Lamellar Bodies Form Solid Three-dimensional Films at the Respiratory Air-Liquid Interface

Pulmonary surfactant is essential for lung function. It is assembled, stored and secreted as particulate entities (lamellar body-like particles; LBPs). LBPs disintegrate when they contact an air-liquid interface, leading to an instantaneous spreading of material and a decline in surface tension. Here, we demonstrate that the film formed by the adsorbed material spontaneously segregate into distinct ordered and disordered lipid phase regions under unprecedented near-physiological conditions and, unlike natural surfactant purified from bronchoalveolar lavages, dynamically reorganized into highly viscous multilayer domains with complex three-dimensional topographies. Multilayer domains, in coexistence with liquid phases, showed a progressive stiffening and finally solidification, probably driven by a self-driven disassembly of LBPs from a sub-surface compartment. We conclude that surface film formation from LBPs is a highly dynamic and complex process, leading to a more elaborated scenario than that observed and predicted by models using reconstituted, lavaged, or fractionated preparations.     

Optical measurement of surface tension in a miniaturized air-liquid interface and its application in lung physiology

We have previously shown that lamellar body-like particles, the form in which pulmonary surfactant is secreted, spontaneously disintegrate when they contact an air-liquid interface, eventually creating an interfacial film. Here, we combined these studies with a new technique enabling the simultaneous and non-invasive measurement of surface tension (gamma). This method is a refinement of the pendant-drop principle. A sapphire cone with a 300-microm aperture keeps the experimental fluid by virtue of surface coherence in a fixed and nearly planar position above the objective of an inverted microscope. The radius of curvature of the fluid meniscus is related to gamma and determines the pattern of light back-reflection upon epi-illumination. This method, which we name "inverted interface", has several novel aspects, in particular its microscopic dimensions. When using lamellar body-like particles freshly released by alveolar type II cells, we found that their conversion at the interface resulted in gamma-reduction close to 30 mN/m. After a fast initial decay, gamma-decrease proceeded slowly and in proportion to single particle conversions. These conversions ceased with time whereas gamma decreased further, probably due to reorganization of the already deposited material. The present investigation indicates that surface film formation by adsorption of large surfactant aggregates is an important mechanism by which gamma is reduced in the lung.     

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.     

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.     

An air-liquid interface promotes the differentiation of gastric surface mucous cells (GSM06) in culture

The gastric surface epithelium is situated at an air-liquid interface because the luminal surface of the alimentary tract is in continuity with the air phase. However, the effects of this microenvironment on the gastric epithelium remain unclear. The aim of this study was to clarify the effects of an air-liquid interface on gastric epithelial cell biology. Gastric surface mucous cells (GSM06) were cultured at an air-liquid interface. Cultured cells were examined by histology, histochemistry, and transmission electron microscopy. When the cells were cultured at an air-liquid interface, the surface cells on the collagen gel became tall columnar and secreted periodic acid-Shiff-positive substances at the apical surface. These cells indicated many mucous granules in the apical cytoplasm and organized the basal lamina at the contact side with the gel. In contrast, under immersed condition, the surface cells showed immature features. This is the first report of an air-liquid interface promoting the differentiation of gastric surface mucous cells in a reconstruction culture of the gastric surface epithelial layer, suggesting that an air-liquid interface may function as a crucial luminal factor to maintain the homeostasis of gastric mucosa.     

Cell deformation at the air-liquid interface induces Ca2+-dependent ATP release from lung epithelial cells

Extracellular nucleotides regulate mucociliary clearance in the airways and surfactant secretion in alveoli. Their release is exquisitely mechanosensitive and may be induced by stretch as well as airflow shear stress acting on lung epithelia. We hypothesized that, in addition, tension forces at the air-liquid interface (ALI) may contribute to mechanosensitive ATP release in the lungs. Local depletion of airway surface liquid, mucins, and surfactants, which normally protect epithelial surfaces, facilitate such release and trigger compensatory mucin and fluid secretion processes. In this study, human bronchial epithelial 16HBE14o(-) and alveolar A549 cells were subjected to tension forces at the ALI by passing an air bubble over the cell monolayer in a flow-through chamber, or by air exposure while tilting the cell culture dish. Such stimulation induced significant ATP release not involving cell lysis, as verified by ethidium bromide staining. Confocal fluorescence microscopy disclosed reversible cell deformation in the monolayer part in contact with the ALI. Fura 2 fluorescence imaging revealed transient intracellular Ca(2+) elevation evoked by the ALI, which did not entail nonspecific Ca(2+) influx from the extracellular space. ATP release was reduced by ～40 to ～90% from cells loaded with the Ca(2+) chelator BAPTA-AM and was completely abolished by N-ethylmalemide (1 mM). These experiments demonstrate that in close proximity to the ALI, surface tension forces are transmitted directly on cells, causing their mechanical deformation and Ca(2+)-dependent exocytotic ATP release. Such a signaling mechanism may contribute to the detection of local deficiency of airway surface liquid and surfactants on the lung surface.     

Epithelial cell interaction in air-liquid interface culture

Summary A novel culture method has been developed to study the interaction of epithelial cells in the absence of a solid substratum. Starting with either a single cell suspension or aggregates, cells were floated at the interface of air and liquid culture medium. Two epithelial cell lines have been studied in this system: Madin-Darby canine kidney cells (MDCK), and a rat bladder tumor cell line (NBT-II). Starting with a single cell suspension of MDCK, the floating cells coalesced in 24 h into sheets of cells. The cells were morphologically polarized with the apical surface facing the liquid medium. Domes were observed regularly in these sheets of cells. NBT-II cells migrated actively from aggregates at the air-liquid interface. In this floating culture, NBT-II cells produced extensive cell processes similar to those seen in cells grown on a solid surface. Because cells at the air-liquid interface lack a solid substratum for adhesion, cell membrane processes such as lamellapodia, retraction fibers, pseudopods, and long, intercellular connections can only exert a tension equal to or less than the surface tension of the liquid. Dimethyl sulfoxide 2% stimulated desmosome formation in floating NBT-II cells, resulting in a cribriform pattern in the sheet of cells. This method of interface can lead to new understanding of morphogenesis of epithelial cells, and the mechanism, of cell motility and formation of cell processess. This research was supported by research grant CA14137 from the National Institutes of Health, Bethesda, MD, and in part by the W. W. Smith Charitable Trust, Rosemont, PA     

Alveolar surface forces and lung architecture

The entire alveolar surface is lined by a thin fluid continuum. As a consequence, surface forces at the air-liquid interface are operative, which in part are transmitted to the delicate lung tissue. Morphologic and morphometric analyses of lungs show that the alveolar surface forces exert a moulding effect on alveolar tissue elements. In particular, in lungs at low degrees of inflation, equivalent to the volume range of normal breathing, there is a derecruitment of alveolar surface area with increasing surface tensions which reflects equilibrium configurations of peripheral air spaces where the sum of tissue energy and surface energy is minimum. Thus, changes in surface tension alter the recoil pressure of the lung directly and indirectly by deforming lung tissue and hence changing tissue tensions. However, the interplay between tissue and surface forces is rather complex, and there is a marked volume dependence of the shaping influence of surface forces. With increasing lung volumes the tissue forces transmitted by the fiber scaffold of the lung become the predominant factor of alveolar micromechanics: at lung volumes of 80% total lung capacity or more, the alveolar surface area-volume relation is largely independent of surface tension. Most important, within the range of normal breathing, the surface tension, its variations and the associated variations in surface area are small. The moulding power of surface forces also affects the configuration of capillaries, and hence the microcirculation, of free cellular elements such as the alveolar macrophages beneath the surface lining layer, and of the surfaces of the peripheral airways. Still enigmatic is the coupling mechanism between the fluid continua of alveoli and airways which might also be of importance for alveolar clearance. As to the surface active lining layer of peripheral air spaces, which determines alveolar surface tension, its structure and structure-function relationship are still ill-defined owing to persisting problems of film preservation and fixation. Electron micrographs of alveolar tissue, of lining layers of captive bubbles, and scanning force micrographs of surfactant films transferred on mica plates reveal a complex structural pattern which precludes so far the formulation of an unequivocal hypothesis.     

A biophysical mechanism by which plasma proteins inhibit lung surfactant activity

These in vitro experiments study a potential mechanism by which plasma proteins, found in the alveoli during pulmonary edema and hemorrhage, may act to inhibit the surface activity of pulmonary surfactant. The results indicate that the inhibition of the adsorption facility and surface tension lowering ability of a calf lung surfactant extract (CLSE) by albumin, hemoglobin, or fibrinogen may be completely abolished by centrifugation of the protein-surfactant mixture at 12,500 x g. Furthermore, albumin, hemoglobin and fibrinogen (1.25 mg/ml) were shown to inhibit the adsorption of high concentrations of CLSE (0.32 mg/ml), normally unaffected by the addition of exogenous proteins, when the CLSE was injected into the subphase under a preformed protein surface film. Similarly, injection of large amounts of these proteins (2.5 mg/ml) into the subphase beneath a preformed CLSE surface film was without effect, even though the CLSE concentration was only 0.06 mg/ml, a surfactant concentration which is normally inhibited by even small amounts of exogenous protein. Taken together, the data suggest that some proteins may inhibit surfactant function by preventing the surfactant phospholipids from adsorbing to the air-liquid interface, possibly by a competition between the proteins and CLSE phospholipids for space at the air-liquid interface rather than direct molecular interactions between proteins and surfactant.     

Transient Exposure of Pulmonary Surfactant to Hyaluronan Promotes Structural and Compositional Transformations into a Highly Active State

Pulmonary surfactant is a lipid-protein complex that lowers surface tension at the respiratory air-liquid interface, stabilizing the lungs against physical forces tending to collapse alveoli. Dysfunction of surfactant is associated with respiratory pathologies such as acute respiratory distress syndrome or meconium aspiration syndrome where naturally occurring surfactant-inhibitory agents such as serum, meconium, or cholesterol reach the lung. We analyzed the effect of hyaluronan (HA) on the structure and surface behavior of pulmonary surfactant to understand the mechanism for HA-promoted surfactant protection in the presence of inhibitory agents. In particular, we found that HA affects structural properties such as the aggregation state of surfactant membranes and the size, distribution, and order/packing of phase-segregated lipid domains. These effects do not require a direct interaction between surfactant complexes and HA and are accompanied by a compositional reorganization of large surfactant complexes that become enriched with saturated phospholipid species. HA-exposed surfactant reaches very high efficiency in terms of rapid and spontaneous adsorption of surfactant phospholipids at the air-liquid interface and shows significantly improved resistance to inactivation by serum or cholesterol. We propose that physical effects pertaining to the formation of a meshwork of interpenetrating HA polymer chains are responsible for the changes in surfactant structure and composition that enhance surfactant function and, thus, resistance to inactivation. The higher resistance of HA-exposed surfactant to inactivation persists even after removal of the polymer, suggesting that transient exposure of surfactant to polymers like HA could be a promising strategy for the production of more efficient therapeutic surfactant preparations.     

Effects of surface tension and intraluminal fluid on mechanics of small airways

Hill, Mark J., Theodore A. Wilson, and Rodney K. Lambert.Effects of surface tension and intraluminal fluid on the mechanicsof small airways. J. Appl. Physiol.82(1): 233-239, 1997.Airway constriction is accompanied byfolding of the mucosa to form ridges that run axially along the innersurface of the airways. The muscosa has been modeled (R. K. Lambert.J. Appl. Physiol. 71: 666-673,1991) as a thin elastic layer with a finite bending stiffness, and thecontribution of its bending stiffness to airway elastance has beencomputed. In this study, we extend that work by including surfacetension and intraluminal fluid in the model. With surface tension, thepressure on the inner surface of the elastic mucosa is modified by thepressure difference across the air-liquid interface. As folds form inthe mucosa, intraluminal fluid collects in pools in the depressionsformed by the folds, and the curvature of the air-liquid interfacebecomes nonuniform. If the amount of intraluminal fluid is small,<2% of luminal volume, the pools of intraluminal fluid are small, the air-liquid interface nearly coincides with the surface of themucosa, and the area of the air-liquid interface remains constant asairway cross-sectional area decreases. In that case, surface energy isindependent of airway area, and surface tension has no effect on airwaymechanics. If the amount of intraluminal fluid is >2%, the area ofthe air-liquid interface decreases as airway cross-sectional areadecreases, and surface tension contributes to airway compression. Themodel predicts that surface tension plus intraluminal fluid can causean instability in the area-pressure curve of small airways. Thisinstability provides a mechanism for abrupt airway closure and abruptreopening at a higher opening pressure.

     

Shear sensitivity of insect cells

Conclusions While insect cells can be easily damaged in bioreactors as a result of hydrodynamic forces, it is also relatively easy to prevent this damage. Of several possible damage mechanisms, the best understood and preventable is the attachment of cells to gas-liquid interfaces and the subjection of these attached cells to the hydro-dynamic forces and/or physical forces associated with these interfaces. For example, cells attached to gas bubbles in a bioreactor can be transported into the foam layer where they are physically removed from the cell suspension, or they can be killed when the gas bubble they are attached to ruptures at the medium-air interface at the top of the bioreactor. The easiest method to prevent this damage is through the use of specific surface active compounds, such as Pluronic F-68 or Methocel E-50 which prevent the cells from attaching to the gas-medium interface.     

A ToF-SIMS study of the lateral organization of lipids and proteins in pulmonary surfactant systems

Pulmonary surfactant is a complex lipid-protein mixture whose main function is to reduce the surface tension at the air-liquid interface of alveoli to minimize the work of breathing. The exact mechanism by which surfactant monolayers and multilayers are formed and how they lower surface tension to very low values during lateral compression remains uncertain. We used time-of-flight secondary ion mass spectrometry to study the lateral organization of lipids and peptide in surfactant preparations ranging in complexity. We show that we can successfully determine the location of phospholipids, cholesterol and a peptide in surfactant Langmuir-Blodgett films and we can determine the effect of cholesterol and peptide addition. A thorough understanding of the lateral organization of PS interfacial films will aid in our understanding of the role of each component as well as different lipid-lipid and lipid-protein interactions. This may further our understanding of pulmonary surfactant function.     

FLO11-based model for air-liquid interfacial biofilm formation by Saccharomyces cerevisiae

Sardinian wine strains of Saccharomyces cerevisiae used to make sherry-like wines form a biofilm at the air-liquid interface at the end of ethanolic fermentation, when grape sugar is depleted and further growth becomes dependent on access to oxygen. Here, we show that FLO11, which encodes a hydrophobic cell wall glycoprotein, is required for the air-liquid interfacial biofilm and that biofilm cells have a buoyant density greater than the suspending medium. We propose a model for biofilm formation based on an increase in cell surface hydrophobicity occurring at the diauxic shift. This increase leads to formation of multicellular aggregates that effectively entrap carbon dioxide, providing buoyancy. A visible biofilm appears when a sufficient number of hydrophobic cell aggregates are carried to and grow on the liquid surface.     

In vitro development and characterization of a manatee bronchial cell line

In the present study, culture conditions that promote the growth and differentiation of manatee respiratory tract epithelial cells toward a mucociliary phenotype were determined. Characterization of a manatee-specific cell line enables investigators to conduct in vitro testing where live-animal experimentation is not possible. Cell cultures were established from both explants and enzymatically dissociated cells that were isolated from manatee bronchial tissue. To modulate their differentiation, bronchial epithelial cells were grown on Transwell collagen membranes either submerged or at an air-liquid interface. Growth on a collagen membrane at an air-liquid interface and medium supplemented with retinoic acid was required to promote a mucociliary phenotype. When cells were grown in submerged cultures without retinoic acid, they appeared more squamous and were not ciliated. Intracellular keratin proteins were detected in both submerged and interface cultures. Cultured manatee bronchial epithelial cells will facilitate future studies to investigate their potential role in pulmonary disease associated with brevetoxicosis after exposure to the red-tide organism, Karenia brevis.     

Role of lipid ordered/disordered phase coexistence in pulmonary surfactant function

The respiratory epithelium has evolved to produce a complicated network of extracellular membranes that are essential for breathing and, ultimately, survival. Surfactant membranes form a stable monolayer at the air-liquid interface with bilayer structures attached to it. By reducing the surface tension at the air-liquid interface, surfactant stabilizes the lung against collapse and facilitates inflation. The special composition of surfactant membranes results in the coexistence of two distinct micrometer-sized ordered/disordered phases maintained up to physiological temperatures. Phase coexistence might facilitate monolayer folding to form three-dimensional structures during exhalation and hence allow the film to attain minimal surface tension. These folded structures may act as a membrane reserve and attenuate the increase in membrane tension during inspiration. The present review summarizes what is known of ordered/disordered lipid phase coexistence in lung surfactant, paying attention to the possible role played by domain boundaries in the monolayer-to-multilayer transition, and the correlations of biophysical inactivation of pulmonary surfactant with alterations in phase coexistence.     

Protein-lipid interactions and surface activity in the pulmonary surfactant system

Pulmonary surfactant is a lipid-protein complex, synthesized and secreted by the respiratory epithelium of lungs to the alveolar spaces, whose main function is to reduce the surface tension at the air-liquid interface to minimize the work of breathing. The activity of surfactant at the alveoli involves three main processes: (i) transfer of surface active molecules from the aqueous hypophase into the interface, (ii) surface tension reduction to values close to 0 mN/m during compression at expiration and (iii) re-extension of the surface active film upon expansion at inspiration. Phospholipids are the main surface active components of pulmonary surfactant, but the dynamic behaviour of phospholipids along the breathing cycle requires the necessary participation of some specific surfactant associated proteins. The present review summarizes the current knowledge on the structure, disposition and lipid-protein interactions of the hydrophobic surfactant proteins SP-B and SP-C, the two main actors participating in the surface properties of pulmonary surfactant. Some of the methodologies currently used to evaluate the surface activity of the proteins in lipid-protein surfactant preparations are also revised. Working models for the potential molecular mechanism of SP-B and SP-C are finally discussed. SP-B might act in surfactant as a sort of amphipathic tag, directing the lipid-protein complexes to insert and re-insert very efficiently into the air-liquid interface along successive breathing cycles. SP-C could be essential to maintain association of lipid-protein complexes with the interface at the highest compressed states, at the end of exhalation. The understanding of the mechanisms of action of these proteins is critical to approach the design and development of new clinical surfactant preparations for therapeutical applications.     

FLO11-Based Model for Air-Liquid Interfacial Biofilm Formation by Saccharomyces cerevisiae

Sardinian wine strains of Saccharomyces cerevisiae used to make sherry-like wines form a biofilm at the air-liquid interface at the end of ethanolic fermentation, when grape sugar is depleted and further growth becomes dependent on access to oxygen. Here, we show that FLO11, which encodes a hydrophobic cell wall glycoprotein, is required for the air-liquid interfacial biofilm and that biofilm cells have a buoyant density greater than the suspending medium. We propose a model for biofilm formation based on an increase in cell surface hydrophobicity occurring at the diauxic shift. This increase leads to formation of multicellular aggregates that effectively entrap carbon dioxide, providing buoyancy. A visible biofilm appears when a sufficient number of hydrophobic cell aggregates are carried to and grow on the liquid surface.     

Actin filaments function as a tension sensor by tension-dependent binding of cofilin to the filament

Intracellular and extracellular mechanical forces affect the structure and dynamics of the actin cytoskeleton. However, the underlying molecular and biophysical mechanisms, including how mechanical forces are sensed, are largely unknown. Actin-depolymerizing factor/cofilin proteins are actin-modulating proteins that are ubiquitously distributed in eukaryotes, and they are the most likely candidate as proteins to drive stress fiber disassembly in response to changes in tension in the fiber. In this study, we propose a novel hypothesis that tension in an actin filament prevents the filament from being severed by cofilin. To test this, we placed single actin filaments under tension using optical tweezers. When a fiber was tensed, it was severed after the application of cofilin with a significantly larger delay in comparison with control filaments suspended in solution. The binding rate of cofilin to an actin bundle decreased when the bundle was tensed. These results suggest that tension in an actin filament reduces the cofilin binding, resulting in a decrease in its effective severing activity.