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Citation Braunschweig A, Poehlmann TG, Busch S, Schleussner E, Markert UR. Signal transducer and activator of transcription 3 (STAT3) and suppressor of cytokine signaling 3 (SOCS3) balance controls cytotoxicity and IL-10 expression in decidual-like natural killer cell line NK-92. Am J Reprod Immunol 2011; 66: 329–335 Objectives In previous studies, we have shown that sHLA-G reduces cytotoxicity of decidual NK cells, which was dependent upon reduction in signal transducer and activator of transcription 3 (STAT3) and perforin. In this study, we aimed to confirm the role of STAT3 for induction of cytotoxicity and to analyze the regulative role of its antagonist suppressor of cytokine signaling 3 (SOCS3). Furthermore, the influence of both factors on cytokine expression should be analyzed. Methods All experiments were performed on NK-92 cells. STAT3 and SOCS3 have been silenced using two different small interfering RNA sequences each. Silencing efficiency and STAT3 tyrosine phosphorylation have been analyzed by Western blotting. Cytotoxicity to K562 target cells has been assessed by flow cytometry. Expression of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-α, and TNF-β has been measured using cytometric bead arrays for flow cytometry. Results STAT3 and SOCS3 have been successfully silenced. STAT3 silencing reduced cytotoxicity. SOCS3 silencing induced increase in STAT3 tyrosine phosphorylation and cytotoxicity. STAT3 silencing reduced IL-10 expression significantly, while SOCS3 silencing induced, also significantly, the opposite effect. The other cytokines were expressed at very low concentration or not constantly affected. Conclusion STAT3 and SOCS3 are involved in regulation of NK cell cytotoxicity and IL-10 expression.

New multifunctional copoly(2-oxazoline) nanoparticles were prepared for cell studies. The polymer contains double-bond side chains as potential reaction sites for "thio"-click reactions as well as a fluorescein label covalently bound to the polymer backbone. Using the nanoprecipitation technique, spherical nanoparticles of 200-800 nm were obtained. Confocal laser scanning microscopy measurements revealed the cellular uptake of the nanoparticles.

Problem During pregnancy, it is crucially important that the mother’s immune system tolerates the developing embryo. Although a number of mechanisms of immunological tolerance have been described, little is known about intracellular signaling events, causing a decrease in the mother’s leukocyte activity. Method of study We investigated the expression and activity of phosphoinositide 3-kinases (PI3K) in maternal blood cells of healthy volunteers by Reverse Transcription PCR and Western blotting. Results Our data reveal a selective downregulation of the p110α catalytic isoform. This correlated with a slight decrease in PI3K activity as judged by the levels of phosphorylated Akt. Conclusion As PI3K are involved in signal transduction of various leukocyte receptors, this downregulation may comprise a means of holding immune functions at bay.

A simple RP-HPLC method based on fluorescence detection was developed for the quantitation of 7-amino-4-trifluoro methylcoumarin (AFC) in cell lysates from JEG-3 choriocarcinoma cells for determination of caspase-4 activity. In contrast to the established methods of AFC detection using a fluorescence microplate reader or using a fluorescence photometer, the separation of AFC-signals from interfering fluorescence signals by a reversed phase column affords more precise quantitation of released AFC. This can be important for analyses of cell lysates with low caspase activity or experimental series with marginal differences among samples. By applying this new method, a linear dynamic range of 40pmol/mL to 3nmol/mL with a correlation coefficient of 0.9996 was achieved. Due to the short retention time ( approximately 7min), the determination of AFC by RP-HPLC under isocratic conditions requires small amounts of samples (50microL injection volume), and allows increased sample throughput. This method should be easily applied with little or no modification to other caspase assays by using the same fluorophore.

Trophoblast cells display a very unique capability: they physiologically invade into the surrounding tissue. This capability is widely associated with tumours, and, indeed, the invasive behaviour of both is rather similar. The imposing difference is that trophoblast cell invasion is temporally and locally controlled in contrast to unlimited tumour invasion. It initiates immediately after embryo implantation into the endometrium. Parallel to tumours, trophoblasts secrete proteases, such as matrix metalloproteinases, which dissolve the extracellular matrix and the surrounding tissue. Thereby, these proteases prepare and allow true invasion of trophoblasts. The invasive capacities of trophoblasts are positively and negatively regulated by numerous cytokines including leukaemia inhibitory factor (LIF), interleukin-6, hepatocyte growth factor, granulocyte macrophage-colony stimulating factor and others. They interact via specific receptors with the trophoblast cells, in which they activate intracellular signalling cascades. These will then induce expression of invasion relevant genes. One of these signalling pathways is the Janus kinase/signal transducers and activators of transcription (STAT) pathway. Especially phosphorylated STAT3 enhances invasiveness of tumours and trophoblast cells, where it is mainly activated by LIF. One of its most efficient physiological antagonists is suppressor of cytokine signalling 3. The balance of these two intracellular molecules seems to be a key regulator of tumour and trophoblast invasion.

Pregnancy is one of the most challenging experiences for the immune system. It entails the confrontation and cooperation of maternal cells with allogeneic (sperm) and semi-allogeneic (fetal) cells and factors. On the one hand, it must actively acquire a specific tolerance towards the foreign cells and organism (the fetus) to avoid harming reactions, while recognizing the very same to support and control their development and growth. On the other hand, the immune system simultaneously may not reduce its capability to defend mother and fetus from microorganisms and pathogens. Almost all branches of the immune system are claimed to react and adapt in order to fulfill these complex duties. In this review, current knowledge concerning the most important cellular and soluble immunological components in the decidua is presented. Special regards are made to decidual NK, T and dendritic cells as well as to trophoblast cells, representing the fetal counterpart of most bilateral interactions. Furthermore, the role and functions of soluble factors, including HLA-G, PIBF, IDO and a variety of cytokines, are described.

Extravillous trophoblast cells resemble cancer cells with regard to their intrinsic invasiveness. They invade decidual tissue, but, unlike tumor cells, shut down their invasive properties, when they become inappropriate. Stimuli involved in the modulation of invasion, as well as their underlying signaling mechanisms require further clarification. We were especially interested in discovering signals capable of stimulating invasion in otherwise low-invasive cells involved in reproduction. Using the choriocarcinoma cell line Jeg-3 as a model, we have addressed the potential role of cytokine/growth factor-driven activation of signal transducer and activator of transcription 3 (STAT3) in this process. Jeg-3 cells were treated with various factors known to induce trophoblast proliferation, differentiation, migration, or invasiveness (insulin-like-growth-factor-II (IGF-II), hepatocyte growth factor (HGF), interleukin-6 (IL-6), and leukemia inhibitory factor (LIF)). Only LIF elicited strong tyrosine phosphorylation and specific DNA-binding activity of STAT3. It induced a significant acceleration of cell proliferation and promoted the capability of Jeg-3 cells to invade into an artificial extracellular matrix. Moreover, LIF influenced the expression pattern of proteases and protease inhibitors with potential relevance for invasiveness (downregulation of mRNA for tissue inhibitor of metalloproteinase 1 (TIMP-1) and upregulation of mRNA for caspase-4). In conjunction with earlier work, in which we found that STAT3 DNA-binding activity was increased in invasive cells (choriocarcinoma, first trimester trophoblasts) and absent in non-invasive cells (term trophoblasts), these findings suggest a connection between LIF-driven STAT3 activity and invasiveness of choriocarcinoma and trophoblast cells.

Aberrant activity of the signal transducer and activator of transcription 3 (Stat3) is believed to be essential for neoplastic cell behaviour and thus for the malignancy of tumor cells [Bowman T, Garcia R, Turkson J, Jove R. STATs in oncogenesis. Oncogene 2000;19:2474-88]. Extravillous trophoblast cells resemble malignancies in their invasive and destructive features, excluding the fact of sequential restriction to the first trimester of pregnancy. Trophoblast cells from term placentas have reduced invasive capacity [Hohn HP, Denker HW. Experimental modulation of cell-cell adhesion, invasiveness and differentiation in trophoblast cells. Cells Tissues Organs 2002;172:218-36]. Constitutively activated Stat3 DNA-binding activity in choriocarcinoma cells, carcinomatous derivates of trophoblast cells, have been reported to correlate with its invasiveness [Corvinus FM, Fitzgerald JS, Friedrich K, Markert UR. Evidence for a correlation between trophoblast invasiveness and STAT3 activity. Am J Reprod Immunol 2003;50:316-21]. Here we demonstrate using RNAi that Stat3 activation is necessary in the invasive phenotype of trophoblast cells and can be controlled via Leukemia Inhibitory Factor (LIF). LIF provides a soluble extracellular signal that stimulates invasion in trophoblast and Jeg-3 choriocarcinoma cells. Loss of LIF-mediated invasion in these cells subsequent to STAT3 knock-down strongly suggests that STAT3 plays a crucial role in mediating this invasion. © 2005 Published by IFPA and Elsevier Ltd.

Human leukocyte antigen-G (HLA-G) plays a major role in escape of trophoblast cells from maternal cytotoxicity. Unless its malignant transformation, Jeg-3 choriocarcinoma cell line maintained the capacity of HLA-G production. For the analysis of function and mechanisms of HLA-G-induced immune regulation, a human cellular model with suppressed HLA-G would be very helpful. RNA interference (RNAi) is a very elegant method for this approach, but the design of appropriate oligonucleotide sequences may provide difficulties. We designed oligonucleotides to interfere exclusively with HLA-G mRNA, which were applied at different concentrations to 50% confluent Jeg-3 cells. After 36 hr, the HLA-G content in Jeg-3 cells was analyzed by Western blots. Applying the described RNAi method and oligonucleotides the cellular content of HLA-G was dose-dependently reduced as assessed in several independent Western blots. This method provides a tool for extensive investigation of HLA-G functions in vitro and if vivo.

Trophoblast invasion is a temporally and locally restricted process, which regulates implantation and oxygen arrival to the embryo through the dialog with spiral artery endothelium. Trophoblast factors with angiogenic potential are activated by hypoxia. Their capacities to induce proliferation, migration, and invasion of trophoblastic cells have been investigated.The expression of interleukin (IL)-6, CD126, CD130, vascular endothelial growth factor (VEGF), and hypoxia inducible factor-1alpha (HIF-1alpha) has been silenced in JEG-3 choriocarcinoma cells by using siRNA. Silencing efficacy has been assessed by ELISA, PCR or Western blotting. Proliferation has been measured by flow cytometry, migration by a transwell assay, and invasion by a Matrigel assay.Proliferation was significantly reduced by silencing of CD126 or CD130, migration by silencing of IL-6, VEGF, or HIF-1alpha, and invasion by silencing of IL-6 and HIF-1alpha.The expression of IL-6, VEGF, and HIF-1alpha in trophoblastic cells is involved in the control of trophoblast invasion and migration.

During pregnancy, it is crucially important that the mother's immune system tolerates the developing embryo. Although a number of mechanisms of immunological tolerance have been described, little is known about intracellular signaling events, causing a decrease in the mother's leukocyte activity.We investigated the expression and activity of phosphoinositide 3-kinases (PI3K) in maternal blood cells of healthy volunteers by Reverse Transcription PCR and Western blotting.Our data reveal a selective downregulation of the p110alpha catalytic isoform. This correlated with a slight decrease in PI3K activity as judged by the levels of phosphorylated Akt. CONCLUSION As PI3K are involved in signal transduction of various leukocyte receptors, this downregulation may comprise a means of holding immune functions at bay.

Amongst the interleukin-6 (IL-6) family of cytokines, leukemia inhibitory factor (LIF) has been shown to promote trophoblast invasion and proliferation. In the present study interleukin-11 (IL-11), another member of the IL-6 family, was investigated for its role in regulating invasion, migration and proliferation of JEG-3 choriocarcinoma cells. JEG-3 cells, like extra villous trophoblast (EVT), express mRNA transcripts encoding IL-11 and IL-11 receptor-alpha (IL-11Ralpha). Treatment of JEG-3 cells with IL-11 led to an increase in invasion across Matrigel extracellular matrix without an increase in proliferation. There was a dose-dependent increase in activation of STAT3 under the influence of IL-11 with maximum Tyr705 phosphorylation by 10min. In addition, treatment of JEG-3 cells with IL-11 for 24h led to an increase in expression of unphosphorylated STAT1 and STAT3. Analysis of the nuclear fraction showed an increased localization of STAT3 following IL-11 treatment while STAT1 was absent. Silencing the expression of STAT3 by siRNA caused a 25% reduction in invasion compared to control cells, however this was not significant. Furthermore, treatment of STAT3-silenced JEG-3 cells with IL-11 led to a significant increase in invasion compared to STAT3-silenced cells without cytokine, but this was not significant compared to non-transfected control cells. Silencing the expression of gp130 but not of IL-6R abrogated the increase in invasiveness of JEG-3 cells following IL-11 treatment. In conclusion, activation and upregulation of STAT3 appears to be critical for the IL-11-mediated increase in invasiveness of JEG-3 cells.

Pregnancy is accompanied by a Th2-prone immune modulation, which is a major puzzle piece among maternofetal tolerance-promoting factors. A large number of cytokines is physiologically or pathologically present in the decidua and is potentially able to act on lymphocytes and NK cells, which express a variety of respective receptors. Intracellular signals from these receptors are to a major part transduced via the Janus kinases (JAK) and signal transducers and activators of a transcription (STAT) system, which consists of at least 4 different kinases and 7 STATs plus several subtypes and splicing variants. A network of suppressors of cytokine signaling (SOCS) controls their balance. The interactions of all these intracellular factors and cross-linking with further signaling systems seem to be crucial for the maintenance of a maternal cytokine profile which promotes the tolerance of the fetus.

During the first trimester of pregnancy, well-differentiated primary cells of the placenta known as trophoblast cells grow in an invasive and destructive fashion similar to malignancies, but limited in space and time. The comparison of trophoblast cells with their malignant counterpart, human choriocarcinoma cells, offers an attractive model to understand the origin or development of malignant growth. Several cytokines and growth factors are known to influence trophoblast migration (e.g. EGF, IGF-2, HGF), proliferation (e.g. leptin, HGF, GM-CSF) and/or invasion (e.g. leukemia inhibitory factor, LIF), each factor utilizing at least one pathway for intracellular signaling in the trophoblast. Two pathways that are crossed especially often mediate the signals of these factors and are simultaneously well established in terms of tumor invasion: the Janus kinase-signal transducers and activators of transcription (Jak-Stat) and receptor-associated tyrosine kinase-mitogen-activated protein kinase (RTK-MAPK) pathways. These two pathways are detrimental for reproduction in general, and in part for placenta development, as a series of knockout experiments demonstrate. Aspects of each pathway are also implicated to be involved in trophoblast invasion, e.g. STAT3 is constitutively activated in invasive first trimester trophoblast cells, and activated ERK is detectable in intermediate trophoblast cells, an invasive phenotype. Interaction at several intersection points between the pathways has been described in several cell systems so that the same would seem to be possible in trophoblast cells. In this review, some of the possible areas of interaction are alluded to.

During the first trimester of pregnancy, well-differentiated primary cells of the placenta known as trophoblast cells grow in an invasive and destructive fashion similar to malignancies, but limited in space and time. The comparison of trophoblast cells with their malignant counterpart, human choriocarcinoma cells, offers an attractive model to understand the origin or development of malignant growth. Several cytokines and growth factors are known to influence trophoblast migration (e.g. EGF, IGF-2, HGF), proliferation (e.g. leptin, HGF, GM-CSF) and/or invasion (e.g. leukemia inhibitory factor, LIF), each factor utilizing at least one pathway for intracellular signaling in the trophoblast. Two pathways that are crossed especially often mediate the signals of these factors and are simultaneously well established in terms of tumor invasion: the Janus kinase-signal transducers and activators of transcription (Jak-Stat) and receptor-associated tyrosine kinase-mitogen-activated protein kinase (RTK-MAPK) pathways. These two pathways are detrimental for reproduction in general, and in part for placenta development, as a series of knockout experiments demonstrate. Aspects of each pathway are also implicated to be involved in trophoblast invasion, e.g. STAT3 is constitutively activated in invasive first trimester trophoblast cells, and activated ERK is detectable in intermediate trophoblast cells, an invasive phenotype. Interaction at several intersection points between the pathways has been described in several cell systems so that the same would seem to be possible in trophoblast cells. In this review, some of the possible areas of interaction are alluded to.

Pregnancy is accompanied by a Th2-prone immune modulation, which is a major puzzle piece among maternofetal tolerance-promoting factors. A large number of cytokines is physiologically or pathologically present in the decidua and is potentially able to act on lymphocytes and NK cells, which express a variety of respective receptors. Intracellular signals from these receptors are to a major part transduced via the Janus kinases (JAK) and signal transducers and activators of a transcription (STAT) system, which consists of at least 4 different kinases and 7 STATs plus several subtypes and splicing variants. A network of suppressors of cytokine signaling (SOCS) controls their balance. The interactions of all these intracellular factors and cross-linking with further signaling systems seem to be crucial for the maintenance of a maternal cytokine profile which promotes the tolerance of the fetus.

Human leukocyte antigen-G (HLA-G) plays a major role in escape of trophoblast cells from maternal cytotoxicity. Unless its malignant transformation, Jeg-3 choriocarcinoma cell line maintained the capacity of HLA-G production. For the analysis of function and mechanisms of HLA-G-induced immune regulation, a human cellular model with suppressed HLA-G would be very helpful. RNA interference (RNAi) is a very elegant method for this approach, but the design of appropriate oligonucleotide sequences may provide difficulties. We designed oligonucleotides to interfere exclusively with HLA-G mRNA, which were applied at different concentrations to 50% confluent Jeg-3 cells. After 36 hr, the HLA-G content in Jeg-3 cells was analyzed by Western blots. Applying the described RNAi method and oligonucleotides the cellular content of HLA-G was dose-dependently reduced as assessed in several independent Western blots. This method provides a tool for extensive investigation of HLA-G functions in vitro and if vivo.

Background: Signal Transducer and Activator of Transcription 3 (STAT3) is an intracellular signalling molecule, which is used by several cytokines, including leukemia inhibitory factor (LIF), epithelial growth factor (EGF), and interleukin-6 (IL-6). It induces a variety of gene transcripts and cell functions. In trophoblast cells and in tumor cells, its tyrosine phosphorylation is directly linked to their invasiveness. The regulation and function of STAT3 serine phosphorylation is still widely unclear. Material and Methods: Jeg-3 choriocarcinoma cells were stimulated with different concentrations of EGF, IL-6 and LIF. STAT3 serine (727) and tyrosine (705) phosphorylation were analyzed 5–60 min after stimulation by SDS-PAGE electrophoresis followed by Western blotting. Results: Jeg-3 cells display spontaneous STAT3 serine phosphorylation. 100 ng/mL EGF induces a time-dependent reduction starting 15 min after stimulation. Tyrosine phosphorylation does not occur spontaneously, but is strongly induced by EGF at all analyzed time points. LIF induces tyrosine phosphorylation, but affects serine phosphorylation only very slightly. IL-6 did not influence neither serine phosphorylation nor tyrosine phosphorylation. Discussion: The EGF induced STAT3 tyrosine phosphorylation may be responsible for its invasion triggering capacities. The parallel reduction of serine phosphorylation may enhance this effect. LIF was formerly shown to enhance trophoblast invasion via STAT3 tyrosine phosphorylation. IL-6 displays very little effects on STAT3 and seems to use other pathways for signalling.