Published in : Current medicinal chemistry (2011), vol. 18, pp. 1415-1422.
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Induced Sputum in Asthma: From Bench to Bedside
P. Bakakos1, F. Schleich2, M. Alchanatis1 and R. Louis2
1 st
1 Respiratory Medicine Department, University of Athens, Medical School, Athens, Greece
2
Department of Pneumology CHU Liège, GIGA I3 research group, University of Liège, Belgium
Abstract
During recent years there has been a growing interest in using non-invasive biomarkers to understand and
monitor the airway inflammation in subjects with respiratory tract disorders and mainly asthma and chronic
obstructive pulmonary disease (COPD). Sputum induction is generally a well-tolerated and safe procedure and a
European Respiratory Society Task Force has published a comprehensive review on sputum methodology.
Induced sputum cell count and, to a lesser extent, mediator measurements have been particularly well validated.
In asthma, the sputum and the cell culture supernatant can be used for the measurement of a variety of soluble
mediators, including eosinophil-derived proteins, nitric oxide (NO) derivatives, cytokines and remodellingassociated proteins. Sputum eosinophilia (> 3%) is a classic feature of asthma although half of the patients seems
to be non eosinophilic. Measuring the percentage of sputum eosinophils has proved to be useful in the clinical
arena in helping to predict short term response to inhaled corticosteroids (ICS) and tailor the dose of ICS in the
severe patients but there is scope for the application of other induced sputum markers potentially useful in
clinical practice. The widespread application of induced sputum in asthma across the spectrum of disease
severity has given insight into the relationship between airway function and airway inflammation, proposed new
disease phenotypes and defined which of these phenotypes respond to current therapy, and perhaps most
importantly provided an additional tool to guide the clinical management of asthmatic patients. To date sputum
induction is the only non-invasive measure of airway inflammation that has a clearly proven role in asthma
management.
Keywords : Induced sputum ; asthma ; biomarkers ; clinical applications.
INTRODUCTION
During recent years there has been a growing interest in using non-invasive biomarkers to understand and
monitor the airway inflammation in subjects with respiratory tract disorders. Currently available data seem to
underline the robustness of induced sputum as a method for assessing airway inflammation in diseases such as
asthma and chronic obstructive pulmonary disease (COPD).
Induced sputum samples the central airways and its cellular components (e.g. eosinophils and neutrophils),
protein components (e.g. mucins and cytokines) and microbiological components (e.g. viruses and bacteria) can
be used as markers of disease severity, exacerbation or progression [1].
Induced sputum cell count and, to a lesser extent, mediator measurements have been particularly well validated
[2]. Normal ranges for sputum cell counts from a relatively large adult population have been published [3-5].
Sputum induction is generally a well-tolerated and safe procedure even in patients with severe obstructive airway
diseases assessed either in stable condition or during an exacerbation. However, some differences in
methodology still exist between various research groups. An important question, therefore, is whether those
differences in methodology influence the validity and reliability of induced sputum in the assessment of airway
inflammation.
The widespread application of induced sputum in asthma, and across the spectrum of disease severity has given
an insight into the relationship between airway function and airway inflammation, proposed new disease
phenotypes and defined which of these phenotypes respond to current therapy, and perhaps most importantly
provided an additional tool to guide the clinical management of asthmatic patients [6].
Published in : Current medicinal chemistry (2011), vol. 18, pp. 1415-1422.
Status : Postprint (Author’s version)
The aim is to identify through non-invasive or minimally invasive methods of assessment of airway
inflammation the future risk of poor asthma control or exacerbations. Although induced sputum eosinophils and
exhaled nitric oxide are the most widely investigated candidates for use in the clinical arena, there is scope for a
great deal of improvement in their application and other biomarkers may prove to be useful or even better [7].
METHODOLOGY
Since the first description of a standardised method to induce and process sputum in asthma in 1992 by Pin et al.
[8], there has been an impressive increase in the number of papers in which researchers have used induced
sputum to study various aspects of airways inflammation. Thus, in response to the interest in sputum analysis, a
European Respiratory Society Task Force has published a comprehensive review on sputum methodology [912].
Sputum Induction and Collection
Induced sputum is usually collected in the morning. Induction is performed using an ultrasonic nebuliser. Two
different approaches for induction have been used:
a) inhalation of the same (3-4.5%) or increasing (3, 4 and 5%) concentrations of aerosolized hypertonic saline
over fixed time periods [8, 13]
b) inhalation of the same concentration of hypertonic saline (4.5%) over increasing time periods [14].
The choice of technique does not seem to influence the differential sputum cell count. The duration of sputum
induction has to be kept standard as it may influence the sputum cell composition. It generally ranges from 10-20
min. A sputum cell count resulting from an induction of 5 min can definitely not be compared with that of an
induction of 20 min. The early sample contains more granulocytes while the proportion of mononuclear cells
increases with the duration of the induction [15].
Irrespective of the induction technique used, the challenge procedure should be performed in a standardized way
that includes the necessary safety procedures, as hypertonic saline can cause severe airway constriction in
asthmatic subjects. Subjects should be pre-treated with inhaled short-acting β2-agonists. It has been shown that
obtaining sputum from the same asthmatic subjects with or without pretreatment with salbutamol, does not
influence the cellular composition [16]. It is also recommended to use isotonic instead of hypertonic saline when
post bronchodilator FEV1 is < 65% predicted [17]. Using either hypertonic or isotonic saline does not change the
cellular or the biochemical readouts [18,19]. The ERS Task Force conclusions regarding the safety of sputum
induction could serve as guidelines, particularly for those who are inexperienced in performing sputum induction
procedures [9]. Adding salbutamol into the cup of the nebuliser during the sputum induction has been shown to
improve bronchoprotection in moderate to severe asthmatics [20].
Sputum Processing and Analysis
Once a sputum sample has been obtained, it should be processed within 2 hours in order to ensure optimum cell
counting and staining. However, placing the sample in the refrigerator at 4°C enables to delay the processing up
to 9 hours after the collection without altering the cellular and some biochemical (ECP,IL-8) readouts [21].
Sputum processing and cellular analysis can be further delayed until 72 hours when the sample is fixed in
dithiothreitol-formaldehyde mixture before dispersal with trypsin [22]. Basically, two techniques for processing
have been described. The first approach, which effectively minimizes contamination with saliva, consists of
selecting all viscid or denser portions from the expectorated sample [8,23]. The second approach involves the
whole expectorate, comprising sputum plus saliva [13]. An adaptation of this method consists of trying to collect
sputum and saliva separately, so as to reduce the salivary contamination of induced sputum. Whatever technique
is used, the sample is put into preweighed polystyrene tube and weighed. Complete homogenisation can be
achieved by the addition of equal volume a dithiothreitol (DTT) solution to the whole sample or 4 x the volume
of a selected plug. Dithiothreitol breaks the disulphide bonds in mucin molecules, allowing cells to be released
[24]. This is important because cells that are incompletely released from mucus tend to stain darkly, making
correct identification difficult. DTT (0.1%) or its equivalent DTE (dithio-erythritol), commonly known as 10%
sputalysin solution, has been shown to be more effective at dispersing cells than phosphate-buffered saline (PBS)
[25].
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The sample is aspirated and dispensed several times with disposable pipette and then agitated on vortex mixer
for about 30 seconds. The duration of homogenisation varies between 10-30 minutes (usually 15 minutes).
Homogenisation is feasible by using either a shaking water bath at 37 °C (and removing the sample periodically
for brief aspiration) or a tube rocker at 22 °C [14,26]. The sample filtration is strongly recommended. Filtration
through a 48-mm nylon mesh is commonly used to remove mucus and debris. A single filtration step results in a
slight reduction in the total cell count (TCC). However, slide quality is improved and the differential cell count
(DCC) remains unchanged. The TCC is performed manually using a haemocytometer. The cell viability is
determined by the trypan blue exclusion method [13,23]. Some investigators perform the TCC before
centrifugation and others after centrifugation. Because centrifugation by itself causes a reduction in total cell
count, it is recommended to perform TCC before centrifugation in order to facilitate standardisation of this
measurement and to allow meaningful comparisons of counts between centres and studies.
In order to separate sputum cells from the fluid phase, the sample must be centrifuged at 300-1,500Xg. The
duration of centrifugation is about 5-10 min. This appears adequate for the purpose of separating cells from the
supernatant [13,23,25]. The supernatant can then be stored at -70°C.
The next step is the cytospin preparation. The cell concentration is adjusted to 1.0X106 cells.mL-1. Then, 40-65
µL of the sample (or 400-650X103 cells) is added to each cytospin. Cytocentrifugation speeds range from 1051Xg with the most common condition being 22 Xg for 6 min [23]. There is always a risk of losing lymphocytes
at lower speeds [27,28].
Cytospin staining for DCCs can be achieved using Giemsa stain. The DCC is determined by counting a
minimum of 400 non-squamous cells and is reported as the relative numbers of macrophages, neutrophils,
lymphocytes, eosinophils, and bronchial epithelial cells, expressed as a percentage of total nonsquamous cells.
The percentage of squamous cells should always be reported separately.
SPUTUM AND CELL CULTURE SUPERNATANT ANALYSIS
The sputum supernatant can be used for the measurement of a variety of soluble mediators (Table 1), including
eosinophil-derived proteins, tryptase, myeloperoxidase, deoxyribonucleic acid (DNA), albumin, fibrinogen,
nitric oxide (NO) derivatives and cytokines, such as interleukin (IL)-5, IL-8 and tumour necrosis factor- α (TNFα) [13,29-36]. However, it has to be born in mind that dithiothreitol (DTT) causes reduction of disulfide bonds
and denaturation of proteins and thus can affect the expression of cellular markers [37] or measurements of
soluble mediators in sputum. After incubation of the standard solution with DTT there was loss of detectable
protein mediators on immunoassay. The technique of optimized dialysis and protease inhibition of sputum DTT
supernatants aids the detection of chemokines and cytokines. Thus, significantly elevated levels of IL-4, IL-5,
IL-13, TNF-α, IL-6, granulocyte-macrophage colony-stimulating factor, and IL-12 were detected in sputum from
subjects with severe asthma, after optimized dialysis [38]. Among severe asthmatics those with frequent
exacerbations were characterized by higher levels of IL-5 and GM-CSF as compared to those with persistent
airway obstruction [39].
Asthma is commonly characterised by eosinophilic airway inflammation. Several groups have reported increased
levels of eosinophil cationic protein (ECP) in sputum from asthmatics when compared to healthy controls
[2,13,14]. Likewise tryptase levels as a marker of mast cell activation and those of albumin as a marker of
plasma exudation were found to be raised in sputum supernatant from asthmatics [35].
Cysteinyl-leukotrienes have been detected in sputum supernatants and montelukast (a leukotriene antagonist)
inhibited the eosinophilic chemotactic activity in both corticosteroid-naïve and corticosteroid-treated asthmatics.
Accordingly, it was suggested that cysteinyl-leukotrienes actively participate in sputum eosinophil chemotactic
activity found in asthmatics irrespective of whether they are or not under treatment with inhaled corticosteroids
[40].
A study, including 48 consecutive stable mild to moderate asthmatics, compared cytokine production from
sputum cells in eosinophilic versus non-eosinophilic asthma. Sputum cells from asthmatics exhibiting
eosinophilic airway inflammation released more IL-4 and less TNF-alpha than those of healthy subjects. By
contrast, non-eosinophilic asthmatics did not distinguish from healthy subjects by abnormal cytokine release
from their sputum cells [41]. Furthermore sputum cells from moderate to severe asthmatics treated with inhaled
corticosteroids release less IL-6 than those of healthy subjects which could contribute to a local impaired innate
immunity [42].
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Table 1. Soluble Markers in Sputum Supernatant from Patients with Asthma
Cytokines
↑ Tumour necrosis factor-α (TNF-α) [31,34,38]
↓ Tumour necrosis factor-α (TNF-α) (in cell culture supernatant) [41]
↑ Interleukm-4 (IL-4) [38]
↑ Interleukin-4 (IL-4) (in cell culture supernatant) [41]
↑ Interleukm-5 (IL-5) [30,34,38,39,54,71]
↑ Interleukm-6 (IL-6) [38,80]
↑ Interleukm-8 (IL-8) [14,39,61,71,80]
↑ Interleukm-12 (IL-12) [38]
↑ Interleukm-13 (IL-13) [38,54]
↑ Granulocyte/macrophage colony-stimulating factor (GM-CSF) [38,39]
Mast cell-derived mediators
↑ Histamme [93]
↑ Tryptase [35,59,80]
Eosinophil-derived mediators
↑ Eosinophil cationic protein (ECP) [2,13,30,33,35,36,39,59,61,64,68,71,81,82,85,93,97]
↑ Eosinophil-derived neurotoxin (EDN) [2]
↑ Eosinophil peroxidase (EPO) [33]
Neutrophil-derived mediators
↑ Myeloperoxidase (MPO) [33,59,61,71]
↑ Neutrophil elastase [80,82]
↑ Human neutrophil lipocalin (HNL) [33]
Remodelling-associated proteins
Matrix metalloproteinases (MMPs)
↑ MMP-2 [46,47]
↑ MMP-9 [47,48,49]
Tissue inhibitors of metalloproteinases (TIMPs)
↑ TIMP-1 [47,48]
~ TIMP-2 [47]
↑ Procollagen type I C-termrnal peptide (PICP) [50]
~ Collagen type I C-termrnal telopeptide (ICTP) [50]
↑ Vascular endothelial growth factor (VEGF) [51]
↑ Transforming growth factor beta 1 (TGF-β1) [52]
Others
↑ Fibrmogen [2,30]
↑ Albumin [2,13,59]
↑ Mucin-like glycoprotein [88]
↑ Deoxyribonucleic acid (DNA) [32]
↑ Nitric oxide (NO) derivatives [36]
↑ Intercellular adhesion molecule-1 (ICAM-1) [35]
↑ 8-iso-PGF(2alpha) [43]
↑ = increased levels, ↓ = decreased levels, ~ = at normal range
Induced sputum 8-iso-PGF(2alpha) concentrations were found to be elevated in subjects with stable asthma
versus control subjects and also increased as clinical asthma pattern worsened. Moreover, sputum 8-isoPGF(2alpha) concentrations were elevated during acute asthma and decreased with recovery [43].
Patients with asthma show lower induced sputum pH than healthy subjects and those with uncontrolled asthma
lower than those with controlled asthma. The pH in induced sputum may reflect a different aspect of asthma
from sputum eosinophils and be related to different pathophysiologic factors [44]. Patients with refractory
asthma have more nitrative stress in their airways compared with patients with well-controlled asthma as shown
by enhanced xanthine oxidase activities and 3-nitrotyrosine in sputum from the refractory asthma group
compared with the well-controlled group [45].
Another fundamental feature of asthma is airway remodelling. Soluble remodelling-associated proteins, such as
procollagen synthesis peptides, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases
(TIMPs) and cytokines, can also be detected in the sputum supernatants. Furthermore, induced sputum samples
can be obtained before and after specific allergen challenge and/or therapeutic trial. Matrix degradation enzymes
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and inhibitors have been measured in the induced sputum, such as MMP-2, MMP-9, TIMP-1, TIMP-2, elastase
and a1-antitrypsin [46-49]. MMP-9 in induced sputum is elevated in severe asthmatics, after allergen challenge
and is not affected by ICS treatment [49]. An imbalance in the MMP-9/TIMP-1 ratio may lead to excessive
degradation of extracellular matrix (ECM) proteins that participate in the injury-repair process. The turnover of
collagen I can also be indirectly detected in sputum supernatant through the quantification of procollagen type I
C-terminal peptide (PICP) and collagen type I C-terminal telopeptide (ICTP). PICP represents synthesis of
collagen while ICTP reflects collagen degradation. It has been previously reported that sputum PICP level is
increased during asthma exacerbations and is correlated with sputum eosinophils [50]. Angiogenic factor,
vascular endothelial growth factor (VEGF) and anti-angiogenic endostatin were measured in the sputum
supernatants of asthmatics. VEGF was found to be elevated in asthmatic sputum suggesting angiogenesis [51].
TGF-betal is also involved in the pathogenesis of airway remodeling. In a recent study including 27 patients with
moderate-to-severe, but stable asthma, levels of TGF-betal in induced sputum were elevated despite treatment
with inhaled corticosteroids and were associated with airflow obstruction and airway wall thickening [52].
The release of GM-CSF, RANTES and IL-8 from induced sputum cells, cultured with beclomethasone
dipropionate (BDP), salbutamol and formoterol either alone or in combination was significantly reduced by BDP
plus salbutamol or formoterol as compared with either drug alone in 20 mild to moderate asthmatic patients. This
study demonstrated a complementary mode of action between BDP and salbutamol or formoterol leading to an
enhanced anti-inflammatory activity [53].
IL-13 has been detected in sputum supernatant and has been inversely correlated with the provocative
concentration of methacholine causing a 20% fall in FEV1 in asthmatic patients, indicating a relationship
between IL-13 and airway hyperresponsiveness (AHR) [54].
When biomarkers obtained from induced sputum were compared with those from other non-invasive methods,
such as exhaled breath condensate (EBC), total protein and surfactant protein A (SPA) EBC levels were at least
100-fold lower than those measured in induced sputum [55]. This indicates that the detection of inflammatory
mediators through different non-invasive methods is quite variable.
CELL COUNTS
The cell fraction of induced sputum clearly differs between healthy subjects and asthmatics [8,13,31,56-58].
Asthma is commonly associated with sputum eosinophilia. Overall, compared to healthy subjects, sputum from
asthmatics contains increased numbers of eosinophils. The normal value for eosinophils in healthy nonsmokers
has been reported as 0.4% with a 90th percentile of up to 1.1%. It is accepted that a sputum eosinophil ≥ 3% has
to be considered as abnormal. The range of eosinophil counts in asthma is wide going from 0 up to 90 % [59].
Up to 70% of corticosteroid-naive subjects [60] and 40- 50% of corticosteroid-treated subjects [61,62] with
currently symptomatic asthma have a sputum eosinophil count that is outside the normal range. There is at best a
weak relationship between the severity of asthma as defined by lung function, airway responsiveness or
symptoms and sputum eosinophil count [59,62-64]. In a cross sectional study performed in daily practice,
uncontrolled asthma was shown to be associated with raised sputum eosinophilia with approximately 60 % of
uncontrolled asthmatics having sputum eosinophil count ≥ 3% irrespective of their maintenance treatment [65].
High sputum eosinophil count, however, is not always associated with poor asthma control. Using a cluster
analysis on a large population of asthmatics encountered in both primary and secondary care, Haldar et al. found
a peculiar asthma phenotype characterized by intense eosinophilic airway inflammation but relatively poor
symptomatic expression [66]. Occupational asthma is associated with similar sputum characteristics to non
occupational asthma, and occupational challenges are associated with an increase in sputum eosinophilia [67,68]
in much the same way as allergen challenge is.
Although sputum eosinophilia is a typical feature of asthma, the increasing application of this technique has led
to the recognition that inflammation in asthma is more heterogeneous than previously believed with
identification of non eosinophilic asthma. A cut-off point of 3% sputum eosinophils has been suggested to
distinguish eosinophilic from non-eosinophilic airway inflammation in asthma [69]. Non eosinophilic asthma is
common, accounting for 25 to 55% of corticosteroid-naive asthmatics [61,62,70]. Part of, but not all, these
patients are characterised by neutrophilic asthma. In contrast to eosinophil count, the sputum neutrophil cell
count in healthy subjects is highly variable, usually averaging 30-35%. Consequently, what has to be considered
as an abnormally high sputum neutrophil count is actually a count which is certainly above 60%. In a large
population of asthmatics seen in clinical practice the proportion of neutrophilic asthma defined as a sputum
neutrophil count greater than 60-65% was close to 20% [62]. Some studies [59,71], but not all [72], pointed out
to a raised sputum neutrophilia in severe asthmatics. Both the percentage and the absolute sputum neutrophil
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counts have been associated with low postbronchodilator FEV1 which lends support to the hypothesis that
neutrophilic airway inflammation has a role in the progression of persistent airflow limitation in asthma [73,74].
Based on sputum eosinophil and neutrophil proportions, airway inflammation in asthma could be categorized
into four inflammatory subtypes. These subtypes were neutrophilic asthma, eosinophilic asthma, mixed
granulocytic asthma and paucigranulocytic asthma. Subjects with increased neutrophils (neutrophilic asthma and
mixed granulocytic asthma) were older and had increased total cell count and cell viability compared with other
subtypes [75].
It is also likely that some of non eosinophilic asthmatics treated with inhaled corticosteroids have in fact residual
eosinophilic inflammation in their airway wall. It seems that having macrophages with large red hued area would
predict a relapse of eosinophilic airway inflammation when weaning off [76].
Menopausal asthma, which shares several characteristics of the severe asthma phenotype, has been associated
with high sputum neutrophils whereas women with premenopausal asthma presented an essentially eosinophilic
inflammatory pattern [77]. Obese people with asthma have poorer asthma control than non obese asthmatics. A
retrospective study has found no relationship between BMI and sputum cell counts [78]. A cluster of obese
women characterised by high symptom expression without sputum eosinophilia has been described recently,
highlighting the possible discordance between symptom expression and airway eosinophilic inflammation [66].
Furthermore, in a recent prospective study, sputum eosinophil and neutrophil counts were found similar in obese
and non obese asthma although there was an inverse correlation between sputum eosinophils and waist
circumference and a trend for a similar relationship for body mass index (BMI) [79].
Subjects with severe acute asthma may exhibit marked sputum eosinophilia or predominant neutrophilia
depending on the triggers that precipitate asthma attack [80]. Massive allergen exposure is associated with raised
sputum eosinophil count [81] while viral infections more often result in neutrophilic infiltration [82].
Compared to COPD patients, patients with chronic asthma usually showed higher sputum eosinophil and lower
neutrophil percentages [33,83] even though some COPD patients exhibited high sputum eosinophil count
[84,85].
Induced sputum has been compared to bronchial wash, bronchoalveolar lavage (BAL) and bronchial biopsies. It
has to be realized that these techniques might sample different locations within the bronchial tree, and that it is,
therefore, difficult to establish with certainty what the "gold standard" is, to which sputum has to be compared.
Comparative studies indicate that sputum contains more neutrophils but fewer macrophages and lymphocytes
than BAL fluid [58,86-88]. However, the cellular distribution of sputum, and especially the relative eosinophil
numbers, correlates relatively well with the eosinophil numbers in bronchial washes or BAL.
INDUCED SPUTUM IN CHILDREN
Non-invasive techniques are the ideal, especially in children, given the necessity of safe and repeatable
measurements to monitor treatment efficacy and disease progression. Although perhaps less successful than in
adults, the technique of induced sputum has been applied in children. The inflammatory response in childhood
asthma is characterized by elevated numbers of sputum eosinophils, and eosinophil cationic protein
concentration, as well as increased nitric oxide and hydrogen peroxide levels in exhaled breath. Sputum
eosinophils correlate with objective markers of disease severity in steroid-naive children with asthma, and in
severe asthma [89].
DISCUSSION
From bench to bedside - association of induced sputum with diagnosis, response to treatment, level of control
and exacerbations.
Knowledge of inflammatory phenotype is useful because it relates to treatment response, mechanistic pathways
involved in disease pathogenesis and future disease risk. Clinically useful applications of induced sputum
analysis have been suggested to be the detection of non-adherence to corticosteroid therapy, assessment of
adequacy of inhaled corticosteroid therapy, long-term therapy management in asthma and oral corticosteroid
dose adjustment in refractory asthma. It can also be used to study the dose-response effect of inhaled
corticosteroids and may be useful to establish the relative potency of different corticosteroid formulations and
delivery devices [6].
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Induced sputum eosinophil percentage has been shown to be a good aid in the diagnosis of mild to moderate
asthma with normal baseline lung function [90]. Under these circumstances sputum eosinophils with a cut-off of
1 % clearly performed better than the variation of peak flow, the extent of bronchodilation after salbutamol or
the blood eosinophil count but slightly less than methacholine challenge. In another study using the same type of
patients, sputum eosinophils with a cut-off of 3% performed equally to FeNO but once again much better than
peak flow variation [91]. However, latest GINA guidelines do not suggest yet the routine use of induced sputum
in the diagnostic approach of bronchial asthma, claiming that sputum eosinophilia has not been evaluated
prospectively as an aid in asthma diagnosis.
An important element in the content validity of induced sputum is the responsiveness to intervention or the
capacity of the technique to reflect changes in the degree of airway inflammation. The composition of sputum
has indeed been shown to respond to factors known to affect the degree of airway inflammation. Following an
allergen inhalation challenge, an increase in the percentage of eosinophils in sputum from atopic asthmatics has
been shown [92-96]. Conversely, treatment with systemic or inhaled steroids reduces sputum eosinophil numbers
and ECP levels [30,97,98].
The short-term response to inhaled corticosteroids in corticosteroid naive patients differs markedly according to
the sputum eosinophil count, with little evidence of improvement in symptoms and airway responsiveness in
subjects with a baseline sputum eosinophil count of < 3% [70]. The response to inhaled corticosteroids appears
to be particularly poor in patients with high sputum neutrophil count [62]. In contrast, asthmatics with sputum
eosinophilia have a favourable response to corticosteroids, even in ex-smokers and current smokers [99]. Sputum
eosinophilia was positively correlated with the degree of clinical improvement to inhaled corticosteroids and was
more closely related to clinical response than FeNO, sputum or peripheral blood eosinophilic cationic protein
[100]. A recent placebo-controlled cross-over trial of inhaled mometasone, 400 µg qd for 8 weeks, in subjects
characterized as either having eosinophilic or non eosinophilic asthma prior to study entry has confirmed that a
favorable response to corticosteroids was reserved to asthmatics with sputum eosinophilia [101]. These findings
suggest that measuring the underlying airway inflammation might provide a better guide regarding the need for
corticosteroid treatment than the assessment of functional abnormality. However, long term studies looking at
other parameters such as exacerbation rate are urgently required before denying the utility of inhaled
corticosteroids in non eosinophilic asthmatics.
Categorising subjects according to the presence of sputum eosinophilia might be particularly useful in asthmatics
that are symptomatic despite treatment with inhaled corticosteroids since the additional treatment options (long
acting beta2-agonists, higher-dose inhaled corticosteroids, leukotriene antagonists and theophylline) differ in
their effects on eosinophilic airway inflammation [102-105]. Sputum cell counts may be useful to study the
potential anti-inflammatory effects of drugs like theophylline, long-acting betaadrenoceptor agonists, leukotriene
antagonists and newer drugs in development. Little et al. found that in patients with asthma, a sputum eosinophil
count >4% had a 68% positive predictive value with a sensitivity of 59% and specificity of 76% for an
improvement in FEV1 >15% after a 2-week course of oral corticosteroids [106]. In a recent study, high-dose oral
corticosteroids reduced sputum eosinophil counts in patients with severe refractory asthma but this effect was
observed only in asthmatics with baseline eosinophilia [107].
A recent study conducted in daily practice has shown that uncontrolled asthmatics were associated with raised
sputum eosinophilia and raised bronchial hyperresponsiveness to methacholine but not raised FeNO.
Approximately 60% of asthmatics with ACQ (Asthma Control Questionnaire) >1.5 exhibit sputum eosinophil
count > 3% [65]. Both FeNO and sputum eosinophils were found to be useful for titrating inhaled corticosteroids
in asthmatic patients in order to achieve better long-term control of the disease. In a study in which unstable
eosinophilic asthmatics were treated by raising the dose of inhaled corticosteroids according to FeNO and
induced sputum eosinophil levels, both inflammatory markers decreased over time but with a different kinetic,
FeNO decrease being more dramatic than that of sputum eosinophil count after 3 months. The change in FeNO
and sputum eosinophil count correlated after 6 months. Concomitant to these changes, a reduction in symptoms
and bronchial hyperresponsiveness were observed compared to baseline [108].
Another important part of the natural history of asthma is exacerbations. There is now strong evidence from two
independent studies supporting the importance of monitoring airway inflammation by sputum induction to
reduce asthma exacerbations. In a randomized placebo-controlled trial, 74 subjects with asthma were assigned to
either a management strategy aimed at normalizing their sputum eosinophil count or standard clinical care.
Patients in the sputum management group had significantly fewer severe asthma exacerbations than patients in
the control group, and significantly fewer patients were admitted to hospital with asthma. The reduction in
exacerbations was achieved without an increase in the total corticosteroid dose in the sputum management group.
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The monitoring of airway inflammation in the sputum management group identified a group of patients with non
eosinophilic asthma whose sputum eosinophil counts remained within the normal range and in this group the
dose of corticosteroids was reduced without evidence of deterioration [109]. In another 2-year follow-up
multicenter, randomized, parallel-group effectiveness study of 117 asthmatics, patients who entered into a
treatment directed at normalizing the sputum eosinophil count also showed a reduction in exacerbations and
increased the time to first exacerbation by 213 days. This benefit was not at the expense of increased therapy in
the intervention group. In this study, the inflammatory phenotype of the exacerbations was characterized and it
was in the sputum management group where eosinophilic, but not non eosinophilic exacerbations were reduced.
Interestingly, non eosinophilic exacerbations were more common. The reduction in exacerbations was more
apparent in those with severe disease [110]. This suggests that it is probably most appropriate to apply this
technique to the management of difficult-to-treat or refractory asthma. In another study, including patients with
refractory eosinophilic asthma (sputum eosinophils >2% despite inhaled steroid treatment) it was shown that
intramuscular administration of triamcinolone almost completely abolished sputum eosinophils. This suggests
that persistent sputum eosinophilia despite extensive antiasthma treatment is not a refractory phenomenon but
may still be sensitive to high-dose systemic corticosteroids and implies that these patients with severe asthma
need additional or alternative anti-inflammatory treatment to combat the eosinophilia and associated poor
prognosis [111]. This was confirmed by two recent placebo controlled studies using mepolizumab (anti-IL-5) in
refractory eosinophilic asthma, showing a consistent reduction of asthma exacerbations in the mepolizumab
group [112,113] together with a corticosteroid sparing effect in one study [112].
A field that attracts attention is the relationship between induced sputum cell counts and other non invasive
markers such as FeNO and EBC pH. Although a FeNO level of 42 ppb generally reflects sputum eosinophil
count ≥ 3% with a satisfying accuracy, high dose of inhaled corticosteroids, atopy and smoking may alter the
FeNO thresholds. Those patients receiving high dose of inhaled corticosteroids (1000 µg fluticasone/day) had a
threshold of 27 ppb and those who were current smokers a threshold of 28 ppb while the best threshold in non
atopic patients was 30 ppb. Combining these variables together leads to the conclusion that the thresholds
predicting significant sputum eosinophilia range from 15 ppb in a smoking non atopic asthmatic receiving high
doses of inhaled corticosteroids to 58 ppb in a non smoking atopic asthmatic not treated with high doses of
inhaled corticosteroids [114]. In another recent study including patients with severe refractory asthma, FeNO
levels > 19 ppb may already predict sputum eosinophilia > 3% while values < 19 ppb suggest a sputum
neutrophilia > 40% irrespective of the presence of eosinophils [115].
The inclusion of sputum induction in the management of asthma is cost-effective. Economic analysis shows that
the healthcare-related savings as a consequence of the reduction in asthma exacerbations outweighs the cost of
sputum induction and processing [109]. Sputum induction requires laboratory support, but all of the equipment is
available in a routine pathology laboratory and minimal training is required. Therefore, sputum induction should
be available as a routine test in specialist centers to evaluate and manage patients with severe or difficult-to-treat
asthma, as its value seems to be greater in those with severe asthma.
To date sputum induction is the only non-invasive measure of airway inflammation that has a proven role in the
management of moderate-to-severe asthma. Regular monitoring of airway inflammation in this disease group
may be required for optimal treatment and in particular in those patients with discordance between airway
inflammation and symptom expression.
ABBREVIATIONS
ACQ
AHR
BAL
BDP
COPD
DCC
DNA
DTT
DTE
EBC
ECP
FeNO
FEV1
GINA
=
=
=
=
=
=
=
=
=
=
=
=
=
=
Asthma Control Questionnaire
Airway hyperresponsiveness
Bronchoalveolar lavage
Beclomethasone dipropionate
Chronic obstructive pulmonary disease
Differential cell count
Deoxyribonucleic acid
Dithiothreitol
Dithioerythritol
Exhaled breath condensate
Eosinophil cationic protein
Fraction Exhaled Nitric Oxide
Forced Expiratory Volume in 1 second
Global INitiative for Asthma
Published in : Current medicinal chemistry (2011), vol. 18, pp. 1415-1422.
Status : Postprint (Author’s version)
GM-CSF
ICS
ICTP
ILMMPs
NO
PBS
8-iso-PGF
PICP
TCC
TGF-beta
TIMPs
TNF-α
VEGF
=
=
=
=
=
=
=
=
=
=
=
=
=
=
Granulocyte macrophage - colony stimulating factor
Inhaled corticosteroids
Collagen type I C-terminal telopeptide
InterleukinMatrix metalloproteinases
Nitric oxide
Phosphate buffered saline
8-iso-prostaglandin F
Procollagen type I C-terminal peptide
Total cell count
Tumor growth factor- beta
Tissue inhibitors of metalloproteinases
Tumor necrosis factor alpha
Vascular endothelial growth factor
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