Histamine influence on apoptosis in trophoblast cell cultures

Introduction

It has been demonstrated that histamine plays an important role in the pathogenesis of preeclampsia. Histamine regulates the process of differentiation of trophoblast cells; it also acts as a growth factor in malignant melanoma cells, and prevents monocytic apoptosis. Trophoblast research has shown that in preeclampsia placentas, trophoblast apoptosis is significantly increased.

Aim of the study

The aim of our study was to demonstrate the influence of histamine on the process of apoptosis in human trophoblast cell cultures.

Materials and methods

Placentas were obtained after vaginal delivery. Tissue samples were excised from placentas and, with the use of modified Kliman’s method, trophoblast cell cultures were established. The cultures were incubated with dexamethasone as an apoptosis inducer 48 hours prior to apoptosis detection assays. Along with dexamethasone, selected cell cultures were incubated with histamine (1 μmol/l) or histamine (1 μmol/l) and terfenadine (from 1 to 5 μmol/l), a H₁ receptor antagonist. For apoptotic activity detection, and quantitative analysis, we used an ELISA assay. M30‐Apoptosense ELISA Kit is based on the M30 monoclonal antibody that binds only the caspase‐cleaved cytokeratin 18 formed during apoptosis in trophoblast cells.

Results

Our investigation showed significantly (p < 0.05) increased apoptotic activity in cultures incubated with dexamethasone, histamine and terfenadine (% of reference value, ±SEM): up to 113.1 ± 4.33%. Cell cultures incubated with dexamethasone and histamine only showed significantly lower apoptotic activity 90.2 ± 5.17%. We suggest that histamine may inhibit apoptotic activity in trophoblast cell cultures via H₁ receptor. Thus histamine may regulate the process of trophoblast differentiation (via integrin aV‐b3 expression, as we previously suggested), and influence cell turnover in the placenta.

     

Histamine prevents apoptosis in human monocytes

BACKGROUND: As chronic atopic dermatitis (AD) is associated with activation of circulating and infiltrating monocytes, monocytes are considered to play a pivotal role in the establishment of chronic lesions in AD. Histamine is an important mediator of inflammatory and allergic responses. Although new immunomodulatory functions of histamine have recently become apparent, the effect of histamine on the life span of monocytes remains unclear. OBJECTIVE: In the present study, we investigated the effect of histamine on the life span of human monocytes from normal healthy donors and patients with AD. METHODS: Monocyte apoptosis was induced by serum deprivation, CD95/Fas ligation, or dexamethasone in the presence of histamine, and measured using annexin V-and propidium iodide-staining. Bcl-2 protein and activated caspase-3 were determined by flow cytometry. We also examined the effect of soluble, histamine-induced factors produced by monocytes on apoptosis. Furthermore, we examined whether monocytic apoptosis is dependent on the cAMP pathway. RESULTS: Histamine prevented monocytic apoptosis induced by serum deprivation, CD95/Fas ligation, or dexamethasone in a dose- and time-dependent fashion. The inhibitory effects of histamine on monocytic apoptosis were blocked by an H2R antagonist, and mimicked by an H2R agonist. Histamine also up-regulated the expression of Bcl-2 and Mcl-1, and inhibited the activation of caspase-3. The culture supernatants from histamine-treated monocytes inhibited monocytic apoptosis, which was partly reversed by the removal of IL-10. Monocytes cultured with anti-IL-10 mAb and histamine did not exhibit an inhibitory effect on apoptosis. The histamine-induced anti-apoptotic effect was attenuated when monocytes were cultured in the presence of a cAMP inhibitor. CONCLUSIONS: These results indicate that the H2R signals induced by histamine allow monocytes to prolong their life span and infiltrate to the site of inflammation. This process may contribute to the establishment of chronic allergic disorders, such as AD.     

Reactivity of human trophoblast monoclonal antibodies with marmoset monkey trophoblast cultures

The aim of this study was to determine whether cultured trophoblast tissues, derived from the trophectoderm of marmoset monkey blastocysts, contain homologues of human trophoblast antigens. This is an essential prerequisite to determine whether the marmoset may be a suitable model for preclinical testing of a human antitrophoblast antigen for fertility regulation. Previously evaluated monoclonal antibodies from the Flinders University laboratory, which reacted with human trophoblast with a high degree of specificity, were tested for immunohistochemical reactivity using an immunoperoxidase detection method on both frozen and paraformaldehyde-fixed sections of the cultured marmoset monkey trophoblast. All monoclonal antibodies raised against human placenta reacted positively, when compared to controls, suggesting that human and marmoset trophoblast cells share common epitopes. The specificity of the monoclonal antibodies was investigated by determining whether there was cross-reactivity with other marmoset monkey tissues, including adrenal, spleen, kidney, liver, muscle, ovary and testis. The specificities of the monoclonal antibodies on these marmoset tissues were similar to those previously found on the corresponding human tissues. We have concluded that marmoset monkey trophoblast exhibits homologues of human trophoblast antigens. The findings also suggest that marmoset monkeys should be evaluated further as a primate model to test suitable target antigens for antitrophoblast vaccines that may be useful contragestation agents in humans.      

X-linked inhibitor of apoptosis (XIAP) confers human trophoblast cell resistance to Fas-mediated apoptosis

Apoptosis occurs in the placenta throughout gestation, with a greater frequency near term in comparison to the first trimester. The Fas/FasL system represents one of the main apoptotic pathways controlling placental apoptosis. Although first trimester trophoblast cells express both Fas and FasL, they are resistant to Fas-induced apoptosis. Therefore, trophoblast resistance to Fas-mediated apoptosis may be due to the inhibition of the pathway downstream of Fas stimulation. Expression levels of X-linked inhibitor of apoptosis (XIAP) were recently shown to decrease in third trimester placentas, correlating with an increase in placental apoptosis. As a potent caspase inhibitor, XIAP prevents the activation of caspase-9 through its BIR3 domain and caspase-3 activation via the linker-BIR2 domain. In the present study, high levels of the active form of XIAP were detected in first trimester trophoblast cells, whereas term placental tissue samples predominantly expressed the inactive form of XIAP. Using a XIAP inhibitor, phenoxodiol, we demonstrate that XIAP inactivation sensitizes trophoblast cells to Fas stimulation, as evidenced by the anti-Fas mAb-induced decrease in trophoblast cell viability and increase in caspase-8, caspase-9 and caspase-3 activation. This suggests a functional role for XIAP in the regulation of the Fas apoptotic cascade in trophoblast cells during pregnancy.     

Induction of cell cycle arrest and apoptosis in JAR trophoblast by antimalarial drugs

Chloroquine, quinine, artemisinin, and pyrimethamine are generally considered safe drugs for treatment of malaria during pregnancy; however, high doses of these drugs are detrimental with adverse outcome of pregnancy. Since antimalarial drugs interaction with placental cells has not been addressed, in this study, we employed a non-radioactive proliferation assay and lactate dehydrogenase (LDH) release assays to investigate the effect of these drugs on JAR trophoblastic cell survival. All drug treatment resulted in inhibition of cell proliferation in a dose-dependent fashion (p < 0.05) with IC50 at 6.96, 6.49, 6.69, and 6.89 microg/mL for chloroquine, quinine, artemisinin and pyrimethamine, respectively. In addition, the inhibition of cell proliferation was accompanied by increased cytotoxicity. Analysis of the progression of the cell cycle showed that these drugs triggered G0/G1 and S phase arrest. Furthermore, these antimalarial drugs induced apoptotic cell death as visualized by DNA fragmentation analysis techniques. Findings in this study revealed that cytotoxicity of these drugs on human placental trophoblast is mediated by both cell cycle arrest and induction of cell death and this could have important implications for the use of antimalarial drugs for treating malaria during pregnancy.     

First trimester trophoblast cells secrete Fas ligand which induces immune cell apoptosis

Since the invading trophoblast represents a semi-allograft, it should be rejected by the mother. It has, therefore, been postulated that during normal pregnancy the trophoblast evades the maternal immune system though the establishment of immune privilege by triggering the death of activated lymphocytes which may be sensitized to paternal alloantigens. Such peripheral tolerance may be directed through the Fas/Fas ligand (FasL) apoptotic pathway and mediated by FasL expressed by the trophoblast. However, in vivo studies show that membrane-associated expression of FasL may instead promote allograft rejection, rather than protection. The aim of this study was to determine if there is a role for FasL in trophoblast immune privilege. In this study, we demonstrate that isolated first trimester trophoblast cells lack membrane-associated FasL, but express a cytoplasmic form in association with a specialized secretory lysosomal pathway. Furthermore, this intracellular FasL is constitutively secreted by trophoblast cells via the release of microvesicles. Following disruption of these microvesicles, the whole 37 kDa secreted FasL is able to induce T-cell death by apoptosis through activation of the Fas pathway. Therefore, we propose that secretion of FasL may be one mechanism by which trophoblast cells promote a state of immune privilege and, therefore, protect themselves from maternal immune recognition.     

Transmissible gastroenteritis virus induced apoptosis in swine testes cell cultures

Summary.  Transmissible gastroenteritis virus (TGEV) is a coronavirus which causes severe gastroenteritis and atrophy of intestinal villous epithelial cells in piglets. However, the mechanism of cell death caused by TGEV is not known. In this study, we report that TGEV induces cell death by apoptosis. TGEV-induced apoptosis was demonstrated by agarose gel electrophoresis, electron microscopy, and terminal deoxytransferase digoxigenin-dUTP nick end labeling (TUNEL). Double labeling experiment confirmed the result from electron microscopy and showed that most of the apoptotic cells were bystander cells as they were negative for TGEV nucleic acids. Results of this study indicate that TGEV induces apoptosis in vitro and that most of the cells undergoing apoptosis are bystander cells, thus amplifying the cytopathic effect of TGEV. Received February 23, 1998 Accepted June 8, 1998     

Effect of folic acid on homocysteine-induced trophoblast apoptosis

In trophoblast cells exposed to homocysteine (Hcy) we observed cellular apoptosis and the inhibition of trophoblast functions. Because folate and Hcy, linked in the same metabolic pathway, are inversely related, we investigated the role of folic acid in reversing the Hcy effect in human placenta. In primary trophoblast cells we examined the cytosolic release of cytochrome c, both M30 and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and DNA laddering. Hcy (20 micromol/l) treatment resulted in cytochrome c release from mitochondria to the cytosol, and an increased number of M30-positive trophoblast cells and TUNEL positive nuclei. Furthermore, DNA cleavage in agarose gel and the determination of histone-associated DNA fragments have been investigated. Homocysteine induced DNA fragmentation and significantly reduced hCG secretion. The addition of folic acid (20 nmol/l) resulted in inhibition of the effects of Hcy on human trophoblast. These results suggest a protective role of folic acid in the prevention of trophoblast apoptosis linked to Hcy.     

The influence of infectious factors on dendritic cell apoptosis

Pathogens can have a negative influence on dendritic cells (DCs), causing their apoptosis, which prevents active presentation of foreign antigens. It results in a state of immunosuppression which makes the body susceptible to secondary infections. Infected immature DCs have lower expression of co-stimulatory and adhesion molecules, reduced ability to secrete cytokines and an inhibited maturation process and are incapable of effective antigen presentation and activation of T-lymphocytes. In some cases, the ability of DCs to undergo rapid apoptosis is important for the body defense, which is probably because of DCs’ ability to cross-present and cooperate with other cells. Apoptotic bodies released from the infected DCs are phagocytosed by other DCs, which then stimulate the effector cells and present antigens more efficiently than infected cells. The aim of this article is to review how the DCs respond to viral and bacterial factors and which biochemical mechanisms are responsible for their apoptosis.     

Nitric oxide inhibits polyamine-induced apoptosis in the human extravillous trophoblast cell line SGHPL-4

BACKGROUND: Polyamines are regulators of proliferation and differentiation in mammalian cells. They are also known to regulate cell survival and apoptosis, although their precise function varies between cell types. We have investigated the effect of polyamines on the apoptosis of human extravillous trophoblasts. METHODS: Using the extravillous trophoblast-derived cell line SGHPL-4 we performed time-lapse microscopy studies to evaluate the induction of apoptosis following exposure to polyamines. RESULTS: The polyamines spermine, and to a lesser extent spermidine, were able to induce apoptosis in extravillous trophoblasts. The induction of apoptosis occurred rapidly and was accompanied by DNA fragmentation and morphological changes consistent with the onset of apoptosis. Apoptosis was inhibited by the broad-spectrum caspase inhibitor Z-VAD-fmk, although no activity was detected using assays for caspase-2, -3, -6, -8 or -9 activity. We demonstrated that an oxidation product of spermine accounted for the induction of apoptosis and implicated the formation of hydrogen peroxide as this oxidation product. We have also demonstrated that exposure to nitric oxide inhibited the onset of spermine-induced apoptosis. CONCLUSIONS: Spermine and spermidine induce apoptosis in extravillous trophoblasts following their oxidation and the production of hydrogen peroxide. Nitric oxide is able to inhibit this apoptosis.     

妊娠期糖尿病中胎盘外泌体对滋养细胞增殖、细胞周期及凋亡的影响

目的：探讨妊娠期糖尿病（GDM）中胎盘外泌体对滋养细胞增殖、细胞周期及细胞凋亡的影响。方法： 选取2018 年3 月至2019 年4 月间重庆市第七人民医院50 例诊断为GDM患者（GDM组）,另选取50 例正常孕产 妇作为对照组。分离纯化GDM组和对照组孕产妇外周血血浆中的胎盘外泌体,通过透射电镜观察外泌体的形 态,纳米粒径分析检测外泌体的直径,免疫印迹检测外泌体标志性蛋白CD63、TSG101 及胎盘标志性分子胎盘 碱性磷酸酶（PLAP）的表达。PKH67染色后荧光共聚焦显微镜观察体外培养滋养细胞系对外泌体的摄取情况; 应用MTT实验检测外泌体对滋养细胞增殖能力的影响,流式细胞术检测外泌体对滋养细胞周期的影响,Annexin V-FITC/PI 双染色结合流式细胞术检测外泌体对滋养细胞凋亡的影响。结果：成功从外周血中分离获得了胎盘外 泌体,形态呈典型的杯状或双凹状,直径为40 ～ 120 nm,CD63、TSG101、PLAP 表达呈阳性;与对照组相比, GDM组胎盘外泌体以浓度依赖性促进滋养细胞的增殖、细胞周期进展,并抑制滋养细胞的凋亡。结论：GDM中 胎盘外泌体可能通过促进滋养细胞的增殖、细胞周期进展,抑制滋养细胞凋亡等参与GDM的发生和发展。     

Histamine influences the expression of IFN-γ in atopic and nonatopic human Th1 cell cultures

Inflammation Research -     

Histamine inhibits proliferation of a pancreatic carcinoma cell line without inducing apoptosis significantly

Inflammation Research -     

The influence of lododeoxyuridine pretreatment of various cell cultures on adenovirus multiplication

Treatment of cell cultures with iododeoxyuridine (IUdR) before virus inoculation may enhance the subsequent virus multiplication. This effect was studied in seven kinds of cell cultures with seven human adenoviruses from six subgenera. IUdR (50 micrograms/ml) was added 24 h after the seeding of cells, left for 2 days, and removed before virus inoculation. IUdR had no effect on cell proliferation. Viral cytopathic effect was enhanced in many instances by IUdR pretreatment. However, virus multiplication was enhanced weakly in only four cases [adenovirus 7 in Vero, African green monkey kidney, cynomolgus monkey kidney; adenovirus 4 (Ad4) in HeLa cells], and strongly only for Ad4 in Vero cells, which are semipermissive for Ad4. IUdR pretreatment of Vero cells shortened the replication cycle for Ad4 and increased sensitivity for minimal virus concentrations about 1000-fold. From immunofluorescence experiments it appears that more than one infectious particle is required to infect an untreated Vero cell.     

The influence of temperature on interferon in cell cultures of homoiothermic and heterothermic mammals

Subnormal temperature was found to depress the production of interferon by cultures of fibroblasts of homoiotherms and heterotherms after virus or poly I.poly C induction. However, in comparison to human (HEF) and mouse (MEF) fibroblasts (homoiotherms) induced with NDV-R or poly I.poly C, interferon production in fibroblasts of spotted sousliks (SL) (heterotherm) and in the aneuploid line of mouse origin (L929) exhibits a greater cold resistance. In contrast to HEF and MEF cells in SL and L929 cells interferon was produced even at 21 degrees C after induction with NDV-R and at 26 degrees C after induction with poly I.poly C. A comparison of alpha and gamma interferon production by mouse and spotted souslik leukocytes did not reveal such distinct differences. At a low temperature (26 degrees C) the production of NDV-R induced interferon was depressed, but it was higher after induction with LPS in both types of leukocytes. PHA and Con A induced interferon was produced in both types of leukocytes only at 37 degrees C. These data confirm that alpha, beta and gamma interferon induction is triggered by different mechanisms and suggest a resistance to cold of beta interferon production in heterothermic animals.     

Does histamine influence differentiation of trophoblast in preeclampsia?

Inflammation Research -     

Bax抑制因子1对人妊娠期肝内胆汁淤积症胎盘滋养细胞凋亡的影响及其机制

目的：探讨Bax 抑制因子1（BI-1）对人妊娠期肝内胆汁淤积症（ICP）胎盘滋养细胞凋亡的影响及其机 制。方法：选取2018 年5 月至2019 年5 月新疆维吾尔自治区人民医院产科住院分娩的ICP 孕产妇15 例为研究对 象,设为ICP 组,另取15 例正常孕产妇作为对照组,免疫组织化学显色检测ICP 组和对照组孕妇胎盘组织BI-1 的 表达;体外培养滋养细胞系HTR8,应用不同浓度（10、50、100 μmol/L）的牛磺胆酸（TCA）进行刺激,RTPCR 及免疫印迹检测HTR8细胞BI-1 mRNA 及蛋白的表达;用过表达BI-1 的慢病毒载体感染HTR8细胞,荧光显 微镜及RT-PCR 检测感染效率后,将细胞分为对照组（NC组）、TCA处理的感染LV-NC 细胞组（TCA+LV-NC 组）、 TCA处理感染LV-BI-1 细胞组（TCA+LV-BI-1 组）;分别用Annexin V-FITC、透射电镜及JC-1 流式线粒体膜电位 检测试剂盒检测3 组滋养细胞凋亡、线粒体超微结构及线粒体膜电位;免疫印迹检测3 组细胞Cyt-C 及凋亡相关 蛋白Bcl-2、Bax 及cleaved caspase-3 的表达。结果：与对照组相比,ICP 组胎盘组织BI-1 表达显著降低;体外实 验结果显示,TCA能够显著降低HTR8细胞BI-1 mRNA及蛋白表达,且呈浓度依赖性;应用慢病毒成功建立过 表达BI-1 的滋养细胞系。与NC组细胞相比,TCA+LV-NC 组与TCA+LV-BI-1 组细胞凋亡率和线粒体损伤水平均 显著增加,线粒体膜电位明显降低;而与TCA+LV-NC 组相比,LV+BI-1 组细胞凋亡率与线粒体损伤水平均明显 降低,线粒体膜电位显著增加;过表达BI-1 能够有效阻止TCA导致的滋养细胞Bcl-2/Bax 比值的降低,以及线粒 体Cyt-C 的释放及cleaved caspase-3 表达的增加。结论：BI-1 在ICP 患者胎盘组织中的表达显著降低,而体外过 表达BI-1 可能通过上调滋养细胞中Bcl-2/Bax 比值,抑制细胞线粒体膜电位降低及结构损伤,从而减少Cyt-C 的 释放,最终降低凋亡蛋白caspase-3 活化而改善TCA诱导的凋亡作用。     

Study of trophoblast cell culturesin vitro

A culture of the trophoblast was obtained from an early human embryonic chorion. Trophoblastic cells of four main types are described: giant, branched, fibroblastlike, and round. All the cells were rich in RNA and glycoproteins and had the complement of enzymes for the Krebs' pentose cycle, but had low succinate dehydrogenase activity. A marked cytotoxic effect of the lymphocytes of the healthy pregnant woman on allogeneic cells of the trophoblast culture and no interaction between neonatal lymphocytes and these trophoblastic cells were found.Laboratory of Immunology, Department of Immunology, Institute of Clinical and Experimental Medicine, Siberian Branch, Academy of Medical Sciences of the USSR, Novosibirsk. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 4, pp. 467–470, April, 1977.