Surfactant protein D is proatherogenic in mice

Surfactant protein D (SP-D) is an important innate immune defense molecule that mediates clearance of pathogens and modulates the inflammatory response. Moreover, SP-D is involved in lipid homeostasis, and pulmonary accumulation of phospholipids has previously been observed in SP-D-deficient (Spd-/-) mice. Atherogenesis involves both inflammation and lipid deposition, and we investigated the role of SP-D in the development of atherosclerosis. SP-D synthesis was localized to vascular endothelial cells. Atherosclerotic lesion areas were 5.6-fold smaller in the aortic roots in Spd-/- mice compared with wild-type C57BL/6N mice on an atherogenic diet. HDL cholesterol (HDL-C) was significantly elevated in Spd-/- mice. Treatment of Spd-/- mice with a recombinant fragment of human SP-D resulted in decreases of HDL-C (21%) as well as total cholesterol (26%), and LDL cholesterol (28%). Plasma TNF-alpha was reduced in Spd-/- mice (45% difference). SP-D was proatherogenic in the mouse model used. The effect is likely to be due to the observed disturbances of plasma lipid metabolism and alteration of the inflammatory process, which underlie the reduced susceptibility to atherosclerosis in Spd-/- mice.     

Surfactant protein D is a divalent cation-dependent carbohydrate-binding protein

Surfactant protein D (SP-D, CP4) is a collagenous surfactant-associated glycoprotein synthesized by lung type II epithelial cells. SP-D can be selectively and efficiently eluted from isolated rat surfactant with glucose, maltose, and certain other saccharides. We therefore examined the ability of the purified protein to interact with carbohydrates in vitro. Saccharide-substituted bovine serum albumins (BSA neoglycoproteins) were adsorbed to plastic wells, and binding of purified SP-D was quantified with monospecific antibodies to SP-D using an indirect immunoassay. SP-D showed specific calcium-dependent binding to alpha-D-glucosidophenyl isothiocyanate-BSA and maltosyl-BSA, but negligible binding to beta-D-glucosidophenyl isothiocyanate-BSA or unconjugated BSA. The most efficient inhibitors of SP-D binding were alpha-glucosyl-containing saccharides (e.g. isomaltose, maltose, malotriose). SP-D showed quantitative binding to maltosyl-agarose and was specifically eluted with maltose or EDTA. High affinity binding to maltosyl-BSA was also demonstrated using a solution-phase polyethylene glycol precipitation assay. These studies demonstrate that SP-D is a calcium dependent lectin-like protein and that the association of SP-D with surfactant is mediated by carbohydrate-dependent interactions with specificity for alpha-glucosyl residues.     

A mutant deficient in the plastid lipid DGD is defective in protein import into chloroplasts

Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol with N-terminal extensions called transit peptides. Transit peptides function as the import signal to chloroplasts. The import process requires several protein components in the envelope and stroma and also requires the hydrolysis of ATP. Lipids have been implicated in the import process based on theories or experiments with in vitro model systems. We show here that chloroplasts isolated from an Arabidopsis mutant deficient in the plastid lipid digalactosyl diacylglycerol (DGD) were normal in importing a chloroplast outer membrane protein, but were defective in importing precursor proteins targeted to the interior of chloroplasts. The impairment includes the binding, or docking, step of the import process that is supported by 100 μM ATP.     

Induced sputum is a reproducible method to assess airway inflammation in asthma

To evaluate the reproducibility of induced sputum analysis, and to estimate the sample size required to obtained reliable results, sputum was induced by hypertonic saline inhalation in 29 asthmatic subjects on two different days. The whole sample method was used for analysis, and inflammatory cells were counted on cytospin slides. Reproducibility, expressed by intra-class correlation coefficients, was good for macrophages (+0.80), neutrophils (+0.85), and eosinophils (+0.87), but not for lymphocytes (+0.15). Detectable differences were 5.5% for macrophages, 0.6% for lymphocytes, 5.2% for neutrophils, and 3.0% for eosinophils. We conclude that analysis of induced sputum is a reproducible method to study airway inflammation in asthma. Sample sizes greater than ours give little improvement in the detectable difference of eosinophil percentages.     

Surfactant protein A is localized at the corners of the pulmonary tubular myelin lattice

Immunogold labeling on sections of a freeze-substituted tubular myelin-enriched fraction isolated from a bronchoalveolar lavage of rat lung showed that surfactant protein A (SP-A) occurs predominantly at the corners of the tubular myelin lattice. Seventy-nine percent of the gold particles were located within 20 nm from a corner. Extracellular SP-A was detected only in the tubular myelin lattice and not in vesicles or secreted lamellar bodies. Ultra-thin cryosections of rat lung fixed in vivo showed that intracellular SP-A was distributed homogeneously over the stacked membranes of lamellar bodies in alveolar Type II cells. The presence of SP-A at the corners of the tubular myelin lattice suggests an important role of this protein in the formation and/or maintenance of this highly ordered lattice.     

Small airway inflammation in asthma

Asthma was originally described as an inflammatory disease that predominantly involves the central airways. Pathological and physiological evidence reported during the past few years suggests that the inflammatory process extends beyond the central airways to the peripheral airways and the lung parenchyma. The small airways are capable of producing T-helper-2 cytokines, as well as chemokines, and they have recently been recognized as a predominant site of airflow obstruction in asthmatic persons. The inflammation at this distal site has been described as more severe than large airway inflammation. These findings are of great clinical significance, and highlight the need to consider the peripheral airways as a target in any therapeutic strategy for treatment of asthma.     

Surfactant protein A is a principal and oxidation-sensitive microbial permeabilizing factor in the alveolar lining fluid

We have reported that surfactant protein A kills some Gram-negative organisms by increasing membrane permeability. In this study, we investigated the physiologic importance of this activity and the effect of oxidative stress on the antimicrobial functions of SP-A in vitro and in vivo. Concentrated bronchoalveolar lavage fluids from SP-A+/+ mice increased the permeability of the Escherichia coli K12 cell membrane to a greater extent than lavage from SP-A-/- animals. Similarly, calcium-dependent surfactant-binding proteins of SP-A+/+ mice increased membrane permeability more than those from SP-A-/- mice and produced greater zonal killing of agar-embedded bacteria in a radial diffusion assay. Exposure of human SP-A to copper-initiated surfactant phospholipid peroxidation or to free radicals generated by human neutrophils in vitro increased the level of SP-A-associated carbonyl moieties and blocked the permeabilizing function of the protein. We also found that exposure of mice to 90% O2 for 4 days, sufficient to lead to consumption of glutathione, oxidation of protein thiols, and accumulation of airspace protein-associated carbonyl moieties, blocked the permeabilizing activity of lavage fluid from SP-A+/+ mice. We conclude that SP-A is a major microbial permeablizing factor in lavage fluid and that oxidative stress inhibits the antibacterial activity of SP-A by a mechanism that includes oxidative modification and functional inactivation of the protein.     

Surfactant protein A regulates complement activation.

Complement proteins aid in the recognition and clearance of pathogens from the body. C1, the first protein of the classical pathway of complement activation, is a calcium-dependent complex of one molecule of C1q and two molecules each of C1r and C1s, the serine proteases that cleave complement proteins. Upon binding of C1q to Ag-bound IgG or IgM, C1r and C1s are sequentially activated and initiate the classical pathway of complement. Because of structural and functional similarities between C1q and members of the collectin family of proteins, including pulmonary surfactant protein A (SP-A), we hypothesized that SP-A may interact with and regulate proteins of the complement system. Previously, SP-A was shown to bind to C1q, but the functional significance of this interaction has not been investigated. Binding studies confirmed that SP-A binds directly to C1q, but only weakly to intact C1. Further investigation revealed that the binding of SP-A to C1q prevents the association of C1q with C1r and C1s, and therefore the formation of the active C1 complex required for classical pathway activation. This finding suggests that SP-A may share a common binding site for C1r and C1s or Clq. SP-A also prevented C1q and C1 from binding to immune complexes. Furthermore, SP-A blocked the ability of C1q to restore classical pathway activity to C1q-depleted serum. SP-A may down-regulate complement activity through its association with C1q. We hypothesize that SP-A may serve a protective role in the lung by preventing C1q-mediated complement activation and inflammation along the delicate alveolar epithelium.     

A mutant of Arabidopsis which is defective in seed development and storage protein accumulation is a new abi3 allele

In order to investigate the role of the plant hormones gibberellin (GA) and abscisic acid (ABA) in seed development and germination the GA biosynthetic inhibitor, Uniconazol, was used to isolate mutants with abnormal germination profiles. In one of these mutants, the ability to germinate on Uniconazol is due to a mutation in the ABI3 gene. However, unlike the previously reported abi3 mutant, this line displays an array of seed-specific developmental defects. The accumulation of seed reserve proteins is dramatically reduced due to reduced levels of the storage protein mRNA. The embryos remain green throughout development and are desiccation intolerant. However, immature seeds are completely non-dormant and grow normally. These results suggest the ABI3 gene is essential for the synthesis of seed storage proteins and for the protection of the embryo during desiccation.     

A T7 amber mutant defective in DNA-Binding protein

Summary A T7 amber mutant, UP-2, in the gene for T7 DNA-binding protein was isolated from mutants that could not grow on sup ⁺ ssb-1 bacteria but could grow on glnU ssb-1 and sup ⁺ ssb ⁺bacteria. The mutant phage synthesized a smaller amber polypeptide (28,000 daltons) than T7 wild-type DNA-dinding protein (32,000 daltons). DNA synthesis of the UP-2 mutant in sup ⁺ ssb-1 cells was severely inhibited and the first round of replication was found to be repressed. The abilities for genetic recombination and DNA repair were also low even in permissive hosts compared with those of wild-type phage. Moreover, recombination intermediate T7 DNA molecules were not formed in UP-2 infected nonpermissive cells. The gene that codes for DNA-binding protein is referred to as gene 2.5 since the mutation was mapped between gene 2 and gene 3.     

Protectin D1 is generated in asthma and dampens airway inflammation and hyperresponsiveness

Protectins are newly identified natural chemical mediators that counter leukocyte activation to promote resolution of inflammation. In this study, we provide the first evidence for protectin D1 (PD1, 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid) formation from docosahexaenoic acid in human asthma in vivo and PD1 counterregulatory actions in allergic airway inflammation. PD1 and 17S-hydroxy-docosahexaenoic acid were present in exhaled breath condensates from healthy subjects. Of interest, levels of PD1 were significantly lower in exhaled breath condensates from subjects with asthma exacerbations. PD1 was also present in extracts of murine lungs from both control animals and those sensitized and aerosol challenged with allergen. When PD1 was administered before aeroallergen challenge, airway eosinophil and T lymphocyte recruitment were decreased, as were airway mucus, levels of specific proinflammatory mediators, including IL-13, cysteinyl leukotrienes, and PGD(2), and airway hyperresponsiveness to inhaled methacholine. Of interest, PD1 treatment after aeroallergen challenge markedly accelerated the resolution of airway inflammation. Together, these findings provide evidence for endogenous PD1 as a pivotal counterregulatory signal in allergic airway inflammation and point to new therapeutic strategies for modulating inflammation in asthmatic lung.     

Surfactant protein A regulates IgG-mediated phagocytosis in inflammatory neutrophils

Surfactant proteins (SP)-A and SP-D have been shown to affect the functions of a variety of innate immune cells and to interact with various immune proteins such as complement and immunoglobulins. The goal of the current study is to test the hypothesis that SP-A regulates IgG-mediated phagocytosis by neutrophils, which are major effector cells of the innate immune response that remove invading pathogens by phagocytosis and by extracellular killing mediated by reactive oxygen and nitrogen. We have previously shown that SP-A stimulates chemotaxis by inflammatory, but not peripheral, neutrophils. To evaluate the ability of SP-A to modulate IgG-mediated phagocytosis, polystyrene beads were coated with BSA and treated with anti-BSA IgG. SP-A significantly and specifically enhanced IgG-mediated phagocytosis by inflammatory neutrophils, but it had no effect on beads not treated with IgG. SP-A bound to IgG-coated beads and enhanced their uptake via direct interactions with the beads as well as direct interactions with the neutrophils. SP-A did not affect reactive oxygen production or binding of IgG to neutrophils and had modest effects on polymerization of actin. These data suggest that SP-A plays an important role in mediating the phagocytic response of neutrophils to IgG-opsonized particles.     

Surfactant protein A is a required mediator of keratinocyte growth factor after experimental marrow transplantation

We reported an association between the ability of recombinant human keratinocyte growth factor (rHuKGF) to upregulate the expression of surfactant protein A (SP-A) and to downregulate pulmonary inflammation that occurs after allogeneic bone marrow transplantation (BMT). To establish a causal relationship, rHuKGF (5 mg/kg) was administered subcutaneously for three consecutive days before irradiation to SP-A-sufficient and -deficient [SP-A(+/+) and SP-A(-/-), respectively] mice given inflammation-inducing allogeneic spleen T cells at the time of BMT. In contrast with SP-A(+/+) mice, rHuKGF failed to suppress the high levels of TNF-alpha, IFN-gamma, and nitric oxide contained in bronchoalveolar lavage fluids collected on day 7 after BMT from SP-A(-/-) mice. Early post-BMT weight loss was attenuated by rHuKGF in both SP-A(+/+) and SP-A(-/-) recipients. In the absence of supportive respiratory care, however, SP-A deficiency eventually abolished the ability of rHuKGF to prevent weight loss and to improve survival monitored for 1 mo after allogeneic BMT. In further experiments, the addition of cyclophosphamide (which is known to cause severe injury to the alveolar epithelium in donor T cell-recipient mice) to the conditioning regimen prevented rHuKGF-induced upregulation of SP-A and suppression of lung inflammation in both SP-A(+/+) and SP-A(-/-) mice. We conclude that endogenous baseline SP-A levels and optimal upregulation of SP-A are required for the anti-inflammatory protective effects of KGF after allogeneic transplantation.     

Apoptosis and airway inflammation in asthma

Asthma is a disease characterized by a chronic inflammation of the airways and by structural alterations of bron-chial tissues, often referred to as airway remodelling. The development of chronic airway inflammation in asthma depends upon the continuous recruitment of inflammatory cells from the bloodstream towards the bronchial mucosa and by their subsequent activation. It is however increasingly accepted that mechanisms involved in the regulation of the survival and apoptosis of inflammatory cells may play a central role in the persistent inflammatory process characterizing this disease. Increased cellular recruitment and activation, enhanced cell survival and cell:cell interactions are therefore the key steps in the development of chronic airway inflammation in asthma, and represent the major causes for tissue damge, repair and remodelling.     

Surfactant function in children with chronic airway inflammation.

Pulmonary surfactant is necessary to keep the terminal conducting airways patent. It is unknown whether mild to moderate airway inflammation may influence surfactant function and thus contribute to the pathogenesis of chronic airway inflammation in children. To answer this question, 21 children with chronic obstructive bronchitis and 19 asymptomatic children with long-term tracheostomy and increased numbers of neutrophils in their airways were compared with 15 healthy controls. Bronchoalveolar lavage fluid was separated into large surfactant aggregates (LA) and a supernatant containing inhibitory constituents. Surfactant function of LA, recombinations of LA and supernatant, and recombinations of a defined bovine surfactant and supernatant was assessed in a capillary surfactometer. Compared with controls, the function of the LA surfactant was reduced and there was no difference between children with tracheostomy and chronic obstructive bronchitis. The function of LA-supernatant recombinations was poor in all subjects. This may be explained by the well-known protein influx during the lavage procedure. The activity of bovine surfactant-supernatant reconstitutions was impaired in children with tracheostomy. In all surfactant mixtures assessed, surfactant function was inversely correlated to the number of neutrophils in the lavage fluid. Chronic lower airway inflammation with mild or no clinical symptoms is associated with impaired surfactant function. The dysfunction may contribute to airflow restrictions frequently observed in these children.     

S100A8 protein in inflammation

S100A8, an important member of the S100 protein family, is a low-molecular-weight (10.8 kDa) calcium-binding protein containing conserved EF-hand structural motifs. Previous studies have shown that the biological function of S100A8 protein is associated with a variety of inflammatory diseases, for example asthma. S100A8 protein plays important roles in the regulation of inflammation. It can activate inflammatory cells and cytokines via chemotactic activity for neutrophils, and bind to the receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4), thus mediating intracellular inflammatory signaling transduction. Additionally, recent studies have reported the anti-inflammation activity of S100A8 protein, which indicates that S100A8 may have a more complex function of biological regulation in the different pathophysiological conditions. In this review, we summarized the studies on the functions and molecular mechanisms of S100A8 protein in inflammation, which would propose a novel strategy for the prophylaxis and treatment of asthma and other inflammatory diseases.     

The maize high-lysine mutant opaque7 is defective in an acyl-CoA synthetase-like protein

Maize (Zea mays) has a large class of seed mutants with opaque or nonvitreous endosperms that could improve the nutritional quality of our food supply. The phenotype of some of them appears to be linked to the improper formation of protein bodies (PBs) where zein storage proteins are deposited. Although a number of genes affecting endosperm vitreousness have been isolated, it has been difficult to clone opaque7 (o7), mainly because of its low penetrance in many genetic backgrounds. The o7-reference (o7-ref) mutant arose spontaneously in a W22 inbred, but is poorly expressed in other lines. We report here the isolation of o7 with a combination of map-based cloning and transposon tagging. We first identified an o7 candidate gene by map-based cloning. The putative o7-ref allele has a 12-bp in-frame deletion of codons 350-353 in a 528-codon-long acyl-CoA synthetase-like gene (ACS). We then confirmed this candidate gene by generating another mutant allele from a transposon-tagging experiment using the Activator/Dissociation (Ac/Ds) system in a W22 background. The second allele, isolated from ～1 million gametes, presented a 2-kb Ds insertion that resembles the single Ds component of double-Ds, McClintock's original Dissociation element, at codon 496 of the ACS gene. PBs exhibited striking membrane invaginations in the o7-ref allele and a severe number reduction in the Ds-insertion mutant, respectively. We propose a model in which the ACS enzyme plays a key role in membrane biogenesis, by taking part in protein acylation, and that altered PBs render the seed nonvitreous.     

The neutrophil-activating protein of Helicobacter pylori down-modulates Th2 inflammation in ovalbumin-induced allergic asthma

The Helicobacter pylori neutrophil-activating protein (HP-NAP) is able in vitro to elicit IL-12 and IL-23 production via agonistic interaction with toll-like receptor 2, and to promote Th1 polarization of allergen-specific T-cell responses. This study was aimed to assess whether systemic/intraperitoneal and/or mucosal HP-NAP administration inhibited the Th2-mediated bronchial inflammation using a mouse model of allergic asthma induced by inhaled ovalbumin (OVA). Systemic HP-NAP delivery markedly reduced the lung eosinophilia in response to repeated challenge with aerosolized OVA. Likewise, the production of IL-4, IL-5 and GM-CSF was significantly lower in the bronchoalveolar lavage of animals treated with systemic HP-NAP plus OVA than that of animals treated with OVA alone. Systemic HP-NAP also significantly resulted in both reduction of total serum IgE and increase of IL-12 plasma levels. Mucosal administration of HP-NAP was equally successful as the systemic delivery in reducing eosinophilia, IgE and Th2 cytokine levels in bronchoalveolar lavage. However, no suppression of lung eosinophilia and bronchial Th2 cytokines was observed in toll-like receptor 2-knock-out mice following HP-NAP treatment. These results identify HP-NAP as a candidate for novel strategies of prevention and treatment of allergic diseases.