Effect of adrenaline on transmesothelial resistance of isolated sheep pleura

The effect of adrenaline on the transmesothelial resistance (RTM) of sheep's visceral and parietal pleura was studied using the Ussing chamber technique. Basal transmesothelial resistance of visceral pleura was found to be 20.71 +/- 0.31 Omega cm2, whereas that of parietal pleura was found to be 19.53 +/- 0.34 Omega cm2. Immediately after the addition of adrenaline (10(-7) M) both apically and basolaterally on the visceral and parietal pleura, these values were significantly increased (P < 0.05). Addition of the nonselective beta-receptor blocker, propranolol (10(-5) M), suppressed this effect in both visceral and parietal pleura, while addition of the nonselective alpha-receptor blocker, phentolamine (10(-5) M), partly suppressed the above-mentioned increase in the parietal pleura. In conclusion, our results show that adrenaline has a rapid effect on both pleurae. This rapid effect is mediated by the stimulation of beta-adrenergic receptors in the case of visceral pleura, while in the case of parietal pleura this effect seems to be due to a stimulation of alpha- and beta-adrenergic receptors. On the visceral pleura the effect of adrenaline vanishes after some minutes and on the parietal this effect is more permanent than the visceral's one, suggesting differences in the distribution of the adrenergic receptors between the visceral and parietal pleura.     

Acid modulates the squamous epithelial barrier function by modulating the localization of claudins in the superficial layers

Acid is a major cause of gastro-esophageal reflux disease. However, the influence of acid on the esophageal stratified epithelial barrier function and tight junction (TJ) proteins is not fully understood. Here, we explore the influence of acid on barrier function and TJ proteins using a newly developed model of the esophageal-like squamous epithelial cell layers that employs an air-liquid interface (ALI) system. Barrier function was determined by measuring trans-epithelial electrical resistance (TEER) and diffusion of paracellular tracers. TJ-related protein (claudin-1, claudin-4, occludin and ZO-1) expression and localization was examined by immunofluorescent staining, and by western blotting of 1% NP-40 soluble and insoluble fractions. We also examined the influence of acid (pH 2-4) on the barrier created by these cells. The in vitro ALI culture system showed a tight barrier (1500-2500 Ω·cm(2)) with the expression of claudin-1, claudin-4, occludin and ZO-1 in the superficial layers. Claudin-1, claudin-4, occludin and ZO-1 were detected as dots and whisker-like lines in the superficial layers, and as a broad line in the suprabasal layers. These localization patterns are similar to those in the human esophagus. On day 7 under ALI culture, TJ proteins were detected in the superficial layers with functional properties, including decreased permeability and increased TEER. Dilated intercellular spaces were detected at the suprabasal cell layers even under the control conditions of ALI cells. pH 2 acid on the apical side significantly reduced the TEER in ALI-cultured cells. This decrease in TEER by the acid was in parallel with the decreased amount of detergent-insoluble claudin-4. Claudin-4 delocalization was confirmed by immunofluorescent staining. In conclusion, TJs are located in the superficial layers of the esophagus, and acid stimulation disrupts barrier function, at least in part by modulating the amount and localization of claudin-4 in the superficial layers.     

Tight junction composition is altered in the epithelium of polycystic kidneys

Kidney cysts in autosomal dominant polycystic kidney disease (ADPKD) undergo progressive enlargement together with luminal fluid secretion. This involves active, uphill transcellular Cl(-) transport which drives passive Na(+) and water secretion. Implicit in this mechanism is the assumption that the paracellular permeability of the cyst epithelium to Cl(-) must be very low. Claudins are tight junction (TJ) transmembrane proteins that determine the ion selectivity of paracellular barriers. The aim of this study was to determine the expression and localization of claudins within renal cysts in a mouse hypomorphic model of ADPKD and in human patients. We found that the majority of cysts were of collecting duct origin. Claudins normally expressed in collecting duct (3, 4, 7, 8, and 10) were found in small cysts. However, only claudin-7 persisted at substantive levels in the dedifferentiated epithelium of large, presumably late-stage cysts, where it was localized both at the TJ and basolaterally. The constitutively expressed TJ proteins, ZO-1 and occludin, were also abundantly expressed and correctly localized, suggesting that the basic infrastructure of the TJ is preserved. A previous study suggested that claudin-7 may function as a paracellular Cl(-) barrier. We postulate that the role of claudin-7 in ADPKD is to seal the paracellular route in Cl(-)-secreting cyst epithelium, preventing backleak of Cl(-), and that it thereby plays a permissive role in fluid secretion and cyst growth.     

A key claudin extracellular loop domain is critical for epithelial barrier integrity

Intercellular tight junctions (TJs) regulate epithelial barrier properties. Claudins are major structural constituents of TJs and belong to a large family of tetra-spanning membrane proteins that have two predicted extracellular loops (ELs). Given that claudin-1 is widely expressed in epithelia, we further defined the role of its EL domains in determining TJ function. The effects of several claudin-1 EL mimetic peptides on epithelial barrier structure and function were examined. Incubation of model human intestinal epithelial cells with a 27-amino acid peptide corresponding to a portion of the first EL domain (Cldn-1(53-80)) reversibly interfered with epithelial barrier function by inducing the rearrangement of key TJ proteins: occludin, claudin-1, junctional adhesion molecule-A, and zonula occludens-1. Cldn-1(53-80) associated with both claudin-1 and occludin, suggesting both the direct interference with the ability of these proteins to assemble into functional TJs and their close interaction under physiological conditions. These effects were specific for Cldn-1(53-80), because peptides corresponding to other claudin-1 EL domains failed to influence TJ function. Furthermore, the oral administration of Cldn-1(53-80) to rats increased paracellular gastric permeability. Thus, the identification of a critical claudin-1 EL motif, Cldn-1(53-80), capable of regulating TJ structure and function, offers a useful adjunct to treatments that require drug delivery across an epithelial barrier.     

Disruption of tight junctions during polymicrobial sepsis in vivo

The disruption of intestinal epithelial tight junctions may result in barrier function dysfunction during polymicrobial sepsis. The pathophysiology of sepsis involves breakdown of barrier integrity, which correlates with adverse outcome during sepsis. However, the mechanisms underlying loss of barrier function in sepsis remain unknown. In the present study in mice, tight junction (TJ) structure was analysed by transmission electron microscopy; intestinal permeability was assessed using molecular tracer measurement; and the distribution of TJ proteins was investigated by immunofluorescence microscopy. The membrane microdomains of TJs were isolated using discontinuous sucrose density gradients and the expression of TJ proteins in these was determined by western blot. Immunofluorescence microscopy revealed that claudins 1, 3, 4, 5, and 8 were present predominantly in the microvillous surface of epithelial cells and along the lateral membranes of the cells; in sepsis, however, labelling of these proteins was present diffusely within cells and was no longer focused at the lateral cell boundaries. Moreover, the expression of claudin‐2 was markedly up‐regulated in sepsis. Using western blot analysis, we found that occludin and claudins were displaced from raft fractions to non‐raft fractions in membrane microdomains of TJs in sepsis. In addition, the disruption of TJ structure was accompanied by increased intestinal permeability. Our results demonstrate for the first time that redistribution of TJ proteins in TJ membrane microdomains and redistribution of claudins in epithelial cells of the colon lead to alteration of TJ architecture and TJ barrier dysfunction during the development of polymicrobial sepsis. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Different changes in the expression of multiple kinds of tight-junction proteins during ischemia-reperfusion injury of the rat ileum

Dysfunction of tight junctions (TJs), located at the most apical part of the intestinal epithelium, is believed to result in various complications in intestinal disease. However, the behaviors of multiple kinds of TJ proteins during ischemia-reperfusion injury are not understood in detail. To determine changes in expression and localization of TJ proteins during intestinal-barrier recovery, we induced intestinal ischemia-reperfusion injury in rats, measured mucosa-to-blood permeability of fluorescein isothiocyanate-dextran-4 kDa, and compared it with spatiotemporal changes of ZO-1, occludin, and claudin-1, -2, -3, -4, and -5 by immunoconfocal microscopy. At 1 hour post-reperfusion, villi were denuded and intestinal-barrier function was lost. From 6 to 24 hours post-reperfusion, villous epithelium was restored by cell migration, and barrier function together with reticular pattern expression of ZO-1, occludin, and claudin-1, -3, and -5, recovered time-dependently. To the contrary, after ischemia-reperfusion injury, the localized expression of claudin-2 and claudin-4 observed in the non-treated control was lost and replaced with broader expression from crypts to villi with increased basolateral claudin-4 expression in epithelial cells. These data demonstrated that recovery of intestinal barrier function is associated with expression of ZO-1, occludin, and claudin-1, -3, and -5, whereas claudin-2 and claudin-4 show unique changes in expression and localization.     

Diagnostic utility of expression of claudins in non-small cell lung cancer: Different expression profiles in squamous cell carcinomas and adenocarcinomas

The claudins, members of a large family of adherent junction proteins, regulate the integrity and function of tight junctions. At present, at least 23 different claudins are known to exist in humans, and claudin gene expression is frequently altered in several human cancers. However, few studies have examined the expression of claudins in lung cancer. This study examined the expression of claudin-1, claudin-3, claudin-4, and claudin-5 proteins using immunohistochemical analysis in 14 normal lung tissue samples and 171 NSCLC samples. All of the claudin proteins examined were expressed in normal bronchial epithelial cells. In the normal peripheral parenchyma, only claudin-5 strongly stained most of the pneumocytes. Claudin-1 expression was stronger in squamous cell carcinomas than in adenocarcinomas, whereas claudin-4 and claudin-5 expression was stronger in adenocarcinomas. Clinically, expression of claudin proteins was not found to be associated with patient survival. These data suggest that the disruption of tight junction protein might be involved in the development of these tumors. Immunohistochemical evaluation of the different expression patterns of claudins in NSCLC suggests that claudin-4, in addition to 1 and 5, might be a useful differential diagnostic marker in Korean people.     

Yersinia enterocolitica induces epithelial barrier dysfunction through regional tight junction changes in colonic HT-29/B6 cell monolayers

Yersinia enterocolitica is a common cause of acute gastroenteritis. This study aimed to clarify the mechanisms leading to barrier dysfunction and diarrhea. Exposure of human colonic HT-29/B6 cells to Y. enterocolitica resulted in a decrease in transepithelial resistance from 404±23 to 163±21 Ω cm2 (P<0.001) in parallel with an increase in mannitol (182?Da) and fluorescein (332?Da) permeability, whereas short circuit current did not change. This effect was time dependent, required the presence of living bacteria, could not be triggered by bacterial supernatants and was not due to Yersinia outer proteins. Concomitantly, Y. enterocolitica induced necrosis as indicated by an increase in lactate dehydrogenase-release, whereas epithelial apoptosis was not upregulated. Local changes in conductivity were detected by conductance scanning, indicating 'leaky regions' within the epithelium that were visualized by biotinylation and confocal microscopy. In these regions, claudin-3 and -4 and, especially claudin-8, were redistributed off the tight junction (TJ) into the cytoplasm. In addition, the expression of claudin-2, -3, -8, -10 and ZO-1 was diminished as quantified by immunoblotting. Moreover, we found claudin-8 to be regulated by the c-Jun N-terminal kinase, the inhibition of which attenuated the Y. enterocolitica-induced decrease in transepithelial resistance and restored claudin-8 protein level. In conclusion, barrier dysfunction in Y. enterocolitica infection is due to circumscribed epithelial TJ protein changes and necrotic cell loss, as a consequence of which leak flux diarrhea and antigen-uptake provoking extraintestinal arthritis may be triggered.     

Loss of claudins-1 and -7 and expression of claudins-3 and -4 correlate with prognostic variables in prostatic adenocarcinomas

Claudins, members of a large family of adherent junction proteins, regulate the integrity and function of tight junctions and influence tumorigenesis. Recent studies have shown that altered levels of the different claudins may be related to invasion and progression of carcinoma cells in several primary neoplasms. However, there is no reported study documenting the pattern of claudin expression in prostatic adenocarcinomas (PACs). Formalin-fixed paraffin-embedded sections from 141 PACs were immunostained by manual and automated methods on the Xmatrx (BioGenex, San Ramon, CA) using antihuman claudin-1, -3, -4, -5, and -7 antibodies (Zymed, San Francisco, CA). Membranous immunoreactivity for each protein was semiquantitatively scored in both the tumor and adjacent benign epithelium in each case. Results were correlated with clinicopathologic variables. Variable membranous positivity was noted in the adjacent benign glands for all 5 proteins in all cases. PACs showed variable membranous positivity ranging from decreased, similar to, and increased in relation to the adjacent benign epithelium for all claudins. Decreased expression of claudin-1 correlated with high tumor grade (P = .001) and biochemical disease recurrence (P = .01), whereas decreased claudin-7 correlated with high tumor grade (P < .0001). In contrast, expression of claudin-3 correlated with advanced stage tumors (P = .03) and recurrence (P = .02), and expression of claudin-4 correlated with advanced stage (P = .02). On multivariate analysis, advanced stage (P = .026) and decreased claudin-1 protein expression (P = .005) independently predicted disease recurrence. Immunohistochemical expression and prognostic significance of claudins are variable in PACs, with decreased expression of claudin-1 emerging as an independent prognostic variable warranting further study.     

Expression of occludin and claudins 1, 3, 4, and 7 in urothelial carcinoma of the upper urinary tract

The contacts between epithelial cells are maintained mainly by adherens junctions and tight junctions (TJs). However, the role of TJ proteins in cancer is not well understood. We studied the expression of occludin and 4 claudins to assess their importance in the progression of urothelial carcinoma of the upper urinary tract (UC-UUT). In 129 cases, we examined their expression using immunohistochemical analysis and also their relationships to clinicopathologic parameters and clinical outcome. Positive expression of occludin and claudins 1, 3, 4, and 7 were recognized in 117 (90.7%), 113 (87.6%), 95 (73.6%), 127 (98.4%), and 123 (95.3%) of tumor samples, respectively. Claudin-3 expression was significantly associated with stage, grade, and pattern of growth. Claudins 1 and 4 expression was significantly associated with stage. However, neither occludin nor claudin-7 expression was associated with clinicopathologic findings. When tumors with scores below the median for a given protein were classified as the "low expression group," univariate analysis of overall survival revealed that claudins 1 and 3 had a significant effect on overall survival. Detection of claudins 1, 3, and 4 would seem to provide valuable information about the progression of UC-UUT.     

Changes in the phosphorylation of claudins during the course of experimental colitis

The phosphorylation of the tight-junction protein claudin causes allosterism, endocytosis and changes in the polarity of the epithelium, thus affecting the barrier function. The phosphorylation status of claudin during the course of colitis has not been demonstrated. In the present study, we found that the phosphorylated claudin-4 and claudin-7 contents were increased in experimental colitis at days 6 and 8, and colonic phosphorylated claudin-6 was found to be increased at day 4 and day 8. Colonic phosphorylated claudin-5 was found to be decreased at day 4 but increased at day 6. These changes were accompanied by increases in intestinal permeability. In T84 cells, phosphorylated claudin-3 was increased at 48 h but decreased at 72 h after lipopolysaccharide (LPS) treatment. Phosphorylated claudin-5 and claudin-7 were decreased 72 h after LPS treatment, while phosphorylated claudin-6 was increased at 72 h after LPS treatment. We conclude that the phosphorylation of colonic claudins was changed during the course of colitis, which may be related to the change in the intestinal barrier function. Cytokine such as LPS was found to affect the phosphorylation of colonic claudins.     

Na+-glucose cotransporter is also expressed in mesothelium of species with thick visceral pleura

Molecular evidence for Na+-glucose cotransporter (SGLT1) in rabbit pleural mesothelium has been recently provided, confirming earlier functional findings on solute-coupled liquid absorption from rabbit pleural space. In this research we checked whether SGLT1 is also expressed in pleural mesothelium of species with thick visceral pleura, which receives blood from systemic circulation, but drains it into pulmonary veins. To this end immunoblot assays were performed on total protein extract of scraped visceral and parietal mesothelium of lambs and adult sheep, and of a human mesothelial cell line. All of them showed SGLT1 specific bands. Moreover, confocal immunofluorescence images of lamb pleural mesothelium showed that SGLT1 is located in apical membrane. Therefore, a solute-coupled liquid absorption should also occur from pleural space of species with thick visceral pleura. Because of this protein-free liquid entering interstitium between visceral mesothelium and capillaries, inherent Starling forces should be different than hitherto considered, and visceral pleura capillaries could absorb liquid even in these species.     

Neutrophil Transmigration in Inflammatory Bowel Disease Is Associated with Differential Expression of Epithelial Intercellular Junction Proteins

Inflammatory bowel disease (IBD) consisting of ulcerative colitis (UC) and Crohn's (CD) typically displays a waxing and waning course punctuated by disease flares that are characterized by transepithelial migration of neutrophils (PMN) and altered barrier function. Since epithelial barrier function is primarily regulated by the apical most intercellular junction referred to as the tight junction (TJ), our aim was to examine expression of TJ and adherens junction (AJ) proteins in relation to PMN infiltration in mucosal tissue samples from patients with active IBD. Expression of epithelial intercellular TJ proteins (occludin, ZO-1, claudin-1, and JAM) and subjacent AJ (beta-catenin and E-cadherin) proteins were examined by immunoflourescence/confocal microscopy, immunohistochemistry, and Western blotting. Colonic mucosa from patients with UC revealed dramatic, global down-regulation of the key TJ transmembrane protein occludin in regions of actively transmigrating PMN and in quiescent areas in the biopsy samples. Significant decreases in occludin expression were observed at the protein and mRNA levels by Western and Northern blotting. In contrast, expression of other TJ and AJ proteins such as ZO-1, claudin-1, JAM, beta-catenin, and E-cadherin were down-regulated only in epithelial cells immediately adjacent to transmigrating PMN. Analysis of inflamed mucosa from Crohn's disease patients mirrored the results obtained with UC patients. No change in TJ and AJ protein expression was observed in colonic epithelium from patients with collagenous colitis or lymphocytic colitis that are respectively characterized by a thickened subepithelial collagen plate and increased intraepithelial lymphocytes. These results suggest that occludin expression is diminished in IBD by mechanisms distinct from those regulating expression of other intercellular junction proteins. We speculate that down-regulation of epithelial occludin may play a role in enhanced paracellular permeability and PMN transmigration that is observed in active inflammatory bowel disease.     

The contribution of ascorbic acid and dehydroascorbic acid to the protective role of pleura during inflammatory reactions

It is well-known that parapneumonic effusions lead to the formation of inflammatory exudates which contain an increasing amount of inflammatory cells, especially polymorphonuclear. At these pathological conditions characterized by oxidative stress, ascorbic acid (AA) plays an important role in quenching free radicals, so that it could protect neutrophils and mesothelial cells from oxidative damage. Besides that ascorbic acid and its metabolite dehydroascorbic acid (DHA) alters the sheep visceral and parietal pleura permeability. More specific ascorbic acid as well as dehydroascorbic acid decreases the permeability of pleura after addition on apical and basolateral side in both visceral and parietal pleurae. It seems that, AA and DHA have an opposite action upon pleura from that of the inflammatory mediators, like VEGF, which increases the permeability of pleura and causes mesothelial barrier dysfunction. The decrease of pleura permeability induced by AA and DHA suggest the hypothesis that AA and/or its metabolite DHA during inflammatory reactions not only protects mesothelial cells from oxidative damage, but also contributes to maintaining the mesothelial barrier function. Consequently, the inflammatory pleural fluid may be trapped in pleural space and the inflammation may be restricted, and have extension avoided.     

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.     

Comparative analysis of theophylline and cholera toxin in rat colon reveals an induction of sealing tight junction proteins

Claudin tight junction proteins have been identified to primarily determine intestinal epithelial barrier properties. While functional contribution of single claudins has been characterized in detail, information on the interplay with secretory mechanisms in native intestinal epithelium is scarce. Therefore, effects of cholera toxin and theophylline on rat colon were analyzed, including detection of sealing claudins. Tissue specimens were stripped off submucosal tissue layers and mounted in Ussing chambers, and short-circuit current (I_SC) and transepithelial resistance (TER) were recorded. In parallel, expression and localization of claudins was analyzed and histological studies were performed employing hematoxylin-eosin staining and light and electron microscopy. Theophylline induced a strong increase of I_SC in colon tissue specimens. In parallel, a decrease of TER was observed. In contrast, cholera toxin did not induce a significant increase of I_SC, whereas an increase of TER was detected after 120 min. Western blots of membrane fractions revealed an increase of claudin-3 and -4 after incubation with cholera toxin, and theophylline induced an increase of claudin-4. In accordance, confocal laser-scanning microscopy exhibited increased signals of claudin-3 and -4 after incubation with cholera toxin, and increased signals of claudin-4 after incubation with theophylline, within tight junction complexes. Morphological analyses revealed no general changes of tight junction complexes, but intercellular spaces were markedly widened after incubation with cholera toxin and theophylline. We conclude that cholera toxin and theophylline have different effects on sealing tight junction proteins in native colon preparations, which may synergistically contribute to transport functions, in vitro.     

The structure of the parietal pleura and its relationship to pleural liquid dynamics in sheep

We studied the parietal pleura of six sheep to obtain information on pleura structure, blood supply, and lymphatic drainage. In the strict sense, the parietal pleura is composed of a single layer of mesothelial cells and a uniform layer of loose, irregular connective tissue (about 23 μm in width) subjacent to the mesothelial cells. The parietal pleural blood vessels are 10–15 μm from the pleural space. Tracer substances put in the pleural space are removed at specific locations. Colloidal carbon and chick red blood cells are cleared by teh parietal pleural lymphatics located over the intercostal spaces at the caudal end of the thoracic wall and over the lateral sides of the pericardial sac. In these areas the mesothelial cells have specialized openings, the stomata, that directly communicate with the underlying lymphatic lacunae. Cells and particulate matter in the pleural space are cleared only by the parietal pleural lymphatics. Compared to the visceral pleura, we believe the thinness of the parietal pleura, the closeness of its blood vessels to the pleural space, and its specialized lymphatic clearance pathways, together indicate that the parietal pleura plays a major role in pleural liquid and protein dynamics in sheep.