Airway surface liquid composition in mice

Elizabeth A. Cowley, Karuthapillai Govindaraju, Claudine Guilbault, Danuta Radzioch, and David H. Eidelman

Meakins-Christie Laboratories, Montreal Chest Institute Research Centre, McGill University Health Centre, McGill University, Montreal, Quebec, Canada H2X 2P2


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Airway surface liquid (ASL) lines the conducting airways of the respiratory tract. We collected small samples of this liquid from the lower tracheae of anesthetized C57BL/6 mice and determined its ionic composition (in mM: 87.2 Na+, 4.7 K+, and 57.0 Cl-). Intravenous methacholine produced significant increases in the concentrations of Na+, K+, and Cl- within ASL. A limited analysis of liquid from cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice revealed no significant differences compared with littermate controls; however, Pseudomonas aeruginosa infection led to an increase in the salt concentration of ASL in cftr(+/+) mice. Morphometric measurements of tracheal submucosal gland volume revealed significant differences between inbred mouse strains, corresponding to ease of ASL collection. We conclude that although submucosal glands may be responsible for the production of some ASL, the ionic composition of this liquid is actively regulated by the underlying epithelial cells.

Pseudomonas aeruginosa; cystic fibrosis; submucosal gland


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AIRWAY SURFACE LIQUID (ASL) is a low-viscosity medium lining the conducting airways of the respiratory tract. Efficient mucociliary clearance of inhaled particles toward the oropharynx depends on the quantity of ASL (also termed periciliary fluid or the "sol" layer) because variations in liquid height will interfere with the mechanical coupling between cilia and the overlying mucus (or "gel") blanket. ASL also contains a number of proteins, including lactoferrin (28, 41), lysozyme (6, 41), and the defensins (15, 35), that contribute to pulmonary host defense by virtue of their potent antibacterial properties. There is evidence that the ionic composition of ASL may also play a fundamental role in protecting the lung from bacterial insult, inasmuch as an ASL of high ionic strength has been reported to reduce the ability of airway epithelial cells to kill bacteria (36). Such a mechanism could explain the excessive bacterial colonization and infection of the lungs of cystic fibrosis (CF) patients with bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus, infections that continue to be the major cause of morbidity and mortality among these patients. CF is associated with mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), the product of which is a Cl- channel present on the apical surface of a variety of epithelial cells. Lack of functional CFTR Cl- channels and the consequent defective ion conductances across airway epithelia could lead to alterations in the ionic composition of the liquid lining the airways and inhibit constitutive host defense activity.

We have reported previously the ionic composition of rat ASL and found it to be lower in salt compared with plasma (10, 17). This supports earlier observations of ASL composition in humans (20) but differs from data reported from analysis of liquid from dogs and ferrets (4, 31), which found ASL hypertonic or isotonic. In the present study, we investigated the ionic composition of mouse ASL in order to make use of the various murine models that have been developed. We measured the ionic composition of ASL from mice infected with P. aeruginosa as well as from a limited number of CFTR knockout mice (B6-CftrUNC-/-) in which the gene encoding the CFTR protein has been disrupted. Additionally, we systematically examined the presence of submucosal glands in different inbred mice strains to determine whether these glands could be a major source of ASL and whether genotypic differences between strains could lead to differences in the quantity of ASL produced.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mice. Male specific pathogen-free inbred mice (C57BL/6, A/J, and BALB/c), 8-12 wk old, were obtained from a commercial animal facility (Harlan Sprague Dawley; Indianapolis, IN).

CF mice. CFTR-knockout mice developed at the University of North Carolina (37) were established on a C57BL/6 background, as described by Gosselin et al. (16). Briefly, heterozygous CFTR knockout (+/-) mice were backcrossed for several generations to C57BL/6 mice, and at each generation mice with the highest level of homozygosity for C57BL/6 background were selected by single sequence length polymorphism analysis and again backcrossed to C57BL/6. These mice are designated B6-CftrUNC-/- and have previously been demonstrated to be more susceptible than littermate controls to infection with P. aeruginosa (16).

Infection with P. aeruginosa. Beads were prepared as previously described (11). Briefly, a suspension of P. aeruginosa 508 grown to late log phase (~103 to 104 colony-forming units) was diluted in trypticase soy agar at 52°C, added to heavy mineral oil (Fisher Scientific; Ottawa, ON) and stirred for 6 min at 20°C. This mixture was then cooled with ice for 10 min. This cooling resulted in bacteria-containing beads less than 200 µm in diameter. These beads were then isolated by centrifugation at 9,000 g for 20 min at 4°C and suspended in phosphate-buffered saline. The density of viable bacteria within the beads was determined by plating serial dilutions of homogenized bead suspension onto plates containing trypticase soy agar. The bacteria-bead suspension was then diluted to a density of 2-10 × 105 colony-forming units immediately before infection of the mice. C57BL/6 mice were anesthetized with an intramuscular injection of ketamine hydrochloride (75 mg/kg) and xylazine (30 mg/kg). The trachea was exposed, and a 22-gauge catheter (Criticon; Tampa, FL) was inserted so that 50 µl of the bead suspension followed by 50 µl of air could be introduced into the animal. The incision was sutured after the inoculation procedure, and ASL was collected from mice 5 days after the infection protocol.

Sampling murine ASL. All mice (C57BL/6, A/J, BALB/c, B6-CftrUNC-/-, B6-CftrUNC+/+, and P. aeruginosa-infected B6-CftrUNC-/- and B6-CftrUNC+/+) were sedated with xylazine (0.08 ml/100 g body wt) and then anesthetized with pentobarbital sodium (0.053 ml/100 g body wt) injected intraperitoneally. Mice were then tracheotomized, and the sampling capillary (a length of PE-10 tubing; Becton Dickinson; Sparks, MD) was inserted into the trachea so that its end lay in contact with the epithelium at the base of the trachea. This sampling capillary remained in situ for ~30 min, after which it was removed from the animal and frozen at -80°C for later analysis of the contained liquid by capillary electrophoresis (CE). In preliminary experiments, no difference was found in salt concentrations when freshly harvested or frozen samples were used (data not shown).

To exclude the possibility of damage by the sampling capillary, we examined the tracheae of 5 C57BL/6 mice histologically. We found no evidence of damage to the epithelium (data not shown). The volume of ASL typically collected in this time period was ~100-200 nl, with preliminary experiments indicating that 30 min was the minimum time required for a large enough sample to accumulate for analysis by CE. Volume was approximated by determining the length of accumulated ASL within the PE-10 tubing and comparing it to known measured volumes.

Administration of methacholine. To determine the contribution of stimulated submucosal glands to ASL composition, 33 or 100 ng methacholine (MCh)/g body wt (ICN Chemicals; Mississauga, ON) were injected into the tail vein of C57BL/6 mice. We previously demonstrated that the larger of these doses produced a significant change in the ionic composition of rat ASL (10). The collection capillary was inserted immediately after administration of the drug, and ASL was collected for the next 30 min.

CE analysis of ASL. The ionic composition of ASL was determined using CE with two different detection systems: CE with a conductivity detector and indirect ultraviolet (UV) detection for anions, whereas CE with indirect UV detection alone was performed for cations. All analysis buffers were prepared fresh daily in deionized water (Milli-Q unit, Millipore; Montreal, PQ), degassed by sonication, and filtered through 0.45-µm membrane filters (Gelman Sciences; Montreal, PQ). The capillary oven temperature was set at 30°C.

Anion analysis. ASL was analyzed using a Crystal CE system with a Concap capillary and Contip conductivity sensor (ATI Unicam; Boston, MA) modified to allow direct injection of ASL samples (~5 nl of sample per injection) or with a CE unit (270A; Applied Biosystems; Foster City, CA) with an indirect UV detector. Data were collected with an integrator (model SP4600, Spectra-Physics; San Jose, CA) and analyzed using Spectra-Physics Winner software (see Ref. 17 for technical details of CE setup). The buffer for conductivity detection contained 100 mM 2-(N-cyclohexylamino)ethanesulfonic acid, 40 mM lithium hydroxide (Sigma), and 2-propanol (Fisher Scientific; Nepean, ON) at 92:8 (vol/vol), pH 9.3, with 80 µM spermine (Sigma) as an electro-osmotic flow modifier. The separation potential was -278 V/cm (current = 8 µA). For analysis with indirect UV detection, the buffer contained 5 mM chromate and 1.5 mM hexamethonium hydroxide at pH 7.0, adjusted with 1 M H2SO4. A vacuum of 17 kPa was applied for 1.5 s for sample injection (~3 nl). Separation was carried out at -347 V/cm (current = 12 µA) with detection at 273 nm.

Cation analysis. ASL was analyzed with a 270A unit (Applied Biosystems; Foster City, CA). A vacuum of 17 kPa was applied for 1.5 s for sample injection (~3 nl), and analysis was performed in 10 mM imidazole with 8% (vol/vol) 2-propanol adjusted to pH 3.5 with 1 M HCl. The separation potential was 347 V/cm (current = 11 µA) with detection at 214 nm.

Calibration was carried out by determining the peak area under the curve of known concentrations of the ion in question. This method was linear over the concentration range of interest, and regression coefficients between r = 0.997 and 0.999 were obtained in all cases. In addition to running calibration standards, samples of mouse plasma were also collected from C57BL/6 mice and analyzed by CE as described above.

Morphometric measurement of submucosal glands. In the course of developing our technique for harvesting ASL, we observed differences in our ability to harvest ASL among inbred mouse strains. Preliminary studies suggested that inbred strains of mice differed in the amount of tracheal gland tissue and that strains difficult to harvest fluid from had small tracheal glands. To confirm this formally, we investigated the volume of tracheal submucosal glands as follows. After ASL was harvested, five mice from the C57BL/6, A/J, and BALB/c strains were terminally anesthetized (pentobarbital sodium) and the tracheae were removed and fixed in 10% buffered Formalin. After paraffin embedding, 5-µm serial sections were cut and stained with Masson's trichrome. Every 20th section was examined with a Leitz light microscope equipped with a drawing tube attachment, and the image was projected onto a digitizing board (Jandel Scientific; Corte Madera, CA). Submucosal gland area was then calculated from each digitized image using commercially available software (Sigma Scan, Jandel Scientific). The total glandular volume of each animal was calculated by assuming a cylindrical model, i.e., by multiplying the glandular area at each level by the total distance to which glandular tissue extended along the trachea.

Statistics. All data are expressed as means ± SE. A difference was considered statistically significant at P < 0.05 as analyzed by one-way ANOVA or t-test as appropriate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Rate of success of collection technique. Harvesting ASL from mice presents a major technical problem because of the small size of these animals. In preliminary experiments, we screened three different inbred strains of mice (C57BL/6, A/J, and BALB/c) but could only reproducibly obtain sufficient liquid for analysis from C57BL/6 mice. Of 24 A/J mice, samples of ASL were collected from only 7 (25% success rate), whereas for BALB/c mice, ASL was collected from only 2 of 11 animals (18%). This is in comparison to a success rate of 48% for C57BL/6 mice (25 samples from 52 mice). However, this rate of success reflects only the presence of ASL within the sampling capillary. For C57BL/6 mice, the amount of ASL collected was considerably larger than that harvested from A/J or BALB/c mice. Preliminary experiments with standard solutions as well as with ASL samples indicated that a minimum volume of ~100 nl of ASL was necessary for accurate and reproducible analysis. Although some samples of ASL from A/J and BALB/c mice were collected, they were less than the minimum volume required for analysis by CE using our protocols.

Normal murine ASL composition. The values for the ions detected in murine ASL and plasma are shown in Table 1. Although results in plasma were as expected, the major ionic species Na+ and Cl- were significantly lower in ASL than they were in plasma. This finding is consistent with previous reports of low ASL salinity in other species, including measurements from the rat collected in vivo (10) and the horse collected in vitro (21). Perhaps more importantly, ASL collected from human subjects has been reported repeatedly to have lower Na+ and Cl- levels than those in plasma (14, 18, 20, 24). In all of these reports (except Ref. 14, which reports only Cl- values), the Na+ from normal ASL was reported to be between 80 and 85 mM. Therefore, our finding of 87.2 ± 3.0 mM Na+ is in agreement with previous reports. We did, however, find a considerably lower Cl- value than has been reported by others (57.0 ± 3.0 mM vs. a range of 84-108 mM) (14, 18, 20, 24), with the presence of a considerable anion gap (30 mM). Additionally, we found considerably lower values for K+ in ASL than previously reported (4.7 ± 0.4 vs. 20-29 mM), with approximately equivalent values between ASL and plasma. Given that osmolarity is often determined from two times Na+ and K+ concentration, the apparent osmolarity of this liquid was 183 mosM compared with ~250 mosM for plasma. However, it is important to note that we were not able to directly measure osmolarity. We additionally report values for HCO-3 and PO3-4 to be lower in mouse ASL than in plasma, while SO2-4 levels are higher.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Composition of mouse ASL from C57BL/6 mice

Ionic composition of ASL from B6-CftrUNC-/- and B6-CftrUNC+/+ mice. ASL from CF patients has been reported to exhibit either elevated salinity (14, 20) or identical levels of Na+ or Cl- compared with normal subjects (18, 24). To evaluate the contribution of functional CFTR Cl- channels to ASL composition, we attempted to collect ASL from a knockout mouse strain raised on a C57BL/6 background in which the gene encoding the CFTR protein has been disrupted. Although we successfully sampled ASL from four of eight B6-CftrUNC-/- mice (a similar success rate to normal C57BL/6 mice), we were only able to collect a sufficient quantity of ASL for reproducible CE analysis from two of these mice. Results were similar between B6-CftrUNC-/- mice (Na+, 84.8 ± 5.7 mM; K+, 4.8 ± 0.01 mM; Cl-, 49.1 ± 18.9 mM; HCO-3, 4.8 ± 1.6 mM) and their littermate B6-CftrUNC+/+ controls (Na+, 85.6 ± 15.8 mM; K+, 5.2 ± 1.1 mM; Cl-, 66.2 ± 7.8 mM; HCO-3, 5.1 ± 0.7 mM).

Ionic composition of ASL from P. aeruginosa-infected mice. CF lungs are commonly infected with the bacteria P. aeruginosa. To determine whether the presence of Pseudomonas and the consequent inflammatory environment it induces affect the ionic composition of ASL, we compared the composition of liquid from infected B6-CftrUNC-/- (n = 3) and B6-CftrUNC+/+ mice (n = 3) vs. noninfected controls. As shown in Fig. 1, the values for ions (in mM) in infected B6-CftrUNC+/+ and B6-CftrUNC-/- mice, respectively, were 131.76 ± 20.01 and 116.13 ± 23.43 for Na+, 12.97 ± 1.77 and 31.52 ± 3.52 for K+, and 91.95 ± 8.65 and 63.0 ± 2.5 for Cl-. Thus higher values tended to be seen in infected vs. noninfected animals, although there were no significant differences between B6-CftrUNC+/+ and B6-CftrUNC-/- mice. Only the values in B6-CftrUNC+/+ mice were significantly different from those in control animals.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of Pseudomonas aeruginosa (PA) infection on ionic concentration (conc) of murine airway surface liquid (ASL; n = 3 mice/group). * P < 0.05 vs. control determined by Student's t-test. CFTR, cystic fibrosis transmembrane conductance regulator.

Treatment with MCh. There are conflicting reports on whether cholinergic stimulation affects ASL composition (4, 10, 21). We examined the effects of MCh (33 and 100 ng/g body wt) on Na+, K+, and Cl- concentrations within ASL. The 33 ng MCh/g body wt had no effect on the ionic composition of the ASL (Cl-, 58.12 ± 3.4 mM vs. the control value of 57.0 ± 2.9 mM). As shown in Fig. 2, administration of 100 ng MCh/g body wt (n = 6) increased the Na+ concentration 69% (87.2 ± 3.0 to 147.6 ± 3.3 mM); the K+ concentration 223% (4.7 ± 0.4 to 15.2 ± 1.8 mM); and the Cl- concentration 77% (57.0 ± 2.9 to 101.0 ± 3.0 mM). MCh administration also resulted in an increased quantity of liquid, as judged by the size of the samples obtained and the relative ease of collection. However, it was not possible to quantitate this apparent increase in liquid production.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of intravenous administration of methacholine (MCh; 100 ng MCh/g body wt) on ionic concentration of ASL from C57BL/6 mice (n = 6). * P < 0.05 determined by Student's t-test.

Submucosal glands. Submucosal glands (Fig. 3, A and B) were detected in all animals examined, situated immediately below the larnyx at the most proximal portion of the trachea. There were significant differences in submucosal gland volume and the length to which those glands extended along the trachea between mouse strains. C57BL/6 mice had a significantly higher volume of glandular material present in their tracheae (0.12 ± 0.02 mm3) compared with both A/J (0.04 ± 0.01 mm3) and BALB/c (0.06 ± 0.01 mm3) mice (Fig. 4A). Additionally, this glandular material extended further along the trachea in C57BL/6 mice compared with A/J mice (0.81 ± 0.07 vs. 0.60 ± 0.09 mm, respectively; Fig. 4B). The glands of BALB/c mice extended as far down the length of the trachea as did those of the C57BL/6 animals (0.80 ± 0.08 vs. 0.81 ± 0.07 mm).


View larger version (157K):
[in this window]
[in a new window]
 
Fig. 3.   Submucosal gland tissue in C57BL/6 (A) and A/J (B) mice at 350 µm below larynx. Paraffin sections (5 µm) were cut and stained with Masson's trichrome. Scale bar, 100 µm.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4.   Strain-related differences in total submucosal gland volume (A) and distance (B) that those submucosal glands extend into trachea when C57BL/6, A/J, and BALB/c mice (5/group) are compared. * P < 0.05 between each group determined by one-way ANOVA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Before discussing our results, it is important to consider the technical limitations of harvesting ASL from the mouse. In any study of ASL composition, the method used for sample collection is an important consideration. The widely used method of sampling with filter paper (4, 18, 20, 24) is not easily adaptable to small-animal work. After careful consideration of a number of approaches, we chose to adapt our previously described technique for collecting rat ASL (10) to the mouse. Despite the previous success with rats, the much smaller caliber airways of these animals presented a series of problems that forced us to alter our initial procedure and devise the protocol outlined here. Most notably, the length of time required to collect ASL was increased by a factor of 10 (from 3 to 30 min). It was also necessary to tracheotomize the mice because the presence of a catheter inserted via an intubation tube presented too high an airway resistance to permit continued spontaneous breathing. Even with these modifications, the success rate of ASL collection was considerably lower in the present study than in that done in the rat. Thus interpretation of our results must be done in the context of imperfect data collection. Nevertheless, we believe that the novel results reported here provide important information concerning ASL composition and are reproducible and consistent with those in other species.

Our principal observation is that murine ASL has a lower salinity than plasma. This finding is in general accordance with previous results from other species both from our (10, 17) and other laboratories (18, 20, 24), although some studies of dog and ferret tracheal ASL have reported higher salinity (4, 31). The consistent finding of subplasma salt concentrations in the airway tree implies that ASL composition is actively regulated, presumably by the respiratory epithelial cells lining the airways. Nevertheless, there is considerable variability from study to study with regard to the precise salt concentrations found. Although variations in the collection methods and analytic techniques employed may account for some of these discrepancies, it is likely that differences do occur in ASL composition both among different anatomic sites and also between species. With regard to the importance of anatomic differences, fluid harvested from the nose in humans with filter paper exhibits salinity close to that in plasma (24). In contrast, in the same study, the salt concentrations in ASL from the central airways were less than that in plasma (24), although not as low as we have found in rodents. Thus differences among anatomic sites as well as species differences seem likely to account for the differences in ASL salt concentrations in different studies. Indeed, our previously reported values for rat tracheal ASL (Na+, 40.57 ± 3.08 mM, and Cl-, 45.16 ± 1.81 mM; Ref. 10) differ from those reported here, even though samples were collected from the same part of the airway using a similar collection technique and an identical analytic technique.

An important difference between our findings and those made with other techniques is that we consistently observe equivalent levels of K+ within plasma and ASL harvested from healthy rodents. In contrast, most other studies report higher K+ values within ASL (4, 20, 21, 24). This discrepancy may reflect physiological effects, particularly species differences. There have been reports from in vitro preparations that respiratory epithelia actively secrete K+ (5, 7, 9). However, there have been no reports to date of K+ measurements across rodent airway epithelia. Another possibility is that the high K+ values reported in other studies may result from differences in sampling techniques. For example, the more commonly used approach of applying thin strips of filter paper directly onto the surface of epithelial cells may promote the release of intracellular K+. We have not observed increases in ASL K+ except after MCh or in Pseudomonas-infected mice in which some element of epithelial dysfunction or damage might be expected. The narrow-bore capillary tubing we used to collect ASL appears to exert a low hydrostatic pressure gradient on the epithelium (~1 cmH2O; Ref. 25). Direct application of filter paper to the epithelium to collect ASL results in a significant hydrostatic pressure gradient, which has been reported to disturb the barrier function of the epithelium and result in a large flow of subepithelial (i.e., interstitial) liquid to the epithelial cell surface (12). Because of our use of the narrow-bore tubing and the extended length of time that was necessary to collect this liquid, we are confident that our results reflect the composition of ASL rather than that of other liquid compartments.

In the limited number of ASL samples from B6-CftrUNC-/- that we were able to measure, we found no apparent differences in the ionic composition of ASL compared with that from littermate controls. Unfortunately, of the mutant mice we were able to screen, we were only able to collect sufficient ASL for analysis from two B6-CftrUNC-/- mice. The difficulties associated with harvesting ASL from these mice in part reflect the smaller size of these mice compared with others. Alternatively, it is possible that lack of functional CFTR somehow reduces the quantity of ASL without affecting its composition. In this regard, Ballard and colleagues (1) recently reported that in porcine bronchi, cholinergic-induced gland liquid secretion is mediated by CFTR. Thus to the extent that gland secretion contributes to the volume of ASL available for harvest, difficulties in harvesting ASL from B6-CftrUNC-/- mice potentially could reflect a physiological rather than an anatomic limitation in harvesting fluid from these mice. In any case, it is unclear whether murine data can ever be used to determine the importance of CFTR in determining ASL salt concentrations. The various murine models of CF, developed by either interruption or deletion of the cftr gene, do not spontaneously display the pulmonary pathology characteristic of the human disease. Mice may possess compensatory Cl- conductances, such as a Ca2+-activated Cl- channel, that negate the functional loss of CFTR (8). It is also possible that other types of "modifier genes" act to prevent full development of the pulmonary CF phenotype in the mouse (32).

Our observation that infection with P. aeruginosa results in higher levels of Na+, K+, and Cl- within ASL is consistent with the earlier report of Joris et al. (20) who found increased levels of these ions in ASL from acutely infected patients. P. aeruginosa decreases both short-circuit current across airway epithelia (39) and amiloride-sensitive Na+ absorption of liquid (13), although the precise mechanism responsible for such alterations in ion transport pathways is unknown. Additionally, the presence of P. aeruginosa results in the production of proinflammatory cytokines such as tumor necrosis factor-alpha (3), which is implicated in the increased intestinal permeability seen during hypoxia (40). Therefore, P. aeruginosa infection may also act to alter airway epithelial integrity in response to infection, resulting in the presence of plasmalike levels of Na+ and Cl- in the ASL. Alterations in epithelial permeability and ion transport pathways could account for differences in the ionic composition of ASL after P. aeruginosa infection, the effects of which may be to further impede the innate antimicrobial activity of this liquid (see below) and encourage bacterial proliferation.

Our finding that intravenous administration of the cholinergic agonist MCh resulted in the production of an increased volume of isotonic ASL also suggests that submucosal glands contribute to the composition of ASL, at least in the region we sampled. Among the many reported effects of cholinergic stimulation are increases in active ion transport (33) and production of liquid from submucosal glands (26). Wu et al. (43) recently reported that application of MCh to bovine trachea stimulated glands to produce copious amounts of liquid, significantly increasing the depth of ASL. This increase in liquid depth was, however, transient and was followed by a slow return to baseline values driven by reabsorption of liquid across the epithelium. In our study, MCh application resulted in an increased production of liquid together with the release of mucus from submucosal glands present immediately below the larynx. The increases we report in the tonicity and volume of MCh-stimulated liquid are consistent with increased glandular secretion.

Although ASL was generally difficult to harvest from mice, the rate of ASL collection was critically related to the strain chosen. We found significant differences in the overall volume of submucosal glands among different mouse strains, and these differences correlated with the ease of collection of ASL. This correlation strongly suggests that tracheal submucosal glands contribute to the volume of ASL produced under basal conditions. Although the source of ASL remains to be conclusively determined, there would appear to be a limited number of possibilities. These include the lung periphery where ASL is swept along by mucociliary clearance, being reabsorbed as it moves more distally; the underlying airway epithelial cells themselves; or submucosal glands. Our data suggest that the glands may play an important role, at least in determining the volume of ASL available for harvest. The total volume and distribution of airway submucosal glands are known to be species related; for example, whereas numerous glands are present throughout the human respiratory tract, in other species (e.g., rat and rabbit) glands are thought to be scarce or even absent (34). In the mouse, submucosal glands are present only in the upper trachea (30) proximal to the distal tip of our sampling capillary. Nevertheless, the apparent relationship between gland volume and ease of harvesting of fluid suggests that the submucosal glands may be responsible for producing the ASL we analyzed. Inasmuch as any technique for harvesting ASL potentially stimulates reflex gland secretion, it is difficult to determine with confidence whether this reflects physiological submucosal gland ASL production or is an artifact of inadvertent gland stimulation (19).

The importance of accurately defining the ionic composition of ASL relates to its potential role in the pathogenesis of certain lung diseases, of which CF is the most obvious example. In the CF lung, it has been proposed that loss of functional CFTR Cl- channels leads to excessive Na+ absorption coupled with a decrease in Cl- secretion (22, 23, 38), resulting in an overall loss of liquid at the apical surface of the respiratory epithelium. Thus lung defense mechanisms are impaired in CF because loss of liquid results in a less hydrated mucus and decreased mucociliary clearance. This model therefore predicts that ASL will be isotonic to plasma. However, an alternate hypothesis (36) suggests that Na+ and Cl- are absorbed across the respiratory epithelium in excess of water such that ASL is normally hypotonic. CF therefore may be marked by an inability to reabsorb Cl- via CFTR (thus reducing the driving force for Na+ reabsorption), resulting in an ASL with elevated salinity. The salinity of ASL may be crucially important because this medium contains various salt-dependent antimicrobial factors such as the defensin hBD-1 (15) and the cathelicidin LL-37 (2). These factors display decreasing activity with increased salinity; therefore, an ASL with increased NaCl would predispose the CF lung to bacterial colonization. A recent report from Zabner et al. (44) supports this scenario. This group found that the ASL lining cultured human CF epithelial cells contains more NaCl than that from normal cells, leading them to suggest that the CFTR Cl- channel seems to be required for maximal transcellular absorption of Cl-. Nevertheless, this hypothesis remains controversial. Although elevated salt levels have been found in the ASL of CF patients (14, 20), these data conflict with more recent reports that ASL salinity in CF infants (18) and adults (24) is similar to that in healthy controls. Furthermore, the mechanism by which a relatively water-permeable epithelium maintains a hypotonic solution at its surface remains to be elucidated. The potential capillarity effects of the cilia to retain water (42) and the possible role of undefined osmolytes in the ASL must be considered as potential explanations.

In summary, we report an analysis of the ionic composition of murine ASL. Although we found no differences in the ASL salinity from normal vs. CFTR knockout mice, significant increases in the concentration of Na+, K+, and Cl- became apparent after infection with P. aeruginosa. Additionally, we report that interstrain differences exist between mouse strains in terms of the amount of tracheal submucosal gland tissue present. A correlation between the ease of collection of ASL and the overall volume of these glands suggested that tracheal submucosal glands may contribute to the production of basal ASL. When glands were stimulated with MCh, a copious isotonic liquid was produced. Our finding of a lower-salinity ASL suggests that the liquid produced by submucosal glands is actively regulated by the underlying epithelial cells. However, when the system is maximally stimulated with a large dose of MCh, the regulatory mechanisms are temporarily overwhelmed, allowing the collection of a higher tonicity liquid than normal.


    ACKNOWLEDGEMENTS

This work was supported by the Canadian Cystic Fibrosis Foundation. D. H. Eidelman is the recipient of a Chercheur-Boursier Award from the Fonds de la Recherche en Santé du Québec, and E. A. Cowley is a Canadian Cystic Fibrosis Foundation Fellow.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. H. Eidelman, Meakins-Christie Laboratories, Montreal Chest Inst. Research Centre, McGill Univ. Health Centre, McGill Univ., 3626 St. Urbain St., Montreal, PQ, H2X 2P2 Canada (E-mail: david{at}meakins.lan.mcgill.ca).

Received 16 April 1999; accepted in final form 17 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ballard, ST, Trout L, Bebok Z, Sorscher EJ, and Crews A. CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands. Am J Physiol Lung Cell Mol Physiol 277: L694-L699, 1999[Abstract/Free Full Text].

2.   Bals, R, Wang X, Zasloff M, and Wilson JM. The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proc Natl Acad Sci USA 95: 9541-9546, 1998[Abstract/Free Full Text].

3.   Bonfield, TL, Panuska JR, Konstan MW, Hilliard KA, Hilliard JB, Ghnaim H, and Berger M. Inflammatory cytokines in cystic fibrosis lungs. Am J Respir Crit Care Med 152: 2111-2118, 1995[Abstract].

4.   Boucher, RC, Stutts MJ, Bromberg PA, and Gatzy JT. Regional differences in airway surface liquid composition. J Appl Physiol 50: 613-620, 1981[Abstract/Free Full Text].

5.   Boucher, RC, Stutts MJ, and Gatzy JT. Regional differences in bioelectric properties and ion flow in excised canine airways. J Appl Physiol 51: 706-714, 1981[Abstract/Free Full Text].

6.   Bowes, D, and Corrin B. Ultrastructural immunocytochemical localization of lysozyme in human bronchial glands. Thorax 32: 163-170, 1977[Abstract].

7.   Clarke, LL, Chinet T, and Boucher RC. Extracellular ATP stimulates K+ secretion across cultured human airway epithelium. Am J Physiol Lung Cell Mol Physiol 272: L1084-L1091, 1997[Abstract/Free Full Text].

8.   Clarke, LL, Grubb BR, Yankaskas JR, Cotton CU, Mc- Kenzie A, and Boucher RC. Relationship of a noncystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in Cftr(-/-) mice. Proc Natl Acad Sci USA 91: 479-483, 1994[Abstract].

9.   Cotton, CU, Lawson EE, Boucher RC, and Gatzy JT. Bioelectric properties and ion transport of airways excised from adult and fetal sheep. J Appl Physiol 55: 1542-1549, 1983[Abstract/Free Full Text].

10.   Cowley, EA, Govindaraju K, Lloyd DK, and Eidelman DH. Airway surface fluid composition in the rat determined by capillary electrophoresis. Am J Physiol Lung Cell Mol Physiol 273: L895-L899, 1997[Abstract/Free Full Text].

11.   Cowley, EA, Wang C-G, Gosselin D, Radzioch D, and Eidelman DH. Measurements of mucociliary clearance in transgenic cystic fibrosis mice infected with Pseudomonas aeruginosa. Eur Respir J 10: 2312-2318, 1997[Abstract/Free Full Text].

12.   Erjefalt, I, and Persson CG. On the use of absorbing discs to sample mucosal surface liquids. Clin Exp Allergy 20: 193-197, 1990[ISI][Medline].

13.   Evans, DJ, Matsumoto PS, Widdicombe JH, Li-Yun C, Maminishkis AA, and Miller SS. Pseudomonas aeruginosa induces changes in fluid transport across airway surface epithelia. Am J Physiol Cell Physiol 275: C1284-C1290, 1998[Abstract/Free Full Text].

14.   Gillijam, H, Ellin A, and Strandvik B. Increased bronchial chloride concentration in cystic fibrosis. Scand J Clin Lab Invest 49: 121-124, 1989[ISI][Medline].

15.   Goldman, MJ, Anderson GM, Stolzenberg ED, Kari UP, Zasloff M, and Wilson JM. Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 88: 553-560, 1997[ISI][Medline].

16.   Gosselin, D, Stevenson MM, Cowley EA, Greisenbach U, Eidelman DH, Boule M, Tam M-F, Kent G, Skamene E, Tsui L-C, and Radzioch D. Impaired ability of Cftr knockout mice to control pulmonary infection with Pseudomonas aeruginosa. Am J Respir Crit Care Med 157: 1253-1262, 1998[Abstract/Free Full Text].

17.   Govindaraju, K, Cowley EA, Eidelman DH, and Lloyd DK. Microanalysis of lung airway surface fluid by capillary electrophoresis with conductivity detection. Anal Chem 69: 2793-2797, 1997[ISI][Medline].

18.   Hull, J, Skinner W, Robertson C, and Phelan P. Elemental content of airway surface liquid from infants with cystic fibrosis. Am J Respir Crit Care Med 157: 10-14, 1998[Abstract/Free Full Text].

19.   Johnson, JG, Robinson J, Foy C, Gatzy JT, Boucher RC, and Knowles MR. Bronchial airway surface liquid (ASL) ion composition in normal subjects (Abstract). Am J Respir Crit Care Med 159: A293, 1999[ISI].

20.   Joris, L, Dab I, and Quinton PM. Elemental composition of human airway surface fluid in healthy and diseased airways. Am Rev Respir Dis 148: 1633-1637, 1993[ISI][Medline].

21.   Joris, L, and Quinton PM. Filter paper equilibration as a novel technique for in vitro studies of the composition of airway surface fluid. Am J Physiol Lung Cell Mol Physiol 263: L243-L248, 1992[Abstract/Free Full Text].

22.   Knowles, MR, Oliver K, Hohneker KW, Robinson J, Bennett WD, and Boucher RC. Pharmacologic treatment of abnormal ion transport in the airway epithelium in cystic fibrosis. Chest 107: 71S-76S, 1995[Abstract/Free Full Text].

23.   Knowles, MR, Oliver K, Noone P, and Boucher RC. Pharmacologic modulation of salt and water in the airway epithelium in cystic fibrosis. Am J Respir Crit Care Med 151: S65-S69, 1995[ISI][Medline].

24.   Knowles, MR, Robinson JM, Wood RE, Pue CA, Mentz WM, Wager GC, Gatzy JT, and Boucher RC. Ion composition of airway surface liquid of patients with cystic fibrosis compared with normal and disease-control subjects. J Clin Invest 100: 2588-2595, 1997[Abstract/Free Full Text].

25.   Landry, J, Cowley EA, and Eidelman DH. Techniques for ASL collection: role of capillarity (Abstract). Am J Respir Crit Care Med 159: A678, 1999[ISI].

26.   Leikauf, GD, Ueki IF, and Nadel JA. Autonomic regulation of viscoelasticity of cat tracheal gland secretions. J Appl Physiol 56: 426-430, 1984[Abstract/Free Full Text].

27.   Lentner, C. Geigy Scientific Tables. Basel: Ciba-Geigy, 1984, p. 78-88.

28.   Masson, PL, Heremans JF, Prignon JJ, and Wauters G. Immunohistochemical localization and bacteriostatic properties of an iron-binding protein from bronchial glands. Thorax 21: 538-544, 1977[ISI][Medline].

29.   Mitrauka, BM, and Rawnsley HM. Clinical Biochemical and Hematological Reference Values in Normal Experimental Animals. New York: Masson, 1977, p. 121-126.

30.   Pack, RJ, Al-Ugaily LH, Morris G, and Widdicombe JG. The distribution and structure of cells in the tracheal epithelium of the mouse. Cell Tissue Res 208: 65-84, 1980[ISI][Medline].

31.   Robinson, NP, Kyle H, Webber SE, and Widdicome JG. Electrolyte and other chemical concentrations in tracheal airway surface fluid and mucus. J Appl Physiol 66: 2129-2135, 1989[Abstract/Free Full Text].

32.   Rozhamel, R, Wilschanski M, Matin A, Plyte S, Oliver M, Auerbach W, Moore A, Forstner J, Durie P, Nadeau J, Bear C, and Tsui L-C. Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor. Nat Genet 12: 274-280, 1996[ISI][Medline].

33.   Sasaki, T, Shimura S, Ikeda K, Sasaki H, and Takishima T. Sodium efflux from tracheal submucosal glands in feline trachea. Am J Physiol Lung Cell Mol Physiol 258: L112-L117, 1990[Abstract/Free Full Text].

34.   Shimura, S, and Takishima T. Airway Secretion. New York: Dekker, 1994, p. 325-398.

35.   Singh, PK, Jia HP, Wiles K, Hesselberth J, Liu L, Conway BA, Greenberg EP, Valore EV, Welsh MJ, Ganz T, Tack BF, and McCray PB, Jr. Production of beta-defensins by human airway epithelia. Proc Natl Acad Sci USA 95: 14961-14966, 1998[Abstract/Free Full Text].

36.   Smith, JJ, Travis SM, Greenberg EP, and Welsh MJ. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85: 229-236, 1996[ISI][Medline].

37.   Snouwaert, JN, Brigman KK, Latour AM, Malouf NN, Boucher RC, Smithies O, and Koller BH. An animal model for cystic fibrosis made by gene targeting. Science 257: 1083-1088, 1992[ISI][Medline].

38.   Stutts, MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC, and Boucher RC. CFTR as a cAMP-dependent regulator of sodium channels. Science 269: 847-850, 1995[ISI][Medline].

39.   Stutts, MJ, Schwab JH, Chen MG, Knowles MR, and Boucher RC. Effects of Pseudomonas aeruginosa on bronchial epithelial ion transport. Am Rev Respir Dis 134: 17-21, 1986[ISI][Medline].

40.   Taylor, CT, Dzus AL, and Colgan SP. Autocrine regulation of epithelial permeability by hypoxia: role for polarized release of tumor necrosis factor alpha. Gastroenterology 114: 657-668, 1998[ISI][Medline].

41.   Thompson, AB, Bohling T, Payvandi F, and Rennard SI. Lower respiratory tract lactoferrin and lysozyme arise primarily in the airways and are elevated in association with chronic bronchitis. J Lab Clin Med 115: 148-158, 1990[ISI][Medline].

42.   Widdicombe, JH, and Widdicombe JG. Regulation of human airway surface fluid. Respir Physiol 99: 3-12, 1995[ISI][Medline].

43.   Wu, DX-Y, Lee CYC, Uyekebo SN, Choi HK, Bastacky SJ, and Widdicombe JH. Regulation of the depth of surface liquid in bovine trachea. Am J Physiol Lung Cell Mol Physiol 274: L388-L395, 1998[Abstract/Free Full Text].

44.   Zabner, J, Smith JJ, Karp PH, Widdicome JH, and Welsh MJ. Loss of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels alters salt absorption by cystic fibrosis airway epithelia in vitro. Mol Cells 2: 397-403, 1998.


Am J Physiol Lung Cell Mol Physiol 278(6):L1213-L1220
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society