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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2819-2826
By
From the Department of Cell Biology, Faculty of Biology and Medicine,
Complutense University, Madrid, Spain.
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
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
(CD4 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 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
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) 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).
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.
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).
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
CD4
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 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 TcR
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.
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
(CD3 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.
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.
1.
Cole TJ, Blendy JA, Monaghan AP, Krieglstein K, Schmid W, Aguzzi A, Fantuzzi G, Hummler E, Unsicker K, Schutz G:
Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation.
Genes Dev
9:1608, 1995
2.
Kitraki E, Kittas C, Stylianopoulou F:
Glucocorticoid receptor gene expression during rat embryogenesis. An in situ hybridization study.
Differentiation
62:21, 1997[Medline]
[Order article via Infotrieve]
3.
Dupouy JP, Coffigny H, Magre S:
Maternal and foetal corticosterone levels during late pregnancy in rats.
J Endocrinol
65:347, 1975
4.
Milkovic S, Milkovic K, Paunovic J:
The initiation of fetal adrenocorticotrophic activity in the rat.
Endocrinology
92:380, 1973[Medline]
[Order article via Infotrieve]
5.
Muglia L, Jacobson L, Dikkes P, Majzoub JA:
Corticotropin-releasing hormone deficiency reveals major fetal but not adult glucocorticoid need.
Nature
373:427, 1995[Medline]
[Order article via Infotrieve]
6.
Wessely O, Deiner EM, Beug H, von Lindern M:
The glucocorticoid receptor is a key regulator of the decision between self-renewal and differentiation in erythroid progenitors.
EMBO J
16:267, 1997[Medline]
[Order article via Infotrieve]
7.
Reichardt HM, Kaestner KH, Tuckermann J, Kretz O, Wessely O, Bock R, Gass P, Schmid W, Herrlich P, Angel P, Schutz G:
DNA binding of the glucocorticoid receptor is not essential for survival.
Cell
93:531, 1998[Medline]
[Order article via Infotrieve]
8.
Garvy BA, King LE, Telford WG, Morford LA, Fraker PJ:
Chronic elevation of plasma corticosterone causes reductions in the number of cycling cells of the B lineage in murine bone marrow and induces apoptosis.
Immunology
80:587, 1993[Medline]
[Order article via Infotrieve]
9.
Shezen E, Shirman M, Goldman R:
Opposing effects of dexamethasone on the clonal growth of granulocyte and macrophage progenitor cells and on the phagocytic capability of mononuclear phagocytes at different stages of differentiation.
J Cell Physiol
124:545, 1985[Medline]
[Order article via Infotrieve]
10.
Kitajima T, Ariizumi K, Bergstresser PR, Takashima A:
A novel mechanism of glucocorticoid-induced immune suppression: The inhibiton of T cell-mediated terminal maturation of a murine dendritic cell line.
J Clin Invest
98:142, 1996[Medline]
[Order article via Infotrieve]
11.
Vacchio MS, Ashwell JD, King LB:
A positive role for thymus-derived steroids in formation of the T-cell repertoire.
Ann N Y Acad Sci
840:317, 1998[Medline]
[Order article via Infotrieve]
12.
Jondal M, Okret S, McConkey D:
Killing of immature CD4+ CD8+ thymocytes in vivo by anti-CD3 or 5'-(N-ethyl)-carboxamide adenosine is blocked by glucocorticoid receptor antagonist RU-486 [published erratum appears in Eur J Immunol 23:2734, 1993].
Eur J Immunol
23:1246, 1993[Medline]
[Order article via Infotrieve]
13.
Iwata M, Hanaoka S, Sato K:
Rescue of thymocytes and T cell hybridomas from glucocorticoid-induced apoptosis by stimulation via the T cell receptor/CD3 complex: A possible in vitro model for positive selection of the T cell repertoire.
Eur J Immunol
21:643, 1991[Medline]
[Order article via Infotrieve]
14.
Zacharchuk CM, Mercep M, Chakraborti PK, Simons SS Jr, Ashwell JD:
Programmed T lymphocyte death. Cell activation- and steroid-induced pathways are mutually antagonistic.
J Immunol
145:4037, 1990[Abstract]
15.
King LB, Vacchio MS, Dixon K, Hunziker R, Margulies DH, Ashwell JD:
A targeted glucocorticoid receptor antisense transgene increases thymocyte apoptosis and alters thymocyte development.
Immunity
3:647, 1995[Medline]
[Order article via Infotrieve]
16.
Sacedón R, Vicente A, Varas A, Jiménez E, Muñoz J, Zapata AG:
Early differentiation of thymic dendritic cells in the absence of glucocorticoids.
J Neuroimmunol
94:103, 1999[Medline]
[Order article via Infotrieve]
17.
Sacedón R, Varas A, Jiménez E, JJ Muñoz, Vicente A, Zapata AG:
Accelerated maturation of the thymic stroma in the progeny of adrenalectomized pregnant rats.
Neuroimmunomodulation
6:23, 1999[Medline]
[Order article via Infotrieve]
18.
Jenkinson EJ, Franchi LL, Kingston R, Owen JJ:
Effect of deoxyguanosine on lymphopoiesis in the developing thymus rudiment in vitro: Application in the production of chimeric thymus rudiments.
Eur J Immunol
12:583, 1982[Medline]
[Order article via Infotrieve]
19.
Kingston R, Jenkinson EJ, Owen JJ:
A single stem cell can recolonize an embryonic thymus, producing phenotypically distinct T-cell populations.
Nature
317:811, 1985[Medline]
[Order article via Infotrieve]
20.
McCarthy KF, Hale ML, Fehnel PL:
Rat colony-forming unit spleen is OX7 positive, W3/13 positive, OX1 positive, and OX22 negative.
Exp Hematol
13:847, 1985[Medline]
[Order article via Infotrieve]
21.
Poggi A, Sargiacomo M, Biassoni R, Pella N, Sivori S, Revello V, Costa P, Valtieri M, Russo G, Mingari MC, Peschel C, Moretta L:
Extrathymic differentiation of T lymphocytes and natural killer cells from human embryonic liver precursors.
Proc Natl Acad Sci USA
90:4465, 1993
22.
Spangrude GJ, Klein J, Heimfeld S, Aihara Y, Weissman IL:
Two monoclonal antibodies identify thymic-repopulating cells in mouse bone marrow.
J Immunol
142:425, 1989[Abstract]
23.
Alonso C, L-M, Vicente A, Varas A, Zapata AG:
Development of rat CD45 13 day-old fetal liver cells in SCID-mouse fetal thymic organ cultures.
Int Immunol
11:1119, 1999
24.
Vicente A, Varas A, Sacedon R, Jimenez E, Munoz JJ, Zapata AG:
Appearance and maturation of T-cell subsets during rat thymus ontogeny.
Dev Immunol
5:319, 1998[Medline]
[Order article via Infotrieve]
25.
Penit C, Vasseur F:
Cell proliferation and differentiation in the fetal and early postnatal mouse thymus.
J Immunol
142:3369, 1989[Abstract]
26.
Ranelletti FO, Maggiano N, Aiello FB, Carbone A, Larocca LM, Musiani P, Piantelli M:
Glucocorticoid receptors and corticosensitivity of human thymocytes at discrete stages of intrathymic differentiation.
J Immunol
138:440, 1987[Abstract]
27.
Morale MC, Batticane N, Gallo F, Barden N, Marchetti B:
Disruption of hypothalamic-pituitary-adrenocortical system in transgenic mice expressing type II glucocorticoid receptor antisense ribonucleic acid permanently impairs T cell function: Effects on T cell trafficking and T cell responsiveness during postnatal development.
Endocrinology
136:3949, 1995[Abstract]
28.
Vacchio MS, Papadopoulos V, Ashwell JD:
Steroid production in the thymus: Implications for thymocyte selection.
J Exp Med
179:1835, 1994
29.
Spits H:
Early stages in human and mouse T-cell development.
Curr Opin Immunol
6:212, 1994[Medline]
[Order article via Infotrieve] |
TOP
ABSTRACT
INTRODUCTION
CD8
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.
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.
.05; ** P 
