Estrogen-dependent regulation of human uterine natural killer cells promotes vascular remodelling via secretion of CCL2.

STUDY QUESTION: Does intrauterine biosynthesis of estrogen play an important role in early pregnancy by altering the function of uterine natural killer (uNK) cells? SUMMARY ANSWER: Estrogens directly regulate the function of human uNK cells by increasing uNK cell migration and secretion of uNK cell-derived chemokine (C-C motif) ligand 2 (CCL2) that critically facilitates uNK-mediated angiogenesis. WHAT IS KNOWN ALREADY: uNK cells are a phenotypically distinct population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. Recently we discovered that decidualisation of human endometrial stromal cells results in the generation of an estrogen-rich microenvironment in areas of decidualised endometrium. We hypothesize that intrauterine biosynthesis of estrogens plays an important role in early pregnancy by altering the function of uNK cells. STUDY DESIGN, SIZE, DURATION: This laboratory-based study used primary human uNK cells which were isolated from first trimester human decidua (n = 32). PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary uNK cells were isolated from first trimester human decidua using magnetic cell sorting. The impact of estrogens on uNK cell function was assessed. Isolated uNK cells were treated with estrone (E1, 10(-8) M) or estradiol (E2, 10(-8) M) alone or in combination with the anti-estrogen ICI 182 780 (ICI, 10(-6) M). uNK cell motility was assessed by transwell migration assay and time-lapse microscopy. Expression of chemokine receptors was assessed by quantitative PCR (qPCR) and immunohistochemistry, and angiogenic factors were assessed by qPCR and cytokine array. Concentrations of CCL2 in supernatants were measured by enzyme-linked immunosorbent assay. Angiogenesis was assessed in a human endometrial endothelial cell network formation assay. MAIN RESULTS AND THE ROLE OF CHANCE: Treatment with either E1 or E2 increased uNK cell migration (P = 0.0092 and P = 0.0063, respectively) compared with control. Co-administration of the anti-estrogen ICI blocked the effects of E1 and E2 on cell migration. Concentrations of C-X-C chemokine receptor type 4 (CXCR4) mRNA in uNK cells were increased by E2 treatment. The network formation assay revealed that conditioned media from uNK cells treated with E2 significantly increased human endometrial endothelial cell (HEEC) angiogenesis (P = 0.0029 versus control). Analysis of media from uNK cells treated with E2 using an antibody array identified CCL2 as the most abundant cytokine. Validation assays confirmed concentrations of CCL2 mRNA and protein were increased by E2 in uNK cells (P < 0.05 versus controls). Compared with the control, recombinant human CCL2 was found to increase HEEC network formation (P < 0.05) and neutralization of CCL2 in uNK conditioned media significantly decreased E2-dependent uNK-mediated network formation (P = 0.0006). LIMITATIONS, REASONS FOR CAUTION: Our results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in vivo in humans. Primary human uNK cells were isolated from first trimester decidua at a range of gestations (8-12 weeks), which may be a source of variation. Primary human uNK cells from non-pregnant endometrium were not assessed and therefore the responses of uNK cells to E2 treatment described in this study may be distinct to uNK cells from first trimester decidua. WIDER IMPLICATIONS OF THE FINDINGS: E2 is an essential regulator of reproductive competence. This study demonstrates a critical role for E2 in regulating cellular cross-talk within the endometrium during early pregnancy. We provide the first evidence that E2 directly regulates the function of human uNK cells by altering uNK cell migration and the secretion of uNK-derived angiogenic factors. We describe a novel mechanism of estrogen-dependent secretion of CCL2 which critically mediates uNK-dependent endometrial angiogenesis. Dysregulation of uNK cell function has been implicated in the aetiology of early implantation disorders and disorders of pregnancy. These novel findings provide unique insight into the regulation of uNK cell activity during the establishment of pregnancy in women and highlight key processes which may be targeted in future therapeutic strategies. STUDY FUNDING/COMPETING INTERESTS: Studies undertaken in the authors' laboratory were supported by MRC Programme Grant G1100356/1 to P.T.K.S. The authors have no conflicts of interest to disclose.

Rate of replenishment and microenvironment contribute to the sexually dimorphic phenotype and function of peritoneal macrophages.

Macrophages reside in the body cavities where they maintain serosal homeostasis and provide immune surveillance. Peritoneal macrophages are implicated in the etiology of pathologies including peritonitis, endometriosis, and metastatic cancer; thus, understanding the factors that govern their behavior is vital. Using a combination of fate mapping techniques, we have investigated the impact of sex and age on murine peritoneal macrophage differentiation, turnover, and function. We demonstrate that the sexually dimorphic replenishment of peritoneal macrophages from the bone marrow, which is high in males and very low in females, is driven by changes in the local microenvironment that arise upon sexual maturation. Population and single-cell RNA sequencing revealed marked dimorphisms in gene expression between male and female peritoneal macrophages that was, in part, explained by differences in composition of these populations. By estimating the time of residency of different subsets within the cavity and assessing development of dimorphisms with age and in monocytopenic Ccr2 -/- mice, we demonstrate that key sex-dependent features of peritoneal macrophages are a function of the differential rate of replenishment from the bone marrow, whereas others are reliant on local microenvironment signals. We demonstrate that the dimorphic turnover of peritoneal macrophages contributes to differences in the ability to protect against pneumococcal peritonitis between the sexes. These data highlight the importance of considering both sex and age in susceptibility to inflammatory and infectious diseases.

Cell rigidity: Effect on concanavalin A-mediated agglutinability of fibroblasts after fixation.

A quantitative hemadsorption assay distinguishes the effects of membrane fixation on concanavalin A-mediated agglutinability of fixed cells with unfixed cells. We observed undiminished adherence of unfixed erythrocytes to glutaraldehyde-fixed normal and virus-transformed hamster fibroblasts coated with concanavalin A. Fixation of the erythrocytes abolished agglutination with fixed fibroblasts. The agglutinability of fixed cells is more likely related to increased cell rigidity than to decreased membrane fluidity.

Activities of types A and B MAO and catechol-o-methyltransferase in blood cells and skin fibroblasts of normal and chronic schizophrenic subjects.

We assayed activities of monoamine oxidase (MAO) type B in blood platelets and type A (and B) in fibroblasts cultured from punch biopsy specimens of skin, as well as of catechol-O-methyltransferase (COMT) in erythrocytes and fibroblasts. Fibroblasts contained moderate amounts of both forms of MAO (types A and B) found in human brain and large amounts of COMT activity. Activities of both enzymes correlated poorly between fibroblasts and blood cells. Comparing carefully diagnosed chronic schizophrenics with age-matched normal young men, we found no difference in these biochemical variables, nor could we distinguish patients with paranoid symptoms. In contrast, we confirmed markedly lower MAO activities in platelet samples from chronic patients provided by colleagues at the National Institute of Mental Health. Results concerning MAO and COMT activities are now sufficiently inconsistently characteristic of schizophrenics as to question their clinical applicability and to indicate a need for further critical evaluation, with special attention to diagnosis, matching of subjects, and effects of possible spurious environmental variables.

Hippocampal CA1 region neurodegeneration produced by ethanol withdrawal requires activation of intrinsic polysynaptic hippocampal pathways and function of N-methyl-D-aspartate receptors.

Long-term intake of ethanol produces adaptive alterations in multiple transmitter systems in the hippocampal formation that likely contribute to ethanol withdrawal-induced seizure and excitotoxicity. The present studies were designed to examine the role of N-methyl-d-aspartate receptor activation and cytosolic Ca(2+) accumulation in the neurotoxic effects of ethanol withdrawal. Further, these studies investigated the role of hippocampal network excitation in promoting both Ca(2+) accumulation and neurotoxicity during ethanol withdrawal. Chronic, continuous (11 day) exposure to ethanol (91 mM starting concentration) did not produce neurotoxicity in any region of organotypic hippocampal explants, as measured by uptake of the non-vital fluorescent marker propidium iodide. Withdrawal from chronic (10 day) ethanol exposure was associated with rapid (30 min) and significant increases in intracellular Ca(2+), assessed by visualization of Calcium-Orange fluorescence, in each region of hippocampal explants. However, neurotoxicity was observed 24 h after initiation of withdrawal and was only seen in the cornu ammonis 1 (CA1) region. Exposure to MK-801 (20 microM) at the start of ethanol withdrawal markedly attenuated Ca(2+) entry in all regions, as well as, CA1 region neurodegeneration. Further, treatment of explants with tetrodotoxin (500 nM) as well as surgical transection of mossy fiber or Schaffer collateral projections immediately prior to ethanol withdrawal blocked both regional increases in Ca(2+) accumulation and CA1 neurotoxicity. These data suggest that neurodegeneration observed during ethanol withdrawal is dependent upon polysynaptic propagation of action potentials ("network excitation") and whole-hippocampal excitation of glutamatergic systems.

Effect of morphine on breathing and behavior in fetal sheep.

To define the dose response of apnea and breathing to morphine we studied 12 fetuses at 116-141 days of gestation using our window technique. We instrumented the fetus to record electrocortical activity (ECoG), eye movements (EOG), diaphragmatic activity (integral of EMGdi), heart rate, carotid blood pressure, and amniotic pressure. Saline and morphine in doses of 0.03, 0.1, 0.5, 1, and 3 mg/kg were injected in random order in the jugular vein of the fetus during low-voltage ECoG. Fetuses were videotaped for evaluation of fetal behavior. We found 1) that saline did not elicit a response; 2) apnea, associated with a change from low- to high-voltage ECoG, increased from 2.2 +/- 1.5 (SE) min in two fetuses at a dose of 0.03 mg to 20 +/- 6.3 min in seven fetuses at 3 mg/kg (P less than 0.005); 3) the length of the breathing responses, associated with a change from high- to low-voltage ECoG, were 15 +/- 1.8 and 135.9 +/- 18.1 min (P less than 0.0005); 4) integral of EMGdi X frequency, an index equivalent to minute ventilation, increased from 1,763 +/- 317 arbitrary units to 10,658 +/- 1,843 at 1.0 mg/kg and then decreased to 7,997 +/- 1,335 at 3.0 mg/kg. These changes were related to a steady increase in integral of EMGdi, whereas frequency decreased at 3 mg/kg. There was an increase in breathing response to morphine plasma concentrations or morphine doses.(ABSTRACT TRUNCATED AT 250 WORDS)

[3H]Etorphine binding activity in early chick embryos: brain and body tissue.

Stereospecific [3H]etorphine binding has been detected in chick embryos as early as day 4 of incubation in both brain and body tissue. By day 10 of incubation [3H]etorphine stereospecific binding activity is not detectable in nonneuronal tissue. The ubiquitous opiate binding sites early in embryogenesis are high affinity and respond to ion and GTP regulation in a manner similar to adult brain tissue. We interpret our observations to indicate all embryonic cells prior to cell differentiation contain opiate receptors. Therefore, we propose that opiate receptors play a dual role; one function early in embryogenesis not associated with neurotransmitter regulation, and another function later in embryonic development and in the adult: the classical neurotransmitter regulatory function.

Endogenous indoles as novel polyamine site ligands at the N-methyl-D-aspartate receptor complex.

High-throughput ligand displacement screens of a series of endogenous indoles revealed that tryptamine, serotonin and 5-methoxytryptamine readily displace [3H]spermidine and [3H]MK-801 from their respective binding sites in rat brain homogenate. These data, coupled with their potent inhibition of spermidine-potentiated [3H]MK-801 binding, suggest that certain endogenous indoles may act as ligands to one or more polyamine binding sites in the brain, including those on the N-methyl-D-aspartate receptor complex.

Monoamine oxidase activity in cultured human skin fibroblasts.

Skin fibroblast cultures were prepared from 21 men, and found to contain types A and B activity of monoamine oxidase, with a possible slight predominance of type A, as evaluated by substrate preferences and differential inhibition by clorgyline and deprenyl. Three women had similar activities. There was a close correlation of activities with different substrates, but there was no quantitative correlation between fibroblast and blood platelet enzyme (type B) activities. The fibroblasts also contained catechol-O-methyltransferase activity exceeding, but poorly correlated with, that in erythrocytes. Fibroblasts may be advantageous in studies of monoamine oxidase in man by providing both types of enzyme as found, for example, in the central nervous system, and by providing a means of removing many in vivo chemical influences from the cells in culture. Nevertheless, great caution must be exercised in generalizing results of this "model" to other tissues, since activities of both enzymes correlated poorly with those in blood cells of the same individuals.

Geographic variations in Medicare utilization of short-stay hospital services, 1981-88.

The change in Federal fiscal year 1984 from cost-based reimbursement to prospective payment at a fixed price for a known and defined product--the hospital stay--represents a fundamental change in the role of the Medicare program within the health care delivery system. In this article, national and selected geographic trends in Medicare short-stay hospital inpatient discharges since 1981 are presented, and they show the impact of the implementation of the prospective payment system.

Medicare discharges by facility status under the prospective payment system, 1984-86.

When the Health Care Financing Administration implemented the Medicare prospective payment system (PPS), several types of hospitals and hospital units were excluded from the new reimbursement system, and they remained under the reasonable cost reimbursement system, subject to the target rate of increase limits. The implementation of PPS has been accompanied by several changes in hospital classification and in utilization patterns. This article examines some of these changes based on excluded facility counts and discharges by facility status under the PPS for fiscal years 1984-86.

The patterns of spinal deformity in Duchenne muscular dystrophy.

In a clinical and roentgenographic study of spinal deformities in sixty-two patients in the later stages of Duchenne muscular dystrophy, many patients had marked scoliosis and kyphosis, while others with hyperextended spines had comparatively little scoliosis. Based on an analysis of the data, it is suggested that the development of spinal deformity in patients with Duchenne muscular dystrophy may progress in two ways: one leading to the early establishment of a position of extension and a maximum intrinsic stability with minor deformity, and the other leading to progressive deformity. It was concluded that management for these patients should be designed to guide the early straight spine toward the late extended pattern by attempting to prevent kyphosis and pelvic obliquity.

The orthopaedic aspects of spinal muscular atrophy.

To clarify the role of the orthopaedic surgeon in the management of spinal muscular atrophy, the records of 130 patients were reviewed. Seventy-three had died. Of the remaining fifty-seven, fifty were re-examined. The clinical manifestations of the disease, particularly those giving rise to orthopaedic problems, were studied. At the time of review the average age of the patients was 11.5 years. Thirty-five could not walk and all had some degree of muscle weakness, more marked proximally and in the lower limbs. The most common medical problem was repeated respiratory infection and the major orthopaedic problem was scoliosis, often severe. Nine of the thirty-five patients with scoliosis had had spine fusion. Most of them suffered some functional loss in attaining spinal stability.

Surgical stabilization of the spine in Duchenne muscular dystrophy.

The problem of real distress from the discomfort of collapsing scoliosis is predictable in Duchenne muscular dystrophy (DMD). Once the lumbar curve has exceeded 35 degrees, further progression is inevitable. A vital capacity, then, of 35% or more permits consideration of spinal surgery. Using these indications, 24 patients with DMD had long Harrington instrumentations and spinal fusions from S1 up to the upper thoracic spine (T4, 5, or 6). After two weeks recumbent, they were mobilized wearing a light spinal support in their wheelchairs. The complications encountered are described in detail. One patient died two years after his operation from dystrophic cardiomyopathy. With a follow-up period of four months to 42 months, the rest of these patients are well and sitting with comfort. The authors think that this experience indicates that prophylactic spinal fusion deserves consideration in the care planned for these patients.

Media for the education of health professionals.

The benefits and pitfalls of applying media and communications techniques to the education of health professionals are considered in the context of their use in the classroom, for independent study and for distance education. The difficulties are emphasized for managing learning materials of this kind, and for keeping them up-to-date.

Endometrial Intracrinology--generation of an estrogen-dominated microenvironment in the secretory phase of women.

CONTEXT: The human endometrium is a complex multicellular tissue subject to cyclical fluctuations in ovarian-derived steroid hormones. Fertile cycles are characterized by differentiation (decidualization) of endometrial stromal cells (ESC). OBJECTIVE: To determine the impact of human stromal cell decidualization on biosynthesis and secretion of estrogens. DESIGN: Primary cell culture was used. Cells were decidualized in vitro. Some cultures were treated with an aromatase inhibitor. SETTING: A University Research Institute. PATIENTS: Primary ESC were derived from women with normal menstrual cycles (n = 12). None of the women were receiving hormonal therapy or suffering from endometriosis. MAIN OUTCOME MEASUREMENTS: Expression of mRNA and protein encoded by the aromatase (CYP19A1) and 3-ß-hydroxysteroid dehydrogenase (HSD3B1) genes was assessed. Aromatase activity was measured using a tritiated water assay; steroid metabolism was determined using thin layer chromatography. Estrone (E1) and estradiol (E2) in cell culture media were measured by ELISA. RESULTS: Decidualization induced a two-fold increase in aromatase mRNA. Aromatase protein was only detected in decidualized ESC. 3-ß-Hydroxysteroid dehydrogenase protein was present in ESC both before and after decidualization; concentrations appeared unchanged. The existence of functional aromatase in decidualized ESC was confirmed; E1 and E2 were secreted into culture media in decidualized ESC and concentrations were reduced when cells were incubated with an aromatase inhibitor. Decidualization resulted in reduced metabolism of E2 and an increase in the ratio of E2:E1. CONCLUSIONS: Decidualization is characterized by an increase in aromatase expression/activity favoring the generation of an E2-dominated estrogen microenvironment within the endometrial stroma.