Adam Barczyk, MD, PhD; Ewa Sozañska, MA; Marzena Trzaska, MD; and Władysław Pierzchała, MD, PhD

Decreased Levels of Myeloperoxidase in Induced Sputum of Patients With COPD After Treatment With Oral Glucocorticoids* Adam Barczyk, MD, PhD; Ewa Sozañska, MA; Marzena Trzaska, MD; and Władysław Pierzchała, MD, PhD Background: Inhaled glucocorticoids may decrease exacerbations in some patients with COPD, and oral glucocorticoids may improve FEV 1 and shorten hospital stay during exacerbations. The mechanism of these improvements is unknown. This study examines the effect of oral glucocorticoids on markers of neutrophilic airway inflammation. Methods: Eighteen patients with COPD received oral prednisone, 0.5 mg/kg/d for 2 weeks. Clinical status, lung function measurements, and sputum induction were performed before and after treatment with oral prednisone. Levels of the neutrophil chemoattractant (interleukin-8 [IL-8]) and neutrophil activation marker (myeloperoxidase [MPO]) were measured in the supernatant of induced sputum by enzyme-linked immunosorbent assay. Results: Levels of MPO decreased significantly after treatment with prednisone (p 0.0004): before treatment median, 2.54 g/ml (range, 1.49 to 12.58 g/ml); after treatment median, 1.79 g/ml (range, 1.32 to 3.57 g/ml). Treatment with prednisone did not influence the levels of IL-8. Conclusions: The treatment of patients with COPD with oral glucocorticoids decreases the activation of neutrophils, which may be partially responsible for clinical improvement in these patients. (CHEST 2004; 126:389 393) Key words: COPD; induced sputum; myeloperoxidase; oral glucocorticoids treatment Abbreviations: DTT dithiothreitol; IL-8 interleukin-8; MPO myeloperoxidase Corticosteroids are regarded as a first-line therapy for asthma, but their role in the treatment of patients with COPD is still controversial. Inhaled corticosteroids seem to be clinically far less effective in treating COPD than asthma. Some patients with COPD show a decreased frequency of exacerbations after long-term treatment with inhaled corticosteroids and a small improvement in lung function measurements. 1,2 Oral corticosteroids are usually administered to patients with COPD for the treatment of exacerbations of the disease. They effectively improve FEV 1 and shorten hospital stay. 3,4 However, the benefits of this treatment are most *From the Department of Pneumology, Silesian Medical Academy, Katowice, Poland. Manuscript received June 10, 2003; revision accepted March 31, 2004. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@chestnet.org). Correspondence to: Adam Barczyk, MD, PhD, Department of Pneumology, Silesian Medical Academy, Katowice, ul. Medykow 14, Poland; e-mail: adagne@mp.pl prominent during the first days, and are usually limited to the first 2 weeks only. 4,5 Approximately 20 to 40% of stable patients with COPD respond to 2 weeks of treatment with oral prednisolone, when a positive response is defined as an increase in FEV 1 by at least 15% above baseline. 6,7 Airway inflammation can be studied in COPD by examination of induced sputum, which shows clear differences compared to asthma. Smokers and exsmokers with COPD have neutrophilic airway inflammation. This is characterized by an increased number of neutrophils in sputum along with a higher level of markers of neutrophil activation, such as myeloperoxidase (MPO), and neutrophilic chemokines, such as interleukin-8 (IL-8). These changes are in contrast to eosinophilic airway inflammation seen in asthma. 8 Corticosteroids effectively diminish eosinophilic airway inflammation in asthma, 9,10 but their influence on neutrophils, which predominate in COPD, is uncertain. In one study, 11 2 months of treatment with inhaled corticosteroids in patients with COPD reduced neutrophilic airway inflamma- www.chestjournal.org CHEST / 126 / 2/ AUGUST, 2004 389

tion, but this was in disagreement with the results of other studies. 10,12 At the time our study began, the results of only one other study 10 on the effects of oral corticosteroids on indices of neutrophilic airway inflammation in patients with COPD had been published. Although Keatings et al 10 concluded that the inflammatory process in COPD was unresponsive to a 2-week course of high-dose oral prednisone, the results could have been influenced by the small size of the study (n 8). In fact, the level of MPO was decreased, but it was not statistically significant. The aim of our study was to measure the effect of treatment with oral corticosteroids on indices of neutrophilic airway inflammation in COPD in a larger population of patients. Subjects Materials and Methods The study protocol was approved by the ethics committee of the Silesian Medical Academy. All subjects gave their written informed consent before they entered the study. Twenty-three stable patients with COPD (18 men and 5 women) were recruited into the study. The inclusion criteria were as follows: FEV 1 /vital capacity 70%, FEV 1 80% predicted value, FEV 1 reversibility 10% of predicted value 15 min after 200 g of inhaled salbutamol, smoking history of at least 10 pack-years, and no use of inhaled or systemic steroids in the last month. Patients with any history of asthma or patients with an exacerbation of the disease in the previous 4 weeks were excluded. Bronchodilator drugs were allowed, provided that they were not changed during the study and in the previous 4 weeks. Five patients were treated with 2 -agonists, anticholinergics, and methylxanthines. Four patients were treated with a combination of two drugs (two patients received anticholinergics and methylxanthines, one patient received 2 -agonists with anticholinergics, and one patient received 2 -agonists with methylxanthines). Eight patients were treated with one medication (five patients with anticholinergics alone, two patients with methylxanthines alone, and one patient with 2 -agonists alone). Six patients received no treatment at all. Study Design The study consisted of two visits. On the first visit, the clinical history according to a study questionnaire was recorded. Spirometry was recorded at baseline and 15 min after inhalation of 200 L of salbutamol, and a sputum induction was performed. All patients were treated for 2 weeks with oral prednisone, 0.5 mg/kg/d. On the second visit, the clinical history for the treatment period was recorded followed by spirometry, reversibility of bronchial obstruction after salbutamol, and sputum induction. Sputum Production and Processing Sputum induction and processing were performed according to a previously described method, 13 with small modifications. 14 First, spirometry was performed before and 15 min after inhalation of 200 g of salbutamol. Then, the subjects inhaled 7 ml of 5% saline solution at room temperature from an ultrasonic nebulizer (Thomex MB; Medbryt; Warsaw, Poland). After mouth washing, they were encouraged to cough sputum. If unsuccessful, they repeated (no more than twice) the above procedure and performed spirometry every time. Sputum was poured into a Petri dish, and all portions that appeared free of salivary contamination macroscopically were separated from saliva using blunted forceps. This selected portion was weighed and a fourfold volume of dithiothreitol (DTT) [Invitrogen; Paisley, UK] was added to the sputum. The mixture was vortexed and repeatedly aspirated, and then placed on a bench rocker for 20 min. Next, a volume of Dulbecco phosphate-buffered saline solution (Gibco-BRL), equal to the volume of sputum, and DTT was added and the mixture was rocked for 5 min. The mixture was then centrifuged at 790g for 10 min, and the supernatant was aspirated and stored in Eppendorf tubes at 70 C for later assay. The cell pellets from sputum centrifugation were resuspended in Dulbecco phosphate-buffered saline solution. Using a Neubauer hemocytometer (Heinz-Herenz; Hamburg, Germany), the following values were measured: total cell count of leukocytes, percentage of epithelial cells, and cell viability (by the Trypan blue exclusion method). Slides were prepared using a Cytospin instrument (MPW 342; Instruments Co-operative; Warsaw, Poland). Two slides were stained with May-Grunwald-Giemsa stain for differential cell counts. The percentages of macrophages, neutrophils, lymphocytes, and eosinophils were determined after the counting of 400 leukocytes. Sputum Assays IL-8 Assay: IL-8 was measured with an enzyme-linked immunosorbent assay using a commercially available kit (Bender MedSystems; San Bruno, CA) according to the instructions of the manufacturer. The lower limit of detection was 11 pg/ml. The results of the assay were linearly affected by DTT; thus, to compensate for this effect, a concentration of DTT equivalent to that in the sputum samples was added to the standards. MPO Assay: MPO concentrations were determined using a commercially available enzyme-linked immunosorbent assay kit (Hycor Biomedical; Garden Grove, CA). The detection limit of the assay was 0.9 U/mL. DTT was added to all standards at a concentration equivalent to that in the samples, as the results of the assay were affected by the presence of DTT. Statistical Analysis Data are expressed as medians values (maximal and minimal values), and are presented as baseline values (prior to prednisone treatment) followed by the end of prednisone treatment values. Comparisons between these two groups were made using the Wilcoxon signed rank-sum test; p 0.05 was accepted as significant. Results Five patients were withdrawn from the study: three of them did not tolerate the prednisone treatment (the first patient complained about malaise and fatigue; the second patient had high BP, tachycardia, and sight difficulties; and the third patient had rash, erythema, and fever). As for the two other patients, one subject was unwilling to continue the study and the other patient did not produce an adequate 390 Clinical Investigations

sputum sample on the second visit. The subject characteristics and the results of the study were based on an analysis of 18 patients who finished the study (age range, 48 to 77 years; median age, 67 years; 2 women and 16 men). The subjects had mild-to-severe airflow limitation (median FEV 1, 53.2% of predicted). The characteristics of patients who completed the study are summarized in Table 1. Two weeks of treatment with prednisone did not significantly change the FEV 1 or any of the other spirometry parameters. There was a significant decrease in the percentage of eosinophils (1.5%; range, 0.1 to 53.0%; vs 0.2%; range, 0 to 3.6%; p 0.005) and the absolute number of eosinophils (0.09 10 6 / g; range, 0.001 to 1.68 10 6 /g; vs 0.01 10 6 /g; range, 0 to 0.18 10 6 /g; p 0.005) in induced sputum after prednisone treatment. Total inflammatory cell counts were unchanged after prednisone treatment. Similarly, the percentage and the absolute numbers of macrophages, neutrophils, and lymphocytes did not differ before and after the treatment period. Cell viability did not change significantly after prednisone treatment. The effects of prednisone treatment on clinical and sputum parameters are summarized in Table 2. MPO was detectable in all samples. There was a significant decrease in the level of MPO in induced sputum after prednisone treatment: 2.54 g/ml; range, 1.49 to 12.58 g/ml; vs 1.79 g/ml; range, 1.32 to 3.57 g/ml (p 0.0004) [Fig 1]. IL-8 was not measurable in the sputum of five patients before and six patients after treatment. Levels of IL-8 did not differ significantly between the treatment periods (Fig 2). Table 2 Effect of Prednisone Treatment on Clinical and Sputum Parameters* Variables Baseline End of Treatment FEV 1, % predicted 53.2 (27.8 79.7) 49.4 (28.8 78.0) Total cells, 10 6 /g of 5.1 (1.3 39.6) 4.3 (0.5 21.5) sputum Viability, % 88 (69 96) 91.5 (77 100) Macrophages, % 18.8 (4.2 43.6) 18.3 (6.1 27.8) Neutrophils, % 77.4 (31.6 93.1) 80.8 (71.5 91.7) Eosinophils, % 1.5 (0.1 53.0) 0.2 (0 3.6) Lymphocytes, % 0.8 (0 2.8) 0.6 (0 3.2) Epithelial cells, % 2.2 (0.3 18.9) 2.1 (0 18.7) *Data are expressed as medians (range). p 0.005 from baseline. sputum of stable patients with COPD. Although the effect of oral prednisone on MPO levels was checked before in one study, 10 the study only showed a nonsignificant decrease in MPO in sputum, probably because of too few included patients (n 8). Neutrophilic airway inflammation is one of the features of COPD. Neutrophils are increased in the airway lumen, but also to a lesser extend in bronchial epithelium of patients with COPD. They are implicated in the development of this disease, as an increased number of neutrophils in induced sputum correlates inversely with FEV 1 and positively with the rate of decline in FEV 1. 8,15,16 Neutrophilic airway inflammation seen in the sputum of patients with COPD may be characterized by an increased number of neutrophils, and also by increased levels of neutrophilic chemokines, such as IL-8, and markers of neutrophil activation, such as MPO, in the Discussion To our knowledge, this is the first study that shows that 2 weeks of treatment with oral corticosteroids significantly decreases the levels of MPO in induced Table 1 Characteristics of Study Subjects* Characteristics Data Subjects, No. 18 Age, yr 67 (48 77) Male/female gender, No. 16/2 Current smoker/ex-smoker, No. 6/12 FEV 1 % predicted 53.2 (27.8 79.7) FEV 1 /vital capacity, % 55.3 (37.3 68.1) FEV 1 after salbutamol % predicted 4.9 (0 8.9) *Data are expressed as median (range) unless otherwise indicated. FEV 1 after salbutamol % predicted reversibility of airway obstruction 15 min after delivering 200 g of salbutamol expressed as a percentage of the predicted value. Figure 1. Effect of treatment with prednisone on levels of IL-8 in induced sputum of patient with COPD after treatment with oral glucocorticosteroids. www.chestjournal.org CHEST / 126 / 2/ AUGUST, 2004 391

Figure 2. Effect of treatment with prednisone on levels of MPO in induced sputum of patients with COPD after treatment with oral glucocorticosteroids. supernatant of induced sputum. Most studies 17,18 that evaluated the effect of treatment with oral corticosteroids on the features of neutrophilic airway inflammation in the sputum of patients with COPD examined only the number of neutrophils and levels of neutrophilic chemokines (mainly IL-8). They showed that neither of these parameters were affected by prednisone treatment. We also confirmed these results in our study, as the percentage and number of neutrophils as well as levels of IL-8 did not differ before or after steroid treatment. In our study, MPO levels decreased after glucocorticoid treatment without a corresponding reduction in neutrophil counts. Although MPO levels in fluids (like BAL fluid or supernatant from induced sputum) tend to be in poor correlation with the number or percentage of neutrophils in these fluids, there are many articles 19,20 showing no such relation between these two measurements. Hence, we think that measurements of MPO in supernatant of induced sputum cannot be viewed as a surrogate for the neutrophil count. This is in opposition to measurements of MPO activity in lung tissue, where this parameter is used to quantify the total neutrophil sequestration to the lungs, because a strong correlation between the number of intravascular neutrophils and the lung content in the enzyme marker MPO has been described. 21 Two factors may be responsible for the weak correlation between MPO levels and the number of neutrophils in BAL or induced sputum. The first factor is the level of granulocyte degranulation, which can also be viewed as a degree of activation. Neutrophils can secrete azurophilic MPO-containing granules within minutes of application of different stimuli, eg, lipopolysaccharide, exercise, and others. 22 Therefore, the same amount of neutrophils can secrete different amounts of MPO into the epithelial lining fluid depending on the existence of external stimuli. The influence of neutrophils situated in bronchial wall may be the second possible reason. These cells are not counted in BAL or induced sputum, but can be a source of additional and varying amount of MPO in these fluids. Most animal or in vitro studies that evaluated the effect of glucocorticoids on the MPO activity in the lung were done in animal models of acute inflammation after lipopolysaccharide tracheal instillation. These studies showed contradictory results, since glucocorticoid resulted in suppression, 23 activation, 24 or had no effects 25 on MPO activity. However, as written above, measurements of MPO activity in the lung parenchyma reflect accumulation of neutrophils in the lung, which is different from measurements of MPO in BAL fluid or in induced sputum, which reflects activation of neutrophils. The effects of glucocorticoids on MPO release from neutrophils after respiratory syncytial virus-induced neutrophil activation were assessed in another in vitro study. 26 Glucocorticoids decreased respiratory syncytial virus-stimulated MPO release from 127 15.3 to 108.8 6.4 ng/ml, but it was not statistically significant. 26 Altogether, these studies do not provide clear information about the effects of glucocorticoids on the neutrophil production of MPO. The levels of MPO in the supernatant of induced sputum are related to the degree of neutrophil activation, the number of neutrophils, and the percentage of dead or dying neutrophils. The number of neutrophils in induced sputum in our study did not change after glucocorticoid treatment. Similarly, we did not observe any changes in cell viability or any correlation between the reduction of MPO and cell viability, so glucocorticoid treatment is highly unlikely to have reduced MPO levels in sputum due to decreased leakage of MPO from dead or dying neutrophils. The most probable explanation for the decreased levels of MPO without a corresponding reduction in neutrophil counts is that glucocorticoid treatment led to the decrease in the degree of neutrophil activation, which resulted in reduced MPO production by neutrophils. The clinical significance of reduced activation of neutrophils after oral steroid treatment in patients with COPD is unknown. The decrease in MPO levels observed in our study was highly significant statistically but relatively low for each of the patients. 392 Clinical Investigations

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