 
Congress 
 
Sponsors
 Search
Write
About
màj 23/07/98 |
Induced sputum to assess airway
inflammation in asthma.
Isabelle Pin, Jerry Dolovich , Frederick E
Hargreave
* Department of Pulmonology, Grenoble, France
** Asthma Research Group, Hamilton, Canada
Samedi 14 Février 1998
|
Présentateurs
|
Modérateurs
|
Experts
|
9h00-10h00
|
Marqueurs de l'inflamation
|
P.
Devillers (Lyon)
I. Vachier (Montpellier)
I. Pin (Grenoble)
Discussion
|
10'
10' 10' 30'
|
M.
Fournier (Paris)
B. Crestani (Paris)
|
S.E.
Dahlen (Stockholm, S)
A.C. Roldaan (Davos, CH)
K. Richter (Grossandorf, D)
|
Airway
inflammation is a major factor in the pathogenesis of asthma. Mast cell and
eosinophil infiltration, epithelial damage and mucus
production are characteristic features. In clinically
stable asthma bronchial biopsies and bronchoalveolar
lavage (BAL) have been used to study the aspects of
airway inflammation, but the discomfort, inconvenience
and risks may limit the use of these techniques. In
people who spontaneously produce sputum during an
exacerbation, analysis of the sputum is a non-invasive
alternative. Patients with controlled asthma do not
spontaneously produce sputum. However, induction of
sputum may be a non-invasive alternative to get
secretions from the lower airways, in order to study the
presence and quality of cells and inflammatory mediators
present in the airways and their changes during the
course of the disease or with treatment.
|
Methodology
Sputum induction
Sputum induction is obtained by
inhaling increasing concentrations of hypertonic saline (3, 4, 5
%), preceded by inhalation of ß2 agonists and monitored carefully with
repeated spirometry to avoid severe bronchoconstriction during
the test. In case of patients presenting with an exacerbation of
asthma, isotonic saline has been shown to be successful and safe.
Ultrasonic nebulisers should be used because they are superior to
jet nebulisers. A moderate output (around 1 ml/min) is
appropriate; higher output nebulisers, whilst there might be a
higher success rate, seem to produce increased adverse effects
including mild bronchoconstriction. Moreover the cellular and
biochemical content of sputum induced by a high output nebuliser
changes with sequential inhalations. In later specimen Therefore
it is recommended to follow the whole sputum induction protocol
each time and to use all the sputum collected for analysis. With
this procedure, the sputum induction is safe. However seldom
moderate to severe bronchoconstrictions can occur, especially in
asthmatic using high doses of inhaled ß2 agonists for an exacerbation. The
success rate is between 70 to 80 % of subjects not producing sputum
spontaneously, including normal subjects. The procedure has been
shown not to altered the cellular and biochemical content of the
sputum, and induced and spontaneous sputum are comparable in the
same individual. The mechanism by which hypertonic saline induces
sputum is incompletely understood but may involve increased
outward water flux across the airway epithelium, stimulation of
cough receptors and perhaps direct stimulation of the mucociliary
escalator.
Sputum processing
This may vary between research
teams. In the " selected " technique, secretions
arising from the lower airways are identified under an inverted
microscope and selected with blunt forceps for analysis, limiting
the squamous cell contamination to a mean of 1.6 %. Other
investigators have suggested that the selected technique might
lead to loss of potential information and thus used the whole
sample, including saliva. The main disadvantage of this approach
is that sputum is inevitably
diluted with saliva and that the
extent of this dilution is difficult to quantify. These
investigators have tried lately to reduce the salivary
contamination by having the patients blow their nose and rinse
their mouse before coughing up the sputum. Comparisons of the
selected and discarded portions of the sample in the selected
technique have shown that the concentration of ECP is 5 to 6 time
higher in the selected portion than in the discarded portion
showing that there is a dilution in the latter. Moreover the
quality of the cytospins is better in the selected portion,
principally because of less salivary contamination.
Dithiothrietol, a mucolytic
agent, is used to disperse the cells and liquefy the sample.
Rocking and filtering help to obtain an homogenous sample. This
can be processed to make cytospins stained to assess the cellular
composition of the sample, to allow immunocytochemical staining
of cellular markers (EG-2, tryptase, HLA-DR..) and to measure the
concentration of fluid phase products such as cytokines (Il-8, TNF a, Il-5) and inflammatory markers (ECP, tryptase, fibrinogen). PCR
techniques can detect mRNA expression of different markers in the
cells.
• Measurements in disease
Induced sputum seems to be a
valid method to assess the airway inflammation in asthma.
Eosinophils and metachromatic cells (mast cells and basophils)
are increased in the asthmatic compared to the normal subjects.
Cell counts are highly reproducible and normal values of cell
counts have been established. Sputum fluid phase mediator (ECP,
tryptase, albumin) concentrations are also increased in the
asthmatics compared to the normal subjects. Changes are more
marked in induced sputum than in broncho-alveolar lavages.
Induced sputum has been compared to other ways of accessing the
airways; its cellular composition seems closer to the bronchial
washes than BAL, probably reflecting its origin in the more
proximal airways.
Sputum cell and fluid phase
indices of inflammation are responsive to changes as indicated by increase in eosinophils, mast cells,
tryptase and ECP after allergen challenges and decrease after
treatmentwith inhaled or oral corticosteroids.
Use of induced sputum has been
already useful to understand certain mechanisms of asthma.
Eosinophils and metachromatic cells are only weekly associated
with bronchial hyperresponsiveness to methacholine but to be
closely associated with changes in airway hyperresponsiveness
after allergen challenge. Exacerbations of asthma induced by
tapering the dose of inhaled
steroids are associated with increased eosinophilic inflammation,
whereas wild exacerbations of asthma are sometimes associated
with neutrophilic inflammation. Responses to corticosteroid
treatment may differ in the two types of inflammation, as shown
in preliminary studies in COPD patients. Experimental infections
with rhinovirus in asthmatics induce an increase in airway
hyperresponsiveness which seems to be associated with an
eosinophilic inflammation and an increase in Il-8 and Il-6.
Therefore induced sputum may be a valuable tool to help
investigating patients in the clnic.
Induced sputum can help to
investigate the anti inflammatory effects of new medications in
asthma. Studies performed after allergen challenge or mild
exacerbations of asthma have shown that long acting ß2 agonists, despite their prolonged effect
on baseline respiratory function and airway hyperresponsiveness,
do not abolish the inflammatory reaction, whereas inhaled
corticosteroid do. These may be valuable models to investigate
the antinflammatory effects of new antilenkotrienes or new
phospho-diestarase inhibitors.
In conclusion, induced sputum is
a reliable non invasive valid and reproducible way to have direct
access to the lower airways. Because it should be performed very
carefully and sputum processing needs more assesament and
improvement, it is still a research tool. However it may become a
clinical tool in the near
future.
. 
Asmanet ENFUMOSA Congrès Conçue et réalisée par: Michel Godard (at) Top
Date de création: 5 Décembre 1997-Dernière mise à jour: 23/07/98
Le secret des correspondances transmises sur le réseau Internet
n'est pas garanti.
Toute personne citée dispose d'un droit d'accès, de
modification, de rectification et de suppression des données le
concernant (art. 34 de la loi
"Informatique et Libertés" n° 78-17 du 6 janvier
1978). Pour l'éxercer adressez-vous à Michel Godard (at)
