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A primary culture model of differentiated murine tracheal epithelium

Donald J. Davidson1, Fiona M. Kilanowski1, Scott H. Randell2, David N. Sheppard3, and Julia R. Dorin1

1 Medical Research Council Human Genetics Unit, Western General Hospital, and 3 Medical Genetics Section, Molecular Medicine Centre, Department of Medical Sciences, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, United Kingdom; and 2 University of North Carolina Cystic Fibrosis Center, Chapel Hill, North Carolina 27599


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

The goal of this study was to develop a primary culture model of differentiated murine tracheal epithelium. When grown on semipermeable membranes at an air interface, dissociated murine tracheal epithelial cells formed confluent polarized epithelia with high transepithelial resistances (~12 kOmega · cm2) that remained viable for up to 80 days. Immunohistochemistry and light and electron microscopy demonstrated that the cells were epithelial in nature (cytokeratin positive, vimentin and alpha -smooth muscle actin negative) and differentiated to form ciliated and secretory cells from day 8 after seeding onward. With RT-PCR, expression of the cystic fibrosis transmembrane conductance regulator (Cftr) and murine beta -defensin (Defb) genes was detected (Defb-1 was constitutively expressed, whereas Defb-2 expression was induced by exposure to lipopolysaccharide). Finally, Ussing chamber experiments demonstrated an electrophysiological profile compatible with functional amiloride-sensitive sodium channels and cAMP-stimulated CFTR chloride channels. These data indicate that primary cultures of murine tracheal epithelium have many characteristics similar to those of murine tracheal epithelium in vivo. This method will facilitate the establishment of primary cultures of airway epithelium from transgenic mouse models of human diseases.

cystic fibrosis; cystic fibrosis transmembrane conductance regulator; defensins; airway surface liquid; airway epithelium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

THE DEVELOPMENT OF ROBUST TECHNIQUES for the primary culture of airway epithelial cells from a variety of species, including human (32), rat (4, 5), dog (14), guinea pig (1), rabbit (17), and hamster (31), has provided invaluable in vitro models for the study of airway physiology in health and disease. However, the development of successful techniques for the culture of mouse airway epithelial cells has remained elusive. The most successful published techniques resulted in the establishment of a relatively undifferentiated epithelium with few, if any, ciliated cells (6, 16). Although transgenic technology in mice has enabled the development of murine models for human pulmonary diseases and knockout mice with prominent pulmonary phenotypes (15, 19, 21, 30), there has been a frustrating inability to establish an adequate system for the primary culture of airway cells from these transgenic models.

Recent studies (20, 28, 33) have utilized primary cultures of human airway epithelium to investigate the pathogenesis of lung disease in cystic fibrosis (CF). This approach has permitted investigation of the characteristics of the airway surface liquid (ASL) that lines the conducting airways. Such studies have suggested that alterations in the ionic composition of the ASL, with consequent impairment of its salt-sensitive antimicrobial properties (28) or its dehydration, with resultant failure of mucociliary clearance (20), may play a key role in the development of the chronic infection and inflammation that characterize this disease (reviewed in Ref. 7).

Despite the prominent role that mouse models of CF have played in recent research into this disorder, studies to parallel and further develop these critical observations have been hampered by the absence of an acceptable primary culture model for murine airway epithelial cells.

To facilitate studies of tracheal epithelium from transgenic mouse models of human disease, we have developed a primary culture model of differentiated mouse tracheal epithelium. Here we describe the establishment of this model and investigate its characteristics using electron microscopy, histochemistry, immunohistochemistry, gene expression analysis, and electrophysiology.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Animals. Specific pathogen-free C57BL/6N mice of both sexes, age 5-7 wk, were used (Charles River UK, Margate, UK). All experimental work complied with the UK Animal (Scientific Procedures) Act 1986. The animals were killed by asphyxiation with CO2.

Isolation and culture of tracheal epithelial cells. On day 1, the mice were killed by asphyxiation with CO2 and doused with 70% ethanol, and the tracheae were excised and severed at the proximal surface of the thyroid cartilage and at the bifurcation of the bronchi. The thyroid glands and other adherent tissue were removed from the superolateral surfaces (while retaining the underlying proximal tracheae). The tracheae were cut open lengthwise, washed in phosphate-buffered saline (PBS), and transferred to collection medium (see Formulations) prewarmed to 37°C. Batches of eight tracheae were transferred to 20 ml of prewarmed dissociation medium containing Pronase and DNase (see Formulations) and incubated at 37°C for 60 min. To stop enzymatic digestion, 5 ml of sterile fetal calf serum were added. To dissociate epithelial cells, the tracheae were gently agitated (the tube containing the tracheae was carefully inverted 12 times, not shaken). The tracheal "husks" were removed from the suspension, placed in 10 ml of culture medium (see Formulations), and gently agitated 12 times as before to release more epithelial cells. The resultant cell suspensions were pooled and centrifuged at 1,000 rpm (120 g) for 5 min at room temperature (21-23°C). After the supernatants were removed, the pellets were resuspended and washed in 10 ml of culture medium, centrifuged at 1,000 rpm for 5 min, and resuspended in 5 ml of culture medium. This suspension was incubated at 37°C for 2 h in a 100-mm culture dish (Premaria, Becton Dickinson UK, Oxford, UK) to remove contaminating nonepithelial cells. After this incubation, the medium was removed with a fine-tip flexible pipette to collect the unattached cells, centrifuged at 1,000 rpm for 5 min, and resuspended in culture medium (200 µl for every two tracheae used). The dissociated cells from two tracheae (~4 × 105 cells) were seeded onto one tissue culture insert semipermeable support membrane (Costar Transwell clear, tissue culture-treated polyester membrane 24-well plate inserts, 0.4-µm pore; Corning Costar, High Wycombe, UK) in 200 µl of culture medium, with 600 µl outside the insert. To precoat these inserts with type VI acid-soluble human placental collagen (Sigma-Aldrich, Poole, UK), 100 µl of collagen solution (0.5 mg/ml of human placental collagen in distilled water with 0.2% glacial acetic acid) were added to the semipermeable membrane, air-dried overnight, and then washed twice with PBS before use. The cells were incubated at 37°C in 6% CO2 in a humidified incubator for 3 days. On day 4, the medium on the apical surface of the cultured cells was removed along with any nonadherent cells and debris, and the medium on the outside of the insert (bathing the basolateral surface) was replaced with 600 µl of Ultroser G (USG) medium (see Formulations). Once the cells had reached confluence, the apical surface of the insert appeared dry; this normally occurred from day 4 onward. The medium bathing the basolateral surface was replaced twice weekly. Primary cultures grown at an air-liquid interface remained viable for up to 80 days.

Formulations. Collection medium consisted of a 1:1 mix of Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL, Life Technologies, Paisley, UK) and Ham's F-12 medium (GIBCO BRL) containing penicillin (100 IU/ml)-streptomycin (100 µg/ml). Dissociation medium consisted of calcium- and magnesium-free minimum essential medium (44 mM NaHCO3, 54 mM KCl, 110 mM NaCl, 0.9 mM NaH2PO4, 0.25 µM FeN3O9, 1 µM sodium pyruvate, and 42 µM phenol red, pH 7.5), 60 IU/ml of penicillin-60 µg/ml of streptomycin, 1.4 mg/ml of Pronase (Boehringer Mannheim UK, Lewes, UK), and 0.1 mg/ml of DNase (Sigma-Aldrich). Culture medium consisted of a 1:1 mixture of DMEM and Ham's F-12 medium containing 100 IU/ml of penicillin-100 µg/ml of streptomycin, 5% fetal calf serum, and 120 IU/l of insulin (Nova Human Actrapid, AAH Pharmaceuticals, Glasgow, UK). USG medium consisted of a 1:1 mixture of DMEM and Ham's F-12 medium containing 100 IU/ml of penicillin-100 µg/ml of streptomycin and 2% USG serum substitute (GIBCO BRL).

Electron microscopy. Scanning (SEM) and transmission (TEM) electron microscopy were performed on primary cultures of murine tracheal epithelium and tracheal specimens from 5-wk-old mice. For SEM, the specimens were washed with PBS, then fixed first in 2.5% glutaraldehyde in PBS and second in 1% osmium tetroxide in PBS. Subsequently, the specimens were dehydrated through an alcohol series, treated with hexamethyldisilazane, and allowed to air-dry overnight. Primary culture specimens (cut from the plastic insert) and tracheal specimens were mounted on stubs with carbon adhesive disks and silver DAG (Agar Scientific, Stansted, UK), sputter coated, and viewed in a Cambridge S250 scanning electron microscope. Images were recorded on Kodak Tmax 100 film. For TEM, the specimens were fixed as for SEM. After dehydration, the specimens were infiltrated with Spurr resin, and serial sections of 80-110 nm were cut with a Leica UCT Ultramicrotome. Sections were viewed on a Philips CM120 Biotwin. TEM plates were Kodak SO 163.

To quantitatively assess the different cell populations present in the primary cultures of murine tracheal epithelium, morphometric analyses were performed on SEM images of primary cultures on days 4, 8, 14, and 28 after seeding and on tracheal specimens from 5-wk-old mice. We counted the number of ciliated and nonciliated cells in >= 4 fields of 100 cells and >= 2 specimens for each time point. TEM sections were used to provide additional information about the ultrastructure of primary cultures of murine tracheal epithelium and the morphology of cell types present. Quantitative analyses of TEM sections were not performed.

Histochemistry. Primary cultures of murine tracheal epithelium were washed in PBS and fixed with 1:1 acetone-methanol for 5 min. A standard periodic acid-Schiff stain was performed to indicate the presence of goblet cells.

Fluorescence immunohistochemistry. Primary cultures of murine tracheal epithelium and tracheal sections from 5-wk-old mice were characterized with antibodies to different cytokeratins, vimentin, and alpha -smooth muscle actin. Similar methods were used for both cultured epithelium and mouse tracheal sections. We used the following antibodies: mouse monoclonal anti-human pan cytokeratin (1:100 dilution; Sigma-Aldrich), mouse monoclonal anti-human alpha -smooth muscle actin (1:400 dilution; Sigma-Aldrich), mouse monoclonal anti-human vimentin (1:40 dilution; Sigma-Aldrich), mouse monoclonal anti-proliferating cell nuclear antigen (1:2,000 dilution; Sigma-Aldrich), and rabbit anti-mouse cytokeratin 14 and rabbit anti-mouse cytokeratin 18. The antibodies cytokeratin 14 and cytokeratin 18 were raised in rabbits immunized with peptide sequences present in the mouse homologs of human cytokeratins 14 and 18 (CGKWSTHEQVLRTKN-COOH and CGRWSETNDTRVLRH-COOH, respectively), conjugated to maleimide-activated ovalbumin or bovine serum albumin (Pierce, Rockford, IL), and affinity purified on peptides linked to maleimide-activated Sepharose (Pierce). Negative controls were performed by omitting the primary antibodies. Tracheal specimens were fixed in 1:1 acetone-methanol, processed to paraffin wax blocks, cut to 4-µm sections with a microtome, deparaffinized in xylene, and hydrated through an alcohol series. Primary culture specimens were washed in PBS, fixed in 1:1 acetone-methanol for 5 min, washed in PBS, and cut from the culture insert. When a mouse primary antibody was used, the specimens were preblocked with blocking solution [PBS with 2% normal animal serum (of the species in which the secondary antibody was raised), 0.2% Tween, 20 mg/ml of bovine serum albumin, and 7% glycerol] with 1% unlabeled sheep anti-mouse IgG [Scottish Antibody Production Unit (SAPU), Carluke, UK] or 1% unlabeled sheep anti-mouse immunoglobulins (SAPU) for 20 min and then washed with PBS. The specimens were incubated with blocking solution for 20 min before incubation for 1 h at room temperature with the primary antibody diluted in blocking solution. After a wash with PBS, a 30-min incubation was performed with the secondary antibody [FITC-labeled sheep anti-mouse IgG (SAPU) or FITC-labeled donkey anti-rabbit (SAPU)] diluted 1:100 in blocking solution. After being washed in PBS, the specimens were mounted in Vectashield (Vector Laboratories, Peterborough, UK) containing 4,6-diamidino-2-phenylindole nuclear stain.

Specimens were viewed with an Axioplan 2 microscope (Zeiss, Welwyn Garden City, UK). Images were acquired with a Pentamax digital camera (Princeton Instruments, Marlow, UK). In-house capture routine scripts and IPLab scientific image processing software, version 3.1.1c (Signal Analytics, Vienna, VA) were used to process images and standardize the representation of fluorescence levels between specimens. To quantify immunohistochemistry data, morphometric analyses were performed with primary cultures of murine tracheal epithelium on days 4, 8, 14, and 28 after seeding. The number of positive-staining cells per 100 nuclei was counted in 3 fields/sample (n >=  2 samples at each time point). Analyses of sections of tracheal specimens was also performed (n >=  3 samples for each antibody). Cell density was assessed by counting the number of nuclei in three fields of view at ×630 magnification (n = 4 samples at each time point).

Gene expression analysis. The gene expression profile of primary cultures of murine tracheal epithelium was investigated with reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA was isolated with RNAzol B (Biogenesis, Poole, UK) and cDNA synthesis was performed with Ready-To-Go You-Prime First-Strand Beads (Amersham Pharmacia Biotech, St. Albans, UK) as described by the manufacturers. The resultant cDNAs were used as templates for intron-spanning PCRs with primers designed to detect the murine CF transmembrane conductance regulator (Cftr) and mouse beta -defensin-1 (Defb-1) and beta -defensin-2 (Defb-2) genes, with parallel amplification of the murine hypoxanthine phosphoribosyltransferase (Hprt) gene as an internal control, all as previously described (8, 22, 23). The amplified products were analyzed by electrophoresis on 2% agarose gels with a Phi X147HaeIII marker, amplified plasmid-positive controls, and negative controls, with either heat-inactivated RT or distilled water substituted for RNA during cDNA synthesis. Defb-2 PCR products were not visible on a stained agarose gel after 34 cycles of PCR but were detectable by hybridization with a Defb-2 internal oligonucleotide probe as previously described (22). Primary cultures were analyzed after a 30-min exposure to either 80 µg of Escherichia coli lipopolysaccharide (LPS; serotype 026:B6; Sigma-Aldrich) in PBS or ~2 × 105 colony-forming units of Pseudomonas aeruginosa (CF clinical strain J1385; generous gift from Prof. J. R. W. Govan, University of Edinburgh, Edinburgh, UK). For both LPS and P. aeruginosa, the vehicle was in PBS. Untreated control epithelia were simultaneously exposed to PBS alone.

Electrophysiological studies. To monitor the transepithelial resistance (Rte) of cultured murine tracheal epithelium, we used an EVOM epithelial voltohmmeter (World Precision Instruments, Stevenage, UK) after the addition and equilibration of 200 µl of USG medium prewarmed to 37°C to the apical surface. Rte was measured on days 4, 8, 14, and 28 after seeding.

Primary cultures of murine tracheal epithelium were mounted in a modified Ussing chamber apparatus (Jim's Instruments Manufacturing, Iowa City, IA), and both the apical and basolateral surfaces were bathed with a physiological solution containing 140 mM NaCl, 5 mM KCl, 0.36 mM K2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 0.5 mM MgCl2, 10 mM HEPES, 4.2 mM NaHCO3, and 5 mM D-glucose, titrated to pH 7.2 with Tris. Physiological solutions were maintained at 37°C and gassed with 95% CO2-5% O2. The voltage (ELEC/CV, ADInstruments, Hastings, UK) and current (Ag-AgCl) electrode pairs were connected to the apical and basolateral solutions by polyethylene tubing (PE-160; Garridor) filled with 3 M KCl-3% (wt/vol) Noble agar (Sigma-Aldrich).

Transepithelial voltage was clamped at 0 mV with a Physiological Instruments VCCMC2 dual-channel voltage-current clamp (ADInstruments). Short-circuit current (Isc) was continuously recorded with a MacLab/4s recorder and Chart for Macintosh (version 3) data acquisition and analysis software (both supplied by ADInstruments); Rte was monitored with 10-mV voltage steps (duration 0.1 s, period 10 s) and calculated with Ohm's law. A positive Isc value represents a flow of positive charge from the luminal (apical) to the basolateral solution (anions moving in the opposite direction).

To investigate the electrophysiological characteristics of primary cultures of murine tracheal epithelium, the following agents were sequentially added: 1) amiloride (10 µM) to the apical reservoir to inhibit the epithelial Na+ channel (ENaC), 2) cAMP agonists [10 µM forskolin, 100 µM 3-isobutyl-1-methylxanthine (IBMX), and 500 µM 8-(4-chlorophenylthio)-cAMP (CPT-cAMP)] to the apical reservoir to activate the CFTR Cl- channel, and 3) bumetanide (100 µM) to the basolateral reservoir to inhibit the Na+-K+-2Cl- cotransporter and hence transepithelial Cl- secretion. Each reservoir contained both the test drug and the previously studied drugs within the sequence. The addition of each agent was performed only after stabilization of Isc. This protocol was performed on primary cultures of murine airway epithelium on day 28 after seeding (n = 6 specimens).

Amiloride hydrochloride, bumetanide, CPT-cAMP, forskolin, HEPES, and IBMX were all supplied by Sigma-Aldrich. All other chemicals were of reagent grade.

CPT-cAMP and amiloride were dissolved in distilled water, forskolin and bumetanide were dissolved in dimethyl sulfoxide (DMSO), and IBMX was dissolved in ethanol. Stock solutions were stored at -20°C and diluted in the physiological solution to achieve the final concentrations immediately before use.

Statistical analysis. Results are expressed as means ± SD; n is the number of observations. To compare sets of data, we used Student's t-test. Differences were considered significant when P < 0.05.


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Primary cultures of murine tracheal epithelium were established on semipermeable support membranes, grown at an air-liquid interface, and characterized to assess differentiation.

The short enzymatic dissociation of pooled tracheae at 37°C as described in MATERIALS AND METHODS generated ~2 × 105 cells/trachea, with cell viability of ~95% as assessed by trypan blue dye exclusion. Isolated cells were seeded at high density on collagen-coated semipermeable membranes. After 72 h of submerged culture, the apical medium along with any nonadherent cells and debris was removed to create an air-liquid interface at the apical surface of the cells. This air-liquid interface was retained for as long as the cultures remained viable (up to 80 days). Phase-contrast light microscopy demonstrated that primary cultures of murine tracheal epithelium had a smooth "cobblestone" appearance. Consistent with this finding, high Rte values indicated confluence, with the formation of tight junctions from day 4 onward. During the second week of culture, the development of beating cilia was observed, with the gradual appearance of scattered foci of "cell stacking" noted over the next few weeks.

Further morphological studies were performed with SEM, TEM, histochemistry, and immunohistochemistry. Additional characterization was performed with RT-PCR to analyze gene expression and with Ussing chamber experiments to study the electrophysiological profile of the primary cultures.

Electron microscopy. SEM was performed on primary cultures of murine tracheal epithelium on days 4, 8, 14, and 28 (n >=  2 observations for each time point) and tracheal specimens from 5-wk-old animals (n = 2 observations) of the same strain used to isolate epithelial cells for primary culture (Fig. 1).


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Fig. 1.   Scanning electron micrographs of a 5-wk-old mouse trachea (a) and primary cultures of murine tracheal epithelium on days 4 (b), 8 (c), and 28 (d). Arrows, cilia.

On day 4, a flattened epithelium was observed (Fig. 1b), with no features of differentiated cells. This was characteristic of early stage primary culture models with other species and is well described for cultured rat tracheal epithelial cells, which lose cilia and secretory granules to become morphologically indistinct, poorly differentiated (PD) cells over the first 48 h (4, 5). In these rat models, only a small proportion of ciliated cells attached to the semipermeable support membrane. These cells lost their cilia but were not observed to proliferate in culture (4). The other cell types were observed to dedifferentiate into PD cells and proliferate before the reappearance of differentiated cell types (4, 5). Both basal and nonciliated columnar cells appear to have clonogenic ability and broad differentiation potential (18, 25). It seems likely that in our model, the murine tracheal epithelial cells on day 4 also represent a population of undifferentiated cells. On day 4 after seeding, proliferating cell nuclear antigen antibodies stained 78 ± 9% (n = 2 observations) of the cultured cells, whereas on day 28, they stained only 25 ± 8% (n = 2 observations) of cultured cells. These results suggest that the majority of cells attached to the support membrane proliferate within the first few days. They also suggest that before the formation of a differentiated epithelium, most cells, rather than a small subset of stem cells, dedifferentiate and proliferate. Similar observations have been reported in studies of primary cultures of rat tracheal epithelium (4, 5).

No ciliated cells were observed on day 4 after seeding (n = 3 observations; Fig. 1b). However, SEM analyses on days 8 and 14 revealed 16 ± 3 (n = 2 observations) and 15 ± 4% (n = 2 observations) ciliated cells, respectively (Fig. 1c). By day 28, the percentage of ciliated cells was 34 ± 9% (n = 2 observations; Fig. 1d). This value agrees well with the percentage of ciliated cells that we observed in murine tracheal specimens by SEM (33 ± 5%; n = 2 observations; Fig. 1a). Moreover, it is consistent with a previous quantitative study (24) detailing the distribution of cell types in the murine trachea. These authors reported that the percentage of ciliated cells was ~38%. Interestingly, the nonciliated cells were observed to be flatter and broader in culture compared with the domed appearance seen in the murine tracheal specimens (Fig. 1, a and d).

TEM was performed to provide additional information about the cell types present in primary cultures of murine tracheal epithelium and their ultrastructure. Primary cultures of murine tracheal epithelium were processed on day 19 (n = 4 observations; Figs. 2b and 3), by which time differentiation had been initiated based on results of the SEM analyses (see above). The results were compared with a previously published morphometric study (24) that reported the distribution and ultrastructure of cell types in the murine trachea. In addition, a tracheal specimen from a 5-wk-old mouse of the same strain used for the preparation of primary cultures of murine tracheal epithelium was analyzed (Fig. 3).


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Fig. 2.   Transmission electron micrographs of a 5-wk-old mouse trachea (a) and primary culture of murine tracheal epithelium on day 19 (b). Arrows, sections through cilia, identified at the apical surface of the cells by electron-dense basal bodies, interspersed with microvilli.



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Fig. 3.   Transmission electron micrographs of primary cultures of murine tracheal epithelium on day 19 demonstrating a layer of cuboidal epithelial cells (a), an apical junctional complex and basolateral interdigitations (b), and a flattened "basal-like" cell underlying nonciliated epithelial cells (c). Solid arrow, microvilli and several cilia; open arrow, a desmosome at the apical junction between two epithelial cells; arrowheads, lateral interdigitations between the basolateral membranes.

A confluent, polarized epithelium was observed in primary cultures of murine tracheal epithelium. Cultured cells were observed to be broader and shorter than native murine epithelial cells, with a cuboidal rather than columnar shape, consistent with a study (1) of primary cultures of guinea pig tracheal epithelium. The murine cultures were composed predominantly of a single layer of ciliated and nonciliated cells (Figs. 2b and 3a), with apical microvilli of varying proportions. Both short immature cilia and long mature cilia were noted, with cytoplasmic basal bodies and classical microtubule organization. Numerous complex interdigitations of the basolateral cell membrane were observed at the boundaries between the cells (Fig. 3b). Interestingly, in primary cultures of human airway epithelium, the appearance of complex interdigitations correlated with improved cellular differentiation and transepithelial ion transport properties (32). Many apical junctional complexes containing tight junctions and desmosomes were noted (Fig. 3b). Larger flattened cells with a more squamous morphology were also observed underlying this cuboidal epithelium. These cells had large oval nuclei, a high nucleus-to-cytoplasm ratio, and long projections (lamellipodiae) that extended over the surface of the insert and provided multiple points of attachment for the cells above them (Fig. 3c). The tall columnar epithelial cells seen in native mouse trachea (Fig. 2a) were not observed in culture. However, the morphology described above was similar to that observed in 11-day-old rat tracheal primary cultures (13), which later developed a morphology that more closely resembled native rat tracheal epithelium. No goblet cells were observed by TEM.

Thus electron microscopy analyses of primary cultures of murine tracheal epithelium demonstrated the presence of ciliated cells, nonciliated cells, and putative basal cells. Importantly, the abundance of ciliated and nonciliated cells observed was similar to that of murine tracheal epithelium (24; present study). However, the ultrastructure of cultured cells showed some differences from native murine tracheal epithelial cells. These differences might represent a variation in the differentiated state and function of cultured epithelial cells or the presence or absence of unknown factors in the primary cultures.

Histochemical characterization. A periodic acid-Schiff stain was performed on days 4, 8, 14, and 28 (n >=  4 observations for each time point). This stain revealed occasional positively stained, magenta-colored cells, suggesting the presence of mucus-producing goblet cells (Fig. 4). These cells were not always present and were variable in number, with no pattern observed on the basis of culture age. The reason for this irregularity is not clear. Given the scarcity of these cells in the cultures, it is perhaps not surprising that they were not detected by TEM. This minimal presence of goblet cells reflected their paucity in the native tracheal epithelium of healthy mice (24).


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Fig. 4.   Histochemical characterization of primary cultures of murine tracheal epithelium with periodic acid-Schiff stain. a: bright-field light micrograph with hematoxylin counterstain. b: phase-contrast micrograph without counterstain. Arrows, magenta-staining, periodic acid-Schiff-positive cells.

Immunohistochemical characterization. To characterize the cell types that constituted the primary cultures of murine tracheal epithelia, immunohistochemical analyses were performed on days 4, 8, 14, and 28 (n >=  3 observations for each antibody at each time point) with a panel of antibodies and compared with murine tracheal sections (n >=  3 specimens for each antibody) (Figs. 5 and 6). Data were quantified as described in MATERIALS AND METHODS.


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Fig. 5.   Fluorescent immunohistochemical characterization of murine tracheal sections (a, d, and g) and primary cultures of murine tracheal epithelium on days 4 (b, e, and h) and 28 (c, f, and i) with anti-human pan cytokeratin antibody (a-c), no primary antibody (negative control; d-f), or anti-mouse cytokeratin 18 antibody (g-i). Positive FITC signal is represented in green, with 4,6-diamidino-2-phenylindole (DAPI) nuclear stain in blue. E, epithelium, S, submucosal glands.



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Fig. 6.   Fluorescent immunohistochemical characterization of murine tracheal sections (a, d, and g) and primary cultures of murine tracheal epithelium on days 4 (b, e, and h) and 28 (c, f, and i) with anti-mouse cytokeratin 14 antibody (a-c), anti-human alpha -smooth muscle actin antibody (d-f), or anti-human vimentin antibody (g-i). Positive FITC signal is represented in green, with DAPI nuclear stain in blue. SM, smooth muscle; LP, lamina propria.

The density of cells in primary cultures of murine airway epithelium was observed to undergo a two- to threefold increase between days 4 and 28. The average number of cells per field of view were 51 ± 11 (n = 4 observations) on day 4, 112 ± 17 (n = 4 observations) on day 8, 123 ± 26 (n = 4 observations) on day 14, and 150 ± 30 (n = 4 observations) on day 28.

Epithelial cells were characterized with antibodies raised against different cytokeratins, a family of intermediate filaments involved in the cytoskeleton of epithelial cells. The expression profile of these cytokeratins is variable and dependent on epithelial cell subtype and the stage of differentiation (reviewed in Ref. 9). However, these expression patterns can be altered in wound healing, influenced by extracellular regulators, and affected by cell culture (reviewed in Ref. 12). Antibodies raised against alpha -smooth muscle actin (expressed in smooth muscle cells) and vimentin (expressed in cells of mesenchymal origin) were used to establish the contribution of nonepithelial cells to the primary cultures of murine tracheal epithelia.

The anti-human pan cytokeratin antibody (anti-cytokeratins 1, 4, 5, 6, 8, 10, 13, 18, and 19) detected all the cells of the tracheal epithelium and submucosal gland epithelium in sections of mouse tracheae (Fig. 5a). This antibody was used to demonstrate the epithelial nature of the primary cultures of murine tracheal epithelium (Fig. 5, b and c). All cultured cells were detected at all time points (n = 3 observations at each time point). Variable intensity of staining was observed in different cells within the same cultures. Small patches of intensely stained cells on a focal plane above the majority of the cells were observed with greater frequency in the older cultures.

Subtypes of epithelial cells were demonstrated in murine tracheal sections with antibodies raised against the murine homologs of human cytokeratins 18 (Fig. 5, g-i) and 14 (Fig. 6, a-c).

Cytokeratin 18 was detected in all the ciliated and nonciliated columnar cells in the tracheal epithelium and acinar epithelial cells in the submucosal glands in sections of murine tracheae (Fig. 5g). The anti-cytokeratin 18 antibody detected 91 ± 4% (n = 2 observations) of the cells in primary cultures on day 4. Estimation of the proportion of cytokeratin 18-positive cells in primary cultures at later time points was complicated by the increased cell density and high proportion of positive staining. However, no gross alteration was observed. A range of intensities of positive signals was observed at all time points (Fig. 5, h and i). These cells were observed to have a largely confluent cobblestone appearance, similar to the staining apparent with the anti-pan cytokeratin antibody, although "gaps" representing unstained cells were observed. No significant alteration was seen in the staining pattern during the PD stage or at the time of cellular differentiation to produce ciliated cells. This was in contrast to the tracheal xenograft model of rat airway epithelium. This model system involves repopulation of a denuded rat tracheal graft with isolated rat epithelial cells (26) and closely parallels rat tracheal cell primary cultures on coated support membranes. In xenografts, the PD cells initially maintain their original cytokeratin 18 and 14 expression patterns when other markers of differentiated cell types are lost. However, on day 3, regardless of their original profile, all PD cells express cytokeratin 14 but not cytokeratin 18 (18). The expression of cytokeratin 18 is not restored until the second week, coincident with the first evidence of ciliary and secretory differentiation of columnar cells (26). The murine tracheal cells in our culture system did not show such dramatic dedifferentiation in the process of establishing a model epithelium.

Cytokeratin 14 was detected in the myoepithelial cells and cuboidal epithelial cells of the proximal duct of the submucosal glands in murine tracheal sections (Fig. 6a). It was also detected, with an irregular distribution pattern, in <5% of the basal cells in the epithelium of tracheal specimens. In primary cultures, the anti-cytokeratin 14 antibody detected 24 ± 8% (n = 2 observations) of cells on day 4. The proportion of cytokeratin 14-positive cells remained constant at 23 ± 8 (n = 2 observations), 23 ± 10 (n = 2 observations), and 23 ± 7% (n = 2 observations) on days 8, 14, and 28, respectively. These cells were observed to have a fairly scattered distribution, with less contact between positive-staining cells than the cytokeratin 18-positive cells (Fig. 6, b and c). They were observed to have an irregular shape with cytoplasmic protrusions and to lie in a focal plane below the majority of the epithelial cells. It is possible that these cells represent the flat underlying "basal-like" cells observed by TEM. Although basal cells constitute only 5-10% of the native murine tracheal epithelium (24), an enrichment of basal cells may have occurred if only a small proportion of ciliated cells were able to attach to the support membrane as observed in the rat culture model (4). However, the origin of these cells remains unclear. The great majority of cells, both myoepithelial and ductal cells, detected by the anti-cytokeratin 14 antibody in murine tracheal sections were, in fact, of submucosal gland origin. However, immunohistochemical analyses of tracheal "husks" after the dissociation stage demonstrated disruption but minimal dissociation of submucosal gland cells as a result of this procedure (data not shown). These results suggest that it is unlikely that a large proportion of the cultured cells originate from the submucosal glands.

However, other data suggested that cells from submucosal glands might be required to establish a differentiated epithelium. We observed that removal of the most proximal portion of the trachea (to which the thyroid gland is adherent) before epithelial cell dissociation resulted in a failure to differentiate and produce ciliated cells in culture. This was despite the standard number of cells being seeded (data not shown). This suggests that cells from the proximal trachea [the main site of murine submucosal glands (3)] are important in the development of a fully differentiated primary culture of murine tracheal epithelium. Furthermore, Borthwick et al. (2) have previously demonstrated that the ciliated duct of murine submucosal glands may be a specific niche for airway stem cells.

The studies presented with anti-cytokeratin antibodies demonstrate the existence of distinct subgroups of epithelial cells within the primary cultures. However, the alteration of cytokeratin expression patterns in cultured epithelium necessitates caution when comparisons are made between the expression profiles of specific cell subtypes in native tracheal epithelium and primary cultures of murine tracheal epithelium.

The anti-human alpha -smooth muscle actin antibody detected smooth muscle but not epithelial cells in murine tracheal sections (Fig. 6d). In day 4 primary cultures of murine tracheal epithelium (n = 3 observations), this antibody demonstrated the presence of a variable number (<1% of cells) of scattered single cells staining positive and observed to lie in a focal plane below the majority of cultured cells (Fig. 6e). These cells were observed to proliferate gradually, with larger clumps of cells detected by day 28 (n = 3 observations), suggesting outgrowth from the single cells contaminating the original cell preparation (Fig. 6f). The proportion of these contaminating cells was variable and largely dependent on the length of the dissociation step and the vigor with which tracheal suspensions were agitated. Primary cultures prepared from dissociated cell suspensions with large numbers of contaminating cells progressively developed lower Rte values and lost their ability to maintain an air-liquid interface. This suggests that growth of these cells may eventually disrupt and "puncture" the confluent epithelial membrane when initial contamination is excessive.

The anti-human vimentin antibody detected connective tissue cells of the lamina propria in murine tracheal sections but also a slight punctate background staining of the epithelial cells (Fig. 6g). Immunostaining of primary cultures (n = 3 observations at each time point) demonstrated the same slight background staining throughout but no evidence of genuine positive signal at any time point (Fig. 6, h and i). This suggests that there was no contamination with connective tissue cells in primary cultures of murine tracheal epithelium.

No positive staining was observed in negative controls (n >=  3 observations at each time point) treated in an identical manner to the other samples but with the primary antibodies omitted (Fig. 5, d-f).

Gene expression. Recent investigations of the pathogenesis of CF lung disease have focused on the characteristics of the ASL and its role in mucociliary clearance and innate lung defenses. The analysis of ASL in vivo has proven to be extremely difficult technically and has produced conflicting results (reviewed in Ref. 11). Consequently, key studies (20, 28, 33) addressing the ionic composition and antibacterial activity of the ASL have been performed in primary cultures of human airway epithelium. To maximize the opportunities presented by the availability of mouse models of CF, it is critical to develop a primary culture model for murine airway epithelial cells. Toward this goal, primary cultures of murine tracheal epithelium on days 12-13 after seeding (n >=  3 observations for each condition) were characterized with RT-PCR. We investigated the expression of Cftr and mouse Defb-1 and Defb-2, mouse homologs of the human beta -defensins, which have been implicated in the pathogenesis of CF lung disease (10, 27). Because Defb-2 expression is upregulated in response to proinflammatory stimuli in vivo (22), these studies were performed with both untreated cultures and cultures exposed to E. coli LPS. Parallel amplification of Hprt was performed as a control (Fig. 7). Figure 7 demonstrates that the expression pattern of these genes replicated the in vivo situation (22, 23). Cftr and Defb-1 were both constitutively expressed (n = 5 observations; Fig. 7a), whereas Defb2 was induced by exposure to E. coli LPS (Fig. 7b). Defb-2 expression was not observed without prior stimulation (n = 3 observations) but was observed in response to LPS exposure (n = 4 observations). Consistent with the in vivo results of Morrison et al. (22), the level of expression of Defb-2 after induction was too low to be observed on a stained agarose gel after 34 cycles of PCR. However, it was detected by hybridization to an internal oligonucleotide probe.


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Fig. 7.   RT-PCR of murine hypoxanthine phosphoribosyltransferase (Hprt), murine cystic fibrosis transmembrane conductance regulator (Cftr), and murine beta -defensin-1 (Defb-1) genes (a) and Hprt and Defb-2 genes (b) from primary cultures of murine tracheal epithelium. Cultures were utilized after 30 min of exposure to either lipopolysaccharide (LPS) or Pseudomonas aeruginosa (a) and LPS (b) in PBS. Untreated cultures were exposed to PBS alone. Amplified products were analyzed on a 2% agarose gel with a Phi X147HaeIII marker, amplified plasmid-positive (+ve) controls, and negative controls with either heat-inactivated reverse transcriptase (-RT) or distilled water substituted for RNA during cDNA synthesis. Defb-2 PCR products were observed only after hybridization to a Defb-2 internal probe.

Electrophysiological characterization. To assess the confluence of primary cultures of murine tracheal epithelium, Rte was monitored on days 4, 8, 14, and 28. Table 1 shows that the maximum value of Rte, 28.4 kOmega · cm2, was measured on day 4 immediately after the establishment of an air-liquid interface. Subsequently, Rte values decreased to ~12 kOmega · cm2 by day 14, after which they remained stable at about this value (Table 1). These values of Rte are markedly higher than those reported for cultures of airway epithelium from other species including human [200-3,000 Omega  · cm2 (32)] and dog [200-400 Omega  · cm2 (14)]. They also greatly exceed those reported for PD primary cultures of murine tracheal epithelium [~400 Omega  · cm2 (6)]. Interestingly, we found that although primary cultures of murine tracheal epithelium that failed to achieve elevated values of Rte were able to maintain an air-liquid interface, they did not develop ciliated epithelial cells.

                              
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Table 1.   Rte of primary cultures of murine tracheal epithelium

To investigate transepithelial ion transport by cultured murine tracheal epithelium, we mounted day 28 primary cultures in modified Ussing chambers and recorded Isc and Rte. Under baseline conditions, Rte was 14.3 ± 1.7 kOmega · cm2 (n = 6 observations) and Isc was 12.9 ± 1.0 µA/cm2 (n = 6 observations). Figure 8 demonstrates that the addition of amiloride (10 µM) to the apical solution inhibited Isc (P < 0.001). Subsequent addition of cAMP agonists (10 µM forskolin, 100 µM IBMX, and 500 µM CPT-cAMP) stimulated Isc (P < 0.001; Fig. 8). Finally, the addition of bumetanide (100 µM) to the basolateral solution inhibited Isc (P < 0.001; Fig. 8). These data are consistent with a study (29) of native murine epithelium. They suggest the presence of ENaC, the amiloride-sensitive sodium channel, and CFTR, the cAMP-stimulated chloride channel. Thus primary cultures established from the tracheae of mouse models of CF, lacking a functional CFTR, would be expected to demonstrate the electrophysiological consequences of CFTR dysfunction together with any resultant abnormalities in the ionic composition and antibacterial activity of the ASL. With the primary culture model of differentiated murine tracheal epithelium that we describe, these abnormalities might be investigated.


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Fig. 8.   Short-circuit current (Isc) measurements in primary cultures of murine tracheal epithelium. a: representative recording of Isc. Amiloride (10 µM), cAMP agonists [10 µM forskolin, 100 µM IBMX, and 500 µM 8-(4-chlorophenylthio)-cAMP], and bumetanide (100 µM) were added at time points 1, 2, and 3, respectively. b: magnitude of changes in Isc (Delta Isc) stimulated by amiloride, cAMP agonists, and bumetanide. Delta Isc was calculated as current measured at the peak of the response (~2 min after the addition of amiloride or cAMP agonists and ~7 min after the addition of bumetanide) minus current immediately before agonist addition. Values are mean ± SD; n = 6 observations. * Significant change in Isc, P < 0.001.

In conclusion, established techniques exist for the primary culture of airway epithelial cells from a variety of species. However, similar methods have not previously allowed the successful culture of mouse airway epithelium. This anomaly has hindered the realization of the full potential of transgenic mouse models in studies of pulmonary disease.

We have established and characterized a novel primary culture model of differentiated murine tracheal epithelium. Using this technique, we have generated confluent, polarized epithelial cultures with a high Rte, minimal contamination with nonepithelial cell types, differentiation to produce ciliated cells and a small number of goblet cells, expression of the Cftr and mouse Defb genes, and an electrophysiological profile consistent with the presence of ENaC and CFTR. However, ultrastructural differences do remain between primary cultures of murine epithelium and native murine tracheal epithelium. Further studies are ongoing to provide greater functional analysis of this model system. Nevertheless, this culture model demonstrates important features of native murine tracheal epithelium, particularly with respect to the study of mouse models of CF. In addition to the study of CF, this model system will be invaluable in the study of the growth and differentiation dynamics of murine tracheal epithelium, bacterial interactions with epithelial cells, the pathophysiology of asthma and other airway diseases, and the evaluation of novel therapies.


    ACKNOWLEDGEMENTS

We thank Gillian Morrison, Paul Perry, Wendy Hannant, Prof. John Govan, Euan Slorach, Brendan Doe, Norman Davidson, and Sheila Webb for advice and assistance with this work and John Findlay at the Biosem Unit at the Institute of Cell and Molecular Biology (Edinburgh University, Edinburgh, UK) for electron microscopy. Special thanks go to Prof. David Porteous for all his advice and support and to Aurita Puga, Phil Karp, and Prof. Michael Welsh (University of Iowa, Iowa City, IA) for their kind hospitality and invaluable tuition and assistance in the initial stages of these studies.


    FOOTNOTES

This work was supported by the Medical Research Council (UK), Biotechnology and Biological Sciences Research Council, Cystic Fibrosis Trust (UK), and National Heart, Lung, and Blood Institute Grant HL-58345.

Address for reprint requests and other correspondence: J. R. Dorin, MRC Human Genetics Unit, Western General Hospital, Crewe Rd., Edinburgh EH4 2XU, UK (E-mail: julia.dorin{at}hgu.mrc.ac.uk).

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.

Received 29 November 1999; accepted in final form 8 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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