Expression of the T helper 17-associated cytokines IL-17A and IL-17F in asthma and COPD

Article (PDF Available)inChest 138(5):1140-7 · November 2010with27 Reads
DOI: 10.1378/chest.09-3058 · Source: PubMed
Abstract
Asthma and COPD are characterized by airway dysfunction and inflammation. Neutrophilic airway inflammation is a common feature of COPD and is recognized in asthma, particularly in severe disease. The T helper (Th) 17 cytokines IL-17A and IL-17F have been implicated in the development of neutrophilic airway inflammation, but their expression in asthma and COPD is uncertain. We assessed IL-17A and IL-17F expression in the bronchial submucosa from 30 subjects with asthma, 10 ex-smokers with mild to moderate COPD, and 27 nonsmoking and 14 smoking control subjects. Sputum IL-17 concentration was measured in 165 subjects with asthma and 27 with COPD. The median (interquartile range) IL-17A cells/mm² submucosa was increased in mild to moderate asthma (2.1 [2.4]) compared with healthy control subjects (0.4 [2.8]) but not in severe asthma (P = .04). In COPD, IL-17A(+) cells/mm² submucosa were increased (0.5 [3.7]) compared with nonsmoking control subjects (0 [0]) but not compared with smoking control subjects (P = .046). IL-17F(+) cells/mm² submucosa were increased in severe asthma (2.7 [3.6]) and mild to moderate asthma (1.6 [1.0]) compared with healthy controls subjects (0.7 [1.4]) (P = .001) but was not increased in subjects with COPD. IL-17A and IL-17F were not associated with increased neutrophilic inflammation, but IL-17F was correlated with the submucosal eosinophil count (rs = 0.5, P = .005). The sputum IL-17 concentration in COPD was increased compared with asthma (2 [0-7] pg/mL vs 0 [0-2] pg/mL, P < .0001) and was correlated with post-bronchodilator FEV₁% predicted (r = -0.5, P = .008) and FEV(1)/FVC (r = -0.4, P = .04). Our findings support a potential role for the Th17 cytokines IL-17A and IL-17F in asthma and COPD, but do not demonstrate a relationship with neutrophilic inflammation.
1140
CHEST
Original Research
ASTHMA
Original Research
A
sthma and COPD are common conditions that
account for substantial morbidity and mortality
worldwide. Asthma affects 5% to 10% of adults, of
whom 10% have severe disease.
1,2
Severe asthma rep-
resents a disproportionate health-care burden as it
leads to debilitating chronic symptoms despite opti-
mal standard asthma treatment and contributes to
more than half of the health-care costs attributed to
asthma.
1-4
COPD is predicted to be the third leading
cause of death in 2030.
5,6
Both conditions are charac-
terized by airfl ow obstruction with airway infl amma-
tion and remodeling.
COPD is considered a neutrophilic airway dis-
ease with increased infi ltration of the airway with
CD8
1
T cells,
7
whereas asthma is characterized by
Th2 cytokine expression and eosinophilic infl amma-
tion.
8
However, there is increasing recognition that
asthma and COPD are diseases with phenotypic
heterogeneity in terms of clinical expression, airway
dysfunction, and immunopathology.
9,10
Indeed, the
Background: Asthma and COPD are characterized by airway dysfunction and infl ammation. Neu-
trophilic airway infl ammation is a common feature of COPD and is recognized in asthma, particu-
larly in severe disease. The T helper (Th) 17 cytokines IL-17A and IL-17F have been implicated
in the development of neutrophilic airway infl ammation, but their expression in asthma and
COPD is uncertain.
Methods: We assessed IL-17A and IL-17F expression in the bronchial submucosa from 30 subjects
with asthma, 10 ex-smokers with mild to moderate COPD, and 27 nonsmoking and 14 smoking
control subjects. Sputum IL-17 concentration was measured in 165 subjects with asthma and
27 with COPD.
Results: The median (interquartile range) IL-17A cells/mm
2
submucosa was increased in mild to
moderate asthma (2.1 [2.4]) compared with healthy control subjects (0.4 [2.8]) but not in severe
asthma ( P 5 .04). In COPD, IL-17A
1
cells/mm
2
submucosa were increased (0.5 [3.7]) compared
with nonsmoking control subjects (0 [0]) but not compared with smoking control subjects ( P 5 .046).
IL-17F
1
cells/mm
2
submucosa were increased in severe asthma (2.7 [3.6]) and mild to moderate
asthma (1.6 [1.0]) compared with healthy controls subjects (0.7 [1.4]) ( P 5 .001) but was not
increased in subjects with COPD. IL-17A and IL-17F were not associated with increased neutro-
philic infl ammation, but IL-17F was correlated with the submucosal eosinophil count ( r s 5 0.5,
P 5 .005). The sputum IL-17 concentration in COPD was increased compared with asthma
(2 [0-7] pg/mL vs 0 [0-2] pg/mL, P , .0001) and was correlated with post-bronchodilator FEV
1
%
predicted ( r 5 2 0.5, P 5 .008) and FEV
1
/FVC ( r 5 2 0.4, P 5 .04).
Conclusions: Our fi ndings support a potential role for the Th17 cytokines IL-17A and IL-17F in
asthma and COPD, but do not demonstrate a relationship with neutrophilic infl ammation.
CHEST 2010; 138(5):1140–1147
Abbreviations: DTT 5 dithiothreitol; ELISA 5 enzyme-linked immunosorbent assay; GINA 5 Global Initiative for
Asthma; IQR 5 interquartile range; PC
20
5 provocation concentration causing a 20% fall in FEV
1
; r 5 Pearson correlation
coeffi cient; rs 5 Spearman rank correlation coeffi cient; Th 5 T helper
Expression of the T Helper 17-Associated
Cytokines IL-17A and IL-17F in Asthma
and COPD
Camille Doe, MSc; Mona Bafadhel, MBChB; Salman Siddiqui, PhD;
Dhananjay Desai, MBBS; Vijay Mistry, BSc; Paul Rugman, PhD;
Margaret McCormick, BSc; Joanne Woods , PhD ; Richard May, PhD;
Matthew A. Sleeman, PhD; Ian K. Anderson, PhD; and Christopher E. Brightling, PhD, FCCP
www.chestpubs.org
CHEST / 138 / 5 / NOVEMBER, 2010
1141
sured the IL-17A sputum concentration and the num-
ber of IL-17A and F
1
cells in the bronchial mucosa
and assessed the degree of neutrophilic infl ammation
in the airway in asthma and COPD.
Materials and Methods
Subjects
Subjects were recruited from hospital staff and general respira-
tory and “Diffi cult Asthma” clinics at Glenfi eld Hospital, Leicester;
local primary health care; and by local advertising. Asthma was
defi ned according to the current Global Initiative for Asthma
(GINA) guidelines.
2
Subjects with asthma had typical symptoms
and the presence of one or more of the following objective crite-
ria: signifi cant bronchodilator reversibility of FEV
1
. 200 mL, a
provocation concentration of methacholine causing a 20% fall in
FEV
1
(PC
20
) of , 8 mg/mL, or a peak fl ow amplitude percent
mean over 2 weeks of . 20%. Asthma severity was classifi ed using
the GINA treatment steps (mild to moderate 1-3, severe 4-5).
2
COPD was diagnosed and severity categorized by using Global
Initiative for Chronic Obstructive Lung Disease criteria.
6
Sub-
jects with COPD who demonstrated partial bronchodilator
reversibility were not excluded. Subjects were recruited as three
independent cross-sectional groups, to assess IL-17 A, F, and IL-17R
expression in proximal airways in asthma (group 1) and COPD
(group 2), and sputum IL-17 concentration in asthma and COPD
(group 3). Subjects were free from exacerbations for at least
6 weeks. Healthy control subjects had normal spirometry and some
smokers with . 10 pack-year history were included to enable com-
parisons between healthy smokers and subjects with COPD. Sixty-
eight of 295 subjects had participated in an earlier report.
20
All
subjects gave written informed consent with study approval from
the Leicestershire ethics committee.
Protocol
Demographics and spirometry were recorded for all subjects.
Subjects with asthma and healthy control subjects in group 1
and 2 also underwent methacholine inhalation test using
the tidal breathing method
21
and allergen skin prick tests for
Dermatophagoides pteronyssinus , dog, cat, and grass pollen. Spu-
tum induction was performed in all subjects in groups 1 and 2.
22
In group 1, subjects underwent bronchoscopy conducted
according to the British Thoracic Society guidelines,
23
and
biopsies were taken from the right middle and lower lobe cari-
nae. In group 2, proximal airway samples were collected from
surgical specimens. All bronchial mucosal specimens were fi xed
in acetone and embedded in glycomethacrylate as described
previously.
24
Sputum IL-17 Measurement
Sputum was selected, dispersed using the mucolytic dithio-
threitol (DTT), and processed to generate a sputum differential
cell count, and cell-free supernatants were stored at -80°C for
later analysis as described previously.
25
Sputum IL-17 was mea-
sured by enzyme-linked immunosorbent assay (ELISA) (R&D
Systems Europe Ltd; Abingdon, England). The lower limit of
detection was 15 pg/mL. The IL-17 assay was validated in line
with European Respiratory Society recommendations to assess
the effect of DTT and the recovery of exogenous spiking with
recombinant cytokine.
25
IL-17 recovery was not affected by DTT
and mean recovery of exogenous spiked IL-17 was 93% (n 5 16).
application of induced sputum to study large groups
of patients with airway disease has suggested that
there is considerable overlap between these condi-
tions, with neutrophilic infl ammation observed in up
to 40% of patients with asthma, particularly in those
with severe disease.
11
Therefore, there is a pressing
need to further understand the potential mechanisms
involved in the initiation and persistence of neutro-
philic infl ammation in airways disease.
A distinct T-cell lineage, called T-helper (Th)17 cells,
has been identifi ed and characterized by the pro-
duction of IL-17A, IL-17F, and IL-22.
12
In addition
to CD4
1
cells, IL-17A and F can be released by
neutrophils, eosinophils, CD8
1
T cells, basophils,
and mast cells.
13
Both cytokines can induce the
expression of a variety of proinfl ammatory cytokines
and chemokines in epithelial and vascular endothelial
cells, fi broblasts, neutrophils, and eosinophils, includ-
ing IL-6, granulocyte macrophage colony-stimulating
factor, CXCL10, and CXCL8.
14
The induction of
CXCL8, a potent neutrophil chemokine, has impli-
cated these Th17 cytokines in the development of
neutrophilic airway infl ammation.
13,14
In support of
this view recent evidence from animal models dem-
onstrates that allergic sensitization through the airway
primes strong Th17 responses that promote airway
neutrophilia and airway hyperresponsiveness,
15
whereas
IL-17F-defi cient mice have an impaired neutrophilic
response to allergen.
16
In humans there is emerging
evidence to support an increase in IL-17A and IL-17F
expression in moderate to severe asthma
17,18
and
COPD.
19
However, whether this expression is associ-
ated with granulocytic infl ammation in the airway wall
or lumen is uncertain.
We hypothesized that IL-17A and IL-17F expres-
sion is increased in asthma and COPD and is related
to disease severity and the intensity of neutrophilic
infl ammation. To test our hypothesis we have mea-
Manuscript received December 21, 2009; revision accepted May
12, 2010.
Affiliations: From the Institute of Lung Health (Ms Doe;
Drs Bafadhel, Siddiqui, Desai, and Brightling; and Mr Mistry),
Leicester; AstraZeneca Charnwood (Dr Rugman and Ms McCormick),
Loughborough; and MedImmune Ltd (Drs Woods, May, Sleeman,
and Anderson), Cambridge, England.
Funding/Support: This study was funded by Asthma UK, Med-
Immune Ltd, and a Wellcome Senior Clinical Fellowship (C. B.).
Ms Doe and Dr Bafadhel contributed equally to this article.
Correspondence to: Christopher E. Brightling, PhD, FCCP,
Institute for Lung Health, University of Leicester, Clinical Sciences
Wing, Glenfi eld Hospital, Groby Rd, Leicester, LE3 9QP, England;
e-mail: ceb17@le.ac.uk
© 2010 American College of Chest Physicians. This is an
Open Access article distributed under the terms of the Creative
Commons Attribution-Noncommercial License (http://creative
commons.org/licenses/by-nc/3.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided the
original work is properly cited. Information for commercial entities
is available online ( http://www.chestpubs.org/site/misc/reprints.xhtml ).
DOI: 10.1378/chest.09-3058
1142
Original Research
not compared with smoking control subjects, ( P 5 .046,
Kruskal-Wallis; P , .05 COPD vs nonsmoking control
subjects) ( Fig 2D ). There were no differences in the
number of IL-17F
1
cells/mm
2
submucosa in lung resec-
tion tissue from subjects with COPD and control
subjects with and without a signifi cant smoking his-
tory ( Fig 2E , Table 2 ).
The number of IL-17A
1
cells was correlated with
FEV
1
% predicted ( R s 5 0.38; P 5 .04), and the sputum
neutrophil count ( R s 5 2 0.43, P 5 .03) in asthma but
was not associated with the number of neutrophils or
eosinophils in the bronchial mucosa ( Table 3 ). The
number of IL-17F
1
was positively correlated with the
number of eosinophils in tissue ( rs 5 0.50; P 5 .005)
but not signifi cantly correlated with lung function or
neutrophil number in tissue or sputum ( Table 3 ).
Sputum IL-17 Asthma and COPD
Sputum IL-17 was below the limit of detection in
all samples from subjects with asthma (n 5 22), sub-
jects with COPD (n 5 22), and control subjects (n 5 12)
measured by ELISA. Using the mesoscale device
IL-17 was also measured in a further independent population
of subjects with COPD and asthma using the mesoscale device
platform.
26
IL-17A and IL-17F Expression in Endobronchial Biopsies
Two-micrometer sections were cut and stained using poly-
clonal antibodies against IL-17A and IL-17F (R&D Systems
Europe Ltd), monoclonal antibodies against eosinophils (major
basic protein), neutrophils (neutrophil elastase), and mast cells
(tryptase), with appropriate isotype controls mouse IgG1 (Dako
UK Ltd; Cambridge, England) and goat immunoglobulins (R&D
Systems Europe Ltd). The number of positive nucleated cells
was enumerated per mm
2
of bronchial submucosa by a blinded
observer as described previously.
27,28
In a subgroup of six subjects
with asthma, IL-17A and IL-17F were colocalized with neutrophils
and eosinophils using sequential sections as previously described.
27
Statistical Analysis
Statistical analysis was performed using PRISM Version 4
(GraphPad; La Jolla, CA). Parametric data were expressed as mean
(SEM), data that had a normal log distribution were log transformed
and described as geometric mean (95% CI), and nonparametric
data were described as median (interquartile range [IQR]). One-way
analysis of variance (Kruskal-Wallis for nonparametric data) was
used for across-group comparisons with Tukey and Dunn post hoc
tests for between-group comparisons, respectively. Correlations
were assessed by Spearman rank ( rs ) and Pearson ( r ) correlation
coeffi cients. No corrections were made for multiple comparisons.
Results
IL-17A, IL-17F, and IL-17R Expression
in Large Airway Tissue Specimens
Clinical characteristics of group 1 are as shown
in Table 1 . Examples of IL-17A
1
and IL-17F
1
cells in
the bronchial submucosa in asthma are as shown in
Figures 1A-1C . The median (IQR) IL-17A cells/mm
2
submucosa was increased in mild to moderate asthma
(2.1 [2.4]) compared with healthy control subjects
(0.4 [2.8]) but not compared with asthma (0.9 [4.2])
( P 5 .04, Kruskal-Wallis; P , .05 mild to moderate
asthma vs control subjects) ( Fig 1D ). The number
of IL-17F
1
cells/mm
2
submucosa was increased
in severe asthma (2.7 [3.6]) and mild to moderate
asthma (1.6 [1.0]) compared with healthy control
subjects (0.7 [1.4]) ( P 5 .001 Kruskal-Wallis; P , .01
severe asthma vs control subjects, and P , .05 mild to
moderate vs control subjects) ( Fig 1E ). The median
(IQR) proportion 50% (27%) and 24% (19%) of the
IL-17A
1
and 44% (40%) and 0 (25%) of IL-17F cells
colocalized to neutrophils and eosinophils, respectively.
Clinical characteristics of group 2 are as shown in
Table 2 . All of the COPD subjects were ex-smokers.
Examples of IL-17A
1
and IL-17F
1
cells in the
bronchial submucosa in COPD are as shown in
Figures 2A-2C . The median (IQR) IL-17A
1
cells/mm
2
submucosa was increased in COPD (0.5 [3.7]) com-
pared with nonsmoking control subjects (0 [0]), but
Table 1Clinical and Sputum Characteristics of Biopsy
Group Asthma
Characteristic Normal
Mild to
Moderate
Asthma
(GINA 1-3)
Severe Asthma
(GINA 4-5)
No. 17 17 13
Age
a
45 (5) 49 (4) 50 (4)
Men/women 7/10 8/9 6/7
Never smokers/current
smokers/ex-smokers
15/2/0 12/5/0 8/5/0
Pack-years
a
0.4 (0.3) 3 (1) 5 (3)
Atopy, No. (%) 6 (35) 9 (53) 7 (54)
PC
20
FEV
1
, mg/mL
b
. 16
0.33 (0.14-0.78)
c
0.38 (0.1-1.4)
c
FEV
1
, % predicted
a
99 (4) 87 (5) 80 (7)
c
Pre-BD FEV
1
/FVC, %
a
78 (3) 74 (3) 74(3)
BD response, %
a
1 (1) 9 (4)
c
13 (6)
c
Sputum cell counts
TCC
a
1.9 (0.4) 2.7 (0.7) 2.7 (0.6)
Eosinophil, %
d
0.3 (0.9) 0.5 (3.2) 6.6 (14.6)
c
Neutrophil, %
a
43 (12) 54 (6) 59 (10)
Macrophage, %
a
51 (9) 38 (6) 25 (6)
c
Lymphocyte, %
a
1.8 (0.8) 1.0 (0.2) 1.5 (0.7)
Epithelial cells, %
a
3 (2) 4 (1) 7 (3)
Cells/mm
2
submucosa
IL-17A
d
0.4 (2.8) 2.1 (2.4)
c
0.9 (4.2)
IL-17F
d
0.7 (1.4) 1.6 (1.0)
c
2.7 (3.6)
c
Mast cells
d
24.4 (23) 28.8 (31.8) 25.8 (56.5)
Neutrophils
d
8 (13) 9.3 (12.7) 8.2 (11.5)
Eosinophils
d
3.0 (9) 6.2 (18.9) 10.0 (41.2)
BD 5 bronchodilator; GINA 5 Global Initiative for Asthma; IQR 5 inter-
quartile range; PC
20
5 provocation concentration causing a 20% fall in
FEV
1
; SE 5 standard error ; TCC 5 total cell count .
a
Mean (SE).
b
Geometric mean (95% CI).
c
P , .05 compared with control subjects.
d
Median (IQR).
www.chestpubs.org
CHEST / 138 / 5 / NOVEMBER, 2010
1143
post-bronchodilator FEV
1
% predicted ( r 5 2 0.5,
P 5 .008) and FEV
1
/FVC ( r 5 2 0.4, P 5 .04) ( Figs 3A-3C ).
The clinical characteristics of these subjects with
asthma and COPD are as shown ( Table 4 ).
Discussion
We report here that IL-17A expression in the bron-
chial submucosa was increased in mild to moderate
platform the sputum IL-17 concentration in COPD
was increased compared with asthma (2 [0-7] pg/mL
vs 0 [0-2] pg/mL, P , .0001) and was correlated with
Figure 1. IL-17A and IL-17F expression in the submucosa in
asthma. Representative photomicrographs of bronchial biopsy
sections from subjects with severe asthma, illustrating isotype
control. A, Goat IgG. B, IL-17A
1
cells present in the bronchial
submucosa. C, IL-17F
1
cells in the submucosa ( 3 400). IL-17A/F
1
cells highlighted by arrows. D, The number of IL-17A
1
and
E, IL-17F
1
cells in the bronchial submucosa of healthy control sub-
jects, subjects with mild to moderate asthma (Global Initiative for
Asthma [GINA] 1-3), and subjects with severe asthma (GINA 4-5).
P values for across-group comparison by Kruskal-Wallis test and
Dunn post hoc test for between-group comparisons are as shown.
Table 2Clinical Characteristics and IL-17A and
IL-17F Expression in COPD
Characteristic Normal Smoker COPD
No. 10 14 10
Age
a
59 (3) 53 (5) 65 (3)
Men (women) 8 (2) 10 (4) 7 (3)
Never smokers/current
smokers/ex-smokers
8/0/2 0/0/14 0/0/10
Pack-years
a
2 (1) 28 (5) 37 (6)
b
FEV
1
a
2.7 (0.2) 2.6 (0.2) 1.7 (0.2)
b
FEV
1
, % predicted
a
86 (3) 84 (3) 62 (3)
b
Pre-BD FEV
1
/FVC
a
78 (2) 78 (1) 56 (3)
b
Cells/mm
2
submucosa
IL-17A 0 (0) 0 (1.1) 0.5 (3.7)
b
IL-17F 0 (1.7) 0 (0) 0.2 (2)
See Table 1 legend for expansion of the abbreviation.
a
Mean (SE).
b
P , .05 compared with nonsmoking control subjects.
Figure 2. IL-17A and IL-17F expression in the submucosa in
COPD. Representative photomicrographs of proximal airway
resections from COPD tissue, illustrating isotype control. A, Goat
IgG. B, IL-17A
1
cells present in the submucosa. C, IL-17F
1
cells
in the submucosa ( 3 400). IL-17A/F
1
cells highlighted by arrows.
D, The number of IL-17A
1
and E, IL-17F
1
cells in the submucosa
of healthy control subjects (never smokers), healthy control sub-
jects (smokers), and subjects with COPD. P values for across-
group comparison by Kruskal-Wallis test and Dunn post hoc test
for between-group comparisons are as shown.
Table 3— Correlations Between IL-17A/F, Granulocytic
Infl ammation, and Lung Function in Asthma
Characteristic IL-17A IL-17F
Neutrophils/mm
2
submucosa
r s 5 2 0.05, P 5 .8 r s 5 0.32, P 5 .078
Sputum neutrophils (%)
r s 5 2 0.43, P 5 .03 r s 5 2 0.15, P 5 .45
Eosinophils/mm
2
submucosa
r s 5 0.2, P 5 .3 r s 5 0.50, P 5 .005
Sputum eosinophils, %
r s 5 0.09, P 5 .7 r s 5 0.08, P 5 .7
FEV
1
, % predicted
r s 5 0.38, P 5 .04 r s 5 2 0.06, P 5 .8
FEV
1
/FVC, %
r s 5 0.35, P 5 .06 r s 5 2 0.17, P 5 .39
Methacholine PC
20
FEV
1
r s 5 2 0.17, P 5 .5 r s 5 2 0.25, P 5 .25
rs 5 Spearman rank correlation coeffi cient. See Table 1 legend for
expansion of the other abbreviation.
1144
Original Research
submucosa. However, there was a good correlation
between the number of IL-17F
1
cells and eosino-
phils in the bronchial submucosa. In COPD there
was a highly signifi cant, albeit small, increase in spu-
tum IL-17 compared with asthma, and this was cor-
related with airflow obstruction and lung function
impairment, but not airway infl ammation. Our study
therefore supports our hypothesis that IL-17 may
play a role in asthma and COPD.
There is an increasing body of evidence supporting
a role for Th17 cells in asthma.
13,14
Animal models of
asthma suggest that Th17 cytokines promote neutro-
philic infl ammation,
15,16
which in concert with Th2 cells
is important in the development of airway hyperre-
sponsiveness.
17
Previous reports have demonstrated
increased IL-17A and IL-17F bronchial submucosa
expression in moderate to severe asthma,
17,18
which
were attenuated by systemic corticosteroids.
17
The
data for sputum IL-17 is confl icting with one study
demonstrating no differences in the sputum IL-17 con-
centration between asthma and healthy volunteers,
29
whereas in another study IL-17 was increased in
moderate to severe asthma.
30
In the latter study spu-
tum IL-17 was measured before and after inhaled
corticosteroids. Importantly, consistent with our fi nd-
ings previous studies have found that the sputum
IL-17 concentration in sputum is low and in some
studies samples have needed to be concentrated to
detect IL-17. We found that IL-17A and IL-F were
increased in mild to moderate disease, whereas IL-17F
but not IL-17A was increased in severe asthma. Our
subjects with severe asthma were treated with high-
dose inhaled and/or oral corticosteroids, and there-
fore this may have attenuated the IL-17A expression
in this group. IL-17A and IL-17F expression was not
associated with smoking status but importantly this
study was not powered to fully explore the effects of
asthma, and IL-17F was increased in mild to moderate
and severe asthma. In contrast, in ex-smoking subjects
with mild to moderate COPD the number of IL-17A
1
cells in the bronchial submucosa, but not IL-17F,
was increased compared with nonsmoking control
subjects. In asthma there was a weak relationship
between the number of IL-17A
1
cells and the FEV
1
%
predicted, and the sputum neutrophil count. There
was no association between neutrophilic infl amma-
tion and IL-17A or F expression in the bronchial
Figure 3. Sputum IL-17 in asthma and COPD. A, Sputum IL-17
concentration in asthma and COPD. Horizontal bar represents the
median. B, Correlations between sputum IL-17 concentration in
subjects with COPD and postbronchodilator FEV
1
% predicted.
C, Correlations between sputum IL-17 concentration in subjects
with COPD and FEV
1
/FVC.
Table 4Clinical Characteristics and IL-17A and
IL-17F Sputum Concentration in Asthma and COPD
Characteristic COPD (n 5 27) Asthma (n 5 165)
Age
a
68 (1.6) 50 (1.1)
Men, % 74 41
Never smokers/current
smokers/ex-smokers
0/18/9 109/44/12
Pack years
a
47 (6) 15 (3)
Post-BD FEV
1
, L
a
1.1 (0.1) 2.2 (0.1)
Post-BD FEV
1
, % predicted
a
42 (3) 76 (2)
Post-BD FEV
1
/FVC %
a
44 (2) 70 (1)
Sputum neutrophil count, %
a
65 (5) 63 (2)
Sputum eosinophil count, %
b
1.5 (0.9-2.7) 2.7 (2.1-3.5)
Sputum IL-17, pg/mL
c
2 (0-7) 0 (0-2)
See Table 1 legend for expansion of the abbreviation.
a
Mean (SE).
b
Geometric mean (95% CI).
c
Median (IQR).
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CHEST / 138 / 5 / NOVEMBER, 2010
1145
IL-17 concentration in COPD was related to the
degree of airfl ow limitation and obstruction. Therefore,
the potential role of IL-17, particularly in severe dis-
ease, and its synergy with the development of airway
infl ammation in response to cigarette smoke exposure
or alternatively through interactions with other cyto-
kines, such as tumor necrosis factor
35
or IL-1 b ,
36
needs
to be further explored.
Neutrophils were the predominant source of
IL-17 as evidenced by colocalization using immuno-
histochemistry. However, there was no correlation
between the number of IL-17
1
cells and neutrophils
in the bronchial submucosa. In contrast to the estab-
lished role of IL-17A and IL-17F in the initiation of
neutrophilic infl ammation, neither has been impli-
cated in the development of eosinophilic infl am-
mation. Indeed, IL-17F transgenic mice develop an
airway neutrophilia after allergen challenge, whereas
knockout mice have an impaired neutrophilic response
and enhanced Th2 cytokine production.
16
Therefore,
we were surprised by the relationship between the
number of IL-17F
1
cells and eosinophils in the bron-
chial submucosal. However, a similar relationship has
been described between sputum IL-17 mRNA and
IL-5 mRNA,
33
possibly suggesting a hitherto unde-
scribed association between eosinophilic airway infl am-
mation and the Th17 axis. Eosinophils express the
IL-17 receptors and have enhanced cytokine release
after IL-17F activation.
37
Thus, the potential interactions
between eosinophilic infl ammation and up-regulation
of the Th17 axis require further study.
Our study has a number of possible criticisms. This
is a cross-sectional observational study. Whether
IL-17A and IL-17F expression is related to longitudi-
nal clinical outcomes, such as disease progression,
lung function decline, and exacerbations, requires fur-
ther examination. Similarly, we are unable to deter-
mine whether differences observed between mild and
severe asthma refl ect disease severity or are a conse-
quence of differences in treatment. Current litera-
ture suggests that IL-17A is sensitive to corticosteroid
therapy,
17,30
in contrast to neutrophilic infl ammation,
which is corticosteroid resistant.
31,32
Importantly, the
number of IL-17A or IL-17F cells was expressed as
cells/mm
2
of submucosa rather than as a proportion
of the total number of cells in the submucosa. There-
fore, whether the changes in cell number between
groups is a refl ection of increased expression or an
increase in the total number of cells needs to be
examined in future studies. We are confi dent that our
assays to assess IL-17 were robust as recovery was
unaffected by the mucolytic DTT and recovery of
exogenous spikes of IL-17 to sputum samples was
good. However, a number of samples were below
the limit of detection; therefore, the sputum data do
need to be interpreted cautiously. Furthermore, no
smoking in asthma. We report for the fi rst time, to
our knowledge, the relationship between IL-17A and
F expression and neutrophilic infl ammation in tissue
and sputum. We found that there was no associa-
tion between IL-17A and IL-17F expression neutro-
philic infl ammation in tissue. Intriguingly, there was a
weak inverse correlation between submucosal IL-17A
expression and the sputum neutrophil count. Corti-
costeroids attenuate IL-17 expression in tissue
17
but
do not modulate sputum neutrophil counts.
31,32
Spu-
tum IL-17 was also markedly attenuated by inhaled
corticosteroids, but the sputum neutrophil count was
unchanged, questioning the role of IL-17 in per-
sistent neutrophilic airway infl ammation. However,
others have demonstrated an association between
IL-17 and IL-8 mRNA in sputum cells and the num-
ber of sputum neutrophils,
33
suggesting that the site
of IL-17 expression may be important. Interestingly,
we also found a weak relationship between FEV
1
%
predicted and IL-17A expression. Consistent with
our fi nding in a recent comparison between subjects
with severe asthma with and without persistent air-
ow obstruction, IL-17 was among the dominant
mediators associated with the absence of persistent
airfl ow obstruction.
34
Together these fi ndings do not
support a role for IL-17 in the development of fi xed
airfl ow obstruction in asthma.
To date there are few data examining the expression
of IL-17A or IL-17F in COPD. Neutrophilic infl am-
mation is a common feature of COPD.
7,11
Hence, it is
predictable that IL-17 may play an important role in
this disease. There is a single report of IL-17A and F
expression in bronchoscopic biopsies obtained across
the spectrum of COPD disease severity compared
with smoking and nonsmoking control subjects.
15
IL-17A
was increased equally across the severity of COPD
compared with the nonsmoking control subjects, but
was not different from the smoking control subjects.
IL-17F was not signifi cantly different across all the
disease and control groups. In this study the number
of neutrophils in tissue was increased in the COPD
groups with a marked increase in severe COPD. There
was no relationship between IL-17A and IL-F expres-
sion and neutrophilic infl ammation. Our fi ndings
of increased IL-17A expression in mild to moderate
COPD compared with nonsmoking control subjects,
but not smoking control subjects, is therefore entirely
consistent with these earlier reports. We found that
the sputum IL-17 concentration was increased in COPD
compared with asthma. However, we were unable to
detect IL-17 by ELISA, and with a more sensitive
mesoscale device platform a substantial proportion
still remained below the limit of detection. The differ-
ence between COPD and asthma was highly signifi -
cant, but small, questioning whether this difference
is biologically important. Interestingly, the sputum
1146
Original Research
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corrections for multiple comparisons were made.
Therefore, fi ndings with a marginal level of signifi -
cance also need to be interpreted with caution.
In conclusion, we found that IL-17A expression
in bronchial submucosa was increased in mild to
moderate asthma and in COPD, although this was not
independent of smoking. IL-17F expression was
increased in mild to moderate and severe asthma,
but not in COPD. The increased IL-17A and IL-17F
expression was not associated with increased neutro-
philic infl ammation. Sputum IL-17 was increased in
COPD and was related to airfl ow obstruction. Our
ndings therefore do support a potential role for
IL-17A and IL-17F in asthma and possibly COPD.
Effi cacy studies of therapeutic strategies targeted at
the IL-17 axis are eagerly awaited that will further
defi ne the functional importance of IL-17A and IL-17F
in airways disease.
Acknowledgments
Author contributions: Ms Doe: contributed to immunohis-
tochemistry analysis and drafting the original manuscript, and
contributed to and approved the fi nal manuscript.
Dr Bafadhel: contributed to supervising clinical characterization,
coordinating the sputum sampling and analysis, and performing
bronchoscopies, and contributed to and approved the fi nal manu-
script.
Dr Siddiqui: contributed to supervising clinical characterization,
coordinating the sputum sampling and analysis, and performing
bronchoscopies, and contributed to and approved the fi nal manu-
script.
Dr Desai: contributed to supervising clinical characterization, coor-
dinating the sputum sampling and analysis, and performing bron-
choscopies, and contributed to and approved the fi nal manu script.
Mr Mistry: contributed to undertaking laboratory characteriza-
tion of patients and ELISAs, and contributed to and approved the
nal manuscript.
Dr Rugman: contributed to performing mesoscale measurements
and contributed to and approved the fi nal manuscript.
Ms McCormick: contributed to performing mesoscale measure-
ments and contributed to and approved the fi nal manuscript.
Dr Woods: contributed to performing the mesoscale measurements
and contributed to and approved the fi nal manuscript.
Dr May: contributed to performing mesoscale measurements
and contributed to and approved the fi nal manuscript.
Dr Sleeman: contributed to conception of the project design and
contributed to and approved the fi nal manuscript.
Dr Anderson: contributed to conception of the project design and
contributed to and approved the fi nal manuscript.
Dr Brightling: contributed to conception of the project design
and contributed to and approved the fi nal manuscript.
Financial/nonfi nancial disclosures: The authors have reported
to CHEST the following confl icts of interest: Dr Brightling
has received consultancy fees from MedImmune, AstraZeneca,
Glaxo SmithKline, Roche, and Genentech Inc, and research grants
from AstraZeneca, MedImmune, and GlaxoSmithKline. Dr Woods
owns stock in AstraZeneca Pharmaceuticals. Drs Woods, May,
Sleeman, and Anderson are employees of MedImmune. Dr Rugman
and Ms McCormick are employees of AstraZeneca. Ms Doe,
Drs Bafadhel, Siddiqui, Desai, and Mr Mistry have reported to
CHEST that no potential confl icts of interest exist with any compa-
nies/organizations whose products or services may be discussed in
this article.
Other contributions: We thank Mss Sue Mckenna and Beverley
Hargadon for assistance with clinical characterization of the sub-
jects and Mr William Monteiro and Ms Natalie Neale for techni-
cal support.
www.chestpubs.org
CHEST / 138 / 5 / NOVEMBER, 2010
1147
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    • "Furthermore , IL-17A promoted stabilization of CXCL8 mRNA in BEAS-2B cells treated with poly (I:C). IL-17A was reported to be highly expressed in lung tissues of patients with chronic inflammatory respiratory diseases compared with healthy persons, and to be associated with lung function decline [22, 23]. IL-17A is suggested to play a key role in neutrophilic inflammation. "
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    Full-text · Article · Oct 2015
    • "Nevertheless, the enhanced expression of Il17 in the lungs of CD2CREMαtg mice does not result in a more severe asthma phenotype. This is interesting since IL-17 has been considered to be a central cytokine in asthma pathogenesis454647. On the other hand, Schnyder-Candrian et. "
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    Eva VerjansEva VerjansKim OhlKim OhlLucy K ReissLucy K Reiss+1more author...[...]
    • "The IL-17 cytokine superfamily triggers the production of different cytokines resulting in the recruitment of inflammatory cells. There is increasing evidence for elevated IL-17 expression in chronic obstructive lung disorders [9,10]. IL-17A is not only expressed by T-cells but also other inflammatory cells including mast cells [11] and it is up-regulated by cigarette smoke exposure in human lung tissue explants [12] being likely involved in inflammatory cell recruitment in smoking-induced lung injury. "
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