Superoxide release and cellular gluthatione peroxidase activity in leukocytes from children with persistent asthma

Marçal, L.E.; Rehder, J.; Newburger, P.E.; Condino-Neto, A.

doi:10.1590/S0100-879X2004001100003

Abstract

Asthma is an inflammatory condition characterized by the involvement of several mediators, including reactive oxygen species. The aim of the present study was to investigate the superoxide release and cellular glutathione peroxidase (cGPx) activity in peripheral blood granulocytes and monocytes from children and adolescents with atopic asthma. Forty-four patients were selected and classified as having intermittent or persistent asthma (mild, moderate or severe). The spontaneous or phorbol myristate acetate (PMA, 30 nM)-induced superoxide release by granulocytes and monocytes was determined at 0, 5, 15, and 25 min. cGPx activity was assayed spectrophotometrically. The spontaneous superoxide release by granulocytes from patients with mild (N = 15), moderate (N = 12) or severe (N = 6) asthma was higher at 25 min compared to healthy individuals (N = 28, P < 0.05, Duncan test). The PMA-induced superoxide release by granulocytes from patients with moderate (N = 12) or severe (N = 6) asthma was higher at 15 and 25 min compared to healthy individuals (N = 28, P < 0.05 in both times of incubation, Duncan test). The spontaneous or PMA-induced superoxide release by monocytes from asthmatic patients was similar to healthy individuals (P > 0.05 in all times of incubation, Duncan test). cGPx activity of granulocytes and monocytes from patients with persistent asthma (N = 20) was also similar to healthy individuals (N = 10, P > 0.05, Kruskal-Wallis test). We conclude that, under specific circumstances, granulocytes from children with persistent asthma present a higher respiratory burst activity compared to healthy individuals. These findings indicate a risk of oxidative stress, phagocyte auto-oxidation, and the subsequent release of intracellular toxic oxidants and enzymes, leading to additional inflammation and lung damage in asthmatic children.

Children; Asthma; Respiratory burst; Granulocytes; Monocytes; Inflammation

Braz J Med Biol Res, November 2004, Volume 37(11) 1607-1613

Superoxide release and cellular gluthatione peroxidase activity in leukocytes from children with persistent asthma

L.E. Marçal¹, J. Rehder¹, P.E. Newburger² and A. Condino-Neto¹

¹Departamento de Pediatria e Centro de Investigação em Pediatria, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brasil

²Department of Pediatrics, University of Massachusetts Medical School, Worcester, MA, USA

References

Acknowledgments ^{Correspondence and Footnotes} Acknowledgments

Abstract

Asthma is an inflammatory condition characterized by the involvement of several mediators, including reactive oxygen species. The aim of the present study was to investigate the superoxide release and cellular glutathione peroxidase (cGPx) activity in peripheral blood granulocytes and monocytes from children and adolescents with atopic asthma. Forty-four patients were selected and classified as having intermittent or persistent asthma (mild, moderate or severe). The spontaneous or phorbol myristate acetate (PMA, 30 nM)-induced superoxide release by granulocytes and monocytes was determined at 0, 5, 15, and 25 min. cGPx activity was assayed spectrophotometrically. The spontaneous superoxide release by granulocytes from patients with mild (N = 15), moderate (N = 12) or severe (N = 6) asthma was higher at 25 min compared to healthy individuals (N = 28, P < 0.05, Duncan test). The PMA-induced superoxide release by granulocytes from patients with moderate (N = 12) or severe (N = 6) asthma was higher at 15 and 25 min compared to healthy individuals (N = 28, P < 0.05 in both times of incubation, Duncan test). The spontaneous or PMA-induced superoxide release by monocytes from asthmatic patients was similar to healthy individuals (P > 0.05 in all times of incubation, Duncan test). cGPx activity of granulocytes and monocytes from patients with persistent asthma (N = 20) was also similar to healthy individuals (N = 10, P > 0.05, Kruskal-Wallis test). We conclude that, under specific circumstances, granulocytes from children with persistent asthma present a higher respiratory burst activity compared to healthy individuals. These findings indicate a risk of oxidative stress, phagocyte auto-oxidation, and the subsequent release of intracellular toxic oxidants and enzymes, leading to additional inflammation and lung damage in asthmatic children.

Key words: Children, Asthma, Respiratory burst, Granulocytes, Monocytes, Inflammation

Introduction

The major characteristics of asthma are reversible airflow obstruction, bronchial hyperresponsiveness, and airway inflammation (1). This is a complex inflammatory disease that involves leukocytes, airway epithelial and smooth muscle cells, and several inflammatory mediators with multiple effects. The airway remains edematous and infiltrated with inflammatory cells, which are predominantly eosinophils and lymphocytes. Mast cells play a key role in asthma symptoms, whereas eosinophils, macrophages and T-helper 2 cells are involved in the chronic inflammation that underlies airway hyperresponsiveness (2). Resident or infiltrating inflammatory phagocytes in the airways release reactive oxygen species and other mediators with pleiotropic effects (1).

The role of phagocytes in the pathophysiology of asthma is not completely understood. They release enzymes, including extracellular matrix-degrading proteases, elastase, cytokines, and reactive oxygen species, which have been implicated in lung injury (3). The reported effects of reactive oxygen species in asthma include a decrease of beta-adrenergic function in lungs, airway smooth muscle contraction, increased vascular permeability, bronchial hyperresponsiveness, increased mucus secretion, impaired ciliary activity, generation of chemotactic factors, lipid peroxidation, and secondary production of mediators with a bronchoconstrictor effect (4,5). Airway inflammation is also associated with increased activity of the inducible nitric oxide synthase found in respiratory epithelium and activated macrophages (6). Superoxide and nitric oxide may rapidly combine to form peroxynitrite, a potent oxidant, leading to the depletion of antioxidants. For instance, the antioxidant glutathione (GSH), which is 100-fold more concentrated in the airway epithelial lining fluid compared with plasma, may be converted to its oxidized form (GSSG) (7). Thus, subsequent lung injury and more inflammation may take place.

Glutathione peroxidases (GPx) play an important role in the detoxificationof various hydroperoxides. Four types of GPx have been identified: cellularGPx (cGPx), gastrointestinal GPx, extracellular GPx,and phospholipid hydroperoxide GPx (8,9). cGPx (EC 1.11.1.9), also termed GPX1, is ubiquitously distributed. It reduces hydrogen peroxide as well as a wide range of organic peroxides derived from unsaturated fatty acids, nucleic acids, and other important biomolecules (8). At peroxide concentrations encountered under physiological conditions, it is more active than catalase (which has a higher K_m for hydrogen peroxide) and is also active against organic peroxides. Thus, cGPx represents a major cellular defense against toxic oxidant species (9).

The ability of cGPx to reduce peroxides and hydroperoxides plays a particularly important role in the anti-oxidant defense system of phagocytes, which are subject to auto-oxidation by their own respiratory burst products (10,11), leading to damage of the surrounding tissues when a great amount of oxygen radicals are released (12). Thus, oxidative stress is a relevant risk factor for lung damage in chronic inflammatory diseases such as asthma (6,13).

The aim of the present study was to investigate superoxide release and cGPx activity of granulocytes and monocytes from children and adolescents with atopic asthma, classified according to the Global Initiative for Asthma (GINA) criteria for evaluation of disease severity.

Subjects and Methods

Subjects and study design

The study included atopic asthmatic patients with the following characteristics: 22 males and 22 females, 20 Caucasians and 24 Blacks. The age ranged from 6 to 16 years (mean = 10.8 ± 2.85 years, median 11 years). The height ranged from 115 to 173 cm, and weight from 21 to 59 kg. Our clinical protocol included patients with a combination of clinical history of recurrent and reversible symptoms of airway obstruction, high serum IgE levels, positive skin tests to recognized allergens, and a family history of allergy (14). We were not allowed to collect blood samples from healthy children. Thus, we selected 28 healthy individuals for the comparative group, with an age range of 22-43 years.

The experimental protocol included assays of superoxide production and cGPx activity in peripheral blood granulocytes and monocytes. Written informed consent was obtained from all patient's parents and healthy individuals prior to the study. The Medical School Ethics Committee approved the experimental protocol in accordance to the Ministry of Health of Brazil (resolution 196/96) and the Helsinki Convention.

Severity of disease was classified according to GINA criteria into mild intermittent, mild persistent, moderate persistent, and severe persistent (15). Patients with moderate persistent asthma used inhaled beta₂-adrenergic and/or inhaled steroids (equivalent to 400-800 µg/day budesonide). Patients with severe persistent asthma used inhaled beta₂-adrenergic and inhaled, but not systemic steroids (equivalent to 800-1600 µg/day budesonide).

None of the patients had received non-steroidal anti-inflammatory drugs, theophylline, leukotriene modifiers, systemic steroids, systemic beta₂-adrenergic agents, or blood transfusion for at least one month prior to the study. Patients with infections, airway foreign bodies, or known systemic or pulmonary chronic diseases such as immunodeficiency, diabetes, ciliary dyskinesia syndromes, cystic fibrosis, or alpha₁-anti-trypsin deficiency were excluded. Smokers were excluded from both patient and control groups.

Isolation of granulocytes and monocytes

Granulocytes and monocytes were isolated by centrifugation of blood samples over a discontinuous density gradient (Histopaque 1.077 and 1.119 g/ml; Sigma, St. Louis, MO, USA) followed by adherence of monocytes to polystyrene plates (16). Contaminating erythrocytes were removed by hypotonic lysis. Both cell populations were washed three times in Hank's balanced salt solution (HBSS) without phenol red and the final leukocyte count was adjusted to 2 x 10⁷ cells/ml. Trypan blue exclusion showed greater than 90% cell viability.

Superoxide anion production

The spontaneous or phorbol myristate acetate (PMA, 30 nM)-induced superoxide anion production by leukocytes was assayed by a spectrophotometric method based on the superoxide dismutase inhibitable reduction of cytochrome c, according to McCord and Fridovich (17), as modified (18). The absorbance at 550 nm of the supernatants was monitored at 0, 5, 15, and 25 min. The amount of superoxide was calculated using an extinction coefficient of 21,100 M/cm. The results are reported as nmol of superoxide released per 10⁶ cells per sampling time. This assay was performed in granulocytes and monocytes from 44 patients and 28 healthy individuals.

Cellular glutathione peroxidase activity

cGPx activity was assayed in peripheral blood granulocytes and monocytes according to the method of Beutler (19), as modified (20). Leukocytes (10⁶) were incubated for 5 to 10 min in cuvettes containing 0.05% Triton X-100, 0.2 nmol GSH, 1 U/ml GSH reductase and HBSS, pH 7.4. The reaction was initiated by the addition of t-butyl-hydroperoxide. The change in absorbance at 340 nm was monitored at 0 and 1 min. Results are reported as nmol of oxidized NADPH min^-1 10⁶ cells^-1. This assay was performed in granulocytes and monocytes from 20 patients and 10 healthy individuals according to cell number availability.

Statistical analysis

Comparisons between the patient groups and healthy individuals were made using the Duncan or Kruskal-Wallis (21) test as indicated, with the level of significance set at P < 0.05.

Results

The spontaneous superoxide production by granulocytes from severe, moderate or mild persistent asthmatic patients was higher at 25 min of incubation compared to healthy individuals (Figure 1A, P < 0.05, Duncan test).

The PMA-stimulated superoxide production by granulocytes from severe and moderate persistent asthmatic patients was higher compared to healthy individuals at 15 and 25 min of incubation (Figure 1B, P < 0.05, Duncan test).

However, neither spontaneous nor PMA-stimulated superoxide production by monocytes from asthmatic patients, grouped according to GINA criteria, differed statistically from healthy individuals (P > 0.05, Duncan test).

The cGPx activity of granulocytes from mild persistent (N = 12), moderate persistent and severe persistent asthmatic children (N = 8), and healthy individuals (N = 10) were (mean ± SD, nmol of oxidized NADPH min^-1 10⁶ cells^-1): 171 ± 75, 181 ± 33, and 197 ± 68, respectively. The cGPx activity of monocytes from mild persistent (N = 12), moderate persistent and severe persistent asthmatic children (N = 8), and healthy individuals (N = 10) were (mean ± standard deviation, nmol of oxidized NADPH min^-1 10⁶ cells^-1): 142 ± 52, 151 ± 75, and 136 ± 36, respectively.

Thus, the cGPx activity of granulocytes or monocytes from children and adolescents with persistent asthma did not differ from that of healthy individuals (P > 0.05 in all situations, Kruskal-Wallis test).

Figure 1.
Superoxide release by granulocytes and monocytes from patients with asthma, grouped according to GINA diagnostic criteria. Panel A, Spontaneous superoxide (O₂^-) production was measured in granulocytes from patients with asthma grouped according to GINA criteria and from healthy individuals at the indicated time points. The spontaneous superoxide production by granulocytes from SP, MOP or MIP asthmatic patients was higher at 25 min of incubation, compared to healthy individuals (*P < 0.05, Duncan test). Panel B, Phorbol myristate acetate (PMA)-stimulated O₂^- production was measured in granulocytes from patients with asthma grouped according to GINA criteria and from healthy individuals at the indicated time points. The PMA-stimulated superoxide production by granulocytes from SP and MOP asthmatic patients was higher compared to healthy individuals at 15 and 25 min of incubation (*P < 0.05, Duncan test). SP = severe persistent (N = 6), MOP = moderate persistent (N = 12), MIP = mild persistent (N = 15), and MI = mild intermittent asthma (N = 11). Healthy individuals (N = 28).

Discussion

Our results show that the spontaneous or stimulated superoxide production by granulocytes from children and adolescents with persistent asthma is elevated compared to healthy individuals.

Ethical restrictions were much greater than ordinary because the study concerned samples from children. We were not allowed to obtain bronchoalveolar lavage cells from our patients with asthma, an apparently more sensitive approach to the questions we asked. Thus, we chose to investigate the superoxide release and cGPx activity of blood leukocytes. Indeed, one must consider that both bronchoalveolar lavage cells and blood leukocytes are not intralesional lung tissue cells.

In addition, we were also not allowed to collect blood samples from healthy children. Thus, we selected 28 healthy individuals for the comparative group, which could not be age matched. Previous studies have shown that the respiratory burst activity of leukocytes from children and adolescents does not differ significantly from that of adults (22,23). This similarity allowed us to compare the NADPH oxidase activity of leukocytes from children with asthma to measurements in healthy adult individuals.

Considering the limitation of volume when collecting blood samples from children, we were unable to separate neutrophils (predominant cells), eosinophils and basophils from the granulocyte pool. Thus, we chose to perform those tests in granulocytes. It is generally accepted that eosinophils and neutrophils produce equivalent amounts of superoxide, although this is still a controversial issue. Because of differences in the assembly of the NADPH oxidase components, eosinophils show a trend to release more extracellular superoxide compared to neutrophils (24).

Other studies have demonstrated that leukocytes from asthmatic patients show higher superoxide production compared to healthy individuals, particularly during asthma attacks (6,24-31). In the present, more detailed, study, pediatric patients with chronic asthma were for the first time carefully classified according to GINA criteria. In addition, we have assessed both respiratory burst and cGPx activity in granulocytes and monocytes. Only patients with persistent asthma showed a significant increase in superoxide release by granulocytes. These findings demonstrate at least a partial relationship between asthma severity and respiratory burst activity in peripheral blood granulocytes from pediatric patients.

Several studies have demonstrated that GPx activity in asthmatic patients varies according to cell lineage or body fluid. GPx activity may be lower in platelets, normal or lower in red blood cells, lower or increased in plasma, and normal or lower in whole blood (6,13,26,32,33). These conflicting data may be the result of assaying distinct isoforms of GPx in different blood components from heterogeneous asthmatic patients. To date, we have shown that cGPx activity of leukocytes from pediatric asthmatic patients, carefully classified according to GINA criteria, was similar to healthy controls and did not follow the up-regulation of NADPH oxidase activity in the same cells.

We conclude that under specific circumstances granulocytes from children and adolescents with persistent asthma produce more reactive oxygen species compared to healthy individuals. These reactive species have the potential to overwhelm the antioxidant system, with resultant oxidative stress and damage of surrounding pulmonary tissue. This process may be a relevant and still uncontrolled risk factor in childhood asthma pathophysiology. In children with asthma, key markers of inflammation are present early in life, highlighting the importance of early intervention and the appropriate use of drugs that modulate the NADPH oxidase activity (34), preventing the irreversible lung remodeling that contributes to this chronic disease.

The authors thank Drs. Marluce Vilela, José Dirceu Ribeiro, Adyléia Toro, and Cristina Sartorelli for helpful discussions and patient care.

Correspondence and Footnotes

Address for correspondence: A. Condino-Neto, Departamento de Pediatria e Centro de Investigação em Pediatria, FCM, UNICAMP, Caixa Postal 6111, 13081-970, Campinas, SP, Brasil. Fax: +55-19-3289-8638. E-mail: condino@lexxa.com.br or condino@unicamp.br

Research supported by FAPESP (No. 98/12144-5) and Fogarty International Center, United States National Institutes of Health (RO3-TW00883). L.E. Marçal was supported by a FAPESP fellowship (No. 97/13954-9). Received October 31, 2003. Accepted August 17, 2004.

1. Busse WW & Lemanske Jr RF (2001). Advances in immunology: asthma. New England Journal of Medicine, 344: 350-362.
2. Barnes PJ (2003). Pathophysiology of asthma. European Respiratory Monograph, 23: 84-113.
3. Vignola AM, Bonanno A, Mirabella A, Riccobono L, Mirabella F, Profita M, Bellia V, Bousquet J & Bonsignore G (1998). Increased levels of elastase and alpha₁-antitrypsin in sputum of asthmatic patients. American Journal of Respiratory and Critical Care Medicine, 157: 505-511.
4. Hancock JT (1997). Superoxide, hydrogen peroxide and nitric oxide as signaling molecules; their production and role in disease. British Journal of Biomedical Science, 54: 38-46.
5. Abraham WM (1997). Reactive oxygen species. In: Barnes PJ, Grunstein MM, Leff AR & Woolcock AJ (Editors), Asthma Lippincott-Raven Publishers, Philadelphia, PA, USA, 627-638.
6. Bowler RP & Crapo JD (2002). Oxidative stress in airways. Is there a role for extracellular superoxide dismutase? American Journal of Respiratory and Critical Care Medicine, 166: 38-43.
7. Van der Vliet A, O'Neill CA, Cross CE, Koostra JM, Volz WG, Halliwell B & Louie S (1999). Determination of low-molecular-mass antioxidant concentrations in human respiratory tract lining fluids. American Journal of Physiology, 276: L289-L296.
8. Tappel AL (1984). Selenium-glutathione peroxidase: properties and synthesis. Current Topics in Cellular Regulation, 24: 87-97.
9. Takebe G, Yarimizu J, Saito Y, Hayashi T, Nakamura H, Yodoi J, Nagasawa S & Takahasi K (2002). A comparative study on the hydroperoxide and thiol specificity of the glutathione peroxidase family and selenoprotein P. Journal of Biological Chemistry, 277: 41254-41258.
10. Baehner RL, Boxer LA, Allen JM & Davis J (1977). Autooxidation as a basis for altered function by polymorphonuclear leukocytes. Blood, 50: 327-335.
11. Ito Y, Kajkenova O, Feuers RJ, Udupa KB, Desai VG, Epstein J, Hart RW & Lipschitz DA (1998). Impaired glutathione peroxidase activity accounts for the age-related accumulation of hydrogen peroxide in activated human neutrophils. Journal of Gerontology. Series A, Biological Sciences and Medical Sciences, 53: M169-M175.
12. Boxer LA (1990). The role of antioxidants in modulating neutrophil functional responses. Advances in Experimental Medicine and Biology, 262: 19-33.
13. Rahman I & MacNee W (2000). Oxidative stress and regulation of glutathione in lung inflammation. European Respiratory Journal, 16: 534-554.
14. Condino-Neto A, Vilela MM, Cambiucci EC, Ribeiro JD, Guglielmi AAG, Magna LA & De Nucci G (1991). Theophylline therapy inhibits neutrophil and mononuclear cell chemotaxis from chronic asthmatic children. British Journal of Clinical Pharmacology, 32: 557-561.
15. National Heart, Lung and Blood Institute (1997). Practical Guide for the Diagnosis and Management of Asthma Expert Panel Report 2, Publication 97-4053.
16. Boyum A (1968). Isolation of mononuclear cells and granulocytes from human blood. Scandinavian Journal of Clinical and Laboratory Investigation, 21 (Suppl 97): 77-89.
17. McCord J & Fridovich I (1969). Superoxide dismutase: An enzymatic function for erythrocuprein (hemocuprein). Journal of Biological Chemistry, 244: 6049-6055.
18. Condino-Neto A & Newburger PE (1988). NADPH oxidase activity and cytochrome b₅₅₈ content of human Epstein-Barr-virus-transformed B lymphocytes correlate with expression of genes encoding components of the oxidase system. Archives of Biochemistry and Biophysics, 360: 158-164.
19. Beutler E (1984). Glutathione peroxidase. In: Beutler E (Editor), Red Cell Metabolism Grune & Stratton, Orlando, CA, USA, 74-76.
20. Speier C, Baker SS & Newburger PE (1985). Relationships between in vitro selenium supply, glutathione peroxidase activity, and phagocytic function in the HL-60 human myeloid cell line. Journal of Biological Chemistry, 260: 8951-8955.
21. Montgomery DC (1991). Design and Analysis of Experiments 3rd edn. John Wiley & Sons, New York.
22. Drossou V, Kanakoudi F, Tzimouli V, Sarafidis K, Taparkou A, Bougiouklis D, Petropulou T & Kremenipoulos G (1997). Impact of prematurity, stress and sepsis on the neutrophil respiratory burst activity of neonates. Biology of the Neonate, 72: 201-209.
23. Speer CP, Ambruso DR, Grimsley J & Johnston Jr RB (1985). Oxidative metabolism in cord blood monocytes and monocyte-derived macrophages. Infection and Immunity, 50: 919-921.
24. Lacy P, Abdel-Latif D, Steward M, Musat-Marcu S, Man SF & Moqbel R (2003). Divergence of mechanisms regulating respiratory burst in blood and sputum eosinophils and neutrophils from atopic subjects. Journal of Immunology, 170: 2670-2679.
25. Jarjour NN & Calhoun WJ (1994). Enhanced production of oxygen radicals in asthma. Journal of Laboratory and Clinical Medicine, 123: 131-136.
26. Bowler RP & Crapo JD (2002). Oxidative stress in allergic respiratory diseases. Journal of Allergy and Clinical Immunology, 110: 349-356.
27. Kanazawa H, Kurihara N, Hirata K & Takeda T (1991). The role of free radicals in airway obstruction in asthmatic patients. Chest, 100: 1319-1322.
28. Cluzel M, Damon M, Chanez P, Bousquet J, Crastes de Paulet A, Michel FB & Godard P (1987). Enhanced alveolar cell luminol-dependent chemiluminescence in asthma. Journal of Allergy and Clinical Immunology, 80: 195-201.
29. Vachier I, Damon M, Le Doucen C, Crastes de Paulet A, Chanez P, Michel FB & Godard P (1992). Increased oxygen species generation in blood monocytes of asthmatic patients. American Review of Respiratory Diseases, 146: 1161-1166.
30. Sedgwick JB, Geiger KM & Busse WW (1990). Superoxide generation by hypodense eosinophils from patients with asthma. American Review of Respiratory Diseases, 142: 120-125.
31. Monteseirin J, Camacho MJ, Bonilla I, De la Calle A, Guardia P, Conde J & Sobrino F (2002). Respiratory burst in neutrophils from asthmatic patients. Journal of Asthma, 39: 619-624.
32. Shanmugasundaram KR, Kumar SS & Raajjee S (2001). Excessive free radical generation in the blood of children suffering from asthma. Clinica Chimica Acta, 305: 107-114.
33. Nadeem A, Chhabra SK, Masood A & Raj HG (2003). Increased oxidative stress and altered levels of antioxidants in asthma. Journal of Allergy and Clinical Immunology, 111: 72-78.
34. Condino-Neto A, Whitney C & Newburger PE (1998). Dexamethasone but not indomethacin inhibits human phagocyte nicotinamide adenine dinucleotide phosphate oxidase activity by down-regulating expression of genes encoding oxidase components. Journal of Immunology, 161: 4960-4967.

Acknowledgments

Publication Dates

Publication in this collection
26 Oct 2004
Date of issue
Nov 2004

History

Accepted
17 Aug 2004
Received
31 Oct 2003

This work is licensed under a Creative Commons Attribution 4.0 International License.

[1] 1. Busse WW & Lemanske Jr RF (2001). Advances in immunology: asthma. New England Journal of Medicine, 344: 350-362.

[2] 2. Barnes PJ (2003). Pathophysiology of asthma. European Respiratory Monograph, 23: 84-113.

[3] 3. Vignola AM, Bonanno A, Mirabella A, Riccobono L, Mirabella F, Profita M, Bellia V, Bousquet J & Bonsignore G (1998). Increased levels of elastase and alpha₁-antitrypsin in sputum of asthmatic patients. American Journal of Respiratory and Critical Care Medicine, 157: 505-511.

[4] 4. Hancock JT (1997). Superoxide, hydrogen peroxide and nitric oxide as signaling molecules; their production and role in disease. British Journal of Biomedical Science, 54: 38-46.

[5] 5. Abraham WM (1997). Reactive oxygen species. In: Barnes PJ, Grunstein MM, Leff AR & Woolcock AJ (Editors), Asthma Lippincott-Raven Publishers, Philadelphia, PA, USA, 627-638.

[6] 6. Bowler RP & Crapo JD (2002). Oxidative stress in airways. Is there a role for extracellular superoxide dismutase? American Journal of Respiratory and Critical Care Medicine, 166: 38-43.

[7] 7. Van der Vliet A, O'Neill CA, Cross CE, Koostra JM, Volz WG, Halliwell B & Louie S (1999). Determination of low-molecular-mass antioxidant concentrations in human respiratory tract lining fluids. American Journal of Physiology, 276: L289-L296.

[8] 8. Tappel AL (1984). Selenium-glutathione peroxidase: properties and synthesis. Current Topics in Cellular Regulation, 24: 87-97.

[9] 9. Takebe G, Yarimizu J, Saito Y, Hayashi T, Nakamura H, Yodoi J, Nagasawa S & Takahasi K (2002). A comparative study on the hydroperoxide and thiol specificity of the glutathione peroxidase family and selenoprotein P. Journal of Biological Chemistry, 277: 41254-41258.

[10] 10. Baehner RL, Boxer LA, Allen JM & Davis J (1977). Autooxidation as a basis for altered function by polymorphonuclear leukocytes. Blood, 50: 327-335.

[11] 11. Ito Y, Kajkenova O, Feuers RJ, Udupa KB, Desai VG, Epstein J, Hart RW & Lipschitz DA (1998). Impaired glutathione peroxidase activity accounts for the age-related accumulation of hydrogen peroxide in activated human neutrophils. Journal of Gerontology. Series A, Biological Sciences and Medical Sciences, 53: M169-M175.

[12] 12. Boxer LA (1990). The role of antioxidants in modulating neutrophil functional responses. Advances in Experimental Medicine and Biology, 262: 19-33.

[13] 13. Rahman I & MacNee W (2000). Oxidative stress and regulation of glutathione in lung inflammation. European Respiratory Journal, 16: 534-554.

[14] 14. Condino-Neto A, Vilela MM, Cambiucci EC, Ribeiro JD, Guglielmi AAG, Magna LA & De Nucci G (1991). Theophylline therapy inhibits neutrophil and mononuclear cell chemotaxis from chronic asthmatic children. British Journal of Clinical Pharmacology, 32: 557-561.

[15] 15. National Heart, Lung and Blood Institute (1997). Practical Guide for the Diagnosis and Management of Asthma Expert Panel Report 2, Publication 97-4053.

[16] 16. Boyum A (1968). Isolation of mononuclear cells and granulocytes from human blood. Scandinavian Journal of Clinical and Laboratory Investigation, 21 (Suppl 97): 77-89.

[17] 17. McCord J & Fridovich I (1969). Superoxide dismutase: An enzymatic function for erythrocuprein (hemocuprein). Journal of Biological Chemistry, 244: 6049-6055.

[18] 18. Condino-Neto A & Newburger PE (1988). NADPH oxidase activity and cytochrome b₅₅₈ content of human Epstein-Barr-virus-transformed B lymphocytes correlate with expression of genes encoding components of the oxidase system. Archives of Biochemistry and Biophysics, 360: 158-164.

[19] 19. Beutler E (1984). Glutathione peroxidase. In: Beutler E (Editor), Red Cell Metabolism Grune & Stratton, Orlando, CA, USA, 74-76.

[20] 20. Speier C, Baker SS & Newburger PE (1985). Relationships between in vitro selenium supply, glutathione peroxidase activity, and phagocytic function in the HL-60 human myeloid cell line. Journal of Biological Chemistry, 260: 8951-8955.

[21] 21. Montgomery DC (1991). Design and Analysis of Experiments 3rd edn. John Wiley & Sons, New York.

[22] 22. Drossou V, Kanakoudi F, Tzimouli V, Sarafidis K, Taparkou A, Bougiouklis D, Petropulou T & Kremenipoulos G (1997). Impact of prematurity, stress and sepsis on the neutrophil respiratory burst activity of neonates. Biology of the Neonate, 72: 201-209.

[23] 23. Speer CP, Ambruso DR, Grimsley J & Johnston Jr RB (1985). Oxidative metabolism in cord blood monocytes and monocyte-derived macrophages. Infection and Immunity, 50: 919-921.

[24] 24. Lacy P, Abdel-Latif D, Steward M, Musat-Marcu S, Man SF & Moqbel R (2003). Divergence of mechanisms regulating respiratory burst in blood and sputum eosinophils and neutrophils from atopic subjects. Journal of Immunology, 170: 2670-2679.

[25] 25. Jarjour NN & Calhoun WJ (1994). Enhanced production of oxygen radicals in asthma. Journal of Laboratory and Clinical Medicine, 123: 131-136.

[26] 26. Bowler RP & Crapo JD (2002). Oxidative stress in allergic respiratory diseases. Journal of Allergy and Clinical Immunology, 110: 349-356.

[27] 27. Kanazawa H, Kurihara N, Hirata K & Takeda T (1991). The role of free radicals in airway obstruction in asthmatic patients. Chest, 100: 1319-1322.

[28] 28. Cluzel M, Damon M, Chanez P, Bousquet J, Crastes de Paulet A, Michel FB & Godard P (1987). Enhanced alveolar cell luminol-dependent chemiluminescence in asthma. Journal of Allergy and Clinical Immunology, 80: 195-201.

[29] 29. Vachier I, Damon M, Le Doucen C, Crastes de Paulet A, Chanez P, Michel FB & Godard P (1992). Increased oxygen species generation in blood monocytes of asthmatic patients. American Review of Respiratory Diseases, 146: 1161-1166.

[30] 30. Sedgwick JB, Geiger KM & Busse WW (1990). Superoxide generation by hypodense eosinophils from patients with asthma. American Review of Respiratory Diseases, 142: 120-125.

[31] 31. Monteseirin J, Camacho MJ, Bonilla I, De la Calle A, Guardia P, Conde J & Sobrino F (2002). Respiratory burst in neutrophils from asthmatic patients. Journal of Asthma, 39: 619-624.

[32] 32. Shanmugasundaram KR, Kumar SS & Raajjee S (2001). Excessive free radical generation in the blood of children suffering from asthma. Clinica Chimica Acta, 305: 107-114.

[33] 33. Nadeem A, Chhabra SK, Masood A & Raj HG (2003). Increased oxidative stress and altered levels of antioxidants in asthma. Journal of Allergy and Clinical Immunology, 111: 72-78.

[34] 34. Condino-Neto A, Whitney C & Newburger PE (1998). Dexamethasone but not indomethacin inhibits human phagocyte nicotinamide adenine dinucleotide phosphate oxidase activity by down-regulating expression of genes encoding oxidase components. Journal of Immunology, 161: 4960-4967.