Expression of the neonatal Fc-receptor in placental-fetal endothelium and in cells of the placental immune system.

INTRODUCTION: Starting from the second trimester of pregnancy, passive immunity is provided to the human fetus by transplacental transfer of maternal IgG. IgG transfer depends on the neonatal Fc receptor, FcRn. While FcRn localization in the placental syncytiotrophoblast (STB) has been demonstrated unequivocally, FcRn expression in placental-fetal endothelial cells (pFECs), which are part of the materno-fetal barrier, is still unclear. Therefore, this study aimed to elucidate the spatio-specific expression pattern of FcRn in placental tissue. METHODS: FcRn expression was investigated by western blotting in term placentas and in isolated human placental arterial and venous endothelial cells (HPAEC, HPVEC) using a validated affinity-purified polyclonal anti-peptide antibody against the cytoplasmic tail of FcRn α-chain. In situ localization of FcRn and IgG was studied by immunofluorescence microscopy on tissue sections of healthy term placentas. RESULTS: FcRn expression was demonstrated in placental vasculature particularly, in HPAEC, and HPVEC. FcRn was localized in cytokeratin 7+ STB and in CD31+ pFECs in terminal as well as stem villi in situ. Additionally, CD68+ placental macrophages exhibited FcRn expression in situ. Endogenous IgG partially co-localized with FcRn in STB, pFECs, and in placental macrophages. DISCUSSION: Placental FcRn expression in endothelial cells and macrophages is analogous to the expression pattern in other organs. FcRn expression in pFECs suggests an involvement of FcRn in IgG transcytosis and/or participation in recycling/salvaging of maternal IgG present in the fetal circulation. FcRn expression in placental macrophages may account for recycling of monomeric IgG and/or processing and presentation of immune complexes.

ICAM-1 Binding Rhinoviruses Enter HeLa Cells via Multiple Pathways and Travel to Distinct Intracellular Compartments for Uncoating.

Of the more than 150 human rhinovirus (RV) serotypes, 89 utilize intercellular adhesion molecule-1 (ICAM-1) for cell entry. These belong either to species A or B. We recently demonstrated that RV-B14 and RV-A89, despite binding this same receptor, are routed into distinct endosomal compartments for release of their RNA into the cytosol. To gain insight into the underlying mechanism we now comparatively investigate the port of entry, temperature-dependence of uncoating, and intracellular routing of RV-B3, RV-B14, RV-A16, and RV-A89 in HeLa cells. The effect of various drugs blocking distinct stages on the individual pathways was determined via comparing the number of infected cells in a TissueFaxs instrument. We found that RV-B14 and RV-A89 enter via clathrin-, dynamin-, and cholesterol-dependent pathways, as well as by macropinocytosis. Drugs interfering with actin function similarly blocked entry of all four viruses, indicating their dependence on a dynamic actin network. However, uniquely, RV-A89 was able to produce progeny when internalized at 20 °C followed by neutralizing the endosomal pH and further incubation at 37 °C. Blocking dynein-dependent endosomal transport prevented uncoating of RV-A16 and RV-A89, but not of RV-B3 and RV-B14, indicative for routing of RV-A16 and RV-A89 into the endocytic recycling compartment for uncoating. Our results call for caution when developing drugs aimed at targeting entry or intracellular trafficking of all rhinovirus serotypes.

Mechanism of human rhinovirus infections.

About 150 human rhinovirus serotypes are responsible for more than 50 % of recurrent upper respiratory infections. Despite having similar 3D structures, some bind members of the low-density lipoprotein receptor family, some ICAM-1, and some use CDHR3 for host cell infection. This is also reflected in the pathways exploited for cellular entry. We found that even rhinovirus serotypes binding the same receptor can travel along different endocytic pathways and release their RNA genome into the cytosol at different locations. How this may account for distinct immune responses elicited by various rhinoviruses and the observed symptoms of the common cold is briefly discussed.

ICAM-1 Binding Rhinoviruses A89 and B14 Uncoat in Different Endosomal Compartments.

UNLABELLED: Human rhinovirus A89 (HRV-A89) and HRV-B14 bind to and are internalized by intercellular adhesion molecule 1 (ICAM-1); as demonstrated earlier, the RNA genome of HRV-B14 penetrates into the cytoplasm from endosomal compartments of the lysosomal pathway. Here, we show by immunofluorescence microscopy that HRV-A89 but not HRV-B14 colocalizes with transferrin in the endocytic recycling compartment (ERC). Applying drugs differentially interfering with endosomal recycling and with the pathway to lysosomes, we demonstrate that these two major-group HRVs productively uncoat in distinct endosomal compartments. Overexpression of constitutively active (Rab11-GTP) and dominant negative (Rab11-GDP) mutants revealed that uncoating of HRV-A89 depends on functional Rab11. Thus, two ICAM-1 binding HRVs are routed into distinct endosomal compartments for productive uncoating. IMPORTANCE: Based on similarity of their RNA genomic sequences, the more than 150 currently known common cold virus serotypes were classified as species A, B, and C. The majority of HRV-A viruses and all HRV-B viruses use ICAM-1 for cell attachment and entry. Our results highlight important differences of two ICAM-1 binding HRVs with respect to their intracellular trafficking and productive uncoating; they demonstrate that serotypes belonging to species A and B, but entering the cell via the same receptors, direct the endocytosis machinery to ferry them along distinct pathways toward different endocytic compartments for uncoating.

Localization of the human neonatal Fc receptor (FcRn) in human nasal epithelium.

The airway epithelium is a central player in the defense against pathogens including efficient mucociliary clearance and secretion of immunoglobulins, mainly polymeric IgA, but also IgG. Pulmonary administration of therapeutic antibodies on one hand, and intranasal immunization on the other, are powerful tools to treat airway infections. In either case, the airway epithelium is the primary site of antibody transfer. In various epithelia, bi-polar transcytosis of IgG and IgG immune complexes is mediated by the human neonatal Fc receptor, FcRn, but FcRn expression in the nasal epithelium had not been demonstrated, so far. We prepared affinity-purified antibodies against FcRn α-chain and confirmed their specificity by Western blotting and immunofluorescence microscopy. These antibodies were used to study the localization of FcRn α-chain in fixed nasal tissue. We here demonstrate for the first time that ciliated epithelial cells, basal cells, gland cells, and endothelial cells in the underlying connective tissue express the receptor. A predominant basolateral steady state distribution of the receptor was observed in ciliated epithelial as well as in gland cells. Co-localization of FcRn α-chain with IgG or with early sorting endosomes (EEA1-positive) but not with late endosomes/lysosomes (LAMP-2-positive) in ciliated cells was observed. This is indicative for the presence of the receptor in the recycling/transcytotic pathway but not in compartments involved in lysosomal degradation supporting the role of FcRn in IgG transcytosis in the nasal epithelium.

Release of Vesicular Stomatitis Virus Spike Protein G-Pseudotyped Lentivirus from the Host Cell Is Impaired upon Low-Density Lipoprotein Receptor Overexpression.

Production of a vesicular stomatitis virus spike protein G (VSVG)-pseudotyped lentiviral expression vector in HEK293 cells decreased on overexpression of low-density lipoprotein receptor (LDLR) but not that of ICAM1 or TfR1. Reverse transcription-quantitative PCR (RT-qPCR) revealed a reduction in vector RNA as a function of LDLR expression. Decreased syncytium formation suggested diminished surface expression of VSVG. Intracellular VSVG granules colocalized with LDLR, ER-Golgi intermediate compartment protein 53 (ERGIC53), LAMP2, and vimentin but not with GM130 or calnexin, suggesting that VSVG interacts with LDLR within the ERGIC, resulting in rerouting into the aggresome/autophagosome pathway.

Role of endocytic uptake in transfection efficiency of solid lipid nanoparticles-based nonviral vectors.

BACKGROUND: As has been shown for different vector systems, the entry pathway(s) impacts upon the transfection efficiency. The present study aimed to explore the cellular uptake mechanisms of three different vectors based on solid lipid nanoparticles (SLN) in HeLa cells. The use of endocytosis inhibitors that affect specific internalization pathways provides a tool for the study of these routes. METHODS: We prepared three vectors based on solid lipid nanoparticles: without protamine, with protamine, and with protamine and dextran. Uptake, percentage of transfected HeLa cells and enhanced green fluorescent protein (EGFP) production were all analyzed in the presence or absence of different endocytosis inhibitors. In addition, co-localization studies using lysosomal markers were carried out to determine the influence of the trafficking to late endosomal compartments on the transfection capacity of the vectors. RESULTS: Uptake and transfection of each vector was affected differently by each endocytosis inhibitor. Ethylisopropylamiloride (EIPA) did not affect uptake of the DNA-SLN vector, whereas all of the inhibitors affected transfection. In the case of protamine-DNA-SLN and dextran-protamine-DNA-SLN vectors, EIPA affected uptake and dynasore did not decrease transfection. CONCLUSIONS: DNA-SLN vector appear to enter productively by multiple pathways in HeLa cells. By contrast, dynamin does not appear to be essential in the productive entry of protamine-containing vectors. In addition, enhancement of the macropinocytic route increases EGFP production when dextran is added to the vector.

Productive entry pathways of human rhinoviruses.

Currently, complete or partial genome sequences of more than 150 human rhinovirus (HRV) isolates are known. Twelve species A use members of the low-density lipoprotein receptor family for cell entry, whereas the remaining HRV-A and all HRV-B bind ICAM-1. HRV-Cs exploit an unknown receptor. At least all A and B type viruses depend on receptor-mediated endocytosis for infection. In HeLa cells, they are internalized mainly by a clathrin- and dynamin-dependent mechanism. Upon uptake into acidic compartments, the icosahedral HRV capsid expands by ~4% and holes open at the 2-fold axes, close to the pseudo-3-fold axes and at the base of the star-shaped dome protruding at the vertices. RNA-protein interactions are broken and new ones are established, the small internal myristoylated capsid protein VP4 is expelled, and amphipathic N-terminal sequences of VP1 become exposed. The now hydrophobic subviral particle attaches to the inner surface of endosomes and transfers its genomic (+) ssRNA into the cytosol. The RNA leaves the virus starting with the poly(A) tail at its 3'-end and passes through a membrane pore contiguous with one of the holes in the capsid wall. Alternatively, the endosome is disrupted and the RNA freely diffuses into the cytoplasm.

HFcRn-mediated transplacental immunoglobulin G transport: protection of and threat to the human fetus and newborn.

In human newborns, endogenous levels of plasma immunoglobulin G (IgG) begin to rise slowly after birth following exposure to the environment. For immunoprotection during fetal and early neonatal life, maternal IgG is provided by transplacental transport. While cellular immunoprotective IgG effects are mainly triggered by FcγRI, -RII and -RIII, transplacental IgG transfer is mediated by the MHC class I-like neonatal Fc-receptor, hFcRn. This compact review explains the mechanism of hFcRn-mediated IgG transcytosis across the placental barrier - syncytiotrophoblast and fetal endothelial cells. Restrictions of this IgG transport are summarized. These include IgG subclass discrimination and limited IgG transport before the third trimester that can cause insufficient protection from infections of preterm (≤ 35 th week) delivered babies. As hFcRn does not discriminate beneficial from hazardous IgGs, maternal auto- and alloimmune as well as therapeutic antibodies can reach the fetus. The consequences including severe diseases of the newborn are summarized in this article.

Entry of a heparan sulphate-binding HRV8 variant strictly depends on dynamin but not on clathrin, caveolin, and flotillin.

The major group human rhinovirus type 8 can enter cells via heparan sulphate. When internalized into ICAM-1 negative rhabdomyosarcoma (RD) cells, HRV8 accumulated in the cells but caused CPE only after 3 days when used at high MOI. Adaptation by three blind passages alternating between RD and HeLa cells resulted in variant HRV8v with decreased stability at acidic pH allowing for productive infection in the absence of ICAM-1. HRV8v produced CPE at 10 times lower MOI within 1 day. Confocal fluorescence microscopy colocalization and the use of pharmacological and dominant negative inhibitors revealed that viral uptake is clathrin, caveolin, and flotillin independent. However, it is blocked by dynasore, amiloride, and EIPA. Furthermore, HRV8v induced FITC-dextran uptake and colocalized with this fluid phase marker. Except for the complete inhibition by dynasore, the entry pathway of HRV8v via HS is similar to that of HRV14 in RD cells that overexpress ICAM-1.

Uncoating of human rhinoviruses.

Human rhinoviruses (HRVs) are a major cause of the common cold. The more than one hundred serotypes, divided into species HRV-A and HRV-B, either bind intercellular adhesion molecule 1 (major group viruses) or members of the low-density lipoprotein receptor (minor group viruses) for cell entry. Some major group HRVs can also access the host cell via heparan sulphate proteoglycans. The cell attachment protein(s) of the recently discovered phylogenetic clade HRV-C is unknown. The respective receptors direct virus uptake via clathrin-dependent or independent endocytosis or via macropinocytosis. Triggered by ICAM-1 and/or the low pH environment in endosomes the virions undergo conformational alterations giving rise to hydrophobic subviral particles. These are handed over from the receptors to the endosomal membrane. According to the current view, the RNA genome is released through an opening at one of the fivefold axes of the icosahedral capsid and crosses the membrane through a pore presumably formed by viral proteins. Alternatively, the membrane may be ruptured allowing subviral particles and RNA to enter the cytosol. Whether a channel is formed or the membrane is disrupted most probably depends on the respective HRV receptor.

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.

Human rhinovirus 14 enters rhabdomyosarcoma cells expressing icam-1 by a clathrin-, caveolin-, and flotillin-independent pathway.

Intercellular adhesion molecule 1 (ICAM-1) mediates binding and entry of major group human rhinoviruses (HRVs). Whereas the entry pathway of minor group HRVs has been studied in detail and is comparatively well understood, the pathway taken by major group HRVs is largely unknown. Use of immunofluorescence microscopy, colocalization with specific endocytic markers, dominant negative mutants, and pharmacological inhibitors allowed us to demonstrate that the major group virus HRV14 enters rhabdomyosarcoma cells transfected to express human ICAM-1 in a clathrin-, caveolin-, and flotillin-independent manner. Electron microscopy revealed that many virions accumulated in long tubular structures, easily distinguishable from clathrin-coated pits and caveolae. Virus entry was strongly sensitive to the Na(+)/H(+) ion exchange inhibitor amiloride and moderately sensitive to cytochalasin D. Thus, cellular uptake of HRV14 occurs via a pathway exhibiting some, but not all, characteristics of macropinocytosis and is similar to that recently described for adenovirus 3 entry via alpha(v) integrin/CD46 in HeLa cells.

Low pH-triggered beta-propeller switch of the low-density lipoprotein receptor assists rhinovirus infection.

Minor group human rhinoviruses (HRVs) bind three members of the low-density lipoprotein receptor (LDLR) family: LDLR proper, very-LDLR (VLDLR) and LDLR-related protein (LRP). Whereas ICAM-1, the receptor of major group HRVs actively contributes to viral uncoating, LDLRs are rather considered passive vehicles for cargo delivery to the low-pH environment of endosomes. Since the Tyr-Trp-Thr-Asp beta-propeller domain of LDLR has been shown to be involved in the dissociation of bound LDL via intramolecular competition at low pH, we studied whether it also plays a role in HRV infection. Human cell lines deficient in LDLR family proteins are not available. Therefore, we used CHO-ldla7 cells that lack endogenous LDLR. These were stably transfected to express either wild-type (wt) human LDLR or a mutant with a deletion of the beta-propeller. When HRV2 was attached to the propeller-negative LDLR, a lower pH was required for conversion to subviral particles than when attached to wt LDLR. This indicates that high-avidity receptor binding maintains the virus in its native conformation. HRV2 internalization directed the mutant LDLR but not wt LDLR to lysosomes, resulting in reduced plasma membrane expression of propeller-negative LDLR. Infection assays using a CHO-adapted HRV2 variant showed a delay in intracellular viral conversion and de novo viral synthesis in cells expressing the truncated LDLR. Our data indicate that the beta-propeller attenuates the virus-stabilizing effect of LDLR binding and thereby facilitates RNA release from endosomes, resulting in the enhancement of infection. This is a nice example of a virus exploiting high-avidity multimodule receptor binding with an intrinsic release mechanism.

Human rhinovirus type 2 uncoating at the plasma membrane is not affected by a pH gradient but is affected by the membrane potential.

The minor receptor group human rhinovirus type 2 enters host cells by endocytosis via members of the low-density-lipoprotein receptor family. In late endosomes, it undergoes a conformational change solely induced by a pH of < or =5.6, resulting in RNA transfer across the endosomal membrane into the cytoplasm. To determine potential driving forces of this process, we investigated whether RNA penetration might depend on the pH gradient and/or the membrane potential between the acidic endosome lumen and the neutral cytoplasm. Since these parameters are difficult to assess in endosomes, we took advantage of the possibility of inducing structural changes, RNA release, and consequently infection from the plasma membrane. To manipulate the pH gradient, cell-bound virus was exposed to membrane-permeant or -impermeant acidic buffers at 4 degrees C, and this was followed by a shift to 34 degrees C in medium containing bafilomycin to prevent RNA release from endosomes. To manipulate the plasma membrane potential, similar experiments were carried out, but these included K(+) diffusion potentials in the presence of the K(+) ionophore valinomycin. We demonstrated that infection does not depend on a pH gradient but is enhanced by plasma membrane hyperpolarization compared to plasma membrane depolarization.

Site of human rhinovirus RNA uncoating revealed by fluorescent in situ hybridization.

By using fluorescent in situ hybridization (FISH), we visualized viral RNA of human rhinovirus type 2 (HRV2) during its entry into HeLa cells. RNA uncoating of HRV2 is entirely dependent on low endosomal pH (< or =5.6). When internalized into cells treated with bafilomycin, which results in neutralization of the endosomal pH, no FISH signal was recorded, whereas in the absence of the drug, fluorescent dots were seen. Therefore, FISH detects the genomic viral RNA only upon its release from the capsid. Free viral RNA was first seen at 10 min postinfection (p.i.) in the perinuclear area of the cell, which is indicative of RNA release in/from late endosomal compartments. Pulse-chase experiments and observation of HRV2 RNA and capsid proteins via microscopy, Western blotting, and reverse transcription-PCR revealed that the RNA signal persisted whereas the protein signal disappeared. This demonstrates transport of capsids to lysosomes and degradation. In contrast, viral RNA that had already been transferred into the cytoplasm escaped lysosomal breakdown as indicated by a persistent FISH signal. Taken together, our results demonstrate by direct means RNA arrival in the cytosol within 10 min p.i. Based on persistence of the FISH signal and productive infection in the presence of the microtubule-depolymerizing drug nocodazole, we localized this process to endosomal carrier vesicles/late endosomes.

Biliary secretion of fluid phase markers is modified under post-cholestatic conditions.

Hepatocytes take up macromolecules from the circulation by receptor-mediated and/or fluid-phase endocytosis. These molecules are either selectively or nonspecifically transported through the cell (transcytosis) and are subsequently secreted into bile. As transcytosis of diverse fluid-phase markers (FPM) is still poorly characterized, biliary secretion of two FPMs (horseradish peroxidase (HRP), FITC-Dextran) was studied in the isolated perfused rat liver following short-term (1 min) single-pulse administration. HRP was secreted into bile with a fast (5 min) and slow (15 min) transit time, while FITC-dextran appeared in bile in a single peak at 7 min. Short-time reversible cholestasis, induced by bile duct ligation (BDL), had been shown to affect HRP secretion. Here, we compare the influence of 2 h BDL on post-cholestatic biliary secretion of HRP and FITC-dextran. BDL drastically stimulated the fast component of HRP secretion into bile, but had an effect neither on the second HRP peak nor on the appearance of FITC-dextran in bile. Perfusion at low temperature (16 degrees C) under control and post-cholestatic conditions suppressed both, the second HRP peak and the appearance of FITC-dextran in bile, but uptake of FPM by endocytosis was not inhibited as the markers were secreted upon re-warming to 37 degrees C. In addition, perfusion at low temperature under control and post-cholestatic conditions delayed the appearance of the fast HRP peak in bile and it abrogated the stimulating effect of BDL on the first HRP peak. These data indicate that BDL boosts HRP secretion along a temperature-sensitive transcellular pathway and/or a paracellular route. This fast route is taken only by HRP but not by FITC-dextran, the latter being exclusively transported by a transcellular route under all conditions investigated.

Histochemistry for placenta research: theory and application.

Histochemical techniques have contributed significantly to advances in placental biology and cell biology. In this mini-review, we describe recent advances in histochemical technologies and show how these technologies can profoundly improve our understanding of placenta morphological function related to health and disease. Fundamental theories and applications of five separate methods discussed here are 1) tissue-based polymerase chain reaction by laser microdissection, 2) a novel antigen retrieval method using citraconic anhydride plus heating, 3) immunohistochemical detection of Lewis-related antigen expression and galectin-1 binding in the human placenta, 4) confocal microscopy analysis of IgG transport in placental trophoblasts, and 5) high-resolution immunofluorescence and correlative microscopy using ultrathin cryosections in placental research. This review article is based on a presentation given in a workshop entitled Histochemistry: Theory and Application at the 12th International Federation of Placenta Associations Meeting held in Kobe, Japan, on September 9, 2006.