Neutrophil-mediated epithelial injury during transmigration: role of elastase

Neutrophil-mediated injury to gut epithelium may lead to disruption of the epithelial barrier function with consequent organ dysfunction, but the mechanisms of this are incompletely characterized. Because the epithelial apical junctional complex, comprised of tight and adherens junctions, is responsible in part for this barrier function, we investigated the effects of neutrophil transmigration on these structures. Using a colonic epithelial cell line, we observed that neutrophils migrating across cell monolayers formed clusters that were associated with focal epithelial cell loss and the creation of circular defects within the monolayer. The loss of epithelial cells was partly attributable to neutrophil-derived proteases, likely elastase, because it was prevented by elastase inhibitors. Spatially delimited disruption of epithelial junctional complexes with focal loss of E-cadherin, beta-catenin, and zonula occludens 1 was observed adjacent to clusters of transmigrating neutrophils. During neutrophil transmigration, fragments of E-cadherin were released into the apical supernatant, and inhibitors of neutrophil elastase prevented this proteolytic degradation. Addition of purified leukocyte elastase also resulted in release of E-cadherin fragments, but only after opening of tight junctions. Taken together, these data demonstrate that neutrophil-derived proteases can mediate spatially delimited disruption of epithelial apical junctions during transmigration. These processes may contribute to epithelial loss and disruption of epithelial barrier function in inflammatory diseases.     

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na(+),K(+)-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors.     

Hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC in conjunction with Lgl

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here, we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell–cell contacts. In 3D Matrigel culture of human hepatocytic HepG2 cells, which mimics a process of liver development and regeneration, depletion of Par3, Cdc42, or aPKC results in an impaired establishment of apicobasolateral polarity and a loss of subsequent apical lumen formation. The aPKC activity is also required for bile canalicular (apical) elongation in mouse primary hepatocytes. The lateral membrane-associated proteins Lgl1 and Lgl2, major substrates of aPKC, seem to be dispensable for hepatocyte polarity establishment because Lgl-depleted HepG2 cells are able to form a single apical lumen in 3D culture. On the other hand, Lgl depletion leads to lateral invasion of aPKC, and overexpression of Lgl1 or Lgl2 prevents apical lumen formation, indicating that they maintain proper lateral integrity. Thus, hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC; Par3 cooperates with Cdc42 to recruit aPKC, which plays a crucial role in apical membrane development and regulation of the lateral maintainer Lgl.     

The septate junction limits mobility of lipophilic markers in plasma membranes ofHydra vulgaris (attenuata)

Summary Fluorescent lipophilic probes were used to study the role of septate junctions in maintaining distinct apical and basolateral domains of plasma membranes in epithelial cells of hydra. In short-term experiments, a 16-carbon chain aminofluorescein probe (AFC₁₆) was localized to the apical plasma membranes of ectodermal and endodermal epithelial cells when presented in the culture medium or injected into the gastric lumen, but did not demarcate basolateral membranes. In longer term experiments, basolateral membranes were stained and the staining was independent of temperature conditions. A dual 18-carbon chain indocarbocyanine probe (DiIC₁₈) gradually diffused across the septate junction to label basolateral membranes at room temperature, but not at 4°C. DiIC₁₈ also filled and stained certain mounted nematocytes. The results indicate that in hydra, lipophilic probes may be limited in mobility within the membrane plane by the septate junctions in a manner similar to vertebrate tight junctions, and that apical membranes of mature nematocytes are differentially permeable.     

Comparative studies of the effect of thermal stimulation on the permeability of the luminal cell junctions of the sweat gland to lanthanum

Lanthanum injected intradermally in vivo into the skin of cattle, sheep, goats and ponies penetrated the intercellular spaces of the sweat glands. It was not, however, detected in the glandular lumen either visually or by electron probe microanalysis even at elevated ambient temperatures when the animals were sweating. It is concluded that the luminal intercellular connections between epithelial cells in these glands are tight junctions, which remain so during sweating despite the occurrence of cell death and extrusion into the lumen.     

Junctional complexes in fetal rat small intestine during morphogenesis

During the 7 days prior to birth (Days 15–22), the small-intestinal epithelium of the fetal rat changes from primitive stratified to simple columnar epithelium which lines villi at 19 days. As seen in thin sections, this remodeling involves rapid formation of new junctional complexes and secondary lumens between epithelial cells deep in the stratified epithelium. We have examined the formation and reorganization of junctional complexes in proximal small intestine of 15- to 19-day fetal rats using freeze-fracture techniques. On Days 15 and 16 the epithelial cells surrounding the primary lumen are joined by conventional apical junctional complexes. Additionally, macular junctional complexes are located on deeper epithelial cells. These display no polarity and consist of tight-junction strands intermixed with gap junction-like arrays and desmosomes. On Days 17 and 18 nonluminal, macular junctional complexes enlarge and secondary lumens develop within their centers. As the secondary lumens expand, microvilli appear and the junctional complex polarizes about the secondary lumen; tight-junction strands become parallel to the luminal surface, desmosomes migrate basolaterally, and gap junction-like arrays disappear. By Day 19, secondary lumens have fused with the primary lumen; concomitant loss of apical cells results in formation of villi lined by simple columnar epithelium with polarized apical tight junctions. The observed pattern of junctional complex formation may play a role in maintaining barrier function and establishing epithelial cell polarity as the epithelium is remodeled.     

Morphology of the ampullae of Lorenzini in juvenile freshwater Carcharhinus leucas

Ampullae of Lorenzini were examined from juvenile Carcharhinus leucas (831–1,045 mm total length) captured from freshwater regions of the Brisbane River. The ampullary organ structure differs from all other previously described ampullae in the canal wall structure, the general shape of the ampullary canal, and the apically nucleated supportive cells. Ampullary pores of 140–205 µm in diameter are distributed over the surface of the head region with 2,681 and 2,913 pores present in two sharks that were studied in detail. The primary variation of the ampullary organs appears in the canal epithelial cells which occur as either flattened squamous epithelial cells or a second form of pseudostratified contour‐ridged epithelial cells; both cell types appear to release material into the ampullary lumen. Secondarily, this ampullary canal varies due to involuted walls that form a clover‐like canal wall structure. At the proximal end of the canal, contour‐ridged cells abut a narrow region of cuboidal epithelial cells that verge on the constant, six alveolar sacs of the ampulla. The alveolar sacs contain numerous receptor and supportive cells bound by tight junctions and desmosomes. Pear‐shaped receptor cells that possess a single apical kinocilium are connected basally by unmyelinated neural boutons. Opposed to previously described ampullae of Lorenzini, the supportive cells have an apical nucleus, possess a low number of microvilli, and form a unique, jagged alveolar wall. A centrally positioned centrum cap of cuboidal epithelial cells overlies a primary afferent lateral line nerve. J. Morphol. 276:481–493, 2015. © 2014 Wiley Periodicals, Inc.     

Endocytosis in the uterine luminal and glandular epithelial cells of mice during early pregnancy

We have localized horseradish peroxidase (HRP) in the mouse uterus after intravenous administration on days 1 and 5 of pregnancy in an effort to understand how serum proteins reach the uterine lumen. Direct movement of HRP into uterine and glandular lumina was blocked by the epithelial tight junctions on both days. In luminal and glandular epithelial cells at both times, HRP was localized in endocytic vesicles along the basolateral membranes, multivesicular bodies (mvb), elongated dense bodies below the nucleus (bdb), and many small vesicles near the apical surface of the cells. The uptake of HRP was most extensive in the luminal epithelium on day 1: the number of tracer-containing apical vesicles and bdb was largest, and there were also clusters of vesicles containing the tracer above the nucleus. Acid phosphatase was localized on day 1 in mvb and bdb in both cell types, indicating that these structures are lysosomes. It appeared that HRP followed two pathways after basolateral endocytosis by the epithelial cells: it was transported to the apical region of the cells, where it was present in small vesicles that may release their contents into the uterine or glandular lumina, or it was transported to lysosomes. To investigate whether macromolecules may be transported from the uterine lumen to the stroma, we also studied endocytosis at the apical pole of luminal epithelial cells after intraluminal injection of HRP. There was no detectable uptake of HRP from the lumen on day 1, and no tracer was detected in the intercellular spaces or basement membrane region. On day 5, a large amount of HRP was taken up from the lumen into apical endocytic vesicles, mvb, and dense bodies, but tracer was not present in the Golgi apparatus, lateral intercellular spaces, or the basement membrane region at the times studied. These observations indicate that there was no transport of luminal macromolecules to the uterine stroma on day 1, while the possibility of transport on day 5 requires further study.     

Listeria monocytogenes invades the epithelial junctions at sites of cell extrusion

Listeria monocytogenes causes invasive disease by crossing the intestinal epithelial barrier. This process depends on the interaction between the bacterial surface protein Internalin A and the host protein E-cadherin, located below the epithelial tight junctions at the lateral cell-to-cell contacts. We used polarized MDCK cells as a model epithelium to determine how L. monocytogenes breaches the tight junctions to gain access to this basolateral receptor protein. We determined that L. monocytogenes does not actively disrupt the tight junctions, but finds E-cadherin at a morphologically distinct subset of intercellular junctions. We identified these sites as naturally occurring regions where single senescent cells are expelled and detached from the epithelium by extrusion. The surrounding cells reorganize to form a multicellular junction that maintains epithelial continuity. We found that E-cadherin is transiently exposed to the lumenal surface at multicellular junctions during and after cell extrusion, and that L. monocytogenes takes advantage of junctional remodeling to adhere to and subsequently invade the epithelium. In intact epithelial monolayers, an anti-E-cadherin antibody specifically decorates multicellular junctions and blocks L. monocytogenes adhesion. Furthermore, an L. monocytogenes mutant in the Internalin A gene is completely deficient in attachment to the epithelial apical surface and is unable to invade. We hypothesized that L. monocytogenes utilizes analogous extrusion sites for epithelial invasion in vivo. By infecting rabbit ileal loops, we found that the junctions at the cell extrusion zone of villus tips are the specific target for L. monocytogenes adhesion and invasion. Thus, L. monocytogenes exploits the dynamic nature of epithelial renewal and junctional remodeling to breach the intestinal barrier.     

Programmed cell death and heterolysis of larval epithelial cells by macrophage-like cells in the anuran small intestine in vivo and in vitro.

The degenerative processes in the larval small intestine of Xenopus laevis tadpoles during spontaneous metamorphosis and during thyroid hormone-induced metamorphosis in vitro were examined by electron microscopy. Around the beginning of spontaneous metamorphic climax (stages 59-61), both apoptotic bodies derived from larval epithelial cells and intraepithelial macrophage-like cells suddenly increase in number. The macrophage-like cells become rounded and enlarged because of numerous vacuoles containing the apoptotic bodies. Mitotic profiles of the macrophage-like cells, however, are localized in the connective tissue where different developmental stages of macrophage-like cells are present. After stage 62, the intraepithelial macrophage-like cells decrease in number, while large macrophage-like cells which include the apoptotic bodies and retain intact cell membranes and nuclei appear in the lumen. Degenerative changes similar to those during spontaneous metamorphosis described above could be reproduced in vitro. In tissue fragments isolated from the small intestine of stage 57 tadpoles and cultured in the presence of thyroid hormone, the number of intraepithelial macrophage-like cells reaches its maximum around the 3rd day of cultivation when the larval epithelial cells most rapidly decrease in number. These results suggest that the rapid degeneration of larval epithelial cells occurs not only because of apoptosis of the epithelial cells themselves but also from heterolysis by macrophages. The macrophages probably originate in the connective tissue, actively proliferate, migrate into the larval epithelium around the beginning of metamorphic climax, and are finally extruded into the lumen.     

A Mechanical Instability in Planar Epithelial Monolayers Leads to Cell Extrusion

In cell extrusion, a cell embedded in an epithelial monolayer loses its apical or basal surface and is subsequently squeezed out of the monolayer by neighboring cells. Cell extrusions occur during apoptosis, epithelial-mesenchymal transition, or precancerous cell invasion. They play important roles in embryogenesis, homeostasis, carcinogenesis, and many other biological processes. Although many of the molecular factors involved in cell extrusion are known, little is known about the mechanical basis of cell extrusion. We used a three-dimensional (3D) vertex model to investigate the mechanical stability of cells arranged in a monolayer with 3D foam geometry. We found that when the cells composing the monolayer have homogeneous mechanical properties, cells are extruded from the monolayer when the symmetry of the 3D geometry is broken because of an increase in cell density or a decrease in the number of topological neighbors around single cells. Those results suggest that mechanical instability inherent in the 3D foam geometry of epithelial monolayers is sufficient to drive epithelial cell extrusion. In the situation in which cells in the monolayer actively generate contractile or adhesive forces under the control of intrinsic genetic programs, the forces act to break the symmetry of the monolayer, leading to cell extrusion that is directed to the apical or basal side of the monolayer by the balance of contractile and adhesive forces on the apical and basal sides. Although our analyses are based on a simple mechanical model, our results are in accordance with observations of epithelial monolayers in vivo and consistently explain cell extrusions under a wide range of physiological and pathophysiological conditions. Our results illustrate the importance of a mechanical understanding of cell extrusion and provide a basis by which to link molecular regulation to physical processes.     

Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical- basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein

Tight junctions, the most apical of the intercellular junctions that connect individual cells in a epithelial sheet, are thought to form a seal that restricts paracellular and intramembrane diffusion. To analyze the functioning of tight junctions, we generated stable MDCK strain 2 cell lines expressing either full-length or COOH-terminally truncated chicken occludin, the only known transmembrane component of tight junctions. Confocal immunofluorescence and immunoelectron microscopy demonstrated that mutant occludin was incorporated into tight junctions but, in contrast to full-length chicken occludin, exhibited a discontinuous junctional staining pattern and also disrupted the continuous junctional ring formed by endogenous occludin. This rearrangement of occludin was not paralleled by apparent changes in the junctional morphology as seen by thin section electron microscopy nor apparent discontinuities of the junctional strands observed by freeze-fracture. Nevertheless, expression of both wild-type and mutant occludin induced increased transepithelial electrical resistance (TER). In contrast to TER, particularly the expression of COOH-terminally truncated occludin led to a severalfold increase in paracellular flux of small molecular weight tracers. Since the selectivity for size or different types of cations was unchanged, expression of wild-type and mutant occludin appears to have activated an existing mechanism that allows selective paracellular flux in the presence of electrically sealed tight junctions. Occludin is also involved in the formation of the apical/basolateral intramembrane diffusion barrier, since expression of the COOH-terminally truncated occludin was found to render MDCK cells incapable of maintaining a fluorescent lipid in a specifically labeled cell surface domain.     

Shigella flexneri regulates tight junction-associated proteins in human intestinal epithelial cells

Shigella spp. are a group of Gram-negative enteric bacilli that cause acute dysentery in humans. We demonstrate that Shigella flexneri has evolved the ability to regulate functional components of tight junctions after interaction at the apical and basolateral pole of model intestinal epithelia. In the regulation of tight junctional protein assemblies, S. flexneri can engage serotype-specific mechanisms, which targets not only expression, but also cellular distribution and membrane association of components of tight junctions. Distinct mechanisms resulting in the regulation of tight junction-associated proteins are initiated after either apical or basolateral interactions. S. flexneri serotype 2a has the ability to remove claudin-1 from Triton X-insoluble protein fractions upon apical exposure to T-84 cell monolayers. S. flexneri serotype 2a and 5, but not the non-invasive Escherichia coli strain F-18, share the ability to regulate expression of ZO-1, ZO-2, E-cadherin and to dephosphorylate occludin. The disruption of tight junctions is dependent on direct interaction of living Shigella with intestinal epithelial cells and is supported by heat-stable secreted bacterial products. Intestinal epithelial cells have the ability to compensate in part for S. flexneri induced regulation of tight junction-associated proteins.     

Fine structure of the spermatheca and of the accessory glands in Orchesella villosa (Collembola, Hexapoda)

The spermatheca and the accessory glands of the collembolan Orchesella villosa are described for the first time. Both organs exhibit ultrastructural differences, according to the time of the intermolt in which the specimens were observed. A thick cuticular layer lines the epithelial cells of the accessory glands. In the reproductive phase, they are involved in secretory activity; a moderately dense secretion found in the apical cell region opens into the gland lumen. Cells with an extracellular cistern are intermingled with the secretory cells. These cells could be involved in fluid secretion, with the secretory product opening into the cistern which is filled with an electron-transparent material. After the reproductive phase, the gland lumen becomes filled with a dense secretion. The accessory gland secretion may play a protective role towards the eggs. The spermatheca is located between the accessory glands; its epithelium is lined by a thin cuticle forming spine-like projections into the lumen and consists of cells provided with an extracellular cistern. Secretory cells, similar to those seen in the accessory glands, are missing. Cells with a cistern could be involved in the production of a fluid secretion determining sperm unrolling and sperm motility.     

Polarity complex proteins

The formation of functional epithelial tissues involves the coordinated action of several protein complexes, which together produce a cell polarity axis and develop cell-cell junctions. During the last decade, the notion of polarity complexes emerged as the result of genetic studies in which a set of genes was discovered first in Caenorhabditis elegans and then in Drosophila melanogaster. In epithelial cells, these complexes are responsible for the development of the apico-basal axis and for the construction and maintenance of apical junctions. In this review, we focus on apical polarity complexes, namely the PAR3/PAR6/aPKC complex and the CRUMBS/PALS1/PATJ complex, which are conserved between species and along with a lateral complex, the SCRIBBLE/DLG/LGL complex, are crucial to the formation of apical junctions such as tight junctions in mammalian epithelial cells. The exact mechanisms underlying their tight junction construction and maintenance activities are poorly understood, and it is proposed to focus in this review on establishing how these apical polarity complexes might regulate epithelial cell morphogenesis and functions. In particular, we will present the latest findings on how these complexes regulate epithelial homeostasis.     

Cell polarity development and protein trafficking in hepatocytes lacking E-cadherin/beta-catenin-based adherens junctions

Using a mutant hepatocyte cell line in which E-cadherin and beta-catenin are completely depleted from the cell surface, and, consequently, fail to form adherens junctions, we have investigated adherens junction requirement for apical-basolateral polarity development and polarized membrane trafficking. It is shown that these hepatocytes retain the capacity to form functional tight junctions, develop full apical-basolateral cell polarity, and assemble a subapical cortical F-actin network, although with a noted delay and a defect in subsequent apical lumen remodeling. Interestingly, whereas hepatocytes typically target the plasma membrane protein dipeptidyl peptidase IV first to the basolateral surface, followed by its transcytosis to the apical domain, hepatocytes lacking E-cadherin-based adherens junctions target dipeptidyl peptidase IV directly to the apical surface. Basolateral surface-directed transport of other proteins or lipids tested was not visibly affected in hepatocytes lacking E-cadherin-based adherens junctions. Together, our data show that E-cadherin/beta-catenin-based adherens junctions are dispensable for tight junction formation and apical lumen biogenesis but not for apical lumen remodeling. In addition, we suggest a possible requirement for E-cadherin/beta-catenin-based adherens junctions with regard to the indirect apical trafficking of specific proteins in hepatocytes.     

Polarity complex proteins

The formation of functional epithelial tissues involves the coordinated action of several protein complexes, which together produce a cell polarity axis and develop cell-cell junctions. During the last decade, the notion of polarity complexes emerged as the result of genetic studies in which a set of genes was discovered first in Caenorhabditis elegans and then in Drosophila melanogaster. In epithelial cells, these complexes are responsible for the development of the apico-basal axis and for the construction and maintenance of apical junctions. In this review, we focus on apical polarity complexes, namely the PAR3/PAR6/aPKC complex and the CRUMBS/PALS1/PATJ complex, which are conserved between species and along with a lateral complex, the SCRIBBLE/DLG/LGL complex, are crucial to the formation of apical junctions such as tight junctions in mammalian epithelial cells. The exact mechanisms underlying their tight junction construction and maintenance activities are poorly understood, and it is proposed to focus in this review on establishing how these apical polarity complexes might regulate epithelial cell morphogenesis and functions. In particular, we will present the latest findings on how these complexes regulate epithelial homeostasis.     

Hepatocyte nuclear factor (HNF)-4alpha triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells

F9 murine embryonal carcinoma cells provide an attractive system for facilitating molecular mechanisms for epithelial morphogenesis, since they have the capability of differentiating into polarized epithelial cells bearing an apical junctional complexes. We previously showed that a specific retinoid X receptor-retinoic acid receptor heterodimer transduced retinoid signals for biogenesis of functional tight junctions in F9 cells (Exp. Cell Res. 263, (2001) 163). In the present study we generated F9 cells expressing doxycycline-inducible hepatocyte nuclear factor (HNF)-4alpha, a nuclear receptor. We herein show that induction of HNF-4alpha initiates differentiation of F9 cells to polarized epithelial cells, in which tight-junction proteins occludin, claudin-6, claudin-7, and ZO-1 are concentrated at the apical-most regions of lateral membranes. Expression of occludin, claudin-6, and claudin-7 was induced in the cells by doxycycline treatment in a dose- and time-dependent manner, in terms of the amount of HNF-4alpha. In contrast, expression levels of ZO-1, ZO-2, E-cadherin, and beta-catenin were not altered by HNF-4alpha. We also demonstrate, by analysis of diffusion of labeled sphingomyelin, that the fence function of tight junctions is achieved by induction of HNF-4alpha. These findings indicate that HNF-4alpha triggers de novo formation of functional tight junctions and establishment of epithelial cell polarity.