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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2819-2826
Early Maturation of T-Cell Progenitors in the Absence of
Glucocorticoids
By
Rosa Sacedón,
Angeles Vicente,
Alberto Varas,
Eva Jiménez,
Juan José Muñoz, and
Agustín G. Zapata
From the Department of Cell Biology, Faculty of Biology and Medicine,
Complutense University, Madrid, Spain.
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ABSTRACT |
In the present work, we demonstrated that both fetal liver and
thymic T-cell precursors express glucocorticoid receptors (GRs) indirectly suggesting a role for glucocorticoids (GCs) in the earliest
events of T-cell differentiation. To evaluate this issue, we analyzed
the thymic ontogeny in the progeny of adrenalectomized pregnant rats
(Adx fetuses), an in vivo experimental model, which ensures the absence
of circulating GCs until the establishment of the fetal
hypothalamus-pituitary-adrenal (HPA) axis. In the absence of maternal
GCs, T-cell development was significantly accelerated, the process
being reversed by in vivo GC replacement. Mature single positive
thymocytes (both CD4 and CD8) appeared in 16-day old fetal Adx thymus
when in the control fetuses, most thymocytes still remained in
the double-negative (DN) CD4 CD8 cell
compartment. In addition, emigration of T-cell receptor (TcR) 
positive cells to the spleen also occurred earlier in Adx
fetuses than in control ones. In vitro recolonization of cultured deoxiguanosine-treated mouse fetal thymus lobes with 13-day-old fetal
liver cell suspensions from both Adx and control fetuses demonstrated
changes in the developmental capabilities of fetal liver T-cell
precursors from embryos grown in the absence of GCs. Furthermore, a
precocious lymphoid colonization of the thymic primordium from Adx
fetuses was evidenced by ultrastructural analysis of both Adx and Sham
early thymus. Both findings accounted for the accelerated T-cell
differentiation observed in Adx fetuses. Together, these results
support a role for GCs not only in the thymic cell death, but also in
the early steps of T-cell differentiation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE IN VIVO INVOLVEMENT of
glucocorticoids (GCs) in the normal development of mammalian embryos
has been demonstrated by disruption of the GC receptor (GR)
gene1 and, indirectly, by the presence of GR in most fetal
tissues.2 In the earliest stages of development, until the
establishment of the hypothalamus-pituitary-adrenal gland (HPA) axis,
the maternal adrenal glands are the only source of circulating GCs for
fetuses.3,4 In this regard, Muglia et al5
demonstrated that mating of heterozygous mice for a null
corticotropin-releasing hormone (CRH) allele, with preservation of
their normal maternal supply of corticosterone, produces viable homozygous CRH-deficient mice with normal growth, fertility, and longevity, despite the low levels of hormone produced by their own
adrenal glands during adulthood. On the contrary, littermates from
homozygous CRH-deficient mothers die within the first 12 hours of life.
The role of GCs in the development of distinct blood cell lineages, a
complex process that entails the regulation of an intricate network of
genes, is poorly known. GCs seem to be involved in the decision of
erythroblast between self-renewal and differentiation.6 Accordingly, the blocking of GR binding to DNA impairs the long-term proliferation of erythroid progenitors.7 In contrast, these steroids appear to have a different effect on B-cell maturation. Corticosterone in vivo reduces the cycling B-cell precursors and induces their apoptosis, giving rise to a drastic decrease in the
number of developing B-lineage cells in the bone marrow.8 Interestingly, in vitro GCs shift the balance of granulocyte versus macrophage formation at early stages of precursor cell
differentiation9 and prevent the T-cell-mediated terminal
maturation of an epidermal-derived dendritic cell (DC)
line.10
The involvement of GCs in the generation of T-cell repertoire has been
repeatedly invoked,11-14 but their role in early T-cell differentiation has been little studied. King et al15
suggested that GCs are necessary for the normal progression of DN
(CD4CD8) to the double-positive
(DP) CD4+CD8+ cell compartment. However, other
in vivo experimental studies indicate that total or partial blockage of
GC signaling has no relevant effects on adult mouse
thymus.1,7 In an attempt to clarify the role of GCs in the
early T-cell maturation, we studied the thymocyte development in the
progeny of adrenalectomized pregnant rats, an experimental model that
ensures the absence of circulating GCs during early embryonic
development, previously used by our group.16,17 Our results
demonstrate an in vivo accelerated T-cell maturation in these
experimental conditions largely due to both changes of the maturation
capabilities of fetal liver lymphoid precursors and a precocious
lymphoid colonization of the fetal thymic primordium.
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MATERIALS AND METHODS |
Animals and treatment.
Wistar rats and Swiss mice were maintained in our laboratory
facilities. Rat and mouse fetuses were obtained from timed pregnancies. The day of finding of a vaginal plug was designated day 0 of gestation. All studies were performed in accordance with the Guide for the Care
and Use of Laboratory Animals, as adopted and promulgated by the
National Institutes of Health (NIH).
Surgery procedure.
Wistar rats were either adrenalectomized or sham adrenalectomized on
the first day of pregnancy. From this moment, the pregnant rats were
transferred to individual cages. Bilateral adrenalectomy (Adx) or sham
adrenalectomy (Sham) was performed using the dorsal approach under
ether anesthesia. The Adx mothers received 0.9% NaCl to drink instead
of water until sacrifice. To reconstitute the fetal circulating
corticosterone levels, 1 osmotic minipump (2ML4, ALZET; Alza Corp, Palo
Alto, CA) was subcutaneously implanted in pregnant rats
during adrenalectomy surgery procedure. The osmotic minipumps were
prepared following instructions supplied by the commercial distributor
to continuously infuse 23 µg/hour of corticosterone (Sigma Chemical,
St Louis, MO) in a volume of 0.25 µL of propylene glycol in 0.9 % NaCl (1:1).
Corticosterone levels.
The blood samples were collected in nonheparinized tubes and after 4 hours at room temperature centrifuged at 2,200 rpm for 15 minutes at
4°C. Sera were stored at  70°C until assayed. Steroid extraction using methylene chloride was performed before testing. A
double antibody commercial radioimmunoassay (RIA) kit
(Gamma-B-125I-Corticosterone RIA; IDS, Boldon, UK), which
provides a highly sensitive method (0.04 ng/mL), was used for the
determination of serum corticosterone levels from both mothers and fetuses.
GR analysis.
The GR expression was analyzed on both fetal liver cell precursors and
early immature thymocytes. For the study of fetal liver precursors,
CD45+ cells were isolated from liver cell suspensions of
13-day-old rat fetuses. Briefly, cell suspensions were stained with a
fluorescein isothiocyanate (FITC)-labeled monoclonal antibody (MoAb)
against rat CD45 (OX1, Pharmingen, San Diego, CA) and CD45+
cells sorted with a FACStar plus (Centro de Citometría de Flujo y Microscopía Confocal, UCM, Madrid, Spain). Sorted fetal liver CD45+ cells and total thymic cells from 15-day-old rat
fetuses were cytospun, fixed in acetone for 5 minutes at
20°C, and incubated with a biotin-conjugated MoAb specific
for rat GR (BuGR2) (ABR, Golden, CO) for 1 hour followed by
avidin-Texas red staining for 45 minutes (Amersham Iberica S.A.,
Madrid, Spain). An isotype-matched irrelevant antibody was
used as negative control to define background fluorescence. The cells
were observed and photographed under a fluorescence Labophot-2
microscope (Nikon, Tokyo, Japan).
Flow cytometry.
Cell suspensions were stained with specific MoAbs during 15 minutes in
phosphate-buffered saline (PBS)/2% fetal calf serum (FCS) at 4°C.
Labelled MoAbs with either phycoerythrin (PE), FITC, or
Cychrome against rat CD4 (OX38), CD8 (OX8), T-cell receptor (TcR)
(R73), CD45 (OX1) were obtained from Pharmingen. Cytometry analysis was
performed in a FACScan, (Becton Dickinson, San Jose, CA) from the
"Servicio Común de Investigación" (Faculty of
Biology, UCM, Madrid, Spain). Debris and dead cells were excluded from the analysis by forward and side scatter gating, and in most cases, 10,000 to 30,000 thymocytes or splenocytes were scored. The data were
analyzed using PC-lysis research software (Becton Dickinson).
Cell cycle and apoptosis analysis.
Cell cycle analysis was performed by staining with 7-AAD (Sigma
Chemicals). Briefly, cell suspensions were stained with FITC- or
PE-labeled MoAbs. After washing, the cells were permeabilized with 30%
ethanol (10 minutes, 4°C) and incubated with 1 mL of RNAse (1 mg/mL) (Sigma Chemicals) for 30 minutes. Finally, cells were incubated
with 7-AAD (7.5 µg/mL) during 30 minutes at 5°C protected from
light. Cells were analyzed by flow cytometry and the number of cycling
cells was determined from individual gated populations on the basis of
surface marker expression. Analysis was performed in a FACScan using
Cell Fit and PC-lysis software (Becton Dickinson). In vivo thymic basal
apoptosis was determined on freshly isolated thymocytes either from
Sham or Adx fetuses by using the Annexin-V-Fluos kit
(Boehringer, Mannheim, Germany) for detecting apoptotic cells by flow
cytometry. Briefly, cells were washed with PBS-2% FCS and incubated
with propidium iodide and FITC-labelled Annexin-V for 15 minutes at
4°C and immediately analyzed in a FACScan flow cytometer. In most
cases, 20,000 cells were scored.
Fetal thymus organ cultures (FTOC) technique.
Thymic lobes were placed on autoclaved polycarbonate membranes
(Millipore, Iberica, Madrid, Spain) suspended by metal grids over the
inner well of Falcon 3037 tissue culture plates. RPMI 1640 supplemented
with 10% FCS (Biosys, Compiégne, France), piruvate (1 mmol/L),
penicilin/streptomicicin (100 mg/mL), and glutamine (2 mmol/L) (all
reagents: GIBCO-BRL, Eragny, France) was used as culture medium and
replaced daily. Distilled H2O was used in the outer well to
maintain a humid environment. Organ cultures were kept at 37°C and
5% CO2.
Recolonization assays.
Alymphoid lobes were prepared by culturing thymic lobes from 15-day old
fetal Swiss mice in FTOC in the presence of 1.35 mmol/L 2'deoxiguanosine (dGuo) (Sigma, Madrid, Spain) for 5 days as previously described.18 After extensively washing,
single depleted lobes were plated with 5 × 104 cells
from 13-day old fetal liver in a total volume of 30 µL in Terasaki
plates (Nalge Nunc International, Naperville, IL). Plates
were then inverted to allow lobe and cells to combine at the bottom of
the hanging drop.19 After 48 hours, recolonized lobes were
cultured in FTOC for 12 days before harvesting. Donor cells were
prepared as follows: fetal livers were dissected either from Sham or
Adx rat embryos and carefully disrupted. Debris were removed by
filtering through a cotton mesh and viable cells determined by trypan
blue exclusion. In all the experiments, fetal liver cell suspensions
from both Sham and Adx fetuses were examined for OX-1 expression to
determine the proportions of rat cell precursors used in the
recolonization assays.
Electron microscopy.
Thirteen-day-old fetuses aseptically isolated from either control Sham
or adrenalectomized pregnant rats were fixed by immersion in 2.5%
glutaraldehyde, buffered to pH 7.3 with Milloning's fluid, postfixed
in 1% osmium tetroxide in the same buffer, and dehydrated in acetone
for embedding in Araldite (Fluka Chemie AG, Neu-Ulm, Switzerland). Semithin sections stained with an alkaline solution of
toluidine blue were used to identify and isolate the thymic primordia.
Ultrathin sections of the selected areas were obtained with a Reichert
OM-U3 ultratome (Reichert-Jung, Wein, Austria), double-stained with
uranyl acetate and lead citrate, and examined and photographed with a
JE0L 1010 electron microscope (Jeol, Tokyo, Japan) of the "Servicio
Común de Investigación" (Faculty of Biology, UCM).
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RESULTS |
GR expression in fetal cell precursors and early thymocytes.
To determine whether early cell progenitors, including T-cell
precursors are able to respond to GCs as previously demonstrated for
other blood lineages,7-10 we analyzed the GR expression in both fetal liver cell precursors, included within CD45+
cell population,20-23 from 13-day-old rat fetuses and
15-day-old fetal thymocytes. As shown in
Fig 1, earliest fetal liver
CD45+ cell precursors (Fig 1b) and immature thymocytes (Fig
1d), which have just arrived at the thymic primordium, are
expressing GRs.
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| Fig 1.
Glucocorticoid receptor expression in 13-day old rat
fetal liver cell progenitors (CD45+) (b) and 15-day old
rat fetal thymocytes (d). Both sorted 13-day old fetal liver
CD45+ cells and 15-day old fetal total thymic cells were
cytospun, fixed, and incubated with a biotin-conjugated MoAb specific
for rat GR (BuGR2) followed by avidin-Texas red staining. An
isotype-matched irrelevant antibody was used as negative control to
define background fluorescence (a and c). The cells were observed and
photographed under a fluorescence Labophot-2 microscope. Original
magnification × 300.
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Effect of maternal adrenalectomy on corticosterone levels of the
progeny.
Because early cell progenitors, present in both fetal liver and thymus,
expressed GRs, we tested the role that these steroids could exert on
early T-cell maturation by performing an in vivo experimental model,
maternal adrenalectomy, that ensures the GC absence in the progeny
until the establishment of fetal HPA axis.3,4 In fact,
using this experimental approach, statistically significant lower
values of circulating corticosterone (near to 0 ng/mL), as measured by
RIA, occurred until fetal day 18 in the serum of the progeny of
adrenalectomized rats (Adx fetuses) compared with control, Sham fetuses
(Fig 2). After the establishment of fetal HPA axis on days 17 to 18 of fetal life, the significant differences disappeared (data not shown).
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| Fig 2.
Circulating corticosterone levels in fetuses either from
adrenalectomized pregnant rats (Adx), control, Sham rats (Sham), and
adrenalectomized pregnant rats provided with corticosterone
(Adx+GCs). To reconstitute the corticosterone serum levels in the
Adx-fetuses, 1 osmotic minipump (2ML4, ALZET, Alza Corporation) was
subcutaneously implanted in pregnant rats during adrenalectomy surgery
procedure, which regularly infused 23 µg/hour of corticosterone. Data
represent the average values of 3 to 4 experiments ± standard
deviation (SD). Significant differences to Sham fetuses are marked as*
P  .05; ** P .01,*** P .001.
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In vivo acceleration of thymocyte differentiation in Adx fetuses.
In the absence of circulating GCs, the multiparametric flow cytometry
analysis demonstrated an acceleration of in vivo T-cell development
throughout thymic ontogeny. On day 15 of fetal life, the most immature
thymic population
CD4CD8TcR
in Sham control animals represented more than 95% of thymocytes (Fig 3A), whereas in Adx thymuses, this
constituted less than 40% of cells. Moreover, the remaining cells
belonged to the intermediate CD4CD8+TcR/lo
cell population (40%) and around 20% of thymocytes were DP
(CD4+CD8+) cells that appeared for the first
time in control Sham thymuses on day 18 of fetal life. At fetal day 15, a small proportion of thymocytes of Adx fetuses expressed, therefore,
TcR molecule in their membranes (Fig 3B), whereas in control Sham
animals, all of the cells were TcR until day
18 of fetal life.24 One day later, on fetal day 16, in Adx
thymuses most cells had reached the DP
(CD4+CD8+) stage and there was even an
important percentage of mature SP (both
CD4+CD8 and
CD4CD8+) TcRhi cells
(Fig 3A and B), lymphocyte populations which under control conditions
appeared in fetal rat thymus on days 20 to 21 of fetal life (data not
shown).
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| Fig 3.
(A) Flow cytometry analysis of thymic T-cell populations
defined by CD4/CD8 expression in 15-(up) and 16-(down) day old Sham
(left) and Adx (right) fetuses. (B) Histograms represent TcR
expression in thymocytes from 15-(up) and 16-(down) day old Sham (open)
and Adx (shadow) fetuses. (C) Thymic populations defined by CD4/CD8
expression in 15- to 16-day-old Adx fetuses from adrenalectomized
mothers subcutaneously implanted with an osmotic minipump that
reconstituted the corticosterone serum levels in the progeny. Results
shown are representative of 3 to 4 experiments.
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The absolute number of thymocytes in Adx fetuses was slightly lower on
day 15 of development, but significantly reduced 1 day later. To
determine the contribution of the intrathymic cell death and/or cell
proliferation in the decrease of thymic cellularity observed in 15 and
16-day-old Adx fetuses, we analyzed, by flow cytometry, the frequency
of apoptotic cells and cycling cells in both groups of embryos. No
significant differences occurred in the percentages of cycling cells
between control and Adx rats at fetal days 15 and 16. However, the
percentage of apoptotic cells was significantly higher in the
16-day-old Adx fetuses than in control ones of the same age, as
estimated by using an Annexin-V binding assay
(Table 1)
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Table 1.
Thymic Size and Percentages of Cycling and Apoptotic
Cells Throughout Early Ontogeny (15 to 17 F) of Sham and Adx
Fetuses
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On day 17, in accordance with a significant increased reduction of
thymic cellularity observed in Adx fetuses (Table 1), DP
(CD4+CD8+), SP
(CD4+CD8 and
CD4CD8+), and TcR+
thymocytes were not detected in the thymus of these fetuses
(Fig 4A). Cell emigration from the thymus
and/or in situ cell death could explain this finding. To examine the
first possibility, flow cytometry analysis of spleen cell content of
18-day-old fetal Adx rats was performed. The study demonstrated the
presence of 6% of TcRhi splenocytes, as well as both
DP (CD4+CD8+) and SP
(CD4+CD8 and
CD4CD8+) T cells in the spleen of Adx
rats (Fig 4B). In control Sham rats, the lymphocyte colonization of
spleen did not occur, however, until day 21 of embryonic life. On the
other hand, the thymus of 17-day-old fetal Adx rats contained, as those
from 16-day-old Adx fetuses, significantly increased percentages of
apoptotic cells (Table 1). We can, therefore, conclude that between
days 16 and 17 of fetal life an important percentage of thymocytes undergoes in situ apoptosis or migrates from thymus to spleen in Adx
rats. Remarkably, in this period, the absolute number of DN
(CD4CD8) cells increased 10 times
in Adx thymus, an event that was not found in the control thymus of any
of the stages studied. This event was not correlated with an increase
in the percentage of cycling DN cells compared with the values
observed in Sham rats (data not shown), suggesting that a new wave of
cell progenitors had colonized the thymus of 17-day- old Adx
fetuses and were beginning to differentiate. In support of that, the
percentage of DN cells was higher in Adx fetuses than in control ones
and these later contained more
CD4CD8+TcRcells
(Fig 4A). Interestingly, in this stage, there was a significant reduction of the percentage of total cycling cells in Adx fetuses presumably due to the smaller proportion of immature CD8+
cells, the thymocyte population that exhibits the highest proliferative index during ontogeny,24,25 found in these animals.
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| Fig 4.
(A, B) Expression of CD4 (up) and TcR (down) versus
CD8 (horizontal axis) in thymocytes from 17-day old (A) and splenocytes
from 18-day old (B), Sham (left) and Adx (right) fetuses. Results shown
are representative of 3 to 4 experiments.
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The recovery of control GC levels prevents the accelerated T cell
maturation in Adx fetuses.
To ensure that the observed changes in the T-cell development of Adx
fetuses were caused by the absence of GCs rather than by the lack of
other biological mediators produced by maternal adrenal glands,
corticosterone replacement of Adx fetuses was performed using osmotic
minipumps (2ML4, Alzet). After infusion of 23 µg/hour of
corticosterone in a volume of 0.25 µL, the levels of hormone measured
by RIA in the serum of 16- and 17-day-old fetuses were around 70 ng/mL.
In these fetuses, both thymic cellularity and percentage of thymocyte
populations (Fig 3C) defined by the CD4/CD8/TcR expression were
normal, demonstrating the relevance of the absence of circulating GC
for the observed accelerated T-cell maturation in Adx fetuses.
The GC absence affects fetal liver thymic precursors.
To investigate whether the acceleration of T-cell maturation in vivo
observed in Adx fetuses was due to an effect of GC absence on the early
progenitors, in vitro recolonization assays were performed.
dGuo-pretreated thymic lobes from 15-day-old fetal mice were
reconstituted with fetal liver cells either from 13-day-old Sham, and
Adx fetuses. Previously, flow cytometry analysis showed similar numbers
of CD45+(OX1+) cells in the fetal liver of both
groups of rats (data not shown). After 12 days of culture, more than
90% of yielded cells were rat thymocytes (OX1+) without
significant differences between the cultures supplied with fetal liver
cells from control fetuses and those receiving fetal liver precursors
from Adx fetal rats. However, there was a significantly higher
proportion of TcRhi cells (around 3-fold) in the
thymic lobes reconstituted with fetal liver Adx precursors (RL-Adx)
than in those reconstituted with Sham ones (RL-Sham)
(Table 2,
Fig 5). Furthermore, this increased
proportion of TcRhi cells found in the RL-Adx
cultures included mature SP (both CD4 and CD8) cells, but also DP
TcRhi thymocytes. To determine the origin of the
increase of TcRhi thymocytes yielded in RL-Adx, we
examined the proportion of cycling cells within this cell subset. Flow
cytometry analysis of DNA cell content and TcR expression showed
that there were no significant differences between cycling cells among
TcRhi thymocytes from RL-Adx and RL-Sham (Table 2).
These results suggested that the higher numbers of mature thymocytes
are not generated by expansion of preexisting cells, but presumably as a consequence of their accelerated production in the RL-Adx cultures.
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| Fig 5.
The GC absence affects fetal liver thymic precursors.
CD4/CD8/TcR expression in thymocytes generated in dGuo-treated
murine thymic lobes after 12 days of in vitro recolonization with fetal
liver cells from either 13-day-old control Sham (up) or Adx (down) rat
fetuses. Histograms represent TcR expression in the gated DN
(CD4CD8),
DP(CD4+CD8+), and SP
(CD4CD8+ and
CD4+CD8) thymic cell populations shown in
the dot plots. Results shown are a representative experiment with cell
recoveries from RL-Sham of 85,000 cells/lobe and of 90,000 cells/lobe
in the case of RL-Adx.
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In summary, the recolonization assays confirm a significant accelerated
maturation of T-cell progenitors from Adx fetal liver, suggesting that
T-cell precursors were affected by the absence of GC before their
arrival at the early thymic primordium.
Early colonization of thymic primordium in Adx fetuses.
As another possible cause accounting for the acceleration of T-cell
maturation, the time of colonization of the early thymic primordium by
lymphoid cells was examined comparatively in Adx and Sham fetuses. The
ultrastructural results demonstrated that the thymic primordium of Adx
fetuses was colonized earlier by lymphoid progenitors and
developed faster than that of control, Sham fetal rats. At day 13 of
gestation, the thymic primordium of control, Sham fetuses, consisted of
homogeneous primitive epithelial cells, as previously
reported,24 many of which were in the process of division
(Fig 6a), which began to establish a
continuous supporting meshwork through incipient cell-to-cell contacts
(Fig 6c), although wide intercellular spaces were still present (Fig
6a). On the contrary, the 13-day old Adx fetal thymus was largely
invaded by lymphoid progenitors and contained numerous, more
differentiated epithelial cells (Fig 6b). Invading lymphoid cells were
round, electron-dense elements with patent nucleoli, a few round,
electron lucent mitochondria, and numerous polyribosomes. Thymic
epithelial cells were irregular elements joined together by incipient
cell junctions (Fig 6d), which seemed to represent 2 distinct cell populations (Fig 6e). The most abundant epithelial cell type was an
electron dense cell type, which contained long profiles of rough
endoplasmic reticulum, numerous mitochondria, and occasional lipid
droplets. In addition, a few irregular, electron lucent epithelial
cells, containing a poor developed endoplasmic reticulum, an incipient
Golgi complex, and some mitochondria occurred in the thymic primordium
of 13-day-old Adx embryos.
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| Fig 6.
(a) Round or polygonal primitive epithelial cells
(arrows), some of them in division (m), constitute the homogeneous
thymic stroma devoid of lymphoid cells of a 13-day-old Sham fetal rat.
Note the lack in these cells of prominent cytoplasmic organelles,
except for a few, elongated mitochondria (arrow heads), and the
enlarged intercellular spaces. Original magnification × 3,700. (b)
Thymic primordium of a 13-day old Adx fetal rat. Numerous lymphoid
progenitors (L) occur between a well constituted network of irregular
thymic epithelial cells (TEC). Nucleolus (stars). Original
magnification × 3,700. (c) Incipient cell-to-cell contact (arrows)
between 2 primitive thymic epithelial cells of a 13-day- old Sham fetal
rats. Mitochondria (Mt). Original magnification × 56,000. (d)
Incipient cell junction (arrows) between 2 thymic epithelial cells of a
13-day-old Adx fetal thymus. Note the increased amount of cisternae of
rough endoplasmic reticulum (RER) and mitochondria (Mt) as compared
with the condition of Sham fetal rats of the same age (c). Original
magnification × 35,700. (e) Electron-dense (DEC) and electron-lucent
thymic epithelial cells (LEC), 13-day old Adx fetal thymus, rough
endoplasmic reticulum (RER), lipid droplets (Li), mitochondria
(arrows), Golgi complex (G), and incipient cell junctions (arrow
heads). Original magnification × 6,000.
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DISCUSSION |
GC function is mediated via specific receptors located in the cytoplasm
of target cells. In the present work, we show that 13-day-old rat
CD45+ fetal liver cells, a population of cell progenitors
that contains thymic precursors,20-23 express GRs and
that this expression is maintained in 15-day-old fetal
thymocytes. In agreement, Ranelletti et al26 demonstrated
that human intrathymic precursors
(CD3CD1 thymocytes) contain
higher levels of GR sites per cell compared with the more mature CD3
positive population. More recently, an in situ hybridization study
demonstrated that rat fetal liver contains GR mRNA as early as day 12 of fetal development when the hematopoietic activity of the organ
begins.2 Moreover, GR-specific gene expression was detected
by these investigators in rat thymic primordium in 13-day-old fetuses,
which increased on day 15. Although recently some studies have analyzed
the effects of lack of GR signaling on thymus
function,1,7,15,27 these are largely devoted to the GC
involvement in thymocyte selection and the physiologic relevance of GR
expression in early thymic precursors has not been investigated.
The analysis of thymocyte ontogeny in the progeny of adrenalectomized
pregnant rats allows an in vivo approach to this problem in which
impairment of GR signaling is induced without blocking the expression
of GR gene, but by eliminating the source of the hormone from the first
day of fetal development. Vacchio et al28 demonstrated that
a fraction of mouse thymic epithelial cells could produce in vitro
pregnelone and deoxicorticosterone that increases under the influence
of adrenocorticotropic hormone (ACTH). Accordingly, this endogenous
production could influence locally the thymocyte development after cell
progenitors colonize the thymic primordia, but not before. Because an
endogenouus production of GCs has not been demonstrated in fetal liver,
where thymic cell progenitors are generated, we could conclude that
maternal GCs are the only source of hormone for fetal liver cell
progenitors until the establishment of the HPA axis3,4 and
its absence should be, therefore, responsible for the changes observed
in the early development of thymic precursors of Adx fetuses. In fact,
as shown in the current study, adrenalectomized pregnant rats provided
with osmotic minipumps, which regularly release corticosterone, permit
recovery of GC levels in early Adx fetuses and the normal development
of thymocytes.
In mammalian fetuses, the thymic primordium is initially colonized by
T-cell precursors from fetal liver, which rearrange the TcR genes to
produce an initial repertoire that is rigorously selected resulting in
T-cell effectiveness and tolerance.29 In control rats,
thymic primordium is colonized between 13 and 14 days of fetal life and
the first DP (CD4+CD8+) cells appear on day 18. However, until day 20, mature T cells are not detected in the thymus
and 1 day later in periphery.24 In the progeny of Adx rats,
this chronology is, however, profoundly altered. As shown in our
electron microscopy study, as early as day 13 of gestation, the thymic
primordia is already colonized by lymphoid progenitors, and the thymic
epithelial cells have begun to differentiate, whereas in control
fetuses of the same age, an alymphoid thymic stroma consists of an
homogeneous mass of primitive epithelial cells. This is followed by the
appearance of DP thymocytes on day 15 of fetal life and 1 day later of
mature SP, both CD4 and CD8, cells that colonize the spleen on day 18. Furthermore, in vitro recolonization assays demonstrate an altered behavior of thymic precursors occurring in the 13-day- old fetal liver
which, together with the above-mentioned early colonization of thymic
primordium, accounts for the faster development observed in the Adx
fetuses. In agreement with these results, Castellanos et
al30 observed a faster recovery of thymic cellularity after irradiation in adrenalectomized compared with control adult rats.
On the other hand, the involvement of GCs in these processes is clearly
demonstrated by the total recovery of normal T-cell differentiation in
Adx fetuses in vivo receiving corticosterone. Moreover, after the
establishment of fetal HPA axis, the thymocyte differentiation is
progressively normalized, although the new wave of cell progenitors,
which colonizes the thymus of Adx fetuses on day 17, exhibits
accelerated maturation (Sacedón et al, manuscript in preparation).
As mentioned above, the role of GCs in the development of T-cell
precursors has been little studied and the results obtained are
incomplete and contradictory. Targeted disruption of the GR gene, which
blocks the chromaffin tissue development and severely retards lung
maturation, does not seem to affect, however, the thymus histology,
although newborn and adult thymocytes are totally resistant to
dexamethasone-induced apoptosis.1 Recently, this same group
has generated mice carrying a dimerization-defective GR.7
The mutants lack inductibility of GC response elements (GRE) and show
impairment of several important physiological functions, including
GC-mediated thymocyte apoptosis. Surprisingly, despite this impairment
of thymocyte apoptosis, a process associated with intrathymic T-cell
selection, they were unable to detect any difference in the relative
abundance of distinct thymocyte subsets defined by the CD4/CD8 cell
profiles. Investigators recognize, however, that these are preliminary
results, which need further confirmation, and in both studies, the
ontogenetical development has not been analyzed. On the contrary, King
et al15 found that the partial blockage of GR expression in
the thymus of transgenic mice expressing an antisense RNA for the GR
under the control of lck proximal promoter, specifically expressed in
thymus, triggered a drastic increase of apoptosis. This elevated cell
death produced an important reduction of thymic cellularity from day 16 of fetal development, mainly due to a reduction of DP population, and a
partial impairment of the normal progression of
CD4CD8 cells to the
CD4+CD8+ compartment. There are, however,
important differences between this experimental model and that used in
the current study. In the transgenic mice, the expression of antisense
transcripts was specifically targeted at immature thymocytes using the
lck proximal promoter. In this respect, during days 14 and 15 of fetal
development, when early
CD3CD4CD8
thymocyte precursors occupy the thymus, the cell recovery from control,
nontransgenic and transgenic mice was remarkably
identical.15 On the contrary, our recolonization assays
conclusively demonstrates that cell precursors are affected by the lack
of circulating GCs before their arrival to the thymic primordium. In
this regard, decreased numbers of the cell progenitors, which colonize
the thymic anlagen, could be also contributing to the lower numbers of
thymocytes observed in the Adx thymuses. Morale et al27
explained, in agreement with the current results, changes in thymic
cellularity and cell proliferation observed in the thymus of transgenic
mice with a blockade of GR gene by alterations in the arrival of cell precursors into the thymus and/or their intrathymic
proliferation.27 Furthermore, the in vitro migration to the
thymic supernatants of bone marrow cell progenitors obtained from
dexamethasone or hydrocortisone long-term treated mice was
significantly increased.31 In both 16- and 17-day-old Adx
thymuses, there are also increased percentages of apoptotic cells,
which could account for reduced thymic cellularity observed in these
animals. In agreement, King et al15 reported an increase of
apoptotic thymocytes in the absence of GCs. Nevertheless, because, as
mentioned above, thymic epithelial cells are able to endogenously
produce GCs,28 it is possible to speculate that high levels
of intrathymic cell death observed in the Adx fetuses could be due to
an increased local production of GCs.
The present study provides, therefore, new information about the
crucial role of GCs on T-cell differentiation. GCs not only regulate
intrathymic T-cell maturation, but also exert a function on fetal liver
cell precursors before thymic colonization and TcR expression.
| |
ACKNOWLEDGMENT |
The technical assistance of Alfonso Cortés and Catalina Escribano
is greatly appreciated. We also thank the Centro Común de
Investigación of the Faculty of Biology of UCM for the use of the facilities.
| |
FOOTNOTES |
Submitted March 23, 1999; accepted June 15, 1999.
Supported by Grant No. PR181/96-6824 from the Complutense University of
Madrid, Grants No. PB94-0332 and PB97-0332 from the Spanish Ministry of
Education and Culture, Grant No. 98/0041 from the Fondo de
Investigaciones Sanitarias (FIS), and Grant No. 08/30014/1997 from the
Comunidad de Madrid. R.S., J.J.M., and E.J. are recipients of a
fellowship from the Spanish Ministry of Education and Culture.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Agustín G. Zapata,
PhD, Department of Cell Biology, Faculty of Biology,
Complutense University, 28040 Madrid, Spain; e-mail:
zapata{at}eucmax.sim.ucm.es.
| |
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