Eosinophils generate brominating oxidants in allergen-induced asthma

Abstract

Eosinophils promote tissue injury and contribute to the pathogenesis of allergen-triggered diseases like asthma, but the chemical basis of damage to eosinophil targets is unknown. We now demonstrate that eosinophil activation in vivo results in oxidative damage of proteins through bromination of tyrosine residues, a heretofore unrecognized pathway for covalent modification of biologic targets in human tissues. Mass spectrometric studies demonstrated that 3-bromotyrosine serves as a specific "molecular fingerprint" for proteins modified through the eosinophil peroxidase-H(2)O(2) system in the presence of plasma levels of halides. We applied a localized allergen challenge to model the effects of eosinophils and brominating oxidants in human lung injury. Endobronchial biopsy specimens from allergen-challenged lung segments of asthmatic, but not healthy control, subjects demonstrated significant enrichments in eosinophils and eosinophil peroxidase. Baseline levels of 3-bromotyrosine in bronchoalveolar lavage (BAL) proteins from mildly allergic asthmatic individuals were modestly but not statistically significantly elevated over those in control subjects. After exposure to segmental allergen challenge, lung segments of asthmatics, but not healthy control subjects, exhibited a >10-fold increase in BAL 3-bromotyrosine content, but only two- to threefold increases in 3-chlorotyrosine, a specific oxidation product formed by neutrophil- and monocyte-derived myeloperoxidase. These results identify reactive brominating species produced by eosinophils as a distinct class of oxidants formed in vivo. They also reveal eosinophil peroxidase as a potential therapeutic target for allergen-triggered inflammatory tissue injury in humans.

Figures

Figure 1

Activated eosinophils utilize plasma levels of bromide to covalently modify protein tyrosine residues. (a) Human eosinophils (1 × 106/mL) were incubated at 37°C in Hanks’ balanced salt solution supplemented with DTPA (100 μM), BSA, 1 mg/mL, and bromide (100 μM NaBr). Eosinophils were activated with PMA (200 nM) and maintained in suspensions by intermittent inversion (Complete System). At the indicated times, eosinophils were removed by centrifugation. Supernatants were delipidated, desalted, and hydrolyzed, and the content of protein-bound 3-bromotyrosine and 3,5-dibromotyrosine was determined by stable isotope dilution GC-MS analysis. In a parallel set of experiments, superoxide production (in the absence of bromide and BSA) was determined as the superoxide dismutase-inhibitable reduction of ferricytochrome c. Data represent the mean ± SD of triplicate determinations (3-bromotyrosine and 3,5-dibromotyrosine) or a representative time course of O2•– production from an experiment performed at least four times. (b) The content of 3-bromotyrosine generated on target proteins after 2-hour incubation in the Complete System was determined by stable isotope dilution GC-MS analysis. Additions or deletions to the Complete System were as indicated. The final concentrations of additions were: catalase and heat inactivated catalase (hi Catalase), 10 μg/mL; NaN3, 1 mM; 3-aminotriazole (Atz), 10 mM. Values are the mean ± SD of triplicate determinations. (c and d) Electron capture negative ion chemical ionization mass spectrum of n-propyl, per pentafluoroproprionyl derivatized (c) 3-bromotyrosine and (d) 3,5-dibromotyrosine recovered in amino acid hydrolysates of BSA exposed to activated eosinophils. (Insets) structures and proposed fragmentation pathways for derivatized (c) 3-bromotyrosine and (d) 3,5-dibromotyrosine.

Figure 2

Specificity of 3-bromotyrosine and 3-chlorotyrosine formation on proteins exposed to activated human eosinophils and neutrophils. Human eosinophils and neutrophils (1 × 106/mL) were individually incubated for 2 hours at 37°C in Hanks’ balanced salt solution supplemented with DTPA (100 μM), BSA (1 mg/mL), and the indicated concentrations of bromide (0–100 μM NaBr). Leukocytes were activated with PMA (200 nM) and maintained in suspensions by intermittent inversion. The content of protein-bound 3-bromotyrosine and 3-chlorotyrosine was then determined by stable isotope dilution GC-MS analysis. Data represent the mean ± SD of triplicate determinations.

Figure 3

Effect of localized allergen challenge on allergic asthmatic airway and BAL proteins. An allergic asthmatic subject underwent fiberoptic bronchoscopy and ragweed allergen was instilled into a specific segment of one lung. A segment in the contralateral lung was similarly challenged with normal saline. Forty-eight hours later, fiberoptic bronchoscopy was repeated and both allergen- and normal saline–challenged lung segments were lavaged with normal saline and biopsied. (a and b) Hematoxylin and eosin staining of (a) normal saline- and (b) allergen-challenged lung segments reveals intense leukocyte infiltration and red granular debris from eosinophils in the allergen-challenged segment. High-power magnification view (data not shown) demonstrated that the majority of leukocytes recruited to the allergen-challenged airways were eosinophils. (c and d) Histological analysis of (c) normal saline– and (d) allergen-challenged lung segments by in situ fluorescence microscopy under conditions specific for the heme moiety of EPO reveals intense fluorescence signal in the allergen-challenged segment. (e and f) Protein recovered in BAL fluid from (e) normal saline– and (f) allergen-challenged lung segments of an asthmatic subject were analyzed by stable isotope dilution GC-MS for the presence of 3-bromotyrosine (BrY) using selected ion monitoring mode. The chromatograms shown were monitored at m/z 445, the base ion for the n-propyl, per pentafluoroproprionyl derivative of 3-bromotyrosine (Figure 1c), as well as the corresponding isotopically enriched counterpart at m/z 451 derived from the internal standard, 3-bromo[13C6]tyrosine.

Figure 4

Quantification of 3-bromotyrosine content in proteins recovered from nonasthmatic and asthmatic subjects at baseline and after segmental allergen challenge. Healthy control and allergic asthmatic subjects underwent fiberoptic bronchoscopy, and a specific segment of one lung was lavaged with normal saline to obtain a baseline sample (t = 0 hours). Two specific segments in the contralateral lung were then each exposed to allergen. One of these was lavaged 10 minutes later with normal saline (10 minutes) to assess the immediate effect of allergen challenge. Forty-eight hours later, fiberoptic bronchoscopy was repeated and the other allergen-challenged lung segment was lavaged with normal saline (48 hours). Cells in the BAL were removed by centrifugation, and the content of 3-bromotyrosine on proteins recovered in the supernatant at baseline and after segmental allergen challenge was then determined by stable isotope dilution GC/MS. P values represent the comparison between t = 0 versus 48 hours for allergen-challenged lung segments in asthmatic subjects (n = six per group).

Figure 5

Comparison of protein modification by brominating versus chlorinating oxidants in proteins recovered 48 hours after segmental allergen-challenge. The contents of 3-bromotyrosine and 3-chlorotyrosine generated on proteins in BAL fluid recovered from normal saline (NS) and allergen-challenged (Ag) lung segments (48 hours after challenge) of nonasthmatic and asthmatic subjects were determined by stable isotope dilution GC/MS. Data represent the mean ± SD (n = six for each group). Exposure to allergen only caused significant increases in bromination and chlorination of proteins recovered from asthmatic subjects. n = six per group.