Surfactant Protein A Protein Research Articles

An internal amino-terminal FLAG-tag octapeptide alters oligomerization of expressed surfactant protein-A

Pulmonary surfactant protein-A (SP-A) is expressed by lung alveolar and bronchiolar epithelial cells and plays a critical role in innate immunity of the lung. Exposure of the lung to various environmental insults alters SP-A homeostasis. To investigate the cellular mechanisms involved in these alterations, we added the FLAG octapeptide (DYKDDDDK) to the carboxy-terminus (SP-A/C-FLAG) or near the amino-terminus (SP-A/N-FLAG) of mouse SP-A (WT-SP-A) to tag specific pools of protein. We hypothesized that addition of FLAG would have negligible effects on SP-A expression, oligomerization and secretion. Analysis of Chinese hamster ovary cells expressing these proteins indicated that tagged SP-A mRNA could be distinguished from WT-SP-A by northern analysis and RT-PCR using sequence-specific oligonucleotides. Tagged SP-A protein could be differentiated from WT-SP-A by western analysis using antibodies specific for the FLAG epitope. Subcellular fractionation and immunocytochemistry indicated the majority of each protein was present in punctuate (presumably endocytic) vesicles, and all forms of SP-A protein were secreted. These results suggest that a FLAG epitope added to the carboxy-terminus or inserted into the amino-terminus of the mature SP-A protein has little effect on its expression and cellular processing. However, disruptions of the amino-terminal end of SP-A prevents proper oligomerization, suggesting that this region of mature SP-A is critical in proper oligomeric assembly and is not useful for studies intended to define mechanisms underlying SP-A homeostasis.

Structural and Functional Determinants of Rodent and Human Surfactant Protein A: A Synthesis of Binding and Computational Data.

Surfactant protein A (SP-A) provides surfactant stability, first line host defense, and lung homeostasis by binding surfactant phospholipids, pathogens, alveolar macrophages (AMs), and epithelial cells. Non-primates express one SP-A protein whereas humans express two: SP-A1 and SP-A2 with core intra- and inter-species differences in the collagen-like domain. Here, we used macrophages and solid phase binding assays to discern structural correlates of rat (r) and human (h) SP-A function. Binding assays using recombinant rSP-A expressed in insect cells showed that lack of proline hydroxylation, truncations of amino-terminal oligomerization domains, and site-directed serine (S) or alanine (A) mutagenesis of cysteine 6 (C6S), glutamate 195 (E195A), and glutamate 171 (E171A) in the carbohydrate recognition domain (CRD) all impaired SP-A binding. Replacement of arginine 197 with alanine found in hSP-A (R197A), however, restored the binding of hydroxyproline-deficient rSP-A to the SP-A receptor SP-R210 similar to native rat and human SP-A. In silico calculation of Ca++ coordination bond length and solvent accessibility surface area revealed that the “humanized” R197A substitution alters topology and solvent accessibility of the Ca++ coordination residues of the CRD domain. Binding assays in mouse AMs that were exposed to either endogenous SP-A or hSP-A1 (6A2) and hSP-A2 (1A0) isoforms in vivo revealed that mouse SP-A is a functional hybrid of hSP-A1 and hSP-A2 in regulating SP-A receptor occupancy and binding affinity. Binding assays using neonatal and adult human AMs indicates that the interaction of SP-A1 and SP-A2 with AMs is developmentally regulated. Furthermore, our data indicate that the auxiliary ion coordination loop encompassing the conserved E171 residue may comprise a conserved site of interaction with macrophages, and SP-R210 specifically, that merits further investigation to discern conserved and divergent SP-A functions between species. In summary, our findings support the notion that complex structural adaptation of SP-A regulate conserved and species specific AM functions in vertebrates.

Adverse impact of ambient PM2.5 on expression and trafficking of surfactant protein A through reactive oxygen species damage to lamellar bodies

Particulate matter with a diameter of less than 2.5 μm (PM2.5) easily deposits on lung alveoli and degrades human health. Surfactant protein A (SP-A) is the most abundant pulmonary surfactant protein stored in lamellar bodies (LBs) of alveolar epithelial type II cells. The impacts of PM2.5 on SP-A are multifaceted and intractable, and the underlying mechanism remains unclear. In this study, the expression and distribution of SP-A in Balb/c mice and A549 cells under PM2.5 exposure were investigated. The results showed that the low and medium concentration of PM2.5 gradually enhanced SP-A protein and mRNA expression, whereas the high concentration of PM2.5 conspicuously decreased SP-A protein but not its mRNA compared with the control. The trafficking of SP-A to LBs was gradually disturbed, and concomitantly, the lesions of LBs responsible for the transport and storage of SP-A protein were exacerbated with increased PM2.5 concentration. Reactive oxygen species production abundantly increased upon PM2.5 exposure, and it was antagonized by the oxidant inhibitor N-acetylcysteine. Subsequently, the injured LBs and the decrease in SP-A expression under exposure to the high concentration of PM2.5 were well rescued. The present study provides a new perspective to investigate the adverse effects of PM2.5 or diesel exhaust particles on other proteins transported to and stored in LBs.

P-310 - A peptide derived from naturally occurring surfactant protein A (SP-A) inhibits the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6) and NOX2 activation and prevents mouse lung injury following intratracheal LPS

We have developed a peptide based on the naturally occurring 26 kDa SP-A protein that inhibits the PLA2 but not the GPx activity of Prdx6. A 16 aa sequence was predicted by the ZDOCK program as the molecular site for binding of SP-A to Prdx6 and was confirmed using ITC and CD measurements.The peptide (called PLA2-inhibitory peptide or PIP) inhibited the PLA2 activity of recombinant human Prdx6 by ~70% and similarly inhibited Prdx6-associated PLA2 activity in homogenates of mouse lungs while presence of the peptide prevented the activation of lung NOX2.We have found since that a 9 aa peptide, named PIP-2,retains the Prdx6-inhibitory properties of both the protein and PIP. PIP was tested in a mouse model of acute lung injury (ALI) generated by the intratracheal instillation of bacterial lipopolysaccharide (LPS).PIP was encapsulated in liposomes for intracellular delivery and administered IV to mice either co-incidentally with or at 12–14 h after LPS. Mouse lungs evaluated at 24 h after LPS showed a marked decrease of lung leukocytic infiltration, of altered permeability, and of the oxidation of tissue macromolecules in the animals receiving PIP. These studies suggest that PIP might be a useful therapeutic for the prevention and treatment of ALI.

Lipopolysaccharide Stimulates Surfactant Protein-A in Human Renal Epithelial HK-2 Cells through Upregulating Toll-like Receptor 4 Dependent MEK1/2-ERK1/2-NF-κB Pathway.

Background:Surfactant protein-A (SP-A) contributes to the regulation of sepsis-induced acute kidney injury. In a previous study, we demonstrated that the expression of SP-A in the human renal tubular epithelial (HK-2) cells can be stimulated by lipopolysaccharide (LPS). The present study evaluated the possible signal-transducing mechanisms of LPS-induced SP-A biosynthesis in the HK-2 cells.Methods:Tetrazolium salt colorimetry (MTT) assay was used to detect cell viability of HK-2 cells after LPS stimulation on different time points. HK-2 cells were stimulated with 100 ng/ml of LPS for different durations to determine the effects of LPS on SP-A and toll-like receptor 4 (TLR4) messenger RNA (mRNA) expression, as well as phosphorylation of mitogen-activated/extracellular signal-regulated kinase (MEK) 1, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38MAPK), and nuclear factor-kappa B (NF-κB) inhibitor-alpha (IkB-α). Then, HK-2 cells were pretreated with CLI-095, a TLR4 inhibitor, to analyze mRNA and protein levels of SP-A and TLR4 and expression of NF-κB in the cytoplasm and nucleus of HK-2 before LPS exposure.Results:HK-2 cells exposed to 100 ng/ml of LPS for 1, 6, and 24 h did not affect cell viability which showed no toxic effect of 100 ng/ml LPS on cells (P = 0.16); however, the biosynthesis of SP-A mRNA and protein in HK-2 cells was significantly increased (P = 0.02). As to the mechanism, LPS enhanced transmembrane receptor TLR4 protein expression. Sequentially, LPS time dependently augmented phosphorylation of MEK1, ERK1/2, and p38MAPK. In addition, levels of phosphorylated IκB-α and nuclear NF-κB were augmented with LPS exposure for 2 h. LPS-induced SP-A and TLR4 mRNA as well as NF-κB expression were significantly inhibited by pretreatment with CLI-095.Conclusions:The present study exhibited that LPS can increase SP-A synthesis in human renal epithelial cells through sequentially activating the TLR4-related MEK1-ERK1/2-NF-κB-dependent pathway.

Surfactant protein A is expressed in the central nervous system of rats with experimental autoimmune encephalomyelitis, and suppresses inflammation in human astrocytes and microglia.

The collectin surfactant protein-A (SP-A), a potent host defense molecule, is well recognized for its role in the maintenance of pulmonary homeostasis and the modulation of inflammatory responses. While previous studies have detected SP-A in numerous extrapulmonary tissues, there is still a lack of information regarding its expression in central nervous system (CNS) and potential effects in neuroinflammatory diseases, such as multiple sclerosis (MS). The present study used experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS, to investigate the expression of SP-A in the CNS at different stages of disease progression. In addition, in vitro experiments with lipopolysaccharide (LPS)-stimulated human astrocytes and microglia were performed to investigate the potential role of SP-A in the modulation of CNS inflammatory responses. The results of the present study demonstrated widespread distribution of SP-A in the rat CNS, and also identified specific expression patterns of SP-A at different stages of EAE. In vitro, the current study revealed that treatment of human astrocytes and microglia with LPS promoted SP-A expression in a dose-dependent manner. Furthermore, exogenous SP-A protein significantly decreased Toll-like receptor 4 and nuclear factor-κB expression, and reduced interleukin-1β and tumor necrosis factor-α levels. The results of the current study indicate a potential role for SP-A in the modulation of CNS inflammatory responses.

The protective effect of different airway humidification liquids to lung after tracheotomy in traumatic brain injury: The role of pulmonary surfactant protein-A (SP-A)

The purpose of this study was to establish a rat model of a brain injury with tracheotomy and compared the wetting effects of different airway humidification liquids, afterward, the best airway humidification liquid was selected for the clinical trial, thus providing a theoretical basis for selecting a proper airway humidification liquid in a clinical setting. Rats were divided into a sham group, group A (0.9% NaCl), group B (0.45% NaCl), group C (0.9% NaCl+ambroxol) and group D (0.9% NaCl+Pulmicort). An established rat model of traumatic brain injury with tracheotomy was used. Brain tissue samples were taken to determine water content, while lung tissue samples were taken to determine wet/dry weight ratio (W/D), histological changes and expression levels of SP-A mRNA and SP-A protein. 30 patients with brain injury and tracheotomy were selected and divided into two groups based on the airway humidification liquid instilled in the trachea tube, 0.45% NaCl and 0.9% NaCl+ambroxol. Blood was then extracted from the patients to measure the levels of SP-A, interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-α (TNF-α). The difference between group C and other groups in lung W/D and expression levels of SP-A mRNA and SP-A protein was significant (P<0.05). In comparison, the histological changes showed that the lung tissue damage was smallest in group C compared to the three other groups. Aspect of patients, 0.45% NaCl group and 0.9% NaCl+ambroxol group were significantly different in the levels of SP-A, IL-6, IL-8 and TNF-α (P<0.01). In the present study, 0.9% NaCl+ambroxol promote the synthesis and secretion of pulmonary surfactant, and has anti-inflammatory and antioxidant effects, which inhibit the release of inflammatory factors and cytokines, making it an ideal airway humidification liquid.

DNA methylation profile and expression of surfactant protein A2 gene in lung cancer

ABSTRACTKnowledge of the methylation profile of genes allow for the identification of biomarkers that may guide diagnosis and effective treatment of disease. Human surfactant protein A (SP-A) plays an important role in lung homeostasis and immunity, and is encoded by two genes (SFTPA1 and SFTPA2). The goal of this study was to identify differentially methylated CpG sites in the promoter region of the SFTPA2 gene in lung cancer tissue, and to determine the correlation between the promoter's methylation profile and gene expression. For this, we collected 28 pairs of cancerous human lung tissue and adjacent noncancerous (NC) lung tissue: 17 adenocarcinoma (AC), 9 squamous cell carcinoma (SCC), and 2 AC with SCC features, and we evaluated DNA methylation of the SFTPA2 promoter region by bisulfite conversion. Our results identified a higher methylation ratio in one CpG site of the SFTPA2 gene in cancerous tissue versus NC tissue (0.36 versus 0.11, p = 0.001). When assessing AC samples, we also found cancerous tissues associated with a higher methylation ratio (0.43 versus 0.10, p = 0.02). In the SCC group, although cancerous tissue showed a higher methylation ratio (0.22 versus 0.11), this difference was not statistically significant (p = 0.35). Expression of SFTPA2 mRNA and total SP-A protein was significantly lower in cancer tissue when compared to adjacent NC tissue (p < 0.001), and correlated with the hypermethylated status of an SFTPA2 CpG site in AC samples. The findings of this pilot study may hold promise for future use of SFTPA2 as a biomarker for the diagnosis of lung cancer.

Knockdown of Drosha in human alveolar type II cells alters expression of SP-A in culture: A pilot study

ABSTRACTHuman surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innate immunity. SP-A is synthesized and secreted by alveolar type II (ATII) cells, one of the two cell types of the distal lung epithelium (ATII and ATI). We have shown that miRNA interactions with sequence polymorphisms on the SP-A mRNA 3'UTRs mediate differential expression of SP-A1 and SP-A2 gene variants in vitro. In the present study, we describe a physiologically relevant model to study miRNA regulation of SP-A in human ATII. For these studies, we purified and cultured human ATII on an air-liquid interface matrix (A/L) or plastic wells without matrix (P). Gene expression analyses confirmed that cells cultured in A/L maintained the ATII phenotype for over 5 days, whereas P-cultured cells differentiated to ATI. When we transfected ATII with siRNAs to inhibit the expression of Drosha, a critical effector of miRNA maturation, the levels of SP-A mRNA and protein increased in a time dependent manner. We next characterized cultured ATII and ATI by studying expression of 1,066 human miRNAs using miRNA PCR arrays. We detected expression of >300 miRNAs with 24 miRNAs differentially expressed in ATII versus ATI, 12 of which predicted to bind SP-A 3'UTRs, indicating that these may be implicated in SP-A downregulation in ATI. Thus, miRNAs not only affect SP-A expression, but also may contribute to the maintenance of the ATII cell phenotype and/or the trans-differentiation of ATII to ATI cells, and may represent new molecular markers that distinguish ATII and ATI.

Identification and Quantitation of Coding Variants and Isoforms of Pulmonary Surfactant Protein A.

Pulmonary surfactant protein A (SP-A), a heterooligomer of SP-A1 and SP-A2, is an important regulator of innate immunity of the lung. Nonsynonymous single nucleotide variants of SP-A have been linked to respiratory diseases, but the expressed repertoire of SP-A protein in human airway has not been investigated. Here, we used parallel trypsin and Glu-C digestion, followed by LC–MS/MS, to obtain sequence coverage of common SP-A variants and isoform-determining peptides. We further developed a SDS-PAGE-based, multiple reaction monitoring (GeLC-MRM) assay for enrichment and targeted quantitation of total SP-A, the SP-A2 isoform, and the Gln223 and Lys223 variants of SP-A, from as little as one milliliter of bronchoalveolar lavage fluid. This assay identified individuals with the three genotypes at the 223 position of SP-A2: homozygous major (Gln223/Gln223), homozygous minor (Lys223/Lys223), or heterozygous (Gln223/Lys223). More generally, our studies demonstrate the challenges inherent in distinguishing highly homologous, copurifying protein isoforms by MS and show the applicability of MRM mass spectrometry for identification and quantitation of nonsynonymous single nucleotide variants and other proteoforms in airway lining fluid.

Surfactant Protein-A Concentration in Sera from Dogs with Pulmonary Parenchymal Diseases

Pulmonary surfactant protein A (SP-A) is used as a biomarker to understand the clinical features of pulmonary diseases and associated prognostic indices in human medicine. This study was conducted to investigate whether or not serum SP-A concentration can be used as a biomarker for identifying pulmonary parenchymal diseases in dogs. Thirty-two dogs with pulmonary parenchymal diseases, 34 with nonrespiratory diseases and 57 healthy dogs were included. Serum SP-A concentration was measured in all dogs using sandwich enzyme linked immunosorbent assay with an anti-dog SP-A polyclonal antibody. Median serum SP-A concentration in healthy dogs was <2.0 ng/ml, whereas that in dogs with aspiration pneumonia (n=11), primary lung tumors (n=9) and blunt traumatic lung injury (BTLI; n=12) was 3.1, 7.2 and 2.6 ng/ml, respectively; these values were significantly higher than those in healthy dogs. The serum SP-A concentration in dogs with nonrespiratory diseases was comparable with that in healthy dogs. No correlation was observed between the serum SP-A and plasma C-reactive protein concentrations in dogs with aspiration pneumonia and BTLI. There was a significant correlation between the serum SP-A concentration and thoracic radiographic changes in dogs with BTLI. These findings suggest that the serum SP-A concentration may be a useful clinical biomarker of alveolar damage that can be used for differential diagnosis of pulmonary parenchymal diseases and nonrespiratory diseases in dogs.

Staphylococcus aureus Proteases Degrade Lung Surfactant Protein A Potentially Impairing Innate Immunity of the Lung

The pulmonary surfactant is a complex mixture of lipids and proteins that is important for respiratory lung functions, which also provides the first line of innate immune defense. Pulmonary surfactant protein-A (SP-A) is a major surfactant component with immune functions with importance during Staphylococcus aureus infections that has been demonstrated in numerous studies. The current study showed that S. aureus can efficiently cleave the SP-A protein using its arsenal of proteolytic enzymes. This degradation appears to be mediated by cysteine proteases, in particular staphopain A (ScpA). The staphopain-mediated proteolysis of SP-A resulted in a decrease or complete abolishment of SP-A biological activity, including the promotion of S. aureus phagocytosis by neutrophils, aggregation of Gram-negative bacteria and bacterial cell adherence to epithelium. Significantly, ScpA has also efficiently degraded SP-A in complete bronchi-alveolar lavage fluid from human lungs. This indicates that staphopain activity in the lungs is resistant to protease inhibitors, thus suggesting that SP-A can be cleaved in vivo. Collectively, this study showed that the S. aureus protease ScpA is an important virulence factor that may impair innate immunity of the lungs.

Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-κB pathway

BackgroundLipoteichoic acid (LTA), a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that the gram-negative endotoxin, lipopolysaccharide (LPS), could induce surfactant protein-A (SP-A) production in human alveolar epithelial (A549) cells.ObjectivesIn this study, we further evaluated the effect of LTA on SP-A biosynthesis and its possible signal-transducing mechanisms.MethodsA549 cells were exposed to LTA. Levels of SP-A, nuclear factor (NF)-κB, extracellular signal-regulated kinase 1/2 (ERK1/2), and mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1 were determined.ResultsExposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability. Meanwhile, when exposed to 30 μg/ml LTA for 1, 6, and 24 h, the biosynthesis of SP-A mRNA and protein in A549 cells significantly increased. As to the mechanism, LTA enhanced cytosolic and nuclear NF-κB levels in time-dependent manners. Pretreatment with BAY 11–7082, an inhibitor of NF-κB activation, significantly inhibited LTA-induced SP-A mRNA expression. Sequentially, LTA time-dependently augmented phosphorylation of ERK1/2. In addition, levels of phosphorylated MEK1 were augmented following treatment with LTA.ConclusionsTherefore, this study showed that LTA can increase SP-A synthesis in human alveolar type II epithelial cells through sequentially activating the MEK1-ERK1/2-NF-κB-dependent pathway.

Surfactant protein A modulates the lipopolysaccharide-induced inflammatory response related to preterm birth

Surfactant protein A (SP-A) functions in homeostasis of lung surfactant and in innate immunity. SP-A is secreted by the fetal lung into amniotic fluid. Additionally it has been detected in gestational tissues. We propose that SP-A influences intrauterine inflammation that is commonly associated with preterm birth, the main underlying cause of neonatal mortality and morbidity. We used our previously established mouse model of LPS-induced preterm birth of live-born pups to investigate the role of SP-A in preterm birth. Mice overexpressing rat SP-A (rSP-A) under the control of human SP-C promoter were used. Cytokine concentrations in maternal and fetal serum and in amniotic fluid and mRNA levels of several inflammatory mediators in lungs and in intrauterine tissues were quantified using Cytometric Bead Array and RNase Protection Assay, respectively. Higher levels of SP-A mRNA were observed in fetal lungs and intrauterine tissues of rSP-A mice compared with wild-type. Using Western blot we detected excess of SP-A protein in fetal lung and in amniotic fluid of rSP-A animals. Despite some differences in the basal levels of TNF-α and IL-10 between rSP-A and wild-type animals, there were no differences in the duration of pregnancy. However, the levels of TNF-α, IL-10 and some other inflammatory mediators in intrauterine tissues and in amniotic fluid differed significantly between the mouse lines after maternal LPS given at 17dpc. We conclude that SP-A modulates the levels of intrauterine inflammatory mediators involved in preterm birth and may contribute to inflammatory processes related to spontaneous preterm labor.

Infant formula alters surfactant protein A (SP‐A) and SP‐B expression in pulmonary epithelial cells

Surfactant proteins A (SP-A) and SP-B are critical in the ability of pulmonary surfactant to reduce alveolar surface tension and provide innate immunity. Aspiration of infant milk formula can lead to lung dysfunction, but direct effects of aspirated formula on surfactant protein expression in pulmonary cells have not been described. The hypothesis that infant formula alters surfactant protein homeostasis was tested in vitro by assessing surfactant protein gene expression in cultured pulmonary epithelial cell lines expressing SP-A and SP-B that were transiently exposed (6 hr) to infant formula. Steady-state levels of SP-A protein and mRNA and SP-B mRNA in human bronchiolar (NCI-H441) and mouse alveolar (MLE15) epithelial cells were reduced in a dose-dependent manner 18 hr after exposure to infant formula. SP-A mRNA levels remained reduced 42 hr after exposure, but SP-B mRNA levels increased 10-fold. Neither soy formula nor non-fat dry milk affected steady-state SP-A and SP-B mRNA levels; suggesting a role of a component of infant formula derived from cow milk. These results indicate that infant formula has a direct, dose-dependent effect to reduce surfactant protein gene expression. Ultimately, milk aspiration may potentially result in a reduced capacity of the lung to defend against environmental insults.

A Toll-Like Receptor-4-Interacting Surfactant Protein-A-Derived Peptide Suppresses Tumor Necrosis Factor-α Release from Mouse JAWS II Dendritic Cells

Surfactant protein-A (SP-A) and Toll-like receptor-4 (TLR4) proteins are recognized as pathogen-recognition receptors. An exaggerated activation of TLR4 induces inflammatory response, whereas SP-A protein down-regulates inflammation. We hypothesized that SP-A-TLR4 interaction may lead to inhibition of inflammation. In this study, we investigated interaction between native baboon lung SP-A and baboon and human TLR4-MD2 proteins by coimmunoprecipitation/immunoblotting and microwell-based methods. The interaction between SP-A and TLR4-MD2 proteins was then analyzed using a bioinformatics approach. In the in silico model of SP-A-TLR4-MD2 complex, we identified potential binding regions and amino acids at the interface of SP-A-TLR4. Using this information, we synthesized a library of human SP-A-derived peptides that contained interacting amino acids. Next, we tested whether the TLR4-interacting SP-A peptides would suppress inflammatory cytokines. The peptides were screened for any changes in the tumor necrosis factor-α (TNF-α) response against lipopolysaccharide (LPS) stimuli in the mouse JAWS II dendritic cell line. Different approaches used in this study suggested binding between SP-A and TLR4-MD2 proteins. In cells pretreated with peptides, three of seven peptides increased TNF-α production against LPS. However, two of these peptides (SPA4: GDFRYSDGTPVNYTNWYRGE and SPA5: YVGLTEGPSPGDFRYSDFTP) decreased the TNF-α production in LPS-challenged JAWS II dendritic cells; SPA4 peptide showed more pronounced inhibitory effect than SPA5 peptide. In conclusion, we identify a human SP-A-derived peptide (SPA4 peptide) that interacts with TLR4-MD2 protein and inhibits the LPS-stimulated release of TNF-α in JAWS II dendritic cells.

Effect of recruitment maneuver on pulmonary surfactant in young piglets with acute lung injury

Objective To investigate the effect of recruitment maneuver(RM) on surfactant proteins in young piglets with acute lung injury and the possible mechanisms of lung recruitment after RM.Methods The piglet model of ALI was established by lipopolysaccharide intravenous injection,12 male piglets were randomly divided into two groups:conventional ventilation group(control group) and RM with low tidal volume group(RM group).After 8 hours of ventilation,mRNA expression of surfactant protein-A(SP-A),SP-B,SP-C,SP-D in the piglet lungs were determined by real time PCR and SP-A protein distribution was assessed by immunohistochemistry.Biochemical analyses of TP,total phospholipids(TPL),DSPC were conducted as well.SP-A levels in bronchoalveolar lavage fluid(BALF) and plasma were measured by enzymelinked immunosorbent assay(ELISA).Results As compared with control group,RM group had higher expression of SP-A,SP-B,SP-C and SP-D.SP-A average gray values of control group and RM group were 97.8±6.4 and 106.3±8.5,and there was significant difference(P＜0.01).RM group showed significant increase of TPL,DSPC and DSPC/TP.The concentration of SP-A in BALF was higher in RM group than that of the control group,however,SP-A plasma level was lower in RM group than that of the control group.Conclusion RM can increase suffactant protein expression in ALI animals,alleviate surfactant protein dysfunction and regulate the concentration of SP-A,which may improve alveolar recruitment following the RM and alleviate ventilator-induced lung injury. Key words: Acute lung injury; Recruitment maneuver; Pulmonary suffactant

Surfactant protein A and D gene polymorphisms and protein expression in victims of sudden infant death

To investigate the innate immune components surfactant protein A (SP-A) and D (SP-D) in victims of sudden infant death syndrome (SIDS). Ten common single nucleotide polymorphisms (SNPs) in the exons of SP-A1, SP-A2 and SP-D genes were analysed in 42 cases of SIDS and 46 explained sudden infant deaths. SP-A and SP-D protein expression in tissue from the aerodigestive tract was semi-quantitatively evaluated by immunohistochemistry. SP-D immunoreactivity was found in lungs and tissue from submandibular gland, palatine tonsils and duodenum. Positive SP-A immune staining was found exclusively in lung tissue. Neither the allele nor the haplotype distribution of the SP-A and SP-D genes was significantly different in SIDS compared to explained deaths. The most common SP-A haplotype, 6A2/1A0, tended to be overrepresented in the cases with low immunohistochemical SP-A expression (61%) compared to cases with high expression (49%), p = 0.08. The SP-D expression was not influenced by the 11 C/T or 160 A/G polymorphisms. No significant association between the common genetic variants of SP-A and SP-D and SIDS is disclosed by the present study. However, low SP-A protein expression may possibly be determined by the 6A2/1A0 SP-A haplotype, this should be subject for further investigation.

Proteomics of Human Lung Tissue Identifies Surfactant Protein A as a Marker of Chronic Obstructive Pulmonary Disease

Chronic Obstructive Pulmonary Disease (COPD), a lung disease related to smoking, is one of the leading causes of chronic morbidity and mortality around the world. One goal in COPD research is the identification of biomarkers for early diagnosis of the disease. Here, we sought COPD-specific changes in the proteome from human lung tissue. This revealed increased levels of surfactant protein A (SP-A) in COPD but not in the normal or fibrotic lung. The results were confirmed by immunohistochemistry, morphometry and Western blotting. Furthermore, elevated SP-A protein levels were detected from the induced sputum supernatants of COPD patients. The levels of other surfactant proteins (SP-B, SP-C, SP-D) were not altered. Our results suggest that SP-A is linked to the pathogenesis of COPD and could be considered as a potential COPD biomarker.

CC10 mRNA and protein expression in Clara cells of CD-1 mice following exposure to styrene or its metabolites styrene oxide or 4-vinylphenol

Styrene, widely used in manufacturing, has both acute and chronic effects in humans. In mice, styrene is both hepato- and pneumo-toxic and causes lung tumors. The primary site for styrene metabolism and its effects in mouse lung is the Clara cell, which secretes Clara cell 10 kDa protein (CC10) and surfactant protein A (SPA). Both play important roles in host defenses and inflammation prevention. The mode of action for styrene-induced lung tumor formation has yet to be elicited, yet one possibility relates to oxidative stress and decreased CC10 levels. CC10 mRNA and protein expression were measured in isolated Clara cells 3, 12, and 24 h following in vivo administration of styrene (600 mg/kg i.p.) or its metabolites [ R-, S-, racemic styrene oxide (SO) (300 mg/kg i.p.), 4-vinylphenol (100 mg/kg i.p.)]. The largest decreases in CC10 mRNA expression were seen with R-SO and racemic SO at 24 h. To determine if rebound effects would be seen, CC10 mRNA and protein expression were determined 48, 120, and 240 h following styrene and R-SO administration. The CC10 protein level did not reach its lowest point to correlate with mRNA expression until 120 h after R-SO administration. Styrene exposure caused a significant decrease in CC10 protein after 24 h, rebounding through 240 h. SPA protein expression showed little change from control levels, indicating a more specific effect on CC10 in the Clara cell by styrene and its metabolites. These studies demonstrate that acute changes in lung CC10 protein and mRNA expression do occur following in vivo treatment with styrene and its metabolites. These changes may be early indicators for a potential mechanism for lung tumor formation in mice as it relates to oxidative stress and the possibility deserves further study.