Download Green Fluorescent Protein-Glucocorticoid Receptor Knockin Mice

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Green Fluorescent Protein-Glucocorticoid
Receptor Knockin Mice Reveal Dynamic
Receptor Modulation During Thymocyte
Development
Judson A. Brewer, Barry P. Sleckman, Wojciech Swat and
Louis J. Muglia
J Immunol 2002; 169:1309-1318; ;
doi: 10.4049/jimmunol.169.3.1309
http://www.jimmunol.org/content/169/3/1309
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Copyright © 2002 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
The Journal of Immunology
Green Fluorescent Protein-Glucocorticoid Receptor Knockin
Mice Reveal Dynamic Receptor Modulation During Thymocyte
Development1
Judson A. Brewer,* Barry P. Sleckman,† Wojciech Swat,† and Louis J. Muglia2*
G
lucocorticoids (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-␬B, and others, although how and when endogenous GCs specifically affect thymocyte development remains unclear (5–7).
GCs exert their effects on tissues outside the brain primarily by
activating the type-II GC receptor (GR). This receptor is abun-
Departments of *Pediatrics, Molecular Biology, and Pharmacology and †Pathology
and Immunology, Washington University School of Medicine, St. Louis, MO 63110
Received for publication April 10, 2002. Accepted for publication May 29, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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.).
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: [email protected]
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.
Copyright © 2002 by The American Association of Immunologists, Inc.
dantly 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 –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, 13–17). 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 –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.
0022-1767/02/$02.00
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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 CD25ⴙCD4ⴚCD8ⴚ thymocytes and returns to
basal levels in CD4ⴙCD8ⴙ 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 2ⴚ/ⴚ thymocytes, GR expression is downregulated 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 CD8ⴙ thymocytes as novel apoptosis-sensitive GC targets, and separate receptor
abundance from susceptibility to apoptosis across thymocyte populations. The Journal of Immunology, 2002, 169: 1309 –1318.
1310
GFP-GR KNOCKIN MICE REVEAL NOVEL REGULATION IN THYMUS
Materials and Methods
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 ⫻ 129/Sv genetic background.
ceau 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-␮m 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).
Generation and in vitro testing of GFP-GR construct
Restraint stress and LPS administration
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 ␮M 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).
Mice were restrained for 30 min as previously described (25) or injected
i.p. with 100 ␮g LPS (Escherichia coli serotype 0111:B4; Sigma-Aldrich)
dissolved in 100 ␮l PBS.
Animal husbandry and plasma sampling
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-␮m mesh, washed, resuspended in DMEM plus 10% FCS, and
grown on cover slips. Where indicated, cells were incubated in 0.1 ␮M
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 Pon-
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␤; 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 ␮g DEX phosphate, 250 ␮g anti-CD3⑀ 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
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.
1A). 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.
1B). 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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Generation of GFP-GR mice
Corticosterone assay
The Journal of Immunology
1311
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. 1C). 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. 1D), 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 local-
izing 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. 1E). Additionally, thymocytes from
homozygous mice fluoresced twice as brightly as heterozygotes at
each stage of development (shown in Fig. 4H 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.
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
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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.
1312
GFP-GR KNOCKIN MICE REVEAL NOVEL REGULATION IN THYMUS
showed green fluorescence in the CA1 region that corresponded exactly with GFP-GR protein expression evaluated immunohistochemically in serial sections (Fig. 2A). 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. 2B). Similar patterns of fluorescence were seen in dispersed thymocytes (Fig. 2C).
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. 3), suggesting that GFP-GR responds normally to physiologic regulatory feedback.
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 ␮g). Data represent the
mean ⫾ SEM of three mice per group.
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, ⫻40). Green fluorescence of serial hippocampal
sections as directly analyzed by fluorescence microscopy (lower panels;
magnification, ⫻100). 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,
⫻600). 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, ⫻600). The insets in B and C show the
same cells viewed by phase contrast microscopy.
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. 4A). 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. 4B), 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. 4, 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. 4G).
In the CD8⫹ compartment, surface staining for TCR␤ 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␤low
and HSAhigh, indicating that they were ISP thymocytes (Fig. 4, C
and F). Additionally, GR was quickly down-regulated to basal
levels in TCR␤low DP thymocytes (Fig. 4D).
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␤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
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Thymocyte GR expression is tightly regulated
The Journal of Immunology
1313
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. 5A). Newborn (day of life (P)1) thymocytes also expressed an intermediate level of GR protein within
both the DN and CD8⫹ compartments (Fig. 5B). Most of the
CD8⫹ cells (93 ⫾ 0.9%, n ⫽ 4) expressed low levels of TCR␤,
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␤; Fig. 5C). 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. 5D). 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.
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, 32–34). 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. 5, 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. 6, A and D)
while CD25⫹ and mature SP thymocytes resisted GC-induced apoptosis (Fig. 6, B and C). In contrast, we detected virtually no ISP
cells after treatment (Fig. 6, B and C). Additionally, TCR␤low DP
thymocytes succumbed to GC-induced killing to a greater degree
than did their TCR␤int counterparts (Fig. 6C).
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
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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␤ (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␤ (upper panel) and green fluorescence was measured (lower panel, solid line,
TCR␤low; dashed line, TCR␤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.
1314
GFP-GR KNOCKIN MICE REVEAL NOVEL REGULATION IN THYMUS
with saline-injected controls. All other subsets showed a 1.5- to
2.5-fold induction at this early time point (Fig. 6E). 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⑀ 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 preTCR complex can down-regulate GR expression, we administered
anti-CD3⑀ 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⑀ Ab administration (Fig. 7A). Interestingly, anti-CD3⑀ administration resulted in down-regulation of GR, suggesting that pre-TCR signaling orchestrates the down-regulation of GR expression in
developing thymocytes (Fig. 7B).
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:
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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␤, 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␤ 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 (E; n ⫽ 6), and P1 (F; n ⫽ 4) thymocyte subpopulations (ISP ⫽ CD8⫹TCR␤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.
The Journal of Immunology
1315
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. 7D). 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. 7D). Taken together, these data suggest not
only that thymocyte GR expression is tightly regulated but also
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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 ␮g)
or saline injection and incubated with allophycocyanin-conjugated anti-CD4 Abs, PerCP-conjugated anti-CD8 Abs, and PE-conjugated anti-CD25 or TCR␤
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␤low) and TCR␤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 ␮g) 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.
1316
GFP-GR KNOCKIN MICE REVEAL NOVEL REGULATION IN THYMUS
that pre-TCR signaling events play an important role in this
process.
Discussion
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␤low DP thymocytes,
and returns to intermediate levels in TCR␤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⑀ 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).
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FIGURE 7. GR expression is down-regulated by activation of CD3⑀ 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⑀ Ab, and incubated
with allophycocyanin-conjugated anti-CD44 and PEconjugated 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⑀ 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.
The Journal of Immunology
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.
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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 (antiCD3⑀ 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 GCinduced 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.
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leaving the question of the effects of GR action unanswered (18 –
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.
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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 GCsensitive cell population, and identified pre-TCR signaling as a
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GFP-GR KNOCKIN MICE REVEAL NOVEL REGULATION IN THYMUS
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