Murine lung epithelial (MLE) cell lines representing the distal bronchiolar and alveolar epithelium were produced from lung tumors generated in transgenic mice harboring the viral oncogene simian virus 40 (SV40) large tumor antigen under transcriptional control of a promoter region from the human surfactant protein C (SP-C) gene. The cell lines exhibited rapid growth, lack ofcontact inhibition, and an epithelial cel morphology for 30-40 passages in culture. MicroviUi, cytoplasmic multivesicular bodies, and multilamellar inclusion bodies (morphologic characteristics of alveolar type II cells) were detected in some of the MLE cell lines by electron microscopic analysis. The MLE cells also maintained functional characteristics of distal respiratory epithelial cells including the expression ofsurfactant proteins and mRNAs and the ability to secrete phospholipids. Expression of the exogenous SV40 large tumor antigen gene was detected in al of the generated cell lines. The SP-C/SV40 large tumor antigen transgenic mice and the MLE cell lines will be useful for the study of pulmonary surfactant production and regulation as wel as lung development and tumorigenesis. (4), and synthesis of SPs (5) in culture.Clara cells and type II epithelial cells also serve as progenitor cells of the distal respiratory epithelium in the adult lung (6, 7) and are believed to be the cell of origin forThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. pulmonary adenocarcinomas, a subtype of non-small-cell lung cancer (8). We have previously produced transgenic mice harboring the simian virus 40 (SV40) large tumor antigen (TAg) under the transcriptional control of regulatory sequences derived from the human SP-C promoter region to study the development of pulmonary adenocarcinomas in vivo (9). Transgenic mice bearing the exogenous SP-C/TAg chimeric gene developed pulmonary tumors within 4-6 months of age. The presence ofmicrovilli and lamellar bodies by electron microscopy, as well as the presence of respiratory epithelial cell markers by in situ hybridization, were consistent with the identification of the tumor cells as both bronchiolar and alveolar subtypes in vivo. While cells of the proximal respiratory epithelium and lung epithelial cells of fetal origin (10) have been established in culture, distal respiratory epithelial cell lines that maintain a differentiated phenotype in culture have not been previously produced. In the current study, we describe the production and characterization of distal respiratory epithelial cell lines derived from the SP-C/TAg transgenic mice.

/ajplung. 00302.2001.-The purpose of this study was to identify culture conditions for maintenance of isolated mouse type II cells with intact surfactant protein (SP) and phospholipid production. Type II cells were isolated from 6-wk-old mice and cultured on Matrigel matrix-rat tail collagen (70:30 vol/ vol) in bronchial epithelial cell growth medium minus hydrocortisone plus 5% charcoal-stripped FBS and 10 ng/ml keratinocyte growth factor. Under these conditions, type II cells actively produced surfactant phospholipids and proteins for at least 7 days. Synthesis and secretion of surfactant phospholipids and SP-A, -B, -C, and -D declined on day 1 of culture but recovered by day 3, reaching levels comparable to or exceeding freshly isolated cells by day 5. Abundant lamellar bodies were readily apparent in cells examined on days 5 and 7, and a surfactant pellet was recovered by centrifugation of media harvested on each day of culture. Secretion of SP-B, SP-C, and phosphatidylcholine was stimulated by phorbol 12-myristate 13-acetate and was inhibited by compound 48/80. When tested with a bubble surfactometer, surfactant secreted by type II cells on day 5 of culture lowered surface tension to 5.2 Ϯ 2.3 mN/m. This is the first description of the synthesis and secretion of a functional surfactant complex by mouse type II cells after 7 days in primary culture.surfactant; secretion; lung ISOLATED ALVEOLAR type II cells in primary culture have provided insight into the function of this important cell type in the lung. However, the rapid loss of the type II cell phenotype has limited the usefulness of this system. Manipulation of culture substratum and media has identified conditions that support the synthesis of surfactant proteins (SPs) and phospholipids in primary cultures of rat type II cells. Key substratum components include elements of extracellular matrix, such as the basement membrane extracted from EngelbrethHolm-Swarm (EHS) tumor (commercially available as Matrigel), which contains laminin, type IV collagen, and heparin sulfate proteoglycan (24, 26). Interaction of type II cells with extracellular matrix is thought to promote a native cuboidal cell shape, which is important for type II cell function in vitro (25,27). Keratinocyte growth factor (KGF; fibroblast growth factor-7) has been identified as a critical component of the culture medium, which likely reflects the importance of epithelial-mesenchymal interactions in vivo (28,36). In addition to effects of media and substratum, the culture of type II cells at an air-liquid interface (by limiting the amount of apical medium and rocking the culture dish) has also been shown to enhance maintenance of the rat type II cell phenotype in vitro (7,35).Although considerable advances have been made in optimizing culture conditions for rat type II cells, comparable progress for mouse type II cell culture is lacking. The development of such a culture system is important, since it would allow the study of type II cells from a large number of transgenic mouse lines in whi...

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Surfactant protein B (SP-B

Secretion of [3H]phosphatidylcholine ([3H]PC) from isolated rat pulmonary type II epithelial cells was inhibited by the surfactant-associated protein of Mr = 35,000 (SAP-35) purified from canine lung surfactant. SAP-35 inhibited [3H]PC secretion in a dose-dependent manner and significantly inhibited basal, phorbol ester, beta-adrenergic, and P2-purinergic agonist-induced [3H]PC secretion. SAP-35 significantly inhibited [3H]PC secretion from 1 to 3 h after treatment. The IC50 for inhibition of [3H]PC secretion by canine SAP-35 was 1-5 X 10(-6) g/ml and was similar for inhibition of both basal and secretagogue-stimulated release. Heat denaturation of SAP-35, addition of monoclonal anti-SAP-35 antibody, reduction and alkylation of SAP-35, or association of SAP-35 with phospholipid vesicles reversed the inhibitory effect on secretagogue-induced secretion. Inhibitory effects of SAP-35 were observed 3 h after cells were washed with buffer that did not contain SAP-35. Although SAP-35 enhanced reassociation of surfactant phospholipid with isolated type II cells, its inhibitory effect on secretion of [3H]PC did not result from stimulation of reuptake of secreted [3H]PC by type II cells. The inhibition of phospholipid secretion by SAP-35 was also not due to inhibition of PC or disaturated PC synthesis by SAP-35. SAP-35, the major phospholipid-associated protein in pulmonary surfactant, is a potent inhibitor of surfactant secretion from type II cells in vitro and may play an important role in homeostasis of surfactant in the alveolar space.