Pulmonary Surfactant Proteins SP-A Research Articles

Surfactant Proteins SP-A and SP-D Modulate Uterine Contractile Events in ULTR Myometrial Cell Line.

Pulmonary surfactant proteins SP-A and SP-D are pattern recognition innate immune molecules. However, there is extrapulmonary existence, especially in the amniotic fluid and at the feto-maternal interface. There is sufficient evidence to suggest that SP-A and SP-D are involved in the initiation of labour. This is of great importance given that preterm birth is associated with increased mortality and morbidity. In this study, we investigated the effects of recombinant forms of SP-A and SP-D (rhSP-A and rhSP-D, the comprising of trimeric lectin domain) on contractile events in vitro, using a human myometrial cell line (ULTR) as an experimental model. Treatment with rhSP-A or rhSP-D increased the cell velocity, distance travelled and displacement by ULTR cells. rhSP-A and rhSP-D also affected the contractile response of ULTRs when grown on collagen matrices showing reduced surface area. We investigated this effect further by measuring contractility-associated protein (CAP) genes. Treatment with rhSP-A and rhSP-D induced expression of oxytocin receptor (OXTR) and connexin 43 (CX43). In addition, rhSP-A and rhSP-D were able to induce secretion of GROα and IL-8. rhSP-D also induced the expression of IL-6 and IL-6 Ra. We provide evidence that SP-A and SP-D play a key role in modulating events prior to labour by reconditioning the human myometrium and in inducing CAP genes and pro-inflammatory cytokines thus shifting the uterus from a quiescent state to a contractile one.

Surfactant Replacement Therapy in Influenza A H1N1

To the Editors: A 22-month-old boy was admitted to the Clinic for Infective Diseases, University Clinical Center, Tuzla (Bosnia and Herzegovina), because of fever, cough, loss of appetite, and vomiting. An influenza virus type A H1N1 infection quick test (QuickVue, Influenza A+B—QuickVue Influenza A+B—bioMérieux) had a positive result, and 48 hours later infection with Influenza type A H1N1 was confirmed by polymerase chain reaction (multiplex real-time PCR, firma Roche Diagnostics, Laval, Quebec, Canada) at the University Clinical Center, Sarajevo. A chest radiograph showed bilateral pneumonia. During the first 4 days, he had attacks of hypoxemia, which finally became irremediable on oxygen therapy via nasal cannula. The boy was afebrile, unconscious, periodically agitated, cyanotic, dehydrated, pale, tachycardic and tachydyspnoic with clear signs of cardial decompensation, and worsening of the chest radiograph. He was transferred to the pediatric intensive care unit (PICU, Clinic for Pediatric Disease, University Clinic Center in Tuzla) for mechanical support. After 4 hours of mechanical ventilation with low lung compliance, it was decided to give bovine surfactant (Survanta, Abbott Laboratories) endotracheally. After 10 minutes, there was a significant improvement in lung compliance, oxygen saturation, and normalization of the heart rate. Therapy with surfactant was repeated every 8 hours. After 48 hours, auscultatory examination and chest radiograph of the boy were considerably improved. The role of surfactant in treating respiratory failure is doubtful in children, except in the newborn, even though surfactant can improve oxygenation and significantly decrease mortality in infants, children, and adolescents with acute lung injury and respiratory distress syndrome.1 That kind of therapy has been found ineffective in adults.2 Some investigations have shown that pulmonary surfactant proteins SP-A and SP-D can act as opsonins and neutralize influenza virus.3 In treatment of this child, we used medication that consisted of only surfactant proteins SP-B and SP-C. The medication led to significant improvement in lung function and the general condition, which was probably the result of lower alveolar surface tension and better lung compliance, but it is not possible to exclude a neutralizing effect of surfactant on influenza virus type A H1N1 by opsonization. Fahrija Skokić, MD, PhD Clinic of Pediatrics Igor Hudić, MD, PhD Clinic of Gynecology and Obstetrics Nešad Hotić, MD, PhD Belkisa Cǒlić, MD, PhD Clinic of Pediatrics Mirsada Prašo, MD, PhD Clinic of Anesthesiology and Reanimation Dubravka Bačaj, MD, PhD Clinic of Pediatrics University Clinical Center Tuzla, Bosnia and Herzegovina

IRRAS Studies of the Host Defense Effect of Pulmonary Surfactants SP-A and SP-D

The pulmonary surfactant proteins SP-A and SP-D have been proven to play a role in host defense. SP-A binds phosphatidylcholine, galactosylceramide, and the lipid A portion of lipopolysaccharide (LPS), which is found in Gram-negative bacterial cell walls. SP-D binds phosphatidylinositol, glucosylceramide and LPS. Although X-ray crystallography and NMR spectroscopy have provided atomic level information about SP-A and SP-D structures, molecular level information about their binding to biological ligands is lacking. IR reflection-absorption spectroscopy (IRRAS) is used in the current work to study the properties of SP-A and SP-D at air/water interfaces and their interaction with biological ligands. The monolayer properties of recombinant rat neck + carbohydrate recognition domain (NCRD) SP-A and its point mutant D215A SP-A are compared. Both NCRD SP-A and D215A SP-A adsorb to the air/water interface, DPPC monolayers, and Lipid A monolayers, but D215A/PL SP-A displays higher affinity and greater stability. Measurements of surface pressure, Amide 1 intensity, and lipid acyl chain conformational ordering can help to elucidate the mechanism of interaction of SP-A and its ligands. Similar experiments have been carried out with SP-D.

Differential Partitioning of Pulmonary Surfactant Protein SP-A into Regions of Monolayers of Dipalmitoylphosphatidylcholine and Dipalmitoylphosphatidylcholine/Dipalmitoylphosphatidylglycerol

The interaction of the pulmonary surfactant protein SP-A fluorescently labeled with Texas Red (TR-SP-A) with monolayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC/dipalmitoylphosphatidylglycerol 7:3 w/w has been investigated. The monolayers were spread on aqueous subphases containing TR-SP-A. TR-SP-A interacted with the monolayers of DPPC to accumulate at the boundary regions between liquid condensed (LC) and liquid expanded (LE) phases. Some TR-SP-A appeared in the LE phase but not in the LC phase. At intermediate surface pressures (10–20 mN/m), the protein caused the occurrence of more, smaller condensed domains, and it appeared to be excluded from the monolayers at surface pressure in the range of 30–40 mN/m. TR-SP-A interaction with DPPC/dipalmitoylphosphatidylglycerol monolayers was different. The protein did not appear in either LE or LC but only in large aggregates at the LC-LE boundary regions, a distribution visually similar to that of fluorescently labeled concanavalin A adsorbed onto monolayers of DPPC. The observations are consistent with a selectivity of interaction of SP-A with DPPC and for its accumulation in boundaries between LC and LE phase.

Developmental changes of neutral glycosphingolipids as receptors for pulmonary surfactant protein SP-A in the alveolar epithelium of murine lung.

A dramatic change in the glycosphingolipid composition in murine lung occurred between 1 day and 1 week after birth. GlcCer and LacCer were the predominant neutral glycosphingolipids prenatally and 1 day after birth, and the concentrations of globo- and ganglio-series glycosphingolipids increased abruptly from 1 week after birth, reaching maxima at 2-3 weeks. To explore the functional significance of the change, we examined the role of neutral glycosphingolipids as receptors for the murine pulmonary surfactant protein, SP-A, and found that SP-A bound to LacCer, Gg3Cer, and Gg1Cer, but not to Gb3Cer, Gb4Cer, IV3GalNAc alpha-Gb4Cer, sulfatide, or several gangliosides. On TLC-blotting with 125I-labeled SP-A, the binding of SP-A to Gg3Cer and Gg4Cer was 5 times higher than that to LacCer, and on immunohistochemical staining Gg4Cer and Gg3Cer was mainly observed in the alveolar epithelium. Thus, the capacity to retain SP-A of glycolipid receptors per gram dry weight of lung at 1 week after birth was 1.6 times higher than that at 1 day after birth, and reached a maximum 3 weeks after birth. These findings suggest that the enhanced synthesis of the ganglio-series neutral glycosphingolipids 1 week after birth results in an increase in the binding capacity for SP-A on the epithelial cell surface of alveoli.

Binding of host collectins to the pathogenic yeast Cryptococcus neoformans: human surfactant protein D acts as an agglutinin for acapsular yeast cells

Cryptococcus neoformans is an opportunistic pathogen in AIDS patients causing disseminated disease and lethal meningitis after inhalation of acapsular or sparsely encapsulated yeast cells. In this study we have investigated whether a recently described family of primitive opsonins, termed collectins, contribute to innate resistance against C. neoformans. The pulmonary surfactant proteins SP-A and SP-D as well as the serum collectins mannose-binding protein and CL-43 bound in a calcium-dependent manner to acapsular C. neoformans in vitro. Binding was concentration dependent and abolished by competition with defined mono- and oligosaccharides. In contrast, no binding of the collectins was observed with the encapsulated form of the yeast. Furthermore, binding of purified collectin SP-D, but not SP-A, mannose-binding protein, or CL-43, led to a concentration-dependent agglutination of acapsular C. neoformans. These data indicate that collectins recognize carbohydrate structures in the cell wall of an initial infectious form of C. neoformans and may play a role in early antifungal defenses in the lung.

Pulmonary Surfactant Protein SP-A with Phospholipids in Spread Monolayers at the Air-Water Interface

Spread monolayers of pulmonary surfactant protein SP-A, alone or mixed with phospholipid(s), were formed at the air-water interface. Binary monolayers of SP-A plus dipalmitoylphosphatidylcholine (DPPC) showed positive deviations from ideal behavior of the mean areas in the films consistent with partial miscibility and interaction between the protein and lipid. During compression of SP-A/DPPC films which contained > or = 5 wt % SP-A, properties were displayed which were consistent with the protein being partially squeezed out at surface pressures of about 30 mN/m. Some protein appeared to remain in the monolayers even when they were compressed to high surface pressures of about 65-70 mN/m, and it was possibly included in the collapse phase(s) that was (were) formed at 72 mN/m. During dynamic cyclic compression-expansion of SP-A/DPPC monolayers initially formed at low surface pressures, SP-A enhanced the respreading of the films compressed beyond collapse compared to the respreading after collapse of films containing DPPC alone. Spread monolayers of SP-A plus either dipalmitoylphosphatidylglycerol (DPPG) or a mixture of DPPC plus DPPG (7:3, mol/mol) displayed additivity of the mean areas in the films, consistent with complete immiscibility (or ideal miscibility, an unlikely effect) between the protein and lipid components. Electrostatic repulsion between SP-A and DPPG, both negatively charged at physiological pH, possibly governed the behavior of these lipid-protein films. Calcium ions in the subphase did not alter the properties of SP-A/DPPC films, whereas they improved the ability of SP-A to mix with DPPG and DPPC/DPPG. Binding of calcium to the negatively charged DPPG and SP-A may account for association of the protein with DPPG and DPPC/DPPG in the monolayers in the presence of the divalent ions.

Intermolecular cross-links mediate aggregation of phospholipid vesicles by pulmonary surfactant protein SP-A

As the most abundant glycoprotein component of pulmonary surfactant, SP-A (Mr = 30,000-36,000) plays a central role in the organization of phospholipid bilayers in the alveolar air space. SP-A, isolated from lung lavage, exists in oligomeric forms (N = 6, 12, 18, ...), mediated by collagen-like triple helices and intermolecular disulfide bonds. These protein-protein interactions, involving the amino-terminal domain of SP-A, are hypothesized to facilitate the alignment of surfactant lipid bilayers into unique tubular myelin structures. SP-A reorganization of surfactant lipid was assessed in vitro by quantitating the calcium-dependent light scattering properties of lipid vesicle suspensions induced by SP-A. Accelerated aggregation of unilamellar vesicles required SP-A and at least 3 mM free calcium. The initial rate of aggregation was proportional to the concentration of canine SP-A over lipid:protein molar ratios ranging from 200:1 to 5000:1. Digestion with bacterial collagenase or incubation with dithiothreitol (DTT) completely blocked lipid aggregation activity. Both treatments decreased the binding of SP-A to phospholipids. The conditions used in the DTT experiments (10 mM DTT, nondenaturing Tris buffer, 37 degrees C) resulted in the selective reduction and 14C-alkylation of the intermolecular disulfide bond involving residue 9Cys, whereas the four cysteines found in the noncollagenous domain of SP-A were inefficiently alkylated with [14C]-iodoacetate. HPLC analysis of tryptic SP-A peptides revealed that these four cysteine residues participate in intramolecular disulfide bond formation (138Cys-229Cys and 207Cys-221Cys). Our data demonstrate the importance of the quaternary structure (triple helix and intermolecular disulfide bond) of SP-A for the aggregation of unilamellar phospholipid vesicles.

Structural Comparison of Recombinant Pulmonary Surfactant Protein SP-A Derived from Two Human Coding Sequences: Implications for the Chain Composition of Natural Human SP-A

The pulmonary surfactant-associated protein SP-A is encoded by presumably two different genes, resulting in slightly different amino acid sequences. Both gene products were expressed in Chinese hamster ovary cells. Their macromolecular structure differed significantly. SP-A alpha 3 exhibited a much higher amount of tetrameric to hexameric structures than SP-A alpha 2, for which dimeric structures predominate. These differences may be caused by the higher expression rates of SP-A alpha 3 presumably due to the presence of introns in the sequence. The occurrence of irregular disulfide links between individual oligomeric SP-A molecules composed of alpha 3-chains together with the demonstrated presence of both gene products in natural human SP-A suggest that the subunits of SP-A are heterotrimers of one alpha 2- and two alpha 3-chains.

Tumor necrosis factor-alpha inhibits expression of pulmonary surfactant protein.

Tumor necrosis factor-alpha (TNF-alpha) decreased the expression of pulmonary surfactant proteins SP-A and SP-B in human pulmonary adenocarcinoma cell lines. The effect of TNF alpha on SP-A content and mRNA in the pulmonary adenocarcinoma cell line, H441-4, was concentration and time dependent. TNF alpha decreased the cellular content of SP-A to less than 10% of control 48 h after addition. TNF alpha decreased de novo synthesis of SP-A and decreased the accumulation of SP-A in media. SP-A mRNA was decreased within 12 h of addition of TNF alpha, with nearly complete loss of SP-A mRNA observed after 24 h. Inhibitory effects of TNF alpha on SP-A mRNA were dose-related with nearly complete inhibition of SP-A mRNA caused by 25 ng/ml TNF alpha. The effects of TNF alpha on SP-A were distinct from the effects of interferon gamma which increased SP-A content approximately twofold in H441-4 cells. TNF alpha also decreased the content of SP-B mRNA. In contrast to the inhibitory effect of TNF alpha on SP-A and SP-B mRNA, TNF alpha increased mRNA encoding human manganese superoxide dismutase (Mn-SOD). TNF alpha did not inhibit growth, alter cell viability or beta-actin mRNA in either cell line. These in vitro studies demonstrate the marked pretranslational inhibitory effects of the cytokine, TNF alpha, on the expression of pulmonary surfactant proteins, SP-A and SP-B. The results support the concept that macrophage-derived cytokines may control surfactant protein expression.

Glucocorticoids regulate surfactant protein synthesis in a pulmonary adenocarcinoma cell line.

Synthesis of pulmonary surfactant proteins SP-A, SP-B, and SP-C was demonstrated in a cell line derived from a human adenocarcinoma of the lung. The cells contained numerous lamellar inclusion bodies and formed organized groups of cells containing well-developed junctional complexes and apical microvillous membranes. Synthesis of SP-A was detected in the cells by enzyme-linked immunoabsorbent assay and by immunoprecipitation of [35S]methionine-labeled protein. SP-A was identified as an Mr 31,000-36,000 polypeptide containing asparagine-linked carbohydrate. Northern blot analysis detected SP-A mRNA of 2.2 kb. Dexamethasone (1-10 nM) enhanced the relative abundance of SP-A mRNA. Despite stimulation of SP-A mRNA, intracellular SP-A content was unaltered or inhibited by dexamethasone. SP-B and SP-C mRNAs and synthesis of the SP-B and SP-C precursors were markedly induced by dexamethasone. ProSP-B was synthesized and secreted primarily as an Mr 42,000-46,000 polypeptide. Proteolysis of the proSP-B resulted in the generation of endoglycosidase F-sensitive Mr = 19,000-21,000 and 25,000-27,000 peptides, which were detected both intra- and extracellularly. SP-C proprotein of Mr = 22,000 and smaller SP-C fragments were detected intracellularly but were not detected in the media. Mature forms of SP-B (Mr = 8,000) and SP-C (Mr = 4,000) were not detected. Glucocorticoids directly enhance the relative synthesis and mRNA of the surfactant proteins SP-A, SP-B, and SP-C. Discrepancies among SP-A mRNA, its de novo synthesis, and cell content suggest that glucocorticoid may alter both pre- and posttranslational factors modulating SP-A expression.

Human Pulmonary Surfactant Protein (SP-A), a Protein Structurally Homologous to C1q, Can Enhance FcR- and CR1-mediated Phagocytosis

C1q, a subunit of the first component (C1) of the classical complement pathway, and the pulmonary surfactant protein SP-A are structurally homologous molecules, each having an extended collagen-like domain contiguous with a non-collagenous domain. It is the collagen-like region of C1q that binds to mononuclear phagocytes and mediates the enhancement of phagocytosis of opsonized particles by these cells. Because SP-A enhances the endocytosis of phospholipids by alveolar type II cells and alveolar macrophages, we examined whether these two molecules were functionally interchangeable. The phagocytosis of sheep erythrocytes opsonized with IgG or with IgM and complement was enhanced by the adherence of monocytes or macrophages, respectively, to SP-A. The enhanced response was dependent on the concentration of SP-A used for coating the surfaces, similar to that seen when monocytes were adhered to C1q-coated surfaces. Both the percentage of cells ingesting the opsonized targets and the number of targets ingested per cell increased with increasing concentrations of SP-A. No such enhancement was seen with cells adhered to albumin, iron-saturated transferrin, or uncoated surfaces. However, SP-A did not substitute for C1q in the formation of hemolytically active C1. C1q did not stimulate lipid uptake by alveolar type II cells or alveolar macrophages and had only a slight inhibitory effect on the binding of SP-A to alveolar type II cells. Thus, these results suggested that a function which requires interactions of both the collagenous and the non-collagenous regions (i.e. initiation of the classic complement cascade) could not be mimicked by a protein sharing structural macromolecular similarity but lacking sequence homology in the non-collagen-like region. However, SP-A could substitute for C1q in stimulating a function previously shown to be mediated by the collagen-like domains of the C1q molecule.