Meakins-Christie Laboratories, Montreal Chest Institute Research Centre, McGill University Health Centre, McGill University, Montreal, Quebec, Canada H2X 2P2
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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).
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.
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RESULTS |
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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.
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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.
|
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.
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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).
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DISCUSSION |
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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-
(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.
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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.
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FOOTNOTES |
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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.
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REFERENCES |
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