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Cell Mol Immunol. Author manuscript; available in PMC Apr 19, 2011.
Published in final edited form as:
PMCID: PMC3079746
CAMSID: CAMS1672
Natural Killer Cell Triggered Vascular Transformation: Maternal Care Before Birth?
Jianhong Zhang,1 Zhilin Chen,1 Graeme N. Smith,1,2 and Anne Croy1
1 Department of Anatomy and Cell Biology, Queen’s University, Kingston, ON K7L 3N6, Canada
2 Department of Obstetrics & Gynecology, Queen’s University, Kingston, ON K7L 3N6, Canada
For Correspondence: Dr. Anne Croy, Department of Anatomy and Cell Biology, 924 Botterell Hall, 18 Stuart Street, Queen’s University, Kingston, Ontario Canada K7L 3N6, Phone: 1-613-533-2859, FAX: 1-613-533-2566, croya/at/queensu.ca
Natural killer (NK) cells are found in lymphoid and non-lymphoid organs. In addition to important roles in immune surveillance, some NK cells contribute to angiogenesis and circulatory regulation. The uterus of early pregnancy is a non lymphoid organ enriched in NK cells that are specifically recruited to placental attachment sites. In species with invasive hemochorial placenta ion, these uterine (u)NK cells, via secretion of cytokines, chemokines, mucins, enzymes and angiogenic growth factors contribute to the physiological change of mesometrial endometrium into the unique stromal environment called decidua basalis. In humans, uNK cells have the phenotype CD56bright CD16dim and they appear in great abundance in the late secretory phase of the menstrual cycle and early pregnancy. Gene expression studies indicate CD56bright CD16dim uterine and circulating cells are functionally distinct. In humans but not mice or other species with post implantation decidualization, uNK cells may contribute to blastocyst implantation and are of interest as therapeutic targets in female infertility. Histological and genetic studies in mice first identified triggering of the process of gestation spiral arterial modification as a major uNK cell function, achieved via IFN-γ secretion. During spiral arterial modification, branches from the uterine artery that traverse the endometrium/decidua transiently lose their muscular coat and ability to vasoconstrict. The expression of vascular markers changes from arterial to venous as these vessels dilate and become low resistance, high volume channels. Full understanding of the vascular interactions of human uNK cells is difficult to obtain because endometrial time course studies are not possible in pregnant women. Here we briefly review key information concerning uNK cell functions from studies in rodents, summarize highlights concerning human uNK cells and describe our preliminary studies on development of a humanized, pregnant mouse model for in vivo investigations of human uNK cell functions.
Keywords: Decidua, Humanized mice, Pregnancy, Uterine Natural Killer cell
Natural killer (NK) cells are classically viewed as innate lymphocytes with high cytolytic potential against virus-infected and tumor-transformed cells. More recently, NK cells were found to share traits with the adaptive immune system such as memory, repertoire and dynamic trafficking 1,2. NK cells are now known to have important physiological roles in mucosal tissue including lymphoid-tissue induction. Uterus is a mucosal tissue that undergoes massive steroid-hormone promoted restructuring during pregnancy to support conceptus development (Fig 1). These changes are accompanied by the differentiation and proliferation of a unique, transient NK cell lineage, uterine (u)NK cells. UNK cells are terminally differentiated cells of limited life span that decline in number after mid gestation in humans, rats and mice. This has made uNK cells refractory to most in vitro study approaches. UNK cells reach 70% of all decidual leukocytes in early human gestation suggesting they have important roles. Because investigative manipulations of pregnant patients to fully define these roles is not possible, the understanding of human uNK cell functions has been extrapolated from alternative approaches (Table 1) with rodent uNK cell studies (ie mouse, rat and others) providing important information.
Fig 1
Fig 1
Structure of mouse implantation sites in cross section. The upper panel drawings illustrate regions of the mouse uterus as virgin (A), gd6–8 (B) and gd10–12 (C). The lower panel presents matched photomicrographs of midsaggital sections (more ...)
Table 1
Table 1
Approaches for the study of human uNK cell functions.
The rodent cells now called uNK cells have had a variety of names which readers should remember in order to obtain full bibliographies of previous work. For mice and rats, the term granulated metrial gland (GMG) cell was in use from the 1930’s to 1990’s. It still appears occasionally today. The most comprehensive monograph on earlier terminologies and on the first century of histological study of these cells was published by S. Peel in 1989 3. Human uNK cells also have a number of synonyms. These include decidual or dNK cell and endometrial or eNK cell to distinguish gestational from pre-conception intervals 4. A common antecedent term was endometrial granulocyte 5.
i) Life history, origins and subsets of uNK cells
Cells of the NK lineage are first detected in mouse uterus by immunohistochemistry in infancy (~2 weeks of age) 6. This precedes the appearance of uterine T cells by ~1 wk 7. Puberty with 4–5 day estrous cycles onsets over the next two wks but brings no changes in the location or relative numbers of NK cells. The NK cells detected in non pregnant cycling mice are randomly-distributed, small, agranular lymphocytes that might be more appropriately called pre-uNK cells.
In naturally-mated mice, conception occurs in the uterine tube and early blastocysts, still enclosed within the zona pellucida, arrive in the uterus 3.5 days later. These embryos expand, hatch and implant by gestation day (gd)4, triggering the primary decidual response. Initial attachment and decidualization occur on the anti-mesometrial side of the uterus. This positions polar trophoblast which forms the placental primordium called the ectoplacental cone, for growth towards the mesometrial side of the uterus where the mesentery delivers the uterine blood supply. As the endometrium undergoes secondary decidualization around the ectoplacental cone, differentiation of uNK cells is induced within a region of secondary decidual called the decidua basalis (DB).
It is important to realize that models of artificial decidualization, induced for example by small beads in hormone-primed mice 8, create a microenvironment that promotes uNK cell differentiation. Establishing this microenvironment requires progesterone but mouse uNK cells do not express the progesterone receptor 9. Studies using artificial decidualization clearly show that uNK cell differentiation and their primary functions are independent of a conceptus and thus of trophoblast 3,8,10. Mouse pregnancy uNK cells first appear in a region of the DB with the unique addressin expression of Vcam1 alone 11. Discrete microdomains for recruitment of other hematopoietic cell linages are also present in early decidua and, over the next few days, the domains blend in this constantly changing tissue. Stromal cell markers that are abundantly expressed in lymphoid organs, particularly the thymus, are strongly expressed by early post-implantation decidual cells. These include thymopoientin (TSLP), ERTR7 and gp38/podoplanin (JH Zhang, J Fritz, Y Rochman, WJ Leonard, J Gommerman, BA Croy, MS submitted). IL-15 is expressed in mouse uterus from the onset of decidualization until gd11.5 12.
It is unclear where the progenitors of uNK cells reside. In situ proliferation and recruitment from the circulation are the hypotheses usually considered; both mechanisms may participate At gd5.5, uNK cells are difficult to identify histologically and it has been suggested from 3H-TdR incorporation estimates of the high rate of early uNK cell proliferation that only five progenitor cells would be needed to give the abundant uNK cell numbers present at midpregnancy 3 (Fig 1B, C, E, F). We orthotopically transplanted uterine segments from normal (+/+) mice into mice genetically deficient in T and NK cells using end to end anastomoses, then bred the recipients. Neither decidualized grafts nor decidualized host uterus differentiated uNK cells, strongly suggesting most uNK cell progenitors are recruited from the circulation 13. In that study and most studies reported prior to 1995, mouse uNK cells were recognized by their lymphoid shape and the reactivity of their cytoplasmic granules with Periodic Acid Schiff’s (PAS) reagent, a histochemical stain for glycoproteins, especially mucin 14. Over the past decade, most investigators of mouse uNK cells have switched to use of the lectin Dolichos biflorus agglutinin (DBA), which reacts with terminal N-acetyl-d-galactosamine for uNK cell identification. This lectin reactivity is not applicable for uNK cells across species (for example, rat uNK cells lack reactivity) and must be tested in each species of interest. Mouse uNK cells display DBA lectin reactivity over their plasma membranes and the membranes covering their cytoplasmic granules. This dual reactivity is not seen in lymphocytes in any other organ of unmated or gd0.5–7.5 mice 15. Use of DBA lectin was widely adopted because it is more visually distinct than PAS in histological sections and is compatible with RNA isolation after cell collection from tissues by laser capture microdissection while PAS is not 16. Additionally, DBA lectin can be magnetically-tagged for isolation of uNK cells from decidual cell suspensions for FACS, short-term culture or RNA isolation. 16
We asked if PAS and DBA lectin were fully coincident stains and documented that they were not. Two subtypes of uNK cells were identified; PAS+DBA− and PAS+DBA+ (Fig 1). At gd6.5 these subsets were equally abundant. As gestation continued and uNK cells increased, the percentage of PAS+DBA+ uNK cells also increased. UNK cells achieve peak numbers between gd10.5–12.5 and, at these times, ~90% of uNK cells were PAS+DBA+. When alymphoid recipients (genotype Rag2−/−/Il2rg−/−; NK−T−B−) were engrafted by congenic +/+ marrow however, PAS+DBA− cells are difficult to find (<1%), even at gd6.5 17. This clearly shows that PAS+DBA+ uNK cells arise from circulating progenitors and suggests that PAS+DBA− uNK cells may be endogenous progenitors. Colucci and his colleagues also identified two distinct subsets of uNK cells in mid-gestation mouse decidual cell suspensions using flow cytometry 18. Their smaller diameter CD3−CD122+ uNK cells had the phenotype of peripheral NK cells (NK1.1+ or DX5+) while cells of larger size were NKp46+Ly49+NK1.1− (in C57BL/6) or NKp46+Ly49+DX5− (in BALB/c) 18. Using their marker strategy, we flow sorted uNK cells from midgestation randombred mice, isolated RNA from each uNK cell subset and conducted realtime PCR analyses. This has revealed that DBA+ and DBA− uNK cell subsets differ functionally in their production of cytokines and angiogenic factors (Chen et al, MS in preparation).
Between gd6.5–11.5, proliferating uNK cells occur in decidua basalis and, from morphological criteria, 4 stages of uNK cell maturation have been proposed. These are i) non granulated, ii) a few cytoplasmic granules, iii) numerous cytoplasmic granules and greatly expanded radius and iv) senescent which is a large, heavily granulated cell with nuclear changes. Senescent cells then break apart and scatter their granules. Active granule secretion by uNK cells has not been documented and it is thought that less mature uNK cells store their granule cargos of perforin and other cytolytic compounds. From gd8.5, all 4 uNK cell subtypes are found, no doubt complicating attempts to culture freshly isolated uNK cells. Also between gd8.5 and late pregnancy, a dense lymphoid structure full of uNK cells is found in the uterine wall at each implantation site. It separates the two smooth muscle layers of the uterus and rings the branches of the uterine artery entering to each implantation site. This donut-like ring, referred to as the mesometrial lymphoid aggregate of pregnancy (MLAp), becomes the site sustaining proliferative uNK cells while larger, post mitotic uNK cells dominate in the midgestation decidua basalis.
ii) Functions of mouse uNK cells
The original histological studies of implantation sites in mice genetically deficient in NK cells made several key observations. There were no uNK cells, decidual and midgestational myometrial structure were abnormal and the spiral arterial branches of the uterine arteries were not modified. These features have been consistently found across a number of different immune deficient mouse strains and their correction by transplantation of NK+T−B− marrow has confirmed that normal uNK cell functions include decidual stromal and vascular remodeling. Reciprocal congenic transfers of IL-15−/− and +/+ marrows between mice who were subsequently mated showed that the interactions between uNK cells and stromal cells are cross regulatory. Absence of IL-15 in decidual stroma blocked uNK cell differentiation from +/+ marrow while +/+ decidua promoted uNK cell differentiation from IL-15−/− marrow. Under barrier husbandry conditions, most strains of mice lacking NK cells breed successfully 19.
Absence of structural change to the maternal arteries feeding into implantation sites becomes apparent when uNK cell-sufficient and -insufficient mice are compared. This led us to examine more closely the relationship between uNK cells and the implantation site vasculature. Morphometry showed that normal vascular remodeling occurred after gd8.5 and was usually completed by gd10.5. This is a physiologically important time interval during which placental structure is completed and placental blood flow begins. At this time ~10% of the very large heavily granulated uNK cells are within lumens of decidual vessels, particularly small capillaries. About 25% of uNK cells are embedded within arterial walls and the remainder associate with decidual cells. It is very unusual to find significant numbers of uNK cells in the placenta but rare cells occur that may kill individual trophoblast cells 20,21.
Mouse uNK cells produce most (~90%) of the IFN-γ in the mesometrial decidua and MLAp. IFN-γ can fully replace uNK cell in inducing spiral arterial modification 22,23. Because IFN-γ acts on hundreds of genes, we suspect it influences the transcription of different molecules in endothelial, vascular smooth muscle, myometrial and decidual cells to orchestrate highly-regulated arterial changes. Eomes regulates Ifng transcription in uNK cells while Tbet regulates Ifng transcription in peripheral NK cells 16. UNK cells also differ by acquiring IL7Rα (CD127) after reaching maturity. This receptor appears to participate in maintaining IFN-γ production by uNK cells (Zhang submitted). Not all mid gestation mouse uNK cells synthesize IFN-γ (Fig 2A).
Fig 2
Fig 2
Fluorescence photomicrographs show co-localization of IFN-γ (A) and PGF (B) to uNK cells in gd10 C57BL/6 decidua basalis. Mouse uNK cells in 4% PFA fixed, paraffin-embedded tissue were tagged by FITC-conjugated DBA lectin, then stained with anti-PGF (more ...)
UNK cells associate only with the arterial side of the vasculature. Typically, nucleated cells enter and exit tissue via the venous side. One explanation for the special positioning of uNK cells, in addition to the addressin profiles expressed by endothelial cells of decidua basalis, comes from studies of the Ephrin family. EphrinB2 (EFNB2) is a signaling tyrosine kinase associated with arteries. Cells expressing EFNB2 associate together during arterial development. EphrinB4 (EPHB4) ligates EFNB2 and is characteristically expressed by veins. EPHB4 is also a tyrosine kinase and cells that express it dissociate from BFNB2+ cells in a “push-pull” interaction seen in differentiating capillaries. We found the expected expression of EFNB2 in implantation site spiral arteries at gd8.5 when these vessels have an arterial appearance but expression was lost over the next 4 days and by gd12.5 SA did not express this marked but had acquired EPHB4 suggesting they were now functioning as veins. Unexpectedly, uNK cells showed dynamic expression of both markers and this occurred in a time course manner that preceded the vascular changes. At gd6.5–12.5 uNK cells expressed EFNB2 which would prevent their association with veins and promote arterial associations. At gd6.5 and 8.5, uNK cells were EPHB4 negative but they co-expressed this ligand with its receptor at gd10.5 and 12.5. At these times the lymphocytes were the most strongly stained cells for both molecules in normal implantation sites. These studies highlight significant functional changes in the lymphocytes as spiral arteries transform and the placental circulation opens 24.
Mouse uNK cells express angiogenic molecules. This was first recognized by Wang et al, who showed immunoreactive vascular endothelial growth factor (VEGF) in uNK cells. By co-staining for endothelial cells, they deduced that the peak of neoangiogenesis in mouse implantation sites was at gd8.5 and that the vasculature was maximal at gd13.5 25,26. UNK cells are one of several cell types in implantation sites that make placenta growth factor (PGF; Fig 2B). This marker has a higher affinity than VEGF for VEGFR1 and accelerates angiogenesis by displacing VEGF from this receptor making it more bioavailable 27. The highest numbers of Pgf transcripts are found in immature uNK cells with few granules 28. We hypothesized that the role of angiogenic uNK cells is to locate the site of blastocyst implantation and to move towards it, thereby creating a “custom made” guidance system for maternal vasculature growth into an implantation site. We examined decidual expression of epidermal growth factor-like domain 7 (EGFL7), a key molecule in endothelial progenitor cell guidance and movement during blood vessel formation 29,30. Between gd6.5 to 12.5, EGLF7 was expressed by venous (EPHB4+) endothelium with peak expression at gd10 but it was not expressed by uNK cells (Fig. 3).
Fig 3
Fig 3
Fluorescence photomicrographs showing EGFL7 expression at gd6 (A), gd8 (B), gd10 (C) and gd12 (D) in C57Bl/6J implantation sites. PFA fixed paraffin-embedded implantation sites were sectioned, stained with anti-EGFL7 (a kind gift from Dr. Huilian Ye, (more ...)
In humans, NK cells and T cells express all components of the renin angiotensin system (RAS). Mouse T cells express RAS and mouse T cell deletion reduces drug-induced vasoconstriction. We asked if uNK cells express vasoactive molecules that could contribute to blood pressure control during pregnancy. We identified uNK and splenic NK cell subsets immunoreactive with antibodies against type 1 and type 2 receptors for angiotensin II (Hatta et al, submitted). Nitric oxide synthase, the enzyme producing NO, a potent vasodilating gas, is also synthesized by uNK cells 31. Recently, we reported a detailed examination of hemodynamic outcomes in pregnancy in normal and alymphoid (Rag2−/−Il2rg−/−) mice 32. The former have midgestational spiral arterial modification; the latter do not. Unexpectedly there were no differences between the strains in mean arterial pressure patterns, in hypoxia of the placenta or fetus (Leno-Duran et al, manuscript INPRESS, DOI# cannot get now) or in placental growth 32. These studies do not give insight into the potential hemodynamic roles for either NK or T cells because opposing, lymphocyte-based regulatory interactions cannot be assessed. Repetition of these studies in mice lacking only NK or only T cells is warranted as are drug challenges of the pregnant NK 33,34 and/or T cell deficient mice to evaluate if pregnancy has modified normal pathways of peripheral vasoactivity. Short term adoptive transfer of B versus T cells in non pregnancy Rag1−/− mice has shown B cells do not participate in hemodynamic alterations 35.
i) Life history and origins of human uNK cells
Human uNK cells are not found in infants or children 36 but appear in every post-pubertal menstrual cycle after ovulation and are sustained by pregnancy in the endometrial decidua, even if the pregnancy is ectopic 5,37. The presence of NK cells in the cycling uterus and when the implantation site is outside of the uterus, clearly indicates that human uNK cells, like those in rats and mice, are induced by endocrine-regulated stromal signals and not by the presence of trophoblast or of a conceptus. During the progesterone-dominant phase of the menstrual cycle, uNK cells associate with elongating spiral arteries 3840 and with basal components of uterine glands. During this interval, human uNK cells may have actions not seen in mice due to the lack of mouse uNK cells prior to conception.
Human uNK cells are highly proliferative in late secretory phase endometrium and in early decidua 41 and reach 70% of all nucleated decidual leukocytes 37. Their phenotype, CD3CD56brightCD16, is displayed by only a very small proportion of blood leukocytes but is associated with mucosal NK cells 4244. Human uNK cells are much less frequent in term decidua but it is difficult to evaluate their pattern of decline due to the need to study pregnancies continuing beyond times typical for elective terminations. In one study with a small number of across pregnancy samples, peak human uNK cell numbers were found between 8–13 wks of gestation 45.
While uNK cells in mice are considered to be activated cells because of their secretion of IFN-γ, human uNK cells are considered activated because they constitutively express Killer cell immunoglobulin-like receptors (KIR). In both species, uNK cells are considered to be cytokine producing cells armed for but not effecting cytotoxicity 4648. In humans, extravillous trophoblast cells that invade the decidua and maternal vasculature express HLA-C, E and G. Detailed characterization of KIR-HLA-C relationships in couples with gestational complications such as recurrent miscarriage or acute onset hypertension with proteinuria (pre-eclampsia), suggests that activation of uNK cells is a component of normal pregnancy and that genetic blockade to this elevates risk for pregnancy complications.
Human uNK cell origins are not defined; a number of sites are postulated. These include endometrium, where CD34+CD45+ hematopoietic stem cells as well as pluripotent mesenchymal and epithelial stem cells are found 49, thymus 50, PLN 51 and blood 52. Differentiation of uNK cells from thymus and PLN has been demonstrated in mice 13. Interestingly, LN draining a non pregnant but not pregnant (gd 3.5–7.5 were tested) mouse uterus had transplantable uNK progenitor cells, suggesting that the gestational uterus retains mobilized uNK progenitor cells. Pregnancy induces changes in lymphoid organs, inducing for example thymic depletion 53,54 and blockade of dendritic cell movement to uterine draining LN 55. Several investigators suggest that in women, as in mice, uNK cell progenitors are of mixed endometrial and peripheral origins 4,56.
ii) Functions of human uNK cells
Studies of timed endometrial biopsies support the hypothesis that human uNK cells have a significant angiogenic role. Over the progesterone-dominated phase of the menstrual cycle, uNK cells show changes in abundance of transcripts for Vegfc (a molecule that promotes lymphatic vessel development), Pgf, and angiopoietin 2 (Ang2). Protein array studies of CD56+ uNK cells collected at 8–10 wks gestational age show that uNK cells are major producers of angiogenic growth factors. This is not true of uNK cells collected at 12–14 wks gestation. The later cells (12–14 wks) are major producers of cytokines 5. Matrigel-supported co-cultures that contain a trophoblast cell line or umbilical cord endothelial cells plus isolated or cloned human blood or uNK cells reveal that VEGFC-producing uNK cells induce TAP-1 expression and MHC class I assembly in trophoblast and endothelial cells and facilitate capillary tube formation 46. Peripheral blood NK cells do not produce VEGFC and do not have these properties but show cytotoxicity 57. An ex vivo chorionic plate artery model has been developed to investigate the role of human first trimester uNK cells and angiogenic growth factors in spiral artery remodeling 58. In this system, uNK cell culture supernatants stimulated the separation of vascular smooth muscle cells from each other, disrupted their organization and initiated their de-differentiation as assessed by decreased immunoreactivity to the vascular smooth muscle cell markers 5. Using similarly conditioned medium and the extravillous cytotrophoblast cell line HTR8/SVneo, others showed that uNK cell products promoted vessel-like assembly of the trophoblast cell line 59,60. This was associated with increased expression of the adhesion molecule ICAM-1 which was identified as a major molecule participating the migration and network formation of the trophoblasts 60. Human uNK cell supernatants also promote angiogenesis and tube formation in human umbilical vein endothelial cells (HUVECs) and in aortic ring assays 46. These in vitro data support the conclusions from in vivo xenogeneic engraftment of the human trophoblast tumor cell line (JEG-3) into nude (Foxn1) mice in matrigel plugs. Surrounding the plug with uterine but not with blood CD56+ NK cells promoted a 5 fold more dense vasculature in the resulting tumor. Expression of Mmp7 and Mmp9 by uNK cells and by the macrophages that co-infiltrate into spiral arterial vascular smooth muscles is also considered important for early initiation of trophoblast-independent spiral arterial remodeling 61.
In humans, CD56bright NK cells are the NK cell subset associated with the synthesis of immunoregulatory cytokines, particularly IFN-γ 62. IFN-γ significantly up-regulates chemokines (CXCL9, CXCL10, CCL8, and CCL5), enzymes (GBP5, TAP1, CYP27B1, SOD2, MX1, CASP1, and PTGES) and transcription factors (Tfap2c, Irf1, Nfe2l3) but down-regulates cytokine genes such as Csf2, Il1r2, Spp1, Wisp2 and Igfbp3 63. These actions, combined with uNK cell production of the chemokines CXCL10, and CXCR2 direct migration and invasion of CXCR1+, CXCR3+, CXCR4+, CCR3+ trophoblast 46, and promote physiological angiogenesis in the placental bed 37,64. Insufficient uNK cell activation would reduce these processes, and contribute to poor decidual artery remodeling. Such histopathologic diagnosis frequently associated with clinical pre-eclampsia and with intrauterine fetal growth restriction 65. Because NK cell functions reflect the sum of signals from their multiple activating and inhibitory receptors 66, it is a complex question to fully understand the pathways that could modulate uNK cell activation in the pregnant uterus. Additionally, outcomes from inappropriately activated uNK cells may not be predicted immunologically. For example, recent studies of women categorized as superfertile but who have repeatedly early pregnancy loss suggest that defective early decidualization extends the window of endometrial receptivity for a blastocyst. This permits karyotypically abnormal or developmentally-delayed embryos to implant; they subsequently fail, often before 6 wks gestation. Elevated uNK cell numbers were previously reported in biopsies of patients during the progesterone-dominated phase of their menstrual cycles. The strong two-way interactions defined between mouse decidual cells and uNK cells suggest that human uNK cells will be shown to be an in vivo factor contributing to pathological elongation of the window of endometrial receptivity
In women with recurrent spontaneous abortion, the high numbers of uNK cells in endometrial biopsies taken in the late secretory (i.e. progesterone dominant) phase of the menstrual cycle, correlated positively with the formation of blood and lymphatic vessels, spiral arteriole smooth muscle differentiation and endometrial edema. It has been postulated that exposing implanted blastocysts to excessive oxidative stress, may induce embryonic loss 67. Opposite clinical thinking is also reported and it must be remembered that female infertility is a heterogeneous problem. For some infertility patients planning to undergo embryo transfer, a clinical protocol has been developed to stimulate uNK cells. This involves the collection of two serial endometrial biopsies in cycles prior to the embryo transfer 6870. This protocol, reported to double the rate of “take home baby”, is designed to enhance the inflammatory milieu of the uterus and to increase numbers of peri-implantation uNK cells.
Because detailed, time course studies on human uNK cells is not feasible and endometrial sampling is limited to small regions in biopsies or hysterectomy specimens in which tissue not affected by disease, and is still not representative of normal uterus 71. Thus many questions remained outstanding regarding the biology, origins, functions and regulation of human uNK cells. We hypothesized that answers to some of these questions could be provided by studying implantation sites in humanized mice and therefore embarked on experiments to develop an appropriate model.
Since T-cell deficient “nude” (Foxn1 mutation) mice were identified in 1970, xenogeneic grafting of normal and pathologic human cells and tissues to immune deficient mice has been embraced as an approach to move in vitro models to more complex in vivo modeling 72. Success in humanizing mice with normal tissues moved significantly forward when the severe combined immunodeficient T-B-mice (Scid, Prkdc mutation) were identified and found to support human hematopoietic cells and lymphoid organs 73,74. Sequential improvements have given several relatively simple and reproducible mouse models for generation of a ‘human immune system’ (HIS) mice. Strains now commonly used to prepare HIS mice are NOD-SCID-Il2rg−/− (NOG) and BALB/c-Rag2−/−Il2rg−/− 75,76 and protocol refinements continue 75,77.
HIS mice have been useful for studying pathogens such as HIV that directly target the human lymphohematopoietic cells, including cells found in the female reproductive tract 78. However, human lymphocyte lineage differentiation in HIS mice differs markedly. B cell reconstitution is robust, T cell reconstitution is reasonable, but NK cell and myeloid lineage reconstitutions are generally poor to undetectable 79. Since IL-15 trans-presentation regulates endogenous human NK cell homeostasis, use of IL-15 receptor agonists has been recommended to improve xenogeneic NK cell engraftment 77. No current model is suitable for addressing the question of whether xenogeneically engrafted human NK cells home to the gestational uterus and effect spiral arterial modification. This is because the models employ recipient preconditioning by irradiation (between 320 and 375 cGy) rendering the recipient reproductively sterile. We turned to 5-fluorouracil (FU), a thymidylate synthase inhibitor as a preconditioning agent for of 6 wk old female BALB/c-Rag2−/−Il2rg−/− mice, at a dose of 150mg/kg 80. After 24 hr, CD34+ cord blood cells enriched by negative selection were inoculated. Six weeks later the females were bred and euthanized for study.
The choice of xenograft recipient is important. Because our research question is focused on promotion of decidual angiogenesis and spiral arterial modification, we are not able to use recipients with a NOD background because the decidual arterial in NOD mice is abnormal 81. Our syngeneic mouse to mouse grafting of Rag2−/−Il2rg−/− mice on either the C57BL/6 or BALB/c is consistently successful in establishing fully functional, graft-derived uNK cells 82,83 that effect quantifiable spiral arterial modification, we selected preconditioned BALB/c-Rag2−/−Il2rg−/− for study and used 16 as recipients for 1×105 human CD34+ cord blood cells. Variables compared were:
  • Administration of freshly isolated cells versus cells expanded in culture (24 hr in StemSpan SFEM medium with CC100 Cytokine Cocktail) 84,85.
  • Tail vein (IV) versus intrafemoral (IF) cell injection 86,87.
  • Without or with human IL15/IL15Rα complex treatment at 6 and 7 wks of age and at gd6.5 after mating 77. These four females were paired with males immediately after their 2nd injection and bred within a few days.
Two additional variables are present in these studies. The first is placenta donor variability—three placentae were used. The second is whether the prepared females successfully mated and subsequently conceived.
All mice were killed at gd12.5 with sera and organs collection for quantification of human IgG by ELISA, RT-PCR for human chromosome 17-specific α-satellite DNA and histology. Table 2 summarizes our progress to date. Of the 16 females used in this study, 14 became pregnant and carried a viable litter to gd12.5. There were no differences in implantation site numbers between unmanipulated BALB/c-Rag2−/−Il2rg−/− and hu-CD34+ cell inoculated BALB/c-Rag2−/−Il2rg−/− (Figure 4A). Of the 14 pregnant females, 3 were identified as chimeric in their implantation sites (decidual and MLAp) and peripheral organs (maternal liver and spleen) and their sera contained huIgG (Fig 3, table 2). One of the two mice who mated but were not pregnant was also chimeric in spleen and liver and had circulating hu-IgG. Cells prepared from one of the 3 donor placentae gave no reconstitution.
Table 2
Table 2
Summary of BALB/c-Rag2−/−Il2rg−/− mice engrafted with human cord blood CD34+ cells at gestation day 12
Fig 4
Fig 4
Implantation sites outcomes after human CD34+ cell inoculation. A total of 16 BALB/c-Rag2−/−Il2rg−/− mice were pretreated with 5-FU (150mg/kg) and inoculated with 1×105 with human CD34+ cord blood cells. Six wks (more ...)
Implantation site histology in the pregnant, chimeric mice was quite variable between littermates, an observation we have never made in unmanipulated or syngeneically-transplanted females. It also differed to what was anticipated (Fig 5). Cells with a lymphoid appearance were not present in the decidua basalis and there was a gain in spiral arterial pathology. In the most severely altered sites (Fig 5), a greatly enlarged vascular wall surrounded the spiral arteries. This region showed a localized loss of reactivity with many histochemical stains and appeared to have lots all of its collagen fibers which are eosin-reactive in normal BALB/c and BALB/c-Rag2−/−Il2rg−/− mice (Fig 5A, B, a, b). This region is not an artifact and has been seen multiple times in another series of intrahepaticly inoculated neonatal recipients (M. Bilinski and B. A. Croy, data not shown). A littermate implantation site to the one illustrated had much milder changes. In it, individual large, irregularly shaped, pale staining mononuclear cells with low nuclear to cytoplasmic ratio were seen that appeared to have specifically homed to the spiral arterial walls. The unusual cells could not be stained by PAS, Alcian blue or Masson’s Trichrome indicating lack of glycoprotein, collagen and mucopolysaccharides (not shown). We anticipate but as yet have no evidence that the unusual cells and tissue are of human origin because 5-FU treatment followed by syngeneic grafting does not induce this appearance. Neither does administration of 3 doses of the IL15/IL15Rα complex alone without cells (not shown). The unusual cells we report are not reactive with antibodies to human CD45 and may represent progeny of circulating human mesenchymal or other stem cells not removed by the CD34 cord blood selection procedure. It is of interest that these proven chimeric implantation sites do not reveal an immune graft versus placenta pathology and that if the fetus imaged in (Fig. 5C, c1–3) was doomed to die in late gestation, its compromise would have been effected through the maternal vasculature with particular involvement at the spiral arterials. That components of human decidual tissue beyond immune cells and circulating endothelial progenitor cells might arise from circulating progenitor cells is a novel hypothesis suggested by these preliminary studies. Might it also be possible that the unusual, spiral artery-homed cells we detected are the early progenitors of uNK cells and that uNK cells arise from committed lymphoid cells, and in humans from circulating CD56bright cells is in error? Readers will realize that we have not yet shown that the unusual cells depicted in Fig. 5 are human in origin and that we remain far from our goal of a humanized pregnant mouse model that will enable in vivo studies of the interactions between human uNK cells and the stromal cells comprising the spiral arteries.
Fig 5
Fig 5
Photomicrographs comparing gd12 implantation sites from wildtype BALB/c (A, a), BALB/c-Rag2−/−Il2rg−/− (alymphoid; B, b) and human CD34+ cell-engrafted BALB/c-Rag2−/−Il2rg−/− mice (iv injection, (more ...)
An individual’s life long health is determined by the quality of his or her environment during pregnancy 88. Rapid and massive changes are induced in the maternal cardiovascular early in pregnancy to support the conceptus and its nutritional welfare. These changes occur systemically and include functional changes in the heart and kidneys and locally within the reproductive tract. Evolution of mammalian pregnancy, a much more recent event than evolution of the immune system, appears to have shaped at least some elements of the immune system into specialized tools to promote and optimize successful pregnancy. Uterine NK cells are an example of these specialized niche cells. They are transient cells endowed with angiogenic and, potentially, circulatory regulatory activities that participate in the early optimization of maternal care of the fetus before birth. This role of lymphocytes appears to be conserved across species that have forms of placentation other than the hemochorial placentae discussed in this review 28. Understanding of the potential for immune cells to function in angiogenesis and in circulatory regulation and the mechanisms the physiologically link the immune and cardiovascular systems is an important new horizon in health research.
Acknowledgments
We thank Ms. Valérie Barrette and Mr. Michael Bilinski, Queen’s University for technical assistance and helpful discussions, Dr. Huilian Ye (Genentech Inc. South San Francisco, USA) for providing anti-Egfl7 antibody and Dr. Aureo T. Yamada (UNICAMP, Campinas Brazil) for histological consultations, Mr. Richard C. Casselman and Ms. Heather Ramshaw for sample collection. These studies were supported by awards from the Natural Sciences and Engineering Research Council, Canada, the Canadian Institutes of Health Research and the Canada Research Chairs Program to BAC and a Province of Ontario/Queen’s Postdoctoral Fellowship awarded to JHZ.
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