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* From the Airways Research Centre, Department of Respiratory and Sleep Medicine, John Hunter Hospital, Newcastle, Australia.
Correspondence to: Peter G. Gibson, MBBS (Hons), Department of Respiratory and Sleep Medicine, John Hunter Hospital, Locked Bag 1 Hunter Mail Centre, Newcastle NSW 2310, Australia; e-mail: mdpgg{at}mail.newcastle.edu.au
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Abstract |
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Methods: Nonsmoking adults (n = 56) with persistent asthma and healthy control subjects (n = 8) underwent hypertonic saline solution challenge and sputum induction. Selected sputum portions were dispersed with dithiothreitol and assayed for total cell count, cellular differential, supernatant eosinophil cationic protein (ECP), myeloperoxidase, and interleukin (IL)-8.
Results: We identified two distinct inflammatory patterns. Typical eosinophilic inflammation occurred in 41% of subjects, whereas the remainder exhibited noneosinophilic asthma (59%). Both neutrophil percentage and absolute neutrophil counts were increased in subjects with noneosinophilic asthma (64%, 283 x 106/mL) compared to eosinophilic asthma (14%, 41 x 106/mL) and control subjects (34%, 49 x 106/mL; p = 0.0001). Myeloperoxidase was elevated in both noneosinophilic (280 ng/mL) and eosinophilic groups (254 ng/mL) compared with control subjects (82 ng/mL; p = 0.002). Sputum IL-8 levels were highest in subjects with noneosinophilic asthma (45 ng/mL) compared to eosinophilic asthma (9.6 ng/mL) and control subjects (3.5 ng/mL; p = 0.0001). Neutrophils correlated with IL-8 levels (r = 0.72). ECP was highest in subjects with eosinophilic asthma (2,685 ng/mL) compared with noneosinophilic asthma (1,081 ng/mL) and control subjects (110 ng/mL; p = 0.0001).
Conclusion: Induced-sputum analysis in persistent asthma identifies two different inflammatory patterns. The most common pattern is noneosinophilic, associated with a neutrophil influx and activation, which may be mediated by IL-8 secretion. There is heterogeneity of airway inflammation in persistent asthma, which indicates differing mechanisms and may impact on treatment responses.
Key Words: asthma • eosinophil • induced sputum • inflammation • interleukin-8 • neutrophil
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Persistent asthma comprises up to 15% of cases of asthma, and is more common than severe refractory asthma.1 The characteristics of airway inflammation in subjects with persistent asthma are not well defined. In particular, the frequency of noneosinophilic disease and neutrophil influx is not known. We tested the hypothesis that induced-sputum analysis could be used to identify two different inflammatory subtypes in persistent asthma: a typical eosinophilic response and a noneosinophilic pattern. Furthermore, we hypothesized that persistent noneosinophilic asthma would be associated with evidence of neutrophilic inflammation characterized by increased sputum neutrophils, release of neutrophil myeloperoxidase, and the presence of increased amounts of IL-8 in sputum supernatant.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1,000 µg, fluticasone
propionate 500 µg, or budesonide 1,000 µg. All patients
were receiving inhaled, short-acting,
ß2-agonist rescue medication on an as-needed
basis for the relief of asthma symptoms. We excluded subjects if they
failed to meet the inclusion criteria or if they had received any
systemic corticosteroids during the previous 4 weeks, had any serious
concurrent diseases, or had a recent (past 4 weeks) asthma
exacerbation, clinical evidence of a respiratory tract infection, or
oral candidiasis. Control subjects were asymptomatic, healthy subjects
with normal spirometry findings and airway responsiveness. The Hunter
Area Health Service Research Ethics Committee approved the protocol,
and informed written consent was obtained from each participant.
Clinical Assessment
At the study visit, demographic details of age, sex, ethnic
origin, height, weight, and history of atopic disease were collected.
An asthma history was taken, including asthma duration, smoking
history, the number of hospitalizations for asthma, and courses of oral
steroids during the last year. Recent asthma symptoms (in the past 2
weeks) were assessed, including morning asthma, nocturnal waking due to
asthma, asthma symptoms overall, any severe attacks, and frequency of
rescue medication use. Any concurrent medical conditions and
medications were recorded. Subjects were also assessed by history and
clinical examination for the presence of a respiratory tract infection
and oral candidiasis, and were excluded if either was present. A
hypertonic saline solution bronchial provocation challenge and sputum
induction were performed. Subjects subsequently recorded symptoms and
peak expiratory flow in a daily diary for 2 weeks.
The severity of persistent asthma was classified as mild persistent (step 2), moderate persistent (step 3), or severe persistent (step 4), using National Heart, Lung, and Blood Institute guidelines.1 Subjects were placed in the highest severity category based on assessment of clinical and spirometric criteria. Subjects with mild persistent asthma exhibited any of the following: FEV1 > 80% predicted, nighttime symptoms more than twice per month, and/or symptoms more than twice per week but less than once a day. Moderate persistent asthma was defined by daily symptoms, and short-acting ß2agonist requirements, nighttime symptoms more than once per week, FEV1 > 60% and < 80% predicted, or peak expiratory flow variability > 30%. In severe persistent asthma, there were continual symptoms, limited physical activity, frequent nighttime symptoms, or FEV1 < 60% predicted.
Spirometry
Subjects withheld taking short-acting
ß2-agonists for 4 h before testing.
Baseline spirometry was performed using a Minato Autospiro AS-600
(Minato Medical Science; Osaka, Japan). Subjects were asked to perform
three reproducible FEV1 and FVC maneuvers while
in a sitting position and wearing nose clips. Results were compared
with published predicted values.14
Saline Solution Challenge
Hypertonic saline solution (4.5%) was inhaled for doubling time
periods (30 s, 1 min, 2 min, 4 min) from a DeVilbiss 2000 ultrasonic
nebulizer (DeVilbiss Health Care; Somerset, PA) with 23-cm
corrugated tubing and a Hans Rudolph 2700, two-way nonrebreathing valve
box (Hans Rudolph; Kansas City, MO) with a rubber mouthpiece and nose
clips, as described.15
The nebulizer output was 1.8 mL/min
and particle size (mass median aerosol diameter) < 5 µm.
FEV1 was measured 60 s after each saline
solution dose. The test was stopped when the FEV1
had fallen by 20% or 15 min (cumulative) of nebulization time had
elapsed. If FEV1 had fallen 20% during the
challenge, ß2-agonist was administered using a
metered-dose inhaler, and the challenge continued when
FEV1 had recovered to within 10% of baseline.
The dose of saline solution delivered to the patient was determined by
weighing the nebulizer cup and tubing before and after the challenge.
Sputum Induction
Sputum induction was conducted concurrently with the saline
solution challenge, as described.15
Before the hypertonic
saline solution challenge was commenced, the sputum-induction procedure
was explained to the subject. The technician demonstrated how to cough
and clear the throat, in order to propel mucus from the lungs into the
mouth, and then to empty these contents into a sterile sputum
container. Subjects were asked to rinse their mouth with water before
the procedure to help eliminate squamous cell contamination of the
sputum sample. They were asked to cough between each dose of nebulized
saline solution to clear their throats and expectorate into the
container. This procedure continued until an adequate sample,
containing > 0.5 mL visible mucocellular material was obtained. If a
satisfactory sputum sample was not obtained at the time the
FEV1 had fallen 20%, nebulization with 4.5%
saline solution was continued for 4-min periods once the
FEV1 had returned to within 10% of baseline.
Sputum Processing
Sputum was selected from saliva and processed as
described.15
Briefly, sputum was treated by adding four
volumes of 0.1% dithiothreitol (Sputolysin 10%; Calbiochem; La Jolla,
CA) and mixed by rotating for 30 min at 37°C, followed by four
volumes of phosphate-buffered saline solution. The suspension was
filtered through a 60-µm nylon gauze (Millipore; North Ryde, NSW,
Australia) and a total cell count of leukocytes and viability were
determined. The cell suspension was centrifuged at 200g for
10 min, and supernatant was aspirated and stored at - 70°C. The
cell pellet was resuspended in phosphate-buffered saline solution to
attain a concentration of 1 x 106 cells/mL and
70 µL placed into cups of a Shandon III cytocentrifuge (Shandon
Cytospin; Sewickey, PA) for slide preparation.
Cytochemistry
A differential count was obtained from 400 cells counted
on May-Grunwald-Giemsa–stained cytopreps. Eosinophils were enumerated
from slides stained with Chromotrope 2R in the same fashion. Cells
staining positive for the secreted form of eosinophil cationic protein
(ECP) were identified using a monoclonal antibody (EG2+; Pharmacia;
Cambridge, MA) and detected using the alkaline phosphatase antialkaline
phosphatase (APAAP) technique, as described.12
These cells
were further differentiated as eosinophils or macrophages based on
conventional morphologic criteria, and the proportion of macrophages
staining positive for EG2+ was evaluated to
identify whether enhanced eosinophil apoptosis and macrophage ingestion
could explain the paucity of eosinophils in subjects with
noneosinophilic asthma.16
IL-8 positive cells were
detected using a monoclonal antibody (mouse anti-human IL-8;
Pharmingen; San Diego, CA) and detected by the APAAP technique.
Positive and antibody (mouse IgG1) controls were
included in each staining run.
Fluid Phase Measurements
The concentrations of ECP, myeloperoxidase, and IL-8 were
determined in sputum supernatant, by radioimmunoassay (ECP RIA; Kabi
Pharmacia Diagnostics AB; Uppsala Sweden) and by enzyme-linked
immunosorbent assay (MPO; Oxis International; Portland, OR, and IL-8;
R&D Systems; Minneapolis, MN) with standard curves based on dilutions
of purified ECP, myeloperoxidase, or recombinant IL-8, respectively.
The limits of detection of the fluid phase assays were 2 ng/mL, 1.6
ng/mL, and 32 pg/mL, respectively.
Analysis
Airways responsiveness was assessed as the
PD20 and was log transformed for analysis.
Results are presented as mean/median with SD/SE/interquartile range
(IQR) as appropriate. Fluid phase measures and absolute cell counts
were log transformed for analysis. Group comparisons were conducted
using analysis of variance with Bonferonni post hoc testing
for normally distributed variables and Kruskall-Wallis testing for
nonparametric data. Associations between variables were examined using
Pearson’s or Spearman correlation coefficients as appropriate.
Significance was accepted when p < 0.05. A priori, we
defined subjects as having sputum eosinophils within the normal range
(< 2.5%, noneosinophilic), or as having increased sputum eosinophils
(> 2.5%), based on our previously reported sputum normal
values.8
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Discussion |
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Airway inflammation in asthma may be more heterogeneous than previously thought. Wenzel et al2 3 described increased airway neutrophils in patients with severe, refractory prednisone-dependent asthma, where neutrophils were increased in the airway lumen, airway wall, and lung interstitium. These changes occurred in both eosinophilic and noneosinophilic refractory asthma.2 3 Pavord et al4 reported noneosinophilic asthma in mild asthmatic subjects, and Turner et al5 described noneosinophilic asthma during a study of exacerbations of asthma. Neutrophils were not increased in these subjects. Our results confirm the existence of noneosinophilic asthma, and extend prior observations by reporting the comparatively high prevalence of noneosinophilic inflammation in persistent asthma. Future work should evaluate the frequency of noneosinophilic asthma in steroid-naïve asthmatic patients.
We have identified a role for neutrophilic inflammation in noneosinophilic persistent asthma. There were increases in both relative and absolute neutrophil numbers, myeloperoxidase, and the potent neutrophil chemoattractant, IL-8. The role of neutrophils in symptomatic asthma is controversial. In stable asthma, there is no clear indication of increased neutrophils8 9 ; however, in viral-induced exacerbations, increased neutrophils are seen.6 10 11 12 13 Neutrophil products can cause airway narrowing, increased mucus secretion,17 and increased airway smooth-muscle responsiveness.18
The sputum neutrophilia in noneosinophilic asthma could represent an effect of the disease or of its treatment.2 3 Corticosteroids do not reduce neutrophilic inflammation in airway disease,19 and persistent airway neutrophilia could even be maintained by corticosteroid-induced inhibition of neutrophil apoptosis.20 Alternatively, IL-8 could mediate increased neutrophil influx and reduced apoptosis.20 It is also possible that structural airway wall changes such as chronic bronchitis, bronchiectasis, or subepithelial fibrosis could be responsible for the airway neutrophilia, since neutrophilic inflammation is characteristic of these diseases.19 21
Our data support a role for IL-8 in mediating the neutrophil influx in noneosinophilic asthma, since IL-8 levels were increased in noneosinophilic asthma and were correlated with neutrophil numbers. IL-8 is a potent chemokine that acts as a chemoattractant and activating agent for both neutrophils and IL-5 primed eosinophils. IL-8 seems to be the main chemoattractant for neutrophils in the lungs.22 Although IL-8 is produced by a large number of cells, in this study we found that the neutrophil was the main airway lumenal cell containing IL-8, suggesting autocrine secretion of IL-8 in persistent asthma. Levels of IL-8 correlated with ECP and myeloperoxidase in sputum supernatant, which is consistent with a role for IL-8 as a degranulating agent for eosinophils and neutrophils. Recently, Page et al23 reported that eosinophil major basic protein could induce IL-8 production by neutrophils. Thus, the correlation of ECP with IL-8 is also consistent with eosinophil degranulation contributing to neutrophil IL-8 release in noneosinophilic asthma. IL-8 levels were high despite the use of inhaled corticosteroids. The data on suppression of IL-8 by corticosteroids are variable, but suggest that there is some, although incomplete suppression. In vitro IL-8 release from epithelium and smooth muscle is reduced by up to 30% with corticosteroid treatment,24 whereas steroids do not inhibit IL-8 release from a human mast cell line. In vivo, inhaled steroid can reduce sputum and serum IL-8 by 30 to 60% in bronchiectasis and COPD.25 26 The persistence of high levels of sputum IL-8 is consistent with incomplete suppression of IL-8 release by corticosteroids. There is clearly a need to identify alternative treatments to target IL-8 in airway inflammation.
The observation of high levels of sputum ECP in noneosinophilic asthma is interesting. Typically, ECP is associated with eosinophil inflammation, and in this study the highest levels were observed in the group with eosinophilic asthma.27 However, increased ECP has been seen in the absence of eosinophils in other neutrophil-mediated airway diseases such as bronchiectasis28 and chronic obstructive airway disease.29 This raises the possibility that neutrophils may contain ECP, which has been demonstrated by Sur et al.30
Another interesting feature was the persistence of clinically active disease and AHR in the absence of eosinophilia. We investigated the possibility that the known ability of corticosteroids to induce eosinophil apoptosis16 and consequent macrophage ingestion could have caused rapid clearance of eosinophils from the airway in noneosinophilic asthma. The data do not support this hypothesis, as there were few, and in some cases, no macrophages containing eosinophil proteins in noneosinophilic asthma. In contrast, the eosinophil group had strong evidence of active eosinophil turnover with 15% of macrophages staining positive for ECP. This figure is higher than previously observed in moderate asthma.16
In conclusion, this study demonstrates that there is heterogeneity of airway inflammation in persistent asthma. The typical eosinophilic pattern occurs in a minority of subjects. The majority of subjects have noneosinophilic asthma with neutrophil degranulation and a neutrophil influx that may be mediated by IL-8. These data support the use of induced sputum to identify the pattern of airway inflammation in asthma, and suggest opportunities to base therapy on the underlying mechanisms in persistent asthma.
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Acknowledgements |
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Footnotes |
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Support has been provided by Glaxo Wellcome Australia, Ltd.
Received for publication February 24, 2000. Accepted for publication December 5, 2000.
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