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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 446-454, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4776

Decreased Distribution of Lung Epithelial Junction Proteins after Intratracheal Antigen or Lipopolysaccharide Challenge

Correlation with Neutrophil Influx and Levels of BALF sE-Cadherin

Steven M. Evans, David I. Blyth, Tony Wong, Shahin Sanjar and Michael R. West

GlaxoSmithKline Research and Development, Stevenage, Hertfordshire, United Kingdom

Address correspondence to: Dr. S. M. Evans, GlaxoSmithKline Research and Development, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK. E-mail: sme40185{at}gsk.co.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Distribution of airway junctional complex proteins after antigen or lipopolysaccharide challenge in sensitized or naive mice, respectively, was investigated. E-cadherin immunoreactivity was detected continuously along neighboring epithelial cell borders and between adjacent alveolar epithelial cells in naive and saline-challenged mice. Occludin and ZO-1 immunoreactivity were observed in the tight junction areas. Both challenges induced changes in epithelial morphology and phenotype, accompanied initially by focal loss of epithelial E-cadherin that increased in size with time and number of allergen challenges. Allergen challenge also led to focal loss of occludin and ZO-1. Western blot analysis revealed increased levels of sE-cadherin in lavage fluid after either challenge, and this increase correlated with lavage neutrophil numbers (P = 0.002). Immunocytochemistry of lavage cells 6 h after either challenge revealed E-cadherin epitopes within cytoplasmic vacuoles of neutrophils, the major cell type. In contrast, peripheral blood neutrophils or tissue neutrophils before epithelial transmigration were negative, suggesting that in airway inflammation, E-cadherin extracellular domain is cleaved by neutrophils during epithelial penetration, instigating the destabilization of adherens and tight junctions. This junctional deterioration could lead to a progressive decrease in epithelial integrity and induce alterations in epithelial morphology, with consequent enhanced paracellular transit of antigens and pathogens.

Abbreviations: adherens junction, AJ • bronchoalveolar lavage, BAL • bronchoalveolar lavage fluid, BALF • ethyleneglycol-bis-(ß-aminoethyl ether)-N,N'-tetraacetic acid, EGTA • lipopolysaccharide, LPS • ovalbumin, OVA • optimal cutting temperature, OCT • phosphate-buffered saline, PBS • standard error of the mean, SEM • tris-buffered saline, TBS • tris-buffered saline with 0.1% tween, TBST • tight junction, TJ


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The epithelium of the lung functions as a selective barrier, maintaining the distinct integrity of tissue compartments and obstructing entry of inhaled allergens, irritants, and microorganisms. The dynamic adhesion and interaction of epithelial cells is mediated via the junctions formed by a variety of cell adhesion molecules. The zonula occludens, or tight junction (TJ), is typically found at the apical region of lateral membranes of epithelial cells isolating apical and basolateral plasma membrane domains to create and maintain their polarity (1). TJs also function as a primary barrier to regulate the diffusion of solutes through the paracellular pathway (2). Occludin is an integral membrane protein specifically localized at TJs (3). More recently, junction adhesion molecules (4) and other TJ-specific integral membrane proteins, termed claudins, have been identified (5). Linkage to the actin cytoskeleton is achieved by association with a number of TJ undercoat proteins, including ZO-1, ZO-2 (6, 7), cingulin (8), 7H6 antigen (9), and symplekin (10). ZO-1 is important in linking claudins and occludin to the actin-based cytoskeleton (11–14).

Adherens junctions (AJs) are another type of intercellular junction, in which cadherins function as adhesion molecules (15–18). In simple epithelial cells, this type of junction appears belt-like, located just basally to TJ, and is involved in the mechanical linkage to adjacent cells (19–22). At least two cytoplasmic proteins, {alpha}- and ß- or {gamma}-catenins, are tightly associated with the cytoplasmic domain of cadherins (20, 23), the former indirectly by binding to the latter. These catenins show similarity to vinculin and the Drosophila armadillo gene product, respectively (24–26), and constitute the undercoat of AJ together with other cytoplasmic proteins, such as vinculin (27, 28), {alpha}-actinin (29, 30), and afadin (31). These two distinct intercellular junctions show an intimate relationship spatially and functionally in epithelial cells. For example, when AJs are disrupted by treating cultured epithelial cells with ethyleneglycol-bis-(ß-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) or function-blocking anti-cadherin monoclonal antibodies, TJ are also disrupted, indicating that the mechanical adhesion activity of AJ is required for structural integrity of the TJ (32–35). Blocking antibodies and decreased Ca2+ concentration has shown inhibition of E-cadherin homotypic adhesion, resulting in functional disruption of the TJ (32, 33). Therefore, loss of E-cadherin localization in the AJ perturbs TJ function and the ability to regulate the paracellular permeability of the epithelial barrier (36). The shedding of E-cadherin in its soluble form, which results in loss of adhesive function, has been correlated with tumor progression (37, 38), systemic inflammation, multiple-organ dysfunction (39), and cutaneous disorders (40).

Phenotypic changes and altered permeability of epithelial cells are associated with airway disease such as asthma, chronic bronchitis, and cystic fibrosis (41, 42). In patients with asthma, the wounded bronchial epithelium is characterized by disruption of TJs and widening of intercellular spaces (43, 44). In this study, we have used immunocytochemical techniques to probe airway epithelium for changes in junctional protein localization following lipopolysaccharide (LPS) or antigen challenge in sensitized mice. In addition, infiltrating cells and luminal proteins recovered by bronchoalveolar lavage (BAL) from these mice have been analyzed for the presence of soluble E-cadherin, given that increases in this molecule would be consistent with epithelial damage or activation and perturbed paracellular transit.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Murine Model of Atopic Asthma
Male BALB/c mice (Charles River, Ltd, Manston, Kent, UK) were sensitized (45) and challenged as previously described (46). Briefly, mice weighing 16–18 g were immunized by intraperitoneal injection of 10 µg ovalbumin (OVA) (grade V; A-5503; Sigma Chemical Co., Poole, Dorset, UK) in 0.1 ml endotoxin-free saline (sodium chloride for injection BP; Evans Medical Ltd, Langhurst, Horsham, West Sussex, UK) on each of 7 alternate days. To avoid nonspecific T-cell and inflammatory responses, no adjuvants were used during sensitization (47, 48). Sham-sensitized mice received 0.1 ml saline intraperitoneally, using the same protocol. The mice (weight 25–28 g) were used for challenge on or after 40 d from the start of the OVA sensitization. Terminal anesthesia was achieved using sodium pentobarbitone (0.1 ml Euthatal, 200 mg/ml; Rhone Merieux, Harlow, Essex, UK).

Intratracheal Instillation
In humans, the cellular response following segmental challenge of the lung with allergen solution more closely approximates the spontaneous asthmatic exacerbations in comparison to that seen after aerosol challenge. For this reason, the segmental technique has been proposed as a better model for the study of human asthma (49). Nonsurgical intratracheal dosing (50) has been adopted in this study because it affords the ability to administer consistent amounts of challenge material to the lungs without involvement of the nasopharynx. Mice were anesthetized with 0.2 ml Saffan (alphaxolone, 0.9% wt/vol + alphadalone, 0.3% wt/vol; Vet Drug Ltd, Dunnington, York, UK) intraperitoneally. Challenge material was then administered in a volume of 10 µl directly into the trachea by the dosing technique described previously (50, 46). The challenge volume of 10 µl has been shown, using radio-opaque barium sulfate contrast medium, to distribute to the periphery of both lungs by 20 min (5).

OVA Challenge
On or after Day 40, when mice weighed ~ 25–26 g, challenge of sensitized mice (normally in groups of five) was performed either as a single dose (20 µg OVA in 10 µl intratracheally) or as the standard protocol (challenge on 3 d, each 3 d apart; 20 µg OVA in 10 µl intratracheally) as used previously (46). After induction of terminal anesthesia, BAL fluid (BALF) was taken at 6 or 24 h after the single challenge or 24 h after three challenges given at 3-d intervals. Sham-challenged mice received 10 µl of endotoxin-free saline only.

LPS Challenge
LPS (endotoxin from Escherichia coli serotype 055:B5; Sigma No. L-6529) was dissolved in endotoxin-free saline and administered intratracheally at a dose of 50 µg in 10 µl; the dose was derived from a previous study (46). BALF was taken at 6 h, as defined from previous studies (51), after a single LPS challenge.

BAL
At the relevant time points after OVA or LPS challenge, mice were killed by terminal anesthesia. Following deflection of the submandibular salivary glands and sternohyoid muscles to one side by blunt dissection, a blunted 21-guage hypodermic needle was inserted as a cannula through an incision in the upper trachea and secured with suture. Lavage was performed by introduction of 1 ml phosphate-buffered saline (PBS) into the lungs via the tracheal cannula and slowly withdrawing the fluid. This was repeated four times. The BALF was collected in a polystyrene tube containing protease inhibitors (Complete; F. Hoffman-La Roche Ltd., Diagnostics Division, Basel, Switzerland) and kept at 4°C.

Western Blot Analysis of BALF Supernatant
Cells were removed from BALF by centrifugation (300 x g for 5 min at 4°C). The remaining BALF supernatant was centrifuged in an Eppendorf microfuge (13,000 rpm for 1 min at 4°C), after which protein was precipitated from the BALF supernatant by incubation with 5% (final concentration) trichloroacetic acid at 4°C for 30 min. Following centrifugation (13,000 rpm for 5 min at 4°C), the supernatant was removed, and the pellet was resuspended in diethyl ether and recentrifuged (13,000 rpm for 5 min at 4°C). After two washes in diethyl ether, the pellet was air dried and solubilized in 40 µl of sodium dodecyl sulfate sample buffer. BALF was normalized to whole lung by maintaining accurate sample volumes during recovery, preparation, and gel loading. Samples (10 µl) were electrophoretically separated on 4% to 20% precast gradient gels (Novex, Invitrogen Ltd., Paisley, UK) run at constant voltage (100 V). Protein was transferred to nitrocellulose membrane (LC2000; Novex) in transfer buffer (Novex). The membrane was blocked overnight at 4°C in 5% marvel in tris-buffered saline (TBS) with 0.1% tween (TBST). The membrane was then incubated (1 h) with the anti-mouse E-cadherin antibodies ECCD-2 (Takara Bio Inc., Japan; 0.3 µg/ml) and DECMA-1 (Sigma; 0.3 µg/ml). After three 5-min washes with TBST, the membrane was incubated (1 h) with an anti-rat peroxidase-linked secondary antibody (anti-Rat POD, Roche Diagnostics) in TBST with 5% Marvel. The membrane was rinsed, and bands were detected using Supersignal luminol enhancer (Perbio Science UK Ltd., Cheshire, UK) followed by exposure on blue-light–sensitive film (Hyperfilm; Amersham Biosciences UK Limited, Little Chalfont, UK). Bands were quantified using densitometry (Leica Q600S; Quantimet 6,000 v01.06A, Leica Microsystems Inc., Chantilly, VA).

Immunocytochemistry of Lung Tissue
At the appropriate time points after OVA or LPS challenge, mice were killed by terminal anesthesia. After surgical exposure of the trachea, a blunted 21-guage hypodermic needle was inserted as a cannula through an incision in the upper trachea and tied in place with suture thread. Mouse lungs were gently inflated in situ by the introduction of 1 ml of an optimal cutting temperature (OCT)-based cryo-preservative (10% sucrose in OCT). After removal of the lungs, the individual lobes were placed in separate metal trays, overlaid with OCT, and snap-frozen using isopentane cooled by dry ice. Cryo-sections were fixed in buffered neutral formalin for 15 min and exposed to one of the following antibodies: ECCD-2 (1/6,000) (Takara), DECMA-1 (1/6,000) (Sigma), ZO-1 (1/400) (MAB1520; Chemicon Europe, Ltd., Hampshire, UK), Occludin (1/1,600) (OC-3F10; Zymed Laboratories, Inc., South San Francisco, CA), or serotype-matched control antibodies (rat IgG and rabbit IgG) (Serotec Ltd., Oxford, UK). After careful washing, the sections were exposed to anti-Rat-FITC or anti-rabbit FITC antibodies for 1 h. After further washing, the sections were mounted and studied using a fluorescence microscope (Nikon Optiphot, Nikon UK Limited, Surrey, UK).

Immunocytochemistry of Cells
The BALF was centrifuged (300 x g for 6 min at 4°C), after which the supernatant was removed for Western blot analysis and the cell pellet resuspended in 0.5 ml PBS. White blood cells were prepared by sedimentation of red cells in a combination of 0.6% methyl cellulose and 15% sodium diatrizoate for 30 min, followed by centrifugation of the supernatant on a Ficoll gradient (400 x g for 20 min at 20°C). White blood cells were removed and washed four times in PBS. Total BAL or blood cells were counted in a Sysmex K1000 counter (Sysmex, Buckinghamshire, UK) and an air-dried slide preparation was made of each sample (Cytospin 3; Shandon Scientific, Runcorn, Cheshire, UK). The slide preparations were used for differential cell counting or immunocytochemical analysis. For differential counts, cells were stained using May-Grünwald-Giemsa stain, and a count of over 200 cells was made according to standard morphologic criteria. The numbers of cells recovered per mouse were then calculated (Sysmex) and expressed as the mean and standard error of the mean (SEM) for each treatment group (normally four mice per group). For immunocytochemistry, cells were fixed in a 1:1 ratio of methanol and acetone for 10 min and allowed to air dry for 30 min. After encircling the cell deposit with a wax pen, ECCD-2 (0.3 µg/ml) or the serotype-matched control rat-IgG antibody was added and incubated for 1 h in a humidity chamber. The slides were repeatedly washed in PBS before the secondary FITC-linked anti-rat immunoglobulin antibody (anti-rat IgG1FITC; F4412; Sigma) was added for a further 1 h incubation.

Statistical Methods
The data are expressed as the mean ± SEM of four to eight mice per experimental group. Statistical analysis was performed on cellular infiltration data using Student's t test for unpaired data; P < 0.05 was taken as significant. Analysis of variance with Dunnett's analysis was performed on groups where required, and linear regression analysis was used to quantify correlations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E-Cadherin Distribution in Mouse Lung
Because LPS and OVA challenge of mice result in gross changes of epithelial morphology and phenotype, we examined mouse lung tissue for the effects of these stimuli on junctional complex proteins. Sections from cryopreserved mouse lungs were examined for E-cadherin localization using ECCD-2 or DECMA-1 antibodies, with both antibodies providing equivalent results. In lung tissue from sensitized but unchallenged mice and naive mice, strong E-cadherin localization was observed along the lateral edges of the epithelial cells as shown by DECMA-1 (Figures 1A and 3A) and ECCD-2 (Figure 1E) antibodies. E-cadherin was not seen on the apical borders of epithelial cells, but faint localization was occasionally seen along basal borders (Figures 1A, 3A, and 1E). In some airways 6 h after a single OVA challenge, loss of intensity of E-cadherin localization from the lateral edges of epithelial cells occurred in very discrete foci (Figure 1B). This focal loss of E-cadherin localization was more evident 24 h after a single OVA challenge and was accompanied by some morphologic changes in the epithelium (Figure 1C). The most obvious changes were seen 24 h after three antigen challenges, when there was an extensive loss of E-cadherin throughout the airway sections as shown using DECMA-1 in Figures 1D and 3B. The comparatively sparse amounts of E-cadherin remaining were localized irregularly along the lateral cell borders with no apical or basal localization. Airway sections probed with serotype-matched control IgG showed no fluorescence.



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  		Figure 1. Immunofluorescent localization of E-cadherin on sections of cryopreserved mouse lung using DECMA-1 (A–D) or ECCD-2 (E and F) antibody. Airway sections from naive mice (A) or OVA-sensitized mice challenged with saline (E) show normal E-cadherin localization at lateral and some basal edges of epithelial cells. However, 6 h (B) and 24 h (C) after a single OVA challenge, there are discrete focal changes in E-cadherin localization, with a more extensive loss of localization seen 24 h after three challenges of OVA (D) and 6 h after LPS challenge (F).

 
In mice challenged with LPS, variable loss of E-cadherin was observed, ranging from normal expression, to discrete focal losses at lateral edges of airway epithelial cells, to extensive airway loss (Figure 1F). Fluorescence that was normally seen at the borders of adjacent alveolar cells was also markedly decreased in the tissue sections from LPS challenge (Figure 2) .



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  		Figure 2. Immunofluorescent localization of E-cadherin on sections of cryopreserved mouse lung using ECCD-2 antibody, with corresponding light microscopy. Alveoli sections from mice challenged with saline (A) show normal E-cadherin localization at cell-to-cell contacts, not apparent in sections from mice challenged with LPS (B).

 
In LPS-challenged mice and at each time point of OVA-challenged mice, occasional unaffected airways were observed within the same lung sections, having normal morphology and exhibiting normal distribution and intensity of E-cadherin.

Occludin and ZO-1 Distribution in Mouse Lung
The localization of occludin in airways of sensitized but unchallenged mice was strongest at the apical surface of the epithelium, particularly at the lateral borders between cells (Figure 3C) . However, a small amount of occludin localization was sometimes observed lower down the lateral edge toward the basal surface. In the same unchallenged mice, the tight-junction–associated protein ZO-1 was discretely localized near the apical surface, consolidated between cells where the tight junctions are found. In contrast to occludin, little or no ZO-1 was observed along the lateral edges (Figure 3e).



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  		Figure 3. Immunofluorescent epithelial localization of E-cadherin (A; ECCD-2 monoclonal antibody), occludin (C; OC-3F10 polyclonal antibody), and ZO-1 (E; MAB1520 monoclonal antibody) in sections of saline-challenged cryopreserved mouse lung. Localization of E-cadherin (B), occludin (D), and ZO-1 (F) in airway epithelium of mice 24 h after three challenges of OVA, demonstrating marked and extensive loss of cadherin and occludin, with discrete focal loss of ZO-1.

 
In lung sections taken 6 and 24 h after a single OVA challenge, occludin and ZO-1 localization was lost at discrete foci. In lung sections taken 24 h after three antigen challenges, occludin localization in affected airways seemed confined to the apical surface, with the remaining lateral localization in close proximity to apical borders. The apical occludin localization was irregular in comparison to normal airways (Figure 3D). In the similarly affected airways of mice 24 h after three challenges of OVA, ZO-1 localization was irresolute, with discrete foci demonstrating complete loss of localization (Figure 3F). Some areas of airways had damaged epithelium and some loss of epithelial cells, which also added to the overall loss. In contrast, in other areas of the same tissue in which airways looked morphologically normal, occludin and ZO-1 were distributed normally.

Similar loss of occludin localization occurred in mice challenged with LPS, resulting in negligible lateral localization and disordered apical localization (not shown). However, in the same LPS-challenged mice, no obvious changes in ZO-1 localization were observed.

Western Blot Analysis of BALF Supernatants
Total BALF protein was elevated in mice challenged with LPS (118 ± 28% increase) and in mice after three challenges of OVA (162 ± 47% increase), in comparison to saline control mice. Bands of 80 kD were identified by ECCD-2 (Figure 4) or DECMA-1 in mouse BALF recovered from all treatment groups. Densitometry of these bands confirmed higher cadherin levels in LPS-challenged mice (50 µg intratracheally; 6 h; 800 ± 79% increase; P > 0.01), in comparison to saline controls. In OVA-sensitized mice, these bands were found to be of higher density in OVA (20 µg intratracheally)-challenged mice, 6 h (500 ± 152% increase; P > 0.01) and 24 h (910 ± 36% increase; P > 0.01) after a single OVA challenge, in comparison to saline-sensitized controls. In mouse BAL supernatants, 24 h after the third challenge, band density was also increased in comparison to levels in mice administered saline (870 ± 120% increase; P > 0.01).



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  		Figure 4. Western blot detection of E-cadherin in supernatants of mouse BALF. ECCD-2 detected bands at 80 kD in samples taken from mice 6 h after challenge with control saline (A), LPS (B), or OVA (C) or 24 h after a single OVA challenge (D) or three challenges of OVA (E).

 
Samples were taken from a separate study in which mice had been challenged as late as 115 d after initial sensitization, as opposed to the standard 40 d, to assess the stability of the sensitization protocols. A decreased response to antigen was observed, possibly caused by a deterioration of the sensitization. The levels of soluble E-cadherin and the number of neutrophils in the BAL samples showed large variation. However, on comparison, a significant correlation (R = 0.96; P = 0.002) was found between the numbers of neutrophils and the level of soluble E-cadherin in the BAL (Figure 5A) . To investigate further this observed correlation between neutrophil influx and levels of soluble E-cadherin, we performed LPS-challenges over a range of concentrations (0.1, 1, 10 and 100 µg intratracheally; 6 h). This range of LPS-challenges produced a concentration-related increase in the number of neutrophils in the BALF. This neutrophil influx correlated significantly to the levels of soluble E-cadherin in the BAL supernatants (R = 0.93; P = 0.002) (Figure 5B).



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  		Figure 5. Comparison of neutrophil numbers with soluble E-cadherin (quantified by Western blot densitometry) recovered via BAL of mouse lungs (A) 6 h after a single OVA challenge 115 d after initial sensitization (R = 0.96; P = 0.002) and (B) 6 h after LPS challenge at concentrations of 0.1 (plus signs), 1 (black squares), 10 (asterisks), and 100 (black triangles) µg (R = 0.93; P = 0.002). Black circles, saline.

 
Immunocytochemical Analysis of Cells
In the BALF of mice receiving only saline challenge, no changes were seen in the total cell numbers or differential cell counts 6 and 24 h after a single challenge or 24 h after three challenges. After a single administration of OVA, a significant increase of neutrophils was seen in the BAL of mice at 6 h (3.7 ± 2.0 x 10-5 per mouse; P < 0.01), although no significant change in eosinophils, lymphocytes, or macrophages was observed at this time. In comparison to 6 h after OVA, the BAL neutrophil numbers were significantly lower at 24 h after OVA (2.5 ± 1.4 x 10-4 per mouse; P < 0.01), with no significant changes in eosinophil, lymphocyte, or macrophage numbers. In mice that had received three challenges of OVA, neutrophils (1.0 ± 0.9 x 10-5 per mouse; P < 0.05) were still significantly elevated above basal levels, although not significantly different from levels seen 24 h after a single challenge. After three challenges, there was a significant increase in eosinophils (3.5 ± 1.8 x 10-5 per mouse; P < 0.01) seen in the BALF 24 h after the final challenge.

Mice that had received LPS showed a significant elevation in BAL neutrophils (16.0 ± 3.0 x 10-5 per mouse; P < 0.001) after 6 h but showed no changes in eosinophils, lymphocytes, or macrophages.

The cellular material from the BALF of these mice challenged with antigen or LPS was investigated via immunocytochemistry using ECCD-2 or its IgG serotype control. At 6 h after OVA challenge (Figure 6A) or LPS (Figure 6 B), neutrophils were found to contain E-cadherin epitopes recognized by ECCD-2. The E-cadherin distribution had a punctate, extra-nuclear appearance consistent with neutrophil phagosome localization, although only confocal microscopy establishes precise cellular localization. Occasional fluorescence was seen in neutrophils 24 h after a single OVA challenge. Infiltrating neutrophils were also observed in the tissue sections taken from LPS-challenged mice, although no granular fluorescence above background levels was observed during immunocytochemistry with ECCD-2.



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  		Figure 6. Specific E-cadherin immunofluorescence in neutrophils prepared from BALF or blood detected by ECCD-2. BALF was taken 6 h after treatment with OVA (A) or LPS (B). For comparison, neutrophils from blood of LPS-treated mice (C) were prepared, demonstrating that no fluorescence is seen before recruitment and infiltration.

 
As a further control, neutrophils were purified from the blood of naive mice and mice challenged with LPS. Immunocytochemistry using ECCD-2 revealed no fluorescence of neutrophils or any other white blood cells (Figure 6C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report the effects of intratracheal antigen or LPS challenge on the distribution of the cell adhesion molecule E-cadherin and the TJ proteins, occludin and ZO-1, expressed in airways and alveolar spaces of mouse lung. Intratracheal challenge with allergen induces a significant neutrophil influx at 6 h, which diminishes over 24 h. Then, after a further two challenges of antigen, a predominantly eosinophilic infiltration is observed (46).

In the present study, in control mice, E-cadherin was localized continuously along the boundaries between neighboring epithelial cells. Six hours after antigen challenge, at a time when neutrophil influx is at a peak, E-cadherin localization was lost at discrete foci. After a further 18 h, this loss of E-cadherin localization was more obvious, as was the modification in epithelial morphology at these foci. The focal nature of this early allergenic response has also been reported in human bronchial asthma and other animal models of asthma (52). In mice that received a further two antigen challenges, the loss of E-cadherin was more extensive, involving the full circumference of some airways. These data suggest a correlation of E-cadherin loss with number of allergen exposures.

LPS is a potent stimulus of neutrophil recruitment, and in this study, the dose of LPS used induced a neutrophil influx that was 4-fold higher than that induced by allergen over a 6 h period. This greater neutrophilia also correlated with a greater loss of E-cadherin localization in the airway epithelium. These data suggested a correlation between neutrophil migration across the epithelium with the severity of E-cadherin loss from the epithelium. However, it is unknown whether neutrophils remove E-cadherin to facilitate translocation or whether these events are a coordinated shedding response by the epithelium to facilitate cell or protein flux. Leukocyte adhesion to the vascular endothelium has been shown to trigger disorganization of the endothelial AJ (53). It is also known that activated neutrophils induce hyperpermeability and phosphorylation of AJ proteins in coronary venular endothelial cells (54). In turn, inhibition of VE-cadherin–mediated adhesion using blocking antibodies has been shown to cause increased monolayer permeability and enhanced neutrophil transendothelial migration across endothelial cells (55). One candidate for proteolysis of endothelial cadherins is neutrophil elastase, which promotes lung microvascular injury (56). Neutrophil elastase can also degrade catenins (57), which would subsequently disrupt the stability of the AJ, although epithelial cell penetration by elastase would be required for this effect.

During inflammation, neutrophils and endothelial cells produce oxidants, which are known to bring about a loss of vascular cadherin localization and an increase in solute permeability (58). Noncytotoxic oxidative stress has also been shown to disrupt the E-cadherin/catenin cell adhesion complex (59).

The present study, using immunocytochemistry, has found that focal changes in epithelial morphology after antigen or LPS challenge are associated not only with a perturbation of the normal localization of E-cadherin but also of occludin and ZO-1. It has been shown previously that disruption of cadherin–cadherin interaction using blocking antibodies or EGTA subsequently disrupts the tight junction, indicating that a stable AJ is required to maintain the structural integrity of the tight junction (32–34). Moreover, loss of E-cadherin function in the intestine caused loss of epithelial barrier function and a marked inflammatory pathology (60). Thus, the early loss of AJ component proteins in the present study may lead to dysfunctional tight junctions and increased paracellular transit, which, combined with increased vascular extravasation, may determine the increased total luminal protein seen after LPS or OVA challenge.

Mice showed a more extensive loss of E-cadherin 6 h after LPS administration compared with 6 h after OVA administration. E-cadherin loss seen 6 h after LPS was similar to that seen after three challenges with OVA, but this was not reflected in the loss of localization of ZO-1 and occludin, which were only mildly disturbed in comparison to the three challenges of OVA. Although localization is not an absolute indicator of TJ function, it suggests that prolonged disruption of the TJ will in time lead to loss of TJ protein localization. Alternatively, it could simply be part of the epithelial phenotype changes seen after three OVA challenges but not at 6 h after a single LPS challenge.

In contrast to the figures shown in this paper, morphologically normal airways were also occasionally observed within these inflamed lungs and displayed a localization of junctional proteins similar to saline controls. With the low volume of challenge given in this study, it is possible that incomplete diffusion led to a proportion of airways receiving insufficient challenge to stimulate a response. Whatever the reason for the lack of response, airways that are not affected serve as good internal controls, further validating the loss of E-cadherin seen in affected airways in the same section.

Through Western blot analysis of BAL protein using two antibodies recognizing different epitopes of the extracellular domain of E-cadherin (61), the present study has shown increased levels of E-cadherin at a fragment size correlating with known length of the extracellular domain (80 kD) of E-cadherin (62). These findings suggest that after airway challenge, the extracellular domain of E-cadherin is cleaved and shed into the lumen. The cleaving of the extracellular component of E-cadherin, and therefore the loss of functional adherens junctions, may in turn sanction the changes in epithelial morphology seen in this study. Comparatively low levels of the E-cadherin extracellular domain were also seen in saline-treated groups. This suggests that there could be a basal level of E-cadherin shed from the epithelium or that shear stress or turbulence produced during BAL collection disturbed the epithelium.

The profile of cellular influx into the luminal compartment of the lung was comparable to previous studies using OVA (46, 51). In the first 6 hours after LPS or OVA, the cellular influx was comprised entirely of neutrophils. Immunocytochemistry of this early cell infiltrate using an anti–E-cadherin antibody revealed fluorescence isolated to specific vacuole-like domains within the cytoplasm of the neutrophil. The immunocytochemistry of tissue sections from LPS-challenged mice showed no fluorescence above background in the neutrophils located between the vasculature and airway epithelium. To establish the normal E-cadherin constitution of circulating neutrophils from unchallenged naive mouse blood or LPS-challenged mouse blood, white blood cells were purified from the blood of normal mice and probed by immunocytochemistry. No fluorescence was seen in any circulating white blood cells from normal or challenged mice. These findings suggest that neutrophils acquire the E-cadherin–positive immunofluorescence after they have migrated from the vasculature and either during or after transmigration of the epithelial barrier. Neutrophil granules contain elastase, an enzyme capable of cleaving endothelial cadherin to release its extracellular domain (56), which supports the assumption that neutrophils disrupt E-cadherin during their translocation through the epithelium. We have previously shown that human elastase (10 U) administered to mice can release similar levels of E-cadherin into the BALF within 5 min (63).

In the present study, a correlation in variability of neutrophil recruitment and BAL-soluble E-cadherin was seen in mice challenged with OVA 115 d after initiation of sensitization. This correlation was confirmed in a LPS dose-response experiment (Figure 5b) and further supports the hypothesis that neutrophils may cleave E-cadherin on translocation of the epithelial barrier. However, it is possible that shedding of sE-cadherin is an epithelium-driven response. Whatever the mechanism underlying the E-cadherin loss, the consequent disruption of AJs would probably facilitate neutrophil transmigration. The appearance of E-cadherin epitopes only in BAL neutrophils rather than circulating neutrophils was a surprising result. The most likely time of E-cadherin–specific labeling is during transmigration, whereby the neutrophils have intimate contact with epithelial cells expressing high levels of E-cadherin. However, we cannot exclude possible nonspecific uptake of sE-cadherin from the epithelial lining fluid once neutrophils have entered the airway lumen. Whatever the source of E-cadherin epitopes, neutrophil activation seems to be important for labeling because, in the circulation where sE-cadherin exposure is also expected (40), neutrophils are E-cadherin negative. Although we have concentrated on E-cadherin labeling in BAL neutrophils, it would be equally valid to probe for extracellular fragments derived from other epithelial intracellular junctions. Thus, appearances of desmoglein, connexin, occludin, and claudin fragments within BAL neutrophils might indicate whether these cells affect desmosomes, gap junctions, TJs, and AJs during epithelial transmigration. However, it does not conflict with the possibility that an epithelium-driven shedding of E-cadherin and subsequent loss of AJ could also facilitate the translocation of neutrophils. In the present study, using Western blotting, we have shown the presence of the E-cadherin extracellular domain in the BAL correlating to inflammatory states. Therefore, neutrophils may alternatively acquire the E-cadherin epitopes by specific or nonspecific phagocytosis after translocation into the lumen.

This study has shown that challenge of mouse lungs with LPS or OVA leads to a loss of E-cadherin localization from the airway epithelium. These challenge materials, administered via intratracheal instillation, have previously been shown to induce airway inflammation (46, 51). Loss of E-cadherin seen in these studies correlates with the quantity of neutrophil influx. After migration through the epithelium, these neutrophils have been shown to acquire the extracellular domain of E-cadherin. These findings suggest that, during airway inflammation, the E-cadherin extracellular domain is cleaved, instigating the destabilization of adherens and tight junction. The loss of E-cadherin may also lead to a progressive decrease in epithelial integrity and induce alterations in epithelial morphology. Such a loss of barrier function may permit enhanced paracellular transit of antigens and pathogens. Therefore, therapeutic modulation of intercellular junctional operation may allow beneficial regulation of epithelial permeability, supporting respiratory defense.


   Acknowledgments
 
The authors thank Phillipa Proctor for photographic support.

Received in original form November 16, 2001

Received in final form May 22, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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