© 2002 American Thoracic Society
Effects of Recombinant Human DNase and Hypertonic Saline on Airway Inflammation in Children with Cystic FibrosisDepartment of Respiratory Paediatrics, Royal Brompton Hospital, London; Respiratory Unit, Great Ormond Street Hospital, London; School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth; and Medical Research Council Biostatistics Unit, Institute of Public Health, Cambridge, United Kingdom Correspondence and requests for reprints should be addressed to Dr. Ranjan Suri, MRCPCH, Department of Respiratory Paediatrics, Royal Brompton and Harefield NHS Trust, Sydney Street, London SW3 6NP, UK. E-mail: suriranjan{at}hotmail.com
Recombinant human DNase (rhDNase) is an established treatment in cystic fibrosis (CF), but it may liberate cationic mediators bound to DNA in the airways. An alternative mucolytic therapy is hypertonic saline (HS); however, HS may potentiate neutrophilic inflammation. We compared the effect of rhDNase and HS on cationic proinflammatory mediators in CF sputum. In a randomized, crossover trial, 48 children with CF were allocated consecutively to 12 weeks of once-daily 2.5 mg rhDNase, alternate-day 2.5 mg rhDNase, and twice-daily 7% HS. Sputum levels of total interleukin-8 (IL-8), free IL-8, myeloperoxidase, eosinophil cationic protein, and neutrophil elastase (NE) activity were measured before and after each treatment. The change in mediator levels from baseline with daily rhDNase and HS was not significant; however, with alternate-day rhDNase, there was an increase in free IL-8. When changes in mediator levels with daily rhDNase were compared with alternate-day rhDNase and HS, no significant differences were detected. Only changes in NE activity were associated with changes in lung function. In summary, we were unable to show that rhDNase or HS promote airway inflammation in CF.
Key Words: cystic fibrosis • inflammation • rhDNase • saline
In cystic fibrosis (CF), impaired clearance of abnormally viscoelastic airway secretions promotes infection and inflammation (1). The inflammatory response is characterized by a massive influx of neutrophils into bronchial tissue and their accumulation in the airway epithelium, as well as by aggregates of B and T cells in the bronchial mucosa (2). Neutrophils are recruited in response to high concentrations of interleukin (IL)-8 in the airways (3), and possibly other  -chemokines such as epithelial neutrophil-activating peptide 78 (4).
Within the CF airways, high concentrations of neutrophil elastase (NE), a major effector of tissue damage and impaired host defense in the lungs of patients with CF, have been reported (5). High sputum concentrations of extracellular neutrophil-derived myeloperoxidase (MPO) have also been detected in patients with CF (6, 7). MPO has considerable potential for producing lung damage by transforming hydrogen peroxide into highly reactive oxygen radicals. Recent evidence suggests that MPO contributes to bronchial injury and respiratory failure in CF (8), and it has also been shown to mediate inactivation of the elastase inhibitor The role of eosinophils and their products in CF airway disease remains uncertain. Eosinophils are increasingly believed to be proinflammatory cells in chronic respiratory disorders, with tissue damaging capacities (11). They contain a number of basic proteins, including eosinophil cationic protein (ECP), a highly potent cytotoxic molecule with the capacity to kill mammalian as well as nonmammalian cells such as parasites, bacteria, and viruses. High levels of ECP are found in the serum and sputum of patients with CF (6, 12). As ECP concentrations in sputum have been shown to reach levels above those needed to destroy pulmonary tissue in vitro (13), it has been suggested that activated eosinophils participate in progressive pulmonary damage in CF (6). DNA, derived from the degeneration of neutrophils (14), is a major contributor to the viscosity of airway secretions and is present at high concentrations (3–14 mg/ml) in the sputum of patients with CF (15, 16). The histones that normally provide the "charge shield" for DNA are cleaved by NE (17) and, in the airways, extracellular DNA is an anionic polymer known to bind the cationic proteases, cathepsin G, and NE (18). Perks and colleagues (19) have shown in vitro that DNA binds to IL-8, and prevents it from binding to neutrophil receptors. Bovine DNase in vitro increased the proportion of active IL-8 10-fold, and also increased significantly the IL-8–dependent neutrophil chemotactic activity of the sputum supernatants. They suggested that an electrostatic interaction between DNA and IL-8 limits the inflammatory potential of the latter but that this interaction was weakened by DNA cleavage by DNase. Similarly, ECP and MPO are highly cationic proteins, which may also bind to DNA, forming inactive complexes in the airways. Worlitzsch and colleagues (20) found that when MPO was incubated with negatively charged substances, including DNA, its cytotoxic effect was inhibited in a dose dependent manner. The functional effects of ECP are neutralized by heparin, also an anionic polymer (21). Extracellular DNA may therefore have a protective role in binding to and inhibiting the activity of cationic proteins found in the airways. This has implications for nebulized recombinant human DNase (rhDNase) (Roche Pharamceuticals, Welwyn Garden City, UK), which is a mucolytic treatment widely used in CF management. rhDNase cleaves DNA in CF sputum, decreasing viscosity in vitro (22) and improving airway clearance in vivo (23, 24); however, this may potentially release bound cationic proteins in an active form, which could actually worsen lung damage. These potential effects of rhDNase have not previously been studied in vivo. Nebulized hypertonic saline (HS) represents a potential alternative mucolytic therapy in CF. In short-term studies in CF, HS appears to have a beneficial effect on lung function and mucociliary clearance (25, 26), which are comparable to those of rhDNase. However, there have been no studies assessing the effect of HS on airway inflammation in CF. Previous studies from patients with asthma have demonstrated that repeated doses of nebulized HS induce increased numbers of neutrophils in sputum (27, 28). If HS leads to an influx of neutrophils, and thus to increased NE or MPO in the CF airway, this would potentially promote further lung damage. The aim of the study was to test the hypothesis that treatment with daily 2.5 mg rhDNase in vivo for 12 weeks leads to an increase in free IL-8, MPO, ECP, and NE activity in the sputum of children with CF. The effects of reducing the dose of rhDNase to alternate day and the effects of HS on airway inflammation compared with daily rhDNase were also investigated.
The study was conducted within a crossover trial comparing the effectiveness of daily rhDNase with HS and alternate-day rhDNase in children with CF, which has been reported in greater detail elsewhere (29). Children with CF aged between 5 and 18 years were enrolled from Great Ormond Street Hospital and the Royal Brompton Hospital, London, UK. Inclusion criteria were the ability to perform spirometry and to be either currently on rhDNase therapy or have a FEV1 of less than 70% of predicted, a severity of lung disease at which most CF centers would consider starting rhDNase. The patients recruited were therefore those who were eligible for daily rhDNase in clinical practice and in whom alternate-day rhDNase and HS could be used as alternative therapies. Exclusion criteria were the inability to take the trial medication, known hypersensitivity to rhDNase or HS, isolation of Burkholderia cepacia in the sputum, pregnancy, and breastfeeding. To ensure that patients were enrolled when they were clinically stable, they had to be free of a lower respiratory tract infection, requiring a change in antibiotics, steroids, or bronchodilator treatment, during the 14 days before randomization. A prospective open, randomized, crossover trial was performed. Patients already on rhDNase or HS before the study discontinued the treatment at least 2 weeks before commencing the trial. Two weeks has been shown to be sufficient time for complete washout to occur for both rhDNase and HS (23, 25). Each patient was allocated to receive, in random order, consecutive 12-week treatments of once-daily 2.5 mg rhDNase, alternate-day 2.5 mg rhDNase, and twice-daily 5 ml of 7% HS. There was a 2-week washout period between treatments, and patients were seen at the beginning and end of each treatment period for a study visit. rhDNase and HS were administered using a Durable Sidestream nebulizer and Porta-Neb compressor (Medic-Aid, Bognor Regis, UK). HS was inhaled twice daily immediately before the patient's regular physiotherapy. rhDNase was administered once a day or once every other day, at least 1 hour before physiotherapy. The primary outcome was change in FEV1, and secondary outcomes included FVC, exercise tolerance, quality of life, and the number of pulmonary exacerbations.
Measurement of Inflammatory Mediators The samples were mixed thoroughly and centrifuged at 20,000 x g for 20 minutes. The following inflammatory mediators were measured in the supernatant: total IL-8, free IL-8, MPO, ECP, and NE activity. Total IL-8 was measured using a commercially available enzyme-linked immunosorbent assay kit (Pelikine kit; Eurogenetics, Hampton, UK). Free IL-8 was measured using an in-house enzyme-linked immunosorbent assay as previously described (3). ECP and neutrophil-derived MPO were assayed using commercially available radioimmunoassays (Pharmacia and UpJohn, Uppsala, Sweden). For NE activity measurement, samples were diluted 1:10 in assay buffer (0.3 M TRIS–HCl, containing 1.5 M NaCl, pH 8.0). Sputum samples (10 µl) and NE standards (human leukocyte elastase [Sigma, Poole, UK], 10, 50, 100 milliunits in 10-µl assay buffer) were then placed in the wells of a 96-well microtiter plate, and preincubated for 1 minute at 37°C. Then 90-µl substrate (0.555 mM N-methoxysuccinyl-ala-ala-pro-val-p-nitroanilide [Sigma] in assay buffer) was added, and the plate was incubated for 5 minutes at 37°C. The color change was read as an increase in absorbance at 410 nm using a microtiter plate reader (Dynatech, Billingshurst, UK).
Statistical Methods
Forty-eight children were recruited to the study, eight to each of the six possible treatment orders. Eight children were unable to complete all three treatment periods.
Effectiveness
Effects of the Treatments on Mediator Levels
The effects of the treatments on inflammatory mediators after 12 weeks of therapy are shown in Table 2 . Neither daily rhDNase nor HS resulted in a change in inflammatory mediator levels over baseline. With alternate-day rhDNase there was a significant increase over baseline in free IL-8, but not in the other mediators. However, when changes in mediator levels with daily rhDNase were compared with alternate-day rhDNase and HS, no significant differences were detected.
Association of Changes in Mediator Levels and Lung Function Any association between the change in inflammatory mediator level and change in FEV1 was assessed using data from all the treatment periods. There was a weak negative correlation between change in FEV1 and change in NE activity (r = -0.25, p = 0.03) (Figure 1) . However, change in FEV1 did not correlate with changes in total IL-8 (r = -0.14, p = 0.24), free IL-8 (r = -0.02, p = 0.87), MPO (r = -0.10, p = 0.41), or ECP (r = 0.06, p = 0.61).
Daily rhDNase and HS did not cause a significant change in the concentration of any mediator over baseline. Furthermore, we were unable to demonstrate a difference in effect on mediator levels with daily rhDNase compared with HS or alternate-day rhDNase. Change in FEV1 inversely correlated with change in NE activity but not with any of the other mediators. Trials of rhDNase have indicated that lung function improves in about 30% but deteriorates in one-third to one-half of patients (30, 31); the reasons for which are unclear. In view of the in vitro observations that DNase I increases IL-8–dependent neutrophil chemotactic activity in CF sputa (19), we assessed whether 12 weeks of rhDNase therapy would lead to increased levels of free IL-8 in vivo. Our data do not support this hypothesis. Furthermore changes in free and total IL-8 levels did not correlate with changes in FEV1. The increase in free IL-8 from baseline following alternate-day rhDNase was just significant. However, there were wide CI about the mean indicating that sampling error may have influenced observed results, and we consider this was a chance occurrence. The results of this study are based on a sample of modest size, and consequently some caution must be exercised when drawing conclusions. The sputum samples in our study were taken at least 18 hours after administering the last dose of rhDNase, whereas an in vitro study measured increased free IL-8 levels 4 hours after incubating CF sputa with DNase I (22). Thus, an effect on free IL-8 concentrations may have occurred within the first few hours following administration of rhDNase that was not detected 18 hours later. At this later time point, free IL-8 levels may have returned to normal following expectoration of free IL-8–rich sputum or by rebinding of released IL-8 to intact DNA, or other IL-8–binding molecules such as actin (19), as a result of cell death of newly recruited neutrophils in the airways. Further studies are needed to address this possibility. Hyperosmolar solutions have been reported to stimulate cytokine production by bronchial epithelial cells in vitro (32). Furthermore, a recent in vitro study (33) showed that exposing human CF bronchial gland cells to hypertonic sodium chloride solutions induces an increase of IL-8 release. In our study HS therapy did not increase any of the inflammatory mediators measured, possibly reflecting the rapid dilution of HS in the airways (34) and the short-lived effect of HS on human airway epithelia (35). However, we were unable to confirm the lack of a proinflammatory effect by measuring neutrophil counts in the sputum, as the samples were frozen before analysis. We could also not exclude a transient proinflammatory effect of HS around the time of inhalation. However, the absence of any effect on neutrophil products mitigates the possibility that an important effect has been missed. There was an inverse correlation between change in NE activity and change in FEV1. Sputum levels of NE have previously been shown to correlate with disease severity, as judged by lung function and chest X-ray scores (36). However, there was no functionally significant effect of changes in MPO on FEV1. In a study by Regelmann and colleagues (7), sputum MPO activity, rather than immunogenic protein, correlated with FEV1 in patients with CF. It is conceivable that we found no correlation between MPO and FEV1 because we measured total immunogenic MPO, including both inactive (DNA-bound) and active (unbound) MPO. Increased serum and sputum levels of eosinophil granule proteins, including ECP, have been reported in CF despite normal eosinophil numbers (6, 37, 38). We detected the same high concentrations of ECP as previously reported (39), which are sufficient potentially to damage the airway. However, although previous studies have shown a correlation of FEV1 with serum ECP levels (37) and that serum levels of ECP are representative of pulmonary levels (6), we found no correlation between changes in FEV1 and sputum ECP. Again, total ECP, rather than ECP activity, was measured and this may be one reason why no correlation with FEV1 was found. In summary, we have shown that medium-term treatment with daily rhDNase and HS does not increase airway inflammation. NE activity inversely correlates with changes in lung function and is an important mediator. However, the role of ECP and MPO in CF lung disease remains unclear.
The authors are grateful to the contribution of Marcus Flather, Belinda Lees, and Pauline Dooley from the Clinical Trials and Evaluations Unit, the Royal Brompton Hospital, London, UK, for their assistance with randomization, database design, and management. Supported by the National Health Service Health Technology Assessment Program. Received in original form October 3, 2001; accepted in final form May 8, 2002
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