Isolation and immortalization of rat pre-type II cell lines

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 E1A gene product. The immortalized pre-type II cells retained many of the ultrastructural 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 lectinMaclura 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.     

Culture of fetal alveolar epithelial type II cells in serum-free medium

Summary A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle’s minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistance of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment.     

Characterization of mouse fetal lung cells cultured on a pigskin substrate

Summary Lung organ bits taken from full-term mice were explanted on the dermal surface of sterile, dead pigskin. The cells migrated onto the pigskin dermis and proliferated to form an organoid culture consisting of ductular structures separated by a matrix of epithelial cells. Cells within the ductular structures were ciliated, produced mucin, and exhibited the activities of nonspecific esterase and gamma-glutamyl transferase; therefore they were considered to be derived from bronchial epithelium. Cells forming the matrix possessed the activities of nonspecific esterase and alkaline phosphatase and contained lamellar structures typical of surfactant-producing pneumocyte Type II cells; therefore they were considered to be derived from alveolar precursor cells. This research was supported by Grant-in-Aid 1203 M from the Council for Tobacco Research, awarded to Aaron E. Freeman.     

Accelerated fetal lung maturation by estrogen is associated with an epithelial-fibroblast interaction

Summary The role of epithelial-mesenchymal interactions in the stimulation of lung development by estrogen is now investigated using organ cultures of lung from male and female fetal rats taken from Days 17 to 21 of gestation. Estradiol at 1 μg/ml was found to reduce cell proliferation in explants taken during a rapid growth phase (Day 18) and to stimulate surfactant synthesis in both males and females only in Day 20 explants when cell division is much slower. At this time more epithelial cells from estrogen-treated explants contained lamellar bodies, which were also secreted to fill the air sacs. These cultures also showed a significant increase in the frequency of cell-to-cell contacts between epithelial cells and fibroblasts. Uptake of tritiated estradiol by explants increased from Day 18 onward, and by autoradiography, labeling was located predominantly over fibroblasts. Using pure cultures of fetal and adult cells, uptake of labeled estradiol was significantly higher in fibroblasts than in corresponding epithelial cells, and estradiol did not directly enhance palmitate incorporation into epithelial cells. The results suggest that the earlier maturation and increased surfactant synthesis in female fetal lung is related at least in part to enhanced binding of estrogen by the fibroblast with subsequent transfer of a maturation factor to the fetal epithelium. This research project was supported by grants from the Medical Research Council of Canada and the Council for Tobacco Research, U.S.A., Inc.     

Fine structural identification of organoid mouse lung cells cultured on a pigskin substrate

Summary Mouse full-term embryonic lung tissue was cultured as organ bits using dead, sterile pigskin dermal collagen as a substrate. Explanted organ bits grew on the surface of, and into, the pigskin dermal collagen for at least 9 weeks after the initiation of culture. The out-growth consisted of a thick cellular sheet containing various sizes of ductular structures within a cellular matrix that did not show any particular structure. Electron microscopic observation revealed that the larger ductular structures consisted largely of ciliated cells. The smaller ductular structure consisted largely of Type II pneumocytes containing lamellar hodies. The cellular matrix consisted of Type II pneumonocytes and other cell types including fibroblasts and macrophages in the early stage of cultivation. Macrophages invaded the pigskin dermal collagen. An intermediate cell type, which has never been observed in vivo, possessing both cilia and lamellar bodies was identified in the larger ductular structures. Upon comparison of the ultrastructure of the organoid in vitro cultures in pigskin with the components and structure of the cultured cells more closely resembled adult lung than the fetal lung used to initiate the cultures. This work was supported by the Council for Tobacco Research Grant 1203M, American Cancer Society Grant RD-65 (for the equipment), and the National Cancer Institute Grant CA 25392.     

Differentiation-arrested rat fetal lung in primary monolayer cell culture

Summary Differentiation-arrested lung cell cultures were developed from fetal rats of various gestational ages. In contrast to previously published observations with cultures in a pO₂ of ∼142 mm Hg, cultures developed in a pO₂ of ∼30 mm Hg, close to the normal fetal arterial pO₂, have improved plating efficiency and a slightly increased growth rate. They did not, however, show gestation-dependent increases of choline incorporation into phospholipids, nor did immature lung cell cultures respond to dexamethasone or triiodothyronine, singly or in combination, by increased choline incorporation into saturated lecithin. The incorporation of choline and glycerol into lipids suggested a mature rate of lipid synthesis by immature cultures at a pO₂ ∼30 mm Hg, despite preservation of an immature morphology. Electron microscope observations revealed no gross differences between immature cultures developed at either pO₂. The cellular mechanisms underlying these differences are unclear but suggest that oxygen tension may significantly influence results obtained with in vitro studies of lipid synthesis by immature lung. This work was supported by grants from the Medical Research Council of Canada, the Ontario Thoracic Society, and the Physicians' Services Incorporated Foundation.     

A novel lysophosphatidylcholine acyl transferase activity is expressed by peroxiredoxin 6

The phospholipase A₂ (PLA₂) activity of peroxiredoxin (Prdx)6 has important physiological roles in the synthesis of lung surfactant and in the repair of peroxidized cell membranes. These functions require the activity of a lysophospholipid acyl transferase as a critical component of the phospholipid remodeling pathway. We now describe a lysophosphatidylcholine acyl transferase (LPCAT) activity for Prdx6 that showed a strong preference for lysophosphatidylcholine (LPC) as the head group and for palmitoyl CoA in the acylation reaction. The calculated kinetic constants for acylation were K_m 18 μM and V_max 30 nmol/min/mg protein; the V_max was increased 25-fold by phosphorylation of the protein while K_m was unchanged. Study of recombinant protein in vitro and in mouse pulmonary microvascular endothelial cells infected with a lentiviral vector construct indicated that amino acid D31 is crucial for LPCAT activity. A linear incorporation of labeled fatty acyl CoA into dipalmitoyl phosphatidylcholine (PC) indicated that LPC generated by Prdx6 PLA₂ activity remained bound to the enzyme for the reacylation reaction. Prdx6 is the first LPCAT enzyme with demonstrated cytoplasmic localization. Thus, Prdx6 is a complete enzyme comprising both PLA₂ and LPCAT activities for the remodeling pathway of PC synthesis or for repair of membrane lipid peroxidation.     

Proliferating cell nuclear antigen (PCNA/cyclin) immunocytochemistry as a labeling index in mouse lung tissues

Summary Proliferating cell nuclear antigen is expressed in cells from late G₁ through the S-phase of the cell cycle. Therefore, antibodies directed against this molecule should provide a probe for labeling immunocytochemically the nuclei of proliferating cells. Herein we demonstrate the feasibility and reliability of this technique by quantifying immunostained pulmonary nuclei. We applied polyclonal and monoclonal antisera to alveolar and bronchiolar pulmonary epithelial cells in various proliferative states in tissue-sections and in vitro. A/J mice had a slightly higher labeling index than C57BL/6J mice, and proliferation in both strains increased dramatically after butylated hydroxytoluene treatment produced compensatory hyperplasia of Type-II pneumocytes. Immunostaining in fetal and neonatal lung samples from mice was higher than in adults. Spontaneous lung adenomas had a higher labeling index than the surrounding normal lung tissue. In addition, new data contained herein demonstrate a strain difference in proliferation of bronchiolar epithelial cells, and quantify the extent to which BHT-induced lung damage increases these proliferative rates. This mammalian nuclear antigen did not cross-react with antiserum to a functionally related bacterial protein, the beta subunit of E. coli DNA polymerase-III holoenzyme.     

Tissue interactions pattern the mesenchyme of the embryonic mouse lung

The mechanisms that control proliferation and differentiation of embryonic lung mesenchyme are largely unknown. We describe an explant system in which exogenous recombinant N-Sonic Hedgehog (N-Shh) protein sustains the survival and proliferation of lung mesenchyme in a dose-dependent manner. In addition, Shh upregulates several mesenchymal cell markers, including its target gene Patched (Ptc), intercellular signaling genes Bone Morphogenetic Protein-4 (Bmp4) and Noggin (Nog), and smooth muscle actin and myosin. In explants exposed to N-Shh in the medium, these products are upregulated throughout the mesenchyme, but not in the periphery. This exclusion zone correlates with the presence of an overlying mesothelial layer, which, as in vivo, expresses Fibroblast Growth Factor 9 (Fgf9). Recombinant Fgf9 protein inhibits the differentiation response of the mesenchyme to N-Shh, but does not affect proliferation. We propose a model for how factors made by two epithelial cell populations, the inner endoderm and the outer jacket of mesothelium, coordinately regulate the proliferation and differentiation of the lung mesoderm.     

Airway branching patterns and cytodifferentiation in cultured fetal hamster lung

Intact fetal hamster lungs were taken for culture on gestational day 12, when only lobar bronchi and primary bronchioles are established and the epithelial cells are undifferentiated. Explants were maintained on Transwell collagen membranes for 2 and 4 days in BGJb medium alone, with 5% FBS, or with the following additives: insulin, transferrin, hydrocortisone, cholera toxin, EGF, and vitamin A. Development of the respiratory tree was affected differently by each medium formulation. BGJb medium with 5% FBS permitted near normal branching of airways and presumptive alveoli. In contrast, BGJb medium alone permitted only limited branching of these structures. BGJb medium with additives permitted branching but markedly altered normal development. The differentiation of endocrine and secretory cells was monitored by immunolabeling for serotonin and calcitonin gene-related peptide, and Clara cell protein, respectively. Ciliated cells were identified by morphology. All medium formulations supported the timely differentiation of endocrine, secretory, and ciliated cells. The ultimate goal of our studies is to characterize factors that influence airway branching and cytodifferentiation during fetal lung development. This study showed that near normal airway branching and cytodifferentiation were supported in vitro by BGJb medium with 5% FBS. Although cytodifferentiation occurred with the two other formulations, airway development was impaired.     

Expression of Carcinoembryonic Cell Adhesion Molecule 6 and Alveolar Epithelial Cell Markers in Lungs of Human Infants with Chronic Lung Disease

The membrane protein carcinoembryonic antigen cell adhesion molecule (CEACAM6) is expressed in the epithelium of various tissues, participating in innate immune defense, cell proliferation and differentiation, with overexpression in gastrointestinal tract, pancreatic and lung tumors. It is developmentally and hormonally regulated in fetal human lung, with an apparent increased production in preterm infants with respiratory failure. To further examine the expression and cell localization of CEACAM6, we performed immunohistochemical and biochemical studies in lung specimens from infants with and without chronic lung disease. CEACAM6 protein and mRNA were increased ~4-fold in lungs from infants with chronic lung disease as compared with controls. By immunostaining, CEACAM6 expression was markedly increased in the lung parenchyma of infants and children with a variety of chronic lung disorders, localizing to hyperplastic epithelial cells with a ~7-fold elevated proliferative rate by PCNA staining. Some of these cells also co-expressed membrane markers of both type I and type II cells, which is not observed in normal postnatal lung, suggesting they are transitional epithelial cells. We suggest that CEACAM6 is both a marker of lung epithelial progenitor cells and a contributor to the proliferative response after injury due to its anti-apoptotic and cell adhesive properties.     

Hoxb-5 control of early airway formation during branching morphogenesis in the developing mouse lung

Hox proteins control structural morphogenesis, pattern formation and cell fate in the developing embryo. To determine if Hoxb-5 participates in patterning of early airway branching during lung morphogenesis, gestational day 11.5 embryonic lung cultures were treated with retinoic acid (RA) to up-regulate and antisense oligonucleotides to down-regulate Hoxb-5 protein expression. RA (10^?6 M) and Hoxb-5 antisense oligonucleotide (20 μM) treatment each significantly decreased branching morphogenesis (P<0.001), but the morphology of branching under these conditions was very different. RA-treated lungs had elongated primary branches but decreased further branching with increased Hoxb-5 immunostaining in subepithelial regions underlying these elongated airways. Western blots confirmed that Hoxb-5 protein was increased by 189±20% (mean±S.E.M., P<0.05) in RA-treated lungs compared to controls. In contrast, lungs treated with Hoxb-5 antisense oligos plus RA had foreshortened primary branches with rudimentary distal clefts resulting in decreased numbers of primary and subsequent branches. Immunohistochemistry confirmed that Hoxb-5 antisense oligos inhibited Hoxb-5 protein expression even in the presence of RA. We conclude that regional and quantitative changes in Hoxb-5 protein expression influence morphogenesis of the first airway divisions from the mainstem bronchi. RA-induced alterations in branching are mediated in part through regulated Hoxb-5 expression.     

Clonal isolation of differentiated rat lung cells

Summary A number of diploid clones have been isolated from an enzymatic dispersion of normal rat lung. Four of these clones are epithelial in morphology, the remainder fibroblastic. On the basis of electron microscopic observations, two of the epithelial clones appear to have originated from type II alveolar pneumonocytes. Supported by funds from the W. Alton Jones Foundation and the American Lung Association.     

Dual secretion locations on type II cells in the avian lung suggest local as well as general roles of surfactant

Transmission electron microscopy indicates that the avian lung surfactant may be secreted in two directions: a) into air passages of parabronchus, atrium and infundibulum where it forms a trilaminar substance serving the respiratory role and b) to the basolateral surface—intercellular space—of type II pneumocytes, contributing to the innate and adoptive immune responses of lung. Basolateral secretion may be confirmed by the presence of trilaminal substance in the intercellular space of type II pneumocytes. Fusion of surfactant containing vesicles with the lateral plasma membrane may result in membrane fusion of neighboring cells and subsequently formation of multinucleated giant cell. The indistinct and in some places discontinuous basal lamina in the parabronchial atrium and infundibulum permits the hydrophilic surfactant proteins to spread into the interstitium of air‐blood capillary region. The hydrophilic surfactant proteins may activate lung interstitial macrophages to migrate into the air passages where they appear as “free avian respiratory macrophages.” Therefore, in the interstitium the hydrophilic surfactant proteins are essential soluble components of innate immunity. J. Morphol. 277:1062–1071, 2016. © 2016 Wiley Periodicals, Inc.     

The formation of histotypic structures from monodisperse fetal rat lung cells cultured on a three-dimensional substrate

Summary Enzymatically dissociated lungs from rat fetuses at 19-days gestation yield single cells which reaggregate to form alveolar-like structures when cultured on gelatin sponge discs. These structures form within 2 days and have been maintained in vitro for as long as 6 weeks. They are composed primarily of type II pneumonocytes as characterized by large, lightly stained nuclei and cytoplasmic inclusion bodies. The lamellar structure of these inclusion bodies has been confirmed by electron microscopy. The dynamic formation of inclusion bodies is suggested by the presence of lamellar bodies in the extra-cellular space and the appearance of new inclusions in the cytoplasm of the type II pneumonocytes. The formation and long-term maintenance of histotypic lung structures in vitro provides a model system for the study of lung development and synthesis of surfactant by type II alveolar pneumonocytes. This work was supported by funds from the American Lung Association, National Heart and Lung Institute (grant HL-17110-01) and the W. Alton Jones Foundation.     

Quantitative characteristics of the Feyrter cells and neuroepithelial bodies of the fetal rabbit lung in normoxia and short term chronic hypoxia

We report here quantitative data on the Feyrter (single) cells (APUD cells) and neuroepithelial bodies (grouped Feyrter cells), in the lungs of rabbit fetuses at 26, 27.5 and 29 days gestational age, during normoxia and short term chronic hypoxia. The apparent number of these cells declines during this period; we suggest that this might be due to increased hypoxemia. Moreover, the number of cells in the lungs of fetuses from short term chronically hypoxic mothers is lower than in the normoxic animals. These findings are in agreement with our previous studies in short term chronically hypoxic neonatal rabbits, and suggest that the increased hypoxemia in the fetus, caused by the induction of hypoxia in the mother, constitutes a stimulus for secretory activity of the Feyrter cells and neuroepithelial bodies (NEBs). This in turn could be part of the mechanism responsible for maintaining the pulmonary vasoconstriction due to hypoxemia. Our results from fetuses of normoxic does provide base line data on the chronological importance of the Feyrter cells and NEBs.     

Mature Surfactant Protein-B Expression by Immunohistochemistry as a Marker for Surfactant System Development in the Fetal Sheep Lung

Evaluation of the number of type II alveolar epithelial cells (AECs) is an important measure of the lung’s ability to produce surfactant. Immunohistochemical staining of these cells in lung tissue commonly uses antibodies directed against mature surfactant protein (SP)-C, which is regarded as a reliable SP marker of type II AECs in rodents. There has been no study demonstrating reliable markers for surfactant system maturation by immunohistochemistry in the fetal sheep lung despite being widely used as a model to study lung development. Here we examine staining of a panel of surfactant pro-proteins (pro–SP-B and pro–SP-C) and mature proteins (SP-B and SP-C) in the fetal sheep lung during late gestation in the saccular/alveolar phase of development (120, 130, and 140 days), with term being 150 ± 3 days, to identify the most reliable marker of surfactant producing cells in this species. Results from this study indicate that during late gestation, use of anti-SP-B antibodies in the sheep lung yields significantly higher cell counts in the alveolar epithelium than SP-C antibodies. Furthermore, this study highlights that mature SP-B antibodies are more reliable markers than SP-C antibodies to evaluate surfactant maturation in the fetal sheep lung by immunohistochemistry.     

Characterization and binding properties of human fetal lung fatty acid-binding proteins

When delipidated Mr>10,000 cut-off human fetal lung cytosol was separated on gel filtration and ion-exchange chromatography on Auto-FPLC system, two fatty acid-binding proteins (FABPs) of pI 6.9 and pI 5.4 were purified to homogeneity. On Western blotting analysis with the anti-human fetal lung pI 6.9 FABP, these two proteins showed immunochemical cross reactivity with each other and with purified hepatic FABPs but not with cardiac or gut FABP. These two FABPs have identical molecular mass of 15.2 kDa, which is slightly higher than that of the hepatic proteins (14.2 kDa). Carbohydrate covalently linked to FABPs, that may substantially add to the molecular mass, was not detected in the purified protein preparations. Amino acid analysis revealed that both the proteins have same amino acid composition each containing one Trp residue that is lacking in hepatic FABP. Different isoforms of lung FABP exhibited different binding ability for their natural ligands. These proteins bind palmitoyl CoA with higher affinity than oleic acid. pI 6.9 FABP can more rapidly and efficiently transfer fatty acid than can pI 5.4 FABP from unilammelar liposomes. Thus these FABPs may play a critical role in fatty acid transport during human fetal lung development.Abbreviations AO anthroyloxy - 12-AS 12-(9-anthroyloxy)stearic acid - FABP fatty acid-binding protein - NBD-PE [N-(4-nitrobenzo-2-oxa-1,3-diazole)phosphatidylethanolamine - Pal-CoA palmitoyl coenzyme A - PITC phenylisothiocyanate - PBS phosphate-buffered saline - PtdCho phosphatidylcholine - SUV small unilamellar vesicle - Tris tris(hydroxymethyl) amino methane     

Optimization of fetal lung organ culture for surfractant biosynthesis

Summary Lung organ culture has been a widely used system for studying differentiation and maturation of alveolar epithelium through various culture conditions. The purpose of this work was to carefully characterize in vitro lung biochemical diffeentiation through isolation of surfactant fraction from tissue and to search for optimal culture conditions. Fetal rat lung was explanted on the 18th gestational day for studying glycogen storage, and on the 20th gestational day for studying surfactant accretion, and cultivated for 48 h. Morphologic differentiation was studies byelectron microscopy tissue explanted on the 17th or 18th gestational days and cultivated for various times. Glycogen storage was greater on fluid medium, although less than occurring in vivo. Cellular integrity and surfactant accumulation were maximal on a semisolid medium containing 0.5% agar. Use of O₂-CO₂ instead of air-CO₂ for gassing the explants slighlty decreased phospholipid accumulation. Among media used in previous lung culture studies, Waymouth MB 752/1 was the only one to allow net glycogen accumulation in vitro. The most favorable media for surfactant phospholipid accretion were Waymouth MB 752/1, Eagle’s minimum essential and its Dulbeccco’s modification, CMRL 1066, and NCTC 109. They allowed a 12- to 14-fold increase of surfactant fraction phospholipids in vitro, which is similar to the increase occurring in vivo during the same peiod. Ham’s F10 and F12 media allowed a six fold increase. RPMI 1640 and medium 199 (M199) allowed only a three fold increase. Phospholipid concentration in nonsurfactant fraction only doubled during culture, and differences between various media were much less marked. DNA concentration changed little during culture. Morphologic differentiation of epithelial cells was advanced as compared with in vivo timing in a medium allowing maximal surfactant accretion (Waymouth MB 752/1) but not in a medium allowing low surfactant increase (RPMI 1640). The possible role of compositional differences between media is discussed.     

Effects of monensin on ATP levels and cell functions in rat liver and lung in vitro

Summary Effects of the proton-alkali cation-exchanging ionophore, monensin, on aspects of cellular metabolism and ionic exchanges have been studied in rat tissues in vitro. Incubation of liver slices at 38°C with 0.1 m monensin induced timedependent vesiculation, initially in the Golgi region, reduction of ATP content and of protein synthesis. At 1 m, monensin also reduced net, active movements of K⁺, Na⁺, Cl^– and water in liver slices and inhibited state 3 respiration in isolated mitochondria. The respiratory inhibitor, amytal, similarly reduced ATP content and protein synthesis at concentrations lower than those inhibiting ion transport in slices. Low concentrations of monensin (0.1–1.0 m) had similar effects on ATP and ion transport in slices of adult lung. By contrast, late-fetal liver and lung were much less sensitive to monensin; in these tissues, glycolysis sustained substantial levels of ATP. Monensin also induced vesiculation of the Golgi apparatus in fetal lung cells. It is concluded that by lowering ATP levels, monensin can markedly alter various metabolic activities in those cells which depend primarily on oxidative phosphorylation for their metabolic energy.