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Published ahead of print on October 15, 2004, doi:10.1165/rcmb.2003-0456OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 59-64, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2003-0456OC

Elemental Composition of Airway Surface Liquid in the Pig Determined by X-Ray Microanalysis

Inna Kozlova, Viengphet Vanthanouvong, Birgitta Almgren, Marieann Högman and Godfried M. Roomans

Department of Medical Cell Biology, University of Uppsala, Uppsala, Sweden

Correspondence and requests for reprints should be addressed to Inna Kozlova, Department of Medical Cell Biology, University of Uppsala, Box 571, SE-75123 Uppsala, Sweden. E-mail: inna.kozlova{at}medcellbiol.uu.se


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The ionic composition of the airway surface liquid (ASL) is of importance in cystic fibrosis and exercise-induced asthma. However, literature data on the composition of the ASL vary markedly. The aim of the study was to determine the composition of the ASL, using two different methods involving minimal manipulation. In one method, the composition of the ASL was measured by X-ray microanalysis of frozen-hydrated samples. In the second method, small dextran beads were equilibrated with the ASL in a moisture chamber, isolated, dried, and analyzed. Plasma or serum from the same pigs was also analyzed. Both methods showed that the Na and Cl concentrations in the ASL are close to the concentrations of these ions in plasma. X-ray microanalysis of frozen-hydrated ASL showed significantly higher K, P, and S because here the upper layer (containing cell debris and secreted mucus) is sampled, whereas the bead method samples the watery component of the ASL. Ultrastructural analysis of the epithelium at various osmotic values showed evident damage at concentrations of 50 mM or less. These data support the notion that the physiologically important watery component of the pig ASL has an ionic composition close to that of plasma.

Key Words: airway surface liquid • cystic fibrosis • ion content • pig • X-ray microanalysis


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The airway surface liquid (ASL) is a thin layer of fluid covering the airways. The ASL secreted by airway epithelium and submucosal glands is the first line of defense of the airway epithelium and ensures the mucociliary transport of inhaled particles. ASL consists of mucus and an underlying periciliary watery layer, and 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 (1). and antimicrobial activity (2) in the airways. Studies of patients with genetic diseases of airway epithelial ion transport, e.g., cystic fibrosis (CF), have strongly suggested that the volume and/or composition of the liquid that lines the airway surface are important components of lung defense. The ionic composition of the airway surface liquid is assumed to be of considerable importance in diseases such as CF (2–6) and exercise-induced asthma (7, 8).

There are a number of theories regarding possible changes in composition and/or volume of the ASL in CF (reviewed in Ref. 9). The two most dominant theories are the "high salt hypothesis" and the "low volume hypothesis." The first (2, 4) claims that the ASL normally is hypotonic, which provides an optimal environment for the defensins, proteins that play a role in the defense against bacteria. According to this view, the ASL in patients with CF, while still hypotonic, would have a higher salt content than normal, and therefore a reduced activity of defensins. The second, on the other hand, claims that the ASL is normally isotonic, and that it is isotonic in patients with CF, but has a reduced volume, leading to the formation of viscous mucus that facilitates bacterial colonization (10; reviewed in Refs. 5 and 6).

However, it has been difficult to determine the exact composition of the ASL. Published data on the composition of the ASL are divergent and show a range from very hypotonic to hypertonic. Tracheal and/or bronchial ASL has been collected both from humans, and from other species, mainly from mouse (reviewed in Ref. 9). Published values for the ionic composition of human ASL are fairly closely together and range from 82–91 mM for Na+ and 82–108 mM for Cl– (11–14). For human xenographs in immunodeficient mice, however, lower values (64 mM Na+, 65 mM Cl–) have been reported (15). For the mouse the variation is very large: 6 mM Na+ and 1 mM Cl– was reported by Baconnais and coworkers (16), 87 mM Na+ and 57 mM Cl– by Cowley and colleagues (17), and values in the isotonic range (~ 115–120 mM Cl–) by several groups (18–21). For monkey and rabbit, values for Cl– of 112 and 114 mM have been found (21). High values were found for dogs, where Na+ concentrations ranged from 153–173 mM and the Cl– concentration was 134 mM (22), and in ferret where Na+ was 167 mM and Cl– 121 mM (23). Clearly hypotonic values were found for the rat, where Na+ was 41 mM and Cl– 45 mM (24). A number of different techniques for sampling and analysis have been used, and it would appear that, apart from possible species differences, one source of variation between the results is the sampling technique. In addition to the studies on mice mentioned above, Zahm and associates (25) give data in mmol/kg dry weight, which is not comparable with the absolute data in mM given in the other studies. Furthermore, studies have been performed on cell cultures of airway epithelial cells, but also these studies did not result in agreement being obtained. According to Matsui and coworkers (10), values for Na and Cl were nearly isotonic, whereas Zabner and colleagues (4) found values for Na and Cl around 50 mM, and McCray and associates (26) found a value for Cl of 18 mM. Widdicombe and coworkers (27) used X-ray microanalysis to measure the composition of ASL on top of cultured airway epithelial cells, but this study was purely qualitative.

Pig airways share many structural and physiologic similarities with human airways (28, 29) and are more similar to human airways than mouse airways (30). In the present study we sought to determine the composition of the ASL in pig airways, which to our knowledge has not been measured previously, by two different techniques: X-ray microanalysis of frozen-hydrated specimens, and X-ray microanalysis of ASL absorbed into small ion exchange beads. We also attempted to find indirect evidence for the osmotic value of normal ASL by experimentally exposing pig trachea to fluids with different osmotic values.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animals and Anesthesia
Ten healthy pigs of mixed breed (Hamshire, Yokeshire, and Swedish landrace; Skaggesta Gård, Uppsala, Sweden) with a body weight of 20–30 kg were used in this study. Before transport to the laboratory, premedication 40 mg azaperon (Stresnil; Janssen Pharmaceutical, Beerse, Belgium) was given by intramuscular injection. Anesthesia was induced with 0.5 mg atropine (Atropin; NM Pharma AB, Stockholm, Sweden) in a mixture of 100 mg tiletamin and 100 mg zolazepam (Zoletile forte vet; Virbac Laboratories, Carros, France) diluted in 5 ml medetomidin 1 mg ml–1 (Domitor; Orion Corporation, Farmos, Finland); 1 ml per 20 kg body weight intramuscularly. The animals were placed in supine position on a heating pad and intubated with a cuffed endotracheal tube 6.0 mm i.d. and ventilated mechanically in a volume- or pressure-controlled mode with 3 cm H2O PEEP (Siemens Servo 900C; Siemens-Elema AB, Solna, Sweden). The I:E ratio was 1:2. The tidal volume was 14 ml/kg plus compensation for dead space volume and the frequency adjusted to an end tidal CO2 of 5.5 kPa. A bolus injection of 0.2 mg fentanyl (Fentanyl; Antigen Pharmaceuticals, Roscrea, Ireland) was given intravenously after the intubation. Anesthesia was maintained by infusion of 5 ml kg/h of 4 g ketamin (Ketamin Veterinaria, Zrich, Switzerland), 1 mg fentanyl in 1,000 ml Rehydrex with glucose (Pharmacia and Upjohn, Stockholm, Sweden). The animals had no signs of respiratory infections or inflammation on visual inspection.

Frozen-Hydrated Pig Tracheae or Bronchi
The tracheae or bronchi were removed and dissected in a specially designed chamber, in which the humidity could be kept constant at close to 100%. The chamber consisted of a Perspex box (manufactured by the workhop, Biomedical Center, Uppsala University, Sweden), with a retractable plastic sheath on one side, which could be opened for handling of the specimen. A water reservoir at 37°C was placed at the bottom of the chamber, and the specimen was placed on a perforated shelf. Humidity was monitored with a hygrometer. Tissue pieces were frozen in liquid propane cooled by liquid nitrogen. The pieces were stored in liquid nitrogen until analysis. For analysis, the tissue pieces were placed with the mucosal side up onto a specially designed holder and transferred to a Philips (Philips Electron Optics, Eindhoven, The Netherlands) 525 scanning electron microscope equipped with a Bio-Rad (Hemel Hempstead, UK) Polaron 7400E cold stage. The samples were coated with a thin carbon layer in the cold stage, at a temperature of –190°C, and kept at this temperature throughout analysis. The lowest possible temperature was selected to minimize the risk of sublimation. After preliminary experiments, an accelerating voltage of 9 or 10 kV was chosen to minimize overpenetration of the beam. The samples were analyzed by a LINK (Oxford Instruments, Oxford, UK) AN 10000 energy-dispersive spectrometer system. Analysis was performed for 500 s with a beam size of 200 nm, a beam emission current of ~ 15 µA, a count rate of 230–235 cps, and a detector dead time of 5%. Typically, 8–10 analyses were performed per sample. For quantitative analysis, the data were compared with the results obtained on a standard. The standard consisted of a solution of 150 mM NaCl containing 5% albumin. The solution was smeared over an aluminum planchet to achieve a relative thin fluid layer, to mimic the situation for the ASL. In this way, mass loss due to irradiation by the electron beam can be assumed to be comparable between specimen and standard. The standard was shock-frozen, transferred to the cold stage of the electron microscope, and analyzed under the same instrumental conditions as the specimen. Quantitative analysis was performed using the ratio of characteristic to continuum intensity and by comparing this ratio with that obtained by analysis of the standard salt solution (31). Concentrations of elements other than Na and Cl were determined from binary standards containing these elements and Na or Cl, using the ratio method (31).

Pig Plasma and Serum
Venous and arterial blood was collected from the pigs, and plasma or serum was collected either after centrifugation or after clotting for 24 h at 4°C. Analysis of Na+, Mg2+, phosphate, Cl–, K+, and Ca2+ in the plasma or serum was performed using a Konelab 30 (Espoo, Finland) analyzer in the Laboratory of Clinical Chemistry, Swedish Agricultural University, Uppsala.

X-Ray Microanalysis of Sections of Trachea
For X-ray microanalysis of the epithelium, 16-µm-thick cryosections were cut on a conventional cryostat at –30°C, mounted on a carbon plate, freeze-dried inside the cryostat for 3 d, and gradually brought to room temperature during a period of 3 h. The sections were coated with a thin conductive carbon layer to prevent charging in the electron microscope (32). The sections were analyzed at room temperature at 20 kV with the instrumentation described above, using a beam size of 100 nm, an emission current of ~ 8 µA, a count rate of 500–700 cps, and a detector dead time of 8%. Quantitative analysis of the freeze-dried sections was performed using a standard consisting of known concentrations of salts in a 20% gelatin matrix, frozen, sectioned, and freeze-dried in the same way as the tissue (31).

Ion-Exchange Beads
Sephadex G-25 (diameter 20–40 µm; Pharmacia, Uppsala, Sweden) were spread evenly on the surface of the dissected pieces of the pig trachea and left during 30 min in the humidity chamber described above. Sephadex is the trade name for a cross-linked dextran gel with ion-exchange capacity. Saturation of the beads with a salt solution was obtained after 5 min (33). After absorption of the ASL, the beads were recovered by flushing with a hydrophobic volatile silicone oil (DC 200, 0.65 cSt; Dow Corning, Midland, MI) and collecting the beads in a watch-glass (34, 35). Under a preparation microscope, all adhering fluid and debris was removed from the beads, and single beads were transferred onto specimen grids, which had been submerged into the oil. The specimen grids used were nylon grids (Agar Scientific, Stansted, UK) covered with a thin Formvar (Merck, Darmstadt, Germany) film. The grid with beads was slowly lifted out of the oil bath and mounted onto an aluminum holder covered with round carbon adhesive tape and left at room temperature for evaporation of the oil. Grids with Sephadex beads were carbon coated before analysis. X-ray microanalysis of the beads was performed with the instrumentation described above, at 20 kV for 100 s with a beam size of 100 nm. Typically 10–12 beads were analyzed from each sample. For quantitative analysis, the data were compared with the results obtained on beads soaked in salt solutions of different concentrations (50–250 mM), and with beads soaked in serum from the same pigs and analyzed chemically.

Morphologic Studies
For morphologic studies, tissue was removed from the anesthetized animal and immediately fixed in 2.5% glutaraldehyde in water or different concentrations of sodium cacodylate buffer (0.025, 0.05, 0.1, or 0.15 mM). The tissues were kept in fixative for 24 h at 4°C and then postfixed with osmium tetroxide, dehydrated in a graded ethanol series, and embedded in epoxy resin. Sections (2 µm thick) were cut perpendicularly to the epithelium and stained with toluidine blue for light microscopy. The height of epithelial cells (50 cells for each fixative concentration) was measured by a semi-automatic image analysis system (VIDS; Synoptics, Cambridge, UK). Ultrathin sections were cut for electron microscopy, contrasted with uranyl acetate and lead citrate, and viewed at 75 kV in a Hitachi 7100 transmission electron microscope.

Pieces of pig trachea exposed for 30 min to Sephadex beads as described above were fixed in 2.5% glutaraldehyde in 0.1 mM sodium cacodylate overnight. Tissues were postfixed and embedded as described above. Thin sections (2 µm) were cut and stained with toluidine blue for light microscopy.

Statistics
Data are presented as mean ± SE. Differences between more than two groups were determined by ANOVA, and differences between two groups were determined using Student's t test.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
The results of the X-ray microanalysis of frozen-hydrated pig trachea, placed with the mucosal side up, are shown in Figure 1. Figure 2 shows an image of frozen-hydrated pig trachea, mounted sideways. This image allows an estimation of the thickness of the ASL layer, which varies between 50 and 100 µm. X-ray microanalysis of the specimen mounted sideways (in the frozen-hydrated state) gave results close to those obtained with the ASL pointing upwards (Figure 1). Results on pig bronchi (measured in the frozen-hydrated state with the mucosal side up) show somewhat higher concentrations for Na, Cl, and K compared with the trachea (Figure 1). In contrast, as expected, according to data from X-ray microanalysis of the epithelial cells in the frozen-dried state, P and K are the main intracellular elements, and intracellular Na and Cl concentrations are low (data not shown).



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  		Figure 1. Results of X-ray microanalysis of frozen-hydrated specimens. The data are given in mmol/kg wet weight and the bars represent the standard error. The following experiments are shown: pig trachea with ASL-covered epithelium upwards (black bars, data based on experiments from 9 pigs), pig trachea with ASL-covered epithelium sideways (white bars, data based on experiments from 4 pigs), and pig principal bronchi with ASL-covered epithelium upwards (gray bars, data based on experiments from 4 pigs). At least 10 measurements were performed on each pig.

 


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  		Figure 2. Scanning electron micrograph of frozen-hydrated pig trachea (mounted sideways). The ASL can be identified. Bar = 1 mm.

 
Transmission electron microscopy of pig trachea fixed in solutions of different osmolarity showed that at concentrations of 50 mM sodium cacodylate and less, evident damage to cellular organelles occurred, in comparison to the normal morphology at the highest concentrations (100–150 mM) of sodium cacodylate (Figure 3). At 50 mM, fluid filled vesicles are formed in the epithelial cells and at lower osmolarity, the mitochondria show clear signs of damage. In addition, it was observed that the cell size was dependent on the osmolarity of the fixative solution (Figure 4).



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  		Figure 3. Effect of the osmolarity of the buffer in the fixative on the ultrastructure of the cilated epithelial cells of pig trachea. The fixative was 2.5% glutaraldehyde in the vehicle: (A) 150 mM Na cacodylate buffer, (B) 50 mM cacodylate buffer, (C) water. At 50 mM Na cacodylate and below, fluid filled vesicles (v) are present in the cell, and the mitochondria (m) appear damaged. Bar = 1 µm.

 


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  		Figure 4. Effect of the osmolarity of the buffer in the fixative on the relative size of the ciliated epithelial cells of pig trachea. Results are expressed as relative length, with the control (150 mM Na cacodylate) as 100. Data are given as mean and standard error of five separate experiments, with 50 cells measured in each (n = 5). Significant differences from control are indicated by asterisks: * p < 0.01, ** p < 0.05.

 
When Sephadex beads are placed onto the trachea and left there for 30 min, the beads sink through the ASL layer and reach the tips of the cilia (Figure 5). Minor mechanical compression of the epithelium due to the weight of the beads can be observed, but the epithelium is still intact and continuous. It can be seen in this figure that the mucus layer contains cells or cell debris. Figure 6 shows the results of the X-ray microanalysis of Sephadex G-25 beads that had absorbed ASL for 30 min, compared with beads that had absorbed plasma or serum from the same pigs. The elemental concentrations in plasma were not significantly different from those in serum and the values were pooled. The Na and Cl concentration in the ASL are only little lower than in plasma. The K concentration is higher than in plasma, but lower than in the measurements on frozen-hydrated ASL. The concentrations of P and S are similar to the values from plasma but lower than in the measurements on frozen-hydrated ASL.



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  		Figure 5. Light microscopical image of a Sephadex G-25 bead (B) embedded in the mucus layer on pig tracheal epithelium (E). The mucus layer, especially the upper part, contains cells and cell debris (arrows).

 


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  		Figure 6. Results of the X-ray microanalysis of cross-linked Sephadex G-25 dextran beads after absorption of the ASL from surface of pig trachea (black bars), compared with the composition of pig plasma (white bars). The results are given in mM and the bars represent the standard error. The data are based on experiments from six pigs. Asterisks indicate significant differences between pig plasma and pig ASL (p < 0.001).

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The results indicate that in the pig, which is close to the human with respect to fluid transport in the airway, the Na+ concentration of the ASL is close to iso-osmolar. There are, however, major differences in the results of the ASL measured in situ in frozen-hydrated specimens, and the results obtained on the Sephadex beads. The ASL in situ contains substantially higher concentrations of, for example, P, S, and K than the ASL in the Sephadex beads. The elements P, S, and K are "cellular," rather than "extracellular" elements. It should be noted that in the data given in Figure 5, because of insufficient spatial resolution of analysis some extracellular space may be sampled, giving too high concentrations for Na and Cl. One could suspect that the measurements on the ASL in the frozen-hydrated state, underlying tissue, or at least cilia, are excited by the electron beam, and thus contribute to the spectrum. There are, however, several arguments against this notion.

First, the spatial resolution of analysis at 9 or 10 kV in a frozen-hydrated specimen can be calculated to be in the order of 2–3 µm (36). This is much less than the actual thickness of the ASL that can be measured from micrographs where the trachea is mounted sideways. Second, the results from the analysis where the trachea is mounted sideways, rather than with the ASL pointing upwards, give the same result, and in this situation overpenetration would not excite underlying tissue. Finally, if the results were due to overpenetration of the electron beam and excitation of the underlying epithelium, one would expect a negative correlation between, e.g., Na and P, or Na and K. A measurement with little or no overpenetration would sample the overlying ASL, and show high Na and low P and K, whereas a measurement with much overpenetration would mainly excite the epithelium and show high P and K and lower Na. Analysis of the data, however, shows that the correlation between Na and P is not negative (Figure 7), and the same result is obtained for the relation between Na and K (not shown).



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  		Figure 7. Correlation between Na and P in the X-ray microanalytical measurements of frozen-hydrated trachea (epithelium pointing upwards). Single data points from none pigs. The line drawn was determined by linear correlation.

 
The difference, then, between the measurements of ASL in situ in frozen hydrated specimens and in the beads must be due to the fact that actually different substances are measured. The measurements of the ASL in the frozen-hydrated specimens measure the upper (mucous) layer of the fluid, whereas the beads absorb the watery component of the layer. The upper part of the ASL likely contains glycoproteins and exfoliated cells and cell debris that could add "cellular" elements such as P, S, and K to the layer. The exfoliated cells and cell debris can be clearly seen in Figure 6. If mucus and debris are left on the beads, higher values for P, S, and K are obtained.

The results unequivocally confirm the notion that the Na and Cl concentrations in the watery phase of the ASL of the pig are close to those found in plasma. The Cl value of around 90 mM in the ASL in the beads agrees reasonably well with the data of Gilljam and coworkers (11), Joris and associates (12), Knowles and colleagues (13), and Hull and coworkers (14) for the human and confirms that the pig is a good model. The fact that the chloride concentration in the upper layer is somewhat lower might be due to the presence of negatively charged macromolecules in this layer that would tend to attract cations and repel anions. The total ion concentration in the ASL appears to be slightly higher than in serum. It could be argued that during analysis of the frozen-hydrated samples there is a risk for sublimation of the ice in the vacuum of the electon microscope, which would result in an increase of the ionic concentrations during analysis. To minimize this risk, the samples were kept at the lowest possible temperature, and the standard solution (a salt solution containing albumin to mimic the organic part of the ASL) was spread out in a thin layer to resemble the analytical conditions for the ASL. The fact that the concentrations of Na + K in the ASL are higher than in serum does not necessarily mean that the fluid is hypertonic, because the presence of large negatively charged organic macromolecules from mucus and cell debris will bind cations and lower their activity.

The morphologic data show that the cells swell when they are subjected to solutions with lower osmolarity than extracellular fluid, and even more important, show clear signs of damage at concentrations of 50 mM and less. Transmission electron microscopy shows the formation of vacuoles and the swelling of mitochondria, both typical but unspecific signs of cellular damage. The effective osmotic value of the fixatives used is close to the nominal osmotic value of the respective buffer solutions, because glutaraldehyde is a membrane-penetrating molecule and therefore does not contribute to the effective osmolarity of the solution. These data argue strongly against the notion that the ASL in the pig could be as hypotonic (less than 50 mM NaCl) as found by some groups for mouse (16) or rat (24). However, given the difference in airway wall structure between mouse and rat on one side and pig and human on the other, data for the pig cannot be transferred to mouse or rat.

The elemental composition of the watery layer of the ASL does, however, differ from that of plasma in a number of respects. Concentrations of Mg, P, and K are higher than in plasma. For K, where we find a concentration of 20 mM, some literature data are available. In human ASL, Joris and coworkers (12) found 29 mM and Knowles and colleagues (13) found 18 mM, which agrees well with our data. Published values for rat (24), mouse (16), and human xenografts in mice (15) are much lower, but these papers also give much lower values for the other ions. No literature data are available for other elements. Possibly, the source of the K in the fluid component of the ASL is the cell debris in the mucous layer that could leak K+ ions as well as small phosphate- and sulfur-containing molecules. A K+ concentration higher than plasma was also found in uterine secretions (37).

It can be concluded that the elemental composition of the ASL is different for the upper mucous layer, and the watery part. Physiologically, the most relevant data would be those from the watery layer close to the cell membrane. Hence, at least for the pig, our data would support the notion of Boucher (5, 6) that the ASL normally is isotonic.


   Acknowledgments
 
The authors gratefully acknowledge the skillful technical assistance of Leif Ljung, Marianne Ljungkvist, and Anders Ahlander.


   Footnotes
 
The study was supported by the Cystic Fibrosis Foundation, the Swedish Heart-Lung Association, the Swedish Science Research Council, the Swedish Asthma and Allergy Association, and the Swedish Association for Cystic Fibrosis.

Received in original form December 30, 2003

Received in final form August 19, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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I. Kozlova, H. Nilsson, M. Phillipson, B. Riederer, U. Seidler, W. H. Colledge, and G. M. Roomans
X-ray microanalysis of airway surface liquid in the mouse
Am J Physiol Lung Cell Mol Physiol, May 1, 2005; 288(5): L874 - L878.
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