Symmetric infection of rotavirus on polarized human intestinal epithelial (Caco-2) cells.

When rotavirus infects the mature villus tip cells of the small intestine, it encounters a highly polarized epithelium. In order to understand this virus-cell interaction more completely, we utilized a cell culture-adapted rhesus rotavirus (RRV) to infect human intestinal (Caco-2) and Madin-Darby canine kidney (MDCK-1) polarized epithelial cells grown on a permeable support. Filter-grown Caco-2 cells and MDCK-1 cells, producing a transepithelial resistance of 300 to 500 and greater than 1,000 omega . cm2, respectively, were infected from either the apical or basolateral domain with RRV or Semliki Forest virus. Whereas Semliki Forest virus infection only occurred when input virions had access to the basolateral domain of MDCK-1 or Caco-2 cells, RRV infected MDCK-1 and Caco-2 monolayers in a symmetric manner. The effect of rotavirus infection on monolayer permeability was analyzed by measuring the transepithelial electrical resistance. Rotavirus infection on filter-grown Caco-2 cells caused a transmembrane leak at 18 h postinfection, before the development of the cytopathic effect (CPE) and extensive virus release. Electrical resistance was completely abolished between 24 and 36 h postinfection. Although no CPE could be detected on RRV-infected MDCK cells, the infection caused a transmembrane leak that totally abolished the electrical resistance at 18 to 24 h postinfection. Cell viability and the CPE analysis together with immunohistochemistry and immunofluorescence data indicated that the abolishment of resistance across the monolayer was due not to an effect on the plasma membrane of the cells but to an effect on the paracellular pathway limited by tight junctions. Attachment and penetration of rotavirus onto Caco-2 cells caused no measurable transmembrane leak during the first hour of infection.     

Effect of neuronal PC12 cells on the functional properties of intestinal epithelial Caco-2 cells

The effect of neuronal cells on the functional properties of intestinal epithelial cells was examined by using an in vitro coculture system. Two cell lines, Caco-2 and PC12, were respectively used as intestinal epithelial and enteric neuronal cell models. Coculture of differentiated Caco-2 cells with PC12 caused a significant decrease in the transepithelial electrical resistance (TER) value of the Caco-2 monolayer. The permeability to lucifer yellow (LY) was also significantly increased, suggesting that the tight junction (TJ) of the Caco-2 monolayers was modulated by coculturing with PC12. To identify the TJ-modulating factor presumably secreted from PC12, the effects of the major neurotransmitters on the TER value and LY transport were examined, but no influence was apparent. The TJ-modulating effect of PC12 was prevented by exposing PC12 to cycloheximide, suggesting that new protein synthesis in PC12 was necessary for this regulation.     

Interference ofSalmonella enteritidis andLactobacillus spp. with IL-8 levels and transepithelial electrical resistance of enterocyte-like caco-2 cells

Caco-2 cells (exhibiting characteristics of mature villus enterocytes) were used to determine bacteria (Salmonella enteritidis causing human gastroenteritis)-intestinal cell interactions. The interference of bacteria with the transepithelial electrical resistance (TEER) of filter-grown Caco-2 cells and the production of IL-8 after exposure of the cells to S. enteritidis 857 and/or Lactobacillus strains (L. gasseri LF221 and L. rhamnosus BGT10) was evaluated. The strain 857 decreased TEER of filter-grown Caco-2 cells; in contrast, lactobacilli had a little or no effect. The effect of S. enteritidis on the TEER decreased if Caco-2 cells were pre-incubated with lactobacilli. This strain induced high levels of IL-8 (which can lead to cell damage). Compared to the IL-8 synthesis after exposure of Caco-2 cells to S. enteritidis 857, simultaneous exposure of Caco-2 cells to S. enteritidis and lactobacilli inhibited the IL-8 synthesis after short recovery periods.     

Effects of capsaicin on human intestinal cell line Caco-2

The influence of capsaicin processing on human intestinal cell line Caco-2 was examined by measuring transepithelial electrical resistance (TER). There was an increase in permeability at high concentration (200 to 500 μM) of capsaicin, and the effect was inhibited by pretreatment of capsazepine, which is a competitive antagonist of the vanilloid receptor 1 (VR1). LDH-activity as well as changes in intracellular Ca²⁺ were determined to know whether or not capsaicin affected TER activity through its influence on the tight junction. We also determined the expression of the VR1-like protein on Caco-2 cells in time-dependent manner by western blotting using vanilloid receptor (VR1) antiserum. Our results showed that the permeability increase by capsaicin was through binding to VR1-like protein of Caco-2 cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Virulent strains of Salmonella enteritidis disrupt the epithelial barrier of Caco-2 and HEp-2 cells

To confirm the existence in nature of Salmonella enteritidis strains of different degrees of virulence and to elucidate the mechanisms underlying the effects of such strains on the epithelial barrier function, the consequences of infection of Caco-2 cells and HEp-2 cells with 15 S. enteritidis strains in a chicken infection model were examined. The more virulent strains of S. enteritidis, which are biofilm producers in adherence test medium, were able to disrupt HEp-2 and Caco-2 monolayers, as shown by transmonolayer electrical resistance and lactate dehydrogenase activity. In contrast, the low-virulence strains of S. enteritidis, which do not produce biofilms in adherence test medium, had no effect on the same cells. An avirulent rough mutant of Salmonella minnesota exhibited a pattern of behaviour similar to that of the low virulence strains of S. enteritidis, whilst a clinical Salmonella typhi strain caused rapid injury to the monolayers. The effect of supernatants of Salmonella cultures in adherence test medium on the integrity of Caco-2 cell monolayers indicated that the high-virulence S. enteritidis strains, but not the low-virulence strains, release a soluble factor when incubated under optimum biofilm-forming conditions, which enables the disruption of the integrity of Caco-2 monolayers.     

The changes in the neuronal PC12 and the intestinal epithelial Caco-2 cells during the coculture. The functional analysis using an in vitro coculture system

The interaction between intestinal epithelial cells andperipheral neuronal cells were examined using an invitro coculture system. Two cell lines, Caco-2 and PC12, were usedfor this experiment as an intestinal epithelial and entericneuronal cell model, respectively. By coculturing with fullydifferentiated Caco-2 cells, the neurite outgrowth was inducedin PC12 cells. This neurite outgrowth in PC12 was blocked byanti-nerve growth factor (NGF) polyclonal antibodies,suggesting that the neurite outgrowth in PC12 during thecoculture with Caco-2 cells was due to NGF secreted fromCaco-2 cells. On the other hand, coculturing with fullydifferentiated PC12 cells induced the decrease oftransepithelial electrical resistance in Caco-2 cellmonolayers. The permeability of lucifer yellow alsosignificantly increased, suggesting that the barrier functionand paracellular permeability of Caco-2 monolayers werealtered by coculturing with PC12 cells. The present studysuggests that this in vitro coculture system is a good modelfor the functional analysis of interaction among intestinalepithelial cells with different cell types.     

Epithelial properties of human intestinal Caco-2 cells cultured in a serum-free medium

Human intestinal Caco-2 cells were cultured under serum-free conditions on an insoluble collagen and FCS matrix (Caco-2-SF), and a comparison was made between several characteristics of Caco-2 and Caco-2-SF cells. Their morphological appearance was identical. Slight differences were found in cell growth and expression of brush border enzymes between Caco-2 and Caco-2-SF cells. Similar levels of activity of Gly-Gly transport were expressed in both types of cell. Caco-2 cells cultured on permeable filters showed high transepithelial electrical resistance (TEER), indicating the high monolayer integrity. The transepithelial transport activity for glucose, alanine and Gly-Gly was detected by measuring the change in short-circuit current (Isc) after adding each of these nutrients to the apical chamber. In Caco-2-SF cells, such parameters as TEER and Isc were reduced drastically, suggesting that the monolayer integrity and cell polarity that are important for transepithelial transport were not attained. These parameters, however, could be restored by adding FCS or by milk whey. The result suggested that FCS and milk whey contain factors which regulate the formation of the tight junctions and, consequently, the development of cell polarity. Thus the Caco-2-SF cell-culture system will provide a useful model for studying factors which regulate the intestinal transepithelial transport functions.Abbreviations BCECF 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein - TEER transepithelial electrical resistance - LY lucifer yellow CH lithium salt     

In vitro model to estimate gut inflammation using co-cultured Caco-2 and RAW264.7 cells

A system for assessing the anti-inflammatory effect of food factors was developed by establishing a co-culture system with intestinal epithelial Caco-2 cells (apical side) and macrophage RAW264.7 cells (basolateral side). In this system, the stimulation of RAW264.7 cells with lipopolysaccharide was followed by a decrease in transepithelial electrical resistance, which is a marker of the integrity of the Caco-2 monolayer and an increase in TNF-α production from RAW264.7 cells and IL-8 mRNA expression in Caco-2 cells. Treatment with anti-TNF-α antibodies or budesonide suppressed in increase in TNF-α production and IL-8 mRNA expression. These results indicated that this co-culture model could imitate the gut inflammation in vivo. In addition, fucoidan, sulphated polysaccharides from brown algae, was employed as a candidate of evolution and added to the apical side of this model. Fucoidan suppressed IL-8 gene expression through a reduction in TNF-α production from RAW264.7 cells stimulated with lipopolysaccharide.     

Investigation of the Cytotoxic Effects of Titanate Nanotubes on Caco-2 Cells

Titanate nanotubes can be used as drug delivery systems, but limited information is available on their interactions with intestinal cells. In this study, we investigated the cytotoxicity and cellular uptake of titanate nanotubes on Caco-2 monolayers and found that up to 5 mg/ml concentration, these nanotubes are not cytotoxic and not able to permeate through the intestinal cell layer. Transmission electron microscopic experiments showed that titanate nanotubes are not taken up by cells, only caused a high-density granulation on the surface of the endoplasmic reticulum. According to these results, titanate nanotubes are suitable systems for intestinal drug delivery.     

Expression levels of heat shock proteins in enterocyte-like Caco-2 cells after exposure to Salmonella enteritidis

The enterocytes of the small intestine are occasionally exposed to pathogenic bacteria, such as Salmonella enteritidis 857, an etiologic agent of intestinal infections in humans. The expression of the heat shock response by enterocytes may be part of a protective mechanism developed against pathogenic bacteria in the intestinal lumen. We aimed at investigating whether S. enteritidis 857 is able to induce a heat shock response in crypt- and villus-like Caco-2 cells and at establishing the extent of the induction. To establish whether S. enteritidis 857 interfered with the integrity of the cell monolayer, the transepithelial electrical resistance (TEER) of filter-grown, differentiated (villus-like) Caco-2 cells was measured. We clearly observed damage to the integrity of the cell monolayer by measuring the TEER. The stress response was screened in both crypt- and villus-like Caco-2 cells exposed to heat (40-43 degrees C) or to graded numbers (10(1)-10(8)) of bacteria and in villus-like cells exposed to S. enteritidis 857 endotoxin. Expression of the heat shock proteins Hsp70 and Hsp90 was analyzed by polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibodies. Exposure to heat or Salmonella resulted in increased levels of Hsp70 and Hsp90 in a temperature-effect or Salmonella-dose relationship, respectively. Incubation of Caco-2 cells with S. enteritidis 857 endotoxin did not induce heat shock gene expression. We conclude that S. enteritidis 857 significantly increases the levels of stress proteins in enterocyte-like Caco-2 cells. However, our data on TEER clearly indicate that this increase is insufficient to protect the cells.     

Protease inhibitors suppress the formation of tight junctions in gastrointestinal cell lines.

Tight junctions (TJ) of the fascia occludens type can be induced in the human colon adenocarcinoma cell lines HT29 and Caco-2 by treatment with 320 mM cesium sulfate. This process can be completely inhibited by the protease inhibitors leupeptin and antipain. The concentration for 50% inhibition was 32 microM leupeptin and 270 microM antipain, respectively. In the polarized colon carcinoma cell line Caco-2, the spontaneous formation of histotypical TJ and the development of transepithelial electrical resistance do not occur when the cells are cultured in medium containing 400 microM leupeptin. Following the removal of leupeptin, zonula occludens type TJ and electrical resistance develop synchronously during a period of 4 h. Dihydroleupeptin, the alcohol analog of leupeptin, inhibits neither the spontaneous nor the induced assembly of TJ fibrils. Thus, the aldehyde group of leupeptin is essential for activity. These data suggest that the salt-induced as well as the spontaneous formation of TJ involve cellular proteases which are susceptible to protease inhibitors.     

Modulation of tight junction barrier function by outer membrane proteins of enteropathogenic Escherichia coli: role of F-actin and junctional adhesion molecule-1

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea. In this work we investigated the effect of outer membrane proteins (OMP) of EPEC on barrier integrity and the role of actin, junctional adhesion molecule (JAM) and signaling pathways contributing to these changes. Barrier function was assessed by transepithelial electrical resistance (TER). OMP of wild type EPEC, eaeA and maltoporin mutants decreased TER levels of Caco-2 cells. The OMP of espB mutant was deficient in decreasing TER of Caco-2 cells. The proteinase K-digested wild type OMP and EAF mutant OMP did not cause any change in barrier function. Our previous studies have demonstrated that EPEC OMP induced changes in cadherin junctions of Caco-2 cells. Immunofluorescence revealed disruption in actin cytoskeleton by EPEC OMP. However, no change in expression of junctional adhesion molecule-1 was observed. NF-kappaB inhibitor slightly blocked the decrease in TER and protected against actin disruption while ERK1/2 inhibitor had no effect in blocking these changes. In conclusion, our data suggest that the OMP of EPEC alter intestinal barrier function by disrupting actin cytoskeleton and signaling pathways like NF-kappaB may have a role in regulating barrier changes.     

An inter-laboratory study to evaluate the effects of medium composition on the differentiation and barrier function of Caco-2 cell lines

Differentiated human intestinal Caco-2 cells are frequently used in toxicology and pharmacology as in vitro models for studies on intestinal barrier functions. Since several discrepancies exist among the different lines and clones of Caco-2 cells, comparison of the results obtained and optimisation of models for use for regulatory purposes are particularly difficult, especially with respect to culture conditions and morphological and biochemical parameters. An inter-laboratory study has been performed on the parental cell line and on three clonal Caco-2 cell lines, with the aim of standardising the culture conditions and identifying the best cell line with respect to parameters relevant to barrier integrity, namely, trans-epithelial electrical resistance (TEER) and mannitol passage, and of epithelial differentiation (alkaline phosphatase activity). Comparison of the cell lines maintained in traditional serum-supplemented culture medium or in defined medium, containing insulin, transferrin, selenium and lipids, showed that parameter performance was better and more reproducible with the traditional medium. The maintenance of the cell lines for 15 days in culture was found to be sufficient for the development of barrier properties, but not for full epithelial differentiation. Caco-2/TC7 cells performed better than the other three cell lines, both in terms of reproducibility and performance, exhibiting low TEER and mannitol passage, and high alkaline phosphatase activity.     

Exogenous HIV-1 Nef upsets the IFN-γ-induced impairment of human intestinal epithelial integrity

Background

The mucosal tissues play a central role in the transmission of HIV-1 infection as well as in the pathogenesis of AIDS. Despite several clinical studies reported intestinal dysfunction during HIV infection, the mechanisms underlying HIV-induced impairments of mucosal epithelial barrier are still unclear. It has been postulated that HIV-1 alters enterocytic function and HIV-1 proteins have been detected in several cell types of the intestinal mucosa. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on human epithelial cell line.

Methodology/Principal Findings

We used unstimulated or IFN-γ-stimulated Caco-2 cells, as a model for homeostatic and inflamed gastrointestinal tracts, respectively. We investigated the effect of exogenous recombinant Nef on monolayer integrity analyzing its uptake, transepithelial electrical resistance, permeability to FITC-dextran and the expression of tight junction proteins. Moreover, we measured the induction of proinflammatory mediators. Exogenous Nef was taken up by Caco-2 cells, increased intestinal epithelial permeability and upset the IFN-γ-induced reduction of transepitelial resistance, interfering with tight junction protein expression. Moreover, Nef inhibited IFN-γ-induced apoptosis and up-regulated TNF-α, IL-6 and MIP-3α production by Caco-2 cells while down-regulated IL-10 production. The simultaneous exposure of Caco-2 cells to Nef and IFN-γ did not affect cytokine secretion respect to untreated cells. Finally, we found that Nef counteracted the IFN-γ induced arachidonic acid cascade.

Conclusion/Significance

Our findings suggest that exogenous Nef, perturbing the IFN-γ-induced impairment of intestinal epithelial cells, could prolong cell survival, thus allowing for accumulation of viral particles. Our results may improve the understanding of AIDS pathogenesis, supporting the discovery of new therapeutic interventions.     

Expression of SLC30A10 and SLC23A3 Transporter mRNAs in Caco-2 Cells Correlates with an Increase in the Area of the Apical Membrane

Drug bioavailability studies commonly employ in vitro barrier tissue models consisting of epithelial and endothelial cells. These experiments require that the cell barrier quality be assessed regularly, which is usually performed using various labeled substrates and/or evaluation of transepithelial (transendothelial) electrical resistance (TEER). This technique provides information on the integrity of the monolayer, but not on differentiation-induced changes in the cell morphology. The present work shows that impedance spectroscopy can be applied to monitor both the integrity of the monolayer and the morphological changes of Caco-2 cells. The growth kinetics of the apical membrane was determined by calculating the electrical capacitance of the cell monolayer. In the course of differentiation, the most pronounced changes in the expression levels were observed for the mRNAs that encode SLC30A10 and SLC23A3 transporters. Their increase correlated with an increase in the apical membrane area, indicating that SLC30A10 and SLC23A3 mRNA levels assessed by qRT-PCR may be employed as cell differentiation biomarkers in Caco-2 models.     

Methylated N-(4-N,N-Dimethylaminobenzyl) Chitosan, a Novel Chitosan Derivative, Enhances Paracellular Permeability Across Intestinal Epithelial Cells (Caco-2)

The aim of this study was to investigate the effect of methylated N-(4-N,N-dimethylaminobenzyl) chitosan, TM-Bz-CS, on the paracellular permeability of Caco-2 cell monolayers and its toxicity towards the cell lines. The factors affecting epithelial permeability, e.g., degree of quaternization (DQ) and extent of dimethylaminobenzyl substitution (ES), were evaluated in intestinal cell monolayers of Caco-2 cells using the transepithelial electrical resistance and permeability of Caco-2 cell monolayers, with fluorescein isothiocyanate dextran 4,400 (FD-4) as a model compound for paracellular tight-junction transport. Cytotoxicity was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide viability assay. The results revealed that, at pH 7.4, TM-Bz-CS appeared to increase cell permeability in a concentration-dependent manner, and this effect was relatively reversible at lower doses of 0.05–0.5 mM. Higher DQ and the ES caused the permeability of FD-4 to be higher. The cytotoxicity of TM-Bz-CS depended on concentration, %DQ, and %ES. These studies demonstrated that this novel modified chitosan has potential as an absorption enhancer.     

Pseudomonas aeruginosa quorum sensing molecule N-(3 oxododecanoyl)-l-homoserine lactone disrupts epithelial barrier integrity of Caco-2 cells

Acyl-homoserine lactone (HSL) quorum sensing molecules play an important role in regulation of virulence gene expression in Pseudomonas aeruginosa. Here, we show that 3O-C(12)-HSL can disrupt barrier integrity in human epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER), increased paracellular flux, reduction in the expression and distribution of ZO-1 and occludin, and reorganization of F-actin. P. aeruginosa 3O-C(12)-HSL activate p38 and p42/44 kinases, and inhibition of these kinases partly prevented 3O-C(12)-HSL-induced changes in TER, paracellular flux and expression of occludin and ZO-1. These findings demonstrate that P. aeruginosa 3O-C(12)-HSL can modulate tight junction integrity of Caco-2 cells.     

Induction by activated macrophage-like THP-1 cells of apoptotic and necrotic cell death in intestinal epithelial Caco-2 monolayers via tumor necrosis factor-alpha

Intestinal epithelial cells interact with immune cells located in the intestinal epithelium via soluble factors. An in vitro model system using coculture was constructed to analyze the effect of macrophages on intestinal epithelial cells, and human intestinal epithelial-like Caco-2 monolayers and activated macrophage-like THP-1 cells were used in this study. Coculturing with THP-1 cells resulted in an increase of lactate dehydrogenase release from Caco-2 and a decrease in the transepithelial electrical resistance of the monolayers, showing that coculturing with THP-1 induced cell damage to Caco-2 cells. This disruption was significantly suppressed by adding anti-TNF-alpha antibody and etanercept, strongly suggesting that TNF-alpha secreted from THP-1 had caused cell damage to Caco-2 monolayers. The disrupted Caco-2 monolayers showed both apoptotic and necrotic characteristics by morphological and biochemical analyses. TNFRI and NF-kappaB seem to have been involved in this regulation. It is suggested that this phenomenon is similar in some respects to that observed with IBD and that this in vitro coculture system could be a good model for searching for the drugs or food substances that can be used to treat or prevent IBD.     

Disruption of epithelial barrier function by H2O2: distinct responses of Caco-2 and Madin-Darby canine kidney (MDCK) strains.

Modulation of epithelial function by oxidants such as hydrogen peroxide may contribute to a variety of disease processes. The effects of hydrogen peroxide (H2O2) on epithelial barrier function and tight junction protein distribution were compared in three distinct types of polarised epithelial cell, each of which was found to respond differently to H2O2. Of the cell-lines examined, Madin-Darby canine kidney (MDCK) stain II was the most susceptible to H2O2 and Caco-2 the least H2O2 induced a decrease in transepithelial electrical resistance in all three epithelial cell-lines which was accompanied by redistribution of the tight junction protein occludin in Caco-2 and MDCK II but not MDCK I, cell layers. The effects of H2O2 on epithelia displayed marked asymmetry, each cell-line being affected more by basal than apical application of H2O2. Genistein partially prevented the effects of H2O2 on Caco-2 cells suggesting a role for protein tyrosine phosphorylation in H2O2-induced epithelial barrier dysfunction. However, genistein was without effect on the responses of MDCK cells to H2O2.Taken together these data indicate that H2O2 has distinct effects onthe tight junctions of epithelial cells from different origins and suggest that enhanced tyrosine phosphorylation is a contributory factor inthe enhanced permeability of some, but possibly not all, epithelial cell-lines.     

Candida albicans and Saccharomyces cerevisiae induce interleukin-8 production from intestinal epithelial-like Caco-2 cells in the presence of butyric acid

Intestinal epithelial cells (IEC) are important in initiation and regulation of immune responses against numerous foreign substances including food, microorganisms and their metabolites in the intestine. Since the responses of IEC against yeasts have not yet been well understood, we investigated the effects of Candida albicans, Saccharomyces cerevisiae, and their cell wall components on interleukin-8 (IL-8) secretion by the IEC-like Caco-2 cells. Live cells of both yeast species stimulated Caco-2 cells to produce IL-8 only in the presence of butyric acid, which is a metabolite produced by intestinal bacteria. S. cerevisiae zymosan and glucan also enhanced IL-8 secretion. Treatment of Caco-2 cells with butyric acid increased the expression of mRNAs coding for Toll-like receptor 1 (TLR1), TLR6 and dectin-1, which recognize zymosan. C. albicans induced more IL-8 secretion and also decreased transepithelial electrical resistance more rapidly than S. cerevisiae. These results suggest that both yeasts in the intestine stimulate the host's mucosal immune systems by interacting with IEC.