Lung surfactant proteins in the early human placenta

Pulmonary surfactant is a complex mixture of phospholipids and four surfactant-associated proteins (SP-A, SP-B, SP-C and SP-D). The biological functions of SP-A and SP-D are primarily twofold, namely surfactant homeostasis and host defense. The hydrophobic surfactant proteins, SP-B and SP-C, are required for achieving the optimal surface tension reducing properties of surfactant by promoting the rapid adsorption of surfactant phospholipids along the alveolar surface. Despite the promising findings, only little is known about the extrapulmonary distribution of these proteins. Therefore, in this study, the presence of SP-A, SP-B, SP-C and SP-D in early human placenta has been investigated. First-trimester placental tissues (22–56 days) were obtained from women undergoing curettage during normal pregnancies. In parallel tissue sections, vimentin, cytokeratin-7 and CD-68 immunostainings were used for the identification of mesenchymal cells, trophoblast cells and Hofbauer cells, respectively. According to immunohistochemistry (IHC) results, SP-A, SP-B, SP-C and SP-D immunoreactivities with different staining intensities were observed in trophoblastic layers of chorionic villous tree, trophoblastic cell columns, stromal cells, Hofbauer cells, angiogenic cell cords and vascular endothelium. Fetal hematopoietic cells showed a variable staining pattern for all four surfactant proteins ranging from none to strong intensity. Western blotting of tissue extracts confirmed our IHC results. The presence of surfactant glycoproteins in early human placenta may yield a very important feature of surfactants during first trimester and enables further studies of the role of surfactants in various pregnancy complications.     

Expression and localization of surfactant proteins in human nasal epithelium

Surfactant proteins (SPs), designated SP-A, SP-B, SP-C, and SP-D, play an important role in surfactant metabolism and host defense mechanisms in the lung. This study investigates expression of the different SP types in human nasal mucosa and cultured normal human nasal epithelial (NHNE) cells and whether the expression of SP mRNA is influenced by the degree of mucociliary differentiation. RT-PCR was performed with mRNA from cultured NHNE cells and nasal mucosa. Immunohistochemical staining for SPs was performed on nasal mucosa specimens. Western blot analysis was performed on cell lysates from cultured NHNE cells. SP-A2, SP-B, and SP-D mRNAs were expressed in normal NHNE cells and human nasal mucosa. SPs were localized in ciliated cells of the surface epithelium and serous acini of the submucosal glands. SP-A, SP-B, and SP-D proteins were expressed in cultured NHNE cells. The degree of mucociliary differentiation influenced expression of the SP gene. We demonstrate that SP-A, SP-B, and SP-D are expressed in human nasal mucosa and cultured NHNE cells. Further study of the functional role of SPs in the upper airway is required.     

Surfactant proteins A and D in the genital tract of mares

The presence of surface-active material in the lung alveolus has been known for several decades as being essential for normal lung function. Surfactant is essential for reducing the surface tension at the alveolar air-liquid interface. Pulmonary surfactant is composed of 90% lipids and 10% proteins. There are four non-serum proteins surfactant protein-A (SP-A), surfactant protein-B (SP-B), surfactant protein-C (SP-C) and surfactant protein-D (SP-D) named in chronologic order of discovery. Lung SP-A and SP-D belong to a family of collagen-containing C-type lectin family called collectins. The host defence and controlling inflammatory processes of the lung are the major functions of SP-A and SP-D. SP-A and SP-D were originally demonstrated in alveolar type II cells, but recent studies have shown extrapulmonary expression of SP-A and SP-D indicating systemic roles of these proteins. Present study describes the presence of SP-A and SP-D in the mare genital tract, vulva, vagina, ovarium, uterus and tuba uterina using immunohistochemistry and Western blotting. The aim of this study was to characterize surfactant proteins in terms of: (i) whether surfactant proteins were present in the various structures of the mare genital system, (ii) if so, identifying and locating the surfactant proteins and finally (iii) determining the differences from those previously characterized for the lung. Although beyond the scope of this report, it is recognized that there are also some potential implications for better defining the reproductive defence mechanisms in mare. Therefore, genital system organs and tissues from mares were examined. We were able to show that proteins reactive with surfactant-specific antibodies were present in the mare genital tract. Thus, surfactant proteins are present not in just lamellar bodies associated with lung, but also genital system of mare.     

Surfactant-associated proteins: functions and structural variation

Pulmonary surfactant is a barrier material of the lungs and has a dual role: firstly, as a true surfactant, lowering the surface tension; and secondly, participating in innate immune defence of the lung and possibly other mucosal surfaces. Surfactant is composed of approximately 90% lipids and 10% proteins. There are four surfactant-specific proteins, designated surfactant protein A (SP-A), SP-B, SP-C and SP-D. Although the sequences and post-translational modifications of SP-B and SP-C are quite conserved between mammalian species, variations exist. The hydrophilic surfactant proteins SP-A and SP-D are members of a family of collagenous carbohydrate binding proteins, known as collectins, consisting of oligomers of trimeric subunits. In view of the different roles of surfactant proteins, studies determining the structure-function relationships of surfactant proteins across the animal kingdom will be very interesting. Such studies may reveal structural elements of the proteins required for surface film dynamics as well as those required for innate immune defence. Since SP-A and SP-D are also present in extrapulmonary tissues, the hydrophobic surfactant proteins SP-B and SP-C may be the most appropriate indicators for the evolutionary origin of surfactant. SP-B is essential for air-breathing in mammals and is therefore largely conserved. Yet, because of its unique structure and its localization in the lung but not in extrapulmonary tissues, SP-C may be the most important indicator for the evolutionary origin of surfactant.     

Swim bladder gas gland cells produce surfactant: in vivo and in culture

Electron microscopical examination of gas gland cells of the physostome European eel (Anguilla anguilla) and of the physoclist perch (Perca fluviatilis) revealed the presence of significant numbers of lamellar bodies, which are known to be involved in surfactant secretion. In the perch, in which the gas gland is a compact structure and gas gland cells are connected to the swim bladder lumen via small canals, lamellar bodies were also found in flattened cells forming the swim bladder epithelium. Flat epithelial cells are absent in the eel swim bladder, in which the whole epithelium consists of cuboidal gas gland cells. In both species, Western blot analysis using specific antibodies to human surfactant protein A (SP-A) showed a cross-reaction with swim bladder tissue homogenate proteins of approximately 65 kDa and in the eel occasionally of approximately 120 kDa, probably representing SP-A-like proteins in a dimeric and a tetrameric state. An additional band was observed at approximately 45 kDa. Western blots using antibodies to rat SP-D again resulted in a single band at approximately 45 kDa in both species, suggesting that there might be a cross-reaction of the antibody to human SP-A with an SP-D-like protein of the swim bladder tissue. To localize the surfactant protein, eel gas gland cells were cultured on permeable supports. Under these conditions, the gas gland cells regain their characteristic polarity. Electron microscopy confirmed the presence of lamellar bodies in cultured cells, and occasionally, exocytotic events were observed. Immunohistochemical staining using an antibody to human SP-A demonstrated the presence of surfactant protein only in luminal membranes and in adjacent lateral membranes. Only occasionally, evidence was found for the presence of surfactant protein in lamellar bodies.     

Expression and localization of lung surfactant protein B in Eustachian tube epithelium

Surfactant protein (SP) B is an essential component of the pulmonary surfactant complex, which participates in reducing the surface tension across the alveolar air-liquid interface. The Eustachian tube (ET) connects the upper respiratory tract to the middle ear, serving as an intermittent airway between the pharynx and the middle ear. Recently, we described the expression of SP-A and SP-D in the ET, suggesting their role in middle ear host defense. Our present aim was to detect whether the expression of SP-B is evident in the porcine ET. With Northern blot analysis, RT-PCR, and in situ hybridizations, SP-B mRNA was identified and localized in the ET epithelium. The cellular localization of SP-B was revealed with immunohistochemistry, electron microscopy, and immunoelectron microscopy. The protein was found in the secretory granules of epithelial cells and also attached to the microvilli at the luminal side of these cells. The SP-B immunoreactivity of aggregates isolated from ET lavage fluid was similar to that isolated from bronchoalveolar lavage fluid. We conclude that there are specialized cells in the ET epithelium expressing and secreting SP-B and propose that SP-B may facilitate normal opening of the tube and mucociliary transport.     

Serum surfactant protein D is increased in acute and chronic inflammation in mice

Surfactant protein A (SP-A) and surfactant protein D (SP-D) are important components of innate immunity that can modify the inflammatory response. However, alterations and regulation of SP-A and SP-D in acute and chronic inflammation are not well defined. In addition, serum SP-D may serve as a biomarker of lung inflammation. We determined the expression of SP-A and SP-D in murine models. To study acute inflammation, we instilled bleomycin intrabronchially. To study chronic lung inflammation, we used a transgenic mouse that overexpresses tumor necrosis factor (TNF)-alpha under the control of the SP-C promoter. These mice have a chronic mononuclear cell infiltration, airspace enlargement, pulmonary hypertension, and focal pulmonary fibrosis. In acute inflammation model, levels of mRNA for all surfactant proteins were reduced after bleomycin administration. However, serum SP-D was increased from days 7 to 28 after instillation. In chronic inflammation model, SP-D mRNA expression was increased, whereas the expression of SP-A, SP-B and SP-C was reduced. Both serum and lung SP-D concentrations were increased in chronic lung inflammation. These data clarified profile of SP-A and SP-D in acute and chronic inflammation and indicated that serum SP-D can serve as a biomarker of lung inflammation in both acute and chronic lung injury in mice.     

Evolution of the lung surfactant proteins in birds and mammals

Phylogenetic analyses of the families of mammalian lung surfactant proteins (SP-A, SP-B, SP-C, and SP-D) supported the hypothesis that these proteins have diverged between birds and mammals as a result of lineage-specific gene duplications and deletions. Homologs of mammalian genes encoding SP-B, SP-C, and SP-D appear to have been deleted in chickens, whereas there was evidence of avian-specific duplications of the genes encoding SP-A and presaposin. Analysis of the genes closely linked to human SP-B, SP-C, and SP-D genes revealed that all three of these genes are closely linked to genes having orthologs on chicken chromosome 6 and also to genes lacking chicken orthologs. These relationships suggest that all of the lung surfactant protein genes, as well as certain related genes, may have been linked in the ancestor of humans and chickens. Further, they imply that the loss of surfactant protein genes in the avian lineages formed part of major genomic rearrangement events that involved the loss of other genes as well. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Human parotid and submandibular glands express and secrete surfactant proteins A,B, C and D

The oral cavity and the salivary glands are open to the oral environment and are thus exposed to multiple microbiological, chemical and mechanical influences. The existence of an efficient defense system is essential to ensure healthy and physiological function of the oral cavity. Surfactant proteins play an important role in innate immunity and surface stability of fluids. This study aimed to evaluate the expression and presence of surfactant proteins (SP) A, B, C, and D in human salivary glands and saliva. The expression of mRNA for SP-A, -B, -C and -D was analyzed by RT-PCR in healthy parotid and submandibular glands. Deposition of all surfactant proteins was determined with monoclonal antibodies by means of Western blot analysis and immunohistochemistry in healthy tissues and saliva of volunteers. Our results show that all four surfactant proteins SP-A, SP-B, SP-C and SP-D are peptides of saliva and salivary glands. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D appear to be involved in immune defense inside the oral cavity. Furthermore, by lowering surface tension between saliva and the epithelial lining of excretory ducts, SP-B and SP-C may assist in drainage and outflow into the oral cavity. Further functions such as pellicle formation on teeth have yet to be determined.     

Regulation of SP-B and SP-C secretion in rat type II cells in primary culture

Secretion of lung surfactant phospholipids is a highly regulated process. A variety of physiological and pharmacological agents stimulate surfactant phospholipid secretion in isolated type II cells. Although the lipid and hydrophobic protein components of surfactant are believed to be secreted together by exocytosis of lamellar body contents, regulation of surfactant protein (SP) B and SP-C secretion has not previously been examined. To address the question of whether secretion of SP-B and SP-C is stimulated by the same agonists that stimulate phospholipid secretion, we measured secretion of all four SPs under the same conditions used to measure phosphatidylcholine secretion. Freshly isolated rat type II cells were cultured overnight and exposed to known surfactant phospholipid secretagogues for 2.5 h, after which the amounts of SP-A, SP-B, SP-C, and SP-D in the medium were measured with immunoblotting. Secretion of SP-B and SP-C was stimulated three- to fivefold by terbutaline, 5'-(N-ethylcarboxyamido)adenosine, ATP, 12-O-tetradecanoylphorbol 13-acetate, and ionomycin. Similar to their effects on phospholipid secretion, the stimulatory effects of the agonists were abolished by Ro 31-8220. Secretion of SP-A and SP-D was not stimulated by the secretagogues tested. We conclude that secretion of the phospholipid and hydrophobic protein components of surfactant is similarly regulated, whereas secretion of the hydrophilic proteins is regulated differently.     

Surfactant proteins A and D in Eustachian tube epithelium

Surfactant protein (SP) A and SP-D are collectins that have roles in host defense. The Eustachian tube (ET) maintains the patency between the upper airways and the middle ear. Dysfunction of local mucosal immunity in ET may predispose infants to recurrent otitis media. We recently described preliminary evidence of the expression of SP-A and SP-D in the ET. Our present aim was to establish the sites of SP-A and SP-D expression within the epithelium of the ET in vivo. With in situ hybridization, electron microscopy, and immunoelectron microscopy, the cells responsible for SP-A and SP-D expression and storage were identified. SP-A expression was localized within the ET epithelium, and the protein was found in the electron-dense granules of microvillar epithelial cells. Being concentrated in the epithelial lining, only a few cells revealed intracellular SP-D, and it was not associated with granules. The SP-A and SP-D immunoreactivities in ET lavage fluid, as shown by Western blot analyses, were similar to those in bronchoalveolar lavage fluid. We propose that there are specialized cells in the ET epithelium expressing and secreting SP-A and SP-D. SP-A and SP-D may be important for antibody-independent protection of the middle ear against infections.     

Surfactant protein A and D in the reproductive tract of stallion

The presence of surface-active material in the lung alveolus has been known for several decades as being essential for normal lung function. The host defense and controlling inflammatory processes of the lung are the major functions of SP-A and SP-D. SP-A and SP-D were originally demonstrated in alveolar type II cells, but recent studies have shown extrapulmonary expression of SP-A and SP-D indicating systemic roles of these proteins. Present study describes the presence of SP-A and SP-D in the stallion genital tract, prepuce, prostate, testis, and seminal vesicle using Western blotting and immunohistochemistry. This paper presents the first evidence for the existence of SP-A and SP-D glycoproteins in the stallion genital tract. We examined genital system organs and tissues from stallion and were able to show that surfactant protein A and D reactive with surfactant-specific antibodies were present in the stallion genital tract tissues and organs. On the basis of results, it can be postulated that surfactant proteins in the stallion reproductive tract contribute to the immune surveillance and to active barrier defense mechanism.     

Isolation and characterization of the cDNA for pulmonary surfactant-associated protein-B (SP-B) in the rabbit

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.     

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 (6A(2)) and SP-A2 (1A(0)), and the coexpressed SP-A1/SP-A2 (6A(2)/1A(0)) 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 (1A(0)) 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 (6A(2)) showed different distribution of the proteins. The morphology of lipid films containing SP-B and the coexpressed SP-A1/SP-A2 (6A(2)/1A(0)) 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.     

Proteolytic inactivation of dog lung surfactant-associated proteins by neutrophil elastase

The adsorption of pulmonary surfactant to an air/fluid interface is influenced by calcium-dependent interactions between its lipid and protein components. The latter include a glycoprotein of 28-36 kDa (SP-A) and two smaller hydrophobic proteins of 5-8 kDa (SP-B, SP-C). Neutrophil elastase and other proteolytic enzymes found in the alveolar washings in a variety of acute lung injuries may cleave the protein components of lung surfactant. To examine the hypothesis that free airspace elastolytic activity may thereby impair surfactant function, we analyzed the effect of neutrophil elastase on surfactant activity in vitro. The adsorption characteristics of dog surfactant and of complexes reassembled from purified surfactant components were examined after incubations with active or heat-inactivated neutrophil elastase. Surfactant preincubated with the active enzyme showed a marked concentration-dependent slowing of adsorption associated with proteolytic cleavage of SP-A. To determine whether elastase also decreases surface activity by affecting the hydrophobic proteins SP-B and SP-C, we studied the effect of incubating elastase with liposomes prepared from surfactant lipid fractions which contain SP-B and SP-C. The addition of intact SP-A to these liposomes incubated with inactive enzyme immediately enhanced adsorption speed. This enhancement was greatly attenuated in liposomes treated with active elastase, suggesting that one or both of the hydrophobic surfactant proteins had been affected by elastase. We conclude that proteolytic cleavage of surfactant proteins reduces adsorption speed in vitro and may disturb surfactant function in vivo.     

Differential effects of glucocorticoid on expression of surfactant proteins in a human lung adenocarcinoma cell line

Synthesis of pulmonary surfactant-associated glycoproteins of Mr 28,000-36,000 (SP-A) and Mr 42,000-46,000 (proSP-B) has been identified in a continuous cell line derived from a human lung adenocarcinoma. SP-A was detected by immunoblot analysis, ELISA assay and by [35S]methionine labelling of the cells. SP-A was secreted into the media as an endoglycosidase F sensitive glycoprotein which co-migrated with the isoforms of SP-A identified in human lavage fluid by 2D-IEF-SDS-PAGE. Hybridization of cellular RNA with SP-A-specific cDNA identified an abundant 2.2 kb mRNA species, identical to that observed in human lung. SP-A RNA and protein content were markedly inhibited by dexamethasone in a dose-dependent fashion. Under identical culture conditions, synthesis of a distinct surfactant protein, SP-B, was markedly stimulated by the glucocorticoid. The SP-B precursor was secreted into the media as heterogeneous Mr 42,000-46,000 protein, pI 4.6-5.1, and was sensitive to endoglycosidase F. Synthesis of proSP-B was enhanced by the glucocorticoid in a dose-dependent fashion and was associated with increased SP-B mRNA of 2.0 kb detected by Northern blot analysis. The cell line secreted proSP-B as Mr 42,000-46,000 glycosylated protein and did not process the precursor to the Mr 7000-8000 surfactant peptide. In summary, a human adenocarcinoma cell line has been identified which synthesizes and secretes two surfactant-associated proteins, SP-A and proSP-B. Glucocorticoid enhanced SP-B but inhibited SP-A expression in this cell line. The identification of a continuous cell line secreting surfactant proteins may be useful in the study of synthesis and secretion of these important proteins and for production of the proteins for clinical uses.     

Antisense inhibition of surfactant protein A decreases tubular myelin formation in human fetal lung in vitro

Surfactant protein A (SP-A) is the most abundant of the surfactant-associated proteins. SP-A is involved in the formation of tubular myelin, the modulation of the surface tension-reducing properties of surfactant phospholipids, the metabolism of surfactant phospholipids, and local pulmonary host defense. We hypothesized that elimination of SP-A would alter the regulation of SP-B gene expression and the formation of tubular myelin. Midtrimester human fetal lung explants were cultured for 3-5 days in the presence or absence of an antisense 18-mer phosphorothioate oligonucleotide (ON) complementary to SP-A mRNA. After 3 days in culture, SP-A mRNA was undetectable in antisense ON-treated explants. After 5 days in culture, levels of SP-A protein were also decreased by antisense treatment. SP-B mRNA levels were not affected by the antisense SP-A ON treatment. However, there was decreased tubular myelin formation in the antisense SP-A ON-treated tissue. We conclude that selective elimination of SP-A mRNA and protein results in a decrease in tubular myelin formation in human fetal lung without affecting SP-B mRNA. We speculate that SP-A is critical to the formation of tubular myelin during human lung development and that the regulation of SP-B gene expression is independent of SP-A gene expression.     

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.