Airway hyperresponsiveness is dissociated from airway structural remodeling

Article (PDF Available)inThe Journal of allergy and clinical immunology 122(2):335-41, 341.e1-3 · July 2008with31 Reads
DOI: 10.1016/j.jaci.2008.05.020 · Source: PubMed
Abstract
Nonasthmatic eosinophilic bronchitis (EB) has emerged as a useful tool to study the structural and inflammatory mechanisms of airway hyperresponsiveness (AHR) in asthma. We have previously shown that vascular remodeling and reticular basement membrane (RBM) thickening are present in EB. However, it is not known whether other features of structural remodeling including increased airway smooth muscle (ASM) mass, matrix deposition, and glandular hyperplasia are also present in EB. We sought to determine whether structural remodeling occurs in EB and is associated with AHR and airflow limitation. Forty-two patients with asthma, 21 patients with EB, and 19 healthy volunteers were recruited. ASM area, RBM thickness, collagen 3 deposition, glandular area, mast cells, and granulocytes were assessed in bronchial biopsy samples. Nonasthmatic eosinophilic bronchitis and asthma were associated with a significant increase in ASM mass and RBM thickness compared with healthy subjects. In contrast, we did not observe any significant differences in collagen 3 deposition in the lamina propria and ASM or the % area of glands in the lamina propria. Univariate analysis demonstrated that mast cell numbers in the ASM were the only feature of remodeling associated with AHR (beta = -0.51; P = .004). Stepwise linear regression revealed that a combination of mast cell numbers in the ASM (beta = -0.43) and disease duration (beta = -0.25; model-adjusted R(2) = 0.26; P = .027) best modeled AHR. Mast cell localization to the ASM bundle, but not structural remodeling of the airway wall, is associated with AHR in asthma.

Figures

Original article
Airway hyperresponsiveness is dissociated from airway
wall structural remodeling
Salman Siddiqui, MRCP, Vijay Mistry, BSc, Camille Doe, BSc, Katy Roach, BSc, Angela Morgan, MRCP, MD,
Andrew Wardlaw, PhD, FRCP, Ian Pavord, DM, FRCP, Peter Bradding, DM, FRCP, and Christopher Brightling, PhD,
MRCP Leicester, United Kingdom
Background: Nonasthmatic eosinophilic bronchitis (EB) has
emerged as a useful tool to study the structural and
inflammatory mechanisms of airway hyperresponsiveness
(AHR) in asthma. We have previously shown that vascular
remodeling and reticular basement membrane (RBM)
thickening are present in EB. However, it is not known whether
other features of structural remodeling including increased
airway smooth muscle (ASM) mass, matrix deposition, and
glandular hyperplasia are also present in EB.
Objectives: We sought to determine whether structural
remodeling occurs in EB and is associated with AHR and
airflow limitation.
Methods: Forty-two patients with asthma, 21 patients with EB,
and 19 healthy volunteers were recruited. ASM area, RBM
thickness, collagen 3 deposition, glandular area, mast cells, and
granulocytes were assessed in bronchial biopsy samples.
Results: Nonasthmatic eosinophilic bronchitis and asthma were
associated with a significant increase in ASM mass and RBM
thickness compared with healthy subjects. In contrast, we did
not observe any significant differences in collagen 3 deposition
in the lamina propria and ASM or the % area of glands in the
lamina propria. Univariate analysis demonstrated that mast cell
numbers in the ASM were the only feature of remodeling
associated with AHR (b5–0.51; P 5 .004). Stepwise linear
regression revealed that a combination of mast cell numbers in
the ASM (b5–0.43) and disease duration (b5–0.25; model-
adjusted R
2
5 0.26; P 5 .027) best modeled AHR.
Conclusion: Mast cell localization to the ASM bundle, but not
structural remodeling of the airway wall, is associated with
AHR in asthma. (J Allergy Clin Immunol nnnn;nnn:nnn-nnn.)
Key words: Asthma, nonasthmatic eosinophilic bronchitis, airway
hyperresponsiveness, mast cell, remodeling
Asthma is a common airway disease that accounts for signif-
icant healthcare cost.
1
It is characterized by variable airflow limi-
tation, airway inflammation, and airway hyperresponsiveness
(AHR). Structural and cellular changes within the airway wall in
asthma, notably increased airway smooth muscle (ASM) mass,
2
vascular remodeling,
3
thickening of the reticular basement mem-
brane (RBM), and fibroblast numbers in the lamina propria,
4
have
been shown to correlate with airflow limitation. Furthermore, cel-
lular infiltrationof the airway wall in asthma is related to decline in
lung function.
5
The association of structural change in the airway
wall in asthma with AHR is much more tenuous. A number of re-
ports have drawn conflicting conclusions about the association of
AHR with epithelial desquamation/loss of tight junctions,
6-8
RBM
thickening,
9-13
vascular remodeling,
2,14
and ASM mass.
2,15
Nonasthmatic eosinophilic bronchitis (EB) has emerged as a
powerful disease control model to study the mechanisms of AHR in
asthma outside the confounding influence of eosinophilic airway
inflammation.
16
EB is a common cause of chronic cough, account-
ing for approximately 10% of referral to a specialist cough clinic
17
and is characterized by eosinophilic airway inflammation. How-
ever, in contrast with asthma, there is an absence of variable airflow
obstruction and AHR.
18
We have previously shown that EB is
characterized by RBM thickening
19
and vascular remodeling
3
to
a similar degree as patients with asthma and eosinophilic airway
inflammation. In contrast, mast cell infiltration of the ASM bundle
was a defining feature of the asthma phenotype being absent in sub-
jects with EB and healthy subjects.
20
Importantly, studies examin-
ing the natural history of EB have demonstrated that fixed airflow
obstruction may occur,
21
which would again support the notion that
structural remodeling of the airway wall may occur in EB.
We therefore hypothesized that (1) structural remodeling of the
airway wall does occur in EB, (2) structural remodeling of the airway
wall is dissociated from AHR in asthma, and (3) mast cell locali-
zation to the ASM bundle is a key determinant of AHR in asthma.
METHODS
Detailed information on methods is available in this article’s Online
Repository at www.jacioline.org.
From the Institute of Lung Health.
Supported by Asthma UK, the Department of Health Clinician Scientist award, and a
Wellcome Senior Clinical Fellowship (C.B.).
Disclosure of potential conflict of interest: I. Pavord is on the speakers’ bureau for
GlaxoSmithKline and AstraZeneca and has received research support from Glaxo-
SmithKline. C. Brightling is on the speakers’ bureau for AstraZeneca, GlaxoSmith-
Kline, and MSD; has received research support from GlaxoSmithKline, AstraZeneca,
and Medimmune; and is on the scientific board at Medimmune. The rest of the authors
have declared that they have no conflict of interest.
Received for publication January 31, 2008; revised May 7, 2008; accepted for publication
May 9, 2008.
Reprint requests: Christopher Brightling, PhD, MRCP, Institute of Lung Health, University
of Leicester, Leicester, LE3 9QP, United Kingdom. E-mail: ceb17@le.ac.uk.
0091-6749/$34.00
Ó 2008 American Academy of Allergy, Asthma & Immunology
doi:10.1016/j.jaci.2008.05.020
Abbreviations used
AHR: Airway hyperresponsiveness
ASM: Airway smooth muscle
EB: Nonasthmatic eosinophilic bronchitis
GINA: Global Initiative for Asthma
RBM: Reticular basement membrane
UK: United Kingdom
1
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Subjects
Twenty-one subjects with EB, 42 subjects with asthma, and 19 normal
controls were recruited from Glenfield Hospital outpatients and staff and by
local advertising.
Asthma was defined by 1 or more of the following objective criteria:
significant bronchodilator reversibility of FEV
1
>200 mL, PC
20
<8 mg/mL, or
a peak flow amplitude percentage mean over 2 weeks of more than 20%.
Asthma severity was classified by using the current Global Initiative for
Asthma (GINA) guidelines based on the GINA treatment steps
22
; GINA
1to2(n5 17), intermittent to mild persistent asthma; GINA 3 to 4 (n 5
15), moderate to severe persistent asthma; and the American Thoracic Society
criteria for refractory asthma (n 5 10).
23
Normal subjects had no history of
respiratory disease and normal spirometry and methacholine responsiveness.
EB was defined by the American College of Chest Physicians criteria.
18
In
brief, patients had a history of chronic cough without symptoms of variable
airflow obstruction, a sputum eosinophil count >3%, and normal lower airway
responsiveness to methacholine (PC
20
>16 mg/mL). All subjects were non-
smokers with a past smoking history of less than 10 pack-years.
Twelve patients with EB, 12 patients with asthma, and 7 healthy controls
were included in previous studies.
19,24,25
The Leicestershire ethics commit-
tee approved the study, and all patients gave their written informed consent.
Protocol and clinical measurements
Subjects attended on 2 occasions. At the first visit, exhaled nitric oxide
(measured at 50 mL/s NIOX; Aerocrine, Stockholm, Sweden), spirometric pa-
rameters before and after bronchodilator (400 mg inhaled albuterol), allergen
skin prick tests, and methacholine airway responsiveness using the tidal
breathing method (0.03-16 mg/mL)
26
were measured, followed on recovery
by sputum induction.
27
At the second visit 1 week later, the subjects under-
went bronchoscopy.
28
Mucosal biopsy specimens were processed into the wa-
ter soluble resin glycol methacrylate (Polysciences, Northampton, United
Kingdom [UK]) for embedding.
29
Immunohistochemistry
Two-micrometer sections were cut, floated on 0.2% ammonia solution in
water for 1 minute, and dried at room temperature for 1 to 4 hours. The
following mouse IgG
1
mAbs were used: a-smooth muscle actin (Dako), col-
lagen type 3 (Sigma, Gillingham, UK), tryptase (Dako), major basic protein
(Caltag, Paiseley, UK), and neutrophil elastase (Dako), with appropriate iso-
type controls (Dako). The technique of immunostaining applied to glycol
methacrylate–embedded tissue has been described previously.
29
Quantification of airway structure and cellular
localization
Morphometry was assessed by using computer-assisted image analysis.
The percentage of the bronchial lamina propria occupied by ASM, glands, and
collagen 3 was determined. RBM thickening (in micrometers) was assessed by
using the 50-point method as previously described.
30
Nucleated immunos-
tained cells were enumerated in the lamina propria/ASM and expressed as
cells per square millimeter of the lamina propria and ASM.
20
Analysis
Statistical analysis was performed by using PRISM Version 4 (Prism, La
Jolla, Calif) and regression analysis by using SPSS Version 13.0 (SPSS Inc,
Chicago, Ill). Parametric data were expressed as means (SEMs), data that
had a log-normal distribution were log-transformed and described as geomet-
ric means (95% CIs), and nonparametric data were described as medians
(ranges). One-way ANOVA and Student t tests were used for across-group
and between-group comparisons, respectively. x
2
Tests were used to compare
categorical data. We chose to examine the relationship between disordered air-
way physiology (postbronchodilator FEV
1
and AHR) and structural remodel-
ing by univariate analysis Pearson correlation coefficients. Stepwise linear
regression was performed to assess which variables best correlated with
AHR in asthma. Independent variables entered into the regression model
were age of onset, daily equivalent dose of inhaled beclomethasone dipropio-
nate, duration of disease, log transformed percentage sputum/lamina propria
eosinophil count, percentage sputum/lamina propria neutrophil count, RBM
thickness, percentage area of ASM and glands in the lamina propria, percent-
age area of lamina propria matrix, mast cell numbers/square millimeter ASM,
and dependent variable of log-transformed methacholine PC
20
. Regression
data are presented as model-adjusted Pearson correlations alongside the
TABLE I. Baseline clinical characteristics
Asthma (n 5 42)
Control (n 5 19)
Eosinophilic
bronchitis (n 5 21)
GINA 1
(n 5 17)
GINA 2-4
(n 5 15)
Refractory
(n 5 10)
Age (y) 36.4 (3.2) 49.8 (2.6)§ 45.4 (3.9) 52.9 (4.4)§ 47.1 (2.1)à
Sex (M:F) 8:11 10:11 7:10 9:6 4:6
Atopy (%) 44.4 53.3 75 70 50#
Disease duration (y) NA 5.0 (1.3) 12.0 (2.5) 15.9 (3.9)k 18.0 (5.1)àk
Inhaled BDP (mg/24 h) NA 200 (84) 0 1067 (122){ 1404 (179)à{
Oral prednisolone (number; dose) NA 0/21; 0 0/10; 0 0/15; 0 8/10; 12.2 (3.2)
Postbronchodilator FEV
1
(L) 3.0 (0.16) 3.8 (0.26) 2.9 (0.28) 2.8 (0.2)k 2.5 (0.25)àk
Postbronchodilator FEV
1
% 105.5 (2.6) 99.6 (2.9) 96.3 (4.0) 94.6 (4.6) 80.7 (6.5)à§k
FEV
1
/FVC postbronchodilator 85.0 (2.7) 81.4 (1.2) 75.4 (2.8) 76.5 (2.0) 70.2 (2.8)à§k
PC
20
(mg/mL)* >16 >16 0.62 (0.25-1.5) 0.44 (0.17-1.1) 0.35 (0.02-5.5)à
Induced sputum
Eosinophils (%)* 0.33 (0.25-0.43) 6.9 (3.9-12)§ 2.0 (0.7-5.2)§ 1.7 (0.66-4.6)§ 6.9 (1.2-38.5)à§
Neutrophils (%) 52.9 (10.4) 53.4 (5.5) 45.6 (7.3) 55.0 (8.3) 44.9 (10.0)
Lamina propria
Mast cells/mm
2
15.9 (10.1-21.7) 28.1 (16.0-35.6) 19.5 (15.7-25.3) 19.0 (11.2-28.0) 17.0 (13.2-29.8)à
Eosinophils/mm
2
2.3 (1.1-5.4) 31.7 (18.3-57.7)§ 20.9 (7.8-38.8)§ 11.3 (4.9-19.7) 28.1 (6.6-31.9)à§
Neutrophils/mm
2
7.7 (2.1-10.3) 35.5 (16.8-46)§ 15.4 (10.6-27.2) 19.5 (5.4-40.7) 15.6 (3.2-22.3)à
M, Male; F, female; FVC, forced vital capacity; NA, not applicable.
Data expressed as means (SEMs), *geometric mean (95% CI), median (interquartile range).
Beclomethasone dipropionate (BDP) equivalents (BDP/24 h): fluticasone 2:1, budesonide 1.25:1, mometasone 1.25:1.
àP < .05 1-way ANOVA ; normal vs EB/GINA 1, GINA 2-4, refractory asthma.
§P < .05 vs control (Bonferroni/Dunn correction for multiple comparisons).
kP < .05 vs EB (Bonferroni /Dunn correction for multiple comparisons).
{P < .05 vs EB and GINA 1 asthma (Bonferroni /Dunn correction for multiple comparisons).
#P < .0001 x
2
test.
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standardized regression coefficient (b) of the modeled independent variable.
For a 1-SD increase in the independent variable, b represents the average
amount of SD by which the dependent variable increases when the other inde-
pendent variables are kept constant. A P value <.05 was taken as the threshold
for statistical significance.
RESULTS
Clinical characteristics of patients and healthy subjects are
shown in Table I.
Airway structural remodeling is present in both
eosinophilic bronchitis and asthma
Nonasthmatic eosinophilic bronchitis and asthma were asso-
ciated with a significant increase in ASM mass and RBM
thickness compared with healthy subjects (Table II; Fig 1). In con-
trast, we did not observe any significant differences in lamina
propria or ASM collagen 3 deposition. We observed a trend for
increased percentage glands in the lamina propria in refractory
asthma ( P 5 .08; Table II; Fig 2).
TABLE II. Airway structural changes in asthma and EB
Asthma (n 5 42)
Control (n 5 19)
Eosinophilic
bronchitis (n 5 21)
GINA 1
(n 5 17)
GINA 2-4
(n 5 15)
Refractory
(n 5 10)
RBM thickness (mm) 6.6 (0.4) 11.1 (0.96) 9.5 (0.77) 8.9 (1.1) 11.0 (0.7)*
Percent ASM 8.5 (1.3) 17.9 (3.4) 17.2 (1.9) 16.1 (3.6) 16.3 (2.8)*
Percent glands 1.6 (0.61) 3.1 (1.4) 0.82 (0.3) 2.4 (1.5) 4.9 (1.6)
Percent collagen 3 lamina propria 8.9 (2.3)à 7.4 (2.1)à 2.6 (1.3) 1.4 (0.4) 6.9 (2.3)*à
Collagen 3 SQS lamina propria 2.02 (0.2) 2.36 (0.15) 1.85 (0.27) 1.68 (0.16)§ 2.23 (0.30)
Collagen 3 SQS ASM (mean score) 0.56 (0.1) 0.68 (0.08) 0.58 (0.14) 0.43 (0.11) 0.68 (0.13)
Collagen 3 SQS ASM (number positive) 7/18 6/14 5/10 7/15 7/10
Mast cells/mm
2
ASM 3.1 (0.63) 1.03 (0.41) 8.8 (2.3)§ 11.7 (2.3)§ 15.6 (2.6)*§
SQS, Semiquantitative score.
Data expressed as means (SEMs).
*P < .05 1-way ANOVA; normal vs EB/GINA 1, GINA 2-4, refractory asthma.
P < .05 vs control (Mann-Whitney tests/t tests for intergroup comparisons).
àP < .05 vs GINA 2-4 asthma (Mann-Whitney tests/t tests for intergroup comparisons).
§P < .05 vs EB (Mann-Whitney tests/t tests for intergroup comparisons).
FIG 1. Increased ASM area in asthma and EB. Representative photomicrographs of smooth muscle actin–
immunostained mucosal biopsies (3100 magnification) in healthy subject (A),EB(B), and refractory asthma
(C). D, Dot plot of percentage ASM in subjects with asthma with severity defined according to GINA, sub-
jects with EB, and healthy subjects.
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Mast cell localization to the ASM bundle is present
in asthma and with increasing asthma severity
The mean (SEM) of mast cells per millimeter ASM was
significantly increased in asthma 11.6 (1.4) compared with patients
with EB 1.03 (0.41) and healthy subjects 3.1 (0.63; P < .0001; Ta-
ble II and Fig 3, A). The mean (SEM) of mast cells per millimeter
ASM was significantly increased in asthma independent of sever-
ity GINA 1 (8.8 [2.3]), GINA 2 to 4 (11.7 [2.3]), and refractory
asthma (15.6 [2.6]) compared with patients with EB and healthy
subjects (P < .0001, ANOVA; P < .05, intergroup comparisons;
Fig 3, A). We did not observe localization of neutrophils to the
ASM bundle in subjects with asthma, subjects with EB, or healthy
subjects. In contrast, eosinophils were present in the ASM bundle
in low numbers in refractory asthma (2.4 [0.84]/mm
2
), in contrast
with subjects with GINA 1 to 4 asthma, subjects with EB, and
healthy subjects, who had no evidence of ASM eosinophilia.
Mast cell localization to the ASM bundle and not
structural remodeling correlates with AHR in
asthma
Univariate analysis demonstrated that mast cell number in the
ASM was the only feature of remodeling that was associated with
log PC
20
(b 5 –0.51; P 5 .004; Table III and Fig 3, B). Stepwise
linear regression revealed that a combination of mast cell num-
bers in the ASM (b 5 –0.43) and disease duration (b 5 –0.25;
model-adjusted R
2
5 0.26; P 5 .027) best modeled log PC
20
.
Eosinophilic airway inflammation is associated
with thickeni ng of the RBM in asthma and EB
Univariate analysis revealed that RBM thickening in the pooled
asthma and EB cohort correlated with log sputum eosinophil
percentage (b 5 0.29; P 5 .03) but not lamina propria eosino-
philia (b 5 0.18; P 5 .20). Stepwise linear regression revealed
that this association was independent of age of onset, disease
duration, and the beclomethasone dipropionate/24 hour dose of
inhaled corticosteroid (b 5 0.36; model-adjusted R
2
5 0.1;
P 5 .04).
DISCUSSION
We have demonstrated for the first time that structural
remodeling of the airway wall, notably increased ASM mass,
occurs to similar degrees in EB and asthma. In addition, we did
not observe differences in glandular area or collagen 3 deposition
between EB and asthma, and we confirm that RBM thickening is
a feature of both conditions. This suggests that all of these
features of the remodeling process in asthma can be dissociated
from AHR. In contrast, mast cell numbers were increased in the
ASM bundle in asthma only, independent of disease severity and
treatment, and were shown to correlate with the degree of AHR.
Finally, eosinophilic inflammation in the airway wall and in
induced sputum was related to RBM thickening in asthma
and EB.
Our data add further support to the growing body of evidence
that indicates a key pathophysiological role for mast cells in the
ASM bundle in asthma (see review
31
) and strengthens our previ-
ously reported association of mast cell infiltration of the ASM
bundle and AHR in asthma.
21
Furthermore, in keeping with a re-
cent report,
32
we have demonstrated that mast cell localization to
the ASM bundle was present independent of disease severity and
treatment. We observed a nonsignificant trend for increased mast
cells in the ASM bundle in severe disease. This would suggest that
this aspect of the remodeling process may not be modulated by
inhaled or oral corticosteroids.
In addition to the relationship between AHR and mast cell
localization to the ASM bundle, AHR was also associated with
disease duration. Indeed, the combination of these 2 factors best
modeled AHR. In subjects with the greatest disease duration,
disease onset often occurred in childhood. It is recognized that
AHR may precede the development of asthma symptoms in
children and persist into adulthood.
33,34
Thus, whether the asso-
ciation between disease duration and AHR reflects increased
AHR in early-onset disease or the disease burden over time
FIG 3. Mast cell numbers in the ASM in asthma correlate with AHR. A, Dot
plot of mast cell numbers (horizontal bar, mean) in the ASM in controls,
subjects with asthma with severity defined according to GINA, subjects
with refractory asthma, and patients with EB. B, Correlation of mast cell
numbers in the ASM (x-axis) and methacholine PC
20
in asthma (y-axis).
FIG 2. Reduced collagen 3 deposition in the lamina propria in moderate asthma. Representative photomi-
crographs of collagen 3 staining (left) and corresponding thresholded collagen 3 (right) in the lamina prop-
ria in mucosal biopsies in healthy subject (A and B),EB(C and D), and asthma (E and F) (325 magnification).
G, Dot plot of percent collagen 3 expression in the lamina propria in subjects with asthma with severity de-
fined according to GINA, subjects with EB, and healthy controls.
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remains to be determined. Importantly, the multivariate regres-
sion coefficient between mast cells in the ASM, disease duration,
and AHR in asthma indicated that a substantial proportion of
the variance in the model was a result of other factors. AHR
is a consequence of complex interactions between a number of
factors, and further work is required to define their relative
contribution.
We did not find any association between structural remodeling
(increased ASM mass, glandular hyperplasia, RBM thickening,
collagen 3 deposition, and glandular hyperplasia) and AHR in
asthma. Furthermore, the presence or structural remodeling of
these airway compartments to a similar degree in EB strengthens
our view that structural remodeling of the proximal airway wall
can be dissociated from AHR. In keeping with this observation,
we have previously demonstrated that vascular remodeling of the
proximal airway wall and expression of VEGF
3
are present to
similar degrees in asthma and EB, again supporting the notion
that compartmental remodeling of the airway wall can be dissoci-
ated from AHR.
Importantly, bronchial biopsies enable the detailed assess-
ment of the structural components of the proximal airway but
cannot determine whether these features of remodeling are
associated with changes in the airway geometry. Imaging studies
of the airway wall provide global measures of airway geometry.
Niimi et al
35
have demonstrated that thickening of the right
bronchus segment 1 was inversely associated with airway reac-
tivity (the slope of the methacholine–airway resistance dose re-
sponse curve), suggesting that global remodeling of the airway
wall may be protective against AHR. In contrast, Boulet et al
36
have demonstrated that thickening of the airway wall in asthma
is associated with increased airway responsiveness in patients
with fixed airflow obstruction but not in patients with near-nor-
mal lung function. The apparent discordance between these
imaging studies may lie in the fact that airway reactivity and
AHR may be linked to different mechanical properties of the
airway wall. Further studies linking measures of static airway
geometry by imaging and mucosal biopsies in the same patients
are required to define further the association of structure and
function.
We did not find an association between RBM thickening,
matrix deposition, and increased ASM mass with airflow limitation
in asthma. In contrast, glandular hyperplasia was significantly
associated with airflow limitation in asthma. We and others have
demonstrated that vascula r remodeling, increased expression of
sputum VEGF,
3
and remodeling of the right bronchus segment
1 is associated with postbronchodilator lung function in asthma.
37-39
Importantly, our subjects with se v ere refractory asthma did not have
fixed airflow obstruction, and this may e xplain the lack of
association between structural remodeling and postbronchodilator
lung function reported in other studies.
4
We have confirmed that eosinophilic airway inflammation is
independently associated with thickening of the RBM in both
asthma and EB. This would support the growing body of evidence
from animal models of asthma
40
and from immunopathological
studies of eosinophilic versus noneosinophilic asthma
41
that
have linked this feature of the remodeling process to eosinophilic
airway inflammation.
One potential limitation of the current study is the cross-
sectional design and the need to confirm our findings in longitu-
dinal studies of airway wall remodeling. However, we are
confident that our findings are robust. We used nonstereologic
measures of ASM mass and quantified collagen 3 deposition as a
proxy for total matrix deposition in the lamina propria and ASM.
However, the measurements of ASM area correlated well with the
ASM volume fraction measured by using the stereologic
Cavalieri method.
42
Furthermore, the measurement of collagen
3 using a validated and automated repeatable thresholding proce-
dure correlated well with a previous study that demonstrated sim-
ilar collagen 3, collagen 1, and total collagen deposition in the
lamina propria in subjects with asthma across the spectrum of se-
verity and healthy subjects, using an automated thresholding pro-
cedure.
12
Interestingly, in keeping with our findings, this study
demonstrated a similar trend for reduced collagen 3 deposition
in moderate asthma.
In conclusion, we have shown for the first time that
remodeling of the airway wall is similar in asthma and EB
and dissociated from AHR. In contrast, mast cell localization to
the ASM bundle was associated with AHR in asthma indepen-
dent of disease severity, treatment, and airway wall structure.
Further studies are required to establish how global airway wall
thickening and compartment remodeling of the airway wall
differentially alter the micromechanical properties of the
airway wall in asthma and translate into disordered airway
function.
TABLE III. Univariate and multivariate correlation of airway structure and function in asthma
Postbronchodilator
FEV
1
%
Univariate analysis
Bronchodilator response FEV
1
(%) Methacholine PC
20
Univariate
analysis
N b
(mg/mL)
Univariate analysis
3b P value b P value b P value N b
RBM thickness (mm) 0.22 .18 20.14 .40 x 0.16 .34 x
Percent ASM 0.20 .22 20.53 .74 x 0.16 .93 x
Percent glands 20.26 .10 0.10 .55 x 0.12 .50 x
Percent collagen 3 lamina propria 0.18 .92 20.072 .69 x 0.05 .81 x
Lamina propria eosinophil
cells/mm
2
20.09 .60 0.32 .048 0.31; P 5 .013 0.32 .06 x
Lamina propria neutrophil
cells/mm
2
0.20 .20 20.064 .70 x 0.13 .45 x
Mast cells/mm
2
ASM 20.03 .89 20.024 .89 x 20.51 .004 20.43 P < .0001
b, Standardized regression coefficient. N, Stepwise linear regression model–adjusted standardized regression coefficient.
Significant values are in boldface and italics.
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We thank Dr Sarah Bolton and Dr Martyn Foster at AstraZeneca, Charn-
wood, UK, for their help in devising the thresholding protocol and access to
microscopy equipment and software.
Key messages
d Structural remodeling of the airway wall is dissociated
from AHR in asthma.
d Mast cell infiltration of the ASM is independently associ-
ated with AHR in asthma.
d Eosinophilic airway inflammation is associated with RBM
thickening in both asthma and EB.
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J ALLERGY CLIN IMMUNOL
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SIDDIQUI ET AL 7
ARTICLE IN PRESS
METHODS
Clinical characterization
Asthma was defined by 1 or more of the following objective criteria:
significant bronchodilator reversibility of >200 mL, PC
20
<8 mg/mL, or a
peak flow amplitude percent mean over a period of 2 weeks of more than
20%. Asthma sev erity was classified using the current GIN A guidelines based
on the GINA treatment steps
E1
GINA1to2(n5 17), intermittent to mild per-
sistentasthma;GINA3to4(n5 15), moderate to severe persistent asthma—and
the American Thoracic Society criteria for refractory asthma (n 5 10).
E2
Normal
subjects had no history of respiratory disease and normal spirometry and meth-
acholine responsiveness. EB was defined by the American College of Chest Phy-
sicians Criteria.
E3
In brief, patients had a history of chronic cough without
symptoms of variable airflow obstruction, a sputum eosinophil count >3%,
and normal lower airway responsiveness to methacholine (PC
20
>16 mg/mL).
All subjects were nonsmokers with a smoking history of less than 10 pack-years.
Spirometric tests were performed by using a dry bellows spirometer
(Vitalograph, Buckingham, UK) with FEV
1
recorded as the best of successive
readings within 100 mL. Allergen skin prick tests were performed to Derma-
tophagoides pteronyssinus, cat fur, dog, grass pollen, and Aspergillus fumiga-
tus solutions with normal saline and histamine controls (Bencard, Sussex,
UK). A positive response to an allergen on the skin prick tests was recorded
by the presence of a wheal of >2 mm more than the negative control.
Bronchoscopy was undertaken by using an Olympus fiber optic broncho-
scope (Olympus Co, Tokyo, Japan) in accordance with British Thoracic
Society guidelines.
E4
Bronchial mucosal biopsy specimens were taken from
the right middle and lower lobe carinae.
Mucosal biopsies were immediately transferred into ice-cooled acetone
containing the protease inhibitors iodoacetamide (20 mmol/L) and phenylme-
thylsulfonyl fluoride (2 mmol/L) for fixation, stored at 208C for 24 hours, and
then processed into the water soluble resin glycol methacrylate (Polysciences,
Northampton, UK) for embedding.
E5
Image analysis and quantitative morphometry
Morphometry was assessed by computer-assisted image analysis on
hematoxylin-stained sections and a-smooth muscle actin–stained sections
for ASM analysis. Total biopsy area was determined in at least 2 noncontig-
uous tissue sections from the same biopsy, and values are expressed in square
millimeters. The area of the lamina propria was derived by subtracting the
ASM area, glandular area, epithelial area, and area of vessels and lymphatics
from the total biopsy area
Measurement of ASM area. We used the Cavalieri method to estimate the
volume of ASM in 8 patients with EB.
E6
In brief, a single biopsy was cut into
28 sections 2 mm apart, giving a reference volume of 56 mm, and sections were
counterstained with Mayers hematoxylin. The reference volume was then in-
terrogated at 5 systematically uniform random sections (SURSs) by generat-
ing a random number between 1 and 11.2 and subsequently adding 11.2
iteratively 4 times to the original random number. A point counting system
was used to determine the total area of the section and the ASM area for the
5 SURSs. The top right quadrant of each cross was used as the 0-dimensional
reference point. Point counts were converted into section areas by multiplying
the total number of points counted Sp, by the area per test point a(p).
The Cavalieri volume was then estimated by multiplying the distance
between sections t by their total cross-sectional area:
Vre f 5 t
aðpÞ
+ p:
Using this method, the mean (SEM) volume fraction of ASM was 0.23 (0.05)
mm
3
/mm
3
.
We found an excellent correlation between this volume fraction and the
area of ASM measured by using image analysis planimetry in the individual
SURSs (r values 5 0.85; P 5 .01).
We also found an excellent correlation between ASM area estimated by
using a-smooth muscle actin staining and hematoxylin staining in the SURSs
(r 5 0.97; P < .0001).
We therefore assumed that a-smooth muscle actin staining of ASM was a
reasonable estimate of ASM mass for the purpose of this study and used the
ASM area in a-smooth muscle actin–stained sections as an estimate of ASM
mass. This is in line with previous reports.
E7,E8
Measurement of collagen 3 deposition. Intensity of matrix deposition in the
lamina propria of collagen 3 was assessed qualitatively using a semiquanti-
tative score (SQS; 0-3) as previously described and in the ASM as either
present (1) or absent(0) by 2 blind investigators (V.M., S.S.).
E7
The mean of
the 2 investigators’ scores was taken as the final score.
For quantitative assessment of matrix deposition in the lamina propria, a
thresholding technique was developed on the basis of the hue, saturation, and
intensity (HSI) of collagen 3 staining. The HSI color system was defined by a
scale of 0 to 255 for HSI. Initially the background noise was defined by
assessing the HSI of collagen 3 deposition in the lamina propria in 4 patients
with a lamina propria SQS of 3, 2, and 1, respectively. Sections were acquired
at 325 magnification, white balance corrected, and pixels of representative
matrix staining selected until high-intensity matrix staining in the lamina
propria was appropriately thresholded. Areas of matrix staining in glands and
the ASM were manually removed to give the area of matrix in the lamina
propria. A minimum of 2 noncontiguous sections were thresholded for each
patient. The median (interquartile range) of lower and upper limit of HSI were
then defined from the 12 validation patients. The lower quartile of hue (upper
limit, 10; lower limit, 0), maximum saturation (upper limit, 255; lower limit,
33) and median intensity (upper limit, 169; lower limit, 94) were then selected
as the final threshold to give a threshold that appropriately captured highly sat-
urated red light. The final threshold was then applied to the isotype control
slides of each of the 12 validation patients and the percent area of matrix in
the lamina propria in the isotypes defined. We then calculated the mean per-
cent isotype staining and the set the limit of noise with the final threshold as
the mean 1 2SD of the final percentage. The mean 1 2SD image noise was
0.88%.
All biopsies were subsequently thresholded using the final threshold, and
the noise percent was subtracted from each biopsy. The mean percent area of
collagen 3 in the lamina propria on 2 sections at least 20 mm apart was taken as
the final percent area of collagen 3 in the lamina propria.
We tested the final threshold by comparing the semiquantitative matrix
score with the thresholded percent area of matrix in the lamina propria. We
found that there was an excellent correlation (r values 5 0.83; P < .0001) and
that the regression line crossed the x-axis at a SQS value of 1.01, suggesting
that our final validated threshold identified matrix staining corresponding to
a SQS of 1 as background noise (Fig E1).
Repeatability. There was excellent intraobserver and interobserver agreement
for the quantitative thresholded matrix area (r values 5 1 and 0.99), respec-
tively, and for the qualitative matrix score (r values 5 0.74 and 0.83). The be-
tween-observer repeatability of collagen 3 scores within the ASM was assessed
using the Cohen k statistic as a binary outcome measure and was found to be
moderate (k 5 0.45) and moderate within observer (k 5 0.47).
Measurement of RBM thickening. RBM thickness was measured by a
single observer at 3400 magnification at 50 points orientated perpendicular to
the epithelium separated by 20 mm as previously described and validated by
Sullivan et al.
E9
The mean of these measurements was derived and quoted as
the final RBM thickness.
The interobserver variability was determined by measuring RBM thickness
by 2 observers (S.S., C.B.) in n 5 12 subjects, and good agreement was found
(r 5 0.79).
Determination of mast cell numbers in the ASM. We determined mast cell
infiltration in the ASM in 2 sections at least 10 mm apart.
The minimum area of ASM used to enumerate mast cells was 0.1 mm
2
as
previously described.
E10
Tryptase-positive cells within the ASM bundle were
expressed in square millimeters of ASM, and the mean of the 2 sections was
used to determine the final count.
Fifteen healthy subjects, 37 subjects with asthma (15 GINA 1, 12 GINA 2 to
4, and 10 refractory) and 18 patients with EB had sufficient ASM for mast cell
quantification.
Quantification of submuscosal cell counts. The area of the lamina propria
was derived by subtracting the area of glands, ASM, epithelium, lymphatics,
and vessels from the total biopsy area. Nucleated immunostained cells
staining for major basic protein (eosinophils), elastase (neutrophils), and tryp-
tase (mast cells) were enumerated in the subepithelium and expressed in
ARTICLE IN PRESS
J ALLERGY CLIN IMMUNOL
nnn 2008
7.e1 SIDDIQUI ET AL
square millimeters of the lamina propria. The mean cell count in 2 sections at
least 20 mm apart was used to determine the final count.
REFERENCES
E1. Global Initiative for Asthma Guidelines. 2007. Available at: http://www.ginasthma.
com/Guidelineitem.asp??l152&l2 51&intId560. Accessed December 29,
2007.
E2. American Thoracic Society. Proceedings of the ATS workshop on refractory
asthma: current understanding, recommendations, and unanswered questions.
Am J Respir Crit Care Med 2000;162:2341-51.
E3. Brightling CE. Chronic cough due to nonasthmatic eosinophilic bronchi tis:
ACCP evidence-based clinical practice guidelines. Chest 2006;129:116S-21S.
E4. British Thoracic Society. British Thoracic Society guidelines on diagnostic flex-
ible bronchoscopy. Thorax 2001;56(suppl):i1-21.
E5. Britten KM, Howarth PH, Roche WR. Immunohistochemistry on resin sections: a
comparison of resin embedding techniques for small mucosal biopsies. Biotech
Histochem 1993;68:271-80.
E6. Gundersen HJ, Jensen EB. The efficiency of systematic sampling in stereology
and its prediction. J Microsc 1987;147:229-63.
E7. Benayoun L, Druilhe A, Dombret MC, Aubier M, Pretolani M. Airway structural
alterations selectively associated with severe asthma. Am J Respir Crit Care Med
2003;167:1360-8.
E8. Trian T, Benard G, Begueret H, Rossignol R, Girodet P-O, Ghosh D, et al. Bronchial
smooth muscle remodeling involves calcium-dependant enhanced mitochondrial
biogenesis in asthma. J Exp Med 2007;204:3173-81.
E9. Sullivan P, Stephens D, Ansari T, Costello J, Jeffery P. Variation in the measure-
ments of basement membrane thickness and inflammatory cell number in bronchial
biopsies. Eur Respir J 1998;12:811-5.
E10. Brightling CE, Bradding P, Symon FA, Holgate ST, Wardlaw AJ, Pavord ID.
Mast-cell infiltration of airway smooth muscle in asthma. N Engl J Med
2002;346:1699-705.
ARTICLE IN PRESS
J ALLERGY CLIN IMMUNOL
VOLUME nnn, NUMBER nn
SIDDIQUI ET AL 7.e2
FIG E1. Correlation between SQS (x-axis) and thresholded collagen 3
deposition (y-axis) in the lamina propria. The regression line crosses the
x-axis at 1, suggesting that the threshold appropriately excluded weak or
absent staining assessed visually as noise.
ARTICLE IN PRESS
J ALLERGY CLIN IMMUNOL
nnn 2008
7.e3 SIDDIQUI ET AL
    • "Though it is difficult to associate aspects of remodeling with disease severity or degree of airways obstruction and hyperresponsiveness (Mauad et al., 2007), some investigators indicated that smooth muscle remodeling is related to the severity of asthma (James et al., 2009 ). It has shown that the clinical expression of asthma (Brightling et al., 2002), AHR (Siddiqui et al., 2008) and impaired airway relaxation (Slats et al., 2007) are associated with mast cell counts in the ASM layer in asthma. The deposition of extracellular matrix inside and outside the smooth muscle layer in asthma also seems to be related to its clinical severity and is altered as compared to healthy controls (Araujo et al., 2008; Klagas et al., 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: Asthma is a serious health and socioeconomic issue all over the world, affecting more than 300 million individuals. The disease is considered as an inflammatory disease in the airway, leading to airway hyperresponsiveness, obstruction, mucus hyper-production and airway wall remodeling. The presence of airway inflammation in asthmatic patients has been found in the 19th century. As the information in patients with asthma increase, paradigm change in immunology and molecular biology have resulted in an extensive evaluation of inflammatory cells and mediators involved in the pathophysiology of asthma. Moreover, it is recognized that airway remodeling into detail, characterized by thickening of the airway wall, can be profound consequences on the mechanics of airway narrowing and contribute to the chronic progression of the disease. Epithelial to mesenchymal transition (EMT) plays an important role in airway remodeling. These epithelial and mesenchymal cells cause persistence of the inflammatory infiltration and induce histological changes in the airway wall, increasing thickness of the basement membrane, collagen deposition and smooth muscle hypertrophy and hyperplasia. Resulting of airway inflammation, airway remodeling leads to the airway wall thickening and induces increased airway smooth muscle mass, which generate asthmatic symptoms. Asthma is classically recognized as the typical Th2 disease, with increased IgE levels and eosinophilic inflammation in the airway. Emerging Th2 cytokines modulates the airway inflammation, which induces airway remodeling. Biological agents, which have specific molecular targets for these Th2 cytokines, are available and clinical trials for asthma are ongoing. However, the relatively simple paradigm has been doubted because of the realization that strategies designed to suppress Th2 function are not effective enough for all patients in the clinical trials. In the future, it is required to understand more details for phenotypes of ast
    Full-text · Article · Sep 2013
    • "Airway smooth muscle cells are the major effector cells regulating bronchomotor tone in response to several mediators [98]. Some authors have reported that increased vascularity, reticular basement membrane thickening, and increased airway smooth muscle mass are features of both diseases [99, 100]. However, the same authors have recently reported that patients with asthma had airway wall thickening, as opposed to subjects with NAEB, who maintained airway patency without wall thickening [101]. "
    [Show abstract] [Hide abstract] ABSTRACT: Eosinophilic bronchitis is a common cause of chronic cough, which like asthma is characterized by sputum eosinophilia, but unlike asthma there is no variable airflow obstruction or airway hyperresponsiveness. Several studies suggest that prostaglandins may play an important role in orchestrating interactions between different cells in several inflammatory diseases such as asthma. PGE(2) is important because of the multiplicity of its effects on immune response in respiratory diseases; however, respiratory system appears to be unique in that PGE(2) has beneficial effects. We described that the difference in airway function observed in patients with eosinophilic bronchitis and asthma could be due to differences in PGE(2) production. PGE(2) present in induced sputum supernatant from NAEB patients decreases BSMC proliferation, probably due to simultaneous stimulation of EP2 and EP4 receptors with inhibitory activity. This protective effect of PGE(2) may not only be the result of a direct action exerted on airway smooth-muscle proliferation but may also be attributable to the other anti-inflammatory actions.
    Full-text · Article · Mar 2012
    • "Our study provides further evidence to support the view that, in asthma, mast cell localization to the ASM bundle is important in the pathogenesis of AHR. Several studies have consistently reported increased mast cell numbers in the ASM bundle in asthma (7, 8, 13, 16, 17, 24, 25). Using stepwise logistic regression to explore the association between AHR and immunopathological features of asthma, we observed that mast cell infiltration of the ASM bundle was the strongest independent predictor of AHR (8). "
    [Show abstract] [Hide abstract] ABSTRACT: Mast cell microlocalization to the airway smooth muscle (ASM) bundle is a key feature of asthma, but whether these mast cells have an altered phenotype is uncertain. In this paper, we report that in vivo, mast cells within the ASM bundle, in contrast to mast cells in the bronchial submucosa, commonly expressed fibroblast markers and the number of these cells was closely related to the degree of airway hyperresponsiveness. In vitro human lung mast cells and mast cell lines cultured with fibronectin or with primary human ASM cells acquired typical fibroblastic markers and morphology. This differentiation toward a fibroblastoid phenotype was mediated by ASM-derived extracellular matrix proteins, independent of cell adhesion molecule-1, and was attenuated by α5β1 blockade. Fibroblastoid mast cells demonstrated increased chymase expression and activation with exaggerated spontaneous histamine release. Together these data indicate that in asthma, ASM-derived extracellular matrix proteins mediate human mast cell transition to a fibroblastoid phenotype, suggesting that this may be pivotal in the development of airway dysfunction in asthma.
    Article · Oct 2010
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