Airway smooth muscle thickness in asthma
is related to severity but not duration of
asthma
A.L. James*,#, T.R. Bai", T. Mauad+, M.J. Abramson1, M. Dolhnikoff+, K.O. McKaye,
P.S. Maxwell*, J.G. Elliot* and F.H. Green**
ABSTRACT: Asthma is characterised by an increased airway smooth muscle (ASM) area
(ASMarea) within the airway wall. The present study examined the relationship of factors including
severity and duration of asthma to ASMarea.
The perimeter of the basement membrane (PBM) and ASMarea were measured on transverse
sections of large and small airways from post mortem cases of fatal (n5107) and nonfatal asthma
(n537) and from control subjects (n569). The thickness of ASM (ASMarea/PBM) was compared
between asthma groups using multivariate linear regression.
When all airways were considered together, ASMarea/PBM (in millimetres) was increased in
nonfatal (median 0.04; interquartile range 0.013–0.051; p50.034) and fatal cases of asthma (0.048;
0.025–0.078; p,0.001) compared with controls (0.036; 0.024–0.042). Compared with cases of
nonfatal asthma, ASMarea/PBM was greater in cases of fatal asthma in large (p,0.001) and
medium (p,0.001), but not small, airways. ASMarea/PBM was not related to duration of asthma,
age of onset of asthma, sex or smoking. No effect due to study centre, other than that due to
sampling strategy, was found.
The thickness of the ASM layer is increased in asthma and is related to the severity of asthma
but not its duration.
KEYWORDS: Airway smooth muscle, asthma, severity
T
he airway smooth muscle (ASM) layer area
(ASMarea) seen on transverse sections is
increased up to five-fold in cases of
asthma [1]. The post mortem study of CARROLL
et al. [2] showed that the thickness of the ASM
layer in people with clinically mild or moderately
severe asthma was less than that in patients who
died of asthma and who had clinically severe
disease. This suggests a relationship between
asthma severity and ASMarea. In biopsy studies,
the only structural change that distinguished
severe from mild/moderate cases of asthma was
the ASMarea [3, 4]. In addition to clinical severity, a
number of other factors, such as sex, age, cigarette
smoking, treatment, age at onset of asthma and its
duration, might be related to the ASMarea. BAI et al.
[5] examined the effects of age on ASMarea and the
relative area fractions of smooth muscle and
extracellular matrix by comparing younger (17–
23 yrs) and older (40–49 yrs) adults with fatal
asthma. The amount of ASM was greater in older
versus younger subjects with fatal asthma after
correction for the fraction of smooth muscle within
the ASM layer.
The Melbourne longitudinal study of asthma
followed patients with mild and severe asthma
from childhood [6]. In general, individuals tended
to show stable clinical severity from childhood
through to middle age. Similarly, reductions in
lung function have been shown to track with
persistence of symptoms from childhood to
adulthood [7]. These studies suggest that the
severity of asthma may be determined early in
life. Therefore, we hypothesised that the ASMarea
would not be related to age or duration of asthma
but to severity of asthma. This hypothesis was
tested by examining the thickness of the ASM
layer in a large number of cases of asthma and
control subjects from six study centres.
MATERIAL AND METHODS
Further details regarding methods are available
in the online supplementary material.
AFFILIATIONS
*Dept of Pulmonary Physiology,
West Australian Sleep Disorders
Research Institute, Queen Elizabeth II
Medical Centre, Nedlands,
#School of Medicine and
Pharmacology, University of Western
Australia, Crawley,
1Dept of Epidemiology & Preventive
Medicine, Monash University,
Melbourne,
eChildren’s Hospital at Westmead,
Sydney, Australia.
"University of British Columbia,
Vancouver, BC,
**University of Calgary, Calgary, AB,
Canada.
+Dept of Pathology, Sa˜o Paulo
University Medical School, Sa˜o
Paulo, Brazil.
CORRESPONDENCE
A.L. James
Dept of Pulmonary Physiology
West Australian Sleep Disorders
Research Institute
Queen Elizabeth II Medical Centre
Nedlands
WA
Australia
E-mail: alj@westnet.com.au
Received:
Nov 30 2008
Accepted after revision:
March 05 2009
First published online:
March 12 2009
European Respiratory Journal
Print ISSN 0903-1936
Online ISSN 1399-3003This article has supplementary material accessible from www.erj.ersjournals.com
1040 VOLUME 34 NUMBER 5 EUROPEAN RESPIRATORY JOURNAL
Eur Respir J 2009; 34: 1040–1045
DOI: 10.1183/09031936.00181608
Copyright�ERS Journals Ltd 2009
Subjects
Post mortem tissues from subjects with or without asthma
contained in six tissue banks (table 1) were studied. These
included a prospective study of fatal asthma [2]; a study of
asthma management and mortality [8]; a study of airway
development [9]; a study of fatal asthma [10]; a study from
New Zealand of fatal asthma [11]; and a Canadian study of
fatal asthma [12]. Subjects were classified as: control (death due
to nonrespiratory causes; history of asthma excluded); nonfatal
asthma (death due to nonrespiratory causes; history of asthma
confirmed); or fatal asthma (cause of death consistent with a
fatal attack of asthma; history of previous asthma confirmed).
Approval for these studies and for the collaborative analysis
was obtained from the Institutional Ethics Committees of the
participating centres and from the Sir Charles Gairdner
Hospital (Nedlands, Australia) Human Research Ethics
Committee. Demographic data, including age at death, sex
and, where available, smoking history, frequency of asthma
symptoms, duration of asthma, age of onset of asthma and
current treatment requirements were recorded. Smoking was
categorised as: ever-smoker (current or ex-smoker); or never-
smoker (at time of death).
Asthma severity score
Those with a history of asthma and with available information
were assigned a category for asthma severity, unrelated to the
cause of death, based on guidelines from the Global Initiative
for Asthma (GINA) [13]. Subjects were classified as having
severe asthma if they were using oral corticosteroids, reported
hospitalisations for asthma (ever) or had daily symptoms.
Subjects were classified as having moderate asthma if they had
none of the above, but had symptoms on most days or nights
(.3 days?week-1), used regular inhaled corticosteroids or used
reliever medications on most days or nights. The remaining
cases were, therefore, classified as mild.
Tissue preparation and measurement
Post mortem tissues (ranging from whole lungs to dissected air-
ways or parenchyma) were fixed by inflation or immersion. Air-
ways had been sampled systematically in some centres [2, 9, 12],
and at the discretion of the pathologist, for diagnostic purposes,
at others [8, 10, 11]. Cases were excluded if there was macroscopic
or microscopic evidence of lung injury or disease (such as
pneumonia). Sections of large and small airways were cut at a
thickness of either 4 or 5 mm and stained with haematoxylin and
eosin or using either Masson’s or Gomori’s trichrome technique.
Planimetry was perfomed on each transverse section of airway in
order to measure the length of the basement membrane (PBM),
and either planimetry or point counts were used to measure the
ASMarea [14].
Analysis
Analyses were conducted using SPSS version 13.0 (SPSS, Chicago
IL, USA), primarily for ASMarea corrected for PBM as a marker of
airway size. As ASMarea/PBM was not normally distributed,
univariate differences in log10ASMarea/PBM between asthma
categories were tested using ANOVA and Tukey’s test post hoc.
Correlations were tested using Pearson’s correlation.
Associations between categories were tested using the Chi-
squared test. In multivariate analyses, general linear models were
used to examine the effects of study centre, age, duration of
asthma, age of onset of asthma, sex, asthma group and PBM on
ASMarea/PBM (logarithmically transformed). Analyses were also
repeated for airways categorised as small (PBM,4 mm),
medium (PBM 4–10 mm) or large (PBM.10 mm) airways.
Probabilities of ,5% were considered significant and adjusted
r2 are presented.
RESULTS
The baseline characteristics of the cases are shown in table 2.
There were 213 subjects with airways available for analysis.
Clinical data were not available for all cases (table 2). The age
distribution was similar for the three case groups, but there
were relatively more females in the asthma groups (p50.013).
In the 57 cases for whom data were available, there was a
strong relationship between nonfatal asthma and clinically
mild or moderate asthma, and fatal asthma and clinically
severe asthma (Chi-squared515.3; p,0.001). There were no
significant differences between groups with regard to smoking
(information available in 99 cases), age, age at onset or
duration of asthma.
When all airways were considered together (fig. 1a), the thickness
(ASMarea/PBM) of ASM was increased in the nonfatal (median
0.040 mm; interquartile range 0.013–0.051 mm; p50.034) and
fatal cases of asthma (0.048 mm; 0.025–0.078 mm; p,0.001)
compared with controls (0.036 mm; 0.024–0.042 mm). Fatal and
nonfatal cases of asthma were not significantly different
(p50.105). ASMarea/PBM increased with airway size and the
TABLE 1 Sources and preparation of tissue for study
Centre Subjects n Groups Fixation Airways samples Sections
Thickness mm Stain
Perth (AU) 65 C, NFA, FA Inflation; formaldehyde Large and small airways 5 H&E
Melbourne (AU) 17 FA Immersion; formaldehyde Sample of random blocks 5 H&E
Sydney (AU) 24 C, NFA, FA Immersion; formaldehyde Large and small airways 4 Gomori’s
Sa˜o Paulo (BR) 23 C, FA Immersion; formaldehyde Sample of blocks 4 Masson
Auckland (NZ) 41 FA Immersion; formaldehyde Sample of blocks 4 Gomori’s
Calgary (CA) 48 C, NFA, FA Inflation; glutaraldehyde Large and small airways 5 Masson
AU: Australia; BR: Brazil; NZ: New Zealand; CA: Canada; C: control; NFA: nonfatal asthma; FA: fatal asthma; H&E: haematoxylin–eosin.
A.L. JAMES ET AL. ASTHMA AND ALLERGY
c
EUROPEAN RESPIRATORY JOURNAL VOLUME 34 NUMBER 5 1041
relationships of ASMarea/PBM and asthma group for different
airway sizes are shown in figure 1b–d. In nonfatal cases of
asthma, compared with controls, ASMarea/PBM was signifi-
cantly increased in large (p50.001), medium (p50.003) and small
airways (p50.001). In cases of fatal asthma, ASMarea/PBM was
greater compared with controls for each airway size group
(p,0.001 for all comparisons) and compared with nonfatal cases
of asthma for large and medium airways (p,0.001), but not for
small airways (p50.399). Univariate analysis showed an effect of
centre, but not sex or cigarette smoking. ASMarea/PBM was
associated weakly with age (r250.166; p50.041) and with
duration of asthma (r250.226; p50.032), but not with age at
onset of asthma (r250.081; p50.78). These relationships and
differences between groups were similar when airways were
grouped by size. Analysis by asthma group showed an effect of
age (fig. 2) in the nonfatal cases of asthma (r250.387; p,0.001).
However no effect of age was apparent for controls or fatal
asthma cases.
The results of the general linear models are summarised in
table 3. The primary model explained 50% of the variance in
ASMarea/PBM. Only subject group (control or nonfatal or fatal
asthma) showed a significant effect on ASMarea/PBM. There
was no significant effect of age, sex or smoking, and there were
no significant interactions between age, asthma group or
0.3a)
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FIGURE 1. Area of the airway smooth muscle (ASMarea) relative to basement membrane perimeter (PBM) in controls (C) and cases of nonfatal asthma (NFA) and fatal
asthma (FA) in: a) all airways combined; b) small airways (PBM ,4 mm); c) medium airways (PBM 4–10 mm); and d) large airways (PBM .10 mm). Boxes represent median
and interquartile range and whiskers represent 95% confidence interval. #: outliers. ***: p,0.001; ###: p50.001; #: p50.034; ": p50.003.
TABLE 2 Subject characteristics
Controls Asthma cases
Nonfatal Fatal
Subjects n 69 37 107
Age yrs 35 (13–90) 27 (12–69) 35 (10–76)
Sex#
Male 46 (67) 20 (54) 47 (44)
Female 23 (33) 17 (46) 60 (56)
Smoking status
Ever 12 (33) 14 (54) 15 (40)
Never 24 (67) 12 (46) 22 (60)
Asthma severity***
Mild/moderate 18 (64) 4 (14)
Severe 10 (36) 25 (86)
Asthma duration yrs 16 (10–26)" 18 (7–27.5)+
Age at asthma onset yrs 9 (2–20)" 12.5 (5–26.8)+
Data are presented as median (interquartile range) or n (%), unless otherwise
stated. Data for age are presented as median (range). ***: p,0.001 for group
effect; #: p50.013 (Chi-squared test). ": n525; +: n544.
ASTHMA AND ALLERGY A.L. JAMES ET AL.
1042 VOLUME 34 NUMBER 5 EUROPEAN RESPIRATORY JOURNAL
smoking. In separate models, no effects of duration of asthma
or age at onset of asthma were observed on ASMarea/PBM. If
PBM was included in the model, it had a strong independent
effect on ASMarea/PBM (p,0.001), but did not significantly
change the effects of other variables. When cases of nonfatal
asthma were analysed separately, there was a significant but
weak effect of age on ASMarea/PBM for all airways (r250.06;
p50.044) and a stronger effect if the analysis was confined to
adults aged 21–60 yrs (r250.255; p50.046). There was no effect
of age in the fatal asthma or control groups.
DISCUSSION
In the present study, the thickness of the ASM layer (ASMarea/
PBM) was increased in cases of asthma compared with
controls, and increased in cases of fatal asthma (clinically
severe) compared with nonfatal cases of asthma (mild or
moderate clinical severity). In multivariate analyses, there was
no relation to duration of asthma, age at onset of asthma,
smoking or sex. These findings suggest that the increased
thickness of the ASM layer is present at an early stage in the
natural history of asthma and may determine clinical severity.
We found a strong relationship between clinical severity and
asthma group (nonfatal or fatal asthma). The underlying severity
of asthma is generally constant over long periods [6, 7], and the
data from the present study suggest that ASM thickness and
asthma severity are associated. Most previous studies have
examined ASM in cases categorised as fatal or nonfatal asthma,
but have not reported grades of clinical severity [1]. In mild-to-
moderate cases studied during life, only small biopsy specimens
can be obtained [3, 15]. These studies have consistently shown
that the relative ASMarea is increased in asthma, and at least
three studies have shown that the amount of ASM distinguished
mild/moderate from severe cases of asthma [3, 4, 16]. The
clinical severity of asthma in the present study was based upon
GINA guidelines [13] and used available data on these post
mortem cases. Data on lung function were not evaluable for
enough subjects to be included in the model. Therefore, severity
may have been generally underestimated since cases were
categorised by their most severe criteria. The presence of lung
function abnormalities would have placed more subjects into
more severe categories. However, even in the absence of lung
function data, the nonfatal cases that were scored as severe
showed similar amounts of ASM to those that were mild to
moderate (fig. E1 of online supplementary material).
The thickness of the ASM layer was found to be independent
of age at onset and duration of asthma. This raises the
possibility that the thickness of the ASM layer is determined
early in the course of asthma. Although asthma severity is
related to lung function [6, 7], the relationships between lung
function, severity of asthma and ASM thickness could not be
assessed in the present study. Others have found a relation
between duration of asthma and lung function [17], and
between age and airway wall thickness, with a similar trend
for ASM [5]. In the present study, a relationship between ASM
thickness and age was found on univariate analysis in the
nonfatal asthma group. There was a small overall effect of age
in the same group in the general linear model. It is possible
that the effects of age are small relative to the effect of severity
and are, therefore, only evident in cases of mild-to-moderate
severity.
The effects of age were not observed if PBM (the marker of
airway size) was included in the model. Although we found
that the results were similar if analyses were confined to
specific airway size groups, there is potential for confounding
by airway size since small airways have a thinner layer of ASM
than do larger airways. Children, having smaller airways, have
a thinner ASM layer in airways at the same anatomical site as
adults [9]. Although the present study included cases over a
wide age range (10–.80 yrs), there were only 13 children aged
,15 yrs, too few for separate analysis. Analyses confined to
adults showed a stronger effect of age in the nonfatal asthma
cases. Therefore, a small effect of ageing on the thickness of the
ASM layer cannot be excluded. There was a small increase in
ASMarea with age in the nonfatal asthma group. The lack of
change with age in the control group suggests that ageing per se
does not increase the thickness of the ASM. The effect in the
nonfatal asthma group may have been apparent because subtle
changes, possibly related to matrix deposition or ASM cell
enlargement or proliferation over time, are discernible against
a background of mild remodelling, whereas, in the fatal cases
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FIGURE 2. Area of the airway smooth muscle (ASMarea) relative to basement
membrane perimeter (PBM) by age for asthma cases categorised as control (n),
nonfatal asthma ($) or fatal asthma (.).
TABLE 3 Parameter estimates for general linear model for
airway smooth muscle area/basement
membrane perimeter
B coefficient¡SEM (95% CI) p-value
Sex (versus male) 0.001¡0.014 (-0.027–0.028) 0.947
Age 0.001¡0.000 (-0.0003–0.0004) 0.790
Duration of asthma# 0.001¡0.000 (-0.0004–0.0013) 0.259
Age at onset of asthma# 0.001¡0.000 (-0.0012–0.0004) 0.285
Asthma group (versus
fatal)
Control -0.040¡0.016 (-0.064– -0.015) 0.002
Nonfatal -0.040¡0.016 (-0.072– -0.009) 0.013
#: assessed in separate models with asthma cases alone.
A.L. JAMES ET AL. ASTHMA AND ALLERGY
c
EUROPEAN RESPIRATORY JOURNAL VOLUME 34 NUMBER 5 1043
of asthma, these subtle changes may be masked by the marked
remodelling seen with such cases.
The findings of the present study and the stability of asthma
severity from early childhood to middle age seen in longi-
tudinal studies suggest that the increased thickness of the ASM
layer in asthma is an early event that changes little with the
duration of asthma and possibly very little with age. The
increase in the thickness of the ASM layer might occur at the
time of, or prior to, the appearance of the first symptoms of
asthma. One study has suggested that some of the inflamma-
tory and remodelling changes (increased thickness of the
basement membrane) in childhood asthma develop at an age
of 2–5 yrs [18], and a recent s
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