flow cytometry (FACSscan) and the CellQuest program (both from BD Biosciences, Immunocytometry Systems, San Jose, CA).

Analysis of thymocyte and T-cell populations
Thymocytes and purified leukocytes from spleen were stained with anti-CD4-FITC and anti-CD8-phycoerythrin (PE) monoclonal antibodies (mAbs) in ice-cold complete RPMI 1640 medium for 1 h on ice and washed. The 10,000 gated cells were analyzed by flow cytometry.

Analysis of apoptosis in the thymus
Apoptosis in the thymus was detected in situ by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay. Thymuses were fixed in 4% paraformaldehyde, and 4-µm-thick paraffin-embedded sections were mounted on glass slides, deparaffinized, and dehydrated. The TUNEL assay was performed using an in situ cell death detection kit, POD (Roche Molecular Biochemicals, Indianapolis, IN). Apoptosis also was detected in isolated thymocytes with 7-AAD (43, 44). Briefly, cells were stained with 10 µg/ml of 7-AAD in PBS + 1% BSA for 30 min on ice under protection from light and were analyzed by flow cytometry.

Cell cycle analysis of thymocyte and T-cell populations
For cell cycle analysis of thymocyte and T-cell populations, 1 x 106 cells were stained with anti-CD4-FITC and anti-CD8-PE, washed, and DNA content determined by costaining with 10 µg/ml 7-AAD in permeabilizing buffer (PBS containing 0.04% saponin and 0.5% BSA) for 1 h on ice; 30,000 gated cells were analyzed by flow cytometry.

Statistical methods
All results are expressed as mean ± SD, unless otherwise stated. For data analysis, a two-tailed homoscedastic Student t test was used with P <= 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Age-associated thymic involution is delayed in Lck(Pr)-sGR transgenic mice
To study the effects of endogenous GCs on thymocytes in regulating the age-associated thymic involution process, we analyzed the Lck(Pr)-sGR transgenic mice with increased GC sensitivity restricted to the T-cell lineage (18) by determining thymocyte number at different ages. At 4 months of age, thymocyte number in transgenic Lck(Pr)-sGR mice was 155 ± 10 million. It reached 214 ± 18 million cells at 6 months of age, whereas at 9.5 months of age, the thymocyte number in Lck(Pr)-sGR transgenic mice had decreased to 132 ± 14 million cells. After 9.5 months the thymocyte number decreased further (Fig. 1AGo). This showed that thymocyte number in Lck(Pr)-sGR transgenic mice peaked around 6 months of age and did not start to decline until after that time, demonstrating that the initiation of age-associated thymic involution was delayed in the transgenic mice. In WT mice, a continuous decrease in the number of thymocytes was observed during this time period as occurs in a normal age-associated thymic involution process. The very late initiation of the age-associated involution process in Lck(Pr)-sGR transgenic mice resulted in a higher number of thymocytes at 6 months of age, compared with the WT mice (214 ± 18 million vs. 123 ± 12 million). The higher number of thymocytes in Lck(Pr)-sGR transgenic in comparison with WT mice was sustained at all ages 6 months of age or older. Analysis of different thymocyte populations revealed a contribution of all four populations to this phenotype (Fig. 1BGo). In addition, flow cytometric analysis of thymocytes from aged Lck(Pr)-sGR mice demonstrated an enhanced percentage of mature CD4+ and CD8+ SP thymocytes as exemplified in Fig. 1CGo for 12- and 18-month-old mice. The values for the percentage of CD4+ SP thymocytes were 9.83 ± 0.38 for Lck(Pr)-sGR transgenic mice vs. 5.98 ± 0.61 for WT mice (P < 0.001) at the age of 12 months and 9.71 ± 0.46 for transgenic vs. 6.09 ± 0.6 for WT mice (P < 0.01) at the age of 18 months. The values for the percentage of CD8+ SP thymocytes were 3.1 ± 0.0.3 for transgenic vs. 1.9 ± 0.14 for WT mice (P < 0.001) at the age of 12 months and 4.04 ± 0.29 for transgenic vs. 1.35 ± 0.16 for WT mice (P < 0.001) at the age of 18 months. This observation also suggests that differentiation of DP population into SP thymocytes or alternatively expansion of CD4+ and CD8+ SP thymocytes was enhanced by endogenous GCs in aged transgenic mice.



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  		FIG. 1. Thymocyte number in aged Lck(Pr)-sGR transgenic and WT mice. A, Absolute number of thymocytes in aged WT and Lck(Pr)-sGR transgenic mice. Six mice collected from at least two experimental occasions were analyzed for each age. B, Number of thymocyte populations in aged WT and Lck(Pr)-sGR transgenic mice at different ages. The number of thymocyte populations was calculated by multiplying the percentage of each population by total number of thymocytes. The absence of SD for some bars are due to their low value, not allowing them to be detected in the graphs C, Flow cytometric analysis of thymocyte subpopulations. Thymocytes from WT and homozygous Lck(Pr)-sGR transgenic were collected, counted, stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 mAbs, and analyzed by flow cytometry. Shown are representative flow cytometric analysis of CD4 vs. CD8 staining of thymocytes from one of six mice at 12 and 18 months of age. The numbers indicate the percentage of cells in each quadrant.

 
GR expression and GC sensitivity in thymocytes and serum GC level in aged Lck(Pr)-sGR mice
We have previously shown that thymocytes from young Lck(Pr)-sGR transgenic mice were significantly more sensitive to GCs (18). To investigate whether the phenotype in the aged transgenic mice was due to a possible change in GR expression, inactivation of the transgene or resistance of transgenic thymocytes to GCs, we analyzed the transgenic and total GR levels (Fig 2AGo) and also the sensitivity of thymocytes from aged Lck(Pr)-sGR transgenic mice to GC-induced apoptosis (Fig. 2Go, B and C). To check whether transgenic or endogenous GR levels are regulated by age, we compared both the transgenic rat GR and endogenous mouse GR expression levels in thymocytes of young and aged transgenic and WT mice by Western immunoblotting (Fig. 2AGo). Our results demonstrated a 2-fold increase in total GR level in thymocytes from both young and aged transgenic mice, compared with the WT mice. This 2-fold increase in total GR expression in thymocytes from aged transgenic mice is in line with 2-fold increase in total GR expression in thymocytes from young transgenic mice as analyzed by ligand binding assay (18). Furthermore, there was no age-dependent regulation of either endogenous or transgenic GR expression in thymocytes (Fig. 2AGo). This demonstrates that age does not influence transgene expression from the Lck promoter.



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  		FIG. 2. GR expression and GC sensitivity in thymocyte, and serum GC level in aged Lck(Pr)-sGR transgenic and WT mice. A, Western immunoblotting analysis of endogenous and transgenic GR expression in thymocytes from young (6 wk) and aged (12 months) mice (1, young WT; 2, aged WT; 3, young transgenic; 4, aged transgenic). Transgenic rat GR was detected with an anti-GR antibody that recognizes the rat but not the mouse GR (upper panel). Total GR level was also detected using an anti-GR antibody that recognizes both rat and mouse GR (middle panel). Membranes were also incubated with an antibody against ß-tubulin as a control for loading (lower panel). Note that the slightly lower intensity of the GR band in lane 4 is due to a slightly lower loading of protein as visualized by the ß-tubulin staining. Similar result was obtained in two separate experiments. B and C, Percentage of viable thymocytes from aged (12 months) Lck(Pr)-sGR and WT mice after treating the cells with the indicated concentrations of corticosterone (CS) for 20 h was determined. B, Shown is one representative flow cytometric analysis of three of treated thymocytes based on the morphological characteristics of living and apoptotic thymocytes using the forward vs. side scatter, representing the size and density of the cells, respectively. The apoptotic cells give lower forward scatter (FSC) and higher side scatter (SSC) values. The percentages of living cells are given in the figures. C, Shown are representative histograms for rhodamine 123 staining of thymocytes from one of three experiments. Apoptotic cells show a decreased rhodamine 123 fluorescence as a result of lack of mitochondrial retention of rhodamine 123 due to the loss of mitochondrial membrane potential (41 42 ). The percentages of living cells are given in the figures. D, Serum corticosterone level in aged (12 months) WT and Lck(Pr)-sGR transgenic mice. Shown are mean ± SD from four mice in each group.

 
To investigate the GR activity in transgenic thymocytes, the sensitivity of these cells to GC-induced apoptosis was investigated. Thymocytes were treated with increasing concentrations of corticosterone, followed by flow cytometric analysis of morphological characteristics associated with apoptotic cells (Fig. 2BGo). Apoptosis of the treated thymocytes was also analyzed by staining with rhodamin 123 (42, 45) (Fig. 2CGo). Both methods showed that thymocytes from the transgenic mice were still more sensitive to the apoptotic effects of GCs, compared with thymocytes from the WT mice. Of note is that no significant difference in serum corticosterone levels between aged WT and Lck(Pr)-sGR mice was detected (Fig. 2DGo), hence excluding altered corticosterone concentration as a cause for the phenotype observed.

Analysis of thymic apoptosis and thymocyte cell cycle in aged Lck(Pr)-sGR mice
The number of thymocytes at a defined time are highly regulated by both cell proliferation and apoptosis (46, 47). To investigate whether the higher cell number in the thymus of aged Lck(Pr)-sGR transgenic mice was due to reduction in thymic apoptosis, TUNEL assay was performed for in situ analysis of apoptosis. The results revealed no difference in the degree of apoptosis in the thymus of aged Lck(Pr)-sGR transgenic mice, compared with the thymus from WT mice (Fig. 3AGo). As expected, apoptotic cells are mainly scattered throughout the cortex (46). Flow cytometric analysis of freshly isolated thymocytes from aged transgenic and WT mice also confirmed the lack of a significant difference in the percentage of apoptotic cells (data not shown). Histological analysis of thymus showed a normal thymic architecture containing normal cortical and medullary domains in both WT and transgenic mice (Fig. 3BGo).



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  		FIG. 3. Detection of thymic apoptosis and histological analysis of the thymus in the aged Lck(Pr)-sGR transgenic and WT mice. A, Apoptosis in the thymuses of 1-yr-old WT and Lck(Pr)-sGR transgenic mice. Sections were prepared from thymuses obtained from WT and transgenic mice, and apoptosis was detected in situ by the TUNEL assay. The number of TUNEL-positive cells in 10 different fields in each section was evaluated. B, Histological analysis (hematoxylin-eosin staining) of the thymuses in WT and transgenic mice. In the lower panel, medulla surrounded by the cortex is magnified. M, medulla; C, cortex. A and B, Shown is one representative picture of nine sections derived from three thymuses.

 
An alternative mechanism explaining the higher number of thymocytes in aged transgenic mice is an increased proliferation of thymocytes. This was investigated by analyzing the percentage of cycling cells among different thymocyte populations by simultaneous staining of cell surface CD4/CD8 molecules and DNA. As seen in Fig. 4Go, A and B, DN and DP populations constitute the majority of cycling thymocytes in both the WT and transgenic mice. Interestingly, the percentage of cycling DN and CD4+ and CD8+ SP thymocytes from aged transgenic mice was significantly higher, compared with thymocyte populations from the WT mice (Fig. 4BGo).



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  		FIG. 4. Analysis of percentage of cycling thymocytes in aged Lck(Pr)-sGR transgenic and WT mice. The percentage of cycling cells in different thymocyte populations from 1-yr-old mice was determined by costaining thymocytes with anti-CD4-FITC and anti-CD8-PE. DNA content was determined by costaining with 7-AAD. Cells were analyzed by flow cytometry. A, Representative flow cytometric cell cycle analysis of thymocytes from one of six mice showing CD4, CD8, and 7-AAD staining. The cell cycle analysis of gated DN, DP, CD4+ SP, and CD8+ SP thymocyte populations is shown in the figures. B, Statistical analysis (mean ± SD) of the percentage of cells in the S/G2/M phase of the cell cycle for different thymocyte populations. The results are based on data from six mice in the control and transgenic group, respectively, obtained from two independent experimental occasions (P < 0.005 for DN thymocytes; P < 0.01 for CD4 SP thymocytes; P < 0.01 for CD8 SP thymocytes).

 
Decreased number of peripheral T cells in aged Lck(Pr)-sGR mice
As described above, due to the delayed initiation of the age-associated thymic involution process in the Lck(Pr)-sGR transgenic, a higher thymocyte number was observed in these mice, compared with WT mice. To investigate whether GCs exerted a similar effect on T-cell homeostasis, spleen T cells in aged mice were analyzed. Interestingly, in contrast to the GC effects seen on thymocyte homeostasis, endogenous GCs significantly decreased the number of both CD4+ and CD8+ T cells in the aged transgenic mice, compared with the T-cell populations in the WT mice (Fig. 5Go).



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  		FIG. 5. T-cell number in the spleen of aged Lck(Pr)-sGR transgenic and WT mice. Spleen leukocytes from WT and homozygous Lck(Pr)-sGR transgenic mice were purified, counted, stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 mAbs, and analyzed by flow cytometry. Shown are numbers (mean ± SD) for the CD4+ and CD8+ T-cell populations derived from six WT and transgenic mice obtained at two independent occasions at 9.5 and 18 months of age. The number of T-cell subsets was calculated by multiplying the percentage of each population by the total number of leukocytes. P < 0.005 for CD4+ and P < 0.01 for CD8+ T cell at 9.5 months of age. P < 0.005 for both CD4+ and CD8+ T cells at 18 months of age.

 
Decreased number of cycling T cells in Lck(Pr)-sGR mice
Homeostatic proliferation is an important mechanism involved in homeostasis of T cells (48). To investigate a possible alteration in the proliferation of T cells in transgenic vs. WT mice, we analyzed the percentage of cycling T cells. Results obtained from flow cytometric analysis demonstrated a significant decrease in the percentage of cycling CD4+ T cells in the aged transgenic mice, compared with WT mice (Figs. 6Go, A and B).



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  		FIG. 6. Analysis of percentage of cycling T cells from aged Lck(Pr)-sGR transgenic and WT mice. Purified spleen leukocytes were stained with anti-CD4-FITC and anti-CD8-PE, washed, and DNA content determined by costaining with 7-AAD. Cells were then analyzed by flow cytometry. A, Representative flow cytometric cell cycle analysis of CD4+ and CD8+ T cells from one of three experiments showing 7-AAD, CD4, and CD8 staining. B, The percentage of gated CD4+ and CD8+ T-cell subsets in S/G2/M phase of the cell cycle is given in the figures. Shown are mean ± SD (n = 3). P < 0.005 for CD4+ T cells; P not significant for CD8+ T cells. The experiment was repeated twice with similar results.

 
CD4+/CD8+ T-cell ratio in aged Lck(Pr)-sGR transgenic mice
Flow cytometric analysis of spleen T lymphocytes from 9.5- and 18-month-old Lck(Pr)-sGR transgenic mice demonstrated a decreased percentage of both CD4+ and CD8+ T cells, compared with WT mice (Fig. 7Go). The values for the percentage CD4+ T cells were 12.26 ± 3.06 for transgenic mice vs. 24.21 ± 0.42 for WT mice (P < 0.005) at the age of 9.5 months and 9.22 ± 0.17 for transgenic mice vs. 17.72 ± 0.95 for WT mice (P < 0.01) at the age of 18 months. The values for the percentage of CD8+ T cells were 9.6 ± 1.6 for transgenic mice vs. 11.17 ± 1.21 for WT mice (P < 0.01) at the age of 9.5 months and 12.38 ± 0.14 for transgenic mice vs. 13.83 ± 0.64 for WT mice (P, not significant) at the age of 18 months. As evident from the figures and similar to what we observed regarding the percentage of cycling T cells, the suppressive effects of endogenous GCs was more pronounced on CD4+ than on CD8+ T cells, resulting in a decreased CD4+/CD8+ T cell ratio. The values for the CD4+/CD8+ T-cell ratio shown in Fig. 7Go were 1.26 ± 0.16 for transgenic mice vs. 2.19 ± 0.25 for WT mice (P < 0.05) at 9.5 months of age and 0.74 ± 0.05 for transgenic mice vs. 1.28 ± 0.12 for WT mice (P < 0.01) at 18 months of age. Notably, this altered ratio had a decreasing rate during aging, so that, surprisingly, in the very old Lck(Pr)-sGR mice, the CD4+/CD8+ T-cell ratio was reversed, resulting in an unusual higher number of CD8+ than CD4+ T cells (Fig. 5Go). Interestingly, a decreased CD4+/CD8+ T-cell ratio could also be observed in aged WT mice, even though less prominent, confirming a good correlation between GC sensitivity of T cells and the decreased CD4+/CD8+ T-cell ratio.



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  		FIG. 7. CD4+/CD8+ T-cell ratio in aged Lck(Pr)-sGR transgenic and WT mice. Spleen leukocytes from WT and homozygous Lck(Pr)-sGR transgenic mice were purified, counted, stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 mAbs, and analyzed by flow cytometry. Shown are representative flow cytometric analysis of CD4 vs. CD8 staining of spleen T cells from one of three experiments at 9.5 and 18 month of age. The numbers indicate the percentage of cells in each quadrant. The statistical analysis is based on n = 3. The experiment was repeated twice with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GCs play a regulatory role in the development and homeostasis of thymocytes (17, 18). However, a possible role of endogenous GCs in regulating the age-associated thymic involution process has not been established. We have for the first time demonstrated that endogenous GCs delay the initiation of the age-associated thymic involution process through directly affecting the thymocytes. In our Lck(Pr)-sGR transgenic mice with increased GC sensitivity restricted to the T-cell lineage, the initiation of the age-associated thymic involution was delayed from around 6 wk to around 6 months of age. Interestingly, thymocyte number in transgenic mice peaked at 6 months of age (~220 million cells), and at this time the total number of thymocytes in Lck(Pr)-sGR transgenic mice was similar to the peak thymocyte number seen in normal mice at the time when thymic involution normally starts (4–6 wk of age). Because thymocyte number in Lck(Pr)-sGR mice reached its maximal number at 6 months of age, the results also showed that thymus development was delayed by GCs in these mice. At 4 months of age, the thymocyte number in Lck(Pr)-sGR and WT mice was similar (~160 million cells). This is explained by an already initiated age-associated involution in WT mice, whereas thymocyte number in Lck(Pr)-sGR mice was still increasing (thymocyte number in Lck(Pr)-sGR mice at 6 wk of age was 135 million (cf. Ref. 18).

The delayed initiation of the age-associated thymic involution in Lck(Pr)-sGR mice resulted in a higher thymocyte number at 6 months of age or older, compared with WT mice. This was in contrast to our previous report in which we showed that thymocyte number in young (4–6 wk old) Lck(Pr)-sGR transgenic mice is lower, compared with WT mice (17, 18). The reversed ratio of thymocyte number in Lck(Pr)-sGR transgenic vs. WT mice in young (4–6 wk of age) and aged mice (>=6 months of age) is explained by an impeded thymus development in young Lck(Pr)-sGR transgenic mice, whereas the effect on the delayed involution is not yet visible. However, in aged Lck(Pr)-sGR transgenic mice, the delayed initiation of the age-associated thymic involution gave rise to the higher number of thymocytes, compared with WT mice. Of note is that the above difference in young vs. aged mice is not due to an altered GR expression (endogenous or transgenic, Fig. 2AGo) or a difference in GC sensitivity (Fig 2Go, B and C) (18), demonstrating that GR activity was unaltered in young and aged thymocytes. Furthermore, serum corticosterone concentration was similar in young and aged mice (Fig. 2DGo) (18).

In the thymus of Lck(Pr)-sGR transgenic mice, the increased GC sensitivity was restricted to the thymocytes. Hence, the results provided clear evidence that the above effects of the GCs on the age-associated thymic involution was primed by direct GC effects on thymocytes and not on other cell types present in the thymus such as thymic epithelial cells, macrophages, and dendritic cells, which are GC responsive and affect the development of thymocytes (49, 50, 51). Thus, our results clarify that thymocytes play an important role in delaying the thymic involution process in Lck(Pr)-sGR transgenic mice. This idea that thymocytes are involved in the regulation of age-associated thymic involution agrees with reports showing that thymic atrophy occurs in severe combined immunodeficient mice (52), recombinase-activating gene knockout mice (53), TCR knockout mice (54), and anti-CD3 treated mice (53), in which thymocytes are absent, demonstrating an important role for the thymocytes in maintaining the thymus architecture.

In the aged Lck(Pr)-sGR transgenic mice, the higher number of thymocytes was associated with increased percentage of cycling DN population. No increase in the percentage of cycling DP thymocytes was detected. Furthermore, no difference in thymocyte apoptosis between aged WT and Lck(Pr)-sGR transgenic mice was observed. This suggests that the higher number of thymocytes in aged Lck(Pr)-sGR transgenic mice, and particularly in the DP population, was primarily a result of a higher number of the precursor DN population. Support for this conclusion comes from a recent study showing that the number of DP cell is proportional to the number of DN cells and that the number of the DP population is not under homeostatic control (55). Furthermore, in aged transgenic mice, it seems unlikely that the higher number of thymocytes at one age accounts for the higher number of thymocytes in later ages. This conclusion is based on the fact that the thymocytes are replaced by new cells within 5–7 d (56), resulting in a complete renewal of the thymocytes within a week. Another interesting observation in the thymus of the aged transgenic mice was the increased percentage of mature CD4+ and CD8+ SP thymocytes (Fig. 1BGo). The molecular mechanism explaining this effect of endogenous GCs on SP thymocytes is not clear. However, our results demonstrated an increase in the percentage of cycling CD4+ and CD8+ SP thymocytes. This suggests that the increased percentage of mature CD4+ and CD8+ SP thymocytes could be due to increased proliferation of these cells. Alternatively, the differentiation of DP into SP thymocytes might be enhanced.

Despite the discovery of age-associated thymic involution decades ago, the mechanism for this process is still not well understood. However, the results from the literature support the notion that intrinsic changes to the thymus are responsible for the age-related thymic involution (9). A factor that has been reported to influence the thymic involution is IL-7, which has been shown to decline with age in mice (7) and inhibit thymic involution after injection (57). Other factors suggested to be involved in regulating thymic aging include leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor. The mRNA expression of these factors have been shown to be elevated in aged thymuses (58). Interestingly, in vivo administration of these factors to mice-induced thymic atrophy (58). At the molecular level, it is not clear how GR is involved in delaying initiation of the age-associated thymic involution. As a transcription factor, GR activates and represses transcription of target genes by either direct interaction with GC response elements or interference with the activities of other transcription factors. This latter mechanism, referred to as cross-talk, is usually mutual, so that in general, age-dependent changes in the expression of proteins in the thymocytes may affect the GC response. Relevant to our results is that GCs have been shown to inhibit the expression or function of some of the above factors suggested to be involved in controlling thymus involution (59, 60). Furthermore, GCs can change the expression levels of GC-regulated genes in the thymocytes, making the cells respond differently to the stimuli of other cells such as thymic epithelial cells and bone marrow-derived antigen-presenting cells in the thymic environment. As an example, GCs has been shown to regulate the response of thymocytes to the TCR stimuli, which controls the development and selection of the thymocytes at different stages (17). Additional research is required to identify target genes that are involved in the delayed thymic involution. However, supporting our result that endogenous GCs indeed can delay thymic involution is a report demonstrating that repetitive hydrocortisone acetate injections maintained the number of thymocytes (61).

An alternative mechanism other than a continuous GC regulation of target genes that could affect the initiation of the thymic involution in transgenic mice may relate to a change in the thymus of young mice that persist in aged mice. However, this is less likely because short-term treatment of mice with GCs has been shown to exert only a temporary effect on thymocyte number because thymopoiesis recovered rapidly after discontinuation of treatment (62). Furthermore, new cells produced by the bone marrow-derived precursor cells replace all thymocytes within a week.

An additional interesting observation in the aged Lck(Pr)-sGR transgenic mice was the opposite effects exerted by GCs on the number of thymocytes and peripheral T cells. Whereas the number of thymocytes in aged transgenic mice was higher than in WT mice, the number of peripheral T cells in aged transgenic mice was lower than in WT mice, thus demonstrating a differential regulation by GCs on thymocyte and peripheral T-cell homeostasis in aged mice. Mechanistically this could be explained by increased percentage of cycling DN and SP thymocytes, whereas the percentage of cycling peripheral CD4+ T cells was repressed. The different response between thymocytes and peripheral T cells in aged mice could be due to a different stage of differentiation or dissimilar microenvironment. Supporting this are studies in GR transactivation-deficient mice (GRdim mice), showing that GCs use different GR signaling mechanisms for inducing apoptosis in thymocytes and peripheral T cells (63). In line with our results, recent studies report that the number of peripheral T cells are largely independent of the number of thymocytes (31, 55, 64, 65). It should be noted that lymphocyte numbers in the periphery is tightly controlled through various homeostatic mechanisms. Recent data from a number of groups have demonstrated that a reduction in the number of peripheral T cells is mainly compensated by proliferation of remaining lymphocytes, a process called homeostatic expansion (65). The repressed cycling of peripheral Lck(Pr)-sGR transgenic CD4+ T cells is in line with the reports demonstrating that GCs inhibit proliferation of T cells (11, 17) and agrees with our observation that T cells with a 50% reduction in GR content expanded more than WT T cells after transfer into T-cell-deficient severe combined immunodeficient mice (our unpublished observations). This observation clearly confirms a direct regulatory effect of endogenous GCs on expansion of T cells in vivo. Thus, endogenous GCs seem to have an important function in regulating expansion of both activated and resting T cells. A possible explanation for this effect could be that GCs attenuate the TCR signaling provided by TCR recognition of peptide/major histocompatibility complex (28), which is a prerequisite for both homeostatic expansion and survival of T cells (65).

An interesting observation in the aged Lck(Pr)-sGR mice was the alteration in the CD4+/CD8+ T-cell ratio. Evidence has been presented that the CD4+ and CD8+ T cells are independently controlled and that the CD4+/CD8+ T-cell ratio is highly consistent within a given strain (65). We previously reported that endogenous GCs have a more pronounced suppressive effects on CD4+ than CD8+ T cells in the young mice, resulting in a decreased CD4+/CD8+ T-cell ratio (18). Of note is that this is not due to different expression levels of the transgene because the lckPr promoter shows a similar activity in both CD4+ and CD8+ cells (66). In this report we also demonstrate that the suppression of CD4+ T-cell number is more pronounced with age, so that in the very old Lck(Pr)-sGR transgenic mice, the CD8+ T cells were unusually more abundant than CD4+ T cells. This can be explained by our results showing a more pronounced suppressive effect of GCs on CD4+ than on CD8+ T-cell cycling. Thus, our results further elucidate the involvement of endogenous GCs in the regulation of the CD4+/CD8+ T-cell ratio via differential effects on CD4+ and CD8+ T cells. Interestingly, the reversed CD4+/CD8+ peripheral T-cell ratio could not be observed when CD4+ and CD8+ SP thymocytes were analyzed. This observation demonstrates that endogenous GCs independently regulate the CD4+/CD8+ T-cell ratios in the thymus and periphery.

In summary, in the present study, we have clarified some novel biological effects of endogenous GCs. First, it is shown that endogenous GCs delay the initiation of age-associated thymic involution. However, no age-dependent alteration of total T-cell number by endogenous GCs was observed. This showed that GCs exert different effects on thymocyte and peripheral T-cell homeostasis in aged mice. Second, we have shown that endogenous GCs play a crucial role in controlling the ratio between the CD4+ and CD8+ T cells. Finally, by using transgenic mice with a restricted change in the GC sensitivity to the T lineage, we could clarify that direct actions of endogenous GCs on the T-cell lineage prime all the above-described effects. The phenotype observed in the aged Lck(Pr)-sGR transgenic mice was not a consequence of the integration site of the transgene because the same results were seen in two independently generated transgenic mice lines. It remains to be seen whether the delayed age-associated thymic involution by GCs could be used to promote immune function particularly in cases when T-cell replenishment is desired, e.g. in situations of large depletion of T cells by disease such as AIDS and cytotoxic therapy in malignancy or after cytoreduction in connection to bone marrow transplantation and in aged individuals (67).


   Acknowledgments
 
The Embryo and Genome Research Core Facility and the animal care unit at the Huddinge University Hospital, Karolinska Institutet, are acknowledged for the pronuclear injections and animal care. We thank Dr. Wolfgang Schmid (DFZK, Heidelberg, Germany) for valuable comments on the manuscript.


   Footnotes
 
This work was supported by the Swedish Cancer Society (to S.O.) and the Swedish Research Council (to M.J.). Part of this work was supported by the European Community Program Quality of Life and Management of Living Resources under contract number QLG1-CT-2001-01574.

Abbreviations: 7-AAD, 7-Aminoactinomycin D; DN, double negative; DP, double positive; FITC, fluorescein isothiocyanate; GC, glucocorticoid; GR, GC receptor; HRP, horseradish peroxidase; mAb, monoclonal antibody; PE, phycoerythrin; SP, single positive; TCR, T-cell receptor; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling; WT, wild-type.

Received December 8, 2003.

Accepted for publication January 7, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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