In Vitro Cell. Dev. Biol. 28A:181-187, March 1992 © 1992 Tissue Culture Associalion 0883-8364/92 $01.50+0.00
ISOLATION AND IMMORTALIZATION OF RAT PRE-TYPE II CELL LINES RAMA K. MALLAMPALLI, CONNIE S. FLOERCHINGER, AND GARY W. HUNNINGHAKE1
Pulmonary Division, Department of Internal Medicine, Department of Veterans Affairs Medical Center and The Universityof lowa College of Medicine, Iowa City, Iowa 52242 (Received 12 July 1991; accepted 10 October 1991)
SUMMARY
The fetal respiratory distress syndrome is due, in part, to the presence of abundant pre-type II alveolar epithelial cells that have not yet differentiated into mature type II cells. Studies of this syndrome have been limited somewhat by the lack of an adequate in vitro model. In the present study we immortalized pre-type II cells by infecting primary isolates obtained from fetal rat lung with a retroviral construct expressing the adenoviral 12S EIA gene product. The immortalized pre-type II cells retained many of the uhrastructural features typical of pre-type II cells in primary culture, most notably lamellar bodies were not detected and the cells contained abundant stores of glycogen, expressed cytokeratin filaments, and bound the lectin Maclura pomifera. Karyotyping revealed that the cells are diploid. Growth studies demonstrate log phase growth in the presence of serum with a markedly decreased growth rate shortly after the cells reach confluence. Exposure of the immortalized pre-type II cells to hydrocortisone and dibutryl cAMP resulted in the induction of lamellar bodylike organelles; however, these cells did not secrete surfactant or express surfactant protein A. These cells may serve as useful models for some in vitro studies of fetal type II cell maturation or the fetal respiratory distress syndrome, or both.
Key words: fetal rat lung; type II cells; lamellar bodies; lung surfactant.
characteristics over time would add significantly to our study of the fetal respiratory distress syndrome. Recently it has been demonstrated that a retroviral vector encoding the adenovirus 12S EIA gene sequence can be used to immortalize a wide variety of epithelial cells in primary culture (9). The cell lines generated retained the essential features of epithelial cells in primary culture, continued to express the E 1A gene product, and yet did not exhibit a tumorogenic pbenotype. In the present study we have applied these methods to establish lines of pre-type II alveolar epithelial cells isolated from the lungs of the fetal rat. We report that these cell lines maintain the essential characteristics typical of primary isolates of pre-type II cells.
INTRODUCTION
The fetal respiratory distress syndrome continues to carry substantial morbidity and mortality in the pre-term infant (24,45). A central feature of this syndrome seems to be the presence in the lung of abundant pre-type II alveolar epithelial cells which have not yet matured into surfactant producing type II cells (2,13,14). Pretype II cells do not express lamellar bodies, which are a marker of type II cell maturity (34,40) and an intraceUular source of surfacrant lipid (1,15,32,33,39,46). Deficiency of surfactant subsequently leads to diffuse atelectasis, widespread edema, and pulmonary dysfunction in the neonate. Studies of the fetal respiratory distress syndrome have been limited by the lack of an adequate in vitro model. Existing cell culture systems have contributed significantly to our understanding of fetal type II cell morphology and maturation; however, the physiologic studies performed using these models have been limited by mixed cell populations (11,12,16,41,44), inadequate numbers of fetal cells for study (22,26), or of failure of the cell culture systems to display the differentiated function of type 11 epithelial cells (26,27). Ideally, a cell model consisting of a cell line of pure type II cells which responds to hormonal control and manifests stable in vitro
MATERIALSANDMETHODS
Materials. Pregnant Sprague-Dawley rats were obtained from Charles River Laboratories, Wilmington, MA. Plasticware was obtained from Corning, Corning, NY. Tissue culture chamber slides for immunofluorescence studies were obtained from Miles Laboratories, Naperville, IL. Cells were cultured in Waymouth's 752/1 medium (Universityof Iowa Tissue Culture and Hybridoma Facility, Iowa City, IA), and Dulbecco's minimal essential medium (DMEM) from Sigma Chemical Co., St. Louis, MO. G418 was obtained from GIBCO Laboratories, Grand Island, NY. Polybreen, dibutryl cAMP, hydrocortisone, trypsin, and fluorescein conjugated goat anti-mouse IgG were obtained from Sigma. Monoclonal anticytokeratin antibody was purchased from Labsystems Inc., Helsinki, Finland, and Maclura pomifera lectin conjugated to fluorescein from E-Y Laboratories, San Mateo, CA. The ~421256 cells were kindly provided by Dr. R. D. Cone (Vollum Inst. for Advanced Biomedical Research, Portland, OR). The monoclonal M-73
l To whom correspondence should be addresscd at Pulmonary Division C33-GH, Department of Internal Medicine, College of Medicine, Iowa City, IA 52242.
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antibody was a generous gift of Dr. E. Harlow (Massachusetts General Hospital Cancer Center, Charlestown, MA). Cell culture. Rat fetuses were surgically removed from pregnant Sprague-Dawley rats of timed gestations of 18 and 19 days. These gestational ages correspond to the embryonal (Day 181 and early canalicular (Day 191 stages of fetal lung maturation. Fetal pre-type II cells were isolated as described by Post et at. (35). Briefly, fetal lungs were resected from rat fetuses and were minced into - 1-mm fragments in Hanks' balanced salt solution (HBSS). Cells were dispersed using trypsin 0.05% and 10 gg/ml DNAse, and the resulting cell suspension was gravity filtered using a 50-#g Nitex filter. The single cell filtrate was then centrifuged to obtain a pellet and resuspended in Waymouth's 752/1 medium with 10% carbon stripped fetal bovine serum (CS-FBS). The harvested cells were initially plated at a density of 3 X 106 cells/75-cm flask for 1 h to allow for attachment of fibroblasts. The nonadherent cells were replated at a density of 1.2 × l 0 s to 8 X 104 cells/cm 2 on 25-cm 2 tissue culture flasks in Waymouth's 752/1 medium with 10% CS-FBS. Immortalization of cells. Fetal pre-type II cells in primary cultures were immortalized as previously described (9). ff4221256 Cells, which produce a retrovirns containing the adenovirus 125 E1 a gene and neomycin phosphotransferase gene, were grown to confluence in DMEM-FBS. The medium was changed and harvested after an additional 24 h of culture. Medium (viral stock) from these cultures contains 104 to l 0 s viruses per ml (9). After 24 h in culture, each 25-cm 2 flask of rat pre-type II cells was incubated for 2.5 h with 0.3 ml of 12S viral stock in the presence of Polybreen (8 #g/ml). The medium was then changed and the cells were maintained in Waymouth's 752/1 medium. G418-resistant isolates were selected by growth in the presence of G418 (0.25 mg/ml) after 2 wk in culture. Fetal pre-type II cell isolates were grown to confluence and expanded in culture. Confluent cultures were harvested by exposure to trypsin (0.25% with 0.1% EDTA) and replated after a 1:4 split of cells. Scanning and transmission electron microscopy. Immortalized pre-type I1 cells were cultured on 35-mm tissue culture plates in control medium (1.5 ml of Waymouth's 752/1 medium with 10% CS-FBS), or the same medium supplemented with 1 X 10 s M hydrocortisone and 1 mM dibutryl cAMP. Tissue culture plates were prepared and examined by transmission and scanning electron microscopy as previously described (37). Detection of intermediate filaments. Cells were grown to confluence on glass tissue culture chamber slides, fixed with 10% buffered formalin, and permeabilized with 0.1% Triton X-100 in phosphate buffered saline (PBS). For detection of intermediate filaments, the cells were exposed to a 1:10 dilution of monoclonal anticytokeratin antibody in PBS-triton (PBS-T) (0.1%). After rinsing with PBS-T, the ceils were exposed to a 1:50 dilution of fluorescein conjugated goat anti-mouse IgG, rinsed, and mounted in glycerol-PBS (9:1). Specimens were viewed under a Nikon Microphot fluorescent microscope. Detection of the EIA gene product. Immortalized pre-type It cells were fixed and permeabilized as described above and exposed to 50 gl of culture medium containing the mouse monoclonal antibody M-73. Specimens were examined using a fluorescent microscope as previously described. Lectin binding. Cells grown on tissue culture chamber slides were washed with PBS and incubated for 1 h at 22 ° C with M. pomifera lectin conjugated to fluorescein (50 mg/ml) in PBS. The cells were then washed with PBS and examined with a fluorescent microscope. Karyotyping. Karyotyping analysis of Giesma-banded cells was performed by the University of Iowa Gytogenetics Laboratory as described by Yoo et al. (47). Electrical resistance. Cells were grown to confluence in Miflicell-HA filter inserts suspended in DMEM-FBS. They were plated at a density of 2.5 X lO s cells per insert. Electrical resistance was then measured as previously described (37). Resistance was measured over 10 to 15 min to ensure that the values remained stable. Growth rate. Immortalized pre-type It cells of Day 18 and Day 19 gestation were plated at a density of 10 s cells per 35-mm tissue culture dish in DMEM with 10% CS-FBS. At each of 8 time points (1,2,4,6,8,10,12,14 days after plating), four plates were washed 3 times with medium and the cells from each plate were harvested with trypsin as described above. The cells were suspended in medium and counted using a hemacytometer and a phase microscope. Glycogen. Whole lungs from rat fetuses of Day 18 and Day 20 gesta-
Fro. 1. Transmission electron micrograph of immortalized Day 18 pretype II cells harvested after 28 wk in culture. >(10 000. Note the presence of apical microvilli, a junctional complex, and electron dense glycogen granules. LameUar bodies are not present.
tion and adult lungs and liver from Sprague-Dawley rats were surgically removed, sectioned, and processed for staining with periodic acid leufuchsin (PAS) stain (10), and counterstained with Harris' hematoxylin. Immortalized pre-type II cells from Day 18 gestation, adult primary type II cell isolates, and an adult immortalized type It cell line were also stained as above. Detection of lamellar bodylike structures. Immortalized pre-type II cells were cultured on tissue culture chamber slides in control medium (Waymouth's 752/1 medium with 10% CF-FBS) or the same medium supplemented with 1 × 10 -5 M hydrocortisone and 1 mM dibutryl cAMP. After 7 days in culture the cell monolayers were stained with tannic acid and polychrome stain as described by Mason et al. (29). Primary isolates of adult type lI cells were stained as above. The cells were also examined by electron microscopy, as described above.
RESULTS Immortalization ofpre-type H cells. Colonies of pre-type II cells appeared 10 to 18 days after exposure to viral stock. The G418-resistant colonies remained viable and under light microscopy and morphologically resembled primary isolates of pre-type II cells. Immortalized cells were maintained in Waymouth's 7 5 2 / 1 medium with 1 0 % CS-FBS and reach confluence on 1 0 0 - m m tissue culture dishes after 7 to 9 days. The cells have been maintained 60 wk in culture and have been passaged over 4 0 times. Scanning and transmission electron microscopy. Transmission electron microscopy of confluent monolayers of immortalized Day 18 and Day 19 pre-type II ceils revealed a low cuboidal epithehum with apical microvilli and cell-to-cell contacts (Fig. 1). Scanning electron microscopy confirms numerous surface microvilli (data not
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Karyotyping. Immortalized pre-type II cells were shown to be diploid by karyotyping analysis at 50 days in culture (Fig. 3). Electrical resistance. Confluent cell monolayers were tested for the presence of tight junctions by measuring electrical resistance after plating at high density on filter inserts. The cells displayed only low levels of electrical resistance (10 ohm • cm 2) after 5 days in culture, relative to control filters (data not shown). Growth rate. The pre-type It cells lines demonstrated a log phase growth in culture 2 to 5 days after plating. Doubling times for Day 18 and Day 19 pre-type II cell lines are approximately 44 and 53 h, respectively. The seeding efficiency after replating is approximately 67 and 76% for Day 18 and Day 19 pre-type II cell lines, respectively. Cell growth rate diminished after 6 days in culture when cells had reached a stationary phase and were confluent in culture. In addition, after 80 passages the ceils continue to rapidly proliferate in culture reaching confluence within a week after replating. Immortalized pre-type II cells remain viable after freezing ( - 7 0 ° C) and thawing. Detection of lamellar bodylike structures. Tannic acid and polychrome staining of adult type II primary isolates revealed large numbers of lamellar bodies as represented by dark staining intracellular inclusions (Fig. 4 A). In contrasting, Day 18 and Day 19 pre-type II cell lines expressed few lamellar bodies when these cells were cultured in control medium (Fig. 4 B). The number of lamellar bodylike structures significantly increased when immortalized pretype II cells (at Passage 50) were co-cultured in hydrocortisone and dibutryl cAMP (Fig. 4 C). After 1 wk of exposure, numerous lamellar inclusions were detected in both cell fines. Cells stimulated with hydrocortisone and dibutryl cAMP were confirmed by electron microscopy to contain typical lameHar bodylike structures (Fig. 5). These studies were repeated after approximately 80 passages in culture. The cells cultured in the presence of hydrocortisone and dibutryl cAMP contained significantly more lamellar bodies than cells cultured in control medium (Fig. 4 D), however, the inclusions were less prominent when compared to our initial studies. Addi-
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FIG. 2. Fluorescence micrographs of immortalized Day 18 pre-type II cells showing that: A, the cells bind the M. pomifera lectin and B, express cytokeratius; see Methods for details. shown). Cells cultured in control medium did not express lamellar bodies but contained numerous intracellular glycogen granules (Fig. 1). Presence of intermediate filaments, E1A protein and lectin binding. The cells stained positive with monoclonal antibodies to cytokeratin 8, 18, and 19 (Fig. 2 A). In addition, both pre-type II cell lines demonstrated positive immunofluorescent staining with M73, a monoclonal antibody to the adenoviral E1A protein (data not shown). Immortalized pre-type II cells exhibited surface binding to the lectin M. pomifera, a characteristic of type II cells in primary culture (Fig. 2 B).
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FIG. 3. Karyotyping analysis of Day 19 pre-type II cell line. Note that the cells are diploid. Day 18 pre-type II ceils were also diploid (data not shown).
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FIG. 4. Tannic acid stains. A, adult type II primary isolates; note the presence of multiple lamellar inclusions. B, Day 18 immortalized pre-type II cells cultured in control medium. Ceils exhibit no lamellar bodies. C, Day 18 immortalized cells cultured in hydrocortisone (1 × 10 -5 M) and 1 mM dibutryl cAMP for 7 days (staining performed after 26 wk in culture). Note the induction of dark staining cytoplasmic inclusions. D, Day 18 immortalized cells cultured in hydrocortisone (1 × 10-s M) and 1 mM dibutryl cAMP for 7 days (staining performed after 56 wk in culture). Ceils re-induced at this time demonstrated less pronounced cytoplasmic inclusions.
tional studies on ceils at Passage 80 showed that the cells did not secrete surfactant lipids or express surfactant protein A (data not shown). Cells at earlier passages were not evaluated. Glycogen. Fetal rat lungs isolated at Day 18 gestation consisted of cuboidal cells forming primitive tubular structures when examined under light microscopy. Pre-type lI cells lining to these structures contained abundant intracellular stores of glycogen similar to liver tissue when stained in situ with PAS (Fig. 6 A,B). Immortalized Day 18 pre-type II cells also demonstrated intense staining for glycogen (Fig. 6 C). Fetal rat lung at Day 20 gestation showed less pronounced staining with PAS as glycogen content gradually diminished with lung maturation (data not shown). Type II cells isolated from an adult rat lung (data not shown) and an immortalized adult type II cell line both demonstrated weak staining for glycogen (Fig. 6D).
DISCUSSION In the present study we demonstrate that a retroviral vector conraining the adenovirus 12S E1A cDNA sequence can be used to immortalized rat pre-type II alveolar epithelial cells. Initially we isolated primary cultures of type II cells from the fetal rat lung at Day 18 and Day 19 gestational age. During these early stages of lung development alveolar epithelial cells have not matured sufficiently to express lamellar bodies; an important feature which would permit studies assessing pre-type II cell maturation and hormonal regulation. Our studies demonstrate that cell lines of pre-type II alveolar epithelial cells generated after exposure to the retroviral construct morphologically resemble primary isolates of pre-type II cells. Transmission electron microscopy of cell monolayers reveals low cuboidal epithelial cells with apical microvilli and junctional corn-
PRE-TYPE II CELL LINES
Fro. 5. Transmission electron micrograph of immortalized Day 18pretype II cells stimulated with hydrocortisone and dibutryl cAMP. ×60 000. Note the presence of a typical lamellar body.
plexes. More importantly, lamellar bodies were not detected by electron microscopy of immortalized pre-type II cells. Immortalized cells isolated from Day 18 and Day 19 gestation cultured in the absence of hormones contained few lamellar bodies as detected by tannic acid staining. These features are all consistent with previous reports characterizing fetal pre-type II cells in primary culture (34,40,43). Another important marker of pre-type II cells is the presence of abundant intracellular stores of glycogen (34). We observed a relatively high cellular content of glycogen in Day 18, but not Day 19, immortalized pre-type II cells as demonstrated by PAS staining and electron microscopy. Our studies are consistent with prior reports describing depletion of glycogen stores in type II cells as lung maturation proceeds (4,12,21,34). Immunocytochemical studies to further characterize the immortalized cells confirm the presence of cytokcratins, identifying their epithelial nature. The cells also exhibit binding to the lectin M. pomifera, a characteristic of both fetal and adult type II cells (34). Both Day 18 and Day 19 cell lines were studied physiologically to determine the presence of tight junctions. The cells exhibited only a very low electrical resistance ( ~ 10 ohm >(cm-1), below values reported for adult type II cell monolayers cultured on porous surfaces (28,37), and significantly lower than monolayers plated on tissue culture treated filters (7). Inasmuch as these differences may be attributed to factors involving substrata and seeding density, additional studies may be required to identify an intact epithelial barrier. Finally, we have detected the E1A protein in pre-type lI cell lines, confirming that these cells express the adenoviral E1A gene product. Previous reports have described the oncogenic potential of human adenoviruses (5,31). Expression of E1A protein alone allows for immortalization of epithelial cells in culture (38). A second
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adenoviral gene product, E1B, is required for full transformation of rodent cells into a malignant phenotype (30). Pre-type II alveolar epithelial cells immortalized with the adenoviral E1A gene as described in this study proliferate rapidly in culture but do not seem to display malignant characteristics. Tumorogenic assays have not been performed; however, both cell lines showed a normal karyotype. Growth studies reveal that cells cultured in the presence of serum exhibit exponential growth and eventually reach a stationary phase of growth shortly after reaching confluence. This suggests that cell replication is suppressed by contact inhibition. Our studies further demonstrate that immortalized pre-type II alveolar epithelial cells can be hormonally induced to form lamellar bodylike structures similar to prior observations using in vivo models (19,21,34,41,43)~ Glucocorticoids, for example, are known to accelerate puhnonary maturation in a number of animal models (6,17,21,35) and are currently being administered prenatally (8,23). Other studies also suggest that glucocorticoids and cAMP are required for maintenance of surfactant synthesis (3,18,20). To further evaluate the in vitro effect of these soluble factors on fetal type II cell differentiation, we cultured immortalized pre-type II cells in control medium and medium supplemented with hydrocortisone and dibutryl cAMP. Cells cultured for 7 days in control medium expressed few lamellar bodies as detected by tannic acid staining. The numbers of these osmiophilic inclusions markedly increased when cells were cultured in hydrocortisone and dibutryl cAMP. Electron microscopy confirmed that these structures were typical lamellar bodylike structures, as evidenced by multiple lamellae layered layered concentrically and located within a membrane. Recently, we have attempted to re-induced expression of lamellar bodies in both cell lines after the cells underwent a number of additional passages in culture. These immortalized cells stimulated with hydrocortisone and dibutryl cAMP expressed significantly more lamellar bodylike structures than control cells; however, the inclusions were noted to be less prominent and fewer relative to our initial observations. The significance of the presence of lamellar bodylike structures in these stimulated cells in unclear. In this regard, we could not demonstrate the presence of SP-A or the release of surfactantlike lipids. It seems that additional studies may be necessary to define the optimal culture enviromcnt to preserve the long-term functional stability of these pre-type II cell lines. In addition to hormonal requirements, for example, expression of lamellar bodies by type II cells is influenced by an adequate supply of critical nutrients including glutathione (25), and various extracellular substrates (20,36). An epithelial-mesenchymal interaction may also play a role in maintaining type II cell morphology (42). Modulation of these factors may be important in generating stable cell lines which may serve as potentially useful in vitro models for studies of surfactant lipid and protein synthesis. In summary, our studies demonstrate that the adenoviral 12S E1A gene can be used to immortalize pre-type II alveolar epithelial cells isolated from the fetal rat. Cells described in this study express few lamellar bodies and contain abundant stores of glycogen, and therefore retain the essential features of pre-type 1I cells in primary culture. Our studies also suggest that immortalized pre-type II cells can be induced to form lamellar bodylike structures when exposed to agents that are known to promote the differentiated state of pretype II cells. These observations should stimulate interest in generating stable cell lines which may ultimately serve as useful models
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FIG. 6. PAS stain for glycogen. A, in situ staining of adult liver. Note presence of abundant PAS-positive material in hepatocytes. B, fetal rat lung, Day 18 gestational age; pre-type II cells lining tubules demonstrate intense staining for glycogen. C, immortalized Day 18 pre-type II cells also demonstrate intense staining for glycogen. Immortahzed Day 19 pre-type II cells stained less intensively with PAS (data not shown). D, an immortalized adult type II cell line demonstrates weak staining for glycogen.
for the morphologic study of fetal type II cell maturation and surfactant regulation. ACKNOWLEDGEMENTS
The authors thank Dr. Shivanand Patil of the University of Iowa Cytogenetics Laboratory for performing the karyotyping analysis. We also thank Debbie Jarrard and Peggy Kuenster for their secretarial assistance; and thank Dr. Jeffrey Whitsett and Dr. James Fisher for evaluating these cells for the presence of surfactant proteins and their mRNAs. This study was supported by the following grants: Institutional NRSA fellowship training grant (HL07121) from the National Heart, Lung, and Blood Institute, Bethesda, MD; a Specialized Center of Research grant (SCOR, HL37121) from the National Heart, Lung, and Blood Institute; an RO-1 (HL43883) from the National Heart, Lung, and Blood Institute; a Merit Review grant from the Department of Veteran Affairs, Washington, DC; and RR59 from the Clinical Research Center, National Institutes of Health, Bethesda, MD.
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27. Mason, R. J.; Williams, M. C.; Widdicombe, J. H. Cell culture systems for studying RDS. In: Raivio, K. O., ed. Respiratory distress syndrome. London: Academic Press; 1984:87-96. 28. Mason, R. J.; Wiliams, M. C.; Widdicombe, J. H., ct al. Transepithelial transport by pulmonary alveolar type II cells in primary culture. Proc. Natl. Acad. Sci. USA 79:6033-6037; 1982. 29. Mason, R. J.; Walker, S. R.; Shields, B. A., et al. Identification of rat alveolar type II epithelial ceils with a tannic acid and polychrome stain. Am. Rev. Respir. Dis. 131:786-792; 1985. 30. Montell, C.; Courtois, G.; Eng, C., et at. Complete transformation by adenovirus 2 requires both E 1A proteins. Cell 36:951-961; 1984. 31. Paraskeva, C.; Gallimore, P. H. Tumorogenicity and in vitro characteristics of rat liver epithelial cells and their adenovirus-transformed derivatives. Int. J. Cancer 25:631-639; 1980. 32. Paul, G. W.; Hassettand, R. J.; Reiss, O. K. Formation of lung surfactant films from intact lamellar bodies. Proc. Natl. Acad. Sci. USA 74:3617-3620; 1977. 33. Possmayer, F.; Yu, S. H.; Weber, J. M., et al. Pulmonary surfactant. Can. J. Biochem. Cell Biol. 62:1121-1133; 1984. 34. Post, M.; Smith, B. T. Histochemical and immunocytochemical identification of alveolar type II epithelial ceils isolated from fetal rat lung. Am. Rev. Respir. Dis. 137:525-530; 1988. 35. Post, M.; Torday, J. S,; Smith, B. T. Alveolar type II cells isolated from fetal rat lung organotypic cultures synthesize and secrete surfactantassociated phospholipids and respond to fibroblast pneumocyte factor. Exp. Lung Res. 7:53-65; 1984. 36. Rannels, D. E.; Rannels, S. R. Influence of the extracellular matrix on type II cell differentiation. Chest 96:165-173; 1989. 37. Rochat, T.; Casale, J.; Hunninghake, G. W., et al. Neutrophil cathespin G increases permeability of cultured type II pneumocytes. Am. J. Physiol. 155:C603-C611; 1988. 38. Ruley, H. E. Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature 304:602-606; 1983. 39. Schlame, M.; Casals, C.; Rostow, B., et al. Molecular species of phosphatidylcholine and phosphatidylglycerol in rat lung surfactant and different pools of pneumocytes type II. Biochem. J. 253:209-215; 1988. 40. Scott, J. E.; Possmayer, F.; Harding, G. R. Alveolar pre-type II cells from the fetal rabbit lung: isolation and characterization. Biochem. Biophys. Acta 753:195-204; 1983. 41. Sevanian, A.; Kaplan, S. A.; Barrett, C. T. Phospholipid synthesis in fetal lung organotypic cultures and isolated type II pneumocytes. Biochem. Biophys. Acta 664:498-512; 1981. 42. Shannon, J. M.; Mason, R. J.; Jennings, S. D. Functional differentiation of alveolar type II epithelial cells in vitro: effects of cell shape, cell-matrix interactions and cell-cell interactions. Biochem. Biophys. Acta 931:143-156; 1987. 43. Sharp, M. J.; Borer, R. C., Jr.; Vadney, L., et at. Choline incorporation into lecithin in response to insulin or dexamethasone in homogenous cell cultures of rat lung epithelial cells and fibroblasts. Pediatr. Res. 14:899-900; 1980. 44. Smith, B. T.; Torday, J. S.; Giround, C. J. P. Evidence for different gestation-dependent effects of cortisol on cultured fetal lung cells, J. Clin. Invest. 53:1518-1526; 1974. 45. Wegman, M. E. Annual summary of vital statistics - 1983. Pediatrics 74:981-990; 1984. 46. Williams, M. Conversion of lamellar body membranes into tubular myelin in alveoli of fetal rat lungs. J. Cell Biol. 72:260-277; 1977. 47. Yoo, T. J.; Kuo, C. Y.; Patiz, S. R., et al. Loss of alpha-fetoprotein in rat hepatoma culture cells. Int. J. Cancer 24:184-192; 1979.
ISOLATION AND IMMORTALIZATION OF RAT PRE-TYPE II CELL LINES RAMA K. MALLAMPALLI, CONNIE S. FLOERCHINGER, AND GARY W. HUNNINGHAKE1
Pulmonary Division, Department of Internal Medicine, Department of Veterans Affairs Medical Center and The Universityof lowa College of Medicine, Iowa City, Iowa 52242 (Received 12 July 1991; accepted 10 October 1991)
SUMMARY
The fetal respiratory distress syndrome is due, in part, to the presence of abundant pre-type II alveolar epithelial cells that have not yet differentiated into mature type II cells. Studies of this syndrome have been limited somewhat by the lack of an adequate in vitro model. In the present study we immortalized pre-type II cells by infecting primary isolates obtained from fetal rat lung with a retroviral construct expressing the adenoviral 12S EIA gene product. The immortalized pre-type II cells retained many of the uhrastructural features typical of pre-type II cells in primary culture, most notably lamellar bodies were not detected and the cells contained abundant stores of glycogen, expressed cytokeratin filaments, and bound the lectin Maclura pomifera. Karyotyping revealed that the cells are diploid. Growth studies demonstrate log phase growth in the presence of serum with a markedly decreased growth rate shortly after the cells reach confluence. Exposure of the immortalized pre-type II cells to hydrocortisone and dibutryl cAMP resulted in the induction of lamellar bodylike organelles; however, these cells did not secrete surfactant or express surfactant protein A. These cells may serve as useful models for some in vitro studies of fetal type II cell maturation or the fetal respiratory distress syndrome, or both.
Key words: fetal rat lung; type II cells; lamellar bodies; lung surfactant.
characteristics over time would add significantly to our study of the fetal respiratory distress syndrome. Recently it has been demonstrated that a retroviral vector encoding the adenovirus 12S EIA gene sequence can be used to immortalize a wide variety of epithelial cells in primary culture (9). The cell lines generated retained the essential features of epithelial cells in primary culture, continued to express the E 1A gene product, and yet did not exhibit a tumorogenic pbenotype. In the present study we have applied these methods to establish lines of pre-type II alveolar epithelial cells isolated from the lungs of the fetal rat. We report that these cell lines maintain the essential characteristics typical of primary isolates of pre-type II cells.
INTRODUCTION
The fetal respiratory distress syndrome continues to carry substantial morbidity and mortality in the pre-term infant (24,45). A central feature of this syndrome seems to be the presence in the lung of abundant pre-type II alveolar epithelial cells which have not yet matured into surfactant producing type II cells (2,13,14). Pretype II cells do not express lamellar bodies, which are a marker of type II cell maturity (34,40) and an intraceUular source of surfacrant lipid (1,15,32,33,39,46). Deficiency of surfactant subsequently leads to diffuse atelectasis, widespread edema, and pulmonary dysfunction in the neonate. Studies of the fetal respiratory distress syndrome have been limited by the lack of an adequate in vitro model. Existing cell culture systems have contributed significantly to our understanding of fetal type II cell morphology and maturation; however, the physiologic studies performed using these models have been limited by mixed cell populations (11,12,16,41,44), inadequate numbers of fetal cells for study (22,26), or of failure of the cell culture systems to display the differentiated function of type 11 epithelial cells (26,27). Ideally, a cell model consisting of a cell line of pure type II cells which responds to hormonal control and manifests stable in vitro
MATERIALSANDMETHODS
Materials. Pregnant Sprague-Dawley rats were obtained from Charles River Laboratories, Wilmington, MA. Plasticware was obtained from Corning, Corning, NY. Tissue culture chamber slides for immunofluorescence studies were obtained from Miles Laboratories, Naperville, IL. Cells were cultured in Waymouth's 752/1 medium (Universityof Iowa Tissue Culture and Hybridoma Facility, Iowa City, IA), and Dulbecco's minimal essential medium (DMEM) from Sigma Chemical Co., St. Louis, MO. G418 was obtained from GIBCO Laboratories, Grand Island, NY. Polybreen, dibutryl cAMP, hydrocortisone, trypsin, and fluorescein conjugated goat anti-mouse IgG were obtained from Sigma. Monoclonal anticytokeratin antibody was purchased from Labsystems Inc., Helsinki, Finland, and Maclura pomifera lectin conjugated to fluorescein from E-Y Laboratories, San Mateo, CA. The ~421256 cells were kindly provided by Dr. R. D. Cone (Vollum Inst. for Advanced Biomedical Research, Portland, OR). The monoclonal M-73
l To whom correspondence should be addresscd at Pulmonary Division C33-GH, Department of Internal Medicine, College of Medicine, Iowa City, IA 52242.
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antibody was a generous gift of Dr. E. Harlow (Massachusetts General Hospital Cancer Center, Charlestown, MA). Cell culture. Rat fetuses were surgically removed from pregnant Sprague-Dawley rats of timed gestations of 18 and 19 days. These gestational ages correspond to the embryonal (Day 181 and early canalicular (Day 191 stages of fetal lung maturation. Fetal pre-type II cells were isolated as described by Post et at. (35). Briefly, fetal lungs were resected from rat fetuses and were minced into - 1-mm fragments in Hanks' balanced salt solution (HBSS). Cells were dispersed using trypsin 0.05% and 10 gg/ml DNAse, and the resulting cell suspension was gravity filtered using a 50-#g Nitex filter. The single cell filtrate was then centrifuged to obtain a pellet and resuspended in Waymouth's 752/1 medium with 10% carbon stripped fetal bovine serum (CS-FBS). The harvested cells were initially plated at a density of 3 X 106 cells/75-cm flask for 1 h to allow for attachment of fibroblasts. The nonadherent cells were replated at a density of 1.2 × l 0 s to 8 X 104 cells/cm 2 on 25-cm 2 tissue culture flasks in Waymouth's 752/1 medium with 10% CS-FBS. Immortalization of cells. Fetal pre-type II cells in primary cultures were immortalized as previously described (9). ff4221256 Cells, which produce a retrovirns containing the adenovirus 125 E1 a gene and neomycin phosphotransferase gene, were grown to confluence in DMEM-FBS. The medium was changed and harvested after an additional 24 h of culture. Medium (viral stock) from these cultures contains 104 to l 0 s viruses per ml (9). After 24 h in culture, each 25-cm 2 flask of rat pre-type II cells was incubated for 2.5 h with 0.3 ml of 12S viral stock in the presence of Polybreen (8 #g/ml). The medium was then changed and the cells were maintained in Waymouth's 752/1 medium. G418-resistant isolates were selected by growth in the presence of G418 (0.25 mg/ml) after 2 wk in culture. Fetal pre-type II cell isolates were grown to confluence and expanded in culture. Confluent cultures were harvested by exposure to trypsin (0.25% with 0.1% EDTA) and replated after a 1:4 split of cells. Scanning and transmission electron microscopy. Immortalized pre-type I1 cells were cultured on 35-mm tissue culture plates in control medium (1.5 ml of Waymouth's 752/1 medium with 10% CS-FBS), or the same medium supplemented with 1 X 10 s M hydrocortisone and 1 mM dibutryl cAMP. Tissue culture plates were prepared and examined by transmission and scanning electron microscopy as previously described (37). Detection of intermediate filaments. Cells were grown to confluence on glass tissue culture chamber slides, fixed with 10% buffered formalin, and permeabilized with 0.1% Triton X-100 in phosphate buffered saline (PBS). For detection of intermediate filaments, the cells were exposed to a 1:10 dilution of monoclonal anticytokeratin antibody in PBS-triton (PBS-T) (0.1%). After rinsing with PBS-T, the ceils were exposed to a 1:50 dilution of fluorescein conjugated goat anti-mouse IgG, rinsed, and mounted in glycerol-PBS (9:1). Specimens were viewed under a Nikon Microphot fluorescent microscope. Detection of the EIA gene product. Immortalized pre-type It cells were fixed and permeabilized as described above and exposed to 50 gl of culture medium containing the mouse monoclonal antibody M-73. Specimens were examined using a fluorescent microscope as previously described. Lectin binding. Cells grown on tissue culture chamber slides were washed with PBS and incubated for 1 h at 22 ° C with M. pomifera lectin conjugated to fluorescein (50 mg/ml) in PBS. The cells were then washed with PBS and examined with a fluorescent microscope. Karyotyping. Karyotyping analysis of Giesma-banded cells was performed by the University of Iowa Gytogenetics Laboratory as described by Yoo et al. (47). Electrical resistance. Cells were grown to confluence in Miflicell-HA filter inserts suspended in DMEM-FBS. They were plated at a density of 2.5 X lO s cells per insert. Electrical resistance was then measured as previously described (37). Resistance was measured over 10 to 15 min to ensure that the values remained stable. Growth rate. Immortalized pre-type It cells of Day 18 and Day 19 gestation were plated at a density of 10 s cells per 35-mm tissue culture dish in DMEM with 10% CS-FBS. At each of 8 time points (1,2,4,6,8,10,12,14 days after plating), four plates were washed 3 times with medium and the cells from each plate were harvested with trypsin as described above. The cells were suspended in medium and counted using a hemacytometer and a phase microscope. Glycogen. Whole lungs from rat fetuses of Day 18 and Day 20 gesta-
Fro. 1. Transmission electron micrograph of immortalized Day 18 pretype II cells harvested after 28 wk in culture. >(10 000. Note the presence of apical microvilli, a junctional complex, and electron dense glycogen granules. LameUar bodies are not present.
tion and adult lungs and liver from Sprague-Dawley rats were surgically removed, sectioned, and processed for staining with periodic acid leufuchsin (PAS) stain (10), and counterstained with Harris' hematoxylin. Immortalized pre-type II cells from Day 18 gestation, adult primary type II cell isolates, and an adult immortalized type It cell line were also stained as above. Detection of lamellar bodylike structures. Immortalized pre-type II cells were cultured on tissue culture chamber slides in control medium (Waymouth's 752/1 medium with 10% CF-FBS) or the same medium supplemented with 1 × 10 -5 M hydrocortisone and 1 mM dibutryl cAMP. After 7 days in culture the cell monolayers were stained with tannic acid and polychrome stain as described by Mason et al. (29). Primary isolates of adult type lI cells were stained as above. The cells were also examined by electron microscopy, as described above.
RESULTS Immortalization ofpre-type H cells. Colonies of pre-type II cells appeared 10 to 18 days after exposure to viral stock. The G418-resistant colonies remained viable and under light microscopy and morphologically resembled primary isolates of pre-type II cells. Immortalized cells were maintained in Waymouth's 7 5 2 / 1 medium with 1 0 % CS-FBS and reach confluence on 1 0 0 - m m tissue culture dishes after 7 to 9 days. The cells have been maintained 60 wk in culture and have been passaged over 4 0 times. Scanning and transmission electron microscopy. Transmission electron microscopy of confluent monolayers of immortalized Day 18 and Day 19 pre-type II ceils revealed a low cuboidal epithehum with apical microvilli and cell-to-cell contacts (Fig. 1). Scanning electron microscopy confirms numerous surface microvilli (data not
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PRE-TYPE II CELL LINES
Karyotyping. Immortalized pre-type II cells were shown to be diploid by karyotyping analysis at 50 days in culture (Fig. 3). Electrical resistance. Confluent cell monolayers were tested for the presence of tight junctions by measuring electrical resistance after plating at high density on filter inserts. The cells displayed only low levels of electrical resistance (10 ohm • cm 2) after 5 days in culture, relative to control filters (data not shown). Growth rate. The pre-type It cells lines demonstrated a log phase growth in culture 2 to 5 days after plating. Doubling times for Day 18 and Day 19 pre-type II cell lines are approximately 44 and 53 h, respectively. The seeding efficiency after replating is approximately 67 and 76% for Day 18 and Day 19 pre-type II cell lines, respectively. Cell growth rate diminished after 6 days in culture when cells had reached a stationary phase and were confluent in culture. In addition, after 80 passages the ceils continue to rapidly proliferate in culture reaching confluence within a week after replating. Immortalized pre-type II cells remain viable after freezing ( - 7 0 ° C) and thawing. Detection of lamellar bodylike structures. Tannic acid and polychrome staining of adult type II primary isolates revealed large numbers of lamellar bodies as represented by dark staining intracellular inclusions (Fig. 4 A). In contrasting, Day 18 and Day 19 pre-type II cell lines expressed few lamellar bodies when these cells were cultured in control medium (Fig. 4 B). The number of lamellar bodylike structures significantly increased when immortalized pretype II cells (at Passage 50) were co-cultured in hydrocortisone and dibutryl cAMP (Fig. 4 C). After 1 wk of exposure, numerous lamellar inclusions were detected in both cell fines. Cells stimulated with hydrocortisone and dibutryl cAMP were confirmed by electron microscopy to contain typical lameHar bodylike structures (Fig. 5). These studies were repeated after approximately 80 passages in culture. The cells cultured in the presence of hydrocortisone and dibutryl cAMP contained significantly more lamellar bodies than cells cultured in control medium (Fig. 4 D), however, the inclusions were less prominent when compared to our initial studies. Addi-
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FIG. 2. Fluorescence micrographs of immortalized Day 18 pre-type II cells showing that: A, the cells bind the M. pomifera lectin and B, express cytokeratius; see Methods for details. shown). Cells cultured in control medium did not express lamellar bodies but contained numerous intracellular glycogen granules (Fig. 1). Presence of intermediate filaments, E1A protein and lectin binding. The cells stained positive with monoclonal antibodies to cytokeratin 8, 18, and 19 (Fig. 2 A). In addition, both pre-type II cell lines demonstrated positive immunofluorescent staining with M73, a monoclonal antibody to the adenoviral E1A protein (data not shown). Immortalized pre-type II cells exhibited surface binding to the lectin M. pomifera, a characteristic of type II cells in primary culture (Fig. 2 B).
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FIG. 3. Karyotyping analysis of Day 19 pre-type II cell line. Note that the cells are diploid. Day 18 pre-type II ceils were also diploid (data not shown).
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FIG. 4. Tannic acid stains. A, adult type II primary isolates; note the presence of multiple lamellar inclusions. B, Day 18 immortalized pre-type II cells cultured in control medium. Ceils exhibit no lamellar bodies. C, Day 18 immortalized cells cultured in hydrocortisone (1 × 10 -5 M) and 1 mM dibutryl cAMP for 7 days (staining performed after 26 wk in culture). Note the induction of dark staining cytoplasmic inclusions. D, Day 18 immortalized cells cultured in hydrocortisone (1 × 10-s M) and 1 mM dibutryl cAMP for 7 days (staining performed after 56 wk in culture). Ceils re-induced at this time demonstrated less pronounced cytoplasmic inclusions.
tional studies on ceils at Passage 80 showed that the cells did not secrete surfactant lipids or express surfactant protein A (data not shown). Cells at earlier passages were not evaluated. Glycogen. Fetal rat lungs isolated at Day 18 gestation consisted of cuboidal cells forming primitive tubular structures when examined under light microscopy. Pre-type lI cells lining to these structures contained abundant intracellular stores of glycogen similar to liver tissue when stained in situ with PAS (Fig. 6 A,B). Immortalized Day 18 pre-type II cells also demonstrated intense staining for glycogen (Fig. 6 C). Fetal rat lung at Day 20 gestation showed less pronounced staining with PAS as glycogen content gradually diminished with lung maturation (data not shown). Type II cells isolated from an adult rat lung (data not shown) and an immortalized adult type II cell line both demonstrated weak staining for glycogen (Fig. 6D).
DISCUSSION In the present study we demonstrate that a retroviral vector conraining the adenovirus 12S E1A cDNA sequence can be used to immortalized rat pre-type II alveolar epithelial cells. Initially we isolated primary cultures of type II cells from the fetal rat lung at Day 18 and Day 19 gestational age. During these early stages of lung development alveolar epithelial cells have not matured sufficiently to express lamellar bodies; an important feature which would permit studies assessing pre-type II cell maturation and hormonal regulation. Our studies demonstrate that cell lines of pre-type II alveolar epithelial cells generated after exposure to the retroviral construct morphologically resemble primary isolates of pre-type II cells. Transmission electron microscopy of cell monolayers reveals low cuboidal epithelial cells with apical microvilli and junctional corn-
PRE-TYPE II CELL LINES
Fro. 5. Transmission electron micrograph of immortalized Day 18pretype II cells stimulated with hydrocortisone and dibutryl cAMP. ×60 000. Note the presence of a typical lamellar body.
plexes. More importantly, lamellar bodies were not detected by electron microscopy of immortalized pre-type II cells. Immortalized cells isolated from Day 18 and Day 19 gestation cultured in the absence of hormones contained few lamellar bodies as detected by tannic acid staining. These features are all consistent with previous reports characterizing fetal pre-type II cells in primary culture (34,40,43). Another important marker of pre-type II cells is the presence of abundant intracellular stores of glycogen (34). We observed a relatively high cellular content of glycogen in Day 18, but not Day 19, immortalized pre-type II cells as demonstrated by PAS staining and electron microscopy. Our studies are consistent with prior reports describing depletion of glycogen stores in type II cells as lung maturation proceeds (4,12,21,34). Immunocytochemical studies to further characterize the immortalized cells confirm the presence of cytokcratins, identifying their epithelial nature. The cells also exhibit binding to the lectin M. pomifera, a characteristic of both fetal and adult type II cells (34). Both Day 18 and Day 19 cell lines were studied physiologically to determine the presence of tight junctions. The cells exhibited only a very low electrical resistance ( ~ 10 ohm >(cm-1), below values reported for adult type II cell monolayers cultured on porous surfaces (28,37), and significantly lower than monolayers plated on tissue culture treated filters (7). Inasmuch as these differences may be attributed to factors involving substrata and seeding density, additional studies may be required to identify an intact epithelial barrier. Finally, we have detected the E1A protein in pre-type lI cell lines, confirming that these cells express the adenoviral E1A gene product. Previous reports have described the oncogenic potential of human adenoviruses (5,31). Expression of E1A protein alone allows for immortalization of epithelial cells in culture (38). A second
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adenoviral gene product, E1B, is required for full transformation of rodent cells into a malignant phenotype (30). Pre-type II alveolar epithelial cells immortalized with the adenoviral E1A gene as described in this study proliferate rapidly in culture but do not seem to display malignant characteristics. Tumorogenic assays have not been performed; however, both cell lines showed a normal karyotype. Growth studies reveal that cells cultured in the presence of serum exhibit exponential growth and eventually reach a stationary phase of growth shortly after reaching confluence. This suggests that cell replication is suppressed by contact inhibition. Our studies further demonstrate that immortalized pre-type II alveolar epithelial cells can be hormonally induced to form lamellar bodylike structures similar to prior observations using in vivo models (19,21,34,41,43)~ Glucocorticoids, for example, are known to accelerate puhnonary maturation in a number of animal models (6,17,21,35) and are currently being administered prenatally (8,23). Other studies also suggest that glucocorticoids and cAMP are required for maintenance of surfactant synthesis (3,18,20). To further evaluate the in vitro effect of these soluble factors on fetal type II cell differentiation, we cultured immortalized pre-type II cells in control medium and medium supplemented with hydrocortisone and dibutryl cAMP. Cells cultured for 7 days in control medium expressed few lamellar bodies as detected by tannic acid staining. The numbers of these osmiophilic inclusions markedly increased when cells were cultured in hydrocortisone and dibutryl cAMP. Electron microscopy confirmed that these structures were typical lamellar bodylike structures, as evidenced by multiple lamellae layered layered concentrically and located within a membrane. Recently, we have attempted to re-induced expression of lamellar bodies in both cell lines after the cells underwent a number of additional passages in culture. These immortalized cells stimulated with hydrocortisone and dibutryl cAMP expressed significantly more lamellar bodylike structures than control cells; however, the inclusions were noted to be less prominent and fewer relative to our initial observations. The significance of the presence of lamellar bodylike structures in these stimulated cells in unclear. In this regard, we could not demonstrate the presence of SP-A or the release of surfactantlike lipids. It seems that additional studies may be necessary to define the optimal culture enviromcnt to preserve the long-term functional stability of these pre-type II cell lines. In addition to hormonal requirements, for example, expression of lamellar bodies by type II cells is influenced by an adequate supply of critical nutrients including glutathione (25), and various extracellular substrates (20,36). An epithelial-mesenchymal interaction may also play a role in maintaining type II cell morphology (42). Modulation of these factors may be important in generating stable cell lines which may serve as potentially useful in vitro models for studies of surfactant lipid and protein synthesis. In summary, our studies demonstrate that the adenoviral 12S E1A gene can be used to immortalize pre-type II alveolar epithelial cells isolated from the fetal rat. Cells described in this study express few lamellar bodies and contain abundant stores of glycogen, and therefore retain the essential features of pre-type 1I cells in primary culture. Our studies also suggest that immortalized pre-type II cells can be induced to form lamellar bodylike structures when exposed to agents that are known to promote the differentiated state of pretype II cells. These observations should stimulate interest in generating stable cell lines which may ultimately serve as useful models
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FIG. 6. PAS stain for glycogen. A, in situ staining of adult liver. Note presence of abundant PAS-positive material in hepatocytes. B, fetal rat lung, Day 18 gestational age; pre-type II cells lining tubules demonstrate intense staining for glycogen. C, immortalized Day 18 pre-type II cells also demonstrate intense staining for glycogen. Immortahzed Day 19 pre-type II cells stained less intensively with PAS (data not shown). D, an immortalized adult type II cell line demonstrates weak staining for glycogen.
for the morphologic study of fetal type II cell maturation and surfactant regulation. ACKNOWLEDGEMENTS
The authors thank Dr. Shivanand Patil of the University of Iowa Cytogenetics Laboratory for performing the karyotyping analysis. We also thank Debbie Jarrard and Peggy Kuenster for their secretarial assistance; and thank Dr. Jeffrey Whitsett and Dr. James Fisher for evaluating these cells for the presence of surfactant proteins and their mRNAs. This study was supported by the following grants: Institutional NRSA fellowship training grant (HL07121) from the National Heart, Lung, and Blood Institute, Bethesda, MD; a Specialized Center of Research grant (SCOR, HL37121) from the National Heart, Lung, and Blood Institute; an RO-1 (HL43883) from the National Heart, Lung, and Blood Institute; a Merit Review grant from the Department of Veteran Affairs, Washington, DC; and RR59 from the Clinical Research Center, National Institutes of Health, Bethesda, MD.
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