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Am J Physiol Lung Cell Mol Physiol 291: L828-L836, 2006. First published June 2, 2006; doi:10.1152/ajplung.00120.2006
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Airway epithelial wound repair: role of carbohydrate sialyl Lewisx

Sima Allahverdian,1 Kimberly R. Wojcik,2 and Delbert R. Dorscheid1

1The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, Faculty of Medicine, St. Paul's Hospital, University of British Columbia, Vancouver, British Columbia, Canada; and 2Section of Pulmonary and Critical Care Medicine, Department of Medicine, Division of Biological Sciences, University of Chicago, Chicago, Illinois

Submitted 1 April 2006 ; accepted in final form 26 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Epithelial repair is a complex cellular and molecular process, the details of which are still not clearly understood. Plasma membrane glycoconjugates can modulate cell function by altering the function of protein and lipids. Sialyl Lewisx (sLex), a fucose-containing tetrasaccharide, decorates membrane-bound and secreted proteins and mediates cell-cell interaction. In the present study we investigated the role of sLex in airway epithelial repair. Using immunohistochemistry, we showed an increased expression of sLex in areas of damaged bronchial epithelium compared with intact regions. Confluent monolayers of airway epithelial cells were mechanically wounded and allowed to close. Wounded monolayers were photographed for wound closure kinetics, fixed for immunocytochemical studies, or subjected to RNA extraction. Examining the expression of different {alpha}1,3-fucosyltransferases (FucT), enzymes that mediate the final step in the synthesis of sLex, we found that FucT-IV was the common gene expressed in all cell lines and primary airway epithelial cells. We demonstrated an increased expression of sLex over time after mechanical injury. Blocking of sLex with an inhibitory antibody completely prevented epithelial repair. Our data suggest an essential functional role for sLex in epithelial repair. Further studies are necessary to explore the exact mechanism for sLex in mediating cell-cell interaction in bronchial epithelial cells to facilitate epithelial migration and repair.

fucosyltransferase; selectin; airway epithelium; epidermal growth factor

AS THE BARRIER to the external environment, the bronchial epithelium is continuously exposed to gaseous and particulate components of inhaled air and therefore is frequently injured. Inflammation is an initial response to tissue injury, which provides immune cells dedicated to debris removal and growth factors to promote tissue repair (7). In addition to this inflammatory response, wound healing involves migration and spreading of epithelial cells into the damaged region and proliferation of new epithelial cells (12, 21). The complete healing of wounds represents an important process by which respiratory epithelial barrier integrity is maintained.

Several proteins essential for normal cell physiology, like membrane-bound receptors for growth factors and cytokines, are glycosylated. Several lines of evidence support the theory that oligosaccharide moieties are crucial for the function of some of those proteins and that variation in their glycosylation pattern often leads to changes in their function (13, 27, 38). Oligosaccharides on cell surface proteins and lipids have functional roles in cell adhesion (36), migration (40), proliferation (4), and growth potential (28). The molecular events that initiate, mediate, and regulate different processes involved in epithelial repair have not been fully elucidated, but a number of studies have suggested that glycoconjugates attached to proteins within the plasma membrane of epithelial cells play a central role in these events. Our laboratory (9) has determined the pattern of cell surface glycosylation in normal human airway epithelial cells. We have shown (47) that glycosylation profiles in airway epithelium change over time during repair of a wound created by mechanical injury. Our data also suggested that cell surface N-glycosylation has a functional role in airway epithelial cell adhesion and migration and that N-glycosylation with terminal fucosylation plays an essential role in the complex process of repair by coordination of certain cell-cell functions (10). In asthma, detailed cellular and ultrastructural examination of bronchial biopsies and bronchoalveolar lavage fluid has provided evidence for epithelial damage, even in mild cases (2, 18, 26, 31). This excessive epithelial damage can arise from an enhanced susceptibility to injury, an inadequate repair response, or a combination of both (7). Kauffmann et al. (20) reported an underrepresentation of carbohydrate structures with terminal fucose in asthmatic patients, with a correlation between this deficiency and the severity of the disease. These data suggest that defects in epithelial repair in asthma patients may be due, in part, to improper glycosylation of airway epithelial cells. Together, these studies indicate an involvement of cell surface carbohydrates, especially those with terminal fucose, in regulation of epithelial repair processes.

Lewis blood group antigens are biosynthetically and structurally related carbohydrate structures used as markers of cell differentiation and embryonic development (34). Expression of these antigens is not limited to erythrocytes, and they can be found in different tissues and organs. It has been shown that these oligosaccharide structures are involved in cell-cell interaction. Sialyl Lewisx (sLex), a fucose-containing tetrasaccharide [NeuAc{alpha}2–3Galbeta1–4(Fuc{alpha}1–3)GlcNAc] belonging to this family, has been recognized as a ligand for E-selectin and therefore has an important role in lymphocyte trafficking (28, 36, 45). This antigen has been detected in various tumors, where it mediates binding of cancerous cells to endothelial selectins, thereby promoting tumor metastasis (14, 30). sLex is also found at the nonreducing termini of N-linked or O-linked oligosaccharides on glycoproteins as well as on glycosphingolipids. The final step in synthesis of sLex is catalyzed by specific {alpha}1,3-fucosyltransferases (FucT). Six human {alpha}1,3-FucT have been cloned and partially characterized (FucT-III, FucT-IV, FucT-V, FucT-VI, FucT-VII, and FucT-IX), which show different patterns of expression among tissues.

Neither the normal processes leading to complete epithelial repair nor the abnormalities that permit chronic damage in disease states of the epithelium are fully understood. Although previous studies in epithelial repair suggested a role for glycoconjugates, none of these studies specified an oligosaccharide structure(s) to be involved in repair. The present study, to our knowledge, demonstrates for the first time a critical role for the tetrasaccharide sLex in airway epithelial repair. We found an overexpression of sLex in vivo and in vitro after injury. In a culture model of epithelial repair, we were able to demonstrate an inhibition of repair after blocking of sLex. These observations have important implications not only for understanding the epithelial injury-repair cycle but also for identifying novel therapies for conditions resulting from impaired epithelial repair, such as asthma.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Collection of airway specimens from normal human subjects. Approval for the use of all human tissue was granted by the University of British Columbia and Providence Health Care Ethics Review Board. Normal bronchial segments were collected from pathological specimens from adults undergoing lung resection in a regional chest hospital in Grosshansdorf, Germany. Samples generally were of fourth-, fifth-, or sixth-generation central airways in transverse section, so that a complete circumference could be examined. Bronchial specimens were fixed in 4% paraformaldehyde for 1 h at room temperature. Samples were then shipped on ice to our laboratory, washed in Dulbecco's phosphate-buffered saline, and stored at 4°C. After paraffin embedding, airways were retrieved and tissue sections were prepared in transverse orientation from the paraffin blocks for preparation of 5-µm-thick sections. No subject had a history of asthma.

Immunohistochemistry. Immunostaining was performed with a mouse monoclonal anti-human sLex antibody (KM 93, Seikagaku America, Ijamsville, MD) or an isotype-matched nonspecific antibody.

Quantification. To study sLex immunoreactivity, the entire epithelium of one airway section was systematically assessed in each subject. Several sections were obtained from the same airway, and more than one airway per donor sample was assessed. Several images were taken from the entire airway section (usually 10–15 images based on the size of airway) in each subject. In this manner, the entire circumference of the airway was documented in images. If a wound was detected in one of these images, this section then became the representative airway section for that donor. Next, from the pool of images for the representative section three numbers were randomly selected, and if they contained damaged area they were further assessed for sLex expression in the areas of damaged and intact epithelium. In this manner selection bias was minimized. Epithelial damage was characterized morphologically by the absence of differentiated ciliated and secretory cells (44). To study the immunoreactivity of sLex in areas of epithelial damage and normal epithelium, the percentage of positively stained basal and columnar cells in three areas exhibiting epithelial damage and three areas of intact epithelium was determined in each subject. A total of six normal airways was assessed. Therefore, 18 areas of intact epithelium were compared with 18 areas of damaged epithelium. Previous studies showed that migratory epithelium presents within 40 µm from the wound edge (12). Therefore, to evaluate sLex immunoreactivity in a damaged area, basal and columnar cells within 40 µm from either wound edge were counted. Next, epithelium farther than 40 µm from the wound edge was considered as intact epithelium if the pseudostratified layer was complete, and 40 µm of this area was evaluated for sLex staining. ImagePro Plus image analysis software (Media Cybernetics) was used to collate the airway images and to determine the positive cells with a point counting grid. Positive counts were confirmed by manual inspection by one author (S. Allahverdian).

Cell culture. 1HAEo– and 16HBE 14o– cells are simian virus 40-transformed normal human airway epithelial cells that have been characterized previously (6, 16) and express multiple surface carbohydrate markers of normal primary basal airway epithelial cells (9). Primary normal human bronchial epithelial (NHBE) cells were collected from subjects undergoing lung resection. These cells were derived from different donors. Epithelial cell purity was determined by examining the typical morphological features of primary epithelial cells in culture and staining the cells with anti-cytokeratin-18 antibody (USBiological, Swampscott, MA). Cells were subcultured and used between passages 3 and 5.

Monolayer wound repair assay. We previously established (10, 22–24) this method using six-well culture dishes. In two experiments NHBE cells grown in a monolayer were treated with an inhibitory anti-sLex antibody (40 ng/ml; KM 93, Seikagaku America) or an isotype-matched nonspecific antibody (40 ng/ml) immediately after mechanical injury. In three experiments, 1HAEo– cells were treated concurrently with both epidermal growth factor (EGF; 15 ng/ml) and 4-deoxy-fucose, a general inhibitor of fucosyltransferases (FuTi) (10–3-10–5 M; Calbiochem, La Jolla, CA). In three experiments, 1HAEo– cells were treated concurrently with both EGF (15 ng/ml) and soluble sLex (10–4-10–7 M; Calbiochem). In two experiments, 1HAEo– cells were treated with either FuTi (10–3-10–5 M) or soluble sLex (10–4-10–7 M) without the addition of EGF. In each experiment, one well was used as a negative control with no treatment, and one well was treated with 15 ng/ml of EGF, which was been demonstrated to be a potent accelerant in models of epithelial monolayer wound closure (22).

Immunocytochemistry. NHBE cells were grown on four-well chamber slides until confluence. Three linear wounds were created with a rubber stylet. Monolayers were then fixed at 0, 2, 6, 12, 24, and 48 h after mechanical injury with Clark's solution (90% ethanol, 10% glacial acetic acid). Expression of sLex was detected with mouse anti-human sLex (KM 93, Seikagaku America).

RNA isolation and real-time PCR. 1HAEo–, 16HBE 14o–, and NHBE cells were grown to confluence and then RNA extracted. Expression of four subtypes of {alpha}1,3-FucT was studied by real-time RT-PCR using primers specific for FucT-III, -IV, -VII, and -IX. RNA was extracted at specific time points after injury with TRIzol reagent (GIBCO BRL), according to the manufacturer's protocol. mRNA expression was quantified by real-time PCR using LightCycler (Roche, Mannheim, Germany). The levels of target mRNAs were normalized to the level of beta-actin mRNA in the same sample.

Flow cytometry analysis. Flow cytometry analysis was performed on 1HAEo– and NHBE cells with anti-E-, -L-, and -P-selectin mouse monoclonal antibodies (RDI, Flanders, NJ).

Statistical analysis. Data were entered in and analyzed by means of SPSS 7.1 for Windows. Wound closure is expressed as a percentage of the area at time 0. In previous videomicroscopy experiments with cell monolayers (24), intraobserver variability was <2% and interobserver variability was <4% for all measurements.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of sLex is higher in areas of epithelial damage compared with intact epithelium. We initiated the investigation of the role of sLex in epithelial repair by studying the expression of sLex in damaged and intact areas of normal airway epithelium. Immunoreactivity of sLex was analyzed in sections of bronchial specimens obtained from normal subjects (n = 6). In each subject three areas of epithelial damage and three areas of intact epithelium were identified as described in MATERIALS AND METHODS. The percentage of positively stained basal and columnar cells was significantly higher in areas of damaged compared with intact epithelium (P < 0.002, Mann-Whitney U-test) (Figs. 1 and 2).


Figure 1
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  		Fig. 1. Expression of sialyl Lewisx (sLex) on airway epithelium in normal subjects. Bronchial segments obtained from pathological specimens of adults undergoing lung resection were processed and sectioned as described in MATERIALS AND METHODS. New fuschin was applied for visualization, and positive sLex detection is noted by red staining. A and B: immunoreactivity for sLex in areas characterized by epithelial damage (A) and intact epithelium (B). C and D: appearance of an isotype-matched nonspecific antibody staining in the areas of damaged (C) and intact (D) epithelium. Scale bar = 10 µm.

 

Figure 2
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  		Fig. 2. Expression of sLex is higher in areas of epithelial damage compared with intact epithelium. Immunostaining of sLex in intact and damaged epithelial zones was assessed in each subject. Epithelial damage was characterized morphologically by the absence of ciliated and secretory cells. Quantification was performed with ImagePro Plus image analysis software. To evaluate sLex immunoreactivity in damaged areas, positive-staining basal and columnar cells within 40 µm from either wound edge were counted. Next, intact epithelium farther than 40 µm from the wound edge was considered as intact epithelium; 40 µm of this area was assessed for sLex staining. Percentage of positively stained basal and columnar cells in 3 areas exhibiting epithelial damage and 3 areas of intact epithelium was determined in each subject. Statistical significance was determined by Mann-Whitney U-test. Horizontal line represents the median. Expression of sLex is significantly higher in areas of epithelial damage compared with intact epithelium (P < 0.002).

 
Mechanical injury enhances the expression of sLex in a culture model of airway epithelium. We examined the effect of mechanical injury on sLex expression in mechanically wounded monolayers of NHBE cells. Epithelial cells were characterized by staining the cells with anti-cytokeratin-18 antibody and examining for typical morphological features of airway epithelial cells in culture. Immunocytochemical staining of the wounded monolayer showed a time-dependent increase in sLex expression coordinate with wound closure and a decrease once the repair was complete (Fig. 3). Increased expression of sLex after mechanical injury was associated with a change in the epithelial cell shape. The majority of epithelial cells expressing sLex exhibit an elongated morphology. These elongated cells appear to be migratory epithelial cells in phenotype. Evaluation of migration and migratory phenotype of these cells was beyond the scope of the present study.


Figure 3
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  		Fig. 3. Mechanical injury induces the expression of sLex in a culture model of epithelial repair. A linear wound was made in confluent monolayers of primary bronchial epithelial cells with a rubber stylet. Monolayers were fixed with Clark's solution at 0, 2, 6, 12, 24, and 48 h after the mechanical injury (T0, T2, T6, T12, T24, and T48, respectively). Expression of sLex was detected, using mouse {alpha}-human sLex and Vector Red for visualization. Detection of sLex increased with repair, denoted by increased red stain. A correlation between increased expression of sLex and the cell shape phenotype also changed over time after mechanical injury. Cells that had higher detection of sLex demonstrated an elongated shape characteristic of migratory cells (see enlarged inset from T12).

 
Blocking of sLex with an anti-sLex inhibitory antibody prevents epithelial monolayer wound repair. To determine whether sLex plays a role in bronchial epithelial repair, we studied kinetics of epithelial wound repair in the presence of a blocking anti-sLex antibody. A linear wound was made in a confluent monolayer of NHBE cells with a rubber stylet. Cells were treated with an anti-sLex antibody (40 ng/ml) or an isotype-matched nonspecific antibody (40 ng/ml) immediately after injury. In each experiment, one well was used as a negative control with no treatment. Corresponding wound areas were determined at 0, 2, 6, 12, and 24 h after wound creation by time-lapse videomicroscopy (Fig. 4A). The remaining wound area 24 h after wounding was significantly higher in monolayers treated with anti-sLex antibody than in nontreated monolayers and those treated with nonspecific antibody (P < 0.05; Fig. 4B). These data showed that blocking of sLex in our culture model of airway epithelial wound repair inhibited wound closure.


Figure 4
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  		Fig. 4. Blocking of sLex with an anti-sLex inhibitory antibody prevents epithelial monolayer wound repair. A linear wound was made in confluent monolayers of primary bronchial epithelial cells as described in MATERIALS AND METHODS, and wounds were treated with an anti-sLex antibody or a control antibody. In each experiment monolayers with no treatment are considered as control group. A: corresponding wound areas determined 2, 6, 12, and 24 h after wound creation by time-lapse videomicroscopy. Data are means ± SE for 24 wounds measured in 2 independent experiments. B: effect of anti-sLex antibody on wound repair. Anti-sLex antibody significantly reduced wound repair compared with controls (*P < 0.05). Statistical significance of the differences between groups was determined by 1-way ANOVA.

 
{alpha}1,3-FucT exhibit a diverse pattern of expression in 1HAEo–, 16HBE 14o–, and NHBE cells. Expression of four subtypes of {alpha}1,3-FucT, the enzyme responsible for the fucosylation step in sLex antigen synthesis, was studied in 1HAEo–, 16HBE 14o–, and NHBE cells by real-time RT-PCR (Table 1). FucT-IX was not expressed in any of the cell lines or primary cells examined. FucT-III and -VII showed a different pattern of expression among two cell lines and primary cells. We found that FucT-IV is the only gene transcribed in both primary cells and cell lines of airway epithelium.


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  		Table 1. {alpha}1,3-Fucosyltransferases show a diverse pattern of expression in 1HAEo–, 16HBE 14o–, and NHBE cells

 
A general FuTi reduces epithelial repair in a culture model of epithelial cell monolayer wound repair in the presence and absence of exogenous EGF. Our previous data provide some evidence on the role of sLex in epithelial repair. We further investigated the role of fucose-containing oligosaccharides in epithelial repair by inhibiting synthesis of fucose-containing oligosaccharides with FuTi. The initial wound area and perimeter for monolayers within each experimental series were equivalent and consistent. In control and EGF-only experiments pooled across experimental series, the remaining wound area after 24 h was 30 ± 3.8% in control cultures and 3.7 ± 1.5% in EGF-stimulated wounds (P < 0.01; n = 10). Concurrent treatment of monolayers with EGF (15 ng/ml) and FuTi inhibited wound repair in a dose-dependent manner compared with EGF alone. Monolayers treated with 10–3 M compared with monolayers treated with 10–4 M and 10–5 M FuTi and monolayers treated with 10–4 M compared with 10–5 M FuTi had higher wound area 24 h after treatment (P < 0.05). The remaining wound areas at 24 h after treatment with 10–4 M FuTi + EGF (35 ± 1.4%) and 10–3 M FuTi + EGF (43 ± 2.6%) were significantly higher compared with the group treated with EGF alone (3.5 ± 1.4%, P < 0.01) (Fig. 5B). In the absence of exogenous EGF, FuTi in its highest doses (10–3 and 10–4 M) inhibited epithelial repair compared with control monolayers. The remaining wound areas at 24 h after treatment with 10–4 M FuTi (34 ± 1.0%) and 10–3 M FuTi (35 ± 3.0%) were significantly higher compared with the control group with no treatment (23 ± 1.6%, P < 0.05) (Fig. 5D).


Figure 5
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  		Fig. 5. Wound repair of 1HAEo– cells is impaired in the presence of a fucosyltransferase inhibitor (FuTi). Confluent monolayers of 1HAEo– were serum starved for 24 h before the creation of a small wound as described in MATERIALS AND METHODS. Cells were treated with 10–3-10–5 M of a FuTi (FuTi-3, -4, and -5) in the presence (A and B) and absence (C and D) of epidermal growth factor (EGF; 15 ng/ml) after mechanical injury. In each experiment, 1 well was used as a negative control and 1 well was treated with EGF (15 ng/ml). Corresponding wound areas determined 0, 2, 6, 12, and 24 h after wound creation by time-lapse videomicroscopy are demonstrated in A and C, with wound repair inhibition at 24 h demonstrated in B and D. The remaining wound area after 24 h was significantly higher in control cultures compared with EGF-treated groups (*P < 0.01, n = 10). In the presence of EGF, monolayers treated with FuTi compared with monolayers treated only with EGF demonstrated a dose-dependent inhibition of repair. At 24 h all FuTi treatments were significantly different ({ddagger}P < 0.05, B) and the 10–3 and 10–4 M FuTi were also significantly different compared with EGF alone (*P < 0.01, B). In the absence of exogenous EGF, 10–3 and 10–4 M FuTi significantly inhibited epithelial repair compared with the control group (*P < 0.01, D). Statistical significance of the differences between groups was determined by 1-way ANOVA.

 
Soluble sLex reduces epithelial repair in a culture model of epithelial cell monolayer wound repair only in the presence of exogenous EGF. We further investigated the role of sLex in epithelial repair by blocking the potential receptors for sLex with soluble sLex. The initial wound area and perimeter for monolayers within each experimental series were equivalent and consistent. In control and EGF-only experiments pooled across experimental series, the remaining wound area after 24 h was 25 ± 4.1% in control cultures and 1 ± 0.3% in EGF-stimulated wounds (P < 0.01; n = 10). Concurrent treatment of monolayers with soluble sLex and EGF inhibited wound repair in a concentration-dependent manner. Monolayers treated with 10–4 M soluble sLex compared with monolayers treated with 10–5, 10–6, and 10–7 M soluble sLex had a higher wound area 24 h after treatment (P < 0.05). The remaining wound area at 24 h after treatment with 10–4, 10–5, and 10–6 M soluble sLex + EGF was significantly higher compared with the group treated with EGF alone (P < 0.01) (Fig. 6B). In the absence of exogenous EGF, soluble sLex had no effect on epithelial repair (data not shown). Altogether our data showed that cotreatment of the injured monolayers with EGF and FuTi or soluble sLex reverses the acceleration effect of EGF on epithelial repair. Although the inhibitory effect of FuTi on epithelial repair remained in the absence of EGF, soluble sLex showed no effect without the concomitant stimulation of EGF. These data provide further evidence on the role of fucose-containing oligosaccharides and sLex in epithelial repair.


Figure 6
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  		Fig. 6. Wound repair of 1HAEo– cells is impaired by soluble sLex only in the presence of exogenous EGF. Confluent monolayers of 1HAEo– were serum starved for 24 h before the creation of a small wound as described in MATERIALS AND METHODS. Cells were treated with 10–4-10–7 M soluble sLex (sLex-4, -5, -6, and -7) and EGF (15 ng/ml) after mechanical injury. In each experiment, 1 well was used as a negative control and 1 well was treated with EGF (15 ng/ml). Corresponding wound areas determined 0, 2, 6, 12, and 24 h after wound creation by time-lapse videomicroscopy (A) and wound repair inhibition at 24 h (B) are demonstrated. The remaining wound area after 24 h was significantly higher in control cultures compared with EGF-treated groups (*P < 0.01, n = 10). The remaining wound area at 24 h after treatment with 10–4, 10–5, and 10–6M soluble sLex + EGF (15 ng/ml) was significantly higher compared with EGF alone (*P < 0.01, B). Monolayers treated with 10–4 M soluble sLex compared with monolayers treated with 10–5, 10–6, and 10–7 M soluble sLex have higher wound area 24 h after treatment ({ddagger}P < 0.05, B). Statistical significance of the differences between groups was determined by 1-way ANOVA.

 
E-selectin is expressed by a subset of airway epithelial cells. Our previous studies identified that fucose-containing ligands are essential for repairing airway epithelium (10). Overexpression of sLex may promote closure of wounds by increasing the selectin (CD62) receptor-ligand interaction in airway epithelial cells. We next examined the expression of P-, E-, and L-selectin by 1HAEo– cells by cytofluorometric analysis. We found that E-, but not P- and L-, selectin was expressed by a subset of airway epithelial cells (data not shown). We also examined the expression of E-selectin by NHBE and 1HAEo– cells before (n = 5) and 24 h after (n = 3) mechanical injury, both in the presence of EGF (15 ng/ml). There was no statistically significant difference in the percentage of cells expressing E-selectin receptor before and after mechanical injury (Table 2).


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  		Table 2. Expression of E-selectin is not changed after mechanical injury in 1HAEo– and NHBE cells

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In providing the physical barrier against the external environment, the bronchial epithelium is continuously exposed to injuries. The airway epithelium is therefore routinely challenged as part of normal function. Epithelial wound healing represents an important process by which the respiratory epithelial barrier restores the physical barrier and tissue integrity is maintained. Epithelial repair involves a series of ordered events including migration, spreading, proliferation, and differentiation of epithelial cells. Cell surface glycoconjugates have a crucial function in a variety of normal and disease states. It has been shown that glycans can modulate the function of proteins and lipids they are attached to and are involved in cell-cell and cell-matrix interaction (13, 27, 36, 38, 42). There is a growing interest in exploring the role of cell surface carbohydrates in epithelial repair. Using lectins in an in vitro model of wound repair, Adam et al. (1) recently demonstrated that N-acetylglucosamine, which is recognized by the lectin wheat germ agglutinin, is required for epithelial repair. Our previous work demonstrated that cell surface N-glycosylation has a functional role in airway epithelial cell adhesion and migration. N-glycans with terminal fucosylation play an essential role in the complex process of repair by coordination of cell-cell functions, including migration (10). In the present study, we examined for the first time the role of a specific fucose-containing carbohydrate structure, sLex, in bronchial epithelial repair.

sLex is a member of the Lewis blood group structures that are found at the nonreducing termini of N-linked or O-linked glycans on glycoproteins and glycolipids. sLex has been identified as a necessary component of selectin-ligand interaction, which mediates cell-cell interaction and migration in several systems (14, 28, 30, 36, 45). To investigate the role of sLex in epithelial repair we examined the expression of this antigen on bronchial epithelium by immunostaining. There was increased expression of sLex in areas of damaged epithelium compared with intact regions. This finding suggests a possible contribution of sLex in epithelial repair. Gipson et al. (15) showed previously that cell surface carbohydrates on epithelial cells that are spreading and/or migrating to cover a wound are different from cell surface carbohydrate structures found on normal epithelial cells. Our laboratory has shown (47) that glycosylation profiles in airway epithelium change over time during repair of a wound created by mechanical injury. It has been shown that injury of the respiratory epithelium enhances bacterium Pseudomonas aeruginosa adhesion to the epithelium, and it has been speculated that changes of cell surface glycoconjugates related to wound repair, cell migration, and/or spreading may favor P. aeruginosa adhesion (37).

The final step in synthesis of sLex is catalyzed by specific {alpha}1,3-FucT. The FucT gene family encodes for a group of proteins that show a complex tissue- and cell type-specific expression pattern. FucT-IV and -VII are expressed in human leukocytes, where they modify carbohydrate motifs that can act as E- and P-selectin ligands (17, 32, 39). In contrast, FucT-III, -V, and -VI are not expressed in leukocytes (5) and FucT-IX is abundantly expressed in brain, stomach, spleen, and peripheral blood leukocytes (19). Among six {alpha}1,3-FucT responsible for synthesis of sLex, we examined the expression of FucT-III, -IV, -VII, and -IX in two bronchial epithelial cell lines (1HAEo– and 16HBE 14o–) and primary cells. FucT-V and -VI do not appear to have an essential biological role, and not all humans have functional forms of these enzymes (8). We found that FucT-IV is the only gene expressed in all airway epithelial cells examined (Table 1); therefore, FucT-IV is to be considered the main FucT in the study of airway epithelial repair. It has been shown that expression of FucT varies during development and malignant transformation (3, 25, 35). This may explain the diversity of FucT expression between the transformed bronchial epithelial cell lines and primary cells studied. Our data showed a time-dependent increase in the expression of FucT-IV after mechanical injury coordinate with both sLex expression and wound closure (data not shown). Several studies have pointed out the similarities between pathways and genes activated during development, malignant transformation, and tissue healing. It has been shown by several studies that FucT-IV expression is significantly higher in tumors than in adjacent normal cells (25, 33, 35). Cailleau-Thomas et al. (3) examined the expression of FucT during human development. They found that FucT-IV and -IX are the only FucT strongly expressed during the first 2 mo of embryogenesis.

In the present study, we demonstrated an increased expression of sLex during repair of a wounded in vitro monolayer. To our knowledge, there is no other report indicating expression of specific carbohydrate structure after injury. This finding confirms our in vivo observation of overexpression of sLex in the area of epithelial damage. Overexpression of sLex by the epithelial cells distant from the wound edge suggests involvement of a soluble factor(s) released by the injured epithelium. This soluble factor would initiate the repair process, including migration of distant cells.

To confirm the role of fucose-containing oligosaccharides and specifically sLex in epithelial repair, we treated human airway epithelial cells in monolayer culture with a FuTi or soluble sLex in the presence and absence of EGF, a potent accelerator of epithelial repair (22). Wounded monolayers were followed for closure by use of time-lapse videomicroscopy. Our data demonstrated that prevention of the synthesis of fucosylated glycans with FuTi inhibited epithelial repair in the presence and absence of EGF. However, blocking of potential receptors for sLex with soluble sLex inhibited epithelial repair only in the presence of EGF. There are several mechanisms by which sLex can participate in epithelial repair. First, sLex is a decorating motif for many membrane-bound and secreted proteins and can modulate the function of certain glycoproteins. Second, sLex has been shown to act as a common ligand for the selectin family of receptors (41, 45). Selectins are a family of three adhesion molecules (L-, E-, and P-selectin) initially described as receptors specialized for capturing leukocytes from the bloodstream on the blood vessel endothelium. It seems that interaction of selectins with their ligands mediates cell adhesion and migration in several cell systems, including leukocyte adhesion on the endothelium and cancer cell metastasis through interaction with E-selectin presented on vascular endothelial cells (11, 29, 32, 43, 46). Our finding that soluble sLex only inhibited accelerated repair, with no effect on nonaccelerated repair, suggests that interaction of sLex with its selectin receptor does not have a prominent role in epithelial repair and is not the sole mechanism utilizing sLex to affect repair. It also suggests that sLex binding to CD62E during repair requires pathways activated by EGF. FuTi, on the other hand, inhibited epithelial repair in either the presence or the absence of EGF. This demonstrates that fucose-containing structures have an essential role in epithelial repair. The universal fucosyltransferase inhibitor, FuTi, prevents the synthesis of fucose-containing structures and thus sLex on the surface of the repairing airway epithelial cells. To address the specific role of sLex in epithelial repair we inhibited sLex motifs with an inhibitory antibody, KM 93, that demonstrated an inhibitory effect on epithelial repair. The exact structure carrying the sLex structure needed for repair remains to be identified.

Our data suggest that binding of sLex to its receptor in part contributes to epithelial repair after mechanical injury, so we investigated the expression of E-, P- and L-selectins on bronchial epithelial cells by flow cytometry. We showed that only E-selectin is expressed by a subset of airway epithelial cells. We also demonstrated that expression of E-selectin by bronchial epithelial cells does not change during repair in the presence of exogenous EGF. Constant expression of E-selectin by a subset of airway epithelial cells in response to injury suggests that this receptor does not play an essential role in epithelial repair, whereas the regulation of the synthesis of the ligand, (sLex) rather than E-selectin expression itself, is the essential link during repair to affect closure. We previously demonstrated (10) the role for N-linked fucosylation but now more specifically demonstrate the role for the fucose-containing sLex.

In conclusion, our data demonstrate that the oligosaccharide sLex plays an essential role in airway epithelial repair. Our data may explain the previous observation of underrepresentation of fucose-containing carbohydrate structures in asthmatic patients reported by Kauffmann et al. (20). In that report severity of asthma and thus epithelial damage were inversely related to the amount of detected fucose-containing antigens. As such, these results and reports suggest that defects in epithelial repair in asthma patients may be due, in part, to improper glycosylation of airway epithelial cells. We demonstrated that FucT-IV is the main FucT expressed in bronchial epithelial cells. The expression of FucT-IV is increased on mechanical injury. sLex has been identified as a tumor-specific antigen that promotes tumor cell motility through interaction with endothelial E-selectins. Another unexplored possibility is that sLex as a carbohydrate modification of another protein structure controls cell motility. Our data showed an important role for the carbohydrate structure sLex in epithelial repair; however, the interaction of sLex with E-selectin receptor, only in part, plays a role in epithelial repair. Further investigation is required to elucidate how sLex as a posttranslational modification of cell protein(s) may alter protein binding or receptor activity in bronchial epithelial cells to affect migration and repair.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by the Canadian Institutes for Health Research (D. R. Dorscheid), British Columbia Lung Association (D. R. Dorscheid), Parker B. Francis Fellowship in Pulmonary Research (D. R. Dorscheid), Michael Smith Foundation for Health Research (Scholar) (D. R. Dorscheid), and the Cordula and Gunter Paetzold Fellowship, University of British Columbia (S. Allahverdian).


   ACKNOWLEDGMENTS
 
The authors gratefully thank Dr. Gurpreet K. Singhera and Dr. Ryo Atsuta for technical assistance and intellectual input and Dr. Maziar Rahmani and Gillian Kent for fruitful discussions and critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. R. Dorscheid, The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St. Paul's Hospital, Rm. 166, 1081 Burrard St., Vancouver, BC, Canada V6Z 1Y6 (e-mail: ddorscheid{at}mrl.ubc.ca)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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