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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 286-295, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4887

Interleukin-9 Induces Goblet Cell Hyperplasia during Repair of Human Airway Epithelia

Paola D. Vermeer*, Robert Harson*, Lisa A. Einwalter, Tom Moninger and Joseph Zabner

Department of General Surgery, Rush-Presbyterian St. Luke's Medical Center, Chicago, Illinois; and Department of Internal Medicine, Central Microscopy Research Facility, University of Iowa, Iowa City, Iowa

Address correspondence to: Joseph Zabner, M.D., University of Iowa College of Medicine, 500 EMRB, Iowa City, IA 52242. E-mail: joseph-zabner{at}uiowa.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Asthma is characterized by airway inflammation, smooth muscle hyperreactivity, and airway remodeling with excessive mucus production. The effect cytokines like interleukin (IL)-9 have on airway epithelia has been addressed using murine models of asthma, as well as transgenic and knockout mice. Though highly informative, differences exist between mouse and human airway epithelia, including cellular composition (e.g., Clara cells) and stem cell/plasticity capabilities. Therefore, to address cytokine effects on human airway epithelia, we have used a primary model system to ask whether IL-9 can alter cell fates of human airway epithelia. Here, we show that IL-9 has little effect on fully differentiated ciliated human airway epithelia. However, in the setting of airway injury repair, IL-9 results in goblet cell hyperplasia. A similar response was observed when the epithelium was exposed to IL-9 before it became fully differentiated. Moreover, exposure to IL-9 resulted in increased lysozyme and mucus production by the epithelia. Thus, a combination of IL-9 and mechanical injury can explain, in part, goblet cell hyperplasia that is evident in the lungs of individuals with asthma. These data suggest that interventions that limit airway epithelial damage, block IL-9, or modulate the repair process should result in decreased airway remodeling and prevent the chronic manifestations of this disease.

Abbreviations: diaminobenzidine, DAB • fluorescein isothiocyanate, FITC • interleukin, IL • Jacalin lectin, JAC • periodic acid-Schiff, PAS • phosphate-buffered saline, PBS • scanning electron microscopy, SEM • transmission electron microscopy, TEM


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Increased mucus production is commonly present in inflammatory diseases of the airway, including sinusitis, asthma, chronic bronchitis, and cystic fibrosis (1–6). Multiple inflammatory mediators have been implicated in the overproduction of mucus in these conditions. However, recent evidence indicates that mucus overproduction could partly be accounted for by interleukin (IL)-9 (7).

IL-9 is a pleiotropic Th2 (8) cytokine released by CD4+ T cells along with IL-4 and IL-5. In their study of IL-9 transgenic mice, Louahed and coworkers demonstrated robust staining for mucus glycoprotein in the airways compared with control mice. Furthermore, treatment of wild-type C57BL/6 mice that have undetectable IL-9 levels in their lungs with exogenous recombinant IL-9 by tracheal instillation resulted in increased mucus production as well as an induction of goblet cell hyperplasia (9). A similar effect was evident in vitro using a human pulmonary cell line, NCI-H292 (9).

IL-13, another pleiotropic cytokine, has also been implicated in goblet cell hyperplasia in mice (10). Interestingly, IL-13 levels were normal in the IL-9 knockout mice generated by Townsend and colleagues (11). However, in the absence of IL-9, IL-13 did not activate goblet cell hyperplasia after parasite challenge. This finding emphasizes the central role that IL-9 plays in goblet cell proliferation. Furthermore, Kung and coworkers demonstrated that anti–IL-9 antibodies reduced goblet cell hyperplasia in mice (47). In contrast to the murine data, a recent publication showed a role of IL-13 in goblet cell differentiation in human nasal airway epithelia in vitro in the absence of IL-9 (13). Furthermore, a recent study by Longphre and colleagues using a dog model for mucin synthesis demonstrated that mucin secretion was blocked by anti–IL-9 antibodies, but not by antibodies directed against either IL-4 or IL-5. Conversely, exposure to recombinant IL-9 increased mucus production (7). Taken together, these data support a role for IL-9 directly regulating goblet cell proliferation as well as airway epithelial mucus production. In addition, the above studies also serve to highlight limitations of animal models. Clearly, the roles of IL-9 and IL-13 in murine and canine airway epithelia may be different from those in human. Though much information can be gained from murine model systems, a study of primary human airway epithelia, though with its own limitations, may be more applicable for humans.

The role of IL-9 in asthma has been previously studied. Strain differences in mice suggest a role for IL-9 in airway hyperresponsiveness, a characteristic of asthma (14). C576BL/6 mice have undetectable IL-9 levels in their lungs and their airways are hyporesponsive, whereas DBA/2J mice, which have high levels of IL-9 protein in their lungs, have hyperresponsive airways (14). Temann and coworkers (2) demonstrated pathological changes characteristic of asthma in IL-9 transgenic mice with increased expression restricted to the lungs. These changes included bronchial hyperresponsiveness to methocholine, cellular infiltrates, and mast cell hyperplasia. In addition, the conducting airways of these transgenic mice stained positively for mucins with alcian blue/periodic acid Schiff, whereas transgene-negative mice did not. Moreover, Shimbara and coworkers (1) demonstrated elevated IL-9 mRNA levels from cells isolated from asthmatic lungs compared with that isolated from patients with chronic bronchitis and sarcoidosis. Though Bhathena and colleagues (15) demonstrated a lack of IL-9 receptor (IL-9R) in human airway cells isolated from normal patients by bronchoscopy, receptor levels may be altered under chronic inflammatory conditions.

Remodeling of the airways is evident in various lung disease states, including chronic bronchiolitis obliterans syndrome (16), cystic fibrosis (17), and asthma (18). This remodeling includes: injury and loss of surface epithelia (19–22), airway oxidative damage (23), increased airway permeability (24–27), reticular membrane thickening (18), increased collagen deposition, and increased airway smooth muscle and blood vessels (28–31). One might imagine that such remodeling would activate repair mechanisms, and that these mechanisms may become altered in an inflammatory environment. We therefore tested the hypothesis that IL-9 has a direct effect on the cell fates chosen during the injury repair process of differentiated human airway epithelia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Staining for Goblet Cells
Human tracheal tissue was isolated from donor lungs and fixed in 4% paraformaldehyde. Tissue was then embedded in paraffin, sectioned, deparaffinized, and rehydrated. Biotinylated Jacalin lectin (JAC; Vector Laboratories, Burlingame, CA) was applied for 1 h, amplified with an avidin biotin system (Vector Laboratories) for 30 min, and bound lectin was visualized with 3,3'-diaminobenzidine (DAB; Sigma, St. Louis, MO). A brown precipitate forms over lectin binding cells. Samples were next stained with periodic acid Schiff (PAS) to identify mucin and goblet cells and then rehydrated prior to imaging with a Leitz Diaplan Microscope.

Primary cultures of human airway epithelia were stained for fluorescence microscopy as follows. Cultures were fixed in 4% paraformaldehyde followed by three washes in phosphate-buffered saline (PBS). JAC lectin directly conjugated to FITC (1:200; Vector Laboratories) or mouse anti-MUC5AC (1:100, n = 8 from two donors; NeoMarkers, Inc., Fremont, CA) was applied to the apical surface for 30 min. Following washes in PBS, JAC-FITC–labeled cultures were mounted onto glass slides and coverslipped with Vectashield mounting medium (Vector Laboratories), whereas MUC5AC-labeled cultures were further incubated with apically applied anti-mouse Alexa-586 (1:200; Molecular Probes, Eugene, OR) for 30 min at room temperature. Cultures were mounted onto glass slides and coverslipped as described above. Epithelia were analyzed using a scanning laser confocal microscope (MRC-1024; BioRad, Richmond, CA). Images were obtained en face. The percent of JAC binding cells from 6–12 different primary cultures were analyzed by paired Student's t test. Cultures from the same donor have to be paired for statistical analysis when more than one donor's epithelia are used.

Primary Human Airway Epithelial Cell Culture Model
Airway epithelial cells were isolated from trachea and bronchi of donor lungs. Cells were seeded onto collagen-coated, semipermeable membranes (0.6 cm2; Millicell-HA, Millipore, Bedford, MA) and grown at an air–liquid interface as previously described (32–34). Twenty-four to 48 h after seeding, the airway cells form a confluent culture with electrically tight junctions. On the third day after seeding, scanning electron microscopy reveals a mostly flat, undifferentiated sheet of cells. Between Days 3 and 14, the epithelial cells differentiate with a predominantly ciliated phenotype (35). Epithelial cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's media and Ham's F12 media (DMEM/F12), that was supplemented with 2% Ultroser G (BioSepra; Villeneuve, La Garenne, France) and 100 mU/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml gentamicin, 25 µg/ml colimycin, 75 µg/ml ceftazidime, 25 µg/ml imipenem, 25 µg/ml cilastin, and 2 µg/ml fluconazole. Basolateral culture media was changed every 2–4 d. Samples were collected with approval from the University of Iowa Institutional Review Board.

For primary cultures treated with IL-9, recombinant human IL-9 (R&D Systems, Inc., Minneapolis, MN) was added to the basolateral media of differentiating primary cultures (n = 15 from five donors) or fully differentiated (n = 15 from five donors) primary cultures. A concentration of 50 ng/ml IL-9 was used and replaced every 3–4 d throughout the course of the experiment. Control and IL-9–treated cultures were harvested 7 and 14 d after commencement of IL-9 treatment. IL-13–treated cultures (50 ng/ml) (n = 8 from two donors; R&D Systems) were processed similarly. To quantify the number of JAC-positive cells per epithelia, epithelial cultures were counterstained with Dapi (Vector Laboratories) to label total number of cells. As results for 7- and 14-d treatments were similar, only 14-d treatment data is presented. To determine whether IL-9 treatment altered epithelial integrity, a few epithelia from each condition were mounted on Ussing chambers, and measurement of transepithelial electrical properties were taken as previously described. No differences were seen (data not shown) (36).

Electron Microscopy
Transmission electron microscopic (TEM) analysis of several different primary cultures was performed (n = 20 from one donor). To avoid removal of mucus by solvent, osmium tetroxide (1%) was dissolved in perfluorocarbon (Fluorinert FC-72; 3M Corp., St. Paul, MN). Samples were gently immersed in this perfluorocarbon fixative for 1 h, followed by three rinses in pure perfluorocarbon. Samples were then dehydrated in three washes of 100% ethanol, transitioned to Eponate –12 (Ted Pella Inc., Redding, CA), and cured overnight at 65oC. Ultrathin sections were stained with uranyl acetate and lead citrate and imaged on a Hitachi H-7000 transmission electron microscope (Hitachi, Tokyo, Japan). Sections were taken from the center of the epithelia in IL-9–treated and control specimens. Multiple micrographs were examined randomly from 20 different specimens, and the thickness of the mucus layer and entire sol/gel layers was measured. Values for control and IL-9–treated samples were analyzed by Student's t test. For scanning electron microscopy, samples were fixed with 2.5% gluteraldehye followed by 1% osmium tetroxide. Samples were dehydrated through a graded series of ethanol and finally with HMDS (hexamethyl-disilizane; Ted Pella). Samples were mounted onto grids, sputter coated with gold, and imaged on a Hitachi S-4000 scanning electron microscope.

Lysozyme Assay
The effect of IL-9 on lysozyme secretion by differentiated airway epithelia was quantified by ELISA (n = 18 from three donors; Biomedical Technologies, Inc., Stoughton, MA). Following 14 d of IL-9 treatment, control and IL-9–treated primary cultures were lysed with lysis buffer (25 mM Tris phosphate pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetreaacetic acid, 10% glycerol, 1% triton X-100). Cell lysates were spun at 8,000 rpm for 10 min to pellet membranes, and soluble fraction collected and diluted 10-fold. Lysozyme was quantified by ELISA and final concentrations calculated based on a standard curve (as described by manufacturer). Control and IL-9–treated lysozyme levels were analyzed by Student's t test.

Effect of IL-9 on Mechanically Injured Primary Cultures
Two days before injury, basolateral IL-9 (n = 15 from five donors) or IL-13 (n = 6 from two donors) treatment of differentiated primary cultures was started. To control for wound area, a wounding apparatus was designed such that the length of the probe contacting the epithelia remained constant. This, in combination with the slightly flexible nature of the filter onto which the epithelia were seeded, ensured that the pressure exerted upon wounding was also constant. In addition, once in contact with the epithelia the probe could not move in the lateral direction; thus the area wounded was reproducible. To wound the epithelia, the probe was lowered onto the cells and turned 360° four times. In doing so, a ring of cells was scraped off the filter, generating a wound. The area of cells scraped was 0.025 cm2 (total area of filter was 0.64 cm2).

Injury of the epithelia should alter transepithelial conductance. To ensure that the injury generated disrupted epithelial integrity in this way, transepithelial conductance measurements were taken with an ohmmeter (World Precision Instruments, New Haven, CT). Measurements were taken every 2–4 h after wounding. Eagle's minimal essential media (EMEM) was apically applied to each culture to take a conductance measurement. Following measurement, EMEM was removed from the apical surface and cultures were returned to the incubator. Transepithelial conductance increased immediately following injury and returned to baseline within 24 h (data not shown). After injury, epithelia were maintained under IL-9 treatment for different periods of time. At each time point, primary cultures were fixed and processed for staining.

PCNA, IL-9R, and IL-13R Staining of Primary Cultures of Differentiated Airway Epithelia
Primary cultures were fixed with 4% paraformaldehyde followed by three washes in PBS. Cells were permeabilized with 0.2% Triton X-100 (Pierce, Rockford, IL) followed by washes in PBS. Biotinylated mouse anti-PCNA antibody (n = 4 from two donors, 1:100; Zymed, San Francisco, CA) was applied to the apical surface of primary cultures for 30 min at 37°C. Following PBS washes, streptavidin-FITC (1:200; Vector Laboratories) was applied to the apical surface for 30 min at room temperature. For IL-9R immunolocalization, mouse anti–IL-9R antibody (n = 6 from two specimens, 1:100; R&D Systems, Inc., Minneapolis, MN) was apically applied for 30 min at 37°C. Following PBS washes, antibody was detected using an anti-mouse IgG directly conjugated to FITC (1:200; Biomeda, Foster City, CA) for 30 min at room temperature. Isotype matched primary antibody (mouse IgG1) served as an additional control (n = 15 from five donors, 1:100; Sigma). Cultures were counter stained with ethidium bromide (10 µg/ml) for 5 min and mounted onto glass slides, coverslipped with Vectashield (Vector Laboratories), and analyzed by confocal microscopy (MRC-1024; BioRad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
JAC Lectin Binding Identifies Goblet Cells in Tracheal Human Airway Epithelia
To quantitatively examine the effect of IL-9 treatment on cell populations within our primary cultures, we first required a cell surface marker to identify goblet cells. Though cellular stains such as PAS can serve to label secretory cells, such staining procedures require sectioning of tissue and cell samples, making quantification difficult and inaccurate. In addition, certain cellular populations can potentially be missed when analyzing sections in this manner (37). Alternatively, a cell surface marker would allow us to quantify the number of goblet cells using an en face view of the stained epithelia. Previous studies have demonstrated that the lectin JAC specifically binds secretory cells, including goblet cells (38–40). Therefore, we examined JAC binding of tracheal explants and, in addition, counterstained with PAS to verify goblet cell identity. Figure 1 demonstrates positive JAC staining (surface brown DAB precipitate) co-localized with PAS positively stained cells (intracellular magenta stain), confirming our ability to use a cell surface marker to identify goblet cells. A quantitative analysis on 10 different sections revealed that 97.1 ± 2% of PAS positively stained cells were also stained with JAC, and none of the JAC-positive cells stained negatively with PAS. We further characterized JAC-lectin binding as a marker for goblet cells by co-localization with an antibody directed against a respiratory gel-forming mucin, MUC5AC (41, 42). As shown in Figures 1C–1E, all JAC-lectin binding cells were also MUC5AC immunopositive (n = 8 from two donors). Based on these results, all experiments described below use JAC staining to identify goblet cells keeping in mind that JAC may not be a perfect marker in all cases.



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  		Figure 1. (A and B) Sections (1 µm) of tracheal human airway epithelia were processed for JAC-lectin binding. Lectin binding was visualized with DAB (brown precipitate over JAC binding cells; plain arrow). Goblet cell identity was confirmed by PAS staining (magenta stain, black arrow). Co-localization demonstrates that JAC binding functions as a marker for goblet cells. (C–E) En face confocal images taken of primary cultures processed for JAC-FITC binding and MUC5AC immunolocalization. C shows MUC5AC-immunopositive cells (red), whereas D shows JAC-lectin binding cells (green). The merged images are shown in E. Yellow label demonstrates co-localization of JAC-lectin binding cells with MUC5AC-expressing cells further confirming the use of JAC-lectin binding as a surface marker for goblet cells (n = 8 from two donors).

 
IL-13 Stimulates Goblet Cell Proliferation on Differentiated Human Airway Epithelia, Whereas IL-9 Has No Effect
Previous work suggests that IL-9 and IL-13 can each stimulate goblet cell hyperplasia or metaplasia in murine and canine airways both in vitro and in vivo (7, 9–11, 43). In addition, studies of IL-9 transgenic mice suggest that this cytokine stimulates goblet cell hyperplasia in the lungs (9). One caveat to these studies is that IL-9 is present throughout development of these transgenic lines. Children are not born with asthma, but rather develop this disease either in early childhood or later on (44), suggesting that the in vivo effects of IL-9 occur on epithelia that is no longer actively developing but rather epithelia that is already fully differentiated. Likewise, transgenic mice suggest that IL-13 plays a role in goblet cell hyperplasia of the airways (10, 45), though the same caveats apply. Therefore, to determine their effects on differentiated human airway epithelia, we asked whether IL-9 or IL-13 treatment of primary cultures of differentiated human airway epithelia could stimulate goblet cell proliferation. Epithelia were treated with IL-9 or IL-13 and then processed for JAC-lectin binding. We found that primary cultures of differentiated human airway epithelia contained on average less than 5% goblet cells (Figure 2). Furthermore, in concordance with the literature, cultures responded to IL-13 treatment with a dramatic increase in the number of JAC-lectin binding goblet cells (Figure 2A, n = 8 from two specimens). In addition, the cellular staining looked qualitatively different compared with baseline and IL-9 treatment. Surprisingly, treatment with IL-9 had no significant effect on the goblet cell population (Figure 2B, n = 15 from five donors). This suggested that once the epithelia were differentiated, IL-9 had no effect. However, because airway epithelia have a low turnover rate (46), we cannot exclude the possibility that IL-9 may have an effect following longer treatment.



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  		Figure 2. Differentiated primary cultures of human airway epithelia were treated in the presence or absence of IL-9 (n = 15 from five donors) or IL-13 (n = 8 from two donors) for up to 14 d. Cultures were stained to identify goblet cells with JAC lectin-FITC. Percent of goblet cells was quantitated as number of JAC-positive cells per total Dapi-stained cells.

 
IL-9 Stimulates Goblet Cell Proliferation in Differentiating Primary Cultures of Human Airway Epithelia
The inability of IL-9 to affect the goblet cell population of differentiated primary cultures of human airway epithelia, together with the literature demonstrating IL-9–mediated goblet cell hyperplasia (11, 47), prompted us to determine whether IL-9 treatment of differentiating cultures would elicit a goblet cell response. Therefore, differentiating primary cultures were treated basolaterally with IL-9 and processed for JAC-lectin binding. The confocal images in Figure 3 demonstrate that IL-9 treatment resulted in an increased number of JAC binding goblet cells compared with untreated controls (compare 3A and 3C to 3B and 3D; C and D are higher magnifications). When quantified (E), IL-9 treatment led to a significant increase in the number of goblet cells (P < 0.001, n = 15 from five donors). These data suggest that IL-9 stimulates goblet cell proliferation in differentiating primary cultures of human airway epithelia. When analyzed by scanning electron microscopy, a prevalence of ciliated cells was evident in control cultures (Figure 3F) and fewer ciliated cells were apparent in the IL-9–treated condition (Figure 3G; the same number of cells are present in each image). Therefore, the decreased number of ciliated cells in the IL-9 condition suggests that a different nonciliated cell type replaced them. Our data suggests this cell type would be goblet cells and that IL-9 treatment results in an increased goblet cell population at the expense of ciliated cells.



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  		Figure 3. Differentiating primary cultures of human airway epithelia were treated in the presence (B and D) or absence (A and C) of IL-9 for 14 d (n = 15 from five donors). Cultures were stained to identify goblet cells with JAC lectin-FITC. A and B are higher magnifications of C and D, respectively. Percent of goblet cells was quantitated as the number of JAC-positive cells per total Dapi-stained cells (E). Scanning electron microscopy of control (F) and IL-9–treated (G) epithelia demonstrate a decreased number of ciliated cells following IL-9 treatment.

 
Interestingly, TEM revealed another quantitative difference between IL-9–treated cultures and controls. For TEM, cultures were fixed with perfluorocarbon, which preserves the mucus layer, allowing us to quantify the amount of mucus present by measuring the height of this layer. More goblet cells and more mucus were present in IL-9–treated epithelia when compared with untreated controls (Figures 4A and 4B) and this difference in mucus was quantitatively significant (P < 0.001, Figure 4, panel D, n = 20 from one donor). The mucus layer has a different density than the sol layer, which can be easily discriminated in the TEM micrographs. Figure 4C is a higher magnification image without the dotted line, demonstrating the visually distinct difference between the two layers. Quantitative morphometric analysis further emphasizes the effect of IL-9 on airway epithelial cell type populations. Figure 4F demonstrates that control cultures contain 81.77 ± 3.95% ciliated cells and 8.65 ± 2.12% goblet cells, whereas cultures treated with IL-9 contain 61.94 ± 7.13% ciliated cells and 36.43 ± 4.77% goblet cells.



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  		Figure 4. TEM micrographs of differentiating primary cultures treated with IL-9 (B) or control (A) (n = 20 from one donor). Dotted line outlines mucus layer. C is a high magnification micrograph lacking the dotted line to demonstrate the distinct visual different between the gel and sol layers. IL-9 treatment stimulated mucus secretion, as evidenced by the increased height of the mucus layer (D). Epithelia were lysed and lysozyme levels measured by ELISA (E, n = 18 from three donors). IL-9 treatment resulted in increased lysozyme levels. Morphometric analysis of TEM micrographs further emphasizes the effect of IL-9 on goblet and ciliated cell populations (F). g and gray bars, goblet cell; c and black bars, ciliated cell.

 
In addition, though lysozyme is traditionally thought of as a serous secretory cell product, it is also a product of goblet cells. For this reason, we quantified the amount of lysozyme in IL-9–treated versus control epithelia by ELISA. Figure 4E demonstrates increased levels of lysozyme present in IL-9–treated epithelia compared with untreated control (P < 0.001, n = 18 from three donors). These data suggest that IL-9 treatment of primary cultures not only stimulates goblet cell proliferation, but as a consequence leads to increased goblet cell secretions. Importantly, increased mucus secretion is a key element in the pathogenesis of airway obstruction in asthma.

IL-9 Receptor Is Expressed at the Apical Surface by Well-Differentiated Human Airway Epithelia
Previous studies have demonstrated the ability of IL-9 to increase the goblet cell population; thus, the lack of an effect on differentiated primary cultures prompted us to ask if differentiated human airway epithelia express the receptor for IL-9. The lack of an effect of IL-9 in our experiment could be explained by an apically polarized receptor in differentiated airway epithelia, where basolaterally instilled IL-9 would not access its receptor. Thus, we immunolocalized the IL-9 receptor on differentiated human airway epithelia. Figure 5 shows that the IL-9 receptor is expressed only at the apical surface of differentiated human airway epithelia (n = 6 from two donors). The lack of staining with an isotype-matched primary antibody verifies the specificity of IL-9R immunolocalization (n = 15 from five donors). Therefore, IL-9 could have no effect on the number of goblet cells in differentiated cultures because the receptor is localized apically. However, when we exposed human airway epithelia to 50 ng/ml IL-9 apically we found no significant increase in JAC-stained cells (5.1 ± 1.0 for IL-9 treatment versus 4.8 ± 0.8 for controls, P = 0.8). These data suggest that aside from a ligand–receptor interaction, an additional signal is required for goblet cell proliferation and/or differentiation. Based on our data demonstrating the effect of IL-9 on differentiating epithelia, we speculated that cell division is that additional signal.



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  		Figure 5. Differentiated primary cultures of airway epithelia were immunostained using an anti–IL-9 receptor antibody (differentiated, n = 6 from two donors). Bound antibody was detected with a FITC-conjugated secondary antibody (green fluorescence). Epithelia were counterstained with ethidium bromide (red fluorescence). Omission of primary antibody in control cultures resulted in an absence of FITC fluorescence (control). Use of an isotype-matched primary antibody (mouse IgG1) and an FITC-conjugated secondary antibody resulted in no FITC signal, demonstrating the specificity of IL-9R staining (n = 15 from five donors). Immunocytochemistry performed on epithelia 4 d after seeding demonstrated IL-9R localization all over the membrane of expressing cells (n = 6 from two donors). Immunolocalization was visualized by confocal microscopy. All panels are confocal images taken in the XZ plane.

 
Moreover, when the IL-13–specific chain of the IL-13 receptor (IL-4R{alpha}) was localized on differentiated primary cultures, it too was found only at the apical membrane (data not shown), though basolateral treatment with IL-13 resulted in a response by the epithelia. Taken together, these data suggest that in addition to IL-9, airway epithelia require cellular proliferation and/or differentiation to respond to this interleukin with goblet cell hyperplasia. In contrast, IL-13 alone is sufficient to elicit goblet cell hyperplasia by airway epithelia.

IL-9 Increases the Number of Goblet Cells following Mechanical Injury of Human Airway Epithelia
Based on the literature demonstrating the effect of IL-9 on goblet cell proliferation, the lack of an effect on differentiated primary cultures prompted us to ask under what conditions IL-9 would regain its effect. One scenario could be in response to injury. Injury repair mechanisms include, among other processes, epithelial proliferation and differentiation to repair the damaged site. Thus, we initially asked: at what point following mechanical injury do the epithelia enter the proliferative phase of repair? Epithelia were injured and allowed to repair for different periods of time. Cultures were then immunostained using an anti-PCNA antibody as a marker of cellular proliferation. We found no difference in the number of PCNA-staining cells between injured and control (intact) epithelia up to 24 h after injury (data not shown). However, at 48 h after injury there was an increase in the number of PCNA-staining cells concentrated at the site of repair (Figure 6, n = 4 from two donors).



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  		Figure 6. Primary cultures of differentiated airway epithelial cells were injured and processed for PCNA immunostaining at 24 and 48 h after injury. PCNA-positive cells were apparent at 48 h and appear concentrated at the repair site.

 
We next asked whether during or after this proliferative phase, epithelia might regain their ability to respond to IL-9. To test this hypothesis, primary cultures of differentiated airway epithelia were mechanically injured and treated basolaterally with or without IL-9 (n = 15 from five donors). Injured cultures were maintained under treatment for 4 d, after which they were processed for JAC binding. Figure 7 demonstrates no difference in JAC binding of intact epithelia treated with IL-9 (Figure 7A) or injured untreated epithelia (Figure 7B). These data suggest that the proportion of goblet cells present in the repaired epithelia is not different from intact control. However, injured epithelia treated with IL-9 demonstrated strong JAC binding in the repaired region (Figure 7C). In fact, a circumference of JAC-positive cells was clearly visualized and corresponded to the injured area. These data suggest that the presence of IL-9 during the repair period stimulated a change in the cellular fate of repairing cells, causing them to adopt the goblet cell lineage.



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  		Figure 7. Differentiated primary cultures of airway epithelial cells were mechanically wounded in the presence (C) or absence (B) of IL-9 (n = 15 from five donors). Treatment was continued until 4 d after injury. Cultures were fixed and analyzed for JAC lectin–FITC binding to identify goblet cells by confocal microscopy. Control differentiated intact epithelial cells treated with IL-9 for 4 d served as control (A). Images were taken en face. IL-9 resulted in an increased number of goblet cells present in the repaired region. Similary, primary cultures were injured and treated with (F) or without (E) IL-13 and processed for JAC-lectin binding as described (n = 6 from two donors). Intact untreated controls were similarly stained. IL-13 resulted in an increased goblet cell population in regions outside the site of injury, a mirror image of the effect of IL-9.

 
To determine the role of IL-13 under the same injury repair condition, primary cultures of airway epithelia were mechanically injured and treated basolaterally with or without IL-13 (n = 6 from two donors). Injured cultures were maintained under treatment for 4 d to allow for repair and then processed for JAC binding as described above. Again, control intact epithelia and untreated wounded epithelia contained similar proportions of goblet cells. Injured epithelia treated with IL-13 responded with a dramatic increase in goblet cells in all regions outside the site of injury. In fact, the circumference corresponding to the injured area was completely devoid of JAC-lectin binding, whereas the areas surrounding it were highly positively stained. Taken together, these data support the hypothesis that cellular proliferation and/or differentiation are required in addition to IL-9 for an airway epithelial response. On the other hand, IL-13 alone is sufficient to elicit an increase in the goblet cell population.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent evidence suggests that IL-9 may play a critical role in the pathophysiology of asthma. Its role in mastocytosis, goblet cell hyperplasia, increased mucus production, and airway hyperresponsiveness—all hallmarks of asthma—has been investigated (1, 2, 7, 9, 11, 12, 14, 15). In addition to these, asthmatic airways display epithelial damage with regions of frank denudation (20–22). The ability of the airway to repair these injured areas in an inflammatory environment, including IL-9, has not been investigated. In the present study, we have demonstrated an altered injury repair program initiated by IL-9 in primary cultures of differentiated human airway epithelia.

The ability of IL-9 to stimulate goblet cell proliferation was found to be dependent on the state of differentiation of the epithelia. IL-9 treatment of actively differentiating primary cultures led to an increase in the goblet cell population, supporting previous studies demonstrating the ability of IL-9 to stimulate goblet cell proliferation (Figure 3) (7, 9, 11). In addition, this increase in goblet cell population appeared to be at the expense of ciliated cells. Moreover, as a consequence of an increased goblet cell population, IL-9–treated epithelia also had elevated mucus and lysozyme production (Figure 4).

As previously documented in the literature, IL-13 profoundly increased the goblet cell population of differentiated airway epithelia (Figure 2); however, we found that IL-9 was unable to stimulate goblet cell proliferation of primary cultures that had already differentiated, suggesting that its effect depends on the differentiation state of the epithelia (Figure 2). One hypothesis to explain this finding is that actively differentiating cells express the IL-9 receptor but lose this expression when fully differentiated, or that both actively and fully differentiated epithelia may express the IL-9 receptor, but once polarized, the receptor becomes apically sorted and thus inaccessible to bind basolaterally applied IL-9. The IL-9R immunolocalized to the apical membrane of differentiated primary cultures. When immunolocalized on cultures 4 d after seeding, the IL-9R was found throughout the plasma membrane of expressing cells demonstrating a lack of receptor polarity at this stage of differentiation. Our present data may, however, explain why apically instilled IL-9 in mice resulted in increased number of goblet cells. To our surprise, when we applied IL-9 apically to the human epithelia, we found no significant increase in goblet cells. Though unexpected, this result suggests that IL-9 binding to its receptor requires an additional signal to activate goblet cell proliferation. We further hypothesize that this additional signal is cellular proliferation and/or differentiation.

Irrespective of why IL-9 cannot stimulate goblet cell proliferation in differentiated primary cultures, it is clear that it does activate a program of goblet cell proliferation in responsive (actively proliferating and differentiating) cells. As mentioned, one circumstance in which differentiated airway epithelia are called upon to dedifferentiate, proliferate, and then re-differentiate is during the process of injury repair. From our data, under this condition epithelia regain their responsiveness to IL-9.

The basic steps taken by damaged airway epithelia to initiate repair have been partially characterized though the molecular mechanisms involved remain elusive. After injury, cells at the wound border take on a fibroblast-like flattened characteristic and begin to migrate into the denuded region (48–51). This temporary wound closure does not restore normal epithelial function to the area; cellular proliferation is thus required to reestablish pre-injury functions (50, 51). Our hypothesis was that during the proliferative phase, repairing epithelia regain their responsiveness to IL-9. Our data support this hypothesis. At 24 h after injury, there is no difference in PCNA or JAC staining between IL-9–treated epithelia and controls. Though transepithelial conductance has returned to baseline at this time (data not shown), cell proliferation has not commenced. These data support the literature suggesting that the initial phase of repair consists of cellular migration and not proliferation. PCNA staining indicates that at 2 d after injury, cells are proliferating. Similarly, an increase in the number of JAC binding cells within the repairing region is also evident, suggesting that the differentiation process has commenced and that it required cell proliferation (Figure 7). In the absence of IL-9, the proportion of goblet cells in the repairing region was no different from that of intact untreated controls. These data suggest that though IL-9–treated airway epithelia initiate normal phases of airway epithelial repair—that is, epithelial migration is followed by epithelial proliferation and differentiation—the fate taken by proliferating cells differs from control in that the majority of cells take on the goblet cell lineage. We suggest that these findings may mimic events in the asthmatic lung.

Interestingly, injured epithelia treated with IL-13 also responded with goblet cell hyperplasia, but in a manner that was the mirror image of that of IL-9–treated cultures. That is, the repaired region was devoid of goblet cells, whereas the surrounding intact areas robustly responded to IL-13 with goblet cell hyperplasia. These data suggest differentiated airway epithelia respond to IL-13 itself, whereas cellular proliferation and/or differentiation is required in addition to IL-9 for the epithelia to respond with goblet cell hyperplasia.

Epithelial injury within the inflammatory environment of the asthmatic lung, which includes IL-9 and IL-13, may alter the composition of repaired and intact epithelia, generating an excessive number of goblet cells. Furthermore, we have demonstrated that in our primary culture model, an increase in goblet cells correlates with an increase in both mucus and lysozyme (Figure 4). Taken together, these data suggest that chronic inflammatory disorders of the airways may involve a cycle of inflammation-induced goblet cell hyperplasia and damage followed by repair mechanisms that generate a further increase in goblet cell population, perpetuating a continued excess of mucus secretion.

Other cytokines (IL-4, IL-5), signaling molecules (PDGF, EGF), and proteases play roles in the asthmatic lung that may potentiate or keep the effects of IL-9 attenuated. Thus, the scheme of inflammation, injury, and goblet cell hyperplasia proposed above need not necessarily go unchecked in vivo. Given the ability of IL-13 to stimulate goblet cell hyperplasia (10, 14), it will be of interest to assess its effects in combination with those of IL-9. The present study serves to highlight altered repair mechanisms in the presence of IL-9 and further emphasizes the role of airway remodeling and its consequences in asthma.


   Acknowledgments
 
The authors thank Phil Karp, Jan Launspach, Parry Weber, Tamara Nesselhauf, Lacey Panko, Jessica Renley, David Welsh, Daniel Vermeer, Theresa Mayhew, and Rosanna Smith for excellent assistance. They thank Michael Welsh, Joel Kline, and Dwight Look for insightful discussion. They appreciate the support of the University of Iowa Central Microscopy Research Facility, the Gene Transfer Morphology Core (supported by the NIDDK), the In Vitro Cell Models Core (supported by the National Heart, Lung and Blood Institute, the Cystic Fibrosis Foundation, and the National Institutes of Diabetes and Digestive and Kidney Diseases), and the Iowa Statewide Organ Procurement. This work was supported by the National Institutes of Health, HL61234 and DK60113.


   Footnotes
 
* These authors contributed equally to the work presented in this article. Back

Received in original form April 25, 2002

Received in final form September 13, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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