INTRODUCTION

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Sputum production induced by inhaled hypertonic saline has been increasingly proposed as a noninvasive alternative to bronchoscopy for collecting secretions and inflammatory cells from the airways of patients with asthma or chronic obstructive pulmonary disease (1). However, inhalation of aerosols of hypertonic saline induces bronchoconstriction and hypoxemia in subjects with bronchial hyperreactivity (4, 5), thereby requiring pretreatment with a short-acting ₂-agonist and monitoring of pulmonary function and arterial oxygen saturation (Sa_O2) during the procedure (5, 6).

Uridine 5'-triphosphate (UTP) is a triphosphate nucleotide, and recent studies have shown that inhalation of UTP provides an improvement in airway mucociliary clearance without apparent side effects in normal persons (7) and in patients with cystic fibrosis and primary ciliary dyskinesia (8, 9). This action of UTP is presumably mediated by stimulation of ciliary motility of airway epithelium (10), Cl and water transport across airway mucosa toward the lumen (11), and mucus secretion from submucosal glands and goblet cells via activation of P₂Y₂ receptors (12, 13). On the basis of these activities of UTP as a secretagogue, aerosols of this nucleotide may be used for sputum induction. Therefore, in the present study we aimed to compare the effectiveness and the safety between methods with inhaled UTP and hypertonic saline in healthy subjects and in asthmatic patients. Furthermore, to determine whether the sputum induction by UTP can be used to reliably assess airway inflammation, we examined the validity and the reproducibility of the measurements, including the total and differential cell counts of induced sputum.

	METHODS

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Subjects

The study population comprised 16 healthy subjects and 16 patients with stable asthma. All patients had well-documented histories of asthma as defined by the American Thoracic Society guidelines (14). Other entry criteria included absence of bronchial symptoms at the time of the study, no asthmatic exacerbations in the previous 8 wk, FEV₁ > 60% of predicted, and a documented reversibility > 15% of FEV₁ over baseline at 15 min after inhalation of a ₂-agonist. Six subjects in the healthy group and seven patients in the asthma group were atopic as indicated by one or more positive allergy skin test to common allergen extracts. Written informed consent was obtained from each subject, and the study was approved by the Tokyo Women's Medical University Medical Board.

Study Design

Subjects attended our outpatient clinic three times at the same time of day with 1 wk apart. In each subject, sputum induction was performed using hypertonic saline on the first visit (Day 1) and UTP on the second and third visits (Days 8 and 15). To compare the effectiveness and the safety between the two methods, the volume of expectorated sputum and the changes in Sa_O2 and peak expiratory flow (PEF) during the procedures were determined on Days 1 and 8. For the assessment of the validity in evaluating airway inflammation, sputum cell counts were determined. To study the reproducibility of the measurements, sputum cell counts were compared between first (Day 8) and second (Day 15) trials with UTP inhalation.

Sputum Induction and Processing

Patients with asthma inhaled salbutamol (200 µg) 5 min before sputum induction. Sterile hypertonic saline (3%, 966 mOsm) or UTP (5 mg/ml in 0.9% saline, 314 mOsm) was aerosolized by ultrasonic nebulizer (Type-76; Kawanishi Iryo Co., Tokyo, Japan) with an output of 3 ml/min and a particle size of 5.25 µm aerodynamic mass median diameter. The subjects inhaled aerosols for 5 min and then expectorated sputum, which was immediately examined, as described by Pizzichini and coworkers (2). The sputum sample was weighed and treated with 0.1% dithiothreitol and PBS. The suspension was filtered and a total nonsquamous cell count was performed in a hemocytometer. From the remainder of the filtrate, cytospins were made and stained with Wright's stain, and a 400 differential nonsquamous cell count was performed. The specimen was considered adequate if total and differential cell counts could be obtained; this required as little as 50 mg of selected sputum (15).

Safety Assessments

Each subject's symptoms were monitored by a physician, and Sa_O2 was measured continuously by a pulse oximeter (Pulsox-5; Minolta Co., Tokyo), from 2 min before the start of the sputum induction until the procedure was completed, and for 5 min after completion, or until recovery to baseline value. During the sputum induction the maximal fall in Sa_O2 from the baseline value was determined, and the procedure was prematurely terminated if Sa_O2 fell below 85% or at the request of the subject. The area under the curve (AUC) was also determined by graphics software (Fig P; Fig P Software Corp., Durham, NC), which calculated the area of the Sa_O2-time response. To assess bronchoconstriction, PEF (best of three attempts) was determined 1 min before and immediately after the sputum was expectorated.

Statistical Analysis

Values were expressed as means ± SD. Differences in variables between groups were determined by ANOVA and the Student-Newman-Keuls test. Differences between groups were first analyzed using the Kruskal-Wallis test. When a significant difference between groups was noted, intergroup comparisons were assessed by a nonparametric method using the Mann-Whitney U test. By using the sputum samples obtained by UTP inhalation on Days 8 and 15, the reproducibility of cell counts was assessed by the reliability coefficient (intraclass correlation coefficient, R) as the proportion in the variance in the measures to the true variance between subjects (16) and graphically reported, as proposed by Bland and Altman (17). All statistical tests were performed with Unistat 3.0 statistical software (Megalon S.A., Novato, CA), and a p value of less than 0.05 was considered statistically significant.

	RESULTS

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Subject characteristics are shown in Table 1. There were no differences between healthy subjects and patients with asthma with respect to sex, age, and baseline Sa_O2 values. During the sputum induction procedure, the subjects commonly reported mild nausea with retching and an unpleasant salty taste when they inhaled hypertonic saline aerosols, whereas such side effects were not observed with UTP aerosols. No subject developed side effects sufficiently severe to require a rescue use of ₂-agonist or premature termination of the procedure.

Sufficient amounts (> 50 mg) of sputum samples were obtained from 11 of 16 healthy subjects and 12 of 16 asthmatics who received hypertonic saline, and from all the subjects who received UTP. As shown in Table 2, inhaled UTP produced twofold greater amounts of sputum than did inhaled hypertonic saline in both healthy subjects and patients with asthma (p < 0.001 for each). The cell viability determined by the trypan blue exclusion method was 81 ± 14% in the sputum samples obtained by hypertonic saline and 84 ± 12% in those obtained by UTP. There were no significant differences in the total cell counts between samples obtained with hypertonic saline and UTP. Regarding the differential cell counts, sputum from asthmatics had a higher proportion of eosinophils than did sputum from healthy subjects, and there were no significant differences in the percentage counts of each cell type between the two methods.

During the sputum induction, Sa_O2 fell in healthy subjects from 97.9 ± 0.9% to the maximum value of 92.9 ± 2.4% (p < 0.001) upon hypertonic saline inhalation and from 97.9 ± 1.0% to 95.3 ± 2.1% (p < 0.001) upon UTP inhalation. The decrease in Sa_O2 from the baseline value observed with UTP inhalation was less than that seen with hypertonic saline inhalation in healthy subjects (p < 0.001) and patients with asthma (p < 0.01) (Table 2). In each subject the response of Sa_O2 was transient, and the value returned to baseline within a few minutes after the completion of inhalation. The AUC of the Sa_O2 response was less for UTP inhalation than for hypertonic saline inhalation (p < 0.01 for healthy subjects and asthmatic patients). Healthy subjects showed a decrease in PEF from 501 ± 25 L/min to 471 ± 26 L/min (p < 0.001) after hypertonic saline and from 501 ± 16 L/min to 485 ± 20 L/min (p < 0.001) after UTP, where the change from the baseline value was significantly different between the procedures (p < 0.05). Similarly, the fall in PEF in patients with asthma was less for UTP inhalation than for hypertonic saline inhalation (p < 0.01).

The reproducibility of the measurements with the UTP method was examined by comparing the total and differential cell counts of sputum samples obtained on Days 8 and 15. The overall reproducibility was low for total cell counts (R = 0.50) and high for eosinophils (R = 0.88) and neutrophils (R = 0.83) (Figure 1). The reproducibility was also high for macrophages (R = 0.66) and lower for lymphocytes (R = 0.39), and bronchial epithelial cells (R = 0.35) (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Bland Altman plots (difference between two measurements versus mean of same two measurements) for repeated measurements of total cell counts (top panel  ), eosinophil counts (middle panel ), and neutrophil counts (bottom panel ) in the sputum induced by UTP in healthy subjects (closed circles) (n = 14) and patients with asthma (closed triangles) (n = 15). R is the global intraclass correlation coefficient.

	DISCUSSION

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In this study, sputum induction was performed by giving hypertonic saline or UTP by aerosolized delivery in healthy subjects and patients with mild-to-moderate asthma. We found that, in comparison with hypertonic saline, inhalation of UTP solution produced a greater amount of sputum and less falls in Sa_O2 and PEF during the procedure. Therefore, although each sputum induction method is well tolerated in both groups of subjects, the procedure can be conducted more effectively and safely by the use of UTP aerosols than by hypertonic saline aerosols.

This is the first report to use aerosols of the nucleotide UTP for the sputum induction method. The observed increase in sputum volume expectorated after UTP inhalation may be derived from the integrated effects of stimulation of airway secretion and enhancement of mucociliary clearance. There is increasing evidence that UTP stimulates Cl secretion through apical Ca²⁺-dependent Cl channels in airway epithelial cells via P₂Y₂ receptors and, hence, liquid secretion across airway mucosa toward the luminal side (11, 18). Furthermore, UTP promotes the release of mucin from submucosal glands and goblet cells (12, 13). These effects probably result in the increase in sputum in the airway lumen. In addition, UTP stimulates ciliary motility of airway epithelium (10), which produces an improvement in mucociliary transport of the retained secretions in the respiratory tract. Indeed, inhalation of UTP aerosols has recently been shown to enhance mucociliary transport in patients with impaired airway clearance (8, 9). Thus, UTP not only represents a potential pharmacotherapy for airway diseases but may also be used as a tool for sputum induction for noninvasive assessment of airway inflammation.

One of the important characteristics of any measurement is validity, which can be examined by its ability to detect differences between different clinical conditions. In healthy subjects, the total and differential cell counts of the UTP-induced sputum were not different from the results obtained with inhaled hypertonic saline. Moreover, in patients with asthma the increase in the proportion of eosinophils was similar between sputum samples obtained with UTP and hypertonic saline, suggesting that sputum induction by UTP aerosols is a valid method for the detection of eosinophilic inflammation in asthmatic airways. We also determined the reproducibility of the measurement using cytospins of the UTP-induced sputum obtained on Days 8 and 15, and found good reproducibility of the differential cell counts of eosinophils, neutrophils, and macrophages. The values for reproducibility (R) are close to what was reported previously in the sputum induced by hypertonic saline (2), and the observed high degree of reproducibility may be a reflection of the lack of UTP actions in airway inflammation, and partially because of the stability of the subjects who were studied.

Several studies have reported the safety of sputum induction with hypertonic saline in asthmatic subjects (1, 5, 6). In those studies, pretreatment with ₂-agonist did not prevent bronchoconstriction, especially in patients with low baseline FEV₁. This bronchoconstriction probably involves proinflammatory activity of hypertonic saline solution (19) and airway smooth muscle contraction produced by the release of tachykinins from sensory neurons (20) or the release of mast cell mediators (21). In our study, inhaled UTP similarly produced a fall in PEF. The mechanism for this is unknown, but it could be associated with purinergic receptor-mediated airway smooth muscle contraction (22) and/or airflow limitation caused by the increased intraluminal secretions. However, the change from the baseline PEF was significantly less compared with that seen with hypertonic saline inhalation. In addition, the maximal change in Sa_O2 from baseline and the AUC of the Sa_O2-time response during sputum induction were less with UTP than with hypertonic saline. These findings emphasize the importance of careful monitoring of bronchoconstriction and oxygen saturation during sputum induction and suggest that on the basis of safety aspect UTP seems to be superior to hypertonic saline as a tool of sputum induction.