Maturation of undifferentiated lung epithelial cells into type II cells in vitro: A temporal process that parallels cell differentiation in vivo

Fetal rat lung epithelial cells were isolated on gestational day 17 (term is 22), separated from fibroblasts, and cultured up to 6 days in a serum-free medium on a basement membrane matrix. Surfactant protein (SP) A, barely detectable by immunostaining at the beginning of the culture, considerably increased in cells and subsequently in the lumen of the epithelial cell clusters. SP-A mRNA, already detectable at culture initiation, progressively increased. By contrast, SP-B and its mRNA appeared after 2-3 days. SP-C mRNA appeared only after 4 days of culture. Cells cultured 6 days had a phospholipid composition similar to that of freshly isolated adult rat type II cells. The enhancement of lipid synthesis between the first and the sixth culture days, reported earlier to occur in these cells, was found to be accompanied by a two- to fivefold increase in amount of mRNAs of lipogenic enzymes and choline phosphate cytidylyltransferase. In conclusion, alveolar epithelial type II cells appear to be capable of full differentiation in vitro, and components of the surfactant system are all regulated developmentally at a pretranslational level.

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Clinical surfactant preparations mediate SOD and catalase uptake by type II cells and lung tissue

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1996.270.4.l659 ◽

1996 ◽

Vol 270 (4) ◽

pp. L659-L667 ◽

Cited By ~ 6

Author(s):

B. Nieves-Cruz ◽

A. Rivera ◽

J. Cifuentes ◽

G. Pataki ◽

S. Matalon ◽

...

Keyword(s):

Antioxidant Enzymes ◽

Epithelial Cells ◽

Alveolar Epithelium ◽

Lung Epithelial Cells ◽

Type Ii Cells ◽

Type Ii ◽

Surfactant Mixtures ◽

Significant Augmentation

Pulmonary surfactant mixtures are rapidly taken up by alveolar type II cells and thus may serve as vectors for the pulmonary delivery of antioxidant enzymes to the alveolar epithelium. We prepared emulsions of Survanta with superoxide dismutase (CuZn-SOD) and catalase and quantified their cellular uptake both in vitro and in vivo. Incubations of fetal lung epithelial cells with an emulsion of Survanta plus SOD and catalase mixtures resulted in significant augmentation of SOD and catalase activities (12.8 +/- 4.6 U SOD/microgram DNA; 7.49 +/- 2.21 U catalase/microgram DNA). These numbers were significantly greater than those obtained in controls (1.8 U SOD/microgram DNA; 0.55 +/- 0.52 U catalase/microgram DNA, Survanta alone (0.43 U SOD/microgram DNA; 0.16 U catalase/microgram DNA), and SOD and catalase alone (3.47 +/- 5.2 U SOD/microgram DNA; 4.24 +/- 3.0 U catalase/microgram DNA). Intratracheal instillation of the Survanta plus SOD and catalase mixture resulted in significant augmentation of enzymes by the rat lung homogenates. Confocal microscopic analysis revealed the presence of antioxidant enzymes in the cytoplasm of epithelial cells. We concluded that Survanta supplementation, in addition to replenishing surfactant stores, can also enhance the delivery of antioxidant enzymes to alveolar epithelium both in vitro and in vivo.

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In Vivo and In Vitro Analysis of Factor Binding Sites in Jaagsiekte Sheep Retrovirus Long Terminal Repeat Enhancer Sequences: Roles of HNF-3, NF-I, and C/EBP for Activity in Lung Epithelial Cells

Journal of Virology ◽

10.1128/jvi.80.1.332-341.2006 ◽

2006 ◽

Vol 80 (1) ◽

pp. 332-341 ◽

Cited By ~ 20

Author(s):

Kathleen McGee-Estrada ◽

Hung Fan

Keyword(s):

Epithelial Cells ◽

Long Terminal Repeat ◽

Binding Site ◽

Terminal Repeat ◽

Lung Epithelial Cells ◽

Type Ii ◽

Jaagsiekte Sheep Retrovirus ◽

Efficient System ◽

Lung Epithelial

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma, a contagious lung cancer of sheep that arises from type II pneumocytes and Clara cells of the lung epithelium. Studies of the tropism of this virus have been hindered by the lack of an efficient system for viral replication in tissue culture. To map regulatory regions important for transcriptional activation, an in vivo footprinting method that couples dimethyl sulfate treatment and ligation-mediated PCR was performed in murine type II pneumocyte-derived MLE-15 cells infected with a chimeric Moloney murine leukemia virus driven by the JSRV enhancers (ΔMo+JS Mo-MuLV). In vivo footprints were found in the JSRV enhancers in two regions previously shown to be important for JSRV long terminal repeat (LTR) activity: a binding site for the lung-specific transcription factor HNF-3β and an E-box element in the distal enhancer adjacent to an NF-κB-like binding site. In addition, in vivo footprints were detected in two downstream motifs likely to bind C/EBP and NF-I. Mutational analysis of a JSRV LTR reporter construct (pJS21luc) revealed that the C/EBP binding site is critical for LTR activity, while the putative NF-I binding element is less important; elimination of these sites resulted in 70% and 40% drops in LTR activity, respectively. Electrophoretic mobility shift assays using nuclear extracts from MLE-15 murine Clara cell-derived mtCC1-2 cells with probes corresponding to the NF-I or C/EBP sites revealed several complexes. Antiserum directed against NF-IA, C/EBPα, or C/EBPβ supershifted the corresponding protein-DNA complexes, indicating that these isoforms, which are also important for the expression of several cellular lung-specific genes, may be important for JSRV expression in lung epithelial cells.

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Influenza A Virus Infection Induces Apical Redistribution of Na+, K+-ATPase in Lung Epithelial Cells In Vitro and In Vivo

American Journal of Respiratory Cell and Molecular Biology ◽

10.1165/rcmb.2019-0096le ◽

2019 ◽

Vol 61 (3) ◽

pp. 395-398

Author(s):

Christin Peteranderl ◽

Irina Kuznetsova ◽

Jessica Schulze ◽

Martin Hardt ◽

Emilia Lecuona ◽

...

Keyword(s):

Epithelial Cells ◽

Virus Infection ◽

Influenza A Virus ◽

Influenza A ◽

Lung Epithelial Cells ◽

Lung Epithelial ◽

Influenza A Virus Infection

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Bcl-2 overexpression in type II epithelial cells does not prevent hyperoxia-induced acute lung injury in mice

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00212.2009 ◽

2010 ◽

Vol 299 (3) ◽

pp. L312-L322 ◽

Cited By ~ 8

Author(s):

Isabelle Métrailler-Ruchonnet ◽

Alessandra Pagano ◽

Stéphanie Carnesecchi ◽

Karim Khatib ◽

Pedro Herrera ◽

...

Keyword(s):

Cell Death ◽

Epithelial Cells ◽

Lung Injury ◽

Ex Vivo ◽

Lung Damage ◽

Type Ii Cells ◽

Type Ii ◽

Lung Weight

Bcl-2 is an anti-apoptotic molecule preventing oxidative stress damage and cell death. We have previously shown that Bcl-2 is able to prevent hyperoxia-induced cell death when overexpressed in a murine fibrosarcoma cell line L929. We hypothesized that its specific overexpression in pulmonary epithelial type II cells could prevent hyperoxia-induced lung injury by protecting the epithelial side of the alveolo-capillary barrier. In the present work, we first showed that in vitro Bcl-2 can rescue murine pulmonary epithelial cells (MLE12) from oxygen-induced cell apoptosis, as shown by analysis of LDH release, annexin V/propidium staining, and caspase-3 activity. We then generated transgenic mice overexpressing specifically Bcl-2 in lung epithelial type II cells under surfactant protein C (SP-C) promoter (Tg-Bcl-2) and exposed them to hyperoxia. Bcl-2 did not hinder hyperoxia-induced mitochondria and DNA oxidative damage of type II cell in vivo. Accordingly, lung damage was identical in both Tg-Bcl-2 and littermate mice strains, as measured by lung weight, bronchoalveolar lavage, and protein content. Nevertheless, we observed a significant lower number of TUNEL-positive cells in type II cells isolated from Tg-Bcl-2 mice exposed to hyperoxia compared with cells isolated from littermate mice. In summary, these results show that although Bcl-2 overexpression is able to prevent hyperoxia-induced cell death at single cell level in vitro and ex vivo, it is not sufficient to prevent cell death of parenchymal cells and to protect the lung from acute damage in mice.

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A bilayer tissue culture model of the bovine alveolus

F1000Research ◽

10.12688/f1000research.18696.1 ◽

2019 ◽

Vol 8 ◽

pp. 357

Author(s):

Diane Lee ◽

Mark Chambers

Keyword(s):

Epithelial Cells ◽

Alveolar Type ◽

Type Ii Cells ◽

Type Ii ◽

Host Pathogen Interactions ◽

Alveolar Type Ii Cells ◽

Permeable Membrane ◽

Host Pathogen

The epithelial lining of the lung is often the first point of interaction between the host and inhaled pathogens, allergens and medications. Epithelial cells are therefore the main focus of studies which aim to shed light on host-pathogen interactions, to dissect the mechanisms of local host immunity and study toxicology. If these studies are not to be conducted exclusively in vivo, it is imperative that in vitro models are developed with a high in vitro-in vivo correlation. We describe here a co-culture bilayer model of the bovine alveolus, designed to overcome some of the limitations encountered with mono-culture and live animal models. Our system includes bovine pulmonary arterial endothelial cells (BPAECs) seeded onto a permeable membrane in 24 well Transwell format. The BPAECs are overlaid with immortalised bovine alveolar type II epithelial cells and the bilayer cultured at air-liquid interface for 14 days before use; in our case to study host-mycobacterial interactions. Characterisation of novel cell lines and the bilayer model have provided compelling evidence that immortalised bovine alveolar type II cells are an authentic substitute for primary alveolar type II cells and their culture as a bilayer in conjunction with BPAECs provides a physiologically relevant in vitro model of the bovine alveolus. The bilayer model may be used to study dynamic intracellular and extracellular host-pathogen interactions, using proteomics, genomics, live cell imaging, in-cell ELISA and confocal microscopy. The model presented in this article enables other researchers to establish an in vitro model of the bovine alveolus that is easy to set up, malleable and serves as a comparable alternative to in vivo models, whilst allowing study of early host-pathogen interactions, currently not feasible in vivo. The model therefore achieves one of the 3Rs objectives in that it replaces the use of animals in research of bovine respiratory diseases.

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Carcinoembryonic cell adhesion molecule 6 in human lung: regulated expression of a multifunctional type II cell protein

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.90596.2008 ◽

2009 ◽

Vol 296 (6) ◽

pp. L1019-L1030 ◽

Cited By ~ 13

Author(s):

Venkatadri Kolla ◽

Linda W. Gonzales ◽

Nicole A. Bailey ◽

Ping Wang ◽

Sreedevi Angampalli ◽

...

Keyword(s):

Cell Adhesion ◽

Epithelial Cells ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Fetal Lung ◽

Lung Epithelial Cells ◽

Cell Protein ◽

Type Ii Cells ◽

Type Ii ◽

Lung Epithelial

Carcinoembryonic cell adhesion molecule 6 (CEACAM6) is a glycosylated, glycosylphosphatidylinositol (GPI)-anchored protein expressed in epithelial cells of various human tissues. It binds gram-negative bacteria and is overexpressed in cancers, where it is antiapoptotic and promotes metastases. To characterize CEACAM6 expression in developing lung, we cultured human fetal lung epithelial cells and examined responses to differentiation-promoting hormones, adenovirus expressing thyroid transcription factor-1 (TTF-1), and silencing of TTF-1 with small inhibitory RNA. Glucocorticoid and cAMP had additive stimulatory effects on CEACAM6 content, and combined treatment maximally increased transcription rate, mRNA, and protein ∼10-fold. Knockdown of TTF-1 reduced hormone induction of CEACAM6 by 80%, and expression of recombinant TTF-1 increased CEACAM6 in a dose-dependent fashion. CEACAM6 content of lung tissue increased during the third trimester and postnatally. By immunostaining, CEACAM6 was present in fetal type II cells, but not mesenchymal cells, and localized to both the plasma membrane and within surfactant-containing lamellar bodies. CEACAM6 was secreted from cultured type II cells and was present in both surfactant and supernatant fractions of infant tracheal aspirates. In functional studies, CEACAM6 reduced inhibition of surfactant surface properties by proteins in vitro and blocked apoptosis of electroporated cultured cells. We conclude that CEACAM6 in fetal lung epithelial cells is developmentally and hormonally regulated and a target protein for TTF-1. Because CEACAM6 acts as an antiapoptotic factor and stabilizes surfactant function, in addition to a putative role in innate defense against bacteria, we propose that it is a multifunctional alveolar protein.

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