Similar and differential behaviour between the nectin-afadin-ponsin and cadherin-catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells

BACKGROUND: We have recently identified a novel cell-cell adhesion system, named NAP system, which is localized at cadherin-based cell-cell adherens junctions (AJs). The NAP system is composed of at least nectin, afadin and ponsin. Nectin is an immunoglobulin-like cell adhesion molecule. Afadin is an actin filament-binding protein which associates nectin with the actin cytoskeleton. Ponsin is an afadin-binding protein which furthermore binds to vinculin and provides a possible linkage of nectin-afadin to cadherin-catenin through vinculin. We compared here the behaviour of the NAP and cadherin-catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells. RESULTS: At the early stage of the formation of the polarized junctional alignment in MTD-1 A cells, primordial spot-like junctions were formed at the tips of thin cellular protrusions radiating from adjacent cells. Nectin, afadin, ponsin, cadherin and catenin were simultaneously recruited to these junctions. As the cell polarization proceeded, the spot-like junctions were gradually fused to form belt-like AJs where all these proteins were concentrated. The disruption of cell-cell AJs in MDCK cells by culturing at a low Ca2+ concentration caused rapid endocytosis of cadherin, but not that of nectin or afadin. Addition of 12-O-tetradecanoylphorbol-13-acetate to the cells formed a tight junction-like structure where nectin and afadin, but not cadherin, accumulated. CONCLUSION: These results indicate that the NAP and cadherin-catenin systems show similar and differential behaviour during the formation and disruption of the polarized junctional alignment in epithelial cells.     

Matrigel-embedded 3D culture of Huh-7 cells as a hepatocyte-like polarized system to study hepatitis C virus cycle

Hepatocytes are highly polarized cells where intercellular junctions, including tight junctions (TJs), determine the polarity. Recently, the TJ-associated proteins claudin-1 and occludin have been implicated in hepatitis C virus (HCV) entry and spread. Nevertheless, cell line-based experimental systems that exhibit hepatocyte-like polarity and permit robust infection and virion production are not currently available. Thus, we sought to determine whether cell line-based, Matrigel-embedded cultures could be used to study hepatitis C virus (HCV) infection and virion production in a context of hepatocyte-like polarized cells. In contrast to standard bidimensional cultures, Matrigel-cultured Huh-7 cells adopted hepatocyte polarization features forming a continuous network of functional proto-bile canaliculi structures. These 3D cultures supported HCV infection by JFH-1 virus and produced infective viral particles which shifted towards lower densities with higher associated specific infectivity. In conclusion, our findings describe a novel use of Matrigel to study the entire HCV cycle in a more relevant context.     

Patterned entry and egress by Epstein-Barr virus in polarized CR2-positive epithelial cells

In polarized epithelium direction of viral entry and release correlates with proclivity of a virus to establish local versus systemic infection. The Epstein-Barr virus (EBV), whose principal tissue reservoir is B lymphocytes, also has disease manifestations in epithelium, suggesting intertissue spread potentially influenced by epithelial cell polarity. We stably transfected the B lymphocyte EBV receptor (CR2/CD21) into Madin-Darby canine kidney (MDCK) epithelial cells used extensively to study effects of cell polarity on infection by both DNA and RNA viruses. CR2/CD21 was detected on both apical and basolateral surfaces of polarized MDCK cells, with predominant expression basolaterally. However, infectivity was up to four-fold greater apically, suggesting that endogenous cell surface molecules, sorted asymmetrically onto polarized plasma membranes, may be involved in EBV entry into MDCK cells. EBV gp350/220, a replicative cycle glycoprotein added to the virus envelope on egress through the cell membrane, was immunolocalized by confocal microscopy to basolateral cell surfaces only. Apical entry of EBV with subsequent basolateral release of newly replicated virus favors systemic infection by viral dissemination to underlying lymphocytic aggregations. Under conditions of long-term culture, latent EBV was not stably maintained in these cells, suggesting that the epithelial phase of acute EBV infection may be transient.     

In vitro and in vivo effects of ingavirin on the ultrastructure and infectivity of influenza virus

The aim of this investigation was to study the effect of ingavirin on the structure and properties of influenza virions forming in its presence. The infectious activity of the virus and the morphology of the virions were analyzed by titration in cell culture and electron microscopy, respectively. The use of ingavirin was shown to reduce the proportion of morphologically intact virions and to increase that of filamentous and giant particles. No defects of surface glycoproteins were observed. The effect of the drug did not depend on the chosen model of virus replication and it was similarly shown in both cultured human cells and laboratory animals. In MDCK and A549 cells and in the mouse lungs, viral infectious activity was decreased by 1-2 orders of magnitude in relation to a model. The findings suggest that Ingavirin is able to impair the processes of viral morphogenesis, which in turn leads to a reduction in the infectivity of progeny virions.     

Ultrastructural Changes in Cultured Cells under the Effect of Vibrio Cholerae Hemagglutinin/Protease

Electron microscopic study of changes in cultured cells caused by Vibrio cholerae recombinant hemagglutinin/protease (HA/P) showed significant structural changes, most pronounced in HeLa and L-929 cells not forming a compact monolayer with tight junctions between the cells: formation of numerous vesicles on the cell surface and clasmatosis, vacuolation of the cytoplasm, swelling of mitochondria, clarification of their matrix and crist distortions, and increase in the number of lysosomes. Cytoplasm vacuolation predominated in MDCK culture, while clasmatosis was less intense. Addition of HA/P to CaCo2 cells forming a differentiated polarized monolayer, led to extension of cell-cell spaces not impairing tight junctions, swelling of mitochondria, cytoplasm vacuolation, and clasmatosis on the apical surface. These changes virtually completely coincided with those caused by the so-called NMDCY factor (non-membrane-damaging cytotoxin), described as new Vibrio cholerae toxin. These findings confirm our previous hypothesis about the identity of these factors.     

Enteropathogenic Escherichia coli type III effectors EspG and EspG2 alter epithelial paracellular permeability

Enteropathogenic Escherichia coli (EPEC) delivers a subset of effectors into host cells via a type III secretion system. Here we show that the type III effector EspG and its homologue EspG2 alter epithelial paracellular permeability. When MDCK cells were infected with wild-type (WT) EPEC, RhoA was activated, and this event was dependent on the delivery of either EspG or EspG2 into host cells. In contrast, a loss of transepithelial electrical resistance and ZO-1 disruption were induced by infection with an espG/espG2 double-knockout mutant, as was the case with the WT EPEC, indicating that EspG/EspG2 is not involved in the disruption of tight junctions during EPEC infection. Although EspG- and EspG2-expressing MDCK cells exhibited normal overall morphology and maintained fully assembled tight junctions, the paracellular permeability to 4-kDa dextran, but not the paracellular permeability to 500-kDa dextran, was greatly increased. This report reveals for the first time that a pathogen can regulate the size-selective paracellular permeability of epithelial cells in order to elicit a disease process.     

Enteropathogenic Escherichia coli decreases the transepithelial electrical resistance of polarized epithelial monolayers.

The mechanisms whereby enteropathogenic Escherichia coli (EPEC) causes diarrhea remain undefined. We found that EPEC caused a decrease in transepithelial electrical resistance across polarized monolayers of Caco-2 and MDCK epithelial cells. This occurred approximately 6 to 10 h after bacterial addition and was reversible if the monolayers were treated with tetracycline or gentamicin. Although significant alterations in host actin occurred beneath adherent EPEC, actin filaments supporting tight junctions were not noticeably affected in the epithelial cells, nor was the distribution of ZO-1, a tight junction protein. Despite the decrease in transepithelial electrical resistance, EPEC did not cause an increase in [3H]inulin penetration across MDCK monolayers. Unlike in the parental strain, mutations in any loci involved in adherence or formation of attaching and effacing lesions were unable to cause a decrease in transepithelial resistance. These data indicate that EPEC causes a decrease in transepithelial electrical resistance by disrupting a transcellular (intracellular) pathway rather than by disrupting intercellular tight junctions (paracellular) and that these disruptions occur only when attaching and effacing lesions are formed.     

Localization of dysfunctional tight junctions in Salmonella enterica serovar typhimurium-infected epithelial layers

Infection of polarized MDCK epithelial layers by Salmonella enterica serovar Typhimurium is accompanied by increased tight junction permeability and by contraction of perijunctional actinomyosin. We localized dysfunctional tight junctions in serovar Typhimurium-infected MDCK layers by imaging apical-basolateral intramembrane diffusion of fluorescent lipid and found that loss of the apical-basolateral diffusion barrier (tight junction fence function) was most marked in areas of prominent perijunctional contraction. The protein kinase inhibitor staurosporine prevented perijunctional contraction but did not reverse the effects of serovar Typhimurium on tight junction barrier function. Hence, perijunctional contraction is not required for Salmonella-induced tight junction dysfunction and this epithelial response to infection may be multifactorial.     

SARS-CoV replication and pathogenesis in an in vitro model of the human conducting airway epithelium

SARS coronavirus (SARS-CoV) emerged in 2002 as an important cause of severe lower respiratory tract infection in humans and in vitro models of the lung are needed to elucidate cellular targets and the consequences of viral infection. The severe and sudden onset of symptoms, resulting in an atypical pneumonia with dry cough and persistent high fever in cases of severe acute respiratory virus brought to light the importance of coronaviruses as potentially lethal human pathogens and the identification of several zoonotic reservoirs has made the reemergence of new strains and future epidemics all the more possible. In this chapter, we describe the pathology of SARS-CoV infection in humans and explore the use of two models of the human conducting airway to develop a better understanding of the replication and pathogenesis of SARS-CoV in relevant in vitro systems. The first culture model is a human bronchial epithelial cell line Calu-3 that can be inoculated by viruses either as a non-polarized monolayer of cells or polarized cells with tight junctions and microvilli. The second model system, derived from primary cells isolated from human airway epithelium and grown on Transwells, form a pseudostratified mucociliary epithelium that recapitulates the morphological and physiological features of the human conducting airway in vivo. Experimental results using these lung epithelial cell models demonstrate that in contrast to the pathology reported in late stage cases SARS-CoV replicates to high titers in epithelial cells of the conducting airway. The SARS-CoV receptor, human angiotensin 1 converting enzyme 2 (hACE2), was detected exclusively on the apical surface of cells in polarized Calu-3 cells and human airway epithelial cultures (HAE), indicating that hACE2 was accessible by SARS-CoV after lumenal airway delivery. Furthermore, in HAE, hACE2 was exclusively localized to ciliated airway epithelial cells. In support of the hACE2 localization data, the most productive route of inoculation and progeny virion egress in both polarized Calu-3 and ciliated cells of HAE was the apical surface suggesting mechanisms to release large quantities of virus into the lumen of the human lung. Preincubation of the apical surface of cultures with antisera directed against hACE2 reduced viral titers by two logs while antisera against DC-SIGN/DC-SIGNR did not reduce viral replication levels suggesting that hACE2 is the primary receptor for entry of SARS-CoV into the ciliated cells of HAE cultures. To assess infectivity in ciliated airway cultures derived from susceptible animal species we generated a recombinant SARS-CoV by deletion of open reading frame 7a/7b (ORF 7a/7b) and insertion of the green fluorescent protein (GFP) resulting in SARS-CoV GFP. SARS-CoV GFP replicated to similar titers as wild type viruses in Vero E6, MA104, and CaCo2 cells. In addition, SARS-CoV replication in airway epithelial cultures generated from Golden Syrian hamster tracheas reached similar titers to the human cultures by 72 h post-infection. Efficient SARS-CoV infection of ciliated cell-types in HAE provides a useful in vitro model of human lung origin to study characteristics of SARS-CoV replication and pathogenesis.     

Effects of src kinase and TGFbeta1 on the differentiation and morphogenesis of MDCK cells grown in three-dimensional collagen and Matrigel environments.

This study attempted to analyse in detail the effect of src kinase on the growth and differentiation of MDCK cells in different extracellular matrix (ECM) environments. A method was developed to label the membrane proteins in situ and the distribution of cytoskeletal and junctional proteins was visualized in three-dimensional cell complexes, using optical sections generated by confocal microscopy. Independently of the ECM, non-transformed MDCK cells formed differentiated cell cysts with one or a few lumina, with the apical side facing the lumen; ZO-1 was expressed at the tight junctions close to the apical side and beta-catenin, E-cadherin and fodrin along the entire lateral walls. The phenotype of src kinase activated MDCK cells was strongly dependent on the ECM and varied from an irregular cluster in collagen I, to tubular structures in laminin or proteoglycans, and finally to a polarized cell cyst in Matrigel. In collagen I, E-cadherin and beta-catenin were seen partially along the lateral walls and partially in the cytoplasm of src-transformed MDCK cells; fodrin was released into the cytoplasm and ZO-1 was not visualized. When the src-transformed cells were cultivated in Matrigel, their junctional proteins were recruited to the cell membranes and ZO-1 reappeared at the apical face. Thus, the components of Matrigel could overcome the deleterious effect of src on the polarity of MDCK cells. TGFbeta1, together with its receptors and other soluble factors in Matrigel, were responsible for the induction of differentiation. The results show that tyrosine phosphorylation sensitizes the epithelial MDCK cells to ECM and TGFbeta1.     

An autoradiographic and immunofluorescent study of the infectious cycle of sheeppox virus in cell cultures exposed to 5-fluorodeoxyuridine

Summary No progeny virus is detected by determination of the infectivity titres when embryo muscle cell cultures are exposed to a concentration of 10^–5 M of 5-fluorodeoxyuridine at the time of sheeppox virus infection. The autoradiographic preparations of FUdR treated cells showed cytoplasmic labelling into viral DNA (tritiated deoxyuridine) in 4 to 9% of the cells during the first 24 hours of infection. In cells exposed to FUdR in which viral DNA synthesis was detected by autoradiography, progeny virus not detectable by infectivity test might be produced. The demonstration of viral proteins by immunofluorescence served to establish that viral DNA synthesis is not essential for viral protein synthesis in FUdR treated cells. Under light microscopy, there is no apparent difference between the cytopathic effects of viral infection in treated and in untreated cells.This work was supported by a Calouste Gulbenkian Foundation Grant (C/7/66).     

Fetal calf serum inhibits virus genome expression in Madin-Darby canine kidney cells persistently infected with influenza A virus

A cell line of Madin-Darby canine kidney (MDCK) cells persistently infected with human influenza A virus has been established and designated as MDCK-IVpi cells. Production of progeny virus in MDCK-IVpi cells was suppressed when the cells were incubated in the presence of 10% fetal calf serum (FCS). FCS impaired virus mRNA synthesis in MDCK-IVpi cells, which resulted in a scarcity of virus proteins for virion formation. However, MDCK-IVpi cells well supported the growth of superinfecting heterologous influenza viruses, even in the presence of FCS. A certain fetuin-like substance in FCS might be responsible for the observed inhibition of virus replication.     

Morphogenetic clonal growth of kidney epithelial cell line MDCK

MDCK (Madin-Darby canine kidney) cells were cultured either dispersed within hydrated collagen gel (HCG) or seeded atop a collagen substrate and then immediately overlaid with HCG. Individual cells exhibited clonal growth in three dimensions to form spherical cysts made up of a simple epithelium enclosing a fluid-filled lumen. The cells of MDCK cysts were polarized with the basolateral surface in contact with the collagen gel and the apical surface bordering the lumen. The ultrastructure of MDCK cysts showed similarities to distal nephron. The cells bore apical microvilli and solitary cilia and had occluding junctions and a simple basolateral surface. MDCK cysts increased in size (greater than 800 microns diameter) with continued culture. MDCK cysts grown between layers of HCG were stripped free of the overlying collagen to give direct access to basolateral surface membrane. Unlike monolayer culture, morphogenetic clonal growth of cell line MDCK produces a polarized cell population with a true lumenal and basolateral surface. Collagen-gel-cultured MDCK cysts provide an easily manipulable in vitro cell system that may offer unique advantages for the study of renal cell structure and function.     

Two-dimensional manipulation of differentiated Madin-Darby canine kidney (MDCK) cell sheets: the noninvasive harvest from temperature-responsive culture dishes and transfer to other surfaces

A renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, adheres, spreads, and proliferates to confluency on our developed temperature-responsive culture dishes grafted with a poly(N-isopropylacrylamide) (PIPAAm) at 37 degrees C. In addition to other cell types, including hepatocytes and endothelial cells, MDCK cell sheets noninvasively were harvested from PIPAAm-grafted dishes merely by reducing the temperature. However, during the early stage of culture (up to 3 weeks), confluent MDCK cell detachment is greatly repressed. In the present study, we succeeded in the rapid harvest of confluent MDCK cell sheets and intact transfer to other culture dishes by utilizing hydrophilically modified poly(vinylidene difluoride) (PVDF) membranes as supporting materials. Immunocytochemistry with anti-beta-catenin antibody revealed that the functional cell-cell junctions were well organized in the transferred MDCK cell sheets. The viability assay showed that the transferred cells were not damaged during the two-dimensional cell-sheet manipulation. By transmission electron microscopy it was confirmed that the harvested MDCK cells retained differentiated phenotypes and had many microvilli and tight junctions at the apical and lateral plasma membranes, respectively. This two-dimensional cell-sheet manipulation technique promises to be useful in tissue engineering as well as in the investigation of epithelial cell sheets.     

Interactions of Pseudorabies virus with swine alveolar macrophages I: virus replication

Summary The replication of Pseudorabies virus (PRV) in cultured swine alveolar macrophages (AM) was studied using 6 different virus strains. AM were highly permissive to PRV infection, with progeny virus titres of 10⁷ TCID₅₀/ml from some strains. Virus progeny titres were higher in cultures infected with the field strains S-62 and 4892 than in cultures infected with the strains Bartha or PRV-C. Virus replication, viral DNA synthesis and the concomitant cell damage were dependent upon virus input m.o.i.s. and virus strain. Furthermore, cells from 7 day old pigs yielded higher virus progeny titres than cells of 6 week old pigs. The results from this study provide support to the premise that PRV infection may predispose anials to respiratory disease.     

Epidermal growth factor protects the apical junctional complexes from hydrogen peroxide in bile duct epithelium

The tight junctions of bile duct epithelium form a barrier between the toxic bile and liver parenchyma. Disruption of tight junctions appears to have a crucial role in the pathogenesis of various liver diseases. In this study, we investigated the disruptive effect of hydrogen peroxide and the protective effect of epidermal growth factor (EGF) on the tight junctions and adherens junctions in the bile duct epithelium. Oxidative stress in NRC-1 and Mz-ChA-1 cell monolayers was induced by administration of hydrogen peroxide. Barrier function was evaluated by measuring electrical resistance and inulin permeability. Integrity of tight junctions, adherens junctions and the actin cytoskeleton was determined by imunofluorescence microscopy. Role of signaling molecules was determined by evaluating the effect of specific inhibitors. Hydrogen peroxide caused a rapid disruption of tight junctions and adherens junctions leading to barrier dysfunction without altering the cell viability. Hydrogen peroxide rapidly increased the levels of p-MLC (myosin light chain) and c-Src(pY418). ML-7 and PP2 (MLCK and Src kinase inhibitors) attenuated hydrogen peroxide-induced barrier dysfunction, tight junction disruption and reorganization of actin cytoskeleton. Pretreatment of cell monolayers with EGF ameliorated hydrogen peroxide-induced tight junction disruption and barrier dysfunction. The protective effect of EGF was abrogated by ET-18-OCH(3) and the Ro-32-0432 (PLCγ and PKC inhibitors). Hydrogen peroxide increased tyrosine phosphorylation of ZO-1, claudin-3, E-cadherin and β-catenin, and pretreatment of cells with EGF attenuated tyrosine phosphorylation of these proteins. These results demonstrate that hydrogen peroxide disrupts tight junctions, adherens junctions and the actin cytoskeleton by an MLCK and Src kinase-dependent mechanism in the bile duct epithelium. EGF prevents hydrogen peroxide-induced tight junction disruption by a PLCγ and PKC-dependent mechanism.     

A bacterial protease perturbs the paracellular barrier function of transporting epithelial monolayers in culture.

Tight junctions between cells and adhesion to the substratum maintain the barrier function of epithelia throughout the body. Damage to the epithelial barrier by microbial products allows penetration of bacteria and promotion of infection. We studied the effects of Pseudomonas elastase (PE) on the barrier function of epithelia by using Madin-Darby canine kidney (MDCK) epithelial cells; these cells form tight junctions (zonula occludens [ZO]) in vitro. PE decreased electrical resistance across the monolayers in a concentration- and time-dependent manner. Immunostaining of selected proteins of the ZO and zonula adherens was used to explore the effects of PE on junctional proteins. PE-treated monolayers of MDCK cells had markedly decreased immunostaining of ZO-1, a protein of the ZO, but light microscopy of PE-treated cells revealed no obvious morphologic changes. A chromium release assay indicated that, even with marked changes in transmonolayer electrical resistance, the permeability defect was not due to membrane disruption. Fluorescence staining of F-actin indicated diminution of cellular microfilaments in PE-treated cells, but E cadherin (uvomorulin), a protein of the zonula adherens, was unaffected by the enzyme. Elastases from porcine pancreas and human leukocytes with similar enzymatic activity (6 U/ml) did not decrease transmonolayer electrical resistance or degrade ZO-1. These results suggest that PE disturbs the barrier function of epithelial monolayers, in part, by changing the cell architecture and altering at least one protein of the ZO.     

CD4/CXCR4 co-expression allows productive HIV-1 infection in canine kidney MDCK cells

The Madin-Darby canine kidney (MDCK) cell line has become the prototypic cell type for studying the mechanisms involved in viral glycoproteins transport and viral assembly in polarized cells. This cell line has been used in our laboratories for studying human immunodeficiency virus (HIV-1), despite the fact that MDCK cells cannot be infected by HIV. In transfected MDCK cells, HIV-1 glycoproteins are specifically transported to the basolateral cell surface where viral budding also mostly occurs. However, this model is of limited use when viral propagation, infection of most cells, or larger production of virions, is needed. The initial objective of this work was thus to establish an MDCK-derived cell line that could be productively infected by HIV-1, in order to pursue our studies on the polarization of viral budding. Expression of both receptor and co-receptor for T-tropic strains of the virus showed that canine cells are rendered permissive once virus binding and entry is allowed. In addition, a reduced infectivity of the viral particles released from the basolateral surface was observed. This observation most likely reflects the interference mediated by CD4 molecules that accumulate at the basolateral domain. Accordingly, this effect was largely prevented when using viruses that down-regulate cell surface CD4 by expression of both viral accessory proteins Vpu and Nef. This is a further evidence that the function of different viral proteins depends of the site of viral budding, which is itself determined by the presence of targeting signal(s) harbored by viral envelope glycoproteins.     

Establishment of cell polarity by afadin during the formation of embryoid bodies

Afadin directly links nectin, an immunoglobulin-like cell–cell adhesion molecule, to actin filaments (F-actin) at adherens junctions (AJs). The nectin–afadin complex is important for the formation of not only AJs but also tight junctions (TJs) in epithelial cells. Studies using afadin-knockout mice have revealed that afadin is indispensable for embryonic development by organizing the formation of cell–cell junctions. However, the molecular mechanism of cell–cell junction disorganization during embryonic development in afadin-knockout mice is poorly understood. To address this, we took advantage of embryoid bodies (EBs) as a model system. The formation of cell–cell junctions including AJs and TJs was impaired in afadin-null EBs. The proper accumulation of the Par complex and the activation of Cdc42 and atypical PKC (aPKC), which are crucial for the formation of cell polarity, were also inhibited by knockout of afadin. In addition, the disruption of afadin caused the abnormal deposition of laminin and the dislocalization of its receptors integrin α₆ and integrin β₁. These results indicate that afadin organizes the formation of cell–cell junctions by regulating cell polarization in early embryonic development.