妊娠維持機構としての子宮NK細胞の重要性

木曾　康郎

（山口大学農学部家畜解剖学教室）

UTERINE NK CELLS / GRANULATED METRIAL GLAND CELLS: CRITICAL ROLES AT THE FETO-MATERNAL INTERFACE DURING SUCCESSFUL PREGNANCY

Yasuo KISO

（Department of Veterinary Anatomy, College of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan）

Introduction In the pregnant murine uterus, the metrial gland is transiently formed in the mesometrium opposite the embryo shortly after embryo implantation. It develops at each individual implantation site in the region commonly called the mesometrial triangle. The gland differentiates on days 6.0 to 6.5 of pregnancy and a residual structure remains post partum. The gland contains stromal and vascular elements plus a population of large cells that are characterized by numerous cytoplasmic granules. These cells were first described by Duval in 18911) and the term granulated metrial gland (GMG) cells were provided by Selye and McKeown in 19352). Then these large cells have become known in mice and rats as GMG cells. Recently, GMG cells are established to be a member of NK cells, uterine NK (uNK) cells (see below). Cells analogous to murine GMG cells have been described in the hamsters (endometrial granulocytes)3), gerbils (granulated endometrial cells)4), field voles5), insectivores6), bats7), horses8,9), pigs10), sheep, goats, cows11), deer12), monkeys (endometrial granulocytes)12,13) and humans (endometrial stromal granulocytes or decidual large granular lymphocytes)14-16), which indicates that GMG cells or the analogous cells are common inhabitants of mammalian uteri. Still, little is known regarding the differentiation and immunological function of GMG cells at the feto-maternal interface remain to be established. Other types of bone marrow-derived cells including neutrophils, eosinophils, mast cells and g/d-T cells could be present under certain conditions such as infection and inflammatory conditions. Macrophages, however, are a specific uterine compartment and are suggested to have significant functions during pregnancy. Readers interested in uterine macrophages should refer to Hunt's papers on the role of uterine macrophages17-21). The present seminar will focus on murine GMG cells/uNK cells with emphasis on our recent studies. Background Significant numbers of granulated metrial gland (GMG) cells are found in the pregnant murine uterus only during pregnancy22,23). GMG cells are localized to the metrial gland and the decidua basalis of each implantation site24,25). They are large (up to 50 mm in diameter in mice), lymphoid cells containing numerous large cytoplasmic granules (up to 5 mm in diameter in mice), achieving a frequency of up to 20 % in the metrial gland. GMG cells are rarely present in the uteri of virgin mice and differentiate throughout early murine decidua. Later, the only proliferative population of GMG cells is found in the metrial gland. After day 15 of gestation, GMG cells gradually decrease in number and by day 18 of gestation they disappear from the metrial gland and the decidua basalis22). The bone marrow origin of GMG cells was demonstrated using adoptive cell transfer; mice reconstituted with rat bone marrow cells26,27) and mice reconstituted with mouse transgene-marked bone marrow cells28). Histologic studies revealed that GMG cells are characterized by cytoplasmic projections and granules. The numerous cytoplasmic projections correlate to the migration property of GMG cells. Indeed, GMG cells are found in the placental blood vessels and their migratory ability is readily demonstrated in vitro29). The cytoplasmic granules of GMG cells are azurophilic and periodic acid-Schiff (PAS)-positive, increasing in both number and size until day 14 of gestation. In mice, the granules are usually oval in shape and contain a central, homogenous, electron-dense core and peripheral 'cap'-structure22). Moreover, the granules are demonstrated to contain lytic proteins including pore forming protein (perforin) and serine esterases30-32). Thus, GMG cell granules are morphologically similar to those reported in natural killer (NK) cells. Studies on surface phenotypes of GMG cells revealed that GMG cells share the phenotypic markers asialo-GM1, Ly-49G2 (formerly LGL-1), NK1.1, Thy-1, FcR, 4H12, GMG-1 and CD45, while lack CD3, CD4, CD5, CD8, IgM, a/b-T cell receptor, d/g-T cell receptor, F4/80 and Mac-132-38). These results indicate that GMG cells are a subset of NK cells. GMG cells are reported to possess low levels of cytotoxic activity which can be induced by interleukin (IL)-239) and produced at least three kinds of cytokines including IL-1, colony stimulating factor (CSF)-1 and leukemia inhibitory factor (LIF)40). The differentiation and immunological function of GMG cells at the feto-maternal interface still remain to be established. We will address here some aspects regarding these questions based on our studies. Are GMG cell precursors resident in the uterus? Although GMG cells are bone marrow derived cells, it is not clear whether GMG cells differentiate from precursor cells that reside in the uterus or from migratory cells that home to the uterus in response to signals associated with decidualization. To answer this question, adult uterine tissue grafts from nonpregnant mice were transplanted in an ectopic site of nonpregnant or mated recipients; grafts were placed into the renal subcapsular space or enclosed within a diffusion chamber and transplanted to the caudal abdomen. In both graft experiments, GMG cells were infrequent 3 days after grafting but they increased in number up to day 12 of gestation only in grafts recovered from pregnant recipients41). This indicates the presence and in situ differentiation of GMG cell precursors within nonpregnant uterine tissue. In addition these results directly exclude any requirement for cell recruitment into the uterus and any influence of the conceptus on the differentiation of GMG cells. More importantly, this technique will permit assessment of GMG cell precursors in uterine tissue from females outside the normal reproductive age range. Such experiments should aid in assessing the roles of GMG cells in promotion of pregnancy success. When do GMG cell precursors first appear in the uterus? Mature GMG cells, which are large and granular, are infrequent in the nonpregnant22). GMG cell precursors are characterized morphologically in the uterus as small and agranular lymphocytes that can be detected using the monoclonal antibody Ly-49G2 (formerly LGL-1)42,43), an NK cell phenotype marker44). The Ly-49G2 is the only NK cell marker known to be expressed in the virgin uterine tissue, the other markers being acquired about day 8 of gestation35). Using this antibody, other investigators had reported that GMG cell precursors are resident cells in 8 to 17 wk old mice35). We demonstrated that GMG cell precursors are resident cells in 7 wk old virgin uteri using the grafting technique as mentioned above. To establish when GMG cell precursors first appear in the uterus, Ly-49G2-positive NK cells were enumerated immunohistochemically in the virgin uteri of conventional (CO) CD-1 mice from birth to puberty (7 wk of age)44). Ly-49G2-positive cells were not detected in the uterus during the first week of life and at 2 wk of age they were first found in low numbers. A large increase in frequency of Ly-49G2-positive cells occurred between wks 2 to 3 of age and between wks 5 to 7 of age. The data suggest approximately 10-fold and 3-fold increases in number of Ly-49G2-positive cells respectively when the gain in uterine weight is also considered. This result clearly establishes that the appearance of Ly-49G2-positive cells in the uterus is a postnatal event, unlike that of T-cells45). Decrease of Ly-49G2 was associated with accumulation of perforin and the other expression of other NK cell surface markers including asialo-GM1 and Thy-1. Thus, it is most likely that the appearance of Ly-49G2-positive cells in the early postnatal uterus represents at least one of the earlier steps in establishment of the uterine NK cells which differentiate into GMG cells in the pregnant uterus. Which factors influence the differentiation of GMG cells? To determine whether the microbial environment or the presence of T-cells and B-cells influence the establishment of uterine NK cells, Ly-49G2-positive cells were counted in the uteri of barrier-reared, flora-defined (FD) CD-1 and of immune deficient mice (genotype C.B.17-scid/scid (SCID) between birth and puberty (7 wks in the former and 10 wks in the latter). No differences were noted between CO CD-1 and FD CD-1 mice with respect to first appearance or frequency of Ly-49G2-positive cells in the uterus or periods of rapid increases in cell numbers. In the SCID uterus, Ly-49G2-positive cells were not detected until wk 5 of age and the frequency was less than that observed in age-matched immunocompetent mice. However, Ly-49G2-positive cells in the SCID uterus reached adult levels at 10 wks of age when SCID mice were cycling44). This result indicates that the microbiological environment of the uterus is not essential for establishment of the uterine NK cells and discounts the hypothesis that the GMG cell function is in uterine defense surveillance, particularly against viral agents. The data also demonstrate that neither T cells nor B cells play an important role in the differentiation of uterine NK cells. Moreover, immunodeficiency of SCID mice and NUDE mice did not influence the morphology, distribution and frequency of GMG cells during successful pregnancy. The similarities of GMG cells among mice including immunodeficient mice suggest that they have functional significance during pregnancy46). Since it was suggested that macrophages precursors can differentiate into NK-like cells under the combination of IL-2 and CSF-147,48), GMG cells were counted in the uteri of osteopetrotic (op/op) mice during placentation period. This mice are unable to produce CSF-1 and have a deficiency in macrophages49,50). GMG cells were normal in morphology and frequency , suggesting that CSF-1 is not required for GMG cell differentiation and that GMG cell precursors are independent of the macrophage lineage51). Further, the production of CSF-1 by GMG cells is not among the critical function of these cells during pregnancy. However, since granulocyte-macrophage-CSF (GM-CSF) is present in op/op mice and is greatly elevated during pregnancy and since unusual macrophages are observed in the metrial gland of op/op mice52), it remains possible that macrophages may influence the GMG cell differentiation or that uterine macrophages may respond to the same signals as GMG cells. Recently, we studied GMG cells in the new mutant, alymphoplasia mice (genotype C57BL/6J-aly/aly) that are genetically deficient in lymph nodes and Peyer's patches due to a lymphoid-associated mesenchymal disorder53,54). This immunodeficient mice is completely different from SCID and NUDE mice who differentiate GMG cells during pregnancy. GMG cell differentiation was delayed in pregnant aly/aly mice, which is not due to immunodeficiency, but due to a mesenchymal disorder (see below). What are the interactions between GMG cells and extracellular matrix (ECM)? ECM receptors of very late activation antigen (VLA)-integrin family have been shown to play an important role in the binding NK cells to targets, in NK cell migration, in NK cell viability, in lytic activation and in regulation of cytokine gene expression55-57). GMG cells are known to migrate throughout the placenta and often transverse blood vessels58). It is also known that the trophoblast produce ECM59,60). Thus, there is merit in characterizing the relationship between GMG cells and the ECM. First, we studied the differentiation and distribution pattern of GMG cells in the tight-skin (Tsk/+) mice and the alymphoplasia (aly/aly) mice. Tsk/+ mice produce excess collagens I and III61). GMG cells differentiated in pregnant Tsk/+ females, but the distribution pattern was abnormal. Viable GMG cells were present in the antimesometrial decidua and in the interconceptual regions until midpregnancy62). Loss of GMG cells from these sites normally occurs by day 6.5 of pregnancy22). The abnormal distribution of GMG cells from Tsk/+ mice suggests that alterations to the ECM change the migration ability or life span of GMG cells. On the other hand, aly/aly mice are considered to be a useful model for the study of interactions between lymphocytes and stromal components, since they are genetically deficient in lymph nodes and Peyer's patches due to a lymphoid-associated mesenchymal disorder. The immunodeficiency of aly/aly mice results from an abnormality in the microenvironment of its various lymphoid tissues53). The interaction between homing receptors on lymphocytes with ligands on endothelial cells has been suggested as a possible deficit54). Metrial gland tissue was present and appeared normal in aly/aly mice. The distribution of GMG cells from aly/+ mice was normal. Fewer GMG cells were present in aly/aly mice than aly/+ mice (Table 5) and the features of individual GMG cells were different; i.e., GMG cells in aly/aly mice were small in size and the granules were poorly developed. By day 14 of gestation, however, GMG cells had acquired a mature size and the granules appeared mature. It is likely that GMG cell differentiation was delayed in pregnant aly/aly mice, due to a mesenchymal disorder affecting metrial gland development in this animal63). Second, we cultured GMG cells on several ECMs including fibronectin (FN), laminin (LN), collagen (CO), vitronectin (VN) and matrigel. GMG cells in cultures lacking ECM died within 48 hrs, but GMG cells maintained on ECM were viable for at least 14 days. In particular, GMG cells cultured on LN had the best-maintained organelles, while GMG cells cultured on FN elongated. GMG cells cultured on VN had well-maintained organelles and showed various shapes23,64). These suggest that GMG cells express ECM receptors. Third, GMG cells are found throughout endometrium during early implantation period, but by day 8 of gestation GMG cells are localized to the decidua basalis and metrial gland and proliferate only in the metrial gland22). We studied whether such a highly localization and viability of GMG cells correlate to the ECM contained in these sites. Cultured GMG cells showed the chemotaxis in response to the ECM and the ectoplacental cone that is known to produce FN, while no response to IL-2, LIF nor B cell conditioned. In particular, VN and LN intensively induced the chemotaxis of GMG cells. This suggests that GMG cell chemotaxis is closely related with ECM. Fourth, the expression of ECM receptors on GMG cells was evaluated immunohistochemically at various stages of gestation using anti-VLA-integrin antibodies. On days 3 and 6 of gestation, Ly-49G2-positive cells were employed to identify GMG cells due to their immature morphology. Between days 8 to 15 of gestation, GMG cells are readily recognized by their mature morphology. Both immature and mature GMG cells expressed VLA-b1 chain at all stages of gestation tested. Immature GMG cells on days 3 and 6 were positive for VLA-a1, VLA-a3 (CO/LN receptors), VLA-a4, VLA-a5 (FN receptor) and VLA-a6 (LN receptor) chains. Between days 8 to 15 of gestation, mature GMG cells were positive for VLA-a4 and VLA-a5 chains, but negative for VLA-a1, VLA-a3 and VLA-a6 chains. This result suggests that major changes occur in the uterine NK/GMG cell population during implantation period65). During this period, GMG cells lose expression of Ly-49G2 surface antigen and gain expression of the lytic proteins (perforin and serine esterases) and the surface antigens, 4H12, asialo-GM1 and Thy-1. The differentiation of uterine NK cells is not equivalent to development of cytotoxic activity. Mature GMG cell morphology, i.e., very large with numerous granules, may result from a suppression of lytic activity through stabilization of granule membranes. NK cells have been reported to express FN receptor. FN receptor expression is also not equivalent to lytic activation66,67). FN receptor is reported to be important to the migratory capacity, target cell binding and the differentiation of NK cells67,68). Alterations of uterine NK/GMG cell surface expression must reflect major changes in cell functions. Uterine NK cell functions during pregnancy remain to be fully defined. GMG cells are known to produce some cytokines and to become lytic under certain condition39,40,69). It is unknown whether the Ly-49G2-positive NK cells in the prepubertal uterus share these functions with mature GMG cells. Although the lymphokine activated killer (LAK) cells is the only immune effector cell lytic for trophoblast33,70), the splenic LAK cells derived from NK1.1-positive/Ly-49G2-positive cells show different lytic properties to NK1.1/Ly-49G2-negative cells and to date there is no evidence that uterine Ly-49G2-positive NK cells can differentiate into LAK cells lytic against trophoblast42,43). Thus, uterine Ly-49G2-positive cells may be a cell population with functions distinct from induction of abortion. Does apoptosis account for the loss of GMG cells in late pregnancy? GMG cells increase in number until mid gestation, but disappear from the uterus in late gestation (table 6)22). It is of great interest to determine whether such a decrease of GMG cells is caused from the cell death including apoptosis. After day 15 of gestation, GMG cells decreased in number and their morphology showed pyknosis and vacuolation, indicative of apoptosis. In situ detection for DNA fragmentation revealed that GMG cells on day 16 have DNA fragmented nuclei in large number and that it was not detected on day 12 of gestation. Electrophoresis of genomic DNA extracted from GMG cells on day 15 displayed ladder fragmentation. These results indicate that the loss of GMG cells in late gestation is due to apoptosis71). In pregnant lpr/lpr mice who lack genetically Fas (CD95) gene that is major apoptosis factor for many cells, GMG cells were seen in significant number after day 15 of gestation72). The Fas antigen belongs to the tumor necrosis factor/nerve growth factor receptor family and transmits an apoptotic signal to the nucleus by binding to the Fas ligand73). The Fas antigen has been proved as a mediator of apoptosis on the passway of T lymphocyte differentiation74). Besides, expression of the Fas antigen was detected on the activated NK cells in the peripheral blood75). Expression of Fas and Fas ligand was observed in the normal mouse uterus76). Indeed, we detected the Fas antigen expression on GMG cells on day 15 of gestation, while could not be seen on GMG cells on day 12. Moreover, we evidenced the Fas ligand can cause apoptosis in GMG cells isolated from the metrial gland on day 16 of gestation, while not on day 12. Thus, it is most likely that the Fas/Fas ligand system is the common apoptosis mediator also in GMG cells. Recently, we found that both the metrial gland and GMG cells were present even at the full term (day 19) of gestation in an infertile strain (IF, genetically not yet established) among various strains of Japanese wild mice, Mus musculus molossinus, and that some GMG cells still persisted in the postpartum uterus of this mice. In contrast to normal mice, Japanese wild mice mate on the day of litter birth. Infertility in IF molossinus mice occurred in second pregnancy as a failure of embryo implantation. Thus, persistence of mature GMG cells into the second pregnancy may have detrimental effects on pregnancy outcome, suggesting that GMG cell apoptosis in late pregnancy is an important event. Do GMG cells play crucial roles in pregnancy success? Little is known regarding GMG cell functions since in vivo antibody mediated depletion has not been achieved during pregnancy. Even though Ly-49G2-positive cells, GMG cell precursors, were removed from the virgin uterus by repeated infusions of the Ly-49G2 antibody, they could not be removed from the pregnant uterus77). Recently, Dr. Croy and her colleagues presented a new evidence on GMG cell functions using transgenic mice expressing a human CD3e gene (TgE26 mice)77-79). Depletion of NK cells has been achieved, in combination with T cell depletion, in TgE26 mice. Pregnancy in TgE26 mice was characterized by very small placenta (half size of the control), poor development of the metrial gland and infrequency of GMG cells. In TgE26 mice, a sudden onset of fetal loss began at day 10 of gestation. Such a fetal death was associated with progressive changes in the maternal uterine arterioles, suggestive of localized hypertension. Since the metrial gland and the GMG cell were normal in genetically T cell deficient mice (nu/nu, scid/scid), anomalies in TgE26 mice result from their NK cell deficiency. These data directly evidenced that GMG cells are an NK cell population and suggest that they play a vital role in the promotion of fetal survival and pregnancy success through modifications of the placental vasculature77,78). Moreover, to confirm the role of the NK deficiency in these reproductive deficits, transplantation of TgE26 females was undertaken using bone marrow from scid/scid donors. Engrafted pregnant females have restoration of the uNK cell population, induced metrial gland differentiation, reduced anomalies in the decidua and decidual blood vessels, increased placental size, and restoration of fetal viability at days 10-14 of pregnancy. This clearly indicates that uNK cells have critical function in pregnancy that promote decidual health, the appropriate vascularization of implantation sites, and placental size. Since NK cells are a lymphocyte population that is highly affected by central nervous system mechanisms80,81), characterization of GMG cell functions is impossible without exploration of relationships between the neuroendocrine and immune systems. Further studies are needed to determine the roles of GMG/uNK cells at the feto-maternal interface during successful pregnancy. Acknowledgements We thank Dr. K. Kusakabe (Osaka Pref Univ) for support. 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