Down modulation of TNF-alpha mRNA placental expression by AZT used for the prevention of HIV-1 mother-to-child transmission

Mechanisms of HIV-1 in utero mother-to-child transmission (MTCT) protection provided by AZT are not completely understood. The placental cytokine network is involved in the control of HIV-1 in utero transmission but the effect of AZT on this network is unknown. To evaluate the effects of AZT on placental cytokine expression, the chorionic villi from HIV-1 uninfected women term placentae were cultured with 0, 100, and 2,000 ng/ml AZT. Tissue fragments were harvested at days 1, 4, and 7 to determine the level of cytokine mRNA by real-time RT-PCR. The viability and morphology of the placental histocultures were monitored by the expression of beta-human chorionic gonadotropin (beta-hCG) gene, lipopolysaccharide (LPS) activation, and microscopic examination. AZT at 2,000 ng/ml significantly down-regulated TNF-alpha mRNA expression at day 1 and day 4, but had no effect on beta-hCG, stromal cell-derived factor 1 (SDF-1), and IL-10 gene expression. AZT did not induce any deleterious impact on placental tissue structure. Furthermore, activation of chorionic villi by LPS for 24 h up-regulated IL-10 and TNF-alpha mRNA expression. Down-regulation of TNF-alpha mRNA could represent a mechanism through which AZT can decrease the risk of HIV-1 MTCT, in addition to its direct effect on HIV-1 replication.     

Expression of pregnancy-associated plasma protein-A (PAPP-A) during human villous trophoblast differentiation in vitro

Pregnancy-associated placental protein-A (PAPP-A), first isolated from maternal serum, has been identified as a metalloprotease cleaving insulin-like growth factor binding protein-4 (IGFBP-4). The source of PAPP-A during pregnancy is unclear. We therefore investigated PAPP-A expression during in vitro human villous cytotrophoblast cell (CT) differentiation into syncytiotrophoblast (ST). CT were isolated from normal first trimester, second trimester and term placentae (n=10) and cultured to form ST. PAPP-A mRNA was quantified by real-time PCR, and PAPP-A protein expression was studied by immunocytochemistry and TRACE technology with specific monoclonal antibodies. PAPP-A mRNA expression in total placental extracts increased during the course of pregnancy. PAPP-A protein was detected in the cytoplasm of both CT and ST. ST formation in vitro was associated with a 19-fold increase in PAPP-A mRNA expression and an 8-fold increase in PAPP-A secretion into the culture medium. No significant difference in PAPP-A production was observed between cultured cells isolated from early and term placentae. In conclusion, PAPP-A production in vitro, is associated to the differentiation of villous cytotrophoblast cells into syncytiotrophoblast, independently of the age of gestation.     

Production of granulocyte colony-stimulating factor by the human placenta at various stages of development

The human placenta is capable of producing a variety of haematopoietic growth factors in vitro. It is not clear, however, whether the placenta produces such factors in vivo and if so, whether placental production of haematopoietic growth factors has a physiological role in fetal haernatopoietic development. As a step toward making this determination, we assessed whether the onset of placental production of granulocyte colony-stimulating factor (G-CSF), in vivo, coincides with the onset of granulocytopoiesis in the developing fetus. To make this assessment, we obtained human placentae between 10 weeks of gestation and term and studied production of G-CSF in several ways. First, we sought to determine whether the onset of production of G-CSF mRNA in the placenta immediately precedes the appearance of neutrophil development in the fetus. Second, we assessed the effect of gestational age on the capacity of the placenta to generate G-CSF in vitro, by incubating cubes of placenta, with or without including interleukin-la (IL-1α) in the culture media, and quantifying G-CSF in the cell culture supernatants 24h later. Third, we assessed the rate of G-CSF production by the placenta, by perfusing two normal, term placentae using a membrane-oxygenator system, and quantifying G-CSF, at intervals, in the perfusates. We found: (1) no evidence that placental production of G-CSF is involved in regulating granulocytopoiesis in the fetus, (2) that the healthy placenta contains little or no G-CSF mRNA in vivo, (3) the placenta at term has a far greater capacity to produce G-CSF, when stimulated, than does the placenta before term, and (4) that although the placenta does not normally produce G-CSF in vivo, it has the capacity of generating very large quantities of G-CSF continuously over at least several days.     

Novel model of placental tissue explants infected by cytomegalovirus reveals different permissiveness in early and term placentae and inhibition of indoleamine 2,3-dioxygenase activity

Human cytomegalovirus (HCMV) is the most common cause of viral intrauterine infection. Placental infection suggests hematogenous spread and permissiveness may vary according to the age of pregnancy. We set up and investigate permissivity of early and term placenta to HCMV with an ex vivo model of placental histocultures and evaluate the activity profile of IDO. Fourteen first trimester placentae were obtained following elective abortion and twelve term placentae after elective caesarean section. Fresh placental chorionic villi were isolated, washed and distributed on collagen sponge gels after overnight incubation with the virus. The culture medium was collected and fresh medium renewed regularly. Histology and immunohistochemistry showed preserved villous integrity in cultured placental histocultures. Infection could be seen in tissue sections of both early and term placentae, although early placentae were more permissive. Indoleamine 2,3-dioxygenase (IDO) is highly expressed in the placenta and is known to prevent maternal immune rejection. Constitutive IDO activity was higher in early, compared to term placentae and HCMV infection inhibited IDO activity in early placentae. IFN-γ-induced IDO activity was suppressed by HCMV in both early and term placentae. Our work shows a novel method of placenta organ culture. Our findings suggest that HCMV infects early placentae more strongly than term placentae. Early placental dysfunction through the inhibition of IDO activity may reveal a possible mechanism for miscarriages.     

Placental imbalance of Th1- and Th2-type cytokines in preeclampsia

OBJECTIVES: To characterize the changes in the level of T helper 1 (Th1)- [interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha] and Th2-type cytokine (IL-10) and the ratios of Th1/Th2 (IL-2/IL-10 and TNF-alpha/IL-10) in placentae from women with preeclampsia and women with gestational hypertension. METHODS: Placental levels of IL-2, TNF-alpha, and IL-10 were determined with radioimmunoassay and Th1/Th2 ratios (IL-2/IL-10 and TNF-alpha/IL-10) calculated in the placentae from 22 women with preeclampsia, 15 women with gestational hypertension, and 32 normal term pregnant women. RESULTS: Although preeclampsia had the trend of the increase in the placental levels of IL-2 and TNF-alpha and the trend of the decrease in placental IL-10, there were not significant difference in placental levels of IL-2, IL-10, and TNF-alpha among preeclampsia, gestational hypertension, and normal pregnancy (P > 0.05 for all). Placental ratios of IL-2/IL-10 and TNF-alpha/IL-10 were significantly higher in preeclampsia than in normal pregnancy (P = 0.035 and P = 0.005, respectively). No differences of Th1/Th2 ratios were found between preeclampsia and gestational hypertension and between gestational hypertension and normal pregnancy (P > 0.05 for all). CONCLUSIONS: Alterations of placental balances of cytokines with Th1 predominance were demonstrated in preeclampsia. These associations may offer insights into the pathogenesis of preeclampsia.     

Prostacyclin production by human placental cells in short-term culture

Human placentae of varying gestational ages have been cultured in vitro with little variation in cell type and pattern of growth found. Cell types found are similar morphologically and histochemically to those previously described. The biological specificity of the cells in culture was also confirmed by human placental lactogen production. Placental cells in culture appear to synthesize and release PGI2 which can be identified by gas chromatography-mass spectrometry. Production of PGI2 was routinely measured by radioimmunoassay (RIA) for 6-oxo-PGF1alpha and 13,14-dihydro-6,15-dioxo-PGF1alpha in culture supernatants. Good agreement was found between RIA and gas chromatography-mass spectrometry measurements. PGI2 production in culture was not affected by mode of delivery, and synthetic capability was found to increase with gestational age. Production of PGI2 by cells from preterm and term placentae was similar but significantly greater than that of first-trimester cells. As the proportion of PGI2 produced in culture supernatants as 13,14-dihydro-6,15-dioxo-PGF1alpha changed with time of incubation, it appears pertinent to measure this metabolite when assessing total PGI2 production. Synthesis of PGI2 wa inhibited by the cyclo-oxygenase inhibitors indomethacin and aspirin and PGI2 synthetase inhibitor 15 hydroperoxyarachidonic acid. However, in the culture system tranylcypromine, a putative specific inhibitor of PGI2 synthetase, produced weak inhibition only before becoming cytotoxic. The cell culture system appears to offer a reliable and reproducible means for measuring placental PGI2 production in vitro and in which to study factors controlling its production and metabolism.     

细胞因子IL-6mRNA与IL-10mRNA在早产合并绒毛膜羊膜炎胎盘组织中的表达水平及意义

目的:研究白细胞介素-6 mRNA(IL-6 mRNA)和白细胞介素-10 mRNA(IL-10 mRNA)在早产合并绒毛膜羊膜炎孕妇胎盘组织中的表达水平,探讨IL-6及IL-10在妊娠中的作用,以及其在早产合并绒毛膜羊膜炎中的变化及意义。方法:取足月分娩及早产孕妇部分胎盘胎膜组织做病理检查,根据病检结果分为3组:早产感染组20例、早产非感染组20例和足月组20例。采用二步法RT-PCR法测定3组胎盘组织中IL-6 mRNA和IL-10 mRNA的表达。结果:早产感染组胎盘组织中IL-6 mRNA表达水平明显高于早产非感染组和足月组,差异有显著性(P<0.05);而IL-10 mRNA表达水平明显低于另外两组,差异有显著性(P<0.05)。结论:感染可引起细胞因子IL-6 mRNA、IL-10 mRNA在胎盘组织中的表达发生改变,推测IL-6可促进分娩发动,使妊娠提前终止,而IL-10在维持正常妊娠中可能起重要作用。     

Effect of T-helper 1 cytokines on secretion of T-helper 2 cytokines by term trophoblast cells in culture.

A successful pregnancy has been postulated to be the result of a discrete balance between T-helper 1 (Th1) and T-helper 2 (Th2) type cytokines involved in growth and development of the conceptus. The aim of the present study was to examine the effect of Th1 cytokines (interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha)) on the release of Th2 cytokines including IL-6 and IL-10 by trophoblast cells obtained from term placenta. Trophoblast cells isolated by enzymatic disaggregation and Percoll gradient fractionation were cultured in supplemented medium alone or with varying concentrations of the selected recombinant cytokines. After 48 h of incubation, samples of the culture supernatant were analyzed for the Th2 cytokines IL-6 and IL-10 using specific ELISA assays. Both IL-1 beta and TNF alpha had no effect on the cell number and viability as determined by MTT assay. IL-1 beta significantly stimulated trophoblast release of IL-6 in a dose-dependent manner (3.3-, 5.5-, 10.3- and 22.4-fold higher compared to the control at 10, 50, 100, 500 U IL-1 beta/ml respectively, p < 0.05). TNF alpha also stimulated release of IL-6 by these cells. However, the stimulation at lower concentrations was not very high and a significant (p < 0.05) stimulation was observed only at higher concentrations (1.1-, 1.3-, 2.6- and 5.9-fold higher at 500, 1000, 1500, 2000 U TNF alpha/ml respectively). In contrast, neither IL-1 beta or TNF alpha exerted any significant effect on IL-10 release by term trophoblast cells (p > 0.05). The results of this study provide evidence that production of Th2 cytokines might be under the control of different regulatory pathways.     

Capacity for hormone production of cultured trophoblast cells obtained from placentae at term and in early pregnancy

Problem: There is an increased doubt about the identity of isolated cytotrophoblast cells at term. Therefore, we compared pregnancy serum levels of three hormones [human placental lactogen (hPL), human chorionic gonadotropin (hCG), and leptin] with the capacity for hormone production of early placentae [EP; 8–13 weeks of gestation (WG)] and term placentae (TP; 38–42 WG).Methods: Serum levels of these hormones were determined in 15 paired maternal (7–41 WG) and fetal (37–41 WG) samples. Cytotrophoblast cells were isolated from term (TP; 38–42 weeks) and early (EP; 8–13 weeks) placentae by enzymatic digestion and subsequent purification on a Percoll gradient. These cells were cultured for 6 days. The production of the hormones hPL, hCG, and leptin was determined as release during culture + cell content after culture – cell content before culture.Results: Serum levels (mean ± SD; n ± 15) at 7–12 and 37–41 WG were 89,652 ± 21,431 and 13,620 ± 5854 mIU/ml for hCG, 400 ± 182 and 7088 ± 2030 ng/ml for hPL, and 12,675 ± 4266 and 32,236 ± 10,961 pg/ml for leptin, respectively. For cultured cells from EP and TP, hCG and hPL showed different patterns of release during the first 2–3 days. While the release of these two hormones by EP cytotrophoblast cells continued during 6 days in culture, their concentrations reached a plateau for TP cytotrophoblasts between 4 and 6 days. Leptin was undetectable (<15 pg/ml) in TP cell cultured media, while for EP all three hormones showed the same release profiles. Production calculated for 30,000 TP trophoblast cells cultured for 6 days (n = 8) was 2–31 mIU for hCG and 0.5–2 ng for hPL. For EP (n = 11), it was 50–1070 mIU for hCG, 15–323 ng for hPL, and 137–580 pg for leptin. Net synthesis of hCG and hPL for TP was >10-fold and <1-fold, respectively. In contrast, the production of all three hormones for EP was at least 100 times the initial cell content.Conclusions: These results demonstrate that trophoblasts from early pregnancy show much higher production rates of hCG, hPL, and leptin than at term. However, the in vitro findings are difficult to be reconciled with the different serum concentrations of the two hormones hPL and leptin observed during the course of pregnancy.     

Role of T-cell cytokines in decidua and in cumulus oophorus during pregnancy

We focus on the roles of decidual and cumulus oophorus T cells. It was suggested that Th1-type cytokines (IFN-gamma and TNF-beta), which promote allograft rejection, may compromise pregnancy, whereas the Th2-type cytokines (IL-4, IL-5, IL-10) inhibiting the Th1 responses promote allograft tolerance and therefore may improve fetal survival. We found that T cells' leukemia inhibitory factor (LIF), M-CSF, IL-4 and IL-10 production at the fetomaternal interface could contribute to the maintenance of pregnancy. Interestingly, we did not find an increased production of IFN-gamma by decidual T cells during spontaneous abortion, as expected. We detected T cells in the cumulus oophorus, which surrounds the oocyte during ovulation and the egg before implantation. Cumulus oophorus T cells produce higher levels of IL-4 and LIF than the T cells of peripheral blood or the ovary. We can only speculate what the factors present in the microenvironment of the T cells are that could be responsible for the cytokine profile of decidual and cumulus oophorus T cells. Hormones present in the decidua and in the cumulus oophorus could be the first candidates. In particular, progesterone is a potent inducer of production of Th2-type cytokines, LIF and M-CSF. Other candidates could be molecules produced by the trophoblast or the embryo.     

The effect of coelomic fluid on the production of cytokines by the first trimester human placenta

Objective

To investigate the modulatory effects of coelomic fluid (CF) on the production of tumour necrosis factor-alpha (TNFα) and its receptors, TNF-R1 and TNF-R2, interferon gamma (IFNγ) and interleukin (IL)-10 by placental villous explants cultured under physiological oxygen (O₂) concentration.

Study design

In vitro culture of placental villous explants at atmospheric and physiological (6%) O₂ levels at varying concentrations of CF.

Main outcome measures

Concentration of TNFα, TNF-R1, TNF-R2, IFNγ and IL-10 in culture medium and villous explant homogenates, measured using flowcytometric bead arrays.

Results

The median level and interquartile range of cytokines and receptors present in CF were: TNFα 0.9 pg/ml (0.85, 0.95); IFNγ 5.38 pg/ml (4.96, 6.18); IL-10 1.59 pg/ml (1.45, 1.76); TNF-R1 6043.65 pg/ml (5961.39, 6187.35) and TNF-R2 3563.52 pg/ml (3390.26, 3635.19). The PO₂ of CF was 48.74 mmHg (SEM 1.59), equivalent to 6% O₂.Increasing doses of CF significantly (p < 0.05) increased the levels of TNFα, TNF-R1 and TNF-R2 in the culture medium at both O₂ concentrations.TNF-R1 levels measured in placental homogenate increased up to 2-fold at both O₂ concentrations, but were significantly lower (1.96 fold; p = 0.03) at 6% O₂ compared to 20% O₂.

Conclusions

The exocoelomic cavity in humans contains high levels of both soluble TNF-R1 and TNF-R2. The addition of CF to placental tissue explants in culture modulates the placental secretion of TNFα-receptors and TNFα at both physiological and atmospheric O₂ tension resulting in a concentration much higher than that found in the CF. This may contribute to the maternal inflammatory response previously reported in normal early pregnancy.     

Perfusion with lipopolysaccharide differently affects the secretion of interleukin-1 beta and interleukin-1 receptor antagonist by term and preterm human placentae

The aim of the present study was to examine the effect of lipopolysaccharide (LPS) on the secretion of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) and of its natural inhibitor interleukin-1 receptor antagonist (IL-1Ra), by perfused human term and preterm placental tissue. Eight term and eight preterm placentae were collected immediately after delivery; four term and four preterm placentae were perfused with control medium (without LPS) and the other four term and four preterm placentae were perfused with medium containing LPS. The release of IL-1beta into the maternal compartment by term placenta was significantly higher than the release by preterm placenta (p<0.001). However, there were no significant differences between IL-1beta levels released into the fetal compartments of term and preterm placentae. No significant differences were observed in the release of IL-1Ra into the maternal and fetal compartments of term placenta, when compared to preterm placenta. Exposure to LPS significantly decreased the capacity of term placenta to release IL-1beta into the maternal compartment (p<0.001) and increased the capacity of term placenta to release IL-1Ra into the maternal and fetal compartments (p<0.001 and p=0.017, respectively). However, the capacity of preterm placentae to release IL-1beta and IL-Ra into the maternal and fetal compartments was not affected by LPS. IL-1beta was expressed by both term and preterm placentae before and after perfusion (+/- LPS), by epithelial cells of the amnion, chorion, by syncytiotrophoblast and stromal cells of villous tissue and by the decidua. IL-1Ra in term and preterm placentae was expressed before perfusion mainly in epithelial cells of the amnion. After perfusion of term placentae (+/- LPS), additional IL-1Ra expression was seen in epithelial cells of the amnion and in syncytiotrophoblast and stromal cells of villous tissue and by the decidua. However, perfusion of preterm placentae (+/- LPS) did not affect IL-1Ra expression. The localization of IL-1beta and IL-1Ra in both term and preterm human placental tissue suggests a their physiologic role. The data presented indicates that the IL-1 system in term and preterm placentae seems to be differently affected by LPS. Down-regulation in the release of the pro-inflammatory cytokine IL-1beta and the up-regulation of its antagonist (IL-1Ra) may be a part of the inflammatory response to infection in human term, but not preterm, placentae. The IL-1 system in term and preterm placentae seems to be differently affected by LPS.     

Insulin-induced receptor regulation in early gestation and term human placental cell cultures

Human placentae of different gestational ages have been used to investigate the binding and degradation of insulin as well as the regulation of insulin membrane receptors. Bacitracin was found to be an effective inhibitor of insulin degradation in human early gestation and term placental cell cultures. In the presence of bacitracin, [125I]-insulin bound rapidly and reversibly: maximal binding occurred at 4 degrees C, with a sharp pH optimum at 7.5, and exhibited a high degree of specificity. The extent of binding was proportional to cell protein and [125I]-insulin concentrations. Term (38 to 41 weeks; n = 25) placental cell cultures possessed receptors for insulin that were increased three-fold compared to early gestation (8 to 18 weeks; n = 17). This was due to an increase in receptor number with no significant alteration in affinity. A decrease in insulin binding in both early gestation and term placental cells was related to both the insulin and bacitracin concentrations present during 12 to 20 h of preincubation at 37 degrees C. The receptor loss was due to a decrease in the number of receptors per mg cell protein with no apparent change in their affinity. We conclude that our in vitro system, which utilizes human placental cells in monolayer culture, will permit a more direct study of the metabolic effects of insulin in both early gestation and term placentae.     

The role of SOCS3 in modulating leukaemia inhibitory factor signalling during murine placental development

Cytokines are an integral part of the adaptive and innate immune responses. The signalling pathways triggered by receptor engagement translate exposure to cytokine into a coordinated biological response. To contain these responses, the initiation, duration and magnitude of the signal is controlled at multiple levels. Suppressor of cytokine signalling (SOCS) proteins act in a negative feedback loop to inhibit signal transduction. Mice with a deletion of SOCS3 die at midgestion due to placental insufficiency. SOCS3-null placentae have increased numbers of mature trophoblast giant cells, disruption of the labyrinthine layer and a decrease in the spongiotrophoblast layer. Genetic crosses have revealed that the phenotype is due to dysregulation of signalling downstream of the leukaemia inhibitory factor (LIF) receptor alpha (LIFRalpha) and that the ligand responsible for this, LIF, is produced by embryonic tissues and acts in a paracrine fashion. These observations highlight the role of LIF as an extrinsic factor regulating trophoblast differentiation in vivo. The creation of mice with conditional deletion of SOCS3 in different tissues has also uncovered critical roles for SOCS3 in the regulation of IL-6, G-CSF and leptin signalling.