Clinical Investigations

Montelukast Treatment Does Not Affect Peripheral Blood Neutrophil Functions in Asthma Patients
R. Levy, L. Avnun, F. Shimonovitz, A. Konforty, D. Heimer

Pulmonary Unit, Infectious Diseases Laboratory, Faculty of Health Sciences, Soroka University Medical Center and Ben-Gurion University of the Negev, Beer Sheva, Israel

Address of Corresponding Author

Respiration 2004;71:37-44 (DOI: 10.1159/000075647)

 Outline

• Key Words
• Abstract
• Introduction
• Materials and Methods
• Patients
• Study Design
• Neutrophil Functions
• Results
• Discussion
• Acknowledgment
• References

• Author Contacts
• Article Information
• Publication Details
• Drug Dosage / Copyright

  Key Words

• Neutrophils
• Leukotriene D₄
• Montelukast
• Chemotaxis
• Superoxide production
• Phagocytosis

  Abstract

Background: Leukotriene receptor antagonists have become an integral part of asthma treatment as an add-on therapy for patients with moderate disease activity. Objective: To determine whether in vivo treatment with montelukast, a leukotriene receptor antagonist, at the recommended therapeutic dose affects neutrophil functions which are essential for host defense. Methods: Twenty nonsmoking patients between the ages of 18 and 70 with moderate asthma were recruited to the study. All of them were receiving inhaled corticosteroids for at least 6 months and ₂-agonist as needed. After the 2-week run-in period, in addition to the regular medication, each patient was treated with 10 mg of montelukast for a 6-week period. Pulmonary function test was performed and blood was tested for neutrophil activity twice during the run-in period and twice during the treatment period. Each assay was performed in parallel to a matched control. Results: The 6-week treatment period with montelukast did not significantly affect neutrophil chemotaxis or phagocytosis nor the elevated superoxide production. However, at 2 weeks of treatment some of the patients showed a reduction in neutrophil function. Conclusions: The study demonstrated that the recommended dose of montelukast has no significant effect on peripheral blood neutrophil activities, indicating that montelukast treatment does not induce an inflammatory process and does not interfere with the first line of host defense.

Copyright © 2004 S. Karger AG, Basel

 Introduction

Bronchial asthma is characterized by reversible bronchial obstruction with airway inflammation [1]. These inflammatory processes are complex reactions which involve inflammatory cells such as peripheral blood neutrophils (PMN), lymphocytes and eosinophils [2]. Several authors have reported that oxygen metabolites produced by PMN cause injury to various cells and tissues both in vivo and in vitro [3, 4]. In children with bronchial asthma, the release of histamine was significantly correlated with superoxide anion generation, suggesting that basic intracellular abnormalities are related to increased bronchial hyperresponsiveness (BHR) [5]. Moreover, a close correlation exists between the degree of BHR and oxygen radical production in PMN of patients with chronic airflow obstruction. This may indicate that BHR and airway inflammation may be linked in a similar way in patients with bronchial asthma [6]. Furthermore, some authors have demonstrated the appearance of neutrophils in BAL fluid and in the bronchial biopsies of patients with severe bronchial asthma [7], suggesting that neutrophils may play a role in the inflammatory process of bronchial asthma. In sputum induction technique an increase in neutrophil count was correlated with the severity of the disease and was not changed with inhaled corticosteroid [8].

The treatment of bronchial asthma in recent years has aimed to relieve bronchoconstriction and to decrease inflammation. The leukotriene D₄ (LTD₄) receptor antagonist has been shown to have a bronchodilator effect since LTD₄ causes strong bronchoconstriction [9]. However, little information has been published about the anti-inflammatory effect of montelukast. It has been demonstrated that montelukast causes a decrease in the eosinophil count in peripheral blood, although the mechanism of its action remains unknown. LTD₄ has been shown to facilitate eosinophil migration from the lamina propria to the lumen by the chemoattractant FMLP [10]. Mucosal biopsy of the lamina propria after LTD₄ challenge showed increased numbers of eosinophils and neutrophils [11]. Since LTD₄ recruits granulocytes, the LTD₄ antagonist, montelukast, has the potential to act as an anti-inflammatory agent. Thus, the objective of this study was to determine whether montelukast treatment has any effect on neutrophil activities in peripheral blood of asthmatic patients.

 Materials and Methods

 Patients

Twenty patients, aged between 18 and 70 years, nonsmokers, with moderately stable asthma with FEV₁ between 40 and 80%, who were receiving treatment with inhaled steroid and ₂-agonist as needed, were recruited from the Pulmonary Clinic of our hospital. All patients demonstrated at least 12% improvement in FEV₁ after administration of 200 µg of salbutamol. None of the patients had received oral corticosteroids over the last 3 months. Administration of long-acting ₂-agonist and theophylline preparations was not permitted during the study. Pregnant women were excluded. All patients gave informed consent.

 Study Design

There was an initial 2-week run-in period during which time the patients continued to take their usual medication of inhaled corticosteroid and ₂-agonist as needed. Thereafter, in addition to their usual medication, all patients received 10 mg of montelukast each evening for 6 weeks. All the patients visited the outpatient clinic every 2 weeks for 8 weeks. The diary card was completed daily throughout the study regarding cough, shortness of breath, and sleep disturbances. The symptoms were evaluated by the patients on a scale of 0-3. The use of ₂-agonist was recorded twice daily. Average score in the second week of the run-in period was compared with the average score of the last week of treatment. Pulmonary function test, complete blood count and neutrophil activity were measured 4 times during the study - twice in the run-in period and twice during the treatment period. Neutrophils were separated immediately after blood was drawn. Each patient's blood sample was analyzed in parallel to a blood sample from a sex- and age-matched healthy donor. The laboratory examinations were double-blind (which blood was from the patient and which from the control was not disclosed). The analysis of the results was done at the end of the study. Compliance with the medication was ensured by counting the tablets at every visit. This prospective study was approved by the Helsinki Committee of the Soroka Medical Center.

 Neutrophil Functions

Preparation of Granulocytes. Granulocytes, at 95% purity, were obtained by Ficoll-Hypaque centrifugation, dextran sedimentation, and hypotonic lysis of erythrocytes, as previously described [12]. Cells were counted and their viability was determined by trypan blue exclusion.

Superoxide Generation. The production of the superoxide anion (O₂^-) by neutrophils was measured as the superoxide dismutase-inhibitable reduction of acetyl ferricytochrome c by the microtiter plate technique, as previously described [13] with modifications [14]. Cells (2.5 × 10⁵ cells/well) were suspended in 100 µl Hanks' balanced salt solution (HBSS) containing acetyl ferricytochrome c (150 mM). Superoxide production by the cells was stimulated by the addition of PMA (50 ng/ml), opsonized zymosan (OZ; 1 mg/ml) or FMLP (10^-7M). In addition, superoxide production by nonstimulated cells was determined. The reduction of acetyl ferricytochrome c was followed by a change of absorbance at 550 nm at 2- to 5-min intervals on a Thermomax Microplate Reader (Molecular Devices, Melno Park, Calif., USA). The maximal rates of superoxide generation were determined and expressed as nanomoles O₂^-/10⁶ cells/ 10 min using the extinction coefficient E₅₅₀ = 21 mM^-1 cm^-1.

Chemotaxis. Chemotaxis was assessed as previously described [15]. Agarose was dissolved in sterile, distilled boiling water for 10 min. After cooling to 48°C in a water bath, the agarose was mixed with an equal volume of prewarmed × 2 minimal essential medium with 10% heat-inactivated fetal calf serum (FCS), and 7.5% (w/v) sodium bicarbonate. Five milliliters of the agarose medium were delivered to 60 × 15 mm tissue culture dishes and allowed to harden. Six series of three wells, 2.4 mm in diameter and spaced 2.4 mm apart, were formed. Cells were suspended in HBSS. The center well of each three-well series received a 10-µl aliquot of the cell suspension containing 2.5 × 10⁵ purified cells. The outer well received 10 µl of FMLP (10^-7 M) and the inner well received 10 µl of HBSS. The dishes were subsequently incubated at 37°C in a humidified atmosphere containing 5% CO₂ in air for 2 h. The plates were fixed by the addition of 3 ml methanol at 4°C overnight. After the methanol was poured off, the plates were placed in glutaraldehyde (2.5%) for 30 min at room temperature. The agarose gel was removed intact after fixation and the plates stained by Giemsa stain and air dried. The linear migration toward the chemoattractant (FMLP) was measured under a light microscope.

Phagocytosis. 5 × 10⁵ cells were suspended in RPMI 1640 containing 10% heat-inactivated FCS and incubated at 37°C for 15 min with 5 µl zymosan (1 mg/ml), opsonized by pooled human serum. Subsequently, the cells were smeared and stained with differential Wright-Giemsa. Phagocytosis was determined under the microscope in at least 100 cells, and defined as percent of cells containing more than two phagocytized particles of OZ.

Statistics Analysis. Statistical evaluation was carried out with unpaired Student's t test with 95% confidence interval. For symptoms score and FEV₁ measurement we compared mean values of the second week of the run-in period with those of the last week of the study.

 Results

Patients' baseline characteristics are given in table 1. Mean age was 45 ± 11.4 years; 10 patients were male and 10 were female. FEV₁ was 1.956 ± 0.5 l/sec baseline and increased to 2.16 ± 0.6 l/sec after 6 weeks of treatment (p = 0.07). Asthma scoring during the day decreased from 1.5 to 0.7 (p = 0.05), and during the night from 0.88 to 0.37 (p < 0.002). The use of ₂-agonist during the day was reduced from 2.7 to 1.4 puffs (p < 0.016) and during the night from 2.27 to 0.78 puffs (p < 0.024). After analyzing the data, the patients could be classified into three groups: (1) patients who responded objectively with an increase in FEV₁ of >10% and subjectively by a decreasing symptom score of more than 0.5 points; (2) patients who responded subjectively without a change in spirometry, and (3) patients who did not respond at all. WBC was reduced from 7.5 × 10³ to 6.7 × 10³/µl (p < 0.05). Eosinophil count decreased from 7.7 to 5.9%, a change that did not reach statistical significance.

Table 1. Effect of leukotriene receptor antagonist on clinical parameters

Neutrophil functions were determined before and during montelukast treatment and included superoxide generation stimulated with 50 ng/ml PMA, 1 mg/ml OZ, 10^-7 M FMLP or in unstimulated cells, phagocytosis of OZ, and linear migration toward FMLP. The results are presented as percent of the matched controls. As shown in figure 1, 9 patients had a full spirometry and symptom score response, with rates of stimulated or unstimulated superoxide production higher than those of their matched control before montelukast treatment. While the means ± SEM of the rates of superoxide production in stimulated neutrophils were higher than in the matched control by about 10-20%, the means ± SEM of the unstimulated neutrophils reached high levels of about 200% (table 2). Montelukast treatment did not systematically affect superoxide production; in some patients superoxide production levels were reduced and in others elevated, but the effects were not statistically different from the baseline rates. In this group of patients, neutrophil phagocytosis of OZ particles was similar to that of the matched controls before treatment and was not affected by montelukast treatment (fig. 1, table 2). Similarly, as shown in figure 1, neutrophil chemotaxis in most of the patients from this group was similar to that of the matched controls and was not affected by the treatment. In 3 patients, chemotaxis was elevated before treatment but was significantly reduced after 2 weeks of treatment and remained at these reduced levels after the 6 weeks of treatment. The means ± SEM of neutrophil chemotaxis were similar to that of the matched control before and during LTD₄ antagonist treatment (table 2).

Fig. 1. Neutrophil functions of asthmatic patients in the group with full response before treatment and 2 weeks and 6 weeks after montelukast treatment. Superoxide production of neutrophils stimulated with either 50 ng/ml PMA, 1 mg/ml OZ or 10-7M FMLP and of unstimulated neutrophils, phagocytosis of OZ and chemotaxis toward FMLP are shown. The functions were analyzed twice before treatment and the values are the mean of the two analyses. The results are expressed as % of their matched controls.

Table 2. Effect of montelukast treatment on neutrophil functions in the group of asthmatic patients with full response

Figure 2 demonstrates neutrophil functions of the 7 patients with a subjective response. It can be seen that there was a variation in the baseline rates of stimulated superoxide production before treatment, which in some patients were higher, in some patients lower and in some patients similar to those of the matched controls. However, the means ± SEM of the rates of superoxide production before treatment were not significantly different from those of the matched controls (table 3). Montelukast treatment did not significantly or systematically change the rates of stimulated superoxide production (fig. 2, table 3). Similar to the patients from the group with a full response, the most significant findings were seen in the rates of unstimulated superoxide production which were elevated in most of the patients of this group before treatment (means ± SEM of 163.1 ± 24.7%). Montelukast treatment did not significantly affect the rate of unstimulated superoxide production. Phagocytosis was similar to that of the matched controls and was not affected by montelukast treatment. The means ± SEM of the rates of neutrophil chemotaxis before treatment were not significantly different from the matched control (table 3). In 3 of the 7 patients in this group, 2 weeks of treatment caused a significant reduction of chemotaxis which was recovered after 6 weeks of treatment. Neutrophil functions before and after treatment of the 4 patients in the group with no clinical or subjective responses are presented in figure 3 and the means ± SEM are presented in table 4. Neutrophil functions were not significantly different from those of the matched controls and were not affected by LTD₄ antagonist treatment.

Table 3. Effect of montelukast treatment on neutrophil functions in the group of asthmatic patients with subjective response

Table 4. Effect of montelukast treatment on neutrophil functions in the group of asthmatic patients with no response

Fig. 2. Neutrophil functions of asthmatic patients in the group with subjective response before treatment and 2 weeks and 6 weeks after montelukast treatment. Superoxide production of neutrophils stimulated with either 50 ng/ml PMA, 1 mg/ml OZ or 10-7M FMLP and of unstimulated neutrophils, phagocytosis of OZ and chemotaxis toward FMLP are shown. The functions were analyzed twice before treatment and the values are the mean of the two analyses. The results are expressed as % of their matched controls.

Fig. 3. Neutrophil functions of asthmatic patients in the group with no response before treatment and 2 weeks and 6 weeks after montelukast treatment. Superoxide production of neutrophils stimulated with either 50 ng/ml PMA, 1 mg/ml OZ or 10-7M FMLP and of unstimulated neutrophils, phagocytosis of OZ and chemotaxis toward FMLP are shown. The functions were analyzed twice before treatment and the values are the mean of the two analyses. The results are expressed as % of their matched controls.

 Discussion

Cysteinyl leukotrienes (LTC₄, LTD₄ and LTE₄) are the most important leukotrienes in the pathogenesis of asthma. One way of blocking the action of leukotrienes is to use a competitive antagonist, such as CysLT1. This drug, which has been demonstrated to alleviate asthma symptoms, is directed against the LTD₄ receptor. Recent studies have reported that PMN from asthmatic patients are in an activated state as indicated by increased superoxide production [5, 16, 17], the presence of increased complement receptor expression [18, 19, 20] and increased activity of the 5-lipoxygenase pathway [21, 22]. It has also been shown that increased severity of asthma can be associated with a significant increase in spontaneous superoxide production by airspace cells [4] as well as with chemiluminescence of alveolar macrophages stimulated with OZ [23], indicating that enhancement of oxidative metabolism by phagocytic lung cells is an important mechanism for increased airway obstruction in asthma. In accordance with those studies, the asthmatic patients in our study exhibited elevated superoxide production which was most prominent in unstimulated neutrophils, indicating that the cells are in activated state. The higher rates of neutrophil superoxide production were detected in the group of patients with a full response, while normal rates were found in the group of patients with no response. In contrast to the high levels of superoxide production in the patients, the rates of chemotaxis or phagocytosis in neutrophils of the asthmatic patients were similar to those of the matched controls.

The results of the present study show that LTD₄ antagonist treatment did not have a significant effect on mean ± SE of the neutrophil functions studied. However, analysis of the individuals themselves shows that LTD₄ antagonist treatment caused a slight decrease in neutrophil activities which was more apparent after the first 2 weeks of treatment. Thus, although LTD₄ antagonist treatment did not cause a dramatic effect in reducing neutrophil activities in general, it did reduce neutrophil function in some patients, especially during the first period of the treatment. The lack of a significant effect of LTD₄ antagonist treatment shown in our study can be explained by the fact that the LTD₄, as opposed to LTB₄, has no in vitro effect on neutrophils but does affect eosinophil functions [24]. In contrast to LTB₄ which stimulates NADPH oxidase activity to generate superoxide in neutrophils, LTD₄ had no effect on this enzyme [24]. Eosinophil chemotaxis toward LTD₄ was evident at 10^-10M concentration and was abolished by a selective peptide LT antagonist. In contrast, neutrophil chemotaxis was apparent only with a very high LTD₄ concentration (10^-5M) [24].

Neutrophils were found in BAL fluid from patients who suffered a sudden onset of a severe asthma attack [25, 26, 27], suggesting that they may play an important role with acute exacerbation. Nocturnal asthma is associated with high levels of neutrophils in BAL fluid and correlates with the severity of the disease. Mucosal biopsy of the lamina propria after LTD₄ challenge showed increased numbers of eosinophils and neutrophils [10], and sputum neutrophilia was correlated with the severity of asthma [11]. Thus, although neutrophils were shown to be recruited to the site of inflammation in asthma after LTD₄ challenge, based on our results demonstrating that this treatment did not affect neutrophil activity we may assume that this did not augment the inflammatory process which may be caused by neutrophils. A recent study has reported that no additional anti-inflammatory effect was demonstrated in bronchial biopsy of mild asthmatic patients who used montelukast as add-on therapy to inhaled steroid [28].

Although the patients studied had already been partially controlled with steroid inhalers, in about 50% of the patients FEV₁ increased by more than 10%. This change in the symptom score and the decrease in the amount of ₂-agonist was statistically and clinically significantly similar to other previously published clinical trials [8].

In conclusion, our study demonstrated that there was no significant effect on the activity of peripheral blood neutrophils after 6 weeks of therapy with the recommended dose of montelukast. Nevertheless, montelukast treatment, in addition to its advantages for asthma treatment, did not enhance the activation of resting neutrophils and did not reduce neutrophil host defense mechanisms.

 Acknowledgment

This study was supported by a grant from MSD, USA.

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  Author Contacts

Prof. Dov Heimer, MD
Pulmonary Unit
Soroka University Medical Center
Beer Sheva 84105 (Israel)
Tel. +972 8 6400807, Fax +972 8 6467477, E-Mail ral@bgumail.bgu.ac.il

  Article Information

Received: December 18, 2002
Accepted after revision: July 4, 2003
Number of Print Pages : 8
Number of Figures : 3, Number of Tables : 4, Number of References : 28

  Publication Details

Respiration (International Review of Thoracic Diseases)
Founded 1944 as 'Schweizerische Zeitschrift für Tuberkulose und Pneumonologie' by E. Bachmann, M. Gilbert, F. Häberlin, W. Löffler, P. Steiner and E. Uehlinger, continued 1962-1967 as 'Medicina Thoracalis' as of 1968 as 'Respiration', H. Herzog (1962-1997)
Official Journal of the European Association for Bronchology and Interventional Pulmonology

Vol. 71, No. 1, Year 2004 (Cover Date: January-February 2004)

Journal Editor: C.T. Bolliger, Cape Town
ISSN: 0025-7931 (print), 1423-0356 (Online)

For additional information: http://www.karger.com/journals/res

  Drug Dosage / Copyright

Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.