Airway inflammation in steroid-naïve asthmatics: characteristics of induced sputum

ORIGINAL ARTICLE

Airway inflammation in steroid-naïve asthmatics: characteristics of induced sputum^*

Simone Van de Sande Lee^I; Marcia Margaret Menezes Pizzichini^II; Leila John Marques^III (te sbpt); Samira Cardoso Ferreira^IV; Emilio Pizzichini^V (te sbpt)

^IStudent at the 6^th year at Medical School of Universidade Federal de Santa Catarina.

^IIMaster in Pneumology. Professor at the General Medicine Discipline, Universidade Federal de Santa Catarina.

^IIIMaster in Pneumology. Pneumology Physician at Hospital Universitário, Universidade Federal de Santa Catarina. Title of Specialist by the Brazilian Society of Pneumology and Tysiology.

^IVPharmaceutical and Biochemical at Hospital Universitário, Universidade Federal de Santa Catarina.

^VMaster in Pneumology. Professor at the Pneumology Discipline, Universidade Federal de Santa Catarina, and Nu­paiva’s Coordinator. Title of Specialist by the Brazilian Society of Pneumology and Tysiology.

^{Correspondence}

ABSTRACT

BACKGROUND: Airway inflammation, acknowledged as an important feature of asthma, can be assessed by the examination of induced sputum.

OBJECTIVE: To determine the pattern of inflammatory cells in induced sputum from stable steroid-naïve asthmatics, in Florianópolis, Santa Catarina.

METHOD: The induced sputum from 34 asthmatics using exclusively inhaled bronchodilators on demand was examined. The patients’ clinical characteristics were obtained at visit 1, and sputum was induced at visit 2. Differential cell count was performed on Giemsa-stained cytospins. Sputum was considered to be eosinophilic if there were ³ 3% eosinophils, and neutrophilic if there were ³ 65% neutrophils.

RESULTS: Results are expressed by median and interquartile range. The total cell count was 3.4 (3.7) x 10⁶ cells/ml, and cell viability was 80.0 (16.4) %. The proportion of neutrophils was 14.4 (22.1) %, of eosinophils 6.4 (17.2) %, of macrophages 60.3 (37.5) %, and of lymphocytes 1.1 (1.2) %. Eosinophilic sputum was observed in 24 subjects (70.6%); none of them had neutrophilic sputum. There were no significant differences between the eosinophilic and non-eosinophilic groups concerning the measured clinical outcomes, total cell count and proportions of cells in the sputum, except for the proportion of eosinophils (14.4 [19.3] vs 0.4 [1.1], p < 0.001).

CONCLUSIONS: In our environment, steroid-naïve asthmatics present a higher proportion of sputum eosinophils, as compared to the established reference values. The clinical and physiological parameters analyzed were unable to predict the presence of eosinophilic inflammation of the airways.

Descritores: Asma. Inflamação. Escarro. Eosinófilos. Neutrófilos.

Acronyms and abbreviations used in this study

SQA –Semi-quartile amplitude

ATS – American Thoracic Society

BD – Bronchodilator

TCC –Total cell count

PC₂₀ –Methacoline provocative concentration capable of producing a 20% fall in the FEV₁

FVC – Forced vital capacity

D-PBS –Dulbecco-Phosphate buffered saline

SD – Standard deviation

DTT – Ditiotreitol

ECP – Eosinophilic cationic protein

ISAAC – International Study of Asthma and Allergy in Children

NUPAIVA – Núcleo de Pesquisa em Asma e Inflamação das Vias Aéreas

Pred – Predicted

SUS – Sistema Único de Saúde

FEV₁ – Forced expiratory volume in the 1^st second

Introduction

Asthma is a chronic inflammatory airway disease that causes, in susceptible subjects, recurrent episodes of wheezing, breathlessness, chest tightness and cough, primarily during the night or early in the morning. These episodes are usually associated with diffuse, but variable, airflow limitation (bronchoconstriction), which is usually reversible, spontaneously or after treatment. Inflammation is also responsible for increased airways responsiveness (hyper- responsiveness), and a variety of non-specific stimulii.^(1,2) The importance of seekingr a greater comprehension about asthma pathophysiology resides in its complications in terms of diagnosis, treatment and prevention, which are part of an important public health problem, confirmed by several studies. One of the most recent studies, the Internatio­nal Study of Asthma and Allergy in Children (ISAAC),⁽³⁾ was performed in 56 countries, on 6 to 7 year-old school children and 13 to 14 year-old adolescents, and showed an asthma symptoms’ prevalence that varied from 1.6 to 36.8% among the several countries; Brazil was among the eight countries with highest prevalences (from 17 to 27%).

Until recently, the knowledge about asthma mechanisms was limited to bronchospasm, which was believed to be a consequence of factors primarily related to bronchial smooth muscles. The inflammatory changes that were observed in necropsy studies on patients who had died after a severe exacerbation used to be attributed to terminal events of the disease.⁽⁴⁾ Airways investigation in alive asthmatic patients was only possible after the eighties, following the introduction of bronchoscopy for the obtainment of bronchial tissue samples and bronchoalveolar lavage, and only then, it was observed that many of those changes were present even in the mildest presentations of the disease.^(5,6) This method was shown to be valid for airway inflammation assessment;^(7,8) however, due to its invasive nature and high costs, besides the restriction for stable mild to moderate disease, it didn’t get a prominent role in clinical practice.⁽⁹⁾ From then on, other, less invasive and safer methods, were introduced, and the most important was the assessment of cells and inflammation markers in sputum, which should be obtained spontaneously⁽¹⁰⁾ or through hypertonic saline solution inhalation.⁽¹¹⁾

The methods for obtaining and analyzing sputum have had a great evolution in the last 13 years.

Some studies have shown it to have excellent properties, particularly when sputum is separated from saliva.⁽¹²⁾ The test was considered valid, making it possible to differentiate between several clinical conditions, and symptomatic from asymptomatic asthmatic patients, in addition to its correlation with spirometry and airway responsiveness to non-specific stimuli, such as methacoline.⁽¹¹⁾ Its results could be reproduced, except for the total cell number and the proportion of lymphocites, as shown by the comparison between results of repeated sputum sampling in stable asthmatic patients obtained on different days of the week.⁽¹¹⁾ After some changes in the methodology for sputum processing, total cell count became a reproducible process too. Another observed feature was the responsiveness to intervention of the markers assessed in sputum; it was observed that inflammatory response in sputum increases after exposition to inhaled allergens⁽¹³⁾ and after symptom’s exacerbation,⁽¹⁴⁾ and decreases after treatment with corticosteroids.^(15,16) The method has an additional advantage of possibly being used in severe exacerbation situations.^(15,17) Airway inflammation can also be indirectly assessed, through eosinophil count and eosinophilic cationic protein (ECP) dosage in peripheral blood; however, sensitivity and specificity of sputum assessment have shown to be significantly higher.⁽¹⁸⁾

Several studies have contributed for the elucidation of asthma pathogenesis, through direct assessment of airway inflammation. Therefore, under current knowledge, inflammation is recognized as its primary cause, and determines disease severity, exacerbations and structural changes. These changes may be persistent, and result in a process known as “remodeling”, which ends in an irreversible airflow obstruction.⁽¹⁹⁾

An important feature of asthma inflammation is the presence of eosinophilic infiltrates in the airways.^(11,15) However, not all asthmatic patients present this kind of inflammatory response, and not all asthma exacerbations are accompanied by increase of these cells’ numbers in sputum.^(17,20,21) This observation has therapeutic implications, since there is a tight correlation between eosinophilia and response to corticosteroids therapy. For example, recent studies concluded that patients who had eosinophilic sputum had significantly higher clinical benefit with this type of drug, as compared with those with non-eosinophilic sputum.^(22,23) Otherwise, indirect inflammation indexes (symptoms, airflow obstruction and airways hyper-responsiveness to several stimuli), used in daily practice for therapeutic conduct, are not specific, and correlate to each other and to direct indexes in a variable fashion,^(24-26) a fact that may result in wrong behaviors. Another type of inflammatory response observed in some asthmatic patients is the neutrophilic one, which may be related, for instance, to cigarette smoke exposition, high ozone concentrations, diesel oil, or different types of infections, including viral ones.^(21,27-29)

Non controlled symptomatic asthmatic patients, inhaled corticosteroids-naïve, in use of only symptomatic treatment (bronchodilators) still constitute most of the population of asthmatic patients seen in Brazil, and are very commonly found in our environment. These patients are frequently seen in emergency rooms and outpatient care at our hospitals. In the present study we intended to determine a standard for the observed cellular infiltrate in induced sputum from stable, non- controlled corticosteroids-naïve asthmatic patients in our environment and to compare clinical features between subject groups according to the presence or absence of eosinophilic inflammation, as detected by sputum examination.

Method

Subjects

Thirty four asthmatic adult patients aged from 18 to 67 years, atopic or not, in use of exclusively on demand inhaled bronchodilators were consecutively enrolled in the study. Patients’ features are described on Table 1. Asthma diagnosis was established by the presence of episodic wheezing, chest tightness, cough and dyspnea within the last year, and was confirmed in 33 of them through confirmation of reversible airflow obstruction (forced expiratory volume in the first second [FEV₁] < 70% of the predicted value, and an improvement on FEV₁ of at least 15% after the use of 200mg salbutamol inhalation with the use of a spacer), and in one subject through the presence of airways hyper-responsiveness with methacoline (a lower than 8mg/ml methacoline provocative concentration with power to produce a 20% fall in FEV₁ [CP₂₀]).

All subjects were non-smokers, or had not been smoking for at least a year. None of them had received systemic or inhaled steroid treatment within the last two months, or reported the presence of common cold or any other respiratory infection within the last six weeks before inclusion in the study. Subjects with any other associated pulmonary disease (such as bronchiectasis, pneumonia, etc.) were excluded. All subjects were able to produce non-purulent sputum after induction with hypertonic saline solution.

The study received approval from the Research in Humans Ethics Committee of Universidade Federal de Santa Catarina, and all subjects signed the informed consent.

Study design

This is a cross-sectional descriptive study. All subjects were consecutively enrolled after a screening for a placebo controlled clinical trial that intended to compare the anti-inflammatory effects of fluticasone dipropionate with the leucotrien receptor antagonist montelukast in asthmatic patients with eosinophilic bronchitis.⁽³⁰⁾ All subjects were treated at Núcleo de Pesquisa em Asma e Inflamação das Vias Aéreas (NUPAIVA), Hospital Universitário Professor Polydoro Ernani de São Thiago, Florianópolis, Santa Catarina.

The study was explained to each subject in details. Subjects who accepted to take part were sent to NUPAIVA for two consecutive visits, with an approximate 48-hour interval between them. At visit 1, after signing the informed consent, a questionnaire to obtain each subject’s individual features was applied. Puncture cutaneous tests for allergy were performed for patients who had not have one performed within the last 12 months, and additionally, a spirometry with an airways reversibility test, or bronchoprovocation with methacoline, if needed. At visit 2, subjects were submitted to another spirometry and also the sputum induction. Sputum was analyzed by a professional that was blinded to subjects’ clinical features.

Clinical and laboratory procedures

Subjects’ features were assessed through a standard questionnaire which included data such as age, gender, height, smoking status, respiratory infection within the last six weeks, symptoms and symptom severity, as determined by frequency of symptoms presence or absence of nighttime symptoms causing awakening, frequency of inhaled bronchodilator use; past or current respiratory disease other than asthma, and current and past treatments.

Cutaneous tests for allergy were performed according to a modified puncture technique ⁽³¹⁾ with 19 usual allergenic inhaled strata. Reading was performed after 15 minutes, with registration of the mean circumference diameter in milimeters. The test was considered positive for a determined allergen when a ³ 3 mm diameter papule was seen. Atopy was defined when one or more positive responses were present.

Spirometry was performed according to the American Thoracic Society (ATS)⁽³²⁾ guidelines, with a computerized spirometer (Koko^â Spirome­ter, PDS Instrumentation, Louisville, USA). The best among three reproducible values with a lower than 5% amplitude was recorded as baseline FEV₁. Patients with a < 70% of the predicted FEV₁value had the reversibility test performed 15 minutes after inhaling 200mg salbutamol through a pressurized metered dose inhaler with a spacer (Aerochamber^â, Trudell Medical International, London, On, Canada). Predicted values were obtained from Crapo et al.⁽³³⁾

Methacoline bronchoprovocation tests were performed, when indicated, through the method described by Juniper et al.⁽³⁴⁾ Results were expressed on CP₂₀ non-cumulative units.

Sputum was induced according to the method described by Pizzichini et al.⁽¹⁵⁾ In summary, the procedure consisted of inhalation of aerosol isotonic saline solution (0.9%) and then a hypertonic saline (3, 4 and 5%); the vapor was produced by a Fisoneb^â ultrasonic nebulizer (Canadian Medical Products, Ltd., Markham, Ontario) with a 0.87ml/min output, and median aerodynamic mass diameter of 5.58mm particles. Subjects were supposed to inhale the solution for about two minutes, according to the severity of bronchoconstriction present before the procedure, and then FEV₁ was assessed. Subjects were asked to rinse their mouths, swallow the water and blow their noses in order to minimize contamination with saliva or post-nasal discharge. Then, they were asked to cough and deposit the sputum on a sterile container. These procedures were consecutively repeated, with increasing saline solution concentration every seven minutes, until 21 minutes had passed, or until a > 20% fall in FEV₁ occurred.

Sputum was processed within two hours, using the technique described by Pizzichini et al.⁽¹¹⁾ Firstly, thick portions of the sputum were selected under naked eye or, when necessary, under inverted light microscopy, with a 115 mm forceps for separation of sputum from saliva. The selected fraction was put in a 15 ml polistiren tube and was treated with a volume of 0.1% ditiotreitol (DTT) (Sputalysin 10%; Calbiochem Corp., San Diego, CA) four -fold bigger than its own; the mixture was shaken for 15 seconds and consecutively aspired and ejected through a Pasteur pipette. The polistiren tube was then placed in a desk agitator (Dade Tube Rocker; Baxter Diagnostics Corporation, Miami, FL) and was agitated for 15 minutes. In order to prevent the DTT effect on the cell suspension, an additional four-fold volume of Dulbecco-phosphate buffered saline (D-PBS) was added. The resulting suspension was filtered through a nylon filter (BBS Thompson, Scarborough, Ontario) with 48mm micropores for removal of cell remainder and non-dissolved mucus. Then, the white blood cell total count was performed, excluding squamous cells by means of a modified Neubauer hemocytometer, and cellular viability was determined through the exclusion method using trypan blue (dead cells appear in blue). Sixty to eighty microliter of the suspension, adjusted for 1.0 x 10⁶/ml, were placed in Shandon III centrifuge containers (Shandon Southern Instruments, Sewickley, PA) and four coded cytospins were prepared at 450 rpm for 6 minutes. After drying at open air, they were stained with Giemsa stain for differential cell count; 400 non-squamous cells were countedon the best quality slide for each subject. Results were expressed as a percentage of total non-squamous cells.

Sputum was considered to be eosinophilic when there was a differential eosinophil count ³ 3,0%, and to be neutrophilic, when there was a differential neutrophil count ³ 65%. These cut-off points were defined based on normal values reported by Belda et al.⁽³⁵⁾

Statistical analysis

Normal distribution data are summarized as mean and standard deviation (SD), and those of non normal distribution (such as total and diferential cell count) are summarized as median and semi-quartiles amplitude (SQA). Differences between groups in terms of continual variables were analyzed with non-paired t test; Non normal distribution variables had a logarithmic transformation before the test was applied. For categorical variables (symptoms), differences between groups were estimated by qui-square test. Significance was accepted at a 95% level.

Results

Subjects’ sputum cellular features are described in Table 2. The presence of sputum eosinophilia was observed in 24 (70.6%) of the 34 participant subjects. None of the subjects presented neutrophilic sputum. Subjects were subdivided into two groups, according to the presence or absence of eosinophilia on sputum. Each group’s clinical features were compared (Table 3). The occurrence of daytime and nighttime symptoms was higher in the eosinophilic group (79.2% vs. 50.0%), but this difference was not statistically significant. The frequency of inhaled bronchodilator use, the magnitude of airway obstruction and bronchodilator response were similar between both groups.

Similarly, except for eosinophil proportion, no differences were observed at sputum total cell count and cell proportions at the eosinophilic or at the non-eosinophilic group (Table 2).

Discussion

We describe, in the present study, the features of airways inflammatory process in asthmatic corticosteroid-naïve subjects as measured through the total and diferential sputum cell count. The results showed the presence of eosinophilia in sputum in 70.6% subjects; no subject had sputum neutrophilia. There was no significant difference between eosinophilic and non-eosinophilic groups in terms of symptoms, use of bronchodilatators, presence of airway obstruction or response to bronchodilatator. These results suggest that the analyzed clinical and physiologic parameters not always reflect presence or absence of eosinophilic inflammation in asthma patients.

Although many other studies have included steroid-naïve patients in their samples,^{(16,28,36,37)} this is the first Brazilian study with a primary objective to describe cellular features of sputum in this group of subjects. The power of the study is based on two aspects. The first one is a cautious selection of subjects, which made it possible to for us to have a group of stable asthmatic patients, in use exclusively of inhaled bronchodilators as needed. The second one is the method that was used for sputum assessment, which provides highly reproducible measurements of cell markers, which are valid and discriminative for different conditions, types and severities of airways inflammatory processes.⁽¹¹⁾

One of the limiting factors of our study is that its results cannot be extrapolated to other site’s populations. It is necessary to consider the particularities of the group analyzed by us: it consisted of people who live in a medium sized city, in an island, where industrial activities are not predominant. Thus, the location where the study was conducted presents unique environmental conditions. It is known that atmosphere pollution may lead to an increase in macrophages⁽³⁸⁾ or neutrophila^(39,40) count in sputum. Another limitation of the study is the lack of a control group with healthy subjects, making it impossible to compare the analyzed features between two groups exposed to similar environmental conditions.

Because of this, we compared our results (Figure 1) with the only published reference values on healthy subjects (Belda et al)⁽³⁵⁾ and with values described in other studies.^(11,28) We observed that in several studies, asthmatic subjects cohorts, with or without treatment with corticosteroids, present higher proportions of neutrophils as compared to our sample. One possible explanation for this lower proportion of neutrophils in our sample may be related with environmental factors. Most of these studies were performed in industrial sites, with higher pollution levels, fact which may explain the results. However, we don’t have specific reference values for the cellular induced sputum profile for healthy subjects in our environment; further studies are needed to determine them.

No difference was observed in this study in terms of symptoms, bronchodilators’ use and magnitude of airway obstruction between the eosinophilic and the non-eosinophilic group. Our results are similar to the ones described by Pavord et al,⁽²³⁾ who analyzed the response to corticosteroids in corticosteroid-naïve patients. The eosinophilic and non-eosinophilic groups had similar baseline FEV₁ measurements (predicted percentage), FEV₁and forced vital capacity (FEV₁/FVC) rate and symptom score. Otherwise, Turner et al⁽²⁰⁾ described asthmatic patients in use and not in use of corticosteroids during a mild symptom exacerbation, and although they didn’t observe any differences in terms of symptoms prevalence, airways obstruction was significantly higher at the eosinophilic group. This discrepance is probably due to differences between the studied populations.

It is important to highlight that in our study we only asked about the frequency of symptoms and the presence or absence of night time symptoms causing awakenings, without further discrimination or characterization. This may have been the reason why no differences were seen between the studied groups. As an example, we might mention a disorder called eosinophilic bronchitis, which is featured by the presence of chronic cough and sputum eosinophilia without any evidence of variable limitation to airways flow or bronchial hyper-responsiveness.^(41,42) Treatment with corticosteroids decreases the number of eosinophils in sputum and improves cough symptoms.^(42,43) If we had studied symptoms in a more detailed way, specially “cough”, we might have found significant differences in terms of such variables between the eosinophilic and non-eosinophilic groups. This hypothesis needs further and more detailed investigations.

Another issue is the cut-off point we used to define sputum eosinophilia. We decided to use a 3% value based on some published results. Belda et al⁽³⁵⁾ showed, from the analysis of induced sputum in a group of 96 healthy subjects, that normal sputum eosinophil proportions may reach 2.2%. Additionally, it was observed that in subjects with eosinophil proportions in sputum, ³ 3% respond favorably to corticosteroid therapy, differently from subjects who do not have eosinophilic sputum.^(22,23) Therefore, we can safely assure that values above this cut-off point are related to eosinophilic inflammation of the airways. However, it needs to be taken into consideration \that with the method we used to manage sputum samples, only 400 cells are counted from each Giemsa stained cytospin. With this number, proportions of eosinophils of 2 and 3% may differ by one or two cells among the total counted cells. Therefore, it is possible that proportions slightly lower than 3% were found on some cytospins, even if the sputum were in fact eosinophilic. However, we believe that this error, intrinsic to the method, is not frequent, and is not a confusing factor for results of interventional studies.

We may conclude that steroid-naïve asthmatic patients in our environment present a higher proportion of sputum eosinophils as compared with established reference values, and that the clinical and physiological outcomes are not able to predict the presence or absence of eosinophilic airway inflammation. The eosinophilic infiltrate that we described is usual in asthma and was reported in several studies.^(11,14,15) However, it is important to highlight that most of these studies were performed with the cross-sectional proportion of eosinophils in a given moment as an inclusion criterium, with no special concern in terms of its variability and the factors that may alter its proportions. Therefore, further longitudinal studies may be needed in order to evaluate sputum eosinophils proportion variability. Still, it is mandatory to assess the usefulness of this information, determining in which proportion response to the use of inhaled corticosteroids by asthmatic patients may be predicted by sputum eosinophilia.⁽⁴⁴⁾

Acknowledgements

We would like to thank all the patients who agreed to take part in this study, NUPAIVA’s nurses, Janara Voltolini Maia and Cristiane Cinara Rocha, for their help on data collection and sputum induction, and Mrs. Célia Tania Zimmermann, for having managed sputum samples.

Received for publication on 20/09/02

Approved, after review, on 17/04/03

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*

This study was performed at Núcleo de Pesquisa em Asma e Inflamação das Vias Aéreas (Nupaiva), Universidade Federal de Santa Catarina, Florianópolis, SC.

Publication Dates

• Publication in this collection
  02 Dec 2003
• Date of issue
  Aug 2003

History

• Accepted
  17 Apr 2003
• Received
  20 Sept 2002