Hyperviscous airway periciliary and mucous liquid layers in cystic fibrosis measured by confocal fluorescence photobleaching

Abstract

Airway surface liquid (ASL) volume depletion and mucus accumulation occur in cystic fibrosis (CF). The ASL comprises a superficial mucus layer (ML) overlying a periciliary fluid layer (PCL) that contacts surface epithelial cells. We measured viscosity of the ML and PCL from the diffusion of FITC-dextran dissolved in the ASL of unperturbed, well-differentiated primary cultures of human bronchial epithelia grown at an air-liquid interface. Diffusion was measured by fluorescence recovery after photobleaching, using a perfluorocarbon immersion lens and confocal fluorescence detection. Bleaching of an in-plane 6-μm-wide region was done in which diffusion coefficients were computed using solution standards of specified viscosity and finite-element computations of 2-layer dye diffusion in 3 dimensions. We found remarkably elevated viscosity in both ML and PCL of CF vs. non-CF bronchial epithelial cell cultures. Relative viscosities (with saline=1) were in the range 7-10 in the non-CF ML and PCL, and 25-30 in both ML and PCL in CF, and greatly reduced by amiloride treatment or mucin washout. These data indicate that the CF airway surface epithelium, even without hyperviscous secretions from submucosal glands, produces an intrinsically hyperviscous PCL and ML, which likely contributes to CF lung disease by impairment of mucociliary clearance. Our results challenge the view that the PCL is a relatively watery, nonviscous fluid layer in contact with a more viscous ML, and offer an explanation for CF lung disease in the gland-free lower airways.

Human cystic fibrosis airway epithelia have reduced Cl − conductance but not increased Na + conductance

“…A comparison of CF and non-CF cultured airway epithelia under equilibrium conditions found no difference in periciliary liquid depth (42). Moreover, periciliary liquid depth was not reduced in the trachea of newborn CFTR −/− pigs (22).…”

A Functional Anatomic Defect of the Cystic Fibrosis Airway

“…To measure fluorescence recovery after photobleaching (FRAP) in HBE cells or trachea explants, ASL was stained with fluorescein isothiocyanate (10 ml at 1 mg/ml in PBS) and incubated for 30 minutes at 37 8 C (20). A circular area of approximately 3-to 5-mm diameter on the ASL surface was bleached with a 5-mW argon laser and 30-mW diode laser, and then fluorescence intensity was quantified as fluorescein isothiocyanate labeled ASL filled the bleached area.…”

Targeting a genetic defect: cystic fibrosis transmembrane conductance regulator modulators in cystic fibrosis

“…Absence or dysfunction of CFTR leads to a lack of chloride efflux and unregulated hyperabsorption of sodium ions. This imbalance of the osmotic gradient causes ASL dehydration, increased mucoviscosity and impaired mucociliary transport [30,32]. Without intervention, this situation gradually deteriorates causing plugging of small airways, bacterial infection, chronic local inflammation and eventually bronchiectasis [9,19,21].…”