Pathological features and inhaled corticosteroid response of eosinophilic and non-eosinophilic asthma.
ABSTRACT Non-eosinophilic asthma is a potentially important clinicopathological phenotype since there is evidence that it responds poorly to inhaled corticosteroid therapy. However, little is known about the underlying airway immunopathology and there are no data from placebo-controlled studies examining the effect of inhaled corticosteroids.
Airway immunopathology was investigated using induced sputum, bronchial biopsies, bronchial wash and bronchoalveolar lavage in 12 patients with symptomatic eosinophilic asthma, 11 patients with non-eosinophilic asthma and 10 healthy controls. The patients with non-eosinophilic asthma and 6 different patients with eosinophilic asthma entered a randomised, double-blind, placebo-controlled crossover study in which the effects of inhaled mometasone 400 microg once daily for 8 weeks on airway responsiveness and asthma quality of life were investigated.
Patients with non-eosinophilic asthma had absence of eosinophils in the mucosa (median 4.4 cells/mm(2) vs 23 cells/mm(2) in eosinophilic asthma and 0 cells/mm(2) in normal controls; p = 0.03) and normal subepithelial layer thickness (5.8 microm vs 10.3 microm in eosinophilic asthma and 5.1 microm in controls, p = 0.002). Non-eosinophilic and eosinophilic asthma groups had increased mast cell numbers in the airway smooth muscle compared with normal controls (9 vs 8 vs 0 cells/mm(2), p = 0.016). Compared with placebo, 8 weeks of treatment with inhaled mometasone led to less improvement in methacholine PC(20) (0.5 vs 5.5 doubling concentrations, 95% CI of difference 1.1 to 9.1; p = 0.018) and asthma quality of life (0.2 vs 1.0 points, 95% CI of difference 0.27 to 1.43; p = 0.008).
Non-eosinophilic asthma represents a pathologically distinct disease phenotype which is characterised by the absence of airway eosinophilia, normal subepithelial layer thickness and a poor short-term response to treatment with inhaled corticosteroids.
- [Show abstract] [Hide abstract]
ABSTRACT: Background and objectiveThe non-eosinophilic phenotype of asthma (NEA) is associated with chronic airway inflammation and airway neutrophilia. An accumulation of apoptotic airway epithelial cells, if not efficiently cleared by efferocytosis, can undergo secondary necrosis, with the potential for inflammation of surrounding tissues. Apoptosis may occur via the T cell granzyme B pathway. The role of granzyme B in NEA is not known. The aim of this study was to investigate production of granzyme B and its inhibitor proteinase inhibitor (PI)-9 by T cells from induced sputum and compare expression between eosinophilic, NEA and healthy controls. Methods We investigated T cell intracellular granzyme B and its inhibitor, PI-9, in sputum from healthy control subjects (n = 10), and patients with NEA (n = 22) or eosinophilic asthma (EA) (n = 15) using flow cytometry. ResultsGranzyme B expression and the ratio of granzyme B to PI-9 positive cells were highest in those with NEA for both CD3+ and CD4+ T cells. The expression of granzyme B was not statistically different between patients with NEA and EA; however, the ratio of granzyme B to PI-9 positive cells for CD3+ T cells was significantly higher in those with NEA compared with EA. Conclusions Induced sputum provides a non-invasive tool for investigating T cell cytotoxic mediators in the various asthma subtypes. Granzyme B expression is increased in NEA and the contribution of granzyme B to chronic inflammation requires further study.Respirology 02/2014; 19(2). · 3.50 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Non-eosinophilic asthma is characterized by infiltration of neutrophils into the lung and variable responsiveness to glucocorticoids. The pathophysiological mechanisms have not been characterized in detail. Here we present an experimental asthma model in mice associated with non-eosinophilic airway inflammation and airway hyperresponsiveness (AHR). For this, BALB/c mice were sensitized by biolistic DNA immunization with a plasmid encoding the model antigen β-galactosidase (pFascin-βGal mice). For comparison, eosinophilic airway inflammation was induced by subcutaneous injection of βGal protein (βGal mice). Intranasal challenge of mice in both groups induced AHR to a comparable extent as well as recruitment of inflammatory cells into the airways. In contrast to βGal mice, which exhibited extensive eosinophilic infiltration in the lung, goblet cell hyperplasia and polarization of CD4+ T cells into Th2 and Th17 cells, pFascin-βGal mice showed considerable neutrophilia, but no goblet cell hyperplasia, and a predominance of Th1 and Tc1 cells in the airways. Depletion studies in pFascin-βGal mice revealed that CD4+ and CD8+ cells cooperated to induce maximum inflammation, but that neutrophilic infiltration was not a prerequisite for AHR induction. Treatment of pFascin-βGal mice with dexamethasone before intranasal challenge did not affect neutrophilic infiltration, but significantly reduced AHR, infiltration of monocytes and lymphocytes as well as content of IFN-γ in the bronchoalveolar fluid. Our results suggest that non-eosinophilic asthma associated predominantly with Th1/Tc1 cells is susceptible to glucocorticoid treatment. pFascin-βGal mice might represent a mouse model to study pathophysiological mechanisms proceeding in the subgroup of asthmatics with non-eosinophilic asthma that respond to inhaled steroids.This article is protected by copyright. All rights reserved.Scandinavian Journal of Immunology 08/2014; · 1.88 Impact Factor
- Respirology 12/2014; · 3.50 Impact Factor
Pathological features and inhaled corticosteroid response of
eosinophilic and non-eosinophilic asthma
Mike Berry, Angela Morgan, Dominick E Shaw, Deborah Parker, Ruth Green, Christopher Brightling,
Peter Bradding, Andrew J Wardlaw, Ian D Pavord
............................................................... ............................................................... .....
See end of article for
Professor I D Pavord, Institute
for Lung Health, Glenfield
Hospital, Leicester LE3 9QP,
Received 16 October 2006
Accepted 2 February 2007
Published Online First
13 March 2007
Thorax 2007;62:1043–1049. doi: 10.1136/thx.2006.073429
Background: Non-eosinophilic asthma is a potentially important clinicopathological phenotype since there is
evidence that it responds poorly to inhaled corticosteroid therapy. However, little is known about the
underlying airway immunopathology and there are no data from placebo-controlled studies examining the
effect of inhaled corticosteroids.
Methods: Airway immunopathology was investigated using induced sputum, bronchial biopsies, bronchial
wash and bronchoalveolar lavage in 12 patients with symptomatic eosinophilic asthma, 11 patients with non-
eosinophilic asthma and 10 healthy controls. The patients with non-eosinophilic asthma and 6 different
patients with eosinophilic asthma entered a randomised, double-blind, placebo-controlled crossover study in
which the effects of inhaled mometasone 400 mg once daily for 8 weeks on airway responsiveness and
asthma quality of life were investigated.
Results: Patients with non-eosinophilic asthma had absence of eosinophils in the mucosa (median 4.4 cells/
mm2vs 23 cells/mm2in eosinophilic asthma and 0 cells/mm2in normal controls; p=0.03) and normal
subepithelial layer thickness (5.8 mm vs 10.3 mm in eosinophilic asthma and 5.1 mm in controls, p=0.002).
Non-eosinophilic and eosinophilic asthma groups had increased mast cell numbers in the airway smooth
muscle compared with normal controls (9 vs 8 vs 0 cells/mm2, p=0.016). Compared with placebo, 8 weeks
of treatment with inhaled mometasone led to less improvement in methacholine PC20(0.5 vs 5.5 doubling
concentrations, 95% CI of difference 1.1 to 9.1; p=0.018) and asthma quality of life (0.2 vs 1.0 points, 95%
CI of difference 0.27 to 1.43; p=0.008).
Conclusions: Non-eosinophilic asthma represents a pathologically distinct disease phenotype which is
characterised by the absence of airway eosinophilia, normal subepithelial layer thickness and a poor short-
term response to treatment with inhaled corticosteroids.
lying airway pathology and disordered function between
patients.3 4The development of safe non-invasive induced
sputum techniques has provided the opportunity to study
airway inflammation in a diverse range of patients. Using this
technique, we and a number of other groups have identified a
subset of adults who have clear physiological evidence of
asthma but no induced sputum evidence of eosinophilic airway
inflammation.5–7This asthma phenotype is potentially clinically
important since several uncontrolled studies have suggested
that it is associated with a poor short-term and longer-term
response to inhaled corticosteroid.5 8 9
Non-eosinophilic asthma is present in 53% of patients
presenting to an adult respiratory clinic with symptomatic
asthma.9Other investigators have reported the absence of a
sputum eosinophilia in up to 50% of patients with refractory
asthma,9patients studied during an asthma exacerbation10and
patients taking high doses of inhaled corticosteroids.6In a
recent longitudinal study of patients with severe asthma, the
absence of sputum eosinophils has been reported to be a stable
feature in a number of patients observed over 12 months;11
another study showed that it was present in untreated
symptomatic patients as well as those receiving inhaled
corticosteroid therapy.9These observations suggest that, in
some patients at least, non-eosinophilic asthma is a stable
phenotype that is not solely explained by the effects of
linicians have long regarded asthma as a heterogeneous
disease,1 2although detailed clinicopathological studies
have tended to emphasise the similarities in the under-
Several studies have noted that an airway neutrophilia is
often present in patients with non-eosinophilic asthma, and
Wenzel et al7reported a predominantly neutrophilic airway
inflammatory response with an absence of eosinophils and
normal basement membrane thickness in a subgroup of
patients with refractory asthma from whom bronchial biopsy
specimens were taken. These findings support the concept that
non-eosinophilic asthma is a pathologically distinct entity,
although the extent to which these findings reflect the effects
of treatment remains unclear.
The aim of this study was to compare the immunopathology
of eosinophilic and non-eosinophilic asthma with normal
controls in patients with symptomatic asthma who were not
treated with inhaled corticosteroids. We also set out to compare
the response to 8 weeks of treatment with the inhaled
corticosteroidmometasone in a prospective
double-blind, placebo-controlled crossover trial in patients with
non-eosinophilic asthma and in a subgroup with eosinophilic
Subjects were recruited from Glenfield Hospital clinics and by
local paper advertisement. All subjects with asthma had
Abbreviations: BAL, bronchoalveolar lavage; ECP, eosinophilic cationic
protein; FEV1, forced expiratory volume in 1 s; GMA, glycol methacrylate;
IFNc, interferon c; IL, interleukin; PC20, concentration of methacholine
provoking a 20% fall in FEV1
symptoms of episodic cough, wheeze or breathlessness such
that their Juniper asthma control score was .1.57 points, a
level which is consistent with the Global Initiative for Asthma
(GINA) recommendations for an increase in treatment.
Subjects had at least one of the following objective measures
of airway hyperresponsiveness and/or variable airflow obstruc-
tion: concentration of methacholine provoking a 20% fall in
forced expiratory volume in 1 s (FEV1PC20) of ,8 mg/ml,
increase in FEV1 of 15% or greater following inhalation of
200 mg salbutamol and/or peak flow amplitude as percentage of
mean over 14 days of .20%. Patients with eosinophilic asthma
were recruited in two separate groups, one for the broncho-
scopy study and one for the placebo-controlled study (fig 1).
Normal control subjects had no respiratory symptoms, normal
spirometric values and a methacholine PC20of .16 mg/ml.
Non-eosinophilic asthma was diagnosed in patients who had
a sputum eosinophil count below our normal range (1.9%9) on
at least two occasions separated by 1 month while still
symptomatic with a Juniper asthma control score of .1.57
points and while not receiving inhaled or oral corticosteroids.
All patients with non-eosinophilic asthma had no evidence of
bronchiectasis on a high-resolution CT scan. Patients who had
symptoms due to rhinitis and gastro-oesophageal reflux disease
were excluded. Our criteria for identifying these symptoms has
been previously described.12None of the patients had ever
smoked, had a respiratory tract infection within 6 weeks of
recruitment or had received inhaled or oral corticosteroids for
3 months before entering the study.
The Leicestershire and Rutland ethics committee approved
the study and all patients provided informed written consent.
T cells from bronchoalveolar lavage (BAL) fluid from some of
the subjects in this study were used in a separate study.13
Single flow nitric oxide was recorded at 50 ml/s as previously
described.14Spirometric parameters were measured using a
rolling seal spirometer (Vitalograph, UK). The methacholine
PC20was measured using the tidal breathing method with a
maximum inhaled concentration of 16 mg/ml, as previously
Methacholine PC20 was calculated by linear
interpolation of the change in FEV1/concentration of methacho-
line on a log dose-response curve as the inhaled concentration
of methacholine causing a 20% reduction in FEV1. Change in
methacholine PC20was expressed in doubling concentrations.
A symptom visual analogue score was measured using three
100 mm scales representing cough, wheeze and breathless-
ness.16Asthma quality of life was measured using the Juniper
asthma quality of life score.17Sputum induction was performed
and samples were processed as previously described.11
Sputum levels of interleukin (IL)-8, cysteinyl leucotrienes
and histamine were measured using standard ELISA kits (BD
Pharmagen, Immunotech and Cayman Chemicals, respectively)
and eosinophilic cationic protein (ECP) was measured using a
fluorescence immunoassay (UniCAP test, Pharmacia, Uppsala,
Sweden). These assays have been previously validated for use in
sputum supernatants.18The sensitivity levels of the assays were
2, 1361023, 0.861023and 5061023ng/ml for ECP, cysteinyl
leucotrienes, IL-8 and histamine. The intra-assay coefficient of
variability was 5–10% and the interassay coefficient of
variability was 3–15% across a range of concentrations of
Bronchoscopy was performed according to British Thoracic
Society guidelines. 20 ml of warmed sterile saline solution was
instilled into the bronchus intermedius, aspirated and analysed
as the wash sample. Three sequential samples of 60 ml warmed
sterile saline solution were then instilled into the middle lobe
bronchus and aspirated; the pooled aspirate from these samples
was analysed as the BAL fluid. Biopsy specimens were taken
from the middle and right lower lobe carinae.
The BAL fluid and bronchial wash aspirates were filtered
through 48 mm gauze and diluted to a cell concentration of
0.56106cells per/ml for cytospins and 56106cells per/ml for
AQL, asthma quality of life; PC20,
concentration of methacholine provoking a
20% fall in forced expiratory volume in 1 s.
Plan of study. NO, nitric oxide;
1044 Berry, Morgan, Shaw, et al
flow cytometry. Cytospins were made with 75 ml aspirate and
stained with Romanowski stain before being counted by a
person blind to the details of the subjects. Cell counts were
given as a percentage of at least 400 inflammatory cells
counted. Flow cytometry was used to measure CD4, CD8, IL-4
and interferon gamma (IFNc) on CD3 positive lymphocytes
from peripheral blood and BAL fluid cells using commercially
available antibodies as previously described.19IL-4 and IFNc
were measured using intracellular staining following stimula-
tion for 4 h with phorbal 12-myristate 13-acetate (PMA) and
calcium ionophore as previously described.
Processing of biopsy specimens
Biopsy specimens were taken from the middle and right lower
lobe carinae and fixed by immediately being placed in ice cold
acetone containing the protease inhibitor phenylmethansul-
fanyl fluoride (PMSF) (2 mM) and maintained at 220˚C for
24 h. The fixative was then replaced first with water-free
acetone and then with methyl benzoate for 15 min at room
temperature. The biopsy specimens were then placed in 5%
methyl benzoate in glycol methacrylate (GMA solution A,
Polysciences, Northampton, UK) for three periods of 2 h each.
Embedding was then performed by placing individual biopsies
into Taab tubes and filling with a mixture of 10 ml GMA
solution A, 250 ml GMA solution B (Polysciences) and 45 mg
benzoyl peroxide (Polysciences), replacing the lid to expel any
air. Specimens were left to polymerise overnight at 4˚C and
then stored with silica gel at 220˚C until sectioning. 2 mm
sections were cut and immunostaining performed for major
basic protein, neutrophil elastase and tryptase followed by
counterstaining with haemotoxylin as previously described.20
The biopsies were counted by an individual who was blind to
the clinical status of the patients and recorded as the number of
cells/mm2positive for major basic protein, neutrophil elastase
and tryptase in the submucosa, the number of tryptase positive
cells/mm2in the airway smooth muscle and the subepithelial
layer thickness. Subepithelial layer thickness was recorded as
the mean of 50 measurements taken over a distance of at least
1 mm as previously described.21
Patients were identified as having eosinophilic or non-
eosinophilic asthma on sputum criteria as part of a standard
clinical assessment before recruitment. All patients with non-
eosinophilic asthma had a CT scan as part of their clinical
evaluation to exclude subclinical bronchiectasis. Following
recruitment, patients attended for a screening visit at which
the concentration of exhaled nitric oxide was measured before
skin prick testing, spirometry, methacholine challenge testing
and sputum induction. Patients with non-eosinophilic asthma
attended for a second screening visit at last 4 weeks after the
initial visit, at which symptom scores and the sputum
differential cell count was repeated. Only patients with
persistent non-eosinophilic asthma proceeded to bronchoscopy
and the placebo-controlled study. All assessments were
performed at the same time of day, at least 6 h after the last
dose of short-acting b2agonist. Because of the possibility that
sputum inductioncould alter
bronchoscopy was performed at least 10 days after the screen-
At least 4 weeks after bronchoscopy, patients with non-
eosinophilic asthma were entered into a randomised, double-
blind, placebo-controlled crossover study of inhaled mometa-
sone; a separate group of patients with eosinophilic asthma
entered this study as a comparison group (fig 1). Flow
independent exhaled nitric oxide parameters, spirometry,
methacholine PC20, induced sputum, symptom visual analogue
scores and asthma quality of life were measured at baseline and
after 8 weeks of each treatment, 24 h after the last dose of
study medication and more than 6 h after the last dose of
inhaled short-acting b2agonist. Placebo or mometasone 400 mg
were inhaled once daily via matched Twisthaler devices.
Treatment phases were randomly allocated; they lasted for
8 weeks and were separated by a 4-week washout phase. The
order of treatment was determined using a randomisation
sequence prepared from a random number generator. The study
drugs and randomisation codes were stored in the hospital
pharmacy and could not be accessed by the investigators.
Analysis of data
Data were tested for normality of distribution using the
Kolmogorov-Smirnov test. Data which where log normally
distributed were log transformed before analysis. Between-
group comparisons of three groups were made using one way
ANOVA with Tukey’s post hoc test for individual group
comparison when data were normally distributed or Kruskal-
Wallis test when they were not. Between two groups
comparisons were made using either independent t tests or
the Mann-Whitney U test according to distribution. Descriptive
statistics are given as mean (SE) for normally distributed data,
geometric mean (log SE) for log normally distributed data and
median (interquartile range, IQR) for data that were not
normally distributed. The bronchial biopsy study was a
descriptive hypothesis-generating study, so no power calcula-
tions were performed and no adjustment was made for multiple
Primary outcome measures for the mometasone trial were
difference in doubling concentration change in methacholine
PC20 between placebo and mometasone at 8 weeks and
difference in change in asthma quality of life score between
placebo and mometasone at 8 weeks. Differences in primary
outcomes were compared within groups using paired t tests and
Clinical characteristics of patients with eosinophilic asthma, non-eosinophilic asthma and normal controls
b2agonist reversibilty (%)
Data given as mean (SE) except age which is given as mean (range) and methacholine PC20which is given as geometric mean (log SE).
FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; b2agonist reversibility, percentage increase in FEV1following inhalation of 200 mg salbutamol;
methacholine PC20, concentration of inhaled methacholine which causes a 20% fall in FEV1.
Non-eosinophilic asthma 1045
between groups by unpaired t tests; the period and order effect
were analysed by analysis of covariance (ANCOVA). Secondary
outcome measures were net change in post-bronchodilator FEV1,
symptom visual analogue score, exhaled nitric oxide concentra-
tion, sputum eosinophil count and sputum neutrophil count. This
study was analysed as a mechanistic study and only data from
chose methacholine PC20as a co-primary outcome as it was the
most responsive outcome measure in an earlier study. Asthma
quality of life was chosen as a second primary outcome as it was
felt to be a more patient relevant outcome than PC20 and
potentially allowed us to evaluate a different component of the
corticosteroid response. The study had .80% power to detect a
line PC20between the two treatment groups.
The characteristics of the study subjects are given in table 1.
Patients with asthma had a significantly lower FEV1(percen-
tage predicted), ratio of FEV1to forced vital capacity (FVC) and
methacholine PC20and significantly higher b2reversibility than
normal controls. There was no difference between eosinophilic
and non-eosinophilic asthma. No patients classified as having
non-eosinophilic asthma at the first visit developed a sputum
eosinophilia (.1.9%) at the second visit or at any other point
during the investigation.
Patients with non-eosinophilic asthma had lower sputum
eosinophil counts, bronchial wash eosinophil counts and BAL
fluid eosinophil counts than patients with eosinophilic asthma
(table 2). There was no significant difference between sputum,
bronchial wash and BAL fluid neutrophil counts, although a
trend towards higher sputum neutrophil counts in non-
eosinophilic asthma was noted (table 2). There was no
difference in the percentage of BAL fluid lymphocytes positive
for IL-4 or IFNc, or in the blood or BAL fluid lymphocyte CD4/
CD8 ratio between the groups.
Following embedding, cutting and staining, adequate biop-
sies suitable for counting were obtained from the following
number of normal controls, patients with eosinophilic asthma
and patients with non-eosinophilic asthma: submucosa (9, 7,
10), airway smooth muscle (5, 7, 6) and subepithelial layer (7,
11, 7). There were no significant differences in the clinical
characteristics of patients from whom adequate biopsies were
obtained and those from whom they were not. Patients with
eosinophilic asthma had a median (distance) of 23 (29)
bronchial submucosal cells positive for major basic protein
per mm2which was higher than both normal controls (0
(9.4) cells/mm2) and patients with non-eosinophilic asthma
(4.4 (7.9) cells/mm2; p=0.03, fig 2A). There was no significant
difference between the groups in the number of submucosal
cells positive for tryptase (13 (5.7) vs 11 (15) vs 22 (33) cells/
mm2; p=0.52); however, the number of tryptase positive cells
in the airway smooth muscle was increased in patients with
eosinophilic asthma (8 (12) cells/mm2) and those with non-
eosinophilic asthma 9 (56) cells/mm2) compared with normal
controls (0 (1.8) cells/mm2; p=0.016, fig 2B). There were no
significant differences in the number of submucosal cells
positive for neutrophil elastase. The subepithelial layer thick-
ness was 10.3 (3.1) mm in patients with eosinophilic asthma
compared with 5.8 (3.0) mm in those with non-eosinophilic
asthma and 5.1 (2.1) mm in normal controls (p=0.002, fig 2C).
The sputum supernatant IL-8 concentration was significantly
lower in patients with non-eosinophilic asthma than in normal
controls and patients with eosinophilic asthma (table 2).
One patient with non-eosinophilic asthma withdrew from the
study after bronchoscopy because of work commitments. No
patients withdrew from this part of the study after enrolment.
Treatment period or order did not influence values before
treatment or the change in primary outcome measures.
Compared with placebo, 8 weeks of treatment with inhaled
mometasone led to a net doubling concentration improvement
in methacholine PC20of 5.5 (95% CI 2.0 to 9.0; p=0.01) in
patients with eosinophilic asthma and 0.5 (95% CI 22.4 to 3.3;
p=0.72) in the non-eosinophilic asthma group (mean differ-
ence 5.1 doubling doses, 95% CI 1.1 to 9.1; p=0.018). There
was a net improvement in Juniper asthma quality of life of 1.0
points (95% CI 0.5 to 1.5; p=0.004) following treatment with
inhaled mometasone compared with placebo in the eosinophilic
asthma group and of 0.2 points (95% CI 20.3 to 0.6; p=0.43)
in the non-eosinophilic asthma group (mean difference 0.9,
95% CI 0.27 to 1.43; p=0.008, fig 3).
Secondary outcome measures are given in table 3.
Inflammatory characteristics of patients with eosinophilic asthma, non-eosinophilic asthma and normal controls
p Value (eosinophilic
Sputum eosinophil count (%)?
Sputum neutrophil count (%)
Sputum IL-8 (ng/ml)*
Sputum ECP (ng/ml)*
Sputum Cys-LT (pg/ml)*
Sputum histamine (ng/ml)*
Bronchial wash eosinophil count (%)?
Bronchial wash neutrophil count (%)
BAL eosinophil count (%)?
BAL neutrophil count (%)
BAL IL-4 (% of CD3+ lymphocytes)
BAL IFNc (% of CD3+ lymphocytes)
Submucosal MBP +ve cells (/mm2)*
Submucosal NE +ve cells (/mm2)*
Submucosal tryptase +ve cells (/mm2)*
Smooth muscle tryptase +ve cells (/mm2)*
Subepithelial layer thickness (mm)*
Data given as mean (SE) except *median (interquartile distance) and ?geometric mean (log SE).
IL, interleukin; ECP, eosinophilic cationic protein; Cys-Lt, cysteinyl leucotriene; BAL, bronchoalveolar lavage; IFNc, interferon c; MBP, major basic protein; NE, neutrophil
elastase; ANOVA, analysis of variance; KW, Kruskal-Wallis.
1046 Berry, Morgan, Shaw, et al
We describe 12 patients with symptomatic asthma who had a
distinct sputum and bronchial biopsy pattern characterised by
the absence of eosinophilic airway inflammation and a normal
subepithelial layer thickness. In common with previous reports,
patients tended to be non-atopic middle aged women. Some
had sputum evidence of neutrophilic airway inflammation,
although increased neutrophil numbers were not evident in
bronchoscopy samples. Importantly, 8 weeks of treatment with
inhaled mometasone had no significant effect on airway
responsiveness and asthma quality of life compared with
placebo. This was in contrast to findings in patients with
asthma and eosinophilic airway inflammation where inhaled
steroids were associated with a marked improvement in these
Our patients were sufficiently symptomatic to warrant an
increase in treatment based on their Juniper asthma control
score and all had clear objective evidence of asthma, so the
absence of eosinophilic airway inflammation seen in our
patients does not reflect remission of underlying disease.
Indeed, they had more symptoms than the patients with
eosinophilic asthma who participated in the double-blind
placebo-controlled study. The absence of sputum eosinophils
was evident before entry into the study and was a consistent
feature during the study. No patients with non-eosinophilic
asthma had evidence of eosinophilic airway inflammation on
bronchoscopy or in the six sputum tests done throughout the
study, five of which were done off inhaled corticosteroids.
These observations support the hypothesis that non-eosinophi-
lic asthma represents a stable clinical phenotype which is not
solely explained by the effects of treatment.11Our finding of a
normal subepithelial layer thickness in our patients with non-
eosinophilic asthma is consistent with the finding by Wenzel
and colleagues in severe asthma.7The fact that thickening of
the subepithelial layer is found in patients with rhinitis and in
patients with eosinophilic bronchitis without asthma and not
in those with non-eosinophilic asthma suggests that this
finding may be related to eosinophilic airway inflammation
rather than asthma per se. Longitudinal studies have suggested
that increased subepithelial layer thickness is a longer term
marker of eosinophilic airway inflammation than cell counts,22
so the observation that subepithelial layer thickness is normal
in non-eosinophilic asthma increases our confidence that the
absence of eosinophilic airway inflammation is a stable feature.
We acknowledge that our biopsy study observations were
made on a small number of patients and that we did not adjust
for multiple comparisons. Our findings should therefore be
regarded as hypothesis-generating rather than definitive.
However, biopsies were examined blind to patient status and
the magnitude and consistency of the effect makes a chance
The cause of airway inflammation was not specifically
investigated in our study. None of the patients had CT evidence
of bronchiectasis, symptoms to suggest recent respiratory tract
infection or a significant smoking history, and atopy was
unusual. The association with female gender and onset of
symptoms in middle age is similar to observations made in
chronic cough,23and it is possible that there are some
similarities between these conditions. Other studies have
reported increased sputum neutrophil counts and IL-8 con-
and have speculated that the inflammatory
response is due to stimuli such as environmental endotoxin,
viral infection or ozone leading to activation of the innate
immune system and consequent release of proinflammatory
cytokines.24Our finding of a reduced IL-8 concentration in the
sputum supernatants of patients with non-eosinophilic asthma
was unexpected and in contrast to previously reported
findings;24one possible explanation is that there are different
types on non-eosinophilic asthma with varying degrees of
Perhaps the main point of interest in non-eosinophilic
asthma is the suggestion that it represents a corticosteroid
resistant form of the disease. Our study is the first double-blind
randomised controlled trial to specifically compare the response
to inhaled corticosteroids in eosinophilic and non-eosinophilic
patients with eosinophilic asthma, non-eosinophilic asthma and normal
Inflammatory cell counts in bronchial biopsy specimens from
study of inhaled mometasone versus placebo
in patients with eosinophilic and non-
eosinophilic asthma. (A) Concentration of
methacholine provoking a fall in FEV1of
20%. (B) Juniper asthma quality of life score.
Open symbols indicate mean/geometric
Primary outcome measures in
400 mg once daily and matched placebo
Clinical and inflammatory characteristics of patients before and after 8 weeks of treatment with inhaled mometasone
Eosinophilic asthma Non-eosinophilic asthma
eosinophilic) Week 0Week 8 Week 0Week 8Week 0 Week 8Week 0 Week 8
Asthma QOL score
Symptom VAS (mm) 59 (27)
Exhaled NO (ppb)*
Alveolar NO (ppb)
1.53 (0.17) 0.16 (0.32) 0.46 (0.22)2.13 (0.27)0.01 1.42 (0.18)1.07 (0.27) 1.69 (0.22)1.74 (0.18)0.720.018
26 (5.6)33 (7.2) 40 (8.9)45 (11)0.93 63 (7.7) 67 (7.6)65 (5.7) 55 (6.7)0.20 0.5
Data given as mean (SE) except *geometric mean (log SE).
FEV1, forced expiratory volume in 1 s; PC20, dose of inhaled methacholine causing a 20% fall in FEV1; QOL, quality of life; VAS, visual analogue score; NO, nitric oxide; IL-8, interleukin-8, ECP,
eosinophilic cationic protein; Cys-Lt, cysteinyl leucotriene.
1048Berry, Morgan, Shaw, et al
asthma. The observations were made on a small number of
patients and there was a difference in baseline severity of
symptoms and airway responsiveness between and within the
group which could have compromised our results. In addition,
the small sample size means that we had limited power to
detect carry-over effects. However, there were clear differences
in primary outcome measures that were above the level that the
study was powered to detect, suggesting that they were real
differences. The significantly reduced response to inhaled
mometasone in patients with non-eosinophilic asthma com-
pared with eosinophilic asthma is consistent with the findings
of previous uncontrolled studies.5 8 9In contrast to these
findings, Godon et al26found a similar response to inhaled
corticosteroids in patients who were classified as eosinophilic or
non-eosinophilic on the basis of a single sputum sample.
However, these observations were based on an uncontrolled
study in patients with symptomatic asthma who were recruited
during a period of poor asthma control and it is possible that
the improvement seen in patients with non-eosinophilic
asthma reflected regression to the mean. In addition, patients
were younger and more likely to be atopic than the patients
with non-eosinophilic asthma identified by us, so it is possible
that the populations were different.
Although our study showed a difference in response to
inhaled corticosteroids in eosinophilic compared with non-
eosinophilic asthma, we cannot conclude from our sample size
that there is no significant response to inhaled mometasone in
patients with non-eosinophilic asthma. Similarly, our study
does not allow us to draw any conclusions about the long-term
12 month study has suggested no increase in exacerbation
frequency in non-eosinophilic asthma despite substantial
reductions in the dose of oral and inhaled corticosteroids.11
Further appropriately powered studies should address this
important question in more detail.
Our findings suggest that eosinophilic and non-eosinophilic
asthma represent distinct clinical and pathological phenotypes.
They provide further evidence against a causal relationship
between eosinophilic airway inflammation and variable airflow
obstruction and airway hyperresponsiveness, but provide support
for the concept that airway hyperresponsiveness and variable
airflow obstruction are causally related to the presence of tryptase
positive mast cells in the airway smooth muscle. We have shown
that patients with non-eosinophilic asthma have a significantly
reduced short-term response to inhaled cortico-steroids than
those with eosinophilic asthma. Longer larger studies are required
to determine whether inhaled corticosteroids can be safely
withdrawn in patients with this asthma phenotype.
The authors thank Natalie Neale and Will Monterio who assisted in the
processing of bronchial biopsy and induced sputum samples.
Further information is given in the online supplement
available at http://thorax.bmj.com/supplemental.
Mike Berry, Angela Morgan, Dominick E Shaw, Deborah Parker, Ruth
Green, Christopher Brightling, Peter Bradding, Andrew J Wardlaw, Ian
D Pavord, Institute for Lung Health, Glenfield Hospital, Leicester, UK
This study was sponsored by a project grant from Asthma UK. The
mometasone inhalers were a generous gift from Schering-Plough who had
no role in the study.
Competing interests: None.
1 Rackemann FM. A clinical classification of asthma. Am J Med Sci
2 Aas K. Heterogeneity of bronchial asthma. Sub-populations—or different stages
of the disease. Allergy 1981;36:3–14.
3 Humbert M, Menz G, Ying S, et al. The immunopathology of extrinsic (atopic)
and intrinsic (non-atopic) asthma: more similarities than differences. Immunol
4 Humbert M, Durham SR, Ying S, et al. IL-4 and IL-5 mRNA and protein in
bronchial biopsies from patients with atopic and nonatopic asthma: evidence
against ‘‘intrinsic’’ asthma being a distinct immunopathologic entity. Am J Respir
Crit Care Med 1996;154:1497–504.
5 Pavord ID, Brightling CE, Woltmann G, et al. Non-eosinophilic corticosteroid
unresponsive asthma. Lancet 1999;353:2213–4.
6 Gibson PG, Simpson JL, Saltos N. Heterogeneity of airway inflammation in
persistent asthma : evidence of neutrophilic inflammation and increased sputum
interleukin-8. Chest 2001;119:1329–36.
7 Wenzel SE, Schwartz LB, Langmack EL, et al. Evidence that severe asthma can be
divided pathologically into two inflammatory subtypes with distinct physiologic
and clinical characteristics. Am J Respir Crit Care Med 1999;160:1001–8.
8 Meijer RJ, Postma DS, Kauffman HF, et al. Accuracy of eosinophils and
eosinophil cationic protein to predict steroid improvement in asthma. Clin Exp
9 Green RH, Brightling CE, Woltmann G, et al. Analysis of induced sputum in
adults with asthma: identification of subgroup with isolated sputum neutrophilia
and poor response to inhaled corticosteroids. Thorax 2002;57:875–9.
10 Fahy JV, Kim KW, Liu J, et al. Prominent neutrophilic inflammation in sputum
from subjects with asthma exacerbation. J Allergy Clin Immunol
11 Green RH, Brightling CE, McKenna S, et al. Asthma exacerbations and
sputum eosinophil counts: a randomised controlled trial. Lancet
12 Hunter CJ, Brightling CE, Woltmann G, et al. A comparison of the validity of
different diagnostic tests in adults with asthma. Chest 2002;121:1051–7.
13 Morgan AJ, Symon FA, Berry MA, et al. IL-4-expressing bronchoalveolar T cells
from asthmatic and healthy subjects preferentially express CCR 3 and CCR 4.
J Allergy Clin Immunol 2005;116:594–600.
14 Kharitonov S, Alving K, Barnes PJ. Exhaled and nasal nitric oxide measurements:
recommendations. The European Respiratory Society Task Force. Eur Respir J
15 Juniper E, Cockroft D, Hargreave FE. Histamine and methacholine inhalation
tests: tidal breathing method; laboratory tests and standardisation, 2nd edn.
Lund, Sweden: Astra-Draco, 1994.
16 Brightling CE, Monterio W, Green RH, et al. Induced sputum and other outcome
measures in chronic obstructive pulmonary disease: safety and repeatability.
Respir Med 2001;95:999–1002.
17 Juniper EF, Guyatt GH, Ferrie PJ, et al. Measuring quality of life in asthma. Am
Rev Respir Dis 1993;147:832–8.
18 Birring SS, Parker D, Brightling CE, et al. Induced sputum inflammatory mediator
concentrations in chronic cough. Am J Respir Crit Care Med 2004;169:15–9.
19 Brightling CE, Symon FA, Birring SS, et al. Comparison of airway
immunopathology of eosinophilic bronchitis and asthma. Thorax
20 Brightling CE, Bradding P, Symon FA, et al. Mast-cell infiltration of airway
smooth muscle in asthma. N Engl J Med 2002;346:1699–705.
21 Benayoun L, Druilhe A, Dombret MC, et al. Airway structural alterations
selectively associated with severe asthma. Am J Respir Crit Care Med
22 Ward C, Pais M, Bish R, et al. Airway inflammation, basement membrane
thickening and bronchial hyperresponsiveness in asthma. Thorax
23 Birring SS, Brightling CE, Symon FA, et al. Idiopathic chronic cough: association
with organ specific autoimmune disease and bronchoalveolar lymphocytosis.
24 Douwes J, Gibson P, Pekkanen J, et al. Non-eosinophilic asthma: importance
and possible mechanisms. Thorax 2002;57:643–8.
25 Simpson JL, Scott R, Boyle MJ, et al. Inflammatory subtypes in asthma:
assessment and identification using induced sputum. Respirology
26 Godon P, Boulet LP, Malo JL, et al. Assessment and evaluation of symptomatic
steroid-naive asthmatics without sputum eosinophilia and their response to
inhaled corticosteroids. Eur Respir J 2002;20:1364–9.