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Submucosal gland dysfunction as a primary defect in cystic fibrosis

Danieli Salinas^*,, Peter M. Haggie^*, Jay R. Thiagarajah^*, Yuanlin Song^*, Kristina Rosbe, Walter E. Finkbeiner, Dennis W. Nielson and A. S. Verkman^*,1

Departments of
^* Medicine and Physiology,
Pediatrics,
Surgery, and
Pathology, University of California, San Francisco, California, USA

¹Correspondence: 1246 Health Sciences East Tower, Cardiovascular Research Institute, UCSF, San Francisco, CA 94143-0521, USA. E-mail: verkman{at}itsa.ucsf.edu

SPECIFIC AIMS

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-regulated membrane Cl^– channel in epithelia in the airways, pancreas, and other tissues. How defective CFTR function causes lung disease in CF is an unresolved issue with important consequences for new therapies. A variety of mechanisms have been postulated to relate defective CFTR to CF lung disease, including abnormalities in airway surface liquid (ASL) ionic composition, pH, oxygenation and volume, intrinsic hyperinflammation, and defective airway submucosal gland function. The defective gland hypothesis postulates reduced secretion of watery fluid by submucosal glands in CF with unimpaired or increased protein secretion. The consequent hyperviscous glandular secretions in CF are postulated to produce lung disease by impairing mucociliary clearance and antimicrobial activity, leading to chronic infection, inflammation, and airway destruction. Various lines of evidence support an important role for defective submucosal gland function in CF lung disease, including CFTR expression in gland epithelial cells, impaired fluid secretion in gland cell culture models, and abnormally viscous fluid secretions from glands of CF airways obtained at lung transplantation. The goal of our study was to determine whether submucosal gland function is defective in early CF prior to significant clinical disease and secondary changes in gland physiology. Functional studies were carried out on freshly obtained nasal airway biopsies from pediatric CF and non-CF subjects. Remarkably increased viscosity and reduced fluid secretion were found in secreted fluid in CF vs. non-CF specimens, providing direct evidence for an intrinsic defect in submucosal gland function in CF.

PRINCIPAL FINDINGS

1. Normal nasal submucosal gland histology in early cystic fibrosis
The age range of study subjects was 2–22 years. Three of the six CF subjects had very mild disease as evidenced from the forced expiratory volume at 1 s (FEV₁) of >75% predicted, Shwachman clinical score >75, and Brasfield clinical score >20 (see full paper for table of clinical data). Nasal biopsies from five non-CF subjects were obtained at the time of tonsillectomy/adenoidectomy surgery. Gland histology in all biopsy specimens was assessed by light microscopy of H&E-stained paraffin sections. A lung pathologist blinded to genotype information found no abnormalities except for a biopsy from one CF subject, where dilatation of gland acini was seen. Gland area in biopsy specimens was not significantly different between the control and CF groups.

2. Gland fluid viscosity is increased in cystic fibrosis
Biopsy specimens were processed immediately after procurement for quantitative measurement of gland fluid viscosity and secretion rate. Specimens were oriented with mucosal surface upward and immobilized in an agarose gel. The mucosal surface was covered with mineral oil to visualize freshly secreted fluid droplets over gland orifices. The viscosity of gland fluid secretions was measured from the kinetics of fluorescence recovery after photobleaching of fluorescently stained gland fluid droplets. Figure 1 A (top) shows the principle of this approach. The fluorescence in a cylindrical region of a fluid droplet was irreversibly bleached by a briefly applied intense laser beam. The subsequent increase in fluorescence results from diffusion of unbleached FITC-dextran into the darkened region, providing a quantitative measure of FITC-dextran diffusion coefficient and hence fluid viscosity. Viscosity was computed from the ratio of the recovery rates in saline (Fig. 1A , bottom right) vs. gland fluid. Half-time (t_1/2) for fluorescence recovery of FITC-dextran in saline was 35 ± 1 ms.

View larger version (20K): [in this window] [in a new window]   Figure 1. Increased gland fluid viscosity in CF as measured by fluorescence recovery after photobleaching. A) Top: schematic showing cylindrical bleached region in a fluorescently stained gland fluid droplet. (bottom, right) Fluorescence recovery after bleaching 10 kDa FITC-dextran in saline showing rapid reduction in fluorescence after laser bleaching, followed by increasing fluorescence (recovery) as unbleached dye diffuses into the bleached region. (bottom, left) Fluorescence micrograph of FITC-dextran stained gland fluid droplets. B) Representative fluorescence recovery data for 10 kDa FITC-dextran stained gland fluid droplets from three normal (left) and three CF (right) subjects (6–8 recovery curves averaged per subject). Note the slower recovery in CF. C) Recovery half-times (t1/2, left axis) and corresponding fluid viscosities (right axis) shown for biopsies from labeled normal and CF subjects (mean±SE, 3–6 glands/subject, solid symbols) along with average for all subjects (±SE, open symbols). *P < 10–4.

Individual gland fluid droplets were microinjected with FITC-dextran for photobleaching measurements (see fluorescence micrograph in Fig. 1A , bottom left). During the microinjections, droplets in all CF biopsy specimens were remarkably more adherent to the glass micropipette than those in specimens from normal subjects. Figure 1B shows representative fluorescence recovery curves for fluorescently stained gland fluid droplets in nasal biopsies from three normal and three CF subjects. Fluorescence was bleached to 70% of initial fluorescence intensity by application of a brief (<1 ms) intense laser beam. Fluorescence recovery in gland fluid from normal subjects was relatively rapid (t_1/2=77±5 ms, SE, n=4 subjects) and essentially complete, as seen by the similar fluorescence before and long (2–12 s) after application of the bleach pulse. Fluorescence recovery was significantly slowed in gland fluid from CF subjects with t_1/2 of 172 ± 7 ms (n=6 subjects). Figure 2 C shows averaged t_1/2 and corresponding relative fluid viscosities for each normal and CF subject, together with averaged results. Gland fluid in CF nasal biopsies was 2.2-fold more viscous than that from biopsies from normal subjects.

View larger version (28K): [in this window] [in a new window]   Figure 2. Reduced gland fluid secretion rate in CF. A) Kinetics of volume increase of fluid droplets from normal and CF subjects. Inset: bright-field microscopy showing expansion of fluid droplets in biopsy specimen from normal and CF subject. Zero time corresponds to removal of mucosal surface fluid and covering the surface with oil. B) Averaged secretion rates (mean±SE, 2–4 glands/subject, solid symbols), along with average for all subjects (±SE, open symbols). *P < 0.05.

3. Gland fluid secretion rate is reduced in cystic fibrosis
Secretion rates for individual gland fluid droplets were measured from the kinetics of droplet growth observed by light microscopy. Figure 2A (inset) shows serial micrographs of gland fluid droplets from a normal and CF biopsy specimen. Figure 2A summarizes the kinetics of expansion of all measured fluid droplets summed for each subject. Figure 2B gives corresponding secretion rates determined from linear regression of droplet growth data. Gland fluid secretion rate was reduced by 2.7-fold in CF compared with normal nasal biopsies.

CONCLUSIONS

We found that fluid secretion rate from submucosal glands was reduced 2.7-fold and secreted fluid viscosity was elevated 2.2-fold in early CF prior to significant clinical disease and glandular pathology in most of the subjects. There was no evidence that age or severity of illness affected gland secretion rate or viscosity in CF subjects that did manifest signs of disease; results here are similar to those reported previously in airways from older, more severely affected CF subjects. These results provide evidence for submucosal gland dysfunction as an intrinsic defect in CF, extending previous findings supporting the involvement of CFTR in gland fluid secretion in intact airways and of reduced fluid secretion and hyperviscosity in severely diseased CF airways. Glandular dysfunction may thus be a primary factor in the initiation and progression of CF lung disease, perhaps in combination with other abnormalities that have been proposed to exist in CF, including accelerated airway surface liquid absorption due to ENaC hyperfunctioning and/or airway hyperinflammation.We propose that an absence of functional CFTR in CF causes a reduction in the fluid component of gland serous secretions, resulting in increased viscosity of the fluid secreted onto the airway surface with consequent impaired bacterial clearance by mucociliary mechanisms (Fig. 3 ).

View larger version (40K): [in this window] [in a new window]   Figure 3. Proposed mechanism of lung disease in CF. Schematic of airway submucosal gland showing watery fluid secreted by CFTR-expressing serous acini, addition of glycoproteins by mucous tubules, and passage of fluid onto the airway surface through a collecting duct. We propose that defective CFTR in serous glandular epithelial cells results in secretion of a reduced amount of a hyperviscous solution, which impairs bacterial clearance mechanisms leading to chronic airway infection, inflammation, and destruction.

Our experimental strategy was to use optical methods to measure the properties of freshly secreted fluid from submucosal glands in nasal biopsies. Fluid droplets were visualized during their appearance and growth from gland openings under oil without modification by airway surface cells or contamination by airway surface fluid. Pilocarpine was used as the agonist to stimulate gland fluid secretion, as little fluid is secreted by CF glands in response to cAMP agonists. Biopsy specimens were oriented and immobilized in agarose to access the mucosal surface for visualization of fluid secretion. Tissues remained viable after immobilization in agarose for at least 6 h, as demonstrated by repeated agonist stimulation after washing the mucosal surface.

Our results focus attention on submucosal glands as a target for drug therapy in CF. Approaches that target submucosal glands specifically may be useful, such as activation of non-CFTR chloride or other channels involved in gland fluid secretion or inhibition of mucus production by glands. Correction of the glandular phenotype in CF—reduced fluid secretion and hyperviscosity—may thus reduce the pulmonary manifestations of the disease.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2879fje;

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