Abstract
The aim of this study was to test whether changes in mucus surface properties by rhDNase treatment could be related to an increased recovery of phospholipids. Purulent sputa from 18 patients with cystic fibrosis (CF) were incubated with either rhDNase (4 μ g/ml) or control excipient. The incubation of mucus samples with rhDNase induced a significant increase (p < 0.002) in the sol phase proportion (33.7 ± 24.0%) compared with that obtained with excipient (12.6 ± 12.4%). Phospholipids were recovered in significantly (p < 0.05) greater amounts from both mucus gel and sol phases after incubation with rhDNase. The phosphatidylglycerol content of mucus sol phase was significantly increased by rhDNase (p < 0.03), as well as the mucus gel phase surface properties and transport by ciliary activity and by cough (p < 0.05). The improvement of mucus gel surface properties and transport capacity by ciliary activity were significantly related to the increased recovery of phosphatidylglycerol (r = − 0.74, p < 0.03 and r = 0.94, p < 0.05, respectively). These results suggest that rhDNase is able to increase the free water content and alter the phospholipid profile of mucus, with a related improvement in CF mucus transportability.
Cystic fibrosis (CF) is characterized by a general dysfunction of surface airway epithelial cells and of exocrine glands of the respiratory tract. This dysfunction is secondary to mutations in the gene coding for cystic fibrosis transmembrane conductance regulator (CFTR) protein (1). The ion and water transport dysfunctions (2) associated with persistent infection produce thick and adhesive airway mucus (3). The mucus transport efficiency with respect to ciliary activity and/or the cough is dependent on mucus rheologic and surface properties, both of which are usually abnormal in patients with CF (4, 5). Previous studies have clearly demonstrated that rhDNase is able to significantly alter the rheologic properties of CF respiratory mucus (6, 7). In addition, we have previously shown that the in vitro incubation of airway mucus with rhDNase improves the surface properties of the mucus (8). We hypothesized that the recovery of surface-active molecules such as phospholipids could originate from the dissociation of glycoprotein-DNA macromolecular complexes.
In purulent CF respiratory mucus, large amounts of deoxyribonucleic acid (DNA) are released that form a complex with mucus glycoproteins and are sequestered together with lipids in the gel fraction of the mucus (9). Lipid components of the complexes are extremely hydrophobic and may noncovalently interact with the DNA. The major constituents of the lipid components are phospholipids, some of which are natural constituents of alveolar surfactant such as dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG) (10). These phospholipids are characterized as surface-active agents capable of triggering alterations in the surface properties of respiratory mucus (11). In CF, the high concentration of DNA in purulent airway mucus could react with the hydrophobic phospholipids and induce an impairment of the mucus surface properties, leading to an inability to transport the mucus in a normal way by ciliary activity and/or by coughing.
We have previously hypothesized that the cleavage of DNA by rhDNase could release, in the sol phase, the phospholipids that are contained in the gel phase of the respiratory mucus (8). These released phospholipids could play a role as surface-active molecules and therefore enhance the mucus transport capacity. In the present work, this hypothesis was tested by analyzing the effect of the in vitro incubation of airway mucus with rhDNase on the distribution of the different phospholipid classes in both the sol and the gel phases of the mucus. In addition, we analyzed whether changes in the phospholipid profile induced by rhDNase were related to changes in the rheologic and surface properties of mucus, and to changes in mucus transportability by ciliary activity and by cough.
Eighteen inpatients with CF were included in the study. The disease was well documented, and recent clinical and pulmonary function data were obtained for each patient. The pulmonary function of the patients was evaluated by the FVC and the FEV1 expressed as percentage of predicted normal values. The severity of the clinical condition was determined by the Shwachman score (12). The rhDNase treatment was suppressed in all patients for at least 2 wk prior to airway mucus collection.
Airway mucus was collected by expectoration. In 25 mucus samples, the lipid content was analyzed. Because of inadequate sample volume, the viscoelastic and surface properties were studied in only 11 of the 25 mucus samples in addition to transport properties. After gentle homogenization for 20 min at 4° C, each mucus sample was divided into two aliquots. One aliquot was incubated for 30 min at 37° C with rhDNase (Dornase alpha) at a final concentration of 4 μg/ml of mucus. The second aliquot was incubated with the excipient of rhDNase (NaCl 150 mM + CaCl2 1 mM = control excipient). Each aliquot was thereafter centrifuged at 20,000 g for 20 min at 10° C. After centrifugation, the gel and sol phases of the samples were collected separately, and the lipid and phospholipid contents as well as the physical and transport properties were analyzed.
The extraction of lipids from the mucus samples was done according to the techniques previously described by Galabert and colleagues (10). Briefly, freeze-dried samples were rehydrated, homogenized with chloroform-methanol, and diluted with water. The chloroform extracts containing the lipids were evaporated under nitrogen, dried, and weighed. Individual lipid and phospholipid components were determined in the lipid extracts resolubilized in chloroform. Total phospholipids were estimated by the microdetermination of phosphorus after oxidation with perchloric acid. Individual components of phospholipids were separated by high performance thin layer chromatography (HPTLC). After visualization with dichlorofluorescein, areas containing the different phospholipid components were isolated, oxidized with perchloric acid, and quantitated by the microdetermination of phosphorus (13). Cholesterol was measured enzymatically by adaptation of the procedure outlined by Roschlau and colleagues (14). Glycerides were measured according to Eggstein (15) using saponification with methanolic KOH followed by enzymatic determination of glycerol. Glycolipids were measured by quantitative densitometry according to Svennerholm and colleagues (16). Fatty acids were separated by HPTLC and analyzed by gas chromatography as previously described (10). Results were normalized as the weight of lipid subfraction expressed as a fraction of the dry weight of the lyophilized gel or sol phases of the mucus.
The viscoelastic properties of mucus samples were analyzed on the gel phase of the samples by using a controlled stress rheometer equipped with a cone-plate geometry (17). The angle between the cone and the plate was 1°, and the sample volume required was 20 μl. The measurements were carried out at 25° C using the creep test technique. A constant stress of 100 dynes was applied to the sample, and the resultant strain was recorded versus time. When a steady flow was achieved, the applied stress was suppressed and the recovery angle “γ” of the strain, representative of the mucus elasticity, was measured and expressed as “tgγ”. The slope of the strain-versus-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.
The surface properties of mucus samples were analyzed by measuring the contact angle of a 20-μl drop of gel phase or sol phase 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 (18).
Experiments were performed using a cough machine developed by King and colleagues (19). A tank (volume, 8 L) was used as 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 value was calculated. In a second set of experiments, the cough transport of the gel phase of mucus was measured after depositing 2 μl of sol phase between the glass slide and the 20-μl drop of the gel phase of mucus.
In vitro measurements of mucus transport by ciliary activity were made using the frog palate technique (20). Isolated palates from frog (Rana esculenta) were placed in a Plexiglas chamber at a controlled temperature (25° C) and in 100% relative humidity. The mucociliary transport rate was measured by following the displacement of calibrated aluminum discs (600 μm in diameter) through a stereomicroscope. After 24 h, when the endogenous secretion of mucus was exhausted, a drop of mucus (1 μl) taken from the palate of a recently killed frog was placed on the depleted palate and its transport velocity was measured. The transport velocities of the CF respiratory mucus aliquots were measured in the same manner, and the results were expressed as a relative transport rate corresponding to the ratio of CF respiratory mucus transport rate to the control frog mucus transport rate. Three measurements were made for each mucus aliquot.
Data are expressed as means ± standard deviations. Wilcoxon's nonparametric test was used to compare the data obtained after incubation of the mucus samples with either the control excipient or the rhDNase. Changes in the values for all parameters were calculated from data measured on samples incubated with rhDNase and with control excipient. Spearman's nonparametric regression test was used to analyze the correlation between the changes in phospholipid content and the changes in physical and transport properties after rhDNase and control excipient treatment. Significance was measured at p < 0.05.
The mean age of the 18 patients included in the study (10 male and eight female) was 23.9 ± 5.8 yr. The mean Shwachman score was 67.3 ± 8.0. The FVC and the FEV1 were 55.3 ± 19.4 and 39.2 ± 5.6% of the predicted values, respectively. In the year prior to mucus sample collection, the patients had undergone an average of 3.3 ± 1.7 pulmonary exacerbations necessitating antibiotic therapy.
As shown in Figure 1, the incubation of the mucus samples with rhDNase induced a significant increase (p = 0.002) in the sol phase volume compared with the sol phase volume collected after incubation with control excipient.
Fig. 1. Proportion of mucus sol phase volume recovered after a 30-min incubation with rhDNase or with control excipient. After incubation with rhDNase or excipient each aliquot was centrifuged at 20,000 g for 20 min at 10° C. After centrifugation, the gel and sol phases of the samples were collected separately. Each bar represents the mean ± SD.
[More] [Minimize]The effect of rhDNase on the lipid content of gel and sol phases of mucus is reported in Table 1. Significantly larger concentrations of lipids (p < 0.002), particularly phospholipids (p < 0.05) and cholesterol (p < 0.02), were recovered from the mucus gel phase after incubation with rhDNase. Among the phospholipid subclasses, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were the major subclasses extracted from the gel phase of the mucus. Furthermore, these two components were extracted in greater amounts from the gel phase after the incubation of the mucus samples with rhDNase (p < 0.01 and p < 0.03, respectively).
| Gel Phase | Sol Phase | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Lipid Content of Mucus (mg/g dry weight) | n | Excipient | rhDNase (4 μg/ml ) | Significance | n | Excipient | rhDNase (4 μg/ml ) | Significance | ||||||||
| Total lipid | 25 | 97.15 ± 17.20 | 104.42 ± 19.28 | p = 0.002 | 16 | 45.24 ± 96.34 | 43.67 ± 81.06 | NS | ||||||||
| Cholesterol | 25 | 13.32 ± 3.16 | 13.94 ± 3.22 | p = 0.02 | 7 | 1.80 ± 1.35 | 1.13 ± 1.00 | NS | ||||||||
| Glycerides | 18 | 7.27 ± 1.64 | 7.59 ± 2.00 | NS | 5 | 0.40 ± 0.21 | 0.64 ± 0.53 | NS | ||||||||
| Glycolipids | 18 | 10.76 ± 4.37 | 12.69 ± 4.26 | NS | 16 | 8.31 ± 19.51 | 8.79 ± 19.96 | NS | ||||||||
| Fatty acids | 18 | 9.61 ± 5.10 | 9.16 ± 5.00 | NS | 0 | |||||||||||
| Phospholipids | 25 | 32.32 ± 10.46 | 35.77 ± 9.62 | p = 0.05 | 9 | 11.55 ± 8.71 | 12.51 ± 8.85 | p = 0.02 | ||||||||
| Lysophosphatidylcholine | 25 | 0.50 ± 0.70 | 0.52 ± 0.67 | NS | 9 | 0.03 ± 0.05 | 0.02 ± 0.03 | NS | ||||||||
| Lysophosphatidylethanolamine | 25 | 0.59 ± 0.701 | 0.59 ± 0.63 | NS | 9 | 0.02 ± 0.04 | 0.02 ± 0.04 | NS | ||||||||
| Phosphatidic acid | 25 | 0.17 ± 0.19 | 0.14 ± 0.24 | NS | 9 | 0.83 ± 2.43 | 0.28 ± 0.79 | NS | ||||||||
| Phosphatidylcholine | 25 | 12.81 ± 4.04 | 14.54 ± 4.39 | p = 0.03 | 9 | 5.20 ± 11.89 | 5.19 ± 11.03 | NS | ||||||||
| Phosphatidylethanolamine | 25 | 8.58 ± 3.07 | 10.19 ± 3.52 | p = 0.01 | 9 | 3.09 ± 7.12 | 3.01 ± 6.28 | NS | ||||||||
| Phosphatidylglycerol | 25 | 0.36 ± 0.28 | 0.34 ± 0.21 | NS | 9 | 0.02 ± 0.04 | 0.35 ± 0.78 | p = 0.04 | ||||||||
| Phosphatidylserine + phosphatidylinositol | 25 | 4.78 ± 1.89 | 5.11 ± 1.57 | NS | 9 | 0.40 ± 0.29 | 2.06 ± 4.29 | p = 0.02 | ||||||||
| Sphingomyelin | 25 | 2.73 ± 1.02 | 2.94 ± 1.04 | NS | 9 | 1.09 ± 2.33 | 0.95 ± 1.72 | NS | ||||||||
Incubation of the mucus samples with rhDNase also significantly enriched the total phospholipid concentration in the mucus sol phase (p < 0.02). Among the phospholipid subclasses, phosphatidylserine plus phosphatidylinositol (PS + PI) and phosphatidylglycerol (PG) were significantly increased by incubation of the mucus samples with rhDNase compared with that measured with the control excipient (p < 0.04).
The range of values of the lipid content of the sol phases was extremely variable from one sample to another (range, 7 to 400 mg/g dry weight) as compared with the range of the lipid content observed in the gel phase (range, 57 to 148 mg/g dry weight). It is noteworthy that PG was not identified in the gel or the sol phases in about 40% of the mucus samples incubated with the control excipient. However, after the incubation with rhDNase, PG was observed in the sol phase of all mucus samples tested.
In both the gel and sol phases, the phospholipid distribution was relatively similar.
The mean viscosity of the mucus gel phase after incubation with the control excipient was 708.9 ± 1,646 Pa · s. Although not significant, a threefold decrease in the viscosity of the mucus gel phase was observed after incubation of the gel phase with rhDNase (238.4 ± 549 Pa · s). Values measured for the elastic modulus of the mucus gel phase were not significantly different for samples incubated with rhDNase or control excipient (4.2 ± 4.7 and 3.4 ± 3.3, respectively).
Incubation of the mucus with rhDNase induced a significant decrease (p < 0.01) in the gel phase contact angle (32.5° ± 7.3°) compared with the contact angle measured after incubation with control excipient (38.3° ± 7.3°).
The contact angle of the sol phase was significantly lower (p < 0.05) than the contact angle of the gel phase. After incubation of mucus samples with rhDNase, the contact angle measured in the sol phase (27.8° ± 8.4°) was significantly lower (p < 0.05) than that measured after incubation with the control excipient (30.6° ± 10.3°).
The relative mucociliary transport rate of the mucus gel phase after incubation with rhDNase was significantly enhanced (0.82 ± 0.14, p < 0.02) compared with that measured after incubation with control excipient (0.70 ± 0.16). In the same way, incubation of the mucus samples with rhDNase induced a significant increase (p < 0.01) in the cough transport (32.8 ± 10.1 mm) compared with that measured in mucus samples incubated with control excipient (27.5 ± 7.0 mm).
The presence of a small volume of sol phase between the gel phase and the glass support in the simulated cough machine induced a significant enhancement (p < 0.002) of the gel phase cough transport. The enhancement of mucus gel phase cough transport induced by the presence of sol phase collected after incubation with rhDNase was significantly higher (45.2 ± 11.5 mm, p < 0.05) than the enhancement induced by the sol phase collected after incubation of the mucus samples with control excipient (41.9 ± 10.5 mm).
As shown in Figure 2, a significant and negative correlation (r = −0.74, p < 0.03) was observed between changes in phosphatidylglycerol (PG) content and changes in the contact angle measured on the mucus gel phase after incubation with rhDNase and control excipient. That is, the higher the increase in PG content, the larger the decrease in mucus contact angle. The increase in PG content after incubation with rhDNase was also significantly and positively correlated to the increase in gel phase mucociliary transport (r = 0.94, p < 0.05); the higher the increase in PG content, the higher the increase in mucus transportability by ciliary activity (Figure 3). It was noteworthy that the changes in mucociliary transport versus changes in PG content had a positive Y-intercept of 0.075, which could correspond to the change in mucus transportability caused by the nonlipidic effect of rhDNase.
Fig. 2. Relationship between the changes in gel mucus phosphatidylglycerol content and contact angle measured in mucus samples incubated with excipient and in samples incubated with rhDNase at 4 μl/ml. The decrease in contact angle induced by rhDNase was related to the increase in phosphatidylglycerol content.
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Fig. 3. Relationship between changes in gel mucus phosphatidylglycerol content and mucociliary transport measured on mucus samples incubated with control excipient and in samples incubated with rhDNase (4 μI/ml). The increase in mucociliary transport induced by rhDNase was related to the increase in phosphatidylglycerol content.
[More] [Minimize]The present study demonstrated that the incubation of CF respiratory mucus with rhDNase at a final concentration of 4 μg/ ml significantly enhanced the mucus sol phase volume and the extractible phospholipid proportion from the gel phase of the mucus. A significant improvement in the mucociliary and cough transport associated with a significant decrease in surface properties (contact angle) of respiratory mucus were clearly demonstrated. The lack of significant change in mucus viscoelastic properties after rhDNase treatment was probably due to the fact that a reduced quantity of gel phase was recovered after rhDNase treatment. In addition, the residual gel was presumably less hydrated than the control and could represent some rhDNase-resistant fraction of the total gel. In addition, the first homogenization step, which was necessary for lipid extraction, changed the mucus viscoelastic properties before the addition of rhDNase or control fluid (data not shown) and may have reduced the potential effect of rhDNase.
The association of large quantities of lipids with mucus glycoproteins has been related to the infectious characteristics of airway secretions (10, 21). Nadziejko and colleagues (9) have clearly demonstrated that most of the lipids in purulent mucus are bound to mucus glycoproteins rather than to DNA. However, Lethem and colleagues (22) showed that glycoproteins present in CF mucus interact with other macromolecules such as DNA, which is likely to result in gels of increased viscoelasticity and altered surface properties. Electron microscopic observations of CF mucus reported by Msrny and colleagues (23) suggest that DNA readily disperses and resides in close proximity to the glycoprotein matrix before rhDNase treatment, and they identify a loss of DNA in mucus samples treated by rhDNase. The cleavage of DNA by rhDNase alters the glycoprotein-DNA interaction, which is likely to unmask the associated lipids, allowing them to play a surface-active role at the interface between the airway mucosa and mucus.
The finding that lipids were extractible in higher amounts after the incubation of mucus samples with rhDNase may be explained by an easier access of organic solvents to the hydrophobic regions of the gel bulk after cleavage of the macromolecular complexes. It has been demonstrated by Witas and colleagues (24) that the interaction of intestinal mucus glycoproteins with phospholipids involved a hydrophobic pronase-susceptible region.
The major finding of the present study concerns the total phospholipid content, which was significantly increased in both gel and sol phase after the mucus incubation with rhDNase. After treatment of the mucus samples with rhDNase, most of the lipids still remained strongly associated with the gel phase. This was particularly marked for the main amphoteric phospholipids, i.e., PC and PE, which were still linked to the gel phase and significantly increased after the cleavage by rhDNase. It is likely that they remained noncovalently bound by hydrophobic interactions with mucins-DNA complexes or other peptides and proteins of the mucus gel phase. More interestingly, the PG and PS + PI, which are characterized by a net negative charge, were significantly increased in the sol phase after incubation of the mucus samples with rhDNase. Because of the presence of hydroxyl radicals in their polar head, PG and PI are much more polar than the other phospholipids and are thereby more easily solubilized in a micellar form in the sol phase. It is likely that the release of a surface-active component such as PG in the mucus sol phase is much more efficient in modifying the mucus-mucosa interactions than the increased amount of surface-active fraction still linked to the gel phase.
The change in mucus contact angle induced by rhDNase treatment reflects a decrease in mucus surface tension generally associated with a decrease in the adhesive properties of mucus. We have previously shown that an increase in the adhesive properties of CF mucus is related to a decrease in mucociliary transport capacity (25). The interfacial interaction between the mucus and the cilia is critical to the effectiveness of mucociliary transport and can be predicted to be related to the hydrophobic nature of the mucus surface. Phospholipid fractions at the interface of the respiratory mucosa and the mucus gel are important in governing the adhesive properties of mucus. Girod de Bentzmann and colleagues (11) have demonstrated that the work of adhesion of CF mucus was significantly decreased by the addition of a sol phase containing distearoyl phosphatidylglycerol, with a significant parallel improvement in mucus transport by cough and ciliary activity. In the present work, we confirmed that an increased recovery of PG after mucus incubation with rhDNase was closely associated with a decrease in the adhesive properties of mucus. Phosphatidylglycerol appears to be an important phospholipid fraction governing the mucus surface properties and the associated transport properties of mucus. The mucus samples in which PG was not identified before incubation with rhDNase were characterized by abnormally low mucociliary and cough transport capacities, which were, respectively, 41 and 31% lower than the ciliary and cough transport capacities observed for the mucus samples in which PG was identified.
Apart from the mucus gel phase properties, mucus sol phase properties are also largely involved in the mucus- mucosa interaction. A noteworthy point concerns the considerable recovery of the sol phase volume after incubation of the mucus samples with rhDNase. This increase in sol phase volume could reflect an increase in the free water content in the mucus after DNA cleavage by rhDNase. As demonstrated by Daugherty and colleagues (26), the increase in the sol phase volume after incubation with rhDNase is accompanied by a redistribution of the total DNA content from the gel phase to the sol phase. The sol phase recovered after rhDNase treatment is also characterized by enhanced surface and transport properties compared with the sol phase recovered after mucus incubation with control excipient. The enhancement of sol phase surface properties can be related to the increase in phospholipid content induced by rhDNase incubation. It is noteworthy that PG was systematically recovered in sol phases after the incubation of mucus samples with rhDNase. In addition, the recovery of PG in rhDNase-treated samples was much higher than in the control samples. Therefore, the increase in the sol phase volume associated with a decrease in its surface tension emphasizes the lubricating effect of rhDNase.
The potential use of rhDNase for treating patients with CF has been emphasized in clinical trials. Wilmott and colleagues (27), in a study involving patients hospitalized and treated for an acute excerbation and additionally treated or not with rhDNase for 14 d, did not demonstrate a statistically significant therapeutic effect of rhDNase. These investigators suggested that the lack of statistical significance could result from the greater variation in pulmonary function of the patients with CF included in the study. In addition, they hypothesized that severely affected patients with a FVC < 35% of the predicted value might respond differently to the treatment and that higher doses of rhDNase may be required to achieve efficacy during acute excerbation. However, short-term and long-term clinical studies, generally involving stable outpatients, have demonstrated a modest but significant improvement in pulmonary function of patients with CF treated with rhDNase (28, 29).
According to our present data, we observed that those patients who had no identifiable PG in their airway mucus before rhDNase treatment were characterized by a more severely impaired pulmonary function (FVC = 45.3 ± 6.1% and FEV1 = 28.7 ± 3.3% of predicted values) than patients with airway mucus in which PG was identified (FVC = 56.0 ± 9.7% and FEV1 = 41.8 ± 9.9% of the predicted values). In addition, in the group of patients with a more severely impaired pulmonary function, the recovery of PG in mucus after rhDNase treatment was higher than in the group of patients with superior pulmonary function. It would be of considerable interest to analyze whether patients with CF characterized by airway mucus with low PG content and low surface properties may exhibit after rhDNase treatment a heightened improvement in respiratory function correlated with high phosphatidylglycerol recovery.
In conclusion, the present work elucidates a new mechanism of action of recombinant human DNase in CF mucus. Because of its hydrolytic activity, rhDNase liberates sequestered surface-active lipids allowing them to play a lubricating role at the mucus surface and therefore improve the cough and ciliary clearance. Alterations of mucus surface properties and cough clearance by rhDNase treatment is of interest for chest physical therapy. We speculate that, in severely ill patients with CF and marked abnormalities of mucus surface properties, aerosolization of rhDNase prior to chest percussion and postural drainage could aid these patients in the clearance of their airways of adhesive mucus.
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