By
From the * Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph,
Guelph, Ontario, Canada N1G 2W1; and the Division of Immunology, Department of Medicine,
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
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Abstract |
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A large, transient population of natural killer (NK) cells appears in the murine uterine mesometrial triangle during pregnancy. Depletion of uterine (u) NK cells, recently achieved using
gene-ablated and transgenic mice, results in pathology. Pregnancies from matings of homozygous NK and T cell-deficient tg26 mice have <1% of normal uNK cell frequency, no development of an implantation site-associated metrial gland, and an edematous decidua with vascular pathology that includes abnormally high vessel walls/lumens ratios. Fetal loss of 64% occurs
midgestation and placentae are small. None of these features are seen in pregnant T cell-deficient mice. To confirm the role of the NK cell deficiency in these reproductive deficits, transplantation of tg
26 females was undertaken using bone marrow from B and T cell-deficient
scid/scid donors. Engrafted pregnant females have restoration of the uNK cell population, induced metrial gland differentiation, reduced anomalies in the decidua and decidual blood vessels, increased placental sizes, and restoration of fetal viability at all gestational days studied (days
10, 12, and 14). Thus, uNK cells appear to have critical functions in pregnancy that promote
decidual health, the appropriate vascularization of implantation sites, and placental size.
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Introduction |
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Large, heavily granulated lymphocytes (LGLs)1 are found in the pregnant uteri of many species, including mice, rats, pigs, and humans (1). Due to their dependency on estrogen and progesterone and their absence from the uteri of virgin and postpartum animals, these lymphocytes are expected, but have not yet been proven, to have important, pregnancy-associated functions. By midgestation in rodents (days 10-14 of gestation in mice), up to 35% of the cells in a specialized, pregnancy-induced region within the uterine musculature called the metrial gland are granulated cells commonly called granulated metrial gland cells (2). In pregnant women, uterine LGLs are more widely dispersed throughout decidual tissue and represent 70% of the leukocytes in decidual cell suspensions (3).
Immunophenotyping of the surface and the cytoplasmic
granules of rodent and human pregnancy-associated LGLs
has led to the hypothesis that uterine LGLs are lymphocytes
of the NK cell lineage (4, 5). Studies using immune-deficient mice support this conclusion, since LGLs typical in
morphology, location, and frequency have been reported
in T cell-deficient mice (6, 7), T and B cell-deficient mice
(7), and NK1+ T cell-deficient mice (8). However, LGLs
and a metrial gland structure were absent from the uteri of
pregnant IL-2R null, p56lck null × IL-2R
null, and
tg
26 mice (8, 9). Because an NK cell deficiency accompanies the T cell deficiency in the three latter strains, uterine
LGLs are now regarded as members of the NK cell lineage
and designated uterine (u)NK cells (8, 9).
Depletion of uNK cells during pregnancy has only been
achieved genetically. A transgenic strain, tg26, carrying
high copy numbers (30-35) of the full human (hu) CD3
gene exhibits NK and T cell deficiencies from fetal life (10,
11). In addition to their splenic NK cell deficiency (10),
uNK cells are deficient in pregnant tg
26 (range from 0 to
3% of the frequency found in immune-competent, random-bred H-2b CD1 mice and inbred H-2k CBA/J mice [8]).
Metrial glands also fail to differentiate in tg
26 implantation
sites (8).
In immune-competent mice, the metrial gland is associated with the maternal portion of the placenta that consists
of transformed stromal cells known as the decidua. The fetal portion of the placenta has three layers: giant cell trophoblast, spongiotrophoblast, and labyrinthine trophoblast.
Implantation sites in tg26 mice appear normal, both
grossly and histologically, until day 9 of gestation (counting
from the copulation plug as day 0). By day 10 of gestation,
the decidua lacks normal cellularity and the large ablumenal
decidual blood vessels have thickened walls. There are progressive degenerative changes to both the media and endothelium over the next four d of gestation. Vascular
anomalies are not found in other organs of pregnant or
nonpregnant tg
26 mice (8).
Death of >50% of the tg26 fetuses occurs in each litter
between days 10 and 14 of gestation, a time correlating
with the decidual pathologies. Surviving tg
26 fetuses have
very small placentae (~50% of normal) and from birth to
adulthood tg
26 mice are smaller than immune-competent, age-matched controls (36% smaller at 24 h after birth
and 27% smaller at 7 wk of age) (8). These data suggested
that uNK cells have major functional roles in optimizing pregnancy success.
To understand the role of uNK cells in the reproductive
deficits of tg26 females, three further studies were undertaken. First, ectoplacental cones from tg
26 mice were examined for expression of the huCD3
transgene. No expression was detected. Second, morphometry was applied
to the maternal decidual arterial sinuses, which are murine
equivalents to human spiral arteries (12). Elevated wall to
lumen ratios were found, suggestive of hypertension. Third, bone marrow was transplanted from scid/scid mice into tg
26
females. These donor cells are known to establish peripheral NK cells but do not generate mature T cells (13). The
uNK cell lineage was established and the quantified reproductive deficiencies of tg
26 mice were reversed or significantly reduced in engrafted, pregnant tg
26 mice.
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Materials and Methods |
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Mice.
8-wk-old random-bred CD1 (H-2b) mice (Charles River Laboratories, St. Constant, Québec, Canada), inbred CBA/J mice (H-2k; The Jackson Laboratory, Bar Harbor, ME), C.B-17 scid/scid (SCID) mice (bred at Guelph) and transgenic tgBone Marrow Transplantation.
Bone marrow transplantation was performed in four experiments, as previously described (13, 14). In brief, female recipients (8-wk-old tgHistology.
Uteri were fixed in Bouin's fixative (Fisher Scientific, Whitby, Ontario, Canada) and processed into paraffin. All implantation sites were serially sectioned transversely at 6 µm and stained using standard protocols for hematoxylin and eosin, periodic acid-Schiff (PAS) reaction, and Masson's trichrome connective tissue stain. The PAS reaction stains the granules of uNK cells a bright magenta and makes it possible to distinguish them from other lymphocytes. Masson's trichrome was used for assessment of decidual blood vessels. All serial sections were examined.Image Analysis.
Micrometer-based uNK cell enumeration has been previously described (9). All other histological measurements used Northern Exposure System 2.6a (Imagexperts Inc., Hollywood, CA) running in Microsoft Windows 3.1 (Microsoft Corp., Redmond, WA). Eight median independent tissue sections were analyzed, including the central section and sections on both sides that were at least 36 µm apart. Cross-sectional area measurements of decidual blood vessel wall and vessel lumen and placental cross-sectional areas were measured from a minimum of two implantation sites per pregnancy. The limits used for the definition of placental cross-sectional area were trophoblast populations up to and including the trophoblast giant cell layer. The decidua and metrial gland were excluded.Statistical Analysis.
Means, standard errors, standard deviations, P values, and paired t tests were performed using the computer software program Microsoft Excel 5.0 for Windows (Microsoft Corp.). ![]() |
Results and Discussion |
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One possible explanation for small placentae in
tg26 pregnancy is aberrant expression of the huCD3
transgene in trophoblast cells. We first established that neither CD1 nor tg
26 trophoblast expressed immunoreactive
murine CD3 (data not shown). Then, day 7 tg
26 ectoplacental cone tissue (the fetally derived structure from which
all trophoblast arises) was examined for huCD3
expression by Northern blotting and a more sensitive reverse transcriptase PCR approach. Day 7 was selected because it is
the latest developmental time point at which pure trophoblast can be isolated. The positive control was RNA from
thymocytes of heterozygous tg
600 mice (13), which carry
a different copy number of the same transgene. Human CD3
mRNA was not detected by either method in the
trophoblast tissue but was present in the heterozygote thymocyte RNA (data not shown). Thus, it is unlikely that
the reduced size of placental trophoblast in tg
26 is due to
transgene expression within the trophoblastic lineages.
The mesometrial decidua in tg26 is very unusual, being relatively acellular and containing vessels with
thickened walls. Masson's trichrome staining did not detect
matrix deposition in the cell-deficient regions (data not
shown). Since neutral red staining of cryostat sections has
also eliminated fat deposition as an explanation for this relative acellularity, we interpret the decidual region as edematous (9).
Normal pregnancy is associated with progressive dilation
of uterine blood vessels and their derivative branches. Since
this was apparently not occurring in tg26 pregnancies, the
anomalous vessels were studied morphometrically. Table 1
summarizes the ratios of tg
26 vessel wall to lumen cross-sectional area measurements and compares them to measurements of the similar vessels in the ablumenal decidua of
CBA/J. In immune-competent control animals, a progressive increase in the ratio of the lumen to wall was found. This is consistent with the hypotensive environment promoted in mammalian uterus during mid-gestation (15).
At days 10, 12, and 14 of gestation, tg
26 vessel walls
showed a thickened media. This was measured at 2.7, 2.0, and 4.7 times greater than the media thickness in control,
gestational day-matched CBA/J. The decidual vessels in tg
26
had relatively less lumen, suggesting vasoconstriction or failure to undergo arterial vessel modification that would facilitate vasodilation. Either mechanism would be expected to
promote thickened decidual vessel walls and a more hypertensive environment in the tg
26 uterus at midpregnancy. Masson's trichrome intensely stained the muscular component of the vessel walls (red). There was little evidence of
increased connective tissue reactivity (blue) on days 10, 12, and 14 of gestation. This demonstrates that the media was
the thickened component in tg
26 decidual vessel walls.
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Six features of pregnancies in tg26 females
have been identified as different from pregnancies in immune-competent or in T cell-deficient mice. These features are as follows: loss of uNK cells to <1% of normal;
absence of metrial gland development; acellularity and
edema in the decidua; increased wall/lumen ratios in the
major ablumenal mesometrial vessels of the decidua; small placental size; and midgestational fetal death (8). To determine whether NK cell deficiency makes a major contribution to any or all of these reproductive deficits, females
were grafted with SCID (NK+ T
B
) bone marrow,
which cannot establish the T cell lineage. Half of the recipients received bone marrow that had been pretreated with
-Thy-1 Ab + complement before injection while the
other half received untreated bone marrow. Of the 20 recipients, only 10 mated successfully and these were from
both of the marrow pretreatment protocols (Table 2). Of
these 10, 7 were considered to have splenic NK cell reconstituted as assessed by standard chromium release assays
(mean percentage of specific lysis of 36.2% ± 2.1 at E:T = 100:1 compared to mean percentage of specific lysis of
42.0% ± 5.0 for CD1 at the same E:T ratio). Three females
who had relatively nonlytic spleen cells (mean percentage
of specific lysis of 9.1% ± 2.0 at E:T = 100:1 compared to
mean percentage of specific lysis of 4.8% ± 3.7 for unmanipulated tg
26 at the same E:T) were considered to be
non-reconstituted, and were analyzed separately from the first seven females.
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uNK cells
were present in considerable numbers in the seven NK cell
reconstituted females but not in the remaining three females. Implantation sites from two immune-competent
strains of mice (CD1 and CBA/J) were used to establish the
parameters for normal number, size, and granularity of uNK
cells on each of days 10, 12, and 14 of gestation. The frequency of uNK cells was not significantly different between CD1 and CBA/J strains at any gestational day studied. Therefore, the values from these two strains were
pooled and are referred to as "control" in Table 2. In nonmanipulated tg26, the number of uNK cells were statistically <1% of control at days 10, 12, and 14 of gestation
(reference 8 and Table 2). When compared to control immune-competent females, day 10 NK cell reconstituted
females had 40.2%, day 12 females had 56.1% and day 14 females had 49.8% of normal frequencies of uNK cells (P
<0.01; Table 2 and Fig. 1). Many uNK cells in the reconstituted females were associated with the main decidual
blood vessels (Fig. 1). In contrast, low numbers of uNK cells
(1-3.1% of immune-competent females, which is also
equivalent to the number of uNK cells found in untreated
tg
26 females) were present in the three bone marrow infused, nonreconstituted tg
26 females (Table 2).
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Histologically, the placentae from the NK cell reconstituted females appeared larger than those in tg26 females
and the implantation sites resembled those in immune-competent females, because they included a well developed
metrial gland (Fig. 1). Reference standards for placental
cross-sectional areas were obtained by measurements of
CD1 and CBA/J implantation sites. Since these were not
statistically different from each other at the gestational days
used (10, 12, and 14), the data were pooled to provide a
larger immune competent control data set than that used
for control reference values in our earlier publication (8).
The mean cross-sectional areas of unmanipulated tg
26
placentae were 53% of the mean at day 10 (P <0.01, n = 6 mice), 50% of the mean at day 12 (P <0.01, n = 4 mice)
and 47% of the mean at day 14 (P <0.01, n = 4 mice) control placentae (Fig. 2).
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Placental cross-sectional areas for the NK cell-reconstituted females were significantly larger than cross-sectional
areas measured for time-matched homozygous tg26 females or for SCID bone marrow-infused females who
failed to show evidence for engraftment (Fig. 2). Placentae
from all NK cell reconstituted tg
26 mothers (independent
of the marrow graft preparation protocol) were 28% (day
10), 27% (day 12), and 34% (day 14) larger than placentae from homozygous tg
26 mice (P <0.01) but were 26%
(day 10), 32% (day 12), and 29% (day 14) smaller than placentae from the immune-competent control mice (P
<0.01). Days 10, 12, and 14 placental cross-sectional areas
from bone marrow-infused, nonreconstituted mice were
not statistically different than those from homozygous tg
26
placentae (Fig. 2). Since normal levels of uNK cells were not
established in the engrafted mice, it is unclear whether or not
the placental sizes achieved represent the maximum placental
growth effects that can be promoted by uNK cells.
The vascular anomalies found in unmanipulated pregnant tg26 mice may or may not underlie the limited placental growth seen in these pregnancies. In the NK cell-
reconstituted tg
26 females, no major anomalies of the
decidua or placental vasculature were found (Fig. 1). The
lumen size in decidual vessels of the NK cell-reconstituted females was larger than that in nonreconstituted tg
26 mice
and appeared to increase with gestational length. The wall/
lumen ratios were similar to those in the immune-competent controls (Tables 1 and 3), suggesting that engraftment
had restored normal dilation of the decidual arterioles. This
occurred in spite of the observation that uNK cell numbers
were lower than in immune-competent mice. Minor variations from the immune-competent controls were found in
implantation sites from only two of the seven NK cell- reconstituted females, the two females with the lowest uNK
cell numbers (38.7 and 42.9% of immune-competent controls, respectively), and this suggests a redundancy in the
uNK cell numbers regarding this trait. In these two females,
the decidual region appeared fluid filled, the width of the
myometrial smooth muscle was increased on the mesometrial side with uNK cells scattered within these smooth
muscle fibers, and a small developing metrial gland was present. The trophoblast giant cell layer appeared mildly disorganized, having produced isolated islands of trophoblast
giant cells, and trophoblast giant cells were found in apposition to decidual vessels. This is the location that many
uNK cells occupy in mice. Blood vessels and placentae
from bone marrow-infused but NK cell-nonreconstituted tg
26 females resembled those from homozygously mated,
nonmanipulated tg
26 females (Tables 1 and 3).
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Viability of the fetuses from the NK cell-reconstituted
tg26 females was equivalent to fetal viability in immune-competent mice at all gestational days studied (Table 2).
Viability of the fetuses from the single time-matched pregnancies available in bone marrow-infused, nonreconstituted females was below control levels at days 10 and 12 of
gestation but, unexpectedly, equivalent to control in the
one day 14 female, for which we have no explanation. Thus, reestablishment of 40-56% of uNK cells restored vasodilation in decidual arteries, promoted placental size, and
promoted fetal viability.
One interpretation of the tg26 experiments is that uNK
cells downregulate vascular smooth muscle cell (VSMC)
numbers during pregnancy or promote VSMC relaxation.
IFN-
inhibits proliferation of rat VSMC through the induction of nitric oxide synthase (NOS) activity which in
turn generates NO (18). NO is a major vasodilator that acts
on vascular smooth muscle in many sites, including the uterus (19, 20). Infusion of an inhibitor of NOSs during
pregnancy in rats caused hypertension and fetal growth retardation, suggesting that a reduction in the synthesis of
NO may contribute to the pathogenesis of pregnancy-induced hypertension (21). IFN-
and iNOS are both
products of mouse uNK cells (7, 22), as is TNF-
(23), another hypotensive molecule (24, 25). A number of histologists have noted a strong association of rodent uNK cells
with blood vessels, and this association was reestablished in
bone marrow-reconstituted tg
26 females (Fig. 1 F). It has been previously suggested that uNK cells are involved in
blood pressure regulation because hypertensive rats in
which an artificial deciduomata had been induced experienced a fall of blood pressure at the time uNK cells appeared and an elevation of blood pressure when uNK cells
disappeared despite persistence of the deciduomata (26).
However, preliminary experiments involving serial blood pressure recordings by tail cuff in four conscious pregnant
tg
26 females failed to demonstrate statistically significant
systemic hypertension over the first 16 d of gestation in
comparison to four nonpregnant tg
26 females (Luross,
J.A., and B.A. Croy, unpublished data).
Three explanations have been considered for incomplete
uNK cell reconstitution in tg26 females. First, the number
of bone marrow cells or the proportion NK cell progenitors within the bone marrow suspension used for treatment
in this study may not have been sufficient for full reconstitution of the uNK cell population. Since bone marrow was
pooled from four donors for each recipient, this is not a
strong argument. Second, the 3-wk time interval between
the time of bone marrow treatment and the time of mating, in addition to the 10-14 d of pregnancy may have been too
long, and thus bypassed the optimal interval (3 wk) when
splenic NK cell numbers peak in tg
26 mice reconstituted
with recombination activating gene 2 (RAG-2) null bone
marrow (13). Third, the 5-FU pretreatment may not have
displaced the recipient marrow cells sufficiently to provide
an adequate niche for optimal donor marrow engraftment.
The decidual blood vessel structure and placental morphology were improved or fully restored in the seven reconstituted tg26 mice despite their lower than normal
frequency of uNK cells. Therefore, the placental size increments achieved in the NK cell-reconstituted tg
26 versus
nonmanipulated tg
26 may represent the maximal growth promotion by uNK cells with other mechanisms, accounting for the deficit in size between reconstituted tg
26 and
immune-competent controls. One further requirement to
promote full placental growth may be immune-competence in the fetal compartment. We have reported elsewhere that embryo transfers demonstrate that unilateral
fetal or maternal NK cell deficiency can reduce placental
size (8).
These in vivo studies have better defined the functional
role of the immune system and the role of uNK cells in the
pregnant uterus in particular. They exclude T cells and
NK1 T cells as essential participants in pregnancy success,
supporting earlier studies of nu/nu and IL-2 null × 2m
null mice (6). These studies identify the NK cell lineage
as important for normal development of implantation sites
and highlight the mesometrial decidua and vasculature as
important sites for uNK cell function. These studies do not
support the previously postulated function of uNK cells to
limiting trophoblast cell growth and invasion. On the contrary, they show that maternal uNK cells promote placental
growth and thereby the growth of the developing fetus. It
remains unclear whether promotion of trophoblast growth
is a direct or indirect effect.
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Footnotes |
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Address correspondence to Dr. Marie-Josée Guimond at her present address, Department of Anatomy and Cell Biology, Medical Sciences Bldg, Rm 473, University of Western Ontario, London, Ontario, Canada N6A 5C1. Phone: 519-679-2111 ext. 6808; Fax: 519-661-3936; E-mail: jguimon2{at}julian.uwo.ca
Received for publication 7 August 1997 and in revised form 5 November 1997.
This work was financed by awards from the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture, Food, and Rural Affairs.We thank Dr. Cox Terhorst (Harvard Medical School, Boston, MA) and Dr. R.A. Phillips (Hospital for
Sick Children, Toronto, Ontario, CA) for providing tg26 and scid/scid breeding pairs, and Andrew Moore
(Ontario Ministry of Agriculture, Food and Rural Affairs, Guelph, Ontario, Canada) for support of image
analysis.
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