Isabella Ellinger

IGG TRANSPORT ACROSS TROPHOBLAST-DERIVED BEWO CELLS : A MODEL SYSTEM TO STUDY IGG TRANSPORT IN THE PLACENTA

In primates, prenatal transfer of IgG from mother to offspring occurs predominantly across the placenta. Although a number of Fcγ‐receptors and IgG binding proteins have been detected in human placental tissue, an involvement of any of these receptors in IgG transport across the syncytiotrophoblast remains to be demonstrated. Therefore, we investigated the mechanism of IgG transcytosis in trophoblast‐derived BeWo cells. BeWo cells were not only found to express the MHC class I‐related IgG Fc receptor, human FcRn, but also specifically bound fluorescein isothiocyanate (FITC)‐labeled human IgG (FITC‐hIgG) at the apical surface at mildly acidic pH. The cells preferentially transcytosed FITC‐hIgG from the apical to the basolateral side when compared to the fluid‐phase marker FITC‐dextran and to FITC‐hIgG transcytosis in the opposite direction. However, endocytosis of FITC‐hIgG at the apical plasma membrane at physiological pH required the continuous presence of FITC‐hIgG at concentrations similar to those present in the maternal circulation. These results suggest a mechanism by which IgG is internalized by BeWo cells via fluid‐phase endocytosis. Tight binding of IgG to hFcRn may then occur in acidic endosomes, followed by selective sorting into the transcytotic pathway. Thus, the main function of this receptor is to prevent entry of IgG into the degradative pathway in lysosomes.

Endocytic and Transcytotic Processes in Villous Syncytiotrophoblast: Role in Nutrient Transport to the Human Fetus

The supply of nutrients to the developing fetus is a major function of the human hemochorial placenta, a placenta type in which the fetal chorion is in direct contact with the maternal blood. At term, nutrients have to be transported across two cell layers in chorionic villi, the syncytiotrophoblast (STB) and fetal endothelial cells. The STB is a continuous syncytium covering the entire surface of chorionic villi. This polarized epithelium is specialized in exchange processes and membrane trafficking between the apical membrane facing the maternal blood and the basal membrane facing the fetal endothelium. To meet placental and fetal requirements, the STB selectively takes up and transports a variety of nutrients, hormones, growth factors and cytokines and also transfers passive immunity to the fetus by receptor‐mediated transcytosis. In this review in vivo and in vitro systems currently used to study STB functions are discussed and the potential mechanisms of transplacental IgG, iron, lipoprotein and glucose transport are presented. As revealed in this article, the placenta is a tissue where intensive cell biological research is required to unravel endocytic trafficking pathways in a highly specialized cell such as the STB.

The analysis of organic anion transporting polypeptide (OATP) mRNA and protein patterns in primary and metastatic liver cancer.

Organic anion transporting polypeptides (OATP, SLCO genes) mediate the uptake of endobiotics and drugs. Thus, their expression levels and pattern could be of relevance for cancer therapy. This prompted us to investigate the expression of poorly characterized OATPs, namely OATP2A1, OATP3A1, OATP4A1 and OATP5A1 in hepatic cancer of different origin. First, mRNA levels of all eleven OATPs were determined in paired (cancerous and adjacent non-cancerous) specimens from 43 patients with primary liver cancer (hepatocellular carcinoma, HCC; cholangiocellular carcinoma, CCC) and liver metastases from colon tumors (MLT). Real-time RT-PCR analysis revealed that all OATPs, except OATP1C1 and OATP6A1, are extensively expressed in nearly all samples. In contrast to downregulated OATP1B1, OATP1B3, OATP1A2 and OATP2B1 in cancerous vs. non-cancerous samples, an increase in OATP2A1, OATP3A1, OATP4A1 and OATP5A1 mRNA levels was seen in tumors (up to 40-fold for OATP5A1 in the MLT group). Therefore, OATP2A1, OATP3A1, OATP4A1 and OATP5A1 were further investigated by immunofluorescence microscopy on paraffin-embedded cancerous and non-cancerous sections (seven per group). OATP-derived immunoreactivity was observed in plasma membranes and cytosol of hepatic tumor cells, and additionally, in various cytokeratin 19 positive bile ducts. An increased percentage of immunoreactive cells and a higher staining intensity in cancerous vs. non-cancerous paraffin sections paralleled higher mRNA levels of OATP2A1, OATP3A1, OATP4A1 and OATP5A1 in cancerous tissues of HCC, CCC and MLT patients. The extensive expression of OATP2A1, OATP3A1, OATP4A1 and OATP5A1 in hepatic tumors of different origin suggests that these transporters might be further exploited for the discovery of novel anticancer agents.

Placental Alkaline Phosphatase Expression at the Apical and Basal Plasma Membrane in Term Villous Trophoblasts

Human placental alkaline phosphatase (PLAP) was localized at the apical and basal plasma membrane of syncytiotrophoblasts and at the surface of cytotrophoblasts in term chorionic villi using immunoelectron microscopy. Similarly, apical and basolateral PLAP expression was found in polarized trophoblast-derived BeWo cells. Trophoblasts isolated from term placentas exhibited mainly vesicular PLAP immunofluorescence staining immediately after isolation. After in vitro differentiation into syncytia, PLAP plasma membrane expression was upregulated and exceeded that observed in mononuclear trophoblasts. These data call for caution in using PLAP as a morphological marker to differentiate syncytiotrophoblasts from cytotrophoblasts or as a marker enzyme for placental brush-border membranes. (J Histochem Cytochem 49:1155–1164, 2001)

Different temperature sensitivity of endosomes involved in transport to lysosomes and transcytosis in rat hepatocytes: Analysis by free‐flow electrophoresis

Endocytosis at reduced temperature has been used to define and characterize endosome subpopulations. Thus, the temperature sensitivity of endosome subpopulations involved in transport to lysosomes and transcytosis in rat hepatocytes was analyzed applying endosome labeling in the isolated perfused rat liver with route‐specific ligands in combination with temperature shift protocols. Free‐flow electrophoresis (FFE) that separates membranes and organelles based on their surface charge was then applied to isolate functional endosomes. Using asialoorosomucoid (ASOR) and polymeric immunoglobulin A (pIgA) as specific ligands of the lysosomal and transcytotic route, respectively, two distinct endosome subpopulations along either pathway were separated by FFE. Upon a short (1–3 min) internalization at 37°C, 125I‐ASOR and fluorescein isothiocyanate (FITC)‐pIgA were colocalized in common early endosomes. Following a 5–10 min chase of the ligands at 37°C endosomes labeled with 125I‐ASOR were separated from endosomes labeled with FITC‐pIgA, indicative of two distinct late compartments along the lysosomal and transcytotic route. Internalization at 16°C resulted in accumulation of both ligands in common early endosomes and, consequently, in inhibition of transport to lysosomes and transcytosis. When 125I‐ASOR or 125I‐pIgA were first chased into late compartments at 37°C and the temperature was subsequently lowered to 16°C, biliary secretion of 125I‐ASOR‐derived counts was arrested, while biliary output of 125I‐pIgA continued. In summary, ASOR en route to lysosomes can be blocked in early as well as in late endosomes at 16°C, while biliary secretion of pIgA cannot be prevented by temperature reduction once the ligand had been transferred from early to late compartments.

The Presence and Localization of Melatonin Receptors in the Rat Aorta

Melatonin is involved in blood pressure modulation in rats and humans. Some of the effects of melatonin are presumably mediated via two G-protein-coupled receptors (MT1 and MT2), but the distribution of MT1 and MT2 in the cardiovascular system remains to be explored comprehensively. We investigated the expression of both the receptors in the rat aorta on mRNA level by RT-PCR and real time RT-PCR as well as on protein level via western blotting and immunofluorescence microscopy. We verified MT1 mRNA expression in the rat aorta and demonstrated the absence of MT2 mRNA in this vessel type. MT1 receptors were confirmed also at the protein level, and surprisingly they were preferentially localized to the tunica adventitia. Since no daily changes in MT1 mRNA expression were detected, we suppose that the circadian changes in circulating melatonin concentrations are sufficient to mediate circadian effects of melatonin in the aorta. The localization of MT1 in the tunica adventitia suggests an influence of melatonin on vasa vasorum function and signal transduction in the aorta wall.

Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters.

BACKGROUND The capacity of the human placenta to handle exogenous stressors is poorly understood. The heavy metal mercury is well-known to pass the placenta and to affect brain development. An active transport across the placenta has been assumed. The underlying mechanisms however are virtually unknown. OBJECTIVES Uptake and efflux transporters (17 candidate proteins) assumed to play a key role in placental mercury transfer were examined for expression, localization and function in human primary trophoblast cells and the trophoblast-derived choriocarcinoma cell line BeWo. METHODS To prove involvement of the transporters, we used small interfering RNA (siRNA) and exposed cells to methylmercury (MeHg). Total mercury contents of cells were analyzed by Cold vapor-atomic fluorescence spectrometry (CV-AFS). Localization of the proteins in human term placenta sections was determined via immunofluorescence microscopy. RESULTS We found the amino acid transporter subunits L-type amino acid transporter (LAT)1 and rBAT (related to b(0,+) type amino acid transporter) as well as the efflux transporter multidrug resistance associated protein (MRP)1 to be involved in mercury kinetics of trophoblast cells (t-test P<0.05). CONCLUSION The amino acid transporters located at the apical side of the syncytiotrophoblast (STB) manage uptake of MeHg. Mercury conjugated to glutathione (GSH) is effluxed via MRP1 localized to the basal side of the STB. The findings can well explain why mercury is transported primarily towards the fetal side.

Functional expression of the human neonatal Fc-receptor, hFcRn, in isolated cultured human syncytiotrophoblasts.

Materno-fetal IgG transfer in the mature human placenta involves transport across the syncytiotrophoblast (STB) and fetal endothelial cell layer. The MHC class I-related Fc gamma-receptor (hFcRn) localized in STB as well as in endothelial cells is involved in overall IgG transfer from the maternal into the fetal circulation. Functional hFcRn is a heterodimer of a transmembrane alpha-chain and beta2-microglobulin. To establish the basis for future studies to unravel the mechanism of IgG transport in STB, we investigated hFcRn alpha-chain and beta2-microglobulin expression in cytotrophoblasts (CTB) isolated from human term placentae and cultured in vitro under conditions where differentiation into multinuclear STB takes place (>or=48 h). Northern blot analysis demonstrated up-regulation of alpha-chain mRNA after 48 h of in vitro cultivation. Likewise, hFcRn alpha-chain and beta2-microglobulin were at the limit of detection by immunofluorescence microscopy in CTB immediately after isolation, but their expression increased upon STB formation. hFcRn alpha-chain co-localized with beta2-microglobulin in multinuclear STB and formed a functional, i.e. low pH IgG binding, receptor as shown by affinity isolation. The in vitro differentiated STB exhibited specific, low pH-dependent IgG binding to the plasma membrane. In conclusion, these cultures can now be applied to study the role of hFcRn in IgG transport and trafficking in STB cultures in vitro.

Genetics of the human placenta: implications for toxicokinetics

Exposure to chemicals and environmental pollutants among them cadmium, lead, and mercury can harm reproduction. The metals cross the placenta, accumulate in placental tissue, and pass onto fetal blood and fetal organs to variable amounts. Still, the mechanisms underlying their transplacental passage are largely unknown and the human placenta is the most poorly understood organ in terms of reproduction toxicology. The genetic factors modulating placental toxicokinetics remain unclear just as well. From a genetic perspective, three aspects, which influence capacities of the human placenta to metabolize and transport toxicants, need to be considered. These are 1/presence and interplay of two genotypes, 2/chromosomal aberrations including aneuploidies and sequence variations, and 3/epigenetics and genetic imprinting. In this review, we summarize the current state of knowledge on how genetics and epigenetics affect placental (patho)physiology and thus fetal development and health.

Receptor-Mediated and Fluid-Phase Transcytosis of Horseradish Peroxidase across Rat Hepatocytes

Horseradish peroxidase (HRP) is often used as a fluid-phase marker to characterize endocytic and transcytotic processes. Likewise, it has been applied to investigate the mechanisms of biliary secretion of fluid in rat liver hepatocytes. However, HRP contains mannose residues and thus binds to mannose receptors (MRs) on liver cells, including hepatocytes. To study the role of MR-mediated endocytosis of HRP transport in hepatocytes, we determined the influence of the oligosaccharid mannan on HRP biliary secretion in the isolated perfused rat liver. A 1-minute pulse of HRP was applied followed by marker-free perfusion. HRP appeared in bile with biphasic kinetics: a first peak at 7 minutes and a second peak at 15 minutes after labeling. Perfusion with 0.8 mg/mL HRP in the presence of a twofold excess of mannan reduced the first peak by 41% without effect on the second one. Together with recently published data on MR expression in rat hepatocytes this demonstrates two different mechanisms for HRP transcytosis: a rapid, receptor-mediated transport and a slower fluid-phase transport.