Increased Arginase Activity in Cystic Fibrosis Airways
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《美国呼吸和危急护理医学》
Children's Hospital, University of Duisburg-Essen, Essen
Institute for Pharmacology and Toxicology, University of Bonn, Bonn, Germany
ABSTRACT
Rationale: Airway nitric oxide concentrations are reduced in cystic fibrosis (CF). Arginases compete for L-arginine, the substrate of nitric oxide synthesis.
Objectives: We hypothesized that increased arginase activity may be one factor contributing to nitric oxide deficiency in CF.
Measurements: We therefore studied sputum arginase activity, exhaled nitric oxide, and pulmonary function in patients with cystic fibrosis.
Results: Mean (± SEM) sputum arginase activity was significantly higher in patients admitted for pulmonary exacerbation compared with patients with stable disease (1.032 ± 0.148 vs. 0.370 ± 0.091 U/mg protein, p = 0.004). Fourteen days of intravenous antibiotic treatment resulted in significantly decreased sputum arginase activity in all patients (p = 0.0002). However, arginase activity was still significantly (p = 0.0001) higher in CF sputum after treatment for exacerbation compared with induced sputum from healthy control subjects (0.026 ± 0.006 U/mg protein). Negative correlations were found for sputum arginase activity at admission with FEV1 (r = –0.41, p = 0.01), as well as changes in arginase activity with percent change in FEV1 during antibiotic therapy (r = –0.4, p < 0.01) in CF. Exhaled nitric oxide in CF was positively correlated to FEV1 (r = 0.34, p = 0.007), and in patients admitted for pulmonary exacerbation negatively correlated to sputum arginase activity (r = –0.45, p = 0.03).
Conclusions: These data suggest that increased sputum arginase activity contributes to nitric oxide deficiency in CF lung disease and may be relevant in the pathogenesis of CF airway disease.
Key Words: inflammatory marker nitric oxide pulmonary function
Arginases catalyze the hydrolysis of L-arginine to form L-ornithine and urea. The urea cycle arginase, arginase I (or liver arginase), is cytosolic, while arginase II (or kidney arginase) is located in mitochondria. Arginase activity has been detected in nonhepatic tissues that lack a complete urea cycle, including alveolar macrophages (1–3) and fibroblasts (4). In alveolar macrophages, arginase I appears to be the major contributor to total arginase activity, although both isoenzymes were expressed in this cell type (3). Ornithine, the product of arginase catalytic activity, is the precursor of proline and polyamines, which have multiple effects on connective tissue, smooth muscle, and mucus-producing cells (5–9).
Interest in arginases is growing because of their potential for affecting the availability of L-arginine for nitric oxide synthases (NOSs). For instance, it was shown that arginase can limit the availability of L-arginine for nitric oxide (NO) synthesis in macrophages (10, 11). The potential relevance of competition between arginases and NOS for their common substrate for airway physiology was demonstrated by Meurs and coworkers (12, 13). They showed that limitation of L-arginine bioavailability for NO synthesis from constitutive NOS by arginase contributed to airway hyperreactivity in naive guinea pigs (12). In addition, bronchial hyperresponsiveness could be normalized by pharmacologic inhibition of arginase in a guinea pig model of asthma (13). The relevance of increased arginase activity and reduced L-arginine bioavailability in human asthma has recently been demonstrated (14–17).
Airways disease in cystic fibrosis (CF) is characterized by chronic inflammation, chronic bacterial infection, and recurrent infection-associated pulmonary exacerbations (18–23). These neutrophil-dominated exacerbations are accompanied by significant deterioration in pulmonary function and a rise in inflammatory markers in the airways (24, 25). Of interest, expression of the inducible isoform of NOS, NOS2, is not increased but decreased or even absent in CF airways (26, 27). The mechanisms resulting in NOS2 deficiency in CF are poorly understood, but limited evidence suggests that decreased NOS2 expression is related to CF airway inflammation (28). There is recent evidence suggesting that expression of NOS2 can be downregulated by limitation of extracellular L-arginine concentrations, reduction of cellular arginine uptake capacity, or overexpression of arginase (29–32).
The potential role of arginase for CF airway inflammation and NO synthesis has not been studied. We therefore measured arginase activity in sputum of patients with stable CF and patients with a pulmonary exacerbation before and after intravenous antibiotic treatment. Parts of this work have been published previously in abstract form (33, 34).
METHODS
Study Design
Patients with CF who were seen in the outpatient department or admitted for intravenous antibiotic treatment between June 2003 and September 2004 were eligible for this study if they could spontaneously expectorate sputum. The diagnosis of CF in all participating patients had been confirmed by repeated sweat tests with chloride concentrations exceeding 60 mmol/L and by mutation analysis of the CFTR gene.
Sputum was collected for the measurement of arginase activity at admission and after 14 d of intravenous antibiotic therapy. Sputum from hospital-admitted patients was compared with sputum from patients with clinically stable CF presenting to the CF clinic without signs of acute upper airway infection or pulmonary exacerbation for the last 6 wk. Arginase activity in CF sputum was also compared with that of induced sputum from healthy control subjects, who had to be nonsmokers with no history of asthma or atopy. After pretreatment with a short-acting 2 agonist, 4% saline was administered by nebulizer (Pari Master; Pari GmbH, Starnberg, Germany) for up to 20 min while subjects were encouraged to cough and expectorate sputum into a sterile container.
Written informed consent was obtained from all patients with CF or their parents and all control subjects. The study was approved by the institutional review board of the University of Essen.
Study Population
This study included 66 patients with CF (31 female and 35 male) with a mean (SEM) age of 20.5 (7.2) yr. Mean (SEM) FEV1 was 49.5% (24.2%), and FVC was 66.3% (23.2%) of predicted reference values. Forty patients were homozygous and 13 patients were compound heterozygous for the F508 CFTR mutation (F508 allele frequency, 70.5%). Fifty-four of the patients studied (81.8%) had airway cultures positive for Pseudomonas aeruginosa.
Of the 47 patients with CF admitted to hospital for intravenous antibiotic treatment, 10 received oral steroids as part of their regular treatment (Figure 1). Of the other 37 hospital-admitted patients, 26 were treated for pulmonary exacerbation and 11 received antibiotics routinely every 3 mo. The 19 patients with CF who were seen in the outpatient department of the Children's Hospital in Essen were in clinically stable condition. There were no differences among these three groups in age, sex, or pulmonary function.
CF sputum was compared with induced sputum from 11 healthy control subjects (5 females) who were 25.6 (3.1) yr old.
Sputum Processing
Immediately after expectoration sputum samples were incubated 1:1 in 0.1% Triton X100 containing the protease inhibitors phenylmethylsulfonyl fluoride (1 mM), leupeptin (0.5 μg/ml), pepstatin A (0.7 mg/ml), and ethylenediaminetetraacetic acid (2 mM). Mixed samples were vortexed for homogenization. All sputum lysate samples were stored at –70°C until analyzed.
Arginase Activity Assay
Sputum samples were mixed with dithiothreitol (final concentration of 5 mM), appropriately diluted and analyzed for arginase activity as described previously (4) based on spectrophotometric determination of urea after the addition of L-arginine (3, 35). Arginase activity was expressed as U/mg protein (1 U is the enzyme activity catalyzing the formation of 1 μmol urea/min). Additional details on the specificity and methodology of the arginase assay are provided in an online supplement.
NO Measurement
A chemiluminescence analyzer (NOA 280; Sievers, Boulder, CO) was used to measure fraction of exhaled NO (FENO). Single-breath online measurements for the assessment of lower airway NO were performed at a constant expiratory flow of 50 ml x sec–1 () in accordance with published American Thoracic Society/European Respiratory Society standards (36, 37). The NO analyzer was calibrated before each study with 0 and 185-ppb NO calibration gas (Linde AG, Unterschleissheim, Germany). The mean value of three end-expiratory NO concentrations within a variation of 15% was calculated for each subject.
Statistics
Data were expressed as mean ± SEM. A Kolmogorov-Smirnov test revealed that values for arginase activity were not normally distributed in the groups. Comparisons between groups were therefore made by the Wilcoxon test. Intragroup comparison was done by paired Wilcoxon tests. A p value below 0.05 was considered statistically significant. PC-Statistik version 2.11 (TopSoft, Hannover, Germany) was used for statistical analysis.
RESULTS
High levels of arginase activity were found in all CF sputum samples. The specificity of the arginase activity was confirmed by showing complete inhibition by the specific arginase inhibitor N-hydroxy-nor-L-arginine (nor-NOHA) in a subset of samples (38). Interference of the assay by urease could be excluded (see online supplement). In the hospital-admitted patients, arginase activity before antibiotic therapy was significantly lower in those treated with systemic steroids (n = 10) compared with non–steroid-treated patients (n = 37) (0.321 ± 0.071 vs. 0.853 ± 0.118 U/mg protein, p = 0.025). Therefore, the subsequent analysis was limited to patients with CF not receiving systemic corticosteroid therapy. In non–steroid-treated patients admitted for intravenous antibiotic treatment, sputum arginase activity was significantly higher in patients with exacerbation (n = 26) compared with those admitted for routine intravenous treatment (n = 11; 1.032 ± 0.148 vs. 0.430 ± 0.122 U/mg protein, p = 0.007). Sputum arginase activity from patients with clinically stable CF (0.370 ± 0.091 U/mg protein) did not differ from arginase activity in patients admitted for elective clean-out but was significantly lower than in patients admitted for exacerbation (p < 0.004, Wilcoxon test; Figure 2).
Mean sputum arginase activity decreased significantly in both groups after intravenous antibiotic treatment, but the decline was much more pronounced in patients with pulmonary exacerbations (Table 1 and Figure 2). No statistically significant difference in arginase activity was found between sputum from patients with CF treated for exacerbation and that of patients undergoing elective clean-out after 14 d of intravenous antibiotic therapy. Arginase activity after treatment was also not different from baseline values in patients with clinically stable CF. However, arginase activity in sputum was significantly higher in all CF groups compared with that in induced sputum from control subjects (Figure 2).
We then tested for a possible relationship between sputum arginase activity and pulmonary function in CF. Because sputum was not available from seven patients and pulmonary function tests were not performed by two patients, this analysis was performed for only 57 of the 66 patients in our study. In these patients, a negative correlation was seen between sputum arginase activity and FEV1 (r = –0.38, p = 0.003). This correlation was also seen in the subgroup of 35 patients who were admitted for intravenous antibiotic treatment (r = –0.41, p = 0.014; Figure 3A). FEV1 improved in patients treated for pulmonary exacerbation (37.4 ± 5.2 vs. 44.9 ± 5.7% predicted, p = 0.0004), but not in patients admitted for routine antibiotic treatment (45.1 ± 3.8 vs. 48.9 ± 5.3% predicted, p = 0.25). It is of interest that the change in sputum arginase activity ( arginase) during antibiotic treatment was negatively correlated to FEV1 (r = –0.44, p = 0.009; Figure 3B).
measured in 61 patients either admitted to hospital for intravenous antibiotic treatment or seen in the outpatient department in clinically stable condition was positively correlated to both FVC (r = 0.36, p = 0.005) and FEV1 (r = 0.34, p = 0.007). There were no significant differences in between patients with stable CF, patients with pulmonary exacerbation, and patients admitted for routine antibiotic therapy. In patients admitted for pulmonary exacerbation, there was a significant negative correlation between sputum arginase activity and at admission (r = –0.45, p = 0.03; Figure 4). A similar correlation was not seen in the patients admitted for elective clean-out. Mean in patients with CF did not change significantly during intravenous antibiotic treatment.
DISCUSSION
This is the first study assessing the role of arginase in patients with CF. The finding of high sputum arginase activity during pulmonary exacerbation, which decreased with antibiotic treatment, as well as the negative correlations of arginase activity with exhaled NO and pulmonary function suggest that arginase may be involved in both airway inflammation and NOS deficiency in patients with CF.
Arginase was measured by an assay based on the determination of urea. The specificity of the assay was confirmed in control experiments with selected samples showing that the specific arginase inhibitor nor-NOHA inhibited urea formation in a competitive manner. Presence of urease in the samples would result in an underestimation of arginase activity. Although urease activity could be detected in undiluted sputum samples, urease interference with the arginase assay under the present assay conditions could be excluded because the high arginase activity required a dilution of the samples prior to analysis, which resulted in undetectable levels of urease (see online supplement).
Indirect evidence for increased arginase activity in CF was provided by a recent study assessing the effect of a single dose of oral L-arginine on amino acid concentrations in plasma. The increase in ornithine, a product of arginase activity, was significantly higher in subjects with CF than in control subjects, whereas the increase in FENO was significantly higher in the control subjects (39). Ornithine is the precursor of proline and the polyamines (e.g., putrescine, spermidine, and spermine), which promote collagen production and cell proliferation (40–44). Arginase activity may therefore have direct effects on fibrosis and airway remodeling in CF. It is of interest that polyamine plasma levels have been reported to be increased in CF in an age-dependent manner (45).
Our observations suggest that arginase is a marker of inflammation in CF as we observed the highest activity levels during pulmonary exacerbation, a significant decrease with antibiotic treatment, and elevated activity even in clinically stable patients who are known to have ongoing airway inflammation. This notion is also supported by the fact that arginase activity was lower in patients treated with steroids compared with those untreated, although the number of patients treated with systemic steroids was low. While different cell types have the capability to express arginase, arginase has recently been shown to be constitutively expressed in azurophil granules of human neutrophils (46), the main inflammatory cell in CF airways. High arginase activity in CF sputum may therefore reflect neutrophil airway inflammation in this condition. Interestingly, arginase activity in neutrophils was found not to be inducible by a variety of proinflammatory stimuli (46). Therefore, increased arginase activity during pulmonary exacerbation may simply reflect an increased neutrophilic burden. Alternatively, other cell types from the airways, such as epithelial cells, macrophages, and fibroblasts, may also contribute to total airway arginase.
There is an increasing body of evidence that arginases may contribute to bronchomotor control in asthma by interference with NO synthesis (12, 13, 47, 48). In animal experiments with isolated perfused tracheae from naive guinea pigs, the inhibition of arginase with specific inhibitors caused a concentration- dependent decrease in methacholine-induced airway constriction. This effect of arginase inhibition could be reversed by the NOS-inhibitor L-NAME (12). Effects of an imbalance of this system induced by inflammation was demonstrated in a subsequent study by the same group (13). In a guinea pig model of allergic asthma, incubation with the NOS inhibitor L-NAME in unchallenged control animal tracheae resulted in hyperresponsiveness similar to that observed in allergen-challenged animals. Addition of the arginase inhibitor nor-NOHA normalized the hyperresponsiveness of challenged airways to basal controls. This effect was fully reversible by L-NAME. Arginase activity in the hyperresponsive airways was significantly increased (13). Electrical-field-stimulation–induced relaxation in guinea pig tracheas precontracted with histamine was significantly reduced with the NOS inhibitor N-nitro-L-arginine (L-NNA), whereas the arginase inhibitor nor-NOHA increased electrical-field-stimulation–induced relaxation similarly to L-arginine (47).
Similar experiments in CF animal models have, to our knowledge, not been published. However, relaxation of precontracted tracheae to electrical field stimulation and substance P was reduced in cftr–/– mice compared with wild-type animals, but the relaxation defect in CF airways could be reversed by addition of exogenous NO and L-arginine (49). This study shows that, in CF, the relative absence of NO compromises airway relaxation, and contributes to bronchial obstruction. We observed in patients with CF that FEV1 was negatively correlated with sputum arginase activity, suggesting that patients with more pronounced airway obstruction have higher arginase activity in their airways than patients with better pulmonary function. In addition, arginase activity significantly decreased during antibiotic treatment in patients with CF. Positive correlations were found between arginase activity before antibiotic treatment and improvement in pulmonary function during the 14 d of antibiotic treatment. These findings may suggest that high arginase activity, possibly by substrate competition, contributes to NO deficiency and thereby to airway obstruction in patients with CF.
To date, at least four mechanisms have been proposed to explain low FENO in patients with CF: (1) reduced formation of NO due to a lack of NOS2 expression in airway epithelium (26, 27); (2) conversion of NO to metabolites such as peroxynitrite by, for instance, neutrophil-derived superoxide (50); (3) consumption of NO by denitrifying bacteria (51); and (4) mechanical retention of NO in CF mucus (52, 53). Likely, the relative contribution of distinct mechanisms to low FENO differs between patients and may change with disease activity in an individual patient. The present study suggests that increased arginase activity may also have an effect on NO formation (and NO-mediated processes) in the CF airways. This regulatory effect of arginases on NO formation may not be confined to the simple concept of substrate limitation for NO synthesis, as it has been demonstrated that the expression of NOS2 is inhibited when arginine availability is reduced. This was shown for limitation of extracellular L-arginine concentrations, reduction of cellular arginine uptake capacity, and overexpression of arginase (29–32). In these studies, translation of NOS2 mRNA was inhibited by low concentrations of L-arginine (29, 30). There may be multiple mechanisms by which L-arginine mediates NOS2 expression, such as control of NOS protein stability or induction of NOS2 mRNA, as recently reviewed (54). NOS2 from airway epithelium is currently thought to be the major contributor to exhaled NO as shown in children with asthma in whom the level of NOS2 protein in airway cells obtained by bronchoalveolar lavage correlated with the FENO (55).
High arginase activity is expected to have different pathophysiologic consequences depending on whether NO production via NOS2 is up- or down-regulated. In asthma, in which NOS2 is known to be overexpressed in airway epithelium, increased arginase activity is thought to cause depletion of substrate for constitutive NOSs with L-arginine being predominantly transformed into reactive NO metabolites via NOS2 (13–17). In contrast, NOS2 is downregulated or lacking in CF airway epithelial cells (26, 27) and substrate deficiency for NOSs is expected to further decrease NO production.
Studies in CF airway cells suggest that decreased NOS2 expression in the airways may be detrimental and contribute to the impaired defense against P. aeruginosa in CF (26–28, 56). Of interest, the expression of NOS2 in CF airways was recently shown to correlate with markers of inflammation, suggesting that an increase in inflammation results in a decrease of NOS2 expression (28). This parallels the observation of a positive correlation between FENO and pulmonary function reported by us and others (57–59). In the present study, we observed that arginase activity was negatively correlated to pulmonary function in CF. Taking these observations together, one could speculate that inflammation results in induction of arginase, followed by a decrease of NOS2 expression and consequently in low FENO (and worsening of pulmonary function, as discussed above). The observation that antibiotic treatment for pulmonary function results in an increase of FENO in CF (51, 60) supports this theory. In contrast to these previous studies, we did not observe an increase of FENO with antibiotic treatment. However, changes of FENO may not adequately reflect NO production by airway cells in patients with large amounts of airway secretions as it is the case during an acute exacerbation.
Arginases are not only expressed in human cells but also by bacteria, such as P. aeruginosa and Staphylococcus aureus. A contribution of bacterial arginase to total sputum arginase activity can, therefore, not be excluded. Of interest, it has recently been shown for Helicobacter pylori that spermine, which is synthesized from the arginase product L-ornithine, significantly attenuated NOS2 protein translation and NO production of macrophages in a concentration-dependent manner. This observation has been postulated to be a mechanism by which H. pylori persists in the gastric mucosa (61). Whether similar factors are relevant in CF airways is currently unknown.
In conclusion, our results demonstrate increased arginase activity in CF sputum. Patients with more advanced disease as well as patients with a pulmonary exacerbation have higher arginase activity, suggesting that arginase activity is directly related to the degree of airway inflammation. Increased arginase activity may be of pathophysiologic relevance as it decreases availability of L-arginine, the substrate for NO synthesis, thereby contributing to airway obstruction in patients with CF.
Acknowledgments
The authors thank Ms. Margarita Fuhrmann for excellent technical assistance.
FOOTNOTES
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200502-253OC on September 15, 2005
Conflict of Interest Statement: H.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.R. received royalties of $15,000 in 2004 for speaking at conferences and lecturing at CF centers sponsored by Chiron and received a grant of 40,000 for a study sponsored by Chiron in 2003–2004.
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Institute for Pharmacology and Toxicology, University of Bonn, Bonn, Germany
ABSTRACT
Rationale: Airway nitric oxide concentrations are reduced in cystic fibrosis (CF). Arginases compete for L-arginine, the substrate of nitric oxide synthesis.
Objectives: We hypothesized that increased arginase activity may be one factor contributing to nitric oxide deficiency in CF.
Measurements: We therefore studied sputum arginase activity, exhaled nitric oxide, and pulmonary function in patients with cystic fibrosis.
Results: Mean (± SEM) sputum arginase activity was significantly higher in patients admitted for pulmonary exacerbation compared with patients with stable disease (1.032 ± 0.148 vs. 0.370 ± 0.091 U/mg protein, p = 0.004). Fourteen days of intravenous antibiotic treatment resulted in significantly decreased sputum arginase activity in all patients (p = 0.0002). However, arginase activity was still significantly (p = 0.0001) higher in CF sputum after treatment for exacerbation compared with induced sputum from healthy control subjects (0.026 ± 0.006 U/mg protein). Negative correlations were found for sputum arginase activity at admission with FEV1 (r = –0.41, p = 0.01), as well as changes in arginase activity with percent change in FEV1 during antibiotic therapy (r = –0.4, p < 0.01) in CF. Exhaled nitric oxide in CF was positively correlated to FEV1 (r = 0.34, p = 0.007), and in patients admitted for pulmonary exacerbation negatively correlated to sputum arginase activity (r = –0.45, p = 0.03).
Conclusions: These data suggest that increased sputum arginase activity contributes to nitric oxide deficiency in CF lung disease and may be relevant in the pathogenesis of CF airway disease.
Key Words: inflammatory marker nitric oxide pulmonary function
Arginases catalyze the hydrolysis of L-arginine to form L-ornithine and urea. The urea cycle arginase, arginase I (or liver arginase), is cytosolic, while arginase II (or kidney arginase) is located in mitochondria. Arginase activity has been detected in nonhepatic tissues that lack a complete urea cycle, including alveolar macrophages (1–3) and fibroblasts (4). In alveolar macrophages, arginase I appears to be the major contributor to total arginase activity, although both isoenzymes were expressed in this cell type (3). Ornithine, the product of arginase catalytic activity, is the precursor of proline and polyamines, which have multiple effects on connective tissue, smooth muscle, and mucus-producing cells (5–9).
Interest in arginases is growing because of their potential for affecting the availability of L-arginine for nitric oxide synthases (NOSs). For instance, it was shown that arginase can limit the availability of L-arginine for nitric oxide (NO) synthesis in macrophages (10, 11). The potential relevance of competition between arginases and NOS for their common substrate for airway physiology was demonstrated by Meurs and coworkers (12, 13). They showed that limitation of L-arginine bioavailability for NO synthesis from constitutive NOS by arginase contributed to airway hyperreactivity in naive guinea pigs (12). In addition, bronchial hyperresponsiveness could be normalized by pharmacologic inhibition of arginase in a guinea pig model of asthma (13). The relevance of increased arginase activity and reduced L-arginine bioavailability in human asthma has recently been demonstrated (14–17).
Airways disease in cystic fibrosis (CF) is characterized by chronic inflammation, chronic bacterial infection, and recurrent infection-associated pulmonary exacerbations (18–23). These neutrophil-dominated exacerbations are accompanied by significant deterioration in pulmonary function and a rise in inflammatory markers in the airways (24, 25). Of interest, expression of the inducible isoform of NOS, NOS2, is not increased but decreased or even absent in CF airways (26, 27). The mechanisms resulting in NOS2 deficiency in CF are poorly understood, but limited evidence suggests that decreased NOS2 expression is related to CF airway inflammation (28). There is recent evidence suggesting that expression of NOS2 can be downregulated by limitation of extracellular L-arginine concentrations, reduction of cellular arginine uptake capacity, or overexpression of arginase (29–32).
The potential role of arginase for CF airway inflammation and NO synthesis has not been studied. We therefore measured arginase activity in sputum of patients with stable CF and patients with a pulmonary exacerbation before and after intravenous antibiotic treatment. Parts of this work have been published previously in abstract form (33, 34).
METHODS
Study Design
Patients with CF who were seen in the outpatient department or admitted for intravenous antibiotic treatment between June 2003 and September 2004 were eligible for this study if they could spontaneously expectorate sputum. The diagnosis of CF in all participating patients had been confirmed by repeated sweat tests with chloride concentrations exceeding 60 mmol/L and by mutation analysis of the CFTR gene.
Sputum was collected for the measurement of arginase activity at admission and after 14 d of intravenous antibiotic therapy. Sputum from hospital-admitted patients was compared with sputum from patients with clinically stable CF presenting to the CF clinic without signs of acute upper airway infection or pulmonary exacerbation for the last 6 wk. Arginase activity in CF sputum was also compared with that of induced sputum from healthy control subjects, who had to be nonsmokers with no history of asthma or atopy. After pretreatment with a short-acting 2 agonist, 4% saline was administered by nebulizer (Pari Master; Pari GmbH, Starnberg, Germany) for up to 20 min while subjects were encouraged to cough and expectorate sputum into a sterile container.
Written informed consent was obtained from all patients with CF or their parents and all control subjects. The study was approved by the institutional review board of the University of Essen.
Study Population
This study included 66 patients with CF (31 female and 35 male) with a mean (SEM) age of 20.5 (7.2) yr. Mean (SEM) FEV1 was 49.5% (24.2%), and FVC was 66.3% (23.2%) of predicted reference values. Forty patients were homozygous and 13 patients were compound heterozygous for the F508 CFTR mutation (F508 allele frequency, 70.5%). Fifty-four of the patients studied (81.8%) had airway cultures positive for Pseudomonas aeruginosa.
Of the 47 patients with CF admitted to hospital for intravenous antibiotic treatment, 10 received oral steroids as part of their regular treatment (Figure 1). Of the other 37 hospital-admitted patients, 26 were treated for pulmonary exacerbation and 11 received antibiotics routinely every 3 mo. The 19 patients with CF who were seen in the outpatient department of the Children's Hospital in Essen were in clinically stable condition. There were no differences among these three groups in age, sex, or pulmonary function.
CF sputum was compared with induced sputum from 11 healthy control subjects (5 females) who were 25.6 (3.1) yr old.
Sputum Processing
Immediately after expectoration sputum samples were incubated 1:1 in 0.1% Triton X100 containing the protease inhibitors phenylmethylsulfonyl fluoride (1 mM), leupeptin (0.5 μg/ml), pepstatin A (0.7 mg/ml), and ethylenediaminetetraacetic acid (2 mM). Mixed samples were vortexed for homogenization. All sputum lysate samples were stored at –70°C until analyzed.
Arginase Activity Assay
Sputum samples were mixed with dithiothreitol (final concentration of 5 mM), appropriately diluted and analyzed for arginase activity as described previously (4) based on spectrophotometric determination of urea after the addition of L-arginine (3, 35). Arginase activity was expressed as U/mg protein (1 U is the enzyme activity catalyzing the formation of 1 μmol urea/min). Additional details on the specificity and methodology of the arginase assay are provided in an online supplement.
NO Measurement
A chemiluminescence analyzer (NOA 280; Sievers, Boulder, CO) was used to measure fraction of exhaled NO (FENO). Single-breath online measurements for the assessment of lower airway NO were performed at a constant expiratory flow of 50 ml x sec–1 () in accordance with published American Thoracic Society/European Respiratory Society standards (36, 37). The NO analyzer was calibrated before each study with 0 and 185-ppb NO calibration gas (Linde AG, Unterschleissheim, Germany). The mean value of three end-expiratory NO concentrations within a variation of 15% was calculated for each subject.
Statistics
Data were expressed as mean ± SEM. A Kolmogorov-Smirnov test revealed that values for arginase activity were not normally distributed in the groups. Comparisons between groups were therefore made by the Wilcoxon test. Intragroup comparison was done by paired Wilcoxon tests. A p value below 0.05 was considered statistically significant. PC-Statistik version 2.11 (TopSoft, Hannover, Germany) was used for statistical analysis.
RESULTS
High levels of arginase activity were found in all CF sputum samples. The specificity of the arginase activity was confirmed by showing complete inhibition by the specific arginase inhibitor N-hydroxy-nor-L-arginine (nor-NOHA) in a subset of samples (38). Interference of the assay by urease could be excluded (see online supplement). In the hospital-admitted patients, arginase activity before antibiotic therapy was significantly lower in those treated with systemic steroids (n = 10) compared with non–steroid-treated patients (n = 37) (0.321 ± 0.071 vs. 0.853 ± 0.118 U/mg protein, p = 0.025). Therefore, the subsequent analysis was limited to patients with CF not receiving systemic corticosteroid therapy. In non–steroid-treated patients admitted for intravenous antibiotic treatment, sputum arginase activity was significantly higher in patients with exacerbation (n = 26) compared with those admitted for routine intravenous treatment (n = 11; 1.032 ± 0.148 vs. 0.430 ± 0.122 U/mg protein, p = 0.007). Sputum arginase activity from patients with clinically stable CF (0.370 ± 0.091 U/mg protein) did not differ from arginase activity in patients admitted for elective clean-out but was significantly lower than in patients admitted for exacerbation (p < 0.004, Wilcoxon test; Figure 2).
Mean sputum arginase activity decreased significantly in both groups after intravenous antibiotic treatment, but the decline was much more pronounced in patients with pulmonary exacerbations (Table 1 and Figure 2). No statistically significant difference in arginase activity was found between sputum from patients with CF treated for exacerbation and that of patients undergoing elective clean-out after 14 d of intravenous antibiotic therapy. Arginase activity after treatment was also not different from baseline values in patients with clinically stable CF. However, arginase activity in sputum was significantly higher in all CF groups compared with that in induced sputum from control subjects (Figure 2).
We then tested for a possible relationship between sputum arginase activity and pulmonary function in CF. Because sputum was not available from seven patients and pulmonary function tests were not performed by two patients, this analysis was performed for only 57 of the 66 patients in our study. In these patients, a negative correlation was seen between sputum arginase activity and FEV1 (r = –0.38, p = 0.003). This correlation was also seen in the subgroup of 35 patients who were admitted for intravenous antibiotic treatment (r = –0.41, p = 0.014; Figure 3A). FEV1 improved in patients treated for pulmonary exacerbation (37.4 ± 5.2 vs. 44.9 ± 5.7% predicted, p = 0.0004), but not in patients admitted for routine antibiotic treatment (45.1 ± 3.8 vs. 48.9 ± 5.3% predicted, p = 0.25). It is of interest that the change in sputum arginase activity ( arginase) during antibiotic treatment was negatively correlated to FEV1 (r = –0.44, p = 0.009; Figure 3B).
measured in 61 patients either admitted to hospital for intravenous antibiotic treatment or seen in the outpatient department in clinically stable condition was positively correlated to both FVC (r = 0.36, p = 0.005) and FEV1 (r = 0.34, p = 0.007). There were no significant differences in between patients with stable CF, patients with pulmonary exacerbation, and patients admitted for routine antibiotic therapy. In patients admitted for pulmonary exacerbation, there was a significant negative correlation between sputum arginase activity and at admission (r = –0.45, p = 0.03; Figure 4). A similar correlation was not seen in the patients admitted for elective clean-out. Mean in patients with CF did not change significantly during intravenous antibiotic treatment.
DISCUSSION
This is the first study assessing the role of arginase in patients with CF. The finding of high sputum arginase activity during pulmonary exacerbation, which decreased with antibiotic treatment, as well as the negative correlations of arginase activity with exhaled NO and pulmonary function suggest that arginase may be involved in both airway inflammation and NOS deficiency in patients with CF.
Arginase was measured by an assay based on the determination of urea. The specificity of the assay was confirmed in control experiments with selected samples showing that the specific arginase inhibitor nor-NOHA inhibited urea formation in a competitive manner. Presence of urease in the samples would result in an underestimation of arginase activity. Although urease activity could be detected in undiluted sputum samples, urease interference with the arginase assay under the present assay conditions could be excluded because the high arginase activity required a dilution of the samples prior to analysis, which resulted in undetectable levels of urease (see online supplement).
Indirect evidence for increased arginase activity in CF was provided by a recent study assessing the effect of a single dose of oral L-arginine on amino acid concentrations in plasma. The increase in ornithine, a product of arginase activity, was significantly higher in subjects with CF than in control subjects, whereas the increase in FENO was significantly higher in the control subjects (39). Ornithine is the precursor of proline and the polyamines (e.g., putrescine, spermidine, and spermine), which promote collagen production and cell proliferation (40–44). Arginase activity may therefore have direct effects on fibrosis and airway remodeling in CF. It is of interest that polyamine plasma levels have been reported to be increased in CF in an age-dependent manner (45).
Our observations suggest that arginase is a marker of inflammation in CF as we observed the highest activity levels during pulmonary exacerbation, a significant decrease with antibiotic treatment, and elevated activity even in clinically stable patients who are known to have ongoing airway inflammation. This notion is also supported by the fact that arginase activity was lower in patients treated with steroids compared with those untreated, although the number of patients treated with systemic steroids was low. While different cell types have the capability to express arginase, arginase has recently been shown to be constitutively expressed in azurophil granules of human neutrophils (46), the main inflammatory cell in CF airways. High arginase activity in CF sputum may therefore reflect neutrophil airway inflammation in this condition. Interestingly, arginase activity in neutrophils was found not to be inducible by a variety of proinflammatory stimuli (46). Therefore, increased arginase activity during pulmonary exacerbation may simply reflect an increased neutrophilic burden. Alternatively, other cell types from the airways, such as epithelial cells, macrophages, and fibroblasts, may also contribute to total airway arginase.
There is an increasing body of evidence that arginases may contribute to bronchomotor control in asthma by interference with NO synthesis (12, 13, 47, 48). In animal experiments with isolated perfused tracheae from naive guinea pigs, the inhibition of arginase with specific inhibitors caused a concentration- dependent decrease in methacholine-induced airway constriction. This effect of arginase inhibition could be reversed by the NOS-inhibitor L-NAME (12). Effects of an imbalance of this system induced by inflammation was demonstrated in a subsequent study by the same group (13). In a guinea pig model of allergic asthma, incubation with the NOS inhibitor L-NAME in unchallenged control animal tracheae resulted in hyperresponsiveness similar to that observed in allergen-challenged animals. Addition of the arginase inhibitor nor-NOHA normalized the hyperresponsiveness of challenged airways to basal controls. This effect was fully reversible by L-NAME. Arginase activity in the hyperresponsive airways was significantly increased (13). Electrical-field-stimulation–induced relaxation in guinea pig tracheas precontracted with histamine was significantly reduced with the NOS inhibitor N-nitro-L-arginine (L-NNA), whereas the arginase inhibitor nor-NOHA increased electrical-field-stimulation–induced relaxation similarly to L-arginine (47).
Similar experiments in CF animal models have, to our knowledge, not been published. However, relaxation of precontracted tracheae to electrical field stimulation and substance P was reduced in cftr–/– mice compared with wild-type animals, but the relaxation defect in CF airways could be reversed by addition of exogenous NO and L-arginine (49). This study shows that, in CF, the relative absence of NO compromises airway relaxation, and contributes to bronchial obstruction. We observed in patients with CF that FEV1 was negatively correlated with sputum arginase activity, suggesting that patients with more pronounced airway obstruction have higher arginase activity in their airways than patients with better pulmonary function. In addition, arginase activity significantly decreased during antibiotic treatment in patients with CF. Positive correlations were found between arginase activity before antibiotic treatment and improvement in pulmonary function during the 14 d of antibiotic treatment. These findings may suggest that high arginase activity, possibly by substrate competition, contributes to NO deficiency and thereby to airway obstruction in patients with CF.
To date, at least four mechanisms have been proposed to explain low FENO in patients with CF: (1) reduced formation of NO due to a lack of NOS2 expression in airway epithelium (26, 27); (2) conversion of NO to metabolites such as peroxynitrite by, for instance, neutrophil-derived superoxide (50); (3) consumption of NO by denitrifying bacteria (51); and (4) mechanical retention of NO in CF mucus (52, 53). Likely, the relative contribution of distinct mechanisms to low FENO differs between patients and may change with disease activity in an individual patient. The present study suggests that increased arginase activity may also have an effect on NO formation (and NO-mediated processes) in the CF airways. This regulatory effect of arginases on NO formation may not be confined to the simple concept of substrate limitation for NO synthesis, as it has been demonstrated that the expression of NOS2 is inhibited when arginine availability is reduced. This was shown for limitation of extracellular L-arginine concentrations, reduction of cellular arginine uptake capacity, and overexpression of arginase (29–32). In these studies, translation of NOS2 mRNA was inhibited by low concentrations of L-arginine (29, 30). There may be multiple mechanisms by which L-arginine mediates NOS2 expression, such as control of NOS protein stability or induction of NOS2 mRNA, as recently reviewed (54). NOS2 from airway epithelium is currently thought to be the major contributor to exhaled NO as shown in children with asthma in whom the level of NOS2 protein in airway cells obtained by bronchoalveolar lavage correlated with the FENO (55).
High arginase activity is expected to have different pathophysiologic consequences depending on whether NO production via NOS2 is up- or down-regulated. In asthma, in which NOS2 is known to be overexpressed in airway epithelium, increased arginase activity is thought to cause depletion of substrate for constitutive NOSs with L-arginine being predominantly transformed into reactive NO metabolites via NOS2 (13–17). In contrast, NOS2 is downregulated or lacking in CF airway epithelial cells (26, 27) and substrate deficiency for NOSs is expected to further decrease NO production.
Studies in CF airway cells suggest that decreased NOS2 expression in the airways may be detrimental and contribute to the impaired defense against P. aeruginosa in CF (26–28, 56). Of interest, the expression of NOS2 in CF airways was recently shown to correlate with markers of inflammation, suggesting that an increase in inflammation results in a decrease of NOS2 expression (28). This parallels the observation of a positive correlation between FENO and pulmonary function reported by us and others (57–59). In the present study, we observed that arginase activity was negatively correlated to pulmonary function in CF. Taking these observations together, one could speculate that inflammation results in induction of arginase, followed by a decrease of NOS2 expression and consequently in low FENO (and worsening of pulmonary function, as discussed above). The observation that antibiotic treatment for pulmonary function results in an increase of FENO in CF (51, 60) supports this theory. In contrast to these previous studies, we did not observe an increase of FENO with antibiotic treatment. However, changes of FENO may not adequately reflect NO production by airway cells in patients with large amounts of airway secretions as it is the case during an acute exacerbation.
Arginases are not only expressed in human cells but also by bacteria, such as P. aeruginosa and Staphylococcus aureus. A contribution of bacterial arginase to total sputum arginase activity can, therefore, not be excluded. Of interest, it has recently been shown for Helicobacter pylori that spermine, which is synthesized from the arginase product L-ornithine, significantly attenuated NOS2 protein translation and NO production of macrophages in a concentration-dependent manner. This observation has been postulated to be a mechanism by which H. pylori persists in the gastric mucosa (61). Whether similar factors are relevant in CF airways is currently unknown.
In conclusion, our results demonstrate increased arginase activity in CF sputum. Patients with more advanced disease as well as patients with a pulmonary exacerbation have higher arginase activity, suggesting that arginase activity is directly related to the degree of airway inflammation. Increased arginase activity may be of pathophysiologic relevance as it decreases availability of L-arginine, the substrate for NO synthesis, thereby contributing to airway obstruction in patients with CF.
Acknowledgments
The authors thank Ms. Margarita Fuhrmann for excellent technical assistance.
FOOTNOTES
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200502-253OC on September 15, 2005
Conflict of Interest Statement: H.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.R. received royalties of $15,000 in 2004 for speaking at conferences and lecturing at CF centers sponsored by Chiron and received a grant of 40,000 for a study sponsored by Chiron in 2003–2004.
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