Interaction Of Surfactant Protein A Research Articles

Exposure to traffic-related particle matter and effects on lung function and potential interactions in a cross-sectional analysis of a cohort study in west Sweden

ObjectivesTo investigate the long-term effects of source-specific particle matter (PM) on lung function, effects of Surfactant Protein A (SP-A) and glutathione S-transferase (GST) genes GSTP1 and GSTT1 gene variants and...

Surfactant protein A (SP-A) inhibits agglomeration and macrophage uptake of toxic amine modified nanoparticles

The lung provides the main route for nanomaterial exposure. Surfactant protein A (SP-A) is an important respiratory innate immune molecule with the ability to bind or opsonise pathogens to enhance phagocytic removal from the airways. We hypothesised that SP-A, like surfactant protein D, may interact with inhaled nanoparticulates, and that this interaction will be affected by nanoparticle (NP) surface characteristics. In this study, we characterise the interaction of SP-A with unmodified (U-PS) and amine-modified (A-PS) polystyrene particles of varying size and zeta potential using dynamic light scatter analysis. SP-A associated with both 100 nm U-PS and A-PS in a calcium-independent manner. SP-A induced significant calcium-dependent agglomeration of 100 nm U-PS NPs but resulted in calcium-independent inhibition of A-PS self agglomeration. SP-A enhanced uptake of 100 nm U-PS into macrophage-like RAW264.7 cells in a dose-dependent manner but in contrast inhibited A-PS uptake. Reduced association of A-PS particles in RAW264.7 cells following pre-incubation of SP-A was also observed with coherent anti-Stokes Raman spectroscopy. Consistent with these findings, alveolar macrophages (AMs) from SP-A−/− mice were more efficient at uptake of 100 nm A-PS compared with wild type C57Bl/6 macrophages. No difference in uptake was observed with 500 nm U-PS or A-PS particles. Pre-incubation with SP-A resulted in a significant decrease in uptake of 100 nm A-PS in macrophages isolated from both groups of mice. In contrast, increased uptake by AMs of U-PS was observed after pre-incubation with SP-A. Thus we have demonstrated that SP-A promotes uptake of non-toxic U-PS particles but inhibits the clearance of potentially toxic A-PS particles by blocking uptake into macrophages.

Pulmonary surfactant protein A-induced changes in the molecular conformation of bacterial deep-rough LPS lead to reduced activity on human macrophages

The lung is constantly exposed to immune stimulation by LPS from inhaled microorganisms. A primary mechanism to maintain immune homeostasis is based on anti-inflammatory regulation by surfactant protein A (SP-A), a secreted component of lung innate immunity. The architecture of LPS aggregates is strongly associated with biological activity. We therefore investigated whether SP-A affects the physico-chemical properties of LPS. Determination of the three-dimensional aggregate structure of LPS by small-angle X-ray scattering demonstrated that SP-A induced the formation of multi-lamellar aggregate structures. Determination of the acyl-chain-fluidity of LPS aggregates by Fourier transform infrared (FTIR) spectroscopy showed that the phase transition temperature of LPS was reduced in the presence of SP-A. The phosphate groups at the diglucosamine backbone of LPS represent important functional groups for the bioactivity of LPS. FTIR analysis revealed changes in the vibrational bands νas PO-(2), indicating an interaction of SP-A with the 1-phosphate, but not with the 4'-phosphate. The physico-chemical changes induced by SP-A were associated with up to 90% reduction in LPS-induced TNF-α-production by human macrophages. In conclusion, our data demonstrate that the SP-A/LPS interaction induces conformational changes in LPS aggregates leading to biologically less active structures, thereby providing a new molecular mechanism of immune modulation by SP-A.

Effect of Smooth Bacterial Lipopolysaccharide on the Behavior of DPPC Films

The airspaces are lined with a DPPC-rich film called pulmonary surfactant, named for its ability to maintain normal respiratory mechanics by reducing surface tension at the air/liquid interface. Inhaled airborne particles containing smooth bacterial lipopolysaccharide (s-LPS) might incorporate into the surfactant monolayer. In this study, we evaluated the effect of s-LPS on the behavior of DPPC films by using epifluorescence microscopy combined with a surface balance. In addition, we investigated whether LPS effects could be counteracted by surfactant protein A (SP-A), which is a LPS binding protein, with the peculiarity that this protein is associated to the surfactant monolayer. Thus SP-A is in the initial defence barrier against inhaled airborne particles. Our data show that s-LPS injected in the aqueous subphase penetrated into DPPC films to form mixed DPPC/s-LPS monolayers. Low amounts of s-LPS fluidized the DPPC monolayer, as demonstrated by fluorescence microscopy and changes in the compressibility modulus. This promoted early collapse and prevented the attainment of high surface pressures. The interaction of SP-A with DPPC/s-LPS film further fluidized the monolayers and facilitated the extraction of s-LPS at surface pressures where SP-A was expelled from the mixed films, suggesting that SP-A is an LPS scavenger. A better understanding of the biophysical properties of lung surfactant monolayer and its susceptibility to LPS inhibition is important for the development of new surfactant formulations for respiratory diseases.

Interaction of Surfactant Protein A with the Intermediate Filaments Desmin and Vimentin

Surfactant protein A (SP-A), a member of the collectin family that modulates innate immunity, has recently been involved in the physiology of reproduction. Consistent with the activation of ERK-1/2 and COX-2 induced by SP-A in myometrial cells, we reported previously the presence of two major proteins recognized by SP-A in these cells. Here we identify by mass spectrometry one of these SP-A targets as the intermediate filament (IF) desmin. In myometrial preparations derived from desmin-deficient mice, the absence of binding of SP-A to any 50 kDa protein confirmed the identity of this SP-A-binding site as desmin. Our data based on partial chymotrypsin digestion of pure desmin suggested that SP-A recognizes especially its rod domain, which is known to play an important role during the assembly of desmin into filaments. In line with that, electron microscopy experiments showed that SP-A inhibits in vitro the polymerization of desmin filaments. SP-A also recognized in vitro polymerized filaments in a calcium-dependent manner at a physiological ionic strength but not the C1q receptor gC1qR. Furthermore, Texas Red-labeled SP-A colocalized with desmin filaments in myometrial cells. Interestingly, vimentin, the IF characteristic of leukocytes, is one of the major proteins recognized by SP-A in protein extracts of U937 cells after PMA-induced differentiation of this monocytic cell line. Interaction of SP-A with vimentin was further confirmed using recombinant vimentin in solid-phase binding assays. The ability of SP-A to interact with desmin and vimentin, and to prevent polymerization of desmin monomers, shed light on unexpected and wider biological roles of this collectin.

Surfactant Protein A Forms Extensive Lattice-Like Structures on 1,2-Dipalmitoylphosphatidylcholine/Rough-Lipopolysaccharide- Mixed Monolayers

Due to the inhalation of airborne particles containing bacterial lipopolysaccharide (LPS), these molecules might incorporate into the 1,2-dipalmitoylphosphatidylcholine (DPPC)-rich monolayer and interact with surfactant protein A (SP-A), the major surfactant protein component involved in host defense. In this study, epifluorescence microscopy combined with a surface balance was used to examine the interaction of SP-A with mixed monolayers of DPPC/rough LPS (Re-LPS). Binary monolayers of Re-LPS plus DPPC showed negative deviations from ideal behavior of the mean areas in the films consistent with partial miscibility and attractive interaction between the lipids. This interaction resulted in rearrangement and reduction of the size of DPPC-rich solid domains in DPPC/Re-LPS monolayers. The adsorption of SP-A to these monolayers caused expansion in the lipid molecular areas. SP-A interacted strongly with Re-LPS and promoted the formation of DPPC-rich solid domains. Fluorescently labeled Texas red-SP-A accumulated at the fluid-solid boundary regions and formed networks of interconnected filaments in the fluid phase of DPPC/Re-LPS monolayers in a Ca 2+-independent manner. These lattice-like structures were also observed when TR-SP-A interacted with lipid A monolayers. These novel results deepen our understanding of the specific interaction of SP-A with the lipid A moiety of bacterial LPS.

Surfactant Protein A Directly Interacts with TLR4 and MD-2 and Regulates Inflammatory Cellular Response: IMPORTANCE OF SUPRATRIMERIC OLIGOMERIZATION

The purpose of the current study was to examine the binding of pulmonary surfactant protein A (SP-A) to TLR4 and MD-2, which are critical signaling receptors for lipopolysaccharides (LPSs). The direct binding of SP-A to the recombinant soluble form of extracellular TLR4 domain (sTLR4) and MD-2 was detected using solid-phase binding, immunoprecipitation, and BIAcore. SP-A bound to sTLR4 and MD-2 in a Ca2+-dependent manner, and an anti-SP-A monoclonal antibody whose epitope lies in the region Thr184-Gly194 blocked the SP-A binding to sTLR4 and MD-2, indicating the involvement of the carbohydrate recognition domain (CRD) in the binding. SP-A avidly bound to the deglycosylated forms of sTLR4 and MD-2, suggesting a protein/protein interaction. In addition, SP-A attenuated cell surface binding of smooth LPS and smooth LPS-induced NF-kappaB activation in TLR4/MD-2-expressing cells. To know the role of oligomerization in the interaction of SP-A with TLR4 and MD-2, the collagenase-resistant fragment (CRF), which consisted of CRD plus neck domain of SP-A, was isolated. CRF assembled as a trimer, whereas SP-A assembled as a higher order oligomer. Although CRD was suggested to be involved in the binding, CRF exhibited approximately 600- and 155-fold higher KD for the binding to TLR4 and MD-2, respectively, when compared with SP-A. Consistently significantly higher molar concentrations of CRF were required to inhibit smooth LPS-induced NF-kappaB activation and tumor necrosis factor-alpha secretion. These results demonstrate for the first time the direct interaction between SP-A and TLR4/MD-2 and suggest the importance of supratrimeric oligomerization in the immunomodulatory function of SP-A.

Interaction of Surfactant Protein A with Peroxiredoxin 6 Regulates Phospholipase A2 Activity

Peroxiredoxin 6 (Prdx6) is a "moonlighting" protein with both GSH peroxidase and phospholipase A(2) (PLA(2)) activities. This protein is responsible for degradation of internalized dipalmitoylphosphatidylcholine, the major phospholipid component of lung surfactant. The PLA(2) activity is inhibited by surfactant protein A (SP-A). We postulate that SP-A regulates the PLA(2) activity of Prdx6 through direct protein-protein interaction. Recombinant human Prdx6 and SP-A isolated from human alveolar proteinosis fluid were studied. Measurement of kinetic constants at pH 4.0 (maximal PLA(2) activity) showed K(m)0.35 mm and V(max) 138 nmol/min/mg of protein. SP-A inhibited PLA(2) activity non-competitively with K(i) 10 mug/ml and was Ca(2+) -independent. Activity at pH 7.4 was approximately 50% less, and inhibition by SP-A was partially dependent on Ca(2+). Interaction of SP-A and Prdx6 at pH 7.4 was shown by Prdx6-mediated inhibition of SP-A binding to agarose beads, a pull-down assay using His-tagged Prdx6 and Ni(2) -chelating beads, co-immunoprecipitation from lung epithelial cells and from a binary mixture of the two proteins, binding after treatment with a trifunctional cross-linker, and size-exclusion chromatography. Analysis by static light scattering and surface plasmon resonance showed calcium-independent SP-A binding to Prdx6 at pH 4.0 and partial Ca(2+) dependence of binding at pH 7.4. These results indicate a direct interaction between SP-A and Prdx6, which provides a mechanism for regulation of the PLA(2) activity of Prdx6 by SP-A.

Pulmonary surfactant proteins and lipids as modulators of inflammation and innate immunity

The pulmonary surfactant system of the human lung consists of unique lipids and proteins that contribute to the biophysical and innate immune properties of the organ. Surfactant protein A (SP-A) is an oligomeric protein consisting of 18 protomers with collagen and lectin-like domains that recognizes glycoconjugates, lipids and protein determinants on both host cells and invading microorganisms. The authors examined the interaction of SP-A with Mycoplasma pneumoniae and the influence of the protein upon the innate immune response to the bacteria. The authors quantified SP-A interaction with bacteria using ELISA, and identified the major surface ligand by thin layer chromatography, HPLC and mass spectrometry. The inflammatory response of human and rat macrophages was measured by quantifying tumour necrosis factor-alpha secretion using ELISA, and nitric oxide production. SP-A bound the bacteria with high affinity and enhanced the inflammatory response of human and rat macrophages to the organism and its membranes. Analysis of the interaction of SP-A with the bacteria revealed that the major ligand was a phospholipid. The lipid ligand was purified by a combination of thin layer and HPLC, and identified by mass spectrometry. The mass spectrometry demonstrated that the SP-A reactive lipid consisted of several disaturated molecular species of phosphatidylglycerol (PtdGro). Additional experiments were performed to determine if disaturated PtdGro was capable of interfering with the action of SP-A as an inhibitor of bacterial lipopolysaccharide-induced inflammatory mediator production by macrophages. The disaturated PtdGro failed to alter the anti-inflammatory action of SP-A but unexpectedly these same studies revealed that unsaturated PtdGro can modify the host response to lipopolysaccharide. These findings reveal that both the lipids and proteins of pulmonary surfactant play a role in regulating the host response to invading microorganisms.

Collectin-mediated innate immune defense in the lung

The focus of attention in immunology has classically been the adaptive immune system. The adaptive immune system is most effective when the host has had prior exposure to the pathogen. Accruing evidence suggests that the innate immune system plays an important role in the regulation of the adaptive immune response and in filling the temporal gap in host immunity. Pattern recognition molecules such as collectins are important components of the innate immune system. Herein we describe the structural aspects and immunological functions of the two lung collectins surfactant protein A (SP-A) and SP-D. Important clinical applications of pulmonary collectin research include the use of SP-A and -D in diagnosis and therapy. This survey focuses on the interactions of SP-A and -D with a wide variety of respiratory pathogens and the regulation of the immune cell response by these collectins.

Interaction of SP-A (surfactant protein A) with bacterial rough lipopolysaccharide (Re-LPS), and effects of SP-A on the binding of Re-LPS to CD14 and LPS-binding protein

SP-A (surfactant protein A) is a lipid-binding collectin primarily involved in innate lung immunity. SP-A interacts with the bacterial rough LPS (lipopolysaccharide) Re-LPS (Re595 mutant of LPS from Salmonella minnesota), but not with smooth LPS. In the present study, we first examined the characteristics of the interaction of human SP-A with Re-LPS. Fluorescence intensity and anisotropy measurements of FITC-labelled Re-LPS in the presence and absence of SP-A indicated that SP-A bound to Re-LPS in solution in a Ca2+-independent manner, with a dissociation constant of 2.8x10(-8) M. In the presence of calcium, a high-mobility complex of SP-A and [3H]Rb-LPS (Rb mutant of LPS from Escherichia coli strain LCD 25) micelles was formed, as detected by sucrose density gradients. Re-LPS aggregation induced by SP-A was further characterized by light scattering. On the other hand, human SP-A inhibited TNF-alpha (tumour necrosis factor-alpha) secretion by human macrophage-like U937 cells stimulated with either Re-LPS or smooth LPS. We further examined the effects of human SP-A on the binding of Re-LPS to LBP (LPS-binding protein) and CD14. SP-A decreased the binding of Re-LPS to CD14, but not to LBP, as detected by cross-linking experiments with 125I-ASD-Re-LPS [125I-labelled sulphosuccinimidyl-2-(p-azidosalicylamido)-1,3-dithiopropionate derivative of Re-LPS] and fluorescence analysis with FITC-Re-LPS. When SP-A, LBP and CD14 were incubated together, SP-A reduced the ability of LBP to transfer 125I-ASD-Re-LPS to CD14. These SP-A effects were not due to the ability of SP-A to aggregate Re-LPS in the presence of calcium, since they were observed in both the absence and the presence of calcium. These studies suggest that SP-A could contribute to modulate Re-LPS responses by altering the competence of the LBP-CD14 receptor complex.

Endocytic pathway for surfactant protein A in human macrophages: binding, clathrin-mediated uptake, and trafficking through the endolysosomal pathway

In the noninflamed lung, surfactant protein A (SP-A) acts as an anti-inflammatory molecule through its effects on macrophage (MPhi) function, modulating cytokine and reactive oxygen and nitrogen intermediate production. The receptors responsible for these effects of SP-A on human MPhi are not clear, although SP-A binding to several proteins has been described. In this study, we demonstrate high-affinity specific binding of SP-A to primary human MPhi. SP-A binding was inhibited by EGTA, indicating calcium dependence. However, mannan did not inhibit SP-A binding, suggesting that binding is mediated by a direct protein-protein interaction that does not involve carbohydrate recognition. Our laboratory has previously shown that SP-A is rapidly endocytosed by human MPhi into discrete vesicles. Although previous work indicates that SP-A is ultimately degraded by murine MPhi over time, the trafficking pathway of SP-A through MPhi after uptake has not been reported and is of potential biological importance. We examined trafficking of SP-A in human MPhi by electron and confocal microscopy and show for the first time that SP-A is endocytosed by primary human MPhi through clathrin-coated pits and colocalizes sequentially over time with the early endosome marker EEA1, late endosome marker lamp-1, and lysosome marker cathepsin D. We conclude that SP-A binds to receptor(s) on human MPhi, is endocytosed by a receptor-mediated, clathrin-dependent process, and trafficks through the endolysosomal pathway. These studies provide further insight into the interactions of SP-A with the MPhi cell surface and intracellular compartments that play important roles in SP-A modulation of lung MPhi biology.

Lactoferrin and surfactant protein A exhibit distinct binding specificity to F protein and differently modulate respiratory syncytial virus infection.

Surfactant protein A (SP-A) and lactoferrin (LF) play important roles in innate immune systems in the respiratory mucous membranes. We investigated how SP-A and LF act against respiratory syncytial virus (RSV) infection. The present study indicated that RSV-induced IL-8 secretion from HEp-2 cells was up-regulated by SP-A (170% of control) but down-regulated by LF (23% of control). RSV infectivity determined by viral titers and the uptake of FITC-labeled RSV were also increased by SP-A, but decreased by LF. To clarify the mechanism of these opposite effects, we examined the interactions of SP-A and LF with RSV F protein, the most important surface glycoprotein for viral penetration. RSV F protein was found to be the ligand for both SP-A and LF, but the manners of binding were different. LF directly interacted with the F(1) subunit, which involved antigenic sites of F protein. Contrarily, SP-A associated with the F(2) subunit, which was highly glycosylated. SP-A but not LF failed to interact with deglycosylated F protein. Moreover, SP-A initiated the hemolyzing fusion activity of F protein. These results suggest that SP-A and LF modulate RSV infection by different binding specificity to F protein.

Surfactant Protein A Inhibits Peptidoglycan-induced Tumor Necrosis Factor-α Secretion in U937 Cells and Alveolar Macrophages by Direct Interaction with Toll-like Receptor 2

Pulmonary surfactant protein A (SP-A) plays an important role in modulation of the innate immune system of the lung. Peptidoglycan (PGN), a cell wall component of Gram-positive bacteria, is known to elicit excessive proinflammatory cytokine production from immune cells. In this study we investigated whether SP-A interacts with PGN and alters PGN-elicited cellular responses. Binding studies demonstrate that PGN is not a ligand for SP-A. However, SP-A significantly reduced PGN-elicited tumor necrosis factor alpha (TNF-alpha) secretion by U937 cells and rat alveolar macrophages. The inhibitory effect on TNF-alpha secretion was dependent upon SP-A concentrations in physiological range. Coincubation of SP-A and PGN with human embryonic kidney 293 cells that had been transiently transfected with the cDNA of Toll-like receptor 2 (TLR2), a cell signaling receptor for PGN, significantly attenuated PGN-induced nuclear factor-kappaB activity. SP-A directly bound to a soluble form of the recombinant extracellular TLR2 domain (sTLR2). Coincubation of sTLR2 with SP-A significantly reduced the binding of sTLR2 to PGN. These results indicate that the direct interaction of SP-A with TLR2 alters PGN-induced cell signaling. We propose that SP-A modulates inflammatory responses against the bacterial components by interactions with pattern-recognition receptors.

ROLE OF SURFACTANT PROTEIN A (SP-A)/LIPID INTERACTIONS FOR SP-A FUNCTIONS IN THE LUNG

Surfactant protein A (SP-A), an oligomeric glycoprotein, is a member of a group of proteins named collections that contain collagen-like and Ca2+ -dependent carbohydrate recognition domains. SP-A interacts with a broad range of amphipathic lipids (glycerophospholipids, sphingophospholipids, glycosphingolipids, lipid A, and lipoglycans) that are present in surfactant or microbial membranes. This review summarizes SP-A/lipid interaction studies regarding the lipid system used (i.e., phospholipid vesicles, phospholipid monolayers, and lipids immobilized on silica or adsorbed on a solid support). The effect of calcium, ionic strength, and pH on the binding of SP-A to lipids and the subsequent lipid aggregation process is discussed. Current evidence suggests that hydrophobic-binding forces are involved in the peripherical association of SP-A to membranes. It is also proposed that fluid and liquid-ordered phase coexistence in surfactant membranes might favor partition of SP-A into those membranes. The binding of SP-A to surfactant membranes containing hydrophobic surfactant peptides makes possible the formation of a membrane reservoir in the alveolar fluid that is protected by SP-A against inactivation and improves the rate of surfactant film formation. In addition, the interaction of SP-A with membranes might enhance the affinity of SP-A for terminal carbohydrates of glycolipids or glycoproteins on the surface of invading microorganisms.

Role of Surfactant Protein a (SP-A)/Lipid Interactions for SP-A Functions in the Lung

Surfactant protein A (SP-A), an oligomeric glycoprotein, is a member of a group of proteins named collectins that contain collagen-like and Ca(2+)-dependent carbohydrate recognition domains. SP-A interacts with a broad range of amphipathic lipids (glycerophospholipids, sphingophospholipids, glycosphingolipids, lipid A, and lipoglycans) that are present in surfactant or microbial membranes. This review summarizes SP-A/lipid interaction studies regarding the lipid system used (i.e., phospholipid vesicles, phospholipid monolayers, and lipids immobilized on silica or adsorbed on a solid support). The effect of calcium, ionic strength, and pH on the binding of SP-A to lipids and the subsequent lipid aggregation process is discussed. Current evidence suggests that hydrophobic-binding forces are involved in the peripherical association of SP-A to membranes. It is also proposed that fluid and liquid-ordered phase coexistence in surfactant membranes might favor partition of SP-A into those membranes. The binding of SP-A to surfactant membranes containing hydrophobic surfactant peptides makes possible the formation of a membrane reservoir in the alveolar fluid that is protected by SP-A against inactivation and improves the rate of surfactant film formation. In addition, the interaction of SP-A with membranes might enhance the affinity of SP-A for terminal carbohydrates of glycolipids or glycoproteins on the surface of invading microorganisms.

Interaction of surfactant protein A with lipopolysaccharide and regulation of inflammatory cytokines in the THP-1 monocytic cell line.

Pulmonary surfactant protein A (SP-A) is involved in innate immunity in the lung. In this study we investigated the interaction of SP-A with different serotypes of lipopolysaccharide (LPS) on the regulation of inflammatory cytokines in vitro. In the human monocytic cell line, THP-1, combining SP-A with lipid A or rough LPS further enhanced lipid A- or rough LPS-stimulated tumor necrosis factor alpha (TNF-alpha) mRNA levels, while SP-A-elicited increases in TNF-alpha mRNA levels were partially neutralized. In contrast, the combination of smooth LPS and SP-A resulted in additive effects on TNF-alpha mRNA levels. We also demonstrated that there was cross-tolerance between SP-A and LPS in THP-1 cells. Pretreatment of THP-1 cells with LPS modestly inhibited the response of these cells to subsequent challenge with SP-A, with regard to the production of TNF-alpha, whereas there was no or little effect on the production of interleukin-1beta (IL-1beta) and IL-8. Conversely, pretreatment of THP-1 cells with SP-A markedly increased the response to subsequent challenge with LPS with regard to the production of IL-1beta and IL-8, although the production of TNF-alpha was modestly decreased. However, a synergistic stimulatory effect was observed when the two agents were added simultaneously to the cells. NF-kappaB formation was downregulated in SP-A- but not in LPS-induced tolerant cells. These results suggested that SP-A exhibits different interactions with distinct serotypes of LPS. In addition, SP-A is different from LPS with regard to the induction of cross-tolerance, and these actions may be mediated, at least in part, through different mechanisms.

Activation of surfactant protein-B transcription: signaling through the SP-A receptor utilizing the PI3 kinase pathway.

This study describes receptor-activated signaling initiated by surfactant protein-A (SP-A), and the means by which it activates transcription of surfactant protein-B. Pulmonary surfactant is a mixture of lipids and associated proteins produced by type II pneumocytes. Interaction of SP-A with its cognate receptor (SPAR) on type II cells is involved in regulating surfactant secretion. This interaction also increases transcription of surfactant proteins and several other genes. To study SP-A cytokine activity, we used as a model surfactant-protein (SP-B) transcription, the activators of which have been characterized. HNF-3 and TTF-1 transcription factors are known to stimulate SP-B transcription. SP-A caused increased phosphorylation and nuclear localization of both. Corresponding increases in protein binding to the SP-B promoter were demonstrated by gel shift analysis. SP-A increased protein binding to HNF-3 and TTF-1 consensus recognition elements. Footprinting analysis indicated that SP-A-induced protein binding to SP-B promoter was greater in amount, but not different in location, from that seen in control cells, which normally transcribe SP-B. SP-A caused transient increases in PI3 kinase localization at the plasma membrane, and SP-A signaling to elicit increased SP-B transcription was blocked by LY294002, an inhibitor of PI3 kinase. Therefore, SP-A signals through PI3 kinase to increase SP-B transcription in type II pneumocytes by enhancing TTF-1 and HNF-3 activation of the SP-B promoter. SP-A activation of this signaling pathway, which affects many cellular functions and has not previously been implicated in type II cell transcriptional activity, has profound import for understanding type II cell biology.

Interactions of Pulmonary Surfactant Protein A with Phospholipid Monolayers Change with pH

The interaction of pulmonary surfactant protein A (SP-A) labeled with Texas Red (TR-SP-A) with monolayers containing zwitterionic and acidic phospholipids has been studied at pH 7.4 and 4.5 using epifluorescence microscopy. At pH 7.4, TR-SP-A expanded the π-A isotherms of film of dipalmitoylphosphatidylcholine (DPPC). It interacted at high concentration at the edges of condensed-expanded phase domains, and distributed evenly at lower concentration into the fluid phase with increasing pressure. At pH 4.5, TR-SP-A expanded DPPC monolayers to a slightly lower extent than at pH 7.4. It interacted primarily at the phase boundaries but it did not distribute into the fluid phase with increasing pressure. Films of DPPC/dipalmitoylphosphatidylglycerol (DPPG) 7:3 mol/mol were somewhat expanded by TR-SP-A at pH 7.4. The protein was distributed in aggregates only at the condensed-expanded phase boundaries at all surface pressures. At pH 4.5 TR-SP-A caused no expansion of the π-A isotherm of DPPC/DPPG, but its fluorescence was relatively homogeneously distributed throughout the expanded phase at all pressures studied. These observations can be explained by a combination of factors including the preference for SP-A aggregates to enter monolayers at packing dislocations and their disaggregation in the presence of lipid under increasing pressure, together with the influence of pH on the aggregation state of SP-A and the interaction of SP-A with zwitterionic and acidic lipid.

Pulmonary Surfactant Protein A Modulates the Cellular Response to Smooth and Rough Lipopolysaccharides by Interaction with CD14

Pulmonary surfactant protein A (SP-A) plays an important part in Ab-independent host defense mechanisms of the lung. In this study we investigated how SP-A interacts with distinct serotypes of bacterial LPS and modulates LPS-elicited cellular responses. SP-A bound to rough forms but not to smooth forms of LPS. In the macrophage-like cell line U937, SP-A inhibited mRNA expression and secretion of TNF-alpha induced by smooth LPS, but rough LPS-induced TNF-alpha expression was unaffected by SP-A. When U937 cells and rat alveolar macrophages were preincubated with SP-A, smooth LPS failed to induce TNF-alpha secretion, whereas rough LPS-induced TNF-alpha secretion was modestly increased. To clarify the mechanism by which SP-A modulates LPS-elicited cellular responses, we further examined the interaction of SP-A with CD14, which is known as a major LPS receptor. Western blot analysis revealed that CD14 was one of the SP-A binding proteins isolated from solubilized U937 cells. In addition, SP-A directly bound to recombinant soluble CD14 (rsCD14). When rsCD14 was preincubated with SP-A, the binding of rsCD14 to smooth LPS was significantly reduced but the association of rsCD14 with rough LPS was augmented. These results demonstrate the different actions of SP-A upon distinct serotypes of LPS and indicate that the direct interaction of SP-A with CD14 constitutes a likely mechanism by which SP-A modulates LPS-elicited cellular responses.