Surfactant-associated Proteins SP-A Research Articles

Impact of a Met(11)Thr single nucleotide polymorphism of surfactant protein D on allergic airway inflammation in a murine asthma model

ABSTRACTThe surfactant-associated proteins SP-A and D are pattern recognition molecules with collectin structure. A single nucleotide polymorphism (SNP) exchanging a methionine (Met) for a threonine (Thr) in the amino-terminal SP-D domain influences the oligomeric structure and function of the protein. In this study, we investigated the susceptibility of mice transgenic for the human SP-D Met(11)Thr SNP to allergic airway inflammation and consequences for microRNA (miRNA, miR) expression. Mice expressing either human Met or Thr SP-D were sensitized and challenged with ovalbumin (OVA) in an acute model of allergic asthma. The influence of the SP-D polymorphism on the allergic airway inflammation was evaluated by lung function measurement, pulmonary inflammation parameters, morphological analysis and miRNA expression. Airway hyperresponsiveness, allergic inflammation, and mucus metaplasia were not significantly different between mice expressing one or the other allelic variant of SP-D. OVA sensitization and challenge led to significant airway hyperresponsiveness in wildtype mice and significantly lower eosinophil numbers and interleukin 5 levels in Thr SP-D mice. OVA challenge induced an upregulation of miR-21 and 155 in Thr SP-D mice and a downregulation of miR-21 in Met SP-D mice. Our results show that murine expression of human polymorphic SP-D variants does not significantly influence the severity of allergic airway inflammation. MiR-21 and 155 are differentially regulated in transgenic mice in response to allergic inflammation. Further studies are required to elucidate the impact of this SNP on inflammatory conditions of the lung.

Detection and Localization of the Hydrophobic Surfactant Proteins B and C in Human Tear Fluid and the Human Lacrimal System

Purpose: To evaluate the expression and presence of the surfactant proteins (SP) B and C in the lacrimal apparatus at the ocular surface and in tear fluid. Methods: Expression of SP-B and SP-C was analyzed by RT-PCR in healthy lacrimal gland, conjunctiva, meibomian gland, accessory lacrimal glands, cornea, and nasolacrimal ducts. The deposition of the hydrophobic proteins SP-B and SP-C was determined by Western blot and immunohistochemistry in healthy tissues, tear fluid, and aqueous humor. Results: The presence of both SP-B and SP-C on mRNA and protein level was evidenced in healthy human lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts. Moreover, both proteins were present in tear fluid but were absent in aqueous humor. Immunohistochemical investigations revealed production of both peptides by acinar epithelial cells of the lacrimal gland and additionally by accessory lacrimal glands of the eyelid as well as epithelial cells of the conjunctiva and nasolacrimal ducts. Immunohistochemically, healthy cornea and goblet cells revealed no reactivity. Conclusions: Besides the recently detected surfactant-associated proteins SP-A and SP-D, our results show that SP-B and SP-C are also peptides of the tear film, the ocular surface, and the lacrimal apparatus. Based on the current knowledge of lowering surface tension in alveolar lung cells, a similar effect of SP-B and SP-C may be assumed concerning the tear film.

Poly(ethylene glycol) (PEG) enhances dynamic surface activity of a bovine lipid extract surfactant (BLES)

Shortage or malfunction of pulmonary surfactant in alveolar space leads to a critical condition termed respiratory distress syndrome (RDS). Surfactant replacement therapy, the major method to treat RDS, is an expensive treatment. In this paper, the effect of poly(ethylene glycol) (PEG) to improve dynamic surface activity of a bovine lipid extract surfactant (BLES) was studied by axisymmetric drop shape analysis (ADSA) and a captive bubble method. The activity of BLES + PEG mixtures was compared to that of a natural surfactant containing surfactant proteins A and D. When PEG was added into BLES mixtures, the surface tension hysteresis of BLES films was minimized when the films were compressed by more than 50%. PEG also helps to quickly restore surfactant films after film collapse. Thus, as far as surface tension effects go, the findings suggest that PEG might be used as a substitute for surfactant-associated protein SP-A in therapeutic surfactant products, and might also be used to reduce the amount of BLES required in clinical applications.

Bench-to-bedside review: Paediatric viral lower respiratory tract disease necessitating mechanical ventilation – should we use exogenous surfactant?

Treatment of infants with viral lower respiratory tract disease (LRTD) necessitating mechanical ventilation is mainly symptomatic. The therapeutic use of surfactant seems rational because significantly lower levels of surfactant phospholipids and proteins, and impaired capacity to reduce surface tension were observed among infants and young children with viral LRTD. This article reviews the role of pulmonary surfactant in the pathogenesis of paediatric viral LRTD. Three randomized trials demonstrated improved oxygenation and reduced duration of mechanical ventilation and paediatric intensive care unit stay in young children with viral LRTD after administration of exogenous surfactant. This suggest that exogenous surfactant is the first beneficial treatment for ventilated infants with viral LRTD. Additionally, in vitro and animal studies demonstrated that surfactant associated proteins SP-A and SP-D bind to respiratory viruses, play a role in eliminating these viruses and induce an inflammatory response. Although these immunomodulating effects are promising, the available data are inconclusive and the findings are unconfirmed in humans. In summary, exogenous surfactant in ventilated infants with viral LRTD could be a useful therapeutic approach. Its beneficial role in improving oxygenation has already been established in clinical trials, whereas the immunomodulating effects are promising but remain to be elucidated.

Collectins and pulmonary innate immunity.

The surfactant-associated proteins SP-A and SP-D are members of a family of host defense lectins, designated collectins. There is increasing evidence that these pulmonary, epithelial-derived proteins are important components of the innate immune response to microbial challenge and participate in other aspects of immune and inflammatory regulation within the lung. Both proteins bind to glycoconjugates and/or lipid moieties expressed by a wide variety of microorganisms, and to certain organic particles, such as pollens. SP-A and SP-D have the capacity to modulate leukocyte function and, in some circumstances; to opsonize and enhance the killing of microorganisms. The biologic activity of cell wall components, such as Gram-negative bacterial polysaccharides, or viral glycoproteins, such as the hemagglutinin of influenza viruses, may be altered by interactions with collectins. In addition, complementary or cooperative interactions between SP-A, SP-D and other host defense lectins could contribute to the efficiency of this defense system. Collectins could play particularly important roles in settings of inadequate or impaired specific immunity, and acquired alterations in the levels of active collectins within the airspaces and distal airways may increase susceptibility to infection.

Collectins and pulmonary host defense.

The surfactant-associated proteins SP-A and SP-D are members of a family of collagenous host defense lectins, designated collectins. There is increasing evidence that these pulmonary epithelial-derived proteins are important components of the innate immune response to microbial challenge, and that they participate in other aspects of immune and inflammatory regulation within the lung. The collectins bind to glycoconjugates and/or lipid moieties expressed by a wide variety of microorganisms and certain other organic particles in vitro. Although binding may facilitate microbial clearance through aggregation or other direct effects on the organism, SP-A and SP-D also have the capacity to modulate leukocyte function and, in some circumstances, to enhance their killing of microorganisms. The biologic activity of cell wall components, such as gram-negative bacterial polysaccharides, may be altered by interactions with collectins. Complementary or cooperative interactions between SP-A and SP-D could contribute to the efficiency of this defense system. Collectins may play particularly important roles in settings of inadequate or impaired specific immunity. Acquired or genetic alterations in the levels of active proteins within the airspaces and distal airways may increase susceptibility to infection.

Impaired surfactant synthesis in fetal type II lung cells from gsd/gsd rats.

The importance of intracellular glycogen for surfactant synthesis was investigated in fetal type II lung cells isolated from rats with a glycogen storage disorder, designated gsd/gsd. Compared to cells from a control Wistar strain, cultured gsd/gsd pneumocytes were glycogenrich and contained fewer and smaller lamellar inclusions. Freshly isolated cells from day 19-21 fetuses of control rats demonstrated the expected gestational rise and fall of cellular glycogen seen in intact fetal lungs. At day 20, when tissue glycogen peaks, cellular glycogen content was 48 and 70 nmol glucose/micrograms DNA in isolated type II cells of control and gsd/gsd lungs, respectively. In control cells, while active glycogen phosphorylase changed from 35 to 65% of total during 24 h of culture, glycogen fell 85%. In contrast, gsd/gsd cell phosphorylase was not activated, phosphorylase kinase activity was nondetectable, and glycogen per cell remained unchanged. [3H]Choline incorporation into total PC and disaturated PC (DSPC) was 50 and 62% lower, respectively, in gsd/gsd type II cells compared to controls in the absence of exogenous substrate. Cellular content of the surfactant-associated protein SP-A was similar in control and gsd/gsd cells at day 20, and increased 3- to 4-fold during a subsequent 24-h interval of tissue culture. These results suggest that PC synthesis is dramatically impaired in type II cells in which glycogen cannot be mobilized, but SP-A is synthesized at normal rates. This work characterizes the isolated gsd/gsd fetal type II cell and supports its use in future studies to determine the importance and relative utilization of specific nonglycogen substrates.

Retinoic acid induces changes in the pattern of airway branching and alters epithelial cell differentiation in the developing lung in vitro.

Retinoids have been shown to influence pattern formation during development and regeneration in numerous systems such as limbs, vertebrae, and neural tube although there is little information about the effects of retinoids on pattern formation in visceral organs. We investigated the effects of exogenous retinoic acid on the in vitro pattern of airway branching and on lung epithelial cell differentiation. Histology, [3H]thymidine autoradiographies and reverse transcriptase/polymerase chain reaction (RT/PCR) amplification were used to assess the effects of retinoids and the expression of lung epithelial markers of differentiation. We found that retinoic acid interferes, in a dose-dependent fashion, with the expression of epithelial genes that are found in distal segments of the fetal lung (surfactant-associated proteins SP-A, SP-B, and SP-C). At high concentrations, retinoic acid (RA) dramatically altered the developmental pattern of the lung, favoring growth of structures that resemble proximal airways and concomitantly suppressing distal epithelial buds. We hypothesize that this in vitro "proximalizing" effect on the developing lung may be related to alterations in the expression of pattern-related genes.

In VitroandIn VivoTransfer and Expression of Human Surfactant SP-A- and SP-B-Associated Protein cDNAs Mediated by Replication-Deficient, Recombinant Adenoviral Vectors

Congenital pulmonary alveolar proteinosis (CPAP) is a fatal disease of full-term infants that is unresponsive to current medical therapy. It is now recognized that at least some forms of this disorder are associated with a deficiency of SP-B, one of the surfactant-associated proteins, as well as probable aberrations in the surfactant-associated proteins SP-A and SP-C. Given these developments, it is logical to hypothesize that CPAP may be amenable to gene therapy, in which the human SP-B cDNA, and possibly the cDNAs of the other surfactant associated proteins, are transferred to the epithelium of the lower respiratory tract. We constructed replication-deficient, recombinant adenovirus vectors in which a constitutive viral promoter drives the expression of the DNAs for the surfactant-associated proteins, SP-B (AdCMV.SP-B) and SP-A (AdCMV.SP-A). Following infection of the human lung A549 epithelial cell line with these vectors in vitro, the appropriately sized mRNAs for these cDNAs were detected, whereas cells infected with a control virus or uninfected cells produced none. Western blots demonstrated expression of these proteins, including appropriate processing of the hydrophobic protein, SP-B. Following in vivo intratracheal infection of rats with these vectors, Northern analysis of the lungs revealed appropriately sized mRNAs for these cDNAs whereas rats infected with control virus or uninfected rats show no hybridization with the human surfactant-associated protein probes. In the AdCM-V.SP-A-infected rats, Western blots confirmed the overproduction of the human SP-A protein in both the bronchoalveolar lavage and lung homogenates compared to controls. Thus, it is feasible to utilize adenovirus vectors to transfer and express the human surfactant associated protein cDNAs in vitro and in vivo, presenting a possible mode of therapy for CPAP, as well as other surfactant deficiency states such as the neonatal respiratory distress syndrome and possibly the adult respiratory distress syndrome.

Regulation of mRNA levels for pulmonary surfactant-associated proteins in developing rabbit lung

Gene transcriptional activities and steady-state mRNA levels have been examined for the surfactant-associated proteins SP-A, SP-B and SP-C in developing rabbit lung. It was observed SP-C mRNA levels increase early in gestation, while SP-A and SP-B mRNA levels increase rapidly between 26 and 30 days gestation. Transcriptional activities for all three surfactant apoproteins increase between 26 and 30 days. Studies conducted with fetal lung explants of 26 days gestation demonstrated exposure to low doses of dexamethasone increases SP-A and SP-C mRNA levels, while high doses stimulate transcription, although this was only significant for SP-C. Time course studies revealed different temporal patterns and glucocorticoid responses for SP-A and SP-C mRNAs. SP-A and SP-C mRNA production and steady-state levels were reduced after treatment with cycloheximide. In contrast, SP-B gene transcription was selectively stimulated, suggesting involvement of a labile negative regulatory factory. It is concluded that expression of the three surfactant apoproteins is independently regulated. Early in gestation, SP-C mRNA levels may be regulated in vivo through message stabilization. Glucocorticoids can affect SP-A and SP-C mRNA levels in culture at both transcriptional and post-transcriptional levels. The ability of glucocorticoids to influence these processes declines during fetal development.

Retinoic acid differentially regulates expression of surfactant- associated proteins in human fetal lung

Retinoic acid is known to play an essential role in maintaining the differentiation of a wide variety of epithelial cell types. However, its effects on the differentiation of lung alveolar epithelium have not been described. In the present study, we examined the effects of retinoic acid on the differentiation of human fetal lung tissue maintained in vitro. Human fetal lung explants were cultured in serum-free medium for 6 days in the absence or presence of all-trans retinoic acid at concentrations from 0.3 nM to 3 microM. Explant content of the surfactant-associated protein SP-A was measured using a specific enzyme-linked immunosorbent assay. Retinoic acid reduced SP-A protein levels in a concentration-dependent manner [analysis of variance (ANOVA), P < 0.01]. To evaluate possible cytotoxic effects of retinoic acid, culture media were assayed for lactate dehydrogenase (LDH), a cytoplasmic enzyme. LDH levels in media from retinoic acid-treated explants were not significantly different than LDH levels in media from control explants, indicating that retinoic acid is not cytotoxic in human fetal lung explants. Changes in messenger RNA (mRNA) levels for surfactant-associated proteins SP-A, SP-B, and SP-C were measured by Northern blot analysis. Retinoic acid reduced SP-A mRNA levels in a concentration-dependent manner (ANOVA, P < 0.02) and reduced SP-C mRNA levels at 3 microM. In contrast, retinoic acid increased SP-B mRNA levels in a concentration-dependent manner (ANOVA, P < 0.03). Morphometric analysis showed that retinoic acid decreased epithelial volume density in the explants by approximately 17% and increased connective tissue volume density by approximately 20% when compared to dimethyl sulfoxide vehicle controls. These data indicate that retinoic acid regulates type II cell surfactant protein gene expression in human fetal lung tissue.

Retinoic acid differentially regulates expression of surfactant-associated proteins in human fetal lung.

Retinoic acid is known to play an essential role in maintaining the differentiation of a wide variety of epithelial cell types. However, its effects on the differentiation of lung alveolar epithelium have not been described. In the present study, we examined the effects of retinoic acid on the differentiation of human fetal lung tissue maintained in vitro. Human fetal lung explants were cultured in serum-free medium for 6 days in the absence or presence of all-trans retinoic acid at concentrations from 0.3 nM to 3 microM. Explant content of the surfactant-associated protein SP-A was measured using a specific enzyme-linked immunosorbent assay. Retinoic acid reduced SP-A protein levels in a concentration-dependent manner [analysis of variance (ANOVA), P < 0.01]. To evaluate possible cytotoxic effects of retinoic acid, culture media were assayed for lactate dehydrogenase (LDH), a cytoplasmic enzyme. LDH levels in media from retinoic acid-treated explants were not significantly different than LDH levels in media from control explants, indicating that retinoic acid is not cytotoxic in human fetal lung explants. Changes in messenger RNA (mRNA) levels for surfactant-associated proteins SP-A, SP-B, and SP-C were measured by Northern blot analysis. Retinoic acid reduced SP-A mRNA levels in a concentration-dependent manner (ANOVA, P < 0.02) and reduced SP-C mRNA levels at 3 microM. In contrast, retinoic acid increased SP-B mRNA levels in a concentration-dependent manner (ANOVA, P < 0.03). Morphometric analysis showed that retinoic acid decreased epithelial volume density in the explants by approximately 17% and increased connective tissue volume density by approximately 20% when compared to dimethyl sulfoxide vehicle controls. These data indicate that retinoic acid regulates type II cell surfactant protein gene expression in human fetal lung tissue.

Localization of surfactant-associated proteins SP-A and SP-B mRNA in rabbit fetal lung tissue by in situ hybridization.

Pulmonary surfactant is a lipoprotein substance, comprised of approximately 80% phospholipid and approximately 10% protein, that lowers surface tension at the air-alveolar aqueous interface. Surfactant is synthesized and secreted by alveolar type II epithelial cells where it is stored intracellularly in lamellar bodies. In the present study, we used the technique of in situ hybridization to localize the mRNA for two surfactant-associated proteins, SP-A and SP-B, in developing rabbit fetal lung tissue. We found that SP-A mRNA was first localized in rabbit fetal lung alveolar type II cells on day 26 of gestation, the time at which lamellar bodies are first observed within fetal lung type II cells. On day 28 of gestation, a very small amount of SP-A mRNA was also detectable in the epithelial cells of some bronchioles. In neonatal and adult rabbit lung tissue, SP-A mRNA was primarily restricted to alveolar type II cells; however, the epithelial cells of some bronchioles contained small amounts of SP-A mRNA. SP-B mRNA was first detected in cuboidal epithelial cells in the prealveolar region of the rabbit fetal lung tissue on day 24 of gestation, i.e., at least 2 days before the appearance of SP-A mRNA and lamellar bodies within differentiated alveolar type II cells. SP-B mRNA was detected in most bronchiolar epithelial cells of the rabbit fetal lung tissue at day 28 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary structure differences of human surfactant-associated proteins isolated from normal and proteinosis lung

The primary structures of human pulmonary surfactant-associated proteins SP-A, SP-B and SP-C isolated from lung lavage of patients with alveolar proteinosis exhibit significant differences from lung surfactant proteins isolated from lungs of healthy individuals. In contrast to SP-A from normal lungs, proteinosis SP-A was shown by SDS gel electrophoresis to contain large amounts of unreducibly cross-linked beta chains. Specific primary structure modifications of SP-C and SP-B proteins were established by direct molecular weight and structural analysis, using [ 252Cf]plasma desorption mass spectrometry (PD/MS) as the principal method. In comparison to normal lung surfactant SP-B, proteinosis SP.B showed a significantly increased molecular weight by approx. 500 Da for the unreduced protein dimer. SP-C proteins from normal lungs were identified to possess a bis-cysteinyl-5,6-(thioester)palmitoylated structure, and to contain a frayed N-terminus resulting in two sequences of 34 and 35 amino acid residues. In contrast, SP-C from proteinosis patients was modified by (i) partial or even complete removal of palmitate residues and (ii) additional N-terminal proteolytic degradation. These results indicate the presence of pathophysiological structure modifications, which are likely to occur in the alveolar space, and may lead to a reduced surfactant function.

Immunoassay for the determination of surfactant apoprotein (SP-A) in human amniotic fluid: comparison with other indices of assessing foetal lung maturity.

The concentration of the major surfactant-associated protein SP-A (28-36 kDa) was determined in 73 amniotic fluid samples obtained from normal (n = 40) and complicated (n = 33) pregnancies. Lecithin/sphingomyelin (L/S) ratio and phosphatidylglycerol (PG) levels were also determined in all the samples by one-dimensional step-wise thin-layer chromatography. An enzyme-linked immunosorbent assay was used to determine human lung surfactant apoprotein SP-A. The amount of SP-A in human amniotic fluid increased as a function of gestational age from 8 mg l-1 at 36 weeks to 11.75 mg l-1 at 40-41 weeks of gestation. There was a significant difference (p less than 0.01) in amniotic fluid SP-A concentration from female (9.93 +/- 0.60 micrograms ml-1) compared to male (9.10 +/- 0.52 micrograms ml-1) foetuses. In amniotic fluid samples obtained from a group of complicated pregnancies, SP-A levels were significantly lower than in the normal group when adjusted for gestational age and sex of the foetus (p less than 0.05).

Surfactant lipid uptake and secretion in type II cells in response to lectins and secretagogues

Secretion of surfactant phospholipids can be inhibited by the surfactant-associated protein SP-A. SP-A was reported to stimulate lipid uptake by type II cells, whereas surfactant secretagogues were reported to have the same effect in isolated perfused lungs. We examined the effect of such secretagogues on uptake of liposomes containing L-alpha-[2-palmitoyl-9,103H(N)]-dipalmitoyl phosphatidylcholine in primary cultures of type II cells. As SP-A contains a lectinlike domain and other lectins were reported to inhibit surfactant secretion, we also examined the effect of such lectins on lipid uptake. At concentrations at which they maximally stimulate phosphatidylcholine secretion in type II cells, several secretagogues had no effect on liposome uptake. Maclura pomifera agglutinin (MPA) stimulated uptake approximately 10-fold with a concentration eliciting 50% maximum stimulation (EC50) of 17 micrograms/ml. The effect of MPA on uptake was considerably greater than that of SP-A. However, although the stimulatory effect of ATP on phosphatidylcholine secretion was almost completely antagonized by SP-A, it was maximally inhibited only 75% by MPA. The concentration eliciting 50% maximum inhibition (IC50) for MPA inhibition of secretion was 0.5 micrograms/ml. Concanavalin A, another lectin, had no effect on lipid uptake but completely inhibited secretion. These data show that a lectin other than SP-A can stimulate phospholipid uptake by type II cells cultured on plastic and suggest that surfactant secretion and reuptake are independently regulated processes.

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.