Inhalation treatment with glutathione in patients with cystic fibrosis. A randomized clinical trial.

RATIONALE: Glutathione is the major antioxidant in the extracellular lining fluid of the lungs and depleted in patients with cystic fibrosis (CF). OBJECTIVES: We aimed to assess glutathione delivered by inhalation as a potential treatment for CF lung disease. METHODS: This randomized, double-blind, placebo-controlled trial evaluated inhaled glutathione in subjects with CF 8 years of age and older and FEV1 of 40-90% of predicted. Subjects were randomized to receive 646 mg glutathione in 4 ml (n = 73) or placebo (n = 80) via an investigational eFlow nebulizer every 12 hours for 6 months. MEASUREMENTS AND MAIN RESULTS: FEV1 (absolute values), both as pre-post differences (P = 0.180) and as area under the curves (P = 0.205), were the primary efficacy endpoints, and were not different between the glutathione group and the placebo group over the 6-month treatment period. Exploratory analysis showed an increase of FEV1 from baseline over placebo of 100 ml or 2.2% predicted; this was significant at 3 months, but not later. Subjects receiving glutathione had neither fewer pulmonary exacerbations, nor better scores for quality of life. Whereas increased glutathione and metabolites in sputum demonstrated significant delivery to the lungs, there was no indication of diminished oxidative stress to proteins or lipids, and no evidence for anti-inflammatory or antiproteolytic actions of glutathione supplemented to the airways. The adverse event incidence was similar between glutathione and placebo. CONCLUSIONS: Inhaled glutathione in the dose administered did not demonstrate clinically relevant improvements in lung function, pulmonary exacerbation frequency, or patient-reported outcomes. Glutathione delivery to the airways was not associated with changes in markers of oxidation, proteolysis, or inflammation. Clinical trial registered with www.clinicaltrials.gov (NCT00506688) and https://eudract.ema.europa.eu/index.html (EudraCT 2005-003870-88).

Ultrafine particles affect the balance of endogenous pro- and anti-inflammatory lipid mediators in the lung: in-vitro and in-vivo studies.

BACKGROUND: Exposure to ultrafine particles exerts diverse harmful effects including aggravation of pulmonary diseases like asthma. Recently we demonstrated in a mouse model for allergic airway inflammation that particle-derived oxidative stress plays a crucial role during augmentation of allergen-induced lung inflammation by ultrafine carbon particle (UfCP) inhalation. The mechanisms how particle inhalation might change the inflammatory balance in the lungs, leading to accelerated inflammatory reactions, remain unclear. Lipid mediators, known to be immediately generated in response to tissue injury, might be strong candidates for priming this particle-triggered change of the inflammatory balance. METHODS: We hypothesize that inhalation of UfCP may disturb the balance of pro- and anti-inflammatory lipid mediators in: i) a model for acute allergic pulmonary inflammation, exposing mice for 24 h before allergen challenge to UfCP inhalation (51.7 nm, 507 µg/m3), and ii) an in-vitro model with primary rat alveolar macrophages (AM) incubated with UfCP (10 µg/1 x 106 cells/ml) for 1 h. Lungs and AM were analysed for pro- and anti-inflammatory lipid mediators, namely leukotriene B4 (LTB4), prostaglandin E2 (PGE2), 15(S)-hydroxy-eicosatetraenoic acid (15(S)-HETE), lipoxin A4 (LXA4) and oxidative stress marker 8-isoprostane by enzyme immunoassays and immunohistochemistry. RESULTS: In non-sensitized mice UfCP exposure induced a light non-significant increase of all lipid mediators. Similarly but significantly in rat AM all lipid mediators were induced already within 1 h of UfCP stimulation. Also sensitized and challenge mice exposed to filtered air showed a partially significant increase in all lipid mediators. In sensitized and challenged mice UfCP exposure induced highest significant levels of all lipid mediators in the lungs together with the peak of allergic airway inflammation on day 7 after UfCP inhalation. The levels of LTB4, 8-isoprostane and PGE2 were significantly increased also one day after UfCP exposure. Immunohistochemistry localized highest concentrations of PGE2 especially in AM one day after UfCP exposure. CONCLUSION: Our results suggest that UfCP exposure affects the balance between pro- and anti-inflammatory lipid mediators. In allergic mice, where the endogenous balance of pro- and anti-inflammatory mediators is already altered, UfCP exposure aggravates the inflammation and the increase in anti-inflammatory, pro-resolving lipid mediators is insufficient to counterbalance the extensive inflammatory response. This may be a contributing mechanism that explains the increased susceptibility of asthmatic patients towards particle exposure.

Soluble iron modulates iron oxide particle-induced inflammatory responses via prostaglandin E(2 )synthesis: In vitro and in vivo studies.

BACKGROUND: Ambient particulate matter (PM)-associated metals have been shown to play an important role in cardiopulmonary health outcomes. To study the modulation of PM-induced inflammation by leached off metals, we investigated intracellular solubility of radio-labeled iron oxide ((59)Fe(2)O(3)) particles of 0.5 and 1.5 mum geometric mean diameter. Fe(2)O(3 )particles were examined for the induction of the release of interleukin 6 (IL-6) as pro-inflammatory and prostaglandin E(2 )(PGE(2)) as anti-inflammatory markers in cultured alveolar macrophages (AM) from Wistar Kyoto (WKY) rats. In addition, we exposed male WKY rats to monodispersed Fe(2)O(3 )particles by intratracheal instillation (1.3 or 4.0 mg/kg body weight) to examine in vivo inflammation. RESULTS: Particles of both sizes are insoluble extracellularly in the media but moderately soluble in AM with an intracellular dissolution rate of 0.0037 +/- 0.0014 d(-1 )for 0.5 mum and 0.0016 +/- 0.0012 d(-1 )for 1.5 mum (59)Fe(2)O(3 )particles. AM exposed in vitro to 1.5 mum particles (10 mug/mL) for 24 h increased IL-6 release (1.8-fold; p < 0.05) and also PGE(2 )synthesis (1.9-fold; p < 0.01). By contrast, 0.5 mum particles did not enhance IL-6 release but strongly increased PGE(2 )synthesis (2.5-fold, p < 0.005). Inhibition of PGE(2 )synthesis by indomethacin caused a pro-inflammatory phenotype as noted by increased IL-6 release from AM exposed to 0.5 mum particles (up to 3-fold; p < 0.005). In the rat lungs, 1.5 but not 0.5 mum particles (4.0 mg/kg) induced neutrophil influx and increased vascular permeability. CONCLUSIONS: Fe(2)O(3 )particle-induced neutrophilic inflammatory response in vivo and pro-inflammatory cytokine release in vitro might be modulated by intracellular soluble iron via PGE(2 )synthesis. The suppressive effect of intracellular released soluble iron on particle-induced inflammation has implications on how ambient PM-associated but soluble metals influence pulmonary toxicity of ambient PM.

Oxymetazoline inhibits and resolves inflammatory reactions in human neutrophils.

The nasal decongestant oxymetazoline (OMZ) exhibits anti-oxidative and anti-inflammatory properties (I. Beck-Speier et al., J Pharmacol Exp Ther. 2006;316:842-851). In a follow up study, we hypothesized that OMZ generates pro-resolving lipoxins being paralleled by production of immune-modulating prostaglandin E(2) (PGE(2)) and anti-inflammatory 15(S)-hydroxy-eicosatetraenoic acid [15(S)-HETE] and depletion of pro-inflammatory leukotriene B(4) (LTB(4)). Human neutrophils (PMN) were chosen as the cellular system. The effect of OMZ on these parameters as well as on respiratory burst activity and oxidative stress marker 8-isprostane was analyzed in unstimulated and co-stimulated PMN by ultrafine carbon particles (UCP) or opsonized zymosan (OZ), respectively. In unstimulated cells, OMZ induced formation of PGE(2), 15(S)-HETE, and LXA(4). The levels of LTB(4) and 8-isoprostane were not affected, whereas respiratory burst activity was drastically inhibited. In UCP- and OZ-stimulated control cells, all parameters were elevated. Here, OMZ maintained the increased levels of PGE(2), 15(S)-HETE, and LXA(4), but substantially suppressed levels of LTB(4) and 8-isoprostane and inhibited the respiratory burst activity. These findings suggest a switch from the pro-inflammatory eicosanoid class LTB(4) to the pro-resolving LXA(4). Since LXA(4) is most relevant in returning inflamed tissue to homeostasis, OMZ is postulated to terminate rhinitis-related inflammation, thus contributing to shortening of disease duration.

Role of oxidative stress in ultrafine particle-induced exacerbation of allergic lung inflammation.

RATIONALE: The effects of ultrafine particle inhalation on allergic airway inflammation are of growing interest. The mechanisms underlying these effects are currently under investigation. OBJECTIVES: To investigate the role of oxidative stress on the adjuvant activity of inhaled elemental carbon ultrafine particles (EC-UFPs) on allergic airway inflammation. METHODS: Ovalbumin-sensitized mice were exposed to EC-UFPs (504 microg/m(3) for 24 h) or filtered air immediately before allergen challenge and systemically treated with N-acetylcysteine or vehicle before and during EC-UFP inhalation. Allergic inflammation was measured up to 1 week after allergen challenge by means of bronchoalveolar lavage, cytokine/total protein assays, lung function, and histology. Isoprostane levels in lung tissue served to measure oxidative stress. Transmission electron microscopy served to localize EC-UFPs in lung tissue and both electrophoretic mobility shift assay and immunohistochemistry to quantify/localize nuclear factor-kappaB (NF-kappaB) activation. MEASUREMENTS AND MAIN RESULTS: In sensitized and challenged mice EC-UFP inhalation increased allergen-induced lung lipid peroxidation and NF-kappaB activation in addition to inflammatory infiltrate, cytokine release, and airway hyperresponsiveness. Prominent NF-kappaB activation was observed in the same cell types in which EC-UFPs were detected. N-acetylcysteine treatment significantly reduced the adjuvant activity of EC-UFPs. In nonsensitized or sensitized but not challenged mice EC-UFP exposure induced a moderate increase in isoprostanes but no significant effect on other parameters of lung inflammation. CONCLUSIONS: Our findings demonstrate a critical role for oxidative stress in EC-UFP-induced augmentation of allergen-induced lung inflammation, where EC-UFP exposure has potentiating effects in lung allergic inflammation. Our data support the concept that allergic individuals are more susceptible to the adverse health effects of EC-UFPs.

Health effects of ambient particulate matter--biological mechanisms and inflammatory responses to in vitro and in vivo particle exposures.

In this article, we review and analyze different modes of exposure to ultrafine particles in order to assess particle-induced inflammatory responses and the underlying mechanisms in vitro and in vivo. Based on results from monocytic cells cultured under submerged conditions, we discuss (1) the impact of particle properties such as surface area and oxidative potential on lipid metabolism as a highly sensitive regulatory pathway and (2) the interference of diesel exhaust particles with toll-like receptor-mediated inflammatory responses. Furthermore, new developments of air-liquid interface exposure used as an alternative approach to simulate cell particle interactions are presented. In addition to the in vitro approaches, animal exposure studies are described that apply selected mouse models to elucidate potential allergic and inflammatory pulmonary responses and mast-cell-related mechanisms after particle exposure. Long-term inhalation of ultrafine particles might lead to irreversible changes in lung structure and function. Clinical studies addressing the characteristics of inflammatory airway cells are a promising approach to understand underlying pathophysiological mechanisms in chronic obstructive pulmonary disease. Finally, a potential outcome of human particle exposure is chronic cough in children. Here, discrimination between asthmatic and nonasthmatic cough by means of immunological parameters appears to be an important step toward improving diagnosis and therapy.

Oxymetazoline inhibits proinflammatory reactions: effect on arachidonic acid-derived metabolites.

The nasal decongestant oxymetazoline effectively reduces rhinitis symptoms. We hypothesized that oxymetazoline affects arachidonic acid-derived metabolites concerning inflammatory and oxidative stress-dependent reactions. The ability of oxymetazoline to model pro- and anti-inflammatory and oxidative stress responses was evaluated in cell-free systems, including 5-lipoxygenase (5-LO) as proinflammatory, 15-lipoxygenase (15-LO) as anti-inflammatory enzymes, and oxidation of methionine by agglomerates of ultrafine carbon particles (UCPs), indicating oxidative stress. In a cellular approach using canine alveolar macrophages (AMs), the impact of oxymetazoline on phospholipase A(2) (PLA(2)) activity, respiratory burst and synthesis of prostaglandin E(2) (PGE(2)), 15(S)-hydroxy-eicosatetraenoic acid (15-HETE), leukotriene B(4) (LTB(4)), and 8-isoprostane was measured in the absence and presence of UCP or opsonized zymosan as particulate stimulants. In cell-free systems, oxymetazoline (0.4-1 mM) inhibited 5-LO but not 15-LO activity and did not alter UCP-induced oxidation of methionine. In AMs, oxymetazoline induced PLA(2) activity and 15-HETE at 1 mM, enhanced PGE(2) at 0.1 mM, strongly inhibited LTB(4) and respiratory burst at 0.4/0.1 mM (p < 0.05), but did not affect 8-isoprostane formation. In contrast, oxymetazoline did not alter UCP-induced PLA(2) activity and PGE(2) and 15-HETE formation in AMs but inhibited UCP-induced LTB(4) formation and respiratory burst at 0.1 mM and 8-isoprostane formation at 0.001 mM (p < 0.05). In opsonized zymosan-stimulated AMs, oxymetazoline inhibited LTB(4) formation and respiratory burst at 0.1 mM (p < 0.05). In conclusion, in canine AMs, oxymetazoline suppressed proinflammatory reactions including 5-LO activity, LTB(4) formation, and respiratory burst and prevented particle-induced oxidative stress, whereas PLA(2) activity and synthesis of immune-modulating PGE(2) and 15-HETE were not affected.

Oxidative stress and lipid mediators induced in alveolar macrophages by ultrafine particles.

In ambient aerosols, ultrafine particles (UFP) and their agglomerates are considered to be major factors contributing to adverse health effects. Reactivity of agglomerated UFP of elemental carbon (EC), Printex 90, Printex G, and diesel exhaust particles (DEP) was evaluated by the capacity of particles to oxidize methionine in a cell-free in vitro system for determination of their innate oxidative potential and by alveolar macrophages (AMs) to determine production of arachidonic acid (AA), including formation of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), reactive oxygen species (ROS), and oxidative stress marker 8-isoprostane. EC exhibiting high oxidative potential induced generation of AA, PGE2, LTB4, and 8-isoprostane in canine and human AMs. Printex 90, Printex G, and DEP, showing low oxidative capacity, still induced formation of AA and PGE2, but not that of LTB4 or 8-isoprostane. Aging of EC lowered oxidative potential while still inducing production of AA and PGE2 but not that of LTB4 and 8-isoprostane. Cellular ROS production was stimulated by all particles independent of oxidative potential. Particle-induced formation of AA metabolites and ROS was dependent on mitogen-activated protein kinase kinase 1 activation of cytosolic phospholipase A2 (cPLA2) as shown by inhibitor studies. In conclusion, cPLA2, PGE2, and ROS formation was activated by all particle types, whereas LTB4 production and 8-isoprostane were strongly dependent on particles' oxidative potential. Physical and chemical parameters of particle surface correlated with oxidative potential and stimulation of AM PGE2 and 8-isoprostane production.

Diesel exhaust particles increase LPS-stimulated COX-2 expression and PGE2 production in human monocytes.

Little is known about health effects of ultrafine particles (UFP) found in ambient air, but much of their action may be on cells of the lung, including cells of the monocyte/macrophage lineage. We have analyzed the effects of diesel exhaust particles (DEP; SRM1650a) on human monocytes in vitro. DEP, on their own, had little effect on cyclooxygenase (COX)-2 gene expression in the Mono Mac 6 cell line. However, when cells were preincubated with DEP for 1 h, then stimulation with the Toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) induced an up-to fourfold-higher production of COX-2 mRNA with an average twofold increase. This costimulatory effect of DEP led to enhanced production of COX-2 protein and to increased release of prostaglandin E(2) (PGE(2)). The effect was specific in that tumor necrosis factor gene expression was not enhanced by DEP costimulation. Furthermore, costimulation with the TLR2 ligand Pam3Cys also led to enhanced COX-2 mRNA. DEP and LPS showed similar effects on COX-2 mRNA in primary blood mononuclear cells, in highly purified CD14-positive monocytes, and in monocyte-derived macrophages. Our data suggest that UFP such as DEP may exert anti-inflammatory effects mediated by enhanced PGE(2) production.

Sulfur-related air pollutants induce the generation of platelet-activating factor, 5-lipoxygenase- and cyclooxygenase-products in canine alveolar macrophages via activation of phospholipases A2.

Recent studies have shown that long-term in vivo exposure of dogs to neutral sulfur(IV)/sulfite aerosols induces mild inflammatory reactions, whereas the combination of neutral sulfite with acidic sulfur(VI)/sulfate aerosols evokes less pronounced effects. To understand underlying mechanisms, we studied in vitro the role of lipid mediators in the responses of alveolar macrophages (AMs) to sulfur-related compounds under neutral (pH 7) or moderate acidic (pH 6) conditions. Canine AMs incubated with sulfite at pH 7 released threefold higher amounts of platelet-activating factor than control (P < 0.005). Generation of arachidonic acid, leukotriene B4, 5-hydroxy-eicosatetraenoic acid, prostaglandin E2, thromboxane B2 and 12-hydroxyheptadecatrienoic acid increased twofold (P < 0.0005). However, these metabolites remained unchanged following incubation of AMs with sulfite at pH 6 or with sulfate at pH 7 or pH 6. Mediator release by sulfite-treated AMs at pH 7 stimulated respiratory burst activity of neutrophils. Inhibition of MAPK pathway by PD 98059, of cytosolic (cPLA2) and secretory phospholipases A2 by AACOCF3 and thioetheramide-PC, respectively, reduced sulfite-induced eicosanoid formation in AMs. Sulfite activated cPLA2 activity twofold at pH 7. This mechanism of sulfite-stimulated responses in phospholipid metabolism predicts that chronic exposure to sulfur(IV)/sulfite is associated with a considerable health risk.