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Departments of
* Pediatrics, Molecular Biology, and Pharmacology, and
Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Ab as WT thymocytes in vitro. Surprisingly,
GRko thymocytes are significantly more resistant than WT thymocytes to
anti-CD3-mediated thymocyte apoptosis in vivo. Consistent with
this finding, in vivo TCR complex activation induces sustained high
levels of glucocorticoids that correlate strongly with thymocyte
apoptosis in WT mice. We find that while direct engagement of the TCR
complex may cause death of a subset of thymocytes, glucocorticoids are
required for deletion of the majority of thymocytes. Thus, TCR
stimulation by Ab administration may more accurately reflect polyclonal
T cell activation than negative selection in
vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Beginning with the observation over three-quarters of a century ago
that removal of systemic glucocorticoids results in increased thymus
size and cellularity (2), and more recently with the
demonstration that TCR activation rescues T cell hybridomas and primary
thymocytes from steroid-mediated apoptosis in vitro (3),
glucocorticoids have been postulated to influence thymocyte
development. Consistent with these observations, glucocorticoids
modulate developmental pathways critical for thymocyte ontogeny, with
effects on ZAP-70, linker for activation of T cells, and NF-
B
signaling (1, 4, 5). However, the consequences of these
interactions for the make-up of T cell repertoire remain controversial.
Studies performed in fetal thymic organ culture
(FTOC),3 using
pharmacologic blockade of steroid biosynthesis, have suggested that
glucocorticoids are important for thymocyte survival (6).
More recent experiments have used FTOC and dispersed cell culture of
thymocytes from mice in which a neomycin resistance cassette was
inserted into exon 2 of the glucocorticoid receptor (GR) gene locus.
Studies with this GR hypomorph allele did not find glucocorticoids
essential for fetal thymocyte survival (7, 8), but were
unable to assess whether the increases in thymocyte number and changes
in subpopulation distribution normally found in postnatal and adult
animals occurred in the absence of GR activity. Glucocorticoid
contributions to thymocyte development in the adult animal have also
been studied, using antisense transgenic mouse models. In this approach
too, results have differed between systems, as evaluation of one model
showed a decrease, while another showed an increase in thymic
cellularity, with reduction of GR expression (9, 10).
In the periphery, glucocorticoids are critical for down-regulating the inflammatory response to pathogens and their toxins. Induced by cytokine stimulation of the hypothalamic-pituitary-adrenal axis, glucocorticoids potentiate the acute phase response while acting in a negative feedback loop to suppress further cytokine production (11, 12, 13). In instances of massive peripheral immune activation, such as in septic shock, endogenous glucocorticoids have been shown to be critical for the prevention of cytokine-mediated multiorgan failure and associated mortality (14). Interestingly, with polymicrobial sepsis, glucocorticoid levels sufficient to induce thymocyte apoptosis are achieved, suggesting glucocorticoid actions on the thymus may contribute to immunoregulation in instances of polyclonal or nonspecific peripheral activation (15). However, links between direct T cell activation and thymocyte killing have not been established.
In this report, we generated mice deficient in the GR and studied thymocyte development in wild-type (WT) mice reconstituted with WT and GR-deficient fetal liver cells. We found that direct GR signaling in thymocytes is not critical for normal development. Surprisingly, using a standard model of in vivo negative selection, we showed that systemic glucocorticoids induced by TCR activation are necessary and sufficient to cause double-positive (DP) thymocyte apoptosis, suggesting that TCR activation induced by Ab administration may more accurately reflect the effects of glucocorticoids stimulated after polyclonal T cell activation than negative selection in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. Matings of estrous females with males were determined by detection of a copulation plug on the morning following introduction into the male cage. After detection of a copulation plug, females were removed from the male cage with the morning of plug detection designated as 0.5 days gestation to obtain accurate gestational timing. Unless otherwise noted, all mice used were 6–10 wk old and of a C57BL/6 x 129/Sv genetic background.
Generation of GRko mice
To build our GRko targeting vector (pGRloxPneo), we inserted a loxP site into the unique SacI site in the GR gene region upstream of exon 2 between exons 1B and 1C using synthetic oligonucleotides with SacI-compatible single-strand overhangs. A PGKneo cassette containing flanking loxP sites was then subcloned into an SpeI restriction site in intron 2 using oligonucleotide linkers. To obtain embryonic stem (ES) clones having replaced one copy of the endogenous murine GR locus with the GRloxPneo allele, TC1 ES cells underwent electroporation with linearized pGRloxPneo as we have previously described (16). DNA from 87 G418-resistant clones was subjected to Southern blot analysis using a probe external to the flanking regions within our targeting vector. Five 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. ES cells heterozygous for the GRloxPneo allele containing the distal loxP site were transiently transfected with the Cre-expression vector pMC Cre. DNA isolated from colonies of individually plated cells following transfection was analyzed by Southern blot. Clones having deleted both exon 2 and the neocassette were identified (GRko allele). One clone heterozygous for the knockout allele was injected into C57BL/6 blastocysts and resulted in germline transmission of the ES genome.
Fetal liver reconstitution
Female C57BL/6 mice were lethally irradiated (900 rad) and reconstituted with fetal liver cells from heterozygous GRko matings as previously described (17). Briefly, embryos were harvested 14.5–16.5 days postcoitus, and fetal livers were dispersed in DMEM using a 20-gauge needle, filtered through 70-µm mesh, washed, resuspended in DMEM, and injected into mice that had been irradiated the previous day. Two to four mice were reconstituted from each embryo. Embryos were genotyped by PCR. Experiments were performed 6 wk after reconstitution. Reconstitution was confirmed by FACS analysis of thymocytes for surface expression of Ly9.1 (>99% of cells were Ly9.1+ in representative WT and GRko reconstituted mice).
Corticosterone assay
Plasma concentration of corticosterone was determined by RIA (ICN Pharmaceuticals, Costa Mesa, CA) from blood collected by retroorbital phlebotomy at circadian nadir in singly housed adult male mice as previously described (18).
In vitro thymocyte apoptosis
Whole thymocytes (5 x 105) were
cultured in 0.2 ml of RPMI supplemented with 5 x
10-5 M 2-ME, L-glutamine, and 10%
heat-inactivated FCS in 96-well tissue culture plates coated with
monoclonal anti-CD3 Ab (145-2C11) at the indicated
concentrations of corticosterone (Sigma-Aldrich, St. Louis, MO) for
20 h. After harvesting, cells were washed and analyzed for
apoptosis using an annexin-V apoptosis detection kit (BD PharMingen,
San Diego, CA) on a FACSCalibur (BD Biosciences, Mountain View, CA).
Percentage of specific killing was calculated using the following
formula: (experimental apoptosis - spontaneous
apoptosis)/(100 - spontaneous apoptosis).
In vivo treatment for deletion of thymocytes
Mice were injected i.p. with 50 µg monoclonal anti-CD3
Ab (145-2C11, prepared from mouse ascites, endotoxin level: 59 pg/mg),
50 µg anti-TCR
Ab (H57-597), 50 µg isotype control Ab (both
from BD PharMingen, endotoxin level: 
10 ng/mg) diluted in 200 µl
PBS, 200 µg dexamethasone (DEX) phosphate, or normal saline, using a
30-gauge needle. Thymocytes were harvested 24 (DEX) or 48 (Ab) h
after injection, dispersed through nylon mesh into PBS, washed, counted
on a hemocytometer, stained for cell surface markers
(FITC-anti-CD25, PerCP-anti-CD4, PE-anti-CD8,
FITC-anti-CD69, and PE-anti-TCR from BD PharMingen), and
analyzed by FACS. Nonviable cells were gated out based on forward and
side scatter profile.
Western blot analysis
Total liver protein from newborn mice was hybridized with amino terminus (M-20; Santa Cruz Biotechnology, Santa Cruz, CA) or DNA-binding domain (BuGR 2; Affinity BioReagents, Golden, CO) Abs at a 1/200 dilution and developed using HRP-conjugated anti-rabbit antisera at a 1/1000 dilution using ECL detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were then stained with Ponceau S solution (Sigma-Aldrich) to ensure equal loading of protein.
Histology
Lungs from newborn mice were isolated and fixed in 4% paraformaldehyde in PBS, embedded in paraffin, cut into 5-µm sections, and stained with H&E.
Statistical methods
Results are expressed as mean ± SEM unless otherwise indicated. Statistical analysis was done by ANOVA with p < 0.05 considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To functionally inactivate the GR, we generated mice which harbor
a deletion of both exons 1C and 2 of the GR gene (GRko; Fig. 1
A). Heterozygous GRko mice
appeared grossly normal and were fertile. However, in >100 progeny
arising from GRko heterozygous x GRko heterozygous matings that
were genotyped at 3 wk of age, none were homozygous GRko. To
determine whether homozygous GRko mice die in utero or at birth, we
evaluated timed pregnancies from GRko heterozygous matings. We noted
litters of normal size, but 5/22 (23%) of the resulting offspring died
within 24 h of birth. When genotyped, these all proved to be
homozygous GRko.
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C). As glucocorticoids have been shown to be
important in pulmonary maturation (19, 20), we examined
the lungs of newborn mice. Consistent with previous reports, histologic
analysis of GRko mice revealed thickened alveolar septae, poor air
space formation, and hypercellularity compared with WT littermates
confirming the GR functional deficiency in these mice (Fig. 1D). GR-deficient thymocytes develop normally in vivo
Glucocorticoids have been postulated to be important for the development of normal thymocyte numbers and subpopulations (6, 9). However, in the embryo, glucocorticoids have also been reported to be dispensable for normal development (8). To determine the role of GR signaling in utero, we analyzed thymocyte numbers and subpopulations at embryonic day 16.5, at which time CD4/CD8 DP thymocytes begin to accumulate in the thymus. Consistent with previous reports (8), we noted no difference in thymocyte numbers and subpopulation percentages between GR-deficient and GR-intact embryos (total thymocytes (x 106): GRko = 1.33 ± 0.22, WT/GR heterozygous (GRhet) = 1.4 ± 0.48; CD4/CD8+ (%): GRko = 28.0 ± 4, WT/GRhet = 32.5 ± 4, n = 2 and 4, respectively). Additionally, thymocyte numbers and subpopulations in GR-deficient mice were no different from their GR-intact counterparts at birth (total thymocytes (x 106): GRko = 13.6 ± 3.4, WT/GRhet = 11.0 ± 4.4; CD4/CD8+ (%): GRko = 80.0 ± 4.2, WT/GRhet = 78.1 ± 7.0; CD4+ (%): GRko = 3.7 ± 0.78, WT/GRhet = 3.7 ± 1.1; CD8+ (%): GRko = 3.2 ± 0.49, WT/GRhet = 3.2 ± 1.0 n = 2 and 7, respectively). These data suggest no requirement for GR in embryonic thymocyte development.
To determine the role of glucocorticoids in adult thymocyte development
in vivo, we reconstituted WT mice with hematopoietic stem cells from
GRko fetal liver. Thymocyte numbers and CD4/CD8 subpopulations in
reconstituted WT, heterozygous, and GRko mice did not differ (Fig. 2). Additionally, TCR and CD69
expression in GRko thymocytes was no different from WT thymocytes (data
not shown). These data suggest that thymocyte GR activation is not
required for expansion and differentiation to DP and single-positive
cells in vivo.
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Glucocorticoids reliably induce substantial DP thymocyte apoptosis
(21). To confirm that GRko thymocytes resist
glucocorticoid killing in vivo, we reconstituted lethally irradiated WT
mice with GRko fetal liver cells and assessed thymocyte numbers and
subpopulations after administration of DEX, a selective GR agonist.
GRko thymocytes were resistant to DEX, in contrast to heterozygous and
WT mice, which showed a large reduction in DP populations (Fig. 2).
Glucocorticoids kill thymocytes more efficiently than TCR stimulation in vitro
Because mice with GR-deficient thymocytes have no reduction in
total thymus cellularity, suggesting that GR is dispensable for normal
expansion, we next tested whether GRko thymocytes could undergo
efficient deletion with TCR activation in vitro. As negative selection
of DP thymocytes can be mimicked using anti-TCR stimulation, we
cultured reconstituted thymocytes with increasing concentrations of
anti-CD3 Ab. Both WT and GRko thymocytes died in a similar
dose-dependent manner with Ab activation (Fig. 3A). Taken together with
normal expression of TCR on the surface of GRko thymocytes, these
data indicate that apoptosis induced by TCR complex activation is not
altered by the absence of GR signaling.
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Abs. Again,
TCR complex activation induced 
20–40% apoptosis in thymocytes.
However, this apoptotic response was minimal compared with that induced
by physiologic levels of corticosterone (Fig. 3B). As a
relative comparison, the highest levels of corticosterone caused
minimal apoptosis of GRko thymocytes. These results indicate that
endogenous steroids induce WT thymocyte apoptosis to a greater
magnitude than TCR complex activation in vitro. Additionally, the
predominant effect of corticosterone, which is less selective for the
GR than DEX, occurs through the GR rather than mineralocorticoid
receptors or other members of the nuclear hormone receptor
superfamily. Glucocorticoids mediate thymocyte apoptosis from TCR complex activation in vivo
As GRko thymocytes die normally with activation in vitro, we
sought to determine whether this was also true in vivo. Using a
standard model of in vivo negative selection, we administered
anti-CD3 Ab to fetal liver-reconstituted mice and measured
thymocyte survival and distribution 48 h later. As expected, we
found that Ab administration killed 90% of WT and heterozygous DP
thymocytes. Surprisingly, GRko thymocytes were markedly resistant to
anti-CD3-induced apoptosis (Fig. 4). Because GRko thymocytes do not show a
defect in killing caused by CD3 cross-linking in vitro, these
results suggest that GR function is not required for apoptosis induced
by TCR complex activation intrathymically. Instead, our results suggest
that mature T cell activation may be causing an abnormal and sustained
peripheral glucocorticoid surge sufficient for killing thymocytes in a
process that does not reflect normal negative selection.
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Ab administration.
Corticosterone levels shown to kill 90% of thymocytes in vitro were
achieved by 24 h and maintained at 48 h after Ab
administration (Fig. 5A).
Additionally, blood corticosterone levels at both 24 and 48 h
correlated strongly with thymocyte apoptosis (Fig. 5B).
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administration or generally applicable to T cell activation, we
administered the same amount of anti-TCR Ab, which at higher
doses (100 µg) has been shown to cause thymocyte apoptosis
(22). We observed a large rise in plasma
corticosterone after Ab injection, although of a lower magnitude than
after anti-CD3 Ab administration (Fig. 5A). Again,
this corticosterone rise directly correlated (p
< 0.01 at both 24 and 48 h) with a significant, but less
pronounced reduction in DP thymocytes (isotype control
(x106) = 139.1 ± 17.4, H57-597 =
74.1 ± 15.9, n = 4–5, p =
0.02). Taken together, these in vitro and in vivo data suggest that anti-TCR Ab administration elicits a strong and prolonged peripheral glucocorticoid surge, which is necessary and sufficient to cause massive DP thymocyte apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although a previous study established normal fetal thymocyte development in GR-hypomorph mice (8), this study was unable to assess the role of GR in mediating the substantial changes in thymocyte number and subpopulation distribution that occur postnatally. We have recently determined GR expression in thymocyte subpopulations, and have noted a surprising difference in GR protein levels not only in different subsets, but also between embryonic and adult thymocytes (J. A. Brewer, B. P. Sleckman, and L. J. Muglia, unpublished observations). These data suggest that in the least, caution should be taken when extrapolating experimental findings from embryo or FTOC to the adult animal. To completely inactivate GR specifically in immune cells at their earliest stages of development and maintain adult mouse viability, while circumventing potential promoter, integration, and specificity issues that can affect anti-sense transgenic models, we reconstituted mice with GR-deficient fetal liver. In adult mice reconstituted with GR-deficient fetal liver, we noted the normal distribution of thymic subpopulations. We did not observe a requirement for GR for normal expansion as previously reported in one line of transgenic antisense GR mice (9).
Although our studies show that thymocyte development is grossly normal in the absence of GR action, they do not prove that mature T cells with normal peptide/MHC avidity are exiting the thymus and generating an unaltered peripheral T cell repertoire. In fact, several lines of evidence suggest that glucocorticoids may be affecting lymphocyte repertoire in ways that our experimental paradigm cannot examine. First, adrenalectomy increases both thymus weight and cellularity (2). Second, corticotropin releasing hormone-deficient animals that lack circadian glucocorticoid surges have a 50% increase in thymus size (23). These observations suggest that systemic glucocorticoids may facilitate clearance of cells destined to die by neglect or negative selective processes. Additionally it is possible, as with any knockout system, that as yet unidentified GR-like receptors are compensating for GR signaling to provide grossly normal thymocyte development.
Because the demonstration that survival of DP thymocytes was sensitive
to administration of anti-CD3 Abs, Ab-induced TCR activation has
become a widely used model of thymocyte negative selection in vivo
(24). The observations that Ab administration induces
CD4/8 expression and thymocyte expansion in
RAG2-/- mice (25) and reduced
surface expression of CD25 on CD4/8- cells in
our mice (anti-CD3 Ab- and saline-treated mice had 7 ± 1
and 43 ± 2%
CD25+/CD4-/CD8-
cells, respectively), suggest that Ab injected i.p. directly effects
the thymus. Based upon the slight reduction in cell number in our GRko
reconstituted mice, direct action of anti-CD3 may be causing
modest DP thymocyte apoptosis in vivo. However, unlike negative
selection experiments in vitro, where the extracellular milieu is well
controlled, cytokines and/or hormones elicited by mature T cell
activation may also be involved in inducing thymocyte apoptosis in vivo
(22). Indeed, we find robust induction of CD69 and IL-2
production in both WT and GR-deficient splenic T cells exposed to
anti-CD3 (J. A. Brewer and L. J. Muglia,
unpublished observations). We and others have shown that administration
of CD3-activating Ab induces many cytokines, several of which
secondarily stimulate high systemic glucocorticoid levels (18, 26). Are these levels sufficient to kill DP cells? Indeed,
sustained corticosterone levels of as little as 100 ng/ml have been
shown to delete a majority of thymocytes in vivo (27).
Additional studies in which WT mice were adrenalectomized and replaced
with normal basal plasma levels of corticosterone (5 or 50 ng/ml)
further support the necessity of a glucocorticoid surge to cause
thymocyte apoptosis after anti-CD3 Ab administration (L.
J. Muglia, unpublished observations). Because of very high
mortality in mice with plasma corticosterone clamped at basal levels
after 12 h of anti-CD3 Ab exposure, reduction in thymocyte
number could not be assessed. However, analysis of DNA laddering as
evidence for early apoptosis revealed robust DNA fragmentation in
sham-adrenalectomized mice that manifest an elevation of plasma
glucocorticoids, while no such fragmentation was observed in those mice
unable to mount a glucocorticoid response above basal levels. Linking
these observations, we show that anti-CD3, as well as
anti-TCR Ab administration, elicits a glucocorticoid response
that far exceeds the threshold necessary for immature thymocyte
apoptosis. As the daily circadian peak in circulating glucocorticoid
concentration approximates the peak level achieved after CD3
activation, but does not cause profound reduction in thymocyte number,
the overall duration of glucocorticoid exposure is likely to be a key
component in causing thymocyte death.
Our findings may also apply to other in vivo models of negative selection where the potential for polyclonal T cell activation is high. Relatively specific deletion of immature and semimature thymocytes occurs with oligoclonal activation by small doses of staphylococcal enterotoxin B (SEB) in WT mice where peripheral T cell contributions are minimal (28). However, SEB has also been shown to activate the hypothalamic-pituitary-adrenal axis in high doses, resulting in rapid corticosterone production to levels capable of depleting DP thymocytes (29). These observations suggest that once a threshold of T cell activation capable of mediating a systemic inflammatory response is achieved, thymocyte killing ceases to be specific, due to the elicitation of systemic glucocorticoids, and no longer models negative selection processes. Past and future studies using anti-TCR Abs, SEB, or other molecules capable of interacting with thymocytes but also activating peripheral T cell responses must be interpreted in this fashion.
Why would the organism have evolved the capacity for the immune system to promote sustained steroid production of a magnitude sufficient to kill the vast majority of immature thymocytes? We postulate that restricted oligoclonal immune responses resulting in limited cytokine production, as exemplified by viral infection, would result only in the low level of adrenal activation needed to dampen a pathogen-specific response. In contrast, nonrestricted polyclonal TCR engagement, as seen in toxic shock, may require a second line of immunomodulation to promote survival of the animal. In this setting, not only down-regulation of activated peripheral T cells, but further blockade of naive lymphocytes, preparing to potentiate and perpetuate uncontrolled inflammatory responses, may be required. Here, apoptosis of DP thymocytes before they mature and emigrate to the periphery, may be critical for removing potential sources of further cytokine production, thus bringing states of immune-mediated shock under control. This two-tiered mechanism of regulation, mediated by glucocorticoids, seems ideally suited to matching requirements of immunosuppression to magnitudes of inflammatory challenge.
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Acknowledgments |
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Footnotes |
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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
3 Abbreviations used in this paper: FTOC, fetal thymic organ culture; DP, double positive; GR, glucocorticoid receptor; ES, embryonic stem; SEB, staphylococcal enterotoxin B; DEX, dexamethasone; GRhet, GR heterozygous; WT, wild type.
Received for publication December 20, 2001. Accepted for publication June 3, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activity through induction of IB synthesis. Science 270:286.
receptor signaling in macrophages and neutrophils. Mol. Cell. Biol. 18:4209.-chain-deficient mutant mice by transmembrane signaling through CD3. Proc. Natl. Acad. Sci. USA 90:11401.-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice. Cell 56:27.[Medline]
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 P. R. Mittelstadt and J. D. Ashwell Disruption of Glucocorticoid Receptor Exon 2 Yields a Ligand-Responsive C-Terminal Fragment that Regulates Gene Expression Mol. Endocrinol., August 1, 2003; 17(8): 1534 - 1542. [Abstract] [Full Text] [PDF]
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N. Tosa, M. Murakami, W. Y. Jia, M. Yokoyama, T. Masunaga, C. Iwabuchi, M. Inobe, K. Iwabuchi, T. Miyazaki, K. Onoe, et al. Critical function of T cell death-associated gene 8 in glucocorticoid-induced thymocyte apoptosis Int. Immunol., June 1, 2003; 15(6): 741 - 749. [Abstract] [Full Text] [PDF]
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Y. Zhan, J. F. Purton, D. I. Godfrey, T. J. Cole, W. R. Heath, and A. M. Lew Without peripheral interference, thymic deletion is mediated in a cohort of double-positive cells without classical activation PNAS, February 4, 2003; 100(3): 1197 - 1202. [Abstract] [Full Text] [PDF]
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