Efficient killing of inhaled bacteria in Delta F508 mice: role of airway surface liquid composition

Paul B. McCray Jr., Joseph Zabner, Hong Peng Jia, Michael J. Welsh, and Peter S. Thorne

Departments of Pediatrics, Internal Medicine, and Occupational and Environmental Health, Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, Iowa 52242


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta F508 cystic fibrosis transmembrane conductance regulator mutation. Naive Delta 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 beta -defensins are salt-sensitive epithelial products, we looked for pulmonary beta -defensin expression. A mouse homolog of human beta -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 Delta F508 -/- mice. The estimated ASL volume was similar for both groups. There were no differences in ASL [Cl-] in Delta 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta 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 Delta 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 Delta 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 Delta F508 mouse model, focusing on the effects of inhaled bacteria on lung inflammation, the antimicrobial properties of mouse ASL, the expression of beta -defensins in the lung, and the electrolyte composition of ASL.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Delta 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, Delta F508 homozygotes (-/-) and Delta F508 heterozygotes (+/-). The rationale for the use of Delta F508 heterozygotes as controls in the studies was as follows. There is no evidence that heterozygous CFTR null mice or Delta 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-alpha (TNF-alpha )]. Cytokine assays were performed using commercially available ELISA kits (TNF-alpha 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 DH5alpha 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 beta -defensin-1 cDNA. The human beta -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 beta -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 Omega  · cm2 (measured by EVOM; World Precision Instruments). The mean Rt for Delta F508 -/- cultures was 664.67 ± 102.5 Omega  · cm2 (n = 12) and for normal epithelia was 613.33 ± 57.83 Omega  · cm2 (n = 12). The differences were not statistically significant (P = 0.4). The amiloride-sensitive short-circuit current for Delta F508 -/- and normal cells was not significantly different and ranged between 5 and 10 µA/cm2 (n = 12 for Delta 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 Delta 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta 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 Delta F508 -/- animals were compared with littermates heterozygous for the Delta F508 mutation. As shown in Table 1, there were no significant differences in the percentage of neutrophils or the proinflammatory cytokines IL-1, TNF-alpha , or IL-6 between the BAL fluids of Delta 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.

                              
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Table 1.   BAL profiles of naive Delta F508 -/- and +/- mice housed under normal conditions

Normal and Delta 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 Delta 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 Delta 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 Delta 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-alpha 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 Delta F508 -/- and +/- mice.



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Fig. 1.   Delta F508 -/- and +/- mice show similar responses to inhaled bacteria. Animals were exposed to aerosolized Pseudomonas aeruginosa for 4 h as described in MATERIALS AND METHODS and were killed at the indicated intervals postexposure, and bronchoalveolar lavage (BAL) was performed. A: BAL polymorphonuclear neutrophil (PMN) counts. B: BAL colony-forming units (CFU) for P. aeruginosa (CFU/ml). Results are means ± SE.

Murine BAL fluid exhibits salt-sensitive antimicrobial activity. The observation that Delta 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.


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Fig. 2.   Antimicrobial activity of murine BAL fluid determined by Escherichia coli luminescence assay. Serial dilutions of BAL were added to the assay buffer as indicated on the x-axis. BAL from normal mice exhibited antimicrobial activity that was inhibited in the presence of 150 mM NaCl. Results are means ± SE for BAL analysis from 4 mice. RLU, relative light units.

Murine lung expresses MBD-1. ASL contains many antimicrobial factors, including the proteins lysozyme (21) and lactoferrin and peptides such as the beta -defensins (20). Recent evidence suggests that low-molecular-weight peptides such as the beta -defensins may play an important role in pulmonary defenses in humans (23, 47, 48). We wondered whether epithelial beta -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.


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Fig. 3.   A: murine beta -defensin-1 (MBD-1) cDNA and deduced amino acid sequences. * Putative mature peptide sequence. B: MBD-1 mRNA expression in mouse tissues by RT-PCR. Transcript of the predicted size was detected in testes, brain, bladder, lung, stomach, and kidney. No transcripts were noted in liver or tongue. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

ASL [Cl-] in normal and Delta 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 beta -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 Delta 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 Delta F508 -/- and normal mice. There were no statistically significant differences between the ASL [Cl-] in Delta F508 -/- and normal mice. These observations show that mice, like rats (53), maintain low NaCl in ASL and suggest that the Delta F508 mutation has no impact on the ASL NaCl concentration.


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Fig. 4.   Comparison of Cl- concentration ([Cl-]) and airway surface liquid (ASL) volume in normal and Delta F508 -/- mice. ASL [Cl-] was 18.29 ± 3.1 and 14.63 ± 1.6 mM in normal and Delta F508 -/- airway epithelia, respectively (P = 0.19). Estimate of ASL volume was 1.43 ± 0.16 and 1.45 ± 0.15 µl in normal and Delta F508 -/- airway epithelia, respectively (P = 0.9); n = 16 epithelia from 3 different experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study shows that Delta 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 Delta 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 beta -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 beta -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 Delta 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 Delta 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 Delta 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 Delta 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.


    NOTE ADDED IN PROOF

Morrison et al. recently reported identification of a second mouse beta -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).


    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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Armstrong, D. S., K. Grimwood, R. Carzino, J. B. Carlin, A. Olinsky, and P. D. Phelan. Lower respiratory infection and inflammation in infants with newly diagnosed cystic fibrosis. Br. Med. J. 310: 1571-1572, 1995[Free Full Text].

2.   Balough, K., M. McCubbin, M. Weinberger, W. Smits, R. Ahrens, and R. Fick. The relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis. Pediatr. Pulmonol. 20: 63-70, 1995[Medline].

3.   Bals, R., M. J. Goldman, and J. M. Wilson. Mouse beta -defensin 1 is a salt-sensitive antimicrobial peptide present in epithelia of the lung and urogenital tract. Infect. Immun. 66: 1225-1232, 1998[Abstract/Free Full Text].

4.   Bensch, K. W., M. Raida, H. J. Magert, P. Schulz-Knappe, and W. G. Forssmann. hBD-1: a novel beta-defensin from human plasma. FEBS Lett. 368: 331-335, 1995[Medline].

5.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

6.   Clarke, L. L., K. A. Burns, J.-Y. Bayle, R. C. Boucher, and M. R. Van Scott. Sodium- and chloride-conductive pathways in cultured mouse tracheal epithelium. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L519-L525, 1992[Abstract/Free Full Text].

7.   Clarke, L. L., B. R. Grubb, S. E. Gabriel, O. Smithies, B. H. Koller, and R. C. Boucher. Defective epithelial chloride transport in a gene-targeted mouse model of cystic fibrosis. Science 257: 1125-1128, 1992[Medline].

8.   Clarke, L. L., B. R. Grubb, J. R. Yankaskas, C. U. Cotton, A. McKenzie, and R. C. Boucher. Relationship of a non-cystic 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.   Colledge, W. H., B. S. Abella, K. W. Sothern, R. Ratcliff, C. Jiang, S. H. Cheng, L. J. MacVinish, J. R. Anderson, A. W. Cuthbert, and M. J. Evans. Generation and characterization of a Delta F508 cystic fibrosis mouse model. Nat. Genet. 10: 445-452, 1995[Medline].

10.   Cowley, E. A., K. Govindaraju, D. K. Lloyd, and D. H. Eidelman. Is mouse airway surface fluid hypotonic? (Abstract). Pediatr. Pulmonol. Suppl. 14: 233, 1997.

11.   Dagenais, A., R. Kothary, and Y. Berthiaume. The alpha  subunit of the epithelial sodium channel in the mouse: developmental regulation of its expression. Pediatr. Res. 42: 327-334, 1997[Abstract].

12.   Davidson, D. J., J. R. Dorin, G. McLachlan, V. Ranaldi, D. Lamb, C. Doherty, J. Govan, and D. J. Porteous. Lung disease in the cystic fibrosis mouse exposed to bacterial pathogens. Nat. Genet. 9: 351-357, 1995[Medline].

13.   Delaney, S. J., E. W. F. W. Alton, S. N. Smith, D. P. Lunn, R. Farley, P. K. Lovelock, S. A. Thomson, D. A. Hume, D. Lamb, D. J. Porteous, J. R. Dorin, and B. J. Wainwright. Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype-phenotype correlations. EMBO J. 15: 955-963, 1996[Abstract].

14.   Diamond, G., M. Zasloff, H. Eck, M. Brasseur, W. L. Maloy, and C. L. Bevins. Tracheal antimicrobial peptide, a cysteine-rich peptide from mammalian tracheal mucosa: peptide isolation and cloning of a cDNA. Proc. Natl. Acad. Sci. USA 88: 3952-3956, 1991[Abstract].

15.   Dorin, J. R., P. Dickinson, E. W. F. W. Alton, S. N. Smith, D. M. Geddes, B. J. Stevenson, W. L. Kimber, S. Fleming, A. R. Clarke, M. L. Hooper, L. Anderson, R. S. P. Beddington, and D. J. Porteous. Cystic fibrosis in the mouse by targeted insertional mutagenesis. Nature 359: 211-215, 1992[Medline].

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

17.   Fleming, A., and V. D. Allison. Observations on a bacteriolytic substance ("lysozyme") found in secretions and tissues. Br. J. Exp. Pathol. 3: 252-260, 1922.

18.   Frackman, S., M. Anhalt, and K. H. Nealson. Cloning, organization, and expression of the bioluminescence genes of Xenorhabdus luminescens. J. Bacteriol. 172: 5767-5773, 1990[Medline].

19.   Franken, C., C. J. Meijer, and J. H. Dijkman. Tissue distribution of antileukoprotease and lysozyme in humans. J. Histochem. Cytochem. 37: 493-498, 1989[Abstract].

20.   Ganz, T., and J. Weiss. Antimicrobial peptides of phagocytes and epithelia. Semin. Hematol. 34: 343-354, 1997[Medline].

21.   Gibson, K. F., and S. Phadke. Intracellular distribution of lysozyme in rat alveolar type II epithelial cells. Exp. Lung Res. 20: 595-611, 1994[Medline].

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

23.   Goldman, M. J., M. G. Anderson, E. D. Stolzenberg, P. U. Kari, M. Zasloff, and J. M. Wilson. Human beta -defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 88: 1-9, 1997[Medline].

24.   Grubb, B. R., A. M. Paradiso, and R. C. Boucher. Anomalies in ion transport in CF mouse tracheal epithelium. Am. J. Physiol. 267 (Cell Physiol. 36): C293-C300, 1994[Abstract/Free Full Text].

25.   Grubb, B. R., R. N. Vick, and R. C. Boucher. Hyperabsorption of Na+ and raised Ca2+-mediated Cl- secretion in nasal epithelia of CF mice. Am. J. Physiol. 266 (Cell Physiol. 35): C1478-C1483, 1994[Abstract/Free Full Text].

26.   Harder, J., J. Bartels, E. Christophers, and J.-M. Schroder. A peptide antibiotic from human skin. Nature 387: 861-862, 1997[Medline].

27.   Hull, J., W. Skinner, C. Robertson, and P. Phelan. 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].

28.   Hummler, E., P. Barker, J. Gatzy, F. Beermann, C. Verdumo, A. Schmidt, R. Boucher, and B. C. Rossier. Early death due to defective neonatal lung liquid clearance in alpha ENaC-deficient mice. Nat. Genet. 12: 325-328, 1996[Medline].

29.   Huttner, K. M., C. A. Kozak, and C. L. Bevins. The mouse genome encodes a single homolog of the antimicrobial peptide human beta -defensin 1. FEBS Lett. 413: 45-49, 1997[Medline].

30.   Huttner, K. M., M. E. Selsted, and A. J. Ouellette. Structure and diversity of the murine cryptdin gene family. Genomics 19: 448-453, 1994[Medline].

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

32.   Kent, G., R. Lles, C. E. Bear, L.-J. Huan, U. Griesenbach, C. McKerlie, H. Frndova, C. Ackerley, D. Gosselin, D. Radzioch, H. O'Brodovich, L.-C. Tsui, M. Buchwald, and A. K. Tanswell. Lung disease in mice with cystic fibrosis. J. Clin. Invest. 100: 3060-3069, 1997[Abstract/Free Full Text].

33.   Kent, G., M. Oliver, K. Foskett, H. Frndova, P. Durie, J. Forstner, G. G. Forstner, J. R. Riordan, D. Percy, and M. Buchwald. Phenotypic abnormalities in long-term surviving cystic fibrosis mice. Pediatr. Res. 40: 233-241, 1996[Abstract].

34.   Khan, T. Z., J. S. Wagener, T. Bost, J. Martinez, F. J. Accurso, and D. W. H. Riches. Early pulmonary inflammation in infants with cystic fibrosis. Am. J. Respir. Crit. Care Med. 151: 1075-1082, 1995[Abstract].

35.   Knowles, M. R., J. M. Robinson, R. E. Wood, C. A. Pue, W. M. Mentz, G. C. Wagner, J. T. Gatzy, and R. C. Boucher. Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects. J. Clin. Invest. 100: 2588-2595, 1997[Abstract/Free Full Text].

36.   Lange, J. L., P. S. Thorne, and N. Lynch. Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria. Appl. Environ. Microbiol. 63: 1557-1563, 1997[Abstract].

37.   Mahoney, M. M., A. Y. Lee, D. J. Brezinski-Caliguri, and K. M. Huttner. Molecular analysis of the sheep cathelin family reveals a novel antimicrobial peptide. FEBS Lett. 377: 519-522, 1995[Medline].

38.   McCray, P. B., Jr., and L. Bentley. Human airway epithelia express a beta -defensin. Am. J. Respir. Cell Mol. Biol. 16: 343-349, 1997[Abstract].

39.   Morrison, G. M., D. J. Davidson, F. M Kilanowski, D. W. Borthwick, K. Crook, A. I. Maxwell, J. R. W. Govan, and J. R. Dorin. Mouse beta defensin-1 is a functional homolog of human beta defensin-1. Mamm. Genome 9: 453-457, 1998[Medline].

40.   O'Neal, W. K., P. Hasty, P. B. McCray, Jr., B. Casey, J. Rivera-Perez, M. J. Welsh, A. L. Beaudet, and A. Bradley. A severe phenotype in mice with a duplication of exon 3 in the cystic fibrosis locus. Hum. Mol. Genet. 2: 1561-1569, 1993[Abstract].

41.   Ouellette, A. J., D. Pravtcheva, F. H. Ruddle, and M. James. Localization of the cryptdin locus on mouse chromosome 8. Genomics 5: 233-239, 1989[Medline].

42.   Quinton, P. M. Cystic fibrosis: a disease in electrolyte transport. FASEB J. 4: 2709-2717, 1990[Abstract/Free Full Text].

43.   Ratcliff, R., M. J. Evans, A. W. Cuthbert, L. J. MacVinish, D. Foster, J. R. Anderson, and W. H. Colledge. Production of a severe cystic fibrosis mutation in mice by gene targeting. Nat. Genet. 4: 35-41, 1993[Medline].

44.   Reynolds, H. Y. Integrated host defense against infections. In: The Lung, edited by R. G. Crystal. New York: Raven, 1997, p. 2353-2365.

45.   Rozmahel, R., M. Wilschanski, A. Matin, S. Plyte, M. Oliver, W. Auerbach, A. Moore, J. Forstner, P. Durie, J. Nadeau, C. Bear, and L. C. Tsui. Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor. Nat. Genet. 12: 280-287, 1996[Medline].

46.   Russell, J. P., G. Diamond, A. P. Tarver, T. F. Scanlin, and C. L. Bevins. Coordinate induction of two antibiotic genes in tracheal epithelial cells exposed to the inflammatory mediators lipopolysaccharide and tumor necrosis factor alpha. Infect. Immun. 64: 1565-1568, 1996[Abstract].

47.   Singh, P. K., H. P. Jia, K. Wiles, J. Hesselberth, L. Liu, B. D. Conway, E. P. Greenberg, E. V. Valore, M. J. Welsh, T. Ganz, B. F. Tack, and P. B. McCray, Jr. Production of beta -defensins by human airway epithelia. Proc. Natl. Acad. Sci. USA 95: 14961-14966, 1998[Abstract/Free Full Text].

48.   Smith, J. J., S. M. Travis, E. P. Greenberg, and M. J. Welsh. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85: 229-236, 1996[Medline].

49.   Snouwaert, J. N., K. K. Brigman, A. M. Latour, E. Iraj, U. Schwab, M. I. Gilmour, and B. H. Koller. A murine model of cystic fibrosis. Am. J. Respir. Crit. Care Med. 151: S59-S64, 1995[Medline].

50.   Thompson, A. B., T. Bohling, F. Payvandi, and S. I. Rennard. 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[Medline].

51.   Thorne, P. S., J. A. DeKoster, and P. Subramanian. Pulmonary effects of machining fluids in guinea pigs and mice. Am. Ind. Hyg. Assoc. J. 57: 1168-1172, 1996[Medline].

52.   Thorne, P. S., M. S. Kiekhaefer, P. Whitten, and K. J. Donham. Comparison of bioaerosol sampling methods in barns housing swine. Appl. Environ. Microbiol. 58: 2543-2551, 1992[Abstract].

53.   Transfiguracion, J. C., C. Dolman, D. H. Eidelman, and D. K. Lloyd. Determination of the inorganic ion composition of rat airway surface fluid by capillary electrophoresis: direct sample injection to allow multiple analyses from nanoliter volumes. Anal. Chem. 67: 2937-2942, 1995[Medline].

54.   Valore, E. V., C. H. Park, A. J. Quayle, K. R. Wiles, P. B. McCray, Jr., and T. Ganz. Human beta -defensin-1, an antimicrobial peptide of urogenital tissues. J. Clin. Invest. 101: 1633-1642, 1998[Abstract/Free Full Text].

55.   Van Heeckeren, A., R. Walenga, M. W. Konstan, T. Bonfield, P. B. Davis, and T. Ferkol. Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with Pseudomonas aeruginosa. J. Clin. Invest. 100: 2810-2815, 1997[Abstract/Free Full Text].

56.   Yamaya, M., W. E. Finkbeiner, S. Y. Chun, and J. H. Widdicombe. Differentiated structure and function of cultures from human tracheal epithelium. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L713-L724, 1992[Abstract/Free Full Text].

57.   Zabner, J., J. J. Smith, P. H. Karp, J. H. Widdicombe, and M. J. Welsh. Loss of CFTR chloride channels alters salt absorption by cystic fibrosis airway epithelia in vitro. Mol. Cells 2: 397-403, 1998.

58.   Zabner, J., B. G. Zeiher, E. Friedman, and M. J. Welsh. Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time. J. Virol. 70: 6994-7003, 1996[Abstract].

59.   Zeiher, B. G., E. Eichwald, J. Zabner, J. J. Smith, A. P. Puga, P. B. McCray, Jr., M. R. Capecchi, M. J. Welsh, and K. R. Thomas. A mouse model for the Delta F508 allele of cystic fibrosis. J. Clin. Invest. 96: 2051-2064, 1995[Medline].

60.   Zhao, C., T. Ganz, and R. I. Lehrer. The structure of porcine protegrin genes. FEBS Lett. 368: 197-202, 1995[Medline].

61.   Zhao, C., I. Wang, and R. I. Lehrer. Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells. FEBS Lett. 396: 319-322, 1996[Medline].


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