1 Division of Pulmonary/Critical Care Medicine, Department of Internal Medicine, University of California, Davis 95616; 2 Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720; and 3 Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, Alabama 35233
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ABSTRACT |
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Cystic fibrosis (CF) is associated with chronic pulmonary inflammation
and progressive lung dysfunction, possibly associated with the
formation of neutrophil myeloperoxidase (MPO)-derived oxidants.
Expectorated sputum specimens from adult CF patients were analyzed for
MPO characteristic protein modifications and found to contain large
amounts of active MPO as well as high levels of protein-associated
3-chlorotyrosine and 3,3'-dityrosine, products that result from MPO
activity, compared with expectorated sputum from non-CF subjects.
Sputum levels of nitrite (NO2) and nitrate
(NO3
), indicating local production of nitric oxide
(NO·), were not elevated but in fact were slightly reduced in CF.
However, there was a slight increase in protein-associated
3-nitrotyrosine in CF sputum compared with controls, reflecting the
formation of reactive nitrogen intermediates, possibly through
MPO-catalyzed oxidation of NO2
. CF sputum MPO was
found to contribute to oxidant-mediated cytotoxicity toward cultured
tracheobronchial epithelial cells; however, peroxidase-dependent protein oxidation occurred primarily within sputum proteins, suggesting scavenging of MPO-derived oxidants by CF mucus and perhaps formation of
secondary cytotoxic products within CF sputum. Our findings demonstrate
the formation of MPO-derived oxidizing and possibly nitrating species
within the respiratory tract of subjects with CF, which collectively
may contribute to bronchial injury and respiratory failure in CF.
inflammation; hypochlorous acid; 3-chlorotyrosine; 3,3'-dityrosine; nitric oxide; 3-nitrotyrosine
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INTRODUCTION |
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CYSTIC
FIBROSIS (CF) is an autosomal recessive genetic disorder caused
by mutations in the CF transmembrane regulator (CFTR) gene and is
accompanied by altered epithelial Cl and water transport,
increased mucus viscosity, reduced mucociliary clearance, and decreased
antibacterial defense within the respiratory tract. Consequently,
patients with CF are increasingly susceptible to bacterial respiratory
tract infections, most commonly by Staphylococcus aureus,
Haemophilus influenzae, and eventually by Pseudomonas aeruginosa, and often die of respiratory failure resulting from repeated acute pulmonary infections and the ensuing chronic airway inflammatory-immune response (32, 45). Respiratory
secretions from CF patients commonly contain large numbers of
inflammatory-immune cells and high amounts of neutrophil-derived
mediators as well as the neutrophil granule enzyme myeloperoxidase
(MPO) (3, 10, 35, 37, 41, 62).
A major component of neutrophil-mediated host defense is the
production of reactive oxygen metabolites [superoxide anion
(O2·), hydrogen peroxide
(H2O2), or hypochlorous acid (HOCl)], but these oxidants are also thought to contribute to lung epithelial dysfunction observed in patients with CF (6, 56, 60).
Pseudomonas aeruginosa, colonized within the respiratory
tract of CF patients, secrete siderophores (such as pyocyanin and
pyochelin) that can also generate oxidants and contribute to epithelial
injury in CF (5, 12, 24). Additionally, these various
oxidants are also believed to contribute to the documented
proteinase-antiproteinase imbalance within the respiratory tract of
patients with CF (2, 40, 51). The presence of oxidative
stress within the respiratory tract of CF patients is supported by
various reports documenting decreased antioxidant status and elevated
indexes of oxidation of lipids, proteins, or DNA (6, 9, 56,
60). Furthermore, malabsorption of fat-soluble antioxidants such
as vitamin E (56) and lowered levels of GSH in respiratory
tract lining fluids, thought to be related to reduced
CFTR-assisted efflux of GSH from alveolar epithelial cells (17,
46), contribute to a reduced antioxidant status within the
respiratory tract of CF patients. Conversely, prooxidant enzymes such
as MPO may augment oxidative injury to various cellular or
extracellular constituents within inflamed tissues and thus contribute
to pulmonary dysfunction (e.g., Ref. 22). Indeed, respiratory levels of
MPO have been found to correlate with decreases in respiratory
parameters (%forced expiratory volume in 1 s or % forced vital
capacity predicted) or disease severity in CF (41,
61). However, there is as yet minimal direct evidence
for the generation or significance of MPO-derived oxidants within the
respiratory tract of CF patients.
Respiratory conditions associated with inflammation, such as asthma,
adult respiratory distress syndrome, or bronchiectasis, are commonly
associated with induction of nitric oxide synthase (NOS) and increased
production of nitric oxide (NO·) within the lung as part of the host
defense system (18). However, overproduction of NO· also
promotes the formation of more reactive nitrogen intermediates, including dinitrogen trioxide (N2O3), nitrogen
dioxide (NO2), and peroxynitrite (ONOO),
oxidants that are capable of inducing cell and tissue injury (e.g.,
Refs. 1, 20). The formation of characteristic modifications in proteins
such as 3-nitrotyrosine is regarded indicative of endogenous formation
of such NO·-derived oxidants (1, 13, 20, 23, 55), and
elevated levels of 3-nitrotyrosine indeed have been detected in various
diverse lung diseases associated with inflammation (reviewed in Ref.
57). Although inducible NOS (iNOS) is constitutively present within
respiratory epithelial cells, its expression is markedly reduced within
the respiratory epithelium of CF patients (27, 33).
Additionally, levels of exhaled NO· or its metabolites nitrite
(NO2
) and nitrate (NO3
) in airway
secretions are often found to be subnormal in CF unless the patients
suffer from acute pulmonary infections (16, 19, 29, 31).
Hence, a role for NO· or NO·-derived oxidants in the pathology of
CF is still unclear.
In the present investigation, we have examined expectorated sputum specimens from adult CF patients, as well as induced sputum samples from healthy subjects for the presence of active MPO and protein oxidation products that are characteristic of peroxidase- and/or NO·-derived oxidants (3,3'-dityrosine, 3-chlorotyrosine, and 3-nitrotyrosine). We also aimed to explore the potential contribution of MPO to epithelial toxicity by CF sputum in relation to characteristic protein modifications. The results demonstrate the formation of reactive oxygen and nitrogen intermediates within the respiratory tract of CF patients that most likely originates from MPO-dependent mechanisms and indicate that these oxidants may contribute to respiratory epithelial injury associated with CF.
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EXPERIMENTAL PROCEDURES |
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Sputum collection and processing.
Spontaneously expectorated sputum specimens were collected from 21 stable adult CF patients who were seen for routine outpatient visits at
the University of California, Davis Medical Center. Although these
patients were infected with Pseudomonas aeruginosa, they
generally did not suffer from major acute infections and were not
receiving steroid treatment. As controls, airway secretions were
collected from 14 healthy nonsmoking volunteers by sputum induction by
inhalation of aerosolized saline, generated using a DeVilbiss 65 ultrasonic nebulizer (DeVilbiss, Somerset, PA) with an output of 2.4 ml/min and particle size of 4.5 µm aerodynamic mass median diameter
(14). Subjects were asked to cough sputum into a sterile
plastic container every 2 min during the sputum induction procedure for
a total period of 12 min. Because the composition of induced sputum has
been reported to be similar to that of spontaneously expectorated
sputum and dilution of respiratory secretions by sputum induction is
thought to be minimal (39), we considered induced sputum
appropriate as a control for this study. Collected sputum specimens
were immediately stored at 80°C and analyzed within 2 mo. Sections
of frozen CF sputum samples were cut, thawed, and diluted in an equal
volume of 50 mM sodium phosphate buffer (pH 7.4) and (unless indicated
otherwise) incubated for 20 min with 20 mM dithiothreitol (DTT) at
37°C to assist in liquefying and homogenizing the sputum. Induced
sputum samples from control subjects were treated similarly but were
not diluted. Sputum samples were centrifuged for 10 min at 10,000 g to separate the insoluble gel phase from the soluble (sol)
phase, which was used for most analyses.
Analysis of peroxidase activity and MPO levels. Peroxidase activity in sol-phase sputum samples was measured by H2O2-dependent oxidation of guaiacol and is expressed in units per milligram of protein (28). The protein content of sol-phase sputum fractions was determined according to Bradford using bovine serum albumin as a standard. The presence of MPO protein within sol-phase sputum was also analyzed by separation of sputum proteins by 10% SDS-PAGE gel electrophoresis and Western blot analysis using a polyclonal antibody against human MPO (Binding Site, San Diego, CA), compared with purified human MPO (Alexis, San Diego, CA). To determine the contribution of MPO to total sputum peroxidase activity, some samples were analyzed with 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate (53) and the inhibitor 4-aminophenylsulfone (dapsone) was used to distinguish between MPO (insensitive to dapsone inhibition) and other peroxidases such as lactoperoxidase and eosinophil peroxidase, which are more potently inhibited by dapsone (53).
Analysis of NO2 and NO3
.
NO2
and NO3
were determined as an
index of local NO· production according to the procedure outlined by
Braman and Hendrix (4). Briefly, this procedure is based
on acidic reduction of NO2
and NO3
to NO· by vanadium(III) and purging of NO· with helium into a Antek
7020 nitric oxide detector (Antek Instruments, Houston, TX). At room
temperature, vanadium(III) only reduces NO2
, whereas
NO3
and other redox forms of NO· such as
S-nitrosothiols are also reduced after the solution is
heated to 80-90°C, so that both NO2
and total
NOx (primarily NO3
) can be measured. For
comparison, NO2
was measured spectrophotometrically
(543 nm) after reaction with 1% sulfanilamide and 0.1%
naphthylethylenediamine in 2.5% H3PO4 (Griess
reagent). Quantitation was performed by comparision with standard
solutions of NO2
and NO3
.
Determination of protein tyrosine modifications.
Whole sputum specimens from CF patients and healthy subjects
(containing both sol and gel phases) were mixed with equal volumes of
100 mM sodium acetate (pH 7.2), and proteins were precipitated by
addition of two volumes of acetonitrile. Precipitated proteins were
centrifuged (5 min, 3,000 g) and washed four times with 2 ml
of sodium acetate buffer (pH 7.2)-acetonitrile (50:50 vol/vol) to
remove soluble contaminants that would cause artifactual nitration or
chlorination during protein hydrolysis, such as NO2,
NO3
and Cl
. Sputum proteins were
hydrolyzed under vacuum in 6 M HCl for 18 h at 110°C and
subsequently dried under N2. Amino acids were reconstituted
in 100 mM sodium acetate buffer (pH 7.2) and analyzed directly by
reverse-phase HPLC and fluorescence detection (excitation 284 nm;
emission 410 nm) for determination of 3,3'-dityrosine (e.g., Ref.
55). For analysis of 3-nitrotyrosine, amino acids were
derivatized by acetylation, followed by O-deacetylation and dithionite reduction of derivatized nitrotyrosine to the corresponding aminotyrosine derivative and the resulting
N-acetylaminotyrosine (AcATyr) and
N-acetyltyrosine (AcTyr) were analyzed by reverse-phase HPLC
with tandem amperometric electrochemical (detector potential 500 mV)
and ultraviolet (UV; 274 nm) detection (48). Similar sample derivatization without dithionite reduction was performed to
correct for the potential presence of endogenous 3-aminotyrosine (e.g.,
Ref. 49). Sputum specimens also were analyzed for the presence of
3-chlorotyrosine, a specific indicator of MPO-derived chlorinating
oxidants (21, 22), by hydrolysis of sputum proteins in 6 M
HBr for 18 h at 110°C, evaporation under N2, and
derivatization of the resulting amino acids with
N-methyl-N-(tert-butyldimethylsilyl)trifluoracetamide (Pierce) for 90 min at 60°C. Samples were analyzed by gas
chromatography-mass spectrometry (GC-MS) using selected ion monitoring
as described previously (58).
Cytotoxicity assays.
Human tracheobronchial epithelial HBE1 cells (immortalized using
papilloma virus; e.g., Ref. 43) were cultured in serum-free F-12 medium
supplemented with 1.2 g/l NaHCO3, 5 µg/ml insulin, 5 µg/ml transferrin, 10 ng/ml epidermal growth factor, 0.1 µM dexamethasone, 10 ng/ml cholera toxin, and 30 µg/ml bovine
hypothalamus extract (44). Cells were seeded at 100,000 cells/well in 24-well plates 1 day before experimentation. Before
experiments, the medium was replaced with 0.5 ml of Earle's balanced
salt solution (EBSS) and either 20 nM MPO or various dilutions of
sol-phase CF sputum (prepared without DTT) were added to the cells and
preincubated for 1 h. During incubations with CF sputum, soybean
trypsin inhibitor (type I-S, Sigma, 100 µg/ml) was added to minimize
sputum proteolytic activity. When indicated, incubations included
NO2 (0.1-1.0 mM) as a potential MPO substrate or 100 µM of the MPO inhibitor 4-aminobenzoic acid hydrazide (ABAH; Sigma).
Incubations were started by addition of 10 mU/ml glucose oxidase to
generate H2O2 continuously at a flux of ~1
µM/min. After incubation, the cells were rinsed twice with fresh EBSS
and subsequently kept in F-12 medium containing 5% Alamar blue
solution. Reduction of this dye by cellular mitochondrial dehydrogenase
activity reflects viable respiring cells and thus reduction in
mitochondrial respiration or cell viability can be monitored by
decreased conversion of Alamar blue, determined spectrophotometrically
at 570 and 600 nm (50). Cell viability was expressed
relative to untreated control cells. For comparison, similar
experiments were performed with various dilutions of induced sputum
from healthy subjects. Some experiments were also performed with
alveolar epithelial A549 cells, which were grown in Ham's F-12 medium
containing 10% fetal bovine serum and 1.2 g/l NaHCO3.
Determination of MPO-dependent protein oxidation in respiratory
tract epithelial cells.
HBE1 or A549 cells were grown to confluence in 100-mm culture dishes,
and the medium was replaced with 4 ml of EBSS. Cells were subsequently
preincubated for 1 h with purified MPO or with various dilutions
of sol-phase sputum from CF patients in the absence or presence of
0.1-1.0 mM NO2 or 100 µM ABAH, and production
of H2O2 was subsequently induced by addition of
10 mU/ml glucose oxidase. Trypsin inhibitor (100 µg/ml) was again
added to minimize proteolytic cell lysis or detachment. After
incubation for 3 h, the medium (containing CF sputum) was aspirated and the cells were washed twice with cold PBS and collected by scraping with a rubber policeman. The aspirated medium containing sputum proteins was centrifuged for 10 min at 500 g to
remove detached cells, which were pooled with the other collected
cells. The collected supernatant, including CF sputum proteins, may
also contain some cellular proteins that may have leaked from necrotic cells. Cell proteins and CF sputum proteins were precipitated by adding
equal volumes of cold acetonitrile, homogenized in 1 ml of 0.1 M sodium
acetate buffer (pH 7.2), and precipitated again with an equal volume of
acetonitrile. This procedure was repeated twice to rigorously remove
contaminants such as NO2
. Proteins were subsequently
hydrolyzed in 6 M HCl for 18 h and processed for analysis of
tyrosine, dityrosine, and 3-nitrotyrosine as described previously.
Data analysis. Results are expressed as means ± SE, and data were analyzed by standard two-way ANOVA for comparison of various parameters in CF sputum compared with induced sputum from healthy subjects. Differences were considered statistically significant at P < 0.05.
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RESULTS |
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MPO and other peroxidases in CF airway secretions.
As expected, sol fractions of CF sputum specimens were found to contain
high peroxidase activity compared with induced sputum samples obtained
from healthy subjects (9.9 ± 3.6 vs. 0.24 ± 0.06 U/mg
protein, P < 0.005, Fig.
1A), which
is indicative of high levels of MPO or related peroxidases.
Peroxidase-dependent oxidation of TMB by CF sputum was resistant to
inhibition by dapsone, similar to MPO-dependent TMB oxidation (Fig.
1B). Induced sputum specimens from healthy subjects also
contained peroxidase activity that was largely resistant to inhibition
by dapsone (Fig. 1B), indicating that MPO also contributes
importantly to peroxidase activity of normal respiratory secretions
(53). The presence of large amounts of MPO protein in CF
sputum, in contrast to induced sputum from healthy subjects, was also
demonstrated by SDS-PAGE and Western blot analysis with a polyclonal
antibody against human MPO (Fig. 1C). The presence of MPO in
CF sputum indicates the recruitment of polymorphonuclear neutrophils
within the respiratory tract of CF patients.
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CF sputum protein contains elevated levels of oxidation products.
As a general marker of protein oxidation, we analyzed sputum proteins
for the presence of 3,3'-dityrosine, a modification brought about by
either Fenton-like oxidants or peroxidase-catalyzed pathways (e.g.,
Ref. 22). Proteins obtained from CF sputum specimens contained
dramatically elevated levels of 3,3'-dityrosine compared with those
collected from induced sputum samples from healthy subjects (Fig.
2A). Average
protein-associated dityrosine levels were found to be increased about
30-fold, from 0.038 ± 0.019 to 1.14 ± 0.28 mmol/mol
tyrosine (P < 0.005). As a more specific determinant
of the local formation of MPO-derived oxidants, we detected elevated
levels of 3-chlorotyrosine in CF sputum proteins (Fig. 2B),
which were increased more than 10-fold over those in induced sputum
specimens from healthy subjects (0.51 ± 0.10 vs. 5.82 ± 0.76 mmol/mol tyrosine, P < 0.005). Collectively,
these findings indicate the involvement of MPO in oxidative reactions in the respiratory tract of CF patients.
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NO·-derived oxidants in CF.
To determine the potential involvement of NO· and formation of
reactive nitrogen intermediates in CF, sputum proteins were analyzed for the presence of 3-nitrotyrosine. As illustrated in Fig. 3A, the relative levels
of protein 3-nitrotyrosine (expressed relative to unmodified tyrosine
residues) were similar in both CF sputum specimens and in induced
sputum samples from healthy subjects (AcATyr levels were 0.085 ± 0.012 and 0.066 ± 0.022 mmol/mol AcTyr in CF sputum and control
sputum, respectively). However, although it is common practice
to express such oxidative modifications of amino acid side chains
relative to the unmodified precursor, this may yield skewed results if
the total protein content varies dramatically between subject groups.
Because the extent of protein nitration is most likely limited by the
amount of nitrating oxidant generated, it should not increase when more
protein is present, and normalization of protein tyrosine modifications
to unmodified tyrosine residues may in fact yield lower values if the
total protein (and thus unmodified tyrosine) content is higher. Hence, comparison of different subject groups with dramatically different protein content (such as in the present case where CF sputum specimens contained much more protein compared with induced sputum samples from
healthy subjects) based on such relative measures of protein oxidation
might yield misleading results. We therefore also expressed protein
3-nitrotyrosine levels in more absolute amounts (i.e., as pmol/ml
sputum), and in this case found 3-nitrotyrosine at about ninefold
higher levels in CF sputum specimens compared with normal sputum
samples from healthy subjects (175 ± 42 vs. 19.9 ± 6.6 pmol/ml, P < 0.05, Fig. 3B). This apparent
increase in protein-associated 3-nitrotyrosine indicates enhanced
production of reactive nitrogen species within the respiratory tract of
CF patients, which may originate from MPO-mediated oxidation of
NO2 (13, 55).
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MPO in CF sputum can contribute to epithelial injury.
To establish whether MPO-derived oxidants might contribute to
epithelial injury or lung dysfunction in CF, we exposed human tracheobronchial epithelial HBE1 cells to H2O2
in the presence of either purified MPO or aliquots of sol-phase CF
sputum specimens and determined changes in mitochondrial respiration (a
measure of cell viability). Exposure of HBE1 cells to
H2O2 (generated at 1 µM/min by glucose
oxidase) for 3 h resulted in a modest decrease in cell viability,
which was markedly enhanced in the presence of 80 nM MPO, most likely
due to the formation of the cytotoxic oxidant HOCl. As expected, this
additional cytotoxicity was prevented almost completely by the MPO
inhibitor ABAH (Fig. 4A).
Similar experiments with CF sputum specimens revealed epithelial
toxicity by CF sputum (at dilutions less than 1:40), which was
generally enhanced in the presence of H2O2. The
H2O2-dependent decrease in cell viability by CF
sputum could in most cases be inhibited by the MPO inhibitor ABAH (100 µM), indicating the involvement of MPO-dependent mechanisms. Figure
4B illustrates typical results with CF sputum specimens
obtained from two separate individuals. MPO-dependent cytotoxicity by
CF sputum was less pronounced compared with that induced by similar
amounts of purified MPO, and other nonoxidant mechanisms (such as
proteolytic cell injury) most likely contributed significantly to the
epithelial toxicity of CF sputum. In similar experiments with diluted
induced sputum specimens from healthy subjects,
H2O2-induced loss in epithelial viability was found to be reduced (from 12 ± 3 to 4 ± 2%; means ± SE from 3 experiments in duplicate), most likely because of
H2O2 removal by catalase, extracellular GSH
peroxidase, or lactoperoxidase (47, 63). Similar
experiments with alveolar A549 cells instead of HBE1 cells yielded
comparable results (data not shown).
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DISCUSSION |
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In agreement with previous findings (35, 37, 63), expectorated sputum specimens from CF patients were found to contain large amounts of active MPO. Based on measured peroxidase activity compared with purified MPO, the high MPO activity of expectorated CF sputum specimens suggests local enzyme concentrations of 0.5-10 µM. A significant portion of the sputum MPO may have been associated with mucus complexes in the gel-phase sputum or may have been partly inactivated or degraded by local oxidative and proteolytic processes; hence the actual total MPO content of CF sputum is probably even higher. Several recent studies have suggested a role for MPO in various diseases, because they have linked a genetic polymorphism in the MPO promotor that affects MPO expression to acute myeloid leukemia (42), lung cancer risk (30), neurodegenerative diseases (36, 43), and granuloma formation in patients with chronic granulomatous disease (15). Although the biochemical mechanism underlying the involvement of MPO in these various conditions has not been clarified, it is generally assumed that MPO-dependent oxidative processes contribute to these disorders. It is feasible that MPO in combination with elevated oxidant production also contributes to lung dysfunction in CF by the formation of various MPO-derived oxidants, including HOCl. The results of the present study, demonstrating elevated levels of protein tyrosine oxidation products such as 3,3'-dityrosine, 3-chlorotyrosine, and 3-nitrotyrosine in sputum specimens from CF patients, indeed provide evidence for ongoing oxidative processes within the respiratory tract of these patients that may largely involve peroxidase-dependent mechanisms. Specifically, the presence of elevated levels of 3-chlorotyrosine indicates the local formation of MPO-dependent HOCl (21, 22) within the respiratory tract of CF patients. Accordingly, a recent study has also demonstrated the presence of chloramines in CF sputum specimens in relation to MPO activity, consistent with formation of HOCl (57).
MPO is known to be capable of enhancing oxidant-induced injury to
epithelial cells, most likely because of formation of the cytotoxic
oxidant HOCl (7, 8, 63). In the present study, MPO-dependent cytotoxicity was not significantly affected by the presence of NO2, despite substantial nitration of
cellular proteins, which suggests that NO·-derived oxidants may not
play an important role in epithelial toxicity or in the pathophysiology
of CF. A relatively minor role for NO· in CF is also exemplified by
the absence of iNOS within the respiratory epithelium of CF patients
and the subnormal levels of the NO· metabolites NO2
and NO3
in airway secretions. Furthermore, compared
with the marked increase in protein 3-chlorotyrosine and
3,3'-dityrosine levels in airway secretions of CF patients, the
relative increase in protein 3-nitrotyrosine was minimal. The relative
moderate increase in protein nitrotyrosine residues compared with other
oxidative protein modifications could also be due to the potential
presence of enzymatic systems that modify nitrated proteins
(26).
MPO-induced cytotoxicity to endothelial or epithelial cells has been found to depend on the ability of MPO to interact with the epithelial surface, and agents that prevent such epithelial interaction or that can act as local scavengers of HOCl diminish epithelial toxicity by MPO (7, 8, 11, 63). Addition of sol-phase CF sputum to cultured human bronchial epithelial cells has been demonstrated to impair ciliary beating, which could be partly attributed to oxidants generated by bacterial pigments (5, 12, 24, 52, 59), but a potential role for MPO in these cellular effects was not established. In the present study, we have demonstrated that the presence of sol-phase CF sputum enhances oxidant-induced epithelial cytotoxicity by an MPO-dependent mechanism, although MPO present within CF sputum was relatively less effective compared with purified MPO. This is most likely related to association of the highly cationic MPO (pI > 10) with negatively charged mucus glycoproteins or DNA present within CF sputum, thereby minimizing interaction of MPO with the epithelial surface (e.g., Ref. 63). Accordingly, MPO-dependent oxidative inactivation of cell surface proteins or toxicity to epithelial cells were found to be dramatically inhibited in the presence of glycosaminoglycans that minimize interaction of MPO with the cell surface (11, 63).
Analysis of oxidation products in either sputum or cellular proteins
after epithelial cell exposures to H2O2 and CF
sputum demonstrated that oxidation or nitration (when
NO2 was present) of sputum proteins was much more
extensive than that of cellular proteins. This finding confirms the
notion that MPO within CF sputum is largely associated with sputum
proteins, thereby preventing interaction of MPO with the epithelial
surface. Thus MPO-derived oxidants appear to be largely scavenged
by sputum proteins before they are able to induce epithelial cell
injury. Certain extracellular functional components might, however,
present critical targets for MPO-derived reactive oxygen and
nitrogen intermediates, and oxidation products formed within airway
secretions might exert secondary toxicity toward the epithelium. For
instance, oxidation and/or nitration can cause the activation of
metalloproteinases (38) and inactivation of tissue
inhibitors of metalloproteinases or
1-proteinase
inhibitor (e.g., Ref. 34), thus contributing to an imbalance in
proteinase and antiproteinase activities in the respiratory tract.
MPO-derived oxidants could also contribute to the pathology of CF by
promoting cell adhesion via the
M
2-integrin (25) or the
formation of covalent cross-links of immune complexes, which may
contribute to the phenomenon of frustrated phagocytosis (54), thereby further stimulating secretion of noxious
neutrophil granular components within the respiratory tract.
In summary, three general conclusions can be drawn from the present
studies: 1) CF sputum contains high levels of active MPO and
elevated amounts of the MPO characteristic protein oxidation products
3-chlorotyrosine; 3,3'-dityrosine, and to a lesser degree 3-nitrotyrosine; 2) MPO-derived oxidants are able to injure
epithelial cells but appear to react primarily with targets within the
airway secretions (e.g., mucus proteins) rather than with components of
underlying cells; and 3) NO· appears to play a minor role
in CF, as levels of the NO· metabolites NO2 and
NO3
in respiratory secretions were not elevated and
3-nitrotyrosine levels were not dramatically increased. Moreover, the
degree of protein nitration does not necessarily correlate with indexes of epithelial toxicity. MPO-derived reactive oxygen (and nitrogen) intermediates might contribute directly to epithelial toxicity or
dysfunction or affect extracellular targets within airway secretions, either noncritical mucus proteins or functional constituents such as
antiproteinases. Although the involvement of MPO in disease pathology
might relate to its ability to induce characteristic protein
modifications such as protein chlorination (21, 22) or
nitration (23, 57), the precise significance of these
specific modifications still remains to be established.
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ACKNOWLEDGEMENTS |
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We thank Barry Halliwell for the contribution to the discussions, Michelle Kim for technical assistance, and Susan Ebeler and Da-Mi Jung for assistance with the GC-MS analyses.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grants HL-60812 and HL-57542 and by research grants from the American Lung Association of California, the Cystic Fibrosis Foundation, and the University of California Tobacco-Related Disease Research Program.
Address for reprint requests and other correspondence: A. van der Vliet, Center for Comparative Respiratory Biology and Medicine, Dept. of Internal Medicine, Univ. of California, 1121 Surge I Annex, Davis, CA 95616. (E-mail: avandervliet{at}ucdavis.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 30 September 1999; accepted in final form 31 March 2000.
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