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The Journal of Immunology, 2005, 175: 4030-4038.
Copyright © 2005 by The American Association of Immunologists

Interferon-{gamma} Inhibits T84 Epithelial Cell Migration by Redirecting Transcytosis of {beta}1 Integrin from the Migrating Leading Edge1

Qiao Tong2, Elena V. Vassilieva2, Andrei I. Ivanov, Zili Wang, Gregory T. Brown, Charles A. Parkos and Asma Nusrat3

Epithelial Pathology Research Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Intestinal inflammation is associated with epithelial damage and formation of mucosal wounds. Epithelial cells migration is required for wound closure. In inflammatory status, migrating epithelial cells are exposed to proinflammatory cytokines such as IFN-{gamma}. However, influence of such cytokines on intestinal epithelial wound closure remains unknown. The present study was designed to investigate the effect of IFN-{gamma} on migration of model T84 intestinal epithelial cells and recovery of epithelial wounds. IFN-{gamma} significantly inhibited rate of T84 cell migration and closure of epithelial wounds. This effect was accompanied by the formation of large aberrant lamellipodia at the leading edge as well as significant decrease in the number of {beta}1 integrin containing focal adhesions. IFN-{gamma} exposure increased endocytosis of {beta}1 integrin and shifted its accumulation from early/recycling endosomes at the leading edge to a yet unidentified compartment at the cell base. This redirection in {beta}1 integrin transcytosis was inhibited by depolymerization of microtubules with nocodazole and was unaffected by stabilization of microtubules with docetaxel. These results suggest that IFN-{gamma} attenuates epithelial wound closure by microtubule-dependent redirection of {beta}1 integrin transcytosis from the leading edge of migrating cells thereby inhibiting adequate turnover of focal adhesion complexes and cell migration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The epithelial lining of the gastrointestinal tract serves as a barrier that separates luminal contents from the underlying tissue compartments. Disease states such as inflammatory bowel disease (IBD)4 are associated with mucosal wounds that serve as avenues for luminal bacteria and Ag access to underlying tissues. Additionally, epithelial injury observed in patients with IBD is associated with inflammatory cell influx into the mucosa and release of proinflammatory cytokines into the milieu of the epithelium that in turn modulate pathophysiologic events such as epithelial cell migration and wound closure. IFN-{gamma} is one of the key proinflammatory cytokines that is increased in mucosa of patients with IBD. Whereas the effects of IFN-{gamma} on epithelial intercellular junctions and barrier properties have been extensively studied (1, 2, 3, 4), its influence on closure of epithelial wounds has not been well characterized.

Superficial wounds rapidly reseal by migration of cells, an event referred to as "restitution" (5, 6). Restitution is also an important component of larger wound closure. Unlike hemopoietic cells, epithelial cells migrate as a cohesive sheet whereby epithelial cells behind the leading edge maintain cell-cell contact as the epithelium covers denuded surfaces. Polarized epithelial cells induced to migrate undergo dramatic changes in cell shape as they flatten out and extrude F-actin-rich protrusions at the leading edge. Such protrusions called lamellipodia transiently adhere to the underlying matrix at focal adhesion complex sites (7, 8). Dynamic interactions between the intracellular environment and the extracellular matrix occurs at focal adhesion sites and provides an important bidirectional sensing mechanism required for coordinated cell movement (7).

Major components of focal adhesion complexes are transmembrane integrins that link the intracellular cytoskeleton to the extracellular matrix. Although the family of integrins expressed in epithelial cells is large, our previous studies have demonstrated an important role of {beta}1{alpha}6 integrin in mediating movement of a model intestinal epithelial cell line, T84 (9). To continue moving, cell attachment at the leading edge must occur in synchrony with cell detachment at the rear of migrating cells. One mechanism by which this can be achieved is the release of surface bound integrins that become endocytosed and subsequently transported in endocytic vesicles to the cell surface at the leading edge where they can participate in formation of new focal complexes (10, 11). Such polarized integrin trafficking would provide an efficient mechanism by which migrating cells attain and maintain adhesive gradients along the axis of cell movement.

Our present study was designed to analyze the influence of IFN-{gamma} on epithelial restitution. Our findings suggest that IFN-{gamma} retards epithelial cell migration by modulating polarized {beta}1 integrin recycling in endosomes thereby influencing its targeting to focal adhesion complexes, a key event mediating forward movement of epithelial cells and wound closure.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and other reagents

The following primary polyclonal Ab (pAb) and mAb were used to detect focal adhesions and cytoskeletal proteins by immunofluorescence labeling and Western blotting: anti-{beta}1 integrin pAb (Chemicon International), anti-early endosomal Ag (EEA)-1 pAb (Upstate), anti-cathepsin D, Rab11 and LAMP1 pAbs (Zymed Laboratories); anti-{beta}1 integrin, anti-FAK, anti-paxillin and FITC-conjugated anti-paxillin, anti-GM130 mAbs (BD Biosciences); anti-Rab4 and Rab5 pAbs (StressGen Biotechnologies), anti-{alpha}-tubulin mAb (Serotec). Rhodamine-phalloidin, as well as donkey anti-rabbit, goat anti-rat, and goat anti-mouse secondary Abs conjugated to or Alexa-488 or Alexa-568 dyes were obtained from Molecular Probes; HRP-conjugated goat anti-rabbit and anti-mouse secondary Abs were obtained from Jackson ImmunoResearch Laboratories.

IFN-{gamma} was a kind gift from Genentech; cytochalasin D, nocodazole, bafilomycin A, and monensin were obtained from Sigma-Aldrich. NHS-SS-Biotin and ImmunoPure immobilized avidin were purchased from Pierce. Docetaxel was a gift from Sanofi-Aventis. Other reagents were of the highest analytical grade and were obtained from Sigma-Aldrich.

Cell culture

T84 intestinal epithelial cells (American Type Culture Collection) were cultured in a 1:1 mixture of DMEM and Ham’s F-12 medium supplemented with 10 mM HEPES (pH 7.5), 14 mM NaHCO3, 40 µg/ml penicillin, 90 µg/ml streptomycin, 6% newborn calf serum and adjusted to pH 7.4 (designated further as a complete T84 medium). For all experiments, T84 cells were grown for 8–14 days on collagen-coated, permeable polycarbonate filters, 0.4 and 5 µm pore size (Costar) (12). Filters with a surface area of 0.33 and 5 cm2 were used for immunocytochemical and biochemical experiments, respectively. IFN-{gamma} (100 U/ml) was administered to both the apical and the basolateral sides of the filters.

Cell migration assay

Effect of IFN-{gamma} on cell migration was analyzed as dynamics of closure of mechanical wounds in epithelial monolayers as described previously (13). Briefly, a mechanical wound was induced in a monolayer of epithelial cells using a 20-µl microtip attached to a vacuum flask to generate reproducible linear wounds. Cells were washed and incubated with or without IFN-{gamma} (100 U/ml) at 37°C for indicated times. Wound closure was recorded using a camera attached to an inverted microscope (Axiover 35 M; Zeiss). Wound widths were measured using a software program (Axio Vision 4.0; Zeiss). Each experiment was conducted three times in duplicate.

Immunofluorescence labeling

Cells were fixed with 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100 followed by blocking in HEPES-buffered HBSS (HBSS+) or Dulbecco’s PBS with calcium chloride and magnesium chloride (PBS-CM; Sigma-Aldrich) containing 5% normal goat serum or 1% BSA (blocking buffer) for 60 min at room temperature and incubated for 60 min with primary Abs in blocking buffer. Cell monolayers were then washed, incubated for 60 min with Alexa dye-conjugated secondary Abs followed by rinsing and mounting on slides with ProLong Antifade medium (Molecular Probes). Nuclei were stained with TO-PRO-3 iodide (Molecular Probes). F-actin was labeled with Alexa-labeled phalloidin. Stained monolayers were examined using a Zeiss LSM510 laser-scanning confocal microscope (Zeiss Microimaging) coupled to a Zeiss 100M axiovert and 63x or 100x Pan-Apochromat oil lenses. Fluorescent dyes were imaged sequentially in frame-interlace mode to eliminate cross talk between channels. Images shown are representative of at least three experiments, with multiple images taken per slide.

Internalization assays with surface biotinylation labeling

The assay based on a biotin-labeling technique was performed essentially as previously described (14). Confluent T84 cells were wounded using a wounding comb with parallel ports 1 mm apart to enrich for migrating cells and incubated in the presence or absence of IFN-{gamma} (100 U/ml) in serum-free and antibiotic-free medium for 24 h. The cells were placed on ice, washed three times with cold PBS-CM and their surface-exposed proteins on the plasma membrane were biotinylated by incubation with 0.5 mg/ml NHS-SS-biotin (Pierce) at 4°C for 30 min. Biotinylated cells were washed in cold PBS-CM, and free NHS-SS-biotin was quenched by three quick washes with 50 mM NH4Cl, followed by three additional washes with PBS-CM. PBS-CM was replaced by serum-free medium, and cells were incubated at 22°C to induce endocytosis of plasma membrane proteins. The experiments at 22°C were conducted to prevent recycling internalized proteins back to the plasma membrane and to induce their accumulation in the cytosolic compartment (15). At different time points of endocytosis, the monolayers were incubated in two 60-min washes of glutathione solution (60 mM glutathione, 0.83 M NaCl, 1% BSA, pH 8.6) at 4°C, to remove biotin groups on the cell surface. Monolayers were washed three times with PBS-CM and then scraped off filters and lysed in 500 µl of RIPA buffer (20 mM Tris-HCl, pH 7.4, with 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA supplemented with protease and phosphatase inhibitors mixtures (Sigma-Aldrich)). Cell extracts were cleared by centrifugation and obtained supernatants were incubated with avidin beads for 1 h at 4°C. Avidin-bound biotinylated proteins were recovered by boiling in SDS sample buffer and analyzed by Western blotting.

Antibody internalization assay

The assay based on an Ab-labeling technique was performed essentially as previously described (15). Confluent T84 cells were wounded and incubated in the presence or absence of IFN-{gamma} (100 U/ml) in serum-free medium for 24 h. The cells were washed three times in cold PBS-CM, and incubated for 30 min at 4°C with anti-{beta}1 integrin Ab (2 µg/ml). Excess Ab was removed with two rapid washes with cold PBS-CM, and monolayers were transferred into serum-free medium and incubated at 22°C for indicated times. The surface membrane-bound Abs were removed by incubation with acidified PBS-CM (pH 4.0) for 10 min at 4°C. Internalized anti-{beta}1 integrin Abs were visualized in paraformaldehyde-fixed and permeabilized cells using FITC-conjugated rabbit anti-mouse Ab labeling and confocal microscopy.

Pharmacological modulation of intracellular trafficking

Confluent T84 monolayers were wounded and incubated with or without IFN-{gamma} for 24 h. Monolayers were preincubated with monensin (20 µM), bafilomycin A (100 nM), and cytochalasin D (1 µg/ml) for 6 h at 37°C, docetaxel (10 µM) for 1 h at 37°C, nocodazole (30 µM) for 1 h at 4°C before performing the {beta}1 integrin Ab internalization assay. Stock solutions of water-insoluble inhibitors were prepared in DMSO and diluted in cell culture medium immediately before each experiment. The final concentration of DMSO was 0.1%; the same concentration of the vehicle was included in appropriate controls. The inhibitors were administered to both the apical and the basolateral sides of the filters.

Statistics

Numerical values from individual experiments were pooled and expressed as mean ± SD throughout. Values obtained for control and IFN-{gamma}-treated groups were compared using two-tailed Student’s t tests, with statistical significance assumed at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IFN-{gamma} inhibited intestinal epithelial cell migration and induced formation of large aberrant membrane protrusions

To evaluate the effect of IFN-{gamma} on epithelial cell migration, we used a well-established in vitro assay based on closure of mechanically inflicted wounds in confluent epithelial cell monolayers. Comparison of the rate of wound closure in control and IFN-{gamma}-treated cells clearly demonstrated inhibitory effect of IFN-{gamma} on this process and therefore on epithelial cell migration (Fig. 1). Indeed at 8, 24, and 48 h after wounding, migrating control cells covered 50, 70, and 90% of the original wound width, whereas the average wound area covered in IFN-{gamma}-treated monolayers was, respectively, 30, 35, and 40% (Fig. 1).



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  		FIGURE 1. IFN-{gamma} attenuates epithelial cell migration and wound closure. Confluent T84 intestinal epithelial monolayers grown on tissue culture plates were wounded and allowed to migrate for up to 48 h in the presence or absence of IFN-{gamma} (100 U/ml) in serum-free medium. Wound closure was followed with a charge-coupled device camera. A, Representative images taken at time of wounding and 48 h later. B, Quantification results of data representing means ± SD from three separate experiments. *, p < 0.05 compared with control. Scale bar, 100 µm.

 
Migrating epithelial cells extrude lamellipodia membrane protrusions at the leading edge (16, 17). Therefore, we next investigated the effect of IFN-{gamma} on formation of lamellipodia. Two different parameters that were analyzed include the number of lamellipodia at the leading edge and their morphological appearance. We found no differences in the average number of lamellipodia at the leading edge in control and IFN-{gamma}-treated monolayers. Indeed, the number of lamellipodia counted in a x40 microscopy field on one side of the wound was 10 ± 3 and 8 ± 2 in control and IFN-{gamma}-treated monolayers, respectively (p > 0.05). However, IFN-{gamma} treatment affected morphology of protrusions at the leading edge (Fig. 2). In control T84 cells, small lamellipodia contained radial dense F-actin bundles oriented in the direction of cell migration (Fig. 2, arrows). However, in IFN-{gamma}-treated cells, large irregularly shaped membrane protrusions were observed (Fig. 2, arrowhead).



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  		FIGURE 2. IFN-{gamma} induces the formation of large abnormal membrane protrusions at the leading edge of migrating cells. Confluent T84 monolayers were wounded and incubated with or without IFN-{gamma} for 24 h. Cells were fixed and F-actin was visualized with Alexa-568 phalloidin whereas nuclei were stained with TO-PRO3 fluorescent dye. Migrating control cells exhibit small lamellipodia (arrow), whereas IFN-{gamma} induces large abnormal membrane protrusions (arrowhead). Scale bar, 20 µm.

 
IFN-{gamma} inhibited formation of focal adhesions and reduced intracellular depot of {beta}1 integrin at the leading edge of migrating epithelial cells

It is well known that attachment of lamellipodia to substrate is critical to execute forward cell movement (17, 18). This attachment is mediated by special multiprotein complexes called focal adhesions (18). We hypothesized that IFN-{gamma} attenuates epithelial cell migration by influencing formation of focal adhesions. To test this hypothesis, we quantified focal adhesions in randomly selected x63 images at the migrating leading edge 24 h after wounding, a time when the effect of IFN-{gamma} on cell migration and morphology of lamellipodia was already obvious (Figs. 1 and 2). To visualize the focal adhesions, we used an Ab to their major structural component, vinculin (19). As shown in Fig. 3, control cells assembled prominent vinculin-rich focal complexes at the leading edge (Fig. 3, arrows). The number of these structures was dramatically decreased in IFN-{gamma}-treated cells (Fig. 3, arrowheads). Similar results were observed by immunolabeling of other focal adhesion components, focal adhesion kinase (data not shown), {beta}1 integrin and paxillin (Fig. 4). We found the most prominent effect of IFN-{gamma} treatment on localization of {beta}1 integrin (Fig. 4). In control wounded T84 monolayers, we identified two fractions of {beta}1 integrin, one within focal adhesions (Fig. 4, arrows) and another in vesicle-like structures located in close proximity to the leading edge of migrating cells (Fig. 4, arrowheads). IFN-{gamma} treatment resulted not only in a substantial loss of {beta}1 integrin from focal adhesions but also a significant reduction in the number of {beta}1 integrin-containing vesicles, which was accompanied by their enlargement (Fig. 4, arrowhead). In migrating epithelial cells, the major mechanism for {beta}1 integrin accumulation at the migrating front is a transcytosis of this protein from the distal part of the plasma membrane to the leading edge (11, 20). We suggested therefore that {beta}1 integrin-containing vesicles adjacent to the migrating front of the cell represent a temporary depot for the transcytosed protein from where it is rapidly recruited into focal adhesions. We hypothesized that depletion of this {beta}1 integrin depot in IFN-{gamma}-treated cells is responsible for the observed decrease in the number of focal adhesions and the rate of cell migration. Therefore, we decided to characterize in detail the effect of IFN-{gamma} on intracellular trafficking of {beta}1 integrin.



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  		FIGURE 3. IFN-{gamma} reduces formation of focal adhesion at the leading edge of migrating cells. Confluent T84 monolayers were wounded, and cell migration was allowed to proceed with or without IFN-{gamma} for 24 h. Cells were fixed and double-labeled for vinculin (green) and F-actin (red). Note vinculin-rich focal adhesions at the leading edge of control cells (arrows) that are less prominent in IFN-{gamma}-treated cells (arrowheads). The dashed line outlines the leading edge of the migrating cells. A, Representative en face confocal images. B, Quantification of number of focal adhesions at the migration leading edge in five randomly selected x63 microscopy fields from three independent experiments. Data presented as mean ± SD; *, p < 0.05 compared with control. Scale bar, 20 µm.

 


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  		FIGURE 4. IFN-{gamma} reduces amount of {beta}1 integrin in the focal adhesions and the number of {beta}1 integrin-positive vesicles at the leading edge of migrating cells. Confluent T84 monolayers were wounded and incubated with or without IFN-{gamma} for 24 h. Cells were fixed and double labeled for {beta}1 integrin (red) and the focal adhesion protein, paxillin (green). Note {beta}1 integrin in focal adhesions (arrows) and in small vesicles behind the leading edge in control T84 cells (arrowhead). These vesicles disappear or fuse into larger structures in IFN-{gamma}-treated cells (arrowhead). Scale bar, 10 µm.

 
{beta}1 integrin accumulated in early/recycling endosomal compartment at the migrating leading edge

First, we sought to identify intracellular compartments occupied by {beta}1 integrin at the leading edge. To do this, we double immunolabeled {beta}1 integrin with known markers of early/recycling endosomal compartment such as small GTPases Rab4, Rab5, and Rab11, as well as a Rab5 effector protein, early endosomal Ag (EEA)-1. As shown in Fig. 5, in the control migrating T84 cells the {beta}1 integrin-containing vesicles at the leading edge appear to be enriched in EEA-1. In addition, significant colocalization of {beta}1 integrin with other early (Rab5) and recycling (Rab4, Rab11) endosomal markers was observed (data not shown). Taken together, these data strongly suggest accumulation of {beta}1 integrin into early/recycling endosomes that are predominantly localized in the upper part of migrating T84 cells (Fig. 5).



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  		FIGURE 5. {beta}1 integrin-containing vesicles at the leading edge represent early endosomes. Confluent T84 monolayers were wounded and incubated with or without IFN-{gamma} for 24 h. Cells were fixed and double immunolabeled for {beta}1 integrin (green) and early endosomal marker, EEA-1 (red). Note the significant colocalization of {beta}1 integrin with EEA-1 (arrows) in a vesicle-like structures enriched beneath the upper surface of lamellopodia. Scale bar, 20 µm.

 
IFN-{gamma} accelerated internalization and altered intracellular distribution of {beta}1 integrin

Three different mechanisms can be envisioned to explain IFN-{gamma}-mediated decrease in amount of {beta}1 integrin within early/recycling endosomes and focal adhesions at the leading edge of migrating epithelial cells. The first mechanism is accelerated degradation of {beta}1 integrin; the second implies decreased internalization of this protein, whereas the third mechanism may be a redirection of transcytosis of {beta}1 integrin from the leading edge to another intracellular compartment. We sought to test all these putative mechanisms.

To evaluate the effect of IFN-{gamma} on {beta}1 integrin stability, we compared expression levels of this protein in control cells and cells treated for 24 h with the cytokine. To inhibit de novo protein synthesis, all cells were incubated with low concentration (5 µM) of cycloheximide. Western blotting analysis of total cell lysates failed to detect significant difference in {beta}1 integrin expression between control and IFN-{gamma}-treated cells (data not shown), strongly suggesting that the cytokine treatment does not accelerate degradation of this protein.

To analyze endocytosis of {beta}1 integrin in wounded confluent T84 monolayers, we used two different assays, viz., a surface biotinylation and Ab internalization. Comparison of the dynamics of intracellular accumulation of biotinylated {beta}1 integrin in control and IFN-{gamma}-treated monolayers clearly demonstrated that IFN-{gamma} accelerates endocytosis of the protein (Fig. 6A). Similar acceleration was found in the assay using internalization of {beta}1 integrin Ab (Fig. 6B). The IFN-{gamma} treatment not only increased the endocytosis rate for {beta}1 integrin, but also changed intracellular distribution of the internalized protein. Indeed, in control cells, internalized {beta}1 integrin Ab accumulated into small vesicles predominantly located along the migrating leading edge (Fig. 6B, arrows). In these vesicles, {beta}1 integrin significantly colocalized with EEA-1 (Fig. 7, arrows). By contrast, in IFN-{gamma}-treated cells internalized {beta}1 integrin did not accumulate at the leading edge but was visualized in broadly distributed large clusters (Fig. 6B, arrowheads). Analysis of reconstructed xz images revealed that in IFN-{gamma}-treated cells, {beta}1 integrin did not colocalize with EEA-1 at the upper part of the cell but accumulated at the cell base (Fig. 7, arrowheads). These data strongly suggest that IFN-{gamma} depletes the depot for recycling {beta}1 integrin at the leading edge by interfering with normal transcytosis of this protein. To test this hypothesis, we treated control migrating T84 cells with bafilomycin or monensin, two inhibitors that have been shown to block transcytosis in different experimental systems (21, 22). Both inhibitors induced redistribution of internalized {beta}1 integrin Ab from the leading edge (Fig. 7, arrow) to the cell base (Fig. 7, arrowheads), thus mimicking the effect of IFN-{gamma} treatment. To identify the intracellular compartment targeted by {beta}1 integrin in IFN-{gamma}-treated cells we double immunolabeled this protein with known markers of late endosomes (Rab9), lysosomes (LAMP1 and cathepsin D), endoplasmic reticulum (calnexin), and Golgi (GM130 and TGN38). No significant colocalization of {beta}1 integrin with either marker was observed in migrating IFN-{gamma}-treated cells (data not shown).



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  		FIGURE 6. IFN-{gamma} accelerates internalization of {beta}1 integrin. A, Internalization of {beta}1 integrin analyzed by surface biotinylation. Confluent T84 monolayers grown on permeable filters were wounded and cell migration was allowed to proceed with or without IFN-{gamma} for 24 h. Surface cell proteins were biotinylated and allowed to internalize for different times (shown on top of the blot). The internalized biotinylated proteins were isolated from cell lysates using avidin beads and analyzed by Western blotting with anti-{beta}1 integrin Ab. B, {beta}1 integrin Ab internalization assay. Confluent T84 monolayers were wounded and incubated with or without IFN-{gamma} for 24 h. Cells were incubated with anti-{beta}1 integrin Abs and {beta}1 integrin Ab internalization assay was performed as described in Materials and Methods. Cells were fixed and immunolabeled for the internalized anti-{beta}1 integrin Abs using fluorescent secondary antibodies. The dashed line highlights the leading edge of migrating cells. Note increased endocytosis of biotinylated {beta}1 integrin and its Ab in IFN-{gamma}-treated cells. In addition, internalized {beta}1 integrin Abs are localized in small vesicles along the leading edge of control cells (arrows), whereas they are broadly distributed and clustered in IFN-{gamma}-treated cells (arrowheads). Scale bar, 20 µm.

 


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  		FIGURE 7. Pharmacological inhibitors of transcytosis mimic effect of IFN-{gamma} on intracellular distribution of {beta}1 integrin. Confluent T84 monolayers were wounded and incubated with or without IFN-{gamma} for 24 h. Monolayers were preincubated with monensin (20 µM), bafilomycin A (0.1 µM) or vehicle before performing the {beta}1 integrin Ab internalization assay. Cells were double immunolabeled for {beta}1 integrin (green) and early endosomal marker EEA-1 (red). The dashed line outlines the leading edge of the migrating cells. Note that monensin and bafilomycin A shift the intracellular distribution of internalized {beta}1 integrin from the upper membrane of lamellipodia (arrow) to the cell base (arrowheads) thus mimicking the effect observed in IFN-{gamma}-treated cells. Scale bar, 20 µm.  

 
The effect of IFN-{gamma} on intracellular trafficking of {beta}1 integrin depended on stable microtubules

Vesicular transport of internalized proteins is mediated by the cytoskeleton, and two major cytoskeletal components, actin microfilaments and microtubules, are involved in this process (23, 24, 25, 26, 27). Therefore, we sought to determine which cytoskeletal component contributes to the altered transcytosis of {beta}1 integrin in IFN-{gamma}-treated epithelial cells. To do that, we used pharmacological agents to selectively perturb organization of F-actin and microtubules. Disorganization of actin microfilaments in migrating cells with cytochalasin D resulted in profound changes in F-actin in both control and IFN-{gamma}-treated cells manifested by large clusters of actin microfilaments randomly located within the cell (data not shown). Internalized {beta}1-integrin was associated with these F-actin clusters and did not display localization patterns characteristics for control or IFN-{gamma}-treated cells (data not shown). Because of nonspecific effects of cytochalasin D treatment on {beta}1 integrin localization, these data appear to be inconclusive. In contrast, depolymerization of microtubules with nocodazole exerted a selective effect on transcytosis of {beta}1 integrin in IFN-{gamma}-treated cells. In control T84 cells, nocodazole treatment did not affect accumulation of internalized {beta}1 integrin at the upper part of migrating leading edge (Fig. 8, arrow). However, depolymerization of microtubules inhibited transcytosis of {beta}1 integrin to the cell base in IFN-{gamma}-treated cells (Fig. 8). Instead, internalized integrin accumulated at the upper part of the cell similarly to its localization in control monolayers (Fig. 8, arrowhead). Interestingly, only depolymerization of microtubules affected transcytosis of {beta}1 integrin, whereas stabilization of these cytoskeletal structures appeared to be ineffective. Indeed, microtubule-stabilizing drug docetaxel (28, 29) while increasing thickness of microtubules in both control and IFN-{gamma}-treated cells (Fig. 9) did not change intracellular distribution of internalized {beta}1 integrin (Fig. 9). Collectively, these results suggest that IFN-{gamma} redirects transcytosis of {beta}1 integrin from the leading edge by a mechanism involving stable microtubules.



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  		FIGURE 8. Depolymerization of microtubules reverses the effect of IFN-{gamma} on transcytosis of {beta}1 integrin. Confluent T84 monolayers were wounded and incubated with or without IFN-{gamma} for 24 h. Monolayers were preincubated with nocodazole (30 µM) for 1 h at 4°C before the {beta}1 integrin Ab internalization assay. Cells were fixed and labeled for {beta}1 integrin (green) and F-actin (red). Black and white insets show labeling of cells at the leading edge with anti-tubulin antibodies. Note a profound depolymerization of microtubules in nocodazole-treated cells (inset). Disruption of microtubules by nocodazole did not affect internalization of {beta}1 integrin into vesicle-like structures at the leading edge in control cells (arrow) but reversed IFN-{gamma}-induced transcytosis of {beta}1 integrin at the cell base (arrowhead). Scale bar, 10 µm.  

 


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  		FIGURE 9. Stabilization of microtubules does not affect transcytosis of {beta}1 integrin. Confluent T84 monolayers were wounded and incubated with or without IFN-{gamma} for 24 h. Monolayers were preincubated with microtubule-stabilizing drug, docetaxel (10 µM) for 1 h at 37°C before the {beta}1 integrin Ab internalization assay. Cells were fixed and labeled for {beta}1 integrin (green) and tubulin (red). Note that stabilization of microtubules by docetaxel did not affect {beta}1 integrin transcytosis in control (arrow) or IFN-{gamma}-treated cells (arrowhead). Scale bar, 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study targeted an important physiological phenomenon, a restitution of intestinal mucosa in IBD patients that takes place during the active inflammatory phase of the disease. At this stage, multiple epithelial wounds created by activated transmigrated leukocytes and by fragility of the epithelium attempt to rapidly reseal to cover denuded surfaces. The restitution begins when intramucosal level of leukocyte-derived proinflammatory cytokines, particularly IFN-{gamma}, is still high. Therefore, we sought to investigate the influence of IFN-{gamma} on epithelial restitution and to address mechanisms by which this cytokine affects migration of intestinal epithelial cells.

IFN-{gamma} as an inhibitor of epithelial cell migration

We found that IFN-{gamma} significantly slows down the rate of wound closure in confluent T84 epithelial monolayers (Fig. 1). It has been recognized that epithelial wounds are repaired by different mechanisms depending on cell type and size of the wound (30). Small wounds caused by elimination of one or few cells are closed by a so-called purse string mechanism involving assembly and contraction of actomyosin cables at the wound edge (31, 32). Medium-sized wounds, which were induced in the present study, reseal by spreading and migration of cells at the wound edge (30). Appearance of multiple lamellipodia rather than actomyosin cables at the wound edge in T84 monolayers (Fig. 2) suggests that cell migration but not actomyosin purse string mediated wound closure in our experimental model. Hence, we attributed the observed inhibitory effect of IFN-{gamma} on wound closure in T84 monolayers to reduced migration of epithelial cells.

The effect of IFN-{gamma} on cell motility in vitro and in vivo is well documented. This cytokine appears to have a promigratory effect on leukocytes, exhibiting an ameboid-type movement that is characterized by short-lived and relatively weak interaction with the substratum (33, 34, 35) but is antimigratory for cells that use mesenchymal or collective migration modes that require strong adhesion to extracellular matrix (36). IFN-{gamma} was shown to reduce in vitro migration of fibroblasts (37), glioblastoma cells (38), and endothelial cells (39). In addition, IFN-{gamma} knockout mice reportedly exhibit accelerated healing of skin wounds, thus indicating an inhibitory effect of the cytokine on wound closure in vivo (40). Interestingly, such "anti-restitution" effect is not unique for IFN-{gamma} and has been attributed to another proinflammatory cytokine TNF-{alpha} (41).

Cell migration relies on formation of membrane protrusions, lamellipodia, which is driven by actin polymerization (16, 17). Newly formed lamellipodia attach to the substratum and serve as footholds for forward dragging of cell body over the matrix (17). It was logical to suggest therefore that the observed inhibitory effect of IFN-{gamma} on epithelial cell migration is due to its interference with formation of lamellipodia. Indeed, fluorescence labeling and confocal microscopy of migrating cytokine-treated T84 cells revealed the formation of enlarged and abnormally shaped lamellipodia (Fig. 2). Lamellipodia attach to extracellular matrix via specialized regions along the ventral plasma membrane called focal adhesions (18). We suggested that the observed attenuation of motility of IFN-{gamma}-treated T84 cells might be the result of decreased formation of focal adhesions. Indeed, by immunolabeling of focal adhesion proteins vinculin (Fig. 3), paxillin (Fig. 4), FAK (data not shown), and {beta}1 integrin (Fig. 4), we found a dramatic decrease in the number of adhesion sites on lamellipodia of cytokine-treated cells. However, the overall adhesion of T84 cells decreased ~35% after 48 h of incubation with IFN-{gamma} but was not affected at the earlier time points (our unpublished observation). A serendipitous observation made in the immunolabeling experiment was an IFN-{gamma}-induced drastic decrease in the amount of intracellular {beta}1 integrin close to the leading edge of migrating cells (Fig. 4). This observation shifted our focus on characterization of intracellular trafficking of {beta}1 integrin for the reasons that are described below.

IFN-{gamma} alters intracellular trafficking pathway for {beta}1 integrin

Integrins are transmembrane proteins in focal adhesions that provide a physical link of lamellipodia surface to the substrate (42, 43). The bulk of the integrin molecule is exposed outside the cell where it interacts with extracellular matrix via both the {alpha} and {beta} subunits. Most of integrin {alpha} and {beta} subunits have short cytoplasmic segments that serve as a site for the assembly of multimeric signaling complexes and actin-binding proteins, by which integrins interact with actin filaments. The {beta}1 subunit is abundantly expressed in intestinal epithelial cells (9, 44), localized at the leading edge of migrating T84 cells (Fig. 4), and, according to Ab-blocking experiments, is critically involved in T84 cell migration (45).

Cell motility depends on transient interactions of integrins with extracellular matrix components. The {beta}1 subunit-containing integrins readily adhere to collagens I and IV and laminin but have poor affinity to another components of extracellular matrix such as fibronectin and vitronectin (46). During the forward migration, there is a constant assembly of new focal contacts at the leading edge accompanied by disassembly the old focal complexes at cell rear (7). The last process is mediated by endocytosis of integrin-containing adhesion complexes (10). Interestingly, internalized integrins are not targeted for degradation but rather recycle back to the cell membrane where they are reused for formation of new focal contacts. Evidences suggest that in migrating cells there is constitutive vectorial flux of integrin-containing vesicles from the cell rear to the leading edge (10, 11). In agreement to this model, we found accumulation of {beta}1 integrin in intracellular vesicles located at the upper part of the control migrating T84 cells that we identified as the early/recycling endosomal compartment (Fig. 5). The {beta}1 integrin-containing vesicles did not colocalize with Golgi markers and did not disappear after a 2–4 h treatment of the cells with protein synthesis blocker, cycloheximide, thus indicating their origination from internalized rather than from Golgi-derived pool of {beta}1 integrin. Our results agree well with previously published data on accumulation of {beta}1 integrin in recycling endosomal compartment of mouse fibroblasts (15) and human breast carcinoma cells (20).

One of the principal findings of the present study is IFN-{gamma}-induced depletion of {beta}1 integrin from the early/recycling endosomes at the leading edge of migrating T84 cells (Fig. 4). No such finding was reported previously except one study showed decreased labeling for {beta}1 integrin in unspecified compartment at the leading edge of IFN-{gamma}-treated lung epithelial cells. Based on the known role of {beta}1 integrin recycling in formation of focal contacts and cells motility (10, 11), we hypothesize that removal of this integrin from the intracellular depot underlies the inhibitory effects of IFN-{gamma} on adhesion and migration of T84 epithelial cells. Furthermore, our data suggest a mechanism underlying the observed depletion of the recycling depot of {beta}1 integrin. This mechanism comprises redirection of intracellular trafficking of {beta}1 integrin from the leading edge to the cell base (Fig. 7). In control migrating epithelial cells, the endocytosed Ab is delivered into early/recycling compartment at the leading edge similar to that of endogenous protein (Fig. 7). However, after incubation with IFN-{gamma}, the internalized {beta}1 integrin Ab accumulated in a basal compartment that is essentially devoid of early/recycling endosomal markers. Furthermore, pharmacological perturbation of transcytosis in control migrating cells using monensin or bafilomycin A also resulted in the basal mistargeting of the {beta}1 integrin Ab (Fig. 7), thus mimicking effect of IFN-{gamma} treatment. Taken together, our data strongly suggest that IFN-{gamma} alters intracellular trafficking of {beta}1 integrin in migrating epithelial cells, most likely by activating its transcytosis to the basal cytosolic compartment. Identity of this basal compartment remains elusive because it was not labeled with markers of any classical intracellular organelles (data not shown). We speculate that it may resemble an unusual storage compartment previously reported in several epithelial cell lines (47, 48).

Effect of IFN-{gamma} on intracellular trafficking of {beta}1 integrin is mediated by microtubules

It is well known that intracellular vesicle trafficking in epithelial cells is regulated by the cytoskeleton, and two major cytoskeletal components, actin filaments and microtubules, appear to be involved (for review, see Refs.49, 50, 51). To clarify which component of the cytoskeleton is responsible for transcytosis of {beta}1 integrin, we conducted the Ab internalization assay in conditions of selective disorganization of either F-actin or microtubules. Disorganization of actin microfilaments by cytochalasin D induced global and nonselective perturbation on {beta}1 integrin trafficking in control and IFN-{gamma}-treated epithelial cells (data not shown). By contrast, depolymerization of microtubules with nocodazole selectively reversed accumulation of {beta}1 integrin in the basal compartment in IFN-{gamma}-treated cells (Fig. 8, arrowhead) with no effect on this protein trafficking in control cells (Fig. 8, arrow). Importantly, stabilization of microtubules with docetaxel did not alter IFN-{gamma}-induced intracellular redistribution of {beta}1 integrin (Fig. 9, arrowhead), thus indicating that this process is mediated by stable microtubules and does not involve microtubule turnover. Based on results of cytoskeletal disruption, we speculate the existence of two competing intracellular pathways for {beta}1 integrin trafficking. The first is a microtubule-independent pathway mediating protein transcytosis to the early/recycling endosomes at the leading edge. The second is a microtubule-dependent pathway that brings {beta}1 integrin into an unidentified compartment at the cell base. In control migrating cells, the microtubule-independent pathway dominates, causing accumulation of {beta}1 integrin close to the migrating front of the plasma membrane. IFN-{gamma} is likely to redirect {beta}1 integrin transcytosis toward the microtubule-dependent pathway. Our hypothesis is supported by several studies that showed stabilizing effects of IFN-{gamma} on microtubules. Indeed, two previous studies found stimulation of microtubule assembly by IFN-{gamma} in a cell-free system (52) and in squamous carcinoma cells (53).

In conclusion, the present study shows that IFN-{gamma} inhibits migration of T84 intestinal epithelial cells by decreasing the number of focal adhesions at the leading edge and causing formation of large aberrant lamellipodia. These morphological changes are accompanied by the disappearance of {beta}1 integrin from the early/recycling endosomes at the migrating leading edge. The mechanism underlying IFN-{gamma}-induced depletion of the {beta}1 integrin recycling depot involves a microtubule-dependent redirection of integrin trafficking from the leading edge to the cell base. We hypothesize that altered trafficking of {beta}1 integrin represents an important mechanism mediating impaired closure of epithelial wounds in intestinal inflammation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health funding Grants DK55679 (to A.N.), DK61379 (to C.A.P.), DK64399 (to A.N. and C.A.P.) and by a research grant from Sanofi-Aventis (to C.A.P.). Back

2 Q.T. and E.V.V. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Asma Nusrat, Department of Pathology and Laboratory Medicine, Emory University, Whitehead Research Building, Room 105E, 615 Michael Street, Atlanta, GA 30322. E-mail address: anusrat{at}emory.edu Back

4 Abbreviations used in this paper: IBD, inflammatory bowel disease; pAb, polyclonal Ab; EEA, early endosomal Ag. Back

Received for publication May 28, 2004. Accepted for publication June 21, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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