Eosinophils Are a Major Source of Nitric Oxide-Derived Oxidants in Severe Asthma: Characterization of Pathways Available to Eosinophils for Generating Reactive Nitrogen Species

Abstract

Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO2Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480 ± 198 μmol/mol tyrosine; n = 11) compared with nonasthmatic subjects (52.5 ± 40.7 μmol/mol tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093 ± 457 μmol BrY/mol tyrosine; 161 ± 88 μmol ClY/mol tyrosine; n = 11 each) compared with nonasthmatic subjects (13 ± 14.5 μmol BrY/mol tyrosine; 65 ± 69 μmol ClY/mol tyrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO2Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO2−, NO3−, or NO2Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO2− or an exogenous NO source. At low, but not high (e.g., >2 μM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.

Interrelationships among asthma, atopy, rhinitis and exhaled nitric oxide in a population‐based sample of children

“…[42]. These results support the hypothesis of a difference in eNO concentration mediated by cell recruitment, in particular that of eosinophils with a T helper 2 pattern of cytokine activation (IL-4, IL-5) [43,44]. Nevertheless, other studies [45][46][47] have found a close pathophysiologic resemblance between atopic and non-atopic asthma in terms of both bronchial inflammatory cell recruitment and circulating factors.…”

Superoxide Dismutase Inactivation in Pathophysiology of Asthmatic Airway Remodeling and Reactivity

“…ing exposure to nitrating oxidants such as peroxynitrite/ peroxycarboxynitrite or peroxidase-mediated reactive nitrogen species. 14,37 Although the oxidative modifications monitored only represent a subfraction of total oxidative insults experienced by epithelial cell MnSOD within asthmatic airways, the quantification of this diverse array of distinct oxidative modifications provides insight into the potential degree of SOD functional impairment from oxidative processes. Given that there are 10 tyrosine residues per monomer and 4 monomers form an active Mn-SOD tetramer, if modification of only 1 tyrosine per MnSOD tetramer is sufficient to affect activity, then the observed cumulative modification burden of 1.13 to 1.73 mmol/mol tyrosine in isolated MnSOD predicts up to a 6% loss of MnSOD activity in these mild asthmatics.…”

“…Although nonspecific events related to increased levels of reactive oxygen and nitrogen species in the asthmatic airway have been postulated to lead to epithelial cell loss, the precise mechanisms of effect are unknown. [12][13][14][15] In general, the human lung has an effective, well-integrated antioxidant system to combat reactive oxygen species and reactive nitrogen species injury, 12 however, the respiratory tract antioxidant capacity is impaired in asthma. 16 -18 Activity of superoxide dismutase (SOD) is lower in asthmatic lungs as compared to healthy controls, and decreases further during an asthmatic attack.…”

Rapid and Selective Oxygen-regulated Protein Tyrosine Denitration and Nitration in Mitochondria

“…Even though biological nitration yields are relatively low (100 -500 mol of nitrotyrosine/mol of tyrosine under inflammatory conditions) (2), they are an established marker for the extent of the production of nitric oxide ( ⅐ NO) 1 -derived reactive species during both physiological and pathological conditions. Cumulative protein tyrosine nitration, manifesting through multiple mechanisms, may be actively involved in the onset and/or progression of various diseases with an inflammatory component and associated with increased levels of reactive oxygen species and ⅐ NO (1,(3)(4)(5)(6)(7). Cumulative protein tyrosine nitration may also be involved in the pathogenesis of acute pathological conditions such as ischemia-reperfusion (8,9).…”