Main

A number of studies have demonstrated that T helper 2 cells (Th-2) cytokines play a critical role in allergic asthma and that IL-13 is a central mediator in the regulation of this allergic diathesis (1–4). In asthma, goblet cell hyperplasia (GCH) is a major contributing factor to the disease, resulting in excessive mucus production (5) and mucus plugging in the airway lumen (6). This is one of the likely pathways involved in an increased frequency and duration of infection, decline in lung function, and increased morbidity and mortality (7).

The exact role of IL-13 in pediatric asthma has not been studied widely. Animal studies have confirmed an essential role for IL-13 in driving GCH and other correlates of asthma pathology, including airway hyperresponsiveness, lung eosinophilia, and subepithelial fibrosis and thickened smooth muscle (8–12). Previous studies suggest that airway epithelial cells are direct targets for IL-13 (12,13); however, the use of epithelial cell lines in these studies could not really address the question of how IL-13 could contribute to airway remodeling. It has also been suggested that IL-13 could cause epithelial barrier dysfunction, and more recently, interest has increased in relation to the effects of IL-13 on tight junction formation (14). It is currently unknown as to whether IL-13 impairs epithelial “tight junction” formation during the altered differentiation process of bronchial epithelial cells. In addition, there are no data available looking at the effects of IL-13 on pediatric bronchial epithelial cells (PBECs) and whether it influences the epithelial differentiation process in a similar manner to adults.

Investigating the airway epithelium during childhood is important because many of the questions surrounding the initial pathophysiological mechanisms in asthma and the evolution of airways remodeling can be addressed at this stage (15). This is also the time when interventions are more likely to alter the natural history of the disease.

We have established a 3D well-differentiated model of PBECs, which, we believe, mimics the bronchial epithelium in vivo (16). Under basal unstimulated conditions, we have previously demonstrated that cultures from asthmatic PBEC, PBEC(A), children contained significantly more goblet cells and fewer ciliated cells compared with those from normal PBEC, PBEC(N), children (16).

Our hypothesis in this study was that chronic exposure of PBECs to IL-13 during mucociliary differentiation would result in GCH and a reduction in the number of ciliated cells in both PBEC(A) and PBEC(N). In addition, we hypothesized that IL-13 could be the driving factor behind the morphological changes seen in our previously observed unstimulated asthmatic phenotype (16). We were particularly interested in determining whether PBEC(N) could be “transitioned” to an asthmatic type phenotype after IL-13 stimulation and whether IL-13 stimulation affected transepithelial electrical resistance (TEER) as a measure of epithelial tight junction formation.

METHODS

Children younger than 12 y [mean age, 8.2 y (range, 2–12 y)] attending elective surgical procedures at the Royal Belfast Hospital for Sick Children were recruited. A doctor administered semistructured proforma was used to record the clinical history (online supplemental data available at http://links.lww.com/PDR/A64) (17), and children were classified into two groups: asthmatic (recurrent wheezing within the last year) and normal (controls) who had never wheezed. All asthmatic children were on a regular inhaled corticosteroid and rescue short-acting beta-agonist therapy as required for symptoms, whereas the normal children were not on any treatment. Written informed parental consent and where appropriate child assent was obtained. This study was approved by the Office of the Research Ethics Committees of Northern Ireland (ORECNI).

Isolation of primary PBECs.

Nonbronchoscopic bronchial brushings were obtained from asthmatic children (n = 10) and normal children (n = 10) as previously described (18). Briefly, the cells obtained from the bronchial brushes were removed and centrifuged. The pellet was then seeded into one collagen-coated T10 flask until they were 80% confluent before transferring them to a collagen-coated T75 flask and finally onto collagen-coated transwells at 0.8 × 105 seeding density. PBECs were cultured using methods developed in our laboratory, which resulted in homogenous cultures of basal epithelial cells (16). Bronchial brush washings were analyzed for viruses using a multiviral PCR analysis, and only uncontaminated cultures were used.

Air liquid interface cultures for establishment of well-differentiated mucociliary epithelium.

Air liquid interface (ALI) cultures were grown as previously described (16). All cells from subjects used in this study were grown at ALI at passage 3. The cells were grown in transwells submerged for the first 9–14 d, during which time the culture medium was changed on d 1 and every other day thereafter. Once the cells reach 100% confluence, ALI was created by removing the apical medium and restricting the culture feeding to the basolateral compartment. After ALI creation, the culture medium was changed on alternate days, and the cells were then differentiated at ALI for 28 d to ensure full differentiation as assessed by the presence of beating cilia and mucus on the apical surface of the cultures.

Stimulation of PBECs with IL-13.

After establishment of ALI, cells were fed basolaterally every other day with ALI medium supplemented with recombinant human IL-13 (PeptroTech EC, Ltd., London, United Kingdom) at two different concentrations (2 and 20 ng/mL) in line with previous studies, which have used concentrations ranging from 0.1 to 100 ng/mL to demonstrate any potential dose response (19–24). The apical side was washed weekly with PBS at 37°C.

TEER.

We used TEER as a measure of “tight junction” formation in epithelial cultures (25). TEER was measured on d 7, 14, 21, and 28 of ALI culture using an EVOM meter (World Precision Instruments, FL) as previously described (16). The TEER of six inserts per treatment group was measured at each time point, and the average was calculated. Briefly, the apical surface was washed before reading with warm PBS (500 μL). After aspiration, warm DMEM with no additives was applied apically followed by the TEER being measured. The DMEM was then removed, and the cultures were fed as per normal. PBS washings were stored for further analysis.

Immunocytochemistry (ICC) for goblet and ciliated cell markers.

Cytospin slides were made to allow for the detection of MUC5AC-positive (goblet) and acetylated alpha-tubulin-positive (ciliated) cells from the PBEC cultures as previously described (16). Immediately after trypsinization of the PBECs, cytospin slides were prepared with a cell density of 5.0 × 104 cells per slide. Immunostaining was performed using anti-MUC5AC antibody (1:100; Abcam, Cambridge, United Kingdom) and acetylated alpha tubulin (1:700; Abcam) for goblet and ciliated cells, respectively. The reaction between the antigen and the antibody was detected using a perixodase-conjugated anti-mouse secondary antibody. Negative controls were subjected to routine conditions with the omission of the primary antibody. MUC5AC and acetylated alpha tubulin positive cells were counted at a magnification of ×60 blindly in six fields of vision and are expressed as the % of the total cells counted per slide (500 cells/slide). The result of the mean % of goblet cells and ciliated cells from the three slides per stain per insert is expressed in this article.

Confocal microscopy.

For confocal microscopy, cells were permeabilized using 0.2% Triton X-100 (Sigma Chemical Co.-Aldrich, Dorset, United Kingdom) for 1 h at 37°C followed by three washes in PBS. Cells were then exposed to either a 1:100 dilution of rabbit anti-MUC5AC primary antibody (goblet cells; Santa Cruz Biotechnology, Inc., CA) or a 1:100 dilution of mouse MAb against acetylated alpha tubulin (ciliated cells; Abcam) overnight at 4°C. After washing, cells were exposed to a 1:250 dilution of Alexaflour 561 goat-anti mouse IgG (Invitrogen, Ltd., Paisley, United Kingdom) and a 1:100 dilution of goat anti-rabbit IgG FITC (Invitrogen, Ltd.) at 4°C in the dark for 1 h. After a gentle wash with PBS, the membrane was cut out from the insert using a scalpel and then mounted on a slide using Vectashield with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Peterborough, United Kingdom). Fluorescent images were viewed on a Leica SP5 confocal DMI 6000 inverted microscope equipped with a krypton-argon laser as the source for the ion beam using a ×40 oil immersion (numerical aperture, 1.25). Images were captured and viewed using LAS AF (Leica) acquisition software. Negative controls (omission of primary antibody) were used to test the specificity of the antibodies and the staining protocol.

RNA extraction and real-time PCR for MUC5AC mRNA.

Total RNA was isolated from lysates of PBECs using the RNeasy Mini kit (Qiagen, Crawley, United Kingdom) according to the manufacturer's instructions and quantified on a spectrophotometer. One microgram of RNA from each sample was reverse transcribed in a 20-μL reaction using First Strand cDNA synthesis kit (AMV-Roche) according to recommended conditions. For the PCR reaction, the DNA amplification was carried out using the Fast Start Universal SYBR Green Master (Rox; Roche, United Kingdom). For each PCR reaction, separate PCR Master Mixes were prepared containing GAPDH (Tebu-bio, Peterbrough, United Kingdom), MUC5AC, or IL-13 (R&D Systems, Abingdon, United Kingdom) primers. RT-PCR was performed on the ABI Prism 7000 (Applied Biosystems, United Kingdom) sequence detection system using GAPDH as the internal control. An identical threshold cycle (Ct) was applied for each gene of interest. Relative mRNA expression levels were calculated using the delta Ct method. MUC5AC primers (Invitrogen, Ltd.) were designed according to the following description:

Forward 5′ TCC TTT CGT GTT GTC ACC GA 3′ localization on cDNA: 2874 bp.

Reverse 5′ TCT TGA TGG CCT TGG AGC 3′ localization on cDNA: 2943 bp.

MUC5AC ELISA.

Production of MUC5AC protein in the apical washes from PBECs was measured using an in-house MUC5AC ELISA. As no MUC5AC standard was commercially available, this ELISA produced a semi-quantitative analysis of the production of this protein by comparing cultures treated with IL-13 compared with unstimulated cultures. A 96-well high-binding ELISA plate (Corning Costar) was coated with experimental samples at 40°C overnight. The plate was then washed three times with PBS. Wells were blocked with a solution of 2% BSA in PBS for 1 h at room temperature. After three washes with PBS, samples were exposed to primary antibody diluted to 1:200 in PBS containing 0.05% Tween20 (Sigma Chemical Co.-Aldrich). After incubating for 1 h, the plate was washed three times and labeled with an horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Jackson Laboratories) diluted to 1:10,000 in PBS + 0.05% Tween20 and incubated for 1 h. After the secondary antibody incubation, the plate was washed with PBS, and the color reaction was developed with 100 μL of TMB (Millipore, United Kingdom) per well incubated for 15 min at room temperature in the dark. A 1M H2SO4 solution was then added to halt color development, and absorbance was read at 450 nm.

IL-13 ELISA.

We performed an IL-13 ELISA using supernatants from unstimulated cultures to determine whether the PBECs were producing and secreting IL-13. Apical washings and basolateral aliquots taken on d 7, 14, 21, and 28 of ALI culture were analyzed for IL-13 secretion using an ELISA kit (Pierce Biotechnology) as per manufacturer's instructions.

Statistical analysis.

We expressed results as mean (SD), and skewed variables were log transformed before making statistical comparisons. Comparisons between groups (asthma versus normal) were made using one- and two-way repeated measures ANOVA. The repeated values within individual patients was time (d 7, 14, 21, and 28) for TEER values and IL-13 protein production using ELISA testing and the three concentrations of IL-13 (control, 2 and 20 ng/mL IL-13) for immumocytochemistry counts. We tested to see if the clinical group (asthma versus normal) and stimulation concentration (control, 2 and 20 ng/mL IL-13) interactions were statistically significant indicating that PBEC(A) responded differently to PBEC(N) to increasing stimulation concentration of IL13. We tested for the simple factor effects (clinical group and stimulation concentration) and used Bonferroni corrections for post hoc tests. Analysis of fold change in MUC5AC mRNA expression and MUC5AC relative absorbance were made against unstimulated cultures in PBEC(N) and PBEC(A).We used the t test to compare the unstimulated normal versus asthmatic immunocytochemistry values. A p < 0.05 was taken as statistically significant.

RESULTS

Effects of IL-13 on PBEC(A) and PBEC(N).

Both PBEC(A) and PBEC(N) formed a pseudostratified columnar epithelium. The TEER at each time point measured was between 200 and 800 [Omega] cm2 confirming “tight junction” formation (25). In PBEC(N) and PBEC(A), we detected no overall differences or changes in TEER values over time or with IL-13 stimulation (Fig. 1A and B).

Figure 1
figure 1

TEER of PBEC(N) (A) and PBEC(A) (B) cultures for 28 d and treated with 2 or 20 ng/mL IL-13. Values expressed as mean ± SEM. There was no significant difference seen in the TEER between the treatment groups in both PBEC(N) and PBEC(A). •, control; ▴, 2 ng/mL IL-13; and ▪, 20 ng/mL IL-13.

Full size image

The % goblet cells were lower in PBEC(N) compared with PBEC(A) under unstimulated conditions [PBEC(N): mean, 18.9% (SD, 5) versus PBEC(A): mean, 30.4% (SD, 13.9); p < 0.05]. The % goblet cells increased in both PBEC(N) and PBEC(A) with increasing IL-13 stimulation concentration (p < 0.05; Fig. 2A). Interestingly, PBEC(N) when stimulated with 20 ng/mL IL-13, resulted in a similar % of goblet cells when compared with unstimulated PBEC(A) [PBEC(N) treated with 20 ng/mL IL-13: mean, 33.8% (SD, 7.2) versus unstimulated PBEC(A): mean, 30.4% (SD, 13.9)]. The % goblet cells for PBEC(A) stimulated with 20 ng/mL IL-13 were higher than PBEC(N) after stimulation [PBEC(N): mean, 33.8% (SD, 7.2) versus PBEC(A): mean, 44.7% (SD, 16.4); p < 0.05].

Figure 2
figure 2

The % of goblet (A) and ciliated (B) cells of PBEC(N) (□) and PBEC(A) (⋄) treated with 2 and 20 ng/mL IL-13 on d 28 of ALI culture. Values are expressed as mean ± SEM. The % goblet cells is increased (p < 0.05), and % ciliated cells is decreased (p < 0.001) in both PBEC(N) and PBEC(A) with increasing IL-13 stimulation concentration (Fig. 2A and B, respectively).

Full size image

The % ciliated cells were higher in PBEC(N) compared with PBEC(A) under unstimulated conditions [PBEC(N): mean, 22.7% (SD, 7.6) versus PBEC(A): mean, 16.3% (SD, 4.2); p < 0.05; Fig. 2B]. The % ciliated cells decreased in both PBEC(N) and PBEC(A) with increased IL-13 stimulation concentration [p < 0.001; PBEC(N) with 20 ng/mL IL-13 reduced from mean, 22.7% (SD, 7.6) to mean, 8% (SD, 5.6) and PBEC(A) with 20 ng/mL IL-13 reduced from mean, 16.3 (SD, 4.2) to 8.8% (SD, 7.5; Fig. 2B)].

We confirmed this goblet cell hyperplasia and decreased presence of ciliated cells in PBEC(N) (Fig. 3A-D) and PBEC(A) (Fig. 4A–D) using immunofluorescent staining and confocal microscopy.

Figure 3
figure 3

Immunofluorescence and confocal microscopy of PBEC(N) at d 28 culture of ALI. Goblet cells indicated by green staining, ciliated cells indicated by red staining, and nuclei stained blue with DAPI (×40). Representative image of (A) apical surface and (B) z-stack of an unstimulated PBEC(N) and (C) apical surface and (D) z-stack of a PBEC(N) treated with 20 ng/mL IL-13. Images (C) and (D) show an increased presence of goblet cells and decreased presence of ciliated cells after stimulation with 20 ng/mL IL-13 compared with unstimulated PBEC(N).

Full size image
Figure 4
figure 4

Immunofluorescence and confocal microscopy of PBEC(A) at d 28 culture of ALI. Goblet cell indicated by green staining, ciliated cell indicated by red staining, and nuclei stained blue with DAPI (×40).Representative image of (A) apical surface and (B) z-stack of an unstimulated PBEC(A) and (C) apical surface and (D) z-stack of PBEC(A) treated with 20 ng/mL IL-13. Images (C) and (D) show an increased presence of goblet cells and decreased presence of ciliated cells after stimulation with 20 ng/mL IL-13 compared with unstimulated PBEC(A). Image (E) is the negative control of PBEC(A) without the primary antibody.

Full size image

We found MUC5AC mRNA expression increased for both PBEC(N) and PBEC(A) with the increasing concentration of IL-13 [p < 0.001; PBEC(N) with 20 ng/mL IL-13 mean fold increase 5 (SD, 3.2) versus unstimulated and PBEC(A) with 20 ng/mL IL-13 mean fold increase 9.4 (SD, 11.2) versus unstimulated; Fig. 5].

Figure 5
figure 5

Fold increase in expression of MUC5AC mRNA from PBEC(N) (□) and PBEC(A) (⋄) treated with IL-13 measured on d 28 of ALI culture using real-time PCR. Values are expressed as mean ± SEM. There is significant fold increase in MUC5AC mRNA expression in both PBEC(N) and PBEC(A), p < 0.001.

Full size image

In PBEC(N), in parallel with the increased MUC5AC mRNA expression, the relative absorbance of MUC5AC in culture supernatants was increased in response to IL-13 stimulation [p < 0.001; 20 ng/mL IL-13: mean, 3.0 (SD, 0.5); 2 ng/mL IL-13: mean, 2.8 (SD, 0.6) versus unstimulated: mean, 1.9 (SD, 0.7; Fig. 6)]. However, in PBEC(A), despite increased MUC5AC mRNA expression, there was no significant increase in relative absorbance of MUC5AC [20 ng/mL IL-13: mean, 2.4 (SD, 0.9) and 2 ng/mL IL-13: mean, 2.6 (SD, 0.8) versus unstimulated: mean, 2.5 (SD, 0.6; Fig. 6)]. We found that only a weak to moderate correlation existed between numbers of goblet cells, MUC5AC mRNA, and MUC5AC protein measured by ELISA (data not shown).

Figure 6
figure 6

Relative absorbance of MUC5AC of PBEC(N) (□) and PBEC(A) (⋄) treated with IL-13 measured on d 28 of ALI culture using ELISA. Values are expressed as mean ± SEM. The relative absorbance of MUC5AC was increased in PBEC(N) response to IL-13 stimulation, p < 0.001only.

Full size image

Previous studies have suggested that epithelial cells are capable of producing IL-13 under unstimulated and wound repair conditions (26); however, we could not detect IL-13 (mRNA or protein) in either the apical or basolateral washes from both cultures (normal and asthmatic) under unstimulated conditions.

DISCUSSION

In our previous studies, we found that PBEC(A) exhibited a significant GCH and decreased ciliogenesis compared with PBEC(N) under unstimulated conditions (16). It is commonly known that IL-13 is a key cytokine in the pathogenesis of asthma and has been attributed with driving the aforementioned effects in normal airway epithelium, and therefore, our original hypothesis for this study was that IL-13 would not only be responsible for driving PBEC(N) epithelium to that of an asthmatic phenotype but also drive PBEC(A) toward a more adverse asthmatic phenotype.

IL-13 has been shown to cause disruption of the epithelial barrier in in vitro models (14). However, we did not observe any effect of IL-13 in TEER in either PBEC(A) or PBEC(N), suggesting a lack of effect of IL-13 in the integrity of “tight junctions” in asthmatic and normal epithelium. A limitation of our study of tight junction formation is that we did not perform additional measures of “tight junction” integrity for example using FITC dextran to measure permeability or assessing specific tight junction complexes such as claudins.

We have shown that IL-13 stimulation of PBEC(N) produced a dose-dependent increase in the number of goblet cells. At the stimulatory concentration of 20 ng/mL IL-13, the % of goblet cells was indeed similar to that of PBEC(A) under unstimulated conditions, thereby supporting our hypothesis that IL-13 is the cause of driving a GCH in normal epithelium, leading eventually to the airway remodeling associated with asthmatic epithelium (19). Our results also confirm that in PBEC(N), IL-13 resulted in a significant increase in MUC5AC mRNA production with a subsequent and significant effect on MUC5AC protein expression from goblet cells as seen in our ELISA analysis (1,21,27). A number of different stimuli have been shown to regulate MUC5AC expression and cause an increase in the number of mucus-producing goblet cells but this study as with others suggests that IL-13 is the main cytokine implicated (28–30). We also observed a significant decrease in the number of ciliated cells in cultures treated with 20 ng/mL IL-13, again agreeing with previous findings that in normal epithelium IL-13 can drive this anticiliogenic effect.

This anticiliogenic effect was also seen in asthmatic cultures treated with 20 ng/mL IL-13, showing a significant reduction in the number of ciliated cells which may, in part, explain why asthmatic patients have abnormal mucociliary clearance resulting in the buildup of mucus in the airway lumen. However, an important additional finding from this study is that IL-13 stimulation seems to be able to further enhance GCH in bronchial epithelium from asthmatic children. Our findings have also highlighted that although the unstimulated bronchial epithelial cells from normal and asthmatic children differ morphologically, they respond to IL-13 stimulation in a similar manner. This finding further highlights IL-13 as a key therapeutic target in asthma.

We observed that under unstimulated conditions, PBEC(A) already expressed higher quantities of MUC5AC mRNA and already had significantly more goblet cells compared with PBEC(N) (16). This led us to assess whether IL-13 is in fact the driver behind unstimulated GCH in asthmatic epithelium. In an earlier study, Allahverdian et al. (26) suggested that SV40-transformed normal adult human airway epithelial cells could produce 20 ng/mL IL-13 under unstimulated conditions, with this level rising when the epithelium is wounded. If this finding was indeed the case, we postulated that this may be a causative factor in the GCH and decreased ciliogenesis seen in our unstimulated asthmatic epithelium. To test this, we analyzed our unstimulated asthmatic cultures using real-time PCR and ELISA for IL-13 mRNA production and protein secretion. We found our cultures to produce no detectable quantities of IL-13 either at the level of message or protein, leading us to conclude that IL-13 is not the main cause of the asthmatic phenotype seen in our unstimulated cultures. This would suggest that it is via an IL-13 independent effect that our asthmatic cultures are differentiating to this phenotype, which we are currently investigating. The origins of goblet cells are not well understood, although Clara cells (31) and ciliated cells (20,32) have been implicated as goblet cell progenitors, which might explain the increase in goblet cell numbers in our asthmatic cultures. We cannot completely exclude that the difference in unstimulated asthmatic cultures is because of a “carry-over effect” from IL-13 in vivo. However, given the duration of culture, we believe this is unlikely and suggests that an alternative IL13-independent mechanism, such as the EGF pathway, or an inherent mechanism for GCH in asthma may be present, which we are currently exploring in our laboratory.

In conclusion, in vitro stimulation with IL-13 drives PBEC(N) toward an asthmatic phenotype and worsens the asthmatic phenotype in PBEC(A) with a significant GCH and decrease in ciliated cell number and no obvious effect on tight junction integrity. In addition, IL-13 seemed not to be the causative factor in driving GCH and ciliated cell effects in our unstimulated asthmatic epithelium, suggesting a potential IL-13 independent or inherent mechanism. A further understanding of the origin and processes regulating goblet cell formation in human airway epithelial cells needs to be investigated further as they have important implications for identification of therapeutic targets to treat respiratory disease.