Jihee Lee Kang

Anti-inflammatory mechanism of ginseng saponins in activated microglia

In the present study, we investigated the effect of ginseng extract (KRG) and total saponins (GTS) on microglial activation. KRG and GTS inhibited LPS-induced expression of iNOS, MMP-9 and proinflammatory cytokines in microglial cells. Suppression of microglial activation by ginseng was also observed in the mouse brain inflamed by LPS. Furthermore, KRG and GTS significantly suppressed NF-kappaB and MAP kinase activities, which are upstream signaling molecules in inflammation. Among the individual ginsenosides tested, Rh2, Rh3 and compound K significantly inhibited LPS-induced iNOS and cytokine expressions. Therefore, the inhibition of microglial activation by ginseng saponins may a good potential therapeutic modality for neurodegenerative diseases.

IL-17 and IL-22 enhance skin inflammation by stimulating the secretion of IL-1β by keratinocytes via the ROS-NLRP3-caspase-1 pathway

BACKGROUND The pathogenesis of inflammatory skin disease involves the release of cytokines from keratinocytes, and one of these, IL-1β, has been previously implicated in inflammatory skin disease. T(h)17 cells, a subset of T(h) cells involved in autoimmunity and inflammation, possess IL-1β receptors and secrete cytokines such as IL-17 and IL-22 in response to IL-1β stimulation. A mutation in the inflammasome protein NLRP3 (NACHT, LRR and PYD domains-containing protein 3) causes excess production of IL-1β, resulting in an augmentation of T(h)17-dominant pathology. METHODS To determine the feedback effect, if any, of IL-17 and/or IL-22 on the secretion of IL-1β from keratinocytes, we stimulated the human keratinocyte cell line HaCaT, as well as caspase-1-deficient mice, with IL-17 or IL-22. RESULTS We found that treatment with IL-17 and IL-22 causes an increase in IL-1β via the activation of NLRP3 by a process that involves the generation of reactive oxygen species. Moreover, skin inflammation induced by IL-17 and IL-22 was lower in caspase-1 knockout (KO) mice relative to that induced by IL-1β treatment. Additionally, skin inflammation induced by the drug imiquimod was lower in caspase-1 KO mice than in wild-type mice. CONCLUSION These results indicate that cytokines from T(h)17 cells may potentiate IL-1β-mediated skin inflammation and result in phenotypic alterations of keratinocytes via a feedback mechanism.

Comparison of the Biological Activity Between Ultrafine and Fine Titanium Dioxide Particles in RAW 264.7 Cells Associated with Oxidative Stress

Ultrafine or fine titanium dioxide (TiO2) particles are widely used in the production of white pigments, for sunscreens, and in cleanup techniques. However, currently knowledge is deficient concerning cellular responses to these particles. The study evaluated and compared the biological activity of ultrafine and fine TiO2 particles in RAW 264.7 macrophages according to an oxidative stress paradigm. In vitro exposure of macrophages to ultrafine or fine TiO2 in the range of 0.5–200 μg/ml did not significantly alter cell viability. However, ultrafine TiO2 enhanced intracellular generation of reactive oxygen species (ROS) to a greater extent than fine TiO2 at each exposure concentration. Ultrafine TiO2 induced ERK1/2 activation in a concentration-dependent manner, while the fine TiO2-induced changes were minimal. Phosphorylation of ERK1/2 occurred following 10 min exposure to higher concentrations of ultrafine TiO2 (≥25 μg/ml). Similarly, ultrafine TiO2 exposure significantly enhanced tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2 secretion in a concentration-dependent manner, and its potency was higher than fine TiO2. These findings suggest that when exposure concentration is based upon equivalent mass, ultrafine TiO2 exerts greater biological activity as measured by ROS generation, ERK 1/2 activation, and proinflammatory mediator secretion in RAW 264.7 macrophages than fine TiO2.

SILICA-INDUCED NUCLEAR FACTOR- k B ACTIVATION: INVOLVEMENT OF REACTIVE OXYGEN SPECIES AND PROTEIN TYROSINE KINASE ACTIVATION

Nuclear factor-kappaB (NF-kappaB) is a multiprotein complex that may regulate a variety of inflammatory cytokines involved in the initiation and progression of silicosis. The present study documents the ability of in vitro silica exposure to induce DNA-binding activity of NF-kappaB in a mouse peritoneal macrophage cell line (RAW264.7 cells) and investigates the role of reactive oxygen species (ROS) and/or protein tyrosine kinase in this activation. In vitro exposure of mouse macrophages to silica (100 microg/ml) resulted in a twofold increase in ROS production, measured as the generation of chemiluminescence (CL), and caused activation of NF-kappaB. Silica-induced CL was inhibited 100% by superoxide dismutase (SOD) and 75% by catalase, while NF-kappaB activation was inhibited by a variety of antioxidants (catalase, superoxide dismutase, alpha-tocopherol, pyrrolidine dithiocarbamate, or N-acetylcysteine). Further evidence for the involvement of ROS in NF-kappaB activation is that 1 mM H2O2 enhanced NF-kappaB/DNA binding and that this activation was inhibited by catalase. Specific inhibitors of protein tyrosine kinase, such as herbimycin A, genistein, and AG-494, prevented NF-kappaB activation in silica-treated cells. Genistein and AG-494 also reduced NF-kappaB activation in H2O2-treated cells. Results confirm that tyrosine phosphorylation of several cellular proteins (approximate molecular mass of 39, 58-70, and 103 kD) was increased in silica-exposed macrophages and that genistein inhibited this silica-induced phosphorylation. In contrast, inhibitors of protein kinase A or C, such as H89, staurosporin, calphostin C, and H7, had no marked inhibitory effect on silica-induced NF-kappaB activation. The results suggest that ROS may play a role in silica-induced NF-kappaB activation in macrophages and that phosphorylation events mediated by tyrosine kinase may be involved in this activation.Nuclear factor-kB (NF-kB) is a multiprotein complex that may regulate a variety of inflammatory cytokines involved in the initiation and progression of silicosis. The present study documents the ability of in vitro silica exposure to induce DNA-binding activity of NF-kB in a mouse peritoneal macrophage cell line (RAW264.7 cells) and investigates the role of reactive oxygen species (ROS) and/or protein tyrosine kinase in this activation. In vitro exposure of mouse macrophages to silica (100 µg/ml) resulted in a twofold increase in ROS production, measured as the generation of chemiluminescence (CL), and caused activation of NF-kB. Silica-induced CL was inhibited 100% by superoxide dismutase (SOD) and 75% by catalase, while NF-kB activation was inhibited by a variety of antioxidants (catalase, superoxide dismutase, alpha-tocopherol, pyrrolidine dithiocarbamate, or N-acetylcysteine). Further evidence for the involvement of ROS in NF-kB activation is that 1 mM H2O2 enhanced NF-kB/DNA binding and that this activation was inhibited by catalase. Specific inhibitors of protein tyrosine kinase, such as herbimycin A, genistein, and AG-494, prevented NF-kB activation in silica-treated cells. Genistein and AG-494 also reduced NF-kB activation in H2O2-treated cells. Results con firm that tyrosine phosphorylation of several cellular proteins (approximate molecular mass of 39, 58?70, and 103 kD) was increased in silica-exposed macrophages and that genistein inhibited this silica-induced phosphorylation. In contrast, inhibitors of protein kinase A or C, such as H89, staurosporin, calphostin C, and H7, had no marked inhibitory effect on silica-induced NF-kB activation. The results suggest that ROS may play a role in silica-induced NF-kB activation in macrophages and that phosphorylation events mediated by tyrosine kinase may be involved in this activation.

Synthetic RGDS peptide attenuates lipopolysaccharide-induced pulmonary inflammation by inhibiting integrin signaled MAP kinase pathways.

BackgroundSynthetic peptides containing the RGD sequence inhibit integrin-related functions in different cell systems. Here, we investigated the effects of synthetic Arg-Gly-Asp-Ser (RGDS) peptide on key inflammatory responses to intratracheal (i.t.) lipopolysaccharide (LPS) treatment and on the integrin signaled mitogen-activated protein (MAP) kinase pathway during the development of acute lung injury.MethodsSaline or LPS (1.5 mg/kg) was administered i.t. with or without a single dose of RGDS (1, 2.5, or 5 mg/kg, i.p.), anti-αv or anti-β3 mAb (5 mg/kg, i.p.). Mice were sacrificed 4 or 24 h post-LPS.ResultsA pretreatment with RGDS inhibited LPS-induced increases in neutrophil and macrophage numbers, total protein levels and TNF-α and MIP-2 levels, and matrix metalloproteinase-9 activity in bronchoalveolar lavage (BAL) fluid at 4 or 24 h post-LPS treatment. RGDS inhibited LPS-induced phosphorylation of focal adhesion kinase and MAP kinases, including ERK, JNK, and p38 MAP kinase, in lung tissue. Importantly, the inhibition of the inflammatory responses and the kinase pathways were still evident when this peptide was administered 2 h after LPS treatment. Similarly, a blocking antibody against integrin αv significantly inhibited LPS-induced inflammatory cell migration into the lung, protein accumulation and proinflammatory mediator production in BAL fluid, at 4 or 24 h post-LPS. Anti-β3 also inhibited all LPS-induced inflammatory responses, except the accumulation of BAL protein at 24 h post-LPS.ConclusionThese results suggest that RGDS with high specificity for αvintegrins attenuates inflammatory cascade during LPS-induced development of acute lung injury.

Nitric oxide up-regulates DNA-binding activity of nuclear factor-κB in macrophages stimulated with silica and inflammatory stimulants

Nitric oxide (NO), a reactive nitrogen species, plays an important role in inflammatory lung damage. In the present study, we investigated the role of NO in DNA-binding activity of NF-κB in macrophages stimulated with silica or other inflammatory stimulants. Treatment of mouse macrophages (RAW264.7 cells) with a selective inhibitor of inducible nitric oxide synthase (iNOS), L-N6-(1-iminoethyl) lysine (L-NIL), or a nonselective iNOS inhibitor, Nω-nitro-L-arginine methylester (L-NAME), resulted in inhibition of silica-induced nitric oxide production as well as silica-induced NF-κB activation. L-NIL also effectively inhibited NF-κB activation induced by other inflammatory stimulants, such as lipopolysaccharide (LPS) or muramyl dipeptide (MDP). These inhibitory effects of L-NIL and L-NAME on silica- or LPS-induced NF-κB activation were also observed in primary rat alveolar macrophages. Furthermore, NO generating compounds, such as sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), caused a dose-dependent increase in NF-κB activation, which was positively correlated with the level of NO production. Specific inhibitors of protein tyrosine kinase, such as genistein and AG494, prevented NF-κB activation in SNP- or SIN-1 treated cells, suggesting involvement of tyrosine kinase in the NO signaling pathway leading to NF-κB activation. In contrast, inhibitors of protein kinase C or A, such as staurosporine or H89, had no inhibitory effect on SIN-1 induced NF-κB activation. Metalloporphyrins, such as tetrakis (N-methyl-4′-pyridyl) porphyrinato iron (III) (Fe-TMPyP) and Zn-TMPyP which are known to alter NO-dependent activity, markedly inhibited silica- and LPS-induced NF-κB activation. The results suggest that NF-κB activation in macrophages can be induced under certain conditions by nitric oxide and that nitric oxide produced by phagocytes exposed to inflammatory agents may up-regulate the activation of NF-κB.

Inhibiting Mer receptor tyrosine kinase suppresses STAT1, SOCS1/3, and NF-κB activation and enhances inflammatory responses in lipopolysaccharide-induced acute lung injury

Mer signaling participates in a novel inhibitory pathway in TLR activation. The purpose of the present study was to examine the role of Mer signaling in the down‐regulation of TLR4 activation‐driven immune responses in mice, i.t.‐treated with LPS, using the specific Mer‐blocking antibody. At 4 h and 24 h after LPS treatment, expression of Mer protein in alveolar macrophages and lung tissue decreased, sMer in BALF increased significantly, and Mer activation increased. Pretreatment with anti‐Mer antibody did not influence the protein levels of Mer and sMer levels. Anti‐Mer antibody significantly reduced LPS‐induced Mer activation, phosphorylation of Akt and FAK, STAT1 activation, and expression of SOCS1 and ‐3. Anti‐Mer antibody enhanced LPS‐induced inflammatory responses, including activation of the NF‐κB pathway; the production of TNF‐α, IL‐1β, and MIP‐2 and MMP‐9 activity; and accumulation of inflammatory cells and the total protein levels in BALF. These results indicate that Mer plays as an intrinsic feedback inhibitor of the TLR4‐ and inflammatory mediator‐driven immune responses during acute lung injury.

N-acetylcysteine inhibits RhoA and promotes apoptotic cell clearance during intense lung inflammation.

RATIONALE The resolution of pulmonary inflammation seen in various inflammatory lung conditions depends on the clearance of apoptotic cells to prevent permanent tissue damage or progressive disease. Uptake of apoptotic cells by alveolar macrophages is suppressed by oxidants through the activation of Rho signaling. OBJECTIVES We hypothesized that antioxidant exposure would increase the ability of alveolar macrophages to clear pulmonary apoptotic cells through the inhibition of RhoA. METHODS The effects of the antioxidant N-acetylcysteine (NAC) on the pulmonary immune response were seen in mice treated intratracheally with LPS, LPS + NAC, or saline. Apoptotic cell clearance, RhoA activity, and changes in the lung inflammatory responses were analyzed in vivo or ex vivo. MEASUREMENTS AND MAIN RESULTS Neutrophil accumulation, apoptosis, necrosis, and oxidant production peaked at 3 days post LPS treatment. NAC enhanced the clearance of apoptotic cells and inhibited RhoA activity in alveolar macrophages at 3 days post LPS treatment. NAC suppressed LPS-induced proinflammatory mediators, enhanced the production of transforming growth factor-beta1, reduced the accumulation of inflammatory cells, and reduced levels of protein and lactate dehydrogenase in bronchoalveolar lavage fluid. In the presence of ex vivo apoptotic cells, alveolar macrophages exposed to LPS or LPS + NAC had reduced tumor necrosis factor-alpha levels and increased transforming growth factor-beta1 levels. A Rho kinase inhibitor mimicked the effects of NAC on the clearance of apoptotic cells and the inflammatory responses. CONCLUSIONS These results indicate that NAC can expedite the resolution of LPS-induced pulmonary inflammation through the inhibition of RhoA activity and the enhancement of apoptotic cell clearance.

Inhibition of c-Jun NH2-terminal kinase or extracellular signal-regulated kinase improves lung injury

BackgroundAlthough in vitro studies have determined that the activation of mitogen-activated protein (MAP) kinases is crucial to the activation of transcription factors and regulation of the production of proinflammatory mediators, the roles of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in acute lung injury have not been elucidated.MethodsSaline or lipopolysaccharide (LPS, 6 mg/kg of body weight) was administered intratracheally with a 1-hour pretreatment with SP600125 (a JNK inhibitor; 30 mg/kg, IO), or PD98059 (an MEK/ERK inhibitor; 30 mg/kg, IO). Rats were sacrificed 4 hours after LPS treatment.ResultsSP600125 or PD98059 inhibited LPS-induced phosphorylation of JNK and ERK, total protein and LDH activity in BAL fluid, and neutrophil influx into the lungs. In addition, these MAP kinase inhibitors substantially reduced LPS-induced production of inflammatory mediators, such as CINC, MMP-9, and nitric oxide. Inhibition of JNK correlated with suppression of NF-κB activation through downregulation of phosphorylation and degradation of IκB-α, while ERK inhibition only slightly influenced the NF-κB pathway.ConclusionJNK and ERK play pivotal roles in LPS-induced acute lung injury. Therefore, inhibition of JNK or ERK activity has potential as an effective therapeutic strategy in interventions of inflammatory cascade-associated lung injury.

Pulmonary inflammation after intraperitoneal administration of ultrafine titanium dioxide (TiO2) at rest or in lungs primed with lipopolysaccharide.

Nanoparticles are widely used in nanomedicines, including for targeted delivery of pharmacological, therapeutic, and diagnostic agents. Since nanoparticles might translocate across cellular barriers from the circulation into targeted organs, it is important to obtain information concerning the pathophysiologic effects of these particles through systemic migration. In the present study, acute pulmonary responses were examined after intraperitoneal (ip) administration of ultrafine titanium dioxide (TiO2, 40 mg/kg) in mice at rest or in lungs primed with lipopolysaccharide (LPS, ip, 5 mg/kg). Ultrafine TiO2 exposure increased neutrophil influx, protein levels in bronchoalveolar lavage (BAL) fluid, and reactive oxygen species (ROS) activity of BAL cells 4 h after exposure. Concomitantly, the levels of proinflammatory mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and macrophage inflammatory protein (MIP)-2 in BAL fluid and mRNA expression of TNF-α and IL-1β in lung tissue were elevated post ultrafine TiO2 exposure. Ultrafine TiO2 exposure resulted in significant activation of inflammatory signaling molecules, such as c-Src and p38 MAP kinase, in lung tissue and alveolar macrophages, and the nuclear factor (NF)-κB pathway in pulmonary tissue. Furthermore, ultrafine TiO2 additively enhanced these inflammatory parameters and this signaling pathway in lungs primed with lipopolysaccharide (LPS). Contrary to this trend, a synergistic effect was found for TNF-α at the level of protein and mRNA expression. These results suggest that ultrafine TiO2 (P25) induces acute lung inflammation after ip administration, and exhibits additive or synergistic effects with LPS, at least partly, via activation of oxidant-dependent inflammatory signaling and the NF-κB pathway, leading to increased production of proinflammatory mediators.