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
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 bron-
chial 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 as-
sessment. Responses were also compared with commercially avail-
able 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
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 in-
terleukin (IL)-6, prostaglandin E2, and epidermal growth factor,
and equivalent amounts of IL-1? and soluble intracellular adhesion
factor ?1. This profile was maintained through successive passages.
Asthmatic epithelial cells also exhibited greater rates of prolifera-
tion 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
Keywords: airway; asthma; bronchial epithelium; cell; nonbroncho-
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
(Received in original form March 17, 2006; accepted in final form August 4, 2006)
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.
Correspondence and requests for reprints should be addressed to Anthony Kicic,
Ph.D., Department of Respiratory Medicine, Princess Margaret Hospital for Chil-
dren, Perth,6001,WesternAustralia, Australia.E-mail: firstname.lastname@example.org
This article has an online supplement, which is accessible from this issue’s table
of contents at www.atsjournals.org
Am J Respir Crit Care Med
Originally Published in Press as DOI: 10.1164/rccm.200603-392OC on August 14, 2006
Internet address: www.atsjournals.org
Vol 174. pp 1110–1118, 2006
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
sis of asthma. However, these data have almost exclusively
that dysregulated epithelial repair originates in childhood
asthma and is a critical determinant of disease progression
What This Study Adds to the Field
The results obtained provide strong evidence that there are
marked inherent differences between healthy and asth-
matic bronchial epithelium. In particular, the cytokeratin
profiles, the augmented release of antiinflammatory media-
tors, and the markedly diminished production of TGF-?1
support the argument that asthmatic epithelial cells func-
tion abnormally even in the absence of inflammation.
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 ob-
served 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 adult-
cells derivedfrom children. This articlepresentsthe firstdetailed
tions are particularly relevant because epithelial samples were
obtained from children with mild asthma and results compared
Previous pediatric studies have, through necessity, studied chil-
dren 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
asthmaand healthyepithelium. We believethat 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
matic (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,
to pediatric cohorts and have shown that sufficient cells can
Kicic, Sutanto, Stevens, et al.: Asthmatic Epithelium Abnormalities1111
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 re-
peated passage. In addition, constitutively expressed pro- and
antiinflammatory mediators were investigated and their produc-
tion was also monitored over subsequent passage in vitro. Prolif-
eration 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 re-
sultsgeneratedfromthesestudies havebeen previouslyreported
in the form of abstracts (11, 12).
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 condi-
tions. 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
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 Chil-
dren’s Human Ethics Committee.
Established cultures were classified as healthy nonatopic child-
derived human bronchial epithelium (hnaCHBE), atopic nonasthmatic
child-derived human bronchial epithelium (haCHBE), or atopic asth-
matic 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.
Total protein was determined from cells using the bicinchoninic acid
(BCA) protein assay (Pierce, Rockford, IL). A total of 50 ?g 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 Detec-
tion System (Amersham Biosciences, Arlington Heights, IL).
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,
TABLE 1. PATIENT DEMOGRAPHICS
Hay Fever and
Eczema PhenotypeSex Number Hay FeverEczema
Definition of abbreviations: F ? female; M ? male.
CA), and CD68 (Dako Corp.) to identify any mesenchymal cells, den-
driticcells, endothelial cells,or macrophages, respectively. Specificanti-
body 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
expression was analyzed by two-step reverse transcriptase–polymerase
chain (RT-PCR) reactions. cDNA was synthesized using hexanucleo-
tide primers and Multiscribe Reverse Transcriptase (Applied Biosys-
tems, Foster City, CA). Refer to the online supplement for PCR and
quantitative PCR (qPCR) conditions.
Assessment of Proliferation
Expression of proliferating cell nuclear antigen (PCNA) was assessed
using qPCR, whereas proliferation was assessed using a 3-[4,5-
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).
Cytokine concentrations were measured in the supernatants using com-
mercial 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 ?1 (TGF-?1), IL-8, and IL-1? (Bio-
source, Camarillo, CA) as per the manufacturer’s instructions. IL-10,
IL-12p70, and tumor necrosis factor ? (TNF-?) were measured using
a multiplexed cytometric bead assay system (BD Biosciences, Bedford,
MA). Results were normalized to cell number.
Before statistical evaluation, all results were tested for population nor-
mality 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 per-
formed 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.
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 ? 106for hnaCHBE,
and did not significantly differ from haCHBE (3.5 ? 1.56 ? 106,
p ? 0.948) or aaCHBE yields (2.55 ? 1.15 ? 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
1112 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1742006
TABLE 2. OLIGONUCLEOTIDE PRIMERS
Gene Primer Sequence Product Length (bp)
Von Willebrand factor 444
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
Figure 1. Phase contract micrographs showing no morphologic varia-
tion 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.
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 posi-
tive 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 expres-
sion of mesenchymal (vimentin; Figure 2O, lane 2), dendritic
(CD1a; Figure 2O, lane 4), and endothelial (von Willebrand
factor; Figure 2O, lane 6) specific markers.
Further investigation revealed distinct variations in cytokera-
tin 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 inaaCHBEsamples (Figure 3A). CK-19protein
ples 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 aaCHBEsamples of freshly isolated cells and atearly passage.
The rank order of CK-19 protein expression of HNA ? HA ?
AA was confirmed by Western blot analysis (Figure 3B). In
isolated at the time of surgery was virtually absent in primary
isolates of hnaCHBE and was only minimally detectable in pas-
saged cultures (Figures 3A and 3B). However, a higher expres-
sion 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 pheno-
types, cytokeratin expression levels were found to be consistent
among patients of the same phenotype (Figure 3C).
Comparison of Asthmatic and Normal Epithelial Cell Function
Production of proinflammatory mediators from aaCHBE and
hnaCHBE cells is similar. The constitutive production of proin-
flammatory cytokines, including IL-1?, sICAM-1, and IL-8, is
shown in Figure 4. No significant difference was observed in the
amount of IL-1?, sICAM-1, and IL-8 produced by hnaCHBE,
haCHBE or aaCHBE cells. Levels of IL-12p70 and TNF-? were
below limits of detection regardless of disease phenotype.
flammatory mediators. We also measured the production of two
antiinflammatory mediators, namely IL-6 and PGE2, in primary
cultures ofepithelial cells (Figure 4). We observedthat 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;
Kicic, Sutanto, Stevens, et al.: Asthmatic Epithelium Abnormalities 1113
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, 400?. (O) Reverse transcriptase–polymerase chain reac-
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.
?-Actin expression confirmed equal loading of samples. Images pro-
vided 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.
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 pro-
duction but the levels were below limits of detection.
Differentialproduction ofgrowth factorsby aaCHBEcells com-
factors that are associated with asthma, including TGF-?1 and
EGF, was also quantified. Primary cultures of aaCHBE cells
produced significantly less TGF-?1 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;
aaCHBE Cells Proliferate Faster than hnaCHBE Cells
We initially assessed the proliferative capacity of bronchial epi-
thelial 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) comparedwith 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
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 significantlyquicker 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).
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 PGE2over repeated passages. Generally, the differ-
phenotypes were maintained in vitro (Figure 6 and Figure E1
of the online supplement). Epithelial cells derived from all phe-
notypes continued to produce similar levels of IL-1?, 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 pheno-
types were maintained for at least three passages before levels
significantly decreased (p ? 0.001). Similar findings were seen
for both TGF-?1 and EGF. In contrast, PGE2production varied
with repeated passages. Although initial cultures of aaCHBE
cells produced the greatest amount of PGE2, the levels signifi-
cantly decreased with passage. In contrast, PGE2production by
both haCHBE and hnaCHBE cells remained consistent over
three subsequent passages before diminishing (Figure 6).
Comparison of Pediatric, Adult, and Transformed Epithelial
To examine whether established hnaCHBE and aaCHBE cells
exhibitedanydifferencescomparedwith normaladult orimmor-
talized 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 pas-
sages. In addition, 16HBE cells produced little IL-6 in compari-
son with either of the primary bronchial cells, and this difference
was also maintained through repeated passages (Figure 7).
characteristics compared with those of children without asthma,
1114AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 174 2006
Figure 3. (A) Cytokeratin expres-
sion profiles of epithelial cells over
passage. Serial cytospins of cells
from p1–p5 were collected and in-
cubated with primary antibodies
specific for CK-19 and CK-5/14
(1:250) for 24 h at 4?C, followed
by fluorescently conjugated sec-
ondary antibodies for a similar pe-
riod. Cytokeratin expression was
then compared with ex vivo cytos-
pins collected from the same
patient at the time of initial
notypes. 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. Con-
versely, CK-5 was expressed primar-
ily in AA cells and was maintained
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.
epithelial cells from children with asthma exhibit marked intrin-
sic biochemical and functional differences. Asthmatic epithelial
cells appear to be less differentiated in that they have substan-
tially 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 sub-
stantiallygreateramountsofIL-6,PGE2, andEGF,similar levels
of the proinflammatory mediators IL-1?, sICAM-1, and IL-8,
but lower levels of TGF-?1 in culture. The aaCHBE cells also
exhibited faster doubling times than hnaCHBE cells. Impor-
tantly, these differences are maintained despite successive pas-
sages, 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 rele-
vant target organ tissue. As a result, most research has relied
heavilyon commerciallyproducedcells or transformedcell lines.
Primary cultured cells should be used for in vitro analyses be-
cause they are most similar to cells in vivo. We and others have
demonstrated the utilityofbronchial brushingsfrom childrenfor
the isolation, characterization, and successful culture of primary
epithelial cells (8–10). The procedure, which involves the inser-
tion 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 usingsevofluorane forinduction andintravenous pro-
pofol for maintenance. The effects of these agents on airway
epithelial gene expression are unknown. However, sample col-
lection 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
The epithelial lineage of all established cultures was con-
firmed using immunohistochemistry, Western blot analysis, and
semiquantitative RT-PCR. All cultures expressed the high-
tifies 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 undifferenti-
ated 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
healthyepithelialcells.Our datashow that,despiteno difference
in the constitutive production of proinflammatory mediators,
epithelial cells derived from children with asthma differ substan-
tially 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-1?,
sICAM-1, andIL-8. These findings are at oddswith theresults of
others that have demonstrated an increased amount of ICAM-1
expression in asthmatic bronchial epithelial cells (24, 25). Simi-
larly, 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 inthe bronchial
Kicic, Sutanto, Stevens, et al.: Asthmatic Epithelium Abnormalities 1115
Figure 4. Biochemical analysis of estab-
lished bronchial epithelial cells. Healthy
nonatopic human bronchial epithelium
(hnaCHBE), healthy atopic human bron-
chial 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 pro-
duction was assessed from 4–6 separate
expression was normalized against con-
as pg/ml/106cells. 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
detectedbetween phenotypesin the pro-
duction of soluble intracellular adhesion
molecule (sICAM)-1 (p ? 0.995) and
IL-1?(p? 0.425) orIL-8 (p? 0.232). The
aaCHBE cells also were found to produce
significantly greater amounts of epidermal
growthfactor(EGF;p ? 0.005) but signifi-
growth factor (TGF)-?1 (p ? 0.0001).
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.
tors and found that asthmatic epithelial cells constitutively pro-
duce 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 bron-
chial epithelium to synthesize and release augmented levels of
IL-6 and other related cytokines (18, 31, 32). The observations
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 re-
sponses that are inappropriate for the degree of inflammation or
To investigate this further, we examined the production of
the profibrotic growth factor TGF-?1 and found that cultures
established from children with asthma produced significantly
lower levels compared with cultures from atopic or healthy chil-
dren. The blunted production of TGF-?1 by asthmatic epithe-
lium may indicate diminished potential to differentiate (33) and/
or migrate (34, 35). In conjunction with this, asthmatic epithelial
cellsproduced significantlyhigherlevels ofEGF whencompared
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
is known to mediate epithelial mitogenesis, we examined
matic cells. Expression of PCNA was markedly up-regulated
in asthmatic cultures. In addition, even in the absence of any
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 pro-
duced over an equivalent period of time. These data taken in
conjunction with the diminished production of TGF-?1 suggest
that, despite the repair processes being initiated, possibly through
EGF-stimulated proliferation, abnormal differentiation may
also occur. Our findings showing that asthmatic epithelium ex-
presses low-molecular-weight cytokeratins are consistent with
1116 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 1742006
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-carboxymethoxyphe-
nyl]-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
performedin parallelat correspondingtime points,andresultsvalidated
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 differenttohnaCHBE;#haCHBEsignificantly
different to hnaCHBE.
Figure 6. Cytokine expression profiles of the various phenotypes over
passage. The hnaCHBE, haCHBE, and aaCHBE cell cultures were estab-
lished, grown to confluence over subsequent passages, and superna-
tants taken after 48 h incubation and for cytokine production asse-
ssment. Mediators involved in inflammatory (IL-1?, ICAM-1, IL-8),
antiinflammatory/proallergic (IL-6, PGE2), and repair processes (EGF,
TGF-?1) 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/106cells and
are the mean of three replicates averaged among at least three separate
patients. Although absolute values varied, the cytokine production pro-
files for each phenotype initially observed with primary cultures were
maintained until at least p3.
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
astheatopic nonasthmaticcells,butthisis morelikelyassociated
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 PGE2produced 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 col-
leagues (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
Kicic, Sutanto, Stevens, et al.: Asthmatic Epithelium Abnormalities1117
Figure 7. Comparison of IL-6 cytokine
production of pediatric epithelial cells
with adult epithelial cells and an immor-
talized 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 superna-
tants collected after 48 h. IL-6 production
was measured from supernatants col-
lected from each passage and compared
with that produced by 16HBE cells cul-
tured in vitro over five serial passages un-
dersimilar conditions.Data are expressed
as pg/ml/106cells 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.
these children was due to an insufficient production of a mito-
genic 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 sever-
ity (moderate/severe vs. mild), may play a role. This is an area
ofinterest for our researchgroup. Anotherpotential 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 produc-
tion 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 produc-
tion was the greatest between our pediatric subjects with asthma
cells produce significantly greater amounts of IL-6 compared
with either nonasthmatic or asthmatic cells from pediatric do-
little or no IL-6 in comparison. These simple observations high-
light the importance of using primary cells in studying the role
of the epithelium in asthma and highlight the difficulty in inter-
preting 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
provide strong evidence that there are marked inherent differ-
ences between healthy and asthmatic bronchial epithelium in
childhood. In particular, the cytokeratin profile, the augmented
release of antiinflammatory mediators, and the markedly dimin-
epithelial cells function abnormally even in the absence of inflam-
mation. 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 exami-
nation. Our studies further highlight that results obtained from
adult cells and cell lines mightnot alwaysbe relevantto pediatric
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.
Acknowledgment: The authors thank Drs. Amanda Griffiths and Rus Awang for
performing the bronchialbrushings andAngela Fonceca andDr. SiobhanBrennan
for technical assistance. They also thank Dr. Paul McNamara and Professor Tony
Bai for their critical comments on this manuscript.
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