Investigation into the signal transduction pathway via which heat stress impairs intestinal epithelial barrier function

BACKGROUND AND AIMS: Intact protein absorption is thought to be a causative factor in several intestinal diseases, such as food allergy, celiac disease and inflammatory bowel disease. However, the mechanism remains unclear. The aim of this study was to characterize a novel signal transduction pathway via which heat stress compromises intestinal epithelial barrier function. METHODS: Heat stress was carried out by exposing confluent human intestinal epithelial cell line T84 cell monolayers to designated temperatures (37-43 degrees C) for 1 h. Transepithelial electric resistance (TER) and permeability to horseradish peroxidase (HRP, molecular weight = 44 000) were used as indicators to assess the intestinal epithelial barrier function. Phosphorylated myosin light chain (MLC), MLC kinase (MLCK) and protein kinase C (PKC) protein of the T84 cells were evaluated in order to identify the signal transduction pathway in the course of heat stress-induced intestinal epithelial barrier dysfunctions. RESULTS: The results showed that exposure to heat stress significantly increased intact protein transport across the intestinal epithelial monolayer; the amount of phospho-PKC, phospho-MLCK and phospho-MLC proteins in T84 cells decreased significantly at 41 degrees C and 43 degrees C although they increased at 39 degrees C. The heat stress-induced T84 monolayer barrier dysfunction was inhibited by pretreatment with PKC inhibitor, MLCK inhibitor, or HSP70. CONCLUSION: Heat stress can induce intestinal epithelial barrier dysfunction via the PKC and MLC signal transduction pathway.     

Campylobacter and IFNgamma interact to cause a rapid loss of epithelial barrier integrity

BACKGROUND: The intestinal epithelium is a single layer of polarized cells and is the primary barrier separating foreign antigen and underlying lymphoid tissue. IFNgamma alters epithelial barrier function during inflammation by disrupting tight cell junctions and facilitating the paracellular transport of luminal antigens. The aim of this work was to determine whether Campylobacter infection of cells exposed to IFNgamma would lead to greater disruption of cell monolayers and hence increased bacterial translocation. METHODS: Monolayers were polarized on Transwell polycarbonate membranes for 14 days and then cultured in the presence or absence of 100 U/mL IFNgamma. Campylobacter was added to the apical side of the monolayer at an MOI of 30. Transepithelial electrical resistance (TEER) was recorded and bacteria in the basal well counted every 2 hours. Cells were stained for occludin, actin, and nuclear DNA, and cell viability determined by measurement of apoptosis. RESULTS: In the presence of IFNgamma, TEER dropped significantly after 18 hours, indicating a reduction in barrier function. A further significant decrease was seen in the presence of both IFNgamma and Campylobacter, indicating a synergistic effect, and cellular morphology and viability were affected. Bacterial translocation across the monolayer was also significantly greater in the presence of IFNgamma. CONCLUSIONS: These combined effects indicate that Campylobacter infection concomitant with intestinal inflammation would result in a rapid and dramatic loss of epithelial barrier integrity, which may be a key event in the pathogenesis of Campylobacter-mediated colitis and the development of bloody diarrhea.     

Interferon gamma induces translocation of commensal Escherichia coli across gut epithelial cells via a lipid raft-mediated process

BACKGROUND & AIMS: The "leaky gut" hypothesis proposes that leakage of enteric bacteria into the body resulting from disruption of the epithelial barrier is a critical step in the pathophysiology of various disorders such as inflammatory bowel disease and sepsis. However, the pathways and underlying mechanisms by which commensal bacteria cross the epithelial barrier in inflammatory conditions remain unclear. This study investigated the mechanisms of interferon gamma-mediated bacterial translocation across human colonic epithelial monolayers. METHODS: Caco-2 and T84 monolayers were exposed to interferon gamma. Barrier function was assessed by transepithelial electrical resistance and lucifer yellow permeability. Internalization and translocation of Escherichia coli strain C25 were measured by quantitative bacterial culture. Expression and distribution of junctional proteins were assessed by immunoblotting and confocal imaging. RESULTS: Minimal apical to basolateral translocation of C25 was observed in untreated T84 and Caco-2 monolayers. Interferon gamma caused a dramatic, dose-dependent increase in C25 translocation, which was uncoupled from cytokine-induced increases in paracellular permeability and disruption of tight junction proteins at low interferon gamma concentrations. These effects were associated with increased internalization of viable bacteria into, but not adherence to, Caco-2 cells. Interferon gamma-mediated bacterial translocation was abolished by pretreatment with the cholesterol-disrupting drugs filipin and methyl-beta-cyclodextrin, whereas these agents had no effect on infection of Caco-2 by the enteric pathogen Shigella sonnei. CONCLUSIONS: Normally poorly invasive enteric bacteria may, in situations of inflammatory stress, exploit lipid raft-mediated transcytotic pathways to cross the intestinal epithelium, and these effects may precede cytokine-induced disruption of tight junctions.     

Membrane-anchored serine protease matriptase regulates epithelial barrier formation and permeability in the intestine

The intestinal epithelium serves as a major protective barrier between the mammalian host and the external environment. Here we show that the transmembrane serine protease matriptase plays a pivotol role in the formation and integrity of the intestinal epithelial barrier. St14 hypomorphic mice, which have a 100-fold reduction in intestinal matriptase mRNA levels, display a 35% reduction in intestinal transepithelial electrical resistance (TEER). Matriptase is expressed during intestinal epithelial differentiation and colocalizes with E-cadherin to apical junctional complexes (AJC) in differentiated polarized Caco-2 monolayers. Inhibition of matriptase activity using a specific peptide inhibitor or by knockdown of matriptase by siRNA disrupts the development of TEER in barrier-forming Caco-2 monolayers and increases paracellular permeability to macromolecular FITC-dextran. Loss of matriptase was associated with enhanced expression and incorporation of the permeability-associated, “leaky” tight junction protein claudin-2 at intercellular junctions. Knockdown of claudin-2 enhanced the development of TEER in matriptase-silenced Caco-2 monolayers, suggesting that the reduced barrier integrity was caused, at least in part, by an inability to regulate claudin-2 expression and incorporation into junctions. We find that matriptase enhances the rate of claudin-2 protein turnover, and that this is mediated indirectly through an atypical PKCζ-dependent signaling pathway. These results support a key role for matriptase in regulating intestinal epithelial barrier competence, and suggest an intriguing link between pericellular serine protease activity and tight junction assembly in polarized epithelia.     

Protective effects of Lactobacillus plantarum against epithelial barrier dysfunction of human colon cell line NCM460

AIM: To investigate the effects of Lactobacillus plantarum (L. plantarum ) in the intestinal permeability and expression of tight junction (TJ) using the normal human colon cell line NCM460. METHODS: Paracellular permeability of NCM460 monolayers was determined by transepithelial electrical resistance and dextran permeability. Expression of TJ proteins in NCM460 cell monolayers was detected by Western blotting and quantitative real-time polymerase chain reaction.RESULTS: L. plantarum played an important role in increasing transepithelial electrical resistance and decreasing the permeability to macromolecules of NCM460 monolayers against the disruption caused by enteropathogenic Escherichia coli (E. coli ) or enteroinvasive E. coli . L. plantarum also prevented the decrease in the expression of TJ proteins and F-actin in NCM460 cells.CONCLUSION: L. plantarum can protect against dysfunction of NCM460 intestinal epithelial barrier caused by enteropathogenic E. coli or enteroinvasive E. coli , and thus can be a potential candidate of therapeutic agents for the treatment of intestinal diseases.     

Disruption of colonic barrier function and induction of mediator release by strains of Campylobacter jejuni that invade epithelial cells

AIM： To study the mechanisms by which Campylobacter jejuni （C. jejuni） causes inflammation and diarrhea. In particular, direct interactions with intestinal epithelial cells and effects on barrier function are poorly understood.METHODS： To model the initial pathogenic effects of C. jejuni on intestinal epithelium, polarized human colonic HCA-7 monolayerswere grown on permeabilized filters and infected apically with clinical isolates of C. jejuni. Integrity of the monolayer was monitored by changes in monolayer resistance, release of lactate dehydrogenase, mannitol fluxes and electron microscopy. Invasion of HCA-7 cells was assessed by a modified gentamicin protection assay, translocation by counting colony forming units in the basal chamber, stimulation of mediator release by immunoassays and secretory responses in monolayers stimulated by bradykinin in an Ussing chamber.RESULTS： All strains translocated across monolayers but only a minority invaded HCA-7 cells. Strains that invaded HCA-7 cells destroyed monolayer resistance over 6 h, accompanied by increased release of lactate dehydrogenase, a four-fold increase in permeability to [^3 H] mannitol, and ultrastructural disruption of tight junctions, with rounding and lifting of cells off the filter membrane. Synthesis of interleukin （IL）-8 and prostaglandin E2 was increased with strains that invaded the monolayer but not with those that did not.CONCLUSION： These data demonstrate two distinct effects of C. jejuni on colonic epithelial cells and provide an informative model for further investigation of initial host cell responses to C. jejuni.     

Heat-shock protein 72 protects against oxidant-induced injury of barrier function of human colonic epithelial Caco2/bbe cells.

BACKGROUND & AIMS: Barrier function of the inflamed intestinal mucosa can be compromised by reactive oxygen metabolites that increase mucosal permeability and disrupt the actin cytoskeleton, the integrity of which is important for maintaining tight epithelial junctions. Because heat-shock protein 72 (hsp72) protects intestinal epithelial cells against injury, we determined whether resistance of Caco2/bbe (C2) intestinal monolayer barrier function was related to their high endogenous hsp72 expression. METHODS: hsp72 anti-sense (C2/AS) and vector-only transfected C2 (C2/CEP4) clones, lines that exhibit low and high hsp72 expression, respectively, were studied. Permeability was assessed by measuring electrical resistance and mannitol fluxes and actin organization by confocal fluorescein isothiocyanate-phalloidin analysis. RESULTS: Basal transepithelial electrical resistance (TER) and mannitol fluxes were not significantly different between groups. However, the oxidant monochloramine rapidly decreased TER and increased mannitol permeability of C2/AS monolayers compared with C2/CEP4 (50% effective doses at 30 minutes were 0.53 +/- 0.11 and 2.06 +/- 0.34 mmol/L, respectively). Associated with these changes, decreased cell viability, dissociation and aggregation of perijunctional and stress actin filaments, loss of cell height, and increased intercellular separation were observed only in C2/AS cells treated with monochloramine. CONCLUSIONS: hsp72 protects intestinal epithelial barrier function against oxidant-induced stress, in part, by protecting the integrity of the actin cytoskeleton.     

Changes in barrier function of a model intestinal epithelium by intraepithelial lymphocytes require new protein synthesis by epithelial cells.

BACKGROUND: Elements of the mucosal immune system may play an important part in regulating epithelial barrier function in the intestinal tract. Intraepithelial lymphocytes (IELs) represent a subtype of immunocyte which is strategically placed to regulate epithelial function at most mucosal sites. AIMS AND METHODS: An IEL derived cell line (SC1) was used to examine its effects on the model epithelium T84--a tumour derived cell line which retains the phenotype of colonic crypt cells. Transepithelial electrical resistance (TER) was used as a marker of epithelial integrity. RESULTS: Coculture of T84 cells with SC1 produced a significant fall in TER as did exposure of T84 monolayers to IEL derived supernatant. Recombinant interferon-gamma (rIFN gamma) also reduced TER in T84 monolayers. Cycloheximide prevented the effects of IEL supernatant and of rIFN gamma on TER. The fall in TER in response to rIFN gamma was attenuated by blocking antibodies, which did not alter the fall in resistance induced by IEL supernatant. Fractions of IEL supernatant, separated on the basis of size, evoked temporally distinct changes in TER. Ultrastructural studies support the hypothesis that the slow onset but severe fall in TER indicates catastrophic effects on the monolayer. The more rapid onset fall in TER was not associated with gross changes in monolayer morphology. Reduction of TER by IEL supernatant was not influenced by inhibitors of tyrosine phosphatase or of protein kinase C. Although herbimycin did reduce the rapid onset change in TER, the tyrosine kinase inhibitor genistein did not alter responses to IEL supernatant. CONCLUSIONS: Mucosal T cells may influence barrier function by a process involving new protein synthesis by epithelial cells. This model may have relevance in some inflammatory conditions of the gastrointestinal tract.     

Induction of increased permeability of polarized enterocyte monolayers by enterotoxigenic Escherichia coli heat-labile enterotoxin

Enterotoxigenic Escherichia coli (ETEC) is a common cause of acute diarrhea in resource-poor settings. We report that some ETEC strains elicit a reduction in trans-epithelial electrical resistance (TER) in polarized T84 epithelial cell monolayers. The effect was irreversible up to 48 hours after a three-hour infection and was observed with heat-labile enterotoxin (LT)-producing strains, but not with heat-stable enterotoxin (ST)-producing strains. Using purified LT, a mutant with reduced ADP-ribosylating activity, and the LT-B subunit alone, we demonstrate that TER reduction requires a functional enterotoxin. Treatment of monolayers with LT or LT-producing strains of ETEC increases paracellular permeability to fluorescein isothiocyanate-dextran. Our data suggest that LT-producing ETEC strains may induce intestinal barrier dysfunction.     

Clostridium difficile toxin B disrupts the barrier function of T84 monolayers.

The contribution of toxin B to Clostridium difficile-associated infection is undefined. Toxin B induces dramatic phenotypic alterations (cytotoxic effects) in cultured mesenchymal and nonintestinal epithelial cells, yet its effects on intestinal epithelial cells are not clearly understood. The alterations induced by toxin B in nonintestinal cells appear to be secondary to toxin-induced redistribution of filamentous actin. It has not been determined whether toxin B exerts similar effects on cultured intestinal epithelial cells or whether such phenotypic alterations are of any physiological consequence. To address these questions, we examined the effect of C. difficile toxin B on the phenotype and barrier function of T84 cell monolayers. Our studies show that the cytotoxic effects of toxin B, i.e., cell rounding, do extend to cultured intestinal epithelial cells (T84). In addition, toxin B dramatically reduces the barrier function of T84 monolayers grown on collagen-coated filters. Toxin B-induced redistribution of filamentous actin appears to be responsible for the alterations in both intestinal epithelial cell phenotype and barrier function. Specifically, filamentous actin comprising the perijunctional actomyosin ring, known to be important in regulating tight junction permeability, is condensed into discrete plaques. Flux studies confirm that the permeability defect is at the level of the tight junction. We conclude that toxin-induced changes in actin distribution perturb intercellular junctional contacts and thereby ablate epithelial barrier function. There was no evidence of cell death as determined by lactate dehydrogenase release assays.     

Growth and function of isolated canine pancreatic ductal cells

These studies investigated the growth characteristics and functional properties of isolated canine pancreatic ductal epithelial cells. Cells were isolated from the accessory pancreatic duct and cultured by using three conditions: on vitrogen-coated petri dishes with fibroblast conditioned medium (nonpolarized); in vitrogen-coated Transwells above a fibroblast feeder layer (polarized); or as organotypic rafts above a fibroblast-embedded collagen layer (polarized). Growth characteristics, transepithelial resistances, and carbonic anhydrase and cyclic adenosine monophosphate (AMP) responses were evaluated. Under polarized conditions, the cells grew as monolayers with columnar epithelial characteristics. The monolayers developed high transepithelial resistance and became impervious to the passage of horseradish peroxidase. Epithelial growth factor (EGF) (2 ng/ml) stimulated ductal cell growth and accelerated the formation of a high-resistance monolayer. Forskolin (10 microM) rapidly decreased transepithelial resistance. Carbonic anhydrase activity, which was lower in nonpolarized compared with polarized conditions, was stimulated by carbachol (175 microM). Secretin, however, did not stimulate carbonic anhydrase activity in these cells. Although secretin stimulated adenylyl cyclase activity in early-passage cells, this response was lost in later-passage cells. Both vasoactive intestinal polypeptide (VIP; 1 microM) and forskolin (10 microM) consistently increased adenylyl cyclase activity. Isolated canine pancreatic ductal epithelial cells proliferate in vitro, develop high-resistance epithelial monolayers, and respond to stimuli that activate adenylyl cyclase. These cells should provide a useful model for regulatory studies of ductal cell functions.     

Epidermal growth factor prevents acetaldehyde-induced paracellular permeability in Caco-2 cell monolayer

BACKGROUND: Intestinal permeability and endotoxemia play a crucial role in the pathogenesis of alcoholic liver disease. Previous studies showed that acetaldehyde disrupts intestinal epithelial barrier function and increases paracellular permeability by a tyrosine kinase-dependent mechanism. In the present study, the role of epidermal growth factor (EGF) in protection of epithelial barrier function from acetaldehyde was evaluated in Caco-2 intestinal epithelial cell monolayer. METHODS: Caco-2 cells on Transwell inserts were exposed to acetaldehyde in the absence or presence of EGF, and the paracellular permeability was evaluated by measuring transepithelial electrical resistance and unidirectional flux of inulin. Integrity of epithelial tight junctions and adherens junctions was analyzed by confocal immunofluorescence microscopy and immunoblot analysis of occludin, zonula occludens (ZO)-1, E-cadherin, and beta-catenin in the actin cytoskeleton. Reorganization of actin cytoskeletal architecture was examined by confocal microscopy. RESULTS: Acetaldehyde increased paracellular permeability to inulin and lipopolysaccharide, and EGF significantly reduced these effects of acetaldehyde in a time- and dose-dependent manner. EGF prevented acetaldehyde-induced reorganization of occludin, ZO-1, E-cadherin, and beta-catenin from the cellular junctions to the intracellular compartments. Acetaldehyde treatment induced a reorganization of actin cytoskeletal network and reduced the levels of occludin, ZO-1, E-cadherin, and beta-catenin associated with the actin cytoskeleton. EGF effectively prevented acetaldehyde-induced reorganization of actin cytoskeleton and the interaction of occludin, ZO-1, E-cadherin, and beta-catenin with the actin cytoskeleton. CONCLUSION: These results indicate that EGF attenuates acetaldehyde-induced disruption of tight junctions and adherens junctions and prevents acetaldehyde-induced reorganization of actin cytoskeleton and its interaction with occludin, ZO-1, E-cadherin, and beta-catenin.     

Tight junction properties of the immortalized human bronchial epithelial cell lines Calu-3 and 16HBE14o-.

Tight junctions (TJs) make a vital contribution to the barrier properties of the airway lining. Opening of TJs, or their frank cleavage, is suspected as a pathophysiological event in the lung, but research into the cellular and molecular mechanisms involved has been impeded by technical limitations of available experimental models. The authors have compared the properties of two epithelial cell lines derived from bronchial epithelium to explore whether these cell lines could constitute appropriate tools for the study of TJ regulation in bronchial epithelium. Investigations of TJs in 16HBE14o- cells and Calu-3 cells were made by fluorescent antibody labelling in conjunction with wide-field, confocal or 2-photon molecular excitation microscopy (2PMEM). The presence of TJ proteins was confirmed by immunoblotting and functional properties of the monolayers were studied by measurements of transepithelial electrical resistance and mannitol permeability. Cells of both lines formed confluent monolayers in which the cells expressed the TJ proteins occludin and ZO-1 in continuous circumferential patterns suggestive of functional TJs. This interpretation was supported by the development of transepithelial electrical resistances and of low paracellular permeability to solutes. Within the limits of resolution offered by 2PMEM, occludin and ZO-1 appeared to colocalize at TJs. These studies suggest that the 16HBE14o- cells and Calu-3 cell lines are potentially useful in vitro models to study how tight junction opening or cleavage changes the functional barrier properties of bronchial epithelium.     

Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase-dependent fashion

BACKGROUND & AIMS: Giardiasis causes malabsorptive diarrhea, and symptoms can be present in the absence of any significant morphologic injury to the intestinal mucosa. The effects of giardiasis on epithelial permeability in vivo remain unknown, and the role of T cells and myosin light chain kinase (MLCK) in altered intestinal barrier function is unclear. This study was conducted to determine whether Giardia spp. alters intestinal permeability in vivo, to assess whether these abnormalities are dependent on T cells, and to assess the role of MLCK in altered epithelial barrier function. METHODS: Immunocompetent and isogenic athymic mice were inoculated with axenic Giardia muris trophozoites or sterile vehicle (control), then assessed for trophozoite colonization and gastrointestinal permeability. Mechanistic studies using nontransformed human duodenal epithelial monolayers (SCBN) determined the effects of Giardia on myosin light chain (MLC) phosphorylation, transepithelial fluorescein isothiocyanate-dextran fluxes, cytoskeletal F-actin, tight junctional zonula occludens-1 (ZO-1), and MLCK. RESULTS: Giardia infection caused a significant increase in small intestinal, but not gastric or colonic, permeability that correlated with trophozoite colonization in both immunocompetent and athymic mice. In vitro, Giardia increased permeability and phosphorylation of MLC and reorganized F-actin and ZO-1. These alterations were abolished with an MLCK inhibitor. CONCLUSIONS: Disruption of small intestinal barrier function is T cell independent, disappears on parasite clearance, and correlates with reorganization of cytoskeletal F-actin and tight junctional ZO-1 in an MLCK-dependent fashion.     

<Emphasis Type="Italic">Clostridium difficile</Emphasis> Toxin A Induces Intestinal Epithelial Cell Apoptosis and Damage: Role of Gln and Ala-Gln in Toxin A Effects

The aim of this study was to investigate the effect of Clostridium difficile toxin A (TxA) on intestinal epithelial cell migration, apoptosis, and transepithelial resistance and to evaluate the effect of glutamine (Gln) and its stable derivative, alanyl-glutamine (Ala-Gln), on TxA-induced damage. Migration was measured in rat intestinal epithelial cells (IEC-6) 6 and 24 hr after a razor scrape of the cell monolayer. Cell proliferation was indirectly measured utilizing the tetrazolium salt WST-1. The cells were incubated with TxA (1–100 ng/ml) in medium without Gln or medium containing Gln or Ala-Gln (1–30 mM). Apoptosis was quantified in IEC-6 cells using annexin V assay. Transepithelial resistance was measured using an epithelial voltohmmeter across T84 cells seeded on a transwell filter. TxA-induced a dose-dependent reduction of migration and also caused dose and time-dependent apoptosis in IEC-6 cells. Gln and Aln-Gln significantly enhanced IEC-6 cell migration and proliferation. Gln and Ala-Gln also prevented the inhibition of migration, apoptosis, and the initial drop in transepithelial resistance induced by TxA. In conclusion, both peptides reduced toxin-induced epithelial damage and thus might play an adjunctive role in C. difficile-induced colitis therapy.This work was supported by National Institutes of Health SBIR Grant 1R43-DK58419-01A1 and ABC Grant 5D43 TW01136-04.     

Exogenous sphingomyelinase causes impaired intestinal epithelial barrier function

AIM: To test the hypothesis that hydrolysis of sphingomyelin to ceramide changes the composition of tight junctions (TJs) with increasing permeability of the intestinal epithelium. METHODS: Monolayers of Caco-2 cells were used as an in vitro model for the intestinal barrier. Permeability was determined by quantifi cation of transepithelialflux and transepithelial resistance. Sphingolipid-rich membrane microdomains were isolated by a discontinuous sucrose gradient and characterized by Western-blot. Lipid content of microdomains was analysed by tandem mass spectrometry. Ceramide was subcellularly localized by immunofluorescent staining.RESULTS: Exogenous sphingomyelinase increased transepithelial permeability and decreased transepithelial resistance at concentrations as low as 0.01 U/mL. Lipid analysis showed rapid accumulation of ceramide in the membrane fractions containing occludin and claudin-4, representing TJs. In these fractions we observed a concomitant decrease of sphingomyelin and cholesterol with increasing concentrations of ceramide. Immunofluorescent staining confirmed clustering of ceramide at the sites of cell-cell contacts. Neutralization of surface ceramide prevented the permeability-increase induced by platelet activating factor. CONCLUSION: Our findings indicate that changes in lipid composition of TJs impair epithelial barrier functions. Generation of ceramide by sphingomyelinases might contribute to disturbed barrier function seen in diseases such as inflammatory, infectious, toxic or radiogenic bowel disease.     

Phosphatidylcholine Reverses Ethanol-Induced Increase in Transepithelial Endotoxin Permeability and Abolishes Transepithelial Leukocyte Activation

Background:  Chronic alcohol abuse increases both intestinal bacterial overgrowth and intestinal permeability to macromolecules. Intestinal permeability of endotoxin, a component of the outer cell membrane of Gram-negative bacteria, plays a crucial role in the development of alcohol-induced liver disease (ALD). As impaired bile flow leads to endotoxemia and the bile component phosphatidylcholine (PC) is therapeutically active in ALD, we tested the hypothesis that conjugated primary bile salts (CPBS) and PC inhibit ethanol-enhanced transepithelial permeability of endotoxin and the subsequent transepithelial activation of human leukocytes.
Methods:  For this purpose, we used a model in which intestinal epithelial cells (Caco-2) were basolaterally cocultivated with mononuclear leukocytes. Cells were challenged apically with endotoxin from Escherichia coli K12 and were incubated with or without the addition of CPBS (1.5 mM), PC (0.38 mM), pooled human bile (2%) in combination with ethanol (0 to 66 mM).
Results:  Ethanol decreased integrity of intestinal epithelial cell monolayer and enhanced transepithelial permeability of endotoxin. Both the transepithelial permeability of endotoxin and the transepithelial stimulation of leukocytes were nearly completely abolished after the apical supplementation of PC with CPBS, but not by CPBS alone. Ethanol up to 66 mM was not able to reverse this effect.
Conclusions:  A considerable part of the therapeutic and preventive effect of PC supplementation in ALD might result from a reduction of ethanol-enhanced permeability of endotoxin through the intestinal barrier.     

The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction

The coxsackievirus and adenovirus receptor (CAR) mediates viral attachment and infection, but its physiologic functions have not been described. In nonpolarized cells, CAR localized to homotypic intercellular contacts, mediated homotypic cell aggregation, and recruited the tight junction protein ZO-1 to sites of cell-cell contact. In polarized epithelial cells, CAR and ZO-1 colocalized to tight junctions and could be coprecipitated from cell lysates. CAR expression led to reduced passage of macromolecules and ions across cell monolayers, and soluble CAR inhibited the formation of functional tight junctions. Virus entry into polarized epithelium required disruption of tight junctions. These results indicate that CAR is a component of the tight junction and of the functional barrier to paracellular solute movement. Sequestration of CAR in tight junctions may limit virus infection across epithelial surfaces.