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Sample records for macrophage cells raw

  1. Direct Effects of Activin A on the Activation of Mouse Macrophage RAW264.7 Cells

    Institute of Scientific and Technical Information of China (English)

    Jingyan Ge; Yinan Wang; Ye Feng; Haiyan Liu; Xueling Cui; Fangfang Chen; Guixiang Tai; Zhonghui Liu

    2009-01-01

    Macrophages play critical roles in innate immune and acquired immune via secreting pro-inflammatory mediators, phagocytosing microorganisms and presenting antigens. Activin A, a member of transforming growth factor β (TGF-β) superfamily, is produced by macrophages and microglia cells. In this study, we reported a direct effect of activin A as a pro-inflammatory factor on mouse macrophage cell line RAW264.7 cells. Our data revealed that activin A could not only increase IL-1v and IL-6 production from RAW264.7 cells, but also promote pinocytic and phagocytic activities of RAW264.7 cells. In addition, activin A obviously up-regulated MHC Ⅱ expression on the surface of RAW264.7 cells, whereas did not influence MHC I expression. Activin A also enhanced CD80 expression, which is a marker of activated macrophages, but did not influence RAW264.7 cell proliferation. These data suggest that activin A may regulate primary macrophage-mediated innate and acquired immune response via promoting the activation of rest macrophages. Cellular & Molecular Immunology.

  2. Endoplasmic Reticulum Stress Cooperates in Zearalenone-Induced Cell Death of RAW 264.7 Macrophages

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    Fenglei Chen

    2015-08-01

    Full Text Available Zearalenone (ZEA is a fungal mycotoxin that causes cell apoptosis and necrosis. However, little is known about the molecular mechanisms of ZEA toxicity. The objective of this study was to explore the effects of ZEA on the proliferation and apoptosis of RAW 264.7 macrophages and to uncover the signaling pathway underlying the cytotoxicity of ZEA in RAW 264.7 macrophages. This study demonstrates that the endoplasmic reticulum (ER stress pathway cooperated in ZEA-induced cell death of the RAW 264.7 macrophages. Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT and flow cytometry assay. Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78 and CCAAT/enhancer binding protein homologous protein (CHOP, two ER stress-related marker genes. Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs, significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death. In summary, our results suggest that ZEA induces the apoptosis and necrosis of RAW 264.7 macrophages in a dose- and time-dependent manner via the ER stress pathway in which the activation of CHOP plays a critical role.

  3. Endoplasmic Reticulum Stress Cooperates in Zearalenone-Induced Cell Death of RAW 264.7 Macrophages.

    Science.gov (United States)

    Chen, Fenglei; Li, Qian; Zhang, Zhe; Lin, Pengfei; Lei, Lanjie; Wang, Aihua; Jin, Yaping

    2015-08-20

    Zearalenone (ZEA) is a fungal mycotoxin that causes cell apoptosis and necrosis. However, little is known about the molecular mechanisms of ZEA toxicity. The objective of this study was to explore the effects of ZEA on the proliferation and apoptosis of RAW 264.7 macrophages and to uncover the signaling pathway underlying the cytotoxicity of ZEA in RAW 264.7 macrophages. This study demonstrates that the endoplasmic reticulum (ER) stress pathway cooperated in ZEA-induced cell death of the RAW 264.7 macrophages. Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry assay. Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), two ER stress-related marker genes. Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA) or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs), significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death. In summary, our results suggest that ZEA induces the apoptosis and necrosis of RAW 264.7 macrophages in a dose- and time-dependent manner via the ER stress pathway in which the activation of CHOP plays a critical role.

  4. Macropinocytosis contributes to the macrophage foam cell formation in RAW264.7 cells

    Institute of Scientific and Technical Information of China (English)

    Wenqi Yao; Ke Li; Kan Liao

    2009-01-01

    The key event in the atherosclerosis development is the lipids uptake by macrophage and the formation of foam cell in subendothelial arterial space. Besides the uptake of modified low-density lipoprotein (LDL) by scavenger receptor-mediated endocytosis, macrophages possess constitutive macropinocytosis, which is capable of taking up a large quantity of solute. Macrophage foam cell formation could be induced in RAW264.7 cells by increasing the serum concentration in the culture medium. Foam cell formation induced by serum could be blocked by phosphoinositide 3-kinase inhibi-tor, LY294002 or wortmannin, which inhibited macro-pinocytosis but not receptor-mediated endocytosis. Further analysis indicated that macropinocytosis took place at the gangliosides-enriched membrane area. Cholesterol depletion by β-methylcyclodextrin-blocked macropinocytosis without affecting scavenger receptor-mediated endocytosis of modified LDLs. These results suggested that macropinocytosis might be one of the important mechanisms for lipid uptake in macrophage. And it made significant contribution to the lipid accumulation and foam cell formation.

  5. 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.

  6. Herp depletion inhibits zearalenone-induced cell death in RAW 264.7 macrophages.

    Science.gov (United States)

    Chen, Fenglei; Lin, Pengfei; Wang, Nan; Yang, Diqi; Wen, Xin; Zhou, Dong; Wang, Aihua; Jin, Yaping

    2016-04-01

    Herp is an endoplasmic reticulum (ER) membrane protein and strongly induced by the ER stress that not only participates in the unfolded protein response (UPR) under the ER stress, but also in cell autophagy under glucose starvation (GS). However, we do not know whether Herp plays any roles in other responses, such as zearalenone (ZEA). In this study, we constructed recombinant lentiviral vectors for Herp shRNA expression and generated stable Herp knockdown RAW 264.7 macrophages. Flow cytometry analysis showed Herp depletion could inhibit cell death induced by ZEA. Western blot analysis revealed that Herp depletion could up-regulate autophagy-related protein LC3-I conversion into LC3-II and the expression of ER stress-related protein CHOP. These results suggest that Herp depletion inhibits cell death by up-regulating autophagy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Suppression of cell division-associated genes by Helicobacter pylori attenuates proliferation of RAW264.7 monocytic macrophage cells.

    Science.gov (United States)

    Tan, Grace Min Yi; Looi, Chung Yeng; Fernandez, Keith Conrad; Vadivelu, Jamuna; Loke, Mun Fai; Wong, Won Fen

    2015-06-16

    Helicobacter pylori at multiplicity of infection (MOI ≥ 50) have been shown to cause apoptosis in RAW264.7 monocytic macrophage cells. Because chronic gastric infection by H. pylori results in the persistence of macrophages in the host's gut, it is likely that H. pylori is present at low to moderate, rather than high numbers in the infected host. At present, the effect of low-MOI H. pylori infection on macrophage has not been fully elucidated. In this study, we investigated the genome-wide transcriptional regulation of H. pylori-infected RAW264.7 cells at MOI 1, 5 and 10 in the absence of cellular apoptosis. Microarray data revealed up- and down-regulation of 1341 and 1591 genes, respectively. The expression of genes encoding for DNA replication and cell cycle-associated molecules, including Aurora-B kinase (AurkB) were down-regulated. Immunoblot analysis verified the decreased expression of AurkB and downstream phosphorylation of Cdk1 caused by H. pylori infection. Consistently, we observed that H. pylori infection inhibited cell proliferation and progression through the G1/S and G2/M checkpoints. In summary, we suggest that H. pylori disrupts expression of cell cycle-associated genes, thereby impeding proliferation of RAW264.7 cells, and such disruption may be an immunoevasive strategy utilized by H. pylori.

  8. Nanosized silver (II) pyridoxine complex to cause greater inflammatory response and less cytotoxicity to RAW264.7 macrophage cells

    Science.gov (United States)

    Paul, Avijit; Ju, Hee; Rangasamy, Sabarinathan; Shim, Yumi; Song, Joon Myong

    2015-03-01

    With advancements in nanotechnology, silver has been engineered into a nanometre size and has attracted great research interest for use in the treatment of wounds. Silver nanoparticles (AgNPs) have emerged as a potential alternative to conventional antibiotics because of their potential antimicrobial property. However, AgNPs also induce cytotoxicity, generate reactive oxygen species (ROS), and cause mitochondrial damage to human cells. Pyridoxine possesses antioxidant and cell proliferation activity. Therefore, in the present investigation, a nanosilver-pyridoxine complex (AgPyNP) was synthesized, and its cytotoxicity and immune response was compared with AgNPs in macrophage RAW264.7 cells. Results revealed that AgPyNPs showed less cytotoxicity compared with AgNPs by producing a smaller amount of ROS in RAW264.7 cells. Surprisingly, however, AgPyNPs caused macrophage RAW264.7 cells to secrete a larger amount of interleukin-8 (IL-8) and generate a more active inflammatory response compared to AgNPs. It activated TNF-α, NF-κB p65, and NF-κB p50 to generate a more vigorous immune protection that produces a greater amount of IL-8 compared to AgNPs. Overall findings indicate that AgPyNPs exhibited less cytotoxicity and evoked a greater immune response in macrophage RAW264.7 cells. Thus, it can be used as a better wound-healing agent than AgNPs.

  9. Peroxiredoxin-1, a possible target in modulating inflammatory cytokine production in macrophage like cell line RAW264.7.

    Science.gov (United States)

    Tae Lim, Young; Sup Song, Dong; Joon Won, Tae; Lee, Yun-Jung; Yoo, Jong-Sun; Eun Hyung, Kyeong; Won Yoon, Joo; Park, So-Young; Woo Hwang, Kwang

    2012-06-01

    Peroxiredoxin (PRX), a scavenger of H(2) O(2) and alkyl hydroperoxides in living organisms, protects cells from oxidative stress. Contrary to its known anti-oxidant roles, the involvement of PRX-1 in the regulation of lipopolysaccharide (LPS) signaling is poorly understood, possible immunological functions of PRX-1 having been uncovered only recently. In the present study, it was discovered that the PRX-1 deficient macrophage like cell line (RAW264.7) has anti-inflammatory activity when stimulated by LPS. Treatment with LPS for 3 hrs resulted in increased gene expression of an anti-inflammatory cytokine, interleukin-10 (IL-10), in PRX-1 knock down RAW264.7 cells. Gene expression of pro-inflammatory cytokines IL-1β and tumor necrosis factor- α (TNF-α) did not show notable changes under the same conditions. However, production of these cytokines significantly decreased in PRX-1 knock down RAW264.7 cells with 12 hrs of stimulation. Production of IL-10 was also increased in PRX-1 knock down RAW264.7 cells with 12 hrs of stimulation. We predicted that higher concentrations of IL-10 would result in decreased expression of IL-1β and TNF-α in PRX-1 knock-down cells. This was confirmed by blocking IL-10, which reestablished IL-1β and TNF-α secretion. We also observed that increased concentrations of IL-10 do not affect the NF-κB pathway. Interestingly, STAT3 phosphorylation by LPS stimulation was significantly increased in PRX-1 knockdown RAW264.7 cells. Up-regulation of IL-10 in PRX-1 knockdown cells and the resulting downregulation of proinflammatory cytokine production seem to involve the STAT3 pathway in macrophages. Thus, down-regulation of PRX-1 may contribute to the suppression of adverse effects caused by excessive activation of macrophages through affecting the STAT3 signaling pathway.

  10. Enniatin B-induced cell death and inflammatory responses in RAW 267.4 murine macrophages

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    Gammelsrud, A. [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway); Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway); Solhaug, A. [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway); Dendelé, B. [EA 4427 SeRAIC, IRSET, Université de Rennes 1, IFR 140, Rennes (France); Sandberg, W.J. [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway); Ivanova, L. [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway); Kocbach Bølling, A. [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway); Lagadic-Gossmann, D. [EA 4427 SeRAIC, IRSET, Université de Rennes 1, IFR 140, Rennes (France); Refsnes, M.; Becher, R. [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway); Eriksen, G. [Norwegian Veterinary Institute, P.O. Box 750, Centrum, N-0106 Oslo (Norway); Holme, J.A., E-mail: jorn.holme@fhi.no [Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo (Norway)

    2012-05-15

    The mycotoxin enniatin B (EnnB) is predominantly produced by species of the Fusarium genera, and often found in grain. The cytotoxic effect of EnnB has been suggested to be related to its ability to form ionophores in cell membranes. The present study examines the effects of EnnB on cell death, differentiation, proliferation and pro-inflammatory responses in the murine monocyte–macrophage cell line RAW 264.7. Exposure to EnnB for 24 h caused an accumulation of cells in the G0/G1-phase with a corresponding decrease in cyclin D1. This cell cycle-arrest was possibly also linked to the reduced cellular ability to capture and internalize receptors as illustrated by the lipid marker ganglioside GM1. EnnB also increased the number of apoptotic, early apoptotic and necrotic cells, as well as cells with elongated spindle-like morphology. The Neutral Red assay indicated that EnnB induced lysosomal damage; supported by transmission electron microscopy (TEM) showing accumulation of lipids inside the lysosomes forming lamellar structures/myelin bodies. Enhanced levels of activated caspase-1 were observed after EnnB exposure and the caspase-1 specific inhibitor ZYVAD-FMK reduced EnnB-induced apoptosis. Moreover, EnnB increased the release of interleukin-1beta (IL-1β) in cells primed with lipopolysaccharide (LPS), and this response was reduced by both ZYVAD-FMK and the cathepsin B inhibitor CA-074Me. In conclusion, EnnB was found to induce cell cycle arrest, cell death and inflammation. Caspase-1 appeared to be involved in the apoptosis and release of IL-1β and possibly activation of the inflammasome through lysosomal damage and leakage of cathepsin B. -- Highlights: ► The mycotoxin EnnB induced cell cycle arrest, cell death and inflammation. ► The G0/G1-arrest was linked to a reduced ability to internalize receptors. ► EnnB caused lysosomal damage, leakage of cathepsin B and caspase-1 cleavage. ► Caspase-1 was partly involved in both apoptosis and release of IL-1

  11. Riboflavin deprivation inhibits macrophage viability and activity - a study on the RAW 264.7 cell line.

    Science.gov (United States)

    Mazur-Bialy, Agnieszka Irena; Buchala, Beata; Plytycz, Barbara

    2013-08-28

    Riboflavin, or vitamin B2, as a precursor of the coenzymes FAD and FMN, has an indirect influence on many metabolic processes and determines the proper functioning of several systems, including the immune system. In the human population, plasma riboflavin concentration varies from 3·1 nM (in a moderate deficiency, e.g. in pregnant women) to 10·4 nM (in healthy adults) and 300 nM (in cases of riboflavin supplementation). The purpose of the present study was to investigate the effects of riboflavin concentration on the activity and viability of macrophages, i.e. on one of the immunocompetent cell populations. The study was performed on the murine monocyte/macrophage RAW 264.7 cell line cultured in medium with various riboflavin concentrations (3·1, 10·4, 300 and 531 nM). The results show that riboflavin deprivation has negative effects on both the activity and viability of macrophages and reduces their ability to generate an immune response. Signs of riboflavin deficiency developed in RAW 264.7 cells within 4 d of culture in the medium with a low riboflavin concentration (3·1 nM). In particular, the low riboflavin content reduced the proliferation rate and enhanced apoptotic cell death connected with the release of lactate dehydrogenase. The riboflavin deprivation impaired cell adhesion, completely inhibited the respiratory burst and slightly impaired phagocytosis of the zymosan particles. In conclusion, macrophages are sensitive to riboflavin deficiency; thus, a low riboflavin intake in the diet may affect the immune system and may consequently decrease proper host immune defence.

  12. Substance P Induces HO-1 Expression in RAW 264.7 Cells Promoting Switch towards M2-Like Macrophages

    Science.gov (United States)

    Montana, Giovanna

    2016-01-01

    Substance P (SP) is a neuropeptide that mediates many physiological as well as inflammatory responses. Recently, SP has been implicated in the resolution of inflammation through induction of M2 macrophages phenotype. The shift between M1-like and M2-like, allowing the resolution of inflammatory processes, also takes place by means of hemeoxygenase-1 (HO-1). HO-1 is induced in response to oxidative stress and inflammatory stimuli and modulates the immune response through macrophages polarisation. SP induces HO-1 expression in human periodontal ligament (PDL), the latter potentially plays a role in cytoprotection. We demonstrated that SP promotes M2-like phenotype from resting as well as from M1 macrophages. Indeed, SP triggers the production of interleukine-10 (IL-10), interleukine-4 (IL-4) and arginase-1 (Arg1) without nitric oxide (NO) generation. In addition, SP increases HO-1 expression in a dose- and time-dependent manner. Here we report that SP, without affecting cell viability, significantly reduces the production of pro-inflammatory cytokines and enzymes, such as tumor necrosis factor-alpha (TNF-α), interleukine-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and ameliorates migration and phagocytic properties in LPS-stimulated RAW 264.7 cells. M2-like conversion required retention of NF-κB p65 into the cytoplasm and HO-1 induced expression. Silencing of the HO-1 mRNA expression reversed the induction of pro-inflammatory cytokines in RAW 264.7 stimulated by LPS and down-regulated anti-inflammatory hallmarks of M2 phenotype. In conclusion, our data show that SP treatment might be associated with anti-inflammatory effects in LPS-stimulated RAW 264.7 cells by suppressing NF-κB activation and inducing HO-1 expression. PMID:27907187

  13. Toxic response of HIPCO single-walled carbon nanotubes in mice and RAW264.7 macrophage cells.

    Science.gov (United States)

    Park, Eun-Jung; Zahari, Nur Elida M; Kang, Min-Sung; Lee, Sang jin; Lee, Kyuhong; Lee, Byoung-Seok; Yoon, Cheolho; Cho, Myung-Haing; Kim, Younghun; Kim, Jae-Ho

    2014-08-17

    In this study, we identified the toxic response of pristine single-walled carbon nanotubes (P-SWCNTs) synthesized by HIPCO method in mice and RAW264.7 cells, a murine peritoneal macrophage cell line. P-SWCNT contained a large amount of Fe ion (36 wt%). In the lungs of mice 24 h after intratracheal administration, P-SWCNTs increased the secretion of IL-6 and MCP-1, and the number of total cells, the portion of neutrophils, lymphocytes, and eosinophils, also significantly increased at a 100 μg/mL of concentration. In RAW264.7 cells, cell viability and ATP production decreased in a dose-dependent manner at 24 h after exposure, whereas the generations of ROS and NO were enhanced at all concentrations together with the activation of the MAP kinase pathway. Moreover, the levels of both apoptosis- and autophagy-related proteins and ER stress-related proteins clearly increased, and the concentrations of Fe, Cu, and Zn ions, but not of Mn ions, increased in a dose-dependent manner. TEM images also revealed that P-SWCNTs induced the formation of autophagosome-like vacuoles, the dilatation of the ER, the generation of mitochondrial flocculent densities, and the separation of organelle by disappearance of the cell membrane. Taken together, we suggest that P-SWCNTs cause acute inflammatory response in the lungs of mice, and induce autophagy accompanied with apoptosis through mitochondrial dysfunction and ER stress in RAW264.7 cells. Furthermore, further study is required to elucidate how the physicochemical properties of SWCNTs determine the cell death pathway and an immune response.

  14. Mycelial Extract of Phellinus linteus Induces Cell Death in A549 Lung Cancer Cells and Elevation of Nitric Oxide in Raw 264.7 Macrophage Cells.

    Science.gov (United States)

    Lee, Jong-Jin; Kwon, Ho-Kyun; Lee, Dong-Soo; Lee, Seung-Woo; Lee, Kye-Kwan; Kim, Kyu-Joong; Kim, Jong-Lae

    2006-09-01

    In the present study, in order to investigate the anti-proliferative phenomenon of PLME, the effects of mycelial extract of Phellinus linteus (PLME) on the growth of human lung carcinoma cell line A549 was examined. We studied on the effects of PLME on the release of nitric oxide (NO) in mouse macrophage Raw 264.7 cells. Treatment of PLME to A549 cells resulted in the growth inhibition, morphological change and induction of apoptotic cell death in a dose-dependent manner as measured by MTT assay. We found that PLME stimulated a dose-dependent increase in NO production. These findings suggest that PLME enhances the anti-tumoral activity of macrophage and may be a potential therapeutic agent for the control of human lung carcinoma cells.

  15. 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.

  16. Prevention of copper-induced cell death by GC-rich DNA oligomers in murine macrophage-like RAW264.7 cells.

    Science.gov (United States)

    Matsushita, Sakiko; Mochizuki, Shinichi; Sakurai, Kazuo; Kawano, Tomonori

    2015-01-01

    Impact of redox active transition metals on activation of cell death signaling in plant cells have been documented to date. We have recently reported that GC-rich DNA oligomers with high affinity for binding of copper and catalytic activity for removal of ROS as novel plant cell-protecting agents. Here, we show that similar DNA oligomers protect the mouse macrophage-like RAW264.7 cells from copper-induced cell death, suggesting that the phenomenon firstly observed in plant model can be expanded to a wider range of cells and/or organisms including mammalian cells.

  17. Determining Effects of Elaidic Acid on PPAR- Gamma Expression in RAW 264.7 Macrophage Cell Line

    Directory of Open Access Journals (Sweden)

    M Doosti

    2012-08-01

    Full Text Available Background: Several dietary factors are involved in cardiovascular coronary heart diseases, including trans fatty acids, which are generally formed during hydrogenation of vegetable oils, a process that causes conversion of liquid oils into semisolid fats. Nowadays, it is well-known that trans fatty acids form a major risk factor in the occurrence and progression of atherosclerosis. On the other hand, it has been identified that some nuclear receptors, such as PPARs, are involved and play important roles in lipid homeostasis and pathogenesis of cardiovascular diseases. Therefore, we studied the effect of elaidic acid on gene expression of peroxisome proliferator activated receptor gamma (PPARγ.Methods: Murine macrophage RAW264.7 cells were treated by 0.5, 1, and 2 mM concentrations of elaidic acid for 6 h. The control group was treated by 50% ethanol (as solvent, equivalent to the amount of ethanol used in 2 mM concentration of elaidic acid. Later, the total RNA was extracted and its cDNA was synthesized. Finally, the quantity of PPARγ gene expression was measured by real-time PCR.Results: Overall, 0.5, 1, and 2 mM concentrations of elaidic acid decreased PPARγ gene expression in RAW264.7 macrophage cell line by -1.36, -1.68, and -3.24 folds compared with the control group, respectively.Conclusion: By decreasing the expression of nuclear receptor PPARγ, elaidic acid causes, intensifies or accelerates the occurrence of cardiovascular diseases, especially atherosclerosis. This finding shows the importance of reducing the consumption of elaidic acid containing foods.

  18. Sesamin Enhances Cholesterol Efflux in RAW264.7 Macrophages

    OpenAIRE

    Nan Liu; Chongming Wu; Lizhong Sun; Jun Zheng; Peng Guo

    2014-01-01

    Foam cells formation as a result of the uncontrolled cytophagy of modified cholesterol by macrophages plays a key role in the occurrence and development of atherosclerosis. Sesamin is an active constituent of Sesamum indicum which has been shown to possess multiple pharmacological activities. In this work, we investigated the effects of sesamin on foam cell formation and cholesterol efflux in RAW264.7 macrophages. Sesamin dose-dependently inhibited the enhanced cholesterol accumulation elicit...

  19. 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.

  20. Indirect detection of superoxide in RAW 264.7 macrophage cells using microchip electrophoresis coupled to laser-induced fluorescence.

    Science.gov (United States)

    de Campos, Richard P S; Siegel, Joseph M; Fresta, Claudia G; Caruso, Giuseppe; da Silva, José A F; Lunte, Susan M

    2015-09-01

    Superoxide, a naturally produced reactive oxygen species (ROS) in the human body, is involved in many pathological and physiological signaling processes. However, if superoxide formation is left unregulated, overproduction can lead to oxidative damage to important biomolecules, such as DNA, lipids, and proteins. Superoxide can also lead to the formation of peroxynitrite, an extremely hazardous substance, through its reaction with endogenously produced nitric oxide. Despite its importance, quantitative information regarding superoxide production is difficult to obtain due to its high reactivity and low concentrations in vivo. MitoHE, a fluorescent probe that specifically reacts with superoxide, was used in conjunction with microchip electrophoresis (ME) and laser-induced fluorescence (LIF) detection to investigate changes in superoxide production by RAW 264.7 macrophage cells following stimulation with phorbol 12-myristate 13-acetate (PMA). Stimulation was performed in the presence and absence of the superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC) and 2-metoxyestradiol (2-ME). The addition of these inhibitors resulted in an increase in the amount of superoxide specific product (2-OH-MitoE(+)) from 0.08 ± 0.01 fmol (0.17 ± 0.03 mM) in native cells to 1.26 ± 0.06 fmol (2.5 ± 0.1 mM) after PMA treatment. This corresponds to an approximately 15-fold increase in intracellular concentration per cell. Furthermore, the addition of 3-morpholino-sydnonimine (SIN-1) to the cells during incubation resulted in the production of 0.061 ± 0.006 fmol (0.12 ± 0.01 mM) of 2-OH-MitoE(+) per cell on average. These results demonstrate that indirect superoxide detection coupled with the use of SOD inhibitors and a separation method is a viable method to discriminate the 2-OH-MitoE(+) signal from possible interferences.

  1. Echinacea Species and Alkamides Inhibit Prostaglandin E2 Production in RAW264.7 Mouse Macrophage Cells

    Science.gov (United States)

    LaLone, Carlie A.; Hammer, Kimberly D. P.; Wu, Lankun; Bae, Jaehoon; Leyva, Norma; Liu, Yi; Solco, Avery K. S.; Kraus, George A.; Murphy, Patricia A.; Wurtele, Eve S.; kim, Ok-Kyung; Seo, Kwon; Widrlechner, Mark P.; Birt, Diane F.

    2008-01-01

    Inhibition of prostaglandin E2 (PGE2) production in lipopolysaccharide-stimulated RAW264.7 mouse macrophage cells was assessed with an enzyme immunoassay following treatments with Echinacea extracts or synthesized alkamides. Results indicated that ethanol extracts diluted in media to a concentration of 15 μg/mL from E. angustifolia, E. pallida, E. simulata, and E. sanguinea significantly inhibited PGE2 production. In further studies, PGE2 production was significantly reduced by all synthesized alkamides assayed at 50 μM, by Bauer alkamides 8, 12A analogue, and 14, Chen alkamide 2, and Chen alkamide 2 analogue at 25 μM and by Bauer alkamide 14 at 10 μM. Cytotoxicity did not play a role in the noted reduction of PGE2 production in either the Echinacea extracts or synthesized alkamides. High-performance liquid chromatography analysis identified individual alkamides present at concentrations below 2.8 μM in the extracts from the six Echinacea species (15 μg/mL crude extract). Because active extracts contained Echinacea in a synergistic or additive manner. PMID:17696440

  2. Some bioactive potentials of two biflavanols isolated from Garcinia kola on cadmium-induced alterations of raw U937 cells and U937-derived macrophages

    Institute of Scientific and Technical Information of China (English)

    Tebekeme Okoko; Diepreye Ere

    2013-01-01

    Objective: To investigate the abilities of two flavonoids - Garcinia biflavanol-1 (GB-1) and Garcinia biflavanol-2 (GB-2) from Garcinia kola (G. kola) in reducing cadmium-induced effects on raw U937 cells and U937-derived macrophages. Methods: Macrophage U937 cells were incubated with cadmium followed by treatment with the flavonoids and cell viability assessed via trypan blue staining. In the other experiment, the U937 cells were transformed to the macrophage form and treated with cadmium in order to activate them. The cells were later incubated with the flavonoids and finally the supernatant of each cell culture was analysed for the secretion of nitric oxide, catalyse activity, and the release of tumour necrosis factor-alpha, interleukin-1 and interleukin-2 as indices of macrophage activation. Quercetin (a flavonol) was used as the reference flavonoid in all experiments. Results: It revealed that the flavonoids significantly increased the viability of the cells and also reduced the cadmium-induced activation of the macrophage cells in a concentration-dependent manner. The flavanols GB-1 and GB-2 possessed higher activities than quercetin in all cases (P<0.05). Garcinia biflavanol-2 possessed a higher bioactivity than GB-1 significantly (P<0.05). Conclusions: In addition to corroborating the several reported importance of G. kola as a potential neutraceutical and pharmacological condiment, the study also clearly indicates the role hydroxylation especially at the 3´- position of polyphenols could play in enhancing bioactivities of flavonoids.

  3. Scopoletin and scopolin isolated from Artemisia iwayomogi suppress differentiation of osteoclastic macrophage RAW 264.7 cells by scavenging reactive oxygen species.

    Science.gov (United States)

    Lee, Sang-Hyun; Ding, Yan; Yan, Xi Tao; Kim, Young-Ho; Jang, Hae-Dong

    2013-04-26

    Artemisia iwayomogi has been used as a folk medicine for treating various diseases including inflammatory and immune-related diseases. Scopoletin (1) and scopolin (2) were isolated from this species. Scopoletin (1) showed more potent peroxyl radical-scavenging capacity, reducing capacity, and cellular antioxidant capacity compared to scopolin (2). The inhibitory effect of 1 on the receptor activator of nuclear factor κB ligand-induced osteoclastic differentiation of RAW 264.7 macrophage cells was also more potent than that of 2. The production of general reactive oxygen species (ROS) and superoxide anions during differentiation of preosteoclastic RAW 264.7 cells into osteoclasts was attenuated by compounds 1 and 2. These findings indicate that the suppressive effects of 1 and 2 on the differentiation of preosteoclastic RAW 264.7 cells is partially due to their intracellular antioxidant capacity, as they can scavenge ROS and play an important signaling role in the differentiation process.

  4. Inhibitory effects of methanol extract of Cyperus rotundus rhizomes on nitric oxide and superoxide productions by murine macrophage cell line, RAW 264.7 cells.

    Science.gov (United States)

    Seo, W G; Pae, H O; Oh, G S; Chai, K Y; Kwon, T O; Yun, Y G; Kim, N Y; Chung, H T

    2001-06-01

    The rhizomes of Cyperus rotundus (C. rotundus) have been used in oriental traditional medicines for the treatment of stomach and bowel disorders, and inflammatory diseases. Nitric oxide (NO) and superoxide (O2-) are important mediators in the pathogenesis of inflammatory diseases. This study was undertaken to address whether the metanol (MeOH) extract of rhizomes of C. rotundus could modulate NO and O2- productions by murine macrophage cell line, RAW 264.7 cells. The MeOH extract of rhizomes of C. rotundus showed the inhibition of NO production in a dose-dependent manner by RAW 264.7 cells stimulated with interferon-gamma plus lipopolysaccharide. The inhibition of NO production by the extract was due to the suppression of iNOS protein, as well as iNOS mRNA expression, determined by Western and Northern blotting analyses, respectively. In addition, the MeOH extract suppressed the production of O2- by phorbol ester-stimulated RAW 264.7 cells in dose- and time-dependent manners. Collectively, these results suggest that the MeOH extract of rhizomes of C. rotundus could be developed as anti-inflammatory candidate for the treatment of inflammatory diseases mediated by overproduction of NO and O2-.

  5. Glycine tomentella Hayata inhibits IL-1β and IL-6 production, inhibits MMP-9 activity, and enhances RAW264.7 macrophage clearance of apoptotic cells

    Directory of Open Access Journals (Sweden)

    Sun Yu-Shu

    2010-11-01

    Full Text Available Abstract Background To assess the effects of Glycine tomentella Hayata (GTH, a traditional herbal medicine for treatment of rheumatic diseases on the expression of the proinflammatory cytokines and on the clearance of apoptotic cells by macrophages. Methods RAW264.7 cells were cultured with lipopolysaccharide (LPS in the presence or absence of ethanol extract of GTH. The expression of proinflammatory cytokines IL-1β, IL-6, and TNF-α, and inducible nitric oxide synthase (iNOS and transglutaminase 2 (TG2 were assayed by reverse transcriptase-polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA. Matrix metalloproteinase (MMP-2 and MMP-9 were assayed by gelatin zymography. For detecting uptake of apoptotic cells, RAW264.7 cells were cultured with carboxyfluorescein diacetate (CFDA-stained apoptotic cells and assayed by flow cytometry. Results The major components of GTH analyzed by high-performance liquid chromatography (HPLC chromatogram were daidzein (42.5%, epicatechin (28.8%, and naringin (9.4%. GTH treatment inhibited the expression of proinflammatory cytokines IL-1β, IL-6 and MMP-9 but did not affect the expression of TNF-α and iNOS. GTH significantly enhanced the expression of TG2 and the clearance of apoptotic cells by RAW264.7 macrophages. Conclusions GTH inhibits proinflammatory cytokine secretion and MMP-9 activity, enhances apoptotic cell uptake and up-regulates TG2 expression. Our data show that GTH might have beneficial effects on rheumatic diseases.

  6. Effects of ultrafine petrol exhaust particles on cytotoxicity, oxidative stress generation, DNA damage and inflammation in human A549 lung cells and murine RAW 264.7 macrophages.

    Science.gov (United States)

    Durga, Mohan; Nathiya, Soundararajan; Rajasekar, Abbu; Devasena, Thiyagarajan

    2014-09-01

    Air pollution has persistently been the major cause of respiratory-related illness and death. Environmental pollutants such as diesel and petrol exhaust particles (PEPs) are the major contributors to urban air pollution. The aim of the present study was to characterize and investigate the in vitro cytotoxicity, oxidative stress, DNA damage and inflammation induced by PEPs. Cultured type II epithelium cells (human A549 lung cells) and alveolar macrophages (murine RAW 264.7 cells) were exposed to control, vehicle control and to different concentrations of PEPs for up to 24h. Each treatment was evaluated by cell viability, cytotoxicity, oxidative stress, DNA damage and inflammatory parameters. Overall in vitro studies demonstrated that both cell lines showed similar patterns in response to the above studies induced by petrol exhaust nanoparticles (PENPs). Vehicle control showed no changes compared with the control. In both cell lines, significant changes at the dose of 20 and 50μg/mL (A549 cell lines) and 10and 20μg/mL (macrophages) for PENPs were found. The reactive oxygen species production in both cell lines shot up in minutes, reached the maximum within an hour and came down after 4h. Hence, exposure to PENPs resulted in dose-dependent toxicity in cultured A549 cells and RAW 264.7 cells and was closely correlated to increased oxidative stress, DNA damage and inflammation. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Suppressive Effect on Lipopolysaccharide-Induced Proinflammatory Mediators by Citrus aurantium L. in Macrophage RAW 264.7 Cells via NF-κB Signal Pathway

    Directory of Open Access Journals (Sweden)

    Sang-Rim Kang

    2011-01-01

    Full Text Available Citrus fruits have been used as an edible fruit and a traditional medicine since ancient times. In particular, the peels of immature citrus fruits are used widely in traditional herbal medicine in Korea, as they are believed to contain bioactive components exerting anti-inflammatory activity. This study examined whether the crude methanol extract of Citrus aurantium L. (CME has a suppressive effect on inducible enzymes and proinflammatory cytokines by inhibiting the NF-κB pathway in LPS-stimulated macrophage RAW 264.7 cells. The cells were pretreated with the indicated concentrations of CME (5, 10, 20, and 50 μg/mL and then treated with LPS (1 μg/mL. The results showed that CME (10, 20, and 50 μg/mL inhibited the LPS- (1 μg/mL induced mRNA and protein expression of iNOS in macrophage Raw 264.7 cells. In addition, the expression of COX-2 was inhibited at the mRNA and protein levels by CME in a dose-dependent manner. The mRNA expression of proinflammatory cytokines, such as TNF-α and IL-6, were markedly reduced by CME (10, 20, and 50 μg/mL. Moreover, CME clearly suppressed the nuclear translocation of the NF-κB p65 subunits, which was correlated with its inhibitory effect on I-κB phosphorylation. These results suggest that CME has anti-inflammatory properties by modulating the expression of COX-2, iNOS, and proinflammatory cytokines, such as TNF-α and IL-6, in macrophage RAW 264.7 cells via the NF-κB pathway.

  8. Anti-inflammatory phenolics isolated from Juniperus rigida leaves and twigs in lipopolysaccharide-stimulated RAW264.7 macrophage cells.

    Science.gov (United States)

    Jeong, Eun Ju; Seo, Hanee; Yang, Heejung; Kim, Jinwoong; Sung, Sang Hyun; Kim, Young Choong

    2012-12-01

    Inflammation is an essential host defense system particularly in response to infection and injury; however, excessive or undesirable inflammatory responses contribute to acute and chronic human diseases. A high-throughput screening effort searching for anti-inflammatory compounds from medicinal plants deduced that the methanolic extract of Juniperus rigida S. et L. (Cupressaceae) inhibited significantly nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Activity-guided fractionation and isolation yielded 13 phenolic compounds, including one new phenylpropanoid glycosides, 3,4-dimethoxycinnamyl 9-O-β-D-glucopyranoside (1). Among the isolated compounds, phenylpropanoid glycosides with p-hydroxy group (2, 4) and massoniaside A (7), (+)-catechin (10), amentoflavone (11) effectively inhibited LPS-induced NO production in RAW264.7 cells.

  9. Downregulation of pro-inflammatory mediators by a water extract of Schisandra chinensis (Turcz.) Baill fruit in lipopolysaccharide-stimulated RAW 264.7 macrophage cells.

    Science.gov (United States)

    Dilshara, Matharage Gayani; Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Kang, Chang-Hee; Lee, Seungheon; Park, Sang Rul; Jeong, Jin-Woo; Choi, Yung Hyun; Seo, Yong Taek; Jang, Young Pyo; Kim, Gi-Young

    2013-09-01

    Schisandra chinensis has a long-standing history of medicinal use as a tonic, a sedative, an anti-tussive, and an anti-aging drug. Nevertheless, the antagonistic effects of S. chinensis against lipopolysaccharide (LPS)-stimulated responses have not yet been studied. In this study, we investigated whether water extract of S. chinensis fruit (WESC) has the ability to attenuate the expression of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α) in LPS-stimulated RAW 264.7 macrophage cells. WESC inhibited the expression of LPS-induced pro-inflammatory mediators, namely, NO, PGE2, and TNF-α. Furthermore, gene expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α was inhibited both at mRNA and protein synthesis levels, without any cytotoxic effect. Moreover, WESC significantly suppressed LPS-induced DNA-binding activity of NF-κB by inhibiting degradation of IκBα. It was found that pyrrolidine dithiocarbamate (PDTC), a specific NF-κB inhibitor, downregulates the expression of these pro-inflammatory genes to be closely regulated by NF-κB activity. Furthermore, we found that WESC retains dephosphorylation of Akt in response to LPS, and consequently suppressed the DNA-binding activity of NF-κB in RAW 264.7 macrophage cells. LY294002, a specific Akt inhibitor, attenuated LPS-induced pro-inflammatory gene expression via suppression of NF-κB activity. Taken together, our results indicate that WESC downregulates the expression of pro-inflammatory genes involved in the synthesis of NO, PGE2, and TNF-α in LPS-stimulated RAW 264.7 macrophage cells by suppressing Akt-dependent NF-κB activity.

  10. 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),...

  11. Sesquiterpenes from an Egyptian herbal medicine, Pulicaria undulate, with inhibitory effects on nitric oxide production in RAW264.7 macrophage cells.

    Science.gov (United States)

    Hegazy, Mohamed-Elamir F; Matsuda, Hisashi; Nakamura, Seikou; Yabe, Mikuko; Matsumoto, Tomoko; Yoshikawa, Masayuki

    2012-01-01

    The methylene chloride-methanol (1 : 1) extract from the air-dried aerial parts of wild Pulicaria undulata collected in North Sinia, Egypt, showed inhibitory effects on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) in RAW264.7 macrophages. From the extract, three new sesquiterpenes named 5α-hydroperoxyivalin, 8-epi-xanthanol, and 8-epi-isoxanthanol were isolated together with four known sesquiterpenes. The structure of each new sesquiterpenes was determined on the basis of physicochemical and chemical evidence. In addition, all the sesquiterpenoids significantly inhibited the production of NO. Ivalin (IC50=2.0 μM) and 2α-hydroxyalantolactone (1.8 μM) showed particularly strong inhibitory effects, but had strong cytotoxic effects as well. Furthermore, ivalin and 2α-hydroxyalantolactone concentration-dependently reduced inducible NO synthase (iNOS) protein levels in RAW264.7 cells.

  12. Microparticles from apoptotic RAW 264.7 macrophage cells carry tumour necrosis factor-α functionally active on cardiomyocytes from adult mice

    Directory of Open Access Journals (Sweden)

    Edward Milbank

    2015-10-01

    Full Text Available After ischaemic injury and in patients with atherosclerosis, the pool of inflammatory macrophages is enlarged in the heart and in atherosclerotic plaques. Monocyte/macrophage-derived microparticles (MPs are part of the pathological process of unstable atherosclerotic plaques. The present study focused on effects of MPs, produced by apoptotic murine RAW 264.7 macrophage cell line, in adult murine cardiomyocytes. Flow cytometry and western blot analysis showed that these MPs contained the soluble form of tumour necrosis factor alpha (TNF-α. Cardiomyocyte sarcomere shortening amplitudes and kinetics were reduced within 5 min of exposure to these MPs. Conversely, Ca2+ transient amplitude and kinetics were not modified. The contractile effects of MPs were completely prevented after pretreatment with nitric oxide synthase, guanylate cyclase or TNF-α inhibitors as well as blocking TNF-α receptor 1 with neutralizing antibody. Microscopy showed that, after 1 h, MPs were clearly surrounding rod-shaped cardiomyocytes, and after 2 h they were internalized into cardiomyocytes undergoing apoptosis. After 4 h of treatment with MPs, cardiomyocytes expressed increased caspase-3, caspase-8, Bax and cytochrome C. Thus, MPs from apoptotic macrophages induced a negative inotropic effect and slowing of both contraction and relaxation, similar to that observed in the presence of TNF-α. The use of specific inhibitors strongly suggests that TNF-α receptors and the guanylate cyclase/cGMP/PKG pathway were involved in the functional responses to these MPs and that the mitochondrial intrinsic pathway was implicated in their proapoptotic effects. These data suggest that MPs issued from activated macrophages carrying TNF-α could contribute to propagation of inflammatory signals leading to myocardial infarction.

  13. Control of microorganisms of oral health interest with Arctium lappa L. (burdock) extract non-cytotoxic to cell culture of macrophages (RAW 264.7).

    Science.gov (United States)

    de Oliveira, Jonatas Rafael; de Aguiar Almeida, Rosilene Batista; das Graças Figueiredo Vilela, Polyana; de Oliveira, Felipe Eduardo; da Rocha, Rosilene Fernandes; Jorge, Antonio Olavo Cardoso; de Oliveira, Luciane Dias

    2014-08-01

    To evaluate the antimicrobial activity of Arctium lappa L. extract on Staphylococcus aureus, S. epidermidis, Streptococcus mutans, Candida albicans, C. tropicalis and C. glabrata. In addition, the cytotoxicity of this extract was analyzed on macrophages (RAW 264.7). By broth microdilution method, different concentrations of the extract (250-0.4 mg/mL) were used in order to determine the minimum microbicidal concentration (MMC) in planktonic cultures and the most effective concentration was used on biofilms on discs made of acrylic resin. The cytotoxicity A. lappa L. extract MMC was evaluated on RAW 264.7 by MTT assay and the quantification of IL-1β and TNF-α by ELISA. The most effective concentration was 250 mg/mL and also promoted significant reduction (log₁₀) in the biofilms of S. aureus (0.438 ± 0.269), S. epidermidis (0.377 ± 0.298), S. mutans (0.244 ± 0.161) and C. albicans (0.746 ± 0.209). Cell viability was similar to 100%. The production of IL-1β was similar to the control group (p>0.05) and there was inhibition of TNF-α (plappa L. extract was microbicidal for all the evaluated strains in planktonic cultures, microbiostatic for biofilms and not cytotoxic to the macrophages. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Sesamin Enhances Cholesterol Efflux in RAW264.7 Macrophages

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    Nan Liu

    2014-06-01

    Full Text Available Foam cells formation as a result of the uncontrolled cytophagy of modified cholesterol by macrophages plays a key role in the occurrence and development of atherosclerosis. Sesamin is an active constituent of Sesamum indicum which has been shown to possess multiple pharmacological activities. In this work, we investigated the effects of sesamin on foam cell formation and cholesterol efflux in RAW264.7 macrophages. Sesamin dose-dependently inhibited the enhanced cholesterol accumulation elicited by oxidized low-density lipoprotein cholesterol (oxLDL in RAW264.7 cells. Treatment with sesamin (10 μM significantly enhanced cholesterol efflux mediated by high-density lipoprotein (HDL. Realtime quantitative PCR and luciferase assays showed that sesamin significantly increased the mRNA levels of PPARγ, LXRα, and ABCG1, and increased the transcriptional activity of PPARγ. The stimulating effect of sesamin on cholesterol efflux was substantially inhibited by the co-treatment with GW9662, a potent inhibitor of PPARγ. These results suggest that sesamin is a new inhibitor of foam cell formation that may stimulate cholesterol efflux through upregulation of the PPARγ-LXRα-ABCG1 pathway.

  15. Sesamin enhances cholesterol efflux in RAW264.7 macrophages.

    Science.gov (United States)

    Liu, Nan; Wu, Chongming; Sun, Lizhong; Zheng, Jun; Guo, Peng

    2014-06-06

    Foam cells formation as a result of the uncontrolled cytophagy of modified cholesterol by macrophages plays a key role in the occurrence and development of atherosclerosis. Sesamin is an active constituent of Sesamum indicum which has been shown to possess multiple pharmacological activities. In this work, we investigated the effects of sesamin on foam cell formation and cholesterol efflux in RAW264.7 macrophages. Sesamin dose-dependently inhibited the enhanced cholesterol accumulation elicited by oxidized low-density lipoprotein cholesterol (oxLDL) in RAW264.7 cells. Treatment with sesamin (10 μM) significantly enhanced cholesterol efflux mediated by high-density lipoprotein (HDL). Realtime quantitative PCR and luciferase assays showed that sesamin significantly increased the mRNA levels of PPARγ, LXRα, and ABCG1, and increased the transcriptional activity of PPARγ. The stimulating effect of sesamin on cholesterol efflux was substantially inhibited by the co-treatment with GW9662, a potent inhibitor of PPARγ. These results suggest that sesamin is a new inhibitor of foam cell formation that may stimulate cholesterol efflux through upregulation of the PPARγ-LXRα-ABCG1 pathway.

  16. 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.

  17. 2-Phenylnaphthalene Derivatives Inhibit Lipopolysaccharide-Induced Pro-Inflammatory Mediators by Downregulating of MAPK/NF-κB Pathways in RAW 264.7 Macrophage Cells

    Science.gov (United States)

    Chang, Chi-Fen; Liao, Kang-Chun; Chen, Chung-Hwan

    2017-01-01

    The anti-inflammatory pharmacological effect of eight 2-phenylnaphthalenes (PNAP-1−PNAP-8) on lipopolysaccharide (LPS)-induced RAW 264.7 (a mouse cell line) was investigated. Among them, 6,7-dihydroxy-2-(4′-hydroxyphenyl)naphthalene (PNAP-6) and 2-(4′-aminophenyl)-6,7-dimethoxynaphthalene (PNAP-8) exhibited the best anti-inflammatory activity in this study. PNAP-6 and PNAP-8 not only significantly decreased the expression of inducible nitric oxide synthase and cyclooxygenase-II, but also inhibited the production of nitric oxide, interleukin-6, and tumor necrosis factor-α in LPS stimulated cells. Moreover, PNAP-6 and PNAP-8 inhibited nuclear factor (NF)-κB activation by decreasing the degradation of IκB and nuclear translocation of NF-κB subunit (p65). In addition, PNAP-6 and PNAP-8 also attenuated the phosphorylation of ERK, p38, and JNK. These results suggest that PNAP-6 and PNAP-8 exert anti-inflammatory activities by down regulating NF-κB activation and the mitogen-activated protein kinase signaling pathway in LPS-stimulated Raw 264.7 cells. This is the first study demonstrating that PNAPs can inhibit LPS-induced pro-inflammatory mediators in macrophages cells. PMID:28060845

  18. Acetyl Eburicoic Acid from Laetiporus sulphureus var. miniatus Suppresses Inflammation in Murine Macrophage RAW 264.7 Cells.

    Science.gov (United States)

    Saba, Evelyn; Son, Youngmin; Jeon, Bo Ra; Kim, Seong-Eun; Lee, In-Kyoung; Yun, Bong-Sik; Rhee, Man Hee

    2015-06-01

    The basidiomycete Laetiporus sulphureus var. miniatus belongs to the Aphyllophorales, Polyporaceae, and grows on the needleleaf tree. The fruiting bodies of Laetiporus species are known to produce N-methylated tyramine derivatives, polysaccharides, and various lanostane triterpenoids. As part of our ongoing effort to discover biologically active compounds from wood-rotting fungi, an anti-inflammatory triterpene, LSM-H7, has been isolated from the fruiting body of L. sulphureus var. miniatus and identified as acetyl eburicoic acid. LSM-H7 dose-dependently inhibited the NO production in RAW 264.7 cells without any cytotoxicity at the tested concentrations. Furthermore it suppressed the production of proinflammatory cytokines, mainly inducible nitric oxide synthase, cyclooxygenase-2, interleukin (IL)-1β, IL-6 and tumor necrosis factor α, when compared with glyceraldehyde 3-phosphate dehydrogenase. These data suggest that LSM-H7 is a crucial component for the anti-inflammatory activity of L. sulphureus var. miniatus.

  19. Anti-inflammatory mechanism of Kaempferia parviflora in murine macrophage cells (RAW 264.7) and in experimental animals.

    Science.gov (United States)

    Sae-wong, Chutha; Tansakul, Pimpimon; Tewtrakul, Supinya

    2009-07-30

    The rhizomes of Kaempferia parviflora Wall. ex Baker have been used in Thailand for treatment of gout, apthous ulcer, peptic ulcer and abscesses. In our previous study, the crude ethanol extract of Kaempferia parviflora and its compound (5, 5-hydroxy-3,7,3',4'-tetramethoxyflavone), was reported to show nitric oxide (NO) inhibition in RAW 264.7 cells. The present study is thus investigated the anti-inflammatory mechanism of Kaempferia parviflora extract and compound 5 against inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expressions. The extract of Kaempferia parviflora and its compound were tested against NO and prostaglandin E(2) (PGE(2)) releases using RAW264.7 cells as well as studied on anti-inflammatory activity in carrageenan-induced rat paw edema and acute toxicity in mice. The results revealed that the ethanol extract of Kaempferia parviflora markedly inhibited PGE(2) release with an IC(50) value of 9.2 microg/ml. This plant extract and compound 5 also suppressed mRNA expression of iNOS in dose-dependent manners, whereas COX-2 mRNA expression was partly affected. According to the in vivo study, chloroform and hexane fractions greater decreased rat paw edema than ethanol, ethyl acetate and water fractions. The mechanisms for anti-inflammatory activity of Kaempferia parviflora and compound 5 are mainly due to the inhibition of iNOS mRNA expression but partly through that of COX-2 mRNA.

  20. Suppressive effects of acetone extract from the stem bark of three Acacia species on nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells

    Institute of Scientific and Technical Information of China (English)

    Kandhasamy Sowndhararajan; Rameshkumar Santhanam; Sunghyun Hong; Jin-Woo Jhoo; Songmun Kim

    2016-01-01

    Objective: To compare the inhibitory effects of acetone extracts from the stem bark of three Acacia species(Acacia dealbata, Acacia ferruginea and Acacia leucophloea) on nitric oxide production.Methods: The lipopolysaccharide(LPS)-stimulated RAW 264.7 macrophage cells were used to investigate the regulatory effect of acetone extracts of three Acacia stem barks on nitric oxide production and the expression of inducible nitric oxide synthase,cyclooxygenase-2 and tumor necrosis factor-a. Further, the phenolic profile of acetone extracts from the Acacia barks was determined by liquid chromatography-mass spectrometry/mass spectrometry analysis.Results: All the three extracts significantly decreased LPS-induced NO production as well as the expression of inducible nitric oxide synthase, cyclooxygenase-2 and tumor necrosis factor-a in a concentration dependent manner(25, 50 and 75 mg/m L). In the liquid chromatography-mass spectrometry/mass spectrometry analysis, acetone extract of Acacia ferruginea bark revealed the presence of 12 different phenolic components including quercetin, catechin, ellagic acid and rosmanol. However, Acacia dealbata and Acacia leucophloea barks each contained 6 different phenolic components.Conclusions: The acetone extracts of three Acacia species effectively inhibited the NO production in LPS-stimulated RAW 264.7 cells and the presence of different phenolic components in the bark extracts might be responsible for reducing the NO level in cells.

  1. 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,...

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

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    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.

  3. The uptake of tocopherols by RAW 264.7 macrophages

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    Papas Andreas M

    2002-10-01

    Full Text Available Abstract Background Alpha-Tocopherol and gamma-tocopherol are the two major forms of vitamin E in human plasma and the primary lipid soluble antioxidants. The dietary intake of gamma-tocopherol is generally higher than that of alpha-tocopherol. However, alpha-tocopherol plasma levels are about four fold higher than those of gamma-tocopherol. Among other factors, a preferential cellular uptake of gamma-tocopherol over alpha-tocopherol could contribute to the observed higher plasma alpha-tocopherol levels. In this investigation, we studied the uptake and depletion of both alpha-tocopherol and gamma-tocopherol (separately and together in cultured RAW 264.7 macrophages. Similar studies were performed with alpha-tocopheryl quinone and gamma-tocopheryl quinone, which are oxidation products of tocopherols. Results RAW 264.7 macrophages showed a greater uptake of gamma-tocopherol compared to alpha-tocopherol (with uptake being defined as the net difference between tocopherol transported into the cells and loss due to catabolism and/or in vitro oxidation. Surprisingly, we also found that the presence of gamma-tocopherol promoted the cellular uptake of alpha-tocopherol. Mass balance considerations suggest that products other than quinone were formed during the incubation of tocopherols with macrophages. Conclusion Our data suggests that gamma-tocopherol could play a significant role in modulating intracellular antioxidant defence mechanisms. Moreover, we found the presence of gamma-tocopherol dramatically influenced the cellular accumulation of alpha-tocopherol, i.e., gamma-tocopherol promoted the accumulation of alpha-tocopherol. If these results could be extrapolated to in vivo conditions they suggest that gamma-tocopherol is selectively taken up by cells and removed from plasma more rapidly than alpha-tocopherol. This could, in part, contribute to the selective maintenance of alpha-tocopherol in plasma compared to gamma-tocopherol.

  4. Heterogeneities in inflammatory and cytotoxic responses of RAW 264.7 macrophage cell line to urban air coarse, fine, and ultrafine particles from six European sampling campaigns

    Energy Technology Data Exchange (ETDEWEB)

    Jalava, P.I.; Salonen, R.O.; Pennanen, A.S.; Sillanpaa, M.; Halinen, A.I.; Happo, M.S.; Hillamo, R.; Brunekreef, B.; Katsouyanni, K.; Sunyer, J.; Hirvonen, M.R. [National Public Health Institute, Kuopio (Finland). Dept. for Environmental Health

    2007-03-15

    We investigated the cytotoxic and inflammatory activities of size-segregated particulate samples (particulate matter, PM) from contrasting air pollution situations in Europe. Coarse (PM10-2.5), fine (PM2.5-0.2), and ultrafine (PM0.2) particulate samples were collected with a modified Harvard high-volume cascade impactor (HVCI). Mouse RAW 264.7 macrophages were exposed to the samples for 24 h. Selected inflammatory mediators, nitric oxide (NO) and cytokines (tumor necrosis factor alpha (TNF alpha), interleukin 6 (IL-6), macrophage inflammatory protein-2 (MIP-2)), were measured together with cytotoxicity (MTT test), and analysis of apoptosis and cell cycle (propidium iodide staining). The PM10-2.5 samples had a much higher inflammatory activity than the PM2.5-0.2 and PM0.2 samples, but the PM2.5-0.2 samples showed the largest differences in inflammatory activity, and the PM0.2 samples in cytotoxicity, between the sampling campaigns. The PM2.5-0.2 samples from traffic environments in springtime Barcelona and summertime Athens had the highest inflammatory activities, which may be related to the high photochemical activity in the atmosphere during the sampling campaigns. The PM0.2 sample from wintertime Prague with proven impacts from local coal and biomass combustion had very high cytotoxic and apoptotic activities and caused a distinct cell cycle arrest. Thus, particulate size, sources, and atmospheric transformation processes affect the toxicity profile of urban air particulate matter. These factors may explain some of the heterogeneity observed in particulate exposure-response relationships of human health effects in epidemiological studies.

  5. Effect of Niacin on Cholesterol Efflux in Macrophage Raw264.7 Cells%烟酸对巨噬细胞 raw264.7胆固醇流出的影响

    Institute of Scientific and Technical Information of China (English)

    董静; 施文剑; 赵水平

    2013-01-01

    [Objective]To observe the effect of niacin on cholesterol efflux in macrophages,and to explore the possible mechanism.[Methods]Macrophage 264.7 cells were incubated in the medium containing various concentration of niacin (0~5.0mM)for 24h.Reverse transcription polymerase chain reaction(RT-PCR)was used to determine the expression of adenosine triphosphate-binding cassette transporter G1(ABCG1),liver X-receptor a(LXRa)and peroxisome proliferator-activated receptor-γ(PPARγ),and Western blot was used to detect the protein expression of ABCG1 and LXRa in macrophages.Liquid scintillation counter was used to de-tect cholesterol efflux.[Results]Niacin dose-dependently increased the mRNA and protein expression of AB-CG1 and promoted ABCG1-mediated cholesterol efflux in Raw264.7 cells.The expression of LXRa and PPARγin Raw264.7 cells was high.Compared with control group,the expression of LXRa and PPARγinter-vened by niacin 5.0mM increased obviously.[Conclusion]Niacin can promote cholesterol efflux pathway me-diated by ABCG1,which may be associated with the up-regulation of LXRa and PPARγ expression.It can provide the clue to explain the mechanisms of high density lipoprotein cholesterol raised by niacin.%[目的]观察烟酸对巨噬细胞胆固醇流出的影响并探讨其机制。[方法]对巨噬细胞(raw 264.7细胞株)给予不同浓度的烟酸(0~5.0 mM)干预24 h 后,收集细胞,反转录聚合酶链反应测定巨噬细胞三磷酸腺苷结合盒转运体 G1(ABCG1),肝 X 受体 a(LXRa)和过氧化物酶增殖物激活受体γ(PPARγ)mRNA 表达及 Western blot 印迹检测 ABCG1和 LXRa 蛋白表达,采用液体闪烁计数器检测细胞内胆固醇流出。[结果]烟酸呈剂量依赖性增加巨噬细胞 ABCG1 mRNA 及蛋白的表达,并促进其介导的胆固醇流出。LXRa 和PPARγ在巨噬细胞有较高水平的表达,与空白组比较,LXRa,PPARγ表达在5.0 mM 浓度的烟酸干预时明显升高。[

  6. Inhibitory effect of Jeju endemic seaweeds on the production of pro-inflammatory mediators in mouse macrophage cell line RAW 264.7*

    Science.gov (United States)

    Yang, Eun-Jin; Moon, Ji-Young; Kim, Min-Jin; Kim, Dong Sam; Kim, Chan-Shick; Lee, Wook Jae; Lee, Nam Ho; Hyun, Chang-Gu

    2010-01-01

    Seaweed has been used in traditional cosmetics and as a herbal medicine in treatments for cough, boils, goiters, stomach ailments, and urinary diseases, and for reducing the incidence of tumors, ulcers, and headaches. Despite the fact that seaweeds are frequently used in the practice of human health, little is known about the role of seaweed in the context of inflammation. This study aimed to investigate the influence of Jeju endemic seaweed on a mouse macrophage cell line (RAW 264.7) under the stimulation of lipopolysaccharide (LPS). Ethyl acetate extracts obtained from 14 different kinds of Jeju seaweeds were screened for inhibitory effects on pro-inflammatory mediators. Our results revealed that extracts from five seaweeds, Laurencia okamurae, Grateloupia elliptica, Sargassum thunbergii, Gloiopeltis furcata, and Hizikia fusiformis, were potent inhibitors of the production of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Based on these results, the anti-inflammatory effects and low cell toxicity of these seaweed extracts suggest potential therapeutic applications in the regulation of the inflammatory response. PMID:20443209

  7. The α-cyclodextrin complex of the Moringa isothiocyanate suppresses lipopolysaccharide-induced inflammation in RAW 264.7 macrophage cells through Akt and p38 inhibition.

    Science.gov (United States)

    Giacoppo, Sabrina; Rajan, Thangavelu Soundara; Iori, Renato; Rollin, Patrick; Bramanti, Placido; Mazzon, Emanuela

    2017-03-13

    In the last decades, a growing need to discover new compounds for the prevention and treatment of inflammatory diseases has led researchers to consider drugs derived from natural products as a valid option in the treatment of inflammation-associated disorders. The purpose of the present study was to investigate the anti-inflammatory effects of a new formulation of Moringa oleifera-derived 4-(α-L-rhamnopyranosyloxy)benzyl isothiocyanate as a complex with alpha-cyclodextrin (moringin + α-CD) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells, a common model used for inflammation studies. In buffered/aqueous solution, the moringin + α-CD complex has enhanced the water solubility and stability of this isothiocyanate by forming a stable inclusion system. Our results showed that moringin + α-CD inhibits the production of inflammatory mediators in LPS-stimulated macrophages by down-regulation of pro-inflammatory cytokines (TNF-α and IL-1β), by preventing IκB-α phosphorylation, translocation of the nuclear factor-κB (NF-κB), and also via the suppression of Akt and p38 phosphorylation. In addition, as a consequence of upstream inhibition of the inflammatory pathway following treatment with moringin + α-CD, the modulation of the oxidative stress (results focused on the expression of iNOS and nitrotyrosine) and apoptotic pathway (Bax and Bcl-2) was demonstrated. Therefore, moringin + α-CD appears to be a new relevant helpful tool to use in clinical practice for inflammation-associated disorders.

  8. MicroRNA-181b regulates endotoxin tolerance by targeting IL-6 in macrophage RAW264.7 cells.

    Science.gov (United States)

    Zhang, Wenjun; Shen, Xiaojun; Xie, Luyang; Chu, Maoping; Ma, Yanmei

    2015-01-01

    Interleukin 6 (IL-6) is a major pro-inflammatory cytokine and dysregulation of IL-6 is relevant to many inflammatory diseases. Endotoxin induced tolerance of IL-6 is an important mechanism to avoid the excessive immune reaction. But to date, the molecular mechanisms of endotoxin tolerance of IL-6 remain unclear. Here we reported that IL-6 secretion and microRNA-181b (miR-181b) expression were inversely correlated following LPS stimulation. We also demonstrated that miR-181b targeting the 3'-UTR of IL-6 transcripts and up-regulation of miR-181b was associated with NF-kB. We further demonstrated that up-regulation of miR-181b in response to LPS was required for inducing IL-6 tolerance in macrophage. Our results suggested that the post-transcriptional control mediated by miR-181b could be involved in fine tuning the critical level of IL-6 expression in endotoxin tolerance.

  9. MRSA 对巨噬细胞系 RAW264.7自噬的影响%Effect of MRSA on the autophagy of RAW264.7 macrophage cell line

    Institute of Scientific and Technical Information of China (English)

    朱军; 吴本权; 杨洋; 朱家馨; 张天托

    2016-01-01

    Objective To evaluate the effect of methicillin-resistant staphylococcus aureus (MRSA) infection upon the autophagy of macrophages.Methods Mouse monocyte-macrophage cell line RAW264.7 was used.In the experimental groups,RAW264.7 cells were infected with Gram-positive MRSA for different time points including 0,30,60,90,1 20,1 50 and 1 80 min.In the control group,the cells were left untreat-ed.After MRSA infection,the expression of ATG5,ATG7 and ATG1 2 was measured by fluorescence quantita-tive PCR,and the expression levels of autophagy-related protein (Beclin1 )and microtubule-associated protein light chain 3 (LC3)were measured by Western blot.Results At 3 h after MRSA infection,the expression levels of ATG5,ATG7 and ATG1 2 mRNA in the experimental group were significantly higher compared with those in the control group (all P 0.05).The relative expression levels of ATG7,ATG1 2 mRNA,LC3 and Beclin1 proteins peaked at 1 80 min following MRSA infection,and the level of ATG5 mRNA achieved the peak at 1 50 min after MRSA infection.Conclusions MRSA could induce autophagy in macrophages.The au-tophagy genes were activated in a certain sequence.Autophagy probably acted as an alternative immune process of phagocytosis of MRSA by macrophages.%目的:探讨耐甲氧西林金黄色葡萄球菌(MRSA)感染对于巨噬细胞自噬的影响。方法以小鼠单核巨噬细胞系 RAW264.7为研究对象,以革兰阳性菌 MRSA 感染3 h 组作为实验组,设立空白对照组(对照组),另设 MRSA 感染0、30、60、120、150、180 min 组。以 MRSA 感染细胞后,荧光定量 PCR 检测自噬相关基因5(ATG5)、ATG7、ATG12的表达情况,蛋白免疫印迹法检测自噬相关蛋白 Beclin1及微管相关蛋白轻链3(LC3)的表达情况。结果感染 MRSA 3 h 后,实验组RAW264.7细胞中 ATG5、ATG7、ATG12的 mRNA 表达水平均高于对照组(P 均<0.01),LC3蛋白相对表达量也比对照组增加(P <0.01),2

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

    Science.gov (United States)

    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.

  11. 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.

  12. Lipoxin A4 negatively regulates lipopolysaccharide-induced differentiation of RAW264.7 murine macrophages into dendritic-like cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Li; WU Ping; JIN Sheng-wei; YUAN Ping; WAN Jing-yuan; ZHOU Xiao-yan; XIONG Wei; FANG Feng; YE Du-yun

    2007-01-01

    Background Lipoxins (LXs), endogenous anti-inflammatory and pro-resolving eicosanoids generated during various inflammatory conditions, have novel immunomodulatory properties. Because dendritic cells (DCs) play crucial roles in the initiation and maintenance of immune response, we determined whether LXs could modulate the maturation process of DCs and investigated the effects of lipoxin A4 (LXA4) on lipopolysaccharide (LPS)-induced differentiation of RAW264.7cells into dendritic-like cells.Methods RAW264.7 cells were cultured in vitro with 1 μg/ml LPS in the absence or presence of LXA4 for 24 hours, and cellular surface markers (MHC-Ⅱ, CD80 (B7-1), CD86(B7-2)) were measured by flow cytometry (FCM). Mixed lymphocyte reaction was performed to evaluate the allostimulatory activity. Cytoplastic IκB degradation and nuclear factor kappa B (NF-κB) translocation were detected by Western blotting. Luciferase reporter plasmid was transiently transfected into RAW264.7 cells, and luciferase activity was determined to measure the transcriptional activity of NF-κB.Results LXA4 reduced the ratio of LPS-treated RAW264.7 cells to DCs with morphological characteristics and inhibited the expression of MHC Ⅱ. LPS-induced up-regulation of CD86 was moderately suppressed by LXA4 but no obvious change of CD80 was observed. Moreover, LXA4 weakened the allostimulatory activity of LPS-treated RAW264.7 cells.These alterations of LPS+LXA4-treated cells were associated with a marked inhibition of IκB degradation, NF-κB translocation and then the transcriptional activity of NF-κB.Conclusions LXA4 negatively regulates LPS-induced differentiation of RAW264.7 cells into dendritic-like cells.This activity reveals an undescribed mechanism of LXA4 to prevent excessive and sustained immune reaction by regulating maturation of DCs.

  13. Suppressive effects of acetone extract from the stem bark of three Acacia species on nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells

    Directory of Open Access Journals (Sweden)

    Kandhasamy Sowndhararajan

    2016-08-01

    Conclusions: The acetone extracts of three Acacia species effectively inhibited the NO production in LPS-stimulated RAW 264.7 cells and the presence of different phenolic components in the bark extracts might be responsible for reducing the NO level in cells.

  14. Neuroprotection of Neuro2a cells and the cytokine suppressive and anti-inflammatory mode of action of resveratrol in activated RAW264.7 macrophages and C8-B4 microglia.

    Science.gov (United States)

    Steiner, Nicole; Balez, Rachelle; Karunaweera, Niloo; Lind, Joanne M; Münch, Gerald; Ooi, Lezanne

    2016-05-01

    Chronic inflammation is a hallmark of neurodegenerative disease and cytotoxic levels of nitric oxide (NO) and pro-inflammatory cytokines can initiate neuronal death pathways. A range of cellular assays were used to assess the anti-inflammatory and neuroprotective action of resveratrol using murine microglial (C8-B4), macrophage (RAW264.7) and neuronal-like (Neuro2a) cell lines. We examined the release of NO by Griess assay and used a Bioplex array to measure a panel of pro- and anti-inflammatory cytokines and chemokines, in response to the inflammatory stimuli lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Resveratrol was a potent inhibitor of NO and cytokine release in activated macrophages and microglia. The activity of resveratrol increased marginally in potency with longer pre-incubation times in cell culture that was not due to cytotoxicity. Using an NO donor we show that resveratrol can protect Neuro2a cells from cytotoxic concentrations of NO. The protective effect of resveratrol from pro-inflammatory signalling in RAW264.7 cells was confirmed in co-culture experiments leading to increased survival of Neuro2a cells. Together our data are indicative of the potential neuroprotective effect of resveratrol during nitrosative stress and neuroinflammation.

  15. Effect of Viili exopolysaccharides on the activation, cell proliferation and cytokine production of murine macrophage cell line RAW264.7%Viili多糖对巨噬细胞RAW264.7激活及分泌细胞因子的影响

    Institute of Scientific and Technical Information of China (English)

    武俊华; 罗成

    2013-01-01

    This study aims to study the activation of RAW264. 7 cells upon stimulation of viili exopolysaccharides (VEPS). Cell proliferation in response to different concentrations of VEPS was analyzed by MTT, phagocytosis was detected by neutral red. The secretion of. IL-6 and IL-1β in the supernatant of cell culture was measured by Griess kit and Elisa kit, respectively. Cell morphology was observed by scanning electronic microscopy (SEM), and cell cycles by flow cytometry. VEPS at 25 through 100 (g/ml promoted cell proliferation, phagocytosis as evidenced by uptake of neutral red, and release of NO, IL-6 and IL-1β by RAW264. 7 cells. The RAW264. 7 cells treated with LPS or VEPS alone, or in combination became flattened demonstrated by SEM, a strong indication of activation of macrophages. VEPS also increased cell number in phase G1. VEPS promoted activation of macrophages in which NO, IL-1β, IL-6 were involved in a very similar way as LPS, VEPS activate macrophages and may eventually activates lymphocytes as well, and increases both innate and specific immunity.%探讨viili胞外多糖(Viili exopolysaccharides,VEPS)对小鼠巨噬细胞RAW264.7激活和增殖的影响.噻唑蓝(MTT)比色法检测细胞的生长与增殖;中性红吞噬实验检测吞噬活性;Griess试剂盒检测培养上清液中NO分泌量,ELISA法检测VEPS不同浓度及不同作用时间培养上清中IL-6,IL-1β含量;扫描电子显微镜观察VEPS对细胞形态的影响;碘化丙啶(PI)染色检测VEPS对细胞周期的影响.结果显示,VEPS对RAW264.7细胞的增殖、吞噬能力、分泌NO、IL-6、IL-1β等都有显著的促进作用,VEPS为100 μg/ml时促进作用最明显,呈剂量相关,作用72 h时细胞因子分泌量达到最大,72 h后下降.VEPS激活巨噬细胞并使其变得扁平伸展且形成伪足.VEPS促进G1期细胞增多,提高细胞的增殖能力.VEPS免疫调节作用与其激活RAW264.7细胞,促进NO、IL-6、IL-1β等分泌有关,且VEPS与LPS对RAW

  16. Evaluation of interactions between RAW264.7 macrophages and small molecules by capillary electrophoresis.

    Science.gov (United States)

    Wang, Feng-Qin; Li, Qiao-Qiao; Zhang, Qian; Wang, Yin-Zhen; Hu, Yuan-Jia; Li, Peng; Wan, Jian-Bo; Yang, Feng-Qing; Xia, Zhi-Ning

    2016-12-09

    In this study, the affinity interactions between RAW 264.7 macrophages and three small molecules including naringin, oleuropein and paeoniflorin were evaluated by affinity capillary electrophoresis (ACE), partial filling affinity capillary electrophoresis (PFACE) and frontal analysis capillary electrophoresis (FACE), respectively. The result indicated that ACE (varying concentrations of cell suspension were filled in the capillary as receptor) may not be suitable for the evaluation of interactions between cell and small molecules due to the high viscosity of cell suspension; PFACE can qualitatively evaluate the interaction, but the difference in viscosity between RAW264.7 suspension and buffer effects on the liner relationship between filling length and injection time, which makes the calculation of binding constant difficult. Furthermore, based on the PFACE results, naringin showed stronger interaction with macrophages than the other two molecules; taking advantage of the aggregation phenomenon of cell induced by electric field, FACE was successfully used to determine the stoichiometry (n = 5×10(9) ) and binding constant (Kb = 1×10(4) L/mol) of the interaction between RAW264.7 and naringin.

  17. Does squalene alter the antioxidant potential of astaxanthin and fucoxanthinol? In vitro evidence in RAW 264.7 cells, a murine macrophage.

    Science.gov (United States)

    Ravi Kumar, Sangeetha; Narayan, Bhaskar; Kizawa, Yuki; Hosokawa, Masashi; Miyashita, Kazuo

    2016-04-01

    Astaxanthin (Ax) and fucoxanthin/fucoxanthinol (FuOH) are marine xanthophylls exhibiting anti-oxidant effects. Squalene (SQ) is a triterpenoid and is a precursor of sterols. This study aimed to determine if SQ can improve the effect of Ax/FuOH on lipid peroxidation. RAW 264.7 cells were treated with different concentrations of Ax, FuOH and SQ and corresponding rate of cell survival was noted. In addition,combination groups - Ax + SQ and FuOH + SQ- were also run. Cells treated with Ax, FuOH, SQ, Ax + SQ and FuOH + SQ were stimulated with lipopolysaccharide and lipid hydroperoxides were estimated. Results showed that 5 μM Ax, 2 μM FuOH and 10 μM SQ supported cell survival. In presence of SQ, cell viability improved for higher concentrations of FuOH (5, 10 μM). Lipid hydroperoxides were supressed by Ax, FuOH, Ax + SQ and FUOH +SQ and were significantly lower in Ax + SQ, indicating the synergistic effect of Ax and SQ. To conclude, combination of Ax with SQ enhances its ability to supress lipid peroxidation while with FuOH, SQ attenuates the toxic effect at higher doses. Moreover, this is the first time that the combined effect of SQ and carotenoids has been studied and reported.

  18. Regulation of sterile α- and armadillo motif (SARM) containing protein expression in Pam2CSK4- and Pam3CSK4-activated mouse macrophage cell line (RAW264.7) requires TLR9.

    Science.gov (United States)

    Pudla, Matsayapan; Kulsantiwong, Panthong; Srisaowakarn, Chanya; Utaisincharoen, Pongsak

    2017-09-09

    We aimed to investigate the involvement of surface TLRs and endosomal TLRs in the regulation of SARM expression by TLR2 ligands (Pam2CSK4 and Pam3CSK4). Mouse macrophage cell line (RAW264.7) was treated with either Pam2CSK4 or Pam3CSK4 (TLR2 ligands) at a concentration of 100 ng/ml. At indicated time points, the treated cells were lysed. The gene and protein expression of SARM were determined by RT-PCR and immunoblotting, respectively. For silencing of TLR9 function, the cells were transfected with TLR9 siRNAs before stimulation by these two TLR2 ligands RESULTS: The SARM expression was upregulated at both transcriptional and translational levels in time-dependent manner during activation of Pam2CSK4 and Pam3CSK4 in mouse macrophages. Blocking of ligand internalization by cytochalasin D showed interference effect with SARM expression. Moreover, our results also demonstrated that endosomal acidification and TLR9 were required for SARM expression suggesting the essential role of endosomal compartment acidification and TLR9 in regulating SARM expression. Our findings suggested the collaboration of TLR2-TLR9 at least in the regulation of SARM expression. However, the underlying mechanism that participated in these two TLRs cooperation is underinvestigated.

  19. Subtoxic Doses of Cadmium Modulate Inflammatory Properties of Murine RAW 264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Sina Riemschneider

    2015-01-01

    Full Text Available Cadmium (Cd is a toxic heavy metal that exhibits various adverse effects in the human and animal organism. Its resemblance to essential metals such as calcium, iron, and zinc leads to an unintended uptake in cells after intake through inhalation and ingestion. In this study we investigated the toxicity and the immunomodulatory potential of Cd in nonactivated and activated murine macrophages (i.e., cell line RAW 264.7. Cadmium alone caused a dose-dependent decreased viability of exposed cells. Subtoxic Cd concentrations delayed cell death in macrophages, resulting from cytotoxic storm, producing reactive oxygen species (ROS and nitric oxide (NO, in response to their stimulation by bacterial antigens via pattern-recognition receptors (PRRs. In addition, production of selected pro- and anti-inflammatory cytokines, the chemokine CXCL1 (KC, and NO was determined. We observed that proinflammatory IL-1β and also CXCL1 were highly upregulated whereas anti-inflammatory or regulatory cytokines IL-6 and IL-10 were suppressed by 10 µM Cd. Also production of antibacterial NO was significantly reduced through exposure to 10 µM Cd, maybe explaining better survival of macrophages. Additionally, we could show by analysis via ICP-MS that different effects of Cd in nonactivated and activated macrophages definitely did not result from different Cd uptake rates.

  20. Effects of compounds from Kaempferia parviflora on nitric oxide, prostaglandin E2 and tumor necrosis factor-alpha productions in RAW264.7 macrophage cells.

    Science.gov (United States)

    Tewtrakul, Supinya; Subhadhirasakul, Sanan

    2008-10-30

    Kaempferia parviflora Wall. ex Baker, is one of the plants in the Zingiberaceae family, locally known in Thai as kra-chai-dam. The rhizome of this plant has been used for treatment of gout, apthous ulcer and abscesses. Since K. parviflora rhizomes have long been used for treatment of inflammation and possessed marked nitric oxide (NO) inhibitory activity (IC(50)=7.8microg/ml), we thus investigated the inhibitory activity of compounds isolated from this plant against lipopolysaccharide (LPS)-induced NO release in RAW264.7 cells. From bioassay-guided fractionation of K. parviflora, seven methoxyflavones were isolated from the hexane fraction and were tested for their anti-inflammatory effects. Among the isolated compounds, compound 5 (5-hydroxy-3,7,3',4'-tetramethoxyflavone) exhibited the highest activity against NO release with an IC(50) value of 16.1microM, followed by 4 (IC(50)=24.5microM) and 3 (IC(50)=30.6microM). Compound 5 was also tested on LPS-induced prostaglandin E(2) (PGE(2)) and tumor necrosis factor-alpha (TNF-alpha) releases from RAW264.7 cells. It was revealed that 5 showed appreciable inhibitory effect on PGE(2) release (IC(50)=16.3microM), but inactive on TNF-alpha (IC(50)>100microM). These findings may support the use in Thai traditional medicine of K. parviflora for treatment of inflammatory-related diseases through the inhibition of NO and PGE(2) releases but partly due to that of TNF-alpha.

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

    Science.gov (United States)

    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.

  2. Toxicity of Aluminum Silicates Used in Hemostatic Dressings Toward Human Umbilical Veins Endothelial Cells, HeLa Cells, and RAW267.4 Mouse Macrophages

    Science.gov (United States)

    2011-09-01

    Std Z39-18 Their study demonstrated a severe toxic effect (cell lysis) of clay minerals such as montmorillonite and bentonite toward human endothelial...of WS in different cell types that may be exposed to this mineral and compared the results with other minerals such as bentonite , kaolin, and QuikClot... bentonite and higher than kaolin and QC. Neither cell type survived for 24 hours in the presence of 100 g/mL WS or bentonite . These minerals, however

  3. 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.

  4. Anti-Inflammatory Cytokine Interleukin-4 Inhibits Inducible Nitric Oxide Synthase Gene Expression in the Mouse Macrophage Cell Line RAW264.7 through the Repression of Octamer-Dependent Transcription

    Directory of Open Access Journals (Sweden)

    Miki Hiroi

    2013-01-01

    Full Text Available Inducible nitric oxide synthase (iNOS is a signature molecule involved in the classical activation of M1 macrophages and is induced by the Nos2 gene upon stimulation with Th1-cell derived interferon-gamma (IFNγ and bacterial lipopolysaccharide (LPS. Although the anti-inflammatory cytokine IL-4 is known to inhibit Nos2 gene expression, the molecular mechanism involved in the negative regulation of Nos2 by IL-4 remains to be fully elucidated. In the present study, we investigated the mechanism of IL-4-mediated Nos2 transcriptional repression in the mouse macrophage-like cell line RAW264.7. Signal transducer and activator of transcription 6 (Stat6 knockdown by siRNA abolished the IL-4-mediated inhibition of Nos2 induced by IFNγ/LPS. Transient transfection of a luciferase reporter gene containing the 5′-flanking region of the Nos2 gene demonstrated that an octamer transcription factor (OCT binding site in the promoter region is required for both positive regulation by IFNγ/LPS and negative regulation by IL-4. Although IL-4 had no inhibitory effect on the DNA-binding activity of constitutively expressed Oct-1, IL-4-induced Nos2-reporter transcriptional repression was partially attenuated by overexpression of the coactivator CREB-binding protein (CBP. These results suggest that a coactivator/cofactor that functionally interacts with Oct-1 is a molecular target for the IL-4-mediated inhibition of Nos2 and that IL-4-activated Stat6 represses Oct-1-dependent transcription by competing with this coactivator/cofactor.

  5. The polysaccharide isolated from Pleurotus nebrodensis (PN-S) shows immune-stimulating activity in RAW264.7 macrophages.

    Science.gov (United States)

    Cui, Hai-Yan; Wang, Chang-Lu; Wang, Yu-Rong; Li, Zhen-Jing; Zhang, Ya-Nan

    2015-05-01

    A novel Pleurotus nebrodensis polysaccharide (PN-S) was purified and characterized, and its immune-stimulating activity was evaluated in RAW264.7 macrophages. PN-S induced the proliferation of RAW264.7 cells in a dose-dependent manner, as determined by the MTT assay. After exposure to PN-S, the phagocytosis of the macrophages was significantly improved, with remarkable changes in morphology being observed. Flow cytometric analysis demonstrated that PN-S promoted RAW264.7 cells to progress through S and G2/M phases. PN-S treatment enhanced the productions of interleukin-6 (IL-6), nitric oxide (NO), interferon gamma (INF-γ), and tumor necrosis factor-α (TNF-α) in the macrophages, with up-regulation of mRNA expressions of interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), interferon gamma(INF-γ) and tumor necrosis factor-α (TNF-α) being observed in a dose-dependent manner, as measured by qRT-PCR. In conclusion, these results suggest that the purified PN-S can improve immunity by activating macrophages.

  6. Effect of P2X7 R gene silencing by RNA interference on proliferation and phagocytosis of murine macrophage cell line RAW264.7%干扰P2X7R基因对RAW264.7细胞增殖和吞噬的影响

    Institute of Scientific and Technical Information of China (English)

    苏程程; 姬文婕; 张译丹; 马永强; 陈雪芬; 向国安; 周欣; 彭守春; 林志春; 魏路清

    2015-01-01

    目的:应用RNA干扰技术抑制小鼠巨噬细胞RAW264.7细胞P2X7受体( P2X7 R)基因的表达,建立稳定干扰细胞株,并观察其对细胞增殖和凋亡的影响。方法:用脂质体法将P2X7 R shRNA重组质粒转染至RAW264.7细胞,经 G418筛选后获得稳定干扰细胞株。细胞分为野生型( WT )组、阴性对照( NC )组和干扰(shP2X7R)组。 Real-time PCR法检测细胞中P2X7R mRNA的表达,Western blot检测细胞中P2X7R蛋白的表达;CCK-8方法检测细胞生长活性,5-乙炔基-2’-脱氧尿苷(5-ethynyl-2’-deoxyuridine,EdU)掺入实验检测细胞增殖活性;流式细胞术分析细胞周期的分布和吞噬情况。结果:P2X7 R shRNA能明显抑制RAW264.7细胞的P2X7 R mR-NA和蛋白的表达,抑制率在80%以上。48 h后,shP2X7R组细胞的生长速度明显高于NC组和WT组(P<0.05),增殖期细胞比例明显升高(P<0.05),说明下调P2X7R基因能明显促进细胞增殖。 shP2X7R组的细胞周期出现改变,S期和G2/M期的比例明显上升,增殖指数增高(P<0.05)。 shP2X7R组的细胞吞噬活性明显高于NC组(P<0.05)。结论:本研究成功构建了稳定干扰P2X7 R基因表达的小鼠巨噬细胞株RAW264.7,shP2X7 R能够明显促进RAW264.7细胞的增殖,改变了细胞的吞噬活性。%AIM: To establish a cell line of stable silencing of P2X7 receptor (P2X7R) expression through short hairpin RNA ( shRNA)-mediated interference in murine RAW264.7 macrophages, and to investigate the proliferation and apoptosis in the cell line.METHODS:Stable silencing of P2X7 R gene in the RAW264.7 cells was achieved by re-combinant shRNA plasmid targeting murine P2X7 R gene via liposome mediated transfection, followed by G418 selection. The efficacy of plasmid transfection and P2X7 R silencing in G418 resistant cells was verified by immunofluorescent micros-copy and real-time PCR

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  8. Anti-Inflammatory Effect of Quercetin on RAW 264.7 Mouse Macrophages Induced with Polyinosinic-Polycytidylic Acid.

    Science.gov (United States)

    Kim, Young-Jin; Park, Wansu

    2016-04-04

    Quercetin (3,3',4',5,6-pentahydroxyflavone) is a well-known antioxidant and a flavonol found in many fruits, leaves, and vegetables. Quercetin also has known anti-inflammatory effects on lipopolysaccharide-induced macrophages. However, the effects of quercetin on virus-induced macrophages have not been fully reported. In this study, the anti-inflammatory effect of quercetin on double-stranded RNA (dsRNA)-induced macrophages was examined. Quercetin at concentrations up to 50 μM significantly inhibited the production of NO, IL-6, MCP-1, IP-10, RANTES, GM-CSF, G-CSF, TNF-α, LIF, LIX, and VEGF as well as calcium release in dsRNA (50 μg/mL of polyinosinic-polycytidylic acid)-induced RAW 264.7 mouse macrophages (p Quercetin at concentrations up to 50 μM also significantly inhibited mRNA expression of signal transducer and activated transcription 1 (STAT1) and STAT3 in dsRNA-induced RAW 264.7 cells (p quercetin had alleviating effects on viral inflammation based on inhibition of NO, cytokines, chemokines, and growth factors in dsRNA-induced macrophages via the calcium-STAT pathway.

  9. 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.

  10. Chemoattractant signaling between tumor cells and macrophages regulates cancer cell migration, metastasis and neovascularization.

    Directory of Open Access Journals (Sweden)

    Chad E Green

    Full Text Available Tumor-associated macrophages are known to influence cancer progression by modulation of immune function, angiogenesis, and cell metastasis, however, little is known about the chemokine signaling networks that regulate this process. Utilizing CT26 colon cancer cells and RAW 264.7 macrophages as a model cellular system, we demonstrate that treatment of CT26 cells with RAW 264.7 conditioned medium induces cell migration, invasion and metastasis. Inflammatory gene microarray analysis indicated CT26-stimulated RAW 264.7 macrophages upregulate SDF-1alpha and VEGF, and that these cytokines contribute to CT26 migration in vitro. RAW 264.7 macrophages also showed a robust chemotactic response towards CT26-derived chemokines. In particular, microarray analysis and functional testing revealed CSF-1 as the major chemoattractant for RAW 264.7 macrophages. Interestingly, in the chick CAM model of cancer progression, RAW 264.7 macrophages localized specifically to the tumor periphery where they were found to increase CT26 tumor growth, microvascular density, vascular disruption, and lung metastasis, suggesting these cells home to actively invading areas of the tumor, but not the hypoxic core of the tumor mass. In support of these findings, hypoxic conditions down regulated CSF-1 production in several tumor cell lines and decreased RAW 264.7 macrophage migration in vitro. Together our findings suggest a model where normoxic tumor cells release CSF-1 to recruit macrophages to the tumor periphery where they secrete motility and angiogenic factors that facilitate tumor cell invasion and metastasis.

  11. Effects of TiO2 nanotube layers on RAW 264.7 macrophage behaviour and bone morphogenetic protein-2 expression.

    Science.gov (United States)

    Sun, S J; Yu, W Q; Zhang, Y L; Jiang, X Q; Zhang, F Q

    2013-12-01

    To investigate behaviour and osteogenic cytokine expression of RAW264.7 macrophages grown on TiO2 nanotube layers. The murine macrophage cell line RAW 264.7 was cultured on TiO2 nanotubes of varying diameter; macrophage morphology was examined using scanning electron microscopy. Cell adhesion and viability were assessed with the aid of the MTT method and BMP-2 and TGF-β gene expression were examined by RT-PCR analysis. Levels of BMP-2, TGF-β1 and ICAM-1 proteins secreted into the supernatant were measured by ELISA assay. Macrophages cultured on nanotube layers had spread out morphology, the largest (120 nm) nanotube layer eliciting an elongation by 24 h. Macrophages adhered significantly less to 120 nm TiO2 nanotubes than to control discs at 4 h after application; after 24 h incubation, macrophages were sufficiently viable (P nanotube layers. Increasing nanotube diameter led to increased BMP-2 protein secretion and increased BMP-2 mRNA expression. These results demonstrate that nanoscale topography of TiO2 nanotube layers can affect macrophage morphology, adhesion, viability and BMP-2 expression. Macrophages grown on layers of large nanotubes had the highest potential to enhance bone formation during bone healing. © 2013 John Wiley & Sons Ltd.

  12. 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

  13. Rab20 regulates phagosome maturation in RAW264 macrophages during Fc gamma receptor-mediated phagocytosis.

    Directory of Open Access Journals (Sweden)

    Youhei Egami

    Full Text Available Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.

  14. Modulation of inducible nitric oxide synthase gene expression in RAW 264.7 murine macrophages by Pacific ciguatoxin

    OpenAIRE

    Kumar-Roine, Shilpa; Matsui, Mariko,; Chinain, M.; Laurent, Dominique; Pauillac, S.

    2008-01-01

    To investigate the possible involvement of the nitric oxide radical (NO) in ciguatera fish poisoning (CFP), the in vitro effects of the main Pacific ciguatoxin (P-CTX-1B) and bacterial lipopolysaccharide (LPS) were comparatively studied on neuroblastoma Neuro-2a and on macrophage RAW 264.7 cell lines. NO accumulation was quantified by measuring nitrite levels in cellular supernatant using Griess reagent while the up-regulation of inducible nitric oxide synthase (iNOS) at the mRNA level was qu...

  15. 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.

  16. 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.

  17. Lanthanum Chloride Inhibiting Expression of Inducible Nitric Oxide Synthase in RAW264.7 Macrophages Induced by Lipopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    Guo Fei; Lou Yuanlei; Wang Yang; Xie An; Li Guohui

    2007-01-01

    Nitric oxide (NO) and its reaction products were key players in the pathophysiology of sepsis and shock. The present study was designed to explore the effects of lanthanum chloride (LaCl3) on inducible nitric oxide synthase (iNOS) expression, at both gene and protein levels, in RAW264.7 macrophages induced by Lipopolysaccharide (LPS). Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence, and western blot were employed to measure iNOS gene expression, localization, and protein expression respectively. NO production in culture supernatants was detected by the nitrate reductase method. The results showed that LaCl3 significantly attenuated the iNOS gene and protein expression, as well as NO production in RAW264.7cells induced by LPS.

  18. Sargachromenol from Sargassum micracanthum Inhibits the Lipopolysaccharide-Induced Production of Inflammatory Mediators in RAW 264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Eun-Jin Yang

    2013-01-01

    Full Text Available During our ongoing screening program designed to determine the anti-inflammatory potential of natural compounds, we isolated sargachromenol from Sargassum micracanthum. In the present study, we investigated the anti-inflammatory effects of sargachromenol on lipopolysaccharide (LPS-induced inflammation in murine RAW 264.7 macrophage cells and the underlying mechanisms. Sargachromenol significantly inhibited the LPS-induced production of nitric oxide (NO and prostaglandin E2 (PGE2 in a dose-dependent manner. It also significantly inhibited the protein expression of inducible NO synthase (iNOS and cyclooxygenase-2 (COX-2 in a dose-dependent manner in LPS-stimulated macrophage cells. Further analyses showed that sargachromenol decreased the cytoplasmic loss of inhibitor κBα (IκBα protein. These results suggest that sargachromenol may exert its anti-inflammatory effects on LPS-stimulated macrophage cells by inhibiting the activation of the NF-κB signaling pathway. In conclusion, to our knowledge, this is the first study to show that sargachromenol isolated from S. micracanthum has an effective anti-inflammatory activity. Therefore, sargachromenol might be useful for cosmetic, food, or medical applications requiring anti-inflammatory properties.

  19. The Impact of Serum Amyloid P-Component on Gene Expression in RAW264.7 Mouse Macrophages

    Directory of Open Access Journals (Sweden)

    Dan Xi

    2016-01-01

    Full Text Available Serum amyloid P-component (SAP contributes to host defense and prevents fibrosis. Macrophages are the most abundant inflammatory cell type in atherosclerotic plaques. In the present study, using 3H-cholesterol-labeled counting radioactivity assay, we demonstrated that the apoAI-mediated cholesterol efflux in RAW264.7 macrophages was increased by SAP treatment in a time- and dose-dependent manner. We analyzed global gene expression changes upon SAP treatment using RNA sequencing. As a result, a total of 175 differentially expressed genes were identified, of which 134 genes were downregulated and 41 genes were upregulated in SAP treated cells compared to control cells. Quantitative RT-PCR analysis confirmed decreased expression of 5 genes and an increase in expression of 1 gene upon SAP treatment. Gene ontology analysis showed that genes involved in response to stimulus were significantly enriched in differentially expressed genes. Beyond protein-coding genes, we also identified 8 differentially expressed long noncoding RNAs. Our study may provide new insights into mechanisms underlying the functional role of SAP in macrophages.

  20. Ethyl acetate extract from Asparagus cochinchinensis exerts anti‑inflammatory effects in LPS‑stimulated RAW264.7 macrophage cells by regulating COX‑2/iNOS, inflammatory cytokine expression, MAP kinase pathways, the cell cycle and anti-oxidant activity.

    Science.gov (United States)

    Lee, Hyun Ah; Koh, Eun Kyoung; Sung, Ji Eun; Kim, Ji Eun; Song, Sung Hwa; Kim, Dong Seob; Son, Hong Joo; Lee, Chung Yeoul; Lee, Hee Seob; Bae, Chang Joon; Hwang, Dae Youn

    2017-04-01

    Asparagus cochinchinesis (A. cochinchinesis) is a medicine traditionally used to treat fever, cough, kidney disease, breast cancer, inflammatory disease and brain disease in northeast Asian countries. Although numerous studies of the anti‑inflammatory effects of A. cochinchinesis have been conducted, the underlying mechanisms of such effects in macrophages remain to be demonstrated. To investigate the mechanism of suppressive effects on the inflammatory response in macrophages, alterations of the nitric oxide (NO) level, the cell viability, inducible nitric oxide synthase (iNOS) and cyclooxygenase‑2 (COX‑2) expression levels, inflammatory cytokine expression, the mitogen-activated protein kinase (MAPK) signaling pathway, cell cycle arrest and reactive oxygen species (ROS) levels were measured in lipopolysaccharide (LPS)-activated RAW264.7 cells following treatment with ethyl acetate extract from A. cochinchinesis root (EaEAC). RAW264.7 cells pretreated two different concentrations of EaEAC prior to LPS treatment exhibited no significant toxicity. The concentration of NO was significantly decreased in the EaEAC + LPS treated group compared with the vehicle + LPS treated group. A similar decrease in mRNA transcript level of COX‑2, iNOS, pro-inflammatory cytokines [tumor necrosis factor‑α and interleukin (IL)‑1β] and anti‑inflammatory cytokines (IL‑6 and IL‑10) was detected in the EaEAC + LPS treated group compared with the vehicle + LPS treated group, although the decrease rate varied. Enhancement of the phosphorylation of MAPK family members following LPS treatment was partially rescued in the EaEAC pretreated group, and the cell cycle was arrested at the G2/M phase. Furthermore, the EaEAC pretreated group exhibited a reduced level of ROS generation compared with the vehicle + LPS treated group. Taken together, these results suggest that EaEAC suppresses inflammatory responses through inhibition of NO production, COX‑2 expression

  1. Jeju seaweeds suppress lipopolysaccharide-stimulated proinflammatory response in RAW 264.7 murine macrophages

    Institute of Scientific and Technical Information of China (English)

    Eun-Jin; Yang; Ji-Young; Moon; Sang; Suk; Kim; Kyong—Wol; Yang; Wook; Jae; Lee; Nam; Ho; Lee; Chang-Gu; Hyun

    2014-01-01

    Objective:To investigate the anti-inflammatory effects of Jeju seaweeds on macrophage RAW264.7 cells under lipopolysaccharide(LPS)stimulation.Methods:Ethyl acetate fractions were prepared from five different types of Jeju seaweeds,Dictyopteris divaricata(D.divaricata),Dictyopteris prolifera(D.prolifefa),Prioutis cornea(P.comea,Grateloupia laceolata(G,lanceolate,and Cralcloupia filicina(G.filicina)They were screened for inhibitory effects on proinflammatory mediators and cytokines such as nitric oxide(NO),prostaglandin E,,tumor necrosis factor-a(TNF-a),and interleukin-6(11.-6).Results:Our results revealed that D.divaricata,D.prolifera,P.cornea,G.lanceolata,and G.filicina potently inhibited I.PS-stimulaled NO production(IC50,values were 18.0,38.36,38.43,32.81 and 37.14μg/mL,respectively).Consistent with these findings,D.divtricata,D.prolifera,P.cornea,and G.fdicina also reduced the IPS-induced and prostaglandin E,production in a concentration-dependent manner.Expectedly,they suppressed the expression of inducible NO synthase and cyclooxygenase-2 at the protein level in a dose-dependent manner in the RAW264.7 cells,as detennined by western blotting.In addition,the levels of TNF-a and IL-6,released into the medium,were also reduced by D.divaricata,D.prolifera,P.cornea,G,lanceolata,and G.fdicina in a dose-dependent manner(IC50values for TNF-a were 16.11,28.21,84.27,45.52 and74.75μg/mL,respectively;IC50,values for IL-6 were 37.35,80.08,103.28,62.53 and 84.28μg/mL,respectively).The total phlorotannin content was measured by the Folin-Ciocalteu method and expressed as phloroglucinol equivalents.The content was 92.0μg/mg for D.divaricata,151.8μg/mg for D.prolifera,57.2μg/mg for P.cornea,53.0 pg/mg for G.lanceolata,and 40.2μg/mg for G.fdicina.Conclusions:Thus,these findings suggest that Jeju seaweed extracts have potential therapeutic applications for inflammatory responses.

  2. Jeju seaweeds suppress lipopolysaccharide-stimulated proinflammatory response in RAW 264.7 murine macrophages

    Institute of Scientific and Technical Information of China (English)

    Eun-Jin Yang; Ji-Young Moon; Sang Suk Kim; Kyong-Wol Yang; Wook Jae Lee; Nam Ho Lee; Chang-Gu Hyun

    2014-01-01

    Objective: To investigate the anti-inflammatory effects of Jeju seaweeds on macrophage RAW 264.7 cells under lipopolysaccharide (LPS) stimulation.Methods:Ethyl acetate fractions were prepared from five different types of Jeju seaweeds, Dictyopteris divaricata (D. divaricata), Dictyopteris prolifera (D. prolifera), Prionitis cornea (P. cornea), Grateloupia lanceolata (G. lanceolata), and Grateloupia filicina (G. filicina). They were screened for inhibitory effects on proinflammatory mediators and cytokines such as nitric oxide (NO), prostaglandin E2, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6).Results:Our results revealed that D. divaricata, D. prolifera, P. cornea, G. lanceolata, and G. filicina potently inhibited LPS-stimulated NO production (IC50 values were 18.0, 38.36, 38.43, 32.81 and 37.14 µg/mL, respectively). Consistent with these findings, D. divaricata, D. prolifera, P. cornea, and G. filicina also reduced the LPS-induced and prostaglandin E2 production in a concentration-dependent manner. Expectedly, they suppressed the expression of inducible NO synthase and cyclooxygenase-2 at the protein level in a dose-dependent manner in the RAW 264.7 cells, as determined by western blotting. In addition, the levels of TNF-α and IL-6, released into the medium, were also reduced by D. divaricata, D. prolifera, P. cornea, G. lanceolata, andG. filicina in a dose-dependent manner (IC 50 values for TNF-α were 16.11, 28.21, 84.27, 45.52 and 74.75 µg/mL, respectively; IC50 values for IL-6 were 37.35, 80.08, 103.28, 62.53 and 84.28 µg/mL, respectively). The total phlorotannin content was measured by the Folin-Ciocalteu method and expressed as phloroglucinol equivalents. The content was 92.0 µg/mg for D. divaricata, 151.8 µg/mg for D. prolifera, 57.2 µg/mg for P. cornea, 53.0 µg/mg for G. lanceolata, and 40.2 µg/mg for G.filicina. Conclusions: Thus, these findings suggest that Jeju seaweed extracts have potential therapeutic applications for

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

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    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.

  4. Soluble Siglec-9 suppresses arthritis in a collagen-induced arthritis mouse model and inhibits M1 activation of RAW264.7 macrophages

    OpenAIRE

    Matsumoto, Takuya; Takahashi, Nobunori; Kojima, Toshihisa; Yoshioka, Yutaka; Ishikawa, Jun; Furukawa, Koichi; Ono, Kenji; Sawada, Makoto; ISHIGURO, NAOKI; Yamamoto, Akihito

    2016-01-01

    Background The aim of this study was to assess the effects of soluble sialic acid-binding immunoglobulin-type lectin (sSiglec)-9 on joint inflammation and destruction in a murine collagen-induced arthritis (CIA) model and in monolayer cultures of murine macrophages (RAW264.7 cells and peritoneal macrophages) and fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis. Methods DBA/1J mice were immunized with type II collagen. Effects of sSiglec-9 were evaluated using...

  5. 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.

  6. 脂多糖刺激RAW264.7和Ana-1形态改变及细胞因子表达的差异%Comparison between two kinds of murine macrophages.Ana-1 and RAW264.7 cell in appearance and cytokines expression

    Institute of Scientific and Technical Information of China (English)

    石榴; 方怡; 袁伟锋; 李理; 黄文杰

    2009-01-01

    Objective To compare the morphologic change and cytokines expression in RAW264.7 and Ana-1 stimulated by lipopolysaccharide(LPS). Methods RAW264.7 and Ana-1 were cultivated with various concentrations of LPS(0.1 mg/L, 1 mg/L, 10 mg/L, 100 mg/L). MTT was performed to evaluate the proliferation ability of cells. Two kinds of cells were cultivated with 1 mg/L. Then,the concentrations of TNF-α,IL-1β,IL-6,IL-10 in culture medium were detected by ELISA in different times(0 h,4 h,8 h,12 h,24 h). Results The survival rates of RAW264.7 and Ana-1 (162.05±28.14)% and (159.92±20.43)% were significantly higher in 1 mg/L group than those of other groups. When stimulated with 1 mg/L LPS, both RAW264.7 and Ana-1 expressed cytokines in time-dependent manner (increased first and decreased finally). The concentrations of TNF-α and IL-10 in RAW264.7 group were higher than those Ana-1 group in 4 h only. IL-1β and IL-6,however,were in higher concentrations in RAW264.7 group than Ana-1 group all the time. Conclusions When stimulated with 1 mg/L LPS, both RAW264.7 and Ana-1 have higher survival rate. RAW264.7 and Ana-1 have different ability in expressing cytokines after being stimulated by LPS. Therefore,RAW264.7 and Ana-1 have variant response to LPS slightly.%目的 比较两株小鼠来源的巨噬细胞株RAW264.7和Ana-1经脂多糖(lipopolysaccharide,LPS)诱导后在形态及细胞因子表达等方面的差异.方法 MTT法检测小同终浓度(0.1 mg/L、1 mg/L、10 mg/L、100 mg/L)LPS作用后两株细胞增殖活力,确定最佳作用浓度.选择1 mg/L LPS刺激RAW264.7和Ana-1细胞,ELISA法分别于0 h、4 h、8 h、12 h、24 h检测两株细胞肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β)、IL-6和IL-10的表达量.结果 LPS终浓度为1 mg/L时两株细胞存活率分别为(162.05±28.14)%、(159.92±20.43)%,显著大于同细胞其他浓度组别(P<0.01);1 mg/L LPS刺激后,两株细胞各细胞因子表达均为随时间呈先增多后减少趋势,峰值出现时间RAW

  7. Modulation of inducible nitric oxide synthase gene expression in RAW 264.7 murine macrophages by Pacific ciguatoxin.

    Science.gov (United States)

    Kumar-Roiné, Shilpa; Matsui, Mariko; Chinain, Mireille; Laurent, Dominique; Pauillac, Serge

    2008-08-01

    To investigate the possible involvement of the nitric oxide radical (NO) in ciguatera fish poisoning (CFP), the in vitro effects of the main Pacific ciguatoxin (P-CTX-1B) and bacterial lipopolysaccharide (LPS) were comparatively studied on neuroblastoma Neuro-2a and on macrophage RAW 264.7 cell lines. NO accumulation was quantified by measuring nitrite levels in cellular supernatant using Griess reagent while the up-regulation of inducible nitric oxide synthase (iNOS) at the mRNA level was quantified via Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR). P-CTX-1B caused a concentration- and time-dependent induction of iNOS in RAW 264.7 cells but not in Neuro-2a cells. NO production was evidenced by increased nitrite levels in the 10 microM range after 48 h of RAW 264.7 cells exposure to LPS and P-CTX-1B (0.05 microg/ml and 6 nM, respectively). The expression of iNOS mRNA peaked at 8h for LPS then gradually decreased to low level at 48 h. In contrast, a sustained level was recorded with P-CTX-1B in the 8-48 h time interval. The addition of N(omega)-nitro-L-arginine methyl ester (L-NAME), a stereoselective NOS inhibitor, strongly diminished NO formation but had no effect on iNOS mRNA synthesis. The implication of NO in CFP paves the way for new therapies for both western and traditional medicines.

  8. Proteomic Investigation of the Time Course Responses of RAW 264.7 Macrophages to Infection with Salmonella enterica

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Liang; Chowdhury, Saiful M.; Smallwood, Heather S.; Yoon, Hyunjin; Mottaz-Brewer, Heather M.; Norbeck, Angela D.; McDermott, Jason E.; Clauss, Therese RW; Heffron, Fred; Smith, Richard D.; Adkins, Joshua N.

    2009-08-01

    Macrophages plan important roles in controlling Salmonella-mediated systemic infection. To investigate the responses of macrophages to Salmonella infection, we infected RAW 264.7 macrophages with Salmonella enterica serovar Typhimurium (STM) and then performed a comparative liquid chromatography-tandem mass spectrometry [LC-MS(/MS)]-based proteomics analysis of the infected macrophages. A total of 1006 macrophage and 115 STM proteins were indentified from this study. Most of STM proteins were found at late stage of the time course of infection, consistent with the fact that STM proliferates inside RAW 264.7 macrophages. Majority of the identified macrophage proteins were house keeping-related, including cytoplasmic superoxide dismutase 1 (SOD1), whose peptide abundances were relatively constant during the time course of infection. Compared to those in no infection control, the peptide abundances of 244 macrophage proteins (or 24% of total indentified macrophage proteins) changed considerably after STM infection. The functions of these STM infection-affected macrophage proteins were diverse and ranged from production of antibacterial nitric oxide (i.e., inducible nitric oxide synthase or iNOS) or production of prostaglandin H2 (i.e., prostaglandin-endoperoxide synthase 2, also know as cyclooxygenase-2 or COX-2) to regulation of intracellular traffic (e.g., sorting nexin or SNX 5, 6 and 9), demonstrating a global impact of STM infection on macrophage proteome. Western-blot analysis not only confirmed the LC-MS(/MS) results of SOD1, COX-2 and iNOS, but also revealed that the protein abundances of mitochondrial SOD2 increased after STM infection, indicating an infection-induced oxidative stress in mitochondria.

  9. Live and Heat-Killed Lactobacillus rhamnosus ATCC 7469 May Induce Modulatory Cytokines Profiles on Macrophages RAW 264.7.

    Science.gov (United States)

    Jorjão, Adeline Lacerda; de Oliveira, Felipe Eduardo; Leão, Mariella Vieira Pereira; Carvalho, Cláudio Antonio Talge; Jorge, Antonio Olavo Cardoso; de Oliveira, Luciane Dias

    2015-01-01

    This study aimed to evaluate the capacity of Lactobacillus rhamnosus and/or its products to induce the synthesis of cytokines (TNF-α, IL-1β, IL-4, IL-6, IL-10, and IL-12) by mouse macrophages (RAW 264.7). Three microorganism preparations were used: live L. rhamnosus (LLR) suspension, heat-killed L. rhamnosus (HKLR) suspension, and the supernatant of a heat-killed L. rhamnosus (SHKLR) suspension, which were cultured with macrophages (37°C, 5% CO2) for 2 h and 30 min. After that, cells were cultured for 16 h. The supernatants were used for the quantitation of cytokines, by ELISA. The results were compared with the synthesis induced by lipopolysaccharide (LPS) and analysed, using ANOVA and Tukey test, 5%. LLR and HKLR groups were able to significantly increase the production of TNF-α, IL-6, and IL-10 (P 0.05). All the L. rhamnosus suspensions were not able to produce detectable levels of IL-1β or significant levels of IL-4 and IL-12 (P > 0.05). In conclusion, live and heat-killed L. rhamnosus suspensions were able to induce the synthesis of different cytokines with proinflammatory (TNF-α and IL-6) or regulatory (IL-10) functions, suggesting the role of strain L. rhamnosus ATCC 7469 in the modulation or in the stimulation of immune responses.

  10. Live and Heat-Killed Lactobacillus rhamnosus ATCC 7469 May Induce Modulatory Cytokines Profiles on Macrophages RAW 264.7

    Directory of Open Access Journals (Sweden)

    Adeline Lacerda Jorjão

    2015-01-01

    Full Text Available This study aimed to evaluate the capacity of Lactobacillus rhamnosus and/or its products to induce the synthesis of cytokines (TNF-α, IL-1β, IL-4, IL-6, IL-10, and IL-12 by mouse macrophages (RAW 264.7. Three microorganism preparations were used: live L. rhamnosus (LLR suspension, heat-killed L. rhamnosus (HKLR suspension, and the supernatant of a heat-killed L. rhamnosus (SHKLR suspension, which were cultured with macrophages (37°C, 5% CO2 for 2 h and 30 min. After that, cells were cultured for 16 h. The supernatants were used for the quantitation of cytokines, by ELISA. The results were compared with the synthesis induced by lipopolysaccharide (LPS and analysed, using ANOVA and Tukey test, 5%. LLR and HKLR groups were able to significantly increase the production of TNF-α, IL-6, and IL-10 (P0.05. All the L. rhamnosus suspensions were not able to produce detectable levels of IL-1β or significant levels of IL-4 and IL-12 (P>0.05. In conclusion, live and heat-killed L. rhamnosus suspensions were able to induce the synthesis of different cytokines with proinflammatory (TNF-α and IL-6 or regulatory (IL-10 functions, suggesting the role of strain L. rhamnosus ATCC 7469 in the modulation or in the stimulation of immune responses.

  11. Anti-inflammatory potential of peat moss extracts in lipopolysaccharide-stimulated RAW 264.7 macrophages.

    Science.gov (United States)

    Choi, Woo-Suk; Jeong, Jin-Woo; Kim, Sung Ok; Kim, Gi-Young; Kim, Byung-Woo; Kim, Cheol Min; Seo, Yong-Bae; Kim, Woe-Yeon; Lee, Sang-Yeol; Jo, Kwon-Ho; Choi, Young Ju; Choi, Yung Hyun; Kim, Gun-Do

    2014-10-01

    The aim of the present study was to identify the anti-inflammatory and anti-oxidative effects of peat moss aqueous extract (PME) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. To demonstrate the anti-inflammatory and antioxidant effects of PME, the levels of nitric oxide (NO) and cytokines were measured using Griess reagent and cytokine ELISA kits, respectively. Reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analysis were conducted to evaluate the expression of genes and proteins. Immunofluorescence was used to measure the expression and translocation of transcription factors. Pre-treatment with PME inhibited the production of prostaglandin E(2) and NO by suppressing the gene expression of cyclooxygenase-2 and inducible NO synthase, respectively. The LPS-stimulated gene expression and the production of tumor necrosis factor-α and interleukin-1β were significantly reduced by PME. In the LPS-stimulated RAW 264.7 cells, nuclear factor‑κB (NF-κB) translocated from the cytosol to the nucleus, while pre-treatment with PME induced the sequestration of NF-κB in the cytosol through the inhibition of IκBα degradation. In the same manner, PME contributed to the inhibition of the activation of mitogen-activated protein kinases. In addition, the PME-treated RAW 264.7 cells facilitated the activation of nuclear factor-like 2 (Nrf2) , and in turn, enhanced heme oxygenase-1 (HO-1) expression. These results indicate that PME exerts anti-inflammatory and antioxidant effects, and suggest that PME may neutralize inflammation and prevent cellular damage by oxidative stress.

  12. Chlorogenic acid protects against atherosclerosis in ApoE-/- mice and promotes cholesterol efflux from RAW264.7 macrophages.

    Science.gov (United States)

    Wu, Chongming; Luan, Hong; Zhang, Xue; Wang, Shuai; Zhang, Xiaopo; Sun, Xiaobo; Guo, Peng

    2014-01-01

    Chlorogenic acid (CGA) is one of the most abundant polyphenols in the human diet and is suggested to be a potential antiatherosclerotic agent due to its proposed hypolipidemic, anti-inflammatory and antioxidative properties. The aim of this study was to evaluate the effect of CGA on atherosclerosis development in ApoE(-/-) mice and its potential mechanism. ApoE(-/-) mice were fed a cholesterol-rich diet without (control) or with CGA (200 and 400 mg/kg) or atorvastatin (4 mg/kg) for 12 weeks. During the study plasma lipid and inflammatory parameters were determined. Treatment with CGA (400 mg/kg) reduced atherosclerotic lesion area and vascular dilatation in the aortic root, comparable to atorvastatin. CGA (400 mg/kg) also significantly decreased plasma levels of total cholesterol, triglycerides and low-density lipoprotein-cholesterol as well as inflammatory markers. Supplementation with CGA or CGA metabolites-containing serum suppressed oxidized low-density lipoprotein (oxLDL)-induced lipid accumulation and stimulated cholesterol efflux from RAW264.7 cells. CGA significantly increased the mRNA levels of PPARγ, LXRα, ABCA1 and ABCG1 as well as the transcriptional activity of PPARγ. Cholesterol efflux assay showed that three major metabolites, caffeic, ferulic and gallic acids, significantly stimulated cholesterol efflux from RAW264.7 cells. These results suggest that CGA potently reduces atherosclerosis development in ApoE(-/-) mice and promotes cholesterol efflux from RAW264.7 macrophages. Caffeic, ferulic and gallic acids may be the potential active compounds accounting for the in vivo effect of CGA.

  13. Chlorogenic acid protects against atherosclerosis in ApoE-/- mice and promotes cholesterol efflux from RAW264.7 macrophages.

    Directory of Open Access Journals (Sweden)

    Chongming Wu

    Full Text Available Chlorogenic acid (CGA is one of the most abundant polyphenols in the human diet and is suggested to be a potential antiatherosclerotic agent due to its proposed hypolipidemic, anti-inflammatory and antioxidative properties. The aim of this study was to evaluate the effect of CGA on atherosclerosis development in ApoE(-/- mice and its potential mechanism. ApoE(-/- mice were fed a cholesterol-rich diet without (control or with CGA (200 and 400 mg/kg or atorvastatin (4 mg/kg for 12 weeks. During the study plasma lipid and inflammatory parameters were determined. Treatment with CGA (400 mg/kg reduced atherosclerotic lesion area and vascular dilatation in the aortic root, comparable to atorvastatin. CGA (400 mg/kg also significantly decreased plasma levels of total cholesterol, triglycerides and low-density lipoprotein-cholesterol as well as inflammatory markers. Supplementation with CGA or CGA metabolites-containing serum suppressed oxidized low-density lipoprotein (oxLDL-induced lipid accumulation and stimulated cholesterol efflux from RAW264.7 cells. CGA significantly increased the mRNA levels of PPARγ, LXRα, ABCA1 and ABCG1 as well as the transcriptional activity of PPARγ. Cholesterol efflux assay showed that three major metabolites, caffeic, ferulic and gallic acids, significantly stimulated cholesterol efflux from RAW264.7 cells. These results suggest that CGA potently reduces atherosclerosis development in ApoE(-/- mice and promotes cholesterol efflux from RAW264.7 macrophages. Caffeic, ferulic and gallic acids may be the potential active compounds accounting for the in vivo effect of CGA.

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

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    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.

  15. Chemically-modified polysaccharide extract derived from Leucaena leucocephala alters Raw 264.7 murine macrophage functions.

    Science.gov (United States)

    Gamal-Eldeen, Amira M; Amer, Hassan; Helmy, Wafaa A; Talaat, Roba M; Ragab, Halla

    2007-06-01

    In this study, a chemical modification of the polysaccharides extract (E) derived from Leucaena leucocephala seeds was performed to prepare C-glycosidic 2-propanol derivative (PE), and its sulphated derivative (SPE). This study aimed to characterize immunomodulatory activities of the original extract and its derivatives by exploring their effects on Raw macrophage 264.7 functions and their antioxidant activity. Our results indicated that PE was an effective radical scavenger to hydroxyl, peroxyl, and superoxide anion radicals, and SPE was a peroxyl radical scavenger. PE and SPE were found to influence the macrophage functions. Both of PE and SPE enhanced the macrophage proliferation and phagocytosis of FITC-zymosan; PE inhibited nitric oxide (NO) generation and tumor necrosis factor-alpha (TNF-alpha) secretion in lipopolysaccharide (LPS)-stimulated Raw macrophage 264.7. In contrast, SPE over-induced NO generation and TNF-alpha secretion. Moreover, PE strongly inhibited the binding affinity of FITC-LPS to Raw 264.7, as indicated by flow cytometry analysis. These findings revealed that PE may act as a potent anti-inflammatory agent; however SPE may act as an inducer of macrophage functions against pathogens.

  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. Cytotoxicity studies of Dynasan 114 solid lipid nanoparticles (SLN) on RAW 264.7 macrophages-impact of phagocytosis on viability and cytokine production.

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    Olbrich, Carsten; Schöler, Nadja; Tabatt, Kerstin; Kayser, Oliver; Müller, Rainer Helmut

    2004-07-01

    Solid lipid nanoparticles (SLN) based on Dynasan 114 (D114) were tested using RAW 264.7 cells. The influence of different surfactants on the cytotoxicity of this type of SLN was examined, expressed as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability and the production of cytokines such as interleukin 6 (IL-6), IL-12 and tumour necrosis factor-alpha (TNF-alpha). Results were compared with previously obtained data when peritoneal mouse macrophages were used. SLN produced with stabilizers/surfactants such as poloxamer 188, sodium cholate, Lipoid S75, Tween 80, Poloxamine 908 and sodium dodecylsulfate were shown to be nontoxic towards RAW 264.7 cells. Cytokine production was reduced and stimulation, expressed in elevated cytokine levels, could not be found. Using cetylpyridinium chloride (CPC) as stabilizing surfactant, SLN became cytotoxic in a concentration-dependent manner. Not only were the viabilities reduced but also cytokine production. Cytotoxic effects of CPC stabilized SLN could be antagonized using cytochalasin B to block phagocytosis. D114-SLN produced with pharmaceutically accepted surfactants for intravenous injection (poloxamer 188, Lipoid S75, sodium cholate, Tween 80) were very well tolerated by the cells. Even sodium dodecylsulfate-stabilized D114-SLN did not exert toxic effects. Comparison of the RAW 264.7 data with previously obtained data from toxicity studies of D114-SLN towards peritoneal mouse macrophages showed similar results. This offers the possibility of using the RAW 264.7 cell line for cytotoxicity studies of colloidal drug carrier systems, rather than using laboratory animals as source of macrophages for these kinds of studies.

  18. Immunomodulatory Efficacy of Standardized Annona muricata (Graviola) Leaf Extract via Activation of Mitogen-Activated Protein Kinase Pathways in RAW 264.7 Macrophages

    OpenAIRE

    Goon-Tae Kim; Nguyen Khoi Song Tran; Eun-Hye Choi; Yoo-Jeong Song; Jae-Hwi Song; Soon-Mi Shim; Tae-Sik Park

    2016-01-01

    Annona muricata, commonly known as Graviola, has been utilized as a traditional medicine to treat various human diseases. The aim of this study was to examine the immune-enhancing activity of Graviola leaf extracts in RAW 264.7 macrophage cells. Active ingredients in Graviola leaf extracts (GE) were identified as kaempferol-3-O-rutinoside and quercetin-3-O-rutinoside by LC-MS/MS. When treated with steam or 50% ethanol GE, cell morphology was altered due to initiation of cell differentiation. ...

  19. Studies on palauan medicinal herbs. II. Activation of mouse macrophages RAW 264.7 by Ongael, leaves of Phaleria cumingii (Meisn.) F. Vill. and its acylglucosylsterols.

    Science.gov (United States)

    Matsuda, Hideaki; Tokunaga, Masashi; Iwahashi, Hiroyasu; Naruto, Shunsuke; Yagi, Hideki; Masuko, Takashi; Kubo, Michinori

    2005-05-01

    The extract of Ongael [leaves of Phaleria cumingii (MEISN.) F. VILL.], a Palauan medicinal herb, enhanced an in vitro phagocytic activity of mouse macrophages RAW 264.7 cells (RAW 264.7). Activity-guided fractionation of the Ongael extract by the in vitro phagocytosis assay using RAW 264.7 led to the isolation of a mixture of acylglucosylsterols (1) as an active constituent along with other inactive constituents, tetracosanol and mangiferin. On the basis of chemical modifications and spectral analyses, the compound 1 was deduced to be a mixture of the known 3-O-(6-O-acyl-beta-D-glucosyl)-beta-sitosterols, the acyl moiety being mainly palmitoyl (57%), oleoyl (12%) and alpha-linolenoyl (12%) with small amount of stearoyl (7%) and linoleoyl (4%).

  20. Chrysin, Apigenin and Acacetin Inhibit Tumor Necrosis Factor-Related Apoptosis—Inducing Ligand Receptor-1 (TRAIL-R1 on Activated RAW264.7 Macrophages

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    Monika Warat

    2014-06-01

    Full Text Available Expression level of Tumor Necrosis Factor—related apoptosis—inducing ligand (TRAIL receptors is one of the most important factors of TRAIL-mediated apoptosis in cancer cells. We here report for the first time data concerning TRAIL-R1 and TRAIL-R2 receptor expression on RAW264.7 macrophages. Three substances belonging to flavones: chrysin, apigenin and acacetin which differ from their substituents at the 4' position in the phenyl ring were used in assays because of the variety of biological activities (e.g., anticancer activity of the polyphenol compounds. The expression of TRAIL-R1 and TRAIL-R2 death receptors on non-stimulated and LPS (lipopolysaccharide-stimulated macrophages was determined using flow cytometry. We demonstrate that RAW264.7 macrophages exhibit TRAIL-R1 surface expression and that the tested compounds: chrysin, apigenin and acacetin can inhibit TRAIL-R1 death receptor expression level on macrophages.

  1. Macrophages overexpressing Aire induce CD4+Foxp3+ T cells.

    Science.gov (United States)

    Sun, Jitong; Fu, Haiying; Wu, Jing; Zhu, Wufei; Li, Yi; Yang, Wei

    2013-01-01

    Aire plays an important role in central immune tolerance by regulating the transcription of thousands of genes. However, the role of Aire in the peripheral immune system is poorly understood. Regulatory T (Treg) cells are considered essential for the maintenance of peripheral tolerance, but the effect of Aire on Treg cells in the peripheral immune system is currently unknown. In this study, we investigated the effects of macrophages overexpressing Aire on CD4+Foxp3+ Treg cells by co-culturing Aire-overexpressing RAW264.7 cells or their supernatant with splenocytes. The results show that macrophages overexpressing Aire enhanced the expression of Foxp3 mRNA and induced different subsets of Treg cells in splenocytes through cell-cell contact or a co-culture supernatants. TGF-β is a key molecule in the increases of CD4+CD45RA+Foxp3hi T cell and activating Treg (aTreg) levels observed following cell‑supernatant co-culturing. Subsets of Treg cells were induced by Aire-overexpressing macrophages, and the manipulation of Treg cells by the targeting of Aire may provide a method for the treatment of inflammatory or autoimmune diseases.

  2. Structural characterization and immunomodulatory effects of polysaccharides from Phellinus linteus and Phellinus igniarius on the IL-6/IL-10 cytokine balance of the mouse macrophage cell lines (RAW 264.7).

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    Suabjakyong, Papawee; Nishimura, Kazuhiro; Toida, Toshihiko; Van Griensven, Leo J L D

    2015-08-01

    Phellinus linteus and igniarius (L.) Quel. have been used in traditional Asian medicine for over two centuries against a variety of diseases. Polysaccharides from their fruiting bodies show strong immunomodulatory activity. In this study we characterized the structure and composition of polysaccharides from Phellinus linteus and Phellinus igniarius by HPLC, GC-MS and NMR (1-H, 13-C, COSY, NOESY and TOCSY). The polysaccharides from P. linteus and P. igniarius mainly contained glucose with minor proportions of mannose, galactose, xylose, arabinose and rhamnose. Methylation analyses showed that the glycosidic linkages were mostly 1 → 3, 1 → 6 or 1 → 3,6. The two-dimensional COSY, NOESY and TOCSY confirmed that these polysaccharides have a main chain of →3)-β-D-Glcp-(1→ with →6)-β-D-Glcp-(1→ side chain. In vitro assays by RT-PCR and ELISA showed that (1 → 3; 1 → 6)-β-D-polysaccharides from P. linteus and P. igniarius decreased TNF-α in RAW 264.7 cells, suggesting an immuno-suppressive activity. Furthermore, these polysaccharides stimulated a high IL-10 response and induced strong suppression of transcription of IL-6. The results suggest that polysaccharides from P. linteus and P. igniarius could possibly find applications in restoring the IL-6/IL-10 balance, the disturbance of which is thought to be related to chronic inflammatory disease, obesity, diabetes type 2, and to mania and depression.

  3. Role of protein tyrosine phosphatase non-receptor type 7 in the regulation of TNF-α production in RAW 264.7 macrophages.

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    Seo, Huiyun; Lee, In-Seon; Park, Jae Eun; Park, Sung Goo; Lee, Do Hee; Park, Byoung Chul; Cho, Sayeon

    2013-01-01

    Protein tyrosine phosphatases play key roles in a diverse range of cellular processes such as differentiation, cell proliferation, apoptosis, immunological signaling, and cytoskeletal function. Protein tyrosine phosphatase non-receptor type 7 (PTPN7), a member of the phosphatase family, specifically inactivates mitogen-activated protein kinases (MAPKs). Here, we report that PTPN7 acts as a regulator of pro-inflammatory TNF-α production in RAW 264.7 cells that are stimulated with lipopolysaccharide (LPS) that acts as an endotoxin and elicits strong immune responses in animals. Stimulation of RAW 264.7 cells with LPS leads to a transient decrease in the levels of PTPN7 mRNA and protein. The overexpression of PTPN7 inhibits LPS-stimulated production of TNF-α. In addition, small interfering RNA (siRNA) analysis showed that knock-down of PTPN7 in RAW 264.7 cells increased TNF-α production. PTPN7 has a negative regulatory function to extracellular signal regulated kinase 1/2 (ERK1/2) and p38 that increase LPS-induced TNF-α production in macrophages. Thus, our data presents PTPN7 as a negative regulator of TNF-α expression and the inflammatory response in macrophages.

  4. Role of protein tyrosine phosphatase non-receptor type 7 in the regulation of TNF-α production in RAW 264.7 macrophages.

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    Huiyun Seo

    Full Text Available Protein tyrosine phosphatases play key roles in a diverse range of cellular processes such as differentiation, cell proliferation, apoptosis, immunological signaling, and cytoskeletal function. Protein tyrosine phosphatase non-receptor type 7 (PTPN7, a member of the phosphatase family, specifically inactivates mitogen-activated protein kinases (MAPKs. Here, we report that PTPN7 acts as a regulator of pro-inflammatory TNF-α production in RAW 264.7 cells that are stimulated with lipopolysaccharide (LPS that acts as an endotoxin and elicits strong immune responses in animals. Stimulation of RAW 264.7 cells with LPS leads to a transient decrease in the levels of PTPN7 mRNA and protein. The overexpression of PTPN7 inhibits LPS-stimulated production of TNF-α. In addition, small interfering RNA (siRNA analysis showed that knock-down of PTPN7 in RAW 264.7 cells increased TNF-α production. PTPN7 has a negative regulatory function to extracellular signal regulated kinase 1/2 (ERK1/2 and p38 that increase LPS-induced TNF-α production in macrophages. Thus, our data presents PTPN7 as a negative regulator of TNF-α expression and the inflammatory response in macrophages.

  5. Uterine NK cells and macrophages in pregnancy

    NARCIS (Netherlands)

    Faas, Marijke M.; de Vos, Paul

    The presence of immune cells in the placental bed is important for both mother and child. Although various immune cells can be found in the placental bed, such as regulatory T cells and dendritic cells, uterine NK cells and macrophages are the most prominent immune cells in the placental bed in

  6. Uterine NK cells and macrophages in pregnancy

    NARCIS (Netherlands)

    Faas, Marijke M; de Vos, Paul

    2017-01-01

    The presence of immune cells in the placental bed is important for both mother and child. Although various immune cells can be found in the placental bed, such as regulatory T cells and dendritic cells, uterine NK cells and macrophages are the most prominent immune cells in the placental bed in earl

  7. Macrophages and Dendritic Cells: Partners in Atherogenesis.

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    Cybulsky, Myron I; Cheong, Cheolho; Robbins, Clinton S

    2016-02-19

    Atherosclerosis is a complex chronic disease. The accumulation of myeloid cells in the arterial intima, including macrophages and dendritic cells (DCs), is a feature of early stages of disease. For decades, it has been known that monocyte recruitment to the intima contributes to the burden of lesion macrophages. Yet, this paradigm may require reevaluation in light of recent advances in understanding of tissue macrophage ontogeny, their capacity for self-renewal, as well as observations that macrophages proliferate throughout atherogenesis and that self-renewal is critical for maintenance of macrophages in advanced lesions. The rate of atherosclerotic lesion formation is profoundly influenced by innate and adaptive immunity, which can be regulated locally within atherosclerotic lesions, as well as in secondary lymphoid organs, the bone marrow and the blood. DCs are important modulators of immunity. Advances in the past decade have cemented our understanding of DC subsets, functions, hematopoietic origin, gene expression patterns, transcription factors critical for differentiation, and provided new tools for study of DC biology. The functions of macrophages and DCs overlap to some extent, thus it is important to reassess the contributions of each of these myeloid cells taking into account strict criteria of cell identification, ontogeny, and determine whether their key roles are within atherosclerotic lesions or secondary lymphoid organs. This review will highlight key aspect of macrophage and DC biology, summarize how these cells participate in different stages of atherogenesis and comment on complexities, controversies, and gaps in knowledge in the field.

  8. Ketamine inhibits tumor necrosis factor secretion by RAW264.7 murine macrophages stimulated with antibiotic-exposed strains of community-associated, methicillin-resistant Staphylococcus aureus

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    Aguirre Carlos

    2011-01-01

    Full Text Available Abstract Background Infections caused by community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA are associated with a marked and prolonged host inflammatory response. In a sepsis simulation model, we tested whether the anesthetic ketamine inhibits the macrophage TNF response to antibiotic-exposed CA-MRSA bacteria via its antagonism of N-methyl-D-aspartate (NMDA receptors. RAW264.7 cells were stimulated for 18 hrs with 105 to 107 CFU/mL inocula of either of two prototypical CA-MRSA isolates, USA300 strain LAC and USA400 strain MW2, in the presence of either vancomycin or daptomycin. One hour before bacterial stimulation, ketamine was added with or without MK-801 (dizocilpine, a chemically unrelated non-competitive NMDA receptor antagonist, APV (D-2-amino-5-phosphono-valerate, a competitive NMDA receptor antagonist, NMDA, or combinations of these agents. Supernatants were collected and assayed for TNF concentration by ELISA. Results RAW264.7 cells exposed to either LAC or MW2 in the presence of daptomycin secreted less TNF than in the presence of vancomycin. The addition of ketamine inhibited macrophage TNF secretion after stimulation with either of the CA-MRSA isolates (LAC, MW2 in the presence of either antibiotic. The NMDA inhibitors, MK-801 and APV, also suppressed macrophage TNF secretion after stimulation with either of the antibiotic-exposed CA-MRSA isolates, and the effect was not additive or synergistic with ketamine. The addition of NMDA substrate augmented TNF secretion in response to the CA-MRSA bacteria, and the addition of APV suppressed the effect of NMDA in a dose-dependent fashion. Conclusions Ketamine inhibits TNF secretion by MRSA-stimulated RAW264.7 macrophages and the mechanism likely involves NMDA receptor antagonism. These findings may have therapeutic significance in MRSA sepsis.

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

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    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.

  10. Aged red garlic extract reduces lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages and acute pulmonary inflammation through haeme oxygenase-1 induction.

    Science.gov (United States)

    Park, H-J; Jeon, B T; Kim, H C; Roh, G S; Shin, J-H; Sung, N-J; Han, J; Kang, D

    2012-05-01

    It is known that garlic has antioxidative and anti-inflammatory properties. Aged red garlic (ARG), a novel aged garlic formulation, has higher antioxidant effects than fresh raw garlic. This study was performed to examine the anti-inflammatory effects of ARG extract (ARGE). The anti-inflammatory effects of ARGE were evaluated in the lipopolysaccharide (LPS)-treated Raw 264.7 macrophages and acute lung inflammatory mice. NO production was determined by the Griess method, and iNOS, HO-1 and COX-2 expressions were measured using Western blot analysis. Histology and inflammation extent of lung were analysed using haematoxylin-eosin staining and immunohistochemistry. ARGE treatment markedly reduced LPS-induced nitrite production in RAW 264.7 macrophages and reduced inducible nitric oxide synthase (iNOS) expression. Treatment of cells with ARGE led to a significant increase in haeme oxygenase-1 (HO-1) protein expression, which was mediated by stimulating the expression of nuclear factor erythroid 2-related factor 2 (Nrf2). Treatment with zinc protoporphyrin, a selective inhibitor of HO-1, significantly reversed the ARGE-mediated inhibition of nitrite production (P < 0.05). In LPS-induced inflammatory mice, ARGE treatment down-regulated iNOS and COX-2 expressions, while it up-regulated HO-1 expression. These results show that ARGE reduces LPS-induced nitric oxide production in RAW 264.7 macrophages through HO-1 induction and suggest that ARGE may have potential effects on prevention and treatment of acute inflammatory lung injury. © 2012 The Authors Acta Physiologica © 2012 Scandinavian Physiological Society.

  11. Effect of Quercetin on Paraoxonase 2 Levels in RAW264.7 Macrophages and in Human Monocytes—Role of Quercetin Metabolism

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    Manfred James Mueller

    2009-09-01

    Full Text Available There is increasing evidence that the intracellular antioxidant enzyme paraoxonase 2 (PON2 may have a protective function in the prevention of atherogenesis. An enhancement of PON2 activity by dietary factors including flavonoids is therefore of interest. In the present study we determined the effect of quercetin on paraoxonase 2 levels in cultured murine macrophages in vitro and in overweight subjects with a high cardiovascular risk phenotype supplemented with 150 mg quercetin/day for 42 days in vivo. Supplementation of murine RAW264.7 macrophages in culture with increasing concentrations of quercetin (1, 10, 20 μmol/L resulted in a significant increase in PON2 mRNA and protein levels, as compared to untreated controls. Unlike quercetin, its glucuronidated metabolite quercetin-3-glucuronide did not affect PON2 gene expression in cultured macrophages. However the methylated quercetin derivative isorhamnetin enhanced PON2 gene expression in RAW264.7 cells to similar extent like quercetin. Although supplementing human volunteers with quercetin was accompanied by a significant increase in plasma quercetin concentration, dietary quercetin supplementation did not change PON2 mRNA levels in human monocytes in vivo. Current data indicate that quercetin supplementation increases PON2 levels in cultured monocytes in vitro but not in human volunteers in vivo.

  12. Streptococcus pyogenes CAMP factor attenuates phagocytic activity of RAW 264.7 cells.

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    Kurosawa, Mie; Oda, Masataka; Domon, Hisanori; Saitoh, Issei; Hayasaki, Haruaki; Terao, Yutaka

    2016-02-01

    Streptococcus pyogenes produces molecules that inhibit the function of human immune system, thus allowing the pathogen to grow and spread in tissues. It is known that S. pyogenes CAMP factor increases erythrocytosis induced by Staphylococcus aureus β-hemolysin. However, the effects of CAMP factor for immune cells are unclear. In this study, we investigated the effects of CAMP factor to macrophages. Western blotting analysis demonstrated that all examined strains expressed CAMP factor protein. In the presence of calcium or magnesium ion, CAMP factor was significantly released in the supernatant. In addition, both culture supernatant from S. pyogenes strain SSI-9 and recombinant CAMP factor dose-dependently induced vacuolation in RAW 264.7 cells, but the culture supernatant from Δcfa isogenic mutant strain did not. CAMP factor formed oligomers in RAW 264.7 cells in a time-dependent manner. CAMP factor suppressed cell proliferation via G2 phase cell cycle arrest without inducing cell death. Furthermore, CAMP factor reduced the uptake of S. pyogenes and phagocytic activity indicator by RAW 264.7 cells. These results suggest that CAMP factor works as a macrophage dysfunction factor. Therefore, we conclude that CAMP factor allows S. pyogenes to escape the host immune system, and contribute to the spread of streptococcal infection.

  13. Suppression of the lipopolysaccharide-induced expression of MARCKS-related protein (MRP) affects transmigration in activated RAW264.7 cells.

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    Chun, Kwang-Rok; Bae, Eun Mi; Kim, Jae-Kwan; Suk, Kyoungho; Lee, Won-Ha

    2009-01-01

    The molecular action mechanism of MRP, one of the protein kinase C (PKC) substrates, has been under intense investigation, but reports on its role in macrophage function remain controversial. The treatment of macrophage cell lines with bacterial lipopolysaccharide (LPS) induced a high level of MRP expression suggesting that MRP plays a role in the function of activated macrophages. In order to investigate the role of MRP in activated RAW264.7 cells, we stably transfected MRP-specific shRNA expression constructs and tested for alterations in macrophage-related functions. The down-regulation of MRP expression resulted in a marked reduction in chemotaxis toward MCP-1 or extracellular matrix proteins. Furthermore, pharmacological inhibitors of PKC significantly inhibited the chemotaxis in RAW264.7 cells. These data reveals the pivotal role of MRP in the transmigration of activated RAW264.7 cells.

  14. 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.

  15. Traditional Herbal Formula Banhasasim-tang Exerts Anti-Inflammatory Effects in RAW 264.7 Macrophages and HaCaT Keratinocytes

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    Seong Eun Jin

    2015-01-01

    Full Text Available Banhasasim-tang (BHSST is a Korean traditional herbal formula comprising eight medicinal herbs. The aim of the present study was to investigate the anti-inflammatory effect of BHSST using macrophage and keratinocyte cell lines. First, we evaluated the effects of BHSST on inflammatory mediator and cytokine production in lipopolysaccharide- (LPS- stimulated RAW 264.7 macrophages. BHSST markedly inhibited the production of nitric oxide (NO, prostaglandin E2 (PGE2, and interleukin- (IL- 6. BHSST significantly suppressed the protein expression of toll-like receptor 4 (TLR4 and phosphorylated nuclear factor-kappa B (NF-κB p65 in RAW 264.7 cells. Second, we examined whether BHSST influences the production of chemokines and STAT1 phosphorylation in tumor necrosis factor-α/interferon-γ TI-stimulated HaCaT keratinocytes. BHSST significantly suppressed the production of RANTES/CCL5, TARC/CCL17, MDC/CCL22, and IL-8 in TI-stimulated HaCaT cells. BHSST also suppressed TI-induced phosphorylation of STAT1 in HaCaT cells. These results suggest that BHSST may be useful as an anti-inflammatory agent, especially for inflammatory skin diseases.

  16. Anti-inflammatory effect of fucoxanthin derivatives isolated from Sargassum siliquastrum in lipopolysaccharide-stimulated RAW 264.7 macrophage.

    Science.gov (United States)

    Heo, Soo-Jin; Yoon, Weon-Jong; Kim, Kil-Nam; Oh, Chulhong; Choi, Young-Ung; Yoon, Kon-Tak; Kang, Do-Hyung; Qian, Zhong-Ji; Choi, Il-Whan; Jung, Won-Kyo

    2012-09-01

    In this study, the anti-inflammatory effect of fucoxanthin (FX) derivatives, which was isolated from Sargassum siliquastrum were evaluated by examining their inhibitory effects on pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells. The FX derivatives were isolated from activity-guided chloroform fraction using inhibition of nitric oxide (NO) production and identified as 9'-cis-(6'R) fucoxnathin (FXA), and 13-cis and 13'-cis-(6'R) fucoxanthin complex (FXB) on the basis of a comparison of NMR spectroscopic data. Both FXA and FXB significantly inhibited the NO production and showed slightly reduce the PGE2 production. However, FXB exhibited cytotoxicity at the whole tested concentration, therefore, the results of FXA was only illustrate for further experiments. FXA induced dose-dependent reduction in the inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) proteins as well as mRNA expression. In addition, FXA reduced the LPS-stimulated production and mRNA expressions of TNF-α and IL-6 in a dose-dependent manner whereas IL-1β production do not inhibit by addition of FXA. Taken together, these findings indicate that the anti-inflammatory properties of FXA may be due to the inhibition of iNOS/NO pathway which associated with the attenuation of TNF-α and IL-6 formation. Thus FXA may provide a potential therapeutic approach for inflammation related diseases.

  17. Antioxidant and anti-inflammatory effects in RAW264.7 macrophages of malvidin, a major red wine polyphenol.

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    Eszter Bognar

    Full Text Available BACKGROUND: Red wine polyphenols can prevent cardiovascular and inflammatory diseases. Resveratrol, the most extensively studied constituent, is unlikely to solely account for these beneficial effects because of its rather low abundance and bioavailability. Malvidin is far the most abundant polyphenol in red wine; however, very limited data are available about its effect on inflammatory processes and kinase signaling pathways. METHODS FINDINGS: The present study was carried out by using RAW 264.7 macrophages stimulated by bacterial lipopolysaccharide in the presence and absence of malvidin. From the cells, activation of nuclear factor-kappaB, mitogen-activated protein kinase, protein kinase B/Akt and poly ADP-ribose polymerase, reactive oxygen species production, mitogen-activated protein kinase phosphatase-1 expression and mitochondrial depolarization were determined. We found that malvidin attenuated lipopolysaccharide-induced nuclear factor-kappaB, poly ADP-ribose polymerase and mitogen-activated protein kinase activation, reactive oxygen species production and mitochondrial depolarization, while upregulated the compensatory processes; mitogen-activated protein kinase phosphatase-1 expression and Akt activation. CONCLUSIONS: These effects of malvidin may explain the previous findings and at least partially account for the positive effects of moderate red wine consumption on inflammation-mediated chronic maladies such as obesity, diabetes, hypertension and cardiovascular disease.

  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. Phellinus linteus inhibits inflammatory mediators by suppressing redox-based NF-kappaB and MAPKs activation in lipopolysaccharide-induced RAW 264.7 macrophage.

    Science.gov (United States)

    Kim, Ho Gyoung; Yoon, Deok Hyo; Lee, Won Ho; Han, Sang Kuk; Shrestha, Bhushan; Kim, Chun Hoi; Lim, Mi Hee; Chang, Woochul; Lim, Soyeon; Choi, Sunga; Song, Won O; Sung, Jae Mo; Hwang, Ki Chul; Kim, Tae Woong

    2007-12-03

    The mushroom Phellinus linteus has been known to exhibit potent biological activity. In contrast to the immuno-potentiating properties of Phellinus linteus, the anti-inflammatory properties of Phellinus linteus have rarely been investigated. Recently, ethanol extract and n-BuOH fractions from Phellinus linteus were deemed most effective in anti-inflammatory activity in RAW 264.7 macrophages. The regulatory mechanisms of Phellinus linteus butanol fractions (PLBF) on the pharmacological and biochemical actions of macrophages involved in inflammation have not been clearly defined yet. In the present study, we tested the role of PLBF on anti-inflammation patterns in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. To investigate the mechanism by which PLBF inhibits NO and PGE2 production as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, we examined the activation of IkappaB and MAPKs in LPS-activated macrophages. PLBF clearly inhibited nuclear translocation of NF-kappaB p65 subunits, which correlated with PLBF's inhibitory effects on IkappaBalpha phosphorylation and degradation. PLBF also suppressed the activation of mitogen-activated protein (MAP) kinases including p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Furthermore, macrophages stimulated with LPS generated ROS via activation of membrane-bound NADPH oxidase, and ROS played an important role in the activation of nuclear factor-kappaB (NF-kappaB) and MAPKs. We demonstrated that PLBF directly blocked intracellular accumulation of reactive oxygen species in RAW 264.7 cells stimulated with LPS much as the NADPH oxidase inhibitors, diphenylene iodonium, and antioxidant pyrrolidine dithiocarbamate did. The suppression of NADPH oxidase also inhibited NO production and iNOS protein expression. Cumulatively, these results suggest that PLBF inhibits the production of NO and PGE2 through the down-regulation of iNOS and COX-2 gene

  20. Immunostimulatory Activity of Opuntia ficus-indica var. Saboten Cladodes Fermented by Lactobacillus plantarum and Bacillus subtilis in RAW 264.7 Macrophages.

    Science.gov (United States)

    Hwang, Joon-Ho; Lim, Sang-Bin

    2017-02-01

    To increase the functionality of Opuntia ficus-indica var. saboten cladodes, it was fermented by Lactobacillus plantarum and Bacillus subtilis. Eighty percent methanol extracts were investigated for their effects on nitric oxide (NO) production, cytokine secretion, nuclear factor-κB (NF-κB) activity, and mitogen-activated protein kinase (MAPK) phosphorylation in RAW 264.7 cells. Methanol extracts of L. plantarum culture medium (LPCME) and B. subtilis culture medium (BSCME) did not affect lipopolysaccharide (LPS)-induced NO production but, at 500 μg/mL, increased interferon (IFN)-γ-induced NO production by 55.2 and 66.5 μM, respectively, in RAW 264.7 cells. In RAW 264.7 cells not treated with LPS and IFN-γ, LPCME did not affect NO production, but BSCME increased NO production significantly in a dose-dependent manner. In addition, BSCME induced the expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW 264.7 cells in a dose-dependent manner. BSCME at 500 μg/mL increased TNF-α and IL-1β mRNA levels by 83.8% and 82.2%, respectively. BSCME increased NF-κB-dependent luciferase activity in a dose-dependent manner; 500 μg/mL BSCME increased activity 9.1-fold compared with the control. BSCME induced the phosphorylation of p38, c-JUN NH2-terminal protein kinase (JNK), and extracellular signal-regulated kinase (ERK) in a dose-dependent manner, but did not affect total ERK levels. In conclusion, BSCME exerted immunostimulatory effects, which were mediated by MAPK phosphorylation and NF-κB activation, resulting in increased TNF-α and IL-1β gene expression in RAW 264.7 macrophages. Therefore, BSCM shows promise for use as an immunostimulatory therapeutic.

  1. Lipopolysaccharide induces multinuclear cell from RAW264.7 line with increased phagocytosis activity

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    Nakanishi-Matsui, Mayumi, E-mail: nakanim@iwate-med.ac.jp [Department of Biochemistry, Faculty of Pharmaceutical Sciences, Iwate Medical University, Futai Special Laboratory, Yahaba, Iwate 028-3694 (Japan); Yano, Shio; Matsumoto, Naomi; Futai, Masamitsu [Department of Biochemistry, Faculty of Pharmaceutical Sciences, Iwate Medical University, Futai Special Laboratory, Yahaba, Iwate 028-3694 (Japan)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer LPS induces multinuclear cells from murine macrophage-derived RAW264.7 cells. Black-Right-Pointing-Pointer The multinuclear cells are formed through cell-cell fusion in the presence of Ca{sup 2+}. Black-Right-Pointing-Pointer The multinuclear cells do not express osteoclast-specific enzymes. Black-Right-Pointing-Pointer They internalized more and larger beads than mononuclear cells and osteoclasts. -- Abstract: Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, induces strong proinflammatory responses, including the release of cytokines and nitric oxide from macrophage. In this study, we found that a murine macrophage-derived line, RAW264.7, became multinuclear through cell-cell fusion after incubation with highly purified LPS or synthetic lipid A in the presence of Ca{sup 2+}. The same cell line is known to differentiate into multinuclear osteoclast, which expresses a specific proton pumping ATPase together with osteoclast markers on stimulation by the extracellular domain of receptor activator of nuclear factor {kappa}B ligand (Toyomura, T., Murata, Y., Yamamoto, A., Oka, T., Sun-Wada, G.-H., Wada, Y. and Futai, M., 2003). The LPS-induced multinuclear cells did not express osteoclast-specific enzymes including tartrate-resistant acid phosphatase and cathepsin K. During multinuclear cell formation, the cells internalized more and larger polystyrene beads (diameter 6-15 {mu}m) than mononuclear cells and osteoclasts. The internalized beads were located in lysosome-marker positive organelles, which were probably phagolysosomes. The LPS-induced multinuclear cell could be a good model system to study phagocytosis of large foreign bodies.

  2. Macrophages, Dendritic Cells, and Regression of Atherosclerosis

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    Jonathan E. Feig

    2012-07-01

    Full Text Available Atherosclerosis is the number one cause of death in the Western world. It results from the interaction between modified lipoproteins and monocyte-derived cells such as macrophages, dendritic cells, T cells, and other cellular elements of the arterial wall. This inflammatory process can ultimately lead to the development of complex lesions, or plaques, that protrude into the arterial lumen. Ultimately, plaque rupture and thrombosis can occur leading to the clinical complications of myocardial infarction or stroke. Although each of the cell types plays roles in the pathogenesis of atherosclerosis, in this review, the focus will be primarily on the monocyte derived cells- macrophages and dendritic cells. The roles of these cell types in atherogenesis will be highlighted. Finally, the mechanisms of atherosclerosis regression as it relates to these cells will be discussed.

  3. Cytoprotective and enhanced anti-inflammatory activities of liposomal piroxicam formulation in lipopolysaccharide-stimulated RAW 264.7 macrophages

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    Chiong HS

    2013-03-01

    Full Text Available Hoe Siong Chiong,1 Yoke Keong Yong,1 Zuraini Ahmad,1 Mohd Roslan Sulaiman,1 Zainul Amiruddin Zakaria,1 Kah Hay Yuen,2 Muhammad Nazrul Hakim1,31Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia; 2School of Pharmaceutical Sciences, Universiti Sains Malaysia, Gelugor, Malaysia; 3Sports Academy, Universiti Putra Malaysia, Serdang, MalaysiaBackground: Liposomal drug delivery systems, a promising lipid-based nanoparticle technology, have been known to play significant roles in improving the safety and efficacy of an encapsulated drug.Methods: Liposomes, prepared using an optimized proliposome method, were used in the present work to encapsulate piroxicam, a widely prescribed nonsteroidal anti-inflammatory drug. The cytotoxic effects as well as the in vitro efficacy in regulation of inflammatory responses by free-form piroxicam and liposome-encapsulated piroxicam were evaluated using a lipopolysaccharide-sensitive macrophage cell line, RAW 264.7.Results: Cells treated with liposome-encapsulated piroxicam demonstrated higher cell viabilities than those treated with free-form piroxicam. In addition, the liposomal piroxicam formulation resulted in statistically stronger inhibition of pro-inflammatory mediators (ie, nitric oxide, tumor necrosis factor-α, interleukin-1β, and prostaglandin E2 than piroxicam at an equivalent dose. The liposome-encapsulated piroxicam also caused statistically significant production of interleukin-10, an anti-inflammatory cytokine.Conclusion: This study affirms the potential of a liposomal piroxicam formulation in reducing cytotoxicity and enhancing anti-inflammatory responses in vitro.Keywords: liposomes, nitric oxide, cytokines, prostaglandin E2, interleukin-1β, piroxicam

  4. Cell-death-mode switch from necrosis to apoptosis in hydrogen peroxide treated macrophages

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Cell death is typically defined either as apoptosis or necrosis. Because the consequences of apoptosis and necrosis are quite different for an entire organism, the investigation of the cell-death-mode switch has considerable clinical significance. The existence of a necrosis-to-apoptosis switch induced by hydrogen peroxide in macrophage cell line RAW 264.7 cells was confirmed by using flow cytometry and fluorescence microscopy. With the help of computational simulations, this study predicted that negative feedbacks between NF-κB and MAPKs are implicated in converting necrosis into apoptosis in macrophages exposed to hydrogen peroxide, which has significant implications.

  5. 沉默TNFAIP8抑制小鼠巨噬细胞RAW264.7的迁移功能%TNFAIP8 gene silencing inhibits the migration of mouse RAW264. 7 macrophages

    Institute of Scientific and Technical Information of China (English)

    杨菲; 吴素霞; 冯世明; 刘广超; 柴立辉

    2016-01-01

    macrophage cell line, and to investigate the effects of TNFAIP8 gene silencing on the functions of mouse macrophages. Methods The shRNA sequence targeting TNFAIP8 gene was designed and DNA oligos containing small hairpin frame was synthesized. The double-stranded DNA was cloned into pLKO. 1-TRC vector after annealing. The recombi-nant vector was verified by using double enzyme digestion and gene sequencing. Lentiviruses were prepared by transfecting the constructed vector into 293T cells. Fluorescent quantitative RT-PCR and Western blot as-say were performed to detect the expression of TNFAIP8 at mRNA and protein levels after infecting the RAW264. 7 cells with lentiviruses. Flat dish adhesion experiment and wound-healing assay were used to evaluate the effects of TNFAIP8 gene silencing on the adhesion and migration of RAW264. 7 cells. Results The recombinant lentiviral vector was successfully constructed as indicated by double enzyme di-gestion and gene sequencing analysis. The expression of TNFAIP8 in RAW264. 7 cells at both mRNA and protein levels were significantly down-regulated after lentivirus infection (P<0. 05). Moreover, TNFAIP8 gene silencing significantly impaired the cell adhesion ability of RAW264. 7 cells after 15 min, 30 min or 2 hours of culture. Compared with the cells in control group, the RAW264. 7 cells harboring silenced TN-FAIP8 gene looked round with a smaller number of cellular extensions. The wound-healing assay showed that less TNFAIP8 gene-silenced RAW264. 7 cells migrated into the wounded area as compared with the cells in control group after 24 hours of culture (P<0. 05). The wound-healing rates of the experimental and control groups were 25% and 50%, respectively. Conclusion The recombinant lentiviral vector containing shRNA targeting the TNFAIP8 gene was successfully constructed. Transfecting the RAW264. 7 cells with the con-structed vector significantly silenced the expression of TNFAIP8 gene and inhibited the adhesion and migra-tion of these

  6. Murine RAW 264.7 cell line as an immune target: are we missing something?

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    Merly, Liza; Smith, Sylvia L

    2017-04-01

    The popular murine macrophage cell line, RAW 264.7, is often used to initially screen natural products for bioactivity and to predict their potential effect in vivo or on primary cells. The cell line response is considered to reflect the potential human de novo response, and is used to evaluate the effective bioactivity of the product. Here, we compared the cytokine response of RAW 264.7 cells to shark cartilage (SC) with that of human leukocytes to determine whether the cell line response was a reliable predictor of the cytokine response one can expect from similarly stimulated human primary cells. Results not only revealed significant differences in the nature and level of TNFα produced by cells in vitro, but also showed that while the primary cell response included an upregulation in the production of IL-1β such a response was absent in RAW 264.7 cells. This suggests that had we relied on RAW 264.7 cells alone to assess the cytokine-inducing capacity of SC, the comprehensive Th1 response (shown in an earlier study) induced by SC in primary cells, consisting of release of several proinflammatory cytokines and chemokines, would not have been revealed. We conclude, therefore, that assays using only RAW 264.7 cells to initially screen for and assess immune reactivity of test products will not necessarily provide a comprehensive picture of the immunomodulatory properties of the substance under investigation, and can in fact be misleading with regard to the overall bioactive potential of the substance on an initial screen.

  7. Anti-inflammatory Effect of Erdosteine in Lipopolysaccharide-Stimulated RAW 264.7 Cells.

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    Park, Jong Sun; Park, Mi-Young; Cho, Young-Jae; Lee, Jae Ho; Yoo, Chul-Gyu; Lee, Choon-Taek; Lee, Sang-Min

    2016-08-01

    Erdosteine is widely used as a mucolytic agent and also has free radical scavenging and antioxidant activities. However, little is known about the mechanisms of the anti-inflammatory effect of erdosteine. We investigated the effect of erdosteine on the activation of the nuclear factor (NF)-kB/inhibitor of NFkB (IkB), and the mitogen-activated protein kinase (MAPK) and Akt pathways in the mouse macrophage cell line RAW 264.7. Cultured RAW 264.7 cells were pretreated with erdosteine and stimulated with lipopolysaccharide (LPS). In Western blotting, pretreatment with erdosteine inhibited the IkBα degradation induced in RAW 264.7 cells by LPS. LPS-induced IkB kinase (IKK) activity and NF-kB transcription were inhibited by pretreatment with erdosteine. Production of IL-6 and IL-1β was also inhibited by erdosteine pretreatment. However, erdosteine did not inhibit LPS-induced phosphorylation of Akt and MAPKs. These results suggest that the anti-inflammatory effect of erdosteine in mouse macrophages is mediated through inhibition of LPS-induced NF-kB activation.

  8. NPFF2 receptor is involved in the modulatory effects of neuropeptide FF for macrophage cell line.

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    Sun, Yu-long; Sun, Tao; Zhang, Xiao-yuan; He, Ning; Zhuang, Yan; Li, Jing-yi; Fang, Quan; Wang, Kai-rong; Wang, Rui

    2014-05-01

    Neuropeptide FF (NPFF) interacts with specific receptors to regulate diverse biological processes. Its modulatory effect in the immune field, however, has not been fully explored yet. Here, we report that NPFF2 receptors may be functionally expressed in two immune cell models, the primary peritoneal macrophage and RAW 264.7 macrophage. Firstly, the mRNA levels of NPFF2 receptor were up-regulated in macrophages when treated with LPS for 24 to 72 h. Subsequently, our data hinted that NPFF regulates the viability of both kinds of macrophages. After treatment with RF9, a reported antagonist for both NPFF receptors, delayed or inhibited the NPFF-induced macrophages viability augmentation, suggesting the involvement of NPFF2 receptor. Furthermore, down-regulation of nitric oxide (NO) synthases (NOSs) partially significantly inhibited the viability augmentation of macrophages induced by NPFF, implying a nitric oxide synthases- dependent pathway is involved. However, the NOSs are not the only route by which NPFF affects the viability of macrophages. Pharmacological inhibitors of NF-κB signal pathway also blocked the NPFF-induced macrophages growth, suggesting the involvement of the NF-κB signal pathway. The regulation activity of NPFF for macrophages suggests that NPFF could act as a potential hormone in the control of immune system. Collectively, our data provide new evidence about the immune modulatory effect of NPFF, which will be helpful in extending the scope of NPFF functions.

  9. Preparation of procyanidin B2 from apple pomace and its inhibitory effect on the expression of cyclooxygenase-2 in lipopolysaccharide-treated RAW264.7 macrophages

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    Huawei Zhang

    2011-06-01

    Full Text Available Dimeric procyanidin B2 (PB2 is one of phenolic compounds in apple pomace, an agro-industrial byproduct in apple juice processing. This work focused on purification of PB2 from apple pomace using sephadex column chromatography and its potential effect on lipopolysaccharide (LPS-induced inflammation using RAW264.7 macrophages. PB2 with the purity of 72.28 ± 1.85% was successfully afforded using resin and gel column chromatographic technique. Anti-inflammatory tests suggested that the expression of cyclooxygenase-2 (COX-2 in LPS-induced murine RAW264.7 macrophages was suppressed in a PB2 concentration-dependent manner. PB2 at no less than 50 μg·mL-1 could significantly suppress inflammation in the LPS-induced cells. Moreover, this suppressive effect was not correlated with PB2 pretreating. However, the COX-2 expression was not reduced in LPS pretreatment way followed by PB2 exposure, which suggested that PB2 has no repairing function. The results showed that high pure PB2 prepared from apple pomace has a remarkable anti-inflammatory property.

  10. Evaluation of anti-proliferative and anti-inflammatory activities of Pelagia noctiluca venom in Lipopolysaccharide/Interferon-γ stimulated RAW264.7 macrophages.

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    Ayed, Yosra; Sghaier, Rabiaa Manel; Laouini, Dhafer; Bacha, Hassen

    2016-12-01

    Components of Pelagia noctiluca (P. noctiluca) venom were evaluated for their anticancer and nitric Oxide (NO) inhibition activities. Three fractions, out of four, obtained by gel filtration on Sephadex G75 of P. noctiluca venom revealed an important selective anti-proliferative activity on several cell lines such as human bladder carcinoma (RT112), human glioblastoma (U87), and human myelogenous leukemia (K562) but not on mitogen-stimulated peripheral blood mononuclear cells. Interestingly, P. noctiluca components showed an important dose-dependent anti-inflammatory activity, through inhibition of NO production via transcriptional regulation of Inducible NO Synthase (iNOS), in IFN-γ/LPS stimulated RAW 264.7 macrophages. These data strongly suggest that P. noctiluca venom could be used as a natural inhibitor of cancer cell lines and a potent anti-inflammatory agent for the treatment of anti-inflammatory diseases.

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

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    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.

  12. Koumine Attenuates Lipopolysaccaride-Stimulated Inflammation in RAW264.7 Macrophages, Coincidentally Associated with Inhibition of NF-κB, ERK and p38 Pathways

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    Zhihang Yuan

    2016-03-01

    Full Text Available Medicinal herbal plants have been commonly used for intervention of different diseases and health enhancement worldwide. Koumine, an alkaloid monomer found abundantly in Gelsemium plants, can be effectively used as an anti-inflammatory medication. In this study, the mechanisms associated with the preventative effect of koumine on lipopolysaccharide (LPS-mediated inflammation in RAW264.7 macrophages were investigated. Koumine induced a decrease in the level of inducible nitric oxide synthase (iNOS protein, concomitant reduction in the production of nitric oxide (NO and reduction of the levels of interleukin (IL-6, tumor necrosis factor-α (TNF-α and IL-1β. Furthermore, koumine decreased the phosphorylation of p65 and inhibited nuclear factor κ Bα (IκBα proteins, resulting in lower production of nuclear factor (NF-κB transactivation. Koumine also induced a decrease in the phosphorylation of extracellular-signal-regulated kinases (ERK and p38 in RAW264 cells. In conclusion, these findings reveal that koumine decreases the productions of pro-inflammatory mediators though the suppression of p38 and ERK MAPK phosphorylation and the inhibition of NF-κB activation in RAW264.7 cells.

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

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    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.

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

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    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.

  15. Effect of pulsed electromagnetic field on inflammatory pathway markers in RAW 264.7 murine macrophages

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    Ross CL

    2013-03-01

    Full Text Available Christina L Ross,1,2 Benjamin S Harrison2 1Akamai University, Department of Energy Medicine, Hilo, HI, USA; 2Wake Forest Institute for Regenerative Medicine, Wake Forest Baptist Health, Winston-Salem, NC, USA Abstract: In the treatment of bacterial infections, antibiotics have proven to be very effective, but the way in which antibiotics are dosed can create a lag time between the administration of the drug and its absorption at the site of insult. The time it takes an antibiotic to reach therapeutic levels can often be significantly increased if the vascular system is compromized. Bacteria can multiply pending the delivery of the drug, therefore, developing treatments that can inhibit the inflammatory response while waiting for antibiotics to take effect could help prevent medical conditions such as septic shock. The aim of this study was to examine the effect of a pulsed electromagnetic field on the production of inflammatory markers tumor necrosis factor (TNF, transcription factor nuclear factor kappa B (NFkB, and the expression of the A20 (tumor necrosis factor-alpha-induced protein 3, in an inflamed-cell model. Lipopolysaccharide-challenged cells were exposed to a pulsed electromagnetic field at various frequencies in order to determine which, if any, frequency would affect the TNF-NFkB-A20 inflammatory response pathway. Our study revealed that cells continuously exposed to a pulsed electromagnetic field at 5 Hz demonstrated significant changes in the downregulation of TNF-α and NFkB and also showed a trend in the down regulation of A20, as compared with controls. This treatment could be beneficial in modulating the immune response, in the presence of infection. Keyword: TNFAIP3, pulsed electromagnetic field, macrophages, TNF, NFkB

  16. Interactions between neutrophils and macrophages promote macrophage killing of rat muscle cells in vitro

    Science.gov (United States)

    Nguyen, Hal X.; Tidball, James G.

    2003-01-01

    Current evidence indicates that the physiological functions of inflammatory cells are highly sensitive to their microenvironment, which is partially determined by the inflammatory cells and their potential targets. In the present investigation, interactions between neutrophils, macrophages and muscle cells that may influence muscle cell death are examined. Findings show that in the absence of macrophages, neutrophils kill muscle cells in vitro by superoxide-dependent mechanisms, and that low concentrations of nitric oxide (NO) protect against neutrophil-mediated killing. In the absence of neutrophils, macrophages kill muscle cells through a NO-dependent mechanism, and the presence of target muscle cells causes a three-fold increase in NO production by macrophages, with no change in the concentration of inducible nitric oxide synthase. Muscle cells that are co-cultured with both neutrophils and macrophages in proportions that are observed in injured muscle show cytotoxicity through a NO-dependent, superoxide-independent mechanism. Furthermore, the concentration of myeloid cells that is necessary for muscle killing is greatly reduced in assays that use mixed myeloid cell populations, rather than uniform populations of neutrophils or macrophages. These findings collectively show that the magnitude and mechanism of muscle cell killing by myeloid cells are modified by interactions between muscle cells and neutrophils, between muscle cells and macrophages and between macrophages and neutrophils.

  17. Wpiegulation of macrophage colony-stimulating factor on protease secretion in RAW 264.7 cell and its possible mechanism%M-CSF促进RAW 264.7细胞MMP-9的分泌及其可能机制

    Institute of Scientific and Technical Information of China (English)

    王春; 许灿新; 彭翠英; 秦旭平; 李凯; 廖端芳

    2006-01-01

    目的 探讨巨噬细胞集落刺激因子(M-CSF)致动脉粥样硬化的作用是否与其影响基质金属蛋白酶(MMP)表达和活性改变有关及其可能机制.方法 应用明胶酶图分析方法观察M-CSF和(或)PD98059对体外培养的RAW 264.7细胞MMP-9活性的影响;Wester blot检测M-CSF和(或)PD98059对体外培养的RAW 264.7细胞p-ERK1/2表达的影响.结果 M-CSF能增强RAW 264.7细胞MMP-9的活性,并呈一定的剂量依赖性;同时M-CSF也呈时间、浓度依赖性促进p-ERK1/2的表达;PD98059不仅阻断了ERK1/2的磷酸化,而且也降低了MMP-9的活性.结论 M-CSF可诱导RAW 264.7细胞MMP-9的活性增加,其机制可能与M-CSF激活MAPK-ERK1/2通路有关.

  18. 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.

  19. Inhibitory effects of salidroside on nitric oxide and prostaglandin E₂ production in lipopolysaccharide-stimulated RAW 264.7 macrophages.

    Science.gov (United States)

    Song, Bocui; Huang, Guoren; Xiong, Ying; Liu, Jingbo; Xu, Linli; Wang, Zhenning; Li, Gen; Lu, Jing; Guan, Shuang

    2013-11-01

    The aim of this study was to evaluate the effect of salidroside on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in RAW 264.7 macrophages and related anti-inflammatory mechanism. PGE₂ production was measured by enzyme-linked immunosorbent assay (ELISA); NO production was tested by Griess reagent. Inducible nitric oxidesynthase (iNOS) and COX-2 were determined by RT-PCR and Western blot analysis; IκB and P-IκB protein express were detected by Western blot analysis; cytosolic free Ca²⁺ ([Ca²⁺](i)) was measured by a fluorescent microscope. The data showed salidroside inhibited LPS-induced NO and PGE₂ production and reduced iNOS and COX-2 protein expression in RAW 264.7 macrophages. Consistent with these observations, salidroside inhibited LPS-induced cytosolic free Ca²⁺ concentration ([Ca²⁺](i)) elevation. In addition, we further investigated signal transduction mechanisms and found that the activation of NF-κB was suppressed by salidroside in a dose-dependent manner. These results suggest that salidroside suppresses NO and PGE₂ production by inhibiting iNOS and COX-2 protein expression, level of [Ca²⁺](i), and activation of NF-κB signal transduction pathway.

  20. 13-hydroxy linoleic acid increases expression of the cholesterol transporters ABCA1, ABCG1 and SR-BI and stimulates apoA-I-dependent cholesterol efflux in RAW264.7 macrophages

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    Kämmerer Ines

    2011-11-01

    Full Text Available Abstract Background Synthetic activators of peroxisome proliferator-activated receptors (PPARs stimulate cholesterol removal from macrophages through PPAR-dependent up-regulation of liver × receptor α (LXRα and subsequent induction of cholesterol exporters such as ATP-binding cassette transporter A1 (ABCA1 and scavenger receptor class B type 1 (SR-BI. The present study aimed to test the hypothesis that the hydroxylated derivative of linoleic acid (LA, 13-HODE, which is a natural PPAR agonist, has similar effects in RAW264.7 macrophages. Methods RAW264.7 macrophages were treated without (control or with LA or 13-HODE in the presence and absence of PPARα or PPARγ antagonists and determined protein levels of LXRα, ABCA1, ABCG1, SR-BI, PPARα and PPARγ and apolipoprotein A-I mediated lipid efflux. Results Treatment of RAW264.7 cells with 13-HODE increased PPAR-transactivation activity and protein concentrations of LXRα, ABCA1, ABCG1 and SR-BI when compared to control treatment (P Conclusion 13-HODE induces cholesterol efflux from macrophages via the PPAR-LXRα-ABCA1/SR-BI-pathway.

  1. Comparison of cytotoxic and inflammatory responses of photoluminescent silicon nanoparticles with silicon micron-sized particles in RAW 264.7 macrophages.

    Science.gov (United States)

    Choi, Jonghoon; Zhang, Qin; Reipa, Vytas; Wang, Nam Sun; Stratmeyer, Melvin E; Hitchins, Victoria M; Goering, Peter L

    2009-01-01

    Photoluminescent silicon nanoparticles have a bright and stable fluorescence and are promising candidates for bio-imaging, cell staining and drug delivery. With increasing development of nanotechnology applications for biomedicine, an understanding of the potential toxicity of nanoparticles is needed to assess safety concerns for clinical applications. The objective of this study was to compare biological responses of silicon nanoparticles (SNs, 3 nm diameter) with silicon microparticles (SMs, approximately 100-3000 nm diameter) in cultured murine macrophages (RAW 264.7) using standard protocols for assessing cytotoxicity/cell viability and inflammatory responses developed for micron-sized particles. SNs and SMs were exposed to macrophages with and without addition of endotoxin lipopolysaccharide (LPS), a positive inducer of tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), and nitric oxide (NO). Cytotoxicity was assayed using the dye exclusion and MTT assays. Cell supernatants were assayed for production TNF-alpha, IL-6 and NO. SNs at concentrations 20 and 200 microg ml(-1), respectively, increased cytotoxicity compared with controls. SMs induced concentration-related increases in TNF-alpha and IL-6 production; in contrast, the production of these cytokines was shown to decrease with increasing concentrations of SNs. NO production was not induced by SNs or SMs alone. Fluorescence microscopy demonstrated that SNs were associated with the macrophages, either internalized or attached to cell membranes. In conclusion, evaluating differences in biological responses for nanoparticles compared with microparticles of the same material may help improve tests to assess biological responses of nanoparticles that may be used in biomedical applications.

  2. The Role of Selected Flavonols in Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor–1 (TRAIL-R1 Expression on Activated RAW 264.7 Macrophages

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    Monika Warat

    2015-01-01

    Full Text Available Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptors (TRAIL-R are an important factor of apoptosis in cancer cells. There are no data about the effect of flavonols on the receptor expression on a surface of macrophage like cells. In this study, the expression level of TRAIL-R1 on murine RAW264.7 macrophages in the presence of selected flavonols: galangin, kaempferol, kaempferide and quercetin, which differ from their phenyl ring substituents, were studied. The expression of TRAIL-R1 death receptors on non-stimulated and lipopolysaccharide (LPS-stimulated macrophages was determined using flow cytometry. The results suggested that compounds being tested can modulate TRAIL-R1 expression and can enhance TRAIL-mediated apoptosis.

  3. Low Dose BCG Infection as a Model for Macrophage Activation Maintaining Cell Viability

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    Leslie Chávez-Galán

    2016-01-01

    Full Text Available Mycobacterium bovis BCG, the current vaccine against tuberculosis, is ingested by macrophages promoting the development of effector functions including cell death and microbicidal mechanisms. Despite accumulating reports on M. tuberculosis, mechanisms of BCG/macrophage interaction remain relatively undefined. In vivo, few bacilli are sufficient to establish a mycobacterial infection; however, in vitro studies systematically use high mycobacterium doses. In this study, we analyze macrophage/BCG interactions and microenvironment upon infection with low BCG doses and propose an in vitro model to study cell activation without affecting viability. We show that RAW macrophages infected with BCG at MOI 1 activated higher and sustained levels of proinflammatory cytokines and transcription factors while MOI 0.1 was more efficient for early stimulation of IL-1β, MCP-1, and KC. Both BCG infection doses induced iNOS and NO in a dose-dependent manner and maintained nuclear and mitochondrial structures. Microenvironment generated by MOI 1 induced macrophage proliferation but not MOI 0.1 infection. In conclusion, BCG infection at low dose is an efficient in vitro model to study macrophage/BCG interactions that maintains macrophage viability and mitochondrial structures. This represents a novel model that can be applied to BCG research fields including mycobacterial infections, cancer immunotherapy, and prevention of autoimmunity and allergies.

  4. Anti‑inflammatory and antioxidant activity of the traditional herbal formula Gwakhyangjeonggi‑san via enhancement of heme oxygenase‑1 expression in RAW264.7 macrophages.

    Science.gov (United States)

    Jeong, Soo-Jin; Kim, Ohn-Soon; Yoo, Sae-Rom; Seo, Chang-Seob; Kim, Yeji; Shin, Hyeun-Kyoo

    2016-05-01

    Gwakhyangjeonggi‑san (GHJGS) is a mixture of herbal plants, including Agastache rugosa, Perilla frutescens, Angelica dahurica, Areca catechu, Poria cocos, Magnolia officinalis, Atractylodes macrocephala, Citrus reticulata, Pinellia ternata, Platycodon grandiflorum, Glycyrrhiza uralensis, Ziziphus jujuba and Zingiber officinale. GHJGS has been used for treating diarrhea‑predominant irritable bowel syndrome in traditional Korean medicine. In the present study, the anti‑inflammatory and antioxidant effects of GHJGS were investigated using the RAW 264.7 murine macrophage cell line. GHJGS significantly reduced production of the proinflammatory cytokines, tumor necrosis factor‑α, interleukin‑6 and prostaglandin E2 in lipopolysaccharide (LPS)‑stimulated macrophages. GHJGS markedly suppressed LPS‑induced phosphorylation of mitogen‑activated protein kinases, whereas it had no effect on nuclear factor‑κB activation. Furthermore, GHJGS enhanced expression of heme oxygenase‑1 and prevented the generation of reactive oxygen species in RAW 264.7 cells. These results indicate that GHJGS is a viable therapeutic agent against inflammation and oxidative stress‑associated disorders.

  5. Comparison of Anti-Inflammatory Effects of Flavonoid-Rich Common and Tartary Buckwheat Sprout Extracts in Lipopolysaccharide-Stimulated RAW 264.7 and Peritoneal Macrophages

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    Tae Gyu Nam

    2017-01-01

    Full Text Available Buckwheat sprouts have been widely consumed all around world due to their great abundance of bioactive compounds. In this study, the anti-inflammatory effects of flavonoid-rich common buckwheat sprout (CBS and tartary buckwheat sprout (TBS extracts were evaluated in lipopolysaccharide- (LPS- stimulated RAW 264.7 murine macrophages and primary peritoneal macrophages from male BALB/c mice. Based on the reversed-phase HPLC analysis, the major flavonoids in CBS were determined to be C-glycosylflavones (orientin, isoorientin, vitexin, and isovitexin, quercetin-3-O-robinobioside, and rutin, whereas TBS contained only high amounts of rutin. The TBS extract exhibited higher inhibitory activity as assessed by the production of proinflammatory mediators such as nitric oxide and cytokines including tumor necrosis factor-α, interleukin- (IL- 6, and IL-12 in LPS-stimulated RAW 264.7 macrophages than CBS extract. In addition, TBS extract suppressed nuclear factor-kappa B activation by preventing inhibitor kappa B-alpha degradation and mitogen-activated protein kinase phosphorylation in LPS-stimulated RAW 264.7 macrophages. Moreover, the TBS extract markedly reduced LPS-induced cytokine production in peritoneal macrophages. Taken together, these findings suggest that TBS extract can be a potential source of anti-inflammatory agents that may influence macrophage-mediated inflammatory disorders.

  6. IM-133N modulates cytokine secretion by RAW264.7 and THP-1 cells.

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    Varma, R Sandeep; Guruprasad, Kanive P; Satyamoorthy, Kapaettu; Kumar, L M Sharath; Babu, U Venkanna; Patki, S Pralhad

    2016-01-01

    An indigenous herbal extract IM-133N containing extracts of Prosopis glandulosa Torr and Symplocos racemosa Roxb were evaluated for potential immunomodulatory effects using RAW264.7 and THP-1 cells. The incubation of the cells for 24 h with IM-133N over a dose range 0-125 µg/ml did not cause cytotoxicity that exceeded 10%. The results indicated that non-cytotoxic doses of IM-133N effectively up-regulated iNOS, TNFα, IL-6, IL-10, IL-8 and IFNγ gene expression in both the RAW264.7 and THP-1 cells. The results also indicated IM-133N elicited dose-related increases in nitric oxide (NO) and tumor necrosis factor (TNF)-α production by RAW264.7 or THP-1 cells. These results demonstrated that IM-133N could stimulate NO and induced pro-inflammatory cytokine expression by monocytes/macrophages. As clinical studies have shown IM-133N to be an effective immunomodulator without any adverse effects, the results of the present study provide further support for the potential use of this agent as an immunostimulant or as an immunotherapy adjuvant.

  7. Multi-walled carbon nanotubes induce COX-2 and iNOS expression via MAP Kinase-dependent and -independent mechanisms in mouse RAW264.7 macrophages

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    Lee Jong

    2012-05-01

    Full Text Available Abstract Background Carbon nanotubes (CNTs are engineered graphene cylinders with numerous applications in engineering, electronics and medicine. However, CNTs cause inflammation and fibrosis in the rodent lung, suggesting a potential human health risk. We hypothesized that multi-walled CNTs (MWCNTs induce two key inflammatory enzymes in macrophages, cyclooxygenase-2 (COX-2 and inducible nitric oxide synthase (iNOS, through activation of extracellular signal-regulated kinases (ERK1,2. Methods RAW264.7 macrophages were exposed to MWCNTs or carbon black nanoparticles (CBNPs over a range of doses and time course. Uptake and subcellular localization of MWCNTs was visualized by transmission electron microscopy (TEM. Protein levels of COX-2, iNOS, and ERK1,2 (total ERK and phosphorylated ERK were measured by Western blot analysis. Prostaglandin-E2 (PGE2 and nitric oxide (NO levels in cell supernatants were measured by ELISA and Greiss assay, respectively. Results MWCNTs, but not CBNPs, induced COX-2 and iNOS in a time- and dose-dependent manner. COX-2 and iNOS induction by MWCNTs correlated with increased PGE2 and NO production, respectively. MWCNTs caused ERK1,2 activation and inhibition of ERK1,2 (U0126 blocked MWCNT induction of COX-2 and PGE2 production, but did not reduce the induction of iNOS. Inhibition of iNOS (L-NAME did not affect ERK1,2 activation, nor did L-NAME significantly decrease COX-2 induction by MWCNT. Nickel nanoparticles (NiNPs, which are present in MWCNTs as a residual catalyst, also induced COX-2 via ERK-1,2. However, a comparison of COX-2 induction by MWCNTs containing 4.5 and 1.8% Ni did not show a significant difference in ability to induce COX-2, indicating that characteristics of MWCNTs in addition to Ni content contribute to COX-2 induction. Conclusion This study identifies COX-2 and subsequent PGE2 production, along with iNOS induction and NO production, as inflammatory mediators involved in the macrophage response to

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

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    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

  9. Effect of manassantin B, a lignan isolated from Saururus chinensis, on lipopolysaccharide-induced interleukin-1β in RAW 264.7 cells

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    Park, Hwan Chul; Bae, Hong-Beom; Jeong, Cheol-Won; Lee, Seong Heon; Jeung, Hye Jin; Kwak, Sang-Hyun

    2012-01-01

    Background Elevated systemic levels of pro-inflammatory cytokines cause hypotension during septic shock and induce capillary leakage in acute lung injury. Manassantin B has anti-inflammatory and anti-plasmoidal properties. This study examined the effects of manassantin B on lipopolysaccharide (LPS)-induced inflammatory response in murine macrophages. Methods RAW 264.7 macrophage cells were incubated without or with (1, 3 and 10 µM) manassantin B and without or with (100 ng/ml) LPS. Manassanti...

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

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    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.

  11. Moringa oleifera Flower Extract Suppresses the Activation of Inflammatory Mediators in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages via NF-κB Pathway.

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    Tan, Woan Sean; Arulselvan, Palanisamy; Karthivashan, Govindarajan; Fakurazi, Sharida

    2015-01-01

    Aim of Study. Moringa oleifera Lam. (M. oleifera) possess highest concentration of antioxidant bioactive compounds and is anticipated to be used as an alternative medicine for inflammation. In the present study, we investigated the anti-inflammatory activity of 80% hydroethanolic extract of M. oleifera flower on proinflammatory mediators and cytokines produced in lipopolysaccharide- (LPS-) induced RAW 264.7 macrophages. Materials and Methods. Cell cytotoxicity was conducted by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide (NO) production was quantified through Griess reaction while proinflammatory cytokines and other key inflammatory markers were assessed through enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Results. Hydroethanolic extract of M. oleifera flower significantly suppressed the secretion and expression of NO, prostaglandin E2 (PGE2), interleukin- (IL-) 6, IL-1β, tumor necrosis factor-alpha (TNF-α), nuclear factor-kappa B (NF-κB), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2). However, it significantly increased the production of IL-10 and IκB-α (inhibitor of κB) in a concentration dependent manner (100 μg/mL and 200 μg/mL). Conclusion. These results suggest that 80% hydroethanolic extract of M. oleifera flower has anti-inflammatory action related to its inhibition of NO, PGE2, proinflammatory cytokines, and inflammatory mediator's production in LPS-stimulated macrophages through preventing degradation of IκB-α in NF-κB signaling pathway.

  12. 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.

  13. Morin, a Bioflavonoid Suppresses Monosodium Urate Crystal-Induced Inflammatory Immune Response in RAW 264.7 Macrophages through the Inhibition of Inflammatory Mediators, Intracellular ROS Levels and NF-κB Activation.

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    Chitra Dhanasekar

    Full Text Available Our previous studies had reported that morin, a bioflavanoid exhibited potent anti-inflammatory effect against adjuvant-induced arthritic rats. In this current study, we investigated the anti-inflammatory mechanism of morin against monosodium urate crystal (MSU-induced inflammation in RAW 264.7 macrophage cells, an in vitro model for acute gouty arthritis. For comparison purpose, colchicine was used as a reference drug. We have observed that morin (100-300 μM treatment significantly suppressed the levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1 and VEGF, inflammatory mediators (NO and PEG2, and lysosomal enzymes (acid phosphatase, β-galactosidase, N-acetyl glucosamindase and cathepsin D in MSU-crystals stimulated macrophage cells. The mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1, inflammatory enzymes (iNOS and COX-2, and NF-κBp65 was found downregulated in MSU crystal stimulated macrophage cells by morin treatment, however, the mRNA expression of hypoxanthine phospho ribosyl transferse (HPRT was found to be increased. The flow cytometry analysis revealed that morin treatment decreased intracellular reactive oxygen species levels in MSU crystal stimulated macrophage cells. The western blot analysis clearly showed that morin mainly exerts its anti-inflammatory effects by inhibiting the MSU crystal-induced COX-2 and TNF-α protein expression through the inactivation of NF-κB signaling pathway in RAW 264.7 macrophage cells similar to that of BAY 11-7082 (IκB kinase inhibitor. Our results collectively suggest that morin can be a potential therapeutic agent for inflammatory disorders like acute gouty arthritis.

  14. Differential Role of Rapamycin and Torin/KU63794 in Inflammatory Response of 264.7 RAW Macrophages Stimulated by CA-MRSA

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    Rebekah K. H. Shappley

    2014-01-01

    Full Text Available Background. Rapamycin suppresses the RAW264.7 macrophage mediated inflammatory response but in lower doses induces it. In the present study, we tested the suppression of the inflammatory response in the presence of mTOR 1 and 2 inhibitors, Torin and KU63794. Methods. RAW264.7 cells were stimulated for 18 hrs with 106 to 107 CFU/mL inocula of community-acquired- (CA- MRSA isolate, USA400 strain MW2, in the presence of Vancomycin. Then, in sequential experiments, we added Torin, KU63794, and Rapamycin alone and in various combinations. Supernatants were collected and assayed for TNF, IL-1, IL-6, INF, and NO. Results. Rapamycin induces 10–20% of the inflammatory cascade at dose of 0.1 ng/mL and suppresses it by 60% at dose of 10 ng/mL. The induction is abolished in the presence of Torin KU63794. Torin and KU63794 are consistently suppressing cytokine production 50–60%. Conclusions. There is a differential response between Rapamycin (mTOR-1 inhibitor and Torin KU63794 (mTOR 1 and 2 inhibitors. Torin and KU63794 exhibit a dose related suppression. Rapamycin exhibits a significant induction-suppression biphasic response. Knowledge of such response may allow manipulation of the septic inflammatory cascade for clinical advantages.

  15. Macrophages in cardiac homeostasis, injury responses and progenitor cell mobilisation

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    Alexander R. Pinto

    2014-11-01

    Full Text Available Macrophages are an immune cell type found in every organ of the body. Classically, macrophages are recognised as housekeeping cells involved in the detection of foreign antigens and danger signatures, and the clearance of tissue debris. However, macrophages are increasingly recognised as a highly versatile cell type with a diverse range of functions that are important for tissue homeostasis and injury responses. Recent research findings suggest that macrophages contribute to tissue regeneration and may play a role in the activation and mobilisation of stem cells. This review describes recent advances in our understanding of the role played by macrophages in cardiac tissue maintenance and repair following injury. We examine the involvement of exogenous and resident tissue macrophages in cardiac inflammatory responses and their potential activity in regulating cardiac regeneration.

  16. Immunostimulatory activity of polysaccharides isolated from Caulerpa lentillifera on macrophage cells.

    Science.gov (United States)

    Maeda, Reiko; Ida, Tomoaki; Ihara, Hideshi; Sakamoto, Tatsuji

    2012-01-01

    Polysaccharides were extracted from Caulerpa lentillifera by treating with water and then purified by size-exclusion chromatography. The purified polysaccharides, termed SP1, were found to be sulfated xylogalactans with a molecular mass of more than 100 kDa. Adding SP1 to murine macrophage RAW 264.7 cells increased the production of nitric oxide (NO) in a dose-dependent manner. NO was found by immunoblotting and RT-PCR analyses to be synthesized by an inducible NO synthase. SP1 caused the degradation of IκB-α and the nuclear translocation of nuclear factor (NF)-κB subunit p65 in macrophage cells. SP1 also increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK). These results demonstrate that SP1 activated macrophage cells via both the NF-κB and p38 MAPK signaling pathways. Moreover, SP1 increased the expression of various genes encoding cytokines, and the phagocytic activity of macrophage cells. These combined results show that SP1 immunostimulated the activity of macrophage cells.

  17. An extract of Phellinus linteus grown on germinated brown rice inhibits inflammation markers in RAW264.7 macrophages by suppressing inflammatory cytokines, chemokines, and mediators and up-regulating antioxidant activity.

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    Park, Hye-Jin; Han, Eun Su; Park, Dong Ki; Lee, Chan; Lee, Ki Won

    2010-12-01

    The immunomodulatory activity of an organic extract of Phellinus linteus grown on slightly germinated brown rice (PBR) was previously demonstrated. Here, we investigated the possible anti-inflammatory activity of the PBR extract by analyzing its effect on the expression of macrophage-derived cytokines, chemokines, and mediator genes that participate in immune and inflammatory responses and diseases. The extract profoundly inhibited the induction of cytokines and chemokines, including tumor necrosis factor-α, chemokine (C-X-C motif) ligand-10, granulocyte-macrophage colony-stimulating factor, and interleukin-6, in lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage cells. It also greatly inhibited LPS-stimulated production of nitric oxide (NO) and prostaglandin E(2) in RAW264.7 cells by suppressing the expression of inducible NO synthase and cyclooxygenase-2. PBR extract inhibited NO production with a twofold lower half-maximal inhibitory concentration value than P. linteus extract. To elucidate the underlying mechanism of action, we examined the effect of the PBR extract on the LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs) in RAW264.7 cells. PBR extract greatly inhibited extracellular signal-regulated kinase and c-Jun N-terminal kinase phosphorylation and slightly inhibited p38 MAPK phosphorylation. It also significantly increased intracellular glutathione peroxidase activity and heme oxygenase-1 protein expression. Thus, the PBR extract has anti-inflammatory activity in LPS-stimulated RAW264.7 cells by virtue of its ability to suppress the production of inflammatory cytokines and chemokines via inhibition of MAPK activation and up-regulation of antioxidant activities.

  18. 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.

  19. Beryllium-stimulated apoptosis in macrophage cell lines.

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    Sawyer, R T; Fadok, V A; Kittle, L A; Maier, L A; Newman, L S

    2000-08-21

    In vitro stimulation of bronchoalveolar lavage cells from patients with chronic beryllium disease (CBD) induces the production of TNF-alpha. We tested the hypothesis that beryllium (Be)-stimulated TNF-alpha might induce apoptosis in mouse and human macrophage cell lines. These cell lines were selected because they produce a range of Be-stimulated TNF-alpha. The mouse macrophage cell line H36.12j produces high levels of Be-stimulated TNF-alpha. The mouse macrophage cell line P388D.1 produces low, constitutive, levels of TNF-alpha and does not up-regulate Be-stimulated TNF-alpha production. The DEOHS-1 human CBD macrophage cell line does not produce constitutive or Be-stimulated TNF-alpha. Apoptosis was determined by microscopic observation of propidium iodide stained fragmented nuclei in unstimulated and BeSO(4)-stimulated macrophage cell lines. BeSO(4) induced apoptosis in all macrophage cell lines tested. Beryllium-stimulated apoptosis was dose-responsive and maximal after 24 h of exposure to 100 microM BeSO(4). In contrast, unstimulated and Al(2)(SO(4))(3)-stimulated macrophage cell lines did not undergo apoptosis. The general caspase inhibitor BD-fmk inhibited Be-stimulated macrophage cell line apoptosis at concentrations above 50 microM. Our data show that Be-stimulated macrophage cell line apoptosis was caspase-dependent and not solely dependent on Be-stimulated TNF-alpha levels. We speculate that the release of Be-antigen from apoptotic macrophages may serve to re-introduce Be material back into the lung microenvironment, make it available for uptake by new macrophages, and thereby amplify Be-stimulated cytokine production, promoting ongoing inflammation and granuloma maintenance in CBD.

  20. Inositol hexakisphosphate inhibits osteoclastogenesis on RAW 264.7 cells and human primary osteoclasts.

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    Arriero, María del Mar; Ramis, Joana M; Perelló, Joan; Monjo, Marta

    2012-01-01

    Inoxitol hexakisphosphate (IP6) has been found to have an important role in biomineralization and a direct effect inhibiting mineralization of osteoblasts in vitro without impairing extracellular matrix production and expression of alkaline phosphatase. IP6 has been proposed to exhibit similar effects to those of bisphosphonates on bone resorption, however, its direct effect on osteoclasts (OCL) is presently unknown. The aim of the present study was to investigate the effect of IP6 on the RAW 264.7 monocyte/macrophage mouse cell line and on human primary osteoclasts. On one hand, we show that IP6 decreases the osteoclastogenesis in RAW 264.7 cells induced by RANKL, without affecting cell proliferation or cell viability. The number of TRAP positive cells and mRNA levels of osteoclast markers such as TRAP, calcitonin receptor, cathepsin K and MMP-9 was decreased by IP6 on RANKL-treated cells. On the contrary, when giving IP6 to mature osteoclasts after RANKL treatment, a significant increase of bone resorption activity and TRAP mRNA levels was found. On the other hand, we show that 1 µM of IP6 inhibits osteoclastogenesis of human peripheral blood mononuclear cells (PBMNC) and their resorption activity both, when given to undifferentiated and to mature osteoclasts. Our results demonstrate that IP6 inhibits osteoclastogenesis on human PBMNC and on the RAW264.7 cell line. Thus, IP6 may represent a novel type of selective inhibitor of osteoclasts and prove useful for the treatment of osteoporosis.

  1. Inositol hexakisphosphate inhibits osteoclastogenesis on RAW 264.7 cells and human primary osteoclasts.

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    María del Mar Arriero

    Full Text Available BACKGROUND: Inoxitol hexakisphosphate (IP6 has been found to have an important role in biomineralization and a direct effect inhibiting mineralization of osteoblasts in vitro without impairing extracellular matrix production and expression of alkaline phosphatase. IP6 has been proposed to exhibit similar effects to those of bisphosphonates on bone resorption, however, its direct effect on osteoclasts (OCL is presently unknown. METHODOLOGY/PRINCIPAL FINDINGS: The aim of the present study was to investigate the effect of IP6 on the RAW 264.7 monocyte/macrophage mouse cell line and on human primary osteoclasts. On one hand, we show that IP6 decreases the osteoclastogenesis in RAW 264.7 cells induced by RANKL, without affecting cell proliferation or cell viability. The number of TRAP positive cells and mRNA levels of osteoclast markers such as TRAP, calcitonin receptor, cathepsin K and MMP-9 was decreased by IP6 on RANKL-treated cells. On the contrary, when giving IP6 to mature osteoclasts after RANKL treatment, a significant increase of bone resorption activity and TRAP mRNA levels was found. On the other hand, we show that 1 µM of IP6 inhibits osteoclastogenesis of human peripheral blood mononuclear cells (PBMNC and their resorption activity both, when given to undifferentiated and to mature osteoclasts. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that IP6 inhibits osteoclastogenesis on human PBMNC and on the RAW264.7 cell line. Thus, IP6 may represent a novel type of selective inhibitor of osteoclasts and prove useful for the treatment of osteoporosis.

  2. Selenium Supplementation of Amaranth Sprouts Influences Betacyanin Content and Improves Anti-Inflammatory Properties via NFκB in Murine RAW 264.7 Macrophages.

    Science.gov (United States)

    Tyszka-Czochara, Malgorzata; Pasko, Pawel; Zagrodzki, Pawel; Gajdzik, Ewelina; Wietecha-Posluszny, Renata; Gorinstein, Shela

    2016-02-01

    Sprouts contain potent compounds which while influencing crucial transduction pathways in cell reveal anti-inflammatory and anticancer activities. In this study, we report the biological activity for seeds and colourful sprouts of four types of edible amaranth, as amaranth has recently attracted interest due to its appreciable nutritional value. MTT assay conducted for the amaranth seeds and sprouts did not show any adverse effect on the viability of murine RAW 264.7 cells. As amaranth accumulates selenium, the sprouts were supplemented with this trace element (10 mg/L; 15 mg/L Se as sodium selenite) while growing. Selenium concentration in sprouts was observed to be significantly correlated with betacyanins content of the tested species. The amounts of Se and betacyanins in sprouts varied for various Amaranth species. In the present study, Amaranthus cruentus sprouts with the highest betacyanins (19.30 ± 0.57-28.85 ± 2.23 mg of amaranthin/100 g of fresh weight) and high total selenium (22.51 ± 1.57-1044.75 ± 73.08 μg/L in methanol extracts) content prevented NFκB translocation to the cell nucleus and subsequently exerted an anti-inflammatory effect by significant decreasing inflammatory interleukin 6 production (587.3 ± 34.2-710.0 ± 88.1 pg/mL) in the cell culture of activated RAW 264.7 macrophages (vs LPS control 1520 ± 114 pg/mL).

  3. Immunomodulatory Efficacy of Standardized Annona muricata (Graviola) Leaf Extract via Activation of Mitogen-Activated Protein Kinase Pathways in RAW 264.7 Macrophages.

    Science.gov (United States)

    Kim, Goon-Tae; Tran, Nguyen Khoi Song; Choi, Eun-Hye; Song, Yoo-Jeong; Song, Jae-Hwi; Shim, Soon-Mi; Park, Tae-Sik

    2016-01-01

    Annona muricata, commonly known as Graviola, has been utilized as a traditional medicine to treat various human diseases. The aim of this study was to examine the immune-enhancing activity of Graviola leaf extracts in RAW 264.7 macrophage cells. Active ingredients in Graviola leaf extracts (GE) were identified as kaempferol-3-O-rutinoside and quercetin-3-O-rutinoside by LC-MS/MS. When treated with steam or 50% ethanol GE, cell morphology was altered due to initiation of cell differentiation. While the cell viability was not altered by the steam GE, it was reduced by the ethanol GE. Both steam and ethanol GE induced the transcriptional expression of cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1β, but only the steam extract upregulated inducible nitric oxide synthase (iNOS). In consistence with mRNA expression, the production of TNF-α and nitrite was elevated by both steam and ethanol extracts of Graviola leaves. This is mainly due to activation of mitogen-activated protein (MAP) kinase signaling pathways. These results suggest that Graviola leaves enhance immunity by activation of the MAP kinase pathways. These bioactive properties of Graviola indicate its potential as a health-promoting ingredient to boost the immune system.

  4. Immunomodulatory Efficacy of Standardized Annona muricata (Graviola Leaf Extract via Activation of Mitogen-Activated Protein Kinase Pathways in RAW 264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Goon-Tae Kim

    2016-01-01

    Full Text Available Annona muricata, commonly known as Graviola, has been utilized as a traditional medicine to treat various human diseases. The aim of this study was to examine the immune-enhancing activity of Graviola leaf extracts in RAW 264.7 macrophage cells. Active ingredients in Graviola leaf extracts (GE were identified as kaempferol-3-O-rutinoside and quercetin-3-O-rutinoside by LC-MS/MS. When treated with steam or 50% ethanol GE, cell morphology was altered due to initiation of cell differentiation. While the cell viability was not altered by the steam GE, it was reduced by the ethanol GE. Both steam and ethanol GE induced the transcriptional expression of cytokines, including tumor necrosis factor-α (TNF-α and interleukin-1β, but only the steam extract upregulated inducible nitric oxide synthase (iNOS. In consistence with mRNA expression, the production of TNF-α and nitrite was elevated by both steam and ethanol extracts of Graviola leaves. This is mainly due to activation of mitogen-activated protein (MAP kinase signaling pathways. These results suggest that Graviola leaves enhance immunity by activation of the MAP kinase pathways. These bioactive properties of Graviola indicate its potential as a health-promoting ingredient to boost the immune system.

  5. Resident macrophages influence stem cell activity in the mammary gland

    NARCIS (Netherlands)

    Gyorki, D.E.; Asselin-Labat, M.L.; Rooijen, van N.; Lindeman, G.J.; Visvader, J.E.

    2009-01-01

    Introduction Macrophages in the mammary gland are essential for morphogenesis of the ductal epithelial tree and have been implicated in promoting breast tumor metastasis. Although it is well established that macrophages influence normal mammopoiesis, the mammary cell types that these accessory cells

  6. Efficient replication of pneumonia virus of mice (PVM in a mouse macrophage cell line

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    Martin Brittany V

    2007-06-01

    Full Text Available Abstract Pneumonia virus of mice (PVM; family Paramyxoviridae, subfamily Pneumovirinae is a natural respiratory pathogen of rodent species and an important new model for the study of severe viral bronchiolitis and pneumonia. However, despite high virus titers typically detected in infected mouse lung tissue in vivo, cell lines used routinely for virus propagation in vitro are not highly susceptible to PVM infection. We have evaluated several rodent and primate cell lines for susceptibility to PVM infection, and detected highest virus titers from infection of the mouse monocyte-macrophage RAW 264.7 cell line. Additionally, virus replication in RAW 264.7 cells induces the synthesis and secretion of proinflammatory cytokines relevant to respiratory virus disease, including tumor necrosis factor-α (TNF-α, interferon-β (IFN-β, macrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β and the functional homolog of human IL-8, mouse macrophage inflammatory peptide-2 (MIP-2. Identification and characterization of a rodent cell line that supports the replication of PVM and induces the synthesis of disease-related proinflammatory mediators will facilitate studies of molecular mechanisms of viral pathogenesis that will complement and expand on findings from mouse model systems.

  7. Novel interactions between erythroblast macrophage protein and cell migration.

    Science.gov (United States)

    Javan, Gulnaz T; Can, Ismail; Yeboah, Fred; Lee, Youngil; Soni, Shivani

    2016-09-01

    Erythroblast macrophage protein is a novel protein known to mediate attachment of erythroid cells to macrophages to form erythroblastic islands in bone marrow during erythropoiesis. Emp-null macrophages are small with round morphologies, and lack cytoplasmic projections which imply immature structure. The role of Emp in macrophage development and function is not fully elucidated. Macrophages perform varied functions (e.g. homeostasis, erythropoiesis), and are implicated in numerous pathophysiological conditions such as cellular malignancy. The objective of the current study is to investigate the interaction of Emp with cytoskeletal- and cell migration-associated proteins involved in macrophage functions. A short hairpin RNA lentiviral system was use to down-regulate the expression of Emp in macrophage cells. A cell migration assay revealed that the relocation of macrophages was significantly inhibited when Emp expression was decreased. To further analyze changes in gene expression related to cell motility, PCR array was performed by down-regulating Emp expression. The results indicated that expression of mitogen-activated protein kinase 1 and thymoma viral proto-oncogene 1 were significantly higher when Emp was down-regulated. The results implicate Emp in abnormal cell motility, thus, warrants to assess its role in cancer where tumor cell motility is required for invasion and metastasis.

  8. 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.

  9. Effects of Soft Denture Liners on L929 Fibroblasts, HaCaT Keratinocytes, and RAW 264.7 Macrophages

    Science.gov (United States)

    Chaves, Carolina de Andrade Lima; de Souza Costa, Carlos Alberto; Vergani, Carlos Eduardo; Chaves de Souza, Pedro Paulo; Machado, Ana Lucia

    2014-01-01

    The effects of six soft liners (Ufi Gel P (UG), Sofreliner S (SR), Durabase Soft (D), Trusoft (T), Coe Comfort (CC), and Softone (ST)) on L929, HaCat, and RAW 264.7 cells were investigated. Eluates (24 and 48 h) from the materials were applied on the cells and the viability, type of cell death, and morphology were evaluated. Cells were also seeded on the specimens' surfaces (direct contact) and incubated (24 or 48 h), and viability was analyzed. Controls were cells in culture medium without eluates or specimens. For cell viability, no significant differences were found among materials or between extraction periods, and the liners were noncytotoxic or slightly cytotoxic. Morphology of RAW 264.7 cells was altered by the 24 h eluates from CC and D and the 48 h eluates from SR, CC, and D. The 24 and 48 h eluates from all materials (except T) increased the percentages of L929 necrotic cells. For direct contact tests, the lowest cytotoxicity was observed for UG and SR. Although eluates did not reduce viability, morphology alterations and increase in necrosis were seen. Moreover, in the direct contact, effects on viability were more pronounced, particularly for D, T, CC and ST. Thus, the use of UG and SR might reduce the risk of adverse effects. PMID:25295276

  10. Muscle cells challenged with saturated fatty acids mount an autonomous inflammatory response that activates macrophages

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    Pillon Nicolas J

    2012-10-01

    Full Text Available Abstract Obesity is associated with chronic low-grade inflammation. Within adipose tissue of mice fed a high fat diet, resident and infiltrating macrophages assume a pro-inflammatory phenotype characterized by the production of cytokines which in turn impact on the surrounding tissue. However, inflammation is not restricted to adipose tissue and high fat-feeding is responsible for a significant increase in pro-inflammatory cytokine expression in muscle. Although skeletal muscle is the major disposer of dietary glucose and a major determinant of glycemia, the origin and consequence of muscle inflammation in the development of insulin resistance are poorly understood. We used a cell culture approach to investigate the vectorial crosstalk between muscle cells and macrophages upon exposure to physiological, low levels of saturated and unsaturated fatty acids. Inflammatory pathway activation and cytokine expression were analyzed in L6 muscle cells expressing myc-tagged GLUT4 (L6GLUT4myc exposed to 0.2 mM palmitate or palmitoleate. Conditioned media thereof, free of fatty acids, were then tested for their ability to activate RAW264.7 macrophages. Palmitate -but not palmitoleate- induced IL-6, TNFα and CCL2 expression in muscle cells, through activation of the NF-κB pathway. Palmitate (0.2 mM alone did not induce insulin resistance in muscle cells, yet conditioned media from palmitate-challenged muscle cells selectively activated macrophages towards a pro-inflammatory phenotype. These results demonstrate that low concentrations of palmitate activate autonomous inflammation in muscle cells to release factors that turn macrophages pro-inflammatory. We hypothesize that saturated fat-induced, low-grade muscle cell inflammation may trigger resident skeletal muscle macrophage polarization, possibly contributing to insulin resistance in vivo.

  11. Interaction of apoptotic cells with macrophages upregulates COX-2/PGE2 and HGF expression via a positive feedback loop.

    Science.gov (United States)

    Byun, Ji Yeon; Youn, Young-So; Lee, Ye-Ji; Choi, Youn-Hee; Woo, So-Yeon; Kang, Jihee Lee

    2014-01-01

    Recognition of apoptotic cells by macrophages is crucial for resolution of inflammation, immune tolerance, and tissue repair. Cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) and hepatocyte growth factor (HGF) play important roles in the tissue repair process. We investigated the characteristics of macrophage COX-2 and PGE2 expression mediated by apoptotic cells and then determined how macrophages exposed to apoptotic cells in vitro and in vivo orchestrate the interaction between COX-2/PGE2 and HGF signaling pathways. Exposure of RAW 264.7 cells and primary peritoneal macrophages to apoptotic cells resulted in induction of COX-2 and PGE2. The COX-2 inhibitor NS-398 suppressed apoptotic cell-induced PGE2 production. Both NS-398 and COX-2-siRNA, as well as the PGE2 receptor EP2 antagonist, blocked HGF expression in response to apoptotic cells. In addition, the HGF receptor antagonist suppressed increases in COX-2 and PGE2 induction. The in vivo relevance of the interaction between the COX-2/PGE2 and HGF pathways through a positive feedback loop was shown in cultured alveolar macrophages following in vivo exposure of bleomycin-stimulated lungs to apoptotic cells. Our results demonstrate that upregulation of the COX-2/PGE2 and HGF in macrophages following exposure to apoptotic cells represents a mechanism for mediating the anti-inflammatory and antifibrotic consequences of apoptotic cell recognition.

  12. Interaction of Apoptotic Cells with Macrophages Upregulates COX-2/PGE2 and HGF Expression via a Positive Feedback Loop

    Directory of Open Access Journals (Sweden)

    Ji Yeon Byun

    2014-01-01

    Full Text Available Recognition of apoptotic cells by macrophages is crucial for resolution of inflammation, immune tolerance, and tissue repair. Cyclooxygenase-2 (COX-2/prostaglandin E2 (PGE2 and hepatocyte growth factor (HGF play important roles in the tissue repair process. We investigated the characteristics of macrophage COX-2 and PGE2 expression mediated by apoptotic cells and then determined how macrophages exposed to apoptotic cells in vitro and in vivo orchestrate the interaction between COX-2/PGE2 and HGF signaling pathways. Exposure of RAW 264.7 cells and primary peritoneal macrophages to apoptotic cells resulted in induction of COX-2 and PGE2. The COX-2 inhibitor NS-398 suppressed apoptotic cell-induced PGE2 production. Both NS-398 and COX-2-siRNA, as well as the PGE2 receptor EP2 antagonist, blocked HGF expression in response to apoptotic cells. In addition, the HGF receptor antagonist suppressed increases in COX-2 and PGE2 induction. The in vivo relevance of the interaction between the COX-2/PGE2 and HGF pathways through a positive feedback loop was shown in cultured alveolar macrophages following in vivo exposure of bleomycin-stimulated lungs to apoptotic cells. Our results demonstrate that upregulation of the COX-2/PGE2 and HGF in macrophages following exposure to apoptotic cells represents a mechanism for mediating the anti-inflammatory and antifibrotic consequences of apoptotic cell recognition.

  13. Cannabinoid CB2 Receptors are Involved in the Protection of RAW264.7 Macrophages Against the Oxidative Stress: An in Vitro Study

    Science.gov (United States)

    Giacoppo, Sabrina; Gugliandolo, Agnese; Trubiani, Oriana; Pollastro, Federica; Grassi, Gianpaolo; Bramanti, Placido; Mazzon, Emanuela

    2017-01-01

    Research in the last decades has widely investigated the anti-oxidant properties of natural products as a therapeutic approach for the prevention and the treatment of oxidative-stress related disorders. In this context, several studies were aimed to evaluate the therapeutic potential of phytocannabinoids, the bioactive compounds of Cannabis sativa. Here, we examined the anti-oxidant ability of Cannabigerol (CBG), a non-psychotropic cannabinoid, still little known, into counteracting the hydrogen peroxide (H2O2)-induced oxidative stress in murine RAW264.7 macrophages. In addition, we tested selective receptor antagonists for cannabinoid receptors and specifically CB1R (SR141716A) and CB2R (AM630) in order to investigate through which CBG may exert its action. Taken together, our in vitro results showed that CBG is able to counteract oxidative stress by activation of CB2 receptors. CB2 antagonist pre-treatment indeed blocked the protective effects of CBG in H2O2 stimulated macrophages, while CB1R was not involved. Specifically, CBG exhibited a potent action in inhibiting oxidative stress, by down-regulation of the main oxidative markers (iNOS, nitrotyrosine and PARP-1), by preventing IκB-α phosphorylation and translocation of the nuclear factor-κB (NF-κB) and also via the modulation of MAP kinases pathway. On the other hand, CBG was found to increase anti-oxidant defense of cells by modulating superoxide dismutase-1 (SOD-1) expression and thus inhibiting cell death (results focused on balance between Bax and Bcl-2). Based on its anti-oxidant activities, CBG may hold great promise as an anti-oxidant agent and therefore used in clinical practice as a new approach in oxidative-stress related disorders.

  14. Cannabinoid CB2 receptors are involved in the protection of RAW264.7 macrophages against the oxidative stress: an in vitro study

    Directory of Open Access Journals (Sweden)

    Sabrina Giacoppo

    2017-01-01

    Full Text Available Research in the last decades has widely investigated the anti-oxidant properties of natural products as a therapeutic approach for the prevention and the treatment of oxidative-stress related disorders. In this context, several studies were aimed to evaluate the therapeutic potential of phytocannabinoids, the bioactive compounds of Cannabis sativa. Here, we examined the anti-oxidant ability of Cannabigerol (CBG, a non-psychotropic cannabinoid, still little known, into counteracting the hydrogen peroxide (H2O2-induced oxidative stress in murine RAW264.7 macrophages. In addition, we tested selective receptor antagonists for cannabinoid receptors and specifically CB1R (SR141716A and CB2R (AM630 in order to investigate through which CBG may exert its action. Taken together, our in vitro results showed that CBG is able to counteract oxidative stress by activation of CB2 receptors. CB2 antagonist pre-treatment indeed blocked the protective effects of CBG in H2O2 stimulated macrophages, while CB1R was not involved. Specifically, CBG exhibited a potent action in inhibiting oxidative stress, by down-regulation of the main oxidative markers (iNOS, nitrotyrosine and PARP-1, by preventing IκB-α phosphorylation and translocation of the nuclear factor-κB (NF-κB and also via the modulation of MAP kinases pathway. On the other hand, CBG was found to increase anti-oxidant defense of cells by modulating superoxide dismutase-1 (SOD-1 expression and thus inhibiting cell death (results focused on balance between Bax and Bcl-2. Based on its antioxidant activities, CBG may hold great promise as an anti-oxidant agent and therefore used in clinical practice as a new approach in oxidative-stress related disorders.

  15. Inhibitory effects of 4-hydroxyderricin and xanthoangelol on lipopolysaccharide-induced inflammatory responses in RAW264 macrophages.

    Science.gov (United States)

    Yasuda, Michiko; Kawabata, Kyuichi; Miyashita, Miki; Okumura, Mayu; Yamamoto, Norio; Takahashi, Masakazu; Ashida, Hitoshi; Ohigashi, Hajime

    2014-01-15

    The Japanese herb, Ashitaba (Angelica keiskei Koidzumi), contains two prenylated chalcones, 4-hydroxyderricin and xanthoangelol, which are considered to be the major active compounds of Ashitaba. However, their effects on inflammatory responses are poorly understood. In the present study, we investigated the effects and underlying molecular mechanisms of 4-hydroxyderricin and xanthoangelol on lipopolysaccharide (LPS)-induced inflammatory responses in RAW264 mouse macrophages. LPS-mediated production of nitric oxide (NO) was markedly reduced by 4-hydroxyderricin (10 μM) and xanthoangelol (5 μM) compared with their parent compound, chalcone (25 μM). They also inhibited LPS-induced secretion of tumor necrosis factor-alpha (TNF-α) and expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Although chalcone decreased the DNA-binding activity of both activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB), 4-hydroxyderricin and xanthoangelol suppressed only AP-1 and had no effect on NF-κB. On the other hand, all of the tested chalcones reduced the phosphorylation (at serine 536) level of the p65 subunit of NF-κB. 4-Hydroxyderricin and xanthoangelol may be promising for the prevention of inflammatory diseases.

  16. Entrance and Survival of Brucella pinnipedialis Hooded Seal Strain in Human Macrophages and Epithelial Cells

    Science.gov (United States)

    Briquemont, Benjamin; Sørensen, Karen K.; Godfroid, Jacques

    2013-01-01

    Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis) and cetaceans (B. ceti) from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis strains (reference strain; NTCT 12890 and a hooded seal isolate; B17) by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1), two murine macrophage cell lines (RAW264.7 and J774A.1), and a human malignant epithelial cell line (HeLa S3) were challenged with bacteria in a gentamicin protection assay. Our results show that B. pinnipedialis is internalized, but is then gradually eliminated during the next 72 – 96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by B. pinnipedialis do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary. PMID:24376851

  17. Entrance and survival of Brucella pinnipedialis hooded seal strain in human macrophages and epithelial cells.

    Directory of Open Access Journals (Sweden)

    Anett K Larsen

    Full Text Available Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis and cetaceans (B. ceti from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis strains (reference strain; NTCT 12890 and a hooded seal isolate; B17 by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1, two murine macrophage cell lines (RAW264.7 and J774A.1, and a human malignant epithelial cell line (HeLa S3 were challenged with bacteria in a gentamicin protection assay. Our results show that B. pinnipedialis is internalized, but is then gradually eliminated during the next 72-96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3, suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by B. pinnipedialis do not release nitric oxide (NO and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary.

  18. Of macrophages and red blood cells; a complex love story.

    Directory of Open Access Journals (Sweden)

    Djuna Zoe de Back

    2014-01-01

    Full Text Available Macrophages tightly control the production and clearance of red blood cells (RBC. During steady state haematopoiesis, approximately 1010 red blood cells are produced per hour within erythroblastic islands in humans. In these erythroblastic islands, resident bone marrow macrophages provide erythroblasts with interactions that are essential for erythroid development. New evidence suggests that not only under homeostasis but also under stress conditions, macrophages play an important role in promoting erythropoiesis. Once RBC have matured, these cells remain in circulation for about 120 days. At the end of their life span, RBC are cleared by macrophages residing in the spleen and the liver. Current theories about the removal of senescent RBC and the essential role of macrophages will be discussed as well as the role of macrophages in facilitating the removal of damaged cellular content from the RBC. In this review we will provide an overview on the role of macrophages in the regulation of RBC production, maintenance and clearance. In addition, we will discuss the interactions between these two cell types during transfer of immune complexes and pathogens from RBC to macrophages.

  19. Stylopine from Chelidonium majus inhibits LPS-induced inflammatory mediators in RAW 264.7 cells.

    Science.gov (United States)

    Jang, Seon Il; Kim, Byung Hee; Lee, Woo-Yiel; An, Sang Jin; Choi, Han Gil; Jeon, Byung Hun; Chung, Hun-Taeg; Rho, Jung-Rae; Kim, Young-Jun; Chai, Kyu-Yun

    2004-09-01

    Stylopine is a major component of the leaf of Chelidonium majus L. (Papaveraceae), which has been used for the removal of warts, papillomas and condylomas, as well as the treatment of liver disease, in oriental countries. Stylopine per se had no cytotoxic effect in unstimulated RAW 264.7 cells, but concentration-dependently reduced nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), and the IL-6 production and cyclooxygenase-2 (COX-2) activity caused by the LPS stimulation. The levels of inducible nitric oxide synthase (iNOS) and COX-2 protein expressions were markedly suppressed by stylopine in a concentration dependent manner. These results suggest that stylopine suppress the NO and PGE2 production in macrophages by inhibiting the iNOS and COX-2 expressions. These biological activities of stylopine may contribute to the anti-inflammatory activity of Chelidonium majus.

  20. 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.

  1. Unique proteomic signatures distinguish macrophages and dendritic cells.

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    Lev Becker

    Full Text Available Monocytes differentiate into heterogeneous populations of tissue macrophages and dendritic cells (DCs that regulate inflammation and immunity. Identifying specific populations of myeloid cells in vivo is problematic, however, because only a limited number of proteins have been used to assign cellular phenotype. Using mass spectrometry and bone marrow-derived cells, we provided a global view of the proteomes of M-CSF-derived macrophages, classically and alternatively activated macrophages, and GM-CSF-derived DCs. Remarkably, the expression levels of half the plasma membrane proteins differed significantly in the various populations of cells derived in vitro. Moreover, the membrane proteomes of macrophages and DCs were more distinct than those of classically and alternatively activated macrophages. Hierarchical cluster and dual statistical analyses demonstrated that each cell type exhibited a robust proteomic signature that was unique. To interrogate the phenotype of myeloid cells in vivo, we subjected elicited peritoneal macrophages harvested from wild-type and GM-CSF-deficient mice to mass spectrometric and functional analysis. Unexpectedly, we found that peritoneal macrophages exhibited many features of the DCs generated in vitro. These findings demonstrate that global analysis of the membrane proteome can help define immune cell phenotypes in vivo.

  2. Moringa oleifera Flower Extract Suppresses the Activation of Inflammatory Mediators in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages via NF-κB Pathway

    Directory of Open Access Journals (Sweden)

    Woan Sean Tan

    2015-01-01

    Full Text Available Aim of Study. Moringa oleifera Lam. (M. oleifera possess highest concentration of antioxidant bioactive compounds and is anticipated to be used as an alternative medicine for inflammation. In the present study, we investigated the anti-inflammatory activity of 80% hydroethanolic extract of M. oleifera flower on proinflammatory mediators and cytokines produced in lipopolysaccharide- (LPS- induced RAW 264.7 macrophages. Materials and Methods. Cell cytotoxicity was conducted by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. Nitric oxide (NO production was quantified through Griess reaction while proinflammatory cytokines and other key inflammatory markers were assessed through enzyme-linked immunosorbent assay (ELISA and immunoblotting. Results. Hydroethanolic extract of M. oleifera flower significantly suppressed the secretion and expression of NO, prostaglandin E2 (PGE2, interleukin- (IL- 6, IL-1β, tumor necrosis factor-alpha (TNF-α, nuclear factor-kappa B (NF-κB, inducible NO synthase (iNOS, and cyclooxygenase-2 (COX-2. However, it significantly increased the production of IL-10 and IκB-α (inhibitor of κB in a concentration dependent manner (100 μg/mL and 200 μg/mL. Conclusion. These results suggest that 80% hydroethanolic extract of M. oleifera flower has anti-inflammatory action related to its inhibition of NO, PGE2, proinflammatory cytokines, and inflammatory mediator’s production in LPS-stimulated macrophages through preventing degradation of IκB-α in NF-κB signaling pathway.

  3. Pro-inflammatory responses of RAW264.7 macrophages when treated with ultralow concentrations of silver, titanium dioxide, and zinc oxide nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Giovanni, Marcella [Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585 (Singapore); Yue, Junqi; Zhang, Lifeng [PUB, 40 Scotts Road, Singapore 228231 (Singapore); Xie, Jianping [Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585 (Singapore); Ong, Choon Nam [Saw Swee Hock School of Public Health, National University of Singapore, 12 Science Drive 2, Singapore 117549 (Singapore); NUS Environmental Research Institute, National University of Singapore, 5A Engineering Drive 1, Singapore 117411 (Singapore); Leong, David Tai, E-mail: cheltwd@nus.edu.sg [Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585 (Singapore)

    2015-10-30

    Highlights: • Ultralow levels of common nanoparticles exist in environment and consumer products. • Common nanoparticles at ultralow levels induce mild pro-inflammation by macrophages. • The nanoparticles are cytotoxic only at high doses. - Abstract: To cellular systems, nanoparticles are considered as foreign particles. Upon particles and cells contact, innate immune system responds by activating the inflammatory pathway. However, excessive inflammation had been linked to various diseases ranging from allergic responses to cancer. Common nanoparticles, namely silver, titanium dioxide, and zinc oxide exist in the environment as well as in consumer products at ultralow level of 10{sup −6}–10{sup −3} μg mL{sup −1}. However, so far the risks of such low NPs concentrations remain unexplored. Therefore, we attempted to screen the pro-inflammatory responses after ultralow concentration treatments of the three nanoparticles on RAW264.7 macrophages, which are a part of the immune system, at both cellular and gene levels. Even though cytotoxicity was only observed at nanoparticles concentrations as high as 10 μg mL{sup −1}, through the level of NF-κB and upregulation of pro-inflammatory genes, we observed activation of the induction of genes encoding pro-inflammatory cytokines starting already at 10{sup −7} μg mL{sup −1}. This calls for more thorough characterization of nanoparticles in the environment as well as in consumer products to ascertain the health and safety of the consumers and living systems in general.

  4. Moringa oleifera Flower Extract Suppresses the Activation of Inflammatory Mediators in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages via NF-κB Pathway

    Science.gov (United States)

    Tan, Woan Sean; Arulselvan, Palanisamy; Karthivashan, Govindarajan; Fakurazi, Sharida

    2015-01-01

    Aim of Study. Moringa oleifera Lam. (M. oleifera) possess highest concentration of antioxidant bioactive compounds and is anticipated to be used as an alternative medicine for inflammation. In the present study, we investigated the anti-inflammatory activity of 80% hydroethanolic extract of M. oleifera flower on proinflammatory mediators and cytokines produced in lipopolysaccharide- (LPS-) induced RAW 264.7 macrophages. Materials and Methods. Cell cytotoxicity was conducted by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide (NO) production was quantified through Griess reaction while proinflammatory cytokines and other key inflammatory markers were assessed through enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Results. Hydroethanolic extract of M. oleifera flower significantly suppressed the secretion and expression of NO, prostaglandin E2 (PGE2), interleukin- (IL-) 6, IL-1β, tumor necrosis factor-alpha (TNF-α), nuclear factor-kappa B (NF-κB), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2). However, it significantly increased the production of IL-10 and IκB-α (inhibitor of κB) in a concentration dependent manner (100 μg/mL and 200 μg/mL). Conclusion. These results suggest that 80% hydroethanolic extract of M. oleifera flower has anti-inflammatory action related to its inhibition of NO, PGE2, proinflammatory cytokines, and inflammatory mediator's production in LPS-stimulated macrophages through preventing degradation of IκB-α in NF-κB signaling pathway. PMID:26609199

  5. Berberine promotes the development of atherosclerosis and foam cell formation by inducing scavenger receptor A expression in macrophage

    Institute of Scientific and Technical Information of China (English)

    Ke Li; Wenqi Yao; Xiudan Zheng; Kan Liao

    2009-01-01

    Berberine is identified to lower the serum cholesterol level in human and hamster through the induction of low density lipoproteins (LDL) receptor in hepatic cells. To evaluate its potential in preventing atherosclerosis, the effect of berberine on atherosclerosis development in apolipoprotein E-deficient (apoE-/-) mice was investigated, in apoE-/-mice, berberine induced in vivo foam cell formation and promoted atherosclerosis development. The foam cell for-mation induced by berberine was also observed in mouse RAW264.7 cells, as well as in mouse and human primary macrophages. By inducing scavenger receptor A (SR-A) expression in macrophages, berberine increased the uptake of modified LDL (DiO-Ac-LDL). Berberine-induced SR-A expression was also observed in macrophage foam cells in vivo and in the cells at atherosclerotic lesion. Analysis in RAW264.7 cells indicated that berberine induced SR-A ex-pression by suppressing PTEN expression, which led to sustained Akt activation. Our results suggest that to evaluate the potential of a cholesterol-reducing compound in alleviating atherosclerosis, its effect on the cells involved in ath-erosclerosis development, such as macrophages, should also be considered. Promotion of foam cell formation could counter-balance the beneficial effect of lowering serum cholesterol.

  6. Dengue tropism for macrophages and dendritic cells : the host cell effect

    NARCIS (Netherlands)

    Flipse, Jacky; Torres, Silvia; Diosa-Toro, Mayra; van der Ende-Metselaar, Heidi; Herrera-Rodriguez, Jose; Urcuqui-Inchima, Silvio; Huckriede, Anke; Rodenhuis-Zybert, Izabela A; Smit, Jolanda M

    2016-01-01

    Dengue virus infects immune cells, including monocytes, macrophages and dendritic cells (DC). We compared virus infectivity in macrophages and DC, and found that the virus-origin determined the cell tropism of progeny virus. The highest efficiency of re-infection was seen for macrophage-derived deng

  7. Calmodulin Mediates DNA Repair Pathways Involving H2AX in Response to Low-Dose Radiation Exposure of RAW 264.7 Macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Smallwood, Heather S.; Lopez Ferrer, Daniel; Eberlein, P. Elis; Watson, David J.; Squier, Thomas C.

    2009-02-05

    Understanding the molecular mechanisms that modulate macrophage radioresistance is necessary for the development of effective radiation therapies, as tumor-associated macrophages promote both angiogenesis and matrix remodeling that, in turn, enhance metastasis. In this respect, we have identified a dose-dependent increase in the abundance of the calcium regulatory protein calmodulin (CaM) in RAW 264.7 macrophages upon irradiation. CaM overexpression results in increased macrophage survival following radiation exposure, acting to diminish the sensitivity to low-dose exposures. Increases in CaM abundance also result in an increase in the number of phosphorylated histone H2AX protein complexes associated with DNA repair following macrophage irradiation, with no change in the extent of double-stranded DNA damage. In comparison, when NFκB-dependent pathways are inhibited, through the expression of a dominant-negative IκB construct, there is no significant increase in phosphorylated H2AX upon irradiation. These results indicate that the molecular basis for the up-regulation of histone H2AX mediated DNA-repair pathways is not the result of nonspecific NFκB-dependent pathways or a specific threshold of DNA damage. Rather, increases in CaM abundance act to minimize the low-dose hypersensitivity to radiation to enhance macrophage radioresistance through processes that include the upregulation of DNA repair pathways involving histone protein H2AX phosphorylation.

  8. Nuclear DAMP complex-mediated RAGE-dependent macrophage cell death

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ruochan [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); Fu, Sha; Fan, Xue-Gong [Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); Lotze, Michael T.; Zeh, Herbert J. [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Tang, Daolin, E-mail: tangd2@upmc.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Kang, Rui, E-mail: kangr@upmc.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States)

    2015-03-13

    High mobility group box 1 (HMGB1), histone, and DNA are essential nuclear components involved in the regulation of chromosome structure and function. In addition to their nuclear function, these molecules act as damage-associated molecular patterns (DAMPs) alone or together when released extracellularly. The synergistic effect of these nuclear DNA-HMGB1-histone complexes as DAMP complexes (nDCs) on immune cells remains largely unexplored. Here, we demonstrate that nDCs limit survival of macrophages (e.g., RAW264.7 and peritoneal macrophages) but not cancer cells (e.g., HCT116, HepG2 and Hepa1-6). nDCs promote production of inflammatory tumor necrosis factor α (TNFα) release, triggering reactive oxygen species-dependent apoptosis and necrosis. Moreover, the receptor for advanced glycation end products (RAGE), but not toll-like receptor (TLR)-4 and TLR-2, was required for Akt-dependent TNFα release and subsequent cell death following treatment with nDCs. Genetic depletion of RAGE by RNAi, antioxidant N-Acetyl-L-cysteine, and TNFα neutralizing antibody significantly attenuated nDC-induced cell death. These findings provide evidence supporting novel signaling mechanisms linking nDCs and inflammation in macrophage cell death. - Highlights: • Nuclear DAMP complexes (nDCs) selectively induce cell death in macrophages, but not cancer cells. • TNFα-mediated oxidative stress is required for nDC-induced death. • RAGE-mediated Akt activation is required for nDC-induced TNFα release. • Blocking RAGE and TNFα inhibits nDC-induced macrophage cell death.

  9. Anti-Inflammatory Effect of Neoechinulin A from the Marine Fungus Eurotium sp. SF-5989 through the Suppression of NF-кB and p38 MAPK Pathways in Lipopolysaccharide-Stimulated RAW264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Kyoung-Su Kim

    2013-10-01

    Full Text Available In the course of a bioassay-guided study of metabolites from the marine fungus Eurotium sp. SF-5989, two diketopiperazine type indole alkaloids, neoechinulins A and B, were isolated. In this study, we investigated the anti-inflammatory effects of neoechinulins A (1 and B (2 on lipopolysaccharide (LPS-stimulated RAW264.7 macrophages. Neoechinulin A (1 markedly suppressed the production of nitric oxide (NO and prostaglandin E2 (PGE2 and the expression of inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2 in a dose dependent manner ranging from 12.5 µM to 100 µM without affecting the cell viability. On the other hand, neoechinulin B (2 affected the cell viability at 25 µM although the compound displayed similar inhibitory effect of NO production to neoechinulin A (1 at lower doses. Furthermore, neoechinulin A (1 decreased the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α and interleukin-1β (IL-1β. We also confirmed that neoechinulin A (1 blocked the activation of nuclear factor-kappaB (NF-κB in LPS-stimulated RAW264.7 macrophages by inhibiting the phosphorylation and degradation of inhibitor kappa B (IκB-α. Moreover, neoechinulin A (1 decreased p38 mitogen-activated protein kinase (MAPK phosphorylation. Therefore, these data showed that the anti-inflammatory effects of neoechinulin A (1 in LPS-stimulated RAW264.7 macrophages were due to the inhibition of the NF-κB and p38 MAPK pathways, suggesting that neoechinulin A (1 might be a potential therapeutic agent for the treatment of various inflammatory diseases.

  10. 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

  11. Neocryptotanshinone inhibits lipopolysaccharide-induced inflammation in RAW264.7 macrophages by suppression of NF-κB and iNOS signaling pathways

    Directory of Open Access Journals (Sweden)

    Chuanhong Wu

    2015-07-01

    Full Text Available Neocryptotanshinone (NCTS is a natural product isolated from traditional Chinese herb Salvia miltiorrhiza Bunge. In this study, we investigated its anti-inflammatory effects in lipopolysaccharide (LPS-stimulated mouse macrophage (RAW264.7 cells. MTT results showed that NCTS partly reversed LPS-induced cytotoxicity. Real-time PCR results showed that NCTS suppressed LPS-induced mRNA expression of inflammatory cytokines, including tumor necrosis factor α (TNFα, interleukin-6 (IL-6 and interleukin-1β (IL-1β. Moreover, NCTS could decrease LPS-induced nitric oxide (NO production. Western blotting results showed that NCTS could down-regulate LPS-induced expression of inducible nitric oxide synthase (iNOS, p-IκBα, p-IKKβ and p-NF-κB p65 without affecting cyclooxygenase-2 (COX-2. In addition, NCTS inhibited LPS-induced p-NF-κB p65 nuclear translocation. In conclusion, these data demonstrated that NCTS showed anti-inflammatory effect by suppression of NF-κB and iNOS signaling pathways.

  12. Zuonin B Inhibits Lipopolysaccharide-Induced Inflammation via Downregulation of the ERK1/2 and JNK Pathways in RAW264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Mee-Young Lee

    2012-01-01

    Full Text Available We investigated whether Zuonin B exerts immunological effects on RAW264.7 cells. Zuonin B, isolated from flower buds of Daphne genkwa, suppressed the levels of nitric oxide and prostaglandin E2, as well as proinflammatory cytokines, such as tumor necrosis factor-α and interleukin-(IL- 6, in lipopolysaccharide-stimulated macrophages. Moreover, the compound inhibited cyclooxygenase-2 and inducible nitric oxide synthase expression. Zuonin B attenuated NF-kappaB (NF-κB activation via suppressing proteolysis of inhibitor kappa B-alpha (IκB-α and p65 nuclear translocation as well as phosphorylation of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase. Additionally, IL-4 and IL-13 production in ConA-induced splenocytes was inhibited by Zuonin B. In conclusion, the anti-inflammatory effects of Zuonin B are attributable to the suppression of proinflammatory cytokines and mediators via blockage of NF-κB and AP-1 activation. Based on these findings, we propose that Zuonin B is potentially an effective functional chemical candidate for the prevention of inflammatory diseases.

  13. Commercial Honeybush (Cyclopia spp.) Tea Extract Inhibits Osteoclast Formation and Bone Resorption in RAW264.7 Murine Macrophages-An in vitro Study.

    Science.gov (United States)

    Visagie, Amcois; Kasonga, Abe; Deepak, Vishwa; Moosa, Shaakirah; Marais, Sumari; Kruger, Marlena C; Coetzee, Magdalena

    2015-10-28

    Honeybush tea, a sweet tasting caffeine-free tea that is indigenous to South Africa, is rich in bioactive compounds that may have beneficial health effects. Bone remodeling is a physiological process that involves the synthesis of bone matrix by osteoblasts and resorption of bone by osteoclasts. When resorption exceeds formation, bone remodeling can be disrupted resulting in bone diseases such as osteoporosis. Osteoclasts are multinucleated cells derived from hematopoietic precursors of monocytic lineage. These precursors fuse and differentiate into mature osteoclasts in the presence of receptor activator of NF-kB ligand (RANKL), produced by osteoblasts. In this study, the in vitro effects of an aqueous extract of fermented honeybush tea were examined on osteoclast formation and bone resorption in RAW264.7 murine macrophages. We found that commercial honeybush tea extract inhibited osteoclast formation and TRAP activity which was accompanied by reduced bone resorption and disruption of characteristic cytoskeletal elements of mature osteoclasts without cytotoxicity. Furthermore, honeybush tea extract decreased expression of key osteoclast specific genes, matrix metalloproteinase-9 (MMP-9), tartrate resistant acid phosphatase (TRAP) and cathepsin K. This study demonstrates for the first time that honeybush tea may have potential anti-osteoclastogenic effects and therefore should be further explored for its beneficial effects on bone.

  14. 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.

  15. Networked T cell death following macrophage infection by Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Stephen H-F Macdonald

    Full Text Available BACKGROUND: Depletion of T cells following infection by Mycobacterium tuberculosis (Mtb impairs disease resolution, and interferes with clinical test performance that relies on cell-mediated immunity. A number of mechanisms contribute to this T cell suppression, such as activation-induced death and trafficking of T cells out of the peripheral circulation and into the diseased lungs. The extent to which Mtb infection of human macrophages affects T cell viability however, is not well characterised. METHODOLOGY/PRINCIPAL FINDINGS: We found that lymphopenia (<1.5 × 10(9 cells/l was prevalent among culture-positive tuberculosis patients, and lymphocyte counts significantly improved post-therapy. We previously reported that Mtb-infected human macrophages resulted in death of infected and uninfected bystander macrophages. In the current study, we sought to examine the influence of infected human alveolar macrophages on T cells. We infected primary human alveolar macrophages (the primary host cell for Mtb or PMA-differentiated THP-1 cells with Mtb H37Ra, then prepared cell-free supernatants. The supernatants of Mtb-infected macrophages caused dose-dependent, caspase-dependent, T cell apoptosis. This toxic effect of infected macrophage secreted factors did not require TNF-α or Fas. The supernatant cytotoxic signal(s were heat-labile and greater than 50 kDa in molecular size. Although ESAT-6 was toxic to T cells, other Mtb-secreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures. Furthermore, supernatants from Mycobacterium bovis Bacille de Calmette et Guerin (BCG- infected macrophages also elicited T cell death suggesting that ESAT-6 itself, although cytotoxic, was not the principal mediator of T cell death in our system. CONCLUSIONS: Mtb-Infected macrophages secrete heat-labile factors that are toxic to T cells, and may contribute to the immunosuppression seen in tuberculosis as well as

  16. Effects of Phosvitin on the Growth and Pinocytosis of RAW246.7 Cell%卵黄高磷蛋白对RAW264.7生长及其吞饮作用的影响

    Institute of Scientific and Technical Information of China (English)

    王亮; 吴子健; 刘爱国; 李建颖

    2013-01-01

    The objective was to investigate the effects of phosvitin(PVS) on the growth and pinocytosis of RAW264.7 cell by MTT assay and neutral red. Exposure of RAW 264.7 cells to 0-100μg/mL of PVS significantly promoted their proliferation in the presence or absence of LPS. When cultured with PVS in the absence of LPS, RAW264.7 cells pinocytosis of neutral red was significantly improved;but in the presence of LPS this ability was inhibited and produced antagonism. PVS significantly inhibited LPS-induced macrophage activation according to the analysis of microscope. It is suggested that the impacts of PVS on the pinocytosis capacity of RAW264.7 cell shows a two-way adjustment.%  经MTT法和中性红吞饮法研究了卵黄高磷蛋白(PVS)对RAW264.7细胞生长及其吞饮作用的影响.研究结果表明,在0~100μg/mL浓度范围内,PVS(或与LPS共培养)可促进RAW264.7细胞增殖,PVS可提高RAW264.7细胞吞饮中性红的能力,但与LPS共作用时,却几乎未能使细胞的吞饮能力发生改变;细胞形态显微观察结果表明PVS明显抑制LPS诱导的巨噬细胞的活化.以上两方面结果表明PVS对RAW264.7巨噬细胞吞饮能力的影响呈双向调节作用.

  17. 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.

  18. Identification of cell surface receptors for murine macrophage inflammatory protein-1 alpha.

    Science.gov (United States)

    Oh, K O; Zhou, Z; Kim, K K; Samanta, H; Fraser, M; Kim, Y J; Broxmeyer, H E; Kwon, B S

    1991-11-01

    We have produced recombinant proteins for a cytokine, L2G25BP (macrophage inflammatory protein-1 alpha) (MIP-1 alpha). By using the recombinant protein (rMIP-1 alpha), receptors for MIP-1 alpha were identified on Con A-stimulated and unstimulated CTLL-R8, a T cell line, and LPS-stimulated RAW 264.7, a macrophage cell line. The 125I-rMIP-1 alpha binds to the receptor in a specific and saturable manner. Scatchard analysis indicated a single class of high affinity receptor, with a Kd of approximately 1.5 x 10(-9) M and approximately 1200 binding sites/Con A-stimulated CTLL-R8 cell and a Kd of 0.9 x 10(-9) M and approximately 380 binding sites/RAW 264.7 cell. 125I-rMIP-1 alpha binding was inhibited by unlabeled rMIP-1 alpha in a dose-dependent manner, but not by IL-1 alpha or IL-2. rMIP-1 alpha inhibited the proliferation of unstimulated CTLL-R8 cells. Rabbit anti-rMIP-1 alpha antibodies blocked the growth-inhibitory effect of the rMIP-1 alpha on CTLL-R8 cells.

  19. 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.

  20. 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.

  1. Molecular Weight-Dependent Immunostimulative Activity of Low Molecular Weight Chitosan via Regulating NF-κB and AP-1 Signaling Pathways in RAW264.7 Macrophages

    Science.gov (United States)

    Zheng, Bin; Wen, Zheng-Shun; Huang, Yun-Juan; Xia, Mei-Sheng; Xiang, Xing-Wei; Qu, You-Le

    2016-01-01

    Chitosan and its derivatives such as low molecular weight chitosans (LMWCs) have been found to possess many important biological properties, such as antioxidant and antitumor effects. In our previous study, LMWCs were found to elicit a strong immunomodulatory response in macrophages dependent on molecular weight. Herein we further investigated the molecular weight-dependent immunostimulative activity of LMWCs and elucidated its mechanism of action on RAW264.7 macrophages. LMWCs (3 kDa and 50 kDa of molecular weight) could significantly enhance the mRNA expression levels of COX-2, IL-10 and MCP-1 in a molecular weight and concentration-dependent manner. The results suggested that LMWCs elicited a significant immunomodulatory response, which was dependent on the dose and the molecular weight. Regarding the possible molecular mechanism of action, LMWCs promoted the expression of the genes of key molecules in NF-κB and AP-1 pathways, including IKKβ, TRAF6 and JNK1, and induced the phosphorylation of protein IKBα in RAW264.7 macrophage. Moreover, LMWCs increased nuclear translocation of p65 and activation of activator protein-1 (AP-1, C-Jun and C-Fos) in a molecular weight-dependent manner. Taken together, our findings suggested that LMWCs exert immunostimulative activity via activation of NF-κB and AP-1 pathways in RAW264.7 macrophages in a molecular weight-dependent manner and that 3 kDa LMWC shows great potential as a novel agent for the treatment of immune suppression diseases and in future vaccines. PMID:27657093

  2. Radiation effects and radioprotection by Thai medicinal plants in mouse macrophage cell line

    Institute of Scientific and Technical Information of China (English)

    Cheeraratana Cheeramakara; Kriyaporn Songmueng; Wanyarat Nakosiri; Montri Chairojana; Arag Vitittheeranon; Nopchai Suthisai; Nongnuch Jangsawang; Channarong Sanghiran; Apichart Nontprasert

    2009-01-01

    Objective:To investigate the effects of radiation on growth-arrested (GA)and micronucleus-production (MP) rates,and the radioprotective properties of Thai medicinal plants in mouse macrophage cell line RAW264.7 in vitro.Methods:Mouse macrophage cell line (RAW264.7)was cultured in vitro.Various radiation expo-sures,growth-arrested rate assay,micronucleus production assay,and radioprotection by Thai medicinal plants were performed.Results:The results showed that GA and MP rates for γ-rays and UV were dose-dependent. The 50%-affected dose of γand UV radiation for the GA rate was 10 Gy and 159 microwatt/cm2 for 0.5 sec-onds,respectively.After X-ray exposure,there was no apparent effect on RAW264.7 cells,even with a forty-fold human diagnostic dose.Two exposures to γradiation at 20 Gy resulted in a significantly higher MP rate than 20 Gy single exposure or control (P <0.05).The Thai medicinal plants (Kamin-chun capsules,Curcu-ma longa Linn;Hed lingeu,Ganoderma lucidum;Ya Pakking capsule,Murdannia loriformis)could not pre-vent cell damage,but epigallocatechin gallate and L-cysteine could provide protection from 2 Gy γ-ray expo-sure.Conclusion:γradiation caused chromosomal damage during cell division and UV caused cell death, while X-ray radiation was safe.The radioprotective effects of Thai medicinal plants,Kamin-chun,Hed lingeu, and Ya Pakking,could not prevent cell damage in this study.

  3. Chromene suppresses the activation of inflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 cells.

    Science.gov (United States)

    Heo, Soo-Jin; Jang, Jiyi; Ye, Bo-Ram; Kim, Min-Sun; Yoon, Weon-Jong; Oh, Chulhong; Kang, Do-Hyung; Lee, Ji-Hyeok; Kang, Min-Cheol; Jeon, You-Jin; Kang, Sung-Myung; Kim, Daekyung; Kim, Kil-Nam

    2014-05-01

    Inflammation is complex process involving a variety of immune cells that defend the body from harmful stimuli. However, pro-inflammatory cytokines and inflammatory mediators can also exacerbate diseases such as cancer. The aim of this study was to identify a natural effective remedy for inflammation. We isolated a functional algal chromene compound from Sargassum siliquastrum, named sargachromanol D (SD). We evaluated the anti-inflammatory effect of SD on lipopolysaccharide (LPS)-exposed RAW 264.7 cells by measuring cell viability, cytotoxicity, and production of inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. SD inhibited production of NO and PGE2 from LPS-induced cells by preventing the expression of inflammatory mediators such as iNOS and COX-2 in a dose-dependent manner. Concurrently, levels of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were reduced with increasing concentrations of SD. In addition, SD inhibited the activation of NF-κB and mitogen-activated protein kinases (MAPKs) pathways in a concentration-dependent manner. These results indicate that SD inhibits LPS-stimulated inflammation by inhibition of the NF-κB and MAPKs pathways in macrophages. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Comparative effects of metal oxide nanoparticles on human airway epithelial cells and macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Rotoli, Bianca Maria; Bussolati, Ovidio [University of Parma, Department of Experimental Medicine (Italy); Costa, Anna Luisa; Blosi, Magda [National Research Council, Institute of Science and Technology for Ceramics (Italy); Di Cristo, Luisana [University of Parma, Department of Pharmacological, Biological and Applied Chemical Sciences (Italy); Zanello, Pier Paolo; Bianchi, Massimiliano G.; Visigalli, Rossana [University of Parma, Department of Experimental Medicine (Italy); Bergamaschi, Enrico, E-mail: enrico.bergamaschi@unipr.it [University of Parma, Unit of Occupational Medicine, Department of Clinical Medicine, Nephrology and Health Sciences (Italy)

    2012-09-15

    Among nanomaterials of industrial relevance, metal-based nanoparticles (NPs) are widely used, but their effects on airway cells are relatively poorly characterized. To compare the effects of metal NPs on cells representative of the lung-blood barrier, Calu-3 epithelial cells and Raw264.7 macrophages were incubated with three industrially relevant preparations of TiO{sub 2} NPs (size range 4-33 nm), two preparations of CeO{sub 2} NPs (9-36 nm) and CuO NPs (25 nm). While Raw264.7 were grown on standard plasticware, Calu-3 cells were seeded on permeable filters, where they form a high-resistance monolayer, providing an in vitro model of the airway barrier. Metal NPs, obtained from industrial sources, were characterized under the conditions adopted for the biological tests. Cytotoxicity was assessed with resazurin method in both epithelial and macrophage cells, while epithelial barrier permeability was monitored measuring the trans-epithelial electrical resistance (TEER). In macrophages, titania and ceria had no significant effect on viability in the whole range of nominal doses tested (15-240 {mu}g/cm{sup 2} of monolayer), while CuO NPs produced a marked viability loss. Moreover, only CuO NPs, but not the other NPs, lowered TEER of Calu-3 monolayers, pointing to the impairment of the epithelial barrier. TEER decreased by 30 % at the dose of 10 {mu}g/cm{sup 2} of CuO NPs, compared to untreated control, and was abolished at doses {>=}80 {mu}g/cm{sup 2}, in strict correlation with changes in cell viability. These results indicate that (1) CuO NPs increase airway epithelium permeability even at relatively low doses and are significantly toxic for macrophages and airway epithelial cells, likely through the release of Cu ions in the medium; (2) TiO{sub 2} and CeO{sub 2} NPs do not affect TEER and exhibit little acute toxicity for airway epithelial cells and macrophages; and (3) TEER measurement can provide a simple method to assess the impairment of in vitro airway

  5. Comparative effects of metal oxide nanoparticles on human airway epithelial cells and macrophages

    Science.gov (United States)

    Rotoli, Bianca Maria; Bussolati, Ovidio; Costa, Anna Luisa; Blosi, Magda; Di Cristo, Luisana; Zanello, Pier Paolo; Bianchi, Massimiliano G.; Visigalli, Rossana; Bergamaschi, Enrico

    2012-09-01

    Among nanomaterials of industrial relevance, metal-based nanoparticles (NPs) are widely used, but their effects on airway cells are relatively poorly characterized. To compare the effects of metal NPs on cells representative of the lung-blood barrier, Calu-3 epithelial cells and Raw264.7 macrophages were incubated with three industrially relevant preparations of TiO2 NPs (size range 4-33 nm), two preparations of CeO2 NPs (9-36 nm) and CuO NPs (25 nm). While Raw264.7 were grown on standard plasticware, Calu-3 cells were seeded on permeable filters, where they form a high-resistance monolayer, providing an in vitro model of the airway barrier. Metal NPs, obtained from industrial sources, were characterized under the conditions adopted for the biological tests. Cytotoxicity was assessed with resazurin method in both epithelial and macrophage cells, while epithelial barrier permeability was monitored measuring the trans-epithelial electrical resistance (TEER). In macrophages, titania and ceria had no significant effect on viability in the whole range of nominal doses tested (15-240 μg/cm2 of monolayer), while CuO NPs produced a marked viability loss. Moreover, only CuO NPs, but not the other NPs, lowered TEER of Calu-3 monolayers, pointing to the impairment of the epithelial barrier. TEER decreased by 30 % at the dose of 10 μg/cm2 of CuO NPs, compared to untreated control, and was abolished at doses ≥80 μg/cm2, in strict correlation with changes in cell viability. These results indicate that (1) CuO NPs increase airway epithelium permeability even at relatively low doses and are significantly toxic for macrophages and airway epithelial cells, likely through the release of Cu ions in the medium; (2) TiO2 and CeO2 NPs do not affect TEER and exhibit little acute toxicity for airway epithelial cells and macrophages; and (3) TEER measurement can provide a simple method to assess the impairment of in vitro airway epithelial barrier model by manufactured nanomaterials.

  6. Macrophages as APC and the dendritic cell myth.

    Science.gov (United States)

    Hume, David A

    2008-11-01

    Dendritic cells have been considered an immune cell type that is specialized for the presentation of Ag to naive T cells. Considerable effort has been applied to separate their lineage, pathways of differentiation, and effectiveness in Ag presentation from those of macrophages. This review summarizes evidence that dendritic cells are a part of the mononuclear phagocyte system and are derived from a common precursor, responsive to the same growth factors (including CSF-1), express the same surface markers (including CD11c), and have no unique adaptation for Ag presentation that is not shared by other macrophages.

  7. Mimicking the tumor microenvironment to regulate macrophage phenotype and assessing chemotherapeutic efficacy in embedded cancer cell/macrophage spheroid models.

    Science.gov (United States)

    Tevis, Kristie M; Cecchi, Ryan J; Colson, Yolonda L; Grinstaff, Mark W

    2017-03-01

    Tumor associated macrophages (TAMs) are critical stromal components intimately involved with the progression, invasion, and metastasis of cancer cells. To address the need for an in vitro system that mimics the clinical observations of TAM localizations and subsequent functional performance, a cancer cell/macrophage spheroid model is described. The central component of the model is a triple negative breast cancer spheroid embedded in a three-dimensional collagen gel. Macrophages are incorporated in two different ways. The first is a heterospheroid, a spheroid containing both tumor cells and macrophages. The heterospheroid mimics the population of TAMs infiltrated into the tumor mass, thus being exposed to hypoxia and metabolic gradients. In the second model, macrophages are diffusely seeded in the collagen surrounding the spheroid, thus modeling TAMs in the cancer stroma. The inclusion of macrophages as a heterospheroid changes the metabolic profile, indicative of synergistic growth. In contrast, macrophages diffusely seeded in the collagen bear the same profile regardless of the presence of a tumor cell spheroid. The macrophages in the heterospheroid secrete EGF, a cytokine critical to tumor/macrophage co-migration, and an EGF inhibitor decreases the metabolic activity of the heterospheroid, which is not observed in the other systems. The increased secretion of IL-10 indicates that the heterospheroid macrophages follow an M2/TAM differentiation pathway. Lastly, the heterospheroid exhibits resistance to paclitaxel. In summary, the collagen embedded heterospheroid model promotes TAM-like characteristics, and will be of utility in cancer biology and drug discovery.

  8. β3-Adrenoceptor activation upregulates apolipoprotein A-I expression in HepG2 cells, which might further promote cholesterol efflux from macrophage foam cells.

    Science.gov (United States)

    Gao, Xia-Qing; Li, Yan-Fang; Jiang, Zhi-Li

    2017-01-01

    The aim of this study was to explore the effects of β3-adrenoceptor (β3-AR) activation on HepG2 cells and its influence on cholesterol efflux from macrophage foam cells. HepG2 cells were cultured and treated with the β3-AR agonist, BRL37344, and antagonist, SR52390A, and the expression of apolipoprotein (Apo) A-I, ApoA-II, ApoB, and β3-AR in the supernatants and cells was determined. The expression of peroxisome proliferator-activated receptor (PPAR) γ and PPARα in the HepG2 cells was also assessed. Next, using the RAW264.7 macrophage foam cell model, we also assessed the influence of the HepG2 cell supernatants on lipid efflux. The cholesterol content of the foam cells was also measured, and the cholesterol efflux from the macrophages was examined by determining (3)H-labeled cholesterol levels. Expression of ATP-binding cassette transporter (ABC) A1 and ABCG1 of the macrophage foam cells was also assessed. β3-AR activation increased ApoA-I expression in both the HepG2 cells and the supernatants; PPARγ expression was upregulated, but PPARα expression was not. Treatment with GW9662 abolished the increased expression of ApoA-I induced by the β3-AR agonist. The HepG2 cell supernatants decreased the lipid accumulation and increased the cholesterol efflux from the macrophage foam cells. ABCA1 expression, but not ABCG1 expression, increased in the macrophage foam cells treated with BRL37344-treated HepG2 cell supernatants. Activation of β3-AR in HepG2 cells upregulates ApoA-I expression, which might further promote cholesterol efflux from macrophage foam cells. PPARγ might be required for the induction of ApoA-I expression.

  9. Macrophages and Leydig Cells in Testicular Biopsies of Azoospermic Men

    Directory of Open Access Journals (Sweden)

    Trpimir Goluža

    2014-01-01

    Full Text Available A number of studies have indicated that testicular macrophages play an important role in regulating steroidogenesis of Leydig cells and maintain homeostasis within the testis. The current paper deals with macrophages (CD68 positive cells and Leydig cells in patients with nonobstructive azoospermia (NOA. Methods employed included histological analysis on semi- and ultrathin sections, immunohistochemistry, morphometry, and hormone analysis in the blood serum. Histological analysis pointed out certain structural changes of macrophages and Leydig cells in NOA group of patients when compared to controls. In the testis interstitium, an increased presence of CD68 positive cells has been noted. Leydig cells in NOA patients displayed a kind of a mosaic picture across the same bioptic sample: both normal and damaged Leydig cells with pronounced vacuolisation and various intensity of expression of testosterone have been observed. Stereological analysis indicated a significant increase in volume density of both CD68 positive and vacuolated Leydig cells and a positive correlation between the volume densities of these cell types. The continuous gonadotropin overstimulation of Leydig cells, together with a negative paracrine action of macrophages, could result in the damage of steroidogenesis and deficit of testosterone in situ.

  10. Macrophages: supportive cells for tissue repair and regeneration.

    Science.gov (United States)

    Chazaud, Bénédicte

    2014-03-01

    Macrophages, and more broadly inflammation, have been considered for a long time as bad markers of tissue homeostasis. However, if it is indisputable that macrophages are associated with many diseases in a deleterious way, new roles have emerged, showing beneficial properties of macrophages during tissue repair and regeneration. This discrepancy is likely due to the high plasticity of macrophages, which may exhibit a wide range of phenotypes and functions depending on their environment. Therefore, regardless of their role in immunity, macrophages play a myriad of roles in the maintenance and recovery of tissue homeostasis. They take a major part in the resolution of inflammation. They also exert various effects of parenchymal cells, including stem and progenitor cell, of which they regulate the fate. In the present review, few examples from various tissues are presented to illustrate that, beyond their specific properties in a given tissue, common features have been described that sustain a role of macrophages in the recovery and maintenance of tissue homeostasis.

  11. Repression of proinflammatory gene expression by lipid extract of Nostoc commune var sphaeroides Kützing, a blue-green alga, via inhibition of nuclear factor-kappaB in RAW 264.7 macrophages.

    Science.gov (United States)

    Park, Young-Ki; Rasmussen, Heather E; Ehlers, Sarah J; Blobaum, Kara R; Lu, Fan; Schlegal, Vicki L; Carr, Timothy P; Lee, Ji-Young

    2008-02-01

    We investigated whether lipid extract from a blue-green alga, N commune, modulates proinflammatory gene expression in RAW 264.7 macrophages. The cells were incubated with N commune lipid extract (0-100 microg/mL) and subsequently activated by LPS (100 ng/mL). Quantitative real-time PCR analysis showed that mRNA abundance of proinflammatory mediators, including TNF-alpha, COX-2, IL-1beta, IL-6, and iNOS, was significantly reduced by N commune lipid extract in a dose-dependent manner. Secretion of TNF-alpha and IL-1beta into cell culture medium was also significantly decreased by N commune lipid extract. Thin-layer chromatography-densitometry analysis showed that N commune lipid extract contained approximately 15% of fatty acids. To determine whether the inhibition of proinflammatory mediator production by N commune lipid extract is primarily conferred by fatty acids in the lipid extract, macrophages were incubated with 100 microg/mL of N commune lipid extract or 15 microg/mL of a fatty acid mixture, which was formulated to reflect the fatty acid composition of N commune lipid extract. The fatty acid mixture significantly reduced RNA abundance of TNF-alpha and COX-2, but to a lesser extent than did the N commune lipid extract, suggesting the presence of additional bioactive compounds with an antiinflammatory property in the lipid extract. As NF-kappaB is a major regulator for the proinflammatory gene expression, we measured its DNA-binding activity. DNA-binding activity of NF-kappaB was significantly reduced by N commune lipid extract. In conclusion, our study suggests that N commune lipid extract represses the expression of proinflammatory genes in RAW 264.7 macrophages, at least in part, by inhibiting the activation of NF-kappaB pathway.

  12. Anti-Inflammatory Effect of Methylpenicinoline from a Marine Isolate of Penicillium sp. (SF-5995: Inhibition of NF-κB and MAPK Pathways in Lipopolysaccharide-Induced RAW264.7 Macrophages and BV2 Microglia

    Directory of Open Access Journals (Sweden)

    Dong-Cheol Kim

    2014-11-01

    Full Text Available In the course of a search for anti-inflammatory metabolites from marine-derived fungi, methylpenicinoline (1 was isolated from a marine isolate of Penicillin sp. Compound 1 inhibited lipopolysaccharide (LPS-stimulated nitric oxide (NO production by suppressing the expression of inducible NO synthase (iNOS in RAW264.7 macrophages and BV2 microglia. It also attenuated prostaglandin E2 (PGE2 production by suppressing cyclooxygenase-2 (COX-2 expression in a concentration-dependent manner (from 10 μM to 80 μM without affecting cell viability. In addition, compound 1 reduced the production of the pro-inflammatory cytokine interleukin-1β (IL-1β. In a further study designed to elucidate the mechanism of its anti-inflammatory effects, compound 1 was shown to block nuclear factor-kappa B (NF-κB activation in LPS-induced RAW264.7 macrophages and BV2 microglia by inhibiting the phosphorylation of inhibitor kappa B-α (IκB-α, thereby suppressing the nuclear translocation of NF-κB dimers, namely p50 and p65, that are known to be crucial molecules associated with iNOS and COX-2 expression. In addition, compound 1 inhibited the activation of mitogen-activated protein kinase (MAPK pathways. Taken together, the results suggest that compound 1 might be a valuable therapeutic agent for the treatment of anti-inflammatory and anti-neuroinflammatory diseases.

  13. Alkaloids from Chelidonium majus and their inhibitory effects on LPS-induced NO production in RAW264.7 cells.

    Science.gov (United States)

    Park, Ji Eun; Cuong, To Dao; Hung, Tran Manh; Lee, IkSoo; Na, MinKyun; Kim, Jin Cheol; Ryoo, SungWoo; Lee, Jeong Hyung; Choi, Jae Sue; Woo, Mi Hee; Min, Byung Sun

    2011-12-01

    A new alkaloid, methyl 2'-(7,8-dihydrosanguinarine-8-yl)acetate (1), together with six known alkaloids, stylopine (2), protopine (3), norchelidonine (4), chelidonine (5), berberine (6), and 8-hydroxydihydrosanguinarine (7), were isolated from Chelidonium majus. Their chemical structures were primarily established using 1D and 2D NMR techniques and mass spectrometry. The anti-inflammatory activity of the isolates was examined for their inhibitory effects on LPS-induced NO production in macrophage RAW264.7 cells. Among them, compounds 5 and 7 showed strong inhibitory activities toward the LPS-induced NO production in macrophage RAW264.7 cells with IC(50) values of 7.3 and 4.5 μM, respectively. In addition, compounds 5 and 7 inhibited the inductions of COX-2 and iNOS mRNA in dose-dependent manners, indicating that these compounds attenuated the syntheses of these transcripts at the transcriptional level. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. 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.

  15. DUAL AND DISTINCT ROLES FOR SPHINGOSINE KINASE 1 AND SPHINGOSINE 1 PHOSPHATE IN THE RESPONSE TO INFLAMMATORY STIMULI IN RAW MACROPHAGES

    OpenAIRE

    Hammad, Samar M.; Crellin, Heather G.; Wu, Bill; Melton, Jessica; Anelli, Viviana; Obeid, Lina M.

    2007-01-01

    Sphingosine kinase 1 (SK1) and its product sphingosine-1-phosphate (S1P) have been implicated in the regulation of many cellular processes including growth regulation, protection from apoptosis, stimulation of angiogenesis, and most recently as mediators of the TNF alpha inflammatory response. In this study we set out to examine the role of SK1/S1P in the RAW macrophage response to the potent inflammatory stimulus LPS. We show that LPS increases cellular levels of SK1 message and protein. Thi...

  16. Reduced transcript stabilization restricts TNF-alpha expression in RAW264.7 macrophages infected with pathogenic mycobacteria: evidence for an involvement of lipomannan.

    Science.gov (United States)

    Basler, Tina; Holtmann, Helmut; Abel, Jens; Eckstein, Torsten; Baumer, Wolfgang; Valentin-Weigand, Peter; Goethe, Ralph

    2010-01-01

    Despite the critical role that TNF-alpha plays in the containment of mycobacterial infection, the mechanisms involved in regulation of its expression by mycobacteria are poorly defined. We addressed this question by studying MAP, which causes a chronic enteritis in ruminants and is linked to human Crohn's disease. We found that in MAP infected macrophages, TNF-alpha gene expression was substantially lower than in macrophages infected with nonpathogenic MS or stimulated with LPS. TNF-alpha transcriptional one could not fully explain the differential TNF-alpha mRNA expression, suggesting that there must be a substantial contribution by post-transcriptional mechanisms.Accordingly, we found reduced TNF-alpha mRNA stability in MAP-infected macrophages. Further comparison of MAP- and MS-infected macrophages revealed that lower TNF-alpha mRNA stability combined with lower mRNA and protein expression in MAP-infected macrophages correlated with lower p38 MAPK phosphorylation. These findings were independent of viability of MAP and MS. We demonstrate that the major mycobacterial cell-wall lipoglycan LM of MAP and MS induced TNF-alpha mRNA transcription,but only the MS-LM induced p38 MAPK-dependent transcript stabilization. Overall, our data suggest that pathogenic mycobacteria cause weak p38 and TNF-alpha mRNA stabilization as a result of their structural cell-wall components such as LM and thereby, restrict TNF-alpha expression in macrophages.

  17. The antioxidative effect of bread crust in a mouse macrophage reporter cell line.

    Science.gov (United States)

    Pötzsch, Sandy; Dalgalarrondo, Michele; Bakan, Benedicte; Marion, Didier; Somoza, Veronika; Stangl, Gabriele; Silber, Rolf-Edgar; Simm, Andreas; Navarrete Santos, Alexander

    2014-10-01

    Free radicals and oxidative stress are important factors in the biology of aging and responsible for the development of age-related diseases. One way to reduce the formation of free radicals is to boost the antioxidative system by nutrition. Heat treatment of food promote the Maillard reaction which is responsible for their characteristic color and taste. During the Maillard reaction reducing sugars react with proteins in a non-enzymatic way leading to the formation of advanced glycation end products (AGEs). As an AGE-rich source our group used bread crust (BCE) to investigate the effect of AGEs on the antioxidant defense. It is well known that the NF-kB pathway is activated by treatment of cells with AGEs. Therefore for stimulation with the BCE we used the macrophage reporter cell line RAW/NF-kB/SEAPorter™. Amino acid analysis and LC-MS/MS by Orbitrap Velo was used to determine the bioactive compounds in the soluble BCE. The radical scavenging effect was conducted by the DPPH-assay. BCE induced the NF-kB pathway in RAW/NF-kB/SEAPorter™ cells and also showed a concentration-dependent antioxidative capacity by the DPPH-assay. With the LC/MS and amino acid analyses, we identified the presence of gliadin in BCE confirmed by using specific gliadin antibodies. By immunoprecipitation (IP) with an antibody against γ-gliadin and western blot probing against the AGE carboxymethyllysine (CML) the presence of AGE-gliadin in BCE was confirmed. Stimulation of the RAW/NF-kB/SEAPorter™ cells with the γ-gliadin depleted fractions did not activate the NF-kB pathway. CML-modified gliadin in the BCE is a bioactive compound of the bread crust which is responsible for the antioxidative capacity and for the induction of the NF-kB pathway in mouse macrophages. Copyright © 2014. Published by Elsevier Inc.

  18. 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.

  19. Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells.

    Science.gov (United States)

    Mahamed, Deeqa; Boulle, Mikael; Ganga, Yashica; Mc Arthur, Chanelle; Skroch, Steven; Oom, Lance; Catinas, Oana; Pillay, Kelly; Naicker, Myshnee; Rampersad, Sanisha; Mathonsi, Colisile; Hunter, Jessica; Wong, Emily B; Suleman, Moosa; Sreejit, Gopalkrishna; Pym, Alexander S; Lustig, Gila; Sigal, Alex

    2017-01-28

    A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states.

  20. The inhibition of macrophage foam cell formation by 9-cis β-carotene is driven by BCMO1 activity.

    Directory of Open Access Journals (Sweden)

    Noa Zolberg Relevy

    Full Text Available Atherosclerosis is a major cause of morbidity and mortality in developed societies, and begins when activated endothelial cells recruit monocytes and T-cells from the bloodstream into the arterial wall. Macrophages that accumulate cholesterol and other fatty materials are transformed into foam cells. Several epidemiological studies have demonstrated that a diet rich in carotenoids is associated with a reduced risk of heart disease; while previous work in our laboratory has shown that the 9-cis β-carotene rich alga Dunaliella inhibits atherogenesis in mice. The effect of 9-cis β-carotene on macrophage foam cell formation has not yet been investigated. In the present work, we sought to study whether the 9-cis β-carotene isomer, isolated from the alga Dunaliella, can inhibit macrophage foam cell formation upon its conversion to retinoids. The 9-cis β-carotene and Dunaliella lipid extract inhibited foam cell formation in the RAW264.7 cell line, similar to 9-cis retinoic acid. Furthermore, dietary enrichment with the algal powder in mice resulted in carotenoid accumulation in the peritoneal macrophages and in the inhibition of foam cell formation ex-vivo and in-vivo. We also found that the β-carotene cleavage enzyme β-carotene 15,15'-monooxygenase (BCMO1 is expressed and active in macrophages. Finally, 9-cis β-carotene, as well as the Dunaliella extract, activated the nuclear receptor RXR in hepa1-6 cells. These results indicate that dietary carotenoids, such as 9-cis β-carotene, accumulate in macrophages and can be locally cleaved by endogenous BCMO1 to form 9-cis retinoic acid and other retinoids. Subsequently, these retinoids activate the nuclear receptor RXR that, along with additional nuclear receptors, can affect various metabolic pathways, including those involved in foam cell formation and atherosclerosis.

  1. 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

  2. DNA-PKcs interacts with Aire and regulates the expression of toll-like receptors in RAW264.7 cells.

    Science.gov (United States)

    Wu, J; Zhu, W; Fu, H; Zhang, Y; Sun, J; Yang, W; Li, Y

    2012-05-01

    The autoimmune regulator (Aire) is a key mediator of the central tolerance for peripheral tissue self-antigen (PTAs) and is involved in the transcriptional control of many antigens in thymic medullary epithelial cells (mTECs). However, the function of Aire in peripheral lymphoid tissues and haematopoietic cells, particularly in monocytes and macrophages, remains poorly understood. We previously found that the expression of Toll-like receptor (TLR) 1, TLR3 and TLR8 was notably upregulated in pEGFPC1/Aire stably transfected RAW264.7 (GFP-Aire/RAW) cells, while the expressions of other TLRs were not significantly changed. The mechanism by which Aire affects TLR1, TLR3 and TLR8 expression is not clear. Interactions with other proteins, such as DNA-dependent protein kinase (DNA-PK), are crucial for regulating the transcriptional activity of Aire. In this study, we found that Aire and DNA-PK catalytic subunit (DNA-PKcs) were co-located in the nucleus of GFP-Aire/RAW cells, and they interact with each other. Small interfering RNA knock-down of DNA-PKcs in these cells decreased the expression of TLR1, TLR3 and TLR8, but no change was observed in pEGFPC1 stably transfected RAW264.7 (GFP/RAW) cells. We did not observe any change in the expressions of other TLRs after DNA-PKcs knock-down in GFP-Aire/RAW or GFP/RAW cells. A similar observation has been made in pEGFPC1/Aire or pEGFPC1 transiently transfected primary peritoneal macrophages. Using a luciferase activity assay, we found the that the transcriptional activity of TLR1, TLR3 and TLR8 promoters was also decreased after knock-down of DNA-PKcs in GFP-Aire/RAW cells. In conclusion, our results suggest that DNA-PKcs may interact with Aire to promote the expression of TLRs in RAW264.7 cells. © 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.

  3. In vitro biodegradation of three brushite calcium phosphate cements by a macrophage cell-line.

    Science.gov (United States)

    Xia, Zhidao; Grover, Liam Michael; Huang, Yizhong; Adamopoulos, Iannis E; Gbureck, Uwe; Triffitt, James T; Shelton, Richard M; Barralet, Jake E

    2006-09-01

    Depending upon local conditions, brushite (CaHPO4 x 2 H2O) cements may be largely resorbed or (following hydrolysis to hydroxyapatite) remain stable in vivo. To determine which factors influence cement resorption, previous studies have investigated the solution-driven degradation of brushite cements in vitro in the absence of any cells. However, the mechanism of cell-mediated biodegradation of the brushite cement is still unknown. The aim of the current study was to observe the cell-mediated biodegradation of brushite cement formulations in vitro. The cements were aged in the presence of a murine cell line (RAW264.7), which had the potential to form osteoclasts in the presence of the receptor for nuclear factor kappa B ligand (RANKL) in vitro, independently of macrophage colony stimulating factor (M-CSF). The cytotoxicity of the cements on RAW264.7 cells and the calcium and phosphate released from materials to the culture media were analysed. Scanning electron microscopy (SEM) and focused ion beam (FIB) microscopy were used to characterise the ultrastructure of the cells. The results showed that the RAW264.7 cell line formed multinucleated TRAP positive osteoclast-like cells, capable of ruffled border formation and lacunar resorption on the brushite calcium phosphate cement in vitro. In the osteoclast-like cell cultures, ultrastructural analysis by SEM revealed phenotypic characteristics of osteoclasts including formation of a sealing zone and ruffled border. Penetration of the surface of the cement, was demonstrated using FIB, and this showed the potential demineralising effect of the cells on the cements. This study has set up a useful model to investigate the cell-mediated cement degradation in vitro.

  4. Acanthopanax koreanum roots inhibit the expression of pro-inflammatory cytokines, inducible nitric oxide synthase, and cyclooxygenase-2 in RAW 264.7 macrophages

    Directory of Open Access Journals (Sweden)

    Eun-Jin Yang

    2016-03-01

    Full Text Available Acanthopanax koreanum is a popular plant found on Jeju Island, Korea and is commonly used to prevent the side effects of consumption of alcoholic beverages. However, this plant has not been properly utilized as a medicinal material. In this study, we investigated the anti-inflammatory effects of the 70% ethanol extract of A. koreanum roots (AKR-E. The results indicated that the AKR-E (200 μg/mL inhibited the lipopolysaccharide (LPS-induced production of nitric oxide (NO and prostaglandin E2 (PGE2 in RAW 264.7 macrophages by 41.2% and 78.9%, respectively. These effects were accompanied by concentration-dependent decreases in the expression levels of inducible NO synthase (iNOS and cyclooxygenase-2 (COX-2 proteins. Additionally, the AKR-E inhibited the expression of pro-inflammatory cytokines, including interleukin (IL-6 (22.7% and IL-1β (74%. These data showed that the AKR-E had protective effects against the induction of LPS-induced inflammation in RAW 264.7 macrophages.

  5. p47phox Directs Murine Macrophage Cell Fate Decisions

    Science.gov (United States)

    Yi, Liang; Liu, Qi; Orandle, Marlene S.; Sadiq-Ali, Sara; Koontz, Sherry M.; Choi, Uimook; Torres-Velez, Fernando J.; Jackson, Sharon H.

    2012-01-01

    Macrophage differentiation and function are pivotal for cell survival from infection and involve the processing of microenvironmental signals that determine macrophage cell fate decisions to establish appropriate inflammatory balance. NADPH oxidase 2 (Nox2)–deficient chronic granulomatous disease (CGD) mice that lack the gp91phox (gp91phox−/−) catalytic subunit show high mortality rates compared with wild-type mice when challenged by infection with Listeria monocytogenes (Lm), whereas p47phox-deficient (p47phox−/−) CGD mice show survival rates that are similar to those of wild-type mice. We demonstrate that such survival results from a skewed macrophage differentiation program in p47phox−/− mice that favors the production of higher levels of alternatively activated macrophages (AAMacs) compared with levels of either wild-type or gp91phox−/− mice. Furthermore, the adoptive transfer of AAMacs from p47phox−/− mice can rescue gp91phox−/− mice during primary Lm infection. Key features of the protective function provided by p47phox−/− AAMacs against Lm infection are enhanced production of IL-1α and killing of Lm. Molecular analysis of this process indicates that p47phox−/− macrophages are hyperresponsive to IL-4 and show higher Stat6 phosphorylation levels and signaling coupled to downstream activation of AAMac transcripts in response to IL-4 stimulation. Notably, restoring p47phox protein expression levels reverts the p47phox-dependent AAMac phenotype. Our results indicate that p47phox is a previously unrecognized regulator for IL-4 signaling pathways that are important for macrophage cell fate choice. PMID:22222227

  6. Ethanol extract of Elaeocarpus petiolatus inhibits lipopolysaccharide-induced inflammation in macrophage cells.

    Science.gov (United States)

    Kwon, Ok-Kyoung; Ahn, Kyung-Seop; Park, Ji-Won; Jang, Ha-Young; Joung, Hyouk; Lee, Hyeong-Kyu; Oh, Sei-Ryang

    2012-04-01

    Elaeocarpus petiolatus is known to exert active oxygen scavenging, anti-aging, and whitening actions. However, the biological effects of E. petiolatus on inflammation and the underlying mechanisms are yet to be established. In the present study, we investigated the anti-inflammatory effects of the ethanol extract from E. petiolatus (EPE) bark in murine Raw264.7 macrophages stimulated with lipopolysaccharide (LPS). EPE inhibited the production of PGE(2), TNF-α, and IL-1β in a dose-dependent manner in Raw264.7 cells stimulated with LPS. The decrease in PGE(2) production was correlated with reduced COX-2 expression. Furthermore, EPE suppressed the phosphorylation of extracellular signal-related kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 as well as translocation of the NF-κB p65 subunit from the cytosol to nucleus. Our results suggest that EPE exerts anti-inflammatory activity through inhibition of inflammatory mediators, such as PGE(2), TNF-α, and IL-1β, and downregulation of COX-2 via suppression of NF-κB translocation and phosphorylation of ERK, JNK, and p38 in LPS-stimulated Raw264.7 cells.

  7. Staining of Langerhans Cells with Monoclonal Antibodies to Macrophages and Lymphoid Cells

    Science.gov (United States)

    Haines, Kathleen A.; Flotte, Thomas J.; Springer, Timothy A.; Gigli, Irma; Thorbecke, G. Jeanette

    1983-06-01

    Langerhans cells are Ia-bearing antigen-presenting cells in the epidermis that share many functions with macrophages. We have used monoclonal antibodies to the macrophage antigens, Mac-2 and -3, Ia antigen, Fc fragment receptor, and the common leukocyte antigen CLA to compare the cell surface antigens of these cells with those of interdigitating and follicular dendritic cells and of macrophages in lymphoid tissues. Immunoperoxidase staining was carried out with epidermal sheets from BALB/c mice and epidermal cell suspensions enriched for Langerhans cells by Fc rosetting. Langerhans cells stained for all of these antigens. Comparison with the staining properties of other dendritic cells and macrophages, in combination with previous observations, indicates a close relationship of Langerhans cells to the interdigitating cells of lymphoid tissues.

  8. Vascular endothelial growth factor enhances macrophage clearance of apoptotic cells

    Science.gov (United States)

    Dalal, Samay; Horstmann, Sarah A.; Richens, Tiffany R.; Tanaka, Takeshi; Doe, Jenna M.; Boe, Darren M.; Voelkel, Norbert F.; Taraseviciene-Stewart, Laimute; Janssen, William J.; Lee, Chun G.; Elias, Jack A.; Bratton, Donna; Tuder, Rubin M.; Henson, Peter M.; Vandivier, R. William

    2012-01-01

    Efficient clearance of apoptotic cells from the lung by alveolar macrophages is important for the maintenance of tissue structure and function. Lung tissue from humans with emphysema contains increased numbers of apoptotic cells and decreased levels of vascular endothelial growth factor (VEGF). Mice treated with VEGF receptor inhibitors have increased numbers of apoptotic cells and develop emphysema. We hypothesized that VEGF regulates apoptotic cell clearance by alveolar macrophages (AM) via its interaction with VEGF receptor 1 (VEGF R1). Our data show that the uptake of apoptotic cells by murine AMs and human monocyte-derived macrophages is inhibited by depletion of VEGF and that VEGF activates Rac1. Antibody blockade or pharmacological inhibition of VEGF R1 activity also decreased apoptotic cell uptake ex vivo. Conversely, overexpression of VEGF significantly enhanced apoptotic cell uptake by AMs in vivo. These results indicate that VEGF serves a positive regulatory role via its interaction with VEGF R1 to activate Rac1 and enhance AM apoptotic cell clearance. PMID:22307908

  9. BIGH3 protein and macrophages in retinal endothelial cell apoptosis.

    Science.gov (United States)

    Mondragon, Albert A; Betts-Obregon, Brandi S; Moritz, Robert J; Parvathaneni, Kalpana; Navarro, Mary M; Kim, Hong Seok; Lee, Chi Fung; LeBaron, Richard G; Asmis, Reto; Tsin, Andrew T

    2015-01-01

    Diabetes is a pandemic disease with a higher occurrence in minority populations. The molecular mechanism to initiate diabetes-associated retinal angiogenesis remains largely unknown. We propose an inflammatory pathway of diabetic retinopathy in which macrophages in the diabetic eye provide TGFβ to retinal endothelial cells (REC) in the retinal microvasculature. In response to TGFβ, REC synthesize and secrete a pro-apoptotic BIGH3 (TGFβ-Induced Gene Human Clone 3) protein, which acts in an autocrine loop to induce REC apoptosis. Rhesus monkey retinal endothelial cells (RhREC) were treated with dMCM (cell media of macrophages treated with high glucose and LDL) and assayed for apoptosis (TUNEL), BIGH3 mRNA (qPCR), and protein (Western blots) expressions. Cells were also treated with ΤGFβ1 and 2 for BIGH3 mRNA and protein expression. Inhibition assays were carried out using antibodies for TGFβ1 and for BIGH3 to block apoptosis and mRNA expression. BIGH3 in cultured RhREC cells were identified by immunohistochemistry (IHC). Distribution of BIGH3 and macrophages in the diabetic mouse retina was examined with IHC. RhRECs treated with dMCM or TGFβ showed a significant increase in apoptosis and BIGH3 protein expression. Recombinant BIGH3 added to RhREC culture medium led to a dose-dependent increase in apoptosis. Antibodies (Ab) directed against BIGH3 and TGFβ, as well as TGFβ receptor blocker resulted in a significant reduction in apoptosis induced by either dMCM, TGFβ or BIGH3. IHC showed that cultured RhREC constitutively expressed BIGH3. Macrophage and BIGH3 protein were co-localized to the inner retina of the diabetic mouse eye. Our results support a novel inflammatory pathway for diabetic retinopathy. This pathway is initiated by TGFβ released from macrophages, which promotes synthesis and release of BIGH3 protein by REC and REC apoptosis.

  10. Leishmania Donovani Cell Surface Sialoglycans Regulate Susceptibility for Siglec Mediated Macrophage Invasion and Parasite Survival

    Directory of Open Access Journals (Sweden)

    Tripti De

    2012-02-01

    Full Text Available Glycoconjugates play a pivotal role in the survival of Leishmania parasites in destructive surroundings. An important constituent present on many glycoconjugates is sialic acid. By virtue of their peripheral position on oligosaccharide chains of glycoconjugates, sialic acids are well suited as molecular determinants of specific biological processes, including the interaction of pathogenic microorganisms with sialylated cellular receptors. Differences in a2,3- and a2,6-sialoglycan patterns detected in clonal virulent Leishmania donovani promastigotes, correlated with the level of a2,3- and a2,6-sialyltransferase activity present in these parasites. The role of macrophage sialic acid-receptors in uptake and survival of L.donovani was studied in the murine macrophage cell line raw 264.7. Macrophage invasion was dependent on the binding to Siglec-1, while suppression of MAPK signaling was mediated through Siglec-5. Sialic acid removal by neuraminidase treatment reduced parasite infectivity. The presence of trypsin resistant sialic acid residues in the neuraminidase treated parasites grown in a serum free medium in presence of sialoglycoconjugates indicated that the parasites could salvage sialic acid from exogenous sialoglycans and reutilize it for de novo glycoprotein sialylation in L.donovani parasites. Thus, our results demonstrate the involvement of sialoglycans in the invasion as well as the survival process of L.donovani parasites.

  11. Fucoidan reduced the invasion of oral squamous cell carcinoma cells and modified their effects to macrophages.

    Science.gov (United States)

    Lin, Junda; Wang, Ketao; Wang, Huayang; Shao, Qianqian; Luan, Yijun; Xu, Yan; Song, Xiaobin; Tan, Wanye; Liu, Shaohua; Wei, Fengcai; Qu, Xun

    2017-01-01

    Fucoidan is a complex of polysaccharides showing antitumor and immunomodulation properties. Our previous studies found its regulation to myeloid immune cells, including macrophages. Aberrant infiltration and functions of macrophages are commonly found in oral squamous cell carcinoma (OSCC). In this study, we analyzed the effects of fucoidan on invasion of OSCC cells, and their regulation to macrophages, trying to evaluate its role as a potential therapy for OSCC. CAL27 and THP-1-derived macrophages were used as models for OSCC cells and tumor-infiltrated macrophages in the in vitro study, respectively. The effects of fucoidan on invasion of OSCC cells and their recruitment to macrophages were analyzed by transwell assay. KIF4A siRNA transfection was performed to investigate its role in fucoidan-modulated OSCC cells invasion. CCL3-neutralizing antibody was added into the conditioned medium of OSCC cells to evaluate its role in fucoidan-mediated macrophages recruitment and re-education. Fucoidan reduced the invasive potential of CAL27 cells with a decrease of MMP-2 and KIF4A transcription. KIF4A knockdown in CAL27 cells led to decreased invasion and MMP-2 expression. The conditioned medium of fucoidan-treated CAL27 cells promoted recruitment and inflammatory cytokines secretion on THP-1-derived macrophages. Further analysis found that fucoidan increased CCL3 production in CAL27 cells. Blocking CCL3 expression reversed the effects of fucoidan on macrophage recruitment and re-education. Our study found that fucoidan regulated the invasion of OSCC cells and also their recruiting and re-educating effects on macrophages, suggesting it could be a complementary approach in the treatment of OSCC.

  12. Mitochondrial function is involved in regulation of cholesterol efflux to apolipoprotein (apoA-I from murine RAW 264.7 macrophages

    Directory of Open Access Journals (Sweden)

    Allen Anne Marie

    2012-12-01

    Full Text Available Abstract Background Mitochondrial DNA damage, increased production of reactive oxygen species and progressive respiratory chain dysfunction, together with increased deposition of cholesterol and cholesteryl esters, are hallmarks of atherosclerosis. This study investigated the role of mitochondrial function in regulation of macrophage cholesterol efflux to apolipoprotein A-I, by the addition of established pharmacological modulators of mitochondrial function. Methods Murine RAW 264.7 macrophages were treated with a range of concentrations of resveratrol, antimycin, dinitrophenol, nigericin and oligomycin, and changes in viability, cytotoxicity, membrane potential and ATP, compared with efflux of [3H]cholesterol to apolipoprotein (apo A-I. The effect of oligomycin treatment on expression of genes implicated in macrophage cholesterol homeostasis were determined by quantitative polymerase chain reaction, and immunoblotting, relative to the housekeeping enzyme, Gapdh, and combined with studies of this molecule on cholesterol esterification, de novo lipid biosynthesis, and induction of apoptosis. Significant differences were determined using analysis of variance, and Dunnett’s or Bonferroni post t-tests, as appropriate. Results The positive control, resveratrol (24 h, significantly enhanced cholesterol efflux to apoA-I at concentrations ≥30 μM. By contrast, cholesterol efflux to apoA-I was significantly inhibited by nigericin (45%; ppAbca1 mRNA. Oligomycin treatment did not affect cholesterol biosynthesis, but significantly inhibited cholesterol esterification following exposure to acetylated LDL, and induced apoptosis at ≥30 μM. Finally, oligomycin induced the expression of genes implicated in both cholesterol efflux (Abca1, Abcg4, Stard1 and cholesterol biosynthesis (Hmgr, Mvk, Scap, Srebf2, indicating profound dysregulation of cholesterol homeostasis. Conclusions Acute loss of mitochondrial function, and in particular Δψm, reduces

  13. Nimbolide Inhibits Nuclear Factor-КB Pathway in Intestinal Epithelial Cells and Macrophages and Alleviates Experimental Colitis in Mice.

    Science.gov (United States)

    Seo, Ji Yeon; Lee, Changhyun; Hwang, Sung Wook; Chun, Jaeyoung; Im, Jong Pil; Kim, Joo Sung

    2016-10-01

    Nimbolide is a limonoid extracted from neem tree (Azadirachta indica) that has antiinflammatory properties. The effect of nimbolide on the nuclear factor-kappa B (NF-κB) pathway in intestinal epithelial cells (IECs), macrophages and in murine colitis models was investigated. The IEC COLO 205, the murine macrophage cell line RAW 264.7, and peritoneal macrophages from interleukin-10-deficient (IL-10(-/-) ) mice were preconditioned with nimbolide and then stimulated with tumor necrosis factor-α (TNF-α) or lipopolysaccharide. Dextran sulfate sodium-induced acute colitis model and chronic colitis model in IL-10(-/-) mice were used for in vivo experiments. Nimbolide significantly suppressed the expression of inflammatory cytokines (IL-6, IL-8, IL-12, and TNF-α) and inhibited the phosphorylation of IκBα and the DNA-binding affinity of NF-κB in IECs and macrophages. Nimbolide ameliorated weight loss, colon shortening, disease activity index score, and histologic scores in dextran sulfate sodium colitis. It also improved histopathologic scores in the chronic colitis of IL-10(-/-) mice. Staining for phosphorylated IκBα was significantly decreased in the colon tissue after treatment with nimbolide in both models. Nimbolide inhibits NF-κB signaling in IECs and macrophages and ameliorates experimental colitis in mice. These results suggest nimbolide could be a potentially new treatment for inflammatory bowel disease. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

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    Lifescience Database Archive (English)

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  15. File list: His.Bld.10.AllAg.Granulocyte-Macrophage_Progenitor_Cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. Signaling events in pathogen-induced macrophage foam cell formation.

    Science.gov (United States)

    Shaik-Dasthagirisaheb, Yazdani B; Mekasha, Samrawit; He, Xianbao; Gibson, Frank C; Ingalls, Robin R

    2016-08-01

    Macrophage foam cell formation is a key event in atherosclerosis. Several triggers induce low-density lipoprotein (LDL) uptake by macrophages to create foam cells, including infections with Porphyromonas gingivalis and Chlamydia pneumoniae, two pathogens that have been linked to atherosclerosis. While gene regulation during foam cell formation has been examined, comparative investigations to identify shared and specific pathogen-elicited molecular events relevant to foam cell formation are not well documented. We infected mouse bone marrow-derived macrophages with P. gingivalis or C. pneumoniae in the presence of LDL to induce foam cell formation, and examined gene expression using an atherosclerosis pathway targeted plate array. We found over 30 genes were significantly induced in response to both pathogens, including PPAR family members that are broadly important in atherosclerosis and matrix remodeling genes that may play a role in plaque development and stability. Six genes mainly involved in lipid transport were significantly downregulated. The response overall was remarkably similar and few genes were regulated in a pathogen-specific manner. Despite very divergent lifestyles, P. gingivalis and C. pneumoniae activate similar gene expression profiles during foam cell formation that may ultimately serve as targets for modulating infection-elicited foam cell burden, and progression of atherosclerosis.

  17. Neutrophils and macrophages: The main partners of phagocyte cell systems

    Directory of Open Access Journals (Sweden)

    Manuel T. Silva

    2012-07-01

    Full Text Available Biological cellular systems are groups of cells sharing a set of characteristics, mainly key function and origin. Phagocytes are crucial in the host defense against microbial infection. The previously proposed phagocyte cell systems including the most recent and presently prevailing one, the Mononuclear Phagocyte System (MPS, grouped mononuclear cells but excluded neutrophils, creating an unacceptable situation. As neutrophils are archetypical phagocytes that must be members of comprehensive phagocyte systems, M. T. Silva recently proposed the creation of a Myeloid Phagocyte System (MYPS that adds neutrophils to the MPS. The phagocytes grouped in the MYPS include the leukocytes neutrophils, inflammatory monocytes, macrophages and immature myeloid DCs. Here the justifications behind the inclusion of neutrophils in a phagocyte system is expanded and the MYPS are further characterized as a group of dedicated phagocytic cells that function in an interacting and cooperative way in the host defense against microbial infection. Neutrophils and macrophages are considered the main arms of this system.

  18. Metabolic and Epigenetic Coordination of T Cell and Macrophage Immunity.

    Science.gov (United States)

    Phan, Anthony T; Goldrath, Ananda W; Glass, Christopher K

    2017-05-16

    Recognition of pathogens by innate and adaptive immune cells instructs rapid alterations of cellular processes to promote effective resolution of infection. To accommodate increased bioenergetic and biosynthetic demands, metabolic pathways are harnessed to maximize proliferation and effector molecule production. In parallel, activation initiates context-specific gene-expression programs that drive effector functions and cell fates that correlate with changes in epigenetic landscapes. Many chromatin- and DNA-modifying enzymes make use of substrates and cofactors that are intermediates of metabolic pathways, providing potential cross talk between metabolism and epigenetic regulation of gene expression. In this review, we discuss recent studies of T cells and macrophages supporting a role for metabolic activity in integrating environmental signals with activation-induced gene-expression programs through modulation of the epigenome and speculate as to how this may influence context-specific macrophage and T cell responses to infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Removal of hematopoietic cells and macrophages from mouse bone marrow cultures: isolation of fibroblastlike stromal cells.

    Science.gov (United States)

    Modderman, W E; Vrijheid-Lammers, T; Löwik, C W; Nijweide, P J

    1994-02-01

    A method is described that permits the removal of hematopoietic cells and macrophages from mouse bone marrow cultures. The method is based on the difference in effect of extracellular ATP4- ions (ATP in the absence of divalent, complexing cations) on cells of hematopoietic origin, including macrophages, and of nonhematopoietic origin, such as fibroblastlike stromal cells. In contrast to fibroblastlike cells, hematopoietic cells and macrophages form under the influence of ATP4- lesions in their plasma membranes, which allows the entrance of molecules such as ethidium bromide (EB) and potassium thiocyanate (KSCN), which normally do not easily cross the membrane. The lesions can be rapidly closed by the addition of Mg2+ to the incubation medium, leaving the EB or KSCN trapped in the cell. This method allows the selective introduction of cell-toxic substances such as KSCN into hematopoietic cells and macrophages. By using this method, fibroblastlike stromal cells can be isolated from mouse bone marrow cultures.

  20. A common carp (Cyprinus carpio L.) leucocyte cell line shares morphological and functional characteristics with macrophages.

    NARCIS (Netherlands)

    Weyts, F.A.A.; Rombout, J.H.W.M.; Flik, G.; Verburg-van Kemenade, B.M.L.

    1997-01-01

    A carp leucocyte cell line (CLC), originating from peripheral blood, was characterised to assess its suitability for studies into carp macrophage functions. The cells reacted with a monoclonal antibody raised against carp head kidney macrophages. Other macrophage characteristics observed were: bindi

  1. Stimulation of nitric oxide synthesis by the aqueous extract of Panax ginseng root in RAW 264.7 cells

    Science.gov (United States)

    Friedl, Roswitha; Moeslinger, Thomas; Kopp, Brigitte; Spieckermann, Paul Gerhard

    2001-01-01

    In this study, we investigated the effect of Panax ginseng root aqueous extracts upon inducible nitric oxide synthesis in RAW 264.7 cells. Panax ginseng root extract has been used in the Asian world for centuries as a traditional herb to enhance physical strength and resistance and is becoming more and more popular in Europe and North America. Incubation of murine macrophages (RAW 264.7 cells) with increasing amounts of aqueous extracts of Panax ginseng (0.05 – 0.8 μg μl−1) showed a dose dependent stimulation of inducible nitric oxide synthesis. Polysaccharides isolated from Panax ginseng showed strong stimulation of inducible nitric oxide synthesis, whereas a triterpene-enriched fraction from an aqueous extract of Panax ginseng did not show any stimulation. Inducible nitric oxide synthase protein expression was enhanced in a dose dependent manner as revealed by immunoblotting when cells were incubated with increasing amounts of Panax ginseng extract. This was associated with an incline in inducible nitric oxide synthase mRNA-levels as determined by semiquantitative polymerase chain reaction and electromobility shift assay studies indicated enhanced nuclear factor-κB DNA binding activity. As nitric oxide plays an important role in immune function, Panax ginseng treatment could modulate several aspects of host defense mechanisms due to stimulation of the inducible nitric oxide synthase. PMID:11739242

  2. Anti-Inflammatory Effects and Mechanisms of Action of Coussaric and Betulinic Acids Isolated from Diospyros kaki in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

    Science.gov (United States)

    Kim, Kyoung-Su; Lee, Dong-Sung; Kim, Dong-Cheol; Yoon, Chi-Su; Ko, Wonmin; Oh, Hyuncheol; Kim, Youn-Chul

    2016-09-09

    Diospyros kaki Thunb. is widely distributed in East Asian countries, its leaves being mainly used for making tea. In this study, coussaric acid (CA) and betulinic acid (BA), both triterpenoid compounds, were obtained from D. kaki leaf extracts through bioassay-guided isolation. CA and BA showed anti-inflammatory effects via inhibition of the nuclear factor-κB (NF-κB) pathway, providing important information on their anti-inflammatory mechanism. Furthermore, they markedly inhibited nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages, and suppressed tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) levels. Furthermore, they decreased protein expression of inducible nitric oxide synthase and cyclooxygenase-2. Pre-treatment with CA and BA inhibited LPS-induced NF-κB. We further examined the effects of CA and BA on heme oxygenase (HO)-1 expression in RAW 264.7 macrophages: BA induced HO-1 protein expression in a dose-dependent manner, while CA had no effect. We also investigated whether BA treatment induced nuclear translocation of Nrf2. BA inhibited LPS-induced NF-κB-binding activity, as well as pro-inflammatory mediator and cytokine production (e.g., NO, PGE₂, TNF-α, IL-1β, IL-6), by partial reversal of this effect by SnPP, an inhibitor of HO-1. These findings further elucidate the anti-inflammatory mechanism of CA and BA isolated from D. kaki.

  3. Anti-Inflammatory Effects and Mechanisms of Action of Coussaric and Betulinic Acids Isolated from Diospyros kaki in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Kyoung-Su Kim

    2016-09-01

    Full Text Available Diospyros kaki Thunb. is widely distributed in East Asian countries, its leaves being mainly used for making tea. In this study, coussaric acid (CA and betulinic acid (BA, both triterpenoid compounds, were obtained from D. kaki leaf extracts through bioassay-guided isolation. CA and BA showed anti-inflammatory effects via inhibition of the nuclear factor-κB (NF-κB pathway, providing important information on their anti-inflammatory mechanism. Furthermore, they markedly inhibited nitric oxide (NO and prostaglandin E2 (PGE2 production in lipopolysaccharide (LPS-activated RAW 264.7 macrophages, and suppressed tumor necrosis factor-α (TNF-α, interleukin-6 (IL-6, and interleukin-1β (IL-1β levels. Furthermore, they decreased protein expression of inducible nitric oxide synthase and cyclooxygenase-2. Pre-treatment with CA and BA inhibited LPS-induced NF-κB. We further examined the effects of CA and BA on heme oxygenase (HO-1 expression in RAW 264.7 macrophages: BA induced HO-1 protein expression in a dose-dependent manner, while CA had no effect. We also investigated whether BA treatment induced nuclear translocation of Nrf2. BA inhibited LPS-induced NF-κB-binding activity, as well as pro-inflammatory mediator and cytokine production (e.g., NO, PGE2, TNF-α, IL-1β, IL-6, by partial reversal of this effect by SnPP, an inhibitor of HO-1. These findings further elucidate the anti-inflammatory mechanism of CA and BA isolated from D. kaki.

  4. Cytotoxicity studies of Dynasan 114 solid lipid nanoparticles (SLN) on RAW 264.7 macrophages-impact of phagocytosis on viability and cytokine production

    NARCIS (Netherlands)

    Olbrich, C.; Scholer, N.; Tabatt, K.; Kayser, Oliver; Muller, R.H.

    2004-01-01

    Solid lipid nanoparticles (SLN) based on Dynasan 114 (D114) were tested using RAW 264.7 cells. The influence of different surfactants on the cytotoxicity of this type of SLN was examined, expressed as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability and the production of

  5. Influence of superparamagnetic iron oxide nano-particles in different concentration on macrophage line RAW264.7 in mice%不同浓度超顺磁性氧化铁纳米粒子对小鼠RAW264.7巨噬细胞的影响

    Institute of Scientific and Technical Information of China (English)

    韩莎莎; 李俊峡

    2013-01-01

    Objective To review the influence of superparamagnetic iron oxide nano-particles ( SPIO ) in different concentration on the cell viability and phagocytic capacity of macrophage line RAW264.7 in mice. Methods RAW264.7 were cultured by using routine method, and then labeled with SPIO in different concentration ( 0 μg/mL, 14 μg/mL, 28μg/mL, 56 μg/mL, 84 μg/mL, 140 μg/mL, 280 μg/mL, 560 μg/mL and 840 μg/mL ) . The labeling yield was detected by using Prussian blue staining, cell viability was detected by using trypan blue, cell reproductive capacity was detected by using MTT assay, and cell phagocytic capacity was detected by using neutral red phagocytosis test. Results The labeling rate reached to 100% after labeling for 24 hours in SPIO with iron concentration of 84 μg/mL, and then as iron concentration in SPIO increased, the iron particles phagocytosed by RAW264.7 increased. When the iron concentration in SPIO was 280 μg/mL, the iron particles phagocytosed by RAW264.7 reached to saturation. When the iron concentration in SPIO was over 280 μg/mL, cell viability and phagocytic capacity decreased, and the iron concentration in SPIO was over 140 μg/mL, cell reproductive capacity decreased. Conclusion When the iron concentration in SPIO is ( 84-140 ) μg/mL and after labeling RAW 264.7 lor 24 hours, the labeling rale is 100% and cell viability, cell reproductive capacity and phagocytic capacity are not influenced.%目的 评价不同浓度超顺磁性氧化铁纳米粒子(superparamagnetic iron oxides,SPIO)对小鼠RAW264.7巨噬细胞的细胞活性及吞噬功能的影响.方法 常规方法培养小鼠RAW264.7巨噬细胞,应用不同浓度SPIO(0 μg/ml、14μg/ml、28μg/ml、56μg/ml、84μg/ml、140μg/ml、280μg/ml、560μg/ml、840μg/ml)标记小鼠RAW264.7巨噬细胞,采用普鲁士蓝染色检测细胞标记率,台盼蓝染色检测细胞活性,四唑盐(MTT)比色实验检测细胞增殖能力,中性红吞噬实验

  6. Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors.

    Science.gov (United States)

    Wyckoff, Jeffrey B; Wang, Yarong; Lin, Elaine Y; Li, Jiu-feng; Goswami, Sumanta; Stanley, E Richard; Segall, Jeffrey E; Pollard, Jeffrey W; Condeelis, John

    2007-03-15

    Although the presence of macrophages in tumors has been correlated with poor prognosis, until now there was no direct observation of how macrophages are involved in hematogenous metastasis. In this study, we use multiphoton microscopy to show, for the first time, that tumor cell intravasation occurs in association with perivascular macrophages in mammary tumors. Furthermore, we show that perivascular macrophages of the mammary tumor are associated with tumor cell intravasation in the absence of local angiogenesis. These results show that the interaction between macrophages and tumor cells lying in close proximity defines a microenvironment that is directly involved in the intravasation of cancer cells in mammary tumors.

  7. 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.

  8. Inhibitory Effects of Benzaldehyde Derivatives from the Marine Fungus Eurotium sp. SF-5989 on Inflammatory Mediators via the Induction of Heme Oxygenase-1 in Lipopolysaccharide-Stimulated RAW264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Kyoung-Su Kim

    2014-12-01

    Full Text Available Two benzaldehyde derivatives, flavoglaucin (1 and isotetrahydro-auroglaucin (2, were isolated from the marine fungus Eurotium sp. SF-5989 through bioassay- and 1H NMR-guided investigation. In this study, we evaluated the anti-inflammatory effects of these compounds in lipopolysaccharide (LPS-stimulated RAW264.7 macrophages. We demonstrated that compounds 1 and 2 markedly inhibited LPS-induced nitric oxide (NO and prostaglandin E2 (PGE2 production by suppressing inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2 protein expression without affecting cell viability. We also demonstrated that the compounds reduced the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α, interleukin-1β (IL-1β and interleukin-6 (IL-6. Furthermore, compounds 1 and 2 inhibited LPS-induced nuclear factor-κB (NF-κB activation by suppressing phosphorylation of IkappaB (IκB. These results indicated that the anti-inflammatory effects of these benzaldehyde derivatives in LPS-stimulated RAW264.7 macrophages were due to the inactivation of the NF-κB pathway. In addition, compounds 1 and 2 induced heme oxygenase-1 (HO-1 expression through the nuclear transcription factor-E2–related factor 2 (Nrf2 translocation. The inhibitory effects of compounds 1 and 2 on the production of pro-inflammatory mediators and on NF-κB binding activity were reversed by HO-1 inhibitor tin protoporphyrin (SnPP. Thus, the anti-inflammatory effects of compounds 1 and 2 also correlated with their ability of inducing HO-1 expression.

  9. Inhibitory effects of benzaldehyde derivatives from the marine fungus Eurotium sp. SF-5989 on inflammatory mediators via the induction of heme oxygenase-1 in lipopolysaccharide-stimulated RAW264.7 macrophages.

    Science.gov (United States)

    Kim, Kyoung-Su; Cui, Xiang; Lee, Dong-Sung; Ko, Wonmin; Sohn, Jae Hak; Yim, Joung Han; An, Ren-Bo; Kim, Youn-Chul; Oh, Hyuncheol

    2014-12-19

    Two benzaldehyde derivatives, flavoglaucin (1) and isotetrahydro-auroglaucin (2), were isolated from the marine fungus Eurotium sp. SF-5989 through bioassay- and 1H NMR-guided investigation. In this study, we evaluated the anti-inflammatory effects of these compounds in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. We demonstrated that compounds 1 and 2 markedly inhibited LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production by suppressing inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression without affecting cell viability. We also demonstrated that the compounds reduced the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Furthermore, compounds 1 and 2 inhibited LPS-induced nuclear factor-κB (NF-κB) activation by suppressing phosphorylation of IkappaB (IκB). These results indicated that the anti-inflammatory effects of these benzaldehyde derivatives in LPS-stimulated RAW264.7 macrophages were due to the inactivation of the NF-κB pathway. In addition, compounds 1 and 2 induced heme oxygenase-1 (HO-1) expression through the nuclear transcription factor-E2-related factor 2 (Nrf2) translocation. The inhibitory effects of compounds 1 and 2 on the production of pro-inflammatory mediators and on NF-κB binding activity were reversed by HO-1 inhibitor tin protoporphyrin (SnPP). Thus, the anti-inflammatory effects of compounds 1 and 2 also correlated with their ability of inducing HO-1 expression.

  10. Ascofuranone inhibits lipopolysaccharide-induced inflammatory response via NF-kappaB and AP-1, p-ERK, TNF-α, IL-6 and IL-1β in RAW 264.7 macrophages.

    Science.gov (United States)

    Park, Jun-Young; Chung, Tae-Wook; Jeong, Yun-Jeong; Kwak, Choong-Hwan; Ha, Sun-Hyung; Kwon, Kyung-Min; Abekura, Fukushi; Cho, Seung-Hak; Lee, Young-Choon; Ha, Ki-Tae; Magae, Junji; Chang, Young-Chae; Kim, Cheorl-Ho

    2017-01-01

    The natural fungal compound ascofuranone (5-chloro-3-[(2E,6E)-7-[(2S)-5,5-dimethyl-4-oxo-tetrahydrofuran-2-yl]-3-methyl-octa-2,6-dienyl]-2,4-dihydroxy-6-methyl-benzaldehyde, MW 420.93) (AF) isolated from Ascochyta viciae has been known to promote cell cycle arrest and inhibit invasion of tumor cells. We have previously studied a structurally similar compound ascochlorin (ASC; MW 404.93) with regard to its anti-inflammatory activity in LPS- stimulated RAW 264.7 macrophages. In order to examine the relationship between the anti-inflammatory activities and the molecular differences between AF and ASC, the activity of AF is herein studied, because ASC has a unique trimethyl oxocyclohexyl structure, while AF has a unique dimethyl-oxo-tetrahydrofuran structure. AF dose-dependently inhibited the production of NO and iNOS and the COX-2 mRNA and protein levels in RAW 264.7 cells. In addition, AF suppressed mRNA expression levels of inflammatory cytokines such as TNF-α, IL-6, and IL-1β, as assessed by RT-PCR. AF (30-50 μg/ml) treatment clearly inhibited the nuclear translocation of NF-κB, AP-1 (p-c-Jun) from the cytosolic space. Phosphorylation of IκB, which functions to maintain the activity of NF-κB, was decreased by AF treatment. Moreover, AF suppressed the binding of NF-κB (p65). Inhibition of IkBa phosphorylation and degradation inhibits nuclear translocation of p65. Immunofluorescence confocal microscopy analysis also revealed that translocation of NF-κB and AP-1 (p-c-Jun) was decreased upon AF treatment. AF specifically decreased the expression level of p-ERK, but not the expression level of p-p38 or p-JNK. Given these results, we suggest that AF suppresses the inflammatory response by targeting p-ERK. This indicates that AF is a negative regulator of LPS-stimulated nuclear translocation of NF-κB and AP-1 (p-c-Jun) in RAW 264.7 macrophages, and specifically it targets p-ERK. Therefore, AF and ASC exert their effects in different ways, most probably because

  11. Anti-inflammatory activity of the active components from the roots of Cosmos bipinnatus in lipopolysaccharide-stimulated RAW 264.7 macrophages.

    Science.gov (United States)

    Sohn, Sang-Hyun; Yun, Bong-Sik; Kim, So-Young; Choi, Wahn-Soo; Jeon, Hyun-Soo; Yoo, Jun-Sik; Kim, Si-Kwan

    2013-01-01

    We isolated a sesquiterpene lactone from the methanol extract of the roots of Cosmos bipinnatus, namely, MDI (a mixture of dihydrocallitrisin and isohelenin). The anti-inflammatory activity of MDI was evaluated using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. MDI significantly inhibited the expression of inducible nitric oxide synthase and cyclooxygenase-2. Consistent with these results, the production of NO and prostaglandin E2 (PGE2) was suggested to be suppressed by MDI in a concentration-dependent manner (IC50 value was 0.94 and 2.88 µg mL(-1) for NO and PGE2, respectively). In addition, MDI significantly inhibited the expressions of pro-inflammatory cytokines such as IL-1β, IL-6, IFN-γ and TNF-α. Furthermore, MDI attenuated DNA-binding activity of NF-κB by inhibiting the phosphorylation of IκB. These results indicate that MDI isolated from the roots of C. bipinnatus shows anti-inflammatory activity in LPS-stimulated murine macrophages by modulating the NF-κB pathway.

  12. The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages.

    Directory of Open Access Journals (Sweden)

    Marc Daigneault

    Full Text Available Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. The phenotype of mature tissue macrophages represents the composite of environmental and differentiation-dependent imprinting. Phorbol-12-myristate-13-acetate (PMA and 1,25-dihydroxyvitamin D3 (VD(3 are stimuli commonly used to induce macrophage differentiation in monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. We have compared the phenotype of the promonocytic THP-1 cell line after various protocols of differentiation utilising VD(3 and PMA in comparison to primary human monocytes or monocyte-derived macrophages (MDM. Both stimuli induced changes in cell morphology indicative of differentiation but neither showed differentiation comparable to MDM. In contrast, PMA treatment followed by 5 days resting in culture without PMA (PMAr increased cytoplasmic to nuclear ratio, increased mitochondrial and lysosomal numbers and altered differentiation-dependent cell surface markers in a pattern similar to MDM. Moreover, PMAr cells showed relative resistance to apoptotic stimuli and maintained levels of the differentiation-dependent anti-apoptotic protein Mcl-1 similar to MDM. PMAr cells retained a high phagocytic capacity for latex beads, and expressed a cytokine profile that resembled MDM in response to TLR ligands, in particular with marked TLR2 responses. Moreover, both MDM and PMAr retained marked plasticity to stimulus-directed polarization. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells.

  13. Macrophages eat cancer cells using their own calreticulin as a guide: roles of TLR and Btk.

    Science.gov (United States)

    Feng, Mingye; Chen, James Y; Weissman-Tsukamoto, Rachel; Volkmer, Jens-Peter; Ho, Po Yi; McKenna, Kelly M; Cheshier, Samuel; Zhang, Michael; Guo, Nan; Gip, Phung; Mitra, Siddhartha S; Weissman, Irving L

    2015-02-17

    Macrophage-mediated programmed cell removal (PrCR) is an important mechanism of eliminating diseased and damaged cells before programmed cell death. The induction of PrCR by eat-me signals on tumor cells is countered by don't-eat-me signals such as CD47, which binds macrophage signal-regulatory protein α to inhibit phagocytosis. Blockade of CD47 on tumor cells leads to phagocytosis by macrophages. Here we demonstrate that the activation of Toll-like receptor (TLR) signaling pathways in macrophages synergizes with blocking CD47 on tumor cells to enhance PrCR. Bruton's tyrosine kinase (Btk) mediates TLR signaling in macrophages. Calreticulin, previously shown to be an eat-me signal on cancer cells, is activated in macrophages for secretion and cell-surface exposure by TLR and Btk to target cancer cells for phagocytosis, even if the cancer cells themselves do not express calreticulin.

  14. Maleylated-BSA suppresses lipopolysaccharide-induced IL-6 production by activating the ERK-signaling pathway in murine RAW264.7 cells.

    Science.gov (United States)

    Tada, Rui; Koide, Yusuke; Yamamuro, Mitsuaki; Tanaka, Riki; Hidaka, Akira; Nagao, Koichiro; Aramaki, Yukihiko

    2014-03-01

    Macrophages are well known for their ability to induce diverse beneficial immune responses, especially in the defense against pathogens. However, an excessive activation of macrophages may cause harmful inflammation. In this context, the suppression of excessive macrophage activation would be a promising therapeutic strategy for treating inflammatory diseases. We have previously found that maleylated-bovine serum albumin (maleylated-BSA) suppresses the production of inflammatory mediators in murine macrophages. However, the immunosuppressive effects and underlying mechanism(s) of maleylated-BSA remain unclear. Here, we report that pretreatment with maleylated-BSA strongly inhibited the production of interleukin 6 (IL-6) induced by bacterial lipopolysaccharide (LPS) in murine RAW264.7 cells. This inhibitory effect of maleylated-BSA on LPS-induced IL-6 production was eliminated by treatment with an extracellular signal-regulated kinase (ERK) inhibitor, U0126, indicating the involvement of ERK pathways. Taken together, we have shown that maleylated-BSA suppresses LPS-induced production of IL-6 via the activation of an ERK signaling pathway in murine macrophages. The findings of this study imply the possibility of a novel therapeutic strategy for inflammatory diseases.

  15. A study of the effects of citrate-coated silver nanoparticles on RAW 264.7 cells using a toolbox of cytotoxic endpoints

    Science.gov (United States)

    Bastos, V.; Duarte, I. F.; Santos, C.; Oliveira, H.

    2017-05-01

    Citrate-coated silver nanoparticles (citrate-AgNPs) are among the most commonly used nanomaterials, widely present in industrial and biomedical products. In this study, the cytotoxicity of 30-nm citrate-AgNPs on the macrophage cell line RAW 264.7 was evaluated, using a battery of cytotoxicity endpoints (viability, oxidative stress, and cytostaticity/clastogenicity), at 24 and 48 h of exposure. Citrate-AgNPs decreased cell proliferation and viability only at 75 μg/mL, suggesting a low sensitivity of RAW cells to lower doses of these AgNPs. After 24 h of exposure, ROS content decreased in cells exposed to 60 μg/mL AgNPs (IC20 value), corroborating the high tolerance of these cells to citrate-AgNPs. However, these cells suffered an impairment of the cell cycle, shown by an increase at the sub-G1 phase. This increase of the sub-G1 population was correlated with an increase of DNA fragmentation, suggesting an increase of apoptosis. Thus, our data are important to understand the effects of low concentrations (IC20) of citrate-AgNPs on in vitro vital macrophage functions.

  16. Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages

    OpenAIRE

    Casadevall Arturo; Alvarez Mauricio

    2007-01-01

    Abstract Background The interaction between macrophages and Cryptococcus neoformans (Cn) is critical for containing dissemination of this pathogenic yeast. However, Cn can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. Both events result in live extracellular yeasts capable of reproducing and disseminating in the extracellular milieu. Another method of exiting the intracellular confines of cells is through host cell-to-cell transfer of the ...

  17. Soluble factor from murine bladder tumor-2 cell elevates nitric oxide production in macrophages and enhances the taxol-mediated macrophage cytotoxicity on tumor cells.

    Science.gov (United States)

    Choi, Suck-Chei; Oh, Hyun-Mee; Park, Jae-Sung; Han, Weon-Cheol; Yoon, Kwon-Ha; Kim, Tae-Hyeon; Yun, Ki-Jung; Kim, Eun-Cheol; Nah, Yong-Ho; Cha, Young-Nam; Chung, Hun-Taeg; Jun, Chang-Duk

    2003-01-01

    The therapeutic mechanism of taxol is believed to reside primarily in its ability to stabilize microtubules and prevent cell progression through mitosis. Taxol also can activate macrophage-mediated antitumor mechanism through a nitric oxide (NO)-dependent pathway. To address whether any mechanisms account for superficial urinary bladder tumor cell killing, we evaluated the effects of taxol on the growth and viability of murine bladder tumor-2 (MBT-2) cells in vitro, both in the absence and presence of murine macrophages. In addition, we evaluated whether a soluble factor generated from MBT-2 cells could modulate the antitumor activity of the taxol-activated macrophages. Although taxol inhibited the growth of MBT-2 cells, it did not kill the tumor cells. However, preincubation of macrophages with taxol significantly decreased the viability of MBT-2 cells. Secretion of NO correlated with MBT-2 cell killing, and the activated macrophages failed to kill tumor cell targets in the presence of NG-monomethyl-L-arginine, a competitive inhibitor of NO synthase. By the co-culture of macrophages and MBT-2 cells, untreated macrophages also released modest amount of NO and this was synergistically augmented by the treatment with taxol, indicating that MBT-2 tumor cells released some unknown factor that activated the macrophages and enhanced NO production. We named this factor the tumor-derived macrophage activating factor (TMAF). The TMAF-mediated activation of macrophages to enhance the NO production was not blocked by treatment of macrophages with oxidized low-density lipoprotein (Ox-LDL), implying that the scavenger receptor of macrophages is not involved. Sodium nitroprusside (SNP), an NO donor given to the MBT-2 cells, increased the activities of c-Jun N-terminal kinase and caspase-3 in MBT-2 cells and associated with nucleosomal fragmentation or apoptosis, whereas taxol had no direct effect on these parameters. Collectively, our results strongly suggest that taxol kills

  18. Generation of dendritic cells and macrophages from human induced pluripotent stem cells aiming at cell therapy.

    Science.gov (United States)

    Senju, S; Haruta, M; Matsumura, K; Matsunaga, Y; Fukushima, S; Ikeda, T; Takamatsu, K; Irie, A; Nishimura, Y

    2011-09-01

    This report describes generation of dendritic cells (DCs) and macrophages from human induced pluripotent stem (iPS) cells. iPS cell-derived DC (iPS-DC) exhibited the morphology of typical DC and function of T-cell stimulation and antigen presentation. iPS-DC loaded with cytomegalovirus (CMV) peptide induced vigorous expansion of CMV-specific autologous CD8+ T cells. Macrophages (iPS-MP) with activity of zymosan phagocytosis and C5a-induced chemotaxis were also generated from iPS cells. Genetically modified iPS-MPs were generated by the introduction of expression vectors into undifferentiated iPS cells, isolation of transfectant iPS cell clone and subsequent differentiation. By this procedure, we generated iPS-MP expressing a membrane-bound form of single chain antibody (scFv) specific to amyloid β (Aβ), the causal protein of Alzheimer's disease. The scFv-transfectant iPS-MP exhibited efficient Aβ-specific phagocytosis activity. iPS-MP expressing CD20-specific scFv engulfed and killed BALL-1 B-cell leukemia cells. Anti-BALL-1 effect of iPS-MP in vivo was demonstrated in a xeno-transplantation model using severe combined immunodeficient mice. In addition, we established a xeno-free culture protocol to generate iPS-DC and iPS-MP. Collectively, we demonstrated the possibility of application of iPS-DC and macrophages to cell therapy.

  19. Adenosine derived from Staphylococcus aureus-engulfed macrophages functions as a potent stimulant for the induction of inflammatory cytokines in mast cells

    DEFF Research Database (Denmark)

    Ma, Ying Jie; Kim, Chan-Hee; Ryu, Kyoung-Hwa;

    2011-01-01

    In this study, we attempted to isolate novel mast cell-stimulating molecules from Staphylococcus aureus. Water-soluble extract of S. aureus cell lysate strongly induced human interleukin- 8 in human mast cell line-1 and mouse interleukin-6 in mouse bone marrow-derived mast cells. The active...... adenosine receptor blocker, verified that purified adenosine can induce interleukin-8 production via adenosine receptors on mast cells. Moreover, adenosine was purified from S. aureusengulfed RAW264.7 cells, a murine macrophage cell line, used to induce phagocytosis of S. aureus. These results show a novel...

  20. The nucleotide receptor P2X7 mediates actin reorganization and membrane blebbing in RAW 264.7 macrophages via p38 MAP kinase and Rho.

    Science.gov (United States)

    Pfeiffer, Zachary A; Aga, Mini; Prabhu, Usha; Watters, Jyoti J; Hall, David J; Bertics, Paul J

    2004-06-01

    Extracellular nucleotides regulate macrophage function via P2X nucleotide receptors that form ligand-gated ion channels. In particular, P2X7 activation is characterized by pore formation, membrane blebbing, and cytokine release. P2X7 is also linked to mitogen-activated protein kinases (MAPK) and Rho-dependent pathways, which are known to affect cytoskeletal structure in other systems. As cytoskeletal function is critical for macrophage behavior, we have tested the importance of these pathways in actin filament reorganization during P2X7 stimulation in RAW 264.7 macrophages. We observed that the P2X7 agonists adenosine 5'-triphosphate (ATP) and 3'-O-(4-benzoylbenzoyl) ATP (BzATP) stimulated actin reorganization and concomitant membrane blebbing within 5 min. Disruption of actin filaments with cytochalasin D attenuated membrane blebbing but not P2X7-dependent pore formation or extracellular-regulated kinase (ERK)1/ERK2 and p38 activation, suggesting that these latter processes do not require intact actin filaments. However, we provide evidence that p38 MAPK and Rho activation but not ERK1/ERK2 activation is important for P2X7-mediated actin reorganization and membrane blebbing. First, activation of p38 and Rho was detected within 5 min of BzATP treatment, which is coincident with membrane blebbing. Second, the p38 inhibitors SB202190 and SB203580 reduced nucleotide-induced blebbing and actin reorganization, whereas the MAPK kinase-1/2 inhibitor U0126, which blocks ERK1/ERK2 activation, had no discernable effect. Third, the Rho-selective inhibitor C3 exoenzyme and the Rho effector kinase, Rho-associated coiled-coil kinase, inhibitor Y-27632, markedly attenuated BzATP-stimulated actin reorganization and membrane blebbing. These data support a model wherein p38- and Rho-dependent pathways are critical for P2X7-dependent actin reorganization and membrane blebbing, thereby facilitating P2X7 involvement in macrophage inflammatory responses.

  1. Biotransformation products of phellopterin by rat liver microsomes and the inhibition on NO production in LPS-activated RAW264.7 cells.

    Science.gov (United States)

    Zhao, Ai-Hong; Yang, Xin-Bao; Yang, Xiu-Wei; Zhang, You-Bo; Xu, Wei; Liu, Jian-Xun

    2012-01-01

    Four new coumarins (2',3'-dihydroxyphellopterin, E-5-methoxytrichoclin acetate, Z-5-methoxytrichoclin acetate, and E-5-methoxytrichoclin) and three known coumarins (byakangelicol, byakangelicin, and Z-5-methoxytrichoclin) were produced by liver microsomes from rats pre-treated with sodium phenobarbital. The chemical structures were elucidated on the basis of their spectroscopic data. The inhibitory activities of nitric oxide (NO) production in lipopolysaccharide-activated macrophage-like cell line RAW264.7 were tested. The main biotransformation product, byakangelicin, showed inhibitory activities of NO production with the IC₅₀ value of 217.83 μM, whereas the parent compound phellopterin showed cytotoxic effect on RAW264.7 cell at the concentration from 40 to 400 μM.

  2. A novel pro-inflammatory protein of Streptococcus suis 2 induces the Toll-like receptor 2-dependent expression of pro-inflammatory cytokines in RAW 264.7 macrophages via activation of ERK1/2 pathway.

    Science.gov (United States)

    Zhang, Qiang; Yang, Yujie; Yan, Shuxian; Liu, Jiantao; Xu, Zhongmin; Yu, Junping; Song, Yajing; Zhang, Anding; Jin, Meilin

    2015-01-01

    Streptococcus suis 2 is an important swine pathogen and an emergent zoonotic pathogen. Excessive inflammation caused by S. suis is responsible for the high levels of early mortality observed in septic shock-like syndrome cases. However, the mechanisms through which S. suis 2 (SS2) causes excessive inflammation remain unclear. Thus, this study aimed to identify novel pro-inflammatory mediators that play important roles in the development of therapies against SS2 infection. In this study, the novel pro-inflammatory protein HP0459, which was encoded by the SSUSC84_0459 gene, was discovered. The stimulation of RAW 264.7 macrophages with recombinant HP0459 protein induced the expression of pro-inflammatory cytokines (IL-1β, MCP-1 and TNF-α). Compared with the wild-type (WT) strain, the isogenic knockout of HP0459 in SS2 led to reduced production of pro-inflammatory cytokines in RAW264.7 macrophages and in vivo. The pro-inflammatory activity of HP0459 was significantly reduced by an antibody against Toll-like receptor 2 (TLR2) in RAW264.7 macrophages and was lower in TLR2-deficient (TLR2-/-) macrophages than in WT macrophages. Furthermore, specific inhibitors of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathways significantly decreased the HP0459-induced pro-inflammatory cytokine production, and a western blot assay showed that HP0459 stimulation induced the activation of the ERK1/2 pathway. Taken together, our data indicate that HP0459 is a novel pro-inflammatory mediator of SS2 and induces TLR2-dependent pro-inflammatory activity in RAW264.7 macrophages through the ERK1/2 pathway.

  3. Anti-inflammatory effects of Viola yedoensis and the application of cell extraction methods for investigating bioactive constituents in macrophages.

    Science.gov (United States)

    Jeong, Yun Hee; Oh, You-Chang; Cho, Won-Kyung; Shin, Hyeji; Lee, Ki Yong; Ma, Jin Yeul

    2016-06-14

    Viola yedoensis (VY, Violaceae) is a popular medicinal herb used in traditional eastern medicine for treating lots of diseases, including inflammation and its related symptoms. However, the anti-inflammatory properties of VY have not been demonstrated. In the present study, we investigated the anti-inflammatory effects of VY ethanol extract (VYE) on macrophages and attempted to identify the bioactive components of VYE. We assessed the effects of VYE on secretion of nitric oxide (NO) and inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β. In addition, we explored the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and changes in heme oxygenase (HO)-1, nuclear factor (NF)-kB, and mitogen-activated protein kinase (MAPK) signaling pathways in RAW 264.7 macrophages stimulated by lipopolysaccharide (LPS). In addition, a rapid and useful approach to identify potential bioactive components in VYE with anti-inflammatory effects was developed using murine macrophage cell extraction coupled with high-performance liquid chromatography tandem mass spectrometry (LC-MS). We found that VYE exerted anti-inflammatory activity by inhibiting the production of key inflammation mediators and related products, as well as suppression of HO-1, NF-kB, and MAPK signaling pathway activation in RAW 264.7 cells. In addition, we identified two compounds in VYE via the cell extraction method. Our results revealed that VYE exerts anti-inflammatory activities and its detailed inhibitory mechanism in macrophages. Furthermore, we identified bioactive components of VYE.

  4. Escherichia coli Prevents Phagocytosis-Induced Death of Macrophages via Classical NF-κB Signaling, a Link to T-Cell Activation

    OpenAIRE

    Groesdonk, Heinrich V.; Schlottmann, Silke; Richter, Friederike; Georgieff, Michael; Senftleben, Uwe

    2006-01-01

    NF-κB is a crucial mediator of macrophage inflammatory responses, but its role in the context of pathogen-induced adaptive immune responses has yet to be elucidated. Here, we demonstrate that classical NF-κB activation delays phagocytosis-induced cell death (PICD) in Raw 264.7 and bone marrow-derived macrophages (BMDMs) upon ingestion of bacteria from the Escherichia coli laboratory strain Top10. By expression of a nondegradable form of IκBα (superrepressor) and pyrrolidine dithiocarbamate tr...

  5. Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages

    Directory of Open Access Journals (Sweden)

    Casadevall Arturo

    2007-08-01

    Full Text Available Abstract Background The interaction between macrophages and Cryptococcus neoformans (Cn is critical for containing dissemination of this pathogenic yeast. However, Cn can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. Both events result in live extracellular yeasts capable of reproducing and disseminating in the extracellular milieu. Another method of exiting the intracellular confines of cells is through host cell-to-cell transfer of the pathogen, and this commonly occurs with the human immuno-deficiency virus (HIV and CD4+ T cells and macrophages. In this report we have used time-lapse imaging to determine if this occurs with Cn. Results Live imaging of Cryptococcus neoformans interactions with murine macrophages revealed cell-to-cell spread of yeast cells from infected donor cells to uninfected cells. Although this phenomenon was relatively rare its occurrence documents a new capacity for this pathogen to infect adjacent cells without exiting the intracellular space. Cell-to-cell spread appeared to be an actin-dependent process. In addition, we noted that cryptococcal phagosomal extrusion was followed by the formation of massive vacuoles suggesting that intracellular residence is accompanied by long lasting damage to host cells. Conclusion C. neoformans can escape the intracellular confines of macrophages in an actin dependent manner by cell-to-cell transfer of the yeast leading to infection of adjacent cells. In addition, complete extrusion of internalized Cn cells can lead to the formation of a massive vacuole which may be a sign of damage to the host macrophage. These observations document new outcomes for the interaction of C. neoformans with host cells that provide precedents for cell biological effects that may contribute to the pathogenesis of cryptococcal infections.

  6. Macrophage polarization in nerve injury:do Schwann cells play a role?

    Institute of Scientific and Technical Information of China (English)

    Jo Anne Stratton; Prajay T Shah

    2016-01-01

    In response to peripheral nerve injury, the inlfammatory response is almost entirely comprised of inifltrat-ing macrophages. Macrophages are a highly plastic, heterogenic immune cell, playing an indispensable role in peripheral nerve injury, clearing debris and regulating the microenvironment to allow for efifcient regen-eration. There are several cells within the microenvironment that likely interact with macrophages to support their function –most notably the Schwann cell, the glial cell of the peripheral nervous system. Schwann cells express several ligands that are known to interact with receptors expressed by macrophages, yet the effects of Schwann cells in regulating macrophage phenotype remains largely unexplored. This review discusses macrophages in peripheral nerve injury and how Schwann cells may regulate their behavior.

  7. Effect of Berberine on Mice RAW264.7 Macrophages Polarization%小檗碱对小鼠RAW264.7巨噬细胞极化的影响

    Institute of Scientific and Technical Information of China (English)

    朱瑞丽; 吴阳阳; 罗进芳; 易浪; 董燕

    2014-01-01

    Objective To investigate the effect of berberine on the polarization of mice RAW264.7 macrophages induced separately by lipopolysaccharide (LPS) and interleukin-4 (IL-4). Methods Mice RAW 264.7 macrophages cultured in vitro were divided into model group, medication group, and blank control group. Both model group and medication group were given either LPS (in final dose of 100 ng/mL) or IL-4 (in final dose of 10 ng/mL). Additionally, the medication group was treated with berberine in final dose of 20 μmol/L. The blank control group was given the same volume of phosphate buffered saline ( PBS). Real-time fluorescence quantitative polymerase chain reaction (PCR) was used to detect the mRNA expression of arginase-1 (Arg-1), inducible nitric oxide synthase ( iNOS) , suppressor of cytokine signaling2 ( SOCS2) and SOCS3. Enzyme-linked immunosorbent assay (ELISA) was used to determine the contents of tumor necrosis factor alpha (TNF-α) and IL-10. Results The content of TNF-αand the mRNA expression levels of iNOS and SOCS3 in macrophages induced by LPS were increased, and then were down-regulated by berberine (P0.05). Conclusion Berberine has an effect on inhibiting the M1 and M2 polarization of macrophages in vitro, suggesting that berberine may play a regulatory role in the dynamic balance of M1/M2.%目的观察小檗碱对脂多糖(LPS)、白细胞介素-4(IL-4)诱导的小鼠RAW264.7细胞极化的影响。方法体外培养小鼠巨噬细胞RAW264.7,模型组和给药组分别加入LPS (终浓度100 ng/mL)或IL-4(终浓度10 ng/mL),给药组用终浓度20μmol/L的小檗碱分别进行干预,同时空白对照组给予等体积磷酸盐缓冲液(PBS)。采用荧光定量聚合酶链反应检测精氨酸酶-1( Arg-1)、一氧化氮合酶( iNOS)、细胞因子信号传导抑制蛋白2( SOCS2)、细胞因子信号传导抑制蛋白3(SOCS3)的mRNA表达水平,采用酶联免疫吸附(ELISA)法检测

  8. A comparison of cytotoxicity and oxidative stress from welding fumes generated with a new nickel-, copper-based consumable versus mild and stainless steel-based welding in RAW 264.7 mouse macrophages.

    Directory of Open Access Journals (Sweden)

    Melissa A Badding

    Full Text Available Welding processes that generate fumes containing toxic metals, such as hexavalent chromium (Cr(VI, manganese (Mn, and nickel (Ni, have been implicated in lung injury, inflammation, and lung tumor promotion in animal models. While federal regulations have reduced permissible worker exposure limits to Cr(VI, this is not always practical considering that welders may work in confined spaces and exhaust ventilation may be ineffective. Thus, there has been a recent initiative to minimize the potentially hazardous components in welding materials by developing new consumables containing much less Cr(VI and Mn. A new nickel (Ni and copper (Cu-based material (Ni-Cu WF is being suggested as a safer alternative to stainless steel consumables; however, its adverse cellular effects have not been studied. This study compared the cytotoxic effects of the newly developed Ni-Cu WF with two well-characterized welding fumes, collected from gas metal arc welding using mild steel (GMA-MS or stainless steel (GMA-SS electrodes. RAW 264.7 mouse macrophages were exposed to the three welding fumes at two doses (50 µg/ml and 250 µg/ml for up to 24 hours. Cell viability, reactive oxygen species (ROS production, phagocytic function, and cytokine production were examined. The GMA-MS and GMA-SS samples were found to be more reactive in terms of ROS production compared to the Ni-Cu WF. However, the fumes from this new material were more cytotoxic, inducing cell death and mitochondrial dysfunction at a lower dose. Additionally, pre-treatment with Ni-Cu WF particles impaired the ability of cells to phagocytize E. coli, suggesting macrophage dysfunction. Thus, the toxic cellular responses to welding fumes are largely due to the metal composition. The results also suggest that reducing Cr(VI and Mn in the generated fume by increasing the concentration of other metals (e.g., Ni, Cu may not necessarily improve welder safety.

  9. Short Communication: HIV Controller T Cells Effectively Inhibit Viral Replication in Alveolar Macrophages.

    Science.gov (United States)

    Walker-Sperling, Victoria E; Merlo, Christian A; Buckheit, Robert W; Lambert, Allison; Tarwater, Patrick; Kirk, Greg D; Drummond, M Bradley; Blankson, Joel N

    Macrophages are targets of HIV-1 infection, and control of viral replication within these cells may be an important component of a T-cell-based vaccine. Although several studies have analyzed the ability of CD8(+) T cells to inhibit viral replication in monocyte-derived macrophages, the effect of T cells on HIV-1-infected tissue macrophages is less clear. We demonstrate here that both CD4(+) and CD8(+) T-cell effectors from HIV controllers are capable of suppressing viral replication in bronchoalveolar lavage-derived alveolar macrophages. These findings have implications for HIV-1 vaccine and eradication strategies.

  10. Secondary Metabolites from Fungal Endophytes of Echinacea purpurea Suppress Cytokine Secretion by Macrophage-Type Cells.

    Science.gov (United States)

    Kaur, Amninder; Oberhofer, Martina; Juzumaite, Monika; Raja, Huzefa A; Gulledge, Travis V; Kao, Diana; Faeth, Stanley H; Laster, Scott M; Oberlies, Nicholas H; Cech, Nadja B

    2016-01-01

    Botanical extracts of Echinacea purpurea have been widely used for the treatment of upper respiratory infections. We sought to chemically examine fungal endophytes inhabiting E. purpurea, and to identify compounds produced by these endophytes with in vitro cytokine-suppressive activity. Twelve isolates from surface sterilized seeds of E. purpurea were subjected to fractionation and major components were isolated. Sixteen secondary metabolites belonging to different structural classes were identified from these isolates based on NMR and mass spectrometry data. The compounds were tested for their influence on cytokine secretion by murine macrophage-type cells. Alternariol (1), O-prenylporriolide (4), porritoxin (10) β-zearalenol (13), and (S)-zearalenone (14) inhibited production of TNF-α from RAW 264.7 macrophages stimulated with LPS in the absence of any significant cytotoxicity. This is the first report of a cytokine-suppressive effect for 4. The results of this study are particularly interesting given that they show the presence of compounds with cytokine-suppressive activity in endophytes from a botanical used to treat inflammation. Future investigations into the role of fungal endophytes in the biological activity of E. purpurea dietary supplements may be warranted.

  11. Inhibitory effects of Enteromorpha prolifera on the production of nitric oxide, prostaglandin E2, and pro-inflammatory cytokines in RAW 264.7 cells

    OpenAIRE

    Yoon, Weon-Jong; Kim, Dong Sam; Yang, Eun-Jin; Moon, Ji-Young; Kim, Min-Jin; Lee, Wook Jae; Lee, Nam Ho; Hyun, Chang-Gu

    2010-01-01

    Inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 have been used as tools for the screening of anti-inflammatory agents. In a search for inhibitors of COX-2 and iNOS, we found that extracts of Enteromorpha prolifera inhibit the production of nitric oxide (NO) and prostaglandin (PG)E2 in LPS-stimulated RAW 264.7 macrophage cells. We first extracted E. prolifera with 80% ethanol and the extract was partitioned with hexane, dichloromethane, ethyl acetate, butanol, and water, succ...

  12. Dynamics of Salmonella infection of macrophages at the single cell level.

    Science.gov (United States)

    Gog, Julia R; Murcia, Alicia; Osterman, Natan; Restif, Olivier; McKinley, Trevelyan J; Sheppard, Mark; Achouri, Sarra; Wei, Bin; Mastroeni, Pietro; Wood, James L N; Maskell, Duncan J; Cicuta, Pietro; Bryant, Clare E

    2012-10-07

    Salmonella enterica causes a range of diseases. Salmonellae are intracellular parasites of macrophages, and the control of bacteria within these cells is critical to surviving an infection. The dynamics of the bacteria invading, surviving, proliferating in and killing macrophages are central to disease pathogenesis. Fundamentally important parameters, however, such as the cellular infection rate, have not previously been calculated. We used two independent approaches to calculate the macrophage infection rate: mathematical modelling of Salmonella infection experiments, and analysis of real-time video microscopy of infection events. Cells repeatedly encounter salmonellae, with the bacteria often remain associated with the macrophage for more than ten seconds. Once Salmonella encounters a macrophage, the probability of that bacterium infecting the cell is remarkably low: less than 5%. The macrophage population is heterogeneous in terms of its susceptibility to the first infection event. Once infected, a macrophage can undergo further infection events, but these reinfection events occur at a lower rate than that of the primary infection.

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

    Directory of Open Access Journals (Sweden)

    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

  14. Microvesicles secreted by macrophages shuttle invasion-potentiating microRNAs into breast cancer cells

    Directory of Open Access Journals (Sweden)

    Lin Ling

    2011-09-01

    Full Text Available Abstract Background Tumor-associated macrophages (TAMs are alternatively activated cells induced by interleukin-4 (IL-4-releasing CD4+ T cells. TAMs promote breast cancer invasion and metastasis; however, the mechanisms underlying these interactions between macrophages and tumor cells that lead to cancer metastasis remain elusive. Previous studies have found microRNAs (miRNAs circulating in the peripheral blood and have identified microvesicles, or exosomes, as mediators of cell-cell communication. Therefore, one alternative mechanism for the promotion of breast cancer cell invasion by TAMs may be through macrophage-secreted exosomes, which would deliver invasion-potentiating miRNAs to breast cancer cells. Results We utilized a co-culture system with IL-4-activated macrophages and breast cancer cells to verify that miRNAs are transported from macrophages to breast cancer cells. The shuttling of fluorescently-labeled exogenous miRNAs from IL-4-activated macrophages to co-cultivated breast cancer cells without direct cell-cell contact was observed. miR-223, a miRNA specific for IL-4-activated macrophages, was detected within the exosomes released by macrophages and was significantly elevated in the co-cultivated SKBR3 and MDA-MB-231 cells. The invasiveness of the co-cultivated breast cancer cells decreased when the IL-4-activated macrophages were treated with a miR-223 antisense oligonucleotide (ASO that would inhibit miR-223 expression. Furthermore, results from a functional assay revealed that miR-223 promoted the invasion of breast cancer cells via the Mef2c-β-catenin pathway. Conclusions We conclude that macrophages regulate the invasiveness of breast cancer cells through exosome-mediated delivery of oncogenic miRNAs. Our data provide insight into the mechanisms underlying the metastasis-promoting interactions between macrophages and breast cancer cells.

  15. DMPD: A role for caspases in the differentiation of erythroid cells and macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17905508 A role for caspases in the differentiation of erythroid cells and macropha...;90(2):416-22. Epub 2007 Sep 2. (.png) (.svg) (.html) (.csml) Show A role for caspases in the differentiatio...n of erythroid cells and macrophages. PubmedID 17905508 Title A role for caspases

  16. 姜黄素促进 RAW264.7源性 M1巨噬细胞向替代激活 M2表型极化%Curcumin induces M1 phenotype derived from murine RAW264.7 macrophages polarization to alternatively activated M2 phenotype

    Institute of Scientific and Technical Information of China (English)

    陈方圆; 袁祖贻; 周娟; 王欢; 薛丽; 郭宁

    2015-01-01

    目的:观察姜黄素对 LPS 和 IFNγ诱导的 RAW264.7巨噬细胞(M1)极化的影响及机制。方法不同浓度姜黄素(6.25、12.5、25μmol/L)干预 LPS 和 IFNγ诱导的 RAW264.7巨 噬 细 胞 (M1)12 h,同时再加入20μmol/L GW9662与25μmol/L 姜黄素共同干预 LPS 和 IFNγ诱导的 RAW264.7巨 噬 细 胞(M1)12 h,采用 Real-time PCR、ELISA 及 Western blot 方法检测 IL-1β、IL-6和 M2表型标志分子 KLF4、FIZZ1、MGL1、PPARγ的表达,及阻断PPARγ后 KLF4和 FIZZ1的表达。结果不同浓度姜黄素均能上调 LPS 和 IFNγ诱导的 RAW264.7巨噬细胞(M1)的 M2标志分子的表达,并且抑制炎症因子 IL-1β和 IL-6的分泌;阻断 PPARγ后,RAW264.7巨噬细胞源性 M1表型巨噬细胞表达 M2标志分子下调。结论姜黄素通过活化 PPARγ促进 LPS 和 IFNγ诱导的 RAW264.7巨噬细胞(M1)向 M2表型极化。%ABSTRACT:Objective To observe the effect of curcumin on RAW264.7 macrophages induced with LPS and IFNγ(M1)and the mechanisms involved.Methods Curcumin of different concentrations (6.25 μmol/L,12.5μmol/L and 25 μmol/L)was used to treat RAW264.7 macrophages induced with LPS and IFNγ(M1)for 12 h,and RAW264.7 macrophages induced with LPS and IFNγ(M1)were incubated with 20μmol/L GW9662 and 25 μmol/L curcumin for 12 h.Using Real-time PCR,ELISA and Western blotting analysis,we examined the expressions of IL-1β,IL-6,PPARγand phenotype markers M2 (KLF4,FIZZ1,and MGL1 )and the expressions of KLF4 and FIZZ1 when PPARγwas inhibited.Results Curcumin of different concentrations all could inhibit the expressions of IL-1βand IL-6 in RAW264.7 macrophages induced with LPS and IFNγ(M1).Curcumin of different concentra-tions could upregulate the expression of M2 markers (KLF4,FIZZ1 and MGL1)and PPARγin RAW264.7 macro-phages induced with LPS and IFNγ(M1).When M1 macrophages were incubated with curcumin and GW9662,the expression of the M2 phenotype markers was reduced.Conclusion Curcumin polarized the M

  17. Coriandrum sativum L. seed extract mitigates lipotoxicity in RAW 264.7 cells and prevents atherogenic changes in rats.

    Science.gov (United States)

    Patel, Dipak; Desai, Swati; Gajaria, Tejal; Devkar, Ranjitsinh; Ramachandran, A V

    2013-01-01

    This study was designed to assess the efficacy of Coriandrum sativum L. (CS) in preventing in vitro low density lipoprotein (LDL) oxidation mediated macrophage modification. Further, an in vivo study was also conducted to confirm upon the efficacy of CS seed extract in alleviating pathophysiological alterations of high cholesterol diet induced atherosclerosis in rats. Copper mediated cell free oxidation of LDL accounted for elevated indices of malondialdehyde (MDA), lipid hydroperoxide (LHP)and protein carbonyl (PC) and a progressive increment in conjugate diene (CD) levels whereas, reverse set of changes were recorded in presence of CS extract. Cell mediated LDL oxidation (using RAW 264.7 cells) accounted for lowered MDA production and oxidized LDL (Ox-LDL) mediated cell death in presence of CS extract and the same was attributed to its potent antioxidant and free radical scavenging potentials. High cholesterol fed atherogenic rats showed elevated lipid indices, evidences of LDL oxidation, plaque formation in thoracic aorta. The same was further validated with immunostaining of cell adhesion molecules and hematoxylin and eosin (HXE) staining. However, co-supplementation of CS to atherogenic rats recorded significant lowering of the above mentioned parameters further strengthening the claim that CS extract is instrumental in preventing onset and progression of atherosclerosis.

  18. Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells

    Science.gov (United States)

    Mahamed, Deeqa; Boulle, Mikael; Ganga, Yashica; Mc Arthur, Chanelle; Skroch, Steven; Oom, Lance; Catinas, Oana; Pillay, Kelly; Naicker, Myshnee; Rampersad, Sanisha; Mathonsi, Colisile; Hunter, Jessica; Sreejit, Gopalkrishna; Pym, Alexander S; Lustig, Gila; Sigal, Alex

    2017-01-01

    A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states. DOI: http://dx.doi.org/10.7554/eLife.22028.001 PMID:28130921

  19. Biphasic influence of PGE2 on the resorption activity of osteoclast-like cells derived from human peripheral blood monocytes and mouse RAW264.7 cells.

    Science.gov (United States)

    Lutter, Anne-Helen; Hempel, Ute; Anderer, Ursula; Dieter, Peter

    2016-08-01

    Osteoclasts are large bone-resorbing cells of hematopoietic origin. Their main function is to dissolve the inorganic component hydroxyapatite and to degrade the organic bone matrix. Prostaglandin E2 (PGE2) indirectly affects osteoclasts by stimulating osteoblasts to release factors that influence osteoclast activity. The direct effect of PGE2 on osteoclasts is still controversial. To study the influence of PGE2 on osteoclast activity, human peripheral blood monocytes (hPBMC) and mouse RAW264.7 cells were cultured on osteoblast-derived extracellular matrix. hPBMC and RAW264.7 cells were differentiated by the addition of macrophage colony-stimulation factor and receptor activator of NFκB ligand and treated with PGE2 before and after differentiation induction. The pit area, an indicator of resorption activity, and the activity of tartrate-resistant acid phosphatase were dose-dependently inhibited when PGE2 was present ab initio, whereas the resorption activity remained unchanged when the cells were exposed to PGE2 from day 4 of culture. These results lead to the conclusion that PGE2 treatment inhibits only the differentiation of precursor osteoclasts whereas differentiated osteoclasts are not affected.

  20. Hyphal growth of phagocytosed Fusarium oxysporum causes cell lysis and death of murine macrophages.

    Science.gov (United States)

    Schäfer, Katja; Bain, Judith M; Di Pietro, Antonio; Gow, Neil A R; Erwig, Lars P

    2014-01-01

    Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host.

  1. Reciprocal interactions between endothelial cells and macrophages in angiogenic vascular niches

    Energy Technology Data Exchange (ETDEWEB)

    Baer, Caroline; Squadrito, Mario Leonardo [The Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne (Switzerland); Iruela-Arispe, M. Luisa, E-mail: arispe@mcdb.ucla.edu [The Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne (Switzerland); Department of Molecular, Cell and Developmental Biology and Molecular Biology Institute, University of California, Los Angeles 90095, CA (United States); De Palma, Michele, E-mail: michele.depalma@epfl.ch [The Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne (Switzerland)

    2013-07-01

    The ability of macrophages to promote vascular growth has been associated with the secretion and local delivery of classic proangiogenic factors (e.g., VEGF-A and proteases). More recently, a series of studies have also revealed that physical contact of macrophages with growing blood vessels coordinates vascular fusion of emerging sprouts. Interestingly, the interactions between macrophages and vascular endothelial cells (ECs) appear to be bidirectional, such that activated ECs also support the expansion and differentiation of proangiogenic macrophages from myeloid progenitors. Here, we discuss recent findings suggesting that dynamic angiogenic vascular niches might also exist in vivo, e.g. in tumors, where sprouting blood vessels and immature myeloid cells like monocytes engage in heterotypic interactions that are required for angiogenesis. Finally, we provide an account of emerging mechanisms of cell-to-cell communication that rely on secreted microvesicles, such as exosomes, which can offer a vehicle for the rapid exchange of molecules and genetic information between macrophages and ECs engaged in angiogenesis. -- Highlights: • Macrophages promote angiogenesis by secreting proangiogenic factors. • Macrophages modulate angiogenesis via cell-to-cell contacts with endothelial cells. • Endothelial cells promote the differentiation of proangiogenic macrophages. • Macrophages and endothelial cells may cooperate to form angiogenic vascular niches.

  2. Macrophages and mast cells in dystrophic masseter muscle: a light and electron microscopic study

    DEFF Research Database (Denmark)

    Kirkeby, S; Mikkelsen, H

    1988-01-01

    Macrophages and mast cells in masseter muscle from normal and dystrophic mice were studied by light and electron microscopy. Acid phosphatase activity and FITC-dextran were used to identify and describe macrophages. Toluidine blue was used as a marker for mast cells. In dystrophic muscle, the num......Macrophages and mast cells in masseter muscle from normal and dystrophic mice were studied by light and electron microscopy. Acid phosphatase activity and FITC-dextran were used to identify and describe macrophages. Toluidine blue was used as a marker for mast cells. In dystrophic muscle...

  3. Immunostimulatory effect of spinach aqueous extract on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages.

    Science.gov (United States)

    Ishida, Momoko; Ose, Saya; Nishi, Kosuke; Sugahara, Takuya

    2016-07-01

    We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.

  4. Fisetin antagonizes cell fusion, cytoskeletal organization and bone resorption in RANKL-differentiated murine macrophages.

    Science.gov (United States)

    Kim, Yun-Ho; Kim, Jung-Lye; Lee, Eun-Jung; Park, Sin-Hye; Han, Seon-Young; Kang, Soon Ah; Kang, Young-Hee

    2014-03-01

    Osteoclastogenesis is comprised of several stage s including progenitor survival, differentiation to mononuclear preosteoclasts, cell fusion to multinuclear mature osteoclasts, and activation to osteoclasts with bone resorbing activity. Botanical antioxidants are now being increasingly investigated for their health-promoting effects on bone. This study investigated that fisetin, a flavonol found naturally in many fruits and vegetables, suppressed osteoclastogenesis by disturbing receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated signaling pathway and demoting osteoclastogenic protein induction. Nontoxic fisetin at ≤10 μM inhibited the induction of RANK, tumor necrosis factor receptor associated factor 6 (TRAF6) and the activation of NF-κB in RANKL-stimulated RAW 264.7 macrophages. In RANKL-differentiated osteoclasts cell fusion protein of E-cadherin was induced, which was dampened by fisetin. The formation of tartrate-resistance acid phosphatase-positive multinucleated osteoclasts was suppressed by adding fisetin to RANKL-exposed macrophages. It was also found that fisetin reduced actin ring formation and gelsolin induction of osteclasts enhanced by RANKL through disturbing c-Src-proline-rich tyrosine kinase 2 signaling. Fisetin deterred preosteoclasts from the cell-cell fusion and the organization of the cytoskeleton to seal the resorbing area and to secret protons for bone resorption. Consistently, the 5 day-treatment of fisetin diminished RANKL-induced cellular expression of carbonic anhydrase II and integrin β3 concurrently with a reduction of osteoclast bone-resorbing activity. Therefore, fisetin was a natural therapeutic agent retarding osteoclast fusion and cytoskeletal organization such as actin rings and ruffled boarder, which is a property of mature osteoclasts and is required for osteoclasts to resorb bone. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Comparative analysis of the internalization of the macrophage receptor sialoadhesin in human and mouse primary macrophages and cell lines.

    Science.gov (United States)

    De Schryver, Marjorie; Leemans, Annelies; Pintelon, Isabel; Cappoen, Davie; Maes, Louis; Caljon, Guy; Cos, Paul; Delputte, Peter L

    2016-11-21

    Sialoadhesin (Sn) is a surface receptor expressed on resident macrophages with the ability to bind with sialic acids. During inflammation, an upregulation of Sn is observed. Upon binding of monoclonal antibodies to Sn, the receptor becomes internalized and this has been observed in multiple species. The latter characteristic, combined with the strong upregulation of Sn on inflammatory macrophages and the fact that Sn-positive macrophages contribute to certain inflammatory diseases, makes Sn an interesting entry portal for phenotype-modulating or cytotoxic drugs. Such drugs or toxins can be linked to Sn-specific antibodies which should enable their targeted uptake by macrophages. However, the activity of such drugs depends not only on their internalization but also on the intracellular trafficking and final fate in the endolysosomal system. Although information is available for porcine Sn, the detailed mechanisms of human and mouse Sn internalization and subsequent intracellular trafficking are currently unknown. To allow development of Sn-targeted therapies, differences across species and cellular background need to be characterized in more detail. In the current report, we show that internalization of human and mouse Sn is dynamin-dependent and clathrin-mediated, both in primary macrophages and CHO cell lines expressing a recombinant Sn. In primary macrophages, internalized Sn-specific F(ab')2 fragments are located mostly in the early endosomes. With Fc containing Sn-specific antibodies, there is a slight shift towards lysosomal localization in mouse macrophages, possibly because of an interaction with Fc receptors. Surprisingly, in CHO cell lines expressing Sn, there is a predominant lysosomal localization. Our results show that the mechanism of Sn internalization and intracellular trafficking is concurrent in the tested species. The cellular background in which Sn is expressed and the type of antibody used can affect the intracellular fate, which in turn can

  6. Data fusion-based assessment of raw materials in mammalian cell culture.

    Science.gov (United States)

    Lee, Hae Woo; Christie, Andrew; Xu, Jin; Yoon, Seongkyu

    2012-11-01

    In mammalian cell culture producing therapeutic proteins, one of the important challenges is the use of several complex raw materials whose compositional variability is relatively high and their influences on cell culture is poorly understood. Under these circumstances, application of spectroscopic techniques combined with chemometrics can provide fast, simple, and non-destructive ways to evaluate raw material quality, leading to more consistent cell culture performance. In this study, a comprehensive data fusion strategy of combining multiple spectroscopic techniques is investigated for the prediction of raw material quality in mammalian cell culture. To achieve this purpose, four different spectroscopic techniques of near-infrared, Raman, 2D fluorescence, and X-ray fluorescence spectra were employed for comprehensive characterization of soy hydrolysates which are commonly used as supplements in culture media. First, the different spectra were compared separately in terms of their prediction capability. Then, ensemble partial least squares (EPLS) was further employed by combining all of these spectral datasets in order to produce a more accurate estimation of raw material properties, and compared with other data fusion techniques. The results showed that data fusion models based on EPLS always exhibit best prediction accuracy among all the models including individual spectroscopic methods, demonstrating the synergetic effects of data fusion in characterizing the raw material quality.

  7. Suppression of lipopolysaccharide-induced of inducible nitric oxide synthase and cyclooxygenase-2 by Sanguis Draconis, a dragon's blood resin, in RAW 264.7 cells.

    Science.gov (United States)

    Choy, Cheuk-Sing; Hu, Chien-Ming; Chiu, Wen-Ta; Lam, Carlos-Shu Kei; Ting, Yih; Tsai, Shin-Han; Wang, Tzu-Chien

    2008-02-12

    Sanguis Draconis (SD) is a kind of dragon's blood resin that is obtained from Daemomorops draco (Palmae). It is used in traditional medicine and has shown anti-inflammatory activity in some diseases. In this study, we examined the effects of Sanguis Dranonis ethanol extract (SDEE) on LPS-induced inflammation using RAW 264.7 cells. Our data indicated that SDEE inhibits LPS-stimulated NO, PGE2, IL-1 beta and TNF-alpha release, and iNOS and COX-2 expression. Furthermore, SDEE suppressed the LPS-induced p65 expression of NF-kappa B, which was associated with the inhibition of I kappa B-alpha degradation. We also found that the expression of HO-1 was significantly increased in RAW 264.7 cells by SDEE. These results suggest among possibilities of anti-inflammation that SDEE inhibits the production of NO and PGE2 by the down-regulation of iNOS and COX-2 gene expression via the suppression of NF-kappaB (p65) activation. SDEE can induce HO-1 over-expression in macrophage cells, which indicates that it may possess antioxidant properties. This result means that SEDD its anti-inflammatory effects in macrophages may be through a novel mechanism that involves the action of HO-1. Thus, SD could provide a potential therapeutic approach for inflammation-associated disorders.

  8. Salidroside Regulates Inflammatory Response in Raw 264.7 Macrophages via TLR4/TAK1 and Ameliorates Inflammation in Alcohol Binge Drinking-Induced Liver Injury

    Directory of Open Access Journals (Sweden)

    Peng Sun

    2016-11-01

    Full Text Available The current study was designed to investigate the anti-inflammatory effect of salidroside (SDS and the underlying mechanism by using lipopolysaccharide (LPS-stimulated RAW 264.7 macrophages in vitro and a mouse model of binge drinking-induced liver injury in vivo. SDS downregulated protein expression of toll-like receptor 4 (TLR4 and CD14. SDS inhibited LPS-triggered phosphorylation of LPS-activated kinase 1 (TAK1, p38, c-Jun terminal kinase (JNK, and extracellular signal-regulated kinase (ERK. Degradation of IκB-α and nuclear translocation of nuclear factor (NF-κB were effectively blocked by SDS. SDS concentration-dependently suppressed LPS mediated inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2 protein levels, as well as their downstream products, NO. SDS significantly inhibited protein secretion and mRNA expression of of interleukin (IL-1β and tumor necrosis factor (TNF-α. Additionally C57BL/6 mice were orally administrated SDS for continuous 5 days, followed by three gavages of ethanol every 30 min. Alcohol binge drinking caused the increasing of hepatic lipid accumulation and serum transaminases levels. SDS pretreatment significantly alleviated liver inflammatory changes and serum transaminases levels. Further investigation indicated that SDS markedly decreased protein level of IL-1β in serum. Taken together, these data implied that SDS inhibits liver inflammation both in vitro and in vivo, and may be a promising candidate for the treatment of inflammatory liver injury.

  9. Krebs cycle rewired for macrophage and dendritic cell effector functions.

    Science.gov (United States)

    Ryan, Dylan Gerard; O'Neill, Luke A J

    2017-07-07

    The Krebs cycle is an amphibolic pathway operating in the mitochondrial matrix of all eukaryotic organisms. In response to proinflammatory stimuli, macrophages and dendritic cells undergo profound metabolic remodelling to support the biosynthetic and bioenergetic requirements of the cell. Recently, it has been discovered that this metabolic shift also involves the rewiring of the Krebs cycle to regulate cellular metabolic flux and the accumulation of Krebs cycle intermediates, notably, citrate, succinate and fumarate. Interestingly, a new role for Krebs cycle intermediates as signalling molecules and immunomodulators that dictate the inflammatory response has begun to emerge. This review will discuss the latest developments in Krebs cycle rewiring and immune cell effector functions, with a particular focus on the regulation of cytokine production. © 2017 Federation of European Biochemical Societies.

  10. Z-100, extracted from Mycobacterium tuberculosis strain Aoyama B, promotes TNF-α production via nucleotide-binding oligomerization domain containing 2 (Nod2)-dependent NF-κB activation in RAW264.7 cells.

    Science.gov (United States)

    Katsunuma, Kokichi; Yoshinaga, Koji; Ohira, Yuta; Eta, Runa; Sato, Takanori; Horii, Takayuki; Tanaka, Takao; Takei, Mineo; Seto, Koichi

    2015-03-01

    Macrophages are a major component of the innate immune system, and the cytokines they secrete are involved in antitumor responses. Z-100 is obtained from hot-water extract of human-type Mycobacterium tuberculosis strain Aoyama B and activates the innate immune response. However, while Z-100 is known to modulate macrophage activity, the mechanism behind this modulation is not fully understood. We evaluated the effects of Z-100 on the murine macrophage cell line RAW264.7. Tumor necrosis factor-alpha (TNF-α) production from RAW264.7 cells was strongly induced by Z-100 and interferon-gamma (IFN-γ) stimulation but only weakly induced by Z-100 alone. Quantitative gene expression analysis showed that nucleotide-binding oligomerization domain containing 2 (Nod2) expression was up-regulated by IFN-γ treatment in RAW264.7 cells while Z-100-induced TNF-α production was attenuated by Nod2 gene silencing. Further, componential analysis demonstrated that muramic acid and amino acids distinctive of muramyl dipeptide (MDP) were contained within Z-100 and Z-100Fr I, the low-molecular-weight fraction containing components Z-100Fr I enhanced TNF-α production in RAW264.7 cells and promoted NOD2-dependent nuclear factor-kappa B (NF-κB) activation in murine NOD2-expressing SEAP reporter HEK293 (HEK-Blue-mNOD2) cells. Taken together, these results suggest that Z-100 contains MDP-like molecules and augments NF-κB signaling via the direct activation of Nod2 in macrophages, which might be one mechanism driving the innate immune responses induced by Z-100 in cancer immunotherapy.

  11. Characterization of macrophage-like cells in the external layers of human small and large intestine

    DEFF Research Database (Denmark)

    Mikkelsen, H B; Rumessen, J J

    1992-01-01

    -DR-positive (expressing the MHC class-II antigen), in contrast to macrophage-like cells in the subserosa and submucosa. Macrophage-like cells in the external muscle layer were mostly acid phosphatase-negative, and at the electron-microscopic level they were found to have features of macrophages: primary lysosomes, coated...... vesicles and pits. However, very few secondary lysosomes were present. Birbeck granules were not observed. It is concluded that in the external muscle layer of human small and large intestine numerous macrophages of a special type are present. It is discussed whether this cell type plays a role...

  12. 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.

  13. 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.

  14. 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

  15. The nuclear factor kappa B (NF-κB) activation is required for phagocytosis of staphylococcus aureus by RAW 264.7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Fei, E-mail: zhufei@zju.edu.cn; Yue, Wanfu; Wang, Yongxia

    2014-10-01

    Nuclear factor kappa B (NF-κB) is a ubiquitous transcription factor which controls the expression of various genes involved in immune responses. However, it is not clear whether NF-κB activation is critical for phagocytosis when Staphylococcus aureus is the pathogen. Using oligonucleotide microarrays, we investigated whether NF-κB cascade genes are altered in a mouse leukemic monocyte macrophage cell line (RAW 264.7) when the cells were stimulated to activate a host innate immune response against live S. aureus or heat-inactivated S. aureus (HISA). NF-κB cascade genes such as Nfκb1, Nfκbiz, Nfκbie, Rel, Traf1 and Tnfaip3 were up-regulated by all treatments at one hour after incubation. NF-κB play an important role in activating phagocytosis in RAW 264.7 cells infected with S. aureus. Inhibition of NF-κB significantly blocked phagocytosis of fluorescently labeled S. aureus and decreased the expression of NFκB1, IL1α, IL1β and TLR2 in this cell line. Our results demonstrate that S. aureus may activate the NF-κB pathway and that NF-κB activation is required for phagocytosis of S. aureus by macrophages. - Highlights: • NF-κB cascade genes such as Nfκb1 and Traf1 were up-regulated by heat-inactivated S. aureus. • Inhibition of NF-κB significantly blocked phagocytosis of fluorescently labeled S. aureus. • NF-κB activation is required for phagocytosis of S. aureus by macrophages.

  16. Effect of apigenin, kaempferol and resveratrol on the gene expression and protein secretion of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) in RAW-264.7 macrophages.

    Science.gov (United States)

    Palacz-Wrobel, Marta; Borkowska, Paulina; Paul-Samojedny, Monika; Kowalczyk, Malgorzata; Fila-Danilow, Anna; Suchanek-Raif, Renata; Kowalski, Jan

    2017-09-01

    Polyphenols such as apigenin, kaempferol or resveratrol are typically found in plants, including fruits, vegetables, herbs and spices, which have a wide range of biological functions such as antioxidative, anti-inflammatory, vasodilative, anticoagulative and proapoptotic. Discovering such multifunctional compounds in widely consumed plant-based products - ones that both inhibit the release of TNF-α from tissue macrophages and at the same time enhance the secretion of IL-10 - would be an important signpost in the quest for effective pharmacological treatment of numerous diseases that have an inflammatory etiology. The aim of the study is to investigate the impact of biologically active polyphenols such as apigenin, resveratrol and kaempferol on gene expression and protein secretion of IL-10 and TNF-α in line RAW-264.7. Cells were cultured under standard conditions. IL-10 and TNF-α genes expression were examined using QRT-PCR and to assess cytokines concentration ELISA have been used. Apigenin, kaempferol and resveratrol at a dose 30μM significantly decrease the TNF-α expression and secretion. Apigenin decrease the IL-10 expression and secretion. Furthermore, increase in IL-10 secretion after administration of kaempferol and resveratrol were observed. In the process of administration of tested compounds before LPS, which activate macrophages, decrease of TNF-α secretion after apigenin and kaempferol and increase of IL-10 secretion after resveratrol were observed. The results of present work indicate that 1) apigenin, resveratrol and kaempferol may reduce the intensity of inflammatory processes by inhibiting the secretion of proinflammatory cytokine TNF-α, and resveratrol and kaempferol additionally by increasing the secretion of anti-inflammatory cytokine IL-10 2) the studies indicate the potentially beneficial - anti-inflammatory - impact of diet rich in products including apigenin, resveratrol and kaempferol. Copyright © 2017 Elsevier Masson SAS. All rights

  17. XH-14, a novel danshen methoxybenzo[b]furan derivative, exhibits anti-inflammatory properties in lipopolysaccharide-treated RAW 264.7 cells

    Directory of Open Access Journals (Sweden)

    Park Geun-Mook

    2013-01-01

    Full Text Available Abstract Background XH-14 isolated from Salvia miltiorrhiza is a bioactive component and adenosine antagonist. In the present study, we evaluated anti-inflammatory properties of XH-14 in murine macrophages. Methods RAW 264.7 murine macrophage cell line was cultured with various concentrations of XH-14 in the absence or presence of lipopolysaccharide (LPS. LPS-induced release and mRNA expression of inflammatory mediators were examined by ELISA and real-time PCR. The modification of signal pathways involved in inflammatory reactions was determined by Western blotting analysis. Results XH-14 suppressed the generation of nitric oxide (NO and prostaglandin E2, and the expression of inducible NO synthase and cyclooxygenase-2 induced by LPS. Similarly, XH-14 inhibited the release of pro-inflammatory cytokines induced by LPS in RAW 264.7 cells. The underlying mechanism of XH-14 on anti-inflammatory action was correlated with down-regulation of mitogen-activated protein kinase and activator protein-1 activation. Conclusions XH-14 inhibits the production of several inflammatory mediators and so might be useful for the treatment of various inflammatory diseases.

  18. Isorhamnetin-3-O-Glucuronide Suppresses JNK and p38 Activation and Increases Heme-Oxygenase-1 in Lipopolysaccharide-Challenged RAW264.7 Cells.

    Science.gov (United States)

    Park, Jin-Young; Kim, Song-In; Lee, Hee Jae; Kim, Sung-Soo; Kwon, Yong-Soo; Chun, Wanjoo

    2016-05-01

    Preclinical Research Isorhanmetin (ISH) exhibits a wide range of biological properties including anticancer, anti-oxidant and anti-inflammatory activities. However, the pharmacological properties of isorhamnetin-3-O-glucuronide (IG), a glycoside derivative of ISH, have not been extensively examined. The objective of this study was to examine the anti-inflammatory properties of IG and its underlying mechanism in lipopolysaccharide (LPS)-challenged RAW264.7 macrophage cells in comparison with its aglycone, ISH. IG suppressed LPS-induced extracellular secretion of the proinflammatory mediators, nitric oxide (NO) and PGE2 , and proinflammatory protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2. IG also increased expression of heme oxygenase-1 (HO-1). IG attenuated LPS-induced activation of c-Jun N-terminal kinase (JNK) and p38 in a concentration-dependent manner with negligible suppression of extracellular signal-regulated kinases (ERK) phosphorylation. In conclusion, this study demonstrates that IG exerts anti-inflammatory activity by increasing HO-1 expression and by suppressing JNK and p38 signaling pathways in LPS-challenged RAW264.7 macrophage cells. Drug Dev Res 77 : 143-151, 2016. © 2016 Wiley Periodicals, Inc.

  19. Terminalia chebula Fructus Inhibits Migration and Proliferation of Vascular Smooth Muscle Cells and Production of Inflammatory Mediators in RAW 264.7

    Directory of Open Access Journals (Sweden)

    Hyun-Ho Lee

    2015-01-01

    Full Text Available Pathogenesis of atherosclerosis and neointima formation after angioplasty involves vascular smooth muscle cells (VSMCs migration and proliferation followed by inflammatory responses mediated by recruited macrophages in the neointima. Terminalia chebula is widely used traditional medicine in Asia for its beneficial effects against cancer, diabetes, and bacterial infection. The study was designed to determine whether Terminalia chebula fructus water extract (TFW suppresses VSMC migration and proliferation and inflammatory mediators production in macrophage (RAW 264.7. Our results showed that TFW possessed strong antioxidative effects in 1,1-diphenyl-2-picryl hydrazyl (DPPH scavenging and lipid peroxidation assays. In addition, TFW reduced nitric oxide (NO production, inducible nitric oxide synthase (iNOS, and cyclooxygenase-2 (COX-2 expression in RAW 264.7 cells. Also, TFW inhibited platelet-derived growth factor (PDGF-BB induced VSMC migration as determined by wound healing and Boyden chamber assays. The antimigratory effect of TFW was due to its inhibitory effect on metalloproteinase-9 (MMP-9 expression, focal adhesion kinase (FAK activation, and Rho-family of small GTPases (Cdc42 and RhoA expression in VSMCs. Furthermore, TFW suppressed PDGF-BB induced VSMC proliferation by downregulation of mitogen activated protein kinases (MAPKs signaling molecules. These results suggest that TFW could be a beneficial resource in the prevention of atherosclerosis.

  20. Alginic acid cell entrapment: a novel method for measuring in vivo macrophage cholesterol homeostasis

    Science.gov (United States)

    Sontag, Timothy J.; Chellan, Bijoy; Bhanvadia, Clarissa V.; Getz, Godfrey S.; Reardon, Catherine A.

    2015-01-01

    Macrophage conversion to atherosclerotic foam cells is partly due to the balance of uptake and efflux of cholesterol. Cholesterol efflux from cells by HDL and its apoproteins for subsequent hepatic elimination is known as reverse cholesterol transport. Numerous methods have been developed to measure in vivo macrophage cholesterol efflux. Most methods do not allow for macrophage recovery for analysis of changes in cellular cholesterol status. We describe a novel method for measuring cellular cholesterol balance using the in vivo entrapment of macrophages in alginate, which retains incorporated cells while being permeable to lipoproteins. Recipient mice were injected subcutaneously with CaCl2 forming a bubble into which a macrophage/alginate suspension was injected, entrapping the macrophages. Cells were recovered after 24 h. Cellular free and esterified cholesterol mass were determined enzymatically and normalized to cellular protein. Both normal and cholesterol loaded macrophages undergo measureable changes in cell cholesterol when injected into WT and apoA-I-, LDL-receptor-, or apoE-deficient mice. Cellular cholesterol balance is dependent on initial cellular cholesterol status, macrophage cholesterol transporter expression, and apolipoprotein deficiency. Alginate entrapment allows for the in vivo measurement of macrophage cholesterol homeostasis and is a novel platform for investigating the role of genetics and therapeutic interventions in atherogenesis. PMID:25465389

  1. Bauer Ketones 23 and 24 from Echinacea paradoxa var. paradoxa Inhibit Lipopolysaccharide-induced Nitric Oxide, Prostaglandin E2 and Cytokines in RAW 264.7 Mouse Macrophages

    Science.gov (United States)

    Zhang, Xiaozhu; Rizshsky, Ludmila; Hauck, Catherine; Qu, Luping; Widrlechner, Mark P.; Nikolau, Basil J.; Murphy, Patricia A.; Birt, Diane F.

    2011-01-01

    Among the nine Echinacea species, E. purpurea, E. angustifolia and E. pallida, have been widely used to treat the common cold, flu and other infections. In our study, ethanol extracts of these three Echinacea species and E. paradoxa, including its typical variety, E. paradoxa var. paradoxa, were screened in lipopolysaccharide (LPS)-stimulated macrophage cells to assess potential anti-inflammatory activity. Echinacea paradoxa var. paradoxa, rich in polyenes/polyacetylenes, was an especially efficient inhibitor of LPS-induced production of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) by 46%, 32%, 53% and 26%, respectively, when tested at 20 μg/ml in comparison to DMSO control. By bioactivity-guided fractionation, pentadeca-8Z-ene-11, 13-diyn-2-one (Bauer ketones 23, compound 1) and pentadeca-8Z, 13Z-dien-11-yn-2-one (Bauer ketone 24, compound 2) from E. paradoxa var. paradoxa were found primarily responsible for inhibitory effects on NO and PGE2 production. Moreover, Bauer ketone 24 (compound 2) was the major contributor to inhibition of inflammatory cytokine production in LPS-induced mouse macrophage cells. These results provide a rationale for exploring the medicinal effects of the Bauer ketone-rich taxon, E. paradoxa var. paradoxa, and confirm the anti-inflammatory properties of Bauer ketones 23 and 24. PMID:22133644

  2. Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages

    Directory of Open Access Journals (Sweden)

    Youhei Egami

    2015-01-01

    Full Text Available Phagocytosis of zymosan by phagocytes is a widely used model of microbial recognition by the innate immune system. Live-cell imaging showed that fluorescent protein-fused Rab35 accumulated in the membranes of phagocytic cups and then dissociated from the membranes of newly formed phagosomes. By our novel pull-down assay for Rab35 activity, we found that Rab35 is deactivated immediately after zymosan internalization into the cells. Phagosome formation was inhibited in cells expressing the GDP- or GTP-locked Rab35 mutant. Moreover, the simultaneous expression of ACAP2—a Rab35 effector protein—with GTP-locked Rab35 or the expression of plasma membrane-targeted ACAP2 showed a marked inhibitory effect on phagocytosis through ARF6 inactivation by the GAP activity of ACAP2. ARF6, a substrate for ACAP2, was also localized on the phagocytic cups and dissociated from the membranes of internalized phagosomes. In support of the microscopic observations, ARF6-GTP pull-down experiments showed that ARF6 is transiently activated during phagosome formation. Furthermore, the expression of GDP- or GTP-locked ARF6 mutants also suppresses the uptake of zymosan. These data suggest that the activation-inactivation cycles of Rab35 and ARF6 are required for the uptake of zymosan and that ACAP2 is an important component that links Rab35/ARF6 signaling during phagocytosis of zymosan.

  3. Extract of buckwheat sprouts scavenges oxidation and inhibits pro-inflammatory mediators in lipopolysaccharide-stimulated macrophages (RAW264.7)

    Institute of Scientific and Technical Information of China (English)

    Rajendra Karki; Cheol-Ho Park; Dong-Wook Kim

    2013-01-01

    OBJECTIVE:Buckwheat has been considered as a potential source of nutraceutical components on the world market of probiotic foodstuffs.The purpose of this study was to evaluate the effects of tartary buckwheat (Fagopyrum tataricum) sprouts on oxidation and pro-inflammatory mediators.METHODS:The anti-oxidant effects of buckwheat extract (BWE) and rutin were evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH)-and nitric oxide (NO)-scavenging activities,serum peroxidation and chelating assays.Lipopolysaccharide (LPS)-stimulated RAW264.7 cells were used to evaluate anti-inflammatory activities of buckwheat and rutin.NO production in LPS-stimulated RAW264.7 cells was determined by using Griess reagent.The expressions of inducible nitric oxide synthase (iNOS),cyclooxygenase-2 (COX-2),nuclear factor-kappa B (NF-κB) p65 subunit in cytosolic and nuclear portions were determined by Western blot analysis.Also,the production of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was determined by enzyme-linked immunosorbent assay.RESULTS:Inhibitory concentration 50 values for DPPH-and NO-scavenging activities of BWE were 24.97 and 72.54 μg/mL respectively.BWE inhibited serum oxidation and possessed chelating activity.Furthermore,BWE inhibited IL-6 and TNF-α production in LPS-stimulated RAW264.7 cells.Also,BWE inhibited iNOS and COX-2 expression and NF-κB p65 translocation.CONCLUSION:Buckwheat sprouts possessed strong antioxidant activity and inhibited production of pro-inflammatory mediators in the applied model systems.Thus,buckwheat can be suggested to be beneficial in inflammatory diseases by inhibiting the free radicals and inflammatory mediators.

  4. Macrophage-like cells in the muscularis externa of mouse small intestine

    DEFF Research Database (Denmark)

    Mikkelsen, H B; Thuneberg, L; Rumessen, J J;

    1985-01-01

    In muscularis externa of mouse small intestine, cells with ultrastructural features of macrophages were invariably observed in three layers: in the subserosal layer, between the circular and longitudinal muscle layers, and in association with the deep circular plexus. These macrophage-like cells...

  5. Salmonella typhimurium Invasion Induces Apoptosis in Infected Macrophages

    Science.gov (United States)

    Monack, Denise M.; Raupach, Barbel; Hromockyj, Alexander E.; Falkow, Stanley

    1996-09-01

    Invasive Salmonella typhimurium induces dramatic cytoskeletal changes on the membrane surface of mammalian epithelial cells and RAW264.7 macrophages as part of its entry mechanism. Noninvasive S. typhimurium strains are unable to induce this membrane ruffling. Invasive S. typhimurium strains invade RAW264.7 macrophages in 2 h with 7- to 10-fold higher levels than noninvasive strains. Invasive S. typhimurium and Salmonella typhi, independent of their ability to replicate intracellularly, are cytotoxic to RAW264.7 macrophages and, to a greater degree, to murine bone marrow-derived macrophages. Here, we show that the macrophage cytotoxicity mediated by invasive Salmonella is apoptosis, as shown by nuclear morphology, cytoplasmic vacuolization, and host cell DNA fragmentation. S. typhimurium that enter cells causing ruffles but are mutant for subsequent intracellular replication also initiate host cell apoptosis. Mutant S. typhimurium that are incapable of inducing host cell membrane ruffling fail to induce apoptosis. The activation state of the macrophage plays a significant role in the response of macrophages to Salmonella invasion, perhaps indicating that the signal or receptor for initiating programmed cell death is upregulated in activated macrophages. The ability of Salmonella to promote apoptosis may be important for the initiation of infection, bacterial survival, and escape of the host immune response.

  6. Compound FLZ inhibits lipopolysaccharide-induced inflammatory effects via down-regulation of the TAK-IKK and TAK-JNK/ p38MAPK pathways in RAW264.7 macrophages

    Institute of Scientific and Technical Information of China (English)

    Hongyan PANG; Gang LIU; Gengtao LIU

    2009-01-01

    Aim:The aim of this study was to investigate the effect of the squamosamide derivative FLZ (N-2-(4-hydroxy-phenyl)-ethyl-2-(2,5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide) on.lipopolysaccharide (LPS)-induced inflam-matory mediator production and the underlying mechanism in RAW264.7 macrophages.Methods: RAW264.7 cells were preincubated with non-toxic concentrations of compound FLZ (1,5,and 10 μmol/L) for 30 min and then stimulated with 10 μg/L LPS.The production of nitric oxide (NO),the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2),and the activation of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways were examined.Results: FLZ significantly inhibited the LPS-induced production of NO,as well as the expression of iNOS and COX-2 at both the RNA and the protein levels in RAW264.7 cells.The LPS-induced increase in the DNA binding activity of NF-κBand activator protein I (AP-1),the nuclear translocation of NF-κB p65,the degradation of the inhibitory κBα protein (IκBα)and the phosphorylation of IκBα,IκB kinase (IKK) α/β,c-Jun NH2-terminal kinase (JNK) and p38 MAPKs were all sup-pressed by FLZ.However,the phosphorylation of extracellular signal-regulated kinase (ERK) was not affected.Further study revealed that FLZ inhibited the phosphorylation of transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1),which is an upstream signaling molecule required for IKKα/β,JNK and p38 activation.Conclusion: FLZ inhibited the LPS-induced production of inflammatory mediators at least partly through the downregula-tion of the TAK-IKK and TAK-JNK/p38MAPK pathways.

  7. BCG对小鼠RAW264.7巨噬细胞miR-203、miR-142-3p、miR-21表达的影响%Effect of BCG on expression of miR-203, miR-142-3p and miR-21 in mouse RAW264.7 macrophages

    Institute of Scientific and Technical Information of China (English)

    张兆波; 魏军; 汤建中; 赵志军; 张一琳; 张瑞芹; 徐广贤

    2013-01-01

    目的:检测卡介苗(Bacillus Calmette-Guerin,BCG)刺激巨噬细胞后miR-203、miR-142-3p、miR-21表达量的变化,为研究microRNA(miRNA)在巨噬细胞抗结核分枝杆菌免疫应答中的调控作用提供依据.方法:利用浓度为1.0 ×107ml-1的BCG刺激培养的小鼠RAW264.7细胞,分别在4、8、12、24小时提取细胞small RNA,并利用相应的茎环反转录引物,反转录成cDNA,同时构建成熟miR-203、miR-142-3p、miR-21的T载体,绘制标准曲线,利用Real-Time PCR检测miR-203、miR-142-3p、miR-21的表达量.结果:BCG作用RAW264.7细胞4、8、12、24小时后,miR-21表达显著性上调(10倍以上,P<0.05),miR-142-3p表达显著性下调(20倍以上,P<0.05),miR-203在4、8、12小时表达下调(3倍以上,P<0.05),24小时后表达上调(2倍以上,P<0.05).结论:BCG刺激RAW264.7巨噬细胞后,miR-203、miR-142-3p、miR-21的表达发生显著性变化,说明这些miRNA可能通过调控免疫相关基因在巨噬细胞抗结核分枝杆菌的免疫应答中发挥着重要的作用.%Objective: To detect the influence of Bacillus Calmette-Guerin (BCG) on the expression of miit-203, miR-142-3p and miR-21 in the macrophages and provide the basis to study the regulation of miRNA in the immune response of macrophages to My-cobacterium tuberculosis. Methods:Small RNA was extracted at different times after stimulated with a concentration of 1.0 × 107 ml"1 of BCG in cultured mouse RAW264. 7 cells. After using stem-loop reverse transcription primers to reverse transcribed into cDNA, the expression of miR-203, miR-142-3p and miR-21 was detected by Real-time PCR. At the same time, building the T vector of mature miR-203, miR-142-3p and miR- 21 to make the standard curve. Results:After RAW264.7 cells was treated by BCG for 4, 8, 12 and 24 h, miR-21 expression was up-regulated significantly (More than 10 times, P < 0. 05). However, miR-142-3 p was significantly down-regulated at the same time( More than 20 times, P <0. 05

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  10. MAP kinase phosphatase 2 regulates macrophage-adipocyte interaction.

    Directory of Open Access Journals (Sweden)

    Huipeng Jiao

    Full Text Available Inflammation is critical for the development of obesity-associated metabolic disorders. This study aims to investigate the role of mitogen-activated protein kinase phosphatase 2 (MKP-2 in inflammation during macrophage-adipocyte interaction.White adipose tissues (WAT from mice either on a high-fat diet (HFD or normal chow (NC were isolated to examine the expression of MKP-2. Murine macrophage cell line RAW264.7 stably expressing MKP-2 was used to study the regulation of MKP-2 in macrophages in response to saturated free fatty acid (FFA and its role in macrophage M1/M2 activation. Macrophage-adipocyte co-culture system was employed to investigate the role of MKP-2 in regulating inflammation during adipocyte-macrophage interaction. c-Jun N-terminal kinase (JNK- and p38-specific inhibitors were used to examine the mechanisms by which MKP-2 regulates macrophage activation and macrophage-adipocytes interaction.HFD changed the expression of MKP-2 in WAT, and MKP-2 was highly expressed in the stromal vascular cells (SVCs. MKP-2 inhibited the production of proinflammatory cytokines in response to FFA stimulation in macrophages. MKP-2 inhibited macrophage M1 activation through JNK and p38. In addition, overexpression of MKP-2 in macrophages suppressed inflammation during macrophage-adipocyte interaction.MKP-2 is a negative regulator of macrophage M1 activation through JNK and p38 and inhibits inflammation during macrophage-adipocyte interaction.

  11. 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.

    Science.gov (United States)

    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.

  12. Glycosyl glycerides from hydroponic Panax ginseng inhibited NO production in lipopolysaccharide-stimulated RAW264.7 cells

    Directory of Open Access Journals (Sweden)

    Byeong-Ju Cha

    2015-04-01

    Results and conclusion: The glycosyl glycerides were identified to be (2S-1-O-7(Z,10(Z,13(Z-hexadecatrienoyl-3-O-β-d-galactopyranosyl-sn-glycerol (1, (2S-1-O-linolenoyl-3-O-β-d-galactopyranosyl-sn-glycerol (2, (2S-1-O-linolenoyl-2-O-linolenoyl-3-O-β-d-galactopyranosyl-sn-glycerol (3, and 2(S-1-O-linoleoyl-2-O-linoleoyl-3-O-β-d-galactopyranosyl-sn-glycerol (4. Compounds 1 and 2 showed moderate inhibition activity on NO production in LPS-stimulated RAW264.7 cells [half maximal inhibitory concentration (IC50: 63.8 ± 6.4μM and 59.4 ± 6.8μM, respectively] without cytotoxicity at concentrations < 100μM, whereas Compounds 3 and 4 showed good inhibition effect (IC50: 7.7 ± 0.6μM and 8.0 ± 0.9μM, respectively without cytotoxicity at concentrations < 20μM. All isolated compounds showed reduced messenger RNA (mRNA expression of interleukin-1β (IL-1β, IL-6, and tumor necrosis factor-α in LPS-induced macrophage cells with strong inhibition of mRNA activity observed for Compounds 3 and 4.

  13. RESEARCHES REGARDING THE SOMATIC CELLS NUMBER FROM RAW MILK USED IN TELEMEA CHEESE TECHNOLOGICAL PROCESS

    Directory of Open Access Journals (Sweden)

    ANDRA SULER

    2013-12-01

    Full Text Available It is known that by milk production hygiene must be assure: milk microbiological security, increase the sensorial and nutritive properties, increase term of availability and consumption. The milk hygienic national strategies involved: raw material risk contamination avoiding and reducing as can is possible and the microorganisms destroying or stopping development of those. In this paper it is presented the results of somatic cells number determination by raw milk used in Telemea cheese technological processes within 5 research stations. Determinations were effectuated on 2 series with 57 samples each of them, prelevated in reception phase in summer and winter seasons.

  14. Melanoma cell-derived exosomes alter macrophage and dendritic cell functions in vitro.

    Science.gov (United States)

    Marton, Annamaria; Vizler, Csaba; Kusz, Erzsebet; Temesfoi, Viktoria; Szathmary, Zsuzsa; Nagy, Krisztina; Szegletes, Zsolt; Varo, Gyorgy; Siklos, Laszlo; Katona, Robert L; Tubak, Vilmos; Howard, O M Zack; Duda, Erno; Minarovits, Janos; Nagy, Katalin; Buzas, Krisztina

    2012-01-01

    To clarify controversies in the literature of the field, we have purified and characterized B16F1 melanoma cell derived exosomes (mcd-exosomes) then we attempted to dissect their immunological activities. We tested how mcd-exosomes influence CD4+ T cell proliferation induced by bone marrow derived dendritic cells; we quantified NF-κB activation in mature macrophages stimulated with mcd-exosomes, and we compared the cytokine profile of LPS-stimulated, IL-4 induced, and mcd-exosome treated macrophages. We observed that mcd-exosomes helped the maturation of dendritic cells, enhancing T cell proliferation induced by the treated dendritic cells. The exosomes also activated macrophages, as measured by NF-κB activation. The cytokine and chemokine profile of macrophages treated with tumor cell derived exosomes showed marked differences from those induced by either LPS or IL-4, and it suggested that exosomes may play a role in the tumor progression and metastasis formation through supporting tumor immune escape mechanisms.

  15. Effects of rapamycin on expression of ten kinds autophagy-related miRNAs in RAW264.7 macrophages%雷帕霉素对RAW264.7巨噬细胞10种与细胞自噬相关的miRNAs表达的影响

    Institute of Scientific and Technical Information of China (English)

    张涛; 郑锡铭; 周林林; 刘鑫; 赵瑾; 徐广贤

    2014-01-01

    目的:为了探讨雷帕霉素对RAW264.7细胞miR-30b、miR-200a和miR-17-5p等10种与细胞自噬相关的miRNAs表达水平的影响,为进一步研究miRNAs调控细胞自噬的机制提供理论依据。方法:雷帕霉素刺激RAW264.7巨噬细胞后,分别在2、4、6、8 h提取细胞small RNA,利用miRNA特异性的茎环引物反转录成cDNA,采用Real-Time PCR检测miR-30b、miR-30c、miR-106a、miR-214、miR-183、miR-200a、miR-376c、miR-17-5p、miR-142-3p、miR-377分子的表达情况。结果:雷帕霉素作用RAW264.7细胞后,miR-17-5p、miR-106在2、4、6 h表达上调(大于2.1倍P<0.05),miR-214在2、8小时表达上调(>2.4倍,P<0.05),miR-30b、miR-30c、miR-183、miR-200a、miR-376c、miR-142-3p在2、6、8 h表达上调(>2.4倍,P<0.05),而miR-183、miR-200a在4 h表达下调(>2.1倍,P<0.05),miR-30b在8小时则是显著性表达下调(大于50倍,P<0.05),miR-377在4 h则是表达上调(>2.5倍,P<0.05),但在2、8 h则是显著表达下调(>50倍,P<0.05)。结论:雷帕霉素刺激RAW264.7巨噬细胞后miR-200a、miR-30b、miR-377、miR-30c、miR-376c、miR-17-5p有显著性变化,说明这些miRNAs可能通过调控某些自噬相关基因在细胞自噬过程中发挥着重要作用。%To detect the influence of rapamycin on the expression of miR-30b,miR-200a and miR-17-5p etc in macrophages and provide the basis to study the regulation of miRNA in autophagy mechanism of macrophages .Methods: Small RNA was extracted at different times after stimulated with rapamycin in cultured RAW 264.7 cells.After using the stem-loop reverse transcription primers to reverse transcribed into cDNA ,the expression of miR-30b ,miR-30c,miR-106a,miR-214,miR-183,miR-200a, miR-376c,miR-17-5p, miR-142-3p, miR-377 was detected by Real-Time PCR.Results: After RAW264.7 cells was treated by rapamycin for 2,4,6 and 8 hours,the expression of miR-17-5p and miR-106

  16. Primary macrophages and J774 cells respond differently to infection with Mycobacterium tuberculosis

    Science.gov (United States)

    Andreu, Nuria; Phelan, Jody; de Sessions, Paola F.; Cliff, Jacqueline M.; Clark, Taane G.; Hibberd, Martin L.

    2017-01-01

    Macrophages play an essential role in the early immune response to Mycobacterium tuberculosis and are the cell type preferentially infected in vivo. Primary macrophages and macrophage-like cell lines are commonly used as infection models, although the physiological relevance of cell lines, particularly for host-pathogen interaction studies, is debatable. Here we use high-throughput RNA-sequencing to analyse transcriptome dynamics of two macrophage models in response to M. tuberculosis infection. Specifically, we study the early response of bone marrow-derived mouse macrophages and cell line J774 to infection with live and γ-irradiated (killed) M. tuberculosis. We show that infection with live bacilli specifically alters the expression of host genes such as Rsad2, Ifit1/2/3 and Rig-I, whose potential roles in resistance to M. tuberculosis infection have not yet been investigated. In addition, the response of primary macrophages is faster and more intense than that of J774 cells in terms of number of differentially expressed genes and magnitude of induction/repression. Our results point to potentially novel processes leading to immune containment early during M. tuberculosis infection, and support the idea that important differences exist between primary macrophages and cell lines, which should be taken into account when choosing a macrophage model to study host-pathogen interactions. PMID:28176867

  17. Moracin C, A Phenolic Compound Isolated from Artocarpus heterophyllus, Suppresses Lipopolysaccharide-Activated Inflammatory Responses in Murine Raw264.7 Macrophages

    Science.gov (United States)

    Yao, Xue; Wu, Dang; Dong, Ningning; Ouyang, Ping; Pu, Jiaqian; Hu, Qian; Wang, Jingyuan; Lu, Weiqiang; Huang, Jin

    2016-01-01

    Artocarpus heterophyllus, a popular tropical fruit commonly known as the jackfruit tree, is normally planted in subtropical or tropical areas. Since a variety of phytochemicals isolated from A. heterophyllus have been found to possess potently anti-inflammatory, antiviral and antimalarial activities, researchers have devoted much interest to its potential pharmaceutical value. However, the exact mechanism underlying its anti-inflammatory activity is not well characterized. In this study, seven natural products isolated from A. heterophyllus, including 25-Hydroxycycloart-23-en-3-one (HY), Artocarpin (AR), Dadahol A (DA), Morachalcone A (MA), Artoheterophyllin B (AB), Cycloheterophyllin (CY) and Moracin C (MC) were collected. Lipopolysaccharide (LPS)-stimulated inflammatory response in RAW264.7 macrophages were used in this study. Among these compounds, MC significantly inhibited LPS-activated reactive oxygen species (ROS) and nitric oxide (NO) release without marked cytotoxicity. Furthermore, MC effectively reduced LPS stimulated up-regulation of mRNA and protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and serval pro-inflammatory cytokines (interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α)). Mechanistic studies revealed that the anti-inflammatory effect of MC was associated with the activation of the mitogen activated protein kinases (MAPKs) (including p38, ERK and JNK) and nuclear factor-κB (NF-κB) pathways, especially reducing the nuclear translocation of NF-κB p65 subunit as revealed by nuclear separation experiment and confocal microscopy. PMID:27463712

  18. Moracin C, A Phenolic Compound Isolated from Artocarpus heterophyllus, Suppresses Lipopolysaccharide-Activated Inflammatory Responses in Murine Raw264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Xue Yao

    2016-07-01

    Full Text Available Artocarpus heterophyllus, a popular tropical fruit commonly known as the jackfruit tree, is normally planted in subtropical or tropical areas. Since a variety of phytochemicals isolated from A. heterophyllus have been found to possess potently anti-inflammatory, antiviral and antimalarial activities, researchers have devoted much interest to its potential pharmaceutical value. However, the exact mechanism underlying its anti-inflammatory activity is not well characterized. In this study, seven natural products isolated from A. heterophyllus, including 25-Hydroxycycloart-23-en-3-one (HY, Artocarpin (AR, Dadahol A (DA, Morachalcone A (MA, Artoheterophyllin B (AB, Cycloheterophyllin (CY and Moracin C (MC were collected. Lipopolysaccharide (LPS-stimulated inflammatory response in RAW264.7 macrophages were used in this study. Among these compounds, MC significantly inhibited LPS-activated reactive oxygen species (ROS and nitric oxide (NO release without marked cytotoxicity. Furthermore, MC effectively reduced LPS stimulated up-regulation of mRNA and protein expression of inducible nitric oxide synthase (iNOS, cyclooxygenase-2 (COX-2, and serval pro-inflammatory cytokines (interleukin-1β (IL-1β, interleukin-6 (IL-6 and tumor necrosis factor α (TNF-α. Mechanistic studies revealed that the anti-inflammatory effect of MC was associated with the activation of the mitogen activated protein kinases (MAPKs (including p38, ERK and JNK and nuclear factor-κB (NF-κB pathways, especially reducing the nuclear translocation of NF-κB p65 subunit as revealed by nuclear separation experiment and confocal microscopy.

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  17. Acetylcholine Inhibits LPS-Induced MMP-9 Production and Cell Migration via the a7 nAChR-JAK2/STAT3 Pathway in RAW264.7 Cells

    Directory of Open Access Journals (Sweden)

    Yong-Hua Yang

    2015-07-01

    Full Text Available Background: Excessive activation of matrix metalloproteinase 9 (MMP-9 has been found in several inflammatory diseases. Previous studies have shown that acetylcholine (ACh reduced the levels of pro-inflammatory cytokines and decreased tissue damage. Therefore, this study was designed to explore the potential effects and mechanisms of ACh on MMP-9 production and cell migration in response to lipopolysaccharide (LPS stimulation in RAW264.7 cells. Methods: MMP-9 expression and activity were induced by LPS in RAW264.7 cells, and examined by real-time PCR, western blotting and gelatin zymography, respectively. ELISA was used to determine the changes in MMP-9 secretion among the groups. Macrophage migration was evaluated using transwell migration assay. Knockdown of a7 nicotinic acetylcholine receptor (a7 nAChR expression was performed using siRNA transfection. Results: Pre-treatment with ACh inhibited LPS-induced MMP-9 production and macrophage migration in RAW264.7 cells. These effects were abolished by the a7 nAChR antagonist methyllycaconitine (MLA and a7 nAChR siRNA. The a7 nAChR agonist PNU282987 was found to have an effect similar to that of ACh. Moreover, ACh enhanced the expression of JAK2 and STAT3, and the JAK2 inhibitor AG490 and the STAT3 inhibitor static restored the effect of ACh. Meanwhile, ACh decreased the phosphorylation and nuclear translocation of NF-κB, and this effect was abrogated in the presence of MLA. In addition, the JAK2 and STAT3 inhibitor abolished the inhibitory effects of ACh on phosphorylation of NF-κB. Conclusions: Activation of a7 nAChR by ACh inhibited LPS-induced MMP-9 production and macrophage migration through the JAK2/STAT3 signaling pathway. These results provide novel insights into the anti-inflammatory effects and mechanisms of ACh.

  18. 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.

  19. Macrophage Recruitment and Epithelial Repair Following Hair Cell Injury in the Mouse Utricle

    Directory of Open Access Journals (Sweden)

    Tejbeer eKaur

    2015-04-01

    Full Text Available The sensory organs of the inner ear possess resident populations of macrophages, but the function of those cells is poorly understood. In many tissues, macrophages participate in the removal of cellular debris after injury and can also promote tissue repair. The present study examined injury-evoked macrophage activity in the mouse utricle. Experiments used transgenic mice in which the gene for the human diphtheria toxin receptor (huDTR was inserted under regulation of the Pou4f3 promoter. Hair cells in such mice can be selectively lesioned by systemic treatment with diphtheria toxin (DT. In order to visualize macrophages, Pou4f3-huDTR mice were crossed with a second transgenic line, in which one or both copies of the gene for the fractalkine receptor CX3CR1 were replaced with a gene for GFP. Such mice expressed GFP in all macrophages, and mice that were CX3CR1GFP/GFP lacked the necessary receptor for fractalkine signaling. Treatment with DT resulted in the death of ~70% of utricular hair cells within seven days, which was accompanied by increased numbers of macrophages within the utricular sensory epithelium. Many of these macrophages appeared to be actively engulfing hair cell debris, indicating that macrophages participate in the process of ‘corpse removal’ in the mammalian vestibular organs. However, we observed no apparent differences in injury-evoked macrophage numbers in the utricles of CX3CR1+/GFP mice vs. CX3CR1GFP/GFP mice, suggesting that fractalkine signaling is not necessary for macrophage recruitment in these sensory organs. Finally, we found that repair of sensory epithelia at short times after DT-induced hair cell lesions was mediated by relatively thin cables of F-actin. After 56 days recovery, however, all cell-cell junctions were characterized by very thick actin cables.

  20. Capsaicin protects endothelial cells and macrophage against oxidized low-density lipoprotein-induced injury by direct antioxidant action.

    Science.gov (United States)

    Chen, Kuo-Shuen; Chen, Pei-Ni; Hsieh, Yih-Shou; Lin, Chin-Yin; Lee, Yi-Hsun; Chu, Shu-Chen

    2015-02-25

    Atherosclerosis is a chronic inflammatory vascular disease. It is characterized by endothelial dysfunction, lipid accumulation, leukocyte activation, and the production of inflammatory mediators and reactive oxygen species (ROS). Capsaicin, a biologically active compound of the red pepper and chili pepper, has several anti-oxidant, anti-inflammatory, anti-cancer, and hypolipidemic biological effects. However, its protective effects on foam cell formation and endothelial injury induced by oxidized low-density lipoprotein (oxLDL) remain unclear. In this study, we evaluated the anti-oxidative activity of capsaicin, and determined the mechanism by which capsaicin rescues human umbilical vein endothelial cells (HUVECs) from oxLDL-mediated dysfunction. The anti-oxidative activity of capsaicin was defined by Apo B fragmentation and conjugated diene production of the copper-mediated oxidation of LDL. Capsaicin repressed ROS generation, as well as subsequent mitochondrial membrane potential collapse, cytochrome c expression, chromosome condensation, and caspase-3 activation induced by oxLDL in HUVECs. Capsaicin also protected foam cell formation in macrophage RAW 264.7 cells. Our results suggest that capsaicin may prevent oxLDL-induced cellular dysfunction and protect RAW 264.7 cells from LDL oxidation.

  1. Mycobacterium bovis Bacillus Calmette-Guérin-Induced Macrophage Cytotoxicity against Bladder Cancer Cells

    Directory of Open Access Journals (Sweden)

    Yi Luo

    2010-01-01

    Full Text Available Many details of the molecular and cellular mechanisms involved in Mycobacterium bovis bacillus Calmette-Guérin (BCG immunotherapy of bladder cancer have been discovered in the past decades. However, information on a potential role for macrophage cytotoxicity as an effector mechanism is limited. Macrophages play pivotal roles in the host innate immunity and serve as a first line of defense in mycobacterial infection. In addition to their function as professional antigen-presenting cells, the tumoricidal activity of macrophages has also been studied with considerable interest. Studies have shown that activated macrophages are potent in killing malignant cells of various tissue origins. This review summarizes the current understanding of the BCG-induced macrophage cytotoxicity toward bladder cancer cells with an intention to inspire investigation on this important but underdeveloped research field.

  2. Inhibitory effect of red ginseng acidic polysaccharide from Korean red ginseng on phagocytic activity and intracellular replication of Brucella abortus in RAW 264.7 cells

    Science.gov (United States)

    Bernardo Reyes, Alisha Wehdnesday; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon; Min, Won Gi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee

    2016-01-01

    Korean red ginseng (KRG) has long been used in traditional Korean and Oriental medicine. However, the anti-bacterial mechanism and therapeutic efficiency of KGR for intracellular Brucella infection are still unclear. In this study, the bactericidal activity of Korean red ginseng acidic polysaccharide (RGAP) on Brucella (B.) abortus and its cytotoxic effects on RAW 264.7 cells were evaluated. In addition, B. abortus internalization and intracellular replication in macrophages were investigated after RGAP treatment. RGAP-incubated cells displayed a marked reduction in the adherence, internalization and intracellular growth of B. abortus in macrophages. Furthermore, decreased F-actin fluorescence was observed relative to untreated B. abortus-infected cells. Western blot analysis of intracellular signaling proteins revealed reduced ERK, JNK and p38α phosphorylation levels in B. abortus-infected RGAP-treated cells compared to the control. Moreover, elevated co-localization of B. abortus-containing phagosomes with lysosome-associated membrane protein 1 (LAMP-1) were observed in RGAP-treated cells compared with the control. Overall, the results of this study suggest that RGAP can disrupt phagocytic activity of B. abortus via suppression of mitogen-activated protein kinases (MAPKs) signaling proteins ERK, JNK and p38 levels and inhibit intracellular replication of B. abortus by enhancing phagolysosome fusion, which may provide an alternative control of brucellosis. PMID:26726017

  3. Inhibitory effect of red ginseng acidic polysaccharide from Korean red ginseng on phagocytic activity and intracellular replication of Brucella abortus in RAW 264.7 cells.

    Science.gov (United States)

    Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon; Min, Won Gi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

    2016-09-30

    Korean red ginseng (KRG) has long been used in traditional Korean and Oriental medicine. However, the anti-bacterial mechanism and therapeutic efficiency of KGR for intracellular Brucella infection are still unclear. In this study, the bactericidal activity of Korean red ginseng acidic polysaccharide (RGAP) on Brucella (B.) abortus and its cytotoxic effects on RAW 264.7 cells were evaluated. In addition, B. abortus internalization and intracellular replication in macrophages were investigated after RGAP treatment. RGAP-incubated cells displayed a marked reduction in the adherence, internalization and intracellular growth of B. abortus in macrophages. Furthermore, decreased F-actin fluorescence was observed relative to untreated B. abortus-infected cells. Western blot analysis of intracellular signaling proteins revealed reduced ERK, JNK and p38α phosphorylation levels in B. abortus-infected RGAP-treated cells compared to the control. Moreover, elevated co-localization of B. abortus-containing phagosomes with lysosome-associated membrane protein 1 (LAMP-1) were observed in RGAP-treated cells compared with the control. Overall, the results of this study suggest that RGAP can disrupt phagocytic activity of B. abortus via suppression of mitogen-activated protein kinases (MAPKs) signaling proteins ERK, JNK and p38 levels and inhibit intracellular replication of B. abortus by enhancing phagolysosome fusion, which may provide an alternative control of brucellosis.

  4. Embryonic stem cell-derived M2-like macrophages delay cutaneous wound healing.

    Science.gov (United States)

    Dreymueller, Daniela; Denecke, Bernd; Ludwig, Andreas; Jahnen-Dechent, Willi

    2013-01-01

    In adults, repair of deeply injured skin wounds results in the formation of scar tissue, whereas in embryos wounds heal almost scar-free. Macrophages are important mediators of wound healing and secrete cytokines and tissue remodeling enzymes. In contrast to host defense mediated by inflammatory M1 macrophages, wound healing and tissue repair involve regulatory M2/M2-like macrophages. Embryonic/fetal macrophages are M2-like, and this may promote scar-free wound healing. In the present study, we asked whether atopical application of ex vivo generated, embryonic stem cell-derived macrophages (ESDM) improve wound healing in mice. ESDM were tested side by side with bone marrow-derived macrophages (BMDM). Compared to BMDM, ESDM resembled a less inflammatory and more M2-like macrophage subtype as indicated by their reduced responsiveness to lipopolysaccharide, reduced expression of Toll-like receptors, and reduced bacterial phagocytosis. Despite this anti-inflammatory phenotype in cell culture, ESDM prolonged the healing of deep skin wounds even more than BMDM. Healed wounds had more scar formation compared to wounds receiving BMDM or cell-free treatment. Our data indicate that atopical application of ex vivo generated macrophages is not a suitable cell therapy of dermal wounds.

  5. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing.

    Directory of Open Access Journals (Sweden)

    Liwen Chen

    Full Text Available Bone marrow derived mesenchymal stem cells (BM-MSCs have been shown to enhance wound healing; however, the mechanisms involved are barely understood. In this study, we examined paracrine factors released by BM-MSCs and their effects on the cells participating in wound healing compared to those released by dermal fibroblasts. Analyses of BM-MSCs with Real-Time PCR and of BM-MSC-conditioned medium by antibody-based protein array and ELISA indicated that BM-MSCs secreted distinctively different cytokines and chemokines, such as greater amounts of VEGF-alpha, IGF-1, EGF, keratinocyte growth factor, angiopoietin-1, stromal derived factor-1, macrophage inflammatory protein-1alpha and beta and erythropoietin, compared to dermal fibroblasts. These molecules are known to be important in normal wound healing. BM-MSC-conditioned medium significantly enhanced migration of macrophages, keratinocytes and endothelial cells and proliferation of keratinocytes and endothelial cells compared to fibroblast-conditioned medium. Moreover, in a mouse model of excisional wound healing, where concentrated BM-MSC-conditioned medium was applied, accelerated wound healing occurred compared to administration of pre-conditioned or fibroblast-conditioned medium. Analysis of cell suspensions derived from the wound by FACS showed that wounds treated with BM-MSC-conditioned medium had increased proportions of CD4/80-positive macrophages and Flk-1-, CD34- or c-kit-positive endothelial (progenitor cells compared to wounds treated with pre-conditioned medium or fibroblast-conditioned medium. Consistent with the above findings, immunohistochemical analysis of wound sections showed that wounds treated with BM-MSC-conditioned medium had increased abundance of macrophages. Our results suggest that factors released by BM-MSCs recruit macrophages and endothelial lineage cells into the wound thus enhancing wound healing.

  6. Interstitial cells of Cajal, macrophages and mast cells in the gut musculature: morphology, distribution, spatial and possible functional interactions

    DEFF Research Database (Denmark)

    Mikkelsen, Hanne B

    2010-01-01

    Interstitial cells of Cajal (ICC) are recognized as pacemaker cells for gastrointestinal movement and are suggested to be mediators of neuromuscular transmission. Intestinal motility disturbances are often associated with a reduced number of ICC and/or ultrastructural damage, sometimes associated...... with immune cells. Macrophages and mast cells in the intestinal muscularis externa of rodents can be found in close spatial contact with ICC. Macrophages are a constant and regularly distributed cell population in the serosa and at the level of Auerbach's plexus (AP). In human colon, ICC are in close contact...... with macrophages at the level of AP, suggesting functional interaction. It has therefore been proposed that ICC and macrophages interact. Macrophages and mast cells are considered to play important roles in the innate immune defence by producing pro-inflammatory mediators during classical activation, which may...

  7. Lignan derivatives from Selaginella tamariscina and their nitric oxide inhibitory effects in LPS-stimulated RAW 264.7 cells.

    Science.gov (United States)

    Dat, Le Duc; Zhao, Bing Tian; Hung, Nguyen Duc; Lee, Jeong Hyung; Min, Byung Sun; Woo, Mi Hee

    2017-02-01

    The chemical characterization of Selaginella tamariscina leaves resulted in the isolation of five lignanoside derivatives (1-4 and 6) and one neolignan (5). These compounds include three new lignanosides, tamariscinosides D-F (1-3), and one liriodendrin (4) that were isolated for the first time from this plant, together with two known compounds, (2R,3S)-dihydro-2-(3,5-dimethoxy-4-hydroxyphenyl)-7-methoxy-5-acetyl-benzofuran (5) and moellenoside B (6). The chemical structures of these isolated compounds were determined using 1D and 2D NMR, MS, and CD spectroscopic data, and the results were compared to data previously reported in the literatures. These compounds were also evaluated in terms of their inhibition of NO production in lipopolysaccharide (LPS)-stimulated activity in the macrophage cell line RAW 264.7. Among them, compounds 1, 2, 5, and 6 exhibited a significant inhibition with IC50 values ranging from 32.3 to 55.8μM.

  8. Antioxidant and Anti-Inflammatory Effects of Chaenomeles sinensis Leaf Extracts on LPS-Stimulated RAW 264.7 Cells.

    Science.gov (United States)

    Han, Young-Ki; Kim, Yon-Suk; Natarajan, Sithranga Boopathy; Kim, Won-Suk; Hwang, Jin-Woo; Jeon, Nam-Joo; Jeong, Jae-Hyun; Moon, Sang-Ho; Jeon, Byong-Tae; Park, Pyo-Jam

    2016-03-28

    The fruit of Chaenomeles sinensis has been traditionally used in ethnomedicine for the treatment of various human ailments, including pneumonia, bronchitis, and so on, but the pharmacological applications of the leaf part of the plant have not been studied. In this study, we evaluated the various radical scavenging activities and anti-inflammatory effects of different Chaenomeles sinensis leaf (CSL) extracts. The water extract showed a higher antioxidant and radical scavenging activities. However the ethanolic extracts showed higher NO scavenging activity than water extract, therefore the ethanolic extract of CSL was examined for anti-inflammatory effects on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The 70% ethanol extract of CSL (CSLE) has higher anti-inflammatory activity and significantly inhibited the production of nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In addition, CSLE suppressed LPS-stimulated inducible nitric oxide synthase (iNOS) and NO production, IL-1β and phospho-STAT1 expression. In this study, we investigated the effect of CSLE on the production of inflammatory mediators through the inhibition of the TRIF-dependent pathways. Furthermore, we evaluated the role of CSLE on LPS-induced expression of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6. Our results suggest that CSLE attenuates the LPS-stimulated inflammatory responses in macrophages through regulating the key inflammatory mechanisms, providing scientific support for its traditional uses in treating various inflammatory diseases.

  9. Anti-Inflammatory Effect of By-Products from Haliotis discus hannai in RAW 264.7 Cells

    Directory of Open Access Journals (Sweden)

    Ho-Seok Rho

    2015-01-01

    Full Text Available Several reports promoted the potential of shellfish due to their ability to act as antioxidant, anti-inflammatory, and antimicrobial agents. Pacific abalone, Haliotis discus hannai viscera is, reported to possess bioactivities such as antioxidative stress and anti-inflammatory. In this study, anti-inflammatory potential of mucus-secreting glands from shell-shucking waste of H. discus hannai was evaluated using RAW 264.7 mouse macrophage cell model. Results indicated that presence of H. discus hannai mucosubstance by-products (AM significantly lowered the nitric oxide (NO production along the expressional suppression of inflammatory mediators such as cytokines TNF-α, IL-1β, and IL-6 and enzymes iNOS and COX-2. Also, AM was shown to increase expression of anti-inflammatory response mediator HO-1. Presence of AM also scavenged the free radicals in vitro. In conclusion, by-products of H. discus hannai are suggested to possess notable anti-inflammatory potential which promotes the possibility of utilization as functional food ingredient.

  10. Taurine chloramine inhibits NO and TNF-α production in zymosan plus interferon-γ activated RAW 264.7 cells.

    Science.gov (United States)

    Kim, Bo Sook; Cho, In Soo; Park, Seung Yong; Schuller-Levis, Georgia; Levis, William; Park, Eunkyue

    2011-06-01

    Taurine is present abundantly in various tissues, especially in leukocytes embattled to foreign invaders such as microorganisms or oxidants. Taurine-chloramine (Tau-Cl) is produced from taurine at the site of inflammation via the myeloperoxidase-halide pathway in leukocytes induced by oxidants and/or infectious materials. Previously, our data demonstrated that Tau-Cl inhibited nitric oxide (NO) production and TNF-α secretion induced by lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR-4) ligand or lipoarabinomannan (LAM), a TLR-2 ligand plus interferon-γ (IFN-γ) in peritoneal macrophages or RAW 264.7 cells. Zymosan, a β-glucan of yeast cell wall, is a ligand for TLR-2 and dectin-1 and stimulates macrophages to produce proinflammatory mediators such as NO and TNF-α. Based on our previous data, we examined the effect of zymosan and IFN-γ induced production of NO and TNF-α in the absence or presence of Tau-Cl or taurine using RAW 264.7 cells. Production of NO and secretion of TNF-α is increased when zymosan is combined with IFN-γ. Tau-Cl inhibited production of NO and secretion of TNF-α in zymosan plus IFN-γ activated RAW 264.7 cells in a dose-dependent manner (99% vs. 48% using 0.8mM Tau-Cl). Taurine was without effect. Nitric oxide synthase protein (iNOS), induced by zymosan plus IFN-γ, was inhibited by Tau-Cl (0.8mM) as measured using western blot analysis. NOS mRNA was inhibited by Tau-Cl at four, eight and 16 hours post activation, but not at 24 hours. TNF-α mRNA was inhibited at four hours and eight hours, but not at 16 and 24 hours. These data suggest that expression of both iNOS and TNF-α mRNAs are inhibited by treatment with Tau-Cl within four and eight hours, but not at later time points. Transient suppression of activation of RAW 264.7 cells induced by zymosan may play a critical physiological role for taurine in protecting against tissue injury from initial overt inflammation. This study indicates that tropical treatment of taurine may

  11. REACTIVE MILIEU OF HODGKIN LYMPHOMA WITH EMPHASIS ON MAST CELLS AND MACROPHAGES

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    Nidhish Kumar

    2016-08-01

    Full Text Available AIM OF STUDY To study the clinical importance of reactive microenvironment inHodgkin Lymphoma (HL with special reference to macrophages and mast cells. MATERIALS AND METHODS The present prospective and retrospective study was undertaken for a period ranging from January 2011 to June 2015 at the Department of Pathology, Kasturba Medical College, Mangalore. The Haematoxylin and Eosin (H and E stained slides were reviewed and classified using WHO (2008 classification. Six immuno-histochemical markers were used in the study. CD 68 was for the macrophage count. Giemsa stain was done to highlight the mast cells. RESULTS AND ANALYSIS Thirty cases of HL were studied. Out of the 5 cases of Lymphocyte Depleted (LD Classical Hodgkin Lymphoma (cHL, all cases showed high macrophage count. Out of 30 cases of HL, only 6 cases showed increased mast cell count. DISCUSSION Mast cells act actively in various types of cancers. They can either have a pro-tumorigenic function or an anti-tumorigenic function depending on the type of cancer. Four (80% cases of LD-cHL showed macrophage count between 25-50% and 1 (20% case showed macrophage count >50% correlating with the aggressive nature and advanced stage of the disease. CONCLUSION In this study of microenvironment of HL mast cells and macrophages were analysed in each subtype. Though the mast cells were seen in all cases, an increased count of >10/10 (High power field HPF was observed only in 6 cases. The macrophage count was highest in LD-cHL and was statistically significant and thus correlated with this aggressive subtype of HL. The mast cell and macrophage count did not correlate with B-symptoms and stage of the disease a conclusion on survival versus the macrophage count and mast cell count was not possible in this study because of shorter follow up. A longer follow up and more number of cases are needed for a significant outcome.

  12. Tumor-promoting macrophages induce the expression of the macrophage-specific receptor CD163 in malignant cells.

    Science.gov (United States)

    Maniecki, Maciej Bogdan; Etzerodt, Anders; Ulhøi, Benedicte Parm; Steiniche, Torben; Borre, Michael; Dyrskjøt, Lars; Orntoft, Torben Falck; Moestrup, Søren Kragh; Møller, Holger Jon

    2012-11-15

    Tumor-associated macrophages (TAMs) represent a distinct malignancy-promoting phenotype suggested to play a key role in tumor formation and metastasis. We aimed to investigate the expression of the monocyte/macrophage-restricted receptor CD163 in bladder tumor biopsies and assess the potential mechanism inducing the CD163 expression in tumor cells. A high CD163 mRNA expression (n = 87) was significantly associated with a poor 13-year overall survival (log-rank test, χ(2) = 8.931; p = 0.0028). Moreover, CD163 mRNA expression was significantly increased in muscle invasive (T2-T4), p = 0.017, and aggressive (grade III/IV) cancers (p = 0.015). The expression strongly correlated with local expression of IL-6 (r = 0.72; p CD163 expression in vitro. CD163 immunostaining (n = 46) confirmed the association between dense TAM infiltration and histologically advanced disease. In 39% of the biopsies, CD163 immunoreactivity was also observed in tumor cells, and CD163-expressing metastatic cells were identified in lymph node biopsies (n = 8). Bladder cancer cell lines did not express CD163; however, when cocultured with macrophages the bladder cancer cell expression of CD163 was significantly induced in an IL-6/IL-10 independent manner. In conclusion, we show a strong association between CD163 mRNA expression in bladder cancer biopsies and poor patient outcome. CD163 expression was not confined to the infiltrating TAMs, but was also expressed by a significant portion of the malignant cells in both tumors and lymph nodes. CD163 expressing tumor cells may constitute a subpopulation of tumor cells with a phenotypic shift associated with epithelial-to-mesenchymal transition (EMT) and increased metastatic activity induced by TAMs. Copyright © 2012 UICC.

  13. Proteomic analysis of pycnogenol effects in RAW 264.7 macrophage reveals induction of cathepsin D expression and enhancement of phagocytosis.

    Science.gov (United States)

    Wu, Ting-Feng; Hsu, Chiung-Yueh; Huang, Huei-Sheng; Chou, Siao-Ping; Wu, Hung

    2007-11-28

    Pycnogenol, polyphenolic compounds extracted from the pine bark, is beneficial for human health. To understand more of its effects, the present study is to explore the protein expression pattern induced by pycnogenol in RAW 264.7 cells. Global analysis using two-dimensional gel electrophoresis indicated that treatment with pycnogenol induces upregulation of four proteins, whose identities were revealed by mass spectrometry as cathepsin D, keratinocyte lipid-binding protein, proteasome subunit alpha type 1, and annexin IV. The pycnogenol effect displayed a time- and concentration-dependent manner. Unlike pycnogenol, N-acetyl cysteine and vitamin C had no effect on cathepsin D expression. Further studies showed that cathepsin D induction is correlated with an increase of lysosomal staining and enhancement of phagocytosis. These results reveal the novel effects of pycnogenol on protein expression and phagocytic functions and illustrate the advantage of proteomics-based strategy in unveiling the molecular basis of phytochemicals.

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  17. Structural environment built by AKAP12+ colon mesenchymal cells drives M2 macrophages during inflammation recovery

    Science.gov (United States)

    Yang, Jun-Mo; Lee, Hye Shin; Seo, Ji Hae; Park, Ji-Hyeon; Gelman, Irwin H.; Lo, Eng H.; Kim, Kyu-Won

    2017-01-01

    Macrophages exhibit phenotypic plasticity, as they have the ability to switch their functional phenotypes during inflammation and recovery. Simultaneously, the mechanical environment actively changes. However, how these dynamic alterations affect the macrophage phenotype is unknown. Here, we observed that the extracellular matrix (ECM) constructed by AKAP12+ colon mesenchymal cells (CMCs) generated M2 macrophages by regulating their shape during recovery. Notably, rounded macrophages were present in the linear and loose ECM of inflamed colons and polarized to the M1 phenotype. In contrast, ramified macrophages emerged in the contracted ECM of recovering colons and mainly expressed M2 macrophage markers. These contracted structures were not observed in the inflamed colons of AKAP12 knockout (KO) mice. Consequently, the proportion of M2 macrophages in inflamed colons was lower in AKAP12 KO mice than in WT mice. In addition, clinical symptoms and histological damage were more severe in AKAP12 KO mice than in WT mice. In experimentally remodeled collagen gels, WT CMCs drove the formation of a more compacted structure than AKAP12 KO CMCs, which promoted the polarization of macrophages toward an M2 phenotype. These results demonstrated that tissue contraction during recovery provides macrophages with the physical cues that drive M2 polarization. PMID:28205544

  18. Jellyfish skin polysaccharides: extraction and inhibitory activity on macrophage-derived foam cell formation.

    Science.gov (United States)

    Zhang, Hai-Lin; Cui, Shao-Hua; Zha, Xue-Qiang; Bansal, Vibha; Xue, Lei; Li, Xiao-Long; Hao, Ran; Pan, Li-Hua; Luo, Jian-Ping

    2014-06-15

    In this work, response surface methodology was used to determine optimum conditions for extraction of polysaccharides from jellyfish skin (JSP). The optimum parameters were found to be raw material to water ratio 1:7.5 (w/v), extraction temperature 100°C and extraction time 4h. Under these conditions, the JSP yield reached 1.007 mg/g. Papain (15 U/mL) in combination with Sevag reagent was beneficial in removing proteins from JSP. After precipitation with ethanol at final concentration of 40%, 60% and 80% in turn, three polysaccharide fractions of JSP1, JSP2 and JSP3 were obtained from JSP, respectively. The three fractions exhibited different physicochemical properties with respect to molecular weight distribution, monosaccharide composition, infrared absorption spectra, and glycosyl bond composition. In addition, JSP3 showed strong inhibitory effects on oxidized low-density lipoprotein (oxLDL) induced conversion of macrophages into foam cells, which possibly attributed to the down-regulation of some atherogenesis-related gene expressions.

  19. Estimation of raw material performance in mammalian cell culture using near infrared spectra combined with chemometrics approaches.

    Science.gov (United States)

    Lee, Hae Woo; Christie, Andrew; Liu, Jun Jay; Yoon, Seongkyu

    2012-01-01

    Understanding variability in raw materials and their impacts on product quality is of critical importance in the biopharmaceutical manufacturing processes. For this purpose, several spectroscopic techniques have been studied for raw material characterization, providing fast and nondestructive ways to measure quality of raw materials. However, investigations of correlation between spectra of raw materials and cell culture performance have been scarce due to their complexity and uncertainty. In this study, near-infrared spectra and bioassays of multiple soy hydrolysate lots manufactured by different vendors were analyzed using chemometrics approaches in order to address variability of raw materials as well as correlation between raw material properties and corresponding cell culture performance. Principal component analysis revealed that near-infrared spectra of different soy lots contain enough physicochemical information about soy hydrolysates to allow identification of lot-to-lot variability as well as vendor-to-vendor differences. The identified compositional variability was further analyzed in order to estimate cell growth and protein production of two mammalian cell lines under the condition of varying soy dosages using partial least square regression combined with optimal variable selection. The performance of the resulting models demonstrates the potential of near-infrared spectroscopy as a robust lot selection tool for raw materials while providing a biological link between chemical composition of raw materials and cell culture performance.

  20. Phototherapy-treated apoptotic tumor cells induce pro-inflammatory cytokines production in macrophage

    Science.gov (United States)

    Lu, Cuixia; Wei, Yanchun; Xing, Da

    2014-09-01

    Our previous studies have demonstrated that as a mitochondria-targeting cancer phototherapy, high fluence low-power laser irradiation (HF-LPLI) induces mitochondrial superoxide anion burst, resulting in oxidative damage to tumor cells. In this study, we further explored the immunological effects of HF-LPLI-induced apoptotic tumor cells. When macrophages were co-incubated with apoptotic cells induced by HF-LPLI, we observed the increased levels of TNF-α secretion and NO production in macrophages. Further experiments showed that NF-κB was activated in macrophages after co-incubation with HF-LPLI-induced apoptotic cells, and inhibition of NF-κB activity by pyrrolidinedithiocarbamic acid (PDTC) reduced the elevated levels of TNF-α secretion and NO production. These data indicate that HF-LPLI-induced apoptotic tumor cells induce the secretion of pro-inflammatory cytokines in macrophages, which may be helpful for better understanding the biological effects of cancer phototherapy.

  1. microRNA-150 inhibits the formation of macrophage foam cells through targeting adiponectin receptor 2

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jing [Department of Geratory, Linzi District People’s Hospital of Zibo City, Zibo, Shandong (China); Zhang, Suhua, E-mail: drsuhuangzhang@qq.com [Department of HealthCare, Qilu Hospital of Shandong University (Qingdao), Qingdao City, Qingdao (China)

    2016-08-05

    Transformation of macrophages into foam cells plays a critical role in the pathogenesis of atherosclerosis. The aim of this study was to determine the expression and biological roles of microRNA (miR)-150 in the formation of macrophage foam cells and to identify its functional target(s). Exposure to 50 μg/ml oxidized low-density lipoprotein (oxLDL) led to a significant upregulation of miR-150 in THP-1 macrophages. Overexpression of miR-150 inhibited oxLDL-induced lipid accumulation in THP-1 macrophages, while knockdown of miR-150 enhanced lipid accumulation. apoA-I- and HDL-mediated cholesterol efflux was increased by 66% and 43%, respectively, in miR-150-overexpressing macrophages relative to control cells. In contrast, downregulation of miR-150 significantly reduced cholesterol efflux from oxLDL-laden macrophages. Bioinformatic analysis and luciferase reporter assay revealed adiponectin receptor 2 (AdipoR2) as a direct target of miR-150. Small interfering RNA-mediated downregulation of AdipoR2 phenocopied the effects of miR-150 overexpression, reducing lipid accumulation and facilitating cholesterol efflux in oxLDL-treated THP-1 macrophages. Knockdown of AdipoR2 induced the expression of proliferator-activated receptor gamma (PPARγ), liver X receptor alpha (LXRα), ABCA1, and ABCG1. Moreover, pharmacological inhibition of PPARγ or LXRα impaired AdipoR2 silencing-induced upregulation of ABCA1 and ABCG1. Taken together, our results indicate that miR-150 can attenuate oxLDL-induced lipid accumulation in macrophages via promotion of cholesterol efflux. The suppressive effects of miR-150 on macrophage foam cell formation are mediated through targeting of AdipoR2. Delivery of miR-150 may represent a potential approach to prevent macrophage foam cell formation in atherosclerosis. -- Highlights: •miR-150 inhibits macrophage foam cell formation. •miR-150 accelerates cholesterol efflux from oxLDL-laden macrophages. •miR-150 suppresses macrophage foam cell

  2. Gravity separation of fat, somatic cells, and bacteria in raw and pasteurized milks.

    Science.gov (United States)

    Caplan, Z; Melilli, C; Barbano, D M

    2013-04-01

    The objective of experiment 1 was to determine if the extent of gravity separation of milk fat, bacteria, and somatic cells is influenced by the time and temperature of gravity separation or the level of contaminating bacteria present in the raw milk. The objective of experiment 2 was to determine if different temperatures of milk heat treatment affected the gravity separation of milk fat, bacteria, and somatic cells. In raw milk, fat, bacteria, and somatic cells rose to the top of columns during gravity separation. About 50 to 80% of the fat and bacteria were present in the top 8% of the milk after gravity separation of raw milk. Gravity separation for 7h at 12°C or for 22h at 4°C produced equivalent separation of fat, bacteria, and somatic cells. The completeness of gravity separation of fat was influenced by the level of bacteria in the milk before separation. Milk with a high bacterial count had less (about 50 to 55%) gravity separation of fat than milk with low bacteria count (about 80%) in 22h at 4°C. Gravity separation caused fat, bacteria, and somatic cells to rise to the top of columns for raw whole milk and high temperature, short-time pasteurized (72.6°C, 25s) whole milk. Pasteurization at ≥76.9°C for 25s prevented all 3 components from rising, possibly due to denaturation of native bovine immunoglobulins that normally associate with fat, bacteria, and somatic cells during gravity separation. Gravity separation can be used to produce reduced-fat milk with decreased bacterial and somatic cell counts, and may be a critical factor in the history of safe and unique traditional Italian hard cheeses produced from gravity-separated raw milk. A better understanding of the mechanism of this natural process could lead to the development of new nonthermal thermal technology (that does not involve heating the milk to high temperatures) to remove bacteria and spores from milk or other liquids. Copyright © 2013 American Dairy Science Association. Published by

  3. Vancomycin-modified mesoporous silica nanoparticles for selective recognition and killing of pathogenic gram-positive bacteria over macrophage-like cells.

    Science.gov (United States)

    Qi, Guobin; Li, Lili; Yu, Faquan; Wang, Hao

    2013-11-13

    Rapid, reliable recognition and detection of bacteria from an authentic specimen have been gained increasing interests in the past decades. Various materials have been designed and prepared for implementation of bacterial recognition and treatment in the artificial systems. However, in the complicated physiological condition, the macrophages always compromise the outcomes of bacterial detection and/or treatment. In this work, we demonstrated the vancomycin-modified mesoporous silica nanoparticles (MSNs is a subset of Van) for efficiently targeting and killing gram-positive bacteria over macrophage-like cells. Owing to the specific hydrogen bonding interactions of vancomycin toward the terminal d-alanyl-d-alanine moieties of gram-positive bacteria, the MSNs is a subset of Van exhibited enhanced recognition for gram-positive bacteria due to the multivalent hydrogen binding effect. Furthermore, the fluorescent molecules (FITC) were covalently decorated inside of mesopores of MSNs for tracking and visualizing the MSNs is a subset of Van during the detection/treatment processes. Upon incubation of FITC decorated MSNs with bacteria (i.e., S. aureus and E. coli as gram-positive and gram-negative bacteria, respectively) or macrophage-like cells (Raw 264.7), the fluorescence signals in S. aureus were 2-4 times higher than that in E. coli and no detectable fluorescence signals were observed in Raw 264.7 cells under the same condition. Finally, the MSNs is a subset of Van showed unambiguous antibacterial efficacy without decrease in cell viability of macrophage-like cells. This new strategy opens a new door for specific detection and treatment of pathogenic bacteria with minimized side effects.

  4. Tumor-promoting macrophages induce the expression of the macrophage-specific receptor CD163 in malignant cells

    DEFF Research Database (Denmark)

    Maniecki, Maciej Bogdan; Etzerodt, Anders; Ulhøi, Benedicte Parm

    2012-01-01

    Tumor-associated macrophages (TAMs) represent a distinct malignancy-promoting phenotype suggested to play a key role in tumor formation and metastasis. We aimed to investigate the expression of the monocyte/macrophage-restricted receptor CD163 in bladder tumor biopsies and assess the potential...... mechanism inducing the CD163 expression in tumor cells. A high CD163 mRNA expression (n = 87) was significantly associated with a poor 13-year overall survival (log-rank test, χ(2) = 8.931; p = 0.0028). Moreover, CD163 mRNA expression was significantly increased in muscle invasive (T2-T4), p = 0.......017, and aggressive (grade III/IV) cancers (p = 0.015). The expression strongly correlated with local expression of IL-6 (r = 0.72; p CD163 expression in vitro. CD163 immunostaining (n = 46) confirmed the association between dense TAM infiltration...

  5. 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.

  6. Caffeine prevents LPS-induced inflammatory responses in RAW264.7 cells and zebrafish.

    Science.gov (United States)

    Hwang, Ji-Hyun; Kim, Kui-Jin; Ryu, Su-Jung; Lee, Boo-Yong

    2016-03-25

    Caffeine is a white crystalline xanthine alkaloid found in the seeds of coffee plants and leaves of the tea bush. In this study, we evaluated whether caffeine exerts anti-inflammatory effects on lipopolysaccharide (LPS)-induced inflammation both in vitro and in vivo. RAW264.7 cells were treated with various concentrations of caffeine in the presence or absence of LPS. Caffeine decreased the LPS-induced inflammatory mediator, nitric oxide (NO). Caffeine treatment also reduced the expression of pro-inflammatory genes, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-3, IL-6 and IL-12, and decreased both IL-6 secretion and phosphorylated p38MAPK expression in LPS-treated RAW264.7 cells. Caffeine inhibited nuclear translocation of nuclear factor κB (NF-κB) via IκBα phosphorylation. In addition, caffeine inhibited LPS-induced NO production in zebrafish. These results suggest that caffeine may suppress LPS-induced inflammatory responses in RAW264.7 cells by regulating NF-κB activation and MAPK phosphorylation.

  7. Tumor associated macrophage × cancer cell hybrids may acquire cancer stem cell properties in breast cancer.

    Directory of Open Access Journals (Sweden)

    Jingxian Ding

    Full Text Available Breast cancer is one of the most frequently diagnosed cancers among women, and metastasis makes it lethal. Tumor-associated macrophages (TAMs that acquire an alternatively activated macrophage (M2 phenotype may promote metastasis. However, the underlying mechanisms are still elusive. Here, we examined how TAMs interact with breast cancer cells to promote metastasis. Immunohistochemistry was used to examine the expression of the M2-specific antigen CD163 in paraffin-embedded mammary carcinoma blocks to explore fusion events in breast cancer patients. U937 cells were used as a substitute for human monocytes, and these cells differentiated into M2 macrophages following phorbol 12-myristate 13-acetate (PMA and M-CSF stimulation. M2 macrophages and the breast cancer cell lines MCF-7 and MDA-MB-231 fused in the presence of 50% polyethylene glycol. Hybrids were isolated by fluorescence-activated cell sorting, and the relevant cell biological properties were compared with their parental counterparts. Breast cancer stem cell (BCSC-related markers were quantified by immunofluorescence staining, RT-PCR, quantitative RT-PCR and/or western blotting. The tumor-initiating and metastatic capacities of the hybrids and their parental counterparts were assessed in NOD/SCID mice. We found that the CD163 expression rate in breast cancer tissues varied significantly and correlated with estrogen receptor status (p0.05. Characterization of the fusion hybrids revealed a more aggressive phenotype, including increased migration, invasion and tumorigenicity, but reduced proliferative ability, compared with the parental lines. The hybrids also gained a CD44(+CD24(-/low phenotype and over-expressed epithelial-mesenchymal transition-associated genes. These results indicate that TAMs may promote breast cancer metastasis through cell fusion, and the hybrids may gain a BCSC phenotype.

  8. Prostaglandin E2 receptor EP4 as the common target on cancer cells and macrophages to abolish angiogenesis, lymphangiogenesis, metastasis, and stem-like cell functions.

    Science.gov (United States)

    Majumder, Mousumi; Xin, Xiping; Liu, Ling; Girish, Gannareddy V; Lala, Peeyush K

    2014-09-01

    We previously established that COX-2 overexpression promotes breast cancer progression and metastasis. As long-term use of COX-2 inhibitors (COX-2i) can promote thrombo-embolic events, we tested an alternative target, prostaglandin E2 receptor EP4 subtype (EP4), downstream of COX-2. Here we used the highly metastatic syngeneic murine C3L5 breast cancer model to test the role of EP4-expressing macrophages in vascular endothelial growth factor (VEGF)-C/D production, angiogenesis, and lymphangiogenesis in situ, the role of EP4 in stem-like cell (SLC) functions of tumor cells, and therapeutic effects of an EP4 antagonist RQ-15986 (EP4A). C3L5 cells expressed all EP receptors, produced VEGF-C/D, and showed high clonogenic tumorsphere forming ability in vitro, functions inhibited with COX-2i or EP4A. Treating murine macrophage RAW 264.7 cell line with COX-2i celecoxib and EP4A significantly reduced VEGF-A/C/D production in vitro, measured with quantitative PCR and Western blots. Orthotopic implants of C3L5 cells in C3H/HeJ mice showed rapid tumor growth, angiogenesis, lymphangiogenesis (CD31/LYVE-1 and CD31/PROX1 immunostaining), and metastasis to lymph nodes and lungs. Tumors revealed high incidence of EP4-expressing, VEGF-C/D producing macrophages identified with dual immunostaining of F4/80 and EP4 or VEGF-C/D. Celecoxib or EP4A therapy at non-toxic doses abrogated tumor growth, lymphangiogenesis, and metastasis to lymph nodes and lungs. Residual tumors in treated mice revealed markedly reduced VEGF-A/C/D and phosphorylated Akt/ERK proteins, VEGF-C/D positive macrophage infiltration, and proliferative/apoptotic cell ratios. Knocking down COX-2 or EP4 in C3L5 cells or treating cells in vitro with celecoxib or EP4A and treating tumor-bearing mice in vivo with the same drug reduced SLC properties of tumor cells including preferential co-expression of COX-2 and SLC markers ALDH1A, CD44, OCT-3/4, β-catenin, and SOX-2. Thus, EP4 is an excellent therapeutic target to block

  9. Bioethanol Production from Uncooked Raw Starch by Immobilized Surface-engineered Yeast Cells

    Science.gov (United States)

    Chen, Jyh-Ping; Wu, Kuo-Wei; Fukuda, Hideki

    Surface-engineered yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis α-amylase on the cell surface was used for direct production of ethanol from uncooked raw starch. By using 50 g/L cells during batch fermentation, ethanol concentration could reach 53 g/L in 7 days. During repeated batch fermentation, the production of ethanol could be maintained for seven consecutive cycles. For cells immobilized in loofa sponge, the concentration of ethanol could reach 42 g/L in 3 days in a circulating packed-bed bioreactor. However, the production of ethanol stopped thereafter because of limited contact between cells and starch. The bioreactor could be operated for repeated batch production of ethanol, but ethanol concentration dropped to 55% of its initial value after five cycles because of a decrease in cell mass and cell viability in the bioreactor. Adding cells to the bioreactor could partially restore ethanol production to 75% of its initial value.

  10. Effects of everolimus on macrophage-derived foam cell behavior

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, Steven, E-mail: steven.hsu@av.abbott.com [Abbott Vascular, 3200 Lakeside Drive, Santa Clara, CA 95054 (United States); Koren, Eugen; Chan, Yen; Koscec, Mirna; Sheehy, Alexander [Abbott Vascular, 3200 Lakeside Drive, Santa Clara, CA 95054 (United States); Kolodgie, Frank; Virmani, Renu [CVPath Institute, Inc., 19 Firstfield Road, Gaithersburg, MD 20878 (United States); Feder, Debra [Abbott Vascular, 3200 Lakeside Drive, Santa Clara, CA 95054 (United States)

    2014-07-15

    Purpose: The purpose of this study was to investigate the effects of everolimus on foam cell (FC) viability, mRNA levels, and inflammatory cytokine production to better understand its potential inhibitory effects on atheroma progression. Methods and materials: Human THP1 macrophage-derived FC were formed using acetylated LDL (acLDL, 100 μg/mL) for 72 hours, followed by everolimus treatment (10{sup -5}–10{sup -11} M) for 24 hours. FC viability was quantified using fluorescent calcein AM/DAPI staining. FC lysates and media supernatants were analyzed for apoptosis and necrosis using a Cell Death ELISA{sup PLUS} assay. FC lysates and media supernatants were also analyzed for inflammatory cytokine (IL1β, IL8, MCP1, TNFα) mRNA levels and protein expression using quantitative reverse transcription real-time polymerase chain reaction (QPCR) and a Procarta® immunoassay, respectively. mRNA levels of autophagy (MAP1LC3), apoptosis (survivin, clusterin), and matrix degradation (MMP1, MMP9) markers were evaluated by Quantigene® Plex assay and verified with QPCR. Additionally, hypercholesterolemic rabbits received everolimus-eluting stents (EES) for 28 or 60 days. RAM-11 immunohistochemical staining was performed to compare %RAM-11 positive area between stented sections and unstented proximal sections. Statistical significance was calculated using one-way ANOVA (p ≤ 0.05). Results: Calcein AM/DAPI staining showed that FC exposed to everolimus (10{sup -5} M) had significantly decreased viability compared to control. FC apoptosis was significantly increased at a high dose of everolimus (10{sup -5} M), with no necrotic effects at any dose tested. Everolimus did not affect endothelial (HUVEC) and smooth muscle (HCASMC) cell apoptosis or necrosis. Everolimus (10{sup -5} M) significantly increased MAP1LC3, caused an increased trend in clusterin (p = 0.10), and significantly decreased survivin and MMP1 mRNA levels in FC. MCP1 cytokine mRNA levels and secreted protein

  11. 三种成体干细胞对脂多糖诱导RAW264.7细胞炎症状态的影响%Effects of three different adult stem cells on inflammatory status of lipopolysaccharide-induced RAW264.7 cells

    Institute of Scientific and Technical Information of China (English)

    何妲; 彭琳; 黄生建; 陆文玲; 王建

    2014-01-01

    目的:比较人羊膜上皮细胞((human amniotic epithelial cell, H-AEC))、羊膜间充质细胞(human amniotic mesenchymal cell, HA-MSC)和脐带间充质细胞(Umbilical cord mesenchymal stem cells, UC-MSC)分泌的细胞因子对脂多糖刺激巨噬细胞系RAW264.7炎症状态的影响。方法将LPS刺激的RAW264.7细胞炎症模型作为对照组,比较H-AEC、HA-MSC、UC-MSC和RAW264.7共培养或条件培养基培养RAW264.7对RAW264.7炎症状态的影响。比较各组RAW264.7细胞的迁移能力;检测各组细胞分泌一氧化氮(NO)的水平;用实时定量多聚酶链反应(RT-PCR)检测各组细胞经典激活的巨噬细胞(classically activated macrophage, M1 macrophage)相关的促炎基因如白介素-1β(IL-1β)、肿瘤坏死基因а(TNFа)、一氧化氮合成酶-2(NOS-2)以及M2 macrophage相关的抑炎基因如精氨酸酶(Arg-1)、甘露糖受体基因CD206、B类清道夫受体CD36)表达情况。结果(1)H-AEC、HA-MSC、UC-MSC处理后RAW264.7的迁移率分别为14.7%±4.5%、9.6%±0.7%、13.0%±0.9%,与对照组(31.1%±11.0%)相比,3种细胞的条件培养基处理后RAW264.7的迁移率均降低,差异具有显著性(P<0.05);(2)H-AEC、HA-MSC、UC-MSC共培养后RAW264.7细胞分泌NO的水平分别为24.26±0.72、44.52±2.51、42.25±0.76μmol/L,与对照组(45.65±1.78μmol/L)相比,H-AEC组细胞分泌的NO有显著性下降(P<0.05);(3)促炎基因与抑炎基因的表达改变:(A)H-AEC处理组促炎基因IL-1β、TNFа、NOS-2、INFβ的表达下调,与对照组相比有显著差别;HA-MSC、UC-MSC处理组促炎基因INFβ表达下调显著,其余基因均上调表达;抑炎相关基因如Arg-1、CD206、CD36均上调;(B)3组细胞干预后抑炎症相关基因Arg-1、CD206、CD36表达均上调,与对照组有显著差异。结论人羊膜上皮细胞、羊膜间充质细胞和脐带间充质细胞

  12. Raw data used to generate figures 2 through 6 in Biological Responses of Raw 264.7 Macrophage Exposed to Two Strains of Stachybotrys chartarum Spores Grown on Four Different Wallboard Types manuscript.

    Data.gov (United States)

    U.S. Environmental Protection Agency — Excel files containing raw data used to generate figures throughout manuscript. This dataset is associated with the following publication: Dean , T., D. Betancourt ,...

  13. Different cell death modes of pancreatic acinar cells on macrophage activation in rats

    Institute of Scientific and Technical Information of China (English)

    LIANG Tao; LIU Tie-fu; XUE Dong-bo; SUN Bei; SHI Li-jun

    2008-01-01

    Background The pathogenesis of acute pancreatitis is complex and largely unclear. The aim of this study was to explore the relationship between modes of cell death in pancreatic acinar cells, the release of cell contents and the inflammatory response of macrophagas.Methods Our experiment included four groups: group A (the control group), group B (AR42J cells overstimulated by caerulein), group C (AR42J cells treated with lipopolysaccharide and caerulein), and group D (AR42J cells treated with octreotide and caerulein). Apoptosis and oncosis, and the release of amylase and lactate dehydrogenase (LDH) from AR42J cells were detected. Rat macrophages were stimulated by 1 ml supematant of culture medium of AR42J cells.Finally, NF-кB activation and TNF-α and IL-1β secretion by macrophages were detected.Results Oncotlc cells in group C increased while apoptctic cells decreased (P <0.05); cells in group D had the inverse reaction. The release of amylase and LDH changed directly with the occurrence of oncosis. The transcription factor NF-кB was activated and secretion of TNF-α and IL-1β were significantly higher in group C than in group B (P <0.05); in group D, these actions were significantly lower than in group B (P<0.05). This trend was in line with changes in amylase and LDH production.Conclusion There is a close relationship between modes of pancreatic acinar cell death, the release of cell contents and the inflammatory reaction of macrophages.

  14. Ultra-High Molecular Weight Polyethylene Reinforced with Multiwall Carbon Nanotubes: In Vitro Biocompatibility Study Using Macrophage-Like Cells

    Directory of Open Access Journals (Sweden)

    Nayeli Camacho

    2015-07-01

    Full Text Available Carbon nanotubes are highly versatile materials; new applications using them are continuously being developed. Special attention is being dedicated to the possible use of multiwall carbon nanotubes in biomaterials contacting with bone. This study describes the response of murine macrophage-like Raw 264.7 cells after two and six days of culture in contact with artificially generated particles from both, ultra-high molecular weight polyethylene polymer and the composite (multiwall carbon nanotubes and ultra-high molecular weight polyethylene. This novel composite has superior wear behavior, having thus the potential to reduce the number of revision knee arthroplasty surgeries required by wear failure of tibial articulating component and diminish particle-induced osteolysis. The results of an in vitro study of viability, and interleukin-6 and tumor necrosis factor-alpha production suggest good cytocompatibility, similar to that of conventional ultra-high molecular weight polyethylene.

  15. An inflammatory gene signature distinguishes neurofibroma Schwann cells and macrophages from cells in the normal peripheral nervous system

    Science.gov (United States)

    Choi, Kwangmin; Komurov, Kakajan; Fletcher, Jonathan S.; Jousma, Edwin; Cancelas, Jose A.; Wu, Jianqiang; Ratner, Nancy

    2017-01-01

    Neurofibromas are benign peripheral nerve tumors driven by NF1 loss in Schwann cells (SCs). Macrophages are abundant in neurofibromas, and macrophage targeted interventions may have therapeutic potential in these tumors. We generated gene expression data from fluorescence-activated cell sorted (FACS) SCs and macrophages from wild-type and mutant nerve and neurofibroma to identify candidate pathways involved in SC-macrophage cross-talk. While in 1-month-old Nf1 mutant nerve neither SCs nor macrophages significantly differed from their normal counterparts, both macrophages and SCs showed significantly altered cytokine gene expression in neurofibromas. Computationally reconstructed SC-macrophage molecular networks were enriched for inflammation-associated pathways. We verified that neurofibroma SC conditioned medium contains macrophage chemo-attractants including colony stimulation factor 1 (CSF1). Network analysis confirmed previously implicated pathways and predict novel paracrine and autocrine loops involving cytokines, chemokines, and growth factors. Network analysis also predicted a central role for decreased type-I interferon signaling. We validated type-I interferon expression in neurofibroma by protein profiling, and show that treatment of neurofibroma-bearing mice with polyethylene glycolyated (PEGylated) type-I interferon-α2b reduces the expression of many cytokines overexpressed in neurofibroma. These studies reveal numerous potential targetable interactions between Nf1 mutant SCs and macrophages for further analyses. PMID:28256556

  16. Macrophage specific overexpression of the human macrophage scavenger receptor in transgenic mice, using a 180-kb yeast artificial chromosome, leads to enhanced foam cell formation of isolated peritoneal macrophages

    NARCIS (Netherlands)

    Winther, M.P.J. de; Dijk, K.W. van; Vlijmen, B.J.M. van; Gijbels, M.J.J.; Heus, J.J.; Wijers, E.R.; Bos, A.C. van den; Breuer, M.; Frants, R.R.; Havekes, L.M.; Hofker, M.H.

    1999-01-01

    Macrophage scavenger receptors class A (MSR) are thought to play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins by macrophages in the vessel wall leading to foam cell formation. To investigate the in vivo role of the MSR in this process, a transgenic

  17. Disruption of Lipid Rafts Interferes with the Interaction of Toxoplasma gondii with Macrophages and Epithelial Cells

    Science.gov (United States)

    Cruz, Karla Dias; Cruz, Thayana Araújo; Veras de Moraes, Gabriela; Paredes-Santos, Tatiana Christina; Attias, Marcia; de Souza, Wanderley

    2014-01-01

    The intracellular parasite Toxoplasma gondii can penetrate any warm-blooded animal cell. Conserved molecular assemblies of host cell plasma membranes should be involved in the parasite-host cell recognition. Lipid rafts are well-conserved membrane microdomains that contain high concentrations of cholesterol, sphingolipids, glycosylphosphatidylinositol, GPI-anchored proteins, and dually acylated proteins such as members of the Src family of tyrosine kinases. Disturbing lipid rafts of mouse peritoneal macrophages and epithelial cells of the lineage LLC-MK2 with methyl-beta cyclodextrin (MβCD) and filipin, which interfere with cholesterol or lidocaine, significantly inhibited internalization of T. gondii in both cell types, although adhesion remained unaffected in macrophages and decreased only in LLC-MK2 cells. Scanning and transmission electron microscopy confirmed these observations. Results are discussed in terms of the original role of macrophages as professional phagocytes versus the LLC-MK2 cell lineage originated from kidney epithelial cells. PMID:24734239

  18. The interplay between monocytes/macrophages and CD4+ T cell subsets in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Ceri A. Roberts

    2015-11-01

    Full Text Available Rheumatoid arthritis (RA is a chronic inflammatory disease characterized by inflammation of the synovial lining (synovitis. The inflammation in the RA joint is associated with and driven by immune cell infiltration, synovial hyperproliferation and excessive production of pro-inflammatory mediators, such as TNFα, IFNγ, IL-1β, IL-6 and IL-17, eventually resulting in damage to the cartilage and underlying bone. The RA joint harbors a wide range of immune cell types, including monocytes, macrophages and CD4+ T cells (both pro-inflammatory and regulatory. The interplay between CD14+ myeloid cells and CD4+ T cells can significantly influence CD4+ T cell function and conversely, effector vs. regulatory CD4+ T cell subsets can exert profound effects on monocyte/macrophage function. In this review, we will discuss how the interplay between CD4+ T cells and monocytes/macrophages may contribute to the immunopathology of RA.

  19. 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.

  20. 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.

  1. Cellular uptake of a dexamethasone palmitate-low density lipoprotein complex by macrophages and foam cells.

    Science.gov (United States)

    Tauchi, Yoshihiko; Chono, Sumio; Morimoto, Kazuhiro

    2003-04-01

    To evaluate the utility of a dexamethasone palmitate (DP)-low density lipoprotein (LDL) complex to transport drug into foam cells, the cellular uptake of DP-LDL complex by macrophages and foam cells was examined. The DP-LDL complex was prepared by incubation with DP and LDL, and the DP-LDL complex and murine macrophages were incubated. No cellular uptake of the DP-LDL complex by macrophages was found until 6 h after the start of incubation, but this gradually increased from 12 to 48 h. On the other hand, the cellular uptake of the oxidized DP-LDL complex was already apparent at 3 h after the start incubation, and then markedly increased until 48 h incubation along with that of the lipid emulsion (LE) containing DP (DP-LE). The cellular uptake of DP-LE by foam cells was significantly lower than that by macrophages. However, the cellular uptake of DP-LDL complex by foam cells was similar to that by macrophages. These findings suggest that the DP-LDL complex is oxidatively modified, and then incorporated into macrophages and foam cells through the scavenger receptor pathway. Since selective delivery of drugs into foam cells in the early stage of atherosclerosis is a useful protocol for antiatherosclerosis treatment, the DP-LDL complex appears to be a potentially useful drug-carrier complex for future antiatherosclerotic therapy.

  2. Cypermethrin Induces Macrophages Death through Cell Cycle Arrest and Oxidative Stress-Mediated JNK/ERK Signaling Regulated Apoptosis

    Directory of Open Access Journals (Sweden)

    Fang Huang

    2016-06-01

    Full Text Available Cypermethrin is one of the most highly effective synthetic pyrethroid insecticides. The toxicity of cypermethrin to the reproductive and nervous systems has been well studied. However, little is known about the toxic effect of cypermethrin on immune cells such as macrophages. Here, we investigated the cytotoxicity of cypermethrin on macrophages and the underlying molecular mechanisms. We found that cypermethrin reduced cell viability and induced apoptosis in RAW 264.7 cells. Cypermethrin also increased reactive oxygen species (ROS production and DNA damage in a dose-dependent manner. Moreover, cypermethrin-induced G1 cell cycle arrest was associated with an enhanced expression of p21, wild-type p53, and down-regulation of cyclin D1, cyclin E and CDK4. In addition, cypermethrin treatment activated MAPK signal pathways by inducing c-Jun N-terminal kinase (JNK and extracellular regulated protein kinases 1/2 ERK1/2 phosphorylation, and increased the cleaved poly ADP-ribose polymerase (PARP. Further, pretreatment with antioxidant N-acetylcysteine (NAC effectively abrogated cypermethrin-induced cell cytotoxicity, G1 cell cycle arrest, DNA damage, PARP activity, and JNK and ERK1/2 activation. The specific JNK inhibitor (SP600125 and ERK1/2 inhibitor (PD98059 effectively reversed the phosphorylation level of JNK and ERK1/2, and attenuated the apoptosis. Taken together, these data suggested that cypermethrin caused immune cell death via inducing cell cycle arrest and apoptosis regulated by ROS-mediated JNK/ERK pathway.

  3. Effect of Three-spot Seahorse Petroleum Ether Extract on Lipopolysaccharide Induced Macrophage RAW264.7 Inflammatory Cytokine Nitric Oxide and Composition Analysis.

    Science.gov (United States)

    Chen, LiPing; Shen, XuanRi; Chen, GuoHua; Cao, XianYing; Yang, Jian

    2015-01-01

    Three-Spot seahorse is a traditional medicine in Asian countries. However, the alcohol extract is largely unknown for its anti-inflammatory activity. This study aimed at elucidating fraction of potent anti-inflammatory activity of seahorse. A systematic solvent extraction method of liquid-liquid fractionation of ethanol crude extract gave four fractions petroleum ether (PE), and ethyl acetate (EtOAc), water saturated butanol (n-BuOH), water (H2O). In this study, PE extract was selected for further study after preliminary screening test, and was connected to silica column chromatography and eluted with different polarity of mobile phases, and obtained four active fractions (Fr I, Fr II, Fr III, Fr IV). Effect of separated fractions on inflammation was investigated in lipopolysaccharide (LPS) stimulated murine RAW264.7 cells in vitro. The result shows that seahorse extract was capable of inhibiting the production of nitric oxide (NO) significantly in a dose dependent manner and exhibited no notable cytotoxicity on cell viability. IC50 of fraction IV was 36.31 μg/mL, indicating that separated fraction possessed potent NO inhibitory activity against LPS-induced inflammatory response, thus, demonstrated its in vitro anti-inflammatory potentiality, it may be at least partially explained by the presence of anti-inflammation active substances, phenolic compounds, phospholipids and polyunsaturated fatty acids, especially phospholipids and polyunsaturated fatty acids. It could be suggested that seahorse lipid-soluble components could be used in functional food and anti-inflammatory drug preparations.

  4. Matrix metalloproteinase-9 and cell division in neuroblastoma cells and bone marrow macrophages.

    Science.gov (United States)

    Sans-Fons, M Gloria; Sole, Sonia; Sanfeliu, Coral; Planas, Anna M

    2010-12-01

    Matrix metalloproteinases (MMPs) degrade the extracellular matrix and carry out key functions in cell development, cancer, injury, and regeneration. In addition to its well recognized extracellular action, functional intracellular MMP activity under certain conditions is supported by increasing evidence. In this study, we observed higher gelatinase activity by in situ zymography and increased MMP-9 immunoreactivity in human neuroblastoma cells and in bone marrow macrophages undergoing mitosis compared with resting cells. We studied the pattern of immunoreactivity at the different stages of cell division by confocal microscopy. Immunostaining with different monoclonal antibodies against MMP-9 revealed a precise, dynamic, and well orchestrated localization of MMP-9 at the different stages of cell division. The cellular distribution of MMP-9 staining was studied in relation to that of microtubules. The spatial pattern of MMP-9 immunoreactivity suggested some participation in both the reorganization of the nuclear content and the process of chromatid segmentation. We then used several MMP-9 inhibitors to find out whether MMP-9 might be involved in the cell cycle. These drugs impaired the entry of cells into mitosis, as revealed by flow cytometry, and reduced cell culture growth. In addition, the silencing of MMP-9 expression with small interfering RNA also reduced cell growth. Taken together, these results suggest that intracellular MMP-9 is involved in the process of cell division in neuroblastoma cells and in primary cultures of macrophages.

  5. Interactions between colon cancer cells and tumor-infiltrated macrophages depending on cancer cell-derived colony stimulating factor 1.

    Science.gov (United States)

    Wang, Huayang; Shao, Qianqian; Sun, Jintang; Ma, Chao; Gao, Wenjuan; Wang, Qingjie; Zhao, Lei; Qu, Xun

    2016-04-01

    Tumor-infiltrated macrophages were potential targets of the immune therapy for patients with colon cancer. Colony stimulating factor 1 (CSF1) is a primary chemoattractant and functional regulator for macrophages, and therefore would be a feasible intervention for the macrophage-targeting therapeutics. However, the expression of CSF1 in colon cancer microenvironment and its roles in cancer development is largely unknown. In the present study, we found that CSF1 was over-expressed exclusively in colon cancer cells and was correlated with macrophages infiltration. The high CSF1 expression and macrophages infiltration were related to the tumor-node-metastasis (TNM) stage of colon cancer, and suggested to be positively associated with survival of colon cancer patients. In the in vitro studies based on an indirect Transwell system, we found that co-culture with macrophage promoted CSF1 production in colon cancer cells. Further investigation on regulatory mechanisms suggested that CSF1 production in colon cancer cells was dependent on PKC pathway, which was activated by IL-8, mainly produced by macrophages. Moreover, colon cancer cell-derived CSF1 drove the recruitment of macrophages and re-educated their secretion profile, including the augment of IL-8 production. The mice tumor xenografts study also found that over-expression of CSF1 in colon cancer cells promoted intratumoral infiltration of macrophages, and partially suppressed tumor growth. In all, our results demonstrated that CSF1 was an important factor in the colon cancer microenvironment, involving in the interactions between colon cancer cells and tumor-infiltrated macrophages.

  6. The Effect of Bee Venom on COX-2, P38, ERK and JNK in RAW 264.7 Cells

    Directory of Open Access Journals (Sweden)

    Jae-Young Sim

    2003-06-01

    Full Text Available Objectives : The purpose of this study was to investigate the effect of Bee Venom on the lipopolysaccharide(LPS, sodium nitroprusside(SNP, hydrogen peroxide(H2O2-induced expressions of cyclooxygenase-2(COX-2, p38, jun N-terminal Kinase(JNK and extra-signal response kinase(ERK in RAW 264.7 cells, a murine macrophage cell line. Methods : The expressions of COX-2, p38, JNK and ERK were determined by western blotting with corresponding antibodies.\\ Results : 1. The 0.5, 1 and 5 ㎍/㎖ of bee venom inhibited significantly LPS and SNP-induced expression of COX-2 compared with control, respectively. The 0.5, 1 and 5 ㎍/㎖ of bee venom inhibited insignificantly H2O2-induced expression of COX-2 compared with control, respectively. 2. The 0.5, 1 and 5 ㎍/㎖ of bee venom inhibited significantly LPS, SNP and H2O2-induced expression of p38 compared with control, respectively. 3. The 1 and 5 ㎍/㎖ of bee venom inhibited significantly SNP-induced expression of JNK compared with control, respectively. All of bee venom inhibited insignificantly LPS and H2O2-induced expression of JNK compared with control, respectively. 4. The 5 ㎍/㎖ of bee venom inhibited significantly SNP-induced expression of ERK, the 0.5 ㎍/㎖ of bee venom increased significantly H2O2-induced expression of ERK compared with control. The 0.5, 1 and 5 ㎍/㎖ of bee venom inhibited insignificantly LPS-induced expression of ERK compared with control, respectively.

  7. Anti-inflammatory effect of Taraxacum officinale leaves on lipopolysaccharide-induced inflammatory responses in RAW 264.7 cells.

    Science.gov (United States)

    Koh, Yoon-Jeoung; Cha, Dong-Soo; Ko, Je-Sang; Park, Hyun-Jin; Choi, Hee-Don

    2010-08-01

    To investigate the efficacy and the mechanism of the anti-inflammatory effect of Taraxacum officinale leaves (TOLs), the effect of a methanol extract and its fractions recovered from TOLs on lipopolysaccharide (LPS)-induced responses was studied in the mouse macrophage cell line, RAW 264.7. Cells were pretreated with various concentrations of the methanol extract and its fractions and subsequently incubated with LPS (1 microg/mL). The levels of nitric oxide (NO), prostaglandin (PG) E(2), and pro-inflammatory cytokines including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 were determined using enzyme-linked immunosorbent assays. Expressions of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 and activation of mitogen-activated protein (MAP) kinases were analyzed using western blotting. The methanol extract and its fractions inhibited LPS-induced production of NO, pro-inflammatory cytokines, and PGE(2) in a dose-dependent manner. The chloroform fraction significantly suppressed production of NO, PGE(2), and two pro-inflammatory cytokines (TNF-alpha and IL-1beta) in a dose-dependent manner with 50% inhibitory concentration values of 66.51, 90.96, 114.76, and 171.06 microg/mL, respectively. The ethyl acetate fraction also inhibited production of the inflammatory molecules. The chloroform and ethyl acetate fractions reduced LPS-induced expressions of iNOS and COX-2 and activation of MAP kinases in a dose-dependent manner. Among the fractions of the methanol extract, the chloroform and ethyl acetate fractions exhibited the most effective anti-inflammatory activities. These results show that the anti-inflammatory effects of TOLs are probably due to down-regulation of NO, PGE(2), and pro-inflammatory cytokines and reduced expressions of iNOS and COX-2 via inactivation of the MAP kinase signal pathway.

  8. 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

  9. CRISPR/CAS9-Mediated Genome Editing of miRNA-155 Inhibits Proinflammatory Cytokine Production by RAW264.7 Cells

    Directory of Open Access Journals (Sweden)

    Weixia Jing

    2015-01-01

    Full Text Available MicroRNA 155 (miR-155 is a key proinflammatory regulator in clinical and experimental rheumatoid arthritis (RA. Here we generated a miR-155 genome knockout (GKO RAW264.7 macrophage cell line using the clustered regulatory interspaced short palindromic repeat (CRISPR/CRISPR-associated protein 9 (CAS9 technology. While upregulating the Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1, the miR-155 GKO line is severely impaired in producing proinflammatory cytokines but slightly increased in osteoclastogenesis upon treatment with receptor activator of nuclear factor-κB ligand (RANKL. Taken together, our results suggest that genome editing of miR-155 holds the potential as a therapeutic strategy in RA.

  10. Bone marrow-derived cells serve as proangiogenic macrophages but not endothelial cells in wound healing.

    Science.gov (United States)

    Okuno, Yuji; Nakamura-Ishizu, Ayako; Kishi, Kazuo; Suda, Toshio; Kubota, Yoshiaki

    2011-05-12

    Bone marrow-derived cells (BMDCs) contribute to postnatal vascular growth by differentiating into endothelial cells or secreting angiogenic factors. However, the extent of their endothelial differentiation highly varies according to the angiogenic models used. Wound healing is an intricate process in which the skin repairs itself after injury. As a process also observed in cancer progression, neoangiogenesis into wound tissues is profoundly involved in this healing process, suggesting the contribution of BMDCs. However, the extent of the differentiation of BMDCs to endothelial cells in wound healing is unclear. In this study, using the green fluorescent protein-bone marrow chim-eric experiment and high resolution confocal microscopy at a single cell level, we observed no endothelial differentiation of BMDCs in 2 acute wound healing models (dorsal excisional wound and ear punch) and a chronic wound healing model (decubitus ulcer). Instead, a major proportion of BMDCs were macrophages. Indeed, colony-stimulating factor 1 (CSF-1) inhibition depleted approximately 80% of the BMDCs at the wound healing site. CSF-1-mutant (CSF-1(op/op)) mice showed significantly reduced neoangiogenesis into the wound site, supporting the substantial role of BMDCs as macrophages. Our data show that the proangiogenic effects of macrophages, but not the endothelial differentiation, are the major contribution of BMDCs in wound healing.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Dihydroaustrasulfone Alcohol (WA-25 Impedes Macrophage Foam Cell Formation by Regulating the Transforming Growth Factor-β1 Pathway

    Directory of Open Access Journals (Sweden)

    Yi-Chen Wang

    2015-05-01

    Full Text Available Atherosclerosis is considered an inflammatory disease. However, clinically used anti-atherosclerotic drugs, such as simvastatin, have many side effects. Recently, several unique marine compounds have been isolated that possess a variety of bioactivities. In a previous study, we found a synthetic precursor of the marine compound (austrasulfone, which is dihydroaustrasulfone alcohol (WA-25, has anti-atherosclerotic effects in vivo. However, the detailed mechanisms remain unclear. Therefore, to clarify the mechanisms through which WA-25 exerts anti-atherosclerotic activity, we used RAW 264.7 macrophages as an in vitro model to evaluate the effects of WA-25. In lipopolysaccharide (LPS-stimulated RAW 264.7 cells, WA-25 significantly inhibited expression of the pro-inflammatory proteins, inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2. In contrast, simvastatin increased the COX-2 expression compared to WA-25. In addition, WA-25 impedes foam cell formation and up-regulated the lysosomal and cyclic adenosine monophosphate (cAMP signaling pathway. We also observed that transforming growth factor β1 (TGF-β1 was up-regulated by WA-25 and simvastatin in LPS-induced RAW 264.7 cells, and the promising anti-atherosclerosis effects of WA-25 were disrupted by blockade of TGF-β1 signaling. Besides, WA-25 might act through increasing lipolysis than through alteration of lipid export. Taken together, these data demonstrate that WA-25 may have potential as an anti-atherosclerotic drug with anti-inflammatory effects.

  13. The Interaction of Adrenomedullin and Macrophages Induces Ovarian Cancer Cell Migration via Activation of RhoA Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Xiaoyan Pang

    2013-01-01

    Full Text Available Tumor-associated macrophages (TAMs are correlated with poor prognosis in many human cancers; however, the mechanism by which TAMs facilitate ovarian cancer cell migration and invasion remains unknown. This study was aimed to examine the function of adrenomedullin (ADM in macrophage polarization and their further effects on the migration of ovarian cancer cells. Exogenous ADM antagonist and small interfering RNA (siRNA specific for ADM expression were treated to macrophages and EOC cell line HO8910, respectively. Then macrophages were cocultured with HO8910 cells without direct contact. Flow cytometry, Western blot and real-time PCR were used to detect macrophage phenotype and cytokine production. The migration ability and cytoskeleton rearrangement of ovarian cancer cells were determined by Transwell migration assay and phalloidin staining. Western blot was performed to evaluate the activity status of signaling molecules in the process of ovarian cancer cell migration. The results showed that ADM induced macrophage phenotype and cytokine production similar to TAMs. Macrophages polarized by ADM promoted the migration and cytoskeleton rearrangement of HO8910 cells. The expression of RhoA and its downstream effector, cofilin, were upregulated in macrophage-induced migration of HO8910 cells. In conclusion, ADM could polarize macrophages similar to TAMs, and then polarized macrophages promote the migration of ovarian cancer cells via activation of RhoA signaling pathway in vitro.

  14. Recent progress in understandıng the function of intestinal macrophages and dendritic cells

    Science.gov (United States)

    Kelsall, Brian

    2016-01-01

    Mucosal immune responses must be tightly controlled, particularly in the intestine, As members of the mononuclear phagocyte family, dendritic cells and macrophages, are well represented in intestinal tissues, and have developed unique functional niches. This review will focus on recent findings on antigen uptake and processing in the intestine, and the role of DCs in the imprinting homing receptors on T and B cells, the induction of IgA B cell responses, and the differentiation of regulatory T cells (Tregs). It will also address the unique phenotype of intestinal macrophages and briefly what is known regarding the relationships between these cell types. PMID:19079213

  15. Diamagnetic levitation promotes osteoclast differentiation from RAW264.7 cells.

    Science.gov (United States)

    Sun, Yu-Long; Chen, Zhi-Hao; Chen, Xiao-Hu; Yin, Chong; Li, Di-Jie; Ma, Xiao-Li; Zhao, Fan; Zhang, Ge; Shang, Peng; Qian, Ai-Rong

    2015-03-01

    The superconducting magnet with a high magnetic force field can levitate diamagnetic materials. In this study, a specially designed superconducting magnet with large gradient high magnetic field (LGHMF), which provides three apparent gravity levels (μg, 1 g, and 2 g), was used to study its influence on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation from preosteoclast cell line RAW264.7. The effects of LGHMF on the viability, nitric oxide (NO) production, morphology in RAW264.7 cells were detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, the Griess method, and the immunofluorescence staining, respectively. The changes induced by LGHMF in osteoclast formation, mRNA expression, and bone resorption were determined by tartrate-resistant acid phosphatase staining, semiquantity PCR, and bone resorption test, respectively. The results showed that: 1) LGHMF had no lethal effect on osteoclast precursors but attenuated NO release in RAW264.7 cells. 2) Diamagnetic levitation (μg) enhanced both the formation and bone resorption capacity of osteoclast. Moreover, diamagnetic levitation up-regulated mRNA expression of RANK, Cathepsin K, MMP-9, and NFATc1, while down-regulated RunX2 in comparison with controls. Furthermore, diamagnetic levitation induced obvious morphological alterations in osteoclast, including active cytoplasmic peripheral pseudopodial expansion, formation of pedosome belt, and aggregation of actin ring. 3) Magnetic field produced by LGHMF attenuated osteoclast resorption activity. Collectively, LGHMF with combined effects has multiple effects on osteoclast, which attenuated osteoclast resorption with magnetic field, whereas promoted osteoclast differentiation with diamagnetic levitation. Therefore, these findings indicate that diamagnetic levitation could be used as a novel ground-based microgravity simulator, which facilitates bone cell research of weightlessness condition.

  16. Wear particles generated from studded tires and pavement induces inflammatory reactions in mouse macrophage cells.

    Science.gov (United States)

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

    2007-06-01

    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. These health risks are of increasing concern in society, and to protect public health, a clarification of the toxic properties of particles from different sources is of importance. Lately, wear particles generated from traffic have been recognized as a major contributing source to the overall particle load, especially in the Nordic countries where studded tires are used. The aim of this study was to further investigate and compare the ability to induce inflammatory mediators of different traffic-related wear particles collected from an urban street, a subway station, and studded tire-pavement wear. Inflammatory effects were measured as induction of nitric oxide (NO), IL-6, TNF-alpha, arachidonic acid (AA), and lipid peroxidation after exposure of the murine macrophage like cell line RAW 264.7. In addition, the redox potential of the particles was measured in a cell-free system. The results show that all particles tested induce IL-6, TNF-alpha, and NO, and those from the urban street were the most potent ones. In contrast, particles collected from a subway station were most potent to induce lipid peroxidation, AA release, and formation of ROS. Particles from studded tire-pavement wear, generated using a road simulator, were able to induce inflammatory cytokines, NO, lipid peroxidation, and ROS formation. Interestingly, particles generated from pavement containing granite as the main stone material were more potent than those generated from pavement containing quartzite as the main stone material.

  17. 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.

  18. 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

  19. Hoxb8 conditionally immortalised macrophage lines model inflammatory monocytic cells with important similarity to dendritic cells.

    Science.gov (United States)

    Rosas, Marcela; Osorio, Fabiola; Robinson, Matthew J; Davies, Luke C; Dierkes, Nicola; Jones, Simon A; Reis e Sousa, Caetano; Taylor, Philip R

    2011-02-01

    We have examined the potential to generate bona fide macrophages (MØ) from conditionally immortalised murine bone marrow precursors. MØ can be derived from Hoxb8 conditionally immortalised macrophage precursor cell lines (MØP) using either M-CSF or GM-CSF. When differentiated in GM-CSF (GM-MØP) the resultant cells resemble GM-CSF bone marrow-derived dendritic cells (BMDC) in morphological phenotype, antigen phenotype and functional responses to microbial stimuli. In spite of this high similarity between the two cell types and the ability of GM-MØP to effectively present antigen to a T-cell hybridoma, these cells are comparatively poor at priming the expansion of IFN-γ responses from naïve CD4(+) T cells. The generation of MØP from transgenic or genetically aberrant mice provides an excellent opportunity to study the inflammatory role of GM-MØP, and reduces the need for mouse colonies in many studies. Hence differentiation of conditionally immortalised MØPs in GM-CSF represents a unique in vitro model of inflammatory monocyte-like cells, with important differences from bone marrow-derived dendritic cells, which will facilitate functional studies relating to the many 'sub-phenotypes' of inflammatory monocytes.

  20. Macrophage-independent T cell infiltration to the site of injury-induced brain inflammation

    DEFF Research Database (Denmark)

    Fux, Michaela; van Rooijen, Nico; Owens, Trevor

    2008-01-01

    We have addressed the role of macrophages in glial response and T cell entry to the CNS after axonal injury, by using intravenous injection of clodronate-loaded mannosylated liposomes, in C57BL6 mice. As expected, clodronate-liposome treatment resulted in depletion of peripheral macrophages which...... was confirmed by F4/80(-) and MOMA-1(-) stainings in spleen. Sequential clodronate-liposome treatment 4, 2 and 0 days before axotomy resulted in significant reduction of infiltrating CD45(high) CD11b(+) macrophages in the hippocampus at 1, 2 and 3 days post-lesion, measured by flow cytometry. There was a slight...

  1. Impact of alginate-producing Pseudomonas aeruginosa on alveolar macrophage apoptotic cell clearance.

    Science.gov (United States)

    McCaslin, Charles A; Petrusca, Daniela N; Poirier, Christophe; Serban, Karina A; Anderson, Gregory G; Petrache, Irina

    2015-01-01

    Pseudomonas aeruginosa infection is a hallmark of lung disease in cystic fibrosis. Acute infection with P. aeruginosa profoundly inhibits alveolar macrophage clearance of apoptotic cells (efferocytosis) via direct effect of virulence factors. During chronic infection, P. aeruginosa evades host defense by decreased virulence, which includes the production or, in the case of mucoidy, overproduction of alginate. The impact of alginate on innate immunity, in particular on macrophage clearance of apoptotic cells is not known. We hypothesized that P. aeruginosa strains that exhibit reduced virulence impair macrophage clearance of apoptotic cells and we investigated if the polysaccharide alginate produced by mucoid P. aeruginosa is sufficient to inhibit alveolar macrophage efferocytosis. Rat alveolar or human peripheral blood monocyte (THP-1)-derived macrophage cell lines were exposed in vitro to exogenous alginate or to wild type or alginate-overproducing mucoid P. aeruginosa prior to challenge with apoptotic human Jurkat T-lymphocytes. The importance of LPS contamination and that of structural integrity of alginate polymers was tested using alginate of different purities and alginate lyase, respectively. Alginate inhibited alveolar macrophage efferocytosis in a dose- and time-dependent manner. This effect was augmented but not exclusively attributed to lipopolysaccharide (LPS) present in alginates. Alginate-producing P. aeruginosa inhibited macrophage efferocytosis by more than 50%. A mannuronic-specific alginate lyase did not restore efferocytosis inhibited by exogenous guluronic-rich marine alginate, but had a marked beneficial effect on efferocytosis of alveolar macrophages exposed to mucoid P. aeruginosa. Despite decreased virulence, mucoid P. aeruginosa may contribute to chronic airway inflammation through significant inhibition of alveolar clearance of apoptotic cells and debris. The mechanism by which mucoid bacteria inhibit efferocytosis may involve alginate

  2. Total bacterial count and somatic cell count in refrigerated raw milk stored in communal tanks

    Directory of Open Access Journals (Sweden)

    Edmar da Costa Alves

    2014-09-01

    Full Text Available The current industry demand for dairy products with extended shelf life has resulted in new challenges for milk quality maintenance. The processing of milk with high bacterial counts compromises the quality and performance of industrial products. The study aimed to evaluate the total bacteria counts (TBC and somatic cell count (SCC in 768 samples of refrigerated raw milk, from 32 communal tanks. Samples were collected in the first quarter of 2010, 2011, 2012 and 2013 and analyzed by the Laboratory of Milk Quality - LQL. Results showed that 62.5%, 37.5%, 15.6% and 27.1% of the means for TBC in 2010, 2011, 2012 and 2013, respectively, were above the values established by legislation. However, we observed a significant reduction in the levels of total bacterial count (TBC in the studied periods. For somatic cell count, 100% of the means indicated values below 600.000 cells/mL, complying with the actual Brazilian legislation. The values found for the somatic cell count suggests the adoption of effective measures for the sanitary control of the herd. However, the results must be considered with caution as it highlights the need for quality improvements of the raw material until it achieves reliable results effectively.

  3. Wear particle-mediated expressions of pro-inflammatory cytokines,NF-κB and RANK were impacted by lanthanum chloride in RAW264.7 cells

    Institute of Scientific and Technical Information of China (English)

    DAI Min; JIANG Chuan; LIU Xiang; LI Zhe; CHENG Xigao; ZOU Yang; NIE Tao

    2013-01-01

    To explore the impact of different concentrations of lanthanum chloride (LaCl3) on critical components of wear particle-mediated signaling pathways in inflammation and osteoclastogenesis,RAW264.7 cells were naturally divided into eight groups and analyzed by CCK-8 assay,flow cytometry,ELISA,RT-PCR and western blot after treatments.The results showed that three concentrations of LaCl3 had no influence on viability of RAW264.7 cells and down-regulated receptor activator of nuclear factor κB (RANK) instead of macrophage colony-stimulating factor receptor (M-CSFR).Additionally,2.5 and 10 μmol/L LaCl3 could signifi-cantly inhibit gene and protein levels of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-lβ,i.e.,TNF-α and IL-1β) and NF-κB/p65,but 100 μrnol/L LaCl3 did not exert an obvious inflammation-inhibiting effect,and even induced inflammation.In conclusion,these findings demonstrated that LaC13 was able to suppress wear particle-induced inflammation and activation of NF-κB in a certain range of concentrations in vitro and mainly decrease the expression of RANK,but not M-CSFR,all of which were generally recognized to play a pivotal role in osteoclastogenesis.

  4. Arctigenin inhibits lipopolysaccharide-induced iNOS expression in RAW264.7 cells through suppressing JAK-STAT signal pathway.

    Science.gov (United States)

    Kou, Xianjuan; Qi, Shimei; Dai, Wuxing; Luo, Lan; Yin, Zhimin

    2011-08-01

    Arctigenin has been demonstrated to have an anti-inflammatory function, but the precise mechanisms of its action remain to be fully defined. In the present study, we determined the effects of arctigenin on lipopolysaccharide (LPS)-induced production of proinflammatory mediators and the underlying mechanisms involved in RAW264.7 cells. Our results indicated that arctigenin exerted its anti-inflammatory effect by inhibiting ROS-dependent STAT signaling through its antioxidant activity. Arctigenin also significantly reduced the phosphorylation of STAT1 and STAT 3 as well as JAK2 in LPS-stimulated RAW264.7 cells. The inhibitions of STAT1 and STAT 3 by arctigenin prevented their translocation to the nucleus and consequently inhibited expression of iNOS, thereby suppressing the expression of inflammation-associated genes, such as IL-1β, IL-6 and MCP-1, whose promoters contain STAT-binding elements. However, COX-2 expression was slightly inhibited at higher drug concentrations (50 μM). Our data demonstrate that arctigenin inhibits iNOS expression via suppressing JAK-STAT signaling pathway in macrophages.

  5. 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.

  6. Antioxidant and Anti-Inflammatory Effects of Various Cultivars of Kiwi Berry (Actinidia arguta) on Lipopolysaccharide-Stimulated RAW 264.7 Cells.

    Science.gov (United States)

    An, Xiangxue; Lee, Sang Gil; Kang, Hee; Heo, Ho Jin; Cho, Youn-Sup; Kim, Dae-Ok

    2016-08-28

    The present study evaluated the total phenolic and flavonoid contents as well as total antioxidant capacity (TAC) of three cultivars of Actinidia arguta Planch. kiwi berries; cv. Mansoo (Mansoo), cv. Chiak (Chiak), and cv. Haeyeon (Haeyeon). In addition, the anti-inflammatory effects of the three cultivars of kiwi berries were investigated using a lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cell line. Mansoo had the highest total phenolic content and TAC among the three cultivars, whereas Chiak had the highest total flavonoid content. The total antioxidant capacities of the kiwi berry extracts were more strongly correlated with total phenolic content than with total flavonoid content. The kiwi berry extracts suppressed the secretion of pro-inflammatory cytokines, including interleukin-6 and tumor necrosis factor-α, from LPS-stimulated RAW 264.7 cells. The release of nitrite, an indirect indicator of nitric oxide, was also ameliorated by pre-treatment with the kiwi berry extracts in a dose-dependent manner. Cellular-based measurements of antioxidant capacity exhibited that the kiwi berry extracts had cellular antioxidant capacities. Such cellular antioxidant effects are possibly attributed to their direct antioxidant capacity or to the inhibition of reactive oxygen species generation via anti-inflammatory effects. Our findings suggest that kiwi berries are potential antioxidant and anti-inflammatory agents.

  7. Coculture with intraocular lens material-activated macrophages induces an inflammatory phenotype in lens epithelial cells.

    Science.gov (United States)

    Pintwala, Robert; Postnikoff, Cameron; Molladavoodi, Sara; Gorbet, Maud

    2015-03-01

    Cataracts are the leading cause of blindness worldwide, requiring surgical implantation of an intraocular lens. Despite evidence of leukocyte ingress into the postoperative lens, few studies have investigated the leukocyte response to intraocular lens materials. A novel coculture model was developed to examine macrophage activation by hydrophilic acrylic (poly(2-hydroxyethyl methacrylate)) and hydrophobic acrylic (polymethylmethacrylate) commercial intraocular lens. The human monocytic cell line THP-1 was differentiated into macrophages and cocultured with human lens epithelial cell line (HLE-B3) with or without an intraocular lens for one, two, four, or six days. Using flow cytometry and confocal microscopy, expression of the macrophage activation marker CD54 (intercellular adhesion molecule-1) and production of reactive oxygen species via the fluorogenic probe 2',7'-dichlorodihydrofluorescein diacetate were examined in macrophages. α-Smooth muscle actin, a transdifferentiation marker, was characterized in lens epithelial cells. The poly(2-hydroxyethyl methacrylate) intraocular lens prevented adhesion but induced significant macrophage activation (p intraocular lens), while the polymethylmethacrylate intraocular lens enabled adhesion and multinucleated fusion, but induced no significant activation. Coculture with either intraocular lens increased reactive oxygen species production in macrophages after one day (p intraocular lens, with hydrophilic surfaces inducing higher activation than hydrophobic surfaces. These findings provide a new method of inquiry into uveal biocompatibility, specifically through the quantification of cell-surface markers of leukocyte activation.

  8. Listeria monocytogenes infection of HD11, chicken macrophage-like cells.

    Science.gov (United States)

    Jarvis, N A; Donaldson, J R; O'Bryan, C A; Ricke, S C; Crandall, P G

    2017-04-01

    Listeria monocytogenes can be carried by and infect poultry, although the clinical disease in birds is rare. Escape from macrophage phagocytosis is a key step in pathogenesis for L. monocytogenes. Therefore, we investigated the infection of the chicken macrophage-like cell line HD11 with 2 strains of L. monocytogenes EGD-e and Scott A. After infection, L. monocytogenes was quantified by spread plating and HD11 was quantified with trypan blue exclusion stain before enumeration. The standard macrophage killing protocols require washing the cell monolayers 3 times with PBS, which was found to negatively influence HD11 monolayers. Maximum bacterial densities within macrophages were not different between the 2 Listeria strains. HD11 required more than 11 h to effectively reduce intracellular L. monocytogenes Scott A, and Scott A was more susceptible to HD11 killing than EGD-e. It appears that Listeria infection initially causes attenuation of HD11 growth, and infected HD11 cells do not begin to lyse until at least 11 h post infection. These results suggest that there are subtle strain to strain differences in response to HD11 macrophage phagocytosis. The long lead-time required for HD11 to kill L. monocytogenes cells means that there is sufficient time available for chicken macrophages to circulate in the blood and transfer the intracellular Listeria to multiple tissues. © 2016 Poultry Science Association Inc.

  9. Combined 17β-Estradiol with TCDD Promotes M2 Polarization of Macrophages in the Endometriotic Milieu with Aid of the Interaction between Endometrial Stromal Cells and Macrophages.

    Directory of Open Access Journals (Sweden)

    Yun Wang

    Full Text Available The goal of this study is to elucidate the effects of 17β-estradiol and TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin on macrophage phenotypes in the endometriotic milieu. Co-culture of endometrial stromal cells (ESCs and U937 cells (macrophage cell line was performed to simulate the endometriotic milieu and to determine the effects of 17β-estradiol and/or TCDD on IL10, IL12 production and HLA-DR, CD86 expression by U937 macrophages. We found that combining 17β-estradiol with TCDD has a synergistic effect on inducing M2 activation when macrophages are co-cultured with ESCs. Moreover, the combination of 17β-estradiol and TCDD significantly enhanced STAT3 and P38 phosphorylation in macrophages. Differentiation of M2 macrophages induced by 17β-estradiol and TCDD were effectively abrogated by STAT3 and P38MAPK inhibitors, but not by ERK1/2 and JNK inhibitors. In conclusion, 17β-estradiol and TCDD in the ectopic milieu may lead to the development of endometriosis by inducing M2 polarization of macrophages through activation of the STAT3 and P38MAPK pathways.

  10. Combined 17β-Estradiol with TCDD Promotes M2 Polarization of Macrophages in the Endometriotic Milieu with Aid of the Interaction between Endometrial Stromal Cells and Macrophages.

    Science.gov (United States)

    Wang, Yun; Chen, Hong; Wang, NingLing; Guo, HaiYan; Fu, Yonglun; Xue, Songguo; Ai, Ai; Lyu, Qifeng; Kuang, Yanping

    2015-01-01

    The goal of this study is to elucidate the effects of 17β-estradiol and TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) on macrophage phenotypes in the endometriotic milieu. Co-culture of endometrial stromal cells (ESCs) and U937 cells (macrophage cell line) was performed to simulate the endometriotic milieu and to determine the effects of 17β-estradiol and/or TCDD on IL10, IL12 production and HLA-DR, CD86 expression by U937 macrophages. We found that combining 17β-estradiol with TCDD has a synergistic effect on inducing M2 activation when macrophages are co-cultured with ESCs. Moreover, the combination of 17β-estradiol and TCDD significantly enhanced STAT3 and P38 phosphorylation in macrophages. Differentiation of M2 macrophages induced by 17β-estradiol and TCDD were effectively abrogated by STAT3 and P38MAPK inhibitors, but not by ERK1/2 and JNK inhibitors. In conclusion, 17β-estradiol and TCDD in the ectopic milieu may lead to the development of endometriosis by inducing M2 polarization of macrophages through activation of the STAT3 and P38MAPK pathways.

  11. Dead Lactobacillus plantarum Stimulates and Skews Immune Responses toward T helper 1 and 17 Polarizations in RAW 264.7 Cells and Mouse Splenocytes.

    Science.gov (United States)

    Lee, Hyun Ah; Kim, Hyunung; Lee, Kwang-Won; Park, Kun-Young

    2016-03-01

    This study was undertaken to evaluate the immunomodulatory effect of dead nano-sized Lactobacillus plantarum (nLp) in RAW 264.7 cells and murine primary splenocytes. nLp is a dead, shrunken, processed form of L. plantarum nF1 isolated from kimchi (a traditional Korean fermented cabbage) and is less than 1 μm in size. It was found that nLp treatment stimulated nitric oxide (NO) production more in RAW 264.7 macrophages than pure live L. plantarum (pLp), and that the stimulatory properties were probably largely derived from its cell wall. In addition, nLp induced murine splenocyte proliferation more so than pLp; in particular, a high dose of nLp (1.0 X 10(11) CFU/ml) stimulated proliferation as much as lipopolysaccharide at 2 μg/ml. Moreover, according to our cytokine profile results in splenocytes, nLp treatment promoted Th1 (TNF-α, IL-12 p70) responses rather than Th2 (IL-4, IL-5) responses and also increased Th17 (IL-6, IL-17A) responses. Thus, nLp stimulated NO release in RAW 264.7 cells and induced splenocyte proliferation more so than pLp and stimulated Th1 and Th17 cytokine production. These findings suggested that dead nLp has potential use as a functional food ingredient to improve the immune response, and especially as a means of inducing Th1/Th17 immune responses.

  12. MiR-16 regulates mouse peritoneal macrophage polarization and affects T-cell activation.

    Science.gov (United States)

    Jia, Xiaoqin; Li, Xiaomin; Shen, Yating; Miao, Junjun; Liu, Hao; Li, Guoli; Wang, Zhengbing

    2016-10-01

    MiR-16 is a tumour suppressor that is down-regulated in certain human cancers. However, little is known on its activity in other cell types. In this study, we examined the biological significance and underlying mechanisms of miR-16 on macrophage polarization and subsequent T-cell activation. Mouse peritoneal macrophages were isolated and induced to undergo either M1 polarization with 100 ng/ml of interferon-γ and 20 ng/ml of lipopolysaccharide, or M2 polarization with 20 ng/ml of interleukin (IL)-4. The identity of polarized macrophages was determined by profiling cell-surface markers by flow cytometry and cytokine production by ELISA. Macrophages were infected with lentivirus-expressing miR-16 to assess the effects of miR-16. Effects on macrophage-T cell interactions were analysed by co-culturing purified CD4(+) T cells with miR-16-expressing peritoneal macrophages, and measuring activation marker CD69 by flow cytometry and cytokine secretion by ELISA. Bioinformatics analysis was applied to search for potential miR-16 targets and understand its underlying mechanisms. MiR-16-induced M1 differentiation of mouse peritoneal macrophages from either the basal M0- or M2-polarized state is indicated by the significant up-regulation of M1 marker CD16/32, repression of M2 marker CD206 and Dectin-1, and increased secretion of M1 cytokine IL-12 and nitric oxide. Consistently, miR-16-expressing macrophages stimulate the activation of purified CD4(+) T cells. Mechanistically, miR-16 significantly down-regulates the expression of PD-L1, a critical immune suppressor that controls macrophage-T cell interaction and T-cell activation. MiR-16 plays an important role in shifting macrophage polarization from M2 to M1 status, and functionally activating CD4(+) T cells. This effect is potentially mediated through the down-regulation of immune suppressor PD-L1.

  13. Schisandrin B inhibits cell growth and induces cellular apoptosis and autophagy in mouse hepatocytes and macrophages: implications for its hepatotoxicity.

    Science.gov (United States)

    Zhang, Yi; Zhou, Zhi-Wei; Jin, Hua; Hu, Chengbin; He, Zhi-Xu; Yu, Zhi-Ling; Ko, Kam-Ming; Yang, Tianxin; Zhang, Xueji; Pan, Si-Yuan; Zhou, Shu-Feng

    2015-01-01

    A number of drugs and herbal compounds have been documented to cause hepatoxicity. Schisandrin B (Sch B) is an active dibenzocyclooctadiene isolated from Schisandrae fructus, with a wide array of pharmacological activities. However, the potential hepatotoxicity of Sch B is a major safety concern, and the underlying mechanism for Sch B-induced liver toxic effects is not fully elucidated. In the present study, we aimed to investigate the liver toxic effects and the molecular mechanisms of Sch B in mouse liver and macrophage cells. The results have shown that Sch B exhibits potent grow inhibitory, proapoptotic, and proautophagic effects in AML-12 and RAW 264.7 cells. Sch B markedly arrested cells in G1 phase in both cell lines, accompanied by the down-regulation of cyclin dependent kinase 2 (CDK2) and cyclin D1 and up-regulation of p27 Kip1 and checkpoint kinase 1. Furthermore, Sch B markedly increased the apoptosis of AML-12 and RAW 264.7 cells with a decrease in the expression of B-cell lymphoma-extra-large and (Bcl-xl) B-cell lymphoma 2 (Bcl-2), but an increase in the expression of B-cell lymphoma 2-associated X protein (Bax). Sch B promoted the cleavage of caspase 3 and poly-adenosine diphosphate-ribose polymerase (PARP) in both cell lines. Additionally, Sch B significantly induced autophagy of AML-12 and RAW 264.7 cells. Sch B inhibited the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, as indicated by their altered phosphorylation, contributing to the proautophagic effect of Sch B. Taken together, our findings show that the inducing effects of Sch B on cell cycle arrest, apoptosis, and autophagy may contribute to its liver toxic effects, which might provide a clue for the investigation of the molecular toxic targets and underlying mechanisms for Sch B-induced hepatotoxicity in herbal consumers. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and

  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. Pure populations of murine macrophages from cultured embryonic stem cells. Application to studies of chemotaxis and apoptotic cell clearance.

    Science.gov (United States)

    Zhuang, Lihui; Pound, John D; Willems, Jorine J L P; Taylor, A Helen; Forrester, Lesley M; Gregory, Christopher D

    2012-11-30

    Embryonic stem cells provide a potentially convenient source of macrophages in the laboratory. Given the propensity of macrophages for plasticity in phenotype and function, standardised culture and differentiation protocols are required to ensure consistency in population output and activity in functional assays. Here we detail the development of an optimised culture protocol for the production of murine embryonic stem cell-derived macrophages (ESDM). This protocol provides improved yields of ESDM and we demonstrate that the cells are suitable for application to the study of macrophage responses to apoptotic cells. ESDM so produced were of higher purity than commonly used primary macrophage preparations and were functional in chemotaxis assays and in phagocytosis of apoptotic cells. Maturation of ESDM was found to be associated with reduced capacity for directed migration and increased capacity for phagocytic clearance of apoptotic cells. These results show ESDM to be functionally active in sequential phases of interaction with apoptotic cells and establish these macrophage populations as useful models for further study of molecular mechanisms underlying the recognition and removal of apoptotic cells. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Imaging-based analysis of liposome internalization to macrophage cells: Effects of liposome size and surface modification with PEG moiety.

    Science.gov (United States)

    Lee, Jae Sun; Hwang, Sang Youn; Lee, E K

    2015-12-01

    Liposome is one of the frequently used carriers for active targeting systems in vivo. Such parameters as its size, surface charge, and surface modifiers are known to influence the liposome uptake by macrophage cells. In this study, we investigated the effects of liposome size and polyethylene glycol (PEG) surface modifier on the liposomal internalization to murine macrophage (RAW-264.7), by using an imaging analysis technique. Three different sized liposomes (100, 200, and 400 nm in nominal diameter) labeled with rhodamine fluorescence were used. Liposome internalization appeared to reach a pseudo-steady plateau in about 5h incubation, and most of the internalized liposomes were seen to accumulate in the cytosol including cellular extensions. The maximum fluorescent density from the internalized liposomes was similar between 100 nm and 200 nm liposomes. However, that of the larger 400 nm liposome was approximately 1.7 times higher than the others, confirming the previous report that the larger the liposomes are the higher the degree of internalization is. When the outside of the 200 nm liposomes was modified with biocompatible anchor molecule (BAM) consisting of PEG (ca. 2kD molecular weight) moiety, the endocytosis was indeed reduced by about 2.1-fold, despite the increase of the hydrodynamic size due to BAM conjugation. This fluorescence-based cellular imaging analysis can be used to quantitatively monitor and optimize cellular internalization systems.

  17. Macrophage migration inhibitory factor promotes cell death and aggravates neurologic deficits after experimental stroke

    OpenAIRE

    Inácio, Ana R; Ruscher, Karsten; Leng, Lin; Bucala, Richard; Deierborg, Tomas

    2010-01-01

    Multiple mechanisms contribute to tissue demise and functional recovery after stroke. We studied the involvement of macrophage migration inhibitory factor (MIF) in cell death and development of neurologic deficits after experimental stroke. Macrophage migration inhibitory factor is upregulated in the brain after cerebral ischemia, and disruption of the Mif gene in mice leads to a smaller infarct volume and better sensory-motor function after transient middle cerebral artery occlusion (tMCAo)....

  18. Hemosiderin laden macrophages and hemosiderin within follicular cells distinguish benign follicular lesions from follicular neoplasms

    Directory of Open Access Journals (Sweden)

    Jaffar Reema

    2009-01-01

    Full Text Available Background: Published criteria to distinguish benign colloid nodules from follicular neoplasms emphasize only three interdependent features: size of follicles, amount of colloid, and cellularity. There is a need for the validation of other independent criteria. Methods: This study quantified the significance of cystic change, defined as presence of macrophages, and the presence of hemosiderin in either the macrophages or follicular cells. The cohort consisted of 165 patients with fine needle aspiration (FNA and histologic follow-up of either goiter (101, follicular adenoma (47, or follicular carcinoma (17. Papillary thyroid carcinomas and Hürthle cell neoplasms were excluded from the cohort, because these categories are known to show cystic change and hemosiderin. FNAs were reviewed blindly with the most cellular slide scored for the presence of macrophages and/or hemosiderin. Results: Hemosiderin within macrophages were seen in 67% (68 of 101 of the goiters and only 6% (four of 64 of follicular neoplasms ( P < .0001. All four follicular neoplasms with hemosiderin in macrophages were adenomas. Three of these four had equivocal features of a benign colloid nodule histologically. None of the 17 follicular carcinomas had hemosiderin in macrophages ( P < .12. Macrophages without hemosiderin also strongly distinguished goiters from neoplasms (83% vs 17% but appears less useful as a criterion since macrophages were present within 3 of 17 follicular carcinomas. Hemosiderin within follicular epithelial cells was present in 18% (18 of 101 of goiters, whereas none of the 64 follicular neoplasms had intraepithelial hemosiderin ( P < .0003. Conclusions: If papillary thyroid carcinoma and Hürthle cell neoplasm are ruled out, our findings indicate that the presence of hemosiderin virtually excludes a clinically significant follicular neoplasm.

  19. Immune-Stimulatory Effects of Althaea rosea Flower Extracts through the MAPK Signaling Pathway in RAW264.7 Cells

    Directory of Open Access Journals (Sweden)

    Yon-Suk Kim

    2017-04-01

    Full Text Available Althaea rosea (Linn. is a medicinal plant from China and Korea that has been traditionally used to control inflammation, to stop bedwetting and as a mouthwash in cases of bleeding gums. Its flowers are employed medicinally for their emollient, demulcent and diuretic properties, which make them useful in chest complaints. Furthermore, a flower extract decoction is used to improve blood circulation, for the treatment of constipation, dysmenorrhoea, haemorrhages, etc. However, the possible mechanisms of the immune-stimulatory effect remains to be elucidated. Therefore, we investigated the role of Althaea rosea flower (ARF extracts in the immune-stimulatory effect of macrophages and the underlying mechanisms of action. ARF water extract (ARFW could dose-dependently increase NO production and cytokines (IL-6 and TNF-α. We also found that ARFW significantly increased the expression of iNOS and COX-2 proteins in RAW264.7 cells. Consistent with these results, MAPK protein (JNK, ERK, p38 expression levels were induced after treatment with ARFW. Additionally, ARFW showed a marked increase in the phosphorylation level of IκBα and subsequent IκBα degradation allowing NF-κB nuclear translocation. These results suggest that the immune-stimulatory effect of A. rosea flower extracts is mediated through the translocation of NF-κB p65 subunit into the nucleus from the cytoplasm and subsequent activation of pro-inflammatory cytokines (IL-6 and TNF-α and other mediators (iNOS and COX-2, which occurs mainly through MAPK signalling pathway. Thus, we suggest that ARFW could be considered as a potential therapeutic agent useful in the development of immune-stimulatory compounds.

  20. Chitosan drives anti-inflammatory macrophage polarisation and pro-inflammatory dendritic cell stimulation

    Directory of Open Access Journals (Sweden)

    MI Oliveira

    2012-07-01

    Full Text Available Macrophages and dendritic cells (DC share the same precursor and play key roles in immunity. Modulation of their behaviour to achieve an optimal host response towards an implanted device is still a challenge. Here we compare the differentiation process and polarisation of these related cell populations and show that they exhibit different responses to chitosan (Ch, with human monocyte-derived macrophages polarising towards an anti-inflammatory phenotype while their DC counterparts display pro-inflammatory features. Macrophages and DC, whose interactions with biomaterials are frequently analysed using fully differentiated cells, were cultured directly on Ch films, rather than exposed to the polymer after complete differentiation. Ch was the sole stimulating factor and activated both macrophages and DC, without leading to significant T cell proliferation. After 10 d on Ch, macrophages significantly down-regulated expression of pro-inflammatory markers, CD86 and MHCII. Production of pro-inflammatory cytokines, particularly TNF-α, decreased with time for cells cultured on Ch, while anti-inflammatory IL-10 and TGF-β1, significantly increased. Altogether, these results suggest an M2c polarisation. Also, macrophage matrix metalloproteinase activity was augmented and cell motility was stimulated by Ch. Conversely, DC significantly enhanced CD86 expression, reduced IL-10 secretion and increased TNF-α and IL-1β levels. Our findings indicate that cells with a common precursor may display different responses, when challenged by the same biomaterial. Moreover, they help to further comprehend macrophage/DC interactions with Ch and the balance between pro- and anti-inflammatory signals associated with implant biomaterials. We propose that an overall pro-inflammatory reaction may hide the expression of anti-inflammatory cytokines, likely relevant for tissue repair/regeneration.

  1. Study on application of electrochemical microsensor detecting NO released from macrophage stimulated by Escherichia coli%电化学微传感器实时检测大肠埃希菌激活的RAW264.7细胞释放NO的研究

    Institute of Scientific and Technical Information of China (English)

    吴汪泽; 汤纪路; 甘甜; 卢忠心; 乔治

    2014-01-01

    Objective To apply nitric oxide(NO) electrochemical microsensor in the real time detection of NO released from RAW 264 .3 cells infected by E .coli ,and to explore the application value of this NO microsensor in the research area of infection im‐munity against bacterium .Methods Taking NO microsensor to detect NO released from RAW 264 .3 cells respectively stimulated by E .coli of different densities and of 1 × 107 mL -1 for different time .Results The level of NO released from RAW 264 .3 cells was enhanced obviously when incubated with E .coli as compared with that of normal cells and the extent of incersase depended on the density of E .coli (P<0 .01) .The released level of NO increased gradually from the beginning and reached its peal at the time of 12 h then decreased slowly when incubated with E .coli of 1 × 107 mL -1 .Conclusion The electrochemical microsensor was applied in the real time detection of NO released from macrophages activated by E .coli successfully .%目的:探讨NO电化学微传感器在抗细菌感染免疫研究中的应用价值。方法应用前期制备的基于纳米金(nano‐Au)修饰玻璃纤维的新型NO电化学微传感器实时检测大肠埃希菌(E .coli)不同浓度刺激组、不同时间刺激组的小鼠巨噬细胞(RAW264.7细胞)NO的释放水平。结果与对照组相比,RAW264.7细胞受到E .coli刺激后,NO的释放水平明显上调( P<0.01),且对E .coli的刺激具有浓度依耐性。随着E .coli作用时间的延续,RAW264.7细胞的 NO释放水平逐渐上升,作用12 h时达到高峰,然后开始下降。结论 NO电化学微传感器成功应用于E .coli激活的巨噬细胞释放NO过程的实时检测。

  2. Raw and thermally treated cement asbestos exerts different cytotoxicity effects on A549 cells in vitro.

    Science.gov (United States)

    Pugnaloni, Armanda; Lucarini, Guendalina; Rubini, Corrado; Smorlesi, Arianna; Tomasetti, Marco; Strafella, Elisabetta; Armeni, Tatiana; Gualtieri, Alessandro F

    2015-01-01

    Raw cement asbestos (RCA) undergoes a complete solid state transformation when heated at high temperatures. The secondary raw material produced, high temperatures-cement asbestos (HT-CA) is composed of newly-formed crystals in place of the asbestos fibers present in RCA. Our previous study showed that HT-CA exerts lower cytotoxic cell damage compared to RCA. Nevertheless further investigations are needed to deepen our understanding of pathogenic pathways involving oxidative and nitrative damage. Our aim is to deepen the understanding of the biological effects on A549 cells of these materials regarding DNA damage related proteins (p53, its isoform p73 and TRAIL) and nitric oxide (NO) production during inducible nitric oxide synthase (iNOS)-mediated inflammation. Increments of p53/p73 expression, iNOS positive cells and NO concentrations were found with RCA, compared to HT-CA and controls mainly at 48 h. Interestingly, ferrous iron causing reactive oxygen species (ROS)-mediated DNA damage was found in RCA as a contaminant. HT-CA thermal treatment induces a global recrystallization with iron in a crystal form poorly released in media. HT-CA slightly interferes with genome expression and exerts lower inflammatory potential compared to RCA on biological systems. It could represent a safe approach for storing or recycling asbestos and an environmentally friendly alternative to asbestos waste.

  3. Macrophages retain hematopoietic stem cells in the spleen via VCAM-1

    Science.gov (United States)

    Hoyer, Friedrich Felix; Grigoryeva, Lubov S.; Sager, Hendrik B.; Leuschner, Florian; Courties, Gabriel; Borodovsky, Anna; Novobrantseva, Tatiana; Ruda, Vera M.; Fitzgerald, Kevin; Iwamoto, Yoshiko; Wojtkiewicz, Gregory; Sun, Yuan; Da Silva, Nicolas; Libby, Peter; Anderson, Daniel G.; Swirski, Filip K.; Weissleder, Ralph

    2015-01-01

    Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)+ macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE−/− mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques. PMID:25800955

  4. Interstitial cells of Cajal, macrophages and mast cells in the gut musculature: morphology, distribution, spatial and possible functional interactions.

    Science.gov (United States)

    Mikkelsen, Hanne B

    2010-04-01

    Interstitial cells of Cajal (ICC) are recognized as pacemaker cells for gastrointestinal movement and are suggested to be mediators of neuromuscular transmission. Intestinal motility disturbances are often associated with a reduced number of ICC and/or ultrastructural damage, sometimes associated with immune cells. Macrophages and mast cells in the intestinal muscularis externa of rodents can be found in close spatial contact with ICC. Macrophages are a constant and regularly distributed cell population in the serosa and at the level of Auerbach's plexus (AP). In human colon, ICC are in close contact with macrophages at the level of AP, suggesting functional interaction. It has therefore been proposed that ICC and macrophages interact. Macrophages and mast cells are considered to play important roles in the innate immune defence by producing pro-inflammatory mediators during classical activation, which may in itself result in damage to the tissue. They also take part in alternative activation which is associated with anti-inflammatory mediators, tissue remodelling and homeostasis, cancer, helminth infections and immunophenotype switch. ICC become damaged under various circumstances - surgical resection, possibly post-operative ileus in rodents - where innate activation takes place, and in helminth infections - where alternative activation takes place. During alternative activation the muscularis macrophage can switch phenotype resulting in up-regulation of F4/80 and the mannose receptor. In more chronic conditions such as Crohn's disease and achalasia, ICC and mast cells develop close spatial contacts and piecemeal degranulation is possibly triggered.

  5. Clonal analysis of proliferation and differentiation of paired daughter cells: action of granulocyte-macrophage colony-stimulating factor on granulocyte-macrophage precursors.

    OpenAIRE

    Metcalf, D.

    1980-01-01

    Mouse granulocyte-macrophage progenitor cells were stimulated to divide by the granulocyte-macrophage colony-stimulating factor (GM-CSF). The two daughter cells were separated; one daughter was transferred to medium containing a high concentration of GM-CSF, the other to medium containing a low concentration. Daughter cell-derived clones in the presence of 2500 units of GM-CSF had average cell cycle times 3.5 +/- 2.5 (SEM) hr shorter than clones derived from the paired daughter cell stimulate...

  6. Comparative Analysis of the Interaction of Helicobacter pylori with Human Dendritic Cells, Macrophages, and Monocytes

    Science.gov (United States)

    Fehlings, Michael; Drobbe, Lea; Moos, Verena; Renner Viveros, Pablo; Hagen, Jana; Beigier-Bompadre, Macarena; Pang, Ervinna; Belogolova, Elena; Churin, Yuri; Schneider, Thomas; Meyer, Thomas F.; Aebischer, Toni

    2012-01-01

    Helicobacter pylori may cause chronic gastritis, gastric cancer, or lymphoma. Myeloid antigen-presenting cells (APCs) are most likely involved in the induction and expression of the underlying inflammatory responses. To study the interaction of human APC subsets with H. pylori, we infected monocytes, monocyte-derived dendritic cells (DCs), and monocyte-derived (classically activated; M1) macrophages with H. pylori and analyzed phenotypic alterations, cytokine secretion, phagocytosis, and immunostimulation. Since we detected CD163+ (alternatively activated; M2) macrophages in gastric biopsy specimens from H. pylori-positive patients, we also included monocyte-derived M2 macrophages in the study. Upon H. pylori infection, monocytes secreted interleukin-1β (IL-1β), IL-6, IL-10, and IL-12p40 (partially secreted as IL-23) but not IL-12p70. Infected DCs became activated, as shown by the enhanced expression of CD25, CD80, CD83, PDL-1, and CCR7, and secreted IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, and IL-23. However, infection led to significantly downregulated CD209 and suppressed the constitutive secretion of macrophage migration inhibitory factor (MIF). H. pylori-infected M1 macrophages upregulated CD14 and CD32, downregulated CD11b and HLA-DR, and secreted mainly IL-1β, IL-6, IL-10, IL-12p40, and IL-23. Activation of DCs and M1 macrophages correlated with increased capacity to induce T-cell proliferation and decreased phagocytosis of dextran. M2 macrophages upregulated CD14 and CD206 and secreted IL-10 but produced less of the proinflammatory cytokines than M1 macrophages. Thus, H. pylori affects the functions of human APC subsets differently, which may influence the course and the outcome of H. pylori infection. The suppression of MIF in DCs constitutes a novel immune evasion mechanism exploited by H. pylori. PMID:22615251

  7. 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.

  8. Differential Modulation of Lipopolysaccharide-Induced Inflammatory Cytokine Production by and Antioxidant Activity of Fomentariol in RAW264.7 Cells.

    Science.gov (United States)

    Seo, Dong-Won; Yi, Young-Joo; Lee, Myeong-Seok; Yun, Bong-Sik; Lee, Sang-Myeong

    2015-12-01

    Medicinal mushrooms have been used worldwide to treat cancer and modulate the immune system. Over the last several years, there has been increasing interest in isolating bioactive compounds from medicinal mushrooms and evaluating their health beneficial effects. Fomes fomentarius is used in traditional oriental medicine and is known to possess antioxidant, anti-inflammatory, antidiabetic, and antitumor effects. In the present study, we isolated fomentariol from Fomes fomentarius and investigated its anti-inflammatory effect in murine macrophages (RAW264.7 cells) stimulated with lipopolysaccharides. Fomentariol inhibited the production of nitric oxide and intracellular reactive oxygen species triggered by lipopolysaccharides. Interestingly, fomentariol differentially regulated cytokine production triggered by lipopolysaccharides. Fomentariol effectively suppressed the production of interleukin-1β and interleukin-6 but not tumor necrosis factor-α. The inhibitory effect of fomentariol against nitric oxide, interleukin-1β, and interleukin-6 production was possibly mediated by downregulation of the extracellular signal-regulated kinase signaling pathway. Taken together, our results suggest that fomentariol differentially modulated inflammatory responses triggered by lipopolysaccharides in macrophages and is one of the bioactive compounds that mediate the physiological effects of Fomes fomentarius.

  9. Macrophage Infiltration in Tumor Stroma is Related to Tumor Cell Expression of CD163 in Colorectal Cancer.

    Science.gov (United States)

    Shabo, Ivan; Olsson, Hans; Elkarim, Rihab; Sun, Xiao-Feng; Svanvik, Joar

    2014-08-01

    The scavenger receptor, CD163, is a macrophage-specific marker. Recent studies have shown that CD163 expression in breast and rectal cancer cells is associated with poor prognosis. This study was conducted to evaluate the relationship between CD163 expression as a macrophage trait in cancer cells, and macrophage infiltration and its clinical significance in colorectal cancer. Immunostaining of CD163 and macrophage infiltration were evaluated in paraffin-embedded specimens, earlier analyzed for CD31, D2-40 and S-phase fraction, from primary tumors and normal colorectal mucosa of 75 patients with colorectal carcinoma. The outcomes were analyzed in relation to clinical-pathological data. CD163 expression was positive in cancer cells in 20 % of colorectal cancer patients and was related to advanced tumor stages (P = 0.008) and unfavorable prognosis (p = 0.001). High macrophage infiltration was related to shorter survival and positive CD163 expression in tumor cells. The prognostic impact of macrophage infiltration was independent of tumor stage and CD163 expression in cancer cells (p = 0.034). The expression of macrophage phenotype in colorectal cancer cells is associated with macrophage density in tumor stroma and lower survival rates. Macrophage infiltration has an independent prognostic impact on mortality in colorectal cancer. In accordance with previous experimental studies, these findings provide new insights into the role of macrophages in colorectal cancer.

  10. Media from macrophages co-incubated with Enterococcus faecalis induces epithelial cell monolayer reassembly and altered cell morphology.

    Science.gov (United States)

    Belogortseva, Natalia; Krezalek, Monika; Guyton, Kristina; Labno, Christine; Poroyko, Valeriy; Zaborina, Olga; Alverdy, John C

    2017-01-01

    Signal exchange between intestinal epithelial cells, microbes and local immune cells is an important mechanism of intestinal homeostasis. Given that intestinal macrophages are in close proximity to both the intestinal epithelium and the microbiota, their pathologic interactions may result in epithelial damage. The present study demonstrates that co-incubation of murine macrophages with E. faecalis strains producing gelatinase (GelE) and serine protease (SprE) leads to resultant condition media (CM) capable of inducing reassembly of primary colonic epithelial cell monolayers. Following the conditioned media (CM) exposure, some epithelial cells are shed whereas adherent cells are observed to undergo dissolution of cell-cell junctions and morphologic transformation with actin cytoskeleton reorganization resulting in flattened and elongated shapes. These cells exhibit marked filamentous filopodia and lamellipodia formation. Cellular reorganization is not observed when epithelial monolayers are exposed to: CM from macrophages co-incubated with E. faecalis GelE/SprE-deficient mutants, CM from macrophages alone, or E. faecalis (GelE/SprE) alone. Flow cytometry analysis reveals increased expression of CD24 and CD44 in cells treated with macrophage/E. faecalis CM. This finding in combination with the appearance colony formation in matrigel demonstrate that the cells treated with macrophage/E. faecalis CM contain a higher proportion progenitor cells compared to untreated control. Taken together, these findings provide evidence for a triangulated molecular dialogue between E. faecalis, macrophages and colonic epithelial cells, which may have important implications for conditions in the gut that involve inflammation, injury or tumorigenesis.

  11. Media from macrophages co-incubated with Enterococcus faecalis induces epithelial cell monolayer reassembly and altered cell morphology

    Science.gov (United States)

    Belogortseva, Natalia; Krezalek, Monika; Guyton, Kristina; Labno, Christine; Poroyko, Valeriy; Zaborina, Olga

    2017-01-01

    Signal exchange between intestinal epithelial cells, microbes and local immune cells is an important mechanism of intestinal homeostasis. Given that intestinal macrophages are in close proximity to both the intestinal epithelium and the microbiota, their pathologic interactions may result in epithelial damage. The present study demonstrates that co-incubation of murine macrophages with E. faecalis strains producing gelatinase (GelE) and serine protease (SprE) leads to resultant condition media (CM) capable of inducing reassembly of primary colonic epithelial cell monolayers. Following the conditioned media (CM) exposure, some epithelial cells are shed whereas adherent cells are observed to undergo dissolution of cell-cell junctions and morphologic transformation with actin cytoskeleton reorganization resulting in flattened and elongated shapes. These cells exhibit marked filamentous filopodia and lamellipodia formation. Cellular reorganization is not observed when epithelial monolayers are exposed to: CM from macrophages co-incubated with E. faecalis GelE/SprE-deficient mutants, CM from macrophages alone, or E. faecalis (GelE/SprE) alone. Flow cytometry analysis reveals increased expression of CD24 and CD44 in cells treated with macrophage/E. faecalis CM. This finding in combination with the appearance colony formation in matrigel demonstrate that the cells treated with macrophage/E. faecalis CM contain a higher proportion progenitor cells compared to untreated control. Taken together, these findings provide evidence for a triangulated molecular dialogue between E. faecalis, macrophages and colonic epithelial cells, which may have important implications for conditions in the gut that involve inflammation, injury or tumorigenesis. PMID:28793333

  12. Induction of Monocyte Chemoattractant Proteins in Macrophages via the Production of Granulocyte-macrophage Colony Stimulating Factor by Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Teizo eYoshimura

    2016-01-01

    Full Text Available Monocyte chemoattractant protein-1 (MCP-1/CCL2 plays an important role in the initiation and progression of cancer. We previously reported that in 4T1 murine breast cancer, non-tumor stromal cells, including macrophages, were the major source of MCP-1. In the present study, we analyzed the potential mechanisms by which MCP-1 is upregulated in macrophages infiltrating 4T1 tumors. We found that cell-free culture supernatants of 4T1 cells (4T1-sup markedly upregulated MCP-1 production by peritoneal inflammatory macrophages. 4T1-sup also upregulated other MCPs, such as MCP-3/CCL7 and MCP-5/CCL12, but modestly neutrophil chemotactic chemokines, such as KC/CXCL1 or MIP-2/CXCL2. Physicochemical analysis indicated that an approximately 2 to 3 kDa 4T1 cell product was responsible for the capacity of 4T1-sup to upregulate MCP-1 expression by macrophages. A neutralizing antibody against granulocyte-macrophage-colony stimulating factor (GM-CSF, but not macrophage-colony stimulating factor, almost completely abrogated MCP-1-inducing activity of 4T1-sup, and recombinant GM-CSF potently up-regulated MCP-1 production by macrophages. The expression levels of GM-CSF in 4T1 tumors in vivo were higher than other tumors, such as Lewis lung carcinoma. Treatment of mice with anti-GM-CSF antibody significantly reduced the growth of 4T1 tumors at the injection sites but did not reduce MCP-1 production or lung metastasis in tumor-bearing mice. These results indicate that 4T1 cells have the capacity to directly up-regulate MCP-1 production by macrophages by releasing GM-CSF; however, other mechanisms are also involved in increased MCP-1 levels in the 4T1 tumor microenvironment.

  13. 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

  14. 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.

  15. Juxtacrine interaction of macrophages and bone marrow stromal cells induce interleukin-6 signals and promote cell migration

    Institute of Scientific and Technical Information of China (English)

    Jia Chang; Amy J Koh; Hernan Roca; Laurie K McCauley

    2015-01-01

    The bone marrow contains a heterogeneous milieu of cells, including macrophages, which are key cellular mediators for resolving infection and inflammation. Macrophages are most well known for their ability to phagocytose foreign bodies or apoptotic cells to maintain homeostasis;however, little is known about their function in the bone microenvironment. In the current study, we investigated the in vitro interaction of murine macrophages and bone marrow stromal cells (BMSCs), with focus on the juxtacrine induction of IL-6 signaling and the resultant effect on BMSC migration and growth. The juxtacrine interaction of primary mouse macrophages and BMSCs activated IL-6 signaling in the co-cultures, which subsequently enhanced BMSC migration and increased BMSC numbers. BMSCs and macrophages harvested from IL-6 knockout mice revealed that IL-6 signaling was essential for enhancement of BMSC migration and increased BMSC numbers via juxtacrine interactions. BMSCs were the main contributor of IL-6 signaling, and hence activation of the IL-6/gp130/STAT3 pathway. Meanwhile, macrophage derived IL-6 remained important for the overall production of IL-6 protein in the co-cultures. Taken together, these findings show the function of macrophages as co-inducers of migration and growth of BMSCs, which could directly influence bone formation and turnover.

  16. Phenotypic and functional plasticity of cells of innate immunity: macrophages, mast cells and neutrophils

    DEFF Research Database (Denmark)

    Galli, Stephen J; Borregaard, Niels; Wynn, Thomas A

    2011-01-01

    Hematopoietic cells, including lymphoid and myeloid cells, can develop into phenotypically distinct 'subpopulations' with different functions. However, evidence indicates that some of these subpopulations can manifest substantial plasticity (that is, undergo changes in their phenotype and functio...... a common mechanism for modulating innate or adaptive immunity.......). Here we focus on the occurrence of phenotypically distinct subpopulations in three lineages of myeloid cells with important roles in innate and acquired immunity: macrophages, mast cells and neutrophils. Cytokine signals, epigenetic modifications and other microenvironmental factors can substantially...... and, in some cases, rapidly and reversibly alter the phenotype of these cells and influence their function. This suggests that regulation of the phenotype and function of differentiated hematopoietic cells by microenvironmental factors, including those generated during immune responses, represents...