ecf-oct-prolastin
Prolastin aerosol therapy and sputum taurine in cystic fibrosis
Abstract
Background. Neutrophil elastase in the cystic fibrosis airways inhibits opsonophagocytosis and induces the expression of interleukin-8, a neutrophil chemoattractant. Prolastin is a therapeutic preparation of alpha-1 proteinase inhibitor (α^sub 1^-PI), a neutrophil elastase inhibitor. The objective of this study was to determine the effects of Prolastin aerosol therapy on airway inflammation in cystic fibrosis.
Methods. The primary endpoint of this study was sputum taurine, an amino-acid present in high concentrations in neutrophils. Sputum taurine correlates with respiratory exacerbations of cystic fibrosis. Seventeen patients with cystic fibrosis were each assigned to three sequential 10-day periods including first, aerosol therapy of 5 ml saline solution bid; second, aerosol therapy of 250 mg Prolastin bid; third, no aerosol therapy. On days 8, 9 and 10 of each period, early morning sputum was collected for the quantification of α^sub 1^-PI, neutrophil elastase activity, IL-8 and taurine.
Results. During Prolastin therapy, a 3-fold increase in sputum α^sub 1^-PI was observed (P = 0.002). Baseline values of sputum α^sub 1^-PI correlated with the values obtained after Prolastin aerosol (R = 0.77, P < 0.01). Sputum neutrophil elastase activity remained unchanged but taurine decreased after Prolastin therapy (during therapy P = 0.052, after therapy P = 0.026). Prolastin aerosol therapy had no adverse effect on pulmonary function.
Conclusions. Aerosol therapy with Prolastin in patients with cystic fibrosis leads to a progressive decrease in sputum taurine. This suggests that even in the absence of sustained elastase inhibition, Prolastin aerosol therapy may have a beneficial effect on airway inflammation in patients with cystic fibrosis.
Chronic lung infection with Pseudomonas aeruginosa is associated with a worsening prognosis in patients with cystic fibrosis (CF).1 In young children it is possible to suppress airway P. aerugtnosa by intense aerosol antibiotic therapy.2 In addition, standards of care for CF include chronic antibiotic aerosol therapy and use of intravenous wide-spectrum antibiotics to suppress P. aeruginosa growth.3,4 While these interventions are associated with clinical improvement, they also carry the risk of increasing the selection pressure for the emergence of antibiotic resistant bacteria. In order to decrease the emergence of multi-resistant bacteria, therapeutic strategies other than antibiotics should be considered. One of these approaches is to improve host defences.
Patients with CF have large numbers of neutrophils in their airways.5 The neutrophils present in the CF airways are unable to ingest and kill bacteria, due to a defect in phagocytic killing related to an abundance of free clastase activity in the extracellular space. Neutrophil clastase in the extracellular space has been shown to cleave C3bi from opsonized P. aeruginosa and CRI from neutrophils, creating an "opsonin-receptor mismatch".6,7 Neutrophil elastase also induces expression of the neutrophil chemoattractant IL-8 and cleaves the phosphatidyl serine receptor on mononuclear phagocytes, further increasing the airway neutrophil and elastase burdens.
One proposed approach to correct this elastasedependent host defence defect is aerosol therapy with α^sub 1^-proteinase inhibitor (α^sub 1^-PI).8,9 α^sub 1^-Proteinase inhibitor aerosol therapy has been shown to be safe and effective at suppressing neutrophil elastase activity in the CF lower respiratory tract as sampled by bronchoalveolar lavage.9,10 Bronchoalveolar lavage (BAL) primarily reflects the alveolar compartment. However, CF is primarily an airway not an alveolar respiratory disease. In addition, the impact of α^sub 1^-PI aerosol therapy on neutrophil density in CF airways has not been determined. The goals of this study were to determine whether aerosol therapy with Ct1-PI (Prolastin) alone is sufficient to suppress neutrophil elastase activity and to decrease neutrophil density in the airway secretions of patients with CF. We have previously shown that sputum taurine, a non-protein amino-acid derived mainly from neutrophils, correlates with the response to anti-P. neruginosa therapy in CF patients, and sputum taurine was therefore used as an index of airway inflammation.11
Methods
Patients. The study was approved by the ethics review boards of the Centre Hospitalier Universitaire de Sherbrooke and of the Centre Hospitalier de l'Université de Montréal. All participating patients provided informed consent prior to enrolment. Nineteen patients with CF were enrolled and 17 completed the study, while two were excluded during the trial because of signs of acute respiratory exacerbations. The diagnosis of CF was based on published criteria 12. All patients had stable disease with an FEV^sub 1^ of > 35% predicted. Exclusion criteria included a respiratory exacerbation or a change in antibiotic therapy within two weeks of enrolment. Patients receiving DNase aerosol therapy were also excluded since neutrophil elastase activity increases significantly within one hour of such therapy13. Details of patient characteristics are provided in Table 1.
Study design. Each patient was assigned to three sequential 10-day treatment periods during which they received the following therapies:
period 1, 5 ml normal saline aerosol therapy twice daily, period 2, 5 ml, 50 mg/ml Prolastin twice daily and, period 3, no intervention other than usual therapy.
Table 1. Characteristics of patients upon enrolment into the study.
Table 2. Pulmonary function at the end of each treatment period.
Aerosol delivery was assured by a PARI-LC Plus nebulizer. On days 8, 9 and 10 of each of the three periods, patients were requested to produce sputum samples before their morning aerosol therapy. Sputum was shipped at 4° C to the University of Sherbrooke for α^sub 1^-PI quantification, elastase activity, interleukin-8 (IL-8) and taurine quantification.
Laboratory investigations
Pulmonary function testing. Forced vital capacity manoeuvres were performed upon enrolment and also on day 10 of each of the three periods in all participants. The forced vital capacity and the forced expiratory volume in 1 sec (FEV^sub 1^) were recorded in absolute values and as a percentage of the predicted values. The highest value of FEV^sub 1^ was chosen in each measurement.
Sputum α^sub 1^-PI and elastase activity. Sputum was diluted 1:1 (vol:wt) with sterile phosphate buffered saline and vortexed before being centrifuged at 25,000 x g for 15 minutes. Supernatants were collected for the determination of Ct1-PI using radial immunodiffusion plates (Dade Behring Inc., Newark, DE) and neutrophil elastase activity. Neutrophil elastase activity was determined by recording the increase in absorbance of the specific neutrophil elastase chromogenic substrate methoxy-succinyl-ala-alapro-val-nitro-anilide (Sigma Chemical Co., St. Louis, MO) in a spectrophotometer (DU7 model, Beckman Instruments, Mississauga, Ontario, Canada) as previously described14. Neutrophil elastase activity was plotted against a standard curve prepared from commercial human neutrophil elastase (Elastin Products Limited, St-Louis, Missouri).
Taurine and cytokine assays. Sputum taurine content was determined by hydrolyzing the sputum in an equal volume of 1 N hydrochloric acid and boiling the sample for 15 min. Taurine was determined as previously described.11 Briefly, the supernatant of the hydrolyzed material was passed through an anion (AG1-X8, Bio-Rad Laboratories, Hercules, CA, USA) and a cation (AG50W-X8, Bio-Rad) exchanger. Since taurine is a zwitterion, its neutral charge allowed it to pass through both ion exchangers. This partially purified material was then pre-derivatized with the fluorescent marker O-phthalaldehyde before being separated on a CIS column using high pressure liquid chromatography and a fluorescence detector (Shimadzu, Kyoto, Japan). The concentration of taurine was expressed in µmol/g of sputum. One pmol of taurine was found to represent approximately 10 million neutrophils. The assay for IL-8 was performed on the sputum supernatants prepared as described previously for α^sub 1^-PI and elastase activity measurements. The IL8 ELISA assay kit was obtained from R & D Systems Inc. (Minneapolis, MN). The IL-8 measurements were performed on supernatants of sputum that had been immediately supplemented with an anti-protease and DNase cocktail (4 mM Pefabloc, 100 mM EDTA, 6.5 mg/ml Prolastin, 1 mg/ml DNase) in order to help prevent proteolytic degradation and liquefy the sputum. All samples were shipped at 4° C and then frozen at -80° C until measurements were performed.
Statistics. The data are expressed as the mean ± the standard error of the mean. Comparisons of data between periods were performed using a paired student's t test with a P value < 0.05 considered as significant.
Results
Characteristics of patients. Demographic data are presented in Table 1. The patients included seven women and 12 men, one of which was a cigarette smoker. The mean age of the study patients was 27.7 ±1.5 years. The mean FEV^sub 1^ upon enrolment of patients into the study was 61.9 ± 3.7% predicted.
The pulmonary function as measured by the FEV^sub 1^ did not differ between the saline aerosol and the Prolastin aerosol therapy periods (table 1; saline FEV^sub 1^ % predicted = 60.7 ± 4.2% vs Prolastin FEV^sub 1^ - 60.7 ± 4.5 %, P = 0.90). Although the FEV^sub 1^ values at the end of the 10-day washout period were slightly higher, this was not statistically significant (FEV^sub 1^ - 63.1 ± 4.4%, P = 0.23 vs saline; P = 0.12 vs Prolastin). One patient did not compile the study due to increased cough and sputum production during the Prolastin period and was treated for a respiratory exacerbation, and a second patient developed fever and hemoptysis attributed to an acute respiratory exacerbation during the trial. Both were excluded from the trial.
FIGURE 1. Effect of Prolastin aerosol therapy on sputum alpha 1 protcinase inhibitor. Patients with cystic fibrosis received for the first 10 day period aerosolized saline, for the second 10 day period aerosolized Prolastin (250 mg bid), and for the last 10 day period no aerosol treatment (washout). Sputum samples were collected on days 8, 9 and 10 of each period, and the means of the 3 days are reported. *P - 0.002 saline vs Prolastin; [dagger] p - 0.002 Prolastin vs washout.
FIGURE 2. Effect of Prolastin therapy on sputum human neutrophil elastase activity. Patients received for the first 10 day period aerosolized saline, for the second 10 day period aerosolized Prolastin (250 mg bid), and for the last 10 day period no aerosol treatment (washout). Sputum samples were collected on days 8, 9 and 10 of each period, and neutrophil elastase activity was measured using the specific substrate MeO-Succ ala-ala-pro-val p-nitroanilide. The means of the 3 days are reported.
FIGURE 3. Sputum taurine levels were determined as a surrogate marker for neutrophil density in airway secretions after 10 days of aerosolized saline, 10 days Prolastin and after 10 days without aerosol therapy (washout). One pmol taurine is equivalent to 107 neutrophils. Saline vs Prolastin, P = 0.052; saline vs washout, P = 0.026.
Alphal-proteinasc inhibitor and clastase activity. The α^sub 1^-PI levels were measured in sputum that was collected immediately before an aerosol therapy. The last aerosol therapy preceded die sputum collection by 12 hr. The mean sputum concentration of α^sub 1^-PI at the end of the saline aerosol therapy period was 2.41 ± 0.54 µM and increased to 8.07 ± 1.88 µM at the end of the 10-day Prolastin aerosol therapy period (P = 0.002, fig 1). Patients with the highest sputum α^sub 1^-PI levels during the saline period tended to be the ones who showed the greatest increase in sputum α^sub 1^-PI during the Prolastin therapy period (correlation coefficient R = 0.78 for saline vs Prolastin, P< 0.01). There was no correlation between α^sub 1^-PI and elastase activity in the sputum of patients during the saline and Prolastin aerosol therapy periods. Sputum α^sub 1^-PI levels of the washout period were similar to those of the saline period (α^sub 1^-PI: washout = 1.98 ± 0.36 µM, saline = 2.41 ± 0.54 µM, P= 0.98). Elastase activity was high during the saline period and did not change significantly at the end of the Prolastin aerosol therapy period nor during the washout period (elastase activity: saline = 5.24 ± 0.86 µM, Prolastin = 5.00 ± 1.24 µM, washout = 5.91 ± 1.69 µM, P > 0.6 all comparisons, fig 2).
Taurine neutrophils and IL-8. Sputum taurine was quantified as a surrogate marker of neutrophil density11. Analysis of purified peripheral blood neutrophils allowed us to determine that 10^sup 7^ neutrophils contained 1 µmole taurine. The taurine content of sputum during the saline aerosol therapy period was 1.12 ± 0.20 µM/g which represents approximately 107 neutrophils/g of sputum. Patients for which data were analyzed were stable without clinical evidence of respiratory exacerbations during the trial. A trend towards a decrease in sputum taurine levels was observed during the Prolastin aerosol therapy (fig 3) which reached statistical significance at the end of the washout period (sputum taurine during saline = 1.05 ± 0.20 µmol/g, Prolastin = 0.79 ± 0.12 µmol/g, washout = 0.69 ± 0.04 µg/g: saline vs Prolastin, P = 0.052; saline vs washout, P = 0.026). The sputum IL-8 levels were not significantly different during the Prolastin aerosol therapy period and the washout periods compared to the saline period (IL-8: saline = 304.4 ± 53.4 ng/ml; Prolastin = 257.2 ± 44.5 ng/ml; washout = 282.2 ± 38.6 ng/ml; P > 0.4, all comparisons).
Discussion
The elastase activity of sputum remained high after ten days of Prolastin aerosol therapy at doses sufficient to increase the nadir levels of sputum α^sub 1^-PI to 8.07 ± 1.88 µM, a 3-fold increase. Despite the apparent absence of suppression of sputum neutrophil elastase activity, we observed a decrease in sputum taurine, a surrogate marker for respiratory exacerbations in CF, suggesting a possible effect on airway inflammation. These results are less marked but remain consistent with previous observations in a rat model of chronic P. aeruginosa lung infection in which treatment with aerosolized Prolastin decreased neutrophil density.15 One patient presented signs of a respiratory exacerbation while receiving Prolastin. Although respiratory exacerbations are a common occurrence in this patient population we cannot exclude the possibility that Prolastin may have contributed to the exacerbation. However, the current study demonstrates that Prolastin aerosol therapy in patients with CF does not adversely affect pulmonary function tests as determined by the FEV^sub 1^.
Airway secretions of patients with CF have very high levels of active neutrophil elastase.16-19 Neutrophil elastase has been shown to hydrolyze several proteins that play a key role in opsonic phagocytosis of P. aeruginosa.6,7,20 McElvaney et al. have shown that extracellular neutrophil elastase present in the bronchoalveolar lavage fluid of CF patients inhibits neutrophil opsonophagocytosis ex vivo, and that the capacity of epithelial lining fluid to support neutrophil-mediated killing of P. neruginosa, in vitro can be restored by Prolastin aerosol.10 In addition, these investigators demonstrated that suppression of neutrophil elastase by aerosolized Prolastin is complete when the epithelial lining fluid concentrations reached 8 µmol/l. However, in the current study despite an increase in the nadir levels of airway secretion α^sub 1^-PI to greater than 8.07 µmol/l, we found no evidence of neutrophil elastase inhibition. A likely explanation for these differences lies in the sampling methods. Bronchoalveolar lavage was used in the previous study whereas sputum was collected in the current study. Fluid obtained by bronchoalveolar lavage primarily reflects the alveolar compartment. In contrast, sputum is comprised of secretions generated in the airway compartment. Since CF is an airway rather than an alveolar disease, sputum is likely to demonstrate a more severe inflammatory milieu, thus increasing the likelihood that sputum α^sub 1^-PI can be proteolytically or oxidatively inactivated.
A decrease in sputum taurine cojcentrations was observed despite the persistence of active neutrophil elastase after Prolastin therapy. One possible explanation for this result may be that α^sub 1^-PI is affecting bacterial clearance through mechanisms that are independent of elastase inhibition. This is unlikely since we have previously shown that Prolastin has no direct bactericidal activity against P. aeruginosa.15 It is also possible that immediately after Prolastin therapy the elastase inhibition is sufficient to allow some transient suppression of neutrophil elastase with positive consequences on airway host defences and inflammation. Sampling of sputum 12 hr after Prolastin therapy may have been too late to allow the observation of decreased neutrophil elastase activity, since neutrophil migration and degranulation likely provides a continuous renewal of fresh active elastase in CF airway secretions. Finally it is important to note that the discrepancy between taurine and neutrophil elastase activity in CF sputum has been observed by others.21 Neutrophil elastase has been shown to bind to material in the centrifuged pellet of CF sputum, thus introducing a source of variability in its measure. The CF sputum supernatant neutrophil elastase activity does not correlate closely with FEV^sub 1^, suggesting that it may not be a reliable surrogate marker of CF lung disease.21 In contrast, sputum taurine is increased during respiratory exacerbations and decreases markedly during antiobiotic therapy of CF patients.11 In addition, concentrations of sputum taurine are closely correlated with those of proteinase 3 (PR3), a neutrophil serine proteinase21 that shows a close inverse correlation with FEV^sub 1^, suggesting that sputum PR3 and taurine may be of interest as markers of CF airway inflammation and disease severity. Sputum taurine concentration as a surrogate marker in CF does need to be further validated before one can clearly define its clinical significance.
Airway neutrophilia is currently thought to be harmful to the lung, contributing to airway neutrophil influx, tissue damage and host defence alterations in patients with CF.22 However proper neutrophil function is essential for normal antibacterial host defences, and paradoxically, neutrophil elastase is an essential component of this defence.23-25 One potential concern with neutrophil elastase inhibition in the CF lung is that such therapy could hinder key host defence responses. Cellular elastase is essential for optimal neutrophil-mediated killing of several gram-negative bacteria. In addition, neutrophil elastase can cleave P. neruginosn flagellin protein and thus cause a decrease in the inflammatory response of epithelial cells to this bacteria in vitro.26 These studies raise the possibility that α^sub 1^-PI could actually increase lung infection and inflammation. However, the current study clearly indicates that Prolastin aerosol therapy does not increase airway inflammation, but rather shows a tendency to decrease the airway neutrophil burden in CF patients.
An intriguing finding of this study was the tight correlation between sputum α^sub 1^-PI levels before and after Prolastin aerosol therapy. These results suggest that inter-individual variation exists in the rate of clearance of α^sub 1^-PI and that it may be possible to predict from baseline sputum α^sub 1^-PI levels which patients are likely to need higher doses of α^sub 1^-PI aerosol.
In order to improve the efficacy of neutrophil elastase inhibition, several options may be considered. First, an increase in the dose and/or in the frequency of α^sub 1^-PI aerosol therapy may be a simple solution, but is not necessarily practical. Second, simultaneous therapies aimed at reducing the burden of airway mucus using mucolytic agents such as Pulmozyme, and physiotherapy which were not used in this study, may help improve the efficacy of Prolastin aerosol. Third, strategies aimed at modifying α^sub 1^-PI to prolong its biological half-life or to resist oxidative inactivation may be helpful.27,28 Finally, other neutrophil elastase inhibitors such as synthetic molecules, EPI-hNE4 and recombinant monocyte neutrophil elastase inhibitor are in various phases of development and may prove to be of therapeutic interest alone or in combination with other therapies29-32.
In summary, we have demonstrated that Prolastin aerosol therapy in patients with CF is followed by a decrease in sputum taurine, a marker of respiratory exacerbations in CF. The decrease in taurine occurred despite the persistence of active neutrophil elastase in the sputum and suggests that elastase inhibitory therapy in CF may be promising, particularly if neutrophil elastase can be suppressed more effectively. Further studies of longer duration including more patients would seem warranted to determine the longterm effects of Prolastin aerosol therapy not only on inflammation but also on the bacterial burden in the CF airways.
Cantin, André M; Berthiaume, Yves; Cloutier, Diane; Martel, Marc
Clinical & Investigative Medicine
August 2006
