Interferon-gamma decreases barrier function in T84 cells by reducing ZO-1 levels and disrupting apical actin

Adel Youakim and Minoo Ahdieh

Department of Biochemistry, Immunex Corporation, Seattle, Washington 98101


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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The effects of interferon-gamma (IFN-gamma ) on tight junctions in T84 human intestinal epithelial cells were investigated. Treatment of T84 cells with IFN-gamma caused a dose- and time-dependent increase in monolayer permeability as assessed by transepithelial electrical resistance measurements. Examination of specific proteins associated with tight junctions by immunoblotting and confocal microscopy revealed changes in the expression levels and localization of some of these proteins after exposure of the cells to IFN-gamma . Specifically, IFN-gamma treatment resulted in an almost total loss of zonula occludens (ZO)-1, whereas the levels of ZO-2 and occludin showed relatively modest decreases compared with untreated cells. Loss of ZO-1 was associated with the altered localization of ZO-2 and occludin. In IFN-gamma -treated cells, ZO-2 and occludin were diffusely distributed, whereas, in control cells, they, along with ZO-1, were predominantly localized to the tight junctions. Analysis of ZO-1 protein and RNA by pulse chase and RT-PCR, respectively, showed an increase in protein turnover, a decrease in protein synthesis, and a reduction in RNA levels following IFN-gamma treatment. In contrast to ZO-1, ZO-2 and occludin did not show any major changes in these parameters. In addition, the organization of actin in the apical and tight junction regions, but not in the mid- or basal regions, of the cells was also perturbed by IFN-gamma treatment of cells. Time-course analysis of IFN-gamma -induced alterations in ZO-1 expression and apical actin perturbation indicated that these two effects were intimately linked and could not be dissociated. These results suggest that IFN-gamma affects barrier function in T84 cells by decreasing the levels of ZO-1 and perturbing apical actin organization, which leads to a disorganization of the tight junction and an increase in paracellular permeability.

tight junctions; epithelial barrier; zonula occludens-1; zonula occludens-2; occludin; actin


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INTRODUCTION
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THE INTESTINAL EPITHELIUM forms a relatively impermeable barrier between the lumen and the submucosa. This barrier function is maintained by a complex of proteins composing the tight junction that is located at the subapical aspect of the lateral membranes. Tight junctions comprise numerous proteins, with the best characterized being zonula occludens (ZO)-1, ZO-2, and occludin (for reviews, see Refs. 2 and 3). ZO-1 and ZO-2 are cytoplasmic proteins (18, 32), whereas occludin is a putative transmembrane protein (9). Recently, several new proteins, ZO-3 (12), claudin-1 and -2 (8), and junctional adhesion molecule (JAM) (25), have also been shown to be associated with tight junctions; the first is cytoplasmic, and the latter two are transmembrane proteins. Although the precise interplay of these proteins within the tight junction complex has not been fully elucidated, recent findings suggest a complex series of interactions among them. ZO-1 and ZO-2 appear to interact, as they coimmunoprecipitate when using either anti-ZO-1 or anti-ZO-2 antibodies (18). ZO-1, ZO-3, and occludin also appear to associate, based on in vitro binding studies (10, 12). The COOH terminus of occludin is necessary for the localization of this molecule to the tight junction of polarized cells (9). Therefore, it is possible that this portion of occludin contains a targeting sequence necessary for delivery to the tight junction and that binding of the COOH terminus to ZO-1 is required to retain occludin at the tight junction. The association of ZO-1 with tight junctions and binding to occludin is mediated by two separate domains within the NH2-terminal half of ZO-1 (7). In addition to associating with other tight junction proteins, ZO-1 also interacts with the cytoskeleton either by binding to spectrin (16) or by binding directly to actin (15). The actin binding activity of ZO-1 has been mapped to the COOH-terminal half of the molecule (7). Thus ZO-1 may act as a direct or indirect link between the transmembrane component of the tight junction, occludin, and the cytoskeleton. The interaction of tight junctions with the cytoskeleton is critical to the regulation of barrier function, and modulation of the cytoskeleton may influence paracellular flow through the tight junctions (20, 21, 30).

Disruption of the epithelial barrier in the gut is a hallmark of inflammatory bowel disease and intestinal infections. Although it remains unclear as to whether barrier breakdown is an initiating event or a consequence of inflammation, it is readily apparent that loss of the barrier contributes to propagation and exacerbation of inflammation. Barrier breakdown can be elicited by a number of agents, including bacteria, cells of the immune system, and proinflammatory cytokines. In the complex milieu of the gut, it is likely that a combination of factors contributes to barrier disruption. This is an issue of significant importance for the treatment of gut inflammation because an understanding of how the epithelial barrier is regulated in disease may allow the development of therapeutic agents that prevent breakdown or enhance recovery of normal barrier function. A number of in vitro model systems, including the T84 human intestinal epithelial cell line, have been developed that enable the study of epithelial barrier regulation. T84 cells form a polarized, impermeable monolayer that possesses many of the functional characteristics of intestinal epithelial cells in vivo, including vectorial solute transport and barrier function (6, 22). Previous studies have demonstrated that T84 cells treated with cytokines, such as interferon-gamma (IFN-gamma ) (1, 15), show decreased barrier function. IFN-gamma is greatly elevated in human intestinal disease and undoubtedly contributes to the inflammatory cascade, which includes barrier disruption. However, the effects of IFN-gamma on tight junctions, the cellular complex responsible for barrier function, have yet to be determined. The objective of this study was to determine if IFN-gamma alters epithelial barrier of T84 cells by influencing the organization and composition of the tight junctions. The results show that, indeed, IFN-gamma disrupts barrier function by dramatically decreasing the levels of ZO-1, perturbing the actin cytoskeleton in the region of the tight junction, and causing the mislocalization of ZO-2 and occludin.


    MATERIALS AND METHODS
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Cell culture. T84 cells were maintained and grown in DMEM-Ham's F-12 (DMEM-F12; 1:1) containing 10% heat-inactivated fetal bovine serum and penicillin, streptomycin, and glutamine (Life Technologies, Gaithersburg, MD). Cells were routinely passaged when they reached 50-75% confluence. For growth on porous filters, cells were grown in the same medium and plated at 3 × 105 cells/100 µl or 5 × 106 cells/2 ml on 6.5-mm or 24-mm Transwell filters (clear polystyrene, 0.4 µm pore size; Costar, Cambridge, MA), respectively. For growth on the 6.5-mm filters, cells were plated "upside down" so that their basolateral membranes were exposed to the upper chamber of the filter cups. The transepithelial electrical resistance (TER) of cells grown on filters was measured with an epithelial voltohmmeter (World Precision Instruments, Sarasota, FL). Cells were used only if their TER was >1,000 Omega  · cm2. Cells with stable TER readings >1,000 Omega  · cm2 were treated with IFN-gamma (107 U/mg; Genzyme, Boston, MA) added to the basolateral side of the filters for the indicated times. In some instances, cells were allowed to recover from IFN-gamma treatment by washing three times in medium and by maintaining in complete medium without IFN-gamma .

Preparation of detergent-soluble and -insoluble fractions. T84 cell lysates were prepared by differential detergent solubility as previously described (29).

Immunoblotting of protein fractions. NP-40-soluble and -insoluble protein fractions were separated on a 10% Tris-glycine polyacrylamide gel (Novex, San Diego, CA), transferred to nitrocellulose (0.45 µm; Bio-Rad, Richmond, CA), and blocked for 4-6 h in PBS-0.1% Tween 20-5% dried milk (PTM). The blots were then incubated overnight at 4°C with primary antibodies (ZO-1, ZO-2, occludin, and E-cadherin; Zymed Laboratories, South San Francisco, CA) diluted 1:1,000-1:2,000 in PTM. The blots were then washed in PBS, incubated at room temperature for 90 min with biotinylated secondary antibody (Molecular Probes, Eugene, OR) diluted 1:1,000 in PTM, followed by horseradish peroxidase-conjugated streptavidin (Sigma, St. Louis, MO) diluted 1:10,000 in PTM, and then processed for enhanced chemiluminescence detection (Amersham, Arlington Heights, IL).

Lambda phosphatase treatment of proteins. Protein (25-100 µg) from the NP-40-soluble and -insoluble fractions was immunoprecipitated with 5 µg of antibody (anti-ZO-1, anti-ZO-2, or anti-occludin) and 50 µl of protein G-agarose (Pierce Chemicals, Rockford, IL) for 4 h at 4°C. The antibody-protein G complex was washed three times in NP-40-insoluble protein buffer without phosphatase inhibitors and then brought up in 100 µl of the same buffer. Manganese chloride was added to a final concentration of 2 mM, and 800 units of lambda phosphatase (400,000 U/ml, New England Biolabs, Beverly, MA) were added. The samples were incubated at 30°C for 90 min with frequent mixing and washed three times with NP-40-insoluble protein, and the bound proteins were eluted from the protein G beads by boiling in reducing SDS-PAGE sample buffer. The samples were then processed as described above for immunoblotting of proteins.

Pulse-chase analysis and immunoprecipitation of ZO-1. T84 cells were grown on 75-mm Transwell filter inserts and treated with 100 ng/ml IFN-gamma for various times. Cells were metabolically labeled for 4 h with Redivue Pro-mix 35S cell labeling mix (180 µCi/ml, sp act <1,000 Ci/mmol, Amersham) in 20 ml cysteine- and methionine-free DMEM (Life Technologies) containing 5% dialyzed fetal bovine serum. One-half of the filter inserts from each sample (0 h chase group) was processed as described in Immunoblotting of protein fractions to isolate the detergent-soluble and -insoluble fractions. The other half of the inserts was washed three times with complete DMEM-F12 containing 10% serum and five times the normal concentration of cysteine and methionine and then maintained in this medium for an additional 12 h (12-h chase group) before being processed for isolation of the different protein fractions. IFN-gamma was present during the entire pulse-chase period in the appropriate samples. Protein concentration was determined by microbicinchoninic acid procedure (Pierce Chemicals). Incorporation of radioactivity was measured by cold TCA precipitation.

ZO-1 and occludin were immunoprecipitated from the NP-40-insoluble protein fraction, whereas ZO-2 was immunoprecipitated from the NP-40-soluble protein fraction. Briefly, 50-100 µg of protein were incubated with 10 µg of antibody overnight at 4°C. This material was added to 50 µl of protein G-agarose (Pierce Chemicals) and incubated for 2 h. The antibody-protein G complex was washed three times in NP-40-insoluble protein buffer and two times in RIPA buffer (150 mM NaCl containing 1% NP-40, 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0). The bound proteins were eluted with 30 µl of SDS-PAGE reducing sample buffer and fractionated on a 10% Tris-glycine polyacrylamide gel, which was subsequently fixed and dried and quantified on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Analysis of ZO-1 and ZO-2 expression by RT-PCR. Total RNA was extracted from T84 cells with TRIzol reagent (Life Technologies) using the manufacturer's protocol. RNA samples were treated with DNase (10 units; Genhunter, Nashville, TN) before the RT-PCR procedure. For RT-PCR, 0.5 µg of total RNA was reverse transcribed with 200 units Moloney murine leukemia virus RT (Life Technologies) that was primed with either 100 ng of random hexamer (Life Technologies) or 0.5 µg of oligo(dT) (Life Technologies) in 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 2 mM DTT containing 10 units RNase inhibitor (Boehringer Mannheim, Indianapolis, IN), and 0.5 mM each dNTPs for 1 h at 42°C. The reaction mixture was heat inactivated at 72°C for 15 min and then brought to 100 µl final volume with water. PCR reactions were performed with 6.25 µl of cDNA from the RT reaction, 10 units Taq polymerase (Boehringer Mannheim), 1.5 mM MgCl2, 0.4 µM each primer, 200 µM each dNTP, and 5 µCi [alpha -32P]dCTP (sp act 3,000 Ci/mmol; Amersham) in 250 µl total volume divided into 25-µl aliquots. The primer sets for ZO-1 (forward primer, 5'-CGGTCCTCTGAGCCTGTAAG-3'; reverse primer, 5'-GGATCTACATGCGACGACAA-3') and for ZO-2 (forward primer, 5'-GCCAAAACCCAGAACAAAGA-3'; reverse primer, 5'-ACTGCTCTCTCCCACCTCCT-3') were both designed to amplify the long and short isoforms of both proteins (16, 17). PCR was performed in a Stratagene Robocycler Gradient 96 (Stratagene, La Jolla, CA) by heating the samples at 94°C for 5 min for one cycle, followed by 94°C for 30 s, 57°C for 30 s, and 72°C for 1 min for the indicated number of cycles. Aliquots were taken after every other cycle between cycles 14 and 30. Ten microliters of each PCR reaction was electrophoresed on a 6% or 10% polyacrylamide-Tris-borate-EDTA gel, dried, and quantified on a PhosphorImager.

Immunofluorescence confocal analysis. Cells were analyzed by confocal microscopy as previously described (14).


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IFN-gamma treatment of T84 cells decreased TER. T84 cells grown on filter inserts formed polarized, impermeable monolayers that possessed relatively high TER. Treatment of the cells with different doses (10, 30, and 100 ng/ml) of IFN-gamma added to the basolateral side of the monolayer resulted in a time- and dose-dependent decrease in TER (Fig. 1). Monolayers treated with the highest concentration of IFN-gamma (100 ng/ml) showed a 40% and 80% decrease in TER after 24 and 48 h of treatment, respectively, relative to control cells. In contrast, cells treated with the lower doses of IFN-gamma showed less of a decrease in TER at 24 and 48 h, but, by 72 h, both showed an overall decline (~80%) in TER that was comparable to the high dose of IFN-gamma . To confirm that the changes in TER were a reflection of altered paracellular permeability, [3H]mannitol flux measurements were performed. In cells treated with 100 ng/ml IFN-gamma , mannitol flux was increased ~10fold above untreated cells at 48 h (5.2 vs. 0.55 nmol · h-1 · cm-2, respectively; data not shown). Cells treated with the other two doses of IFN-gamma showed a similar increase in mannitol flux relative to untreated cells at 72 h (data not shown). Thus the decrease in TER induced by IFN-gamma is at least partially due to increased paracellular permeability.


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Fig. 1.   Interferon-gamma (IFN-gamma ) treatment of T84 cells decreases transepithelial electrical resistance (TER) in a time- and dose-dependent manner. T84 cells grown on polystyrene filters were treated with different concentrations of IFN-gamma added basolaterally. At the indicated times, TER measurements were taken. Results represent an average of 9 filters per time point. In all instances, baseline TER at time 0 was >1,000 Omega  · cm2. , Untreated; , 10 ng/ml IFN-gamma ; black-triangle, 30 ng/ml IFN-gamma ; black-diamond , 100 ng/ml IFN-gamma .

IFN-gamma treatment reduced the levels of ZO-1 in T84 cells. To examine the effects of IFN-gamma on proteins associated with the tight junctions, ZO-1, ZO-2, and occludin were analyzed by immunoblotting. Cells were separated into NP-40-soluble and -insoluble fractions as previously described (29). According to this method, cells are first solubilized with NP-40 to remove proteins that are not associated with the cytoskeleton. Subsequently, the NP-40-insoluble material is solubilized with SDS. This latter fraction contains the cytoskeleton and associated proteins, including those found in tight junctions. Proteins in these two fractions were collected from untreated T84 cells and cells treated for 72 h with different doses of IFN-gamma and processed for immunoblotting. The blots were probed with antibodies to ZO-1, ZO-2, occludin, and E-cadherin. Although E-cadherin is not associated with tight junctions, its expression was analyzed because disruption of E-cadherin-mediated adhesion events has been shown to prevent tight junction formation (11). As shown in Fig. 2, a number of changes were apparent in the various proteins following IFN-gamma treatment. In untreated cells, ZO-1 was associated almost exclusively in the insoluble fraction. Treatment of the cells with IFN-gamma resulted in a substantial decrease (7- to 15-fold, 3 experiments) in ZO-1 levels in the insoluble fraction. The fact that ZO-1 protein was not detected in the soluble fraction after IFN-gamma treatment suggests that this protein was not being "chased" from the insoluble fraction; rather, the expression of this protein was reduced. In contrast to ZO-1, ZO-2 in untreated cells was found in both the soluble and insoluble fractions. However, there appeared to be two forms of ZO-2 in the soluble fraction and only one, corresponding to the higher-molecular-weight form, in the insoluble fraction. The possible structural significance of the different species of ZO-2 will be addressed later. Treatment of the cells with IFN-gamma appeared to have only relatively modest effects (20-40% decrease, 3 experiments) on the amount of ZO-2 in the soluble fraction. In contrast, ZO-2 in the insoluble fraction decreased dramatically, similar to what was observed with ZO-1. Quantitation of ZO-2 in the soluble fraction of the IFN-gamma -treated samples did not show an increase in the proportion of the upper band as would be expected if this protein were being displaced from the insoluble to the soluble fraction. However, this could simply be a reflection of the modest decrease in overall ZO-2 levels induced by IFN-gamma , which may have masked any accumulation of the higher-molecular-weight species in the soluble fraction.


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Fig. 2.   Differential detergent solubility of tight junction and adhesion proteins and effects of IFN-gamma treatment on protein expression. T84 cells were treated for 72 h with the indicated concentrations of IFN-gamma and then extracted as described in MATERIALS AND METHODS. NP-40-soluble (S) and NP-40-insoluble (I) fractions were immunoblotted with antibodies to zonula occludens (ZO)-1, ZO-2, occludin, and E-cadherin.

Occludin, the only known transmembrane component of tight junctions, was also distributed in the soluble and insoluble fractions. However, the nature of the proteins in the two fractions was quite different. A major protein species was found in the soluble fraction, whereas, in the insoluble fraction, a heterogeneous mixture of discrete, higher-molecular-weight polypeptides was detected. IFN-gamma treatment of the cells showed a relatively modest reduction (25-40%, 3 experiments) in occludin in the soluble and insoluble fractions. Furthermore, there did not appear to be a change in the pattern of the protein bands in either fraction following IFN-gamma treatment.

The cell adhesion molecule E-cadherin was found primarily in the soluble fraction, although a significant proportion was also found in the insoluble fraction. These results are somewhat surprising, since the adhesive properties of E-cadherin are dependent on its cytoskeletal association; they suggest that, in T84 cells, only a portion of E-cadherin is functionally involved in mediating cell adhesion or simply that the association of E-cadherin with the cytoskeleton in these cells may not be as strong as that of tight junction proteins under the conditions used for extraction. Nonetheless, treatment with IFN-gamma did not seem to alter the expression of E-cadherin in either fraction. Thus it appears that IFN-gamma treatment of T84 cells induces a dramatic decrease in ZO-1 levels, whereas the effects on other proteins are relatively modest.

Previous studies have shown that a number of the tight junction proteins are phosphorylated. To determine the contribution of phosphorylation to the complexity of the protein patterns observed, particularly with regard to ZO-2 and occludin, the samples were treated with lambda phosphatase before immunoblotting (Fig. 3). Lambda phosphatase cleaves phosphates on serine, threonine, and tyrosine residues (34). ZO-1, present in only the insoluble fraction, showed no change in mobility after phosphatase treatment, indicating either an absence or a low level of phosphorylation. This observation is consistent with an earlier study showing that ZO-1 in Madin-Darby canine kidney (MDCK) cells with high transepithelial resistance was not highly phosphorylated (30). Similarly, neither ZO-2 in the soluble nor ZO-2 in the insoluble fraction was altered by treatment with the phosphatase. This result raises the possibility that the two species of ZO-2 protein in the soluble fraction may represent the long (beta +) and short (beta -) isoforms of the protein (4) or that the lower-molecular-weight band is simply a proteolytic product of the higher-molecular-weight band. These possibilities will be addressed in later sections of this study.


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Fig. 3.   Phosphatase treatment of tight junction proteins. NP-40-soluble (S) and NP-40-insoluble (I) fractions from untreated T84 cells were immunoprecipitated with the appropriate antibodies, treated without (-) or with (+) lambda phosphatase (PPase) and then immunoblotted as described in MATERIALS AND METHODS. Results shown are with proteins from untreated cells, but similar results were obtained with samples from IFN-gamma -treated cells.

Occludin showed the most dramatic effect following phosphatase treatment. Although occludin in the soluble fraction was unaffected by the phosphatase, occludin in the insoluble fraction was reduced from a broad, heterogeneous protein profile to a lower-molecular-weight protein that migrated similarly to the one in the soluble fraction. Thus these results show that occludin in the insoluble fraction of T84 cells is heavily phosphorylated, as has been shown previously for occludin in the tight junction fraction of MDCK cells (29).

To further assess how IFN-gamma affects ZO-1 expression, the levels of ZO-1 were measured at different times after treatment with 100 ng/ml IFN-gamma . Within 24 h of IFN-gamma treatment, ZO-1 levels decreased by ~50% (Fig. 4), which correlates with the decrease in TER (Fig. 1). By 48 and 72 h of treatment, ZO-1 levels were undetectable by immunoblotting (Fig. 4). Thus it appears that the loss of ZO-1 coincides with the decrease in barrier function. However, barrier function was not totally abolished even in the absence of detectable ZO-1 (cf. Figs. 1 and 4). These results suggest that other proteins may compensate for the loss of ZO-1 and may thus contribute to the maintenance of barrier function, albeit at a reduced level.


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Fig. 4.   ZO-1 levels decrease with increasing time of treatment with IFN-gamma . T84 cells were treated with 100 ng/ml IFN-gamma for the indicated times. Cells were extracted as in Fig. 1, and immunoblotting to detect ZO-1 was performed.

IFN-gamma treatment decreases ZO-1 protein synthesis and stability. To determine how IFN-gamma treatment reduces ZO-1 expression, the biosynthesis of ZO-1 protein was examined. T84 cells were analyzed by pulse-chase metabolic labeling with 35S-labeled amino acids and immunoprecipitation of ZO-1, ZO-2, and occludin (Fig. 5). In untreated cells, a single band corresponding to ZO-1 was detected at 0 and 12 h of chase (Fig. 5, top). Quantitation of these bands showed that the intensity of the 12-h chase sample was ~0.75 times that of the 0-h chase sample. Thus the approximate half-life of ZO-1 in untreated T84 cells is ~24 h. T84 cells treated with IFN-gamma for 24 h before metabolic labeling also showed a single band corresponding to ZO-1. However, the intensity ratio of the 12-h chase to the 0-h chase was 0.60, indicating a half-life of ~15 h. Thus IFN-gamma treatment of the T84 cells appears to increase the turnover rate of ZO-1 by almost 40%. No detectable ZO-1 was immunoprecipitated from cells pretreated with IFN-gamma for 48 h (or 72 h, data not shown). In contrast to ZO-1, ZO-2 (Fig. 5, middle) showed no change in half-life between untreated cells and cells treated with IFN-gamma for 24 h (half-life of ~24 h in both cases). Interestingly, in cells treated for 48 h with IFN-gamma , the half-life of ZO-2 decreased to ~15 h. The fact that ZO-2 shows a more rapid turnover rate at about the same time that ZO-1 levels become undetectable may indicate that ZO-2 is somewhat more unstable in the absence of ZO-1. Occludin (Fig. 5, bottom) showed no change in protein turnover rate between untreated and IFN-gamma -treated cells at any of the time points. In all cases, a broad, heterogeneous collection of bands corresponding to occludin was detected. The ratio of signal intensity at the 12-h chase compared with the 0-h chase was about equal in all cases and indicated a half-life of ~24 h, similar to ZO-1 in untreated cells. These results indicate that the stability of ZO-1, ZO-2, and occludin are affected differently by treatment with IFN-gamma .


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Fig. 5.   Pulse-chase analysis of tight junction proteins in untreated and IFN-gamma -treated T84 cells. T84 cells, treated for indicated times with 100 ng/ml IFN-gamma , were analyzed following pulse-chase metabolic labeling, and immunoprecipitation of tight junction proteins was performed as described in MATERIALS AND METHODS. Relative intensity refers to the ratio of signal intensity at the 12-h chase to 0-h chase in each sample. Note that in the ZO-1 48-h IFN-gamma treatment no protein band was detected. OCC, occludin.

The rate of protein biosynthesis can also be measured in metabolically labeled cells, by comparing the intensity of the radiolabeled protein bands at 0 h of chase between untreated and IFN-gamma -treated cells. In the case of ZO-1, the ratio of signal intensity at the 0-h chase between cells treated with IFN-gamma for 24 h and untreated cells was ~0.5, indicating a 50% decrease in the rate of ZO-1 biosynthesis. The dramatic reduction in the biosynthetic rate of ZO-1 is made more apparent by the fact that cells treated with IFN-gamma for 48 h had no detectable ZO-1 protein. In contrast, the ratios of ZO-2 signal intensities of cells treated for 24 and 48 h with IFN-gamma were 1.0 and 0.95, respectively, relative to untreated cells. Similarly for occludin, relative to untreated cells, the ratios were 0.93 and 0.84 for cells treated with IFN-gamma for 24 and 48 h, respectively. Thus the biosynthesis of ZO-1 is severely reduced, whereas ZO-2 and occludin are relatively unaffected by IFN-gamma treatment. These observations are consistent with the immunoblotting data that showed that only ZO-1 protein levels are reduced by IFN-gamma treatment, whereas ZO-2 and occludin are not. Furthermore, these results show that IFN-gamma treatment of T84 cells appears to specifically repress ZO-1 biosynthesis.

IFN-gamma treatment decreases the transcription of ZO-1. The effect of IFN-gamma treatment on the mRNA levels of ZO-1 was also examined. The levels of RNA were measured using a semiquantitative RT-PCR rather than Northern blotting because RT-PCR is useful for measuring small changes in mRNA levels and also because it can resolve splice variants that differ only slightly in size. This latter point is an issue for ZO-1 and ZO-2 because each gene encodes at least two splice variants that differ by ~240 and 108 nt, respectively. These variants are very difficult to resolve by Northern analysis given the relatively large sizes of the mRNAs for these genes (~7.5 and 5 kb for ZO-1 and ZO-2, respectively). [32P]dCTP was incorporated into the PCR reaction, and the labeled product that was synthesized was analyzed at various cycle numbers. The cycle number at which the labeled product appeared was a measure of the relative abundance of that product. In untreated cells, the labeled DNA fragment of ZO-1 was first detectable at cycle 26 but did not appear until cycle 28 in the IFN-gamma -treated cells (Fig. 6A). With increasing cycle number, this difference remained apparent (Fig. 6). Thus the decrease in ZO-1 mRNA levels was consistent with the decrease observed in the levels of ZO-1 protein. In contrast, and as a control for possible sample variation, when levels of ZO-2 mRNA were measured by RT-PCR (Fig. 6), a labeled product of equal intensity was observed in both control and IFN-gamma -treated samples beginning at cycle 26. In subsequent cycles, the levels of ZO-2 remained about equal. This result indicates that transcription of ZO-2 mRNA is unaffected by IFN-gamma and is consistent with the relatively modest changes observed in ZO-2 protein levels. The primers chosen for the RT-PCR analysis flank the DNA sequences that encode the splice variants of both ZO-1 and ZO-2 and are thus able to detect the long and short isoforms of each protein. In both instances, however, only one major DNA fragment was amplified that corresponds to the long isoform of each protein (4, 33). Thus, in T84 cells, only the alpha +-form of ZO-1 and the beta +-form of ZO-2 are expressed. In the case of ZO-2, this observation, along with the fact that phosphatase treatment had no effect on the protein, suggests that the lower-molecular-weight band seen in the soluble fraction of the immunoblot analysis may be a product of proteolysis.


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Fig. 6.   RT-PCR analysis of ZO-1 and ZO-2 mRNA in untreated and IFN-gamma -treated T84 cells. RNA from either untreated T84 cells or cells treated for 72 h with 100 ng/ml IFN-gamma was processed for RT-PCR as described in MATERIALS AND METHODS. A: autoradiographs of the 32P-labeled PCR products. Expected sizes of the amplified fragments are 371 and 212 bp for ZO-1 and ZO-2, respectively. Numbers above the gels indicate the number of PCR cycles used to obtain the product. B: quantitation of the autoradiographs. , Untreated cells; , IFN-gamma -treated cells.

Loss of ZO-1 alters the subcellular distribution of ZO-2 and occludin. To examine if the loss of ZO-1 affects the localization of ZO-2 and occludin, a confocal microscopic analysis was undertaken (Fig. 7). In untreated cells, en face (x-y) sections showed that ZO-1 was localized to the cell boundaries (Fig. 7A), and, in vertical (x-z) sections through the monolayer, it was localized to a discrete subapical region of the lateral membranes (Fig. 7a). This immunostaining pattern is characteristic of proteins that are found at tight junctions. ZO-2 and occludin showed very similar staining patterns (Fig. 7Bb and Cc, respectively). E-cadherin also stained cell boundaries in en face sections (Fig. 7D), but, in vertical sections, it was located on the lateral membranes boundaries as expected for a protein that mediates cell-to-cell interactions (Fig. 7d). Analysis of IFN-gamma -treated T84 cells showed that immunostaining for ZO-1 was very faint to undetectable in en face and vertical sections (Fig. 7Ee). ZO-2 staining in treated cells appeared more diffuse in en face staining (Fig. 7F) compared with untreated cells. In vertical sections, the staining of ZO-2 was no longer localized to the tight junctions; rather, it was diffusely scattered in the cells (Fig. 7f). Occludin showed a similar diffuse staining in en face sections (Fig. 7G), and it was also distributed along the apical and lateral membranes in vertical sections (Fig. 7g). E-cadherin staining in IFN-gamma -treated cells was similar to untreated cells, remaining at regions of cell contact (Fig. 7Hh). Thus, in untreated cells, ZO-1, ZO-2, and occludin were all localized to tight junctions. However, after IFN-gamma treatment, ZO-1 was lost and the distribution of ZO-2 and occludin was no longer restricted to the tight junctions. Thus these results raise the intriguing possibility that the proper subcellular localization of ZO-2 and occludin does not occur in the absence of ZO-1.


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Fig. 7.   Confocal analysis of untreated and IFN-gamma -treated T84 cells stained for ZO-1, ZO-2, occludin, and E-cadherin. Untreated T84 cells or cells treated for 72 h with 100 ng/ml IFN-gamma were processed for immunofluorescence as described in MATERIALS AND METHODS. Cells were then analyzed by confocal microscopy. Shown are the en face (x-y) sections (A-H) and the corresponding vertical (x-z) sections (a-h). Untreated T84 cells (A-D and a-d) and IFN-gamma -treated cells (E-H and e-h) were stained for ZO-1 (A, E and a, e), ZO-2 (B, F and b, f), occludin (C, G and c, g), and E-cadherin (D, H and d, h). Arrows by the x-z sections indicate the apical (Ap) and basal (Ba) aspects of the cells. Bar in a = 10 µm.

IFN-gamma treatment perturbs actin in the apical-tight junction region of T84 cells. It is well established that epithelial barrier function can also be regulated by the actin-containing cytoskeleton (2). To determine if IFN-gamma treatment of T84 cells also affects the cytoskeleton, the distribution of actin was examined by confocal microscopy using rhodamine-phalloidin (Fig. 8). In polarized epithelial cells, actin is found in the microvillar projections, in the terminal web, in a belt at the level of the adherens junctions, at cortical regions below the plasma membranes, and in stress fibers on the basal surface of the cells (26). There is also evidence that actin is associated with the tight junctions (20). In control cells, actin staining at the apical-tight junction level revealed a bright, dense, punctate pattern representing staining in the microvilli and a dense network of actin at the cell boundaries (Fig. 8A). At the midpoint of the cell, a cortical actin ring was seen (Fig. 8B), whereas, at the basal end of the cells, a dense network of stress fibers was detected (Fig. 8C). In IFN-gamma -treated cells, the most striking differences observed in actin staining were seen at the apical-tight junction level (Fig. 8D), where the punctate staining seen in controls was absent. In addition, actin at the cell periphery appeared discontinuous and not as sharply defined as in untreated cells. In many instances, no actin staining was detected at the apical-tight junction level (Fig. 8D). At the midcell and basal levels, actin was found at the cell cortex and in a dense network of stress fibers, (Fig. 8, E and F, respectively), reminiscent of what was seen in untreated cells. These results indicate that the effects of IFN-gamma on the actin cytoskeleton in T84 cells is limited to the apical-tight junction-associated actin. The actin cytoskeleton in the more basal regions of the cells is unaffected. This effect is not simply due to a decrease in actin levels in IFN-gamma -treated cells, since immunoblotting with anti-actin antibodies showed comparable levels of actin in untreated and treated cells (data not shown). Thus IFN-gamma may be perturbing the barrier function because of its effects on actin as well as on ZO-1.


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Fig. 8.   Effects of IFN-gamma treatment on the actin cytoskeleton. Untreated T84 cells or cells treated for 72 h with 100 ng/ml IFN-gamma were stained with rhodamine-phalloidin and analyzed by confocal microscopy. Vertical sections (x-z) were taken off the monolayers to define the top and bottom of the monolayer. En face sections (x-y) were then generated from selected planes in the vertical section. Untreated cells (A-C) and IFN-gamma -treated cells (D-F) are shown at the apical-tight junction level (A, D), midcell level (B, E), and basal level (C, F). Arrows in D indicate regions where no actin staining is detected. Bar in A = 10 µm.

The alterations in apical actin in IFN-gamma -treated T84 cells observed in this study were more dramatic than previously described (5, 24). This difference may be a reflection of clonal variability in the T84 cells used by the various laboratories. Alternatively, the stability of the cytoskeleton may have been influenced by the matrices on which the cells were grown (tissue culture-treated polystyrene filters in this study vs. collagen-coated filters in the previous studies).

Loss of ZO-1 and apical actin disruption coincide temporally. In an attempt to dissociate the effects of IFN-gamma on ZO-1 and actin and also to determine if the effects of IFN-gamma were reversible, the expression of ZO-1 and actin was examined by confocal microscopy at various times during IFN-gamma treatment and after removal of IFN-gamma (Fig. 9). Disruption and recovery of the epithelial barrier were assessed by TER measurements of cells treated with 100 ng/ml IFN-gamma for 72 h and subsequent removal of IFN-gamma from the medium for an additional 72 h (Fig. 9A). Treatment of the cells with IFN-gamma showed a time-dependent decrease in TER, whereas removal of the IFN-gamma showed a time-dependent recovery in TER. The fact that the cells could reestablish epithelial barriers with high electrical resistance when IFN-gamma was removed demonstrates that IFN-gamma is not toxic to cells (1). Concurrent confocal analysis showed that changes in ZO-1 and actin expression correlated well with barrier breakdown and recovery (Fig. 9, B-G). Untreated T84 cells showed the characteristic ZO-1 and actin staining pattern around the cell periphery (Fig. 9B). After 24 h of treatment with IFN-gamma , the staining pattern was similar but of a decreased intensity (Fig. 9C). At 48 and 72 h, however, ZO-1 staining was almost undetectable as was actin staining at the apical portion of the cells (Fig. 9D). Twenty-four hours after removal of IFN-gamma , ZO-1 staining was very weak and discontinuous; however, wherever there was ZO-1 staining, apical actin staining was also detected (Fig. 9E). After 48 and 72 h of recovery, ZO-1 and apical actin staining increased concomitantly (Fig. 9, F and G). Thus, by temporal analysis, the expression of ZO-1 at tight junctions and the reorganization of actin at the apical/tight junction region of the cells coincide.


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Fig. 9.   ZO-1 and actin expression during barrier breakdown and recovery. Cells were treated with IFN-gamma for 0-72 h. At 72 h, IFN-gamma was removed from the medium and the cells were allowed to recover for 24-72 h. A: TER measurements during the course of IFN-gamma treatment (solid bar along x-axis) and recovery after IFN-gamma removal from the culture medium (open bar along x-axis). B-G: confocal analyses of ZO-1 and actin were performed during the IFN-gamma -induced barrier breakdown and subsequent recovery. Shown are the en face sections (x-y) from the apical region of cells stained for ZO-1 (green) and actin (red). B: untreated cells. C: 24-h IFN-gamma treatment. D: 48 and 72 h IFN-gamma treatment. E: 24 h after removal of IFN-gamma . F: 48 h after removal of IFN-gamma . G: 72 h after removal of IFN-gamma . Bar in F = 5 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Degradation of the intestinal epithelial barrier is a characteristic of numerous gut disorders, including inflammatory bowel disease. The cause of the breakdown is multifactorial and involves bacteria, cells of the immune system, and cytokines. To better understand the contribution of proinflammatory cytokines to regulation of the epithelial barrier, we have examined the effects of IFN-gamma on T84 intestinal epithelial cells. The findings in this study indicate that IFN-gamma acts on at least two elements critical to barrier function. The first is to cause a decrease in the expression of ZO-1, a key component of the tight junction. The second is to alter the organization of the actin cytoskeleton in the apical region of the cells.

The pivotal role of ZO-1 in the tight junction complex is reflected by its binding to numerous other tight junction and cytoskeletal proteins. ZO-1 has been shown to bind occludin (10), ZO-2 (18), ZO-3 (12), and actin (7, 15). As such, ZO-1 may serve to organize the tight junction complex and also to connect it to the actin cytoskeleton. A recent study has demonstrated that ZO-1 does indeed bind to occludin, via an NH2-terminal domain, and actin, via a COOH-terminal domain, thus acting as a bridge between the plasma membrane and the cytoskeleton (7). Given its role in connecting numerous proteins, one might predict that loss of ZO-1 would result in a disorganization of the tight junction, such as aberrant localization of ZO-2 and occludin. Similarly, if the organization of tight junction-associated actin requires binding to ZO-1, then loss of ZO-1 should perturb actin at that site. These alterations are, in fact, what is observed when T84 cells are treated with IFN-gamma . As the barrier function of T84 cells is eliminated, there is an associated loss of ZO-1 and a subsequent mislocalization of both ZO-2 and occludin as observed by confocal microscopy. In the case of ZO-2, alterations in subcellular distribution were also reflected in biochemical assays in which the protein showed almost complete loss from the detergent-insoluble, tight junction-enriched fraction after IFN-gamma treatment. This result supports the notion that ZO-2 association with the tight junction can only occur in the presence of ZO-1. Occludin shows a similar disorganized subcellular localization in the IFN-gamma -treated cells. However, in contrast to ZO-2, occludin remains in the detergent-insoluble fraction even in the absence of ZO-1. This suggests that the insolubility of occludin may be due to its inherent propensity to form oligmers (29). In addition to remaining in the insoluble fraction, occludin from IFN-gamma -treated cells did not show gross alterations in phosphorylation, suggesting that this modification occurs independently of ZO-1 association and tight junction localization. The mislocalization of occludin in cells with decreased levels of ZO-1, however, is consistent with a previous report showing that interaction of occludin with ZO-1 is required for the proper subcellular localization of occludin to the tight junctions (10). Thus it appears that ZO-1 plays an active role in maintaining the localization of ZO-2 and occludin. Whether these proteins are targeted to the tight junction but are unable to remain there because ZO-1 is not present to anchor them at that site remains to be determined.

Treatment of T84 cells with IFN-gamma also altered the actin cytoskeleton in the region of the tight junction. The importance of actin to the maintenance and regulation of barrier function has been demonstrated in numerous studies (20, 21, 30). It has been suggested that expansion or contraction of the actin cytoskeleton may exert forces that tighten or loosen the tight junctions, respectively (20). The tight junction regulatory activity of actin appears to be localized to the apical portion of the cytoskeleton. Thus, in MDCK cells treated with high concentrations of cytochalasin D, only basal actin was perturbed and barrier function was only modestly affected. In contrast, MDCK cells treated with low concentrations of cytochalasin D in which both the apical and basal pools of actin were perturbed show decreased barrier function (31). Bacterial infection of epithelial cells by Clostridium difficile and Salmonella typhymurium also alters apical and tight junction-associated actin and subsequently decreases epithelial barrier function (13, 17). With C. difficle, the effect is most likely due to inactivation of Rho, a small GTP binding protein required for actin assembly (19). Similar results are obtained by directly targeting Rho by transfection of T84 cells with C. botulinum toxin C3 exoenzyme (27). Interestingly, in this study, ZO-1 was also lost from the tight junctions of toxin-transfected cells and was redistributed in the cell cytoplasm; ZO-1 levels, however, were not reduced compared with control cells. Thus, whereas the effects on apical actin may be similar with bacterial infection and IFN-gamma treatment of T84 cells, the effects on ZO-1 are quite different, suggesting that the mechanism of barrier disruption induced by these various agents is not the same. Whether the effects of IFN-gamma on apical actin are the result of Rho inactivation remains to be determined.

On the basis of the experiments in this study, it has not been possible to dissociate the effects of IFN-gamma on ZO-1 from those on actin. Because ZO-1 levels in IFN-gamma -treated cells are reduced and protein is not simply mislocalized, it is tempting to speculate that loss of ZO-1 is critical to the disorganization of apical actin. Further support for this hypothesis comes from the fact that agents whose mode of action is primarily on actin organization disrupt barrier function rapidly (i.e., minutes to hours). In contrast, IFN-gamma -induced barrier breakdown requires significantly longer periods (i.e., hours to days), suggesting that the perturbation of actin, in this case, is secondary to the loss of ZO-1. Nonetheless, because the two events, loss of ZO-1 and apical actin disruption, are temporally linked, it is not possible to unequivocally state that one event is responsible for the occurrence of the other. In future studies, it may be possible to test whether loss of ZO-1 influences apical actin organization by expressing ZO-1 from a heterologous promoter and then measuring the effects of IFN-gamma treatment on barrier function. One would predict that if loss of ZO-1 was the initiating event in barrier disruption then overexpression of ZO-1 should prevent barrier breakdown and actin reorganization.

It is intriguing that, despite the apparent lack of ZO-1, IFN-gamma -treated T84 cells can still maintain some barrier function. This observation suggests that other proteins may contribute to barrier function despite the loss of a critical component of the tight junction. Redundancy of function in tight junctions was shown in cells lacking occludin; these cells could still form tight junctions and form impermeable barriers (28). In this case, proteins, such as claudin-1 and -2 (8) and JAM (24), may functionally substitute for occludin. What is unclear is whether these proteins function at tight junctions within the same complex as does occludin (i.e., they bind to ZO-1) or as part of a "parallel" complex (i.e., ZO-1 independent). Similarly, in this study, is the residual barrier activity seen in the absence of ZO-1 the result of redundancy in ZO-1 function or is it due to a ZO-1-independent complex of proteins? Further analysis of the composition and dynamics of tight junctions will allow us to resolve these questions.


    ACKNOWLEDGEMENTS

We thank Lori Whittaker, Helen Hathaway, Douglas Williams, and Linda Park for critical reading of the manuscript, Ann Bannister for editorial assistance, and Mari Hall for assistance with graphics.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. Youakim, Dept. of Biochemistry, Immunex Corporation, 51 University St., Seattle, WA 98101 (E-mail: ayouakim{at}immunex.com).

Received 7 December 1998; accepted in final form 3 February 1999.


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