1 Institut National de la Santé et de la Recherche Médicale Unité 514, Institut Federatif de Recherche 53, Reims 51092; 2 Laboratoire de Microscopie Electronique, Unité de Formation et de Recherche Sciences, Reims 51685, France; and 3 Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, United Kingdom
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ABSTRACT |
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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 Cftrtm1Hgu/Cftrtm1Hgu mice compared with that in control littermates. ASL ion content from eight Cftrtm1Hgu/Cftrtm1Hgu 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.
cryoprobe; ion concentration
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INTRODUCTION |
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THE AIRWAY SURFACE LIQUID (ASL) is a thin layer of fluid covering the airways that contains proteins, glycoproteins, lipids, peptides, ions, and water. The ionic composition of the ASL plays a crucial role in airway host defense by controlling the ciliary activity, mucin release (16), and antimicrobial activity (15) and regulating the volume and/or ionic composition of the ASL by ion transport across the airway epithelium. The major active ion transport process across human airway epithelia is amiloride-sensitive absorption of Na, with Cl acting as the major counteranion (9, 16).
Two hypotheses linking cystic fibrosis (CF) transmembrane conductance regulator (CFTR) dysfunction to lung disease pathogenesis have emerged and have focused investigation on salt and fluid absorption mechanisms across the airway epithelium. The first hypothesis proposes that active transcellular absorption of Na is accompanied by the diffusion of Cl ions through tight junctions between the cells and that water rapidly follows, thus maintaining osmolarity (11). As a consequence, this hypothesis predicts that the NaCl concentration in ASL should be similar to that in plasma. According to this hypothesis, the pathogenesis of lung disease in CF is related to a reduction in ASL volume due to an increased rate of isotonic ion and water absorption. Consequently, ciliary transport of ASL is impaired and airway infection develops.
The second hypothesis considers that Cl ions follow Na, transported transcellularly via Cl channels in the apical membrane (17). This hypothesis predicts that the NaCl concentration in ASL should be lower than that in plasma, with the volume maintained constant by capillary pressure from the cilia, osmotic pressure from nondiffusable osmolytes, or impermeability of the epithelium to water. Among the Cl channels regulating these ion effluxes, the CFTR protein has been heavily implicated. Hence this theory predicts that CFTR dysfunction compromises the ability of the cells to absorb Cl ions from the ASL. This failure and the consequent impairment of Na absorption would result in a raised salt concentration in the ASL of CF individuals. This hypothesis is supported by the observation that ASL displays broad-spectrum antibacterial activity that is impaired by a high-salt environment and is defective in CF (15). More recently, using a noninvasive in vivo fluorescence measurement of salt concentration, Jayaraman et al. (7) have shown that the Cl and Na contents in ASL were similar in wild-type and CFTR-null mice and that the ASL was approximately isotonic.
In view of these emergent but contradictory hypotheses, it is critical that the precise ASL composition be determined. However, the depth of this layer is so small in normal airways that it is difficult to sample ASL in vivo without disturbing the underlying epithelium. In addition, ASL is a complex mixture produced by the secretory cells of the surface epithelium and by glands in the trachea and bronchi and may also include contributions from the bronchiolar and alveolar liquids. Thus physiological studies on isolated or cultured epithelium do not appear to be the most appropriate methods for analyzing ASL composition.
Baconnais et al. (1a) have recently developed methods to collect and analyze the elemental composition of tracheal ASL in mice. The collection is carried out under conditions that do not induce any stimulatory, morphological, or functional alterations of the airway cells that produce ASL. After collection, the complete ionic composition is determined by X-ray microanalysis. The aim of this study was to use these techniques to compare the ionic composition of ASL collected from wild-type and transgenic CF mutant mice (6).
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MATERIALS AND METHODS |
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CF mouse model. The Cftrtm1Hgu/Cftrtm1Hgu transgenic mouse model used in this study was generated after targeted insertional mutagenesis into exon 10 of the murine Cftr gene in embryonic stem cells (6). The mutation is slightly "leaky," resulting in the production of a low level of wild-type CFTR as a result of exon skipping and aberrant splicing. Nevertheless, these mutant mice display the electrophysiological defect in the gastrointestinal and respiratory tracts that is characteristic of CF and can be unequivocally distinguished from their non-CF littermates on this basis (6). The mutant mice also display significantly reduced pulmonary clearance of Staphylococcus aureus and Burkholderia cepacia and significantly more severe lung pathology after repeated challenge with these same pathogens (5).
ASL was collected from pathogen-free mice: eight Cftrtm1Hgu/Cftrtm1Hgu on an outbred MF1/129 strain background (mean age and weight, 87.3 ± 22.9 days and 27.8 ± 1.5 g, respectively) and eight non-CF littermate controls (mean age and weight, 108.9 ± 43.1 days and 28.5 ± 1.8 g, respectively). The mice were housed in germ-free conditions in an isolator unit under positive pressure and maintained with sterilized food and bedding. The diet was the same for the CF mice and the littermate controls. The mice were anesthetized with an intraperitoneal injection of 100 µl of pentobarbital sodium. The trachea of each mouse was incised longitudinally along 5 mm, and the ASL was collected within 2 min with a specially designed cryoprobe.ASL collection.
A special steel probe was designed to collect the native ASL at the
tracheal surface. The tip of the probe had a curvature less important
(radius = 0.6 mm) compared with the internal curvature of the
mouse trachea (radius = 1-1.5 mm) so only the extreme bottom part of the probe came in contact with the tracheal mucosa. In addition, the use of mice of consistent weight (25-30 g) led to a
similar surface contact between the probe and the tracheal mucosa from
one mouse to the other. Before the collection of ASL, the probe was
washed with ultrapure water (Fluka, St. Quentin Fallavier, France) and
pure methanol (Sigma, St. Quentin Fallavier, France), dried at room
temperature, and cooled by plunging it into a liquid nitrogen bath
(180°C) until the thermal balance was reached. The top of the probe
consisted of a small liquid nitrogen tank, allowing the probe tip to be
maintained at a low temperature during the experiment. The cryoprobe
was then attached to a transducer that controlled the pressure at which
the cryoprobe was applied on the tracheal mucosa during ASL collection
(Fig. 1). The mouse, placed in the supine
position, was then moved upward at a constant speed (1 cm/min) until a
contact pressure of <2,000 Pa between the cryoprobe and the tracheal
mucosa was reached. Under such conditions, the ASL making contact with
the cryoprobe was immediately frozen, resulting in a small amount (<10
pl) of ASL adhering to the cryoprobe tip. With warming to room
temperature, the ASL adhering to the cryoprobe was thawed and deposited
on an electron microscope copper grid (Maxtaform 200 mesh, Touzard et
Matignon, Vitry sur Seine, France) that had successively been coated
with a collodion membrane and a 10-nm carbon film.
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X-ray microanalysis. The samples were analyzed in a scanning transmission electron microscope (CM30, Philips, Limeil-Brevannes, France) equipped with a 30-mm2 (0.13-steradian subtending solid angle, 172-eV resolution) Edax Si(Li) detector with a beryllium window.
The elemental composition of dehydrated ASL samples was determined with the continuum method (1), which implies the measurement of the specimen background intensity. The experimental background may be due to the specimen itself but may also be related to the grid fluorescence. In our experiments, we used pure copper grids. The X-ray spectra were therefore characterized by the presence of the KStatistical analysis. Means ± SE were calculated for ionic concentrations for the non-CF and CF groups. The two groups of mice were compared by the nonparametric Mann-Whitney test. The values were considered significantly different at P < 0.05.
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RESULTS |
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Standard solutions.
The results obtained from the analysis of the standard solutions are
presented in Table 1. The measured
concentrations of elements in the NaCl, MgCl2,
CaCl2, and KCl solutions were not significantly different
compared with the expected values.
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Ionic composition of airway liquid in
Cftrtm1Hgu/Cftrtm1Hgu
mice.
A typical spectrum obtained from ASL X-ray microanalysis is presented
in Fig. 2. This spectrum clearly
identifies the major ions in ASL, i.e., Na, Cl, S, and Ca. The copper
peak observed at a high-energy level corresponds to the copper grid on
which the ASL sample was deposited. Table
2 shows the concentration of the
different ions analyzed in ASL samples collected in
Cftrtm1Hgu/Cftrtm1Hgu and
wild-type mice. The data show that in ASL collected from mice tracheae,
Na and Cl represent the two major unbound ions. The mean concentrations
of Na, Cl, Mg, S, P, K, and Ca were not significantly different in
Cftrtm1Hgu/Cftrtm1Hgu
mice compared with wild-type mice. A huge interindividual variability was observed for the different ion concentrations in both groups of
mice.
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DISCUSSION |
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In the present study, we report an original noninvasive method for analyzing the ionic composition of the in situ ASL collected with a specially designed cryoprobe from non-CF and CF mice. The complete ionic composition of the ASL collected from the tracheal surface of pathogen-free mice was determined by X-ray microanalysis. The main advantage of the X-ray microanalysis technique is that it requires a very low sample volume (10-50 pl), which can be easily collected with the cryoprobe described in the present paper. This small sample volume is therefore easily collectable without any mechanical stimulation, even in healthy animals. The technique also allows the simultaneous, nondestructive quantitative analysis of the different ions of interest, whereas other methods like radiotracer dilution or selective microelectrode methods (2, 12) are restricted to the measurement of one or two ions, generally Cl and Na. Moreover, the radiotracer dilution technique can only be applied to culture models. A novel noninvasive fluorescence microscopy technique has been recently described (7) to measure ASL thickness, salt concentration, and pH either in cell culture models, in vivo in the mouse trachea, or in freshly excised human bronchi. The main advantage of this technique is that it permits continuous quantitative measurements of ASL properties, although it is at present only applied to Na and Cl measurement. The cryoprobe technique that we used, associated with the X-ray microanalysis technique, allows the sampling of native ASL, but there is a limitation in the expression mode of the data that are expressed in millimoles per kilogram of dry mass.
The sampling technique may be a key factor in the ionic composition determination of ASL. The cryotechnique used in the present study allowed the rapid collection of ASL under conditions that did not induce any stimulatory, morphological, or functional alterations of the airways cells that produce ASL (1a). We carefully checked the pressure under which the probe was applied to the tracheal mucosa. Having previously shown that a pressure of <2,000 Pa did not damage the surface epithelium, we paid particular attention to limit the pressure to this level during ASL collection (1a). When analyzing with scanning electron microscopy the tracheal area where the cryoprobe had been applied, we did not observe any damage to the Clara cells and ciliated cells that cover the mouse tracheal epithelium. Moreover, we did not observe any change in the ciliary beating frequency of the ciliated cells present in the area where ASL had been collected (1a). This suggests that the ASL is mainly collected from the upper gel layer and is instantaneously frozen, thus preventing any metabolic or histological alterations of the underlying epithelial cells. In contrast, most of the techniques described in the literature (3, 8) require that the collection probe be in contact with the airway mucosa for a prolonged period, thus potentially stimulating ASL secretion during the sampling process. Such variables may well be the explanation for the apparent incompatibility of many of the published results in this field to date and suggest that the two most prominent models describing the ASL composition may not be mutually exclusive but may reflect key differences in the experimental model systems used.
We demonstrate that when housed in germ-free conditions, i.e., in the absence of any previous bacterial infection, CF mice exhibit a NaCl content in their ASL that is not significantly different compared with that in their littermate controls. Our results are consistent with the recent data reported by Cowley et al. (3), who demonstrated no difference in NaCl composition in ASL from CF and non-CF mice except when the mice were infected with Pseudomonas aeruginosa. These results are emphasized by previous data demonstrating that ASL collected from CF and non-CF xenografts of human fetal airways did not show a significant difference in the ion composition (1). Here, again, the lack of difference could be related to the perfect sterility of the mature fetal tracheal xenografts. The huge variability in the ion concentrations that we observed in both groups of mice could be related to any genes capable of modifying the genotype present in the inbred 129 or MF1 outbred genetic background. The fact that the Cftrtm1Hgu/Cftrtm1Hgu mice are not genetically identical may well account for the variation that we observed in the measured phenotypes. Genetic modifiers may influence the degree of wild-type CFTR transcription from the Cftrtm1Hgu allele and/or the level of non-CFTR-mediated Cl ion transport (14).
Having previously observed no alteration in beat frequency of ciliated
cells from
Cftrtm1Hgu/Cftrtm1Hgu
mice but a decreased mucociliary transport rate (18), we
proposed that this latter could be related to a decreased ASL water
content. Data reported by Zhang and Engelhardt (19), using
a human bronchial xenograft model, suggest that the hydration in CF
airways may be impaired. Loss of water from the ASL is likely
responsible for an increase in the mucin content of ASL, thus reducing
the transport capacity of ASL (13). The "isotonic volume
transport/mucus clearance" hypothesis proposed by Matsui and
colleagues (10, 11) supports the proposal that in CF, the
primary defect is characterized by an increased water absorption with
an excessive isotonic volume absorption, leading to alterations in ASL
properties. Widdicombe and Widdicombe (16), using
primary cultures of human tracheal epithelium in unstimulated and
uninfected conditions, obtained a hypotonic ASL (120 meq/l), and Cowley
and colleagues (3, 4) reported hypotonic ASL collected by
capillary electrophoresis in both mice and rats. McCray et al.
(12), using a new radiotracer technique to measure the
NaCl content of the ASL produced by primary cultures of murine
tracheal epithelium from
Cftrtm1F508Uta/Cftrtm1
F508Uta
and wild-type control mice, also reported that murine ASL is hypotonic.
However, neither Cowley et al. (3) nor McCray et al.
(12) found any difference between the ASL composition of CF mice and control littermates. These results are emphasized by
Jayaraman et al. (7), who reported no difference in ASL salt concentrations in CF and non-CF mice.
In summary, we report an original technique to collect in vivo murine ASL and to analyze its ionic composition. We found no differences in the salinity of ASL from CF mice compared with wild-type littermates. Our results suggest that it would be of major interest to develop a technique allowing to quantify the water content in parallel with the ion content of the murine ASL.
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ACKNOWLEDGEMENTS |
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We thank Laurence Killian and Alain Perchet for help and contribution to this work and Prof. Thierry Chinet for critically reading the manuscript.
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FOOTNOTES |
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* Jean-Marie Zahm and Sonia Baconnais contributed equally to this work.
This work was supported by the Association Française de Lutte contre la Mucoviscidose and Région Champagne Ardenne (France), the Cystic Fibrosis Research Trust, and the Medical Research Council (United Kingdom).
Address for reprint requests and other correspondence: E. Puchelle, INSERM U514, 45 rue Cognacq-Jay, 51092 Reims Cedex, France (E-mail: epuche{at}worldnet.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 August 2000; accepted in final form 28 February 2001.
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