Abstract
In cystic fibrosis (CF), actin and DNA originating from inflammatory cells contribute to the thickness of airway secretions. Actin can bind to DNA-rich fibers and potently inhibit the enzymatic activity of rhDNase. The in vitro effects of the actin-resistant rhDNase variant (A114R) were analyzed and compared with those of the wild-type rhDNase. Frozen and thawed CF airway secretions were incubated for 30 min with different concentrations (0.1, 0.5, 1, 5, or 10 μ g/ml) of either actin-resistant rhDNase or wild-type rhDNase. We observed that both the wild-type and the actin-resistant rhDNase significantly decreased (p < 0.05 and p < 0.001, respectively) the airway secretion viscosity. The decrease in airway secretion viscosity was significant even at low concentrations (0.1 μ g/ml) of the actin-resistant variant. Incubation with the actin-resistant variant resulted in a significant decrease (p < 0.02) of the airway secretion contact angle and cough transport. A significantly higher (p < 0.01) increase in contact angle and cough transport of airway secretions was observed at 10 μ g/ml with the actin-resistant variant as compared with the wild-type rhDNase. The present study had demonstrated that the actin-resistant rhDNase variant (A114R) has an enhanced capacity to improve the physical properties and cough transport of airway secretions from patients with cystic fibrosis.
The mucus that lines the airways provides a renewable and transportable protective barrier that, in normal conditions, eliminates the toxic particles and materials inhaled. One of the main missions ascribed to airway mucus is to protect the epithelial cells from invasion and injury by microorganisms and viruses. In numerous pulmonary diseases, the pathophysiologic changes of airway mucus (volume, rheology, altered ion transport, and water content) result in decreased mucociliary clearance and altered barrier function. This is particularly the case in cystic fibrosis (CF) where these alterations are thought to be caused by mutations in the gene coding the CF transmembrane conductance regulator protein (CFTR). CFTR gene transfer into CF airway cells represents an important goal for CF therapy. However, direct application of gene transfer vectors to the airways may be impeded by the presence of hyperviscous airway secretions, as demonstrated by Stern and colleagues (1) who have observed that the in vitro transfection of cell lines is markedly inhibited in the presence of airway mucus.
In purulent CF respiratory mucus, large amounts of deoxyribonucleic acid (DNA), released by inflammatory and desquamated cells, has long been known to form a complex with mucus glycoproteins responsible for the rheologic properties of respiratory mucus (2, 3). Among the other proteins originating from inflammatory cells, actin in its filamentous form (F-actin) is another polymer that may also contribute to the thickness of CF mucus (4). Recombinant human deoxyribonuclease I (rhDNase) has been demonstrated to degrade DNA to lower molecular weight fragments, thus reducing the viscosity and improving the surface properties of CF sputum (5, 6). The use of rhDNase clinically has demonstrated its efficacy on the pulmonary function and symptoms of patients with CF (7). In addition, Stern and colleagues (1) have demonstrated that the in vitro treatment of mucous-covered cells with rhDNase significantly improve gene transfer. However, because DNase I can also depolymerize F-actin (4), this suggests an alternative mechanism for reducing viscosity or possibly improving gene transfer. This hypothesis is supported by experiments using gelsolin, a protein that severs actin filaments and decreases the viscosity of CF sputum (4), but is strongly disfavored using engineered mutants of rhDNase (8) and biochemical methods showing that gelsolin serves only to relieve the inhibition of DNase I by actin (9).
To potentially improve the efficacy of rhDNase as well as address its mechanism of action, several classes of variants have recently been engineered. These include actin-resistant variants that no longer bind to globular actin (G-actin) (8), a potent inhibitor (K i ∼ 1 nM) of the enzymatic activity of DNase I (10), and hyperactive variants that have increased enzymatic activity and can digest DNA much more efficiently relative to wild-type (11-13). Both the actin-resistant and the hyperactive variants have increased activity relative to wild-type rhDNase in their ability to reduce the airway mucus viscosity in CF sputum (8, 13). But to date, the effect of the rhDNase variants on mucus surface properties and transport properties has not been demonstrated.
In this report, we address various properties of the actin-resistant rhDNase variant (A114R), where alanine at residue 114 has been replaced by an arginine, and compare them with those of wild-type rhDNase. In order to elucidate the specific role of actin on the inhibition of rhDNase activity, we amplified the effect of actin on the inhibition of rhDNase activity by freezing and thawing the CF mucus samples. This procedure, which led to the lysis of cells present in the mucus and to the release of actin within the mucus, mimics the marked increase of DNA and actin in the airway mucus after severe infection. We further compared, versus control, the in vitro dose-dependent effects of both wild-type rhDNase and actin-resistant rhDNase variant on mucus surface and cough transport properties. We also compared head-to-head the effect of the wild-type and the actin-resistant rhDNase. We show that the actin-resistant rhDNase variant (A114R) has an enhanced capacity to improve the physical and transport properties of airway secretions from patients with CF.
Fourteen inpatients, with a well documented CF disease and examined during clinical and pulmonary function follow-up, were included in the study. The pulmonary function of the patients was evaluated by the FVC and the FEV1, which were expressed as a percentage of predicted normal values. The severity of the clinical condition was determined by the Schwachman score (14). We checked that no rhDNase treatment was given in all patients for at least 2 wk prior to airway mucus collection. Mucus was collected by expectoration after physiotherapy maneuvers (percussion, vibration, breathing exercises) for cytobacteriologic study included in the clinical follow-up of the patients. A part of the mucus sample was used for the present in vitro study, which followed international guidelines.
Wild-type rhDNase and actin-resistant rhDNase (A114R), where the alanine at residue 114 has been replaced by an arginine, were expressed transiently in human embryonic kidney cells (ATCC CRL 1573) and expressed and characterized essentially as previously described (8). rhDNase concentrations in cell culture supernatants were quantified by an ELISA technique (8).
In the first set of experiments, mucus samples were collected by expectoration after a 30-min period of physiotherapy maneuvers in seven patients with CF not treated with rhDNase. The mucus samples were immediately sent to the laboratory for analysis. Each sample of mucus was divided into two aliquots. The viscosity of the first aliquot (native aliquot) was analyzed immediately. The second aliquot (frozen aliquot) was frozen at −20° C for 7 d, thawed at room temperature, and the viscosity was measured.
Before and after freezing, 100 μl of airway secretions were added to 900 μl of a 10-fold diluted solution of 2,3-dihydroxy-1,4-dithiolbutane (Digest'Eur; Eurobio, Paris, France), vortexed and incubated for 1 h at 37° C. The total number of cells present in the airway secretion samples was quantified by using a Malassez hemocytometer.
In a second set of experiments, samples of mucus were collected by expectoration after a 30-min period of physiotherapy maneuvers (percussion, vibration, breathing exercises) in 10 patients with CF not treated with rhDNase. The mucus samples were immediately frozen at −20° C until analysis.
After thawing, each mucus sample was divided into 11 aliquots. Each aliquot was incubated with different concentrations (0.1, 0.5, 1, 5, or 10 μg/ml) of either the actin-resistant rhDNase variant or the wild-type rhDNase at 37° C for 30 min. The control used for dose-dependent effects of wild-type and actin-resistant rhDNase was mock-transfected cell culture medium.
The viscous properties of the mucus samples were analyzed by using a controlled stress rheometer (TA Instruments, Guyancourt, France) equipped with a cone-plate geometry. The angle between the cone and the plate was 1 degree, and the sample volume required was 20 μl. The measurements were carried out at 25° C using the creep test technique at a constant stress of 10 Pa (15), and the resultant strain was recorded. When a steady flow was achieved, the slope of the strain versus the time curve was representative of the shear rate applied to the mucus sample. The ratio of shear stress to shear rate was used to calculate the viscosity of the mucus. For each wild-type or actin-resistant rhDNase concentration, one measurement of viscosity was performed.
The surface properties of mucus samples were analyzed by measuring the contact angle of a 20-μl drop of mucus, which was deposited on a glass slide in a small chamber with 100% relative humidity. An image analysis technique was used to measure the angle between the tangent to the mucus-air interface and the horizontal at the contact point of the drop of mucus with the glass slide (16). According to the sample volume, one to three measurements were performed on each sample, and the mean value was calculated.
Experiments were performed using a cough machine developed by King and colleagues (17). A tank (volume, 8 L) was used as a reservoir for pressurized air and was connected through a solenoid valve to a plastic tube simulating the trachea. The floor of this tube was made from a glass slide, on which was deposited the 20-μl drop of mucus gel phase used for contact angle measurement. A cough was simulated by opening the solenoid valve, releasing the pressurized air at a flow rate of 8 L/s through the model trachea. The distance traveled by the mucus under the effect of the airflow was measured and represented as the mucus cough transport (expressed in millimeters). According to the volume of mucus collected, one to three measurements were made for each aliquot, and the mean valued was calculated.
Data in the figures are expressed as mean ± SEM. The paired t test was used to analyze the effect of the freeze/thaw procedure on mucus viscosity and to compare the effect of the wild-type rhDNase with that of the actin-resistant rhDNase at each concentration. The variance analysis test (ANOVA) was used to test the dose-dependent effect of wild-type rhDNase and actin-resistant rhDNase. Fisher's test was used to compare the effect of each rhDNase concentration versus control. Significance was measured at p < 0.05.
The mean age of the 14 patients with CF included in the study (six male and eight female) was 23.7 ± 4.8 yr. The mean Shwachman score was 59.1 ± 9.5. The FVC and the FEV1 were 48.7 ± 12.8 and 32.8 ± 11.3% of the predicted values, respectively.
To increase the amount of free actin and DNA within the mucus samples, aliquots were frozen for 7 d at −20° C and thawed at room temperature. We analyzed the effect of the freeze/thaw procedure on the number of inflammatory cells, the release of actin and DNA, and the viscous properties.
The mean number of cells quantified in seven mucus samples before freezing was 19.9 ± 10.5 × 106 cells/ml. The amount of cells decreased dramatically to 7 ± 3 × 104 cells/ml after the freeze/thaw procedure (p < 0.001). The freeze/thaw procedure applied to the seven mucus samples significantly (p < 0.05) increased the viscosity by 2-fold as compared with the native samples (Figure 1).
Fig. 1. Effect of freezing and thawing on viscosity of seven airway mucus samples. The freeze/thaw procedure significantly (p < 0.05) enhanced the viscosity of airway mucus samples.
[More] [Minimize]The results above demonstrated that the freeze/thaw procedure resulted in an increase in mucus viscosity; this could potentially exacerbate the effect of the actin-resistant rhDNase compared with wild-type rhDNase. Using 10 frozen/thawed mucus samples, we further analyzed the dose-dependent effect of the actin-resistant rhDNase on mucus viscosity, surface properties, and cough transport capacity, compared with the effect of wild-type rhDNase.
The effect of the actin-resistant rhDNase variant on viscosity of airway mucus is shown in Figure 2. We observed that the actin-resistant variant at concentrations varying from 0.1 to 10 μg/ml, significantly decreased (p < 0.001) the airway mucus viscosity (−82.9% to −99.9% of decrease as compared with control). A less significant (p < 0.05) dose-dependent effect was observed on viscosity when the mucus samples were incubated with wild-type rhDNase. It is noteworthy that the effect of the wild-type rhDNase became significant at concentrations higher than 0.5 μg/ml, whereas the actin-resistant had a significant effect at 0.1 μg/ml. Although there was a significant reduction in the viscosity at higher concentrations of both wild-type and actin-resistant variant compared with control, there were no significant differences between them.
Fig. 2. Effect of the actin-resistant rhDNase variant and wild-type rhDNase on viscosity of 10 airway mucus samples. The actin-resistant variant at concentrations varying from 0.1 to 10 μg/ml significantly decreased (***p < 0.001 compared with control without rhDNase) the airway mucus viscosity. The wild-type rhDNase at high concentrations also significantly decreased (*p < 0.05 compared with control without rhDNase) the viscosity of airway mucus. At each rhDNase concentration, the viscosity measured in presence of the wild-type or the actin-resistant variant was not significantly different.
[More] [Minimize]The effect of the actin-resistant rhDNase variant on airway mucus contact angle is shown in Figure 3. The actin-resistant variant significantly decreased (p < 0.05) in a dose-dependent manner the mucus contact angle. Compared with control, the effect of the actin-resistant variant was significant at 1, 5, and 10 μg/ml. No significant effect was observed when the mucus samples were incubated with increasing concentrations of wild-type rhDNase. At 10 μg/ml, the effect of the actin-resistant rhDNase was significantly higher (p < 0.05) as compared with the effect of the wild-type rhDNase.
Fig. 3. Effect of the actin-resistant rhDNase variant and wild-type rhDNase on contact angle of 10 airway mucus samples. No significant effect was observed when the mucus samples were incubated with the wild-type rhDNase. The actin-resistant variant at 1 to 10 μg/ml significantly (*p < 0.05 and **p < 0.01 compared with control without rhDNase) decreased the mucus contact angle. At 10 μg/ml, the effect of the actin-resistant variant was significantly higher (p < 0.05) as compared with the effect of the wild-type rhDNase.
[More] [Minimize]As shown in Figure 4, the actin-resistant rhDNase variant significantly increased (p < 0.05) the mucus cough transport. The increase in cough transport, compared with control, was significant for each actin-resistant rhDNase concentration. At 0.1 to 1 μg/ml, the wild-type rhDNase did not significantly enhance cough transport, but it had a significant effect at high concentrations (5 and 10 μg/ml). It is noteworthy that at 10 μg/ml, the actin-resistant rhDNase significantly increased (p < 0.01) the mucus cough transport as compared with the effect of the wild-type at the same concentration.
Fig. 4. Effect of the actin-resistant rhDNase variant and wild-type rhDNase on cough transport of 10 airway mucus samples. A significant (*p < 0.05) increase in mucus cough transport was observed when the mucus samples were incubated with the actin-resistant variant at 0.1 to 10 μg/ml. For each actin-resistant rhDNase concentration, the increase was significant (*p < 0.05 and **p < 0.01 compared with control without rhDNase). The wild-type rhDNase at low concentration did not significantly alter the mucus cough clearance. At 10 μg/ml, the effect of the actin-resistant variant was significantly higher (p < 0.01) as compared with the effect of the wild-type rhDNase.
[More] [Minimize]In the present work, we have demonstrated that in CF airway secretions, rupture and release of content of inflammatory cells may contribute to the viscous properties of airway secretions. We observed that the decrease in the number of inflammatory cells caused by cell lysis is accompanied by an increase in the mucus viscosity. These results are consistent with the findings reported by Sheils and colleagues (18) who visualized for the first time filamentous actin in all sputum samples examined from patients with mild or severe CF or chronic bronchitis. Interestingly, these investigators also demonstrated that the in vitro polymerization of actin in the presence of DNA produces fibers bundles identical to those seen in airway mucus samples, whereas purified DNA does not form large fibers alone in vitro. The presence of actin in CF airway secretions has previously been demonstrated by Vasconcellos and colleagues (4). Despite the absence of any correlation between actin concentration and airway secretion viscosity, these investigators demonstrated that gelsolin was able to sever noncovalent bonds between monomers within actin filaments, with a parallel decrease in mucus viscosity. Taken together, these data and our present results confirm the important role of polymers released by inflammatory cells on the physical structure of airway mucus.
Although the enzymatic hydrolysis of DNA is widely accepted as the mechanism of action for decreasing the viscosity, an alternative mechanism has been suggested since DNase I can also depolymerize F-actin (4). Furthermore, since G-actin is a potent inhibitor (K i ∼ 1 nM) of the enzymatic activity of DNase I (10), actin could adversely affect the biologic activity of rhDNase. The apparent contradiction between the ability of gelsolin to reduce mucus viscosity and the lack of correlation between actin concentration and mucus viscosity has been addressed by two studies. First, two classes of rhDNase variants have been engineered—actin-resistant variants that catalyze DNA hydrolysis comparable to wild-type, but no longer inhibited by actin, and active site variants that bind actin with affinity comparable to wild-type, but no longer catalyze DNA hydrolysis (8). This protein engineering strategy showed that the reduction of viscosity of DNase I in CF sputum resulted from DNA hydrolysis and not from depolymerization of F-actin. It also showed that G-actin is a significant inhibitor of DNase I in CF sputum, and that actin-resistant variants were 10- to 50-fold more potent than wild-type in CF sputum (8, 13). Second, experiments showing that gelsolin can bind to actin and compete for DNase I binding, thus relieving the inhibition of DNase I by actin, also support the commonly accepted mechanism for rhDNase of DNA degradation rather than actin depolymerization (9). Of course, the mechanism by which rhDNase is efficacious in vivo is still an open question.
It has been recently demonstrated (19) that Pseudomonas aeruginosa, an opportunistic pathogen frequently encountered in CF, is able to resist the bactericidal action of polymorphonuclear neutrophils and to induce their cellular death. This cellular death, occurring mainly through necrosis, liberates large amounts of cellular proteins in the mucus. To mimic cellular necrosis in vitro, we submitted CF mucus samples to a freeze/thaw procedure, which clearly decreased the number of inflammatory cells in the samples, presumably because of cell lysis. In fact, the fluorescent staining of actin and DNA clearly demonstrated that these two proteins are major components of the fiber bundles that form mucus samples (data not shown). Perkins and colleagues (20) have shown that pure DNA, in ionic conditions found in the airways, does not form large fibers. However, the work of Sheils and colleagues (18) clearly demonstrated that polymerization of actin in the presence of DNA produces fiber bundles identical to those found in airway secretions. The interaction of actin and DNA likely plays a key role in the formation of the hyperviscous structure of airway secretions in patients superinfected with CF and would therefore contribute to the formation of a physical barrier that could decrease the efficiency of gene transfer in the airways.
Inasmuch as pretreatment of CF airways with mucolytic agents such as rhDNase may be appropriate before viral or non-viral-mediated gene therapy and may be particularly useful for improving mucus transport, we analyzed the effect of an actin-resistant rhDNase variant on mucus physical properties and transport properties. The present study has demonstrated that the actin-resistant rhDNase variant has an enhanced capacity to decrease the physical properties, particularly the surface properties, of airway mucus from patients with CF as compared with the effect of the wild-type rhDNase. In fact, with regard to actin concentration, it appears that the mean inflammatory cell number of the native mucus samples from the present study was high. This is likely due to a worse clinical status of the patients included in the present study, which could lead to a greater amount of cellular debris in the airway mucus, particularly actin. Galabert and colleagues (21) have previously shown that in patients with CF with superinfection, assessed by an increase in inflammatory cells and bacteria, the viscosity of mucus was higher, compared with nonsuperinfected patients. This increase in mucus viscosity in superinfected patients could be related to the increase of the amount of cellular debris released by the numerous inflammatory cells present in the mucus.
The decrease in mucus viscosity induced by the actin-resistant variant was associated with a decrease in mucus contact angle, reflecting a decrease in mucus surface tension. A decrease in the surface tension of mucus is generally associated with a decrease in the adhesive properties of the mucus and a parallel increase in the transport capacity by ciliary activity and by cough (6). The decrease in mucus surface properties caused by DNA fragmentation by rhDNase could be related to a recovery of surface-active molecules such as phospholipids that might be dissociated from DNA by rhDNase, as previously shown (22). Apart from the mucus gel phase properties, mucus sol phase properties are also largely involved in the mucus-mucosa interaction and might have been improved by the actin-resistant rhDNase variant (22).
An important feature of the present results concerns the effect of the actin-resistant rhDNase variant on the mucus transport by cough. Inasmuch as in patients with pulmonary pathologies, cough clearance represents the major mechanism allowing these patients to clear their tracheobronchial tree, drugs able to improve such a mechanism are important to consider. In a recent study on patients with CF who achieved a 10% or greater improvement in FEV1, Robinson and colleagues (23) did not demonstrate any improvements in either ciliary or cough in vivo clearance in response to a short course of wild-type rhDNase. Our results show that the actin-resistant variant, even at low concentration, renders more efficient the in vitro cough clearance and would therefore be of potential benefit in patients with CF in whom excessively viscous and adherent secretions accumulate. The improvement of cough clearance by rhDNase can be attributed to both a decrease in mucus viscosity and an improvement in mucus surface properties. The better efficiency for the actin-resistant rhDNase to improve the surface and transport properties of airway mucus, as compared with the effect of the wild-type, could be related to a better capacity of the actin-resistant variant to dissociate surface-active phospholipids, which might have been sequestered within the actin-DNA fibrillar network.
In conclusion, the present study demonstrates that the actin-resistant rhDNase variant has an enhanced capacity to improve the physical properties of airway mucus from patients with CF as compared with the effect of the wild-type rhDNase. The improvement of the airway mucus physical properties was accompanied by an improvement in the mucus transport capacity by cough. The mean value of rhDNase concentrations in the airway mucus 15 min after aerosolization of the recommended therapeutic 2.5-mg dose has been reported to be 2.9 μg/ml of mucus (24) and it is, therefore, in the range of the concentrations that have been shown to be efficient in the present study. The result presented herein suggest that the actin-resistant rhDNase variant may be an appropriate mucolytic for CF airway obstruction. The actin-resistant rhDNase variant could be particularly useful in pretreating patients with CF in order to improve the efficiency of gene therapy vectors delivered through aerosols.
The writers thank C. Grosskopf and S. Shak for fruitful discussions, and they are grateful to C. Perrot-Minot and C. Rouget (CF center, CHU Reims) who helped them collect sputum samples during physiotherapy.
Supported by Grant No. 95089 from INSERM/Roche/Genentech.
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