Myeloperoxidase and protein oxidation in cystic fibrosis

Albert Van der Vliet1, Mai N. Nguyen1, Mark K. Shigenaga2, Jason P. Eiserich3, Gregory P. Marelich1, and Carroll E. Cross1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Cystic fibrosis (CF) sputum specimens contain large amounts of active myeloperoxidase (MPO). A: soluble (sol)-phase sputum from 15 CF patients and induced sputum specimens from 12 healthy control subjects were analyzed for MPO activity using guaiacol as a substrate. Horizontal lines, averages ± SE. n, no. of subjects. B: susceptibility of peroxidase-dependent tetramethylbenzidine (TMB) oxidation to inhibition by 4-aminophenylsulfone (dapsone). Purified bovine milk lactoperoxidase (3 nM; ), MPO (3 nM; ), sol-phase CF sputum (2-5 µl; open circle ), or induced sputum from healthy subjects (10-20 µl; ) were incubated in the presence of TMB, H2O2, and the indicated concentrations of 4-aminophenylsulfone (dapsone), and the rate of TMB oxidation was followed spectrophotometrically. Average results from 2-3 CF or non-CF sputum samples are shown. C: analysis of MPO protein in CF and non-CF sputum by 10% SDS-PAGE and Western blot analysis using a polyclonal antibody against human MPO. The 59-kDa marker indicates the presence of the heavy subunit of MPO.

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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Fig. 2.   CF sputum specimens contain elevated levels of protein 3,3'-dityrosine (DiTyr) and 3-chlorotyrosine (Cl-Tyr). Proteins from CF or non-CF sputum specimens were collected, hydrolyzed, and subsequently analyzed for 3,3'-dityrosine (A) and 3-chlorotyrosine (B) by HPLC with tandem ultraviolet and fluorescence detection or gas chromatography-mass spectrometry, respectively. Tyr, tyrosine. Average values ± SE are indicated. n, No. of subjects.

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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Fig. 3.   Protein 3-nitrotyrosine in CF sputum. Sputum proteins were collected and hydrolyzed, and amino acids were derivatized for 3-nitrotyrosine analysis by HPLC with electrochemical detection. Protein 3-nitrotyrosine levels are expressed either relative to unmodified tyrosine residues [N-acetylaminotyrosine (AcATyr) and N-acetyltyrosine (AcTyr); mmol/mol tyrosine, A] or as absolute concentrations (pmol/ml sputum, B). Average values ± SE are indicated. n, No. of subjects.

The observed increase in protein nitration was only moderate compared with the more dramatic increases in protein dityrosine and 3-chlorotyrosine. In fact, similar expression of 3-chlorotyrosine or dityrosine levels as picomoles per milliliter sputum would have indicated even more dramatic increases in these protein modifications. The results therefore suggest that NO· plays a relatively minor role in oxidative lung injury in CF. Indeed, levels of the NO· metabolites NO2- and NO3- within sol-phase CF sputum specimens were not increased compared with those found in induced sputum samples from healthy subjects and were in fact somewhat lower (Table 1). Although contamination of sputum specimens with saliva, known to be rich in both NO3- and NO2- (e.g., Ref. 55), might have obscured potential differences in NO2-/NO3- levels within airway secretions, our results are comparable to those reported by others (18, 29) and do not provide evidence of induction of NO· synthesis in CF.

                              
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Table 1.   Levels of NO2- and NO3- in sputum specimens from CF patients and healthy subjects

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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Fig. 4.   Cytotoxicity of MPO and CF sputum to human tracheobronchial epithelial cells. A: human bronchial epithelial HBE1 cells [100,000 cells/well in 0.5 ml Earle's balanced salt solution (EBSS)] were preincubated with 80 nM (1 U/ml) MPO in the absence or presence of 100 µM nitrite (NO2-) or 100 µM aminobenzoic acid hydrazide (ABAH) for 60 min. Subsequently, 10 mU/ml glucose oxidase were added to initiate the formation of H2O2 at ~1 µM/min for 3 h. Effects on cell viability were assessed by measurement of mitochondrial respiration using Alamar blue. B: aliquots of sol-phase CF sputum from two individuals (CF-1 and CF-2, containing MPO at 47.8 and 12.8 U/ml, respectively) were added to HBE1 cells at 1:40 dilution in EBSS in the absence or presence of 100 µM ABAH, and H2O2 production was initiated by addition of glucose oxidase. After 3 h, cell viability was measured using Alamar blue. Results are means ± SE of quadruplicate determinations from a representative experiment.

During such exposures of HBE1 cells to CF sputum and H2O2, sputum proteins were substantially oxidized. For instance, sputum protein 3,3'-dityrosine content increased from 0.65 ±0.46 to 1.58 ±0.34 mmol/mol tyrosine, and this increase was largely abolished in the presence of 100 µM ABAH (0.79 ±0.43 mmol dityrosine/mol tyrosine), indicating involvement of MPO. In contrast, there was no detectable increase in dityrosine content in cellular proteins under these conditions. Comparable results were obtained with regard to MPO-dependent protein chlorination, which occurred primarily within sputum proteins. Analysis by HPLC with UV detection (e.g., Ref 13) revealed levels of 15-30 mmol chlorotyrosine/mol tyrosine in sputum proteins in two typical experiments. Chlorination of cellular proteins was not significantly increased over background chlorination, which was relatively high (1-4 mmol/mol tyrosine) due to protein hydrolysis in HCl. Similar exposure of HBE1 cells to CF sputum and H2O2 in the presence of 0.1-1.0 mM NO2- resulted in nitration of cellular proteins as determined by HPLC analysis (Fig. 5) and by SDS-PAGE and Western blot analysis using alpha -nitrotyrosine antibodies (data not shown), but sputum proteins were nitrated to a nearly 100-fold larger extent under these conditions (Fig. 5). Protein nitration was in each case almost completely prevented in the presence of 100 µM ABAH (data not shown), indicating involvement of MPO. The degree of sputum protein nitration under these experimental conditions was much higher than that found in vivo (Fig. 3), most likely because NO2- was used at levels well above those found in vivo (Table 1), and dilution of CF sputum for these experiments similarly resulted in dilution of other potential endogenous MPO substrates. When experiments were performed with NO2- diluted to a similar extent (to ~1 µM), no significant increase in sputum protein nitration above endogenous levels was observed. Overall, these results indicate that MPO present within CF sputum has the capacity of catalyzing protein oxidation, chlorination, and nitration, but this occurs primarily in CF sputum proteins rather than in proteins from underlying epithelial cells.


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Fig. 5.   Oxidant-induced protein modification by CF sputum. Confluent HBE1 cells in 100-mm culture dishes were incubated with sol-phase CF sputum (diluted 1:40 in EBSS) in the absence or presence of 100 µM NO2- or 100 µM ABAH, and H2O2 generation (1 µM/min) was initiated by addition of 10 mU/ml glucose oxidase. Protein nitration in cellular proteins (solid bars) and in CF sputum proteins (open bars) was assessed by HPLC. Data are mean values ± SE from 3-4 separate experiments.

Experiments with purified MPO under otherwise identical conditions also demonstrated substantial tyrosine nitration of cellular proteins in the presence of 0.1 or 1.0 mM NO2- (increasing from 0.01 to 0.2 and 1.5 mmol/mol tyrosine, respectively; Fig. 5). However, despite increased protein nitration, the presence of NO2- at pathophysiological levels (10-100 µM) did not significantly affect MPO-dependent cytotoxicity (Fig. 4A), nor did it affect epithelial toxicity by CF sputum and H2O2. High concentrations of NO2- (>500 µM) were found to inhibit epithelial toxicity by MPO-H2O2, perhaps by inhibiting HOCl formation by outcompeting Cl- for oxidation by MPO or by scavenging of HOCl (e.g., Ref. 13), even though the extent of tyrosine nitration was increased. These findings imply that tyrosine nitration is not directly related to changes in cell viability in this model system.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 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 alpha Mbeta 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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