Immunogold labeling of perforin and serine esterases in granulated metrial gland cells

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by creating large pores on the target plasma membrane. Besides perforin, the granules of CTL and NK cells contain a family of serine esterases. Perforin has also been localized in granulated metrial gland (GMG) cells of the murine embryo implantation site by light microscopic immunostaining. Ultrastructural immunogold labeling with antibodies against perforin and a serine esterase (MTSP 1 or granzyme A) shows that GMG cells contain both perforin and serine esterases in the fine granular matrix of their granules. Perforin has been located in all of the granules, whereas gold particles corresponding to serine esterases have been found in most of the granules. Results from the double immunogold technique indicate that perforin and serine esterases colocalize to most of the same granules in GMG cells. This study supports the view that GMG cells are related to cytolytic lymphocytes.     

Demonstration of YAC target cell lysis by murine granulated metrial gland cells

Granulated metrial gland (GMG) cells are a consistently observed but poorly understood feature of the murine uterus during successful pregnancy. From morphological studies and antibody phenotyping it has been suggested that GMG cells may be members of the natural killer (NK) cell lineage. However, lysis of murine NK cell targets by GMG cells has not been observed although lysis of freshly dissociated trophoblast cells by GMG cells has been recorded using timelapse video. We failed to demonstrate significant interactions between migrating GMG cells, collected from explant cultures under previously reported cultures conditions, and YAC target cells. However, YAC cell lysis did occur if hrIL-2 was present throughout the periods of explant culture and lysis assay. Furthermore, lysis was enhanced if the pregnant females were treated with the interferon inducer poly I.C. 24 hr before metrial gland collection. GMG cells expressed perforin and serine protease mRNA. Consistent with the lysis experiments, expression of these genes was enhanced when the cells were incubated with hrIL-2. Our data provide further support for a relationship between GMG cells and NK cells, but do not establish a relationship of identity since hrIL-2, a growth factor sufficient for the culture of NK cells, cannot support growth or prolong survival of GMG cells.     

Granulated metrial gland cells of pregnant mouse uterus are natural killer-like cells that contain perforin and serine esterases

The mouse uterus during pregnancy contains a large population of lymphoid cells termed granulated metrial gland (GMG) cells. Our observations suggest that these cells are highly activated cytolytic lymphocytes related to NK or lymphokine-activated killer cells. Immunostaining demonstrated asialo GM1 and Thy-1 on GMG cells, both of which are expressed by NK cells. Decidua basalis tissue and isolated GMG cells contained three proteins that are characteristic of activated cytolytic lymphocyte granules: perforin, serine esterase 1, and serine esterase 2. These mediators were demonstrated in GMG cells by Western blot analysis using polyclonal antisera and by Northern blot analysis using specific cDNA probes for their mRNA. The proteins were not detected in normal spleen or liver or in asialo GM1+ cells isolated from those organs, consistent with the absence of these mediators from resting cytolytic cells. The amount of perforin in GMG cells was similar to that present in cloned, IL-2-stimulated, CTL shown previously to contain a large amount of this protein. A large population of NK cells bearing the surface marker LGL-1 was demonstrated at the implantation site by labeling with monoclonal antibody 4D11, but T cells were not detected. Many LGL-1+ cells at the implantation site expressed the GMG cell markers asialo GM1, Thy-1, and perforin. Staining intensities were inversely correlated, with LGL-1-bright cells showing little or no staining of GMG cell markers and LGL-1-faint cells showing more obvious staining of GMG cell markers. This suggests that LGL-1+ NK cells may differentiate in situ to GMG cells, losing LGL-1 and gaining a high concentration of GMG cell markers in the process. Activated cytolytic cells related to NK or lymphokine-activated killer cells may function in the pregnant rodent uterus to intercept and kill aberrant placental or embryonic cells that might otherwise enter the female and proliferate.     

Uptake of immunoglobulin and albumin by granulated metrial gland cells in vitro

Single cell suspensions of metrial gland tissue from rats at Day 14 of pregnancy were prepared for maintenance in vitro. During the first 2 days of culture IgG was detected in glycoprotein granule-containing granulated metrial gland (GMG) cells. Albumin was also detected in GMG cells at the same stages. The IgG and albumin were not detected during the next 4 days in culture. When metrial gland cells, maintained in vitro for 5 days, were incubated with rat serum for a further 24 h, IgG and albumin were detected in GMG cells. When similar cultures were incubated for 24 h with purified rat IgG or purified rat albumin, GMG cells were positive for IgG and albumin respectively. Albumin was not detected in GMG cells in wax sections of metrial gland tissue, although IgG has previously been demonstrated. The uptake of serum proteins by GMG cells in vitro has been clearly shown but the difference in IgG and albumin content of these cells in paraffin-wax sections indicates that the means by which IgG accumulates intracellularly may be different in vitro and in vivo.     

Expression of perforin by natural killer cells within first trimester endometrium in humans.

The lymphocyte pore-forming protein (PFP)--perforin, also named cytolysin--is a potent mediator of cytotoxicity found in the granules of cytotoxic T lymphocytes and natural killer (NK) cells. Granulated metrial gland (GMG) cells found in the pregnant mouse uterus express perforin and are thought to be highly activated cytolytic lymphocytes related to NK cells. Their role in pregnancy is unknown. Human endometrial granulocytes (EGs) are phenotypically similar to murine GMG cells and, like them, express NK cell markers. However, up to now it was not known whether EGs also express perforin. By means of immunohistochemical analysis, using antisera specific for perforin and monoclonal antibodies to CD56 (NKH-1), CD2 (T11), CD3 (Leu-4), CD4 (Leu-3a), and CD8 (OKT-8), we demonstrated that perforin is present in EGs in the decidualized endometrial stroma and decidual tissue of first-trimester gestational endometrium. In fact, double immunohistochemical labeling demonstrated the co-expression of perforin and NKH-1. This population also expressed some T-cell surface antigens (Leu-4 and T11), but not Leu-3a or OKT-8. Chorionic villi, in contrast, lack perforin+ cells. The presence of a potent cytolytic mediator in NK-like cells in both murine and human pregnant uterus raises the issue of the function of such cells in pregnancy.     

Measurement of natural killer activity and target cell binding by mouse metrial gland cells isolated by enzymic or mechanical methods

Cells of the metrial glands of mice were isolated by enzymic or mechanical dissociation procedures. Morphological observations indicated that up to half of the enzymically dissociated cells and nearly all of the mechanically dissociated cells were granulated metrial gland cells, but the presence of some fibroblast-like stromal cells among the latter population was not ruled out. Moreover, the granulated metrial gland cells had lost a substantial part of their granule content during isolation. Both cell preparations had little or no natural killer (NK) activity, indicating either that granulated metrial gland cells are not NK-like or that their NK activity was impaired by loss of granule-associated lytic substances or by other factors. Enzymically dissociated metrial gland cells did not bind significantly to the NK target cell YAC-1, nor did they develop granules, NK activity, or the ability to bind YAC-1 cells during culture in vitro, either in normal medium or with the addition of indomethacin or lymphokines. Mechanically dissociated metrial gland cells bound avidly to YAC-1 cells but not to P815 cells or adult thymus cells, which are not NK target cells. Since many if not most of the mechanically dissociated metrial gland cells appeared morphologically to be granulated metrial gland cells, their selective binding to an NK target cell suggests that granulated metrial gland cells may be related in some way to NK cells.     

Murine granulated metrial gland cells at uterine implantation sites are natural killer lineage cells

Granulated metrial gland (GMG) cells, a population of morphologically distinct, bone marrow-derived cells in murine decidua that react with mAb 4H12, are shown in this report to express NK-specific Ag and to become cytolytic to the NK cell target YAC-1 when cultured in the lymphokine IL-2. When 1-mm3 explants of 8-day decidual tissue were cultured with IL-2, large numbers of 4H12+ GMG cells migrated out of the tissue. Migration was dependent on the amount of IL-2 used. This explant technique was used to isolate a pure population of GMG cells. The migratory activated GMG cells were phenotypically 4H12+, NK1.1+, LGL-1+/-, CD3-, and MAC-1-. Furthermore, the IL-2-activated GMG cells killed YAC-1 but not P815 cells in a 4-h 51Cr-release cytotoxicity assay. 4H12+ GMG cells from collagenase-digested decidual tissue also were analyzed for the presence of NK lineage Ag by flow cytometry and shown to coexpress the NK-associated Ag NK1.1 and ASGM1 but not the T cell Ag CD3 or macrophage Ag MAC-1 or F4/80. GMG cells isolated by collagenase digestion did not express LGL-1, an Ag associated with lytic NK cells. Our results demonstrate that GMG cells express Ag and functions characteristic of NK cells, and thus GMG cells can be assigned to the NK lineage. The possible relevance of NK cells at implantation sites is discussed.     

妊娠小鼠子宫腺细胞区的碱性磷酸酶与酸性磷酸酶的研究

小鼠子宫系膜三角区在妊娠后出现上皮样细胞群,群内的细胞称颗粒子宫腺细胞(granu-lated metriial gland cells,GMG细胞),该区改称子宫腺细胞区(metriial gland cell area,MGCA).取孕12～19天MGCA,液氮速冻,恒冷箱切片,偶氮偶联法显示碱性磷酸酶(ALP)与酸性磷酸酶(ACP).结果为,ALP主要分布于GMG细胞群间的疏松结缔组织中;GMG细胞为阴性反应.ACP主要位于GMG细胞内,群间结缔组织含量较少.两种酶的活性随胎龄增加而减弱.ALP与ACP的定位与活性变化特性显示它们与GMG细胞功能关系密切.     

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.     

The metrial gland

The information available about the metrial gland of the pregnant rodent uterus with its content of granulated metrial gland (GMG) cells is reviewed. Recent research shows that GMG cells differentiate from bone marrow cells and supports the suggestion that GMG cells are involved in the immunological relationship between mother and foetus. There is probably a complex association between GMG cells and stromal cells of the metrial gland, and it is suggested that the association between GMG cells and the placental labyrinthine cells represents a functional interaction.     

Granulated metrial gland cells in the pregnant uterus of mice expressing the collagen mutation tight-skin (Tsk/+)

Summary Influences of the extracellular matrix (ECM) on the differentiation and distribution of granulated metrial gland (GMG) cells, a uterine natural killer (NK)-like cell subset, were studied by histological examination of implantation sites in the mouse mutant Tsk/+. Tsk/+ mice overproduce collagens I and III. GMG cell differentiation appeared to progress normally in Tsk/+ mice between days 6.5 and 12.5 of gestation. The distribution of GMG cells, however, was abnormal. Significant numbers of GMG cells were found in the antimesometrial and lateral decidual regions at day 8.5 of gestation and in the regions between implantation sites until day 10.5 of gestation. Loss of GMG cells from these regions normally occurs by day 6.5 of gestation. These data suggest that alterations to the ECM change the migration properties or life span of GMG cells.     

Migration of granulated metrial gland cells from cultured explants of mouse metrial gland tissue

Summary The migration of granulated metrial gland (GMG) cells from cultured explants of metrial gland tissue obtained from mice killed between days 10 and 16 of pregnancy has been studied. GMG cells migrated from all of the explants but more GMG cells were found around explants obtained from mice at day 10 of pregnancy than around explants obtained at later stages of pregnancy. The number of GMG cells found around each explant reached a peak at days 1, 2 or 3 of culture but only a few GMG cells were found around the explants by day 7 of culture.     

Mouse granulated metrial gland cells originate by local activation of uterine natural killer lymphocytes

Natural killer (NK) lymphocytes were identified in the mouse uterus by immunostaining their surface membrane marker, LGL-1. The cells were present in large numbers from before mating through Day 14 of pregnancy. Double immunostaining indicated that uterine NK cells began to contain the pore-forming protein, perforin, on Day 6 of pregnancy in mesometrial decidua. Perforin is a probable mediator of cellular cytotoxicity found in lymphokine-activated NK and cytotoxic T lymphocytes. Activation of NK cells to produce perforin continued in mesometrial decidua on Days 8 and 10 of pregnancy and in the peripheral portion of metrial glands (MGs) on Days 12 and 14 of pregnancy, where cells at 3 stages of activation were simultaneously present: small cells with bright surface membrane staining of LGL-1 but no perforin (nonactivated), larger cells with intermediate staining of both markers (partially activated), and large cells with bright staining of perforin but no LGL-1 (fully activated). These observations indicate that activation of uterine NK cells involves loss of membrane LGL-1 as perforin accumulates in the cytoplasm, that the zone of activation shifts from mesometrial decidua to the MG on about Day 11 of pregnancy, and that nonactivated NK cells probably enter activation zones continuously during this period. Resting NK cells may enter activation zones by proliferation and/or migration from other regions of the uterus, rather than from blood, because depletion of circulating NK cells during pregnancy by treatment with NK-1.1 or asialo GM1 antibodies had no effect or only a small effect on the numbers of LGL-1-or perforin-positive cells seen in the uterus later in pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

颗粒子宫腺细胞的分离、纯化与细胞化学研究

胰蛋白酶消化小鼠子宫腺细胞区,制成单细胞悬液,滴片后进行细胞学及细胞化学研究。结果：有多种类型细胞存在于悬液中,其中中等大小细胞（直径２０μｍ左右）大部分为颗粒子宫腺细胞（ＧｒａｎｕｌａｔｅｄＭｅｔｒｉａｌＧｌａｎｄＣｅｌｌｓ,ＧＭＧｃｅｌｌｓ）。ＧＭＧ细胞约占所有细胞的５０％。利用Ｐｅｒｃｏｌｌ非连续性密度梯度离心可以提高ＧＭＧ细胞的纯度,使之达８０％以上。细胞化学研究显示ＧＭＧ细胞含ＡＣＰ,ＮＳＥ,ＬＮＡｓｅ及糖蛋白成份。     

Eosinophilic globule cells in mouse MFH-like sarcomas: lectin histochemistry.

Lectin binding patterns in ten mouse malignant fibrous histiocytoma (MFH)-like sarcomas containing eosinophilic globule (EG) cells and in granular metrial gland (GMG) cells of mouse placenta were stained with nine lectins (Con A, LCA, WGA, DBA, SBA, e-PHA, PNA, RCA-I and UEA-I) by an avidin-biotin-peroxidase-complex method. EG cells stained strongly with DBA, SBA and PNA which are specific for N-acetyl-D-galactosamine and/or D-galactose. DBA and SBA bound throughout the cytoplasm including the globules; PNA reacted preferentially at the cell surface. There was no evidence that these three lectins were reactive for immature EG cells. WGA, RCA-I and e-PHA also gave a slightly to moderately positive reaction to globules of EG cells. The results indicate that the globules contain abundant O-linked sequences of sugars, but also a few N-linked residues. MFH tumor cells showed a variable degree of binding with Con A, RCA-I, and WGA, but did not react with DBA, SBA and PNA. On the other hand, GMG cells exhibited specific affinities for DBA, SBA and PNA with staining patterns similar to those of EG cells. These findings suggest that EG and GMG cells may be of the same cellular lineage.     

Reduced release and binding of perforin at the immunological synapse underlies the age-related decline in natural killer cell cytotoxicity

Physiological aging is accompanied by a marked reduction in natural killer (NK) cell cytotoxicity (NKCC) at the single cell level, but the underlying mechanisms are unknown. To address this issue, we isolated NK cells from healthy young (≤ 35 years) and old (≤ 60 years) subjects and examined the effect of age on events fundamental to the process of NKCC. Simultaneous assessment of NKCC and NK cell–target cell conjugate formation revealed a marked age‐associated decline in NK cell killing but comparable conjugate formation, indicating a post‐target cell binding defect was responsible for impaired NKCC. Despite a reduction in the proportion of NK cells expressing the activatory receptor NKp46, NK cells from old donors were not hyporesponsive to stimulation, as no age‐associated difference was observed in the expression of the early activation marker CD69 following target cell coculture. Furthermore, intracellular levels of the key cytotoxic effector molecules perforin and granzyme B, and the fusion of secretory lysosomes with the NK cell membrane were also similar between the two groups. However, when we examined the binding of the pore‐forming protein perforin to the surface of its target cell, an event that correlated strongly with target cell lysis, we found the percentage of perforin positive target cells was lower following coculture with NK cells from old subjects. Underlying this reduction in binding was an age‐associated impairment in perforin secretion, which was associated with defective polarization of lytic granules towards the immunological synapse. We propose that reduced perforin secretion underlies the reduction in NKCC that accompanies physiological aging.     

In situ localization and characterization of bone marrow-derived cells in the decidua of normal murine pregnancy.

The study reported here was designed to examine the in situ distribution and characteristics of hemopoietically derived decidual cells during normal pregnancy in mice prenatally reconstituted with bone marrow cells carrying a transgenic marker. Bone marrow cells from a transgenic CD-1 strain (CD-1 beta; carrying 1000 copies of beta-globin genes in tandem) were injected into the yolk sac of Day 17 conventional CD-1 embryos. The pregnant females were allowed to deliver normally, and the female offspring raised to puberty were mated with CD-1 males and then killed on Day 12 of gestation. The extent of chimerism in sections of their spleens, uteri, and other organs was evaluated by in situ hybridization of the sections with a biotinylated cDNA probe specific for the beta-globin genes followed by avidin-biotin-peroxidase staining. Tissue controls were provided by CD-1 beta and CD-1 mice, respectively. Tissues were also processed without the application of the probe or with the application of biotinylated lambda DNA as specificity controls. Reconstituted mice exhibited variable degrees of hemopoietic chimerism as indicated by labeling of their splenic lymphocytes (18-54%; mean 42%) as well as hemopoietic cells in other organs. Variable cellular labeling was also noted in their decidua basalis and metrial glands. Labeled cells in these tissues were identified as typical decidual cells, macrophages, and granulated metrial gland (GMG) cells. Labeling of typical decidual cells varied extensively among implantation sites in the same chimera, the average labeling ranging from 17% to 33% (mean 24%) in various chimeras. Labeling was also noted in GMG cells, lymphocytes, and some decidual cells migrating out of metrial gland explants after 24-h culture. The non-pregnant uterus of a chimeric mouse revealed significant labeling of endometrial stromal cells indicative of their hemopoietic origin. These results revealed a hemopoietic origin of certain typical decidual cells and GMG cells identified in situ during normal murine pregnancy and a hemopoietic origin of certain endometrial stromal cells that may represent precursors of decidual cells. The precise timing of the predecidual stem cell migration from the bone marrow to the uterus remains to be defined.     

Degranulation enhances presynaptic membrane packing,which protects NK cells from perforin-mediated autolysis

Natural killer (NK) cells kill a target cell by secreting perforin into the lytic immunological synapse, a specialized interface formed between the NK cell and its target. Perforin creates pores in target cell membranes allowing delivery of proapoptotic enzymes. Despite the fact that secreted perforin is in close range to both the NK and target cell membranes, the NK cell typically survives while the target cell does not. How NK cells preferentially avoid death during the secretion of perforin via the degranulation of their perforin-containing organelles (lytic granules) is perplexing. Here, we demonstrate that NK cells are protected from perforin-mediated autolysis by densely packed and highly ordered presynaptic lipid membranes, which increase packing upon synapse formation. When treated with 7-ketocholesterol, lipid packing is reduced in NK cells making them susceptible to perforin-mediated lysis after degranulation. Using high-resolution imaging and lipidomics, we identified lytic granules themselves as having endogenously densely packed lipid membranes. During degranulation, lytic granule–cell membrane fusion thereby further augments presynaptic membrane packing, enhancing membrane protection at the specific sites where NK cells would face maximum concentrations of secreted perforin. Additionally, we found that an aggressive breast cancer cell line is perforin resistant and evades NK cell–mediated killing owing to a densely packed postsynaptic membrane. By disrupting membrane packing, these cells were switched to an NK-susceptible state, which could suggest strategies for improving cytotoxic cell-based cancer therapies. Thus, lipid membranes serve an unexpected role in NK cell functionality protecting them from autolysis, while degranulation allows for the inherent lytic granule membrane properties to create local ordered lipid “shields” against self-destruction.

Natural killer cells mediate largely unidirectional potent cytotoxicity against diseased cells while sparing themselves. The authors show that the NK cell membrane contains and focuses lipids of high density which shield against self-destruction, and a similar densely packed postsynaptic membrane is responsible for the perforin resistance and NK cell-mediated killing evasion of an aggressive breast cancer cell line.     

Immunosuppressor activity of rat endometrial granulated cells and their differentiation

Natural killer and natural suppressor activities of the rat endometrial granulated cells were assayed on day 13 of pregnancy or pseudopregnancy. Metrial gland granulated cells were used as endometrial granulated cells. The natural killer activities of metrial gland granulated cells and other cells were determined by means of Hashimoto-Sudo test with K562 cells as targets. The estimation of natural killer activity included removal of the cells sticking to glass from a suspension of material gland granulated cells. Cytochemically, metrial gland granulated cells were identified by the presence of PAS-positive granules in the cytoplasm after treatment of the cells with diastase and identification of a specific antigen with the help of specific antisera. The natural killer activity of metrial gland granulated cells was twice weaker than that of splenocytes from the same pregnant or pseudopregnant females. The level of natural killer activity was proportional to the content of metrial gland granulated cells in a cell system. These data suggest that the natural killer activity of metrial gland granulated cells is realized via their contact with cell targets. Natural killer and suppressor activities were determined simultaneously for metrial gland granulated cells and splenocytes of the same rat with common cell targets. When estimating the nuclear suppressor activity of metrial gland granulated cells, the splenocytes of the same rat were used as an effector in a natural killer test. Various amounts of metrial gland granulated cells were added to the effector : target system at a ratio of 50:1. The natural suppressor activity of metrial gland granulated cells did not depend on the amount of metrial gland granulated cells present in a natural killer system. After fractionation in a Percoll gradient, the highest natural killer activity was recorded in a 30% Percoll fraction. The highest and lowest natural suppressor activities were recorded in 30% and 60% Percoll fractions, respectively. The culture medium was characterized by natural suppressor activity as well. The differences in mean areas of metrial gland granulated cells in 30 and 60% Percoll fractions between the pregnant (144.7 +/- 13.4 and 75.0 +/- 12.5 microm2, respectively) and pseudopregnant (97.5 +/- 4.9 and 69.2 +/- 3.5 microm2) females were reliable. The natural killer activity was estimated in all studied 23 samples of metrial gland granulated cells, among which 18 (79.6 +/- 7.8%) displayed the natural suppressor activity as well. The absence of natural suppressor activity in five samples was combined with the absence of this activity in their culture medium and with a reduction in the mean area of metrial gland granulated cells in 30% Percoll fraction to 109.1 +/- 5.2 microm2. The data obtained confirm the known data on a low activity of metrial gland granulated cells and demonstrated for the first time the natural suppressor activity of these cells. It was concluded that the natural suppressor activity of metrial gland granulated cells is due to their differentiation from metrial gland granulated cells with natural killer activity.