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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 1+1 National Library of Canada Bibliothèque nationale du Canada Acquisitions and Bibliographie Services Acquisisitons et services bibliographiques 395 Wellington Street Ottawa ON K1A DN4 Canada 395, rue Wellington Ottawa ON K1A DN4 Canada Your file Votre référence ISBN: Q-612-85812-X Our file Notre référence ISBN: Q-612-85812-X The author has granted a nonexclusive licence allowing the National Library of Canada to reproduce, loan, distribute or sell copies of this thesis in microform, paper or electronic formats. L'auteur a accordé une licence non exclusive permettant à la Bibliothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou aturement reproduits sans son autorisation. Canada 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. 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