Enhanced Glucocorticoid Receptor Signaling in T Cells Impacts Thymocyte Apoptosis and Adaptive Immune Responses
2007; Elsevier BV; Volume: 170; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2007.060804
ISSN1525-2191
AutoresJens van den Brandt, Fred Lühder, Kirsty McPherson, Katrien L. de Graaf, Denise Tischner, Stefan Wiehr, Thomas Herrmann, Robert Weissert, Ralf Gold, Holger M. Reichardt,
Tópico(s)Immunotherapy and Immune Responses
ResumoTo study the effect of enhanced glucocorticoid signaling on T cells, we generated transgenic rats overexpressing a mutant glucocorticoid receptor with increased ligand affinity in the thymus. We found that this caused massive thymocyte apoptosis at physiological hormone levels, which could be reversed by adrenalectomy. Due to homeostatic proliferation, a considerable number of mature T lymphocytes accumulated in the periphery, responding normally to costimulation but exhibiting a perturbed T-cell repertoire. Furthermore, the transgenic rats showed increased resistance to experimental autoimmune encephalomyelitis, which manifests in a delayed onset and milder disease course, impaired leukocyte infiltration into the central nervous system and a distinct cytokine profile. In contrast, the ability of the transgenic rats to mount an allergic airway response to ovalbumin was not compromised, although isotype switching of antigen-specific immunoglobulins was altered. Collectively, our findings suggest that endogenous glucocorticoids impact T-cell development and favor the selection of Th2- over Th1-dominated adaptive immune responses. To study the effect of enhanced glucocorticoid signaling on T cells, we generated transgenic rats overexpressing a mutant glucocorticoid receptor with increased ligand affinity in the thymus. We found that this caused massive thymocyte apoptosis at physiological hormone levels, which could be reversed by adrenalectomy. Due to homeostatic proliferation, a considerable number of mature T lymphocytes accumulated in the periphery, responding normally to costimulation but exhibiting a perturbed T-cell repertoire. Furthermore, the transgenic rats showed increased resistance to experimental autoimmune encephalomyelitis, which manifests in a delayed onset and milder disease course, impaired leukocyte infiltration into the central nervous system and a distinct cytokine profile. In contrast, the ability of the transgenic rats to mount an allergic airway response to ovalbumin was not compromised, although isotype switching of antigen-specific immunoglobulins was altered. Collectively, our findings suggest that endogenous glucocorticoids impact T-cell development and favor the selection of Th2- over Th1-dominated adaptive immune responses. Glucocorticoids (GCs) belong to a class of steroid hormones that are synthesized by the adrenal gland and released in response to stimuli such as stress and inflammation. Their secretion is under the control of the hypothalamus-pituitary-adrenal axis, a neuroendocrine cascade that involves positive and negative feedback loops. Once in the circulation, GCs exert pleiotropic effects ranging from the regulation of energy metabolism and the control of cognitive functions to the modulation of the immune system. Due to their lipophilic nature, they can passively diffuse into the cytoplasm and bind to the glucocorticoid receptor (GR). In turn, the GR translocates into the nucleus and interacts directly or indirectly via other transcription factors with promoter and enhancer elements of responsive genes.1Beato M Herrlich P Schütz G Steroid hormone receptors: many actors in search of a plot.Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1642) Google Scholar, 2Zhou J Cidlowski JA The human glucocorticoid receptor: one gene, multiple proteins and diverse responses.Steroids. 2005; 70: 407-417Crossref PubMed Scopus (315) Google Scholar This ultimately leads to altered gene expression, forming the basis for most of the immunomodulatory activities of GCs. Although application of pharmacological doses of synthetic GCs has strong anti-inflammatory and immunosuppressive effects, endogenous GCs seem to modulate rather than outright suppress the immune system.3Barnes PJ Anti-inflammatory actions of glucocorticoids: molecular mechanisms.Clin Sci. 1998; 94: 557-572Crossref PubMed Scopus (1253) Google Scholar, 4Tuckermann JP Kleiman A McPherson KG Reichardt HM Molecular mechanisms of glucocorticoids in the control of inflammation and lymphocyte apoptosis.Crit Rev Clin Lab Sci. 2005; 42: 71-104Crossref PubMed Scopus (163) Google Scholar The role of the GR in these processes has been investigated in cell culture and animal models, implicating it in lymphocyte development, apoptosis, and the control of innate and adaptive immunity.5Herold MJ McPherson KG Reichardt HM Glucocorticoids in T cell apoptosis and function.Cell Mol Life Sci. 2006; 63: 60-72Crossref PubMed Scopus (292) Google Scholar, 6Winoto A Littman DR Nuclear hormone receptors in T lymphocytes.Cell. 2002; 109: S57-S66Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar Nevertheless, many aspects of the function that endogenous GCs play in the thymus and the modulation of immune responses remain controversial. In the thymus, immunocompetent T cells develop from pluripotent progenitors through a series of differentiation and selection steps.7Bommhardt U Beyer M Hünig T Reichardt HM Molecular and cellular mechanisms of T cell development.Cell Mol Life Sci. 2004; 61: 263-280Crossref PubMed Scopus (46) Google Scholar Whereas the ability of GCs to induce apoptosis in thymocytes is widely recognized, it is still controversial as to whether they are also involved in T-cell maturation.8Vacchio MS Ashwell JD Glucocorticoids and thymocyte development.Semin Immunol. 2000; 12: 475-485Crossref PubMed Scopus (52) Google Scholar, 9Brewer JA Kanagawa O Sleckman BP Muglia LJ Thymocyte apoptosis induced by T cell activation is mediated by glucocorticoids in vivo.J Immunol. 2002; 169: 1837-1843Crossref PubMed Scopus (122) Google Scholar More than a decade ago, GR signaling was proposed to determine the outcome of positive and negative selection. Although mice expressing an antisense GR in the thymus were found to possess a T-cell repertoire with altered specificity, arguing that GC signaling impacts thymocyte selection, the analysis of hypomorphic GR knockout mice failed to provide any support for this model.10Tolosa E King LB Ashwell JD Thymocyte glucocorticoid resistance alters positive selection and inhibits autoimmunity and lymphoproliferative disease in MRL-lpr/lpr mice.Immunity. 1998; 8: 67-76Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 11Purton JF Boyd RL Cole TJ Godfrey DI Intrathymic T cell development and selection proceeds normally in the absence of glucocorticoid receptor signaling.Immunity. 2000; 13: 179-186Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar Furthermore, there is also debate regarding the degree to which the thymus synthesizes GCs in addition to its common source, the adrenal gland.12Jondal M Pazirandeh A Okret S Different roles for glucocorticoids in thymocyte homeostasis?.Trends Immunol. 2004; 25: 595-600Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Corticosterone synthesis was demonstrated in the thymus,13Vacchio MS Papadopoulos V Ashwell JD Steroid production in the thymus: implications for thymocyte selection.J Exp Med. 1994; 179: 1835-1846Crossref PubMed Scopus (322) Google Scholar, 14Pazirandeh A Xue Y Rafter I Sjovall J Jondal M Okret S Paracrine glucocorticoid activity produced by mouse thymic epithelial cells.FASEB J. 1999; 13: 893-901PubMed Google Scholar, 15Lechner O Wiegers GJ Oliveira-Dos-Santos AJ Dietrich H Recheis H Waterman M Boyd R Wick G Glucocorticoid production in the murine thymus.Eur J Immunol. 2000; 30: 337-346Crossref PubMed Scopus (98) Google Scholar but studies using the inhibitor metyrapone led to ambiguous conclusions.16Purton JF Zhan Y Liddicoat DR Hardy CL Lew AM Cole TJ Godfrey DI Glucocorticoid receptor deficient thymic and peripheral T cells develop normally in adult mice.Eur J Immunol. 2002; 32: 3546-3555Crossref PubMed Scopus (42) Google Scholar Moreover, various functions were attributed to these GCs, ranging from T-cell development and thymic selection8Vacchio MS Ashwell JD Glucocorticoids and thymocyte development.Semin Immunol. 2000; 12: 475-485Crossref PubMed Scopus (52) Google Scholar, 12Jondal M Pazirandeh A Okret S Different roles for glucocorticoids in thymocyte homeostasis?.Trends Immunol. 2004; 25: 595-600Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar to the control of thymic involution.17Pazirandeh A Jondal M Okret S Glucocorticoids delay age-associated thymic involution through directly affecting the thymocytes.Endocrinology. 2004; 145: 2392-2401Crossref PubMed Scopus (34) Google Scholar In summary, thymus-derived steroids and their relevance remain a matter of debate. Beyond a role in T-cell development, it is also believed that GCs impact the type of immune responses generated.18Elenkov IJ Chrousos GP Stress hormones, Th1/Th2 patterns, pro/anti-inflammatory cytokines and susceptibility to disease.Trends Endocrinol Metab. 1999; 10: 359-368Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar In particular, it was observed that elevated levels of endogenous GCs, such as experienced during prolonged periods of stress, can suppress cellular immunity while boosting humoral immunity. This has led to the concept that GCs govern the outcome of autoimmune and atopic diseases via their influence on cytokine production.19Hill N Sarvetnick N Cytokines: promoters and dampeners of autoimmunity.Curr Opin Immunol. 2002; 14: 791-797Crossref PubMed Scopus (107) Google Scholar, 20Elenkov IJ Glucocorticoids and the Th1/Th2 balance.Ann NY Acad Sci. 2004; 1024: 138-146Crossref PubMed Scopus (508) Google Scholar A link between the activity of the hypothalamus-pituitary-adrenal axis and disease susceptibility is suggested by both animal experiments and human studies. Lewis rats, which have a hypoactive stress system, are extremely prone to the induction of Th1-mediated diseases such as experimental autoimmune encephalomyelitis.21Wilder RL Neuroendocrine-immune system interactions and autoimmunity.Annu Rev Immunol. 1995; 13: 307-338Crossref PubMed Scopus (450) Google Scholar, 22El-Etr M Vukusic S Gignoux L Durand-Dubief F Achiti I Baulieu EE Confavreux C Steroid hormones in multiple sclerosis.J Neurol Sci. 2005; 233: 49-54Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Conversely, women in the third trimester of pregnancy, who have increased levels of cortisol, often experience remission of Th1-mediated autoimmune diseases including multiple sclerosis and rheumatoid arthritis.22El-Etr M Vukusic S Gignoux L Durand-Dubief F Achiti I Baulieu EE Confavreux C Steroid hormones in multiple sclerosis.J Neurol Sci. 2005; 233: 49-54Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar This was explained by increased production of IL-4 and IL-10 and a reduction in IL-12. In line with this notion, Th2-mediated autoimmune disorders such as systemic lupus erythematosus can flare up under conditions of chronically elevated cortisol levels.18Elenkov IJ Chrousos GP Stress hormones, Th1/Th2 patterns, pro/anti-inflammatory cytokines and susceptibility to disease.Trends Endocrinol Metab. 1999; 10: 359-368Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar In summary, despite good evidence that the strength of GR signaling impacts autoimmune and atopic diseases, the causal relationship to altered T-cell function is not yet well established. Although many studies have addressed the question of what occurs when the GR is lacking, only a few reports have so far explored the physiological effects of increased GR levels in vivo.23Reichardt HM Immunomodulatory activities of glucocorticoids: insights from transgenesis and gene targeting.Curr Pharm Des. 2004; 10: 2797-2805Crossref PubMed Scopus (38) Google Scholar, 24Reichardt HM Umland T Bauer A Kretz O Schütz G Mice with an increased glucocorticoid receptor gene dosage show enhanced resistance to stress and endotoxic shock.Mol Cell Biol. 2000; 20: 9009-9017Crossref PubMed Scopus (186) Google Scholar In an approach by Pazirandeh and colleagues, 25Pazirandeh A Xue Y Prestegaard T Jondal M Okret S Effects of altered glucocorticoid sensitivity in the T cell lineage on thymocyte and T cell homeostasis.FASEB J. 2002; 16: 727-729Crossref PubMed Scopus (85) Google Scholar GR overexpression was directed to the T-cell lineage, resulting in approximately twofold elevated receptor levels. This was accompanied by a moderate increase in GC sensitivity and a reduction in the thymic and peripheral T-cell pool. Analysis of aged mice further suggested that increased GC signaling interferes with thymic involution.17Pazirandeh A Jondal M Okret S Glucocorticoids delay age-associated thymic involution through directly affecting the thymocytes.Endocrinology. 2004; 145: 2392-2401Crossref PubMed Scopus (34) Google Scholar Although these studies provided valuable new insight into the consequences of GR overexpression in T cells, the effects were comparably mild. Therefore, we decided to reevaluate the role that enhanced GR signaling plays in thymocyte development and adaptive immune responses by generating transgenic rats expressing a mutant GR with enhanced ligand affinity.26Chakraborti PK Garabedian MJ Yamamoto KR Simons Jr, SS Creation of "super" glucocorticoid receptors by point mutations in the steroid binding domain.J Biol Chem. 1991; 266: 22075-22078Abstract Full Text PDF PubMed Google Scholar These animals express strongly elevated receptor levels resulting in an extraordinarily high GC sensitivity. Consequently, thymocyte and mature T-cell numbers are strongly reduced, the T-cell repertoire perturbed, and the susceptibility to autoimmune and atopic diseases altered. This suggests that the modulation of T-cell development and the selection of Th2- over Th1-dominated adaptive immune responses are primary functions of endogenous GCs. In this sense, these rats represent a valuable model to assess effects of increased GR signaling such as observed during chronic stress. Two lentiviral vectors were cloned by inserting either the wild-type or a mutant mouse GR cDNA (carrying the point mutation C656G) along with an IRES-eGFP cassette into the plasmid FUW.27Lois C Hong EJ Pease S Brown EJ Baltimore D Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors.Science. 2002; 295: 868-872Crossref PubMed Scopus (1640) Google Scholar Virus particles were generated following published protocols and were used to transduce Jurkat E6.1 cells by spinocculation.27Lois C Hong EJ Pease S Brown EJ Baltimore D Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors.Science. 2002; 295: 868-872Crossref PubMed Scopus (1640) Google Scholar To obtain cell lines that uniformly express the wild-type GR (line J.Gr) or the mutant GR (line J.Gr*), the transduced cells were further sorted based on their eGFP levels by preparative flow cytometry using a FACSDiVa machine (BD Biosciences, San Jose, CA). Transgenesis was performed by pronucleus injection of a purified NotI fragment into (Crl:CDxLew/Crl)F1 zygotes as previously described.28van den Brandt J Kwon SH Hünig T McPherson KG Reichardt HM Sustained pre-TCR expression in Notch1IC-transgenic rats impairs T cell maturation and selection.J Immunol. 2005; 174: 7845-7852PubMed Google Scholar, 29van den Brandt J Wang D Kwon S-H Heinkelein M Reichardt HM Lentivirally generated eGFP-transgenic rats allow efficient cell tracking in vivo.Genesis. 2004; 39: 94-99Crossref PubMed Scopus (65) Google Scholar The transgene vector was constructed by cloning the mutant mouse GR (mGR C656G) into p1017, which consists of the proximal lck promoter and a human growth hormone (hGH) minigene.30Chaffin KE Beals CR Wilkie TM Forbush KA Simon MI Perlmutter RM Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase.EMBO J. 1990; 9: 3821-3829Crossref PubMed Scopus (218) Google Scholar Analyses requiring a defined MHC haplotype were performed in rats that had been backcrossed to the inbred Lewis strain (RT1l) for a minimum of seven generations. All animal experiments were performed on male rats and approved by the Bavarian and Lower Saxony state authorities. All antibodies were obtained from BD Biosciences unless otherwise indicated: Ox34 (CD2), Ox35 and Ox38 (CD4), Ox8 (CD8α), 3.4.1. (CD8β), JJ319 (CD28), Ox22 (CD45RC), Ox33 (CD45RA), P4/16 (RT6.1), Ox7 (Thy1), R73 (TCRβ), C-A11 (Vβ3.3), R78 (Vβ8.2), B73 (Vβ8.5), G101 (Vβ10), 18b1 (Vβ13), His42 (Vβ16) and V65 (TCRγδ). Yuggu-F6 (CD69) was a kind gift from Dr. Jung-Hyun Park (Experimental Immunology Branch, National Cancer Institute, Bethesda, MD). The polyclonal antibody against the GR was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Cells from the draining lymph nodes or magnetically purified T cells were cultured in 96-well plates in the presence of plate-bound anti-TCRβ (R73, 2 μg/ml) plus soluble anti-CD28 (JJ319, 0.5 μg/ml) monoclonal antibodies, ConA (2.5 μg/ml) or gpMBP (20 μg/ml). After 48 hours, 9.25 kBq of [3H]thymidine was added to the cultures, and 18 hours later the amount of incorporated radioactivity was determined using a β-plate reader. Lymph node cDNA was amplified by PCR using a set of 23 different TCRBV primers together with a 6-FAM-tagged CB primer. All sequences have been reported previously.31de Graaf KL Berne GP Herrmann MM Hansson GK Olsson T Weissert R CDR3 sequence preference of TCRBV8S2+ T cells within the CNS does not reflect single amino acid dependent avidity expansion.J Neuroimmunol. 2005; 166: 47-54Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar Cycling conditions for PCR were as follows: 95°C for 3 minutes and 30 seconds for denaturation, 57°C for 40 seconds for annealing, and 72°C for 1 minute for extension, followed by 34 cycles of 94°C for 40 seconds, 57°C for 40 seconds, and 72°C for 1 minute. PCR products were analyzed on a high-resolving polyacrylamide gel electrophoresis system in an automatic DNA sequencer model 377 (Applied Biosystems, Foster City, CA). The fluorescence-labeled DNA profile on the gel was analyzed using the Genescan software program (Applied Biosystems), which records the size and the intensity of each band. Spectratypes revealed by this analysis usually consist of five to seven bands. The rats were weighed and anesthetized using ketamine and Rompun. For ADX, the adrenals were quickly removed through two small dorsal incisions, the wounds sealed with tissue glue, and the rats placed into their home cage. For sham surgery the same procedure was performed, except that the adrenals were merely exposed instead of removed. To compensate for the disturbed salt homeostasis, the rats were offered ad libitum a 0.9% NaCl solution for drinking. Total thymocytes or lymph node cells were cultured at 1 × 106 cells/ml RPMI containing 10% charcoal/dextran-treated FCS (HyClone, Logan, UT) in 48-well plates for 24 hours as described previously.32van den Brandt J Wang D Reichardt HM Resistance of single-positive thymocytes to glucocorticoid-induced apoptosis is mediated by CD28 signaling.Mol Endocrinol. 2004; 18: 687-695Crossref PubMed Scopus (22) Google Scholar The cells were analyzed by flow cytometry using Annexin V and monoclonal antibodies against TCRβ, CD4, and CD8. Blood was collected retro-orbitally into precooled SST Microtainers (BD Biosciences) between 9:00 AM and 11:00 AM and kept on crushed ice. The samples were centrifuged and the serum stored at −20°C until analysis. The corticosterone RIA was performed according to the instructions of the manufacturer (MP Biomedicals, Eschwege, Germany). Active EAE in Lewis rats was induced as previously described.33Schmidt J Metselaar JM Wauben MH Toyka KV Storm G Gold R Drug targeting by long-circulating liposomal glucocorticosteroids increases therapeutic efficacy in a model of multiple sclerosis.Brain. 2003; 126: 1895-1904Crossref PubMed Scopus (197) Google Scholar Briefly, transgenic rats and wild-type littermates were immunized subcutaneously with 100 μg of guinea pig MBP in 100 μg of CFA in the hind paws and limb. Disease severity was assessed as follows: 0 = no disease; 1 = limp tail; 2 = whole tail paralysis; 3 = beginning gait ataxia; 4 = more severe gait ataxia; 5 = mild paraparesis of the hind limbs; 6 = paraparesis of both hind limps or paraplegia of one hind limb; 7 = paraplegia of both hind limbs; 8 = mild tetraparesis; 9 = severe tetraparesis, moribund; and 10 = death.34Hartung HP Schafer B Heininger K Stoll G Toyka KV The role of macrophages and eicosanoids in the pathogenesis of experimental allergic neuritis: serial clinical, electrophysiological, biochemical and morphological observations.Brain. 1988; 111: 1039-1059Crossref PubMed Scopus (147) Google Scholar The rats were perfused with 4% paraformaldehyde and the spinal cords prepared, dehydrated, and embedded in paraffin as described.33Schmidt J Metselaar JM Wauben MH Toyka KV Storm G Gold R Drug targeting by long-circulating liposomal glucocorticosteroids increases therapeutic efficacy in a model of multiple sclerosis.Brain. 2003; 126: 1895-1904Crossref PubMed Scopus (197) Google Scholar Five-micrometer sections were stained with hematoxylin and eosin (H&E), and infiltration was assessed by two independent investigators. Cytokine secretion by draining lymph node cells was determined by cytokine bead array (CBA, BD Biosciences) after culture for 72 hours in the presence of either ConA or gpMBP. Culture supernatant (50 μl) was incubated with beads specific for IFNγ, IL-4, or IL-10 according to the manufacturer's instructions and analyzed using the supplied software. Rats were sensitized using 2 mg/ml ovalbumin (OVA, grade V; Sigma Chemical, St. Louis, MO) in PBS, precipitated at a 1:1 ratio with alum (Sigma). One hundred micrograms of OVA/alum suspension was applied intraperitoneally. Four weeks later, the rats were challenged by two intranasal applications of 500 μg of OVA in PBS. Eighteen hours after the second treatment, the bronchoalveolar lavage and the draining lymph nodes were isolated. Serum was collected at weekly intervals over the whole experimental period to allow for the determination of the antibody titer. Serum titers of IgG1, IgG2a, IgG2b, and IgE OVA-specific antibodies were measured by ELISA. Ninety-six-well plates were coated with 10 μg/ml OVA in carbonate buffer. After blocking with 10% FCS, 1:1000 serum dilutions were added. OVA-specific immunoglobulins were detected using 1 μg/ml biotinylated anti-rat IgG1, IgG2a, IgG2b, and IgE antibodies (clones RG11/39.4, RG7/1.30, RG7/11.1, and B41-3; BD Biosciences) followed by incubation with streptavidin-AP conjugate (diluted 1:1000; Roche Diagnostics GmbH, Mannheim, Germany). The assay was developed using fast p-nitrophenyl phosphate (Sigma) and measured at 405 nm. OVA-specific positive and negative sera were used as controls and the results expressed as optical densities. The data were analyzed by Student's t-test and are presented as the mean ± SEM (n.s., not significant, *P < 0.05, **P < 0.01). Residue C656 of the GR is involved in ligand binding and interpretation.26Chakraborti PK Garabedian MJ Yamamoto KR Simons Jr, SS Creation of "super" glucocorticoid receptors by point mutations in the steroid binding domain.J Biol Chem. 1991; 266: 22075-22078Abstract Full Text PDF PubMed Google Scholar, 35Sarlis NJ Bayly SF Szapary D Simons Jr, SS Quantity of partial agonist activity for antiglucocorticoids complexed with mutant glucocorticoid receptors is constant in two different transactivation assays but not predictable from steroid structure.J Steroid Biochem Mol Biol. 1999; 68: 89-102Crossref PubMed Scopus (28) Google Scholar When mutated to a glycine, the modified GR transactivates genes at lower hormone concentrations compared with the wild-type receptor and was therefore designated as a "super" GR. To investigate whether this mutation leads to a similar shift in the dose-response curve in the context of T-cell apoptosis, we tested its characteristics in cell culture. Jurkat E6.1 cells that are devoid of endogenous GR protein were transduced with lentiviruses coding for either the wild-type GR or the mutant GR(C656G). Analysis of the newly established cell lines J.Gr and J.Gr* by Western blot confirmed equal levels of GR expression (Figure 1A). To investigate their sensitivity to GC-induced apoptosis, the two cell lines were cultured in the presence of 10−11 to 10−8 mol/L dexamethasone (dex) for 48 hours and subsequently analyzed by flow cytometry using Annexin V staining. The dose-response curve for GC-induced cell death was shifted toward lower hormone concentrations, confirming that the point mutation C656G of the GR indeed increased the sensitivity of T cells to apoptosis (Figure 1B). Having established that the C656G point mutation increases the GR's sensitivity to GC-induced apoptosis, we questioned whether such a modified receptor would be constitutively active in the presence of basal GC levels when overexpressed in thymocytes. Therefore, we constructed a transgene vector encompassing the proximal lck promoter, the mouse GR cDNA carrying the point mutation C656G, and a human growth hormone minigene to allow for correct polyadenylation and splicing (Figure 1C). This construct was injected into Lew/CD F1 rat zygotes and the offspring screened for transgene integration. Two founder rats were identified, one of which was shown to have a high copy number and was used for further breeding and analysis (data not shown). These rats were designated superTGR in analogy with the initial description of the point mutation used to establish this transgenic line.26Chakraborti PK Garabedian MJ Yamamoto KR Simons Jr, SS Creation of "super" glucocorticoid receptors by point mutations in the steroid binding domain.J Biol Chem. 1991; 266: 22075-22078Abstract Full Text PDF PubMed Google Scholar First, we studied expression of the transgene in thymocytes and lymph node T cells by RT-PCR using primers derived from regions of the GR gene that were conserved between mouse, the origin of the transgene, and rat. A restriction site exclusively present in the amplicon derived from the transgene allowed us to determine the ratio between the mutant and the endogenous GR mRNA. Interestingly, the mutant receptor was predominantly expressed in both thymocytes and lymph node T cells (Figure 1D). Subsequently, we analyzed GR protein levels by Western blot. In transgenic thymocytes, the GR was dramatically overexpressed as compared with wild-type cells, whereas its increase in lymph node T cells from superTGR rats was more moderate (Figure 1E). This is in line with the initial goal to generate a thymus-specific transgenic rat. The impact of enhanced GR signaling on thymocyte apoptosis and development in superTGR rats was investigated by flow cytometry. Most impressively, the thymus as an anatomical structure was virtually absent in transgenic animals. Enumeration revealed that thymocyte cellularity was reduced by more than 90% in young adult superTGR rats (Figure 2A; note the split axis). This was accompanied by a strong reduction in the percentages of CD4+CD8+ DP, αβTCRint, and αβTCRhigh cells (Figure 2B). Because previous studies had indicated that GCs delay thymus involution,17Pazirandeh A Jondal M Okret S Glucocorticoids delay age-associated thymic involution through directly affecting the thymocytes.Endocrinology. 2004; 145: 2392-2401Crossref PubMed Scopus (34) Google Scholar we extended our studies until 20 weeks of age. Despite its overall reduced size, the thymus in transgenic rats clearly underwent involution (Figure 2A). Importantly, this process appears to occur even earlier in superTGR rats compared with wild-type controls. Taken together, the mutant GR causes massive thymocyte apoptosis at basal hormone levels. Next, we analyzed the extent to which the different thymocyte subtypes were affected in young adult superTGR rats. First, we determined the absolute number of the four major thymocyte subpopulations in transgenic rats. Although the cellularity of the DN cell population was moderately affected (approximately 40% of control levels), there was a strong reduction in the number of DP, CD4 SP, and CD8 SP cells (Figure 2C). Second, we further dissected the effect on DN cells. The most immature cells in the rat thymus are CD45RC+CD2− DN cells (referred to as DN1), which subsequently progress to the CD45RC+CD2+ stage (referred to as DN2).36van den Brandt J Voss K Schott M Hünig T Wolfe MS Reichardt HM Inhibition of Notch signaling biases rat thymocyte development towards the NK cell lineage.Eur J Immunol. 2004; 34: 1405-1413Crossref PubMed Scopus (34) Google Scholar This is also the time point when the lck promoter becomes active in the rat.28van den Brandt J Kwon SH Hünig T McPherson KG Reichardt HM Sustained pre-TCR expression in Notch1IC-transgenic rats impairs T cell maturation and selection.J Immunol. 2005; 174: 7845-7852PubMed Google Scholar The number of DN1 and γδ T cells was only mildly affected in superTGR rats, whereas considerably less DN2 cells were present in transgenic compared with wild-type animals (Figure 2C). Thus, enhanced GR signaling decreases the cellularity of all thymocyte subsets from the DN2 stage on, except for γδ T cells. In light of the pronounced alterations observed in thymocytes, we wondered whether peripheral T lymphocytes were also affected in superTGR rats. The number of lymph node T cells was indeed diminished, although approximately 25% of wild-type levels were reached in older transgenic animals (Figure 3A). Because the number of B cells was similar in both genotypes, the relative number of lymph node T cells was also reduced in superTGR rats (Figure 3A). To characterize the peripheral T cells in more detail, we performed a number of flow cytometric analyses. CD8+ T cells were more affected by the expression of the mutant GR than CD4+ T cells (Figure 3B). In addi
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