Surfactant lipid peroxidation damages surfactant protein A and inhibits interactions with phospholipid vesicles

The goal of these studies was to examine the effect of lipid peroxidation (LPO) on the function of surfactant protein A (SP-A). First, the optimal dialysis conditions for quantitative removal of EDTA and redoxactive metals from reagents were established. Surfactant phospholipids were incubated with free radical generators in the absence or presence of the SP-A or with BSA as a control. We found that SP-A inhibited copper-initiated LPO to an extent similar to BSA (P < 0.05). Exposure of SP-A to LPO was associated with an increase in the level of SP-A-associated carbonyl moieties and a marked reduction in SP-A-mediated aggregation of liposomes. LPO initiated by an azo-compound also resulted in enhanced protein oxidation and markedly inhibited SP-A-mediated liposome aggregation. The kinetics of aggregation of auto-oxidized and nonoxidized liposomes by nonoxidized SP-A was similar, suggesting that SP-A has similar affinities for oxidized and nonoxidized lipids. Oxidative inactivation of SP-A did not occur upon direct incubation of the protein with malondialdehyde alone. We conclude that exposure of SP-A to LPO results in oxidative modification and functional inactivation of SP-A by phospholipid radicals.     

Enhanced clearance of surfactant protein D during LPS-induced acute inflammation in rat lung

Pulmonary surfactant participates in the regulation of alveolar compliance and lung host defense. Surfactant homeostasis is regulated through a combination of synthesis, secretion, clearance, recycling, and degradation of surfactant components. The extracellular pool size of surfactant protein (SP) D fluctuates significantly during acute inflammation. We hypothesized that changes in SP-D levels are due, in part, to altered clearance of SP-D. Clearance pathways in rats were assessed with fluorescently labeled SP-D that was instilled into control lungs or lungs that had been treated with lipopolysaccharide (LPS) 16 h earlier. SP-D clearance from lavage into lung tissue was time dependent from 5 min to 1 h and 1.7-fold greater in LPS-treated lungs than in control lungs. Analysis of cells isolated by enzymatic digestion of lung tissue revealed differences in the SP-D-positive cell population between groups. LPS-treated lungs had 28.1-fold more SP-D-positive tissue-associated neutrophils and 193.6-fold greater SP-D association with those neutrophils compared with control lungs. These data suggest that clearance of SP-D into lung tissue is increased during inflammation and that tissue-associated neutrophils significantly contribute to this process.     

Surfactant protein D enhances clearance of influenza A virus from the lung in vivo.

Mice lacking surfactant protein surfactant protein D (SP-D(-/-)) and wild-type mice (SP-D(+/+)) were infected with influenza A virus (IAV) by intranasal instillation. IAV infection increased the endogenous SP-D concentration in wild-type mice. SP-D-deficient mice showed decreased viral clearance of the Phil/82 strain of IAV and increased production of inflammatory cytokines in response to viral challenge. However, the less glycosylated strain of IAV, Mem/71, which is relatively resistant to SP-D in vitro, was cleared efficiently from the lungs of SP-D(-/-) mice. Viral clearance of the Phil/82 strain of IAV and the cytokine response were both normalized by the coadministration of recombinant SP-D. Since the airway is the usual portal of entry for influenza A virus and other respiratory pathogens, SP-D is likely to play an important role in innate defense responses to IAV.     

Surfactant protein A regulates complement activation.

Complement proteins aid in the recognition and clearance of pathogens from the body. C1, the first protein of the classical pathway of complement activation, is a calcium-dependent complex of one molecule of C1q and two molecules each of C1r and C1s, the serine proteases that cleave complement proteins. Upon binding of C1q to Ag-bound IgG or IgM, C1r and C1s are sequentially activated and initiate the classical pathway of complement. Because of structural and functional similarities between C1q and members of the collectin family of proteins, including pulmonary surfactant protein A (SP-A), we hypothesized that SP-A may interact with and regulate proteins of the complement system. Previously, SP-A was shown to bind to C1q, but the functional significance of this interaction has not been investigated. Binding studies confirmed that SP-A binds directly to C1q, but only weakly to intact C1. Further investigation revealed that the binding of SP-A to C1q prevents the association of C1q with C1r and C1s, and therefore the formation of the active C1 complex required for classical pathway activation. This finding suggests that SP-A may share a common binding site for C1r and C1s or Clq. SP-A also prevented C1q and C1 from binding to immune complexes. Furthermore, SP-A blocked the ability of C1q to restore classical pathway activity to C1q-depleted serum. SP-A may down-regulate complement activity through its association with C1q. We hypothesize that SP-A may serve a protective role in the lung by preventing C1q-mediated complement activation and inflammation along the delicate alveolar epithelium.     

Surfactant protein A regulates pulmonary surfactant secretion via activation of phosphatidylinositol 3-kinase in type II alveolar cells

Pulmonary surfactant is secreted by the type II alveolar cells of the lung, and this secretion is induced by secretagogues of several types (e.g., ionomycin, phorbol esters, and terbutaline). Secretagogue-induced secretion is inhibited by surfactant-associated protein A (SP-A), which binds to a specific receptor (SPAR) on the surface of type II cells. The mechanism of SP-A-activated SPAR signaling is completely unknown. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 rescued surfactant secretion from inhibition by SP-A. In order to directly demonstrate a role for PI3K in SPAR signaling, PI3K activity was immunoprecipitated from type II cell extracts. PI3K activity increased rapidly after SP-A addition to type II cells. Since many receptors that activate PI3K do so through tyrosine-specific protein phosphorylation, antisera to phosphotyrosine, insulin-receptor substrate-1 (IRS-1), or SPAR were also examined. These antisera coimmunoprecipitated PI3K activity that was stimulated by SP-A. In addition, the tyrosine-specific protein kinase inhibitors genistein and herbimycin A blocked the action of SP-A on surfactant secretion. We conclude that SP-A signals to regulate surfactant secretion through SPAR, via pathways that involve tyrosine phosphorylation, include IRS-1, and entail activation of PI3K. This activation leads to inhibition of secretagogue-induced secretion of pulmonary surfactant.     

Surfactant protein A regulates IgG-mediated phagocytosis in inflammatory neutrophils

Surfactant proteins (SP)-A and SP-D have been shown to affect the functions of a variety of innate immune cells and to interact with various immune proteins such as complement and immunoglobulins. The goal of the current study is to test the hypothesis that SP-A regulates IgG-mediated phagocytosis by neutrophils, which are major effector cells of the innate immune response that remove invading pathogens by phagocytosis and by extracellular killing mediated by reactive oxygen and nitrogen. We have previously shown that SP-A stimulates chemotaxis by inflammatory, but not peripheral, neutrophils. To evaluate the ability of SP-A to modulate IgG-mediated phagocytosis, polystyrene beads were coated with BSA and treated with anti-BSA IgG. SP-A significantly and specifically enhanced IgG-mediated phagocytosis by inflammatory neutrophils, but it had no effect on beads not treated with IgG. SP-A bound to IgG-coated beads and enhanced their uptake via direct interactions with the beads as well as direct interactions with the neutrophils. SP-A did not affect reactive oxygen production or binding of IgG to neutrophils and had modest effects on polymerization of actin. These data suggest that SP-A plays an important role in mediating the phagocytic response of neutrophils to IgG-opsonized particles.     

Surfactant protein A mediates pulmonary clearance of Staphylococcus aureus

Surfactant protein A has been shown to enhance opsonization and clearance of Staphylococcus aureus in vitro. Here, the phagocytosis of alveolar S. aureus was investigated in vivo using intravital microscopy. Fluorescence labelled S. aureus Newman cells were intratracheally administered to anesthetized mice and the alveolar surface was observed for fifteen minutes. Confirming previously reported in vitro data, surfactant protein A-deficient mice showed a significantly reduced uptake of bacteria compared to wild-type mice.     

Inhibitory effects of surfactant protein A on surfactant phospholipid hydrolysis by secreted phospholipases A2

Hydrolysis of surfactant phospholipids by secreted phospholipases A(2) (sPLA(2)) contributes to surfactant dysfunction in acute respiratory distress syndrome. The present study demonstrates that sPLA(2)-IIA, sPLA(2)-V, and sPLA(2)-X efficiently hydrolyze surfactant phospholipids in vitro. In contrast, sPLA(2)-IIC, -IID, -IIE, and -IIF have no effect. Since purified surfactant protein A (SP-A) has been shown to inhibit sPLA(2)-IIA activity, we investigated the in vitro effect of SP-A on the other active sPLA(2) and the consequences of sPLA(2)-IIA inhibition by SP-A on surfactant phospholipid hydrolysis. SP-A inhibits sPLA(2)-X activity, but fails to interfere with that of sPLA(2)-V. Moreover, in vitro inhibition of sPLA(2)-IIA-induces surfactant phospholipid hydrolysis correlates with the concentration of SP-A in surfactant. Intratracheal administration of sPLA(2)-IIA to mice causes hydrolysis of surfactant phosphatidylglycerol. Interestingly, such hydrolysis is significantly higher for SP-A gene-targeted mice, showing the in vivo inhibitory effect of SP-A on sPLA(2)-IIA activity. Administration of sPLA(2)-IIA also induces respiratory distress, which is more pronounced in SP-A gene-targeted mice than in wild-type mice. We conclude that SP-A inhibits sPLA(2) activity, which may play a protective role by maintaining surfactant integrity during lung injury.     

Mitochondrial phospholipid bilayer structure is ruined after liver oxidative injury in vivo

The purpose of this study was to investigate whether, after oxidative injury in vivo, liver mitochondrial phospholipids suffered from structural defects similar to those we have previously observed after either chemical oxidation or respiration state IV incubation of isolated mitochondria in vitro. Oxidative injury of the liver was simulated by endogastric administration of CCl4 to rats in variable amounts for different times, under various conditions. Measurements of the phospholipid bilayer packing order were carried out by electron paramagnetic resonance (EPR) spectrometry of oriented planar samples of phospholipids extracted from liver mitochondria, spin labeled with 5-doxylstearoyl-lecithin. Disordering of the bilayer was revealed by the anisotropy loss of EPR spectra and reached a maximum value 4.5 h after CCl4 administration, vanishing thereafter. The observed disorder also increased with the amount of CCl4 administered, showing distinct dose-dependence, while administration of resveratrol soon after carbon tetrachloride decreased bilayer disordering by 50%. On the contrary, the order parameter S of spin labeled lecithin in isolated mitochondrial membranes from intoxicated rats revealed no change in membrane fluidity after oxidative stress. It is concluded that the phospholipid damage leading to disturbed bilayer geometry after oxidative attack already observed in model membranes and in isolated mitochondria in vitro also occurs in a simulated pathological state in vivo, indicating its possible occurrence also in real oxidative stress-linked pathologies as a contribution to the onset/sustaining of related diseases.     

Pathways for clearance of surfactant protein A from the lung

Uptake and degradation of (125)I-surfactant protein A (SP-A) over a 1-h period was studied in alveolar cells in culture and in isolated perfused lungs to elucidate the mechanism for clearance of the protein from the alveolar space. Specific inhibitors of clathrin- and actin-dependent endocytosis were utilized. In type II cells, uptake of SP-A, compared with controls, was decreased by 60% on incubation with clathrin inhibitors (amantadine and phenylarsine oxide) or with the actin inhibitor cytochalasin D. All agents reduced SP-A metabolism by alveolar macrophages. Untreated rat isolated perfused lungs internalized 36% of instilled SP-A, and 56% of the incorporated SP-A was degraded. Inhibitors of clathrin and actin significantly reduced SP-A uptake by approximately 54%, whereas cytochalasin D inhibited SP-A degradation. Coincubation of agents did not produce an additive effect on uptake of SP-A by cultured pneumocytes or isolated perfused lungs, indicating that all agents affected the same pathway. Thus SP-A clears the lung via a clathrin-mediated pathway that requires the polymerization of actin.     

Surfactant protein A differentially regulates peripheral and inflammatory neutrophil chemotaxis

Surfactant protein A (SP-A), a pulmonary lectin, plays an important role in regulating innate immune cell function. Besides accelerating pathogen clearance by pulmonary phagocytes, SP-A also stimulates alveolar macrophage chemotaxis and directed actin polymerization. We hypothesized that SP-A would also stimulate neutrophil chemotaxis. With the use of a Boyden chamber assay, we found that SP-A (0.5-25 microg/ml) did not stimulate chemotaxis of rat peripheral neutrophils or inflammatory bronchoalveolar lavage (BAL) neutrophils isolated from LPS-treated lungs. However, SP-A affected neutrophil chemotaxis toward the bacterial peptide formyl-met-leu-phe (fMLP). Surprisingly, the effect was different for the two neutrophil populations: SP-A reduced peripheral neutrophil chemotaxis toward fMLP (49 +/- 5% fMLP alone) and enhanced inflammatory BAL neutrophil chemotaxis (277 +/- 48% fMLP alone). This differential effect was not seen for the homologous proteins mannose binding lectin and complement protein 1q but was recapitulated by type IV collagen. SP-A bound both neutrophil populations comparably and did not alter formyl peptide binding. These data support a role for SP-A in regulating neutrophil migration in pulmonary tissue.     

IL-4 increases surfactant and regulates metabolism in vivo

Lung lavage fluid of patients with acute lung injury (ALI) has increased levels of interleukin-1 (IL-1) and neutrophils, but their relationship to the lung leak that characterizes these patients is unclear. To address this concern, we investigated the role of the neutrophil agonist platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF)] in the development of the acute neutrophil-dependent lung leak that is induced by giving IL-1 intratracheally to rats. We found that PAF acetyltransferase and PAF activities increased in lungs of rats given IL-1 intratracheally compared with lungs of sham-treated rats given saline intratracheally. The participation of PAF in the development of lung leak and lung neutrophil accumulation after IL-1 administration was suggested when treatment with WEB-2086, a commonly used PAF-receptor antagonist, decreased lung leak, lung myeloperoxidase activity, and lung lavage fluid neutrophil increases in rats given IL-1 intratracheally. Additionally, neutrophils recovered from the lung lavage fluid of rats given IL-1 intratracheally reduced more nitro blue tetrazolium (NBT) in vitro than neutrophils recovered from control rats or rats that had been given WEB-2086 and then IL-1. Histological examination indicated that the endothelial cell-neutrophil interfaces of cerium chloride-stained lung sections of rats given IL-1 contained increased cerium perhydroxide (the reaction product of cerium chloride with hydrogen peroxide) compared with lungs of control rats or rats treated with WEB-2086 and then given IL-1 intratracheally. These in vivo findings were supported by parallel findings showing that WEB-2086 treatment decreased neutrophil adhesion to IL-1-treated cultured endothelial cells in vitro. We concluded that PAF contributes to neutrophil recruitment and neutrophil activation in lungs of rats given IL-1 intratracheally.     

Differential effects of surfactant protein A on regional organization of phospholipid monolayers containing surfactant protein B or C

Epifluorescence microscopy combined with a surface balance was used to study monolayers of dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylglycerol (PG) (8:2, mol/mol) plus 17 wt % SP-B or SP-C spread on subphases containing SP-A in the presence or absence of 5 mM Ca(2+). Independently of the presence of Ca(2+) in the subphase, SP-A at a bulk concentration of 0.68 microg/ml adsorbed into the spread monolayers and caused an increase in the molecular areas in the films. Films of DPPC/PG formed on SP-A solutions showed a pressure-dependent coexistence of liquid-condensed (LC) and liquid-expanded (LE) phases. Apart from these surface phases, a probe-excluding phase, likely enriched in SP-A, was seen in the films between 7 mN/m < or = pi < or = 20 mN/m. In monolayers of SP-B/(DPPC/PG) spread on SP-A, regardless of the presence of calcium ions, large clusters of a probe-excluding phase, different from probe-excluding lipid LC phase, appeared and segregated from the LE phase at near-zero surface pressures and coexisted with the conventional LE and LC phases up to approximately 35 mN/m. Varying the levels of either SP-A or SP-B in films of SP-B/SP-A/(DPPC/PG) revealed that the formation of the probe-excluding clusters distinctive for the quaternary films was influenced by the two proteins. Concanavalin A in the subphase could not replace SP-A in its ability to modulate the textures of films of SP-B/(DPPC/PG). In films of SP-C/SP-A/(DPPC/PG), in the absence of calcium, regions consisting of a probe-excluding phase, likely enriched in SP-A, were detected at surface pressures between 2 mN/m and 20 mN/m in addition to the lipid LE and LC phases. Ca(2+) in the subphase appeared to disperse this phase into tiny probe-excluding particles, likely comprising Ca(2+)-aggregated SP-A. Despite their strikingly different morphologies, the films of DPPC/PG that contained combinations of SP-B/SP-A or SP-C/SP-A displayed similar distributions of LC and LE phases with LC regions occupying a maximum of 20% of the total monolayer area. Combining SP-A and SP-B reorganized the morphology of monolayers composed of DPPC and PG in a Ca(2+)-independent manner that led to the formation of a separate potentially protein-rich phase in the films.     

Fatty acid-binding protein regulates LPS-induced TNF-alpha production in mast cells

There has been increasing evidence for the involvement of fatty acid-binding proteins (FABPs) in the cytokine production of macrophages and dendritic cells probably through the control of cellular lipid metabolism and signal transduction. Since mast cells (MCs) are recently shown to be involved in immune response through modification of cytokine production, it is possible that some FABPs could also be involved in the immune response of MCs. In this study, we found that epidermal-type FABP (E-FABP) was expressed in murine bone marrow-derived MCs (BMMCs). Using BMMCs from genetically E-FABP-null mutated mice, we demonstrated that E-FABP in BMMCs plays a key role in the production of TNF-alpha following lipopolysaccharide (LPS) stimulation. In the in vivo septic peritonitis model (cecal ligation and puncture model), E-FABP-null mice showed a significantly increased mortality compared to wild-type mice. However, no significant difference in antigen-induced cytokine production was observed between wild-type and E-FABP-null BMMCs, and systemic anaphylaxis was equally induced in vivo in both wild-type and E-FABP-null mice. These results suggest that E-FABP is specifically involved in the LPS-induced cytokine production of MCs, and could play a role in the host-defense against bacterial infection, possibly through regulation of TNF-alpha production.     

Surfactant protein-A plays an important role in lung surfactant clearance: evidence using the surfactant protein-A gene-targeted mouse

Previous studies with the isolated perfused rat lung showed that both clathrin- and actin-mediated pathways are responsible for endocytosis of dipalmitoylphosphatidylcholine (DPPC)-labeled liposomes by granular pneumocytes in the intact lung. Using surfactant protein-A (SP-A) gene-targeted mice, we examined the uptake of [(3)H]DPPC liposomes by isolated mouse lungs under basal and secretagogue-stimulated conditions. Unilamellar liposomes composed of [(3)H]DPPC: phosphatidylcholine:cholesterol:egg phosphatidylglycerol (10:5:3:2 mol fraction) were instilled into the trachea of anesthetized mice, and the lungs were perfused (2 h). Uptake was calculated as percentage of instilled disintegrations per minute in the postlavaged lung. Amantadine, an inhibitor of clathrin and, thus, receptor-mediated endocytosis via clathrin-coated pits, decreased basal [(3)H]DPPC uptake by 70% in SP-A +/+ but only by 20% in SP-A -/- lung, data compatible with an SP-A/receptor-regulated lipid clearance pathway in the SP-A +/+ mice. The nonclathrin, actin-dependent process was low in the SP-A +/+ lung but accounted for 55% of liposome endocytosis in the SP-A -/- mouse. With secretagogue (8-bromoadenosine 3',5'-cyclic monophosphate) treatment, both clathrin- and actin-dependent lipid clearance were elevated in the SP-A +/+ lungs while neither pathway responded in the SP-A -/- lungs. Binding of iodinated SP-A to type II cells isolated from both genotypes of mice was similar indicating a normal SP-A receptor status in the SP-A -/- lung. Inclusion of SP-A with instilled liposomes served to "rescue" the SP-A -/- lungs by reestablishing secretagogue-dependent enhancement of liposome uptake. These data are compatible with a major role for receptor-mediated endocytosis of DPPC by granular pneumocytes, a process critically dependent on SP-A.     

Surfactant-associated protein A inhibits LPS-induced cytokine and nitric oxide production in vivo

The role of surfactant-associated protein (SP) A in the mediation of pulmonary responses to bacterial lipopolysaccharide (LPS) was assessed in vivo with SP-A gene-targeted [SP-deficient; SP-A(-/-)] and wild-type [SP-A(+/+)] mice. Concentrations of tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein-2, and nitric oxide were determined in recovered bronchoalveolar lavage fluid after intratracheal administration of LPS. SP-A(-/-) mice produced significantly more TNF-alpha and nitric oxide than SP-A(+/+) mice after LPS treatment. Intratracheal administration of human SP-A (1 mg/kg) to SP-A(-/-) mice restored regulation of TNF-alpha, macrophage inflammatory protein-2, and nitric oxide production to that of SP-A(+/+) mice. Other markers of lung injury including bronchoalveolar fluid protein, phospholipid content, and neutrophil numbers were not influenced by SP-A. Data from experiments designed to test possible mechanisms of SP-A-mediated suppression suggest that neither binding of LPS by SP-A nor enhanced LPS clearance are the primary means of inhibition. Our data and others suggest that SP-A acts directly on immune cells to suppress LPS-induced inflammation. These results demonstrate that endogenous or exogenous SP-A inhibits pulmonary LPS-induced cytokine and nitric oxide production in vivo.     

Surfactant protein interactions with neutral and acidic phospholipid films

The captive bubble tensiometer was employed to study interactions of phospholipid (PL) mixtures of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) at 50 microg/ml with physiological levels of the surfactant protein (SP) A SP-B, and SP-C alone and in combination at 37 degrees C. All surfactant proteins enhanced lipid adsorption to equilibrium surface tension (gamma), with SP-C being most effective. Kinetics were consistent with the presence of two adsorption phases. Under the conditions employed, SP-A did not affect the rate of film formation in the presence of SP-B or SP-C. Little difference in gamma(min) was observed between the acidic POPG and the neutral POPC systems with SP-B or SP-C with and without SP-A. However, gamma(max) was lower with the acidic POPG system during dynamic, but not during quasi-static, cycling. Considerably lower compression ratios were required to generate low gamma(min) values with SP-B than SP-C. DPPC-POPG-SP-B was superior to the neutral POPC-SP-B system. Although SP-A had little effect on film formation with SP-B, surface activity during compression was enhanced with both PL systems. In the presence of SP-C, lower compression ratios were required with the acidic system, and with this mixture, SP-A addition adversely affected surface activity. The results suggest specific interactions between SP-B and phosphatidylglycerol, and between SP-B and SP-A. These observations are consistent with the presence of a surface-associated surfactant reservoir which is involved in generating low gamma during film compression and lipid respreading during film expansion.     

Surfactant protein B inhibits secretory phospholipase A2 hydrolysis of surfactant phospholipids

Hydrolysis of surfactant phospholipids (PL) by secretory phospholipases A(2) (sPLA(2)) contributes to surfactant damage in inflammatory airway diseases such as acute lung injury/acute respiratory distress syndrome. We and others have reported that each sPLA(2) exhibits specificity in hydrolyzing different PLs in pulmonary surfactant and that the presence of hydrophilic surfactant protein A (SP-A) alters sPLA(2)-mediated hydrolysis. This report tests the hypothesis that hydrophobic SP-B also inhibits sPLA(2)-mediated surfactant hydrolysis. Three surfactant preparations were used containing varied amounts of SP-B and radiolabeled tracers of phosphatidylcholine (PC) or phosphatidylglycerol (PG): 1) washed ovine surfactant (OS) (pre- and postorganic extraction) compared with Survanta (protein poor), 2) Survanta supplemented with purified bovine SP-B (1-5%, wt/wt), and 3) a mixture of dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) (DPPC:POPC:POPG, 40:40:20) prepared as vesicles and monomolecular films in the presence or absence of SP-B. Hydrolysis of PG and PC by Group IB sPLA(2) (PLA2G1A) was significantly lower in the extracted OS, which contains SP-B, compared with Survanta (P = 0.005), which is SP-B poor. Hydrolysis of PG and PC in nonextracted OS, which contains all SPs, was lower than both Survanta and extracted OS. When Survanta was supplemented with 1% SP-B, PG and PC hydrolysis by PLA2G1B was significantly lower (P < 0.001) than in Survanta alone. When supplemented into pure lipid vesicles and monomolecular films composed of PG and PC mixtures, SP-B also inhibited hydrolysis by both PLA2G1B and Group IIA sPLA2 (PLA2G2A). In films, PLA2G1B hydrolyzed surfactant PL monolayers at surface pressures ≤30 mN/m (P < 0.01), and SP-B lowered the surface pressure range at which hydrolysis can occur. These results suggest the hydrophobic SP, SP-B, protects alveolar surfactant PL from hydrolysis mediated by multiple sPLA(2) in both vesicles (alveolar subphase) and monomolecular films (air-liquid interface).     

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.