Modification of surfactant metabolizing cells in rat lung by clofibrate, a hypolipidemic peroxisome proliferating agent. Evidence to suggest that clofibrate influences pulmonary surfactant metabolism

The influence of clofibrate (ethyl-alpha-p-chlorophenoxy-isobutyrate), a hypolipidemic peroxisome proliferating agent, has been tested on the lungs of adult male rats. Drug administration for 7 days caused structural changes in two types of lung cells, both of which are involved in the metabolism of the pulmonary surfactant. By light microscopy the prominent features were the presence of enlarged type II alveolar epithelial cells and foamy intraalveolar macrophages. Compared with controls, type II cells in treated rats apparently contained more numerous surfactant-containing lamellar bodies, as visualized in semi-thin sections of Epon-embedded tissue. This difference was quantified morphometrically by light microscopy: the number of lamellar bodies was estimated as the profile number per individual type II alveolar cell, transsected at its nucleus. Clofibrate administration for 7 days resulted in a significant increase in the number of the lamellar inclusions. In contrast the number of type II alveolar cells per area of lung remained unchanged. There was no evidence of atelectasis or inflammatory infiltration in the drug-treated lungs, a finding confirmed in sections of perfusion-fixed, paraffin-embedded whole lung-lobes. By electron microscopy the lamellar inclusion bodies in the type II alveolar cells in treated rats, apart from being more numerous and sometimes smaller, were morphologically identical to those in controls. The vacuolated alveolar macrophages seen in treated rats also contained various lamellar phospholipid inclusions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arguments against and alternatives for an extracellular surfactant layer in the alveoli of mammalian lung

It is generally believed that lung alveoli contain an extracellular aqueous layer of surfactant material, which is allegedly required to prevent alveolar collapse at small lung volume; the surfactant's major constituent is a fully saturated phospholipid, referred to as dipalmitoyl lecithin or DPL. I herein demonstrate that the surfactant hypothesis of alveolar stability is fundamentally wrong. Although DPL is synthesized inside type II epithelial cells and stored in the typical inclusion bodies therein and lowers surface tension to zero in the surface balance, there is no evidence to the effect that type II cells secrete the DPL surfactant into the aqueous intra-alveolar layer which is shown by electron microscopy in support of the surfactant theory. To the contrary, all the evidence indicates that, when seen, such an extracellular layer is an artifact. This is probably upon the damage glutaraldehyde inflicts onto alveolar structures during fixation of air-inflated lung tissue. Furthermore, several cogent arguments invalidate the belief that an extracellular layer of DPL and serum proteins is present in the alveoli of normal lung. In light of these arguments, a surface tension role of DPL in alveolar stability is excluded. Three hypotheses for an alternative role of DPL in respiration mechanics are proposed. They are: (a) alveolar clearance by viscolytic and surfactant action (bubble or foam formation) on the aqueous systems which are present in lung alveoli during edema and in prenatal life and which would otherwise be impervious to air; (b) homeostasis of blood palmitate in normal lung; (c) modulation of the elasticity of terminal lung tissue by the intact inclusion bodies and parts thereof inside type II cells in normal lung.     

Properties of freshly isolated type II alveolar epithelial cells

The biochemical characteristics of type II alveolar epithelial cells dissociated from adult rabbit lung by instillation of low concentrations of an elastase trypsin mixture are reported. Cells studied immediately (within 4 h) after isolation were found to incorporate the radioactively labelled precursors [U-¹⁴C]glucose, [methyl-³H]choline and [³H]palmitate into cellular phosphatidylcholine at rates 2–10-fold higher than previously reported for cells not subject to short-term cell culture. Secretion of phosphatidylcholine was stimulated by beta-adrenergic agonists. Measurement of specific activities of enzymes of phospholipid biosynthesis in subcellular fractions of isolated lung cells showed a significant enrichment of acyl coenzyme A-lysophosphatidylcholine acyltransferase, an enzyme believed to be involved in pulmonary surfactant phosphatidylcholine remodeling, in the endoplasmic reticulum of type II cells. These observations support the utility of freshly isolated type II cells as a model system for the study of the functions of the alveolar epithelium.     

光,电镜观察小鼠呼吸道合胞病毒性肺炎的免疫病理改变

Ｂａｌｂ／ｃ小鼠经鼻吸入呼吸道合胞病毒（ＲＳＶ）悬液感染成ＲＳＶ肺炎。于感染第５天后连续隔日取肺,光镜与透射电镜检查。感染第５～７天,肺组织病理改变最严重,多数小鼠表现为间质淋巴细胞（ＬＣ）套状浸润,肺泡隔增宽;少数小鼠出现间质内大量ＬＣ浸润与肺泡内大量单个核细胞渗出的两种病理改变。病毒包涵体出现于肺泡上皮细胞内,细胞受感染后发生肿胀、坏死。Ⅰ型细胞核周胞质内有核衣壳复制,表面病毒芽生形成长短不等的丝状体。第９天,肺泡隔增宽与间质ＬＣ浸润逐渐减轻。第１２天,病毒包涵体明显减少。     

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro : A novel system for type II pneumocyte culture

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 micron thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane. On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.     

Density and substrata are important in lung type II cell transdifferentiation in vitro.

Morphological techniques and metabolic cell marker assays were used to study the transdifferentiation of pulmonary type II epithelial cells to type I-like cells in vitro. In the lung this process is important during remodelling and alveolar repair. Type II cell phenotype was best maintained over eight days when densely packed cells were plated out on a commercially available extracellular matrix. Such cells retained type II cell characteristics (lamellar bodies, high activities of gamma glutamyl transpeptidase and alkaline phosphatase) but expressed low levels of rT1(40) a surface protein marker of type I cells. In contrast, low density cultures, irrespective of substratum, exhibited rapid cell spreading, loss of lamellar bodies, loss of type II cell enzyme markers and expressed high levels or rT1(40). Conditions have been described whereby the same isolate of type II cells can be used to produce differential epithelial phenotypes and use can be made of this for further characterisation or to investigate the effect of toxins on different lung cell types in vitro.     

Immunocytochemical distribution of E-cadherin in normal and injured lung tissue of the rat

Affinity purified rabbit anti-mouse E-cadherin antibodies, reacting with diverse rat epithelia, were used to characterize epithelial changes in a radiation-induced fibrosis model of rat lung by immunoblotting techniques, immunoperoxidase and immunofluorescence microscopy. Immunostaining of normal rat lung tissues revealed a predominant staining of type II pneumocytes. Immunoelectron microscopy confirmed the immunohistochemical data of normal lung tissue obtained at the light microscopic level. In severely injured rat lung, we found enhanced immunoreactivity for E-cadherin at the surface of type I alveolar epithelial cells. The results suggest that E-cadherin is an adhesion molecule that is modulated after pathological alteration of the alveolar epithelium and that the antiserum may be useful for the characterization of normal and diseased rat epithelia.     

Fine structural localization of alkaline phosphatase in the granular pneumonocytes of hamster lung.

The fine structural localization of nonspecific alkaline phosphatase was studied in the granular pneumonocytes (type II alveolar epithelial cells) of hamster lung by incubating sections of glutaraldehyde-fixed tissues in a medium containing lead ions and sodium beta-glycerophosphate or alpha-naphthyl acid phosphate. The specificity of the reaction was tested by exposing the sections to inhibitors of alkaline phosphatase. The results showed that alkaline phosphatase activity was present in the inclusion bodies of granular pneumonocytes. The enzyme reaction was strong in the membrane lining the inclusion bodies and a weaker reaction was generally detectable in the inclusion contents. Although only a proportion of the inclusion bodies showed enzyme activity, there was no obvious correlation between the reactivity of the inclusions and their intracellular position or size. The other organelles were unreactive. The finding of alkaline phosphatase activity within the inclusion bodies of granular pneumonocytes is an enigma as these organelles are generally considered to be lyosomes.     

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

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.     

Uptake and intracellular transport of cationic ferritin in the bronchiolar and alveolar epithelia of the rat

Summary Cationic ferritin was used as a marker to reveal the processes of endocytosis and intracellular transport in bronchiolar and alveolar epithelia. The marker was injected into the lung via the trachea, and ultrastructural observation of the distribution of ferritin particles in bronchiolar and alveolar epithelial cells was carried out at intervals of 5, 15, 30 and 60 min after the injection. The luminal surface of the airway and the alveolar epithelium showed diffuse labeling with cationic ferritin. In general, ferritin particles were observed in vesicles and vacuoles of the bronchiolar and alveolar epithelial cells within 5 min of injection; they appeared in multivesicular bodies within 15 min. Multivesicular bodies and secondary lysosomes containing ferritin particles, some of which showed a positive reaction for acid phosphatase, were seen in the basal cytoplasm within 30 min; ferritin particles appeared in the basal lamina below the Clara cells, ciliated cells and type 2 alveolar cells within 30 min. Ferritin particles were seen in ovoid granules of some Clara cells and in lamellar inclusion bodies of many type 2 alveolar cells. Brush cells and type 1 alveolar cells took up only a small quantity of ferritin particles.     

Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development

Apoptosis plays a central role in the cellular remodeling of the developing lung. We determined the spatiotemporal patterns of the cell death regulators Fas and Fas ligand (FasL) during rabbit lung development and correlated their expression with pulmonary and type II cell apoptosis. Fetal rabbit lungs (25-31 days gestation) were assayed for apoptotic activity by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and DNA size analysis. Fas and FasL expression were analyzed by RT-PCR, immunoblot, and immunohistochemistry. Type II cell apoptosis increased significantly on gestational day 28; the type II cell apoptotic index increased from 0.54 +/- 0.34% on gestational day 27 to 3.34 +/- 1.24% on day 28, P < 0.01 (ANOVA). This corresponded with the transition from the canalicular to the terminal sac stage of development. The day 28 rise in epithelial apoptosis was synchronous with a robust if transient 20-fold increase in FasL mRNA and a threefold increase in FasL protein levels. In contrast, Fas mRNA levels remained constant, suggestive of constitutive expression. Fas and FasL proteins were immunolocalized to alveolar type II cells and bronchiolar Clara cells. The correlation of this highly specific pattern of FasL expression with alveolar epithelial apoptosis and remodeling implicates the Fas/FasL system as a potentially important regulatory pathway in the control of postcanalicular alveolar cytodifferentiation.     

Monoclonal antibodies specific to apical surfaces of rat alveolar type I cells bind to surfaces of cultured, but not freshly isolated, type II cells

The alveolar surface of the lung is lined by two classes of epithelial cells, type I and type II cells. Type I cells cover more than 97% of the alveolar surface. Although this cell type is felt to be essential for normal gas exchange, neither unique identifying characteristics nor functions have been described for the type I cell. We have produced monoclonal antibodies to (a) component(s) of molecular weight 40,000 and 42,000 of the apical surface of rat alveolar type I cells. The antibodies are specific to the lung in Western blots of organ homogenates. In immunocytochemical studies of frozen lung at the level of both light and electron microscopy, the monoclonal antibodies appear to react specifically with the apical plasma membrane of type I cells. Airway, vascular, interstitial cells, type II cells and macrophages are not immunoreactive. Western blots of isolated type I cells (approx. 70% pure) also demonstrate immunoreactivity at molecular weights of 40,000 and 42,000. When the lung is injured, type I cells may be damaged and sloughed from the alveolar surface. Alveolar repair occurs when the second type of alveolar cell, the type II cell, divides. Cell progeny may retain type II cell morphology or may differentiate into type I cells. Western blots of freshly isolated type II cells (approx. 85% pure) do not display immunoreactivity with our monoclonal antibodies. However, type II cells maintained in culture acquire immunoreactivity to monoclonal antibodies, demonstrating that type II cells in vitro have the capacity to develop a characteristic associated with type I cells in situ. The availability of markers for a specific membrane component of type I cells should facilitate the study of many questions on alveolar functions, development and response to injury.     

Isolation of viable type II alveolar epithelial cells by flow cytometry

We developed a new method for isolating viable type II cells from fractionated and unfractionated lung cell suspensions by flow cytometry using acridine orange (AO). Fischer-344 rat lungs were dispersed into single-cell suspensions by a technique that yields a high number of cells (4-5 X 10(8) cells/lung, congruent to 85% viable), congruent to 11% of which are type II cells. Elutriated fractions from the lung cell preparation and parent, unfractionated cell suspensions were incubated with 1.0-0.02 micrograms/ml AO and analyzed by flow cytometry. Parameters analyzed included axial light loss (ALL) and red fluorescence (RF). Based on their unique RF, attributable to AO staining of type II cell lamellar bodies, and their ALL characteristics, type II pneumocytes were sorted from elutriated fractions to greater than 95% purity. Using the same approach, type II pneumocytes were sorted from unfractionated lung cell suspensions at greater than or equal to 85% purity. The viabilities of the type II alveolar epithelial cells isolated by this method range from 85% to 95%, and the ultrastructural features of the sorted cells were unaltered by AO labeling or sorting.     

Phospholipid biosynthesis and secretion by a cell line (A549) which resembles type II aleveolar epithelial cells.

The A549 cell line is a continuous cell line derived from a human adenocarcinoma of the lung. At low cell population density the cells contain relatively few lamellar bodies, but in mature cells in very confluent cultures lamellar bodies are abundant. The lamellar bodies from these cells are enriched for phosphatidylcholine and disaturated phosphatidylcholine. In mature cells, 45% of newly synthesized phosphatidylcholine is disaturated. Stimulation with the calcium ionophore A23187 produces exocytosis of phosphatidylcholine (46% disaturated). The A549 cell synthesizes, stores in lamellar bodies, and secretes phosphatidylcholine, and thus has many important biological properties of the alveolar epithelial type II cell.     

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.     

Laminin-311 (Laminin-6) fiber assembly by type I-like alveolar cells.

Two epithelial cell types cover the alveolar surface of the lung. Type II alveolar epithelial cells produce surfactant and, during development or following wounding, give rise to type I cells that are involved in gas exchange and alveolar fluid homeostasis. In culture, freshly isolated alveolar type II cells assume a more squamous (type I-like) appearance within 4 days after plating. They assemble numerous focal adhesions that associate with the actin cytoskeleton at the cell margins. These alveolar epithelial cells lose expression of type II cell markers including SP-C and after 4 days in culture express the type I cell marker T1alpha. Those cells that express T1alpha also deposit fibers of laminin-311 in their matrix. The latter appears to be related to their development of a type I phenotype because freshly isolated, primary type I cells also assemble laminin-311-rich fibers in vitro. A beta1 integrin antibody antagonist inhibits the assembly of laminin-311 matrix fibers. Moreover, the formation of laminin fibers is dependent on the activity of the small GTPases and is perturbed by ML-7, a myosin light chain kinase inhibitor. In summary, our data indicate that assembly of laminin-311 fibers by lung epithelial cells is integrin and actin cytoskeleton dependent, and that these fibers are characteristic of type I alveolar cells.     

Role of lamellar inclusions in surfactant production: studies on phospholipid composition and biosynthesis in rat and rabbit lung subcellular fractions.

Lamellar inclusion bodies in the type II alveolar epithelial cell are believed to be involved in pulmonary surfactant production. However, it is not clear whether their role is that of synthesis, storage, or secretion. We have examined the phospholipid composition and fatty acid content of rabbit lung wash, lamellar bodies, mitochondria, and microsomes. Phosphatidylcholine and phosphatidylglycerol, the surface-active components of pulmonary surfactant, accounted for over 80% of the total phospholipid in lung wash and lamellar bodies but for only about 50% in mitochondria and microsomes. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin accounted for over 40% of the total in mitochondria and microsomes but for only 6% in lung wash and 15% in lamellar bodies. The fatty acid composition of lamellar body phosphatidylcholine was similar to that of lung wash, but different from that of mitochondria and microsomes, in containing palmitic acid as a major component with little stearic acid and few fatty acids of chain length greater than 18 carbon atoms. The biosynthesis of phosphatidylcholine and phosphatidylglycerol was examined in the mitochondrial, microsomal, and lamellar body fractions from rat lung. Cholinephosphotransferase was largely microsomal. The activity in the lamellar body fraction could be attributed to microsomal contamination. The activity of glycerolphosphate phosphatidyltransferase, however, was high in the lamellar body fraction, although it was highest in the mitochondria and was also active in the microsomes. These data suggest that the lamellar bodies are involved both in the storage of the lipid components of surfactant and in the synthesis of at least one of those components, phosphatidylglycerol.     

A quantitative study of the development of type II pneumocytes in fetal lung

Cell populations dissociated from fetal rabbit lungs were analyzed by laser flow cytometry for the presence of type II pneumocytes. These cells are distinguishable by the staining of their lamellar bodies with the fluorescent lipophilic dye, phosphine-3R and by their intensity of low-angle light scatter. Lung cells were obtained by enzymatic dissociation from fetal rabbits at gestational ages of 24 d, 27 d, and from 2-d newborn rabbits. Flow cytometric analysis was sufficiently sensitive to discriminate between fetuses. Quantitative analysis of type II pneumocytes showed that newborn rabbits had a distinct cell subpopulation in a region of low-angle light scatter and phosphine-3R fluorescence intensity similar to that previously reported on type II cells from adult rabbits. By contrast, 24-d gestation rabbits had a negligible type II cell subpopulation. Fetuses of 27 and 30 d gestation showed a slow but progressive increase in the numbers of cells in the type II region. Mathematical analyses of light scatter and fluorescence intensity distributions were used to define statistically significant (P less than .05) boundaries that characterize the development of the type II cell subpopulation in fetal rabbit lung. The methods employed offer new possibilities for quantification of developing lung cell subpopulations of particular interest to the problem of respiratory distress syndrome in human neonates.     

Time-dependent effects of maternal continuous propranolol on fetal lung development in rats

Previous studies have demonstrated a role for the beta-adrenergic system in the maturation of the fetal alveolar epithelium. Chronic blockade of beta-adrenergic binding sites has been shown to adversely effect physiologic and biochemical indices of fetal lung maturation. In the present study timed-pregnant female Sprague-Dawley rats were treated with a continuous 0.5 mg/hr dose of propranolol HCl, or saline, via an osmotic pump. The treatment periods were days 18-21, or 20-23 of gestation. Fetal body weights were obtained, and the morphology of the fetal lungs studied by light and electron microscopy. Cytoplasmic volume densities of lamellar inclusion bodies and glycogen within developing type II alveolar epithelial cells were also determined. In addition, total phospholipids (as phosphorus) and glycogen content were determined biochemically. The fetuses from females treated from day 20-23 demonstrated no differences between saline-treated and propranolol-treated groups, in either fetal weight or the morphologic appearance of the developing lung. In contrast, the fetuses from mothers treated from day 18-21 with propranolol were significantly smaller, and their lungs appeared less mature than saline-treated counterparts. The glycogen content of developing type II alveolar epithelial cells was significantly more abundant (as judged by stereologic and biochemical analyses) in the propranolol-treated fetuses. In addition, total phospholipids were decreased in the propranolol-treated 21-day fetuses. The results of the present study suggest that the development of the alveolar epithelium is sensitive to continuous beta-adrenergic blockade by propranolol during a critical time late in gestation.