Download The Role of Natural Killer Cells in Murine Early Embryo Loss.

The Role of Natural Killer Cells in Murine Early
Embryo Loss.
By
Christopher Ng Thow Hing
Department of Microbiology and Immunology
McGill University
Submitted to the Faculty of Graduate Studies and Research
in partial fulfillment of the requirements for the degree of
Master of Science.
Submitted: July 2002
© Copyright by Christopher Ng Thow Hing, 2002
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Abstract
About 40% of human pregnancies are unsuccessful and many of these are thought to be
genetically normal. The murine mating of a DBAJ2 male with a CBAJJ female provides
an ideal model by which to study these losses. In this combination, the CBAJJ female has
a 20-30% rate of embryo resorption before day 12 of gestation. It is thought that the
major events in early embryo loss are macrophages and natural killer (NK) cells
infiltrating the decidua with subsequent production of a Th1 type pro-inflammatory
cytokine profile.
Previous work has shown that in vitro incubation of decidual
macrophages with lipopolysaccharide leads to the production of the cytotoxins TNP-Cl
and nitric oxide. Significant production of these effector molecules requires priming of
the macrophages and this is mediated by IFN-y. The increased expression of TNP-Cl,
iNOS and IFN-y rnRNA in single embryos has also been correlated to the incidence of
resorption in our murine model. Our hypothesis is that decidual NK cells in abortion
prone pregnancies produce IFN-y which primes macrophages.
These primed
macrophages are then triggered by a second signal to become the major effectors in early
embryo resorption. Analysis of individual implantation sites was performed at day 9 of
pregnancy to determine the cytokine profile ofthese NK cells and ifNK cells selectively
infiltrate the decidua of embryos that will undergo resorption. Use of a pan-NK cell
marker (DX5) allowed labeling of decidual NK cells for flow cytometric analysis.
Magnetic labeling and isolation of DX5+ cells from individual embryos was followed by
RT-PCR and southem blot analysis. This thesis prevents evidence that a number of the
embryos are infiltrated by higher numbers of DX5+ NK-cells with an incidence that is
similar to the occurrence of early embryo loss in this experimental model. Furthermore,
DX5+ cells produce significant amounts ofIFN-y and TNP-Cl rnRNA suggesting that NK
cells may not only prime but trigger macrophages. DX5- cells were found to produce
sorne IFN-y but also large quantities of TNF-Cl rnRNA. Perforin expression in both
DX5+ and DX5- cells was similar indicating other cellular sources such as T cells and
GMG cells.
The results support the concept that NK cells play a major role in the
mechanisms of early embryo loss.
11
Résumé
Environ 40% des grossesses humaines sont inéffectueses, dont la plupart sont le résultat
des mécanismes génétiques.
L'accouplement entre un mâle murine DBA/2 et une
femelle murine CBA/J répresente un modèle idéal pour étudier la perte des embryons.
Douze jours après gestation, le taux de résorption de l'embryon chez la femelle est de 20
à 30%. L'infiltration par les macrophages et les cellules NK dans les caduques basales et
la production subséquente de cytokines inflammatoires type Thl, sont les causes
majeures du rejet des embryons.
Il a été avancé que l'incubation -en vitro des
macrophages trouvées dans la caduque basale, avec des liposaccharides, résulte dans la
production de cytokines TNF-a et de l'oxyde d'azote (iNOS). La production de ces
molécules effecteures dépend sur les étapes intialles chez le dévéloppement des
macrophages. Cette phénomène de 'primer' les macrophages est controlé par l'INF-y.
L'expression de TNF-a, iNOS et l'ARN messager (ARNm) de IFN-y dans les embryons
solitaires est corrélée aux taux de résorption dans la modèle murine. Notre hypothèse est
que les cellules NK localisées dans la caduque basale produisent IFN-y lors des
avortements. Cette production de IFN-y résulte dans la génération des macrophages, qui
par suite, sont activées par une deuxième signal moléculaire.
L'analyse des sites
d'implantation étaient faite pendant la neuvième jour de grossesse, afin de déterminer la
profile cytokinétique des cellules NK et d'évaluer l'infiltration sélective du caduque
basale chez les embryons résorbés. Le marquer DXS était implimenté lors de l'analyse
flot cytométrique des cellules NK. Des marquers magnétiques étaient employés pendant
l'isolation des cellules marquées DXS+. Ces cellules étaient utilisées pour l'extirpation
de l'ARNm. Cette thèse mette en évidence l'infiltration par les cellules DXS+ dans des
sites d'implantation à un taux similaire du modèle murine. De même, les cellules DXS+
produisent des quantités significantes d'IFN-y et de l'ARNm TNF-Œ. Cela sugère que les
cellules NK ne serent pas à primer les macrophages. Les cellules DXS- produisent peu
de l'IFN-y, mais ils générent en grande quantité l'ARNm TNF-Œ.
L'expression du
perforin est presque identique chez les cellules DXS+ et DXS-. Cet résultat indique la
présence des sources alternatives pour la production des cellules, par exemple, des
III
cellules T ou GMG. Finalement, les résultats supportent l'idée que les cellules NKjouent
une rôle majeure dans les mécanismes du perte embryonique.
IV
Acknowledgements
First and foremost, 1 would like to thank my supervisor Dr. Malcolm Baines for allowing
me the opportunity to work in his laboratory. Entering the lab with a little immunological
background, your patience and encouragement has enabled me to learn a lot about
immunology in a short time.
My eternal gratitude also goes out to Dr. Emilia Antecka whose te~~!1ical skills
are unmatched. Thank you for the assistance and generosity without which 1 would have
surely not completed this work.
Thanks to Ken and Jaime for the technical assistance in using the FACS facilities.
1 would also like to thank my fellow lab mates Genevieve, Natasha, Cheryl and
Kris for making the lab a fun place to work.
A special thanks goes out to Deborah Stewart and Sean Hughes for proof-reading
and translation of the abstracto
Final1y, 1 would like to express my thanks to my family and close friends who
have supported me throughout my studies.
v
Table ofContents
Abstract
ii
Résumé
iii
Acknowledgements
v
List ofFigures
viii
List of Tables
lx
1
L
Literature Review
IL
Rationale and Objectives ofthe Study
18
IlL
Materials and Methods
20
1. Mice and Matings
20
2. Isolation and Preparation of Tissues
20
3. Cell Counts of Suspensions
21
4. Immunofluorescent Staining ofNatural Killer (NK) Cells in
Embryonic and Splenic Cell Suspensions
21
5. Magnetic Cell Sorting ofNK Cells
22
6. RNA Extraction
23
7. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR)
Analysis
IV.
24
8. Southem Blotting
25
9. Radiolabeling of Gene Specifie Oligonucleotide Probes
26
10. Membrane Hybridization with Radiolabeled Probes
26
Il. Statistics
27
Results
1. Sample Populations
30
30
2. FACS Analysis of Day 9 Embryos Shows Elevated Natural
Killer Cell Numbers
30
3. Cytokine Expression in Natural Killer Cell Enriched and
Depleted Populations from Individual Embryos
34
VI
4. DX5+ Cells from Individual Embryos Express Significant Levels
ofIFN-y, TNF-a. and Perforin
40
5. DX5- Cells from Individual Embryos Express Significant
Amounts ofPerforin and TNF-a.
41
Jt:
Discussion
46
VI.
References
56
Vil
List ofFigures
Figure 1:
Quantitation ofDX5 Positive Natural Killer Cells in
Individual Embryos at Day 9 ofPregnancy
Figure 2:
Expression ofIFN-y, Perforin and TNF-a. rnRNA in
Individual Embryos at Day 9 ofPregnancy
Figure 3:
42
Frequency Distribution ofTNF-a. rnRNA Expression in
DX5+ and DXS- cens at Day 9 ofPregnancy
Figure 8:
39
Frequency Distribution ofIFN-y rnRNA Expression in
DX5+ and DX5- cens at Day 9 ofPregnancy
Figure 7:
38
Relative Expression ofPerforin rnRNA at Day 9 of
Pregnancy
Figure 6:
37
Relative Expression ofTNF-a. rnRNA at Day 9 of
Pregnancy
Figure 5:
36
Relative Expression ofIFN-y rnRNA at Day 9 of
Pregnancy
Figure 4:
32
43
Frequency Distribution ofPerforin rnRNA Expression in
DX5+ and DX5- cens at Day 9 ofPregnancy
44
Vl1l
List of Tables
Table 1:
Oligonucleotide Primer and Probe Sequences
29
Table 2:
NK-cell Statistics from FACS Analysis
33
Table 3:
Summary of Cytokine Expression Levels
45
IX
Literature Review
In terms of transplantation immunology, whether a graft is accepted or not is dependent
on the matching of tissue antigens such as those determined by the major
histocompatibility complex (MHC) (Martin and Dyer, 1993). From this observation,
mammalian pregnancy presents a unique situation to the maternaI immune system. For
pregnancy to be successful, the mother must accept what is basically a fetal allograft for
an extended period of time. It can be termed an allograft since the fetus and placenta
both express paternal antigens that come into direct contact with maternaI immune
effectors such as T cells. Consequently, one would anticipate immune rejection of the
fetal 'allograft' via MHC-restricted T cell mediated cytotoxicity much in the same way a
tissue graft is rejected.
However, this is not the case since many pregnancies are
obviously successful and thus the fetus is somehow tolerated.
In 1953, Medawar proposed 4 explanations for the ability of the mother to tolerate
the fetus. He suggested that the fetus may somehow be immunologically invisible to the
mother and will thus not evoke an immune response. Second, he proposed that if an
immune response is generated then the response will be suppressed. His third proposaI
was that the uterus is an immunologically privileged site much in the same way as the
human eye and hamster cheek pouch.
Finally, Medawar proposed that the placenta
provides a physical barrier between the mother and the fetus. Research since then has
shown that the fetus does cause an immune response and that the uterus is not an
immunologically privileged site.
Consequently, the focus of research has been in
1
examining the maternaI immune response and in looking at the components forming the
placental barrier.
The development of the feto-maternal interface begins when the newly fertilized
ovum differentiates into the blastocyst over a period of 4-6 days. Implantation of the
blastocyst into the uterine wall causes the formation of contacts with maternaI cells by
fetal trophoblast cells (Torry et al., 1997). These trophoblast cells are the embryonic
cells that come into direct contact with the maternaI tissues.
Consequently, the
trophoblast cells are important as a poterttial inducer and regulator of the maternaI
immune response. Examination of these trophoblast cells has provided sorne insight into
the way the fetus avoids maternaI immune attack. Normally, tissue allografts express
conventional MHC molecules. Host T cells can then bind these non-self MHC antigens,
become activated resuiting in graft rejection. In successful pregnancy, there is a lack of
this T cell mediated rejection and this may be due to a lack of trophoblast
immunogenicity. It has been shown that trophoblast cells have altered or decreased
expression of conventional MHC class 1 and II antigens (Wood, 1994). In humans, there
is an absence of polymorphic MHC 1 and II on trophoblast cells and in mice, there are no
class II antigens.
The importance of this observation was shown when mice were
induced to express MHC II and fetal survival decreased (Vassiliadis et al., 1994).
Furthermore, addition of antibodies to these MHC II antigens restored normal embryo
viability. The absence of MHC 1 and II on human trophoblasts would give it similar
properties to sorne tumors and virally infected cells which downregulate MHC to protect
themselves from T cell recognition and subsequent cytotoxic attack.
In both these
examples, NK cells can recognize this absence of MHC (missing self hypothesis,
2
Ljunggren and Karre, 1990) and cause NK cell mediated cytolysis. However, trophoblast
cells express a non-conventional, minimally polymorphie MHC 1 antigen, HLA-G
(Carosella et al., 1996) which will bind NK cells killer inhibitory receptor (KIR) and
downregulate NK cell function and thus avoid NK cell mediated lysis (Munz et al.,
1997).
Trophoblast cells also express Fas ligand that is thought to confer on the uterus
immune privilege-like properties (Hammer et al., 1999). Fas is usually expressed on
activated T cells and when bound to its naturalligand, Fas ligand (FasL), will transmit a
signal to the T cell to induce apoptosis (Suda et al., 1994). Conventionally, the Fas/FasL
system is used to downregulate immune responses thereby avoiding excessive damage to
host tissues during an immune event (Alderson et al., 1995). Furthermore, expression of
FasL on certain cells may help in the maintenance of immune privilege in sites like the
eye. Invading inflammatory cells expressing Fas receptor would undergo apoptosis and
thus prevent an episode of inflammation thereby avoiding tissue damage (Griffith et al.,
1995). Although not truly an immune privileged site, Fas/FasL interactions at the fetomaternai interface may provide a mechanism by which the fetus avoids sorne immune
effectors. Specifically, the abundant presence of FasL on trophoblast cells may proteet
the fetus by binding the Fas death receptor (CD95) on aetivated maternalleukocytes and
cause apoptosis of these cells (Sakata et al., 1998). Furthermore, expression of a nonfunctional FasL as seen in homozygous matings of gld mice resulted in the infiltration of
the decidual-placental interface with maternai leukocytes (Hunt et al., 1997).
The
consequence of this infiltration is increased resorption due to necrosis and a subsequent
decrease in the size of the liUer. However, successful pregnancy has been observed in
3
mice with non-functional FasL indicating that although it may be important, FasL does
not seem to be essential.
The complement regulatory pathway is also a mechanism by which the
trophoblast cells may protect the fetus. Normally, complement is the method by which
the immune system responds to extracellular infection resulting in opsonization and
killing of the pathogen. Antibodies with specificities for foreign antigen may trigger the
complement pathway and trophoblast specific antibodies are seen in placentae of normal
pregnant and secondary aborting women (Kajino et al., 1988).
Therefore, close
surveillance of the complement pathway is needed to prevent complement mediated lysis
oftrophoblast (Zuckermann et al., 1987). Additionally, complement activation results in
the recruitment of inflammatory mediators that are inevitably inhospitable to the
conceptus. To this end, the trophoblast expresses CD46 (membrane cofactor protein),
CD55 (decay accelerating factor) and CD59. CD46 and CD55 inhibit C3 convertase
activity while CD59 prevents membrane attack complex (MAC) formation.
These
proteins prevent activation of maternaI C3 via the alternative and classical pathway and
prevent complement mediated injury due to maternaI immunoglobulins with fetal
specificities (Torry et al., 1997).
Complementary to these evasion and neutralization tactics that the trophoblast
employs is the observation that trophoblast itself is highly resistant to direct immune
attack.
In keeping with Medawar's postulate that the placenta acts as a barrier is
experimental data showing that trophoblast cells are resistant to lysis by many ditTerent
immune cells and mediators (Ferry et al., 1991, Abadia-Molina et al., 1996).
Trophoblast cells exposed to NK cells, macrophages, cytotoxic T lymphocytes and tumor
4
necrosis factor (TNF) have shown to be resistant to lysis. Thus far, the only cells capable
of killing trophoblast cells is lymphokine activated killer cells (LAK) both in humans
(King & Loke, 1990) and mice (Drake & Head, 1989). However, generation ofLAKs
requires interleukin-2 and rô T cells may produce this cytokine in vitro but it has not yet
been detected in the decidua during normal pregnancy.
Additionally, trophoblast recognition by the maternaI immune system may in fact
promote survival of the embryo. Presentation offetal antigen may stimulate decidual cell
production of cytokines beneficial for placentation (Raghupathy & Tangri, 1996).
Colony stimulating factor 1 (CSF-l), interleukin 3 (IL-3) and granulocyte macrophage
colony stimulating factor (GM-CSF) are examples of cytokines which may augment
placental development. A possible source of these factors may be T cells and anti-CD4
and CD8 treatment reduced placental growth and decreased embryos survival
(Athanassakis et al., 1990). Furthermore alloimmunization of a mother with paternal
leukocytes before mating enhanced embryo viability (Chaouat et al., 1985, Clark et al.,
1987). Indeed, maternaI T cells specific for paternal alloantigens engage in a transient
state of tolerance for the duration of pregnancy (Tafuri et al., 1995). Although T cells
seem to play an important role in embryo survival, they may not be the only effectors in
play since T cell deficient
scm mice are capable of successful pregnancy.
This would
suggest that potential local immunomodulatory events are occurring in successful
pregnancy rather than outright systemic immunosuppression as postulated by Medawar.
Immunomodulation rather than immune suppression makes sense and is backed
by two simple observations. The first is that pregnant women are not more susceptible to
overwhelming infection (Sacks et al., 1999) than their non-pregnant counterparts and
5
second, that antibody mediated immunity (Wegmann et al., 1993) is relatively normal
during pregnancy.
These striking observations led to the proposaI that successful
pregnancy can be accounted for by the ThllTh2-3 paradigm. The CD4 subset ofT ceUs
can be broken down into two major groups based on their role in an immune response
and in their respective cytokine production profile.
The rationale behind this
classification is that each subset of Th1 or Th2 ceUs may be effective for a particular
insult to the host but relatively useless or detrimental to the other. Th1 ceUs secrete IFN-
y, IL-2, TNF-a and TNF-f3. Th1 responses usuaUy activate inflammatory processes and
the Th1 cytokines that are released activate macrophages and ceU mediated reactions that
are important in clearing intraceUular infections as weU as in delayed type
hypersensitivity (DTH) reactions.
Furthermore, Th1 cytokines such as IFN-y have a
suppressive effect against Th2 type responses. Conversely, Th2 cytokines include IL-4,
5, 6, 10 and 13 and are important in promoting the production of antibodies against
extraceuUular organisms. A third Th-ceU subset classified as Th3 ceUs differ from Th1
and Th2 ceUs in that they secrete TGF-f3 but do not secrete traditional Th1 or Th2
cytokines such as IL-2, IFN-y, IL-4 or IL-lO. However, similarly to Th2 responses, Th3
ceUs downregulate Th1 type reactions and are thus commonly grouped with Th2 ceUs.
The effect of a type 1 or type 2/3 response is ceU-mediated (Th1) or humoral (Th2)
immunity respectively. Wegmann and coUeagues (1993) proposed a model in which
pregnancy is governed by this ThllTh2 paradigm and that a successful pregnancy
depends on the balance of the pro-inflammatory Th1 type cytokines and the antiinflammatory Th2 type cytokines.
They proposed that a less damaging local Th2
cytokine pattern is invoked in response to the developing fetus. This Th2 bias of an
6
antibody mediated response would be preferable over a Thl cell mediated attack against
the fetus and the potentially more damaging nonspecific innate inflammatory processes
promoted by Thl cytokines.
Supporting this theory is work done by Dudley and
colleagues (1993) on normal pregnant mice showing an increase in IL-4 and IL-6 from
activated lymphocytes and a reduction in levels of IL-2. Additionally, peripheral blood
mononuclear cells (PBMC) from humans show that pregnant women have significantly
higher levels of the Th2 type cytokine IL-l 0 than their non-pregnant counterparts (Hanna
et al., 2000).
PBMCs.
Also shown was a decrease in IL-2 and IFN-y mRNA expression in
These and additional studies show a definite shift to a Th2 type status in
pregnant women. If there is an opposite shift in the Thl/Th2 balance, this may have
adverse effects on gestation. If pregnant C57Bl/6 mice are infected with Leishmania
major, whether or not the pregnancy is successful depends on the mice' response to this
parasite. Rejecting the parasite requires a shift to Thl type immunity and ifthis occurs so
does abortion.
However, if a Th2 type status is maintained, then the pregnancy is
successful but clearing of the infection fails (Krishnan et al., 1996)
Contributing to this Th lITh2 proposai is the presence of immunosuppressive
hormones and blocking antibodies present during normal pregnancy.
In general,
hormonal changes are a typical part of pregnancy and the female will see increases in
progesterone, estrogen and corticosteroids (Wilder, 1998). In particular, these hormonal
changes may directly influence the Thl/Th2 balance by promoting a Th2 cell
development under the influence of progesterone.
Antibodies generated during a Th2 response may be beneficial to the developing
conceptus due to the production ofblocking or protective antibodies. More specifically,
7
antibodies may be generated against host antigens which may block a cytokine response.
For example, generation of an antibody against the antigen binding site of a T cell
receptor (Chaouat & Lankar, 1988) or similar immune effector may block a potential
response against the fetus.
These anti-idiotypic antibodies were able to reduce the
incidence of resorption in the CBA/J X DBA/2 model when injected early in gestation.
Additionally, protective antibodies to non-host targets such as trophoblast expressed
antigens can be generated. Indeed, antibodies against fetal, placental and paternal targets
are normally found in the serum of women who have had successful pregnancy
(Billington, 1992). During normal pregnancy an immunoglobulin G antibody is found
bound to trophoblast (Mowbray et al., 1997)
Elution and characterization of this
antibody revealed that it bound to a trophoblast expressed protein, R80K. Although the
exact function of this 80-kOa protein is unknown, it is found expressed on the placentae
of both humans and mice (Jalali et al., 1995) and its masking seems to be a normal facet
of pregnancy. Exposure of unmasked R80K to maternaI effectors generates an NK cell
mediated response against the trophoblast. Protective IgG bound to R80K prevents this
particular recognition and response.
AlI these mechanisms discussed may be required in the maintenance of successful
pregnancy but failure does occur in a significant proportion of pregnancies. Between 30
and 50% of all implanted embryos spontaneously abort before the l4th week of gestation
(Baines & Gendron, 1993). As much as 60% ofthese losses are attributed to an unknown
etiology and cannot be explained by chromosomal anomalies, endocrinological
abnormalities, infection or anatomie problems (Warburton et al., 1987, Coulam et al.,
1996). The existence of such a large number of cases with unknown causes has focused
8
investigation on the possibility of an immunological etiology. Study of this problem is
difficult from a human perspective due to ethical and logistical constraints. Most early
abortions occur unbeknownst to the mother and the products of conception will be
expelled during the normal menstrual cycle. Also, any tissues obtained from later stages
of gestation are not particularly useful for the study of early embryo loss.
These
limitations led to the discovery of a useful mouse model for the study of early embryo
loss. The mating of a DBN2 male with a CBNJ female (Clark et al., 1980) has provided
sorne valuable insight into the immunology of early embryo loss. In this particular
model, 20-30% of embryos are lost before the 12th day of gestation. Interestingly, this
incidence of early embryo loss is similar to that seen in humans. Normally, CBNJ
females mated with males from other mouse strains including those with the same MHC
haplotype (BALB/c) show low incidences of resorption around 5-7%. Evidence that the
embryo loss in the high loss mating is occurring due to the maternai response is
supported by various observations.
The presence of various cells involved in host
defense at resorption sites is quite evident. Increased numbers of NK cells are found at
the feto-maternal interface at sites where resorption is occurring (Gendron & Baines,
1988).
Furthermore, injection with anti-asiaio-GMI to deplete NK cells resulted in
reduction ofresorption (de Fougerolles & Baines, 1987). A striking experiment was that
if CBNJ females were injected with BALB/c cells before mating with DBN2 males then
embryo loss was completely averted (Clark et al., 1987).
This response to
immunotherapy along with other data on embryo loss would indicate the presence of an
immunological rather than a genetic etiology. Consequently, research has focused on
maternai trophic factors such as immune effectors which may play a role in these
9
embryos losses.
During gestation, the decidua is infiltrated by a variety of immune
effectors and examination of these effectors in high and low loss mating combinations
have provided sorne insight into their role in early embryo loss.
The major immune cens present in the decidua during pregnancy are
macrophages, NK cens, granulated metrial gland (GMG) cens and T cens. GMG cens
are large granulated lymphocytes which are charaeterized as NK-like due to their
expression ofNK surface markers Ly49G2, NK1.1, asialo-GM1 (Croy & Kiso, 1993) and
the presence of lytic granules containing perforin and granzymes. They are localized to
the metrial gland in the basal area of the murine uterus during pregnancy. During early
implantation in humans, the GMG cens seem to undergo proliferation (pace et al., 1989)
and make up the majority of decidual leukocytes during the first trimester. However, at
the time of delivery GMG cens are not found (Searle et al., 1999) suggesting that their
role may be in the early modification of uterine vasculature (Croy et al., 2000) and the
maintenance of the early decidua. Their function is not clear but the absence of GMG
cens jeopardizes the survival of the embryo. In strains of mice lacking GMG cens, the
decidua fails to properly develop (Greenwood et al., 2000).
Specificany, structural
abnormalities were seen in decidual arteries and this may be related to the condition
known as pre-eclampsia, where pregnant women fail to remodel decidual spiral arteries
(Ashkar & Croy, 2001).
However, in both cases, embryo loss is not immediately
observed although placental growth may be compromised subsequently resulting in
abortion. Evidence exists to suggest that these GMG cens may be a major source ofIFN-
y (Platt & Hunt, 1998).
Furthermore, production of additional molecules such as
angiopoietin-2 may also assist in uterine remodeling by destabilizing arteries
10
(Maisonpierre et al., 1997).
Suggested functions of GMG cens include trophoblast
migration, placentation and vascularization due to the release of immune mediators such
as IFN-r (Redline, 2000). However more work has to be done to further clarify their role
especially in regard to the molecules they secrete and their specific targets.
Sorne T cells are also present in the decidua during pregnancy. Both CD4 and
CD8 T cens do not seem to recognize or react to trophoblast but their role in normal
pregnancy may be to maintain a Th2 type environment favorable to the embryo (Arck et
al., 1999).
However, neutralization of CD4+ cens does not affect the incidence of
resorption in high loss matings (Chaouat & Menu, 1997) indicating that they are not a
major effector in early embryo loss. The role of CD8+ cens is somewhat confusing with
contradictory data.
An abortive effect was shown when CD8+ cells that had been
stimulated by placentae from the high loss CBA/J X DBA/2 matings were adoptively
transferred to pregnant mice (Raghupathy, 1997). When placentae from low loss CBA/J
X BALB/c females were used as the stimulator, the adoptive transfer of the CD8+ cens
resulted in normal pregnancy. However, injection with anti-CD8 antibodies in CBA/J X
DBA/2 pregnancies can either increase the incidence of resorption or have no effect
(Chaouat & Menu., 1997). These varied effects may be explained by the fact that CD8+
T cens may play a role in the formation of either a Thl or Th2 profile. The major T cell
population in the uterus is comprised of rB T cells that don't express the more common
af3 T cell receptor (Heybome et al.,
1994). The rB T cells make up approximately 60%
of the T cens in the uteru.~ and their function, like the af3 T cens, may be to contribute to
the Th2 bias. Unlike <Xp T cells, rB T cens can recognize non-conventional MHC 1
antigens such as the IH...A-G expressed on human trophoblast. This recognition may
11
selectively trigger the y8 T cell to produce
TGF-~2
and a-10, which are Th2 type
cytokines (Arck et al., 1997). Numerically, there are more y8 T cells in the uterus of
allogeneic as compared to syngeneic pregnancies suggesting that their presence is normal
and perhaps required for successful gestation (Suzuki et al., 1995). Furthermore, the
recognition and activation of the y8 T cell causes the expression of a cell surface
progesterone receptor (Szkeres-Bartho et al., 1990). Using this as an activation marker, it
is found that almost all y8 T cells in the decidua are in an activated state_{MinchevaNilsson et al., 1994). Progesterone binding to its receptor causes production of a 34KD
protein called progesterone induced blocking factor (PffiF) which mediates the
immunoregulatory effects of progesterone (Szkeres-Bartho et al., 1989).
PffiF
contributes to an anti-inflammatory response by downregulating NK cell activity
(Szkeres-Bartho et al., 1997) and these y8 T cells may be major source of progesterone
induced suppression in the early placenta. Therefore, recognition of fetal antigens by y8
T cells leads to the activation of these cells causing upregulation of the progesterone
receptor. In the presence of progesterone, activated y8 T cells synthesize and release
PffiF which modulates the immune response in favor of successful pregnancy (polgar et
al., 1999). More recent work has shown the presence oftwo distinct populations ofy8 T
cells in the murine decidua (Barakonyi et al., 1999) which may have opposing functions
in respect to Th1 or Th2 cytokine secretion patterns. This may influence which particular
population dominates and whether the activation of one population will adversely affect
the outcome of pregnancy. Furthermore, accumulation of y8 T cells prior to decidual
formation may have abortive effects due to the early release of pro-inflammatory TNF-cx.
12
and IFN-y which may influence the local environment and promote a Thl type
phenotype.
Macrophages are components of the innate immune system which have microbial
and cytotoxic activity. They are capable of release of reactive and oxygen and nitrogen
intermediates (ROI, RNI) along with an ensemble of enzymes and cytokines. They also
function as professional antigen presenting cells (APC) presenting peptides in the context
of MHC II. Macrophages along with natural killer cells are thought to participate in the
rejection of the fetus in high loss matings.
Treatment with anti-macrophage Mac-l
antibody decreases the incidence of resorption in the CBA/J X DBA/2 model (Baines et
al., 1997). Immunohistochemistry has shown that macrophages infiltrate 20-30% of
implantation sites in high loss CBA female X DBA male matings correlating to the
incidence of resorption (Duclos et al., 1995). Furthermore, this infiltration is not seen in
the control 10w loss mating of CBA/J X BALB/c.
A similar proportion of these
infiltrating macrophages were also shown to be activated due to the increased expression
of the class II MHC antigen. The infiltration of these macrophages occurs at day 8 just
before embryo pathology occurs. If these macrophages are involved in embryo loss then
their mechanism of action is likely to be related to the mediators they release. Activated
macrophages found in implantation sites destined to resorb, are a major source ofTNF-ex.
rnRNA (Merkouris, 1999 Haddad et al., 1997) and this Thl cytokine has been implicated
in the normal processes of pregnancy since uterine and trophoblast cells also show TNFex. production. It may also be involved in regulation of trophoblast growth by stimulating
apoptosis (Yui et al., 1994). However, overproduction of TNF-ex. may cause damage to
the early embryo and administration of TNF-ex. to pregnant mice before day 10 of
13
gestation led to fetal death (Chaouat et al., 1990).
Conversely use of the TNF-a
suppressing drug pentoxifylline (PXF) prevented abortion induced by lipopolysaccharide
(LPS) (Gendron et al., 1990).
Furthermore, LPS, which is known to increase the
incidence of resorption, may function by triggering the production and release of TNF-a
from primed macrophages. In the CBA/J female X DBA/2 male mating, increased TNF-
a rnRNA expression at implantation sites correlates to the incidence of resorption
(Haddad et al., 1997) indicating that TNF-a may be a necessary factor in fe!~l death. In
the same study, inducible nitric oxide synthase (iNOS) rnRNA was seen at elevated levels
to the same embryos with increased TNF-a rnRNA suggesting a similar or related role.
Activation of macrophages leads to the synthesis of nitric oxide (NO) from L-arginine by
iNOS (Knowles & Moncada, 1994). This free radical short range mediator may exert its
effects directly through cytotoxicity or by increasing local cytotoxic activity of TNF-a.
The association between NO production and embryo loss can be seen by the increase in
levels of NO production by LPS stimulated decidual cens from resorbing embryos
(Haddad et al., 1995).
Furthermore, treatment with aminoguanidine that selectively
inhibits the inducible form of nitric oxide synthase, effectively lowers the incidence of
resorption. From these studies, NO along with TNF-a has been suggested to mediate
lethality to the embryo. However, release ofthese molecules from macrophages requires
2 signaIs. The first signal will prime resting macrophages and the second signal will
trigger production of NO and TNF-a (Baines at al. 1997). Priming of macrophages may
be achieved by IFN-y (Haddad et al., 1997), a Th1 cytokine produced by Th1 cens and
natural killer cens (Merkouris, 1999). Administration of IFN-y increases the abortion
rate presumably by priming macrophages (Baines et al. 1997). The dependence on IFN-y
14
for priming suggests that macrophages are not the sole immune effectors participating in
early embryo loss
The fact that decidual macrophages respond to LPS triggering in vivo suggests
that they are primed during the implantation of the embryo. Priming of macrophages
requires IFN-y and a potent source ofthis cytokine is the natural killer cell. NK cells are
components of innate resistance and are important in killing tumor cells, viroses and
other intracellular pathogens. In such infections, MHC 1 expression is downregulated
(Moretta et al., 1996) and NK cells can recognize this via cell surface receptors that
recognize MllC 1 (Yokoyama, 1995). NK cell receptors that recognize MHC 1 belong to
the Ig superfamily or the C-type lectin family. In humans, the killer inhibitory receptor
(KIR) on NK cells belongs to the Ig superfamily (Long et al., 1997) whereas the
NKG2/CD94 receptors belong to the C-type lectin family (Lopez-Botet et al., 1997). In
mice, the Ly-49 family of cell surface receptors is involved in recognition of class 1
molecules (Brennan et al., 1996). The receptors from both species function in a similar
fashion and binding of the receptor transmits a negative signal which downregulates NK
cell function. Without this negative signal, NK cells exert their cytotoxic effects upon
target cells suggesting that NK cells are always active. However, more recent data has
shown the existence ofNK cell receptors responsible for the delivery of a positive signal.
These natural cytotoxicity receptors (NCR) are the first evidence of actual triggering
receptors (Moretta et al., 2000). This can be seen by using monoclonal antibodies to
NCRs such as NKp46, NKp30 and NKp44 and observing a reduction in the lysis of
tumor target cells (Moretta et al., 2000). Unlike macrophages, NK cells do not require
priming or activation to exert their effects (Moretta et al., 2002).
The trophoblast
15
expresses non-conventional MHC and thus avoids direct perforin and granzynie mediated
cytolysis by NK cells and this is supported by in vitro data (Zuckermann & Head, 1988).
Furthermore, NK cells isolated from early implantation sites show only weak cytotoxicity
indicating that they probably do not directly attack the embryo (Baines et al., 1997).
However NK cells also employ cytokine production as a major effector mechanism.
Consequently, NK cell derived IFN-r is thought to consitute the major signal for priming
of macrophages (Haddad et al., 1997). Supporting this theory are studies relating NK
cell numbers and activity to embryo resorption. In humans, elevated numbers of NK
cells in peripheral blood of women with a history of recurrent spontaneous abortion
indicate an increased risk of abortion (Emmer et al., 1999). In the high loss mating
DBA/2 X CBA/J, infiltration of the decidua by natural killer cells is observed at sites of
resorption (Gendron & Baines, 1988). Administration of IFN-r has a similar effect as
TNF-a and demonstrates an abortive effect in mice (Chaouat et al., 1990). Neutralizing
the IFN-r via injection of anti-IFN-r anti-sera reduces the incidence of abortion. Haddad
and colleagues (1997) showed that pregnant IFN-r deficient GKO mice were more
resistant to the effects ofLPS than were wild type mice. This result suggested that IFN-r
was the major and perhaps exclusive cytokine involved in priming of macrophages in the
decidua. Consequently, the non-primed macrophages could not be triggered by LPS to
exert their effector functions. The result also supported the theory that the majority of
macrophages in the decidua are already primed. Injection of pregnant mice with the
double stranded synthetic RNA poly I:C significantly raises the incidence of resorption
(de Fougerolles & Baines, 1987). A proposed mechanism of action is that poly I:C
boosts the activity ofNK cells such that IFN-r production is increased. This increase in
16
IFN-y production will lead to increased priming of macrophages.
Since LPS will
stimulate abortion in normal pregnancy, macrophages may be a part of normal
implantation and placental development even though they are in a primed state.
Overproduction of IFN-y and perhaps TNF-a such as that by poly I:C stimulated NK
cells may indicate that the triggering signal is in fact due to the high levels of IFN-y or
TNF-a.
Indeed, normal gestational levels of IFN-y may be important for placental
growth and may trigger HLA-G expression on the trophoblast (Lefebvre et al., 2000).
Consequently, the role of natural killer cells during successful pregnancy may be to
secrete cytokines beneficial to pregnancy.
These may include GM-CSF, CSF-1,
leukemia inhibitory factor, TGF-I31, TNF-a and most importantly IFN-y.
17
Raâonale and Objectives orthe Studv
Although Th! cytokines are present at relatively low levels, a Th2 type profile is the
predominant one in normal pregnancy. So in abortion prone pregnancies, NK cells that
have been activated to secrete more IFN-y will contribute to a reverse in the Thl/Th2
balance and may start a series of events leading to a predominance ofThl type cytokines.
Our principal hypothesis related to the mechanisms of early embryo loss
i~._as
follows.
NK cells in resorption prone pregnancies increase in numbers and are somehow
stimulated to become fully active.
In this state, there is a significant increase in
production of Thl type cytokines such as IFN-y and possibly TNF-a. NK cell derived
IFN-y will prime resting decidual macrophages inducing the transcription of proinflammatory genes including iNOS and TNF-a. A second signal such as TNP-a, LPS
or IFN-y will then trigger. these macrophages to a fully active state.
In this state,
macrophages will produce NO and TNP-a which will subsequently exert their cytolytic
effects on fetal targets such as the placenta. Previous work in our lab has shown that in
vivo depletion of NK cells or macrophages can abrogate embryo loss in the DBN2 X
CBNJ model (de Fougerolles & Baines, 1987 Duclos et al., 1994).
Further, the
incidence of resorption in this high loss mating coincides with the increased expression of
macrophage activation markers, TNP-a and iNOS rnRNA (Haddad et al., 1997). RTPCR analysis also showed a strong association between IFN-y rnRNA and resorption
(Haddad et al., 1997). Normal but elevated distribution of perforin rnRNA across all
embryos in the high loss mating in combination with weakened cytotoxicity against
trophoblast suggests that direct
NI(
cytotoxicity was not responsible for embryo loss
18
(Merkouris, 1999). Finally, examination of pooled decidual DX5+ NK cells showed that
they may be the major source of decidual IFN-y.
From these results we asked the
following questions:
1. Do maternaI NK cells infiltrate the decidua in greater number in resorbing
embryos than in non-resorbing embryos?
2. Do these infiltrating NK cells produce the macrophage priming and activating
cytokines that are thought to participate in embryo loss?
3.
Do these NK cells participate in the production of a Th1 type profile in
resorbing embryos and in a Th2 type profile in non-resorbing embryos?
19
Materials and Methods
1. Mice and Matings
CBAlJ (H2-k) female mlce were purchased from Jackson Laboratories (Bar
Harbor, ME, USA) and DBAl2 (H2-d) males from Charles River (St-Constant, Quebec,
Canada). AlI mice were obtained between 8-9 weeks old and acclimatized to the animal
facilities for a minimum of 4 weeks. Mice were housed in open-top wire cages and
provided food and water as necessary. Illumination of the animal facility followed a 12
hour light/dark cycle.
Matings were performed by placing 4 females with 1 male
ovemight. Pregnancy was indicated by the presence of a copulatory plug and this was
arbitrarily designated as day 0 ofpregnancy.
2. Isolation and Preparation of Tissues
Day 9 pregnant mice were sacrificed by cervical dislocation.
Individual
implantation sites containing the embryos and surrounding deciduum were removed and
placed in tubes containing Mg2+ and Ca2+ free phosphate buffered saline (PBS) pH 7.2
(137mM NaCl, 2.7mM KCI, 4.3mM Na2HP04·7H20, 1.4mM KH2P04) supplemented
with 2.5% fetal calf serum (FCS).
Individual implantation sites were then quickly
homogenized with a Tissuemizer homogenizer (Tekmar, Cincinnati, OH, USA) at low
power output (10/100) on the Tekamer TR-I0 Power Control to disperse the tissue and
yield a single cell suspension. The cell suspension was spun down at 140xg for 10
minutes and resuspended in PBS + 2.5% FCS ready for labeling by fluorescent or
magnetic markers. Splenic tissue was also acquired and prepared in a similar fashion.
20
Due to the increased population of red blood ce11s (RBC), an additional RBC Iysis step
was performed. Briefly, the splenic ce11 suspension was spun down and resuspended in
ACK lysis buffer (0.15M ammonium chloride, lOmM potassium bicarbonate, 0.1mM
sodium EDTA, pH 7.3) for 3 minutes at room temperature. This was fo11owed by 2 cell
washings in PBS + 2.5% FCS and final resuspension.
3. Cell Counts ofSuspensions
The number of cells m each ce11 suspenSiOn was determined usmg a
haemacytometer. Ce11s were stained with white blood ce11 (WBC) counting fluid (0.01%
gentian violet in 3% acetic acidlwater) and counted under a microscope.
Cell
concentration was determined using the formula:
x cells/ml = total ce11 count in white squares x 104/mL x l
number of white squares counted
dilution factor
Viability of ce11 populations was assessed using Trypan blue exclusion.
4. lmmunofluorescent Staining ofNatural Ki/1er (NK) Cells in Embryonic and Splenic
Cell Suspensions
NK cells in suspensions were labeled with a phycoerythrin (PE) conjugated rat
anti-mouse pan-NK cell monoclonal antibody (Pharmingen) specifie for the DX5 surface
marker. For the splenic ce11 suspension, 106 total cells were taken for labeling. In the
embryonic ce11 suspension, a11 the cells were taken for labeling. Pre-incubation of cells
21
for 5 minutes on ice with 11lg purified rat anti-mouse CD16/CD32 monoclonal antibody
(pharmingen) was necessary to prevent non-specific binding of immunoglobulins to
mouse B cells via FcyII receptors. Following pre-incubation, 11lg ofPE-linked antibody
was added and allowed to incubate for 15 minutes on ice in the dark.
The cell
suspensions were then washed twice in PBS + 2.5% FCS and finally resuspended in
500llL PBS + 2.5% FCS. Finally, 2.5llg of propidium iodide was added to discriminate
against dead cells.
Analysis of cell suspensions was performed by
flo~_cytometry.
Percentages and total numbers of DX5 positive cells were calculated using the FACS
analysis software.
5. Magnetic Cell Sorting ofNK cells
Natural killer cells from individual embryonic and splenic cell suspensions were
isolated using MACS technology (Miltenyi Biotec).
Cellular suspensions were
centrifuged at 300xg for 10 minutes and resuspended in 90 IlL degassed PBS + 2.5%
FCS per lOx106 cells.
Ten microliters of MACS super-paramagnetic microbeads
conjugated to monoclonal rat anti-mouse NK cell antibody (DX5) per 107 cells was
added and allowed to incubate for 20 minutes at 4°C. The cells were washed twice with
1O-20X labeling volume of buffer and centrifuged at 300xg for 10 minutes. After the
final wash, the cells were resuspended in 500 ilL degassed buffer per 100x1 06 cells.
These cells were then used for magnetic separation of the NK cells.
The magnetic
separation apparatus consisted of MACS stand, magnetic column holder and single use
sterile separation columns. Briefly, the magnetically labeled cell suspension is passed
through the column and the positively labeled cells (DX5+) are retained in the column.
22
The negative fraction (DX5-) is eluted and the column washed three times with degassed
buffer. Following the washes, the column is removed from the magnetic holder and the
positive fraction is flushed out using a plunger. The positive fraction is then reapplied to
a new column and re-eluted as above. The positive and negative fractions were then
stained by fluorescent markers to verify purity or directly used for RT-PCR analysis.
6. RNA Extraction
Total RNA from cells was extracted using Trizol reagent (Life Technologies).
10,000 cells were delivered into a microfuge tube containing 250~ ofTrizol reagent and
pipetted up and down to ensure complete lysis of cells. This sample was stored at -SO°C
until ready for extraction. At time of extraction, the sample was thawed and incubated at
room temperature for 5 minutes.
Addition of 50~ of chloroform was followed by
vigorous shaking by hand for 15 seconds and subsequent incubation at room temperature
for 3 minutes. The sample was centrifuged at 12,OOOxg for 15 minutes at 4°C to separate
the sample into a lower organic phase, an interphase and an upper aqueous phase. The
RNA-containing upper aqueous phase was transferred into a new microfuge tube.
Precipitation of the RNA was aided by adding lOllg oflinear polyacryalmide carrier prior
to 125~ of isopropyl alcohol. The sample was mixed thoroughly and stored at -20°C
ovemight. The following day, the RNA was pelleted by centrifugation at 12,000xg for
15 minutes at 4°C. The RNA pellet was washed with 250~ of chilled 75% ethanol and
mixed by voretxing. The pellet was spun down again at 7,50Oxg for 5 minutes at 4°C.
The supematant was removed and the pellet allowed to air-dry for 20 minutes. Finally,
the RNA was resuspended by dissolving the pellet in
1O~
of 0.1% diethylene
23
pyrocarbonate (DEPC) treated H20 and incubated for 15 minutes at 65°C. The total
RNA solution was then used in RT-PCR analysis.
7. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) Analysis
Total RNA extracted from DX5 positive and negative cells were reverse
transcribed to cDNA. To the
1O~
total RNA solution was added
Oligo dT primers (Life Technologies),
l~
1~
of SOOIlg/mL
of 100nM random hexamer primers (Life
Technologies) and lllL of lOmM dNTP mix (Amersham Pharmacia Biotech).
The
mixture was heated to 65°C for 5 minutes followed by a quick chill on ice. After a brief
centrifugation to collect the contents of the tube, 4~ of 5x 1st strand buffer (Life
Technologies),
2~
of O.lM DTT (Life Technologies),
RNase Inhibitor (Amersham Pharmacia Biotech) and
0.5~
0.5~
II reverse transcriptase (Life Technologies) was added.
of 36 units/~ RNAguard
of 200 units/llL Superscript
The reaction was allowed to
proceed for 60 minutes at 50°C followed by inactivation at 70°C for 15 minutes. Finally,
the mixture was diluted to a total of 100llL with sterile distilled water. This cDNA was
then used for amplification in PCR reactions. As a control, each experiment included a
negative control in which no reverse transcriptase enzyme was added.
Gene specifie primers and probes (Table 1) for G6PDH and TNP-a. were taken
from previously published papers (Haddad et al., 1997).
IFN-r and perforin primers and
probes were taken from a previous Master's student's thesis (Merkouris, 1999).
Oligonucleotides were designed using Oligo 4S software with cDNA sequences retrieved
from Genbank. Synthesis of the oligonucleotides was performed in-house at the Sheldon
24
Biotechnology Center (McGill University) and they were stored at a concentration of
1IlglJJL till use.
Once cDNA had been produced, gene specifie primers were used to detect
G6PDH, IFN-y, TNF-a and perforin rnRNA via PCR (Table 1). Each reaction sample
consisted of 35JJL of sterile, distilled water, 5J.1L of the diluted cDNA and 10 J.1L of a
PCR reaetion mixture for a total reaction volume 50J.1L.
The PCR reaction mixture
consisted of 51lL lOX PCR buffer (50mM KCI, lOmM TrisHCI, 1.5mM MgÇh, 0.01%
Triton X), 2J.1L of lOmM dNTP mix (Amersham Pharmacia Biotech), IJJL each of
0.15IlglJ.1L sense and anti-sense gene specifie primers and IJ.1L of 2.2 units/J.1L Taq
polymerase (Life Technologies).
The amplification reaetion was an initial 4 minutes
strand denaturing at 94°C followed by 40 cycles of: 1 minute denaturing at 94°C, 1.5
minutes at the primer annealing temperature and 1 minute elongation at 72°C. After the
final cycle, an additional 10 minute elongation step at 72°C was performed.
As a
negative control, each experiment included a reaction mixture in which no cDNA was
added.
8. Southern Blotting
The amplification products of the RT-PCR reaction were visualized under ultraviolet light on a 1% agarose gel containing ethidium bromide. Amplification bands were
documented using Kodak Digital Science digital photography software. If necessary,
amplification produets were transferred to a solid support by gel transfer hybridization.
For this, the gel was first immersed in a denaturation buffer (87.66g NaCI and 20g NaOH
in IL distilled water) and placed on a shaker for 30 minutes. The denaturation step was
25
followed by immersion in a neutralization buffer (87.66g NaCI and 60.5g Trizina Base in
IL distilled water) for 30 minutes on a shaker. The DNA from the gel was then allowed
to transfer overnight to a Hybond-N nylon membrane (Amersham Pharmacia Biotech)
via capillary action as described by Southern (1975). The next day, the immobilized
DNA was crosslinked to the membrane by a UV Stratlinker (Stratagene). Membranes
were then kept sandwiched dry in blotting paper till further use.
9. Radiolabeling ofGene Specifie Oliognucleotide Probes
The 5' ends of the oligonucleotide probes for G6PDH, IFN-y, TNF-cx. and perforin
(Table 1) were radiolabeled using Ready-To-Go T4 Polynucleotide Kinase (Amersham
Pharmacia Biotech). Briefly, the tube T4 PNK was reconstituted using 25J,LL of sterile
water and incubated for 5 minutes at room temperature. To this tube, 10pmoles of 5'end
oligonucleotides was added and the reaction volume was brought up to 49J,LL with sterile
water. Finally, IJ,LL of 1OIlCi/IlL [y_32p]_ATP was added and the reaction was incubated
at 37°C for 30 minutes.
2S0mM EDTA.
The reaction was subsequendy stopped by adding 51lL of
Unbound [y)2p ]_ATP was removed from the reaction using a
ProbeQuant Sephadex G-SO Micro Column (Amersham Pharmacia Biotech) and
centrifuging the reaction mixture at 735xg for 2 Ill:inutes. The purified radioactive probe
was then ready for use in hybridization reactions.
10. Membrane Hybridization with Radiolabeled Probes
Detection of amplification products immobilized on nylon membrane with
radiolabeled probes followed a protocol described by Haddad et al. (1997).
A pre-
26
hybridization solution consisting of 8.9mL water, 7.5mL 20X SSC (88.23g Tri-sodium
citrate and 175.32g NaCL in IL of distilled water), 3mL lOOX Denhardt's solution (2.0g
bovine serum albumin, 2.0g Ficoll 400 and 2.0g ofpolyvinylpyrrolivolone in 100mL of
distilled water) and lO.5mL 20% SDS was made. This pre-hybridization mixture was
placed in a 65°C water bath for 15 minutes followed by the addition of 100J.1,L of boiled
salmon sperm DNA (Amersham Pharmacia Biotech). The mixture was then added to
specifie hybridization tubes containing membranes which had been pre-wetted in 5X
SSC. The tubes were then incubated at the probe's annealing temperature for 2 hours in a
rotary hybridization oven. Following the pre-hybridization, 50J.1,L of radioactive probe
was added to the tube and the hybridization was allowed to proceed ovemight. The
following day, the membranes were washed in 2X SCC and 0.1% SDS in a volume of
400mL of water for 20 minutes. This wash was repeated and then washed twice further
in O.lX SSC and 0.1% SDS in a volume of 360mL water. After the final wash, the
membranes were dried between blotting paper, wrapped in plastic film and placed in a
Molecular Dynamics phosphorimaging screen cassette. Exposure of the membranes to
the screen proceeded ovemight and bands were visualized using a Storm phosphorimager
(Molecular Dynamics).
Quantitation of the bands was performed by ImageQuant
software (Molecular Dynamics).
11. Statistics
Statistical analysis of FACS data and phosphorimaging bands was computed
using Microsoft Excel and SPSS for Windows (SPSS Inc.). ImageQuant data files were
comprised of intensity of bands for each specifie cytokine. Band intensity of IFN-y,
27
TNF -(l and perforin were normalized against the expression of the housekeeping gene,
G6PDH. K-means cluster analysis was used to determine intra-group means of a sample
population which had a bimodal distribution. The upper 95% confidence limit (K means
of cluster + (1.96 x standard deviation)) of a normal population was calculated using Kmeans to determine significance in a sample data set.
28
Table 1. Oligonucleotide Primer and Probe
Sequences
Gene
Sequence (5'
~
3')
Annealing
Temperature
Expected
Amplification
Product Size
(Basepairs)
G6PDH
Sense
Anti-8ense
Probe
CTAAAC TCAGAAAAC ATC ATG GC
TAG GAA TTA CGG GCA AAG AAC TC
GAG CAG GTG GCC CTG AGC CG
55.6"C
338
IFN-y
Sense
Anti-8ense
Probe
ACA CTG CAT CTT GGC TTT GC
CGA CTC CTT TTC CGC TTC CT
GGA GGAACT GGC AAA AGG ATG G
60CC
450
TNF-a
Sense
Anti-8ense
Probe
CCA GAC CCT CAC ACT CAG AT
AAC ACC CAT TCC CTT CAC AG .
CCA AGT ooA GGA GCA GCT GGA G
580C
498
Perforln
Sense
Anti-8ense
Probe
m TCC TOC TGC TGC CAC GAC CTG
GCCGTGATAAAG TGCGTGCCATAG
ACA GAG ooT GCA GGT GCG GTC AGG
60CC
646
29
Results
1. Sample Populations
Individual implantation sites from day 9 pregnancies from DBA/2 male mated
CBA/J female mice were taken for analysis in these experiments. At this particular day
of gestation, embryos can be rescued from the process of resorption normally complete
by day 12 of gestation. Embryos were analyzed for the number of natural killer cells
(DX5+) and purified into natural killer cell enriched and depleted populations. Magnetic
sorting of natural killer cells from individual embryos yielded purities for enriched and
depleted populations of >90% and >99% respectively.
The 6 pregnancies taken for
analysis contained an average of7.8±J.3 embryos per female mouse.
2. FACS Analysis ofDay 9 Embryos Shows Elevated Natural Killer Cell Numbers
Individual implantation sites from DBA/2 mated CBA/J females were assessed at
day 9 of pregnancy to determine whether there were more NK cells in sorne
presumptively resorbing embryos than in the majority of normal embryos. NK cells were
stained with a PE-linked rat anti-mouse pan-NK cell monoclonal antibody.
F ACS
analysis (Figure 1) indicated the presence of 2 distinct populations of embryos. Cells
displayed a broad range of fluorescence intensity indicating the presence of larger or
activated cells. K-means cluster analysis of the bimodal distribution was calculated and
the mean of the normal population was used to determine the 95% upper confidence limit
(DCL) for the majority of the embryos. The 95% DCL was calculated at 7.70% ofDX5+
cells with 14 embryos displaying elevated percentages of NK cells (Figure lA). This
30
corresponds to about 22% of embryos which is in the range of the incidence of embryos
resorption (20-30%) in this model. In terms of absolute numbers (Figure lB), the 95%
DeL was calculated at 15.8x104 of DX5+ cells with 16 embryos out of 37 showing
elevated numbers of NK cells. This corresponds to about 43% of embryos which is
above the incidence of resorption in the model. Analysis of the 37 embryos (Table 2)
after bimodal distribution showed that the major group of embryos (21) with low NK cell
numbers gave a lower total cell yield of 2.90x106 ± 1.05x106 and a lower percentage of
NK cells at 2.34% ± 2.19%. The minor group of embryos (16) with high NK cell
numbers gave a higher total cell yield of 4.86x106 ± 1.43x106 and a higher percentage of
NK cells at 7.32% ± 4.41%. AlI the embryos appeared healthy and therefore the NK cell
infiltration occurs before any apparent embryo destruction.
31
Figure 1. Quantitation of DX5 Positive Natural Killer Cells in Individual Embryos
at Day 9 of Pregnancy
Individual implantation sites from CBA/J female X DBA/2 male matings were
taken at day 9 of pregnancy and assayed for numbers of natural killer cells expressing the
DX5 marker.
A total of 64 embryos were labeled with DX5 antibody and NK cell
numbers were quantitated via FACS analysis on an acquisition of 10,000 cells for each
embryo. The histogram in figure lA shows the frequency of embryos expressing varying
percentages of DX5 positive NK cells. The histogram in figure lB shows the frequency
of embryos containing numbers of DX5+ cells. Due to technical reasons, numbers of
DX5+ cells (Figure lB) were only obtained for a total of37 embryos.
32
Figure 1
A.
Natural Killer CeUs in Individuallmplantation S~es
10
:g
91:::::}}}}}::::}:1
8 -!":':':':::::':':':':':::::::':':~
>~ 7 -K{:;:::{f.l!3}::~
~ 6
'0 5
G> 4
~ 3
~ 2
1
O.filli:I4lli$llit+m$llil'+illt+mtmrifimifiliill:jill>LfS:~p:mP&fillill'pillpill~~pillj
%DX5+ ceUs
B.
Natural Killer Cells in Individualll'lllianlation Sites
7
6
o
~
~
~
~
~
~
~
~
~
4
)
Nurrilers of OX5+ cells (10
~
~
~
Table 2. NK-cell Statistics trom FACS Analysis
Cell Populations in
the Deciduallissue
of Individual
Major Group of 21
Embryos with lOIN NKœil NumbersJEmbryo
Minor Group of 16
EnDyos with high NKcel 1NumberslEmbryo
Total Cell
YieidiEmbryo
2.90X1Q6 ± 1.05X1Q6
4.86X1Q6 ± 1.43X1Q6
Number of NKcellslEmbryo
6.02X1Q4 ± 4.36X1Q4
3. 15X1 05 ± 1.23X105
Percentage of NK-cells
(%)
2.34±2.19
7.32±4.41
Ermryos
33
3.
Cytokine Expression in Natural Killer Cell Enriched and Depleted Populations
/rom Individual Embryos
Magnetic cell separation (MACS) allowed for the sorting of DX5+ cells from
individual embryos.
After two passes through a magnetic column, the purity of the
DX5+ enriched sample was shown to be greater than 90% by FACS analysis. Similarly,
the DX5 negative cells in the column flow-through was efficiently depleted of DX5+
cells after 1 pass to greater than 99% non-DX5+ cells. RT-PCR analysis was performed
on the DX5- and DX5+ fractions for 47 embryos. Primers were used to amplify cDNA
for the housekeeping gene G6PDH and the cytokines IFN-y, perforin and TNF-a.. A
representative example for the analysis of one pregnancy is shown in Figure 2.
Radioactive hybridization demonstrated constitutive expression of the housekeeping gene
G6PDH in aIl 9 embryos and in the spleen control in both DX5+ and DX5- fractions.
Normalization of IFN-y, perforin and TNF-a. signal to the housekeeping signal enabled
the calculation of relative units of cytokine expression. Comparative analysis of IFN-y
rnRNA expression (Figure 3) showed that 6 of9 embryos expressed more IFN-y rnRNA
in DX5+ samples as opposed to the DX5- sample. The remaining 3 embryos showed
similar levels of IFN-y rnRNA or slightly higher levels in DX5- samples. However, in
terms of relative units, aIl embryos displayed varying levels of IFN-y expression across
both DX5+ and DX5- fractions. For TNF-a. rnRNA expression (Figure 4), DX5+ and
DX5- cells showed similar levels of expression within each individual embryo based on
10,000 cells. Similarly to IFN-y expression, embryos also displayed varying levels of
TNF-a. in an embryo to embryo comparison. FinaIly, perforin rnRNA expression (Figure
5) was similar to TNF-a. expression with both DX5+ and DX5- cells showing similar
34
levels of expression at the individual embryo level. In a comparison across "embryos, 2
embryos displayed elevated levels of perforin expression. Furthermore, the data aIso
shows that in the DX5- population, IFN-y and TNF-cx. rnRNA expression was also notable
in 2 and 3 embryos respectively. However, none of the embryos displayed both IFN-y
and TNF-cx. rnRNA expression simultaneously
35
Figure 2. Expression of IFN-y, Perforin and TNF-a. mRNA in Individual Embryos
at Day 9 of Pregnancy
Individual embryos from CBA/J female X DBA/2 male matings were taken at day
9 of pregnancy and labeled with magnetic beads conjugated to DX5 specifie monoclonal
antibody. Cell populations from individual embryos were sorted into DX5+ and DX5fractions and analyzed. The figure shows the Southem blot of a pregnancy containing 9
embryos and a spleen control.
36
Figure 2
SPLEEN
OX5 Marker
EMBRYOS
+_ +_ +_ +_ +_ + _ + _ +_ +_ +_
G6PDH
IFN-y
TNF-a
CEE
1
2
E
3
E
4
E
5
E
6
E
7
E
8
Individual Embryos at Day 9 of
Pregnancy
E
9
Figure 3. Relative Expression of IFN-y mRNA at Day 9 of Pregnancy
Individual embryos from CBA/J female X DBA/2 male matings were taken at day
9 of pregnancy and labeled with magnetic beads conjugated to DX5 specifie monoclonal
antibody. Cell populations from individual embryos were sorted into DX5+ and DX5fractions and analyzed.
The histogram shows the relative levels of IFN-y rnRNA
expression in DX5+ and DX5- fractions from one pregnancy containing 9 embryos. Note
that embryos 1, 2, 4, 6 and 8 express large amounts of IFN-y rnRNA in their DX5+
fraction as compared to their DX5- fraction.
37
Figure 3
Relative IFN Gamma Expression at Day 9 of Gestation
0.16
0.14
0.12
,l(!
c:
0.1
I~DX5+1
.DX5-
::J
.~ 0.08
ilQ)
a:
0.06
0.04
0.02
2
3
4
5
Indvidual Embryos
6
7
8
9
Figure 4. Relative Expression ofTNF-a. mRNA at Day 9 ofPregnancy
Individual embryos from CBA/J female X DBA/2 male matings were taken at day
9 of pregnancy and labeled with magnetic beads conjugated to DX5 specifie monoclonal
antibody. Cell populations from individual embryos were sorted into DX5+ and DX5fractions and analyzed.
The histogram shows the relative levels of TNF-a. rnRNA
expression in DX5+ and DX5- fractions from one pregnancy containing 9 embryos. Note
that most embryos express similar amounts of TNF-a. rnRNA in their DX5+ and DX5fractions.
38
Figure 4
Relative TNF Alpha Expression at Day 9 of Gestation
0.08
0.07
0.06
en
0.05
""
<:
::;)
IS0X5+1
BOX5-
.~ 0.04
:iii
Q)
a:
0.03
0.02
0.01
a
2
3
4
5
Individual Embryos
6
7
8
9
Figure 5. Relative Expression ofPerforin mRNA at Day 9 ofPregnancy
Individual embryos from CBA/J female X DBA/2 male matings were taken at day
9 of pregnancy and labeled with magnetic beads conjugated to DX5 specifie monoclonal
antibody. Cell populations from individual embryos were sorted into DX5+ and DX5fractions and analyzed.
The histogram shows the relative levels of perforin rnRNA
expression in DX5+ and DX5- fractions from one pregnancy containing 9 embryos. Note
that most embryos express similar amounts of perforin rnRNA in their DX5+ and DX5fractions.
39
Figure 5
Relative Perforin Expression at Day 9 of Gestation
I~D~+I
.D~-
2
3
4
5
Individual Embryos
6
7
8
9
4. DX5+ Cells from lndividual Embryos Express Significant Levels of IFN-'F, TNF-a
and Perforin
It was shown that decidual infiltration of NK ceIls increase in a proportion of
embryos similar to that of the incidence of resorption in the CBA/J female X DBA/2
male mating. Further, expression of IFN-y was higher in DX5+ ceIls than in DX5- ceIls
implicating NK cells as a major source of this cytokine. Frequency distribution plots of
IFN-y, TNP-a and perforin were created to examine further the relationship of cytokines
to NK cens. For plots with apparent bimodal distributions, K-means cluster analysis was
used to calculate the means of the 2 modes. Dsing the mean of cluster 1 and its standard
deviates, a 95% upper confidence limit (DCL) was calculated. Any values above the
95% DCL were classified as significant.
A histogram examining IFN-y rnRNA
expression in DX5 + cens (Figure 6A) showed an apparent bimodal distribution and its
95% DCL was calculated to be 0.327. From aIl 47 embryos analyzed, 18 embryos (38%)
showed significant expression ofIFN-y. This data indicated that not aIl NK ceIls from aIl
embryos produced equivalent amounts of IFN-y indicating that only sorne embryos were
infiltrated by active cytokine producing NK ceIls. Similarly to IFN-y, TNP-a expression
was also significant in 14 embryos or 30% in DX5+ cens (Figure 7A) indicating that NK
cens may be participating in the production ofthis Th1 cytokine. A 95% DCL of 0.679
was calculated and many embryos were expressing very large amounts of TNP-a in
DX5+ cens. Perforin rnRNA also seemed to be statisticany significant (Figure 8A) after
a 95% DCL of 0.521 was calculated. A total of Il embryos or 23% were expressing
increased amounts of perforin in DX5+ cens.
40
5. DX5- Cells Irom Individual Embryos Express Significant Amounts 01 Perforin and
TNF-a
Since NK cells were thought to be the major source ofIFN-y, non-NK cells were
thought to participate minitnally in the expression of this Th1 cytokine. In DX5- cells
(Figure 6B), IFN-y expression was significant in only 6 embryos or 13% indicating that
non-NK cells are not producing large levels of IFN-y.
There was however a large
separation of modes with the 6 embryos displaying large relative expression levels of
IFN-y indicating the possibility of non-NK cens participating in increased IFN-y
expression. TNF-a expression in DX5- cens (Figure 7B) was significant in 9 embryos or
19%. Perforin expression in DX5- cens (Figure 8B) was also significant in 13 embryos
or 28 %. Interestingly, this was a greater number of embryos than in DX5+ populations.
The 95% DeLs for IFN-y, TNF-a and perforin were calculated to be 0.727, 0.963 and
0.701 respectively.
41
Figure 6. Frequency Distribution of IFN-y mRNA Expression in DX5+ and DX5cells at Day 9 of Pregnancy
Individual embryos from CBA/J female X DBA/2 male matings were taken at day
9 of pregnancy and labeled with magnetic beads conjugated to DX5 specifie monoclonal
antibody. Cell populations from individual embryos were sorted into DX5+ and DX5fractions. A total of 6 pregnancies containing 47 embryos were analyzed. In figure 6A,
IFN-y expression in DX5+ cells shows a skewed distribution and the 95% DCL was
calculated as 0.327 above which 38% of embryos had significant expression ofIFN-y. In
figure 6B, IFN-y expression in DX5- cells also is more evidently bimodal and the 95%
DCL was calculated as 0.727 indicating that 13% of embryos had significant expression
ofIFN-y.
42
Figure 6
A.
Frequency distribution of IFN Gamma mRNA Expression in DX5+ CeUs at
Day 9 of Gestation
Relative Expression
B.
Frequency Distribution of IFN Gamma mRNA Expression in DX5- ceUs at Day
9 of Gestation
al··
······Î·[1·1·····:··,_
7
~
..Q
JI
.. :.:.: :::..:;.::.:..
6
5
'0 4
11
3
z
2
5
q
o
~
0
d
~
d
~
~
d
~
0
~
d
~
0
~
d
~
0
m
~
d 0
~
d
Relative Expression
~
0
~
d
~
0
~
d
~
0
~
d
Figure 7. Frequency Distribution of TNF-a. mRNA Expression in DX5+ and DX5cells at Day 9 of Pregnancy
Individual embryos from CBA/J female X DBA/2 male matings were taken at day
9 of pregnancy and labeled with magnetic beads conjugated to DX5 specifie monoclonal
antibody. Cell populations from individual embryos were sorted into DX5+ and DX5fractions. A total of 6 pregnancies containing 47 embryos were analyzed. In figure 7 A,
TNP-a. expression in DX5+ cells shows a skewed distribution and the 95% DCL was
calculated at 0.679 above which 30% of embryos had significant expression of TNP-a..
In figure 7B, TNP-a. expression in DX5- cells also shows a skewed distribution and the
95% DCL was calculated at 0.963 indicating that 19% of embryos had significant
expression of TNP-a..
43
Figure 7
A.
Frequency Distribution ofTNF Alpha mRNA Expression in DX5+ Cells at
Day90fGestation
Relative Expression
B.
Frequency Distribution ofTNF Alpha mRNA Expression in DX5- Cells at Day 9 of
Gestation
9
8
l
Ji
7
6
5
'ë
1i
§
z
4
3
2
o
Relative Expression
Figure 8. Frequency Distribution of Perforin mRNA Expression in DX5+ and DX5cells at Day 9 of Pregnancy
Individual embryos from CBNJ female X DBN2 male matings were taken at day
9 of pregnancy and labeled with magnetic beads conjugated to DX5 specifie monoclonal
antibody. Cell populations from individual embryos were sorted into DX5+ and DX5fractions. A total of 6 pregnancies containing 47 embryos were analyzed. In figure 8A,
perforin expression in DX5+ cells shows a skewed distribution and the 95% UCL was
calculated at 0.521 above which 23 % of enibryos had significant expression of perforin.
In figure 8B, perforin expression in DX5- cells also shows a skewed distribution and the
95% UCL was calculated at 0.707 above which 28% of embryos had significant
expression of perforin.
44
Figure 8
A.
Frequency Distribution of Perforin mRNA Expression in DX5+ cells al Day 9 of Gestation
7
o -Jllilli$M$OOl$M$OOl$M$OOl$M$OOltEt$OOltEtP
~~~~~.~~~~~~~~~~~.~.
l;)'
l;)'
l;)'
l;)'
l;)'
l;)'
l;)'
l;)'
l;)'
l;)'
l;)'
Relative Expression
B.
Frequency Distribution of Perforin mRNA Expression in DX5- Cells at Day 9 of
Gestation
6
5
Ul
o
~ 4
E
w
'0 3
,g
§ 2
z
0
.
"-
".
"-
~.
Relative Expression
Table 3. Summary of Cytokine Expression Levels
0/0
Cytokine
Cell
of embryos with
increased expression
IFN-y
TNF-u
Perforin
DX5+
38
.DX5-
13
DX5+
30
DX5DX5+
19
23
DX5-
28
45
Discussion
Examination of the immune effectors involved in early embryo loss has revealed that
macrophages and natural killer ceUs play major roles in mediating lethality to the
compromised embryo (Baines et al., 1997). In the CBNJ female X DBN2 male model,
immunohistochemical analysis has shown an infiltration of macrophages and natural
killer ceUs prior to embryo loss (Duclos et al., 1995). Moreover, production ofThl proinflammatory cytokines by these ceUs such as IFN-y and TNF-a. has been shown to be
embryotoxic (Hill et al., 1995). Regulation of this Thlrrh2 balance may be vital in the
maintenance of successful pregnancy. Previous work has demonstrated that natural killer
cells may be the primary source ofIFN-y in the deciduas (Merkouris, 1999). Ifthis is the
case, then activation of decidual NK ceUs could very weU produce the Thl type cytokine
profile leading to early embryo loss.
Furthermore, priming of resting decidual
macrophages is thought to be wholly due to NK cell derived IFN-y (Haddad et al., 1997).
To this end, the primary objective ofthese experiments was to better characterize the role
of NK cells in the high loss mating, CBNJ female X DBN2 male and to distinguish
whether augmented IFN-y production is related to increased NK ceU numbers, relative
activity or both.
Previously, immunohistochemical analysis of individual implantation sites from
CBNJ female X DBN2 male matings showed that sorne implantation sites are infiltrated
by asialo-GM1+ NK ceUs at day 8 of pregnancy (Gendron & Baines, 1988).
The
percentage of embryos that display increased NK cells is proportional to the percentage
of embryos that subsequently resorb. Quantitation of infiltrating NK cells was achieved
46
by counting asialo-GM1 + NK ceUs under the microscope. Rapid quantitation of NK
ceUs from individual embryos was accomplished using FACS analysis specific for the
DXS marker. The results (Figure lA) demonstrate that the percentage ofNK ceUs varies
in individual embryos up to 200,/0. A majority ofthe implantation sites displayed between
1-10% DXS+ NK ceUs and this is presumably normal. The histogram analysis displayed
an apparent bimodal distribution and K-means cluster analysis indicated that 22% of the
64 embryos examined showed a significant increase in the percentage ofNK ceUs. This
is equivalent to the incidence of early embryo loss in the CBA/J female X DBA/2 male
model.
Furthermore, in terms of numbers (Figure lB), 43% of implantation sites
displayed significant numbers of DXS+ ceUs. Implantation sites with total DXS+ ceUs
greater than lS.8x104 ceUs was deemed significant by K-means cluster analysis.
Statistical analysis ofthese major and minor groups of embryos (Table 2) showed that the
major group of 21 embryos with low NK ceU numbers also had a lower total ceU yield of
6
2.90x10 ± 1.0Sx106 . Conversely, the minor group of 16 embryos with high NK ceU
numbers also had a higher total ceU yield per embryo with 4.86x106 ± 1.43x106 .
Although the total ceU yield from the minor group was higher, the percentage ofNK ceUs
was still higher at 7.32% ± 4.41% as compared to 2.34% ± 2.19% from the major group.
The data demonstrates that inflammation in the embryo increases NK ceUs numbers
whilst also increasing total ceU yield. Reduction of other ceUs in the implantation site is
therefore unlikely since the total ceU yield in the normal major group is lower as
compared to the presumptively resorbing minor group of embryos. Thus, this FACS data
corroborates what was previously observed and reinforces the notion that infiltration of
NK ceUs is an accurate precursor to early embryo loss. Indeed, in women with recurrent
47
spontaneous abortion (RSA), increased numbers of NK cells prior to pregnancy indicate
an increased risk of abortion (Emmer et al., 1999). However, NK cell number is not
necessarily an indicator of cytotoxicity. Gilman-Sachs and colleagues (1999) showed
that there was no correlation between NK cell number and cytotoxicity. Also Baines and
colleagues (1997) showed that decidual NK cells are not actively cytolytic in vitro unlike
spleen NK cells. The lytic potential of NK cells may be regulated by their content of
perforin, recycling capacity and developmental stage.
Therefore, although the
relationship between infiltrating NK cells and embryo loss is established, are these NK
cells more active in resorbing embryos than in non-resorbing embryos?
Earlier work has shown that the incidence of resorption coincided with the
increased expression of TNF-a, iNOS and IFN-y rnRNA (Haddad et al., 1997). These
were thought to be produced by activated decidual NK cells and macrophages and this
notion was reinforced by the abrogation of early embryo loss following in vivo depletion
ofthese cells (Duclos et al., 1994). Furthermore, perforin displayed a normal distribution
across all embryos with perforin positive cells constitutively present in normal deciduum
indicating that there may be more than one population of perforin positive cell present
(Merkouris, 1999). Finally, preliminary evidence showed that DX5+ NK cells from the
decidua were perhaps the sole source ofIFN-y (Merkouris, 1999). Ifthat was the case,
NK cells and IFN-y may participate in the normal processes ofplacental development and
that the overproduction of IFN-y may lead to embryo loss. Certainly, IFN-y is produced
by pre-implantation embryos and its production may be to trigger HLA-G expression on
embryos (Ozomek et al., 1997). AlI previous data had indicated that NK cells were not
involved in direct lethality to the embryo.
This led to the notion that NK cells are
48
involved in the shaping of a Thl type environment unfavorable to the developing
embryo.
Indeed, Thl cytokines boost abortion rates whereas Th2 cytokines lower
abortion rates when administered in mice (Chaouat et al., 1995).
If the Thl/Th2
paradigm is important in explaining embryo loss then a balance of this profile or even
Th2 dominance may be critical in maintaining successful pregnancy (Raghupathy, 2001).
If as hypothesized that decidual NK cells are the major producers of IFN-y during
implantation and placental development then their active cytokine production may play a
large part in tipping the Thl/Th2 balance.. To examine the nature of these NK cells in
both resorbing and non-resorbing embryos, the cytokine expression levels of IFN-y,
perforin and TNF-a were examined. IFN-y and TNF-a are pro-inflammatory Thl type
cytokines and presumably their expression is important in the shaping of the local
cytokine environment.
Furthermore, peforin expression may be an indicator of the
cytolytic capability of the cells that produce it although NK cells constitutively express
perforin. To test the hypothesis that NK cells are the major producers ofIFN-y, Southem
blot and radioactive hybridization allowed for study of gene expression in both NK cell
enriched and depleted populations.
The results confirmed that NK cells express
significantly larger amounts ofIFN-y than in NK cell depleted populations (Figure 3). In
many embryos, expression levels ofIFN-y in DX5+ NK cells were more than double that
in DX5- cells. However sorne embryos displayed comparable levels of IFN-y in DX5cells and all DX5- populations did express sorne levels ofIFN-y. Thus, NK cells are not
the sole source ofIFN-y but they may he a major source. Since the magnetic purification
procedure of DX5+ cells did not achieve 100% purity in either depletion or enrichment
fractions, one cannot discount the possibility of a small contamination of a DX5-
49
population by NK cells. More likely another decidual cell may be producing IFN-y such
as T-cells or granulated metrial gland (GMG) cells. In contrast to the results obtained for
IFN-y expression, TNF-a (Figure 4) and perforin (Figure 5) expression was comparable
in both DXS+ and DXS- samples. TNF-a expression, although varied across embryos
was similar within DXS+ and DXS- populations in each individual embryo suggesting
that TNF-a expression in DXS- populations may correlate with macrophage activation.
Perforin expression was analogous which indicates the participation of other cells in
perforin expression.
Our principal hypothesis in early embryo loss is that NK cells actively infiltrate
implantation sites and are stimulated to become active and produce cytokines.
Production of these Th!
environment.
cytokines leads to an unfavorable pro-inflammatory
Moreover, local production of IFN-y by NK cells stimulates resting
macrophages into a primed state. Full activation of these macrophages is triggered by a
second signal, TNF-a or LPS. Macrophages then produce nitric oxide and TNF-a at
much higher levels which together mediate lethality to the embryo. Our initial results
show that sorne NK cells are producing a large amount of IFN-y and thus may not only
be the major primer of macrophages but the activator also. If our hypothesis is correct
then the question remains as to whether priming by NK cells is a function of number or
activity. If early embryo loss is a function of the number ofNK cells as the FACS data
indicate then presumably aIl NK cells are in a general state of readiness. If it is a case of
increased cellular activity then sorne NK cells may remain in a resting state and not
participate in the mechanics of early embryo loss. Thus, in a given population of NK
cells, sorne may be more prone to cytokine release.
We have already shown that
50
infiltration of NK cens coincides with early embryo loss in the CBAlJ female X DBAl2
male model. Our experiments aimed to purify NK cens from individual implantation
sites and assess their cytokine production. A total of 47 embryos were sorted into NK
cell enriched and depleted populations and analyzed for expression ofIFN-y, TNF-a and
perforin rnRNA.
A frequency distribution plot of IFN-y expression in DX5+ cens (Figure 6A)
showed that 38% of embryos had increased expression. Furthermore, in DX5- cens
(Figure 6B), only 13% of embryos had significant expression. These observations led to
the proposaI that NK cells are in various states of activation in individual implantation
sites. DX5+ cens in 38% of the implantation sites showed increased expression which is
above the expected 20-3()O/c) incidence of resorption. However, the data would tend to
indicate that in resorbing embryos, NK cells are fully activated to produce Thl cytokines
such as IFN-y and TNF-a whereas, in non-resorbing embryos, NK cells are in a less
active condition even though they too express IFN-y though in lesser quantities.
Certainly the data demonstrates that DX5+ NK cell populations from different embryos
express varying amounts ofIFN-y. The significant expression ofIFN-y in 13% ofDX5cells was surprising since expression was not only significant but very large also relative
to expression of the housekeeping gene. The production ofIFN-y by DX5- cells may be
due to an NK-like cell, the granulated metrial gland (GMG) cell which have not been
well characterized to this date but this is unlikely since these are large fragile cells which
are difficult to isolate. Because of the NK-like properties ofthese cells, IFN-y production
may be significant but whether it is detrimental to the embryo is unknown. One can also
not exclude T-cells as a source of IFN-y but participation in early embryo loss has not
51
been observed. Therefore, although DX5+ NK cells are not exclusively producing the
IFN-y in the decidua, a considerable percentage of them are expressing significant
quantities of IFN-y proportional to the incidence of resorption in our mice model. This
data reaffirms a role for the NK cell in the priming of resting macrophages and also
introduces the question of whether differing populations of NK cells exist between
healthy and resorbing embryos. That is, are the majority of NK cells in a resting or
inactive state in normal embryos?
Interestingly, TNF-a expression was significant in both DX5+ and DX5populations with 30% and 19% embryos displaying increased expression respectively.
Thus, NK cells are also producing major amounts of this pro-inflammatory cytokine that
can also serve as a macrophage triggering factor. Similarly to IFN-y producing DX5+
cells, 30% are expressing significant amounts of TNF-a (Figure 7A) reinforcing the
theory that NK cells in resorbing embryos are more active. The data also supports the
notion that NK cells are participating in the production of a Th1 type profile in resorbing
embryos.
Thus, our hypothesis that NK cells produce both IFN-y and TNF-a is
confirmed although the linkage is not consistent and the role of TNF-a is less certain.
Unlike IFN-y, TNF-a expression in DX5- cells is quite high at 19% (Figure 7B)
indicating that NK cells are not the sole source of TNF-a and that other cells are
producing this cytokine.
Indeed, macrophages are a major source of TNF-a and
macrophage production of this cytokine may be stimulated by the activation of decidual
NK cells.
The percentage of embryos with increased perforin expression was similar in
DX5+ and DX5- cells (Figure 8A,B) at 23% and 28% respectively. This was not a
52
surprising result since perforin has been previously shown to have a normal distribution
across aIl embryos in the CBA/J female X DBA/2 male model (Merkouris, 1999).
Further, perforin is thought to confer protective rather than abortive effects in pregnancy
(Rukavina & Podack, 2000). It was then puzzling why perforin expression was increased
at a level similar to the incidence of embryo loss. Interestingly, DX5- ceIls had a higher
percentage of increased expression and therefore the involvement of another ceIl is
implicated. Perforin expressing ceIls in the decidua include NK. ceIls, T ceIls and GMG
ceIls. Of these, NK. ceIls and T cens are potentiany harmful whereas GMG cens are
beneficial to pregnancy (Ashkar & Croy, 2001). Both NK. cens and GMGs constitutively
express perforin whereas T ceIls must be activated to express perforin. The observation
that increased perforin expression is seen in 28% of embryos could be explained in part
by an increased response by GMG cens or activation ofT cens by Th1 cytokines. IfNK.
ceIls are activated to produce Th1 cytokines, perforin production could coincide with the
elevated expression ofIFN-y and TNF-a. Furthermore, perforin rnRNA expression is not
indicative of actual perforin release into the local environment.
Our experiments have shown that DX5+ ceIls express increased amounts ofIFN-y
and TNF-a rnRNA suggesting that NK. ceIls are a major source of these Th1 cytokines.
The percentages of embryos with this increased expression in DX5+ cens are
proportional to the embryo losses seen in the CBA/J female X DBA/2 male model. This
corroborates previous studies examining the increased expression ofIFN-y and TNF-a in
embryos destined to resorb.
Furthermore, NK ceIls seem to be in various states of
activation as DX5+ populations express varying levels of IFN-y, TNF-a and perforin.
While only 13% ofDX5- ceIl populations expressed significant levels ofIFN-y, this does
53
indicate the presence of an additional cen involved in IFN-y production. TNF-ex. and
perforin expression was significantly elevated in DX5- populations demonstrating that
other cens such as macrophages produce major amounts of these cytokines. If the results
indicate that NK. cells are possibly in various states of activation, then future studies
should address this proposaI. Optimization of the rnRNA extraction protocol to the single
cell level should allow for determination of expression of Thl cytokines from DX5+
cells. Furthermore, future work should address the DX5- population producing large
amounts of IFN-y. Granulated metrial gland cens are a good candidate for these studies
although their isolation has proven to be difficult. Recent work has shown that GMG
cells may play a beneficial role in pregnancy by enhancing placental development and
more recently, produce IFN-y. If past observations about GMG cells are correct then
IFN-y from these cens are part of normal gestation and hence the lower percentage of
embryos expressing significant amounts of IFN-y in our studies. The role of other Thl
and Th2 cytokines produced by DX5+ and DX5- cells should be investigated.
In
particular, expression of switch cytokines such as IL-12, 15 and 10 by macrophages and
T cells may promote the production of a local Th1 type environment. Furthermore, IL-12
may be able to substitute for IL-2 in the-generation oflymphokine activated killer (LAK)
cells from NK cens (Salvucci et al., 1996). This would be important since LAKs are the
only effectors capable of killing trophoblast cells. Finally, since trophoblast has been
shown to be highly resistant to cytotoxic effeetors, future work should attempt to identify
the cellular target which eventually leads to embryo loss. Clark and colleagues (1998)
have proposed that production of a Th1 cytokine environment causes embryo loss by
acting on the mother not the fetus. That is, TNF-ex. and IFN-y act on maternaI uterine
54
vascular endothelium to stimulate expression of procoagulant, fg12 prothrombinase.
Vascular injury due to fg12 may be the causative factor in abortion in both humans and
mice.
In conclusion, the results presented in this thesis demonstrate the participation of
natural killer cells in early embryo loss. The data presented supports the notion that
infiltrating NK cens are an important precursor to fetal resorption. Furthermore, NK
cells in resorbing embryos may have higher activity with elevated Thl cytokine
production. Previously, NK cells were implicated as the major and perhaps exclusive
source of decidual IFN-y. Our results have shown that NK cens do express elevated IFNy rnRNA but that they are not the exclusive source. These results support the concept of
a key role for NK cens in the immunological mechanisms of early embryo loss. Further
research will hopefully allow for better understanding of these mechanisms in mice with
consequent broader implications in women susceptible to recurrent spontaneous abortion.
55
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