The collagen-like region of surfactant protein A (SP-A) is required for correction of surfactant structural and functional defects in the SP-A null mouse

Pulmonary surfactant isolated from gene-targeted surfactant protein A null mice (SP-A(-/-)) is deficient in the surfactant aggregate tubular myelin and has surface tension-lowering activity that is easily inhibited by serum proteins in vitro. To further elucidate the role of SP-A and its collagen-like region in surfactant function, we used the human SP-C promoter to drive expression of rat SP-A (rSPA) or SP-A containing a deletion of the collagen-like domain (DeltaG8-P80) in the Clara cells and alveolar type II cells of SP-A(-/-) mice. The level of the SP-A in the alveolar wash of the SP-A(-/-,rSP-A) and SP-A(-/-,DeltaG8-P80) mice was 6.1-and 1.3-fold higher, respectively, than in the wild type controls. Tissue levels of saturated phosphatidylcholine were slightly reduced in the SP-A(-/-,rSP-A) mice compared with SP-A(-/-) littermates. Tubular myelin was present in the large surfactant aggregates isolated from the SP-A(-/-,rSP-A) lines but not in the SP-A(-/-,DeltaG8-P80) mice or SP-A(-/-) controls. The equilibrium and minimum surface tensions of surfactant from the SP-A(-/-,rSP-A) mice were similar to SP-A(-/-) controls, but both were markedly elevated in the SP-A(-/-,DeltaG8-P80) mice. There was no defect in the surface tension-lowering activity of surfactant from SP-A(+/+,DeltaG8-P80) mice, indicating that the inhibitory effect of DeltaG8-P80 on surface activity can be overcome by wild type levels of mouse SP-A. The surface activity of surfactant isolated from the SP-A(-/-,rSP-A) but not the SP-A(-/-,DeltaG8-P80) mice was more resistant than SP-A(-/-) littermate control animals to inhibition by serum proteins in vitro. Pressure volume relationships of lungs from the SP-A(-/-), SP-A(-/-,rSP-A), and SP-A(-/-,DeltaG8-P80) lines were very similar. These data indicate that expression of SP-A in the pulmonary epithelium of SP-A(-/-) animals restores tubular myelin formation and resistance of isolated surfactant to protein inhibition by a mechanism that is dependent on the collagen-like region.     

Pulmonary surfactant function is abolished by an elevated proportion of cholesterol

A molecular film of pulmonary surfactant strongly reduces the surface tension of the lung epithelium-air interface. Human pulmonary surfactant contains 5-10% cholesterol by mass, among other lipids and surfactant specific proteins. An elevated proportion of cholesterol is found in surfactant, recovered from acutely injured lungs (ALI). The functional role of cholesterol in pulmonary surfactant has remained controversial. Cholesterol is excluded from most pulmonary surfactant replacement formulations, used clinically to treat conditions of surfactant deficiency. This is because cholesterol has been shown in vitro to impair the surface activity of surfactant even at a physiological level. In the current study, the functional role of cholesterol has been re-evaluated using an improved method of evaluating surface activity in vitro, the captive bubble surfactometer (CBS). Cholesterol was added to one of the clinically used therapeutic surfactants, BLES, a bovine lipid extract surfactant, and the surface activity evaluated, including the adsorption rate of the substance to the air-water interface, its ability to produce a surface tension close to zero and the area compression needed to obtain that low surface tension. No differences in the surface activity were found for BLES samples containing either none, 5 or 10% cholesterol by mass with respect to the minimal surface tension. Our findings therefore suggest that the earlier-described deleterious effects of physiological amounts of cholesterol are related to the experimental methodology. However, at 20%, cholesterol effectively abolished surfactant function and a surface tension below 15 mN/m was not obtained. Inhibition of surface activity by cholesterol may therefore partially or fully explain the impaired lung function in the case of ALI. We discuss a molecular mechanism that could explain why cholesterol does not prevent low surface tension of surfactant films at physiological levels but abolishes surfactant function at higher levels.     

The role of palmitic acid in pulmonary surfactant: enhancement of surface activity and prevention of inhibition by blood proteins

The surface activity of two surfactant preparations, Lipid Extract Surfactant (LES) and Survanta, was examined during adsorption and dynamic compression using a pulsating bubble surfactometer. At low surfactant phospholipid concentrations (1-2.5 mg/ml), Survanta reduces surface tension at minimum bubble radius faster than LES: however, with continued pulsation LES obtains a lower surface tension. Addition of surfactant-associated protein A (SP-A) to LES significantly reduces the time required to reduce surface tension. Survanta is completely unresponsive to the addition of SP-A in that no further reduction of surface tension is observed. Addition of various blood components has been previously shown to inactivate surfactants in vitro. Addition of fibrinogen to Survanta causes an increase in surface tension when measured in the absence of calcium. When assayed in the presence of calcium, inhibition by fibrinogen is not observed possibly due to aggregation of this protein. Albumin and alpha-globulin strongly inhibit Survanta at physiological serum concentrations both in the presence and absence of calcium. The surface activity of Survanta is also inhibited by lysophosphatidylcholine (lyso-PC). The role of palmitic acid in the surface activity of pulmonary surfactant was examined by adding palmitic acid to LES. At low phospholipid concentrations addition of palmitic acid (10% w/w of the surfactant phospholipid) greatly enhances the surface activity of LES. Maximal enhancement of surface activity and adsorption was observed at or above 7.5% added palmitic acid (w/w of surfactant lipid). LES supplemented with palmitic acid is more resistant to inhibition by fibrinogen, albumin, alpha-globulin and lyso-PC than LES alone, however, the counteraction of blood protein inhibition is not as pronounced as that observed with SP-A.     

Dipalmitoylphosphatidylcholine and cholesterol in monolayers spread from adsorbed films of pulmonary surfactant

Pulmonary surfactant forms a surface film that consists of a monolayer and a monolayer-associated reservoir. The extent to which surfactant components including the main component, dipalmitoylphosphatidylcholine (DPPC), are adsorbed into the monolayer, and how surfactant protein SP-A affects their adsorptions, is not clear. Transport of cholesterol to the surface region from dispersions of bovine lipid extract surfactant [BLES(chol)] with or without SP-A at 37 degrees C was studied by measuring surface radioactivities of [4-(14)C]cholesterol-labeled BLES(chol), and the Wilhelmy plate technique was used to monitor adsorption of monolayers. Results showed that transport of cholesterol was lipid concentration dependent. SP-A accelerated lipid adsorption but suppressed the final level of cholesterol in the surface. Surfactant adsorbed from a dispersion with or without SP-A was transferred via a wet filter paper to a clean surface, where the surface radioactivity and surface tension were recorded simultaneously. It was observed that 1) surface radioactivity was constant over a range of dispersion concentrations; 2) cholesterol and DPPC were transferred simultaneously; and 3) SP-A limited transfer of cholesterol.These results indicate that non-DPPC components of pulmonary surfactant can be adsorbed into the monolayer. Studies in the transfer of [1-(14)C]DPPC-labeled BLES(chol) to an equal or larger clean surface area revealed that SP-A did not increase selective adsorption of DPPC into the monolayer. Evaluation of transferred surfactant with a surface balance indicated that it equilibrated as a monolayer. Furthermore, examination of transferred surfactants from dispersions with and without prespread BLES(chol) monolayers revealed a functional contiguous association between adsorbed monolayers and reservoirs.     

Chemical modification of surfactant protein A alters high affinity binding to rat alveolar type II cells and regulation of phospholipid secretion

Alveolar type II cells express a high affinity receptor for pulmonary surfactant protein A (SP-A), and the interaction of SP-A with these cells leads to inhibition of surfactant lipid secretion. We have investigated the binding of native and modified forms of SP-A to isolated rat alveolar type II cells. Native and deglycosylated forms of SP-A readily competed with 125I-SP-A for cell surface binding. Alkylation of SP-A with excess iodoacetamide yielded forms of SP-A that did not inhibit surfactant lipid secretion and did not compete with 125I-SP-A for cell surface binding. Reductive methylation of SP-A with H2CO and NaCNBH3 yielded forms of SP-A with markedly reduced receptor binding activity that also exhibited significantly reduced capacity to inhibit lipid secretion. Modification of SP-A with cyclohexanedione reversibly altered cell surface binding and the activity of SP-A as an inhibitor of lipid secretion. Two monoclonal antibodies that block the function of SP-A as an inhibitor of lipid secretion completely prevented the high affinity binding of SP-A to type II cells. A monoclonal antibody that recognizes epitopes on SP-A but failed to block the inhibition of secretion also failed to completely attenuate high affinity binding to the receptor. Concanavalin A inhibits phospholipid secretion of type II cells by a mechanism that is reversed in the presence of excess alpha-methylmannoside. Concanavalin A did not block the high affinity binding of 125I-SP-A to the receptor. Neither the high affinity binding nor the inhibitor activity of SP-A was prevented by the presence of mannose or alpha-methylmannoside. The SP-A derived from humans with alveolar proteinosis is a potent inhibitor of surfactant lipid secretion but failed to completely displace 125I-SP-A binding from type II cells. From these data we conclude that: 1) cell surface binding activity of rat SP-A is directly related to its capacity to inhibit surfactant lipid secretion; 2) monoclonal antibodies directed against SP-A can be used to map binding domains for the receptor; 3) the lectin activity of SP-A against mannose ligands does not appear to be essential for cell surface binding; 4) concanavalin A does not compete with SP-A for receptor binding; and 5) the human SP-A derived from individuals with alveolar proteinosis exhibits different binding characteristics from rat SP-A.     

A comparative study of mechanisms of surfactant inhibition

Pulmonary surfactant spreads to the hydrated air-lung interface and reduces the surface tension to a very small value. This function fails in acute respiratory distress syndrome (ARDS) and the surface tension stays high. Dysfunction has been attributed to competition for the air-lung interface between plasma proteins and surfactant or, alternatively, to ARDS-specific alterations of the molecular profile of surfactant. Here, we compared the two mechanisms in vitro, to assess their potential role in causing respiratory distress. Albumin and fibrinogen exposure at or above blood level concentrations served as the models for testing competitive adsorption. An elevated level of cholesterol was chosen as a known adverse change in the molecular profile of surfactant in ARDS. Bovine lipid extract surfactant (BLES) was spread from a small bolus of a concentrated suspension (27 mg/ml) to the air-water interface in a captive bubble surfactometer (CBS) and the bubble volume was cyclically reduced and increased to assess surface activity of the spread material. Concentrations of inhibitors and the concentration and spreading method of pulmonary surfactant were chosen in an attempt to reproduce the exposure of surfactant to inhibitors in the lung. Under these conditions, neither serum albumin nor fibrinogen was persistently inhibitory and normal near-zero minimum surface tension values were obtained after a small number of cycles. In contrast, inhibition by an increased level of cholesterol persisted even after extensive cycling. These results suggest that in ARDS, competitive adsorption may not sufficiently explain high surface tension, and that disruption of the surfactant film needs to be given causal consideration.     

Enhancement of biophysical activity of lung surfactant extracts and phospholipid-apoprotein mixtures by surfactant protein A

The effects of surfactant protein (SP)-A on the dynamic surface tension lowering and resistance to inhibition of dispersions of calf lung surfactant extract (CLSE) and mixtures of synthetic phospholipids combined with SP-B,C hydrophobic apoproteins were studied at 37 degrees C and rapid cycling rate (20 cycles/min). Addition of SP-A to CLSE, which already contains SP-B and -C, gave a slight improvement in the time course of surface tension lowering on an oscillating bubble apparatus in the absence of inhibitory protein molecules such as albumin or hemoglobin. However, when these proteins were present at concentrations of 10-50 mg/ml, SP-A substantially improved the resistance of CLSE to their inhibitory effects. The beneficial effect of SP-A required the presence of Ca2+ ions, and disappeared when EDTA was substituted for this divalent cation in the subphase. The effect was also retained when SP-A was heated to 50 degrees C prior to addition to CLSE, but was abolished by heating SP-A to 99 degrees C. Additional studies showed that similar improvements in resistance to inhibition were found when SP-A was added to synthetic mixtures of dipalmitoyl phosphatidylcholine (DPPC):egg phosphatidylglycerol (PG) (80:20 by weight) reconstituted with 1% SP-B or SP-B and -C, but not to phospholipid mixtures containing only SP-C. The requirements for SP-B and calcium for the beneficial effects of SP-A on surface activity suggest that the formation of ordered, larger phospholipid-apoprotein aggregates may be involved in the process. The finding that SP-A enhances the ability of CLSE and other surfactant mixtures containing SP-B to resist inhibition is an advantage that will need to be weighed against other factors such as increased antigenicity and heat sensitivity in therapeutic applications in surfactant replacement therapy.     

Interaction of pulmonary surfactant protein SP-A with DPPC/egg-PG bilayers

In the mixture of lipids and proteins which comprise pulmonary surfactant, the dominant protein by mass is surfactant protein A (SP-A), a hydrophilic glycoprotein. SP-A forms octadecamers that interact with phospholipid bilayer surfaces in the presence of calcium. Deuterium NMR was used to characterize the perturbation by SP-A, in the presence of 5 mM Ca²⁺, of dipalmitoyl phosphatidylcholine (DPPC) properties in DPPC/egg-PG (7:3) bilayers. Effects of SP-A were uniformly distributed over the observed DPPC population. SP-A reduced DPPC chain orientational order significantly in the gel phase but only slightly in the liquid-crystalline phase. Quadrupole echo decay times for DPPC chain deuterons were sensitive to SP-A in the liquid-crystalline mixture but not in the gel phase. SP-A reduced quadrupole splittings of DPPC choline β-deuterons but had little effect on choline α-deuteron splittings. The observed effects of SP-A on DPPC/egg-PG bilayer properties differ from those of the hydrophobic surfactant proteins SP-B and SP-C. This is consistent with the expectation that SP-A interacts primarily at bilayer surfaces.     

Dysfunction of pulmonary surfactant mediated by phospholipid oxidation is cholesterol-dependent

Pulmonary surfactant forms a cohesive film at the alveolar air-lung interface, lowering surface tension, and thus reducing the work of breathing and preventing atelectasis. Surfactant function becomes impaired during inflammation due to degradation of the surfactant lipids and proteins by free radicals. In this study, we examine the role of reactive nitrogen (RNS) and oxygen (ROS) species on surfactant function with and without physiological cholesterol levels (5–10%). Surface activity was assessed in vitro in a captive bubble surfactometer (CBS). Surfactant chemistry, monolayer fluidity and thermodynamic behavior were also recorded before and after oxidation. We report that physiologic amounts of cholesterol combined with oxidation results in severe impairment of surfactant function. We also show that surfactant polyunsaturated phospholipids are the most susceptible to oxidative alteration. Membrane thermodynamic experiments showed significant surfactant film stiffening after free radical exposure in the presence of cholesterol. These results point to a previously unappreciated role for cholesterol in amplifying defects in surface activity caused by oxidation of pulmonary surfactant, a finding that may have implications for treating several lung diseases.     

Cation-mediated conformational variants of surfactant protein A

Surfactant protein A (SP-A) is the major protein of pulmonary surfactant. This protein is implicated in regulating surfactant secretion, alveolar processing, recycling, and in non-serum-induced immune response. An increasing body of work indicates the importance of cations, particularly calcium, on SP-A function. However, little information exists on the effects of cations on SP-A quaternary structure. Here, the quaternary organisation of bovine surfactant protein A in the presence of cations has been quantitatively and systematically studied by transmission electron microscopy. The conformation of SP-A is altered by the presence of cations, especially calcium, then sodium, and to a small extent, magnesium. There is a transition concentration, unique for each cation, at which a conformational switch occurs. These transition concentrations are: 5 mM for CaCl₂, 100 mM for NaCl and 1 mM for MgCl₂. Below these concentrations, SP-A exists primarily in an opened form with a large head diameter of 20 nm; above it, SP-A is mostly in a closed form due to a compaction of the headgroups resulting in a head diameter of 11 nm. There is a corresponding increase in particle length from 17 nm for opened SP-A to 20 nm for closed SP-A. The fact that the transition concentrations are within physiological range suggests that cation-mediated conformational changes of SP-A could be operative in vivo.     

A comparative study of mechanisms of surfactant inhibition

Pulmonary surfactant spreads to the hydrated air-lung interface and reduces the surface tension to a very small value. This function fails in acute respiratory distress syndrome (ARDS) and the surface tension stays high. Dysfunction has been attributed to competition for the air-lung interface between plasma proteins and surfactant or, alternatively, to ARDS-specific alterations of the molecular profile of surfactant. Here, we compared the two mechanisms in vitro, to assess their potential role in causing respiratory distress. Albumin and fibrinogen exposure at or above blood level concentrations served as the models for testing competitive adsorption. An elevated level of cholesterol was chosen as a known adverse change in the molecular profile of surfactant in ARDS. Bovine lipid extract surfactant (BLES) was spread from a small bolus of a concentrated suspension (27 mg/ml) to the air-water interface in a captive bubble surfactometer (CBS) and the bubble volume was cyclically reduced and increased to assess surface activity of the spread material. Concentrations of inhibitors and the concentration and spreading method of pulmonary surfactant were chosen in an attempt to reproduce the exposure of surfactant to inhibitors in the lung. Under these conditions, neither serum albumin nor fibrinogen was persistently inhibitory and normal near-zero minimum surface tension values were obtained after a small number of cycles. In contrast, inhibition by an increased level of cholesterol persisted even after extensive cycling. These results suggest that in ARDS, competitive adsorption may not sufficiently explain high surface tension, and that disruption of the surfactant film needs to be given causal consideration.     

The effect of diet-induced serum hypercholesterolemia on the surfactant system and the development of lung injury

Acute respiratory distress syndrome (ARDS) is a pulmonary disorder associated with alterations to the pulmonary surfactant system. Recent studies showed that supra-physiological levels of cholesterol in surfactant contribute to impaired function. Since cholesterol is incorporated into surfactant within the alveolar type II cells which derives its cholesterol from serum, it was hypothesized that serum hypercholesterolemia would predispose the host to the development of lung injury due to alterations of cholesterol content in the surfactant system.Wistar rats were randomized to a standard lab diet or a high cholesterol diet for 17–20 days. Animals were then exposed to one of three models of lung injury: i) acid aspiration ii) ventilation induced lung injury, and iii) surfactant depletion. Following physiological monitoring, lungs were lavaged to obtain and analyze the surfactant system.The physiological results showed there was no effect of the high cholesterol diet on the severity of lung injury in any of the three models of injury. There was also no effect of the diet on surfactant cholesterol composition. Rats fed a high cholesterol diet had a significant impairment in surface tension reducing capabilities of isolated surfactant compared to those fed a standard diet exposed to the surfactant depletion injury. In addition, only rats that were exposed to ventilation induced lung injury had elevated levels of surfactant associated cholesterol compared to non-injured rats.It is concluded that serum hypercholesterolemia does not predispose rats to altered surfactant cholesterol composition or to lung injury. Elevated cholesterol within surfactant may be a marker for ventilation induced lung damage.     

Effects of cholesterol on surface activity and surface topography of spread surfactant films

Pulmonary surfactant forms a monolayer of lipids and proteins at the alveolar air/liquid interface. Although cholesterol is a natural component of surfactant, its function in surface dynamics is unclear. To further elucidate the role of cholesterol in surfactant, we used a captive bubble surfactometer (CBS) to measure surface activity of spread films containing dipalmitoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylglycerol (DPPC/POPC/POPG, 50/30/20 molar percentages), surfactant protein B (SP-B, 0.75 mol %), and/or surfactant protein C (SP-C, 3 mol %) with up to 20 mol % cholesterol. A cholesterol concentration of 10 mol % was optimal for reaching and maintaining low surface tensions in SP-B-containing films but led to an increase in maximum surface tension in films containing SP-C. No effect of cholesterol on surface activity was found in films containing both SP-B and SP-C. Atomic force microscopy (AFM) was used, for the first time, to visualize the effect of cholesterol on topography of SP-B- and/or SP-C-containing films compressed to a surface tension of 22 mN/m. The protrusions found in the presence of cholesterol were homogeneously dispersed over the film, whereas in the absence of cholesterol the protrusions tended to be more clustered into network structures. A more homogeneous dispersion of surfactant lipid components may facilitate lipid insertion into the surfactant monolayer. Our data provide additional evidence that natural surfactant, containing SP-B and SP-C, is superior to surfactants lacking one of the components, and furthermore, this raises the possibility that the cholesterol found in surfactant of warm-blooded mammals does not have a function in surface activity.     

Physiological and inflammatory response to instillation of an oxidized surfactant in a rat model of surfactant deficiency.

Pulmonary surfactant is a mixture of phospholipids ( approximately 90%) and surfactant-associated proteins (SPs) ( approximately 10%) that stabilize the lung by reducing the surface tension. One proposed mechanism by which surfactant is altered during acute lung injury is via direct oxidative damage to surfactant. In vitro studies have revealed that the surface activity of oxidized surfactant was impaired and that this effect could be overcome by adding SP-A. On the basis of this information, we hypothesized that animals receiving oxidized surfactant preparations would exhibit an inferior physiological and inflammatory response and that the addition of SP-A to the oxidized preparations would ameliorate this response. To test this hypothesis, mechanically ventilated, surfactant-deficient rats were administered either bovine lipid extract surfactant (BLES) or in vitro oxidized BLES of three doses: 10 mg/kg, 50 mg/kg, or 10 mg/kg + SP-A. When instilled with 10 mg/kg normal surfactant, the rats had a significantly superior arterial Po2 responses compared with the rats receiving oxidized surfactant. Interestingly, increasing the dose five times mitigated this physiological effect, and the addition of SP-A to the surfactant preparation had little impact on improving oxygenation. There were no differences in alveolar surfactant pools and the indexes of pulmonary inflammation between the 10 mg/kg dose groups, nor was there any differences observed between either of the groups supplemented with SP-A. However, there was significantly more surfactant and more inflammatory cytokines in the 50 mg/kg oxidized BLES group compared with the 50 mg/kg BLES group. We conclude that instillation of an in vitro oxidized surfactant causes an inferior physiological response in a surfactant-deficient rat.     

Serum surfactant protein-A,but not surfactant protein-D or KL-6, can predict preclinical lung damage induced by smoking

Serum surfactant protein (SP)-A offers a useful clinical marker for interstitial lung disease (ILD). However, SP-A is occasionally elevated in non-ILD pulmonary patients. The present study was conducted to investigate factors that affect serum SP-A levels in respiratory medicine. Serum SP-A, serum SP-D, serum Klebs von den Lungen (KL)-6 and pulmonary function tests were evaluated in 929 patients (current smokers, n=255; ex-smokers, n=242; never-smokers, n=432) without ILD or pulmonary alveolar proteinosis. Serum SP-A was significantly higher in current smokers than in never- or ex-smokers (p<0.01 and p<0.05, respectively). Serum SP-A was significantly higher in chronic obstructive pulmonary disease (COPD) and pulmonary thromboembolism than in other diseases (p<0.01). Serum SP-A correlated positively with amount of smoking (p<0.01) and negatively with forced expiratory volume in 1 s/forced vital capacity (p<0.05). Serum SP-D and KL-6 were unaffected by smoking. Smoking should be taken into account when evaluating serum SP-A levels, and different baseline levels of serum SP-A should be established for smokers and non-smokers. Serum SP-A may also represent a useful marker for predicting COPD in the preclinical stage.     

The role of pulmonary collectin N-terminal domains in surfactant structure,function, and homeostasis in vivo

The N-terminal domains of the lung collectins, surfactant proteins A (SP-A) and D (SP-D), are critical for surfactant phospholipid interactions and surfactant homeostasis, respectively. To further assess the importance of lung collectin N-terminal domains in surfactant structure and function, a chimeric SP-D/SP-A (D/A) gene was constructed by substituting nucleotides encoding amino acids Asn(1)-Ala(7) of rat SP-A with the corresponding N-terminal sequences from rat SP-D, Ala(1)-Asn(25). Recombinant D/A migrated as a 35-kDa band on reducing SDS-PAGE and as a ladder of disulfide-linked multimers under nonreducing conditions. The recombinant D/A bound and aggregated phosphatidylcholine containing vesicles as effectively as rat SP-A. Mice in which endogenous pulmonary collectins were replaced with D/A were developed by human SP-C promoter-driven overexpression of the D/A gene in SP-A(-/-) and SP-D(-/-) animals. Analysis of lavage fluid from SP-A(-/-,D/A) mice revealed that glycosylated, oligomeric D/A was secreted into the air spaces at levels that were comparable with the authentic collectins and that the N-terminal interchange converted SP-A from a "bouquet" to a cruciform configuration. Transmission electron microscopy of surfactant from the SP-A(-/-,D/A) mice revealed atypical tubular myelin containing central "target-like" electron density. Surfactant isolated from SP-A(-/-,D/A) mice exhibited elevated surface tension both in the presence and absence of plasma inhibitors, but whole lung compliance of the SP-A(-/-,D/A) animals was not different from the SP-A(-/-) littermates. Lung-specific overexpression of D/A in the SPD(-/-) mouse resulted in hetero-oligomer formation with mouse SP-A and did not correct the air space dilation or phospholipidosis that occurs in the absence of SP-D. These studies indicate that the N terminus of SP-D 1) can functionally replace the N terminus of SP-A for lipid aggregation and tubular myelin formation, but not for surface tension lowering properties of SP-A, and 2) is not sufficient to reverse the structural and metabolic pulmonary defects in the SP-D(-/-) mouse.     

Differential effects of human SP-A1 and SP-A2 variants on phospholipid monolayers containing surfactant protein B

Surfactant protein A (SP-A), the most abundant protein in the lung alveolar surface, has multiple activities, including surfactant-related functions. SP-A is required for the formation of tubular myelin and the lung surface film. The human SP-A locus consists of two functional SP-A genes, SP-A1 and SP-A2, with a number of alleles characterized for each gene. We have found that the human in vitro expressed variants, SP-A1 (6A²) and SP-A2 (1A⁰), and the coexpressed SP-A1/SP-A2 (6A²/1A⁰) protein have a differential influence on the organization of phospholipid monolayers containing surfactant protein B (SP-B). Lipid films containing SP-B and SP-A2 (1A⁰) showed surface features similar to those observed in lipid films with SP-B and native human SP-A. Fluorescence images revealed the presence of characteristic fluorescent probe-excluding clusters coexisting with the traditional lipid liquid-expanded and liquid-condensed phase. Images of the films containing SP-B and SP-A1 (6A²) showed different distribution of the proteins. The morphology of lipid films containing SP-B and the coexpressed SP-A1/SP-A2 (6A²/1A⁰) combined features of the individual films containing the SP-A1 or SP-A2 variant. The results indicate that human SP-A1 and SP-A2 variants exhibit differential effects on characteristics of phospholipid monolayers containing SP-B. This may differentially impact surface film activity.     

Lamellar bodies of cultured human fetal lung: content of surfactant protein A (SP-A), surface film formation and structural transformation in vitro

Lamellar bodies were isolated from dexamethasone and T3-treated explant cultures of human fetal lung, using sucrose density-gradient centrifugation. We examined their content of surfactant apoprotein A (SP-A), and their ability to form surface films and to undergo structural transformation in vitro. SP-A measured by ELISA composed less than 2% of total protein within lamellar bodies; this represented, as a minimum estimate, a 2-12-fold enrichment over homogenate. One- and two-dimensional gel electrophoresis also suggested that SP-A was a minor protein component of lamellar bodies. Adsorption of lamellar bodies to an air/water interface was moderately rapid, but accelerated dramatically upon addition of exogenous SP-A in ratios of 1:2-16 (SP-A:phospholipid, w/w). Similar adsorption patterns were seen for lamellar bodies from fresh adult rat and rabbit lung. Lamellar bodies incubated under conditions that promote formation of tubular myelin underwent structural rearrangement only in the presence of exogenous SP-A, with extensive formation of multilamellate whorls of lipid bilayers (but no classical tubular myelin lattices). We conclude that lamellar bodies are enriched in SP-A, but have insufficient content of SP-A for structural transformation to tubular myelin and rapid surface film formation in vitro.     

TGF-alpha perturbs surfactant homeostasis in vivo

To determine potential relationships between transforming growth factor (TGF)-alpha and surfactant homeostasis, the metabolism, function, and composition of surfactant phospholipid and proteins were assessed in transgenic mice in which TGF-alpha was expressed in respiratory epithelial cells. Secretion of saturated phosphatidylcholine was decreased 40-60% by expression of TGF-alpha. Although SP-A, SP-B, and SP-C mRNA levels were unchanged by expression of TGF-alpha, SP-A and SP-B content in bronchoalveolar lavage fluid was decreased. The minimum surface tension of surfactant isolated from the transgenic mice was significantly increased. Incubation of cultured normal mice type II cells with TGF-alpha in vitro did not change secretion of surfactant phosphatidylcholine and SP-B, indicating that TGF-alpha does not directly influence surfactant secretion. Expression of a dominant negative (mutant) EGF receptor in the respiratory epithelium blocked the TGF-alpha-induced changes in lung morphology and surfactant secretion, indicating that EGF receptor signaling in distal epithelial cells was required for TGF-alpha effects on surfactant homeostasis. Because many epithelial cells were embedded in fibrotic lesions caused by TGF-alpha, changes in surfactant homeostasis may at least in part be influenced by tissue remodeling that results in decreased surfactant secretion. The number of nonembedded type II cells was decreased 30% when TGF-alpha was expressed during development and was increased threefold by TGF-alpha expression in adulthood, suggesting possible alteration of type II cells on surfactant metabolism in the adult lung. Abnormalities in surfactant function and decreased surfactant level in the airways may contribute to the pathophysiology induced by TGF-alpha in both the developing and adult lung.     

Effect of surfactant protein A on the physical properties and surface activity of KL4-surfactant

SP-A, the major protein component of pulmonary surfactant, is absent in exogenous surfactants currently used in clinical practice. However, it is thought that therapeutic properties of natural surfactants improve after enrichment with SP-A. The objective of this study was to determine SP-A effects on physical properties and surface activity of a new synthetic lung surfactant based on a cationic and hydrophobic 21-residue peptide KLLLLKLLLLKLLLLKLLLLK, KL(4). We have analyzed the interaction of SP-A with liposomes consisting of DPPC/POPG/PA (28:9:5.6, w/w/w) with and without 0.57 mol % KL(4) peptide. We found that SP-A had a concentration-dependent effect on the surface activity of KL(4)-DPPC/POPG/PA membranes but not on that of an animal-derived LES. The surface activity of KL(4)-surfactant significantly improved after enrichment with 2.5-5 wt % SP-A. However, it worsened at SP-A concentrations > or =10 wt %. This was due to the fluidizing effect of supraphysiological SP-A concentrations on KL(4)-DPPC/POPG/PA membranes as determined by fluorescence anisotropy measurements, calorimetric studies, and confocal fluorescence microscopy of GUVs. High SP-A concentrations caused disappearance of the solid/fluid phase coexistence of KL(4)-surfactant, suggesting that phase coexistence might be important for the surface adsorption process.