Inhibition of lymphoma vascularization and dissemination by estrogen receptor ß agonists.

Most lymphomas show an increased incidence and poorer prognosis in males vs females, suggesting endocrine regulation. We have previously shown that tumor growth in vivo of a murine T-cell-derived lymphoma is repressed following activation of estrogen receptor ß (ERß, ESR2). By using ERß-deficient mice, we now demonstrate that this inhibition is mediated via a direct effect on the tumor cells and not on the microenvironment. Furthermore, we show that the growth-suppressing effects of ERß agonist are also valid for human B-cell lymphomas as demonstrated in tumors derived from Granta-519 mantle cell lymphoma (MCL) and Raji Burkitt lymphoma (BL) cells. In Granta-519 MCL tumors, activation of ERß reduced expression of BAFF and GRB7, 2 important molecules involved in B-cell proliferation and survival. Importantly, activation of ERß inhibited angiogenesis and lymphangiogenesis, possibly mediated by impaired vascular endothelial growth factor C expression. Furthermore, using disseminating Raji BL cells, we show that ERß activation reduces dissemination of grafted Raji BL tumors. We also show by immunohistochemistry that ERß is expressed in primary MCL tissue. These results suggest that targeting ERß with agonists may be valuable in the treatment of some lymphomas, affecting several aspects of the malignant process, including proliferation, vascularization, and dissemination.

ACTH controls thymocyte homeostasis independent of glucocorticoids.

It has been known for decades that lowering the circulating glucocorticoid (GC) concentration as in Addison's disease or after removing the adrenals results in thymus enlargement, largely due to thymocyte expansion. This has been attributed to the loss of the proapoptotic effects on thymocytes by adrenal GCs. Here, we test this concept and report that ACTH directly controls thymic growth post-adrenalectomy (ADX) independent of the proapoptotic effect of GCs. This was supported by the finding that ADX caused thymus enlargement and a 1.7-fold (P < 0.001) increase in thymocyte number in GR(LckCre) mice resistant to GC-induced thymocyte apoptosis, similar to the increase seen in wild-type mice (2.2-fold; P < 0.01). We show by immunostaining that melanocortin receptor subtype 2, which selectively binds ACTH, is partly expressed on the thymic epithelium. Furthermore, ACTH in comparison to vehicle induced a 2.0-fold (P < 0.01) increase in fetal thymic organ culture thymocyte numbers in vitro and enhanced 2.2-fold (P < 0.05) the expression of delta-like ligand 4, a factor that supports T-cell development. Additionally, adrenalectomized GR(LckCre) mice treated with ACTH under conditions that repressed endogenous ACTH secretion showed increased thymocyte cellularity (1.9-fold; P < 0.01) and splenic naive T-cell numbers (2.5-fold; P < 0.001) compared to when treated with PBS. Altogether, our results show that ACTH directly controls thymocyte homeostasis independent of GCs. These results revise the old paradigm behind compensatory thymus growth following ADX, now demonstrating that ACTH has a central role in regulating thymocyte expansion when systemic GC concentration is low.

Sex- and Female Age-Dependent Differences in Gene Expression in Diffuse Large B-Cell Lymphoma-Possible Estrogen Effects.

For most lymphomas, including diffuse large B-cell lymphoma (DLBCL), the male incidence is higher, and the prognosis is worse compared to females. The reasons are unclear; however, epidemiological and experimental data suggest that estrogens are involved. With this in mind, we analyzed gene expression data from a publicly available cohort (EGAD00001003600) of 746 DLBCL samples based on RNA sequencing. We found 1293 genes to be differentially expressed between males and females (adj. p-value < 0.05). Few autosomal genes and pathways showed common sex-regulated expression between germinal center B-cell (GCB) and activated B-cell lymphoma (ABC) DLBCL. Analysis of differentially expressed genes between pre- vs. postmenopausal females identified 208 GCB and 345 ABC genes, with only 5 being shared. When combining the differentially expressed genes between females vs. males and pre- vs. postmenopausal females, nine putative estrogen-regulated genes were identified in ABC DLBCL. Two of them, NR4A2 and MUC5B, showed induced and repressed expression, respectively. Interestingly, NR4A2 has been reported as a tumor suppressor in lymphoma. We show that ABC DLBCL females with a high NR4A2 expression showed better survival. Inversely, MUC5B expression causes a more malignant phenotype in several cancers. NR4A2 and MUC5B were confirmed to be estrogen-regulated when the ABC cell line U2932 was grafted to mice. The results demonstrate sex- and female reproductive age-dependent differences in gene expression between DLBCL subtypes, likely due to estrogens. This may contribute to the sex differences in incidence and prognosis.

A MYC-controlled redox switch protects B lymphoma cells from EGR1-dependent apoptosis.

Refractory and relapsed B cell lymphomas are often driven by the difficult-to-target oncogene MYC. Here, we report that high MYC expression stimulates proliferation and protects B lymphoma cells from apoptosis under normal oxidative stress levels and that compounds including N-acetylcysteine (NAC) and vitamin C (VitC) induce apoptosis by reducing oxidative stress. NAC and VitC injections effectively reduce tumor growth in lymphoma cells with high MYC expression but not in those with low MYC expression. MYC knockdown confers tumor resistance to NAC and VitC, while MYC activation renders B cells sensitive to these compounds. Mechanistically, NAC and VitC stimulate MYC binding to EGR1 through Cys117 of MYC, shifting its transcriptional output from cell cycle to apoptosis gene expression. These results identify a redox-controlled mechanism for MYC's role in maintaining proliferation and preventing apoptosis, offering a potential therapeutic rationale for evaluating NAC or VitC in patients with MYC-driven B cell lymphoma.

Estrogen Receptor ß (ESR2) Transcriptome and Chromatin Binding in a Mantle Cell Lymphoma Tumor Model Reveal the Tumor-Suppressing Mechanisms of Estrogens.

Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma with one of the highest male-to-female incidence ratios. The reason for this is not clear, but epidemiological as well as experimental data have suggested a role for estrogens, particularly acting through estrogen receptor ß (ESR2). To study the ESR2 effects on MCL progression, MCL cells sensitive and resistant to the Bruton tyrosine kinase inhibitor ibrutinib were grafted to mice and treated with the ESR2-selective agonist diarylpropionitrile (DPN). The results showed that the DPN treatment of mice grafted with both ibrutinib-sensitive and -resistant MCL tumors resulted in impaired tumor progression. To identify the signaling pathways involved in the impaired tumor progression following ESR2 agonist treatment, the transcriptome and ESR2 binding to target genes were investigated by genome-wide chromatin immunoprecipitation in Granta-519 MCL tumors. DPN-regulated genes were enriched in several biological processes that included cell-cell adhesion, endothelial-mesenchymal transition, nuclear factor-kappaB signaling, vasculogenesis, lymphocyte proliferation, and apoptosis. In addition, downregulation of individual genes, such as SOX11 and MALAT1, that play a role in MCL progression was also observed. Furthermore, the data suggested an interplay between the lymphoma cells and the tumor microenvironment in response to the ESR2 agonist. In conclusion, the results clarify the mechanisms by which estrogens, via ESR2, impair MCL tumor progression and provide a possible explanation for the sex-dependent difference in incidence. Furthermore, targeting ESR2 with a selective agonist may be an additional option when considering the treatment of both ibrutinib-sensitive and -resistant MCL tumors.

Thymus-derived glucocorticoids mediate androgen effects on thymocyte homeostasis.

Androgens contribute to the involution process of the aging thymus gland. However, molecular mechanisms behind this effect remain largely unknown. We have investigated the influence of testosterone on the ectopic synthesis of glucocorticoids (GCs) in thymocytes, an activity recently shown by us to be important for the homeostatic regulation of these cells. Castration, which leads to a strong increase in thymus tissue and function, was associated with a reduced GC release from thymocytes caused by down-regulated expression of several enzymes involved in GC synthesis, without affecting GC synthesis in the adrenals. Testosterone treatment of castrated male mice reversed these effects, also without affecting adrenal GC synthesis. The effects of testosterone in castrated mice on thymocyte homeostasis and GC release were strongly reduced in mice pretreated with the CYP11B1 enzyme inhibitor metyrapone, acting on the last step in the corticosterone synthesis. The androgen-induced thymic involution was dependent on GC action, because this was completely absent in mice lacking GC receptor (GR) expression specifically in thymocytes. We provide here an unrecognized mechanism how androgens contribute to thymic involution by stimulating local synthesis and release of GCs in the thymus.

Inhibition of extracellular signal-regulated kinase (ERK) signaling participates in repression of nuclear factor (NF)-kappaB activity by glucocorticoids.

Glucocorticoid (GC) effects are mediated via the GC-receptor (GR), which either stimulates or represses gene expression. Repression of target genes often involves negative cross-talk between the GR and other transcription factors e.g. NF-kappaB, important for gene activation. Using HEK293 cells we here describe that repression of NF-kappaB requires functions of the GR that are dependent on the signaling pathways employed to activate NF-kappaB. While a GR mutant was able to repress NF-kappaB activity following activation by TNFalpha, it did not so following activation by the phorbol ester TPA. In these cells, TPA stimulation but not TNFalpha, activated extracellular signal-regulated kinase (ERK). We demonstrated that the ability of the dexamethasone activated GR mutant to repress TPA-induced NF-kappaB activity was restored in conjunction with ERK1/2 inhibition. Previous reports have shown GC-mediated inhibition of ERK1/2 phosphorylation to involve GC induction of MAPK phosphatase-1 (MKP-1). Here, we demonstrated that the GRR488Q mutant was incapable of inducing gene expression of endogenous MKP-1 following dexamethasone treatment, in contrast to the GRwt. However, TPA treatment alone resulted in much stronger MKP-1 expression in both GRwt and GRR488Q containing cells than that of dexamethasone suggesting that the inability of GRR488Q to inhibit TPA-induced NF-kappaB activity did not involve a lack of MKP-1 expression. In line with this, RNAi targeted towards MKP-1 did not abolish or inhibit the ability of the GRwt to repress NF-kappaB activity. Importantly, we observed no difference in activated ERK1/2 (phospho-ERK1/2) expression over time between GRwt and GRR488Q containing cells following co-treatment with TPA and dexamethasone. Based on these results we suggest that GRwt does not directly regulate ERK1/2 but rather alters ERK1/2-mediated effects allowing it to repress NF-kappaB activity, a capacity lacked by the GRR488Q mutant.

Age-related synthesis of glucocorticoids in thymocytes.

Glucocorticoids (GCs) are primarily synthesized in the adrenal glands but an ectopic production has also been reported in the brain, the gastrointestinal tract and in thymic epithelial cells (TEC). Here we show that thymocytes express genes encoding for all enzymes required for de novo GC synthesis and produce the hormone as demonstrated by both a GC specific reporter assay and a corticosterone specific ELISA assay. Interestingly, GC synthesis is detectable in cells from young mice (4 weeks) and thereafter increases during aging (14-22 weeks) together with an increased gene expression of the rate-limiting enzymes StAR and CYP11A1. Hormone production occurred at a thymocyte differentiation stage characterized by being double positive for the CD4 and CD8 surface markers but was found to be unrelated to CD69 expression, a marker for thymocytes undergoing positive selection. No GC synthesis was found in resting or anti-CD3 activated CD4 and CD8 positive T cells isolated from the spleen. Thymocyte-derived GC had an anti-proliferative effect on a GR-transfected cell line and induced apoptosis in thymocytes. The age- and differentiation stage-related GC synthesis in thymocytes may play a role in the involution process that the thymus gland undergoes.

p110α Inhibition Overcomes Stromal Cell-Mediated Ibrutinib Resistance in Mantle Cell Lymphoma.

Acquired resistance to cancer drugs is common, also for modern targeted drugs like the Bruton tyrosine kinase (BTK) inhibitor ibrutinib, a new drug approved for the treatment of the highly aggressive and relapsing mantle cell lymphoma (MCL). The tumor microenvironment often impacts negatively on drug response. Here, we demonstrate that stromal cells protect MCL cells from ibrutinib-induced apoptosis and support MCL cell regrowth after drug removal by impairing ibrutinib-mediated downregulation of PI3K/AKT signaling. Importantly, the stromal cell-mediated ibrutinib resistance was overcome in vitro by inhibiting AKT activity using the PI3K catalytic p110α subunit-specific inhibitor BYL719. This was seen both for MCL cell lines and primary MCL cells. Furthermore, inhibition of p110α activity by BYL719 potentiated the ability of ibrutinib to inhibit MCL tumor growth in vivo in a mouse xenograft model. The stromal cell-mediated ibrutinib resistance was found to be due to a direct interaction with MCL cells and involves the integrin VLA-4, as disrupting stromal cell-MCL cell interaction using a VLA-4 blocking antibody abrogated the ibrutinib resistance. This suggests that combined treatment with ibrutinib and a p110α inhibitor, alternatively by disrupting stromal cell-MCL cell interaction, may be a promising therapeutic strategy to overcome stromal cell-mediated ibrutinib resistance in MCL. Mol Cancer Ther; 17(5); 1090-100. ©2018 AACR.

Suppression of lymphoma growth by the xenoestrogens bisphenol A and genistein.

Well-defined physiological functions of estrogens are mediated via nuclear estrogen receptors α (ESR1) and ß (ESR2). With regard to hematological malignancies, expression of ESR2 has been found in both B and T cell lymphomas. In addition to endogenous estrogens or selective ESR2 agonists, ESR2 signaling may be affected by both environmental synthetic estrogen-mimicking compounds and dietary phytoestrogens. In the present study, we demonstrate that oral exposure with either the synthetic compound bisphenol A (BPA) or the dietary phytoestrogen genistein reduced the growth of grafted murine T cell (EG7) and human B cell (Granta-519 mantle cell) lymphomas which both express ESR2. Suppression of lymphoma growth was due to reduced proliferation (BPA and genistein) and induction of apoptosis (genistein). Inhibition of lymphoma growth was seen at a BPA dose of 50 µg/kg body weight (BW)/day considered to be safe human exposure dose or a genistein dose of 1 mg/kg BW/day orally, which is reached in soy-rich diets. Thus, our study indicates that the environmental xenoestrogens BPA and genistein have anti-proliferative effects on ESR2-expressing lymphomas. Our data suggest that phytoestrogens may be considered as a dietary supplement for lymphoma patients and possibly for prevention of lymphoid malignancies.

Estrogen receptor ß1 in diffuse large B-cell lymphoma growth and as a prognostic biomarker.

Diffuse large B-cell lymphoma (DLBCL) shows a higher incidence in males versus females. Epidemiological studies have shown that female gender is a favorable prognostic factor, which may be explained by estrogens. Here we show that when grafting human DLBCL cells to immunocompromised mice, tumor growth in males is faster. When treating mice grafted with either germinal center or activated B-cell like DLBCL cells with the selective estrogen receptor ß (ERß) agonist diarylpropionitrile, tumor growth was significantly inhibited. Furthermore, nuclear ERß1 expression analysis in primary DLBCL's by immunohistochemistry revealed expression in 89% of the cases. Nuclear ERß1 expression was in a univariate and multivariate analysis, an independent prognostic factor for adverse progression-free survival in Rituximab-chemotherapy treated DLBCL (p = 0.02 and p = 0.04, respectively). These results suggest that estrogen signaling through ERß1 is an interesting future therapeutic target for treatment of DLBCL, and that ERß1 expression can be used as a prognostic marker.

Identification of target genes involved in the antiproliferative effect of glucocorticoids reveals a role for nuclear factor-(kappa)B repression.

Glucocorticoid hormones (GCs) exert an antiproliferative effect on most cells. However, the molecular mechanism is still largely unclear. We investigated the antiproliferative mechanism by GCs in human embryonic kidney 293 cells with stably introduced glucocorticoid receptor (GR) mutants that discriminate between cross-talk with nuclear factor-(kappa)B (NF-(kappa)B) and activator protein-1 signaling, transactivation and transrepression, and antiproliferative vs. non-antiproliferative responses. Using the GR mutants, we here demonstrate a correlation between repression of NF-(kappa)B signaling and antiproliferative response. Gene expression profiling of endogenous genes in cells containing mutant GRs identified a limited number of genes that correlated with the antiproliferative response. This included a GC-mediated up-regulation of the NF-(kappa)B-inhibitory protein I(kappa)B(alpha), in line with repression of NF-(kappa)B signaling being important in the GC-mediated antiproliferative response. Interestingly, the GC-stimulated expression of I(kappa)B(alpha) was a direct effect despite the inability of the GR mutant to transactivate through a GC-responsive element. Selective expression of I(kappa)B(alpha) in human embryonic kidney 293 cells resulted in a decreased percentage of cells in the S/G2/M phase and impaired cell proliferation. These results demonstrate that GC-mediated inhibition of NF-(kappa)B is an important mechanism in the antiproliferative response to GCs.

Inhibition of estrogen biosynthesis enhances lymphoma growth in mice.

Most lymphomas show higher incidence and poorer prognosis in males compared to females. However, the endocrine contribution to this gender difference is not entirely known. Here we show that castration accelerates lymphoma growth in C57BL6 male mice grafted with murine EG7 T cell lymphoma cells. However, the androgen receptor antagonist Bicalutamide did not affect lymphoma growth, suggesting no impact of androgen receptor signaling on lymphoma progression. In contrast, inhibition of androgen-to-estrogen conversion by the aromatase inhibitor (AI) Letrozole induced faster lymphoma growth in mice, suggesting that androgens impact lymphoma growth through its conversion to estrogens. This was supported by the inability of dihydrotestosterone, which is not converted to estrogens by aromatase, to influence lymphoma growth in castrated male mice. Lymphoma growth was also stimulated in immunocompromised mice grafted with human B cell lymphoma (Granta-519) and treated with either reversible or irreversible AIs, showing that the blockage of estrogen synthesis caused enhanced growth of both murine T and human B cell lymphomas and with different AIs. Additionally, AI-treated EG7 lymphomas showed accelerated growth not only in male but also in intact female mice. Altogether, our results demonstrate that aromatase inhibition accelerates lymphoma growth but not androgens per se, highlighting a protective role of estrogens in lymphoma pathogenesis. These results also raise concern that the use of AIs in women with breast cancer might enhance lymphoma progression.

Conditional expression of a glucocorticoid receptor transgene in thymocytes reveals a role for thymic-derived glucocorticoids in thymopoiesis in vivo.

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.

Aged transgenic mice with increased glucocorticoid sensitivity in pancreatic beta-cells develop diabetes.

Glucocorticoids are diabetogenic hormones because they decrease glucose uptake, increase hepatic glucose production, and inhibit insulin release. To study the long-term effects of increased glucocorticoid sensitivity in beta-cells, we studied transgenic mice overexpressing the rat glucocorticoid receptor targeted to the beta-cells using the rat insulin I promoter. Here we report that these mice developed hyperglycemia both in the fed and the overnight-fasted states at 12-15 months of age. Progression from impaired glucose tolerance, previously observed in the same colony at the age of 3 months, to manifest diabetes was not associated with morphological changes or increased apoptosis in the beta-cells. Instead, our current results suggest that the development of diabetes is due to augmented inhibition of insulin secretion through alpha(2)-adrenergic receptors (alpha(2)-ARs). Thus, we found a significantly higher density of alpha(2)-ARs in the islets of transgenic mice compared with controls, based on binding studies with the alpha(2)-AR agonist UK 14304. Furthermore, incubation of islets with benextramine, a selective antagonist of the alpha(2)-AR, restored insulin secretion in response to glucose in isolated islets from transgenic mice, whereas it had no effect on control islets. These results indicate that the chronic enhancement of glucocorticoid signaling in pancreatic beta-cells results in hyperglycemia and impaired glucose tolerance. This effect may involve signaling pathways that participate in the regulation of insulin secretion via the alpha(2)-AR.

Effects of ethanol on cytokine generation and NFkappaB activity in human lung epithelial cell.

Alcohol abuse is associated with enhanced risk for pulmonary infections, but the mechanisms remain obscure. We assessed whether ethanol reduced generation of cytokines from a human lung epithelial cell line (A549) in vitro and if effects on the NFkappaB transcription factor were involved. Exposure of A549 to ethanol (0.1-1%) dose-dependently inhibited (by 15-49%) the release of G-CSF and IL-8, but not of M-CSF, triggered by IL1beta or TNFalpha. Ethanol also inhibited by 49% the IL-1beta stimulated translocation of the p65 subunit of NFkappaB from the cytoplasm into the nucleus. Using a kappaB binding and luciferase coupled construct, transfected into A549 cells, we found that 1% ethanol specifically reduced IL-1beta and TNFalpha induced luciferase activity with 34 and 40%, respectively. Thus, in vitro exposure of lung epithelial cells to ethanol reduced the generation of cytokines, as well as translocation and gene activation by NFkappaB.

Effects of altered glucocorticoid sensitivity in the T cell lineage on thymocyte and T cell homeostasis.

The homeostatic regulation that controls total thymocyte and peripheral T-cell numbers is not clearly understood. We describe here a direct hormonal influence of endogenous levels of glucocorticoids (GCs) on thymocyte and peripheral T-cell homeostasis independent of indirect systemic effects of GCs. The results were obtained by generating transgenic mice with an altered GC sensitivity targeted to thymocytes and peripheral T cells by increasing or decreasing glucocorticoid receptor (GR) expression specifically in thymocytes and peripheral T cells. A twofold increase in GC sensitivity resulted in a major decrease in thymocyte number, affecting all subpopulations, although single-positive CD8+ cells were less influenced. In the thymus, this was due to increased apoptosis in the organ, whereas proliferation of thymocyte populations was unaffected. In the periphery, a pronounced reduction in T-cell number was seen, demonstrating an effect of endogenous GCs also on T-cell homeostasis. The effects were confirmed in transgenic mice with reduced GR expression, which showed increased thymocyte and T-cell numbers. Thus, our data demonstrate that physiological GC levels are directly involved in controlling the size of both thymocyte and T-cell pools.

Local glucocorticoid production in the thymus.

Besides generating immunocompetent T lymphocytes, the thymus is an established site of de novo extra-adrenal glucocorticoid (GC) production. Among the compartments of the thymus, both stromal thymic epithelial cells (TECs) and thymocytes secrete biologically active GCs. Locally produced GCs secreted by the various thymic cellular compartments have been suggested to have different impact on thymic homeostasis. TEC-derived GCs may regulate thymocyte differentiation whereas thymocyte-derived GCs might regulate age-dependent involution. However the full biological significance of thymic-derived GCs is still not fully understood. In this review, we summarize and describe recent advances in the understanding of local GC production in the thymus and immunoregulatory steroid production by peripheral T cells and highlight the possible role of local GCs for thymus function.

The selective impact of transgenically expressed glucocorticoid receptor on T cells.

Glucocorticoids (GCs) strongly impact on different T cell subsets inducing generally immunosuppressive effects, whereas much less is known about the effect of GC on natural killer (NK) cells. The aims of this study were to investigate the effects of GC on T cell functions, including T cell-mediated anti-tumor immune response, and on NK cells. We have used lck-GR mice, which overexpress a transgenic rat GR in both T and NK cells. These mice were found to have decreased both CD4(+) and CD8(+) T cell populations in the periphery. In contrast, both NK and NKT cells were found in normal numbers in lck-GR mice. To identify genes and pathways affected by GR overexpression in our system in T cells, we have compared gene expression profiles in wild-type and lck-GR T cells. Among the genes upregulated in T cells from lck-GR mice, the microarray analysis has identified genes regulating expansion of regulatory T cells. The analysis of genes downregulated in lck-GR mice has identified genes and gene associated with the regulation of immune response. With regard to the effects on T cell functions in lck-GR mice, transgenic expression of GR had a suppressive effect on killer cell activity in vitro. In addition, lck-GR mice showed an increased tumor growth in murine tumor model in vivo, which may be a possible consequence of reduced T cell numbers and activity. We conclude that an increased expression of the GR strongly affects numbers and possibly functions of T cell subsets, but has little effect on NK cells.

Glucocorticoids delay age-associated thymic involution through directly affecting the thymocytes.

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.