Identification and isolation of mouse type II cells on the basis of intrinsic expression of enhanced green fluorescent protein

The unique morphology and cell-specific expression of surfactant genes have been used to identify and isolate alveolar type II epithelial cells. Because these attributes can change during lung injury, a novel method was developed for detecting and isolating mouse type II cells on the basis of transgenic expression of enhanced green fluorescence protein (EGFP). A line of transgenic mice was created in which EGFP was targeted to type II cells under control of the human surfactant protein (SP)-C promoter. Green fluorescent cells that colocalized by immunostaining with endogenous pro-SP-C were scattered throughout the parenchyma. EGFP was not detected in Clara cell secretory protein-expressing airway epithelial cells or other nonlung tissues. Pro-SP-C immunostaining diminished in lungs exposed to hyperoxia, consistent with decreased expression and secretion of intracellular precursor protein. In contrast, type II cells could still be identified by their intrinsic green fluorescence, because EGFP is not secreted. Type II cells could also be purified from single-cell suspensions of lung homogenates using fluorescence-activated cell sorting. Less than 1% of presorted cells exhibited green fluorescence compared with >95% of the sorted population. As expected for type II cells, ultrastructural analysis revealed that the sorted cells contained numerous lamellar bodies. SP-A, SP-B, and SP-C mRNAs were detected in the sorted population, but T1alpha and CD31 (platelet endothelial cell adhesion molecule) were not, indicating enrichment of type II epithelial cells. This method will be invaluable for detecting and isolating mouse type II cells under a variety of experimental conditions.     

Surface-expressed lamellar body membrane is recycled to lamellar bodies

Monoclonal antibody (MAb) 3C9, an antibody generated to the lamellar body of rat lung type II pneumocytes, specifically labels the luminal face of the lamellar body membrane. To follow the retrieval of lamellar body membrane from the cell surface in these cells, MAb 3C9 was instilled into rat lungs. In vivo, it was endocytosed by type II cells but not by other lung cells. In type II cells that were isolated from rat lungs by elastase digestion and cultured on plastic for 24 h, MAb 3C9 first bound to the cell surface, then was found in endosomes, vesicular structures, and multivesicular bodies and, finally, clustered on the luminal face of lamellar body membranes. The amount internalized reached a plateau after 1.5 h of incubation and was stimulated with the secretagogue ATP. In double-labeling experiments, internalized MAb 3C9 did not completely colocalize with NBD-PC liposomes or the nonspecific endocytic marker TMA-DPH, suggesting that lamellar body membrane is retrieved back to existing lamellar bodies by a pathway different from that of bulk membrane and may be one pathway for surfactant endocytosis. The lamellar body membrane components are retrieved as subunits that are redistributed among the preexisting lamellar bodies in the cell.     

Lamellar body membrane turnover is stimulated by secretagogues

Lamellar bodies are specialized cellular organelles used for storage of surfactant by alveolar type II cells of the lung. We utilized monoclonal antibody (MAb) 3C9, which recognizes an integral lamellar body-limiting membrane protein of 180 kDa, to follow lamellar body trafficking. (125)I-labeled MAb 3C9 bound to the surface of type II cells and was internalized by the cells in a time- and concentration-dependent manner that was inhibitable by excess unlabeled antibody. The internalized antibody remained undegraded over a 4-h time period. The L2 rat lung cell line that does not have lamellar bodies did not bind iodinated 3C9. Exposure of type II cells to the secretagogues ATP, phorbol 12-myristate 13-acetate, and cAMP resulted in a 1.5- to 2-fold enhancement of binding and uptake of MAb 3C9. Calphostin C inhibited phorbol 12-myristate 13-acetate-stimulated phospholipid secretion and also reduced binding and uptake of MAb 3C9 by type II cells. Treatment of type II cells with phenylarsine oxide to obstruct clathrin-mediated endocytosis had no effect on the internalization of MAb 3C9 while markedly blocking the uptake of surfactant protein A and transferrin. An actin-mediated process was important for lamellar body membrane uptake because incubation with cytochalasin D partially inhibited MAb 3C9 incorporation by type II cells. These studies are compatible with enhanced lamellar body membrane turnover associated with surfactant secretion and indicate that this process can be monitored by the trafficking of the antigen reporter MAb 3C9.     

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.     

Colocalization of cytokeratin 18 and villin in type III alveolar cells (brush cells) of the rat lung

Alveoli of the rat lung are lined by three different cell types, the flat type I cells and the cuboidal type II and type III cells. Type III cells differ from type II cells by the presence of an apical tuft of microvilli and the absence of lamellar type secretory granules. In the present study we show by double immunolabelling that type III cells of the rat lung can be identified at the light-and electron microscope level by antibodies against both cytokeratin 18 and the actin-crosslinking protein villin. At the ultrastructural level, microvilli and their rootlets in the apical cytoplasm were labelled by the anti-villin antibodies, whereas a monoclonal antibody against cytokeratin 18 (Ks18.04) labelled bundles of intermediate filaments. In conclusion, antibodies against villin and certain monoclonal antibodies specific for cytokeratin 18 can be used as tools for selective visualization of type III cells in the rat lung.     

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.     

An improved method for the isolation of type II and clara cells from mice

Identifying the causal events and temporal aspects of lung cancer development requires the ability to isolate target and nontarget cells for comparative analyses. Current methodology can either isolate only one pure specific cell population from a lung or multiple cell types at lower purity. Previous studies in our laboratory have identified the alveolar type II cell as the progenitor cell for tumor development in the A/J mouse. The purpose of this study was to develop new protocols for the isolation and culture of type II and Clara cells from the mouse lung. Both type II and Clara cells were obtained in high purity using a sequential centrifugal elutriation protocol. In the first elutriation, cell fractions were collected using a Standard chamber. The type II and Clara cell fractions were then elutriated separately (two different separations) using a Sanderson chamber. The final purity of the type II and Clara cell preparations was 73% and 76%, respectively. Colonies of 4 to 20 Clara cells exhibiting epithelial morphology were evident 1 wk after plating in low serum medium. The growth of type II cells required the addition of bronchioalveolar lavage fluid and acidic fibroblast growth factor to the medium. The isolation of viable mouse type II and Clara cells in high purity should facilitate the identification of cell-specific changes in gene expressions or in enzymatic pathways following in vivo or in vitro exposure to environmental carcinogens.     

Isolation of alveolar type II cells from fetal rat lung by differential adherence in monolayer culture

Type II alveolar epithelial cells were isolated from fetal rat lung by differential adherence in monolayer culture. The preparation had a high degree of purity, as assessed by phase contrast microscopy and immunocytochemistry. Purity, based on reactivity with specific anti-adult lung serum (SAALS), which recognizes only type II cells, was 91% for cells isolated from 19-day fetal lungs and 79% for cells isolated from 21-day fetal lungs. The lower purity of type II cells in cultures derived from 1-day postnatal rat lungs (51% cells reactive with SAALS) is probably due to a lower tendency of the type II cells from neonatal rats to adhere to culture dishes than of type II cells from fetal rats. Type II cells isolated from 21-day fetal lungs contained a higher percentage phosphatidylglycerol and incorporated [Me-3H]choline faster into phosphatidylcholine (PC) than type II cells isolated from 19-day fetal lungs. Moreover, in cell preparations derived from lungs at fetal day 21, a higher percentage of epithelial cells contained lamellar bodies than in preparations derived from lungs at fetal day 19. The observation of these differences in the stage of maturation indicates that these differences, which are typical features of the original material, are not obliterated by differentiation during the culture. Type II cells isolated according to the present procedure were capable of synthesizing PC with a high percentage of the disaturated species. This method for the isolation of fetal type II cells may be a useful tool in studies concerning surfactant synthesis and its regulation in the fetal lung.     

Characterization of the Niemann-Pick C pathway in alveolar type II cells and lamellar bodies of the lung

The Niemann-Pick C (NPC) pathway plays an essential role in the intracellular trafficking of cholesterol by facilitating the release of lipoprotein-derived sterol from the lumen of lysosomes. Regulation of cellular cholesterol homeostasis is of particular importance to lung alveolar type II cells because of the need for production of surfactant with an appropriate lipid composition. We performed microscopic and biochemical analysis of NPC proteins in isolated rat type II pneumocytes. NPC1 and NPC2 proteins were present in the lung, isolated type II cells in culture, and alveolar macrophages. The glycosylated and nonglycosylated forms of NPC1 were prominent in the lung and the lamellar body organelles. Immunocytochemical analysis of isolated type II pneumocytes showed localization of NPC1 to the limiting membrane of lamellar bodies. NPC2 and lysosomal acid lipase were found within these organelles, as confirmed by z-stack analysis of confocal images. All three proteins also were identified in small, lysosome-like vesicles. In the presence of serum, pharmacological inhibition of the NPC pathway with compound U18666A resulted in doubling of the cholesterol content of the type II cells. Filipin staining revealed a striking accumulation of cholesterol within lamellar bodies. Thus the NPC pathway functions to control cholesterol accumulation in lamellar bodies of type II pneumocytes and, thereby, may play a role in the regulation of surfactant cholesterol content.     

Actin in peripheral rat lung: S1 labeling and structural changes induced by cytochalasin

Actin was identified with S1-labeling in type I and II cells, pericytes, and capillary endothelial cells in the peripheral lung of the adult rat. In type II cells abundant actin was present in the apex of cells, in microvilli, and in close association with lamellar bodies near the cell surface. Lamellar bodies secreting their content into the lumen of alveoli were always surrounded by a thick layer of actin-like material. In specimens treated with cytochalasin D the surface of type I, type II and contractile interstitial cells became irregular. In type II cells, lamellar bodies were no longer surrounded by actin-like material and no exocytic profiles of lamellar bodies were encountered. In type II cells actin filaments may be involved in moving lamellar bodies through the cytoplasm and/or in their secretion into alveoli. These observations also suggest that intact actin filaments may be required for maintenance of cell shape in various lung cells and that cells containing actin may be capable of limited contraction.     

Growth characteristics, morphology, and phospholipid composition of human type II pulmonary alveolar cells grown in a collagen-free microenvironment

Human lung epithelial cells have been isolated and maintained in pure culture and characterized during their time in culture. Any residual fibroblasts were removed by selective trypsinization within the first 48 h in culture and the residual epithelial cells from the primary culture grew to confluent density. The epithelial cells at Passage 2 or greater were serially subpassaged when cultures reached ca. 80% confluency. This procedure permitted us to conduct biochemical and structural studies of starting materials and subsequent population doublings. Electron microscope evaluation of both initial monolayers and cell suspensions showed cultures to be composed of a single cell type. These cells had microvilli on their free or apical surface. Subsequent population doubling level 1 up to 5 exhibited the same structures. They contained lamellar inclusions, which are typical of Type II alveolar epithelial cells. Fetal lung (age 18 to 20 wk) cell suspensions processed for electron microscopy before culturing showed cells to be undifferentiated, epithelial-like with small microvilli along cell borders, and with desmosomes at cell junctions. Lamellar inclusions were not observed in these cells. Ultrastructural studies of the cultured epithelial cells demonstrated that the lamellar inclusions had a slightly positive reaction when tested for acid phosphatase. Phospholipid analysis of these lung epithelial cells showed a phospholipid composition consistent with that found in surfactant-containing Type II cells. Cultured epithelial cells stained with phosphine 3-R demonstrated a green fluorescent cytoplasm and nucleus with brightly fluorescent yellow-orange perinuclear particles. The preceding characterization of these cells leads us to conclude that they exhibit structural and biochemical features commensurate with Type II epithelial cells from human lung. Moreover, these selection techniques applied to the isolation of human lung Type II cells from the tissue permit us to study the differentiative function of these cells routinely under conditions of growth in vitro.     

Aberrant lung structure,composition, and function in a murine model of Hermansky-Pudlak syndrome

Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous inherited disease causing hypopigmentation and prolonged bleeding times. An additional serious clinical problem of HPS is the development of lung pathology, which may lead to severe lung disease and premature death. No cure for the disease exists, and previously, no animal model for the HPS lung abnormalities has been reported. A mouse model of HPS, which is homozygously recessive for both the Hps1 (pale ear) and Hps2 (pearl) genes, exhibits striking abnormalities of lung type II cells. Type II cells and lamellar bodies of this mutant are greatly enlarged, and the lamellar bodies are engorged with surfactant. Mutant lungs accumulate excessive autofluorescent pigment. The air spaces of mutant lungs contain age-related elevations of inflammatory cells and foamy macrophages. In vivo measurement of lung hysteresivity demonstrated aberrant lung function in mutant mice. All these features are similar to the lung pathology described in HPS patients. Morphometry of mutant lungs indicates a significant emphysema. These mutant mice provide a model to further investigate the lung pathology and therapy of HPS. We hypothesize that abnormal type II cell lamellar body structure/function may predict future lung pathology in HPS.     

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.     

Partial SP-B deficiency perturbs lung function and causes air space abnormalities

Surfactant protein B (SP-B) is required for function of newborn and adult lung, and partial deficiency has been associated with susceptibility to lung injury. In the present study, transgenic mice were produced in which expression of SP-B in type II epithelial cells was conditionally regulated. Concentrations of SP-B were maintained at 60-70% of that normally present in control. Immunostaining for SP-B demonstrated cellular heterogeneity in expression of the protein. In subsets of type II cells in which SP-B staining was decreased, immunostaining for pro-SP-C was increased and lamellar body ultrastructure was disrupted, consistent with focal SP-B deficiency. Fluorescence-activated cell sorting analyses of freshly isolated type II cells identified a population of cells with low SP-B content and a smaller population with increased SP-B content, confirming nonuniform expression of the SP-B transgene. Focal air space enlargement, without cellular infiltration or inflammation, was observed. Pressure-volume curves indicated that maximal tidal volume was unchanged; however, hysteresis was modestly altered and residual volumes were significantly decreased in the SP-B-deficient mice. Chronic, nonuniform SP-B deficiency perturbed pulmonary function and caused air space enlargement.     

Growth characteristics,morphology, and phospholipid composition of human type II pulmonary alveolar cells grown in a collagen-free microenvironment

Summary Human lung epithelial cells have been isolated and maintained in pure culture and characterized during their time in culture. Any residual fibroblasts were removed by selective trypsinization within the first 48 h in culture and the residual epithelial cells from the primary culture grew to confluent density. The epithelial cells at Passage 2 or greater were serially subpassaged when cultures reached ca. 80% confluency. This procedure permitted us to conduct biochemical and structural studies of starting materials and subsequent population doublings. Electron microscope evaluation of both initial monolayers and cell suspensions showed cultures to be composed of a single cell type. These cells had microvilli on their free or apical surface. Subsequent population doubling level 1 up to 5 exhibited the same structures. They contained lamellar inclusions, which are typical of Type II alveolar epithelial cells. Fetal lung (age 18 to 20 wk) cell suspensions processed for electron microscopy before culturing showed cells to be undifferentiated, epithelial-like with small microvilli along cell borders, and with desmosomes at cell junctions. Lamellar inclusions were not observed in these cells. Ultrastructural studies of the cultured epithelial cells demonstrated that the lamellar inclusions had a slightly positive reaction when tested for acid phosphatase. Phospholipid analysis of these lung epithelial cells showed a phospholipid composition consistent with that found in surfactant-containing Type II cells. Cultured epithelial cells stained with phosphine 3-R demonstrated a green fluorescent cytoplasm and nucleus with brightly fluorescent yellow-orange perinuclear particles. The preceding characterization of these cells leads us to conclude that they exhibit structural and biochemical features commensurate with Type II epithelial cells from human lung. Moreover, these selection techniques applied to the isolation of human lung Type II cells from the tissue permit us to study the differentiative function of these cells routinely under conditions of growth in vitro. This work was supported in part by grants from EPA, R 806638-01 and 131-640-1599A1     

Demonstration of lung type II cell differentiation by polarized light microscopy

Birefringence is shown to be a property of lamellar bodies in living lung type II cells and is used to demonstrate the appearance of differentiated type II cells in cultures of fetal rat lung explanted as early as Day 13. The culture medium was F-12 K without added serum, hormones, or growth factors. This system is considered to be potentially useful in analyzing factors and cell interactions involved in determination and cytodifferentiation of the type II cell from lung bud epithelium.     

大鼠肺泡Ⅱ型上皮细胞的原代分离

目的建立大鼠肺泡Ⅱ型上皮细胞(alveolar epithelial cells typeⅡ,AECⅡ)分离、纯化、原代培养及鉴定的方法。方法用4.2U/ml的弹性蛋白酶通过气管插管注入肺泡内,消化分离AECⅡ。把细胞悬液接种到包被有大鼠IgG的塑料平皿中纯化细胞。用电镜、碱性磷酸酶显色法、改良巴氏染色法、单宁酸染色法、免疫组化染色法鉴定AECⅡ。结果细胞纯度达到90%以上,倒置显微镜下可见细胞呈岛屿状生长。电镜下可见细胞内有大量板层小体,包膜上有绒毛结构。碱性磷酸酶染色法(BCIP/NBT)可见胞浆内有蓝色颗粒。改良巴氏染色法、单宁酸染色法可见胞浆内有黑色颗粒。抗大鼠肺泡表面活性蛋白A(surfactant protein A,SP-A)免疫组化染色呈阳性反应。结论弹性蛋白酶作用温和,不损伤胞膜,分离所得细胞活力好;IgG免疫粘附纯化法操作简单,纯化效率高。电镜、BCIP/NBT、巴氏染色、单宁酸染色及免疫组化染色等鉴定方法稳定可靠,特异性高。     

Calcium-dependent self-association of annexin II: a possible implication in exocytosis.

Alveolar type II cells secrete lung surfactant through exocytosis of lamellar bodies. We previously showed that the annexin II tetramer (Anx IIt) mediates the fusion of lamellar bodies with liposomes. The present study examined the possible involvement of membrane proteins in this process. Pre-treatment of lamellar bodies with trypsin and alpha-chymotrypsin reduced Anx IIt-mediated membrane fusion. With the use of an Anx IIt-conjugated Sepharose column, three Anx IIt-binding proteins with molecular weights of 67,000, 36,000 and 34,000 were isolated froM the Triton X-100 extract of bovine lung tissue membranes. These proteins were identified as annexins VI, II and IV by Western blot. The interaction of Anx IIt with annexins II and IV was confirmed by ligand blot assay. An EGTA-resistant membrane-bound annexin II was present in lung type II cells. Anx IIt preferentially hound to membranous annexin II compared with cytosolic annexin II of type II cells. With the use of immunofluorescence, annexin II was found to translocate from cytoplasm to plasma membranes in type II cells upon stimulation with phorbol 12-myristate 13-acetate. These results suggest that cytosolic annexin II may bind to membranous annexin II and form a protein-protein bridge to bring two membranes together.     

Intermediate filaments in the developing type II cell of fetal monkey lung

Anticytokeratin monoclonal antibody was used to study epithelial cell development in fetal monkey lungs taken from animals of different ages. It is well established that the overall maturity of fetal lung depends greatly on the maturation of type II epithelial cells in the alveolus. In this study, we have correlated the cytokeratin phenotype of mammalian epithelial cells with pneumocyte maturation. We show that differentiation and maturation of the type II cell is related to intermediate filament expression. Twenty-four fetal monkeys (Macaca nemestrina) were delivered by cesarean section at a gestational age of 135-140 days (term = 168 days) and divided into two groups. One group of animals was sacrificed during the first 3 hr of life, and the other group was maintained in incubators for 92-120 hr. Anticytokeratin monoclonal antibody recognizes only alveolar type I and type II epithelial cells. In the first 3 hr of life, the cytokeratin was localized only at the alveolar surface and at the cytoplasmic periphery of the type II cells of these premature animals. However, at the age of 92-120 hr, the epithelia in the lungs reacted more intensely than they did during the first 3 hr. Electron microscopy revealed and confirmed that the type II cells were matured and abundant intermediate filaments appeared in the cytoplasm. The filaments appeared to form either aggregates or parallel filament bundles and few were closely associated with the lamellar bodies. In the immature type II cells at 0-3 hr of life, few intermediate filaments could be localized in the cytoplasm, and no parallel filament bundle was observed, though many appeared in the 92-120 hr lungs. This suggests that the intermediate filaments have a functional significance in the development and maturation of the type II cell. The location and stability of keratin filaments in type II cells may confer the structural strength necessary for cells covering a free surface in the alveoli during lung maturation.     

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.