Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells.

The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na(+) absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na(+), Mg(2+), P, S and Cl(-)) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR(inh)-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.

X-ray microanalysis of airway surface liquid collected in cystic fibrosis mice.

The airway surface liquid (ASL) that lines the airway surface epithelium plays a major role in airway antibacterial defense and mucociliary transport efficiency, two key factors in cystic fibrosis (CF) disease. A major difficulty is to collect ASL in native conditions without stimulation or alteration of the underlying airway epithelium. Using a cryoprobe specifically adapted to collect native ASL from the tracheal mouse surface, we analyzed by X-ray microanalysis the complete ASL and plasma ion content in Cftr(tm1Hgu)/Cftr(tm1Hgu) mice compared with that in control littermates. ASL ion content from eight Cftr(tm1Hgu)/Cftr(tm1Hgu) mice and eight control littermates did not appear significantly different. The mean (+/-SE) concentrations were 2,352 +/- 367 and 2,058 +/- 401 mmol/kg dry weight for Na, 1,659 +/- 272 and 1,448 +/- 281 mmol/kg dry weight for Cl, 357 +/- 57 and 337 +/- 38 mmol/kg dry weight for S, 1,066 +/- 220 and 787 +/- 182 mmol/kg dry weight for K, 400 +/- 82 and 301 +/- 58 mmol/kg dry weight for Ca, 105 +/- 31 and 105 +/- 20 mmol/kg dry weight for Mg, 33 +/- 15 and 29 +/- 9 mmol/kg dry weight for P in non-CF and CF mice, respectively. This cryotechnique appears to be a promising technique for analyzing the complete elemental composition of native ASL in CF and non-CF tissues.

Ion composition and rheology of airway liquid from cystic fibrosis fetal tracheal xenografts.

The composition of airway surface liquid (ASL) is partly determined by active ion and water transport through the respiratory epithelium. It is usually stated that in cystic fibrosis (CF), CF transmembrane conductance regulator protein abnormality results in imbalanced ion composition and dehydration of ASL, leading to abnormal rheologic and transport properties. To explore the relationship between ion composition, water content, and viscosity of airway liquid (AL), we used a human xenograft model of fetal airways developed in severe combined immunodeficiency (SCID) mice. Six non-CF and six CF portions of fetal tracheas were engrafted subcutaneously in the flanks of SCID mice raised in pathogen-free conditions. AL accumulated in the closed cylindric grafts was harvested 9 to 17 wk after implantation. At the time of AL sampling, all tracheal grafts displayed well-differentiated pseudostratified surface epithelium and submucosal glands. The viscosity of AL was measured using a controlled-stress rheometer. The ion composition of AL was quantified by X-ray microanalysis. No significant difference was observed for AL viscosity between non-CF (0.6 +/- 0.5 Pa. s) and CF (0.2 +/- 0.1 Pa. s) samples. In AL from non-CF and CF samples, the ion concentrations were Na: 63.9 +/- 7.6, 79.7 +/- 11.6; Cl: 64.9 +/- 13.2, 82.6 +/- 15.7; Mg: 1.9 +/- 0.3, 2.2 +/- 0.4; S: 4.9 +/- 1. 3, 4.8 +/- 0.5; K: 2.4 +/- 0.5, 3.2 +/- 1.6; and Ca: 1.2 +/- 0.3, 2.6 +/- 0.8 mmol/liter, respectively. The ion composition of AL from CF versus non-CF xenografts was not significantly different. These results suggest that prior to inflammation and infection, the viscosity and ion composition of the fetal AL do not differ in CF and non-CF.

X-ray microanalysis of native airway surface liquid collected by cryotechnique.

The airway surface liquid (ASL) that lines the surface epithelium of the tracheobronchial tree is of vital importance to the airway defence against microbial invasion and damage due to environmental factors. Little is known about the ASL collected in situ in native conditions, owing to difficulties in collecting ASL without causing damage to the airway mucosa. We have developed a method to collect and analyse the elemental composition of tracheal ASL in pathogen-free mice. A specially designed cryoprobe, adapted to the internal curvature of the mouse trachea, was used to collect the native ASL from the tracheal surface. The complete ASL elemental composition including [Na] = 5.5 +/- 0.3, [Cl] = 1.3 +/- 0.3, [K] = 1.1 +/- 0.2, [Ca] = 1.2 +/- 0.3, [P] = 1.5 +/- 0.8, [S] = 1.7 +/- 0.4 and [Mg] = 1.3 +/- 0.4 mmol L-1 was determined by X-ray micro-analysis. We demonstrate here that the technique that we used for ASL collection maintained perfectly the airway epithelial integrity and functionality.

X-ray microanalysis of native airway surface liquid collected by cryotechnique.

The airway surface liquid (ASL) that lines the surface epithelium of the tracheobronchial tree is of vital importance to the airway defence against microbial invasion and damage due to environmental factors. Little is known about the ASL collected in situ in native conditions, owing to difficulties in collecting ASL without causing damage to the airway mucosa. We have developed a method to collect and analyse the elemental composition of tracheal ASL in pathogen-free mice. A specially designed cryoprobe, adapted to the internal curvature of the mouse trachea, was used to collect the native ASL from the tracheal surface. The complete ASL elemental composition including [Na] = 5.5 +/- 0.3, [Cl] = 1.3 +/- 0.3, [K] = 1.1 +/- 0.2, [Ca] = 1.2 +/- 0.3, [P] = 1.5 +/- 0.8, [S] = 1.7 +/- 0.4 and [Mg] = 1.3 +/- 0.4 mmol L-1 was determined by X-ray micro analysis. We demonstrate here that the technique that we used for ASL collection maintained perfectly the airway epithelial integrity and functionality.