A near-field microscopy study of submicron domain structure in a model lung surfactant monolayer.

The submicron domain structure of coexisting liquid condensed (LC) and liquid expanded (LE) phases in monolayers composed of palmitic acid and 20 wt% of a lung surfactant protein B fragment has been investigated. Near-field microscopy was used to simultaneously measure topography and fluorescence images of monolayers that were prepared at a surface pressure of 15 mN/m and a temperature of 22 degrees C. The use of a fluorescently tagged peptide allowed for unambiguous determination of the peptide location in the two-component system. The LC and LE phases in the monolayers are measured on the submicron length scale. A 6-11 A height difference between the LC and LE phases was evident in the height images. Gradual transitions between the LC and LE domains were observed across a 1.3 microm length scale in the near-field fluorescence images, but were significantly sharper in the simultaneously collected topography images and in the separately measured AFM images. These results may reflect the occurrence of peptide encroachment into the LC domains.

Enhanced efficacy of porcine lung surfactant extract by utilization of its aqueous swelling dynamics.

This study investigates the interactions between a porcine lung surfactant (PLS) extract and distilled water, saline solution or Ringer solution. The phases which coexist in equilibrium with water or electrolyte solutions were analysed by X-ray diffraction and cryo transmission electron microscopy (cryo-TEM). A lamellar phase with a structure unit consisting of double bilayers was observed in water, whereas lamellar phases with the usual bilayer structure unit were formed in saline and in Ringer solutions. At 25 degrees C the presence of a 4.2-A peak in the X-ray diffraction wide-angle region of these three maximally swollen phases showed that most of the hydrocarbon chains were organized in a crystalline packing. At 42 degrees C the chains in all three phases were melted which, in combination with the low-angle diffraction, shows that they were liquid-crystalline. Polyhedral-like vesicles and spherically shaped multilamellar vesicles were observed in cryo-TEM. The bilayer unit structures were consistent with the periodicity seen by X-ray diffraction. The dynamic swelling behaviour was followed in the polarizing microscope. A remarkable growth of birefringent networks was seen at the air interface of samples swollen in Ringer solution and saline solution. No such interfacial growth phenomena were observed during swelling in water without electrolytes. Then, these dynamics were analysed in relation to time-dependent pulmonary administration of the surfactant extract in rats. Variation in the time of administration (20 and 60 min) after mixing the extract with saline or Ringer solution showed clear differences in physiological effects. At pulmonary administration when the swelling behaviour in vitro showed a maximum in dynamics, the arterial oxygenation was superior to that of administration at a time after a steady-state had been reached. This means that the clinical performance of mammalian lung surfactant extracts can be significantly improved by taking the time-dependent aqueous swelling of the extract into account.

Scanning force microscopy at the air-water interface of an air bubble coated with pulmonary surfactant.

To study the structure-function relationship of pulmonary surfactant under conditions close to nature, molecular films of a model system consisting of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and surfactant-associated protein C were prepared at the air-water interface of air bubbles about the size of human alveoli (diameter of 100 microm). The high mechanical stability as well as the absence of substantial film flow, inherent to small air bubbles, allowed for scanning force microscopy (SFM) directly at the air-water interface. The SFM topographical structure was correlated to the local distribution of fluorescent-labeled dipalmitoylphosphatidylcholine, as revealed from fluorescence light microscopy of the same bubbles. Although SFM has proven before to be exceptionally well suited to probe the structure of molecular films of pulmonary surfactant, the films so far had to be transferred onto a solid support from the air-water interface of a film balance, where they had been formed. This made them prone to artifacts imposed by the transfer. Moreover, the supported monolayers disallowed the direct observation of the structural dynamics associated with expansion and compression of the films as upon breathing. The current findings are compared in this respect to our earlier findings from films, transferred onto a solid support.

Imaging of monolayers composed of palmitic acid and lung surfactant protein B.

Near-field scanning optical microscopy and atomic force microscopy are used to probe the sub-micrometre phase structure in palmitic acid monolayers containing the 25 peptide amino terminus of lung surfactant protein B (SP-B(1-25)). Monolayers deposited onto mica substrates at a surface pressure of 15 mN m-1 exhibit a two-phase coexistence across a broad range of SP-B(1-25) concentrations. Monolayers containing 5 wt.% SP-B(1-25) or less exhibit an expanse of liquid condensed phase in which elliptical liquid expanded (LE) domains with areas of approximately 25 microm2 coexist. By contrast, monolayers containing 20 wt.% SP-B(1-25) exhibit an expanse of liquid expanded phase in which circular liquid condensed domains coexist. The phase distribution dependence on SP-B(1-25) concentration suggests that the peptide induces disorder in the monolayer.

Commercial versus native surfactants. Surface activity, molecular components, and the effect of calcium.

Despite their broad clinical use, there is no standardized comparative study on the functional, biochemical, and morphologic differences of the various commercial surfactants in relation to native surfactant. We investigated these parameters in Alveofact, Curosurf, Exosurf, and Survanta, and compared them with native bovine (NBS) and porcine (NPS) surfactant. For Curosurf and Alveofact the concentrations necessary for minimal surface tensions < 5 mN/m were six to 12 times higher (1.5 and 3 mg/ml, respectively) than with NPS and NBS. Exosurf and Survanta only reached 22 and 8 mN/m, respectively. Increasing calcium to nonphysiologic concentrations artificially improved the function of Alveofact and Curosurf, but it had little effect on Exosurf and Survanta. Impaired surface activity of commercial versus native surfactants corresponded with their lack in surfactant protein SP-A and decreased SP-B/C. The higher surface activity of Curosurf compared with Alveofact corresponded with its higher concentration of dipalmitoylphosphatidylcholine (DPPC). Despite their enrichment in DPPC Survanta and Exosurf exhibited poor surface activity because of low or absent SP-B/C. Ultrastructurally, Curosurf and Alveofact consisted mainly of lamellar and vesicular structures, which were also present in NPS and NBS. Exosurf contained crystalline structures only, whereas the DPPC-enriched Survanta contained separate lamellar/vesicular and crystalline structures. We conclude that in vitro surface activity of commercial surfactants is impaired compared with native surfactants at physiologic calcium concentrations. In the presence of SP-B/C, surface activity corresponds to the concentration of DPPC. Our data underscore the importance of a standardized protocol at physiologic calcium concentrations for the in vitro assessment of commercial surfactants.

Formation of three-dimensional protein-lipid aggregates in monolayer films induced by surfactant protein B.

This study focuses on the structural organization of surfactant protein B (SP-B) containing lipid monolayers. The artificial system is composed of the saturated phospholipids dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) in a molar ratio of 4:1 with 0.2 mol% SP-B. The different "squeeze-out" structures of SP-B were visualized by scanning probe microscopy and compared with structures formed by SP-C. Particularly, the morphology and material properties of mixed monolayers containing 0.2 mol% SP-B in a wide pressure range of 10 to 54 mN/m were investigated revealing that filamentous domain boundaries occur at intermediate surface pressure (15-30 mN/m), while disc-like protrusions prevail at elevated pressure (50-54 mN/m). In contrast, SP-C containing lipid monolayers exhibit large flat protrusions composed of stacked bilayers in the plateau region (app. 52 mN/m) of the pressure-area isotherm. By using different scanning probe techniques (lateral force microscopy, force modulation, phase imaging) it was shown that SP-B is dissolved in the liquid expanded rather than in the liquid condensed phase of the monolayer. Although artificial, the investigation of this system contributes to further understanding of the function of lung surfactant in the alveolus.

Three-dimensional structure of rat surfactant protein A trimers in association with phospholipid monolayers.

Surfactant protein A (SP-A) is a C-type lectin found primarily in the lung and plays a role in innate immunity and the maintenance of surfactant integrity. To determine the three-dimensional (3D) structure of SP-A in association with a lipid ligand, we have used single particle electron crystallography and computational 3D reconstruction in combination with molecular modeling. Recombinant rat SP-A, containing a deletion of the collagen-like domain, was incubated with dipalmitoylphosphatidylcholine:egg phosphatidylcholine (1:1, wt/wt) lipid monolayers in the presence of calcium, negatively stained, and examined by transmission electron microscopy. Images of SP-A-lipid complexes with different angular orientations were used to reconstruct the 3D structure of the protein. These results showed that SP-A subunits readily formed trimers and interacted with lipid monolayers exclusively via the globular domains. A homology-based molecular model of SP-A was generated and fitted into the electron density map of the protein. The plane of the putative lipid-protein interface was relatively flat and perpendicular to the hydrophobic neck region, and the cleft region in the middle of the trimer had no apparent charge clusters. Amino acid residues that are known to affect lipid interactions, Glu(195) and Arg(197), were located at the protein-lipid interface. The molecular model indicated that the hydrophobic neck region of the SP-A did not interact with lipid monolayers but was instead involved in intratrimeric subunit interactions. The glycosylation site of SP-A was located at the side of each subunit, suggesting that the covalently linked carbohydrate moiety probably occupies the spaces between the adjacent globular domains, a location that would not sterically interfere with ligand binding.

Beneficial effect of lung preservation is related to ultrastructural integrity of tubular myelin after experimental ischemia and reperfusion.

Ischemia/reperfusion (I/R) injury results in the impairment of surfactant activity. The hypothesis that the differences in lung preservation quality obtained by EuroCollins (EC) and Celsior (CE) solutions were related to surfactant alterations was tested. To avoid extensive structural damage and edema formation, which can secondarily affect the surfactant system, lungs were stored for a short ischemic period (2 h at 10 degrees C) and reperfused (50 min) in an isolated perfused rat lung model after preservation with either potassium-reduced (40 mmol) EC40 or with CE. Using a modified stereological approach ultrastructure, total amount and distribution of phospholipid membranes composing tubular myelin (tm) and small (s) and large (l) unilameliar vesicles (ul) were investigated in the organ in lungs fixed by vascular perfusion either in situ (controls) or after I/R (n = 5 per group). The total amount of intraalveolar surfactant was increased after I/R. However, a significant amount (p = 0.008) of tm was displaced into the alveolar lumen and showed wider meshes of the tm lattices than did the controls (p = 0.023) where almost all tm was epithelial. In lungs preserved with EC40, epithelial tm was significantly reduced (p = 0.018), resulting in a higher ratio (p = 0.034) of surface-inactive small ul (0.05 to 0.3 microm) to surface-active epithelial tm. In the CE group approximately 50% of the total tm pool was epithelial. This was accompanied by higher parenchymal air space and improved functional parameters. Epithelial and endothelial cell-specific immunostaining did not reveal any gross damage of the blood-gas barrier. In summary, improved lung function during reperfusion was associated with beneficial effects of lung preservation on tm integrity after I/R. These observations suggest that preservation solutions ameliorate events leading to surfactant disturbance even before extensive lung injury is manifested.

Distribution of the surfactant-associated protein C within a lung surfactant model film investigated by near-field optical microscopy.

Lung surfactant films at the air/water interface exhibit the particularity that surfactant molecules are expelled from the surface monolayer into a surface associated multilamellar phase during compression. They are able to re-enter the surface film during the following expansion. The underlying mechanism for this behavior is not fully understood yet. However, an important role is ascribed to the surfactant-associated protein C (SP-C). Here, we studied a model lung surfactant, consisting of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and SP-C, by means of scanning near-field optical microscopy (SNOM). Attaching a fluorescent dye to the protein allowed the localization of its lateral distribution at various surface pressures with high resolution. At an early stage of compression, the film appears demixed into a pure lipid phase and a protein-enriched phase. Within the latter phase, protein aggregations are revealed. They show a uniform density, having three times the fluorescence intensity of their surroundings. Across the phase boundary between the lipid phase and the protein-rich phase, there is a protein density gradient rather than an abrupt border. When the film is highly compressed, we observe the formation of multilamellar structures that are fluorescent. They are often surrounded by a slightly fluorescent monolayer. The fluorescence of the multilayer stacks (i. e., the protein content per unit area) is proportional to the height of the stacks.

Chemoprevention of vinyl carbamate-induced lung tumors in strain A mice.

The ability of potential chemopreventive agents to prevent vinyl carbamate-induced lung tumors was determined in 2 different experiments. Female strain A mice administered intraperitoneally either a single injection of 60 mg/kg vinyl carbamate that induced 24.0 +/- 1.72 tumors/mouse at 24 weeks or 2 injections of 16 mg/kg vinyl carbamate each (32 mg/kg total dose) that induced 43.2 +/- 3.2 tumors/mouse at 20 weeks. Lung carcinomas were found as early as 16 weeks. Dexamethasone and piroxicam provided in the diet were found to significantly inhibit lung tumors induced by 60 mg/kg vinyl carbamate at 24 weeks whereas myo-inositol also provided in the diet, did not significantly inhibit tumor formation. In animals given 6 16-mg/kg doses of vinyl carbamate, tumor multiplicity was reduced roughly 25% by alpha-difluoromethylornithine and green tea and reduced 50% by dexamethasone and piroxicam. Combinations of these agents were also tested using a total dose of 32 mg/kg of vinyl carbamate. Although alpha-difluoromethylornithine and green tea did not result in a significant inhibition of lung tumor formation if used alone, the combination of alpha-difluoromethylornithine and green tea resulted in a significant reduction of tumor multiplicity. The combinations of alpha-difluoromethylornithine or green tea with either dexamethasone or piroxicam or the combination of dexamethasone and piroxicam did not decrease tumor multiplicity greater than achieved by dexamethasone and piroxicam alone. In summary, selected chemopreventive agents previously shown to inhibit lung tumors by other chemical carcinogens also inhibited vinyl carbamate-induced lung tumors.

[Morpho-functional changes of surfactant system in pulmonary tuberculosis]. / Morfofunktsional'nye izmeneniia surfaktantnoi sistemy pri tuberkuleze legkikh.

A complex study of different cellular and extracellular surfactant elements in the dynamics of the spontaneous course of generalized tuberculosis was made in 240 guinea-pigs and 77 patients with different forms of active pulmonary tuberculosis by using electron microscopy, biochemistry, and physical chemistry. A diagnostic scheme was proposed for life-time assessment of surfactant in bronchoalveolar lavage specimens. Three degrees of changes in surfactant were identified; 1) adaptive rearrangement of its elements developing in local surfactant damages; 2) significantly impaired type 2 alveolocyte production of surfactant, which affects its biochemical composition and capacity of forming characteristic structures; 3) common morphological, biochemical, surface-active signs of surfactant decompensation, profound metabolic disturbances, alveolar epithelial destruction with cleared alveolocytes found in the lavage.

The use of nonparametric statistics in quantitative electron microscopy.

Parametric statistical methods assume samples that have a normal distribution and representative sample sizes (i.e. n >20). Quantitative electron microscopy is inherently restricted to small sample sizes and a priori there is no way to know if the expression of the ligand being studied has a normal distribution. Thus to make statistical inferences based on data generated by quantitative electron microscopy using parametric methods may not be justified. Nonparametric statistical methods offer a tool for the evaluation of data that do not meet the criteria for analysis by parametric methods. In this report I show the utility of using nonparametric statistical methods for the analysis of data generated by quantitative electron microscopy.

Estrogen-induced microvilli and microvillar channels and entrapment of surfactant-lipids by alveolar type I cells of bovine lung.

The ATI cells are simple, flat squamous epithelial cells, which are evolved to function as a component of the alveolar-capillary membrane, ideally designed for gaseous exchange. They inherently lack an active metabolic machinery and lead a precarious existence in the face of hostile environment. On the other hand, the ATI cells of the lung of ruminating animals are endowed with structure-functional properties which enable them to exert a selective barrier function against a wide range of osmotic pressure gradients at their luminal surface. Such gradients are created by a complex gaseous homeostasis due to expectoration of several gases and volatile fatty acids originating from the complex stomach of the ruminants. The purpose of this study is to examine the effect of estradiol propionate on the ultrastructure of the ATI cells and their interaction with the surfactant lipids. The lungs of estrogen and dexamethasone treated male calves were harvested for electromicroscopic examination. The evidence is presented that estradiol induced the formation of microvilli and microvillar channels at the luminal surface. At these regional modifications, intense interactions with the surfactant lipids and their entrapment into the pathways of endocytosis, took place in the squamous part of the ATI cells. Concurrently, large basal protrusions ended up as long lamellipods deep into the alveolar interstitium. The filamentous cytoskeletal network and microtubules intermixed with the translocated organelles such as Golgi apparatus and associated coated and uncoated vesicles. The results of this study support the hypothesis that estrogen regulate the selective barrier-function of the ATI cells. The entrapment of surfactant lipids under the influence of estrogen by ATI cells is a significant change perhaps in response to extracellular stimuli and expression of transmembrane receptors. It implies that these epithelial cells are specially evolved to adapt to a complex gaseous homeostasis in the lung of the ruminating ungulates.

Ultrastructural alterations in intraalveolar surfactant subtypes after experimental ischemia and reperfusion.

Ischemia and reperfusion (I/R) result in surfactant dysfunction. Whether the impairment of surfactant is a consequence or a cause of intraalveolar edema formation is still unknown. The cumulative effects of lung perfusion, ischemic storage, and subsequent reperfusion on surfactant ultrastructure and pulmonary function were studied in a rat isolated perfused lung model. The left lungs were fixed for electron microscopy by vascular perfusion either immediately after excision (control; n = 5) or after perfusion with modified Euro-Collins solution (EC), storage for 2 h at 4 degrees C in EC, and reperfusion for 40 min (n = 5). A stereological approach was chosen to discriminate between intraalveolar surfactant subtypes of edematous regions and regions free of edema. Intraalveolar edema seen after I/R in the EC group occupied 36 +/- 6% (mean +/- SEM) of the gas exchange region as compared with control lungs (1 +/- 1%; p = 0.008). Relative intraalveolar surfactant composition showed a decrease in surface active tubular myelin (3 +/- 1 versus 12 +/- 0%; p = 0.008) and an increase in inactive unilamellar forms (83 +/- 2 versus 64 +/- 5%; p = 0.008) in the EC group. These changes occurred both in edematous (tubular myelin, 3 +/- 1%; unilamellar forms, 88 +/- 6%) and in nonedematous regions (tubular myelin, 4 +/- 3%; unilamellar forms, 77 +/- 5%). The ultrastructural changes in surfactant were associated with an increase in peak inspiratory pressure during reperfusion. In conclusion, surfactant alterations seen after I/R are not directly related to the presence of edema fluid in the alveoli. Disturbances in intraalveolar surfactant after I/R are not merely the result of inactivation due to plasma protein leakage but may instead be responsible for an increased permeability of the blood-air barrier, resulting in a vicious cycle of intraalveolar edema formation and progressing surfactant impairment.

Ultrastructural and protein analysis of surfactant in the Australian lungfish Neoceratodus forsteri: evidence for conservation of composition for 300 million years.

The Australian lungfish Neoceratodus forsteri is the most primitive member of the lungfish family, with a surfactant lipid composition similar to the actinopterygiian fishes, which evolved 400 million years ago. We have analysed the proteins associated with surfactant isolated from lung lavage of this species, and used electron microscopy and immunohistochemistry to examine the surfactant structures and the subcellular localisation of these proteins. The epithelial lining of the gas-exchange region of the lungfish lung consists of one basic cell type, which has characteristics of both mammalian alveolar type I and type II cells and may be the common ancestor of both. It has long cytoplasmic plates containing microvilli, large osmiophilic bodies resembling mammalian lamellar bodies and a cytoplasm rich in metabolic organelles. Extracellular structures reminiscent of mammalian surfactant forms, but not including tubular myelin, were observed in the airspaces. Immunochemical analysis of the lungfish surfactant and lung tissue, using antibodies to human SP-A and SP-B, showed a similar staining pattern to human surfactant, indicating that SP-A- and SP-B-like proteins are present. Immunohistochemistry revealed that both SP-A and SP-B reactivity was present in the secretory cell osmiophilic bodies. In conclusion, our results suggest that, despite the great diversity in present day lung structures, a common cellular mechanism may have evolved to overcome fundamental problems associated with air-breathing.

Correlated atomic force and transmission electron microscopy of nanotubular structures in pulmonary surfactant.

Pulmonary surfactant stabilizes the lung by reducing surface tension at the air-water interface of the alveoli. Surfactant is present in the lung in a number of morphological forms, including tubular myelin (TM). TM is composed of unusual 40 x 40 nm square elongated proteolipid tubes. Atomic force microscopy (AFM) was performed on polymer-embedded Lowicryl and London Resin-White (LR-White) unstained thin sections. AFM was used in imaging regions of the sections where TM was detected by transmission electron microscopy (EM) of corresponding stained sections. Tapping- and contact-mode AFM imaging of the unstained sections containing TM indicated a highly heterogeneous surface topography with height variations ranging from 10 to 100 nm. In tapping-mode AFM, tubular myelin was seen as hemispherical protrusions of 30-70 nm in diameter, with vertical dimensions of 5-8 nm. In contact-mode AFM and with phase imaging using a sharper (>10 nm nominal radius) probe, square open-ended tubes which resembled typical electron micrographs of such regions were observed. The cross-hatch structures observed inside the tubes using EM were not observed using AFM, although certain multilobe structures and topographic heterogeneity were detected inside some tubes. Other regions of multilamellar bodies and some regions where such bilayer lamella appear to fuse with the tubes were found in association with TM using AFM. EM of acetone-delipidated tubes in LR-White revealed rectangular tubular cores containing cross-hatched structures, presumably protein skeletons. AFM surface topography of these regions showed hollow depressions at positions at which the protein was anticipated instead of the protrusions seen in the lipid-containing sections. Gold-labeled antibody to surfactant protein A was found associated somewhat randomly within the regions containing the protein skeletons. The topography of the gold particles was observed as sharp peaks in contact-mode AFM. This study suggests a method for unambiguous detection of three-dimensional nanotubes present in low abundance in a biological macromolecular complex. Only limited detection of proteins and lipids in surfaces of embedded tubular myelin was possible. EM and AFM imaging of such unusual biological structures may suggest unique lipid-protein associations and arrangements in three dimensions.

Filaments of surfactant protein A specifically interact with corrugated surfaces of phospholipid membranes.

Pulmonary surfactant, a mixture of lipids and surfactant proteins (SPs), plays an important role in respiration and gas exchange. SP-A, the major SP, exists as an octadecamer that can self-associate to form elongated protein filaments in vitro. We have studied here the association of purified bovine SP-A with lipid vesicle bilayers in vitro with negative staining with uranyl acetate and transmission electron microscopy. Native bovine surfactant was also examined by transmission electron microscopy of thinly sectioned embedded material. Lipid vesicles made from dipalmitoylphosphatidylcholine and egg phosphatidylcholine (1:1 wt/wt) generally showed a smooth surface morphology, but some large vesicles showed a corrugated one. On the smooth-surfaced vesicles, SP-As primarily interacted in the form of separate octadecamers or as multidirectional protein networks. On the surfaces of the striated vesicles, SP-As primarily formed regularly spaced unidirectional filaments. The mean spacing between adjacent striations and between adjacent filaments was 49 nm. The striated surfaces were not essential for the formation of filaments but appeared to stabilize them. In native surfactant preparations, SP-A was detected in the dense layers. This latter arrangement of the lipid bilayer-associated SP-As supported the potential relevance of the in vitro structures to the in vivo situation.

Formation of membrane lattice structures and their specific interactions with surfactant protein A.

Biological membranes exist in many forms, one of which is known as tubular myelin (TM). This pulmonary surfactant membranous structure contains elongated tubes that form square lattices. To understand the interaction of surfactant protein (SP) A and various lipids commonly found in TM, we undertook a series of transmission-electron-microscopic studies using purified SP-A and lipid vesicles made in vitro and also native surfactant from bovine lung. Specimens from in vitro experiments were negatively stained with 2% uranyl acetate, whereas fixed native surfactant was delipidated, embedded, and sectioned. We found that dipalmitoylphosphatidylcholine-egg phosphatidylcholine (1:1 wt/wt) bilayers formed corrugations, folds, and predominantly 47-nm-square latticelike structures. SP-A specifically interacted with these lipid bilayers and folds. We visualized other proteolipid structures that could act as intermediates for reorganizing lipids and SP-As. Such a reorganization could lead to the localization of SP-A in the lattice corners and could explain, in part, the formation of TM-like structures in vivo.

Structures of surfactant films: a scanning force microscopy study.

The alveolar lining layer is thought to consist of a continuous duplex layer, i.e., an aqueous hypophase covered by a thin surfactant film which is a monolayer with dipalmitoyl-phosphatidylcholine (DPPC) as its most important component. Findings obtained by electron microscopy and results from in vitro experiments suggest, however, that the structure and hence the structure-function relations of surfactant films are more complex. In order to better define their structures films of surfactants were studied by scanning force microscopy. Four different surfactants were spread on a Langmuir-Wilhelmy balance, and then transferred onto a solid mica plate by the Langmuir-Blodgett technique, under various states of film compression. Imaging of the films by scanning force microscopy was performed in the contact (repulsive) mode in air. The scanning force micrographs revealed that surfactant films are not homogeneous, but rather undergo phase transitions depending on the surface pressures. Even at comparable surface pressures different surfactants show quite different surface patterns. Differences in surface structure can even be observed in films containing surfactant proteins (SP)-B and SP-C. These observations give further evidence that the widely accepted hypothesis of a regular monolayer of phospholipids governing the surface tension probably does not hold true, but that the structure-function relationship of surface active surfactant films is even more complex than hitherto thought.

Surfactant protein-D regulates surfactant phospholipid homeostasis in vivo.

Surfactant protein D (SP-D) is a 43-kDa member of the collectin family of collagenous lectin domain-containing proteins that is expressed in epithelial cells of the lung. The SP-D gene was targeted by homologous recombination in embryonic stem cells that were used to produce SP-D (+/-) and SP-D (-/-) mice. Both SP-D (-/-) and SP-D (+/-) mice survived normally in the perinatal and postnatal periods. Whereas no abnormalities were observed in SP-D (+/-) mice, alveolar and tissue phosphatidylcholine pool sizes were markedly increased in SP-D (-/-) mice. Increased numbers of large foamy alveolar macrophages and enlarged alveoli were also observed in SP-D (-/-) mice. Phospholipid composition was unaltered in SP-D (-/-) mice, but surfactant morphology was abnormal, consisting of dense phospholipid membranous arrays with decreased tubular myelin. The pulmonary lipoidosis in the SP-D (-/-) mice was not associated with accumulation of surfactant proteins B or C, or their mRNAs, distinguishing the disorder from alveolar proteinosis syndromes. Surfactant protein A mRNA was reduced and, SP-A protein appeared to be reduced in SP-D (-/-) compared with wild type mice. Targeting of the mouse SP-D gene caused accumulation of surfactant lipid and altered phospholipid structures, demonstrating a previously unsuspected role for SP-D in surfactant lipid homeostasis in vivo.