Download Local versus systemic control of numbers of endometrial T cells

Immunology 2001 102 317±322
Local versus systemic control of numbers of endometrial T cells
during pregnancy in sheep
A. C. MAJEWSKI, S. TEKIN & P. J. HANSEN Department of Animal Sciences, University of Florida,
Gainesville,USA
SUMMARY
Pregnancy in sheep is associated with changes in numbers of speci®c T-lymphocyte populations in
the uterine endometrium. These changes probably contribute to evasion by the conceptus of
maternal immunological rejection and indicate a possible role for T cells in placental growth,
parturition and post-parturient uterine defence against infection. The purpose of the present
experiment was to evaluate the relative importance of systemic signals (i.e. those present throughout
the uterus or from the circulation, including conceptus hormones secreted into the maternal blood)
versus locally acting conceptus signals for regulating changes in numbers of endometrial
lymphocytes during pregnancy. The approach taken was to surgically con®ne pregnancy to one
uterine horn and compare differences in lymphocyte numbers between the two uterine horns as
well as between both horns of pregnant ewes with those of ovariectomized ewes. As compared with
ovariectomized ewes, there was a decline in numbers of CD45R+ lymphocytes within glandular
epithelium and an increase in cd T-cell number within the luminal epithelium. These changes
occurred in both the pregnant and non-pregnant uterine horns of unilaterally pregnant ewes.
Moreover, there were no signi®cant differences in lymphocyte numbers between the two uterine
horns of unilaterally pregnant ewes. Expression of CD25 was absent in tissues from both uterine
horns. In conclusion, changes in numbers of endometrial lymphocytes during pregnancy, rather
than due to locally acting signals of conceptus origin, are the result of hormonal signals of maternal
or conceptus origin that either act directly on endometrial lymphocytes or stimulate the uterine
endometrium to induce synthesis of regulatory molecules that affect lymphocyte dynamics.
INTRODUCTION
favours conceptus survival. In mice, for example, abortion can
be increased by administration of anti-CD8 early in pregnancy6
and cd T-cell subsets that produce transforming growth factorb (TGF-b) have been identi®ed in the mouse uterus that can
suppress other lymphocytes.7,8 Also, NK cells have been
implicated in the regulation of murine placenta growth.9
In sheep, there are pregnancy-associated changes in
endometrial lymphocyte numbers that suggest differential
regulation of speci®c lymphocyte populations by conceptus
and maternal factors. The endometrial portion of the
placentome, which is formed by the fusion of maternal
caruncles and the fetal cotyledons, is nearly devoid of
lymphocytes. Rather, lymphocytes are con®ned primarily to
epithelial areas of the interplacentomal endometrium.10
Approximately half of all lymphocytes from the luminal
epithelium of non-pregnant ewes are CD8+ CD45R± cd±. The
other lymphocytes are roughly equally divided into a population of CD8+ CD45R+ cd± cells and a population of
CD8+ CD45R+ cd+ T cells.11 During pregnancy, the number
of non-granulated T cells decreases within endometrial
glands, while numbers in the epithelium lining the lumen of
the uterus initially decline and then become similar to values
The uterus contains populations of T cells suf®cient to induce
graft rejection.1,2 Proper adjustments in uterine lymphocyte
function to inhibit anti-fetal responses are therefore critical for
pregnancy success; disruption of this regulation can lead to
pregnancy loss.3 That certain lymphocyte populations are
involved in anti-fetal responses is indicated by passive
immunization experiments. For example, administration of
antibody to asialoGM1 reduces abortion in the CBA/JrDBA/
2 mouse abortion model4 and following activation of natural
killer (NK) cells by injection of poly(I).poly(C).5 Some
lymphocyte populations, in contrast, may play a role that
Received 12 April 2000; revised 23 October 2000; accepted 6
November.
Abbreviations: NK, natural killer cells; NPH, non-pregnant horn;
OvUS, ovine uterine serpin; PH, pregnant horn; TGF-b, transforming
growth factor-b.
Correspondence: Dr P. J. Hansen, PO Box 110920, University of
Florida, Gainesville, FL 32611±0920, USA.
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2001 Blackwell Science Ltd
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318
A. C. Majewski et al.
for non-pregnant ewes.12 Beginning in mid-pregnancy, in
contrast, there is a large increase in the number and degree of
granulation of a population of granulated lymphocytes in the
lumen.11,12 These granulated T cells have been identi®ed as cd
T cells.11 Given the roles of cd T cells in regulation of cell
growth13 and immunoglobulin A (IgA) secretion,14 these cells
may promote fetal or placental development or contribute
to anti-microbial defence postpartum. Recently, it has been
reported that endometrial cd T cells express perforin;15 perhaps
they are cytotoxic and contribute to placental detachment from
the endometrium during parturition.
Evidence suggests that these changes in lymphocyte
numbers are regulated by progesterone and local conceptus
signals. Treatment of ovariectomized ewes with progesterone
decreased numbers of CD45R+ lymphocytes in glandular
and luminal epithelium.16 In addition, progesterone induces
endometrial secretion of ovine uterine serpin (OvUS),17 a
protein that can inhibit lymphocyte proliferation and natural
killer activity and can block immune responses in vivo.18
Other immunosuppressants produced in sheep include prostaglandin E2 (PGE2) from uterus and placenta,19 partially
identi®ed glycoproteins from placenta20 and TGF-b from the
endometrium.21
The fact that cd T cells increase in number during
pregnancy despite the presence of several molecules with antiproliferative activity may re¯ect their resistance to inhibition by
these regulators. For example, OvUS inhibited concanavalin
A-induced up-regulation of CD25 expression on cd± T cells but
not on cd+ T cells.22 In addition, endometrial cd T cells may be
activated by some locally acting signal from the conceptus.
Using a unilaterally pregnant model, in which the conceptus
was restricted to one uterine horn, there was an increased
expression of activation antigens such as CD25 on cd T cells
from the endometrium ipsilateral to the conceptus.23 This
®nding, which is consistent with data in humans that the
placenta can activate cd T cells,24 has been called into question
by recent ®ndings that CD25 expression was absent from
endometrial lymphocytes.25
The purpose of the present experiment was to evaluate the
relative importance of systemic signals (i.e. those present
throughout the uterus or from the circulation, including
conceptus hormones secreted into the maternal blood) versus
locally acting conceptus signals for regulating changes in
lymphocyte numbers in the endometrium during pregnancy.
The approach taken was to con®ne pregnancy surgically to one
uterine horn and compare differences in lymphocyte number
between the two uterine horns (where only the horn ipsilateral
to the conceptus would be exposed to locally acting conceptus
factors) and between both horns of the unilaterally pregnant
ewes with uteri of ovariectomized ewes (where both horns of
pregnant animals would be exposed to changes in circulating
hormones of pregnancy as well as uterine-derived factors
induced by those hormones while the uterus of ovariectomized
animals would be exposed to neither).
MATERIALS AND METHODS
Materials
Hybridoma cells producing antibodies to CD8 (7C2), CD45R
(20±96) and cd T-cell receptor (TCR) (86D) were obtained
from the European Collection of Animal Cell Culture
(Salisbury, UK) and were maintained at the Hybridoma Core
Laboratory, University of Florida Interdisciplinary Center for
Biotechnology Research. Antibody to CD8 was puri®ed using
a Hi-Trap Protein G Sepharose column (Pharmacia & Upjohn,
Kalamazoo, MI). The antibodies towards CD45R and cd
T cells were used as ascites ¯uid. Monoclonal antibody towards
CD25 (clone 155.2) was obtained as an ascites ¯uid from the
Centre for Animal Biotechnology, University of Melbourne,
Parkville, Victoria, Australia. The immunohistochemistry kit
(HistoScan Universal Monoclonal Detector Kit) was obtained
from Biomeda (Foster City, CA), Tissue-Tek OCT Compound
was from Miles Diagnostic (Elkhart, IN) and mouse IgG1,
control mouse ascites ¯uid (clone NS-1) and normal goat serum
were from Sigma (St Louis, MO). Other reagents were from
Sigma or Fisher Scienti®c (Pittsburgh, PA).
Animals
Rambouillet-type ewes, maintained on a diet of Bermuda hay
ad libitum, were used for the experiment. Six ewes were
unilaterally ovariectomized via midventral laparotomy and a
single uterine ligation was made on the side ipsilateral to the
side of the ovariectomy.26 After approximately 3 weeks, ewes
were bred to a fertile ram at the next oestrus. Pregnant ewes
were slaughtered via captive-bolt stunning and exsanguination
at day 140 of pregnancy and reproductive tracts were removed.
Four additional ewes were bilaterally ovariectomized and a
uterine ligation placed around each uterine horn. Ewes were
slaughtered 8 months after ovariectomy. These animals served
as controls that were not exposed to any ovarian steroids that
might contribute to systemic effects of pregnancy on endometrial lymphocyte number. Ligations were placed around the
uterus of ovariectomized animals to parallel the presence of
a ligation in pregnant animals.
Tissue collection and embedding
Tissue samples were excised from randomly chosen regions of
the intercaruncular endometrium of all ewes. For pregnant
ewes, tissues were collected from the ligated, non-pregnant
horn (NPH) and from the pregnant horn (PH). Tissue samples
(y3 mm3) were frozen in aluminium boats (1 cm3) containing
OCT freezing medium using isopentane cooled with liquid
nitrogen. Tissue blocks were stored at x70u until sectioning
was performed.
Histochemistry
Immunohistochemistry was performed on tissues from two
separate blocks from each uterine horn (pregnant ewes) or
uterus (ovariectomized ewes). Frozen blocks of tissue were
sectioned on a cryostat at 5-mm thickness. Sections were placed
on polylysine-coated slides and ®xed with 95% ethanol.
Sections were air dried onto the slide and then rehydrated
with phosphate-buffered saline (PBS) containing 1% (w/v)
normal goat serum (PBSG) and kept at 4u until processed. All
histochemical steps were performed at room temperature and
slides were rinsed with PBSG and blotted dry between each
step. Slides were treated with PBSG, pH 7.4 containing 0.6%
(w/v) H2O2 for 3 min to eliminate endogenous peroxides.
Tissues were then treated with conditioner from the Biomeda
kit for 5 min. Sections were incubated for 6 hr with antibody to
CD8 (6 mg/ml), CD45R (1 : 800), cd T cells (1 : 300) or CD25
(1 : 500) diluted in PBS. Mouse IgG1 (5 mg/ml) and control
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2001 Blackwell Science Ltd, Immunology, 102, 317±322
Changes in endometrial lymphocytes during pregnancy
mouse ascites ¯uid (1 : 300) were used as negative controls.
Other steps, including incubation with anti-mouse IgG linked
to biotin (30 min), streptavidin-peroxidase (30 min) and
3-amino-9-ethyl-carbazole (10 min) were conducted using
reagents supplied in the kit. Slides were rinsed with deionized
water for several minutes and carefully blotted before applying
mounting medium and coverslips.
Quanti®cation of positive cells
Slides were observed at r400 magni®cation in a bright-®eld
microscope containing a graticule in one eyepiece. The number
of positive cells was counted in three tissue types: luminal
epithelium, glandular epithelium and the stroma. For each
tissue type, three randomly selected sites were chosen for
morphometric analysis. The number of positive cells in 30
squares of the graticule (each square having a surface area of
625 mm2) were counted. For epithelium, squares were chosen
for counting so as to ensure that the entire area of the squares
was within the epithelium. For stroma, squares were chosen for
counting so as to evaluate a cross-sectional area extending from
the lumen to the deep stroma.
Statistical analysis
Data were analysed by least square analysis of variance using
the General Linear Models procedure of SAS.27 For the
analysis, each uterine horn of the pregnant ewes was considered
as an independent experimental unit. Data were analysed
separately for each of three sites (luminal epithelium, glandular
epithelium and stroma). The mathematical model included
effects of treatment (ovariectomized; NPH of pregnant ewes;
PH of pregnant ewes) and error. Orthogonal contrasts were
used to partition variance due to the overall effect of pregnancy
(ovariectomized versus NPH+PH) and the effect of local
presence of the conceptus (NPH vs. PH).
RESULTS
Distribution and appearance of lymphocytes
For CD45R, cd T cells and CD8, positive cells were located
predominantly in the luminal and glandular epithelium (Fig. 1)
and only a few scattered lymphocytes were present in the
stroma. There were no cells positive for CD25 detected in
endometrium although the antibody did react with some cells
in the spleen and a proportion of mitogen-activated peripheral
blood lymphocytes (results not shown).
Pregnancy was associated with an increase in the thickening
of the epithelial lining of the endometrial glands and lumen
(compare Fig. 1e with Fig. 1f and Fig. 1g with Fig. 1h) but the
distribution of lymphocytes was unaffected by pregnancy
status. In general, the appearance of positive-staining cells was
similar for all tissues except that intraepithelial CD45R+ cells
from ovariectomized ewes stained less intensely with the
reaction product than did cells from either the PH or NPH of
pregnant ewes (Fig. 1e versus Figure 1f). Except for CD45R+
cells from ovariectomized ewes, staining intensity of CD8+
cells was lower than for the other two markers.
Local and systemic effects on numbers of lymphocytes
Data are shown in Fig. 2. The number of CD45R+
lymphocytes in luminal epithelium did not differ between the
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2001 Blackwell Science Ltd, Immunology, 102, 317±322
319
three uterine types. In contrast, there was a reduction
(P<0.0001) in numbers of CD45R+ cells in the glandular
epithelium of both uterine horns from pregnant ewes as
compared to ovariectomized ewes. Values were 1.69t0.15
cells/625 mm2 for ovariectomized ewes versus 0.26t0.12 cells/
625 mm2 for PH and 0.46t0.12 cells/625 mm2 for NPH. The
difference between PH and NPH was not signi®cant.
Numbers of cd T cells in the luminal epithelium were higher
(P=0.05) for both horns of pregnant ewes as compared to the
ovariectomized ewes and there was no difference between PH
and NPH. Numbers of cells were 0.28t0.15 cells/625 mm2 for
ovariectomized ewes, 0.70t0.13 cells/625 mm2 for PH and
0.62t0.13 cells/625 mm2 for NPH. Numbers of cd T cells
in the glandular epithelium were unaffected by uterine type.
Although there was a tendency for numbers of CD8+ cells
in luminal epithelium to be higher in pregnant ewes, there
was no signi®cant effect of uterine type on numbers of CD8+
cells in either luminal or glandular epithelium.
DISCUSSION
As compared to ovariectomized ewes, the pregnancy-associated decrease in CD45R+ cells in glandular epithelium and
increase in luminal cd T cells mirror earlier observations in
pregnant ewes.12 Present results indicate that these changes,
rather than due to locally acting conceptus factors, are instead
the result of hormonal signals of maternal or fetal origin that
either act directly on endometrial lymphocytes or stimulate the
uterine endometrium to induce synthesis of regulatory
molecules that affect lymphocyte dynamics. This is shown by
the fact that pregnancy-associated changes in lymphocyte
populations occurred in both the pregnant and non-pregnant
uterine horn of pregnant ewes and there were no differences in
numbers of lymphocytes between the pregnant and nonpregnant horns.
The pregnancy-associated decrease in intraepithelial
CD45R+ cells that occurred in both uterine horns may be
largely the result of the immunosuppressive actions of
progesterone. Uterine ¯uid is rich in progesterone-induced
immunosuppressive substances28 and progesterone treatment
of ovariectomized ewes decreased numbers of CD45R+ cells in
glandular and luminal epithelium.16 Chief among these
immunosuppressive molecules is OvUS, which can inhibit a
variety of T-cell- and NK-cell-mediated phenomena.18 In
addition, PGE2 and TGF-b, both of which can inhibit
lymphocyte proliferation in sheep,29,30 are produced by the
endometrium of pregnant ewes.19,21 The failure of a decrease in
CD45R+ cells in the luminal epithelium, despite the fact that
progesterone treatment reduced numbers of CD45R+ cells in
both glandular and luminal epithelium,16 probably re¯ects the
increase in cd T cells in the lumen, since these cells are
CD45R+.11 It was also observed that the staining intensity of
CD45R+ cells was greater for pregnant ewes than ovariectomized ewes, suggesting that there is up-regulation of CD45R
expression on at least some populations of CD45R+ cells.
It was hypothesized that the increase in numbers of
cd T cells seen in the luminal epithelium duing mid- and
late-pregnancy12 represents stimulatory effects of local signals
from the conceptus because trophoblast can activate cd T cells
in humans24 and expression of CD25 and other activation
markers on intraepithelial cd T cells in luminal epithelium of
320
A. C. Majewski et al.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Figure 1. Immunohistochemical localization of T cells in the sheep endometrium. The ®rst three panels are low magni®cation images
that illustrate the distribution of cells staining for CD45R (a), cd (b) and CD8 (c). A section of endometrium in which antibody was
replaced by control mouse IgG1 is shown in (d). Panels (e)±(h) are higher magni®cation images to demonstrate effects of pregnancy on
numbers of CD45R+ cells in the glandular epithelium: (e) is from an ovariectomized ewe and (f) is from a pregnant ewe; and on
numbers of cd T cells in luminal epithelium: (g) is from an ovariectomized ewe and (h) is from a pregnant ewe.
unilaterally pregnant ewes was higher for cells from the
uterine horn ipsilateral to the conceptus.23 This hypothesis
is not supported by the data in two respects. First,
immunohistochemistry failed to identify cells in the endometrium that stained for CD25. This result is similar to another
recent report of lack of expression of CD25 on endometrial
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2001 Blackwell Science Ltd, Immunology, 102, 317±322
Changes in endometrial lymphocytes during pregnancy
2
CD45R
1·5
1
0·5
0
No. of positive T cells/625 µm2
1
cd
0·8
0·6
0·4
0·2
0
1
CD8
0·8
0·6
0·4
0·2
0
PH
NPH
Glandular
epithelium
OVX
PH
NPH
OVX
PH
NPH
OVX
Luminal
epithelium
Stroma
Figure 2. Numbers of endometrial lymphocytes. Data are least-squares
means t SEM for endometrial tissue collected from ovariectomized
ewes (OVX) and from the non-pregnant horn (NPH) and pregnant
horn (PH) of unilaterally pregnant ewes at day 140 of pregnancy.
cd T cells.25 Thus, the detection of CD25 on endometrial cd
T cells by Liu et al.23 may have represented up-regulation of
expression after cells had been harvested. The present data also
failed to support the hypothesis of local stimulation of
endometrial lymphocyte populations because there were more
luminal cd T cells in both the non-pregnant and pregnant
uterine horns of unilaterally pregnant ewes as compared
to endometrium from ovariectomized ewes. Moreover, the
number of cd T cells in the luminal epithelium was similar
for the pregnant and non-pregnant uterine horns.
Thus, as for CD45R+ cells, the signal for the increase in
luminal cd T-cell number is likely to be hormonally regulated.
Maternal or conceptus hormones could act directly on luminal
cd T cells or induce synthesis of locally acting endometrial
signals involved in cd T-cell growth such as interleukin-7 and
interleukin-15.31,32 Interestingly, the increase in cd T-cell
numbers seen in the luminal epithelium was not observed in
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2001 Blackwell Science Ltd, Immunology, 102, 317±322
321
glandular epithelium. This indicates that either the stimulatory
signal is absent in endometrial glands, the population of cd
T cells in the glandular epithelium are functionally distinct
from those in the lumen, or that other regulatory signals in the
glands block activation of cd T cells.
The absence of a difference between pregnant and
ovariectomized ewes in numbers of CD8+ cells may re¯ect
the fact that CD8 is expressed by both cd T cells and ab T cells11
and increases in some CD8+ subpopulations may be offset by
decreases in other subpopulations. In addition, some pregnancy-induced changes in CD8+ populations in endometrium
may not be apparent using immunohistochemistry because
only a portion of endometrial CD8+ cells appear to be detected
by immunohistochemistry. Indeed, although analysis by ¯ow
cytometry suggests that all CD45R+ and cd T cells express
CD8,11 the numbers of CD8+ cells determined by immunohistochemistry were less than for CD45R+ cells (present study;
see also refs. 10, 16). Although not signi®cant, there was a
tendency for CD8+ cells to increase in the luminal epithelium
in unilaterally pregnant ewes, particularly in the pregnant horn.
These increases may re¯ect changes in numbers of cd T cells
and, perhaps, activation of a subpopulation of CD8+ cells by
some local conceptus factor.
Changes in lymphocyte number in the endometrium could
represent regulation of lymphocyte proliferation, differentiation, migration, or apoptosis. Several uterine-derived molecules
that can inhibit lymphocyte proliferation have been described
in the sheep including OvUS,18 PGE219 and TGF-b.21 In
addition, suppressor cells in the ovine uterus that have
characteristics distinct from T cells33 are increased in number
by both oestradiol-17b and progesterone.34 The possibility that
endometrial lymphocyte dynamics involve changes in the rate
of differentiation in situ exists because, at least in the human,
lymphocyte differentiation markers (recombinase-activating
genes 1 and 2) are expressed in the endometrium.35 A recent
paper has demonstrated migration of lymphocytes into the
ovine uterine endometrium.36 The degree to which migration
occurs may be low, however, since there are few lymphocytes
resident in the endometrial stroma through which lymphocytes
migrating to and from the vasculature must traverse. Yet
another potential explanation for the pregnancy-associated
decrease in lymphocyte numbers (in particular the decrease in
CD45R+ cells in glandular epithelium) may be the hypertrophy of endometrial glands during pregnancy. The increase
in volume of the glands would result in a corresponding
reduction in the density of lymphocytes within the epithelium if
the rate of lymphocyte growth was lower than the rate of
growth of the endometrial epithelium.
In conclusion, the present experiment points out the
importance of hormonally derived signals for regulating
lymphocyte numbers during pregnancy. Further work should
focus on identi®cation of these endocrine signals, their origin
(fetal versus maternal) and of the local, endometrial cytokine
and growth factor networks through which they regulate
endometrial lymphocytes.
ACKNOWLEDGMENTS
The authors thank Fabiola Paula-Lopes, for technical advice on
histochemistry; the entire laboratory for assistance during surgery;
322
A. C. Majewski et al.
Gary Barcham, Els Meeusen and the Centre for Animal Biotechnology,
University of Melbourne, Parkville, Victoria, for donation of antiCD25; Dean Glicco for animal care; and the University of Florida
Interdisciplinary Center for Biotechnology Research for technical
support including Scott Whittaker and the Electron Microscopy
Laboratory for help with cryostat sectioning and computer imaging
and the Hybridoma Core Laboratory for production of antibodies.
Research was supported in part by USDA NRI 96±35203±3304 and a
grant from the Florida Milk Checkoff Program. This is Journal Series
Number R-07512 of the Florida Agricultural Experiment Station.
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