Inflammatory and oxidative stress responses of an alveolar epithelial cell line to airborne zinc oxide nanoparticles at the air-liquid interface: a comparison with conventional, submerged cell-culture conditions.

The biological effects of inhalable nanoparticles have been widely studied in vitro with pulmonary cells cultured under submerged and air-liquid interface (ALI) conditions. Submerged exposures are experimentally simpler, but ALI exposures are physiologically more realistic and hence potentially biologically more meaningful. In this study, we investigated the cellular response of human alveolar epithelial-like cells (A549) to airborne agglomerates of zinc oxide (ZnO) nanoparticles at the ALI, compared it to the response under submerged culture conditions, and provided a quantitative comparison with the literature data on different types of particles and cells. For ZnO nanoparticle doses of 0.7 and 2.5 µ g ZnO/cm(2) (or 0.09 and 0.33 cm(2) ZnO/cm(2)), cell viability was not mitigated and no significant effects on the transcript levels of oxidative stress markers (HMOX1, SOD-2 and GCS) were observed. However, the transcript levels of proinflammatory markers (IL-8, IL-6, and GM-CSF) were induced to higher levels under ALI conditions. This is consistent with the literature data and it suggests that in vitro toxicity screening of nanoparticles with ALI cell culture systems may produce less false negative results than screening with submerged cell cultures. However, the database is currently too scarce to draw a definite conclusion on this issue.

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.

Direct crosstalk between mast cell-TNF and TNFR1-expressing endothelia mediates local tissue inflammation.

Signaling through tumor necrosis factor receptor 1 (TNFR1) controls bacterial infections and the induction of inflammatory Th1 cell-mediated autoimmune diseases. By dissecting Th1 cell-mediated delayed-type hypersensitivity responses (DTHRs) into single steps, we localized a central defect to the missing TNFR1 expression by endothelial cells (ECs). Adoptive transfer and mast cell knockin experiments into Kit(W)/Kit(W-v), TNF(-/-), and TNFR1(-/-) mice showed that the signaling defect exclusively affects mast cell-EC interactions but not T cells or antigen-presenting cells. As a consequence, TNFR1(-/-) mice had strongly reduced mRNA and protein expression of P-selectin, E-selectin, ICAM-1, and VCAM-1 during DTHR elicitation. In consequence, intravital fluorescence microscopy revealed up to 80% reduction of leukocyte rolling and firm adhesion in TNFR1(-/-) mice. As substitution of TNF(-/-) mice with TNF-producing mast cells fully restored DTHR in these mice, signaling of mast cell-derived TNF through TNFR1-expressing ECs is essential for the recruitment of leukocytes into sites of inflammation.

SWCNT suppress inflammatory mediator responses in human lung epithelium in vitro.

Single-walled carbon nanotubes have gained enormous popularity due to a variety of potential applications which will ultimately lead to increased human and environmental exposure to these nanoparticles. This study was carried out in order to evaluate the inflammatory response of immortalised and primary human lung epithelial cells (A549 and NHBE) to single-walled carbon nanotube samples (SWCNT). Special focus was placed on the mediating role of lung surfactant on particle toxicity. The toxicity of SWCNT dispersed in cell culture medium was compared to that of nanotubes dispersed in dipalmitoylphosphatidylcholine (DPPC, the main component of lung lining fluid). Exposure was carried out for 6 to 48 h with the latter time-point showing the most significant responses. Moreover, exposure was performed in the presence of the pro-inflammatory stimulus tumour necrosis factor-alpha (TNF-alpha) in order to mimic exposure of stimulated cells, as would occur during infection. Endpoints evaluated included cell viability, proliferation and the analysis of inflammatory mediators such as interleukin (IL)-8, IL-6, TNF-alpha and macrophage chemoattractant protein-1 (MCP-1). Crocidolite asbestos was included as a well characterised, toxic fibre control. The results of this study showed that HiPco SWCNT samples suppress inflammatory responses of A549 and NHBE cells. This was also true for TNF-alpha stimulated cells. The use of DPPC improved the degree of SWCNT dispersion in A549 medium and in turn, leads to increased particle toxicity, however, it was not shown to modify NHBE cell responses.

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.