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,*,*
Departments of
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Cell Biology,
Cardiology,
Cancer Biology,
Pulmonary and Critical Care Medicine, and
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Center for Cardiovascular Diagnostics, Cleveland Clinic Foundation, Cleveland, OH 44195;
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Cleveland State University, Department of Chemistry, Cleveland, OH 44115;
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Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM 87131; and
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Quest Diagnostics, San Juan Capistrano, CA 92690
Department of Cell Biology,
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Department of Cardiology,
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Department of Cancer Biology,
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Department of Pulmonary and Critical Care Medicine, and
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Center for Cardiovascular Diagnostics, Cleveland Clinic Foundation, Cleveland, OH 44195;
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Cleveland State University, Department of Chemistry, Cleveland, OH 44115;
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Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM 87131; and
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Quest Diagnostics, San Juan Capistrano, CA 92690
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eosinophils are unique among circulating leukocytes in their prodigious capacity to wage chemical warfare. As mentioned above, they are endowed with numerous highly basic and cytotoxic granule proteins that are released upon activation or during cell necrosis. They also possess an arsenal of enzymes designed to inflict oxidative damage upon biological targets (1, 2, 3). During eosinophil activation, a respiratory burst occurs where O2·- and its dismutation product, H2O2, are formed (1, 2, 11). The respiratory burst of human eosinophils produces anywhere from 3 to 10 times as much O2·- as a corresponding number of similarly treated neutrophils (12, 13). Although reduced oxygen species such as O2·- and H2O2 do not effectively oxidize biological targets, one of the most abundant proteins secreted upon eosinophil activation, EPO, amplifies the oxidizing potential of H2O2 by using it as cosubstrate to generate potent reactive oxidants and diffusible radical species (2, 14, 15, 16).
EPO is a member of the mammalian peroxidase superfamily
(17) and has a high degree of sequence homology with
myeloperoxidase (MPO), an abundant peroxidase in neutrophils,
monocytes, and certain tissue macrophages (18). Both EPO
and MPO share the unique ability to use halides and pseudohalides as
substrates to make highly reactive oxidants, hypohalous acids (HOX)
(Equation 1
).
 | (1) |
At normal plasma levels of halides and pseudohalides (100 mM Cl-, 20–150 µM Br-, <69 µM SCN-, 0.1–0.6 µM I-; Ref. 19), EPO preferentially uses the pseudohalide SCN- (20) and Br- as substrates (15, 21), whereas MPO uses Cl- (22, 23). Recent gas chromatography-mass spectrometry (GC-MS) studies demonstrate that eosinophils use EPO to generate oxidants in allergen-triggered asthma (24). BrY (3-bromotyrosine), a specific marker of protein modification by reactive brominating species (25), was markedly enriched in bronchoalveolar lavage (BAL) proteins recovered from asthmatic subjects following exposure to segmental allergen challenge (24). Thus, one chemical pathway used by eosinophils to promote oxidative modification of proteins during asthma is through EPO-generated reactive brominating species.
Another potential pathway for oxidative modification of tissues in
asthma by eosinophils may involve formation of reactive nitrogen
species (RNS). NO production is increased in asthma
(26, 27, 28). Although NO is a relatively long-lived radical
that does not nitrate biological targets directly, it can be converted
into more potent RNS. The most commonly studied NO-derived oxidant is
peroxynitrite (ONOO-), a product formed by the
near diffusion-limited interaction of NO and
O2·- (29).
ONOO- nitrates protein tyrosine residues
(29, 30, 31), and immunohistochemical studies suggest that
3-nitrotyrosine (NO2Y) is produced in asthmatic
airways (32, 33). At physiological levels of
CO2/HCO3-,
ONOO- exists predominantly as the more potent
nitrating species, peroxycarboxynitrite
(ONOOCO2-) (Refs.
34, 35, 36 ; Equation 2).
 | (2) |
However, recent studies have identified alternative pathways for generating NO-derived oxidants that involve leukocyte peroxidases. van der Vliet and colleagues first demonstrated that MPO could use nitrite (NO2-), a major end-product of NO metabolism, as a substrate to nitrate tyrosine residues (38). Subsequent studies with isolated neutrophils (39, 40) and monocytes (41, 42, 43) have demonstrated that MPO-generated RNS can play a significant, and even predominant, role in oxidative modification of biomolecules. We recently demonstrated that EPO is significantly more effective than MPO at promoting protein nitration at physiologically relevant concentrations of halides and NO2- (44). Although increased levels of NO, NO2-, H2O2, and eosinophils are present in asthmatic airways, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma are reported.
In this study we demonstrate that RNS play a significant role in oxidative modification of proteins in asthma. Using a combination of approaches including mass spectrometry, immunohistochemistry, and studies with isolated eosinophils, we show that eosinophils generate NO-derived oxidants both in vitro and in vivo. Finally, we examine the chemical pathways available to human eosinophils for generating NO-derived oxidants and demonstrate that the leukocytes form RNS by at least two mechanistically distinct pathways.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All solvents were purchased from Fisher Scientific (Pittsburgh, PA) and were Optima or HPLC grade. PAPA NONOate (Z)-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate was obtained from Alexis (San Diego, CA). Glucose oxidase (grade II) and catalase were acquired from Roche Molecular Biochemicals (Indianapolis, IN). L-[13C915N]tyrosine and L-[13C6]tyrosine were obtained from Cambridge Isotope Laboratories (Andover, MA). Unless otherwise stated, all other chemicals were purchased from Sigma (St. Louis, MO).
Subjects and sample collection
Human endotracheal/bronchial aspirates were obtained from
patients (n = 11) who required mechanical ventilatory
assistance due to respiratory failure from a severe asthmatic
exacerbation. All asthmatic subjects had a history of a >12% increase
in absolute forced expiratory volume in 1 s
(FEV1) and a 200-cc increase in forced expiratory
volume either spontaneously or after bronchodilator within 1 year of
hospitalization, and satisfied the definition of severe asthma as
defined by the National Institutes of Health guidelines
(45). Samples were collected within 12 h of admission
because asthmatic subjects received i.v. corticosteroids upon
presentation to the emergency department and subsequent transfer to the
intensive care unit, and the effects of corticosteroids on protein
oxidation products is unknown. All asthmatic individuals had a history
of using inhaled 
2-agonists either regularly (n = 8)
or on an as-needed basis (n = 3) in the month preceding
admission. Several also used inhaled corticosteroids (n
= 8) and/or had received oral corticosteroids within 1 month of
presentation (n = 4). Control subjects
(n = 12) were age- and sex-matched, nonsmokers, and had
no prior history of asthma or other lung disease.
Endotracheal/bronchial aspirates were obtained from several control
subjects who were either undergoing elective surgery (n
= 3) or were admitted to the intensive care unit for a noninflammatory,
nonrespiratory process (i.e., airway protection secondary to either
head trauma (n = 2) or drug overdose (n
= 2)). The remaining clinical specimens from healthy nonasthmatic
controls (n = 5) were obtained as residual specimens
collected as baseline BAL samples for a separate clinical study
(46). Healthy control subjects in that study all had a
negative methacholine challenge test. Thus, all 11 specimens from
asthmatic subjects were obtained as endotracheal/bronchial aspirates,
and the 12 specimens from nonasthmatic controls were obtained as
endotracheal/bronchial aspirates (n = 7) and baseline
BAL (n = 5) specimens. No differences were noted in
levels of NO2Y, BrY, or 3-chlorotyrosine (ClY)
(per mol tyrosine) in endotracheal/bronchial aspirates vs BAL;
consequently, specimens from the nonasthmatic subjects were combined as
a single control group. Cells in clinical specimens were removed by
centrifugation, the supernatants were supplemented with a mixture of
antioxidants and peroxidase inhibitors (final concentration: 200 µM
diethylenetriaminepentaacetic acid (DTPA), 100 µM butylated
hydroxytoluene, 50 mM sodium phosphate (pH 7.0), 1 mM aminotriazole),
capped in vials under argon atmosphere, snap frozen in liquid
N2, and stored at -80°C until time of sample
preparation and mass spectrometry analysis.
Histological analysis of lung and bronchial tissues
Histological sections were cut from paraffin blocks of lung and bronchial tissues from four individuals who died from asthma and four nonasthmatic age-matched individuals who died of nonpulmonary processes. For NO2Y immunostaining, slides were initially incubated at 37°C with 0.01 mg/ml protease K for 15 min. Following wash with PBS containing 0.5 mM levamisole, the tissue was treated with 1% BSA in PBS to block nonspecific binding, then incubated for 2 h with immunopurified polyclonal Ab directed against NO2Y (1:150 diluted in 1% BSA/PBS; Upstate Biotechnology, Lake Placid, NY). Following wash with PBS/0.5 mM levamisole, the tissue was incubated with a biotin-conjugated secondary Ab (Dako, Carpinteria, CA) for 10 min. Washing was followed by another 10-min incubation with alkaline phosphatase-labeled streptavidin (Dako). Immunostaining was visualized with an alkaline phosphate substrate solution containing napthol AS-MX phosphatase, Fast Red, and levamisole in Tris buffer (pH 8.2) (Dako) and counterstained with the nuclear stain, hematoxylin. Negative control experiments involved either immunoabsorption of NO2Y Ab with 3.75 mM NO2Y before incubation with tissue sections or incubating the section with isotype-control nonimmune Ig instead of the primary Ab. In separate studies, the specificity of the primary Ab was confirmed by observing loss of nitrotyrosine-specific recognition following reduction of nitrotyrosine-containing protein with dithionite. All biopsies were stained on the same day. Specific autofluorescence imaging of EPO in biopsies was performed as described (47, 48, 49). EPO-specific in situ peroxidase staining of tissues was performed on anti-nitrotyrosine-stained tissue sections following treatment of slides with 0.01 M KCN to inhibit MPO (50, 51).
Eosinophil and neutrophil isolation
Human eosinophils were isolated by negative selection using CD16 microbeads (Miltenyi Biotec, Auburn, CA), as described (52). Human neutrophils were isolated by buoyant density centrifugation (53), and low levels of contaminating eosinophils were then removed by fluorescence-activated cell sorting (54). The final purity of cell preparations was confirmed by flow cytometry using selective Abs for cell surface Ag on eosinophils (CD49d) and neutrophils (CD16), respectively (54). No detectable cross-contamination of peroxidase activity in detergent extracts of leukocyte preparations was observed following SDS-PAGE with in-gel tetramethylbenzidine peroxidase staining (55). Trypan blue exclusion tests demonstrated over 97% viability in eosinophil and neutrophil preparations.
Cell experiments
These studies were performed in the presence of
CO2 (5% gas phase) and
HCO2- in the medium (4.2 mM) to
more closely mimic a biologically relevant situation. Leukocytes
(1 x 106/ml) were incubated at 37°C under
95% air, 5% CO2, in Medium A
(Ca2+/Mg2+/phenol-free
HBSS; Life Technologies, (Gaithersburg, MD) supplemented with 100 µM
NaBr, 50 µM L-arginine, and 200 µM DTPA, pH 7.4 final)
in the absence or presence of NaNO2 (0–50 µM,
as indicated in the figure legends). Where indicated, 100 µM of
either L-tyrosine or its deaminated analog,
3-(4-hydroxyphenyl)propanoic acid (HPA) was included. Cells were
activated by addition of PMA (200 nM) and incubated for either 1 h
or the indicated time interval. In some experiments, eosinophils were
activated in the presence of an exogenous NO source by addition of PAPA
NONOate (Alexis) for 1 min following PMA addition. Rates of NO flux
were determined spectrophotometrically by reaction of NO with
oxyhemoglobin (56) under the identical conditions used for
experiments, but in the absence of any added cells. To maintain a final
pH of 7.4 during experiments with PAPA NONOate, incubations were
performed in Medium B (Medium A containing only 100 mM NaCl and
supplemented with 20 mM sodium phosphate, pH 7.4).
NO2- levels accumulated to over
50 µM in medium incubated with the NO-generating system (2 µM/min)
in the absence of cells. In some cases, leukocyte reaction mixtures
also contained one of the following: 1 mM NaN3,
10 mM 3-aminotriazole (Atz), 300 nM catalase (Cat), 300 nM
heat-inactivated catalase (hiCat), 10 µg/ml superoxide dismutase
(SOD), 1 mM methionine (Met), or 1 mM
N
-acetyl lysine.
Quantification of leukocyte-generated products in vitro
NO2Y production by isolated eosinophils was quantified by reversed phase HPLC with photodiode array detection (44). Peak identity was routinely confirmed by demonstrating the appropriate UV-VIS absorbance spectrum of the peak that comigrated with authentic NO2Y. In preliminary studies, NO2Y production by eosinophils was also independently confirmed by HPLC with on-line electrospray ionization mass spectrometry, similar to prior studies using isolated EPO (44). The nitrated (NO2-HPA; 3-(4-hydroxy-3-nitrophenyl)propanoic acid), brominated (Br-HPA; 3-(3-bromo-4-hydroxyphenyl)propanoic acid), and chlorinated (Cl-HPA; 3-(3-chloro-4-hydroxyphenyl)propanoic acid) products of the tyrosine analog, HPA, were routinely quantified by reversed phase HPLC with electrochemical (coulometric) detection on an ESA CoulArray HPLC (Cambridge, MA) equipped with UV detector and electrochemical cells (eight channels) (44). Peak identity was established by demonstrating the appropriate retention time, redox potential, ratio of integrated currents in adjacent channels, and by the method of standard additions for each analyte. Authentic standards of NO2-HPA, Br-HPA, and Cl-HPA were prepared by reaction of HPA with a molar equivalent of ONOO-, HOBr, or HOCl, respectively. Standards were then isolated by reversed phase HPLC, and their structures were confirmed by electrospray ionization mass spectrometry by demonstrating the appropriate mass-to-charge ratio (and isotopic cluster, where applicable) of the anticipated molecular ion of the isolated product.
Sample preparation and mass spectrometry
The contents of BrY and ClY in proteins present in clinical specimens were determined by stable isotope dilution GC-MS using 3-bromo[13C6]tyrosine and 3-chloro[13C6]tyrosine as internal standards (24). The NO2Y content of lavage proteins was quantified by stable isotope dilution GC-MS following reduction to aminotyrosine (57). All results were normalized to the content of the precursor amino acid, L-tyrosine, which was similarly quantified by GC-MS (58) using L-[13C915N]tyrosine as internal standard. Intrapreparative formation of 3-bromo[13C915N]tyrosine, 3-chloro[13C915N]tyrosine, or 3-nitro[13C915N]tyrosine was routinely monitored and found to be negligible (i.e., <5% of the level of the natural abundance product observed) under the conditions used.
General procedures
All water used to prepare buffers and medium was pretreated with Chelex-100 resin (Bio-Rad, Hercules, CA) and supplemented with 100 µM DTPA to remove trace levels of potential redox-active transition metal ions. Superoxide generation by activated human eosinophils was measured as the SOD-inhibitable reduction of ferricytochrome c (59). Quantification of NO2- and NO3- was performed by anion exchange HPLC with UV detection at 210 nm under argon atmosphere. Products were resolved on a Spherisorb S5 SAX column (24 cm x 4.6 mm, 5 µm; Phase Separations, Norwalk, CT) under isocratic conditions using 45 mM sodium phosphate (pH 3.0) as the mobile phase.
Leukocytes were isolated from whole blood of healthy volunteers after obtaining informed consent. Tissue sections were obtained from the New Mexico Office of the Medical Examiner and the Anatomic Pathology Department at the Cleveland Clinic Foundation. Sections were anonymized by the forensic pathologists for use in these studies. All protocols were in accordance with institutional guidelines of either the University of New Mexico School of Medicine or the Cleveland Clinic Foundation and were approved by their respective Institutional Review Committees.
Statistics
Data represent the mean ± SD of the indicated number of samples. Statistical analyses were made using a paired Student’s t test. For all hypotheses the significance level was 0.05. When multiple comparisons were made, a Bonferroni correction to the significance criterion for each test was made.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To quantify the potential role of RNS in promoting protein
oxidation in asthma, we used stable isotope dilution GC-MS to compare
the protein content of NO2Y recovered from
airways of subjects with severe asthma vs nonasthmatic subjects. A
significant (p < 0.0001) 10-fold
increase in protein NO2Y levels was observed in
samples from severe asthmatic patients compared with levels present in
nonasthmatic subjects (480 ± 198 vs 52.5 ± 40.7 µmol/mol
tyrosine; asthmatic vs nonasthmatic, respectively; Fig. 1).
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To identify the potential cellular source(s) of NO-derived
oxidants in severe asthma, specimens from subjects who died from asthma
(status asthmaticus) were examined using affinity-purified
Ab specific for NO2Y. Intense staining that
colocalized with eosinophils was typically observed in the majority of
specimens (Fig. 2). Diffuse staining of
epithelial cells was also commonly observed, as has previously been
reported (32, 33). Both in situ fluorescence microscopy
specific for the heme group of EPO (data not shown) and in situ
peroxidase staining specific for EPO were also abundant in
eosinophil-rich areas of specimens from asthmatics. Double staining of
sections for both NO2Y- and EPO-specific in situ
peroxidase staining confirmed colocalization of
NO2Y with eosinophils in the submucosa of airways
from severe asthmatic subjects (Fig. 2).
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To assess the relative contributions of eosinophils and
neutrophils in the oxidative modification of proteins in severe asthma,
the protein content of BrY and ClY, molecular fingerprints for
eosinophil- and neutrophil-mediated tissue damage, respectively
(24, 60), were determined in the same clinical specimens
evaluated for protein NO2Y content in Fig. 1
.
There was a striking 84-fold elevation (p <
0.0001) in the content of BrY observed in proteins recovered from
airways of asthmatic (1093 ± 457 µmol BrY/mol tyrosine) vs
nonasthmatic subjects, whose levels were near the limit of detection
(13 ± 14.5 µmol BrY/mol tyrosine) (Fig. 3A). There was also a
significant 3-fold increase (p < 0.05) in ClY
in airway proteins recovered from severe asthmatics (161 ± 88
µmol ClY/mol tyrosine) over nonasthmatics (65 ± 69 µmol
ClY/mol tyrosine) (Fig. 3B). A comparison of the BrY/ClY
ratios, an indication of the relative preferential contribution of
eosinophils vs neutrophils toward oxidation of proteins, revealed a
30-fold difference in asthmatics compared with nonasthmatics (ratio of
6.8 vs 0.2 for asthmatic vs nonasthmatic, respectively).
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We recently demonstrated that isolated EPO effectively uses
NO2- at concentrations
comparable to those observed in inflammatory tissues and fluids (
50
µM; Ref. 38 and references therein) in cell-free systems
to generate a RNS capable of promoting tyrosine nitration
(44). Based upon these studies, we hypothesized that
eosinophils might use EPO to contribute to protein oxidation through
nitration in asthma. However, the ability of human eosinophils to
generate NO-derived oxidants has not yet been demonstrated. To
determine whether eosinophils can generate NO-derived oxidants, we
isolated peripheral cells from normal healthy donors and incubated them
in medium containing L-tyrosine, plasma levels of halides
(Medium A, see Materials and Methods), and the agonist PMA.
Analysis of medium revealed that no significant
NO2Y was formed (Fig. 4A). Moreover, we were also
unable to detect any endogenous NO production or significant (i.e., >1
µM)
NO2-/NO3-
accumulation by human eosinophils freshly isolated from peripheral
blood, with or without phorbol ester activation, under the conditions
and time course used (data not shown). In contrast, eosinophils
activated in Medium A supplemented with pathophysiologically relevant
levels of NO2- (50 µM)
readily produced NO2Y (Fig. 4A). The
time course for NO2Y formation paralleled the
time course for O2·- production during
a respiratory burst (Fig. 4B). Finally, in separate studies,
eosinophils were activated with an alternative agonist,
N-formyl-methionyl-leucyl-phenylalanine (100 nM).
Cell-dependent NO2Y formation again demonstrated
an absolute requirement for exogenous
NO2-, although
NO2Y levels produced were 
8-fold less than
that observed with eosinophils stimulated with PMA (data not
shown).
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).
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) eosinophils preferentially nitrated
HPA, generating 3-fold more NO2-HPA than
Br-HPA, and no detectable Cl-HPA (Fig. 6).
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). Similar
reaction requirements were observed for eosinophil-mediated bromination
reactions (i.e., sensitivity to
H2O2 scavengers and
peroxidase inhibitors) (Fig. 6). One distinguishing feature between
eosinophil-mediated nitration and bromination reactions is the
inability of the HOBr scavenger methionine to affect nitration by the
cells. The lack of inhibition in cell-dependent bromination observed in
the presence of the primary amine-containing species,
N-acetyl lysine, is consistent with our
prior observation that N-bromoamines serve as excellent
mediators of aromatic bromination reactions (25). Eosinophils are more efficient than neutrophils at promoting aromatic nitration reactions
In the next series of experiments we compared the ability of
purified human eosinophils and neutrophils to nitrate, chlorinate, and
brominate the tyrosine analog HPA in the presence of plasma levels of
halides over the (patho)physiologically relevant range of
NO2- (0–50 µM). Eosinophils
generated significantly more NO2-HPA (>5-fold)
than an equivalent number of neutrophils at all concentrations of
NO2- examined (Fig. 7). At levels of
NO2- observed in epithelial
lining fluid from normal individuals (i.e., <10 µM
NO2-), eosinophils were more
effective at oxidation of phenolic groups through bromination. As
NO2- levels became elevated
into the pathophysiological range (>10 µM), nitration of targets
predominated (Fig. 7A). In stark contrast to neutrophils, no
significant oxidation of HPA through chlorination was observed under
all conditions examined (Fig. 7). Finally, neutrophils were ineffective
at oxidizing the tyrosine analog through bromination under all
conditions examined (Fig. 7).
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One striking feature of the results thus far described was that we
could find no evidence that isolated eosinophils generate nitrating
intermediates through formation of ONOO-
(Equation 2). Based upon the inability to detect NO,
NO2-, or
NO3- accumulation in cell
medium (see above), this likely reflects the limited capacity of
freshly isolated peripheral blood eosinophils from healthy
(nonallergic) donors to generate NO, particularly within the brief time
period of the respiratory burst (1 h, Fig. 4B). However,
enhanced levels of NO in expired breath of asthmatics is well
documented, and expression of NO synthase isoforms in the airways is
likewise established (26, 27, 28). Therefore, we performed a
series of experiments in which eosinophils were activated in the
presence of an exogenous NO-generating system, PAPA NONOate, and plasma
levels of Br- (100 µM). These conditions
should more closely mimic a physiological mechanism for
NO2- formation, as well as
provide an environment where eosinophil-generated
O2·- might react with NO
before it dismutates into the EPO substrate,
H2O2. Although eosinophils
activated in the absence of the NO donor failed to mediate nitration
reactions, cells stimulated in the presence of a continuous NO source
readily formed NO2-HPA (Fig. 8). Aromatic nitration by eosinophils
required cell activation, consistent with a requirement for reduced
oxygen species (O2·-
and/or H2O2) for oxidation.
Moreover, nitration was the favored biochemical pathway for oxidative
modification at all but the lowest levels of NO flux examined. At the
higher fluxes of NO (>2 µM/min) examined, the overall extent of
NO2-HPA formation diminished (Fig. 8).
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). Under these same
conditions, catalase, but not hiCat, significantly attenuated
NO2-HPA production, consistent with a role for
H2O2 in the aromatic
nitration reaction. In contrast, at high rates of NO flux (>2
µM/min), aromatic nitration by activated eosinophils became
increasingly less sensitive to inhibition by either azide or catalase.
Finally, while addition of SOD to reactions demonstrated a modest
increase in NO2-HPA formation at low levels of NO
flux, inhibition in nitration was observed at the higher rates of NO
flux examined (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of this study directly demonstrate that RNS contribute to
oxidative modification of proteins in asthma. They also strongly
support a role for eosinophils in generating NO-derived oxidants in
severe asthma. Using a specific and sensitive mass spectrometric
method, we observe a 10-fold increase in the content of
NO2Y in proteins recovered from airways of
patients with severe asthma compared with nonasthmatic subjects (Fig. 1). Eosinophil activation and subsequent protein oxidation in the
asthmatic airways is also supported by the dramatic increase in BrY
content observed in airway proteins recovered from asthmatic subjects
admitted to the intensive care unit (Fig. 3). Moreover,
immunohistochemical studies implicate eosinophils as a major cellular
generator of NO-derived oxidants in severe asthma because epitopes
demonstrating NO2Y-specific immunostaining
substantially colocalize with the leukocyte (Fig. 2). Finally, studies
with isolated human eosinophils reveal that aromatic nitration
reactions are a preferred oxidation pathway mediated by the leukocytes
at plasma concentrations of halides and levels of nitrite observed in
airway lining fluid of asthmatics (Figs. 6 and 7).
One of the remarkable findings of these studies is that human
eosinophils can use distinct chemical mechanisms for generating
NO-derived oxidants and that the relative preference of eosinophils for
promoting aromatic nitration vs halogenation reactions varies depending
upon their environment during activation. Moreover, the environment in
which eosinophil activation occurs (e.g., rate of NO production) also
influences the relative contribution that chemically distinct oxidation
pathways play in contributing to protein nitration. On the basis of
these results and recently published studies (24, 25, 44),
we have generated the following model (Fig. 10) of pathways available to human
eosinophils for oxidizing proteins through formation of reactive
halogen and RNS. During eosinophil activation, such as following
allergen challenge, the phagocyte undergoes a respiratory burst,
generating O2·- and its
dismutation product, H2O2.
Concomitantly, the contents of the secretory granules are released,
including EPO. EPO (15 µg/106 eosinophils)
constitutes 25% of the total protein mass of specific granule
protein, and is thus one of the most abundant proteins in eosinophils
(61). When human eosinophils are activated in medium
possessing plasma levels of halides and low levels of NO (and its
autoxidation product, NO2-),
eosinophils preferentially use the
EPO-H2O2 system to generate
brominating oxidants (Figs. 6 and 8). Aromatic bromination reactions
mediated by the activated cells are inhibited in the presence of the
HOBr scavenger methionine, a thiolether, but not by large molar
excesses of primary amines (Fig. 6). These results are consistent with
our prior observations that N-mono-bromoamines are preferred
brominating intermediates for protein tyrosine residues in cell-free
systems at neutral pH (44). The ability of
N-mono-bromoamines to promote phenolic bromination reactions
across the (patho)physiological pH range also likely contributes to the
relatively high levels of protein BrY observed in
endotracheal/bronchial aspirates recovered from patients with
severe asthma (Fig. 3).
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).
The ability of human eosinophils to use this pathway is strongly
supported by the demonstration that activated eosinophils (this study)
and isolated EPO (44) use physiologically relevant levels
of NO2- to promote nitration of
phenolic targets as a preferred activity. A primary role for EPO- and
H2O2-dependent formation of
RNS by eosinophils is further supported by 1) the absolute requirement
of NO2- for aromatic nitration
reactions (Figs. 4 and 7); and 2) the ability of EPO inhibitors
(NaN3) and
H2O2 scavengers (catalase,
but not hiCat) to block phenolic nitration (Figs. 6 and 9). The RNS
formed by EPO-catalyzed oxidation of
NO2- has not been identified.
Studies with the related hemoprotein MPO suggest that the one electron
oxidation product, nitrogen dioxide
(·NO2), is a likely
product (38, 40, 62). It should also be noted that
isolated human EPO promotes nitration of free and protein-bound
tyrosine residues at least 4- to 5-fold more efficiently than human MPO
(on an equimolar basis, which corresponds to a 8- to 10-fold difference
per heme group) (44).
Another remarkable finding of this study is the overall high yield of
tyrosine nitration by eosinophils via the
EPO-H2O2-NO2-
pathway. If one assumes that 2 mol of
O2·- are required to form 1 mol of
H2O2 during the respiratory
burst, and 2 mol of H2O2
are required per mol of NO2Y formed (one to
generate ·NO2 and
another to generate tyrosyl radical; Ref. 63), then the
overall yield of eosinophil-dependent nitration of tyrosine via the
EPO-H2O2-NO2-
system was 6.6% under the conditions used in Fig. 4. Finally,
although formation of a nitrating and halogenating species through
secondary oxidation of NO2- by
hypohalous acids has been suggested for the MPO-generated product HOCl
(64), eosinophil-mediated nitration of HPA in the presence
of NO2- was not blocked by
addition of scavengers of halogenating oxidants (thiolethers and
amines, Fig. 6). Thus, eosinophil-generated HOBr does not appear to
play a significant role in subsequent formation of NO-derived oxidants
by these cells.
Isolated peripheral blood eosinophils from normal healthy donors failed
to generate detectable levels of NO,
NO2-, or
NO3- during the brief
incubation periods used in this study. Consistent with these results,
eosinophil activation in the absence of an exogenous source of either
NO2- or NO failed to nitrate
phenolic targets (Figs. 4, 7, and 8). However, eosinophil activation in
vivo occurs in an environment replete in NO, whether generated by
cytokine-stimulated eosinophils or adjacent cells. As noted above, in
the presence of an exogenous NO donor, eosinophils readily promoted
aromatic nitration reactions (Figs. 8 and 9). At high rates of NO flux
(>2 µM/min), eosinophils appear to use a pathway for generating RNS
that is chemically distinct from the
EPO-H2O2-NO2-
system because EPO inhibitors and
H2O2 scavengers do not
significantly inhibit phenolic nitration reactions under these
conditions, yet addition of SOD does (Fig. 9). These results are
consistent with interaction of NO with cell-generated
O2·- forming
ONOO- and
ONOOCO2- (Equation 2; Fig. 10).
The relative contribution of the
ONOO-/ONOOCO2-
vs the
EPO-H2O2-NO2-
system for eosinophil-dependent generation of NO-derived oxidants
during asthma is unknown. However, the high levels of BrY observed in
BAL proteins from severe asthmatics suggest that the EPO pathway may
play a significant role in protein oxidation and, therefore,
NO2Y formation. At the levels of
NO2- typically observed in
epithelial lining fluid during severe asthma (>10 µM), nitration of
phenolic targets by the EPO system of eosinophils is preferred (Fig. 7). Finally, it should be noted that a wide range of steady-state NO
levels are observed in vivo (20 nM-2 µM; Ref. 30), and
NO-dependent protein nitration was mediated by activated eosinophils
over this range (Fig. 8).
One interesting finding in this study is that the overall yields of
nitration and bromination decline at higher fluxes (>2 µM/min) of NO
(Fig. 8). Similar bell-shaped curves for the overall extent of protein
and lipid oxidation mediated by leukocytes (e.g., neutrophils and
monocytes) activated in the presence of varying levels of NO recently
have been reported (39, 41). One possible explanation is
that NO modulates the extent of oxidation by interacting with critical
heme proteins involved in O2·-
formation (e.g., NADPH oxidase) or RNS formation (e.g., EPO). Recent
studies demonstrate that NO modulates the catalytic activity of the
related peroxidase MPO (65). At low levels, NO serves to
enhance catalytic rates by accelerating the rate limiting step in the
peroxidase cycle, reduction of Compound II (65, 66). At
higher levels of NO, MPO activity is inhibited by formation of a
ferric-nitrosyl complex (65). Similar behavior may occur
during NO interactions with EPO. Another potential mechanism accounting
for the inhibition in the extent of oxidation at higher fluxes of NO is
that it may partially act as an antioxidant under these conditions by
scavenging reactive intermediates though radical-radical interactions
(67). Alternatively, decreased formation of nitrotyrosine
at high fluxes of NO may be due to lower overall yields with
ONOO-/ONOOCO2-
compared with the RNS formed by the
EPO-H2O2-NO2-
system.
Recent studies by Gaston and colleagues demonstrate that airway vapor
condensates from severe asthmatic subjects are acidic
(68). This has led to the suggestion that elevated levels
of NO observed in asthmatic subjects may arise in part from protonation
of NO2- (68, 69).
Although the pH of airway lining fluids in the subjects examined in
this study were not determined, it is likely that they were acidic as
well. Therefore, one might speculate that some of the
NO2Y formed on BAL proteins during asthma arose
from nonenzymatic formation of RNS. However, several lines of evidence
suggest that eosinophils are a major source of NO-derived oxidants in
vivo and that EPO plays an active role in NO2Y
formation during asthma. Perhaps the strongest evidence is the intense
focal staining observed in bronchial biopsies probed with Abs specific
for NO2Y where the immunostaining predominantly
colocalized with eosinophils (Fig. 2). Moreover, using mass
spectrometry, we recently demonstrated that significant levels of
bromination (25) and nitration (44) of target
proteins incubated in medium containing plasma levels of halides and
pathophysiologically relevant levels of
NO2- does not occur through
nonenzymatic processes to any significant degree over the pH range
observed in normal and asthmatic human airways (pH 5.5–7.5). Moreover,
analysis of halogenated tyrosine adducts in protein present in lavage
fluids from this study revealed a much higher proportion of BrY than
ClY in severe asthmatics vs controls subjects (Fig. 3), despite the
1000-fold higher concentration of Cl- observed
in plasma. Given that eosinophils selectively brominate while
neutrophils chlorinate proteins at plasma levels of halides (Fig. 7 and
Ref. 24), these in vivo data strongly support the notion
that eosinophils are likely a major leukocyte responsible for promoting
oxidative modification of proteins in asthmatic patients. The
calculation of the BrY/ClY ratio may thus represent an objective and
quantifiable index to estimate the relative contributions of
eosinophils vs neutrophils in oxidative modification of proteins in
tissues. Finally, these studies with isolated human eosinophils clearly
demonstrate that the leukocytes readily form NO-derived oxidants under
physiologically relevant conditions.
Measurement of NO2Y is now widely used as a marker for protein oxidation by RNS. A multitude of techniques for quantification of NO2Y in biological tissues and fluids are used, including immunoassay and a variety of HPLC and mass spectrometry-based methods. The validity of many of these methods has been questioned because of the ease with which artificial phenolic nitration occurs in the presence of nitrite and acid pH. Only mass spectrometry combined with isotope-labeling techniques permits simultaneous monitoring of authentic oxidized amino acids (e.g., NO2Y) and intrapreparative formation of the analyte during sample handling (24, 41, 70, 71). The stable isotope dilution mass spectrometric methods used in this study excluded any significant contribution of ex vivo nitration (as well as bromination and chlorination) of airway proteins. It is interesting to note that levels of NO2Y observed in proteins recovered from airways of nonasthmatic subjects in this study are extremely low, similar to those recently observed on induced sputum of normal subjects (72) and in other tissues and fluids examined using mass spectrometry-based methods that permit development of sample preparation methods that minimize artifactual nitration (70, 71).
It should be noted that the biological consequences of protein
oxidation via nitration or bromination during asthma are not known.
However, it should also be appreciated that the stable covalent adducts
of tyrosine monitored represent only a fraction of the total
modifications incurred during exposure of proteins to reactive nitrogen
or halogenating species. Other nucleophilic targets on proteins
including thiol (cysteine), thiolether (methionine), imidazole
(histidine), and indole (tryptophan) groups are all targets for
oxidation by either brominating or nitrating agents. The chemical
reactivity of EPO-generated oxidants undoubtedly plays a significant
role in the cytotoxic properties of these agents on pathogens and
eukaryotic cells. Thus it is reasonable to speculate that oxidative
modification of critical biological targets in asthmatic airways may
contribute to the pathophysiological sequelae of the disease.
Particularly given the known toxicity of HOBr and the
EPO-H2O2-Br-
system to eukaryotic cells in culture (73, 74), and their
capacity to elicit many of the pathophysiologic features of asthma,
such as epithelial cell damage and sloughing, airway hyperreactivity,
bronchoconstriction, -adrenergic dysfunction, mucus hyper secretion,
microvascular leak, and edema (75, 76, 77, 78, 79, 80).
Collectively, these data suggest that eosinophils contribute to oxidative protein modification in lung and airways of asthmatics. They also further define chemical pathways available to eosinophils for contributing to inflammatory injury in vivo and reveal that these unique effector cells in the host response can use a variety of distinct chemical pathways to promote oxidative modification of targets. Knowledge of the precise chemical pathways used by eosinophils is critical to the development of targeted interventions designed to retard or interrupt these processes. One such target that is suggested by these studies is EPO. Future work is warranted to assess the potential clinical impact of EPO inhibition in human asthma.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stanley L. Hazen, Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, NC-10, Cleveland, OH 44195.
3 Abbreviations used in this paper: EPO, eosinophil peroxidase; BAL, bronchoalveolar lavage; Br-HPA, 3-(3-bromo-4-hydroxyphenyl)propanoic acid; BrY, 3-bromotyrosine; Cl-HPA, 3-(3-chloro-4-hydroxyphenyl)propanoic acid; ClY, 3-cholorotyrosine; DTPA, diethylenetriaminepentaacetic acid; GC-MS, gas chromatography-mass spectrometry; hiCat, heat inactivated catalase; HPA, 3-(4-hydroxyphenyl) propanoic acid; NO2-HPA, 3-(4-hydroxy-3-nitrophenyl)propanoic acid; NO2Y, 3-nitrotyrosine; PAPA NONOate, (Z)-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate; RNS, reactive nitrogen species; SOD, superoxide dismutase.
4 This work was supported by National Institutes of Health Grants HL61878 and HL62526 and by the American Heart Association. J.M. was supported by a National Institutes of Health NRSA Fellowship.
5 Address correspondence and reprint requests to Dr. Stanley L. Hazen, Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, NC-10, Cleveland, OH 44195.
6 Abbreviations used in this paper: EPO, eosinophil peroxidase; BAL, bronchoalveolar lavage; Br-HPA, 3-(3-bromo-4-hydroxyphenyl)propanoic acid; BrY, 3-bromotyrosine; Cl-HPA, 3-(3-chloro-4-hydroxyphenyl)propanoic acid; ClY, 3-cholorotyrosine; DTPA, diethylenetriaminepentaacetic acid; GC-MS, gas chromatography-mass spectro-metry; hiCat, heat inactivated catalase; HPA, 3-(4-hydroxyphenyl) propanoic acid; NO2-HPA, 3-(4-hydroxy-3-nitrophenyl)propanoic acid; NO2Y, 3-nitrotyrosine; PAPA NONOate, (Z)-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate; RNS, reactive nitrogen species; SOD, superoxide dismutase.
Received for publication October 6, 2000. Accepted for publication February 22, 2001.
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