1Section of Gastrointestinal Sciences, Faculty of Medicine, University of Manchester, Salford M6 5HD; and 2School of Informatics, University of Wales, Bangor LL57 1UT, United Kingdom
Submitted 7 January 2004 ; accepted in final form 28 June 2004
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ABSTRACT |
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Caco-2; intestinal permeability
The gut barrier is formed to a large extent by tight junctions (TJ). These are multiprotein complexes that link adjacent epithelial cells near their apical border. Proteins present in the TJ complexes include ZO-1, occludin, and one or more claudin isoforms. TJ seal the luminal end of the intercellular space and limit transport by this paracellular route to relatively small hydrophilic molecules.
Recent data from several laboratories have begun to elucidate the roles of individual TJ proteins. ZO-1, a member of the MAGUK family of kinases, acts as a scaffold to organize transmembrane TJ proteins and recruits various signaling molecules to the complex (1, 11). Occludin binds to ZO-1 and the actin cytoskeleton and appears to have a role in regulating permeability through the TJ (8, 24). However, numerous studies have pointed to the claudin family of TJ proteins as a key determinant of paracellular characteristics. These proteins appear to form the backbone of the TJ and to provide it with selectivity to ions (9, 10, 26, 27).
In vivo the TJ is a dynamic and highly regulated structure. Its permeability changes in response to various physiological and pathophysiological stimuli, including disease and trauma (4). It has also become clear that the integrity of the gut barrier is dependant on the presence of micronutrients within the gut. Parenteral nutrition is not sufficient to maintain the barrier, and this form of feeding can lead to bacterial translocation and increased risk of infection (7, 17, 20, 21). By contrast, enteral feeding preserves TJ integrity and maintains the gut barrier. This suggests that the gut can respond to signals generated by the presence of food within it. The particular dietary constituents required to maintain the gut barrier are as yet unidentified, and the mechanisms by which changes in junctional permeability occur are not generally understood. In addition, to date little attention has been paid to the non-nutritive constituents of food that may have adverse effects on the gut barrier.
In this study, we have examined the effects of ochratoxin A on epithelial barrier function. Ochratoxin A is a small organic toxin that is produced by many of the common fungal molds, such as Aspergillus and Penicillium (19, 22). The toxin has been found in foods such as cereals, coffee, grapes, and meat and poses a serious threat to both human and animal health. Ochratoxin A has been shown to be nephrotoxic, immunotoxic, and carcinogenic to a variety of animals (5, 12, 15). Its principal site of action is the kidneys, where it has been implicated in at least three diseases. However, upon ingestion, its primary interaction is with the gut epithelium. A single previous study by others described the effects of the toxin on transepithelial electrical resistance in cultured intestinal cell lines (18). However, it is not clear from that study whether these effects are due to increase in paracellular permeability or to membrane effects. In this study, using the intestinal cell line Caco-2, we have demonstrated that ochratoxin A alters the permeability of the epithelium by a mechanism involving removal of specific claudin isoforms from the TJ.
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MATERIAL AND METHODS |
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Immunofluorescence microscopy. Caco-2 cells cultured on chamber slides (Nalge Nunc, Naperville, IL) were fixed with methanol for 20 min at 20°C. Cells were then washed three times in Tris-buffered saline (TBS), treated with 0.5% Triton-X-100 in TBS for 5 min, washed in TBS three times, and then soaked in 3% normal serum for 1 h at room temperature. Samples were incubated with the primary antibodies overnight at 4°C. These were rabbit polyclonal anti-claudin-1 (Jay.8), claudin 2 (MH44; Zymed Laboratories, South San Francisco, CA), or goat anti-claudin 3 (C-20) or 4 (C-18) (Santa Cruz Biotechnology, Santa Cruz, CA). FITC-conjugated goat anti-rabbit and rabbit anti-goat IgG (Stratech Laboratories, Cambridge, UK) were used as the secondary antibodies. Cells were then washed three times in TBS. Images were captured using confocal laser scanning microscopy (Bio-Rad MRC1024 MP confocal scanning system mounted on a Nikon Eclipse TE300 fluorescence microscope). A gallery of 30 optical sections (1 µM) through the z-plane was obtained, and composite images were processed using Confocal Assistant version 4.02 (Bio-Rad, Hercules, CA).
Measurement of transepithelial electrical resistance. Cells were seeded on Transwell polycarbonate cell culture inserts with a mean pore size of 0.4 µM (Costar) at 3 x 105 cells/cm2 and were grown for 21 days before experiment or until the transepithelial electrical resistance (TEER) had become stable. TEER was monitored using an Evometer (World Precision Instruments, Stevenage, UK) fitted with Chopstick electrodes. TEER was normalized by the area of the monolayer, and the background TEER of blank filters was subtracted from the TEER of the cell monolayer.
Treatment of cells with ochratoxin A. Cell medium was replaced with phenol red-free, fetal calf serum-free medium 24 h before the start of the experiment. Ochratoxin A (Sigma-Aldrich, Dorset, UK) was dissolved in 100 mM sodium bicarbonate (pH 7.4) The toxin was added routinely at a final concentration of 100 µM to either the apical or the basolateral side of cells growing in Transwell chambers.
MTT assay. 3-[4,5-Dimethylthiazol-2-yl]diphenyltetrazolium bromide (MTT; Sigma-Aldrich, Poole, UK) was prepared as a stock solution of 5 mg/ml in phosphate-buffered saline. Caco-2 cells were grown to 100% confluence in 96-well plates and then treated with ochratoxin A for 24 h as described above. Treatment medium was then replaced by medium containing 10% (vol/vol) MTT stock solution. Plates were incubated for 4 h at 37°C, after which the medium was replaced with dimethyl sulfoxide. Plates were shaken to dissolve the purple formazan producer, and the absorbance of each well at 570 nm was read on a Bio-Tek EL340 plate reader (Bio-Tek Instruments, Winooski, VT). Cell viability was expressed as follows: cell viability = treated wells A570/untreated wells A570.
Paracellular tracer flux assay. For paracellular tracer flux assays, FITC-labeled dextran with a molecular mass of 4, 10, 20 or 40 kDa (Sigma, Dorset, UK) was dissolved in medium at a concentration of 2 mg/ml. FITC-dextran was added at a final concentration of 0.2 mg/ml to Caco-2 monolayers growing in Transwell chambers that had been pretreated with ochratoxin A for 24 h. To evaluate the permeability of the monolayers, basal compartment media were collected after 4-h incubation with the FITC-dextran, and the amount of fluorescence in the media was measured using a Wallac 1420 Victor2 fluorimeter (Wallac, Finland). The excitation and emission wavelengths were 485 and 535 nm, respectively.
Cell extraction, SDS-PAGE, and immunoblotting. Cells were extracted by scraping into 200 µl of buffer containing NaCl (120 mM) and HEPES, pH 7.5 (25 mM), Triton X-100 (1%), EDTA (2 mM), NaF (25 mM), NaVO4 (1 mM), SDS (0.2%), aprotinin (10 µg/ml), leupeptin (10 µg/ml), and pepstatin A (10 µg/ml). The samples were incubated on ice for 30 min and then centrifuged for 15 min at full speed in a microcentrifuge. The supernatant was removed for further analysis.
SDS-PAGE was performed according to the method of Laemmli (16), and proteins were electrophoretically transferred from 12% gels onto PVDF membranes. The membranes were blocked in 5% skimmed milk and then incubated with the primary antibodies (1:1,000 dilution of rabbit anti-claudin 1, 3, or 4, mouse anti-ZO-1, or mouse anti-occludin; Zymed Laboratories). After being washed, the membranes were incubated with the secondary antibodies horseradish peroxidase-conjugated rabbit or mouse IgG as appropriate at a dilution of 1:5,000. The blots were developed using enhanced chemiluminescence (Amersham, Little Chalfont, UK) and were quantified densitometrically. Gels were scanned at 300 dpi in transmission mode using a high-resolution flat bed scanner (Epson Expression 1600) and stored in 16-bit grayscale bitmap format using a nondestructive compression algorithm tagged image file format (TIFF) to preserve image integrity. Stored bitmaps were subsequently analyzed using a program written for the MATLAB technical computing environment (Mathworks, Natick, MA).
The optical density of individual pixels within a gel image scanned in transmission or transparency mode was derived from an adaptation (Eq. 1) of the standard optical density formula
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Statistical analysis. Data are expressed as means ± SD. Statistical analysis was performed using the nonparametric Mann-Whitney U test, with P < 0.05 considered statistically significant. All experiments were conducted more than five times.
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RESULTS |
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We next investigated whether ochratoxin A-induced effects on TEER were reversible by washing the cells three times in PBS at the end of the experiment. However, recovery of the TEER was not possible after 24 h of exposure. Interestingly, recovery of the TEER was possible only after very short (2 h) exposure to the toxin. Under these conditions, cells would subsequently recover 85% (±8%, n = 5) of the control TEER within 12 h of removal of the toxin (data not shown).
Ochratoxin A increases the permeability of Caco-2 cells to FITC-labeled dextrans. Changes in TEER are not always indicative of alterations in epithelial barrier function and can often be explained as alterations in the transcellular permeability of ions. To eliminate this possibility, we studied the flux of the membrane-impermeant paracellular tracer FITC-labeled dextran across Caco-2 monolayers. As shown in Fig. 1C, ochratoxin A added to the apical side of the cells caused an approximately threefold increase in the paracellular flux of 4-kDa dextran (n = 6 with duplicates within each experiment; P < 0.006). Untreated Caco-2 monolayers are not readily permeable to 10-kDa dextrans; however, upon treatment with the mycotoxin, the monolayer became significantly more permeable to this species (Fig. 1C, n = 6; P < 0.002). The paracellular pathway in Caco-2 cells was not found to be available to FITC-labeled dextrans of either 20 or 40 kDa in either the presence or absence of ochratoxin A (data not shown). These findings demonstrate that ochratoxin A is able to modulate the paracellular pathway in Caco-2 cells.
Ochratoxin A removes specific claudin isoforms from the tight junction.
We next examined whether the decrease in TEER and increase in permeability observed were due to effects of ochratoxin A on specific TJ proteins. First, we examined which claudin isoforms are expressed in Caco-2 cells. A total cell lysate was prepared from Caco-2 cells grown to confluence on a Transwell filter, and the lysate was subjected to SDS-PAGE followed by immunoblotting using antibodies specific to claudin 1, 2, 3, or 4. As shown in Fig. 2A, in the total cell lysate of Caco-2 cells, claudins 1, 3, and 4 but not claudin 2 were detected by immunoblotting as bands of the expected molecular mass (22 kDa). To confirm the immunoblotting results and also to analyze the cellular distribution, we obtained a z series of confocal images of Caco-2 cells stained for claudin 1, 2, 3, or 4. Claudins 1, 3, and 4 were appropriately localized in a characteristic chicken wire pattern (11) consistent with their distribution in TJ (Fig. 2B). However, claudin 2 could not be detected in Caco-2 cells (Fig. 2B). Therefore, to control for the antibody, Madin-Darby canine kidney (MDCK) type II cells were stained for claudin 2. In agreement with previous work by others (25), claudin 2 produced the typical chicken wire pattern of staining in MDCK type II cells (Fig. 2C).
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DISCUSSION |
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Previously, it was shown by others that ochratoxin A decreased the TEER of Caco-2 cells (18). However, the mechanism underlying this observation was not explored. In particular, it was not clear whether reduction in TEER was due to effects on the TJ barrier properties or to plasma membrane effects such as differences in transcellular ion transport. The main finding from this study is that ochratoxin A is able to reduce the barrier function of Caco-2 monolayers. The reduction in barrier properties is evident on the basis of two measures. In addition to a time-dependent reduction in TEER, ochratoxin A is able to increase the permeability of Caco-2 cells to paracellular tracers of 4 and 10 kDa. However, the barrier is still preserved with respect to dextrans with greater molecular mass (20 and 40 kDa). This indicates that the toxin does not simply destroy the TJ complex but can effect a "loosening" of the TJ that facilitates the passage of smaller molecules through the paracellular pathway.
The mechanism by which ochratoxin A reduces the barrier properties of Caco-2 cells appears to be via removal of two specific claudin isoforms, 3 and 4, from the TJ. This is clear from our observation that reduction in TEER follows a time scale comparable to that of the disappearance of claudins 3 and 4 from the cells. The levels of other TJ proteins, ZO-1, occludin and claudin 1 stay constant over this time period, which suggests that claudins 3 and 4 may have a central role in TJ barrier function in Caco-2 cells.
The mechanism by which ochratoxin A is able to remove specific claudins from the TJ is at present unclear. The toxin is known to be an inhibitor of protein synthesis in other systems (18). However, the observation that levels of claudins 3 and 4 are depressed, while the levels of other TJ components remain constant, would tend not to favor a general inhibition of protein or RNA synthesis by the toxin. Similarly, it is difficult to explain the effects in terms of general cell damage, because even after 24-h exposure to the toxin, the barrier is still 60% intact, as judged by TEER, and is still able to discriminate small from large paracellular probes. Interestingly, similar results regarding the barrier have been observed with enterotoxin A from Clostridium perfringens (25). The COOH-terminal half of this peptide toxin is able to remove claudins 3 and 4 from TJ strands in MDCK cells, resulting in decreased TEER and increased permeability. The underlying mechanism appears to be direct binding of the toxin to claudin isoforms. However, in these studies, the C. perfringens toxin did not induce temporal differences between removal of claudin 3 and 4, suggesting a different mechanism of action. When we examined a z series of cells treated with ochratoxin and stained with anticlaudin antibodies, we noted a large reduction in the intensity of the junctional staining for claudins 3 and 4 but not for claudin 1. We did not see relocalization of the fluorescent signal to other cellular areas, suggesting that claudins 3 and 4 had disappeared from the cell. This observation is in keeping with the immunoblotting results, which demonstrate a reduction in the levels of claudins 3 and 4 but not claudin 1. The overall morphology of the cells was unchanged by ochratoxin treatment, suggesting specific effects on claudins rather than gross changes to the cells. However, why only claudins 3 and 4 are affected is still a subject for debate.
Ochratoxin A displays similar kinetics, regardless of to which side of the membrane it is added. This indicates that both apical and basolateral membranes are equally susceptible to the effects of the toxin. This is most likely explained by the observation that ochratoxin A is able to diffuse through the Caco-2 cell membrane by virtue of its physicochemical characteristics and is accumulated within the cells (3). Certainly, in our experiments, it was impossible to reverse the effects of the toxin by washing if the cells had been exposed for >2 h. Shorter exposures followed by washing of the cells resulted in the reestablishment of the TEER within 12 h (data not shown). This would tend to suggest that the toxin is accumulated within the cells. At the end point of our studies (24 h), the cells were still viable as judged by MTT assay, and only minor changes in morphology were observed. However, very prolonged (>72 h) exposure to the mycotoxin resulted in the cells lifting from the filter, presumably due to other toxic effects (data not shown).
Overall, our data demonstrate for the first time that ochratoxin A is able to induce a decrease in the barrier function of Caco-2 cells and is associated with the removal of specific claudins from the TJ. This observation may help to explain, at a molecular level, some of the in vivo effects of the toxin on the intestine. This study is also a further demonstration of the importance of the claudin family of proteins in the maintenance of epithelial barrier properties.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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