| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 26, 2002 as doi:10.1096/fj.01-0891fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,2
* Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, Novum, SE-141 86 Huddinge, Sweden; and
Microbiology and Tumor Biology Center (MTC), Karolinska Institutet, SE-171 77 Stockholm, Sweden
3Correspondence: Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, Novum, SE-141 86 Huddinge, Sweden. E-mail: Sam.Okret{at}mednut.ki.se
SPECIFIC AIMS
The aim of the present study was to investigate a direct effect of normal concentrations of endogenous glucocorticoids (GCs) under regular nonstressed conditions on thymocyte and T cell homeostasis independent of secondary systemic effects of GCs. This was achieved by generating transgenic mice with an altered GC sensitivity restricted to the T cell lineage.
PRINCIPAL FINDINGS
1. Increased GC sensitivity in the T cell lineage causes a reduction in thymocyte pool size
Transgenic mice with an increased GC sensitivity in T cell lineage were generated by overexpressing GR cDNA in sense orientation under the control of the p56 proximal lck promoter (lckPr-sGR). Analysis of transgene expression showed it was restricted to lymphoid tissues like the thymus, spleen, and lymph nodes. No expression was seen in nonlymphoid tissues: liver, heart, and kidney. Quantification of the GR concentration in thymocytes from lckPr-sGR mice showed it had increased by 
twofold in mice homogeneous for the transgene. This twofold increase in GR concentration was reflected in a two- to threefold increase in GC sensitivity as measured by the ability of corticosterone to induce apoptosis in thymocytes in an in vitro assay. Thymocyte number in lckPr-sGR mice homozygous for the transgene was reduced by 42 ± 8% compared with control mice (P<0.005). FACS analysis of thymocyte populations from the lckPr-sGR transgenic mice revealed no significant change in the percentage of CD4-8- (DN), CD4+8+ (DP) or CD4 single positive (SP) thymocytes vs. the relative thymocyte distribution seen in control mice. In contrast, the percentage of CD8 SP cells increased from 2.7 to 4.9% (P<0.01) (Fig. 1
A). However, taking into account a 42% loss of total thymocyte number (see above), the total number of DP cells decreased by 47%, DN by 38%, and CD4 SP by 40% whereas the number of CD8 SP cells was not significantly altered (Fig. 1B
). These results showed that in the lckPr-sGR transgenic mice CD8 SP cells are influenced less than CD4 SP cells.
|
To evaluate whether the decreased thymocyte number was due to an increased apoptosis or decreased proliferation, TUNEL assay, 7-aminoactinomycin D (7-AAD) and rhodamine 123 staining for detection of apoptosis and propidium iodide and 7-AAD staining for analysis of cell cycle were performed. We found no significant change in cell cycling in thymocyte subpopulations between lckPr-sGR and control mice. However, after staining of thymocytes by 7-AAD or rhodamine 123, we observed that apoptosis in freshly isolated thymocytes was increased twofold in lckPr-sGR mice vs. control mice (14%±1.6 vs. 6.6%±0.7 n=6, P<0.01). An increased thymic apoptosis in lckPr-sGR mice was confirmed after in situ analysis of apoptosis by TUNEL assay. The increased thymic apoptosis observed in lckPr-sGR transgenic mice was not due to increased circulating serum corticosterone levels. Serum corticosterone levels did not significantly differ between transgenic and control mice (233±47 vs. 261±23 ng/ml, n=5). Thus, the reduced thymocyte pool size in lckPr-sGR transgenic mice was most likely due to increased apoptosis and not to inhibition of cell proliferation.
2. Increased GC sensitivity in the T cell lineage causes a reduction in T cell pool size
We show that the lckPr promoter drives expression of the transgene to peripheral T cells to a level similar to that observed in thymocytes. This was reflected in a two- to threefold increase in GC sensitivity of nonactivated peripheral T cells from lckPr-sGR mice as determined in an in vitro apoptosis assay. A similar increase in GC sensitivity was observed when proliferation of mitogen (concanavalin A or TPA and ionomycin) -stimulated T cells was inhibited by GCs.
We further characterized the effect of the increased GC sensitivity in peripheral T cells on T cell homeostasis. FACS analysis of spleen T lymphocytes demonstrated that the relative CD4+ population of total splenocytes had decreased by 55% in lckPr-sGR mice (14.04±1.5% vs. 31.04±3.9% in control mice). The relative change in the CD8+ population was decreased by only 23% (10.22±1.7% vs. 13.2±1.3%), resulting in a decrease in CD4+:CD8+ T cell ratio from 2.35 to 1.37 (Fig. 2
A). Using the above data, we found that total spleen CD4+ T cells were decreased by 73% and spleen CD8+ T cells were decreased by 52% (Fig. 2B
).
|
FACS analysis of blood T cells from lckPr-sGR mice showed that the CD4+ T cells had decreased by 58% (13.3±3% vs. 31.6±6.2%) whereas CD8+ T cells had decreased by 38% (6.91±2% vs. 11.15±1.1%), resulting in a decrease in CD4+:CD8+ T cell ratio from 2.8 to 1.9. Calculating the total number of blood CD4+ and CD8+ T cells revealed a decrease by 83% and 78%, respectively. Functionally, the reduced peripheral T cell number in lckPr-sGR transgenic mice was reflected in a decreased T cell-dependent antibody response whereas the T cell-independent antibody response was unaffected.
3. Transgenic mice with a reduced GR expression in the T cell lineage show an increase in thymocyte and T cell number
To further confirm that the phenotype of our lckPr-sGR transgenic mice was a consequence of the overexpression of GR, we produced transgenic mice in which the endogenous GR level in the T cell lineage was reduced. This was achieved by expressing a portion of the 3' untranslated region of the GR cDNA in the antisense orientation under the control of the same lckPr promoter. When the construct lckPr-asGR was expressed in heterozygotic transgenic mice, it resulted in a 25% reduction in expression of the endogenous GR in thymocytes. Examination of total thymocyte number in the lckPr-asGR mice revealed a 1.6-fold increase compared with control mice. Analysis of individual thymocyte subpopulations showed that the DP and CD4 SP cells were significantly increased. In the spleen, total T cell number increased by 1.4-fold. The increase was mainly confined to the peripheral CD4+ cells, confirming results from the lckPr-sGR mice that GCs have a differential effect on CD4+ and CD8+ T cells. The overall mirror image phenotype of the lckPr-asGR vs. the lckPr-sGR transgenic mice confirmed the conclusion that basal concentrations of endogenous GCs under nonstressed conditions contribute to the regulation of thymocyte and T cell homeostasis by directly affecting the cells.
CONCLUSIONS
The main conclusion of this work is that normal concentrations of endogenous GCs under regular nonstressed conditions reduce thymocyte and T pools by directly affecting the cells. A modest twofold increase in GC sensitivity in lckPr-sGR transgenic mice was sufficient to cause pronounced phenotypic changes in thymocyte and T cell homeostasis. The phenotype of the lckPr-sGR mice was confirmed by lckPr-asGR mice, which showed an overall mirror image of thymocyte and T cell homeostasis. The main mechanism explaining the reduced thymocyte number in lckPr-sGR mice seems to be primarily due to increased apoptosis rather than impaired proliferation of thymocytes. A more pronounced effect on CD4+ vs. CD8+ T cells was observed showing that GCs regulate the CD4+:CD8+ T cell ratio. Thymocyte and peripheral T cell pools are at least in part independently regulated by GCs. This is based on our observation that the reduction in T cell number in lckPr-sGR mice was much more pronounced than the reduction in mature SP thymocytes. This is in line with several previous reports demonstrating an independent regulation of thymocyte and T cell pools.
Why do we see a reduction in thymocyte and T cell pool size in the lck-sGR mice? It might represent a continuous ‘GC pressure’ in vivo on developing thymocytes and naive peripheral T cells. In this model, thymocytes at different stages of differentiation need to generate protective signals through either stromal interactions, cytokines, or TCR recognition of peptide/MHC complexes in order to escape induction of apoptosis caused by the endogenous GCs. As apoptosis by default (caused by the lack of positive signals) is the major cause of thymocyte deletion, high GC sensitivity in thymocytes will accelerate the death of neglected cells, resulting in reduced thymocyte numbers. The pronounced effect on thymic apoptosis seen in our transgenic mice affirms that GCs are involved in the deletion of neglected cells. We suggest that a similar mechanism operates for regulation of the peripheral T cell pool size by GCs. Recent reports have demonstrated that the survival of naive T cells requires continuous TCR ligation to peptide/MHC complexes, mechanisms similar to those acting during positive selection in the thymus. T cell pool size thus may be regulated by a balanced set of positive and negative signals. In our proposal, GCs would act as such a negative signal, exerting a continuous GC pressure in vivo, reducing the size of the peripheral T cell pool by deleting T cells not generating survival signals or suppressing signals involved in controlling T cell survival (Fig. 3
).
|
Previous data on the effects of a systemic change in GC levels (e.g., after adrenalectomy, stress, or GC administration) on thymocytes and T cells is well studied. In this report, we present data that normal, nonstressed levels of endogenous GCs directly regulate the thymocyte and T cell pool size independent of systemic effects. The results presented here enhance understanding of how the peripheral T cell pool is regulated and of the role of GCs in thymocyte development, a matter of recent debate.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0891fje; to cite this article, use FASEB J. (March 26, 2002) 10.1096/fj.01-0891fje; 
2 Present address: Department of Immunology, School of Medicine, Beijing University, Beijing 100083, PR China.
This article has been cited by other articles:
|
|
 |
|
  J. v. d. Brandt, F. Luhder, K. G. McPherson, K. L. de Graaf, D. Tischner, S. Wiehr, T. Herrmann, R. Weissert, R. Gold, and H. M. Reichardt Enhanced Glucocorticoid Receptor Signaling in T Cells Impacts Thymocyte Apoptosis and Adaptive Immune Responses Am. J. Pathol., March 1, 2007; 170(3): 1041 - 1053. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Pazirandeh, M. Jondal, and S. Okret Conditional Expression of a Glucocorticoid Receptor Transgene in Thymocytes Reveals a Role for Thymic-Derived Glucocorticoids in Thymopoiesis in Vivo Endocrinology, June 1, 2005; 146(6): 2501 - 2507. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. I Freeman, H. L Munn, V. Lyons, A. Dammermann, J. R Seckl, and K. E Chapman Glucocorticoid down-regulation of rat glucocorticoid receptor does not involve differential promoter regulation J. Endocrinol., November 1, 2004; 183(2): 365 - 374. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Jeong, K. Y. Lee, D. Shin, H. Chung, S. H. Jeon, and R. H. Seong Nitric Oxide Inhibits Glucocorticoid-induced Apoptosis of Thymocytes by Repressing the SRG3 Expression J. Biol. Chem., August 13, 2004; 279(33): 34373 - 34379. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ko, J. Jang, J. Ahn, K. Lee, H. Chung, S. H. Jeon, and R. H. Seong T Cell Receptor Signaling Inhibits Glucocorticoid-induced Apoptosis by Repressing the SRG3 Expression via Ras Activation J. Biol. Chem., May 21, 2004; 279(21): 21903 - 21915. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Pazirandeh, M. Jondal, and S. Okret Glucocorticoids Delay Age-Associated Thymic Involution through Directly Affecting the Thymocytes Endocrinology, May 1, 2004; 145(5): 2392 - 2401. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
