Ca2+ induced surfactant secretion in alveolar type II cultures isolated from the H-2Kb-tsA58 transgenic mouse.

BACKGROUND/AIMS: There is a need for the development of transgenic mice to elucidate molecular mechanisms in surfactant secretion. However at present very little is known about the regulation of surfactant exocytosis in murine alveolar type II (AT II) cells. METHODS: We brought AT II cells isolated from the Immorto mouse into culture at 33 degrees C, in the presence of interferon, to generate immortal mouse AT II cells (iMAT II). Surfactant secretion was measured using real-time fluorescence imaging. RESULTS: iMAT II cells stained with lysotracker green (LTG), a dye specific for lamellar body related vesicles in rat AT II cells. Expression of densely layered structures, characteristic of LBs, was confirmed by transmission electron microscopy. Flash photolysis of caged Ca2+, which specifically elevates intracellular Ca2+ concentration ([Ca2+]i), resulted in LB fusion to the plasma membrane, as analysed using the lipid staining dye FM 1-43. Purinergic stimulation with ATP (10 microM), also resulted in a rise in [Ca2+]i (measured by fura-2), which was followed by LB fusion. CONCLUSIONS: iMATII cells maintain the expression of LBs over several passages. Surfactant secretion in these cells is regulated by [Ca2+]i, and exhibits similar characteristics to that of rat AT II cells. These cells will be beneficial in studying the impact of genetic modifications on regulated surfactant secretion.     

The Role of Autophagy in Lamellar Body Formation and Surfactant Production in Type 2 Alveolar Epithelial Cells

The lamellar body (LB), a concentric structure loaded with surfactant proteins and phospholipids, is an organelle specific to type 2 alveolar epithelial cells (AT2). However, the origin of LBs has not been fully elucidated. We have previously reported that autophagy regulates Weibel-Palade bodies (WPBs) formation, and here we demonstrated that autophagy is involved in LB maturation, another lysosome-related organelle. We found that during development, LBs were transformed from autophagic vacuoles containing cytoplasmic contents such as glycogen. Fusion between LBs and autophagosomes was observed in wild-type neonate mice. Moreover, the markers of autophagic activity, microtubule-associated protein 1 light chain 3B (LC3B), largely co-localized on the limiting membrane of the LB. Both autophagy-related gene 7 (Atg7) global knockout and conditional Atg7 knockdown in AT2 cells in mice led to defects in LB maturation and surfactant protein B production. Additionally, changes in autophagic activity altered LB formation and surfactant protein B production. Taken together, these results suggest that autophagy plays a critical role in the regulation of LB formation during development and the maintenance of LB homeostasis during adulthood.     

Physiological variables affecting surface film formation by native lamellar body-like pulmonary surfactant particles

Pulmonary surfactant (PS) is a surface active complex of lipids and proteins that prevents the alveolar structures from collapsing and reduces the work of breathing by lowering the surface tension at the alveolar air–liquid interface (A_LI). Surfactant is synthesized by the alveolar type II (AT II) cells, and it is stored in specialized organelles, the lamellar bodies (LBs), as tightly packed lipid bilayers. Upon secretion into the alveolar lining fluid, a large fraction of these particles retain most of their packed lamellar structure, giving rise to the term lamellar body like-particles (LBPs). Due to their stability in aqueous media, freshly secreted LBPs can be harvested from AT II cell preparations. However, when LBPs get in contact with an A_LI, they quickly and spontaneously adsorb into a highly organized surface film. In the present study we investigated the adsorptive capacity of LBPs at an A_LI under relevant physiological parameters that characterize the alveolar environment in homeostatic or in pathological conditions. Adsorption of LBPs at an A_LI is highly sensitive to pH, temperature and albumin concentration and to a relatively lesser extent to changes in osmolarity or Ca^2 + concentrations in the physiological range. Furthermore, proteolysis of LBPs significantly decreases their adsorptive capacity confirming the important role of surfactant proteins in the formation of surface active films.     

Pneumocytes Assemble Lung Surfactant as Highly Packed/Dehydrated States with Optimal Surface Activity

Pulmonary surfactant (PS) is an essential complex of lipids and specific proteins synthesized in alveolar type II pneumocytes, where it is assembled and stored intracellularly as multilayered organelles known as lamellar bodies (LBs). Once secreted upon physiological stimulation, LBs maintain a densely packed structure in the form of lamellar body-like particles (LBPs), which are efficiently transferred into the alveolar air-water interface, lowering surface tension to avoid lung collapse at end-expiration. In this work, the structural organization of membranes in LBs and LBPs freshly secreted by primary cultures of rat ATII cells has been compared with that of native lung surfactant membranes isolated from porcine bronchoalveolar lavage. PS assembles in LBs as crystalline-like highly ordered structures, with a highly packed and dehydrated state, which is maintained at supraphysiological temperatures. This relatively ordered/packed state is retained in secreted LBPs. The micro- and nanostructural examination of LBPs suggests the existence of high levels of structural complexity in comparison with the material purified from lavages, which may contain partially inactivated or spent structures. Additionally, freshly secreted surfactant LBPs exhibit superior activity when generating interfacial films and a higher intrinsic resistance to inactivating agents, such as serum proteins or meconium. We propose that LBs are assembled as an energy-activated structure competent to form very efficient interfacial films, and that the organization of lipids and proteins and the properties displayed by the films formed by LBPs are likely similar to those established at the alveolar interface and represent the actual functional structure of surfactant as it sustains respiration.     

Effect of surfactant protein A on granular pneumocyte surfactant secretion in vitro

Surfactant secretion by lung type II cells occurs when lamellar bodies (LBs) fuse with the plasma membrane and surfactant is released into the alveolar lumen. Surfactant protein A (SP-A) blocks secretagogue-stimulated phospholipid (PL) release, even in the presence of surfactant-like lipid. The mechanism of action is not clear. We have shown previously that an antibody to LB membranes (MAb 3C9) can be used to measure LB membrane trafficking. Although the ATP-stimulated secretion of PL was blocked by SP-A, the cell association of iodinated MAb 3C9 was not altered, indicating no effect on LB movement. FM1-43 is a hydrophobic dye used to monitor the formation of fusion pores. After secretagogue exposure, the threefold enhancement of the number of FM1-43 fluorescent LBs (per 100 cells) was not altered by the presence of SP-A. Finally, there was no evidence of a large PL pool retained on the cell surface through interaction with SP-A. Thus SP-A exposure does not affect these stages in the surfactant secretory pathway of type II cells.     

The conception of fusion pores as rate-limiting structures for surfactant secretion

It is well established that the release of surfactant phospholipids into the alveolar lumen proceeds by the exocytosis of lamellar bodies (LBs), the characteristic storage organelles of surfactant in alveolar type II cells. Consequently, the fusion of LBs with the plasma membrane and the formation of exocytotic fusion pores are key steps linking cellular synthesis of surfactant with its delivery into the alveolar space. Considering the unique structural organization of LBs or LB-associated aggregates which are found in lung lavages, and the roughly 1-microm-sized dimensions of these particles, we speculated whether the fusion pore diameter of fused LBs might be a specific hindrance for surfactant secretion, delaying or even impeding full release. In this mini-review, we have compiled published data shedding light on a possibly important role of fusion pores during the secretory process in alveolar type II cells.     

Structure of pulmonary surfactant membranes and films: The role of proteins and lipid–protein interactions

The pulmonary surfactant system constitutes an excellent example of how dynamic membrane polymorphism governs some biological functions through specific lipid–lipid, lipid–protein and protein–protein interactions assembled in highly differentiated cells. Lipid–protein surfactant complexes are assembled in alveolar pneumocytes in the form of tightly packed membranes, which are stored in specialized organelles called lamellar bodies (LB). Upon secretion of LBs, surfactant develops a membrane-based network that covers rapidly and efficiently the whole respiratory surface. This membrane-based surface layer is organized in a way that permits efficient gas exchange while optimizing the encounter of many different molecules and cells at the epithelial surface, in a cross-talk essential to keep the whole organism safe from potential pathogenic invaders.The present review summarizes what is known about the structure of the different forms of surfactant, with special emphasis on current models of the molecular organization of surfactant membrane components. The architecture and the behaviour shown by surfactant structures in vivo are interpreted, to some extent, from the interactions and the properties exhibited by different surfactant models as they have been studied in vitro, particularly addressing the possible role played by surfactant proteins. However, the limitations in structural complexity and biophysical performance of surfactant preparations reconstituted in vitro will be highlighted in particular, to allow for a proper evaluation of the significance of the experimental model systems used so far to study structure–function relationships in surfactant, and to define future challenges in the design and production of more efficient clinical surfactants.     

Ca(2+)](i) oscillations regulate type II cell exocytosis in the pulmonary alveolus

Pulmonary surfactant, a critical determinant of alveolar stability, is secreted by alveolar type II cells by exocytosis of lamellar bodies (LBs). To determine exocytosis mechanisms in situ, we imaged single alveolar cells from the isolated blood-perfused rat lung. We quantified cytosolic Ca(2+) concentration ([Ca(2+)](i)) by the fura 2 method and LB exocytosis as the loss of cell fluorescence of LysoTracker Green. We identified alveolar cell type by immunofluorescence in situ. A 15-s lung expansion induced synchronous [Ca(2+)](i) oscillations in all alveolar cells and LB exocytosis in type II cells. The exocytosis rate correlated with the frequency of [Ca(2+)](i) oscillations. Fluorescence of the lipidophilic dye FM1-43 indicated multiple exocytosis sites per cell. Intracellular Ca(2+) chelation and gap junctional inhibition each blocked [Ca(2+)](i) oscillations and exocytosis in type II cells. We demonstrated the feasibility of real-time quantifications in alveolar cells in situ. We conclude that in lung expansion, type II cell exocytosis is modulated by the frequency of intercellularly communicated [Ca(2+)](i) oscillations that are likely to be initiated in type I cells. Thus during lung inflation, type I cells may act as alveolar mechanotransducers that regulate type II cell secretion.     

Surfactant lipid synthesis and lamellar body formation in glycogen-laden type II cells

Pulmonary surfactant is a lipoprotein complex that functions to reduce surface tension at the air liquid interface in the alveolus of the mature lung. In late gestation glycogen-laden type II cells shift their metabolic program toward the synthesis of surfactant, of which phosphatidylcholine (PC) is by far the most abundant lipid. To investigate the cellular site of surfactant PC synthesis in these cells we determined the subcellular localization of two key enzymes for PC biosynthesis, fatty acid synthase (FAS) and CTP:phosphocholine cytidylyltransferase-alpha (CCT-alpha), and compared their localization with that of surfactant storage organelles, the lamellar bodies (LBs), and surfactant proteins (SPs) in fetal mouse lung. Ultrastructural analysis showed that immature and mature LBs were present within the glycogen pools of fetal type II cells. Multivesicular bodies were noted only in the cytoplasm. Immunogold electron microscopy (EM) revealed that the glycogen pools were the prominent cellular sites for FAS and CCT-alpha. Energy-filtering EM demonstrated that CCT-alpha bound to phosphorus-rich (phospholipid) structures in the glycogen. SP-B and SP-C, but not SP-A, localized predominantly to the glycogen stores. Collectively, these data suggest that the glycogen stores in fetal type II cells are a cellular site for surfactant PC synthesis and LB formation/maturation consistent with the idea that the glycogen is a unique substrate for surfactant lipids.     

Characterization of VAMP‐2 in the lung: implication in lung surfactant secretion

Lung surfactant is crucial for reducing the surface tension of alveolar space, thus preventing the alveoli from collapse. Lung surfactant is synthesized in alveolar epithelial type II cells and stored in lamellar bodies before being released via the fusion of lamellar bodies with the apical plasma membrane. SNAREs (soluble N‐ethylmaleimide‐sensitive fusion protein‐attachment protein receptors) play an essential role in membrane fusion. We have previously demonstrated the requirement of t‐SNARE (target SNARE) proteins, syntaxin 2 and SNAP‐23 (N‐ethylmaleimide‐sensitive factor‐attachment protein 23), in regulated surfactant secretion. Here, we characterized the distribution of VAMPs (vesicle‐associated membrane proteins) in rat lung and alveolar type II cells. VAMP‐2, ?3 and ?8 are shown in type II cells at both mRNA and protein levels. VAMP‐2 and ?8 were enriched in LB (lamellar body) fraction. Immunochemistry studies indicated that VAMP‐2 was co‐localized with the LB marker protein, LB‐180. Functionally, the cytoplasmic domain of VAMP‐2, but not VAMP‐8 inhibited surfactant secretion in type II cells. We suggest that VAMP‐2 is the v‐SNARE (vesicle SNARE) involved in regulated surfactant secretion.     

Spatio-temporal aspects, pathways and actions of Ca(2+) in surfactant secreting pulmonary alveolar type II pneumocytes

The type II cell of the pulmonary alveolus is a polarized epithelial cell that secretes surfactant into the alveolar space by regulated exocytosis of lamellar bodies (LBs). This process consists of multiple sequential steps and is correlated to elevations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) required for extended periods of secretory activity. Both chemical (purinergic) and mechanical (cell stretch or exposure to an air-liquid interface) stimuli give rise to complex Ca(2+) signals (such as Ca(2+) peaks, spikes and plateaus) that differ in shape, origin and spatio-temporal behavior. This review summarizes current knowledge about Ca(2+) channels, including vesicular P2X4 purinoceptors, in type II cells and associated signaling cascades within the alveolar microenvironment, and relates stimulus-dependent activation of these pathways with distinct stages of surfactant secretion, including pre- and postfusion stages of LB exocytosis.     

Comparative proteomic analysis of lung lamellar bodies and lysosome-related organelles

Pulmonary surfactant is a complex mixture of lipids and proteins that is essential for postnatal function. Surfactant is synthesized in alveolar type II cells and stored as multi-bilayer membranes in a specialized secretory lysosome-related organelle (LRO), known as the lamellar body (LB), prior to secretion into the alveolar airspaces. Few LB proteins have been identified and the mechanisms regulating formation and trafficking of this organelle are poorly understood. Lamellar bodies were isolated from rat lungs, separated into limiting membrane and core populations, fractionated by SDS-PAGE and proteins identified by nanoLC-tandem mass spectrometry. In total 562 proteins were identified, significantly extending a previous study that identified 44 proteins in rat lung LB. The lung LB proteome reflects the dynamic interaction of this organelle with the biosynthetic, secretory and endocytic pathways of the type II epithelial cell. Comparison with other LRO proteomes indicated that 60% of LB proteins were detected in one or more of 8 other proteomes, confirming classification of the LB as a LRO. Remarkably the LB shared 37.8% of its proteins with the melanosome but only 9.9% with lamellar bodies from the skin. Of the 229 proteins not detected in other LRO proteomes, a subset of 34 proteins was enriched in lung relative to other tissues. Proteins with lipid-related functions comprised a significant proportion of the LB unique subset, consistent with the major function of this organelle in the organization, storage and secretion of surfactant lipid. The lung LB proteome will facilitate identification of molecular pathways involved in LB biogenesis, surfactant homeostasis and disease pathogenesis.     

Structure of pulmonary surfactant membranes and films: the role of proteins and lipid-protein interactions

The pulmonary surfactant system constitutes an excellent example of how dynamic membrane polymorphism governs some biological functions through specific lipid-lipid, lipid-protein and protein-protein interactions assembled in highly differentiated cells. Lipid-protein surfactant complexes are assembled in alveolar pneumocytes in the form of tightly packed membranes, which are stored in specialized organelles called lamellar bodies (LB). Upon secretion of LBs, surfactant develops a membrane-based network that covers rapidly and efficiently the whole respiratory surface. This membrane-based surface layer is organized in a way that permits efficient gas exchange while optimizing the encounter of many different molecules and cells at the epithelial surface, in a cross-talk essential to keep the whole organism safe from potential pathogenic invaders. The present review summarizes what is known about the structure of the different forms of surfactant, with special emphasis on current models of the molecular organization of surfactant membrane components. The architecture and the behaviour shown by surfactant structures in vivo are interpreted, to some extent, from the interactions and the properties exhibited by different surfactant models as they have been studied in vitro, particularly addressing the possible role played by surfactant proteins. However, the limitations in structural complexity and biophysical performance of surfactant preparations reconstituted in vitro will be highlighted in particular, to allow for a proper evaluation of the significance of the experimental model systems used so far to study structure-function relationships in surfactant, and to define future challenges in the design and production of more efficient clinical surfactants.     

Dynamic and regulated association of caveolin with lipid bodies: modulation of lipid body motility and function by a dominant negative mutant

Caveolins are a crucial component of caveolae but have also been localized to the Golgi complex, and, under some experimental conditions, to lipid bodies (LBs). The physiological relevance and dynamics of LB association remain unclear. We now show that endogenous caveolin-1 and caveolin-2 redistribute to LBs in lipid loaded A431 and FRT cells. Association with LBs is regulated and reversible; removal of fatty acids causes caveolin to rapidly leave the lipid body. We also show by subcellular fractionation, light and electron microscopy that during the first hours of liver regeneration, caveolins show a dramatic redistribution from the cell surface to the newly formed LBs. At later stages of the regeneration process (when LBs are still abundant), the levels of caveolins in LBs decrease dramatically. As a model system to study association of caveolins with LBs we have used brefeldin A (BFA). BFA causes rapid redistribution of endogenous caveolins to LBs and this association was reversed upon BFA washout. Finally, we have used a dominant negative LB-associated caveolin mutant (cav^DGV) to study LB formation and to examine its effect on LB function. We now show that the cav^DGV mutant inhibits microtubule-dependent LB motility and blocks the reversal of lipid accumulation in LBs.     

Lipid bodies in coral-dinoflagellate endosymbiosis: proteomic and ultrastructural studies

Gastrodermal lipid bodies (LBs) are organelles involved in the regulation of the mutualistic endosymbiosis between reef‐building corals and their dinoflagellate endosymbionts (genus Symbiodinium). As their molecular composition remains poorly defined, we herein describe the first gastrodermal LB proteome and examine in situ morphology of LBs in order to provide insight into their structure and function. After tissue separation of the tentacles of the stony coral Euphyllia glabrescens, buoyant LBs of the gastroderm encompassing a variety of sizes (0.5–4 μm in diameter) were isolated after two cycles of subcellular fractionation via stepwise sucrose gradient ultracentrifugation and detergent washing. The purity of the isolated LBs was demonstrated by their high degree of lipid enrichment and as well as the absence of contaminating proteins of the host cell and Symbiodinium. LB‐associated proteins were then purified, subjected to SDS‐PAGE, and identified by MS using an LC‐nano‐ESI‐MS/MS. A total of 42 proteins were identified within eight functional groups, including metabolism, intracellular trafficking, the stress response/molecular modification and development. Ultrastructural analyses of LBs in situ showed that they exhibit defined morphological characteristics, including a high‐electron density resulting from a distinct lipid composition from that of the lipid droplets of mammalian cells. Coral LBs were also characterized by the presence of numerous electron‐transparent inclusions of unknown origin and composition. Both proteomic and ultrastructural observations seem to suggest that both Symbiodinium and host organelles, such as the ER, are involved in LB biogenesis.     

Heterocellular cultures of pulmonary alveolar epithelial cells grown on laminin-5 supplemented matrix

The pulmonary alveolar epithelium consists of alveolar type I (AT1) and alveolar type II (AT2) cells. Interactions between these two cell types are necessary for alveolar homeostasis and remodeling. These interactions have been difficult to study in vitro because current cell culture models of the alveolar epithelium do not provide a heterocellular population of AT1 and AT2 cells for an extended period of time in culture. In this study, a new method for obtaining heterocellular cultures of AT1- and AT2-like alveolar epithelial cells maintained for 7 d on a rat tail collagen-fibronectin matrix supplemented with laminin-5 is described. These cultures contain cells that appear by their morphology to be either AT1 cells (larger flattened cells without lamellar bodies) or AT2 cells (smaller cuboidal cells with lamellar bodies). AT1-like cells stain for the type I cell marker aquaporin-5, whereas AT2-like cells stain for the type II cell markers surfactant protein C or prosurfactant protein C. AT1/AT2 cell ratios, cell morphology, and cell phenotype-specific staining patterns seen in 7-d-old heterocellular cultures are similar to those seen in alveoli in situ. This culture system, in which a mixed population of phenotypically distinct alveolar epithelial cells are maintained, may facilitate in vitro studies that are more representative of AT1-AT2 cell interactions that occur in vivo.     

A fluorescent microplate assay for exocytosis in alveolar type II cells

The authors describe a simple, reliable, and quantitative assay to monitor exocytotic fusion of lamellar bodies (LBs) in adherent rat alveolar type II (AT II) cells. The assay is based on fluorescence measurements of LB-plasma membrane (PM) fusions modified for the use in multiwell culture plates to obtain a high-sample throughput. In particular, it is based on the presence of a highly light-absorbing dye in the cell supernatants to increase the specificity of fluorescence signals and to yield pseudo-confocal information from the cells. When the assay was tested with agonist-(ATP) and phorbolester-induced stimulation of LB-PM fusions, the authors found a good correlation with direct microscopic investigations based on single cell recordings. To further validate the assay, they used Curosurf at 10 mg/ml. However, it influenced neither the basal nor the ATP-stimulated rate of LB-PM fusions. This was corroborated by the fact that Curosurf had no effect on resting Ca (2+) levels nor the ATP induced Ca (2+) signals. The results cast new light on previous findings that surfactant phospholipids decrease the rate of secretion in AT II cells in a dose-dependent way. The authors conclude that the inhibitory effect exerted by phospholipids might be due to action on a later step in exocytosis, probably associated with exocytotic fusion pore expansion and content release out of fused vesicles.     

Lipid body formation during maturation of human mast cells

Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.     

Conversion of lamellar body membranes into tubular myelin in alveoli of fetal rat lungs

Fluid-filled lumina of fetal rat lungs contain lamellar bodies (LBs) as well as tubular myelin (TM), both of which are thought to be stores of phospholipid-rich pulmonary surfactant. The alveolar epithelium is believed to secrete LBs, but neither the origin nor the mechanism of TM formation is entirely certain. The main objective of this study was to determine the relationship between secreted LBs and TM and to define membrane phenomena which occur during TM formation. I examined lung tissues of 20-21 day-old fetuses (day 22 = term) using transmission and high voltage transmission electron microscopy and cytochemistry. My findings indicate that secreted LBs, identified by the presence of an acid-phosphatase reactive core, are the precursor of TM. Secreted LBs are highly organized structures which contain structurally specialized areas, one of which is a "mini-lattice" structure similar to TM. During TM formation, fuzzes or 8.0-nm diameter particles appear on transition membranes, although LB membranes appear to lack both structures. Similar particles are present on TM membranes and are generally associated with membrane intersections. My results provide evidence that TM is formed from LBs within the alveolar lumen by mechanisms which may be complex.