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Sample records for macrophages released lps

  1. A novel hybrid aspirin-NO-releasing compound inhibits TNFalpha release from LPS-activated human monocytes and macrophages

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    Fox Sarah

    2008-07-01

    Full Text Available Abstract Background The cytoprotective nature of nitric oxide (NO led to development of NO-aspirins in the hope of overcoming the gastric side-effects of aspirin. However, the NO moiety gives these hybrids potential for actions further to their aspirin-mediated anti-platelet and anti-inflammatory effects. Having previously shown that novel NO-aspirin hybrids containing a furoxan NO-releasing group have potent anti-platelet effects, here we investigate their anti-inflammatory properties. Here we examine their effects upon TNFα release from lipopolysaccharide (LPS-stimulated human monocytes and monocyte-derived macrophages and investigate a potential mechanism of action through effects on LPS-stimulated nuclear factor-kappa B (NF-κB activation. Methods Peripheral venous blood was drawn from the antecubital fossa of human volunteers. Mononuclear cells were isolated and cultured. The resultant differentiated macrophages were treated with pharmacologically relevant concentrations of either a furoxan-aspirin (B8, B7; 10 μM, their respective furazan NO-free counterparts (B16, B15; 10 μM, aspirin (10 μM, existing nitroaspirin (NCX4016; 10 μM, an NO donor (DEA/NO; 10 μM or dexamethasone (1 μM, in the presence and absence of LPS (10 ng/ml; 4 h. Parallel experiments were conducted on undifferentiated fresh monocytes. Supernatants were assessed by specific ELISA for TNFα release and by lactate dehydrogenase (LDH assay for cell necrosis. To assess NF-κB activation, the effects of the compounds on the loss of cytoplasmic inhibitor of NF-κB, IκBα (assessed by western blotting and nuclear localisation (assessed by immunofluorescence of the p65 subunit of NF-κB were determined. Results B8 significantly reduced TNFα release from LPS-treated macrophages to 36 ± 10% of the LPS control. B8 and B16 significantly inhibited monocyte TNFα release to 28 ± 5, and 49 ± 9% of control, respectively. The B8 effect was equivalent in magnitude to that of

  2. RNA from LPS-stirnulated macrophages induces the release of tumour necrosis factor-α and interleukin-1 by resident macrophages

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    R. A. Ribeiro

    1993-01-01

    Full Text Available The effect of exogenous RNA on many cellular functions has been studied in a variety of eukaryotic cells but there are few reports on macrophages. In the present study, it is demonstrated that cytoplasmatic RNA extracted from rat macrophages stimulated with Escherichia coli lipopolysaccharide (LPS, referred to as L-RNA, induced the release of TNF-α and IL-1 from monolayers of peritoneal resident macrophages. The activity of L-RNA was not altered by polymyxin B but was abolished by ribonuclease (RNase pretreatment, indicating the absence of LPS contamination and that the integrity of the polynucleotide chain is essential for this activity. Both the poly A(− and poly A(+ fractions obtained from L-RNA applied to oligo(dT–cellulose chromatography induced TNF-α and IL-1 release. The L-RNA-induced cytokine release was inhibited by dexamethasone and seemed to be dependent on protein synthesis since this effect was abolished by cycloheximide or actinomycin-D. The LPS-stimulated macrophages, when pre-incubated with [5-3H]-uridine, secreted a trichloroacetic acid (TCA precipitable material which was sensitive to RNase and KOH hydrolysis, suggesting that the material is RNA. This substance was also released from macrophage monolayers stimulated with IL-1β but not with TNF-α, IL-6 or IL-8. The substance secreted (3H-RNA sediments in the 4–5S region of a 5–20% sucrose gradient. These results show that L-RNA induces cytokine secretion by macrophage monolayers and support the idea that, during inflammation, stimulated macrophages could release RNA which may further induce the release of cytokines by the resident cell population.

  3. Intracellular NAD+ levels are associated with LPS-induced TNF-α release in pro-inflammatory macrophages.

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    Al-Shabany, Abbas Jawad; Moody, Alan John; Foey, Andrew David; Billington, Richard Andrew

    2016-01-13

    Metabolism and immune responses have been shown to be closely linked and as our understanding increases, so do the intricacies of the level of linkage. NAD(+) has previously been shown to regulate tumour necrosis factor-α (TNF-α) synthesis and TNF-α has been shown to regulate NAD(+) homoeostasis providing a link between a pro-inflammatory response and redox status. In the present study, we have used THP-1 differentiation into pro- (M1-like) and anti- (M2-like) inflammatory macrophage subset models to investigate this link further. Pro- and anti-inflammatory macrophages showed different resting NAD(+) levels and expression levels of NAD(+) homoeostasis enzymes. Challenge with bacterial lipopolysaccharide, a pro-inflammatory stimulus for macrophages, caused a large, biphasic and transient increase in NAD(+) levels in pro- but not anti-inflammatory macrophages that were correlated with TNF-α release and inhibition of certain NAD(+) synthesis pathways blocked TNF-α release. Lipopolysaccharide stimulation also caused changes in mRNA levels of some NAD(+) homoeostasis enzymes in M1-like cells. Surprisingly, despite M2-like cells not releasing TNF-α or changing NAD(+) levels in response to lipopolysaccharide, they showed similar mRNA changes compared with M1-like cells. These data further strengthen the link between pro-inflammatory responses in macrophages and NAD(+). The agonist-induced rise in NAD(+) shows striking parallels to well-known second messengers and raises the possibility that NAD(+) is acting in a similar manner in this model.

  4. Ethyl pyruvate inhibits the acetylation and release of HMGB1 via effects on SIRT1/STAT signaling in LPS-activated RAW264.7 cells and peritoneal macrophages.

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    Kim, Young Min; Park, Eun Jung; Kim, Jung Hwan; Park, Sang Won; Kim, Hye Jung; Chang, Ki Churl

    2016-12-01

    High mobility group box 1 (HMGB1), a cytokine present in the late phase of sepsis, may be a potential target for the treatment of sepsis. For HMGB1 to be actively secreted from macrophages during infections, it must be post-translationally modified. Although ethyl pyruvate (EP), a simple aliphatic ester derived from pyruvic acid, has been shown to inhibit the release of HMGB1 in lipopolysaccharide (LPS)-treated RAW 264.7 cells, the underlying mechanism(s) are not yet clear. We investigated the hypothesis that the upregulation of SIRT1 by EP might promote the deacetylation of HMGB1, which reduces HMGB1 release in LPS-activated macrophages. Our results show that EP induced the expression of the SIRT1 protein in RAW264.7 cells and that it significantly inhibited the LPS-induced acetylation of HMGB1. Transfection with a SIRT1-overexpressing vector resulted in a significant decrease in the acetylation of HMGB1 in LPS-activated RAW264.7 cells relative to control cells. The genetic ablation or the pharmacological inhibition of SIRT1 by sirtinol increased LPS-induced HMGB1 acetylation. Moreover, EP inhibited the acetylation of HMGB1 in peritoneal macrophages treated with LPS. Interestingly, EP significantly reduced the LPS-induced phosphorylation of STAT1, which was significantly reversed by siSIRT1 transfection in RAW264.7 cells, indicating that SIRT1 negatively regulates the phosphorylation of STAT1. Overall, the results show that EP promotes the deacetylation of HMGB1 via the inhibition of STAT1 phosphorylation through the upregulation of SIRT1, which reduces HMGB1 release in LPS-activated RAW264.7 cells. In conclusion, EP might be useful in the treatment of diseases that target HMGB1, such as sepsis. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Soybean-derived Bowman-Birk inhibitor inhibits neurotoxicity of LPS-activated macrophages

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    Persidsky Yuri

    2011-02-01

    Full Text Available Abstract Background Lipopolysaccharide (LPS, the major component of the outer membrane of gram-negative bacteria, can activate immune cells including macrophages. Activation of macrophages in the central nervous system (CNS contributes to neuronal injury. Bowman-Birk inhibitor (BBI, a soybean-derived protease inhibitor, has anti-inflammatory properties. In this study, we examined whether BBI has the ability to inhibit LPS-mediated macrophage activation, reducing the release of pro-inflammatory cytokines and subsequent neurotoxicity in primary cortical neural cultures. Methods Mixed cortical neural cultures from rat were used as target cells for testing neurotoxicity induced by LPS-treated macrophage supernatant. Neuronal survival was measured using a cell-based ELISA method for expression of the neuronal marker MAP-2. Intracellular reactive oxygen species (ROS production in macrophages was measured via 2', 7'-dichlorofluorescin diacetate (DCFH2DA oxidation. Cytokine expression was determined by quantitative real-time PCR. Results LPS treatment of macrophages induced expression of proinflammatory cytokines (IL-1β, IL-6 and TNF-α and of ROS. In contrast, BBI pretreatment (1-100 μg/ml of macrophages significantly inhibited LPS-mediated induction of these cytokines and ROS. Further, supernatant from BBI-pretreated and LPS-activated macrophage cultures was found to be less cytotoxic to neurons than that from non-BBI-pretreated and LPS-activated macrophage cultures. BBI, when directly added to the neuronal cultures (1-100 μg/ml, had no protective effect on neurons with or without LPS-activated macrophage supernatant treatment. In addition, BBI (100 μg/ml had no effect on N-methyl-D-aspartic acid (NMDA-mediated neurotoxicity. Conclusions These findings demonstrate that BBI, through its anti-inflammatory properties, protects neurons from neurotoxicity mediated by activated macrophages.

  6. Lipopolysaccharide (LPS) processing by Kupffer cells releases a modified LPS with increased hepatocyte binding and decreased tumor necrosis factor-alpha stimulatory capacity.

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    Treon, S P; Thomas, P; Broitman, S A

    1993-02-01

    Normal physiological clearance of gut-derived endotoxin lipopolysaccharide [LPS] has been described previously; initially, there is uptake by Kupffer cells (KC), then release of modified LPS, followed by hepatocyte uptake. Previous work in our laboratories indicated that LPS is structurally modified with loss of carbohydrate prior to its release by KC. In this study, we functionally characterize KC modified LPS. KC-modified 125I-LPS was prepared from primary rat KC. Escherichia coli 0127:B8 native 125I-LPS or KC-modified 125I-LPS (40 ng) was incubated for 1 hr with 1 x 10E6 primary hepatocytes. The binding of KC-modified LPS was 4.33-fold higher than native LPS (P = 0.0024). Binding analysis studies were conducted to determine the region of KC-modified LPS responsible for enhanced hepatocyte binding. KC-modified Salmonella minnesota LPS was competed with 100-fold excess native or mutant (Ra, Rc, Rd, or Re) strains of LPS or Lipid A with no decrease to hepatocyte binding. S. minnesota-native 125I-LPS was compared with KC-modified 125I-LPS in a study to assess induction of tumor necrosis factor (TNF)-gamma by rat peritoneal macrophages. Native or KC-modified 125I-LPS (100 ng) was presented to 1 x 10E7 peritoneal macrophages for 6 hr. TNF-alpha was measured in supernatants using the WEHI-164 cytotoxicity assay. Native LPS induced 5.7-fold higher TNF-alpha levels than KC-modified LPS (P < 0.0001). The above data suggest that structural alterations in KC-modified LPS are accompanied by functional alterations resulting in enhanced hepatocyte binding and decreased TNF-alpha release. The latter result implies that an early step in LPS detoxification occurs in the KC in which LPS is modified to prevent elicitation of biologically active cytokines.

  7. Ginger extract inhibits LPS induced macrophage activation and function

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    Bruch David

    2008-01-01

    Full Text Available Abstract Background Macrophages play a dual role in host defence. They act as the first line of defence by mounting an inflammatory response to antigen exposure and also act as antigen presenting cells and initiate the adaptive immune response. They are also the primary infiltrating cells at the site of inflammation. Inhibition of macrophage activation is one of the possible approaches towards modulating inflammation. Both conventional and alternative approaches are being studied in this regard. Ginger, an herbal product with broad anti inflammatory actions, is used as an alternative medicine in a number of inflammatory conditions like rheumatic disorders. In the present study we examined the effect of ginger extract on macrophage activation in the presence of LPS stimulation. Methods Murine peritoneal macrophages were stimulated by LPS in presence or absence of ginger extract and production of proinflammatory cytokines and chemokines were observed. We also studied the effect of ginger extract on the LPS induced expression of MHC II, B7.1, B7.2 and CD40 molecules. We also studied the antigen presenting function of ginger extract treated macrophages by primary mixed lymphocyte reaction. Results We observed that ginger extract inhibited IL-12, TNF-α, IL-1β (pro inflammatory cytokines and RANTES, MCP-1 (pro inflammatory chemokines production in LPS stimulated macrophages. Ginger extract also down regulated the expression of B7.1, B7.2 and MHC class II molecules. In addition ginger extract negatively affected the antigen presenting function of macrophages and we observed a significant reduction in T cell proliferation in response to allostimulation, when ginger extract treated macrophages were used as APCs. A significant decrease in IFN-γ and IL-2 production by T cells in response to allostimulation was also observed. Conclusion In conclusion ginger extract inhibits macrophage activation and APC function and indirectly inhibits T cell activation.

  8. LPS-inducible factor(s) from activated macrophages mediates cytolysis of Naegleria fowleri amoebae

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    Cleary, S.F.; Marciano-Cabral, F.

    1986-03-01

    Soluble cytolytic factors of macrophage origin have previously been described with respect to their tumoricidal activity. The purpose of this study was to investigate the mechanism and possible factor(s) responsible for cytolysis of the amoeba Naegleria fowleri by activated peritoneal macrophages from B6C3F1 mice. Macrophages or conditioned medium (CM) from macrophage cultures were incubated with /sup 3/H-Uridine labeled amoebae. Percent specific release of label served as an index of cytolysis. Bacille Calmette-Guerin (BCG) and Corynebacterium parvum macrophages demonstrated significant cytolysis of amoebae at 24 h with an effector to target ratio of 10:1. Treatment of macrophages with inhibitors of RNA or protein synthesis blocked amoebicidal activity. Interposition of a 1 ..mu..m pore membrane between macrophages and amoebae inhibited killing. Inhibition in the presence of the membrane was overcome by stimulating the macrophages with LPS. CM from SPS-stimulated, but not unstimulated, cultures of activated macrophages was cytotoxic for amoebae. The activity was heat sensitive and was recovered from ammonium sulfate precipitation of the CM. Results indicate that amoebicidal activity is mediated by a protein(s) of macrophage origin induced by target cell contact or stimulation with LPS.

  9. DMPD: Toll receptors, CD14, and macrophage activation and deactivation by LPS. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12106783 Toll receptors, CD14, and macrophage activation and deactivation by LPS. D...ceptors, CD14, and macrophage activation and deactivation by LPS. PubmedID 12106783 Title Toll receptors, CD14, and macrophage activa...tion and deactivation by LPS. Authors Dobrovolskaia MA,

  10. Ghrelin inhibits LPS-induced release of IL-6 from mouse dopaminergic neurones

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    Beynon, Amy L; Brown, M. Rowan; Wright, Rhiannon; Rees, Mark I.; Sheldon, I Martin; Davies, Jeffrey S.

    2013-01-01

    Background Ghrelin is an orexigenic stomach hormone that acts centrally to increase mid-brain dopamine neurone activity, amplify dopamine signaling and protect against neurotoxin-induced dopamine cell death in the mouse substantia nigra pars compacta (SNpc). In addition, ghrelin inhibits the lipopolysaccharide (LPS)-induced release of pro-inflammatory cytokines from peripheral macrophages, T-cells and from LPS stimulated microglia. Here we sought to determine whether ghrelin attenuates pro-in...

  11. Mechanistic study of macrophage activation by LPS stimulation using fluorescence imaging techinques

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    Lu, Cuixia; Zhou, Feifan; Chen, Wei R.; Xing, Da

    2012-03-01

    Lipopolysaccharide (LPS), a structural component of the outer membrane of gram negative bacteria, has been suggested that stimulates macrophages secrete a wide variety of inflammatory mediators, such as nitric oxide (NO). However, the cellular mechanisms of NO generation in macrophage by LPS stimulation are not well known. In this study, LPS stimulated NO generation in macrophage was determined by measuring fluorescence changes with a NO specific probe DAF-FM DA. Using the fluorescence resonance energy transfer (FRET) techniques, we found an increase of protein kinase C (PKC) activation was dynamically monitored in macrophages treated with LPS. Nuclear factor kappa B (NF-κB) translocated from the cytoplasm to the nucleus in macrophage was measured by confocal laser scanning microscopy. Moreover, the PKC inhibitor GÖ6983 inhibited LPS-stimulated NF-κB activation and NO production. These results indicated that LPS stimulated NF-κB mediated NO production by activating PKC.

  12. Differential Cell Sensitivity between OTA and LPS upon Releasing TNF-α

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    Lauy Al-Anati

    2010-06-01

    Full Text Available The release of tumor necrosis factor α (TNF-α by ochratoxin A (OTA was studied in various macrophage and non-macrophage cell lines and compared with E. coli lipopolysaccharide (LPS as a standard TNF-α release agent. Cells were exposed either to 0, 2.5 or 12.5 µmol/L OTA, or to 0.1 µg/mL LPS, for up to 24 h. OTA at 2.5 µmol/L and LPS at 0.1 µg/mL were not toxic to the tested cells as indicated by viability markers. TNF-a was detected in the incubated cell medium of rat Kupffer cells, peritoneal rat macrophages, and the mouse monocyte macrophage cell line J774A.1: TNF-a concentrations were 1,000 pg/mL, 1,560 pg/mL, and 650 pg/mL, respectively, for 2.5 µmol/L OTA exposure and 3,000 pg/mL, 2,600 pg/mL, and 2,115 pg/mL, respectively, for LPS exposure. Rat liver sinusoidal endothelial cells, rat hepatocytes, human HepG2 cells, and mouse L929 cells lacked any cytokine response to OTA, but showed a significant release of TNF-a after LPS exposure, with the exception of HepG2 cells. In non-responsive cell lines, OTA lacked both any activation of NF-κB or the translocation of activated NF-κB to the cell nucleus, i.e., in mouse L929 cells. In J774A.1 cells, OTA mediated TNF-a release via the pRaf/MEK 1/2–NF-κB and p38-NF-κB pathways, whereas LPS used pRaf/MEK 1/2-NF-κB, but not p38-NF-κB pathways. In contrast, in L929 cells, LPS used other pathways to activate NF-κB. Our data indicate that only macrophages and macrophage derived cells respond to OTA and are considered as sources for TNF-a release upon OTA exposure.

  13. LPS counter regulates RNA expression of extracellular proteases and their inhibitors in murine macrophages

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    Hald, Andreas; Rønø, Birgitte; Lund, Leif R

    2012-01-01

    in extracellular matrix metabolism in the mouse derived-macrophage cell line RAW 264.7 following stimulation with LPS. Our results revealed that LPS induces the expression of matrix metalloproteinases while at the same time decreased the expression of matrix metalloproteinase inhibitors. The opposite scenario...

  14. Low pH Environmental Stress Inhibits LPS and LTA-Stimulated Proinflammatory Cytokine Production in Rat Alveolar Macrophages

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    Stanley F. Fernandez

    2013-01-01

    Full Text Available Gastric aspiration increases the risks for developing secondary bacterial pneumonia. Cytokine elaboration through pathogen recognition receptors (PRRs is an important mechanism in initiating innate immune host response. Effects of low pH stress, a critical component of aspiration pathogenesis, on the PRR pathways were examined, specifically toll-like receptor-2 (TLR2 and TLR4, using isolated rat alveolar macrophages (aMØs. We assessed the ability of aMØs after brief exposure to acidified saline to elaborate proinflammatory cytokines in response to lipopolysaccharide (LPS and lipoteichoic acid (LTA stimulation, known ligands of TLR4 and TLR2, respectively. Low pH stress reduced LPS- and LTA-mediated cytokine release (CINC-1, MIP-2, TNF-, MCP-1, and IFN-. LPS and LTA increased intracellular Ca2+ concentrations while Ca2+ chelation by BAPTA decreased LPS- and LTA-mediated cytokine responses. BAPTA blocked the effects of low pH stress on most of LPS-stimulated cytokines but not of LTA-stimulated responses. In vivo mouse model demonstrates suppressed E. coli and S. pneumoniae clearance following acid aspiration. In conclusion, low pH stress inhibits antibacterial cytokine response of aMØs due to impaired TLR2 (MyD88 pathway and TLR4 signaling (MyD88 and TRIF pathways. The role of Ca2+ in low pH stress-induced signaling is complex but appears to be distinct between LPS- and LTA-mediated responses.

  15. Leptomeningeal Cells Transduce Peripheral Macrophages Inflammatory Signal to Microglia in Reponse to Porphyromonas gingivalis LPS

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    Yicong Liu

    2013-01-01

    Full Text Available We report here that the leptomeningeal cells transduce inflammatory signals from peripheral macrophages to brain-resident microglia in response to Porphyromonas gingivalis (P.g. LPS. The expression of Toll-like receptor 2 (TLR2, TLR4, TNF-α, and inducible NO synthase was mainly detected in the gingival macrophages of chronic periodontitis patients. In in vitro studies, P.g. LPS induced the secretion of TNF-α and IL-1β from THP-1 human monocyte-like cell line and RAW264.7 mouse macrophages. Surprisingly, the mean mRNA levels of TNF-α and IL-1β in leptomeningeal cells after treatment with the conditioned medium from P.g. LPS-stimulated RAW264.7 macrophages were significantly higher than those after treatment with P.g. LPS alone. Furthermore, the mean mRNA levels of TNF-α and IL-1β in microglia after treatment with the conditioned medium from P.g. LPS-stimulated leptomeningeal cells were significantly higher than those after P.g. LPS alone. These observations suggest that leptomeninges serve as an important route for transducing inflammatory signals from macrophages to microglia by secretion of proinflammatory mediators during chronic periodontitis. Moreover, propolis significantly reduced the P.g. LPS-induced TNF-α and IL-1 β production by leptomeningeal cells through inhibiting the nuclear factor-κB signaling pathway. Together with the inhibitory effect on microglial activation, propolis may be beneficial in preventing neuroinflammation during chronic periodontitis.

  16. Inflammatory cytokine regulation by LPS and lymphoid cells in human gamma-irradiated monocytes/macrophages; Regulation des cytokines de l`inflammation en presence de LPS ou de lymphocytes dans les monocytes/macrophages humains irradies

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    Pons, I.; Gras, G.; Dormont, D. [Centre de Recherches du Service de Sante des Armees, La Tronche, 38 - Grenoble (France)]|[Centre de Recherches du Service de Sante des Armees - Centre d`Etudes Nucleaires de Fontenay-aux-Roses, 92 (France)]|[Paris-5 Univ., 75 (France)

    1997-12-31

    We have investigated the inflammatory cytokine regulation after ionizing radiation of monocytes/macrophages. We have not evidenced any significant induction of tumour necrosis factor-{alpha}(TNF{alpha}) after irradiation alone. For one donor only out of eight, interleukin-1{beta}(IL-l{beta}) gene expression was affected by {gamma}-irradiation, with a 2-3-fold increase in level, while for two other donors, interleukin-6 (IL-6) mRNA expression was 5-14 fold increased. For one of the eight donors tested, monocytes/macrophages responded to 10 Gy {gamma}-rays by releasing inflammatory cytokines. In the presence of LPS, a significant increase of IL-1{beta} mRNA expression was detected in 10 Gy {gamma}-irradiated cells treated with 1 {mu}g/ml LPS. In most cases, combination of LPS treatment and 10 Gy irradiation down-regulated cytokine secretion except for a TNF{alpha} induction at 6 h post-irradiation. In the presence of lymphoid cells, IL-6 mRNA level was increased in irradiated cells at 24 h. Increases of IL-1{beta} and IL-6 releases were detected at 24 h post-irradiation too. (authors)

  17. Brazilian Red Propolis Attenuates Inflammatory Signaling Cascade in LPS-Activated Macrophages

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    Bueno-Silva, Bruno; Kawamoto, Dione; Ando-Suguimoto, Ellen S.; Alencar, Severino M.; Rosalen, Pedro L.; Mayer, Marcia P. A.

    2015-01-01

    Although previous studies suggested an anti-inflammatory property of Brazilian red propolis (BRP), the mechanisms involved in the anti-inflammatory effects of BRP and its activity on macrophages were still not elucidated. This study aimed to evaluate whether BRP attenuates the inflammatory effect of LPS on macrophages and to investigate its underlying mechanisms. BRP was added to RAW 264.7 murine macrophages after activation with LPS. NO production, cell viability, cytokines profile were evaluated. Activation of inflammatory signaling pathways and macrophage polarization were determined by RT-qPCR and Western blot. BRP at 50 μg/ml inhibited NO production by 78% without affecting cell viability. Cd80 and Cd86 were upregulated whereas mrc1 was down regulated by BRP indicating macrophage polarization at M1. BRP attenuated the production of pro-inflammatory mediators IL-12, GM-CSF, IFN-Ɣ, IL-1β in cell supernatants although levels of TNF- α and IL-6 were slightly increased after BRP treatment. Levels of IL-4, IL-10 and TGF-β were also reduced by BRP. BRP significantly reduced the up-regulation promoted by LPS of transcription of genes in inflammatory signaling (Pdk1, Pak1, Nfkb1, Mtcp1, Gsk3b, Fos and Elk1) and of Il1β and Il1f9 (fold-change rate > 5), which were further confirmed by the inhibition of NF-κB and MAPK signaling pathways. Furthermore, the upstream adaptor MyD88 adaptor-like (Mal), also known as TIRAP, involved in TLR2 and TLR4 signaling, was down- regulated in BRP treated LPS-activated macrophages. Given that BRP inhibited multiple signaling pathways in macrophages involved in the inflammatory process activated by LPS, our data indicated that BRP is a noteworthy food-source for the discovery of new bioactive compounds and a potential candidate to attenuate exhacerbated inflammatory diseases. PMID:26660901

  18. Brazilian Red Propolis Attenuates Inflammatory Signaling Cascade in LPS-Activated Macrophages.

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    Bruno Bueno-Silva

    Full Text Available Although previous studies suggested an anti-inflammatory property of Brazilian red propolis (BRP, the mechanisms involved in the anti-inflammatory effects of BRP and its activity on macrophages were still not elucidated. This study aimed to evaluate whether BRP attenuates the inflammatory effect of LPS on macrophages and to investigate its underlying mechanisms. BRP was added to RAW 264.7 murine macrophages after activation with LPS. NO production, cell viability, cytokines profile were evaluated. Activation of inflammatory signaling pathways and macrophage polarization were determined by RT-qPCR and Western blot. BRP at 50 μg/ml inhibited NO production by 78% without affecting cell viability. Cd80 and Cd86 were upregulated whereas mrc1 was down regulated by BRP indicating macrophage polarization at M1. BRP attenuated the production of pro-inflammatory mediators IL-12, GM-CSF, IFN-Ɣ, IL-1β in cell supernatants although levels of TNF- α and IL-6 were slightly increased after BRP treatment. Levels of IL-4, IL-10 and TGF-β were also reduced by BRP. BRP significantly reduced the up-regulation promoted by LPS of transcription of genes in inflammatory signaling (Pdk1, Pak1, Nfkb1, Mtcp1, Gsk3b, Fos and Elk1 and of Il1β and Il1f9 (fold-change rate > 5, which were further confirmed by the inhibition of NF-κB and MAPK signaling pathways. Furthermore, the upstream adaptor MyD88 adaptor-like (Mal, also known as TIRAP, involved in TLR2 and TLR4 signaling, was down- regulated in BRP treated LPS-activated macrophages. Given that BRP inhibited multiple signaling pathways in macrophages involved in the inflammatory process activated by LPS, our data indicated that BRP is a noteworthy food-source for the discovery of new bioactive compounds and a potential candidate to attenuate exhacerbated inflammatory diseases.

  19. Brazilian Red Propolis Attenuates Inflammatory Signaling Cascade in LPS-Activated Macrophages.

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    Bueno-Silva, Bruno; Kawamoto, Dione; Ando-Suguimoto, Ellen S; Alencar, Severino M; Rosalen, Pedro L; Mayer, Marcia P A

    2015-01-01

    Although previous studies suggested an anti-inflammatory property of Brazilian red propolis (BRP), the mechanisms involved in the anti-inflammatory effects of BRP and its activity on macrophages were still not elucidated. This study aimed to evaluate whether BRP attenuates the inflammatory effect of LPS on macrophages and to investigate its underlying mechanisms. BRP was added to RAW 264.7 murine macrophages after activation with LPS. NO production, cell viability, cytokines profile were evaluated. Activation of inflammatory signaling pathways and macrophage polarization were determined by RT-qPCR and Western blot. BRP at 50 μg/ml inhibited NO production by 78% without affecting cell viability. Cd80 and Cd86 were upregulated whereas mrc1 was down regulated by BRP indicating macrophage polarization at M1. BRP attenuated the production of pro-inflammatory mediators IL-12, GM-CSF, IFN-Ɣ, IL-1β in cell supernatants although levels of TNF- α and IL-6 were slightly increased after BRP treatment. Levels of IL-4, IL-10 and TGF-β were also reduced by BRP. BRP significantly reduced the up-regulation promoted by LPS of transcription of genes in inflammatory signaling (Pdk1, Pak1, Nfkb1, Mtcp1, Gsk3b, Fos and Elk1) and of Il1β and Il1f9 (fold-change rate > 5), which were further confirmed by the inhibition of NF-κB and MAPK signaling pathways. Furthermore, the upstream adaptor MyD88 adaptor-like (Mal), also known as TIRAP, involved in TLR2 and TLR4 signaling, was down- regulated in BRP treated LPS-activated macrophages. Given that BRP inhibited multiple signaling pathways in macrophages involved in the inflammatory process activated by LPS, our data indicated that BRP is a noteworthy food-source for the discovery of new bioactive compounds and a potential candidate to attenuate exhacerbated inflammatory diseases.

  20. Glycyrrhiza glabra L. Extract Inhibits LPS-Induced Inflammation in RAW Macrophages.

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    Li, Chunmei; Eom, Taekil; Jeong, Yoonhwa

    2015-01-01

    Glycyrrhiza glabra has been used in medicine for thousands of years. Our previous study revealed that the methanolic extract of Glycyrrhiza glabra L. (EGGR) exhibits significant nitric oxide (NO) inhibitory effect on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages among 100 other extracts. Accordingly, the aim of the present study was to investigate the potential anti-inflammatory effect of EGGR. The anti-inflammatory effect of EGGR on LPS-stimulated RAW 264.7 macrophages was measured by MTT assay, NO content analysis, reactive oxygen species (ROS) level analysis, RT-PCR, Western blot analysis, and ELISA assay. Low doses of EGGR were non-toxic to macrophages and imparted protective effect against LPS induced cell death. Incubation of LPS-treated macrophages with 100 μg/mL EGGR led to an increase in cell viability from 66.6 to 99%. Moreover, EGGR led to down regulation of NO (NO2+NO3) and ROS productions in a dose-dependent manner. In particular, 100 μg/mL EGGR led to a reduction in NO2+NO3 level from 336.2 to 24.1 pM/mL, and ROS level from 483.5 to 128.4%. Consistent with the result related to NO production, EGGR suppressed the ability of LPS to induce mRNA and protein expressions of nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) cytokines, tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and IL-6 productions which were analyzed by an ELISA assay. These results provide a comprehensive approach into the anti-inflammatory effect of EGGR on LPS-stimulated macrophages; however, efforts are underway on gaining detailed insight into anti-inflammatory signaling pathways.

  1. NAC attenuates LPS-induced toxicity in aspirin-sensitized mouse macrophages via suppression of oxidative stress and mitochondrial dysfunction.

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    Haider Raza

    Full Text Available Bacterial endotoxin lipopolysaccharide (LPS induces the production of inflammatory cytokines and reactive oxygen species (ROS under in vivo and in vitro conditions. Acetylsalicylic acid (ASA, aspirin is a commonly used anti-inflammatory drug. Our aim was to study the effects of N-acetyl cysteine (NAC, an antioxidant precursor of GSH synthesis, on aspirin-sensitized macrophages treated with LPS. We investigated the effects of LPS alone and in conjunction with a sub-toxic concentration of ASA, on metabolic and oxidative stress, apoptosis, and mitochondrial function using J774.2 mouse macrophage cell line. Protection from LPS-induced toxicity by NAC was also studied. LPS alone markedly induced ROS production and oxidative stress in macrophage cells. When ASA was added to LPS-treated macrophages, the increase in oxidative stress was significantly higher than that with LPS alone. Similarly, alteration in glutathione-dependent redox metabolism was also observed in macrophages after treatment with LPS and ASA. The combination of LPS and ASA selectively altered the CYP 3A4, CYP 2E1 and CYP 1A1 catalytic activities. Mitochondrial respiratory complexes and ATP production were also inhibited by LPS-ASA treatment. Furthermore a higher apoptotic cell death was also observed in LPS-ASA treated macrophages. NAC pre-treatment showed protection against oxidative stress induced apoptosis and mitochondrial dysfunction. These effects are presumed, at least in part, to be associated with alterations in NF-κB/Nrf-2 mediated cell signaling. These results suggest that macrophages are more sensitive to LPS when challenged with ASA and that NAC pre-treatment protects the macrophages from these deleterious effects.

  2. Inhibition of CDKS by roscovitine suppressed LPS-induced *NO production through inhibiting NFkappaB activation and BH4 biosynthesis in macrophages.

    Science.gov (United States)

    Du, Jianhai; Wei, Na; Guan, Tongju; Xu, Hao; An, Jianzhong; Pritchard, Kirkwood A; Shi, Yang

    2009-09-01

    In inflammatory diseases, tissue damage is critically associated with nitric oxide ((*)NO) and cytokines, which are overproduced in response to cellular release of endotoxins. Here we investigated the inhibitory effect of roscovitine, a selective inhibitor of cyclin-dependent kinases (CDKs) on (*)NO production in mouse macrophages. In RAW264.7 cells, we found that roscovitine abolished the production of (*)NO induced by lipopolysaccharide (LPS). Moreover, roscovitine significantly inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA and protein expression. Our data also showed that roscovitine attenuated LPS-induced phosphorylation of IkappaB kinase beta (IKKbeta), IkappaB, and p65 but enhanced the phosphorylation of ERK, p38, and c-Jun NH(2)-terminal kinase (JNK). In addition, roscovitine dose dependently inhibited LPS-induced expression of cyclooxygenase-2 (COX)-2, IL-1beta, and IL-6 but not tumor necrosis factor (TNF)-alpha. Tetrahydrobiopterin (BH(4)), an essential cofactor for iNOS, is easily oxidized to 7,8-dihydrobiopterin (BH(2)). Roscovitine significantly inhibited LPS-induced BH(4) biosynthesis and decreased BH(4)-to-BH(2) ratio. Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase-1 (GCH-1), the rate-limiting enzyme for BH(4) biosynthesis. Using other CDK inhibitors, we found that CDK1, CDK5, and CDK7, but not CDK2, significantly inhibited LPS-induced (*)NO production in macrophages. Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited (*)NO production, iNOS, and COX-2 upregulation, activation of NFkappaB, and induction of GCH-1 by LPS. Together, our data indicate that roscovitine abolishes LPS-induced (*)NO production in macrophages by suppressing nuclear factor-kappaB activation and BH(4) biosynthesis, which might be mediated by CDK1, CDK5, and CDK7. Our results also suggest that roscovitine may inhibit inflammation and that CDKs may play important roles in the mechanisms by which

  3. Anti-inflammatory effect of garlic 14-kDa protein on LPS-stimulated-J774A.1 macrophages.

    Science.gov (United States)

    Rabe, Shahrzad Zamani Taghizadeh; Ghazanfari, Tooba; Siadat, Zahra; Rastin, Maryam; Rabe, Shahin Zamani Taghizadeh; Mahmoudi, Mahmoud

    2015-04-01

    Garlic 14-kDa protein is purified from garlic (Allium sativum L.) which is used in traditional medicine and exerts various immunomodulatory activities. The present study investigated the suppressive effect of garlic 14-kDa protein on LPS-induced expression of pro-inflammatory mediators and underlying mechanism in inflammatory macrophages. J774A.1 macrophages were treated with 14-kDa protein (5-30 μg/ml) with/without LPS (1 μg/ml) and the production of inflammatory mediators such as prostaglandin E2 (PGE2), TNF-α, and IL-1β released were measured using ELISA. Nitric oxide (NO) production was determined using the Griess method. The anti-inflammatory activity of 14-kDa protein was examined by measuring inducible nitric oxide synthase and cyclooxygenase-2 proteins using western blot. The expression of nuclear NF-κB p65 subunit was assessed by western blot. Garlic 14-kDa protein significantly inhibited the excessive production of NO, PGE, TNF-α, and IL-1β in lipopolysaccharide (LPS)-activated J774A.1 macrophages in a concentration-related manner without cytotoxic effect. Western blot analysis demonstrated that garlic 14-kDa protein suppressed corresponding inducible NO synthase expression and activated cyclooxygenase-2 protein expression. The inhibitory effect was mediated partly by a reduction in the activity and expression of transcription factor NF-κB protein. Our results suggested, for the first time, garlic 14-kDa protein exhibits anti-inflammatory properties in macrophages possibly by suppressing the inflammatory mediators via the inhibition of transcription factor NF-κB signaling pathway. The traditional use of garlic as anti-inflammatory remedy could be ascribed partly to 14-kDa protein content. This protein might be a useful candidate for controlling inflammatory diseases and further investigations in vivo.

  4. 乳香-没药配伍前后化学成分溶出变化及其对LPS-诱导的巨噬细胞产生NO的影响%Change in dissolution of chemical components of frankincense-myrrh before and after their compatibility and effect on no release of LPS-induced macrophage cells

    Institute of Scientific and Technical Information of China (English)

    陈婷; 宿树兰; 段金廒; 尚尔鑫; 钱大玮; 唐于平

    2013-01-01

    目的:分析乳香-没药配伍前后化学成分的变化,评价差异性化合物对LPS-诱导的小鼠腹腔巨噬细胞产生NO的影响,以期从化学成分变化角度探讨乳香-没药配伍协同增效的物质基础.方法:采用UPLC-Q-TOF-MS/MS联用技术对乳香-没药配伍前后化学成分进行分析,并结合MarkerLynx4.1统计软件分析2味药配伍后的差异性化合物.结果:经PCA分析乳香-没药合提液与合并液色谱图存在明显差异,提示2味药配伍前后化学成分存在显著差异.经OPLS-DA分析及化学成分鉴定,2味药配伍后五环三萜类化合物(α-乳香酸、β-乳香酸等),四环三萜类化合物(榄香酮酸、3-乙酰氧基-16-羟基-24-甲基达玛烷、3-羟基甘遂烷-7,24-二烯-21-酸/3-羟基甘遂烷-8,24-二烯-21-酸等)溶出量显著增加,而环倍半萜类,大环二萜类化合物溶出量均有下降趋势.体外活性评价表明:化合物2-甲氧基-8,12-环氧-吉玛-1(10),7,11-三烯-6-酮,2-甲氧基-5-乙酰氧基-呋喃-吉玛-1(10)-烯-6-酮,3-羰基甘遂-8,24-二烯-21-羧酸显著抑制LPS-诱导的小鼠腹腔巨噬细胞产生NO水平.结论:该研究结果为揭示乳香-没药配伍增强抗炎效应的物质基础提供了一定科学依据与参考.%Objective:To analyze the difference of chemical compounds of frankincense-myrrh before and after their compatibility,and evaluate the effect of differentiated compounds on NO generated by LPS-induced peritoneal macrophage cells in rats,in order to discuss synergetic material basis of frankincense-myrrh compatibility from the prospective of change in chemical constituents.Method:UPLC-Q-TOF-MS/MS combined technology was used to analyze the chemical components of frankincense-myrrh before and after their compatibility.MarkerLynx 4.1 statistical software was used to analyze differentiated compounds before and afte their compatibility.Result:The results of PCA showed that there were significant differences in the combined

  5. Preferential macrophage recruitment and polarization in LPS-induced animal model for COPD: noninvasive tracking using MRI.

    Science.gov (United States)

    Al Faraj, Achraf; Sultana Shaik, Asma; Pureza, Mary Angeline; Alnafea, Mohammad; Halwani, Rabih

    2014-01-01

    Noninvasive imaging of macrophages activity has raised increasing interest for diagnosis of chronic obstructive respiratory diseases (COPD), which make them attractive vehicles to deliver contrast agents for diagnostic or drugs for therapeutic purposes. This study was designed to monitor and evaluate the migration of differently polarized M1 and M2 iron labeled macrophage subsets to the lung of a LPS-induced COPD animal model and to assess their polarization state once they have reached the inflammatory sites in the lung after intravenous injection. Ex vivo polarized bone marrow derived M1 or M2 macrophages were first efficiently and safely labeled with amine-modified PEGylated dextran-coated SPIO nanoparticles and without altering their polarization profile. Their biodistribution in abdominal organs and their homing to the site of inflammation in the lung was tracked for the first time using a free-breathing non-invasive MR imaging protocol on a 4.7T magnet after their intravenous administration. This imaging protocol was optimized to allow both detection of iron labeled macrophages and visualization of inflammation in the lung. M1 and M2 macrophages were successfully detected in the lung starting from 2 hours post injection with no variation in their migration profile. Quantification of cytokines release, analysis of surface membrane expression using flow cytometry and immunohistochemistry investigations confirmed the successful recruitment of injected iron labeled macrophages in the lung of COPD mice and revealed that even with a continuum switch in the polarization profile of M1 and M2 macrophages during the time course of inflammation a balanced number of macrophage subsets predominate.

  6. HSF-1 is involved in attenuating the release of inflammatory cytokines induced by LPS through regulating autophagy.

    Science.gov (United States)

    Tong, Zhongyi; Jiang, Bimei; Zhang, Lingli; Liu, Yanjuan; Gao, Min; Jiang, Yu; Li, Yuanbin; Lu, Qinglan; Yao, Yongming; Xiao, Xianzhong

    2014-05-01

    Autophagy plays a protective role in endotoxemic mice. Heat shock factor 1 (HSF-1) also plays a crucial protective role in endotoxemic mice by decreasing inflammatory cytokines. The purpose of this study was to determine whether HSF-1 is involved in attenuating the release of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated mice and peritoneal macrophages (PMs) through regulating autophagy activity. Autophagosome formation in HSF-1(+/+) and HSF-1(-/-) mice and PMs stimulated by LPS was examined by Western blotting and immunofluorescence. Lipopolysaccharide-induced autophagy and inflammatory cytokines were examined in HSF-1(+/+) and HSF-1(-/-) PMs treated with 3-methyladenine (3-MA) or rapamycin. Results showed that LPS-induced autophagy was elevated transiently at 12 h but declined at 24 h in the livers and lungs of mice. Higher levels of inflammatory cytokines and lower autophagy activity were detected in HSF-1(-/-) mice and PMs compared with HSF-1(+/+) mice and PMs. Interestingly, LPS-induced release of inflammatory cytokines did not further increase in HSF-1(-/-) PMs treated with 3-MA but aggravated in HSF-1(+/+) PMs. Lipopolysaccharide-induced autophagy did not decrease in HSF-1(-/-) PMs treated with 3-MA but decreased in HSF-1 PMs(+/+). Taken together, our results suggested that HSF-1 attenuated the release of inflammatory cytokines induced by LPS by regulating autophagy activity.

  7. DMPD: The role of macrophages in the hypothalamic-pituitary-adrenal activation inresponse to endotoxin (LPS). [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 1315450 The role of macrophages in the hypothalamic-pituitary-adrenal activation in...png) (.svg) (.html) (.csml) Show The role of macrophages in the hypothalamic-pituitary-adrenal activation in...response to endotoxin (LPS). PubmedID 1315450 Title The role of macrophages in th

  8. Friedelane-type triterpenoids as selective anti-inflammatory agents by regulation of differential signaling pathways in LPS-stimulated macrophages.

    Science.gov (United States)

    Villar-Lorenzo, Andrea; Ardiles, Alejandro E; Arroba, Ana I; Hernández-Jiménez, Enrique; Pardo, Virginia; López-Collazo, Eduardo; Jiménez, Ignacio A; Bazzocchi, Isabel L; González-Rodríguez, Águeda; Valverde, Ángela M

    2016-12-15

    A series of 31 pentacyclic triterpenoids isolated from the root barks of Celastrus vulcanicola and Maytenus jelskii were tested for cytotoxicity and inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. Compounds 18 (C18) and 25 (C25) exhibited significant inhibition of LPS-induced NO release at 50 and 25μM concentrations, respectively, and decreased mRNAs of pro-inflammatory cytokines. At the molecular level, C18 neither inhibited LPS-mediated phosphorylation of mitogen activated protein kinases (MAPKs) nor nuclear translocation of nuclear factor kappa beta (NFκB). Instead, C18 enhanced and prolonged nuclear translocation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and increased the expression of its target genes including hemeoxigenase 1 (HO1). C25 efficiently inhibited LPS-mediated phosphorylation of JNK, p38 and ERK, without affecting NFκB or Nrf2 signaling pathways. Both compounds reduced LPS-mediated processing of caspase-1 and the cleavage of interleukin 1β (IL1β) proform, reflecting their ability to target the inflammasome. C25 also counteracted LPS effects on iNOS expression and pro-inflammatory cytokines mRNA levels in Bv-2 microglial cells. The anti-inflammatory effect of both compounds was also assessed in human macrophages. Our results suggest that triterpenoids C18 and C25 possess anti-inflammatory effects, which may be therapeutically relevant for diseases linked to inflammation.

  9. Xanthohumol from Hop (Humulus lupulus L.) Is an Efficient Inhibitor of Monocyte Chemoattractant Protein-1 and Tumor Necrosis Factor-a Release in LPS-Stimulated RAW 264.7 Mouse Macrophages and U937 Human Monocytes

    NARCIS (Netherlands)

    Lupinacci, E.; Meijerink, J.; Vincken, J.P.; Gabriele, B.; Gruppen, H.; Witkamp, R.F.

    2009-01-01

    Activated macrophages in adipose tissue play a major role in the chronic inflammatory process that has been linked to the complications of overweight and obesity. The hop plant (Humulus lupulus L.) has been described to possess both anti-inflammatory and antidiabetic effects. In the present study, t

  10. Reduced caveolin-1 promotes hyper-inflammation due to abnormal heme oxygenase-1 localizationin LPS challenged macrophages with dysfunctional CFTR

    Science.gov (United States)

    Zhang, Ping-Xia; Murray, Thomas S.; Villella, Valeria Rachela; Ferrari, Eleonora; Esposito, Speranza; D'Souza, Anthony; Raia, Valeria; Maiuri, Luigi; Krause, Diane S.; Egan, Marie E.; Bruscia, Emanuela M.

    2013-01-01

    We have previously reported that TLR4 signaling is increased in lipopolysaccharide (LPS) -stimulated Cystic Fibrosis (CF) macrophages (MΦs), contributing to the robust production of pro-inflammatory cytokines. The heme oxygenase (HO-1)/carbon monoxide (CO) pathway modulates cellular redox status, inflammatory responses, and cell survival. The HO-1 enzyme, together with the scaffold protein caveolin 1 (CAV-1), also acts as a negative regulator of TLR4 signaling in MΦs. Here, we demonstrate that in LPS-challenged CF MΦs, HO-1 does not compartmentalize normally to the cell surface and instead accumulates intracellularly. The abnormal HO-1 localization in CF MΦs in response to LPS is due to decreased CAV-1 expression, which is controlled by the cellular oxidative state, and is required for HO-1 delivery to the cell surface. Overexpression of HO-1 or stimulating the pathway with CO-releasing molecules (CORM2)enhancesCAV-1 expression in CF MΦs, suggesting a positive-feed forward loop between HO-1/CO induction and CAV-1 expression. These manipulations reestablished HO-1 and CAV-1 cell surface localization in CF MΦ's. Consistent with restoration of HO-1/CAV-1 negative regulation of TLR4 signaling, genetic or pharmacological (CORM2)-induced enhancement of this pathway decreased the inflammatory response of CF MΦs and CF mice treated with LPS. In conclusion, our results demonstrate that the counter-regulatory HO-1/CO pathway, which is critical in balancing and limiting the inflammatory response, is defective in CF MΦs through a CAV-1-dependent mechanism, exacerbating the CF MΦ's response to LPS. This pathway could be a potential target for therapeutic intervention for CF lung disease. PMID:23606537

  11. Could a B-1 cell derived phagocyte "be one" of the peritoneal macrophages during LPS-driven inflammation?

    Directory of Open Access Journals (Sweden)

    Ana Flavia Popi

    Full Text Available The inflammatory response is driven by signals that recruit and elicit immune cells to areas of tissue damage or infection. The concept of a mononuclear phagocyte system postulates that monocytes circulating in the bloodstream are recruited to inflamed tissues where they give rise to macrophages. A recent publication demonstrated that the large increase in the macrophages observed during infection was the result of the multiplication of these cells rather than the recruitment of blood monocytes. We demonstrated previously that B-1 cells undergo differentiation to acquire a mononuclear phagocyte phenotype in vitro (B-1CDP, and we propose that B-1 cells could be an alternative origin for peritoneal macrophages. A number of recent studies that describe the phagocytic and microbicidal activity of B-1 cells in vitro and in vivo support this hypothesis. Based on these findings, we further investigated the differentiation of B-1 cells into phagocytes in vivo in response to LPS-induced inflammation. Therefore, we investigated the role of B-1 cells in the composition of the peritoneal macrophage population after LPS stimulation using osteopetrotic mice, BALB/Xid mice and the depletion of monocytes/macrophages by clodronate treatment. We show that peritoneal macrophages appear in op/op((-/- mice after LPS stimulation and exhibit the same Ig gene rearrangement (VH11 that is often found in B-1 cells. These results strongly suggest that op/op((-/- peritoneal "macrophages" are B-1CDP. Similarly, the LPS-induced increase in the macrophage population was observed even following monocyte/macrophage depletion by clodronate. After monocyte/macrophage depletion by clodronate, LPS-elicited macrophages were observed in BALB/Xid mice only following the transfer of B-1 cells. Based on these data, we confirmed that B-1 cell differentiation into phagocytes also occurs in vivo. In conclusion, the results strongly suggest that B-1 cell derived phagocytes are a component of

  12. Aspirin inhibits LPS-induced macrophage activation via the NF-κB pathway.

    Science.gov (United States)

    Liu, Yitong; Fang, Silian; Li, Xiaoyan; Feng, Jie; Du, Juan; Guo, Lijia; Su, Yingying; Zhou, Jian; Ding, Gang; Bai, Yuxing; Wang, Songling; Wang, Hao; Liu, Yi

    2017-09-14

    Aspirin (acetylsalicylic acid, ASA) has been shown to improve bone marrow mesenchymal stem cell-based calvarial bone regeneration by promoting osteogenesis and inhibiting osteoclastogenesis. However, it remains unknown whether aspirin influences other immune cells during bone formation. In the present study, we investigated whether ASA treatment influenced macrophage activation during the LPS inducement. We found that ASA could downregulate the expressions of iNOS and TNF-α both in mouse peritoneum macrophages and RAW264.7 cells induced by LPS via the IκK/IκB/NF-κB pathway and a COX2/PGE2/EP2/NF-κB feedback loop, without affecting the expressions of FIZZ/YM-1/ARG1 induced by IL-4. Furthermore, we created a rat mandibular bone defect model and showed that ASA treatment improved bone regeneration by inhibiting LPS-induced macrophage activation in the early stages of inflammation. Taken together, our results indicated that ASA treatment was a feasible strategy for improving bone regeneration, particularly in inflammatory conditions.

  13. Effect of sydnone SYD-1 on certain functions of LPS-stimulated macrophages.

    Science.gov (United States)

    Bizetto, Eduardo Luis; Noleto, Guilhermina Rodrigues; Echevarria, Aurea; Canuto, André Vinicius; Cadena, Silvia Maria Suter Correia

    2012-01-01

    In an earlier article, we demonstrated that sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) inhibits electron transport in the respiratory chain and uncouples oxidative phosphorylation, and postulated that these effects are probably involved in its antitumor activity. We now report the effect of SYD-1 on certain macrophage functions, considering the important role of these cells in inflammatory response and also the relevant anti-inflammatory activity reported for some sydnones. Incubation of macrophages with SYD-1 (5-100 μM) for 48 h did not affect the cell viability up to a concentration of 50 μM. However, at the highest concentration (100 μM), the compound decreased macrophage viability by ~20%. In assays involving 2 h and 24 h of incubation, SYD-1 (5-100 μM) did not affect the cell viability. The incubation of macrophages with the compound for 2 h promoted a dose-dependent reduction of phagocytic activity of up to ~65% (100 μM). SYD-1 (100 μM) was also able to increase the production of superoxide anion (~50%). In the absence of LPS, SYD-1 decreased NO production dose-dependently by up to ~80% (100 μM). When SYD-1 and LPS were incubated concomitantly, the decrease of NO promoted by SYD was the most pronounced, reaching up to ~98% at the same concentration (50 μM). SYD-1 dose-dependently suppressed IL-6 secretion by LPS-stimulated macrophages, reaching up to ~90% of inhibition at the highest concentration (100 μM). These results indicate that SYD-1 promotes effects similar to those described for anti-inflammatory and immunosuppressive drugs, thus motivating further studies to clarify the mechanisms involved in this activity.

  14. Human macrophages chronically exposed to LPS can be reactivated by stimulation with MDP to acquire an antimicrobial phenotype.

    Science.gov (United States)

    Guzmán-Beltrán, Silvia; Torres, Martha; Arellano, Monserrat; Juárez, Esmeralda

    2017-02-21

    Macrophages are important in host defense and can differentiate into functionally distinct subsets named classically (M1) or alternatively (M2) activated. In several inflammatory disorders, macrophages become tolerized to prevent deleterious consequences. This tolerization reduces the ability of macrophages to respond to bacterial components (e.g., LPS) maintaining a low level of inflammation but compromising the ability of macrophages to mount an effective immune response during subsequent pathogen encounters. In this study, we aimed to reactivate human monocyte-derived macrophages chronically exposed to LPS by re-stimulation with muramyl dipeptide (MDP). We observed an undefined profile of cell surface marker expression during endotoxin tolerance and absence of TNFα production. Stimulating macrophages chronically exposed to LPS with LPS+MDP restored TNFα, production together with an increased production of IL1, IL6, IFNγ, IL4, IL5 and IL10. These results suggest that macrophages chronically exposed to LPS possess a mixed M1-M2 phenotype with sufficient antimicrobial and homeostatic potential.

  15. Procalcitonin neutralizes bacterial LPS and reduces LPS-induced cytokine release in human peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Matera Giovanni

    2012-05-01

    Full Text Available Abstract Background Procalcitonin (PCT is a polypeptide with several cationic aminoacids in its chemical structure and it is a well known marker of sepsis. It is now emerging that PCT might exhibit some anti-inflammatory effects. The present study, based on the evaluation of the in vitro interaction between PCT and bacterial lipopolisaccharide (LPS, reports new data supporting the interesting and potentially useful anti-inflammatory activity of PCT. Results PCT significantly decreased (p Salmonella typhimurium (rough chemotype and Escherichia coli (smooth chemotype. Subsequently, the in vitro effects of PCT on LPS-induced cytokine release were studied in human peripheral blood mononuclear cells (PBMC. When LPS was pre-incubated for 30 minutes with different concentrations of PCT, the release of interleukin-10 (IL-10 and tumor necrosis factor alpha (TNFα by PBMC decreased in a concentration-dependent manner after 24 hours for IL-10 and 4 hours for TNFα. The release of monocyte chemotactic protein-1 (MCP-1 exhibited a drastic reduction at 4 hours for all the PCT concentrations assessed, whereas such decrease was concentration-dependent after 24 hours. Conclusions This study provides the first evidence of the capability of PCT to directly neutralize bacterial LPS, thus leading to a reduction of its major inflammatory mediators.

  16. The 5-lipoxygenase inhibitor, zileuton, suppresses prostaglandin biosynthesis by inhibition of arachidonic acid release in macrophages

    Science.gov (United States)

    Rossi, A; Pergola, C; Koeberle, A; Hoffmann, M; Dehm, F; Bramanti, P; Cuzzocrea, S; Werz, O; Sautebin, L

    2010-01-01

    BACKGROUND AND PURPOSE Zileuton is the only 5-lipoxygenase (5-LOX) inhibitor marketed as a treatment for asthma, and is often utilized as a selective tool to evaluate the role of 5-LOX and leukotrienes. The aim of this study was to investigate the effect of zileuton on prostaglandin (PG) production in vitro and in vivo. EXPERIMENTAL APPROACH Peritoneal macrophages activated with lipopolysaccharide (LPS)/interferon γ (LPS/IFNγ), J774 macrophages and human whole blood stimulated with LPS were used as in vitro models and rat carrageenan-induced pleurisy as an in vivo model. KEY RESULTS Zileuton suppressed PG biosynthesis by interference with arachidonic acid (AA) release in macrophages. We found that zileuton significantly reduced PGE2 and 6-keto prostaglandin F1α (PGF1α) levels in activated mouse peritoneal macrophages and in J774 macrophages. This effect was not related to 5-LOX inhibition, because it was also observed in macrophages from 5-LOX knockout mice. Notably, zileuton inhibited PGE2 production in LPS-stimulated human whole blood and suppressed PGE2 and 6-keto PGF1α pleural levels in rat carrageenan-induced pleurisy. Interestingly, zileuton failed to inhibit the activity of microsomal PGE2 synthase1 and of cyclooxygenase (COX)-2 and did not affect COX-2 expression. However, zileuton significantly decreased AA release in macrophages accompanied by inhibition of phospholipase A2 translocation to cellular membranes. CONCLUSIONS AND IMPLICATION Zileuton inhibited PG production by interfering at the level of AA release. Its mechanism of action, as well as its use as a pharmacological tool, in experimental models of inflammation should be reassessed. PMID:20880396

  17. Ca(OH2 action on TNF-alpha and NO release in macrophage culture stimulated by Pseudomonas aeruginosa LPS = Ação do Ca(OH2 sobre a produção de TNF-alfa e NO de cultura de macrófagos estimulada por LPS de Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Queiroz, Celso Emanoel de Souza

    2008-01-01

    Full Text Available Objetivo: A ação do hidróxido de cálcio [Ca(OH2] com o sistema imune e o mecanismo de neutralização das bactérias e seus subprodutos ainda não foi completamente esclarecida. Neste estudo foi avaliada a capacidade do Ca(OH2 em neutralizar o lipopolissacarídeo (LPS de Pseudomonas aeruginosa, utilizando-se duas metodologias: liberação de Óxido Nítrico (NO e Fator de Necrose Tumoral Alfa (TNF-alfa em cultura de macrófagos peritoneais de camundongos. Metodologia: No ensaio do NO, as células peritoneais foram expostas a uma solução de LPS (25mg/mL e 50mg/mL; e à suspensão de LPS/Ca(OH2 em duas concentrações (50mg/ 25mg e 25mg/25mg. Após 8 horas de incubação, foi utilizado reagente de Griess, e a liberação de NO foi quantificada. No ensaio do TNF-alfa, a solução de LPS foi usada na concentração de 25mg/mL e o LPS/Ca(OH2 a 25mg/25mg. Após 24 horas, as células foram fixadas e coradas com cristal violeta, e os valores de absorbância foram obtidos. Os resultados foram expressos em micromols. Todos os testes foram realizados em triplicata. Resultados: A presença de Ca(OH2 nas duas concentrações avaliadas reduziu significativamente a liberação de NO e TNF-alfa. Conclusão: Pode-se concluir que o LPS bacteriano representa um forte estímulo para liberação destas citocinas, mas o hidróxido de cálcio foi capaz de neutralizar este efeito

  18. Inhibition of CDKS by roscovitine suppressed LPS-induced ·NO production through inhibiting NFκB activation and BH4 biosynthesis in macrophages

    Science.gov (United States)

    Wei, Na; Guan, Tongju; Xu, Hao; An, Jianzhong; Pritchard, Kirkwood A.

    2009-01-01

    In inflammatory diseases, tissue damage is critically associated with nitric oxide (·NO) and cytokines, which are overproduced in response to cellular release of endotoxins. Here we investigated the inhibitory effect of roscovitine, a selective inhibitor of cyclin-dependent kinases (CDKs) on ·NO production in mouse macrophages. In RAW264.7 cells, we found that roscovitine abolished the production of ·NO induced by lipopolysaccharide (LPS). Moreover, roscovitine significantly inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA and protein expression. Our data also showed that roscovitine attenuated LPS-induced phosphorylation of IκB kinase β (IKKβ), IκB, and p65 but enhanced the phosphorylation of ERK, p38, and c-Jun NH2-terminal kinase (JNK). In addition, roscovitine dose dependently inhibited LPS-induced expression of cyclooxygenase-2 (COX)-2, IL-1β, and IL-6 but not tumor necrosis factor (TNF)-α. Tetrahydrobiopterin (BH4), an essential cofactor for iNOS, is easily oxidized to 7,8-dihydrobiopterin (BH2). Roscovitine significantly inhibited LPS-induced BH4 biosynthesis and decreased BH4-to-BH2 ratio. Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase-1 (GCH-1), the rate-limiting enzyme for BH4 biosynthesis. Using other CDK inhibitors, we found that CDK1, CDK5, and CDK7, but not CDK2, significantly inhibited LPS-induced ·NO production in macrophages. Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited ·NO production, iNOS, and COX-2 upregulation, activation of NFκB, and induction of GCH-1 by LPS. Together, our data indicate that roscovitine abolishes LPS-induced ·NO production in macrophages by suppressing nuclear factor-κB activation and BH4 biosynthesis, which might be mediated by CDK1, CDK5, and CDK7. Our results also suggest that roscovitine may inhibit inflammation and that CDKs may play important roles in the mechanisms by which roscovitine attenuates inflammation. PMID:19553566

  19. A regulatory role for macrophage class A scavenger receptors in TLR4-mediated LPS responses.

    Science.gov (United States)

    Chen, Yunying; Wermeling, Fredrik; Sundqvist, Johanna; Jonsson, Ann-Beth; Tryggvason, Karl; Pikkarainen, Timo; Karlsson, Mikael C I

    2010-05-01

    Recognition of microbial components by TLR, key sensors of infection, leads to induction of inflammatory responses. We found that, in vivo, TLR4 engagement by LPS induces up-regulation of the class A scavenger receptors (SR) macrophage receptor with a collagenous structure (MARCO) and SR-A, which occurs, at least in the case of MARCO, via both MyD88-dependent and -independent pathways. When challenging mice with a low dose of LPS followed by a high dose, class A SR-deficient mice showed a higher survival rate than WT mice. This was paired with increased production of IL-10 and anti-LPS Ab, as well as increased activation status of marginal zone B cells. However, the receptors were not crucial for survival when challenging mice i.p. with Neisseria meningitidis or Listeria monocytogenes, but they were found to contribute to microbial capture and clearance. This indicates physiological significance for the up-regulation of class A SR during early stages of bacterial infection. Thus, we believe that we have revealed a mechanism where SR regulate the activation status of the immune system and are involved in balancing a proper immune response to infection. This regulation could also be important in maintaining tolerance since these receptors have been shown to be involved in regulation of self-reactivity.

  20. Comparative nitric oxide production by LPS-stimulated monocyte-derived macrophages from Ovis canadensis and Ovis aries.

    Science.gov (United States)

    Sacco, R E; Waters, W R; Rudolph, K M; Drew, M L

    2006-01-01

    Bighorn sheep are more susceptible to respiratory infection by Mannheimia haemolytica than are domestic sheep. In response to bacterial challenge, macrophages produce a number of molecules that play key roles in the inflammatory response, including highly reactive nitrogen intermediates such as nitric oxide (NO). Supernatants from monocyte-derived macrophages cultured with M. haemolytica LPS were assayed for nitric oxide activity via measurement of the NO metabolite, nitrite. In response to LPS stimulation, bighorn sheep macrophages secreted significantly higher levels of NO compared to levels for non-stimulated macrophages. In contrast, levels of NO produced by domestic sheep macrophages in response to M. haemolytica LPS did not differ from levels detected in non-stimulated cell cultures. Nitrite levels detected in supernatants of LPS-stimulated bighorn macrophage cultures treated with an inducible nitric oxide synthase (INOS) inhibitor, N(G)-monomethyl-L-arginine, were similar to that observed in non-stimulated cultures indicating a role for the iNOS pathway.

  1. Neoagaro-oligosaccharide monomers inhibit inflammation in LPS-stimulated macrophages through suppression of MAPK and NF-κB pathways

    Science.gov (United States)

    Wang, Wei; Liu, Pei; Hao, Cui; Wu, Lijuan; Wan, Wenjin; Mao, Xiangzhao

    2017-01-01

    Neoagaro-oligosaccharides derived from agarose have been demonstrated to possess a variety of biological activities, such as anti-bacteria and anti-oxidative activities. In this study, we mainly explored the inhibitory effects and the mechanisms of neoagaro-oligosaccharide monomers against LPS-induced inflammatory responses in mouse macrophage RAW264.7 cells. The results indicated that neoagaro-oligosaccharide monomers especially neoagarotetraose could significantly reduce the production and release of NO in LPS-induced macrophages. Neoagarotetraose significantly suppressed the expression and secretion of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines such as TNF-α and IL-6. The inhibition mechanisms may be associated with the inhibition of the activation of p38MAPK, Ras/MEK/ERK and NF-κB signaling pathways. Thus, neoagarotetraose may attenuate the inflammatory responses through downregulating the MAPK and NF-κB signaling pathways in LPS-stimulated macrophages. In summary, the marine-derived neoagaro-oligosaccharide monomers merit further investigation as novel anti-inflammation agents in the future. PMID:28266652

  2. Release of salusin-beta from human monocytes/macrophages.

    Science.gov (United States)

    Sato, Kengo; Fujimoto, Kazumi; Koyama, Takatoshi; Shichiri, Masayoshi

    2010-06-01

    Salusin-alpha and salusin-beta are related bioactive peptides biosynthesized from the same precursor, prosalusin. Despite the potent hemodynamic and proatherosclerotic activities of salusin-beta, its exact distribution and biological functions remain largely undetermined because of technical difficulties associated with its unique physicochemical characteristics, such as marked adhesiveness to polypropylene and polystyrene. By circumventing these problems, we recently established a specific radioimmunoassay for detecting immunoreactive human salusin-beta. In the current study, we demonstrated the release of salusin-beta from the human monoblastic leukemia cell lines, THP-1 and U937. Dilution curves of extracted conditioned media from both cells were parallel with those of standard human salusin-beta by radioimmunoassay. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection of the culture supernatants revealed a major immunoreactive component that co-eluted with authentic salusin-beta. Both cell lines secreted salusin-beta-like immunoreactivity (LI) into serum-free media as a function of time (1234.3 + or - 122.7 and 186.7 + or - 9.1 fmol/10(5) cells per 24h). When THP-1 and U937 cells differentiated into macrophages after incubation with 2-O-tetradecanoylphorbol-13-acetate (TPA), they secreted far greater amounts of salusin-beta-LI into the culture supernatant (3351.9 + or - 899.3 and 1545.8 + or - 183.3 fmol/10(5) cells per 24h). TPA treatment accelerated the processing of prosalusin into its cleaved fragments, suggesting that the increased secretion of salusin-beta-LI in THP-1-derived macrophages was caused by the enhanced intracellular processing of prosalusin. Stimulation with the inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS), resulted in increased secretion of salusin-beta without inducing expression of the gene for preprosalusin, suggesting that TNF-alpha and LPS stimulated

  3. Anti-myeloperoxidase antibodies attenuate the monocyte response to LPS and shape macrophage development

    Science.gov (United States)

    Popat, Reena J.; Hakki, Seran; Coughlan, Alice M.; Watson, Julie; Little, Mark A.; Spickett, Corinne M.; Lavender, Paul; Afzali, Behdad; Kemper, Claudia; Robson, Michael G.

    2017-01-01

    Anti-neutrophil cytoplasmic antibody (ANCA) vasculitis is characterized by the presence of autoantibodies to myeloperoxidase and proteinase-3, which bind monocytes in addition to neutrophils. While a pathological effect on neutrophils is acknowledged, the impact of ANCA on monocyte function is less well understood. Using IgG from patients we investigated the effect of these autoantibodies on monocytes and found that anti-myeloperoxidase antibodies (MPO-ANCA) reduced both IL-10 and IL-6 secretion in response to LPS. This reduction in IL-10 and IL-6 depended on Fc receptors and enzymatic myeloperoxidase and was accompanied by a significant reduction in TLR-driven signaling pathways. Aligning with changes in TLR signals, oxidized phospholipids, which function as TLR4 antagonists, were increased in monocytes in the presence of MPO-ANCA. We further observed that MPO-ANCA increased monocyte survival and differentiation to macrophages by stimulating CSF-1 production. However, this was independent of myeloperoxidase enzymatic activity and TLR signaling. Macrophages differentiated in the presence of MPO-ANCA secreted more TGF-β and further promoted the development of IL-10– and TGF-β–secreting CD4+ T cells. Thus, MPO-ANCA may promote inflammation by reducing the secretion of antiinflammatory IL-10 from monocytes, and MPO-ANCA can alter the development of macrophages and T cells to potentially promote fibrosis. PMID:28138552

  4. Effects of propofol on lipopolysaccharide-induced expression and release of HMGB1 in macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Wang, T.; Wei, X.Y.; Liu, B.; Wang, L.J.; Jiang, L.H. [Department of Anesthesiology, the Third Affiliated Hospital, Zhengzhou University, Zhengzhou (China)

    2015-02-24

    This study aimed to determine the effects of different concentrations of propofol (2,6-diisopropylphenol) on lipopolysaccharide (LPS)-induced expression and release of high-mobility group box 1 protein (HMGB1) in mouse macrophages. Mouse macrophage cell line RAW264.7 cells were randomly divided into 5 treatment groups. Expression levels of HMGB1 mRNA were detected using RT-PCR, and cell culture supernatant HMGB1 protein levels were detected using enzyme-linked immunosorbent assay (ELISA). Translocation of HMGB1 from the nucleus to the cytoplasm in macrophages was observed by Western blotting and activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus was detected using ELISA. HMGB1 mRNA expression levels increased significantly in the cell culture supernatant and in cells after 24 h of stimulating RAW264.7 cells with LPS (500 ng/mL). However, HMGB1 mRNA expression levels in the P2 and P3 groups, which received 500 ng/mL LPS with 25 or 50 μmol/mL propofol, respectively, were significantly lower than those in the group receiving LPS stimulation (P<0.05). After stimulation by LPS, HMGB1 protein levels were reduced significantly in the nucleus but were increased in the cytoplasm (P<0.05). Simultaneously, the activity of NF-κB was enhanced significantly (P<0.05). After propofol intervention, HMGB1 translocation from the nucleus to the cytoplasm and NF-κB activity were inhibited significantly (each P<0.05). Thus, propofol can inhibit the LPS-induced expression and release of HMGB1 by inhibiting HMGB1 translocation and NF-κB activity in RAW264.7 cells, suggesting propofol may be protective in patients with sepsis.

  5. The LPS-induced neutrophil recruitment into rat air pouches is mediated by TNFα: likely macrophage origin

    Directory of Open Access Journals (Sweden)

    C-D. Arreto

    1997-01-01

    Full Text Available The role of resident cells during the lipopolysaccharide (LPS-induced neutrophil recruitment into rat air pouches was investigated. In this model, LPS (Escherichia coli, O55: B5 strain; 2–2000 ng induced a dose– and time-dependent neutrophil recruitment accompanied by the generation of a tumour necrosis factor-α (TNFα-like activity. Dexamethasone (0.05–5 mug and cycloheximide (6 ng, injected 2 h before LPS into the pouches, inhibited the neutrophil recruitment and the generation of the TNFα-like activity, while the H1-receptor antagonist mepyramine (1 and 4 mg/kg, i.p., 0.5 h before LPS and the PAF-receptor antagonist WEB 2170 (0.05 and 1 mg/kg, i.p., 0.5 h before LPS had no effect. Purified alveolar macrophages (AM were used to replenish the pouches of cycloheximide-treated recipient rats. AM provided by PBS-treated animals led to the recovery of the LPS-induced neutrophil recruitment and of the TNFα-like formation contrasting with those from cycloheximide-treated animals (1 mg/kg, i.p.. When delivered in situ, liposome-encapsulated clodronate, a macrophage depletor, significantly impaired both the LPSinduced neutrophil recruitment and the TNFα-like activity. An anti-murine TNFα polyclonal antibody (0.5 h before LPS was also effective. These results emphasize the pivotal role of macrophages for LPS-induced neutrophil recruitment via the formation of TNFα.

  6. Anti-inflammatory role of microRNA let-7c in LPS treated alveolar macrophages by targeting STAT3

    Institute of Scientific and Technical Information of China (English)

    Ji-Hui Yu; Li Long; Zhi-Xiao Luo; Lin-Man Li; Jie-Ru You

    2016-01-01

    Objective: To explore the expression of microRNA (miRNA) let-7c and its function in chronic obstructive pulmonary disease (COPD) and alveolar macrophage cells. Methods: Real time PCR was performed to detect the expression of miRNA let-7c in the lung tissue of COPD patients and COPD model in mice. MiRNA let-7c was overexpressed in alveolar macrophages isolated from mice and its effect was measured by the production of pro-inflammation cytokines and the protein level of signal transducer and activator of transcription 3 (STAT3) as well as phosphorylation level of STAT3 after LPS stimulation. Luciferase assay was used to detect the binding of miRNA let-7c and 3'UTR of STAT3. Results: MiRNA let-7c expression was significantly lower in patients with COPD compared with control group, and the similar result was found in COPD mice and LPS stimulated alveolar macrophages. Overexpression of miRNA let-7c in alveolar macrophages inhibited LPS-induced increasing of tumor necrosis factor alpha, interleukin-6 and interleukin-1β. Luciferase assay showed STAT3 was a targeting of miRNA let-7c in alveolar macrophages. Conclusions: MiRNA let-7c low expression in COPD can regulate inflammatory responses by targeting STAT3 in alveolar macrophage, which may provide a new target for COPD treatment strategies.

  7. Methods to Prevent or Treat Refractory Diseases by Focusing on Intestinal Microbes Using LPS and Macrophages.

    Science.gov (United States)

    Soma, Gen-Ichiro; Inagawa, Hiroyuki

    2015-08-01

    Intestinal microbes are known to influence host homeostasis by producing various substances. Recently, the presence of a diverse range of intestinal microbiota has been shown to play a key role in the maintenance of health, along with influencing the host's innate immunity towards various diseases. For example, fecal microbiota transplantation (FMT) from healthy individuals was remarkably effective in cases of refractory Clostridium difficile colitis. Conversely, decreased number of intestinal microbes resulting from the oral administration of antibiotics reportedly suppressed the antitumor effects of immunotherapy or anticancer drugs. Furthermore, it has been shown that a change in the intestinal environment triggered by oral administration of antibiotics resulted in increased number of drug-resistant microbes causing nosocomial infections. Intestinal microbes are also shown to be effective in cancer treatment as they activate macrophages at the site of cancer. One of the effects of intestinal microbes on hosts that has been gaining increasing attention is the biological regulation caused by the lipopolysaccharides (LPS) produced by Gram-negative bacteria. Among the intestinal microbiota present in the host, Gram-negative bacteria form the most dominant flora. The administration of antibiotics leads to a decreased number of intestinal microbes, as well as to suppression of cancer immunotherapy effects or anticancer drug effects, and this deterioration has been shown to be improved by oral administration of LPS. In this article, we discuss the functions of intestinal microbiota, that is currently undergoing a paradigm shift in relation to maintenance of health and the validity of LPS as a possible target for bio-treatment in the future.

  8. 粗根荨麻水提取部分对佐剂性关节炎大鼠腹腔巨噬细胞分泌TNF-α及PGE2的影响%The Effects of Aqueous Fraction of Urtica macrorrhiza Hand-Mazz on Production of TNF-α, PGE2 Release from Peritoneal Macrophages Induced by LPS in Adjuvant Arthritis Rats

    Institute of Scientific and Technical Information of China (English)

    李晓红; 赵永娜; 邵晓霞; 李顺英; 张荣平

    2008-01-01

    To investigate the effects of aqueous fraction of Urtica macrorrhiza Hand-Mazz(Ur) on modulating tumor nec- rosis factor-alpha (TNF-α)and prostaglandin E2 (PGE2) production induced by lipopolysaceharide (LPS) in peritoneal macrophages in adjuvant arthritis rats and elucidate the possible mechanisms of anti-inflammatory and antirheumatoid effects of Ur, adjuvant arthritis (AA) rat was used as the model. The PMψ samples were taken at different time after medication. TNF-α, PGE2 levels were :measured by ELISA method. Production of TNF-α, and PGE2 increased in the cul-ture supematant of PMψ in AA model rats. Ur(400 and 200 mg/kg) could inhibit TNF-α and PGE2 release induced by LPS from PMψ in AA rats. The anti-inflaramatory mechanisms of Ur in AA rats might be reIated to its inhibitory effects on the level of TNF-α and PGE2 from PMψ in vivo.%观察滇产粗根荨麻水提取部分对佐剂性关节炎(adjuvant arthritis,AA)大鼠腹腔巨噬细胞(peritoneal macrophages,PMcp)分泌肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)及前列腺素E2(prostaglandin E2,PGE2)的影响.建立大鼠佐剂性关节炎模型,Ur水提取部分连续灌胃给药14或21 d后分次获取大鼠腹腔巨噬细胞,脂多糖(lipopolysacehafide,LPS)诱导大鼠腹腔巨噬细胞,用酶联免疫吸附法检测培养上清液中TNF-α及PGE2水平.从大鼠腹腔巨噬细胞TNF-α及PGE2分泌较正常组升高,Ur水提取部分(400,200 mg/kg)对LPS诱导的AA大鼠腹腔巨噬细胞分泌TNF-α及PGE2水平有明显抑制作用.滇产粗根荨麻水提取部分对佐剂性关节炎的治疗作用可能与其抑制腹腔巨噬细胞分泌TNF-α及PGE2有关.

  9. Inhibition of Emodin on LPS-induced Nitric Oxide Generation by Suppressing PLC-γ Phosphorylation in Rat Peritoneal Macrophages

    Institute of Scientific and Technical Information of China (English)

    WANG Xin-yu; CAI Shou-guang; WU Yi-fen; LI Jun-ying; YANG Wen-xiu; HU Fen

    2010-01-01

    Objective To investigate the inhibitory mechanism of emodin on lipopolysaccharide(LPS)-induced nitric oxide(NO)generation in rat peritoneal macrophages.Methods NO production and iNOS expression were measured through nitrite assay and Western blotting assay,respectively.NF-kB activity and nuclei P65 expression were estimated by dual-luciferase and Western blotting assay,respectively.Intracellular free Ca2+([Ca2+]i)was detected using the ratiometric fluorescent calcium indicator dye,Fura-2,and a microspectrofluorometer.PLC-γphosporylation was analyzed by Western blotting assay.Results First,emodin was found playing active roles in suppressing LPS-induced NF-kB activation in rat peritoneal macrophages.Second,emodin down-regulated transient[Ca2*]i and could increase in NF-kB upstream signal.Finally,emodin suppressed phosphorylation of PLC-γ by LPS stimulation in the upstream of[Ca2+]i.Conclusion Suppression of PLC-γ phosphorylation is involved in emodin inhibiting NO generation by LPS stimulation in rat peritoneal macrophages.

  10. Atorvastatin Attenuates TNF-alpha Production via Heme Oxygenase-1 Pathway in LPS-stimulated RAW264.7 Macrophages

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao Qiao; LUO Nian Sang; CHEN Zhong Qing; LIN Yong Qing; GU Miao Ning; CHEN Yang Xin

    2014-01-01

    ObjectiveTo assess the effect of atorvastatin on lipopolysaccharide(LPS)-inducedTNF-α production in RAW264.7 macrophages. MethodsRAW264.7 macrophageswere treated in different LPS concentrations oratdifferent time points with or without atorvastatin. TNF-α level in supernatant was measured. Expressions of TNF-αmRNA and protein and heme oxygenase-1 (HO-1) were detected by ELISA, PCR, and Western blot, respectively. HOactivity was assayed. ResultsLPS significantly increased the TNF-α expression and secretion in a dose- and time-dependent manner. The HO-1activity and HO-1 expression level were significantly higher after atorvastatin treatment than before atorvastatin treatment and attenuated by SB203580 and PD98059 but not by SP600125, suggesting that the ERK and p38 mitogen-activated protein kinase (MAPK) pathways participate inregulating the above-mentioned effects of atorvastatin. Moreover, the HO-1 activity suppressed by SnPP or the HO-1 expression inhibited by siRNA significantly attenuated the effect of atorvastatin onTNF-α expression and production in LPS-stimulated macrophages. ConclusionAtorvastatin can attenuate LPS-induced TNF-α expression and production by activating HO-1 via the ERK and p38 MAPK pathways,suggesting that atorvastatin can be used in treatment of inflammatory diseases such as sepsis, especially in those with atherosclerotic diseases.

  11. Comparison of non-crystalline silica nanoparticles in IL-1β release from macrophages

    Directory of Open Access Journals (Sweden)

    Sandberg Wiggo J

    2012-08-01

    Full Text Available Abstract Background Respirable crystalline silica (silicon dioxide; SiO2, quartz particles are known to induce chronic inflammation and lung disease upon long-term inhalation, whereas non-crystalline (amorphous SiO2 particles in the submicrometre range are regarded as less harmful. Several reports have demonstrated that crystalline, but also non-crystalline silica particles induce IL-1β release from macrophages via the NALP3-inflammasome complex (caspase-1, ASC and NALP3 in the presence of lipopolysaccharide (LPS from bacteria. Our aim was to study the potential of different non-crystalline SiO2 particles from the nano- to submicro-sized range to activate IL-1β responses in LPS-primed RAW264.7 macrophages and primary rat lung macrophages. The role of the NALP3-inflammasome and up-stream mechanisms was further explored in RAW264.7 cells. Results In the present study, we have shown that 6 h exposure to non-crystalline SiO2 particles in nano- (SiNPs, 5–20 nm, 50 nm and submicro-sizes induced strong IL-1β responses in LPS-primed mouse macrophages (RAW264.7 and primary rat lung macrophages. The primary lung macrophages were more sensitive to Si-exposure than the RAW-macrophages, and responded more strongly. In the lung macrophages, crystalline silica (MinUsil 5 induced IL-1β release more potently than the non-crystalline Si50 and Si500, when adjusted to surface area. This difference was much less pronounced versus fumed SiNPs. The caspase-1 inhibitor zYVAD and RNA silencing of the NALP3 receptor reduced the particle-induced IL-1β release in the RAW264.7 macrophages. Furthermore, inhibitors of phagocytosis, endosomal acidification, and cathepsin B activity reduced the IL-1β responses to the different particles to a similar extent. Conclusions In conclusion, non-crystalline silica particles in the nano- and submicro-size ranges seemed to induce IL-1β release from LPS-primed RAW264.7 macrophages via similar mechanisms as crystalline

  12. DMPD: Function of lipopolysaccharide (LPS)-binding protein (LBP) and CD14, thereceptor for LPS/LBP complexes: a short review. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 1373512 Function of lipopolysaccharide (LPS)-binding protein (LBP) and CD14, thereceptor for LPS/LBP complex....html) (.csml) Show Function of lipopolysaccharide (LPS)-binding protein (LBP) and CD14, thereceptor for LPS/LBP complex...ride (LPS)-binding protein (LBP) and CD14, thereceptor for LPS/LBP complexes: a short review. Authors Schuma

  13. Genome-wide analysis of TIAR RNA ligands in mouse macrophages before and after LPS stimulation

    Directory of Open Access Journals (Sweden)

    Yacine Kharraz

    2016-03-01

    Full Text Available TIA-1 related protein (TIAR is a RNA-binding protein involved in several steps of gene expression such as RNA splicing Aznarez et al. (2008 [1] and translation Piecyk et al. (2000 [2]. TIAR contains three RNA recognition motifs (RRMs allowing its interaction with specific sequences localized in the untranslated regions (UTRs of several mRNAs. In myeloid cells, TIAR has been shown to bind and regulate the translation and stability of various mRNA-encoding proteins important for the inflammatory response, such as TNFα Piecyk et al. (2000, Gueydan et al. (1999 [2,3], Cox-2 Cok et al. (2003 [4] or IL-8 Suswam et al. (2005 [5]. Here, we generated two macrophage-like RAW 264.7 cell lines expressing either a tagged full-length TIAR protein or a RRM2-truncated mutant unable to bind RNA with high affinity Dember et al. (1996, Kim et al. (2013 . By a combination of RNA-IP and microarray analysis (RIP-chip, we identified mRNAs specifically bound by the full-length protein both in basal conditions and in response to LPS (GSE77577.

  14. DMPD: LPS, TLR4 and infectious disease diversity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15608698 LPS, TLR4 and infectious disease diversity. Miller SI, Ernst RK, Bader MW.... Nat Rev Microbiol. 2005 Jan;3(1):36-46. (.png) (.svg) (.html) (.csml) Show LPS, TLR4 and infectious disease diversity.... PubmedID 15608698 Title LPS, TLR4 and infectious disease diversity. Authors Miller SI, Ernst RK,

  15. DMPD: LPS/TLR4 signal transduction pathway. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18304834 LPS/TLR4 signal transduction pathway. Lu YC, Yeh WC, Ohashi PS. Cytokine. ...2008 May;42(2):145-51. Epub 2008 Mar 4. (.png) (.svg) (.html) (.csml) Show LPS/TLR4 signal transduction path...way. PubmedID 18304834 Title LPS/TLR4 signal transduction pathway. Authors Lu YC, Yeh WC, Ohashi PS. Publica

  16. DMPD: LPS induction of gene expression in human monocytes. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 11257452 LPS induction of gene expression in human monocytes. Guha M, Mackman N. Ce...ll Signal. 2001 Feb;13(2):85-94. (.png) (.svg) (.html) (.csml) Show LPS induction of gene expression in human... monocytes. PubmedID 11257452 Title LPS induction of gene expression in human monocytes. Authors Guha M, Ma

  17. Identification of a novel compound that inhibits iNOS and COX-2 expression in LPS-stimulated macrophages from Schisandra chinensis.

    Science.gov (United States)

    Lee, You Jin; Park, Sun Young; Kim, Sun Gun; Park, Da Jung; Kang, Jum Soon; Lee, Sang Joon; Yoon, Sik; Kim, Young Hun; Bae, Yoe-Sik; Choi, Young-Whan

    2010-01-22

    A novel alpha-iso-cubebenol, which has anti-inflammatory effects in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, was isolated from the fruits of Schisandra chinensis. alpha-iso-cubebenolinhibited LPS-induced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production. Consistent with these findings, alpha-iso-cubebenol also reduced the LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 at the protein and mRNA levels in a concentration-dependent manner. alpha-iso-cubebenol also inhibited LPS-induced nuclear translocation of the NF-kappaB p65 subunit. Furthermore, alpha-iso-cubebenol suppressed the phosphorylation of ERK, JNK, and p38 kinase induced by LPS. Since the novel alpha-iso-cubebenol blocked the production of several pro-inflammatory mediators induced by LPS in macrophages, the molecule can be useful material for the development of anti-inflammatory agents against bacterial infections or endotoxin.

  18. Pseuderanthemum palatiferum leaf extract inhibits the proinflammatory cytokines, TNF-α and IL-6 expression in LPS-activated macrophages.

    Science.gov (United States)

    Sittisart, Patcharawan; Chitsomboon, Benjamart; Kaminski, Norbert E

    2016-11-01

    The anti-inflammatory potential and underlying mechanisms of an ethanol extract of Pseuderanthemum palatiferum (EEP) leaves was investigated using LPS-activated macrophages. Our results show EEP produced a concentration-dependent suppression of TNF-α and IL-6 secretion by LPS-activated mouse peritoneal macrophages. EEP also suppressed LPS-induced TNF-α and IL-6 protein and mRNA levels in mouse-derived myeloid cell line RAW264.7. To further elucidate the molecular mechanisms responsible for impaired TNF-α and IL-6 regulation by EEP, the activation of transcription factors, NF-κB, C/EBP, and AP-1, was monitored using electrophoretic mobility shift assays. EEP suppressed LPS-induced NF-κB DNA binding activity within both the TNF-α and IL-6 promoters in RAW264.7 cells with impairment being more pronounced in the IL-6 promoter. In addition, EEP exhibited a concentration-dependent suppression of C/EBP and AP-1 DNA binding activity within the IL-6 promoter. Concordantly, IL-6 luciferase promoter reporter activity was also suppressed by EEP in transiently transfected RAW264.7 cells, upon LPS activation. EEP analysis by GC-MS and HPLC DAD-MSD revealed the presence of β-sitosterol and various polyphenols, respectively, which are known to possess anti-inflammatory activity. Collectively, these results suggest that the anti-inflammatory effects of EEP are mediated, at least in part, by modulating TNF-α and IL-6 expression through impairment of NF-κB, C/EBP, and AP-1 activity.

  19. Metformin Suppresses Lipopolysaccharide (LPS)-induced Inflammatory Response in Murine Macrophages via Activating Transcription Factor-3 (ATF-3) Induction*

    Science.gov (United States)

    Kim, Juyoung; Kwak, Hyun Jeong; Cha, Ji-Young; Jeong, Yun-Seung; Rhee, Sang Dahl; Kim, Kwang Rok; Cheon, Hyae Gyeong

    2014-01-01

    Metformin, a well known antidiabetic agent that improves peripheral insulin sensitivity, also elicits anti-inflammatory actions, but its mechanism is unclear. Here, we investigated the mechanism responsible for the anti-inflammatory effect of metformin action in lipopolysaccharide (LPS)-stimulated murine macrophages. Metformin inhibited LPS-induced production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in a concentration-dependent manner and in parallel induction of activating transcription factor-3 (ATF-3), a transcription factor and member of the cAMP-responsive element-binding protein family. ATF-3 knockdown abolished the inhibitory effects of metformin on LPS-induced proinflammatory cytokine production accompanied with reversal of metformin-induced suppression of mitogen-activated protein kinase (MAPK) phosphorylation. Conversely, AMP-activated protein kinase (AMPK) phosphorylation and NF-κB suppression by metformin were unaffected by ATF-3 knockdown. ChIP-PCR analysis revealed that LPS-induced NF-κB enrichments on the promoters of IL-6 and TNF-α were replaced by ATF-3 upon metformin treatment. AMPK knockdown blunted all the effects of metformin (ATF-3 induction, proinflammatory cytokine inhibition, and MAPK inactivation), suggesting that AMPK activation by metformin is required for and precedes ATF-3 induction. Oral administration of metformin to either mice with LPS-induced endotoxemia or ob/ob mice lowered the plasma and tissue levels of TNF-α and IL-6 and increased ATF-3 expression in spleen and lungs. These results suggest that metformin exhibits anti-inflammatory action in macrophages at least in part via pathways involving AMPK activation and ATF-3 induction. PMID:24973221

  20. Metformin suppresses lipopolysaccharide (LPS)-induced inflammatory response in murine macrophages via activating transcription factor-3 (ATF-3) induction.

    Science.gov (United States)

    Kim, Juyoung; Kwak, Hyun Jeong; Cha, Ji-Young; Jeong, Yun-Seung; Rhee, Sang Dahl; Kim, Kwang Rok; Cheon, Hyae Gyeong

    2014-08-15

    Metformin, a well known antidiabetic agent that improves peripheral insulin sensitivity, also elicits anti-inflammatory actions, but its mechanism is unclear. Here, we investigated the mechanism responsible for the anti-inflammatory effect of metformin action in lipopolysaccharide (LPS)-stimulated murine macrophages. Metformin inhibited LPS-induced production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in a concentration-dependent manner and in parallel induction of activating transcription factor-3 (ATF-3), a transcription factor and member of the cAMP-responsive element-binding protein family. ATF-3 knockdown abolished the inhibitory effects of metformin on LPS-induced proinflammatory cytokine production accompanied with reversal of metformin-induced suppression of mitogen-activated protein kinase (MAPK) phosphorylation. Conversely, AMP-activated protein kinase (AMPK) phosphorylation and NF-κB suppression by metformin were unaffected by ATF-3 knockdown. ChIP-PCR analysis revealed that LPS-induced NF-κB enrichments on the promoters of IL-6 and TNF-α were replaced by ATF-3 upon metformin treatment. AMPK knockdown blunted all the effects of metformin (ATF-3 induction, proinflammatory cytokine inhibition, and MAPK inactivation), suggesting that AMPK activation by metformin is required for and precedes ATF-3 induction. Oral administration of metformin to either mice with LPS-induced endotoxemia or ob/ob mice lowered the plasma and tissue levels of TNF-α and IL-6 and increased ATF-3 expression in spleen and lungs. These results suggest that metformin exhibits anti-inflammatory action in macrophages at least in part via pathways involving AMPK activation and ATF-3 induction. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. DMPD: The Lps locus: genetic regulation of host responses to bacteriallipopolysaccharide. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 10669111 The Lps locus: genetic regulation of host responses to bacteriallipopolysa...ccharide. Qureshi ST, Gros P, Malo D. Inflamm Res. 1999 Dec;48(12):613-20. (.png) (.svg) (.html) (.csml) Show The... Lps locus: genetic regulation of host responses to bacteriallipopolysaccharide. PubmedID 10669111 Title The

  2. Nfkb1 inhibits LPS-induced IFN-β and IL-12 p40 production in macrophages by distinct mechanisms.

    Directory of Open Access Journals (Sweden)

    Xixing Zhao

    Full Text Available BACKGROUND: Nfkb1-deficient murine macrophages express higher levels of IFN-β and IL-12 p40 following LPS stimulation than control macrophages, but the molecular basis for this phenomenon has not been completely defined. Nfkb1 encodes several gene products including the NF-κB subunit p50 and its precursor p105. p50 is derived from the N-terminal of 105, and p50 homodimers can exhibit suppressive activity when overexpressed. The C-terminal region of p105 is necessary for LPS-induced ERK activation and it has been suggested that ERK activity inhibits both IFN-β and IL-12 p40 following LPS stimulation. However, the contributions of p50 and the C-terminal domain of p105 in regulating endogenous IFN-β(Ifnb and IL-12 p40 (Il12b gene expression in macrophages following LPS stimulation have not been directly compared. METHODOLOGY/PRINCIPAL FINDINGS: We have used recombinant retroviruses to express p105, p50, and the C-terminal domain of p105 (p105ΔN in Nfkb1-deficient murine bone marrow-derived macrophages at near endogenous levels. We found that both p50 and p105ΔN inhibited expression of Ifnb, and that inhibition of Ifnb by p105ΔN depended on ERK activation, because a mutant of p105ΔN (p105ΔNS930A that lacks a key serine necessary to support ERK activation failed to inhibit. In contrast, only p105ΔN but not p50 inhibited Il12b expression. Surprisingly, p105ΔNS930A retained inhibitory activity for Il12b, indicating that ERK activation was not necessary for inhibition. The differential effects of p105ΔNS930A on Ifnb and Il12b expression inversely correlated with the function of one of its binding partners, c-Rel. This raised the possibility that p105ΔNS930A influences gene expression by interfering with the function of c-Rel. CONCLUSIONS: These results demonstrate that Nfkb1 exhibits multiple gene-specific inhibitory functions following TLR stimulation of murine macrophages.

  3. Critical roles of the WASP N-terminal domain and Btk in LPS-induced inflammatory response in macrophages.

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    Chisato Sakuma

    Full Text Available While Wiskott-Aldrich syndrome protein (WASP plays critical roles in TCR signaling as an adaptor molecule, how it transduces innate immune signals remains to be elucidated. To investigate the roles of WASP in innate immune cells, we established bone marrow-derived macrophage (BMDM cell lines from WASP15 transgenic (Tg mice overexpressing the WASP N-terminal region (exons 1-5. Upon LPS stimulation, WASP15 Tg BMDM cell lines produce lower levels of inflammatory cytokines, such as TNF-α, IL-6, and IL-12p40 than the wild-type BMDM cell line. In addition, the production of nitric oxide by WASP15 Tg BMDM cells in response to LPS and IFN-γ was significantly impaired. Furthermore, we uncovered that the WASP N-terminal domain associates with the Src homology (SH 3 domain of Bruton's tyrosine kinase (Btk. Overexpression of the WASP N-terminal domain diminishes the extent of tyrosine phosphorylation of endogenous WASP in WASP15 Tg BMDM cells, possibly by interfering with the specific binding between endogenous WASP and Btk during LPS signaling. These observations strongly suggest that the interaction between WASP N-terminal domain and Btk plays important roles in the LPS signaling cascade in innate immunity.

  4. Polyoxygenated steroids from the gorgonian Menella woodin with capabilities to modulate ROS levels in macrophages at response to LPS.

    Science.gov (United States)

    Tu, Vu A; Lyakhova, Ekaterina G; Diep, Chau N; Kalinovsky, Anatoly I; Dmitrenok, Pavel S; Cuong, Nguyen X; Thanh, Nguyen V; Menchinskaya, Ekaterina S; Pislyagin, Evgeny A; Nam, Nguyen H; Kiem, Phan V; Stonik, Valentin A; Minh, Chau V

    2015-12-01

    Four new polyoxygenated sterol derivatives (1-4) along with the compounds (5-7) previously known from other biological sources were isolated from the gorgonian Menella woodin, collected from the Vietnamese waters. Structures of 1-4 were elucidated by the detailed NMR spectroscopic and mass-spectrometric analyses as well as comparison with those reported in literature data. Compounds 1, 4, and 6 decrease the production of reactive oxygen species (ROS) by the murine macrophages of RAW 264.7 line at induction by endotoxic lipopolysaccharide (LPS) from Escherichia coli.

  5. Pyrrolizidine alkaloids from Liparis nervosa with inhibitory activities against LPS-induced NO production in RAW264.7 macrophages.

    Science.gov (United States)

    Huang, Shuai; Zhou, Xian-li; Wang, Cui-juan; Wang, You-song; Xiao, Feng; Shan, Lian-hai; Guo, Zhi-yun; Weng, Jie

    2013-09-01

    Six pyrrolizidine alkaloids were isolated from the whole herb of Liparis nervosa together with two previously known ones. Their structures were elucidated by extensive spectroscopic analyses and chemical reactions. The cytotoxicity of the isolates was evaluated against A549, HepG2, and MCF-7 human cancer cell lines; however, no significant growth inhibition was observed. All compounds were evaluated for the inhibition of LPS-induced nitric oxide (NO) production in RAW264.7 macrophages, and most significantly inhibited NO production with IC50 values in the range of 2.16-38.25 μM.

  6. Niacin attenuates the production of pro-inflammatory cytokines in LPS-induced mouse alveolar macrophages by HCA2 dependent mechanisms.

    Science.gov (United States)

    Zhou, Ershun; Li, Yimeng; Yao, Minjun; Wei, Zhengkai; Fu, Yunhe; Yang, Zhengtao

    2014-11-01

    Niacin has been reported to have potent anti-inflammatory effects in LPS-induced acute lung injury. However, the molecular mechanism of niacin has not been fully understood. The aim of the present study was to investigate the effects of niacin on the production of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β in LPS-induced mouse alveolar macrophages and explore its underlying mechanism. Mouse alveolar macrophages were incubated in the presence or absence of various concentrations of niacin (1, 10, 100 μmol/l) 1h before LPS (1 μg/ml) challenge. The results showed that niacin reduced the levels of TNF-α, IL-6 and IL-1β in LPS-challenged alveolar macrophages. Furthermore, NF-κB activation was inhibited by niacin through blocking the phosphorylation of NF-κB p65 and IκBα. In addition, silencing HCA2 abrogated the effect of niacin on the production of pro-inflammatory cytokines. These findings suggested that niacin attenuated the LPS-induced pro-inflammatory cytokines possibly mediated by HCA2 in LPS-challenged alveolar macrophages.

  7. LPS inhibits caspase 3-dependent apoptosis in RAW264.7 macrophages induced by the AMPK activator AICAR

    Energy Technology Data Exchange (ETDEWEB)

    Russe, Otto Quintus, E-mail: quintus@russe.eu; Möser, Christine V., E-mail: chmoeser@hotmail.com; Kynast, Katharina L., E-mail: katharina.kynast@googlemail.com; King, Tanya S., E-mail: tanya.sarah.king@googlemail.com; Olbrich, Katrin, E-mail: Katrin.olbrich@gmx.net; Grösch, Sabine, E-mail: groesch@em.uni-frankfurt.de; Geisslinger, Gerd, E-mail: geisslinger@em.uni-frankfurt.de; Niederberger, Ellen, E-mail: e.niederberger@em.uni-frankfurt.de

    2014-05-09

    Highlights: • AMPK-activation induces caspase 3-dependent apoptosis in macrophages. • Apoptosis is associated with decreased mTOR and increased p21 levels. • All effects can be significantly inhibited by the TLR4 agonist lipopolysaccharide. - Abstract: AMP-activated kinase is a cellular energy sensor which is activated in stages of increased ATP consumption. Its activation has been associated with a number of beneficial effects such as decreasing inflammatory processes and the disease progress of diabetes and obesity, respectively. Furthermore, AMPK activation has been linked with induction of cell cycle arrest and apoptosis in cancer and vascular cells, indicating that it might have a therapeutic impact for the treatment of cancer and atherosclerosis. However, the impact of AMPK on the proliferation of macrophages, which also play a key role in the formation of atherosclerotic plaques and in inflammatory processes, has not been focused so far. We have assessed the influence of AICAR- and metformin-induced AMPK activation on cell viability of macrophages with and without inflammatory stimulation, respectively. In cells without inflammatory stimulation, we found a strong induction of caspase 3-dependent apoptosis associated with decreased mTOR levels and increased expression of p21. Interestingly, these effects could be inhibited by co-stimulation with bacterial lipopolysaccharide (LPS) but not by other proinflammatory cytokines suggesting that AICAR induces apoptosis via AMPK in a TLR4-pathway dependent manner. In conclusion, our results revealed that AMPK activation is not only associated with positive effects but might also contribute to risk factors by disturbing important features of macrophages. The fact that LPS is able to restore AMPK-associated apoptosis might indicate an important role of TLR4 agonists in preventing unfavorable cell death of immune cells.

  8. Citrus unshiu flower inhibits LPS-induced iNOS and COX-2 via MAPKs in RAW 264.7 macrophage cells

    Directory of Open Access Journals (Sweden)

    Min-Jin Kim

    2015-12-01

    Full Text Available In the present study, we investigated the effects of Citrus unshiu flower on regulatory mechanisms of cytokines and nitric oxide (NO involved in immunological activity of RAW 264.7 macrophages. Our results indicated that ethyl acetate fraction of Citrus unshiu flower (CUF-EA downregulated LPS-induced nitric oxide (NO synthase (iNOS and cyclooxygenase-2 (COX-2 expression, thereby reducing the production of NO and prostaglandin E2 (PGE2 in LPS-activated RAW 264.7 macrophages. Furthermore, CUF-EA suppressed LPS-induced production of pro-inflammatory cytokines such as interleukin IL-6, and tumor necrosis factor (TNF-α. To elucidate its anti-inflammatory mechanisms, CUF-EA was investigated as an inhibitor of phosphorylation of mitogen-activated protein (MAP kinase in LPS-stimulated RAW 264.7 macrophages. As expected, the phosphorylation of MAP kinases (p38, ERK1/2 and JNK in LPS-stimulated RAW 264.7 macrophages was suppressed by CUF-EA in a dose-dependent manner. These results suggest that the anti-inflammatory properties of CUF-EA might results from inhibition of NO, PGE2, iNOS, COX-2, IL-6 and TNF-α expressions through the down-regulation of phosphorylation of MAPKs in RAW 264.7 macrophages.

  9. The Anti-Inflammatory Effect of Algae-Derived Lipid Extracts on Lipopolysaccharide (LPS-Stimulated Human THP-1 Macrophages

    Directory of Open Access Journals (Sweden)

    Ruairi C. Robertson

    2015-08-01

    Full Text Available Algae contain a number of anti-inflammatory bioactive compounds such as omega-3 polyunsaturated fatty acids (n-3 PUFA and chlorophyll a, hence as dietary ingredients, their extracts may be effective in chronic inflammation-linked metabolic diseases such as cardiovascular disease. In this study, anti-inflammatory potential of lipid extracts from three red seaweeds (Porphyra dioica, Palmaria palmata and Chondrus crispus and one microalga (Pavlova lutheri were assessed in lipopolysaccharide (LPS-stimulated human THP-1 macrophages. Extracts contained 34%–42% total fatty acids as n-3 PUFA and 5%–7% crude extract as pigments, including chlorophyll a, β-carotene and fucoxanthin. Pretreatment of the THP-1 cells with lipid extract from P. palmata inhibited production of the pro-inflammatory cytokines interleukin (IL-6 (p < 0.05 and IL-8 (p < 0.05 while that of P. lutheri inhibited IL-6 (p < 0.01 production. Quantitative gene expression analysis of a panel of 92 genes linked to inflammatory signaling pathway revealed down-regulation of the expression of 14 pro-inflammatory genes (TLR1, TLR2, TLR4, TLR8, TRAF5, TRAF6, TNFSF18, IL6R, IL23, CCR1, CCR4, CCL17, STAT3, MAP3K1 by the lipid extracts. The lipid extracts effectively inhibited the LPS-induced pro-inflammatory signaling pathways mediated via toll-like receptors, chemokines and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB signaling molecules. These results suggest that lipid extracts from P. lutheri, P. palmata, P. dioica and C. crispus can inhibit LPS-induced inflammatory pathways in human macrophages. Therefore, algal lipid extracts should be further explored as anti-inflammatory ingredients for chronic inflammation-linked metabolic diseases.

  10. Aloe vera downregulates LPS-induced inflammatory cytokine production and expression of NLRP3 inflammasome in human macrophages.

    Science.gov (United States)

    Budai, Marietta M; Varga, Aliz; Milesz, Sándor; Tőzsér, József; Benkő, Szilvia

    2013-12-01

    Aloe vera has been used in traditional herbal medicine as an immunomodulatory agent inducing anti-inflammatory effects. However, its role on the IL-1β inflammatory cytokine production has not been studied. IL-1β production is strictly regulated both at transcriptional and posttranslational levels through the activity of Nlrp3 inflammasome. In this study we aimed to determine the effect of Aloe vera on the molecular mechanisms of Nlrp3 inflammasome-mediated IL-1β production in LPS-activated human THP-1 cells and monocyte-derived macrophages. Our results show that Aloe vera significantly reduced IL-8, TNFα, IL-6 and IL-1β cytokine production in a dose dependent manner. The inhibitory effect was substantially more pronounced in the primary cells. We found that Aloe vera inhibited the expression of pro-IL-1β, Nlrp3, caspase-1 as well as that of the P2X7 receptor in the LPS-induced primary macrophages. Furthermore, LPS-induced activation of signaling pathways like NF-κB, p38, JNK and ERK were inhibited by Aloe vera in these cells. Altogether, we show for the first time that Aloe vera-mediated strong reduction of IL-1β appears to be the consequence of the reduced expression of both pro-IL-1β as well as Nlrp3 inflammasome components via suppressing specific signal transduction pathways. Furthermore, we show that the expression of the ATP sensor P2X7 receptor is also downregulated by Aloe vera that could also contribute to the attenuated IL-1β cytokine secretion. These results may provide a new therapeutic approach to regulate inflammasome-mediated responses.

  11. Lipopolysaccharide Induces Alveolar Macrophage Necrosis via CD14 and the P2X7 Receptor Leading to Interleukin-1α Release.

    Science.gov (United States)

    Dagvadorj, Jargalsaikhan; Shimada, Kenichi; Chen, Shuang; Jones, Heather D; Tumurkhuu, Gantsetseg; Zhang, Wenxuan; Wawrowsky, Kolja A; Crother, Timothy R; Arditi, Moshe

    2015-04-21

    Acute lung injury (ALI) remains a serious health issue with little improvement in our understanding of the pathophysiology and therapeutic approaches. We investigated the mechanism that lipopolysaccharide (LPS) induces early neutrophil recruitment to lungs and increases pulmonary vascular permeability during ALI. Intratracheal LPS induced release of pro-interleukin-1α (IL-1α) from necrotic alveolar macrophages (AM), which activated endothelial cells (EC) to induce vascular leakage via loss of vascular endothelial (VE)-cadherin. LPS triggered the AM purinergic receptor P2X7(R) to induce Ca(2+) influx and ATP depletion, which led to necrosis. P2X7R deficiency significantly reduced necrotic death of AM and release of pro-IL-1α into the lung. CD14 was required for LPS binding to P2X7R, as CD14 neutralization significantly diminished LPS induced necrotic death of AM and pro-IL-1α release. These results demonstrate a key role for pro-IL-1α from necrotic alveolar macrophages in LPS-mediated ALI, as a critical initiator of increased vascular permeability and early neutrophil infiltration.

  12. Computational modeling and analysis of iron release from macrophages.

    Directory of Open Access Journals (Sweden)

    Alka A Potdar

    2014-07-01

    Full Text Available A major process of iron homeostasis in whole-body iron metabolism is the release of iron from the macrophages of the reticuloendothelial system. Macrophages recognize and phagocytose senescent or damaged erythrocytes. Then, they process the heme iron, which is returned to the circulation for reutilization by red blood cell precursors during erythropoiesis. The amount of iron released, compared to the amount shunted for storage as ferritin, is greater during iron deficiency. A currently accepted model of iron release assumes a passive-gradient with free diffusion of intracellular labile iron (Fe2+ through ferroportin (FPN, the transporter on the plasma membrane. Outside the cell, a multi-copper ferroxidase, ceruloplasmin (Cp, oxidizes ferrous to ferric ion. Apo-transferrin (Tf, the primary carrier of soluble iron in the plasma, binds ferric ion to form mono-ferric and di-ferric transferrin. According to the passive-gradient model, the removal of ferrous ion from the site of release sustains the gradient that maintains the iron release. Subcellular localization of FPN, however, indicates that the role of FPN may be more complex. By experiments and mathematical modeling, we have investigated the detailed mechanism of iron release from macrophages focusing on the roles of the Cp, FPN and apo-Tf. The passive-gradient model is quantitatively analyzed using a mathematical model for the first time. A comparison of experimental data with model simulations shows that the passive-gradient model cannot explain macrophage iron release. However, a facilitated-transport model associated with FPN can explain the iron release mechanism. According to the facilitated-transport model, intracellular FPN carries labile iron to the macrophage membrane. Extracellular Cp accelerates the oxidation of ferrous ion bound to FPN. Apo-Tf in the extracellular environment binds to the oxidized ferrous ion, completing the release process. Facilitated-transport model can

  13. Nogo-B Facilitates LPS-Mediated Immune Responses by Up-Regulation of TLR4-Signaling in Macrophage RAW264.7

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    Ying Zhu

    2017-01-01

    Full Text Available Background/Aims: Nogo-B, a member of the reticulon family of proteins, is mainly located in the endoplasmic reticulum (ER. Here, we investigate the function and mechanism of Nogo-B in the regulation of TLR4-associated immune responses in the macrophage cell line of RAW264.7. Methods: Nogo-B was up- and down-regulated through the use of appropriate adenoviral vectors or siRNA, and the effects of Nogo-B on macrophages under liposaccharide (LPS stimulation were evaluated via western blotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA, flow cytometric analysis, and transwell assay. Results: Our data indicates that the protein of Nogo-B was down-regulated in a time- and dose-dependent manner following LPS administration in the macrophage. Nogo-B overexpression increased the production of inflammatory cytokines (MCP-1, TNF-α, IL-1β, and TGF-β, enhanced macrophage migration activities, activated major histocompatibility complex II (MHC II, and elevated the expression of macrophage scavenger receptor 1(MSR1, all of which suggest that Nogo-B is necessary for immune responses and plays an important role in regulating macrophage recruitment. Mechanistically, Nogo-B may enhance TLR4 expression in macrophage surfaces, activate mitogen-activated protein kinase (MAPK pathways, and initiate inflammatory responses. Conclusion: These findings illustrate the key regulatory functions of Nogo-B in facilitating LPS-mediated immune responses through promoting the phosphorylation of MAP kinase.

  14. Citrus unshiu flower inhibits LPS-induced iNOS and COX-2 via MAPKs in RAW 264.7 macrophage cells

    OpenAIRE

    2015-01-01

    In the present study, we investigated the effects of Citrus unshiu flower on regulatory mechanisms of cytokines and nitric oxide (NO) involved in immunological activity of RAW 264.7 macrophages. Our results indicated that ethyl acetate fraction of Citrus unshiu flower (CUF-EA) downregulated LPS-induced nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, thereby reducing the production of NO and prostaglandin E2 (PGE2) in LPS-activated RAW 264.7 macrophages. Furthermore,...

  15. Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor

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    Qureshi Asaf A

    2012-07-01

    Full Text Available Abstract Background Altered immune function during ageing results in increased production of nitric oxide (NO and other inflammatory mediators. Recently, we have reported that NO production was inhibited by naturally-occurring proteasome inhibitors (quercetin, δ-tocotrienol, and riboflavin in lipopolysaccharide (LPS-stimulated RAW264.7 cells, and thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. In a continuous effort to find more potent, non-toxic, commercially available, naturally-occurring proteasome inhibitors that suppress inflammation, the present study was carried out to describe the inhibition of NF-κB activation and NO, TNF-α, IL-6, IL-1β, and iNOS expression by trans-resveratrol, trans-pterostilbene, morin hydrate, and nicotinic acid in LPS-induced RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice. Results The present results indicate that resveratrol, pterostilbene, and morin hydrate caused significant inhibition (>70% to 90%; P 40%; P 60%; P 40%; P P  Conclusions The present results clearly demonstrate that resveratrol and pterostilbene are particularly potent proteasome inhibitors that suppress expression of genes, and production of inflammatory products in LPS-stimulated RAW 264.7 cells, and macrophages from C57BL/6 and BALB/c mice. Resveratrol and pterostilbene which are present in grapes, blueberries, and red wine, have been implicated as contributing factors to the lower incidence of cardiovascular disease in the French population, despite their relatively high dietary fat intake. Consequently, it appears likely that the beneficial nutritional effects of resveratrol and pterostilbene are due at least in part, to their ability to inhibit NF-κB activation by the proteasome, thereby suppressing activation of pro-inflammatory cytokines and iNOS genes, resulting in decreased secretion of TNF-α, IL-1β, IL-6, and NO levels, in response to inflammatory stimuli

  16. Low doses of LPS and minimally oxidized LDL cooperatively activate macrophages via NF-kappaB and AP-1: Possible mechanism for acceleration of atherosclerosis by subclinical endotoxemia

    Science.gov (United States)

    Wiesner, Philipp; Choi, Soo-Ho; Almazan, Felicidad; Benner, Christopher; Huang, Wendy; Diehl, Cody J.; Gonen, Ayelet; Butler, Susan; Witztum, Joseph L.; Glass, Christopher K.; Miller, Yury I.

    2010-01-01

    Rationale Oxidized low-density lipoprotein (LDL) is an important determinant of inflammation in atherosclerotic lesions. It has also been documented that certain chronic infectious diseases, such as periodontitis and chlamydial infection, exacerbate clinical manifestations of atherosclerosis. In addition, low-level but persistent metabolic endotoxemia is often found in diabetic and obese subjects and is induced in mice fed a high-fat diet. Objective In this study, we examined cooperative macrophage activation by low levels of bacterial LPS and by minimally oxidized LDL (mmLDL), as a model for subclinical endotoxemia-complicated atherosclerosis. Methods and Results We found that both in vitro and in vivo, mmLDL and LPS (Kdo2-LipidA) cooperatively activated macrophages to express pro-inflammatory cytokines Cxcl2 (MIP-2), Ccl3 (MIP-1alpha), and Ccl4 (MIP-1beta). Importantly, the mmLDL and LPS cooperative effects were evident at a threshold LPS concentration (1 ng/ml) at which LPS alone induced only a limited macrophage response. Analyzing microarray data with a de novo motif discovery algorithm, we found that genes transcribed by promoters containing an AP-1 binding site were significantly upregulated by co-stimulation with mmLDL and LPS. In a nuclear factor-DNA binding assay, the cooperative effect of mmLDL and LPS co-stimulation on c-Jun and c-Fos DNA binding, but not on p65 or p50, was dependent on mmLDL-induced activation of ERK1/2. In addition, mmLDL induced JNK-dependent derepression of AP-1 by removing the corepressor NCoR from the chemokine promoters. Conclusions The cooperative engagement of AP-1 and NF-kappaB by mmLDL and LPS may constitute a mechanism of increased transcription of inflammatory cytokines within atherosclerotic lesions. PMID:20489162

  17. Brief Glutamine Pretreatment Increases Alveolar Macrophage CD163/Heme Oxygenase-1/p38-MAPK Dephosphorylation Pathway and Decreases Capillary Damage but Not Neutrophil Recruitment in IL-1/LPS-Insufflated Rats.

    Science.gov (United States)

    Fernandez-Bustamante, Ana; Agazio, Amanda; Wilson, Paul; Elkins, Nancy; Domaleski, Luke; He, Qianbin; Baer, Kaily A; Moss, Angela F D; Wischmeyer, Paul E; Repine, John E

    2015-01-01

    Glutamine (GLN) attenuates acute lung injury (ALI) but its effect on alveolar macrophages is unknown. We hypothesized that GLN pretreatment would induce the anti-inflammatory CD163/heme oxygenase (HO)-1/p38-MAPK dephosphorylation pathway in alveolar macrophages and reduce ALI in rats insufflated with interleukin-1 (IL-1) and lipopolysaccharide (LPS). Male Sprague-Dawley rats were randomized to the following groups: GLN-IL-1/LPS-, GLN+IL-1/LPS-, GLN-IL-1/LPS+, and GLN+IL-1/LPS+. GLN pretreatment was given via gavage (1 g/kg L-alanyl-L-glutamine) daily for 2 days. ALI was subsequently induced by insufflating 50 ng IL-1 followed by 5mg/kg E.coli LPS. After 24h, bronchoalveolar lavage (BAL) protein, lactate dehydrogenase (LDH) and neutrophil concentrations were analyzed. BAL alveolar macrophage CD163+ expression, HO-1 and p38-MAPK concentrations were measured, as well as alveolar macrophage tumor necrosis factor (TNF)-α and interleukin (IL)-10 concentrations. Histology and immunofluorescence studies were also performed. Following IL-1/LPS insufflation, GLN pretreated rats had significantly decreased BAL protein and LDH concentrations, but not BAL neutrophil counts, compared to non-GLN pretreated rats. The number of alveolar macrophages and the number of CD163+ macrophages were significantly increased in GLN pretreated IL-1/LPS-insufflated rats compared to non-GLN pretreated, IL-1/LPS-insufflated rats. GLN pretreatment before IL-1/LPS also significantly increased HO-1 concentrations and dephosphorylated p38-MAPK levels but not cytokine levels in alveolar macrophages. Immunofluorescence localized CD163 and HO-1 in alveolar macrophages. Short-term GLN pretreatment activates the anti-inflammatory CD163/HO-1/p38-MAPK dephosphorylation pathway of alveolar macrophages and decreases capillary damage but not neutrophil recruitment in IL-1/LPS-insufflated rats.

  18. Identification of a novel compound that inhibits iNOS and COX-2 expression in LPS-stimulated macrophages from Schisandra chinensis

    Energy Technology Data Exchange (ETDEWEB)

    Lee, You Jin [Department of Horticultural Bioscience, Pusan National University, Miryang 627-706 (Korea, Republic of); Park, Sun Young [Korea BIO-IT Foundry Center, Pusan National University, Busan 609-735 (Korea, Republic of); Kim, Sun Gun; Park, Da Jung; Kang, Jum Soon [Department of Horticultural Bioscience, Pusan National University, Miryang 627-706 (Korea, Republic of); Lee, Sang Joon [Department of Microbiology, Pusan National University, Busan 609-735 (Korea, Republic of); Yoon, Sik [Department of Anatomy, School of Medicine, Pusan National University, Yangsan 626-770 (Korea, Republic of); Medical Research Center for Ischemic Tissue Regeneration, School of Medicine, Pusan National University, Yangsan 626-770 (Korea, Republic of); Kim, Young Hun [Korea BIO-IT Foundry Center, Pusan National University, Busan 609-735 (Korea, Republic of); Bae, Yoe-Sik, E-mail: yoesik@dau.ac.kr [Department of Biochemistry, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Choi, Young-Whan, E-mail: ywchoi@pusan.ac.kr [Department of Horticultural Bioscience, Pusan National University, Miryang 627-706 (Korea, Republic of)

    2010-01-22

    A novel {alpha}-iso-cubebenol, which has anti-inflammatory effects in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, was isolated from the fruits of Schisandra chinensis. {alpha}-iso-cubebenol inhibited LPS-induced nitric oxide (NO) and prostaglandin E{sub 2} (PGE{sub 2}) production. Consistent with these findings, {alpha}-iso-cubebenol also reduced the LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 at the protein and mRNA levels in a concentration-dependent manner. {alpha}-iso-cubebenol also inhibited LPS-induced nuclear translocation of the NF-{kappa}B p65 subunit. Furthermore, {alpha}-iso-cubebenol suppressed the phosphorylation of ERK, JNK, and p38 kinase induced by LPS. Since the novel {alpha}-iso-cubebenol blocked the production of several pro-inflammatory mediators induced by LPS in macrophages, the molecule can be useful material for the development of anti-inflammatory agents against bacterial infections or endotoxin.

  19. Human lung-resident macrophages express CB1 and CB2 receptors whose activation inhibits the release of angiogenic and lymphangiogenic factors.

    Science.gov (United States)

    Staiano, Rosaria I; Loffredo, Stefania; Borriello, Francesco; Iannotti, Fabio Arturo; Piscitelli, Fabiana; Orlando, Pierangelo; Secondo, Agnese; Granata, Francescopaolo; Lepore, Maria Teresa; Fiorelli, Alfonso; Varricchi, Gilda; Santini, Mario; Triggiani, Massimo; Di Marzo, Vincenzo; Marone, Gianni

    2016-04-01

    Macrophages are pivotal effector cells in immune responses and tissue remodeling by producing a wide spectrum of mediators, including angiogenic and lymphangiogenic factors. Activation of cannabinoid receptor types 1 and 2 has been suggested as a new strategy to modulate angiogenesis in vitro and in vivo. We investigated whether human lung-resident macrophages express a complete endocannabinoid system by assessing their production of endocannabinoids and expression of cannabinoid receptors. Unstimulated human lung macrophage produce 2-arachidonoylglycerol,N-arachidonoyl-ethanolamine,N-palmitoyl-ethanolamine, and N-oleoyl-ethanolamine. On LPS stimulation, human lung macrophages selectively synthesize 2-arachidonoylglycerol in a calcium-dependent manner. Human lung macrophages express cannabinoid receptor types 1 and 2, and their activation induces ERK1/2 phosphorylation and reactive oxygen species generation. Cannabinoid receptor activation by the specific synthetic agonists ACEA and JWH-133 (but not the endogenous agonist 2-arachidonoylglycerol) markedly inhibits LPS-induced production of vascular endothelial growth factor-A, vascular endothelial growth factor-C, and angiopoietins and modestly affects IL-6 secretion. No significant modulation of TNF-α or IL-8/CXCL8 release was observed. The production of vascular endothelial growth factor-A by human monocyte-derived macrophages is not modulated by activation of cannabinoid receptor types 1 and 2. Given the prominent role of macrophage-assisted vascular remodeling in many tumors, we identified the expression of cannabinoid receptors in lung cancer-associated macrophages. Our results demonstrate that cannabinoid receptor activation selectively inhibits the release of angiogenic and lymphangiogenic factors from human lung macrophage but not from monocyte-derived macrophages. Activation of cannabinoid receptors on tissue-resident macrophages might be a novel strategy to modulate macrophage-assisted vascular remodeling

  20. Production of IL-12, IL-23 and IL-27p28 by bone marrow-derived conventional dendritic cells rather than macrophages after LPS/TLR4-dependent induction by Salmonella Enteritidis.

    Science.gov (United States)

    Siegemund, Sabine; Schütze, Nicole; Freudenberg, Marina A; Lutz, Manfred B; Straubinger, Reinhard K; Alber, Gottfried

    2007-01-01

    Induction of the interleukin-12 (IL-12) cytokine family comprising IL-12, IL-23, IL-27, and IL-12p40 by intracellular pathogens is required for orchestration of cell-mediated immune responses. Macrophages (MPhi) have been shown to be a source of IL-12 following TLR4-dependent activation by Salmonella (S.). In this study another antigen-presenting cell type, the conventional dendritic cell (cDC), was analyzed and its cytokine responses compared with those of MPhi. We generated bone marrow-derived conventional dendritic cells (BMDC) and macrophages (BMMPhi) by incubating murine bone marrow cells with supernatants containing granulocyte/macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), respectively. Stimulation of BMDC and BMMPhi with S. enterica serovar Enteritidis (SE) or LPS resulted in the release of IL-12 and IL-23 by BMDC but not by BMMPhi. Furthermore, BMDC secreted approx. 20-fold more IL-12p40 and IL-27p28 than BMMPhi. However, BMDC and BMMPhi produced similar levels of IL-10. Using BMDC originating from wild-type (wt), TLR2(def) and TLR4(def) mice, we show that in BMDC the induction of IL-12, IL-23, and IL-27p28 by SE is dependent on TLR4, whereas low-level production of p40 is also mediated by pattern recognition receptors (PRR) other than TLR4. Interestingly, LPS- and SE-provoked responses of BMDC were remarkably similar indicating that LPS is the primary danger molecule of SE. Taken together, our results point to cDC rather than MPhi as the major producers of the IL-12 family members during in vitro infection with SE. The mechanisms of recognition of SE, however, appear to be the same for cDC and MPhi.

  1. Indirubin-3′-(2,3 dihydroxypropyl)-oximether (E804) is a potent modulator of LPS-stimulated macrophage functions

    Energy Technology Data Exchange (ETDEWEB)

    Babcock, Abigail S. [Department of Biological Sciences, Clemson University, Clemson, SC 29634 (United States); Anderson, Amy L. [Department of Biological Sciences, Clemson University, Clemson, SC 29634 (United States); Graduate Program in Environmental Toxicology, Clemson University, Clemson, SC 29634 (United States); Rice, Charles D., E-mail: cdrice@clemson.edu [Department of Biological Sciences, Clemson University, Clemson, SC 29634 (United States); Graduate Program in Environmental Toxicology, Clemson University, Clemson, SC 29634 (United States)

    2013-01-01

    Indirubin is a deep-red bis-indole isomer of indigo blue, both of which are biologically active ingredients in Danggui Longhui Wan, an ancient Chinese herbal tea mixture used to treat neoplasia and chronic inflammation and to enhance detoxification of xenobiotics. Multiple indirubin derivatives have been synthesized and shown to inhibit cyclin-dependent kinases (CDKs) and glycogen-synthase kinase (GSK-3β) with varying degrees of potency. Several indirubins are also aryl hydrocarbon receptor (AhR) agonists, with AhR-associated activities covering a wide range of potencies, depending on molecular structure. This study examined the effects of indirubin-3′-(2,3 dihydroxypropyl)-oximether (E804), a novel indirubin with potent STAT3 inhibitory properties, on basal and LPS-inducible activities in murine RAW264.7 macrophages. Using a focused commercial qRT-PCR array platform (SuperArray®), the effects of E804 on expression of a suite of genes associated with stress and toxicity were determined. Most genes up-regulated by LPS treatment were suppressed by E804; including LPS-induced expression of pro-inflammatory cytokines and receptors, apoptosis control genes, and oxidative stress response genes. Using qRT-PCR as a follow up to the commercial arrays, E804 treatment suppressed LPS-induced COX-2, iNOS, IL-6 and IL-10 gene expression, though the effects on iNOS and COX-2 protein expression were less dramatic. E804 also inhibited LPS-induced secretion of IL-6 and IL-10. Functional endpoints, including iNOS and lysozyme enzymatic activity, phagocytosis of fluorescent latex beads, and intracellular killing of bacteria, were also examined, and in each experimental condition E804 suppressed activities. Collectively, these results indicate that E804 is a potent modulator of pro-inflammatory profiles in LPS-treated macrophages. -- Highlights: ► RAW 264.7 macrophages were treated with 1 μM Indirubin E804, 1 μg/ml LPS, or both. ► E804 suppresses LPS-induced expression of i

  2. Inhibitory effect of a formulated extract from multiple citrus peels on LPS-induced inflammation in RAW 246.7 macrophages

    Directory of Open Access Journals (Sweden)

    Tadahiro Etoh

    2013-06-01

    Full Text Available ABSTRACTBackground: Formulated Citrus Peel Extract (GL made from the peels of six citrus fruits available in Japan, namely navel oranges, citrus hassaku, citrus limon, citrus natsudaidai, citrus miyauchi and satsuma, was initially developed as a cosmetic product to protect skin from UV irradiation. Anecdotal evidences of anti-cancer property of GL have been reported by consumers based on the cases such as topical application for melanoma, and oral ingestion for prostate, lung and liver cancers.Those anecdotal reports stimulated us to investigate anti-tumorigenesis activity of GL. In the previous study, we reported that the topical application of GL inhibited DMBA/TPA-induced skin tumor formation by decreasing inflammatory gene parameters.Objective: In this study, we mainly investigated the effect of GL on translocation of NF-kB together with production of nitric-oxide and TNF-α induced by LPS in RAW 264.7 cells.Results: This investigation showed that GL decreased the release of TNF-α and nitric oxide from macrophage RAW264.7 cells stimulated by LPS in a dose-dependent manner. In addition, GL suppressed the expression of iNOS and nuclear translocation of NF-kB in RAW264.7 cells, inhibited the degradation of IκB-α, and scavenged hydroxyl radicals (DMPO/OH adduct in vitro.Conclusions: Our findings suggest that GL suppresses the inflammation in vitro, and exerts chemopreventive activity through the inhibition of production of TNF-α and iNOS proteins due to the inhibition of nuclear translocation of NF-kB and oxidative stress. GL appears to be a novel functional natural product capable of preventing inflammation and inflammation-associated tumorigenesis.Keywords: GL, Citrus peel extract, anti-inflammation, Nitric oxide, iNOS, NF-kB, TNF-α

  3. Inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells by the norsesterterpene peroxide, epimuqubilin A.

    Science.gov (United States)

    Cheenpracha, Sarot; Park, Eun-Jung; Rostama, Bahman; Pezzuto, John M; Chang, Leng Chee

    2010-03-01

    Seven norsesterterpene peroxides: epimuqubilin A (1), muqubilone B (2), unnamed cyclic peroxide ester (3), epimuqubilin B (4), sigmosceptrellin A methyl ester (5), sigmosceptrellin A (6), and sigmosceptrellin B methyl ester (7), isolated from the marine sponge Latrunculia sp., were examined with regard to their effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells. The results indicated epimuqubilin A (1) possessed potent NO inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide release with an IC(50) value of 7.4 microM, a level three times greater than the positive control, L-N(G)-monomethyl arginine citrate, followed by 6 (sigmosceptrellin A, IC(50) = 9.9 microM), whereas other compounds exhibited only modest activity (Table 1). These compounds did not show appreciable cytotoxicity at their IC(50) values for NO-inhibitory activity. The structure-activity upon NO inhibition could be summarized as follows: (1) a monocyclic carbon skeleton framework was essential for activity, (2) free acids gave higher activity, (3) the orientation of H3-22 with an equatorial position increased activity, and (4) a bicyclic structure reduced activity. This is the first report of a norsesterterpene peroxide with NO-inhibitory activity. In addition, compounds 1-7 were also evaluated for their inhibitory activities in the yeast glycogen synthase kinase-3beta assay. In summary, several norsesterterpene peroxides showed novel biological activities of inhibition in NO production, suggesting that these might provide leads for anti-inflammatory or cancer chemopreventive agents.

  4. Inflammatory mediator release byBrugia malayi from macrophages of susceptible hostMastomys coucha andTHP-1 andRAW 264.7 cell lines

    Institute of Scientific and Technical Information of China (English)

    Shiv Kumar Verma; Vikas Kushwaha; Vijaya Dubey; Kirti Saxena; Aakanksha Sharma; Puvvada Kalpana Murthy

    2011-01-01

    Objective:To investigate which life stage of the parasite has the ability to stimulate release of pro- or anti-inflammatory mediators from macrophages.Methods: The human macrophage/monocyte cell lineTHP-1, the mouse macrophage cell lineRAW 264.7 and naive peritoneal macrophages(PM)from the rodent hostMastomys coucha (M. coucha)were incubated at37 ℃in 5% CO2atmosphere with extracts of microfilariae(Mf), third stage infective larvae(L3) and adult worms (Ad)ofBrugia malayi. After48 hr post exposure,IL-1β, IL-6, TNF-α, IL-10 and nitric oxide (NO) in cell-free supernatants were estimated.Results: Extracts of all the life stages of the parasite were capable of stimulating pro-(IL-1β, IL-6 andTNF-α) and anti-inflammatory (IL-10)cytokines in both the cell lines and peritoneal macrophages ofM. coucha. Mf was the strongest stimulator of pro-inflammatory cytokines followed by L3 and Ad; however, Ad was a strong stimulator ofIL-10 release. Mf was found to have potential to modulateLPS-inducedNO release inRAW cells. Ad-inducedNO release was concentration dependent with maximum at 20 μg/mL in bothRAW andPMs.Conclusions:The results show that parasites at all life stages were capable of stimulating pro- (IL-1β, IL-6 and TNF-α) and anti-inflammatory(IL-10) cytokines andNO release from macrophages of susceptible hostM. coucha, human and mouse macrophage cell lines.Mf can suppress theLPS-inducedNO release inRAW cells. The findings also show that the two cell lines may provide a convenientin vitro system for assaying parasite-induced inflammatory mediator release.

  5. Refractory ceramic fibers activate alveolar macrophage eicosanoid and cytokine release.

    Science.gov (United States)

    Leikauf, G D; Fink, S P; Miller, M L; Lockey, J E; Driscoll, K E

    1995-01-01

    Refractory ceramic fiber has been developed for industrial processes requiring materials with high thermal and mechanical stability. To evaluate the biological activity of this fiber, rat alveolar macrophages were exposed for < or = 24 h to 0-1,000 micrograms/ml of refractory ceramic fiber, crocidolite asbestos, silica (fibrogenic particles), or titanium dioxide (a nonfibrogenic particle), and eicosanoid, tumor necrosis factor-alpha (TNF), and lactate dehydrogenase release were measured. Particle dimensions were determined by electron microscopy. Radioactivity coeluting with leukotriene B4 (LTB4) and immunoreactive LTB4 and TNF release increased after refractory ceramic fiber and were similar in magnitude after asbestos but less than after silica. For example, the total [3H]eicosanoid release increased 3.9-fold after refractory ceramic fiber, 4.6-fold after asbestos, and 8.7-fold after silica. Refractory ceramic fiber and asbestos also have similar particle dimensions (diameter, length, and surface area). Inasmuch as macrophage-derived LTB4 and TNF are potent mediators in inflammatory events, including migration and activation of neutrophils, these findings suggest that refractory ceramic fiber can activate macrophages in vitro to release mediators relevant to in vivo findings of inflammation and fibrotic lung disease in laboratory animals.

  6. Ultrapotent effects of salvinorin A, a hallucinogenic compound from Salvia divinorum, on LPS-stimulated murine macrophages and its anti-inflammatory action in vivo.

    Science.gov (United States)

    Aviello, Gabriella; Borrelli, Francesca; Guida, Francesca; Romano, Barbara; Lewellyn, Kevin; De Chiaro, Maria; Luongo, Livio; Zjawiony, Jordan K; Maione, Sabatino; Izzo, Angelo A; Capasso, Raffaele

    2011-09-01

    The hallucinogenic compound, salvinorin A, is a potent κ-opioid receptor (KOR) agonist. However, other target(s) than the KOR, such as the cannabinoid CB1 receptor, have been proposed to explain its multiple pharmacological actions. Here, we have evaluated the effect of salvinorin A in lipopolysaccharide (LPS)-stimulated macrophages as well as in models of inflammation in vivo. Salvinorin A (0.1-10 pM) reduced LPS-stimulated nitrite, TNF-α and IL-10 (but not IL-1β) levels as well as iNOS (but not COX-2) LPS-induced hyperexpression. The effect of salvinorin A on nitrite levels was reverted by the opioid antagonist naloxone, the KOR antagonist nor-binaltorphimine and by the CB1 antagonist rimonabant Salvinorin A also prevented KOR and CB1 hyperexpression induced by LPS. In vivo, salvinorin A reduced the LPS- and the carrageenan-induced paw oedema and formalin-induced inflammatory pain, in a nor-binaltorphimine and rimonabant-sensitive manner. It is concluded that salvinorin A-via KORs and CB1 receptors-exerts ultrapotent actions on macrophages and also shows moderate antinflammatory effects in vivo.

  7. Inhibitory effect of opiates on LPS mediated release of TNF and IL-8.

    Science.gov (United States)

    Bastami, Salumeh; Norling, Cecilia; Trinks, Cecilia; Holmlund, Birgitta; Walz, Thomas M; Ahlner, Johan; Uppugunduri, Srinivas

    2013-06-01

    Most patients with advanced cancer experience severe pain and are often treated with opiates. Cancer patients are especially susceptible to opportunistic infections due to treatment with immunosuppressive and cytostatic drugs. Since opiates have been demonstrated to have immunomodulatory effects, it is of clinical importance to evaluate potential differences between commonly used opiates with regard to their effect on the immune system. The aim of this study was to evaluate the effect of morphine, tramadol, fentanyl and ketobemidone on the functioning of the immune system with special reference to TNF and IL-8 release. Method. U-937 cells were preincubated with different concentrations of opioids followed by stimulation with LPS 100 μg/ml for three hours. The effect of opioids on the levels of cytokine mRNA was studied using RT-PCR. Erk and Akt phosphorylation was also measured by Western blot. Results. All opioids with the exception of fentanyl were capable of inhibiting TNF release from U-937 cells. Morphine had no effect on IL-8 release but the effect of other opiates was almost the same as the effect on TNF. All opioids with the exception of fentanyl were capable of inhibiting production of mRNA for TNF and IL-8. The observed effects of opiates were not always reversible by naloxone, suggesting that the effects might be mediated by other receptors or through a non-receptor mediated direct effect. Although preliminary evidence suggests the involvement of Erk and Akt pathways, further studies are needed to unravel the intracellular pathways involved in mediating the effects of opiates. Our data suggests that the order of potency with regard to inhibition of cytokine release is as follows: tramadol > ketobemidone > morphine > fentanyl. Conclusion. Further studies are needed to understand the clinical implications of the observed immunosuppressive effects of tramadol and ketobemidone and to improve opioid treatment strategies in patients with cancer.

  8. Is nitric oxide decrease observed with naphthoquinones in LPS stimulated RAW 264.7 macrophages a beneficial property?

    Directory of Open Access Journals (Sweden)

    Brígida R Pinho

    Full Text Available The search of new anti-inflammatory drugs has been a current preoccupation, due to the need of effective drugs, with less adverse reactions than those used nowadays. Several naphthoquinones (plumbagin, naphthazarin, juglone, menadione, diosquinone and 1,4-naphthoquinone, plus p-hydroquinone and p-benzoquinone were evaluated for their ability to cause a reduction of nitric oxide (NO production, when RAW 264.7 macrophages were stimulated with lipopolysaccharide (LPS. Dexamethasone was used as positive control. Among the tested compounds, diosquinone was the only one that caused a NO reduction with statistical importance and without cytotoxicity: an IC(25 of 1.09±0.24 µM was found, with 38.25±6.50% (p<0.001 NO reduction at 1.5 µM. In order to elucidate if this NO decrease resulted from the interference of diosquinone with cellular defence mechanisms against LPS or to its conversion into peroxynitrite, by reaction with superoxide radical formed by naphthoquinones redox cycling, 3-nitrotyrosine and superoxide determination was also performed. None of these parameters showed significant changes relative to control. Furthermore, diosquinone caused a decrease in the pro-inflammatory cytokines: tumour necrosis factor-alpha (TNF-α and interleukin 6 (IL-6. Therefore, according to the results obtained, diosquinone, studied for its anti-inflammatory potential for the first time herein, has beneficial effects in inflammation control. This study enlightens the mechanisms of action of naphthoquinones in inflammatory models, by checking for the first time the contribution of oxidative stress generated by naphthoquinones to NO reduction.

  9. Anti-inflammatory effect of pomegranate flower in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.

    Science.gov (United States)

    Xu, Jianjun; Zhao, Yongxin; Aisa, Haji Akber

    2017-12-01

    Punica granatum L (Punicaceae) flower is an important diabetes treatment in oriental herbal medicine. This study investigates the inflammation effects of pomegranate flower (PFE) ethanol extract in LPS-induced RAW264.7 cells. PFE (10, 25, 50, 100 μg/mL) was applied to 1 μg/mL LPS-induced RAW 264.7 macrophages in vitro. Levels of nitric oxide (NO), prostaglandin E2 (PGE2) and pro-inflammatory cytokines interleukin (IL)-1β (IL-1β), interleukin (IL)-6 (IL-6) and tumor necrosis factor (TNF-α) in the supernatant fraction were determined using enzyme-linked immunosorbent assay (ELISA). Expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), phosphorylation of mitogen-activated protein kinase (MAPK) subgroups extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and P38, as well as nuclear factor-κB (NF-κB) activation in extracts were detected via Western blot. 10-100 μg/mL PFE decreased the production of NO (IC50 value = 31.8 μg/mL), PGE2 (IC50 value = 54.5 μg/mL), IL-6 (IC50 value = 48.7 μg/mL), IL-1β (IC50 value = 71.3 μg/mL) and TNF-α (IC50 value = 62.5 μg/mL) in LPS-stimulated RAW 264.7 cells significantly. A mechanism-based study showed that phosphorylation of ERK1/2, p38, JNK and translocation of the NF-B p65 subunit into nuclei were inhibited by the PFE treatment. These results show that PFE produced potential anti-inflammatory effect through modulating the synthesis of several mediators and cytokines involved in the inflammatory process.

  10. Induction of Golli-MBP Expression in CNS Macrophages During Acute LPS-Induced CNS Inflammation and Experimental Autoimmune Encephalomyelitis (EAE

    Directory of Open Access Journals (Sweden)

    Tracey L. Papenfuss

    2007-01-01

    Full Text Available Microglia are the tissue macrophages of the CNS. Microglial activation coupled with macrophage infiltration is a common feature of many classic neurodegenerative disorders. The absence of cell-type specific markers has confounded and complicated the analysis of cell-type specific contributions toward the onset, progression, and remission of neurodegeneration. Molecular screens comparing gene expression in cultured microglia and macrophages identified Golli-myelin basic protein (MBP as a candidate molecule enriched in peripheral macrophages. In situ hybridization analysis of LPS/IFNg and experimental autoimmune encephalomyelitis (EAE–induced CNS inflammation revealed that only a subset of CNS macrophages express Golli-MBP. Interestingly, the location and morphology of Golli-MBP+ CNS macrophages differs between these two models of CNS inflammation. These data demonstrate the difficulties of extending in vitro observations to in vivo biology and concretely illustrate the complex heterogeneity of macrophage activation states present in region- and stage-specific phases of CNS inflammation. Taken altogether, these are consistent with the emerging picture that the phenotype of CNS macrophages is actively defined by their molecular interactions with the CNS microenvironment.

  11. Macrophage Deletion of SOCS1 Increases Sensitivity to LPS and Palmitic Acid and Results in Systemic Inflammation and Hepatic Insulin Resistance

    Science.gov (United States)

    Graham, Kate L.; Galic, Sandra; Honeyman, Jane E.; Fynch, Stacey L.; Hewitt, Kimberly A.; Kay, Thomas W.

    2011-01-01

    OBJECTIVE Macrophage secretion of proinflammatory cytokines contributes to the pathogenesis of obesity-related insulin resistance. An important regulator of inflammation is the suppressor of cytokine signaling-1 (SOCS1), which inhibits the JAK-STAT and toll-like receptor-4 (TLR4) pathways. Despite the reported role of SOCS1 in inhibiting insulin signaling, it is surprising that a SOCS1 polymorphism that increases SOCS1 promoter activity is associated with enhanced insulin sensitivity despite obesity. In the current study, we investigated the physiological role of myeloid and lymphoid cell SOCS1 in regulating inflammation and insulin sensitivity. RESEARCH DESIGN AND METHODS We used mice generated by crossing SOCS1 floxed mice with mice expressing Cre recombinase under the control of the LysM-Cre promoter (SOCS1 LysM-Cre). These mice have deletion of SOCS1 in macrophages and lymphocytes. We assessed macrophage inflammation using flow cytometry and serum cytokine levels using Bioplex assays. We then measured insulin sensitivity using glucose tolerance tests and the euglycemic-hyperinsulinemic clamp. Using bone marrow–derived macrophages, we tested the effects of SOCS1 deletion in regulating responses to the TLR4 ligands: lipopolysaccharide (LPS) and palmitic acid. RESULTS SOCS1 LysM-Cre mice had increased macrophage expression of CD11c, enhanced sensitivity to LPS, and palmitic acid and increased serum concentrations of tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein. Increased inflammation was associated with impaired glucose tolerance and hyperinsulinemia as a result of reduced hepatic but not skeletal muscle insulin sensitivity. CONCLUSIONS The expression of SOCS1 in hematopoietic cells protects mice against systemic inflammation and hepatic insulin resistance potentially by inhibiting LPS and palmitate-induced TLR4 signaling in macrophages. PMID:21646388

  12. 金银花提取物含药血清对正常及LPS刺激的大鼠腹腔巨噬细胞释放NO的影响%EFFECT OF SERUM OF LOUICERA JAPONICA THUMB ON RELEASE OF NO FROM NORMAL AND LPS INDUCED PERITONEAL MACROPHAGES

    Institute of Scientific and Technical Information of China (English)

    崔晓燕

    2008-01-01

    目的 考察金银花提取物(louicera japonica thunb,LTE)含药血清对正常的及细菌脂多糖(lipopolysaccharide,LPS)刺激的大鼠腹腔巨噬细胞释放一氧化氮(nitrous oxide,NO)的影响.方法 运用中药血清药理学方法结合Griess比色法、四甲基偶氮唑盐法检测LTE的大鼠含药血清对正常的及LPS(1 μg/mL)刺激的大鼠腹腔巨噬细胞NO释放量的影响.结果 LTE的含药血清在不影响细胞存活率的情况下,可以明显的降低正常的及LPS刺激的大鼠腹腔巨噬细胞NO的释放量.结论 LTE含药血清具有免疫抑制及抗炎活性.

  13. Different effect of glutamine on macrophage tumor necrosis factor-alpha release and heat shock protein 72 expression in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Mengfan Liang; Xuemin Wang; Yuan Yuan; Quanhong Zhou; Chuanyao Tong; Wei Jiang

    2009-01-01

    Macrophage plays a vital role in sepsis. However, the modulatory effect of glutamine (Gln) on macrophage/ monocyte-mediate cytokines release is still controver-sial. Thus, we investigated the effect of Gin on macro-phage tumor necrosis factor (TNF)-α release and heat shock protein (HSP) 72 expression in vivo and in vitro. Data from our study indicated that the increase of HSP72 expression was significant at 8 mM of Gln 4 h after lipopolysaccharide (LPS) stimulation and became independent of Gin concentrations at 24 h, whereas TNF-α release was dose- and time-dependent on Gln. Heat stress (HS) induced more HSP72 and less TNF-α production compared with the non-HS group. However, the production of TNF-α in cells pretreated with HS was increased with increasing concentrations of Gln. Treatment with various concentrations of Gin for 1 h and then 0.5 mM Gin for 4h led to an increase in HSP72 expression, but not in TNF-α production. In sepsis model mice, Gin treatment led to a significantly lower intracellular TNF-α level and an increase in HSP72 expression in mouse peritoneal macrophages. Our results demonstrate that Gin directly increases TNF-α release of LPS-stimulated RAW264.7 macro-phages in a dose-dependent manner, and also decreases mouse peritoneal macrophages TNF-α release in the sepsis model. Taken together, our data suggest that there may be more additional pathways by which Gln modulates cytokine production besides HSP72 expression in macrophage during sepsis.

  14. DMPD: Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15081522 Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signall...ruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? PubmedID 15081522 Title Bruton...'s tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? Authors

  15. Evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to LPS and LTA

    Directory of Open Access Journals (Sweden)

    Cinar Mehmet

    2012-02-01

    Full Text Available Abstract Background To obtain reliable quantitative real-time PCR data, normalization relative to stable housekeeping genes (HKGs is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in pigs are relatively rare and have never been performed in porcine alveolar macrophages (AMs. In this study, expression stability of putative housekeeping genes were identified in the porcine AMs in response to the stimulation with two pathogen-associated molecular patterns (PAMPs lipopolysaccharide (LPS and lipoteichoic acid (LTA. Three different algorithms (geNorm, Normfinder and BestKeeper were applied to assess the stability of HKGs. Results The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ was determined by qRT-PCR in AMs that were stimulated by LPS and LTA in vitro. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P SDHA, B2M and RPL4 showed a high expression stability in the irrespective to the stimulation group, while SDHA, YWHAZ and RPL4 showed high stability in non-stimulated control group. In all cases, GAPDH showed the least stability in geNorm. NormFinder revealed that SDHA was the most stable gene in all the groups. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study. Conclusions There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms. In conclusion, the geometric mean of the SDHA, YWHAZ and RPL4 seemed to be the most appropriate combination of HKGs for accurate normalization of gene expression data in porcine AMs without knowing the type of bacterial pathogenic status of

  16. Endomorphin-suppressed nitric oxide release from mice peritoneal macrophages.

    Science.gov (United States)

    Balog, Tihomir; Sarić, Ana; Sobocanec, Sandra; Kusić, Borka; Marotti, Tatjana

    2010-02-01

    Endomorphins are newly discovered mu-opioid receptor selective immunocompetent opioid peptides. Endomorphin 1 is predominantly distributed in brain, while endomorphin 2 is widely allocated in the spinal cord. Lately, endomorphins have been investigated as modulators of reactive oxygen and nitrogen species. Nitric oxide is short lived radical involved in various biological processes such as regulation of blood vessel contraction, inflammation, neurotransmission and apoptosis. The aim of this work was to investigate the in vivo effects of endomorphins on nitric oxide release and NOS 2 isoenzyme upregulation in mice peritoneal macrophages additionally challenged ex vivo with lipopolysaccharide. The results showed that endomorphin 1 or endomorphin 2 in vitro did not change NO release from peritoneal mouse macrophages during a 48 h incubation period. On the other hand in vivo endomorphins had suppressive effect on NO release as well as on NOS 2 and IL-1 protein concentration. The most of suppressive effect in vivo of both endomorphins was blocked with 30 min pretreatment with mu-receptor selective antagonist beta-FNA, which proved involvement of opioid receptor pathway in suppressive effects of endomorphins.

  17. aged black garlic exerts anti-inflammatory effects by decreasing no and proinflammatory cytokine production with less cytoxicity in LPS-stimulated raw 264.7 macrophages and LPS-induced septicemia mice.

    Science.gov (United States)

    Kim, Min Jee; Yoo, Yung Choon; Kim, Hyun Jung; Shin, Suk Kyung; Sohn, Eun Jeong; Min, A Young; Sung, Nak Yun; Kim, Mee Ree

    2014-10-01

    In this study, the anti-inflammatory and antisepticemic activities of a water extract of aged black garlic (AGE), which is not pungent, were compared with those of raw garlic extract (RGE). The methyl thiazolyl tetrazolium (MTT) assay showed that AGE was not toxic up to 1000 μg/mL and was at least four times less cytotoxic than RGE. AGE significantly suppressed the production of nitric oxide (NO), tumor-necrosis factor-α (TNF-α), and prostaglandin (PG)-E2 in a dose-dependent manner in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Furthermore, the inhibitory effect of AGE on LPS-induced inflammation was confirmed by downregulation of inducible NO synthase and TNF-α mRNA expression, as well as cyclooxygenase-2 protein expression. The anti-inflammatory activities of AGE were similar to those of RGE at nontoxic concentrations up to 250 μg/mL. Signal transduction pathway studies further indicated that both garlic extracts inhibited activation of mitogen-activated protein kinase and nuclear factor-κB induced by LPS stimulation. Treatment with both AGE and RGE in an in vivo experiment of LPS-induced endotoxemia significantly reduced the level of TNF-α and interleukin-6 in serum and completely protected against LPS-induced lethal shock in C57BL/6 mice. The results suggest that AGE is a more promising nutraceutical or medicinal agent to prevent or cure inflammation-related diseases for safety aspects compared with RGE.

  18. The presence of MOMA-2+ macrophages in the outer B cell zone and protection of the splenic micro-architecture from LPS-induced destruction depend on secreted IgM.

    Science.gov (United States)

    Fischer, Michael B; Rüger, Beate; Vaculik, Christine; Becherer, Alexander; Wadsak, Wolfgang; Yanagida, Genya; Losert, Udo M; Chen, Jianzhu; Carroll, Michael C; Eibl, Martha M

    2007-10-01

    The role secretory IgM has in protecting splenic tissue from LPS-induced damage was assessed in mice incapable of secreting IgM but able to express surface IgM and IgD. Within seconds after LPS challenge, 99% of the (131)I-labeled LPS was found in the liver and the spleen of both sIgM-deficient and wild-type mice. In the spleen FITC-labeled LPS was found on the surface of 2F8(+) scavenger receptor macrophages localized in the outer marginal zone, while none of the labeled LPS could be detected on marginal zone ER-TR9(+) and MOMA-1(+) macrophages. An additional population of macrophages, MOMA-2(+), were capable of producing C3 locally in the T and B cell zone after LPS challenge. Local C3 production was regulated, as no C3 was found in splenic tissue of unchallenged mice. Interestingly, in the absence of circulating and locally produced secretory IgM, MOMA-2(+) macrophages of the T and B cell zone failed to establish an additional ring of C3-producing macrophages in the outer B cell zone close to the marginal zone upon LPS challenge. The consequence was a massive destruction of the microarchitecture of the spleen where marginal zones disorganized, lymphoid follicles and T cell zones disrupted and follicular DC (FDC) networks disappeared.

  19. Effect Of α2-Adrenergic Agonists And Antagonists On Cytokine Release From Human Lung Macrophages Cultured In Vitro

    Science.gov (United States)

    Piazza, O.; Staiano, R.I.; De Robertis, E.; Conti, G.; Di Crescenzo, V.; Loffredo, S.; Marone, G.; Marinosci, G. Zito; Cataldi, M. M.

    2016-01-01

    The most trusted hypothesis to explain how α2-adrenergic agonists may preserve pulmonary functions in critically ill patients is that they directly act on macrophages by interfering with an autocrine/paracrine adrenergic system that controls cytokine release through locally synthetized noradrenaline and α1- and α2-adrenoreceptors. We tested this hypothesis in primary cultures of resident macrophages from human lung (HLMs). HLMs were isolated by centrifugation on percoll gradients from macroscopically healthy human lung tissue obtained from four different patients at the time of lung resection for cancer. HLMs from these patients showed a significant expression of α2A, α2B and α2C adrenoreceptors both at the mRNA and at the protein level. To evaluate whether α2 adrenoreceptors controlled cytokine release from HMLs, we measured IL-6, IL-8 and TNF-α concentrations in the culture medium in basal conditions and after preincubation with several α2-adrenergic agonists or antagonists. Neither the pretreatment with the α2-adrenergic agonists clonidine, medetomidine or dexdemetomidine or with the α2-adrenergic antagonist yohimbine caused significant changes in the response of any of these cytokines to LPS. These results show that, different from what reported in rodents, clonidine and dexdemetomidine do not directly suppress cytokine release from human pulmonary macrophages. This suggests that alternative mechanisms such as effects on immune cells activation or the modulation of autonomic neurotransmission could be responsible for the beneficial effects of these drugs on lung function in critical patients. PMID:27896229

  20. Mouse CD84 is a pan-leukocyte cell-surface molecule that modulates LPS-induced cytokine secretion by macrophages.

    Science.gov (United States)

    Sintes, Jordi; Romero, Xavier; de Salort, Jose; Terhorst, Cox; Engel, Pablo

    2010-10-01

    CD84 is 1 of the 9 SLAM family cell-surface receptors involved in leukocyte activation. The CD84 ectodomain is highly glycosylated, and its cytoplasmic tail contains 2 copies of an ITSM, which can be phosphorylated. Here, we report that although mouse CD84 was present on all BM HSCs, its expression declined in developing thymic and BM lymphocytes. However, CD84 expression levels did increase significantly during the later maturation stages and were expressed abundantly on mature B and T cells. Among lymphocyte subsets, the highest expression was found on innate-like lymphocytes; specifically, on NKT and marginal zone B cells. Splenic CD4+ T(FH) cells exhibited higher levels of CD84 compared with the other CD4+ T cell subsets. CD84 was expressed abundantly on monocytes, macrophages, granulocytes, and DCs. Moreover, as the function of CD84 in myeloid cells remains unknown, we focused on the role this receptor plays in mouse macrophage activation. Transfection of CD84 in RAW-264.7 macrophages led to an increase in MAPK phosphorylation and NF-κB activation upon LPS stimulation. Concomitantly, the presence of CD84 increased the LPS-induced secretion of TNF-α and MCP-1 but lowered IL-10 and IL-6 production significantly. This modulatory effect was mediated by Y(300) within the second ITSM of CD84. Additionally, CD84 knock-down decreased TNF-α and IL-6 production in LPS-activated BMDMs. Taken together, these results show that mouse CD84 is a pan-leukocyte receptor, able to modulate signaling pathways downstream of TLR4, and regulates macrophage cell-fate decisions and effector functions.

  1. Preconditioning of Carbon Monoxide Releasing Molecule-derived CO Attenuates LPS-induced Activation of HUVEC

    Directory of Open Access Journals (Sweden)

    Bingwei Sun, Xiangqian Zou, Yueling Chen, Ping Zhang, Gengsheng Shi

    2008-01-01

    Full Text Available Objective: To investigate the effects and potential mechanisms of preconditioning of tricarbonyldichlororuthenium (III dimer (CORM-2-liberated CO on LPS-induced activation of endothelial cells (HUVEC. Methods: HUVEC were pretreated with CORM-2 at the concentration of 50 or 100μM for 2 hrs, washed and stimulated with LPS (10μg/ml for additional 4 hrs. Activation (oxidative stress of HUVEC was assessed by measuring intracellular oxidation of DHR 123 or nitration of DAF-FM, specific H2O2 and NO fluorochromes, respectively. The expression of HO-1, iNOS (Western blot and ICAM-1 (cell ELISA proteins and activation of inflammation-relevant transcription factor, NF-κB (EMSA were assessed. In addition, PMN adhesion to HUVEC was also assessed. Results: The obtained data indicate that pretreatment of HUVEC with CORM-2 results in: 1 decrease of LPS-induced production of ROS and NO; 2 up-regulation of HO-1 but decrease in iNOS at the protein levels; 3 inhibition of LPS-induced activation of NF-κB; and 4 downregulation of expression of ICAM-1, and this was accompanied by a decrease of PMN adhesion to LPS-stimulated HUVEC. Conclusions: Preconditioning of CO liberated by CORM-2 elicited its anti-inflammatory effects by interfering with the induction of intracellular oxidative stress. In addition, it also supports the notion that CO is a potent inhibitor of iNOS and NF-κB.

  2. cAMP elevators inhibit LPS-induced IL-12 p40 expression by interfering with phosphorylation of p38 MAPK in Murine Peritoneal Macrophages

    Institute of Scientific and Technical Information of China (English)

    WEI; GUO; FENG; YI; BING; WANG; JIN; SONG; ZHANG; XING; YU; WANG; CHANG; LIN; LI; ZONG; LIANG; CHANG

    2002-01-01

    cAMP mediated signaling may play a suppressive role in immune response. We previously found thatthe cAMP-elevators (CTx and 8-Br-cAMP) inhibited IL-12, IL-la, IL-6 gene expression, but increasedthe transcriptional levels of IL-10 and IL-1Ra in LPS-treated murine peritoneal macrophages. The presentstudy examined a possible molecular mechanism involved in cAMP elevators-induced inhibition of IL-12 p40expression in response to LPS. Our data demonstrated that cAMP elevators downregulated IL-12 p40 mRNAexpression and IL-12 p70 production in murine peritoneal macrophages. Subsequent studies revealed thatcAMP-elevators blocked phosphorylation of p38 MAPK, but did not affect the activity of NF-κB bindingto IL-12 promoter (-136/-112). This is the first report that cAMP elevators inhibit LPS-induced IL-12production by a mechanism that is associated, at least in part, with p38-dependent inhibition by cAMPsignaling pathways.

  3. Quince (Cydonia oblonga Miller) peel polyphenols modulate LPS-induced inflammation in human THP-1-derived macrophages through NF-{kappa}B, p38MAPK and Akt inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Essafi-Benkhadir, Khadija [Laboratoire d' epidemiologie Moleculaire et Pathologie Experimentale Appliquee Aux Maladies Infectieuses, Institut Pasteur de Tunis (Tunisia); Refai, Amira [Laboratoire de Recherche sur la Transmission, le Controle et l' immunobiologie des Infections, Institut Pasteur de Tunis (Tunisia); Riahi, Ichrak [Laboratoire d' epidemiologie Moleculaire et Pathologie Experimentale Appliquee Aux Maladies Infectieuses, Institut Pasteur de Tunis (Tunisia); Fattouch, Sami [Laboratory LIP-MB National Institute of Applied Sciences and Technology, Tunis (Tunisia); Karoui, Habib [Laboratoire d' epidemiologie Moleculaire et Pathologie Experimentale Appliquee Aux Maladies Infectieuses, Institut Pasteur de Tunis (Tunisia); Essafi, Makram, E-mail: makram.essafi@pasteur.rns.tn [Laboratoire de Recherche sur la Transmission, le Controle et l' immunobiologie des Infections, Institut Pasteur de Tunis (Tunisia)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer Quince peel polyphenols inhibit LPS-induced secretion of TNF-{alpha} and IL-8. Black-Right-Pointing-Pointer Quince peel polyphenols augment LPS-induced secretion of IL-10 and IL-6. Black-Right-Pointing-Pointer Quince peel polyphenols-mediated inhibition of LPS-induced secretion of TNF-{alpha} is partially mediated by IL-6. Black-Right-Pointing-Pointer The anti-inflammatory effects of quince polyphenols pass through NF-{kappa}B, p38MAPK and Akt inhibition. -- Abstract: Chronic inflammation is a hallmark of several pathologies, such as rheumatoid arthritis, gastritis, inflammatory bowel disease, atherosclerosis and cancer. A wide range of anti-inflammatory chemicals have been used to treat such diseases while presenting high toxicity and numerous side effects. Here, we report the anti-inflammatory effect of a non-toxic, cost-effective natural agent, polyphenolic extract from the Tunisian quince Cydonia oblonga Miller. Lipopolysaccharide (LPS) treatment of human THP-1-derived macrophages induced the secretion of high levels of the pro-inflammatory cytokine TNF-{alpha} and the chemokine IL-8, which was inhibited by quince peel polyphenolic extract in a dose-dependent manner. Concomitantly, quince polyphenols enhanced the level of the anti-inflammatory cytokine IL-10 secreted by LPS-treated macrophages. We further demonstrated that the unexpected increase in IL-6 secretion that occurred when quince polyphenols were associated with LPS treatment was partially responsible for the polyphenols-mediated inhibition of TNF-{alpha} secretion. Biochemical analysis showed that quince polyphenols extract inhibited the LPS-mediated activation of three major cellular pro-inflammatory effectors, nuclear factor-kappa B (NF-{kappa}B), p38MAPK and Akt. Overall, our data indicate that quince peel polyphenolic extract induces a potent anti-inflammatory effect that may prove useful for the treatment of inflammatory diseases and that a quince

  4. Mulberry fruit prevents LPS-induced NF-κB/pERK/MAPK signals in macrophages and suppresses acute colitis and colorectal tumorigenesis in mice.

    Science.gov (United States)

    Qian, Zhengjiang; Wu, Zhiqin; Huang, Lian; Qiu, Huiling; Wang, Liyan; Li, Li; Yao, Lijun; Kang, Kang; Qu, Junle; Wu, Yonghou; Luo, Jun; Liu, Johnson J; Yang, Yi; Yang, Wancai; Gou, Deming

    2015-11-30

    Here, we investigated the impact of mulberry fruit (MBF) extracts on lipopolysaccharide (LPS)-induced inflammatory responses in RAW 264.7 macrophages, and the therapeutic efficacy of MBF diet in mice with dextran sulfate sodium (DSS)-induced acute colitis and MUC2(-/-) mice with colorectal cancer. In vitro, LPS-induced nitric oxide (NO) production was significantly inhibited by MBF extracts via suppressing the expression of proinflammatory molecules, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-β) and IL-6. Particularly, a dose-dependent inhibition on LPS-induced inflammatory responses was observed following treatment with MBF dichloromethane extract (MBF-DE), in which linoleic acid and ethyl linolenate were identified as two active compounds. Moreover, we elucidated that MBF-DE attenuated LPS-induced inflammatory responses by blocking activation of both NF-κB/p65 and pERK/MAPK pathways. In vivo, DSS-induced acute colitis was significantly ameliorated in MBF-fed mice as gauged by weight loss, colon morphology and histological damage. In addition, MBF-fed MUC2(-/-) mice displayed significant decrease in intestinal tumor and inflammation incidence compared to control diet-fed group. Overall, our results demonstrated that MBF suppressed the development of intestinal inflammation and tumorgenesis both in vitro and in vivo, and supports the potential of MBF as a therapeutic functional food for testing in human clinical trials.

  5. Ethylacetate extract from Draconis Resina inhibits LPS-induced inflammatory responses in vascular smooth muscle cells and macrophages via suppression of ROS production.

    Science.gov (United States)

    Heo, Sook-Kyoung; Yi, Hyo-Seung; Yun, Hyun-Jeong; Ko, Chang-Hyun; Choi, Jae-Woo; Park, Sun-Dong

    2010-05-01

    Draconis Resina (DR) is a type of dragon's blood resin obtained from Daemomorops draco BL. (Palmae). DR has long been used as a traditional Korean herbal medicine, and is currently used in traditional clinics to treat wounds, tumors, diarrhea, and rheumatism, insect bites and other conditions. In this study, we evaluated fractionated extracts of DR to determine if they inhibited the production of interleukin-1beta (IL-1beta) and the expression of cyclooxygenase (COX)-2. The results of this analysis revealed that the ethylacetate extract of Draconis Resina (DREA) was more potent than that of other extracts. Moreover, DREA inhibited the production of nitric oxide (NO), reactive oxygen species (ROS), prostaglandin E(2) (PGE(2)), tumor necrosis factor-alpha (TNF-alpha), IL-8 and IL-6 in lipopolysaccharide (LPS)-treated human aortic smooth muscle cells (HASMC) and RAW 264.7 macrophages. Furthermore, treatment with an NADPH oxidase assembly inhibitor, AEBSF, efficiently blocked LPS-induced mitogen-activated protein kinases (MAPKs) activation, as did DREA. These findings indicate that DREA inhibits the production of NO, PGE(2), TNF-alpha, IL-8, and IL-6 by LPS via the inhibition of ROS production, which demonstrates that DREA inhibits LPS-induced inflammatory responses via the suppression of ROS production. Taken together, these results indicate that DREA has the potential for use as an anti-atherosclerosis agent.

  6. Evaluation of inhibitory activities of plant extracts on production of LPS-stimulated pro-inflammatory mediators in J774 murine macrophages.

    Science.gov (United States)

    Verma, Nandini; Tripathi, Subhash K; Sahu, Debasis; Das, Hasi R; Das, Rakha H

    2010-03-01

    Whole plant methanolic extracts of 14 traditionally used medicinal herbs were evaluated for their anti-inflammatory activity. Extracts of Grindelia robusta, Salix nigra, Arnica montana, and Quassia amara showed up to 4.5-fold inhibition of nitric oxide (NO) production in the J774 murine macrophage cells challenged with LPS without cytotoxicity. These four selected extracts significantly reduced the protein levels of inducible NO synthase (iNOS) and the cyclooxygenase-2 (COX-2) as observed by Western blot analysis. Culture supernatants from cells treated with these extracts indicated 3-5-fold reduction of tumor necrosis factor-alpha (TNF-alpha). However, only G. robusta and Q. amara extracts significantly inhibited (by 50%) IL-1beta and IL-12 secretions. Furthermore, all these plant extracts were shown to prevent the LPS-mediated nuclear translocation of nuclear factor-kappaB (NF-kappaB). All the above observations indicate the anti-inflammatory potential of these plant extracts.

  7. Endothelin receptor-antagonists suppress lipopolysaccharide-induced cytokine release from alveolar macrophages of non-smokers, smokers and COPD subjects.

    Science.gov (United States)

    Gerlach, Kathrin; Köhler-Bachmann, Stefanie; Jungck, David; Körber, Sandra; Yanik, Sarah; Knoop, Heiko; Wehde, Deborah; Rheinländer, Sonja; Walther, Jörg W; Kronsbein, Juliane; Knobloch, Jürgen; Koch, Andrea

    2015-12-01

    Smoking-induced COPD is characterized by chronic airway inflammation, which becomes enhanced by bacterial infections resulting in accelerated disease progression called exacerbation. Alveolar macrophages (AM) release endothelin-1 (ET-1), IL-6, CCL-2 and MMP-9, all of which are linked to COPD pathogenesis and exacerbation. ET-1 signals via ETA- and ETB-receptors (ETAR, ETBR). This is blocked by endothelin receptor antagonists (ERAs), like bosentan, which targets both receptors, ETAR-selective ambrisentan and ETBR-specific BQ788. Therefore, ERAs could have anti-inflammatory potential, which might be useful in COPD and other inflammatory lung diseases. We hypothesized that ERAs suppress cytokine release from AM of smokers and COPD subjects induced by lipopolysaccharide (LPS), the most important immunogen of gram-negative bacteria. AM were isolated from the broncho-alveolar lavage (BAL) of n=29 subjects (11 non-smokers, 10 current smokers without COPD, 8 smokers with COPD), cultivated and stimulated with LPS in the presence or absence of ERAs. Cytokines were measured by ELISA. Endothelin receptor expression was investigated by RT-PCR and western blot. AM expressed ETAR and ETBR mRNA, but only ETBR protein was detected. LPS and ET-1 both induced IL-6, CCL-2 and MMP-9. LPS-induced IL-6 release was increased in COPD versus non-smokers and smokers. Bosentan, ambrisentan and BQ788 all partially reduced all cytokines without differences between cohorts. Specific ETBR inhibition was most effective. LPS induced ET-1, which was exclusively blocked by BQ788. In conclusion, LPS induces ET-1 release in AM, which in turn leads to CCL-2, IL-6 and MMP-9 expression rendering AM sensitive for ERAs. ERAs could have anti-inflammatory potential in smoking-induced COPD.

  8. Anti-Inflammatory Activity of Heterocarpin from the Salt Marsh Plant Corydalis heterocarpa in LPS-Induced RAW 264.7 Macrophage Cells

    OpenAIRE

    You Ah Kim; Chang-Suk Kong; Hyo Hyun Park; Eunkyung Lee; Mi-Soon Jang; Ki-Ho Nam; Youngwan Seo

    2015-01-01

    The inhibitory effect of three chromones 1–3 and two coumarins 4–5 on the production of nitric oxide (NO) was evaluated in LPS-induced RAW 264.7 macrophage cells. Among the compounds tested heterocarpin (1), a furochromone, significantly inhibited its production in a dose-dependent manner. In addition, heterocarpin suppressed prostaglandin E2 (PGE2) production and expression of cytokines such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α),...

  9. DMPD: New roles for Bcl10 in B-cell development and LPS response. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15049289 New roles for Bcl10 in B-cell development and LPS response. Fischer KD, Te...dford K, Wirth T. Trends Immunol. 2004 Mar;25(3):113-6. (.png) (.svg) (.html) (.csml) Show New roles for Bcl...10 in B-cell development and LPS response. PubmedID 15049289 Title New roles for Bcl10 in B-cell development

  10. Pro-oxidant activity of indicaxanthin from Opuntia ficus indica modulates arachidonate metabolism and prostaglandin synthesis through lipid peroxide production in LPS-stimulated RAW 264.7 macrophages.

    Science.gov (United States)

    Allegra, M; D'Acquisto, F; Tesoriere, L; Attanzio, A; Livrea, M A

    2014-01-01

    Macrophages come across active prostaglandin (PG) metabolism during inflammation, shunting early production of pro-inflammatory towards anti-inflammatory mediators terminating the process. This work for the first time provides evidence that a phytochemical may modulate the arachidonate (AA) metabolism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, promoting the ultimate formation of anti-inflammatory cyclopentenone 15deoxy-PGJ2. Added 1 h before LPS, indicaxanthin from Opuntia Ficus Indica prevented activation of nuclear factor-κB (NF-κB) and over-expression of PGE2 synthase-1 (mPGES-1), but up-regulated cyclo-oxygenase-2 (COX-2) and PGD2 synthase (H-PGDS), with final production of the anti-inflammatory cyclopentenone. The effects were positively related with concentration between 50 and 100 µM. Indicaxanthin did not have any effect in the absence of LPS. A kinetic study investigating the redox status of LPS-stimulated macrophages between 0.5 and 12 h, either in the absence or in the presence of 50-100 µM indicaxanthin, revealed a differential control of ROS production, with early (0.5-3 h) modest inhibition, followed by a progressive (3-12 h) concentration-dependent enhancement over the level induced by LPS alone. In addition, indicaxanthin caused early (0.5-3 h) concentration-dependent elevation of conjugated diene lipid hydroperoxides, and production of hydroxynonenal-protein adducts, over the amount induced by LPS. In LPS-stimulated macrophages indicaxanthin did not affect PG metabolism when co-incubated with either an inhibitor of NADPH oxidase or vitamin E. It is concluded that LPS-induced pro-oxidant activity of indicaxanthin at the membrane level allows formation of signaling intermediates whose accumulation modulates PG biosynthetic pathway in inflamed macrophages.

  11. Pro-oxidant activity of indicaxanthin from Opuntia ficus indica modulates arachidonate metabolism and prostaglandin synthesis through lipid peroxide production in LPS-stimulated RAW 264.7 macrophages

    Science.gov (United States)

    Allegra, M.; D’Acquisto, F.; Tesoriere, L.; Attanzio, A.; Livrea, M.A.

    2014-01-01

    Macrophages come across active prostaglandin (PG) metabolism during inflammation, shunting early production of pro-inflammatory towards anti-inflammatory mediators terminating the process. This work for the first time provides evidence that a phytochemical may modulate the arachidonate (AA) metabolism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, promoting the ultimate formation of anti-inflammatory cyclopentenone 15deoxy-PGJ2. Added 1 h before LPS, indicaxanthin from Opuntia Ficus Indica prevented activation of nuclear factor-κB (NF-κB) and over-expression of PGE2 synthase-1 (mPGES-1), but up-regulated cyclo-oxygenase-2 (COX-2) and PGD2 synthase (H-PGDS), with final production of the anti-inflammatory cyclopentenone. The effects were positively related with concentration between 50 and 100 µM. Indicaxanthin did not have any effect in the absence of LPS. A kinetic study investigating the redox status of LPS-stimulated macrophages between 0.5 and 12 h, either in the absence or in the presence of 50–100 µM indicaxanthin, revealed a differential control of ROS production, with early (0.5–3 h) modest inhibition, followed by a progressive (3–12 h) concentration-dependent enhancement over the level induced by LPS alone. In addition, indicaxanthin caused early (0.5–3 h) concentration-dependent elevation of conjugated diene lipid hydroperoxides, and production of hydroxynonenal-protein adducts, over the amount induced by LPS. In LPS-stimulated macrophages indicaxanthin did not affect PG metabolism when co-incubated with either an inhibitor of NADPH oxidase or vitamin E. It is concluded that LPS-induced pro-oxidant activity of indicaxanthin at the membrane level allows formation of signaling intermediates whose accumulation modulates PG biosynthetic pathway in inflamed macrophages. PMID:25180166

  12. A Novel Herbal Medicine KIOM-MA Exerts an Anti-Inflammatory Effect in LPS-Stimulated RAW 264.7 Macrophage Cells

    Directory of Open Access Journals (Sweden)

    You-Chang Oh

    2012-01-01

    Full Text Available KIOM-MA was recently reported as a novel herbal medicine effective for atopic dermatitis and asthma. In this study, we have demonstrated the inhibitory effect of KIOM-MA on proinflammatory mediator produced in lipopolysaccharide (LPS-stimulated RAW 264.7 cells. KIOM-MA significantly inhibited the expression of inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2 as well as nitric oxide (NO and prostaglandin E2 (PGE2. Consistent with the inhibitory effect on PGE2, KIOM-MA suppresses the LPS-induced migration of macrophages and gelatinase activity and the expression of matrix metalloprotease-9 (MMP-9 in a dose-dependent manner. Additionally, KIOM-MA showed a strong suppressive effect on the inflammatory cytokines production such as tumor necrosis factor-α (TNF-α and interleukin-6 (IL-6. We also found that KIOM-MA inhibits the activation of nuclear factor-κB (NF-κB and represses the activity of extracellular signal-regulated kinase (ERK, p38, and c-Jun NH2-terminal kinase (JNK mitogen-activated protein kinases (MAPKs. Taken together, we elucidated the mechanism of anti-inflammatory effect of KIOM-MA using RAW 264.7 cells stimulated by LPS.

  13. Punicalagin inhibits inflammation in LPS-induced RAW264.7 macrophages via the suppression of TLR4-mediated MAPKs and NF-κB activation.

    Science.gov (United States)

    Xu, Xiaolong; Yin, Peng; Wan, Changrong; Chong, Xinlu; Liu, Mingjiang; Cheng, Peng; Chen, Jiajia; Liu, Fenghua; Xu, Jianqin

    2014-06-01

    Punicalagin (2,3,hexahydroxydiphenoyl-gallagyl-D-glucose and referred to as PUN) is a bioactive ellagitannin isolated from pomegranate, which is widely used for the treatment of inflammatory bowel disease (IBD), diarrhea, and ulcers in Chinese traditional medicine. In this study, we detected the anti-inflammation potentials of PUN in lipopolysaccharide (LPS)-induced macrophages and tried to uncover the underlying mechanism. Results demonstrated that PUN (25, 50, or 100 μM) treatment could significantly decrease the LPS-induced production of nitric oxide), prostaglandin E2 (PGE2), interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in RAW264.7 cells. Molecular research showed that PUN inhibited the activation of upstream mediator nuclear factor-κB by suppressing the phosphorylation of IκBα and p65. Results also indicated that PUN could suppress the phosphorylation of mitogen-activated protein kinase including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase. In conclusion, we observed that PUN could inhibit LPS-induced inflammation, and it may be a potential choice for the treatment of inflammation diseases.

  14. Red ginseng marc oil inhibits iNOS and COX-2 via NFκB and p38 pathways in LPS-stimulated RAW 264.7 macrophages.

    Science.gov (United States)

    Bak, Min-Ji; Hong, Soon-Gi; Lee, Jong-Won; Jeong, Woo-Sik

    2012-11-22

    In this study, we investigated the anti-inflammatory effects of red ginseng marc oil (RMO) in the RAW 264.7 macrophage cell line. RMO was prepared by a supercritical CO(2) extraction of waste product generated after hot water extraction of red ginseng. RMO significantly inhibited the production of oxidative stress molecules such as nitric oxide and reactive oxygen species in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Levels of inflammatory targets including prostaglandin E2, tumor necrosis factor-α, interleukin (IL)-1β and IL-6 were also reduced after the treatment with RMO. In addition, RMO diminished the expressions of inducible nitric oxide synthase and cyclooxygenase 2 at both mRNA and protein levels. Blockade of nuclear translocation of the p65 subunit of nuclear factor κB (NFκB) was also observed after the treatment of RMO. Furthermore, RMO decreased the phosphorylations of p38 mitogen-activated protein kinase (MAPK) and its upstream kinases including MAPK kinases 3/6 (MKK3/6) and TAK 1 (TGF-β activated kinase 1). Gas chromatographic analysis on RMO revealed that RMO contained about 10% phytosterols including sitosterol, stigmasterol and campesterol which may contribute to the anti-inflammatory properties of RMO. Taken together, these results suggest that the anti-inflammatory effect of RMO in LPS-induced RAW 264.7 macrophages could be associated with the inhibition of NFκB transcriptional activity, possibly via blocking the p38 MAPK pathway.

  15. Roxatidine suppresses inflammatory responses via inhibition of NF-κB and p38 MAPK activation in LPS-induced RAW 264.7 macrophages.

    Science.gov (United States)

    Cho, Eu-Jin; An, Hyo-Jin; Shin, Ji-Sun; Choi, Hye-Eun; Ko, Jane; Cho, Young-Wuk; Kim, Hyung-Min; Choi, Jung-Hye; Lee, Kyung-Tae

    2011-12-01

    Roxatidine is a novel, specific, competitive H(2) -receptor antagonist that is used to treat gastric and duodenal ulcers, and which is known to suppress the growth of several tumors by reducing vascular endothelial growth factor (VEGF) expression. Nevertheless, it remains unclear whether roxatidine has anti-inflammatory effects. In this study, we the authors investigated the anti-inflammatory effect of roxatidine in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. It was found that roxatidine dose-dependently inhibited the productions of prostaglandin E(2) (PGE(2)), nitric oxide (NO), and histamine, and the protein and mRNA expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and histidine decarboxylase (HDC). In addition, roxatidine reduced the productions and expressions of VEGF-1 and pro-inflammatory cytokines, including those of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Electrophoretic mobility shift assays (EMSA) and reporter gene assays revealed that treatment with roxatidine attenuated the LPS-induced DNA-binding and transcriptional activity of nuclear factor kappa B (NF-κB). In addition, it was found that pretreatment with roxatidine significantly inhibited the nuclear translocations of the p65 and p50 subunits of NF-κB, and these inhibitions were not found to be associated with decreases in the phosphorylation or degradation of inhibitory kappa B-α (IκBα). Furthermore, roxatidine suppressed the phosphorylation of p38 MAP kinase, but not of IκB kinase-α/β (IKKα/β), c-Jun NH(2) -terminal kinase (JNK), or extracellular signal-regulated kinase (ERK). Taken together, these results indicate that the anti-inflammatory properties of roxatidine in LPS-treated RAW 264.7 macrophages are mediated by the inhibition of NF-κB transcriptional activity and the p38 MAP kinase pathway.

  16. A novel CD14(high) CD16(high) subset of peritoneal macrophages from cirrhotic patients is associated to an increased response to LPS.

    Science.gov (United States)

    Ruiz-Alcaraz, Antonio José; Tapia-Abellán, Ana; Fernández-Fernández, María Dolores; Tristán-Manzano, María; Hernández-Caselles, Trinidad; Sánchez-Velasco, Eduardo; Miras-López, Manuel; Martínez-Esparza, María; García-Peñarrubia, Pilar

    2016-04-01

    The aim of this study was to characterize monocyte-derived macrophages (M-DM) from blood and ascites of cirrhotic patients comparatively with those obtained from blood of healthy controls. The phenotypic profile based on CD14/CD16 expression was analyzed by flow cytometry. Cells were isolated and stimulated in vitro with LPS and heat killed Candida albicans. Phosphorylation of ERK, c-Jun, p38 MAPK, and PKB/Akt was analyzed by Western blotting. A novel CD14(high)CD16(high) M-DM subpopulation is present in ascites (∼33%). The CD14(++)CD16(+) intermediate subset is increased in the blood of cirrhotic patients (∼from 4% to 11%) and is predominant in ascites (49%), while the classical CD14(++)CD16(-) subpopulation is notably reduced in ascites (18%). Basal hyperactivation of ERK and JNK/c-Jun pathways observed in ascites M-DM correlates with CD14/CD16 high expressing subsets, while PI3K/PKB does it with the CD16 low expressing cells. In vitro LPS treatment highly increases ERK1/2, PKB/Akt and c-Jun phosphorylation, while that of p38 MAPK is decreased in M-DM from ascites compared to control blood M-DM. Stimulation of healthy blood M-DM with LPS and C. albicans induced higher phosphorylation levels of p38 than those from ascites. Regarding cytokines secretion, in vitro activated M-DM from ascites of cirrhotic patients produced significantly higher amounts of IL-6, IL-10 and TNF-α, and lower levels of IL-1β and IL-12 than control blood M-DM. In conclusion, a new subpopulation of CD14(high)CD16(high) peritoneal M-DM has been identified in ascites of cirrhotic patients, which is very sensitive to LPS stimulation.

  17. DMPD: Induction of proliferation and cytokine production in human T lymphocytes bylipopolysaccharide (LPS). [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 11090938 Induction of proliferation and cytokine production in human T lymphocytes ... (.png) (.svg) (.html) (.csml) Show Induction of proliferation and cytokine production in human T lymphocyte...and cytokine production in human T lymphocytes bylipopolysaccharide (LPS). Authors Ulmer AJ, Flad H, Rietsch

  18. Wool and grain dusts stimulate TNF secretion by alveolar macrophages in vitro.

    Science.gov (United States)

    Brown, D M; Donaldson, K

    1996-01-01

    OBJECTIVE: The aim of the study was to investigate the ability of two organic dusts, wool and grain, and their soluble leachates to stimulate secretion of tumour necrosis factor (TNF) by rat alveolar macrophages with special reference to the role of lipopolysaccharide (LPS). METHODS: Rat alveolar macrophages were isolated by bronchoalveolar lavage (BAL) and treated in vitro with whole dust, dust leachates, and a standard LPS preparation. TNF production was measured in supernatants with the L929 cell line bioassay. RESULTS: Both wool and grain dust samples were capable of stimulating TNF release from rat alveolar macrophages in a dose-dependent manner. The standard LPS preparation caused a dose-dependent secretion of TNF. Leachates prepared from the dusts contained LPS and also caused TNF release but leachable LPS could not account for the TNF release and it was clear that non-LPS leachable activity was present in the grain dust and that wool dust particles themselves were capable of causing release of TNF. The role of LPS in wool dust leachates was further investigated by treating peritoneal macrophages from two strains of mice, LPS responders (C3H) and LPS non-responders (C3H/HEJ), with LPS. The non-responder mouse macrophages produced very low concentrations of TNF in response to the wool dust leachates compared with the responders. CONCLUSIONS: LPS and other unidentified leachable substances present on the surface of grain dust, and to a lesser extent on wool dust, are a trigger for TNF release by lung macrophages. Wool dust particles themselves stimulate TNF. TNF release from macrophages could contribute to enhancement of inflammatory responses and symptoms of bronchitis and breathlessness in workers exposed to organic dusts such as wool and grain. PMID:8758033

  19. Integrated analysis of COX-2 and iNOS derived inflammatory mediators in LPS-stimulated RAW macrophages pre-exposed to Echium plantagineum L. bee pollen extract.

    Directory of Open Access Journals (Sweden)

    Eduarda Moita

    Full Text Available Oxidative stress and inflammation play important roles in disease development. This study intended to evaluate the anti-inflammatory and antioxidant potential of Echium plantagineum L. bee pollen to support its claimed health beneficial effects. The hydromethanol extract efficiently scavenged nitric oxide ((•NO although against superoxide (O2(•- it behaved as antioxidant at lower concentrations and as pro-oxidant at higher concentrations. The anti-inflammatory potential was evaluated in LPS-stimulated macrophages. The levels of (•NO and L-citrulline decreased for all extract concentrations tested, while the levels of prostaglandins, their metabolites and isoprostanes, evaluated by UPLC-MS, decreased with low extract concentrations. So, E. plantagineum bee pollen extract can exert anti-inflammatory activity by reducing (•NO and prostaglandins. The extract is able to scavenge the reactive species (•NO and O2(•- and reduce markers of oxidative stress in cells at low concentrations.

  20. Anthemis wiedemanniana essential oil prevents LPS-induced production of NO in RAW 264.7 macrophages and exerts antiproliferative and antibacterial activities in vitro.

    Science.gov (United States)

    Conforti, Filomena; Menichini, Federica; Formisano, Carmen; Rigano, Daniela; Senatore, Felice; Bruno, Maurizio; Rosselli, Sergio; Celik, Sezgin

    2012-01-01

    Anthemis wiedemanniana is known in folk medicine for the treatment of microbial infections, cancer and also urinary and pulmonary problems. In this study, the chemical composition of the essential oil from A. wiedemanniana was evaluated and its antibacterial activity was tested against 10 bacterial strains. The oil was also tested for its potentiality to inhibit nitric oxide production in RAW 264.7 macrophages and for its cytotoxicity against four human cancer cell lines. A. wiedemanniana oil, rich of oxygenated monoterpenes (25.4%), showed a good antibacterial activity against Gram-positive bacteria and a good activity against the two Gram-negative bacteria, Escherichia coli and Proteus vulgaris. Besides that, it exhibited a high inhibitory effect on the LPS-induced nitrite production and a strong cytotoxic activity, especially against amelanotic melanoma (C32) and large lung cell carcinoma (COR-L23) cell lines.

  1. Pro-oxidant activity of indicaxanthin from Opuntia ficus indica modulates arachidonate metabolism and prostaglandin synthesis through lipid peroxide production in LPS-stimulated RAW 264.7 macrophages

    Directory of Open Access Journals (Sweden)

    M. Allegra

    2014-01-01

    A kinetic study investigating the redox status of LPS-stimulated macrophages between 0.5 and 12 h, either in the absence or in the presence of 50–100 µM indicaxanthin, revealed a differential control of ROS production, with early (0.5–3 h modest inhibition, followed by a progressive (3–12 h concentration-dependent enhancement over the level induced by LPS alone. In addition, indicaxanthin caused early (0.5–3 h concentration-dependent elevation of conjugated diene lipid hydroperoxides, and production of hydroxynonenal-protein adducts, over the amount induced by LPS. In LPS-stimulated macrophages indicaxanthin did not affect PG metabolism when co-incubated with either an inhibitor of NADPH oxidase or vitamin E. It is concluded that LPS-induced pro-oxidant activity of indicaxanthin at the membrane level allows formation of signaling intermediates whose accumulation modulates PG biosynthetic pathway in inflamed macrophages.

  2. Red Ginseng Marc Oil Inhibits iNOS and COX-2 via NFκB and p38 Pathways in LPS-Stimulated RAW 264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Woo-Sik Jeong

    2012-11-01

    Full Text Available In this study, we investigated the anti-inflammatory effects of red ginseng marc oil (RMO in the RAW 264.7 macrophage cell line. RMO was prepared by a supercritical CO2 extraction of waste product generated after hot water extraction of red ginseng. RMO significantly inhibited the production of oxidative stress molecules such as nitric oxide and reactive oxygen species in lipopolysaccharide (LPS-activated RAW 264.7 cells. Levels of inflammatory targets including prostaglandin E2, tumor necrosis factor-α, interleukin (IL-1β and IL-6 were also reduced after the treatment with RMO. In addition, RMO diminished the expressions of inducible nitric oxide synthase and cyclooxygenase 2 at both mRNA and protein levels. Blockade of nuclear translocation of the p65 subunit of nuclear factor κB (NFκB was also observed after the treatment of RMO. Furthermore, RMO decreased the phosphorylations of p38 mitogen-activated protein kinase (MAPK and its upstream kinases including MAPK kinases 3/6 (MKK3/6 and TAK 1 (TGF-β activated kinase 1. Gas chromatographic analysis on RMO revealed that RMO contained about 10% phytosterols including sitosterol, stigmasterol and campesterol which may contribute to the anti-inflammatory properties of RMO. Taken together, these results suggest that the anti-inflammatory effect of RMO in LPS-induced RAW 264.7 macrophages could be associated with the inhibition of NFκB transcriptional activity, possibly via blocking the p38 MAPK pathway.

  3. Effect of gamma irradiation on the change of solubility and anti-inflammation activity of chrysin in macrophage cells and LPS-injected endotoxemic mice

    Science.gov (United States)

    Byun, Eui-Baek; Jang, Beom-Su; Byun, Eui-Hong; Sung, Nak-Yun

    2016-10-01

    This study evaluated the changes of solubility and anti-inflammatory properties of structurally modified gamma-irradiated chrysin. Chrysin was irradiated at various doses for a physical analysis and determining any structural changes and solubility. As shown through the physical analysis, the main peak of the chrysin was decreased as the irradiation dose increased, and it was concomitant with the appearance of several new peaks, which were highly increased in 50 kGy gamma-irradiated chrysin. The solubility was markedly increased in the gamma-irradiated groups. As shown through a physiological analysis, both gamma-irradiated- (15-50 kGy) and intact-chrysin (0 kGy) did not exert cytotoxicity to bone-marrow derived macrophages. The treatment of LPS-stimulated macrophages with 50 kGy gamma-irradiated chrysin resulted in a dose-dependent decrease in pro-inflammatory mediators, such as iNOS-mediated NO, PGE2, COX-2, and cell surface marker (CD80 and CD86), as well as pro-inflammatory cytokines (TNF-α and IL-6), when compared to the intact-chrysin treated group. Mechanically, we found that the inhibition of these pro-inflammatory mediators induced by gamma-irradiated chrysin occurred through an inhibition of MAPKs (ERK1/2 and p38) and the NF-κB signaling pathways. Furthermore, the anti-inflammatory activity remained in the LPS-injected animal model. In this model, gamma-irradiated chrysin treatment highly increased the mouse survival, and significantly decreased the serum cytokine (TNF-α, IL-6 and IL-1β) levels. From these findings, the anti-inflammatory action by gamma-irradiated chrysin may be closely mediated with structural modification. It seems likely that gamma irradiation can be an effective tool for improvement of the physical and physiological properties of polyphenols.

  4. Anti-inflammatory effect of a selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor via the stimulation of heme oxygenase-1 in LPS-activated mice and J774.1 murine macrophages

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    Sung Bum Park

    2016-08-01

    Full Text Available 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1 converts inactive cortisone to the active cortisol. 11β-HSD1 may be involved in the resolution of inflammation. In the present study, we investigate the anti-inflammatory effects of 2-(3-benzoyl-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR-66344, a selective 11β-HSD1 inhibitor, in lipopolysaccharide (LPS-activated C57BL/6J mice and macrophages. LPS increased 11β-HSD1 activity and expression in macrophages, which was inhibited by KR-66344. In addition, KR-66344 increased survival rate in LPS treated C57BL/6J mice. HO-1 mRNA expression level was increased by KR-66344, and this effect was reversed by the HO competitive inhibitor, ZnPP, in macrophages. Moreover, ZnPP reversed the suppression of ROS formation and cell death induced by KR-66344. ZnPP also suppressed animal survival rate in LPS plus KR-66344 treated C57BL/6J mice. In the spleen of LPS-treated mice, KR-66344 prevented cell death via suppression of inflammation, followed by inhibition of ROS, iNOS and COX-2 expression. Furthermore, LPS increased NFκB-p65 and MAPK phosphorylation, and these effects were abolished by pretreatment with KR-66344. Taken together, KR-66344 protects against LPS-induced animal death and spleen injury by inhibition of inflammation via induction of HO-1 and inhibition of 11β-HSD1 activity. Thus, we concluded that the selective 11β-HSD1 inhibitor may provide a novel strategy in the prevention/treatment of inflammatory disorders in patients.

  5. Cholesterol enrichment of human monocyte/macrophages induces surface exposure of phosphatidylserine and the release of biologically-active tissue factor-positive microvesicles.

    Science.gov (United States)

    Liu, Ming-Lin; Reilly, Michael P; Casasanto, Peter; McKenzie, Steven E; Williams, Kevin Jon

    2007-02-01

    Biologically significant amounts of two procoagulant molecules, phosphatidylserine (PS) and tissue factor (TF), are transported by monocyte/macrophage-derived microvesicles (MVs). Because cellular cholesterol accumulation is an important feature of atherosclerotic vascular disease, we now examined effects of cholesterol enrichment on MV release from human monocytes and macrophages. Cholesterol enrichment of human THP-1 monocytes, alone or in combination with lipopolysaccharide (LPS), tripled their total MV generation, as quantified by flow cytometry based on particle size and PS exposure. The subset of these MVs that were also TF-positive was likewise increased by cellular cholesterol enrichment, and these TF-positive MVs exhibited a striking 10-fold increase in procoagulant activity. Moreover, cholesterol enrichment of primary human monocyte-derived macrophages also increased their total as well as TF-positive MV release, and these TF-positive MVs exhibited a similar 10-fold increase in procoagulant activity. To explore the mechanisms of enhanced MV release, we found that cholesterol enrichment of monocytes caused PS exposure on the cell surface by as early as 2 hours and genomic DNA fragmentation in a minority of cells by 20 hours. Addition of a caspase inhibitor at the beginning of these incubations blunted both cholesterol-induced apoptosis and MV release. Cholesterol enrichment of human monocyte/macrophages induces the generation of highly biologically active, PS-positive MVs, at least in part through induction of apoptosis. Cholesterol-induced monocyte/macrophage MVs, both TF-positive and TF-negative, may be novel contributors to atherothrombosis.

  6. Stress hormone release is a key component of the metabolic response to lipopolysaccharide (LPS): studies in hypopituitary and healthy subjects

    DEFF Research Database (Denmark)

    Bach, Ermina; Møller, Andreas Buch; Jørgensen, Jens Otto Lunde

    2016-01-01

    of stress hormones. We compared the metabolic effects of LPS in hypopituitary patients (HP) (in the absence of pituitary stress hormone responses) and healthy control subjects (CTR) (with normal pituitary stress hormone responses). DESIGN: Single blind randomized. METHODS: We compared effects of LPS...... but not in HP. LPS increased whole body palmitate fluxes (3-fold) and decreased palmitate specific activity 40-50 % in CTR, but not in HP. G(0)/G(1) Switch Gene 2 (G0S2 - an inhibitor of lipolysis) adipose tissue mRNA was decreased in CTR. LPS increased phenylalanine fluxes significantly more in CTR, whereas...... on glucose, protein and lipid metabolism in eight HP and eight matched CTR twice during 4-h basal and 2-h hyperinsulinemic euglycemic clamp conditions with muscle biopsies and fat biopsies in each period during infusion with saline or LPS. RESULTS: LPS increased cortisol and growth hormone (GH) levels in CTR...

  7. Metformin Inhibits the Production of Reactive Oxygen Species from NADH:Ubiquinone Oxidoreductase to Limit Induction of Interleukin-1β (IL-1β) and Boosts Interleukin-10 (IL-10) in Lipopolysaccharide (LPS)-activated Macrophages.

    Science.gov (United States)

    Kelly, Beth; Tannahill, Gillian M; Murphy, Michael P; O'Neill, Luke A J

    2015-08-14

    Metformin, a frontline treatment for type II diabetes mellitus, decreases production of the pro-form of the inflammatory cytokine IL-1β in response to LPS in macrophages. We found that it specifically inhibited pro-IL-1β production, having no effect on TNF-α. Furthermore, metformin boosted induction of the anti-inflammatory cytokine IL-10 in response to LPS. We ruled out a role for AMP-activated protein kinase (AMPK) in the effect of metformin because activation of AMPK with A769662 did not mimic metformin here. Furthermore, metformin was still inhibitory in AMKPα1- or AMPKβ1-deficient cells. The activity of NADH:ubiquinone oxidoreductase (complex I) was inhibited by metformin. Another complex I inhibitor, rotenone, mimicked the effect of metformin on pro-IL-1β and IL-10. LPS induced reactive oxygen species production, an effect inhibited by metformin or rotenone pretreatment. MitoQ, a mitochondrially targeted antioxidant, decreased LPS-induced IL-1β without affecting TNF-α. These results, therefore, implicate complex I in LPS action in macrophages.

  8. Enhanced Inhibitory Effect of Ultra-Fine Granules of Red Ginseng on LPS-induced Cytokine Expression in the Monocyte-Derived Macrophage THP-1 Cells

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    Hong-Yeoul Kim

    2008-08-01

    Full Text Available Red ginseng is one of the most popular traditional medicines in Korea because its soluble hot-water extract is known to be very effective on enhancing immunity as well as inhibiting inflammation. Recently, we developed a new technique, called the HACgearshift system, which can pulverize red ginseng into the ultra-fine granules ranging from 0.2 to 7.0 μm in size. In this study, the soluble hot-water extract of those ultra-fine granules of red ginseng (URG was investigated and compared to that of the normal-sized granules of red ginseng (RG. The high pressure liquid chromatographic analyses of the soluble hot-water extracts of both URG and RG revealed that URG had about 2-fold higher amounts of the ginsenosides, the biologically active components in red ginseng, than RG did. Using quantitative RT-PCR, cytokine profiling against the Escherichia coli lipopolysaccharide (LPS in the monocyte-derived macrophage THP-1 cells demonstrated that the URG-treated cells showed a significant reduction in cytokine expression than the RG-treated ones. Transcription expression of the LPS-induced cytokines such as TNF-α, IL-1β, IL-6, IL-8, IL-10, and TGF-β was significantly inhibited by URG compared to RG. These results suggest that some biologically active and soluble components in red ginseng can be more effectively extracted from URG than RG by standard hot-water extraction.

  9. α-Solanine Isolated From Solanum Tuberosum L. cv Jayoung Abrogates LPS-Induced Inflammatory Responses Via NF-κB Inactivation in RAW 264.7 Macrophages and Endotoxin-Induced Shock Model in Mice.

    Science.gov (United States)

    Shin, Ji-Sun; Lee, Kyoung-Goo; Lee, Hwi-Ho; Lee, Hae Jun; An, Hyo-Jin; Nam, Jung-Hwan; Jang, Dae Sik; Lee, Kyung-Tae

    2016-10-01

    α-Solanine, a trisaccharide glycoalkaloid, has been reported to possess anti-cancer effects. In this study, we investigated the anti-inflammatory effects of α-solanine isolated from "Jayoung" a dark purple-fleshed potato by examining its in vitro inhibitory effects on inducible nitric-oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines in LPS-induced RAW 264.7 macrophages and its in vivo effects on LPS-induced septic shock in a mouse model. α-Solanine suppressed the expression of iNOS and COX-2 both at protein and mRNA levels and consequently inhibited nitric oxide (NO) and prostaglandin E2 (PGE2 ) production in LPS-induced RAW 264.7 macrophages. α-Solanine also reduced the production and mRNA expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) induced by LPS. Furthermore, molecular mechanism studies indicated that α-solanine inhibited LPS-induced activation of nuclear factor-κB (NF-κB) by reducing nuclear translocation of p65, degradation of inhibitory κBα (IκBα), and phosphorylation of IκB kinaseα/β (IKKα/β). In an in vivo experiment of LPS-induced endotoxemia, treatment with α-solanine suppressed mRNA expressions of iNOS, COX-2, IL-6, TNF-α, and IL-1β, and the activation of NF-κB in liver. Importantly, α-solanine increased the survival rate of mice in LPS-induced endotoxemia and polymicrobial sepsis models. Taken together, our data suggest that the α-solanine may be a promising therapeutic against inflammatory diseases by inhibiting the NF-κB signaling pathway. J. Cell. Biochem. 117: 2327-2339, 2016. © 2016 Wiley Periodicals, Inc.

  10. Dehydroepiandrosterone inhibits the spontaneous release of superoxide radical by alveolar macrophages in vitro in asbestosis

    Energy Technology Data Exchange (ETDEWEB)

    Rom, W.N.; Harkin, T. (New York Univ. Medical Center, New York (United States))

    1991-08-01

    Asbestosis is characterized by an alveolar macrophage alveolitis with injury and fibrosis of the lower respiratory tract. Alveolar macrophages recovered by bronchoalveolar lavage spontaneously release exaggerated amounts of oxidants including superoxide anion and hydrogen peroxide that may mediate alveolar epithelial cell injury. Dehydroepiandrosterone (DHEA) is a normally occurring adrenal androgen that inhibits glucose-6-phosphate dehydrogenase, the initial enzyme in the pentose phosphate shunt necessary for NADPH generation and superoxide anion formation. In this regard, the authors hypothesized that DHEA may reduce asbestos-induced oxidant release. DHEA added in vitro to alveolar macrophages lavaged from 11 nonsmoking asbestos workers significantly reduced superoxide anion release. DHEA is an antioxidant and potential anticarcinogenic agent that may have a therapeutic role in reducing the increased oxidant burden in asbestos-induced alveolitis of the lower respiratory tract.

  11. Integrated Analysis of COX-2 and iNOS Derived Inflammatory Mediators in LPS-Stimulated RAW Macrophages Pre-Exposed to Echium plantagineum L. Bee Pollen Extract

    Science.gov (United States)

    Moita, Eduarda; Gil-Izquierdo, Angel; Sousa, Carla; Ferreres, Federico; Silva, Luís R.; Valentão, Patrícia; Domínguez-Perles, Raúl; Baenas, Nieves; Andrade, Paula B.

    2013-01-01

    Oxidative stress and inflammation play important roles in disease development. This study intended to evaluate the anti-inflammatory and antioxidant potential of Echium plantagineum L. bee pollen to support its claimed health beneficial effects. The hydromethanol extract efficiently scavenged nitric oxide (•NO) although against superoxide (O2•−) it behaved as antioxidant at lower concentrations and as pro-oxidant at higher concentrations. The anti-inflammatory potential was evaluated in LPS-stimulated macrophages. The levels of •NO and L-citrulline decreased for all extract concentrations tested, while the levels of prostaglandins, their metabolites and isoprostanes, evaluated by UPLC-MS, decreased with low extract concentrations. So, E. plantagineum bee pollen extract can exert anti-inflammatory activity by reducing •NO and prostaglandins. The extract is able to scavenge the reactive species •NO and O2•− and reduce markers of oxidative stress in cells at low concentrations. PMID:23520554

  12. Anti-inflammatory and antioxidant effects of a combination of cannabidiol and moringin in LPS-stimulated macrophages.

    Science.gov (United States)

    Rajan, Thangavelu Soundara; Giacoppo, Sabrina; Iori, Renato; De Nicola, Gina Rosalinda; Grassi, Gianpaolo; Pollastro, Federica; Bramanti, Placido; Mazzon, Emanuela

    2016-07-01

    Inflammatory response plays an important role in the activation and progress of many debilitating diseases. Natural products, like cannabidiol, a constituent of Cannabis sativa, and moringin, an isothiocyanate obtained from myrosinase-mediated hydrolysis of the glucosinolate precursor glucomoringin present in Moringa oleifera seeds, are well known antioxidants also endowed with anti-inflammatory activity. This is due to a covalent-based mechanism for ITC, while non-covalent interactions underlie the activity of CBD. Since these two mechanisms are distinct, and the molecular endpoints are potentially complementary, we investigated in a comparative way the protective effect of these compounds alone or in combination on lipopolysaccharide-stimulated murine macrophages. Our results show that the cannabidiol (5μM) and moringin (5μM) combination outperformed the single constituents that, at this dosage had only a moderate efficacy on inflammatory (Tumor necrosis factor-α, Interleukin-10) and oxidative markers (inducible nitric oxide synthase, nuclear factor erythroid 2-related factor 2, nitrotyrosine). Significant upregulation of Bcl-2 and downregulation of Bax and cleaved caspase-3 was observed in cells treated with cannabidiol-moringin combination. Treatment with the transient receptor potential vanilloid receptor 1 antagonist was detrimental for the efficacy of cannabidiol, while no effect was elicited by cannabinoid receptor 1 and cannabinoid receptor 2 antagonists. None of these receptors was involved in the activity of moringin. Taken together, our in vitro results testify the anti-inflammatory, antioxidative, and anti-apoptotic effects of the combination of cannabidiol and moringin.

  13. LPS-induced release of IL-6 from glia modulates production of IL-1beta in a JAK2-dependent manner

    LENUS (Irish Health Repository)

    Minogue, Aedín M

    2012-06-14

    AbstractBackgroundCompelling evidence has implicated neuroinflammation in the pathogenesis of a number of neurodegenerative conditions. Chronic activation of both astrocytes and microglia leads to excessive secretion of proinflammatory molecules such as TNFα, IL-6 and IL-1β with potentially deleterious consequences for neuronal viability. Many signaling pathways involving the mitogen-activated protein kinases (MAPKs), nuclear factor κB (NFκB) complex and the Janus kinases (JAKs)\\/signal transducers and activators of transcription (STAT)-1 have been implicated in the secretion of proinflammatory cytokines from glia. We sought to identify signaling kinases responsible for cytokine production and to delineate the complex interactions which govern time-related responses to lipopolysaccharide (LPS).MethodsWe examined the time-related changes in certain signaling events and the release of proinflammatory cytokines from LPS-stimulated co-cultures of astrocytes and microglia isolated from neonatal rats.ResultsTNFα was detected in the supernatant approximately 1 to 2 hours after LPS treatment while IL-1β and IL-6 were detected after 2 to 3 and 4 to 6 hours, respectively. Interestingly, activation of NFκB signaling preceded release of all cytokines while phosphorylation of STAT1 was evident only after 2 hours, indicating that activation of JAK\\/STAT may be important in the up-regulation of IL-6 production. Additionally, incubation of glia with TNFα induced both phosphorylation of JAK2 and STAT1 and the interaction of JAK2 with the TNFα receptor (TNFR1). Co-treatment of glia with LPS and recombinant IL-6 protein attenuated the LPS-induced release of both TNFα and IL-1β while potentiating the effect of LPS on suppressor of cytokine signaling (SOCS)3 expression and IL-10 release.ConclusionsThese data indicate that TNFα may regulate IL-6 production through activation of JAK\\/STAT signaling and that the subsequent production of IL-6 may impact on the release of

  14. Anti-inflammatory action of high molecular weight Mytilus edulis hydrolysates fraction in LPS-induced RAW264.7 macrophage via NF-κB and MAPK pathways.

    Science.gov (United States)

    Kim, Young-Sang; Ahn, Chang-Bum; Je, Jae-Young

    2016-07-01

    Anti-inflammatory Mytilus edulis hydrolysates (MEHs) were prepared by peptic hydrolysis and MEH was further fractionated into three fractions based on molecular weight, namely >5kDa, 1-5kDa, and 5kDa peptide fraction exerted the highest nitric oxide (NO) inhibitory activity and inhibited prostaglandin E2 (PGE2) secretion in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Pretreatment with the >5kDa peptide fraction markedly inhibited LPS-stimulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and gene expressions. Stimulation by LPS induced the production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and -1β (IL-1β), whereas co-treatment with the >5kDa peptide fraction suppressed pro-inflammatory cytokine production. The >5kDa peptide fraction inhibited the translocation of NF-κB (nuclear factor-kappa B) through the prevention of IκBα (inhibitory factor kappa B alpha) phosphorylation and degradation and also inhibited the MAPK signaling pathway in LPS-stimulated RAW264.7 macrophages.

  15. Cyclic AMP enhancing drugs modulate eicosanoid release from human alveolar macrophages

    NARCIS (Netherlands)

    F.D. Beusenberg; H.C. Hoogsteden (Henk); I.L. Bonta; J.G.C. van Amsterdam (Jan)

    1994-01-01

    textabstractThe effect of the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX), salbutamol and sodium nitroprusside was evaluated regarding PGE2 and LTB4 release and cAMP and cGMP level in human alveolar macrophages obtained from controls and COPD patients. Basal levels per five million co

  16. Natural product HTP screening for antibacterial (E.coli 0157:H7) and anti-inflammatory agents in (LPS from E. coli O111:B4) activated macrophages and microglial cells; focus on sepsis.

    Science.gov (United States)

    Mazzio, Elizabeth A; Li, Nan; Bauer, David; Mendonca, Patricia; Taka, Equar; Darb, Mohammed; Thomas, Leeshawn; Williams, Henry; Soliman, Karam F A

    2016-11-15

    Acute systemic inflammatory response syndrome arising from infection can lead to multiple organ failure and death, with greater susceptibility occurring in immunocompromised individuals. Moreover, sub-acute chronic inflammation is a contributor to the pathology of diverse degenerative diseases (Parkinson's disease, Alzheimer's disease and arthritis). Given the known limitations in Western medicine to treat a broad range of inflammatory related illness as well as the emergence of antibiotic resistance, there is a renewed interest in complementary and alternative medicines (CAMs) to achieve these means. A high throughput (HTP) screening of >1400 commonly sold natural products (bulk herbs, cooking spices, teas, leaves, supplement components, nutraceutical food components, fruit and vegetables, rinds, seeds, polyphenolics etc.) was conducted to elucidate anti-inflammatory substances in lipopolysaccharide (LPS) (E. coli serotype O111:B4) monocytes: RAW 264.7 macrophages [peripheral], BV-2 microglia [brain]) relative to hydrocortisone, dexamethasone and L-N6-(1Iminoethyl)lysine (L-NIL). HTP evaluation was also carried out for lethal kill curves against E.coli 0157:H7 1x10(6) CFU/mL relative to penicillin. Validation studies were performed to assess cytokine profiling using antibody arrays. Findings were corroborated by independent ELISAs and NO2-/iNOS expression quantified using the Griess Reagent and immunocytochemistry, respectively. For robust screening, we developed an in-vitro efficacy paradigm to ensure anti-inflammatory parameters were observed independent of cytotoxicity. This caution was taken given that many plants exert tumoricidal and anti-inflammatory effects at close range through similar signaling pathways, which could lead to false positives. The data show that activated BV-2 microglia cells (+ LPS 1μg/ml) release >10-fold greater IL-6, MIP1/2, RANTES and nitric oxide (NO2-), where RAW 264.7 macrophages (+ LPS 1μg/ml) produced > 10-fold rise in sTNFR2

  17. CD14 mediates binding of high doses of LPS but is dispensable for TNF-α production.

    Science.gov (United States)

    Borzęcka, Kinga; Płóciennikowska, Agnieszka; Björkelund, Hanna; Sobota, Andrzej; Kwiatkowska, Katarzyna

    2013-01-01

    Activation of macrophages with lipopolysaccharide (LPS) involves a sequential engagement of serum LPS-binding protein (LBP), plasma membrane CD14, and TLR4/MD-2 signaling complex. We analyzed participation of CD14 in TNF-α production stimulated with 1-1000 ng/mL of smooth or rough LPS (sLPS or rLPS) and in sLPS binding to RAW264 and J744 cells. CD14 was indispensable for TNF-α generation induced by a low concentration, 1 ng/mL, of sLPS and rLPS. At higher doses of both LPS forms (100-1000 ng/mL), TNF-α release required CD14 to much lower extent. Among the two forms of LPS, rLPS-induced TNF-α production was less CD14-dependent and could proceed in the absence of serum as an LBP source. On the other hand, the involvement of CD14 was crucial for the binding of 1000 ng/mL of sLPS judging from an inhibitory effect of the anti-CD14 antibody. The binding of sLPS was also strongly inhibited by dextran sulfate, a competitive ligand of scavenger receptors (SR). In the presence of dextran sulfate, sLPS-induced production of TNF-α was upregulated about 1.6-fold. The data indicate that CD14 together with SR participates in the binding of high doses of sLPS. However, CD14 contribution to TNF α production induced by high concentrations of sLPS and rLPS can be limited.

  18. Ethanol inhibits LPS-induced signaling and modulates cytokine production in peritoneal macrophages in vivo in a model for binge drinking

    Directory of Open Access Journals (Sweden)

    Pruett Stephen B

    2009-09-01

    Full Text Available Abstract Background Previous reports indicate that ethanol, in a binge drinking model in mice, inhibits the production of pro-inflammatory cytokines in vivo. However, the inhibition of signaling through TLR4 has not been investigated in this experimental model in vivo. Considering evidence that signaling can be very different in vitro and in vivo, the present study was conducted to determine if effects of ethanol on TLR4 signaling reported for cells in culture or cells removed from ethanol treated mice and stimulated in culture also occur when ethanol treatment and TLR4 activation occur in vivo. Results Phosphorylated p38, ERK, and c-Jun (nuclear were quantified with kits or by western blot using samples taken 15, 30, and 60 min after stimulation of peritoneal macrophages with lipopolysaccharide in vivo. Effects of ethanol were assessed by administering ethanol by gavage at 6 g/kg 30 min before administration of lipopolysaccharide (LPS. Cytokine concentrations in the samples of peritoneal lavage fluid and in serum were determined at 1, 2, and 6 hr after lipopolysaccharide administration. All of these data were used to measure the area under the concentration vs time curve, which provided an indication of the overall effects of ethanol in this system. Ethanol suppressed production of most pro-inflammatory cytokines to a similar degree as it inhibited key TLR4 signaling events. However, NF-κB (p65 translocation to the nucleus was not inhibited by ethanol. To determine if NF-κB composed of other subunits was inhibited, transgenic mice with a luciferase reporter were used. This revealed a reproducible inhibition of NF-κB activity, which is consistent with the observed inhibition of cytokines whose expression is known to be NF-κB dependent. Conclusion Overall, the effects of ethanol on signalling in vivo were similar to those reported for in vitro exposure to ethanol and/or lipopolysaccharide. However, inhibition of the activation of NF-κB was

  19. A defective TLR4 signaling for IFN-β expression is responsible for the innately lower ability of BALB/c macrophages to produce NO in response to LPS as compared to C57BL/6.

    Directory of Open Access Journals (Sweden)

    Luciana S Oliveira

    Full Text Available C57BL/6 mice macrophages innately produce higher levels of NO than BALB/c cells when stimulated with LPS. Here, we investigated the molecular events that account for this intrinsic differential production of NO. We found that the lower production of NO in BALB/c is not due to a subtraction of L-arginine by arginase, and correlates with a lower iNOS accumulation, which is independent of its degradation rate. Instead, the lower accumulation of iNOS is due to the lower levels of iNOS mRNA, previously shown to be also independent of its stability, suggesting that iNOS transcription is less efficient in BALB/c than in C57BL/6 macrophages. Activation of NFκB is more efficient in BALB/c, thus not correlating with iNOS expression. Conversely, activation of STAT-1 does correlate with iNOS expression, being more prominent in C57BL/6 than in BALB/c macrophages. IFN-β and IL-10 are more highly expressed in C57BL/6 than in BALB/c macrophages, and the opposite is true for TNF-α. Whereas IL-10 and TNF-α do not seem to participate in their differential production of NO, IFN-β has a determinant role since 1 anti-IFN-β neutralizing antibodies abolish STAT-1 activation reducing NO production in C57BL/6 macrophages to levels as low as in BALB/c cells and 2 exogenous rIFN-β confers to LPS-stimulated BALB/c macrophages the ability to phosphorylate STAT-1 and to produce NO as efficiently as C57BL/6 cells. We demonstrate, for the first time, that BALB/c macrophages are innately lower NO producers than C57BL/6 cells because they are defective in the TLR-4-induced IFN-β-mediated STAT-1 activation pathway.

  20. Upregulation of heme oxygenase-1 via PI3K/Akt and Nrf-2 signaling pathways mediates the anti-inflammatory activity of Schisandrin in Porphyromonas gingivalis LPS-stimulated macrophages.

    Science.gov (United States)

    Park, Sun Young; Park, Da Jung; Kim, Young Hun; Kim, Younghee; Kim, Sun Gun; Shon, Kwang Jae; Choi, Young-Whan; Lee, Sang-Joon

    2011-09-30

    The lipopolysaccharide (LPS) of Porphyromonas gingivalis is thought to induce periodontitis. In this study, we isolated Schisandrin from the dried fruits of Schisandra chinensis and examined the anti-inflammatory effect of Schisandrin in macrophages stimulated with LPS from P. gingivalis. First, Schisandrin inhibited LPS-induced pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6. And Schisandrin suppressed the nuclear translocation and activity of NF-κB and phosphorylation of IκBα in LPS-stimulated RAW 264.7 cells. Next, the presence of a selective inhibitor of HO-1 (SnPP) and a siRNA specific for HO-1 inhibited Schisandrin-mediated anti-inflammatory activity. Furthermore, Schisandrin induced HO-1 expression of RAW 264.7 cells through Nrf-2, PI3K/Akt, and ERK activation. Therefore, these results suggest that the anti-inflammatory effects of Schisandrin on P. gingivalis LPS-stimulated RAW 264.7 cells may be due to a reduction of NF-κB activity and induction of the expression of HO-1, leading to TNF-α, IL-1β, and IL-6 down-regulation.

  1. The essential oil isolated from Artemisia capillaris prevents LPS-induced production of NO and PGE(2) by inhibiting MAPK-mediated pathways in RAW 264.7 macrophages.

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    Cha, Jeong-Dan; Moon, Sang-Eun; Kim, Hye-Young; Lee, Jeong-Chae; Lee, Kyung-Yeol

    2009-01-01

    Artemisia capillaris (A. capillaris) is used in traditional Korean herbal medicine for its believedanti-inflammatory activities. Previous studies have suggested that the essential oil of A. capillaris contains the active components responsible for its pharmacological effect, even though the mechanism for its action is unclear. This study examined the inhibitory effects of the essential oil of A. capillaris on the lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)). The essential oil significantly inhibited the production of NO in the LPS-stimulated RAW 264.7 macrophages, which was mediated by the down-regulation of inducible NO synthase (iNOS) expression but not by its direct cytotoxic activity. The essential oil also blocked the secretion of PGE(2) and the expression of cyclooxygenase-2 (COX-2) in the LPS-stimulated cells. Western blot analysis showed that the essential oil inhibited the phosphorylation of IkappaB-alpha, nuclear translocation of p65, and subsequent activation of NF-kappaB. In addition, the essential oil suppressed the LPS-stimulated activation of mitogen-activated protein kinases (MAPKs) as well as the AP-1 DNA-binding activity. Moreover, MAPK inhibitors significantly reduced the LPS-induced production of NO and PGE(2). Collectively, we suggest that the oil inhibits the expression and production of inflammatory mediators by blocking the MAPK-mediated pathways and inhibiting the activation of NF-kappaB and AP-1.

  2. Beta-adrenergic receptor agonists induce the release of granulocyte chemotactic protein-2, oncostatin M, and vascular endothelial growth factor from macrophages

    NARCIS (Netherlands)

    Verhoeckx, K.C.M.; Doornbos, R.P.; Witkamp, R.F.; Greef, de J.; Rodenburg, R.J.T.

    2006-01-01

    Vascular endothelial growth factor (VEGF), oncostatin M (OSM), and granulocyte chemotactic protein-2 (GCP-2/CXCL6) are up-regulated in U937 macrophages and peripheral blood macrophages exposed to LPS, beta-adrenergic receptor (ß2-AR) agonists (e.g. zilpaterol, and clenbuterol) and some other agents

  3. Beta-adrenergic receptor agonists induce the release of granulocyte chemotactic protein-2, oncostatin M, and vascular endothelial growth factor from macrophages

    NARCIS (Netherlands)

    Verhoeckx, K.C.; Doornbos, R.P.; Witkamp, R.F.; Greef, J. van der; Rodenburg, R.J.T.

    2006-01-01

    Vascular endothelial growth factor (VEGF), oncostatin M (OSM), and granulocyte chemotactic protein-2 (GCP-2/CXCL6) are up-regulated in U937 macrophages and peripheral blood macrophages exposed to LPS, beta-adrenergic receptor (beta2-AR) agonists (e.g. zilpaterol, and clenbuterol) and some other agen

  4. Beta-adrenergic receptor agonists induce the release of granulocyte chemotactic protein-2, oncostatin M, and vascular endothelial growth factor from macrophages

    NARCIS (Netherlands)

    Verhoeckx, K.C.M.; Doornbos, R.P.; Witkamp, R.F.; Greef, J. van der; Rodenburg, R.J.T.

    2006-01-01

    Vascular endothelial growth factor (VEGF), oncostatin M (OSM), and granulocyte chemotactic protein-2 (GCP-2/CXCL6) are up-regulated in U937 macrophages and peripheral blood macrophages exposed to LPS, beta-adrenergic receptor (β2-AR) agonists (e.g. zilpaterol, and clenbuterol) and some other agents

  5. Propolis and its constituent caffeic acid suppress LPS-stimulated pro-inflammatory response by blocking NF-κB and MAPK activation in macrophages.

    Science.gov (United States)

    Búfalo, Michelle Cristiane; Ferreira, Isabel; Costa, Gustavo; Francisco, Vera; Liberal, Joana; Cruz, Maria Teresa; Lopes, Maria Celeste; Batista, Maria Teresa; Sforcin, José Maurício

    2013-08-26

    Propolis is a bee product with numerous biological and pharmacological properties, such as immunomodulatory and anti-inflammatory activities. It has been used in folk medicine as a healthy drink and in food to improve health and prevent inflammatory diseases. However, little is known about its mechanism of action. Thus, the goal of this study was to verify the antioxidant activity and to explore the anti-inflammatory properties of propolis by addressing its intracellular mechanism of action. Caffeic acid was investigated as a possible compound responsible for propolis action. The antioxidant properties of propolis and caffeic acid were evaluated by using the 2,2-Diphenyl-1-picrylhydrazyl free radical (DPPH) scavenging method. To analyze the anti-inflammatory activity, Raw 264.7 macrophages were treated with different concentrations of propolis or caffeic acid, and nitric oxide (NO) production, a strong pro-inflammatory mediator, was evaluated by the Griess reaction. The concentrations of propolis and caffeic acid that inhibited NO production were evaluated on intracellular signaling pathways triggered during inflammation, namely p38 mitogen-activated protein kinase (MAPK), c-jun NH2-terminal kinase (JNK1/2), the transcription nuclear factor (NF)-κB and extracellular signal-regulated kinase (ERK1/2), through Western blot using specific antibodies. A possible effect of propolis on the cytotoxicity of hepatocytes was also evaluated, since this product can be used in human diets. Caffeic acid showed a higher antioxidant activity than propolis extract. Propolis and caffeic acid inhibited NO production in macrophages, at concentrations without cytotoxicity. Furthermore, both propolis and caffeic acid suppressed LPS-induced signaling pathways, namely p38 MAPK, JNK1/2 and NF-κB. ERK1/2 was not affected by propolis extract and caffeic acid. In addition, propolis and caffeic acid did not induce hepatotoxicity at concentrations with strong anti-inflammatory potential

  6. Genkwanin inhibits proinflammatory mediators mainly through the regulation of miR-101/MKP-1/MAPK pathway in LPS-activated macrophages.

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    Yuan Gao

    Full Text Available Genkwanin is one of the major non-glycosylated flavonoids in many herbs with anti-inflammatory activities. Although its anti-inflammatory activity in vivo has been reported, the potential molecular mechanisms remain obscure. In this study, by pharmacological and genetic approaches, we explore the anti-inflammatory effects of genkwanin in LPS-activated RAW264.7 macrophages. Genkwanin potently decreases the proinflammatory mediators, such as iNOS, TNF-α, IL-1β and IL-6, at the transcriptional and translational levels without cytotoxicity, indicating the excellent anti-inflammatory potency of genkwanin in vitro. Mechanism study shows that genkwanin significantly suppresses the p38- and JNK-mediated AP-1 signaling pathway and increases the mitogen-activated protein kinase (MAPK phosphatase 1 (MKP-1 expression at the posttranscriptional level. We also confirmed that microRNA-101 (miR-101 is a negative regulator of MKP-1 expression. Moreover, regardless of miR-101-deficient cells or miR-101-abundant cells, the suppression effects of genkwanin on supernatant proinflammatory mediators' levels are far less than that in respective negative control cells, suggesting that genkwanin exerts anti-inflammatory effect mainly through reducing miR-101 production. However, genkwanin can't affect the level of phospho-Akt (p-Akt, indicating that the phosphorylation of Akt may be not responsible for the effect of genkwanin on miR-101 production. We conclude that genkwanin exerts its anti-inflammatory effect mainly through the regulation of the miR-101/MKP-1/MAPK pathway.

  7. System x(c)(-) regulates microglia and macrophage glutamate excitotoxicity in vivo.

    Science.gov (United States)

    Kigerl, Kristina A; Ankeny, Daniel P; Garg, Sanjay K; Wei, Ping; Guan, Zhen; Lai, Wenmin; McTigue, Dana M; Banerjee, Ruma; Popovich, Phillip G

    2012-01-01

    It is widely believed that microglia and monocyte-derived macrophages (collectively referred to as central nervous system (CNS) macrophages) cause excitotoxicity in the diseased or injured CNS. This view has evolved mostly from in vitro studies showing that neurotoxic concentrations of glutamate are released from CNS macrophages stimulated with lipopolysaccharide (LPS), a potent inflammogen. We hypothesized that excitotoxic killing by CNS macrophages is more rigorously controlled in vivo, requiring both the activation of the glutamate/cystine antiporter (system x(c)(-)) and an increase in extracellular cystine, the substrate that drives glutamate release. Here, we show that non-traumatic microinjection of low-dose LPS into spinal cord gray matter activates CNS macrophages but without causing overt neuropathology. In contrast, neurotoxic inflammation occurs when LPS and cystine are co-injected. Simultaneous injection of NBQX, an antagonist of AMPA glutamate receptors, reduces the neurotoxic effects of LPS+cystine, implicating glutamate as a mediator of neuronal cell death in this model. Surprisingly, neither LPS nor LPS+cystine adversely affects survival of oligodendrocytes or oligodendrocyte progenitor cells. Ex vivo analyses show that redox balance in microglia and macrophages is controlled by induction of system x(c)(-) and that high GSH:GSSG ratios predict the neurotoxic potential of these cells. Together, these data indicate that modulation of redox balance in CNS macrophages, perhaps through regulating system x(c)(-), could be a novel approach for attenuating injurious neuroinflammatory cascades.

  8. Euscaphic acid isolated from roots of Rosa rugosa inhibits LPS-induced inflammatory responses via TLR4-mediated NF-κB inactivation in RAW 264.7 macrophages.

    Science.gov (United States)

    Kim, In-Tae; Ryu, Suran; Shin, Ji-Sun; Choi, Jung-Hye; Park, Hee-Juhn; Lee, Kyung-Tae

    2012-06-01

    As an attempt to search for bioactive natural products exerting anti-inflammatory activity, we have evaluated the anti-inflammatory effects of euscaphic acid (19α-hydroxyursane-type triterpenoids, EA) isolated from roots of Rosa rugosa and its underlying molecular mechanisms in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. EA concentration-dependently reduced the production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) induced by LPS in RAW 264.7 macgophages. Consistent with these data, expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and iNOS, COX-2, TNF-α, and IL-1β mRNA were inhibited by EA in a concentration-dependent manner. In addition, EA attenuated LPS-induced DNA binding and transcriptional activity of nuclear factor-kappa B (NF-κB), which was accompanied by a parallel reduction of degradation and phosphorylation of inhibitory kappa Bα (IκBα) and consequently by decreased nuclear translocation of p65 subunit of NF-κB. Pretreatment with EA significantly inhibited the LPS-induced phosphorylation of IκB kinase β (IKKβ), p38, and JNK, whereas the phosphorylation of ERK1/2 was unaffected. Furthermore, EA interfered with the LPS-induced clustering of TNF receptor-associated factor 6 (TRAF6) with interleukin receptor associated kinase 1 (IRAK1) and transforming growth factor-β-activated kinase 1 (TAK1). Taken together, these results suggest that EA inhibits LPS-induced inflammatory responses by interference with the clustering of TRAF6 with IRAK1 and TAK1, resulting in blocking the activation of IKK and MAPKs signal transduction to downregulate NF-κB activations.

  9. Tacaribe virus but not junin virus infection induces cytokine release from primary human monocytes and macrophages.

    Directory of Open Access Journals (Sweden)

    Allison Groseth

    Full Text Available The mechanisms underlying the development of disease during arenavirus infection are poorly understood. However, common to all hemorrhagic fever diseases is the involvement of macrophages as primary target cells, suggesting that the immune response in these cells may be of paramount importance during infection. Thus, in order to identify features of the immune response that contribute to arenavirus pathogenesis, we have examined the growth kinetics and cytokine profiles of two closely related New World arenaviruses, the apathogenic Tacaribe virus (TCRV and the hemorrhagic fever-causing Junin virus (JUNV, in primary human monocytes and macrophages. Both viruses grew robustly in VeroE6 cells; however, TCRV titres were decreased by approximately 10 fold compared to JUNV in both monocytes and macrophages. Infection of both monocytes and macrophages with TCRV also resulted in the release of high levels of IL-6, IL-10 and TNF-α, while levels of IFN-α, IFN-β and IL-12 were not affected. However, we could show that the presence of these cytokines had no direct effect on growth of either TCRV of JUNV in macrophages. Further analysis also showed that while the production of IL-6 and IL-10 are dependent on viral replication, production of TNF-α also occurs after exposure to UV-inactivated TCRV particles and is thus independent of productive virus infection. Surprisingly, JUNV infection did not have an effect on any of the cytokines examined indicating that, in contrast to other viral hemorrhagic fever viruses, macrophage-derived cytokine production is unlikely to play an active role in contributing to the cytokine dysregulation observed in JUNV infected patients. Rather, these results suggest that an early, controlled immune response by infected macrophages may be critical for the successful control of infection of apathogenic viruses and prevention of subsequent disease, including systemic cytokine dysregulation.

  10. IRF8 is the target of SIRT1 for the inflammation response in macrophages.

    Science.gov (United States)

    Jia, Yanhui; Han, Shichao; Li, Jun; Wang, Hongtao; Liu, Jiaqi; Li, Na; Yang, Xuekang; Shi, Jihong; Han, Juntao; Li, Yan; Bai, Xiaozhi; Su, Linlin; Hu, Dahai

    2017-02-01

    The type III histone deacetylase SIRT1 has recently emerged as a critical immune regulator by suppressing T-cell immunity and macrophage activation during inflammation, but its mechanism in regulating inflammatory response in macrophages remains unclear. Here we show that the expression of SIRT1 in macrophage cells decreased following the release of inflammation cytokines when the cells were stimulated with LPS. IRF8, an important regulator in monocyte differentiation and macrophage polarization, showed the opposite trend, with SIRT1 expression levels increasing after the cells treated with LPS. Co-immunoprecipitation and immunofluorescence experiments showed that SIRT1 could not only interact with IRF8, but also deacetylate it. LPS treatment had no effect on the expression of IRF8 in macrophage cells in which sirt1 was specifically deleted. Our results show that IRF8 may be the target of histone deacetylase SIRT1 to regulate the inflammation in the macrophage cells.

  11. Effect of pecan phenolics on the release of nitric oxide from murine RAW 264.7 macrophage cells.

    Science.gov (United States)

    Robbins, Katherine S; Greenspan, Phillip; Pegg, Ronald B

    2016-12-01

    Inflammation is linked to numerous chronic disease states. Phenolic compounds have attracted attention because a number of these compounds possess anti-inflammatory properties. A phenolic crude extract was prepared from pecans and separated by Sephadex LH-20 column chromatography into low- and high-molecular-weight (LMW/HMW) fractions. Anti-inflammatory properties of these fractions were assessed in LPS-stimulated RAW 264.7 murine macrophage cells. NO and reactive oxygen species (ROS) production was monitored after 3 different experimental protocols: (1) pre-treatment with Escherichia coli O111:B4 lipopolysaccharide (LPS); (2) pre-treatment with a pecan crude extract and its fractions; and (3) co-incubation of LPS with a pecan crude extract and its fractions. The LMW fraction displayed a dose-dependent decrease in NO production and a significant decrease from the LPS control in ROS production when cells were either co-incubated with or pre-treated with LPS. The phenolics were characterized by HPLC to help identify those responsible for the observed effect.

  12. A Curcumin Derivative, 2,6-Bis(2,5-dimethoxybenzylidene-cyclohexanone (BDMC33 Attenuates Prostaglandin E2 Synthesis via Selective Suppression of Cyclooxygenase-2 in IFN-g/LPS-Stimulated Macrophages

    Directory of Open Access Journals (Sweden)

    Syahida Ahmad

    2011-11-01

    Full Text Available Our preliminary screening had shown that the curcumin derivative [2,6-bis(2,5-dimethoxybenzylidenecyclohexanone] or BDMC33 exhibited improved anti-inflammatory activity by inhibiting nitric oxide synthesis in activated macrophage cells. In this study, we further investigated the anti-inflammatory properties of BDMC33 on PGE2 synthesis and cyclooxygenase (COX expression in IFN-g/LPS-stimulated macrophages. We found that BDMC33 significantly inhibited PGE2 synthesis in a concentration-dependent manner albeit at a low inhibition level with an IC50 value of 47.33 ± 1.00 µM. Interestingly, the PGE2 inhibitory activity of BDMC33 is not attributed to inhibition of the COX enzyme activities, but rather BDMC33 selectively down-regulated the expression of COX-2. In addition, BDMC33 modulates the COX expression by sustaining the constitutively COX-1 expression in IFN-g/LPS-treated macrophage cells. Collectively, the experimental data suggest an immunodulatory action of BDMC33 on PGE2 synthesis and COX expression, making it a possible treatment for inflammatory disorders with minimal gastrointestinal-related side effects.

  13. A curcumin derivative, 2,6-bis(2,5-dimethoxybenzylidene)-cyclohexanone (BDMC33) attenuates prostaglandin E2 synthesis via selective suppression of cyclooxygenase-2 in IFN-γ/LPS-stimulated macrophages.

    Science.gov (United States)

    Lee, Ka-Heng; Abas, Faridah; Alitheen, Noorjahan Banu Mohamed; Shaari, Khozirah; Lajis, Nordin Haji; Ahmad, Syahida

    2011-11-23

    Our preliminary screening had shown that the curcumin derivative [2,6-bis(2,5-dimethoxybenzylidene)cyclohexanone] or BDMC33 exhibited improved anti-inflammatory activity by inhibiting nitric oxide synthesis in activated macrophage cells. In this study, we further investigated the anti-inflammatory properties of BDMC33 on PGE(2 )synthesis and cyclooxygenase (COX) expression in IFN-γ/LPS-stimulated macrophages. We found that BDMC33 significantly inhibited PGE(2) synthesis in a concentration-dependent manner albeit at a low inhibition level with an IC(50) value of 47.33 ± 1.00 µM. Interestingly, the PGE(2) inhibitory activity of BDMC33 is not attributed to inhibition of the COX enzyme activities, but rather BDMC33 selectively down-regulated the expression of COX-2. In addition, BDMC33 modulates the COX expression by sustaining the constitutively COX-1 expression in IFN-γ/LPS-treated macrophage cells. Collectively, the experimental data suggest an immunodulatory action of BDMC33 on PGE(2) synthesis and COX expression, making it a possible treatment for inflammatory disorders with minimal gastrointestinal-related side effects.

  14. Release of lysosomal enzymes in Candida albicans phagocytosis by rat peritoneal macrophages.

    Science.gov (United States)

    Fontenla de Petrino, S E; Sirena, A

    1984-02-15

    The present paper reports the in vitro release of lysosomal enzymes in the supernatant of cultures of rat peritoneal macrophages, with the addition of Candida albicans cells. Macrophages were taken from the rat peritoneal cavity 72 hr after non-specific activation with Brain-Heart-Infusion (B.H.I.) broth containing 10% proteose-peptone No. 3. They were then cultured in Parker medium No. 199 (TC 199). After 24 hr a suspension of Candida albicans cells, in a determined concentration, was added to the peritoneal macrophage cultures. At that time, and during pre-determined periods, the following enzymes in the culture supernatants were studied using colorimetric methods: beta-glucuronidase, beta-galactosidase and acid phosphatase. It is concluded that, under identical conditions, the release of beta-galactosidase and acid phosphatase is higher than for beta-glucuronidase. The release rate of all three enzymes is the highest at a 6 hr incubation period, after which, a gradual decrease leads to the rate down to 50% at 24 hr.

  15. Responses of macrophages to the danger signals released from necrotic cells.

    Science.gov (United States)

    Kimura, Toshifumi; Kobayashi, Shuhei; Hanihara-Tatsuzawa, Fumito; Sayama, Aoi; MaruYama, Takashi; Muta, Tatsushi

    2014-12-01

    The immune system maintains homeostasis by recognizing and responding to cell death caused by various stresses. The immune response is considered to be elicited by 'danger signals' released from necrotic cells. However, the identity of the danger signals remains elusive. In this study, we focused on the expression of chemokines by macrophages stimulated with necrotic cells. In mouse bone-marrow-derived macrophages, the chemokine monocyte chemoattractant protein (MCP)-3 was induced at both the mRNA and protein levels in response to heat-killed murine cells. The induction of MCP-3 was also observed in MyD88-deficient macrophages, indicating that Toll-like receptors and the IL-1 receptor are not involved in this response. Consistent with this observation, the activation of NF-κB was not detected in RAW264.7 macrophages stimulated with necrotic cells. Treatments with proteinase K, DNaseI or RNaseA did not affect the ' STIMULATING ACTIVITY': of necrotic cells. In contrast, treatment with apyrase, which removes phosphates from nucleoside tri- and di-phosphates, abolished the inducing activity. Purified UDP at 30 µM concentration elicited similar induction of MCP-3 in RAW264.7 macrophages. Small interfering RNA-mediated knock-down of the UDP receptor P2Y6 in RAW264.7 cells significantly reduced the induction of MCP-3 in response to necrotic cells, but not its induction by lipopolysaccharide. Furthermore, ectopic expression of the P2Y6 receptor in HEK293 cells conferred responsiveness to necrotic cells. These results suggest that UDP released by necrotic cells plays a critical role as an endogenous danger signal and that P2Y6 is required for the induction of MCP-3 in response to necrotic cells.

  16. Synthesis of New Tricyclic and Tetracyclic Fused Coumarin Sulfonate Derivatives and Their Inhibitory Effects on LPS-Induced Nitric Oxide and PGE2 Productions in RAW 264.7 Macrophages: Part 2.

    Science.gov (United States)

    El-Gamal, Mohammed I; Lee, Woo-Seok; Shin, Ji-Sun; Oh, Chang-Hyun; Lee, Kyung-Tae; Choi, Jungseung; Myoung, Nohsun; Baek, Daejin

    2016-11-01

    The synthesis of a new series of 21 fused coumarin derivatives is described, and the biological evaluation of their in vitro antiinflammatory effects as inhibitors of lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2 ) production in RAW 264.7 macrophages. The target compounds 1a-u were first tested for cytotoxicity to determine a non-toxic concentration for antiinflammatory screening, so that the inhibitory effects against NO and PGE2 production would not be caused by cytotoxicity. Compounds 1f and 1p were the most active PGE2 inhibitors with IC50 values of 0.89 and 0.95 µM, respectively. Western blot and cell-free COX-2 screening showed that their effects were due to inhibition of both COX-2 protein expression and COX-2 enzyme activity. Their IC50 values against the COX-2 enzyme were 0.67 and 0.85 µM, respectively, which is more potent than etoricoxib. The selectivity indexes of compounds 1f and 1p against COX-2 compared to COX-1 were 41.1 and 42.5, respectively. Compound 1f showed strong inhibitory effects at 5 µM concentration on COX-2 mRNA expression in LPS-induced RAW 264.7 macrophages. Moreover, the tricyclic compounds 1l and 1n as well as the tetracyclic analog 1u were the most potent NO inhibitors, with one-digit micromolar IC50 values. They showed dose-dependent inhibition of inducible nitric oxide synthase (iNOS) protein expression. The tetracyclic derivative 1u was the most potent inhibitor of NO production. It also exhibited a strong inhibitory effect on iNOS mRNA expression in LPS-induced RAW 264.7 macrophages.

  17. Reduced scytonemin isolated from Nostoc commune suppresses LPS/IFNγ-induced NO production in murine macrophage RAW264 cells by inducing hemeoxygenase-1 expression via the Nrf2/ARE pathway.

    Science.gov (United States)

    Itoh, Tomohiro; Koketsu, Mamoru; Yokota, Naoto; Touho, Shota; Ando, Masashi; Tsukamasa, Yasuyuki

    2014-07-01

    Reduced scytonemin (R-scy) and scytonemin (Scy) isolated from Nostoc commune exhibit anti-tumor and ultraviolet-absorbing properties. In this study, we examined the effects of R-scy and Scy on the induction of nitric oxide (NO) production by lipopolysaccharide (LPS) and interferon-γ (IFNγ) in murine macrophage RAW264 cells. While both R-scy and Scy suppressed LPS/IFNγ-induced NO production, R-scy exhibited a stronger inhibitory effect compared with Scy. To further elucidate the mechanisms underlying the anti-inflammatory effects of R-scy, we examined the changes in the intracellular signaling cascade after LPS/IFNγ stimulation in cells. In addition to the attenuation of LPS/IFNγ-induced upregulation of the inducible isoform of NO synthase, R-scy decreased the activity of nuclear factor-κB, phosphatidylinositol 3-kinase (PI3K)/Akt, and mitogen-activated protein kinases (MAPKs) after LPS/IFNγ stimulation. R-scy treatment increased heme oxygenase-1 (HO-1) expression by increasing the intracellular levels of reactive oxygen species and thereby activating nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant response element signaling. The induction of HO-1 by R-scy was inhibited by pretreatment with an antioxidant, N-acetyl-cysteine (NAC), as well as SB203580 and LY294002, inhibitors for p38 MAPK and PI3K/Akt, respectively. Our findings suggest that the anti-inflammatory effects of R-scy could involve both the ROS/PI3K/Akt and the p38 MAPK/Nrf2 signaling pathways. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. CD14 mediated endogenous TNF-alpha release in HL60 AML cells: a potential model for CD14 mediated endogenous cytokine release in the treatment of AML.

    Science.gov (United States)

    Treon, S P; Anand, B; Ulevitch, R; Broitman, S A

    1994-01-01

    In previous studies, HL60 AML cells treated with tumor necrosis factor-alpha (TNF), interferon-gamma (IFN), and lipopolysaccharides (LPS) displayed decreased growth and viability, enhanced monocytic pathway differentiation and endogenous TNF release. Endogenous TNF release by LPS/TNF/IFN treated HL60 cells was postulated to play a role with the above findings. In these studies, HL60 cells expressed CD14 when treated with TNF, IFN, and LPS. CD14 mediates TNF release in monocytes/macrophages in response to binding of LPS with LPS binding protein (LBP). CD14 was not expressed in either untreated or LPS only treated HL60 cells. CD14 expression was present and greater with HL60 cells cultured with LPS/TNF/IFN vs TNF/IFN (47.47% vs 9.07% positive, respectively) suggesting synergism for LPS in CD14 induction. CD14 expression was associated with endogenous TNF release, and with significantly higher levels by HL60 cells treated with LPS/TNF/IFN vs TNF/IFN (p < 0.001). Addition of anti-CD14 antibody significantly reduced release of TNF in TNF/IFN (p < 0.001) and LPS/TNF/IFN (p = 0.0013) treated cells. KG1 and U937 AML cells treated with LPS, TNF, and IFN did not express CD14, nor release TNF. A model for inducing release of endogenous growth inhibitory cytokines by CD14 bearing AML cells is proposed as an approach to AML therapy.

  19. Oral gold compound auranofin triggers arachidonate release and cyclooxygenase metabolism in the alveolar macrophage

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    Peters-Golden, M.; Shelly, C.

    1988-12-01

    We examined the effect of in vitro incubation with the oral gold compound auranofin (AF) on arachidonic acid (AA) release and metabolism by rat alveolar macrophages (AMs). AF stimulated dose- and time-dependent release of /sup 14/C-AA from prelabeled AMs, which reached 4.7 +/- 0.3% (mean +/- SEM) of incorporated radioactivity at 10 micrograms/ml for 90 min, as compared to 0.5 +/- 0.1% release following control incubation for 90 min (p less than 0.001). Similar dose- and time-dependent synthesis of thromboxane (Tx) A2 (measured as TxB2) and prostaglandin (PG) E2 was demonstrated by radioimmunoassay of medium from unlabeled cultures, reaching 18-fold and 9-fold, respectively, of the control values at 10 micrograms/ml AF for 90 min (p less than 0.001 for both). AF-induced TxB2 and PGE2 synthesis was inhibited by indomethacin as well as by pretreatment with methylprednisolone. No increase in the synthesis of immunoreactive leukotrienes (LT) B4 or C4 was noted at any dose or time of AF. High performance liquid chromatographic separation of /sup 14/C-eicosanoids synthesized by prelabeled AMs confirmed that AF induced the release of free AA and its metabolism to cyclooxygenase, but not 5-lipoxygenase, metabolites. The ability of AF to trigger macrophage AA metabolism may be relevant to the exacerbation of certain inflammatory processes which sometimes accompany gold therapy.

  20. Contribution of inflammatory cytokine release to activation of resident peritoneal macrophages after in vivo low-dose {gamma}-irradiation

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    Ibuki, Yuko; Goto, Rensuke [Shizuoka Univ. (Japan). Graduate School of Nutritional and Environmental Sciences

    1999-09-01

    The activation mechanism of resident peritoneal macrophages by in vivo {gamma}-irradiation was investigated. The function of macrophages as accessory cells in concanavalin A-induced proliferation of spleno-lymphocytes (accessory function) was enhanced 4 h after a low-dose irradiation (4 cGy) in vivo, but not in vitro, indicating that low-dose irradiation acts indirectly on the activation of macrophages. Because we expected that macrophages were activated by the recognition of substances damaged by in vivo irradiation, we co-cultured macrophages with oxidized erythrocyte-ghosts. No change was found in their accessory function. The production of inflammatory cytokines, interleukin-1{beta} (IL-1{beta}) and interferon-{gamma} (IFN-{gamma}), in the supernatant of co-cultures of spleno-lymphocytes and macrophages was determined by an ELISA. Production of both increased in the presence of in vivo irradiated macrophages. Furthermore, IL-1{beta} production from in vivo-irradiated macrophages treated with recombinant IFN-{gamma} also was enhanced. The mRNA expression of the cytokines released from macrophages and lymphocytes was determined by RT-PCR. Increased IL-1{beta}mRNA expression were found in both in vivo- and in vitro-irradiated macrophages. In vivo irradiation also enhanced the expression of IFN-{gamma}mRNA in lymphocytes, whereas there was no change after in vitro irradiation. On the basis of these observations, we propose that the activation of macrophages is caused by interaction with neighboring cells, such as lymphocytes, and by paracrine induction of certain cytokines which is initiated by the small amount of IL-1{beta} released by irradiated macrophages. (author)

  1. Oleifolioside A, a New Active Compound, Attenuates LPS-Stimulated iNOS and COX-2 Expression through the Downregulation of NF-κB and MAPK Activities in RAW 264.7 Macrophages.

    Science.gov (United States)

    Yu, Hai Yang; Kim, Kyoung-Sook; Lee, Young-Choon; Moon, Hyung-In; Lee, Jai-Heon

    2012-01-01

    Oleifolioside A, a new triterpenoid compound isolated from Dendropanax morbifera Leveille (D. morbifera), was shown in this study to have potent inhibitory effects on lipopolysaccharide (LPS-)stimulated nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in RAW 264.7 macrophages. Consistent with these findings, oleifolioside A was further shown to suppress the expression of LPS-stimulated inducible nitric oxide synthase (iNOS) and cyclooxigenase-2 (COX-2) in a dose-dependent manner at both the protein and mRNA levels and to significantly inhibit the DNA-binding activity and transcriptional activity of NF-κB in response to LPS. These results were found to be associated with the inhibition of the degradation and phosphorylation of IκB-α and subsequent translocation of the NF-κB p65 subunit to the nucleus. Inhibition of NF-κB activation by oleifolioside A was also shown to be mediated through the prevention of p38 MAPK and ERK1/2 phosphorylation. Taken together, our results suggest that oleifolioside A has the potential to be a novel anti-inflammatory agent capable of targeting both the NF-κB and MAPK signaling pathways.

  2. Oleifolioside A, a New Active Compound, Attenuates LPS-Stimulated iNOS and COX-2 Expression through the Downregulation of NF-κB and MAPK Activities in RAW 264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Hai Yang Yu

    2012-01-01

    Full Text Available Oleifolioside A, a new triterpenoid compound isolated from Dendropanax morbifera Leveille (D. morbifera, was shown in this study to have potent inhibitory effects on lipopolysaccharide (LPS-stimulated nitric oxide (NO and prostaglandin E2 (PGE2 production in RAW 264.7 macrophages. Consistent with these findings, oleifolioside A was further shown to suppress the expression of LPS-stimulated inducible nitric oxide synthase (iNOS and cyclooxigenase-2 (COX-2 in a dose-dependent manner at both the protein and mRNA levels and to significantly inhibit the DNA-binding activity and transcriptional activity of NF-κB in response to LPS. These results were found to be associated with the inhibition of the degradation and phosphorylation of IκB-α and subsequent translocation of the NF-κB p65 subunit to the nucleus. Inhibition of NF-κB activation by oleifolioside A was also shown to be mediated through the prevention of p38 MAPK and ERK1/2 phosphorylation. Taken together, our results suggest that oleifolioside A has the potential to be a novel anti-inflammatory agent capable of targeting both the NF-κB and MAPK signaling pathways.

  3. Aqueous extract of Gracilaria tenuistipitata suppresses LPS-induced NF-κB and MAPK activation in RAW 264.7 and rat peritoneal macrophages and exerts hepatoprotective effects on carbon tetrachloride-treated rat.

    Science.gov (United States)

    Tseng, Chin-Kai; Lin, Chun-Kuang; Chang, Hsueh-Wei; Wu, Yu-Hsuan; Yen, Feng-Lin; Chang, Fang-Rong; Chen, Wei-Chun; Yeh, Chi-Chen; Lee, Jin-Ching

    2014-01-01

    In addition to the previous investigations of bioactivity of aqueous extract of the edible Gracilaria tenuistipitata (AEGT) against H2O2-induced DNA damage and hepatitis C virus replication, the purpose of this study is to evaluate the potential therapeutic properties of AEGT against inflammation and hepatotoxicity using lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 cells, primary rat peritoneal macrophages and carbon tetrachloride (CCl4)-induced acute hepatitis model in rats. AEGT concentration-dependently inhibited the elevated RNA and protein levels of inducible nitric oxide synthase and cyclooxygenase-2, thereby reducing nitric oxide and prostaglandin E2 levels, respectively. Moreover, AEGT significantly suppressed the production of LPS-induced proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor-α. These inhibitory effects were associated with the suppression of nuclear factor-kappa B activation and mitogen-activated protein kinase phosphorylation by AEGT in LPS-stimulated cells. In addition, we highlighted the hepatoprotective and curative effects of AEGT in a rat model of CCl4-intoxicated acute liver injury, which was evident from reduction in the elevated serum aspartate aminotransferase and alanine aminotransferase levels as well as amelioration of histological damage by pre-treatment or post-treatment of AEGT. In conclusion, the results demonstrate that AEGT may serve as a potential supplement in the prevention or amelioration of inflammatory diseases.

  4. Aqueous extract of Gracilaria tenuistipitata suppresses LPS-induced NF-κB and MAPK activation in RAW 264.7 and rat peritoneal macrophages and exerts hepatoprotective effects on carbon tetrachloride-treated rat.

    Directory of Open Access Journals (Sweden)

    Chin-Kai Tseng

    Full Text Available In addition to the previous investigations of bioactivity of aqueous extract of the edible Gracilaria tenuistipitata (AEGT against H2O2-induced DNA damage and hepatitis C virus replication, the purpose of this study is to evaluate the potential therapeutic properties of AEGT against inflammation and hepatotoxicity using lipopolysaccharide (LPS-stimulated mouse RAW 264.7 cells, primary rat peritoneal macrophages and carbon tetrachloride (CCl4-induced acute hepatitis model in rats. AEGT concentration-dependently inhibited the elevated RNA and protein levels of inducible nitric oxide synthase and cyclooxygenase-2, thereby reducing nitric oxide and prostaglandin E2 levels, respectively. Moreover, AEGT significantly suppressed the production of LPS-induced proinflammatory cytokines, including interleukin (IL-1β, IL-6 and tumor necrosis factor-α. These inhibitory effects were associated with the suppression of nuclear factor-kappa B activation and mitogen-activated protein kinase phosphorylation by AEGT in LPS-stimulated cells. In addition, we highlighted the hepatoprotective and curative effects of AEGT in a rat model of CCl4-intoxicated acute liver injury, which was evident from reduction in the elevated serum aspartate aminotransferase and alanine aminotransferase levels as well as amelioration of histological damage by pre-treatment or post-treatment of AEGT. In conclusion, the results demonstrate that AEGT may serve as a potential supplement in the prevention or amelioration of inflammatory diseases.

  5. Short-term heating reduces the anti-inflammatory effects of fresh raw garlic extracts on the LPS-induced production of NO and pro-inflammatory cytokines by downregulating allicin activity in RAW 264.7 macrophages.

    Science.gov (United States)

    Shin, Jung-Hye; Ryu, Ji Hyeon; Kang, Min Jung; Hwang, Cho Rong; Han, Jaehee; Kang, Dawon

    2013-08-01

    Garlic has a variety of biologic activities, including anti-inflammatory properties. Although garlic has several biologic activities, some people dislike eating fresh raw garlic because of its strong taste and smell. Therefore, garlic formulations involving heating procedures have been developed. In this study, we investigated whether short-term heating affects the anti-inflammatory properties of garlic. Fresh and heated raw garlic extracts (FRGE and HRGE) were prepared with incubation at 25 °C and 95 °C, respectively, for 2 h. Treatment with FRGE and HRGE significantly reduced the LPS-induced increase in the pro-inflammatory cytokine concentration (TNF-α, IL-1β, and IL-6) and NO through HO-1 upregulation in RAW 264.7 macrophages. The anti-inflammatory effect was greater in FRGE than in HRGE. The allicin concentration was higher in FRGE than in HRGE. Allicin treatment showed reduced production of pro-inflammatory cytokines and NO and increased HO-1 activity. The results show that the decrease in LPS-induced NO and pro-inflammatory cytokines in RAW 264.7 macrophages through HO-1 induction was greater for FRGE compared with HRGE. Additionally, the results indicate that allicin is responsible for the anti-inflammatory effect of FRGE. Our results suggest a potential therapeutic use of allicin in the treatment of chronic inflammatory disease.

  6. Globular Adiponectin Causes Tolerance to LPS-Induced TNF-α Expression via Autophagy Induction in RAW 264.7 Macrophages: Involvement of SIRT1/FoxO3A Axis.

    Science.gov (United States)

    Pun, Nirmala Tilija; Subedi, Amit; Kim, Mi Jin; Park, Pil-Hoon

    2015-01-01

    Adiponectin, an adipokine predominantly produced from adipose tissue, exhibited potent anti-inflammatory properties. In particular, it inhibits production of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In the present study, we investigated the role of autophagy induction in the suppression of Lipopolysaccharide (LPS) -induced TNF-α expression by globular adiponectin (gAcrp) and its potential mechanisms. Herein, we found that gAcrp treatment increased expression of genes related with autophagy, including Atg5 and microtubule-associated protein light chain (LC3B), induced autophagosome formation and autophagy flux in RAW 264.7 macrophages. Similar results were observed in primary macrophages isolated peritoneum of mice. Interestingly, inhibition of autophagy by pretreatment with Bafilomycin A1 or knocking down of LC3B gene restored suppression of TNF-α expression, tumor necrosis factor receptor- associated factor 6 (TRAF6) expression and p38MAPK phosphorylation by gAcrp, implying a critical role of autophagy induction in the development of tolerance to LPS-induced TNF-α expression by gAcrp. We also found that knocking-down of FoxO3A, a forkhead box O member of transcription factor, blocked gAcrp-induced expression of LC3II and Atg5. Moreover, gene silencing of Silent information regulator 1 (SIRT1) blocked both gAcrp-induced nuclear translocation of FoxO3A and LC3II expression. Finally, pretreatment with ROS inhibitors, prevented gAcrp-induced SIRT1 expression and further generated inhibitory effects on gAcrp-induced autophagy, indicating a role of ROS production in gAcrp-induced SIRT1 expression and subsequent autophagy induction. Taken together, these findings indicate that globular adiponectin suppresses LPS-induced TNF-α expression, at least in part, via autophagy activation. Furthermore, SIRT1-FoxO3A

  7. Extracellular Release of CD11b by TLR9 Stimulation in Macrophages.

    Directory of Open Access Journals (Sweden)

    Dongbum Kim

    Full Text Available CpG-DNA upregulates the expression of pro-inflammatory cytokines, chemokines and cell surface markers. Investigators have shown that CD11b (integrin αM regulates TLR-triggered inflammatory responses in the macrophages and dendritic cells. Therefore, we aimed to identify the effects of CpG-DNA on the expression of CD11b in macrophages. There was no significant change in surface expression of CD11b after CpG-DNA stimulation. However, CD11b was released into culture supernatants after stimulation with phosphorothioate-backbone modified CpG-DNA such as PS-ODN CpG-DNA 1826(S. In contrast, MB-ODN 4531 and non-CpG-DNA control (regardless of backbone type and liposome-encapsulation failed to induce release of CD11b. Therefore, the context of the CpG-DNA sequence and phosphorothioate backbone modification may regulate the effects of CpG-DNA on CD11b release. Based on inhibitor studies, CD11b release is mediated by p38 MAP kinase activation, but not by the PI3K and NF-κB activation. CD11b release is mediated by lysosomal degradation and by vacuolar acidification in response to CpG-DNA stimulation. The amount of CD11b in the exosome precipitant was significantly increased by CpG-DNA stimulation in vivo and in vitro depending on TLR9. Our observations perhaps give more insight into understanding of the mechanisms involved in CpG-DNA-induced immunomodulation in the innate immunity.

  8. Zinc Oxide Nanoparticles Suppress LPS-Induced NF-κB Activation by Inducing A20, a Negative Regulator of NF-κB, in RAW 264.7 Macrophages.

    Science.gov (United States)

    Kim, Min-Ho; Jeong, Hyun-Ja

    2015-09-01

    Zinc contained in solar salt and bamboo salt plays a critical role in various immune responses. Zinc oxide is a source of zinc, and recently it has been reported that zinc oxide nanoparticles (ZO-NP) more effectively decrease allergic inflammatory reactions than zinc oxide bulk material. The aim of this work was to investigate the regulatory effect of ZO-NP on interferon (IFN)-γ plus lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. ZO-NP (0.1-10 μg/mL) did not affect cell viability but toxicity was evident at a ZO-NP concentration of 100 μg/mL. ZO-NP (10 μg/mL) inhibited the IFN-γ plus LPS-induced production of nitric oxide and the protein expressions of inducible nitric oxide synthase and cyclooxygenase-2. The productions of inflammatory cytokines, such as, interleukin (IL)-1β and tumor necrosis factor (TNF)-α were increased by IFN-γ plus LPS but down-regulated by ZO-NP treatment. Furthermore, the up-regulations of IL-1β and TNF-α mRNAs by IFN-γ plus LPS were reduced by ZO-NP at low (0.1 μg/mL) and high (10 μg/mL) concentrations. ZO-NP (0.1, 1, and 10 μg/mL) inhibited the nuclear translocation of nuclear factor-κB by blocking IκBα phosphorylation and degradation. In addition, ZO-NP induced the expression of A20, a zinc finger protein and negative regulator of NF-κB. In conclusion, the present study demonstrated that ZO-NP offer a potential means of treating inflammatory diseases.

  9. Asbestos fibres and man made mineral fibres: induction and release of tumour necrosis factor-alpha from rat alveolar macrophages.

    OpenAIRE

    Ljungman, A G; Lindahl, M.; Tagesson, C

    1994-01-01

    OBJECTIVES--Mounting evidence suggests that asbestos fibres can stimulate alveolar macrophages to generate the potent inflammatory and fibrogenic mediator, tumour necrosis factor-alpha (TNF-alpha), and that this may play an important part in the onset and development of airway inflammation and lung fibrosis due to asbestos fibre inhalation. Little is known, however, about the ability of other mineral fibres to initiate formation and release of TNF-alpha by alveolar macrophages. Therefore the ...

  10. Apoptotic neutrophils containing Staphylococcus epidermidis stimulate macrophages to release the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6.

    Science.gov (United States)

    Wilsson, Asa; Lind, Sara; Ohman, Lena; Nilsdotter-Augustinsson, Asa; Lundqvist-Setterud, Helen

    2008-06-01

    Staphylococcus epidermidis infections are usually nosocomial and involve colonization of biomaterials. The immune defense system cannot efficiently control the bacteria during these infections, which often results in protracted chronic inflammation, in which a key event is disturbed removal of neutrophils by tissue macrophages. While ingesting uninfected apoptotic neutrophils, macrophages release anti-inflammatory cytokines that lead to resolution of inflammation. In clinical studies, we have previously found elevated levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 in synovial fluid from prostheses infected with coagulase negative staphylococci. We show that macrophages phagocytosing apoptotic neutrophils containing S. epidermidis released TNF-alpha and interleukin-6, whereas macrophages phagocytosing spontaneously apoptotic neutrophils did not. This difference was not due to dissimilar phagocytic capacities, because macrophages ingested both types of neutrophils to the same extent. The activation was induced mainly by the apoptotic neutrophils themselves, not by the few remaining extracellular bacteria. Macrophages were not activated by apoptotic neutrophils that contained paraformaldehyde-killed S. epidermidis. Proinflammatory reactions induced by clearance of apoptotic neutrophils containing S. epidermidis might represent an important mechanism to combat the infective agent. This activation of macrophages may contribute to the development of chronic inflammation instead of inflammation resolution.

  11. Nutritionally relevant concentrations of resveratrol and hydroxytyrosol mitigate oxidative burst of human granulocytes and monocytes and the production of pro-inflammatory mediators in LPS-stimulated RAW 264.7 macrophages.

    Science.gov (United States)

    Bigagli, Elisabetta; Cinci, Lorenzo; Paccosi, Sara; Parenti, Astrid; D'Ambrosio, Mario; Luceri, Cristina

    2017-02-01

    The health benefits of bio-active phenolic compounds have been largely investigated in vitro at concentrations which exceed those reachable in vivo. We investigated and compared the anti-inflammatory effects of resveratrol, hydroxytyrosol and oleuropein at physiologically relevant concentrations by using in vitro models of inflammation. Human granulocytes and monocytes were stimulated with phorbol myristate acetate (PMA) and the ability of resveratrol, hydroxytyrosol and oleuropein to inhibit the oxidative burst and CD11b expression was measured. Nitric oxide (NO), prostaglandin E2 (PGE2) levels, COX-2, iNOS, TNFα, IL-1β and miR-146a expression and activation of the transcription factor Nrf2 were evaluated in macrophages RAW 264.7 stimulated with LPS (1μg/ml) for 18h, exposed to resveratrol, hydroxytyrosol and oleuropein (5 and 10μM). Synergistic effects were explored as well, together with the levels of PGE2, COX-2 and IL-1β expression in macrophages after 6h of LPS stimulation. PGE2 and COX-2 expression were also assessed on human monocytes. All the tested compounds inhibited granulocytes oxidative burst in a concentration dependent manner and CD11b expression was also significantly counteracted by resveratrol and hydroxytyrosol. The measurement of oxidative burst in human monocytes produced similar effects being resveratrol more active. Hydroxytyrosol and resveratrol inhibited the production of NO and PGE2 but did not reduce iNOS, TNFα or IL-1β gene expression in LPS-stimulated RAW 264.7 for 18h. Resveratrol slightly decreased COX-2 expression after 18h but not after 6h, but reduced PGE2 levels after 6h. Resveratrol and hydroxytyrosol 10μM induced NRf2 nuclear translocation and reduced miR-146a expression in LPS treated RAW 264.7. Overall, we reported an anti-inflammatory effect of resveratrol and hydroxytyrosol at low, nutritionally relevant concentrations, involving the inhibition of granulocytes and monocytes activation, the modulation of miR-146a

  12. Inflammatory and fibrotic mediator release by alveolar macrophages from coal miners

    Energy Technology Data Exchange (ETDEWEB)

    Kuhn, D.C.; Stauffer, J.L.; Gaydos, L.J.; Demers, L.M. [Pennsylvania State University, Hershey, PA (United States). Milton S. Hershey Medical Center, Dept. of Pathology

    1995-09-01

    Eicosanoids and cytokines produced by alveolar macrophages (AM) are key mediators of pulmonary inflammation and fibrosis. In order to determine if eicosanoid production and cytokine production are altered in AM obtained from coal miners, we compared production of prostaglandin E(2) (PGE (2)), thromboxane A(2) (TXA(2)), leukotriene B-4 (LTB(4)), interleukin-1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF alpha) by cultured AM from normal human subjects and coal miners. The recovery of AM from miners` lungs by bronchoalveolar lavage was significantly greater than that from control subjects. Mean eicosanoid and cytokine production by AM from active miners was also increased compared to AM from control subjects, but this increase was not statistically significant. AM from control subjects produced significantly more TXA (2) and TNF alpha when exposed to lipopolysaccharide than did AM from miners. The cyclooxygenase inhibitor suprofen reduced PGE(2) and TXA(2) production and TNF alpha release but had no effect on LTB (4) production or IL-1 beta release by miners` AM. The lipoxygenase inhibitor nordihydroguaiaretic acid attenuated TNF alpha release, as well as that of LTB(4), but had no effect on IL-1 beta release. Inhibition of thromboxane synthase by UK 38,485 also reduced TNF alpha release by active miners` AM but had no effect on PGE(2), LTB(4) production, or IL-1 beta release. The results of these studies suggest that occupational inhalation of coal dust may increase total lung eicosanoid and cytokine levels and reduce the reactivity of AM to bacterial endotoxin. Furthermore, coal dust-induced changes in both eicosanoid and cytokine release may be subject to pharmacological modulation.

  13. Consistent inhibition of cyclooxygenase drives macrophages towards the inflammatory phenotype.

    Directory of Open Access Journals (Sweden)

    Yi Rang Na

    Full Text Available Macrophages play important roles in defense against infection, as well as in homeostasis maintenance. Thus alterations of macrophage function can have unexpected pathological results. Cyclooxygenase (COX inhibitors are widely used to relieve pain, but the effects of long-term usage on macrophage function remain to be elucidated. Using bone marrow-derived macrophage culture and long-term COX inhibitor treatments in BALB/c mice and zebrafish, we showed that chronic COX inhibition drives macrophages into an inflammatory state. Macrophages differentiated in the presence of SC-560 (COX-1 inhibitor, NS-398 (COX-2 inhibitor or indomethacin (COX-1/2 inhibitor for 7 days produced more TNFα or IL-12p70 with enhanced p65/IκB phosphoylation. YmI and IRF4 expression was reduced significantly, indicative of a more inflammatory phenotype. We further observed that indomethacin or NS-398 delivery accelerated zebrafish death rates during LPS induced sepsis. When COX inhibitors were released over 30 days from an osmotic pump implant in mice, macrophages from peritoneal cavities and adipose tissue produced more TNFα in both the basal state and under LPS stimulation. Consequently, indomethacin-exposed mice showed accelerated systemic inflammation after LPS injection. Our findings suggest that macrophages exhibit a more inflammatory phenotype when COX activities are chronically inhibited.

  14. Chronic cigarette smoking enhances spontaneous release of tumour necrosis factor-α from alveolar macrophages of rats

    Directory of Open Access Journals (Sweden)

    G. P. Pessina

    1993-01-01

    Full Text Available Some biological effects of chronic cigarette smoking (two cigarettes for 2 h, daily for 4 months in rats were evaluated. During the smoking period, body weight of smoker rats was always significantly lower than that of control rats. Immediately after the last smoking session the carboxyhaemoglobin concentration in the blood was about 8.5% and the polymorphonuclear cells in the bronchoalveolar fluid increased significantly. At the same time, enzymatic analyses on the supernatants of bronchoalveolar fluid revealed a significant increase of β-glucuronidase in the smoker group. Alveolar macrophages, collected 0, 8 and 24 h after the last smoking session, significantly increased the generation of superoxide anion and, after incubation for 24 h at 37° C in a humidified atmosphere, released significantly high amounts of TNF-α. When challenged with lipopolysaccharide, alveolar macrophages of smoker rats released much more TNF-α but, in such a case, TNF-α release was about one half of that observed in the control group. Peritoneal macrophages of both control and smoker rats were unable either to generate high levels of superoxide anion or to release significant amounts of TNF-α. The results clearly demonstrated the activated state of alveolar macrophages and the resting state of peritoneal macrophages.

  15. Different particle determinants induce apoptosis and cytokine release in primary alveolar macrophage cultures

    Directory of Open Access Journals (Sweden)

    Schwarze Per E

    2006-06-01

    Full Text Available Abstract Background Particles are known to induce both cytokine release (MIP-2, TNF-α, a reduction in cell viability and an increased apoptosis in alveolar macrophages. To examine whether these responses are triggered by the same particle determinants, alveolar macrophages were exposed in vitro to mineral particles of different physical-chemical properties. Results The crystalline particles of the different stone types mylonite, gabbro, basalt, feldspar, quartz, hornfels and fine grain syenite porphyr (porphyr, with a relatively equal size distribution (≤ 10 μm, but different chemical/mineral composition, all induced low and relatively similar levels of apoptosis. In contrast, mylonite and gabbro induced a marked MIP-2 response compared to the other particles. For particles of smaller size, quartz (≤ 2 μm seemed to induce a somewhat stronger apoptotic response than even smaller quartz (≤ 0.5 μm and larger quartz (≤ 10 μm in relation to surface area, and was more potent than hornfels and porphyr (≤ 2 μm. The reduction in cell viability induced by quartz of the different sizes was roughly similar when adjusted to surface area. With respect to cytokines, the release was more marked after exposure to quartz ≤ 0.5 μm than to quartz ≤ 2 μm and ≤ 10 μm. Furthermore, hornfels (≤ 2 μm was more potent than the corresponding hornfels (≤ 10 μm and quartz (≤ 2 μm to induce cytokine responses. Pre-treatment of hornfels and quartz particles ≤ 2 μm with aluminium lactate, to diminish the surface reactivity, did significantly reduce the MIP-2 response to hornfels. In contrast, the apoptotic responses to the particles were not affected. Conclusion These results indicate that different determinants of mineral/stone particles are critical for inducing cytokine responses, reduction in cell viability and apoptosis in alveolar macrophages. The data suggest that the particle surface reactivity was critical for cytokine responses

  16. Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia.

    Directory of Open Access Journals (Sweden)

    Santosh K Panda

    Full Text Available Sepsis is a consequence of systemic bacterial infections leading to hyper activation of immune cells by bacterial products resulting in enhanced release of mediators of inflammation. Endotoxin (LPS is a major component of the outer membrane of Gram negative bacteria and a critical factor in pathogenesis of sepsis. Development of antagonists that inhibit the storm of inflammatory molecules by blocking Toll like receptors (TLR has been the main stay of research efforts. We report here that a filarial glycoprotein binds to murine macrophages and human monocytes through TLR4 and activates them through alternate pathway and in the process inhibits LPS mediated classical activation which leads to inflammation associated with endotoxemia. The active component of the nematode glycoprotein mediating alternate activation of macrophages was found to be a carbohydrate residue, Chitohexaose. Murine macrophages and human monocytes up regulated Arginase-1 and released high levels of IL-10 when incubated with chitohexaose. Macrophages of C3H/HeJ mice (non-responsive to LPS failed to get activated by chitohexaose suggesting that a functional TLR4 is critical for alternate activation of macrophages also. Chitohexaose inhibited LPS induced production of inflammatory molecules TNF-α, IL-1β and IL-6 by macropahges in vitro and in vivo in mice. Intraperitoneal injection of chitohexaose completely protected mice against endotoxemia when challenged with a lethal dose of LPS. Furthermore, Chitohexaose was found to reverse LPS induced endotoxemia in mice even 6/24/48 hrs after its onset. Monocytes of subjects with active filarial infection displayed characteristic alternate activation markers and were refractory to LPS mediated inflammatory activation suggesting an interesting possibility of subjects with filarial infections being less prone to develop of endotoxemia. These observations that innate activation of alternate pathway of macrophages by chtx through TLR4 has

  17. The β-adrenoceptor agonist clenbuterol is a potent inhibitor of the LPS-induced production of TNF-α and IL-6 in vitro and in vivo

    NARCIS (Netherlands)

    Izeboud, C.A.; Monshouwer, M.; Miert, A.S.J.P.A.M. van; Witkamp, R.F.

    1999-01-01

    Objective and Design: To investigate the suppressive effects of the β-agonist clenbuterol on the release of TNF-α and IL-6 in a lipopolysaccharide (LPS)-model of inflammation, both in vitro and in vivo. Material and Subjects: Human U-937 cell line (monocyte-derived macrophages), and male Wistar rats

  18. Distinct cytokine release profiles from human endothelial and THP-1 macrophage-like cells exposed to different amphotericin B formulations.

    Science.gov (United States)

    Turtinen, Lloyd W; Bremer, Lindsay A; Prall, David N; Schwartzhoff, Jenifer; Hartsel, Scott C

    2005-01-01

    Amphotericin B(AmB) formulations, Fungizone, and Amphotec caused substantially greater proinflammatory cytokine release than AmBisome (L-AMB) and Abelcet in TPA differentiated THP-1 macrophages as determined by antibody based protein arrays. Lipopolysaccharide but not AmB induced significant pro-inflammatory cytokines in human endothelial cells.

  19. Outward potassium current oscillations in macrophage polykaryons: extracellular calcium entry and calcium-induced calcium release

    Directory of Open Access Journals (Sweden)

    Saraiva R.M.

    1997-01-01

    Full Text Available Outward current oscillations associated with transient membrane hyperpolarizations were induced in murine macrophage polykaryons by membrane depolarization in the absence of external Na+. Oscillations corresponded to a cyclic activation of Ca2+-dependent K+ currents (IKCa probably correlated with variations in intracellular Ca2+ concentration. Addition of external Na+ (8 mM immediately abolished the outward current oscillations, suggesting that the absence of the cation is necessary not only for their induction but also for their maintenance. Oscillations were completely blocked by nisoldipine. Ruthenium red and ryanodine reduced the number of outward current cycles in each episode, whereas quercetin prolonged the hyperpolarization 2- to 15-fold. Neither low molecular weight heparin nor the absence of a Na+ gradient across the membrane had any influence on oscillations. The evidence suggests that Ca2+ entry through a pathway sensitive to Ca2+ channel blockers is elicited by membrane depolarization in Na+-free medium and is essential to initiate oscillations, which are also dependent on the cyclic release of Ca2+ from intracellular Ca2+-sensitive stores; Ca2+ ATPase acts by reducing intracellular Ca2+, thus allowing slow deactivation of IKCa. Evidence is presented that neither a Na+/Ca2+ antiporter nor Ca2+ release from IP3-sensitive Ca2+ stores participate directly in the mechanism of oscillation

  20. Kinetics of chemotaxis, cytokine, and chemokine release of NR8383 macrophages after exposure to inflammatory and inert granular insoluble particles.

    Science.gov (United States)

    Schremmer, I; Brik, A; Weber, D G; Rosenkranz, N; Rostek, A; Loza, K; Brüning, T; Johnen, G; Epple, M; Bünger, J; Westphal, G A

    2016-11-30

    Accumulation of macrophages and neutrophil granulocytes in the lung are key events in the inflammatory response to inhaled particles. The present study aims at the time course of chemotaxis in vitro in response to the challenge of various biopersistent particles and its functional relation to the transcription of inflammatory mediators. NR8383 rat alveolar macrophages were challenged with particles of coarse quartz, barium sulfate, and nanosized silica for one, four, and 16h and with coarse and nanosized titanium dioxide particles (rutile and anatase) for 16h only. The cell supernatants were used to investigate the chemotaxis of unexposed NR8383 macrophages. The transcription of inflammatory mediators in cells exposed to quartz, silica, and barium sulfate was analyzed by quantitative real-time PCR. Challenge with quartz, silica, and rutile particles induced significant chemotaxis of unexposed NR8383 macrophages. Chemotaxis caused by quartz and silica was accompanied by an elevated transcription of CCL3, CCL4, CXCL1, CXCL3, and TNFα. Quartz exposure showed an earlier onset of both effects compared to the nanosized silica. The strength of this response roughly paralleled the cytotoxic effects. Barium sulfate and anatase did not induce chemotaxis and barium sulfate as well caused no elevated transcription. In conclusion, NR8383 macrophages respond to the challenge with inflammatory particles with the release of chemotactic compounds that act on unexposed macrophages. The kinetics of the response differs between the various particles. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Contribution of TIR domain-containing adapter inducing IFN-beta-mediated IL-18 release to LPS-induced liver injury in mice

    NARCIS (Netherlands)

    Imamura, M.; Tsutsui, H.; Yasuda, K.; Uchiyama, R.; Yumikura-Futatsugi, S.; Mitani, K.; Hayashi, S.; Akira, S; Taniguchi, S.; Rooijen, van N.; Tschopp, J.; Yamamoto, T.; Fujimoto, J.; Nakanishi, K.

    2009-01-01

    Background/Aims: After treatment with heat-killed Propionibacterium acnes mice show dense hepatic granuloma formation. Such mice develop liver injury in an interleukin (IL)-18-dependent manner after challenge with a sublethal dose LPS. As previously shown, LPS-stimulated Kupffer cells secrete IL-18

  2. Enzymatic hydrolysis of gelatin layers of X-Ray films and release of silver particles using keratinolytic serine proteases from Purpureocillium lilacinum LPS # 876.

    Science.gov (United States)

    Cavello, Ivana Alejandra; Hours, Roque Alberto; Cavalitto, Sebastián Fernando

    2013-08-01

    Enzymatic decomposition of gelatin layers on used X-ray films and repeated utilization of the enzyme for potential application in silver recovery were investigated using keratinolytic serine proteases from Purpureocillium lilacinum LPS # 876. At pH 9.0, the enzymatic reaction was enhanced by the increase of enzyme concentration or by the increase of the temperature up to 60℃. Under the conditions of 6.9 U/ml, 60℃, and pH 9.0, hydrolysis of the gelatin layers and the resulting release of silver particles were achieved within 6 min. The protective effect of polyols against thermal denaturation was investigated. The presence of glycerol and propylene glycol increased enzyme stability. When the reusability of the enzyme for gelatin hydrolysis was tested, it could be seen that it could be effectively reused for more cycles when glycerol was added, compared with the enzyme without protective agents. The results of these repeated treatments suggested that a continuous process of recycling silver from used X-ray is feasible. Keeping in mind that recycling is (at the present time) needed and imperative, it can be remarked that, in this research, three wastes were successfully used: hair waste in order to produce serine proteases; glycerol in order to enhance enzyme thermal stability; and used Xray films in order to recover silver and PET films.

  3. Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.

    Science.gov (United States)

    Hortelano, S; Alvarez, A M; Boscá, L

    1999-12-01

    Treatment of elicited peritoneal macrophages or the macrophage cell line RAW 264.7 with high concentrations of nitric oxide donors is followed by apoptotic cell death. Analysis of the changes in the mitochondrial transmembrane potential (DeltaPsi(m)) with specific fluorescent probes showed a rapid and persistent increase of DeltaPsi(m), a potential that usually decreases in cells undergoing apoptosis through mitochondrial-dependent mechanisms. Using confocal microscopy, the release of cytochrome c from the mitochondria to the cytosol was characterized as an early event preceding the rise of DeltaPsi(m). The cytochrome c from cells treated with nitric oxide donors was modified chemically, probably through the formation of nitrotyrosine residues, suggesting the synthesis of peroxynitrite in the mitochondria. These results indicate that nitric oxide-dependent apoptosis in macrophages occurs in the presence of a sustained increase of DeltaPsi(m), and that the chemical modification and release of cytochrome c from the mitochondria precede the changes of DeltaPsi(m).-Hortelano, S., Alvarez, A. M., Boscá, L. Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.

  4. Long-Time Treatment by Low-Dose N-Acetyl-L-Cysteine Enhances Proinflammatory Cytokine Expressions in LPS-Stimulated Macrophages

    OpenAIRE

    Tomokazu Ohnishi; Kenjiro Bandow; Kyoko Kakimoto; Joji Kusuyama; Tetsuya Matsuguchi

    2014-01-01

    N-acetyl-L-cysteine is known to act as a reactive oxygen species scavenger and used in clinical applications. Previous reports have shown that high-dose N-acetyl-L-cysteine treatment inhibits the expression of proinflammatory cytokines in activated macrophages. Here, we have found that long-time N-acetyl-L-cysteine treatment at low-concentration increases phosphorylation of extracellular signal-regulated kinase 1/2 and AKT, which are essential for the induction of proinflammatory cytokines in...

  5. Depression of alveolar macrophage hydrogen peroxide and superoxide anion release by mineral dusts: correlation with antimony, lead, and arsenic contents.

    Science.gov (United States)

    Gulyas, H; Labedzka, M; Gercken, G

    1990-04-01

    Activated rabbit alveolar macrophages were incubated with airborne dusts from four West German sites (1 to 200 micrograms/10(6) cells) and waste incinerator fly ash fractions (50 to 500 micrograms/10(6) cells). Quartz dust DQ 12 (5 to 200 micrograms/10(6) cells) and Fe2O3 (0.05 to 50 micrograms/10(6) cells) were used as control dusts. The zymosan-stimulated hydrogen peroxide and superoxide anion release of the macrophages were not affected significantly by Fe2O3. All other investigated dusts decreased the two cell functions which were correlated negatively with surfaces, particle numbers, and antimony, lead, and arsenic contents of the dusts. The influence of heavy metal antagonisms and dust surfaces on dust toxicity against alveolar macrophages is discussed.

  6. IL-1α and IL-1β-producing macrophages populate lung tumor lesions in mice.

    Science.gov (United States)

    Terlizzi, Michela; Colarusso, Chiara; Popolo, Ada; Pinto, Aldo; Sorrentino, Rosalinda

    2016-09-06

    Macrophages highly populate tumour microenvironment and are referred to as tumor-associated macrophages (TAMs). The inflammasome is a multiprotein complex responsible of IL-1 like cytokines release, which biology has been widely studied by using bone-marrow-derived macrophages to mimic a physiological and/or host defense condition. To understand the role of this complex in lung tumor-associated macrophages (TAMs), we isolated and cultured broncho-alveolar lavage (BAL)-derived cells of lung tumor-bearing mice. The stimulation of lung TAMs with LPS+ATP increased the release of IL-1β. The inhibition of NLRP3 by means of glybenclamide significantly reduced IL-1β release. Similarly, C3H-derived, caspase-1 ko and caspase-11 ko TAMs released significantly reduced levels of IL-1β. Moreover, the stimulation of lung TAMs with the sole LPS induced a significant release of IL-1α, which was significantly reduced after caspase-1 pharmacological inhibition, and in TAMs genetically lacking caspase-1 and caspase-11. The inhibition of calpain I/II by means of MDL28170 did not alter IL-1α release after LPS treatment of lung TAMs. To note, the inoculation of LPS-treated bone marrow-derived macrophages into carcinogen-exposed mice increased lung tumor formation. In contrast, the depletion of TAMs by means of clodronate liposomes reduced lung tumorigenesis, associated to lower in vivo release of IL-1α and IL-1β.In conclusion, our data imply lung tumor lesions are populated by macrophages which pro-tumor activity is regulated by the activation of the NLRP3 inflammasome that leads to the release of IL-1α and IL-1β in a caspase-11/caspase-1-dependent manner.

  7. Comparison of WTC dust size on macrophage inflammatory cytokine release in vivo and in vitro.

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    Michael D Weiden

    Full Text Available BACKGROUND: The WTC collapse exposed over 300,000 people to high concentrations of WTC-PM; particulates up to ∼50 mm were recovered from rescue workers' lungs. Elevated MDC and GM-CSF independently predicted subsequent lung injury in WTC-PM-exposed workers. Our hypotheses are that components of WTC dust strongly induce GM-CSF and MDC in AM; and that these two risk factors are in separate inflammatory pathways. METHODOLOGY/PRINCIPAL FINDINGS: Normal adherent AM from 15 subjects without WTC-exposure were incubated in media alone, LPS 40 ng/mL, or suspensions of WTC-PM(10-53 or WTC-PM(2.5 at concentrations of 10, 50 or 100 µg/mL for 24 hours; supernatants assayed for 39 chemokines/cytokines. In addition, sera from WTC-exposed subjects who developed lung injury were assayed for the same cytokines. In the in vitro studies, cytokines formed two clusters with GM-CSF and MDC as a result of PM(10-53 and PM(2.5. GM-CSF clustered with IL-6 and IL-12(p70 at baseline, after exposure to WTC-PM(10-53 and in sera of WTC dust-exposed subjects (n = 70 with WTC lung injury. Similarly, MDC clustered with GRO and MCP-1. WTC-PM(10-53 consistently induced more cytokine release than WTC-PM(2.5 at 100 µg/mL. Individual baseline expression correlated with WTC-PM-induced GM-CSF and MDC. CONCLUSIONS: WTC-PM(10-53 induced a stronger inflammatory response by human AM than WTC-PM(2.5. This large particle exposure may have contributed to the high incidence of lung injury in those exposed to particles at the WTC site. GM-CSF and MDC consistently cluster separately, suggesting a role for differential cytokine release in WTC-PM injury. Subject-specific response to WTC-PM may underlie individual susceptibility to lung injury after irritant dust exposure.

  8. Comparison of WTC dust size on macrophage inflammatory cytokine release in vivo and in vitro.

    Science.gov (United States)

    Weiden, Michael D; Naveed, Bushra; Kwon, Sophia; Segal, Leopoldo N; Cho, Soo Jung; Tsukiji, Jun; Kulkarni, Rohan; Comfort, Ashley L; Kasturiarachchi, Kusali J; Prophete, Colette; Cohen, Mitchell D; Chen, Lung-Chi; Rom, William N; Prezant, David J; Nolan, Anna

    2012-01-01

    The WTC collapse exposed over 300,000 people to high concentrations of WTC-PM; particulates up to ∼50 mm were recovered from rescue workers' lungs. Elevated MDC and GM-CSF independently predicted subsequent lung injury in WTC-PM-exposed workers. Our hypotheses are that components of WTC dust strongly induce GM-CSF and MDC in AM; and that these two risk factors are in separate inflammatory pathways. Normal adherent AM from 15 subjects without WTC-exposure were incubated in media alone, LPS 40 ng/mL, or suspensions of WTC-PM(10-53) or WTC-PM(2.5) at concentrations of 10, 50 or 100 µg/mL for 24 hours; supernatants assayed for 39 chemokines/cytokines. In addition, sera from WTC-exposed subjects who developed lung injury were assayed for the same cytokines. In the in vitro studies, cytokines formed two clusters with GM-CSF and MDC as a result of PM(10-53) and PM(2.5). GM-CSF clustered with IL-6 and IL-12(p70) at baseline, after exposure to WTC-PM(10-53) and in sera of WTC dust-exposed subjects (n = 70) with WTC lung injury. Similarly, MDC clustered with GRO and MCP-1. WTC-PM(10-53) consistently induced more cytokine release than WTC-PM(2.5) at 100 µg/mL. Individual baseline expression correlated with WTC-PM-induced GM-CSF and MDC. WTC-PM(10-53) induced a stronger inflammatory response by human AM than WTC-PM(2.5). This large particle exposure may have contributed to the high incidence of lung injury in those exposed to particles at the WTC site. GM-CSF and MDC consistently cluster separately, suggesting a role for differential cytokine release in WTC-PM injury. Subject-specific response to WTC-PM may underlie individual susceptibility to lung injury after irritant dust exposure.

  9. α-Dihydroxychalcone-glycoside (α-DHC) isolated from the heartwood of Pterocarpus marsupium inhibits LPS induced MAPK activation and up regulates HO-1 expression in murine RAW 264.7 macrophage

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Prarthana; Saraswat, Ghungroo; Kabir, Syed N., E-mail: snkabir@iicb.res.in

    2014-05-15

    Three phenolic glycosides isolated from the heartwood of Pterocarpus marsupium showed significant free radical and superoxide ion scavenging activity and antioxidant potential that were comparable to, or several folds higher than those of standard antioxidants, trolox and ascorbic acid. The effective concentrations of these compounds were far below their cytotoxic levels. Compound 3, which was characterized to be α-dihydroxychalcone-glycoside (α-DHC), was the most potent one. Subsequent studies demonstrated that α-DHC effectively reduced nitric oxide and cytokine production by the LPS stimulated RAW 264.7 mouse macrophage cell line. The compound effectively attenuated the expression of inflammation-mediating enzymes COX-2 and iNOS at the mRNA as well as protein levels in a concentration dependent manner. It prevented phosphorylation of all the three MAPKs (JNK, ERK, p38) and eventually blocked the activation of downstream elements contributing to inflammation. Phosphorylation of IκB-α and subsequent translocation of NF-κB into the nucleus were restricted, while the expression of stress responsive gene HO-1 was up-regulated. α-DHC targeted Keap-1 by modifying its cysteine thiols, dissociating it from Nrf-2 and facilitating nuclear entry of the latter; and this in turn induced HO-1 expression. Thus α-DHC exerts its anti-inflammatory activity in a dual manner: by down regulating MAPKs and restricting nuclear stabilization of NF-κB at one end, and by disrupting Nrf-2–Keap-1 complex on the other. In conclusion, the anti-inflammatory potential together with its high therapeutic index envisages α-DHC as a prospective candidate molecule for the development of therapeutic strategy against inflammatory disorders. - Highlights: • α-DHC isolated from Pterocarpus marsupium has significant antioxidant potential. • α-DHC inhibits NO, IL-6, IL-1β, TNF-α production in LPS-stimulated RAW 264.7 cells. • α-DHC down-regulates of COX-2, iNOS expression in LPS

  10. Migration inhibitory factor (MIF) released by macrophages upon recognition of immune complexes is critical to inflammation in Arthus reaction.

    Science.gov (United States)

    Paiva, Claudia N; Arras, Rosa H; Magalhães, Elisabeth S; Alves, Letícia S; Lessa, Luiz Paulo; Silva, Maria Helena; Ejzemberg, Regina; Canetti, Cláudio; Bozza, Marcelo T

    2009-05-01

    Deposition of immune complexes (IC) triggers Fc gamma R-dependent inflammation, leading to tissue damage in rheumatoid arthritis, systemic lupus erythematous, immune glomerulonephritis, and several immune vasculitides. Evidences support a role for macrophage migration inhibitory factor (MIF) in a number of inflammatory diseases, but the triggering of its secretion and its physiopathological role upon IC deposition remain elusive. Herein, we show that human macrophages secreted MIF after IC recognition, which in turn controlled the secretion of TNF. Macrophages from Mif-/- mice produced smaller amounts of TNF when stimulated with IgG-opsonized erythrocytes than wild-type (WT) cells. Using passive reverse Arthus reaction in the peritoneum and lungs as a model for IC-induced inflammation, we demonstrated that Mif-/- mice had a milder response, observed by reduced neutrophil recruitment, vascular leakage, and secretion of TNF, MIP-2, and keratinocyte-derived chemokine compared with WT controls. Adoptive transfer of alveolar macrophages from WT to Mif-/- mice rescued pulmonary neutrophil recruitment and TNF production upon passive reverse Arthus reaction. Our study indicates that Arthus inflammatory reaction is largely dependent on MIF and poses macrophages as a source of the MIF released upon IC recognition. These results give experimental support to the proposition that blockade of MIF might constitute an adjunctive, therapeutic approach to IC disease.

  11. Effects of β-Glucan on the Release of Nitric Oxide by Macrophages Stimulated with Lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    E. Y. Choi

    2016-11-01

    Full Text Available This research analyzed the effect of β-glucan that is expected to alleviate the production of the inflammatory mediator in macrophagocytes, which are processed by the lipopolysaccharide (LPS of Escherichia. The incubated layer was used for a nitric oxide (NO analysis. The DNA-binding activation of the small unit of nuclear factor-κB was measured using the enzyme-linked immunosorbent assay-based kit. In the RAW264.7 cells that were vitalized by Escherichia coli (E. coli LPS, the β-glucan inhibited both the combatant and rendering phases of the inducible NO synthase (iNOS-derived NO. β-Glucan increased the expression of the heme oxygenase-1 (HO-1 in the cells that were stimulated by E. coli LPS, and the HO-1 activation was inhibited by the tin protoporphyrin IX (SnPP. This shows that the NO production induced by LPS is related to the inhibition effect of β-glucan. The phosphorylation of c-Jun N-terminal kinases (JNK and the p38 induced by the LPS were not influenced by the β-glucan, and the inhibitory κB-α (IκB-α decomposition was not influenced either. Instead, β-glucan remarkably inhibited the phosphorylation of the signal transducer and activator of transcription-1 (STAT1 that was induced by the E. coli LPS. Overall, the β-glucan inhibited the production of NO in macrophagocytes that was vitalized by the E .coli LPS through the HO-1 induction and the STAT1 pathways inhibition in this research. As the host immune response control by β-glucan weakens the progress of the inflammatory disease, β-glucan can be used as an effective immunomodulator.

  12. Inorganic polyphosphate suppresses lipopolysaccharide-induced inducible nitric oxide synthase (iNOS expression in macrophages.

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    Kana Harada

    Full Text Available In response to infection, macrophages produce a series of inflammatory mediators, including nitric oxide (NO, to eliminate pathogens. The production of these molecules is tightly regulated via various mechanisms, as excessive responses are often detrimental to host tissues. Here, we report that inorganic polyphosphate [poly(P], a linear polymer of orthophosphate ubiquitously found in mammalian cells, suppresses inducible nitric oxide synthase (iNOS expression induced by lipopolysaccharide (LPS, a cell wall component of Gram-negative bacteria, in mouse peritoneal macrophages. Poly(P with longer chains is more potent than those with shorter chains in suppressing LPS-induced iNOS expression. In addition, poly(P decreased LPS-induced NO release. Moreover, poly(P suppressed iNOS mRNA expression induced by LPS stimulation, thereby indicating that poly(P reduces LPS-induced iNOS expression by down-regulation at the mRNA level. In contrast, poly(P did not affect the LPS-induced release of TNF, another inflammatory mediator. Poly(P may serve as a regulatory factor of innate immunity by modulating iNOS expression in macrophages.

  13. Long-time treatment by low-dose N-acetyl-L-cysteine enhances proinflammatory cytokine expressions in LPS-stimulated macrophages.

    Directory of Open Access Journals (Sweden)

    Tomokazu Ohnishi

    Full Text Available N-acetyl-L-cysteine is known to act as a reactive oxygen species scavenger and used in clinical applications. Previous reports have shown that high-dose N-acetyl-L-cysteine treatment inhibits the expression of proinflammatory cytokines in activated macrophages. Here, we have found that long-time N-acetyl-L-cysteine treatment at low-concentration increases phosphorylation of extracellular signal-regulated kinase 1/2 and AKT, which are essential for the induction of proinflammatory cytokines including interleukin 1β and interleukin 6 in lipopolysaccharide-stimulated RAW264.7 cells. Furthermore, long-time N-acetyl-L-cysteine treatment decreases expressions of protein phosphatases, catalytic subunit of protein phosphatase-2A and dual specificity phosphatase 1. On the other hand, we have found that short-time N-acetyl-L-cysteine treatment at low dose increases p53 expression, which inhibits expressions of proinflammatory cytokines. These observations suggest that long-time low-dose N-acetyl-L-cysteine treatment increases expressions of proinflammatory cytokines through enhancement of kinase phosphorylation.

  14. Inhibition of LPS-induced TNF-α and NO production in mouse macrophage and inflammatory response in rat animal models by a novel Ayurvedic formulation, BV-9238.

    Science.gov (United States)

    Dey, Debendranath; Chaskar, Sunetra; Athavale, Nitin; Chitre, Deepa

    2014-10-01

    Rheumatoid arthritis is a chronic crippling disease, where protein-based tumor necrosis factor-alpha (TNF-α) inhibitors show significant relief, but with potentially fatal side effects. A need for a safe, oral, cost-effective small molecule or phyto-pharmaceutical is warranted. BV-9238 is an Ayurvedic poly-herbal formulation containing specialized standardized extracts of Withania somnifera, Boswellia serrata, Zingiber officinale and Curcuma longa. The anti-inflammatory and anti-arthritic effects of BV-9238 were evaluated for inhibition of TNF-α and nitric oxide (NO) production, in lipopolysaccharide-stimulated, RAW 264.7, mouse macrophage cell line. BV-9238 reduced TNF-α and NO production, without any cytotoxic effects. Subsequently, the formulation was tested in adjuvant-induced arthritis (AIA) and carrageenan-induced paw edema (CPE) rat animal models. AIA was induced in rats by injecting Freund's complete adjuvant intra-dermally in the paw, and BV-9238 and controls were administered orally for 21 days. Arthritic scores in AIA study and inflamed paw volume in CPE study were significantly reduced upon treatment with BV-9238. These results suggest that the anti-inflammatory and anti-arthritic effects of BV-9238 are due to its inhibition of TNF-α, and NO, and this formulation shows promise as an alternate therapy for inflammatory disorders where TNF-α and NO play important roles.

  15. Total bacterial load within Echinacea purpurea, determined using a new PCR-based quantification method, is correlated with LPS levels and in vitro macrophage activity.

    Science.gov (United States)

    Pugh, Nirmal D; Jackson, Colin R; Pasco, David S

    2013-01-01

    Our previous studies indicate that the majority of in vitro monocyte/macrophage activation exhibited by extracts of Echinacea depends on bacterial components. In the present study, total bacterial load was determined within E. purpurea samples and ranged from 6.4 × 10(6) to 3.3 × 10(8) bacteria/g of dry plant material. To estimate total bacterial load, we developed a PCR-based quantification method that circumvents the problems associated with nonviable/nonculturable cells (which precludes using plate counts) or the coamplification of mitochondrial or chloroplast DNA with the use of universal bacterial primers (which precludes the use of qPCR). Differences in total bacterial load within Echinacea samples were strongly correlated with the activity (NF-κB activation in THP-1 cells) and content of bacterial lipopolysaccharides within extracts of this plant material. These results add to the growing body of evidence that bacteria within Echinacea are the main source of components responsible for enhancing innate immune function. Georg Thieme Verlag KG Stuttgart · New York.

  16. Gomisin A decreases the LPS-induced expression of iNOS and COX-2 and activation of RIP2/NF-κB in mouse peritoneal macrophages.

    Science.gov (United States)

    Jeong, Hyun-Ja; Han, Na-Ra; Kim, Kyu-Yeob; Choi, Il-Sook; Kim, Hyung-Min

    2014-06-01

    Gomisin A (GA), a lignan component contained in the fruit of Schisandra chinensis Baillon, improves hepatic cell degeneration, vasodilatory activity and insulin sensitivity. These effects also impact the immune system, including various inflammatory mediators and cytokines. In this study, the anti-inflammatory effect of GA on lipopolysaccharide-stimulated mouse peritoneal macrophages was studied. Pretreatment with GA attenuated the expression of receptor-interacting protein 2 (RIP2) and IκB kinase-β (IKK-β) as well as IKK-β phosphorylation. The activation of nuclear factor-kappa B (NF-κB) in the nucleus, the phosphorylation of IκBα and degradation of IκBα in the cytosol were suppressed by GA. GA decreased the production and mRNA expression of the inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6. In addition, expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and production of nitric oxide were decreased by pretreatment with GA. In conclusion, these results show that the anti-inflammatory properties of GA potentially result from the inhibition of COX-2, iNOS, IL-6, TNF-α and NO through the down-regulation of RIP2 and NF-κB activation. These results impact the development of potential health products for preventing and treating inflammatory diseases.

  17. Interaction with extracellular matrix proteins influences Lsh/Ity/Bcg (candidate Nramp) gene regulation of macrophage priming/activation for tumour necrosis factor-alpha and nitrite release.

    Science.gov (United States)

    Formica, S; Roach, T I; Blackwell, J M

    1994-05-01

    The murine resistance gene Lsh/Ity/Bcg regulates activation of macrophages for tumour necrosis factor-alpha (TNF-alpha)-dependent production of nitric oxide mediating antimicrobial activity against Leishmania, Salmonella and Mycobacterium. As Lsh is differentially expressed in macrophages from different tissue sites, experiments were performed to determine whether interaction with extracellular matrix (ECM) proteins would influence the macrophage TNF-alpha response. Plating of bone marrow-derived macrophages onto purified fibrinogen or fibronectin-rich L929 cell-derived matrices, but not onto mannan, was itself sufficient to stimulate TNF-alpha release, with significantly higher levels released from congenic B10.L-Lshr compared to C57BL/10ScSn (Lshs) macrophages. Only macrophages plated onto fibrinogen also released measurable levels of nitrites, again higher in Lshr compared to Lshs macrophages. Addition of interferon-gamma (IFN-gamma), but not bacterial lipopolysaccharide or mycobacterial lipoarabinomannan, as a second signal enhanced the TNF-alpha and nitrite responses of macrophages plated onto fibrinogen, particularly in the Lshr macrophages. Interaction with fibrinogen and fibronectin also primed macrophages for an enhanced TNF-alpha response to leishmanial parasites, but this was only translated into enhanced nitrite responses in the presence of IFN-gamma. In these experiments, Lshr macrophages remained superior in their TNF-alpha responses throughout, but to a degree which reflected the magnitude of the difference observed on ECM alone. Hence, the specificity for the enhanced TNF-alpha responses of Lshr macrophages lay in their interaction with fibrinogen and fibronectin ECM, while a differential nitrite response was only observed with fibrinogen and/or IFN-gamma. The results are discussed in relation to the possible function of the recently cloned candidate gene Nramp, which has structural identity to eukaryote transporters and an N-terminal cytoplasmic

  18. Lipopolysaccharide Could Be Internalized into Human Peripheral Blood Mononuclear Cells and Elicits TNF-α Release,but not via the Pathway of Toll-Like Receptor 4 on the Cell Surface

    Institute of Scientific and Technical Information of China (English)

    HongZhou; GuofuDing; WeiLiu; LiangxiWang; YonglingLu; HongweiCao; JiangZheng

    2004-01-01

    Lipopolysaccharide (LPS [endotoxin]), the principal component of the outer membrane of gram-negative bacteria, stimulate various cell types to release numerous proinflammatory mediators such as TNF-α, IL-6 and IL-12, which may damage cells and lead to organ injury, even sepsis and septic shock. Toll-like receptor 4(TLR4) has been identified as the receptor involved in the recognition of LPS, but the role of LPS uptake inactivating signal transduction remains controversial. In the present study, TNF-α was used as a marker of macrophages/monocytes activated by LPS, and CQ was used as an inhibitor of endosome mature in order to definitude what stage the signal transduction elicited by LPS was interrupted. We found that there indeed existed internalization of LPS and internalization partially participated in LPS signaling since CQ inhibitedcytokine release, and decreased accumulation of FITC-LPS in hPBMC. In contrast, anti-hTLR4 antibodycould decrease cytokines' release, but no inhibition on accumulation of FITC-LPS. This result revealed thatinhibition of cytokine release was related to reduction of FITC-LPS accumulation in the cells. But TLR4 on thecell surface didn't possibly participated in internalization of LPS. Thus, LPS signaling and internalizationcannot be viewed as mutually independent processes. Cellular & Molecular Immunology. 2004;1(5):373-377.

  19. Krill Oil-In-Water Emulsion Protects against Lipopolysaccharide-Induced Proinflammatory Activation of Macrophages In Vitro

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    Gabriel A. Bonaterra

    2017-03-01

    Full Text Available Background: Parenteral nutrition is often a mandatory therapeutic strategy for cases of septicemia. Likewise, therapeutic application of anti-oxidants, anti-inflammatory therapy, and endotoxin lowering, by removal or inactivation, might be beneficial to ameliorate the systemic inflammatory response during the acute phases of critical illness. Concerning anti-inflammatory properties in this setting, omega-3 fatty acids of marine origin have been frequently described. This study investigated the anti-inflammatory and LPS-inactivating properties of krill oil (KO-in-water emulsion in human macrophages in vitro. Materials and Methods: Differentiated THP-1 macrophages were activated using specific ultrapure-LPS that binds only on the toll-like receptor 4 (TLR4 in order to determine the inhibitory properties of the KO emulsion on the LPS-binding capacity, and the subsequent release of TNF-α. Results: KO emulsion inhibited the macrophage binding of LPS to the TLR4 by 50% (at 12.5 µg/mL and 75% (at 25 µg/mL, whereas, at 50 µg/mL, completely abolished the LPS binding. Moreover, KO (12.5 µg/mL, 25 µg/mL, or 50 µg/mL also inhibited (30%, 40%, or 75%, respectively the TNF-α release after activation with 0.01 µg/mL LPS in comparison with LPS treatment alone. Conclusion: KO emulsion influences the LPS-induced pro-inflammatory activation of macrophages, possibly due to inactivation of the LPS binding capacity.

  20. Exosomes released from M. tuberculosis infected cells can suppress IFN-γ mediated activation of naive macrophages.

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    Prachi P Singh

    Full Text Available BACKGROUND: Macrophages infected with Mycobacterium tuberculosis (M.tb are known to be refractory to IFN-γ stimulation. Previous studies have shown that M.tb express components such as the 19-kDa lipoprotein and peptidoglycan that can bind to macrophage receptors including the Toll-like receptor 2 resulting in the loss in IFN-γ responsiveness. However, it is unclear whether this effect is limited to infected macrophages. We have previously shown that M.tb-infected macrophages release exosomes which are 30-100 nm membrane bound vesicles of endosomal origin that function in intercellular communication. These exosomes contain mycobacterial components including the 19-kDa lipoprotein and therefore we hypothesized that macrophages exposed to exosomes may show limited response to IFN-γ stimulation. METHODOLOGY/PRINCIPAL FINDINGS: Exosomes were isolated from resting as well as M.tb-infected RAW264.7 macrophages. Mouse bone marrow-derived macrophages (BMMØ were treated with exosomes +/- IFN-γ. Cells were harvested and analyzed for suppression of IFN-γ responsive genes by flow cytometry and real time PCR. We found that exosomes derived from M.tb H37Rv-infected but not from uninfected macrophages inhibited IFN-γ induced MHC class II and CD64 expression on BMMØ. This inhibition was only partially dependent on the presence of lipoproteins but completely dependent on TLR2 and MyD88. The exosomes isolated from infected cells did not inhibit STAT1 Tyrosine phosphorylation but down-regulated IFN-γ induced expression of the class II major histocompatibility complex transactivator; a key regulator of class II MHC expression. Microarray studies showed that subsets of genes induced by IFN-γ were inhibited by exosomes from H37Rv-infected cells including genes involved in antigen presentation. Moreover, this set of genes partially overlapped with the IFN-γ-induced genes inhibited by H37Rv infection. CONCLUSIONS: Our study suggests that exosomes, as

  1. Upregulating Nonneuronal Cholinergic Activity Decreases TNF Release from Lipopolysaccharide-Stimulated RAW264.7 Cells

    Directory of Open Access Journals (Sweden)

    Yi Lv

    2014-01-01

    Full Text Available Nonneuronal cholinergic system plays a primary role in maintaining homeostasis. It has been proved that endogenous neuronal acetylcholine (ACh could play an anti-inflammatory role, and exogenous cholinergic agonists could weaken macrophages inflammatory response to lipopolysaccharide (LPS stimulation through activation of α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR. We assumed that nonneuronal cholinergic system existing in macrophages could modulate inflammation through autocrine ACh and expressed α7nAChR on the cells. Therefore, we explored whether LPS continuous stimulation could upregulate the nonneuronal cholinergic activity in macrophages and whether increasing autocrine ACh could decrease TNF release from the macrophages. The results showed that, in RAW264.7 cells incubated with LPS for 20 hours, the secretion of ACh was significantly decreased at 4 h and then gradually increased, accompanied with the enhancement of α7nAChR expression level. The release of TNF was greatly increased from RAW264.7 cells at 4 h and 8 h exposure to LPS; however, it was suppressed at 20 h. Upregulating choline acetyltransferase (ChAT expression through ChAT gene transfection could enhance ACh secretion and reduce TNF release from the infected RAW264. 7cells. The results indicated that LPS stimulation could modulate the activity of nonneuronal cholinergic system of RAW264.7 cells. Enhancing autocrine ACh production could attenuate TNF release from RAW264.7 cells.

  2. α-Dihydroxychalcone-glycoside (α-DHC) isolated from the heartwood of Pterocarpus marsupium inhibits LPS induced MAPK activation and up regulates HO-1 expression in murine RAW 264.7 macrophage.

    Science.gov (United States)

    Chakraborty, Prarthana; Saraswat, Ghungroo; Kabir, Syed N

    2014-05-15

    Three phenolic glycosides isolated from the heartwood of Pterocarpus marsupium showed significant free radical and superoxide ion scavenging activity and antioxidant potential that were comparable to, or several folds higher than those of standard antioxidants, trolox and ascorbic acid. The effective concentrations of these compounds were far below their cytotoxic levels. Compound 3, which was characterized to be α-dihydroxychalcone-glycoside (α-DHC), was the most potent one. Subsequent studies demonstrated that α-DHC effectively reduced nitric oxide and cytokine production by the LPS stimulated RAW 264.7 mouse macrophage cell line. The compound effectively attenuated the expression of inflammation-mediating enzymes COX-2 and iNOS at the mRNA as well as protein levels in a concentration dependent manner. It prevented phosphorylation of all the three MAPKs (JNK, ERK, p38) and eventually blocked the activation of downstream elements contributing to inflammation. Phosphorylation of IκB-α and subsequent translocation of NF-κB into the nucleus were restricted, while the expression of stress responsive gene HO-1 was up-regulated. α-DHC targeted Keap-1 by modifying its cysteine thiols, dissociating it from Nrf-2 and facilitating nuclear entry of the latter; and this in turn induced HO-1 expression. Thus α-DHC exerts its anti-inflammatory activity in a dual manner: by down regulating MAPKs and restricting nuclear stabilization of NF-κB at one end, and by disrupting Nrf-2-Keap-1 complex on the other. In conclusion, the anti-inflammatory potential together with its high therapeutic index envisages α-DHC as a prospective candidate molecule for the development of therapeutic strategy against inflammatory disorders. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Exosome release of ADAM15 and the functional implications of human macrophage-derived ADAM15 exosomes.

    Science.gov (United States)

    Lee, Hee Doo; Koo, Bon-Hun; Kim, Yeon Hyang; Jeon, Ok-Hee; Kim, Doo-Sik

    2012-07-01

    A disintegrin and metalloproteinase 15 (ADAM15), the only ADAM protein containing an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain, is a widely expressed membrane protein that is involved in tumor progression and suppression. However, the underlying mechanism of ADAM15-mediated tumor suppression is not clearly understood. This study demonstrates that ADAM15 is released as an exosomal component, and ADAM15 exosomes exert tumor suppressive activities. We found that exosomal ADAM15 release is stimulated by phorbol 12-myristate 13-acetate, a typical protein kinase C activator, in various tumor cell types, and this results in a corresponding decrease in plasma membrane-associated ADAM15. Exosomes rich in ADAM15 display enhanced binding affinity for integrin αvβ3 in an RGD-dependent manner and suppress vitronectin- and fibronectin-induced cell adhesion, growth, and migration, as well as in vivo tumor growth. Exosomal ADAM15 is released from human macrophages, and macrophage-derived ADAM15 exosomes have tumor inhibitory effects. This work suggests a primary role of ADAM15 for exosome-mediated tumor suppression, as well as functional significance of exosomal ADAM protein in antitumor immunity.

  4. The effects of propolis on cytokine production in lipopolysaccharide-stimulated macrophages

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    Hatice Özbilge

    2011-12-01

    Full Text Available Objectives: Propolis, a bee-product, has attracted researchers’ interest in recent years because of several biological and pharmacological properties. Lipopolysaccharide (LPS is a component of the outer membrane of Gram-negative bacteria and has an important role in the pathogenesis of septic shock and several inflammatory diseases by causing excessive release of inflammatory cytokines. The aim of this study was to investigate the effects of ethanol extract of propolis collected in Kayseri and its surroundings on production of pro-inflammatory cytokines in LPS-stimulated macrophages.Materials and methods: In vitro, U937 human macrophage cells were grown in RPMI-1640 medium supplemented with fetal bovine serum (10% and penicillin-streptomycin (2% and divided into: control, LPS treated, and propolis+LPS treated cell groups. After incubation in an atmosphere of 5% CO2 and at 37°C of cells, interleukin (IL-1β, IL-6 and tumor necrosis factor (TNF-α levels were measured in cell-free supernatants by ELISA.Results: IL-1β, IL-6 and TNF-α levels increased in LPS treated cell group according to control, statistically significant. Each cytokine levels significantly decreased in LPS and propolis treated cell group according to only LPS treated cell group (p<0.05.Conclusion: Propolis is a natural product to be examined for usage when needed the suppression of pro-inflammatory cytokines. J Clin Exp Invest 2011; 2 (4: 366-370

  5. Activation of Macrophages by Lipopolysaccharide for Assessing the Immunomodulatory Property of Biomaterials.

    Science.gov (United States)

    Han, Shengwei; Chen, Zetao; Han, Pingping; Hu, Qingang; Xiao, Yin

    2017-03-24

    The design paradigm of biomaterials has been changed to ones with favorable immunomodulatory effects, indicating the importance of accurately evaluating the immunomodulatory properties of biomaterials. Among all the immune cells macrophages receive most attention, due to their plasticity and multiple roles in the materials and host interactions, and thereby become model immune cells for the evaluation of immunomodulatory properties of biomaterials in many studies. Lipopolysaccharides (LPS), a polysaccharide in the outer membrane of Gram-negative bacteria, elicit strong immune responses, which was often applied to activate macrophages, resulting in a proinflammatory M1 phenotype, and the release of proinflammatory cytokines, including tumor necrosis factor alpha (TNFα), interleukin (IL)-1, and IL-6. However, there is no consensus on how to apply macrophages and LPS to detect the immunomodulatory properties of biomaterials. The lack of scientific consideration of this issue has led to some inaccurate and insufficient conclusions on the immunomodulatory properties of biomaterials, and inconsistences between different research groups. In this study, we carried out a systemic study to investigate the stimulatory effects of LPS with different times, doses, and conditions on the activation of macrophages. An experimental pathway was proposed accordingly for the activation of macrophages using LPS for assessing the immunomodulatory property of biomaterials.

  6. Regulatory role of PI3K-protein kinase B on the release of interleukin-1β in peritoneal macrophages from the ascites of cirrhotic patients.

    Science.gov (United States)

    Tapia-Abellán, A; Ruiz-Alcaraz, A J; Antón, G; Miras-López, M; Francés, R; Such, J; Martínez-Esparza, M; García-Peñarrubia, P

    2014-12-01

    Great effort has been paid to identify novel targets for pharmaceutical intervention to control inflammation associated with different diseases. We have studied the effect of signalling inhibitors in the secretion of the proinflammatory and profibrogenic cytokine interleukin (IL)-1β in monocyte-derived macrophages (M-DM) obtained from the ascites of cirrhotic patients and compared with those obtained from the blood of healthy donors. Peritoneal M-DM were isolated from non-infected ascites of cirrhotic patients and stimulated in vitro with lipopolysaccharide (LPS) and heat-killed Candida albicans in the presence or absence of inhibitors for c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 1 (MEK1), p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). The IL1B and CASP1 gene expression were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The expression of IL-1β and caspase-1 were determined by Western blot. IL-1β was also assayed by enzyme-linked immunosorbent assay (ELISA) in cell culture supernatants. Results revealed that MEK1 and JNK inhibition significantly reduced the basal and stimulated IL-1β secretion, while the p38 MAPK inhibitor had no effect on IL-1β levels. On the contrary, inhibition of PI3K increased the secretion of IL-1β from stimulated M-DM. The activating effect of PI3K inhibitor on IL-1β release was mediated mainly by the enhancement of the intracellular IL-1β and caspase-1 content release to the extracellular medium and not by increasing the corresponding mRNA and protein expression levels. These data point towards the role of MEK1 and JNK inhibitors, in contrast to the PI3K-protein kinase B inhibitors, as potential therapeutic tools for pharmaceutical intervention to diminish hepatic damage by reducing the inflammatory response mediated by IL-1β associated with liver failure.

  7. Role of lysosomal enzymes released by alveolar macrophages in the pathogenesis of the acute phase of hypersensitivity pneumonitis

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    J. L. Pérez-Arellano

    1995-01-01

    Full Text Available Hydrolytic enzymes are the major constituents of alveolar macrophages (AM and have been shown to be involved in many aspects of the inflammatory pulmonary response. The aim of this study was to evaluate the role of lysosomal enzymes in the acute phase of hypersensitivity pneumonitis (HPs. An experimental study on AM lysosomal enzymes of an HP-guinea-pig model was performed. The results obtained both in vivo and in vitro suggest that intracellular enzymatic activity decrease is, at least partly, due to release of lysosomal enzymes into the medium. A positive but slight correlation was found between extracellular lysosomal activity and four parameters of lung lesion (lung index, bronchoalveolar fluid total (BALF protein concentration, BALF LDH and BALF alkaline phosphatase activities. All the above findings suggest that the AM release of lysosomal enzymes during HP is a factor involved, although possibly not the only one, in the pulmonary lesions appearing in this disease.

  8. Activation of AMPA receptor promotes TNF-α release via the ROS-cSrc-NFκB signaling cascade in RAW264.7 macrophages

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    Cheng, Xiu-Li [Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Ding, Fan [Office of Scientific R& D, Tsinghua University, Beijing (China); Li, Hui; Tan, Xiao-Qiu [Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Liu, Xiao [Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Cao, Ji-Min, E-mail: caojimin@126.com [Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Gao, Xue, E-mail: longlongnose@163.com [Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China)

    2015-05-29

    The relationship between glutamate signaling and inflammation has not been well defined. This study aimed to investigate the role of AMPA receptor (AMPAR) in the expression and release of tumor necrosis factor-alpha (TNF-α) from macrophages and the underlying mechanisms. A series of approaches, including confocal microscopy, immunofluorescency, flow cytometry, ELISA and Western blotting, were used to estimate the expression of AMPAR and downstream signaling molecules, TNF-α release and reactive oxygen species (ROS) generation in the macrophage-like RAW264.7 cells. The results demonstrated that AMPAR was expressed in RAW264.7 cells. AMPA significantly enhanced TNF-α release from RAW264.7 cells, and this effect was abolished by CNQX (AMPAR antagonist). AMPA also induced elevation of ROS production, phosphorylation of c-Src and activation of nuclear factor (NF)-κB in RAW264.7 cells. Blocking c-Src by PP2, scavenging ROS by glutathione (GSH) or inhibiting NF-κB activation by pyrrolidine dithiocarbamate (PDTC) decreased TNF-α production from RAW264.7 cells. We concluded that AMPA promotes TNF-α release in RAW264.7 macrophages likely through the following signaling cascade: AMPAR activation → ROS generation → c-Src phosphorylation → NF-κB activation → TNF-α elevation. The study suggests that AMPAR may participate in macrophage activation and inflammation. - Highlights: • AMPAR is expressed in RAW264.7 macrophages and is upregulated by AMPA stimulation. • Activation of AMPAR stimulates TNF-α release in macrophages through the ROS-cSrc-NFκB signaling cascade. • Macrophage AMPAR signaling may play an important role in inflammation.

  9. Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

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    Sundler Roger

    2006-05-01

    Full Text Available Abstract Background Yeast and bacteria elicit arachidonate release in macrophages, leading to the formation of leukotrienes and prostaglandins, important mediators of inflammation. Receptors recognising various microbes have been identified, but the signalling pathways are not entirely understood. Cytosolic phospholipase A2 is a major down-stream target and this enzyme is regulated by both phosphorylation and an increase in intracellular Ca2+. Potential signal components are MAP kinases, phosphatidylinositol 3-kinase and phospholipase Cγ2. The latter can undergo tyrosine phosphorylation, and Src family kinases might carry out this phosphorylation. Btk, a Tec family kinase, could also be important. Our aim was to further elucidate the role of Src family kinases and Btk. Methods Arachidonate release from murine peritoneal macrophages was measured by prior radiolabeling. Furthermore, immunoprecipitation and Western blotting were used to monitor changes in activity/phosphorylation of intermediate signal components. To determine the role of Src family kinases two different inhibitors with broad specificity (PP2 and the Src kinase inhibitor 1, SKI-1 were used as well as the Btk inhibitor LFM-A13. Results Arachidonate release initiated by either Staphylococcus aureus or yeast-derived zymosan beads was shown to depend on members of the Src kinase family as well as Btk. Src kinases were found to act upstream of Btk, phosphatidylinositol 3-kinase, phospholipase Cγ2 and the MAP kinases ERK and p38, thereby affecting all branches of the signalling investigated. In contrast, Btk was not involved in the activation of the MAP-kinases. Since the cytosolic phospholipase A2 in macrophages is regulated by both phosphorylation (via ERK and p38 and an increase in intracellular Ca2+, we propose that members of the Src kinase family are involved in both types of regulation, while the role of Btk may be restricted to the latter type. Conclusion Arachidonate release

  10. Differential response to dexamethasone on the TXB2 release in guinea-pig alveolar macrophages induced by zymosan and cytokines

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    M. E. Salgueiro

    1997-01-01

    Full Text Available Glucocorticosteroids reduce the production of inflammatory mediators but this effect may depend on the stimulus. We have compared the time course of the effect of dexamethasone on the thromboxane B2 (TXB2 release induced by cytokine stimulation and zymosan in guinea-pig alveolar macrophages. Interleukin-1β (IL-1β, tumour necrosis factor-α (TNF-α and opsonized zymosan (OZ, all stimulate TXB2 release. High concentrations of dexamethasone (1–10 μM inhibit the TXB2 production induced by both cytokines and OZ, but the time course of this response is different. Four hours of incubation with dexamethasone reduce the basal TXB2 release and that induced by IL-1β and TNF-α, but do not modify the TXB2 release induced by OZ. However, this stimulus was reduced after 24 h incubation. Our results suggest that the antiinflammatory activity of glucocorticosteroids shows some dependence on stimulus and, therefore, may have more than one mechanism involved.

  11. Protective effect of cyclosporin A and FK506 from nitric oxide-dependent apoptosis in activated macrophages

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    Hortelano, Sonsoles; López-Collazo, Eduardo; Boscá, Lisardo

    1999-01-01

    Activation of macrophages with lipopolysaccharide (LPS) and low doses of interferon-γ (IFN-γ) induced apoptotic death through a nitric oxide-dependent pathway. Treatment of cells with the immunosuppressors cyclosporin A (CsA) or FK506 inhibited the activation-dependent apoptosis. These drugs decreased the up-regulation of p53 and Bax characteristic of activated macrophages. Moreover, incubation of activated macrophages with CsA and FK506 contributed to maintain higher levels of Bcl-2 than in LPS/IFN-γ treated cells. The inhibition of apoptosis exerted by CsA and FK506 in macrophages was also observed when cell death was induced by treatment with chemical nitric oxide donors. Incubation of macrophages with LPS/IFN-γ barely affected caspase-1 but promoted an important activation of caspase-3. Both CsA and FK506 inhibited pathways leading to caspase-3 activation. Moreover, the cleavage of poly(ADP-ribose) polymerase, a well established caspase substrate, was reduced by these immunosuppressive drugs. CsA and FK506 reduced the release of cytochrome c to the cytosol and the activation of caspase-3 in cells treated with nitric oxide donors. These results indicate that CsA and FK506 protect macrophages from nitric oxide-dependent apoptosis and suggest a contribution of the macrophage to innate immunity under conditions of immunosuppression of the host. PMID:10205001

  12. Exposure to wear particles generated from studded tires and pavement induces inflammatory cytokine release from human macrophages.

    Science.gov (United States)

    Lindbom, John; Gustafsson, Mats; Blomqvist, Göran; Dahl, Andreas; Gudmundsson, Anders; Swietlicki, Erik; Ljungman, Anders G

    2006-04-01

    Health risks associated with exposure to airborne particulate matter (PM) have been shown epidemiologically as well as experimentally, pointing to both respiratory and cardiovascular effects. Lately, wear particles generated from traffic have been recognized to be a major contributing source to the overall particle load, especially in the Nordic countries were studded tires are used. In this work, we investigated the inflammatory effect of PM10 generated from the wear of studded tires on two different types of pavement. As comparison, we also investigated PM10 from a traffic-intensive street, a subway station, and diesel exhaust particles (DEP). Human monocyte-derived macrophages, nasal epithelial cells (RPMI 2650), and bronchial epithelial cells (BEAS-2B) were exposed to the different types of particles, and the secretion of IL-6, IL-8, IL-10, and TNF-alpha into the culture medium was measured. The results show a significant release of cytokines from macrophages after exposure for all types of particles. When particles generated from asphalt/granite pavement were compared to asphalt/quartzite pavement, the granite pavement had a significantly higher capacity to induce the release of cytokines. The granite pavement particles induced cytokine release at the same magnitude as the street particles did, which was higher than what particles from both a subway station and DEP did. Exposure of epithelial cells to PM10 resulted in a significant increase of TNF-alpha secreted from BEAS-2B cells for all types of particles used (DEP was not tested), and the highest levels were induced by subway particles. None of the particle types were able to evoke detectable cytokine release from RPMI 2650 cells. The results indicate that PM10 generated by the wear of studded tires on the street surface is a large contributor to the cytokine-releasing ability of particles in traffic-intensive areas and that the type of pavement used is important for the level of this contribution

  13. Neuropeptide FF inhibits LPS-mediated osteoclast differentiation of RAW264.7 cells.

    Science.gov (United States)

    Sun, Yu-Long; Chen, Zhi-Hao; Li, Di-Jie; Zhao, Fan; Ma, Xiao-Li; Shang, Peng; Yang, Tuanming; Qian, Airong

    2014-01-01

    Neuropeptide FF (NPFF) has been implicated in many physiological processes. Previously, we have reported that NPFF modulates the viability and nitric oxide (NO) production of RAW264.7 macrophages. In this study, we investigated the influence of NPFF on lipopolysaccharide (LPS)-mediated osteoclast formation of RAW264.7 cells. Our results suggest that, NPFF dose-dependently (1 nM, 10 nM and 100 nM) inhibited osteoclast formation, TRAP enzyme activity and bone resorption in osteoclasts induced by LPS respectively. Moreover, LPS-provoked NO release was also inhibited by NPFF treatment, indicating a NO-dependent pathway is mainly involved. Furthermore, the alterations of osteoclast marker genes were also assessed including TRAP, Cathepsin K, MMP-9, NFATc1 and Runx2. NPFF downregulated LPS-caused gene augmentations of TRAP, Cathepsin K and MMP-9, whereas showed no influences on NFATc1 and Runx2. In addition, NPFF receptor 2 (NPFFR2) mRNA expression was also augmented in response to NPFF treatment, hinting the involvement of NPFFR2 pathway. It should be mentioned that RF9 (1 µ M), a reported pharmacological inhibitor for NPFF receptors, exerted NPFF-like agonist properties as to attenuate osteoclastogenesis. Collectively, our findings provide new evidence for the in vitro activity of NPFF on osteoclasts, which may be helpful to extend the scope of NPFF functions.

  14. 5-Bromo-2-hydroxy-4-methyl-benzaldehyde inhibited LPS-induced production of pro-inflammatory mediators through the inactivation of ERK, p38, and NF-κB pathways in RAW 264.7 macrophages.

    Science.gov (United States)

    Kim, Kil-Nam; Ko, Seok-Chun; Ye, Bo-Ram; Kim, Min-Sun; Kim, Junseong; Ko, Eun-Yi; Cho, Su-Hyeon; Kim, Daekyung; Heo, Soo-Jin; Jung, Won-Kyo

    2016-10-25

    The aim of the present study was to investigate the effects of 5-bromo-2-hydroxy-4-methyl-benzaldehyde (BHMB) on inflammatory responses to lipopolysaccharide (LPS) in RAW 264.7 cells and the associated mechanism of action. BHMB concentration-dependently suppressed protein and mRNA expressions of iNOS and COX-2, thereby inhibiting the production of NO and PGE2 in LPS-stimulated RAW 264.7 cells. BHMB also reduced the mRNA expression of TNF-α, IL-6, and IL-1β in LPS-stimulated RAW 264.7 cells. To elucidate the mechanism underlying the anti-inflammatory activity of BHMB, we investigated the effects of BHMB on the mitogen-activated protein kinase and nuclear factor-kappa B (NF-κB) pathways. BHMB suppressed the phosphorylation and degradation of IκB-α and markedly inhibited the nuclear translocation of p65 and p50 in LPS-stimulated RAW 264.7 cells. The compound also inhibited the LPS-stimulated phosphorylation of ERK and p38. Taken together, these results illustrated that BHMB suppresses pro-inflammatory mediator and cytokine expression in LPS-stimulated RAW 264.7 cells by inhibiting the phosphorylation of ERK and p38 and the activation of NF-κB.

  15. Effects of Benzalkonium Chloride on THP-1 Differentiated Macrophages In Vitro

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    Michée, Sylvain; Brignole-Baudouin, Françoise; Riancho, Luisa; Rostene, William; Baudouin, Christophe; Labbé, Antoine

    2013-01-01

    Purpose To characterize the effects of benzalkonium chloride (BAK) in THP-1 differentiated cells in vitro. Methods Macrophages were obtained after differentiation of THP-1 cells, a human monocytic leukemia cell line. Macrophages were exposed for 24 h to 33 nM (10−5%) benzalkonium chloride (BAK), 10 nM dinitrochlorobenzene (DNCB), 100 ng/mL lipopolysaccharide (LPS), 5 ng/mL tumor necrosis factor alpha (TNF-α) or phosphate buffered saline (PBS) as controls. The expression of CD11b, CD11c, CD33 and CD54 was evaluated using immunohistochemistry and flow cytometry (FCM). Phagocytosis function was analyzed using carboxylate-modified fluorescent microspheres and quantified by FCM. Migration was evaluated in cocultures with conjunctival epithelial cells. Cytokine production was detected and quantified in culture supernatants using a human cytokine array. Results Stimulation of THP-1-derived macrophages with a low concentration of BAK increased CD11b and CD11c expression and decreased CD33. Macrophages exposed to BAK, LPS and TNF-α had increased phagocytosis. In contrast to LPS, BAK and TNF-α increased macrophage migration. Cytokines in supernatants of macrophages exposed to BAK revealed an increased release of CCL1, CCL4/MIP-1β, TNF-α, soluble CD54/ICAM-1 and IL-1β. Conclusion In vitro, BAK has a direct stimulating effect on macrophages, increasing phagocytosis, cytokine release, migration and expression of CD11b and CD11c. Long-term exposure to low concentrations of BAK should be considered as a stimulating factor responsible for inflammation through macrophage activation. PMID:23991114

  16. Effects of benzalkonium chloride on THP-1 differentiated macrophages in vitro.

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    Sylvain Michée

    Full Text Available PURPOSE: To characterize the effects of benzalkonium chloride (BAK in THP-1 differentiated cells in vitro. METHODS: Macrophages were obtained after differentiation of THP-1 cells, a human monocytic leukemia cell line. Macrophages were exposed for 24 h to 33 nM (10(-5% benzalkonium chloride (BAK, 10 nM dinitrochlorobenzene (DNCB, 100 ng/mL lipopolysaccharide (LPS, 5 ng/mL tumor necrosis factor alpha (TNF-α or phosphate buffered saline (PBS as controls. The expression of CD11b, CD11c, CD33 and CD54 was evaluated using immunohistochemistry and flow cytometry (FCM. Phagocytosis function was analyzed using carboxylate-modified fluorescent microspheres and quantified by FCM. Migration was evaluated in cocultures with conjunctival epithelial cells. Cytokine production was detected and quantified in culture supernatants using a human cytokine array. RESULTS: Stimulation of THP-1-derived macrophages with a low concentration of BAK increased CD11b and CD11c expression and decreased CD33. Macrophages exposed to BAK, LPS and TNF-α had increased phagocytosis. In contrast to LPS, BAK and TNF-α increased macrophage migration. Cytokines in supernatants of macrophages exposed to BAK revealed an increased release of CCL1, CCL4/MIP-1β, TNF-α, soluble CD54/ICAM-1 and IL-1β. CONCLUSION: In vitro, BAK has a direct stimulating effect on macrophages, increasing phagocytosis, cytokine release, migration and expression of CD11b and CD11c. Long-term exposure to low concentrations of BAK should be considered as a stimulating factor responsible for inflammation through macrophage activation.

  17. Bone Marrow Mesenchymal Stem Cells Inhibit Lipopolysaccharide-Induced Inflammatory Reactions in Macrophages and Endothelial Cells

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    Dequan Li

    2016-01-01

    Full Text Available Background. Systemic inflammatory response syndrome (SIRS accompanied by trauma can lead to multiple organ dysfunction syndrome (MODS and even death. Early inhibition of the inflammation is necessary for damage control. Bone marrow mesenchymal stem cells (BMSCs, as a novel therapy modality, have been shown to reduce inflammatory responses in human and animal models. Methods. In this study, we used Western blot, quantitative PCR, and enzyme-linked immunosorbent assay (ELISA to assess the activity of BMSCs to suppress the inflammation induced by lipopolysaccharide (LPS in human umbilical cord endothelial cells (HUVECs and alveolar macrophages. Results. Our results demonstrated that LPS caused an inflammatory response in alveolar macrophages and HUVECs, increased permeability of HUVEC, upregulated expression of toll-like receptor (TLR 2, TLR4, phosphorylated p65, downregulated release of IL10, and promoted release of TNF-α in both cells. Coculture with BMSCs attenuated all of these activities induced by LPS in the two tested cell types. Conclusions. Together, our results demonstrate that BMSCs dosage dependently attenuates the inflammation damage of alveolar macrophages and HUVECs induced by LPS.

  18. Surfactant protein-A modulates LPS-induced TLR4 localization and signaling via β-arrestin 2.

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    Vicky Sender

    Full Text Available The soluble C-type lectin surfactant protein (SP-A mediates lung immune responses partially via its direct effects on alveolar macrophages (AM, the main resident leukocytes exposed to antigens. SP-A modulates the AM threshold of lipopolysaccharide (LPS activity towards an anti-inflammatory phenotype both in vitro and in vivo through various mechanisms. LPS responses are tightly regulated via distinct pathways including subcellular TLR4 localization and thus ligand sensing. The cytosolic scaffold and signaling protein β-arrestin 2 acts as negative regulator of LPS-induced TLR4 activation. Here we show that SP-A neither increases TLR4 abundancy nor co-localizes with TLR4 in primary AM. SP-A significantly reduces the LPS-induced co-localization of TLR4 with the early endosome antigen (EEA 1 by promoting the co-localization of TLR4 with the post-Golgi compartment marker Vti1b in freshly isolated AM from rats and wild-type (WT mice, but not in β-arrestin 2(-/- AM. Compared to WT mice pulmonary LPS-induced TNF-α release in β-arrestin 2(-/- mice is accelerated and enhanced and exogenous SP-A fails to inhibit both lung LPS-induced TNF-α release and TLR4/EEA1 positioning. SP-A, but not LPS, enhances β-arrestin 2 protein expression in a time-dependent manner in primary rat AM. The constitutive expression of β-arrestin 2 in AM from SP-A(-/- mice is significantly reduced compared to SP-A(+/+ mice and is rescued by SP-A. Prolonged endosome retention of LPS-induced TLR4 in AM from SP-A(-/- mice is restored by exogenous SP-A, and is antagonized by β-arrestin 2 blocking peptides. LPS induces β-arrestin 2/TLR4 association in primary AM which is further enhanced by SP-A. The data demonstrate that SP-A modulates LPS-induced TLR4 trafficking and signaling in vitro and in vivo engaging β-arrestin 2.

  19. Interferon regulatory factor-1 mediates the release of high mobility group box-1 in endotoxemia in mice

    Institute of Scientific and Technical Information of China (English)

    PAN Pin-hua; Jon Cardinal; LI Mo-li; HU Cheng-ping; Allan Tsung

    2013-01-01

    Background The extracellular release of the danger signal high mobility group box-1 (HMGB1) has been implicated in the pathogenesis and outcomes of sepsis.Understanding the mechanisms responsible for HMGB1 release can lead to the identification of targets that may inhibit this process.The transcription factor interferon regulatory factor-1 (IRF-1) is an important mediator of innate immune responses and has been shown to participate in mortality associated with endotoxemia; however,its role in mediating the release of HMGB1 in these settings is unknown.Methods Male IRF-1 knockout (KO) and age matched C57BL/6 wild type (WT) mice were given intraperitoneal (IP)injections of lipopolysaccharide (LPS).In some experiments,96 hours survival rates were observed.In other experiments,mice were sacrificed 12 hours after LPS administration and sera were harvested for future analysis.In in vitro study,RAW 264.7 murine monocyte/macrophage-like cells or primary peritoneal macrophage obtained from IRF-1 KO and WF mice were cultured for LPS mediated HMGB1 release analysis.And the mechanism for HMGB1 release was analyzed by immune-precipitation.Results IRF-1 KO mice experienced less mortality,and released less systerric HMGB1 compared to their WT counterparts.Exogenous administration of recombinant HMGB1 to IRF-1 KO mice returned the mortality rate to that seen originally in IRF-1 WT mice.Using cultures of peritoneal macrophages or RAW264.7 cells,in vitro LPS stimulation induced the release of HMGB1 in an IRF-1 dependent manner.And the janus associated kinase (JAK)-IRF-1 signal pathway appeared to participate in the signaling mechanisms of LPS-induced HMGB1 release by mediating acetylation of HMGB1.Conclusion IRF-1 plays a role in LPS induced release of HMGB1 and therefore may serve as a novel target in sepsis.

  20. Pharmacological effects of mitraphylline from Uncaria tomentosa in primary human monocytes: Skew toward M2 macrophages.

    Science.gov (United States)

    Montserrat-de la Paz, S; de la Puerta, R; Fernandez-Arche, A; Quilez, A M; Muriana, F J G; Garcia-Gimenez, M D; Bermudez, B

    2015-07-21

    Uncaria tomentosa (Willdenow ex Roemer & Schultes) DC. (Rubiaceae) is a Peruvian thorny liana, commonly known as "cat׳s claw", and traditionally used in folk medicine to deal with several inflammatory diseases. Mitraphylline (MTP) is the most abundant pentacyclic oxindolic alkaloid (POA) from U. Tomentosa and has been reported to modify the inflammatory response. Herein, we have sought to identify the mechanisms underlying this modulatory effect of MTP on primary human monocytes and its ability to regulate differentiation processes on human primary monocyte and monocyte-derived macrophages. In vitro studies with human primary monocytes and monocyte-derived macrophages were performed. Monocytes and M0 macrophages were exposed to MTP (25μM) and LPS (100ng/mL). M0 macrophages were polarized to M1 and M2 phenotypes in the absence or presence of MTP. The activation state of monocytes/macrophages was assessed by flow cytometry, gene expression and protein analysis of different specific markers. In human primary monocytes, the incubation of MTP for 24h reduced the number of classical (CD14(++)CD16(-)) and intermediate (CD14(++)CD16(+)) subsets when compared to untreated or LPS-treated cells. MTP also reduced the chemotactic capacity of human primary monocytes. In addition, MTP promoted the polarization of M0 macrophages toward an anti-inflammatory M2 phenotype, the abrogation of the release of pro-inflammatory cytokines such as TNFα, IL-6 or IL-1β, as well as the restoration of markers for M2 macrophages in LPS-treated M1 macrophages. Our results suggest that MTP may be a key modulator for regulating the plasticity of monocytes/macrophages and the attenuation of the inflammatory response. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. Cytotoxic macrophage-released tumour necrosis factor-alpha (TNF-α) as a killing mechanism for cancer cell death after cold plasma activation

    Science.gov (United States)

    Kaushik, Nagendra Kumar; Kaushik, Neha; Min, Booki; Choi, Ki Hong; Hong, Young June; Miller, Vandana; Fridman, Alexander; Choi, Eun Ha

    2016-03-01

    The present study aims at studying the anticancer role of cold plasma-activated immune cells. The direct anti-cancer activity of plasma-activated immune cells against human solid cancers has not been described so far. Hence, we assessed the effect of plasma-treated RAW264.7 macrophages on cancer cell growth after co-culture. In particular, flow cytometer analysis revealed that plasma did not induce any cell death in RAW264.7 macrophages. Interestingly, immunofluorescence and western blot analysis confirmed that TNF-α released from plasma-activated macrophages acts as a tumour cell death inducer. In support of these findings, activated macrophages down-regulated the cell growth in solid cancer cell lines and induced cell death in vitro. Together our findings suggest plasma-induced reactive species recruit cytotoxic macrophages to release TNF-α, which blocks cancer cell growth and can have the potential to contribute to reducing tumour growth in vivo in the near future.

  2. Depressant effects of ambroxol and erdosteine on cytokine synthesis, granule enzyme release, and free radical production in rat alveolar macrophages activated by lipopolysaccharide.

    Science.gov (United States)

    Jang, Yoon Young; Song, Jin Ho; Shin, Yong Kyoo; Han, Eun Sook; Lee, Chung Soo

    2003-04-01

    The present study examined the effects of ambroxol and erdosteine, bronchial expectorants, on the cytokine synthesis, granule enzyme release, and free radical production in rat alveolar macrophages activated by lipopolysaccharide. Ambroxol and erdosteine significantly decreased the production of tumour necrosis factors-alpha, interleukin-1beta, and interleukin-6 in alveolar macrophages activated by lipopolysaccharide. These drugs significantly reduced the production of superoxide anion, hydrogen peroxide, and nitric oxide and the release of acid phosphatase and lysozyme in lipopolysaccharide-activated macrophages. Ambroxol and erdosteine showed no scavenging effect on superoxide anion and hydrogen peroxide, whereas both drugs effectively decomposed nitric oxide. The results show that ambroxol and erdosteine may inhibit the responses, including cytokine synthesis and free radical production, in rat alveolar macrophages activated by lipopolysaccharide. Unlike the production of reactive oxygen species, the inhibitory effect of ambroxol and erdosteine on the production of nitric oxide in lipopolysaccharide-activated alveolar macrophages may be accomplished by a scavenging action on the species and inhibition of the respiratory burst.

  3. Dependence of macrophage superoxide release on the pulse amplitude of an applied pressure regime: a potential factor at the soft tissue-implant interface.

    Science.gov (United States)

    Shin, Hainsworth Y; Frechette, Danielle M; Rohner, Nathan; Zhang, Xiaoyan; Puleo, David A; Bjursten, Lars M

    2016-03-01

    Failure of soft tissue implants has been largely attributed to the influence of biomaterial surface properties on the foreign body response, but some implant complications, e.g. macrophage accumulation and necrosis, are still not effectively addressed with surface treatments to minimize deleterious biomaterial effects. We explored an alternative explanation for implant failure, linking biocompatibility with implant micromotion-induced pressure fluctuations at the tissue-biomaterial interface. For this purpose, we used a custom in vitro system to characterize the effects of pressure fluctuations on the activity of macrophages, the predominant cells at a healing implant site. Initially, we quantified superoxide production by HL60-derived macrophage-like cells under several different pressure regimes with means of 5-40 mmHg, amplitudes of 0-15 mmHg and frequencies of 0-1.5 Hz. All pressure regimes tested elicited significantly (p superoxide production by macrophage-like cells relative to parallel controls. Notably, pressure-sensitive reductions in superoxide release correlated (r(2)  = 0.74; p superoxide production and cell viability, we also explored the influence of cyclic pressure on macrophage numbers and death. Compared to controls, adherent macrophage-like cells exposed to 7.5/2.5 mmHg cyclic pressures for 6 h exhibited significantly (p superoxide dismutase. Collectively, our results suggest that pressure pulses are a putative regulator of macrophage adhesion via a superoxide-related effect. Pressure fluctuations, e.g. due to implant micromotion, may, therefore, potentially modulate macrophage-dependent wound healing.

  4. Ginkgolide A Ameliorates LPS-Induced Inflammatory Responses In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Yan Li

    2017-04-01

    Full Text Available Ginkgolide A (GA is a natural compound isolated from Ginkgo biloba and has been used to treat cardiovascular diseases and diabetic vascular complications. However, only a few studies have been conducted on the anti-inflammatory effects of GA. In particular, no related reports have been published in a common inflammation model of lipopolysaccharide (LPS-stimulated macrophages, and the anti-inflammatory mechanisms of GA have not been fully elucidated. In the present study, we extensively investigated the anti-inflammatory potential of GA in vitro and in vivo. We showed that GA could suppress the expression of pro-inflammatory mediators (cyclooxygenase-2 (COX-2 and nitric oxide (NO and pro-inflammatory cytokines (tumor necrosis factor (TNF-α, interleukin (IL-6 and IL-1β in LPS-treated mouse peritoneal macrophages, mouse macrophage RAW264.7 cells, and differentiated human monocytes (dTHP-1 in vitro. These effects were partially carried out via downregulating Nuclear factor kappa-B (NF-κB, Mitogen-activated protein kinases (MAPKs (p38 mitogen-activated protein kinase and extracellular signal-regulated kinase (ERK, but not c-Jun N-terminal kinase (JNK, and activating the AMP-activated protein kinase (AMPK signaling pathway also seems to be important. Consistently, GA was also shown to inhibit the LPS-stimulated release of TNF-α and IL-6 in mice. Taken together, these findings suggest that GA can serve as an effective inflammatory inhibitor in vitro and in vivo.

  5. Decreased pro-inflammatory cytokine production by LPS-stimulated PBMC upon in vitro incubation with the flavonoids apigenin, luteolin or chrysin, due to selective elimination of monocytes/macrophages.

    NARCIS (Netherlands)

    Hougee, S.; Sanders, A.; Faber, J.; Graus, Y.M.; Berg, W.B. van den; Garssen, J.; Smit, H.F.; Hoijer, M.A.

    2005-01-01

    Apigenin and its structural analogues chrysin and luteolin were used to evaluate their capacity to inhibit the production of pro-inflammatory cytokines by lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC). Furthermore, flowcytometric analysis was performed to compar

  6. Molecular mechanism of apigenin on inhibition of LPS-induced inflammatory mediators in murine macrophages%芹菜素抑制脂多糖诱导小鼠巨噬细胞分泌炎症介质的分子机制

    Institute of Scientific and Technical Information of China (English)

    吴广; 符平; 周玉生; 周润梅

    2015-01-01

    Objective:To investigate the effect and the mechanism of Apigenin on lipopolysaccharides ( LPS )-induced inflammatory mediators production in murine macrophages. Methods:The murine macrophage cell line RAW 264. 7 cells were cultured in vitro,and were treated with different concentration of Apigenin followed by LPS administration. Expression of heme oxygenase-1 ( HO-1),cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS),phosphorylation of p38 and IκB,nuclear translocation of Nrf2 were detected by Western blot. Production of Nitrite and nitrate ( NOx) was analyzed by colorimetric technique. Secretion of prosta-glandin E2 (PGE2) was detected by ELISA. Activation of NF-κB was measured by luciferase assay. Results: Western blot indicated that apigenin could induce RAW 264. 7 cells expression of HO-1, and pretreatment of SB203580, an inhibitor of p38 significantly inhibited apigenin induced HO-1 expression. In addition,Apigenin could also decrease the content of nuclear transcription factor Nrf2 in cytoplasm and increase its level in the nucleus. Silencing of Nrf2 by specific siRNA could inhibit apigenin-induced HO-1 expression. Furthermore,apigenin administration significantly inhibited LPS-induced NOx production and PGE2 secretion, COX-2 and iNOS expression,IκB phosphorylation and NF-κB activation,and transfection of HO-1 siRNA could reverse these actions. Conclusion:Apigenin inhibits LPS-induced inflammatory response through induction of HO-1 and inhibition of NF-κB in macrophages.%目的::观察芹菜素对脂多糖( LPS)诱导小鼠巨噬细胞分泌炎症介质的影响,并探讨其分子机制。方法:体外培养小鼠巨噬细胞RAW 264.7,用不同浓度的芹菜素处理后,加入LPS刺激。 Western blot检测血红素氧合酶-1(HO-1)、环氧化酶-2(COX-2)、诱导型一氧化氮合成酶(iNOS)的表达以及p38和IκB的磷酸化;比色法检测亚硝酸盐和硝酸盐(NOx)的含量;ELISA检测PGE2的产生;报

  7. Cell elasticity determines macrophage function.

    Directory of Open Access Journals (Sweden)

    Naimish R Patel

    Full Text Available Macrophages serve to maintain organ homeostasis in response to challenges from injury, inflammation, malignancy, particulate exposure, or infection. Until now, receptor ligation has been understood as being the central mechanism that regulates macrophage function. Using macrophages of different origins and species, we report that macrophage elasticity is a major determinant of innate macrophage function. Macrophage elasticity is modulated not only by classical biologic activators such as LPS and IFN-γ, but to an equal extent by substrate rigidity and substrate stretch. Macrophage elasticity is dependent upon actin polymerization and small rhoGTPase activation, but functional effects of elasticity are not predicted by examination of gene expression profiles alone. Taken together, these data demonstrate an unanticipated role for cell elasticity as a common pathway by which mechanical and biologic factors determine macrophage function.

  8. Mangiferin inhibits macrophage classical activation via downregulating interferon regulatory factor 5 expression.

    Science.gov (United States)

    Wei, Zhiquan; Yan, Li; Chen, Yixin; Bao, Chuanhong; Deng, Jing; Deng, Jiagang

    2016-08-01

    Mangiferin is a natural polyphenol and the predominant effective component of Mangifera indica Linn. leaves. For hundreds of years, Mangifera indica Linn. leaf has been used as an ingredient in numerous traditional Chinese medicine preparations for the treatment of bronchitis. However, the pharmacological mechanism of mangiferin in the treatment of bronchitis remains to be elucidated. Macrophage classical activation is important role in the process of bronchial airway inflammation, and interferon regulatory factor 5 (IRF5) has been identified as a key regulatory factor for macrophage classical activation. The present study used the THP‑1 human monocyte cell line to investigate whether mangiferin inhibits macrophage classical activation via suppressing IRF5 expression in vitro. THP‑1 cells were differentiated to macrophages by phorbol 12‑myristate 13‑acetate. Macrophages were polarized to M1 macrophages following stimulation with lipopolysaccharide (LPS)/interferon‑γ (IFN‑γ). Flow cytometric analysis was conducted to detect the M1 macrophages. Reverse transcription‑quantitative polymerase chain reaction was used to investigate cellular IRF5 gene expression. Levels of proinflammatory cytokines and IRF5 were assessed following cell culture and cellular homogenization using enzyme‑linked immunosorbent assay. IRF5 protein and nuclei co‑localization was performed in macrophages with laser scanning confocal microscope immunofluorescence analysis. The results of the present study demonstrated that mangiferin significantly inhibits LPS/IFN‑γ stimulation‑induced classical activation of macrophages in vitro and markedly decreases proinflammatory cytokine release. In addition, cellular IRF5 expression was markedly downregulated. These results suggest that the inhibitory effect of mangiferin on classical activation of macrophages may be exerted via downregulation of cellular IRF5 expression levels.

  9. Mangiferin inhibits macrophage classical activation via downregulating interferon regulatory factor 5 expression

    Science.gov (United States)

    Wei, Zhiquan; Yan, Li; Chen, Yixin; Bao, Chuanhong; Deng, Jing; Deng, Jiagang

    2016-01-01

    Mangiferin is a natural polyphenol and the predominant effective component of Mangifera indica Linn. leaves. For hundreds of years, Mangifera indica Linn. leaf has been used as an ingredient in numerous traditional Chinese medicine preparations for the treatment of bronchitis. However, the pharmacological mechanism of mangiferin in the treatment of bronchitis remains to be elucidated. Macrophage classical activation is important role in the process of bronchial airway inflammation, and interferon regulatory factor 5 (IRF5) has been identified as a key regulatory factor for macrophage classical activation. The present study used the THP-1 human monocyte cell line to investigate whether mangiferin inhibits macrophage classical activation via suppressing IRF5 expression in vitro. THP-1 cells were differentiated to macrophages by phorbol 12-myristate 13-acetate. Macrophages were polarized to M1 macrophages following stimulation with lipopolysaccharide (LPS)/interferon-γ (IFN-γ). Flow cytometric analysis was conducted to detect the M1 macrophages. Reverse transcription-quantitative polymerase chain reaction was used to investigate cellular IRF5 gene expression. Levels of proinflammatory cytokines and IRF5 were assessed following cell culture and cellular homogenization using enzyme-linked immunosorbent assay. IRF5 protein and nuclei co-localization was performed in macrophages with laser scanning confocal microscope immunofluorescence analysis. The results of the present study demonstrated that mangiferin significantly inhibits LPS/IFN-γ stimulation-induced classical activation of macrophages in vitro and markedly decreases proinflammatory cytokine release. In addition, cellular IRF5 expression was markedly downregulated. These results suggest that the inhibitory effect of mangiferin on classical activation of macrophages may be exerted via downregulation of cellular IRF5 expression levels. PMID:27277156

  10. LPS-induced lung inflammation in marmoset monkeys - an acute model for anti-inflammatory drug testing.

    Directory of Open Access Journals (Sweden)

    Sophie Seehase

    Full Text Available Increasing incidence and substantial morbidity and mortality of respiratory diseases requires the development of new human-specific anti-inflammatory and disease-modifying therapeutics. Therefore, new predictive animal models that closely reflect human lung pathology are needed. In the current study, a tiered acute lipopolysaccharide (LPS-induced inflammation model was established in marmoset monkeys (Callithrix jacchus to reflect crucial features of inflammatory lung diseases. Firstly, in an ex vivo approach marmoset and, for the purposes of comparison, human precision-cut lung slices (PCLS were stimulated with LPS in the presence or absence of the phosphodiesterase-4 (PDE4 inhibitor roflumilast. Pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α and macrophage inflammatory protein-1 beta (MIP-1β were measured. The corticosteroid dexamethasone was used as treatment control. Secondly, in an in vivo approach marmosets were pre-treated with roflumilast or dexamethasone and unilaterally challenged with LPS. Ipsilateral bronchoalveolar lavage (BAL was conducted 18 hours after LPS challenge. BAL fluid was processed and analyzed for neutrophils, TNF-α, and MIP-1β. TNF-α release in marmoset PCLS correlated significantly with human PCLS. Roflumilast treatment significantly reduced TNF-α secretion ex vivo in both species, with comparable half maximal inhibitory concentration (IC(50. LPS instillation into marmoset lungs caused a profound inflammation as shown by neutrophilic influx and increased TNF-α and MIP-1β levels in BAL fluid. This inflammatory response was significantly suppressed by roflumilast and dexamethasone. The close similarity of marmoset and human lungs regarding LPS-induced inflammation and the significant anti-inflammatory effect of approved pharmaceuticals assess the suitability of marmoset monkeys to serve as a promising model for studying anti-inflammatory drugs.

  11. Inhibition of NOS-NO System Prevents Autoimmune Orchitis Development in Rats: Relevance of NO Released by Testicular Macrophages in Germ Cell Apoptosis and Testosterone Secretion.

    Directory of Open Access Journals (Sweden)

    Sabrina Jarazo Dietrich

    Full Text Available Although the testis is considered an immunoprivileged organ it can orchestrate immune responses against pathological insults such as infection and trauma. Experimental autoimmune orchitis (EAO is a model of chronic inflammation whose main histopathological features it shares with human orchitis. In EAO an increased number of macrophages infiltrate the interstitium concomitantly with progressive germ cell degeneration and impaired steroidogenesis. Up-regulation of nitric oxide (NO-NO synthase (NOS system occurs, macrophages being the main producers of NO.The aim of our study was to evaluate the role of NO-NOS system in orchitis development and determine the involvement of NO released by testicular macrophages on germ cell apoptosis and testosterone secretion.EAO was induced in rats by immunization with testicular homogenate and adjuvants (E group and a group of untreated normal rats (N was also studied. Blockage of NOS by i.p. injection of E rats with a competitive inhibitor of NOS, L-NAME (8mg/kg, significantly reduced the incidence and severity of orchitis and lowered testicular nitrite content. L-NAME reduced germ cell apoptosis and restored intratesticular testosterone levels, without variations in serum LH. Co-culture of N testicular fragments with testicular macrophages obtained from EAO rats significantly increased germ cell apoptosis and testosterone secretion, whereas addition of L-NAME lowered both effects and reduced nitrite content. Incubation of testicular fragments from N rats with a NO donor DETA-NOnoate (DETA-NO induced germ cell apoptosis through external and internal apoptotic pathways, an effect prevented by N-acetyl-L-cysteine (NAC. DETA-NO inhibited testosterone released from Leydig cells, whereas NAC (from 2.5 to 15 mM did not prevent this effect.We demonstrated that NO-NOS system is involved in the impairment of testicular function in orchitis. NO secreted mainly by testicular macrophages could promote oxidative stress

  12. LPS-TLR4/NF-kB信号通路与抗炎免疫研究进展

    Institute of Scientific and Technical Information of China (English)

    何金鑫; 黄丽华

    2013-01-01

    脂多糖(lipopolysaccharide, LPS)可以激活许多与炎症发生的相关的细胞(如单核细胞和巨噬细胞等),引起相关细胞的细胞因子的合成以及释放,最终导致生物体全身炎症的发生。LPS可以与巨噬细胞表面上的Tol -4受体(TLR4)受体相结合,引起自噬反应的发生,从而利于巨噬细胞对生物体内病原体的吞噬。近些年来,大量的研究表明LPS-TLR4/NF-kB信号通路与生物的炎症密切相关,本文主要综述了LPS,TLR4,NF - kB以及它们之间形成的通路与抗炎免疫的关系,从而为临床上LPS引起的炎症的治疗提供新的途径。%lipopolysaccharide (LPS) lipopolysaccharide, can activate many cels related to inflammation, (such as monocytes and macrophages, etc.), and cause the related cels and synthesis of cytokines and release, eventualy leading to organism systemic inflammation. LPS can combine macrophage receptor on the surface of Tol - 4 (TLR4) receptors, causing the occurrence of autophagy reaction, thus conducive to macrophage phagocytosis of biological pathogens in the body. In recent years, a large number of studies have shown that LPS - TLR4/ NF - kB signaling pathway is closely related to inflammation of the creatures. This paper mainly summarized the LPS, TLR4, NF - kB and formation of the channel between them and the relationship of them. This paper provides a new way for the clinical treatment of inflammation caused by LPS.

  13. Comparison of various assays to quantitate macrophage activation by biological response modifiers

    Energy Technology Data Exchange (ETDEWEB)

    Schultz, R.M.; Nanda, S.; Altom, M.G.

    1984-01-01

    Macrophages treated with various compounds that enhance host antitumor resistance exhibit measurable changes in metabolism, function, and surface antigens. In this study, murine peptone-induced peritoneal macrophages were stimulated in vitro by bacterial lipopolysaccharide (LPS), muramyl dipeptide (MDP), and poly I.poly C. They were subsequently compared in their ability to release superoxide and act as tumoristatic and tumoricidal effector cells. Superoxide generation was assayed by the reduction of ferricytochrome C. All three compounds failed to induce significant O/sub 2/- release, unless the cells were also treated with phorbol myristate acetate (PMA). MDP was most active in potentiating the PMA response. In the tumor growth inhibition assay, cytostatic activity was comparable for all three compounds and did not exceed 32 percent. The combination of subthreshold levels of these compounds and hybridoma-derived MAF acted synergistically to induce potent cytostatic activity. In the chromium release assay, LPS and poly I.poly C rendered macrophages cytolytic for P815 target cells at concentrations greater than or equal to 1 microgram/ml. In contrast, significant cytolysis was observed with MDP only at 100 micrograms/ml. Defining precisely the effect of various biological response modifiers on several parameters of macrophage function may facilitate use of these agents in cancer therapy.

  14. Differential regulation of acid sphingomyelinase in macrophages stimulated with oxidized low-density lipoprotein (LDL) and oxidized LDL immune complexes: role in phagocytosis and cytokine release.

    Science.gov (United States)

    Truman, Jean-Philip; Al Gadban, Mohammed M; Smith, Kent J; Jenkins, Russell W; Mayroo, Nalini; Virella, Gabriel; Lopes-Virella, Maria F; Bielawska, Alicja; Hannun, Yusuf A; Hammad, Samar M

    2012-05-01

    Oxidized low-density lipoprotein (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to the formation of lipid-laden macrophages (foam cells). Fcγ receptors mediate uptake of oxLDL-IC, whereas scavenger receptors internalize oxLDL. We have previously reported that oxLDL-IC, but not free oxLDL, activate macrophages and prolong their survival. Sphingomyelin is a major constituent of cell membranes and lipoprotein particles and acid sphingomyelinase (ASMase) hydrolyses sphingomyelin to generate the bioactive lipid ceramide. ASMase exists in two forms: lysosomal (L-ASMase) and secretory (S-ASMase). In this study we examined whether oxLDL and oxLDL-IC regulate ASMase differently, and whether ASMase mediates monocyte/macrophage activation and cytokine release. The oxLDL-IC, but not oxLDL, induced early and consistent release of catalytically active S-ASMase. The oxLDL-IC also consistently stimulated L-ASMase activity, whereas oxLDL induced a rapid transient increase in L-ASMase activity before it steadily declined below baseline. Prolonged exposure to oxLDL increased L-ASMase activity; however, activity remained significantly lower than that induced by oxLDL-IC. Further studies were aimed at defining the function of the activated ASMase. In response to oxLDL-IC, heat-shock protein 70B' (HSP70B') was up-regulated and localized with redistributed ASMase in the endosomal compartment outside the lysosome. Treatment with oxLDL-IC induced the formation and release of HSP70-containing and IL-1β-containing exosomes via an ASMase-dependent mechanism. Taken together, the results suggest that oxLDL and oxLDL-IC differentially regulate ASMase activity, and the pro-inflammatory responses to oxLDL-IC are mediated by prolonged activation of ASMase. These findings may contribute to increased understanding of mechanisms mediating macrophage involvement in atherosclerosis.

  15. LPS Catch and Effort Estimation

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Data collected from the LPS dockside (LPIS) and the LPS telephone (LPTS) surveys are combined to produce estimates of total recreational catch, landings, and fishing...

  16. Transfer of cholesterol from macrophages to lymphocytes in culture.

    Science.gov (United States)

    de Bittencourt Júnior, P I; Curi, R

    1998-02-01

    A major feature of macrophage metabolism is its capacity to produce and export cholesterol. Several reports have shown that the manipulation of lymphocyte cholesterol content elicits important changes in lymphocyte proliferation. These findings lead to an inquiry as to whether macrophage-derived cholesterol released into the lymphocyte surroundings may be transferred to the latter thus affecting lymphocyte function. In this study, cholesterol transfer from macrophages to lymphocytes was examined in vitro using rat cells in culture. The findings indicate that there may be a significant transfer of cholesterol from [4-14C]cholesterol labeled resident peritoneal macrophages to mesenteric lymph node resting lymphocytes (up to 173.9 +/- 2.7 pmol/10(7) lymphocytes/10(7) macrophages when co-cultivated for 48 h), in a lipoprotein-dependent manner. This represents the mass transfer of ca. 17 nmoles of cholesterol molecules per 10(7) lymphocytes from 10(7) macrophages (calculated on the basis of specific radioactivity incorporated into macrophages after the pre-labelling period), which suggests that macrophages are capable of replacing the whole lymphocyte cholesterol pool every 21 h. Moreover, an 111%-increase in the total cholesterol content of lymphocytes was found after co-cultivation with macrophages for 48 h. When compared to peritoneal cells, monocytes/macrophages obtained from circulating blood leukocytes presented a much higher cholesterol transfer capacity to lymphocytes (3.06 +/- 0.10 nmol/10(7) lymphocytes/10(7) macrophages co-cultivated for 24 h). Interestingly, inflammatory macrophages dramatically reduced their cholesterol transfer ability (by up to 91%, as compared to resident macrophages). Cholesterol transfer may involve a humoral influence, since it is not only observed when cells are co-cultivated in a single-well chamber system (cells in direct contact), but also in a two-compartment system (where cells can communicate but not by direct contact). Co

  17. VISUALIZATION AND ANALYSIS OF LPS DISTRIBUTION IN BINARY PHOSPHOLIPID BILAYERS

    Science.gov (United States)

    Florencia, Henning María; Susana, Sanchez; Laura, Bakás

    2010-01-01

    Lipopolysaccharide (LPS) is an endotoxin released from the outer membrane of Gram negative bacteria during infections. It have been reported that LPS may play a rol in the outer membrane of bacteria similar to that of cholesterol in eukaryotic plasma membranes. In this article we compare the effect of introducing LPS or cholesterol in liposomes made of dipalmitoylphosphatidylcholine/dioleoylphosphatidylcholine on the solubilization process by Triton X-100. The results show that liposomes containing LPS or Cholesterol are more resistant to solubilization by Triton X-100 than the binary phospholipid mixtures at 4°C. The LPS distribution was analyzed on GUVs of DPPC:DOPC using FITC-LPS. Solid and liquid-crystalline domains were visualized labeling the GUVs with LAURDAN and GP images were acquired using a two-photon microscope. The images show a selective distribution of LPS in gel domains. Our results support the hypothesis that LPS could aggregate and concentrate selectively in biological membranes providing a mechanism to bring together several components of the LPS-sensing machinery. PMID:19324006

  18. A designed TLR4/MD-2 complex to capture LPS.

    Science.gov (United States)

    Brandl, Katharina; Glück, Thomas; Hartmann, Pia; Salzberger, Bernd; Falk, Werner

    2005-01-01

    The family of Toll-like receptors (TLRs) is involved in the defense of an organism to microbial attack. TLR4-induced signaling is involved in infectious diseases, chronic inflammatory diseases and sepsis; therefore, we aimed at modulating TLR4-signaling via ligand-binding soluble receptors. Because recognition of microbial structures shows some species-specific traits, we specifically selected the mouse model for later in vivo studies. We first prepared the N-terminally Flag-tagged mouse (m) recombinant (r) soluble (s) fusion proteins mrsTLR4-IgGFc (T4Fc) and mrsMD-2 in Drosophila melanogaster Schneider 2 (S2) cells. The function of these molecules was tested by inhibition of synthesis of pro-inflammatory cytokines after stimulation of mouse macrophage RAW 264.7 cells with purified lipopolysaccharide (LPS). T4Fc alone had no inhibitory activity; however, a T4Fc/MD-2 complex blocked LPS activity. By analogy with 'cytokine traps', we then prepared a designer molecule (LPS-Trap) by fusing MD-2 to the C-terminus of soluble TLR4 via a flexible linker. LPS-Trap significantly inhibited TNF production by LPS-stimulated RAW 264.7 cells. Thus, the T4Fc/MD-2 complex as well as the LPS-Trap blocked LPS activity in vitro and might thus represent a new therapeutic option in sepsis by neutralization of TLR4-activating ligands.

  19. An alpha-helical cationic antimicrobial peptide selectively modulates macrophage responses to lipopolysaccharide and directly alters macrophage gene expression.

    Science.gov (United States)

    Scott, M G; Rosenberger, C M; Gold, M R; Finlay, B B; Hancock, R E

    2000-09-15

    Certain cationic antimicrobial peptides block the binding of LPS to LPS-binding protein and reduce the ability of LPS to induce the production of inflammatory mediators by macrophages. To gain a more complete understanding of how LPS activates macrophages and how cationic peptides influence this process, we have used gene array technology to profile gene expression patterns in macrophages treated with LPS in the presence or the absence of the insect-derived cationic antimicrobial peptide CEMA (cecropin-melittin hybrid). We found that CEMA selectively blocked LPS-induced gene expression in the RAW 264.7 macrophage cell line. The ability of LPS to induce the expression of >40 genes was strongly inhibited by CEMA, while LPS-induced expression of another 16 genes was relatively unaffected. In addition, CEMA itself induced the expression of a distinct set of 35 genes, including genes involved in cell adhesion and apoptosis. Thus, CEMA, a synthetic alpha-helical peptide, selectively modulates the transcriptional response of macrophages to LPS and can alter gene expression in macrophages.

  20. Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

    Science.gov (United States)

    Alvarado-Vazquez, Perla Abigail; Bernal, Laura; Paige, Candler A; Grosick, Rachel L; Moracho Vilrriales, Carolina; Ferreira, David Wilson; Ulecia-Morón, Cristina; Romero-Sandoval, E Alfonso

    2017-08-01

    M1 macrophages release proinflammatory factors during inflammation. They transit to an M2 phenotype and release anti-inflammatory factors to resolve inflammation. An imbalance in the transition from M1 to M2 phenotype in macrophages contributes to the development of persistent inflammation. CD163, a member of the scavenger receptor cysteine-rich family, is an M2 macrophage marker. The functional role of CD163 during the resolution of inflammation is not completely known. We postulate that CD163 contributes to the transition from M1 to M2 phenotype in macrophages. We induced CD163 gene in THP-1 and primary human macrophages using polyethylenimine nanoparticles grafted with a mannose ligand (Man-PEI). This nanoparticle specifically targets cells of monocytic origin via mannose receptors. Cells were challenged with a single or a double stimulation of lipopolysaccharide (LPS). A CD163 or empty plasmid was complexed with Man-PEI nanoparticles for cell transfections. Quantitative RT-PCR, immunocytochemistry, and ELISAs were used for molecular assessments. CD163-overexpressing macrophages displayed reduced levels of tumor necrosis factor-alpha (TNF)-α and monocytes chemoattractant protein (MCP)-1 after a single stimulation with LPS. Following a double stimulation paradigm, CD163-overexpressing macrophages showed an increase of interleukin (IL)-10 and IL-1ra and a reduction of MCP-1. This anti-inflammatory phenotype was partially blocked by an anti-CD163 antibody (effects on IL-10 and IL-1ra). A decrease in the release of TNF-α, IL-1β, and IL-6 was observed in CD163-overexpressing human primary macrophages. The release of IL-6 was blocked by an anti-CD163 antibody in the CD163-overexpressing group. Our data show that the induction of the CD163 gene in human macrophages under inflammatory conditions produces changes in cytokine secretion in favor of an anti-inflammatory phenotype. Targeting macrophages to induce CD163 using cell-directed nanotechnology is an attractive

  1. 蓝玉簪颗粒抑制脂多糖诱导大鼠肺泡巨噬细胞TNF-α产生及相关机制研究%Gentiana veitchiorum particles inhibited LPS induced pulmonary alveolar macrophages(AM)TNF-α production and the underlying mechanism

    Institute of Scientific and Technical Information of China (English)

    侯颖; 曹蔚; 李涛; 刘水冰; 张晓楠; 李旭波; 田琼; 尤福生

    2011-01-01

    AIM: To investigate the effect of Gentiana veitchiorum particles on the expression of TNF-α in pulmonary alveolar macrophages (AM) which induced by LPS, to explain the mechanism about anti-inflammatory action of Gentiana veitchiorum particles. METHODS: Purification of rat AM, TNF-α level in AM culture supematant was detected by ELISA. Western blot method for detecting the expression of TNF-α and pERK in the AM. While application of ERK antagonist (PD98059) in rat AM and the expression of TNFα was observed by Western blot. RESULTS: Gentiana veitchiorum particles can reduce the LPS induced AM TNF-α increase in dose dependent manner. Gentiana veitchiorum particles (100 mg/L) can significantly reduce the LPS induc ed pERK and TNF-α protein expression in AM. compared with LPS stimulation group, we found that ERK inhibitor ( PD98059 30 mol/L), Gentiana veitchiorum particles intervention and Gentiana veitchiorum particles + PD98059 groups' TNF-α expression were significantly reduced in rat AM. CONCLUSION: Gentiana veitchiorum particles can inhibit the LPS induced pulmonary AM TNF-α expression, one of the possible mechanism is to inhibit the extracailular signal transduction pathway.%目的:探讨蓝玉簪颗粒对脂多糖(LPS)诱导大鼠肺泡巨噬细胞(AM)内TNF-α表达及可能作用机制.方法:分离纯化AM,应用ELISA法检测蓝玉簪颗粒对LPS诱导的大鼠AM培养上清中的TNF-α水平的影响,应用Western blot方法检测大鼠AM内TNF-α及pERK蛋白表达水平,同时应用ERK拮抗剂(PD98059)观察AM内TNF-α蛋白表达.结果:蓝玉簪颗粒可剂量依赖的降低由于LPS刺激导致的AM培养上清内TNF-α含量升高;蓝玉簪(100 mg/L)颗粒可显著降低由于LPS刺激导致的AM细胞内pERK及TNF-α蛋白表达升高;ERK特异性抑制剂(PD98059 30 mol/L)及蓝玉簪颗粒干预,蓝玉簪颗粒+PD98059干预后,我们发现与LPS刺激组相比,大鼠AM中TNF-α表达显著降低.结论:蓝玉

  2. Kaurane diterpenes protect against apoptosis and inhibition of phagocytosis in activated macrophages.

    Science.gov (United States)

    de las Heras, B; Hortelano, S; Girón, N; Bermejo, P; Rodríguez, B; Boscá, L

    2007-09-01

    The kaurane diterpenes foliol and linearol are inhibitors of the activation of nuclear factor kappaB, a transcription factor involved in the inflammatory response. Effects of these diterpenes on apoptosis and phagocytosis have been analysed in cultured peritoneal macrophages and in the mouse macrophage cell line, RAW 264.7. Macrophages were maintained in culture and activated with pro-inflammatory stimuli in the absence or presence of diterpenes. Apoptosis and the phagocytosis in these cells under these conditions were determined. Incubation of macrophages with a mixture of bacterial lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) induced apoptosis through a NO-dependent pathway, an effect significantly inhibited by foliol and linearol in the low muM range, without cytotoxic effects. Apoptosis in macrophages induced by NO donors was also inhibited. The diterpenes prevented apoptosis through a mechanism compatible with the inhibition of caspase-3 activation, release of cytochrome c to the cytosol and p53 overexpression, as well as an alteration in the levels of proteins of the Bcl-2 family, in particular, the levels of Bax. Cleavage of poly(ADP-ribose) polymerase, a well-established caspase substrate, was reduced by these diterpenes. Treatment of cells with foliol and linearol decreased phagocytosis of zymosan bioparticles by RAW 264.7 cells and to a greater extent by peritoneal macrophages. Both diterpenes protected macrophages from apoptosis and inhibited phagocytosis, resulting in a paradoxical control of macrophage function, as viability was prolonged but inflammatory and phagocytic functions were impaired.

  3. Effect of PAR-2 Deficiency in Mice on KC Expression after Intratracheal LPS Administration

    Directory of Open Access Journals (Sweden)

    Julie C. Williams

    2011-01-01

    Full Text Available Protease activated receptors (PAR have been shown to play a role in inflammation. PAR-2 is expressed by numerous cells in the lung and has either proinflammatory, anti-inflammatory, or no effect depending on the model. Here, we examined the role of PAR-2 in a model of LPS-induced lung inflammation. We found that PAR-2-deficient mice had significantly less KC expression in bronchial lavage fluid compared with wild-type mice but there was no difference in MIP-2 or TNF-α expression. We also found that isolated alveolar and resident peritoneal macrophages lacking PAR-2 showed a similar deficit in KC after LPS stimulation without differences in MIP-2 or TNF-α. Infiltration of neutrophils and macrophages into the lung following LPS administration was not affected by an absence of PAR-2. Our results support the notion that PAR-2 plays a role in LPS activation of TLR4 signaling in macrophages.

  4. Tyrosol exhibits negative regulatory effects on LPS response and endotoxemia.

    Science.gov (United States)

    Lu, Jing; Huang, Guoren; Wang, Zhenning; Zhuang, Shuang; Xu, Linli; Song, Bocui; Xiong, Ying; Guan, Shuang

    2013-12-01

    Tyrosol, a phenolic compound, was isolated from wine, olive oil and other plant-derived products. In the present study, we first investigated the negative regulatory effects of tyrosol on cytokine production by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in vitro, and the results showed that tyrosol reduced tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) secretion. This inspired us to further study the effects of tyrosol in vivo. Tyrosol significantly attenuated TNF-α, IL-1β and IL-6 production in serum from mice challenged with LPS, and consistent with the results in vitro. In the murine model of endotoxemia, mice were treated with tyrosol prior to or after LPS challenge. The results showed that tyrosol significantly increased mice survival. We further investigated signal transduction ways to determine how tyrosol works. The data revealed that tyrosol shocked LPS-induced mitogen activated protein kinases (MAPKs) and nuclear transcription factor-κB (NF-κB) signal transduction pathways in RAW 264.7 macrophages. These observations indicated that tyrosol exerted negative regulatory effects on LPS response in vitro and in vivo through suppressing NF-κB and p38/ERK MAPK signaling pathways.

  5. Anti-inflammatory effects of ethanolic extract from Sargassum horneri (Turner) C. Agardh on lipopolysaccharide-stimulated macrophage activation via NF-κB pathway regulation.

    Science.gov (United States)

    Kim, Mi Eun; Jung, Yun Chan; Jung, Inae; Lee, Hee-Woo; Youn, Hwa-Young; Lee, Jun Sik

    2015-01-01

    Inflammation is major symptom of the innate immune response by infection of microbes. Macrophages, one of immune response related cells, play a role in inflammatory response. Recent studies reported that various natural products can regulate the activation of immune cells such as macrophage. Sargassum horneri (Turner) C. Agardh is one of brown algae. Recently, various seaweeds including brown algae have antioxidant and anti-inflammatory effects. However, anti-inflammatory effects of Sargassum horneri (Turner) C. Agardh are still unknown. In this study, we investigated anti-inflammatory effects of ethanolic extract of Sargassum horneri (Turner) C. Agardh (ESH) on RAW 264.7 murine macrophage cell line. The ESH was extracted from dried Sargassum horneri (Turner) C. Agardh with 70% ethanol and then lyophilized at -40 °C. ESH was not cytotoxic to RAW 264.7, and nitric oxide (NO) production induced by LPS-stimulated macrophage activation was significantly decreased by the addition of 200 μg/mL of ESH. Moreover, ESH treatment reduced mRNA level of cytokines, including IL-1β, and pro-inflammatory genes such as iNOS and COX-2 in LPS-stimulated macrophage activation in a dose-dependent manner. ESH was found to elicit anti-inflammatory effects by inhibiting ERK, p-p38 and NF-κB phosphorylation. In addition, ESH inhibited the release of IL-1β in LPS-stimulated macrophages. These results suggest that ESH elicits anti-inflammatory effects on LPS-stimulated macrophage activation via the inhibition of ERK, p-p38, NF-κB, and pro-inflammatory gene expression.

  6. The chemerin/ChemR23 system does not affect the pro-inflammatory response of mouse and human macrophages ex vivo.

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    Benjamin Bondue

    Full Text Available Macrophages constitute a major component of innate immunity and play an essential role in defense mechanisms against external aggressions and in inflammatory responses. Chemerin, a chemoattractant protein, is generated in inflammatory conditions, and recruits cells expressing the G protein-coupled receptor ChemR23, including macrophages. Chemerin was initially expected to behave as a pro-inflammatory agent. However, recent data described more complex activities that are either pro- or anti-inflammatory, according to the disease model investigated. In the present study, peritoneal macrophages were generated from WT or ChemR23(-/- mice, stimulated with lipopolyssaccharide in combination or not with IFN-γ and the production of pro- (TNF-α, IL-1β and IL-6 and anti-inflammatory (IL-10 cytokines was evaluated using qRT-PCR and ELISA. Human macrophages generated from peripheral blood monocytes were also tested in parallel. Peritoneal macrophages from WT mice, recruited by thioglycolate or polyacrylamide beads, functionally expressed ChemR23, as assessed by flow cytometry, binding and chemotaxis assays. However, chemerin had no effect on the strong upregulation of cytokine release by these cells upon stimulation by LPS or LPS/IFN-γ, whatever the concentration tested. Similar data were obtained with human macrophages. In conclusion, our results rule out the direct anti-inflammatory effect of chemerin on macrophages ex vivo, described previously in the literature, despite the expression of a functional ChemR23 receptor in these cells.

  7. The chemerin/ChemR23 system does not affect the pro-inflammatory response of mouse and human macrophages ex vivo.

    Science.gov (United States)

    Bondue, Benjamin; De Henau, Olivier; Luangsay, Souphalone; Devosse, Thalie; de Nadaï, Patricia; Springael, Jean-Yves; Parmentier, Marc; Vosters, Olivier

    2012-01-01

    Macrophages constitute a major component of innate immunity and play an essential role in defense mechanisms against external aggressions and in inflammatory responses. Chemerin, a chemoattractant protein, is generated in inflammatory conditions, and recruits cells expressing the G protein-coupled receptor ChemR23, including macrophages. Chemerin was initially expected to behave as a pro-inflammatory agent. However, recent data described more complex activities that are either pro- or anti-inflammatory, according to the disease model investigated. In the present study, peritoneal macrophages were generated from WT or ChemR23(-/-) mice, stimulated with lipopolyssaccharide in combination or not with IFN-γ and the production of pro- (TNF-α, IL-1β and IL-6) and anti-inflammatory (IL-10) cytokines was evaluated using qRT-PCR and ELISA. Human macrophages generated from peripheral blood monocytes were also tested in parallel. Peritoneal macrophages from WT mice, recruited by thioglycolate or polyacrylamide beads, functionally expressed ChemR23, as assessed by flow cytometry, binding and chemotaxis assays. However, chemerin had no effect on the strong upregulation of cytokine release by these cells upon stimulation by LPS or LPS/IFN-γ, whatever the concentration tested. Similar data were obtained with human macrophages. In conclusion, our results rule out the direct anti-inflammatory effect of chemerin on macrophages ex vivo, described previously in the literature, despite the expression of a functional ChemR23 receptor in these cells.

  8. Effects of quartz, airborne particulates and fly ash fractions from a waste incinerator on elastase release by activated and nonactivated rabbit alveolar macrophages.

    Science.gov (United States)

    Gulyas, H; Labedzka, M; Schmidt, N; Gercken, G

    1988-01-01

    Elastase release from cultured, activated and nonactivated rabbit alveolar macrophages (AM) was investigated after stimulation by different environmentally related mineral dusts (50-1000 micrograms/10(6) cells). Eight different dusts were analyzed for element contents and grain size: one rural and three urban airborne dusts, a coarse and a fine fraction of a sieved waste incinerator fly ash, a sonicated coarse fly ash fraction, and the standard quartz dust DQ 12. The fine fly ash fraction, the sonicated coarse fly ash fraction, and the quartz dust DQ 12 enhanced elastase release by activated AM. Only one of the tested airborne dusts effected a comparable elastase release. The untreated coarse fraction of the fly ash did not cause a significant increase of extracellular elastase activities. Elastase release was dependent on particle numbers and chemical composition and correlated best with barium and tin contents. Nonactivated AM released higher elastase activities than activated AM at low-dose levels. The possible role of dust-induced elastase secretion in the pathogenesis of emphysema is discussed.

  9. Nanoformulated antiretroviral drug combinations extend drug release and antiretroviral responses in HIV-1-infected macrophages: implications for neuroAIDS therapeutics.

    Science.gov (United States)

    Nowacek, Ari S; McMillan, JoEllyn; Miller, Reagan; Anderson, Alec; Rabinow, Barrett; Gendelman, Howard E

    2010-12-01

    We posit that improvements in pharmacokinetics and biodistributions of antiretroviral therapies (ART) for human immunodeficiency virus type one-infected people can be achieved through nanoformulationed drug delivery systems. To this end, we manufactured nanoparticles of atazanavir, efavirenz, and ritonavir (termed nanoART) and treated human monocyte-derived macrophages (MDM) in combination therapies to assess antiretroviral responses. This resulted in improved drug uptake, release, and antiretroviral efficacy over monotherapy. MDM rapidly, within minutes, ingested nanoART combinations, at equal or similar rates, as individual formulations. Combination nanoART ingested by MDM facilitated individual drug release from 15 to >20 days. These findings are noteworthy as a nanoART cell-mediated drug delivery provides a means to deliver therapeutics to viral sanctuaries, such as the central nervous system during progressive human immunodeficiency virus type one infection. The work brings us yet another step closer to realizing the utility of nanoART for virus-infected people.

  10. Alveolar macrophage-epithelial cell interaction following exposure to atmospheric particles induces the release of mediators involved in monocyte mobilization and recruitment

    Directory of Open Access Journals (Sweden)

    Mukae Hiroshi

    2005-08-01

    Full Text Available Abstract Background Studies from our laboratory have shown that human alveolar macrophages (AM and bronchial epithelial cells (HBEC exposed to ambient particles (PM10 in vitro increase their production of inflammatory mediators and that supernatants from PM10-exposed cells shorten the transit time of monocytes through the bone marrow and promote their release into the circulation. Methods The present study concerns co-culture of AM and HBEC exposed to PM10 (EHC-93 and the production of mediators involved in monocyte kinetics measured at both the mRNA and protein levels. The experiments were also designed to determine the role of the adhesive interaction between these cells via the intercellular adhesion molecule (ICAM-1 in the production of these mediators. Results AM/HBEC co-cultures exposed to 100 μg/ml of PM10 for 2 or 24 h increased their levels of granulocyte-macrophage colony-stimulating factor (GM-CSF, M-CSF, macrophage inflammatory protein (MIP-1β, monocyte chemotactic protein (MCP-1, interleukin (IL-6 and ICAM-1 mRNA, compared to exposed AM or HBEC mono-cultures, or control non-exposed co-cultures. The levels of GM-CSF, M-CSF, MIP-1β and IL-6 increased in co-cultured supernatants collected after 24 h exposure compared to control cells (p 10-induced increase in co-culture mRNA expression. Conclusion We conclude that an ICAM-1 independent interaction between AM and HBEC, lung cells that process inhaled particles, increases the production and release of mediators that enhance bone marrow turnover of monocytes and their recruitment into tissues. We speculate that this interaction amplifies PM10-induced lung inflammation and contributes to both the pulmonary and systemic morbidity associated with exposure to air pollution.

  11. Cathelicidin LL-37 induces time-resolved release of LTB4 and TXA2 by human macrophages and triggers eicosanoid generation in vivo.

    Science.gov (United States)

    Wan, Min; Soehnlein, Oliver; Tang, Xiao; van der Does, Anne M; Smedler, Erik; Uhlén, Per; Lindbom, Lennart; Agerberth, Birgitta; Haeggström, Jesper Z

    2014-08-01

    In humans, LL-37 and eicosanoids are important mediators of inflammation and immune responses. Here we report that LL-37 promotes leukotriene B4 (LTB4) and thromboxane A2 (TXA2) generation by human monocyte-derived macrophages (HMDMs). LL-37 evokes calcium mobilization apparently via the P2X7 receptor (P2X7R), activation of ERK1/2 and p38 MAPKs, as well as cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase in HMDMs, leading to an early (1 h) release of LTB4. Similarly, TXA2 production at an early time involved the same signaling sequence along an LL-37-P2X7R-cPLA2-cyclooxygenase-1 (COX-1) axis. However, at later (6-8 h) time points, internalized LL-37 up-regulates COX-2 expression, promoting TXA2 production. Furthermore, intraperitoneal injection of mice with murine cathelicidin-related antimicrobial peptide (mCRAMP) induces significantly higher levels of LTB4 and TXA2 in mouse ascites rich in macrophages. Conversely, cathelicidin-deficient (Cnlp(-/-)) mice produce much less LTB4 and TXB2 in vivo in response to TNF-α compared with control mice. We conclude that LL-37 elicits a biphasic release of eicosanoids in macrophages with early, Ca(2+)-dependent formation of LTB4 and TXA2 followed by a late peak of TXA2, generated via induction of COX-2 by internalized LL-37, thus allowing eicosanoid production in a temporally controlled manner. Moreover, our findings provide evidence that LL-37 is an endogenous regulator of eicosanoid-dependent inflammatory responses in vivo.

  12. Glutamine Modulates Macrophage Lipotoxicity

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    Li He

    2016-04-01

    Full Text Available Obesity and diabetes are associated with excessive inflammation and impaired wound healing. Increasing evidence suggests that macrophage dysfunction is responsible for these inflammatory defects. In the setting of excess nutrients, particularly dietary saturated fatty acids (SFAs, activated macrophages develop lysosome dysfunction, which triggers activation of the NLRP3 inflammasome and cell death. The molecular pathways that connect lipid stress to lysosome pathology are not well understood, but may represent a viable target for therapy. Glutamine uptake is increased in activated macrophages leading us to hypothesize that in the context of excess lipids glutamine metabolism could overwhelm the mitochondria and promote the accumulation of toxic metabolites. To investigate this question we assessed macrophage lipotoxicity in the absence of glutamine using LPS-activated peritoneal macrophages exposed to the SFA palmitate. We found that glutamine deficiency reduced lipid induced lysosome dysfunction, inflammasome activation, and cell death. Under glutamine deficient conditions mTOR activation was decreased and autophagy was enhanced; however, autophagy was dispensable for the rescue phenotype. Rather, glutamine deficiency prevented the suppressive effect of the SFA palmitate on mitochondrial respiration and this phenotype was associated with protection from macrophage cell death. Together, these findings reveal that crosstalk between activation-induced metabolic reprogramming and the nutrient microenvironment can dramatically alter macrophage responses to inflammatory stimuli.

  13. 绵羊Toll样受体家庭在肺泡巨噬细胞的分布及脂多糖(LPS)刺激对TLR2、TLR4表达的影响%Distribution of Toll-like Receptors(TLRs) and TLR2,TLR4 Expression During LPS Stimulus Sheep Alveolar Macrophage

    Institute of Scientific and Technical Information of China (English)

    盛金良; 陈创夫; 杨霞; 王远志; 张辉

    2010-01-01

    为确定toll样受体家族在绵羊肺泡巨噬细胞的分布及脂多糖(LPS)刺激过程中TLR2、4的动态表达规律.通过RT-PCR方法检测绵羊TLR1-TLR10表达分布;以α-tubulin和GAPDH为内标基因,对绵羊TLR2、4进行克隆和测序,将重组质粒作为标准品建立实时定量PCR标准曲线,通过实时定量PCR方法检测LPS诱导0~48 h TLR2、4的动态表达.结果表明,在绵羊肺泡巨噬细胞中检测到TLR1、2、4、6、7、8、9、10的表达,而TLR3、5未见表达;α-tubulin和GAPDH在LPS诱导前后表达量存在显著差异,均不适合作为内标基因,以使用的细胞数和总RNA量进行均一化校正,发现TLR2、4在诱导后20 min就达到高峰,且在整个诱导过程中维持较高水平.绵羊肺泡巨噬细胞TLR2、4对LPS刺激的应答反应很灵敏,参与机体的早期应答反应.

  14. Vibrio cholerae porin OmpU induces LPS tolerance by attenuating TLR-mediated signaling.

    Science.gov (United States)

    Sakharwade, Sanica C; Mukhopadhaya, Arunika

    2015-12-01

    Porins can act as pathogen-associated molecular patterns, can be recognized by the host immune system and modulate immune responses. Vibrio choleraeporin OmpU aids in bacterial survival in the human gut by increasing resistance against bile acids and anti-microbial peptides. V. choleraeOmpU is pro-inflammatory in nature. However, interestingly, it can also down-regulate LPS-mediated pro-inflammatory responses. In this study, we have explored how OmpU-pretreatment affects LPS-mediated responses. Our study indicates that OmpU-pretreatment followed by LPS-activation does not induce M2-polarization of macrophages/monocytes. Further, OmpU attenuates LPS-mediated TLR2/TLR6 signaling by decreasing the association of TLRs along with recruitment of MyD88 and IRAKs to the receptor complex. This results in decreased translocation of NFκB in the nucleus. Additionally, OmpU-pretreatment up-regulates expression of IRAK-M, a negative regulator of TLR signaling, in RAW 264.7 mouse macrophage cells upon LPS-stimulation. Suppressor cytokine IL-10 is partially involved in OmpU-induced down-regulation of LPS-mediated TNFα production in human PBMCs. Furthermore, OmpU-pretreatment also affects macrophage function, by enhancing phagocytosis in LPS-treated RAW 264.7 cells, and down-regulates LPS-induced cell surface expression of co-stimulatory molecules. Altogether, OmpU causes suppression of LPS-mediated responses by attenuating the LPS-mediated TLR signaling pathway.

  15. Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization.

    Science.gov (United States)

    Liu, Kun; Zhao, Enpeng; Ilyas, Ghulam; Lalazar, Gadi; Lin, Yu; Haseeb, Muhammad; Tanaka, Kathryn E; Czaja, Mark J

    2015-01-01

    Recent evidence that excessive lipid accumulation can decrease cellular levels of autophagy and that autophagy regulates immune responsiveness suggested that impaired macrophage autophagy may promote the increased innate immune activation that underlies obesity. Primary bone marrow-derived macrophages (BMDM) and peritoneal macrophages from high-fat diet (HFD)-fed mice had decreased levels of autophagic flux indicating a generalized impairment of macrophage autophagy in obese mice. To assess the effects of decreased macrophage autophagy on inflammation, mice with a Lyz2-Cre-mediated knockout of Atg5 in macrophages were fed a HFD and treated with low-dose lipopolysaccharide (LPS). Knockout mice developed systemic and hepatic inflammation with HFD feeding and LPS. This effect was liver specific as knockout mice did not have increased adipose tissue inflammation. The mechanism by which the loss of autophagy promoted inflammation was through the regulation of macrophage polarization. BMDM and Kupffer cells from knockout mice exhibited abnormalities in polarization with both increased proinflammatory M1 and decreased anti-inflammatory M2 polarization as determined by measures of genes and proteins. The heightened hepatic inflammatory response in HFD-fed, LPS-treated knockout mice led to liver injury without affecting steatosis. These findings demonstrate that autophagy has a critical regulatory function in macrophage polarization that downregulates inflammation. Defects in macrophage autophagy may underlie inflammatory disease states such as the decrease in macrophage autophagy with obesity that leads to hepatic inflammation and the progression to liver injury.

  16. DMPD: Regulation of arachidonic acid release and cytosolic phospholipase A2activation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 10080535 Regulation of arachidonic acid release and cytosolic phospholipase A2activ...ation. Gijon MA, Leslie CC. J Leukoc Biol. 1999 Mar;65(3):330-6. (.png) (.svg) (.html) (.csml) Show Regulation... of arachidonic acid release and cytosolic phospholipase A2activation. PubmedID 10080535 Title Regulation

  17. Glutathione peroxidase-1 primes pro-inflammatory cytokine production after LPS challenge in vivo.

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    Steven Bozinovski

    Full Text Available Reactive oxygen species produced during the innate immune response to LPS are important agents of anti-pathogen defence but may also cause oxidative lung damage. Glutathione peroxidase-1 (gpx-1 is an anti-oxidant enzyme that may protect lungs from such damage. We assessed the in vivo importance of gpx-1 in LPS-induced lung inflammation. Male wild-type (WT or gpx-1 deficient (gpx-1(-/- mice were treated intranasally with PBS or 10 µg LPS and killed 3 and 24 h post LPS. Lungs were lavaged with PBS and then harvested for inflammatory marker expression. LPS caused an intense neutrophilia in WT BALF evident 3 and 24 h post challenge that was reduced in gpx-1(-/- mice. In addition, LPS-treated gpx-1(-/- mice had significantly fewer macrophages than LPS-treated WT mice. To understand the basis for this paradoxical reduction we assessed inflammatory cytokines and proteases at protein and transcript levels. MMP-9 expression and net gelatinase activity in BALF of gpx-1(-/- mice treated with LPS for 3 and 24 h was no different to that found in LPS-treated WT mice. BALF from LPS-treated gpx-1(-/- mice (3 h had less TNF-α, MIP-2 and GM-CSF protein than LPS-treated WT mice. In contrast, LPS-induced increases in TNF-α, MIP-2 and GM-CSF mRNA expression in WT mice were similar to those observed in gpx-1(-/- mice. These attenuated protein levels were unexpectedly not mirrored by reduced mRNA transcripts but were associated with increased 20S proteasome expression. Thus, these data suggest that gpx-1 primes pro-inflammatory cytokine production after LPS challenge in vivo.

  18. 青藤碱对LPS、IL-4诱导的小鼠RAW264.7巨噬细胞极化的影响%Effect of sinomenine on mouse RAW264.7 macrophage cells line polarization induced by LPS or IL-4

    Institute of Scientific and Technical Information of China (English)

    罗进芳; 朱瑞丽; 易浪; 董燕; 王培训

    2015-01-01

    Objective:To investigate sinomenine (Sinomenine,SIN) effect on RAW264.7 cells polarization to M1 or M2 phenotype induced by lipopolysaccharide (LPS) or interleukin-4 (IL-4) .Methods:RAW264.7 cells were induced to polarize to M1 by LPS ,and to M2 by IL-4.Sinomenine effects on LPS or IL-4 induced macrophages:TNF-αand IL-10 secretion induced by different condition were detected by Enzyme linked immunosorbent assay (ELISA);The expression level of mRNA of Arginase1(Arg-1),Nitric oxide synthase(iNOS),suppressor of cytokine signaling protein-2(SOCS2) and suppressor of cytokine signaling protein-3(SOCS3) of M1/M2 phenotypes were detected by real time PCR respectively.Results:Sinomenine inhibited the increase of TNF-αsecretion,iNOS and SOCS3 mRNA expression level induced by LPS.Sinomenine inhibited the increase of IL-10 secretion and Arg-1 mRNA expression level induced by IL-4,but SOCS2 mRNA expression level was not affected by Sinomenine.Conclusion: Sinomenine can inhibite the macrophage polarization to M1 and M2 induced by LPS and IL-4.Sinomenine plays a regulatory role on imbalance of M1/M2,and is conducive to maintain the dynamic balance.%目的:探讨青藤碱(Sinomenine,SIN)对脂多糖(LPS)以及白细胞介素4(IL-4)诱导的RAW264.7细胞向M1、M2型极化的影响。方法:以LPS刺激RAW264.7细胞诱导M1型极化,IL-4刺激RAW264.7细胞诱导M2型极化;青藤碱作用于LPS或IL-4诱导的巨噬细胞后:用酶联免疫法( ELISA)检测不同诱导状态下RAW264.7细胞TNF-α和IL-10的分泌量;荧光定量PCR检测与巨噬细胞极化相关的精氨酸酶-1(Arg-1)、一氧化氮合酶(iNOS)、细胞因子信号转导抑制蛋白-2(SOCS2)和细胞因子信号转导抑制蛋白-3(SOCS3)的mRNA表达水平。结果:青藤碱能抑制LPS诱导下细胞TNF-α的分泌量,抑制细胞iNOS和SOCS3的mRNA表达水平的升高。青藤碱能抑制IL-4诱导下细胞IL-10

  19. Effects of schisandrin on transcriptional factors in lipopolysaccharide-pretreated macrophages.

    Science.gov (United States)

    Guo, Lian Yu; Hung, Tran Manh; Bae, Kihwan; Jang, Sehyun; Shin, Eun Myoung; Chung, Ji Won; Kang, Sam Sik; Kim, Hyun Pyo; Kim, Yeong Shik

    2009-03-01

    Schisandrin is the main active ingredient isolated from Schisandra chinensis Baill. Recent studies have demonstrated that schisandrin exhibits anti-inflammatory effects in vivo and in vitro. In this study, we examined whether the order of lipopolysaccharide (LPS) treatment affects the mechanism of schisandrin anti-inflammatory activity. We found that the antiinflammatory mechanisms are not the same depending on whether macrophages were treated with schisandrin before or after LPS. The main difference is that inhibitor kappaBalpha (IkappaBalpha) degradation was not inhibited when macrophages were pretreated by LPS before schisandrin and was weakly inhibited when macrophages were pretreated by schisandrin before LPS.

  20. Prostaglandin E2, thromboxane B2, and leukotriene B4 release from peritoneal macrophages by different osmotic agents in nonuremic guinea pigs.

    Science.gov (United States)

    Hain, H; Jörres, A; Kögel, B; Mahiout, A; Gahl, G M; Kessel, M

    1988-01-01

    Interleukin-1 (Il-1), prostaglandins, and leukotrienes have been identified as inflammatory parameters in the setting of peritoneal dialysis. Recently, it was postulated that chronic overstimulation of peritoneal macrophages (PM) may result in fibrosis and loss of ultrafiltration. The aim of the present study was to investigate whether alternative osmotic agents (polyglucose, amino acids, glycerol, bicarbonate/glucose, gelatine, hydroxyethyl starch) provoke greater eicosanoid release by PMs than glucose. Fifty milliliters of sterile dialysate containing different osmotic agents were injected intraperitoneally into nonuremic guinea pigs. After 4 hours of dwell time, prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and leukotriene B4 (LTB4) production was analyzed in peritoneal effluents using specific radioimmunoassays (RIA) after liquid extraction. Cyclooxygenase products were generated with all osmotic agents: PGE2 concentrations ranged from 0.9 to 2.8 ng/4h, and TXB2 levels ranged from 39 to 49 ng/4h. In addition, the lipoxygenase product LTB4 was found in concentrations between 1.8 and 3.5 ng/4h. There were no significant differences in eicosanoid release among the osmotic agents. Thus, in this experimental setting, the capacity of PM to release inflammatory mediators did not correlate with the chemical composition of the dialysis solutions.

  1. Effect of UHMWPE particle size, dose, and endotoxin on in vitro macrophage response.

    Science.gov (United States)

    Alley, Carie; Haggard, Warren; Smith, Richard

    2014-01-01

    Ultra-high molecular weight polyethylene wear debris generated by a prosthetic hip or knee has been linked to osteolysis and the limited lifespan of the implant. However, research results are conflicting with regard to which characteristics of the polyethylene wear debris are most inflammatory. The goal of this study was to determine whether particle size, number, and the presence of endotoxin significantly contribute to increased secretion of pro-inflammatory mediators by macrophages in vitro in response to polyethylene wear debris generated by a hip simulator. The results show that the prevailing inflammatory factor is endotoxin. The macrophages released only minimal levels of TNF-α and IL-6 in response to cleaned polyethylene particles, but these cytokines were released in significantly higher amounts in response to particles spiked with lipopolysaccharide (LPS). The number (up to 500 particles per cell) and size of the particles tested in this study had no significant influence on any of the measured outputs (macrophage viability, TNF-α, IL-6, or PGE₂) unless associated with LPS.

  2. Berteroin Present in Cruciferous Vegetables Exerts Potent Anti-Inflammatory Properties in Murine Macrophages and Mouse Skin

    Directory of Open Access Journals (Sweden)

    Yoo Jin Jung

    2014-11-01

    Full Text Available Berteroin (5-methylthiopentyl isothiocyanate is a sulforaphane analog present in cruciferous vegetables, including Chinese cabbage, rucola salad leaves, and mustard oil. We examined whether berteroin exerts anti-inflammatory activities using lipopolysaccharide (LPS-stimulated Raw 264.7 macrophages and 12-O-tetradecanoylphorbol-13-acetate (TPA-induced mouse skin inflammation models. Berteroin decreased LPS-induced release of inflammatory mediators and pro-inflammatory cytokines in Raw 264.7 macrophages. Berteroin inhibited LPS-induced degradation of inhibitor of κBα (IκBα and nuclear factor-κB p65 translocation to the nucleus and DNA binding activity. Furthermore, berteroin suppressed degradation of IL-1 receptor-associated kinase and phosphorylation of transforming growth factor β activated kinase-1. Berteroin also inhibited LPS-induced phosphorylation of p38 MAPK, ERK1/2, and AKT. In the mouse ear, berteroin effectively suppressed TPA-induced edema formation and down-regulated iNOS and COX-2 expression as well as phosphorylation of AKT and ERK1/2. These results demonstrate that berteroin exhibits potent anti-inflammatory properties and suggest that berteroin can be developed as a skin anti-inflammatory agent.

  3. Study of Nitric Oxide production by murine peritoneal macrophages induced by Brucella Lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Kavoosi G

    2001-07-01

    Full Text Available Brueclla is a gram negative bacteria that causes Brucellosis. Lipopolysaccharide (LPS ", the pathogenic agent of Brucella is composed of O-chain, core oligosaccharide and lipid A. in addition, the structural and biological properties of different LPS extracted from different strains are not identical. The first defense system against LPS is nonspecific immunity that causes macrophage activation. Activated macrophages produce oxygen and nitrogen radicals that enhance the protection against intracellular pathogens.In this experiment LPS was extracted by hot phenol- water procedure and the effect of various LPSs on nitric oxide prodution by peritoneal mouse macrophages was examined.Our results demonstrated that the effect of LPS on nitric oxide production is concentration-dependent we observed the maximum response in concentration of 10-20 microgram per milliliter. Also our results demonstrate that LPS extracted from vaccine Brucella abortus (S 19 had a highe effect on nitric oxide production than the LPS from other strains

  4. LYATK1 potently inhibits LPS-mediated pro-inflammatory response

    Energy Technology Data Exchange (ETDEWEB)

    Xi, Feng [Department of Intensive Care Unit, Taixing People" ' s Hospital, Taixing, Jiangsu Province, 225400 (China); Liu, Yuan [Department of Ophthalmology, Nanjing First Hospital, Nanjing Medical University, Nanjing (China); Wang, Xiujuan; Kong, Wei [Department of Intensive Care Unit, Taixing People" ' s Hospital, Taixing, Jiangsu Province, 225400 (China); Zhao, Feng, E-mail: taixingzhaofeng163@163.com [Department of Intensive Care Unit, Taixing People" ' s Hospital, Taixing, Jiangsu Province, 225400 (China)

    2016-01-29

    Lipopolysaccharide (LPS)-primed monocytes/macrophages produce pro-inflammatory cytokines, which could lead to endotoxin shock. TGF-β-activated kinase1 (TAK1) activation is involved in the process. In the current study, we studied the potential effect of a selective TAK1 inhibitor, LYTAK1, on LPS-stimulated response both in vitro and in vivo. We demonstrated that LYTAK1 inhibited LPS-induced mRNA expression and production of several pro-inflammatory cytokines [interleukin 1β (IL-1β), tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6)] in RAW 264.7 macrophages. LYTAK1's activity was almost nullified with TAK1 shRNA-knockdown. Meanwhile, in both primary mouse bone marrow derived macrophages (BMDMs) and human peripheral blood mononuclear cells (PBMCs), LPS-induced pro-inflammatory cytokine production was again attenuated with LYTAK1 co-treatment. Molecularly, LYTAK1 dramatically inhibited LPS-induced TAK1-nuclear factor kappa B (NFκB) and mitogen-activated protein kinase (Erk, Jnk and p38) activation in RAW 264.7 cells, mouse BMDMs and human PBMCs. In vivo, oral administration of LYTAK1 inhibited LPS-induced activation of TAK1-NFκB-p38 in ex-vivo cultured PBMCs, and cytokine production and endotoxin shock in mice. Together, these results demonstrate that LYTAK1 inhibits LPS-induced production of several pro-inflammatory cytokines and endotoxin shock probably through blocking TAK1-regulated signalings. - Highlights: • LYTAK1 inhibits LPS-induced pro-inflammatory cytokine production in RAW 264.7 cells. • The effect by LYTAK1 is more potent than other known TAK1 inhibitors. • LYTAK1 inhibits LPS-induced cytokine production in primary macrophages/monocytes. • LYTAK1 inhibits LPS-induced TAK1-NFκB and MAPK activation in macrophages/monocytes. • LYTAK1 gavage inhibits LPS-induced endotoxin shock and cytokine production in mice.

  5. A new metabolomic assay to examine inflammation and redox pathways following LPS challenge

    Directory of Open Access Journals (Sweden)

    Suh Jung H

    2012-10-01

    Full Text Available Abstract Background Shifts in intracellular arginine (Arg and sulfur amino acid (SAA redox metabolism modulate macrophage activation, polarization and phenotype. Despite their importance in inflammation and redox regulatory pathways, comprehensive analysis of these metabolic networks was not previously possible with existing analytical methods. Methods The Arg/thiol redox LC-MS/MS metabolomics assay permits simultaneous assessment of amino acids and derivative products generated from Arg and SAA metabolism. Using this assay, LPS-induced changes in macrophage amino acid metabolism were monitored to identify pathway shifts during activation and their linkage to cellular redox regulation. Results Metabolite concentrations most significantly changed after treatment of a macrophage-like cell line (RAW with LPS for 24 hrs were citrulline (Cit (48-fold increase, ornithine (Orn (8.5-fold increase, arginine (Arg (66% decrease, and aspartic acid (Asp (73% decrease. The ratio Cit + Orn/Arg + Asp (CO/AA was more sensitive to LPS stimulation than other amino acid ratios commonly used to measure LPS-dependent inflammation (e.g., SAM/SAH, GSH/GSSG and total media NOx. The CO/AA ratio was also the first ratio to change significantly after LPS treatment (4 hrs. Changes in the overall metabolomic profile over time indicated that metabolic pathways shifted from Arg catabolism to thiol oxidation. Conclusions Simultaneous quantification of Arg and SAA metabolic pathway shifts following LPS challenge of macrophage indicate that, in this system, the Arg-Citrulline/NO cycle and arginase pathways are the amino acid metabolic pathways most sensitive to LPS-challenge. The cellular (Cit + Orn/(Arg + Asp ratio, which summarizes this pathway, was more responsive to lower concentrations of LPS and responded earlier than other metabolic biomarkers of macrophage activation including GSH redox. It is suggested that the CO/AA ratio is a redox- independent early biomarker of

  6. TARM1 Is a Novel Leukocyte Receptor Complex-Encoded ITAM Receptor That Costimulates Proinflammatory Cytokine Secretion by Macrophages and Neutrophils

    DEFF Research Database (Denmark)

    Radjabova, Valeria; Mastroeni, Piero; Skjødt, Karsten;

    2015-01-01

    , consistent with association with a signaling adaptor. TARM1 associated with the ITAM adaptor FcRγ but not with DAP10 or DAP12. In healthy mice, TARM1 is constitutively expressed on the cell surface of mature and immature CD11b(+)Gr-1(+) neutrophils within the bone marrow. Following i.p. LPS treatment...... in vitro. Ligation of TARM1 receptor in the presence of TLR ligands, such as LPS, enhanced the secretion of proinflammatory cytokines by macrophages and primary mouse neutrophils, whereas TARM1 stimulation alone had no effect. Finally, an immobilized TARM1-Fc fusion protein suppressed CD4(+) T cell...... activation and proliferation in vitro. These results suggest that a putative T cell ligand can interact with TARM1 receptor, resulting in bidirectional signaling and raising the T cell activation threshold while costimulating the release of proinflammatory cytokines by macrophages and neutrophils....

  7. Compound list: LPS [Open TG-GATEs

    Lifescience Database Archive (English)

    Full Text Available LPS LPS 00A07 ftp://ftp.biosciencedbc.jp/archive/open-tggates/LATEST/Human/in_vitro/LPS.Human.in_vitro....Liver.zip ftp://ftp.biosciencedbc.jp/archive/open-tggates/LATEST/Rat/in_vitro/LPS.Rat.in_vitro

  8. Neuroimmunological communication via CGRP promotes the development of a regulatory phenotype in TLR4-stimulated macrophages.

    Science.gov (United States)

    Baliu-Piqué, Mariona; Jusek, Gabriela; Holzmann, Bernhard

    2014-12-01

    Environmental signals shape the phenotype and function of activated macrophages. Here, we show that the neuropeptide calcitonin gene-related peptide (CGRP), which is released from sensory nerves, modulates the phenotype of TLR4-activated murine macrophages by enhancing expression of the regulatory macrophage markers IL-10, sphingosine kinase 1 (SPHK1), and LIGHT (lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes). In contrast, CGRP inhibits production of cytokines characteristic of inflammatory macrophages and does not affect expression of wound-healing macrophage markers upon TLR4 engagement. In IL-4-stimulated macrophages, CGRP increased LIGHT expression, but failed to induce IL-10 and SPHK1. The stimulatory effect of CGRP on IL-10 production required activation of protein kinase A and was linked to prolonged phosphorylation of CREB and sustained nuclear accumulation of CRTC2 and CRTC3 (where CRTC is CREB-regulated transcriptional cofactor). CGRP enhanced expression of regulatory macrophage markers during the early, but not late, phase of LPS-stimulation and this effect was independent of autocrine type-I IFN activity. In contrast, autocrine type-I IFN activity and treatment of macrophages with IFN-β promoted late-phase IL-10 production, but had only minor influence on LIGHT and SPHK1 expression. Together, the results identify neuroimmunological communication through CGRP as a novel costimulatory pathway promoting the development of a regulatory phenotype of TLR4-stimulated macrophages. CGRP appears to act through a mechanism that involves sustained activation of CREB-dependent gene transcription.

  9. Arginine and glutamine availability and macrophage functions in the obese insulin-resistant Zucker rat.

    Science.gov (United States)

    Blanc, Marie-Céline; Moinard, Christophe; Béziel, Aurélie; Darquy, Sylviane; Cynober, Luc; De Bandt, Jean-Pascal

    2005-01-01

    Increased susceptibility to infections in obese patients may be related to decreased availability of arginine and glutamine, which may affect immune cell functions. Our aim was to evaluate the in vitro effects of these amino acids on the function of macrophages from obese insulin-resistant Zucker rats. Macrophages, isolated from male Zucker obese or lean rats by peritoneal lavage, were incubated in Dulbecco's modified Eagle medium (DMEM) without arginine or glutamine. Arginine or glutamine was added to the medium at increasing final concentrations (0, 0.25, 0.5, 1 or 2 mM). After stimulation by lipopolysaccharide (LPS) from E. coli (40 microg/ml), productions of tumour necrosis factor alpha (TNFalpha) and of nitric oxide (NO) were measured after 3 or 48 h incubation, respectively. NO production, lower in macrophages from obese rats, decreased in macrophages from lean rats (0 mM: 2,423 +/- 1,174 vs. 2 mM: 198 +/- 31 microM/mg protein/24 h; P glutamine was added. TNFalpha production, lower in macrophages from obese rats, was inversely correlated with glutamine concentration. In the presence of arginine, NO production was constantly higher in macrophages from obese rats. It peaked at 0.5 mM arginine and decreased thereafter in both groups. TNFalpha production in macrophages from lean rats was unaffected by arginine, but decreased in macrophages from obese rats (0 mM: 1920 +/- 450 vs. 2 mM: 810 +/- 90 microM/mg protein/3 h; P arginine and glutamine metabolism in macrophages of obese rats, resulting in decreased TNFalpha production and increased NO release, may contribute to increased susceptibility to infection in insulin-resistant states.

  10. Differences in the effects of four TRPV1 channel antagonists on lipopolysaccharide-induced cytokine production and COX-2 expression in murine macrophages.

    Science.gov (United States)

    Ninomiya, Yuki; Tanuma, Sei-Ichi; Tsukimoto, Mitsutoshi

    2017-03-11

    Sepsis is a systemic inflammatory response syndrome triggered by lipopolysaccharide (LPS), an outer membrane component of gram-negative bacteria, and cytokine production via LPS-induced macrophage activation is deeply involved in its pathogenesis. Effective therapy of sepsis has not yet been established. However, it was reported that transient receptor potential vanilloid 1 (TRPV1) channel antagonist capsazepine (CPZ; a capsaicin analogue) attenuates sepsis in a murine model [Ang et al., PLoS ONE 6(9) (2011) e24535; J. Immunol. 187 (2011) 4778-4787]. Here, we profiled the effects of four TRPV1 channel antagonists, AMG9810, SB366791, BCTC and CPZ, on the release of IL-6, IL-1β and IL-18, and on expression of cyclooxygenase 2 (COX-2) in LPS-activated macrophages. Treatment of murine macrophage J774.1 cells or BALB/c mouse-derived intraperitoneal immune cells with LPS induced pro-inflammatory cytokines production and COX-2 expression. Pretreatment with AMG9810 or CPZ significantly suppressed the release of IL-6, IL-1β and IL-18, and COX-2 expression, whereas SB366791 and BCTC were less effective. These results support a role of TRPV1 channel in macrophage activation, but also indicate that only a subset of TRPV1 channel antagonists may be effective in suppressing inflammatory responses. These results suggest that at least some TRPV1 channel antagonists, such as AMG9810 and CPZ, may be candidate anti-inflammatory agents for treatment of sepsis.

  11. The equine alveolar macrophage: Functional and phenotypic comparisons with peritoneal macrophages☆

    Science.gov (United States)

    Karagianni, Anna E.; Kapetanovic, Ronan; McGorum, Bruce C.; Hume, David A.; Pirie, Scott R.

    2013-01-01

    Alveolar macrophages (AMs) constitute the first line of defence in the lung of all species, playing a crucial role in the regulation of immune responses to inhaled pathogens. A detailed understanding of the function and phenotype of AMs is a necessary pre-requisite to both elucidating their role in preventing opportunistic bacterial colonisation of the lower respiratory tract and developing appropriate preventative strategies. The purpose of the study was to characterise this important innate immune cell at the tissue level by making functional and phenotypic comparisons with peritoneal macrophages (PMs). We hypothesised that the tissue of origin determines a unique phenotype of AMs, which may constitute an appropriate therapeutic target for certain equine respiratory diseases. Macrophages isolated from the lung and the peritoneal cavity of 9 horses were stimulated with various toll like receptor (TLR) ligands and the production of nitrite, tumour necrosis factor alpha (TNFα), interleukin (IL) 10 and indoleamine 2,3-dioxygenase (IDO) were measured by the Griess reaction and enzyme linked immunosorbent assay (ELISA) and/or quantitative polymerase chain reaction, respectively. Cells were also compared on the basis of phagocytic-capacity and the expression of several cell surface markers. AMs, but not PMs, demonstrated increased TNFα release following stimulation with LPS, polyinosinic polycytidylic acid (Poly IC) and heat-killed Salmonella typhinurium and increased TNFα and IDO mRNA expression when stimulated with LPS. AMs showed high expression of the specific macrophage markers cluster of differentiation (CD) 14, CD163 and TLR4, whereas PMs showed high expression of TLR4 only. AMs, but not PMs, demonstrated efficient phagocytic activity. Our results demonstrate that AMs are more active than PMs when stimulated with various pro-inflammatory ligands, thus supporting the importance of the local microenvironment in the activation status of the macrophage. This

  12. Yohimbine enhances protection of berberine against LPS-induced mouse lethality through multiple mechanisms.

    Directory of Open Access Journals (Sweden)

    Hui Li

    Full Text Available Sepsis remains a major cause of mortality in intensive care units, better therapies are urgently needed. Gram-negative bacterial lipopolysaccharide (LPS is an important trigger of sepsis. We have demonstrated that berberine (Ber protects against lethality induced by LPS, which is enhanced by yohimbine (Y pretreatment, and Ber combined with Y also improves survival in septic mice. However, the precise mechanisms by which Y enhances protection of Ber against LPS-induced lethality remain unclear. The present study confirmed that simultaneously administered Y also enhanced protection of Ber against LPS-induced lethality. Ber or/and Y attenuated liver injury, but not renal injury in LPS-challenged mice. Ber or/and Y all inhibited LPS-stimulated IκBα, JNK and ERK phosphorylation, NF-κB activation as well as TNF-α production. Ber also increased IL-10 production in LPS-challenged mice, which was enhanced by Y. Furthermore, Ber or/and Y all suppressed LPS-induced IRF3, TyK2 and STAT1 phosphorylation, as well as IFN-β and IP-10 mRNA expression in spleen of mice at 1 h after LPS challenge. Especially, Y enhanced the inhibitory effect of Ber on LPS-induced IP-10 mRNA expression. In vitro experiments further demonstrated that Y significantly enhanced the inhibitory effect of Ber on TNF-α production in LPS-treated peritoneal macrophages, Ber combined with Y promoted LPS-induced IL-10 production and LPS-stimulated IκBα, JNK, ERK and IRF3 phosphorylation and NF-κB activation were also suppressed by Ber or/and Y pretreatment in peritoneal macrophages. Taken together, these results demonstrate that Y enhances the protection of Ber against LPS-induced lethality in mice via attenuating liver injury, upregulating IL-10 production and suppressing IκBα, JNK, ERK and IRF3 phosphorylation. Ber combined with Y may be an effective immunomodulator agent for the prevention of sepsis.

  13. LPS inmobilization on porous and non-porous supports as an approach for the isolation of anti-LPS host-defense peptides.

    Directory of Open Access Journals (Sweden)

    Carlos eLopez-Abarrategui

    2013-12-01

    Full Text Available Lipopolysaccharides (LPS are the major molecular component of the outer membrane of Gram-negative bacteria. This molecule is recognized as a sign of bacterial infection, responsible for the development of local inflammatory response and, in extreme cases, septic shock. Unfortunately, despite substantial advances in the pathophysiology of sepsis, there is no efficacious therapy against this syndrome yet. As a consequence, septic shock syndrome continues to increase, reaching mortality rates over 50% in some cases. Even though many preclinical studies and clinical trials have been conducted, there is no FDA-approved drug yet that interacts directly against LPS. Cationic host defense peptides could be an alternative solution since they possess both antimicrobial and antiseptic properties. Host defense peptides are small, positively charged peptides which are evolutionarily conserved components of the innate immune response. In fact, binding to diverse chemotypes of LPS and inhibition of LPS-induced pro-inflammatory cytokines from macrophages have been demonstrated for different host defense peptides (HDPs. Curiously, none of them have been isolated by their affinity to LPS. A diversity of supports could be useful for such biological interaction and suitable for isolating host defense peptides that recognize LPS. This approach could expand the rational search for anti-LPS host defense peptides.

  14. Arctigenin ameliorates inflammation in vitro and in vivo by inhibiting the PI3K/AKT pathway and polarizing M1 macrophages to M2-like macrophages.

    Science.gov (United States)

    Hyam, Supriya R; Lee, In-Ah; Gu, Wan; Kim, Kyung-Ah; Jeong, Jin-Ju; Jang, Se-Eun; Han, Myung Joo; Kim, Dong-Hyun

    2013-05-15

    Seeds of Arctium lappa, containing arctigenin and its glycoside arctiin as main constituents, have been used as a diuretic, anti-inflammatory and detoxifying agent in Chinese traditional medicine. In our preliminary study, arctigenin inhibited IKKβ and NF-κB activation in peptidoglycan (PGN)- or lipopolysaccharide (LPS)-induced peritoneal macrophages. To understand the anti-inflammatory effect of arctigenin, we investigated its anti-inflammatory effect in LPS-stimulated peritoneal macrophages and on LPS-induced systemic inflammation as well as 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. Arctigenin inhibited LPS-increased IL-1β, IL-6 and TNF-α expression in LPS-stimulated peritoneal macrophages, but increased LPS-reduced IL-10 and CD204 expression. Arctigenin inhibited LPS-induced PI3K, AKT and IKKβ phosphorylation, but did not suppress LPS-induced IRAK-1 phosphorylation. However, arctigenin did not inhibit NF-κB activation in LPS-stimulated PI3K siRNA-treated peritoneal macrophages. Arctigenin suppressed the binding of p-PI3K antibody and the nucleus translocation of NF-κB p65 in LPS-stimulated peritoneal macrophages. Arctigenin suppressed blood IL-1β and TNF-α level in mice systemically inflamed by intraperitoneal injection of LPS. Arctigenin also inhibited colon shortening, macroscopic scores and myeloperoxidase activity in TNBS-induced colitic mice. Arctigenin inhibited TNBS-induced IL-1β, TNF-α and IL-6 expression, as well as PI3K, AKT and IKKβ phosphorylation and NF-κB activation in mice, but increased IL-10 and CD204 expression. However, it did not affect IRAK-1 phosphorylation. Based on these findings, arctigenin may ameliorate inflammatory diseases, such as colitis, by inhibiting PI3K and polarizing M1 macrophages to M2-like macrophages.

  15. Immunoregulation by macrophages II. Separation of mouse peritoneal macrophages having tumoricidal and bactericidal activities and those secreting PGE and interleukin I

    Energy Technology Data Exchange (ETDEWEB)

    Hopper, K.E.; Cahill, J.M.

    1983-06-01

    Macrophage subpopulations having bactericidal or tumoricidal activities and secreting interleukin I (IL1) or prostaglandin E (PGE) were identified through primary or secondary infection with Salmonella enteritidis and separated by sedimentation velocity. Bactericidal activity was measured by (3H)-thymidine release from Listeria monocytogenes and tumoricidal activity by 51Cr-release from C-4 fibrosarcoma or P815 mastocytoma cells. Macrophages with bactericidal activity were distinguished from those with tumoricidal activity a) during secondary infection when cytolytic activity occurred only at days 1-4 post injection and bactericidal activity remained high throughout and b) after sedimentation velocity separation. Cytolysis was consistently greatest among adherent cells of low sedimentation velocity, whereas cells with bactericidal activity increased in size during the infection. Tumour cytostasis (inhibition and promotion of (3H)-thymidine uptake) differed from cytolysis in that the former was more prolonged during infection and was also detected among large cells. Secretion of immunoregulatory molecules PGE and IL1 occurred maximally among different macrophage subpopulations separated by sedimentation velocity and depending on the type of stimulus used in vitro. There was an inverse correlation between IL1 production and PGE production after stimulation with C3-zymosan or lipopolysaccharide (LPS). The development of immunity during infection may therefore be dependent upon the relative proportions of effector and regulatory macrophage subpopulations and the selective effects of environmental stimuli on these functions.

  16. Usefulness of ELISA Methods for Assessing LPS Interactions with Proteins and Peptides

    Science.gov (United States)

    Martínez-Sernández, Victoria; Orbegozo-Medina, Ricardo A.; Romarís, Fernanda; Paniagua, Esperanza; Ubeira, Florencio M.

    2016-01-01

    Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, can trigger severe inflammatory responses during bacterial infections, possibly leading to septic shock. One approach to combatting endotoxic shock is to neutralize the most conserved part and major mediator of LPS activity (lipid A) with LPS-binding proteins or peptides. Although several available assays evaluate the biological activity of these molecules on LPS (e.g. inhibition of LPS-induced TNF-α production in macrophages), the development of simple and cost-effective methods that would enable preliminary screening of large numbers of potential candidate molecules is of great interest. Moreover, it would be also desirable that such methods could provide information about the possible biological relevance of the interactions between proteins and LPS, which may enhance or neutralize LPS-induced inflammatory responses. In this study, we designed and evaluated different types of ELISA that could be used to study possible interactions between LPS and any protein or peptide. We also analysed the usefulness and limitations of the different ELISAs. Specifically, we tested the capacity of several proteins and peptides to bind FITC-labeled LPSs from Escherichia coli serotypes O111:B4 and O55:B5 in an indirect ELISA and in two competitive ELISAs including casein hydrolysate (hCAS) and biotinylated polymyxin B (captured by deglycosylated avidin; PMX) as LPS-binding agents in the solid phase. We also examined the influence of pH, detergents and different blocking agents on LPS binding. Our results showed that the competitive hCAS-ELISA performed under mildly acidic conditions can be used as a general method for studying LPS interactions, while the more restrictive PMX-ELISA may help to identify proteins/peptides that are likely to have neutralizing properties in vitro or in vivo. PMID:27249227

  17. Brazilian red propolis effects on peritoneal macrophage activity: Nitric oxide, cell viability, pro-inflammatory cytokines and gene expression.

    Science.gov (United States)

    Bueno-Silva, Bruno; Kawamoto, Dione; Ando-Suguimoto, Ellen S; Casarin, Renato C V; Alencar, Severino M; Rosalen, Pedro L; Mayer, Marcia P A

    2017-07-31

    Propolis has been used in folk medicine since ancient times and it presented inhibitory effect on neutrophil recruitment previously. However, its effect on macrophage obtained from mice remains unclear. To demonstrate BRP effects on LPS activated peritoneal macrophage. Peritoneal macrophages, obtained from C57BL6 mice and activated with LPS, were treated with 50-80µg/mL of crude extract of Brazilian red propolis (BRP) during 48h. Cell viability, levels of NO, 20 cytokines and expression of 360 genes were evaluated. BRP 60µg/mL reduced NO production by 65% without affecting the cell viability and decreased production IL1α, IL1β, IL4, IL6, IL12p40, Il12p70, IL13, MCP1 and GM-CSF. Molecular mechanism beyond the anti-inflammatory activity may be due to BRP-effects on decreasing expression of Mmp7, Egfr, Adm, Gata3, Wnt2b, Txn1, Herpud1, Axin2, Car9, Id1, Vegfa, Hes1, Hes5, Icam1, Wnt3a, Pcna, Wnt5a, Tnfsf10, Ccl5, Il1b, Akt1, Mapk1, Noxa1 and Cdkn1b and increasing expression of Cav1, Wnt6, Calm1, Tnf, Rb1, Socs3 and Dab2. Therefore, BRP has anti-inflammatory effects on macrophage activity by reducing NO levels and diminished release and expression of pro-inflammatory cytokine and genes, respectively. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  18. STAT1 Antisense Oligonucleotides Attenuate the Proinflammatory Cytokine Release of Alveolar Macrophages in Bleomycin-Induced Fibrosis

    Institute of Scientific and Technical Information of China (English)

    Xianming Fan; Zengli Wang

    2005-01-01

    To investigate the effect of signal transducers and activators of transcription 1 (STAT1) antisense oligonucleotides (ASON) on concentrations of TNF-α, IL-8, NO secreted by alveolar macrophages (AMs) in bleomycin-induced rat pulmonary fibrosis, five adult female Wistar rats were intratracheally instilled with bleomycin. After 7 days, the rats were killed by right ventricle of heart exsanguinations under ketamine anaesthesia and bronchoalveolar lavage (BAL) was performed to obtain AMs. AMs were divided into four groups, treated with STAT1 ASON, STAT1 sense oligonucleotides (SON), dexamethasone (DEX) and medium alone (control), respectively. AMs and media were collected after culture for 36 h. The mRNA and protein expressions of STAT1 and ICAM-1 in AMs were detected by RT-PCR and ELISA, respectively. The concentrations of TNF-α, IL-8, NO in cultured medium were detected.The STAT1 mRNA expression by AMs in the STAT1 ASON group was lower than those of AMs in the STAT1 SON group, the DEX group and the control group (p < 0.05). Moreover, the STAT1 mRNA expression by AMs in the DEX group was also lower than those of AMs in the STAT1 SON group and the control group (p < 0.05), but the STAT1 mRNA expression by AMs in the STAT1 SON group was not different from that of the control group (p >0.05). The protein expressions of STAT1 and ICAM-1 and the mRNA expression of ICAM-1 showed similar changes to the STAT1 mRNA expression by AMs. The concentrations of TNF-α, IL-8, NO in cultured medium from STAT1 ASON group were lower than those from STAT1 SON, DEX and the control groups (p < 0.05). Moreover,the concentrations of TNF-α, IL-8, NO in cultured medium from DEX group were also lower than those from the control and STAT1 SON group (p < 0.05), but no difference between STAT1 SON group and the control (p > 0.05).The results suggest that STAT1 ASON could inhibit the secretion of TNF-α, IL-8, NO in AMs, and STAT1 could become a target of treating pulmonary fibrosis.

  19. Moringa fruit inhibits LPS-induced NO/iNOS expression through suppressing the NF-κ B activation in RAW264.7 cells.

    Science.gov (United States)

    Lee, Hyo-Jin; Jeong, Yun-Jeong; Lee, Tae-Sung; Park, Yoon-Yub; Chae, Whi-Gun; Chung, Il-Kyung; Chang, Hyeun-Wook; Kim, Cheorl-Ho; Choi, Yung-Hyun; Kim, Wun-Jae; Moon, Sung-Kwon; Chang, Young-Chae

    2013-01-01

    In this study, we evaluated the anti-inflammatory effects of moringa (Moringa oleifera Lam.), a natural biologically active substance, by determining its inhibitory effects on pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated macrophage RAW264.7 cells. Extracts from different parts of moringa (root, leaf, and fruit) reduced LPS-induced nitric oxide (NO) release in a dose-dependent manner. The moringa fruit extract most effectively inhibited LPS-induced NO production and levels of inducible nitric oxide synthase (iNOS). The moringa fruit extract also was shown to suppress the production of inflammatory cytokines including IL-1β, TNF-α, and IL-6. Furthermore, moringa fruit extract inhibited the cytoplasmic degradation of I κ B -α and the nuclear translocation of p65 proteins, resulting in lower levels of NF -κ B transactivation. Collectively, the results of this study demonstrate that moringa fruit extract reduces the levels of pro-inflammatory mediators including NO , IL-1β, TNF-α, and IL-6 via the inhibition of NF -κ B activation in RAW264.7 cells. These findings reveal, in part, the molecular basis underlying the anti-inflammatory properties of moringa fruit extract.

  20. Graphene oxide directed in-situ synthesis of Prussian blue for non-enzymatic sensing of hydrogen peroxide released from macrophages.

    Science.gov (United States)

    Qiu, Weiwei; Zhu, Qionghua; Gao, Fei; Gao, Feng; Huang, Jiafu; Pan, Yutian; Wang, Qingxiang

    2017-03-01

    A novel electrochemical non-enzymatic hydrogen peroxide (H2O2) sensor has been developed based on Prussian blue (PB) and electrochemically reduced graphene oxide (ERGO). The GO was covalently modified on glassy carbon electrode (GCE), and utilized as a directing platform for in-situ synthesis of electroactive PB. Then the GO was electrochemically treated to reduction form to improve the effective surface area and electroactivity of the sensing interface. The fabrication process was characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) and atomic force microscopy (AFM). The results showed that the rich oxygen containing groups play a crucial role for the successful synthesis of PB, and the obtained PB layer on the covalently immobilized GO has good stability. Electrochemical sensing assay showed that the modified electrode had tremendous electrocatalytic property for the reduction of H2O2. The steady-state current response increased linearly with H2O2 concentrations from 5μM to 1mM with a fast response time (less than 3s). The detection limit was estimated to be 0.8μM. When the sensor was applied for determination of H2O2 released from living cells of macrophages, satisfactory results were achieved. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Pinellia ternata lectin exerts a pro-inflammatory effect on macrophages by inducing the release of pro-inflammatory cytokines, the activation of the nuclear factor-κB signaling pathway and the overproduction of reactive oxygen species.

    Science.gov (United States)

    Yu, Hong-Li; Zhao, Teng-Fei; Wu, Hao; Pan, Yao-Zong; Zhang, Qian; Wang, Kui-Long; Zhang, Chen-Chao; Jin, Yang-Ping

    2015-10-01

    Pinellia ternata (PT) is a widely used traditional Chinese medicine. The raw material has a throat-irritating toxicity that is associated with the PT lectin (PTL). PTL is a monocot lectin isolated from the tubers of PT, which exhibits mouse peritoneal acute inflammatory effects in vivo. The present study aimed to investigate the pro-inflammatory effect of PTL on macrophages. PTL (50 µg/ml)‑stimulated macrophages enhanced the chemotactic activity of neutrophils. PTL (50, 100, 200 and 400 µg/ml) significantly elevated the production of cytokines [tumor necrosis factor‑α (TNF-α) , interleukin (IL)‑1β and IL‑6]. PTL (25, 50 and 100 µg/ml) induced intracellular reactive oxygen species (ROS) overproduction. PTL also caused transfer of p65 from the macrophage cytoplasm to the nucleus and activated the nuclear factor‑κB (NF‑κB) signaling pathway. Scanning electron microscope images revealed severe cell swelling and membrane integrity defection of macrophages following PTL (100 µg/ml) stimulation, which was also associated with inflammation. PTL had pro‑inflammatory activity, involving induced neutrophil migration, cytokine release, ROS overproduction and the activation of the NF-κB signaling pathway, which was associated with the activation of macrophages.

  2. Modulation of Stat-1 in Human Macrophages Infected with Different Species of Intracellular Pathogenic Bacteria

    Directory of Open Access Journals (Sweden)

    Giuditta Fiorella Schiavano

    2016-01-01

    Full Text Available The infection of human macrophages by pathogenic bacteria induces different signaling pathways depending on the type of cellular receptors involved in the microorganism entry and on their mechanism(s of survival and replication in the host cell. It was reported that Stat proteins play an important role in this process. In the present study, we investigate the changes in Stat-1 activation (phosphorylation in p-tyr701 after uptake of two Gram-positive (Listeria monocytogenes and Staphylococcus aureus and two Gram-negative bacteria (Salmonella typhimurium and Legionella pneumophila characterized by their varying abilities to enter, survive, and replicate in human macrophages. Comparing the results obtained with Gram-negative and Gram-positive bacteria, Stat-1 activation in macrophages does not seem to be related to LPS content. The p-tyr701Stat-1 expression levels were found to be independent of the internalized bacterial number and IFN-γ release. On the contrary, Jak/Stat-1 pathway activation only occurs when an active infection has been established in the host macrophage, and it is plausible that the differences in the expression levels of p-tyr701Stat-1 could be due to different survival mechanisms or to differences in bacteria life cycles within macrophages.

  3. The macrophages in rheumatic diseases

    Directory of Open Access Journals (Sweden)

    Laria A

    2016-02-01

    Full Text Available Antonella Laria, Alfredomaria Lurati , Mariagrazia Marrazza , Daniela Mazzocchi, Katia Angela Re, Magda Scarpellini Rheumatology Unit, Fornaroli Hospital, Magenta, Italy Abstract: Macrophages belong to the innate immune system giving us protection against pathogens. However it is known that they are also involved in rheumatic diseases. Activated macrophages have two different phenotypes related to different stimuli: M1 (classically activated and M2 (alternatively activated. M1 macrophages release high levels of pro-inflammatory cytokines, reactive nitrogen and oxygen intermediates killing microorganisms and tumor cells; while M2 macrophages are involved in resolution of inflammation through phagocytosis of apoptotic neutrophils, reduced production of pro-inflammatory cytokines, and increased synthesis of mediators important in tissue remodeling, angiogenesis, and wound repair. The role of macrophages in the different rheumatic diseases is different according to their M1/M2 macrophages phenotype. Keywords: macrophage, rheumatic diseases

  4. Modulation of macrophage antitumor potential by apoptotic lymphoma cells.

    Science.gov (United States)

    Voss, Jorine J L P; Ford, Catriona A; Petrova, Sofia; Melville, Lynsey; Paterson, Margaret; Pound, John D; Holland, Pam; Giotti, Bruno; Freeman, Tom C; Gregory, Christopher D

    2017-06-01

    In aggressive non-Hodgkin's lymphoma (NHL), constitutive apoptosis of a proportion of the tumor cell population can promote net tumor growth. This is associated with the accumulation of tumor-associated macrophages (TAMs) that clear apoptotic cells and exhibit pro-oncogenic transcriptional activation profiles characteristic of reparatory, anti-inflammatory and angiogenic programs. Here we consider further the activation status of these TAMs. We compare their transcriptomic profile with that of a range of other macrophage types from various tissues noting especially their expression of classically activated (IFN-γ and LPS) gene clusters - typically antitumor - in addition to their previously described protumor phenotype. To understand the impact of apoptotic cells on the macrophage activation state, we cocultured apoptotic lymphoma cells with classically activated macrophages (M(IFN-γ/LPS), also known as M1, macrophages). Although untreated and M(IFN-γ/LPS) macrophages were able to bind apoptotic lymphoma cells equally well, M(IFN-γ/LPS) macrophages displayed enhanced ability to phagocytose them. We found that direct exposure of M(IFN-γ/LPS) macrophages to apoptotic lymphoma cells caused switching towards a protumor activation state (often referred to as M2-like) with concomitant inhibition of antitumor activity that was a characteristic feature of M(IFN-γ/LPS) macrophages. Indeed, M(IFN-γ/LPS) macrophages exposed to apoptotic lymphoma cells displayed increased lymphoma growth-promoting activities. Antilymphoma activity by M(IFN-γ/LPS) macrophages was mediated, in part, by galectin-3, a pleiotropic glycoprotein involved in apoptotic cell clearance that is strongly expressed by lymphoma TAMs but not lymphoma cells. Intriguingly, aggressive lymphoma growth was markedly impaired in mice deficient in galectin-3, suggesting either that host galectin-3-mediated antilymphoma activity is required to sustain net tumor growth or that additional functions of galectin-3

  5. Voluntary exercise attenuates obesity-associated inflammation through ghrelin expressed in macrophages.

    Science.gov (United States)

    Kizaki, Takako; Maegawa, Taketeru; Sakurai, Takuya; Ogasawara, Jun-etsu; Ookawara, Tomomi; Oh-ishi, Shuji; Izawa, Tetsuya; Haga, Shukoh; Ohno, Hideki

    2011-09-30

    Chronic low-level inflammation is associated with obesity and a sedentary lifestyle, causing metabolic disturbances such as insulin resistance. Exercise training has been shown to decrease chronic low-level systemic inflammation in high-fat diet (HFD)-induced obesity. However, the molecular mechanisms mediating its beneficial effects are not fully understood. Ghrelin is a peptide hormone predominantly produced in the stomach that stimulates appetite and induces growth hormone release. In addition to these well-known functions, recent studies suggest that ghrelin localizes to immune cells and exerts an anti-inflammatory effect. The purpose of the current study was to investigate the role of ghrelin expressed in macrophages in the anti-inflammatory effects of voluntary exercise training. Expression of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein (MCP)-1 and F4/80 was increased in adipose tissue from mice fed a HFD (HFD mice) compared with mice fed a standard diet (SD mice), whereas the expression of these inflammatory cytokines was markedly decreased in mice performing voluntary wheel running during the feeding of a HFD (HFEx mice). The expression of TNF-α was also increased in peritoneal macrophages by a HFD and exercise training inhibited the increase of TNF-α expression. Interestingly, expression of ghrelin in peritoneal macrophages was decreased by a HFD and recovered by exercise training. Suppression of ghrelin expression by siRNA increased TNF-α expression and LPS-stimulated NF-κB activation in RAW264 cells, which is a macrophage cell line. TNF-α expression by stimulation with LPS was significantly suppressed in RAW264 cells cultured in the presence of ghrelin. These results suggest that ghrelin exerts potent anti-inflammatory effects in macrophages and functions as a mediator of the beneficial effects of exercise training. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Atomic force microscopy based investigations of anti-inflammatory effects in lipopolysaccharide-stimulated macrophages.

    Science.gov (United States)

    Pi, Jiang; Cai, Huaihong; Yang, Fen; Jin, Hua; Liu, Jianxin; Yang, Peihui; Cai, Jiye

    2016-01-01

    A new method based on atomic force microscopy (AFM) was developed to investigate the anti-inflammatory effects of drugs on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The LPS-stimulated RAW264.7 macrophage cell line is a widely used in vitro cell model for the screening of anti-inflammatory drugs or the study of anti-inflammatory mechanisms. In this work, the inhibitory effects of dexamethasone and quercetin on LPS-CD14 receptor binding in RAW264.7 macrophages was probed by LPS-functionalized tips for the first time. Both dexamethasone and quercetin were found to inhibit LPS-induced NO production, iNOS expression, IκBα phosphorylation, and IKKα/β phosphorylation in RAW264.7 macrophages. The morphology and ultrastructure of RAW264.7 macrophages were determined by AFM, which indicated that dexamethasone and quercetin could inhibit LPS-induced cell surface particle size and roughness increase in RAW264.7 macrophages. The binding of LPS and its receptor in RAW264.7 macrophages was determined by LPS-functionalized AFM tips, which demonstrated that the binding force and binding probability between LPS and CD14 receptor on the surface of RAW264.7 macrophages were also inhibited by dexamethasone or quercetin treatment. The obtained results imply that AFM, which is very useful for the investigation of potential targets for anti-inflammatory drugs on native macrophages and the enhancement of our understanding of the anti-inflammatory effects of drugs, is expected to be developed into a promising tool for the study of anti-inflammatory drugs.

  7. LPS inmobilization on porous and non-porous supports as an approach for the isolation of anti-LPS host-defense peptides

    Science.gov (United States)

    López-Abarrategui, Carlos; del Monte-Martínez, Alberto; Reyes-Acosta, Osvaldo; Franco, Octavio L.; Otero-González, Anselmo J.

    2013-01-01

    Lipopolysaccharides (LPSs) are the major molecular component of the outer membrane of Gram-negative bacteria. This molecule is recognized as a sign of bacterial infection, responsible for the development of local inflammatory response and, in extreme cases, septic shock. Unfortunately, despite substantial advances in the pathophysiology of sepsis, there is no efficacious therapy against this syndrome yet. As a consequence, septic shock syndrome continues to increase, reaching mortality rates over 50% in some cases. Even though many preclinical studies and clinical trials have been conducted, there is no Food and Drug Administration-approved drug yet that interacts directly against LPS. Cationic host-defense peptides (HDPs) could be an alternative solution since they possess both antimicrobial and antiseptic properties. HDPs are small, positively charged peptides which are evolutionarily conserved components of the innate immune response. In fact, binding to diverse chemotypes of LPS and inhibition of LPS-induced pro-inflammatory cytokines from macrophages have been demonstrated for different HDPs. Curiously, none of them have been isolated by their affinity to LPS. A diversity of supports could be useful for such biological interaction and suitable for isolating HDPs that recognize LPS. This approach could expand the rational search for anti-LPS HDPs. PMID:24409171

  8. Sustained nitric oxide delivery delays nitric oxide-dependent apoptosis in macrophages: contribution to the physiological function of activated macrophages.

    Science.gov (United States)

    Hortelano, Sonsoles; Través, Paqui G; Zeini, Miriam; Alvarez, Alberto M; Boscá, Lisardo

    2003-12-01

    Treatment of the macrophage cell line RAW 264.7 with the short-lived NO donor S-nitrosoglutathione triggers apoptosis through the release of mitochondrial mediators. However, continuous supply of NO by long-lived NO donors protected cells from apoptosis through mechanisms that involved the maintenance or an increase in the levels of the inhibitor of apoptosis proteins (IAPs) cIAP-1, cIAP-2, and xIAP and decreases in the accumulation of p53 and in the levels and targeting of Bax to the mitochondria. As a result of these changes, the activation of caspases 9 and 3 was notably delayed, expanding the time of viability of the macrophages. Moreover, inhibition of NO synthase 2 activity after 8 h of stimulation of RAW 264.7 cells with LPS and IFN-gamma accelerated apoptosis via an increase in the processing and activation of caspases. These data suggest that NO exerts an important role in the autoregulation of apoptosis in macrophages.

  9. Intra-amniotic LPS modulation of TLR signaling in lung and blood monocytes of fetal sheep.

    Science.gov (United States)

    Kramer, Boris W; Kallapur, Suhas G; Moss, Timothy J; Nitsos, Ilias; Newnham, John P; Jobe, Alan H

    2009-04-01

    Epidemiological studies suggest that intra-uterine exposure to inflammation may prime postnatal immune responses. In fetal sheep, intra-amniotic injection of lipopolysaccharide (LPS) induced chorioamnionitis, lung inflammation and maturation, matured lung monocytes to macrophages and initiated systemic tolerance of fetal monocytes to subsequent challenge with LPS. We hypothesized that LPS-mediated chorioamnionitis altered the response of lung and blood monocytes to Toll-like receptor (TLR) ligands such as PamCysK4 (TLR2), flagellin (TLR5), and human CpG-DNA (TLR9). Time-mated ewes were given intra-amniotic injections of LPS or saline. Blood and lung monocytes were assessed after 2 days, 7 days and 2 days and 7 days repetitive LPS injections before delivery at 124 days gestational age (term 150 days). Responsiveness of blood and lung monocytes to TLR-ligands in vitro was assessed by interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha) and hydrogen peroxide. Monocytes from preterm controls had minimal responses. Lipopolysaccharide-mediated chorioamnionitis increased IL-6, TNF- alpha and hydrogen peroxide to all TLR agonists in blood and lung monocytes. Repetitive exposure to antenatal LPS reduced IL-6, TNF- alpha and hydrogen peroxide to TLR-ligands suggesting tolerance. Tolerance to TLR-ligands reduced IL-1 receptor associated kinase-4 expression. Thus, repeated fetal exposure to LPS induced tolerance to other TLR-ligands. These modulations of fetal innate immunity have implications for host defense and injury responses in preterm infants.

  10. SIRT2 ameliorates lipopolysaccharide-induced inflammation in macrophages

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    Lee, Ae Sin; Jung, Yu Jin; Kim, Dal; Nguyen-Thanh, Tung [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Kang, Kyung Pyo [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Research Institute of Clinical Medicine of Chonbuk National University, Chonbuk National University Hospital, Jeonju (Korea, Republic of); Lee, Sik [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Park, Sung Kwang [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Research Institute of Clinical Medicine of Chonbuk National University, Chonbuk National University Hospital, Jeonju (Korea, Republic of); Kim, Won, E-mail: kwon@jbnu.ac.kr [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Research Institute of Clinical Medicine of Chonbuk National University, Chonbuk National University Hospital, Jeonju (Korea, Republic of)

    2014-08-08

    Highlights: • Knockout of SIRT2 attenuates lipopolysaccharide-induced iNOS expression. • Lipopolysaccharide-induced NO production is decreased in SIRT2 KO macrophage. • SIRT2 deficiency suppresses lipopolysaccharide-induced ROS production in macrophage. • M1-macrophage related factors are decreased in SIRT2 deficient cells. • SIRT2 deficiency decreases lipopolysaccharide-induced activation of NFκB. - Abstract: Introduction: SIRT2 is a NAD(+)-dependent deacetylases and associated with numerous processes such as infection, carcinogenesis, DNA damage and cell cycle regulation. However, the role of SIRT2 in inflammatory process in macrophage remains unclear. Materials and methods: In the present study, we have evaluated the regulatory effects of SIRT2 in lipopolysaccharide (LPS)-stimulated macrophages isolated from SIRT2 knockout (KO) and wild type (WT) mice or Raw264.7 macrophage cells. As inflammatory parameters, expression of inducible nitric oxide synthase (iNOS), the productions of nitric oxide, reactive oxygen species (ROS) and M1-macrophage-related factors were evaluated. We also examined the effects of SIRT2 on activation of nuclear factor-kappaB (NFκB) signaling. Results: SIRT2 deficiency inhibits LPS-induced iNOS mRNA and protein expression in bone marrow derived macrophages. SIRT2-siRNA transfection also suppressed LPS-induced iNOS expression in Raw264.7 macrophage cells. Bone marrow derived macrophages isolated from SIRT2 KO mice produced lower nitric oxide and expressed lower levels of M1-macrophage related markers including iNOS and CD86 in response to LPS than WT mice. Decrease of SIRT2 reduced the LPS-induced reactive oxygen species production. Deficiency of SIRT2 resulted in inhibition of NFκB activation through reducing the phosphorylation and degradation of IκBα. The phosphorylation and nuclear translocation of p65 was significantly decreased in SIRT2-deficient macrophages after LPS stimulation. Discussion: Our data suggested that

  11. Inhibition effect on LPS-induced TNF-α release by homegrown and Australian Melaleuca alternifolia (Tea Tree) oil%不同来源茶树油抑制LPS诱导TNF-α释放的体外实验

    Institute of Scientific and Technical Information of China (English)

    王懿; 王振维

    2007-01-01

    目的 比较国产和进口茶树油(tea tree oil,TTO)对脂多糖(lipopolysaccharide, LPS)诱导炎症的抑制作用.方法 四唑盐(MTT)比色实验筛选无细胞毒性作用的TTO溶液浓度;各浓度TTO水溶液按量效和时效关系预处理细胞后,测定LPS激活RAW264.7细胞分泌肿瘤坏死因子-α(tumor necrotic factor-alpha, TNF-α)的浓度,以观察国产和进口TTO水溶液对LPS诱导炎症的抑制效果.结果 浓度低于0.001%的国产和进口TTO溶液对细胞无毒性作用;0.000 125%~0.001%进口TTO和0.000 25%~0.001%国产TTO各浓度组均能非常显著地抑制LPS诱导细胞释放TNF-α;0.000 25%进口和国产TTO分别在3 h和6 h内对细胞有显著的保护作用.结论 国产和进口TTO对LPS诱导的炎症均有显著抑制效果.

  12. Effect of resveratrol, tyrosol and beta-sitosterol on oxidised low-density lipoprotein-stimulated oxidative stress, arachidonic acid release and prostaglandin E2 synthesis by RAW 264.7 macrophages.

    Science.gov (United States)

    Vivancos, Marta; Moreno, Juan J

    2008-06-01

    Oxidation of LDL is hypothesised as an early and critical event in atherogenesis. Oxidised LDL (oxLDL) favour the transformation of macrophages into foam cells, an important cell involved in atherosclerosis. Furthermore, oxLDL cause multiple changes in macrophage functions. Thus, oxLDL induces certain genes, suppresses others and alters cell lipid metabolism. Consumption of a Mediterranean diet is associated with a low incidence of atherosclerotic disease, but data about the specific dietary constituents involved and mechanisms conferring cardioprotection are still sparse. The aim of the present study was to determine the effect of representative minor components of wine and olive oil on reactive oxygen species and eicosanoid synthesis induced by oxLDL-stimulated macrophages. We observed that exposure to non-toxic oxLDL concentrations leads to the production of H2O2 by RAW 264.7 macrophages and this effect was reverted by apocynin, a NADPH oxidase inhibitor. Moreover, oxLDL induced arachidonic acid (AA) release, cyclo-oxygenase-2 overexpression and subsequent PGE2 release. We observed that resveratrol and tyrosol revert H2O2 production induced by oxLDL as well as AA release and PGE2 synthesis and that these effects were not as a consequence of these compounds interfering with the oxLDL binding to their receptors. Interestingly, beta-sitosterol presence enhances these polyphenol actions. Thus, we found a synergistic action of polyphenols of olive oil and wine and beta-sitosterol of olive oil led to the modulation of the effects of oxLDL on oxidative stress and PGE2 synthesis.

  13. Visceral adipose tissue activated macrophage content and inflammatory adipokine secretion is higher in pre-eclampsia than in healthy pregnancys.

    Science.gov (United States)

    Huda, Shahzya S; Jordan, Fiona; Bray, Jack; Love, Gillian; Payne, Reba; Sattar, Naveed; Freeman, Dilys J

    2017-07-01

    Obesity increases pre-eclampsia (PE) risk. Adipose tissue inflammation may contribute to the clinical syndrome of PE. We compared adipose tissue macrophage infiltration and release of pro-inflammatory adipokines in PE and healthy pregnancy. Subcutaneous and visceral adipose tissue biopsies were collected from healthy (n=13) and PE (n=13) mothers. Basal and lipopolysaccharide (LPS) stimulated adipocyte TNFα, IL-6, CCL-2, and CRP release was measured. Adipose tissue cell densities of activated (cfms(+)) and total (CD68(+)) macrophages were determined. In PE only, visceral adipose tissue TNFα release was increased after LPS stimulation (57 [76] versus 81 [97] pg/ml/µg DNA, P=0.030). Basal TNFα release was negatively correlated insulin sensitivity of visceral adipocytes (r = -0.61, P=0.030) in PE. Visceral adipocyte IL-6 release was increased after LPS stimulation in PE only (566 [696] versus 852 [914] pg/ml/µg DNA, P=0.019). Visceral adipocyte CCL-2 basal (67 [61] versus 187 [219] pg/ml/µgDNA, P=0.049) and stimulated (46 [46] versus 224 [271] pg/ml/µg DNA, P=0.003) release was greater than in subcutaneous adipocytes in PE only. In PE, median TNF mRNA expression in visceral adipose tissue was higher than controls (1.94 [1.13-4.14] versus 0.8 [0.00-1.27] TNF/PPIA ratio, P=0.006). In visceral adipose tissue, CSF1R (a marker of activated macrophages) mRNA expression (24.8[11.0] versus 51.0[29.9] CSF1R/PPIA ratio, P=0.011) and activated (cfms+) macrophage count (6.7[2.6] versus 15.2[8.8] % cfms+/adipocyte, P=0.031) were higher in PE than in controls. In conclusion, our study demonstrates dysregulation of inflammatory pathways predominantly in visceral adipose tissue in PE. Inflammation of visceral adipose tissue may mediate many of the adverse metabolic effects associated with PE. © 2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution

  14. TLR-Activated Gap Junction Channels Protect Mice against Bacterial Infection through Extracellular UDP Release.

    Science.gov (United States)

    Qin, Juliang; Zhang, Guangxu; Zhang, Xiaoyu; Tan, Binghe; Lv, Zhangsheng; Liu, Mingyao; Ren, Hua; Qian, Min; Du, Bing

    2016-02-15

    Extracellular UDP (eUDP), released as a danger signal by stressed or apoptotic cells, plays an important role in a series of physiological processes. Although the mechanism of eUDP release in apoptotic cells has been well defined, how the eUDP is released in innate immune responses remains unknown. In this study, we demonstrated that UDP was released in both Escherichia coli-infected mice and LPS- or Pam3CSK4-treated macrophages. Also, LPS-induced UDP release could be significantly blocked by selective TLR4 inhibitor Atractylenolide I and selective gap junction inhibitors carbenoxolone and flufenamic acid (FFA), suggesting the key role of TLR signaling and gap junction channels in this process. Meanwhile, eUDP protected mice from peritonitis by reducing invaded bacteria that could be rescued by MRS2578 (selective P2Y6 receptor inhibitor) and FFA. Then, connexin 43, as one of the gap junction proteins, was found to be clearly increased by LPS in a dose- and time-dependent manner. Furthermore, if we blocked LPS-induced ERK signaling by U0126, the expression of connexin 43 and UDP release was also inhibited dramatically. In addition, UDP-induced MCP-1 secretion was significantly reduced by MRS2578, FFA, and P2Y6 mutation. Accordingly, pretreating mice with U0126 and Gap26 increased invaded bacteria and aggravated mice death. Taken together, our study reveals an internal relationship between danger signals and TLR signaling in innate immune responses, which suggests a potential therapeutic significance of gap junction channel-mediated UDP release in infectious diseases. Copyright © 2016 by The American Association of Immunologists, Inc.

  15. Forced IFIT-2 expression represses LPS induced TNF-alpha expression at posttranscriptional levels

    Directory of Open Access Journals (Sweden)

    Autenrieth Ingo B

    2008-12-01

    Full Text Available Abstract Background Interferon induced tetratricopeptide repeat protein 2 (IFIT-2, P54 belongs to the type I interferon response genes and is highly induced after stimulation with LPS. The biological function of this protein is so far unclear. Previous studies indicated that IFIT-2 binds to the initiation factor subunit eIF-3c, affects translation initiation and inhibits protein synthesis. The aim of the study was to further characterize the function of IFIT-2. Results Stimulation of RAW264.7 macrophages with LPS or IFN-γ leads to the expression of IFIT-2 in a type I interferon dependent manner. By using stably transfected RAW264.7 macrophages overexpressing IFIT-2 we found that IFIT-2 inhibits selectively LPS induced expression of TNF-α, IL-6, and MIP-2 but not of IFIT-1 or EGR-1. In IFIT-2 overexpressing cells TNF-α mRNA expression was lower after LPS stimulation due to reduced mRNA stability. Further experiments suggest that characteristics of the 3'UTR of transcripts discriminate whether IFIT-2 has a strong impact on protein expression or not. Conclusion Our data suggest that IFIT-2 may affect selectively LPS induced protein expression probably by regulation at different posttranscriptional levels.

  16. ATF-2 regulates lipopolysaccharide-induced transcription in macrophage cells.

    Science.gov (United States)

    Hirose, Noriyuki; Maekawa, Toshio; Shinagawa, Toshie; Ishii, Shunsuke

    2009-07-17

    The transcription factor ATF-2, a member of the ATF/CREB family, is a target of p38 that are involved in stress-induced apoptosis and in Toll-like receptor (TLR)-mediated signaling. Phosphorylation of ATF-2 at Thr-71 was enhanced by treating of RAW264.7 macrophage cells with either LPS, MALP-2, or CpG-ODN. LPS treatment enhanced the trans-activation capacity of ATF-2. Among multiple LPS-induced genes, the LPS-induced expression of Socs-3 was significantly reduced by the treatment of RAW264.7 cells with an Atf-2 siRNA. Transcription from the Socs-3 promoter was synergistically stimulated by ATF-2 and LPS, whereas it was suppressed by Atf-2 siRNA. Histone deacetylase 1 (HDAC1) interacted with ATF-2 after LPS treatment, but not before treatment. Treatment of RAW264.7 cells with trichostatin A, an inhibitor of HDAC, suppressed the LPS-induced Socs-3 expression, suggesting that HDAC1 positively regulates the LPS-induced transcription of Socs-3. Thus, ATF-2 plays an important role in TLR-mediated transcriptional control in macrophage cells.

  17. Naringin lauroyl ester inhibits lipopolysaccharide-induced activation of nuclear factor κB signaling in macrophages.

    Science.gov (United States)

    Hattori, Hiromi; Tsutsuki, Hiroyasu; Nakazawa, Masami; Ueda, Mitsuhiro; Ihara, Hideshi; Sakamoto, Tatsuji

    2016-07-01

    Naringin (Nar) has antioxidant and anti-inflammatory properties. It was recently reported that enzymatic modification of Nar enhanced its functions. Here, we acylated Nar with fatty acids of different sizes (C2-C18) using immobilized lipase from Rhizomucor miehei and investigated the anti-inflammatory effects of these molecules. Treatment of murine macrophage RAW264.7 cells with Nar alkyl esters inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production, with Nar lauroyl ester (Nar-C12) showing the strongest effect. Furthermore, Nar-C12 suppressed the LPS-induced expression of inducible NO synthase by blocking the phosphorylation of inhibitor of nuclear factor (NF)-κB-α as well as the nuclear translocation of NF-κB subunit p65 in macrophage cells. Analysis of Nar-C12 uptake in macrophage cells revealed that Nar-C12 ester bond was partially degraded in the cell membrane and free Nar was translocated to the cytosol. These results indicate that Nar released from Nar-C12 exerts anti-inflammatory effects by suppressing NF-κB signaling pathway.

  18. Propofol inhibits LPS-induced apoptosis in lung epithelial cell line, BEAS-2B.

    Science.gov (United States)

    Lv, Xiang; Zhou, Xuhui; Yan, Jia; Jiang, Jue; Jiang, Hong

    2017-03-01

    Lipopolysaccharide (LPS) plays an important role in lung endothelial apoptosis which is crucial for lung fibrogenesis in ARDS progression. Reactive oxygen species (ROS) has been reported to be involved in LPS-induced lung epithelial cell apoptosis. Propofol is a commonly used intravenous anesthetic agent in clinic and it could attenuate LPS-induced epithelial cells oxidation and apoptosis. However, the mechanisms are still obscure. In this study, we examined whether and how propofol attenuates LPS-induced oxidation and apoptosis in BEAS-2B cells. Compared with control group, LPS up-regulated Pin-1, phosphatase A2 (PP2A) expression, induced p66(Shc)-Ser(36) phosphorylation, and facilitated p66(Shc) mitochondrial translocation, thus leading to superoxide anion (O2(-)) generation, mitochondrial cytochrome c release, active caspase 3 over-expression and cell viability inhibition. Importantly, propofol was shown to down-regulate LPS-induced PP2A expression, limit p66(Shc) mitochondrial translocation, decrease O2(-) generation, inhibit mitochondrial cytochrome c release, reduce active caspase 3 expression, and recover cells viability, while propofol had no effects on LPS-induced Pin-1 expression and p66(Shc)-Ser(36) phosphorylation. Moreover, the protective effects of propofol on LPS-induced BEAS-2B cells apoptosis were similar to that of calyculin A, which is an inhibitor of PP2A. We also found that FTY720, which is an activator of PP2A, can effectively reverse the protective function of propofol. Our data illustrated that propofol could alleviate LPS-induced BEAS-2B cells oxidation and apoptosis through down-regulating PP2A expression, limiting p66(Shc)-Ser(36) dephosphorylation and p66(Shc) mitochondrial translocation, decreasing O2(-) generation, mitochondrial cytochrome c release, activating caspase 3 expression. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  19. Mycobacterium tuberculosis Synergizes with ATP To Induce Release of Microvesicles and Exosomes Containing Major Histocompatibility Complex Class II Molecules Capable of Antigen Presentation ▿ †

    Science.gov (United States)

    Ramachandra, Lakshmi; Qu, Yan; Wang, Ying; Lewis, Colleen J.; Cobb, Brian A.; Takatsu, Kiyoshi; Boom, W. Henry; Dubyak, George R.; Harding, Clifford V.

    2010-01-01

    Major histocompatibility complex class II (MHC-II) molecules are released by murine macrophages upon lipopolysaccharide (LPS) stimulation and ATP signaling through the P2X7 receptor. These studies show that infection of macrophages with Mycobacterium tuberculosis or M. bovis strain BCG enhances MHC-II release in synergy with ATP. Shed MHC-II was contained in two distinct organelles, exosomes and plasma membrane-derived microvesicles, which were both able to present exogenous antigenic peptide to T hybridoma cells. Furthermore, microvesicles from mycobacterium-infected macrophages were able to directly present M. tuberculosis antigen (Ag) 85B(241-256)-I-Ab complexes that were generated by the processing of M. tuberculosis Ag 85B in infected cells to both M. tuberculosis-specific T hybridoma cells and naïve P25 M. tuberculosis T-cell receptor (TCR)-transgenic T cells. In the presence of prefixed macrophages, exosomes from mycobacterium-infected macrophages provided weak stimulation to M. tuberculosis-specific T hybridoma cells but not naïve P25 T cells. Thus, infection with M. tuberculosis primes macrophages for the increased release of exosomes and microvesicles bearing M. tuberculosis peptide-MHC-II complexes that may generate antimicrobial T-cell responses. PMID:20837713

  20. Anti-inflammatory effects of cordycepin in lipopolysaccharide-stimulated RAW 264.7 macrophages through Toll-like receptor 4-mediated suppression of mitogen-activated protein kinases and NF-κB signaling pathways

    Directory of Open Access Journals (Sweden)

    Choi YH

    2014-10-01

    Full Text Available Yung Hyun Choi,1,2 Gi-Young Kim,3 Hye Hyeon Lee4 1Department of Biochemistry, Dongeui University College of Korean Medicine, Busan, 2Anti-Aging Research Center and Blue-Bio Industry RIC, Dongeui University, Busan, 3Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National University, Jeju, 4Daegu Gyeongbuk Institute of Science and Technology, Daegu, Republic of Korea Abstract: Cordycepin is the main functional component of the Cordyceps species, which has been widely used in traditional Oriental medicine. This compound possesses many pharmacological properties, such as an ability to enhance immune function, as well as antioxidant, antiaging, and anticancer effects. In the present study, we investigated the anti-inflammatory effects of cordycepin using a murine macrophage RAW 264.7 cell model. Our data demonstrated that cordycepin suppressed production of proinflammatory mediators such as nitric oxide (NO and prostaglandin E2 by inhibiting inducible NO synthase and cyclooxygenase-2 gene expression. Cordycepin also inhibited the release of proinflammatory cytokines, including tumor necrosis factor-alpha and interleukin-1-beta, through downregulation of respective mRNA expression. In addition, pretreatment with cordycepin significantly inhibited lipopolysaccharide (LPS-induced phosphorylation of mitogen-activating protein kinases and attenuated nuclear translocation of NF-κB by LPS, which was associated with abrogation of inhibitor kappa B-alpha degradation. Furthermore, cordycepin potently inhibited the binding of LPS to macrophages and LPS-induced Toll-like receptor 4 and myeloid differentiation factor 88 expression. Taken together, the results suggest that the inhibitory effects of cordycepin on LPS-stimulated inflammatory responses in RAW 264.7 macrophages are associated with suppression of mitogen-activating protein kinases and activation of NF-κB by inhibition of the Toll-like receptor 4 signaling pathway. Keywords

  1. Model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation

    Energy Technology Data Exchange (ETDEWEB)

    Bordbar, Aarash; Mo, Monica L.; Nakayasu, Ernesto S.; Rutledge, Alexandra C.; Kim, Young-Mo; Metz, Thomas O.; Jones, Marcus B.; Frank, Bryan C.; Smith, Richard D.; Peterson, Scott N.; Hyduke, Daniel R.; Adkins, Joshua N.; Palsson, Bernhard O.

    2012-06-26

    Macrophages are central players in the immune response, manifesting divergent phenotypes to control inflammation and innate immunity through the release of cytokines and other regulatory factor-dependent signaling pathways. In recent years, the focus on metabolism has been reemphasized as critical signaling and regulatory pathways of human pathophysiology, ranging from cancer to aging, often converge on metabolic responses. Here, we used genome-scale modeling and multi-omics (transcriptomics, proteomics, and metabolomics) analysis to assess metabolic features critical for macrophage functions. We constructed a genome-scale metabolic network for the RAW 264.7 cell line to determine metabolic modulators of macrophage activation. Metabolites well-known to be associated with immunoactivation (e.g., glucose and arginine) and immunosuppression (e.g., tryptophan and vitamin D3) were amongst the most critical effectors. Intracellular metabolic mechanisms linked to critical suppressive effectors were then assessed, identifying a suppressive role for de novo nucleotide synthesis. Finally, the underlying metabolic mechanisms of macrophage activation are identified by analyzing multi-omic data obtained from LPS-stimulated RAW cells in the context of our flux-based predictions. Our study demonstrates metabolism's role in regulating activation may be greater than previously anticipated and elucidates underlying metabolic connections between activation and metabolic effectors.

  2. Mechanism of macrophage injury following traumatic hemorrhagic shock: through PTX-sensitive G-protein-mediated signal transduction pathway

    Institute of Scientific and Technical Information of China (English)

    刘靖华; 刘良明; 陈惠孙; 胡德耀; 刘怀琼

    2002-01-01

    Objective: To study the mechanism of macrophage injury after trauma-hemorrhagic shock.   Methods: Wistar male rats underwent trauma (closed bone fracture) and hemorrhage (mean arterial blood pressure of 35 mm Hg±5 mm Hg for 60 minutes, following fluid resuscitation). Rats without trauma, hemorrhage or fluid resuscitation served as controls. Peritoneal macrophages were harvested at 6 hours and 1, 2, 3, 7 days after traumatic hemorrhagic shock to determine the effects of pertussis toxin (PTX, as a specific inhibitor to Giα) and cholera toxin (CTX, as a stimulant to Gsα) on macrophage-Ia expression and TNF-α production and levels of Giα and Gsα.   Results: The macrophages from the injured rats revealed a significant decrease of Ia positive number and TNF-α release in response to LPS. With pretreatment with PTX 10-100 ng/ml Ia positive cells and LPS-induced TNFα production in both control and impaired macrophages populations were dose-dependently increased. Both macrophages populations were not responding to CTX treatment (10-100 ng/ml). Western blot analyses showed that the levels of Giα protein expression increased as much as 116.5%-148.8% of the control level from 6 hours through 7 days after traumatic hemorrhage. The levels of Gsα protein expression were reduced at 6 hours and decreased to the lowest degree; 36% of the control at day 1, began to return at day 2 and returned to the normal level at day 7, following traumatic hemorrhagic shock.   Conclusions: PTX-sensitive G-protein may participate in the modulation of macrophage-Ia expression and TNF-α release following traumatic hemorrhagic shock. Analyses of the alteration of Giα and Gsα protein expressions further supports the concept that G-protein is involved in trauma-induced macrophage signal transduction pathways.

  3. Synthesis, characterization, antimicrobial activity and LPS-interaction properties of SB041, a novel dendrimeric peptide with antimicrobial properties.

    Science.gov (United States)

    Bruschi, Michela; Pirri, Giovanna; Giuliani, Andrea; Nicoletto, Silvia Fabiole; Baster, Izabela; Scorciapino, Mariano Andrea; Casu, Mariano; Rinaldi, Andrea C

    2010-08-01

    Multimeric peptides offer several advantages with respect to their monomeric counterparts, as increased activity and greater stability to peptidases and proteases. SB041 is a novel antimicrobial peptide with dendrimeric structure; it is a tetramer of pyrEKKIRVRLSA linked by a lysine core, with an amino valeric acid chain. Here, we report on its synthesis, NMR characterization, antimicrobial activity, and LPS-interaction properties. The peptide was especially active against Gram-negative strains, with a potency comparable (on molar basis) to that of lipopeptides colistin and polymixin B, but it also displayed some activity against selected Gram-positive strains. Following these indications, we investigated the efficacy of SB041 in binding Escherichia coli and Pseudomonas aeruginosa LPS in vitro and counteracting its biological effects in RAW-Blue cells, derived from RAW 264.7 macrophages. SB041 strongly bound purified LPS, especially that of E. coli, as proved by fluorescent displacement assay, and readily penetrated into LPS monolayers. However, the killing activity of SB041 against E. coli was not inhibited by increasing concentrations of LPS added to the medium. Checking the SB041 effect on LPS-induced activation of pattern recognition receptors (PRRs) in Raw-Blue cells revealed that while the peptide gave a statistically significant decrease in PRRs stimulation when RAW-Blue cells were challenged with P. aeruginosa LPS, the same was not seen when E. coli LPS was used to activate innate immune defense-like responses. Thus, as previously seen for other antimicrobial peptides, also for SB041 binding to LPS did not translate necessarily into LPS-neutralizing activity, suggesting that SB041-LPS interactions must be of complex nature.

  4. Inhibition of IRAK-4 activity for rescuing endotoxin LPS-induced septic mortality in mice by lonicerae flos extract

    Energy Technology Data Exchange (ETDEWEB)

    Park, Sun Hong; Roh, Eunmiri [College of Pharmacy, Chungbuk National University, Cheongju 361-763 (Korea, Republic of); Kim, Hyun Soo [Pharmaceutical R and D Center, Huons Co., Ltd., Anyang (Korea, Republic of); Baek, Seung-Il [College of Pharmacy, Chungbuk National University, Cheongju 361-763 (Korea, Republic of); Choi, Nam Song [Pharmaceutical R and D Center, Huons Co., Ltd., Anyang (Korea, Republic of); Kim, Narae; Hwang, Bang Yeon; Han, Sang-Bae [College of Pharmacy, Chungbuk National University, Cheongju 361-763 (Korea, Republic of); Kim, Youngsoo, E-mail: youngsoo@chungbuk.ac.kr [College of Pharmacy, Chungbuk National University, Cheongju 361-763 (Korea, Republic of)

    2013-12-13

    Highlights: •Lonicerae flos extract (HS-23) is a clinical candidate, Phase I for sepsis treatment. •Here, HS-23 or its major constituents rescued LPS-induced septic mortality in mice. •As a mechanism, they directly inhibited IRAK-4-catalyzed kinase activity. •Thus, they suppressed LPS-induced expression of NF-κB/AP-1-target inflammatory genes. -- Abstract: Lonicerae flos extract (HS-23) is a clinical candidate currently undergoing Phase I trial in lipopolysaccharide (LPS)-injected healthy human volunteers, but its molecular basis remains to be defined. Here, we investigated protective effects of HS-23 or its major constituents on Escherichia coli LPS-induced septic mortality in mice. Intravenous treatment with HS-23 rescued LPS-intoxicated C57BL/6J mice under septic conditions, and decreased the levels of cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β and high-mobility group box-1 (HMGB-1) in the blood. Chlorogenic acid (CGA) and its isomers were assigned as major constituents of HS-23 in the protection against endotoxemia. As a molecular mechanism, HS-23 or CGA isomers inhibited endotoxin LPS-induced autophosphorylation of the IL-1 receptor-associated kinase 4 (IRAK-4) in mouse peritoneal macrophages as well as the kinase activity of IRAK-4 in cell-free reactions. HS-23 consequently suppressed downstream pathways critical for LPS-induced activation of nuclear factor (NF)-κB or activating protein 1 (AP-1) in the peritoneal macrophages. HS-23 also inhibited various toll-like receptor agonists-induced nitric oxide (NO) production, and down-regulated LPS-induced expression of NF-κB/AP-1-target inflammatory genes in the cells. Taken together, HS-23 or CGA isomers exhibited anti-inflammatory therapy against LPS-induced septic mortality in mice, at least in part, mediated through the inhibition of IRAK-4.

  5. Oral supplementation with non-absorbable antibiotics or curcumin attenuates western diet-induced atherosclerosis and glucose intolerance in LDLR-/- mice--role of intestinal permeability and macrophage activation.

    Directory of Open Access Journals (Sweden)

    Siddhartha S Ghosh

    Full Text Available Association between circulating lipopolysaccharide (LPS and metabolic diseases (such as Type 2 Diabetes and atherosclerosis has shifted the focus from Western diet-induced changes in gut microbiota per se to release of gut bacteria-derived products into circulation as the possible mechanism for the chronic inflammatory state underlying the development of these diseases. Under physiological conditions, an intact intestinal barrier prevents this release of LPS underscoring the importance of examining and modulating the direct effects of Western diet on intestinal barrier function. In the present study we evaluated two strategies, namely selective gut decontamination and supplementation with oral curcumin, to modulate Western-diet (WD induced changes in intestinal barrier function and subsequent development of glucose intolerance and atherosclerosis. LDLR-/- mice were fed WD for 16 weeks and either received non-absorbable antibiotics (Neomycin and polymyxin in drinking water for selective gut decontamination or gavaged daily with curcumin. WD significantly increased intestinal permeability as assessed by in vivo translocation of FITC-dextran and plasma LPS levels. Selective gut decontamination and supplementation with curcumin significantly attenuated the WD-induced increase in plasma LPS levels (3.32 vs 1.90 or 1.51 EU/ml, respectively and improved intestinal barrier function at multiple levels (restoring intestinal alkaline phosphatase activity and expression of tight junction proteins, ZO-1 and Claudin-1. Consequently, both these interventions significantly reduced WD-induced glucose intolerance and atherosclerosis in LDLR-/- mice. Activation of macrophages by low levels of LPS (50 ng/ml and its exacerbation by fatty acids is likely the mechanism by which release of trace amounts of LPS into circulation due to disruption of intestinal barrier function induces the development of these diseases. These studies not only establish the important role

  6. ILK mediates LPS-induced vascular adhesion receptor expression and subsequent leucocyte trans-endothelial migration.

    Science.gov (United States)

    Hortelano, Sonsoles; López-Fontal, Raquel; Través, Paqui G; Villa, Natividad; Grashoff, Carsten; Boscá, Lisardo; Luque, Alfonso

    2010-05-01

    The inflammatory response to injurious agents is tightly regulated to avoid adverse consequences of inappropriate leucocyte accumulation or failed resolution. Lipopolysaccharide (LPS)-activated endothelium recruits leucocytes to the inflamed tissue through controlled expression of membrane-associated adhesion molecules. LPS responses in macrophages are known to be regulated by integrin-linked kinase (ILK); in this study, we investigated the role of ILK in the regulation of the LPS-elicited inflammatory response in endothelium. This study was performed on immortalized mouse endothelial cells (EC) isolated from lung and coronary vasculature. Cells were thoroughly characterized and the role of ILK in the regulation of the LPS response was investigated by suppressing ILK expression using siRNA and shRNA technologies. Phenotypic and functional analyses confirmed that the immortalized cells behaved as true EC. LPS induced the expression of the inflammatory genes E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). ILK knockdown impaired LPS-mediated endothelial activation by preventing the induction of ICAM-1 and VCAM-1. Blockade of the LPS-induced response inhibited the inflammatory-related processes of firm adhesion and trans-endothelial migration of leucocytes. ILK is involved in the expression of cell adhesion molecules by EC activated with the inflammatory stimulus LPS. This reduced expression modulates leucocyte adhesion to the endothelium and the extravasation process. This finding suggests ILK as a potential anti-inflammatory target for the development of vascular-specific treatments for inflammation-related diseases.

  7. Ascorbic acid pre-treated quartz stimulates TNF-α release in RAW 264.7 murine macrophages through ROS production and membrane lipid peroxidation

    Directory of Open Access Journals (Sweden)

    Benvenuto Federica

    2009-03-01

    Full Text Available Abstract Background Inhalation of crystalline silica induces a pulmonary fibrotic degeneration called silicosis caused by the inability of alveolar macrophages to dissolve the crystalline structure of phagocytosed quartz particles. Ascorbic acid is capable of partially dissolving quartz crystals, leading to an increase of soluble silica concentration and to the generation of new radical sites on the quartz surface. The reaction is specific for the crystalline forms of silica. It has been already demonstrated an increased cytotoxicity and stronger induction of pro-inflammatory cyclooxygenase-2 (COX-2 by ascorbic acid pre-treated quartz (QA compared to untreated quartz (Q in the murine macrophage cell line RAW 264.7. Methods Taking advantage of the enhanced macrophage response to QA as compared to Q particles, we investigated the first steps of cell activation and the contribution of early signals generated directly from the plasma membrane to the production of TNF-α, a cytokine that activates both inflammatory and fibrogenic pathways. Results Here we demonstrate that TNF-α mRNA synthesis and protein secretion are significantly increased in RAW 264.7 macrophages challenged with QA as compared to Q particles, and that the enhanced response is due to an increase of intracellular ROS. Plasma membrane-particle contact, in the absence of phagocytosis, is sufficient to trigger TNF-α production through a mechanism involving membrane lipid peroxidation and this appears to be even more detrimental to macrophage survival than particle phagocytosis itself. Conclusion Taken together these data suggest that an impairment of pulmonary macrophage phagocytosis, i.e. in the case of alcoholic subjects, could potentiate lung disease in silica-exposed individuals.

  8. Cytotoxicity and immunomodulatory effects of sol-gel combustion based titanium dioxide (TiO2) particles of large surface area on RAW 264.7 macrophages.

    Science.gov (United States)

    Dinesh, Palani; Suresh Yadav, C; Kannadasan, Sathanandhan; Rasool, Mahaboobkhan

    2017-09-01

    The current study was designed to investigate the cytotoxicity and immunomodulatory effects of sol-gel combustion based TiO2 particles (glycine and l-alanine as reducing agents) of large surface area on RAW 264.7 macrophages. RAW 264.7 macrophages exposed to varying concentrations of TiO2 particles (0.001 to 1000μg/ml) were assessed after 24h and showed a reduced cell viability at 100 and 1000μg/ml and increased LDH release at 10μg/ml. Furthermore, TiO2 particles (0.1, 1 and 10μg/ml) were utilized to assess the immune responses and intracellular ROS levels on RAW 264.7 macrophages. TiO2 particles at 10μg/ml showed increased mRNA expression of inflammatory cytokines (TNFα, IL-1β and IL-6), inflammatory mediators (iNOS and COX-2) and transcription factor (NFκB) similar to that of LPS stimulated macrophages. However, the mRNA expression levels were found near normal levels at lower concentrations (0.1 and 1μg/ml). In addition, TiO2 particles at 10μg/ml also increased the production of inflammatory cytokines (TNFα, IL-1β and IL-6) and intracellular ROS levels in RAW 264.7 macrophages similar to that of LPS stimulated macrophages. Conclusively, TiO2 particles prepared through this method at a concentration≤0.1μg/ml can be used for various biological applications with minimal immunomodulatory effects. Copyright © 2017. Published by Elsevier Ltd.

  9. Lack of TCRalphabeta+ CD8+ and TCRgammadelta+ lymphocytes ameliorates LPS induced orchitis in mice--preliminary histological observations.

    Science.gov (United States)

    Sliwa, Leopold; Macura, Barbara; Majewska-Szczepanik, Monika; Szczepanik, Marian

    2014-01-01

    The inflammation of the reproductive system can affect reproduction causing partial or complete infertility. It is well known that lipopolysaccharide (LPS) triggers an inflammatory response in the whole organism, including immunologically privileged organs, e.g. the testicles. Adult male TCRalpha-/-, TCRdelta-/-, CD1d-/- and beta2m-/- on B10.PL (H-2(u)) and B10.PL control mice were intraperitonealy (i.p.) injected with lipopolysaccharide (LPS). The animals were killed 24h and 10 days post LPS treatment and their gonads were prepared for microscopic examination. Histological changes in the testes after LPS injection were found only in control B10PL and CD1d-/- mice. The experiments revealed disturbances in Leydig's glands structure, blood vessel dilatation in the interstitial tissue as well as degeneration of seminal tubule epithelium, disruption ofspermatogenesis and subsequent decrease of sperm cell number in the tubule lumen. These changes were noticed mainly 10 days after LPS treatment. Lack of either TCRalphabeta+ CD8+ or TCRgammadelta+ lymphocytes diminishes the response of testicular macrophages to LPS whereas the absence of CD1d-dependent NKT cells does not affect macrophage reactivity.

  10. Effects of 4-methylumbelliferone and high molecular weight hyaluronic acid on the inflammation of corneal stromal cells induced by LPS.

    Science.gov (United States)

    Li, Fang; Hao, Peng; Liu, Guangjie; Wang, Weiyi; Han, Ruifang; Jiang, Zhixin; Li, Xuan

    2017-03-01

    To investigate the effects of hyaluronic acid (HA) on the inflammation of corneal fibroblasts induced by lipopolysaccharide (LPS). Primary rabbit corneal keratocytes were isolated with collagenase. The keratocytes were cultured in a serum-containing medium to induce corneal fibroblasts, which represented the wound repair phenotype of corneal keratocytes. Corneal fibroblasts were treated with LPS with or without 4-methylumbelliferone (4-MU) / high molecular weight hyaluronic acid (HMWHA). The gene expression was evaluated via real-time PCR, immunofluorescence, and western blot. The release of inflammatory cytokines and HA was determined by ELISA. Three types of hyaluronan synthase (HAS) were detected in corneal fibroblasts. LPS stimulation caused the up-regulation of HAS1 and HAS2 expression in corneal fibroblasts. LPS-induced HAS2 expression was significantly inhibited by 4-MU, and accompanied by decreased HA release by the corneal fibroblasts. In the corneal fibroblasts, 4-MU reduced the LPS-stimulated up-regulation of inflammatory cytokines including IL-1, IL-6, IL-8, TNF-α, and also attenuated the LPS-induced up-regulation of inflammatory related receptors including TLR2, TLR4, CD44, and CXCR1. HMWHA treatment resulted in a significant decline in the expression of IL-6, IL-8, TLR4, and CXCR1 responded to LPS stimulation. Consistent with mRNA expression of level, the up-regulation of the release of IL-6 and IL-8 induced by LPS in corneal fibroblasts was significantly attenuated by 4-MU and HMWHA. The LPS-induced expression of IL-8 and its receptor CXCR1 at both the mRNA and protein level were significantly attenuated by 4-MU and HMWHA. The inhibitor of HA synthesis 4-MU, and HMWHA successfully reduced LPS-induced inflammation in corneal fibroblasts. The mechanism might be via the inhibition of LPS-induced TLR4 up-regulation.

  11. In Vitro Effects of the Reduced Form of Coenzyme Q(10) on Secretion Levels of TNF-alpha and Chemokines in Response to LPS in the Human Monocytic Cell Line THP-1.

    Science.gov (United States)

    Schmelzer, Constance; Lorenz, Gerti; Rimbach, Gerald; Döring, Frank

    2009-01-01

    Ubiquinol-10 (QH(2)), the reduced form of Coenzyme Q(10) (CoQ(10)) serves as a potent antioxidant of lipid membranes. Because many antioxidants reveal potent anti-inflammatory effects, the influence of QH(2) on lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and chemokines were determined in the human monocytic cell line THP-1. Stimulation of cells with LPS resulted in a distinct release of Tumour necrosis factor-alpha (TNF-alpha), Macrophage inflammatory protein-1 alpha (MIP-1alpha), Regulated upon activation, normal T cell expressed and secreted (RANTES) and Monocyte chemotattractant protein-1 (MCP-1). The LPS-induced responses were significantly decreased by pre-incubation of cells with QH(2) to 60.27 +/- 9.3% (p = 0.0009), 48.13 +/- 6.93% (p = 0.0007) and 74.36 +/- 7.25% (p = 0.008) for TNF-alpha, MIP-1alpha and RANTES, respectively. In conclusion, our results indicate anti-inflammatory effects of the reduced form of CoQ(10) on various proinflammatory cytokines and chemokines in vitro.

  12. Salidroside attenuates LPS-induced pro-inflammatory cytokine responses and improves survival in murine endotoxemia.

    Science.gov (United States)

    Guan, Shuang; Feng, Haihua; Song, Bocui; Guo, Weixiao; Xiong, Ying; Huang, Guoren; Zhong, Weiting; Huo, Meixia; Chen, Na; Lu, Jing; Deng, Xuming

    2011-12-01

    Salidroside is a major component isolated from the Rhodiola rosea. In the present study, we investigated the anti-inflammatory effects of salidroside on cytokine production by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in vitro, and the results showed that salidroside reduced tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) secretions. This inspired us to further study the effects of salidroside in vivo. Salidroside significantly attenuated TNF-α, IL-1β and IL-6 productions in serum from mice challenged with LPS, and consistent with the results in vitro. In the murine model of endotoxemia, mice were treated with salidroside prior to or after LPS challenge. The results showed that salidroside significantly increased mouse survival. Further studies revealed that salidroside could downregulate LPS-induced nuclear transcription factor-қB (NF-қB) DNA-binding activation and ERK/MAPKs signal transduction pathways production in RAW 264.7 macrophages. These observations indicated that salidroside modulated early cytokine responses by blocking NF-қB and ERK/MAPKs activation, and thus, increased mouse survival. These effects of salidroside may be of potential usefulness in the treatment of inflammation-mediated endotoxemia. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Biological characterization of purified macrophage-derived neutrophil chemotactic factor

    Directory of Open Access Journals (Sweden)

    M. Dias-Baruffi

    1995-01-01

    Full Text Available We have recently described the purification of a 54 kDa acidic protein, identified as macrophage-derived neutrophil chemotactic factor (MNCF. This protein causes in vitro chemotaxis as well as in vivo neutrophil migration even in animals treated with dexamethasone. This in vivo chemotactic activity of MNCF in animals pretreated with dexamethasone is an uncommon characteristic which discriminates MNCF from known chemotactic cytokines. MNCF is released in the supernatant by macrophage monolayers stimulated with lipopolysaccharide (LPS. In the present study, we describe some biological characteristics of homogenous purified MNCF. When assayed in vitro, MNCF gave a bell-shaped dose–response curve. This in vitro activity was shown to be caused by haptotaxis. Unlike N-formyl-methionylleucyl- phenylalanine (FMLP or interleukin 8 (IL-8, the chemotactic activity of MNCF in vivo and in vitro, was inhibited by preincubation with D-galactose but not with D-mannose. In contrast with IL-8, MNCF did not bind to heparin and antiserum against IL-8 was ineffective in inhibiting its chemotactic activity. These data indicate that MNCF induces neutrophil migration through a carbohydrate recognition property, but by a mechanism different from that of the known chemokines. It is suggested that MNCF may be an important mediator in the recruitment of neutrophils via the formation of a substrate bound chemotactic gradient (haptotaxis in the inflamed tissues.

  14. Synthetic LPS-Binding Polymer Nanoparticles

    Science.gov (United States)

    Jiang, Tian

    Lipopolysaccharide (LPS), one of the principal components of most gram-negative bacteria's outer membrane, is a type of contaminant that can be frequently found in recombinant DNA products. Because of its strong and even lethal biological effects, selective LPS removal from bioproducts solution is of particular importance in the pharmaceutical and health care industries. In this thesis, for the first time, a proof-of-concept study on preparing LPS-binding hydrogel-like NPs through facile one-step free-radical polymerization was presented. With the incorporation of various hydrophobic (TBAm), cationic (APM, GUA) monomers and cross-linkers (BIS, PEG), a small library of NPs was constructed. Their FITC-LPS binding behaviors were investigated and compared with those of commercially available LPS-binding products. Moreover, the LPS binding selectivity of the NPs was also explored by studying the NPs-BSA interactions. The results showed that all NPs obtained generally presented higher FITC-LPS binding capacity in lower ionic strength buffer than higher ionic strength. However, unlike commercial poly-lysine cellulose and polymyxin B agarose beads' nearly linear increase of FITC-LPS binding with particle concentration, NPs exhibited serious aggregation and the binding quickly saturated or even decreased at high particle concentration. Among various types of NPs, higher FITC-LPS binding capacity was observed for those containing more hydrophobic monomers (TBAm). However, surprisingly, more cationic NPs with higher content of APM exhibited decreased FITC-LPS binding in high ionic strength conditions. Additionally, when new cationic monomer and cross-linker, GUA and PEG, were applied to replace APM and BIS, the obtained NPs showed improved FITC-LPS binding capacity at low NP concentration. But compared with APM- and BIS-containing NPs, the FITC-LPS binding capacity of GUA- and PEG-containing NPs saturated earlier. To investigate the NPs' binding to proteins, we tested the NPs

  15. P2X7R activation drives distinct IL-1 responses in dendritic cells compared to macrophages.

    Science.gov (United States)

    Englezou, Pavlos C; Rothwell, Simon W; Ainscough, Joseph S; Brough, David; Landsiedel, Robert; Verkhratsky, Alexei; Kimber, Ian; Dearman, Rebecca J

    2015-08-01

    The P2X(7)R is a functionally distinct member of the P2X family of non-selective cation channels associated with rapid activation of the inflammasome complex and signalling interleukin (IL)-1β release in macrophages. The main focus of this investigation was to compare P2X(7)R-driven IL-1 production by primary murine bone marrow derived dendritic cells (BMDC) and macrophages (BMM). P2X(7)R expression in murine BMDC and BMM at both transcriptional (P2X(7)A variant) and protein levels was demonstrated. Priming with lipopolysaccharide (LPS) and receptor activation with adenosine triphosphate (ATP) resulted in markedly enhanced IL-1 (α and β) secretion in BMDC compared with BMM. In both cell types IL-1 production was profoundly inhibited with a P2X(7)R-specific inhibitor (A-740003) demonstrating that this release is predominantly a P2X(7)R-dependent process. These data also suggest that P2X(7)R and caspase-1 activation drive IL-1α release from BMDC. Both cell types expressed constitutively the gain-of-function P2X(7)K as well as the full P2X(7)A variant at equivalent levels. LPS priming reduced significantly levels of P2X(7)A but not P2X(7)K transcripts in both BMDC and BMM. P2X(7)R-induced pore formation, assessed by YO-PRO-1 dye uptake, was greater in BMDC, and these cells were protected from cell death. These data demonstrate that DC and macrophages display distinct patterns of cytokine regulation, particularly with respect to IL-1, as a consequence of cell-type specific differences in the physicochemical properties of the P2X(7)R. Understanding the cell-specific regulation of these cytokines is essential for manipulating such responses in health and disease.

  16. The Alternative Faces of Macrophage Generate Osteoclasts

    Directory of Open Access Journals (Sweden)

    N. Lampiasi

    2016-01-01

    Full Text Available The understanding of how osteoclasts are generated and whether they can be altered by inflammatory stimuli is a topic of particular interest for osteoclastogenesis. It is known that the monocyte/macrophage lineage gives rise to osteoclasts (OCs by the action of macrophage colony stimulating factor (M-CSF and receptor activator of nuclear factor-kB ligand (RANKL, which induce cell differentiation through their receptors, c-fms and RANK, respectively. The multinucleated giant cells (MGCs generated by the engagement of RANK/RANKL are typical OCs. Nevertheless, very few studies have addressed the question of which subset of macrophages generates OCs. Indeed, two main subsets of macrophages are postulated, the inflammatory or classically activated type (M1 and the anti-inflammatory or alternatively activated type (M2. It has been proposed that macrophages can be polarized in vitro towards a predominantly M1 or M2 phenotype with the addition of granulocyte macrophage- (GM- CSF or M-CSF, respectively. Various inflammatory stimuli known to induce macrophage polarization, such as LPS or TNF-α, can alter the type of MGC obtained from RANKL-induced differentiation. This review aims to highlight the role of immune-related stimuli and factors in inducing macrophages towards the osteoclastogenesis choice.

  17. Functional Toll-like receptor 4 expressed in lactotrophs mediates LPS-induced proliferation in experimental pituitary hyperplasia

    Energy Technology Data Exchange (ETDEWEB)

    Sabatino, María Eugenia; Sosa, Liliana del Valle; Petiti, Juan Pablo; Mukdsi, Jorge Humberto [Centro de Microscopía Electrónica, Instituto de Investigaciones en Ciencias de la Salud (INICSA-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Av. Enrique Barros y Enfermera Gordillo, Ciudad Universitaria, CP 5000, Córdoba (Argentina); Mascanfroni, Iván Darío; Pellizas, Claudia Gabriela [Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Av. Haya de la Torre y Medina Allende, Ciudad Universitaria, CP 5000, Córdoba (Argentina); Gutiérrez, Silvina; Torres, Alicia Inés [Centro de Microscopía Electrónica, Instituto de Investigaciones en Ciencias de la Salud (INICSA-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Av. Enrique Barros y Enfermera Gordillo, Ciudad Universitaria, CP 5000, Córdoba (Argentina); De Paul, Ana Lucía, E-mail: adepaul@cmefcm.uncor.edu [Centro de Microscopía Electrónica, Instituto de Investigaciones en Ciencias de la Salud (INICSA-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Av. Enrique Barros y Enfermera Gordillo, Ciudad Universitaria, CP 5000, Córdoba (Argentina)

    2013-11-15

    Toll like receptor 4 (TLR4) has been characterized for its ability to recognize bacterial endotoxin lipopolysaccharide (LPS). Considering that infections or inflammatory processes might contribute to the progression of pituitary tumors, we analyzed the TLR4 functional role by evaluating the LPS effect on lactotroph proliferation in primary cultures from experimental pituitary tumors, and examined the involvement of PI3K-Akt and NF-κB activation in this effect. In addition, the role of 17β-estradiol as a possible modulator of LPS-induced PRL cell proliferation was further investigated. In estrogen-induced hyperplasic pituitaries, LPS triggered lactotroph cell proliferation. However, endotoxin failed to increase the number of lactotrophs taking up BrdU in normal pituitaries. Moreover, incubation with anti-TLR4 antibody significantly reduced LPS-induced lactotroph proliferation, suggesting a functional role of this receptor. As a sign of TLR4 activation, an LPS challenge increased IL-6 release in normal and tumoral cells. By flow cytometry, TLR4 baseline expression was revealed at the plasma membrane of tumoral lactotrophs, without changes noted in the percentage of double PRL/TLR4 positive cells after LPS stimulus. Increases in TLR4 intracellular expression were detected as well as rises in CD14, p-Akt and NF-κB after an LPS challenge, as assessed by western blotting. The TLR4/PRL and PRL/NF-κB co-localization was also corroborated by immunofluorescence and the involvement of PI3K/Akt signaling in lactotroph proliferation and IL-6 release was revealed through the PI3K inhibitor Ly-294002. In addition, 17β-estradiol attenuated the LPS-evoked increase in tumoral lactotroph proliferation and IL-6 release. Collectively these results demonstrate the presence of functional TLR4 in lactotrophs from estrogen-induced hyperplasic pituitaries, which responded to the proliferative stimulation and IL-6 release induced by LPS through TLR4/CD14, with a contribution of the PI3K

  18. Sesamin increases heme oxygenase-1 protein in RAW 264.7 macrophages through inhibiting its ubiquitination process.

    Science.gov (United States)

    Fukunaga, Mizuki; Ohnishi, Masatoshi; Shiratsuchi, Ayano; Kawakami, Takuya; Takahashi, Madoka; Motomura, Misato; Egusa, Kyohei; Urasaki, Tomoka; Inoue, Atsuko

    2014-10-15

    Sesamin is a major component in lignans of sesame seed oil, known to possess potent anti-oxidative capacity. In this study, the variation of heme oxygenase (HO)-1, a kind of anti-oxidative enzyme, by sesamin in murine macrophage cell line RAW 264.7 cells was investigated. Lipopolysaccharide (LPS; 10μg/ml) exposure tended to increase HO-1 protein expression. Co-treatment with 100μM sesamin for 12h up-regulated the HO-1 protein level increased by LPS; however, HO-1 mRNA was unaffected. Sesamin delayed the reversal, by the protein synthesis inhibitor cycloheximide (1μM), of the LPS-induced increase of HO-1 protein level. Meanwhile, sesamin suppressed LPS-induced expression of inducible nitric oxide (NO) synthase (iNOS) protein and associated NO release. LPS-induced increase of iNOS protein expression was also reversed by cycloheximide, which was not affected by sesamin, unlike HO-1. To clarify the mechanisms that underlie the up-regulation of HO-1 protein level by sesamin, the human embryonic kidney (HEK) 293T cell line transfected with Flag-tagged HO-1 was used. A proteasome inhibitor, MG-132 (10μM), stabilized HO-1 protein in HEK 293T cells. Co-treatment with sesamin decreased ubiquitinated HO-1 protein accumulation by MG-132. However, sesamin did not affect the proteasome activity. These findings suggest that sesamin disturbs the degradation of HO-1 protein through inhibiting its ubiquitination, resulting in HO-1 protein up-regulation.

  19. LPS-induced NF-{kappa}B expression in THP-1Blue cells correlates with neopterin production and activity of indoleamine 2,3-dioxygenase

    Energy Technology Data Exchange (ETDEWEB)

    Schroecksnadel, Sebastian [Division of Biological Chemistry, Innsbruck Medical University, Innsbruck (Austria); Jenny, Marcel [Division of Biological Chemistry, Innsbruck Medical University, Innsbruck (Austria); Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, Innsbruck (Austria); Kurz, Katharina [Department of Internal Medicine, Innsbruck Medical University, Innsbruck (Austria); Klein, Angela [Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, Innsbruck (Austria); Ledochowski, Maximilian [Department of Internal Medicine, Innsbruck Medical University, Innsbruck (Austria); Uberall, Florian [Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, Innsbruck (Austria); Fuchs, Dietmar, E-mail: dietmar.fuchs@i-med.ac.at [Division of Biological Chemistry, Innsbruck Medical University, Innsbruck (Austria)

    2010-09-03

    Research highlights: {yields} LPS induces NF-{kappa}B, neopterin formation and tryptophan degradation in THP-1 cells. {yields} Close dose- and time-dependent correlations exist between these biochemical events. {yields} Data provides some evidence for a parallel induction of them upon TLR stimulation. {yields} Results can be of considerable relevance also in vivo. -- Abstract: Neopterin production is induced in human monocyte-derived macrophages and dendritic cells upon stimulation with Th1-type cytokine interferon-{gamma} (IFN-{gamma}). In parallel, IFN-{gamma} induces the tryptophan-(trp)-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and triggers the formation of reactive oxygen species (ROS). Translocation of the signal transduction element nuclear factor-{kappa}B (NF-{kappa}B) is induced by ROS and accelerates the pro-inflammatory response by activation of other pro-inflammatory pathways. Therefore, a close relationship between NF-{kappa}B expression, the production of neopterin and the degradation of trp can be assumed, although this has not been demonstrated so far. In the present in vitro study we compared the influence of lipopolysaccharide (LPS) on NF-{kappa}B activation, neopterin formation and the degradation of trp in THP-1Blue cells, which represent the human myelomonocytic cell line THP-1 stably transfected with an NF-{kappa}B inducible reporter system. In cells stimulated with LPS, a significant induction of NF-{kappa}B was observed, and this was paralleled by an increase of kynureunine (kyn) and neopterin concentrations and a decline of trp. The increase of the kyn to trp quotient indicates accelerated IDO activity. Higher LPS concentrations and longer incubation of cells were associated with higher activities of all three biochemical pathways and significant correlations existed between NF-{kappa}B activation, neopterin release and trp degradation (all p < 0.001). We conclude that there is a parallel induction of NF-{kappa}B, neopterin

  20. M2 polarization enhances silica nanoparticle uptake by macrophages

    Directory of Open Access Journals (Sweden)

    Jessica eHoppstädter

    2015-03-01

    Full Text Available While silica nanoparticles have enabled numerous industrial and medical applications, their toxicological safety requires further evaluation. Macrophages are the major cell population responsible for nanoparticle clearance in vivo. The prevailing macrophage phenotype largely depends on the local immune status of the host. Whereas M1-polarized macrophages are considered as pro-inflammatory macrophages involved in host defense, M2 macrophages exhibit anti-inflammatory and wound-healing properties, but also promote tumor growth.We employed different models of M1 and M2 polarization: GM-CSF/LPS/IFN-gamma was used to generate primary human M1 cells and M-CSF/IL-10 to differentiate M2 monocyte-derived macrophages. PMA-differentiated THP-1 cells were polarized towards an M1 type by LPS/IFN-gamma and towards M2 by IL-10. Uptake of fluorescent silica nanoparticles (Ø 26 and 41 nm and microparticles (Ø 1.75 µm was quantified. At the concentration used (50 µg/ml, silica nanoparticles did not influence cell viability as assessed by MTT assay. Nanoparticle uptake was enhanced in M2-polarized primary human monocyte-derived macrophages compared with M1 cells, as shown by flow cytometric and microscopic approaches. In contrast, the uptake of microparticles did not differ between M1 and M2 phenotypes. M2 polarization was also associated with increased nanoparticle uptake in the macrophage-like THP-1 cell line. In accordance, in vivo polarized M2-like primary human tumor-associated macrophages (TAM obtained from lung tumors took up more nanoparticles than M1-like alveolar macrophages isolated from the surrounding lung tissue.In summary, our data indicate that the M2 polarization of macrophages promotes nanoparticle internalization. Therefore, the phenotypical differences between macrophage subsets should be taken into consideration in future investigations on nanosafety, but might also open up therapeutic perspectives allowing to specifically target M2

  1. Mouse ChemR23 is expressed in dendritic cell subsets and macrophages, and mediates an anti-inflammatory activity of chemerin in a lung disease model.

    Science.gov (United States)

    Luangsay, Souphalone; Wittamer, Valérie; Bondue, Benjamin; De Henau, Olivier; Rouger, Laurie; Brait, Maryse; Franssen, Jean-Denis; de Nadai, Patricia; Huaux, François; Parmentier, Marc

    2009-11-15

    Chemerin is the ligand of the ChemR23 receptor and a chemoattractant factor for human immature dendritic cells (DCs), macrophages, and NK cells. In this study, we characterized the mouse chemerin/ChemR23 system in terms of pharmacology, structure-function, distribution, and in vivo biological properties. Mouse chemerin is synthesized as an inactive precursor (prochemerin) requiring, as in human, the precise processing of its C terminus for generating an agonist of ChemR23. Mouse ChemR23 is highly expressed in immature plasmacytoid DCs and at lower levels in myeloid DCs, macrophages, and NK cells. Mouse prochemerin is expressed in most epithelial cells acting as barriers for pathogens but not in leukocytes. Chemerin promotes calcium mobilization and chemotaxis on DCs and macrophages and these functional responses were abrogated in ChemR23 knockout mice. In a mouse model of acute lung inflammation induced by LPS, chemerin displayed potent anti-inflammatory properties, reducing neutrophil infiltration and inflammatory cytokine release in a ChemR23-dependent manner. ChemR23 knockout mice were unresponsive to chemerin and displayed an increased neutrophil infiltrate following LPS challenge. Altogether, the mouse chemerin/ChemR23 system is structurally and functionally conserved between human and mouse, and mouse can therefore be considered as a good model for studying the anti-inflammatory role of this system in the regulation of immune responses and inflammatory diseases.

  2. The TLR Expression Pattern on Monocyte-Derived Macrophages for Lipopolysaccharid Stimulation of Calves

    Institute of Scientific and Technical Information of China (English)

    GUO Yi-jie; ZHAO Guo-Qi; HUO Yong-jiu; Sachi Tana-ka; Hisashi Aso; Takahiro Yamaguchi

    2009-01-01

    In this paper, toll-like receptor expression pattern in monocytes-derived macrophages by lipopolysaccharid (LPS) stimulation was examined. Jugular venous blood samples from 4 Japanese calves were obtained and the peripheral blood mononuclear cells (PBMC) were isolated. The PBMC were cultured for 7 d so as to collect monocytes-derived macrophages in Repcell. The PBMC were stimulated by LPS for 24 h and the mRNA expression pattern of TLR and cytokines in monocytes-derived macrophages (Mod-Mφ) was analyzed. Results showed that LPS stimulation of Mod-Mφ could increase the mRNA levels of the genes of TNF-α, IL-6, and IL-8. In addition, the mRNA levels of the genes of TNF-α and IL-6 in the group of LPS stimulation were most significantly (P<0.01) higher than those in control group and the mRNA levels of TLR1, 3, 5, 8, and 10 were significantly (P<0.05) decreased after LPS stimulation. There was no difference in the mRNA expressions of TLR2, 4, 6, and 7 between the groups of the control and LPS stimulation. Besides, expression of TLR9 was not found. It suggested that monocytes-derived macrophages could respond to LPS and they might take an important role in the innate immunity. The important function of the cells might contribute to better disease treatment.

  3. Endotoxin-induced enhancement of glucose influx into murine peritoneal macrophages via GLUT1.

    Science.gov (United States)

    Fukuzumi, M; Shinomiya, H; Shimizu, Y; Ohishi, K; Utsumi, S

    1996-01-01

    Hypoglycemia is among the most injurious metabolic disorders caused by endotoxemia. In experimental endotoxemia with lipopolysaccharide (LPS) in animals, a marked glucose consumption is observed in macrophage-rich organs. However, the direct effect of LPS on the uptake of glucose by macrophages has not been fully understood, and the present study was undertaken to shed light on this point. The consumption and uptake of glucose, as measured with 2-deoxy-D-[3H]glucose, by murine peritoneal exudate macrophages in culture were accelerated two- to threefold by stimulation with 3 ng of LPS per ml. The rate of glucose uptake reached a plateau after 20 min of stimulation and remained at the maximum as long as LPS was present. Northern (RNA) blot analysis with cDNA probes for five known isoforms of glucose transporter (GLUT) revealed that the expression of GLUT by macrophages was restricted to the GLUT1 isoform during LPS stimulation and the amount of GLUT1 mRNA was increased by the stimulation. These results suggest that macrophage responses to LPS are supported by a rapid and sustained glucose influx via GLUT1 and that this is a participating factor in the development of systemic hypoglycemia when endotoxemia is prolonged. PMID:8557327

  4. The role of the galU gene of uropathogenic Escherichia coli in modulating macrophage TNF-α response.

    Science.gov (United States)

    Meyer, Christian; Hoffmann, Christiane; Haas, Rainer; Schubert, Sören

    2015-12-01

    Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections (UTI). These bacteria are equipped with an arsenal of virulence factors, such as siderophores and adhesins enabling UPECs to sufficiently colonize the urinary tract of humans and animals. Such virulence factors manipulate and impair the recognition of UPECs by the host's innate immune system. Among those, factors like the TIR domain containing proteins in E. coli (TcpC) have been described to interfere with the Toll-like receptor 4 signaling cascade. Nevertheless, some UPECs such as strain UTI89 lack TcpC, but also manipulate the innate immune response. By a random mutant-library approach we identified the galU gene of strain UTI89 to be responsible for a reduced immune response of macrophages. Consequently, we created a site directed knockout mutant of the galU gene in strain UTI89. This mutant caused a significantly increased cytokine response when co-incubated with J774A.1 macrophages. This phenotype could be recomplemented in trans by insertion of a galU-expressing plasmid. No differences in the viability of macrophages co-incubated with either the wild-type (WT) or the ΔgalU mutant strain could be observed. Nor could any growth impairment be detected in the ΔgalU mutant compared to WT strain. Hence, the increased cytokine response was not due to differences in the bacterial cytotoxicity or bacterial counts in the assay. Our results also demonstrated a reduction of intracellular counts of UTI89ΔgalU in the infection model. We were able to show a loss of the O-polysaccharide side chain of the ΔgalU mutant LPS. A comparable LPS structure could be generated by the deletion of the waaL gene in the UTI89. This also caused an impaired immune modulation. In contrast, purified LPS was not sufficient to impair cytokine release of macrophages. Moreover, no differences could be detected by applying bacteria inactivated with heat or formalin treatment. From this, we assume that the

  5. Effects of sulfation pachymaran on the function of mouse peritoneal macrophages%硫酸化茯苓多糖对小鼠腹腔巨噬细胞功能的影响

    Institute of Scientific and Technical Information of China (English)

    高贵珍; 孙冰清; 吴超; 刘小阳

    2015-01-01

    研究硫酸化茯苓多糖(SP)对脂多糖(LPS)诱导的体内外小鼠巨噬细胞氧化损伤的影响.体外实验中,制备小鼠腹腔巨噬细胞,加入不同质量浓度(0~100 mg/mL)的 SP 和1 mg/mL 的 LPS 诱导剂,24 h 后检测细胞活力、一氧化氮(NO)、超氧阴离子自由基(O -2)及肿瘤坏死因子-α(TNF-α)含量.体内实验中,42只 ICR 小鼠随机分为空白组、模型组和预防组,采用腹腔注射法连续给药7天后收集腹腔巨噬细胞,检测多种生化指标.实验结果表明:LPS 可降低巨噬细胞的活性,增加 O -2的释放和 NO、TNF-α的分泌;SP 能够显著降低 NO的水平,提高巨噬细胞抗 O -2的活力,对于 TNF-α的含量没有显著影响.SP 可有效地改善 LPS 对小鼠腹腔巨噬细胞的氧化损伤作用.%The effects of sulfation pachymaran (SP)on the oxidative damage of murine peritoneal macro-phages induced by lipopolysaccharide (LPS)in vitro and vivo models were investigated.In vitro experi-ment,the peritoneal macrophages were harvested from ICR mice and then treated with 1 mg/mL of LPS and 0~100 mg/mL of SP.The cell activity,nitric oxide (NO),superoxide anion redical (O -2 )and tumor necrosis factor-α(TNF-α)were assayed respectively after 24 hours'cultivation.In vivo experiment,42 fe-male ICR mice were randomly divided into control group,prevention group and LPS group.After succes-sive administration of mice by intraperitoneal injection for 7 days,peritoneal macrophages were collected to detect different biochemical indexes.LPS reduced decreased cell viability of macrophages,increased the release of O -2 ,NO,and TNF-α;SP significantly reduced the production of O -2 and NO,No significant effects were observed on the release of TNF-α.SP effectively meliorates the oxidative damage of murine peritoneal macrophages induced by LPS.

  6. FAM19A4 is a novel cytokine ligand of formyl peptide receptor 1 (FPR1) and is able to promote the migration and phagocytosis of macrophages

    Science.gov (United States)

    Wang, Wenyan; Li, Ting; Wang, Xiaolin; Yuan, Wanxiong; Cheng, Yingying; Zhang, Heyu; Xu, Enquan; Zhang, Yingmei; Shi, Shuang; Ma, Dalong; Han, Wenling

    2015-01-01

    FAM19A4 is an abbreviation for family with sequence similarity 19 (chemokine (C–C motif)-like) member A4, which is a secretory protein expressed in low levels in normal tissues. The biological functions of FAM19A4 remain to be determined, and its potential receptor(s) is unclarified. In this study, we demonstrated that FAM19A4 was a classical secretory protein and we verified for the first time that its mature protein is composed of 95 amino acids. We found that the expression of this novel cytokine was upregulated in lipopolysaccharide (LPS)-stimulated monocytes and macrophages and was typically in polarized M1. FAM19A4 shows chemotactic activities on macrophages and enhances the macrophage phagocytosis of zymosan both in vitro and in vivo with noticeable increases of the phosphorylation of protein kinase B (Akt). FAM19A4 can also increase the release of reactive oxygen species (ROS) upon zymosan stimulation. Furthermore, based on receptor internalization, radio ligand binding assays and receptor blockage, we demonstrated for the first time that FAM19A4 is a novel ligand of formyl peptide receptor 1 (FPR1). The above data indicate that upon inflammatory stimulation, monocyte/macrophage-derived FAM19A4 may play a crucial role in the migration and activation of macrophages during pathogenic infections. PMID:25109685

  7. Lipopolysaccharide enhances the inhibition of NF-κB expression in NNK-mediated peritoneal macrophages

    Institute of Scientific and Technical Information of China (English)

    Bin Li; Mei Wu; Xiaoping Liu

    2014-01-01

    The aim of the study was to investigate the efect of lipopolysaccharide (LPS) on the expression of nuclear factor kappa B (NF-κB) in 4-(methylitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-mediated primary mouse peritoneal macrophagesin vitro.Methods: The activity of peritoneal macrophages treated with diferent concentrations of LPS was de-tected by MTT assay in rider to find the optimal concentration. Peritoneal macrophages were also treated with NNK (100-500μM), with or without LPS for 9 h. The expression of NF-κB was demonstrated via immunocytochemistry (ICC) and Western-blot, respectively.Results:The concentration of LPS at 25 μg/mL was found to be the optimal concentration to improve the activity of peritoneal macrophages (P < 0.01). Simultaneously, LPS (25 μg/mL) increased the expression of NF-κB in both the nucleus and cytoplasm and facilitated transfer of NF-κB to the nucleus. NNK treatment significantly inhibited the expression of NF-κB in a concentration-dependent manner, among the LPS-stimulated or unstimulated peritoneal macrophages, espe-cialy when cotreated with LPS (25 μg/mL,P < 0.01 ). Furthermore, NNK treatment (500 μM) with LPS yielded a significant decrease in NF-κB translocation to nucleus and inhibited the expression of NF-κB (P < 0.005).Conclusion: LPS enhances the suppression of NF-κB expression in NNK-mediated mouse peritoneal macrophages, which may provide a theoretical basis for the inhibition of cancer.

  8. Macrophage micro-RNA-155 promotes lipopolysaccharide-induced acute lung injury in mice and rats.

    Science.gov (United States)

    Wang, Wen; Liu, Zhi; Su, Jie; Chen, Wen-Sheng; Wang, Xiao-Wu; Bai, San-Xing; Zhang, Jin-Zhou; Yu, Shi-Qiang

    2016-08-01

    Micro-RNA (miR)-155 is a novel gene regulator with important roles in inflammation. Herein, our study aimed to explore the role of miR-155 in LPS-induced acute lung injury(ALI). ALI in mice was induced by intratracheally delivered LPS. Loss-of-function experiments performed on miR-155 knockout mice showed that miR-155 gene inactivation protected mice from LPS-induced ALI, as manifested by preserved lung permeability and reduced lung inflammation compared with wild-type controls. Bone marrow transplantation experiments identified leukocytes, but not lung parenchymal-derived miR-155-promoted acute lung inflammation. Real-time PCR analysis showed that the expression of miR-155 in lung tissue was greatly elevated in wild-type mice after LPS stimulation. In situ hybridization showed that miR-155 was mainly expressed in alveolar macrophages. In vitro experiments performed in isolated alveolar macrophages and polarized bone marrow-derived macrophages confirmed that miR-155 expression in macrophages was increased in response to LPS stimulation. Conversely, miR-155 gain-of-function in alveolar macrophages remarkably exaggerated LPS-induced acute lung injury. Molecular studies identified the inflammation repressor suppressor of cytokine signaling (SOCS-1) as the downstream target of miR-155. By binding to the 3'-UTR of the SOCS-1 mRNA, miR-155 downregulated SOCS-1 expression, thus, permitting the inflammatory response during lung injury. Finally, we generated a novel miR-155 knockout rat strain and showed that the proinflammatory role of miR-155 was conserved in rats. Our study identified miR-155 as a proinflammatory factor after LPS stimulation, and alveolar macrophages-derived miR-155 has an important role in LPS-induced ALI. Copyright © 2016 the American Physiological Society.

  9. Detection of Adhesion Molecules on Inflamed Macrophages at Early-Stage Using SERS Probe Gold Nanorods

    Institute of Scientific and Technical Information of China (English)

    Dakrong Pissuwan; Yusuke Hattori

    2017-01-01

    In recent years, it has been shown that inflammatory biomarkers can be used as an effective signal for disease diagnoses. The early detection of these signals provides useful information that could prevent the occurrence of severe diseases. Here, we employed surface-enhanced Raman scattering (SERS) probe gold nanorods (GNRs) as a tool for the early detection of inflammatory molecules in inflamed cells. A murine macrophage cell line (Raw264.7) stimulated with lipopolysaccharide (LPS) was used as a model in this study. The prepared SERS probe GNRs containing 4-mercapto-benzoic acid as a Raman reporter to generate SERS signals were used for detection of intracellular adhesion molecule-1 (ICAM-1) in macrophages after treatment with LPS for varying lengths of time. Our results show that SERS probe GNRs could detect significant differences in the expression of ICAM-1 molecules in LPS-treated macrophages compared to those in untreated macrophages after only 1 h of LPS treatment. In contrast, when using fluorescent labeling or enzyme-linked immunosorbent assays (ELISA) to detect ICAM-1, significant differences between inflamed and un-inflamed macrophages were not seen until the cells had been treated with LPS for 5 h. These results indicate that our SERS probe GNRs provide a higher sensitivity for detecting biomarker molecules in inflamed macrophages than the conventional fluorescence and ELISA techniques, and could therefore be useful as a potential diagnostic tool for managing disease risk.

  10. Urokinase plasminogen activator inhibits HIV virion release from macrophage-differentiated chronically infected cells via activation of RhoA and PKCε.

    Directory of Open Access Journals (Sweden)

    Francesca Graziano

    Full Text Available BACKGROUND: HIV replication in mononuclear phagocytes is a multi-step process regulated by viral and cellular proteins with the peculiar feature of virion budding and accumulation in intra-cytoplasmic vesicles. Interaction of urokinase-type plasminogen activator (uPA with its cell surface receptor (uPAR has been shown to favor virion accumulation in such sub-cellular compartment in primary monocyte-derived macrophages and chronically infected promonocytic U1 cells differentiated into macrophage-like cells by stimulation with phorbol myristate acetate (PMA. By adopting this latter model system, we have here investigated which intracellular signaling pathways were triggered by uPA/uPAR interaction leading the redirection of virion accumulation in intra-cytoplasmic vesicles. RESULTS: uPA induced activation of RhoA, PKCδ and PKCε in PMA-differentiated U1 cells. In the same conditions, RhoA, PKCδ and PKCε modulated uPA-induced cell adhesion and polarization, whereas only RhoA and PKCε were also responsible for the redirection of virions in intracellular vesicles. Distribution of G and F actin revealed that uPA reorganized the cytoskeleton in both adherent and polarized cells. The role of G and F actin isoforms was unveiled by the use of cytochalasin D, a cell-permeable fungal toxin that prevents F actin polymerization. Receptor-independent cytoskeleton remodeling by Cytochalasin D resulted in cell adhesion, polarization and intracellular accumulation of HIV virions similar to the effects gained with uPA. CONCLUSIONS: These findings illustrate the potential contribution of the uPA/uPAR system in the generation and/or maintenance of intra-cytoplasmic vesicles that actively accumulate virions, thus sustaining the presence of HIV reservoirs of macrophage origin. In addition, our observations also provide evidences that pathways controlling cytoskeleton remodeling and activation of PKCε bear relevance for the design of new antiviral strategies aimed

  11. Macrophage phenotype modulation by CXCL4 in vascular disease

    Directory of Open Access Journals (Sweden)

    Christian Albert Gleissner

    2012-01-01

    Full Text Available During atherogenesis, blood monocytes transmigrate into the subendothelial space and differentiate towards macrophages and foam cells. The major driver of this differentiation process is macrophage colony-stimulation factor (M-CSF. M-CSF-induced macrophages are important promoters of atherogenesis as demonstrated in M-CSF and M-CSF receptor knock out mice. However, M-CSF is not the only relevant promoter of macrophage differentiation. The platelet chemokine CXCL4 prevents monocyte apoptosis and promotes macrophage differentiation in vitro. It is secreted from activated platelets and has effects on various cell types relevant in atherogenesis. Knocking out the Pf4 gene coding for CXCL4 in Apoe-/- mice leads to reduced atherogenesis. Thus, it seems likely that CXC4-induced macrophages may have specific pro-atherogenic capacities. We have studied CXC4-induced differentiation of human macrophages using gene chips, systems biology and functional in vitro and ex vivo experiments. Our data indicate that CXCL4-induced macrophages are distinct from both their M-CSF-induced counterparts and other known macrophage polarizations like M1 macrophages (induced by LPS and interferon-gamma or M2 macrophages (induced by interleukin-4. CXCL4-induced macrophages have distinct phenotypic and functional characteristics, e.g. the complete loss of the hemoglobin-haptoglobin (Hb-Hp scavenger receptor CD163 which is necessary for effective hemoglobin clearance after plaque hemorrhage. Lack of CD163 is accompanied by the inability to upregulate the atheroprotective enzyme heme oxygenase-1 in response to Hb-Hp complexes.This review covers the current knowledge about CXCL4-induced macrophages, which based on their unique properties we have suggested to call these macrophages M4. CXCL4 may represent an important driver of macrophage heterogeneity within atherosclerotic lesions. Further dissecting its effects on macrophage differentiation may help to identify novel

  12. Anti-inflammatory activity of atractylenolide III through inhibition of nuclear factor-κB and mitogen-activated protein kinase pathways in mouse macrophages.

    Science.gov (United States)

    Ji, Guang-Quan; Chen, Ren-Qiong; Wang, Ling

    2016-01-01

    To elucidate the anti-inflammatory mechanisms involved, we investigated the effects of atractylenolide III (ATL-III) on cytokine expression, extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 mitogen-activated protein kinase (p38), C-Jun-N-terminal protein kinase1/2 (JNK1/2) and nuclear factor-κB (NF-κB) pathways in lipopolysaccharide (LPS)-induced RAW264.7 mouse macrophages. Macrophages were incubated with various concentrations (0, 25, 50, 100 μM) of ATL-III and/or LPS (1 μg/mL) for 24 h. The production of nitric oxide (NO) was determined by the Greiss reagent. The production of tumor necrosis factor alpha (TNF-α), prostaglandin E2 (PGE2) and interleukin 6 (IL-6) was determined by enzyme-linked immunosorbent assay (ELISA). Furthermore, macrophages were treated with ATL-III (0, 25, 100 μM) for 1 h and then stimulated by LPS. NF-κB, p38, JNK1/2 and ERK1/2 were determined by western blotting. We found ATL-III showed no inhibitory effect on cell proliferation at concentrations ranging from 1 μM to 100 μM. In addition, ATL-III decreased the release of NO, TNF-α, PGE2 and IL-6 in a dose-dependent manner and showed statistically significant at concentrations of 50 μM and 100 μM as well as cyclooxygenase-2 (COX-2) expression. Furthermore, ATL-III suppressed the transcriptional activity of NF-κB. ATL-III also inhibited the activation of ERK1/2, p38 and JNK1/2 in LPS-treated macrophages and showed statistically significant at concentrations of 25 μM and 100 μM. These data suggest that ATL-III shows an anti-inflammatory effect by suppressing the release of NO, PGE2, TNF-α and IL-6 related to the NF-κB- and MAPK-signaling pathways.

  13. Contribution of cyclopentenone prostaglandins to the resolution of inflammation through the potentiation of apoptosis in activated macrophages.

    Science.gov (United States)

    Hortelano, S; Castrillo, A; Alvarez, A M; Boscá, L

    2000-12-01

    Activation of the macrophage cell line RAW 264.7 with LPS and IFN-gamma induces apoptosis through the synthesis of high concentrations of NO due to the expression of NO synthase-2. In addition to NO, activated macrophages release other molecules involved in the inflammatory response, such as reactive oxygen intermediates and PGs. Treatment of macrophages with cyclopentenone PGs, which are synthesized late in the inflammatory onset, exerted a negative regulation on cell activation by impairing the expression of genes involved in host defense, among them NO synthase-2. However, despite the attenuation of NO synthesis, the percentage of apoptotic cells increased with respect to activated cells in the absence of cyclopentenone PGs. Analysis of the mechanisms by which these PGs enhanced apoptosis suggested a potentiation of superoxide anion synthesis that reacted with NO, leading to the formation of higher concentrations of peroxynitrite, a more reactive and proapoptotic molecule than the precursors. The effect of the cyclopentenone 15-deoxy-Delta(12,14)-PGJ(2) on superoxide synthesis was dependent on p38 mitogen-activated protein kinase activity, but was independent of the interaction with peroxisomal proliferator-activated receptor gamma. The potentiation of apoptosis induced by cyclopentenone PGs involved an increase in the release of cytochrome c from the mitochondria to the cytosol and in the nitration of this protein. These results suggest a role for cyclopentenone PGs in the resolution of inflammation by inducing apoptosis of activated cells.

  14. Resveratrol and its metabolites inhibit pro-inflammatory effects of lipopolysaccharides in U-937 macrophages in plasma-representative concentrations.

    Science.gov (United States)

    Walker, Jessica; Schueller, Katharina; Schaefer, Lisa-Marie; Pignitter, Marc; Esefelder, Laura; Somoza, Veronika

    2014-01-01

    Resveratrol has been shown to exploit various biological activities, including an anti-inflammatory activity. However, resveratrol is metabolized by phase II enzymes post-absorption to predominantly form glucuronides and sulfates. To investigate the anti-inflammatory effects of resveratrol and its dominating sulfated and glucuronated metabolites formed in vivo, U-937 macrophages were chosen as an immune-competent model system, known to release cytokines upon lipopolysaccharide stimulation. U-937 cells were stimulated with lipopolysaccharides from Escherichia coli (E. coli-LPS) to evoke an inflammatory reaction, and pre- or co-incubated with 1 or 10 μM of resveratrol (RES), resveratrol-3-sulfate (R3S), resveratrol-disulfates (RDS), resveratrol-3-glucuronide or resveratrol-4'-glucuronide. Time dependent gene expression of IL-6, IL-1α/β and IL-1R by qPCR was studied at 1 h, 3 h, 6 h, 9 h, and 24 h of incubation, and the release of IL-6 and TNF-α, after 6 h was analysed by means of non-magnetic or magnetic bead analysis. As a result, 10 μM resveratrol completely inhibited the E. coli-LPS-induced release of IL-6, while resveratrol-3-sulfate and resveratrol-disulfates decreased it by respective 84.2 ± 29.4% and 52.3 ± 39.5%. Whereas TNF-α release was reduced by 48.1 ± 15.4%, 33.0 ± 10.0% and 46.7 ± 8.7% by RES, R3S and RDS, respectively. These results show that not only resveratrol but also resveratrol-3-sulfate and resveratrol-disulfates exhibit an anti-inflammatory potential by counteracting an inflammatory challenge in U-937 macrophages at plasma representative concentrations.

  15. LPS-induced oxidative inflammation and hyperlipidemia in male rats: The protective role of Origanum majorana extract

    Directory of Open Access Journals (Sweden)

    Mayssaa M. Wahby

    2015-12-01

    Full Text Available The antimicrobicidal activity of the phenolic compounds in the methanolic extract of Origanum majorana was recommended. The present study aimed to investigate the protective effect of Origanum majorana against LPS-induced toxicity in rats. Forty-eight male Sprague-Dawley rats were randomly divided into four equal groups, with 12 rats each group. Group C was used as control, while group E was treated with plant extract orally for 10 days (0.5 mg/kg/day. Group I was given LPS at a single i.p. dose (10 mg/kg BW and group E + I was treated with plant extract (0.5 mg/kg/day for 10 days, followed by a single i.p. dose of LPS (10 mg/kg BW. The WBC count and the number of macrophages in addition to the nitric oxide level in the peritoneal fluid were determined. Also, the lipids profile and the levels of urea and creatinine were detected. In addition, the MDA, glutathione and total proteins, as well as AST and ALT activities, were measured in all groups. The results indicated that the LPS injection caused significant decrease in the WBC count, hepatic glutathione and the total proteins, as well as serum HDL-c. On the other hand, LPS injection showed significant increase in the number of peritoneal macrophages, the levels of nitric oxide and MDA. Moreover, the total lipids, total cholesterol, triglycerides, urea, and creatinine concentrations, as well as AST and ALT activities, were significantly elevated. The pretreatment with Origanum majorana extract prior to LPS antagonized and alleviated its toxic effects in the treated animals. The results indicated that the treatment with Origanum majorana extract alone did not affect the tested parameters, except the number of peritoneal macrophages, which were significantly decreased.

  16. CD163 and CD206 expression does not correlate with tolerance and cytokine production in LPS-tolerant human monocytes.

    Science.gov (United States)

    Alves-Januzzi, Amanda Barba; Brunialti, Milena Karina Colo; Salomao, Reinaldo

    2017-05-01

    Lipopolysaccharide (LPS)-tolerant monocytes produce small amounts of inflammatory cytokines, which is one of the characteristics of the alternative activated macrophages (AAM). These cells exhibited an increased expression of CD206 and CD163. Given the functional similarities of AAMs with the modulation of monocytes' functions observed during sepsis and LPS-tolerance, we evaluated whether the inhibition of inflammatory cytokine production by LPS-tolerant monocytes is associated with the phenotype of cells expressing CD206 and CD163. We investigated whether tolerant human monocytes would modulate their expression of CD206 and CD163, markers of alternative activation, and whether the level of their expression would be related to cytokines detection. Tolerance to LPS was induced in peripheral blood mononuclear cell by pre-incubating the cells with increasing concentrations of LPS. The expression of CD206 and CD163 and intracellular TNF-α and IL-6 was determined 24 h after LPS challenge by flow cytometry. No differences in CD163 expression were observed between tolerant and non-tolerant cells, while the expression of CD206, which was decreased following LPS stimulation in non-tolerized cells, was further reduced in tolerant cells. Decreased production of inflammatory cytokines was observed in the tolerized cells, regardless of the expression of CD163 and CD206, with the exception of IL-6 in CD206+ monocytes, which was similarly expressed in both tolerized and non-tolerized cells. The effect of LPS in the expression of CD163 and CD206 on monocytes is not reverted in LPS tolerant cells, and the inhibition of inflammatory cytokines in tolerant cells is not related with modulation of these receptors. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

  17. The phosphoproteome of toll-like receptor-activated macrophages

    DEFF Research Database (Denmark)

    Weintz, Gabriele; Olsen, Jesper Velgaard; Frühauf, Katja;

    2010-01-01

    Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome...

  18. Effect of azithromycin on the LPS-induced production and secretion of phospholipase A2 in lung cells.

    Science.gov (United States)

    Kitsiouli, Eirini; Antoniou, Georgia; Gotzou, Helen; Karagiannopoulos, Michalis; Basagiannis, Dimitris; Christoforidis, Savvas; Nakos, George; Lekka, Marilena E

    2015-07-01

    Azithromycin is a member of macrolides, utilized in the treatment of infections. Independently, these antibiotics also possess anti-inflammatory and immunomodulatory properties. Phospholipase A2 isotypes, which are implicated in the pathophysiology of inflammatory lung disorders, are produced by alveolar macrophages and other lung cells during inflammatory response and can promote lung injury by destructing lung surfactant. The aim of the study was to investigate whether in lung cells azithromycin can inhibit secretory and cytosolic phospholipases A2, (sPLA2) and (cPLA2), respectively, which are induced by an inflammatory trigger. In this respect, we studied the lipopolysaccharide (LPS)-mediated production or secretion of sPLA2 and cPLA2 from A549 cells, a cancer bronchial epithelial cell line, and alveolar macrophages, isolated from bronchoalveolar lavage fluid of ARDS and control patients without cardiopulmonary disease or sepsis. Pre-treatment of cells with azithromycin caused a dose-dependent decrease in the LPS-induced sPLA2-IIA levels in A549 cells. This inhibition was rather due to reduced PLA2G2A mRNA expression and secretion of sPLA2-IIA protein levels, as observed by western blotting and indirect immunofluorescence by confocal microscopy, respectively, than to the inhibition of the enzymic activity per se. On the contrary, azithromycin had no effect on the LPS-induced production or secretion of sPLA2-IIA from alveolar macrophages. The levels of LPS-induced c-PLA2 were not significantly affected by azithromycin in either cell type. We conclude that azithromycin exerts anti-inflammatory properties on lung epithelial cells through the inhibition of both the expression and secretion of LPS-induced sPLA2-IIA, while it does not affect alveolar macrophages.

  19. A comparison of the peritoneal cell population of pregnant rabbits after LPS or TNF-alpha induced septic shock.

    Science.gov (United States)

    Terlikowski, S; Sulkowska, M; Południewski, G; Dziecioł, J; Sobaniec-Lotowska, M; Musiatowicz, B; Kulikowski, M

    1997-01-01

    Septic shock is a catastrophic consequence of invasive infection. Unfortunately, recent advances in surgical and medical sciences have not significantly reduced the overall mortality from septic shock. Bacterial antigens stimulate a cascade of cytokine release; each cytokine helps the host to overcome infection, but their excessive production causes them to trigger events that lead to septic syndrome and shock. Tumour necrosis factor (TNF-alpha) has a pivotal role in orchestrating the events leading to septic shock. Intraperitoneal administration of certain substances can increase the number and phagocytic activity of cells, which reach naturally the site of infection. Activation of the immunity cells in the peritoneal cavity and their immunocompetence are found to be responsible for the organism protection against abdominal cavity infections. Macrophages, lymphocytes and granulocytes of low activity in the non-stimulated peritoneal cavity become significant due to the influence of numerous biologically active substances. This study was designed to determine the peritoneal response to local administration of LPS or TNF-alpha in the course of experimental septic shock.

  20. Resveratrol ameliorates LPS-induced acute lung injury via NLRP3 inflammasome modulation.

    Science.gov (United States)

    Jiang, Lei; Zhang, Lei; Kang, Kai; Fei, Dongsheng; Gong, Rui; Cao, Yanhui; Pan, Shangha; Zhao, Mingran; Zhao, Mingyan

    2016-12-01

    NLRP3 inflammasome plays a pivotal role in the development of acute lung injury (ALI), accelerating IL-1β and IL-18 release and inducing lung inflammation. Resveratrol, a natural phytoalexin, has anti-inflammatory properties via inhibition of oxidation, leukocyte priming, and production of inflammatory mediators. In this study, we aimed to investigate the effect of resveratrol on NLRP3 inflammasome in lipopolysaccharide-induced ALI. Mice were intratracheally instilled with 3mg/kg lipopolysaccharide (LPS) to induce ALI. Resveratrol treatment alleviated the LPS-induced lung pathological damage, lung edema and neutrophil infiltration. In addition, resveratrol reversed the LPS-mediated elevation of IL-1β and IL-18 level in the BAL fluids. In lung tissue, resveratrol also inhibited the LPS-induced NLRP3, ASC, caspase-1 mRNA and protein expression, and NLRP3 inflammasome activation. Moreover, resveratrol administration not only suppressed the NF-κB p65 nuclear translocation, NF-κB activity and ROS production in the LPS-treated mice, but also inhibited the LPS-induced thioredoxin-interacting protein (TXNIP) protein expression and interaction of TXNIP-NLRP3 in lung tissue. Meanwhile, resveratrol obviously induced SIRT1 mRNA and protein expression in the LPS-challenged mice. Taken together, our study suggests that resveratrol protects against LPS-induced lung injury by NLRP3 inflammasome inhibition. These findings further suggest that resveratrol may be of great value in the treatment of ALI and a potential and an effective pharmacological agent for inflammasome-relevant diseases. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  1. Inhibition of 5-Lipoxygenase Pathway Attenuates Acute Liver Failure by Inhibiting Macrophage Activation

    Directory of Open Access Journals (Sweden)

    Lu Li

    2014-01-01

    Full Text Available This study aimed to investigate the role of 5-lipoxygenase (5-LO in acute liver failure (ALF and changes in macrophage activation by blocking it. ALF was induced in rats by administration of D-galactosamine (D-GalN/lipopolysaccharide (LPS. Rats were injected intraperitoneally with AA-861 (a specific 5-LO inhibitor, 24 hr before D-GalN/LPS administration. After D-GalN/LPS injection, the liver tissue was collected for assessment of histology, macrophage microstructure, macrophage counts, 5-LO mRNA formation, protein expression, and concentration of leukotrienes. Serum was collected for detecting alanine aminotransferase (ALT, aspartate transaminase (AST, total bilirubin (Tbil, and tumor necrosis factor- (TNF-α. Twenty-four hours after injection, compared with controls, ALF rats were characterized by widespread hepatocyte necrosis and elevated ALT, AST, and Tbil, and 5-LO protein expression reached a peak. Liver leukotriene B4 was also significantly elevated. However, 5-LO mRNA reached a peak 8 hr after D-GalN/LPS injection. Simultaneously, the microstructure of macrophages was changed most significantly and macrophages counts were increased significantly. Moreover, serum TNF-α was also elevated. By contrast, AA-861 pretreatment significantly decreased liver necrosis as well as all of the parameters compared with the rats without pretreatment. Macrophages, via the 5-LO pathway, play a critical role in ALF, and 5-LO inhibitor significantly alleviates ALF, possibly related to macrophage inhibition.

  2. Macrophage receptors of polysaccharide isolated from a marine filamentous fungus Phoma herbarum YS4108

    Institute of Scientific and Technical Information of China (English)

    Song CHEN; Deng-ke YIN; Wen-bing YAO; Yi-dan WANG; Yi-ran ZHANG; Xiang-dong GAO

    2009-01-01

    Aim:YCP,a novel (1,4)-α-D-glucan,was isolated from the mycelium of the marine filamentous fungus Phoma herbarum YS4108.In this work,we investigated a YCP-binding cellular receptor expressed by macrophages and the intracellular signal transduction pathways involved in YCP-induced macrophage activation.Methods:Fluorescence-labeled YCP (fl-YCP) was prepared using the CDAP-activation method.Fluorescence confocal laser microscopy and fluorescence-activated cell sorting (FACS) were used to analyze the effect of fl-YCP on macrophages.To characterize the properties of the YCP receptor,carbohydrates and antibodies were used to inhibit the binding of fl-YCP to macrophages.Moreover,we investigated the role of membrane receptors Toll-like receptor 2 (TLR2),Toll-like receptor 4 (TLR4),Toll-like receptor 6 (TLR6) and complement receptor 3 (CR3).We also examined the role of the p38 kinase pathway in mediating nitric oxide (NO) production.Results:YCP had an in vitro stimulatory effect on the release of NO in macrophage,and fl-YCP can bind directly to receptors on the surface of macrophages in a time- and dose-dependent manner.Competition studies show that LPS,laminarin,anti-TLR4 antibody and anti-CD11b (CR3) antibody could inhibit fl-YCP binding to macrophages.Conversely,mannose,anti-TLR2 and anti-TLR6 antibody could not.Treatment of RAW264.7 cells with YCP resulted in significant activation of p38 in a time-dependent manner.The specific p38 inhibitor SB203580 abrogated YCP-induced NO generation.Treatment of RAW264.7 cells with anti-TLR4 antibody and anti-CR3 antibody significantly reduced YCP-induced NO production and p38 activation.Conclusion:We have demonstrated that YCP-induced NO production occurs through the TLR4 and CR3 membrane receptors in a p38 kinase-dependent manner in macrophages.

  3. New model of macrophage acquisition of the lymphatic endothelial phenotype.

    Directory of Open Access Journals (Sweden)

    Kelly L Hall

    Full Text Available BACKGROUND: Macrophage-derived lymphatic endothelial cell progenitors (M-LECPs contribute to new lymphatic vessel formation, but the mechanisms regulating their differentiation, recruitment, and function are poorly understood. Detailed characterization of M-LECPs is limited by low frequency in vivo and lack of model systems allowing in-depth molecular analyses in vitro. Our goal was to establish a cell culture model to characterize inflammation-induced macrophage-to-LECP differentiation under controlled conditions. METHODOLOGY/PRINCIPAL FINDINGS: Time-course analysis of diaphragms from lipopolysaccharide (LPS-treated mice revealed rapid mobilization of bone marrow-derived and peritoneal macrophages to the proximity of lymphatic vessels followed by widespread (∼50% incorporation of M-LECPs into the inflamed lymphatic vasculature. A differentiation shift toward the lymphatic phenotype was found in three LPS-induced subsets of activated macrophages that were positive for VEGFR-3 and many other lymphatic-specific markers. VEGFR-3 was strongly elevated in the early stage of macrophage transition to LECPs but undetectable in M-LECPs prior to vascular integration. Similar transient pattern of VEGFR-3 expression was found in RAW264.7 macrophages activated by LPS in vitro. Activated RAW264.7 cells co-expressed VEGF-C that induced an autocrine signaling loop as indicated by VEGFR-3 phosphorylation inhibited by a soluble receptor. LPS-activated RAW264.7 macrophages also showed a 68% overlap with endogenous CD11b(+/VEGFR-3(+ LECPs in the expression of lymphatic-specific genes. Moreover, when injected into LPS- but not saline-treated mice, GFP-tagged RAW264.7 cells massively infiltrated the inflamed diaphragm followed by integration into 18% of lymphatic vessels. CONCLUSIONS/SIGNIFICANCE: We present a new model for macrophage-LECP differentiation based on LPS activation of cultured RAW264.7 cells. This system designated here as the "RAW model" mimics

  4. Inhibition of Cinnamic Aldehyde on Migration and M1 Polarization of Raw264.7 Macrophages Induced by LPS%桂皮醛对LPS诱导的Raw264.7巨噬细胞迁移和M1极化的抑制作用研究

    Institute of Scientific and Technical Information of China (English)

    姜涛; 王剑

    2016-01-01

    目的 研究桂皮醛对脂多糖(LPS)诱导的Raw264.7巨噬细胞迁移和M1极化的抑制作用.方法 培养Raw264.7巨噬细胞,随机分为空白对照组(不加LPS及药物)、LPS组(加入终浓度为20μg· L-1的LPS)、桂皮醛(0.1μmol·L-1,0.3 μmol· L-1)组.应用Transwell小室法观察桂皮醛对LPS诱导的巨噬细胞迁移的影响;应用流式细胞术观察桂皮醛对LPS诱导的巨噬细胞M1极化的影响.结果 Transwell小室迁移实验结果与流式细胞术检测结果均显示,LPS(20 ug· L-1)可明显促进RAW264.7巨噬细胞的迁移及M1极化(P<0.01);与LPS组比较,桂皮醛0.1 μmol· L-1组、桂皮醛0.3 μmol· L-1组可明显抑制LPS诱导的Raw64.7巨噬细胞的迁移和M1极化(P<0.01),且桂皮醛0.3 μmol· L-1组作用优于桂皮醛0.1 μmol· L-1组(P<0.05).结论 桂皮醛对LPS诱导的RAW264.7巨噬细胞的迁移和M1极化有明显抑制作用,且呈浓度依赖性.

  5. Up-regulation of T lymphocyte and antibody production by inflammatory cytokines released by macrophage exposure to multi-walled carbon nanotubes.

    Science.gov (United States)

    Grecco, Ana Carolina P; Paula, Rosemeire F O; Mizutani, Erica; Sartorelli, Juliana C; Milani, Ana M; Longhini, Ana Leda F; Oliveira, Elaine C; Pradella, Fernando; Silva, Vania D R; Moraes, Adriel S; Peterlevitz, Alfredo C; Farias, Alessandro S; Ceragioli, Helder J; Santos, Leonilda M B; Baranauskas, Vitor

    2011-07-01

    Our data demonstrate that multi-walled carbon nanotubes (MWCNTs) are internalized by macrophages, subsequently activating them to produce interleukin (IL)-12 (IL-12). This cytokine induced the proliferative response of T lymphocytes to a nonspecific mitogen and to ovalbumin (OVA). This increase in the proliferative response was accompanied by an increase in the expression of pro-inflammatory cytokines, such as interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα) and IL-6, in mice inoculated with MWCNTs, whether or not they had been immunized with OVA. A decrease in the expression of transforming growth factor-beta (TGFβ) was observed in the mice treated with MWCNTs, whereas the suppression of the expression of both TGFβ and IL-10 was observed in mice that had been both treated and immunized. The activation of the T lymphocyte response by the pro-inflammatory cytokines leads to an increase in antibody production to OVA, suggesting the important immunostimulatory effect of carbon nanotubes.

  6. Up-regulation of T lymphocyte and antibody production by inflammatory cytokines released by macrophage exposure to multi-walled carbon nanotubes

    Science.gov (United States)

    Grecco, Ana Carolina P.; Paula, Rosemeire F. O.; Mizutani, Erica; Sartorelli, Juliana C.; Milani, Ana M.; Longhini, Ana Leda F.; Oliveira, Elaine C.; Pradella, Fernando; Silva, Vania D. R.; Moraes, Adriel S.; Peterlevitz, Alfredo C.; Farias, Alessandro S.; Ceragioli, Helder J.; Santos, Leonilda M. B.; Baranauskas, Vitor

    2011-07-01

    Our data demonstrate that multi-walled carbon nanotubes (MWCNTs) are internalized by macrophages, subsequently activating them to produce interleukin (IL)-12 (IL-12). This cytokine induced the proliferative response of T lymphocytes to a nonspecific mitogen and to ovalbumin (OVA). This increase in the proliferative response was accompanied by an increase in the expression of pro-inflammatory cytokines, such as interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα) and IL-6, in mice inoculated with MWCNTs, whether or not they had been immunized with OVA. A decrease in the expression of transforming growth factor-beta (TGFβ) was observed in the mice treated with MWCNTs, whereas the suppression of the expression of both TGFβ and IL-10 was observed in mice that had been both treated and immunized. The activation of the T lymphocyte response by the pro-inflammatory cytokines leads to an increase in antibody production to OVA, suggesting the important immunostimulatory effect of carbon nanotubes.

  7. Inhibition of Cholesterol Esterification Influences Cytokine Exspression in Lypopolisaccharide-activated P388D1 Macrophages

    Directory of Open Access Journals (Sweden)

    Rosa Rita Bonatesta

    2007-01-01

    Full Text Available Several in vivo and in vitro studies have demonstrated the involvement of infectious agents in the development of atherosclerosis. However, the mechanisms by which micro-organisms induce and/or aggravate atherosclerosis, are so far unclear. Accumulation of cholesterol esters and lipid laden cell formation are hallmark of the atherogenesis, however, the possible relationship between cholesterol esterification and the signal-transducing component of LPS recognition complex inducing cytokine secretion has not been yet investigated. In the present study, we investigated the effect of mevinolin, the ACAT inhibitor, Sandoz 58035, and plasma from statin-treated hypercholesterolemic patients on cholesterol metabolism and cytokine expression in LPS activated P388D1 macrophages. In P388D1 macrophages cholesterol synthesis and uptake, as well as cholesterol ester synthesis, were unchanged following LPS-activation. When cells were grown in presence of serum from patients under statin therapy, cholesterol esterification was lower compared to cells grown with plasma from healthy subjects, independently from the type of statin used. This effect was accompanied by inhibition of IL-1β expression in LPS activated cells. The ACAT inhibitor, Sandoz 58035, which completely blocked cholesterol esterification in normal and LPS-activated macrophages, prevented IL-1β and IL-6 over-expression in LPS activated cells. Although preliminary, these data point to a possible relationship between cholesterol esterification and cytokine production in macrophages, prospecting new possible mechanisms by which microbial or inflammatory agents may induce and/or accelerate the atherosclerotic process.

  8. Increase in the nitric oxide release without changes in cell viability of macrophages after laser therapy with 660 and 808 nm lasers.

    Science.gov (United States)

    Silva, Igor Henrique Morais; de Andrade, Samantha Cardoso; de Faria, Andreza Barkokebas Santos; Fonsêca, Deborah Daniela Diniz; Gueiros, Luiz Alcino Monteiro; Carvalho, Alessandra Albuquerque Tavares; da Silva, Wylla Tatiana Ferreira; de Castro, Raul Manhães; Leão, Jair Carneiro

    2016-12-01

    The aim of this study was to evaluate the influence of low-level laser therapy (LLLT) with different parameters and wavelengths on nitric oxide (NO) release and cell viability. Irradiation was performed with Ga-Al-As laser, continuous mode and wavelengths of 660 and 808 nm at different energy and power densities. For each wavelength, powers of 30, 50, and 100 mW and times of 10, 30, and 60 s were used. NO release was measured using Griess reaction, and cell viability was evaluated by mitochondrial reduction of bromide 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to formazan. LLLT promoted statistically significant changes in NO release and MTT value only at the wavelength of 660 nm (p < 0.05). LLLT also promoted an increase in the NO release and cell viability when the energy densities 64 (p = 0.04) and 214 J/cm(2) (p = 0.012), respectively, were used. LLLT has a significant impact on NO release without affecting cell viability, but the significance of these findings in the inflammatory response needs to be further studied.

  9. The Many Alternative Faces of Macrophage Activation.

    Directory of Open Access Journals (Sweden)

    David A. Hume

    2015-07-01

    Full Text Available Monocytes and macrophages provide the first line of defense against pathogens. They also initiate acquired immunity by processing and presenting antigens and provide the downstream effector functions. In large gene expression datasets from multiple cells and tissues, it is possible to identify sets of genes that are co-regulated with the transcription factors that regulate them. In macrophages, they include lineage-specific genes, interferon-responsive genes, early inflammatory genes, and those associated with endocytosis. Macrophages enter tissues and alter their function to deal with a wide range of challenges related to development and organogenesis, tissue injury, malignancy, sterile or pathogenic inflammatory stimuli. These stimuli alter gene expression to produce activated macrophages that are better equipped to eliminate the cause of their influx, and to restore homeostasis. Activation or polarization states of macrophages have been classified as classical and alternative or M1 and M2. These proposed states of cells are not supported by large-scale transcriptomic data, including macrophage-associated signatures from large cancer tissue datasets, where the supposed markers do not correlate with other. Individual macrophage cells differ markedly from each other, and change their functions in response to doses and combinations of agonists and time. The most studied macrophage activation response is the transcriptional cascade initiated by the TLR4 agonist lipopolysaccharide (LPS. This response is reviewed herein. The network architecture is conserved across species, but many of the target genes evolve rapidly and differ between mouse and human. There is also considerable divergence in the sets of target genes between mouse strains, between individuals and in other species such as pigs. The data and publication deluge related to macrophage activation requires the development of new analytical tools, and ways of presenting information in an

  10. Macrophage responsiveness to light therapy

    Energy Technology Data Exchange (ETDEWEB)

    Young, S.; Bolton, P.; Dyson, M.; Harvey, W.; Diamantopoulos, C. (United Medical School, London (England))

    1989-01-01

    Macrophages are a source of many important mediators of wound repair. It was the purpose of this study to see if light could stimulate the release of these mediators. In this study an established macrophage-like cell line (U-937) was used. The cells were exposed in culture to the following wavelengths of light: 660 nm, 820 nm, 870 nm, and 880 nm. The 820-nm source was coherent and polarised, and the others were non-coherent. Twelve hours after exposure the macrophage supernatant was removed and placed on 3T3 fibroblast cultures. Fibroblast proliferation was assessed over a 5-day period. The results showed that 660-nm, 820-nm, and 870-nm wavelengths encouraged the macrophages to release factors that stimulated fibroblast proliferation above the control levels, whereas the 880-nm wavelength either inhibited the release of these factors or encouraged the release of some inhibitory factors of fibroblast proliferation. These results suggest that light at certain wavelengths may be a useful therapeutic agent by providing a means of either stimulating or inhibiting fibroblast proliferation where necessary. At certain wavelengths coherence is not essential.

  11. Learning in a simple biological system: a pilot study of classical conditioning of human macrophages in vitro

    Directory of Open Access Journals (Sweden)

    Nilsonne Gustav

    2011-11-01

    Full Text Available Abstract Recent advances in cell biology and gene regulation suggest mechanisms whereby associative learning could be performed by single cells. Therefore, we explored a model of classical conditioning in human macrophages in vitro. In macrophage cultures, bacterial lipopolysaccharide (LPS; unconditioned stimulus was paired once with streptomycin (conditioned stimulus. Secretion of interleukin-6 (IL-6 was used as response measure. At evocation, conditioning was not observed. Levels of IL-6 were higher only in those cultures that had been exposed to LPS in the learning phase (p's However, habituation was evident, with a 62% loss of the IL-6 response after three LPS presentations (p

  12. Macrophage migration and invasion is regulated by MMP10 expression.

    Directory of Open Access Journals (Sweden)

    Megan Y Murray

    Full Text Available This study was designed to identify metalloproteinase determinants of macrophage migration and led to the specific hypothesis that matrix metalloproteinase 10 (MMP10/stromelysin-2 facilitates macrophage migration. We first profiled expression of all MMPs in LPS-stimulated primary murine bone marrow-derived macrophages and Raw264.7 cells and found that MMP10 was stimulated early (3 h and down-regulated later (24 h. Based on this pattern of expression, we speculated that MMP10 plays a role in macrophage responses, such as migration. Indeed, using time lapse microscopy, we found that RNAi silencing of MMP10 in primary macrophages resulted in markedly reduced migration, which was reversed with exogenous active MMP10 protein. Mmp10 (-/- bone marrow-derived macrophages displayed significantly reduced migration over a two-dimensional fibronectin matrix. Invasion of primary wild-type macrophages into Matrigel supplemented with fibronectin was also markedly impaired in Mmp10 (-/- cells. MMP10 expression in macrophages thus emerges as an important moderator of cell migration and invasion. These findings support the hypothesis that MMP10 promotes macrophage movement and may have implications in understanding the control of macrophages in several pathologies, including the abnormal wound healing response associated with pro-inflammatory conditions.

  13. Inflammatory Macrophage Phenotype in BTBR T+tf/J Mice

    Directory of Open Access Journals (Sweden)

    Paul eAshwood

    2013-09-01

    Full Text Available Although autism is a behaviorally defined disorder, many studies report an association with increased pro-inflammatory cytokine production. Recent characterization of the BTBR T+tf/J (BTBR inbred mouse strain has revealed several behavioral characteristics including social deficits, repetitive behavior, and atypical vocalizations which may be relevant to autism. We therefore hypothesized that asocial BTBR mice, which exhibit autism-like behaviors, may have an inflammatory immune profile similar to that observed in children with autism. The objectives of this study were to characterize the myeloid immune profile of BTBR mice and to explore their associations with autism-relevant behaviors. C57BL/6J (C57 mice and BTBR mice were tested for social interest and repetitive self-grooming behavior. Cytokine production was measured in bone-marrow derived macrophages incubated for 24 hours in either growth media alone, LPS, IL-4/ LPS, or IFNγ/ LPS to ascertain any M1/M2 skewing. After LPS stimulation, BTBR macrophages produced higher levels of IL-6, MCP-1, and MIP-1α and lower IL-10 (p<0.01 that C57 mice, suggesting an exaggerated inflammatory profile. After exposure to IL-4/LPS BTBR macrophages produced less IL-10 than C57 macrophages and more IL-12p40 (p<0.01 suggesting poor M2 polarization. Levels of IL-12(p70 (p<0.05 were higher in BTBR macrophages after IFNγ/LPS stimulation, suggesting enhanced M1 polarization. We further observed a positive correlation between grooming frequency, and production of IL-12(p40, IL-12p70, IL-6, and TNFα (p<0.05 after treatment with IFNγ/LPS across both strains. Collectively, these data suggest that the asocial BTBR mouse strain exhibits a more inflammatory, or M1, macrophage profile in comparison to social C57 strain. We have further demonstrated a relationship between this relative increase in inflammation and repetitive grooming behavior, which may have relevance to repetitive and stereotyped behavior of autism.

  14. Lignans from Arctium lappa and their inhibition of LPS-induced nitric oxide production.

    Science.gov (United States)

    Park, So Young; Hong, Seong Su; Han, Xiang Hua; Hwang, Ji Sang; Lee, Dongho; Ro, Jai Seup; Hwang, Bang Yeon

    2007-01-01

    A new butyrolactone sesquilignan, isolappaol C (1), together with four known lignans, lappaol C (2), lappaol D (3), lappaol F (4), and diarctigenin (5), were isolated from the methanolic extract of the seeds from the Arctium lappa plant. The structure of isolappaol C (1) was determined by spectral analysis including 1D- and 2D-NMR. All the isolates were evaluated for their inhibitory effects on the LPS-induced nitric oxide production using murine macrophage RAW264.7 cells. Lappaol F (4) and diarctigenin (5) strongly inhibited NO production in the LPS-stimulated RAW264.7 cells with IC(50) values of 9.5 and 9.6 microM, respectively.

  15. Injury-induced GR-1+ macrophage expansion and activation occurs independently of CD4 T-cell influence.

    Science.gov (United States)

    O'Leary, Fionnuala M; Tajima, Goro; Delisle, Adam J; Ikeda, Kimiko; Dolan, Sinead M; Hanschen, Marc; Mannick, John A; Lederer, James A

    2011-08-01

    Burn injury initiates an enhanced inflammatory condition referred to as the systemic inflammatory response syndrome or the two-hit response phenotype. Prior reports indicated that macrophages respond to injury and demonstrate a heightened reactivity to Toll-like receptor stimulation. Since we and others observed a significant increase in splenic GR-1 F4/80 CD11b macrophages in burn-injured mice, we wished to test if these macrophages might be the primary macrophage subset that shows heightened LPS reactivity. We report here that burn injury promoted higher level TNF-α expression in GR-1, but not GR-1 macrophages, after LPS activation both in vivo and ex vivo. We next tested whether CD4 T cells, which are known to suppress injury-induced inflammatory responses, might control the activation and expansion of GR-1 macrophages. Interestingly, we found that GR-1 macrophage expansion and LPS-induced TNF-α expression were not significantly different between wild-type and CD4 T cell-deficient CD4(-/-) mice. However, further investigations showed that LPS-induced TNF-α production was significantly influenced by CD4 T cells. Taken together, these data indicate that GR-1 F4/80 CD11b macrophages represent the primary macrophage subset that expands in response to burn injury and that CD4 T cells do not influence the GR-1 macrophage expansion process, but do suppress LPS-induced TNF-α production. These data suggest that modulating GR-1 macrophage activation as well as CD4 T cell responses after severe injury may help control the development of systemic inflammatory response syndrome and the two-hit response phenotype.

  16. Up-regulation of T lymphocyte and antibody production by inflammatory cytokines released by macrophage exposure to multi-walled carbon nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    Grecco, Ana Carolina P; Mizutani, Erica; Peterlevitz, Alfredo C; Ceragioli, Helder J; Baranauskas, Vitor [Faculdade de Engenharia Eletrica e Computacao, Universidade de Campinas, Campinas, SP (Brazil); Paula, Rosemeire F O; Sartorelli, Juliana C; Milani, Ana M; Longhini, Ana Leda F; Oliveira, Elaine C; Pradella, Fernando; Silva, Vania D R; Moraes, Adriel S; Farias, Alessandro S; Santos, Leonilda M B, E-mail: leonilda@unicamp.br [Laboratorio de Neuroimunologia, Departamento Genetica, Evolucao e Bioagentes, Instituto de Biologia, Universidade de Campinas, Campinas, SP (Brazil)

    2011-07-01

    Our data demonstrate that multi-walled carbon nanotubes (MWCNTs) are internalized by macrophages, subsequently activating them to produce interleukin (IL)-12 (IL-12). This cytokine induced the proliferative response of T lymphocytes to a nonspecific mitogen and to ovalbumin (OVA). This increase in the proliferative response was accompanied by an increase in the expression of pro-inflammatory cytokines, such as interferon-gamma (IFN{gamma}), tumor necrosis factor-alpha (TNF{alpha}) and IL-6, in mice inoculated with MWCNTs, whether or not they had been immunized with OVA. A decrease in the expression of transforming growth factor-beta (TGF{beta}) was observed in the mice treated with MWCNTs, whereas the suppression of the expression of both TGF{beta} and IL-10 was observed in mice that had been both treated and immunized. The activation of the T lymphocyte response by the pro-inflammatory cytokines leads to an increase in antibody production to OVA, suggesting the important immunostimulatory effect of carbon nanotubes.

  17. Vibrio vulnificus MO6-24/O Lipopolysaccharide Stimulates Superoxide Anion, Thromboxane B2, Matrix Metalloproteinase-9, Cytokine and Chemokine Release by Rat Brain Microglia in Vitro

    Directory of Open Access Journals (Sweden)

    Alejandro M. S. Mayer

    2014-03-01

    Full Text Available Although human exposure to Gram-negative Vibrio vulnificus (V. vulnificus lipopolysaccharide (LPS has been reported to result in septic shock, its impact on the central nervous system’s innate immunity remains undetermined. The purpose of this study was to determine whether V. vulnificus MO6-24/O LPS might activate rat microglia in vitro and stimulate the release of superoxide anion (O2−, a reactive oxygen species known to cause oxidative stress and neuronal injury in vivo. Brain microglia were isolated from neonatal rats, and then treated with either V. vulnificus MO6-24/O LPS or Escherichia coli O26:B6 LPS for 17 hours in vitro. O2− was determined by cytochrome C reduction, and matrix metalloproteinase-2 (MMP-2 and MMP-9 by gelatinase zymography. Generation of cytokines tumor necrosis factor alpha (TNF-α, interleukin-1 alpha (IL-1α, IL-6, and transforming growth factor-beta 1 (TGF-β1, chemokines macrophage inflammatory protein (MIP-1α/chemokine (C-C motif ligand 3 (CCL3, MIP-2/chemokine (C-X-C motif ligand 2 (CXCL2, monocyte chemotactic protein-1 (MCP-1/CCL2, and cytokine-induced neutrophil chemoattractant-2alpha/beta (CINC-2α/β/CXCL3, and brain-derived neurotrophic factor (BDNF, were determined by specific immunoassays. Priming of rat microglia by V. vulnificus MO6-24/O LPS in vitro yielded a bell-shaped dose-response curve for PMA (phorbol 12-myristate 13-acetate-stimulated O2− generation: (1 0.1–1 ng/mL V. vulnificus LPS enhanced O2− generation significantly but with limited inflammatory mediator generation; (2 10–100 ng/mL V. vulnificus LPS maximized O2− generation with concomitant release of thromboxane B2 (TXB2, matrix metalloproteinase-9 (MMP-9, and several cytokines and chemokines; (3 1000–100,000 ng/mL V. vulnificus LPS, with the exception of TXB2, yielded both attenuated O2− production, and a progressive decrease in MMP-9, cytokines and chemokines investigated. Thus concentration-dependent treatment of

  18. Vibrio vulnificus MO6-24/O lipopolysaccharide stimulates superoxide anion, thromboxane B₂, matrix metalloproteinase-9, cytokine and chemokine release by rat brain microglia in vitro.

    Science.gov (United States)

    Mayer, Alejandro M S; Hall, Mary L; Holland, Michael; De Castro, Cristina; Molinaro, Antonio; Aldulescu, Monica; Frenkel, Jeffrey; Ottenhoff, Lauren; Rowley, David; Powell, Jan

    2014-03-26

    Although human exposure to Gram-negative Vibrio vulnificus (V. vulnificus) lipopolysaccharide (LPS) has been reported to result in septic shock, its impact on the central nervous system's innate immunity remains undetermined. The purpose of this study was to determine whether V. vulnificus MO6-24/O LPS might activate rat microglia in vitro and stimulate the release of superoxide anion (O₂⁻), a reactive oxygen species known to cause oxidative stress and neuronal injury in vivo. Brain microglia were isolated from neonatal rats, and then treated with either V. vulnificus MO6-24/O LPS or Escherichia coli O26:B6 LPS for 17 hours in vitro. O₂⁻ was determined by cytochrome C reduction, and matrix metalloproteinase-2 (MMP-2) and MMP-9 by gelatinase zymography. Generation of cytokines tumor necrosis factor alpha (TNF-α), interleukin-1 alpha (IL-1α), IL-6, and transforming growth factor-beta 1 (TGF-β1), chemokines macrophage inflammatory protein (MIP-1α)/chemokine (C-C motif) ligand 3 (CCL3), MIP-2/chemokine (C-X-C motif) ligand 2 (CXCL2), monocyte chemotactic protein-1 (MCP-1)/CCL2, and cytokine-induced neutrophil chemoattractant-2alpha/beta (CINC-2α/β)/CXCL3, and brain-derived neurotrophic factor (BDNF), were determined by specific immunoassays. Priming of rat microglia by V. vulnificus MO6-24/O LPS in vitro yielded a bell-shaped dose-response curve for PMA (phorbol 12-myristate 13-acetate)-stimulated O₂⁻ generation: (1) 0.1-1 ng/mL V. vulnificus LPS enhanced O₂⁻ generation significantly but with limited inflammatory mediator generation; (2) 10-100 ng/mL V. vulnificus LPS maximized O₂⁻ generation with concomitant release of thromboxane B2 (TXB2), matrix metalloproteinase-9 (MMP-9), and several cytokines and chemokines; (3) 1000-100,000 ng/mL V. vulnificus LPS, with the exception of TXB2, yielded both attenuated O₂⁻ production, and a progressive decrease in MMP-9, cytokines and chemokines investigated. Thus concentration-dependent treatment of

  19. Fisetin Suppresses Macrophage-Mediated Inflammatory Responses by Blockade of Src and Syk.

    Science.gov (United States)

    Kim, Jun Ho; Kim, Mi-Yeon; Kim, Jong-Hoon; Cho, Jae Youl

    2015-09-01

    Flavonoids, such as fisetin (3,7,3',4'-tetrahydroxyflavone), are plant secondary metabolites. It has been reported that fisetin is able to perform numerous pharmacological roles including anti-inflammatory, anti-microbial, and anti-cancer activities; however, the exact anti-inflammatory mechanism of fisetin is not understood. In this study, the pharmacological action modes of fisetin in lipopolysaccharide (LPS)-stimulated macrophage-like cells were elucidated by using immunoblotting analysis, kinase assays, and an overexpression strategy. Fisetin diminished the release of nitric oxide (NO) and reduced the mRNA levels of inducible NO synthase (iNOS), tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 in LPS-stimulated RAW264.7 cells without displaying cytotoxicity. This compound also blocked the nuclear translocation of p65/nuclear factor (NF)-κB. In agreement, the upstream phosphorylation events for NF-κB activation, composed of Src, Syk, and IκBα, were also reduced by fisetin. The phospho-Src level, triggered by overexpression of wild-type Src, was also inhibited by fisetin. Therefore, these results strongly suggest that fisetin can be considered a bioactive immunomodulatory compound with anti-inflammatory properties through suppression of Src and Syk activities.

  20. Evidence for lipopolysaccharide-induced differentiation of RAW264⋅ 7 murine macrophage cell line into dendritic like cells

    Indian Academy of Sciences (India)

    Rajiv K Saxena; Val Vallyathan; Daniel M Lewis

    2003-02-01

    Effect of lipopolysaccharide (LPS) on RAW264.7 macrophage cell line was studied. LPS-treated RAW264.7 cells increased in cell size and acquired distinct dendritic morphology. At the optimal dose of LPS (1 g/ml), almost 70% RAW264.7 cells acquired dendritic morphology. Flow cytometric studies indicate that the cell surface markers known to be expressed on dendritic cells and involved in antigen presentation and T cell activation (B7.1, B7.2, CD40, MHC class II antigens and CD1d) were also markedly upregulated on LPS-treated RAW264.7 cells. Our results suggest the possibility that LPS by itself could constitute a sufficient signal for differentiation of macrophages into DC-like cells.

  1. The proinflammatory cytokine response to Chlamydia trachomatis elementary bodies in human macrophages is partly mediated by a lipoprotein, the macrophage infectivity potentiator, through TLR2/TLR1/TLR6 and CD14.

    Science.gov (United States)

    Bas, Sylvette; Neff, Laurence; Vuillet, Madeleine; Spenato, Ursula; Seya, Tsukasa; Matsumoto, Misako; Gabay, Cem

    2008-01-15

    Chlamydiae components and signaling pathway(s) responsible for the production of proinflammatory cytokines by human monocytes/macrophages are not clearly identified. To this aim, Chlamydia trachomatis-inactivated elementary bodies (EB) as well as the following seven individual Ags were tested for their ability to induce the production of proinflammatory cytokines by human monocytes/macrophages and THP-1 cells: purified LPS, recombinant heat shock protein (rhsp)70, rhsp60, rhsp10, recombinant polypeptide encoded by open reading frame 3 of the plasmid (rpgp3), recombinant macrophage infectivity potentiator (rMip), and recombinant outer membrane protein 2 (rOmp2). Aside from EB, rMip displayed the highest ability to induce release of IL-1beta, TNF-alpha, IL-6, and IL-8. rMip proinflammatory activity could not be attributed to Escherichia coli LPS contamination as determined by the Limulus Amoebocyte lysate assay, insensitivity to polymyxin B (50 microg/ml), and different serum requirement. We have recently demonstrated that Mip is a "classical" bacterial lipoprotein, exposed at the surface of EB. The proinflammatory activity of EB was significantly attenuated in the presence of polyclonal Ab to rMip. Native Mip was able to induce TNF-alpha and IL-8 secretion, whereas a nonlipidated C20A rMip variant was not. Proinflammatory activity of rMip was unaffected by heat or proteinase K treatments but was greatly reduced by treatment with lipases, supporting a role of lipid modification in this process. Stimulating pathways appeared to involve TLR2/TLR1/TLR6 with the help of CD14 but not TLR4. These data support a role of Mip lipoprotein in pathogenesis of C. trachomatis-induced inflammatory responses.

  2. Effects of neutral sulfate berberine on LPS-induced cardiomyocyte TNF-αsecretion, abnormal calcium cycling, and cardiac dysfunction in rats

    Institute of Scientific and Technical Information of China (English)

    Jing YANG; Hua-dong WANG; Da-xiang LU; Yan-ping WANG; Ren-bin QI; Jing LI; Fei LI; Chu-jie LI

    2006-01-01

    Aim: To evaluate the effect of neutral sulfate berberine on cardiac function, tumornecrosis factor α (TNF-α) release, and intracellular calcium concentration ([Ca2+]i)in cardiomyocytes exposed to lipopolysaccharide (LPS). Methods: Primary cultured rat cardiomyocytes were prepared from ventricles of 3-4-day old SpragueDawley rats. TNF-α concentrations in cell-conditioned media were measured by using a Quantikine enzyme-linked immunosorbent assay kit, and cardiomyocyte [Ca2+]i was measured by using Fura-2/AM. The isolated rat hearts were perfused in the Langendorff mode. Results: LPS at doses of 1, 5, 10, and 20 μg/mL markedly stimulated TNF-α secretion from cardiomyocytes, and neutral sulfate berberine inhibited LPS-induced TNF-α production. Intracellular calcium concentration was significantly decreased after LPS stimulation for 1 h, and increased 2 h after LPS treatment. Pretreatment with neutral sulfate berberine reversed the LPS-induced [Ca2+]i alterations, although neutral sulfate berberine did not inhibit a rapid increase in cardiomyocyte [Ca2+]i induced by LPS. Perfusion of isolated hearts with LPS (100 μg/mL) for 20 min resulted in significantly impaired cardiac performance at 120 min after LPS challenge: the maximal rate of left ventricular pressure rise and fall (±dp/dtmax) decreased compared with the control. In contrast, ±dp/dtmax at 120min in hearts perfused with neutral sulfate berberine (1 μmol/L) for 10 min followed by 20 min LPS (100 μg/mL) was greater than the corresponding value in the LPS group. Conclusion: Neutral sulfate berberine inhibits LPS-stimulated myocardial TNF-α production, impairs calcium cycling, and improves LPS-induced contractile dysfunction in intact heart.

  3. Antioxidant and anti-inflammatory effects of Ruta chalepensis L. extracts on LPS-stimulated RAW 264.7 cells.

    Science.gov (United States)

    Kacem, Mohamed; Simon, Gaëlle; Leschiera, Raphael; Misery, Laurent; ElFeki, Abdelfattah; Lebonvallet, Nicolas

    2015-02-01

    Ruta chalepensis L. is used in the traditional herbal treatment of various diseases. The aim of this work is to investigate the effect of different extracts of R. chalepensis L. on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expressions and their antioxidant capacity on murine RAW 264.7 macrophage challenged with lipopolysaccharide (LPS). In fact, this study shows that the ethanol and ethyl acetate extracts of R. chalepensis L. considerably decreased the nitric oxide (NO) production in murine RAW 264.7 macrophages stimulated with lipopolysaccharide. Thus, the treatment with both extracts significantly suppressed the levels of iNOS and COX-2 gene expressions through the inhibition of the nuclear factor-κB (NF-κB) activation. The preincubation of RAW 264.7 cells with various concentrations of ethanol and ethyl acetate extracts decreased the production of thiobarbituric acid-reactive substances (TBARS) in a dose-dependent manner. It also increased the activities of antioxidative enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in LPS-stimulated macrophages, compared to those in the cells treated only with LPS. Besides, the (1)H NMR spectra of both extracts have demonstrated the presence of aromatic signals, thus confirming the existence of phenolic compounds such as flavonoids and polyphenols. So, the ethanol and ethyl acetate extracts of R. chalepensis L. have been shown to possess enough antioxidant and anti-inflammatory activities to prevent LPS-induced oxidative stress and inflammation in RAW 264.7 macrophages.

  4. Rab6a/a’ Are Important Golgi Regulators of Pro-Inflammatory TNF Secretion in Macrophages

    Science.gov (United States)

    Micaroni, Massimo; Stanley, Amanda C.; Khromykh, Tatiana; Venturato, Juliana; Wong, Colin X. F.; Lim, Jet P.; Marsh, Brad J.; Storrie, Brian; Gleeson, Paul A.; Stow, Jennifer L.

    2013-01-01

    Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are trans-Golgi-localized GTPases known for roles in maintaining Golgi structure and Golgi-associated trafficking. We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6–GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages. PMID:23437303

  5. Rab6a/a' are important Golgi regulators of pro-inflammatory TNF secretion in macrophages.

    Science.gov (United States)

    Micaroni, Massimo; Stanley, Amanda C; Khromykh, Tatiana; Venturato, Juliana; Wong, Colin X F; Lim, Jet P; Marsh, Brad J; Storrie, Brian; Gleeson, Paul A; Stow, Jennifer L

    2013-01-01

    Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are trans-Golgi-localized GTPases known for roles in maintaining Golgi structure and Golgi-associated trafficking. We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6-GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages.

  6. A study to evaluate the effect of nootropic drug-piracetam on DNA damage in leukocytes and macrophages.

    Science.gov (United States)

    Singh, Sarika; Goswami, Poonam; Swarnkar, Supriya; Singh, Sheelendra Pratap; Wahajuddin; Nath, Chandishwar; Sharma, Sharad

    2011-11-27

    Piracetam is a nootropic drug that protects neurons in neuropathological and age-related diseases and the activation and modulation of peripheral blood cells in patients with neuropathological conditions is well known. Therefore, in the present study, in vivo, ex vivo, and in vitro tests were conducted to investigate the effect of piracetam on leukocytes and macrophages. Lipopolysaccharide (LPS) causes oxidative DNA damage; thus, in the present study, LPS was used as a tool to induce DNA damage. In vivo experiments were conducted on Sprague Dawley rats, and piracetam (600mg/kg, oral) was provided for five consecutive days. On the fifth day, a single injection of LPS (10mg/kg, i.p.) was administered. Three hours after LPS injection, blood leukocytes and peritoneal macrophages were collected and processed, and a variety of different assays were conducted. Ex vivo treatments were performed on isolated rat blood leukocytes, and in vitro experiments were conducted on rat macrophage cell line J774A.1. Cell viability and the level of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage were estimated in untreated (control) and piracetam-, LPS- and LPS+piracetam-treated leukocytes and macrophages. In vivo experiments revealed that rats pretreated with piracetam were significantly protected against LPS-induced increases in ROS levels and DNA damage. Ex vivo isolated leukocytes and J774A.1 cells treated with LPS exhibited augmented ROS levels and DNA damage, which were attenuated with piracetam treatment. Thus, the present study revealed the salutary effect of piracetam against LPS-induced oxidative stress and DNA damage in leukocytes and macrophages. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. The pro-inflammatory cytokine, interleukin-6, enhances the polarization of alternatively activated macrophages.

    Directory of Open Access Journals (Sweden)

    Maria Ruweka Fernando

    Full Text Available Macrophages are important innate immune cells that are associated with two distinct phenotypes: a pro-inflammatory (or classically activated subset with prototypic macrophage functions such as inflammatory cytokine production and bactericidal activity, and an anti-inflammatory (or alternatively activated (AAM subset linked with wound healing and tissue repair processes. In this study, we examined the effect of interlukein-6 on human and murine macrophage polarization. The results indicate that despite being commonly associated with pro-inflammatory functions and being implicated in the pathogenesis/pathophysiology of numerous inflammatory diseases, interleukin-6 can enhance the polarization of AAMs, based on increased expression of hallmark markers: arginase-1, Ym1 and CD206; this effect required the AAM differentiating cytokines, IL-4 and IL-13. Co-treatment of AAMs with IL-6 resulted in spontaneous release of IL-10, suppressed LPS-induced nitric oxide production and inhibited cytokine production by activated CD4+ T cells - immunoregulatory features not observed in the 'parent' IL-4+IL-13-induced AAM. The effect of IL-6 required signal transducer and activator of transcription (STAT-3, was partially dependent on up-regulation of the IL4Rα chain, and was independent of autocrine IL-10. In the presence of IFNγ, IL-6 promoted the production of IL-1β and TNFα suggesting that this cytokine can enhance the phenotype to which a macrophage has committed. This finding may explain the pleiotrophic nature of IL-6, where it is associated with the perpetuation and enhancement of disease in inflammatory situations, but is also necessary for resolution of inflammation and adequate wound healing to occur in others. Thus, the potential benefit of IL-6 in promoting an AAM, with its' anti-inflammatory and wound healing ability, may need to be considered in immunotherapies aimed at in vivo modulation or inhibition of IL-6.

  8. The pro-inflammatory cytokine, interleukin-6, enhances the polarization of alternatively activated macrophages.

    Science.gov (United States)

    Fernando, Maria Ruweka; Reyes, Jose Luis; Iannuzzi, Jordan; Leung, Gabriella; McKay, Derek Mark

    2014-01-01

    Macrophages are important innate immune cells that are associated with two distinct phenotypes: a pro-inflammatory (or classically activated) subset with prototypic macrophage functions such as inflammatory cytokine production and bactericidal activity, and an anti-inflammatory (or alternatively activated (AAM)) subset linked with wound healing and tissue repair processes. In this study, we examined the effect of interlukein-6 on human and murine macrophage polarization. The results indicate that despite being commonly associated with pro-inflammatory functions and being implicated in the pathogenesis/pathophysiology of numerous inflammatory diseases, interleukin-6 can enhance the polarization of AAMs, based on increased expression of hallmark markers: arginase-1, Ym1 and CD206; this effect required the AAM differentiating cytokines, IL-4 and IL-13. Co-treatment of AAMs with IL-6 resulted in spontaneous release of IL-10, suppressed LPS-induced nitric oxide production and inhibited cytokine production by activated CD4+ T cells - immunoregulatory features not observed in the 'parent' IL-4+IL-13-induced AAM. The effect of IL-6 required signal transducer and activator of transcription (STAT)-3, was partially dependent on up-regulation of the IL4Rα chain, and was independent of autocrine IL-10. In the presence of IFNγ, IL-6 promoted the production of IL-1β and TNFα suggesting that this cytokine can enhance the phenotype to which a macrophage has committed. This finding may explain the pleiotrophic nature of IL-6, where it is associated with the perpetuation and enhancement of disease in inflammatory situations, but is also necessary for resolution of inflammation and adequate wound healing to occur in others. Thus, the potential benefit of IL-6 in promoting an AAM, with its' anti-inflammatory and wound healing ability, may need to be considered in immunotherapies aimed at in vivo modulation or inhibition of IL-6.

  9. Inhibitory Effects of 12 Hemostatic Chinese Herbs on Nitric Oxide Production in LPS-Activated RAW264.7 Macrophages%12味止血中药对脂多糖诱导小鼠巨噬细胞产生一氧化碳的抑制作用

    Institute of Scientific and Technical Information of China (English)

    廖晖; Linda K Banbury; David N Leach

    2007-01-01

    目的:研究12味止血中药对脂多糖(LPS)诱导小鼠巨噬细胞产生一氧化氮(NO)的抑制作用,并探讨其止血机制.方法:以1μg·mL-1 LPS刺激小鼠巨噬细胞RAW 264.7,22h后以Griess法测定NO的终产物亚硝酸盐的含量,观察LPS对NO生成的影响.结果:12味止血药均可抑制LPS诱导小鼠巨噬细胞RAW 264.7中NO的生成.三七、地榆、仙鹤草、槐花、艾叶、蒲黄、侧柏叶及白茅根的抑制作用显著(P<0.05).结论:本实验为12味止血中药对脂多糖诱导小鼠巨噬细胞产生NO的抑制作用提供了一定的理论依据.

  10. The inhibitory effect of PGE2 on LPS-induced MIP-1 alpha and beta by peritoneal macrophage cells%PGE2抑制LPS诱导腹腔巨噬细胞产生MIP-1α和MIP-1β

    Institute of Scientific and Technical Information of China (English)

    敬华娥; 顾鹏; 敬慧娥

    2006-01-01

    目的:在离体情况下,研究PGE2能否抑制脂多糖(LPS)诱导腹腔巨噬细胞MIP-1α和MIP-1β的产生.方法:制备腹腔巨噬细胞,应用夹心酶联免疫分析法(Sandwich ELISA)检测MIP-1α和MIP-1β的浓度,并应用实时逆转录聚合酶链反应(real-time RT-PCR)法检测MIP-1α和MIP-1βmRNA的表达.结果:PGE2抑制腹腔巨噬细胞产生MIP-1α和MIP-1β,呈时间依赖性和浓度依赖性,抑制发生在mRNA和蛋白质水平,PGE2对巨噬细胞的抑制作用通过EP4和EP2介导.结论:PGE2能抑制MIP-1α和MIP-1β产生而调节免疫反应.

  11. Effects of ischemia on lung macrophages.

    Directory of Open Access Journals (Sweden)

    Aigul Moldobaeva

    Full Text Available Angiogenesis after pulmonary ischemia is initiated by reactive O(2 species and is dependent on CXC chemokine growth factors, and its magnitude is correlated with the number of lavaged macrophages. After complete obstruction of the left pulmonary artery in mice, the left lung is isolated from the peripheral circulation until 5-7 days later, when a new systemic vasculature invades the lung parenchyma. Consequently, this model offers a unique opportunity to study the differentiation and/or proliferation of monocyte-derived cells within the lung. In this study, we questioned whether macrophage subpopulations were differentially expressed and which subset contributed to growth factor release. We characterized the change in number of all macrophages (MHCII(int, CD11C+, alveolar macrophages (MHCII(int, CD11C+, CD11B- and mature lung macrophages (MHCII(int, CD11C+, CD11B+ in left lungs from mice immediately (0 h or 24 h after left pulmonary artery ligation (LPAL. In left lung homogenates, only lung macrophages increased 24 h after LPAL (vs. 0 h; p<0.05. No changes in proliferation were seen in any subset by PCNA expression (0 h vs. 24 h lungs. When the number of monocytic cells was reduced with clodronate liposomes, systemic blood flow to the left lung 14 days after LPAL decreased by 42% (p<0.01 compared to vehicle controls. Furthermore, when alveolar macrophages and lung macrophages were sorted and studied in vitro, only lung macrophages secreted the chemokine MIP-2α (ELISA. These data suggest that ischemic stress within the lung contributes to the differentiation of immature monocytes to lung macrophages within the first 24 h after LPAL. Lung macrophages but not alveolar macrophages increase and secrete the proangiogenic chemokine MIP-2α. Overall, an increase in the number of lung macrophages appears to be critical for neovascularization in the lung, since clodronate treatment decreased their number and attenuated functional angiogenesis.

  12. Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress.

    Directory of Open Access Journals (Sweden)

    Paola Checconi

    Full Text Available Protein cysteines can form transient disulfides with glutathione (GSH, resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX 1, PRDX2, vimentin (VIM, profilin1 (PFN1 and thioredoxin 1 (TXN1. For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX, which also inhibited tumor necrosis factor (TNF-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC, which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions.

  13. Distinct signaling pathways for induction of type II NOS by IFNgamma and LPS in BV-2 microglial cells.

    Science.gov (United States)

    Shen, Siming; Yu, Sue; Binek, Joshua; Chalimoniuk, Malgorzata; Zhang, Xiaolin; Lo, Shih-Ching; Hannink, Mark; Wu, Jinmei; Fritsche, Kevin; Donato, Rosario; Sun, Grace Y

    2005-09-01

    Nitric oxide (NO) release upon microglial cell activation has been implicated in the tissue injury and cell death in many neurodegenerative diseases. Recent studies have indicated the ability of interferon-gamma (IFNgamma) and lipopolysaccharides (LPS) to independently induce type II nitric oxide synthase (iNOS) expression and NO production in BV-2 microglial cells. However, a detailed comparison between the signaling pathways activating iNOS by these two agents has not been accomplished. Analysis of PKC isoforms revealed mainly the presence of PKCdelta, iota and lambda in BV-2 cells. Although both IFNgamma and LPS could specifically enhance the tyrosine phosphorylation of PKCdelta, treatment with IFNgamma induced a steady increase of phospho-PKCdelta for up to 1h, whereas treatment with LPS elevated phospho-PKCdelta levels only transiently, with peak activity at 5 min. Rottlerin, a specific inhibitor for PKCdelta, dose-dependently inhibited IFNgamma- and LPS-induced NO production. Despite the common involvement of PKCdelta, IFNgamma- but not LPS-induced NO production involved extracellular signal-regulated kinases (ERK1/2) cascade and IFNgamma-induced phosphorylation of ERK1/2 was mediated through PKC. On the other hand, LPS- but not IFNgamma-induced NO production was through stimulation of NF-kappaB activation and nuclear translocation to interact with DNA. These results demonstrated distinct signaling pathways for induction of iNOS by IFNgamma and LPS in BV-2 microglial cells.

  14. Delineation of diverse macrophage activation programs in response to intracellular parasites and cytokines.

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    Shuyi Zhang

    Full Text Available BACKGROUND: The ability to reside and proliferate in macrophages is characteristic of several infectious agents that are of major importance to public health, including the intracellular parasites Trypanosoma cruzi (the etiological agent of Chagas disease and Leishmania species (etiological agents of Kala-Azar and cutaneous leishmaniasis. Although recent studies have elucidated some of the ways macrophages respond to these pathogens, the relationships between activation programs elicited by these pathogens and the macrophage activation programs elicited by bacterial pathogens and cytokines have not been delineated. METHODOLOGY/PRINCIPAL FINDINGS: To provide a global perspective on the relationships between macrophage activation programs and to understand how certain pathogens circumvent them, we used transcriptional profiling by genome-wide microarray analysis to compare the responses of mouse macrophages following exposure to the intracellular parasites T. cruzi and Leishmania mexicana, the bacterial product lipopolysaccharide (LPS, and the cytokines IFNG, TNF, IFNB, IL-4, IL-10, and IL-17. We found that LPS induced a classical activation state that resembled macrophage stimulation by the Th1 cytokines IFNG and TNF. However, infection by the protozoan pathogen L. mexicana produced so few transcriptional changes that the infected macrophages were almost indistinguishable from uninfected cells. T. cruzi activated macrophages produced a transcriptional signature characterized by the induction of interferon-stimulated genes by 24 h post-infection. Despite this delayed IFN response by T. cruzi, the transcriptional response of macrophages infected by the kinetoplastid pathogens more closely resembled the transcriptional response of macrophages stimulated by the cytokines IL-4, IL-10, and IL-17 than macrophages stimulated by Th1 cytokines. CONCLUSIONS/SIGNIFICANCE: This study provides global gene expression data for a diverse set of biologically

  15. Piperine Suppresses Pyroptosis and Interleukin-1β Release upon ATP Triggering and Bacterial Infection

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    Liang, Yi-Dan; Bai, Wen-Jing; Li, Chen-Guang; Xu, Li-Hui; Wei, Hong-Xia; Pan, Hao; He, Xian-Hui; Ouyang, Dong-Yun

    2016-01-01

    Piperine is a phytochemical present in black pepper (Piper nigrum Linn) and other related herbs, possessing a wide array of pharmacological activities including anti-inflammatory effects. Previously, we demonstrated that piperine has therapeutic effects on bacterial sepsis in mice, but the underlying mechanism has not been fully elucidated. In this study, we aimed to investigate the influences of piperine on pyroptosis in murine macrophages. The results showed that piperine dose-dependently inhibited ATP-induced pyroptosis, thereby suppressing interleukin-1β (IL-1β) or high mobility group box-1 protein (HMGB1) release in LPS-primed bone marrow-derived macrophages and J774A.1 cells. Accompanying this, ATP-induced AMP-activated protein kinase (AMPK) activation was greatly suppressed by piperine, whereas AMPK agonist metformin counteracted piperine’s inhibitory effects on pyroptosis. Moreover, piperine administration greatly reduced both peritoneal and serum IL-1β levels in the mouse model intraperitoneally infected with Escherichia coli, suggestive of suppressing systemic inflammation and pyroptosis. Our data indicated that piperine could protect macrophages from pyroptosis and reduced IL-1β and HMGB1 release by suppressing ATP-induced AMPK activation, suggesting that piperine may become a potential therapeutic agent against bacterial sepsis. PMID:27812336

  16. Piperine suppresses pyroptosis and interleukin-1β release upon ATP triggering and bacterial infection

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    Yi-Dan Liang

    2016-10-01

    Full Text Available Piperine is a phytochemical present in black pepper (Piper nigrum Linn and other related herbs, possessing a wide array of pharmacological activities including anti-inflammatory effects. Previously, we demonstrated that piperine has therapeutic effects on bacterial sepsis in mice, but the underlying mechanism has not been fully elucidated. In this study, we aimed to investigate the influences of piperine on pyroptosis in murine macrophages. The results showed that piperine dose-dependently inhibited ATP-induced pyroptosis, thereby suppressing interleukin-1β (IL-1β or high mobility group box-1 protein (HMGB1 release in LPS-primed bone marrow-derived macrophages (BMDMs and J774A.1 cells. Accompanying this, ATP-induced AMP-activated protein kinase (AMPK activation was greatly suppressed by piperine, whereas AMPK agonist metformin counteracted piperine’s inhibitory effects on pyroptosis. Moreover, piperine administration greatly reduced both peritoneal and serum IL-1β levels in the mouse model intraperitoneally infected with Escherichia coli, suggestive of suppressing systemic inflammation and pyroptosis. Our data indicated that piperine could protect macrophages from pyroptosis and reduced IL-1β and HMGB1 release by suppressing ATP-induced AMPK activation, suggesting that piperine may become a potential therapeutic agent against bacterial sepsis.

  17. Quercetin prevents progression of disease in elastase/LPS-exposed mice by negatively regulating MMP expression

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    Martinez Fernando J

    2010-09-01

    Full Text Available Abstract Background Chronic obstructive pulmonary disease (COPD is characterized by chronic bronchitis, emphysema and irreversible airflow limitation. These changes are thought to be due to oxidative stress and an imbalance of proteases and antiproteases. Quercetin, a plant flavonoid, is a potent antioxidant and anti-inflammatory agent. We hypothesized that quercetin reduces lung inflammation and improves lung function in elastase/lipopolysaccharide (LPS-exposed mice which show typical features of COPD, including airways inflammation, goblet cell metaplasia, and emphysema. Methods Mice treated with elastase and LPS once a week for 4 weeks were subsequently administered 0.5 mg of quercetin dihydrate or 50% propylene glycol (vehicle by gavage for 10 days. Lungs were examined for elastance, oxidative stress, inflammation, and matrix metalloproteinase (MMP activity. Effects of quercetin on MMP transcription and activity were examined in LPS-exposed murine macrophages. Results Quercetin-treated, elastase/LPS-exposed mice showed improved elastic recoil and decreased alveolar chord length compared to vehicle-treated controls. Quercetin-treated mice showed decreased levels of thiobarbituric acid reactive substances, a measure of lipid peroxidation caused by oxidative stress. Quercetin also reduced lung inflammation, goblet cell metaplasia, and mRNA expression of pro-inflammatory cytokines and muc5AC. Quercetin treatment decreased the expression and activity of MMP9 and MMP12 in vivo and in vitro, while increasing expression of the histone deacetylase Sirt-1 and suppressing MMP promoter H4 acetylation. Finally, co-treatment with the Sirt-1 inhibitor sirtinol blocked the effects of quercetin on the lung phenotype. Conclusions Quercetin prevents progression of emphysema in elastase/LPS-treated mice by reducing oxidative stress, lung inflammation and expression of MMP9 and MMP12.

  18. Myeloid Growth Factors Promote Resistance to Mycobacterial Infection by Curtailing Granuloma Necrosis through Macrophage Replenishment.

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    Pagán, Antonio J; Yang, Chao-Tsung; Cameron, James; Swaim, Laura E; Ellett, Felix; Lieschke, Graham J; Ramakrishnan, Lalita

    2015-07-08

    The mycobacterial ESX-1 virulence locus accelerates macrophage recruitment to the forming tuberculous granuloma. Newly recruited macrophages phagocytose previously infected apoptotic macrophages to become new bacterial growth niches. Granuloma macrophages can then necrose, releasing mycobacteria into the extracellular milieu, which potentiates their growth even further. Using zebrafish with genetic or pharmacologically induced macrophage deficiencies, we find that global macrophage deficits increase susceptibility to mycobacterial infection by accelerating granuloma necrosis. This is because reduction in the macrophage supply below a critical threshold decreases granuloma macrophage replenishment to the point where apoptotic infected macrophages, failing to get engulfed, necrose. Reducing macrophage demand by removing bacterial ESX-1 offsets the susceptibility of macrophage deficits. Conversely, increasing macrophage supply in wild-type fish by overexpressing myeloid growth factors induces resistance by curtailing necrosis. These findings may explain the susceptibility of humans with mononuclear cytopenias to mycobacterial infections and highlight the therapeutic potential of myeloid growth factors in tuberculosis.

  19. The interaction of lipopolysaccharide-coated polystyrene particle with membrane receptor proteins on macrophage measured by optical tweezers

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    Wei, Ming-Tzo; Hua, Kuo-Feng; Hsu, Jowey; Karmenyan, Artashes; Hsu, Hsien-Yeh; Chiou, Arthur

    2006-08-01

    Lipopolysaccharide (LPS) is one of the cell wall components of Gram-positive bacteria recognized by and interacted with receptor proteins such as CD14 on macrophage cells. Such a process plays an important role in our innate immune system. In this paper, we report the application of optical tweezers (λ = 1064nm Gaussian beam focused by a water-immersed objective lens with N.A. = 1.0) to the study of the dynamics of the binding of a LPS-coated polystyrene particle (diameter = 1.5μm) onto the plasma membrane of a macrophage cell. We demonstrated that the binding rate increased significantly when the macrophage cell was pre-treated with the extract of Reishi polysaccharides (EORP) which has been shown to enhance the cell surface expression of CD14 (receptor of LPS) on macrophage cells.

  20. TLR4 mediates LPS-induced HO-1 expression in mouse liver: Role of TNF-α and IL-1β

    Institute of Scientific and Technical Information of China (English)

    Yong Song; Yi Shi; Li-Hua Ao; Alden H; Harken; Xian-Zhong Meng

    2003-01-01

    AIM: Heme oxygenase (HO)-1 catalyzes the conversion of heme to biliverdin, iron and carbon monoxide. HO-1 is induced by many stimuli including heme, Hb, heat stress,lipopolysaccharide (LPS) and cytokines. Previous studies demonstrated that LPS induced HO-1 gene activation and HO-1 expression in liver. However, the mechanisms of LPSinduced HO-1 expression in liver remain unknown. The effect of toil-like receptor-4 (TLR4) on LPS-induced liver HO-1expression and the role of TNF-α and IL-1β in this condition were determined.METHODS: HO-1 expression was determined by immunofluorescent staining and immunoblotting. Double immunofluorescent staining was performed to determine the cell type of HO-1 expression in liver.RESULTS: A low dose of LPS significantly increased HO-1expression in the liver which was localized in Kupffer cells only. Furthermore, HO-1 expression was enhanced by three doses of LPS. HO-1 expression was significantly inhibited in the liver of TLR4 mutant mice. While the liver HO-1expression in TNF KO mice was much lower than that in C57 mice following the same LPS treatment, IL-1β KO had a slight influence on liver HO-1 expression following LPS treatment.CONCLUSION: The present results confirm that macrophages are the major source of HO-1 in the liver induced by LPS.This study demonstrates that TLR4 plays a dominant role in mediating HO-1 expression following LPS. LPS-induced HO-1 expression is mainly mediated by endogenous TNF-α, but only partially by endogenous IL-1β.

  1. The influence of N-acetyl-L-cysteine on oxidative stress and nitric oxide synthesis in stimulated macrophages treated with a mustard gas analogue

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    Smith Milton

    2008-06-01

    Full Text Available Abstract Background Sulphur mustard gas, 2, 2'-dichlorodiethyl sulphide (HD, is a chemical warfare agent. Both mustard gas and its monofunctional analogue, 2-chloroethyl ethyl sulphide (CEES, are alkylating agents that react with and diminish cellular thiols and are highly toxic. Previously, we reported that lipopolysaccharide (LPS significantly enhances the cytotoxicity of CEES in murine RAW 264.7 macrophages and that CEES transiently inhibits nitric oxide (NO production via suppression of inducible NO synthase (iNOS protein expression. NO generation is an important factor in wound healing. In this paper, we explored the hypotheses that LPS increases CEES toxicity by increasing oxidative stress and that treatment with N-acetyl-L-cysteine (NAC would block LPS induced oxidative stress and protect against loss of NO production. NAC stimulates glutathione (GSH synthesis and also acts directly as a free radical scavenger. The potential therapeutic use of the antibiotic, polymyxin B, was also evaluated since it binds to LPS and could thereby block the enhancement of CEES toxicity by LPS and also inhibit the secondary infections characteristic of HD/CEES wounds. Results We found that 10 mM NAC, when administered simultaneously or prior to treatment with 500 μM CEES, increased the viability of LPS stimulated macrophages. Surprisingly, NAC failed to protect LPS stimulated macrophages from CEES induced loss of NO production. Macrophages treated with both LPS and CEES show increased oxidative stress parameters (cellular thiol depletion and increased protein carbonyl levels. NAC effectively protected RAW 264.7 cells simultaneously treated with CEES and LPS from GSH loss and oxidative stress. Polymyxin B was found to partially block nitric oxide production and diminish CEES toxicity in LPS-treated macrophages. Conclusion The present study shows that oxidative stress is an important mechanism contributing to CEES toxicity in LPS stimulated macrophages and

  2. Modulation of human alveolar macrophage properties by ozone exposure in vitro

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    Becker, S.; Madden, M.C.; Newman, S.L.; Devlin, R.B.; Koren, H.S. (ABB Environmental Services, Inc., Chapel Hill, NC (United States))

    1991-09-15

    The authors have investigated changes in human alveolar macrophage (HAM) function after exposure in vitro to ozone (O3). The functions studied reflect concern that O3 is detrimental to host defense mechanisms in the bronchoalveolar spaces. Exposure of HAM to O3 caused a concentration-dependent increase in release of prostaglandin E2 (PGE2), an important modulator of inflammation, phagocytosis, and oxidative burst. Although phagocytosis of particulate immune complexes was decreased by O3, we found no change in the quantity of Fc receptors and complement receptors on the HAM surface. Superoxide (O2-) production in response to phorbol ester was reduced after exposure of HAM to O3 while the basal O2- release in response to plastic adherence was not affected. Growth inhibition of the opportunistic yeast Cryptococcus neoformans by HAM was not affected by O3 exposure. The production of inflammatory mediators and immune modulators such as tumor necrosis factor-alpha, interleukin 1, and interleukin 6 were not induced by exposure to O3. However, compared to controls, O3- exposed HAM produced significantly lower levels of these cytokines when stimulated with bacterial lipopolysaccharide (LPS). Two-dimensional gel electrophoretic analysis of proteins made by HAM following in vitro exposure to O3 identified 11 proteins whose rate of synthesis was significantly altered. Thus, these studies show that exposure to O3 alters the functional competence of HAM. While there is a minimal effect on protein expression or synthesis, the responses of HAM to particulate immune complexes, to bacterial LPS, and to PMA are impaired. The release of arachidonic acid and PGE2 suggest that the effect of O3 is primarily targeted to the HAM cell membrane. These changes may ultimately result in increased susceptibility to inhaled infectious agents in the O3-exposed individual.

  3. Cyclical DNA Methylation and Histone Changes Are Induced by LPS to Activate COX-2 in Human Intestinal Epithelial Cells

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    Brancaccio, Mariarita; Coretti, Lorena; Florio, Ermanno; Pezone, Antonio; Calabrò, Viola; Falco, Geppino; Keller, Simona; Lembo, Francesca; Avvedimento, Vittorio Enrico; Chiariotti, Lorenzo

    2016-01-01

    Bacterial lipopolysaccharide (LPS) induces release of inflammatory mediators both in immune and epithelial cells. We investigated whether changes of epigenetic marks, including selected histone modification and DNA methylation, may drive or accompany the activation of COX-2 gene in HT-29 human intestinal epithelial cells upon exposure to LPS. Here we describe cyclical histone acetylation (H3), methylation (H3K4, H3K9, H3K27) and DNA methylation changes occurring at COX-2 gene promoter overtime after LPS stimulation. Histone K27 methylation changes are carried out by the H3 demethylase JMJD3 and are essential for COX-2 induction by LPS. The changes of the histone code are associated with cyclical methylation signatures at the promoter and gene body of COX-2 gene. PMID:27253528

  4. Mycobacterium paratuberculosis, Mycobacterium smegmatis, and lipopolysaccharide induce different transcriptional and post-transcriptional regulation of the IRG1 gene in murine macrophages.

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    Basler, Tina; Jeckstadt, Sabine; Valentin-Weigand, Peter; Goethe, Ralph

    2006-03-01

    Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic enteritis in ruminants. In addition, MAP is presently the most favored pathogen linked to Crohn's disease. In this study, we were interested in dissecting the molecular mechanisms of macrophage activation or deactivation after infection with MAP. By subtractive hybridization of cDNAs, we identified the immune-responsive gene 1 (IRG1), which was expressed substantially higher in lipopolysaccharide (LPS)-stimulated than in MAP-infected murine macrophage cell lines. A nuclear run-on transcription assay revealed that the IRG1 gene was activated transcriptionally in LPS-stimulated and MAP-infected macrophages with higher expression in LPS-stimulated cells. Analysis of post-transcriptional regulation demonstrated that IRG1 mRNA stability was increased in LPS-stimulated but not in MAP-infected macrophages. Furthermore, IRG1 gene expression of macrophages infected with the nonpathogenic Mycobacterium smegmatis differed from those of LPS-stimulated and MAP-infected macrophages. At 2 h postinfection, M. smegmatis-induced IRG1 gene expression was as low as in MAP-infected, and 8 h postinfection, it increased nearly to the level in LPS-stimulated macrophages. Transient transfection experiments revealed similar IRG1 promoter activities in MAP- and M. smegmatis-infected cells. Northern analysis demonstrated increased IRG1 mRNA stability in M. smegmatis-infected macrophages. IRG1 mRNA stabilization was p38 mitogen-activated protein kinase-independent. Inhibition of protein synthesis revealed that constitutively expressed factors seemed to be responsible for IRG1 mRNA destabilization. Thus, our data demonstrate that transcriptional and post-transcriptional mechanisms are responsible for a differential IRG1 gene expression in murine macrophages treated with LPS, MAP, and M. smegmatis.

  5. Btk regulates macrophage polarization in response to lipopolysaccharide.

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    Joan Ní Gabhann

    Full Text Available Bacterial Lipopolysaccharide (LPS is a strong inducer of inflammation and does so by inducing polarization of macrophages to the classic inflammatory M1 population. Given the role of Btk as a critical signal transducer downstream of TLR4, we investigated its role in M1/M2 induction. In Btk deficient (Btk (-\\- mice we observed markedly reduced recruitment of M1 macrophages following intraperitoneal administration of LPS. Ex vivo analysis demonstrated an impaired ability of Btk(-/- macrophages to polarize into M1 macrophages, instead showing enhanced induction of immunosuppressive M2-associated markers in response to M1 polarizing stimuli, a finding accompanied by reduced phosphorylation of STAT1 and enhanced STAT6 phosphorylation. In addition to STAT activation, M1 and M2 polarizing signals modulate the expression of inflammatory genes via differential activation of transcription factors and regulatory proteins, including NF-κB and SHIP1. In keeping with a critical role for Btk in macrophage polarization, we observed reduced levels of NF-κB p65 and Akt phosphorylation, as well as reduced induction of the M1 associated marker iNOS in Btk(-/- macrophages in response to M1 polarizing stimuli. Additionally enhanced expression of SHIP1, a key negative regulator of macrophage polarisation, was observed in Btk(-/- macrophages in response to M2 polarizing stimuli. Employing classic models of allergic M2 inflammation, treatment of Btk (-/- mice with either Schistosoma mansoni eggs or chitin resulted in increased recruitment of M2 macrophages and induction of M2-associated genes. This demonstrates an enhanced M2 skew in the absence of Btk, thus promoting the development of allergic inflammation.

  6. Emodin Ameliorates LPS-Induced Acute Lung Injury, Involving the Inactivation of NF-κB in Mice

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    Min Xiao

    2014-10-01

    Full Text Available Acute lung injury (ALI and its severe manifestation of acute respiratory distress syndrome (ARDS are well-known illnesses. Uncontrolled and self-amplified pulmonary inflammation lies at the center of the pathology of this disease. Emodin, the bio-active coxund of herb Radix rhizoma Rhei, shows potent anti-inflammatory properties through inactivation of nuclear factor-κB (NF-κB. The aim of this study was to evaluate the effect of emodin on lipopolysaccharide (LPS-induced ALI in mice, and its potential bio-mechanism. In our study, BALB/c mice were stimulated with LPS to induce ALI. After 72 h of LPS stimulation, pulmonary pathological changes, lung injury scores, pulmonary edema, myeloperoxidase (MPO activity, total cells, neutrophils, macrophages, TNF-α, IL-6 and IL-1β in bronchoalveolar lavage fluid (BALF, and MCP-1 and E-selectin expression were notably attenuated by emodin in mice. Meanwhile, our data also revealed that emodin significantly inhibited the LPS-enhanced the phosphorylation of NF-κB p65 and NF-κB p65 DNA binding activity in lung. Our data indicates that emodin potently inhibits LPS-induced pulmonary inflammation, pulmonary edema and MCP-1 and E-selectin expression, and that these effects were very likely mediated by inactivation of NF-κB in mice. These results suggest a therapeutic potential of emodin as an anti-inflammatory agent for ALI/ARDS treatment.

  7. Anti-inflammatory effects of galangin on lipopolysaccharide-activated macrophages via ERK and NF-κB pathway regulation.

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    Jung, Yun Chan; Kim, Mi Eun; Yoon, Ju Hwa; Park, Pu Reum; Youn, Hwa-Young; Lee, Hee-Woo; Lee, Jun Sik

    2014-12-01

    Inflammation is the major symptom of the innate immune response to microbial infection. Macrophages, immune response-related cells, play a role in the inflammatory response. Galangin is a member of the flavonols and is found in Alpinia officinarum, galangal root and propolis. Previous studies have demonstrated that galangin has antioxidant, anticancer, and antineoplastic activities. However, the anti-inflammatory effects of galangin are still unknown. In this study, we investigated the anti-inflammatory effects of galangin on RAW 264.7 murine macrophages. Galagin was not cytotoxic to RAW 264.7 cells, and nitric oxide (NO) production induced by lipopolysaccharide (LPS)-stimulated macrophages was significantly decreased by the addition of 50 μM galangin. Moreover, galangin treatment reduced mRNA levels of cytokines, including IL-1β and IL-6, and proinflammatory genes, such as iNOS in LPS-activated macrophages in a dose-dependent manner. Galangin treatment also decreased the protein expression levels of iNOS in activated macrophages. Galangin was found to elicit anti-inflammatory effects by inhibiting ERK and NF-κB-p65 phosphorylation. In addition, galangin-inhibited IL-1β production in LPS-activated macrophages. These results suggest that galangin elicits anti-inflammatory effects on LPS-activated macrophages via the inhibition of ERK, NF-κB-p65 and proinflammatory gene expression.

  8. Efek ekstrak daun singkong (Manihot utilissima terhadap ekspresi COX-2 pada monosit yang dipapar LPS E.coli (The effect of Manihot utilissima extracts on COX-2 expression of monocytes induced by LPS E. coli

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    Zahara Meilawaty

    2013-12-01

    Full Text Available Background: Periodontal disease is a common and widespread disease in the community. Gram negative bacteria have a role inperiodontitis. These bacteria secrete a variety of products such as endotoxin lipopolysaccharide (LPS, which causes the occurrenceof inflammation or infection. The body defense responses are neutrophils and mononuclear cells (monocytes and macrophages. Inresponse to defense mechanism, the body will be expressed enzyme cyclooxygenase (COX which functions convert arachidonic acidto prostaglandins. Cassava leaf cells known to play a role in reducing inflammation, but the mechanism for inhibiting COX-2, is notknown. Purpose: The study was aimed to determine the effect of cassava leaf extract (Manihot utilissima on expression of enzyme COX-2 in monocytes which were exposed by LPS E. coli. Methods: This study was in vitro experimental studies with the design of posttestonly control group design. The sample was the cassava leaves extract (Manihot utilissima at concentration of 12.5 % and 25 %. Theexpression of COX-2 was determined by immunocytochemistry method. Isolated monocytes were incubated in cassava leaf extract, andthen exposed to LPS, after washing imunostaning procedure was performed using a monoclonal antibody (MAb anti-human COX-2.The research data was the number of monocytes that express COX-2. Results: Expression of COX-2 in the group cassava leaf extractwas higher than the group that induced by LPS E. coli only. Conclusion: Cassava leaf extract did not inhibit the expression of COX-2in monocytes which were exposed by LPS E. coli.Latar belakang: Penyakit periodontal merupakan penyakit umum dan tersebar luas di masyarakat. Bakteri yang banyak berperanpada periodontitis adalah Gram negatif. Bakteri ini mengeluarkan berbagai produk antara lain endotoksin lipopolisakarida (LPS yangmenyebabkan inflamasi atau infeksi. Respon pertahanan tubuh pertama adalah netrofil dan sel mononuklear (monosit dan makrofag.Pada respon

  9. Macrophages transfer antigens to dendritic cells by releasing exosomes containing dead-cell-associated antigens partially through a ceramide-dependent pathway to enhance CD4(+) T-cell responses.

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    Xu, Yingping; Liu, Yi; Yang, Chunqing; Kang, Li; Wang, Meixiang; Hu, Jingxia; He, Hao; Song, Wengang; Tang, Hua

    2016-10-01

    Defects in rapid clearance of apoptotic cells lead to an accumulation of dead cells (late apoptotic or secondary necrotic cells), which results in an aberrant immune response. However, little is known about whether and how macrophages (Mφs) cooperate with dendritic cells (DCs) in the presentation of dead-cell-associated antigens in this process. By transferring high numbers of dead cells to mimic a failure of apoptotic cell clearance in vivo, we found that Mφs and neutrophils were the predominant phagocytes in the uptake of dead cells in the spleen. Moreover, both Mφs and DCs were required for an optimal CD4(+) T-cell response triggered by dead-cell-associated antigens. Importantly, although Mφs alone had a poor capacity for antigen presentation, they could transfer phagocytosed antigens to DCs for potent antigen presentation to enhance T-cell responses. Finally, we found that exosomes released from Mφs acted as a transmitter to convey antigens to DCs partially in a ceramide-dependent manner, since treatment with the neutral sphingomyelinase inhibitor GW4869 and spiroepoxide resulted in a significant reduction of T-cell proliferation in vitro and in vivo. These findings point to a novel pathway of cross-talk between Mφs and DCs, which will be helpful to explain possible mechanisms for autoimmune diseases characterized by increased rates of apoptosis.

  10. Effects of PARP-1 deficiency on airway inflammatory cell recruitment in response to LPS or TNF: differential effects on CXCR2 ligands and Duffy Antigen Receptor for Chemokines.

    Science.gov (United States)

    Zerfaoui, Mourad; Naura, Amarjit S; Errami, Youssef; Hans, Chetan P; Rezk, Bashir M; Park, Jiwon; Elsegeiny, Waleed; Kim, Hogyoung; Lord, Kevin; Kim, Jong G; Boulares, A Hamid

    2009-12-01

    We reported that PARP-1 exhibits differential roles in expression of inflammatory factors. Here, we show that PARP-1 deletion was associated with a significant reduction in inflammatory cell recruitment to mouse airways upon intratracheal administration of LPS. However, PARP-1 deletion exerted little effect in response to TNF exposure. LPS induced massive neutrophilia and moderate recruitment of macrophages, and TNF induced recruitment of primarily macrophages with smaller numbers of neutrophils in the lungs. Following either exposure, macrophage recruitment was blocked severely in PARP-1(-/-) mice, and this was associated with a marked reduction in MCP-1 and MIP-1alpha. This association was corroborated partly by macrophage recruitment in response to intratracheal administration of MCP-1 in PARP-1(-/-) mice. Surprisingly, although neutrophil recruitment was reduced significantly in LPS-treated PARP-1(-/-) mice, neutrophil numbers increased in TNF-treated mice, suggesting that PARP-1 deletion may promote a macrophagic-to-neutrophilic shift in the inflammatory response upon TNF exposure. Neutrophil-specific chemokines mKC and MIP-2 were reduced significantly in lungs of LPS-treated but only partially reduced in TNF-treated PARP-1(-/-) mice. Furthermore, the MIP-2 antagonist abrogated the shift to a neutrophilic response in TNF-exposed PARP-1(-/-) mice. Although CXCR2 expression increased in response to either stimulus in PARP-1(+/+) mice, the DARC increased only in lungs of TNF-treated PARP-1(+/+) mice; both receptors were reduced to basal levels in treated PARP-1(-/-) mice. Our results show that the balance of pro-neutrophilic or pro-macrophagic stimulatory factors and the differential influence of PARP-1 on these factors are critical determinants for the nature of the airway inflammatory response.

  11. The Potential Role of an Endotoxin Tolerance-Like Mechanism for the Anti-inflammatory Effect of Spirulina platensis Organic Extract in Macrophages.

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    Pham, Tho X; Park, Young-Ki; Bae, Minkyung; Lee, Ji-Young

    2017-01-25

    Endotoxin tolerance is a phenomenon where exposure of innate immune cells to lipopolysaccharide (LPS) induces a refractory state to subsequent endotoxin exposures. The goal of this study was to investigate if Spirulina platensis organic extract (SPE) induces an endotoxin tolerance-like state. We used splenocytes and peritoneal macrophages from C57BL/6J mice fed a high-fat/high-sucrose (HF/HS) control or a HF/HS diet containing 0.25% (w/w) SPE for 16 weeks for ex vivo LPS stimulation and endotoxin-tolerant (ET) macrophages to evaluate the effects of SPE on endotoxin tolerance. Cells from SPE-fed mice displayed significantly less expression of proinflammatory genes than those from control mice. ET macrophages were produced in vitro by incubating RAW 264.7 macrophages with low-dose LPS to determine the energy phenotype of naive, SPE-treated, and ET macrophages. Compared to naive macrophages exposed to a high-dose LPS (100 ng/mL) for the first time, ET macrophages showed significantly less proinflammatory gene expression after LPS stimulation, which was also observed with SPE treatment. Consistently, nuclear translocation of p65 was markedly reduced in both ET- and SPE-treated macrophages on LPS stimulation with increase in nuclear protein levels of p50 and B cell lymphoma 3-encoded protein. In conclusion, the anti-inflammatory effect of SPE is at least partly attributable to the induction of an endotoxin tolerance-like state in macrophages, which shares common characteristics of macrophage endotoxin tolerance.

  12. BMP pathway regulation of and by macrophages.

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    Megha Talati

    Full Text Available Pulmonary arterial hypertension (PAH is a disease of progressively increasing pulmonary vascular resistance, associated with mutations of the type 2 receptor for the BMP pathway, BMPR2. The canonical signaling pathway for BMPR2 is through the SMAD family of transcription factors. BMPR2 is expressed in every cell type, but the impact of BMPR2 mutations affecting SMAD signaling, such as Bmpr2delx4+, had only previously been investigated in smooth muscle and endothelium. In the present study, we created a mouse with universal doxycycline-inducible expression of Bmpr2delx4+ in order to determine if broader expression had an impact relevant to the development of PAH. We found that the most obvious phenotype was a dramatic, but patchy, increase in pulmonary inflammation. We crossed these double transgenic mice onto an NF-κB reporter strain, and by luciferase assays on live mice, individual organs and isolated macrophages, we narrowed down the origin of the inflammatory phenotype to constitutive activation of tissue macrophages. Study of bone marrow-derived macrophages from mutant and wild-type mice suggested a baseline difference in differentiation state in Bmpr2 mutants. When activated with LPS, both mutant and wild-type macrophages secrete BMP pathway inhibitors sufficient to suppress BMP pathway activity in smooth muscle cells (SMC treated with conditioned media. Functionally, co-culture with macrophages results in a BMP signaling-dependent increase in scratch closure in cultured SMC. We conclude that SMAD signaling through BMP is responsible, in part, for preventing macrophage activation in both live animals and in cells in culture, and that activated macrophages secrete BMP inhibitors in sufficient quantity to cause paracrine effect on vascular smooth muscle.

  13. CD36 is upregulated in mice with periodontitis and metabolic syndrome and involved in macrophage gene upregulation by palmitate.

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    Lu, Z; Li, Y; Brinson, C W; Kirkwood, K L; Lopes-Virella, M F; Huang, Y

    2017-03-01

    We reported that high-fat diet (HFD)-induced metabolic syndrome (MetS) exacerbates lipopolysaccharide (LPS)-stimulated periodontitis and palmitate, the major saturated fatty acid in the HFD, amplified LPS-stimulated gene expression in vitro. As CD36 is a major receptor for fatty acids, we investigated periodontal CD36 expression in mice with periodontitis and MetS, and the role of CD36 in inflammatory gene expression in macrophages stimulated by palmitate. MetS and periodontitis were induced in mice by HFD and periodontal injection of LPS, respectively. The periodontal CD36 expression and its relationship with alveolar bone loss were studied using immunohistochemistry, real-time PCR, and correlation analysis. The role of CD36 in upregulation of inflammatory mediators by LPS and palmitate in macrophages was assessed using pharmacological inhibitor and small interfering RNA. Periodontal CD36 expression was higher in mice with both MetS and periodontitis than that in mice with periodontitis or MetS alone and was correlated with osteoclastogenesis and alveolar bone loss. In vitro studies showed that CD36 expression in macrophages was upregulated by LPS and palmitate, and targeting CD36 attenuated palmitate-enhanced gene expression. CD36 expression is upregulated in mice with periodontitis and MetS and involved in gene expression in macrophages stimulated by palmitate and LPS. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Intracellular survival and persistence of Chlamydia muridarum is determined by macrophage polarization.

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    Eric Gracey

    Full Text Available Macrophages can display a number of distinct phenotypes, known collectively as polarized macrophages. The best defined of these phenotypes are the classically-activated, interferon gamma (IFNγ/LPS induced (M1 and alternatively-activated, IL-4 induced (M2 macrophages. The goal of this study is to characterize macrophage-Chlamydia interactions in the context of macrophage polarization. Here we use Chlamydia muridarum and murine bone-marrow derived macrophages to show Chlamydia does not induce M2 polarization in macrophages as a survival strategy. Unexpectedly, the infection of macrophages was silent with no upregulation of M1 macrophage-associated genes. We further demonstrate that macrophages polarized prior to infection have a differential capacity to control Chlamydia. M1 macrophages harbor up to 40-fold lower inclusion forming units (IFU than non-polarized or M2 polarized macrophages. Gene expression analysis showed an increase in 16sRNA in M2 macrophages with no change in M1 macrophages. Suppressed Chlamydia growth in M1 macrophages correlated with the induction of a bacterial gene expression profile typical of persistence as evident by increased Euo expression and decreased Omp1 and Tal expression. Observations of permissive Chlamydia growth in non-polarized and M2 macrophages and persistence in M1 macrophages were supported through electron microscopy. This work supports the importance of IFNγ in the innate immune response to Chlamydia. However, demonstration that the M1 macrophages, despite an antimicrobial signature, fail to eliminate intracellular Chlamydia supports the notion that host-pathogen co-evolution has yielded a pathogen that can evade cellular defenses against this pathogen, and persist for prolonged periods of time in the host.

  15. Glucagon-like peptide 1 improves insulin resistance in vitro through anti-inflammation of macrophages

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    C. Guo

    Full Text Available Glucagon-like peptide 1 (GLP-1, a kind of gut hormone, is used in the treatment of type 2 diabetes (T2D. Emerging evidence indicates that GLP-1 has anti-inflammatory activity. Chronic inflammation in the adipose tissue of obese individuals is a cause of insulin resistance and T2D. We hypothesized that GLP-1 analogue therapy in patients with T2D could suppress the inflammatory response of macrophages, and therefore inhibit insulin resistance. Our results showed that GLP-1 agonist (exendin-4 not only attenuated macrophage infiltration, but also inhibited the macrophage secretion of inflammatory cytokines including TNF-β, IL-6, and IL-1β. Furthermore, we observed that lipopolysaccharide (LPS-induced macrophage conditioned media could impair insulin-stimulated glucose uptake. This effect was compensated by treatment with the conditioned media from macrophages treated with the combination of LPS and exendin-4. It was also observed that exendin-4 directly inhibited the activation of NF-κB in macrophages. In conclusion, our results indicated that GLP-1 improved inflammatory macrophage-derived insulin resistance by inhibiting NF-κB pathway and secretion of inflammatory cytokines in macrophages. Furthermore, our observations suggested that the anti-inflammatory effect of GLP-1 on macrophages can contribute to GLP-1 analogue therapy of T2D.

  16. Chikusetsusaponin IVa Methyl Ester Isolated from the Roots of Achyranthes japonica Suppresses LPS-Induced iNOS, TNF-α, IL-6, and IL-1β Expression by NF-κB and AP-1 Inactivation.

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    Lee, Hae-Jun; Shin, Ji-Sun; Lee, Woo-Seok; Shim, Heon-Yong; Park, Ji-Min; Jang, Dae-Sik; Lee, Kyung-Tae

    2016-01-01

    We investigated the effect of chikusetsusaponin IVa (CS) and chikusetsusaponin IVa methyl ester (CS-ME) from the roots of Achyranthes japonica NAKAI on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW264.7 macrophages. CS-ME more potently inhibited LPS-induced NO and PGE2 production than CS. CS-ME concentration-dependently inhibited LPS-induced tumor necrosis factor (TNF)-α and interleukin (IL)-6 and IL-1β production in RAW264.7 macrophages and mouse peritoneal macrophages. Consistent with these findings, CS-ME suppressed LPS-induced expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 at protein level as well as iNOS, COX-2, TNF-α, IL-6, and IL-1β at mRNA level. In addition, CS-ME suppressed LPS-induced transcriptional activity of nuclear factor (NF)-κB and activator protein (AP)-1. The anti-inflammatory properties of CS-ME might result from suppression of iNOS, COX-2, TNF-α, IL-6, and IL-1β expression through downregulation of NF-κB and AP-1 in macrophages.

  17. The Hydroalcoholic Extract Obtained from Mentha piperita L. Leaves Attenuates Oxidative Stress and Improves Survival in Lipopolysaccharide-Treated Macrophages

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    Mariana Oliveira Arruda

    2017-01-01

    Full Text Available Mentha piperita L. (peppermint possesses antimicrobial properties, but little is known of its ability to modulate macrophages. Macrophages are essential in bacterial infection control due to their antimicrobial functions and ability to link the innate and adaptive immune responses. We evaluated the effects of the peppermint leaf hydroalcoholic extract (LHAE on cultured murine peritoneal macrophages stimulated or not with lipopolysaccharide (LPS in vitro. Vehicle-treated cells were used as controls. The constituents of the extract were also identified. Epicatechin was the major compound detected in the LHAE. LPS-induced macrophage death was reversed by incubation with LHAE (1–30 μg/ml. Higher concentrations of the extract (≥100 μg/ml decreased macrophage viability (49–57% in the absence of LPS. LHAE (1–300 μg/ml attenuated H2O2 (34.6–53.4% but not nitric oxide production by these cells. At similar concentrations, the extract increased the activity of superoxide dismutase (15.3–63.5-fold and glutathione peroxidase (34.4–73.6-fold in LPS-treated macrophages. Only LPS-unstimulated macrophages presented enhanced phagocytosis (3.6–6.6-fold increase when incubated with LHAE (3–30 μg/ml. Overall, the LHAE obtained from peppermint modulates macrophage-mediated inflammatory responses, by stimulating the antioxidant pathway in these cells. These effects may be beneficial when the excessive activation of macrophages contributes to tissue damage during infectious disease.

  18. Lobohedleolide induces interleukin-8 production in LPS-stimulated human monocytic cell line THP-1

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    T Oda

    2011-09-01

    Full Text Available Summary: Lobohedleolide (1 has been isolated from a soft coral, Sarcophyton sp., as one of three responsible substances for observed inhibitory activity against TNF-a production in lipopolysaccharide (LPS-stimulated murine macrophage-like RAW 264.7 cells. During the examination of other inflammatory cytokines, we found that only 1 induced the production of interleukin (IL-8 in LPS-stimulated human monocytic THP-1 cells, while the other two compounds did not affect the production of IL-8. Although 1 showed an inhibitory effect on nuclear factor-kappaB (NF-kB activation, this compound induced IL-8 promoter activity, which led to the induction of IL-8 production in LPS-stimulated THP-1 cells. Industrial Relevance: Marine organisms are a rich resource of biologically active secondary metabolites. Two marine natural products and two derivatives of marine sponge compounds have been utilized for medical treatment, and marked numbers of marine natural products and their derivatives are now in clinical and pre-clinical trials. Therefore, marine natural products are attractive sources of medicines and their lead compounds, and elucidation of new bioactivity and the mechanism of action of marine natural products are also a very important study for drug discovery. This report describes a new bioactivity of a diterpene previously obtained from an Indonesian soft corral.

  19. Cordycepin inhibits LPS-induced inflammatory and matrix degradation in the intervertebral disc

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    Yan Li

    2016-05-01

    Full Text Available Cordycepin is a component of the extract obtained from Cordyceps militaris and has many biological activities, including anti-cancer, anti-metastatic and anti-inflammatory effects. Intervertebral disc degeneration (IDD is a degenerative disease that is closely related to the inflammation of nucleus pulposus (NP cells. The effect of cordycepin on NP cells in relation to inflammation and degeneration has not yet been studied. In our study, we used a rat NP cell culture and an intervertebral disc (IVD organ culture model to examine the inhibitory effects of cordycepin on lipopolysaccharide (LPS-induced gene expression and the production of matrix degradation enzymes (MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 and oxidative stress-associated factors (nitric oxide and PGE2. We found a protective effect of cordycepin on NP cells and IVDs against LPS-induced matrix degradation and macrophage infiltration. In addition, western blot and luciferase assay results demonstrated that pretreatment with cordycepin significantly suppressed the LPS-induced activation of the NF-κB pathway. Taken together, the results of our research suggest that cordycepin could exert anti-inflammatory and anti-degenerative effects on NP cells and IVDs by inhibiting the activation of the NF-κB pathway. Therefore, cordycepin may be a potential treatment for IDD in the future.

  20. The Protective Effect of Apamin on LPS/Fat-Induced Atherosclerotic Mice

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    Soo-Jung Kim

    2012-01-01

    Full Text Available Apamin, a peptide component of bee venom (BV, has anti-inflammatory properties. However, the molecular mechanisms by which apamin prevents atherosclerosis are not fully understood. We examined the effect of apamin on atherosclerotic mice. Atherosclerotic mice received intraperitoneal (ip injections of lipopolysaccharide (LPS, 2 mg/kg to induce atherosclerotic change and were fed an atherogenic diet for 12 weeks. Apamin (0.05 mg/kg was administered by ip injection. LPS-induced THP-1-derived macrophage inflammation treated with apamin reduced expression of tumor necrosis factor (TNF-α, vascular cell adhesion molecule (VCAM-1, and intracellular cell adhesion molecule (ICAM-1, as well as the nuclear factor kappa B (NF-κB signaling pathway. Apamin decreased the formation of atherosclerotic lesions as assessed by hematoxylin and elastic staining. Treatment with apamin reduced lipids, Ca2+ levels, and TNF-α in the serum from atherosclerotic mice. Further, apamin significantly attenuated expression of VCAM-1, ICAM-1, TGF-β1, and fibronectin in the descending aorta from atherosclerotic mice. These results indicate that apamin plays an important role in monocyte/macrophage inflammatory processing and may be of potential value for preventing atherosclerosis.

  1. Changes in the relative inflammatory responses in sheep cells overexpressing of toll-like receptor 4 when stimulated with LPS.

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    Shoulong Deng

    Full Text Available BACKGROUND: Many groups of Gram-negative bacteria cause diseases harmful to sheep. TLR4 is an important Toll-like receptor (TLR which responds to common Gram-negative bacterial infections. Activation of TLR4 leads to the induction of inflammatory responses, which is a linkage between the innate and adaptive immune systems. A vector pTLR4-3S was constructed to overexpress TLR4 gene in sheep. In this study, effects of TLR4 overexpression on inflammation response under LPS stimulated were addressed in vivo and in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Sheep fetal fibroblasts were transfected with expression vector pTLR4-3S. Transgenic sheep were produced by microinjection of the constructed plasmids into fertilized eggs. Fetal fibroblasts, monocyte-macrophage and fibroblasts isolated from the transgenic sheep were stimulated by LPS. After that immunoactive factors (TNF-α, IL-10, IL-6, IL-8, IFN-γ, nitric oxide, phagocytize ability and adhesion were detected. Furthermore, transgenic sheep were intradermal injected of LPS in ear and observed pathological changes by HE strain. Overexpression of TLR4 gene was observed on transgenic cells and individuals. In vitro, TLR4 overexpression transgenic cells secreted Th1 and Th2 inducing cytokines with a strong LPS mediated inflammation response and promoting the secretion of nitric oxide, and then recovered to initial level. The phagocytosis index of monocyte/macrophage in transgenic sheep was higher than that of non-transgenic sheep (P<0.05. In vivo, tissue sections showed that transgenic individuals launched inflammation response more quickly. CONCLUSIONS/SIGNIFICANCE: Overexpression of TLR4 in transgenic sheep enhanced the clearance of invaded microbe through secretion of cytokines, activation of macrophage, oxidation damage and infiltration of neutrophil.

  2. The role of visfatin on the regulation of inflammation and apoptosis in the spleen of LPS-treated rats.

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    Xiao, Ke; Zou, Wei-Hua; Yang, Zhi; Zia ur Rehman; Ansari, Abdur Rahman; Yuan, Huai-Rui; Zhou, Ying; Cui, Lu; Peng, Ke-Mei; Song, Hui

    2015-02-01

    The purpose of the present study is to determine if visfatin is involved in inflammation or apoptosis induced by LPS in rat. Forty Wistar rats were divided into four groups: saline group, LPS group, visfatin group and Visfatin + LPS co-stimulated group. Spleen samples from each group of rats were collected for study. The spleen structure was examined by histological imaging. Apoptosis was evaluated with TUNEL reaction. Caspase-3 was detected with immunohistochemistry and western blot. The apoptosis-related genes were detected by qPCR and inflammatory cytokines were tested by ELISA. Our main findings were as follows. (1) Macrophages were markedly increased in the visfatin group compared with the saline group. This finding was confirmed when spleen samples were examined with western blot using CD68 antibody. (2) Visfatin promoted the expression of CD68 and caspase-3 in rat spleen, whereas visfatin could inhibit the expression of CD68 and activated caspase-3 in spleen of LPS-induced acute inflammation. (3) Visfatin had a pro-apoptotic effect on normal rat spleen, whereas it exerted an anti-apoptotic effect during LPS-induced lymphocytes apoptosis in rat spleen. Moreover, the effect of visfatin on cell apoptosis was mediated by the mitochondrial pathway. (4) Visfatin could modulate both the anti-inflammatory cytokines and pro-inflammatory cytokines in rat spleen, such as IL-10, IL-4, IL-6, TNF-α and IL-1β. Taken together, we demonstrate that visfatin could participate in the inflammatory process in rat spleen by modulating the macrophages and inflammatory cytokines. Also, visfatin plays a dual role in the apoptosis in rat spleen, which is mediated by the mitochondrial pathway.

  3. Signaling pathways involved in LPS induced TNFalpha production in human adipocytes

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    Festy Franck

    2010-01-01

    Full Text Available Abstract Background The development of obesity has been linked to an inflammatory process, and the role of adipose tissue in the secretion of pro-inflammatory molecules such as IL-6 or TNFalpha has now been largely confirmed. Although TNFalpha secretion by adipose cells is probably induced, most notably by TLR ligands, the activation and secretion pathways of this cytokine are not yet entirely understood. Moreover, given that macrophagic infiltration is a characteristic of obesity, it is difficult to clearly establish the level of involvement of the different cellular types present within the adipose tissue during inflammation. Methods Primary cultures of human adipocytes and human peripheral blood mononuclear cells were used. Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors. Western blot for p38 MAP Kinase was performed on cell lysates. TNFalpha mRNA was detected in cells by RT-PCR and TNFalpha protein was detected in supernatants by ELISA assays. Results We show for the first time that the production of TNFalpha in mature human adipocytes is mainly dependent upon two pathways: NFkappaB and p38 MAP Kinase. Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway. Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages. Conclusion This study clearly demonstrates that the LPS induced activation pathway is an integral part of the inflammatory process linked to obesity, and that adipocytes are responsible for most of the secreted TNFalpha in inflamed adipose tissue, through TLR4 activation.

  4. Cordycepin Inhibits Lipopolysaccharide (LPS-Induced Tumor Necrosis Factor (TNF-α Production via Activating AMP-Activated Protein Kinase (AMPK Signaling

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    Jian-Li Zhang

    2014-07-01

    Full Text Available Tumor necrosis factor (TNF-α is elevated during the acute phase of Kawasaki disease (KD, which damages vascular endothelial cells to cause systemic vasculitis. In the current study, we investigated the potential role of cordycepin on TNFα expression in both lipopolysaccharide (LPS-stimulated macrophages and ex vivo cultured peripheral blood mononuclear cells (PBMCs of KD patients. We found that cordycepin significantly suppressed LPS-induced TNFα expression and production in mouse macrophages (RAW 264.7 cells and bone marrow-derived macrophages (BMDMs. Meanwhile, cordycepin alleviated TNFα production in KD patients’ PBMCs. PBMCs from healthy controls had a much lower level of basal TNF-α content than that of KD patients. LPS-induced TNF-α production in healthy controls’ PBMCs was also inhibited by cordycepin. For the mechanism study, we discovered that cordycepin activated AMP-activated protein kinase (AMPK signaling in both KD patients’ PBMCs and LPS-stimulated macrophages, which mediated cordycepin-induced inhibition against TNFα production. AMPK inhibition by its inhibitor (compound C or by siRNA depletion alleviated cordycepin’s effect on TNFα production. Further, we found that cordycepin inhibited reactive oxygen species (ROS production and nuclear factor kappa B (NF-κB activation in LPS-stimulate RAW 264.7 cells or healthy controls’ PBMCs. PBMCs of KD patients showed higher basal level of ROS and NF-κB activation, which was also inhibited by cordycepin co-treatment. In conclusion, our data showed that cordycepin inhibited TNFα production, which was associated with AMPK activation as well as ROS and NF-κB inhibition. The results of this study should have significant translational relevance in managing this devastating disease.

  5. Cordycepin inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production via activating amp-activated protein kinase (AMPK) signaling.

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    Zhang, Jian-Li; Xu, Ying; Shen, Jie

    2014-07-08

    Tumor necrosis factor (TNF)-α is elevated during the acute phase of Kawasaki disease (KD), which damages vascular endothelial cells to cause systemic vasculitis. In the current study, we investigated the potential role of cordycepin on TNFα expression in both lipopolysaccharide (LPS)-stimulated macrophages and ex vivo cultured peripheral blood mononuclear cells (PBMCs) of KD patients. We found that cordycepin significantly suppressed LPS-induced TNFα expression and production in mouse macrophages (RAW 264.7 cells and bone marrow-derived macrophages (BMDMs)). Meanwhile, cordycepin alleviated TNFα production in KD patients' PBMCs. PBMCs from healthy controls had a much lower level of basal TNF-α content than that of KD patients. LPS-induced TNF-α production in healthy controls' PBMCs was also inhibited by cordycepin. For the mechanism study, we discovered that cordycepin activated AMP-activated protein kinase (AMPK) signaling in both KD patients' PBMCs and LPS-stimulated macrophages, which mediated cordycepin-induced inhibition against TNFα production. AMPK inhibition by its inhibitor (compound C) or by siRNA depletion alleviated cordycepin's effect on TNFα production. Further, we found that cordycepin inhibited reactive oxygen species (ROS) production and nuclear factor kappa B (NF-κB) activation in LPS-stimulate RAW 264.7 cells or healthy controls' PBMCs. PBMCs of KD patients showed higher basal level of ROS and NF-κB activation, which was also inhibited by cordycepin co-treatment. In conclusion, our data showed that cordycepin inhibited TNFα production, which was associated with AMPK activation as well as ROS and NF-κB inhibition. The results of this study should have significant translational relevance in managing this devastating disease.

  6. Evidence that PGE2 in the dorsal and median raphe nuclei is involved in LPS-induced anorexia in rats.

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    Kopf, Brigitte S; Langhans, Wolfgang; Geary, Nori; Hrupka, Brian; Asarian, Lori

    2011-09-01

    Anorexia is an element of the acute-phase immune response. Its mechanisms remain poorly understood. Activation of inducible cyclooxygenase-2 (COX-2) in blood-brain-barrier endothelial cells and subsequent release of prostaglandins (e.g., prostaglandin E2, PGE2) may be involved. Therefore, we sought to relate the effects of prostaglandins on the anorexia following gram-negative bacterial lipopolysaccharide treatment (LPS) to neural activity in the dorsal and median raphe nuclei (DRN and MnR) in rats. COX-2 antagonist (NS-398, 10mg/kg; IP) administration prior to LPS (100μg/kg; IP) prevented anorexia and reduced c-Fos expression the DRN, MnR, nucleus tractus solitarii and several related forebrain areas. These data indicate that COX-2-mediated prostaglandin synthesis is necessary for LPS anorexia and much of the initial LPS-induced neural activation. Injection of NS-398 into the DRN and MnR (1ng/site) attenuated LPS-induced anorexia to nearly the same extent as IP NS-398, suggesting that prostaglandin signaling in these areas is necessary for LPS anorexia. Because the DRN and MnR are sources of major serotonergic projections to the forebrain, these data suggest that serotonergic neurons originating in the midbrain raphe play an important role in acute-phase response anorexia.

  7. Epigenetic Modulation in Periodontitis: Interaction of Adiponectin and JMJD3-IRF4 Axis in Macrophages.

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    Xuan, Dongying; Han, Qianqian; Tu, Qisheng; Zhang, Lan; Yu, Liming; Murry, Dana; Tu, Tianchi; Tang, Yin; Lian, Jane B; Stein, Gary S; Valverde, Paloma; Zhang, Jincai; Chen, Jake

    2016-05-01

    Emerging evidence suggests an important role for epigenetic mechanisms in modulating signals during macrophage polarization and inflammation. JMJD3, a JmjC family histone demethylase necessary for M2 polarization is also required for effective induction of multiple M1 genes by lipopolysaccharide (LPS). However, the effects of JMJD3 to inflammation in the context of obesity remains unknown. To address this deficiency, we firstly examined the expression of JMJD3 in macrophage isolated from bone marrow and adipose tissue of diet induced obesity (DIO) mice. The results indicated that JMJD3 was down-regulated in obesity. Adiponectin (APN), a factor secreted by adipose tissue which is down-regulated in obesity, functions to switch macrophage polarization from M1 to M2, thereby attenuating chronic inflammation. Intriguingly, our results indicated that APN contributed to JMJD3 up-regulation, reduced macrophage infiltration in obese adipose tissue, and abolished the up-regulation of JMJD3 in peritoneal macrophages isolated from DIO mice when challenged with Porphyromonas gingivalis LPS (pg.lps). To elucidate the interaction of APN and JMJD3 involved in macrophage transformation in the context of inflammation, we designed the loss and gain-function experiments of APN in vivo with APN(-/-) mice with experimental periodontitis and in vitro with macrophage isolated from APN(-/-) mice. For the first time, we found that APN can help to reduce periodontitis-related bone loss, modulate JMJD3 and IRF4 expression, and macrophage infiltration. Therefore, it can be inferred that APN may contribute to anti-inflammation macrophage polarization by regulating JMJD3 expression, which provides a basis for macrophage-centered epigenetic therapeutic strategies.

  8. Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile.

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    Julian Maggini

    Full Text Available In recent years it has become clear that the therapeutic properties of bone marrow-derived mesenchymal stromal cells (MSC are related not only to their ability to differentiate into different lineages but also to their capacity to suppress the immune response. We here studied the influence of MSC on macrophage function. Using mouse thioglycolate-elicited peritoneal macrophages (M stimulated with LPS, we found that MSC markedly suppressed the production of the inflammatory cytokines TNF-alpha, IL-6, IL-12p70 and interferon-gamma while increased the production of IL-10 and IL-12p40. Similar results were observed using supernatants from MSC suggesting that factor(s constitutively released by MSC are involved. Supporting a role for PGE(2 we observed that acetylsalicylic acid impaired the ability of MSC to inhibit the production of inflammatory cytokines and to stimulate the production of IL-10 by LPS-stimulated M. Moreover, we found that MSC constitutively produce PGE2 at levels able to inhibit the production of TNF-alpha and IL-6 by activated M. MSC also inhibited the up-regulation of CD86 and MHC class II in LPS-stimulated M impairing their ability to activate antigen-specific T CD4+ cells. On the other hand, they stimulated the uptake of apoptotic thymocytes by M. Of note, MSC turned M into cells highly susceptible to infection with the parasite Trypanosoma cruzi increasing more than 5-fold the rate of M infection. Using a model of inflammation triggered by s.c. implantation of glass cylinders, we found that MSC stimulated the recruitment of macrophages which showed a low expression of CD86 and the MHC class II molecule Ia(b and a high ability to produce IL-10 and IL-12p40, but not IL-12 p70. In summary, our results suggest that MSC switch M into a regulatory profile characterized by a low ability to produce inflammatory cytokines, a high ability to phagocyte apoptotic cells, and a marked increase in their susceptibility to infection by

  9. Perifosine inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production via regulation multiple signaling pathways: new implication for Kawasaki disease (KD) treatment.

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    Shen, Jie; Liang, Li; Wang, Chunlin

    2013-07-26

    Kawasaki disease (KD) is a multisystem vasculitis of unknown etiology, with coronary artery aneurysms occurring in majority of untreated cases. Tumor necrosis factor (TNF)-α is the pleiotropic inflammatory cytokine elevated during the acute phase of KD, which induces damage to vascular endothelial cells to cause systemic vasculitis. We here investigated the potential role of perifosine, a novel Akt inhibitor, on TNFα expression in LPS-stimulated macrophages and in ex-vivo cultured peripheral blood mononuclear cells (PBMCs) of acute KD patients. Here, we found that perifosine inhibited LPS-induced TNFα expression and production in mouse macrophages (RAW 264.7 cells and bone marrow-derived macrophages (BMDMs)). Meanwhile, perifosine administration down-regulated TNFα production in PBMCs isolated from acute KD patients. For the mechanism study, we found that perifosine significantly inhibited Akt and ERK/mitogen-activated protein kinases (MAPK) signaling, while activating AMP-activated protein kinase (AMPK) signaling in both patients' PBMCs and LPS-stimulated macrophages. Interestingly, although perifosine is generally known as an Akt inhibitor, our data suggested that ERK inhibition and AMPK activation, but not Akt inactivation were possibly involved in perifosine-mediated inhibition against TNFα production in monocytes. In conclusion, our data suggested that perifosine significantly inhibited TNFα production via regulation multiple signaling pathways. The results of this study should have significant translational relevance in managing this devastating disease.

  10. Attenuation of LPS-induced lung inflammation by glucosamine in rats.

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    Chuang, Kun-Han; Peng, Yen-Chun; Chien, Han-Yun; Lu, Meng-Lun; Du, Hsin-I; Wu, Yuh-Lin

    2013-12-01

    Acute inflammation is often observed during acute lung injury (ALI) and acute respiratory distress syndrome. Glucosamine is known to act as an anti-inflammatory molecule. The effects of glucosamine on acute lung inflammation and its associated mechanisms remain unclear. The present study sought to address how glucosamine plays an anti-inflammatory role in acute lung inflammation in vivo and in vitro. Using the LPS intratracheal instillation-elicited rat lung inflammation model, we found that glucosamine attenuated pulmonary edema and polymorphonuclear leukocyte infiltration, as well as the production of TNF-α, IL-1β, cytokine-induced neutrophil chemoattractant (CINC)-1, macrophage inflammatory protein (MIP)-2, and nitric oxide (NO) in the bronchoalveolar lavage fluid (BALF) and in the cultured medium of BALF cells. The expression of TNF-α, IL-1β, IFN-γ, CINC-1, MIP-2, monocyte chemotactic protein-1, and inducible NO synthase (iNOS) in LPS-inflamed lung tissue was also suppressed by glucosamine. Using the rat alveolar epithelial cell line L2, we noted that the cytokine mixture (cytomix)-regulated production and mRNA expression of CINC-1 and MIP-2, NO production, the protein and mRNA expression of iNOS, iNOS mRNA stability, and iNOS promoter activity were all inhibited by glucosamine. Furthermore, glucosamine reduced LPS-mediated NF-κB signaling by decreasing IκB phosphorylation, p65 nuclear translocation, and NF-κB reporter activity. Overexpression of the p65 subunit restored the inhibitory action of glucosamine on cytomix-regulated NO production and iNOS expression. In conclusion, glucosamine appears to act as an anti-inflammatory molecule in LPS-induced lung inflammation, at least in part by targeting the NF-κB signaling pathway.

  11. Inhibition of inducible Nitric Oxide Synthase by a mustard gas analog in murine macrophages

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    Smith Milton

    2006-11-01

    Full Text Available Abstract Background 2-Chloroethyl ethyl sulphide (CEES is a sulphur vesicating agent and an analogue of the chemical warfare agent 2,2'-dichlorodiethyl sulphide, or sulphur mustard gas (HD. Both CEES and HD are alkylating agents that influence cellular thiols and are highly toxic. In a previous publication, we reported that lipopolysaccharide (LPS enhances the cytotoxicity of CEES in murine RAW264.7 macrophages. In the present investigation, we studied the influence of CEES on nitric oxide (NO production in LPS stimulated RAW264.7 cells since NO signalling affects inflammation, cell death, and wound healing. Murine macrophages stimulated with LPS produce NO almost exclusively via inducible nitric oxide synthase (iNOS activity. We suggest that the influence of CEES or HD on the cellular production of NO could play an important role in the pathophysiological responses of tissues to these toxicants. In particular, it is known that macrophage generated NO synthesised by iNOS plays a critical role in wound healing. Results We initially confirmed that in LPS stimulated RAW264.7 macrophages NO is exclusively generated by the iNOS form of nitric oxide synthase. CEES treatment inhibited the synthesis of NO (after 24 hours in viable LPS-stimulated RAW264.7 macrophages as measured by either nitrite secretion into the culture medium or the intracellular conversion of 4,5-diaminofluorescein diacetate (DAF-2DA or dichlorofluorescin diacetate (DCFH-DA. Western blots showed that CEES transiently decreased the expression of iNOS protein; however, treatment of active iNOS with CEES in vitro did not inhibit its enzymatic activity Conclusion CEES inhibits NO production in LPS stimulated macrophages by decreasing iNOS protein expression. Decreased iNOS expression is likely the result of CEES induced alteration in the nuclear factor kappa B (NF-κB signalling pathway. Since NO can act as an antioxidant, the CEES induced down-regulation of iNOS in LPS

  12. Berberine reduces Toll-like receptor-mediated macrophage migration by suppression of Src enhancement.

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    Cheng, Wei-Erh; Ying Chang, Miao; Wei, Jyun-Yan; Chen, Yen-Jen; Maa, Ming-Chei; Leu, Tzeng-Horng

    2015-06-15

    Berberine is an isoquinoline with anti-inflammatory activity. We previously demonstrated that there was a loop of signal amplification between nuclear factor kappa B and Src for macrophage mobility triggered by the engagement of Toll-like receptors (TLRs). The simultaneous suppression of lipopolysaccharide (LPS)-mediated upregulation of inducible nitric oxide synthase, cyclooxygenase 2, and cell mobility in berberine-treated macrophages suggested Src might be a target of berberine. Indeed, th reduced migration, greatly suppressed Src induction in both protein and RNA transcript by berberine were observed in macrophages exposed to LPS, peptidoglycan, polyinosinic-polycytidylic acid, and CpG-oligodeoxynucleotides. In addition to Src induction, berberine also inhibited LPS-mediated Src activation in Src overexpressing macrophages and S-nitroso-N-acetylpenicillamine (a nitric oxide donor) could partly restore it. Moreover, berberine suppressed Src activity in fibronectin-stimulated macrophages and in v-Src transformed cells. These results implied that by effectively reducing Src expression and activity, berberine inhibited TLR-mediated cell motility in macrophages.

  13. Oxidized galectin-1 reduces lipopolysaccharide-induced increase of proinflammatory cytokine mRNA in cultured macrophages

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    Yukie Kogawa

    2011-01-01

    Full Text Available Yukie Kogawa1, Kou Nakajima1, Kenichi Sasaguri1, Nobushiro Hamada2, Haruhisa Kawasaki3, Sadao Sato1, Toshihiko Kadoya4, Hidenori Horie51Department of Orthodontics, 2Department of Oral Microbiology, Kanagawa Dental College, Yokosuka; 3Keio University, Kanagawa; 4Maebashi Institute of Technology, Maebashi; 5Research Center of Brain and Oral Science, Kanagawa Dental College, Yokosuka, JapanBackground: Periodontitis is prevalent in older humans. Limiting the inflammation associated with periodontitis may provide a therapy for this condition, because Gram-negative bacteria expressing lipopolysaccharide (LPS have a key role in initiation of inflammation by activating macrophage functions. Because oxidized galectin-1 regulates macrophage functions in other systems, we sought to establish whether this galectin-1 mRNA is expressed in the oral cavity, and whether it could dampen LPS-induced macrophage activation in vitro.Methods: Using the reverse transcriptase polymerase chain reaction (RT-PCR, we measured galectin-1 mRNA expression to clarify its localization to rat gingival tissues and studied the effect of Porphyromonas gingivalis challenge on galectin-1 expression. Next, we tested the effects of adding oxidized galectin-1 to cultured LPS-activated peritoneal macrophages on mRNA expression of proinflammatory factors by RT-PCR and real-time RT-PCR.Results: We established that galectin-1 mRNA is expressed in gingival tissues and also showed that galectin-1 mRNA was significantly increased by challenge with P. gingivalis, indicating that galectin-1 may regulate oral inflammation. On the other hand, LPS 100 ng/mL in serum-containing medium induced macrophages to upregulate mRNA associated with a proinflammatory response, ie, interleukins 1β and 6, and inducible nitric oxide synthase. We showed that application of 0.1–10 ng/mL of oxidized galectin-1 to LPS-treated macrophages reduced the intense LPS-induced increase by serum in proinflammatory m

  14. Hepatitis B Virus X Protein Up-regulates TNF-α and IL-1β Secretion of Macrophages

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective: To provide the experimental basis for further studying the molecular transformation mechanism of Hepatitis B virus (HBV) X protein (HBx) on hepatocellular carcinoma. Methods: Reconstructed plasmid pcDNA3.1(+)-HBx was transfected into THP-1 macrophages. Expression of HBx was assayed in macrophages lysate by Western-blotting, and TNF-α and IL-1β contents were detected respectively by ELISA. All the data were analyzed by SPSS13.0. Results: In THP-1 macrophages, the pcDNA3.1(+)-HBx plasmid expressed HBx with a molecular weight of about 17 KDa demonstrated by Western-blotting. The secreted TNF-α and IL-1β from macrophages were determined by ELISA, the results from analysis of all groups showed as following: control group was different from LPS group and pcDNA3.1(+) group (P<0.01), and so was pcDNA3.1(+)-HBx group; but there was no obvious difference between pcDNA3.1(+) group and LPS group (P>0.05), all of which indicated that transient overexpression of HBx enhanced LPS-induced production of TNF-α and IL-1β by macrophages.Conclusion: Transient overexpression of HBx up-regulates LPS-induced TNF-α and IL-1β secretion of macrophages.

  15. Role of the endocannabinoid system in the mechanisms involved in the LPS-induced preterm labor.

    Science.gov (United States)

    Bariani, María Victoria; Domínguez Rubio, Ana Paula; Cella, Maximiliano; Burdet, Juliana; Franchi, Ana María; Aisemberg, Julieta

    2015-12-01

    P