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Am J Physiol Lung Cell Mol Physiol 290: L270-L277, 2006. First published September 9, 2005; doi:10.1152/ajplung.00249.2005
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Culture of murine nasal epithelia: model for cystic fibrosis

B. R. Grubb, T. D. Rogers, P. C. Diggs, R. C. Boucher, and L. E. Ostrowski

Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina

Submitted 8 June 2005 ; accepted in final form 7 September 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
The ion transport defects reported for human cystic fibrosis (CF) airways are reproduced in nasal epithelia of the CF mouse. Although this tissue has been studied in vivo using the nasal potential difference technique and as a native tissue mounted in the Ussing chamber, little information is available on cultured murine nasal epithelia. We have developed a polarized cell culture model of primary murine nasal epithelia in which the CF tissue exhibits not only a defect in cAMP-mediated Cl– secretion but also the Na+ hyperabsorption and upregulation of the Ca2+-activated Cl– conductance observed in human airways. Both the wild-type and CF cultures were constituted predominantly of undifferentiated cuboidal columnar cells, with most cultures exhibiting a small number of ciliated cells. Although no goblet cells were observed, RT-PCR demonstrated the expression of Muc5ac RNA after ~22 days in culture. The CF tissue exhibited an adherent layer of mucus similar to the mucus plaques reported in the distal airways of human CF patients. Furthermore, we found that treatment of CF preparations with a Na+ channel blocker for 7 days prevented formation of mucus adherent to epithelial surfaces. The cultured murine nasal epithelial preparation should be an excellent model tissue for gene transfer studies and pharmacological studies of Na+ channel blockers and mucolytic agents as well as for further characterization of CF ion transport defects. Culture of nasal epithelia from {Delta}F508 mice will be particularly useful in testing drugs that allow {Delta}F508 CFTR to traffic to the membrane.

{Delta}F508 mice; cystic transmembrane conductance regulator; epithelial sodium channel; mucus; Na+ hyperabsorption

THE AIRWAYS of the cystic fibrosis (CF) patient are characterized by an absence of cystic fibrosis transmembrane conductance regulator (CFTR) function in the apical membrane (11, 37), leading to hyperabsorption of Na+ (1–3) and a failure of cAMP-regulated Cl– secretion. The in vivo bioelectric phenotype of human CF airway epithelia has been reproduced in cultured human airway epithelia (40, 42), which serves as a valuable model system for electrophysiological studies (37, 41), gene transfer experiments (21, 22, 44), and drug testing (19, 24, 29, 32).

As human CF tissue is often in very short supply, it would be useful to have a CF mouse airway culture system that mimics the human phenotype. Although there are studies that focus on culture of murine lower airway epithelia (trachea) (5, 43), the lower airways of the CF mouse reproduce little of the human CF airway phenotype (15). In contrast to the murine trachea, nasal epithelia of the CF mouse appear to closely mimic the bioelectric phenotype of human CF airway, exhibiting both a defect in cAMP-mediated Cl– secretion and Na+ hyperabsorption (16, 17). Whereas the nasal epithelia of the mouse have been studied both in vivo (16, 17) and as freshly excised cells/tissue (8, 17, 34), little work has been done on cultured mouse nasal epithelia (6). The absence of such studies reflects the difficulty in culturing murine airway epithelia in general (9). Because no murine airway epithelial culture models are available that reproduce the human CF airway epithelial phenotype, we have focused on the culture of normal and CF murine nasal epithelia in an attempt to reproduce the bioelectric phenotype exhibited by CF human airways.

For these studies, we resected nasal epithelia from wild-type (WT) and CF mice and plated disaggregated cells on Transwell-Col (T-Col) membranes. The culture media selected contained bovine pituitary extract and cholera toxin as well as other, more standard hormones and ingredients (12), and an air-liquid interface system was employed to promote cellular differentiation (39). We then characterized the ion transport properties of these preparations in Ussing chambers and examined the relationships between abnormal ion transport and mucus adherence to cellular surfaces using histological approaches.


   METHODS
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Animals. Adult mice of both sexes were used in this study. Most of these mice were of mixed strain (BALB/C, C57BL/6, DBA/2, 129/SvEv). The "normal" WT mice were heterozygous for CFTR and littermates to the CF null mice (Cftrtm1Unc) (31). (We have been unable to distinguish a difference in airway bioelectrics between heterozygotes and homozygous normal mice. Thus, in this paper, we refer to the heterozygous mice as normal, WT mice.) A very limited number of {Delta}F508 mice (Cftrtm1Kth) (45) and littermate controls (on a C57BL/6 background) were also studied. Animals were maintained and studied under protocols approved by the University of North Carolina Institutional Animal Care and Use Committee. Mice were killed by CO2 inhalation, and the nasal epithelia were immediately dissected and removed from the nasal cavity (14).

Cell culture. After the epithelia were removed from the nasal cavity, they were placed in 10 ml of ice-cold F-12 media, 1% protease-0.01% DNase (1.5 ml) was added, the tube containing the specimens was shaken gently for ~1 min and refrigerated for 2 h with occasional agitation (~1 min every 20 min), and the supernatant was poured through a cell strainer (70-µm nylon, BD Falcon) into a 50-ml conical tube. Ten milliliters of 10% FBS in F-12 were added to tissue remaining in the tube, and the tissue was shaken gently and poured through the cell strainer. This wash was repeated, resulting in a final volume of ~30 ml. The harvested nasal cells were spun for 10 min at 1,500 rpm, the supernatant was aspirated, a new volume of 10% FBS in F-12 was added, and the washing procedure was repeated twice. The cells were then resuspended in 5 ml of culture media and counted. We typically obtained ~500,000 nasal cells/mouse. The cells were then seeded on 6.5-mm T-Col (Corning, Acton, MA) substrates at a density of ~250,000 cells/T-Col (~750,000 cells/cm2) in 50 µl of culture media, and 1.5 ml of culture media were placed on the basolateral side of the T-Col. The cells were incubated at 37°C in 5% CO2 in a humidified incubator. On day 2 postseeding, the apical surface of the cultures was washed with PBS (1x) to remove debris and nonadherent cells, and then all apical liquid was removed. The basolateral liquid was also removed, and 2 ml of fresh media were added basolaterally. The cultures were fed (basolateral media was changed), and the apical surface was washed and aspirated to dryness three times per week. Once the cells reached confluence (usually after ~8–10 days), the apical surface remained dry as a result of absorption of the apical liquid. From this point onward, except for washing the apical surface and aspirating the wash liquid (PBS), no liquid was left on the apical surface.

Media. The culture media were composed of 50% Ham's F-12 and 50% conditioned MEM (42), to which the following ingredients were added (all concentrations given as final media concentration): FBS (10%), insulin (10 µl/ml), hydrocortisone (1 µM), triiodothyronine (30 nM), penicillin (1,000 U/ml), streptomycin (100 mg/ml), cholera toxin (10 ng/ml) (all purchased from Sigma), endothelial cell growth supplement (3.75 µg/ml), epidermal growth factor (25 ng/ml), transferrin (5 µg/ml) (last 3 ingredients purchased from Collaborative Research), and bovine pituitary extract (500 µl) (9).

Electrophysiological measurements. The T-Col inserts were studied in Ussing chambers fitted with adaptors to accommodate T-Col membranes (Warner Instruments). The tissues were studied under open-circuit conditions using a Physiologic Instruments voltage clamp (San Diego, CA). The electrical potential difference (PD) across the tissue was continually recorded, and a constant current pulse (2–10 µA) was applied across the tissue at 1-min intervals to calculate tissue resistance. From these measurements, the equivalent short-circuit current (Ieq) was calculated. All other details of Ussing chamber techniques have been previously published (14).

Solutions and drugs. For electrophysiological study, the tissues were bathed bilaterally (10 ml/side) in Krebs-Ringer bicarbonate (gassed with 95% O2/5% CO2) having the following composition (in mM): 140 Na+, 120 Cl–, 5.2 K+, 1.2 Mg2+, 1.2 Ca2+, 2.4 HPO42–, 0.4 H2PO4–, and 25 HCO3–. All preparations contained 5 mM glucose on the basolateral side and 5 mM mannitol in the apical solution.

In these studies, amiloride (10–4 M apical addition) was used to block electrogenic Na+ absorption. Forskolin (10–5 M apical) and UTP (10–4 M apical) were used to induce anion secretion via increases in cAMP and intracellular Ca2+, respectively. Bumetanide (10–4 M), an inhibitor of Na+-K+-Cl– cotransporter (NKCC1), was added to the basolateral bath to block Cl– secretion. All drugs for Ussing chamber studies were purchased from Sigma, with the exception of UTP (Amersham Pharmacia Biotech).

Histology. T-Cols that were to be studied histologically were placed in 20-ml neutral buffered formalin for 24 h before paraffin embedding and processing by standard techniques. Sections were stained with either hematoxylin and eosin, for the determination of general histology of the preparations, or Alcian blue/periodic acid-Schiff (AB-PAS), to identify the presence of mucins. Immunostaining for the detection of cilia and Muc5ac was performed using standard techniques with the following modifications. After deparaffinization, sections were treated with antigen retrieval solution (Dako Cytomation, Carpinteria, CA) and incubated in Fab Fragment goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a 1:65 dilution in PBS, fixed briefly with 4% paraformaldehyde, washed with PBS, and blocked in 10% normal goat serum (Jackson ImmunoResearch Laboratories) in PBS. The sections were incubated with anti-{beta}-IV tubulin (Sigma, St. Louis, MO), anti-Muc5ac (Lab Vision, Fremont, CA), or mouse IgG (Jackson). After washing, immunodetection was performed using biotin-SP-conjugated goat anti-mouse, followed by Texas red dye-conjugated streptavidin (Jackson). Slides were washed with PBS/0.05% Tween, mounted using Vecta-Shield mounting medium containing 4',6-diamidino-2-phenylindole to label nuclei (Vector Laboratories, Burlingame, CA), and sealed with nail polish. Image acquisition and analysis were performed on a Leica SP2 AOBS upright laser scanning confocal microscope (Leica Microsystems, Heidelberg, Germany). Images were processed using Adobe Photoshop software.

RNA isolation and analysis. RNA was isolated from representative T-Cols using the Qiagen RNeasy mini-kit according to the manufacturer's instructions. Reverse transcription of RNA into cDNA was performed using Superscript II (InVitrogen), and PCR was performed using AmpliTaq Gold (Applied Biosystems), both using standard procedures. Primers for Muc5ac and the three subunits of epithelial Na+ channel (ENaC) were as previously published (10, 27). Primers to olfactory marker protein (OMP) [a marker for olfactory receptor neurons (38)] were 5'-ATGGATTGGAATGAGGCAGACG-3' and 5'-CCCAGTGTCTTGAAGGCCCTAA-3' and primers for murine CFTR were 5'-ACGTTCACACCCAACTCAGGCTCC-3' and 5'-GAAGCAGCCACCTCAACCAGAAAAA-3'. These primers amplify a product from both CF and WT mice, although the mutated CF transcript does not produce functional protein.

Effect of Na+ channel blocker on mucus accumulation in CF nasal epithelia. The effect of the Na+ channel blocker (NCB) PS 518 (a gift from Parion, Durham, NC) on mucus accumulation on CF airway epithelia surfaces over 7 days was studied by applying 10 µM NCB in 22 µl of PBS apically and 10 µM compound on the basolateral side of CF T-Cols, starting on day 14, a time that preceded surface mucus accumulation. (PS 518 has an EC50 of ~31 nM and has been found to block apical Na+ absorption when applied from either the apical or basolateral side of the tissue; personal communication with A. Hirsch at Parion.) The effects of PS 518 were compared with two groups of control CF T-Cols. One control group received no treatment (no apical liquid), and the other control group received 22 µl of PBS apically without the NCB. All T-Cols had 2 ml of media on the basolateral side. All T-Cols were placed on a rocker and slowly rocked for the duration of the experiment to keep the NCB on the T-Cols uniformly distributed over the surface. At the end of each 24-h period, all T-Cols were fed, and any remaining apical liquid was aspirated (no wash) and new apical liquid (either with or without the NCB) was added. At the end of 7 days, the T-Cols were fixed in neutral buffered formalin and sectioned for histological study.

Statistics. All data are shown as means ± SE. A Student's t-test was used to compare means between two groups, and P ≤ 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
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Bioelectrics. Bioelectric data of cultured murine nasal epithelia from CF and normal mice are shown in Fig. 1. Unless otherwise noted, all data shown for CF mice are from CF null mice (Cftrtm1Unc). Most measurements were performed on day 22 of culture, and results at other time points were similar. Actual PD recorder traces of a WT and a CF culture are shown in Fig. 1, A and B. Although the basal Ieq did not differ significantly between the genotypes, the electrogenic Na+ absorption (the amiloride-sensitive Ieq) was significantly greater in the CF preparations (P ≤ 0.001; Fig. 1C). Amiloride reduced the basal Ieq to nearly zero in the CF preparations, whereas the normal cultures exhibited a substantial Ieq postamiloride (Fig. 1C, also see Fig. 1, A and B). After amiloride application, forskolin was added to the apical bath of the Ussing chamber. The CF nasal epithelia exhibited little response to forskolin, whereas the nasal epithelia from the normal mice exhibited a robust forskolin response (P ≤ 0.001, Fig. 1, A, B, and D). The response to UTP by the CF epithelia was double that of the WT nasal epithelia (P ≤ 0.01, Fig. 1D). The response to bumetanide (added after UTP) by the WT tissue was approximately threefold greater than that exhibited by the CF tissue (P ≤ 0.001, Fig. 1D). The transepithelial resistance did not differ between the cultures of the two genotypes [100 ± 11.8 (n = 17) vs. 118.9 (n = 20) {Omega}·cm2, WT and CF, respectively]. Although this resistance appears relatively low for cultured airway preparations, it is greater than the resistance reported for native murine nasal epithelia (17). In addition, studies from a variety of species found that the resistance of excised nasal tissue was between 40 and 100 {Omega}·cm2 compared with a resistance of ~360–640 {Omega}·cm2 for cultured nasal epithelia (30). Therefore, our cultured preparations more closely mimic the resistance reported for native tissue.



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  		Fig. 1. Bioelectrics of cultured wild-type (WT) and cystic fibrosis (CF) murine nasal epithelia. A: potential difference (PD) recorder trace of a typical WT nasal culture. Preparations were studied open circuit, and the recorded PD is shown. The change in PD in response to a constant current pulse (pulsed at 1-min intervals, used to calculate resistance) was omitted from the traces. For this preparation, the basal equivalent short-circuit current (Ieq) (preamiloride) was 71 µA·cm–2 with a resistance of 225 {Omega}·cm2. B: recorder trace of a typical CF nasal culture. The basal Ieq (preamiloride) of this particular trace was 115 µA·cm–2 with a resistance of 204 {Omega}·cm2. C: basal Ieq and that portion of the basal Ieq sensitive to amiloride (Amil sensitive) (P ≤ 0.001 CF compared with WT). Post amil is that portion of the Ieq remaining after amiloride treatment. P ≤ 0.001 CF compared with WT. D: forskolin (Forsk) response (added after amiloride) in the normal preparations is very robust and significantly greater (P ≤ 0.001) than the negligible response exhibited by the CF preparations. The response to UTP (added after forskolin) in the CF preparations was double that of the WT preparations, P ≤ 0.01. The response to bumetanide (Bumet; added after UTP) differed significantly between the 2 groups (P ≤ 0.01). All bars are means ± SE, n = 17 normal Transwell-Cols (T-Cols) [representing 6 different groups of mice (6 mice/group)] and n = 20 CF T-Cols [representing 8 different groups of mice (6 mice/group)]. Open bars, tissue from normal WT mice; filled bars, tissue from CF mice. *P < 0.01, **P < 0.001.

 
With a very limited number of {Delta}F508 CF mice and littermate controls (n = 4), we obtained three CF and three WT nasal cultures that we studied on day 21. Similar to the CFTR null mice, the nasal epithelia of the {Delta}F508 CF mice exhibited an elevated response to amiloride [{Delta}short-circuit current ({Delta}Isc) 50.1 ± 12 vs. 27.8 ± 6.2 µA·cm–2]. The forskolin response of the {Delta}F508 CF cultures was not significantly different from zero compared with the robust forskolin response exhibited by the WT control cultures (0.6 ± 0.17 and 58.7 ± 7.3 µA·cm–2, CF and WT, respectively). Whereas the response to UTP was small in cultures of both genotypes, the UTP response was more than four times greater (P ≤ 0.05) in the CF cultures ({Delta}Isc 4.3 ± 0.8 µA·cm–2) compared with the response of reversed polarity ({Delta}Isc –1.3 µA·cm–2) exhibited by the WT cultures. Thus our CF nasal cultures (from both null and {Delta}F508 animals) exhibited hyperabsorption of Na+, absence of cAMP-mediated Cl– secretion, and upregulation of the Ca2+-activated Cl– conductance.

Culture characterization. On day 22 postseeding (the time most typically chosen for bioelectric study), the cultures were variably differentiated. The cultures were always multilayered, with the predominant superficial cell type being cuboidal/columnar (Fig. 2, A and B). Ciliated cells were present in most cultures examined (>80% of CF cultures, ~50% of WT), although the percentage of ciliated cells was typically low (<25%, Fig. 2, C and D). The number of ciliated cells observed did not appear to increase further with the length of time in culture (days 28–35). Both the normal and CF cultures exhibited invaginations of the epithelia that appeared somewhat ductlike (Fig. 2, A and B).



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  		Fig. 2. Histology of WT (A) and CF (B) cultured (22-day) nasal epithelia stained with hematoxylin and eosin or with a monoclonal antibody against {beta}-IV tubulin to identify ciliated cells (C and D). Both cultures exhibit a multilayered epithelium consisting primarily of undifferentiated cells. Ciliated cells (arrows) are clearly visible in WT (C) and CF (D) cultures when stained with the anti-{beta}-IV tubulin antibody. Scale bars in A and B are 20 µm.

 
Although the initial cell inoculum consisted of a mixture of both respiratory and olfactory epithelia, no mature olfactory neurons were observed by histological examination. Furthermore, several cultures were analyzed for the presence of OMP mRNA by RT-PCR (38). These cultures were negative for the presence of OMP (Fig. 3A), indicating that differentiated olfactory neurons were not present. However, both normal and CF cultures were positive for Muc5ac expression by RT-PCR (Fig. 3B). Furthermore, RT-PCR demonstrated the expression of CFTR and ENaC (Fig. 3, C and D), in agreement with the bioelectric data above. Although not quantitative, these results indicate that the cultured epithelia maintain expression of several relevant transcripts.



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  		Fig. 3. RT-PCR analysis of selected transcripts in cultures of CF and WT nasal epithelial cells. RNA was isolated from cultures of nasal epithelial cells and subjected to RT-PCR using specific primers as described in METHODS. RNA from freshly isolated tracheal and nasal tissue was used as positive controls. Reactions were performed in the presence (+) or absence (–) of RT to confirm the specificity of the reaction. Both CF and WT cultures were negative for the expression of olfactory marker protein (OMP; A), whereas both were positive for the expression of Muc5ac (B). Both cultures expressed transcripts for cystic fibrosis transmembrane conductance regulator (CFTR; C) and for the {alpha}- (D), {beta}-, and {gamma}- (not shown) subunits of epithelial Na+ channel (ENaC). Note that the CFTR transcript in the CF null mice does not produce a functional protein.

 
Mucus accumulation. The difference in the appearance of the apical surface between normal and CF nasal cultures with respect to the amount of apical mucus present was striking (Fig. 4, A and B). In general, there was no mucus (AB-PAS-positive material) accumulation on the apical surface of WT nasal cultures (Fig. 4A). In the CF nasal preparations, there was copious mucus (AB-PAS-positive material) present both on the surface and in the epithelial invaginations (Fig. 4B). Many of the epithelial invaginations present in WT cultures were largely devoid of mucus. Cellular debris was clearly visible in the mucus layer, which may represent the accumulation of cells lost from the epithelia through normal turnover. Interestingly, no goblet cells were noted in the AB-PAS-stained sections from nasal cultures of either genotype. Immunostaining of paraffin sections with a monoclonal antibody against Muc5ac revealed patches of reactive material in the apical mucus of CF cultures (Fig. 4C), compared with the lack of staining observed with a control mouse IgG (Fig. 4D). A WT culture stained for Muc5ac, which had limited apical mucus accumulation, showed no specific staining (not shown).



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  		Fig. 4. WT (A) and CF (B) nasal epithelia stained with Alcian blue/periodic acid-Schiff demonstrating that the WT preparations are largely devoid of mucus (blue line is the glycocalyx layer) and the CF preparation exhibits copious apical mucus as well as mucus in the epithelial invaginations. Note the absence of goblet cells in both cultures. Immunostaining of a CF culture with an antibody against Muc5ac demonstrating reactive material in the apical mucus (C, arrows) that was not observed in sections stained with a control IgG (D). Scale bars in A and B are 20 µm.

 
Of the preparations examined histologically (typically between days 19 and 24, Table 1), ~80% of the CF preparations exhibited the mucus phenotype shown in Fig. 4. Fewer than 20% of the CF preparations examined failed to exhibit the mucus phenotype. Of the normal preparations prepared for histological study, >90% were devoid of mucus (Table 1).


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  		Table 1. Distribution of mucus phenotype in cultured murine nasal epithelia

 
Effect of NCB on mucus accumulation in CF nasal epithelia. To determine whether mucus accumulation on the apical surface of the CF nasal epithelia could be prevented by blocking Na+ hyperabsorption, we treated CF preparations daily with PS 518, a potent, long-acting NCB. This compound was chosen instead of amiloride because we have found that amiloride was very rapidly transported across the epithelia, quickly depleting the apical amiloride concentration and diminishing the effectiveness of the compound. In a pilot study, we found that the concentration of amiloride (100 µM in 33 µl of apical volume) on the apical surface of murine nasal epithelia grown on a T-Col (6.5 mm) had declined >50% in 180 min (unpublished observation). The daily application of the NCB PS 518, both apically and basolaterally, was completely effective in preventing the accumulation of apical mucus on the treated CF nasal preparations (Fig. 5A). In the treated preparations, the apical cilia appeared upright and were not adhered to the apical surface (Fig. 5A). In contrast, the vehicle-treated preparations had copious surface mucus, and the barely visible cilia again appeared stuck to the apical surface (Fig. 5B). Whereas the PS 518-treated preparations maintained a volume of visible liquid on the apical surface at the end of each 24-h period, the apical surfaces of the nontreated and vehicle-treated preparations appeared completely dry.



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  		Fig. 5. A: culture of CF nasal epithelia treated with the Na+ channel blocker PS 518 for 7 days (n = 3). B: cultured CF nasal epithelia treated with vehicle only (n = 3). Note the easily visible cilia in A compared with B. The 3 cultures receiving no treatment were indistinguishable from those receiving vehicle only (not shown). Both preparations were from the same group of mice, and photos were taken at day 21 postseeding. Both preparations were stained with Alcian blue/periodic acid-Schiff. Scale bars in A and B are 25 µm.

 

   DISCUSSION
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 ABSTRACT
 METHODS
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GRANTS
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 REFERENCES
 
Because of the limited availability of human airway tissue, it would be useful to have a culture model of murine CF airway epithelia that exhibits the phenotype displayed by human CF tissue. There are a number of studies in the literature that describe the culture and study of murine tracheal epithelia (5, 6, 8, 9, 33, 43). However, the murine CF trachea (native tissue in vitro) does not exhibit Na+ hyperabsorption and also exhibits a non-CFTR cAMP-mediated Cl– conductance that is difficult to distinguish from the cAMP response in WT trachea (7, 15). Consequently, although some investigators have been successful at teasing out a small portion of the cAMP Cl– conductance due to CFTR in WT tracheae (7, 25), distinguishing the CF from normal phenotype in freshly excised murine tracheae has been difficult. Interestingly, it appears easier to discriminate between CF and WT tracheal tissue in culture, because the increase in cAMP may have less effect on intracellular Ca2+ concentrations (6, 33) than seen in native tissue. However, the CF tracheal cultures still fail to exhibit Na+ hyperabsorption (6, 33), which is a hallmark of the human disease, and is thought to play a major role in lung pathology observed in the human CF patient (23, 28).

The nasal epithelia of the CF mouse, unlike tracheal epithelia, exhibit both the defect in cAMP-mediated Cl– secretion and hyperabsorption of Na+. In addition, the CF murine nasal epithelia exhibit an upregulation of Cl– secretion mediated via a Ca2+-activated pathway. However, almost all studies of nasal epithelia of the normal vs. CF mice have employed either freshly excised tissue (17, 26) or used the nasal PD technique to study the tissue in vivo (16, 20).

With the exception of one paper published over a decade ago (6), no information comparing the properties of WT and CF cultured murine nasal epithelia is available. Although the paper published by Clarke et al. (6) found that there was a significant difference in the magnitude of the forskolin response in WT vs. CF murine cultured nasal epithelia, the magnitude of the Cl– secretory response in the WT tissue was small (<10 µA·cm–2), and no quantitative data were given on the magnitude of the amiloride-sensitive Ieq. Furthermore, no histological features of the cultures were described.

Our nasal culture system appears to reproduce much of the transport function observed in vivo and in freshly excised tissue. Although most of our data were obtained on cultures from the CFTR null mouse, we obtained very similar bioelectric data from a limited number {Delta}F508 CF murine nasal cultures. However, the discussion that follows pertains to data in Figs. 1–5 from the CFTR null mouse. Like human CF airway and CF mouse native nasal epithelia, CF cultured murine nasal epithelia exhibit an enhanced response to amiloride consistent with hyperabsorption of Na+. Likewise, the defect in cAMP-mediated Cl– secretion was present in cultured CF mouse nasal epithelia, as evidenced by the virtual absence of a forskolin response in contrast to the robust response in WT cultures. Native CF nasal epithelia (both human and murine) also exhibited a significantly upregulated Ca2+-mediated Cl– secretory response (4, 13, 17, 24). Specifically, our cultured CF nasal epithelia exhibited a similar phenotype, with the UTP response in the CF epithelia being more than 2x that exhibited by the WT preparations. While this Cl– secretory pathway has been studied electrophysiologically, the molecular identity of this Cl– channel remains unknown (13). In our cultures, the response to bumetanide was significantly greater in the WT preparations compared with the CF nasal epithelia. This response likely reflects a component of the forskolin-induced Cl– secretion (via blocking NKCC1 basolateral Cl– entry) in the WT preparations. Also, in the WT preparations, the postamiloride Ieq is likely due to Cl– secretion, which may have been partially blocked by bumetanide.

The CF nasal epithelia cultured under these conditions exhibited significant accumulation of apical mucus, as shown by AB-PAS staining. This observation was intriguing, as we could not identify goblet cells in our cultured nasal preparations, even though goblet cells were clearly found throughout the nasal epithelia of the native tissue (35). Similarly, no goblet cells were observed morphologically in cultures of murine tracheal cells, although Muc5ac expression was detected (43). Both CF and normal cultures were positive by RT-PCR for the presence of the secreted mucin Muc5ac, and immunostaining suggested the presence of Muc5ac reactive material in the apical mucus. Muc5ac is a major respiratory mucin and has been shown to be expressed in murine nasal tissue (36). Thus secretion of this mucin by the nasal epithelia may have contributed to the mucus layer.

In the CF cultures, the accumulated mucus was most likely a result of the elevated rate of Na+ (and accompanying water) absorption, which desiccated the apical surfaces, promoted mucus adhesion, and resulted in failure to remove mucus during the daily aspiration maneuver. This is similar to the airway mucus accumulation observed in mice hyperabsorbing Na+ as a result of overexpression of ENaC (27). Whereas the mucus on the CF cultures could not be easily removed even with vigorous washing, exposing the CF epithelia to an NCB for 7 days (before mucus accumulation had begun) completely prevented mucus accumulation on the CF cultures (Fig. 5). The absence of mucus accumulation presumably reflected persistent dilution of mucus that allowed it to be cleared by daily culture aspiration. The experiments reported here do not allow us to determine whether the CF cultures produce more mucus than the WT cultures. However, in human airway cultures, neither the quantity nor the composition of the mucins produced differed between normal and CF cultures (18). Thus it is likely that mucus accumulation/adhesion observed in CF cultures reflected accelerated Na+ absorption.

In conclusion, we have developed a mouse culture model of normal and CF nasal epithelia. The cultures typically form a multilayered epithelia composed primarily of undifferentiated cells with evidence of ciliated cell differentiation and mucus secretion. The cultures express CFTR and ENaC, and most importantly, the CF cultures exhibit the classic ion transport defects exhibited by human CF airway epithelia. In addition, the CF cultures accumulate mucus on the apical surface and in epithelial invaginations. Although the defect in cAMP-mediated Cl– secretion likely contributed to the accumulation of apical mucus in the CF preparations, blocking electrogenic Na+ absorption was effective in maintaining hydration of the apical surface and preventing the mucus accumulation that characterized these cultures. Therefore, hyperabsorption of Na+ likely played the major role in allowing mucus accumulation in the CF cultures. These cultured preparations will be useful in gene transfer studies and further bioelectric studies of the ion transport defect in CF tissue as well as in pharmacological studies testing NCB. Culture of nasal epithelia from the {Delta}F508 mouse demonstrated similar ion transport defects to those exhibited by cultures of the CFTR null mouse, suggesting that culture of nasal epithelia from {Delta}F508 CF mice would be particularly useful in testing drugs that allowed {Delta}F508 CFTR to traffic to the apical membrane.


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This study was supported in part by the National Institutes of Health (NIH) Specialized Center of Research (SCOR) and NIH SCOR HL-060280 (project IV) and HL-70199 (L. E. Ostrowski).


   DISCLOSURES
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REFERENCES
 
R. C. Boucher owns more than 5% equity interest in Parion Sciences, which holds the patent relevant to PS 518, the molecule used in this paper.


   ACKNOWLEDGMENTS
 
We thank Weining Yin, the University of North Carolina Cystic Fibrosis Center Histology Core (Kim Burns and Elizabeth Andrews), and Michael Hooker Microscopy Facility for helpful technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. R. Grubb, Cystic Fibrosis/Pulmonary Research and Treatment Center, 7011 Thurston-Bowles Bldg., CB#7248, Univ. of North Carolina, Chapel Hill, NC 27599-7248 (e-mail: bgrubb{at}med.unc.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

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