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. 2000;36(6):374–382. doi: 10.1290/1071-2690(2000)036<0374:GOAIDL>2.0.CO;2

Generation of an immortal differentiated lung type-II epithelial cell line from the adult H-2KbtsA58 transgenic mouse

Daphne E deMello 1,2,✉, Sohir Mahmoud 1,2, Philip J Padfield 1, Joseph W Hoffmann 1
PMCID: PMC7101677  PMID: 10949996

Summary

This paper describes a new fully differentiated Type-II alveolar epithelial cell line designated T7, derived from transgenic H-2Kb-tsA58 mice, capable of being passaged as an immortalized cloned cell line in culture. H-2Kb-tsA58 mice harbor a temperature-sensitive (ts) mutant of the simian virus 40 (SV40) large tumor antigen (T antigen) under the control of the γ-interferon (INF)-inducible mouse major histocompatibility complex H-2Kb promoter. When cultured under permissive conditions (33°C and in the presence of γ-INF) cells isolated from H-2Kb-tsA58 mice express the large T antigen, which drives the cells to proliferate. However, upon withdrawal of the γ-INF and transfer of the cells to a higher temperature (39°C), T antigen expression is turned off, the cells stop proliferating and differentiate. The T7 cell line is a clonal cell line originally derived from a Type-II cell-rich fraction isolated from lungs of H-2Kb-tsA58 mice. The T7 cells form confluent monolayers, and have a polarized epithelial cell morphology with tight junctions and apical microvilli. In addition, the T7 cells have distinct cytoplasmic lamellar bodies, which become more numerous and pronounced when the cells are grown under nonpermissive conditions. The T7 cells synthesize and secrete phosphatidylcholine and the three surfactant proteins, SP-A, SP-B, and SP-C. The T7 cell line is unique in that it is the first non-tumor-derived Type-II cell line capable of synthesizing and secreting the major components of surfactant. Based on the criteria studied, the T7 cell line is phenotypically very similar to normal Type-II cells. The T7 cell line, therefore, should prove a valuable experimental system to advance the study of the cell biology/physiology of surfactant metabolism and secretion as well as serve as a model for other studies of Type-II cell physiology.

Key words: pulmonary, surfactant, lipid, protein, secretion, synthesis

References

  1. Avery M.E., Mead J. Surface properties in relation to atelectasis and hyaline membrane disease. Am. J. Dis. Child. 1959;97:517–523. doi: 10.1001/archpedi.1959.02070010519001. [DOI] [PubMed] [Google Scholar]
  2. Beers M.F., Kim C. Y., Dodia C., Fisher A. B. Localization, synthesis, and processing of surfactant protein SP-C in rat lung analyzed by epitope-specific antipeptide antibodies. J. Biol. Chem. 1994;269:20,318–20,328. [PubMed] [Google Scholar]
  3. Beers M. F., Wali A., Eckenhoff M. F., Feinstein S. I., Fisher J. H., Fisher A. B. An antibody with specificity for surfactant protein C precursors: identification of pro-SP-C in rat lung. Am. J. Respir. Cell. Mol. Biol. 1992;7:368–378. doi: 10.1165/ajrcmb/7.4.368. [DOI] [PubMed] [Google Scholar]
  4. Bligh E. G., Dyer W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959;37:911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  5. Chambers T. J., Owens J. M., Hattersley G., Jat P. S., Noble M. D. Generation of osteoclast-inductive and osteoclastogenic cell lines from the H-2KbtsA58 transgenic mouse. Proc. Natl. Acad. Sci. USA. 1993;90:5578–5582. doi: 10.1073/pnas.90.12.5578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chomezynski P., Sacchi N. Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 1987;162:156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Clark J.C., Wert S. E., Bachurski C. J., Stahlman M. Y., Stripp B. R., Weaver T. E., Whitsett J. A. Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice. Proc. Natl. Acad. Sci. USA. 1995;92:7794–7798. doi: 10.1073/pnas.92.17.7794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clement A., Campisi J., Farmer S. R., Brody J. S. Constitutive expression of growth related mRNAs in proliferating and nonproliferating lung epithelial cells in primary culture: evidence for growth-dependent translational control. Proc. Natl. Acad. Sci. USA. 1990;87:318–322. doi: 10.1073/pnas.87.1.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clements J. A. Surface tension of lung extracts. Proc. Soc. Exp. Biol. Med. 1957;95:170–172. doi: 10.3181/00379727-95-23156. [DOI] [PubMed] [Google Scholar]
  10. Coalson J. J. In: Pathology of pulmonary disease. Saldana M. J., editor. Philadelphia, PA: J. B. Lippincott; 1994. pp. 4–4. [Google Scholar]
  11. D'Amore-Bruno M. A., Wikenheiser K. A., Carter J. E., Clark J. C., Whitsett J. A. Sequence, ontogeny, and cellular localization of murine surfactant protein B mRNA. Am. J. Physiol. (Lung Cell. Mol. Physiol. 6) 1991;262:L40–L47. doi: 10.1152/ajplung.1992.262.1.L40. [DOI] [PubMed] [Google Scholar]
  12. deMello D. E., Heyman S., Phelps D. S., Hamvas A., Nogee L., Cole S., Colten H. R. Ultrastructure of lung in surfactant protein B (SP-B) deficiency. Am. J. Respir. Cell. Mol. Biol. 1994;11:230–239. doi: 10.1165/ajrcmb.11.2.8049084. [DOI] [PubMed] [Google Scholar]
  13. deMello D. E., Nogee L. M., Heyman S., et al. Molecular and phenotypic variability in the congenital alveolar proteinosis syndrome associated with inherited surfactant protein B deficiency. J. Pediatr. 1994;125:43–50. doi: 10.1016/S0022-3476(94)70119-9. [DOI] [PubMed] [Google Scholar]
  14. Dobbs L. G. Isolation and culture of alveolar type II cells. Am. J. Physiol. 1990;258:L134–L147. doi: 10.1152/ajplung.1990.258.4.L134. [DOI] [PubMed] [Google Scholar]
  15. Dobbs L. G., Pian M.S., Maglio M., Dumars S., Allen L. Maitenance of the differentiated type II cell phenotype by culture with an apical air surface. Am. J. Physiol. 1997;273(2):L347–L354. doi: 10.1152/ajplung.1997.273.2.L347. [DOI] [PubMed] [Google Scholar]
  16. Freshney R. I. Culture of animal cells: a manual of basic technique. New York, NY: Wiley; 1991. [Google Scholar]
  17. Glasser S. W., Korfhagen T. R., Bruno M. D., Dey C., Whitsett J. A. Structure and expression of the pulmonary surfactant protein SP-C gene in the mouse. J. Biol. Chem. 1990;265:21,986–21,991. [PubMed] [Google Scholar]
  18. Hartog A., Gommers D., Lachmann B. Role of surfactant in the pathophysiology of the acute respiratory distress syndrome (ARDS) Monaldi Arch. Chest Dis. 1995;50:372–377. [PubMed] [Google Scholar]
  19. Jat P. S., Noble M. D., Ataliotis P., Tanaka Y., Yannoutsos N., Larsen L., Kioussis D. Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse. Proc. Natl. Acad. Sci. USA. 1991;88:5096–5100. doi: 10.1073/pnas.88.12.5096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Korfhagen T. R., Bruno M. D., Glasser S. W., Ciraolo P. J., Whitsett J. A., Lattier D. L., Wikenheiser K. A., Clark J. C. Murine pulmonary surfactant SP-A gene: cloning, sequence, and transcriptional activity. Am. J. Physiol. (Lung Cell. Mol. Physiol.) 1992;263:L546–L554. doi: 10.1152/ajplung.1992.263.5.L546. [DOI] [PubMed] [Google Scholar]
  21. Kresch M. J., Dynia D. W., Gross I. Culture of differentiated and undifferentiated type II cells from fetal rat lung. Biochem. Biophys. Acta. 1987;930:19–32. doi: 10.1016/0167-4889(87)90151-0. [DOI] [PubMed] [Google Scholar]
  22. Leslie C. C., McCormick-Shannon K., Mason R. J., Shannon J. M. Proliferation of rat alveolar epithelial cells in low density primary culture. Am. J. Respir. Cell. Mol. Biol. 1993;9:64–72. doi: 10.1165/ajrcmb/9.1.64. [DOI] [PubMed] [Google Scholar]
  23. Lin S., Phillips K. S., Wilder M. R., Weaver T. E. Structural requirements for intracellular transport of pulmonary surfactant protein B (SP-B) Biochim. Biophys. Acta. 1996;1312:177–185. doi: 10.1016/0167-4889(95)00201-4. [DOI] [PubMed] [Google Scholar]
  24. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951;193:265–275. [PubMed] [Google Scholar]
  25. Mallampalli R. K., Floerchinger C. S., Hunninghake G. W. Isolation and immortalization of rat pre-type II cell lines. In Vitro Cell. Dev. Biol. 1992;28A:181–187. doi: 10.1007/BF02631089. [DOI] [PubMed] [Google Scholar]
  26. Mason R. J., Apostolou S., Power J., Robinson P. Human alveolar type II cells: stimulation of DNA synthesis by insulin and endothelial cell growth supplement. Am. J. Respir. Cell. Mol. Biol. 1990;3:571–577. doi: 10.1165/ajrcmb/3.6.571. [DOI] [PubMed] [Google Scholar]
  27. Mason R. J., Dobbs L. G., Greenleaf R. D., Williams M. C. Alveolar Type II cells. Fed. Proc. Fed. Am. Soc. Exp. Biol. 1997;36:2697–2702. [PubMed] [Google Scholar]
  28. Mason R. J., Dobbs L. G. Synthesis of phosphatidylcholine and phosphatidyglycerol by alveolar type II cells in primary culture. J. Biol. Chem. 1980;255:5101–5107. [PubMed] [Google Scholar]
  29. Mason R. J., Williams M. C., Greenleaf R. D., Clements J. A. Isolation and properties of type II alveolar cells from rat lung. Am. Rev. Respir. Dis. 1997;115:1015–1026. doi: 10.1164/arrd.1977.115.6.1015. [DOI] [PubMed] [Google Scholar]
  30. Massey T. E., Geddes B., Forkert P. G. Isolation of nonciliated bronchiolar epithelial (Clara) cells and alveolar type II cells from mouse lungs. Can. J. Physiol. Pharmacol. 1987;65:2368–2372. doi: 10.1139/y87-375. [DOI] [PubMed] [Google Scholar]
  31. Nogee L. M., deMello D. E., Dehner L. P., Colten H. R. Deficiency of pulmonary surfactant protein B in congenital alveolar proteinosis. N. Engl. J. Med. 1993;328:406–410. doi: 10.1056/NEJM199302113280606. [DOI] [PubMed] [Google Scholar]
  32. O'Reilly M. A., Clark J. C., Whitsett J. A. Glucocorticoid enhances pulmonary surfactant protein B gene transcription. Am. J. Physiol. 1991;260:L37–L43. doi: 10.1152/ajplung.1991.260.2.L37. [DOI] [PubMed] [Google Scholar]
  33. O'Reilly M. A., Gazdar A. F., Clark J. C., Pilot-Matias T. J., Wert S. E., Hull W. M., Whitsett J. A. Glucocorticoids regulate surfactant protein synthesis in a pulmonary adenocarcinoma cell line. Am. J. Physiol. 1988;257:L385–L392. doi: 10.1152/ajplung.1989.257.6.L385. [DOI] [PubMed] [Google Scholar]
  34. O'Reilly M.A., Gazdar A. F., Morris R. E., Whitsett J. A. Differential effects of glucocorticoid on expression of surfactant proteins in a human lung adenocarcinoma cell line. Biochem. Biophys. Acta. 1988;970:194–204. doi: 10.1016/0167-4889(88)90179-6. [DOI] [PubMed] [Google Scholar]
  35. Phelps D. S., Taeusch H. W., Benson G., Hawgood S. An electrophoretic and immunochemical characterization of human surfactant-associated proteins. Biochem. Biophys. Acta. 1984;791:226–238. doi: 10.1016/0167-4838(84)90013-x. [DOI] [PubMed] [Google Scholar]
  36. Sen N., Granstein M. M., Chander A. Stimulation of lung surfactant secretion by endothelin-1 from rat alveolar type II cells. Am. J. Physiol. 1994;266:L255–L262. doi: 10.1152/ajplung.1994.266.3.L255. [DOI] [PubMed] [Google Scholar]
  37. Shannon J. M., Jennings S. D., Nielsen L. D. Modulation of alveolar Type II cell differentiated function in vitro. Am. J. Physiol. 1992;262:L427–L436. doi: 10.1152/ajplung.1992.262.4.L427. [DOI] [PubMed] [Google Scholar]
  38. Smith B. T. Cell line A549: a model system for the study of alveolar type II cell function. Am. Rev. Respir. Dis. 1977;115:285–293. doi: 10.1164/arrd.1977.115.2.285. [DOI] [PubMed] [Google Scholar]
  39. Steele M. P., Levine R. A., Joyce-Brady M., Brody J. S. A rat alveolar type II cell line developed by adenovirus 12SE1A gene transfer. Am. J. Respir. Cell. Mol. Biol. 1992;6:50–56. doi: 10.1165/ajrcmb/6.1.50. [DOI] [PubMed] [Google Scholar]
  40. Suzuki Y., Kogishi K., Fujita Y., Kina T., Nishikawa S. A monoclonal antibody to the 15,000 dalton protein associated with porcine pulmonary surfactant. Exp. Lung Res. 1986;11:61–73. doi: 10.3109/01902148609062827. [DOI] [PubMed] [Google Scholar]
  41. Tokunaga K., Taniguchi H., Yoda K., Shimizu M., Sakiyama S. Nucleotide sequence of a full-length cDNA for mouse cytoskeletal β-actin mRNA. Nucleic Acids Res. 1986;14:2829–2829. doi: 10.1093/nar/14.6.2829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Touchstone J. C., Chen J. C., Beaver K. M. Improved separation of phospholipids in thin layer chromatography. Lipids. 1979;15:61–62. doi: 10.1007/BF02534120. [DOI] [Google Scholar]
  43. Van Scott M. R., Lee N. P., Yankaskas J. R., Boucher R. C. Effect of hormones on growth and function of cultured canine tracheal epithelial cells. Am. J. Physiol. 1988;255:C237–C245. doi: 10.1152/ajpcell.1988.255.2.C237. [DOI] [PubMed] [Google Scholar]
  44. Weaver T., Whitsett J. A. Function and regulation of expression of pulmonary surfactant proteins. Biochem. J. 1991;273:249–264. doi: 10.1042/bj2730249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Whitehead R. H., VanEeden P. E., Noble M. D., Ataliotis P., Jat P. S. Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2KbtsA58 transgenic mice. Proc. Natl. Acad. Sci. USA. 1993;90:587–591. doi: 10.1073/pnas.90.2.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wikenheiser K. A., Vorbroker D. K., Rice W. R., Clark J. C., Bachurski C. J., Oie H. K., Whitsett J. A. Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus 40 large tumor antigen transgenic mice. Proc. Natl. Acad. Sci. USA. 1993;90:11,029–11,033. doi: 10.1073/pnas.90.23.11029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yamaya M., Finkbeiner W. E., Chun S. Y., Widdicombe J. H. Differentiated structure and function of cultures from human tracheal epithelium. Am. J. Physiol. (Lung Cell. Mol. Physiol. 6) 1992;262:L713–L724. doi: 10.1152/ajplung.1992.262.6.L713. [DOI] [PubMed] [Google Scholar]

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