Expression Of SP-B Research Articles

Vitamin C rescues in part the effects of nitrofen on cultured human pneumocytes.

One of the mechanisms of action of nitrofen is intracellular oxidative stress. It is therefore likely that anti-oxidant agents can counteract its action. The aim of this study was to examine whether vitamin C mitigates the effects of nitrofen on the proliferation/apoptosis balance and functional maturation of cultured human pneumocytes. Lung aCa type II pneumocytes (NIH-H441) in culture were exposed to 1.5 microM of nitrofen alone or followed 48 h later by 60 microM of vitamin C. The culture dishes were recovered at 72 h for immunohistochemical (PCNA for proliferation, bis-benzimide for apoptosis, anti-SpB and anti-TTF-1 antibodies for SpB and TTF-1 assessment) and molecular studies. Real-time PCR was used to quantitate TTF-1 RNAm, with S26 as internal control. ANOVA or Kruskall-Wallis with appropriate post hoc tests were used for comparison with p<0.05 as threshold of significance. Nitrofen decreased proliferation and TTF/1 and SpB expression with no apparent affect on apoptosis. Additional exposure to vitamin C reverted these effects. Furthermore, nitrofen decreased TTF/1 mRNA and vitamin C tended to rescue normal values. Vitamin C partially rescued proliferation and maturity in nitrofen-treated human pneumocytes. The likely beneficial influence of this prenatal anti-oxidant medication should be further investigated.

Alterations in SP-B and SP-C expression in neonatal lung disease.

The hydrophobic surfactant proteins, SP-B and SP-C, have important roles in surfactant function. The importance of these proteins in normal lung function is highlighted by the lung diseases associated with abnormalities in their expression. Mutations in the gene encoding SP-B result in severe, fatal neonatal lung disease, and mutations in the gene encoding SP-C are associated with chronic interstitial lung diseases in newborns, older children, and adults. This work reviews the current state of knowledge concerning the lung diseases associated with mutations in the SP-B and SP-C genes, and the potential roles of abnormal SP-B and SP-C expression and genetic variation in these genes in other lung diseases.

Effects of vascular endothelial growth factor on isolated fetal alveolar type II cells.

Previous investigations gained from in vivo or lung explant studies suggested that VEGF is an autocrine proliferation and maturation factor for developing alveolar type II cells. The objective of this work was to determine whether VEGF exerted its growth and maturation effects directly on isolated type II cells. These were isolated from 19-day fetal rat lung and cultured in defined medium. The presence of VEGF receptor-2 was assessed in cultured cells at the pre- and posttranslational levels. Recombinant VEGF(165), formerly found to be active on lung explants, failed to enhance type II cell proliferation estimated by thymidine and 5-bromo-2'-deoxy-uridine incorporation. It increased choline incorporation in saturated phosphatidylcholine by 27% but did not increase phospholipid surfactant pool size. VEGF (100 ng/ml) left unchanged the transcript level of surfactant proteins (SP)-A, SP-C, and SP-D but increased SP-B transcripts to four times the control steady-state level. VEGF slightly retarded, but did not prevent, the in vitro transdifferentiation of type II into type I cells, as assessed by immunolabeling of the type I cell marker T1alpha. We conclude that, with the exception of SP-B expression, which appears to be controlled directly, the previously observed effects of this VEGF isoform on type II cells are likely to be exerted indirectly through reciprocal paracrine interactions involving other lung cell types.

FGF signaling is required for pulmonary homeostasis following hyperoxia.

To assess the role of fibroblast growth factor (FGF) signaling in pulmonary function in the postnatal period, we generated transgenic mice in which a soluble FGF receptor (FGFR-HFc) was conditionally expressed in respiratory epithelial cells of the mouse lung, thereby inhibiting FGF activity. Although FGFR-HFc did not alter postnatal lung morphogenesis, male FGFR-HFc transgenic mice were more susceptible to hyperoxia and failed to recover when ambient oxygen concentrations were normalized. Inflammation, alveolar-capillary leak, and mortality were increased following exposure to 95% Fi(O(2)). Expression of surfactant protein (SP)-A and SP-B were significantly decreased in association with decreased immunostaining for thyroid transcription factor-1. FGF signaling is required for maintenance of surfactant homeostasis and lung function during hyperoxia in vivo, mediated, at least in part, by its role in the maintenance of SP-B expression.

Inflammatory and anti-inflammatory responsiveness of surfactant proteins in fetal and neonatal rabbit lung.

Spontaneous preterm birth due to intrauterine infection is associated with increased concentrations of cytokines in amniotic fluid and in the airways at birth. Intra-amniotic IL-1 induces fetal lung maturity, consistent with the decrease in the incidence of respiratory distress syndrome (RDS) in intrauterine inflammation. On the other hand, antenatal corticosteroid decreases the incidence of RDS in infants born prematurely. The aim of the present study was to investigate the interaction between IL-1 and glucocorticoid in the expression of the surfactant proteins SP-A, -B, and -C. Lung explants from rabbit fetuses at 22 (immature), 27 (transitional), and 30 (mature) d of gestation (term, 30-31 d) and on d 1 after term birth were cultured with dexamethasone (Dx), IL-1alpha, or vehicle in the presence or absence of actinomycin D. According to the present results, IL-1alpha and Dx additively increased the expression of SP-A and SP-B on d 22. Later in gestation, SP-B and SP-C were suppressed by IL-1, whereas glucocorticoid tended to increase the expression of SP-B and SP-C and prevented the IL-1-induced suppression of SP. IL-1alpha and steroid interactively increased the stability of SP mRNA compared with the single agonist, possibly explaining the additive effects on the SP mRNA levels. The present results reveal beneficial additive effects of glucocorticoid and cytokine on lung surfactant. They may explain some of the acute beneficial effects of glucocorticoid therapy in chorioamnionitis before premature birth and in inflammatory lung disease after birth.

BR22, a 26 kDa thyroid transcription factor-1 associated protein (TAP26), is expressed in human lung cells.

The current authors have previously identified BR22, a thyroid transcription factor (TTF)-1 associated protein 26 (TAP26), which interacts with TTF-1 to enhance human surfactant protein (SP)-B promoter activity in transfected 293 cells. However, the expression of TAP26 in the lung cells and its biological relevance to the SP-B production under physiological conditions were not examined. In this study, endogenous co-immunoprecipitation and in situ immunohistochemical staining techniques were employed to explore the presence of TAP26 and TTF-1 complex in the lung epithelial cells. The correlation of TAP26, TTF-1 and SP-B expression was inspected in H441 cells in the presence of dexamethasone, a known positive effector of the SP-B promoter. Monoclonal antibody (mAb) against TAP26 can co-immunoprecipitate both TAP26 and TTF-1 from H441 cells. Using this antibody in in situ staining of human lung sections, the data show that TAP26 is present in the lung alveolar epithelial cells. Reverse transcriptase-polymerase chain reaction and Western blot analyses of type-II cells as well as dexamethasone-treated H441 cells suggest that TAP26 expression is modulated coordinately with SP-B and TTF-1 in these cells. In summary, the current study demonstrates that thyroid transcription factor-1 associated protein 26 is an associated protein of thyroid transcription factor-1 in the lung alveolar epithelial cells where surfactant protein gene expressions take place in vivo.

In vitro surfactant protein B deficiency inhibits lamellar body formation.

Surfactant protein (SP) B is essential for normal pulmonary surfactant activity and lamellar body genesis in type 2 cells. However, the role of SP-B in lamellar body genesis is poorly understood. We developed an adenovirus vector expressing antisense SP-B as an alternative in vitro model of SP-B deficiency to begin to explore the role of SP-B in lamellar body genesis. RT-PCR analysis revealed that antisense SP-B expression interfered with translation of endogenous SP-B mRNA. Antisense SP-B expression resulted in reliable in vitro reproduction of many features of SP-B deficiency, including absent mature SP-B and decreased lamellar bodies and SP-C. Light and electron microscopy demonstrated significant reductions in lamellar body number. Western blotting revealed a significant reduction in mature 8-kD SP-B protein and decreased mature SP-C. Our data indicate that antisense SP-B can be effectively used to replicate the SP-B-deficient type 2 cell phenotype in vitro, and provides an attractive alternative to transgenic models for the further study of the role of SP-B in lamellar body genesis.

SP-B deficiency causes respiratory failure in adult mice.

Targeted deletion of the surfactant protein (SP)-B locus in mice causes lethal neonatal respiratory distress. To assess the importance of SP-B for postnatal lung function, compound transgenic mice were generated in which the mouse SP-B cDNA was conditionally expressed under control of exogenous doxycycline in SP-B-/- mice. Doxycycline-regulated expression of SP-B fully corrected lung function in compound SP-B-/- mice and protected mice from respiratory failure at birth. Withdrawal of doxycycline from adult compound SP-B-/- mice resulted in decreased alveolar content of SP-B, causing respiratory failure when SP-B concentration was reduced to <25% of normal levels. Decreased SP-B was associated with low alveolar content of phosphatidylglycerol, accumulation of misprocessed SP-C proprotein in the air spaces, increased protein content in bronchoalveolar lavage fluid, and altered surfactant activity in vitro. Consistent with surfactant dysfunction, hysteresis, maximal tidal volumes, and end expiratory volumes were decreased. Reduction of alveolar SP-B content causes surfactant dysfunction and respiratory failure, indicating that SP-B is required for postnatal lung function.

Surfactant Protein B Inhibits Endotoxin-Induced Lung Inflammation

Transgenic mice, in which the level of surfactant protein (SP)-B mature peptide varied 5.6-fold between SP-B(+/-) and SP-B-overexpressing lines (SP-B+/+/+), were used to test the hypothesis that SP-B protects against endotoxin-induced lung inflammation. Intratracheal administration of endotoxin resulted in significantly lower concentration of SP-B mature peptide and elevated levels of total protein in bronchoalveolar lavage fluid of SP-B(+/-) mice compared with SP-B-overexpressing mice, indicating that endotoxin treatment leads to impairment of SP-B expression coincident with increased lung injury in SP-B(+/-) mice. Recruitment of inflammatory cells and elaboration of proinflammatory cytokines in bronchoalveolar lavage fluid were reduced in SP-B-overexpressing mice compared with SP-B(+/-) mice, suggesting that SP-B inhibited endotoxin-induced lung inflammation. Lung compliance and tissue damping were significantly decreased in SP-B(+/+) and SP-B(+/-) mice, but were not changed in SP-B(+/+/+) mice, consistent with a protective effect of SP-B. The minimum surface tension of large aggregate surfactant was significantly lower for surfactant isolated from SP-B-overexpressing mice, both in the absence and the presence of added plasma proteins. These data suggest that SP-B protected against endotoxin-induced lung inflammation by enhancing surfactant function, resulting in reduced lung injury, decreased influx of inflammatory cells, and lower cytokine levels; in contrast, levels of SP-B in SP-B(+/-) mice were further decreased by endotoxin treatment, likely exacerbating lung injury in this group.

Transgenic overexpression of IGF-II induces spontaneous lung tumors: a model for human lung adenocarcinoma.

Elevated levels of insulin-like growth factor (IGF)-II are associated with a poor prognosis in human pulmonary adenocarcinoma; however, a causal role for IGF-II in pulmonary adenocarcinoma has not been demonstrated. Here, we show that transgenic overexpression of IGF-II in lung epithelium induces lung tumors in 69% of mice older than 18 months of age. These tumors displayed morphological characteristics of human pulmonary adenocarcinoma such as their epithelial origin, tubulo-acinar architecture and expression of TTF-1, SP-B and proSP-C. Examination of signaling molecules downstream of the IGF-IR showed the activation of either the Erk1/Erk2 or p38 MAPK pathways, but not both, within the lung tumors. Notably, all lung tumors contained high levels of phosphorylated CREB, suggesting that both the Erk1/Erk2 and p38 MAPK pathways converged on this transcription factor. Moreover, IGF-II induced proliferation and CREB phosphorylation in human lung cancer cell lines, suggesting that IGF-II and CREB also contribute to the growth of human lung tumors. Thus, IGF-II is an important genetic factor in the development of lung tumorigenesis, in which activation of CREB is a ubiquitous event. The MMTV-IGF-II transgenic mice provide a critical model for elucidating the role of IGF-II in this fatal human disease.

Effect of exogenous surfactant on the development of surfactant synthesis in premature rabbit lung.

Surfactant replacement is an effective therapy for neonatal respiratory distress syndrome. Full recovery from respiratory distress syndrome requires development of endogenous surfactant synthesis and metabolism. The influence of exogenous surfactant on the development of surfactant synthesis in premature lungs is not known. We hypothesized that different exogenous surfactants have different effects on the development of endogenous surfactant production in the premature lung. We treated organ cultures of d 25 fetal rabbit lung for 3 d with 100 mg/kg body weight of natural rabbit surfactant, Survanta, and Exosurf and measured their effects on the development of surfactant synthesis. Additional experiments tested how these surfactants and Curosurf affected surfactant protein (SP) SP-A, SP-B, and SP-C mRNA expression. Surfactant synthesis was measured as the incorporation of 3H-choline and 14C-glycerol into disaturated phosphatidylcholine recovered from lamellar bodies. Randomized-block ANOVA showed significant differences among treatments for incorporation of both labels (p < 0.01), with natural rabbit surfactant less than control, Survanta greater than control, and Exosurf unchanged. Additional experiments with natural rabbit surfactant alone showed no significant effects in doses up to 1000 mg/kg. Survanta stimulated disaturated phosphatidylcholine synthesis (173 +/- 41% of control; p = 0.01), increased total lamellar body disaturated phosphatidylcholine by 22% (p < 0.05), and increased 14C-disat-PC specific activity by 35% (p < 0.05). The response to Survanta was dose-dependent up to 1000 mg/kg. Survanta did not affect surfactant release. No surfactant altered the expression of mRNA for SP-A, SP-B, or SP-C. We conclude that surfactant replacement therapy can enhance the maturation of surfactant synthesis, but this potential benefit differs with different surfactant preparations.

Regulation of surfactant proteins by LPS and proinflammatory cytokines in fetal and newborn lung.

Intra-amniotic lipopolysaccharide (LPS) and cytokines may decrease respiratory distress syndrome (RDS) and increase chronic lung disease in the newborn. The aim was to identify the primary inflammatory mediators regulating the expression of surfactant proteins (SP) in explants from immature (22-day-old fetus) and mature (30-day term fetus and 2-day-old newborn) rabbits. In immature lung, interleukin (IL)-1alpha and IL-1beta upregulated the expression of SP-A and SP-B. These effects of IL-1 were diminished, and SP-C mRNA was suppressed additively in the presence of tumor necrosis factor (TNF)-alpha and either LPS or interferon (IFN)-gamma. LPS, TNF-alpha, or IFN-gamma had no effect alone. In explants from the term fetus and the newborn, LPS, IL-1alpha, and TNF-alpha additively suppressed the SPs. LPS acutely induced IL-1alpha in alveolar macrophages in mature lung but not in the immature lung. IFN-gamma that generally has low expression in intrauterine infection decreased the age dependence of the other agonists' effects on SPs. The present study serves to explain the variation of the pulmonary outcome after an inflammatory insult. We propose that IL-1 from extrapulmonary sources induces the SPs in premature lung and is responsible for the decreased risk of RDS in intra-amniotic infection.

Maintenance of surfactant protein A and D secretion by rat alveolar type II cells in vitro

Secretion of surfactant proteins A and D (SP-A and SP-D) has been difficult to study in vitro because a culture system for maintaining surfactant secretion has been difficult to establish. We evaluated several growth factors, corticosteroids, rat serum, and a fibroblast feeder layer for the ability to produce and maintain a polarized epithelium of type II cells that secretes SP-A and SP-D into the apical medium. Type II cells were plated on a filter insert coated with an extracellular matrix and were cultured at an air-liquid interface. Keratinocyte growth factor (KGF) stimulated type II cell proliferation and secretion of SP-A and SP-D more than fibroblast growth factor-10 (FGF-10), hepatocyte growth factor (HGF), or heparin-binding epidermal-like growth factor (HB-EGF). Cells cultured in the presence of KGF and rat serum with or without fibroblasts had high surfactant protein mRNA levels and exhibited a high level of SP-A and SP-D secretion. Dexamethasone inhibited type II cell proliferation but increased expression of SP-B. In the presence of KGF, rat serum, and dexamethasone, the mRNAs for the surfactant proteins were maintained at high levels. Secretion of SP-A and SP-D was found to be independent of phospholipid secretion.

Nitrate and nitrite in exhaled breath condensate are derived from the lower airway

Nitrate and nitrite in exhaled breath condensate are derived from the lower airway

Mice lacking CCAAt/enhancer-binding protein-alpha show hyperproliferation of alveolar type II cells and increased surfactant protein mRNAs.

The lung-specific surfactant proteins (SP) are essential for normal respiratory function. Transcription factors may play an important role in the regulation of surfactant proteins. The CCAAT/enhancer-binding protein (C/EBP) family consists of transcription factors that can stimulate expression of genes in lipid-metabolizing epithelial cells. C/EBPalpha-deficient mice have been shown to exhibit abnormal pulmonary histopathology. Recently, we demonstrated that C/EBP family members are differentially expressed in alveolar type II cell proliferation and in pulmonary fibrosis. In the present study, to investigate whether the C/EBP family would be involved in the regulation of surfactant proteins, we examined the protein expression of SP-A, and SP-C, and mRNA expression of SP-A, SP-B, and SP-C in the lungs from newborn C/EBPalpha-deficient mice. Using immunohistochemistry, we demonstrated that positive cells for SP-C, specific to alveolar type II cells, in the lungs were more abundant in the newborn C/EBPalpha-deficient mice than in control mice, which suggests the hyperproliferation of alveolar type II cells in the lungs of the C/EBPalpha-deficient mice. In situ hybridization analysis revealed that expression of SP-A, SP-B, and SP-C mRNAs were increased in the lungs of newborn C/EBPalpha-deficient mice. Northern blot analysis revealed that surfactant protein mRNAs were also increased. Thus, these results suggest that C/EBPalpha may play a key role in the proliferation of alveolar type II cells and the regulation of genes of surfactant protein.

Aspergillus fumigatus-induced allergic airway inflammation alters surfactant homeostasis and lung function in BALB/c mice.

The differential regulation of pulmonary surfactant proteins (SPs) is demonstrated in a murine model of Aspergillus fumigatus (Af )-induced allergic airway inflammation and hyperresponsiveness. BALB/c mice were sensitized intraperitoneally and challenged intranasally with Af extract. Enzyme-linked immunosorbent assay analysis of serum immunoglobulin (Ig) levels in these mice showed markedly increased total IgE and Af-specific IgE and IgG1. This was associated with peribronchial/perivascular tissue inflammation, airway eosinophilia, and secretion of interleukin (IL)-4 and IL-5 into the bronchoalveolar lavage fluid (BALF). Functional analysis revealed that in comparison with nonsensitized mice, allergic sensitization and challenge resulted in significant increases in acetylcholine responsiveness. To analyze levels of SPs, the cell-free supernate of the BALF was further fractionated by high-speed (20,000 x g) centrifugation. After sensitization and challenges, the pellet (large-aggregate fraction) showed a selective downregulation of hydrophobic SPs SP-B and SP-C by 50%. This reduction was reflected by commensurate decreases in SP-B and SP-C messenger RNA (mRNA) expression of the lung tissue of these animals. In contrast, there was a 9-fold increase in SP-D protein levels in the 20,000 x g supernate without changes in SP-D mRNA. The increased levels of SP-D showed a significant positive correlation with serum IgE (r = 0.85, P < 0.001). Tissue mRNA and protein levels of SP-A in either the large- or the small-aggregate fractions were unaffected. Our data indicate that allergic airway inflammation induces selective inhibition of hydrophobic SP synthesis accompanied by marked increases in the lung collectin SP-D protein content of BALF. These changes may contribute significantly to the pathophysiology of Af-induced allergic airway hyperresponsiveness.

Decreased surfactant protein-B expression and surfactant dysfunction in a murine model of acute lung injury.

This study examines the relationships between inflammation, surfactant protein (SP) expression, surfactant function, and lung physiology in a murine model of acute lung injury (ALI). 129/J mice received aerosolized endotoxin lipopolysaccharide [LPS] daily for up to 96 h to simulate the cytokine release and acute inflammation of ALI. Lung elastance (E(L)) and resistance, lavage fluid cell counts, cytokine levels, phospholipid and protein content, and surfactant function were measured. Lavage and lung tissue SP content were determined by Western blot and immunohistochemistry, and tissue messenger RNA (mRNA) levels were assessed by Northern blot and in situ hybridization. Tumor necrosis factor-alpha and neutrophil counts in bronchoalveolar lavage fluid increased within 2 h of LPS exposure, followed by increases in total protein, interleukin (IL)-1beta, IL-6, and interferon-gamma. E(L) increased within 24 h of LPS exposure and remained abnormal up to 96 h. SP-B protein and mRNA levels were decreased at 24, 48, and 96 h. By contrast, SP-A protein and mRNA levels and SP-C mRNA levels were not reduced. Surfactant dysfunction occurred coincident with changes in SP-B levels. This study demonstrates that lung dysfunction in mice with LPS-ALI corresponds closely with abnormal surfactant function and reduced SP-B expression.

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

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

Establishment of pulmonary alveolar type II cell line from p53-deficient mice.

We obtained a pulmonary alveolar type II epithelial cell line, MAC7, derived from the lung of p53-deficient mice (p53 -/-). When this cell line was passaged for long periods of time, the epithelial cells grew at a high rate for over 50th passage and never entered the non-growing senescent phase characteristic of the normal cells (p53 +/+). Each pulmonary epithelial outgrowth was characterized morphologically and immunocytochemically, and the growth and viability of the outgrowths were examined by MTT assay. Expression of surfactant-associated protein, SP-B, was detected, and transmission electron microscopy demonstrated a type II phenotype containing lamellar bodies and phospholipids. These findings indicate that MAC7 cells retain the morphological and physiological properties of alveolar type II epithelial cells. This cell line should be useful in experimental systems for studying lung pathology.

Effect of hyperoxia on surfactant protein gene expression in hypoplastic lung in nitrofen-induced diaphragmatic hernia in rats

The hypoplastic lung in congenital diaphragmatic hernia (CDH) has both a quantitative and qualitative reduction in surfactant. Recently, the role of oxygen (O2) as a regulator of pulmonary surfactant-associated protein (SP) gene expression has been reported. The mRNA level of SP has been demonstrated to be increased in the lungs of animals exposed to hyperoxia. The aim of this study was to investigate SP mRNA expression in hypoplastic CDH lung in rats during mechanical ventilation in order to determine the effect of O2 on SP synthesis in CDH. A CDH model was induced in pregnant rats following administration of nitrofen. The newborn rats with CDH and controls were intubated and ventilated. Ventilation was continued for 6 h under 100% oxygen. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to evaluate the relative amounts of mRNA expression of SP-A, SP-B, SP-C, and SP-D. Relative amounts of SP-A, SP-B, and SP-D mRNA expression in CDH lung were significantly decreased compared to controls at birth and 6 h after ventilation. There was no significant difference in SP-C mRNA expression between CDH animals and controls. Upregulated mRNA expression of SP-A, SP-B, and SP-D in lungs of control animals at 6 h after ventilation suggests that oxygenation accelerates postnatal SP synthesis in normal lungs. The inability of O2 to increase SP mRNA expression in hypoplastic CDH lung suggests that the hypoplastic lung is not responsive to increased oxygenation for the synthesis of SP.