Mepolizumab reduced the number of blood and sputum eosinophils and allowed prednisone sparing in patients who had asthma with sputum eosinophilia despite prednisone treatment. (ClinicalTrials.gov number, NCT00292877.)

Methods to examine sputum for indices of airway inflammation are evolving. We have examined the repeatability and the validity of an improved method to measure sputum cells and fluid-phase eosinophil cationic protein (ECP), major basic protein (MBP), eosinophil-derived neurotoxin (EDN), albumin, fibrinogen, tryptase, and interleukin-5 (IL-5). Sputum was induced with hypertonic saline twice within 6 d in 10 healthy subjects, 19 stable asthmatics, and 10 smokers with nonobstructive bronchitis. The method included the processing of freshly expectorated sputum separated from saliva, treatment with a fixed proportion of dithiothreitol 0.1% followed by Dulbecco's phosphate-buffered saline, making cytospins, and collecting the supernatant. The reproducibility of measurements, calculated by the intraclass correlation coefficient, was high for all indices measured with the exception of total cell counts and proportion of lymphocytes. Asthmatics, in comparison with healthy subjects and smokers with bronchitis, had a higher proportion of sputum eosinophils (median percent 5.2 versus 0.5 and 0.3), metachromatic cells (0.3 versus 0.07 and 0.08), ECP (1,040 micrograms/L versus 288 and 352), MBP (1,176 micrograms/L versus 304 and 160), and EDN (1,512 micrograms/L versus 448 and 272). Asthmatics differed from healthy subjects, but not from smokers with bronchitis, in the proportion of neutrophils (46.9% versus 24.1%), albumin (704 versus 288 micrograms/mL), and fibrinogen (2,080 versus 440 ng/mL). Smokers with bronchitis showed a trend for a higher neutrophil count and levels of albumin and fibrinogen than healthy subjects. The proportion of sputum eosinophils correlated positively with ECP, MBP, EDN, albumin and fibrinogen levels, and metachromatic cell counts correlated with tryptase. In asthmatics, IL-5 correlated with eosinophil counts. There was a significant negative correlation between sputum indices and expiratory flows and methacholine PC20. Thus, the methods of measuring cell and fluid phase markers in induced sputum used in this study are reproducible and valid. They can therefore be used to reliably measure these indices of airway inflammation.

We have investigated the time-course of symptoms, forced expiratory volume in one second (FEV1), and the airway inflammatory changes in sputum selected from saliva and blood of 10 patients with severe exacerbation of asthma betwen presentation and after 1, 2, 3, 7, and 21 days of treatment. The sputum was induced by a modified standard protocol, and we examined its safety. The severe exacerbation of asthma was defined by the presence of nocturnal symptoms disturbing sleep and/or the need for inhaled short acting beta2-agonist > or = 8 puffs/d and an FEV1 after bronchodilator < 60% of predicted. The treatment consisted of additional prednisone 30 mg daily for 5 d followed by reduction to zero by day 10. Abnormal findings [median (interquartile range)] in spontaneous and induced sputum included low viability of cells [52.0 (34.0)%]; eosinophilia [20.0 (16.4)%]; many free eosinophil granules; and increased levels of fluid-phase ECP [1960 (9204) microg/L], fibrinogen [6045 (10720) microg/L], and IL-5 [160 (212) pg/ml]. Peripheral blood eosinophils [10.4 (7.6)%] and ECP levels [34.0 (35.0) microg/L] were increased. After treatment, symptoms, FEV1, blood eosinophilia, and serum ECP improved in the first 24 h. Sputum eosinophils and ECP did not improve until 48 h and fibrinogen not until 7 d. The improvement in sputum eosinophils and ECP levels was correlated with improvement of FEV1 and in fluid-phase IL-5. Thirty sputum inductions were performed safely in the majority with inhaled isotonic or 3% saline (23.3% or 63.3%, respectively) over a short duration (mean 8.4 min). The patients who had a fall in FEV1 of > or = 10% (10 occasions) after induction differed from those with a fall of < 10% only in the amount of inhaled beta2-agonist used by the patients in the preceding 24 h [8.0 (5.0) versus 4.0 (3.0) puffs/d, p = 0.01]. The results suggest that spontaneous or induced sputum can be used safely to follow the kinetics of effects of antiinflammatory treatment in a severe exacerbation of asthma. The clinical and blood indices improve before those in sputum, raising the possibility that examination of sputum is a better guide in these patients to follow the effects of treatment.

Sputum examination is being used increasingly as a noninvasive method to assess airway inflammation. Expectorated sputum has variable contamination with saliva. Methods of processing have included the selection of portions of the sample considered to be representative of pulmonary origin versus use of the whole specimen, which is confounded by varying volumes of saliva. We compared cell profiles and eosinophilic cationic protein (ECP) concentration in sputum selected from the expectorate and in the usually discarded residual portion to determine to what degree salivary contamination is minimized and if the results are representative of lower respiratory secretions. Sputum was induced with hypertonic saline in six healthy and nine asthmatic subjects. All portions considered to be of pure lower respiratory tract origin were selected from the residual. The selected and residual portions were treated with dithiothreitol, total cell counts and cell viability were obtained, cytospins were made for differential cell counts and supernatant was collected for ECP assay. Selected portions of the specimens, in comparison with the residual portion showed: little squamous cell contamination (median 1.2 vs 70%; p < 0.001); higher total cell counts.mL-1 (5.1 vs 0.5 x 10(6) cells.mL-1; p < 0.001); higher number of viable nonsquamous cells per sample (1.9 vs 0.6 x 10(6) cells; p < 0.001); higher slide quality score (7 vs 4; p < 0.001); and higher levels of ECP (768 vs 136 micrograms.L-1; p < 0.001). There were no differences in the differential cell counts of eosinophils (1.3 vs 3.8%), neutrophils (44 vs 32%), and lymphocytes (0.6 vs 0.6%). While the proportion of macrophages was lower (36 vs 54%; p < 0.05), the absolute number (41 vs 19 x 10(4) cells; p < 0.05) was higher in the selected portion. In summary, selection of all portions of induced sputum from the expectorate minimized the confounding influence of saliva. Loss of nonsquamous cells in the residual portion was variable but usually less than one third of those in the selected portion. With one exception, this loss had little influence on the differential counts of inflammatory cells. Similar observations apply to eosinophilic cationic protein levels. We conclude that, in healthy subjects and treated asthmatics, inflammatory markers in the selected portion of the expectorate can be used to represent those in the lower respiratory tract in general.

Leukotrienes are pro-inflammatory mediators which may contribute to tissue, sputum, and blood eosinophilia seen in allergic and inflammatory diseases, including asthma. Montelukast is a cysteinyl leukotriene 1 (CysLT 1 ) receptor antagonist which improves asthma control; the aim of this study was to investigate its effect on induced sputum eosinophils.Montelukast 10 mg (n=19) or placebo (n=21) were administered orally once in the evening for 4 weeks to 40 chronic adult asthmatic patients, aged 19±64 yrs, in a double-blind, randomized, parallel group study. Patients were included if, at prestudy, they had >5% sputum eosinophils, symptomatic asthma with a forced expiratory volume in one second $65% of the predicted value and were being treated only with "as needed" inhaled b 2 -agonists. In addition to sputum eosinophils, blood eosinophils and clinical endpoints were also assessed.Four weeks of montelukast treatment decreased sputum eosinophils from 7.5% to 3.9% (3.6% decrease, 95% confidence interval (CI) -16.6±0.4). In contrast, placebo treatment was associated with an increase in sputum eosinophils from 14.5% to 17.9% (3.4% increase, 95% CI -3.5±9.8). The least squares mean difference between groups (-11.3%, 95% CI -21.1± -1.4) was significant (p=0.026). Compared with placebo, montelukast significantly reduced blood eosinophils (p=0.009), asthma symptoms (p=0.001) and b 2 -agonist use (p<0.001) while significantly increasing morning peak expiratory flow (p=0.001). Montelukast was generally well tolerated in this study, with a safety profile similar to the placebo.These results demonstrate that montelukast decreases airway eosinophilic inflammation in addition to improving clinical parameters. Its efficacy in the treatment of chronic asthma may be due, in part, to the effect on airway inflammation.

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Inhalation of hypertonic saline to induce sputum may alter cells and fluid-phase markers in sputum. We have compared indices of inflammation in sputum produced spontaneously with sputum induced by an aerosol of hypertonic saline. Twenty-three asthmatics produced spontaneous followed by induced sputum on the same day. The sputum specimen was separated from saliva within 2 h, dispersed with dithiothreitol (DTT) and processed to obtain cytospins and supernatant. The statistical power to detect a 20% difference in sputum parameters was > 90%. Results are expressed as median and interquartile range [IQR]. Induced sputum had a higher proportion of viable cells (77.0 [19.0] versus 47.0 [38.0]%, p < 0.001), less squamous cell contamination (1.0 [1.2] versus 1.8 [34.0]%, p < 0.001) and better quality cytospins (score of 8.0 [4.0] versus 4.0 [2.0], p < 0.001). It also had lower fluid-phase levels of eosinophil cationic protein (ECP) (1,358 [1,102] versus 1,574 [2,479] microg/L) and fibrinogen (1,560.0 [3,130.0] versus 4,350.0 [5,970.0] ng/ml) but only the latter was significantly different (p = 0.02). Induced sputum was similar to spontaneous sputum in weight (200.0 [219.0] versus 270.0 [227.0] mg), total cell count (3.3 [4.1] versus 3.5 [4.5] x 10(6)/ml), proportion of nonsquamous cells, and levels of tryptase. The agreement between induced and spontaneous measurements was good, but fluid-phase levels were affected by the low viability of some spontaneous samples. We conclude that for the indices measured in asthmatic subjects, induced sputum separated from saliva is similar to lower respiratory secretions expectorated spontaneously and has the advantage of better cell viability.

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