Actions of endothelin-1 on prostaglandin production by gestational tissues

Endothelin-1 (10(-11)M-10(-7)M) was incubated with human umbilical vein endothelial cells and cells derived from amnion and decidua and prostaglandin production was determined. The rates of biosynthesis of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2) by endothelial cells were increased significantly by treatment with endothelin-1. Amnion cell PGE2 production was reduced significantly by endothelin-1 treatment whereas decidual PGE2 and prostaglandin F2 alpha production was unaffected by this treatment. Thus, it is possible that endothelins may play a part in the regulation of uteroplacental hemodynamics and the mechanisms of parturition.     

Endothelin-1 stimulates phospholipid hydrolysis and prostaglandin F2α production in primary human decidua cell cultures

The regulation of prostaglandin (PG) production by the uterine decidua may be an important mechanism controlling the onset and maintenance of human parturition. The present in vitro study has evaluated the potential for endothelin-1 (ET-1) to activate cell signalling and PGE2 alpha production in human primary decidua cell cultures. ET-1 stimulated a dose-dependent increase in inositol phospholipid hydrolysis and PG precursor release as evidenced by respective increases in [3H] inositol monophosphate accumulation and [14C] arachidonate release from radiolabelled decidua cells. PGF2 alpha production was increased in some but not all cell preparations in response to ET-1 alone. Pretreatment of decidua cells with interleukin-1 beta (IL-1 beta) enhanced PGF2 alpha production but not arachidonate release in response to ET-1. These in vitro observations support a possible role for ET-1 in the regulation of decidual PG production during parturition.     

Actions of endothelin-1 on prostaglandin production by gestational tissues

Endothelin-1 (10⁻¹¹M-10⁻⁷M) was incubated with human umbilical vein endothelial cells and cells derived from amnion and decidua and prostaglandin production was determined. The rates of biosynthesis of 6-keto-prostaglandin F₁ (6-keto-PGF₁) and prostaglandin E₂ (PGE₂) by endothelial cells were increased significantly by treatment with endothelin-1. Amnion cell PGE₂ production was reduced significantly by endothelin-1 treatment whereas decidual PGE₂ and prostaglandin F₂ production was unaffected by this treatment. Thus, it is possible that endothelins may play a part in the regulation of uteroplacental hemodynamics and the mechanisms of parturition.     

Decidual adenylate cyclase and prostaglandin production in vitro

Human decidua contains an active adenylate cyclase, and a number of studies indicate that adenylate cyclase is functionally linked to increased in vitro prostaglandin synthesis. Increased decidual prostaglandin synthesis is associated with parturition, and therefore activation of adenylate cyclase may be involved in the control of human parturition. In this study, third trimester human decidual cells were preincubated for no more than 24 h prior to stimulation with a number of reagents which increase cellular cyclic AMP levels. Forskolin rapidly increased intracellular and extracellular cyclic AMP levels, but there was no increase in prostaglandin E₂ biosynthesis during incubations ranging from 5 min up to 24 h. Dibutyryl cyclic AMP or 8-bromo-cyclic AMP were also without effect on PGE₂ production, which suggests that the adenylate cyclase was not linked to the mechanisms regulating prostaglandin production. Cholera toxin increased basal cyclic AMP and PGE₂ synthesis, and was without effect on IL-1β-stimulated PGE₂ levels. PGE₂ synthesis was increased by 24 h culture with IL-1β in all the cell preparations, indicating that the cells were biologically active, and that the lack of effect of changes in cyclic AMP synthesis on PGE₂ levels could not be attributed to a defect in the prostaglandin synthetic pathway. Our findings did not agree with earlier work which showed that changes in cyclic AMP were correlated with changes in PGE₂ production by human decidual cells. It is clear that in the previous studies the decidual cells were preincubated for 4–7 days prior to stimulation, in contrast with 24 h in our investigation. We suggest that the functional link between cyclic AMP and PGE₂ synthesis reported previously may develop during culture, and not be a part of normal decidual cell function, but further studies are needed to test this hypothesis.     

Prostaglandin biosynthesis by human decidual cells: effects of inflammatory mediators

There is substantial evidence that decidual activation, in association with infection, is linked with the onset of both preterm and term labor. We therefore undertook the present study to evaluate prostaglandin production and its potential regulation by inflammatory mediators in human decidual cells in primary monolayer culture. Upon attaining confluence, the cells were incubated with endotoxin, interleukin 1 alpha (IL1 alpha), interleukin 1 beta (IL1 beta); or tumor necrosis factor (TNF). Production of prostaglandin (PG) E2 and PGF2 alpha was determined using specific radioimmunoassays. Endotoxin and these cytokines all induced significant concentration-dependent increases in PGE2 and PGF2 alpha production. Our results suggest that term human decidual cells are responsive to endotoxin and cytokines and that generation of these substances in the decidua or nearby (eg. in response to infection) will lead to increased prostaglandin production and uterine contractions.     

Work in progress. Occurence of phospholipase A1 and A2 in human decidua

Phospholipase A₂, an enzyme which may regulate the formation of polyunsaturated fatty acids utilized for prostaglandin synthesis, was found to have significant higher activity in decidual than in myometrial tissue. The major part of phospholipase A₂ in the decidua had an acid pH optimum, which indicates that most of the enzyme is stored in the lysosomes of this tissue. These findings, together with previous observations, lend further support to the view that lysosomal phospholipase A₂ released within decidual cells might be a trigger of abortion and parturition.     

Work in progress. Occurence of phospholipase A1 and A2 in human decidua.

Phospholipase A2, an enzyme which may regulate the formation of polyunsaturated fatty acids utilized for prostaglandin synthesis, was found to have significant higher activity in decidual than in myometrial tissue. The major part of phospholipase A2 in the decidua had an acid pH optimum, which indicates that most of the enzyme is stored in the lysosomes of this tissue. These findings, together with previous observations, lend further support to the view that lysosomal phospholipase A2 released within decidual cells might be a trigger of abortion and parturition.     

A comparison of two populations of decidual cells by immunocytochemistry and prostaglandin production

Summary The decidua has been implicated in the control of human labour, particularly through changes in prostaglandin production, but this tissue contains a number of different cell types. A density gradient system was used to obtain two populations of cells from term human decidua, and these populations were characterised. The more dense cells (population B) was a mixed population, predominantly macrophages (80%), but small numbers of T- and B-lymphocytes were also present, as identified by immunocytochemistry. Most of these cell types also contained detectable levels of cyclooxygenase enzyme. The less-dense cell population (population A) did not contain significant numbers of the above cell types and released prolactin, suggesting that they were decidual stromal cells. This preparation of decidual stromal cells may be of use in defining the functions of these cells in labour.     

A comparison of two populations of decidual cells by immunocytochemistry and prostaglandin production

The decidua has been implicated in the control of human labour, particularly through changes in prostaglandin production, but this tissue contains a number of different cell types. A density gradient system was used to obtain two populations of cells from term human decidua, and these populations were characterised. The more dense cells (population B) was a mixed population, predominantly macrophages (80%), but small numbers of T- and B-lymphocytes were also present, as identified by immunocytochemistry. Most of these cell types also contained detectable levels of cyclooxygenase enzyme. The less-dense cell population (population A) did not contain significant numbers of the above cell types and released prolactin, suggesting that they were decidual stromal cells. This preparation of decidual stromal cells may be of use in defining the functions of these cells in labour.     

Epidermal growth factor stimulate cell proliferation and inhibits prolactin secretion of the human decidual cells in culture

Epidermal growth factor (EGF) has been found to be mitogenic in a variety of tissues. We investigated the biological effect of EGF on early pregnant human decidua and the non-pregnant decidualized human endometrium in the primary cell culture. EGF had a stimulatory action on cell proliferation in the early pregnant decidual cells and an inhibitory effect on prolactin (PRL) secretion from the decidual cells. The addition of progesterone into culture medium suppressed cell proliferation of decidual cells, whereas it enhanced PRL secretion from decidua. The analysis of the specific receptor for EGF in the early pregnant decidua and non-pregnant decidualized endometrium revealed that both tissues had a single component EGF receptor with a dissociation constant of nM order. These results suggest that EGF may play a role in the growth and function of endometrial stromal cells.     

An inhibitor of prostaglandin biosynthesis from human decidua: partial purification and properties

An inhibitor of prostaglandin synthetase which catalyzes the conversion of arachidonic acid into prostaglandin E2 was partially purified from the 105,000 x g supernatant fraction of the human decidual cell homogenate. By means of ammonium sulfate fractionation, Mono Q ion-exchange chromatography, and gel filtration chromatography, the inhibitor was purified about 15-fold, giving a preparation with a molecular weight of 55-60 KDa. The 50% inhibitory concentration of the purified substance was approximately 0.2 mg/ml. The inhibitor may play a role in suppression of prostaglandin production by decidua in early pregnancy.     

Role of uterine natural killer cells in angiogenesis of human decidua of the first-trimester pregnancy

Decidualization is accompanied by extensive angiogenesis, which is an essential step in the maturation of new blood vessels in mammalian pregnancy. The purpose of this study was to determine a distribution of uNK cells (CD56 uNK or CD56bright cells) in human decidua of the first-trimester pregnancy, and investigate whether uNK cells in human decidua could express vascular endothelial growth factor (VEGF-A) and/or angiopoietin2 (Ang2). Our immunohistochemical staining results demonstrated that a great amount of uNK (CD56 ) cells scattered throughout the decidual stroma and near endometrial gland and spiral vessels in human decidua. The protein expression of VEGF-A and Ang2 was detected in decidual stroma cells, capillary endothelial cells and glandular cells in tissue specimens. There was a positive correlation between microvessel density (MVD) and the number of the CD56-positive uNK cells in decidual stroma, and also between the number of the CD56-positive uNK cells and VEGF-A protein expression in the tissue. In addition, we found that uNK cells in human decidua could express VEGF-A mRNA, but not Ang2 mRNA, in isolated uNK cells in human decidua of the first-trimester gestation by combination of LCM and Nested-PCR. Our study indicated that uNK cells, through expressing VEGF-A, may play an important role in the angiogenic response at the time of human decidualization and early placenta development.     

Role of uterine natural killer cells in angiogenesis of human decidua of the first-trimester pregnancy

Decidualization is accompanied by extensive angiogenesis, which is an essential step in the maturation of new blood vessels in mammalian pregnancy. The purpose of this study was to determine a distribution of uNK cells (CD56⁺ uNK or CD56^bright cells) in human decidua of the first-trimester pregnancy, and investigate whether uNK cells in human decidua could express vascular endothelial growth factor (VEGF-A) and/or angiopoietin2 (Ang2). Our immunohistochemical staining results demonstrated that a great amount of uNK (CD56⁺) cells scattered throughout the decidual stroma and near endometrial gland and spiral vessels in human decidua. The protein expression of VEGF-A and Ang2 was detected in decidual stroma cells, capillary endothelial cells and glandular cells in tissue specimens. There was a positive correlation between microvessel density (MVD) and the number of the CD56-positive uNK cells in decidual stroma, and also between the number of the CD56-positive uNK cells and VEGF-A protein expression in the tissue. In addition, we found that uNK cells in human decidua could express VEGF-A mRNA, but not Ang2 mRNA, in isolated uNK cells in human decidua of the first-trimester gestation by combination of LCM and Nested-PCR. Our study indicated that uNK cells, through expressing VEGF-A, may play an important role in the angiogenic response at the time of human decidualization and early placenta development.     

Immunophenotype and cytokine profiles of rhesus monkey CD56bright and CD56dim decidual natural killer cells

The primate endometrium is characterized in pregnancy by a tissue-specific population of CD56(bright) natural killer (NK) cells. These cells are observed in human, rhesus, and other nonhuman primate decidua. However, other subsets of NK cells are present in the decidua and may play distinct roles in pregnancy. The purpose of this study was to define the surface marker phenotype of rhesus monkey decidual NK (dNK) cell subsets, and to address functional differences by profiling cytokine and chemokine secretion in contrast with decidual T cells and macrophages. Rhesus monkey decidual leukocytes were obtained from early pregnancy tissues, and were characterized by flow cytometry and multiplex assay of secreted factors. We concluded that the major NK cell population in rhesus early pregnancy decidua are CD56(bright) CD16(+)NKp30(-) decidual NK cells, with minor CD56(dim) and CD56(neg) dNK cells. Intracellular cytokine staining demonstrated that CD56(dim) and not CD56(bright) dNK cells are the primary interferon-gamma (IFNG) producers. In addition, the profile of other cytokines, chemokines, and growth factors secreted by these two dNK cell populations was generally similar, but distinct from that of peripheral blood NK cells. Finally, analysis of multiple pregnancies from eight dams revealed that the decidual immune cell profile is characteristic of an individual animal and is consistently maintained across successive pregnancies, suggesting that the uterine immune environment in pregnancy is carefully regulated in the rhesus monkey decidua.     

Characterization of murine decidual natural killer (NK) cells and their relevance to the success of pregnancy

The identification of lytic cells in 6.5-day to 9.5-day murine decidua as NK cells has been extended. The cells with natural killer (NK) activity in early decidua were nonphagocytic and heterogeneous in size as assessed by velocity sedimentation at unit gravity. The numbers of lytic cells were reduced by treatment with anti-asialo GM1 in vivo and they were absent from the decidua of bg/bg mice. Thus, decidual NK cells were not distinct from NK cells in other tissues. The decline in the levels of decidual NK activity as pregnancy progressed was attributed to their regulation by other cells present in decidua by midgestation. The development of NK activity in decidua was dependent upon the presence of an embryo, however, decidual NK cells were not essential for successful pregnancy because viable offspring were obtained from mice lacking decidual NK activity. It was shown that NK cells from either spleen or decidua were unlikely to cause damage to embryos during the first half of pregnancy as freshly dissociated 9.5- and 11.5-day embryonic cells resisted NK lysis. Furthermore, blastocysts were not damaged by coincubation with splenic or decidual NK cells and were viable upon subsequent embryo transfer. These studies indicate that decidual NK cells are not essential for successful pregnancy and are not necessarily detrimental to early embryos. It is suggested that decidual NK cells may play other nonimmunological roles during embryonic development.     

Macrophages infiltrate the human and rat decidua during term and preterm labor: evidence that decidual inflammation precedes labor

Preterm delivery is the leading cause of perinatal mortality and morbidity. Current tocolytics target myometrial contractions, a late step in the labor cascade. Identifying earlier events in parturition may lead to more effective therapeutic strategies. We hypothesized that inflammatory events in decidua (the maternal-fetal interface), characterized by leucocyte infiltration, are an early event during term and preterm labor (PTL). Leucocyte abundance in decidua of human pregnancies was quantified following term labor and PTL (idiopathic and infection associated), in conjunction with investigation of temporal inflammatory events in rat uterus during the perilabor period and in PTL induced by mifepristone. In human decidua, macrophage numbers were 4-fold higher in term labor (P < 0.01) and 2.5-fold higher in non-infection-associated PTL (P < 0.05) than in term nonlaboring samples. Neutrophil abundance was unchanged with labor but elevated in PTL with infection (5- to 53-fold increase; P < 0.01). T and NK cells were more abundant in idiopathic PTL than TL (P < 0.05). In rat, decidual macrophage infiltration increased 4.5-fold 12 h prior to labor and remained elevated during labor and early postpartum (P < 0.01). Decidual infiltration preceded that of the myometrium and was 4-fold higher (P < 0.01). In rat PTL, decidual macrophage numbers were also elevated (P < 0.01) and exceeded those of the myometrium (P < 0.05). These studies show for the first time that leucocytes infiltrate decidua during labor at term and preterm, supporting a role for leucocyte-derived inflammatory mediators in decidual activation. In the rat, this occurred prior to labor, suggesting it is an early event during parturition and thus a potential target for intervention.     

Immunohistochemical localization of prostaglandin endoperoxide synthase in human fetal membranes and decidua

Prostaglandins play an important role during the maintenance of pregnancy and the initiation of parturition. Prostaglandin endoperoxide synthase activity has been demonstrated in human fetal membranes and decidua. Using immunohistochemical techniques, we identified in these tissues the cell types that contain prostaglandin endoperoxide synthase. A total of 33 specimens, ranging from 8 wk to 42 wk gestation, were studied. Decidualized stromal cells stained the most intensely and consistently of all cell types. Cytotrophoblast of the chorion and early placental villi and syncytotrophoblast of all gestational ages demonstrated a lighter, more variable staining pattern. Regardless of gestational age, amnion stained in a heterogeneous fashion, with some cells demonstrating an intense staining and other cells having no staining. There were no observable differences in laboring compared to nonlaboring term specimens. In summary, the specific cell types that contain immunoreactive prostaglandin endoperoxide synthase have been identified in fetal membranes and decidua.     

Activation of maternal killer cells in the pregnant uterus with chronic indomethacin therapy, IL-2 therapy, or a combination therapy is associated with embryonic demise

We have previously shown that NK lineage cells migrate to the murine decidua of pregnancy; but with advancing gestation, they are progressively inactivated in situ by prostaglandins of the E series (PGE2) secreted by decidual cells and decidual macrophages. We have also shown that the same mechanism inactivates all killer lineage cells in the human decidua, and that this inactivation is at least in part due to a down-regulation of IL-2 receptors and an inhibition of IL-2 production in situ. We examined whether chronic indomethacin therapy (to block prostaglandin synthesis), or a systemic administration of a high dose of IL-2, or a combination of both agents administered to pregnant mice could activate killer cells in situ and interfere with the progress of pregnancy; and if so, whether there was a causal relationship between the two events. Pregnant CD1 mice (Day 5 of gestation) were subjected to chronic indomethacin therapy (14 micrograms/ml in drinking water up to Day 15, or 50 micrograms twice daily sc or ip up to Day 10), high dose IL-2 therapy (25,000 Cetus U of human recombinant IL-2, ip every 8 or 12 hr for 3-5 days), or a combination of the two. These treatments led to pregnancy loss in 89-100% of mice, in contrast to 1% loss in control, vehicle-treated mice. Uterine mononuclear cells isolated from the embryo resorption sites exhibited high killer activity against YAC-1 lymphoma as well as murine trophoblast targets, with NK-like phenotype (Asialo GM-1+, Thy-1-) after indomethacin therapy and LAK-like phenotype (AGM-1+, Thy-1+) after IL-2 or indomethacin + IL-2 therapy. That AGM-1+ killer cells resulted in the pregnancy loss was suggested by the findings that in two of three separate experiments, iv injections of AGM-1 ab into pregnant indomethacin + IL-2-treated mice nearly completely prevented the fetoplacental demise (reducing it to 7.7% from 100%). These results reveal that PGE2-mediated inactivation of killer lineage cells in the decidua in situ is conducive to the survival of the conceptus.