Characterization of the tumour necrosis factor receptor in human placenta

A specific receptor for tumour necrosis factor present in a purified plasma membrane preparation from the villous tissue of human placenta has been characterized. The data fit a one-class-of-sites model exhibiting a KD = 28.4 (+/- 0.002) nM, with 8.1 (+/- 0.05) X 10(11) receptors mg-1 membrane protein. These data provide evidence for the existence of a specific receptor for a major endotoxin-induced cytokine in the placenta.     

The regulation of interleukin-6 secretion by prostanoids and members of the tumor necrosis factor superfamily in fresh villous fragments of term human placenta

OBJECTIVE: To determine whether prostanoids, nonsteroidal anti-inflammatory drugs (NSAIDs), and members of the tumor necrosis factor superfamily can regulate placental secretion of interleukin-6 (IL-6) and whether labor influences any such effects. METHODS: Villous fragments of term, human placenta were kept in culture for up to 4 hours, and IL-6 concentrations were measured in the supernatant. We assessed the effects of the following prostanoids: PGE(2), PGF(2alpha), thromboxane A(2) mimetic (U-46619), and carbacyclin, a stable prostacyclin analogue (all at 1 microM); NSAIDs: indomethacin (150 microM) or nimesulide (100 microM); and Fas ligand (5 ng/mL). RESULTS: Secretion (mean +/- standard error) of IL-6 was, for control conditions, 1.92 +/- 0.28 fmol/mg wet weight per 3 hours; for PGE(2), 3.57 +/- 0.29 fmol/mg wet weight per 3 hours, P <.01; and for carbacyclin, 3.11 +/- 0.44 fmol/mg wet weight per 3 hours, P <.01. Incubation with PGF(2alpha) or the thromboxane A(2) analogue, U46619, had no effect on IL-6 secretion under these conditions. Fas ligand stimulated IL-6 secretion (3.06 +/- 0.38 fmol/mg wet weight per 3 hours, P <.05). Labor did not alter the effects of prostanoids or FasL. The effects of NSAIDs were assessed over 4 hours. Secretion (median, interquartile range) was, under control conditions 3.26, 2.83-6.23 fmol/mg wet weight per 4 hours, with indomethacin 1.4, 1.28-3.21 (P <.05), and with nimesulide 0.75, 0.50-1.56 fmol/mg wet weight per 4 hours. The magnitude of the effect of Fas ligand in the presence of NSAIDs depended on whether the placentas were delivered before or after labor. CONCLUSION: Prostanoids, NSAIDs, and the Fas ligand regulate placental IL-6 secretion. Although the effects of individual agents did not vary with the presence or absence of labor, modulation of IL-6 secretion by labor became apparent when agents were combined.     

瘦素与肿瘤坏死因子α在妊娠高血压综合征患者胎盘组织中的表达

妊娠高血压综合征(PIH)是常见的严重危害母婴健康的妊娠并发症,目前其发病机理尚不明确。有文献报道,胎盘组织可合成瘦素;肿瘤坏死因子α(TNF-α）表达异常时,将诱发一系列妊娠相关疾病。因而推测,妊娠期孕妇血清瘦素水平可能受TNF-α的调节。本研究采用免疫组化方法,检测PIH及正常妊娠妇女胎盘瘦素水平及TNF-α的表达情况,并分析两者的相关性,旨在进一步探讨PIH的发病原因。     

Epidermal growth factor binding and receptor distribution in term human placenta

Human placenta has a large number of epidermal growth factor (EGF) receptors when measured either by [125I]iodoEGF binding or by protein yield after purification. To localize EGF receptors in situ in normal human term placenta, two different light microscopic methods were used. To detect unoccupied, accessible EGF binding sites on the extracellular surface of placental cells in intact blocks of tissue, samples were incubated with [125I]iodoEGF, sectioned and autoradiography performed. To detect the total pool of intracellular and extracellular EGF receptors, placental tissue was sectioned, treated with detergent, and then anti-EGF receptor antibody was localized by immunohistoperoxidase techniques. Both [125I]iodoEGF and anti-EGF receptor antibody methods showed that EGF receptors were primarily present on syncytiotrophoblast cells of placental villi. Smooth muscle cells of placental blood vessels also contained EGF receptors. Neither connective tissue cells within the core of terminal chorionic villi nor endothelium of fetal blood vessels had detectable [125I]iodoEGF binding or immunoreactive EGF receptors. Since the quantity of placental smooth muscle cells is only a small fraction compared to trophoblast cells, we conclude that syncytiotrophoblast cells are primarily responsible for the high levels of EGF receptors found in extracts prepared from human term placenta.     

Cellular localization of metallothionein in human term placenta.

Cellular localization of metallothionein (MT) in placenta may provide information on its function as a metal binding protein. Rabbit antibodies to rat liver MT cross-reacted with human MT and were used to localize MT in human term placenta by avidin-biotin peroxidase technique. Serial sections (5 microns) were cut from paraffin-embedded placentae obtained at term from five normal women and incubated with rabbit antibodies to MT. Normal rabbit serum was used as a negative control. The slides were incubated with biotinylated swine anti-rabbit IgG (linking antibody) then with avidin-biotin horseradish peroxidase complex and developed with diaminobenzidine in hydrogen peroxide (0.03 per cent) substrate. The optimum staining of MT was obtained at a 1:800 antibody dilution. MT was identified in fetal amniotic cells, syncytial trophoblasts and villous interstitial cells, and in maternal decidual cells. The presence of MT at specific cellular sites suggests that it may regulate the transplacental transport of metals such as zinc, copper and cadmium. Since the level of cadmium is lower and that of zinc and copper higher in fetal than in maternal blood, this may suggest that placental MT may restrict cadmium while enhancing zinc and copper transport.     

Suppression of human chorionic gonadotropin in the human placenta at term by human thyroid-stimulating hormone in vitro.

Human chorionic gonadotropin (hCG) exerts a clinically apparent negative feedback on the secretion of human thyroid-stimulating hormone (hTSH) in pregnancy, and the two have cross-reactivity for the TSH receptor in membrane preparations of the thyroid. We examined whether hTSH, in turn, has an influence on the secretion and synthesis of hCG in short-term cultures of human placenta at term. A dose- and time-dependent decrease in the extracellular hCG concentration caused by hTSH was demonstrated. To examine whether hTSH inhibits de novo synthesis of hCG or decreases hCG depletion, we determined the amount of hCG secreted and the size of the intracellular pool by using an enzyme immunoassay. By incorporating a radiolabeled amino acid in the hCG molecule, we measured the amount of hCG synthesized de novo. We concluded that hTSH acts by decreasing the rate of de novo synthesis of placental hCG.     

Oestrogen and progesterone receptors in human term placenta. Measurement by binding assays and immunological methods

Oestrogen and progesterone receptors have been implicated in human placental steroidogenesis, but studies developed to measure their concentration in this tissue at term yielded discrepant results. Potential inaccuracy of the binding assays, the technique used in previous studies, might influence the discrepancy. In the present study, oestrogen and progesterone receptors were investigated by two different approaches: binding assays with radiolabelled ligand and immunoassays with monoclonal antibodies against receptors. Binding assays included techniques to quantify free and occupied sites in both the cytosolic and nuclear fraction. Conditions intended to block or decrease potential causes of inaccuracy, such as proteolysis or thermal denaturation of the sites, were included in the assays. Cytosolic specific binding was measured by two different techniques, dextran-coated charcoal and hydroxylapatite, whereas nuclear sites were studied on salt-extracts and by exchange. Negative or moderately positive results were obtained for either oestrogen or progesterone receptors, but high levels of non-specific binding made the technique unreliable. Immunoassays included the use of two techniques, histochemistry and enzyme-linked-immunosorbent-assay (ELISA). Both frozen and paraffin (Bouin solution or buffered formalin as fixatives) sections were stained with monoclonal antibodies against the oestrogen receptor. Results were always negative in all cases. Levels of oestrogen and progesterone receptors were measured in either cytosol or nuclear extracts by ELISA. This technique was highly sensitive and reproducible. Results were negative for both the cytosolic and nuclear fractions of oestrogen receptors. For progesterone receptors, low positivity was obtained in either crude (3.2 +/- 0.4 fmol/mg protein, mean +/- s.e.) or ammonium sulphate precipitated cytosols (12.6 +/- 3.5 fmol/mg protein, mean +/- s.e.), whereas the values for nuclear extracts were 62.0 +/- 9.0 fmol/mg DNA (mean +/- s.e.). We conclude that: (1) oestrogen receptors could not be detected in human term placenta by the methods used in this study, and (2) low levels of progesterone receptors were detected in both cytosolic and nuclear extracts.     

Perfusion with lipopolysaccharide differently affects the secretion of tumor necrosis factor-alpha and interleukin-6 by term and preterm human placenta

This study has compared the functional capacity of term and preterm placentas in terms of production of pro-inflammatory cytokines in a perfusion system reflecting their ability to react to inflammatory agents, such as lipopolysaccharide (LPS), mimicking the situation of chorioamnionitis. We have demonstrated that term placentas secrete higher levels of tumor necrosis factor (TNF)-alpha compared with preterm placentas. Moreover, TNF-alpha secretion was significantly higher after exposure to LPS in the maternal and fetal sides of term placentas. In contrast, in preterm placentas, only the fetal side responded with a significant increase in secretion of TNF-alpha after exposure to LPS. The maternal side of term placentas secreted significantly higher amounts of interleukin (IL)-6 compared with preterm placentas. Exposure to LPS significantly decreased IL-6 secretion from the maternal side in both term and preterm placentas. Moreover, the fetal side of term placentas secreted significantly lower amounts of IL-6 compared with preterm placentas. In summary, this study has indicated that term and preterm placental tissues have a differing responsiveness to LPS stimulation, with term placentas disposed to a higher TNF-alpha:IL-6 ratio. Release of cytokines into fetal circulation is less than into the maternal side. However, TNF-alpha is released into fetal circulation after LPS stimulation and this may be relevant to the etiology of chorioamnionitis.     

Effect of proline rich polypeptide from ovine colostrum on virus replication in human placenta and amniotic membrane at term; possible role of endogenous tumour necrosis factor alpha.

Freshly prepared organ cultures of human placentae and amniotic membranes at term show different sensitivity to vesicular stomatitis virus (VSV) infection. In six of 16 amniotic membranes and seven of 17 placentae VSV replicated to relatively high titres (10(3)-10(6)TCID(50)/ml). The others were partially or completely resistant to virus infection (<10(1)-10(2)TCID(50)/ml). Addition of the immunomodulating agent, proline-rich-polypeptide (PRP) from ovine colostrum to explants freshly obtained from the organs, influenced VSV replication in a manner dependent on the innate immune state of the organ culture. In cultures resistant to the virus, PRP at a concentration of 10 microg/ml increased 10-10 000 times the VSV titre. In contrast, treatment of highly sensitive cultures by PRP hardly influenced viral replication at all. The effect of virus stimulation by PRP was abolished by specific anti-TNF antibodies. The results indicate that endogenous TNF may be one of the mediators of virus stimulation by PRP. Antibodies against TNFalpha, added to VSV infected organ cultures sensitive to the virus reduced viral replication. The antibodies caused stimulation of virus replication in VSV-infected resistant organ cultures. The results indicate the double role of endogenous TNF in viral replication in placenta and the amniotic membrane.     

Tumor necrosis factor in preterm and term labor.

OBJECTIVE: Our objective was to determine if labor (term and preterm) and microbial invasion of the amniotic cavity were associated with changes in amniotic fluid concentrations of tumor necrosis factor. STUDY DESIGN: Amniotic fluid was retrieved by transabdominal amniocentesis from 269 women in the following groups: midtrimester (n = 38), preterm labor with intact membranes (n = 52), preterm premature rupture of membranes (n = 74), term in active labor (n = 84), and term not in labor (n = 21). Fluid was cultured for aerobic and anaerobic bacteria and for Mycoplasma species. Tumor necrosis factor was measured with a commercially available enzyme-linked immunosorbent assay validated for amniotic fluid (sensitivity 60 pg/ml). RESULTS: Amniotic fluid from pregnant women in the second and third trimesters who were not in labor did not contain tumor necrosis factor. Among women in preterm labor, 92.3% (12/13) of patients with a positive amniotic fluid culture had detectable tumor necrosis factor in the amniotic fluid (median 820 pg/ml, range less than 60 to 2340 pg/ml). In contrast, only 10.2% (4/39) of women with a negative amniotic fluid culture had detectable tumor necrosis factor. Histopathologic chorioamnionitis was found in all patients who had a positive amniotic fluid culture, and tumor necrosis factor was detectable in the amniotic fluid of all but one of these patients. Among women in active labor at term, 25% (21/84) had detectable tumor necrosis factor in the amniotic fluid. Tumor necrosis factor was detected more frequently in the amniotic fluid of patients with a positive amniotic fluid culture than in patients with a negative culture (46.6% [7/15] vs 20.2% [14/69], p = 0.047). Amniotic fluid concentrations of tumor necrosis factor were significantly higher in patients with preterm premature rupture of membranes, labor, and a positive amniotic fluid culture than in the other subgroups of patients with preterm premature rupture of membranes. CONCLUSION: Parturition in the setting of microbial invasion of the amniotic cavity is associated with activation of the cytokine network as demonstrated by the detection of tumor necrosis factor in human amniotic fluid.     

In vitro perfusion study of the human placenta at term: preliminary results.

Dual perfusion of the human placental lobule in vitro is a useful method for studying the transfer of molecules across the placenta, including the transfer of endogenous substances and xenobiotics. To establish the first model of in vitro placental dynamic study in our country, we used a dual recirculating perfusion system modified from that described by Miller et al. A placental lobule without tears or gross lesions was chosen. Both the fetal and maternal sides of the placenta were perfused with Medium 199 in addition to heparin, glucose, dextran and antibiotics. Perfusate samples were obtained periodically and analyzed for blood gas, glucose, lactate, human placental lactogen (hPL) and human chorionic gonadotropin (hCG). The stability of this human placental lobule preparation during 10 hours of perfusion was reflected by the stability of the arterial pressure and by the constant volume in the fetal compartment. A constant rate of oxygen was delivered by the maternal circulation system, and a steady rate of oxygen was gained by the fetal circulation system. Neither oxygen nor glucose consumption by the tissue was significantly reduced during the period of perfusion. The releasing rates of hCG and hPL did not change significantly during perfusion. The development of this dual perfusion system for the human placenta can provide for the study of placental hemodynamics and transplacental transport in perinatal medicine in our country.     

Heme oxygenase activity in term human placenta

Carbon monoxide (CO) is a novel gaseous chemical messenger, formed during heme oxygenase (HO)-catalysed oxidation of heme. CO is proposed to play a key role(s) in cell function in many organ systems, including vasodilator action in the cardiovascular system. Recently, it has been demonstrated that there is expression of HO protein in the human placenta and this appears to have a regulatory role in placental perfusion. The objective of the present study was to determine HO enzymatic activity in vitro in five different regions of term human placenta. HO activity was determined in the microsomal fraction of tissue homogenate by measuring the rate of formation of CO from heme, using a gas-chromatographic method. HO activity, expressed as nmol CO formed/g tissue wet weight/h, was higher (P< 0.05) in the chorionic plate, chorionic villi, basal plate and chorio-decidua compared with the amnion. The finding that HO enzymatic activity is present in different regions of term human placenta supports the concept that the heme-CO (HO) pathway plays a complementary role with the L -arginine-nitric oxide (nitric oxide synthase) pathway in the regulation of placental haemodynamics.     

Expression and subcellular localization of TLR-4 in term and first trimester human placenta

Toll-like receptor 4 (TLR-4) mediates Gram-negative bacterial-induced inflammatory responses, including production of pro-inflammatory cytokines. Maternal infection and inflammation play an important role in preterm birth and neonatal brain damage. The localization of placental TLR-4 as well as changes during normal gestation are critical issues in understanding the role of toll-like receptors in defending the placento-fetal unit from maternal infection. We therefore investigated, by immunohistochemistry (IHC) and Western blot, the subcellular localization of TLR-4 in first trimester and term human placenta. In both term placenta (n=4) and first trimester placenta villous samples (n=5), immunoreactivity for TLR-4 was found in the cytoplasm of the syncytiotrophoblast, with darker staining in some areas of the maternal facing plasma membrane (MVM). In addition, TLR-4 was found to be expressed in the first trimester cytotrophoblast cells. Using Western blot analysis, TLR-4 was identified in both placental homogenates and isolated MVM and the fetal facing basal membrane (BM). TLR-4 expression in MVM was significantly higher in term (n=9) as compared to first trimester (n=2) samples. We have shown for the first time that the subcellular localization of TLR-4 in term placenta is preferentially in the MVM compared to BM. The MVM is continuously bathed in maternal blood, suggesting that from this vantage point TLR-4 can initiate a rapid response to maternal bacterial infection.     

Vascular endothelial growth factor, placenta growth factor and their receptors in isolated human trophoblast

The expression of the angiogenic growth factors, vascular endothelial cell growth factor (VEGF) and placenta growth factor (PIGF) was demonstrated in isolated human term cytotrophoblast and in vitro differentiated syncytiotrophoblast. RNase protection assays demonstrated VEGF expression in both cytotrophoblast and syncytiotrophoblast while prominent PIGF expression was detected in both types of trophoblast by Northern blot analyses. VEGF expression increased approximately eightfold in trophoblast cultured under hypoxic conditions (1 per cent O₂) yet PIGF expression decreased 73 ± 5.5 per cent in the same trophoblast. These results suggest distinct regulatory mechanisms govern expression of VEGF and PIGF in trophoblast. Characterization of the VEGF/PIGF receptors, KDR and flt-1, revealed the presence of flt-1 mRNA in isolated cytotrophoblast and in vitro differentiated syncytiotrophoblast. KDR was not detected in the isolated trophoblast. Exogenous rhVEGF induced c-Jun N-terminal kinase (JNK) activity in the normal trophoblast indicating that the flt-1 receptors on trophoblast are functional. Trophoblast-derived VEGF/PIGF could act in a paracrine fashion to promote uterine angiogenesis and vascular permeability within the placental bed. In addition, presence of functional flt-1 on normal trophoblast suggests that VEGF/P1GF functions in an autocrine manner to perform an as yet undefined role in trophoblast invasion, differentiation, and/or metabolic activity during placentation.