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Co-culture of type I and type II pneumocytes as a model of alveolar epithelium.

ABSTRACT: The epithelial tissues of the distal lung are continuously exposed to inhaled air, and are of research interest in studying respiratory exposure to both hazardous and therapeutic materials. Pharmaco-toxicological research depends on the development of sophisticated models of the alveolar epithelium, which better represent the different cell types present in the native lung and interactions between them. We developed an air-liquid interface (ALI) model of the alveolar epithelium which incorporates cell lines which bear features of type I (hAELVi) and type II (NCI-H441) epithelial cells. We compared morphology of single cells and the structure of cell layers of the two lines using light and electron microscopy. Working both in monotypic cultures and cocultures, we measured barrier function by trans-epithelial electrical resistance (TEER), and demonstrated that barrier properties can be maintained for 30 days. We created a mathematical model of TEER development over time based on these data in order to make inferences about the interactions occurring in these culture systems. We assessed expression of a panel of relevant genes that play important roles in barrier function and differentiation. The coculture model was observed to form a stable barrier akin to that seen in hAELVi, while expressing surfactant protein C, and having a profile of expression of claudins and aquaporins appropriate for the distal lung. We described cavities which arise within stratified cell layers in NCI-H441 and cocultured cells, and present evidence that these cavities represent an aberrant apical surface. In summary, our results support the coculture of these two cell lines to produce a model which better represents the breadth of functions seen in native alveolar epithelium.

SUBMITTER: Brookes O

PROVIDER: S-EPMC8475999 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Proteins from primary cultures of type II granular pneumocytes have been examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to identify type II cell-specific proteins. The distribution of Coomassie Blue-stained bands in preparations of cellular proteins, culture medium, lavage and lamellar bodies have been compared. The most prominent stained band in the serum-free medium from type II cell cultures (HS1; Mr 39900) corresponds to a major protein in acellular sedimentable (20000 g for 30 min) crude surfactant obtained from rat lungs by saline (0.9% NaCl) lavage. A second protein (HS2; Mr 12000) is also found both in type II cell-conditioned medium and in lavage. Neither rat serum nor donor calf serum (used in the isolation of the type II cells) contains a protein co-migrating with HS1 or HS2 proteins. HS1 is also found in Coomassie Blue-stained gels of cellular proteins and of lamellar bodies isolated from whole lungs. Cultures of type II cells incorporate [14C]phenylalanine into HS1 and HS2 as shown by autoradiography of sodium dodecyl sulphate/polyacrylamide gels of culture medium. Rat lungs perfused in situ incorporate [35S]methionine into HS1 in the lamellar body fraction. A third protein (HS3; Mr 47000) is observed only in autoradiographs of cell culture medium; no corresponding Coomassie Blue-stained band can be identified in medium, in cells or in lung lavage. No protein bands corresponding to HS1, HS2 or HS3 are found in conditioned media from pulmonary alveolar macrophages, rat fibroblasts or bovine aorta endothelial cells. Two-dimensional gel electrophoresis of HS1 shows a single polypeptide with an isoelectric point of 6.3; HS3 appears as a chain of spots with a range of isoelectric points from 6.3 to 6.6. HS2 has not been identified on two-dimensional gels. The amino acid composition of HS1 does not differ significantly from that of surfactant apoproteins studied previously; however, HS1 is not detected by glycoprotein stains, nor does it appear to be a subunit of a thiol-linked multimer.

Project description:Alveolar surfactant (exposed to air and therefore a prime target of air oxidants) is supplied with antioxidants during its intracellular formation on type-II pneumocytes [Rüstow, Haupt, Stevens and Kunze (1993) Am. J. Physiol. 265, L133-L139]. Plasmalogens can protect animal cells against lipid peroxidation caused by u.v. radiation. It has been suggested that plasmalogens play a direct role in protecting animal cell membranes against oxidative stress [Zoeller, Morand and Raetz (1988) J. Biol. Chem. 263, 11590-11596]. We investigated biosynthesis and secretion of plasmalogens and phospholipids by type-II cells of adult rat lungs. The plasmalogens of type-II cells consist of 93% ethanolamine plasmalogens (EthPlas) and 7% choline plasmalogens (ChoPlas). Plasmalogens isolated from alveolar surfactant, however, consist of 36.5% ChoPlas and 63.5% EthPlas. The different incorporation rates of [14C]hexadecanol into both types of plasmalogen by type-II pneumocytes are reflected in the relative proportions of their total cellular plasmalogen content. Type-II cells cultured in the presence of labelled hexadecanol or labelled hexadecylglycerol and of labelled palmitate secrete labelled ChoPlas and labelled phospholipids, both spontaneously and in response to isoprenaline. The spontaneous and stimulated secretion rates of labelled ChoPlas are 3-6 times higher than those of labelled EthPlas. This higher relative secretion rate of ChoPlas corresponds to its higher proportion in the total plasmalogen content of alveolar surfactant compared with type-II cells. Added extracellular surfactant-specific protein A inhibits the secretion of plasmalogens as well as that of phospholipids by type-II cells. The molecular species of EthPlas and ChoPlas isolated from type-II cells or lung lavage do not differ significantly and consist mainly of molecular species containing poly-unsaturated fatty acids. We conclude that ChoPlas are secreted partly as integral constituents of the alveolar surfactant. Type-II cells select between both types of plasmalogens for secretion as a constituent of surfactant. The intramolecular sorting signal presumably is the choline moiety.

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Co-culture of type I and type II pneumocytes as a model of alveolar epithelium.

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