SP-A Expression Research Articles

Inhibition of Virulence Gene Expression in Staphylococcus aureus by Novel Depsipeptides from a Marine Photobacterium

During a global research expedition, more than five hundred marine bacterial strains capable of inhibiting the growth of pathogenic bacteria were collected. The purpose of the present study was to determine if these marine bacteria are also a source of compounds that interfere with the agr quorum sensing system that controls virulence gene expression in Staphylococcus aureus. Using a gene reporter fusion bioassay, we recorded agr interference as enhanced expression of spa, encoding Protein A, concomitantly with reduced expression of hla, encoding α-hemolysin, and rnaIII encoding RNAIII, the effector molecule of agr. A marine Photobacterium produced compounds interfering with agr in S. aureus strain 8325-4, and bioassay-guided fractionation of crude extracts led to the isolation of two novel cyclodepsipeptides, designated solonamide A and B. Northern blot analysis confirmed the agr interfering activity of pure solonamides in both S. aureus strain 8325-4 and the highly virulent, community-acquired strain USA300 (CA-MRSA). To our knowledge, this is the first report of inhibitors of the agr system by a marine bacterium.

Prognostic value of immunohistochemical surfactant protein A expression in regenerative/hyperplastic alveolar epithelial cells in idiopathic interstitial pneumonias

BackgroundIt is difficult to predict survival in patients with idiopathic pulmonary fibrosis. Recently, several proteins, such as surfactant protein (SP) and KL-6, have been reported to be useful biologic markers for prediction of prognosis for interstitial pneumonias. It is not clear whether there is any relationship between expression of these proteins in regenerative/hyperplastic alveolar epithelial cells and prognosis of idiopathic interstitial pneumonias (IIPs).ObjectivesThis study aimed to elucidate the clinical significance of the expression of such lung secretory proteins as SP-A and KL-6 in lung tissues of patients with IIPs.MethodsWe retrospectively investigated the immunohistochemical expression of SP-A, KL-6, cytokeratin (CK), and epithelial membrane antigen (EMA) in alveolar epithelial cells in lung tissues obtained from surgical lung biopsy in 43 patients with IIPs, and analyzed the correlation between expression of these markers and the prognosis of each IIP patient. CK and EMA were used as general markers for epithelial cells.ResultsIn patients with usual interstitial pneumonia (UIP), the ratio of SP-A positive epithelial cells to all alveolar epithelial cells (SP-A positive ratio) in the collapsed and mural fibrosis areas varied, ranging from cases where almost all alveolar epithelial cells expressed SP-A to cases where only a few did. On the other hand, in many patients with nonspecific interstitial pneumonia (NSIP), many of the alveolar epithelial cells in the diseased areas expressed SP-A. The SP-A positive ratio was significantly lower in patients who died from progression of UIP than in patients with UIP who remained stable or deteriorated but did not die. In NSIP patients, a similar tendency was noted between the SP-A positive ratio and prognosis.ConclusionsThe results suggest that the paucity of immunohistochemical SP-A expression in alveolar epithelial cells in diseased areas (i.e. regenerative/hyperplastic alveolar epithelial cells) may predict a worse prognosis for patients with IIPs, especially patients with UIP. A prospective study is needed to confirm these results.

Keratinocyte Growth Factor (KGF) Gene Therapy Mediated by an Attenuated Form of Salmonella typhimurium Ameliorates Radiation Induced Pulmonary Injury in Rats

The aim of this study is to investigate the effect of KGF (Keratinocyte growth factor) gene therapy mediated by the attenuated Salmonella typhimurium Ty21a on radiation-induced pulmonary injury in rats model. Sprague-Dawley rats were divided into three groups: TPK group (treated with TPK strain, attenuated Salmonella typhimurium Ty21a-recombined human KGF gene); TP group (treated with TP strain, attenuated Salmonella typhimurium Ty21a-recombined blank plasmid); and Saline group (treated with saline). After intraperitoneal administration for 48 h, the thoraxes of the rats were exposed to X-ray (20 Gy), and the rats were administered again two weeks after radiation. On the 3rd, 5th, 7th, 14th and 28th day after radiation, the rats were sacrificed and lung tissues were harvested. Histological analysis was performed, MDA contents and SOD activity were detected, mRNA levels of KGF, TGF-β, SP-A and SP-C were measured by Real-time RT-PCR, and their concentrations in the BALF were quantified with ELISA. Administration of TPK strain improved the pathological changes of the lung on the 28th day. In the TPK group, KGF effectively expressed since the 3rd day, MDA contents decreased and SOD activity increased significantly, on the 7th day and 14th day respectively. SP-A and SP-C expression elevated, whereas TGF-β expression was inhibited in the TPK group. These results suggest that this novel gene therapy of KGF could ameliorate radiation-induced pulmonary injury in rats, and may be a promising therapy for the treatment of radiative pulmonary injury.

Lung-Derived Microvesicles Induce Stable Long-Term Epigenetic Changes In Marrow Cells

AbstractAbstract 4799We have previously shown that microvesicles derived from irradiated murine lung cells enter murine bone marrow cells and induce expression of the pulmonary epithelial cell-specific mRNAs Surfactants A-D (Sp-A-D), Aquaporin-5 (Aq-5) and Clara Cell Specific Protein (CCSP). The early mRNA increases are due to direct delivery of mRNA and due to the transfer of a transcription factor to marrow cells (Aliotta JM, et al, Exp Hematol, 2010). The modulated marrow cells produce pulmonary epithelial cell-specific protein and have an enhanced ability to convert into pulmonary epithelial cells after transplantation into lethally-irradiated mice. In the present studies, we evaluated the stability of pulmonary epithelial cell-specific mRNA expression in cytokine-stimulated liquid culture and after in vivo transplantation into lethally-irradiated mice. Whole bone marrow cells isolated from C57BL/6 mice were cultured for seven days opposite C57BL/6 lung fragments, but separated from them by a 0.4 micron cell-impermeable membrane. Marrow cells were then either infused into lethally-irradiated C57BL/6 mice or established in culture with DMEM supplemented with IL-3, IL-6, IL-11 and steel factor. Engrafted mice were sacrificed six weeks after transplantation and real-time RT-PCR was carried out on marrow, liver, spleen and thymus tissue. Expression of Sp-A, B and D, CCSP and Aq-5 were variably seen in all tissues and increases over control ranged from 12-fold to 58-fold. Liquid culture was analyzed every two weeks out to 12 weeks and high levels of expression of different pulmonary epithelial cell-specific mRNAs and proteins (by western blot and Immunohistochemical staining) were observed. In order to determine the mechanism for the increased expression in culture, we established co-cultures of Fischer-344 rat lung opposite C57BL/6 mouse bone marrow cells and, using species-specific primers, determined whether the increased pulmonary epithelial cell-specific mRNA expression at each time point of secondary cytokine-supported culture was rat or mouse-specific. In every instance, the pulmonary epithelial cell-specific mRNA which was detected was mouse-specific, not rat-specific, and thus the long-term expression of lung-specific mRNA in mouse marrow was due to induced transcription by the mouse marrow cells. These data indicate that lung-derived microvesicles induce long-term stable epigenetic changes in marrow cells and suggests that the genetic changes induced by tissue microvesicles in neighboring cells may be biologically important. Disclosures:No relevant conflicts of interest to declare.

Reduced airway inflammation in CD26/DPP4‐deficient F344 rats is associated with altered recruitment patterns of regulatory T cells and expression of pulmonary surfactant proteins

CD26 is highly expressed on lung epithelial cells as well as on immune cells. Ovalbumin (OVA)-induced airway inflammation induces a further increase of CD26 expression. CD26-deficient rat strains exhibit blunted clinical courses in models of experimental asthma. (1) To investigate the involvement of regulatory T cells (Tregs) and the surfactant system in a rat model of genetic CD26 deficiency. (2) To investigate regulatory mechanisms dependent on the endogenous CD26 expression. (3) To investigate the impact of CD26 on surfactant protein (SP)-levels under inflammatory conditions. Wild-type and CD26-deficient F344 rats were sensitized to and challenged with OVA. Subsequently, airway inflammation, SP levels as well as surface tension of the bronchoalveolar lavage (BAL) fluid were evaluated. CD26 deficiency led to decreased airway inflammation, e.g. reduced numbers of eosinophils and activated T cells in the BAL. Remarkably, the CD26-deficient rats exhibited a significantly increased influx of FoxP3(+) Tregs into the lungs and increased IL-10-secretion/production by draining lymph node cells in culture experiments. Furthermore, in OVA-challenged CD26-deficient rats, the increase of the expression of the collectins SP-A and SP-D as well as of the surface tension-active SP-B was significantly less pronounced than in the CD26-positive strain. Only in the wild-type rats, functional alterations of the surfactant system, e.g. the increased surface tension were obvious after OVA challenge. Reduced airway inflammation in CD26-deficient F344 rats appear to be mediated by differences in the recruitment and activity of Tregs. This altered inflammation is associated with differences in the SP expression as well as function.

491 Recombinant Human Kgf/Palifermin® as An Alternative to Glucocorticoids- Effects on Pneumocyte Proliferation and Gene Expression in Neonatal Rats

Background: Surfactant decreases surface tension of pulmonary air:liquid interfaces. It mainly comprizes characteristic phosphatidylcholine (PC) species and proteins SP-A to -D. Betamethasone used to accelerate “lung maturation” is catabolic, while keratinocyte growth factor (KGF), expressed by fibroblasts, is non-catabolic, acts specifically on type-II-pneumocytes and correlates inversely with bronchopulmonary dysplasia incidence.Aims: To explore the potential of recombinant human KGF (rhKGF, Palifermin®) on neonatal lung development and surfactant metabolism. To contrast rhKGF with betamethasone effects in vivo.Methods: Postnatal rats (d1, d5, d19) were injected with rhKGF (2x5mg/kg), betamethasone (2x1mg/kg) or rhKGF+betamethasone over 48h. Pneumocyte proliferation was detected using bromodeoxyuridine incorporation. Expression of SP-A to -D, adipocyte triglyceride lipase (ATGL), lyso-PC-acyltransferase (LPCAT) and the ATGL activator CGI-58 were measured with PCR.Results: RhKGF, betamethasone and combination treatment increased SP-B expression throughout by +97-117%, +42-51% and +93-309% (P< 0.001), respectively. SP-C was increased by +35-48%, +30- 44% and +74-108% (P< 0.001) after the respective treatments, while SP-A&-D were unchanged in immature lungs. All treatments increased the expression of ATGL (+50-75%), LPCAT (+15-20%) und CGI-58 (+5-10%) (P< 0.05). However, whereas rhKGF effects were accompanied by increased pneumocyte proliferation, betamethasone blocked both basic (d7) and rhKGF-induced (d7+d21) proliferation.Conclusion: Short term medication of neonatal rats with rhKGF/Palifermin® increased the expression of genes relevant for surfactant function and metabolism. Actions were identical or superior to those of betamethasone, with combination treatment exerting maximal effects. However, while betamethasone effects were at the expense of lung anabolism/pneumocyte proliferation, rhKGF enhanced proliferation.

Surfactant Protein (SP)-A and SP-D as Antimicrobial and Immunotherapeutic Agents

Surfactant protein (SP)-A and SP-D belong to the "Soluble C-type Lectin" family of proteins and are collectively known as "Collectins". Based on their ability to recognize pathogens and to regulate the host defense, SP-A and SP-D have been recently categorized as "Secretory Pathogen Recognition Receptors". SP-A and SP-D were first identified in the lung; the expression of SP-A and SP-D has also been observed at other mucosal surfaces, such as lacrimal glands, gastrointestinal mucosa, genitourinary epithelium and periodontal surfaces. Since the role of these proteins is not fully elucidated at other mucosal surfaces, the focus of this article is on lung-SP-A and SP-D. It has become clear from research studies performed over a number of years that SP-A and SP-D are critical for the maintenance of lung homeostasis and the regulation of host defense and inflammation. However, none of the surfactant preparations available for clinical use have SP-A or SP-D. A review is presented here on SP-A- and SP-D-deficiencies in lung diseases, the importance of the administration of SP-A and SP-D, and recent patents and research directions that may lead to the design of novel SP-A- or SP-D-based therapeutics and surfactants.

Early gestational intrauterine infection induces postnatal lung inflammation and arrests lung development in a rat model

Objective. In order to investigate the early gestational inflammation effect on the prenatal and postnatal lung development, identification of the proinflammatory cytokines (IL-1β and TNF-α), genes implicated in angiogenesis (Vascular endothelial growth factor [VEGF], fms-like tyrosine kinase-1 [Flt-1], fetal liver kinase-1 [Flk-1]), and surfactant proteins (SPs) were observed.Methods. Escherichia coli (E. coli) was inoculated into uterine cervix of pregnant rats at embryonic day 15 (E15) during pseudoglandular period of lung development and the control group was inoculated with normal saline. IL-1β, TNF-α, VEGF, Flt-1, Flk-1, SP-A, and SP-B mRNA in pup's lung at E17, 19, 21 and postnatal day (P) 1, 3, 7, 14 were quantified by real-time RT-PCR. Western blot or immunohistochemistry analysis was also performed for the evaluation of VEGF, Flk-1, Flt-1, and SP-A expression in pup's lung.Results. Compared with the control group, the fetal lung of the E. coli-treated group was more immature, the postnatal lung development was impaired marked by less alveoli, fewer secondary septa, and thicker alveolar wall. The lung weight and lung/body weight ratio were lower in the E. coli-treated group pups. IL-1β and TNF-α mRNA were increased significantly in E. coli-treated pup's lung after birth, but no significant difference of IL-1β and TNF-α mRNA levels in fetal lung were found between the two groups. SP-A expression was depressed at E17, E19, and E21 after intrauterine E. coli treated, accompanied with lower SP-B mRNA level at E19 and E21. Furthermore, intrauterine E. coli treated reduced the VEGF mRNA and protein levels in the fetal lung at E17 and E19, while the expression of Flt-1 and Flk-1 were higher at P7, P14 and P1, P7, P14, respectively, compared to the controls.Conclusions. These results suggested early gestational intrauterine E. coli infection could induce a postnatal pulmonary inflammation and might arrest the alveolarization in developing lung which was involved with the VEGF signaling. However, intrauterine E. coli infection could not induce the increase of proinflammatory cytokines in fetal lung and might fail to accelerate the maturation of fetal lung.

Cycloxygenase Inhibition Enhances the Effects of Surfactant Therapy in Endotoxin-Induced Rat Model of ARDS

The present study examines the relationships between inflammation and surfactant protein (SP) expression in a rodent model of acute respiratory distress syndrome (ARDS). Rats were intratracheally instilled with lipopolysaccharide (LPS) for 72 hours to induce ARDS and further treated with exogenous surfactant. Prostaglandin E(2) (PGE(2)) levels, cycloxygenase (COX) activity and alterations in SP-A apoprotein were measured. COX and SP-A expressions in lung tissue and SP-A-positive cells were determined by Western blot and immunofluorescence, respectively. PGE(2) levels and COX activity and its expression were increased with LPS exposure, whereas SP-A protein and percentage of SP-A-positive cells were decreased, which were subsequently reverted back by exogenous surfactant instillation. Because inhibition of COX-2 action is proposed to be useful in various inflammatory lung injuries, these results suggest that COX-2 expression and the possible beneficial effects of its inhibition on lung inflammation and dysfunction with LPS-ARDS corresponds closely with reduced SP-A expression.

Effects of prenatal diesel exhaust inhalation on pulmonary inflammation and development of specific immune responses

There is increasing evidence that exposure to air pollutants during pregnancy can result in a number of deleterious effects including low birth weight and the incidence of allergic asthma. To investigate the in utero effects of DE exposure, timed pregnant BALB/c mice were exposed to 0, 0.8 or 3.1 mg/m 3 of DE during gestation days (GD) 9 to GD 18. The number of successful pregnancies was 15/20 in the air controls and 10/20 in each of the diesel exposures. Immune function in the 6-week-old offspring as determined by development of delayed type hypersensitivity (DTH) reactions to bovine serum albumin (BSA), antibody titers to injected sheep red blood cells (SRBC), splenic T cells expressing CD45 +CD3 +CD8 + and CD3 +CD25 +, and mRNA expression of TNF-α, TLR2, SP-A, TGF-β and Foxp3 in the lung were not affected by prenatal DE exposure. On the other hand, lung TLR4 mRNA expression, the number of neutrophils in the bronchoalveolar lavage fluid (BALF) and splenic T cells expressing CD45 +CD3 +CD4 + and CD4 +CD25 + were differentially affected depending on the DE concentration and gender. When additional groups of mice were sensitized and challenged via the respiratory tract with ovalbumin to induce allergic airway inflammation, female mice had higher protein levels in the BALF compared to males and this was reduced by prenatal exposure to either concentration of DE. No other changes in allergen-induced immunity, lung function or severity of inflammation were noted. Collectively, the results show that in utero exposure to DE altered some baseline inflammatory indices in the lung in a gender-specific manner, but had no effect on development of specific immune responses to experimental antigens, or the severity of allergic lung inflammation.

Secreted surfactant protein A from fetal membranes induces stress fibers in cultured human myometrial cells

In the present study, we investigated the ability of human fetal membranes (amnion and choriodecidua) to regulate human maternal uterine cell functions through the secretion of surfactant protein (SP)-A and SP-D at the end of pregnancy. We detected the expression of both SP-A (SP-A1 and SP-A2) and SP-D by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry revealed that human fetal membranes expressed both SP-A and SP-D. By Western blot analysis, we demonstrated that SP-A protein expression was predominant in choriodecidua, whereas the amnion predominantly expressed SP-D. Only the secretion of SP-A was evidenced in the culture supernatants of amnion and choriodecidua explants by immunodot blot and confirmed by Western blot. Exogenous human purified SP-A induced stress fiber formation in cultured human myometrial cells via a pathway involving Rho-kinase. Conditioned medium from choriodecidua and amnion explants mimicked the SP-A effect. Treatment of myometrial cells with SP-A-depleted conditioned medium from choriodecidua or amnion explants failed to change the actin dynamic. These data indicate that SP-A released by human fetal membranes is able to exert a paracrine regulation of F-actin filament organization in myometrial cells.

Effects of nicotine on pulmonary surfactant proteins A and D in ovine lung epithelia

Maternal smoking during pregnancy increases the incidence and severity of respiratory infections in neonates. Surfactant proteins A and D (SP-A and SP-D, respectively) are components of pulmonary innate immunity and have an important role in defense against inhaled pathogens. The purpose of this study was to determine if nicotine exposure during the third trimester of pregnancy alters the expression of SP-A and SP-D of fetal lung epithelia. Pregnant ewes were assigned to four groups; a nicotine-exposed full-term and pre-term group, and control full-term and pre-term group. Lung tissue was collected for Western blot and IHC analysis of SP-A level, Western blot analysis of SP-D level and qPCR analysis of SP-A and SP-D mRNA expression. Exposure to nicotine significantly decreased SP-A gene expression (P = 0.01) and SP-A protein level in pre-term lambs. This finding suggests that maternal nicotine exposure during the last trimester of pregnancy alters a key component of lung innate immunity in offspring.

Fatty diabetic lung: altered alveolar structure and surfactant protein expression

Pulmonary dysfunction develops in type 2 diabetes mellitus (T2DM) in direct correlation with glycemia and is exacerbated by obesity; however, the associated structural derangement has not been quantified. We studied lungs from obese diabetic (fa/fa) male Zucker diabetic fatty (ZDF) rats at 4, 12, and 36 wk of age, before and after onset of T2DM, compared with lean nondiabetic (+/+) rats. Surfactant proteins A and C (SP-A and SP-C) immunoexpression in lung tissue was quantified at ages 14 and 18 wk, after the onset of T2DM. In fa/fa animals, lung volume was normal despite obesity. Numerous lipid droplets were visible within alveolar interstitium, lipofibroblasts, and macrophages, particularly in subpleural regions. Total triglyceride content was 136% higher. By 12 wk, septum volume was 21% higher, and alveolar duct volume was 36% lower. Capillary basement membrane was 29% thicker. Volume of lamellar bodies was 45% higher. By age 36 wk, volumes of interstitial collagen fibers, cells, and matrix were respectively 32, 25, and 80% higher, and capillary blood volume was 18% lower. ZDF rats exhibited a strain-specific increase in resistance of the air-blood diffusion barrier with age, which was exaggerated in fa/fa lungs compared with +/+ lungs. In fa/fa lungs, SP-A and SP-C expression were elevated at age 14-18 wk; the normal age-related increase in SP-A expression was accelerated, whereas SP-C expression declined with age. Thus lungs from obese T2DM animals develop many qualitatively similar changes as in type 1 diabetes mellitus but with extensive lipid deposition, altered alveolar type 2 cell ultrastructure, and surfactant protein expression patterns that suggest additive effects of hyperglycemia and lipotoxicity.

Protective Mechanism of Ultrafiltration Against Cardiopulmonary Bypass–Induced Lung Injury

Background We previously demonstrated a negative effect of cardiopulmonary bypass (CPB) in a canine model of single-lung graft function and an improved effect with ultrafiltration during CPB. Objective To investigate the mechanism of these effects, focusing on cytokines and pulmonary surfactants using real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR). Materials and Methods Fifteen left-sided single-lung transplant procedures were performed in pairs of dogs. The animals were divided into 3 groups. In one group, transplantation was performed without CPB (non-CPB group); in a second group, transplantation was performed with CPB and CPB flow was decreased slowly and pulmonary artery pressure was controlled (CPB group; and in the third group, transplantation was performed with CPB and ultrafiltration (CPB+UF group). Grafted lung specimens were harvested for RT-PCR of cytokines (IL-6, IL-8, and IL-10) and surfactant proteins (SP-A, SP-B, and SP-C). Results Real-time quantitative RT-PCR demonstrated increased IL-6 expression in the CPB group compared with the non-CPB group. IL-6 gene expression was suppressed and pulmonary surfactant restored using ultrafiltration. Gene expression of surfactant protein (SP)–A, SP-B, and SP-C was decreased in the CPB group compared with normal lung and ultrafiltration groups, which demonstrated sustained gene expression of SP-A and SP-B. Conclusion Cardiopulmonary bypass has negative effects on grafts; however, ultrafiltration attenuates acute lung dysfunction by decreasing the inflammatory response and increasing pulmonary surfactant.

Nucleotide Polymorphism in the Wheat Transcriptional ActivatorSpaInfluences Its Pattern of Expression and Has Pleiotropic Effects on Grain Protein Composition, Dough Viscoelasticity, and Grain Hardness

Storage protein activator (SPA) is a key regulator of the transcription of wheat (Triticum aestivum) grain storage protein genes and belongs to the Opaque2 transcription factor subfamily. We analyzed the sequence polymorphism of the three homoeologous Spa genes in hexaploid wheat. The level of polymorphism in these genes was high particularly in the promoter. The deduced protein sequences of each homoeolog and haplotype show greater than 93% identity. Two major haplotypes were studied for each Spa gene. The three Spa homoeologs have similar patterns of expression during grain development, with a peak in expression around 300 degree days after anthesis. On average, Spa-B is 10 and seven times more strongly expressed than Spa-A and Spa-D, respectively. The haplotypes are associated with significant quantitative differences in Spa expression, especially for Spa-A and Spa-D. Significant differences were found in the quantity of total grain nitrogen allocated to the gliadin protein fractions for the Spa-A haplotypes, whereas the synthesis of glutenins is not modified. Genetic association analysis between Spa and dough viscoelasticity revealed that Spa polymorphisms are associated with dough tenacity, extensibility, and strength. Except for Spa-A, these associations can be explained by differences in grain hardness. No association was found between Spa markers and the average single grain dry mass or grain protein concentration. These results demonstrate that in planta Spa is involved in the regulation of grain storage protein synthesis. The associations between Spa and dough viscoelasticity and grain hardness strongly suggest that Spa has complex pleiotropic functions during grain development.

Molecular mechanisms of lipopolysaccharide-caused induction of surfactant protein-A gene expression in human alveolar epithelial A549 cells

Surfactant proteins (SPs) participate in the physiological and pathophysiological regulation of sepsis-induced acute lung injury. Lipopolysaccharide (LPS), a Gram-negative bacterial outer membrane component, is one of the major causes of septic shock. This study was designed to evaluate the effects of LPS on the regulation of SP-A and SP-D gene expressions in human alveolar epithelial A549 cells. Exposure of A549 cells to LPS increased SP-A mRNA synthesis in concentration and time-dependent manners without affecting SP-D mRNA production. LPS selectively enhanced translocation of transcription factor c-Jun from the cytoplasm to nuclei, but not nuclear factor kappa-B. In parallel, the DNA-binding activity of AP-1 was increased by LPS. Pretreatment of A549 cells with SP600125, an inhibitor of c-Jun N-terminal kinase, decreased c-Jun translocation, and significantly ameliorated LPS-induced SP-A mRNA production. Levels of toll-like receptor (TLR2) mRNA in A549 cells were time-dependently induced following LPS treatment. Application of TLR2 small interference (si)RNA into A549 cells significantly knocked-down the translation of this receptor, and simultaneously alleviated LPS-induced SP-A synthesis. Taken together, this study has shown that LPS selectively induces SP-A gene expression possibly through TLR2-mediated activation of c-Jun in human alveolar epithelial A549 cells.

Cap-independent translation of human SP-A 5′-UTR variants: a double-loop structure andcis-element contribution

Human surfactant protein A (hSP-A), a molecule of innate immunity and surfactant-related functions, consists of two functional genes, SP-A1 and SP-A2. SP-A expression is regulated by several factors including environmental stressors. SP-A1 and SP-A2 5'-untranslated region (5'-UTR) splice variants have a differential impact on translation efficiency and mRNA stability. To study whether these variants mediate internal ribosome entry site (IRES) activity (i.e., cap-independent translation), we performed transient transfection experiments in H441 cells with constructs containing one SP-A1 (A'D', AB'D', or A'CD') or SP-A2 (ABD) 5'-UTR splice variant between the Renilla and firefly luciferase genes of a bicistronic reporter vector. We found that 1) variants A'D', ABD, and AB'D' exhibit significantly higher IRES activities than negative control (no SP-A 5'-UTR) and A'CD' has no activity; the order of highest IRES activity was ABD > A'D' > AB'D; 2) IRES activity of ABD significantly increased in response to diesel particulate matter (20 microg/ml) but not in response to ozone (1 ppm for 1 h); 3) deletion mutants of ABD revealed regulatory elements associated with IRES activity; one at the end of exon A attenuated activity, whereas a region containing a short adenosine-rich motif in the second half of exon B and the start of exon D enhanced activity; 4) elimination of a predicted double-loop structure or increase in free energy significantly reduced IRES activity; 5) elimination of one or both double-loop structures in A'D' did not affect cap-dependent translation activity. Thus several factors, including cis-elements and secondary structure type and stability, are required for hSP-A 5'-UTR variant-mediated cap-independent translation.

Exogenous Surfactant Suppresses Inflammation in Experimental Endotoxin-Induced Lung Injury

Our objective was to evaluate the anti-inflammatory effects of exogenous surfactant and surfactant phospholipids on the lipopolysaccharide (LPS)-induced lung injury. Exogenous surfactant (porcine surfactant) and surfactant phospholipid (dipalmitoyl phospholipid DPPC, hexadecanol, tylaxopol) were instilled intratracheally with LPS in rats. Expression of surfactant apoproteins (SP-A) and the cyclooxygenase enzymes (COX-1 and -2) was studied by immunohistochemistry, and apoptosis was analyzed by in situ terminal dUTP nick end labeling TUNEL assay. The intracellular reactive oxygen species (ROS) was measured in the isolated macrophages by fluorescence measurement with dichlorofluorescein diacetate (DCFH-DA). LPS-induced oxidative burst and apoptosis at 72 hours were reduced by both porcine and synthetic surfactant. SP-A as well as COX-1 and -2 expressions were suppressed with synthetic surfactant treatment, whereas with porcine surfactant (P-SF) the SP-A expression was enhanced in response to LPS administration. These results indicate that exogenous surfactant inhibits LPS-induced inflammation. This anti-inflammatory activity may be an important outcome of surfactant therapy in endotoxin-induced respiratory distress.

HIF1α Is Essential for Normal Intrauterine Differentiation of Alveolar Epithelium and Surfactant Production in the Newborn Lung of Mice

Neonatal respiratory distress syndrome (RDS) is mainly the result of perturbation in surfactant production and is a common complication seen in premature infants. Normal fetal lung development and alveolar cell differentiation is regulated by a network of transcription factors. Functional loss of any of these factors will alter the developmental program and impact surfactant production and normal gas exchange. During development, the fetus is exposed to varying oxygen concentrations and must be able to quickly adapt to these changes in order to survive. Hypoxia-inducible factor 1alpha (HIF1alpha) is the primary transcription factor that is responsible for regulating the cellular response to changes in oxygen tension and is essential for normal development. Its role in lung maturation is not well defined and to address this knowledge gap, a lung-specific HIF1alpha knock-out model has been developed. Loss of HIF1alpha early in lung development leads to pups that die within hours of parturition, exhibiting symptoms similar to RDS. Lungs from these pups display impaired alveolar epithelial differentiation and an almost complete loss of surfactant protein expression. Ultrastructural analysis of lungs from HIF1alpha deletion pups had high levels of glycogen, aberrant septal development, and decreased expression of several factors necessary for proper lung development, including HIF2alpha, beta-catenin, and vascular endothelial growth factor. These results suggest that HIF1alpha is essential for proper lung maturation and alteration in its normal signaling during premature delivery might explain the pathophysiology of neonatal RDS.

Altered expression of antimicrobial molecules in cigarette smoke‐exposed emphysematous mice lungs

The natural history of COPD, a disease usually caused by cigarette smoking, is associated with frequent respiratory infections. Consistent with human COPD, bacterial clearance in the lungs has been reported to be impaired in mice exposed to cigarette smoke. In the airways, several antimicrobial molecules such as surfactant proteins (SP), beta-defensins (BD), secretory leucocyte protease inhibitor (SLPI) and lysozyme play important roles in the defence against invading pathogens. This study evaluated the expression of antimicrobial molecules in mice lungs with cigarette smoke-induced emphysematous changes. Six B6C3F1 mice were exposed to cigarette smoke (2 cigarettes/day/mouse for 6 months) or room air. Gene expression within the lungs of mice in both groups was assessed by RT-PCR. The expression of SP-A, BD2, BD3 and SLPI was significantly elevated in the lungs of cigarette smoke-exposed mice compared with air-exposed mice. BD1 expression decreased in the smoke-exposed mice and lysozyme expression was unchanged. Chronic cigarette smoke exposure did not suppress the expression of antimicrobial molecules in the lung. Altered expression of antimicrobial molecules in this mouse model does not explain the impaired host defence against respiratory microbes seen in patients with COPD.