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* From Pediatric Pulmonology (Drs. Bush, Payne, Pike, and Jenkins), Imperial School of Medicine at National Heart and Lung Institute, London, UK; and Department of Pediatrics (Dr. Henke and Rubin), Wake Forest University School of Medicine, Winston-Salem, NC.
Correspondence to: Bruce K. Rubin, MEngr, MD, MBA, FCCP, Professor and Vice Chair, Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1081; e-mail: brubin{at}wfubmc.edu
Abstract
Objective: It has been assumed that cystic fibrosis (CF) lung disease is due in part to abnormal airway mucus. Primary ciliary dyskinesia (PCD) is a form of bronchiectasis that is similar to CF in many ways but is caused by congenital defects in mucociliary clearance. Our objective was to compare the biophysical and transport properties of CF and PCD sputa in subjects matched for age and degree of lung function impairment.
Design, setting, participants: PCD patients (n = 19; mean age, 9.5 ± 3.0 years [± SD]; FEV1, 65.0 ± 7.8 L) were recruited from the clinic at the Royal Brompton Hospital. Patients with CF (n = 30, mean age, 10.8 ± 2.6 years; FEV1, 61.8 ± 22.8 L) were identified from the Wake Forest University School of Medicine CF Center. Pulmonary function testing and sputum collection were performed as part of routine, scheduled clinic visits.
Measurements: Pulmonary function was measured by spirometry, and sputum was collected during the pulmonary function test maneuver. Some patients were longitudinally assessed at visits during the course of 3 years. Sputum properties measured were dynamic viscoelasticity, wettability, cohesivity, interfacial (surface) tension, solids composition, DNA and interleukin (IL)-8 concentration, in vitro mucociliary transportability, and cough transportability.
Results: Inflammation as measured by IL-8 concentration was three times greater in the PCD sputa (p < 0.0001). There were no significant differences in the sputum biophysical or transport properties comparing CF with PCD sputum.
Conclusions: It is unlikely that established CF lung disease is principally due to abnormal sputum properties, and it is more likely that the biophysical and transport properties reflect disease severity regardless of whether bronchiectasis is due to CF or PCD.
Key Words: cilia • cough • cystic fibrosis • inflammation • interleukin-8 • mucus • primary ciliary dyskinesia • rheology • surface tension
Primary ciliary dyskinesia (PCD) is typically characterized by the neonatal onset of upper and lower respiratory tract symptoms, including chronic and recurrent lower respiratory infection leading to bronchiectasis, chronic sinusitis, and chronic secretory otitis media.1 Half of all persons so affected also have situs inversus. The underlying defect is failure of mucociliary clearance due to primary failure of normal ciliary beating, leading to chronic bronchial infection and inflammation. However, the nature of the chronic inflammatory processes in the lower airway has not been well characterized. Typically PCD is diagnosed late,2 when the airway epithelium is damaged and pulmonary function is decreased. However, a treatment program including airway clearance with physical therapy, and the appropriate use of antibiotics, can stabilize pulmonary function over prolonged periods.34 This is in marked contrast to cystic fibrosis (CF), in which chronic infection and inflammation lead to progressive deterioration and ultimately premature death from respiratory failure. Traditionally, it was thought that failure of mucociliary clearance related to the production of abnormal secretions was the primary cause of lung disease, leading to chronic infection and inflammation. Data from both the CF knockout mouse5 and in human CF67 more clearly suggest that mucociliary dysfunction is secondary to infection and inflammation. It is also unclear whether chronic airway inflammation is a primary feature of CF, or if there is an exaggerated inflammatory response to infection.8
The contrast between the apparent similarities of airway disease but the much better prognosis for PCD compared with CF is striking. Ellerman and Bisgaard9 showed that for the most part, lung function was stable after diagnosis of PCD, irrespective of the level of function at diagnosis, quite unlike the clinical experience with CF. In another study,10 the slope of a regression line of FEV1 plotted vs age for a cohort of patients of all ages with PCD was – 0.8 ± 0.13 (± SD) [a drop of approximately 0.8% per year], as compared with a slope decline of up to approximately 3.6% for patients with CF. Understanding the factors that promote a milder PCD phenotype might lead to the discovery of new therapies for CF. We hypothesized that a comparison between the two diseases might reveal differences in the biophysical or transport properties of sputum or in sputum inflammatory mediator content.
Materials and Methods
Patients Studied
We recruited children attending the PCD clinic at the Royal Brompton Hospital. The diagnosis had been established on standard criteria, including nasal nitric oxide, ciliary beat frequency, and electron microscopy.111 The comparison group were children with CF diagnosed according to the Consensus statement recommendations12 who were attending the Wake Forest University CF Center. In both centers, spirometry was performed at each clinic visit according to American Thoracic Society guidelines.13 Three reproducible flow-volume curves with < 10% variability in FEV1 were recorded. Sputum was collected with the aid of an experienced respiratory therapist when needed. Patients were asked to swallow saliva and to expectorate all secretions during routine pulmonary function testing or respiratory physical therapy. We used sputum that would usually be discarded after removing the amount needed for bacterial culture. Sputum plugs were visually separated from saliva and stored at – 70°C in small, tightly sealed O-ring containers to minimize water loss.
Since 1997, the Wake Forest University CF Center has collected sputum and demographic data on all patients with CF who attended the clinic and were able to expectorate. This database formed the sample from which CF subjects were matched with PCD subjects according to age and primarily by pulmonary function (FVC and FEV1), as these measurements are most commonly used to stratify CF subjects on illness severity. This study was approved by the Wake Forest University Institutional Review Board for Human Research, the Wake Forest University Animal Care and Use Committee, and The Royal Brompton Harefield NHS Trust Ethics Committee.
Measurement of the Physical Properties of Sputum
Viscoelasticity (Rheology):
Viscosity (loss modulus [G"]) is the loss of energy from a rheologic probe (stress) and thus the resistance to flow. Elasticity (storage modulus [G’]) is the recoil energy transmitted back to the probe. Viscoelasticity is a property of non-Newtonian fluids (gels). We used a rheometer (AR1000; TA Instruments; New Castle, DE) to assess the dynamic frequency range of stress strain of a 20-µL sputum sample over driving frequencies of 1 to 100 rad/s. The G’ and the G" of the specimen were determined from these curves after nondestructive creep transformation.14
Sputum Cohesivity:
Cohesivity is defined as interfacial tension multiplied by the new area as after a test substance is subjected to nonshearing stress. For ideal or Newtonian fluids this is equal to two times the interfacial tension, 
. For a gel such as mucus, a distraction device (Filancemeter; constructed by Dr. B. K. Rubin) is used to stretch the mucus until breaking. The measurement is performed with a 25-µL sample at a distraction velocity of 10 mm/s. An electric signal conducted through the sample is interrupted when the thread is broken. A cone with a mean diameter of 1 mm is formed to the length at which it breaks yielding a cohesivity equal to x 
x length in millimeters/100.15
Mucociliary Transportability: Mature leopard frogs (Rana pipiens) were decapitated and the palate removed. The palate was placed on gauze saturated with amphibian Ringer solution prepared by mixing two parts of nonlactated Ringer injection solution with one part sterile water. The palate was allowed to deplete of mucus at 4°C for 12 h and was then placed in a humidified chamber. It was focused under a microscope so that a micrometer scale ran between the optic bulges to the opening of the esophagus. The movement of a 4-µL sputum specimen was timed as the trailing edge moved across a 3-mm segment. Three measurements of mucus transport velocity were taken and the mean normalized to the transport rate for endogenous frog mucus.16
In Vitro Cough Transportability: A simulated cough machine was used to measure the airflow-dependent clearability of sputum. A model Plexiglas trachea, rectangular in cross-section (1.2 x 2 cm) was connected to a 6.4-L tank containing air pressurized to 11 pounds per square inch, giving a flow of approximately 11 L/s. A solenoid-controlled air release through a flow constrictive element was used to mimic the airflow pattern of a natural cough. A sample, 40 µL in volume and 0.5 mm in depth, was placed in a thin line across the base of the Plexiglas trachea. The bulk transport of the sample was measured in millimeters after three cough maneuvers, and the results were averaged.17
Sessile Drop Wettability:
A 15-µL sputum drop was placed on the surface of a glass slide that had been cleaned with chromic sulfuric acid, rinsed with deionized water, and then stored in absolute ethanol to maintain dehydration before use. Sputum was transferred to a sharp needle and kept 3 to 5 mm above the glass surface until it fell. A stabilization time of 1 min was allowed before the image of the sessile drop was captured in the video-processing system, and the glass/sputum contact angle, 
, was calculated.18
Interfacial Tension by Ring Distraction (de Noüy Ring Method):
This test measures the interfacial tension at a sputum/air interface. A specially instrumented platinum-iridium ring, a metal that is completely wettable, was pulled from the mucus sample at a distraction velocity of 10 mm/s until separation was achieved. The force of separation is measured by a strain gauge connected to the ring. This gives the work of adhesion (Wad) in dyne/cm. We used a semiautomated tensiometer for these measurements (Fischer Tensiomat Model 21; Fischer Scientific; Pittsburgh, PA). We calibrated a platinum-iridium ring with a circumference of 1.7145 ± 0.0381 cm and a wire diameter of 1 mm so that with a 0.5-cm depth chamber we needed only a volume of 0.12 mL to accurately measure the adhesion tension. The Wad is calculated from the contact angle and interfacial tension using the Young equation: Wad = (1 + cos ); and tenacity (work of distraction) is the product of cohesivity and Wad.19
Sputum Inflammatory Mediator Content Analysis: Sputum volume was measured and then diluted by adding an equal volume of phosphate-buffered saline solution. This was gently vortexed for 30 s and then left for 30 min at 22 to 24°C. It was then added to an equal volume of phosphate-buffered saline solution and centrifuged at 2,000g for 5 min and the supernatant collected for analysis.20
Measurement of Free IL-8: IL-8 was measured using a commercially available enzyme-linked immunosorbent assay kit. The collected supernatant was diluted 1:30 with diluent containing sodium azide (< 0.1%). After washing three times, the plate was incubated with 50 µL of biotinylated antibody and 100 µL of streptavidin-horseradish peroxidase conjugate for 30 min at room temperature and agitated at 350 revolutions per minute. The plate was then again washed three times. Tetramethylbenzidine substrate, 100 µL, was added and incubated for 20 min at room temperature in the dark. This was stopped by adding 50 µL of 2 N sulfuric acid. Color development was read as absorbance at 450 nm in an enzyme-linked immunosorbent assay reader. The concentration of free IL-8 was calculated against recombinant human standard (8 to 2,000 pg/mL).
Measurement of Sputum DNA: DNA was measured by microfluorimetry.20 The supernatant was diluted 1:800 with saline sodium citrate buffer (0.0154 mol/L NaCl-0.015 mol/L Na3-citrate, pH 7.0). One milliliter of 33258 Hoechst 1.5 x 10– 6 mol/L (Calbiochem; La Jolla, CA) was added, and fluorescence was measured by spectrophotofluorometry with an excitation wavelength at 360 nm and emission at 450 nm. DNA concentration was calculated by comparison with calf thymus DNA (0.25 to 10.0 µg/mL).
Statistical Evaluation
The study was powered to detect an 80% difference in sputum viscosity (G") with a power of 0.80 at the p < 0.05 significance level (unpaired two-tailed t test). Data for the biophysical and transport properties of secretions were tested for normality using the Kolmogorov-Smirnov test. Statistical comparison of the properties of CF and PCD secretions was completed using unpaired, two-tailed t tests with post hoc Bonferroni correction for multiple statistical comparisons. Conventionally, p < 0.05 was considered to be statistically significant.
Results
Patients
We recruited 19 PCD children, mean ± SD age, 9.5 ± 3.0 years. Fourteen children were able to perform spirometry; FEV1 was 65.0 ± 7.8% of predicted and FVC was 78.6 ± 14.5% of predicted. Each subject produced a sputum sample at the same single visit. These subjects were age and pulmonary function matched with 30 children with CF who provided a total of 134 sputum samples at 1 to 11 clinic visits over 38 months. Of these, 39 specimens were large enough in volume for complete biophysical and biochemical analysis. The age of the CF subjects was 10.8 ± 2.6 years (p = 0.6 compared with PCD). Their mean FEV1 was 61.8 ± 22.8% of predicted (p = 0.60) and FVC was 80.0 ± 23.8% of predicted (p = 0.84 compared with PCD). Twenty-five of 30 CF subjects were chronically infected with Pseudomonas aeruginosa, compared with none of 19 children with PCD. Sputum was collected from the CF subjects during periods of clinical stability and during an "exacerbation," defined only by the center physician’s decision to begin antibiotic therapy at the clinic visit. None of the subjects required supplemental oxygen or gastrostomy tube feeding during the course of the study.
Sputum Biophysical and Transport Properties
The biophysical properties were not different comparing PCD and CF sputa (Table 1
). The calculated loss modulus (tangent 
, the ratio of loss to storage moduli) did not significantly differ.
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Relationships Among Measurements
As described previously18 in the PCD set, cough transportability of secretions (CTR) was inversely correlated with cohesivity (R2 = 0.36, p = 0.05) [Fig 1
].
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This is the first study to evaluate the biophysical and transport properties of PCD sputum and one of the most comprehensive analyses conducted of CF sputum properties. The aim of this study was to try to determine whether differences between the properties of sputum from two carefully matched groups of patients with different neutrophilic airway diseases leading to bronchiectasis could account for the generally milder prognosis in PCD. The principal findings of this study are surprising. We report for the first time that the biophysical properties of sputum in the two diseases are the same but that there is evidence of more airway inflammation, at least as manifested by IL-8 levels, in PCD, the condition with the better prognosis.
Mucus is a viscoelastic gel consisting primarily of water and high-molecular-weight, linearly cross-linked, entangled glycoproteins. Respiratory mucus is usually cleared by cilia. Sputum, which is mucus mixed with inflammatory cells, cellular debris, and bacteria, is generally cleared by cough. It is thought that the physical and surface properties of mucus or sputum largely determine mucociliary transportability of secretions and CTR. Early studies21 of expectorated sputa from subjects with CF, bronchiectasis, asthma, or chronic bronchitis showed that as sputum became more visibly purulent in appearance, viscosity was increased regardless of the underlying disease. However, for all degrees of purulence, CF sputum viscosity was nearly identical to that of non-CF bronchiectasis sputum, less viscous than bronchitis sputum, and much less viscous than asthma mucus.2122 Other studies2324 of the properties of CF secretions have not shown these to be abnormally viscous or thick, although there are data1925 suggesting that these secretions may be abnormally hydrated or have increased tenacity (the product of adhesivity and cohesivity). The results presented here are consistent with this finding.
In CF, the cilia are normal. In PCD, the cilia are abnormal, but there have been no published data on the properties of airway secretions. Because of the mild course of the disease in the face of a profound defect of ciliary clearance, it is tempting to speculate that there may be compensatory changes in mucus properties that would favor its clearance by airflow or cough. Although we hypothesized that there may be compensatory changes in the surface properties of PCD secretions that would make them more easily cleared by cough (ie, increased CTR) we found that the CTR of PCD secretions was similar to that of CF secretions and both CF and PCD sputa had decreased CTR due to high tenacity (the product of cohesivity and Wad).
CF sputum contains much less mucin than either normal mucus or bronchitis sputum but large amounts of DNA.26 This present study extends previous reports27282930313233 of sputum properties in CF and other inflammatory lung diseases. The popular belief has long been that CF pulmonary disease is caused, in part, by viscous mucus that is poorly cleared from the airway, despite studies21222324 clearly showing otherwise. It is likely that sputum-surface properties contribute to the relatively fulminant course of CF lung disease when compared with other types of bronchiectasis.1819 Differences between CF and non-CF sputum may be due to several factors including the host response to infecting microorganisms, the recruitment of inflammatory cells into the airway, and to the properties of secretions. However, although we found no difference in biophysical properties, we documented a much greater concentration of DNA in the CF sputa but significantly less IL-8. This may be influenced by IL-8 binding to the abundant DNA and actin in CF sputum.34
There are several implications to these findings. The first is that excessive production of IL-8 cannot be the reason for the worse prognosis in CF. It is known that for a given level of infection, CF sputum has more neutrophils than in chronic bronchitis sputum, although this comparison has not been made for PCD. We hypothesize that other undetermined neutrophil chemoattractants may be responsible for the airway neutrophilia of CF. We further hypothesize that the increased amount of IL-8 reported in previous studies3536373839 in CF are a marker for the presence of neutrophils, and may not be of major pathophysiologic significance. The further implication of this report is that strategies to reduce sputum IL-8 levels in isolation are not likely to be effective therapeutically, since the levels appear to be lower than in a disease with a much better prognosis, namely PCD. Furthermore, the use of decreased sputum IL-8 as a surrogate marker in clinical trials would not appear to be advisable.
This study has several limitations that need to be considered when interpreting the results. Uninfected mucus is difficult to obtain either from healthy people or from patients with CF, so there are few data on the properties of "normal" mucus. This knowledge is crucial for us to understand how mucus (rather than sputum) contributes to the pathogenesis of CF and how abnormalities in the CF gene and protein cause lung disease. A further possible weakness is that the two patient groups came from different centers. However, care was taken to ensure standardization of sputum collection and storage, and the measurement of height, weight, and lung function. A potential bias in this study is the absence of PCD children chronically infected with P aeruginosa, and the fact that some of the CF children had sputum collected during an infective exacerbation. Both biases would tend to lead to the CF group exhibiting more abnormalities and infection, making our findings of much greater IL-8 levels in the PCD group even more surprising.
In summary, we have shown similar biophysical properties of CF and PCD sputum but a more than threefold increase in sputum IL-8 in PCD. The reason for the differences in prognosis between the two conditions remains unclear, but further studies to determine significant differences should be pursued in order to try to identify novel therapeutic targets for CF patients.
Footnotes
Abbreviations: CF = cystic fibrosis; CTR = cough transportability of secretions; G’ = storage modulus; G" = loss modulus; IL = interleukin; PCD = primary ciliary dyskinesia; Wad = work of adhesion
Received for publication May 17, 2005. Accepted for publication July 15, 2005.
References
This article has been cited by other articles:
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  H. Mussaffi, E. M. Fireman, M. Mei-Zahav, D. Prais, and H. Blau Induced Sputum in the Very Young: A New Key to Infection and Inflammation Chest, January 1, 2008; 133(1): 176 - 182. [Abstract] [Full Text] [PDF]
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B. K. Rubin, A. P. Kater, and A. L. Goldstein Thymosin {beta}4 Sequesters Actin in Cystic Fibrosis Sputum and Decreases Sputum Cohesivity in Vitro. Chest, November 1, 2006; 130(5): 1433 - 1440. [Abstract] [Full Text] [PDF]
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