Placental hFcRn/beta-2-microglobulin (b2m) expression

Research project P 14079 Placental hFcRn/beta-2-microglobulin (b2m) expression Renate FUCHS 24.01.2000 Immunity of the newborn human during the first weeks of life is provided by maternal IgG antibodies. During pregnancy fetal IgG levels steadily increase and exceed that in the maternal circulation at term. IgG transfer from the mother to the fetus is thought to be mediated by the epithelial IgG Fc receptor (hFcRn) present in the first cellular barrier, the syncytiotrophoblast (STB). At term, the ST13 is in direct contact with the fetal capillary endothelium that further transport IgG into the fetal circulation. In the immature placenta, IgG has to be transported through the underlaying cytotrophoblast (CTB) cell layer also. It is unknown, however, whether CTB are already endowed with functional hFcRn [i.e. hFcRn heavy chain associated with b2-microglobulin (b2m)] and therefore allow for IgG transport in the Ist trimester. Possibly, low hFcRn expression in CTI3 could be upregulated upon differentiation and fusion of CTB into STB with gestational age of the placenta. In support of this hypothesis, our recent data show that hFcRn protein is expressed to a much higher -extent (based on equal amount of cellular protein) in human term placental villous tissue than in human trophoblastderived BeWo cells. hFcRn isolated from placental tissue and BeWo cells has been shown to be associated with b2m. However, data on b2m protein localization in villous STB are conflicting and in situ hybridization for b2m mRNA has not been carried out. It,remains therefore undetermined whether b2m is synthesized by STB or taken up by endocytosis from the matemal blood and subsequently associate with hFcRn heavy chain. Furthermore, the requirement of b2in for hFcRn biosynthesis, transport to the plasma membrane and hFcRn-mediated IgG transport has not been demonstrated. The proposed project will focus on the comparative analysis of hFcRn expression in CTB and STB in first and third trimester placentae, since these placentae can be easily obtained in sufficient quantities from local hospitals. Based on the present knowledge of the rate of IgG transport across the placenta, one might assume that total hFcRn is upregulated during gestation as well as during differentiation and syncytiurn formation of CTB. The suggested importance of b2m in hFcRn expression and function in the placenta will be evaluated by correlating the expression of hFcRn and b2m mRNAs and protein(s) using in situ and in vitro techniques. Finally, the role of b2m in hFcRn biosynthesis and IgG transport will be investigated in transfected cell lines devoid of endogenous b2m and hFcRn. The clinical significances of the transplacental IgG transport include many aspects that range from immunoprotection in utero to transfer of IgG that is directed against fetal antigens e.g. in rhesus imcompatibility or during maternal autoimmune diseases. Thus, understanding the function of the placental IgG receptor may open new avenues in vaccine design and drug delivery.

In this project the cellular mechanisms involved in transplacental IgG transport were investigated. The human fetus and neonate is incapable of synthesis of its own immunoglobulin G (IgG) antibodies and therefore depends on maternal IgG that is exclusively acquired via the placenta. During pregnancy fetal IgG levels steadily increase and exceed those in the maternal circulation at term to provide passive immunity to the newborn. IgG transfer from the maternal into the fetal circulation occurs transcellularly through various cellular barriers and is thought to be mediated by a specific IgG transport protein (IgG Fc receptor), hFcRn. Functional hFcRn requires association with another protein, beta2-microglobulin (b2m). We could demonstrate that functional hFcRn is present in all three cell barriers early in pregnancy (1 st trimester): 1. In the outer, continuous cell layer (syncytiotrophoblast; STB) in direct contact with maternal blood, followed by the 2. inner cell layer (cytotrophoblasts; CTB) and 3. fetal blood vessels (endothelial cells). In the mature placenta, there are two cell layers only, STB and fetal vessels, both expressing hFcRn. The concentration of total hFcRn is similar in 1st trimester and in mature placentae. Consequently, increasing IgG transport capacity during pregnancy appears to be due to the increasing surface area of the first layer (STB) and the altered architecture of the tissue. At the ultrastructural level, co-localization of IgG and hFcRn/b2m is found in plasma-membrane derived compartments (= endosomes) charateristic for compartments involved in cellular uptake (endocytosis) and transcellular transport (transcytosis) in STB. IgG is taken up from maternal blood into the cells by unspecific endocytosis and delivered to hFcRn-containing endosomes where binding to the receptor can occur. The IgG-hFcRn complexes are then sorted into transcytotic endosomes for IgG delivery to the opposite membrane (fetal side) as well as into compartment mediating IgG recycling to the same plasma membrane. However, at the high maternal IgG concentration (10 - 20 mg/ml), the majority of IgG is present in compartments involved in transport to lysosomes. Thus, hFcRn in villous STB are presumably saturated which results in IgG transport to lysosomes and its degradation in addition to receptor-mediated transcytosis and recycling of intact IgG. This knowledge, the methodologies and in vitro systems established can be applied to determine the transport efficiency of vaccines and drug-coupled IgGs for immunoprotection and delivery in utero against e.g. human immunodeficiency virus (HIV) infections, toxoplasmosis, malaria and cytomegalovirus infections.