Departments of Pediatrics, Internal Medicine, and Occupational and Environmental Health, Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, Iowa 52242
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ABSTRACT |
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Cystic fibrosis mice have been generated by gene
targeting but show little lung disease without repeated exposure to
bacteria. We asked if murine mucosal defenses and airway surface liquid (ASL) Cl were altered by
the
F508 cystic fibrosis transmembrane conductance regulator
mutation. Naive
F508
/
and +/
mice showed no
pulmonary inflammation and after inhaled Pseudomonas
aeruginosa had similar inflammatory responses and
bacterial clearance rates. We therefore investigated components of the
innate immune system. Bronchoalveolar lavage fluid from mice killed
Escherichia coli, and the microbicidal activity was inhibited by NaCl. Because
-defensins are
salt-sensitive epithelial products, we looked for pulmonary
-defensin expression. A mouse homolog of human
-defensin-1
(termed "MBD-1") was identified; the mRNA was expressed in the
lung. Using a radiotracer technique, ASL volume and
Cl
concentration
([Cl
]) were
measured in cultured tracheal epithelia from normal and
F508
/
mice. The estimated ASL volume was similar for both groups. There were no differences in ASL
[Cl
] in
F508
/
and normal mice (13.8 ± 2.6 vs. 17.8 ± 5.6 meq/l). Because ASL
[Cl
] is low in
normal and mutant mice, salt-sensitive antimicrobial factors, including
MBD-1, may be normally active.
cystic fibrosis transmembrane conductance regulator; Pseudomonas aeruginosa; defensin; cystic fibrosis
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INTRODUCTION |
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ONE APPROACH TO understanding the pathogenesis and
pathophysiology of cystic fibrosis (CF) is to develop animal models
that reflect the disease. Toward that end, CF mouse models have been generated using gene targeting to disrupt the murine cystic fibrosis transmembrane conductance regulator (CFTR) locus by homologous recombination (7, 15, 40, 43) and by introducing specific human
mutations into the equivalent mouse loci, including F508 (9, 59) and
G551D (13). However, although intestinal disease manifestations are
prominent in these animals, there is remarkably little evidence of lung
disease in "CF mice" maintained under normal housing conditions.
Explanations put forward to account for the low incidence of lung
infection in CF mice include the presence of alternative
Cl
transport pathways (8,
25) and the presence of modifier genes (45).
Several layers of defenses in the normal lung help prevent infection from inhaled or aspirated microorganisms. These include the mechanical filtering of particulates that occurs in the nasal airway, the trapping of particulates in mucus, and mucociliary clearance. Respiratory epithelia also secrete a number of protein and peptide products that are important in the innate mucosal immunity (17, 19, 44, 50). In addition, macrophages and neutrophils may participate in the clearance of microorganisms from the lung, usually at the cost of some degree of inflammation (44). In humans with CF, the early onset inflammation characterized by neutrophilia and proinflammatory cytokines in bronchoalveolar lavage (BAL) precedes chronic infection (1, 2, 34). In contrast to the characteristic lung disease of humans with CF, mouse models have had more variable pulmonary manifestations. Initial reports of CFTR null mice showed little evidence of lung disease (9, 40, 59). A later report by Davidson and colleagues (12) demonstrated that, with repeated bacterial challenges, it is possible to find evidence of decreased clearance of inhaled bacteria and persistent inflammatory disease. Van Heeckeren et al. (55) found that CFTR null mice showed more inflammation and morbidity when challenged with agar beads coated with Pseudomonas aeruginosa. Kent and colleagues (32) found that an inbred congenic strain of CFTR null mice spontaneously developed lung disease; however, unlike CF in humans, the disease was primarily alveolar. Thus with exposures to large bacterial loads or alteration of the genetic background, CF mouse models may develop lung disease.
The goal of these studies was to learn if the lungs of mice homozygous
for the F508 mutation show any differences in baseline markers of
pulmonary inflammation or develop lung disease when challenged with
aerosolized bacteria. We approached these issues by performing BAL
studies on naive mice before and after exposure to aerosolized
P. aeruginosa. We found no significant
differences between homozygous
F508 mice and their heterozygous littermates.
When we found little difference between the groups, we undertook
further studies to investigate components of the innate immune system
in mice. Recent studies indicate that human airway surface liquid (ASL)
contains salt-sensitive antimicrobial factors that may be important in
lung defenses (23, 48). These factors are secreted products of
epithelia, exhibit broad spectrum activity, and may be inactive in CF
ASL due to its elevated salt concentration (22, 23, 31, 48, 57). Thus
altered electrolyte transport in CF epithelia may increase the salt
concentration in ASL and impair the activity of innate mucosal
antimicrobial factors. Therefore, we evaluated some of these aspects in
the F508 mouse model, focusing on the effects of inhaled bacteria on
lung inflammation, the antimicrobial properties of mouse ASL, the
expression of
-defensins in the lung, and the electrolyte
composition of ASL.
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MATERIALS AND METHODS |
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Animals. F508 mice were generated
by gene targeting in ES cells as previously reported (59). The genetic
background of the mice is a cross of the C57/Bl6 and 129 strains. The
animals were housed in normal conditions (not pathogen free) before
study and were genotyped using tail DNA PCR as previously reported
(59). To ensure that mice were correctly genotyped during the
inhalation challenge and postinhalation procedures, an identifying
microchip was implanted under the skin of each animal, and animals were regenotyped at the end of study (Mini Tracker; Avid, Norco, CA). For
the inhalation challenge, two groups of animals were studied,
F508
homozygotes (
/
) and
F508 heterozygotes (+/
).
The rationale for the use of
F508 heterozygotes as controls in the
studies was as follows. There is no evidence that heterozygous CFTR
null mice or
F508 mice have a pathological or physiological
phenotype. Likewise, with the exception of abnormalities of stimulated
sweat secretion, humans heterozygous for CFTR mutations do not have a
disease phenotype.
Inhalation exposure system. Mice were exposed by inhalation to aerosolized P. aeruginosa in a 40-liter glass whole body exposure chamber for a period of 4 h with a modification of previously described methods (37, 51). For these studies, the P. aeruginosa strain ATCC no. 10145 was used (American Type Culture Collection, Manassas, VA). The bacterial solution for generating the aerosol was obtained by placing one cryovial of laboratory-cultured and lyophilized P. aeruginosa in 50 ml of sterile pyrogen-free saline in a 37°C shaker bath for 16 h. The concentration of bacteria was assessed by spectrophotometry at 600 nm, and the solution was supplied to a Pitt no. 1 nebulizer via a precision syringe pump. The nebulizer was supplied with temperature-controlled, filtered air and was operated at 101.5 kPa gauge pressure. The exposure chamber exhaust rate was set at 20.0 l/min and was metered with a rotometer calibrated against a primary standard. The concentration of P. aeruginosa in the exposure chamber was determined with two all-glass impingers placed in series as described previously (36, 52). The concentration of bacteria in the aerosol generator solutions and the impinger solutions was quantified by serial dilution plating of 0.1-ml aliquots on trypticase soy agar incubated at 37°C, with daily colony counting for 5 days. The mean aerosol generator solution concentration was 2 × 109 colony-forming units (CFU)/ml. This exposure system yielded a mean airborne P. aeruginosa concentration of 3.3 × 108 CFU/m3. Under the assumption of a 0.3 deposition fraction, the estimated inhaled burden from this 4-h exposure was 106 CFU/mouse.
Necropsy and lung lavage. Mice were
killed by cervical dislocation under methoxyflurane anesthesia at 5, 24, and 72 h after the onset of inhalation exposure. Animals were
immediately necropsied, and BAL fluid was recovered (1 ml of PBS 4 times) and assayed for total cells, differential cell count, and
cytokine concentration [interleukin (IL)-1, IL-6, and tumor
necrosis factor- (TNF-
)]. Cytokine assays were performed
using commercially available ELISA kits (TNF-
from Genzyme
Immunologicals, Cambridge, MA; IL-1 and IL-6 from Endogen, Cambridge,
MA). BAL was also assayed quantitatively for P. aeruginosa by dilution plating on trypticase soy agar
as described above.
Assay of mouse BAL for antimicrobial
activity. A quantitative luminescence assay was used to
screen mouse BAL fluid for antimicrobial activity. The assay employs
Escherichia coli DH5 containing the luminescence plasmid pCGLS1 (18) and allows rapid quantitative assessment of bactericidal activity. We validated our assay by showing
a correlation of luminescence (quantitated as relative light units)
with viable cell numbers. Luminescence is an energy-requiring activity,
and this is directly related to bacterial viability as determined by
plate counts (data not shown). Bacteria were grown at 30°C in
Luria-Bertani medium, centrifuged, and resuspended at a concentration
of 107 cells/ml in 10 mM potassium
phosphate, pH 7.2, with 1% Luria-Bertani medium. Bacteria
(106) were then incubated with
mouse BAL fluid in 96-well plates (Optiplate; Packard Instruments) for
4 h, and luminescence was measured with a microtiter dish luminometer
(Anthos). To assess the salt sensitivity of the BAL fluid activity, the
assays were performed in the presence of 0 or 150 mM added NaCl.
Cloning the murine -defensin-1
cDNA. The human
-defensin-1 (HBD-1) mature peptide
sequence (DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCKS) was used to screen the
GenBank dbest data base of expressed sequence tags (ESTs) using the
NCBI BLAST program. The search identified six identical murine ESTs
with homology to HBD-1. Four clones contained the entire 210-bp open
reading frame for a putative murine
-defensin-1 (MBD-1). Primer
sequences were designed to the putative MBD-1 sequence and used to
clone the cDNA from mouse kidney using RT-PCR. Mouse kidney RNA was
treated with DNase I (RQ1 DNase I; Promega, Madison, WI) for 1 h to
remove genomic DNA before the RT reaction, phenol-chloroform extracted,
and precipitated. One microgram of total RNA was reverse transcribed
using the GeneAmp PCR reagents (Perkin-Elmer, Norwalk, CT), Moloney
murine leukemia virus RT, and a poly(A) reverse primer. The resultant
cDNA was used as a template in the PCR. Primer sequences were as
follows: forward primer sequence, CACATCCTCTCTGCACTCTGGACCC; reverse
primer, CCATCGCTCGTCCTTTATGCTCATTC. Each reaction contained ~0.15
µM primers, 2 mM Mg2+, and the
20-µl RT reaction product in a total volume of 100 µl. After an
initial denaturation step (95°C for 3 min), 25 cycles of annealing
(60°C for 30 s) and extending (72°C for 30 s) were performed.
The predicted 287-bp MBD-1 PCR product (bp
39 to 248 of the
cDNA) was isolated and cloned into the PCR Script vector (Stratagene,
La Jolla, CA), and the identity was confirmed by DNA sequencing.
RT-PCR of MBD-1. Total RNA was isolated using the single-step RNazol method (5). One microgram of total RNA was reverse transcribed by random hexamer primers using the SuperScript transcription system (GIBCO BRL) according to the manufacturer's instructions. First-strand MBD-1 cDNA was amplified by PCR as described above. As an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified in the same reaction using the following primers: GAPDH forward primer, GTCAGTGGTGGACCTGACCT; GAPDH reverse primer, AGGGGTCTACATGGCAACTG. A 25-µl aliquot of the PCR product was electrophoresed on a 2% agarose gel and visualized with ethidium bromide.
Cell culture. Mouse tracheal
epithelial cells were cultured at the air-liquid interface using a
modification of previously described methods (56, 58). Tracheal
epithelial cells were plated on collagen-coated
0.6-cm2 Millicell HA filter
inserts at a density of 5 × 104
cells/cm2. The nutrient medium
consisted of DMEM-F-12 with 2% Ultroser G (48). Viability of the
cultures was confirmed visually by verifying that the epithelia
maintained a dry apical surface in culture and by documenting the
bioelectric properties of the cells. Under these conditions, the
epithelia differentiate and develop a ciliated apical surface (56, 58).
All cultures used in measurements of ASL fluid and electrolyte
composition had a transepithelial resistance
(Rt) >500
· cm2 (measured by EVOM; World Precision
Instruments). The mean
Rt for
F508
/
cultures was 664.67 ± 102.5
· cm2 (n = 12) and for normal
epithelia was 613.33 ± 57.83
· cm2
(n = 12). The differences were not
statistically significant (P = 0.4).
The amiloride-sensitive short-circuit current for
F508
/
and normal cells was not significantly different and
ranged between 5 and 10 µA/cm2
(n = 12 for
F508
/
and normal
epithelia). The tracheae from 10 mice were combined to make one
epithelial cell preparation.
Measurement of ASL volume and Cl
concentration by radiotracer technique.
Because knowledge of the salt concentration at the air interface is
critical to understanding CF airway disease, we developed a new
radiotracer method to measure ASL
Cl
concentration
([Cl
]; see Ref.
57). To measure
[Cl
] in ASL, we
added
36Cl
to the basolateral medium together with
3H2O.
After the tracer content of ASL reached equilibrium (48 h), we removed
ASL by rinsing the apical surface with 100 µl of medium. The
basolateral medium was also sampled, and its
[Cl
] was
measured. The ratio of
36Cl
to
3H2O
in each compartment allowed us to calculate ASL
[Cl
]. There
were two key methodological details. First, after isotopes were added,
normal and
F508
/
epithelia were studied at the same
time and sealed in the same chamber. Second, the sealed container was
humidified with water that had the same specific activity of
3H2O
as the culture medium. In this way, water vapor over the surface of
epithelia contained the same ratio of
3H2O
to H2O as that in ASL and the
basolateral medium.
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RESULTS |
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Naive animals have normal BAL
profiles. In infants and children with CF, it appears
that a defect in innate immunity contributes to the chronic airway
inflammation and infection that is a clinical hallmark of the disease.
Asymptomatic infants with CF may have elevations in BAL neutrophils and
proinflammatory cytokines that precede the onset of symptoms (2, 34).
We tested the hypothesis that F508
/
mice housed under
normal conditions from birth develop pulmonary inflammation by
measuring BAL cell counts and cytokine levels. Under such housing
conditions, animals will be chronically exposed to low levels of
inhaled bacteria. Naive
F508
/
animals were compared
with littermates heterozygous for the
F508 mutation. As shown in
Table 1, there were no significant differences in the percentage of neutrophils or the proinflammatory cytokines IL-1, TNF-
, or IL-6 between the BAL fluids of
F508
/
or +/
animals. This result suggests that there
are no differences between animal groups in pulmonary bacterial
clearance mechanisms, which include both filtering mechanisms and
innate immunity.
|
Normal and F508 mice clear inhaled bacteria at
similar rates. The chronic inflammation in infants and
children with CF is characteristically neutrophilic. Because we
detected no differences between
F508
/
and +/
animals with low-level exposure to inhaled bacteria, we tested the
hypothesis that the ability to clear an inhaled bacterial challenge is
impaired in
F508
/
mice compared with heterozygote
littermates. For these experiments, mice were exposed to aerosolized
P. aeruginosa in a whole body exposure chamber for 4 h. In this exposure procedure, the airborne concentration of P. aeruginosa was 3.3 × 108
CFU/m3 for an estimated lung
deposition of 1 × 106
CFU/animal (see MATERIALS AND METHODS). At this dose of
inhaled bacteria, we assume the mechanism of bacterial clearance
included both innate and cellular responses. After the exposure, groups of animals were killed at 5, 24, and 72 h, and BAL studies were performed. As shown in Fig.
1A, both
F508
/
and +/
mice developed marked BAL
neutrophilia after bacterial inhalation. This was greatest at 5 h after
onset of P. aeruginosa inhalation and
gradually declined over 72 h. Quantitative BAL bacterial colony counts,
shown in Fig. 1B, demonstrated that
both groups had similar amounts of P. aeruginosa recovered at 5 h, and the bacteria were
rapidly cleared by 24 h after exposure in both groups. The BAL
neutrophilia was accompanied by a rise in IL-6 and TNF-
at 5 h,
although there were no significant differences between the groups (data
not shown). From these studies, we conclude that the pulmonary
inflammatory responses and bacterial clearance after inhalation
challenge with P. aeruginosa are
equivalent for
F508
/
and +/
mice.
|
Murine BAL fluid exhibits salt-sensitive antimicrobial
activity. The observation that F508
/
and +/
mice cleared bacteria equally efficiently and had no
demonstrable elevations in proinflammatory cytokines or neutrophils in
BAL under normal housing conditions suggests two possible explanations.
First, murine pulmonary antimicrobial factors are salt insensitive,
such as the protegrins from pig neutrophils (60). Second, murine ASL
might maintain a low NaCl concentration even in the face of CFTR
mutations. To address the first possibility, we isolated BAL fluid from
normal mice and tested its ability to kill bacteria in the presence or
absence of added NaCl. As shown in Fig. 2,
murine BAL fluid killed E. coli in a
concentration-dependent manner. When the BAL samples were tested in the
presence of 150 mM NaCl, the antimicrobial activity was lost. Thus
murine BAL fluid, like human ASL (48), exhibited antimicrobial activity
that was inhibited by salt.
|
Murine lung expresses MBD-1. ASL
contains many antimicrobial factors, including the proteins lysozyme
(21) and lactoferrin and peptides such as the -defensins (20).
Recent evidence suggests that low-molecular-weight peptides such as the
-defensins may play an important role in pulmonary defenses in
humans (23, 47, 48). We wondered whether epithelial
-defensins
similar to those reported in bovine (14) and human (23, 26, 38) lungs
were also expressed in the murine lung. Although mice are known to
express a class of intestinal defensins termed cryptdins (30, 41), no MBD had been identified at the time of these studies.
Using the HBD-1 sequence as a query (4), we searched the GenBank dbest
data base of ESTs and found an MBD homolog. The MBD-1 cDNA clone
consisted of a 210-bp open reading frame, predicted to encode a
69-amino acid protein (shown in Fig.
3A). The
cDNA clone isolated from mouse kidney was identical to the MBD-1 cDNA
reported by Huttner and colleagues (29), and the predicted mature
peptide sequence was ~55% identical to HBD-1 (4, 38). To determine
the tissue distribution of MBD-1 mRNA, we performed RT-PCR (Fig.
3B). MBD-1 mRNA was expressed most
abundantly in kidney and testes, with transcripts also detected in
lung, brain, bladder, and stomach.
|
ASL [Cl] in normal and
F508
/
mice is similar.
In humans, one proposed effect of CFTR mutations on airway epithelia is
an impaired ability to absorb NaCl (22, 23, 31, 48, 57). This leads to
an elevation in ASL NaCl concentration and may inhibit the activity of
salt-sensitive antimicrobial factors secreted by epithelia, including
-defensins. Measurements of the ASL composition in normal and CF
subjects suggest that the NaCl concentration in CF ASL is elevated
compared with that in normal mice (22, 23, 31, 57). We performed
experiments to address this issue in mice. Using a radiotracer
technique, we measured
[Cl
] in ASL of
primary cultures of tracheal epithelia from
F508
/
mice and wild-type mice. We added radiolabeled
Cl
and
H2O to the basal media of
confluent cultures of tracheal epithelial cells. After 48 h, the
labeled ions reached equilibrium between the mucosal and basal
solutions, and measurements were made. As shown in Fig.
4, the estimated volume of the apical
solution (ASL) was similar for both
F508
/
and normal
mice. There were no statistically significant differences between the
ASL [Cl
] in
F508
/
and normal mice. These observations show that
mice, like rats (53), maintain low NaCl in ASL and suggest that the
F508 mutation has no impact on the ASL NaCl concentration.
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DISCUSSION |
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This study shows that F508
/
and +/
mice housed
under normal conditions show no significant inflammation on BAL
analysis, suggesting equivalent abilities to clear inhaled bacteria.
Furthermore, when challenged with inhaled P. aeruginosa, both groups developed similar increases in
BAL fluid neutrophilia and cytokines. These inflammatory responses were
transient, and over 24-72 h, bacteria were cleared from the lungs
of both groups with similar rapidity. Thus we were unable to
demonstrate any differences between
F508
/
and
+/
mice in their inflammatory responses and clearance of inhaled bacteria.
Several mouse models of CF have been generated, including CFTR null mice and mice with specific mutations, in the hope that these animals would prove to be useful for the study of CF lung disease (7, 9, 13, 15, 40, 43, 59). A striking feature of some models is the severity of the gastrointestinal disease and minimal evidence of lung disease (9, 40, 59). Kent et al. (33) reported the pathological findings on CFTR null mice surviving >100 days and found no pulmonary abnormalities. Snouwaert and co-workers (49) reported minor pulmonary abnormalities consisting of increases in goblet cells and mucus in cells of the upper airways of CFTR null mice surviving to adulthood. In one animal, marked goblet cell hyperplasia, mucus obstruction, and inflammatory cells were noted. No differences between CFTR null and normal mice were noted in bacterial clearance after repeated exposure to Staphylococcus aureus over a 1-mo period (49). In contrast, Davidson and colleagues (12) found that, on repeated exposure to S. aureus or Burkholderia cepacia, CFTR null mice had delayed clearance of bacteria and developed pathological abnormalities. Recently, van Heeckeren and colleagues (55) reported that CFTR null mice developed increases in proinflammatory cytokines and had diminished survival when challenged by chronic lung infection with agar beads coated with P. aeruginosa. The breeding of CFTR null mice onto different mouse backgrounds can also alter disease severity, suggesting that there are other genetic loci that may modify the severity of CFTR mutations (45). Kent and colleagues (32) observed that CFTR null mice bred onto a C57BL/6J background spontaneously developed progressive lung disease. However, in contrast to the airway disease present in humans, the lung disease in inbred congenic CFTR null mice was primarily alveolar and had little resemblance to CF in humans. Overall, there is little evidence that CF mice develop lung disease unless they are frequently or persistently exposed to a large burden of bacterial pathogens.
As shown in these studies (Fig. 2), BAL fluid from mice exhibited
salt-sensitive antimicrobial activity. This activity probably represents the aggregate effect of several factors, including epithelial -defensin peptides. Similar to HBD-1 (38, 54), MBD-1 mRNA
expression was greatest in the kidney but was also present in lung
(29). Huttner and colleagues (29) first reported the cloning of the
MBD-1 gene and recognized its homology to HBD-1. Bals et al. (3)
recently reported that MBD-1 mRNA is expressed in murine airway
epithelia and found that the antibacterial activity of a synthetic
MBD-1 peptide was salt sensitive. These findings were confirmed by
Morrison and colleagues (39) who, in addition, noted that MBD-1 mRNA
expression in the lung was not induced in response to instilled
lipopolysaccharide. Thus, unlike previous observations with bovine
-defensins tracheal antimicrobial peptide and lingual antimicrobial
peptide (46) and HBD-2 (26), MBD-1 expression did not
change in response to proinflammatory stimuli (39). This constitutive
expression of MBD-1 is similar to HBD-1 (38, 47, 54, 61).
Surprisingly, our results indicate that the F508 CFTR mutation on an
outbred genetic background did not alter the ASL volume or
[Cl
] in
cultured mouse tracheal epithelia. In vivo measurements of tracheal ASL
electrolytes in another rodent, the rat, with a capillary electrophoresis method also showed that the salt concentration was
reduced compared with serum
[Na+ concentration
([Na+]) 40.57 ± 3.08 mM and
[Cl
] 45.16 ± 1.81 mM; see Ref. 53], and preliminary measurements in mice
suggest that normal tracheal ASL also has a low salt concentration (10). We recently used a radiotracer method to measure ASL NaCl concentration in cultured human airway epithelial cells from non-CF and
CF patients (57). These studies showed that, in non-CF epithelia, the
ASL [Na+] was 50 ± 4 mM and [Cl
]
was 37 ± 6 mM. In contrast, ASL covering CF epithelia had
[Na+] and
[Cl
] that were
approximately two times these values (57). Furthermore, the non-CF and
CF epithelia had the same volume of ASL (average ASL depth of ~20
µm). Three additional studies presented evidence that normal ASL has
a low NaCl concentration that is elevated in CF patients (22, 23, 31).
Thus, like the sweat duct (42), ASL salt concentration may be increased
in CF because the loss of CFTR impairs the ability of airway epithelia
to absorb NaCl. The hypothesis that ASL NaCl concentration is increased
in CF is controversial as evidenced by two recent reports finding no differences between non-CF and CF patients (27, 35). However, filter
paper methods of collecting ASL in vivo can introduce significant artifacts (16). Elevations of ASL NaCl concentration in humans could
have a profound impact on the ability of airway epithelia to prevent
bacterial colonization (23, 48).
The physiological basis for the identical ASL
[Cl] in
F508
+/
and
/
mice is not known. It is well documented
that murine airways express a cAMP-activated
Cl
conductance consistent
with CFTR and that this conductance is diminished in CFTR null mice (6,
7, 15, 40, 43) and in mice homozygous for the
F508 mutation (9). The
epithelial sodium channel is also expressed in the lung and presumably
accounts for the amiloride-sensitive
Na+ conductance across murine
airway epithelia (11, 28). However, CFTR-mediated
Cl
transport may be less
important in murine airway epithelia because there are alternative
Cl
transport pathways
available. Compared with human airways, some strains of mice show
evidence of significant
Ca2+-activated
Cl
channel activity that
might compensate for the absence of CFTR (6, 8, 24, 25). Indeed, Grubb
and colleagues (24, 25) reported that respiratory epithelia of CFTR
null mice exhibit increased
Ca2+-activated
Cl
channel activity
compared with that in normal mice. Perhaps murine CFTR plays a minor
role in regulating the composition of ASL because alternative
Cl
channels provide a
pathway for NaCl absorption and facilitate the maintenance of a low
salt concentration. We speculate that one explanation for the lack of
significant lung disease in
F508 mice, and perhaps in other CF mouse
models, is that the low salt environment of murine ASL allows
salt-sensitive components of their pulmonary mucosal defenses,
including MBD-1, to function normally.
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NOTE ADDED IN PROOF |
---|
Morrison et al. recently reported identification of a second mouse
-defensin that is expressed in the lung (Morrison, G. M.,
D. J. Davidson, and J. R. Dorin. A novel mouse beta defensin, Defb2,
which is upregulated in the airways by lipopolysaccharide. FEBS
Lett. 442: 112-116, 1999).
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ACKNOWLEDGEMENTS |
---|
We thank Kerry Wiles, Marsha O'Neill, Norma Hillerts, and Lei Yee Wang for excellent technical assistance. We thank Aurita Puga for assistance in cultures of mouse airway epithelia. We acknowledge the assistance of the Cell Culture Core supported in part by the Cystic Fibrosis Foundation.
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FOOTNOTES |
---|
This work was supported by the Cystic Fibrosis Foundation (McCray97ZO), National Heart, Lung, and Blood Institute Grant HL-42385 (M. J. Welsh), and the Children's Miracle Network Telethon. P. B. McCray is the recipient of a Career Investigator Award from the American Lung Association. J. Zabner is a fellow of the Roy J. Carver Charitable Trust. M. J. Welsh is an Investigator of the Howard Hughes Medical Institute.
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: P. B. McCray, Jr., Dept. of Pediatrics, Univ. of Iowa College of Medicine, Iowa City, IA 52242 (E-mail: paul-mccray{at}uiowa.edu).
Received 18 September 1998; accepted in final form 7 April 1999.
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