CFTR and tight junctions in cultured bronchial epithelial cells

Airway epithelial salt and water transport takes place through paracellular and transcellular pathways. This transport depends critically on the epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane conductance regulator (CFTR), operating in concert with the paracellular pathway through the tight junctions (TJ). Normal (16HBE14o-), cystic fibrosis (CFBE41o-), and corrected CFBE41o- (CFBE41o-pCep4 overexpressing wtCFTR) airway epithelial cell lines were cultured under isotonic conditions. Transepithelial electrical resistance (TEER) was measured as indicator of the tightness of the cultures. Morphology was investigated by immunofluorescence and paracellular permeability by lanthanum nitrate or [¹⁴C] mannitol as permeability markers.The CFTR-defective cell line CFBE41o- developed higher TEER than its corrected counterpart CFBE41o-pCep4. Addition of a specific inhibitor of CFTR (CFTR_inh-172) to 16HBE14o- and CFBE41o-pCep4 cells resulted in a time-dependent increase in TEER, whereas stimulation of CFTR by IBMX and forskolin caused a decrease. Permeability to lanthanum and [¹⁴C] mannitol was lower in CFBE41o- and in 16HBE14o- cells exposed to CFTR_inh-172, compared to untreated 16HBE14o- and CFBE41o-pCep4 cells, respectively. 16HBE14o- cells exposed to IBMX and forskolin showed higher permeability to lanthanum but lower permeability to [¹⁴C] mannitol compared to control. Immunofluorescence revealed a disorganization of F-actin and α-tubulin in 16HBE14o- cells and CFBE41o- pCep4 exposed to CFTR_inh-172 and in CFBE41o- cells. Changes in F-actin and α-tubulin in 16HBE14o- cells exposed to IBMX and forskolin were also seen. These results suggest the possibility of an interaction between CFTR and the TJ protein complex, probably via the cytoskeleton.     

Role of tight junctions of polarized epithelial MDCK cells in the replication of herpes simplex virus type 1

Before completion of polarization, Madin-Darby canine kidney (MDCK) cells showed high infectivity and progeny production of herpes simplex virus type 1 infection. After polarization or formation of tight junctions, the infectivity and virus replication in MDCK cells was restricted significantly. The disruption of tight junctions by depletion of Ca²⁺ resulted in increasing virus infectivity and productivity. Mechanical disruption of tight junctions by scratching the cell monolayers with injection needle allowed markedly the replication of HSV-I in the cells aligned along the injured area. In polarized MDCK cells the progeny were released preferentially from the apical surface of the cells. These data suggest that because polarized MDCK cells mimic the epithelial cell layers, this cell line is helpful for determining the factors which regulate viral transmission in the human body. © Wiley-Liss, Inc.     

E-Cadherin and tight junctions between epithelial cells of different animal species

The assembly and permanent sealing of tight junctions (TJs) depend crucially on cell-cell contacts containing E-cadherin. This poses a puzzling problem because, while TJs can be established between epithelial cells from different tissues and even different animal species ("heterotypic TJs"; Gonzalez-Mariscal et al. 1989, J Membr Biol 107:43), the cell-cell binding mediated by E-cadherin is a highly specific one (Takeichi 1995, Curr Opin Cell Biol 7:619). Yet the demonstration that TJs can be established at heterotypic borders is open to two distinct challenges. First, it is based on transepithelial electrical resistance (TER) and restriction to ruthenium red permeation only, which today are known to be just two of the many characteristics of TJs; and second some attributes of the TJs (e.g. the presence of specific molecules) have been found even in cells that do not establish these structures. This raised the question of whether heterotypic TJs were not true or full TJs. In the present work we demonstrate that heterotypic TJs in mixed monolayers of MDCK cells with a different cell type (LLC-PK1) are true TJs through several criteria, such as TER, the ability to stop the membrane diffusion of fluorescent sphingomyelin from the apical to the lateral domain, the presence of ZO-1, ZO-2, occludin, claudin-1 and claudin-2. We then turn to the presence of E-cadherin at heterotypic borders, and observe that it cannot be detected by the highly specific DECMA-1 antibody, in spite of the fact that this antibody does reveal the presence of E-cadherin at homotypic contacts of the same cell. Yet, ECCD-2, an antibody against another domain of E-cadherin, reveals that this molecule may be present at both types of borders. Thus, E-cadherin is present at heterotypic borders, yet it seems to be in a conformation unable to bind DECMA-1. Our results suggest: (1) that heterotypic borders can establish fully developed TJs; (2) that the sealing of these heterotypic TJs depends on E-cadherin; (3) but that this dependence is mediated through a cascade of chemical reactions involving two different G-proteins, PLC, PKC and calmodulin, which we have characterized elsewhere (Balda et al. 1991, J Membr Biol 122:193); and (4) hence molecules of E-cadherin that trigger junction formation can act from a distant homotypic contact.     

Turner's syndrome patients lack tight junctions between uterine epithelial cells.

Eleven endometrial biopsies, taken from six Turner's syndrome patients receiving hormone replacement therapy prior to treatment by oocyte donation and embryo transfer, were assessed by freeze fracture followed by electron microscopy for epithelial tight junctions. Nine of the eleven biopsies had no discernible tight junctions; the other two biopsies had reduced and disorganized junctional structures. Two patients subsequently became pregnant following embryo transfer. It is concluded that compromised epithelial integrity does not prevent embryo implantation in the human, an observation that is consistent with a barrier role for the epithelium except at times when appropriately conditioned with oestrogen and progesterone to induce receptivity for implantation.     

Complex tight junctions of epithelial and of endothelial cells in early foetal brain

Summary The morphology of epithelial and of endothelial intercellular junctions in human foetal (9–15 weeks gestation) and sheep foetal (50, 60 and 125 days gestation, term 147 days) brain has been studied using the freeze-fracture technique and thin section transmission electronmicroscopy. Freeze-fracture replicas of the choroid plexus of both early human and sheep foetuses showed that the choroidal ependymal cells are linked at the ventricular surface by tight junctions. Freeze-fracture replicas of foetal cortical endothelial cell junctions showed that they are still more complex than those of choroidal epithelial cells, in all specimens so far examined. In some 60 day sheep foetuses the dye Alcian blue, which binds to plasma albumin and which is electrondense when treated with osmium tetroxide, was injected intravenously a few minutes prior to fixation. The dye penetrated from blood into brain extracellular space and c.s.f. but apparentlynot by anintercellular route. The dye was found in a tubular system (endoplasmic reticulum) in both choroidal epithelial and cortical endothelial cells. The possibility that protein penetrates into the foetal brain and c.s.f. by atranscellular route is discussed. The possible significance of these findings in relation to previous ideas and studies of the development of blood-brain barrier mechanisms is also considered.     

尿酸钠结晶影响肾小管上皮细胞的紧密连接

背景：目前研究来看,尿酸性结石与紧密连接蛋白及肾间质纤维化的关系仍未明确。 目的：观察尿酸钠结晶对肾小管上皮细胞紧密连接的影响。 方法：配制单钠尿酸钠晶体。将正常大鼠肾小管上皮细胞随机分为对照组和尿酸钠结晶组,分别用无血清培养基和尿酸钠结晶进行培养。免疫荧光和RT-PCR方法检测24,48,72 h紧密连接蛋白的表达。 结果与结论：与对照组比较,尿酸钠结晶于不同时相刺激NRK-52E细胞后,紧密连接蛋白和mRNA表达下降(P < 0.05),72 h时下降显著(P < 0.01),蛋白表达出现重新分布现象。说明尿酸钠结晶可破坏肾小管上皮细胞紧密连接蛋白的结构、功能及导致分布异常。     

A freeze-fracture electron microscopic study of tight junctions of epithelial cells in the human uterus

Summary Freeze-fracture electron microscopy has been used to study tight junctions of luminal epithelial cells of the human uterus. The junctions are deeper and more extensive in the middle of the menstrual cycle than they are later in the cycle. The results suggest that the contents of the uterine lumen may be more closely regulated at some times than at others.     

Role of stretch on tight junction structure in alveolar epithelial cells.

Previous studies have demonstrated that high tidal volumes can cause interstitial and alveolar edema, with degradation of pulmonary epithelial barrier integrity. Separate studies have shown that F-actin disruption and decreased intracellular ATP (ATP(i)) levels in the nonpulmonary epithelium can increase tight junction (TJ) permeability. We hypothesized that large epithelial stretch perturbs ATP(i) and actin architecture, each of which adversely affects TJ structure, and thus increases TJ permeability. Primary alveolar epithelial cells were subjected to a uniform 25% or 37% change in surface area (DeltaSA), cyclic biaxial stretch (15 cycles/min) for 1 h, or treated with either glycolytic metabolic inhibitors or cytoskeletal disrupting agents. Unstretched, untreated cells served as controls. Changes in the TJ proteins occludin and ZO-1 were determined by immunocytochemical evaluation. A stretch amplitude of 25% DeltaSA did not produce any significant cytologic changes compared with controls, but an amplitude of 37% DeltaSA stretch resulted in significant decreases in the intensity of the peripheral occludin band, the degree of cell-cell attachment (CCA), and total cellular occludin content. ATP depletion significantly diminished the occludin band intensity and decreased CCA. Actin disruption did not affect TJ protein band intensities (although the occludin distribution became punctate) but altered CCA. Untreated cells stretched cyclically at 25% or 50% DeltaSA for 1 h had significantly decreased ATP(i) compared with unstretched controls. These results suggest that stretch-induced ATP(i) reduction and actin perturbation disrupt TJ structure and CCA, which may lead to the alveolar flooding associated with high tidal volumes.     

Matrix metalloproteinase-9-deficient dendritic cells have impaired migration through tracheal epithelial tight junctions

When sampling inhaled antigens, dendritic cells (DC) must penetrate the tight junction (TJ) barrier while maintaining the TJ seal. In matrix metalloproteinase (MMP)-9-deficient mice, in vivo experiments suggest that migration of DC into air spaces is impaired. To examine the underlying mechanisms, we established a well-defined in vitro model using mouse tracheal epithelial cells and mouse bone marrow DC (BMDC). Transmigration was elicited with either macrophage inflammatory protein (MIP)-1alpha or MIP-3beta in a time-dependent manner. Control MMP-9(+/+) BMDC cultured with granulocyte macrophage-colony-stimulating factor for 7 d showed a 30-fold greater transepithelial migration toward MIP-3beta than MIP-1alpha, indicating a more mature DC phenotype. MMP-9(-/-) BMDC as well as MMP-9(+/+) BMDC in the presence of the MMP inhibitor GM6001, although showing a similar preference for MIP-3beta, were markedly impaired in their ability to traverse the epithelium. Expression levels of CCR5 and CCR7, however, were similar in both MMP-9(-/-) and MMP-9(+/+) BMDC. Expression of the integral TJ proteins, occludin and claudin-1, were examined in BMDC before and after transepithelial migration. Interestingly, occludin but not claudin-1 was degraded following transepithelial migration in both MMP-9(-/-) and control BMDC. In addition, there was a > 2-fold increase in claudin-1 expression in MMP-9(-/-) as compared with control BMDC. These observations indicate that occludin and claudin-1 are differentially regulated and suggest that the lack of MMP-9 may affect claudin-1 turnover.     

Disruption of tight junctions and induction of proinflammatory cytokine responses in colonic epithelial cells by Campylobacter jejuni

Campylobacter jejuni is a leading cause of human enterocolitis and is associated with postinfectious complications, including irritable bowel syndrome and Guillain-Barré syndrome. However, the pathogenesis of C. jejuni infection remains poorly understood. Paracellular pathways in intestinal epithelial cells are gated by intercellular junctions (tight junctions and adherens junctions), providing a functional barrier between luminal microbes and host immune cells in the lamina propria. Here we describe alterations in tight junctions in intestinal epithelial monolayers following C. jejuni infection. Apical infection of polarized T84 monolayers caused a time-dependent decrease in transepithelial electrical resistance (TER). Immunofluorescence microscopy revealed a redistribution of the tight junctional transmembrane protein occludin from an intercellular to an intracellular location. Subcellular fractionation using equilibrium sucrose density gradients demonstrated decreased hyperphosphorylated occludin in lipid rafts, Triton X-100-soluble fractions, and the Triton X-100-insoluble pellet following apical infection. Apical infection with C. jejuni also caused rapid activation of NF-kappaB and AP-1, phosphorylation of extracellular signal-regulated kinase, Jun N-terminal protein kinase, and p38 mitogen-activated protein kinases, and basolateral secretion of the CXC chemokine interleukin-8 (IL-8). Basolateral infection with C. jejuni caused a more rapid decrease in TER, comparable redistribution of tight-junction proteins, and secretion of more IL-8 than that seen with apical infection. These results suggest that compromised barrier function and increased chemokine expression contribute to the pathogenesis of C. jejuni-induced enterocolitis.     

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.     

Freeze-fracture morphology and quantification of human bronchial epithelial tight junctions.

A comprehensive investigation of the morphology of human airway epithelial tight junctions was carried out by freeze-fracture electron microscopy using quantitative methods designed to analyze a range of junctional characteristics. Extrapulmonary bronchi that appeared grossly normal were taken at sites distant from tumor in lungs resected for pulmonary carcinoma. The absence of cellular atypia in the samples was confirmed by histology. Airway levels I (main bronchus; n = 7 subjects) and II (lobar bronchus; n = 5 subjects) were compared with respect to junctional depth, strand number, and junctional complexity. Junctional complexity was assessed by frequency of strand interconnection and numbers of strands per interconnection. Comparisons between airway levels I and II for these parameters showed that there were no significant differences in strand number or junctional complexity between the two airway levels. However, junctional depth was slightly but significantly reduced at level II compared with level I (P less than 0.01). The arrangement of strands varied considerably from one junction to the next, irrespective of the cell types involved. "Parallel" and "network" patterns of junctions were observed; the existence of gradations between these two patterns indicated that they represent opposite extremes of a single junctional form rather than distinct categories of junction. These results have allowed us to establish a data pool for normal human bronchi from which the structure of epithelial cell junctions in bronchial diseases can be compared.     

Focal tight junctions between mesenchymal cells of fetal dermis

This freeze fracture study shows the presence of focal tight junctions (maculae occludentes) between the mesenchymal cells in the connective tissue matrix of embryonic and fetal dermis. The overall outline of these unique junctions varies from circular to very angular. The junctional elements are most frequently present in a groove on the E fracture face. The corresponding P fracture face has ridges delineating the junction. These intercellular junctions may provide a means of informational or metabolic coupling between cells, may serve a structural role as scaffolding in the deposition and orientation of extracellular materials, or may be involved in the early stages of angiogenesis.     

Enhanced epithelial gene transfer by modulation of tight junctions with sodium caprate

The airway epithelium is resistant to infection by gene transfer vectors when infected from the luminal surface. One strategy for enhancing airway epithelial gene transfer is to modify paracellular permeability, thereby permitting the diffusion of vectors to the basolateral surface, where uptake receptors are expressed. We investigated the ability of a medium-chain fatty acid known to enhance drug absorption, sodium caprate (C10), to increase airway paracellular permeability in comparison with ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). Apical application of C10 decreased transepithelial resistance by > 90% within minutes, whereas EGTA required an hour or more to produce a similar effect. C10 increased mannitol and dextran permeability by sevenfold, as compared with a twofold increase produced by EGTA. A greater enhancement of adenoviral lacZ gene transfer was mediated by C10 (50-fold over controls) than by EGTA (10-fold over controls). This correlated with a significant enhancement of adenoviral CFTR-mediated correction of Cl(-) transport in polarized human airway epithelial (HAE) cells from cystic fibrosis (CF) patients. Confocal microscopy revealed a redistribution of claudin-1 following C10 but not EGTA treatment as a possible mechanism of gene-transfer enhancement by C10. These data suggest that C10 may be a better agent for enhancing gene transfer than is EGTA, and that this effect occurs through disruption of claudin-1.     

Periderm cells covering palatal shelves have tight junctions and their desquamation reduces the polarity of palatal shelf epithelial cells in palatogenesis

In palatogenesis, bilateral palatal shelves grow and fuse with each other to establish mesenchyme continuity across the horizontal palate. The palatal shelves are covered with the medial edge epithelium (MEE) in which most apical cells are periderm cells. We investigated localization and roles of tight junction (TJ) and adherens junction (AJ) components and an apical membrane marker in the MEE in palatogenesis. Immunofluorescence and immunoelectron microscopy analyses revealed that TJs were located at the boundary between neighboring periderm cells, whereas AJ components were localized at the boundary between all epithelial cells in the MEE. Specifically, typical AJs were observed at the boundaries between neighboring periderm cells and between periderm cells and underlying epithelial cells where the signal for nectin-1 was observed. The TGF-β-induced desquamation of periderm cells reduced the polarity of remaining epithelial cells as estimated by changes of epithelial cell morphology and the staining of the polarity marker and the AJ components. These less polarized epithelial cells then intermingled and finally disappeared at least partly by apoptosis. These results indicate that periderm cells covering growing palatal shelves have bona fide TJs and their desquamation reduces the polarity of palatal shelf epithelial cells in palatogenesis.     

Ammonium affects tight junctions and the cytoskeleton in MDCK cells

In the kidney medulla, tubule cells are exposed not only to elevated NaCl but also to high NH₄Cl concentrations. Although it is well known that long-term exposure to high NaCl concentrations leads to reorganization of the actin-based cytoskeleton and to altered transport properties of renal epithelial cells, there have been no comparable studies on the effects of elevated extracellular NH₄Cl concentrations. We therefore examined the effect of prolonged (up to 72 h) exposure of Madin-Darby canine kidney (MDCK) cells to increased NH₄Cl concentrations on the actin-based cytoskeleton, the transepithelial electrical resistance (TER) and the expression and intracellular distribution of the tight junction protein occludin. NH₄Cl exposure resulted in rarefaction of cytoplasmic stress fibres, formation of intense peripheral actin bands and reduced abundance of both F- and G-actin. While under control conditions occludin staining was restricted to the tight junction region, ample dot-like intracellular staining was apparent after NH₄Cl exposure. These changes in cell structure were associated with an increase in TER and the enhanced expression of an additional putative, 40-kDa occludin isoform. Exposure to elevated extracellular NH₄Cl concentrations thus leads to distinct alterations in the architecture and transepithelial transport properties of MDCK cells that may also be relevant for the tubule cells of the renal inner medulla.     

Vibrio parahaemolyticus disruption of epithelial cell tight junctions occurs independently of toxin production

Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis worldwide. Virulence is commonly associated with the production of two toxins, thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH). Although the majority of clinical isolates produce TDH and/or TRH, clinical samples lacking toxin genes have been identified. In the present study, we investigated the effects of V. parahaemolyticus on transepithelial resistance (TER) and paracellular permeability in Caco-2 cultured epithelial cells. We found that V. parahaemolyticus profoundly disrupts epithelial barrier function in Caco-2 cells and that this disruption occurs independently of toxin production. Clinical isolates with different toxin genotypes all led to a significant decrease in TER, which was accompanied by an increased flux of fluorescent dextran across the Caco-2 monolayer, and profound disruption of actin and the tight junction-associated proteins zonula occludin protein 1 and occludin. Purified TDH, even at concentrations eightfold higher than those produced by the bacteria, had no effect on either TER or paracellular permeability. We used lactate dehydrogenase release as a measure of cytotoxicity and found that this parameter did not correlate with the ability to disrupt tight junctions. As the effect on barrier function occurs independently of toxin production, we used PCR to determine the toxin genotypes of V. parahaemolyticus isolates obtained from both clinical and environmental sources, and we found that 5.6% of the clinical isolates were toxin negative. These data strongly indicate that the effect on tight junctions is not due to TDH and suggest that there are other virulence factors.     

The selective and superoxide-independent disruption of intestinal epithelial tight junctions during leukocyte transmigration

Polymorphonuclear leukocyte (PMN) transmigration across cultured intestinal epithelial monolayers has been shown to be associated with a decrease in transepithelial resistance to the passive flow of ions. Using flux techniques, we show that this effect reflects selective, PMN induced alterations in paracellular, as opposed to transcellular, ion permeability. Enhancement of paracellular permeability due to PMN transmigration is not simply due to expansion of the paracellular space resulting from cell death as cytotoxicity does not occur during this process. Thus, permeability alterations accompanying PMN transmigration can be specifically attributed to altered permeability of the rate limiting barrier of the paracellular pathway, the intercellular tight junction. We have also explored the mechanism by which PMN induce transient tight junction dissolution during transmigration. Use of inhibitors of toxic oxygen metabolites or use of PMN from patients with chronic granulomatous disease show that oxygen metabolites are neither required for transmigration or for the permeability abnormality accompanying transmigration. Similarly, use of protease inhibitors suggest that release of proteases by PMN during transmigration is not the basis by which PMN are able to cross tight junctions. Structural studies show that transient intimate PMN-epithelial cell plasma membrane associations and cytoskeletal specializations preceed junctional impalement by PMN. We speculate that such putative adhesion sites serve as the foothold from which PMN may generate the mechanical force necessary to cross tight junctions during transmigration.     

Type 2 innate lymphoid cells disrupt bronchial epithelial barrier integrity by targeting tight junctions through IL-13 in asthmatic patients

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