Effect of surfactant on pulmonary expression of type IIA PLA(2) in an animal model of acute lung injury

We previously showed that the seminatural surfactant Curosurf inhibits the in vitro synthesis of secretory type IIA phospholipase A(2) (sPLA(2)-IIA) in alveolar macrophages (AM). These cells are the main source of sPLA(2)-IIA in a guinea pig model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Here, we investigate the effect of Curosurf on the pulmonary synthesis of sPLA(2)-IIA in this ALI model. Our results showed that intratracheal administration of LPS (330 microg/kg) induced an increase in pulmonary expression of sPLA(2)-IIA, which was inhibited when animals received Curosurf (16 mg/guinea pig) 30 min or 8 h after LPS instillation. When AM were isolated from LPS-treated animals and cultured in conditioned medium, they expressed higher levels of sPLA(2)-IIA than AM from saline-treated animals. This ex vivo sPLA(2)-IIA expression was significantly reduced when guinea pigs received Curosurf 30 min after LPS instillation. Finally, we examined the effect of Curosurf on pulmonary inflammation measured 8 or 24 h after LPS administration. Curosurf instillation 30 min or 8 h after LPS reversed the increase in tumor necrosis factor-alpha expression, polymorphonuclear cell extravasation, and protein concentration in bronchoalveolar lavage fluids. Curosurf also decreased the bronchial reactivity induced by LPS. We conclude that Curosurf inhibits the pulmonary expression of sPLA(2)-IIA and exhibits palliative anti-inflammatory effects in an animal model of ALI.     

Eicosanoid-mediated proinflammatory activity of Pseudomonas aeruginosa ExoU

As Pseudomonas aeruginosa ExoU possesses two functional blocks of homology to calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)), we addressed the question whether it would exhibit a proinflammatory activity by enhancing the synthesis of eicosanoids by host organisms. Endothelial cells from the HMEC-1 line infected with the ExoU-producing PA103 strain exhibited a potent release of arachidonic acid (AA) that could be significantly inhibited by methyl arachidonyl fluorophosphonate (MAFP), a specific PLA(2) inhibitor, as well as significant amounts of the cyclooxygenase (COX)-derived prostaglandins PGE(2) and PGI(2). Cells infected with an isogenic mutant defective in ExoU synthesis did not differ from non-infected cells in the AA release and produced prostanoids in significantly lower concentrations. Infection by PA103 induced a marked inflammatory response in two different in vivo experimental models. Inoculation of the parental bacteria into mice footpads led to an early increase in the infected limb volume that could be significantly reduced by inhibitors of both COX and lipoxygenase (ibuprofen and NDGA respectively). In an experimental respiratory infection model, bronchoalveolar lavage (BAL) from mice instilled with 10(4) cfu of PA103 exhibited a marked influx of inflammatory cells and PGE(2) release that could be significantly reduced by indomethacin, a non-selective COX inhibitor. Our results suggest that ExoU may contribute to P. aeruginosa pathogenesis by inducing an eicosanoid-mediated inflammatory response of host organisms.     

Pseudomonas aeruginosa LPS or Flagellin Are Sufficient to Activate TLR-Dependent Signaling in Murine Alveolar Macrophages and Airway Epithelial Cells

Background

The human lung is exposed to a large number of airborne pathogens as a result of the daily inhalation of 10,000 liters of air. Innate immunity is thus essential to defend the lungs against these pathogens. This defense is mediated in part through the recognition of specific microbial ligands by Toll-like receptors (TLR) of which there are at least 10 in humans. Pseudomonas aeruginosa is the main pathogen that infects the lungs of cystic fibrosis patients. Based on whole animal experiments, using TLR knockout mice, the control of this bacterium is believed to occur by the recognition of LPS and flagellin by TLRs 2,4 and 5, respectively.

Methodology/Principal Findings

In the present study, we investigated in vitro the role of these same TLR and ligands, in alveolar macrophage (AM) and epithelial cell (EC) activation. Cellular responses to P. aeruginosa was evaluated by measuring KC, TNF-α, IL-6 and G-CSF secretion, four different markers of the innate immune response. AM and EC from WT and TLR2, 4, 5 and MyD88 knockout mice for were stimulated with the wild-type P. aeruginosa or with a mutant devoid of flagellin production.

Conclusions/Significance

The results clearly demonstrate that only two ligand/receptor pairs are necessary for the induction of KC, TNF-α, and IL-6 synthesis by P. aeruginosa-activated cells, i.e. TLR2,4/LPS and TLR5/flagellin. Either ligand/receptor pair is sufficient to sense the bacterium and to trigger cell activation, and when both are missing lung EC and AM are unable to produce such a response as were cells from MyD88^−/− mice.     

The effect of 1-O-alkyl-2-acetyl-Sn-glycero- 3-phosphocholine (paf-acether) on the arterial wall

The effect of a topical paf-acether superfusion over an injured arterial segment was assessed in the guinea-pig, using an opto-electronic in vivo thrombosis model allowing on line quantification of small platelet thrombus dynamics.As compared to control, ADP-induced, thromboformation and behaviour, exogenous paf-acether causes a large, dense platelet thrombus, invaded and surrounded by numerous leukocytes, spreading widely over the adjoining, vacuolized, endothelium. Its embolization has to be forced with prostanoids, mepacrine, EDTA, or with a specific paf-acether antagonist3 (BN 52021). A few minutes after such forced embolization, a new thrombus starts growing at the same site, without renewal of the paf-acether superfusion. This phenomenon of spontaneous reappearance after forced embolization can be followed during several hours. Experiments with labelled paf-acether and the paf-acether antagonist indicate a possible endogenous paf-acether (or paf-acether-like) production triggered by superfusion with exogenous paf-acether.     

Type-IIA secreted phospholipase A2 is an endogenous antibiotic-like protein of the host

Type-IIA secreted phospholipase A₂ (sPLA₂-IIA) has been proposed to play a role in the development of inflammatory diseases. It has been shown to release arachidonic acid, the precursor of proinflammatory eicosanoids, to hydrolyze phospholipids of pulmonary surfactant, and to bind to specific receptors located on cell surface membranes. However, the most established biological role of sPLA₂-IIA is related to its potent bactericidal property in particular toward Gram-positive bacteria. This enzyme is present in animal and human biological fluids at concentrations sufficient to kill bacteria. Human recombinant sPLA₂-IIA is able to kill Gram-positive bacteria at concentrations as low as 1.1 ng/ml. This remarkable property is due to the unique preference of sPLA₂-IIA for anionic phospholipids such as phosphatidylglycerol, the main phospholipid component of bacterial membranes. Much higher concentrations of sPLA₂-IIA are required for its action on host cell membranes and surfactant both of which are mainly composed by phosphatidylcholine, a poor substrate for sPLA₂-IIA. Transgenic mice over-expressing human sPLA₂-IIA are resistant to infection by Staphylococcus aureus, Escherichia coli, and Bacillus anthracis, the etiological agent of anthrax. Conversely, certain bacteria, such as B. anthracis, E. coli and Bordetella pertussis are able to inhibit sPLA₂-IIA expression by host cells, thus highlighting a mechanism by which these bacteria can subvert the host immune system. Intranasal instillation of recombinant sPLA₂-IIA protects mice from mortality caused by pulmonary anthrax. Interestingly, this protective effect was obtained even with B. anthracis strains that down-regulate the expression of endogenous sPLA₂-IIA, indicating that instilled sPLA₂-IIA can overcome the subversive action of B. anthracis. We conclude that sPLA₂-IIA is an efficient endogenous antibiotic of the host and can play a role in host defense against pathogenic bacteria. It can be used as a therapeutic agent in adjunct with current therapy to treat bacteria resistant to multiple antibiotics.     

Implications of oxidative stress in the cytotoxicity of Pseudomonas aeruginosa ExoU

ExoU PLA2-like activity has been shown to account for membrane lysis and acute death of infected cells. Translocation of effector proteins by the type III secretion systems depends on close contact between microbial and host cells. Our finding that both the ExoU-producing PA103 Pseudomonas aeruginosa and its mutant obtained by deletion of exoU adhered poorly to endothelial cells (EC) led to the hypothesis that, in some cells, the amount of injected toxin may not be enough to induce cell lysis but cells would suffer from a long-term effect of ExoU intoxication. To address this question, cells were exposed to both bacteria for 1 h and then treated with gentamicin-containing medium, to eliminate infecting microorganisms. After 24 h, the percentage of viable EC in PA103-infected cultures was significantly lower than in cultures exposed to the mutant, as determined by the MTT assay. Cell death was not likely to depend on the ExoU lytic activity since cell labeling with propidium iodide was similar in cultures infected with both bacterial strains. Bacterial cytotoxicity was significantly reduced by MAFP, a specific inhibitor of cPLA2 and iPLA2. Since the PLA2 activity on membrane phospholipids generates free fatty acid, including arachidonic acid (AA), we next compared the bacterial ability to release AA from infected EC. PA103 was shown to induce a potent AA release that was inhibited by MAFP. AA oxidation by oxygenases generates eicosanoids, known to induce both cell death and proliferation. However neither inhibitors of cyclooxygenases (ibuprofen) nor lipoxygenases (NDGA) reduced the ExoU toxicity. Since non-enzymatic oxidation of AA generates reactive radicals, we next investigated the PA103 ability to induce oxidative stress in infected cells. FACS analysis of cell labeling with the C-11 fluor probe and with anti-4-hydroxynonel antibody revealed a significant peroxidation of cell membrane lipids. These results, together with our finding that PA103-infected EC death was significantly attenuated by alpha-tocopherol, led to the conclusion that AA-induced oxidative stress may be another mechanism of cell damage in the course of infection by ExoU-producing P. aeruginosa.     

Surfactant protein A suppresses lipopolysaccharide-induced IL-10 production by murine macrophages

Upon LPS exposure, mononuclear phagocytes produce TNF-alpha and IL-10, two cytokines with pro- and anti-inflammatory activities, respectively. We previously described that murine resident alveolar macrophages, which play a central role in the immunosurveillance of the lung alveoli, do not synthesize IL-10 in vivo or in vitro when exposed to LPS. In the present report we demonstrate that during lung inflammation induced by the intranasal administration of LPS, bronchoalveolar cells collected between days 3 and 5 are able to synthesize IL-10 when exposed to LPS. We also show that depletion of resident alveolar macrophages by an intratracheal instillation of liposome-encapsulated clodronate is followed by subsequent replenishment of the airspaces by mononuclear phagocytes. This is accompanied by the transient competence of cells for IL-10 production. The cell capacity to produce IL-10 is evident up to 3 days and then decreases. This led us to hypothesize that the alveolar environment contains a down-regulator of LPS-induced IL-10 synthesis by recently emigrating mononuclear phagocytes. We show that the surfactant protein A, an airspace protein that has known immunomodulatory activities, dramatically inhibits LPS-induced IL-10 formation by bone marrow-derived macrophages. These data show a difference between resident and inflammatory macrophages with respect to IL-10 synthesis. Moreover, this study highlights for the first time the inhibitory role of surfactant protein A in the anti-inflammatory activity of macrophages through inhibition of IL-10 production.     

1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine is the precursor of platelet-activating factor in stimulated rabbit platelets. Evidence for an alkylacetyl-glycerophosphorylcholine cycle

The metabolism of [3H]PAF-acether ([1',2'-3H]alkyl-2-acetyl-sn-glycero-3-phosphorylcholine ([3H]alkylacetyl-GPC)) by rabbit platelets was investigated using thin-layer chromatography and high-performance liquid chromatography followed by radioactivity detection. After 2 h of incubation at 37 degrees C, 90 +/- 5.3% of [3H]PAF-acether taken up by the platelets were converted into a product identified as sn-2 long-chain acyl analogue ([3H]alkylacyl-GPC) which was incorporated in the membranes. This conversion was independent from extracellular calcium and was completely inhibited by platelet pre-exposure to 2 mM phenylmethylsulfonyl fluoride, a serine hydrolase inhibitor, which failed to inhibit the uptake of [3H]PAF-acether by the cells. The 2-deacetylated derivative, lyso-[3H]PAF-acether was found to be an intermediate of the conversion of [3H]PAF-acether into [3H]alkylacyl-GPC in platelet homogenates. Platelet stimulation with 2.5 U/ml of thrombin induced a reduction (16.5 +/- 2.2%) of its content of [3H]alkylacyl-GPC, accompanied by the release of [3H]PAF-acether and lyso-[3H]PAF-acether to the medium. These effects were suppressed by the phospholipase A2 inhibitor, p-bromophenacyl bromide. Our results demonstrate that intact platelets convert exogenous PAF-acether into alkylacyl-GPC, which can serve as the precursor of PAF-acether released during stimulation. The existence of a metabolic cycle for the uptake, the release and the inactivation of PAF-acether by platelets is suggested.     

Impact of CFTR DeltaF508 mutation on prostaglandin E2 production and type IIA phospholipase A2 expression by pulmonary epithelial cells

Cystic fibrosis (CF) is characterized by an exacerbated inflammatory pulmonary response with excessive production of inflammatory mediators. We investigated here the impact of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction on prostaglandin E2 (PGE2) production and type IIA secreted phospholipase A2 (sPLA2-IIA) expression. We show that both resting and LPS-stimulated human respiratory epithelial cell line bearing DeltaF508 mutation on CFTR (CF cells) released more PGE2 than control cell line. This was accompanied by enhanced expression and activity of cyclooxygenase-2 in CF cells. PGE2 release was attenuated after experimentally induced retrafficking of the DeltaF508-CFTR at the plasma membrane. sPLA2-IIA expression occurred at higher levels in CF cells than in control cells and was enhanced by LPS and PGE2. Suppression of PGE2 synthesis by aspirin led to an inhibition of LPS-induced sPLA2-IIA expression. Higher activation of NF-kappaB was observed in CF cells compared with control cells and was enhanced by LPS. However, addition of PGE2 or aspirin had no effect on NF-kappaB activation. LPS-induced sPLA2-IIA expression was reduced by an NF-kappaB inhibitor. We suggest that the lack of the CFTR in the plasma membrane results in a PGE2 overproduction and an enhanced sPLA2-IIA expression. This expression is upregulated by NF-kappaB and amplified by PGE2 via a unidentified signaling pathway.