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* From the Lung Research Group (Drs. Starosta and Griese), Children's Hospital of Ludwig Maximilians University, Munich; Department of Pediatric Pneumology and Allergology (Dr. Rietschel), Childrens’ Hospital University of Cologne, Cologne; Department of Pediatric Pneumology and Immunology (Dr. Paul), Charité, Humboldt-University, Berlin; and Department of Pediatric Pulmonology and Neonatology (Dr. Baumann), Hannover Medical School, Hannover, Germany.
Correspondence to: Matthias Griese, Childrens' Hospital, Ludwig-Maximilians-University, Lindwurmstr 4, D-80337 Munich, Germany; e-mail: matthias.griese{at}med.uni-muenchen.de
Abstract
Chronic bacterial infection and severe, polymorphonuclear neutrophil-dominated endobronchial inflammation are characteristic hallmarks of cystic fibrosis (CF) lung disease. The free radicals generated can be deleterious for structure and function of many proteins. The goal of this study was to investigate the degree of oxidation of pulmonary epithelial lining fluid proteins. BAL fluid (BALF) from 55 children with CF and from 11 patients in a control group were investigated by dot-blot assay for content and by two-dimensional electrophoresis and Western blotting for the pattern of distribution of oxidized proteins. The highest level of oxidative stress, as assessed by the level of protein carbonyls, was found in patients with FEV1 < 80% of predicted or with highly elevated neutrophil counts. Compared to control subjects without lung disease, CF patients with normal lung function and CF patients with a normal neutrophil count in their BALF had significantly higher protein carbonyl levels. The extent of protein oxidation was directly related to the neutrophil granulocyte count and inversely to lung function. Our data support the hypothesis that oxidative damage of pulmonary proteins during chronic and excessive neutrophilic endobronchial inflammation may contribute to the decline of lung function in CF patients.
Key Words: cystic fibrosis • lung disease • protein carbonyls
Convincing evidence suggests that oxidative stress and reactive oxygen species (ROS) play an important role in the etiology and progression of a number of human diseases.12 The normal, noninflamed lung is well adapted to the relatively high ambient oxygen concentration, but the extremely high generation of ROS during inflammation overwhelms the protective mechanisms of the lung, and it becomes a particularly vulnerable target during inflammatory processes. Generation of oxidative species is a principal contributor to inflammatory injury, and failure to control inflammation may lead to continuing inflammation and organ dysfunction progressing to organ failure.3
Cystic fibrosis (CF) is the most frequent hereditary disease in white subjects, leading to early death, mainly from respiratory insufficiency. Mutations of the CF transmembrane regulator lead to progressive pulmonary damage and fibrosis. The sequence of events leading from defects in CF transmembrane regulator function to marked inflammation and tissue destruction is not yet precisely known. Mechanisms include viscous mucous and chronic bacterial infection of the airways. The endobronchial inflammation in CF is characterized by large numbers of polymorphonuclear neutrophils (PMNs). In the airspaces, free radicals generated by these cells can be deleterious for structure and function of many proteins.4 In addition, redox-regulated signal transduction can contribute to the apoptosis of lung cells, which is partly determined by cross-talk between the cellular signaling pathways and the redox state.5 Thus, the prevention of excessive oxidative stress through antioxidant therapies may be useful in the prevention and treatment of CF lung disease.
Increased oxidative stress in CF patients has been estimated by measurement of serum parameters.678 Measurement of sputum samples from CF patients who spontaneously produced sputum due to a significant degree of lung damage demonstrated the formation of myeloperoxidase-derived oxidizing and possibly nitrating species within the respiratory tract.9 Expectorated sputum originates mainly from the large airways and contains large amounts of cellular debris releasing proteolytic enzymes and oxidative species, partially leading to secondary oxidative damage within sputum, prior to expectoration, and thus may not be representative for the assessment of protein oxidation in the lung. A more relevant measure of oxidative stress in the lungs may be derived from assessments of samples containing epithelial lining fluid of the distal airways including the alveoli as obtained by BAL. Such measurements have been reported only in young CF patients.10
The goal of this study was to investigate protein oxidation in CF patients with a wide range of endobronchial inflammation in relation to their lung function and degree of pulmonary inflammation. We hypothesized that the level of protein oxidation may be increased even in the presence of normal pulmonary function tests. Our findings demonstrated a close relation between the degree of protein oxidation, neutrophil granulocyte (PMN) count in BAL fluid (BALF), and lung function of the subjects, suggesting a causal relationship among these variables.
Materials and Methods
Subjects, BAL, and Sample Preparation
BALF, which was available as a cell-free supernatant, was analyzed from 55 patients with CF. These patients had participated in the Bronchoalveolar Lavage for the Evaluation of Antiinflammatory Treatment study and the Glutathione study in the Munich, Cologne, and Berlin study centers.1112 The clinical characteristics of the patients are given in Table 1
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Biochemical Methods
The content of protein carbonyls was assessed as a measure of oxidative stress by the sensitive dot-blot assay as described.15 The lower limit of the detection was 2 pmol of carbonyls per microgram of protein. The assay was calibrated by dilutions of different proportions of oxidized and reduced bovine serum albumin (BSA) [Paesel-Lorei; Hanau, Germany]. BSA was oxidized in vitro by the Fenton reaction and completely reduced by NaBH4. The concentration of protein carbonyls in these BSA mixtures was quantified in a colorimetric assay.16
Two-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed on two-dimensional gels (NuPAGE; Invitrogen; Carlsbad, CA) with modification for protein carbonyl identification.17 Briefly, following sample rehydration and isoelectric focusing on the immobilized pH gradient strips (pH 3 to 10; 7 cm; Immobiline; Amersham Biosciences, Uppsala, Sweden) for 2,000 Volt-hours. Samples were placed in 15-mL test tubes and incubated for 15 min in 2 N HCl with 10 mmol/L dinitrophenylhydrazin (DNPH) [Sigma; Taufkirchen, Germany] at 25°C. After the reaction, the samples were washed with 2 mol/L Tris-Base/30% glycerol (Plusone; Uppsala, Sweden) for 15 min. Molecular weight separation was performed on 4 to 12% Bis-Tris gels (Invitrogen). Subsequent proteins were transferred to the polyvinylidene fluoride membrane (Millipore; Bedford, MA) and analyzed with anti-2,4-dinitrophenyl antibodies (Sigma) for protein carbonyls.
Statistical Analysis
Data are given as median values and range. Two groups were compared by Mann-Whitney U test, and three groups were compared by Kruskall-Wallis test with a Dunn post hoc test. The relation values between the different groups are given as a Spearman rank-sum correlation; p < 0.05 was considered significant.
Results
Oxidation of BALF Proteins
The overall degree of protein oxidation in BALF was significantly higher in CF patients than in healthy subjects (Fig 1
). CF patients with pathologic FEV1 (< 80% of predicted) had considerably higher levels of protein carbonyls (median, 3,033 pmol/mL BALF; 15.6 pmol/µg protein) compared to either CF patients with normal pulmonary function values (FEV1 > 80% of predicted; 612 pmol/mL; 6.9 pmol/µg; p < 0.01) or healthy control subjects (0 pmol/mL, p < 0.001) [Fig 1]. There was an inverse correlation between pulmonary function and the level of protein carbonyls in the BALF of CF patients (Fig 2
). Similarly, CF patients with a high degree of neutrophilic inflammation (> 10% of PMNs in BALF) had exceedingly higher values of protein carbonyls (2046 pmol/mL; 11.5 pmol/µg) than those with a low neutrophil count (< 10%; 53.8 pmol/mL; 0.5 pmol/µg; p < 0.001) and the control group (p < 0.001) [Fig 1]. As expected, protein carbonyls and total cell count in BALF (n = 52; rs = 0.54; p < 0.0001), absolute neutrophil count (n = 46; rs = 0.62; p < 0.0001), and relative neutrophil count (n = 47; rs = 0.6; p < 0.0001; Fig 3
) were positively correlated within the group of CF patients.
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Both the number and volume (optical density times area) of oxidized proteins increased with increasing overall degree of oxidation of a sample (see representative gels in Fig 4 ). However, no clear hierarchic sequence of preferential oxidation of certain protein spots could be identified with the gels from CF patients with different degrees of overall oxidation.
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CF patients had significantly higher level of oxidative stress than control subjects, as assessed by protein oxidation from the content of protein carbonyls in their BALF. The highest levels were found in patients with pathologic pulmonary function or with highly elevated neutrophil counts. Compared to control subjects without lung disease, CF patients with normal lung function, defined as FEV1 > 80% of predicted, also had significantly higher protein carbonyl levels. These data show direct a strong association between increased oxidative stress in the lungs and lung function of these patients with CF. The data support the hypothesis that an abundance of ROS may be a major contributor to the progressive pulmonary damage observed in CF patients. Our findings are consistent with the excessive PMN infiltration in the lungs of CF patients.18 The relevance of the PMN infiltration for the oxidative damage to proteins is suggested by our observations that the level of protein carbonyls strongly correlated with the number of PMNs in BALF, and that the oxidative damage was higher in the groups with a higher neutrophil counts. Several lines of evidence suggest that CF patients have inadequate antioxidant defenses to cope with the elevated oxidative stress that they regularly experience. Patients with CF have an impaired absorption of antioxidant nutrients, resulting in lower levels of antioxidants together with increased oxidative stress caused by chronic pulmonary infections.19 Specifically in the lungs decreased levels of reduced glutathione in the epithelial lining fluid have been linked to an impaired export of glutathione by the epithelial cells due to lack of CF conductance regulator.20 It has been suggested that recurring oxidative lung injury can contribute to the decline in pulmonary function in these patients.2122 In our findings, there was also a weak positive correlation between protein carbonyls and the age of the CF patients (n = 51; rs = 0.32; p = 0.02) but not between age and pulmonary function (n = 51; rs = – 0.22; p = 0.12). This may reflect a decreasing antioxidative capacity with age or declining activity of the proteasome.2324 Previously it was demonstrated that the formation of myeloperoxidase-derived oxidizing and possibly nitrating species within the respiratory tract of subjects with CF may contribute to bronchial injury and respiratory failure.9 Nevertheless, this study9 performed on the population of children with CF revealed no correlation between myeloperoxidase activity, neutrophil numbers, and protein carbonylation. Our data directly support the hypothesis that protein oxidation during chronic and excessive neutrophilic inflammation may contribute to the decline of pulmonary function in CF patients. These processes lead to an increase in local concentrations of free oxygen radicals at the sites of inflammation.2526 This may result in the most effective defense against pathogens. However, if the pathogens cannot be eliminated as in CF, a high concentration of free radicals locally and an attenuation of antiproteolytic activity may result in lung tissue damage.827 It is also known that treatment of infective exacerbations in CF patients results in increased plasma levels of some antioxidant vitamins. However, no immediate changes in plasma protein oxidation are observed, while lipid oxidation is decreased.8 Also, local antioxidative treatment of CF patients with inhaled glutathione for 2 weeks did not change the level of oxidized proteins in the lungs.8 This may be due both to an enormously pronounced lung inflammation in CF lungs, which leads to the continuous generation of free radicals by PMNs and consequently to profound oxidative protein damage and an insufficient protective effect of known antioxidants. Based on the correlation between the level of protein carbonyls, neutrophil granulocyte count, and lung function, the carbonylation state of pulmonary proteins may be used as a marker of disease and help to explore pathophysiology of oxidative stress.
The pattern of the proteins that were carbonylated in the lavages was displayed by two-dimensional electrophoresis and Western blotting. The distribution of oxidized proteins on two-dimensional gels was different in the groups of patients with different carbonyl levels. CF patients with low carbonyl content had fewer carbonylated protein spots on their gels. Furthermore, the overall degree of carbonylation of individual, corresponding proteins was obviously less than that in patients with high carbonyl content. Interestingly, in addition to a broad range of serum proteins present in BALF, ie, albumine, Igs, and 
1-antitrypsin, lung-specific proteins such as SP-A were heavily oxidized in CF patients. This directly showed oxidative modification of a protein essential for certain intra-alveolar functions, ie, the formation of tubular myelin and the resistance of surfactant to inhibition of its biophysical activity.2829 Previously it has been shown that oxidation of SP-A also leads to changes in its function; and together with reported proteolytic damage and diminished levels of this protein in CF, additional oxidative changes may result in heavy impairment of SP-A–dependent immune function within the lung.303132333435 Protein oxidation in the lungs of patients with CF may be assessed as protein carbonyls and may be a useful as marker of disease activity and oxidative lung damage. Because of the high level of oxidative stress present in the CF lung, its reduction is a valid therapeutic option. Whether or not a symptomatic-specific antioxidative therapy may be helpful has not been clarified, but it seems to be meaningful in the treatment of CF lung disease. However, clinical trials with natural and synthetic antioxidants must be performed. Until then, monitoring oxidative changes in CF airway secretions may give further insights into pathophysiology and help to define specific treatments. In summary, our results confirm the presence of increased oxidative stress in the lungs of patients with CF, even in subjects with normal lung function.
Footnotes
Abbreviations: BALF = BAL fluid; BSA = bovine serum albumin; CF = cystic fibrosis; DNPH = dinitrophenylhydrazin; PMN = polymorphonuclear neutrophil; ROS = reactive oxygen species; SP-A = surfactant protein A
This study was supported by a German Academic Exchange Service (DAAD) grant to Dr. Starosta.
Received for publication April 21, 2005. Accepted for publication July 3, 2005.
References
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