Recombinant human deoxyribonuclease (rhDNase) has been shown to improve lung function and reduce the number of pulmonary exacerbations in patients with cystic fibrosis (CF), but its long-term effect on airway inflammation remains unknown. In this study, we used bronchoalveolar lavage (BAL) to investigate the long-term effect of rhDNase on inflammation in patients with CF having mild lung disease. A total of 105 patients with CF (⩾ 5 years of age) having normal lung function were randomized to receive rhDNase (2.5 mg/day) or no rhDNase. Patients with a normal percentage of neutrophils in BAL fluid at baseline were not randomized and served as the control group. The percentage of neutrophils in the pooled BAL sample was similar in both randomized groups at baseline. A significant increase in neutrophils was observed over the 3-year study period in both untreated patients and control subjects, whereas neutrophils remained unchanged in patients treated with rhDNase. Elastase activities and interleukin-8 concentrations also increased in untreated patients and remained stable in patients on rhDNase. We conclude that in patients with CF, an increase in neutrophilic airway inflammation is found that is positively influenced by rhDNase treatment.
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane regulator gene that encodes a protein that functions as a chloride channel in epithelial membranes. The disease is characterized by the depletion of the periciliary liquid layer of bronchial epithelial cells that impairs mucociliary transport with retention of thick and viscid mucus that is subsequently invaded by bacterial pathogens (1, 2). The persistent bacterial infections lead to a massive neutrophil-dominated host response with high levels of the proinflammatory cytokine interleukin (IL)-8 and the release of neutrophil granule enzymes such as elastase and myeloperoxidase (3–5). With the use of bronchoalveolar lavage (BAL), neutrophil-dominated airway inflammation has been shown to be present very early in the course of the disease, and in stable patients, with few clinical symptoms (6–8). Because of the invasiveness of the technique, most of these studies have been performed cross-sectionally, and there is only limited information available on the natural course of this inflammatory response in patients with CF.
Invading neutrophils disintegrate during the inflammatory response and release large amounts of intracellular DNA that contributes to the viscid airway secretions in patients with CF (9). Recombinant human deoxyribonuclease (rhDNase) has been shown to reduce sputum viscosity, improve pulmonary function, and reduce the number of pulmonary exacerbations in patients with moderate lung disease (10–12). Similar effects have also been demonstrated in patients with mild disease, making rhDNase the only mucolytic with proven efficacy in CF (12, 13). However, studies using spontaneously expectorated sputum have raised concerns that rhDNase may increase airway inflammation by releasing proinflammatory cytokines such as IL-8 that are bound to DNA in airway secretions (14, 15). This may only be a short-lived effect because a subsequent study was unable to demonstrate a reproducible effect of rhDNase on IL-8 concentrations (16). Currently, there are no data available on the long-term effect of rhDNase on lower airway inflammation in CF. Given the positive effects on both airway clearance and frequency of pulmonary exacerbations, rhDNase could also exhibit an antiinflammatory effect in CF airways. To further define the natural evolution of airway inflammation in CF and to assess the effect of rhDNase on this inflammatory process, we have studied lower airway inflammation over a 3-year period using BAL in a cohort of patients with CF having mild lung disease. Some of the results of this study have been reported previously in the form of abstracts (17, 18).
A total of 105 patients (53 females) aged 5 to 37 years (mean age ± SD, 11.8 ± 5.4 years) were recruited at the five participating centers (Berlin, Cologne, Essen, Hanover, and Munich). Only 10 of these patients were older than 15 years. The diagnosis of CF had been confirmed by repeated sweat test with chloride concentrations exceeding 60 mmol/L and/or CF transmembrane regulator mutation analysis. Patients were eligible for this study if they were able to perform lung function tests, had normal lung function defined as an FEV1 greater than 80% predicted, and were clinically stable. Exclusion criteria were (1) the use of antiinflammatory treatment (Ibuprofen, systemic or inhaled corticosteroids, and α1-antitrypsin), (2) a modified Shwachman score not including X-rays that is less than or equal to 55 points, (3) allergic bronchopulmonary aspergillosis, and (4) other severe organ involvement such as advanced hepatic disease. All patients had to be free of acute respiratory tract infections before bronchoscopy for at least 6 weeks. The study was approved by the local ethic committees of all participating centers. Written informed consent of both parents and/or the patients was obtained in all cases.
Flexible fiberoptic bronchoscopy and BAL were performed at baseline, after 18 and 36 months, respectively. Onsite visits by one investigator (F.R.) were performed for the first procedure to ensure that the same approach was used in every center. The bronchoscope was wedged in the lingula or one of its segments in all patients. The same segment/subsegment was lavaged on all three occasions in every patient on whom BAL was performed, as described previously in the report of the baseline data for this study (19, 20). The laboratory personnel performing the BAL fluid analysis were blinded to the patient's treatment. The first aliquot of the recovered BAL fluid was treated separately; all other samples were pooled for analysis. The total cell count was measured by a hemocytometer and the differential cell count of the BAL by cytocentrifugation at the different sites. Bacterial cultures were performed from the first BAL aliquot.
Total IL-8 was measured in duplicate in pooled BAL samples using a commercially available ELISA kit (Pelikine kit; Eurogenetics, Hampton, UK) (20, 21). The sensitivity of the assay is 1 pg/ml, and a standard curve was prepared in the range 1 to 240 pg/ml. Samples were diluted as appropriate (usually 1:50) in phosphate-buffered saline. The intraassay and interassay variations are both less than 10%.
The myeloperoxidase-catalyzed oxidation of guaiacol to tetraguaiacol in the presence of hydrogen peroxide was used to quantify myeloperoxidase activity in pooled BAL samples of patients with CF (22, 23). The reaction was followed at 470 nm (Ultrospec III; Pharmacia Biotech GmbH, Freiburg, Germany). One myeloperoxidase unit was defined as the consumption of 1 μmol of hydrogen peroxide per minute. The detection limit was 0.02 U. Neutrophil elastase activity was investigated photometrically at 410 nm using the peptide MeOSuc-Ala-Ala-Pro-Val-p-nitroanilide (Bachem, Heidelberg, Germany) as the specific chromogenic substrate. One unit was defined as the release of 1 μmol p-nitroanilide/minute/ml using the extinction coefficient ε410 nm = 8,800 M−1 cm−1 (23, 24). The detection limit was 0.2 U. Measurements for both enzymes were performed six times, and results were expressed as mean values of sixfold measurements.
Patients 8 years or younger with a relative neutrophil count greater than 10% and patients older than 8 years with a relative neutrophil count greater than 5% in pooled BAL fluid were randomized to 2.5 mg rhDNase once daily (n = 46) or no rhDNase (n = 39). A higher cutoff level was used for younger children because we found a higher percentage of neutrophils in children younger than 8 years in our previous study on children without lung disease (19). Patients with a lower percentage of neutrophils (n = 20) were not randomized and followed as a control group. Patients were routinely evaluated clinically every 3 months during the 3-year period.
Statistical analysis was done with SPSS release 11.0.1 and SAS release 8.02. The primary outcome variables of this study were the change in the percentage of neutrophils, IL-8, and neutrophil elastase in pooled BAL samples within the 3-year period. Nonparametric repeated-measures analyses of variance were used to take into account the correlated nature of repeated measurements. For comparison of different treatment groups, we performed a two-factorial analysis for each variable, with group and time point as factors (25). For the detection of decreasing or increasing trends over time, statistics for patterned time effects were computed. A p value less than 0.05 was considered statistically significant.
A total of 105 patients underwent the first BAL; 46 were randomized to rhDNase treatment, 39 to no treatment, and 20 patients were not randomized due to a low percentage of neutrophils in BAL fluid (Table 1)
|Age, yr, mean (SD)||11.3 (5.1)||12.2 (4.4)||12.5 (7.5)|
|DF508 homozygous, %||66||63||45|
|DF508 compound heterozygous, %||9||21||35|
|FVC, mean (SD), %||86 (27)||95 (19)||83 (24)|
|FEV1, mean (SD), %||96 (13)||98 (15)||93 (15)|
|MEF25–75 VC, mean (SD), %||92 (30)||90 (32)||74 (21)|
|Total cell counts, median (range) ×106||4.4 (0–126)||5.9 (0–159)||1.8 (0–13)|
|Macrophages, median (range), %||63.5 (8–91)||63 (8–94)*||89 (62–94)|
|Lymphocytes, median (range), %||5.9 (0–38)||4 (0–32)||8.7 (1–39)|
|Neutrophils, median (range), %||29 (6–87)*||29 (7–91)*||1.5 (0.3–7)|
|Eosinophils, median (range), %||0 (0–3.4)||0.3 (0–10.7)||0.1 (0–1)|
|IL-8, ng/ml, median (range)||0.69 (0.15–7)*||0.51 (0.04–3.5)*||0.07 (0.02–0.32)|
|Elastase, U, median (range)||0.018 (0–0.18)||0.02 (0.01–1.81)||0.017 (0–0.02)|
|MPO, U, median (range)||0.05 (0–0.064)||0.052 (0–0.082)||0 (0–0.059)|
Total cell counts of BAL fluid were highly variable and did not change significantly over time in the three groups. The percentage of neutrophils in pooled BAL samples was similar at baseline in both randomized groups and significantly increased over time in both untreated patients and control subjects (p < 0.02) (Figure 1). There was no detectable age effect, but numbers were too small for older patients to perform a meaningful statistical analysis. No change in the percentage of neutrophils was observed in patients treated with rhDNase (Figure 1). The variability in the annual change of the percentage of neutrophils in the two randomized groups is shown in Figure 2 .
In contrast to the findings in pooled BAL samples, the percentage of neutrophils of the first syringe of BAL increased significantly over time in all groups, although the increase was more pronounced in untreated patients (p < 0.01 for patients treated with rhDNase, p < 0.005 for untreated patients for the percentage of neutrophils) (Figure 3). Total IL-8 concentrations increased in untreated patients (p < 0.02) and remained stable in patients treated with rhDNase (p = 0.58) and in control subjects (p = 0.09) (Figure 4) . Similar results were obtained for free elastase activity in BAL fluid that increased in untreated patients with CF (p < 0.007), whereas it remained stable in rhDNase-treated patients and in control subjects (Figure 5) . A similar trend was observed for myeloperoxidase that failed to reach statistical significance (Figure 6) .
Concomitant therapy was not statistically different between the three groups (data not shown). Chest physiotherapy was routinely performed in all patients, approximately 85% of patients received inhaled albuterol in both groups. Macrolides were only allowed for short treatment periods (2–4 weeks). There was no significant difference in the number of antibiotic courses (oral, intravenous, or inhaled) between the two randomized groups.
The results of bacterial cultures from BAL fluid are shown in Table 2
|P. Aeruginosa, %||37||21||15|
|S. aureus, %||48||51||30|
|H. influenzae, %||22||23||10|
|Other bacteria, %||24||21||20|
|No bacteria, %||13||18||50*|
|P. aeruginosa, %||28||21||19|
|S. aureus, %||30||45||19|
|H. influenzae, %||26||18||25|
|Other bacteria, %||10||24||25|
|No bacteria, %||14||18||31|
|P. aeruginosa, %||38||33||17|
|S. aureus, %||29||54||17|
|H. influenzae, %||38||33||25|
|Other bacteria, %||38||25||25|
|No bacteria, %||3||21||42|
BAL was well tolerated in all but one patient who developed fever and dyspnea in the first 24 hours that required hospitalization but was resolved within 48 hours. No severe side effects of the BAL were noted.
This study was not designed to assess the effect of rhDNase treatment on lung function, and no significant differences were observed in the evolution of lung function parameters over the 3-year period. A significant decline in FEV1 was observed in the two randomized groups (p = 0.001 for patients treated with rhDNase, and p = 0.0003 for untreated patients), whereas the control group with a normal percentage of neutrophils in the pooled BAL fluid sample did not show any significant decline over the 3-year period (p = 0.07) (Figure 7). Similar results were obtained when the analysis was limited to patients homozygous for DF508 (data not shown). The median annual rate of decline for the total study population was −2.12% in control subjects, −1.99% in rhDNase-treated patients, and −3.26% in untreated patients. Although not statistically different, the rate of decline was highest in untreated patients. Maximal expiratory flow between 25 and 75% of the FVC VC significantly decreased in both randomized patient groups (p < 0.01), whereas no change was observed in control subjects (p = 0.51) (Figure 8) . In contrast, FVC remained stable both in control subjects (p = 0.79) and in patients treated with rhDNase (p = 0.26), whereas it decreased significantly only in untreated patients (p < 0.008) (Figure 9) .
In this extensive BAL study, we provide evidence that the majority of patients with CF having normal lung function showed neutrophilic airway inflammation that increases over time. In patients with elevated neutrophil counts, treatment with rhDNase did not reverse the neutrophilia but prevented progression of the increase in BAL fluid neutrophils and, therefore, may be efficacious in modulating the inflammatory process. This is the first study demonstrating that rhDNase is not only an effective mucolytic drug but also affects the progression of airway inflammation in CF.
Short-term studies using sputum analysis have found evidence that rhDNase releases proinflammatory cytokines that are bound to DNA and, therefore, may have a negative effect on airway inflammation (14, 15). These findings were not supported by two subsequent studies using spontaneously expectorated sputum in patients with more severe lung disease, in which rhDNase treatment over 1 to 3 months was found to have no effect on airway inflammation (16, 26). In this study, we have been able to show that rhDNase treatment over 3 years not only has no proinflammatory effect but rather prevents the increase in airway inflammation that is observed in untreated patients.
In a preliminary analysis of the initial BAL performed in each patient, we have shown that the majority of patients in our study population with a normal percentage of neutrophils in pooled BAL sample already had an elevated percentage of neutrophils in their first BAL sample (20). This is a more bronchial sample and, therefore, may be more sensitive for early bronchial inflammation in CF. Longitudinally, we have now found an increase in neutrophils over time in the first sample in all groups with no effect of rhDNase treatment. Whether this is of relevance for the effect of treatment remains unclear. The absolute amount of neutrophils and neutrophil products is much lower in the first BAL sample, and even though this may be a more sensitive marker of a bronchial disease, the absolute inflammatory burden of neutrophil degradation products may be more relevant for the damage caused to the peripheral airways and the lung parenchyma. A large study assessing lung function as a primary outcome measure in patients with CF having mild lung disease showed greater improvement in rhDNase-treated patients in mean forced expiratory flow during the middle half of the FVC compared with FEV1, a test that is more sensitive for changes in peripheral airways (13). In addition, studies using sputum that is derived from more central airways have failed to demonstrate any effect of rhDNase on airway inflammation (16, 26). These findings, therefore, may suggest that rhDNase may have a more pronounced effect on peripheral airway inflammation. Long-term follow-up of the patients in our study may give a clue as to which of the markers of BAL fluid and which BAL sample may be more useful to predict the future course of lung disease in patients with CF.
The mechanism of an antiinflammatory effect of rhDNase is unclear. RhDNase has been shown to release cationic enzymes from complexes with DNA (27) and to reduce the concentration and the size of extracellular DNA in sputum (28). Patients treated with rhDNase produced sputum of significantly lower viscosity and an increased ratio of viscosity in proportion to elasticity at high frequency, consistent with increased clearability of sputum by coughing. This is critically dependent on the adhesiveness of mucous, which is determined by its surface tension and is better with a lower tension and consequently a more efficient air-jet mucus interaction (29). RhDNase has no direct effect on the production of proinflammatory cytokines, the chemotaxis of neutrophils, or the release of neutrophil products. Studies of CF airway infection in CF cell lines and in animal models have demonstrated not only an exaggerated response to infectious agents but also defective downregulation that leads to the persistence of the inflammatory process over time (30, 31). This defective downregulation seems to be linked to decreased production of the antiinflammatory cytokine IL-10 (32). An increase in neutrophilic airway inflammation is not limited to bacterial infection and was also demonstrated in children with CF having viral respiratory tract infection (33). As rhDNase has been found to decrease the number of pulmonary exacerbations in CF, the lack of increase in BAL neutrophils in rhDNase-treated patients may be caused by a lower rate of exacerbations in these patients. Alternatively, the improved clearance of mucous may directly clear neutrophils and their degradation products from the lung.
Although it is well known that CF lung disease is characterized by neutrophilic airway inflammation even in patients with mild lung disease, little progress has been made in the development of effective antiinflammatory treatment. In addition, the long-term effect of airway inflammation in CF remains poorly defined. Indirect evidence from trials using ibuprofen or systemic corticosteroids suggests that treatment of airway inflammation has a positive effect on lung function (34, 35). In these studies, both therapeutic approaches did not improve lung function but rather slowed the decline of lung function over time. Cross-sectional data from a study of infants suggest that neutrophilic airway inflammation shows a negative correlation with lung function (36). In our study, patients with a normal percentage of neutrophils in BAL fluid did not show a decline in lung function over a 3-year period, a finding that would favor the view that airway inflammation per se has a negative impact on the subsequent course of lung disease and that the absence of airway inflammation is a positive prognostic factor in CF lung disease. However, this patient group also showed an increase in neutrophilic airway inflammation over time, raising the question whether a threshold exists for airway inflammation to become deleterious for the further course of lung disease.
Even though BAL has the advantage to sample material directly from the lower respiratory tract, its use in longitudinal studies has some limitations. BAL studies in patients with CF have provided evidence for regional heterogeneity of lung disease with differences in both bacterial colonization and the extent of airway inflammation in different regions of the lung (37, 38). We have tried to address this problem by always performing the BAL in the same region in the lung and by selecting the lingula, which belong to the upper lobe and may be more sensitive to detect airway inflammation in patients with mild disease, because CF is characterized by a more prominent involvement of the upper lobes (37). Currently, there is little information on the longitudinal course of variability in regional airway inflammation in CF and therefore we cannot exclude the fact that this may have influenced the results of this study.
In summary, we have shown that rhDNase has a positive impact on airway inflammation in patients with CF. These data support our preliminary findings that rhDNase has a beneficial effect on metalloproteases in BAL fluid (39) and would favor an early use of this drug in patients with CF having mild lung disease and support the evidence provided by the pulmozyme early intervention trial study that demonstrated a positive effect of rhDNase on both lung function and pulmonary exacerbations in patients with CF having mild disease (13). Further follow-up of these patients will show whether this positive effect on airway inflammation will affect the decline in lung function over time.
The authors thank all the collaborators in the clinics and in the laboratories at the different participating centers for their excellent collaboration in this study.
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