u-0126 and diphenyleneiodonium

CPS-F, a polysaccharide derived from Cordyceps sinensis, is a potential anti-inflammatory and anti-oxidative agent. We demonstrated that CPS-F not only inhibits platelet-derived growth factor BB (PDGF-BB)-induced intracellular reactive oxygen species (ROS) generation, and up-regulation of tumor necrosis factor-α (TNF-α), TNF-α receptor 1 (TNFR1), and monocyte chemotactic protein-1 (MCP-1), but also acts synergistically in combination with MAPK/ERK inhibitor U0126 and PI3K/Akt inhibitor LY294002. Additionally, up-regulation of pro-inflammatory factors was reversed by use of a combination of CPS-F and NADPH oxidase (NOX) inhibitor diphenyleneiodonium chloride (DPI) or silencing of NOX1. Furthermore, CPS-F prevents the PDGF receptor β (PDGFRβ) promoter activity induced by PDGF-BB in transfected cells and ameliorates increased levels of TNF-α, TNFR1, and MCP-1 when PDGFRβ is silenced, thereby suggesting that CPS-F possesses a bidirectional regulatory function. Our findings suggest CPS-F may exert its therapeutic effect for the treatment of glomerulonephritis related to human mesangial cells (HMCs) through the ERK1/2/Akt pathways.

Topics: Anti-Inflammatory Agents; Antioxidants; Becaplermin; Butadienes; Cell Line; Chemokine CCL2; Chromones; Cordyceps; Fungal Polysaccharides; Humans; Mesangial Cells; Morpholines; NADPH Oxidase 1; NADPH Oxidases; Nitriles; Onium Compounds; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-sis; Reactive Oxygen Species; Receptors, Tumor Necrosis Factor; Tumor Necrosis Factor-alpha

Canonical neutrophil antimicrobial effector mechanisms, such as degranulation, production of reactive oxygen species, and release of neutrophil extracellular traps (NETs), can result in severe pathology. Activation of neutrophils through immune complexes (ICs) plays a central role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report that immobilized ICs (iICs), which are hallmarks of several autoimmune diseases, induce the release of NETs from primary human neutrophils. The iIC-induced NET formation was found to require production of reactive oxygen species by NADPH oxidase and myeloperoxidase and to be mediated by FcγRIIIb. Blocking of the β2 integrin macrophage-1 Ag but not lymphocyte function-associated Ag-1 abolished iIC-induced NET formation. This suggests that FcγRIIIb signals in association with macrophage-1 Ag. As intracellular signaling pathways involved in iIC-induced NET formation we identified the tyrosine kinase Src/Syk pathway, which downstream regulates the PI3K/Akt, p38 MAPK, and ERK1/2 pathways. To our knowledge, the present study shows for the first time that iICs induce NET formation. Thus, we conclude that NETs contribute to pathology in autoimmune inflammatory disorders associated with surface-bound ICs.

Topics: Aminopyrine; Antigen-Antibody Complex; Antioxidants; Ascorbic Acid; Autoimmune Diseases; Butadienes; CD11a Antigen; CD18 Antigens; Cell Degranulation; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; GPI-Linked Proteins; Humans; Imidazoles; Inflammation; Intracellular Signaling Peptides and Proteins; Lymphocyte Function-Associated Antigen-1; Macrophage-1 Antigen; Mesalamine; Neutrophil Activation; Neutrophils; Nitriles; Onium Compounds; p38 Mitogen-Activated Protein Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; Reactive Oxygen Species; Receptors, IgG; src-Family Kinases; Syk Kinase

Neutrophils/polymorphonuclear leukocytes (PMNs), an important component of innate immune system, release extracellular traps (NETs) to eliminate invaded pathogens; however understanding of the role of signaling molecules/proteins need to be elucidated. In the present study role of p38 MAPK and extracellular signal regulated kinase (ERK) against phorbol 12-myristate 13-acetate (PMA) induced reactive oxygen species (ROS) generation and NETs formation has been investigated. Human neutrophils were treated with PMA to induce free radical generation and NETs release, which were monitored by NBT reduction and elastase/DNA release, respectively. PMA treatment led to the time dependent phosphorylation of p38 MAPK and ERK in PMNs. Pretreatment of PMNs with SB202190 or U0126 did not significantly reduce PMA induce free radical generation, but prevented NETs release. Pretreatment of PMNs with NADPH oxidase inhibitor (diphenyleneiodonium chloride) significantly reduced free radical generation, p38 MAPK and ERK phosphorylation as well as NETs release, suggesting that p38 MAPK and ERK activation was downstream to free radical generation. The present study thus demonstrates ROS dependent activation of ERK and p38 MAPK, which mediated PMA induced NETs release from human neutrophils.

Topics: Butadienes; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Imidazoles; MAP Kinase Signaling System; NADPH Oxidases; Neutrophils; Nitriles; Onium Compounds; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Reactive Oxygen Species; Tetradecanoylphorbol Acetate

Glycated serum albumin (GSA) promotes vascular complications in diabetes. The aim of this study was to determine if GSA induces chemokine, particularly CXCL8 (IL-8), and to determine intracellular signaling pathways activated by GSA in vascular smooth muscle cells (VSMCs). GSA increased IL-8 transcription via promoter activation and enhanced CXCL8 release from VSMCs. GSA-induced promoter activation of the IL-8 gene was suppressed by dominant-negative mutants of TLR-4, MyD88, and TRIF, but not by a dominant-negative form of TLR-2. In addition, IL-8 up-regulation in response to GSA was inhibited by resveratrol, curcumin, diphenyleneiodium, U0126, and SB202190. Mutation at the NF-kappaB- or C/EBP-binding site, but not at the AP-1-binding site, in the IL-8 promoter region suppressed GSA-induced promoter activation. Moreover, gene delivery of IkappaB suppressed CXCL8 release. This study suggests that GSA induces expression of IL-8 in VSMCs and that TLR-4, mitogen-activated protein kinases, NF-kappaB, and NADPH oxidase are involved in that process.

Topics: Butadienes; Cells, Cultured; Curcumin; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Interleukin-8; Mitogen-Activated Protein Kinase Kinases; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NADPH Oxidases; NF-kappa B; Nitriles; Onium Compounds; Promoter Regions, Genetic; Resveratrol; Serum Albumin; Stilbenes; Toll-Like Receptor 4; Transcriptional Activation; Up-Regulation

Up-regulation of cytosolic phospholipase A2 (cPLA2) by cigarette smoke extract (CSE) may play a critical role in airway inflammatory diseases. However, the mechanisms underlying CSE-induced cPLA2 expression in human tracheal smooth muscle cells (HTSMCs) remain unknown. CSE induced cPLA2 protein and mRNA expression, and ROS generation was attenuated by pretreatment with a reactive oxygen species (ROS) scavenger (N-acetylcysteine), or inhibitors of NADPH oxidase (diphenyleneiodonium chloride, apocynin) and transfection with p47phox siRNA, suggesting that CSE-induced cPLA2 expression was mediated through NADPH oxidase activation and ROS production in HTSMCs. Furthermore, CSE-induced cPLA2 expression was attenuated by pretreatment with the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125), which were further confirmed by transfection with siRNAs of JNK1, p42, and p38 to down-regulate the expression of respective proteins and reduce cPLA2 expression. Induction of cPLA2 by CSE was attenuated by selective inhibitors of NF-kappaB (helenalin) and AP-1 (curcumin). Moreover, promoter assays revealed that increases of cPLA2, NF-kappaB, and AP-1 luciferase activities stimulated by CSE were attenuated by these inhibitors. These results suggest that in HTSMCs, CSE induced NADPH oxidase activation leading to phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK. These reactions induced nuclear transcription NF-kappaB and AP-1 activities which were essential for CSE-induced cPLA2 gene expression.

Topics: Acetophenones; Acetylcysteine; Butadienes; Cells, Cultured; Curcumin; Cytoplasm; Gene Expression Regulation, Enzymologic; Humans; MAP Kinase Kinase Kinases; Myocytes, Smooth Muscle; NADPH Oxidases; NF-kappa B; Nitriles; Onium Compounds; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phospholipases A2; RNA, Small Interfering; Sesquiterpenes; Sesquiterpenes, Guaiane; Smoking; Trachea; Transcription Factor AP-1; Transcriptional Activation

Alveolar macrophages (AM) have an important role in clearing particles from the lungs. In response to different stimuli they can release reactive oxygen species (ROS) and inflammatory mediators and promote pulmonary inflammation. We exposed rat AM to carbon black (CB) particles (0.63-20 microg/ml) and measured the eneration of ROS by using the fluorescent probe 2',7'-dichlorofluorescein diacetate. Fluorescence was elevated in a concentration dependent manner in the AM exposed to CB. Follow-up experiments using a series of enzyme inhibitors indicate that the ERK MAP kinase pathway and the p38 MAP kinase pathway may be involved in the formation of ROS.

Topics: Animals; Butadienes; Dose-Response Relationship, Drug; Enzyme Inhibitors; Imidazoles; In Vitro Techniques; Macrophages, Alveolar; Male; MAP Kinase Kinase 5; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 7; NADPH Oxidases; Nitriles; Onium Compounds; p38 Mitogen-Activated Protein Kinases; Particulate Matter; Pyridines; Rats; Rats, Inbred WKY; Reactive Oxygen Species; Soot; Tacrolimus

Elevated exposure to manganese is known to cause neurodegeneration in the basal ganglia and to induce movement abnormalities called manganism. However, the underlying mechanism of action is not fully understood. Activation of the resident immune cells in the brain, microglia that release a variety of neurotoxic factors, has been implicated to contribute to neurodegeneration. Of the various neurotoxic factors released by activated microglia, reactive oxygen species such as superoxide and hydrogen peroxide are particularly detrimental to the survival of the oxidative damage-prone neurons. In this study, we report that exposure of rat microglia to manganese chloride (MnCl(2)) resulted in a time- and concentration-dependent release of hydrogen peroxide (H(2)O(2)). The MnCl(2)-stimulated microglial H(2)O(2) release was sensitive to inhibitors of mitogen-activated protein kinases (MAPK) but not that of NADPH oxidase. MnCl(2)-induced a rapid activation of the extracellular signal-regulated kinase (ERK) and p38-MAPK in microglia that appeared to precede the MnCl(2)-induced H(2)O(2) release, suggesting that ERK and p38-MAPK influenced the MnCl(2)-induced H(2)O(2) release in microglia. In summary, these results demonstrate that manganese chloride is capable of activating microglia to release ROS and MAPK may, in part, be key regulators of the process. These findings may shed significant light on the potential role of microglia in the manganese-induced neurotoxicity.

Topics: Animals; Animals, Newborn; Anthracenes; Butadienes; Cell Line; Cells, Cultured; Chlorides; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Hydrogen Peroxide; Imidazoles; Manganese Compounds; Microglia; NADPH Oxidases; Nitriles; Onium Compounds; p38 Mitogen-Activated Protein Kinases; Pyridines; Rats; Rats, Inbred F344; Time Factors

Monocyte chemotactic protein-1 (MCP-1), a potent chemoattractant for monocytes, is thought to play a major role in atherosclerosis, but whether its atherogenic effects involve the direct modulation of vascular smooth muscle cell (SMC) functions remains unclear. This study examined the effects of MCP-1 on the migration of cultured A7r5 SMCs and the signaling pathways involved. Addition of recombinant MCP-1 stimulated SMC migration in modified Boyden chambers coated with type I collagen in a concentration-dependent manner, with 10(-9) M being maximally effective. Using untreated A7r5 cells, two MCP-1 receptors, CCR2 and CCR4, were detected and MCP-1 secretion was significantly increased by stimulation with platelet-derived growth factor. MCP-1-stimulated A7r5 migration was completely blocked by the NAD(P)H oxidase inhibitor, diphenylene iodonium (DPI), and dose-dependently inhibited by polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), suggesting a role for reactive oxygen species (ROS) in this process. During MCP-1 stimulation, ROS production increased rapidly, then gradually decayed over 60 min, and this effect was markedly decreased by pretreatment with DPI or PEG-SOD. Interestingly, U0126 and PD98059, which inhibit activation of extracellular signal-regulated kinases 1/2 (ERK 1/2), significantly inhibited MCP-1-activated ROS generation. Furthermore, transfection of an active mutant of MEK1 (ERK 1/2 kinase) markedly increased superoxide production in rat aortic smooth muscle cells, as detected by dihydroethydium staining, suggesting that ERK 1/2 activation stimulates ROS generation. ERK 1/2 activation was increased for at least 30 min in cells incubated with MCP-1, and this effect was abolished by U0126 or DPI pretreatment. These results demonstrate that MCP-1 is a chemoattractant for SMCs and that MCP-1-stimulated migration requires both ROS production and ERK 1/2 activation in a positive activation loop, which may contribute to the atherogenic effects of MCP-1.

Topics: Animals; Aorta; Butadienes; Cell Line; Cell Movement; Cells, Cultured; Chemokine CCL2; Coloring Agents; DNA Primers; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Flavonoids; Immunoblotting; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocytes, Smooth Muscle; Nitriles; Onium Compounds; Polyethylene Glycols; Rats; Reactive Oxygen Species; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Superoxide Dismutase; Superoxides; Temperature; Time Factors; Transfection