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Am J Physiol Lung Cell Mol Physiol 292: L462-L468, 2007. First published October 6, 2006; doi:10.1152/ajplung.00292.2006
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Epithelial repair is inhibited by an {alpha}1,6-fucose binding lectin

Elizabeth C. Adam, Stephen T. Holgate, and Peter M. Lackie

Allergy and Inflammation Research, Division of Infection Inflammation and Repair, University of Southampton, Southampton General Hospital, Southampton, United Kingdom

Submitted 3 August 2006 ; accepted in final form 5 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effective repair of damage to the airway epithelium is essential to maintain the ability to exclude airborne particulates and protect against potential pathogens. Carbohydrates on the cell surface have an important role in cell-cell and cell substrate interactions. Using a model of repair with airway epithelial-derived cells of the 16HBE 14o– cell line, we have examined the effect of the Aleuria aurantia lectin (AAL), which binds very selectively to {alpha}1,6-linked fucose residues. Addition of unconjugated or FITC-labeled AAL reduced the rate of epithelial repair to approximately one-third of control values as measured by image analysis while cell viability was maintained. Pulse labeling with AAL-FITC for 30 min followed by incubation in AAL-free medium caused similar inhibition of repair but could be reversed by addition of fucose up to 7 h after AAL removal. By confocal microscopy, AAL binding was found to be on the apical, but not basolateral, surfaces of cells, and internalization of the labeled lectin was seen. Preincubation of the lectin with fucose prevented this effect. Ulex europeaus I lectin, which is also fucose specific, resulted in similar binding to the cells and internalization, but it did not affect the speed of the repair process. We conclude that {alpha}1,6-fucose binding sites play an important role in epithelial repair. Better understanding of this process will provide a deeper insight into the crucial mechanisms of epithelial repair.

airway; endocytosis; epithelium; inhibition

THE CONDUCTING AIRWAYS PROVIDE an essential passage to the respiratory portion of the lung and are the primary site of impact for environmental particulates, including potential pathogens. The mucosa of these conducting airways is an active protective barrier comprising important elements of both the acquired and innate immune systems (6). It is an integrated structure made up of epithelial and mesenchymal cells (13). The epithelium provides a cellular barrier and facilitates clearance of material impacting on the airway, whereas the mesenchymal elements contribute both support and also a context for infiltrating cells of the immune system. Damage to the epithelium or challenge associated with damage can result in loss of barrier function or structural integrity, associated with local mucosal activation (32). In asthma, epithelial loss and damage has long been recognized as characteristic, and a significant proportion of columnar epithelial cells is shed as Creola bodies (15, 25), thus highlighting the importance of normal epithelial repair processes. Studies in animals have shown that repair of normal airway epithelium after minor damage involves the migration of the remaining epithelial cells to cover the damaged area. This is a rapid process, suggesting an autonomous response by cells in the vicinity of the damage (12).

Although neither the processes governing epithelial repair nor the changes in disease are fully understood, many of the key interactions between epithelial cells as well as their interaction with cells of the immune system are based on glycoconjugate binding or are modified by changes in cell surface glycoconjugates. For example, Pace et al. (27) established that human Galectin I induced apoptosis by binding to a specific set of cell surface glycoproteins resulting in a dramatic redistribution of surface glycoproteins in those cells that subsequently died. Fucose-containing glycoconjugates are widely expressed in mammals, and a number of key biological processes are already attributed to them. The Lewis antigens (containing {alpha}1,3-linked fucose) are essential for the correct functioning of the immune system, whereas H antigen, an {alpha}1,2-fucosylated precursor of the Lewis antigen, is increased in dermal wound healing. Recent studies indicating that the spatial modulation of Notch family receptors during development is mediated by receptor fucosylation by the glycosyltransferase Fringe emphasize the potential for a central role of fucose in cell signaling (23).

The conformation of the fucose linkage is also biologically important and provides a further level of discrimination: the role of glycoconjugates can only be fully understood if we know their structure. During leukocyte binding to endothelium at sites of inflammation, the {alpha}1,3-linked, fucose-containing glycoconjugates sialyl LewisA and sialyl LewisX are specific ligands for the cell adhesion molecule E-selectin, which is expressed on vascular endothelial cells. Selectins tether the leukocytes to the endothelium (22) and are an essential prerequisite for the subsequent cell crawling and extravasation required for their movement to a site of inflammation. Increased H antigen levels in dermal wound healing (9) indicate a wound-related increase in the activity of the secretor-independent {alpha}1,2-fucosyltransferase. Kauffmann and colleagues (18) have shown that the absence of Lewis antigens is associated with higher prevalence of asthma and wheezing.

Lectins are naturally occurring glycoconjugate binding molecules that can be isolated from a wide variety of plants and animals and provide exquisitely selective tools to identify or block specific glycoconjugate motifs. Whereas many lectins bind to a range of sugar structures, some are selective for particular sugars in particular linkages (37). The lectin from the mushroom Aleuria aurantia (AAL) has a high affinity for {alpha}1,6-linked fucose found in disaccharides, oligosaccharides, and glycoproteins. AAL comprises two identical non-glycosylated subunits of 36 kDa. In studies of binding to immobilized AAL, only {alpha}1,6-linked, fucose-containing glycoconjugates and terminal unsubstituted fucose {alpha}1,2-galactose linkages bound to AAL (44). In the same study, passage of {alpha}1,2- and {alpha}1,3-fucose-containing oligosaccharides through an AAL column was retarded, but these sugars did not bind. Thus in practice AAL can be considered to be a highly specific tool for identifying {alpha}1,6-fucose residues, and there have been a number of studies using AAL in this context (34, 38, 42). In human respiratory tract, Dorscheid and colleagues (11) have shown that AAL bound to the surface of nonsecretory columnar and basal epithelial cells but not to the basement membrane. In the same study, Anguilla anguilla and Lotus tetragonolobus lectin binding sites (both fucose specific but with different linkages) were found on columnar, but not basal, cells, implicating a link between {alpha}1,6-fucose linkages and a differentiated epithelial cell phenotype.

In this study, we demonstrate the importance of {alpha}1,6-linked fucose in the repair of damage to epithelial cell layers. These observations have important implications not only in understanding the epithelial injury-repair cycle but also for identifying targets for novel drug development in diseases such as asthma.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue culture. The simian virus 40-transformed epithelial cell line 16HBE 14o– derived from human bronchial epithelial cells (7) was maintained in Eagles Minimum Essential Medium with 10% FCS, 1 mM glutamine, 1% penicillin/streptomycin solution (10,000 U/ml), and 25 mM HEPES (all from Invitrogen, Paisley, UK) at 37°C in a 5% CO2 environment. For assessment of damage repair, the cells were grown in uncoated 35-mm-diameter Petri dishes with 2-mm square grids embossed on the bottom surfaces (Invitrogen) at an initial density of 1.0 x 106 cells/dish. After 3 days, when the cells were fully confluent, they were mechanically damaged by scoring with a pipette tip parallel to the grid bars. Cultures were examined on an Olympus CK2 inverted microscope (Olympus Optical, Middlesex, UK), and digital images of identifiable squares of the damaged areas were recorded at 0 and 24 h using an Olympus 3040Z digital camera. Each experiment was repeated at least three times. In each set of three experiments, the same numbers of squares were measured for each sample (typically 7), thus providing a matched set with equal length of damage. Viability was assessed by incubating the monolayers with Hoechst 33258 (10 µg/ml medium) for 10 min and rinsing briefly in PBS, followed by incubation in 0.01% ethidium bromide in PBS for 5 min with a final rinse in PBS. The total (Hoechst-stained) and nonviable (ethidium bromide-stained) cell nuclei were counted using a Leica DMIRB fluorescent inverted microscope (Leica Microsystems, Milton Keynes, UK), and percentage cell viability was calculated. A total of four fields of view (normally 100–150 cells each) were counted for each sample.

Lectins. Unconjugated lectin from AAL and the lectin from Furze Gorse [Ulex europeaus (UEA I)] conjugated to FITC were supplied by Vector Laboratories (Peterborough, UK). Both of these lectins are fucose specific but have preferences for different fucose linkages. AAL binds preferentially to {alpha}1,6-linked fucose with a secondary specificity for {alpha}1,3-linked fucose (10, 44), whereas UEA I binds to {alpha}1,2-linked fucose and poorly or not at all to {alpha}1,6- and {alpha}1,3-linked fucose (5, 35). AAL-FITC was produced by conjugating AAL with FITC using a Fluorotag FITC conjugation kit (Sigma-Aldrich, Poole, Dorset, UK) following the manufacturer's directions.

Lectin addition. Unconjugated AAL was added to cultures at a concentration of 3 µg/ml medium for 24 h. A dose titration with varying concentrations between 1 and 10 µg/ml showed that at 3 µg/ml, wound repair was maximally inhibited while viability was always above 85% (results not shown). Following a similar titration, AAL conjugated to FITC was added to cultures at a concentration of 5 µg/ml for 24 h. A pulsed addition of AAL-FITC at 50 µg/ml for 30 min had no effect on the viability of the monolayers and was therefore used to saturate the AAL binding sites on the cells. Following this, the monolayer was thoroughly rinsed, and normal medium was replaced and left for 24 h.

UEA I-FITC was added to cultures at the same concentration as AAL-FITC (5 or 50 µg/ml). Media in control cultures were changed at the same time points as the treated cultures but received no lectin additions to the culture medium. Lectin specificity control experiments were undertaken by preincubating the lectins with L-fucose (0.3 M, Sigma-Aldrich) for 30 min before addition to the culture medium. The final concentration of fucose after addition to the culture medium was 12 mM.

Lectin cytochemistry. To determine whether the lectins bound to the surface of the cells, lectins directly conjugated to FITC at a concentration of 50 µg/ml in medium were added to cultures of cells grown on glass coverslips for 30 min at room temperature. The coverslips were either rinsed in PBS at pH 7.4 or washed and placed in normal medium for a further 24 h of culture. After being rinsed in PBS, they were then fixed in cold methanol for 10 min at 5°C, rinsed again in PBS, and mounted in AF1 solution (Citifluor, London, UK).

Fluorescence microscopy. Labeling with fluorescent counterstains and lectins was assessed by viewing cells on coverslips using a Leica DMRBE fluorescence microscope with a Hoechst, FITC, or tetramethyl rhodamine iso-thiocyanate filter block. A Leica TCS SP2 confocal microscope was used for confocal imaging when the cells were fixed and counterstained with propidium iodide (5 µg/ml for 5 min) before examination.

Estimation of growth. KS400 Image Analysis software (Imaging Associates, Thame, UK) was used to measure the areas of initial damage and the area remaining after repair in the cultures. The area of damage in the images was traced using a Calcomp DrawingBoard III digitizer tablet, and the area of repair was calculated by subtracting the area of damage remaining after 24 h from the original area of damage at time 0. Each experiment was repeated a minimum of three times, and statistical comparisons were made using a two-tailed unpaired t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of wound repair by AAL. After addition of AAL-FITC to mechanically damaged monolayer cultures of 16HBE for 24 h, a marked inhibition of wound repair was observed. Figure 1A shows typical damage immediately after wounding. Within 24 h, the control cultures had almost completely repaired (Fig. 1B), whereas those cultures where lectin had been added showed less repair (Fig. 1C). Cell viabilities were always greater than 85%. Results from image analysis (Fig. 2) revealed that on average the control cultures achieved 18.3 mm2 of repair growth, whereas cultures with AAL-FITC additions showed only 6.3 mm2 of growth (P = 0.006). AAL-FITC preincubated with fucose before addition to the culture medium produced results not significantly different to the control (P = 0.23) (Fig. 2). When native AAL was added to the cultures for 24 h, the same inhibition of repair was seen as was observed with the AAL-FITC. The pattern of repair was also the same, indicating that the inhibitory effect was due to the AAL rather than the FITC.


Figure 1
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  		Fig. 1. A: monolayer culture of 16HBE cells immediately after damage. One of the 2-mm squares marked on the Petri dish is shown, and the area of damage can be clearly seen. B: the same area from culture shown in A 24 h after damage. The damaged area has almost repaired. C: a comparable culture, but with Aleuria aurantia lectin (AAL)-FITC added immediately after damage and cultured for 24 h. The area of damage shows little repair. Bar represents 400 µm.

 

Figure 2
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  		Fig. 2. Shown is the area of repair in a monolayer culture of 16HBE cells 24 h after damage. The control culture and the culture with AAL addition preincubated with fucose (fuc) both show substantial repair. Those cultures with AAL and AAL-FITC additions show a marked reduction in repair.

 
Time course for AAL action. Subsequent experiments with the addition of AAL-FITC to monolayer cultures for 30 min only, followed by replacement with normal medium for 24 h, showed a similar marked reduction in wound repair to the 24-h incubations with either unlabeled or FITC-labeled lectin (Fig. 3). The AAL-FITC pulsed cultures only grew 8.6 mm2 (P = 0.03), whereas control cultures achieved an average of 21.9 mm2 of growth. Control cultures where the AAL-FITC had been preincubated with fucose before a 30-min pulse repaired normally in a similar manner to the control cultures (P = 0.11). In these experiments, cell viability was always greater than 90%.


Figure 3
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  		Fig. 3. Damaged cultures with initial AAL-FITC addition, followed by fucose additions at varying time intervals, demonstrate that the damage repair can be returned to normal by addition of fucose up to 4 h after the initial AAL-FITC addition. Differences are not statistically significant (P > 0.05) unless shown.

 
In cultures incubated with AAL-FITC for 30 min and then returned to normal medium, addition of fucose to the culture medium after 1 or 4 h restored the rate of repair to that observed in control cultures [Fig. 3, P = 0.59 (1 h) and P = 0.43 (4 h) compared with control].

AAL binds to the surface of the cells. AAL-FITC was distributed throughout the repairing epithelium (Fig. 4). After addition to the culture medium, AAL-FITC bound rapidly to the surface of the cells. After 30 min of incubation with AAL-FITC, the majority of cells showed surface labeling with some variability in the intensity of labeling and one or two cells in each field without detectable labeling (Fig. 4A). Figure 5A shows a confocal image of a similar monolayer of 16HBE cells, again 30 min after addition of AAL-FITC. Fluorescence was uniformly distributed on the apical surfaces of the cells and occurs above the nuclei. This labeling covered the majority of the apical surfaces but was not visible on the basolateral surfaces. The single x-y confocal sections in Fig. 5A show more apparent variability in apical cell surface labeling, as not all the apical surface is included in this plane. The z-plane reconstruction from the confocal sections in Fig. 5A demonstrates the distribution of labeling more clearly. At this stage, no intracellular fluorescence was visible. Cells observed 24 h after the AAL-FITC was replaced with medium showed that all the AAL-FITC was now within the cells, as seen in the confocal image (Fig. 5B). The fluorescent granules were generally in a perinuclear position and seen as small granules or aggregates of granules. No fluorescence remained on the apical surfaces of the cells as shown by z-plane images constructed from the confocal image stack. This change in pattern and location of the labeled granules below the level of the nuclei indicated that the AAL-FITC had been internalized.


Figure 4
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  		Fig. 4. Distribution of AAL-FITC on layers of confluent epithelial cells unwounded (A, B) or wounded (C) viewed using a Leica DMRBE microscope with an FITC fluorescence filter block. Epithelial layers were incubated with AAL-FITC for 30 min, cells were washed and photographed (A) or transferred to normal medium for a further 24 h, and photomicrographs were taken (B, C). The labeling pattern on damaged layers at 30 min was indistinguishable from that seen in A.

 

Figure 5
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  		Fig. 5. Confocal images showing 16HBE cells labeled with AAL-FITC (A and B) or UEA I-FITC (C and D) (green) counterstained with propidium iodide (red) and imaged at 30 min (A and C) or 24 h. Thirty minutes after AAL-FITC addition, fluorescence was seen over the cell layer (A), and the z-plane (A, bottom) showed AAL-FITC bound to the apical cell surface. At 24 h, a vesicular pattern of AAL-FITC labeling was seen, and the z-plane indicated that the AAL-FITC is now internal, at the level of the nuclei. Thirty minutes after UEA I-FITC addition, the cell layer was labeled (C), and the z-plane (C, bottom) again showed apical surface binding of the lectin. Twenty-four hours after UEA I-FITC addition, the z-plane (D, bottom) showed the lectin labeling was now internal, although the pattern was subtly different from that shown by AAL-FITC in B. Bar represents 10 µm.

 
Effects of other fucose-binding lectins. With addition of the fucose-specific lectin UEA I-FITC to the cultures for 30 min, no inhibition of wound repair took place, and these cultures showed no significant difference to the controls (data not shown, P value 0.25) and repaired normally. Following the addition of UEA I-FITC to the medium, the lectin bound to the apical surfaces of the cells as shown by confocal images and the z-plane image constructed from the confocal stack (Fig. 5C). After a further 24-h incubation in normal medium, the fluorescent pattern had altered so that no fluorescence remained on the surface of the cells but was instead visible within the cells (Fig. 5D). The pattern of surface labeling and internalization of UEA I was similar to that with AAL-FITC, but the internal granules appeared more diffusely distributed in the cytoplasm of the cells than was the case with AAL (Fig. 5, B and D).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the current study indicate that the addition of the {alpha}1,6-fucose binding lectin AAL to damaged monolayers of epithelial cells blocks, or greatly inhibits, the normal ability of the layer to repair. Normally, repair of an experimental wound in this system (1) would be complete in 24 h, but in the presence of AAL, repair is not complete in 48 h, and the rate of repair drops to approximately one-third of the original. Although we have used an in vitro model in these studies, it is known from work in animal systems that similar processes occur in vivo (12). Any delay in repairing minor damage would significantly increase the risk of infection as there is already likely to be an increased danger of infection at sites of trauma. The AAL binding sites therefore appear to have an important role in the normal epithelial repair process.

AAL is an exceptionally well-characterized lectin from the edible orange cup mushroom (Aleuria aurantia). It contains 312 amino acids and is multivalent with a high binding affinity for fucose glycoconjugates (10, 43). It has been reported to have highest affinity for {alpha}1,6-fucose structures, particularly those that also contain an {alpha}1,3-fucose linkage (10). The specificity of the inhibitory effect of AAL on repair is further supported by the ability to block the effect using fucose preincubation and also by the lack of inhibition of repair caused by the binding of the lectin UEA I, which is known to bind predominantly to {alpha}1,2-linked fucose residues but very poorly or not at all to {alpha}1,6- and {alpha}1,3-linked fucose residues (5, 35). AAL preincubation with fucose before addition to cells prevents binding of AAL to the cell surface and indicated that binding was necessary for inhibition. The binding of AAL appears to be complete within 30 min, and sufficient AAL binds within this time to provide maximal inhibition at 24 h even in the absence of any further AAL in the cell medium. Comparison of the effect of native AAL and AAL conjugated to fluorescein (AAL-FITC) indicated that the effect on epithelial repair was indistinguishable in this model. The use of FITC-labeled AAL (AAL-FITC) therefore provides a useful tool to track the location of the AAL during the treatments.

The reduced speed of repair following AAL binding, while cell viability is maintained, suggests that binding affects the regulation or implementation of one or more of the complex steps involved in regenerating an epithelium after damage. The fact that some repair does occur and that in time it is completed confirms the viability of the cells while indicating either that binding is not sufficient to give complete inhibition or that there are multiple redundant processes that can bring about repair, albeit at different rates. If cells incubated with a 30-min pulse of AAL were treated with fucose up to 7 h later, normal repair was seen, whereas after this time, significant inhibition of repair occurred. This unexpected result suggests that AAL has the most significant inhibitory effect on repair during the period starting 7 h after repair is initiated. At 7 h, much of the bound AAL has internalized, implicating an interaction within an internalization compartment of the cell. This pattern and time course of labeling is consistent with AAL endocytosis rather than reaction with fucosylated molecules synthesized de novo within the cell. This might reflect the occurrence of an important fucose-dependent interaction at this site.

To effectively interpret the underlying effects of AAL, it is important to understand not only the dynamics of the effect but also the subcellular localization of the AAL that causes it. The initial AAL-FITC binding pattern is consistent with binding to only the apical epithelial surface, and confocal imaging clearly confirms this. In the subsequent 2–8 h, the fluorescent label shows a progressively more vesicular pattern of staining consistent with endocytosis, and by 24 h, no surface labeling remains, with confocal microscopy showing fluorescent intracellular vesicles. Although it does not inhibit repair, FITC-labeled UEA I (which is also fucose binding) shows a similar surface-labeling pattern initially followed by internalization and the formation of intracellular vesicles. There are some minor differences in the pattern of vesicular labeling between AAL and UEA that could reflect different subcellular compartmentalization, but it seems most likely that differences in the binding affinity between the two lectins are responsible for the differential effects, further supporting a specific role for fucosylated glycoconjugates in repair. Although it is possible for lectin binding to cells to either enhance or inhibit cell proliferation, we know from previous studies that proliferation does not play a significant role in epithelial repair in this system (16). This, together with the localization, time course of inhibition, and sensitivity to fucose blocking suggests a regulatory step in the migratory phase of repair involves fucose-containing glycoconjugates.

In human respiratory tract, Dorscheid and colleagues (11) have shown that AAL bound to the surface of nonsecretory columnar and basal epithelial cells but not to the basement membrane. In the same study, A. anguilla and L. tetragonolobus (both fucose specific) lectin binding sites were found on columnar, but not basal, cells, implicating a link between {alpha}1,6-fucose linkages and a differentiated epithelial cell phenotype. Our results indicating apical localization of AAL binding sites are consistent with this. In studies by the same authors, EGF-enhanced epithelial repair was inhibited by blocking integrin binding to collagen IV (40) or using wheat germ agglutinin lectin potentially via binding to {alpha}-dystroglycan (41). Using {alpha}1,6-fucosyltransferase null mice, Wang and colleagues (39) have shown that immunoprecipitated EGF receptor (EGFR) binds AAL and that this binding is reduced in the null mice, supporting the contention that AAL is binding {alpha}1,6-fucose. In their study, {alpha}1,6-fucosylation of the EGFR in embryonic fibroblasts was required for ligand binding to induce normal EGFR phosphorylation. Given the phosphorylation and increased expression of EGFR following epithelial damage and the associated effect in enhancing repair (29, 30, 40), AAL binding to EGFR could affect epithelial repair. The apical distribution of AAL-binding sites in undamaged cultures, where EGFR might be expected to be basolaterally localized, together with the similarity in distribution with damaged, repairing cultures suggests that other fucosylated molecules may also be important. At the patient level, Kauffmann and colleagues (18) have shown that the absence of Lewis antigens is associated with higher prevalence of asthma and wheezing, whereas Allahverdian et al. (4) suggest an essential function for sialyl LewisX in epithelial repair. The importance of AAL-binding, fucose-containing glycoconjugates in epithelial repair supports and extends our previous studies showing that lectin binding is not sufficient to inhibit damage repair but is dependent on the carbohydrate specificity of the lectin (1).

The importance of fucosylation in regulating protein activity has been shown in a number of studies. Increased H antigen levels in dermal wound healing (9) indicate a wound-related increase in the activity of the secretor-independent {alpha}1,2-fucosyltransferase. Fucose residues have also been implicated in the attachment of the bacterium Pseudomonas aeruginosa to airway epithelium. This bacterium produces a fucose-specific lectin (PA II) that inhibits the beating of airway cilia (2), which in vivo would result in mucus retention, making it easier for the bacteria to attach and replicate without being expelled. This action of PA II was shown to be inhibited by fucose addition. Furthermore, this effect of cessation of ciliary beating was also observed in cystic fibrosis (CF) patients (3). This group of patients normally exhibit colonization by P. aeruginosa at a fairly early age, and there is a constant problem in management of this disease. These results have demonstrated that there is potential for therapeutic treatment of these patients with fucose, as any reduction in the attachment of this bacterium could only be advantageous for CF patients. Using AAL as an immunosensor, fucosylation of {alpha}1-acid glycoprotein has been shown to increase with inflammation and rises in patients with severe burns (20). Serum glycoproteins have been shown to stimulate airway epithelial repair. Patchell and Dorscheid (28) have postulated that glycosylation of asthmatic airways may be altered by the immune response such that interaction with the serum proteins is reduced resulting in persistence of epithelial damage (19). Mouse monoclonal antibodies specific for the {alpha}1,6-fucose linkage (CAB2 and CAB4) have been used to show regulated expression of this fucose linkage in adhesion-related changes in Dictyostelium (8) and tissue-specific patterns of expression (33).

Changes in cell membrane carbohydrate antigens also play an important role in carcinogenesis and metastasis (24). Sialyl LewisA and sialyl LewisX antigens are commonly expressed on human cancer cells and are thought to be specifically involved in the binding of the cancer cells to the endothelium during metastatic tumor spread. As a consequence, the presence of these carbohydrate determinants can be used as a prognostic indicator of metastasis (17). Highly metastatic melanoma cells have an increased expression of the sialyl LewisX tetrasaccharide (26) on the cell surface and can be used to determine the metastatic potential of these cells. Increased expression of sialyl LewisX has also been seen in a number of other cancers including colorectal carcinoma (36), liver cancer (21), lung adenocarcinoma (14), and non-small cell carcinoma (31).

In conclusion, our studies implicate fucose-containing glycoconjugates on the surface of epithelial cells as key molecules in the repair of epithelial damage. This may have important implications for our understanding of respiratory disease and related inflammatory pathologies occurring at mucosal surfaces.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. C. Adam, Allergy and Inflammation Research, Division of Infection Inflammation and Repair, Univ. of Southampton, MP12, Biomedical Imaging Unit, Southampton General Hospital, Southampton SO16 6YD, United Kingdom (e-mail: eca{at}soton.ac.uk)

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
 REFERENCES
 

  1. Adam EC, Holgate ST, Fildew CJ, Lackie PM. Role of carbohydrates in repair of human respiratory epithelium using an in vitro model. Clin Exp Allergy 33: 1398–1404, 2003.[CrossRef][Web of Science][Medline]
  2. Adam EC, Mitchell BS, Schumacher DU, Grant G, Schumacher U. Pseudomonas aeruginosa II lectin stops human ciliary beating: therapeutic implications of fucose. Am J Respir Crit Care Med 155: 2102–2104, 1997.[Abstract]
  3. Adam EC, Schumacher DU, Schumacher U. Cilia from a cystic fibrosis patient react to the ciliotoxic Pseudomonas aeruginosa II lectin in a similar manner to normal control cilia—a case report. J Laryngol Otol 111: 760–762, 1997.[Web of Science][Medline]
  4. Allahverdian S, Wojcik KR, Dorscheid DR. Airway epithelial wound repair: role of carbohydrate sialyl LewisX. Am J Physiol Lung Cell Mol Physiol 291: L828–L836, 2006.[Abstract/Free Full Text]
  5. Baldus SE, Thiele J, Park YO, Hanisch FG, Bara J, Fischer R. Characterization of the binding specificity of Anguilla anguilla agglutinin (AAA) in comparison to Ulex europaeus agglutinin I (UEA-I). Glycoconj J 13: 585–590, 1996.[CrossRef][Web of Science][Medline]
  6. Bals R, Hiemstra PS. Innate immunity in the lung: how epithelial cells fight against respiratory pathogens. Eur Respir J 23: 327–333, 2004.[Abstract/Free Full Text]
  7. Cozens AL, Yezzi MJ, Kunzelmann K, Ohrui T, Chin L, Eng K, Finkbeiner WE, Widdicombe JH, Gruenert DC. CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 10: 38–47, 1994.[Abstract]
  8. Crandall IE, Newell PC. Changes in cell surface glycoproteins during Dictyostelium development analysed using monoclonal antibodies. Development 107: 87–94, 1989.[Abstract]
  9. Dabelsteen E, Gron B, Mandel U, Mackenzie I. Altered expression of epithelial cell surface glycoconjugates and intermediate filaments at the margins of mucosal wounds. J Invest Dermatol 111: 592–597, 1998.[CrossRef][Web of Science][Medline]
  10. Debray H, Montreuil J. Aleuria aurantia agglutinin. A new isolation procedure and further study of its specificity towards various glycopeptides and oligosaccharides. Carbohydr Res 185: 15–26, 1989.[CrossRef][Web of Science][Medline]
  11. Dorscheid DR, Conforti AE, Hamann KJ, Rabe KF, White SR. Characterization of cell surface lectin-binding patterns of human airway epithelium. Histochem J 31: 145–151, 1999.[CrossRef][Web of Science][Medline]
  12. Erjefalt JS, Erjefalt I, Sundler F, Persson CGA. In-vivo restitution of airway epithelium. Cell Tissue Res 281: 305–316, 1995.[Web of Science][Medline]
  13. Evans MJ, Van Winkle LS, Fanucchi MV, Plopper CG. The attenuated fibroblast sheath of the respiratory tract epithelial-mesenchymal trophic unit. Am J Respir Cell Mol Biol 21: 655–657, 1999.[Free Full Text]
  14. Fukuoka K, Narita N, Saijo N. Increased expression of sialyl Lewis(x) antigen is associated with distant metastasis in lung cancer patients: immunohistochemical study on bronchofiberscopic biopsy specimens. Lung Cancer 20: 109–116, 1998.[CrossRef][Web of Science][Medline]
  15. Holgate ST, Lackie P, Wilson S, Roche W, Davies D. Bronchial epithelium as a key regulator of airway allergen sensitization and remodeling in asthma. Am J Respir Crit Care Med 162: S113–S117, 2000.[Free Full Text]
  16. Howat WJ, Holgate ST, Lackie PM. TGF-beta isoform release and activation during in vitro bronchial epithelial wound repair. Am J Physiol Lung Cell Mol Physiol 282: L115–L123, 2002.[Abstract/Free Full Text]
  17. Kannagi R. Carbohydrate-mediated cell adhesion involved in hematogenous metastasis of cancer. Glycoconj J 14: 577–584, 1997.[CrossRef][Web of Science][Medline]
  18. Kauffmann F, Frette C, Pham QT, Nafissi S, Bertrand JP, Oriol R. Associations of blood group-related antigens to FEV1, wheezing, and asthma. Am J Respir Crit Care Med 153: 76–82, 1996.[Abstract]
  19. Lackie PM, Adam EC. Sweet talking cellular carbohydrates and epithelial repair. Clin Exp Allergy 36: 560–562, 2006.[CrossRef][Web of Science][Medline]
  20. Liljeblad M, Ryden I, Ohlson S, Lundblad A, Pahlsson P. A lectin immunosensor technique for determination of alpha(1)-acid glycoprotein fucosylation. Anal Biochem 288: 216–224, 2001.[CrossRef][Web of Science][Medline]
  21. Liu F, Qi HL, Chen HL. Regulation of differentiation- and proliferation-inducers on Lewis antigens, alpha-fucosyltransferase and metastatic potential in hepatocarcinoma cells. Br J Cancer 84: 1556–1563, 2001.[CrossRef][Web of Science][Medline]
  22. McEver RP. Selectin-carbohydrate interactions during inflammation and metastasis. Glycoconj J 14: 585–591, 1997.[CrossRef][Web of Science][Medline]
  23. Moloney DJ, Panin VM, Johnston SH, Chen J, Shao L, Wilson R, Wang Y, Stanley P, Irvine KD, Haltiwanger RS, Vogt TF. Fringe is a glycosyltransferase that modifies Notch. Nature 406: 369–375, 2000.[CrossRef][Medline]
  24. Muramatsu T. Carbohydrate signals in metastasis and prognosis of human carcinomas. Glycobiology 3: 291–296, 1993.[Free Full Text]
  25. Naylor B. The shedding of the mucosa of the bronchial tree in asthma. Thorax 17: 69–72, 1962.[Free Full Text]
  26. Ohyama C, Tsuboi S, Fukuda M. Dual roles of sialyl Lewis X oligosaccharides in tumor metastasis and rejection by natural killer cells. EMBO J 18: 1516–1525, 1999.[CrossRef][Web of Science][Medline]
  27. Pace KE, Lee C, Stewart PL, Baum LG. Restricted receptor segregation into membrane microdomains occurs on human T cells during apoptosis induced by galectin-1. J Immunol 163: 3801–3811, 1999.[Abstract/Free Full Text]
  28. Patchell BJ, Dorscheid DR. Repair of the injury to respiratory epithelial cells characteristic of asthma is stimulated by Allomyrina dichotoma agglutinin specific serum glycoproteins. Clin Exp Allergy 36: 585–593, 2006.[CrossRef][Web of Science][Medline]
  29. Polosa R, Puddicombe SM, Krishna MT, Tuck AB, Howarth PH, Holgate ST, Davies DE. Expression of c-erbB receptors and ligands in the bronchial epithelium of asthmatic subjects. J Allergy Clin Immunol 109: 75–81, 2002.[CrossRef][Web of Science][Medline]
  30. Puddicombe SM, Polosa R, Richter A, Krishna MT, Howarth PH, Holgate ST, Davies DE. Involvement of the epidermal growth factor receptor in epithelial repair in asthma. FASEB J 14: 1362–1374, 2000.[Abstract/Free Full Text]
  31. Satoh H, Ishikawa H, Kamma H, Yamashita YT, Takahashi H, Ohtsuka M, Hasegawa S. Serum sialyl lewis X-i antigen levels in non-small cell lung cancer: correlation with distant metastasis and survival. Clin Cancer Res 3: 495–499, 1997.[Abstract]
  32. Sha Q, Truong-Tran AQ, Plitt JR, Beck LA, Schleimer RP. Activation of airway epithelial cells by toll-like receptor agonists. Am J Respir Cell Mol Biol 31: 358–364, 2004.[Abstract/Free Full Text]
  33. Srikrishna G, Varki NM, Newell PC, Varki A, Freeze HH. An IgG monoclonal antibody against Dictyostelium discoideum glycoproteins specifically recognizes Fucalpha1,6GlcNAcbeta in the core of N-linked glycans. Localized expression of core-fucosylated glycoconjugates in human tissues. J Biol Chem 272: 25743–25752, 1997.[Abstract/Free Full Text]
  34. Stelck S, Robitzki A, Willbold E, Layer PG. Fucose in alpha(1–6)-linkage regulates proliferation and histogenesis in reaggregated retinal spheroids of the chick embryo. Glycobiology 9: 1171–1179, 1999.[Abstract/Free Full Text]
  35. Sughii S, Kabat EA, Baer HH. Further immunochemical studies on the combining sites of Lotus tetragonolobus and Ulex europaeus I and II lectins. Carbohydr Res 99: 99–101, 1982.[CrossRef][Web of Science][Medline]
  36. Tatsumi M, Watanabe A, Sawada H, Yamada Y, Shino Y, Nakano H. Immunohistochemical expression of the sialyl Lewis x antigen on gastric cancer cells correlates with the presence of liver metastasis. Clin Exp Metastasis 16: 743–750, 1998.[CrossRef][Web of Science][Medline]
  37. Vann Damme EJM, Peuman WJ, Pusztai A, Bardocz S. Handbook of Plant Lectins: Properties and Biomedical Applications. London: Wiley & Sons, 1998.
  38. Wang W, Li W, Ikeda Y, Miyagawa JI, Taniguchi M, Miyoshi E, Sheng Y, Ekuni A, Ko JH, Yamamoto Y, Sugimoto T, Yamashita S, Matsuzawa Y, Grabowski GA, Honke K, Taniguchi N. Ectopic expression of alpha1,6 fucosyltransferase in mice causes steatosis in the liver and kidney accompanied by a modification of lysosomal acid lipase. Glycobiology 11: 165–174, 2001.[Abstract/Free Full Text]
  39. Wang X, Gu J, Ihara H, Miyoshi E, Honke K, Taniguchi N. Core fucosylation regulates epidermal growth factor receptor-mediated intracellular signaling. J Biol Chem 281: 2572–2577, 2006.[Abstract/Free Full Text]
  40. White SR, Dorscheid DR, Rabe KF, Wojcik KR, Hamann KJ. Role of very late adhesion integrins in mediating repair of human airway epithelial cell monolayers after mechanical injury. Am J Respir Cell Mol Biol 20: 787–796, 1999.[Abstract/Free Full Text]
  41. White SR, Wojcik KR, Gruenert D, Sun S, Dorscheid DR. Airway epithelial cell wound repair mediated by alpha-dystroglycan. Am J Respir Cell Mol Biol 24: 179–186, 2001.[Abstract/Free Full Text]
  42. Willbold E, Rothermel A, Huhn J, Stelck S, Layer PG. Inhibition of alpha(1–6)-linked fucose decreases inner retinal cells and increases photoreceptors in chicken retinal reaggregates. Dev Neurosci 23: 464–472, 2001.[CrossRef][Web of Science][Medline]
  43. Wimmerova M, Mitchell E, Sanchez JF, Gautier C, Imberty A. Crystal structure of fungal lectin: six-bladed beta-propeller fold and novel fucose recognition mode for Aleuria aurantia lectin. J Biol Chem 278: 27059–27067, 2003.[Abstract/Free Full Text]
  44. Yamashita K, Kochibe N, Ohkura T, Ueda I, Kobata A. Fractionation of L-fucose-containing oligosaccharides on immobilized Aleuria aurantia lectin. J Biol Chem 260: 4688–4693, 1985.[Abstract/Free Full Text]



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