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The Journal of Immunology, 2002, 169: 1309-1318.
Copyright © 2002 by The American Association of Immunologists

Green Fluorescent Protein-Glucocorticoid Receptor Knockin Mice Reveal Dynamic Receptor Modulation During Thymocyte Development1

Judson A. Brewer*, Barry P. Sleckman{dagger}, Wojciech Swat{dagger} and Louis J. Muglia2,*

Departments of * Pediatrics, Molecular Biology, and Pharmacology and {dagger} Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To delineate the cellular targets and mechanisms by which glucocorticoids (GCs) exert their actions, we generated mice in which a green fluorescent protein (GFP)-GC receptor (GR) fusion gene is knocked into the GR locus. In these mice, the GFP-GR protein, which is functionally indistinguishable from endogenous GR, allows the tracking and quantitation of GR expression in single living cells. In GFP-GR thymus, GR expression is uniform among embryonic thymocyte subpopulations but gradually matures over a 3-wk period after birth. In the adult, GR is specifically induced to high levels in CD25+CD4-CD8- thymocytes and returns to basal levels in CD4+CD8+ thymocytes of wild-type and positively selecting female HY TCR-transgenic mice, but not negatively selecting male HY TCR-transgenic mice. In GFP-GR/recombinase-activating gene 2-/- thymocytes, GR expression is down-regulated by pre-TCR complex stimulation. Additionally, relative GR expression is dissociated from GC-induced apoptosis in vivo. Results from these studies define differential GR expression throughout ontogeny, suggest pre-TCR activation as a specific mechanism of GR down-regulation, define immature CD8+ thymocytes as novel apoptosis-sensitive GC targets, and separate receptor abundance from susceptibility to apoptosis across thymocyte populations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoids (GCs)3 have dramatic effects on many aspects of immune system function. One of the most prominent consequences of increased systemic GCs is thymocyte apoptosis. Conversely, removal of all systemic GCs by adrenalectomy, or only the daily circadian elevation of these steroids by genetic means, results in increased thymus size and cellularity (1, 2). Additionally, it is the immature CD4+8+ double positive (DP) thymocyte that demonstrates the greatest apoptotic response to exogenous and endogenous GCs when compared with mature CD4+ and CD8+ single positive (SP) thymocytes (3, 4).

The high sensitivity of developing thymocytes to GCs suggests that GCs may also influence normal thymocyte development, during which potentially autoreactive and nonfunctional T cells are deleted from the developmental repertoire (5). Consistent with this notion, GCs modulate signaling pathways critical for thymocyte ontogeny, with effects on ZAP-70, linker for activation of T cells, NF-{kappa}B, and others, although how and when endogenous GCs specifically affect thymocyte development remains unclear (5, 6, 7).

GCs exert their effects on tissues outside the brain primarily by activating the type-II GC receptor (GR). This receptor is abundantly expressed in the thymus as compared with other organs (8). Several lines of evidence suggest that the relative amount of GR expressed within a cell determines the magnitude and nature of the response to GCs. It has been observed that both overexpression of GR and expression of antisense GR mRNA in transgenic mice alters thymocyte survival in vitro and in vivo (9, 10, 11). Furthermore, the relative ratio of GR to other transcription factors within a given cell type determines whether the predominant consequence will be transcription enhancement or repression for certain target genes (12).

Because DP thymocytes are exquisitely sensitive to GCs and manipulation of GR levels can have an impact on this phenomenon, one testable hypothesis is that relative levels of endogenous GR set the threshold for sensitivity to steroid-induced apoptosis. Studies using receptor binding techniques and intracellular immunofluorescent staining to address this hypothesis have yielded conflicting results (8, 13, 14, 15, 16, 17). Additionally, it remains unknown whether GR expression is associated with selective processes within developing thymocytes. Knowledge about relative GR abundance in specific thymic subpopulations would not only provide mechanistic insight into thymocyte GC sensitivity but would also provide a framework in which to determine the controversial role of these steroids in thymocyte development (9, 18, 19, 20, 21, 22).

To understand the role of GR in modulation of thymocyte development, the precise delineation of the magnitude and compartmentalization of GR expression at critical stages during ontogeny is essential. We have generated knockin mice in which a chimeric green fluorescent protein (GFP)-GR fusion protein is expressed in place of the endogenous GR allele. Analysis of thymocytes from these mice showed a striking GR induction in CD4-CD8- double negative (DN) thymocytes. GR was rapidly down-regulated at the DP stage of development in wild-type and female HY but not male TCR-transgenic mice. Additionally, exogenous GC administration induced robust apoptosis in immature SP (ISP) thymocytes expressing relatively high levels of the receptor, and DP thymocytes expressing basal GR levels but not DN thymocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal husbandry and plasma sampling

All mouse protocols were in accordance with National Institutes of Health guidelines and were approved by the Animal Care and Use Committee of Washington University School of Medicine (St. Louis, MO). Mice were housed on a 12 h/12 h light/dark cycle with ad libitum access to rodent chow. Plasma for measurement of corticosterone was obtained by rapid retroorbital phlebotomy into heparinized capillary tubes with a total time from first handling the animal to completion of bleeding not exceeding 30 s. Blood was collected on ice and plasma was separated by centrifugation and stored at -80°C until assay. Unless otherwise noted, all mice used were 6–10 wk old and were of a C57BL/6 x 129/Sv genetic background.

Generation and in vitro testing of GFP-GR construct

Full-length mouse GR (mGR) cDNA containing an engineered XhoI site at the third amino acid (generous gift of Dr. J. Bodwell, Dartmouth, NH) was inserted into the BglII site of pEGFP-C2 (Clontech Laboratories, Palo Alto, CA) using the oligonucleotide linkers 5'-GATCTCCGGAGGCGGCATGGAC-3' and 5'-AGGCCTCCGCCGTACCTGAGCT-3'. The resulting vector (pGFP-GR), a mGR expression vector (pGR) or GFP expression vector (pEGFP-C2), was transiently cotransfected with a luciferase reporter vector containing two GC response elements (GREs) from tyrosine aminotransferase (pxpG2T; generous gift of Dr. J. Bodwell) into Jurkat cells. Twenty hours after transfection, cells were resuspended in Jurkat medium (RPMI 1640 plus 10% FCS) containing 1 or 0.1 µM dexamethasone (DEX; American Reagent Laboratories, Shirley, NY) for 7 h, and luciferase activity was determined using a luciferase assay system according to the manufacturer’s instructions (Promega, Madison, WI).

Generation of GFP-GR mice

A murine 129/Sv bacterial artificial chromosome library (Incyte Genomics, St. Louis, MO) was screened by PCR using exon 2-specific primers. DNA isolated from positive bacterial artificial chromosome clones was subjected to restriction endonuclease digestion and Southern blot analysis with an exon 2 probe to identify fragments of 10–15 kb in size for subcloning into pBluescript SK II to facilitate detailed characterization. A phosphoglycerate kinase neomycin resistance (PGKneo) cassette containing flanking loxP sites was subcloned into an SpeI restriction site in intron 2 using oligonucleotide linkers (pGRloxPneo). An AgeI/Bsu36 I restriction fragment containing coding sequences for GFP through amino acid 35 of mGR from pGFP-GR was inserted into pGRloxPneo (partially digested with SalI and Bsu 36 I) using oligonucleotide linkers. To obtain embryonic stem (ES) clones having replaced one copy of the endogenous murine GR locus with the GFP-GRneo allele, TC1 ES cells (23) underwent electroporation with linearized pGFP-GRneo as we have previously described (24). Clones surviving 7 days of G418 selection were isolated and expanded for further analysis. DNA from 96 G418-resistant clones was subjected to Southern blot analysis using a probe external to the flanking regions within our targeting vector. Three clones demonstrated homologous recombination of the targeting vector into the endogenous GR locus as evidenced by the appearance of a 4-kb restriction fragment-length polymorphism. Clones were confirmed by Southern blot analysis with a GFP-specific probe, and one GFP-positive clone was injected into C57BL/6 blastocysts and resulted in germline transmission of the ES genome. Heterozygous GFP-GRneo mice were mated to EIIA-Cre recombinase transgenic mice (generated by Dr. H. Westphal, Bethesda, MD, and provided by Dr. M. Bessler, St. Louis, MO) and offspring were screened for deletion of the neomycin resistance cassette by PCR.

Harvest and culture of MEFs

Embryos from wild-type and GFP-GR homozygous mice were harvested 14.5 days postcoitus, and fetal carcasses were minced with razor blades in 0.05% trypsin, dispersed in DMEM using a 20-gauge needle, filtered through 70-µm mesh, washed, resuspended in DMEM plus 10% FCS, and grown on cover slips. Where indicated, cells were incubated in 0.1 µM DEX for 30 min before harvest. Coverslips were then mounted directly on slides and imaged using an Axiovision digital imaging system (Zeiss, Oberkochen, Germany).

Ab detection of GFP-GR protein

Fifteen micrograms of total liver protein from adult mice was harvested, resolved on a 4–12% bis-Tris polyacrylamide gel, probed with anti-GR antisera (M-20; Santa Cruz Biotechnology, Santa Cruz, CA) at a 1/200 dilution, and developed using ECL detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were then stained with Ponceau S solution (Sigma-Aldrich, St. Louis, MO) to ensure equal loading of protein. For localization of GFP-GR within the brain, adult wild-type and GFP-GR heterozygous mice were deeply anesthetized with 1 ml of 2.5% avertin and transcardially perfused with D-PBS followed by 4% paraformaldehyde in D-PBS. Brains were postfixed by immersion in 4% paraformaldehyde for 1 h at 4°C and cryoprotected in 10% sucrose in D-PBS. Detection of GFP fluorescence and immunoreactivity was performed on free-floating sections cut at 35-µm thickness on a cryostat. For GFP immunohistochemistry, after blocking in 3% normal goat serum in PBS for 30 min, sections were incubated with a 1/2000 dilution of a polyclonal rabbit anti-GFP Ab (Clontech Laboratories) in D-PBS with 1% goat serum. Peroxidase staining was visualized with a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA).

Restraint stress and LPS administration

Mice were restrained for 30 min as previously described (25) or injected i.p. with 100 µg LPS (Escherichia coli serotype 0111:B4; Sigma-Aldrich) dissolved in 100 µl PBS.

Corticosterone assay

Plasma concentration of corticosterone was determined by RIA (ICN Pharmaceuticals, Costa Mesa, CA) from blood collected by retroorbital phlebotomy at indicated the time points in singly housed adult male mice as previously described (25).

Flow cytometry

Thymocytes were dispersed through nylon mesh into PBS, washed, counted on a hemocytometer using trypan blue to exclude nonviable cells, stained for cell surface markers (PE-anti-CD25, PerCP-anti-CD8, allophycocyanin-anti-CD4, PE-anti-heat stable Ag (HSA), PE-anti-TCR{beta}; BD PharMingen, San Diego, CA), washed, resuspended in PBS, and analyzed on a FACSCalibur (BD Biosciences, Mountain View, CA). For annexin V analysis, cells were resuspended in binding buffer containing FITC-conjugated annexin V according to the manufacturer’s specifications (BD PharMingen). Unless otherwise indicated, nonviable cells were excluded from analysis based on forward and side scatter profiles.

DEX and Ab treatment

Mice were injected i.p. with 200 µg DEX phosphate, 250 µg anti-CD3{epsilon} Ab (145-2C11), or normal saline, using a 30-gauge needle. Thymocytes were harvested 8, 24, or 48 h after injection for analysis.

PBMC analysis

Blood was obtained by rapid retroorbital phlebotomy via heparinized capillary tubes. Blood was diluted with PBS, layered over 2 ml of Histopaque 1083 (Sigma-Aldrich), and centrifuged for 15 min at 2500 rpm. The white interface was transferred to a new tube, washed with PBS, and analyzed by flow cytometry, gating on PBMCs by forward and side scatter profiles.

FTOC

Fetal thymi were harvested 15.5 days postcoitus and cultured on nitrocellulose filters (Millipore, Bedford, MA) resting on gel-foam (Upjohn, Kalamazoo, MI) in RPMI 1640 plus 10% FCS for 7 days, with one change of medium at day 4.

Statistical methods

All results are expressed as mean ± SD unless otherwise stated. Statistical analysis was done by ANOVA with p < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of GFP-GR knockin mice

We first measured whether the addition of GFP to GR affected its transactivation capacity in vitro. To this end, we generated a construct in which we added the full-length cDNA of mGR via a 5-aa linker (GGSGG) to the C terminus of enhanced GFP (eGFP) (Fig. 1GoA). This GFP-GR fusion protein functioned in a manner similar to normal GR when transiently coexpressed in Jurkat cells with a luciferase reporter gene driven by tandem GREs. In a dose response analysis to the synthetic GC DEX, GFP-GR promoted expression of the luciferase gene to the same extent as mGR (Fig. 1GoB). This result indicated that the addition of GFP to the amino terminus of GR was not inhibiting GR function in vitro.



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  		FIGURE 1. Generation and functional testing of GFP-GR fusion protein in vitro and in vivo. A, Schematic of pGFP-GR expression vector. CMV, CMV immediate early promoter; eGFP, eGFP cDNA; mGR, mGR cDNA. B, GFP-GR transactivates GREs similarly to mGR. GFP-GR, mGR, or eGFP expression vectors were cotransfected with a luciferase reporter vector driven by tandem GREs into Jurkat cells, treated with DEX. Cell extracts were measured for luciferase activity. C, Generation of GFP-GR knockin mice. A targeting vector was designed in which GFP was fused to the initiator methionine in exon 2 of the GR gene. After homologous recombination in ES cells and subsequent germline transmission, the phosphoglycerate kinase neomycin resistance (PGKNeo) gene was removed by mating to EIIA-Cre transgenic mice. D, GFP-GR and wild-type GR are equally expressed. Total protein was harvested from liver of wild-type and GFP-GR heterozygous mice, and steady-state expression of knockin GFP-GR and endogenous GR was detected by Western blot analysis. E, Protein levels are proportional to fluorescence intensity in GFP-GR mice. PBMCs were collected from wild-type (shaded), heterozygous, and homozygous mice, purified by density centrifugation, and analyzed for green fluorescence intensity by flow cytometry. The MFI of representative mice is shown.

 
We introduced a GFP-GR allele into the mouse genome by fusing eGFP (with a 5-aa linker, GGSGG) to the initiator methionine of the mGR gene of our targeting vector, downstream of the exon 2 splice acceptor site, thus maintaining all endogenous regulatory sequences in the GR gene after homologous recombination in ES cells (Fig. 1GoC). Heterozygous GFP-GR knockin mice derived from our targeted ES cells were mated with EIIA-Cre transgenic mice, which express Cre recombinase transiently in the early blastocyst (26), to remove the neomycin resistance gene that was flanked by loxP sites in intron 2 of the GR gene. GFP-GR heterozygous and homozygous mice with and without the neomycin gene behaved identically and were combined in the following studies.

We measured whether the addition of GFP to GR affected protein synthesis or degradation in vivo. To rigorously examine this, we evaluated heterozygous mice in which endogenous GR served as an internal control. Western blot analysis of these mice showed identical steady state levels of protein arising from the endogenous and knockin alleles (Fig. 1GoD), indicating that the GFP fusion was not altering GR half-life or regulation.

GFP fusion proteins have proven remarkably useful in tracking protein localization intracellularly in vitro and recently for localizing expression to cellular subsets in vivo (27). However, GFP fluorescence has not yet been used for direct quantitation of endogenous protein expression within single cells in vivo. As a direct test of whether GFP fluorescence intensity correlated with levels of expression, we measured the mean fluorescence intensity (MFI) of PBMCs and thymocyte subpopulations from GFP-GR heterozygotes and homozygotes by flow cytometry. MFI of homozygous PBMCs was twice that of heterozygous PBMCs (ratio of 1.9 ± 0.06; n = 4 per group; Fig. 1GoE). Additionally, thymocytes from homozygous mice fluoresced twice as brightly as heterozygotes at each stage of development (shown in Fig. 4GoH and discussed in detail next section). These results suggested that GFP fluorescence accurately reflects relative GR gene expression as measured on a single-cell level.



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  		FIGURE 4. GR expression is tightly regulated during thymocyte ontogeny. Adult GFP-GR thymocytes were harvested, dispersed, and incubated with allophycocyanin-conjugated anti-CD4, PerCP-conjugated anti-CD8 Abs, and PE-conjugated anti-TCR{beta} (C), CD25 (E), or HSA (F) Abs, and green fluorescence intensity was quantified via flow cytometry. A, Percentages of subpopulations and gates used for B–F. B, Thymocytes were gated as indicated in A and analyzed for green fluorescence. Open histograms represent wild-type control. D, GR expression does not vary within DP thymocyte subpopulations. DP thymocytes were gated based on surface expression of TCR{beta} (upper panel) and green fluorescence was measured (lower panel, solid line, TCR{beta}low; dashed line, TCR{beta}int). G, GR up-regulation begins early in the DN stage of development. Adult RAG2-/-/GFP-GR heterozygous thymocytes were harvested, dispersed, and incubated with allophycocyanin-conjugated anti-CD44 or PE-conjugated anti-CD25 Abs, and green fluorescence intensity was quantified via flow cytometry. Thymocytes were gated as indicated (upper panel) and analyzed for green fluorescence (lower panel). Shown are GR expression histograms and MFIs from gates 1 (shaded histogram) and 2 (open histogram). As a relative comparison, the MFI of CD25+ thymocytes from a RAG2+/+/GFP-GR heterozygous littermate was 52.5. H, Graphical representation of thymocyte subsets in GFP-GR heterozygous and homozygous mice (mean ± SD; n = 3 per group). Data are representative of three to five independent experiments.

 
We next surveyed GFP-GR mice to determine whether increased GFP fluorescence reflected cell populations normally expressing relatively high levels of GR. To this end, we analyzed the hippocampus, which is known to express high levels of GR in the CA1 region but low levels in the surrounding CA2 and CA3 regions. Fluorescence microscopic analysis of GFP-GR heterozygous brain showed green fluorescence in the CA1 region that corresponded exactly with GFP-GR protein expression evaluated immunohistochemically in serial sections (Fig. 2GoA). Green fluorescence was also readily detectable in a granular pattern in the cytoplasm and to a variable degree in the nucleus of murine embryonic fibroblasts (MEFs) from GFP-GR mice. This fluorescence was restricted to the nucleus when MEFs were treated with DEX (Fig. 2GoB). Similar patterns of fluorescence were seen in dispersed thymocytes (Fig. 2GoC).



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  		FIGURE 2. Green fluorescence is specific to GFP-GR protein expression. A, Immunohistochemical stain of hippocampal sections from brains of wild-type and GFP-GR heterozygous mice using anti-GFP Abs (upper panels; magnification, x40). Green fluorescence of serial hippocampal sections as directly analyzed by fluorescence microscopy (lower panels; magnification, x100). B, Embryonic fibroblasts from wild-type or homozygous GFP-GR mice were treated with medium or DEX for 30 min and analyzed microscopically for green fluorescence (magnification, x600). C, Thymocytes from wild-type or homozygous GFP-GR mice were treated with saline or DEX for 10 min and analyzed microscopically for green fluorescence (magnification, x600). The insets in B and C show the same cells viewed by phase contrast microscopy.

 
We next tested whether GFP-GR protein functioned normally in vivo. Because activity of the hypothalamic-pituitary-adrenal axis accurately reflects GC transrepression acting through the GR at several sites within the CNS and pituitary, we measured sensitive indicators of feedback inhibition: circadian GC modulation, GC responses to psychologic stress, and GC responses to inflammatory stress. No differences were detected in circulating GC levels in any of these paradigms between wild-type and GFP-GR homozygous mice (Fig. 3Go), suggesting that GFP-GR responds normally to physiologic regulatory feedback.



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  		FIGURE 3. GFP-GR mediates normal feedback regulation. Plasma corticosterone was measured in wild-type and homozygous GFP-GR mice in the morning (circadian nadir) or evening (circadian peak), after a 30-min restraint, or 24 h after i.p. injection of LPS (100 µg). Data represent the mean ± SEM of three mice per group.

 
Thymocyte GR expression is tightly regulated

Thymocytes must pass several developmental milestones on their way to becoming functional peripheral T cells. In the thymus, immature cells begin as CD4-CD8- DN thymocytes, which can be further subdivided based on differential expression of CD44 and CD25 (28). Thymocyte survival is reported to be most sensitive to exogenous and endogenous GCs during passage through a CD4+CD8+ DP stage on their way to becoming CD4+ or CD8+ SP cells (3, 4). To test the hypothesis that this increased sensitivity resulted from increased abundance of the GR protein, we analyzed thymocyte subpopulations in GFP-GR mice for differential GR expression. Homozygous GFP-GR thymocytes showed no difference in total cell numbers or subpopulations from their wild-type counterparts (Fig. 4GoA). Histogram analysis of GR expression in thymocytes revealed a relatively low level of GR protein in CD4+ and DP thymocytes (which will subsequently be referred to as basal). Surprisingly, we noted bimodal fluorescence peaks in both CD8+ and DN subpopulations (Fig. 4GoB), indicating differential GR expression in these compartments.

Further analysis of the DN compartment showed that CD25+ thymocytes expressed high levels of GR, an abundance 4-fold greater than DP and CD4+ cells (Fig. 4Go, E and H). To determine where in the DN compartment GR expression begins to increase, we bred GFP-GR mice to recombinase-activating gene (RAG)2-/- mice in which cells are arrested at the CD25+ stage of thymocyte development (29). We noted that GR expression increased concomitant with CD44 expression and peaked at the CD25+CD44- stage of development (Fig. 4GoG).

In the CD8+ compartment, surface staining for TCR{beta} and HSA showed that a subset (19.7 ± 2%, n = 4) of CD8+ cells expressing high levels of GR (but less than CD25+ DN cells) were TCR{beta}low and HSAhigh, indicating that they were ISP thymocytes (Fig. 4Go, C and F). Additionally, GR was quickly down-regulated to basal levels in TCR{beta}low DP thymocytes (Fig. 4GoD).

Taken together, these results suggest that, in a cell cycle-independent manner, GR begins to be up-regulated at the CD44+ stage, reaches highest levels at the CD25+ DN stage, and then is quickly down-regulated at the DP TCR{beta}low stage of development.

Thymocyte GR expression varies during ontogeny

Circulating GCs show modulation during development such that they do not reach peak physiologic levels or begin to vary in a circadian fashion until ~4 wk of age (30, 31). To determine whether developmental regulation of GR expression could also contribute to age-dependent GC actions, we analyzed GFP-GR thymi from embryos in utero (embryonic day (E)15.5) through adulthood. E15.5 thymi expressed an intermediate amount of GR protein compared with high levels seen in CD25+ DN thymocytes in the adult. In contrast to the bimodal pattern of DN and ISP thymocytes, we detected uniform GR expression within the DN subpopulation and relatively small differences among DN, ISP, and DP subpopulations (Fig. 5GoA). Newborn (day of life (P)1) thymocytes also expressed an intermediate level of GR protein within both the DN and CD8+ compartments (Fig. 5GoB). Most of the CD8+ cells (93 ± 0.9%, n = 4) expressed low levels of TCR{beta}, suggesting that these were almost entirely in the ISP stage of thymocyte development. In contrast to embryonic thymocytes, we noted that GR was down-regulated in DP thymocytes to levels similar to those found in adult animals. By P7, the MFI of CD25+ DN thymocytes approached that of adult cells, while a second population expressing basal GR levels appeared in the CD8+ compartment. These proved to be mature CD8+ SP cells (based on high surface expression of TCR{beta}; Fig. 5GoC). At P14, CD25+ DN thymocytes expressed GR levels equal to those in adult mice and CD8+ thymocytes showed the same GR expression and subsequent SP:ISP ratio seen in adult mice (5:1; data not shown). Twenty-one-day-old mice also showed thymocyte GR expression at levels similar to those found in adult mice (Fig. 5GoD). These results indicate not only that GR expression differs widely between the embryo and adult but also that GR expression gradually matures over a 2- to 3-wk period after birth.



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  		FIGURE 5. Thymocyte GR expression differs between embryogenesis and adulthood. A–E, GFP-GR thymocytes were harvested at the indicated time points, dispersed, and incubated with Abs directed against CD4, CD8, and TCR{beta}, and green fluorescence intensity was quantified via flow cytometry. The left side of each panel shows percentages of subpopulations and gates used for GFP MFI quantitation. E15.5 thymocytes were pooled (n = 3) in A, whereas representative plots of multiple GFP-GRhet/homozygotes are shown in B–D. E and F, Thymocytes grown in FTOC resemble newborn cells. Fetal thymocytes were cultured for 7 days, dispersed, and incubated with allophycocyanin-conjugated anti-CD4 and PerCP-conjugated anti-CD8 Abs and PE-conjugated anti-TCR{beta} Abs, and green fluorescence intensity was quantified via flow cytometry. E, Percentages of CD4/8+ subpopulations and gates used for F. F, MFI ± SD of FTOC ({circ}; n = 6), and P1 (•; n = 4) thymocyte subpopulations (ISP = CD8+TCR{beta}low). Adult GFP-GR homozygous thymocytes were harvested in the same experiment and are included as a relative comparison. Results shown are representative of two independent experiments.

 
Thymocytes grown ex vivo express GR levels resembling the newborn

Many studies have used fetal thymic organ culture (FTOC) as a model system to discern GR function in thymocyte development (22, 32, 33, 34). We analyzed GR expression in cells grown in FTOC to determine where along the expression spectrum between embryogenesis and adulthood these thymocyte subpopulations would lie. Interestingly, we noted that cells grown for 7 days in FTOC expressed intermediate GR levels in DN and ISP thymocytes that decreased to basal levels in DP and SP cells (Fig. 5Go, E and F). These expression levels very closely resembled those found in P1 but not adult thymocytes, suggesting that GR actions in FTOC may not accurately model actions in the adult in the context of thymocyte development.

Thymocyte GC sensitivity is dissociated from GR expression

DP thymocytes have been shown to be sensitive to apoptosis induced by exogenous and endogenous GCs (3, 35). Although DN thymocytes have long been known to resist GCs (4), the relative sensitivities of CD25+ DN, ISP, and subpopulations within the DP thymocyte compartment have not been investigated in vivo. We analyzed thymocyte subset sensitivity to DEX in GFP-GR mice. Consistent with previous reports (4), overall thymus cellularity decreased significantly in DEX-treated animals (Fig. 6Go, A and D) while CD25+ and mature SP thymocytes resisted GC-induced apoptosis (Fig. 6Go, B and C). In contrast, we detected virtually no ISP cells after treatment (Fig. 6Go, B and C). Additionally, TCR{beta}low DP thymocytes succumbed to GC-induced killing to a greater degree than did their TCR{beta}int counterparts (Fig. 6GoC).



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  		FIGURE 6. Relative GR expression is dissociated from sensitivity to GCs. A–D, Thymocytes were harvested from GFP-GR 24 h after DEX (200 µg) or saline injection and incubated with allophycocyanin-conjugated anti-CD4 Abs, PerCP-conjugated anti-CD8 Abs, and PE-conjugated anti-CD25 or TCR{beta} Abs and green fluorescence intensity was quantified via flow cytometry. A, Representative percentages of live (based on forward and side scatter) subpopulations and gates used for B. B, ISP thymocytes (arrow), but not DN thymocytes, expressing high levels of GR, are susceptible to DEX-induced apoptosis. Thymocytes were gated as indicated in A and analyzed for GR expression via green fluorescence. Open histograms show a representative saline-injected mouse, and filled histograms show a representative DEX-injected mouse. C, Graphical representation of B. Values of p < 0.01 between saline and DEX-treated ISP (CD8+TCR{beta}low) and TCR{beta}low DP thymocyte groups. D, Total thymocyte viability after indicated treatments (based on trypan blue exclusion). Results shown are averages ± SEM of three (saline) and four (DEX) mice and are representative of two independent experiments. E, Thymocytes from wild-type mice were harvested 8 h after DEX (200 µg) administration, stained as above, but resuspended with annexin V-FITC before FACS analysis. Apoptotic induction was quantitated by dividing annexin V+ thymocyte subsets from DEX-treated mice by control mice. Baseline and DEX-treated annexin V+ cells, respectively: DN, 1.8 ± 0.2 and 4.7 ± 1.1; ISP, 1.5 ± 0.1 and 7.7 ± 0.8; DP, 2.8 ± 0.2 and 16.7 ± 1.1; CD8+, 2.6 ± 1.2 and 5.3 ± 1.5; CD4+, 4.6 ± 0.1 and 8.1 ± 2.9. Results shown are averages ± SEM of three mice per group. Values of p < 0.01 between DN and ISP, DN and DP, SP (both CD4+ and CD8+) and ISP, and SP and DP thymocytes.

 
To prove that ISP thymocytes were dying with DEX treatment and not merely differentiating to DP thymocytes before becoming susceptible to apoptosis, we measured an early indicator of apoptosis: the binding of annexin V to the surface of thymocytes, 8 h after DEX administration. Consistent with the relative paucity of live cells 24 h after DEX, and confirming the relative sensitivity of these thymocyte subsets, ISP and DP thymocytes showed a 5- to 6-fold induction of apoptosis in DEX-treated animals compared with saline-injected controls. All other subsets showed a 1.5- to 2.5-fold induction at this early time point (Fig. 6GoE). These results indicate that relative GR protein levels do not determine sensitivity to GCs. Additionally, these data show that thymocyte apoptotic sensitivity begins before cells reach the DP stage of development.

Signaling through CD3{epsilon} and positive selection down-regulates GR

Our results indicate that GR is expressed at highest levels in CD25+ DN thymocytes and that GR is down-regulated to basal levels at the DP stage of development. This down-regulation coincides temporally with the onset of signaling through the pre-TCR. To test whether signaling through components of the pre-TCR complex can down-regulate GR expression, we administered anti-CD3{epsilon} Abs to RAG2-/-/GFP-GR heterozygous mice. Consistent with previous reports (36), CD25 disappeared from the surface of RAG2-/-/GPF-GR thymocytes 48 h after anti-CD3{epsilon} Ab administration (Fig. 7GoA). Interestingly, anti-CD3{epsilon} administration resulted in down-regulation of GR, suggesting that pre-TCR signaling orchestrates the down-regulation of GR expression in developing thymocytes (Fig. 7GoB).



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  		FIGURE 7. GR expression is down-regulated by activation of CD3{epsilon} in RAG2-/- and in positively selecting HY TCR-transgenic thymocytes. Thymocytes were harvested from RAG2-/-/GFP-GR heterozygotes 48 h after i.p. administration of anti-CD3{epsilon} Ab, and incubated with allophycocyanin-conjugated anti-CD44 and PE-conjugated anti-CD25 Abs, and green fluorescence intensity was quantified via flow cytometry. A, Representative CD44 and CD25 subpopulations from Ab-treated and control mice, and gates used for B. B, Thymocytes were gated as indicated in A and analyzed for GR expression via green fluorescence. Open histograms show a representative control mouse and filled histograms show a representative anti-CD3{epsilon} Ab-injected mouse. Results shown are representative of two independent experiments. C and D, GR expression is down-regulated in positively selecting thymocytes but remains high in negatively selecting thymocytes. Thymocytes were harvested from littermate male and female HY+RAG2-/-/GFP-GR heterozygotes. C, Percentages of CD4+ and CD8+ subpopulations and gates used for GFP MFI quantitation. D, Thymocytes were gated as indicated in C and analyzed for green fluorescence. Open histograms denote female and shaded histograms denote male thymocytes.

 
To further address the hypothesis that pre-TCR signaling causes GR down-regulation in the context of Ag presentation, we studied GR expression in a common model of in vivo thymocyte selection: HY TCR-transgenic mice. We measured GR expression in male and female GFP-GR heterozygous/HY+RAG2-/- mice. Although GR expression was equal in DN thymocytes, we noted a striking difference in both CD8+ and DP thymocytes between the sexes. Female mice, which positively select CD8+ thymocytes, showed a GR expression pattern very similar to that seen in wild-type mice, in which ISP cells express relatively high levels of GR, which is down-regulated to basal levels in DP and SP CD8+ thymocytes (Fig. 7GoD). Male mice, which negatively select CD8+ thymocytes because of the endogenous expression of the HY Ag, express identical GR levels in ISP cells. However, in contrast to females, male DP thymocytes did not down-regulate GR expression to any appreciable degree (Fig. 7GoD). Taken together, these data suggest not only that thymocyte GR expression is tightly regulated but also that pre-TCR signaling events play an important role in this process.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To assess GR gene expression in individual cells in vivo we generated GFP-GR knockin mice. This strategy provided mice that express a GFP-tagged GR protein under entirely endogenous regulatory control. Indeed, expression and function of the chimeric GR in GFP-GR mice are indistinguishable from its endogenous counterpart. Thus, GFP-GR mice provide a unique system in which GR protein expression can simply and reliably be localized and quantitated on a single-cell basis in vivo.

In order for GFP-GR mice to prove a useful model of GR expression and localization, it was critical that the protein was detectable in single living cells with little manipulation. Viewed under the microscope, GFP-GR cells showed cytoplasmic fluorescence in MEFs and dispersed thymocytes. This fluorescence localized to the nucleus when cells were treated with GCs. Additionally, the CA1 region of the hippocampus, which is known to express high GR levels, showed relatively high and specific green fluorescence when compared with surrounding brain regions. Finally, as assessed by flow cytometry, homozygous GFP-GR mice showed a precise 2:1 ratio of fluorescence intensity when compared with heterozygous mice in PBMCs and in six different thymocyte subpopulations expressing different GR levels. These results indicate not only that green fluorescence can be used as a specific marker of GR localization but also that it can be used to directly quantitate GR expression in single living cells in vivo.

As exemplified by RAG2, among other proteins (27), the elucidation of when and to what levels a protein is expressed is critical for determining its contribution to thymocyte development. Using GFP-GR mice, we have sensitively mapped GR expression throughout thymocyte development. GR begins a significant, tightly controlled induction very early in thymocyte development. In fact, thymocytes show a steady rise in GR levels with increasing surface expression of CD25 (J. A. Brewer and L. J. Muglia, unpublished results). Expression of GR returns to basal levels at the DP stage, suggesting a previously unanticipated, nonapoptotic role for GR very early in thymocyte development.

A recent study using intracellular Ab staining of permeabilized thymocytes reported that relative GR expression is high in DN thymocytes, decreases to low levels in TCR{beta}low DP thymocytes, and returns to intermediate levels in TCR{beta}int DP as well as mature SP thymic subpopulations. These results are discordant with the GR expression pattern that GFP-GR mice reveal. One possible limitation to quantitation based upon intracellular staining is variable accessibility of Ab binding across different thymocyte subpopulations, where chaperones and other GR binding proteins may be differentially expressed. Using fluorescence intensity as an intrinsic property of GFP-GR protein, such variables are eliminated when quantitating GR expression.

Although the mechanism for GR up-regulation in DN thymocytes remains to be determined, as shown by anti-CD3{epsilon} Ab administration to RAG2-/-/GFP-GR mice and in female HY/RAG2-/-/GFP-GR TCR-transgenic mice, pre-TCR signaling induces GR down-regulation. The failure to observe a reduction in GR expression in male HY/RAG2-/-/GFP-GR TCR-transgenic mice may reflect clearance of negatively selecting thymocytes before GR down-regulation, rapid clearance precluding detection of thymocytes that have undergone GR reduction, or a protective effect of high levels of GR on cells destined for negative selection, such that apoptosis occurs as GR levels are decreased. In accord with the last possibility, a protective effect of GCs during thymocyte development has been implicated in previous studies in vitro and in vivo (9, 33, 34, 37, 38).

Importantly, we have observed no increase or decrease in GR expression from basal levels in thymocytes or peripheral T cells from GFP-GR mice given LPS, TCR complex stimulation (anti-CD3{epsilon} Ab), DEX, psychologic stressors (acute and chronic restraint), or chronic corticosterone administration, or after removal of systemic GCs via adrenalectomy (J. A. Brewer and L. J. Muglia, unpublished results). Thus, it seems that GR up-regulation in thymocytes reflects a unique developmental program or set of environmental conditions limited to the thymus.

Endogenous and exogenous GCs have been known to modulate thymus cellularity for three-quarters of a century (1). These effects have been ascribed mainly to GC-mediated induction of DP thymocyte apoptosis. Using GFP-GR mice we have shown that ISP, in addition to DP thymocytes, have an increased sensitivity to GC-induced apoptosis compared with other thymocyte subsets. Interestingly, this sensitivity does not correlate with GR expression as shown by a relative resistance to apoptosis by DN thymocytes, which, like ISPs, express high levels of GR, and the relative resistance of SP thymocytes to apoptosis, which express the same low GR levels as their GC-sensitive DP counterparts. Previously, reduction of thymocyte cellularity with GC administration and increase of thymocyte cellularity with adrenalectomy have been ascribed to actions within the DP compartment. Our data show that ISPs are also exquisitely sensitive to GC-mediated apoptosis. These observations may further explain the magnitude and duration of GC-induced thymocyte depletion: the DP thymocyte compartment not only is killed but also is prevented from being repopulated, due to the absence of ISP thymocytes.

Additionally, the dissociation between GR expression and GC killing suggests that factors other than GR protein levels open and close the window for steroid sensitization. For example, SRG3, a mouse homolog of human BAF155, has been reported to bind to the GR complex and modulate GC-induced apoptosis in vitro and in vivo (39, 40). SRG3 seems to be expressed at higher levels in preselection thymocytes (CD3lowCD69-) than positively selected thymocytes and peripheral T cells (CD3highCD69+), both of which are resistant to GC-induced apoptosis, suggesting that SRG3 down-regulation may contribute to GC desensitization after selection (41). In preliminary studies, we have not detected differences in SRG3 expression between DN and DP thymocytes, suggesting that other factors are likely to be involved in the initiation of GC sensitivity (J. A. Brewer and L. J. Muglia, unpublished results).

In addition to finding that the relative abundance of GR does not serve as a primary determinant of sensitivity to GC-mediated apoptosis and that pre-TCR signaling down-regulates GR expression, we also demonstrate striking differences in GR expression between embryogenesis, early postnatal life, and adulthood. This pattern of maturation of GR expression is significant for several reasons. First, many studies addressing the role of GR in thymocyte development have been undertaken using embryonic thymocytes cultured in vitro or FTOC. Results from these studies have conflicted, leaving the question of the effects of GR action unanswered (18, 19, 20). The large differences in GR expression in thymocyte subsets between the embryo, FTOC, and adult mouse may help to explain some of these discrepancies: GC action may be different in each of these systems due at least in part to GR abundance. In light of these new data, caution must be exercised in extrapolating findings from fetal thymus or FTOC to action of GCs on adult thymocytes.

In summary, analysis of GFP-GR mice has defined relative GR expression patterns in thymocyte subpopulations through ontogeny and in the adult animal, dissociated GR expression from GC-induced apoptosis, characterized ISP thymocytes as a novel GC-sensitive cell population, and identified pre-TCR signaling as a mechanism of GR down-regulation. Future studies designed to 1) measure nuclear occupancy of GR during physiological processes in vivo, 2) evaluate GC analogs for cell type-specific receptor translocation in hopes of identifying dissociated steroids that maintain anti-inflammatory actions without the therapy-limiting side effects of standard GCs, or 3) use relative GR expression concentrations to facilitate sorting of specific populations of live cells represent only a small portion of the types of analyses that will now be possible.


   Acknowledgments
 
We thank Sherri Vogt for expert technical assistance, Andrea Wooley for assistance with FTOC experiments, Dr. Jack Bodwell for providing plasmids, Drs. Alec Cheng and Osami Kanagawa for helpful discussions, and Dr. Andrey Shaw for critical review of this manuscript. We also thank Mia Wallace and the Washington University Mouse Genetics Core for ES cell injections.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health, the American Cancer Society, the Burroughs Wellcome Fund (to L.J.M. and B.P.S.), and the Medical Scientist Training Program (to J.A.B.). Back

2 Address correspondence and reprint requests to Dr. Louis J. Muglia, Developmental Biology Unit, Department of Pediatrics, Washington University School of Medicine, Box 8208, St. Louis, MO 63110. E-mail address: muglia_l{at}kids.wustl.edu Back

3 Abbreviations used in this paper: GC, glucocorticoid; DP, double positive; SP, single positive; ISP, immature SP; DN, double negative; RAG, recombinase-activating gene; GR, GC receptor; mGR, mouse GR; GFP, green fluorescent protein; eGFP, enhanced GFP; DEX, dexamethasone; MFI, mean fluorescence intensity; HSA, heat stable Ag; GRE, GC response element; MEF, murine embryonic fibroblast; ES, embryonic stem; FTOC, fetal thymic organ culture; E, embryonic day; P, day of life. Back

Received for publication April 10, 2002. Accepted for publication May 29, 2002.


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 Materials and Methods
 Results
 Discussion
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