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Published ahead of print on August 14, 2006, doi:10.1164/rccm.200603-392OC
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American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 1110-1118, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200603-392OC

Original Article

Intrinsic Biochemical and Functional Differences in Bronchial Epithelial Cells of Children with Asthma

Anthony Kicic, Erika N. Sutanto, Paul T. Stevens, Darryl A. Knight and Stephen M. Stick

Department of Respiratory Medicine, Princess Margaret Hospital for Children, Perth; School of Pediatrics and Child Health, The University of Western Australia, Nedlands; Telethon Institute for Child Health Research, Subiaco, Western Australia, Australia; James Hogg iCAPTURE Center for Cardiovascular and Pulmonary Research, St. Paul's Hospital, and Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, Canada

Correspondence and requests for reprints should be addressed to Anthony Kicic, Ph.D., Department of Respiratory Medicine, Princess Margaret Hospital for Children, Perth, 6001, Western Australia, Australia. E-mail: anthonyk{at}ichr.uwa.edu.au


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Convincing evidence of epithelial damage and aberrant repair exists in adult asthmatic airways, even in the absence of inflammation. However, comparable studies in children have been limited by access and availability of clinical samples.

Objectives: To determine whether bronchial epithelial cells from children with asthma are inherently distinct from those obtained from children without asthma.

Methods: Epithelial cells were obtained by nonbronchoscopic bronchial brushing of children with mild asthma (n = 7), atopic children without asthma (n = 9), and healthy children (n = 12). Cells were subject to morphologic, biochemical, molecular, and functional assessment. Responses were also compared with commercially available epithelial cultures and the transformed cell line 16HBE140.

Results: All epithelial cells exhibited a "cobblestone" morphology, which was maintained throughout culture and repeated passage. Expression of cytokeratin 19 varied, with disease phenotype being greatest in healthy nonatopics and lowest in asthmatics. In contrast, expression of cytokeratin 5/14 was greatest in asthmatic samples and least in healthy nonatopic samples. Asthmatic epithelial cells also spontaneously produced significantly greater amounts of interleukin (IL)-6, prostaglandin E2, and epidermal growth factor, and equivalent amounts of IL-1beta and soluble intracellular adhesion molecule-1, but significantly lower amounts of transforming growth factor beta1. This profile was maintained through successive passages. Asthmatic epithelial cells also exhibited greater rates of proliferation than nonasthmatic cells.

Conclusions: This study has shown that epithelial cells from children with mild asthma are intrinsically different both biochemically and functionally compared with epithelial cells from children without asthma. Importantly, these differences are maintained over successive passages, suggesting that they are not dependent on an in vivo environment.

Key Words: airway • asthma • bronchial epithelium • cell • nonbronchoscopic brushing



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
The airway epithelium plays a central role in the pathogenesis of asthma. However, these data have almost exclusively been generated from studies using adults, whereas it is likely that dysregulated epithelial repair originates in childhood asthma and is a critical determinant of disease progression into adulthood.

What This Study Adds to the Field
The results obtained provide strong evidence that there are marked inherent differences between healthy and asthmatic bronchial epithelium. In particular, the cytokeratin profiles, the augmented release of antiinflammatory mediators, and the markedly diminished production of TGF-beta1 support the argument that asthmatic epithelial cells function abnormally even in the absence of inflammation.

 
Recent evidence supports the assertion that the epithelium plays an important role in the pathogenesis of asthma (1). Because asthma is common in children and often persists into adulthood, determining the nature and extent of epithelial involvement in the pathophysiologic processes that result in persistence of asthma could help identify new targets for intervention. Indeed, marked irregularities within the epithelial layer have been observed both in mild (2, 3) and in more severe cases of asthma (4, 5). However, these data have been largely generated by studies involving adults, whereas we believe that it is likely that dysregulated epithelial repair originates in childhood asthma and is a critical determinant of disease progression into adulthood. There have been no comprehensive studies using epithelial cells derived from children. This article presents the first detailed examination of the epithelium in childhood asthma. The observations are particularly relevant because epithelial samples were obtained from children with mild asthma and results compared with samples from atopic and nonatopic children without asthma. Previous pediatric studies have, through necessity, studied children with more severe disease and are unlikely to reflect the situation in the majority of children with asthma.

The aim of this study was to test the hypothesis that there are intrinsic differences between the epithelium of children with asthma and healthy epithelium. We believe that biochemical and functional properties of the epithelium associated with asthma result in dysregulated epithelial responses to injury. To address this hypothesis, we obtained translaryngeal, nonbronchoscopic brushings to isolate pure populations of epithelial cells from healthy nonatopic nonasthmatic (HNA), healthy atopic nonasthmatic (HA), and atopic asthmatic (AA) children. This approach has been shown to produce a consistently high yield of cells with high viability when obtained from adults (6, 7). More recently, we (8, 9) and others (10) have adapted and applied this technique to pediatric cohorts and have shown that sufficient cells can be routinely harvested for biochemical, protein, genomic, and functional assays. Successfully established cultures were then investigated in detail for the expression of epithelial lineage markers and assessed for any morphologic variation over repeated passage. In addition, constitutively expressed pro- and antiinflammatory mediators were investigated and their production was also monitored over subsequent passage in vitro. Proliferation assays were then performed to determine and compare the rates of growth between the various phenotypes. Finally, the above outcomes were then compared with commercially derived adult and transformed bronchial epithelial cell lines. Some results generated from these studies have been previously reported in the form of abstracts (11, 12).


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Please refer to the online supplement for full details of methods.

Patients and Cell Isolation
Bronchial brushings were obtained from 17 male and 11 age-matched female children undergoing elective surgery for nonrespiratory conditions. Asthma was defined as physician-diagnosed asthma plus wheeze documented by a physician in the past 12 mo. A positive response to relevant questions on the ISAAC (International Study of Asthma and Allergies in Childhood) and American Thoracic Society respiratory questionnaires was used to corroborate the diagnostic label and negative responses used to validate absence of respiratory symptoms reported by parents or subject (13, 14). Atopic status was determined by a positive RAST or skin prick test to common allergens. All AA children had mild disease, such that none were treated with inhaled or oral glucocorticosteroids. Subject demographic data are provided in Table 1. The study was approved by the Princess Margaret Hospital for Children's Human Ethics Committee.


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  		TABLE 1. PATIENT DEMOGRAPHICS

 
Established cultures were classified as healthy nonatopic child-derived human bronchial epithelium (hnaCHBE), atopic nonasthmatic child-derived human bronchial epithelium (haCHBE), or atopic asthmatic child-derived human bronchial epithelium (aaCHBE). Normal human bronchial epithelium (NHBE; Cambrex, Baltimore, MD) and the 16HBE140 (16HBE) transformed epithelial cell line (Dr. Dieter Gruenet, University of Vermont, Burlington, VT) were also used.

Western Blot
Total protein was determined from cells using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). A total of 50 µg of protein was electrophoresed on a 12% (wt/vol) sodium dodecyl sulfate–polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (100 mA; 1 h, 4°C). Protein expression was visualized using the enhanced chemiluminescence (ECL-Plus) Western Blotting Detection System (Amersham Biosciences, Arlington Heights, IL).

Immunocytochemistry
Epithelial cells were cytospun onto glass microscope slides, fixed in 4% paraformaldehyde, washed, and stained for the epithelial markers cytokeratin 5/14 (CK-5/14; Dako Corp., Carpinteria, CA) and CK-19 (Sigma, St. Louis, MO). Cells were also stained with vimentin, CD1a, von Willebrand factor (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and CD68 (Dako Corp.) to identify any mesenchymal cells, dendritic cells, endothelial cells, or macrophages, respectively. Specific antibody staining was then visualized using a fluorescent microscope (Leica Microsystems Pty Ltd, Wetzlar, Germany).

Reverse Transcriptase–Polymerase Chain Reaction and Quantitative Polymerase Chain Reaction
Cellular RNA was extracted using the RNeasy Mini Extraction Kit (Qiagen, Hilden, Germany). Primers used are provided in Table 2. Gene expression was analyzed by two-step reverse transcriptase–polymerase chain (RT-PCR) reactions. cDNA was synthesized using hexanucleotide primers and Multiscribe Reverse Transcriptase (Applied Biosystems, Foster City, CA). Refer to the online supplement for PCR and quantitative PCR (qPCR) conditions.


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  		TABLE 2. OLIGONUCLEOTIDE PRIMERS

 
Assessment of Proliferation
Expression of proliferating cell nuclear antigen (PCNA) was assessed using qPCR, whereas proliferation was assessed using a 3-[4,5-dimethylthiazol-2yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium inner salt (MTS) assay (Promega, Madison, WI) (15). These results were validated by performing cell counts and calculating doubling rates as described previously (16).

ELISA
Cytokine concentrations were measured in the supernatants using commercial ELISA kits. Proteins measured included interleukin (IL)-6 and prostaglandin E2 (PGE2; R&D Systems, Minneapolis, MN), soluble intracellular adhesion molecule-1 (sICAM-1), epidermal growth factor (EGF), transforming growth factor beta1 (TGF-beta1), IL-8, and IL-1beta (Biosource, Camarillo, CA) as per the manufacturer's instructions. IL-10, IL-12p70, and tumor necrosis factor {alpha} (TNF-{alpha}) were measured using a multiplexed cytometric bead assay system (BD Biosciences, Bedford, MA). Results were normalized to cell number.

Statistics
Before statistical evaluation, all results were tested for population normality and homogeneity of variance, and where applicable, a Student t test was performed. One-way analysis of variance and Dunnett's test were performed on all multiple comparisons. Experiments were performed at least in triplicate and using four to six patients of each cohort per experiment. All values presented are means ± SD. All p values less than 0.05 were considered to be significant.


   RESULTS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Culture Establishment and Epithelial Lineage Confirmation
Mean yields of epithelial cells obtained in this study after two passes with the brush were 3.93 ± 1.19 x 106 for hnaCHBE, and did not significantly differ from haCHBE (3.5 ± 1.56 x 106, p = 0.948) or aaCHBE yields (2.55 ± 1.15 x 106, p = 0.424). The overall cell culture success rate was more than 85%. Successfully established cultures used in this study reached confluence after 10 to 14 d in culture and demonstrated the typical polygonal cobblestone pattern characteristic of epithelial cells, which was maintained up to passage 5 (Figure 1). There were no gross morphologic differences between hnaCHBE and aaCHBE cells. Due to the nature of the procedure, samples were interrogated to confirm that they were not contaminated by other cell types, including the following: mesenchymal cells (vimentin; Figures 2B and 2C), macrophages (CD68; Figures 2E and 2F), dendritic cells (CD1a; Figures 2H and 2I), and endothelial cells (von Willebrand factor; Figures 2K and 2L). All cultures stained positive for CK-19 (Figures 2M and 2N), suggesting epithelial cell lineage. RT-PCR further confirmed the expression of CK-19 gene by the cells (Figure 2O, lane 8) and the absence of expression of mesenchymal (vimentin; Figure 2O, lane 2), dendritic (CD1a; Figure 2O, lane 4), and endothelial (von Willebrand factor; Figure 2O, lane 6) specific markers.


Figure 1
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  		Figure 1. Phase contract micrographs showing no morphologic variation in bronchial epithelial cells derived from healthy nonatopic (HNA; A–E) and atopic asthmatic (AA; F–J) patients over their proliferative life of five passages (p1–p5) or typically >= 60 d duration in vitro. All cells grown exhibited a "cobblestone" cell morphology. The overall initial culture success rates for bronchial brushings from each phenotype were 78% for HNA, 100% for healthy atopic, and 86% for AA.

 

Figure 2
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  		Figure 2. (A–N) Characterization of established epithelial cell cultures. Cytospins from a representative HNA (passage [p] 2) were incubated with primary antibodies specific for epithelial (cytokeratin 19 [CK-19]), mesenchymal (vimentin), macrophage (CD68), dendritic (CD1a), and endothelial lineages (von Willebrand factor) for 24 h at 4°C, followed by fluorescently conjugated secondary antibodies for a similar period. Established cultures stained positively for CK-19, and CK-5/14 but did not stain for vimentin, CD68, CD1a, or von Willebrand factor. Original magnification, 400x. (O) Reverse transcriptase–polymerase chain reaction confirming epithelial lineage of bronchial cultures. RNA was isolated from cells at each passage (p1–p3) and epithelial lineage confirmed using cell lineage–specific primers for the above-mentioned potentially contaminating cell types. Cells were found not to express mesenchymal (lane 2), dendritic (lane 4), and endothelial (lane 6)-specific lineage markers, but did express the epithelial lineage–specific marker CK-19 (lane 8). Note: Lane 1: positive control for mesenchymal lineage, Hela cells; lane 3: positive control for dendritic cells, primary cultured human dendritic cells; lane 5: positive control for endothelial lineage, NIH 3T3 fibroblasts; lane 7: positive control for epithelial lineage, ME180 cells. beta-Actin expression confirmed equal loading of samples. Images provided are representative of an HNA sample from p1–p3. Similar staining patterns are observed for healthy atopic (HA) and AA samples, although the intensity of CK gene and protein expression varies.

 
Further investigation revealed distinct variations in cytokeratin expression patterns both between disease phenotypes as well as over continual passage. In cytospins collected at the time of surgery, CK-19 protein expression was observed in all samples, although a much stronger intensity of staining was observed in hnaCHBE than in aaCHBE samples (Figure 3A). CK-19 protein expression continued to be strongly expressed in hnaCHBE samples at early passage and was maintained for their duration in culture (Figure 3A). A similar expression profile was observed in haCHBE cells (data not shown). However, immunostaining of CK-19 protein expression was observed to be markedly lower in aaCHBE samples of freshly isolated cells and at early passage. The rank order of CK-19 protein expression of HNA > HA > AA was confirmed by Western blot analysis (Figure 3B). In contrast, expression of the low-molecular-weight CK-5/14 in cells isolated at the time of surgery was virtually absent in primary isolates of hnaCHBE and was only minimally detectable in passaged cultures (Figures 3A and 3B). However, a higher expression and intensity pattern of CK-5/14 was observed in aaCHBE cells at initial culture and throughout subsequent serial passage (Figure 3A). Despite the variation observed between the phenotypes, cytokeratin expression levels were found to be consistent among patients of the same phenotype (Figure 3C).


Figure 3
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  		Figure 3. (A) Cytokeratin expression profiles of epithelial cells over passage. Serial cytospins of cells from p1–p5 were collected and incubated with primary antibodies specific for CK-19 and CK-5/14 (1:250) for 24 h at 4°C, followed by fluorescently conjugated secondary antibodies for a similar period. Cytokeratin expression was then compared with ex vivo cytospins collected from the same patient at the time of initial processing as well as between phenotypes. Epithelial cells from HA subjects stained strongly for CK-19 early in passage, whereas cells derived from AA only stained for CK-19 much later in passage. Conversely, CK-5 was expressed primarily in AA cells and was maintained through subsequent passages. Original magnification, 400x; CK-19 and CK-5/14 expression profiles were determined in five separate patients from each cohort and a typical profile from one patient from each is represented. (B) Validation of immunocytochemical findings. Cells were collected at p2 and expression of CK-19 and CK-5/14 evaluated by Western blot. (C) Cytokeratin expression profiles between phenotypes of the same passage. p2 cultures of each phenotype were assessed for both CK-19 and CK-5/14 expression via Western blot analysis as described above. Results revealed and confirmed the expression levels of both cytokeratins between subjects of the same phenotype.

 
Comparison of Asthmatic and Normal Epithelial Cell Function
Production of proinflammatory mediators from aaCHBE and hnaCHBE cells is similar.
The constitutive production of proinflammatory cytokines, including IL-1beta, sICAM-1, and IL-8, is shown in Figure 4. No significant difference was observed in the amount of IL-1beta, sICAM-1, and IL-8 produced by hnaCHBE, haCHBE or aaCHBE cells. Levels of IL-12p70 and TNF-{alpha} were below limits of detection regardless of disease phenotype.


Figure 4
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  		Figure 4. Biochemical analysis of established bronchial epithelial cells. Healthy nonatopic human bronchial epithelium (hnaCHBE), healthy atopic human bronchial epithelium (haCHBE), and atopic asthmatic human bronchial epithelium (aaCHBE) cell cultures were established, grown to confluence, and supernatants taken after 48 h incubation for cytokine production assessment. Mediator production was assessed from 4–6 separate cultures of each cohort by ELISA. Cytokine expression was normalized against control media and cell number and expressed as pg/ml/106 cells. Results showed that aaCHBE cells produce significantly more of the inflammatory cytokines interleukin (IL)-6 (p < 0.0001) and prostaglandin E2 (PGE2; p < 0.0001). No difference was detected between phenotypes in the production of soluble intracellular adhesion molecule (sICAM)-1 (p = 0.995) and IL-1beta (p = 0.425) or IL-8 (p = 0.232). The aaCHBE cells also were found to produce significantly greater amounts of epidermal growth factor (EGF; p = 0.005) but significantly lower levels of transforming growth factor (TGF)-beta1 (p < 0.0001).

 
Epithelial cells from AA subjects produce higher levels of antiinflammatory mediators.
We also measured the production of two antiinflammatory mediators, namely IL-6 and PGE2, in primary cultures of epithelial cells (Figure 4). We observed that aaCHBE cells constitutively produced 30-fold more IL-6 than hnaCHBE cells (3,326.6 ± 474.3 pg/ml vs. 130.8.1 ± 61.2 pg/ml, p < 0.0001), whereas the amount of IL-6 produced by haCHBE cells (1,416.4 ± 164 pg/ml) was 10-fold higher than hnaCHBE cells (p < 0.0001; Figure 4). PGE2 production was also significantly elevated in both aaCHBE and haCHBE cells when compared with hnaCHBE (527.5 ± 22 pg/ml, 362.6 ± 22.3 vs. 226.9 ± 54 pg/ml, p < 0.001; Figure 4). We also attempted to measure IL-10 production but the levels were below limits of detection.

Differential production of growth factors by aaCHBE cells compared with hnaCHBE cells.
The production of a number of growth factors that are associated with asthma, including TGF-beta1 and EGF, was also quantified. Primary cultures of aaCHBE cells produced significantly less TGF-beta1 than either haCHBE or hnaCHBE cells (3,052.9 ± 293.4, 8,243.9 ± 264.2, and 11,695.4 ± 1,289.2 pg/ml, respectively; p < 0.0001; Figure 4). In contrast, both aaCHBE and haCHBE cells produced significantly greater amounts of EGF (22,623.1 ± 374.1 and 20,430 ± 1,958.1 pg/ml) than did hnaCHBE cells (11,040 ± 2,610.8 pg/ml, p = 0.005l; Figure 4).

aaCHBE Cells Proliferate Faster than hnaCHBE Cells
We initially assessed the proliferative capacity of bronchial epithelial cells from each cohort by examining the gene expression of PCNA, using Taqman qPCR analysis. Expression of PCNA was increased 42- and 18-fold in aaCHBE (p < 0.05) and haCHBE cells (p < 0.05) compared with hnaCHBE cells (Figure 5A). To determine if this increase in proliferative gene expression correlated with a functional change in the growth characteristics of bronchial epithelial cells from each cohort, we investigated the proliferative capacity of primary cultures of hnaCHBE, haCHBE and aaCHBE cells, as well as of the transformed cell line 16HBE. As shown in Figure 5A, in the absence of exogenous stimuli, both aaCHBE and haCHBE cells proliferated faster than hnaCHBE cells (p < 0.001; Figure 5A). Direct cells counts were also used to determine proliferation rates and doubling times (Figure 5B). In these experiments, aaCHBE and haCHBE cells exhibited similar doubling rates (25.4 ± 2.9 and 23.7 ± 4.2 h, respectively). This was significantly quicker than hnaCHBE cells at 41.6 ± 6.6 h (p < 0.001), though somewhat slower than that of the calculated doubling rate of 16HBE cells (17.9 ± 1.0 h).


Figure 5
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  		Figure 5. Assessment of epithelial proliferative capacity in hnaCHBE, haCHBE, and aaCHBE cells. (A) Expression of a proliferative marker, proliferating cell nuclear antigen (PCNA), was measured by quantitative polymerase chain reaction in freshly isolated cells from 7 HNA, 6 HA, and 6 AA children. Expression of PCNA was significantly up-regulated in both HA and AA children. (B) Growth kinetics of hnaCHBE, haCHBE, and aaCHBE cells from pediatric subjects. Cells were collected from the indicated cohort subjects and cultures established and proliferation assessed via a 3-[4,5-dimethylthiazol-2yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]2H-tetrazolium inner salt (MTS) assay. The aaCHBE cells exhibited a greater rate of proliferation when compared with hnaCHBE cells. Similarly, haCHBE cells also showed a higher rate of proliferation compared with hnaCHBE, but significance was not evident at times up to 72 h (p < 0.05). (C) Direct cell counts were performed in parallel at corresponding time points, and results validated initial findings using the MTS assay. Cell counts were then used to calculate the mean doubling times of each subgroup: aaCHBE cells, 25.4 ± 2.9 h; haCHBE cells, 23.7 ± 4.2 h; and hnaCHBE cells, 41.6 ± 6.6 h. *aaCHBE significantly different to hnaCHBE; #haCHBE significantly different to hnaCHBE.

 
Measurement of Mediator Levels over Passage
To assess whether initially determined cytokine profiles were maintained through serial culture, we measured the production of IL-6 and PGE2 over repeated passages. Generally, the differences in cytokine production observed initially between the three phenotypes were maintained in vitro (Figure 6 and Figure E1 of the online supplement). Epithelial cells derived from all phenotypes continued to produce similar levels of IL-1beta, sICAM-1, and IL-8 (Figure 6 and Figure E1). Similarly, IL-6 production was greatest in aaCHBE cells followed by haCHBE and hnaCHBE cells (Figure 6). Although absolute amounts varied, the magnitudes of the differences observed between the phenotypes were maintained for at least three passages before levels significantly decreased (p < 0.001). Similar findings were seen for both TGF-beta1 and EGF. In contrast, PGE2 production varied with repeated passages. Although initial cultures of aaCHBE cells produced the greatest amount of PGE2, the levels significantly decreased with passage. In contrast, PGE2 production by both haCHBE and hnaCHBE cells remained consistent over three subsequent passages before diminishing (Figure 6).


Figure 6
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  		Figure 6. Cytokine expression profiles of the various phenotypes over passage. The hnaCHBE, haCHBE, and aaCHBE cell cultures were established, grown to confluence over subsequent passages, and supernatants taken after 48 h incubation and for cytokine production assessment. Mediators involved in inflammatory (IL-1beta, ICAM-1, IL-8), antiinflammatory/proallergic (IL-6, PGE2), and repair processes (EGF, TGF-beta1) were chosen, and their production measured at each passage. Mediator production was assessed via ELISA and normalized against the supplemented media; results were expressed as pg/ml/106 cells and are the mean of three replicates averaged among at least three separate patients. Although absolute values varied, the cytokine production profiles for each phenotype initially observed with primary cultures were maintained until at least p3.

 
Comparison of Pediatric, Adult, and Transformed Epithelial Cell Lines
To examine whether established hnaCHBE and aaCHBE cells exhibited any differences compared with normal adult or immortalized cells, we measured IL-6 production over passage. Initial cultures of adult NHBE cells produced more than sevenfold more IL-6 than did hnaCHBE cells and over twofold more than did aaCHBE cells (Figure 7). Although absolute values of IL-6 production increased over passage, the difference between adult and pediatric epithelial cells was retained over repeated passages. In addition, 16HBE cells produced little IL-6 in comparison with either of the primary bronchial cells, and this difference was also maintained through repeated passages (Figure 7).


Figure 7
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  		Figure 7. Comparison of IL-6 cytokine production of pediatric epithelial cells with adult epithelial cells and an immortalized human bronchial epithelial cell line. The hnaCHBE, aaCHBE, and NHBE primary cells were established and grown over five passages. At each passage, cells were grown to confluence and supernatants collected after 48 h. IL-6 production was measured from supernatants collected from each passage and compared with that produced by 16HBE cells cultured in vitro over five serial passages under similar conditions. Data are expressed as pg/ml/106 cells and are the mean of three replicates averaged among at least three separate patients. Primary cultures produce markedly greater amounts of IL-6 compared with cell lines, and adult epithelial cells produce greater amounts than pediatric cells.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study has shown that, despite exhibiting similar morphologic characteristics compared with those of children without asthma, epithelial cells from children with asthma exhibit marked intrinsic biochemical and functional differences. Asthmatic epithelial cells appear to be less differentiated in that they have substantially greater expression of low-molecular-weight CK-5/14 and very low expression of the high-molecular-weight cytokeratin, CK-19. In addition, aaCHBE cells constitutively produced substantially greater amounts of IL-6, PGE2, and EGF, similar levels of the proinflammatory mediators IL-1beta, sICAM-1, and IL-8, but lower levels of TGF-beta1 in culture. The aaCHBE cells also exhibited faster doubling times than hnaCHBE cells. Importantly, these differences are maintained despite successive passages, indicating that they are not dependent on an in vivo environment. These data support the hypothesis that, even in mild asthma where there is an absence of overt inflammation, the epithelium is intrinsically abnormal.

Attempts at understanding asthma, particularly in children, have been severely hampered by the difficulty in obtaining relevant target organ tissue. As a result, most research has relied heavily on commercially produced cells or transformed cell lines. Primary cultured cells should be used for in vitro analyses because they are most similar to cells in vivo. We and others have demonstrated the utility of bronchial brushings from children for the isolation, characterization, and successful culture of primary epithelial cells (8–10). The procedure, which involves the insertion of a soft cytology brush into the airway until the carina is reached, allows for maximal cell retrieval with minimal side effects to the patient (9). Samples were collected after general anesthesia using sevofluorane for induction and intravenous propofol for maintenance. The effects of these agents on airway epithelial gene expression are unknown. However, sample collection was completed within 3 min of induction of anesthesia, regardless of underlying disease, and the phenotypic differences were maintained through serial passages. We believe, therefore, that it is unlikely that the sampling technique could explain the differences observed between cells from children with and without asthma.

The epithelial lineage of all established cultures was confirmed using immunohistochemistry, Western blot analysis, and semiquantitative RT-PCR. All cultures expressed the high-molecular-weight epithelial marker CK-19, which generally identifies differentiated epithelial cells (17, 18). However, the level of expression of CK-19 differed significantly between patient cohorts, with the greatest expression in cells isolated from healthy children. In contrast, expression was lower in cells taken from atopic children and virtually absent in cells from children with asthma. However, we observed the opposite expression profile for the low-molecular-weight CK-5/14, which is generally accepted as a marker of undifferentiated basal or progenitor cells (19–23). Expression of this marker was greatest in children with asthma and virtually absent in cells from healthy children. The fact that these differences were seen in cells extracted at the time of surgery and subsequent cultures, despite a standardized sampling and culture methodology, indicates that epithelial cells from even children with mild asthma are in a more undifferentiated state than cells from healthy children and supports the theory that asthmatic epithelium is unable to undergo normal mechanisms of repair and differentiation.

The next component of this study was to evaluate the profile of cytokines and mediators released by asthmatic, atopic, and healthy epithelial cells. Our data show that, despite no difference in the constitutive production of proinflammatory mediators, epithelial cells derived from children with asthma differ substantially in their release of antiinflammatory cytokines and growth factors. First, we found epithelial cells from all three cohorts produced similar levels of the proinflammatory cytokines IL-1beta, sICAM-1, and IL-8. These findings are at odds with the results of others that have demonstrated an increased amount of ICAM-1 expression in asthmatic bronchial epithelial cells (24, 25). Similarly, increased IL-1 production by bronchial epithelium has been reported in asymptomatic and symptomatic individuals with asthma (26, 27), and IL-8 has been detected in the bronchial tissue of atopic subjects with severe asthma but not in samples from healthy nonatopic subjects (28). There are a number of possible explanations to account for these discrepancies. First, the subjects with asthma recruited for this study had very mild disease. Others have shown that ICAM-1 expression correlates to the severity of the disease (24) and that significantly elevated IL-8 production was not detected in atopic subjects with mild asthma (28–30). Second, in the current study, epithelial cells were derived from children. Most investigations reporting ICAM-1, IL-1, and IL-8 expression levels have been obtained using cells from adults. Additional detailed investigation into this area is necessary to better understand the significance of our observations in children.

We also examined the production of antiinflammatory mediators and found that asthmatic epithelial cells constitutively produce significantly higher amounts of IL-6 and PGE2, compared with cells isolated from atopic and healthy subjects. Our data agree with other studies that have reported the ability of bronchial epithelium to synthesize and release augmented levels of IL-6 and other related cytokines (18, 31, 32). The observations made in this investigation suggest that, despite the lack of obvious inflammatory stimuli, asthmatic epithelial cells are configured to release higher levels of antiinflammatory mediators and that the asthmatic epithelium could be locked in a cycle of reparative responses that are inappropriate for the degree of inflammation or damage.

To investigate this further, we examined the production of the profibrotic growth factor TGF-beta1 and found that cultures established from children with asthma produced significantly lower levels compared with cultures from atopic or healthy children. The blunted production of TGF-beta1 by asthmatic epithelium may indicate diminished potential to differentiate (33) and/or migrate (34, 35). In conjunction with this, asthmatic epithelial cells produced significantly higher levels of EGF when compared with their healthy counterparts. These findings agree with others who have observed that EGF expression is increased in the bronchial epithelium, bronchial glands (36, 37), smooth muscle (36), and submucosa (38) of patients with asthma. Because EGF was significantly elevated in asthmatic cells and this growth factor is known to mediate epithelial mitogenesis, we examined whether asthmatic epithelial cells proliferate faster than nonasthmatic cells. Expression of PCNA was markedly up-regulated in asthmatic cultures. In addition, even in the absence of any exogenously added stimuli, asthmatic epithelial cells proliferated faster than nonasthmatic cells. Asthmatic epithelial cells had a doubling rate of approximately 25 h compared with 40 h for healthy cells, resulting in almost twice the number of cells produced over an equivalent period of time. These data taken in conjunction with the diminished production of TGF-beta1 suggest that, despite the repair processes being initiated, possibly through EGF-stimulated proliferation, abnormal differentiation may also occur. Our findings showing that asthmatic epithelium expresses low-molecular-weight cytokeratins are consistent with this hypothesis.

The high proliferative capacity of asthmatic epithelial cells could be atopy related because all our subjects with asthma were atopic and these cells exhibited a similar proliferative capacity as the atopic nonasthmatic cells, but this is more likely associated with the high EGF produced by these and healthy atopic cells. In addition, the possible autocrine action of the high levels of IL-6 and PGE2 produced by aaCHBE cells, which others have found to promote basal cell proliferation in some lung cancers, may also play a role (39–41). Interestingly, Fedorov and colleagues (42) reported a decreased expression of a marker of cell proliferation, namely Ki-67, in the epithelium of children with asthma at biopsy and suggested that the lack of proliferation in these children was due to an insufficient production of a mitogenic stimulus. In contrast, we have performed real-time gene expression analysis on freshly isolated epithelial cells and shown a substantial increase (42-fold) in the expression of a validated marker of proliferation, PCNA, in asthmatic epithelial cells. The reason for this discrepancy between these two studies remains unknown; however, we believe that issues such as disease severity (moderate/severe vs. mild), may play a role. This is an area of interest for our research group. Another potential confounder may be the use of asthma medication in the study by Fedorov and colleagues. In our study, none of the subjects were receiving corticosteroids, which may influence epithelial proliferation.

Because it is plausible that dysregulated epithelial repair in childhood asthma contributes to the persistence of asthma into adulthood and to nonreversible or difficult-to-reverse structural changes, the specific investigation of the cellular mechanisms involved in asthma using cells derived from children is highly relevant. To reinforce this point, we compared cytokine production between commercially obtained, adult-derived primary bronchial epithelial cells (NHBE cells) and an immortalized bronchial epithelial cell line (16HBE) with that of our pediatric cultures. We initially chose to measure IL-6 because its production was the greatest between our pediatric subjects with asthma and healthy nonatopic subjects. Our findings indicate that NHBE cells produce significantly greater amounts of IL-6 compared with either nonasthmatic or asthmatic cells from pediatric donors. In addition, the immortalized cell line was found to produce little or no IL-6 in comparison. These simple observations highlight the importance of using primary cells in studying the role of the epithelium in asthma and highlight the difficulty in interpreting and in the relevance of observations made in adult samples with regard to asthma in childhood.

In conclusion, we have evaluated in detail the biochemical and functional characteristics of bronchial epithelial cell cultures obtained from HNA, HA, and AA children. The results obtained provide strong evidence that there are marked inherent differences between healthy and asthmatic bronchial epithelium in childhood. In particular, the cytokeratin profile, the augmented release of antiinflammatory mediators, and the markedly diminished production of TGF-beta1 support the argument that asthmatic epithelial cells function abnormally even in the absence of inflammation. That these differences are maintained in culture through repeated passages suggests that the differences are not dependent on an in vivo environment. However, it must be highlighted that these phenotypic differences were obtained using submerged monolayer cultures. The characteristics and responses of these cells grown in air–liquid interface cultures could be qualitatively different, and their functional significance needs further examination. Our studies further highlight that results obtained from adult cells and cell lines might not always be relevant to pediatric airway disease.


   Acknowledgments
 
The authors thank Drs. Amanda Griffiths and Rus Awang for performing the bronchial brushings and Angela Fonceca and Dr. Siobhan Brennan for technical assistance. They also thank Dr. Paul McNamara and Professor Tony Bai for their critical comments on this manuscript.


   FOOTNOTES
 
Supported by a grant from the National Health and Medical Research Council of Australia (303145), the Asthma Foundation of Western Australia, and a Child Health Research Foundation fellowship to A.K. S.M.S. is a National Health and Medical Research Council of Australia Practitioner Fellow.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200603-392OC on August 14, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form March 17, 2006; accepted in final form August 4, 2006


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 AT A GLANCE COMMENTARY
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 RESULTS
 DISCUSSION
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