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1 Depts of Pulmonary Medicine and 2 Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research
CORRESPONDENCE: S.K. Jindal, Dept of Pulmonary Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, 160012, India. Fax: 91 172744401
Keywords: bronchial asthma, neutrophils, nitric oxide
Received: July 12, 1999
Accepted December 28, 2000
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Abstract |
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Thirty asthmatic patients (ten each of mild, moderate and severe asthma) and ten healthy controls were included in the study. Neutrophils from peripheral venous blood were stimulated with latex, and production of nitrite (an NO metabolise) and l-citrulline (a co-product of NO) was studied. It was postulated that peripheral blood neutrophils, being in a primed or activated state in asthma, would reflect the changes occurring in bronchial tree neutrophils.
Nitrite and l-citrulline production by neutrophils was significantly higher in asthmatics (p<0.001) and increased with disease severity. A strong negative correlation was observed between peak expiratory flow and both nitrite (r=–0.87, p<0.001) and l-citrulline (r=–0.88, p<0.001) production.
It is concluded that nitric oxide production by neutrophils is increased in bronchial asthma and can possibly contribute to airway narrowing and disease severity.
Nitric oxide (NO) is implicated in the pathogenesis of bronchial asthma, but its precise role has not been defined 1. NO acts as a neurotransmitter in the bronchodilator nonadrenergic, noncholinergic nerves 2. Several experimental studies have shown that NO administration produces bronchodilation 3, 4. These observations lead to the impression that it is a relative deficiency of endogenous NO that might contribute to airway narrowing in bronchial asthma. Endogenous NO production may, however, act as a double-edged sword. When produced locally by constitutive nitric oxide synthase (NOS) in the bronchodilator neurons, it may be beneficial in relaxing airway smooth muscle in small amounts 2. However, when produced in high concentrations by inducible nitric oxide synthase (iNOS) present in various inflammatory cells, it leads to hyperaemia, oedema and exudation, and can contribute to airway narrowing in bronchial asthma 5.
Various cells and their mediators are involved in airway inflammation and hyperresponsiveness in asthma. These cells include neutrophils, which play an important role in asthma attacks, especially in the late-phase reaction 6. Neutrophils have iNOS and are capable of producing NO 7. This study was designed to look into the endogenous production of NO by neutrophils.
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Material and methods |
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Study design
Detailed history was obtained and physical examination carried out in each subject. Relevant laboratory investigations and PEF measurements were obtained in all patients to establish the diagnosis and grade the severity of asthma. Severity of asthma was assessed using criteria defined by the International Consensus Report on diagnosis and treatment of asthma 8. Patients with intermittent brief symptoms (<1–2 times a week), PEF>80% predicted, and requiring intermittent inhaled short acting ß2-agonists only, were categorized as the mild asthma group. Those with exacerbations >1–2 times a week, PEF 60–80% pred, and requiring a daily inhaled anti-inflammatory agent and a long-acting bronchodilator were categorized as the moderate asthma group.
Patients with more frequent exacerbations or continuous symptoms restricting physical activities, PEF<60% pred, and requiring frequent systemic corticosteroids, were characterized as the severe asthma group. In case of overlap of these features, an individual was assigned to the most severe grade in which any one feature occurred.
Heparinized peripheral venous blood (5 mL) was collected from each subject and the neutrophils were studied for their ability to generate nitrite (an NO metabolize) and l-citrulline (a co-product of NO pathway) upon stimulation with latex.
Estimation of nitrite and l-citrulline
Neutrophils were separated by Ficoll isopaque gradient centrifugation 9 and viability of cells was checked by 0.1% trypan blue dye exclusion 10. Identification of neutrophils was carried out by Giemsa staining. Around 106 neutrophils were stimulated with 20 µL latex and incubated for 2 h.
The accumulation of nitrite in culture supernatant was measured using the method described by Green et al. 11. Absorbance was measured at 546 nm usingan Uvicon Spectrophotometer (Model No. 930, Kontron). l-citrulline in the culture supernatant was estimated using the procedure described by Boyde and Rahmatullah 12, by measuring absorbance at 530 nm. For both tests, each standard and culture supernatant sample was analysed in duplicate and an average absorbance value was calculated. The nitrite and l-citrulline concentrations in the culture supernatant were calculated from absorbance values using standard curves. These standard curves were prepared separately using varying concentrations of sodium nitrite in a range of 10–700 nM, and of dl-citrulline in a range of 10–500 nM.
Statistical analysis
All values were expressed as mean±sd. One way analysis of variance and unpaired t-tests were used to evaluate differences in PEF, nitrite and l-citrulline production among various groups. Nitrite and l-citrulline levels were also correlated with PEF by calculating Pearson's correlation coefficient.
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Results |
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Nitrite and l-citrulline production by neutrophils were significantly higher in asthmatics as compared to controls (fig. 1
). There was a progressive increase in nitrite production as the severity of asthma increased, with a nitrite level in mild, moderate and severe asthma of 161.4±24.8, 347.6±30.9 and 485.6±54.5 nM·10–6 cells, respectively (p<0.001). A similar trend was seen in l-citrulline production in mild, moderate and severe asthma (82.4±18.9, 143.8±27.3, 186.7±14.3 nM·10–6 cells, respectively, p<0.001). PEF among all asthmatics correlated negatively with nitrite and l-citrulline levels (r=–0.87 and –0.88, respectively, p<0.001) (figs. 2 and 3).
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Discussion |
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The production of nitrite and l-citrulline by neutrophils in this study increased significantly as the severity of asthma increased from mild to severe, and a strong negative correlation was noted with PEF, suggesting an association between NO production and progressive airway narrowing. Other investigators have also observed higher NO levels in exhaled air in asthmatics 19, 20. NO is a potent vasodilator and may contribute to hyperaemia and oedema leading to airway narrowing in asthmatics. In fact, inhibition of endogenous NO production was shown to decrease plasma exudation and inflammation in guinea-pig airways in one study 21.
Conversely, it can be argued that an increase in NO levels, found with increasing levels of bronchoconstriction, is a host defence response in asthma to counteract bronchoconstriction, in view of the smooth muscle relaxant effect of NO. However, steroids, which inhibit the iNOS 22 and thus decrease NO production, are well known to have a beneficial role in bronchial asthma. This is another strong pointer that favours the hypothesis that increased NO production might actually contribute to airway narrowing in asthmatics. This study found a lower nitrite production by neutrophils among patients treated with steroids in each group. This finding is consistent with the noted effect of steroids on NO production.
In summary, it is concluded that nitric oxide production by neutrophils is increased in bronchial asthma and could partly contribute to airway narrowing.
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) and l-citrulline (•) levels in asthmatics and controls. Peripheral blood neutrophils of the subjects were stimulated with latex. The nitrite and l-citrulline levels in the culture supernatant are shown (p<0.001 on comparing each group with each of the other groups).
