Granulated Metrial Gland Cells Research Articles

Relationship of Granulated Metrial Gland cells to the trophoblastic barrier of mice and human placenta

Relationship of Granulated Metrial Gland cells to the trophoblastic barrier of mice and human placenta

Granzyme D Is a Novel Murine Mast Cell Protease That Is Highly Induced by Multiple Pathways of Mast Cell Activation

Granzymes are serine proteases known mostly for their role in the induction of apoptosis. Granzymes A and B have been extensively studied, but relatively little is known about granzymes C to G and K to M. T cells, lymphohematopoietic stromal cells, and granulated metrial gland cells express granzyme D, but the function of granzyme D is unknown. Here we show that granzyme D is expressed by murine mast cells and that its level of expression correlates positively with the extent of mast cell maturation. Coculture of mast cells with live, Gram-positive bacteria caused a profound, Toll-like receptor 2 (TLR2)-dependent induction of granzyme D expression. Granzyme D expression was also induced by isolated bacterial cell wall components, including lipopolysaccharide (LPS) and peptidoglycan, and by stem cell factor, IgE receptor cross-linking, and calcium ionophore stimulation. Granzyme D was released into the medium in response to mast cell activation. Granzyme D induction was dependent on protein kinase C and nuclear factor of activated T cells (NFAT). Together, these findings identify granzyme D as a novel murine mast cell protease and implicate granzyme D in settings where mast cells are activated, such as bacterial infection and allergy.

The Metrial Gland in the Rat and Its Similarities to Granular Cell Tumors

Metrial glands are normal structures located in the mesometrial triangle of the pregnant rat uterus from gestational day (GD) 8 through termination of pregnancy. Metrial glands are composed of a dynamic mixed cell population of granulated metrial gland (GMG) cells, endometrial stromal cells, trophoblasts, blood vessels, and fibroblasts. Collections of similar cells may be seen in association with pseudopregnancy and other hormonal disturbances. Granulated metrial gland cells are the hallmark cell of the metrial gland. They are bone-marrow-derived, perforin-positive, natural killer cells that proliferate in the pregnant uterus. Understanding the normal histogenesis of the metrial gland and recognizing the possible existence of GMG cells and a reactive metrial gland in the nonpregnant state are important when examining any uterine lesion that contains granulated cells. This report demonstrates that the cellular composition, morphology, and immunohistochemical staining profile of normal metrial glands are similar to reported granular cell neoplasms in rats and mice. The possibility of a non-neoplastic lesion involving the metrial gland should be considered when proliferative lesions involving granulated cells are observed in the uterus of mice and rats from nonclinical toxicity studies. Positive immunohistochemical staining for perforin and S100 would assist in the classification of such lesions as a reactive metrial gland or decidual reaction.

Vascular endothelial growth factor production by rat granulated metrial gland cells and their morphological features in normal and pathological conditions

Granulated metrial gland (GMG) cells are pregnancy-specific cells that may have many functions in successful placentation and pregnancy. In the present study, changes in the rat GMG cell structure, distribution and vascular endothelial growth factor (VEGF) expression during early pregnancy were evaluated by light microscopy. Implantation sites taken from females with spontaneous abortion were also investigated. On Day 7 of pregnancy, GMG cells were distributed through the implantation and interimplantation sites. They formed metrial glands in the mesometrial triangle on Day 9, and were observed in the decidua basalis on Day 14 of pregnancy. Avidin-biotin complex immunohistochemistry revealed that GMG cells showed moderate staining for VEGF at the beginning of pregnancy and intense staining on Days 9 and 10 of pregnancy. They were localised mostly near the newly formed blood vessels. The implantation sites from spontaneously aborting females showed numerous leucocytes in the lumen of mesometrial blood vessels. In spontaneously aborting females, GMG cells showed a distinct morphology, increased in number and volume, their granules were denser and degranulation was observed. These results suggest that rat GMG cells might be a guide for placental angiogenesis and they might share a role with leucocytes in pathological conditions.

Histological Study of Granulated Metrial Gland (GMG) Cells in Beige (DA-bg/bg) Rats

To clarify the roles of granulated metrial gland (GMG) cells for successful pregnancy in rats, GMG cells in beige rats (genotype: DA-bg/bg), whose NK cells show lysosomal dysfunction because of abnormalities in cytoplasmic granules, were examined in mid- and late-pregnancy by light and electron microscopies. The GMG cells of beige rats were significantly less in number than those of the two controls (genotypes: DA-bg/+ and DA-+/+) in mid- and late-pregnancy, and this accompanied a low reproductive performance in the beige rats. The size of intracellular granules in the GMG cells of the beige rats was larger than for the two controls on each corresponding day of pregnancy. These results suggest that the activity of rat GMG cells and peripheral NK cells might be influenced by the beige gene, which is involved in reproductive performance.

Human uterine natural killer cells: a reappraisal

The presence of granulated cells within the uterus of many species has been recognised for many years but only recently have these been recognised to be a type of NK cell. Various terms have been applied to the cells, including endometrial granulocyte, K cell and, in mouse and rat, granulated metrial gland cell. Although early studies are often based on histology and electron microscopy, these often include important information for current studies. In vitro studies of purified cells have focused particularly on cytotoxicity and cytokine production and roles in the control of trophoblast invasion and spiral artery remodelling in human pregnancy have been proposed. Evidence in mouse has implicated uNK cell production of IFN-γ in vascular remodelling but evidence for such a role for human uNK cells remains to be established. Investigation of uNK cells in human pregnancy is hampered by the lack of availability of tissues from the first half of the second trimester of pregnancy when vascular remodelling occurs and also by possible differences between cells from different regions of decidua. The presence of similar cells in species with no trophoblast invasion into the uterus and epitheliochorial placentation raises the question of whether control of trophoblast invasion by human uNK cells is important in vivo and raises the possibility of another function which is conserved between species.

Development of transferred xenogeneic vole embryos in mouse uteri

ABSTRACTAn experimental model to study interspecific pregnancy using voles, Microtus arvalis, and green fluorescent protein gene‐induced transgenic mice is presented. Xenogeneic blastocysts from the vole were transferred into the uteri of pseudopregnant mice along with allogeneic blastocysts from green fluorescent protein gene‐induced transgenic mice. The uteri containing xeno‐allo combined transfers were examined from day 6 to 13 of gestation. Although the vole embryos implanted, the uteri containing vole embryos were smaller compared with those having allogeneic mouse embryos. On day 8, the uteri containing vole embryos hemorrhaged internally and no vole embryo was found in the pregnant uterus after day 11. Allogeneic mouse embryos developed normally despite the presence and abortion of the vole embryos. In uteri implanted with vole embryos, decidua were formed and numerous blood vessels were distributed around the embryo. Maternal blood cells infiltrated into the celomic cavity of the vole embryo through the discontinuous region of trophoblast. Periodic acid‐Schiff‐positive granulated metrial gland cells were remarkably increased in the decidual sites. These findings suggest that a disorder of embryo–maternal interaction might induce the appearance of numerous granulated metrial gland cells and rejection of the embryos.

Granulated metrial gland cells in the murine uterus: localization, kinetics, and the functional role in angiogenesis during pregnancy.

Granulated metrial gland (GMG) cells are a major immune cell population in the murine pregnant uterus, and contribute to the maintenance of pregnancy by functioning as uterus-specific natural killer (NK) cells. In order to reveal their kinetics, activation, and functional roles in pregnancy, we conducted quantitative and immunohistochemical analyses in normal and immuno-modulator-treated mice. Under a light microscope, GMG cells were identified by red cytoplasmic granules in periodic-acid-Schiff (PAS)-stained sections. They progressively increased in number and size with the peak at day 12-14 of pregnancy in the decidua and metrial gland. New vessel formation was most prominent around day 8, and the total vascular area reached the peak at day 13. GMG cells were often located near the blood vessels, and expressed vascular endothelial growth factor (VEGF), suggesting their possible inducing role in angiogenesis during the development of decidua/metrial gland. While blood vessels in the non-pregnant uterus were negative for vascular cell adhesion molecule (VCAM)-1, those in the pregnant one were positive. Treatment with neutralizing antibody against VCAM-1, however, did not decrease the number of GMG cells. On the other hand, mitosis of GMG cells was frequently observed. These data suggest that the increment of GMG cells during pregnancy may largely result from local proliferation in the uterus rather than an increased influx of precursor cells. Although we attempted to induce in vivo activation of GMG cells by administration of interleukin-12 (IL-12) or alpha-galactosylceramide, a potent activator for natural killer-T (NK-T) cells, the number of GMG cells did not appreciably increase. The present study has demonstrated that GMG cells locally proliferate in the pregnant uterus, not being related to VCAM-1 expression by the uterine vasculature or systemic activation of NK cells and NK-T cells, and seem to be involved in angiogenesis in the pregnant uterus through VEGF production.

Alterations in the expression of homing-associated molecules at the maternal/fetal interface during the course of pregnancy.

One of the most fascinating immunologic questions is how the genetically distinct fetus is able to survive and develop within the mother without provoking an immune rejection response. The pregnant uterus undergoes rapid morphological and functional changes, and these changes may influence the nature of local immune responses at the maternal/fetal interface at different stages of gestation. We hypothesized that specialized mechanisms exist to control access of maternal leukocyte subsets to the decidua and that these mechanisms are modulated during the course of pregnancy. At the critical period of initial placenta development, the maternal/fetal interface displays an unparalleled compartmentalization of microenvironmental domains associated with highly differentiated vessels expressing vascular addressins in nonoverlapping patterns and with recruitment of specialized leukocyte subsets (monocytes, granulated metrial gland cells, and granulocytes) thought to support, modulate, and regulate trophoblast invasion. One of the most striking observations at this time of gestation is the almost complete exclusion of lymphocytes from the maternal/fetal interface. The second half of pregnancy is characterized by a partial loss of microenvironmental specialization and different switches in vascular specificity within the decidua basalis, paralleling dramatic changes in the populations of recruited leukocytes (e.g., a striking influx of lymphocytes, especially T cells). In the term pregnant uterus, the expression of all vascular addressins decreased dramatically; only weakly staining maternal vascular segments remained. These segments may define sites of extremely low residual traffic in the term decidua, which contains remarkably few maternal leukocytes overall. Our results suggest that the maternal/fetal interface represents a situation in which leukocyte trafficking is exquisitely regulated to allow entry of specialized leukocyte subsets that may play a fundamental role in immune regulation during pregnancy.

Spontaneous eosinophilic granulated round cell tumors in rats.

Morphologic and histochemical characteristics were noted for three spontaneous tumors with eosinophilic cytoplasmic granules that occurred in aged Fischer 344 rats. Macroscopic lesions were widely distributed in the body, mainly involving the intra-abdominal adipose tissue, pancreas, and mesenterium. These lesions were generally hard swellings with nodular and sclerosing areas. Bloody ascites was a concomitant finding. Histologically, the tumor cells were round, from 9 to 30 microm in diameter with one or two round to oval nuclei, and characterized by eosinophilic granules (0.5-2.0 microm) that stained definitely to weakly positive with the periodic acid-Schiff reaction and demonstrated no metachromasia with toluidine blue stain. Furthermore, the granules were characterized by a positive reaction with lectin histochemistry for concanavalin A (Con A), wheat germ agglutinin (WGA), phaseolus vulgaris agglutinin (PHA-E4), lens culinaris agglutinin (LCA), and recinus communis agglutinin (RCA-I) in all tumors and for ulex europaeus agglutinin (UEA-I), peanut agglutinin (PNA), and soybean agglutinin (SBA) in one tumor. Positive reactions for anti-rat mast cell protease II and CD8 were not demonstrated immunohistochemically. Abundant glycogen was noted in the large tumor cells from one rat. With electron microscopy, the cytoplasmic granules were identified as electron-dense homogenous bodies bounded by a single unit membrane. These characteristics are similar to those of granulated metrial gland cells, but further study is needed to clarify the cell of origin for these tumors.

Mouse granulated metrial gland cells require contact with stromal cells to maintain viability.

Granulated metrial gland (GMG) cells differentiate in the uterine wall in pregnancy in mice but the mechanisms which control their differentiation and maintenance are unknown. In vivo, GMG cells share an intimate association with fibroblast-like stromal cells. The importance of this association has been assessed by examining the effects of withdrawal of stromal cell contact on GMG cell maintenance in vitro. When single cell suspensions of cells were prepared from mouse metrial glands there was a steady decline in numbers with days of culture but usually some remained at 7 d of culture. The ability of metrial gland cells to kill Wehi 164 target cells in 51Cr-release cytotoxicity assays was retained by cells cultured for at least 3 d. When explants of metrial gland were maintained in culture to allow GMG cell migration onto the culture flask, the attached GMG cells were lost by 1 d later. Overall, these results suggest that a juxtacrine regulatory mechanism maintains GMG cells. The rapid loss of unsupported GMG cells in culture has major implications in the design of assays to examine GMG cell function.

Expression of vascular endothelial growth factor by granulated metrial gland cells in pregnant murine uteri.

Granulated metrial gland (GMG) cells are a characteristic uterine component belonging to a natural killer cell lineage. This study is aimed at revealing their kinetic and spatial relationship with vascular growth during pregnancy and the expression of vascular endothelial growth factor (VEGF). GMG cells and blood vessels were identified by periodic-acid-Schiff-reagent (PAS)-stained granules and positive staining for factor-VIII-related antigen, respectively. GMG cells were widely distributed in the decidua and metrial gland and showed a numerical increase with a peak at day 13 in parallel with the increase of vascular density. Preceding the maximal vascular development at day 13, microvessels with a narrow lumen representative of neovascularization prevailed at days 7-9, and the VEGF content in the decidua/metrial gland was significantly elevated at days 7-13 concurrently with mRNA expression. By immunolight microscopy combined with PAS staining, GMG cells with PAS-stained granules were positive for VEGF. Immunoelectron microscopy demonstrated that immunoreactions were diffuse in the cytoplasm but not localized in the granules. In contrast, fibroblast-like stromal cells were negative. These data indicate that GMG cells express VEGF and may play inducing roles in uterine neovascularization during pregnancy.

Mouse granulated metrial gland cell cytotoxicity of Wehi 164 cells: is there a role for interleukin-3 and tumour necrosis factor-alpha?

Mouse granulated metrial gland (GMG) cells are maternal, cytotoxic cells found in the uterine wall in pregnancy. In vivo, they are cytotoxic to layer 1 labyrinthine placental cells. In an in vitro system, granulated metrial gland cells are highly cytotoxic to Wehi 164 fibrosarcoma cells. In this study the role of interleukin (IL)-3 and tumour necrosis factor (TNF)-alpha in granulated metrial gland cell cytotoxic activity was investigated. Chromium-release cytotoxicity assays using mouse metrial gland effector cells and Wehi 164 target cells with the addition of IL-3 or an antibody to TNF-alpha. An antibody to TNF-alpha reduced the level of cytotoxic activity. IL-3 had no effect on the level of cytotoxicity. Mouse granulated metrial gland cells kill using a TNF-alpha mediated mechanism which is independent of IL-3 stimulation.

Natural killer cells fertile with receptors for HLA-G?

Natural killer (NK) cells are lymphocytes involved in the innate immune response against certain microbial and parasitic infections (1, 2). These cells comprise ≈5–20% of peripheral blood lymphocytes and are present in spleen, liver, bone marrow, and thymus at a lower frequency and are rare in lymphatics and lymph nodes. However, in the mucosal decidual tissues of the maternal uterus, NK cells are the most abundant class of lymphocyte, representing up to 95% of all lymphocytes. Initially identified by their distinct morphology, NK cells in the placenta were designated “granulated metrial gland cells” in rodents (3) and “uterine granular lymphocytes” in humans (4, 5). Why do NK cells preferentially accumulate in the decidua? No clear rationale has emerged to explain this phenomenon. Given the established role of NK cells in innate immunity, protection of the fetus against transplacental infections is a good bet, but nonimmune functions involving fetal sustenance have also been proposed (6).

"Mesenchymal tumor" or "decidual-like reaction"?

For more than 40 yr, an unusual urinary bladder lesion has been known to occur in certain strains of mice, but no consensus has been obtained regarding its etiology, pathogenesis, biology, or classification. The lesion was first assumed to be epithelial and non-neoplastic, then it was called a smooth muscle cell tumor or leiomyosarcoma because of ultrastructural characteristics for smooth muscle cells. Later, the nonspecific term "mesenchymal tumor" was introduced due to histomorphologic differences from all smooth muscle tumors known. Recently, a proposal was made to name it "decidual-like reaction" because of the histomorphologic similarity to the rare spontaneous decidual reaction in the uterus of aging mice. Both lesions are characterized by spindle and large pleomorphic epithelioid cells with large bizarre nuclei; these characteristics mimic anaplasia of malignant tumors and led pathologists to assume a neoplastic nature. The decidual hypothesis is supported by the regular presence of nuclear progesterone receptors, the occasional occurrence of eosinophilic cytoplasmic granules, the rare finding of cells morphologically resembling granulated metrial gland cells (all also observed in the uterine decidual reaction), and the reproducibility through long-term feeding of combinations of estrogens and progestogens. It appears that the new decidual hypothesis can explain many detailed facets of the lesion, with the exception of the reported smooth muscle cell characteristics. The controversy of "mesenchymal tumor versus decidual-like reaction" should be resolved soon, not only as a scientific issue, but also because of consequences for risk assessment.

Evidence of specialized leukocyte-vascular homing interactions at the maternal/fetal interface.

In normal pregnancy, the maternal immune system fails to reject the fetus or the placenta as an allogeneic graft. We hypothesize that specialized mechanisms of leukocyte recruitment might limit access of circulating maternal immune cells to the maternal/fetal interface. During the critical period of initial trophoblast invasion there is an elegantly orchestrated progression of leukocyte homing events in the decidua basalis, associated with highly regulated expression of vascular addressins and segregation of specialized leukocyte subsets into well-defined decidual microdomains. Neutrophils are limited to the region of necrosis associated with enzymatic digestion at the leading edge of the invading trophoblast, where an almost linear array of maternal blood vessels displays the neutrophil ligand E-selectin. Cells with the phenotype of monocytes but expressing alpha4beta7 integrin are localized in the blood vessels of the specialized "vascular zone", which display the unusual combination of P-selectin (partially associated with platelets) and the alpha4beta7 ligand mucosal vascular addressin-1 (MAdCAM-1). Granulated metrial gland cells (alpha4+beta7-, probably alpha4beta1+) constitute a well-defined cluster positioned in the central decidua basalis around venules prominently expressing the alpha4beta1 ligand VCAM-1 (but not MAdCAM-1). T and B lymphocytes are rare. Our results suggest that selective mechanisms for regulating leukocyte access, associated with microdomain specialization within the decidua basalis, may play a fundamental role in immune regulation during the invasive period of placental development.

Effect of serum on mouse granulated metrial gland cell cytotoxicity.

To examine factors affecting mouse granulated metrial gland cell cytotoxicity. Chromium-release assays using mouse metrial gland cell suspensions targeted against Wehi 164 fibrosarcoma cells were used. Modulation of cellular cytotoxicity was investigated using an antibody, anti-asialo-GM1, to attempt depletion of effector cells and sera to determine if inhibitory factors are present in blood in vivo. No reduction in cellular cytotoxicity was found following treatment of effector cells with asialo-GM1 antibody, but there was a reduction following treatment with asialo-GM1 antibody plus guinea pig serum and with guinea pig serum alone. Sera from pregnant, male, and SCID mice also reduced the level of cytotoxicity by metrial gland cell suspensions. The reduction in cytotoxicity in the presence of serum indicates that there are factors in the blood which are inhibitory to granulated metrial gland cell cytotoxic activity in vivo. The resistance of mouse metrial gland cells to asialo-GM1 antibody depletion is supportive of a natural cytotoxic (NC) lineage for this cell type.

Granulated metrial gland cells and interstitial trophoblast in the uterine wall of the bank vole, Clethrionomys glareolus, in early pregnancy.

The morphology and distribution of granulated metrial gland cells and of interstitial trophoblast cells in the uterine wall was studied in the first half of pregnancy in the bank vole, Clethrionomys glareolus. The morphology and distribution of granulated metrial gland cells was generally similar to that found in other members of the Rodentia, although they were absent from the walls of the arterial vessels passing through the decidua basalis. Interstitial trophoblast invaded the decidualising endometrium mesometrial to, and antimesometrial to, the implanted embryos. There was no apparent spatiotemporal relationship between the distribution of granulated metrial gland cells and interstitial trophoblast cells.

Cell death of uterine natural killer cells in murine placenta during placentation and preterm periods.

In the murine uterus granulated metrial gland (GMG) cells appear only during normal pregnancy. GMG cells belong to a member of natural killer (NK) cells and play an important role in fetus survival and placental growth. Our previous study revealed that mouse GMG/uterine NK (uNK) cells in the late pregnancy rapidly disappear from the uterus, due to the degenerative change classified as necrosis. But there are few reports regarding appearance and morphology of uNK cells during late pregnancy. We examined histologically and histochemically how and when uNK cells undergo cell death. The uNK cells in the metrial gland increased in number and reached maximum until day 12 of pregnancy. Sudden disappearance, however, occurred after day 15 and the granules reduced in both number and size. In situ DNA fragmentation detection revealed that DNA fragmented uNK cells increased in number during days 13 to 15 and reached 70.2% at day 15 of pregnancy. From days 13 to 17, uNK cells were positive against anti-perforin antibody. Ultrastructurally, uNK cells at day 15 showed poor organelles and unusual granules in structure. In uNK cells at day 17, condensation of nucleus chromatin, reduction in size and phagocytosis into other uNK cells were observed. These results suggested that uNK cells undergo at least two types of cell death, classified as necrosis and apoptosis, at the different stages of pregnancy, and that perforin is not a mediator for cell death.

Mode of cell death in the rat metrial gland during peripartum regression.

In the rodent uterus, the metrial gland develops during midpregnancy and undergoes regression prior to parturation. The involution of the gland is reported to be accompanied by the loss of gland cells due to their death in situ. Cell death has been classified by using morphological criteria into two types: necrosis and apoptosis. To study the mechanism involved in the peripartum regression of the rat metrial gland, we examined the mode of cell death in the gland during the last week of gestation. We identified apoptotic cells in the regressing metrial gland by using DNA fragmentation, in situ DNA 3'-end labeling, and electron microscopy. Expression of progesterone receptor (PR) and estrogen receptor (ER) was also demonstrated by immunohistochemistry in the gland. The mean weight of metrial gland nodes decreased after day 18 of pregnancy. The apoptotic granulated metrial gland (GMG) cells that were detected by using the in situ DNA 3'-end labeling method were observed on day 16 of pregnancy, and they increased in number after day 20 of pregnancy. Intense fragmentation of DNA was also found from day 20 to day 22 of pregnancy. Electron microscopy demonstrated apoptotic GMG cells in the regressing metrial glands, confirming the results of the labeling studies. Immunohistochemical study revealed that expression of PR and ER, which were localized mainly in fibroblast-like stromal cells but not in GMG cells, was almost unchanged during late pregnancy. Apoptotic cell death is the major mode of rat metrial gland cell death in the peripartum loss of metrial gland cells.