Classical and alternative macrophage activation in the lung following ozone-induced oxidative stress

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Sunil, Vasanthi R., E-mail: sunilva@pharmacy.rutgers.edu [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ 08854 (United States); Patel-Vayas, Kinal; Shen, Jianliang [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ 08854 (United States); Laskin, Jeffrey D. [Department of Environmental and Occupational Medicine, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ (United States); Laskin, Debra L. [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ 08854 (United States)

2012-09-01

Ozone is a pulmonary irritant known to cause oxidative stress, inflammation and tissue injury. Evidence suggests that macrophages play a role in the pathogenic response; however, their contribution depends on the mediators they encounter in the lung which dictate their function. In these studies we analyzed the effects of ozone-induced oxidative stress on the phenotype of alveolar macrophages (AM). Exposure of rats to ozone (2 ppm, 3 h) resulted in increased expression of 8-hydroxy-2′-deoxyguanosine (8-OHdG), as well as heme oxygenase-1 (HO-1) in AM. Whereas 8-OHdG was maximum at 24 h, expression of HO-1 was biphasic increasing after 3 h and 48–72 h. Cleaved caspase-9 and beclin-1, markers of apoptosis and autophagy, were also induced in AM 24 h post-ozone. This was associated with increased bronchoalveolar lavage protein and cells, as well as matrix metalloproteinase (MMP)-2 and MMP-9, demonstrating alveolar epithelial injury. Ozone intoxication resulted in biphasic activation of the transcription factor, NFκB. This correlated with expression of monocyte chemotactic protein‐1, inducible nitric oxide synthase and cyclooxygenase‐2, markers of proinflammatory macrophages. Increases in arginase-1, Ym1 and galectin-3 positive anti-inflammatory/wound repair macrophages were also observed in the lung after ozone inhalation, beginning at 24 h (arginase-1, Ym1), and persisting for 72 h (galectin-3). This was associated with increased expression of pro-surfactant protein-C, a marker of Type II cell proliferation and activation, important steps in wound repair. These data suggest that both proinflammatory/cytotoxic and anti-inflammatory/wound repair macrophages are activated early in the response to ozone-induced oxidative stress and tissue injury. -- Highlights: ► Lung macrophages are highly sensitive to ozone induced oxidative stress. ► Ozone induces autophagy and apoptosis in lung macrophages. ► Proinflammatory and wound repair macrophages are activated

Mesoporous carbon nanomaterials induced pulmonary surfactant inhibition, cytotoxicity, inflammation and lung fibrosis.

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Chen, Yunan; Yang, Yi; Xu, Bolong; Wang, Shunhao; Li, Bin; Ma, Juan; Gao, Jie; Zuo, Yi Y; Liu, Sijin

2017-12-01

Environmental exposure and health risk upon engineered nanomaterials are increasingly concerned. The family of mesoporous carbon nanomaterials (MCNs) is a rising star in nanotechnology for multidisciplinary research with versatile applications in electronics, energy and gas storage, and biomedicine. Meanwhile, there is mounting concern on their environmental health risks due to the growing production and usage of MCNs. The lung is the primary site for particle invasion under environmental exposure to nanomaterials. Here, we studied the comprehensive toxicological profile of MCNs in the lung under the scenario of moderate environmental exposure. It was found that at a low concentration of 10μg/mL MCNs induced biophysical inhibition of natural pulmonary surfactant. Moreover, MCNs at similar concentrations reduced viability of J774A.1 macrophages and lung epithelial A549 cells. Incubating with nature pulmonary surfactant effectively reduced the cytotoxicity of MCNs. Regarding the pro-inflammatory responses, MCNs activated macrophages in vitro, and stimulated lung inflammation in mice after inhalation exposure, associated with lung fibrosis. Moreover, we found that the size of MCNs played a significant role in regulating cytotoxicity and pro-inflammatory potential of this nanomaterial. In general, larger MCNs induced more pronounced cytotoxic and pro-inflammatory effects than their smaller counterparts. Our results provided valuable information on the toxicological profile and environmental health risks of MCNs, and suggested that fine-tuning the size of MCNs could be a practical precautionary design strategy to increase safety and biocompatibility of this nanomaterial. Copyright © 2017. Published by Elsevier B.V.

Asbestos Induces Oxidative Stress and Activation of Nrf2 Signaling in Murine Macrophages: Chemopreventive Role of the Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605

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Ralph A. Pietrofesa

2016-03-01

Full Text Available The interaction of asbestos fibers with macrophages generates harmful reactive oxygen species (ROS and subsequent oxidative cell damage that are key processes linked to malignancy. Secoisolariciresinol diglucoside (SDG is a non-toxic, flaxseed-derived pluripotent compound that has antioxidant properties and may thus function as a chemopreventive agent for asbestos-induced mesothelioma. We thus evaluated synthetic SDG (LGM2605 in asbestos-exposed, elicited murine peritoneal macrophages as an in vitro model of tissue phagocytic response to the presence of asbestos in the pleural space. Murine peritoneal macrophages (MFs were exposed to crocidolite asbestos fibers (20 µg/cm2 and evaluated at various times post exposure for cytotoxicity, ROS generation, malondialdehyde (MDA, and levels of 8-iso Prostaglandin F2α (8-isoP. We then evaluated the ability of LGM2605 to mitigate asbestos-induced oxidative stress by administering LGM2605 (50 µM 4-h prior to asbestos exposure. We observed a significant (p < 0.0001, time-dependent increase in asbestos-induced cytotoxicity, ROS generation, and the release of MDA and 8-iso Prostaglandin F2α, markers of lipid peroxidation, which increased linearly over time. LGM2605 treatment significantly (p < 0.0001 reduced asbestos-induced cytotoxicity and ROS generation, while decreasing levels of MDA and 8-isoP by 71%–88% and 41%–73%, respectively. Importantly, exposure to asbestos fibers induced cell protective defenses, such as cellular Nrf2 activation and the expression of phase II antioxidant enzymes, HO-1 and Nqo1 that were further enhanced by LGM2605 treatment. LGM2605 boosted antioxidant defenses, as well as reduced asbestos-induced ROS generation and markers of oxidative stress in murine peritoneal macrophages, supporting its possible use as a chemoprevention agent in the development of asbestos-induced malignant mesothelioma.

Differential Cytotoxicity of Acetaminophen in Mouse Macrophage J774.2 and Human Hepatoma HepG2 Cells: Protection by Diallyl Sulfide.

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Haider Raza

Full Text Available Non-steroidal anti-inflammatory drugs (NSAIDs, including acetaminophen (APAP, have been reported to induce cytotoxicity in cancer and non-cancerous cells. Overdose of acetaminophen (APAP causes liver injury in humans and animals. Hepatic glutathione (GSH depletion followed by oxidative stress and mitochondrial dysfunction are believed to be the main causes of APAP toxicity. The precise molecular mechanism of APAP toxicity in different cellular systems is, however, not clearly understood. Our previous studies on mouse macrophage J774.2 cells treated with APAP strongly suggest induction of apoptosis associated with mitochondrial dysfunction and oxidative stress. In the present study, using human hepatoma HepG2 cells, we have further demonstrated that macrophages are a more sensitive target for APAP-induced toxicity than HepG2 cells. Using similar dose- and time-point studies, a marked increase in apoptosis and DNA fragmentation were seen in macrophages compared to HepG2 cells. Differential effects of APAP on mitochondrial respiratory functions and oxidative stress were observed in the two cell lines which are presumably dependent on the varying degree of drug metabolism by the different cytochrome P450s and detoxification by glutathione S-transferase enzyme systems. Our results demonstrate a marked increase in the activity and expression of glutathione transferase (GST and multidrug resistance (MDR1 proteins in APAP-treated HepG2 cells compared to macrophages. This may explain the apparent resistance of HepG2 cells to APAP toxicity. However, treatment of these cells with diallyl sulfide (DAS, 200 μM, a known chemopreventive agent from garlic extract, 24 h prior to APAP (10 μmol/ml for 18h exhibited comparable cytoprotective effects in the two cell lines. These results may help in better understanding the mechanism of cytotoxicity caused by APAP and cytoprotection by chemopreventive agents in cancer and non-cancerous cellular systems.

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.

SIRT2 ameliorates lipopolysaccharide-induced inflammation in macrophages

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Lee, Ae Sin; Jung, Yu Jin; Kim, Dal; Nguyen-Thanh, Tung; Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang; Kim, Won

2014-01-01

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

SIRT2 ameliorates lipopolysaccharide-induced inflammation in macrophages

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

2014-08-08

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

Autophagy deficiency in macrophages enhances NLRP3 inflammasome activity and chronic lung disease following silica exposure

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Jessop, Forrest; Hamilton, Raymond F.; Rhoderick, Joseph F.; Shaw, Pamela K.; Holian, Andrij

2016-01-01

Autophagy is an important metabolic mechanism that can promote cellular survival following injury. The specific contribution of autophagy to silica-induced inflammation and disease is not known. The objective of these studies was to determine the effects of silica exposure on the autophagic pathway in macrophages, as well as the general contribution of autophagy in macrophages to inflammation and disease. Silica exposure enhanced autophagic activity in vitro in Bone Marrow derived Macrophages and in vivo in Alveolar Macrophages isolated from silica-exposed mice. Impairment of autophagy in myeloid cells in vivo using Atg5 fl/fl LysM-Cre + mice resulted in enhanced cytotoxicity and inflammation after silica exposure compared to littermate controls, including elevated IL-18 and the alarmin HMGB1 in the whole lavage fluid. Autophagy deficiency caused some spontaneous inflammation and disease. Greater silica-induced acute inflammation in Atg5 fl/fl LysM-Cre + mice correlated with increased fibrosis and chronic lung disease. These studies demonstrate a critical role for autophagy in suppressing silica-induced cytotoxicity and inflammation in disease development. Furthermore, this data highlights the importance of basal autophagy in macrophages and other myeloid cells in maintaining lung homeostasis. - Highlights: • Silica exposure increases autophagy in macrophages. • Autophagy deficient mice have enhanced inflammation and silicosis. • Autophagy deficiency in macrophages results in greater silica-induced cytotoxicity. • Autophagy deficiency in macrophages increases extracellular IL-18 and HMGB1.

Monosodium Urate Crystals Induce Upregulation of NK1.1-Dependent Killing by Macrophages and Support Tumor-Resident NK1.1+ Monocyte/Macrophage Populations in Antitumor Therapy.

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Steiger, Stefanie; Kuhn, Sabine; Ronchese, Franca; Harper, Jacquie L

2015-12-01

Macrophages display phenotypic and functional heterogeneity dependent on the changing inflammatory microenvironment. Under some conditions, macrophages can acquire effector functions commonly associated with NK cells. In the current study, we investigated how the endogenous danger signal monosodium urate (MSU) crystals can alter macrophage functions. We report that naive, primary peritoneal macrophages rapidly upregulate the expression of the NK cell-surface marker NK1.1 in response to MSU crystals but not in response to LPS or other urate crystals. NK1.1 upregulation by macrophages was associated with mechanisms including phagocytosis of crystals, NLRP3 inflammasome activation, and autocrine proinflammatory cytokine signaling. Further analysis demonstrated that MSU crystal-activated macrophages exhibited NK cell-like cytotoxic activity against target cells in a perforin/granzyme B-dependent manner. Furthermore, analysis of tumor hemopoietic cell populations showed that effective, MSU-mediated antitumor activity required coadministration with Mycobacterium smegmatis to induce IL-1β production and significant accumulation of monocytes and macrophages (but not granulocytes or dendritic cells) expressing elevated levels of NK1.1. Our findings provide evidence that MSU crystal-activated macrophages have the potential to develop tumoricidal NK cell-like functions that may be exploited to boost antitumor activity in vivo. Copyright © 2015 by The American Association of Immunologists, Inc.

Yersinia pestis endowed with increased cytotoxicity is avirulent in a bubonic plague model and induces rapid protection against pneumonic plague.

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Ayelet Zauberman

Full Text Available An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica Oratio8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10(7-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD(50 of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed

Evaluation of a nanotechnology-based approach to induce gene-expression in human THP-1 macrophages under inflammatory conditions.

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Bernal, Laura; Alvarado-Vázquez, Abigail; Ferreira, David Wilson; Paige, Candler A; Ulecia-Morón, Cristina; Hill, Bailey; Caesar, Marina; Romero-Sandoval, E Alfonso

2017-02-01

Macrophages orchestrate the initiation and resolution of inflammation by producing pro- and anti-inflammatory products. An imbalance in these mediators may originate from a deficient or excessive immune response. Therefore, macrophages are valid therapeutic targets to restore homeostasis under inflammatory conditions. We hypothesize that a specific mannosylated nanoparticle effectively induces gene expression in human macrophages under inflammatory conditions without undesirable immunogenic responses. THP-1 macrophages were challenged with lipopolysaccharide (LPS, 5μg/mL). Polyethylenimine (PEI) nanoparticles grafted with a mannose receptor ligand (Man-PEI) were used as a gene delivery method. Nanoparticle toxicity, Man-PEI cellular uptake rate and gene induction efficiency (GFP, CD14 or CD68) were studied. Potential immunogenic responses were evaluated by measuring the production of tumor necrosis factor-alpha (TNF-α), Interleukin (IL)-6 and IL-10. Man-PEI did not produce cytotoxicity, and it was effectively up-taken by THP-1 macrophages (69%). This approach produced a significant expression of GFP (mRNA and protein), CD14 and CD68 (mRNA), and transiently and mildly reduced IL-6 and IL-10 levels in LPS-challenged macrophages. Our results indicate that Man-PEI is suitable for inducing an efficient gene overexpression in human macrophages under inflammatory conditions with limited immunogenic responses. Our promising results set the foundation to test this technology to induce functional anti-inflammatory genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

Systematic immunosuppression induced by photodynamic therapy (PDT) is adoptively transferred by macrophages

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Lynch, D.H.; Haddad, S; King, V.J.; Ott, M.J.; Jolles, C.J.; Straight, R. C.

1989-01-01

The purpose of this study was to determine whether photodynamic therapy induced suppression of contact hypersensitivity (CHS) responses was an active phenomenon that could be adoptively transferred by viable splenocytes from PDT-treated mice. Although induction of adoptively transferable suppressor cells in PDT-treated mice required exposure to antigen, the suppressor cells were found to be antigen nonspecific in their function. Furthermore, splenocytes from PDT-treated mice were capable of generating levels of allospecific cytotoxic T lymphocyte (CTL) activity which were comparable to those generated by normal control mice, but the ability of irradiated spleen cells from PDT-treated mice to stimulate a mixed lymphocyte response (MLR) was dramatically impaired. Finally, chromatographic separation of T cells, B cells and macrophages showed that the cell type which mediates adoptively transferable suppression of CHS responsiveness is in the macrophage lineage. (author)

Cytotoxicity and cytokine expression induced by silorane and methacrylate-based composite resins.

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Longo, Daniele Lucca; Paula-Silva, Francisco Wanderley Garcia; Faccioli, Lucia Helena; Gatón-Hernández, Patrícia Maria; Queiroz, Alexandra Mussolino de; Silva, Léa Assed Bezerra da

2016-01-01

The aim of this study was to evaluate cytotoxicity and cytokine production induced by light-cured or non-light-cured methacrylate-based and silorane composite resins in RAW 264.7 macrophages. Cells were stimulated with the extracts from light-cured or non-light-cured composite resins. After incubation for 24 h, cytotoxicity was assessed with the lactate dehydrogenase (LDH) and methyl thiazolyl tetrazolium (MTT) assays, and total protein was quantified using the Lowry method. TNF-α detection was examined with an enzyme-linked immunosorbent assay (ELISA) conducted with cell supernatants after cell stimulation for 6, 12, and 24 h. Data were analyzed using one-way analysis of variance (ANOVA) and Tukey's post hoc test (α=0.05). KaloreTM and FiltekTM Silorane were cytotoxic with or without light curing (p0.05). However, after 24 h FiltekTM Silorane inhibited the production of TNF-α (p<0.05). KaloreTM and FiltekTM Silorane were cytotoxic regardless of light curing. The extract obtained from KaloreTM after 15 days of incubation stimulated the production of TNF-α, unlike that obtained from FiltekTM Silorane.

Inhibition of inducible Nitric Oxide Synthase by a mustard gas analog in murine macrophages

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Smith Milton

2006-11-01

Full Text Available Abstract Background 2-Chloroethyl ethyl sulphide (CEES is a sulphur vesicating agent and an analogue of the chemical warfare agent 2,2'-dichlorodiethyl sulphide, or sulphur mustard gas (HD. Both CEES and HD are alkylating agents that influence cellular thiols and are highly toxic. In a previous publication, we reported that lipopolysaccharide (LPS enhances the cytotoxicity of CEES in murine RAW264.7 macrophages. In the present investigation, we studied the influence of CEES on nitric oxide (NO production in LPS stimulated RAW264.7 cells since NO signalling affects inflammation, cell death, and wound healing. Murine macrophages stimulated with LPS produce NO almost exclusively via inducible nitric oxide synthase (iNOS activity. We suggest that the influence of CEES or HD on the cellular production of NO could play an important role in the pathophysiological responses of tissues to these toxicants. In particular, it is known that macrophage generated NO synthesised by iNOS plays a critical role in wound healing. Results We initially confirmed that in LPS stimulated RAW264.7 macrophages NO is exclusively generated by the iNOS form of nitric oxide synthase. CEES treatment inhibited the synthesis of NO (after 24 hours in viable LPS-stimulated RAW264.7 macrophages as measured by either nitrite secretion into the culture medium or the intracellular conversion of 4,5-diaminofluorescein diacetate (DAF-2DA or dichlorofluorescin diacetate (DCFH-DA. Western blots showed that CEES transiently decreased the expression of iNOS protein; however, treatment of active iNOS with CEES in vitro did not inhibit its enzymatic activity Conclusion CEES inhibits NO production in LPS stimulated macrophages by decreasing iNOS protein expression. Decreased iNOS expression is likely the result of CEES induced alteration in the nuclear factor kappa B (NF-κB signalling pathway. Since NO can act as an antioxidant, the CEES induced down-regulation of iNOS in LPS

DMPD: Pathogen-induced apoptosis of macrophages: a common end for different pathogenicstrategies. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 11207583 Pathogen-induced apoptosis of macrophages: a common end for different path...ml) Show Pathogen-induced apoptosis of macrophages: a common end for different pathogenicstrategies. PubmedI...D 11207583 Title Pathogen-induced apoptosis of macrophages: a common end for diff

The natural compound berberine positively affects macrophage functions involved in atherogenesis.

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Zimetti, F; Adorni, M P; Ronda, N; Gatti, R; Bernini, F; Favari, E

2015-02-01

We investigated the effect of berberine (BBR), an alkaloid showing antiatherogenic properties beyond the cholesterol lowering capacity, on macrophage cholesterol handling upon exposure to human serum and on macrophage responses to excess free cholesterol (FC) loading. Mouse and human macrophages were utilized as cellular models. Cholesterol content was measured by a fluorimetric assay; cholesterol efflux, cytotoxicity and membrane FC distribution were evaluated by radioisotopic assays. Monocyte chemotactic protein-1 (MCP-1) secretion was measured by ELISA; membrane ruffling and macropinocytosis were visualized by confocal microscopy. Exposure of cholesterol-enriched MPM to serum in the presence of 1 μM BBR resulted in a reduction of intracellular cholesterol content twice greater than exposure to serum alone (-52%; p microscope analysis revealed that BBR inhibited macropinocytosis, an independent-receptor process involved in LDL internalization. Macrophage FC-enrichment increased MCP-1 release by 1.5 folds, increased cytotoxicity by 2 fold, and induced membrane ruffling; all these responses were markedly inhibited by BBR. FC-enrichment led to an increase in plasma membrane cholesterol by 4.5 folds, an effect counteracted by BBR. We showed novel potentially atheroprotective activities of BBR in macrophages, consisting in the inhibition of serum-induced cholesterol accumulation, occurring at least in part through an impairment of macropinocytosis, and of FC-induced deleterious effects. Copyright © 2014 Elsevier B.V. All rights reserved.

Brucella Dissociation Is Essential for Macrophage Egress and Bacterial Dissemination

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Thomas A Ficht

2014-03-01

Full Text Available It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic de-tails of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using Brucella strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while Brucella uptake and replication were monitored using an immunofluorescence assay (IFA. Vis-ible plaques were detected at 4-5 days post infection (p.i. with cytotoxic Brucella 16M∆manBA at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating Brucella. Visible plaques were not detected in monolayers infected with non-cytotoxic 16M∆manBA∆virB2 at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci with replicating Brucella in the monolayers infected with 16M∆manBA∆virB2. The size of the foci observed in macrophage monolayers infected with rough Brucella correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for Brucella egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addi-tion of gentamicin to the culture medium inhibited plaque formation, suggesting that the cell-to-cell spreading occurred only following release of the organisms from the cells. Taken together, these results demonstrate that Brucella induced cytotoxicity is critical for Brucella egress and dissemination.

Brucella dissociation is essential for macrophage egress and bacterial dissemination.

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Pei, Jianwu; Kahl-McDonagh, Melissa; Ficht, Thomas A

2014-01-01

It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic details of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using Brucella strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while Brucella uptake and replication were monitored using an immunofluorescence assay (IFA). Visible plaques were detected at 4-5 days post infection (p.i.) with cytotoxic Brucella 16MΔmanBA at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating Brucella. Visible plaques were not detected in monolayers infected with non-cytotoxic 16MΔmanBAΔvirB2 at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci) with replicating Brucella in the monolayers infected with 16MΔmanBAΔvirB2. The size of the foci observed in macrophage monolayers infected with rough Brucella correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for Brucella egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addition of gentamicin to the culture medium inhibited plaque formation, suggesting that cell-to-cell spread occurred only following release of the organisms from the cells. Taken together, these results demonstrate that Brucella-induced cytotoxicity is critical for Brucella egress and dissemination.

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion.

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Li, Peng; Tian, Mingxing; Bao, Yanqing; Hu, Hai; Liu, Jiameng; Yin, Yi; Ding, Chan; Wang, Shaohui; Yu, Shengqing

2017-01-01

Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant Δ rfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion

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Peng Li

2017-09-01

Full Text Available Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the

Autophagy induced by silica nanoparticles protects RAW264.7 macrophages from cell death.

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Marquardt, Clarissa; Fritsch-Decker, Susanne; Al-Rawi, Marco; Diabaté, Silvia; Weiss, Carsten

2017-03-15

Although the technological and economic benefits of engineered nanomaterials are obvious, concerns have been raised about adverse effects if such material is inhaled, ingested, applied to the skin or even released into the environment. Here we studied the cytotoxic effects of the most abundant nanomaterial, silica nanoparticles (SiO 2 -NPs), in murine RAW264.7 macrophages. SiO 2 -NPs dose-dependently induce membrane leakage and cell death without obvious involvement of reactive oxygen species. Interestingly, at low concentrations SiO 2 -NPs trigger autophagy, evidenced by morphological and biochemical hallmarks such as autophagolysosomes or increased levels of LC3-II, which serves to protect cells from cytotoxicity. Hence SiO 2 -NPs initiate an adaptive stress response which dependent on dose serve to balance survival and death and ultimately dictates the cellular fate. Copyright © 2017 Elsevier B.V. All rights reserved.

Impact of Silver and Iron Nanoparticle Exposure on Cholesterol Uptake by Macrophages

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Jonathan H. Shannahan

2015-01-01

Full Text Available Macrophages are central to the development of atherosclerosis by absorbing lipids, promoting inflammation, and increasing plaque deposition. Nanoparticles (NPs are becoming increasingly common in biomedical applications thereby increasing exposure to the immune and vascular systems. This project investigated the influence of NPs on macrophage function and specifically cholesterol uptake. Macrophages were exposed to 20 nm silver NPs (AgNPs, 110 nm AgNPs, or 20 nm Fe3O4 NPs for 2 h and NP uptake, cytotoxicity, and subsequent uptake of fluorescently labeled cholesterol were assessed. Macrophage uptake of NPs did not induce cytotoxicity at concentrations utilized (25 μg/mL; however, macrophage exposure to 20 nm AgNPs reduced subsequent uptake of cholesterol. Further, we assessed the impact of a cholesterol-rich environment on macrophage function following NP exposure. In these sets of experiments, macrophages internalized NPs, exhibited no cytotoxicity, and altered cholesterol uptake. Alterations in the expression of scavenger receptor-B1 following NP exposure, which likely influences cholesterol uptake, were observed. Overall, NPs alter cholesterol uptake, which may have implications in the progression of vascular or immune mediated diseases. Therefore, for the safe development of NPs for biomedical applications, it is necessary to understand their impact on cellular function and biological interactions in underlying disease environments.

Mycobacterium leprae-Infected Macrophages Preferentially Primed Regulatory T Cell Responses and Was Associated with Lepromatous Leprosy.

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Degang Yang

2016-01-01

Full Text Available The persistence of Mycobacterium leprae (M. leprae infection is largely dependent on the types of host immune responses being induced. Macrophage, a crucial modulator of innate and adaptive immune responses, could be directly infected by M. leprae. We therefore postulated that M. leprae-infected macrophages might have altered immune functions.Here, we treated monocyte-derived macrophages with live or killed M. leprae, and examined their activation status and antigen presentation. We found that macrophages treated with live M. leprae showed committed M2-like function, with decreased interleukin 1 beta (IL-1beta, IL-6, tumor necrosis factor alpha (TNF-alpha and MHC class II molecule expression and elevated IL-10 and CD163 expression. When incubating with naive T cells, macrophages treated with live M. leprae preferentially primed regulatory T (Treg cell responses with elevated FoxP3 and IL-10 expression, while interferon gamma (IFN-gamma expression and CD8+ T cell cytotoxicity were reduced. Chromium release assay also found that live M. leprae-treated macrophages were more resistant to CD8+ T cell-mediated cytotoxicity than sonicated M. leprae-treated monocytes. Ex vivo studies showed that the phenotype and function of monocytes and macrophages had clear differences between L-lep and T-lep patients, consistent with the in vitro findings.Together, our data demonstrate that M. leprae could utilize infected macrophages by two mechanisms: firstly, M. leprae-infected macrophages preferentially primed Treg but not Th1 or cytotoxic T cell responses; secondly, M. leprae-infected macrophages were more effective at evading CD8+ T cell-mediated cytotoxicity.

Mycobacterium leprae-Infected Macrophages Preferentially Primed Regulatory T Cell Responses and Was Associated with Lepromatous Leprosy.

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Yang, Degang; Shui, Tiejun; Miranda, Jake W; Gilson, Danny J; Song, Zhengyu; Chen, Jia; Shi, Chao; Zhu, Jianyu; Yang, Jun; Jing, Zhichun

2016-01-01

The persistence of Mycobacterium leprae (M. leprae) infection is largely dependent on the types of host immune responses being induced. Macrophage, a crucial modulator of innate and adaptive immune responses, could be directly infected by M. leprae. We therefore postulated that M. leprae-infected macrophages might have altered immune functions. Here, we treated monocyte-derived macrophages with live or killed M. leprae, and examined their activation status and antigen presentation. We found that macrophages treated with live M. leprae showed committed M2-like function, with decreased interleukin 1 beta (IL-1beta), IL-6, tumor necrosis factor alpha (TNF-alpha) and MHC class II molecule expression and elevated IL-10 and CD163 expression. When incubating with naive T cells, macrophages treated with live M. leprae preferentially primed regulatory T (Treg) cell responses with elevated FoxP3 and IL-10 expression, while interferon gamma (IFN-gamma) expression and CD8+ T cell cytotoxicity were reduced. Chromium release assay also found that live M. leprae-treated macrophages were more resistant to CD8+ T cell-mediated cytotoxicity than sonicated M. leprae-treated monocytes. Ex vivo studies showed that the phenotype and function of monocytes and macrophages had clear differences between L-lep and T-lep patients, consistent with the in vitro findings. Together, our data demonstrate that M. leprae could utilize infected macrophages by two mechanisms: firstly, M. leprae-infected macrophages preferentially primed Treg but not Th1 or cytotoxic T cell responses; secondly, M. leprae-infected macrophages were more effective at evading CD8+ T cell-mediated cytotoxicity.

Macrophage activation by a vanadyl-aspirin complex is dependent on L-type calcium channel and the generation of nitric oxide

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Molinuevo, Maria Silvina; Etcheverry, Susana Beatriz; Cortizo, Ana Maria

2005-01-01

Bone homeostasis is the result of a tight balance between bone resorption and bone formation where macrophage activation is believed to contribute to bone resorption. We have previously shown that a vanadyl(IV)-aspirin complex (VOAspi) regulates cell proliferation and differentiation of osteoblasts in culture. In this study, we assessed VOAspi and VO effects and their possible mechanism of action on a mouse macrophage cell line RAW 264.7. Both vanadium compounds inhibited cell proliferation in a dose-dependent manner. Nifedipine completely reversed the VOAspi-induced macrophage cytotoxicity, while it could not block the effect of VO. VOAspi also stimulated nitric oxide (NO) production, the oxidation of dihydrorhodamine 123 (DHR-123) and enhanced the expression of both constitutive and inducible isoforms of nitric oxide syntases (NOS). All these effects were abolished by nifedipine. Althogether our finding give evidence that VOAspi-induced macrophage cytotoxicity is dependent on L-type calcium channel and the generation of NO though the induction of eNOS and iNOS. Contrary, the parent compound VO exerted a cytotoxic effect by mechanisms independent of a calcium entry and the NO/NOS activation

Oxidized phospholipids induce ceramide accumulation in RAW 264.7 macrophages: role of ceramide synthases.

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Lingaraju M Halasiddappa

Full Text Available Oxidized phospholipids (OxPLs, including 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC and 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine (POVPC are among several biologically active derivatives that are generated during oxidation of low-density lipoproteins (LDLs. These OxPLs are factors contributing to pro-atherogenic effects of oxidized LDLs (OxLDLs, including inflammation, proliferation and death of vascular cells. OxLDL also elicits formation of the lipid messenger ceramide (Cer which plays a pivotal role in apoptotic signaling pathways. Here we report that both PGPC and POVPC are cytotoxic to cultured macrophages and induce apoptosis in these cells which is associated with increased cellular ceramide levels after several hours. In addition, exposure of RAW 264.7 cells to POVPC and PGPC under the same conditions resulted in a significant increase in ceramide synthase activity, whereas, acid or neutral sphingomyelinase activities were not affected. PGPC is not only more toxic than POVPC, but also a more potent inducer of ceramide formation by activating a limited subset of CerS isoforms. The stimulated CerS activities are in line with the C16-, C22-, and C24:0-Cer species that are generated under the influence of the OxPL. Fumonisin B1, a specific inhibitor of CerS, suppressed OxPL-induced ceramide generation, demonstrating that OxPL-induced CerS activity in macrophages is responsible for the accumulation of ceramide. OxLDL elicits the same cellular ceramide and CerS effects. Thus, it is concluded that PGPC and POVPC are active components that contribute to the capacity of this lipoprotein to elevate ceramide levels in macrophages.

Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605 Reduces Asbestos-Induced Cytotoxicity in an Nrf2-Dependent and -Independent Manner

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Ralph A. Pietrofesa

2018-03-01

Full Text Available Asbestos exposure triggers inflammatory processes associated with oxidative stress and tissue damage linked to malignancy. LGM2605 is the synthetic lignan secoisolariciresinol diglucoside (SDG with free radical scavenging, antioxidant, and anti-inflammatory properties in diverse inflammatory cell and mouse models, including exposure to asbestos fibers. Nuclear factor-E2 related factor 2 (Nrf2 activation and boosting of endogenous tissue defenses were associated with the protective action of LGM2605 from asbestos-induced cellular damage. To elucidate the role of Nrf2 induction by LGM2605 in protection from asbestos-induced cellular damage, we evaluated LGM2605 in asbestos-exposed macrophages from wild-type (WT and Nrf2 disrupted (Nrf2−/− mice. Cells were pretreated with LGM2605 (50 µM and 100 µM and exposed to asbestos fibers (20 µg/cm2 and evaluated 8 h and 24 h later for inflammasome activation, secreted cytokine levels (interleukin-1β (IL-1β, interleukin-18 (IL-18, interleukin-6 (IL-6, and tumor necrosis factor alpha (TNFα, cytotoxicity and cell death, nitrosative stress, and Nrf2-regulated enzyme levels. Asbestos exposure induced robust oxidative and nitrosative stress, cell death and cytotoxicity, which were equally mitigated by LGM2605. Inflammasome activation was significantly attenuated in Nrf2−/− macrophages compared to WT, and the protective action of LGM2605 was seen only in WT cells. In conclusion, in a cell model of asbestos-induced toxicity, LGM2605 acts via protective mechanisms that may not involve Nrf2 activation.

Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605) Reduces Asbestos-Induced Cytotoxicity in an Nrf2-Dependent and -Independent Manner

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Pietrofesa, Ralph A.; Chatterjee, Shampa; Park, Kyewon; Arguiri, Evguenia; Albelda, Steven M.; Christofidou-Solomidou, Melpo

2018-01-01

Asbestos exposure triggers inflammatory processes associated with oxidative stress and tissue damage linked to malignancy. LGM2605 is the synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant, and anti-inflammatory properties in diverse inflammatory cell and mouse models, including exposure to asbestos fibers. Nuclear factor-E2 related factor 2 (Nrf2) activation and boosting of endogenous tissue defenses were associated with the protective action of LGM2605 from asbestos-induced cellular damage. To elucidate the role of Nrf2 induction by LGM2605 in protection from asbestos-induced cellular damage, we evaluated LGM2605 in asbestos-exposed macrophages from wild-type (WT) and Nrf2 disrupted (Nrf2−/−) mice. Cells were pretreated with LGM2605 (50 µM and 100 µM) and exposed to asbestos fibers (20 µg/cm2) and evaluated 8 h and 24 h later for inflammasome activation, secreted cytokine levels (interleukin-1β (IL-1β), interleukin-18 (IL-18), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFα)), cytotoxicity and cell death, nitrosative stress, and Nrf2-regulated enzyme levels. Asbestos exposure induced robust oxidative and nitrosative stress, cell death and cytotoxicity, which were equally mitigated by LGM2605. Inflammasome activation was significantly attenuated in Nrf2−/− macrophages compared to WT, and the protective action of LGM2605 was seen only in WT cells. In conclusion, in a cell model of asbestos-induced toxicity, LGM2605 acts via protective mechanisms that may not involve Nrf2 activation. PMID:29498660

Triglyceride-induced macrophage cell death is triggered by caspase-1.

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Son, Sin Jee; Rhee, Ki-Jong; Lim, Jaewon; Kim, Tae Ue; Kim, Tack-Joong; Kim, Yoon Suk

2013-01-01

Triglyceride (TG) induces macrophage cell death which contributes to the development of atherosclerosis. We confirmed that exogenous TG accumulates in human THP-1 macrophages and causes cell death. TG treated THP-1 macrophages exhibited no change in tumor necrosis factor (TNF)-α, interleukin (IL)-18, macrophage inflammatory protein (MIP)-1α, and IL-1R1 receptor mRNA expression. However, there was a marked decrease in IL-1β mRNA expression but an increase in IL-1β protein secretion. Decreased expression of IL-1β mRNA and increased secretion of IL-1β protein was not the direct cause of cell death. Until now, TG was assumed to induce necrotic cell death in macrophages. Since caspase-1 is known to be involved in activation and secretion of IL-1β protein and pyroptotic cell death, next we determined whether caspase-1 is associated with TG-induced macrophage cell death. We found an increase in caspase-1 activity in TG-treated THP-1 macrophages and inhibition of caspase-1 activity using a specific inhibitor partially rescued cell death. These results suggest activation of the pyroptotic pathway by TG. This is the first report implicating the activation of caspase-1 and the triggering of the pyroptosis pathway in TG-induced macrophage cell death.

Specific macrophage subtypes influence the progression of rhabdomyolysis-induced kidney injury.

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Belliere, Julie; Casemayou, Audrey; Ducasse, Laure; Zakaroff-Girard, Alexia; Martins, Frédéric; Iacovoni, Jason S; Guilbeau-Frugier, Céline; Buffin-Meyer, Bénédicte; Pipy, Bernard; Chauveau, Dominique; Schanstra, Joost P; Bascands, Jean-Loup

2015-06-01

Rhabdomyolysis can be life threatening if complicated by AKI. Macrophage infiltration has been observed in rat kidneys after glycerol-induced rhabdomyolysis, but the role of macrophages in rhabdomyolysis-induced AKI remains unknown. Here, in a patient diagnosed with rhabdomyolysis, we detected substantial macrophage infiltration in the kidney. In a mouse model of rhabdomyolysis-induced AKI, diverse renal macrophage phenotypes were observed depending on the stage of the disease. Two days after rhabdomyolysis, F4/80(low)CD11b(high)Ly6b(high)CD206(low) kidney macrophages were dominant, whereas by day 8, F4/80(high)CD11b(+)Ly6b(low)CD206(high) cells became the most abundant. Single-cell gene expression analyses of FACS-sorted macrophages revealed that these subpopulations were heterogeneous and that individual cells simultaneously expressed both M1 and M2 markers. Liposomal clodronate-mediated macrophage depletion significantly reduced the early infiltration of F4/80(low)CD11b(high)Ly6b(high)CD206(low) macrophages. Furthermore, transcriptionally regulated targets potentially involved in disease progression, including fibronectin, collagen III, and chemoattractants that were identified via single-cell analysis, were verified as macrophage-dependent in situ. In vitro, myoglobin treatment induced proximal tubular cells to secrete chemoattractants and macrophages to express proinflammatory markers. At day 30, liposomal clodronate-mediated macrophage depletion reduced fibrosis and improved both kidney repair and mouse survival. Seven months after rhabdomyolysis, histologic lesions were still present but were substantially reduced with prior depletion of macrophages. These results suggest an important role for macrophages in rhabdomyolysis-induced AKI progression and advocate the utility of long-term follow-up for patients with this disease. Copyright © 2015 by the American Society of Nephrology.

Effect of Tityus serrulatus venom on cytokine production and the activity of murine macrophages

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Vera L. Petricevich

2002-01-01

Full Text Available The purpose of this study was to investigate the effects of Tityus serrulatus venom (TSV on murine peritoneal macrophages evaluated in terms of activation. The effects of crude TSV were analysed by detection of cytokines, oxygen intermediate metabolites (H2O2 and nitric oxide (NO in supernatants of peritoneal macrophages. Several functional bioassays were employed including an in vitro model for envenomating: cytotoxicity of TSV was assessed using the lyses percentage. Tumor necrosis factor (TNF activity was assayed by measuring its cytotoxic activity on L-929 cells, and interleukin-6 (IL-6 and interferon-γ (IFN-γ were assayed by enzyme-linked immunosorbent assay, whereas NO levels were detected by Griess colorimetric reactions in culture supernatant of macrophages incubated with TSV and subsequently exposed to either lipopolysaccharide or IFN-γ. Incubation of macrophages with TSV increased production of IL-6 and IFN-γ in a dose-dependent manner. TNF production was not detected in supernatants treated with TSV at any concentration. The increase in IL-6 secretion was not associated with concentration-dependent cytoxicity of TSV on these cells. These data suggest that the cytotoxicity does not appear to be the main cause of an increased cytokine production by these cells. Although NO is an important effector molecule in macrophage microbicidal activity, the inducing potential of the test compounds for its release was found to be very moderate, ranging from 125 to 800 mM. Interestingly, NO levels of peritoneal macrophages were increased after IFN-γ. Moreover, NO production had an apparent effect on macrophage activity. The results obtained here also shown that the TSV induces an important elevation in H2O2 release. These results combined with NO production suggest that TSV possesses significant immunomodulatory activities capable of stimulating immune functions in vitro.

Potential role of an antimicrobial peptide, KLK in inhibiting lipopolysaccharide-induced macrophage inflammation.

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Pornpimon Jantaruk

Full Text Available Antimicrobial peptides (AMPs are attractive alternatives to antibiotics. Due to their immune modulatory properties, AMPs are at present emerging as promising agents for controlling inflammatory-mediated diseases. In this study, anti-inflammatory potential of an antimicrobial peptide, KLK (KLKLLLLLKLK and its analogs was evaluated in lipopolysaccharide (LPS-induced RAW 264.7 macrophages. The results herein demonstrated that KLK peptide as well as its analogs significantly inhibited the pro-inflammatory mediator nitric oxide (NO, interleukin-1β (IL-1β and tumor necrosis factor-α (TNF-α production in LPS-stimulated RAW 264.7 macrophages in dose-dependent manners, and such inhibitory effects were not due to direct cytotoxicity. When considering inhibition potency, KLK among the test peptides exhibited the most effective activity. The inhibitory activity of KLK peptide also extended to include suppression of LPS-induced production of prostaglandin E2 (PGE2. KLK significantly decreased mRNA and protein expression of inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2 as well as mRNA expression of IL-1β and TNF-α. Moreover, KLK inhibited nuclear translocation of nuclear factor-κB (NF-κB p65 and blocked degradation and phosphorylation of inhibitor of κB (IκB. Taken together, these results suggested that the KLK peptide inhibited inflammatory response through the down-regulation of NF-κB mediated activation in macrophages. Since peptide analogs with different amino acid sequences and arrangement were investigated for their anti-inflammatory activities, the residues/structures required for activity were also discussed. Our findings therefore proved anti-inflammatory potential of the KLK peptide and provide direct evidence for therapeutic application of KLK as a novel anti-inflammatory agent.

Extracts of Crinum latifolium inhibit the cell viability of mouse lymphoma cell line EL4 and induce activation of anti-tumour activity of macrophages in vitro.

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Nguyen, Hoang-Yen T; Vo, Bach-Hue T; Nguyen, Lac-Thuy H; Bernad, Jose; Alaeddine, Mohamad; Coste, Agnes; Reybier, Karine; Pipy, Bernard; Nepveu, Françoise

2013-08-26

Crinum latifolium L. (CL) leaf extracts have been traditionally used in Vietnam and are now used all over the world for the treatment of prostate cancer. However, the precise cellular mechanisms of the action of CL extracts remain unclear. To examine the effects of CL samples on the anti-tumour activity of peritoneal murine macrophages. The properties of three extracts (aqueous, flavonoid, alkaloid), one fraction (alkaloid), and one pure compound (6-hydroxycrinamidine) obtained from CL, were studied (i) for redox capacities (DPPH and bleaching beta-carotene assays), (ii) on murine peritoneal macrophages (MTT assay) and on lymphoma EL4-luc2 cells (luciferine assay) for cytotoxicity, (iii) on macrophage polarization (production of ROS and gene expression by PCR), and (iv) on the tumoricidal functions of murine peritoneal macrophages (lymphoma cytotoxicity by co-culture with syngeneic macrophages). The total flavonoid extract with a high antioxidant activity (IC50=107.36 mg/L, DPPH assay) showed an inhibitory action on cancer cells. Alkaloid extracts inhibited the proliferation of lymphoma cells either by directly acting on tumour cells or by activating of the tumoricidal functions of syngeneic macrophages. The aqueous extract induced mRNA expression of tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin 6 (IL-6) indicating differentiation of macrophages into pro-inflammatory M1 polarized macrophages. The total flavonoid, alkaloid extracts and an alkaloid fraction induced the expression of the formyl peptide receptor (FPR) on the surface of the polarized macrophages that could lead to the activation of macrophages towards the M1 phenotype. Aqueous and flavonoid extracts enhanced NADPH quinine oxido-reductase 1 (NQO1) mRNA expression in polarized macrophages which could play an important role in cancer chemoprevention. All the samples studied were non-toxic to normal living cells and the pure alkaloid tested, 6-hydroxycrinamidine, was not

In vitro evaluation of cytotoxic and inflammatory properties of silica nanoparticles of different sizes in murine RAW 264.7 macrophages

International Nuclear Information System (INIS)

Park, Margriet V. D. Z.; Lynch, Iseult; Ramírez-García, Sonia; Dawson, Kenneth A.; Fonteyne, Liset de la; Gremmer, Eric; Slob, Wout; Briedé, Jacob J.; Elsaesser, Andreas; Howard, C. Vyvyan; Loveren, Henk van; Jong, Wim H. de

2011-01-01

The biological response to four well-characterized amorphous silica nanoparticles was investigated in RAW 264.7 macrophages in view of their potential application as drug carriers to sites of inflammation. All silica nanoparticles-induced cell membrane damage, reduced metabolic activity, generated ROS and released various cytokines, but to different extents. Two silica nanoparticles of 34 nm (A and B) with different zetapotentials were more cytotoxic than (aggregated) 11 and 248 nm nanoparticles, while cytokines were mostly induced by the (aggregated) 11 nm and only one of the 34 nm nanoparticles (34A). The results indicate that specific silica nanoparticles may have counterproductive effects, for example when used as carriers of anti-inflammatory drugs. The physicochemical properties determining the response of nanoparticles vary for different responses, implying that a screening approach for the safe development of nanoparticles needs to consider the role of combinations of (dynamic) physicochemical properties and needs to include multiple toxicity endpoints.

Reactive oxygen species (ROS) induced cytokine production and cytotoxicity of PAMAM dendrimers in J774A.1 cells

International Nuclear Information System (INIS)

Naha, Pratap C.; Davoren, Maria; Lyng, Fiona M.; Byrne, Hugh J.

2010-01-01

The immunotoxicity of three generations of polyamidoamine (PAMAM) dendrimers (G-4, G-5 and G-6) was evaluated in mouse macrophage cells in vitro. Using the Alamar blue and MTT assays, a generation dependent cytotoxicity of the PAMAM dendrimers was found whereby G-6 > G-5 > G-4. The toxic response of the PAMAM dendrimers correlated well with the number of surface primary amino groups, with increasing number resulting in an increase in toxic response. An assessment of intracellular ROS generation by the PAMAM dendrimers was performed by measuring the increased fluorescence as a result of intracellular oxidation of Carboxy H 2 DCFDA to DCF both quantitatively using plate reader and qualitatively by confocal laser scanning microscopy. The inflammatory mediators macrophage inflammatory protein-2 (MIP-2), tumour necrosis factor-α (TNF-α) and interleukin-6, (IL-6) were measured by the enzyme linked immunosorbant assay (ELISA) following exposure of mouse macrophage cells to PAMAM dendrimers. A generation dependent ROS and cytokine production was found, which correlated well with the cytotoxicological response and therefore number of surface amino groups. A clear time sequence of increased ROS generation (maximum at ∼ 4 h), TNF-α and IL-6 secretion (maximum at ∼ 24 h), MIP-2 levels and cell death (∼ 72 h) was observed. The intracellular ROS generation and cytokine production induced cytotoxicity point towards the mechanistic pathway of cell death upon exposure to PAMAM dendrimers.

Fibronectin induces macrophage migration through a SFK-FAK/CSF-1R pathway.

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Digiacomo, Graziana; Tusa, Ignazia; Bacci, Marina; Cipolleschi, Maria Grazia; Dello Sbarba, Persio; Rovida, Elisabetta

2017-07-04

Integrins, following binding to proteins of the extracellular matrix (ECM) including collagen, laminin and fibronectin (FN), are able to transduce molecular signals inside the cells and to regulate several biological functions such as migration, proliferation and differentiation. Besides activation of adaptor molecules and kinases, integrins transactivate Receptor Tyrosine Kinases (RTK). In particular, adhesion to the ECM may promote RTK activation in the absence of growth factors. The Colony-Stimulating Factor-1 Receptor (CSF-1R) is a RTK that supports the survival, proliferation, and motility of monocytes/macrophages, which are essential components of innate immunity and cancer development. Macrophage interaction with FN is recognized as an important aspect of host defense and wound repair. The aim of the present study was to investigate on a possible cross-talk between FN-elicited signals and CSF-1R in macrophages. FN induced migration in BAC1.2F5 and J774 murine macrophage cell lines and in human primary macrophages. Adhesion to FN determined phosphorylation of the Focal Adhesion Kinase (FAK) and Src Family Kinases (SFK) and activation of the SFK/FAK complex, as witnessed by paxillin phosphorylation. SFK activity was necessary for FAK activation and macrophage migration. Moreover, FN-induced migration was dependent on FAK in either murine macrophage cell lines or human primary macrophages. FN also induced FAK-dependent/ligand-independent CSF-1R phosphorylation, as well as the interaction between CSF-1R and β1. CSF-1R activity was necessary for FN-induced macrophage migration. Indeed, genetic or pharmacological inhibition of CSF-1R prevented FN-induced macrophage migration. Our results identified a new SFK-FAK/CSF-1R signaling pathway that mediates FN-induced migration of macrophages.

Effects of retinoic acid-inducible gene-I-like receptors activations and ionizing radiation cotreatment on cytotoxicity against human non-small cell lung cancer in vitro.

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Yoshino, Hironori; Iwabuchi, Miyu; Kazama, Yuka; Furukawa, Maho; Kashiwakura, Ikuo

2018-04-01

Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) are pattern-recognition receptors that recognize pathogen-associated molecular patterns and induce antiviral immune responses. Recent studies have demonstrated that RLR activation induces antitumor immunity and cytotoxicity against different types of cancer, including lung cancer. However a previous report has demonstrated that ionizing radiation exerts a limited effect on RLR in human monocytic cell-derived macrophages, suggesting that RLR agonists may be used as effective immunostimulants during radiation therapy. However, it is unclear whether ionizing radiation affects the cytotoxicity of RLR agonists against cancer cells. Therefore, in the present study the effects of cotreatment with ionizing radiation and RLR agonists on cytotoxicity against human non-small cell lung cancer cells A549 and H1299 was investigated. Treatment with RLR agonist poly(I:C)/LyoVec™ [poly(I:C)] exerted cytotoxic effects against human non-small cell lung cancer. The cytotoxic effects of poly(I:C) were enhanced by cotreatment with ionizing radiation, and poly(I:C) pretreatment resulted in the radiosensitization of non-small cell lung cancer. Furthermore, cotreatment of A549 and H1299 cells with poly(I:C) and ionizing radiation effectively induced apoptosis in a caspase-dependent manner compared with treatment with poly(I:C) or ionizing radiation alone. These results indicate that RLR agonists and ionizing radiation cotreatment effectively exert cytotoxic effects against human non-small cell lung cancer through caspase-mediated apoptosis.

Human Induced Pluripotent Stem Cell-Derived Macrophages for Unraveling Human Macrophage Biology.

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Zhang, Hanrui; Reilly, Muredach P

2017-11-01

Despite a substantial appreciation for the critical role of macrophages in cardiometabolic diseases, understanding of human macrophage biology has been hampered by the lack of reliable and scalable models for cellular and genetic studies. Human induced pluripotent stem cell (iPSC)-derived macrophages (IPSDM), as an unlimited source of subject genotype-specific cells, will undoubtedly play an important role in advancing our understanding of the role of macrophages in human diseases. In this review, we summarize current literature in the differentiation and characterization of IPSDM at phenotypic, functional, and transcriptomic levels. We emphasize the progress in differentiating iPSC to tissue resident macrophages, and in understanding the ontogeny of in vitro differentiated IPSDM that resembles primitive hematopoiesis, rather than adult definitive hematopoiesis. We review the application of IPSDM in modeling both Mendelian genetic disorders and host-pathogen interactions. Finally, we highlighted the potential areas of research using IPSDM in functional validation of coronary artery disease loci in genome-wide association studies, functional genomic analyses, drug testing, and cell therapeutics in cardiovascular diseases. © 2017 American Heart Association, Inc.

Alternatively Activated (M2) Macrophage Phenotype Is Inducible by Endothelin-1 in Cultured Human Macrophages.

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Soldano, Stefano; Pizzorni, Carmen; Paolino, Sabrina; Trombetta, Amelia Chiara; Montagna, Paola; Brizzolara, Renata; Ruaro, Barbara; Sulli, Alberto; Cutolo, Maurizio

2016-01-01

Alternatively activated (M2) macrophages are phenotypically characterized by the expression of specific markers, mainly macrophage scavenger receptors (CD204 and CD163) and mannose receptor-1 (CD206), and participate in the fibrotic process by over-producing pro-fibrotic molecules, such as transforming growth factor-beta1 (TGFbeta1) and metalloproteinase (MMP)-9. Endothelin-1 (ET-1) is implicated in the fibrotic process, exerting its pro-fibrotic effects through the interaction with its receptors (ETA and ETB). The study investigated the possible role of ET-1 in inducing the transition from cultured human macrophages into M2 cells. Cultured human monocytes (THP-1 cell line) were activated into macrophages (M0 macrophages) with phorbol myristate acetate and subsequently maintained in growth medium (M0-controls) or treated with either ET-1 (100nM) or interleukin-4 (IL-4, 10ng/mL, M2 inducer) for 72 hours. Similarly, primary cultures of human peripheral blood monocyte (PBM)-derived macrophages obtained from healthy subjects, were maintained in growth medium (untreated cells) or treated with ET-1 or IL-4 for 6 days. Both M0 and PBM-derived macrophages were pre-treated with ET receptor antagonist (ETA/BRA, bosentan 10-5M) for 1 hour before ET-1 stimulation. Protein and gene expression of CD204, CD206, CD163, TGFbeta1 were analysed by immunocytochemistry, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Gene expression of interleukin(IL)-10 and macrophage derived chemokine (CCL-22) was evaluated by qRT-PCR. MMP-9 production was investigated by gel zymography. ET-1 significantly increased the expression of M2 phenotype markers CD204, CD206, CD163, IL-10 and CCL-22, and the production of MMP-9 in both cultures of M0 and PBM-derived macrophages compared to M0-controls and untreated cells. In cultured PBM-derived macrophages, ET-1 increased TGFbeta1 protein and gene expression compared to untreated cells. The ET-1-mediated effects were

Particle Size-Dependent Antibacterial Activity and Murine Cell Cytotoxicity Induced by Graphene Oxide Nanomaterials

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Lin Zhao

2016-01-01

Full Text Available Recent studies have indicated that graphene and its derivative graphene oxide (GO engage in a wide range of antibacterial activities with limited toxicity to human cells. Here, we systematically evaluate the dependence of GO toxicity on the size of the nanoparticles used in treatments: we compare the cytotoxic effects of graphene quantum dots (GQDs, <15 nm, small GOs (SGOs, 50–200 nm, and large GOs (LGOs, 0.5–3 μm. We synthesize the results of bacterial colony count assays and SEM-based observations of morphological changes to assess the antibacterial properties that these GOs bring into effect against E. coli. We also use Live/Dead assays and morphological analysis to investigate changes to mammalian (Murine macrophage-like Raw 264.7 cells induced by the presence of the various GO particle types. Our results demonstrate that LGOs, SGOs, and GQDs possess antibacterial activities and cause mammalian cell cytotoxicity at descending levels of potency. Placing our observations in the context of previous simulation results, we suggest that both the lateral size and surface area of GO particles contribute to cytotoxic effects. We hope that the size dependence elucidated here provides a useful schematic for tuning GO-cell interactions in biomedical applications.

Macrophage triggering by aggregated immunoglobulins. II. Comparison of IgE and IgG aggregates or immune complexes.

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Pestel, J; Dessaint, J P; Joseph, M; Bazin, H; Capron, A

1984-01-01

Macrophages incubated with complexed or aggregated IgE released beta-glucuronidase (beta-G) within 30 min. In contrast in the presence of aggregated or complexed IgG, macrophages liberated equivalent amount of beta-G only after 6 h incubation. In addition the rapid macrophage stimulation induced by aggregated IgE was also followed by a faster 3H-glucosamine incorporation when compared to the delayed activation caused by aggregated IgG. However, macrophages stimulated either by IgG or by IgE oligomers produced the same percentage of plasminogen activator at 24 h. In contrast, while the interaction between macrophages and aggregated IgE was only followed by a peak of cyclic GMP and a beta-G release during the first 30 min of incubation, the interaction between macrophages and IgG oligomers was accompanied by a simultaneous increase of cyclic GMP and AMP nucleotides and by an absence of beta-G exocytosis. Moreover, the beta-G release induced by aggregated IgE was increased when macrophages were preincubated with aggregated IgG. This additive effect was not observed in the reverse situation. Finally macrophages activated by IgG oligomers were demonstrated to exert a cytotoxic effect on tumour cells and to kill schistosomula in the presence of a low level of complement. Taken together these results underline the peculiar ability of aggregated or complexed IgE to trigger rapidly the macrophage activation compared to aggregated IgG and can explain the important role of complexed IgE in some macrophage dependent cytotoxicity mechanisms (i.e. in parasitic diseases). PMID:6088135

Cytotoxic and radioprotective effects of Podophyllum hexandrum.

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Shukla, Sandeep Kumar; Chaudhary, Pankaj; Prem Kumar, Indracanti; Afrin, Farhat; Puri, Satish Chandra; Qazi, Ghulam Nabi; Sharma, Rakesh Kumar

2006-07-01

Podophyllum hexandrum, a herb thriving in Himalayas has already been reported to exhibit antitumor and radioprotective properties. Present study was undertaken to unravel the possible mechanism responsible for the cytotoxic and radioprotective properties of REC-2001, a fraction isolated from the rhizome of P. hexandrum using murine peritoneal macrophages and plasmid DNA as model systems. Cell death, levels of intracellular reactive oxygen species (ROS) and apoptosis were studied employing trypan blue exclusion assay, dichlorofluorescein diacetate and DNA fragmentation assay, respectively. Superoxide anions, hydroxyl radicals and DNA damage were estimated following nitroblue tetrazolium, 2-deoxyribose degradation and plasmid DNA relaxation assays, respectively. Pre-irradiation administration of REC-2001 to peritoneal macrophages in the concentration range of 25-200μg/ml significantly reduced radiation induced ROS generation, DNA damage, apoptosis and cell killing in comparison to radiation control group indicating radioprotective potential. Studies with plasmid DNA indicated the ability of REC-2001 to inhibit 20Gy induced single and double strand breaks further supporting the antioxidative potential. However, REC-2001 in a dose-dependent fashion induced cell death, ROS and DNA fragmentation indicating the cytotoxic nature. REC-2001, in presence of 100μM copper sulfate, generated significant amount of hydroxyl radicals and superoxide anions indicating ability to act as a pro-oxidant in presence of metal ions. The superoxide anion generation was found to be sensitive to metal chelators like EDTA and deferoxamine mesylate (DFR). These results suggest that the ability of REC-2001 to act as a pro-oxidant in presence of metal ions and antioxidant in presence of free radicals might be responsible for cytotoxic and radioprotective properties.

Rhinovirus infection induces distinct transcriptome profiles in polarized human macrophages.

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Rajput, Charu; Walsh, Megan P; Eder, Breanna N; Metitiri, Ediri E; Popova, Antonia P; Hershenson, Marc B

2018-05-01

Infections with rhinovirus (RV) cause asthma exacerbations. Recent studies suggest that macrophages play a role in asthmatic airway inflammation and the innate immune response to RV infection. Macrophages exhibit phenotypes based on surface markers and gene expression. We hypothesized that macrophage polarization state alters gene expression in response to RV infection. Cells were derived from human peripheral blood derived monocytes. M1 and M2 polarization was carried out by using IFN-γ and IL-4, respectively, and RNA was extracted for Affymetrix Human Gene ST2.1 exon arrays. Selected genes were validated by quantitative (q)PCR. Treatment of nonactivated (M0) macrophages with IFN-γ and IL-4 induced the expression of 252 and 153 distinct genes, respectively, including previously-identified M1 and M2 markers. RV infection of M0 macrophages induced upregulation of 232 genes; pathway analysis showed significant overrepresentation of genes involved in IFN-α/β signaling and cytokine signaling in the immune system. RV infection induced differential expression of 195 distinct genes in M1-like macrophages but only seven distinct genes in M2-like-polarized cells. In a secondary analysis, comparison between M0-, RV-infected, and M1-like-polarized, RV-infected macrophages revealed differential expression of 227 genes including those associated with asthma and its exacerbation. qPCR demonstrated increased expression of CCL8, CXCL10, TNFSF10, TNFSF18, IL6, NOD2, and GSDMD and reduced expression of VNN1, AGO1, and AGO2. Together, these data show that, in contrast to M2-like-polarized macrophages, gene expression of M1-like macrophages is highly regulated by RV.

CTLA-4Ig immunotherapy of obesity-induced insulin resistance by manipulation of macrophage polarization in adipose tissues

International Nuclear Information System (INIS)

Fujii, Masakazu; Inoguchi, Toyoshi; Batchuluun, Battsetseg; Sugiyama, Naonobu; Kobayashi, Kunihisa; Sonoda, Noriyuki; Takayanagi, Ryoichi

2013-01-01

Highlights: •CTLA-4Ig completely alleviates HFD-induced insulin resistance. •CTLA-4Ig reduces epididymal and subcutaneous fat tissue weight and adipocyte size. •CTLA-4Ig alters ATM polarization from inflammatory M1 to anti-inflammatory M2. •CTLA-4Ig may lead to a novel anti-obesity/inflammation/insulin resistance agent. •We identified the mechanism of the novel favorable effects of CTLA-4lg. -- Abstract: It has been established that obesity alters the metabolic and endocrine function of adipose tissue and, together with accumulation of adipose tissue macrophages, contributes to insulin resistance. Although numerous studies have reported that shifting the polarization of macrophages from M1 to M2 can alleviate adipose tissue inflammation, manipulation of macrophage polarization has not been considered as a specific therapy. Here, we determined whether cytotoxic T-lymphocyte-associated antigen-4IgG1 (CTLA-4Ig) can ameliorate insulin resistance by induction of macrophages from proinflammatory M1 to anti-inflammatory M2 polarization in the adipose tissues of high fat diet-induced insulin-resistant mice. CTLA4-Ig treatment prevented insulin resistance by changing gene expression to M2 polarization, which increased the levels of arginase 1. Furthermore, flow cytometric analysis confirmed the alteration of polarization from CD11c (M1)- to CD206 (M2)-positive cells. Concomitantly, CTLA-4Ig treatment resulted in weight reductions of epididymal and subcutaneous adipose tissues, which may be closely related to overexpression of apoptosis inhibitors in macrophages. Moreover, proinflammatory cytokine and chemokine levels decreased significantly. In contrast, CCAAT enhancer binding protein α, peroxisome proliferator-activated receptor γ, and adiponectin expression increased significantly in subcutaneous adipose tissue. This novel mechanism of CTLA-4lg immunotherapy may lead to an ideal anti-obesity/inflammation/insulin resistance agent

CTLA-4Ig immunotherapy of obesity-induced insulin resistance by manipulation of macrophage polarization in adipose tissues

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Fujii, Masakazu, E-mail: masakazu731079@yahoo.co.jp [Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Inoguchi, Toyoshi, E-mail: toyoshi@intmed3.med.kyushu-u.ac.jp [Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Innovation Center for Medical Redox Navigation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Batchuluun, Battsetseg, E-mail: battsetseg.batchuluun@gmail.com [Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Sugiyama, Naonobu, E-mail: nao1@intmed1.med.kyushu-u.ac.jp [Department of Medicine and Biosystemic Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Kobayashi, Kunihisa, E-mail: nihisak@fukuoka-u.ac.jp [Department of Endocrinology and Diabetes Mellitus, Fukuoka University Chikushi Hospital, 1-1-1 Zokumyoin, Chikushino, Fukuoka 818-8502 (Japan); Sonoda, Noriyuki, E-mail: noriyuki@intmed3.med.kyushu-u.ac.jp [Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Innovation Center for Medical Redox Navigation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Takayanagi, Ryoichi, E-mail: takayana@intmed3.med.kyushu-u.ac.jp [Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

2013-08-16

Highlights: •CTLA-4Ig completely alleviates HFD-induced insulin resistance. •CTLA-4Ig reduces epididymal and subcutaneous fat tissue weight and adipocyte size. •CTLA-4Ig alters ATM polarization from inflammatory M1 to anti-inflammatory M2. •CTLA-4Ig may lead to a novel anti-obesity/inflammation/insulin resistance agent. •We identified the mechanism of the novel favorable effects of CTLA-4lg. -- Abstract: It has been established that obesity alters the metabolic and endocrine function of adipose tissue and, together with accumulation of adipose tissue macrophages, contributes to insulin resistance. Although numerous studies have reported that shifting the polarization of macrophages from M1 to M2 can alleviate adipose tissue inflammation, manipulation of macrophage polarization has not been considered as a specific therapy. Here, we determined whether cytotoxic T-lymphocyte-associated antigen-4IgG1 (CTLA-4Ig) can ameliorate insulin resistance by induction of macrophages from proinflammatory M1 to anti-inflammatory M2 polarization in the adipose tissues of high fat diet-induced insulin-resistant mice. CTLA4-Ig treatment prevented insulin resistance by changing gene expression to M2 polarization, which increased the levels of arginase 1. Furthermore, flow cytometric analysis confirmed the alteration of polarization from CD11c (M1)- to CD206 (M2)-positive cells. Concomitantly, CTLA-4Ig treatment resulted in weight reductions of epididymal and subcutaneous adipose tissues, which may be closely related to overexpression of apoptosis inhibitors in macrophages. Moreover, proinflammatory cytokine and chemokine levels decreased significantly. In contrast, CCAAT enhancer binding protein α, peroxisome proliferator-activated receptor γ, and adiponectin expression increased significantly in subcutaneous adipose tissue. This novel mechanism of CTLA-4lg immunotherapy may lead to an ideal anti-obesity/inflammation/insulin resistance agent.

Effect of combustion condition on cytotoxic and inflammatory activity of residential wood combustion particles

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Jalava, Pasi I.; Salonen, Raimo O.; Nuutinen, Kati; Pennanen, Arto S.; Happo, Mikko S.; Tissari, Jarkko; Frey, Anna; Hillamo, Risto; Jokiniemi, Jorma; Hirvonen, Maija-Riitta

2010-05-01

Residential heating is an important local source of fine particles and may cause significant exposure and health effects in populations. We investigated the cytotoxic and inflammatory activity of particulate emissions from normal (NC) and smouldering (SC) combustion in one masonry heater. The PM 1-0.2 and PM 0.2 samples were collected from the dilution tunnel with a high-volume cascade impactor (HVCI). Mouse RAW 264.7 macrophages were exposed to the PM-samples for 24 h. Inflammatory mediators, (IL-6, TNFα and MIP-2), and cytotoxicity (MTT-test), were measured. Furthermore, apoptosis and cell cycle of macrophages were analyzed. The HVCI particulate samples were characterized for ions, elements and PAH compounds. Assays of elemental and organic carbon were conducted from parallel low volume samples. All the samples displayed mostly dose-dependent inflammatory and cytotoxic activity. SC samples were more potent than NC samples at inducing cytotoxicity and MIP-2 production, while the order of potency was reversed in TNFα production. SC-PM 1-0.2 sample was a significantly more potent inducer of apoptosis than the respective NC sample. After adjustment for the relative toxicity with emission factor (mg MJ -1), the SC-PM emissions had clearly higher inflammatory and cytotoxic potential than the NC-PM emissions. Thus, operational practice in batch burning of wood and the resultant combustion condition clearly affect the toxic potential of particulate emissions.

Salmonella Typhimurium induces SPI-1 and SPI-2 regulated and strain dependent downregulation of MHC II expression on porcine alveolar macrophages

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Van Parys Alexander

2012-06-01

Full Text Available Abstract Foodborne salmonellosis is one of the most important bacterial zoonotic diseases worldwide. Salmonella Typhimurium is the serovar most frequently isolated from persistently infected slaughter pigs in Europe. Circumvention of the host’s immune system by Salmonella might contribute to persistent infection of pigs. In the present study, we found that Salmonella Typhimurium strain 112910a specifically downregulated MHC II, but not MHC I, expression on porcine alveolar macrophages in a Salmonella pathogenicity island (SPI-1 and SPI-2 dependent way. Salmonella induced downregulation of MHC II expression and intracellular proliferation of Salmonella in macrophages were significantly impaired after opsonization with Salmonella specific antibodies prior to inoculation. Furthermore, the capacity to downregulate MHC II expression on macrophages differed significantly among Salmonella strains, independently of strain specific differences in invasion capacity, Salmonella induced cytotoxicity and altered macrophage activation status. The fact that strain specific differences in MHC II downregulation did not correlate with the extent of in vitro SPI-1 or SPI-2 gene expression indicates that other factors are involved in MHC II downregulation as well. Since Salmonella strain dependent interference with the pig’s immune response through downregulation of MHC II expression might indicate that certain Salmonella strains are more likely to escape serological detection, our findings are of major interest for Salmonella monitoring programs primarily based on serology.

IgM-mediated opsonization and cytotoxicity in the shark.

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McKinney, E C; Flajnik, M F

1997-02-01

Two types of cytotoxic reactions have been observed using cells from the nurse shark: spontaneous cytotoxicity mediated by cells of the macrophage lineage and antibody-dependent killing carried out by a different effector cell population. Previous data showed that removal of phagocytic cells using iron particles abolished macrophage-mediated killing, but not antibody-dependent reactions. The current study used single cell assays and showed that the effector of antibody-driven reactions was the neutrophil. Surprisingly, the mechanism of killing was shown to be phagocytosis mediated by both 7S and 19S immunoglobulin M (IgM). Reactions proceeded with as little as 0.01 microg of purified 19S or 7S IgM and were complete within 4-6 h. In contrast, purified immunoglobulin did not adsorb to macrophages and had no effect on target cell binding or cytotoxicity. Pretreatment of cells with cytochalasin D abolished the phagocytic reaction, but not spontaneous cytotoxicity. These data show that antibody-mediated killing results from opsonization and phagocytosis; the mechanism of macrophage killing is currently unknown. In addition, these data show that the shark neutrophil, not the macrophage lineage, carries a receptor for Fc mu.

Oxidized LDL Induces Alternative Macrophage Phenotype through Activation of CD36 and PAFR

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Francisco J. Rios

2013-01-01

Full Text Available OxLDL is recognized by macrophage scavenger receptors, including CD36; we have recently found that Platelet-Activating Factor Receptor (PAFR is also involved. Since PAFR in macrophages is associated with suppressor function, we examined the effect of oxLDL on macrophage phenotype. It was found that the presence of oxLDL during macrophage differentiation induced high mRNA levels to IL-10, mannose receptor, PPARγ and arginase-1 and low levels of IL-12 and iNOS. When human THP-1 macrophages were pre-treated with oxLDL then stimulated with LPS, the production of IL-10 and TGF-β significantly increased, whereas that of IL-6 and IL-8 decreased. In murine TG-elicited macrophages, this protocol significantly reduced NO, iNOS and COX2 expression. Thus, oxLDL induced macrophage differentiation and activation towards the alternatively activated M2-phenotype. In murine macrophages, oxLDL induced TGF-β, arginase-1 and IL-10 mRNA expression, which were significantly reduced by pre-treatment with PAFR antagonists (WEB and CV or with antibodies to CD36. The mRNA expression of IL-12, RANTES and CXCL2 were not affected. We showed that this profile of macrophage activation is dependent on the engagement of both CD36 and PAFR. We conclude that oxLDL induces alternative macrophage activation by mechanisms involving CD36 and PAFR.

Human Induced Pluripotent Stem Cell-Derived Macrophages Share Ontogeny with MYB-Independent Tissue-Resident Macrophages

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Julian Buchrieser

2017-02-01

Full Text Available Tissue-resident macrophages, such as microglia, Kupffer cells, and Langerhans cells, derive from Myb-independent yolk sac (YS progenitors generated before the emergence of hematopoietic stem cells (HSCs. Myb-independent YS-derived resident macrophages self-renew locally, independently of circulating monocytes and HSCs. In contrast, adult blood monocytes, as well as infiltrating, gut, and dermal macrophages, derive from Myb-dependent HSCs. These findings are derived from the mouse, using gene knockouts and lineage tracing, but their applicability to human development has not been formally demonstrated. Here, we use human induced pluripotent stem cells (iPSCs as a tool to model human hematopoietic development. By using a CRISPR-Cas9 knockout strategy, we show that human iPSC-derived monocytes/macrophages develop in an MYB-independent, RUNX1-, and SPI1 (PU.1-dependent fashion. This result makes human iPSC-derived macrophages developmentally related to and a good model for MYB-independent tissue-resident macrophages, such as alveolar and kidney macrophages, microglia, Kupffer cells, and Langerhans cells.

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. Copyright © 2013 Elsevier Ltd. All rights reserved.

Corn silk induced cyclooxygenase-2 in murine macrophages.

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Kim, Kyung A; Shin, Hyun-Hee; Choi, Sang Kyu; Choi, Hye-Seon

2005-10-01

Stimulation of murine macrophages with corn silk induced cyclooxygenase (COX)-2 with secretion of PGE2. Expression of COX-2 was inhibited by pyrolidine dithiocarbamate (PDTC), and increased DNA binding by nuclear factor kappa B (NF-kappaB), indicating that COX-2 induction proceeds also via the NF-kappaB signaling pathway. A specific inhibitor of COX-2 decreased the expression level of inducible nitric oxide synthase (iNOS) stimulated by corn silk. PGE2 elevated the expression level of iNOS, probably via EP2 and EP4 receptors on the surface of the macrophages.

Uremic Toxins Enhance Statin-Induced Cytotoxicity in Differentiated Human Rhabdomyosarcoma Cells

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Hitoshi Uchiyama

2014-09-01

Full Text Available The risk of myopathy and rhabdomyolysis is considerably increased in statin users with end-stage renal failure (ESRF. Uremic toxins, which accumulate in patients with ESRF, exert cytotoxic effects that are mediated by various mechanisms. Therefore, accumulation of uremic toxins might increase statin-induced cytotoxicity. The purpose of this study was to determine the effect of four uremic toxins—hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, indole-3-acetic acid, and 3-indoxyl sulfate—on statin-induced myopathy. Differentiated rhabdomyosarcoma cells were pre-treated with the uremic toxins for seven days, and then the cells were treated with pravastatin or simvastatin. Cell viability and apoptosis were assessed by viability assays and flow cytometry. Pre-treatment with uremic toxins increased statin- but not cisplatin-induced cytotoxicity (p < 0.05 vs. untreated. In addition, the pre-treatment increased statin-induced apoptosis, which is one of the cytotoxic factors (p < 0.05 vs. untreated. However, mevalonate, farnesol, and geranylgeraniol reversed the effects of uremic toxins and lowered statin-induced cytotoxicity (p < 0.05 vs. untreated. These results demonstrate that uremic toxins enhance statin-induced apoptosis and cytotoxicity. The mechanism underlying this effect might be associated with small G-protein geranylgeranylation. In conclusion, the increased severity of statin-induced rhabdomyolysis in patients with ESRF is likely due to the accumulation of uremic toxins.

The role of autophagy in THP-1 macrophages resistance to HIV- vpr-induced apoptosis

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Zhou, Hua-ying, E-mail: zhouhuaying_2004@126.com; Zheng, Yu-huang; He, Yan; Chen, Zi; He, Bo

2017-02-01

Macrophages are resistant to cell death and are one of HIV reservoirs. HIV viral protein Vpr has the potential to promote infection of and survival of macrophages, which could be a highly significant factor in the development and/or maintenance of macrophage viral reservoirs. However, the impact of vpr on macrophages resistance to apoptosis is yet to be comprehended. Autophagy is a cell survival mechanism under stress state. In this study, we investigated whether autophagy is involved in macrophages resistant to vpr-induced apoptosis. Using the THP1 macrophages, we studied the interconnection between macrophages resistance to apoptosis and autophagy. We found that vpr is able to trigger autophagy in transfected THP-1 macrophages confirmed by electron microscopy (EM) and western blot analysis, and inhibition of autophagy with 3MA increased vpr-induced apoptosis. The results indicate that autophagy may be responsible for maintenance of macrophage HIV reservoirs. - Highlights: • HIV Vpr is able to trigger autophagy in transfected THP-1 macrophages. • Autophagy inhibition increases vpr-transfected THP1-macrophages apoptosis. • Autophagy is involved in THP-1 macrophages resistant to vpr-induced apoptosis.

Kaempferol impedes IL-32-induced monocyte-macrophage differentiation.

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Nam, Sun-Young; Jeong, Hyun-Ja; Kim, Hyung-Min

2017-08-25

Kaempferol possesses a wide range of therapeutic properties, including antioxidant, anti-inflammatory, and anticancer properties. The present study sought to evaluate the effects and possible pharmacological mechanisms of kaempferol on interleukin (IL)-32-induced monocyte-macrophage differentiation. In this study, we performed flow cytometry assay, immunocytochemical staining, quantitative real-time PCR, enzyme-linked immuno sorbent assay, caspase-1 assay, and Western blotting to observe the effects and underlying mechanisms of kaempferol using the human monocyte cell line THP-1. The flow cytometry, immunocytochemical staining, and real-time PCR results show that kaempferol attenuated IL-32-induced monocyte differentiation to product macrophage-like cells. Kaempferol decreased the production and mRNA expression of pro-inflammatory cytokines, in this case thymic stromal lymphopoietin (TSLP), IL-1β, tumor necrosis factor (TNF)-α, and IL-8. Furthermore, kaempferol inhibited the IL-32-induced activation of p38 and nuclear factor-κB in a dose-dependent manner in THP-1 cells. Kaempferol also ameliorated the lipopolysaccharide-induced production of the inflammatory mediators TSLP, IL-1β, TNF-α, IL-8, and nitric oxide of macrophage-like cells differentiated by IL-32. In brief, our findings may provide new mechanistic insights into the anti-inflammatory effects of kaempferol. Copyright © 2017 Elsevier B.V. All rights reserved.

Characteristics of adipose tissue macrophages and macrophage-derived insulin-like growth factor-1 in virus-induced obesity.

Science.gov (United States)

Park, S; Park, H-L; Lee, S-Y; Nam, J-H

2016-03-01

Various pathogens are implicated in the induction of obesity. Previous studies have confirmed that human adenovirus 36 (Ad36) is associated with increased adiposity, improved glycemic control and induction of inflammation. The Ad36-induced inflammation is reflected in the infiltration of macrophages into adipose tissue. However, the characteristics and role of adipose tissue macrophages (ATMs) and macrophage-secreted factors in virus-induced obesity (VIO) are unclear. Although insulin-like growth factor-1 (IGF-1) is involved in obesity metabolism, the contribution of IGF secreted by macrophages in VIO has not been studied. Four-week-old male mice were studied 1 week and 12 weeks after Ad36 infection for determining the characteristics of ATMs in VIO and diet-induced obesity (DIO). In addition, macrophage-specific IGF-1-deficient (MIKO) mice were used to study the involvement of IGF-1 in VIO. In the early stage of VIO (1 week after Ad36 infection), the M1 ATM sub-population increased, which increased the M1/M2 ratio, whereas DIO did not cause this change. In the late stage of VIO (12 weeks after Ad36 infection), the M1/M2 ratio did not change because the M1 and M2 ATM sub-populations increased to a similar extent, despite an increase in adiposity. By contrast, DIO increased the M1/M2 ratio. In addition, VIO in wild-type mice upregulated angiogenesis in adipose tissue and improved glycemic control. However, MIKO mice showed no increase in adiposity, angiogenesis, infiltration of macrophages into adipose tissue, or improvement in glycemic control after Ad36 infection. These data suggest that IGF-1 secreted by macrophages may contribute to hyperplasia and hypertrophy in adipose tissue by increasing angiogenesis, which helps to maintain the 'adipose tissue robustness'.

Conditioned medium from alternatively activated macrophages induce mesangial cell apoptosis via the effect of Fas

International Nuclear Information System (INIS)

Huang, Yuan; Luo, Fangjun; Li, Hui; Jiang, Tao; Zhang, Nong

2013-01-01

During inflammation in the glomerulus, the proliferation of myofiroblast-like mesangial cells is commonly associated with the pathological process. Macrophages play an important role in regulating the growth of resident mesangial cells in the glomeruli. Alternatively activated macrophage (M2 macrophage) is a subset of macrophages induced by IL-13/IL-4, which is shown to play a repair role in glomerulonephritis. Prompted by studies of development, we performed bone marrow derived macrophage and rat mesangial cell co-culture study. Conditioned medium from IL-4 primed M2 macrophages induced rat mesangial cell apoptosis. The pro-apoptotic effect of M2 macrophages was demonstrated by condensed nuclei stained with Hoechst 33258, increased apoptosis rates by flow cytometry analysis and enhanced caspase-3 activation by western blot. Fas protein was up-regulated in rat mesangial cells, and its neutralizing antibody ZB4 partly inhibited M2 macrophage-induced apoptosis. The up-regulated arginase-1 expression in M2 macrophage also contributed to this apoptotic effect. These results indicated that the process of apoptosis triggered by conditioned medium from M2 macrophages, at least is partly conducted through Fas in rat mesangial cells. Our findings provide compelling evidence that M2 macrophages control the growth of mesangial cells in renal inflammatory conditions. - Highlights: • Conditioned-medium from M2 macrophages induces rat mesangial cell (MsC) apoptosis. • M2 macrophage conditioned medium exerts its pro-apoptotic effects via Fas ligand. • Arginase-1 activity in M2 macrophages plays a role in inducing apoptosis in rat MsC

Conditioned medium from alternatively activated macrophages induce mesangial cell apoptosis via the effect of Fas

Energy Technology Data Exchange (ETDEWEB)

Huang, Yuan; Luo, Fangjun; Li, Hui; Jiang, Tao; Zhang, Nong, E-mail: nzhang@fudan.edu.cn

2013-11-15

During inflammation in the glomerulus, the proliferation of myofiroblast-like mesangial cells is commonly associated with the pathological process. Macrophages play an important role in regulating the growth of resident mesangial cells in the glomeruli. Alternatively activated macrophage (M2 macrophage) is a subset of macrophages induced by IL-13/IL-4, which is shown to play a repair role in glomerulonephritis. Prompted by studies of development, we performed bone marrow derived macrophage and rat mesangial cell co-culture study. Conditioned medium from IL-4 primed M2 macrophages induced rat mesangial cell apoptosis. The pro-apoptotic effect of M2 macrophages was demonstrated by condensed nuclei stained with Hoechst 33258, increased apoptosis rates by flow cytometry analysis and enhanced caspase-3 activation by western blot. Fas protein was up-regulated in rat mesangial cells, and its neutralizing antibody ZB4 partly inhibited M2 macrophage-induced apoptosis. The up-regulated arginase-1 expression in M2 macrophage also contributed to this apoptotic effect. These results indicated that the process of apoptosis triggered by conditioned medium from M2 macrophages, at least is partly conducted through Fas in rat mesangial cells. Our findings provide compelling evidence that M2 macrophages control the growth of mesangial cells in renal inflammatory conditions. - Highlights: • Conditioned-medium from M2 macrophages induces rat mesangial cell (MsC) apoptosis. • M2 macrophage conditioned medium exerts its pro-apoptotic effects via Fas ligand. • Arginase-1 activity in M2 macrophages plays a role in inducing apoptosis in rat MsC.

Transferrin-derived synthetic peptide induces highly conserved pro-inflammatory responses of macrophages.

Science.gov (United States)

Haddad, George; Belosevic, Miodrag

2009-02-01

We examined the induction of macrophage pro-inflammatory responses by transferrin-derived synthetic peptide originally identified following digestion of transferrin from different species (murine, bovine, human N-lobe and goldfish) using elastase. The mass spectrometry analysis of elastase-digested murine transferrin identified a 31 amino acid peptide located in the N2 sub-domain of the transferrin N-lobe, that we named TMAP. TMAP was synthetically produced and shown to induce a number of pro-inflammatory genes by quantitative PCR. TMAP induced chemotaxis, a potent nitric oxide response, and TNF-alpha secretion in different macrophage populations; P338D1 macrophage-like cells, mouse peritoneal macrophages, mouse bone marrow-derived macrophages (BMDM) and goldfish macrophages. The treatment of BMDM cultures with TMAP stimulated the production of nine cytokines and chemokines (IL-6, MCP-5, MIP-1 alpha, MIP-1 gamma, MIP-2, GCSF, KC, VEGF, and RANTES) that was measured using cytokine antibody array and confirmed by Western blot. Our results indicate that transferrin-derived peptide, TMAP, is an immunomodulating molecule capable of inducing pro-inflammatory responses in lower and higher vertebrates.

Conditional Macrophage Depletion Increases Inflammation and Does Not Inhibit the Development of Osteoarthritis in Obese Macrophage Fas-Induced Apoptosis-Transgenic Mice.

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Wu, Chia-Lung; McNeill, Jenna; Goon, Kelsey; Little, Dianne; Kimmerling, Kelly; Huebner, Janet; Kraus, Virginia; Guilak, Farshid

2017-09-01

To investigate whether short-term, systemic depletion of macrophages can mitigate osteoarthritis (OA) following injury in the setting of obesity. CSF-1R-GFP+ macrophage Fas-induced apoptosis (MaFIA)-transgenic mice that allow conditional depletion of macrophages were placed on a high-fat diet and underwent surgery to induce knee OA. A small molecule (AP20187) was administrated to deplete macrophages in MaFIA mice. The effects of macrophage depletion on acute joint inflammation, OA severity, and arthritic bone changes were evaluated using histology and micro-computed tomography. Immunohistochemical analysis was performed to identify various immune cells. The levels of serum and synovial fluid cytokines were also measured. Macrophage-depleted mice had significantly fewer M1 and M2 macrophages in the surgically operated joints relative to controls and exhibited decreased osteophyte formation immediately following depletion. Surprisingly, macrophage depletion did not attenuate the severity of OA in obese mice; instead, it induced systemic inflammation and led to a massive infiltration of CD3+ T cells and particularly neutrophils, but not B cells, into the injured joints. Macrophage-depleted mice also demonstrated a markedly increased number of proinflammatory cytokines including granulocyte colony-stimulating factor, interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor in both serum and joint synovial fluid, although the mice showed a trend toward decreased levels of insulin and leptin in serum after macrophage depletion. Our findings indicate that macrophages are vital for modulating homeostasis of immune cells in the setting of obesity and suggest that more targeted approaches of depleting specific macrophage subtypes may be necessary to mitigate inflammation and OA in the setting of obesity. © 2017, American College of Rheumatology.

Decreased inducibility of TNF expression in lipid-loaded macrophages

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Kallin Bengt

2002-10-01

Full Text Available Abstract Background Inflammation and immune responses are considered to be very important in the pathogenesis of atherosclerosis. Lipid accumulation in macrophages of the arterial intima is a characteristic feature of atherosclerosis which can influence the inflammatory potential of macrophages. We studied the effects of lipid loading on the regulation of TNF expression in human monocyte-derived macrophages. Results In macrophages incubated with acetylated low density lipoprotein (ac-LDL for 2 days, mRNA expression of TNF in cells stimulated with TNF decreased by 75%. In cell cultures stimulated over night with IL-1β, lipid loading decreased secretion of TNF into culture medium by 48%. These results suggest that lipid accumulation in macrophages makes them less responsive to inflammatory stimuli. Decreased basal activity and inducibility of transcription factor AP-1 was observed in lipid-loaded cells, suggesting a mechanism for the suppression of cytokine expression. NF-κB binding activity and inducibility were only marginally affected by ac-LDL. LDL and ac-LDL did not activate PPARγ. In contrast, oxidized LDL stimulated AP-1 and PPARγ but inhibited NF-κB, indicating that the effects of lipid loading with ac-LDL were not due to oxidation of lipids. Conclusions Accumulation of lipid, mainly cholesterol, results in down-regulation of TNF expression in macrophages. Since monocytes are known to be activated by cell adhesion, these results suggest that foam cells in atherosclerotic plaques may contribute less potently to an inflammatory reaction than newly arrived monocytes/macrophages.

The role of NF-κB signaling pathway in polyhexamethylene guanidine phosphate induced inflammatory response in mouse macrophage RAW264.7 cells.

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Kim, Ha Ryong; Shin, Da Young; Chung, Kyu Hyuck

2015-03-04

Polyhexamethylene guanidine (PHMG) phosphate is a competitive disinfectant with strong antibacterial activity. However, epidemiologists revealed that inhaled PHMG-phosphate may increase the risk of pulmonary fibrosis associated with inflammation, resulting in the deaths of many people, including infants and pregnant women. In addition, in vitro and in vivo studies reported the inflammatory effects of PHMG-phosphate. Therefore, the aim of the present study was to clarify the inflammatory effects and its mechanism induced by PHMG-phosphate in murine RAW264.7 macrophages. Cell viability, inflammatory cytokine secretion, nuclear factor kappa B (NF-κB) activation, and reactive oxygen species (ROS) generation were investigated in macrophages exposed to PHMG-phosphate. PHMG-phosphate induced dose-dependent cytotoxicity, with LC50 values of 11.15-0.99mg/ml at 6 and 24h, respectively. PHMG-phosphate induced pro-inflammatory cytokines including IL-1β, IL-6, and IL-8. In particular, IL-8 expression was completely inhibited by the NF-κB inhibitor BAY11-7082. In addition, PHMG-phosphate decreased IκB-α protein expression and increased NF-κB-mediated luciferase activity, which was diminished by N-acetyl-l-cystein. However, abundant amounts of ROS were generated in the presence of PHMG-phosphate at high concentrations with a cytotoxic effect. Our results demonstrated that PHMG-phosphate triggered the activation of NF-κB signaling pathway by modulating the degradation of IκB-α. Furthermore, the NF-κB signaling pathway plays a critical role in the inflammatory responses induced by PHMG-phosphate. We assumed that ROS generated by PHMG-phosphate were associated with inflammatory responses as secondary mechanism. In conclusion, we suggest that PHMG-phosphate induces inflammatory responses via NF-κB signaling pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

TRIF promotes angiotensin II-induced cross-talk between fibroblasts and macrophages in atrial fibrosis

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Chen, Xiao-Qing; Zhang, Dao-Liang [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China); Zhang, Ming-Jian; Guo, Meng; Zhan, Yang-Yang; Liu, Fang [National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai (China); Jiang, Wei-Feng; Zhou, Li [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China); Zhao, Liang, E-mail: zhaol_zg@163.com [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China); Wang, Quan-Xing, E-mail: wqxejd@126.com [National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai (China); Liu, Xu, E-mail: liuxu_xk@163.com [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China)

2015-08-14

Aims: Atrial fibroblasts and macrophages have long been thought to participate in atrial fibrillation (AF). However, which specific mediator may regulate the interaction between them remains unclear. Methods and results: We provided the evidence for the involvement of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF), an important inflammation-related molecule, in the pathophysiology of AF. Patients with AF showed higher levels of angiotensin II (AngII) and TRIF expression and larger number of macrophages infiltration in left atria appendage than individuals with sinus rhythm (SR). In the cell study, AngII induced chemokines expressions in mouse atrial fibroblasts and AngII-stimulated atrial fibroblasts induced the chemotaxis of macrophages, which were reduced by losartan and TRIF siRNA. Meanwhile, AngII-stimulated atrial fibroblasts proliferation was enhanced by macrophages. Conclusions: Our data demonstrated that TRIF may be a crucial factor promoting the interaction between atrial fibroblasts and macrophages, leading to atrial fibrosis. - Highlights: • Compared with SR, AF showed higher TRIF expression in left atrial appendage. • TRIF siRNA reversed macrophage chemotaxis induced by AngII-treated fibroblast. • TRIF siRNA reversed chemokines expressions induced by AngII in fibroblast. • AngII-stimulated atrial fibroblast proliferation was enhanced by macrophage.

TRIF promotes angiotensin II-induced cross-talk between fibroblasts and macrophages in atrial fibrosis

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Chen, Xiao-Qing; Zhang, Dao-Liang; Zhang, Ming-Jian; Guo, Meng; Zhan, Yang-Yang; Liu, Fang; Jiang, Wei-Feng; Zhou, Li; Zhao, Liang; Wang, Quan-Xing; Liu, Xu

2015-01-01

Aims: Atrial fibroblasts and macrophages have long been thought to participate in atrial fibrillation (AF). However, which specific mediator may regulate the interaction between them remains unclear. Methods and results: We provided the evidence for the involvement of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF), an important inflammation-related molecule, in the pathophysiology of AF. Patients with AF showed higher levels of angiotensin II (AngII) and TRIF expression and larger number of macrophages infiltration in left atria appendage than individuals with sinus rhythm (SR). In the cell study, AngII induced chemokines expressions in mouse atrial fibroblasts and AngII-stimulated atrial fibroblasts induced the chemotaxis of macrophages, which were reduced by losartan and TRIF siRNA. Meanwhile, AngII-stimulated atrial fibroblasts proliferation was enhanced by macrophages. Conclusions: Our data demonstrated that TRIF may be a crucial factor promoting the interaction between atrial fibroblasts and macrophages, leading to atrial fibrosis. - Highlights: • Compared with SR, AF showed higher TRIF expression in left atrial appendage. • TRIF siRNA reversed macrophage chemotaxis induced by AngII-treated fibroblast. • TRIF siRNA reversed chemokines expressions induced by AngII in fibroblast. • AngII-stimulated atrial fibroblast proliferation was enhanced by macrophage

Characterization of the growth-inhibitory and apoptosis-inducing activities of a triterpene saponin, securioside B against BAC1.2F5 macrophages

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Satoru Yui

2003-01-01

Full Text Available Background: Since the growth state of macrophages in local pathological sites is considered a factor that regulates the processes of many disease, such as tumors, inflammation, and atherosclerosis, the substances that regulate macrophage growth or survival may be useful for disease control. We previously reported that securiosides A and B, novel triterpene saponins, exerted macrophage-oriented cytotoxicity in the presence of a L-cell-conditioned medium containing macrophage colony-stimulating factor (M-CSF, while the compounds did not exhibit an effect on macrophages in the absence of the growth-stimulating factors.

Yersinia pestis and host macrophages: immunodeficiency of mouse macrophages induced by YscW.

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Bi, Yujing; Du, Zongmin; Han, Yanping; Guo, Zhaobiao; Tan, Yafang; Zhu, Ziwen; Yang, Ruifu

2009-09-01

The virulence of the pathogenic Yersinia species depends on a plasmid-encoded type III secretion system (T3SS) that transfers six Yersinia outer protein (Yop) effector proteins into the cytoplasm of eukaryotic cells, leading to disruption of host defence mechanisms. It is shown in this study that Yersinia pestis YscW, a protein of the T3SS injectisome, contributes to the induction of a deficiency in phagocytosis in host macrophages and a reduction in their antigen-presenting capacity. A Y. pestis strain lacking yscW had no effect on uptake by host macrophages. In mice infected with wild-type Y. pestis, the yscW mutant or a complement strain, immunodeficiency was observed in host macrophages compared with those from uninfected mice. However, the phagocytosis and antigen presenting capacities of macrophages infected by yscW mutant strain both in vivo and in vitro were significantly higher than those by wild type strain. Consistent with this finding, when YscW was expressed in the RAW264.7 macrophage cell line, phagocytosis and antigen-presenting capacities were significantly lower than those of the control groups. These results indicate that Y. pestis YscW may directly induce immunodeficiency in murine macrophages by crippling their phagocytosis and antigen-presenting capacities. These data provide evidences to Y. pestis pathogenesis that some proteins in T3SS injectisome, such as YscW protein, might play independent roles in disrupting host defense apart from their known functions.

Networked T cell death following macrophage infection by Mycobacterium tuberculosis.

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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

Mycobacterium avium MAV2052 protein induces apoptosis in murine macrophage cells through Toll-like receptor 4.

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Lee, Kang-In; Choi, Han-Gyu; Son, Yeo-Jin; Whang, Jake; Kim, Kwangwook; Jeon, Heat Sal; Park, Hye-Soo; Back, Yong Woo; Choi, Seunga; Kim, Seong-Woo; Choi, Chul Hee; Kim, Hwa-Jung

2016-04-01

Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨm), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨm loss, and apoptosis were all significantly reduced in TLR4(-/-) macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.

Cells of the J774 macrophage cell line are primed for antibody-dependent cell-mediated cytotoxicity following exposure to γ-irradiation

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Duerst, R.; Werberig, K.

1991-01-01

Activation of macrophages (M phi) for host defense against tumor cells follows a sequence of priming events followed by an initiating stimulus that results in production and release of cytotoxic molecules that mediate target cell killing. The authors have developed a model to study specific macrophage cytotoxicity in vitro utilizing a cultured murine M phi cell line, J774. Specific cytotoxicity of cultured human gastrointestinal tumor cells is achieved in the presence of murine IgG2a monoclonal antibody (mAb) 17-1-A. The ability of these cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) is greatly enhanced following gamma-irradiation. ADCC can be demonstrated at mAb 17-1-A concentrations greater than or equal to 1 microgram/ml and effector/target cell ratios greater than or equal to 2. Exposure to doses greater than or equal to 10 Gy of gamma-irradiation increases ADCC threefold. Varying the duration from J774 M phi exposure to γ-irradiation until addition of antibody-coated target cells showed that the primed state for ADCC is stable for at least 8 days but approximately 24 hr is required for complete development of the primed state. mAb-dependent target cell death begins 8 hr after addition of mAb and labeled target cells to primed effector cells and is complete by 24 hr. Incubation of unirradiated J774 M phi effector cells with recombinant murine interferon-γ (rmIFN-γ) also results in enhanced ADCC, but the extent of target cell killing achieved is less than that following priming by γ-irradiation. Concomitant priming of γ-irradiated J774 M phi with rmIFN-γ increases the extent of ADCC. Further study of irradiated J774 cells may elucidate the molecular pathways utilized by M phi for achieving and maintaining the primed state for ADCC

A Study on Genetic Analysis and Extract Cytotoxicity of Scolopendra subspinipes multilans L. Koch

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Kim Sung-Nam

2006-06-01

Full Text Available Objective : The purpose of this study is to investigate nucleotide sequence and extract cytotoxicity of Scolopendrae corpus. The nature and taste of Scolopendrae corpus is hot, Warm and toxic, and the effect of this is dispelling wind, anti-spasmodic action and detoxication so it has been used for C.V.A, facial palsy, sensory disorder at extremities, wounds and arthritis. Methods : Scolopendrae corpus were collected by locality on the market. They were morphologically classified. Their nucleotide sequence was investigated and compared among them. In addition, the water-alcohol extract cytotoxicity of them was studied by MTT-based cytotoxicity assay. Results : It was shown that the each Scolopendrae corpus by locality is almost identical at genetic result and is identified as Scolopendra subspinipes mutilans L. Koch. Nucleotide sequence of Scolopendra subspinipes mutilans L. Koch in this study will help to discriminate other species of Scolopendrae corpus. The water-alcohol extract of Scolopendra subspinipes mutilans L. Koch did not induce cytotoxicity on Hep G2, L929 cell and peritoneal macrophages. Besides, it did not influence nitrite production of peritoneal macrophages. These results can be used as basic data for genetic discrimination with another species of scolopendrae corpus.

Polyelectrolyte Complex Optimization for Macrophage Delivery of Redox Enzyme Nanoparticles

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Zhao, Yuling; Haney, Matthew J.; Klyachko, Natalia L.; Li, Shu; Booth, Stephanie L.; Higginbotham, Sheila M.; Jones, Jocelyn; Zimmerman, Matthew C.; Mosley, R. Lee; Kabanov, Alexander V.; Gendelman, Howard E.; Batrakova, Elena V.

2011-01-01

Background We posit that cell-mediated drug delivery can improve transport of therapeutic enzymes to the brain and decrease inflammation and neurodegeneration induced during Parkinson’s disease. Our prior work demonstrated that macrophages loaded with nanoformulated catalase (“nanozyme”) protect the nigrostriatum in a murine model of Parkinson’s disease. Packaging of catalase into block ionomer complex with a synthetic polyelectrolyte block copolymers protects the enzyme degradation in macrophages. Methods We examined relationships between the composition and structure of block ionomer complexes, their physicochemical characteristics, and loadings, release rates, and catalase activity in bone marrow-derived macrophages. Results Formation of block-ionomer complexes resulted in improved aggregation stability. Block ionomer complexes with ε-polylisine, and poly-L-glutamic acid -poly(ethylene glycol) demonstrated the least cytotoxicity and high loading and release rates, however, did not efficiently protect catalase inside macrophages. Conclusion nanozymes with polyethyleneimine- and poly(L-lysine)10-poly(ethylene glycol) provided the best protection of enzymatic activity for cell-mediated drug delivery. PMID:21182416

induced acute cytotoxicity in human cervical epithelial carcinoma cells

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Molecular basis of arsenite (As +3 )-induced acute cytotoxicity in human cervical epithelial carcinoma cells. ... Libyan Journal of Medicine ... Methods: After performing cytotoxic assays on a human epithelial carcinoma cell line, expression analysis was done by quantitative polymerase chain reaction, western blotting, and ...

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

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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.

Macrophage-secreted factors induce adipocyte inflammation and insulin resistance

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Permana, Paska A.; Menge, Christopher; Reaven, Peter D.

2006-01-01

Macrophage infiltration into adipose tissue increases with obesity, a condition associated with low-grade inflammation and insulin resistance. We investigated the direct effects of macrophage-secreted factors on adipocyte inflammation and insulin resistance. 3T3-L1 adipocytes incubated with media conditioned by RAW264.7 macrophages (RAW-CM) showed dramatically increased transcription of several inflammation-related genes, greater nuclear factor kappa B (NF-κB) activity, and enhanced binding of U937 monocytes. All of these effects were prevented by co-incubation with pyrrolidinedithiocarbamate, an NF-κB inhibitor. Adipocytes incubated with RAW-CM also released more non-esterified fatty acids and this increased lipolysis was not suppressed by insulin. In addition, RAW-CM treatment decreased insulin-stimulated glucose uptake in adipocytes. Taken together, these results indicate that macrophage-secreted factors induce inflammatory responses and reduce insulin responsiveness in adipocytes. These effects of macrophage-secreted factors on adipocytes may contribute significantly to the systemic inflammation and insulin resistance associated with obesity

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

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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.

Oxidized low density lipoprotein induced caspase-1 mediated pyroptotic cell death in macrophages: implication in lesion instability?

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Jing Lin

Full Text Available BACKGROUND: Macrophage death in advanced lesion has been confirmed to play an important role in plaque instability. However, the mechanism underlying lesion macrophage death still remains largely unknown. METHODS AND RESULTS: Immunohistochemistry showed that caspase-1 activated in advanced lesion and co-located with macrophages and TUNEL positive reaction. In in-vitro experiments showed that ox-LDL induced caspase-1 activation and this activation was required for ox-LDL induced macrophages lysis, IL-1β and IL-18 production as well as DNA fragmentation. Mechanism experiments showed that CD36 and NLRP3/caspase-1/pathway involved in ox-LDL induced macrophage pyroptosis. CONCLUSION: Our study here identified a novel cell death, pyroptosis in ox-LDL induced human macrophage, which may be implicated in lesion macrophages death and play an important role in lesion instability.

Pegylated silica nanoparticles: cytotoxicity and macrophage uptake

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Glorani, Giulia; Marin, Riccardo; Canton, Patrizia; Pinto, Marcella; Conti, Giamaica; Fracasso, Giulio; Riello, Pietro

2017-08-01

Here, we present a thorough study of pegylated silica nanoparticle (SNP) interaction with different biological environments. The SNPs have a mean diameter of about 40 nm and are coated with polyethylene glycol (PEG) of different molecular weights. The physicochemical characterization of SNPs allowed the confirmation of the binding of PEG chains to the silica surface, the reproducibility of the synthesis and the narrow size-dispersion. In view of clarifying the SNP interaction with biological environments, we first assessed the SNP reactivity after the incubation with two cell lines (macrophages RAW 264.7 and primary human fibroblasts), observing a reduced toxicity of pegylated SNPs compared to the bare ones. Then, we investigated the effect of the protein adsorption on the SNP surface using the model serum protein, bovine serum albumin (BSA). We found that the protein adsorption takes place more heavily on poorly pegylated SNPs, promoting the uptake of the latter by macrophages and leading to an increased mortality of these cells. To better understand this mechanism by means of flow cytometry, the dye Ru(bpy)3Cl2 was incorporated in the SNPs. The overall results highlight the SNP potentialities as a drug delivery system, thanks to the low interactions with the macrophages.

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

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Persidsky Yuri

2011-02-01

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

Mesenchymal stem cells ameliorate rhabdomyolysis-induced acute kidney injury via the activation of M2 macrophages

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2014-01-01

Introduction The mortality of rhabdomyolysis-induced acute kidney injury (AKI) is still high, as there is no effective therapy. It has been shown that bone marrow-derived mesenchymal stem cells (MSCs) can induce M2 macrophages, which mediate MSC protection in other experimental inflammation-related organ injury. This study was designed to investigate the protective effects of macrophage activation in MSC therapy of rhabdomyolysis-induced AKI. Methods MSCs were injected into glycerol-induced rhabdomyolysis mice. Renal injury was evaluated using the serum creatinine, urea nitrogen, renal pathology and acute tubular necrosis score. The distribution of MSCs was detected using two-photon fluorescence confocal imaging. Immunofluorescence of anti-F4/80 and anti-CD206 was performed to determine macrophages and M2 macrophages in the tissues of the kidney, and M2 macrophage infiltration was also evaluated using western blotting analyses. After depletion of macrophages using clodronate liposomes at the phase of kidney repair, renal injury was re-evaluated. RAW 264.7 macrophages were incubated with lipopolysaccharide and co-cultured with MSCs and subsequently visualised using immunofluorescence staining and flow cytometry analysis. Finally, disparate phenotype macrophages, including normal macrophages (M0), lipopolysaccharide-stimulated macrophages (M1), and MSC-co-cultured macrophages (M2), were infused into mice with AKI, which were pre-treated with liposomal clodronate. Results In vivo infusion of MSCs protected AKI mice from renal function impairment and severe tubular injury, which was accompanied by a time-dependent increase in CD206-positive M2 macrophage infiltration. In addition, depleting macrophages with clodronate delayed restoration of AKI. In vitro, macrophages co-cultured with MSCs acquired an anti-inflammatory M2 phenotype, which was characterised by an increased expression of CD206 and the secretory cytokine interleukin (IL)-10. The concentrations of IL-10, IL

The cytotoxic activity of miltefosine against Leishmania and macrophages is associated with dynamic changes in plasma membrane proteins.

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Fernandes, Kelly Souza; de Souza, Paulo Eduardo Narcizo; Dorta, Miriam Leandro; Alonso, Antonio

2017-01-01

In this study, we combined electron paramagnetic resonance (EPR) spectroscopy with an analysis of biophysical cellular parameters to study the mechanisms underlying the in vitro anti-leishmanial activity of miltefosine (MT). A thiol-specific spin label attached to membrane-bound proteins of Leishmania amazonensis and peritoneal macrophages indicated that MT may bind to plasma membrane proteins in large quantities via a detergent-like action and cause structural changes associated with a marked increase in dynamics and exposure to an aqueous environment. EPR spectra of a spin-labeled stearic acid indicated strong interactions between the probe and membrane proteins and a marked increase in the membrane fluidity of MT-treated cells. The cytotoxicity of MT was found to depend on the cell concentration used in the assay. This dependence was described by an equation involving the 50% inhibitory concentrations of MT in the aqueous medium (c w50 ) and the cell membrane (c m50 ) and the membrane-aqueous medium partition coefficient of MT (K). With a c w50 of 8.7μM, macrophages were less sensitive to MT than amastigotes and promastigotes of Leishmania, which had c w50 values of 2.4-3.1μM. The estimated c m50 of MT for Leishmania was 1.8M, which appears sufficient to cause ruptures or formation of pores in the plasma membrane. Additionally, we demonstrated that the changes in the plasma membrane detected by EPR spectroscopy occurred at cytotoxic concentrations of MT, as assessed through in vitro assays. Copyright © 2016 Elsevier B.V. All rights reserved.

Induced-Pluripotent-Stem-Cell-Derived Primitive Macrophages Provide a Platform for Modeling Tissue-Resident Macrophage Differentiation and Function.

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Takata, Kazuyuki; Kozaki, Tatsuya; Lee, Christopher Zhe Wei; Thion, Morgane Sonia; Otsuka, Masayuki; Lim, Shawn; Utami, Kagistia Hana; Fidan, Kerem; Park, Dong Shin; Malleret, Benoit; Chakarov, Svetoslav; See, Peter; Low, Donovan; Low, Gillian; Garcia-Miralles, Marta; Zeng, Ruizhu; Zhang, Jinqiu; Goh, Chi Ching; Gul, Ahmet; Hubert, Sandra; Lee, Bernett; Chen, Jinmiao; Low, Ivy; Shadan, Nurhidaya Binte; Lum, Josephine; Wei, Tay Seok; Mok, Esther; Kawanishi, Shohei; Kitamura, Yoshihisa; Larbi, Anis; Poidinger, Michael; Renia, Laurent; Ng, Lai Guan; Wolf, Yochai; Jung, Steffen; Önder, Tamer; Newell, Evan; Huber, Tara; Ashihara, Eishi; Garel, Sonia; Pouladi, Mahmoud A; Ginhoux, Florent

2017-07-18

Tissue macrophages arise during embryogenesis from yolk-sac (YS) progenitors that give rise to primitive YS macrophages. Until recently, it has been impossible to isolate or derive sufficient numbers of YS-derived macrophages for further study, but data now suggest that induced pluripotent stem cells (iPSCs) can be driven to undergo a process reminiscent of YS-hematopoiesis in vitro. We asked whether iPSC-derived primitive macrophages (iMacs) can terminally differentiate into specialized macrophages with the help of growth factors and organ-specific cues. Co-culturing human or murine iMacs with iPSC-derived neurons promoted differentiation into microglia-like cells in vitro. Furthermore, murine iMacs differentiated in vivo into microglia after injection into the brain and into functional alveolar macrophages after engraftment in the lung. Finally, iPSCs from a patient with familial Mediterranean fever differentiated into iMacs with pro-inflammatory characteristics, mimicking the disease phenotype. Altogether, iMacs constitute a source of tissue-resident macrophage precursors that can be used for biological, pathophysiological, and therapeutic studies. Copyright © 2017 Elsevier Inc. All rights reserved.

Cytotoxicity of p-chloroamphetamine in dimethylhydrazine-induced carcinomata of rat colon.

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Tutton, P J; Barkla, D H

1979-01-01

Previous studies have shown that several serotonin-related compounds are cytotoxic to dimethylhydrazine-induced carcinomata of the colon of rat. This paper reports the cytotoxicity of another serotonin-related compound, p-chloroamphetamine.

HIV-1-infected macrophages induce astrogliosis by SDF-1α and matrix metalloproteinases

International Nuclear Information System (INIS)

Okamoto, Mika; Wang, Xin; Baba, Masanori

2005-01-01

Brain macrophages/microglia and astrocytes are known to be involved in the pathogenesis of HIV-1-associated dementia (HAD). To clarify their interaction and contribution to the pathogenesis, HIV-1-infected or uninfected macrophages were used as a model of brain macrophages/microglia, and their effects on human astrocytes in vitro were examined. The culture supernatants of HIV-1-infected or uninfected macrophages induced significant astrocyte proliferation, which was annihilated with a neutralizing antibody to stromal cell-derived factor (SDF)-1α or a matrix metalloproteinase (MMP) inhibitor. In these astrocytes, CXCR4, MMP, and tissue inhibitors of matrix metalloproteinase mRNA expression and SDF-1α production were significantly up-regulated. The supernatants of infected macrophages were always more effective than those of uninfected cells. Moreover, the enhanced production of SDF-1α was suppressed by the MMP inhibitor. These results indicate that the activated and HIV-1-infected macrophages can indirectly induce astrocyte proliferation through up-regulating SDF-1α and MMP production, which implies a mechanism of astrogliosis in HAD

Dextran loading protects macrophages from lipid peroxidation and induces a Keap1/Nrf2/ARE-dependent antioxidant response.

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Chechushkov, Anton; Zaitseva, Natalia; Vorontsova, Elena; Kozhin, Petr; Menshchikova, Elena; Shkurupiy, Vyacheslav

2016-12-01

Linear dextrans are often proposed as drug delivery systems with milder adverse effects and lower effective drug concentrations. Linear dextrans are polysaccharides that can potentially be used to load macrophages with drugs to transport them to a site of inflammation. Recently, it was reported that dextrans may exert a protective effect vis-à-vis drug cytotoxicity and during wound healing. The aim of the current work was to evaluate molecular mechanisms of action of dextrans that may be relevant to the cytoprotective effects. We determined the effect of treatment with 40- or 70-kDa dextran on production of reactive oxygen species, lipid peroxidation, and lysosomal pH in the J774 macrophage cell line. In addition, induction of Keap1/Nrf2/ARE and autophagic activity were evaluated. Dextrans of both molecular weights protected the cells from oxidative stress induced by cumene hydroperoxide and from lysosomal stress induced by ammonium chloride. The effect was associated with induction of the Keap1/Nrf2/ARE signaling pathway. Furthermore, dextran stimulated autophagy in a dose-dependent manner but inhibited the autophagosome-lysosome fusion in a time-dependent manner. This study shows possible cytoprotective effects of dextran under oxidative stress, and these findings may be used for the development of novel (dextran-based) drug delivery approaches. Copyright © 2016 Elsevier Inc. All rights reserved.

PHA-induced cytotoxicity of human lymphocytes against adherent hela-cells

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Huges-Law, G.; de Gast, G. C.; The, T. Hauw

The conditions for a phytohaemagglutinin(PHA)-induced cytotoxicity test of human peripheral blood lymphocytes were investigated. [3H]thymidine prelabelled HeLa cells were used as target cells. Stimulation with 10 μl PHA/ml during 24 h gave the best measure of lymphocyte cytotoxic capacity.

Eliminating Legionella by inhibiting BCL-XL to induce macrophage apoptosis.

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Speir, Mary; Lawlor, Kate E; Glaser, Stefan P; Abraham, Gilu; Chow, Seong; Vogrin, Adam; Schulze, Keith E; Schuelein, Ralf; O'Reilly, Lorraine A; Mason, Kylie; Hartland, Elizabeth L; Lithgow, Trevor; Strasser, Andreas; Lessene, Guillaume; Huang, David C S; Vince, James E; Naderer, Thomas

2016-02-24

Human pathogenic Legionella replicate in alveolar macrophages and cause a potentially lethal form of pneumonia known as Legionnaires' disease(1). Here, we have identified a host-directed therapeutic approach to eliminate intracellular Legionella infections. We demonstrate that the genetic deletion, or pharmacological inhibition, of the host cell pro-survival protein BCL-XL induces intrinsic apoptosis of macrophages infected with virulent Legionella strains, thereby abrogating Legionella replication. BCL-XL is essential for the survival of Legionella-infected macrophages due to bacterial inhibition of host-cell protein synthesis, resulting in reduced levels of the short-lived, related BCL-2 pro-survival family member, MCL-1. Consequently, a single dose of a BCL-XL-targeted BH3-mimetic therapy, or myeloid cell-restricted deletion of BCL-XL, limits Legionella replication and prevents lethal lung infections in mice. These results indicate that repurposing BH3-mimetic compounds, originally developed to induce cancer cell apoptosis, may have efficacy in treating Legionnaires' and other diseases caused by intracellular microbes.

Heterogenous populations of cytotoxic cells in the peritoneal cavity of BALB/c mice immunized with allogeneic EL4 leukemia cells

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Zighelboim, J.; Bonavida, B.; Fahey, J.L.

1974-01-01

Adherent cells, presumably macrophages, obtained from the peritoneal cavity shortly after rejection of the allogeneic leukemia EL4, produced effective cell-mediated cytotoxicity (CMC) in vitro. These cytotoxic cells were sensitive to anti-macrophage serum and resistant to anti-thymocyte serum and 10,000 roentgen irradiation. In contrast, a second population of specifically cytotoxic cells were nonadherent, sensitive to x-rays and anti-thymocyte serum, but not to anti-macrophage serum. The two cell populations had a cooperative cytotoxic effect in vitro against allogeneic tumor cells

The effect of lipid peroxidation products on reactive oxygen species formation and nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophages.

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Ambrozova, Gabriela; Pekarova, Michaela; Lojek, Antonin

2011-02-01

Lipid peroxidation induced by oxidants leads to the formation of highly reactive metabolites. These can affect various immune functions, including reactive oxygen species (ROS) and nitric oxide (NO) production. The aim of the present study was to investigate the effects of lipid peroxidation products (LPPs) - acrolein, 4-hydroxynonenal, and malondialdehyde - on ROS and NO production in RAW 264.7 macrophages and to compare these effects with the cytotoxic properties of LPPs. Macrophages were stimulated with lipopolysaccharide (0.1 μg/ml) and treated with selected LPPs (concentration range: 0.1-100 μM). ATP test, luminol-enhanced chemiluminescence, Griess reaction, Western blotting analysis, amperometric and total peroxyl radical-trapping antioxidant parameter assay were used for determining the LPPs cytotoxicity, ROS and NO production, inducible nitric oxide synthase expression, NO scavenging, and antioxidant properties of LPPs, respectively. Our study shows that the cytotoxic action of acrolein and 4-hydroxynonenal works in a dose- and time-dependent manner. Further, our results imply that acrolein, 4-hydroxynonenal, and malondialdehyde can inhibit, to a different degree, ROS and NO production in stimulated macrophages, partially independently of their toxic effect. Also, changes in enzymatic pathways (especially NADPH-oxidase and nitric oxide synthase inhibition) and NO scavenging properties are included in the downregulation of reactive species formation. Copyright © 2010 Elsevier Ltd. All rights reserved.

Suppression of cytotoxic T lymphocytes by carrageenan-activated macrophage-like cells

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Yung, Y.P.; Cudkowicz, G.

1978-01-01

In the presence of 100 μg/ml of carrageenans (CAR), B6D2F 1 responder spleen cells failed to generate antiparent or anti-allogeneic cytotoxic T lymphocytes in vitro, but instead generated suppressor cells. Cultured CAR-treated cells added to mixtures of B6D2F 1 anti-B6 or B6D2F 1 anti-C3H cytotoxic effectors (induced in vitro) and the appropriate 51 Cr-labeled lymphoma targets reduced or abolished cytolysis (measured as 51 Cr release) depending on the ratio of suppressor to effector cells. Cultured spleen cells not exposed to CAR failed to inhibit both types of cytotoxicity. Presuppressor cells were associated with a splenic subpopulation independent of the thymus (i.e., present in spleens of athymic nude mice), were moderately adherent to Sephadex G-10 columns, but were not phagocytic or ''sticky'' to carbonyl iron particles. Activation of such cells by CAR was not prevented by in vitro exposure to 2000 rads of γ-rays before culture, nor facilitated by antigenic stimulation. The matured suppressor cells remained radioresistant and became strongly adherent to Sephadex G-10. The suppressors lacked surface Thy-1 alloantigen detectable by antibody and rabbit complement. Suppressor cell activity was not restricted by the immunologic specificity and major histocompatibility type of effectors

Identification of Francisella novicida mutants that fail to induce prostaglandin E2 synthesis by infected macrophages.

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Matthew Dale Woolard

2013-02-01

Full Text Available Francisella tularensis is the causative agent of tularemia. We have previously shown that infection with F. tularensis Live Vaccine Strain (LVS induces macrophages to synthesize prostaglandin E2 (PGE2. Synthesis of PGE2 by F. tularensis infected macrophages results in decreased T cell proliferation in vitro and increased bacterial survival in vivo. Although we understand some of the biological consequences of F. tularensis induced PGE2 synthesis by macrophages, we do not understand the cellular pathways (neither host nor bacterial that result in up-regulation of the PGE2 biosynthetic pathway in F. tularensis infected macrophages. We took a genetic approach to begin to understand the molecular mechanisms of bacterial induction of PGE2 synthesis from infected macrophages. To identify F. tularensis genes necessary for the induction of PGE2 in primary macrophages, we infected cells with individual mutants from the closely related strain Francisella tularensis subspecies novicida U112 (U112 two allele mutant library. Twenty genes were identified that when disrupted resulted in U112 mutant strains unable to induce the synthesis of PGE2 by infected macrophages. Fourteen of the genes identified are located within the Francisella pathogenicity island (FPI. Genes in the FPI are required for F. tularensis to escape from the phagosome and replicate in the cytosol, which might account for the failure of U112 with transposon insertions within the FPI to induce PGE2. This implies that U112 mutant strains that do not grow intracellularly would also not induce PGE2. We found that U112 clpB::Tn grows within macrophages yet fails to induce PGE2, while U112 pdpA::Tn does not grow yet does induce PGE2. We also found that U112 iglC::Tn neither grows nor induces PGE2. These findings indicate that there is dissociation between intracellular growth and the ability of F. tularensis to induce PGE2 synthesis. These mutants provide a critical entrée into the pathways used

Heavy metal-induced cytotoxicity to cultured human epidermal keratinocytes and effects of antioxidants.

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Kappus, H; Reinhold, C

1994-04-01

Human epidermal keratinocytes which have been cultured were treated with the heavy metal ions of cadmium, mercury, copper and zinc. Cytotoxicity was measured either by protein estimation or by using the neutral red assay. Antioxidants were added in order to find out whether heavy metal-induced cytotoxicity is related to oxidative stress. All metals used showed considerable cytotoxic effects within 24 h in moderate concentrations. None of the antioxidants vitamin E (alpha-tocopherol), pyrogallol, propyl gallate, BHT or ebselen showed any protective or preventive effect. This indicates that oxidative stress may not be involved in the cytotoxicity induced by heavy metals in human epidermal keratinocytes. The cells used are, however, a valuable tool to study mechanisms of cytotoxicity.

Ginger extract inhibits LPS induced macrophage activation and function

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Bruch David

2008-01-01

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

Alendronate inhalation ameliorates elastase-induced pulmonary emphysema in mice by induction of apoptosis of alveolar macrophages.

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Ueno, Manabu; Maeno, Toshitaka; Nishimura, Satoshi; Ogata, Fusa; Masubuchi, Hiroaki; Hara, Kenichiro; Yamaguchi, Kouichi; Aoki, Fumiaki; Suga, Tatsuo; Nagai, Ryozo; Kurabayashi, Masahiko

2015-03-10

Alveolar macrophages play a crucial role in the pathogenesis of emphysema, for which there is currently no effective treatment. Bisphosphonates are widely used to treat osteoclast-mediated bone diseases. Here we show that delivery of the nitrogen-containing bisphosphonate alendronate via aerosol inhalation ameliorates elastase-induced emphysema in mice. Inhaled, but not orally ingested, alendronate inhibits airspace enlargement after elastase instillation, and induces apoptosis of macrophages in bronchoalveolar fluid via caspase-3- and mevalonate-dependent pathways. Cytometric analysis indicates that the F4/80(+)CD11b(high)CD11c(mild) population characterizing inflammatory macrophages, and the F4/80(+)CD11b(mild)CD11c(high) population defining resident alveolar macrophages take up substantial amounts of the bisphosphonate imaging agent OsteoSense680 after aerosol inhalation. We further show that alendronate inhibits macrophage migratory and phagocytotic activities and blunts the inflammatory response of alveolar macrophages by inhibiting nuclear factor-κB signalling. Given that the alendronate inhalation effectively induces apoptosis in both recruited and resident alveolar macrophages, we suggest this strategy may have therapeutic potential for the treatment of emphysema.

Phagocytosis-induced 51Cr release from activated macrophages and blood mononuclears. Effect of colchicine and antioxidants

International Nuclear Information System (INIS)

McGee, M.P.; Hale, A.H.

1981-01-01

The chromium-release test was adapted to the measurement of the cellular injury induced when activated macrophages phagocytose particulates. Macrophages obtained from rabbit lungs undergoing BCG-induced chronic inflammation released more chromium when incubated in the presence of phagocytosable particles than when incubated under resting conditions. Blood mononuclear cells, 40-60% monocytes, procured from the same BCG-injected animals, were less susceptible to phagocytosis-induced injury than the macrophages obtained from the lungs. The amount of chromium released by the activated macrophages was proportional to the number of particles present during incubation. In the presence of catalase, the amounts of chromium released by phagocytosing and resting macrophages were similar; in the presence of superoxide dismutase and cytochrome c, the amount of chromium released by phagocytosing macrophages was 13-35% less than the amount of chromium released by macrophages incubated without the antioxidants. In addition, colchicine, an inhibitor of degranulation also exerted partial inhibition of the chromium release. These results suggest that oxygen radicals and lysosomal contents contribute to the cellular injury that results from phagocytosis

Virus-specific HLA-restricted lysis of herpes simplex virus-infected human monocytes and macrophages mediated by cytotoxic T lymphocytes

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Torpey, D.J. III

1987-01-01

Freshly-isolated peripheral blood human monocytes and 5 day in vitro cultured macrophages were infected with herpes simplex virus type 1 (HSV-1), labeled with /sup 51/Cr, and used as target cells in a 12-14 hour cell-mediated cytotoxicity assay. Mononuclear leukocytes (MNL) from HSV-1 non-immune individuals, whether unstimulated or stimulated with HSV-1 antigen, did not mediate significant lysis of either target cell. HSV-immune MNL, both freshly-isolated and cultured for 5 days without antigen, demonstrated only low levels of natural killer (NK) cell-mediate lysis. MNL from HSV-immune individuals incubated for 5 days in vitro with HSV-1 antigen mediated significant virus-specific lysis of both target cells. Mean virus-specific lysis of autologous monocytes was 8.5(/+-/2.0)% compared to a three-fold greater virus-specific lysis of autologous macrophages. Greater than 70% of this lytic activity was mediated by Leu-11-negative, T3-positive cytotoxic T lymphocytes (CTL). Allogeneic target cells lacking a common HLA determinant were not significantly lysed while T8-positive CTL mediated infrequent lysis of target cells sharing a common HLA-A and/or HLA-B determinant. T4-positive lymphocytes were demonstrated to be the predominant cell mediating lysis of autologous target cells and allogeneic target cells sharing both HLA-A and/or HLA-B plus HLA-DR determinants with the CTL; the T4-positive cell was the sole CTL mediator of lysis of allogeneic target cells having a common HLA-DR determinant.

Virus-specific HLA-restricted lysis of herpes simplex virus-infected human monocytes and macrophages mediated by cytotoxic T lymphocytes

International Nuclear Information System (INIS)

Torpey, D.J. III.

1987-01-01

Freshly-isolated peripheral blood human monocytes and 5 day in vitro cultured macrophages were infected with herpes simplex virus type 1 (HSV-1), labeled with 51 Cr, and used as target cells in a 12-14 hour cell-mediated cytotoxicity assay. Mononuclear leukocytes (MNL) from HSV-1 non-immune individuals, whether unstimulated or stimulated with HSV-1 antigen, did not mediate significant lysis of either target cell. HSV-immune MNL, both freshly-isolated and cultured for 5 days without antigen, demonstrated only low levels of natural killer (NK) cell-mediate lysis. MNL from HSV-immune individuals incubated for 5 days in vitro with HSV-1 antigen mediated significant virus-specific lysis of both target cells. Mean virus-specific lysis of autologous monocytes was 8.5(/+-/2.0)% compared to a three-fold greater virus-specific lysis of autologous macrophages. Greater than 70% of this lytic activity was mediated by Leu-11-negative, T3-positive cytotoxic T lymphocytes (CTL). Allogeneic target cells lacking a common HLA determinant were not significantly lysed while T8-positive CTL mediated infrequent lysis of target cells sharing a common HLA-A and/or HLA-B determinant. T4-positive lymphocytes were demonstrated to be the predominant cell mediating lysis of autologous target cells and allogeneic target cells sharing both HLA-A and/or HLA-B plus HLA-DR determinants with the CTL; the T4-positive cell was the sole CTL mediator of lysis of allogeneic target cells having a common HLA-DR determinant

Activation of peritoneal macrophages to cytoxicity against B16 melanoma cells by Serratia marcescens polyribosome fractions

International Nuclear Information System (INIS)

Hoover, S.K.

1985-01-01

Serratia marcescens polyribosomes (SMPR) have been shown to elicit an anti-tumor response in vivo. The in-vitro effects of SMPR on macrophages as the nonspecific mediators of the anti-tumor response have not previously been examined. The first objective of this research project is to corroborate and analyze the in-vivo results by the development and application of an in-vitro cytotoxicity assay. The second objective is to examine the effect of SMPR upon previously unstimulated peritoneal macrophages as representing the mechanism of cytotoxicity. The third objective is to identify the minimal effective component of SMPR responsible for an effect on macrophages. Results revealed that SMPR preparations exert a number of effects upon macrophages. Morphologic changes included increased spreading and increased perinuclear vacuolization. Macrophages were shown to be metabolically activate by two lines of evidence. SMPR-treated macrophages exhibited increased cellular metabolism by the increased uptake of 3 H-thymidine and by the increased levels of secreted leucine aminopeptidase as compared to control macrophages. Results also showed that SMPR activates macrophages to cytotoxicity against syngeneic tumor target cells. Buoyant-density fractions were isolated and assayed for macrophage activating ability. Results showed 50S ribosomal subunits to be the smallest fraction effective for macrophage activation. Both the RNA and protein were necessary for complete effectiveness

Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

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Islam, Shamima; Hassan, Ferdaus; Tumurkhuu, Gantsetseg; Dagvadorj, Jargalsaikhan; Koide, Naoki; Naiki, Yoshikazu; Mori, Isamu; Yoshida, Tomoaki; Yokochi, Takashi

2007-01-01

Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-α antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB ligand (RANKL). TNF-α might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-κB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed

Immunological Demyelination Triggers Macrophage/Microglial Cells Activation without Inducing Astrogliosis

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Frank Cloutier

2013-01-01

Full Text Available The glial scar formed by reactive astrocytes and axon growth inhibitors associated with myelin play important roles in the failure of axonal regeneration following central nervous system (CNS injury. Our laboratory has previously demonstrated that immunological demyelination of the CNS facilitates regeneration of severed axons following spinal cord injury. In the present study, we evaluate whether immunological demyelination is accompanied with astrogliosis. We compared the astrogliosis and macrophage/microglial cell responses 7 days after either immunological demyelination or a stab injury to the dorsal funiculus. Both lesions induced a strong activated macrophage/microglial cells response which was significantly higher within regions of immunological demyelination. However, immunological demyelination regions were not accompanied by astrogliosis compared to stab injury that induced astrogliosis which extended several millimeters above and below the lesions, evidenced by astroglial hypertrophy, formation of a glial scar, and upregulation of intermediate filaments glial fibrillary acidic protein (GFAP. Moreover, a stab or a hemisection lesion directly within immunological demyelination regions did not induced astrogliosis within the immunological demyelination region. These results suggest that immunological demyelination creates a unique environment in which astrocytes do not form a glial scar and provides a unique model to understand the putative interaction between astrocytes and activated macrophage/microglial cells.

Macrophage activation induced by the polysaccharides isolated from the roots of Sanguisorba officinalis.

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Tong, Haibin; Mao, Dirui; Zhai, Mingyue; Zhang, Zhuorui; Sun, Guangren; Jiang, Guiquan

2015-01-01

Macrophage, involved at all stages of immune response, is an important component of the host defense system. Polysaccharides exist almost ubiquitously in medical plants and most of them possess immunomodulation and macrophage activation properties. This study elucidates the effects on macrophage activation and molecular mechanism induced by the polysaccharides (SOPs) from the roots of Sanguisorba officinalis Linne (Rosaceae). Polysaccharides (SOPs) from the roots of S. officinalis were obtained by water extraction and ethanol precipitation. Physicochemical characterization of SOPs was analyzed by phenol-sulfuric acid, m-hydroxydiphenyl, Bradford method, and gas chromatography. Phagocytic capacity of RAW 264.7 macrophages incubated with SOPs (25 and 100 μg/ml) was determined by the aseptic neutral red method. Macrophages were incubated with SOPs (25 and 100 μg/ml), and the TNF-α and NO the secretion were measured using ELISA kit and Griess reagent, respectively. In addition, TNF-α and iNOS transcripts were evaluated by semi-quantitative RT-PCR, and NF-κB signaling activation was detected by Western blot assay. SOPs enhanced the phagocytosis capacity of macrophages to aseptic neutral red solution and increased TNF-α and NO secretion. The amounts of TNF-α and iNOS transcript were increased significantly at the mRNA level when macrophages were exposed to SOPs. Meanwhile, the stimulation of macrophages by SOPs induced phosphorylation of p65 at serine 536 and a marked decrease of IκB expression. These results suggested that SOPs exhibited significant macrophage activation properties through NF-κB signaling pathway and could be considered as a new immunopotentiator.

INTRACELLULAR Leishmania amazonensis KILLING INDUCED BY THE GUANINE NUCLEOSIDE 8-BROMOGUANOSINE

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GIORGIO Selma

1998-01-01

Full Text Available In this study we investigated the effect of 8-Bromoguanosine, an immunostimulatory compound, on the cytotoxicity of macrophages against Leishmania amazonensis in an in vitro system. The results showed that macrophages treated with 8-Bromoguanosine before or after infection are capable to reduce parasite load, as monitored by the number of amastigotes per macrophage and the percentage of infected cells (i.e. phagocytic index. Since 8-Bromoguanosine was not directly toxic to the promastigotes, it was concluded that the ribonucleoside induced macrophage activation. Presumably, 8-Bromoguanosine primed macrophages by inducing interferon alpha and beta which ultimately led to L. amazonensis amastigote killing. The results suggest that guanine ribonucleosides may be useful to treat infections with intracellular pathogens.

Inflammation induced mTORC2-Akt-mTORC1 signaling promotes macrophage foam cell formation.

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Banerjee, Dipanjan; Sinha, Archana; Saikia, Sudeshna; Gogoi, Bhaskarjyoti; Rathore, Arvind K; Das, Anindhya Sundar; Pal, Durba; Buragohain, Alak K; Dasgupta, Suman

2018-06-05

The transformation of macrophages into lipid loaded foam cells is a critical and early event in the pathogenesis of atherosclerosis. Several recent reports highlighted that induction of TLR4 signaling promotes macrophage foam cell formation; however, the underlying molecular mechanisms have not been clearly elucidated. Here, we found that the TLR4 mediated inflammatory signaling communicated with mTORC2-Akt-mTORC1 metabolic cascade in macrophage and thereby promoting lipid uptake and foam cell formation. Mechanistically, LPS treatment markedly upregulates TLR4 mediated inflammatory pathway which by activating mTORC2 induces Akt phosphorylation at serine 473 and that aggravate mTORC1 dependent scavenger receptors expression and consequent lipid accumulation in THP-1 macrophages. Inhibition of mTORC2 either by silencing Rictor expression or inhibiting its association with mTOR notably prevents LPS induced Akt activation, scavenger receptors expression and macrophage lipid accumulation. Although suppression of mTORC1 expression by genetic knockdown of Raptor did not produce any significant change in Akt S473 phosphorylation, however, incubation with Akt activator in Rictor silenced cells failed to promote scavenger receptors expression and macrophage foam cell formation. Thus, present research explored the signaling pathway involved in inflammation induced macrophage foam cells formation and therefore, targeting this pathway might be useful for preventing macrophage foam cell formation. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Modulation of allogeneic stimulation in man. I. Characterization of an in vitro induced suppressor macrophage population

International Nuclear Information System (INIS)

Stux, S.V.; Dubey, D.P.; Yunis, E.J.

1981-01-01

Cultured human peripheral blood mononuclear cells suppressed the allogeneic response of fresh autologous lymphocytes. This suppressor activity developed gradually over a period of one week. The cells primarily responsible for this effect were enriched by Ficoll density gradient centrifugation. It was found that the suppressor cell is a large, low density nylon wool adherent, radioresistant, phagocytic, and nonspecific esterase positive mononuclear cell. Moreover, these cells did not form E rosettes and were Fc positive. Electron microscopy confirmed that suppressor cells were macrophage like. Suppressor activity was not due to cytotoxicity, crowding, or steric hinderance by the cultured cells. The suppressor macrophage population did not appear to inhibit the allogeneic response via prostaglandin or arginase release, or interfere with the tritiated thymidine uptake by release of endogenous thymidine. The above system is viewed as an in vitro model of immune regulation by suppressor macrophages, in the context of allogeneic response

Cytotoxicity of Carbon Nanotubes on J774 Macrophages Is a Purification-Dependent Effect

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Silvia Lorena Montes-Fonseca

2012-01-01

Full Text Available The cytotoxicity of the carbon nanotubes (CNTs is an important factor for the manufacture of nanovaccines. The aim of this work was to evaluate the relationship of the purification method of CNTs in cellular toxicity using macrophages (MOs from the J774 cell line. Viability test was performed with MTT assays at 24 h of exposure at concentrations of 0.06, 0.6, and 6 mg/L of unpurified (UP-CNTs or purified (P-CNTs CNTs by two different methods: (1 reflux with 3M HNO3 and (2 sonication in H2SO4/HNO3. Characterization and COOH content of CNTs was performed using scanning electron microscopy, raman spectroscopy, and titration with NaHCO3. P-CNTs1 had lengths >100 μm and 2.76% COOH content, while P-CNTs2 had lengths >1 μm and 7% COOH content. This last particle showed a lower toxic effect. The results suggest that the lenght and COOH content are important factors in the toxicity of the CNTs.

Macrophage antioxidant protection within atherosclerotic plaques.

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Gieseg, Steven P; Leake, David S; Flavall, Elizabeth M; Amit, Zunika; Reid, Linzi; Yang, Ya-Ting

2009-01-01

Macrophage cells within inflammatory lesions are exposed to a wide range of degrading and cytotoxic molecules including reactive oxygen species. Unlike neutrophils, macrophages do not normally die in this environment but continue to generate oxidants, phagocytose cellular remains, and release a range of cyto-active agents which modulate the immune response. It is this potential of the macrophage cell to survive in an oxidative environment that allows the growth and complexity of advanced atherosclerotic plaques. This review will examine the oxidants encountered by macrophages within an atherosclerotic plaque and describe some of the potential antioxidant mechanisms which enable macrophages to function within inflammatory lesions. Ascorbate, a-tocopherol, and glutathione appear to be central to the protection of macrophages yet additional antioxidant mechanisms appear to be involved. Gamma-Interferon causes macrophages to generate 7,8-dihydroneopterin, neopterin and 3-hydroxyanthranilic acid both of which have antioxidant properties. Manganese superoxide dismutase is also upregulated in macrophages. The evidence that these antioxidants provide further protection, so allowing the macrophage cells to survive within sites of chronic inflammation such as atherosclerotic plaques, will be described.

C-reactive protein interaction with macrophages: in vitro induction of tumor cytotoxicity, and characterization of C-reactive protein binding to macrophages

International Nuclear Information System (INIS)

Zahedi, K.A.

1987-01-01

The ability of C-reactive protein (CRP) to activate macrophages to tumoricidal state was examined. CRP was able to activate macrophages to kill tumor cells. The activation was shown to be due to CRP and not to low levels of other activators present in the CRP preparations, since specific removal of CRP led to abrogation of the CRP mediated activation of macrophages. The role of lipopolysaccharide (LPS) as a contaminating activator was eliminated by showing the ability of CRP preparations to activate macrophages from LPS non-responsive strains of mice, and to activate macrophages under conditions which specifically inactivated or removed the contaminating LPS. In order to exclude the possibility of indirect activation of macrophages by other cells present in the peritoneal exudate cell population, effect of CRP on pure macrophages was examined. Bone marrow derived macrophages as well as well as macrophage cell lines exhibited a significant increase in their capacity to kill tumor cells after treatment with CRP. The nature of CRP and macrophage interaction was examined using radioiodinated CRP. Labelled CRP bound specifically to macrophages and macrophage cell lines

EVIDENCE FOR THE MACROPHAGE INDUCING GENE IN MYCOBACTERIUM INTRACELLULARE

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Background: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and possibly others. Organisms belonging to the MAC are phylogenetically closely related, opportunistic pathogens. The macrophage inducing gene (mig) is the only well-des...

Suppression of LPS-induced inflammatory responses in macrophages infected with Leishmania

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Kelly Ben L

2010-02-01

Full Text Available Abstract Background Chronic inflammation activated by macrophage innate pathogen recognition receptors such as TLR4 can lead to a range of inflammatory diseases, including atherosclerosis, Crohn's disease, arthritis and cancer. Unlike many microbes, the kinetoplastid protozoan pathogen Leishmania has been shown to avoid and even actively suppress host inflammatory cytokine responses, such as LPS-induced IL-12 production. The nature and scope of Leishmania-mediated inflammatory cytokine suppression, however, is not well characterized. Advancing our knowledge of such microbe-mediated cytokine suppression may provide new avenues for therapeutic intervention in inflammatory disease. Methods We explored the kinetics of a range of cytokine and chemokine responses in primary murine macrophages stimulated with LPS in the presence versus absence of two clinically distinct species of Leishmania using sensitive multiplex cytokine analyses. To confirm that these effects were parasite-specific, we compared the effects of Leishmania uptake on LPS-induced cytokine expression with uptake of inert latex beads. Results Whilst Leishmania uptake alone did not induce significant levels of any cytokine analysed in this study, Leishmania uptake in the presence of LPS caused parasite-specific suppression of certain LPS-induced pro-inflammatory cytokines, including IL-12, IL-17 and IL-6. Interestingly, L. amazonensis was generally more suppressive than L. major. We also found that other LPS-induced proinflammatory cytokines, such as IL-1α, TNF-α and the chemokines MIP-1α and MCP-1 and also the anti-inflammatory cytokine IL-10, were augmented during Leishmania uptake, in a parasite-specific manner. Conclusions During uptake by macrophages, Leishmania evades the activation of a broad range of cytokines and chemokines. Further, in the presence of a strong inflammatory stimulus, Leishmania suppresses certain proinflammatory cytokine responses in a parasite

Selective macrophage inhibition abolishes warfarin-induced reduction of metastasis

NARCIS (Netherlands)

Maat, B.

1980-01-01

Warfarin administered to tumor-bearing mice reduces the number of spontaneous lung metastases. Both macrophage inhibitors silica and carrageenan abolish the warfarin-induced decrease in tumour metastasis, which strongly supports the concept that the antitumour effect of coumarin derivatives is

Deubiquitinase USP12 promotes LPS induced macrophage responses through inhibition of IκBα

International Nuclear Information System (INIS)

Nayak, Tapan Kumar Singh; Alamuru-Yellapragada, Neeraja P.; Parsa, Kishore V.L.

2017-01-01

Post translational modifications, ubiquitination and its reversal by deubiquitination play an important role in regulating innate immune system. USP12 is a poorly studied deubiquitinase reported to regulate T-cell receptor signalling however the functional role of USP12 in macrophages, the principal architects of inflammation, is unknown. Thus, in this study we probed the involvement of USP12 in macrophage mediated inflammatory responses using bacterial endotoxin, LPS, as the model system. Here, we observed that the expression of USP12 was altered in time dependent manner in LPS stimulated RAW 264.7 macrophages at both mRNA and protein levels as revealed by qPCR and western blot analysis, respectively. Further analysis showed that LPS reduced the levels of Sp1 which enhanced the transcriptional levels of USP12. We observed that siRNA mediated ablation of USP12 expression in mouse macrophages suppressed the induction of LPS-induced iNOS and IL-6 expression but failed to alter IFN-β synthesis, oxidative stress and phagocytic ability of macrophages. Mechanistic analysis suggest that USP12 may be required for the activation of NFκB pathway as knockdown of USP12 reduced the inhibitory phosphorylation of IκBα, a well characterized inhibitor of NFκB nuclear translocation. Further, USP12 was observed to be required for LPS elicited phosphorylation of ERK1/2 and p38. Collectively, our data suggest that USP12 may be a key mediator of LPS stimulated macrophage responses. - Highlights: • USP12 levels are significantly altered in LPS stimulated macrophages. • USP12 is required for LPS induced iNOS and IL6 expression. • USP12 is crucial for LPS induced phosphorylation of IκBα, ERK1/2, p38.

Anti-Inflammatory Effects of Benfotiamine are Mediated Through the Regulation of Arachidonic Acid Pathway in Macrophages

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Shoeb, Mohammad; Ramana, Kota V

2011-01-01

Benfotiamine, a lipid-soluble analogue of vitamin B1, is a potent anti-oxidant that is used as a food supplement for the treatment of diabetic complications. Our recent study indicates a novel role of benfotiamine in the prevention of bacterial endotoxin, lipopolysaccharide (LPS)-induced cytotoxicity and inflammatory response in murine macrophages. Nevertheless, it remains unclear how benfotiamine mediates anti-inflammatory effects. In this study, we investigated the anti-inflammatory role of benfotiamine in regulating the arachidonic acid (AA) pathway generated inflammatory lipid mediators in RAW 264.7 macrophages. Benfotiamine prevented the LPS-induced activation of cPLA2 and release of AA metabolites such as leukotrienes (LTB4), prostaglandin E2 (PGE2), thromboxanes 2 (TXB2) and prostacyclin (PGI2) in macrophages. Further, LPS-induced expressions of AA metabolizing enzymes such as COX-2, LOX-5, TXB synthase and PGI2 synthase were significantly blocked by benfotiamine. Furthermore, benfotiamine prevented the LPS-induced phosphorylation of ERK1/2 and expression of transcription factors NF-kB, and Egr-1. Benfotiamine also prevented the LPS-induced oxidative stress and protein-HNE adducts formation. Most importantly, as compared to specific COX-2 and LOX-5 inhibitors, benfotiamine significantly prevented the LPS-induced macrophage death and monocytes adhesion to endothelial cells. Thus, our studies indicate that the dual regulation of COX and LOX pathways in AA metabolism could be a novel mechanism by which benfotiamine exhibits its potential anti-inflammatory response. PMID:22067901

Protective Effect of Saccharomyces boulardii on Deoxynivalenol-Induced Injury of Porcine Macrophage via Attenuating p38 MAPK Signal Pathway.

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Chang, Chao; Wang, Kun; Zhou, Sheng-Nan; Wang, Xue-Dong; Wu, Jin-E

2017-05-01

The aims of our study were to evaluate the effects of Saccharomyces boulardii (S. boulardii) on deoxynivalenol (DON)-induced injury in porcine alveolar macrophage cells (PAMCs) and to explore the underlying mechanisms. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometric analysis, ELISA, qRT-PCR, and western blot were performed to assess whether S. boulardii could prevent DON-induced injury by p38 mitogen-activated protein kinase (p38 MAPK) signal pathway. The results showed that pretreatment with 8 μM DON could decrease the viability of PAMC and significantly increase the apoptosis rate of PAMC, whereas S. boulardii could rescue apoptotic PAMC cells induced by DON. Further experiments revealed that S. boulardii effectively reversed DON-induced cytotoxicity via downregulating the expression of TNF-α, IL-6, and IL-lβ. In addition, S. boulardii significantly alleviated DON-induced phosphorylation and mRNA expression of p38 and further increased the expression of apoptosis regulation genes Bcl-xl and Bcl-2 and inhibited the activation of Bax. Our results suggest that S. boulardii could suppress DON-induced p38 MAPK pathway activation and reduce the expression of downstream inflammatory cytokines, as well as promote the expression of anti-apoptotic genes to inhibit apoptosis induced by DON in PAMC.

Erythropoietin protects against rhabdomyolysis-induced acute kidney injury by modulating macrophage polarization

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Wang, Shuo; Zhang, Chao; Li, Jiawei; Niyazi, Sidikejiang; Zheng, Long; Xu, Ming; Rong, Ruiming; Yang, Cheng; Zhu, Tongyu

2017-01-01

Erythropoietin (EPO) is a well-known hormone that is clinically used for the treatment of anemia. Very recently, an increasing body of evidence showed that EPO could still regulate bioactivities of macrophages. However, the details about the immunomodulatory effect of EPO on macrophages are not fully delineated, particularly in the setting of renal damages. Therefore, in the present study, we determined whether EPO could exert an impact on the dynamics of macrophages in a well-established model of rhabdomyolysis-induced acute kidney injury and explored the potential mechanisms. EPO was found to ameliorate kidney injuries by reducing macrophages recruitment and promoting phenotype switch toward M2 macrophages in vivo. It was also confirmed that EPO could directly suppress pro-inflammatory responses of M1 macrophages and promote M2 marker expression in vitro. Data indicated the possible involvement of Jak2/STAT3/STAT6 pathway in the augmentation of EPO on M2 polarization. These results improved the understanding of the immunoregulatory capacity of EPO on macrophages, which might optimize the therapeutic modalities of EPO. PMID:28383559

CX3CL1-mediated macrophage activation contributed to paclitaxel-induced DRG neuronal apoptosis and painful peripheral neuropathy.

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Huang, Zhen-Zhen; Li, Dai; Liu, Cui-Cui; Cui, Yu; Zhu, He-Quan; Zhang, Wen-Wen; Li, Yong-Yong; Xin, Wen-Jun

2014-08-01

Painful peripheral neuropathy is a dose-limiting side effect of paclitaxel therapy, which hampers the optimal clinical management of chemotherapy in cancer patients. Currently the underlying mechanisms remain largely unknown. Here we showed that the clinically relevant dose of paclitaxel (3×8mg/kg, cumulative dose 24mg/kg) induced significant upregulation of the chemokine CX3CL1 in the A-fiber primary sensory neurons in vivo and in vitro and infiltration of macrophages into the dorsal root ganglion (DRG) in rats. Paclitaxel treatment also increased cleaved caspase-3 expression, induced the loss of primary afferent terminal fibers and decreased sciatic-evoked A-fiber responses in the spinal dorsal horn, indicating DRG neuronal apoptosis induced by paclitaxel. In addition, the paclitaxel-induced DRG neuronal apoptosis occurred exclusively in the presence of macrophage in vitro study. Intrathecal or systemic injection of CX3CL1 neutralizing antibody blocked paclitaxel-induced macrophage recruitment and neuronal apoptosis in the DRG, and also attenuated paclitaxel-induced allodynia. Furthermore, depletion of macrophage by systemic administration of clodronate inhibited paclitaxel-induced allodynia. Blocking CX3CL1 decreased activation of p38 MAPK in the macrophage, and inhibition of p38 MAPK activity blocked the neuronal apoptosis and development of mechanical allodynia induced by paclitaxel. These findings provide novel evidence that CX3CL1-recruited macrophage contributed to paclitaxel-induced DRG neuronal apoptosis and painful peripheral neuropathy. Copyright © 2014 Elsevier Inc. All rights reserved.

Alveolar Macrophages Play a Key Role in Cockroach-Induced Allergic Inflammation via TNF-α Pathway

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Kim, Joo Young; Sohn, Jung Ho; Choi, Je-Min; Lee, Jae-Hyun; Hong, Chein-Soo; Lee, Joo-Shil; Park, Jung-Won

2012-01-01

The activity of the serine protease in the German cockroach allergen is important to the development of allergic disease. The protease-activated receptor (PAR)-2, which is expressed in numerous cell types in lung tissue, is known to mediate the cellular events caused by inhaled serine protease. Alveolar macrophages express PAR-2 and produce considerable amounts of tumor necrosis factor (TNF)-α. We determined whether the serine protease in German cockroach extract (GCE) enhances TNF-α production by alveolar macrophages through the PAR-2 pathway and whether the TNF-α production affects GCE-induced pulmonary inflammation. Effects of GCE on alveolar macrophages and TNF-α production were evaluated using in vitro MH-S and RAW264.6 cells and in vivo GCE-induced asthma models of BALB/c mice. GCE contained a large amount of serine protease. In the MH-S and RAW264.7 cells, GCE activated PAR-2 and thereby produced TNF-α. In the GCE-induced asthma model, intranasal administration of GCE increased airway hyperresponsiveness (AHR), inflammatory cell infiltration, productions of serum immunoglobulin E, interleukin (IL)-5, IL-13 and TNF-α production in alveolar macrophages. Blockade of serine proteases prevented the development of GCE induced allergic pathologies. TNF-α blockade also prevented the development of such asthma-like lesions. Depletion of alveolar macrophages reduced AHR and intracellular TNF-α level in pulmonary cell populations in the GCE-induced asthma model. These results suggest that serine protease from GCE affects asthma through an alveolar macrophage and TNF-α dependent manner, reflecting the close relation of innate and adaptive immune response in allergic asthma model. PMID:23094102

Fluoromica nanoparticle cytotoxicity in macrophages decreases with size and extent of uptake

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Tee N

2015-03-01

Full Text Available Nicolin Tee,1 Yingdong Zhu,2 Gysell M Mortimer,1 Darren J Martin,2 Rodney F Minchin11School of Biomedical Science, University of Queensland, Brisbane, QLD, Australia; 2Australian Institute of Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD, AustraliaAbstract: Polyurethanes are widely used in biomedical devices such as heart valves, pacemaker leads, catheters, vascular devices, and surgical dressings because of their excellent mechanical properties and good biocompatibility. Layered silicate nanoparticles can significantly increase tensile strength and breaking strain of polyurethanes potentially increasing the life span of biomedical devices that suffer from wear in vivo. However, very little is known about how these nanoparticles interact with proteins and cells and how they might exert unwanted effects. A series of fluoromica nanoparticles ranging in platelet size from 90 to over 600 nm in diameter were generated from the same base material ME100 by high energy milling and differential centrifugation. The cytotoxicity of the resulting particles was dependent on platelet size but in a manner that is opposite to many other types of nanomaterials. For the fluoromicas, the smaller the platelet size, the less toxicity was observed. The small fluoromica nanoparticles (<200 nm were internalized by macrophages via scavenger receptors, which was dependent on the protein corona formed in serum. This internalization was associated with apoptosis in RAW cells but not in dTHP-1 cells. The larger particles were not internalized efficiently but mostly decorated the surface of the cells, causing membrane disruption, even in the presence of 80% serum. This work suggests the smaller fluoromica platelets may be safer for use in humans but their propensity to recognize macrophage scavenger receptors also suggests that they will target the reticulo-endoplasmic system in vivo.Keywords: layered silicates, accumulation, phagocytosis, high

Mycotoxin zearalenone induces AIF- and ROS-mediated cell death through p53- and MAPK-dependent signaling pathways in RAW264.7 macrophages.

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Yu, Ji-Yeon; Zheng, Zhong-Hua; Son, Young-Ok; Shi, Xianglin; Jang, Young-Oh; Lee, Jeong-Chae

2011-12-01

Zearalenone (ZEN) is commonly found in many food commodities and is known to cause reproductive disorders and genotoxic effects. However, the mode of ZEN-induced cell death of macrophages and the mechanisms by which ZEN causes cytotoxicity remain unclear. The present study shows that ZEN treatment reduces viability of RAW264.7 cells in a dose-dependent manner. ZEN causes predominantly necrotic and late apoptotic cell death. ZEN treatment also results in the loss of mitochondrial membrane potential (MMP), mitochondrial changes in Bcl-2 and Bax proteins, and cytoplasmic release of cytochrome c and apoptosis-inducing factor (AIF). Pre-treatment of the cells with either z-VAD-fmk or z-IETD-fmk does not attenuate ZEN-mediated cell death, whereas catalase suppresses the ZEN-induced decrease in viability in RAW264.7 cells. Treating the cells with c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), or p53 inhibitor prevented ZEN-mediated changes, such as MMP loss, cellular reactive oxygen species (ROS) increase, and cell death. JNK or p38 MAPK inhibitor inhibited mitochondrial alterations of Bcl-2 and Bax proteins with attendant decreases in cellular ROS levels. Knockdown of AIF via siRNA transfection also diminished ZEN-induced cell death. Further, adenosine triphosphate was markedly depleted in the ZEN-exposed cells. Collectively, these results suggest that ZEN induces cytotoxicity in RAW264.7 cells via AIF- and ROS-mediated signaling, in which the activations of p53 and JNK/p38 play a key role. Copyright © 2011 Elsevier Ltd. All rights reserved.

MicroRNA-24 Modulates Staphylococcus aureus-Induced Macrophage Polarization by Suppressing CHI3L1.

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Jingjing, Zhang; Nan, Zhang; Wei, Wu; Qinghe, Guo; Weijuan, Wang; Peng, Wang; Xiangpeng, Wang

2017-06-01

Macrophages play a crucial role in host innate anti-Staphylococcus aureus defense, which is tightly regulated by multiple factors, including microRNAs. A recent study showed that miR-24 plays an important role in macrophage polarization. Here, we investigated the biological function of miR-24 in S. aureus-stimulated macrophages. The results revealed that miR-24 expression was significantly decreased in both human and mouse macrophage cell lines with S. aureus stimulation in a time-dependent manner. Moreover, miR-24 overexpression significantly decreased the production of M1 phenotype markers, such as IL-6, iNOS, TNF-α, CD86, and CD80, whereas it increased the production of M2 markers, such as Arg1, CCL17, CCL22, CD163, and CD206, in S. aureus-stimulated macrophages. Conversely, knockdown of miR-24 promoted M1 macrophage polarization but diminished M2 macrophage polarization in S. aureus-stimulated macrophages. Furthermore, CHI3L1 was predicted as a target gene of miR-24 using bioinformatics software and identified by luciferase reporter assay. Additionally, miR-24 overexpression inhibited CHI3L1 expression and downregulated the downstream MAPK pathway in S. aureus-stimulated macrophages. Finally, CHI3L1 overexpression rescued macrophage polarization and MAPK pathway inhibition induced by miR-24 mimic transfection in S. aureus-stimulated macrophages. In conclusion, the data suggest that miR-24 serves as a molecular regulator in S. aureus-induced macrophage polarization through targeting of CHI3L1 and regulation of the MAPK pathway, which may provide a promising therapeutic target for S. aureus-related infections and inflammatory diseases.

Activation of M1 macrophages in sepsis-induced acute kidney injury in response to heparin-binding protein.

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Li Xing

Full Text Available In the early stage of sepsis, M1 macrophages result in the production of inflammatory mediators and AKI. Heparin-binding protein (HBP have been shown to play important roles in sepsis-induced AKI. In this study, we investigate the association of HBP with M1 macrophages in sepsis-induced AKI.Male C57BL6 mice were subjected to cecal ligation and puncture (CLP or sham surgery. Biochemical and histological renal damage was assessed. Macrophage infiltration was assessed by immunohistochemistry. RT-PCR was used to investigate the expression of heparin-binding protein (HBP, the inducible nitric oxide synthase (iNOS and arginase 1 (Arg-1 mRNAs. Western blots were performed to assay the tissue levels of HBP, tumor necrosis factor alpha (TNF-α and interleukin-6 (IL-6.High levels of HBP were obviously detected 24 h after sepsis-induced AKI. Heparin inhibited HBP expression during sepsis-induced AKI. The suppression of HBP expression by heparin injection after the establishment of sepsis-induced AKI resulted in a reduction in renal injury severity accompanied with a significant repression of M1 macrophage activation and expression of TNF-α and IL-6.HBP plays an important role in the initial inflammatory reaction associated with sepsis-induced AKI, presumably by activating M1 macrophages and suppressing TNF-α and IL-6 secretion.

Adventitial fibroblasts induce a distinct proinflammatory/profibrotic macrophage phenotype in pulmonary hypertension.

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El Kasmi, Karim C; Pugliese, Steven C; Riddle, Suzette R; Poth, Jens M; Anderson, Aimee L; Frid, Maria G; Li, Min; Pullamsetti, Soni S; Savai, Rajkumar; Nagel, Maria A; Fini, Mehdi A; Graham, Brian B; Tuder, Rubin M; Friedman, Jacob E; Eltzschig, Holger K; Sokol, Ronald J; Stenmark, Kurt R

2014-07-15

Macrophage accumulation is not only a characteristic hallmark but is also a critical component of pulmonary artery remodeling associated with pulmonary hypertension (PH). However, the cellular and molecular mechanisms that drive vascular macrophage activation and their functional phenotype remain poorly defined. Using multiple levels of in vivo (bovine and rat models of hypoxia-induced PH, together with human tissue samples) and in vitro (primary mouse, rat, and bovine macrophages, human monocytes, and primary human and bovine fibroblasts) approaches, we observed that adventitial fibroblasts derived from hypertensive pulmonary arteries (bovine and human) regulate macrophage activation. These fibroblasts activate macrophages through paracrine IL-6 and STAT3, HIF1, and C/EBPβ signaling to drive expression of genes previously implicated in chronic inflammation, tissue remodeling, and PH. This distinct fibroblast-activated macrophage phenotype was independent of IL-4/IL-13-STAT6 and TLR-MyD88 signaling. We found that genetic STAT3 haplodeficiency in macrophages attenuated macrophage activation, complete STAT3 deficiency increased macrophage activation through compensatory upregulation of STAT1 signaling, and deficiency in C/EBPβ or HIF1 attenuated fibroblast-driven macrophage activation. These findings challenge the current paradigm of IL-4/IL-13-STAT6-mediated alternative macrophage activation as the sole driver of vascular remodeling in PH, and uncover a cross-talk between adventitial fibroblasts and macrophages in which paracrine IL-6-activated STAT3, HIF1α, and C/EBPβ signaling are critical for macrophage activation and polarization. Thus, targeting IL-6 signaling in macrophages by completely inhibiting C/EBPβ or HIF1α or by partially inhibiting STAT3 may hold therapeutic value for treatment of PH and other inflammatory conditions characterized by increased IL-6 and absent IL-4/IL-13 signaling. Copyright © 2014 by The American Association of Immunologists

Inhibition of inducible nitric oxide synthesis by azathioprine in a macrophage cell line.

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

2006-06-20

Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0-210 muM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 muM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0-210 muM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of

Proinsulin-producing, hyperglycemia-induced adipose tissue macrophages underlie insulin resistance in high fat-fed diabetic mice

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Adipose tissue macrophages play an important role in the pathogenesis of obese type 2 diabetes. High-fat diet-induced obesity has been shown to lead to adipose tissue macrophages accumulation in rodents;however, the impact of hyperglycemia on adipose tissue macrophages dynamics in high-fat diet-fed ...

Macrophage JAK2 deficiency protects against high-fat diet-induced inflammation.

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Desai, Harsh R; Sivasubramaniyam, Tharini; Revelo, Xavier S; Schroer, Stephanie A; Luk, Cynthia T; Rikkala, Prashanth R; Metherel, Adam H; Dodington, David W; Park, Yoo Jin; Kim, Min Jeong; Rapps, Joshua A; Besla, Rickvinder; Robbins, Clinton S; Wagner, Kay-Uwe; Bazinet, Richard P; Winer, Daniel A; Woo, Minna

2017-08-09

During obesity, macrophages can infiltrate metabolic tissues, and contribute to chronic low-grade inflammation, and mediate insulin resistance and diabetes. Recent studies have elucidated the metabolic role of JAK2, a key mediator downstream of various cytokines and growth factors. Our study addresses the essential role of macrophage JAK2 in the pathogenesis to obesity-associated inflammation and insulin resistance. During high-fat diet (HFD) feeding, macrophage-specific JAK2 knockout (M-JAK2 -/- ) mice gained less body weight compared to wildtype littermate control (M-JAK2 +/+ ) mice and were protected from HFD-induced systemic insulin resistance. Histological analysis revealed smaller adipocytes and qPCR analysis showed upregulated expression of some adipogenesis markers in visceral adipose tissue (VAT) of HFD-fed M-JAK2 -/- mice. There were decreased crown-like structures in VAT along with reduced mRNA expression of some macrophage markers and chemokines in liver and VAT of HFD-fed M-JAK2 -/- mice. Peritoneal macrophages from M-JAK2 -/- mice and Jak2 knockdown in macrophage cell line RAW 264.7 also showed lower levels of chemokine expression and reduced phosphorylated STAT3. However, leptin-dependent effects on augmenting chemokine expression in RAW 264.7 cells did not require JAK2. Collectively, our findings show that macrophage JAK2 deficiency improves systemic insulin sensitivity and reduces inflammation in VAT and liver in response to metabolic stress.

Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production.

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Liu, Ning; Wang, Lin; Sun, Changjiang; Yang, Li; Tang, Bin; Sun, Wanchun; Peng, Qisheng

2015-12-01

Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival. Copyright © 2015 Elsevier Ltd. All rights reserved.

Drug Trafficking into Macrophages via the Endocytotic Receptor CD163

DEFF Research Database (Denmark)

Graversen, Jonas Heilskov; Moestrup, Søren Kragh

2015-01-01

for cytotoxic or phenotype-modulating drugs in the treatment of inflammatory and cancerous diseases. Such targeting of macrophages has been tried using the natural propensity of macrophages to non-specifically phagocytose circulating foreign particulate material. In addition, the specific targeting...... of macrophage-expressed receptors has been used in order to obtain a selective uptake in macrophages and reduce adverse effects of off-target delivery of drugs. CD163 is a highly expressed macrophage-specific endocytic receptor that has been studied for intracellular delivery of small molecule drugs...... to macrophages using targeted liposomes or antibody drug conjugates. This review will focus on the biology of CD163 and its potential role as a target for selective macrophage targeting compared with other macrophage targeting approaches....

Dexamethasone targeted directly to macrophages induces macrophage niches that promote erythroid expansion

DEFF Research Database (Denmark)

Falchi, Mario; Varricchio, Lilian; Martelli, Fabrizio

2015-01-01

Cultures of human CD34(pos) cells stimulated with erythroid growth factors plus dexamethasone, a model for stress erythropoiesis, generate numerous erythroid cells plus a few macrophages (approx. 3%; 3:1 positive and negative for CD169). Interactions occurring between erythroblasts and macrophages...... in these cultures and the biological effects associated with these interactions were documented by live phase-contrast videomicroscopy. Macrophages expressed high motility interacting with hundreds/thousands of erythroblasts per hour. CD169(pos) macrophages established multiple rapid 'loose' interactions...... with proerythroblasts leading to formation of transient erythroblastic island-like structures. By contrast, CD169(neg) macrophages established 'tight' interactions with mature erythroblasts and phagocytosed these cells. 'Loose' interactions of CD169(pos) macrophages were associated with proerythroblast cytokinesis (the...

Nitric oxide protects macrophages from hydrogen peroxide-induced apoptosis by inducing the formation of catalase.

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Yoshioka, Yasuhiro; Kitao, Tatsuya; Kishino, Takashi; Yamamuro, Akiko; Maeda, Sadaaki

2006-04-15

We investigated the cytoprotective effect of NO on H2O2-induced cell death in mouse macrophage-like cell line RAW264. H2O2-treated cells showed apoptotic features, such as activation of caspase-9 and caspase-3, nuclear fragmentation, and DNA fragmentation. These apoptotic features were significantly inhibited by pretreatment for 24 h with NO donors, sodium nitroprusside and 1-hydroxy-2-oxo-3,3-bis-(2-aminoethyl)-1-triazene, at a low nontoxic concentration. The cytoprotective effect of NO was abrogated by the catalase inhibitor 3-amino-1,2,4-triazole but was not affected by a glutathione synthesis inhibitor, L-buthionine-(S,R)-sulfoximine. NO donors increased the level of catalase and its activity in a concentration-dependent manner. Cycloheximide, a protein synthesis inhibitor, inhibited both the NO-induced increase in the catalase level and the cytoprotective effect of NO. These results indicate that NO at a low concentration protects macrophages from H2O2-induced apoptosis by inducing the production of catalase.

Immunohistochemical study of macrophage migration inhibitory factor in rat liver fibrosis induced by thioacetamide

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Y Hori

2009-06-01

Full Text Available Macrophage migration inhibitory factor (MIF is a molecule known to regulate macrophage accumulation at sites of inflammation. To elucidate the role of MIF in progression of liver fibrosis, the immunohistochemical localization of MIF and macrophages in the liver were examined. Male Wistar rats received thioacetamide (TA injections (200 mg/kg, i.p. for 1 or 6 weeks. In biochemical and histological tests, it was confirmed that liver fibrosis was induced. In immunohistochemical analyses, the expression of MIF protein was seen in hepatocytes in the areas extending out from the central veins to the portal tracts. In particular, at 6 weeks, immunoreactivity was detected in degenerated hepatocytes adjacent to the fibrotic areas but hardly observed in the fibrotic areas. On the other hand, a number of exudate macrophages stained by antibody ED1 were seen in the areas from the central veins to the portal tracts at 1 week and in the fibrotic areas at 6 weeks. Macrophages also showed a significant increase in number as compared with controls. These results revealed that there was a close relationship between the appearance of MIF expression and ED1-positive exudate macrophages in degenerated hepatocytes during the progression of TA-induced liver fibrosis.

Immunohistochemical study of macrophage migration inhibitory factor in rat liver fibrosis induced by thioacetamide.

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Hori, Y; Sato, S; Yamate, J; Kurasaki, M; Nishihira, J; Hosokawa, T; Fujita, H; Saito, T

2003-01-01

Macrophage migration inhibitory factor (MIF) is a molecule known to regulate macrophage accumulation at sites of inflammation. To elucidate the role of MIF in progression of liver fibrosis, the immunohistochemical localization of MIF and macrophages in the liver were examined. Male Wistar rats received thioacetamide (TA) injections (200 mg/kg, i.p.) for 1 or 6 weeks. In biochemical and histological tests, it was confirmed that liver fibrosis was induced. In immunohistochemical analyses, the expression of MIF protein was seen in hepatocytes in the areas extending out from the central veins to the portal tracts. In particular, at 6 weeks, immunoreactivity was detected in degenerated hepatocytes adjacent to the fibrotic areas but hardly observed in the fibrotic areas. On the other hand, a number of exudate macrophages stained by antibody ED1 were seen in the areas from the central veins to the portal tracts at 1 week and in the fibrotic areas at 6 weeks. Macrophages also showed a significant increase in number as compared with controls. These results revealed that there was a close relationship between the appearance of MIF expression and ED1-positive exudate macrophages in degenerated hepatocytes during the progression of TA-induced liver fibrosis.

Andrographolide Inhibits Oxidized LDL-Induced Cholesterol Accumulation and Foam Cell Formation in Macrophages.

Science.gov (United States)

Lin, Hung-Chih; Lii, Chong-Kuei; Chen, Hui-Chun; Lin, Ai-Hsuan; Yang, Ya-Chen; Chen, Haw-Wen

2018-01-01

oxLDL is involved in the pathogenesis of atherosclerotic lesions through cholesterol accumulation in macrophage foam cells. Andrographolide, the bioactive component of Andrographis paniculata, possesses several biological activities such as anti-inflammatory, anti-oxidant, and anticancer functions. Scavenger receptors (SRs), including class A SR (SR-A) and CD36, are responsible for the internalization of oxLDL. In contrast, receptors for reverse cholesterol transport, including ABCA1 and ABCG1, mediate the efflux of cholesterol from macrophage foam cells. Transcription factor liver X receptor [Formula: see text] (LXR[Formula: see text] plays a key role in lipid metabolism and inflammation as well as in the regulation of ABCA1 and ABCG1 expression. Because of the contribution of inflammation to macrophage foam cell formation and the potent anti-inflammatory activity of andrographolide, we hypothesized that andrographolide might inhibit oxLDL-induced macrophage foam cell formation. The results showed that andrographolide reduced oxLDL-induced lipid accumulation in macrophage foam cells. Andrographolide decreased the mRNA and protein expression of CD36 by inducing the degradation of CD36 mRNA; however, andrographolide had no effect on SR-A expression. In contrast, andrographolide increased the mRNA and protein expression of ABCA1 and ABCG1, which were dependent on LXR[Formula: see text]. Andrographolide enhanced LXR[Formula: see text] nuclear translocation and DNA binding activity. Treatment with the LXR[Formula: see text] antagonist GGPP and transfection with LXR[Formula: see text] siRNA reversed the ability of andrographolide to stimulate ABCA1 and ABCG1 protein expression. In conclusion, inhibition of CD36-mediated oxLDL uptake and induction of ABCA1- and ABCG1-dependent cholesterol efflux are two working mechanisms by which andrographolide inhibits macrophage foam cell formation, which suggests that andrographolide could be a potential candidate to prevent

Macrophage migration inhibitory factor counter-regulates dexamethasone-induced annexin 1 expression and influences the release of eicosanoids in murine macrophages.

Science.gov (United States)

Sun, Yu; Wang, Yu; Li, Jia-Hui; Zhu, Shi-Hui; Tang, Hong-Tai; Xia, Zhao-Fan

2013-10-01

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine and glucocorticoid (GC) counter-regulator, has emerged as an important modulator of inflammatory responses. However, the molecular mechanisms of MIF counter-regulation of GC still remain incomplete. In the present study, we investigated whether MIF mediated the counter-regulation of the anti-inflammatory effect of GC by affecting annexin 1 in RAW 264.7 macrophages. We found that stimulation of RAW 264.7 macrophages with lipopolysaccharide (LPS) resulted in down-regulation of annexin 1, while GC dexamethasone (Dex) or Dex plus LPS led to significant up-regulation of annexin 1 expression. RNA interference-mediated knockdown of intracellular MIF increased annexin 1 expression with or without incubation of Dex, whereas Dex-induced annexin 1 expression was counter-regulated by the exogenous application of recombinant MIF. Moreover, recombinant MIF counter-regulated, in a dose-dependent manner, inhibition of cytosolic phospholipase A2α (cPLA2α) activation and prostaglandin E2 (PGE2 ) and leukotriene B4 (LTB4 ) release by Dex in RAW 264.7 macrophages stimulated with LPS. Endogenous depletion of MIF enhanced the effects of Dex, reflected by further decease of cPLA2α expression and lower PGE2 and LTB4 release in RAW 264.7 macrophages. Based on these data, we suggest that MIF counter-regulates Dex-induced annexin 1 expression, further influencing the activation of cPLA2α and the release of eicosanoids. These findings will add new insights into the mechanisms of MIF counter-regulation of GC. © 2013 John Wiley & Sons Ltd.

Immunohistochemical study of macrophage migration inhibitory factor in rat liver fibrosis induced by thioacetamide

OpenAIRE

Y Hori; S Sato; J Yamate; M Kurasaki

2009-01-01

Macrophage migration inhibitory factor (MIF) is a molecule known to regulate macrophage accumulation at sites of inflammation. To elucidate the role of MIF in progression of liver fibrosis, the immunohistochemical localization of MIF and macrophages in the liver were examined. Male Wistar rats received thioacetamide (TA) injections (200 mg/kg, i.p.) for 1 or 6 weeks. In biochemical and histological tests, it was confirmed that liver fibrosis was induced. In immunohistochemical analyses, the e...

Macrophage-induced cytostasis: kinetic analysis of bromodeoxyuridine-pulsed cells

International Nuclear Information System (INIS)

Stevenson, A.P.; Crissman, H.A.; Stewart, C.C.

1985-01-01

The effect of tumoricidal macrophages on the cell cycle progression of six different cell lines was studied using an anti-bromodeoxyuridine (BrdUrd) monoclonal antibody to follow the traverse of BrdUrd-labeled cells. Exponentially growing cultured mammalian cells, from six different cell lines, were prepulsed with BrdUrd before exposure to tumoricidal macrophages. The cultured cells were then analyzed as a function of time for DNA content (by propidium iodide staining) and for BrdUrd incorporation (using a fluoresceini-sothiocyanate [FITC]-conjugated anti-BrdUrd monoclonal antibody). The position of the cells in cycle and the progression of the BrdUrd-labeled cohort was followed using flow cytometry. The cell lines examined were: Colon 26; BALB/c-3T3, ST3T3 (a spontaneously transformed, tumorigenic clone of 3T3), WCHE5 (a clone of whole Chinese hamster embryo cells), RIF (a radiation-induced fibrosarcoma), and A101D (a human melanoma). The bivariate distributions showed that for all six cell lines the BrdUrd-labeled cohort in the control cultures progressed around the cell cycle during the first 12 h of culture, as the cells exponentially increased. In contrast, when each cell line was incubated with tumoricidal macrophages, the BrdUrd-labeled cohort did not progress through cell cycle but remained in S phase throughout the 12-h culture period. There was also no evidence for progression of cells out of G 1 . The data show that cells were arrested in every phase of cell cycle. This study suggests that cytostasis, as manifested by the termination of progression in all phases of the cell cycle, is a universal phenomenon induced by tumoricidal macrophages. 20 references, 4 figures

Butylated Hydroxyanisole Blocks the Occurrence of Tumor Associated Macrophages in Tobacco Smoke Carcinogen-Induced Lung Tumorigenesis

International Nuclear Information System (INIS)

Zhang, Yan; Choksi, Swati; Liu, Zheng-Gang

2013-01-01

Tumor-associated macrophages (TAMs) promote tumorigenesis because of their proangiogenic and immune-suppressive functions. Here, we report that butylated hydroxyanisole (BHA) blocks occurrence of tumor associated macrophages (TAMs) in tobacco smoke carcinogen-induced lung tumorigenesis. Continuous administration of butylated hydroxyanisole (BHA), a ROS inhibitor, before or after NNK treatment significantly blocked tumor development, although less effectively when BHA is administered after NNK treatment. Strikingly, BHA abolished the occurrence of F4/80 + macrophages with similar efficiency no matter whether it was administered before or after NNK treatment. Detection of cells from bronchioalveolar lavage fluid (BALF) confirmed that BHA markedly inhibited the accumulation of macrophages while slightly reducing the number of lymphocytes that were induced by NNK. Immunohistological staining showed that BHA specifically abolished the occurrence of CD206 + TAMs when it was administered before or after NNK treatment. Western blot analysis of TAMs markers, arginase I and Ym-1, showed that BHA blocked NNK-induced TAMs accumulation. Our study clearly demonstrated that inhibiting the occurrence of TAMs by BHA contributes to the inhibition of tobacco smoke carcinogen-induced tumorigenesis, suggesting ROS inhibitors may serve as a therapeutic target for treating smoke-induced lung cancer

SUCNR1-mediated chemotaxis of macrophages aggravates obesity-induced inflammation and diabetes.

NARCIS (Netherlands)

Diepen, van Janna A.; Hooiveld, Guido; Stienstra, Rinke; Deen, Peter M.

2017-01-01

Obesity induces macrophages to drive inflammation in adipose tissue, a crucial step towards the development of type 2 diabetes. The tricarboxylic acid (TCA) cycle intermediate succinate is released from cells under metabolic stress and has recently emerged as a metabolic signal induced by

Mycolactone cytotoxicity in Schwann cells could explain nerve damage in Buruli ulcer.

Directory of Open Access Journals (Sweden)

Junichiro En

2017-08-01

Full Text Available Buruli ulcer is a chronic painless skin disease caused by Mycobacterium ulcerans. The local nerve damage induced by M. ulcerans invasion is similar to the nerve damage evoked by the injection of mycolactone in a Buruli ulcer mouse model. In order to elucidate the mechanism of this nerve damage, we tested and compared the cytotoxic effect of synthetic mycolactone A/B on cultured Schwann cells, fibroblasts and macrophages. Mycolactone induced much higher cell death and apoptosis in Schwann cell line SW10 than in fibroblast line L929. These results suggest that mycolactone is a key substance in the production of nerve damage of Buruli ulcer.

Cisplatin Induces Cytotoxicity through the Mitogen-Activated Protein Kinase Pathways ana Activating Transcription Factor 3

Directory of Open Access Journals (Sweden)

Carly St. Germain

2010-07-01

Full Text Available The mechanisms underlying the proapoptotic effect of the chemotherapeutic agent, cisplatin, are largely undefined. Understanding the mechanisms regulating cisplatin cytotoxicity may uncover strategies to enhance the efficacy of this important therapeutic agent. This study evaluates the role of activating transcription factor 3 (ATF3 as a mediator of cisplatin-induced cytotoxicity. Cytotoxic doses of cisplatin and carboplatin treatments consistently induced ATF3 expression in five tumor-derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response-independent mechanism, all previously implicated in stress-mediated ATF3 induction. Analysis of mitogenactivated protein kinase (MAPK pathway involvement in ATF3 induction by cisplatin revealed a MAPK-dependent mechanism. Cisplatin treatment combined with specific inhibitors to each MAPK pathway (c-Jun N-terminal kinase, extracellularsignal-regulated kinase, and p38 resulted in decreasedATF3 induction at the protein level. MAPK pathway inhibition led to decreased ATF3 messenger RNA expression and reduced cytotoxic effects of cisplatin as measured by the 3-(4,5-dimethylthiazol-2-ylF2,5-diphenyltetrazolium bromide cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific small hairpin RNA also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3-/murine embryonic fibroblasts (MEFs were shown to be less sensitive to cisplatin-induced cytotoxicity compared with ATF3+/+ MEFs. This study identifies cisplatin as a MAPK pathway-dependent inducer of ATF3, whose expression influences cisplatin’s cytotoxic effects.

Activation of Rho GTPases by Cytotoxic Necrotizing Factor 1 Induces Macropinocytosis and Scavenging Activity in Epithelial Cells

Science.gov (United States)

Fiorentini, Carla; Falzano, Loredana; Fabbri, Alessia; Stringaro, Annarita; Logozzi, Mariaantonia; Travaglione, Sara; Contamin, Stéphanette; Arancia, Giuseppe; Malorni, Walter; Fais, Stefano

2001-01-01

Macropinocytosis, a ruffling-driven process that allows the capture of large material, is an essential aspect of normal cell function. It can be either constitutive, as in professional phagocytes where it ends with the digestion of captured material, or induced, as in epithelial cells stimulated by growth factors. In this case, the internalized material recycles back to the cell surface. We herein show that activation of Rho GTPases by a bacterial protein toxin, the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), allowed epithelial cells to engulf and digest apoptotic cells in a manner similar to that of professional phagocytes. In particular, we have demonstrated that 1) the activation of all Rho, Rac, and Cdc42 by CNF1 was essential for the capture and internalization of apoptotic cells; and 2) such activation allowed the discharge of macropinosomal content into Rab7 and lysosomal associated membrane protein-1 acidic lysosomal vesicles where the ingested particles underwent degradation. Taken together, these findings indicate that CNF1-induced “switching on” of Rho GTPases may induce in epithelial cells a scavenging activity, comparable to that exerted by professional phagocytes. The activation of such activity in epithelial cells may be relevant, in mucosal tissues, in supporting or integrating the scavenging activity of resident macrophages. PMID:11452003

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...... delay in the expansion of CD45(dim) CD11b(+) microglia in clodronate-liposome treated mice, but macrophage depletion had no effect on the percentage of infiltrating T cells in the lesion-reactive hippocampus. Lesion-induced TNFalpha mRNA expression was not affected by macrophage depletion, suggesting...... that activated glial cells are the primary source of this cytokine in the axonal injury-reactive brain. This identifies a potentially important distinction from inflammatory autoimmune infiltration in EAE, where macrophages are a prominent source of TNFalpha and their depletion prevents parenchymal T cell...

Specific inhibition of cytotoxic memory cells produced against uv-induced tumors in uv-irradiation mice

International Nuclear Information System (INIS)

Thorn, R.M.

1978-01-01

Cytotoxic responses of uv-irradiated mice against syngeneic uv-induced tumors were measured by using a 51 Cr-release assay to determine if uv treatment induced a specific reduction of cytotoxic activity. The in vivo and in vitro primary responses against syngeneic tumors and allogeneic cells were unaffected, as was the ''memory'' response (in vivo stimulation, in vitro restimulation) against alloantigens. In contrast, the memory response of uv-treated mice against syngeneic, uv-induced tumors was consistently and significantly depressed. The cytotoxicity generated by tumor cell stimulation in vivo or in vitro was tumor-specific and T cell-dependent. Since the primary response against syngeneic uv-induced tumors produces apparently normal amounts of tumor-specific cytotoxic activity, uv-treated mice may not reject transplanted syngeneic tumors because of too few T effector memory cells. These results imply that, at least in this system, tumor rejection depends mostly on the secondary responses against tumor antigens and that at least one carcinogen can, indirectly, specifically regulate immune responses

Cyclosporine A and palmitic acid treatment synergistically induce cytotoxicity in HepG2 cells

Energy Technology Data Exchange (ETDEWEB)

Luo, Yi, E-mail: yi.luo@pfizer.com; Rana, Payal; Will, Yvonne

2012-06-01

Immunosuppressant cyclosporine A (CsA) treatment can cause severe side effects. Patients taking immunosuppressant after organ transplantation often display hyperlipidemia and obesity. Elevated levels of free fatty acids have been linked to the etiology of metabolic syndromes, nonalcoholic fatty liver and steatohepatitis. The contribution of free fatty acids to CsA-induced toxicity is not known. In this study we explored the effect of palmitic acid on CsA-induced toxicity in HepG2 cells. CsA by itself at therapeutic exposure levels did not induce detectible cytotoxicity in HepG2 cells. Co-treatment of palmitic acid and CsA resulted in a dose dependent increase in cytotoxicity, suggesting that fatty acid could sensitize cells to CsA-induced cytotoxicity at the therapeutic doses of CsA. A synergized induction of caspase-3/7 activity was also observed, indicating that apoptosis may contribute to the cytotoxicity. We demonstrated that CsA reduced cellular oxygen consumption which was further exacerbated by palmitic acid, implicating that impaired mitochondrial respiration might be an underlying mechanism for the enhanced toxicity. Inhibition of c-Jun N-terminal kinase (JNK) attenuated palmitic acid and CsA induced toxicity, suggesting that JNK activation plays an important role in mediating the enhanced palmitic acid/CsA-induced toxicity. Our data suggest that elevated FFA levels, especially saturated FFA such as palmitic acid, may be predisposing factors for CsA toxicity, and patients with underlying diseases that would elevate free fatty acids may be susceptible to CsA-induced toxicity. Furthermore, hyperlipidemia/obesity resulting from immunosuppressive therapy may aggravate CsA-induced toxicity and worsen the outcome in transplant patients. -- Highlights: ► Palmitic acid and cyclosporine (CsA) synergistically increased cytotoxicity. ► The impairment of mitochondrial functions may contribute to the enhanced toxicity. ► Inhibition of JNK activity attenuated

Increased expression of interleukin-1β in triglyceride-induced macrophage cell death is mediated by p38 MAP kinase.

Science.gov (United States)

Sung, Ho Joong; Son, Sin Jee; Yang, Seung-ju; Rhee, Ki-Jong; Kim, Yoon Suk

2012-07-01

Triglycerides (TG) are implicated in the development of atherosclerosis through formation of foam cells and induction of macrophage cell death. In this study, we report that addition of exogenous TG induced cell death in phorbol 12-myristate 13-acetate-differentiated THP-1 human macrophages. TG treatment induced a dramatic decrease in interleukin-1β (IL-1β) mRNA expression in a dose- and time-dependent manner. The expression of granulocyte macrophage colony-stimulating factor and platelet endothelial cell adhesion molecule remained unchanged. To identify signaling pathways involved in TG-induced downregulation of IL-1β, we added p38 MAPK, protein kinase C (PKC) or c-Raf1 specific inhibitors. We found that inhibition of p38 MAPK alleviated the TG-induced downregulation of IL-1β, whereas inhibition of PKC and c-Raf1 had no effect. This is the first report showing decreased IL-1β expression during TG-induced cell death in a human macrophage line. Our results suggest that downregulation of IL-1β expression by TG-treated macrophages may play a role during atherogenesis.

Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

Science.gov (United States)

Danelishvili, Lia; McGarvey, Jeffery; Li, Yong-Jun; Bermudez, Luiz E

2003-09-01

Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the

Anti-inflammatory effects of benfotiamine are mediated through the regulation of the arachidonic acid pathway in macrophages.

Science.gov (United States)

Shoeb, Mohammad; Ramana, Kota V

2012-01-01

Benfotiamine, a lipid-soluble analogue of vitamin B1, is a potent antioxidant that is used as a food supplement for the treatment of diabetic complications. Our recent study (U.C. Yadav et al., Free Radic. Biol. Med. 48:1423-1434, 2010) indicates a novel role for benfotiamine in the prevention of bacterial endotoxin, lipopolysaccharide (LPS)-induced cytotoxicity and inflammatory response in murine macrophages. Nevertheless, it remains unclear how benfotiamine mediates anti-inflammatory effects. In this study, we investigated the anti-inflammatory role of benfotiamine in regulating arachidonic acid (AA) pathway-generated inflammatory lipid mediators in RAW264.7 macrophages. Benfotiamine prevented the LPS-induced activation of cPLA2 and release of AA metabolites such as leukotrienes, prostaglandin E2, thromboxane 2 (TXB2), and prostacyclin (PGI2) in macrophages. Further, LPS-induced expression of AA-metabolizing enzymes such as COX-2, LOX-5, TXB synthase, and PGI2 synthase was significantly blocked by benfotiamine. Furthermore, benfotiamine prevented the LPS-induced phosphorylation of ERK1/2 and expression of transcription factors NF-κB and Egr-1. Benfotiamine also prevented the LPS-induced oxidative stress and protein-HNE adduct formation. Most importantly, compared to specific COX-2 and LOX-5 inhibitors, benfotiamine significantly prevented LPS-induced macrophage death and monocyte adhesion to endothelial cells. Thus, our studies indicate that the dual regulation of the COX and LOX pathways in AA metabolism could be a novel mechanism by which benfotiamine exhibits its potential anti-inflammatory response. Copyright © 2011 Elsevier Inc. All rights reserved.

Increased Tim-3 expression alleviates liver injury by regulating macrophage activation in MCD-induced NASH mice.

Science.gov (United States)

Du, Xianhong; Wu, Zhuanchang; Xu, Yong; Liu, Yuan; Liu, Wen; Wang, Tixiao; Li, Chunyang; Zhang, Cuijuan; Yi, Fan; Gao, Lifen; Liang, Xiaohong; Ma, Chunhong

2018-05-07

As an immune checkpoint, Tim-3 plays roles in the regulation of both adaptive and innate immune cells including macrophages and is greatly involved in chronic liver diseases. However, the precise roles of Tim-3 in nonalcoholic steatohepatitis (NASH) remain unstated. In the current study, we analyzed Tim-3 expression on different subpopulations of liver macrophages and further investigated the potential roles of Tim-3 on hepatic macrophages in methionine and choline-deficient diet (MCD)-induced NASH mice. The results of flow cytometry demonstrated the significantly increased expression of Tim-3 on all detected liver macrophage subsets in MCD mice, including F4/80 + CD11b + , F4/80 + CD68 + , and F4/80 + CD169 + macrophages. Remarkably, Tim-3 knockout (KO) significantly accelerated MCD-induced liver steatosis, displaying higher serum ALT, larger hepatic vacuolation, more liver lipid deposition, and more severe liver fibrosis. Moreover, compared with wild-type C57BL/6 mice, Tim-3 KO MCD mice demonstrated an enhanced expression of NOX2, NLRP3, and caspase-1 p20 together with increased generation of IL-1β and IL-18 in livers. In vitro studies demonstrated that Tim-3 negatively regulated the production of reactive oxygen species (ROS) and related downstream pro-inflammatory cytokine secretion of IL-1β and IL-18 in macrophages. Exogenous administration of N-Acetyl-L-cysteine (NAC), a small molecular inhibitor of ROS, remarkably suppressed caspase-1 p20 expression and IL-1β and IL-18 production in livers of Tim-3 KO mice, thus significantly reducing the severity of steatohepatitis induced by MCD. In conclusion, Tim-3 is a promising protector in MCD-induced steatohepatitis by controlling ROS and the associated pro-inflammatory cytokine production in macrophages.

Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

International Nuclear Information System (INIS)

Ristic, Biljana; Bosnjak, Mihajlo; Arsikin, Katarina; Mircic, Aleksandar; Suzin-Zivkovic, Violeta; Bogdanovic, Andrija; Perovic, Vladimir; Martinovic, Tamara; Kravic-Stevovic, Tamara; Bumbasirevic, Vladimir; Trajkovic, Vladimir; Harhaji-Trajkovic, Ljubica

2014-01-01

We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy. - Highlights: • Idarubicin induces autophagy in leukemic cell lines and primary leukemic cells. • Idarubicin induces autophagy by inhibiting mTOR in leukemic cells. • mTOR suppression by idarubicin is associated with AMPK activation and Akt blockade. �

Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

Energy Technology Data Exchange (ETDEWEB)

Ristic, Biljana [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Bosnjak, Mihajlo [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Arsikin, Katarina [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Mircic, Aleksandar; Suzin-Zivkovic, Violeta [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Bogdanovic, Andrija [Clinic for Hematology, Clinical Centre of Serbia, School of Medicine, University of Belgrade, Belgrade (Serbia); Perovic, Vladimir [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Martinovic, Tamara; Kravic-Stevovic, Tamara; Bumbasirevic, Vladimir [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Trajkovic, Vladimir, E-mail: vtrajkovic@med.bg.ac.rs [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Harhaji-Trajkovic, Ljubica, E-mail: buajk@yahoo.com [Institute for Biological Research, University of Belgrade, Belgrade, Despot Stefan Blvd. 142, 11000 Belgrade (Serbia)

2014-08-01

We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy. - Highlights: • Idarubicin induces autophagy in leukemic cell lines and primary leukemic cells. • Idarubicin induces autophagy by inhibiting mTOR in leukemic cells. • mTOR suppression by idarubicin is associated with AMPK activation and Akt blockade. �

Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays

International Nuclear Information System (INIS)

Vieira, J.M.B.D.; Seabra, S.H.; Vallim, D.C.; Americo, M.A.; Fracallanza, S.E.L.; Vommaro, R.C.; Domingues, R.M.C.P.

2009-01-01

Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.

Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays

Energy Technology Data Exchange (ETDEWEB)

Vieira, J.M.B.D., E-mail: jmanya@terra.com.br [Laboratorio de Tecnologia em Cultura de Celulas, UEZO, Rio de Janeiro (Brazil); Laboratorio de Biologia de Anaerobios, IMPPG, UFRJ, Rio de Janeiro (Brazil); Seabra, S.H. [Laboratorio de Tecnologia em Cultura de Celulas, UEZO, Rio de Janeiro (Brazil); Vallim, D.C. [Instituto Oswaldo Cruz, Rio de Janeiro (Brazil); Americo, M.A.; Fracallanza, S.E.L. [Laboratorio de Bacteriologia Medica, IMPPG, UFRJ, Rio de Janeiro (Brazil); Vommaro, R.C. [Laboratorio de Ultra-estrutura Celular Hertha Meyer, IBCCF, UFRJ (Brazil); Domingues, R.M.C.P. [Laboratorio de Biologia de Anaerobios, IMPPG, UFRJ, Rio de Janeiro (Brazil)

2009-10-02

Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.

Anti-Inflammatory Effects of Benfotiamine are Mediated Through the Regulation of Arachidonic Acid Pathway in Macrophages

OpenAIRE

Shoeb, Mohammad; Ramana, Kota V

2011-01-01

Benfotiamine, a lipid-soluble analogue of vitamin B1, is a potent anti-oxidant that is used as a food supplement for the treatment of diabetic complications. Our recent study indicates a novel role of benfotiamine in the prevention of bacterial endotoxin, lipopolysaccharide (LPS)-induced cytotoxicity and inflammatory response in murine macrophages. Nevertheless, it remains unclear how benfotiamine mediates anti-inflammatory effects. In this study, we investigated the anti-inflammatory role of...

Reactions of macrophages exposed to particles <10 μm

International Nuclear Information System (INIS)

Monn, Christian; Naef, Roland; Koller, Theo

2003-01-01

This study describes experiments on cytotoxic effects and the production of oxidative radicals and the proinflammatory cytokine tumor growth factor alpha (TNFα) in a cell line of rat lung macrophages exposed to aqueou extracts from ambient air particles 10 ) collected on Teflon filters. The particles were collected during the four seasons at two urban sites, one rural site, and one alpine site in Switzerland. Cytotoxic effects determined as a reduction in the metabolic activity, were found in particle extracts from all sites and seasons. Taking together the data from all site and seasons, a dose-response function was observed between the particle mass on the filter and toxicity (r 2 =0.633, linear regression). The release of the pro-inflammatory cytokine TNFα as well as of oxidative radicals was most pronounced in particles collected in spring-summer and autumn. While a Montana (alpine), the stimulation of the cells was positively correlated with the particle mass on the filters, this correlation was negative at the urban sites Zuerich and Lugano. It is interpreted that at high PM 10 levels, as in these cities, macrophages are inhibited by increasing air pollution due to toxic effects. Cytotoxic effects and the release of oxidative radicals could be inhibited when the extracts were treated with an endotoxin-neutralizing protein. This suggests that endotoxin, a cell-wall constituent of gram-negative bacteria, is one of the factors which modulates macrophag activity. All together, the experiments indicate that in the PM 10 fraction water-soluble macrophage-toxic and macrophage-stimulating compounds ar present. The data offer an explanation for at least some of the known harmful effects of PM 10 , and confirm endotoxin as a possible reactant

[Characteristic and function of peripheral blood mononuclear cells-induced macrophages in patients with myelodysplastic syndrome].

Science.gov (United States)

Han, Y; Wang, H Q; Fu, R; Qu, W; Ruan, E B; Wang, X M; Wang, G J; Wu, Y H; Liu, H; Song, J; Guan, J; Xing, L M; Li, L J; Jiang, H J; Liu, H; Wang, Y H; Liu, C Y; Zhang, W; Shao, Z H

2017-08-14

Objective: To explore characteristic and function of peripheral blood mononuclear cells (PBMNC) -induced macrophages in patients with myelodysplastic syndrome (MDS) to couple with its progression. Methods: A total of 24 MDS patients (11 low-risk patients and 13 high-risk group patients) referred to Department of Hematology of Tianjin Medical University General Hospital and normal controls were enrolled from September 2014 to December 2015. PBMNC was stimulated with GM-CSF to transform to macrophages. The morphology of macrophages was observed by microscope. The quantity of macrophages, CD206 and SIRPα on surface of macrophages were detected by flow cytometry. The phagocytic function of macrophages was analyzed by fluorescence microscopy and flow cytometry. Results: The morphology of macrophages from MDS patients was abnormal. The percentage of transformed macrophages was (5.17±3.47) % in patients with MDS, which was lower than that in controls significantly[ (66.18±13.43) %, t =3.529, P =0.001]. The expression of CD206 on macrophages from MDS patients was significantly lower than that of controls[ (9.73±2.59) % vs (51.15±10.82) %, t =4.551, P patients was significantly lower than that of controls [ (0.51±0.09) % vs (0.77±0.06) %, t =2.102, P =0.043]. The phagocytic index and the percentage of phagocytic of macrophages from MDS patients were significantly lower than those of macrophages from normal controls[0.45±0.08 vs 0.92±0.07, t =-6.253, P =0.008; (23.69±3.22) % vs (42.75±2.13) %, t =-6.982, P =0.006 respectively]by flow cytometry. The phagocytic index of MDS patients was significantly lower than that of controls (0.24±0.04 vs 0.48±0.96, t =3.464, P =0.001) by fluorescence microscopy. Conclusion: The quantity, recognization receptors and phagocytosis of PBMNC-induced macrophages decreased in MDS patients.

Evaluation of the cytotoxicity of dihydroxytryptamines and 5-hydroxytryptamine antagonists as cytotoxic agents in dimethylhydrazine-induced adenocarcinomata.

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Tutton, P J; Barkla, D H

1978-01-01

The cytotoxicity of 5,6-dihydroxytryptamine (5,6-DHT), 5,7-dihydroxytryptamine (5,7-DHT), bromolysergic acid diethylamide (BOL), methysergide, and cyproheptadine, and also of 5,6-DHT together with either BOL, methysergide, or cyproheptadine in dimethylhydrazine-induced (DMH) carcinomata of rat colon was evaluated by estimating the percentage of necrotic cells in histological sections of tissues taken 15 h after injection of each of the drugs. In addition, the influence of methysergide and cyproheptadine on the tumour cell mitotic rate was estimated by means of a stathmokinetic technique. Both 5,6-DHT and 5,7-DHT were cytotoxic at each dose tested and for each of these agents the percentage of necrotic cells was directly correlated with the dose of drug used. BOL was not found to be cytotoxic to the colonic carcinomata, whereas both methysergide and cyproheptadine did cause detectable tumour cell necrosis. Methysergide was also found to accelerate tumour cell proliferation, whereas cyproheptadine did not. BOL competitively inhibited the cytotoxicity of 5,6-DHT and neither methysergide nor cyproheptadine potentiated the effect of 5,6 DHT.

Carbon black nanoparticles induce type II epithelial cells to release chemotaxins for alveolar macrophages

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Donaldson Ken

2005-12-01

Full Text Available Abstract Background Alveolar macrophages are a key cell in dealing with particles deposited in the lungs and in determining the subsequent response to that particle exposure. Nanoparticles are considered a potential threat to the lungs and the mechanism of pulmonary response to nanoparticles is currently under intense scrutiny. The type II alveolar epithelial cell has previously been shown to release chemoattractants which can recruit alveolar macrophages to sites of particle deposition. The aim of this study was to assess the responses of a type II epithelial cell line (L-2 to both fine and nanoparticle exposure in terms of secretion of chemotactic substances capable of inducing macrophage migration. Results Exposure of type II cells to carbon black nanoparticles resulted in significant release of macrophage chemoattractant compared to the negative control and to other dusts tested (fine carbon black and TiO2 and nanoparticle TiO2 as measured by macrophage migration towards type II cell conditioned medium. SDS-PAGE analysis of the conditioned medium from particle treated type II cells revealed that a higher number of protein bands were present in the conditioned medium obtained from type II cells treated with nanoparticle carbon black compared to other dusts tested. Size-fractionation of the chemotaxin-rich supernatant determined that the chemoattractants released from the epithelial cells were between 5 and 30 kDa in size. Conclusion The highly toxic nature and reactive surface chemistry of the carbon black nanoparticles has very likely induced the type II cell line to release pro-inflammatory mediators that can potentially induce migration of macrophages. This could aid in the rapid recruitment of inflammatory cells to sites of particle deposition and the subsequent removal of the particles by phagocytic cells such as macrophages and neutrophils. Future studies in this area could focus on the exact identity of the substance(s released by the

Tumor associated macrophages protect colon cancer cells from TRAIL-induced apoptosis through IL-1beta-dependent stabilization of Snail in tumor cells.

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Pawan Kaler

2010-07-01

Full Text Available We recently reported that colon tumor cells stimulate macrophages to release IL-1beta, which in turn inactivates GSK3beta and enhances Wnt signaling in colon cancer cells, generating a self-amplifying loop that promotes the growth of tumor cells.Here we describe that macrophages protect HCT116 and Hke-3 colon cancer cells from TRAIL-induced apoptosis. Inactivation of IL-1beta by neutralizing IL-1beta antibody, or silencing of IL-1beta in macrophages inhibited their ability to counter TRAIL-induced apoptosis. Accordingly, IL-1beta was sufficient to inhibit TRAIL-induced apoptosis. TRAIL-induced collapse of the mitochondrial membrane potential (Delta psi and activation of caspases were prevented by macrophages or by recombinant IL-1beta. Pharmacological inhibition of IL-1beta release from macrophages by vitamin D(3, a potent chemopreventive agent for colorectal cancer, restored the ability of TRAIL to induce apoptosis of tumor cells cultured with macrophages. Macrophages and IL-1beta failed to inhibit TRAIL-induced apoptosis in HCT116 cells expressing dnIkappaB, dnAKT or dnTCF4, confirming that they oppose TRAIL-induced cell death through induction of Wnt signaling in tumor cells. We showed that macrophages and IL-1beta stabilized Snail in tumor cells in an NF-kappaB/Wnt dependent manner and that Snail deficient tumor cells were not protected from TRAIL-induced apoptosis by macrophages or by IL-1beta, demonstrating a crucial role of Snail in the resistance of tumor cells to TRAIL.We have identified a positive feedback loop between tumor cells and macrophages that propagates the growth and promotes the survival of colon cancer cells: tumor cells stimulate macrophages to secrete IL-1beta, which in turn, promotes Wnt signaling and stabilizes Snail in tumor cells, conferring resistance to TRAIL. Vitamin D(3 halts this amplifying loop by interfering with the release of IL-1beta from macrophages. Accordingly, vitamin D(3 sensitizes tumor cells to TRAIL-induced

Urtica dioica Induces Cytotoxicity in Human Prostate Carcinoma ...

African Journals Online (AJOL)

In order to evaluate the involvement of caspases in UD-AQ induced cytotoxicity, the activities of caspase 3 and 9 were measured using a colorimetric assay. Following treatment of. LNCaP cells with UD-AQ extract (50 µg/ml) in 6- well plates, cells were collected by centrifugation and lysed with lysis buffer (1 % Triton X-100,.

VEGF-production by CCR2-dependent macrophages contributes to laser-induced choroidal neovascularization.

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Torsten A Krause

Full Text Available Age-related macular degeneration (AMD is the most prevalent cause of blindness in the elderly, and its exsudative subtype critically depends on local production of vascular endothelial growth factor A (VEGF. Mononuclear phagocytes, such as macrophages and microglia cells, can produce VEGF. Their precursors, for example monocytes, can be recruited to sites of inflammation by the chemokine receptor CCR2, and this has been proposed to be important in AMD. To investigate the role of macrophages and CCR2 in AMD, we studied intracellular VEGF content in a laser-induced murine model of choroidal neovascularisation. To this end, we established a technique to quantify the VEGF content in cell subsets from the laser-treated retina and choroid separately. 3 days after laser, macrophage numbers and their VEGF content were substantially elevated in the choroid. Macrophage accumulation was CCR2-dependent, indicating recruitment from the circulation. In the retina, microglia cells were the main VEGF+ phagocyte type. A greater proportion of microglia cells contained VEGF after laser, and this was CCR2-independent. On day 6, VEGF-expressing macrophage numbers had already declined, whereas numbers of VEGF+ microglia cells remained increased. Other sources of VEGF detectable by flow cytometry included in dendritic cells and endothelial cells in both retina and choroid, and Müller cells/astrocytes in the retina. However, their VEGF content was not increased after laser. When we analyzed flatmounts of laser-treated eyes, CCR2-deficient mice showed reduced neovascular areas after 2 weeks, but this difference was not evident 3 weeks after laser. In summary, CCR2-dependent influx of macrophages causes a transient VEGF increase in the choroid. However, macrophages augmented choroidal neovascularization only initially, presumably because VEGF production by CCR2-independent eye cells prevailed at later time points. These findings identify macrophages as a relevant source

Quantification of sterol-specific response in human macrophages using automated imaged-based analysis.

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Gater, Deborah L; Widatalla, Namareq; Islam, Kinza; AlRaeesi, Maryam; Teo, Jeremy C M; Pearson, Yanthe E

2017-12-13

The transformation of normal macrophage cells into lipid-laden foam cells is an important step in the progression of atherosclerosis. One major contributor to foam cell formation in vivo is the intracellular accumulation of cholesterol. Here, we report the effects of various combinations of low-density lipoprotein, sterols, lipids and other factors on human macrophages, using an automated image analysis program to quantitatively compare single cell properties, such as cell size and lipid content, in different conditions. We observed that the addition of cholesterol caused an increase in average cell lipid content across a range of conditions. All of the sterol-lipid mixtures examined were capable of inducing increases in average cell lipid content, with variations in the distribution of the response, in cytotoxicity and in how the sterol-lipid combination interacted with other activating factors. For example, cholesterol and lipopolysaccharide acted synergistically to increase cell lipid content while also increasing cell survival compared with the addition of lipopolysaccharide alone. Additionally, ergosterol and cholesteryl hemisuccinate caused similar increases in lipid content but also exhibited considerably greater cytotoxicity than cholesterol. The use of automated image analysis enables us to assess not only changes in average cell size and content, but also to rapidly and automatically compare population distributions based on simple fluorescence images. Our observations add to increasing understanding of the complex and multifactorial nature of foam-cell formation and provide a novel approach to assessing the heterogeneity of macrophage response to a variety of factors.

Metformin reduces the endotoxin-induced down-regulation of apolipoprotein E gene expression in macrophages

Energy Technology Data Exchange (ETDEWEB)

Stavri, Simona; Trusca, Violeta G.; Simionescu, Maya; Gafencu, Anca V., E-mail: anca.gafencu@icbp.ro

2015-05-29

The atheroprotective role of macrophage-derived apolipoprotein E (apoE) is well known. Our previous reports demonstrated that inflammatory stress down-regulates apoE expression in macrophages, aggravating atherogenesis. Metformin, extensively used as an anti-diabetic drug, has also anti-inflammatory properties, and thus confers vascular protection. In this study, we questioned whether metformin could have an effect on apoE expression in macrophages in normal conditions or under lipopolysaccharide (LPS)-induced stress. The results showed that metformin slightly increases the apoE expression only at high doses (5–10 mM). Low doses of metformin (1–3 mM) significantly reduce the LPS down-regulatory effect on apoE expression in macrophages. Our experiments demonstrated that LPS-induced NF-κB binds to the macrophage-specific distal regulatory element of apoE gene, namely to the multienhancer 2 (ME.2) and its 5′-deletion fragments. The NF-κB binding on ME.2 and apoE promoter has a down-regulatory effect. In addition, data revealed that metformin impairs NF-κB nuclear translocation, and thus, improves the apoE levels in macrophages under inflammatory stress. The positive effect of metformin in the inflammatory states, its clinical safety and low cost, make this drug a potential adjuvant in the therapeutic strategies for atherosclerosis. - Highlights: • High doses of metformin slightly increase apoE expression in macrophages. • Low doses of metformin up-regulate apoE gene in endotoxin-stressed macrophages. • Metformin reduces the negative effect of LPS on apoE expression by NF-κB inhibition.

Metformin reduces the endotoxin-induced down-regulation of apolipoprotein E gene expression in macrophages

International Nuclear Information System (INIS)

Stavri, Simona; Trusca, Violeta G.; Simionescu, Maya; Gafencu, Anca V.

2015-01-01

The atheroprotective role of macrophage-derived apolipoprotein E (apoE) is well known. Our previous reports demonstrated that inflammatory stress down-regulates apoE expression in macrophages, aggravating atherogenesis. Metformin, extensively used as an anti-diabetic drug, has also anti-inflammatory properties, and thus confers vascular protection. In this study, we questioned whether metformin could have an effect on apoE expression in macrophages in normal conditions or under lipopolysaccharide (LPS)-induced stress. The results showed that metformin slightly increases the apoE expression only at high doses (5–10 mM). Low doses of metformin (1–3 mM) significantly reduce the LPS down-regulatory effect on apoE expression in macrophages. Our experiments demonstrated that LPS-induced NF-κB binds to the macrophage-specific distal regulatory element of apoE gene, namely to the multienhancer 2 (ME.2) and its 5′-deletion fragments. The NF-κB binding on ME.2 and apoE promoter has a down-regulatory effect. In addition, data revealed that metformin impairs NF-κB nuclear translocation, and thus, improves the apoE levels in macrophages under inflammatory stress. The positive effect of metformin in the inflammatory states, its clinical safety and low cost, make this drug a potential adjuvant in the therapeutic strategies for atherosclerosis. - Highlights: • High doses of metformin slightly increase apoE expression in macrophages. • Low doses of metformin up-regulate apoE gene in endotoxin-stressed macrophages. • Metformin reduces the negative effect of LPS on apoE expression by NF-κB inhibition

Sulforaphane Protects the Liver against CdSe Quantum Dot-Induced Cytotoxicity.

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Wei Wang

Full Text Available The potential cytotoxicity of cadmium selenide (CdSe quantum dots (QDs presents a barrier to their use in biomedical imaging or as diagnostic and therapeutic agents. Sulforaphane (SFN is a chemoprotective compound derived from cruciferous vegetables which can up-regulate antioxidant enzymes and induce apoptosis and autophagy. This study reports the effects of SFN on CdSe QD-induced cytotoxicity in immortalised human hepatocytes and in the livers of mice. CdSe QDs induced dose-dependent cell death in hepatocytes with an IC50 = 20.4 μM. Pre-treatment with SFN (5 μM increased cell viability in response to CdSe QDs (20 μM from 49.5 to 89.3%. SFN induced a pro-oxidant effect characterized by depletion of intracellular reduced glutathione during short term exposure (3-6 h, followed by up-regulation of antioxidant enzymes and glutathione levels at 24 h. SFN also caused Nrf2 translocation into the nucleus, up-regulation of antioxidant enzymes and autophagy. siRNA knockdown of Nrf2 suggests that the Nrf2 pathway plays a role in the protection against CdSe QD-induced cell death. Wortmannin inhibition of SFN-induced autophagy significantly suppressed the protective effect of SFN on CdSe QD-induced cell death. Moreover, the role of autophagy in SFN protection against CdSe QD-induced cell death was confirmed using mouse embryonic fibroblasts lacking ATG5. CdSe QDs caused significant liver damage in mice, and this was decreased by SFN treatment. In conclusion, SFN attenuated the cytotoxicity of CdSe QDs in both human hepatocytes and in the mouse liver, and this protection was associated with the induction of Nrf2 pathway and autophagy.

Granulocyte-macrophage colony stimulatory factor enhances the pro-inflammatory response of interferon-γ-treated macrophages to Pseudomonas aeruginosa infection.

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Sonali Singh

Full Text Available Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF patients. CF patients have been proposed to have a Th2 and Th17-biased immune response suggesting that the lack of Th1 and/or over exuberant Th17 responses could contribute to the establishment of chronic P. aeruginosa infection and deterioration of lung function. Accordingly, we have observed that interferon (IFN-γ production by peripheral blood mononuclear cells from CF patients positively correlated with lung function, particularly in patients chronically infected with P. aeruginosa. In contrast, IL-17A levels tended to correlate negatively with lung function with this trend becoming significant in patients chronically infected with P. aeruginosa. These results are in agreement with IFN-γ and IL-17A playing protective and detrimental roles, respectively, in CF. In order to explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF, on the ability of human macrophages to control P. aeruginosa growth, resist the cytotoxicity induced by this bacterium or promote inflammation was investigated. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF, failed to alter bacterial growth or macrophage survival upon P. aeruginosa infection, but changed the inflammatory potential of macrophages. IFN-γ caused up-regulation of monocyte chemoattractant protein-1 (MCP-1 and TNF-α and down-regulation of IL-10 expression by infected macrophages. GM-CSF in combination with IFN-γ promoted IL-6 production and further reduction of IL-10 synthesis. Comparison of TNF-α vs. IL-10 and IL-6 vs. IL-10 ratios revealed the following hierarchy in regard to the pro-inflammatory potential of human

Granulocyte-macrophage colony stimulatory factor enhances the pro-inflammatory response of interferon-γ-treated macrophages to Pseudomonas aeruginosa infection.

Science.gov (United States)

Singh, Sonali; Barr, Helen; Liu, Yi-Chia; Robins, Adrian; Heeb, Stephan; Williams, Paul; Fogarty, Andrew; Cámara, Miguel; Martínez-Pomares, Luisa

2015-01-01

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF) patients. CF patients have been proposed to have a Th2 and Th17-biased immune response suggesting that the lack of Th1 and/or over exuberant Th17 responses could contribute to the establishment of chronic P. aeruginosa infection and deterioration of lung function. Accordingly, we have observed that interferon (IFN)-γ production by peripheral blood mononuclear cells from CF patients positively correlated with lung function, particularly in patients chronically infected with P. aeruginosa. In contrast, IL-17A levels tended to correlate negatively with lung function with this trend becoming significant in patients chronically infected with P. aeruginosa. These results are in agreement with IFN-γ and IL-17A playing protective and detrimental roles, respectively, in CF. In order to explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), on the ability of human macrophages to control P. aeruginosa growth, resist the cytotoxicity induced by this bacterium or promote inflammation was investigated. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF, failed to alter bacterial growth or macrophage survival upon P. aeruginosa infection, but changed the inflammatory potential of macrophages. IFN-γ caused up-regulation of monocyte chemoattractant protein-1 (MCP-1) and TNF-α and down-regulation of IL-10 expression by infected macrophages. GM-CSF in combination with IFN-γ promoted IL-6 production and further reduction of IL-10 synthesis. Comparison of TNF-α vs. IL-10 and IL-6 vs. IL-10 ratios revealed the following hierarchy in regard to the pro-inflammatory potential of human macrophages

circRNA Mediates Silica-Induced Macrophage Activation Via HECTD1/ZC3H12A-Dependent Ubiquitination

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Zhou, Zewei; Jiang, Rong; Yang, Xiyue; Guo, Huifang; Fang, Shencun; Zhang, Yingming; Cheng, Yusi; Wang, Jing; Yao, Honghong; Chao, Jie

2018-01-01

Rationale: Phagocytosis of silicon dioxide (SiO2) into lung cells causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Circular RNAs (circRNAs) are a subclass of non-coding RNAs detected within mammalian cells; however, researchers have not determined whether circRNAs are involved in the pathophysiological process of silicosis. The upstream molecular mechanisms and functional effects on cell apoptosis, proliferation and migration were investigated to elucidate the role of circRNAs in SiO2-induced inflammation in pulmonary macrophages. Methods: Primary cultures of alveolar macrophages from healthy donors and patients as well as the RAW264.7 macrophage cell line were used to explore the functions of circHECTD1 (HECT domain E3 ubiquitin protein ligase 1) in macrophage activation. Results: The results of the experiments indicated that 1) SiO2 concomitantly decreased circHECTD1 levels and increased HECTD1 protein expression; 2) circHECTD1 and HECTD1 were involved in SiO2-induced macrophage activation via ubiquitination; and 3) SiO2-activated macrophages promoted fibroblast proliferation and migration via the circHECTD1/HECTD1 pathway. Tissue samples from silicosis patients confirmed the upregulation of HECTD1. Conclusions: Our study elucidated a link between SiO2-induced macrophage activation and the circHECTD1/HECTD1 pathway, thereby providing new insight into the potential use of HECTD1 in the development of novel therapeutic strategies for treating silicosis. PMID:29290828

circRNA Mediates Silica-Induced Macrophage Activation Via HECTD1/ZC3H12A-Dependent Ubiquitination.

Science.gov (United States)

Zhou, Zewei; Jiang, Rong; Yang, Xiyue; Guo, Huifang; Fang, Shencun; Zhang, Yingming; Cheng, Yusi; Wang, Jing; Yao, Honghong; Chao, Jie

2018-01-01

Rationale: Phagocytosis of silicon dioxide (SiO 2 ) into lung cells causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Circular RNAs (circRNAs) are a subclass of non-coding RNAs detected within mammalian cells; however, researchers have not determined whether circRNAs are involved in the pathophysiological process of silicosis. The upstream molecular mechanisms and functional effects on cell apoptosis, proliferation and migration were investigated to elucidate the role of circRNAs in SiO 2 -induced inflammation in pulmonary macrophages. Methods: Primary cultures of alveolar macrophages from healthy donors and patients as well as the RAW264.7 macrophage cell line were used to explore the functions of circHECTD1 (HECT domain E3 ubiquitin protein ligase 1) in macrophage activation. Results: The results of the experiments indicated that 1) SiO 2 concomitantly decreased circHECTD1 levels and increased HECTD1 protein expression; 2) circHECTD1 and HECTD1 were involved in SiO 2 -induced macrophage activation via ubiquitination; and 3) SiO 2 -activated macrophages promoted fibroblast proliferation and migration via the circHECTD1/HECTD1 pathway. Tissue samples from silicosis patients confirmed the upregulation of HECTD1. Conclusions: Our study elucidated a link between SiO 2 -induced macrophage activation and the circHECTD1/HECTD1 pathway, thereby providing new insight into the potential use of HECTD1 in the development of novel therapeutic strategies for treating silicosis.

A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers.

Science.gov (United States)

Jemnitz, Katalin; Bátai-Konczos, Attila; Szabó, Mónika; Ioja, Enikő; Kolacsek, Orsolya; Orbán, Tamás I; Török, György; Homolya, László; Kovács, Eszter; Jablonkai, István; Veres, Zsuzsa

2017-02-01

Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca 2+ level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca 2+ sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca 2+ imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca 2+ oscillation and sustained Ca 2+ signals at 1μM and10 μM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca 2+ signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI. Copyright © 2016 Elsevier B.V. All rights reserved.

PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells

International Nuclear Information System (INIS)

Seo, Kang-Sik; Hwang, Byung-Doo; Kim, Jong-Seok; Park, Ji-Hoon; Song, Kyoung-Sub; Yun, Eun-Jin; Park, Jong-Il; Kweon, Gi Ryang; Yoon, Wan-Hee; Lim, Kyu

2014-01-01

Combination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells. Cell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively. We found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G 1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner. These results suggest that the synergy between PMA and apicularen A is involved by

Involvement of Nitric Oxide on Bothropoides insularis Venom Biological Effects on Murine Macrophages In Vitro.

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Ramon R P P B de Menezes

Full Text Available Viperidae venom has several local and systemic effects, such as pain, edema, inflammation, kidney failure and coagulopathy. Additionally, bothropic venom and its isolated components directly interfere on cellular metabolism, causing alterations such as cell death and proliferation. Inflammatory cells are particularly involved in pathological envenomation mechanisms due to their capacity of releasing many mediators, such as nitric oxide (NO. NO has many effects on cell viability and it is associated to the development of inflammation and tissue damage caused by Bothrops and Bothropoides venom. Bothropoides insularis is a snake found only in Queimada Grande Island, which has markedly toxic venom. Thus, the aim of this work was to evaluate the biological effects of Bothropoides insularis venom (BiV on RAW 264.7 cells and assess NO involvement. The venom was submitted to colorimetric assays to identify the presence of some enzymatic components. We observed that BiV induced H2O2 production and showed proteolytic and phospholipasic activities. RAW 264.7 murine macrophages were incubated with different concentrations of BiV and then cell viability was assessed by MTT reduction assay after 2, 6, 12 and 24 hours of incubation. A time- and concentration-dependent effect was observed, with a tendency to cell proliferation at lower BiV concentrations and cell death at higher concentrations. The cytotoxic effect was confirmed after lactate dehydrogenase (LDH measurement in the supernatant from the experimental groups. Flow cytometry analyses revealed that necrosis is the main cell death pathway caused by BiV. Also, BiV induced NO release. The inhibition of both proliferative and cytotoxic effects with L-NAME were demonstrated, indicating that NO is important for these effects. Finally, BiV induced an increase in iNOS expression. Altogether, these results demonstrate that B. insularis venom have proliferative and cytotoxic effects on macrophages, with

Stimulation of Inducible Nitric Oxide Synthase Expression by Beta Interferon Increases Necrotic Death of Macrophages upon Listeria monocytogenes Infection▿

OpenAIRE

Zwaferink, Heather; Stockinger, Silvia; Reipert, Siegfried; Decker, Thomas

2008-01-01

Murine macrophage death upon infection with Listeria monocytogenes was previously shown to be increased by beta interferon, produced by the infected cells. We saw that interferon-upregulated caspase activation or other interferon-inducible, death-associated proteins, including TRAIL, protein kinase R, and p53, were not necessary for cell death. Macrophage death was reduced when inducible nitric oxide synthase (iNOS) was inhibited during infection, and iNOS-deficient macrophages were less susc...

Cytotoxicity evaluation of ceramic particles of different sizes and shapes.

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Yamamoto, Akiko; Honma, Rieko; Sumita, Masae; Hanawa, Takao

2004-02-01

When artificial hip or knee joints are implanted in the human body, they release metallic, ceramic, and polymeric debris into the surrounding tissues. The toxicity of the released particles is of two types: chemical, caused by the released soluble ions and monomers, and mechanical, a result of mechanical stimulation produced by the insoluble particles. In this study, the cytotoxicity of particles of TiO2, Al2O3, ZrO2, Si3N4, and SiC for murine fibroblasts and macrophages were examined to evaluate just their mechanical toxicity because these particles are not expected to release soluble metal ions. Different sizes and shapes of TiO2 particles were used to evaluate the effect of size and shape on particle cytotoxicity. The results suggest that the cytotoxicity of ceramic particles does not depend on their chemical species. Cytotoxicity levels were lower than those of corresponding metal ions, indicating that the mechanical toxicity of particles is lower than the chemical toxicity of released soluble ions and monomers. The differences in size did not affect the mechanical toxicity of these particles. The dendritic particles had a higher cytotoxicity level for macrophages than did spindle and spheric particles. Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 68A: 244-256, 2004

Critical illness induces alternative activation of M2 macrophages in adipose tissue.

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Langouche, Lies; Marques, Mirna B; Ingels, Catherine; Gunst, Jan; Derde, Sarah; Vander Perre, Sarah; D'Hoore, André; Van den Berghe, Greet

2011-01-01

We recently reported macrophage accumulation in adipose tissue of critically ill patients. Classically activated macrophage accumulation in adipose tissue is a known feature of obesity, where it is linked with increasing insulin resistance. However, the characteristics of adipose tissue macrophage accumulation in critical illness remain unknown. We studied macrophage markers with immunostaining and gene expression in visceral and subcutaneous adipose tissue from healthy control subjects (n = 20) and non-surviving prolonged critically ill patients (n = 61). For comparison, also subcutaneous in vivo adipose tissue biopsies were studied from 15 prolonged critically ill patients. Subcutaneous and visceral adipose tissue biopsies from non-surviving prolonged critically ill patients displayed a large increase in macrophage staining. This staining corresponded with elevated gene expression of "alternatively activated" M2 macrophage markers arginase-1, IL-10 and CD163 and low levels of the "classically activated" M1 macrophage markers tumor necrosis factor (TNF)-α and inducible nitric-oxide synthase (iNOS). Immunostaining for CD163 confirmed positive M2 macrophage staining in both visceral and subcutaneous adipose tissue biopsies from critically ill patients. Surprisingly, circulating levels and tissue gene expression of the alternative M2 activators IL-4 and IL-13 were low and not different from controls. In contrast, adipose tissue protein levels of peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor required for M2 differentiation and acting downstream of IL-4, was markedly elevated in illness. In subcutaneous abdominal adipose tissue biopsies from surviving critically ill patients, we could confirm positive macrophage staining with CD68 and CD163. We also could confirm elevated arginase-1 gene expression and elevated PPARγ protein levels. Unlike obesity, critical illness evokes adipose tissue accumulation of alternatively activated M2

Soluble immune complexes shift the TLR-induced cytokine production of distinct polarized human macrophage subsets towards IL-10.

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Carmen A Ambarus

Full Text Available BACKGROUND: Costimulation of murine macrophages with immune complexes (ICs and TLR ligands leads to alternative activation. Studies on human myeloid cells, however, indicate that ICs induce an increased pro-inflammatory cytokine production. This study aimed to clarify the effect of ICs on the pro- versus anti-inflammatory profile of human polarized macrophages. MATERIALS AND METHODS: Monocytes isolated from peripheral blood of healthy donors were polarized for four days with IFN-γ, IL-4, IL-10, GM-CSF, M-CSF, or LPS, in the presence or absence of heat aggregated gamma-globulins (HAGGs. Phenotypic polarization markers were measured by flow cytometry. Polarized macrophages were stimulated with HAGGs or immobilized IgG alone or in combination with TLR ligands. TNF, IL-6, IL-10, IL-12, and IL-23 were measured by Luminex and/or RT-qPCR. RESULTS: HAGGs did not modulate the phenotypic polarization and the cytokine production of macrophages. However, HAGGs significantly altered the TLR-induced cytokine production of all polarized macrophage subsets, with the exception of MΦ(IL-4. In particular, HAGGs consistently enhanced the TLR-induced IL-10 production in both classically and alternatively polarized macrophages (M1 and M2. The effect of HAGGs on TNF and IL-6 production was less pronounced and depended on the polarization status, while IL-23p19 and IL-12p35 expression was not affected. In contrast with HAGGs, immobilized IgG induced a strong upregulation of not only IL-10, but also TNF and IL-6. CONCLUSION: HAGGs alone do not alter the phenotype and cytokine production of in vitro polarized human macrophages. In combination with TLR-ligands, however, HAGGs but not immobilized IgG shift the cytokine production of distinct macrophage subsets toward IL-10.

Low-Cytotoxic Synthetic Bromorutaecarpine Exhibits Anti-Inflammation and Activation of Transient Receptor Potential Vanilloid Type 1 Activities

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Chi-Ming Lee

2013-01-01

Full Text Available Rutaecarpine (RUT, the major bioactive ingredient isolated from the Chinese herb Evodia rutaecarpa, possesses a wide spectrum of biological activities, including anti-inflammation and preventing cardiovascular diseases. However, its high cytotoxicity hampers pharmaceutical development. We designed and synthesized a derivative of RUT, bromo-dimethoxyrutaecarpine (Br-RUT, which showed no cytotoxicity at 20 μM. Br-RUT suppressed nitric oxide (NO production and tumor necrosis factor-α release in concentration-dependent (0~20 μM manners in lipopolysaccharide (LPS-treated RAW 264.7 macrophages; protein levels of inducible NO synthase (iNOS and cyclooxygenase-2 induced by LPS were downregulated. Br-RUT inhibited cell migration and invasion of ovarian carcinoma A2780 cells with 0~48 h of treatment. Furthermore, Br-RUT enhanced the expression of transient receptor potential vanilloid type 1 and activated endothelial NOS in human aortic endothelial cells. These results suggest that the synthetic Br-RUT possesses very low cytotoxicity but retains its activities against inflammation and vasodilation that could be beneficial for cardiovascular disease therapeutics.

Effect of silica particle size on macrophage inflammatory responses.

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Toshimasa Kusaka

Full Text Available Amorphous silica particles, such as nanoparticles (<100 nm diameter particles, are used in a wide variety of products, including pharmaceuticals, paints, cosmetics, and food. Nevertheless, the immunotoxicity of these particles and the relationship between silica particle size and pro-inflammatory activity are not fully understood. In this study, we addressed the relationship between the size of amorphous silica (particle dose, diameter, number, and surface area and the inflammatory activity (macrophage phagocytosis, inflammasome activation, IL-1β secretion, cell death and lung inflammation. Irrespective of diameter size, silica particles were efficiently internalized by mouse bone marrow-derived macrophages via an actin cytoskeleton-dependent pathway, and induced caspase-1, but not caspase-11, activation. Of note, 30 nm-1000 nm diameter silica particles induced lysosomal destabilization, cell death, and IL-1β secretion at markedly higher levels than did 3000 nm-10000 nm silica particles. Consistent with in vitro results, intra-tracheal administration of 30 nm silica particles into mice caused more severe lung inflammation than that of 3000 nm silica particles, as assessed by measurement of pro-inflammatory cytokines and neutrophil infiltration in bronchoalveolar lavage fluid of mice, and by the micro-computed tomography analysis. Taken together, these results suggest that silica particle size impacts immune responses, with submicron amorphous silica particles inducing higher inflammatory responses than silica particles over 1000 nm in size, which is ascribed not only to their ability to induce caspase-1 activation but also to their cytotoxicity.

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.

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Badding, Melissa A; Fix, Natalie R; Antonini, James M; Leonard, Stephen S

2014-01-01

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.

Adipocyte fetuin-A contributes to macrophage migration into adipose tissue and polarization of macrophages.

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Chatterjee, Priyajit; Seal, Soma; Mukherjee, Sandip; Kundu, Rakesh; Mukherjee, Sutapa; Ray, Sukanta; Mukhopadhyay, Satinath; Majumdar, Subeer S; Bhattacharya, Samir

2013-09-27

Macrophage infiltration into adipose tissue during obesity and their phenotypic conversion from anti-inflammatory M2 to proinflammatory M1 subtype significantly contributes to develop a link between inflammation and insulin resistance; signaling molecule(s) for these events, however, remains poorly understood. We demonstrate here that excess lipid in the adipose tissue environment may trigger one such signal. Adipose tissue from obese diabetic db/db mice, high fat diet-fed mice, and obese diabetic patients showed significantly elevated fetuin-A (FetA) levels in respect to their controls; partially hepatectomized high fat diet mice did not show noticeable alteration, indicating adipose tissue to be the source of this alteration. In adipocytes, fatty acid induces FetA gene and protein expressions, resulting in its copious release. We found that FetA could act as a chemoattractant for macrophages. To simulate lipid-induced inflammatory conditions when proinflammatory adipose tissue and macrophages create a niche of an altered microenvironment, we set up a transculture system of macrophages and adipocytes; the addition of fatty acid to adipocytes released FetA into the medium, which polarized M2 macrophages to M1. This was further confirmed by direct FetA addition to macrophages. Taken together, lipid-induced FetA from adipocytes is an efficient chemokine for macrophage migration and polarization. These findings open a new dimension for understanding obesity-induced inflammation.

Antibody dependent cellular phagocytosis by macrophages is a novel mechanism of action of elotuzumab.

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Kurdi, Ahmed T; Glavey, Siobhan V; Bezman, Natalie A; Jhatakia, Amy; Guerriero, Jennifer L; Manier, Salomon; Moschetta, Michele; Mishima, Yuji; Roccaro, Aldo; Detappe, Alexandre; Liu, Chia-Jen; Sacco, Antonio; Huynh, Daisy; Tai, Yu-Tzu; Robbins, Michael D; Azzi, Jamil; Ghobrial, Irene M

2018-04-13

Elotuzumab, a recently approved antibody for the treatment of multiple myeloma (MM), has been shown to stimulate Fcγ receptor (FcγR)-mediated antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells towards myeloma cells. The modulatory effects of elotuzumab on other effector cells in the tumor microenvironment, however, has not been fully explored. Antibody dependent cellular phagocytosis (ADCP) is a mechanism by which macrophages contribute to anti-tumor potency of monoclonal antibodies. Herein, we studied the NK cell independent effect of elotuzumab on tumor associated macrophages (TAMs) using a xenograft tumor model deficient in NK and adaptive immune cells. We demonstrate significant anti-tumor efficacy of single agent elotuzumab in immunocompromised xenograft models of multiple myeloma, which is in part mediated by Fc-FcγR interaction of elotuzumab with macrophages. Elotuzumab is shown in this study to induce phenotypic activation of macrophages in-vivo and mediates ADCP of myeloma cells though a FcγR dependent manner in-vitro. Together, these findings propose a novel immune mediated mechanism by which elotuzumab exerts anti-myeloma activity and helps to provide rationale for combination therapies that can enhance macrophage activity. Copyright ©2018, American Association for Cancer Research.

Apoptotic death of Listeria monocytogenes-infected human macrophages induced by lactoferricin B, a bovine lactoferrin-derived peptide.

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Longhi, C; Conte, M P; Ranaldi, S; Penta, M; Valenti, P; Tinari, A; Superti, F; Seganti, L

2005-01-01

Listeria monocytogenes, an intracellular facultative food-borne pathogen, was reported to induce apoptosis in vitro and in vivo in a variety of cell types with the exception of murine macrophages. These cells represent the predominant compartment of bacterial multiplication and die as a result of necrosis. In this study we showed that human non-activated and IFN-gamma-activated macrophagic-like (THP-1) cells infected with L. monocytogenes, mainly die by necrosis rather than by an apoptotic process. Two natural products derived from bovine milk, lactoferrin and its derivative peptide lactoferricin B, are capable of regulating the fate of infected human macrophages. Bovine lactoferrin treatment of macrophages protects them from L. monocytogenes-induced death whereas lactoferricin B, its derivative peptide, determines a shifting of the equilibrium from necrosis to apoptosis.

Alternatively activated macrophages (M2 macrophages) in the skin of patient with localized scleroderma.

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Higashi-Kuwata, Nobuyo; Makino, Takamitsu; Inoue, Yuji; Takeya, Motohiro; Ihn, Hironobu

2009-08-01

Localized scleroderma is a connective tissue disorder that is limited to the skin and subcutaneous tissue. Macrophages have been reported to be particularly activated in patients with skin disease including systemic sclerosis and are potentially important sources for fibrosis-inducing cytokines, such as transforming growth factor beta. To clarify the features of immunohistochemical characterization of the immune cell infiltrates in localized scleroderma focusing on macrophages, skin biopsy specimens were analysed by immunohistochemistry. The number of cells stained with monoclonal antibodies, CD68, CD163 and CD204, was calculated. An evident macrophage infiltrate and increased number of alternatively activated macrophages (M2 macrophages) in their fibrotic areas were observed along with their severity of inflammation. This study revealed that alternatively activated macrophages (M2 macrophages) may be a potential source of fibrosis-inducing cytokines in localized scleroderma, and may play a crucial role in the pathogenesis of localized scleroderma.

Quercetin-3-O-glucuronide induces ABCA1 expression by LXRα activation in murine macrophages

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Ohara, Kazuaki, E-mail: Kazuaki_Ohara@kirin.co.jp [Research Laboratories for Health Science and Food Technologies, Kirin Company Limited, 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236-0004 (Japan); Wakabayashi, Hideyuki [Laboratory for New Product Development, Kirin Beverage Company Limited, 1-17-1 Namamugi, Tsurumi-ku, Yokohama 230-8628 (Japan); Taniguchi, Yoshimasa [Research Laboratories for Health Science and Food Technologies, Kirin Company Limited, 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236-0004 (Japan); Shindo, Kazutoshi [Department of Food and Nutrition, Japan Women’s University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681 (Japan); Yajima, Hiroaki [Research Laboratories for Health Science and Food Technologies, Kirin Company Limited, 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236-0004 (Japan); Yoshida, Aruto [Central Laboratories for Key Technologies, Kirin Company Limited, 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236-0004 (Japan)

2013-11-29

Highlights: •The major circulating quercetin metabolite (Q3GA) activated LXRα. •Q3GA induced ABCA1 via LXRα activation in macrophages. •Nelumbo nucifera leaf extracts contained quercetin glycosides. •N. nucifera leaf extract feeding elevated HDLC in mice. -- Abstract: Reverse cholesterol transport (RCT) removes excess cholesterol from macrophages to prevent atherosclerosis. ATP-binding cassette, subfamily A, member 1 (ABCA1) is a crucial cholesterol transporter involved in RCT to produce high density lipoprotein-cholesterol (HDLC), and is transcriptionally regulated by liver X receptor alpha (LXRα), a nuclear receptor. Quercetin is a widely distributed flavonoid in edible plants which prevented atherosclerosis in an animal model. We found that quercetin-3-O-glucuronide (Q3GA), a major quercetin metabolite after absorption from the digestive tract, enhanced ABCA1 expression, in vitro, via LXRα in macrophages. In addition, leaf extracts of a traditional Asian edible plant, Nelumbo nucifera (NNE), which contained abundant amounts of quercetin glycosides, significantly elevated plasma HDLC in mice. We are the first to present experimental evidence that Q3GA induced ABCA1 in macrophages, and to provide an alternative explanation to previous studies on arteriosclerosis prevention by quercetin.

Adventitial Fibroblasts induce a distinct Pro-inflammatory/Pro-fibrotic Macrophage Phenotype in Pulmonary Hypertension

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El Kasmi, Karim C.; Pugliese, Steven C.; Riddle, Suzette R.; Poth, Jens M.; Anderson, Aimee L.; Frid, Maria G.; Li, Min; Pullamsetti, Soni S.; Savai, Rajkumar; Nagel, Maria A.; Fini, Mehdi A.; Graham, Brian B.; Tuder, Rubin M.; Friedman, Jacob E.; Eltzschig, Holger K.; Sokol, Ronald J.; Stenmark, Kurt R.

2014-01-01

Macrophage accumulation is not only a characteristic hallmark but also a critical component of pulmonary artery (PA) remodeling associated with pulmonary hypertension (PH). However, the cellular and molecular mechanisms that drive vascular macrophage activation and their functional phenotype remain poorly defined. Utilizing multiple levels of in vivo (bovine and rat models of hypoxia-induced PH, together with human tissue samples) and in vitro (primary mouse, rat, and bovine macrophages, human monocytes, as well as primary human and bovine fibroblasts) approaches, we observed that adventitial fibroblasts derived from hypertensive Pas (bovine and human) regulate macrophage activation. These fibroblasts activate macrophages through paracrine IL6 and STAT3, HIF1, and C/EBPβ signaling to drive expression of genes previously implicated in chronic inflammation, tissue remodeling, and PH. This distinct fibroblast-activated macrophage phenotype was independent of IL4/IL13-STAT6 and TLR-MyD88 signaling. We found that genetic STAT3 haplodeficiency in macrophages attenuated macrophage activation while complete STAT3 deficiency increased macrophage activation through compensatory upregulation of STAT1 signaling, while deficiency in C/EBPβ or HIF1 attenuated fibroblast driven macrophage activation. These findings challenge the current paradigm of IL4/IL13-STAT6 mediated alternative macrophage activation as the sole driver of vascular remodeling in PH and uncover a crosstalk between adventitial fibroblasts and macrophages in which paracrine IL6 activated STAT3, HIF1, and C/EBPβ signaling is critical for macrophage activation and polarization. Thus, targeting IL6 signaling in macrophages by completely inhibiting C/EBPβ, HIF1a or partially inhibiting STAT3 may hold therapeutic value for treatment of PH and other inflammatory conditions characterized by increased IL6 and absent IL4/IL13 signaling. PMID:24928992

Mechanism of inhibitory effect of atorvastatin on resistin expression induced by tumor necrosis factor-α in macrophages

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Chua Su-Kiat

2009-05-01

Full Text Available Abstract Atorvastatin has been shown to reduce resistin expression in macrophages after pro-inflammatory stimulation. However, the mechanism of reducing resistin expression by atorvastatin is not known. Therefore, we sought to investigate the molecular mechanisms of atorvastatin for reducing resistin expression after proinflammatory cytokine, tumor necrosis factor-α (TNF-α stimulation in cultured macrophages. Cultured macrophages were obtained from human peripheral blood mononuclear cells. TNF-α stimulation increased resistin protein and mRNA expression and atorvastatin inhibited the induction of resistin by TNF-α. Addition of mevalonate induced resistin protein expression similar to TNF-α stimulation. However, atorvastatin did not have effect on resistin protein expression induced by mevalonate. SP600125 and JNK small interfering RNA (siRNA completely attenuated the resistin protein expression induced by TNF-α and mevalonate. TNF-α induced phosphorylation of Rac, while atorvastatin and Rac-1 inhibitor inhibited the phosphorylation of Rac induced by TNF-α. The gel shift and promoter activity assay showed that TNF-α increased AP-1-binding activity and resistin promoter activity, while SP600125 and atorvastatin inhibited the AP-1-binding activity and resistin promoter activity induced by TNF-α. Recombinant resistin and TNF-α significantly reduced glucose uptake in cultured macrophages, while atorvastatin reversed the reduced glucose uptake by TNF-α. In conclusion, JNK and Rac pathway mediates the inhibitory effect of atorvastatin on resistin expression induced by TNF-α.

YC-1 potentiates cAMP-induced CREB activation and nitric oxide production in alveolar macrophages

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Hwang, Tsong-Long, E-mail: htl@mail.cgu.edu.tw [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan, Taiwan (China); Tang, Ming-Chi [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Kuo, Liang-Mou [Department of General Surgery, Chang Gung Memorial Hospital at Chia-Yi, Taiwan (China); Chang, Wen-De; Chung, Pei-Jen; Chang, Ya-Wen; Fang, Yao-Ching [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China)

2012-04-15

Alveolar macrophages play significant roles in the pathogenesis of several inflammatory lung diseases. Increases in exhaled nitric oxide (NO) are well documented to reflect disease severity in the airway. In this study, we investigated the effect of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on prostaglandin (PG)E{sub 1} (a stable PGE{sub 2} analogue) and forskolin (a adenylate cyclase activator) induced NO production and inducible NO synthase (iNOS) expression in rat alveolar macrophages (NR8383). YC-1 did not directly cause NO production or iNOS expression, but drastically potentiated PGE{sub 1}- or forskolin-induced NO production and iNOS expression in NR8383 alveolar macrophages. Combination treatment with YC-1 and PGE{sub 1} significantly increased phosphorylation of the cAMP response element-binding protein (CREB), but not nuclear factor (NF)-κB activation. The combined effect on NO production, iNOS expression, and CREB phosphorylation was reversed by a protein kinase (PK)A inhibitor (H89), suggesting that the potentiating functions were mediated through a cAMP/PKA signaling pathway. Consistent with this, cAMP analogues, but not the cGMP analogue, caused NO release, iNOS expression, and CREB activation. YC-1 treatment induced an increase in PGE{sub 1}-induced cAMP formation, which occurred through the inhibition of cAMP-specific phosphodiesterase (PDE) activity. Furthermore, the combination of rolipram (an inhibitor of PDE4), but not milronone (an inhibitor of PDE3), and PGE{sub 1} also triggered NO production and iNOS expression. In summary, YC-1 potentiates PGE{sub 1}-induced NO production and iNOS expression in alveolar macrophages through inhibition of cAMP PDE activity and activation of the cAMP/PKA/CREB signaling pathway. Highlights: ► YC-1 potentiated PGE1-induced iNOS expression in alveolar macrophages. ► The combination of YC-1 and PGE1 increased CREB but not NFκB activation. �

Modified pectin from Theobroma cacao induces potent pro-inflammatory activity in murine peritoneal macrophage.

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Amorim, Juliana C; Vriesmann, Lucia Cristina; Petkowicz, Carmen L O; Martinez, Glaucia Regina; Noleto, Guilhermina R

2016-11-01

In vitro effects of acetylated pectin (OP) isolated from cacao pod husks (Theobroma cacao L.), its partially deacetylated and de-esterified form (MOP), and a commercial homogalacturonan (PG) were investigated on murine peritoneal macrophages. MOP stood out among the studied pectins. After 48h of incubation, compared with the control group, it was able to promote significant macrophage morphological differentiation from resident to activated stage and also stimulated nitric oxide production, which reached a level of 85% of that of LPS stimulus. In the presence of the highest tested concentration of MOP (200μg·mL -1 ), the levels of the cytokines TNF-α (6h) and IL-12 and IL-10 (48h) increased substantially in relation to untreated cells. Our results show that the partial deacetylation and de-esterification of pectin extracted from cacao pod husks (T. cacao L.) produced a polymer with greater ability than its native form to activate macrophages to a cytotoxic phenotype. Like this, they provide the possibility of a therapeutic application to MOP, which could lead to a decreased susceptibility to microbial infection besides antitumor activity. Additionally, the present results also corroborate with the proposition of that the chemical modifications of the biopolymers can result in an improved molecule with new possibilities of application. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of 14-kDa and 47-kDa protein molecules of age garlic extract on peritoneal macrophages.

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Daneshmandi, Saeed; Hajimoradi, Monire; Ahmadabad, Hasan Namdar; Hassan, Zuhair Mohammad; Roudbary, Maryam; Ghazanfari, Tooba

2011-03-01

Garlic (Allium sativum), traditionally being used as a spice worldwide, has different applications and is claimed to possess beneficial effects in several health ailments such as tumor and atherosclerosis. Garlic is also an immunomodulator and its different components are responsible for different properties. The present work aimed to assess the effect of protein fractions of garlic on peritoneal macrophages. 14-kDa and 47-kDa protein fractions of garlic were purified. Mice peritoneal macrophages were lavaged and cultured in a microtiter plate and exposed to different concentrations of garlic proteins. MTT assay was performed to evaluate the viability of macrophage. The amount of nitric oxide (NO) was detected in culture supernatants of macrophages by Griess reagent and furthermore, the cytotoxicity study of culture supernatants was carried out on WEHI-164 fibrosarcoma cell line as tumor necrosis factor-α bioassay. MTT assay results for both 14-kDa and 47-kDa protein fractions of stimulated macrophages were not significant (P > 0.05). Both 14-kDa and 47-kDa fractions significantly suppressed production of NO from macrophages (P = 0.007 and P = 0.003, respectively). Cytotoxicity of macrophages' supernatant on WEHI-164 fibrosarcoma cells was not affected by garlic protein fractions (P = 0.066 for 14-kDa and P = 0.085 for 47-kDa fractions). according to our finding, 14-kDa and 47-kDa fractions of aged garlic extract are able to suppress NO production from macrophages, which can be used as a biological advantage. These molecules had no cytotoxic effect on macrophages and do not increase tumoricidal property of macrophages.

Multiple Pseudomonas species secrete exolysin-like toxins and provoke Caspase-1-dependent macrophage death.

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Basso, Pauline; Wallet, Pierre; Elsen, Sylvie; Soleilhac, Emmanuelle; Henry, Thomas; Faudry, Eric; Attrée, Ina

2017-10-01

Pathogenic bacteria secrete protein toxins that provoke apoptosis or necrosis of eukaryotic cells. Here, we developed a live-imaging method, based on incorporation of a DNA-intercalating dye into membrane-damaged host cells, to study the kinetics of primary bone marrow-derived macrophages (BMDMs) mortality induced by opportunistic pathogen Pseudomonas aeruginosa expressing either Type III Secretion System (T3SS) toxins or the pore-forming toxin, Exolysin (ExlA). We found that ExlA promotes the activation of Caspase-1 and maturation of interleukin-1β. BMDMs deficient for Caspase-1 and Caspase-11 were resistant to ExlA-induced death. Furthermore, by using KO BMDMs, we determined that the upstream NLRP3/ASC complex leads to the Caspase-1 activation. We also demonstrated that Pseudomonas putida and Pseudomonas protegens and the Drosophila pathogen Pseudomonas entomophila, which naturally express ExlA-like toxins, are cytotoxic toward macrophages and provoke the same type of pro-inflammatory death as does ExlA + P. aeruginosa. These results demonstrate that ExlA-like toxins of two-partner secretion systems from diverse Pseudomonas species activate the NLRP3 inflammasome and provoke inflammatory pyroptotic death of macrophages. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

The response of macrophages to titanium particles is determined by macrophage polarization.

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Pajarinen, Jukka; Kouri, Vesa-Petteri; Jämsen, Eemeli; Li, Tian-Fang; Mandelin, Jami; Konttinen, Yrjö T

2013-11-01

Aseptic loosening of total joint replacements is driven by the reaction of macrophages to foreign body particles released from the implant. It was hypothesized that the macrophages' response to these particles is dependent, in addition to particle characteristics and contaminating biomolecules, on the state of macrophage polarization as determined by the local cytokine microenvironment. To test this hypothesis we differentiated M1 and M2 macrophages from human peripheral blood monocytes and compared their responses to titanium particles using genome-wide microarray analysis and a multiplex cytokine assay. In comparison to non-activated M0 macrophages, the overall chemotactic and inflammatory responses to titanium particles were greatly enhanced in M1 macrophages and effectively suppressed in M2 macrophages. In addition, the genome-wide approach revealed several novel, potentially osteolytic, particle-induced mediators, and signaling pathway analysis suggested the involvement of toll-like and nod-like receptor signaling in particle recognition. It is concluded that the magnitude of foreign body reaction caused by titanium particles is dependent on the state of macrophage polarization. Thus, by limiting the action of M1 polarizing factors, e.g. bacterial biofilm formation, in peri-implant tissues and promoting M2 macrophage polarization by biomaterial solutions or pharmacologically, it might be possible to restrict wear-particle-induced inflammation and osteolysis. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells.

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Yadav, Umesh C S; Ramana, K V; Srivastava, Satish K

2013-12-01

Aldose reductase (AR), a glucose-metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30 µM) relative to glucose. Acrolein, a major endogenous lipid peroxidation product as well as a component of environmental pollutants and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders, but the mechanism of acrolein-induced cytotoxicity is not clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells (SAECs). Exposure of SAECs to varying concentrations of acrolein caused cell death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low-dose (5-10 µM) but not the high-dose (>10 µM) acrolein-induced SAEC death. AR inhibition protected SAECs from low-dose (5 µM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail moment, and annexin V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of the proapoptotic proteins Bax and Bad from the cytosol to the mitochondria and that of Bcl2 and BclXL from the mitochondria to the cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases 1 and 2, stress-activated protein kinase/c-Jun NH2-terminal kinase, and p38MAPK, and c-Jun were transiently activated in airway epithelial cells by acrolein in a concentration- and time-dependent fashion, which was significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells. Copyright © 2013 Elsevier Inc. All rights

Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells

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Yadav, Umesh CS; Ramana, KV; Srivastava, SK

2013-01-01

Aldose reductase (AR), a glucose metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30μM) than glucose. Acrolein, a major endogenous lipid peroxidation product as well as component of environmental pollutant and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders but the mechanism of acrolein-induced cytotoxicity is not clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells SAECs. Exposure of SAECs to varying concentrations of acrolein caused cell-death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low (5 to 10 μM) but not high (>10 μM) concentrations of acrolein-induced SAECs cell death. AR inhibition protected SAECs from low dose (5 μM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail-moment, and annexin-V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of pro-apoptotic proteins Bax and Bad from cytosol to the mitochondria, and that of Bcl2 and BclXL from mitochondria to cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPK) such as extracellular signal-regulated kinases 1 and 2 (ERK1/2), stress-activated protein kinases/c-jun NH2-terminal kinases (SAPK/JNK) and p38MAPK, and c-jun were transiently activated in airway epithelial cells by acrolein in a concentration and time-dependent fashion, which were significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells. PMID:23770200

Hypoxic stress up-regulates the expression of Toll-like receptor 4 in macrophages via hypoxia-inducible factor.

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Kim, So Young; Choi, Yong Jun; Joung, Sun Myung; Lee, Byung Ho; Jung, Yi-Sook; Lee, Joo Young

2010-04-01

Toll-like receptors (TLRs) are germline-encoded innate immune receptors that recognize invading micro-organisms and induce immune and inflammatory responses. Deregulation of TLRs is known to be closely linked to various immune disorders and inflammatory diseases. Cells at sites of inflammation are exposed to hypoxic stress, which further aggravates inflammatory processes. We have examined if hypoxic stress modulates the TLR activity of macrophages. Hypoxia and CoCl(2) (a hypoxia mimetic) enhanced the expression of TLR4 messenger RNA and protein in macrophages (RAW264.7 cells), whereas the messenger RNA of other TLRs was not increased. To determine the underlying mechanism, we investigated the role of hypoxia-inducible factor 1 (HIF-1) in the regulation of TLR4 expression. Knockdown of HIF-1alpha expression by small interfering RNA inhibited hypoxia-induced and CoCl(2)-induced TLR4 expression in macrophages, while over-expression of HIF-1alpha potentiated TLR4 expression. Chromatin immunoprecipitation assays revealed that HIF-1alpha binds to the TLR4 promoter region under hypoxic conditions. In addition, deletion or mutation of a putative HIF-1-binding motif in the TLR4 promoter greatly attenuated HIF-1alpha-induced TLR4 promoter reporter expression. Up-regulation of TLR4 expression by hypoxic stress enhanced the response of macrophages to lipopolysaccharide, resulting in increased expression of cyclooxygenase-2, interleukin-6, regulated on activation normal T cell expressed and secreted, and interferon-inducible protein-10. These results demonstrate that TLR4 expression in macrophages is up-regulated via HIF-1 in response to hypoxic stress, suggesting that hypoxic stress at sites of inflammation enhances susceptibility to subsequent infection and inflammatory signals by up-regulating TLR4.

Streptococcus sanguinis induces foam cell formation and cell death of macrophages in association with production of reactive oxygen species.

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Okahashi, Nobuo; Okinaga, Toshinori; Sakurai, Atsuo; Terao, Yutaka; Nakata, Masanobu; Nakashima, Keisuke; Shintani, Seikou; Kawabata, Shigetada; Ooshima, Takashi; Nishihara, Tatsuji

2011-10-01

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, is a common streptococcal species implicated in infective endocarditis. Herein, we investigated the effects of infection with S. sanguinis on foam cell formation and cell death of macrophages. Infection with S. sanguinis stimulated foam cell formation of THP-1, a human macrophage cell line. At a multiplicity of infection >100, S. sanguinis-induced cell death of the macrophages. Viable bacterial infection was required to trigger cell death because heat-inactivated S. sanguinis did not induce cell death. The production of cytokines interleukin-1β and tumor necrosis factor-α from macrophages was also stimulated during bacterial infection. Inhibition of the production of reactive oxygen species (ROS) resulted in reduced cell death, suggesting an association of ROS with cell death. Furthermore, S. sanguinis-induced cell death appeared to be independent of activation of inflammasomes, because cleavage of procaspase-1 was not evident in infected macrophages. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Immunomodulatory Role of Ocimum gratissimum and Ascorbic Acid against Nicotine-Induced Murine Peritoneal Macrophages In Vitro

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Santanu Kar Mahapatra

2011-01-01

Full Text Available The aim of this present study was to evaluate the immune functions and immune responses in nicotine-induced (10 mM macrophages and concurrently establish the immunomodulatory role of aqueous extract of Ocimum gratissimum (Ae-Og and ascorbic acid. In this study, nitrite generations and some phenotype functions by macrophages were studied. Beside that, release of Th1 cytokines (TNF-α, IL-12 and Th2 cytokines (IL-10, TGF-β was measured by ELISA, and the expression of these cytokines at mRNA level was analyzed by real-time PCR. Ae-Og, at a dose of 10 μg/mL, significantly reduced the nicotine-induced NO generation and iNOSII expression. Similar kinds of response were observed with supplementation of ascorbic acid (0.01 mM. The administration of Ae-Og and ascorbic acid increased the decreased adherence, chemotaxis, phagocytosis, and intracellular killing of bacteria in nicotine-treated macrophages. Ae-Og and ascorbic acid were found to protect the murine peritoneal macrophages through downregulation of Th1 cytokines in nicotine-treated macrophages with concurrent activation of Th2 responses. These findings strongly enhanced our understanding of the molecular mechanism leading to nicotine-induced suppression of immune functions and provide additional rationale for application of anti-inflammatory therapeutic approaches by O. gratissimum and ascorbic acid for different inflammatory disease prevention and treatment during nicotine toxicity.

Study of Galfenol direct cytotoxicity and remote microactuation in cells.

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Vargas-Estevez, Carolina; Blanquer, Andreu; Dulal, Prabesh; Pérez Del Real, Rafael; Duch, Marta; Ibáñez, Elena; Barrios, Leonardo; Murillo, Gonzalo; Torras, Núria; Nogués, Carme; Stadler, Bethanie J H; Plaza, José A; Esteve, Jaume

2017-09-01

Remote microactuators are of great interest in biology and medicine as minimally-invasive tools for cellular stimulation. Remote actuation can be achieved by active magnetostrictive transducers which are capable of changing shape in response to external magnetic fields thereby creating controlled displacements. Among the magnetostrictive materials, Galfenol, the multifaceted iron-based smart material, offers high magnetostriction with robust mechanical properties. In order to explore these capabilities for biomedical applications, it is necessary to study the feasibility of material miniaturization in standard fabrication processes as well as evaluate the biocompatibility. Here we develop a technology to fabricate, release, and suspend Galfenol-based microparticles, without affecting the integrity of the material. The morphology, composition and magnetic properties of the material itself are characterized. The direct cytotoxicity of Galfenol is evaluated in vitro using human macrophages, osteoblast and osteosarcoma cells. In addition, cytotoxicity and actuation of Galfenol microparticles in suspension are evaluated using human macrophages. The biological parameters analyzed indicate that Galfenol is not cytotoxic, even after internalization of some of the particles by macrophages. The microparticles were remotely actuated forming intra- and extracellular chains that did not impact the integrity of the cells. The results propose Galfenol as a suitable material to develop remote microactuators for cell biology studies and intracellular applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of Cytochrome P 450 Inhibitors on Itraconazole and Fluconazole Induced Cytotoxicity in Hepatocytes

International Nuclear Information System (INIS)

Somchit, N.; Ngee, C.S.; Yaakob, A.; Ahmad, Z.; Zakaria, Z.A.

2009-01-01

Itraconazole and fluconazole have been reported to induce hepatotoxicity in patients. The present study was designed to investigate the role of cytochrome P450 inhibitors, SKF 525A, and curcumin pretreatment on the cytotoxicity of antifungal drugs fluconazole and itraconazole. For 3 consecutive days, female rats were administered daily SKF 525A or curcumin (5 and 25?mg/kg). Control rats received an equivalent amount of dosed vehicle. The animals were anaesthetised 24 hours after receiving the last dose for liver perfusion. Hepatocytes were then exposed to various concentrations of antifungal drugs. In vitro incubation of hepatocytes with itraconazole revealed significantly lower viability when compared to fluconazole as assessed by lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase activities. The cytotoxicity of itraconazole was enhanced when incubated with hepatocytes pretreated with SKF 525A. SKF 525A had no effects on the cytotoxicity of fluconazole. Curcumin failed to either increase or decrease the cytotoxicity of both antifungal drugs. ATP levels also showed significant decrease in both itraconazole and fluconazole incubated hepatocytes. However, SKF 525A pretreated hepatocytes had significantly lower ATP levels after itraconazole incubations. Collectively, these results confirm the involvement of cytochrome P450 in the cytoprotection in itraconazole induced hepatocyte toxicity. Differences of the effects of SKF 525A on the cytotoxicity induced by itraconazole and fluconazole may be due to the differences on the metabolism of each antifungal drug in vivo.

Activated human mast cells induce LOX-1-specific scavenger receptor expression in human monocyte-derived macrophages.

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Mervi Alanne-Kinnunen

Full Text Available Activated mast cells in atherosclerotic lesions degranulate and release bioactive compounds capable of regulating atherogenesis. Here we examined the ability of activated human primary mast cells to regulate the expression of the major scavenger receptors in cultured human primary monocyte-derived macrophages (HMDMs.Components released by immunologically activated human primary mast cells induced a transient expression of lectin-like oxidized LDL receptor (LOX-1 mRNA in HMDMs, while the expression of two other scavenger receptors, MSR1 and CD36, remained unaffected. The LOX-1-inducing secretory components were identified as histamine, tumor necrosis factor alpha (TNF-α, and transforming growth factor beta (TGF-β1, which exhibited a synergistic effect on LOX-1 mRNA expression. Histamine induced a transient expression of LOX-1 protein. Mast cell -induced increase in LOX-1 expression was not associated with increased uptake of oxidized LDL by the macrophages.Mast cell-derived histamine, TNF-α, and TGF-β1 act in concert to induce a transient increase in LOX-1 expression in human primary monocyte-derived macrophages. The LOX-1-inducing activity potentially endows mast cells a hitherto unrecognized role in the regulation of innate immune reactions in atherogenesis.

Role of iron overload-induced macrophage apoptosis in the pathogenesis of peritoneal endometriosis.

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Pirdel, Leila; Pirdel, Manijeh

2014-06-01

This article presents an overview of the involvement of iron overload-induced nitric oxide (NO) overproduction in apoptosis of peritoneal macrophages of women with endometriosis. We have postulated that the peritoneal iron overload originated from retrograde menstruation or bleeding lesions in the ectopic endometrium, which may contribute to the development of endometriosis by a wide range of mechanisms, including oxidative damage and chronic inflammation. Excessive NO production may also be associated with impaired clearance of endometrial cells by macrophages, which promotes cell growth in the peritoneal cavity. Therefore, further research of the mechanisms and consequences of macrophage apoptosis in endometriosis helps discover novel therapeutic strategies that are designed to prevent progression of endometriosis. © 2014 Society for Reproduction and Fertility.

Mulberry Fruit Extract Affords Protection against Ethyl Carbamate-Induced Cytotoxicity and Oxidative Stress

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Wei Chen

2017-01-01

Full Text Available Ethyl carbamate (EC is a food and environmental toxicant and is a cause of concern for human exposure. Several studies indicated that EC-induced toxicity was associated with oxidative stress. Mulberry fruits are reported to have a wide range of bioactive compounds and pharmacological activities. The present study was therefore aimed to investigate the protective property of mulberry fruit extract (MFE on EC-induced cytotoxicity and oxidative stress. Chemical composition analysis showed that total phenolic content and total flavonoid content in MFE were 502.43 ± 5.10 and 219.12 ± 4.45 mg QE/100 g FW. Cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside were the major anthocyanins in MFE. In vitro antioxidant studies (DPPH, ABTS, and FRAP assays jointly exhibited the potent antioxidant capacity of MFE. Further study indicated that MFE protected human liver HepG2 cells from EC-induced cytotoxicity by scavenging overproduced cellular ROS. EC treatment promoted intracellular glutathione (GSH depletion and caused mitochondrial membrane potential (MMP collapse, as well as mitochondrial membrane lipid peroxidation, whereas MFE pretreatment significantly inhibited GSH depletion and restored the mitochondrial membrane function. Overall, our study suggested that polyphenolic-rich MFE could afford a potent protection against EC-induced cytotoxicity and oxidative stress.

Chemotherapy-Induced Macrophage Infiltration into Tumors Enhances Nanographene-Based Photodynamic Therapy.

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Zhao, Yang; Zhang, Chenran; Gao, Liquan; Yu, Xinhe; Lai, Jianhao; Lu, Dehua; Bao, Rui; Wang, Yanpu; Jia, Bing; Wang, Fan; Liu, Zhaofei

2017-11-01

Increased recruitment of tumor-associated macrophages (TAM) to tumors following chemotherapy promotes tumor resistance and recurrence and correlates with poor prognosis. TAM depletion suppresses tumor growth, but is not highly effective due to the effects of tumorigenic mediators from other stromal sources. Here, we report that adoptive macrophage transfer led to a dramatically enhanced photodynamic therapy (PDT) effect of 2-(1-hexyloxyethyl)-2-devinyl pyropheophor-bide-alpha (HPPH)-coated polyethylene glycosylated nanographene oxide [GO(HPPH)-PEG] by increasing its tumor accumulation. Moreover, tumor treatment with commonly used chemotherapeutic drugs induced an increase in macrophage infiltration into tumors, which also enhanced tumor uptake and the PDT effects of GO(HPPH)-PEG, resulting in tumor eradication. Macrophage recruitment to tumors after chemotherapy was visualized noninvasively by near-infrared fluorescence and single-photon emission CT imaging using F4/80-specific imaging probes. Our results demonstrate that chemotherapy combined with GO(HPPH)-PEG PDT is a promising strategy for the treatment of tumors, especially those resistant to chemotherapy. Furthermore, TAM-targeted molecular imaging could potentially be used to predict the efficacy of combination therapy and select patients who would most benefit from this treatment approach. Cancer Res; 77(21); 6021-32. ©2017 AACR . ©2017 American Association for Cancer Research.

Effect of Vibrio cholerae neuraminidase on the mitogen response of T lymphocytes. I. Enhancement of macrophage T-lymphocyte cooperation in concanavalin-A-induced lymphocyte activation.

Science.gov (United States)

Knop, J

1980-12-01

Vibrio cholerae neuraminidase (VCN) enhances the immune response of lymphocytes in various systems, such as antigen- and mitogen-induced blastogenesis, mixed lymphocyte culture (MLC) and tumor-cell response. We used macrophage-depleted and reconstituted murine lymph-node T-cells to investigate the effect of VCN on macrophage-T-lymphocyte co-operation in Con-A-induced lymphocyte activation. In unfractionated lymph-node cells VCN enhanced the Con-A-induced lymphocyte activation as measured by 3H-thymidine (3H-dThd) incorporation. Removing macrophages from the cells resulted in a significantly diminished response. In addition the enhancing effect of VCN was greatly reduced. Reconstitution of the lymphocyte cultures with macrophages in increasing numbers and from various sources rstored the lymphocyte response and the enhancing effect of VCN. VCN proved to be most efficient in cultures reconstituted with normal peritoneal macrophages. Some effect was also observed using bone-marrow-derived (BM) macrophages. However, higher numbers of normal PE macrophages in the presence of VCN inhibited lymphocyte activation, and inhibition by thioglycollate-broth-induced macrophages was considerably increased by VCN. These results suggest that VCN acts by increasing the efficiency of macrophage-T lymphocyte interaction.

SUCNR1-mediated chemotaxis of macrophages aggravates obesity-induced inflammation and diabetes

NARCIS (Netherlands)

Diepen, van Janna A.; Robben, Joris H.; Hooiveld, Guido J.; Carmone, Claudia; Alsady, Mohammad; Boutens, Lily; Bekkenkamp-Grovenstein, Melissa; Hijmans, Anneke; Engelke, Udo F.H.; Wevers, Ron A.; Netea, Mihai G.; Tack, Cees J.; Stienstra, Rinke; Deen, Peter M.T.

2017-01-01

Aims/hypothesis: Obesity induces macrophages to drive inflammation in adipose tissue, a crucial step towards the development of type 2 diabetes. The tricarboxylic acid (TCA) cycle intermediate succinate is released from cells under metabolic stress and has recently emerged as a metabolic signal

The Impaired Function of Macrophages Induced by Strenuous Exercise Could Not Be Ameliorated by BCAA Supplementation.

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Xiao, Weihua; Chen, Peijie; Liu, Xiaoguang; Zhao, Linlin

2015-10-21

The aim of this study was to evaluate the effect of strenuous exercise on the functions of peritoneal macrophages in rats and to test the hypothesis that branched-chain amino acid (BCAA) supplementation will be beneficial to the macrophages of rats from strenuous exercise. Forty male Wistar rats were randomly divided into five groups: (C) Control, E) Exercise, (E1) Exercise with one week to recover, (ES) Exercise + Supplementation and (ES1) Exercise + Supplementation with 1 week to recover. All rats except those of the sedentary control were subjected to four weeks of strenuous exercise. Blood hemoglobin, serum testosterone and BCAA levels were tested. Peritoneal macrophages functions were also determined at the same time. The data showed that hemoglobin, testosterone, BCAA levels, and body weight in group E decreased significantly as compared with that of group C. Meanwhile, phagocytosis capacity (decreased by 17.07%, p = 0.031), reactive oxygen species (ROS) production (decreased by 26%, p = 0.003) and MHC II mRNA (decreased by 22%, p = 0.041) of macrophages decreased in the strenuous exercise group as compared with group C. However, the chemotaxis of macrophages did not change significantly. In addition, BCAA supplementation could slightly increase the serum BCAA levels of rats from strenuous exercise (increased by 6.70%, p > 0.05). Moreover, the body weight, the blood hemoglobin, the serum testosterone and the function of peritoneal macrophages in group ES did not change significantly as compared with group E. These results suggest that long-term intensive exercise impairs the function of macrophages, which is essential for microbicidal capability. This may represent a novel mechanism of immunosuppression induced by strenuous exercise. Moreover, the impaired function of macrophage induced by strenuous exercise could not be ameliorated by BCAA supplementation in the dosing and timing used for this study.

The Impaired Function of Macrophages Induced by Strenuous Exercise Could Not Be Ameliorated by BCAA Supplementation

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Weihua Xiao

2015-10-01

Full Text Available The aim of this study was to evaluate the effect of strenuous exercise on the functions of peritoneal macrophages in rats and to test the hypothesis that branched-chain amino acid (BCAA supplementation will be beneficial to the macrophages of rats from strenuous exercise. Forty male Wistar rats were randomly divided into five groups: (C Control, E Exercise, (E1 Exercise with one week to recover, (ES Exercise + Supplementation and (ES1 Exercise + Supplementation with 1 week to recover. All rats except those of the sedentary control were subjected to four weeks of strenuous exercise. Blood hemoglobin, serum testosterone and BCAA levels were tested. Peritoneal macrophages functions were also determined at the same time. The data showed that hemoglobin, testosterone, BCAA levels, and body weight in group E decreased significantly as compared with that of group C. Meanwhile, phagocytosis capacity (decreased by 17.07%, p = 0.031, reactive oxygen species (ROS production (decreased by 26%, p = 0.003 and MHC II mRNA (decreased by 22%, p = 0.041 of macrophages decreased in the strenuous exercise group as compared with group C. However, the chemotaxis of macrophages did not change significantly. In addition, BCAA supplementation could slightly increase the serum BCAA levels of rats from strenuous exercise (increased by 6.70%, p > 0.05. Moreover, the body weight, the blood hemoglobin, the serum testosterone and the function of peritoneal macrophages in group ES did not change significantly as compared with group E. These results suggest that long-term intensive exercise impairs the function of macrophages, which is essential for microbicidal capability. This may represent a novel mechanism of immunosuppression induced by strenuous exercise. Moreover, the impaired function of macrophage induced by strenuous exercise could not be ameliorated by BCAA supplementation in the dosing and timing used for this study.

Silica-induced initiation of circular ZC3H4 RNA/ZC3H4 pathway promotes the pulmonary macrophage activation.

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Yang, Xiyue; Wang, Jing; Zhou, Zewei; Jiang, Rong; Huang, Jie; Chen, Lulu; Cao, Zhouli; Chu, Han; Han, Bing; Cheng, Yusi; Chao, Jie

2018-01-22

Phagocytosis of silicon dioxide (SiO 2 ) into lung cells causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Circular RNAs (circRNAs) are a subclass of noncoding RNAs that are present within mammalian cells; however, researchers have not determined whether circRNAs are involved in the pathophysiologic process of silicosis. To elucidate the role of these RNAs in SiO 2 -induced inflammation in pulmonary macrophages, we investigated the upstream molecular mechanisms and functional effects of circRNAs on cell apoptosis, proliferation, and migration. Primary cultures of alveolar macrophages from healthy donors and from patients and the RAW264.7 macrophage cell line were used to explore the functions of circZC3H4 RNA in macrophage activation. The experimental results indicated the following: 1) SiO 2 concomitantly increased circZC3H4 RNA expression and increased ZC3H4 protein levels; 2) circular ZC3H4 (circZC3H4) RNA and ZC3H4 protein participated in SiO 2 -induced macrophage activation; and 3) SiO 2 -activated macrophages promoted fibroblast proliferation and migration via the circZC3H4 RNA/ZC3H4 pathway. The up-regulation of the ZC3H4 protein was confirmed in tissue samples from patients with silicosis. Our study elucidates a link between SiO 2 -induced macrophage activation and the circZC3H4 RNA/ZC3H4 pathway, thereby providing novel insight into the potential use of ZC3H4 to develop novel therapeutic strategies for silicosis.-Yang, X., Wang, J., Zhou, Z., Jiang, R., Huang, J., Chen, L., Cao, Z., Chu, H., Han, B., Cheng, Y., Chao, J. Silica-induced initiation of circular ZC3H4 RNA/ZC3H4 pathway promotes the pulmonary macrophage activation.

Dopamine induces growth inhibition and vascular normalization through reprogramming M2-polarized macrophages in rat C6 glioma

International Nuclear Information System (INIS)

Qin, Tian; Wang, Chenlong; Chen, Xuewei; Duan, Chenfan; Zhang, Xiaoyan; Zhang, Jing; Chai, Hongyan; Tang, Tian; Chen, Honglei; Yue, Jiang; Li, Ying; Yang, Jing

2015-01-01

Dopamine (DA), a monoamine catecholamine neurotransmitter with antiangiogenic activity, stabilizes tumor vessels in colon, prostate and ovarian cancers, thus increases chemotherapeutic efficacy. Here, in the rat C6 glioma models, we investigated the vascular normalization effects of DA and its mechanisms of action. DA (25, 50 mg/kg) inhibited tumor growth, while a precursor of DA (levodopa) prolonged the survival time of rats bearing orthotopic C6 glioma. DA improved tumor perfusion, with significant effects from day 3, and a higher level at days 5 to 7. In addition, DA decreased microvessel density and hypoxia-inducible factor-1α expression in tumor tissues, while increasing the coverage of pericyte. Conversely, an antagonist of dopamine receptor 2 (DR2) (eticlopride) but not DR1 (butaclamol) abrogated DA-induced tumor regression and vascular normalization. Furthermore, DA improved the delivery and efficacy of temozolomide therapy. Importantly, DA increased representative M1 markers (iNOS, CXCL9, etc.), while decreasing M2 markers (CD206, arginase-1, etc.). Depletion of macrophages by clodronate or zoledronic acid attenuated the effects of DA. Notably, DA treatment induced M2-to-M1 polarization in RAW264.7 cells and mouse peritoneal macrophages, and enhanced the migration of pericyte-like cells (10T1/2), which was reversed by eticlopride or DR2-siRNA. Such changes were accompanied by the downregulation of VEGF/VEGFR2 signaling. In summary, DA induces growth inhibition and vascular normalization through reprogramming M2-polarized macrophages. Thus, targeting the tumor microvasculature by DA represents a promising strategy for human glioma therapy. - Highlights: • Dopamine induces tumor growth inhibition and vascular normalization in rat C6 glioma. • Dopamine switches macrophage phenotype from M2 to M1. • Dopamine-induced vascular normalization is mediated by macrophage polarization. • Dopamine is a promising agent targeting the microvasculature in tumor

Dopamine induces growth inhibition and vascular normalization through reprogramming M2-polarized macrophages in rat C6 glioma

Energy Technology Data Exchange (ETDEWEB)

Qin, Tian; Wang, Chenlong; Chen, Xuewei; Duan, Chenfan; Zhang, Xiaoyan [Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Zhang, Jing [Animal Experimental Center of Wuhan University, Wuhan 430071 (China); Chai, Hongyan [Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Tang, Tian [Department of Oncology, Renmin Hospital of Wuhan University, Wuhan 430060 (China); Chen, Honglei [Department of Pathology and Pathophysiology, School of Medicine, Wuhan University, Wuhan 430071 (China); Yue, Jiang [Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Li, Ying, E-mail: lyying0@163.com [Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Yang, Jing, E-mail: yangjingliu2013@163.com [Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China)

2015-07-15

Dopamine (DA), a monoamine catecholamine neurotransmitter with antiangiogenic activity, stabilizes tumor vessels in colon, prostate and ovarian cancers, thus increases chemotherapeutic efficacy. Here, in the rat C6 glioma models, we investigated the vascular normalization effects of DA and its mechanisms of action. DA (25, 50 mg/kg) inhibited tumor growth, while a precursor of DA (levodopa) prolonged the survival time of rats bearing orthotopic C6 glioma. DA improved tumor perfusion, with significant effects from day 3, and a higher level at days 5 to 7. In addition, DA decreased microvessel density and hypoxia-inducible factor-1α expression in tumor tissues, while increasing the coverage of pericyte. Conversely, an antagonist of dopamine receptor 2 (DR2) (eticlopride) but not DR1 (butaclamol) abrogated DA-induced tumor regression and vascular normalization. Furthermore, DA improved the delivery and efficacy of temozolomide therapy. Importantly, DA increased representative M1 markers (iNOS, CXCL9, etc.), while decreasing M2 markers (CD206, arginase-1, etc.). Depletion of macrophages by clodronate or zoledronic acid attenuated the effects of DA. Notably, DA treatment induced M2-to-M1 polarization in RAW264.7 cells and mouse peritoneal macrophages, and enhanced the migration of pericyte-like cells (10T1/2), which was reversed by eticlopride or DR2-siRNA. Such changes were accompanied by the downregulation of VEGF/VEGFR2 signaling. In summary, DA induces growth inhibition and vascular normalization through reprogramming M2-polarized macrophages. Thus, targeting the tumor microvasculature by DA represents a promising strategy for human glioma therapy. - Highlights: • Dopamine induces tumor growth inhibition and vascular normalization in rat C6 glioma. • Dopamine switches macrophage phenotype from M2 to M1. • Dopamine-induced vascular normalization is mediated by macrophage polarization. • Dopamine is a promising agent targeting the microvasculature in tumor

Cytotoxic and cytoprotective activities of curcumin. Effects on paracetamol-induced cytotoxicity, lipid peroxidation and glutathione depletion in rat hepatocytes

NARCIS (Netherlands)

Donatus, I A; Sardjoko,; Vermeulen, N P

1990-01-01

The cytoprotective effect of curcumin, a natural constituent of Curcuma longa, on the cytotoxicity of paracetamol in rat hepatocytes was studied. Paracetamol was selected as a model-toxin, since it is known to be bioactivated by 3-methylcholanthrene inducible cytochromes P450 presumably to

Antibody-dependent cellular cytotoxicity and skin disease

International Nuclear Information System (INIS)

Norris, D.A.; Lee, L.A.

1985-01-01

Antibody dependent cellular cytotoxicity (ADCC) is a recently described mechanism of immunologic lysis in which cellular targets sensitized by specific antibodies are efficiently and selectively lysed by Fc receptor (FcR) bearing nonspecific effectors. Immunoglobulins of various classes (IgG, IgM, IgA, IgE) and various cellular effectors (large granular lymphocytes, monocyte/macrophages, T lymphocytes, neutrophils, and eosinophils) can induce ADCC in vitro, and the importance of ADCC in vivo is being tested experimentally in resistance to viral, bacterial, and parasitic infection, in tumor surveillance, in allograft rejection, and in inflammatory diseases. There is much indirect evidence that ADCC may be the mechanism of damage of different cellular targets in skin diseases, but the best direct evidence concerns immunologic keratinocyte damage, especially in cutaneous lupus erythematosus (LE). The authors have shown that keratinocytes of several species are highly susceptible to lymphocyte and monocyte-mediated ADCC, but not to neutrophil or eosinophil ADCC in vitro using two different cytotoxicity assays. In contrast, complement was a relatively ineffective mediator of lysis of metabolically intact keratinocyte targets. Patients with certain cutaneous lupus syndromes have serum antibodies capable of inducing monocyte and lymphocyte ADCC of targets coated with extractable nuclear antigens. The authors have shown that these antigens apparently move to the cell membrane of keratinocytes in vitro following ultraviolet irradiation. In an animal model, they have shown that antibodies to SSA/Ro bind to human keratinocytes in vivo, especially after ultraviolet irradiation

Role of Macrophage-Induced Inflammation in Mesothelioma

Science.gov (United States)

2011-07-01

macrophages). Normal pleura becomes available intermittently , serving to slow the completion of this task. All is set in place for us to complete the...GFP) regulated by a Csf1r-promoter (Sasmono et al. 2003) show that macrophages travel up and down these fibers at a fast rate and also “jump” between...2010). Macrophages have also recently been shown to be important in adipogenesis at least during obesity , through their secretion of adipocyte growth

CYLD Proteolysis Protects Macrophages from TNF-Mediated Auto-necroptosis Induced by LPS and Licensed by Type I IFN

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Diana Legarda

2016-06-01

Full Text Available Tumor necrosis factor (TNF induces necroptosis, a RIPK3/MLKL-dependent form of inflammatory cell death. In response to infection by Gram-negative bacteria, multiple receptors on macrophages, including TLR4, TNF, and type I IFN receptors, are concurrently activated, but it is unclear how they crosstalk to regulate necroptosis. We report that TLR4 activates CASPASE-8 to cleave and remove the deubiquitinase cylindromatosis (CYLD in a TRIF- and RIPK1-dependent manner to disable necroptosis in macrophages. Inhibiting CASPASE-8 leads to CYLD-dependent necroptosis caused by the TNF produced in response to TLR4 ligation. While lipopolysaccharides (LPS-induced necroptosis was abrogated in Tnf−/− macrophages, a soluble TNF antagonist was not able to do so in Tnf+/+ macrophages, indicating that necroptosis occurs in a cell-autonomous manner. Surprisingly, TNF-mediated auto-necroptosis of macrophages requires type I IFN, which primes the expression of key necroptosis-signaling molecules, including TNFR2 and MLKL. Thus, the TNF necroptosis pathway is regulated by both negative and positive crosstalk.

Curcumin ameliorates macrophage infiltration by inhibiting NF-κB activation and proinflammatory cytokines in streptozotocin induced-diabetic nephropathy

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Suzuki Kenji

2011-06-01

Full Text Available Abstract Background Chronic inflammation plays an important role in the progression of diabetic nephropathy (DN and that the infiltration of macrophages in glomerulus has been implicated in the development of glomerular injury. We hypothesized that the plant polyphenolic compound curcumin, which is known to exert potent anti-inflammatory effect, would ameliorate macrophage infiltration in streptozotocin (STZ-induced diabetic rats. Methods Diabetes was induced with STZ (55 mg/kg by intraperitoneal injection in rats. Three weeks after STZ injection, rats were divided into three groups, namely, control, diabetic, and diabetic treated with curcumin at 100 mg/kg/day, p.o., for 8 weeks. The rats were sacrificed 11 weeks after induction of diabetes. The excised kidney was used to assess macrophage infiltration and expression of various inflammatory markers. Results At 11 weeks after STZ injection, diabetic rats exhibited renal dysfunction, as evidenced by reduced creatinine clearance, increased blood glucose, blood urea nitrogen and proteinuria, along with marked reduction in the body weight. All of these abnormalities were significantly reversed by curcumin. Hyperglycemia induced the degradation of IκBα and NF-κB activation and as a result increased infiltration of macrophages (52% as well as increased proinflammatory cytokines: TNF-α and IL-1β. Curcumin treatment significantly reduced macrophage infiltration in the kidneys of diabetic rats, suppressed the expression of above proinflammatory cytokines and degradation of IκBα. In addition, curcumin treatment also markedly decreased ICAM-1, MCP-1 and TGF-β1 protein expression. Moreover, at nuclear level curcumin inhibited the NF-κB activity. Conclusion Our results suggested that curcumin treatment protect against the development of DN in rats by reducing macrophage infiltration through the inhibition of NF-κB activation in STZ-induced diabetic rats.

Inhibition of macrophage oxidative stress prevents the reduction of ABCA-1 transporter induced by advanced glycated albumin.

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de Souza Pinto, Raphael; Castilho, Gabriela; Paim, Bruno Alves; Machado-Lima, Adriana; Inada, Natalia M; Nakandakare, Edna Regina; Vercesi, Aníbal Eugênio; Passarelli, Marisa

2012-05-01

We investigated the role of aminoguanidine and benfotiamine on the inhibition of reactive oxygen species (ROS) generation in macrophages induced by advanced glycated albumin (AGE-albumin) and its relationship with cell cholesterol homeostasis, emphasizing the expression of the ATP binding cassette transporter A-1 (ABCA-1). AGE-albumin was made by incubating fatty acid-free albumin with 10 mM glycolaldehyde. ROS production and ABCA-1 protein level were determined by flow cytometry in J774 macrophages treated along time with control (C) or AGE-albumin alone or in the presence of aminoguanidine or benfotiamine. Mitochondrial function was evaluated by oxygraphy. Compared to C-albumin, AGE-albumin increased ROS production in macrophages, which was ascribed to the activities of NADPH oxidase and of the mitochondrial system. Mitochondrial respiratory chain activity was reduced in cells incubated with AGE-albumin. ROS generation along time was associated with the reduction in macrophage ABCA-1 protein level. Aminoguanidine prevented ROS elevation and restored the ABCA-1 content in macrophages; on the other hand, benfotiamine that promoted a lesser reduction in ROS generation was not able to restore ABCA-1 levels. Inhibition of oxidative stress induced by AGE-albumin prevents disturbances in reverse cholesterol transport by curbing the reduction of ABCA-1 elicited by advanced glycation in macrophages and therefore may contribute to the prevention of atherosclerosis in diabetes mellitus.

Macrophage Immune Response Suppression by Recombinant Mycobacterium tuberculosis Antigens, the ESAT-6, CFP-10, and ESAT-6/CFP-10 Fusion Proteins

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Seghatoleslam, Atefeh; Hemmati, Mina; Ebadat, Saeedeh; Movahedi, Bahram; Mostafavi-Pour, Zohreh

2016-01-01

Background: Macrophage immune responses are affected by the secretory proteins of Mycobacterium tuberculosis (Mtb). This study aimed to examine the immune responses of macrophages to Mtb secretory antigens, namely ESAT-6, CFP-10, and ESAT-6/CFP-10. Methods: THP-1 cells (a human monocytic cell line) were cultured and differentiated to macrophages by phorbol 12-myristate 13-acetate. The cytotoxicity of the recombinant Mtb proteins was assessed using the MTT assay. Two important immune responses of macrophages, namely NO and ROS production, were measured in response to the ESAT-6, CFP-10, and ESAT-6/CFP-10 antigens. The data were analyzed using one-way ANOVA with SPSS, version 16, and considered significant at Pproteins markedly reduced macrophage immune response. The treatment of the THP-1-differentiated cells with ESAT-6, CFP-10, and ESAT-6/CFP-10 reduced NO and ROS production. The treated THP-1-differentiated cells exhibited less inducible NO synthase activity than did the untreated cells. No toxic effect on macrophage viability was observed for the applied proteins at the different concentrations. Conclusion: It seems that the decline in macrophage immune response is due to the suppression of NO and ROS production pathways without any effect on cell viability. PMID:27365551

Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A2-induced degranulation in mast cells

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Nishikawa, Hirofumi; Kitani, Seiichi

2011-01-01

Sting accident by honeybee causes severe pain, inflammation and allergic reaction through IgE-mediated anaphylaxis. In addition to this hypersensitivity, an anaphylactoid reaction occurs by toxic effects even in a non-allergic person via cytolysis followed by similar clinical manifestations. Auto-injectable epinephrine might be effective for bee stings, but cannot inhibit mast cell lysis and degranulation by venom toxins. We used connective tissue type canine mast cell line (CM-MC) for finding an effective measure that might inhibit bee venom toxicity. We evaluated degranulation and cytotoxicity by measurement of β-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation and cytotoxicity, which were strongly inhibited by mono-sialoganglioside (G M1 ), di-sialoganglioside (G D1a ) and tri-sialoganglioside (G T1b ). In contrast, honeybee venom-derived phospholipase A 2 induced the net degranulation directly without cytotoxicity, which was not inhibited by G M1 , G D1a and G T1b . For analysis of distribution of Gα q and Gα i protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of Gα q and Gα i at lipid raft, but gangliosides stabilized the rafts. As a result from this cell-based study, bee venom-induced anaphylactoid reaction can be explained with melittin cytotoxicity and phospholipase A 2 -induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A 2 -induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting.

Insulin-Like Growth Factor-I Induces Arginase Activity in Leishmania amazonensis Amastigote-Infected Macrophages through a Cytokine-Independent Mechanism

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Celia Maria Vieira Vendrame

2014-01-01

Full Text Available Leishmania (Leishmania amazonensis exhibits peculiarities in its interactions with hosts. Because amastigotes are the primary form associated with the progression of infection, we studied the effect of insulin-like growth factor (IGF-I on interactions between L. (L. amazonensis amastigotes and macrophages. Upon stimulation of infected macrophages with IGF-I, we observed decreased nitric oxide production but increased arginase expression and activity, which lead to increased parasitism. However, stimulation of amastigote-infected macrophages with IGF-I did not result in altered cytokine levels compared to unstimulated controls. Because IGF-I is present in tissue fluids and also within macrophages, we examined the possible effect of this factor on phosphatidylserine (PS exposure on amastigotes, seen previously in tissue-derived amastigotes leading to increased parasitism. Stimulation with IGF-I induced PS exposure on amastigotes but not on promastigotes. Using a PS-liposome instead of amastigotes, we observed that the PS-liposome but not the control phosphatidylcholine-liposome led to increased arginase activity in macrophages, and this process was not blocked by anti-TGF-β antibodies. Our results suggest that in L. (L. amazonensis amastigote-infected macrophages, IGF-I induces arginase activity directly in amastigotes and in macrophages through the induction of PS exposure on amastigotes in the latter, which could lead to the alternative activation of macrophages through cytokine-independent mechanisms.

Autophagy plays a critical role in ChLym-1-induced cytotoxicity of non-hodgkin's lymphoma cells.

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Jiajun Fan

Full Text Available Autophagy is a critical mechanism in both cancer therapy resistance and tumor suppression. Monoclonal antibodies have been documented to kill tumor cells via apoptosis, antibody-dependent cellular cytotoxicity (ADCC and complement-dependent cytotoxicity (CDC. In this study, we report for the first time that chLym-1, a chimeric anti-human HLA-DR monoclonal antibody, induces autophagy in Raji Non-Hodgkin's Lymphoma (NHL cells. Interestingly, inhibition of autophagy by pharmacological inhibitors (3-methyladenine and NH4Cl or genetic approaches (siRNA targeting Atg5 suppresses chLym-1-induced growth inhibition, apoptosis, ADCC and CDC in Raji cells, while induction of autophagy could accelerate cytotoxic effects of chLym-1 on Raji cells. Furthermore, chLym-1-induced autophagy can mediate apoptosis through Caspase 9 activation, demonstrating the tumor-suppressing role of autophagy in antilymphoma effects of chLym-1. Moreover, chLym-1 can activate several upstream signaling pathways of autophagy including Akt/mTOR and extracellular signal-regulated kinase 1/2 (Erk1/2. These results elucidate the critical role of autophagy in cytotoxicity of chLym-1 antibody and suggest a potential therapeutic strategy of NHL therapy by monoclonal antibody chLym-1 in combination with autophagy inducer.

Carbon monoxide induced PPARγ SUMOylation and UCP2 block inflammatory gene expression in macrophages.

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Arvand Haschemi

Full Text Available Carbon monoxide (CO dampens pro-inflammatory responses in a peroxisome proliferator-activated receptor-γ (PPARγ and p38 mitogen-activated protein kinase (MAPK dependent manner. Previously, we demonstrated that CO inhibits lipopolysaccharide (LPS-induced expression of the proinflammatory early growth response-1 (Egr-1 transcription factor in macrophages via activation of PPARγ. Here, we further characterize the molecular mechanisms by which CO modulates the activity of PPARγ and Egr-1 repression. We demonstrate that CO enhances SUMOylation of PPARγ which we find was attributed to mitochondrial ROS generation. Ectopic expression of a SUMOylation-defective PPARγ-K365R mutant partially abolished CO-mediated suppression of LPS-induced Egr-1 promoter activity. Expression of a PPARγ-K77R mutant did not impair the effect of CO. In addition to PPARγ SUMOylation, CO-activated p38 MAPK was responsible for Egr-1 repression. Blocking both CO-induced PPARγ SUMOylation and p38 activation, completely reversed the effects of CO on inflammatory gene expression. In primary macrophages isolated form C57/BL6 male mice, we identify mitochondrial ROS formation by CO as the upstream trigger for the observed effects on Egr-1 in part through uncoupling protein 2 (UCP2. Macrophages derived from bone marrow isolated from Ucp2 gene Knock-Out C57/BL6 mice (Ucp2(-/-, produced significantly less ROS with CO exposure versus wild-type macrophages. Moreover, absence of UCP2 resulted in a complete loss of CO mediated Egr-1 repression. Collectively, these results indentify p38 activation, PPARγ-SUMOylation and ROS formation via UCP2 as a cooperative system by which CO impacts the inflammatory response.

Macrophage migration inhibitory factor induces vascular leakage via autophagy

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Hong-Ru Chen

2015-01-01

Full Text Available Vascular leakage is an important feature of acute inflammatory shock, which currently has no effective treatment. Macrophage migration inhibitory factor (MIF is a pro-inflammatory cytokine that can induce vascular leakage and plays an important role in the pathogenesis of shock. However, the mechanism of MIF-induced vascular leakage is still unclear. In this study, using recombinant MIF (rMIF, we demonstrated that MIF induced disorganization and degradation of junction proteins and increased the permeability of human endothelial cells in vitro. Western blotting analysis showed that rMIF treatment induced LC3 conversion and p62 degradation. Inhibition of autophagy with a PI3K inhibitor (3-MA, a ROS scavenger (NAC or autophagosomal-lysosomal fusion inhibitors (bafilomycin A1 and chloroquine rescued rMIF-induced vascular leakage, suggesting that autophagy mediates MIF-induced vascular leakage. The potential involvement of other signaling pathways was also studied using different inhibitors, and the results suggested that MIF-induced vascular leakage may occur through the ERK pathway. In conclusion, we showed that MIF triggered autophagic degradation of endothelial cells, resulting in vascular leakage. Inhibition of MIF-induced autophagy may provide therapeutic targets against vascular leakage in inflammatory shock.

Inhibitory effects on the production of inflammatory mediators and reactive oxygen species by Mori folium in lipopolysaccharide-stimulated macrophages and zebrafish

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DA HYE KWON

Full Text Available ABSTRACT Mori folium, the leaf of Morus alba L. (Moraceae, has been traditionally used for various medicinal purposes from ancient times to the present. In this study, we examined the effects of water extract of Mori folium (WEMF on the production of inflammatory mediators, such as nitric oxide (NO and prostaglandin E2 (PGE2, and reactive oxygen species (ROS in lipopolysaccharide (LPS-stimulated murine RAW 264.7 macrophages. Our data indicated that WEMF significantly suppressed the secretion of NO and PGE2 in RAW 264.7 macrophages without any significant cytotoxicity. The protective effects were accompanied by a marked reduction in their regulatory gene expression at the transcription level. WEMF attenuated LPS-induced intracellular ROS production in RAW 264.7 macrophages. It inhibited the nuclear translocation of the nuclear factor-kappa B p65 subunit and the activation of mitogen-activated protein kinases in LPS-treated RAW 264.7 macrophages. Furthermore, WEMF reduced LPS-induced NO production and ROS accumulation in zebrafish. Although more efforts are needed to fully understand the critical role of WEMF in the inhibition of inflammation, the findings of the present study may provide insights into the approaches for Mori folium as a potential therapeutic agent for inflammatory and antioxidant disorders.

Periodontitis promotes the diabetic development of obese rat via miR-147 induced classical macrophage activation.

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Xu, Ran; Zeng, Guang; Wang, Shuyong; Tao, Hong; Ren, Le; Zhang, Zhe; Zhang, Qingna; Zhao, Jinxiu; Gao, Jing; Li, Daxu

2016-10-01

Emerging evidence has indicated the bad effect of periodontal inflammation on diabetes control. However, the exact regulatory mechanisms within the association between periodontitis and diabetic development remain unclear. This study aims to investigate the function of microRNAs in regulating periodontitis-induced inflammation in an obese rat model. Experimental periodontitis was introduced into OLETF and LETO rat. Intraperitoneal glucose tolerance test was performed to detect diabetic development. Serum cytokines levels and microRNAs expression were detected by ELISA and RT-PCR analysis respectively. And, macrophages were isolated for gain- and loss-of-function studies, to investigate the regulatory mechanism of miR-147 in periodontitis-induced inflammation. Periodontitis induced proinflammatory response with classical activated macrophages in both rats, but distinctively aggravated the impaired glucose tolerance of OLETF rat with spontaneous type 2 diabetes. Analysis for serum microRNAs expression showed the distinctive and synergistic upregulation of miR-147 with periodontitis-induced effects in rats, while further experiments demonstrated the positive regulatory mechanism of miR-147 on classical activated macrophages with overexpressed proinflammatory markers, showing M1 phenotype. This study provided new evidence for the positive effect of periodontal inflammation on diabetic development, while the regulatory mechanism of miR-147 on classical macrophage activation, was verified, and presumed to contribute to the impaired glucose tolerance aggravated by periodontitis in obese rats. Besides, this study indicated the application of miR-147 for therapeutic approach in the treatment of diabetes with periodontitis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Streptococcus gordonii induces nitric oxide production through its lipoproteins stimulating Toll-like receptor 2 in murine macrophages.

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Kim, Hyun Young; Baik, Jung Eun; Ahn, Ki Bum; Seo, Ho Seong; Yun, Cheol-Heui; Han, Seung Hyun

2017-02-01

Streptococcus gordonii, a Gram-positive commensal in the oral cavity, is an opportunistic pathogen that can cause endodontic and systemic infections resulting in infective endocarditis. Lipoteichoic acid (LTA) and lipoprotein are major virulence factors of Gram-positive bacteria that are preferentially recognized by Toll-like receptor 2 (TLR2) on immune cells. In the present study, we investigated the effect of S. gordonii LTA and lipoprotein on the production of the representative inflammatory mediator nitric oxide (NO) by the mouse macrophages. Heat-killed S. gordonii wild-type and an LTA-deficient mutant (ΔltaS) but not a lipoprotein-deficient mutant (Δlgt) induced NO production in mouse primary macrophages and the cell line, RAW 264.7. S. gordonii wild-type and ΔltaS also induced the expression of inducible NO synthase (iNOS) at the mRNA and protein levels. In contrast, the Δlgt mutant showed little effect under the same condition. Furthermore, S. gordonii wild-type and ΔltaS induced NF-κB activation, STAT1 phosphorylation, and IFN-β expression, which are important for the induction of iNOS gene expression, with little activation by Δlgt. S. gordonii wild-type and ΔltaS showed an increased adherence and internalization to RAW 264.7 cells compared to Δlgt. In addition, S. gordonii wild-type and ΔltaS, but not Δlgt, substantially increased TLR2 activation while none of these induced NO production in TLR2-deficient macrophages. Triton X-114-extracted lipoproteins from S. gordonii were sufficient to induce NO production. Collectively, we suggest that lipoprotein is an essential cell wall component of S. gordonii to induce NO production in macrophages through TLR2 triggering NF-κB and STAT1 activation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Toxoplasma gondii exposes phosphatidylserine inducing a TGF-β1 autocrine effect orchestrating macrophage evasion

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Seabra, Sergio H.; Souza, Wanderley de; Matta, Renato A. da

2004-01-01

Toxoplasmosis is a worldwide disease caused by Toxoplasma gondii. Activated macrophages control T. gondii growth by nitric oxide (NO) production. However, T. gondii active invasion inhibits NO production, allowing parasite persistence. Here we show that the mechanism used by T. gondii to inhibit NO production persisting in activated macrophages depends on phosphatidylserine (PS) exposure. Masking PS with annexin-V on parasites or activated macrophages abolished NO production inhibition and parasite persistence. NO production inhibition depended on a transforming growth factor-β 1 (TGF-β 1 ) autocrine effect confirmed by the expression of Smad 2 and 3 in infected macrophages. TGF-β 1 led to inducible nitric oxide synthase (iNOS) degradation, actin filament (F-actin) depolymerization, and lack of nuclear factor-κB (NF-κB) in the nucleus. All these features were reverted by TGF-β 1 neutralizing antibody treatment. Thus, T. gondii mimics the evasion mechanism used by Leishmania amazonensis and also the anti-inflammatory response evoked by apoptotic cells

Autophagy Inhibition Contributes to ROS-Producing NLRP3-Dependent Inflammasome Activation and Cytokine Secretion in High Glucose-Induced Macrophages.

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Dai, Jiezhi; Zhang, Xiaotian; Li, Li; Chen, Hua; Chai, Yimin

2017-01-01

Type 2 diabetes is a persistent inflammatory response that impairs the healing process. We hypothesized that stimulation with high glucose following a pro-inflammatory signal would lead to autophagy inhibition, reactive oxygen species (ROS) production and eventually to the activation of the Nod-like receptor protein (NLRP) -3. Macrophages were isolated from human diabetic wound. We measured the expression of NLRP3, caspase1 and interleukin-1 beta (IL-1β) by western blot and real-time PCR, and the surface markers on cells by flow cytometry. THP-1-derived macrophages exposed to high glucose were applied to study the link between autophagy, ROS and NLRP3 activation. LC3-II, P62, NLRP3 inflammation and IL-1β expression were measured by western blot and real-time PCR. ROS production was measured with a Cellular Reactive Oxygen Species Detection Assay Kit. Macrophages isolated from diabetic wounds exhibited a pro-inflammatory phenotype, including sustained NLRP3 inflammasome activity associated with IL-1β secretion. Our data showed that high glucose inhibited autophagy, induced ROS production, and activated NLRP3 inflammasome and cytokine secretion in THP-1-derived macrophages. To study high glucose-induced NLRP3 inflammasome signalling, we performed studies using an autophagy inducer, a ROS inhibitor and a NLRP3 inhibitor and found that all reduced the NLRP3 inflammasome activation and cytokine secretion. Sustained NLRP3 inflammasome activity in wound-derived macrophages contributes to the hyper-inflammation in human diabetic wounds. Autophagy inhibition and ROS generation play an essential role in high glucose-induced NLRP3 inflammasome activation and cytokine secretion in macrophages. © 2017 The Author(s). Published by S. Karger AG, Basel.

Oligosaccharide modification by N-acetylglucosaminyltransferase-V in macrophages are involved in pathogenesis of bleomycin-induced scleroderma.

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Kato, Arisa; Yutani, Mizuki; Terao, Mika; Kimura, Akihiro; Itoi, Saori; Murota, Hiroyuki; Miyoshi, Eiji; Katayama, Ichiro

2015-08-01

Oligosaccharide modification by N-acetylglucosaminyltransferase-V (GnT-V), which catalyses the formation of β1,6 GlcNAc (N-acetylglucosamine) branches on N-glycans, is associated with various pathologies, such as cancer metastasis, multiple sclerosis and liver fibrosis. In this study, we demonstrated the involvement of GnT-V in the pathophysiology of scleroderma. High expression of GnT-V was observed in infiltrating cells in skin section samples from systemic and localized patients with scleroderma. Most of the infiltrating cells were T cells and macrophages, most of which were CD163(+) M2 macrophages. To determine the role of GnT-V in scleroderma, we next investigated skin sclerosis in GnT-V knockout (MGAT5(-/-) ) mice. Expression of GnT-V was also elevated in bleomycin (BLM)-injected sclerotic skin, and MGAT5(-/-) mice were resistant to BLM-induced skin sclerosis with reduced collagen type 1 α1 content, suggesting the biological significance of GnT-V in skin sclerosis. Furthermore, the number of CD163(+) M2 macrophages and CD3-positive T cells in BLM-induced skin sclerosis was significantly fewer in MGAT5(-/-) mice. In bone marrow-derived macrophages (BMDMs), IL-4-induced expressions of Fizz1 and Ym1 were significantly reduced in MGAT5(-/-) mice-derived BMDMs. Taken together, these results suggest the induction of GnT-V in skin sclerosis progression is possibly dependent on increased numbers of M2 macrophages in the skin, which are important for tissue fibrosis and remodelling. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hypoxia-induced pulmonary vascular remodeling requires recruitment of circulating mesenchymal precursors of a monocyte/macrophage lineage.

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Frid, Maria G; Brunetti, Jacqueline A; Burke, Danielle L; Carpenter, Todd C; Davie, Neil J; Reeves, John T; Roedersheimer, Mark T; van Rooijen, Nico; Stenmark, Kurt R

2006-02-01

Vascular remodeling in chronic hypoxic pulmonary hypertension includes marked fibroproliferative changes in the pulmonary artery (PA) adventitia. Although resident PA fibroblasts have long been considered the primary contributors to these processes, we tested the hypothesis that hypoxia-induced pulmonary vascular remodeling requires recruitment of circulating mesenchymal precursors of a monocyte/macrophage lineage, termed fibrocytes. Using two neonatal animal models (rats and calves) of chronic hypoxic pulmonary hypertension, we demonstrated a dramatic perivascular accumulation of mononuclear cells of a monocyte/macrophage lineage (expressing CD45, CD11b, CD14, CD68, ED1, ED2). Many of these cells produced type I collagen, expressed alpha-smooth muscle actin, and proliferated, thus exhibiting mesenchymal cell characteristics attributed to fibrocytes. The blood-borne origin of these cells was confirmed in experiments wherein circulating monocytes/macrophages of chronically hypoxic rats were in vivo-labeled with DiI fluorochrome via liposome delivery and subsequently identified in the remodeled pulmonary, but not systemic, arterial adventitia. The DiI-labeled cells that appeared in the vessel wall expressed monocyte/macrophage markers and procollagen. Selective depletion of this monocytic cell population, using either clodronate-liposomes or gadolinium chloride, prevented pulmonary adventitial remodeling (ie, production of collagen, fibronectin, and tenascin-C and accumulation of myofibroblasts). We conclude that circulating mesenchymal precursors of a monocyte/macrophage lineage, including fibrocytes, are essential contributors to hypoxia-induced pulmonary vascular remodeling.

Evaluation of the Leishmanicidal and Cytotoxic Potential of Essential Oils Derived from Ten Colombian Plants

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JF Sanchez-Suarez

2013-03-01

Full Text Available Background: The leishmanicidal and cytotoxic activity of ten essential oils obtained from ten plant specimens were evaluated.Methods: Essential oils were obtained by the steam distillation of plant leaves without any prior processing. Cytotoxicity was tested on J774 macrophages and leishmanicidal activity was assessed against four species of Leishmania associated with cutaneous leishmaniasis. Results: Seven essential oils exhibited activity against Leishmania parasites, five of which were toxic against J774 macrophages. Selectivity indices of >6 and 13 were calculated for the essential oils of Ocimum basilicum and Origanum vulgare, respectively.Conclusion: The essential oil of Ocimum basilicum was active against promastigotes of Leishmania and innocuous to J774 macrophages at concentrations up to 1600 µg/mL and should be further investi­gated for leishmanicidal activity in others in vitro and in vivo experimental models.

Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

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Civini, Sara; Pacelli, Consiglia; Dieng, Mame Massar; Lemieux, William; Jin, Ping; Bazin, Renée; Patey, Natacha; Marincola, Francesco M.; Moldovan, Florina; Zaouter, Charlotte; Trudeau, Louis-Eric; Benabdhalla, Basma; Louis, Isabelle; Beauséjour, Christian; Stroncek, David; Le Deist, Françoise; Haddad, Elie

2016-01-01

Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation. PMID:27070086

TRPV4 calcium-permeable channel is a novel regulator of oxidized LDL-induced macrophage foam cell formation.

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Goswami, Rishov; Merth, Michael; Sharma, Shweta; Alharbi, Mazen O; Aranda-Espinoza, Helim; Zhu, Xiaoping; Rahaman, Shaik O

2017-09-01

Cardiovascular disease is the number one cause of death in United States, and atherosclerosis, a chronic inflammatory arterial disease, is the most dominant underlying pathology. Macrophages are thought to orchestrate atherosclerosis by generating lipid-laden foam cells and by secreting inflammatory mediators. Emerging data support a role for a mechanical factor, e.g., matrix stiffness, in regulation of macrophage function, vascular elasticity, and atherogenesis. However, the identity of the plasma membrane mechanosensor and the mechanisms by which pro-atherogenic signals are transduced/maintained are unknown. We have obtained evidence that TRPV4, an ion channel in the transient receptor potential vanilloid family and a known mechanosensor, is the likely mediator of oxidized low-density lipoprotein (oxLDL)-dependent macrophage foam cell formation, a critical process in atherogenesis. Specifically, we found that: i) genetic ablation of TRPV4 or pharmacologic inhibition of TRPV4 activity by a specific antagonist blocked oxLDL-induced macrophage foam cell formation, and ii) TRPV4 deficiency prevented pathophysiological range matrix stiffness or scratch-induced exacerbation of oxLDL-induced foam cell formation. Mechanistically, we found that: i) plasma membrane localization of TRPV4 was sensitized to the increasing level of matrix stiffness, ii) lack of foam cell formation in TRPV4 null cells was not due to lack of expression of CD36, a major receptor for oxLDL, and iii) TRPV4 channel activity regulated oxLDL uptake but not its binding on macrophages. Altogether, these findings identify a novel role for TRPV4 in regulating macrophage foam cell formation by modulating uptake of oxLDL. These findings suggest that therapeutic targeting of TRPV4 may provide a selective approach to the treatment of atherosclerosis. Copyright © 2017. Published by Elsevier Inc.

The Role of Macrophage Migration Inhibitory Factor (MIF) in Ultraviolet Radiation-Induced Carcinogenesis

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Shimizu, Tadamichi [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, 930-0194, Toyama (Japan)

2010-08-09

Ultraviolet (UV) radiation is the most common cause of physical injury to the skin due to environmental damage, and UV exposure substantially increases the risk of actinic damage to the skin. The inflammatory changes induced by acute UV exposure include erythema (sunburn) of the skin, while chronic exposure to solar UV radiation causes photo-aging, immunosuppression, and ultimately, carcinogenesis of the skin. After skin damage by UV radiation, the cells are known to secrete many cytokines, including interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α. and macrophage migration inhibitory factor (MIF). MIF was originally identified as a lymphokine that concentrates macrophages at inflammatory loci, and is known to be a potent activator of macrophages in vivo. MIF is considered to play an important role in cell-mediated immunity. Since the molecular cloning of MIF cDNA, MIF has been re-evaluated as a proinflammatory cytokine and pituitary-derived hormone that potentiates endotoxemia. MIF is ubiquitously expressed in various tissues, including the skin. Recent studies have suggested a potentially broader role for MIF in growth regulation because of its ability to antagonize p53-mediated gene activation and apoptosis. This article reviews the latest findings on the roles of MIF with regard to UV-induced skin cancer.

The Role of Macrophage Migration Inhibitory Factor (MIF) in Ultraviolet Radiation-Induced Carcinogenesis

International Nuclear Information System (INIS)

Shimizu, Tadamichi

2010-01-01

Ultraviolet (UV) radiation is the most common cause of physical injury to the skin due to environmental damage, and UV exposure substantially increases the risk of actinic damage to the skin. The inflammatory changes induced by acute UV exposure include erythema (sunburn) of the skin, while chronic exposure to solar UV radiation causes photo-aging, immunosuppression, and ultimately, carcinogenesis of the skin. After skin damage by UV radiation, the cells are known to secrete many cytokines, including interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α. and macrophage migration inhibitory factor (MIF). MIF was originally identified as a lymphokine that concentrates macrophages at inflammatory loci, and is known to be a potent activator of macrophages in vivo. MIF is considered to play an important role in cell-mediated immunity. Since the molecular cloning of MIF cDNA, MIF has been re-evaluated as a proinflammatory cytokine and pituitary-derived hormone that potentiates endotoxemia. MIF is ubiquitously expressed in various tissues, including the skin. Recent studies have suggested a potentially broader role for MIF in growth regulation because of its ability to antagonize p53-mediated gene activation and apoptosis. This article reviews the latest findings on the roles of MIF with regard to UV-induced skin cancer

The Role of Macrophage Migration Inhibitory Factor (MIF in Ultraviolet Radiation-Induced Carcinogenesis

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Tadamichi Shimizu

2010-08-01

Full Text Available Ultraviolet (UV radiation is the most common cause of physical injury to the skin due to environmental damage, and UV exposure substantially increases the risk of actinic damage to the skin. The inflammatory changes induced by acute UV exposure include erythema (sunburn of the skin, while chronic exposure to solar UV radiation causes photo-aging, immunosuppression, and ultimately, carcinogenesis of the skin. After skin damage by UV radiation, the cells are known to secrete many cytokines, including interleukin (IL-1, IL-6, tumor necrosis factor (TNF-α. and macrophage migration inhibitory factor (MIF. MIF was originally identified as a lymphokine that concentrates macrophages at inflammatory loci, and is known to be a potent activator of macrophages in vivo. MIF is considered to play an important role in cell-mediated immunity. Since the molecular cloning of MIF cDNA, MIF has been re-evaluated as a proinflammatory cytokine and pituitary-derived hormone that potentiates endotoxemia. MIF is ubiquitously expressed in various tissues, including the skin. Recent studies have suggested a potentially broader role for MIF in growth regulation because of its ability to antagonize p53-mediated gene activation and apoptosis. This article reviews the latest findings on the roles of MIF with regard to UV-induced skin cancer.

27-Oxygenated cholesterol induces expression of CXCL8 in macrophages via NF-κB and CD88

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Kim, Sun-Mi, E-mail: lala1647@hanmail.net [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of); Lee, Chung Won, E-mail: vasculardoctorlee@gmail.com [Department of Thoracic and Cardiovascular Surgery, Pusan National University Hospital, Pusan 602-739 (Korea, Republic of); Kim, Bo-Young, E-mail: kimboyoung@pusan.ac.kr [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of); Jung, Young-Suk, E-mail: youngjung@pusan.ac.kr [College of Pharmacy, Pusan National University, Busan 609-735 (Korea, Republic of); Eo, Seong-Kug, E-mail: vetvirus@chonbuk.ac.kr [College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, Jeonbuk 570-752 (Korea, Republic of); Park, Young Chul, E-mail: ycpark@pusan.ac.kr [Department of Microbiology & Immunology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of); Kim, Koanhoi, E-mail: koanhoi@pusan.ac.kr [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of)

2015-08-07

We attempted to determine the effects of a milieu rich in cholesterol molecules on expression of chemokine CXCL8. A high-cholesterol diet led to an increased transcription of the IL-8 gene in the arteries and elevated levels of CXCL8 in sera of ApoE{sup −/−} mice, compared with those of wild-type C57BL/6 mice. Treatment of THP-1 monocyte/macrophage cells with 27-hydroxycholesterol (27OHChol) resulted in transcription of the IL-8 gene and increased secretion of its corresponding gene product whereas cholesterol did not induce expression of CXCL8 in THP-1 cells. 27OHChol-induced transcription of the IL-8 gene was blocked by cycloheximide, but not by polymyxin B. Treatment of THP-1 cells with 27OHChol caused translocation of p65 NF-κB subunit into the nucleus and up-regulation of CD88. Inhibition of NF-κB and CD88 using SN50 and W-54011, respectively, resulted in reduced transcription of the IL-8 gene and attenuated secretion of CXCL8 induced by 27OHChol. We propose that oxidatively modified cholesterol like 27OHChol, rather than cholesterol, is responsible for sustained expression of CXCL8 in monocytes/macrophages in atherosclerotic arteries. - Highlights: • Consumption of a high-cholesterol diet leads to increased CXCL8 expression in ApoE{sup −/−} mice. • 27OHChol, but not cholesterol, up-regulates expression of CXCL8 in macrophages. • 27OHChol enhances nuclear translocation of NF-κB and expression of CD88 in macrophages. • Inhibition of NF-κB or CD88 results in decreased CXCL8 expression induced by 27OHChol. • 27OHChol up-regulates CXCL8 expression via NF-κB and CD88 in macrophages.

Simvastatin induces caspase-independent apoptosis in LPS-activated RAW264.7 macrophage cells

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Kim, Yong Chan; Song, Seok Bean; Lee, Mi Hee; Kang, Kwang Il; Lee, Hayyoung; Paik, Sang-Gi; Kim, Kyoon Eon; Kim, Young Sang

2006-01-01

Macrophages participate in several inflammatory pathologies such as sepsis and arthritis. We examined the effect of simvastatin on the LPS-induced proinflammatory macrophage RAW264.7 cells. Co-treatment of LPS and a non-toxic dose of simvastatin induced cell death in RAW264.7 cells. The cell death was accompanied by disruption of mitochondrial membrane potential (MMP), genomic DNA fragmentation, and caspase-3 activation. Surprisingly, despite caspase-dependent apoptotic cascade being completely blocked by Z-VAD-fmk, a pan-caspase inhibitor, the cell death was only partially repressed. In the presence of Z-VAD-fmk, DNA fragmentation was blocked, but DNA condensation, disruption of MMP, and nuclear translocation of apoptosis inducing factor were obvious. The cell death by simvastatin and LPS was effectively decreased by both the FPP and GGPP treatments as well as mevalonate. Our findings indicate that simvastatin triggers the cell death of LPS-treated RAW264.7 cells through both caspase-dependent and -independent apoptotic pathways, suggesting a novel mechanism of statins for the severe inflammatory disease therapy

Galectin-2 induces a proinflammatory, anti-arteriogenic phenotype in monocytes and macrophages.

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Cansu Yıldırım

Full Text Available Galectin-2 is a monocyte-expressed carbohydrate-binding lectin, for which increased expression is genetically determined and associated with decreased collateral arteriogenesis in obstructive coronary artery disease patients. The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown. In this study we aimed to explore the effects of galectin-2 on monocyte/macrophage phenotype in vitro and vivo, and to identify the receptor by which galectin-2 exerts these effects. We now show that the binding of galectin-2 to different circulating human monocyte subsets is dependent on monocyte surface expression levels of CD14. The high affinity binding is blocked by an anti-CD14 antibody but not by carbohydrates, indicating a specific protein-protein interaction. Galectin-2 binding to human monocytes modulated their transcriptome by inducing proinflammatory cytokines and inhibiting pro-arteriogenic factors, while attenuating monocyte migration. Using specific knock-out mice, we show that galectin-2 acts through the CD14/toll-like receptor (TLR-4 pathway. Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire. This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1. In a murine model we show that galectin-2 administration, known to attenuate arteriogenesis, leads to increased numbers of CD40-positive (M1 and reduced numbers of CD206-positive (M2 macrophages surrounding actively remodeling collateral arteries. In conclusion galectin-2 is the first endogenous CD14/TLR4 ligand that induces a proinflammatory, non-arteriogenic phenotype in monocytes/macrophages. Interference with CD14-Galectin-2 interaction may provide a new intervention strategy to stimulate growth of collateral arteries in genetically compromised cardiovascular

RORα Induces KLF4-Mediated M2 Polarization in the Liver Macrophages that Protect against Nonalcoholic Steatohepatitis

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Yong-Hyun Han

2017-07-01

Full Text Available The regulation of M1/M2 polarization in liver macrophages is closely associated with the progression of nonalcoholic steatohepatitis (NASH; however, the mechanism involved in this process remains unclear. Here, we describe the orphan nuclear receptor retinoic-acid-related orphan receptor α (RORα as a key regulator of M1/M2 polarization in hepatic residential Kupffer cells (KCs and infiltrated monocyte-derived macrophages. RORα enhanced M2 polarization in KCs by inducing the kruppel-like factor 4. M2 polarization was defective in KCs and bone-marrow-derived macrophages of the myeloid-specific RORα null mice, and these mice were susceptible to HFD-induced NASH. We found that IL-10 played an important role in connecting the function of M2 KCs to lipid accumulation and apoptosis in hepatocytes. Importantly, M2 polarization was controlled by a RORα activator, JC1-40, which improved symptoms of NASH. Our results suggest that the M2-promoting effects of RORα in liver macrophages may provide better therapeutic strategies against NASH.

Cytosolic NADP(+)-dependent isocitrate dehydrogenase protects macrophages from LPS-induced nitric oxide and reactive oxygen species.

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Maeng, Oky; Kim, Yong Chan; Shin, Han-Jae; Lee, Jie-Oh; Huh, Tae-Lin; Kang, Kwang-il; Kim, Young Sang; Paik, Sang-Gi; Lee, Hayyoung

2004-04-30

Macrophages activated by microbial lipopolysaccharides (LPS) produce bursts of nitric oxide and reactive oxygen species (ROS). Redox protection systems are essential for the survival of the macrophages since the nitric oxide and ROS can be toxic to them as well as to pathogens. Using suppression subtractive hybridization (SSH) we found that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is strongly upregulated by nitric oxide in macrophages. The levels of IDPc mRNA and of the corresponding enzymatic activity were markedly increased by treatment of RAW264.7 cells or peritoneal macrophages with LPS or SNAP (a nitric oxide donor). Over-expression of IDPc reduced intracellular peroxide levels and enhanced the survival of H2O2- and SNAP-treated RAW264.7 macrophages. IDPc is known to generate NADPH, a cellular reducing agent, via oxidative decarboxylation of isocitrate. The expression of enzymes implicated in redox protection, superoxide dismutase (SOD) and catalase, was relatively unaffected by LPS and SNAP. We propose that the induction of IDPc is one of the main self-protection mechanisms of macrophages against LPS-induced oxidative stress.

Stimulation of alveolar macrophages by BCG vaccine enhances the process of lung fibrosis induced by bleomycin.

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Chyczewska, E; Chyczewski, L; Bańkowski, E; Sułkowski, S; Nikliński, J

1993-01-01

It was found that the BCG vaccine injected subcutaneously to the rats enhances the process of lung fibrosis induced by bleomycin. Pretreatment of rats with this vaccine results in accumulation of activated macrophages in lung interstitium and in the bronchoalveolar spaces. It may be suggested that the activated macrophages release various cytokines which may stimulate the proliferation of fibroblasts and biosynthesis of extracellular matrix components.

Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A(2)-induced degranulation in mast cells.

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Nishikawa, Hirofumi; Kitani, Seiichi

2011-05-01

Sting accident by honeybee causes severe pain, inflammation and allergic reaction through IgE-mediated anaphylaxis. In addition to this hypersensitivity, an anaphylactoid reaction occurs by toxic effects even in a non-allergic person via cytolysis followed by similar clinical manifestations. Auto-injectable epinephrine might be effective for bee stings, but cannot inhibit mast cell lysis and degranulation by venom toxins. We used connective tissue type canine mast cell line (CM-MC) for finding an effective measure that might inhibit bee venom toxicity. We evaluated degranulation and cytotoxicity by measurement of β-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation and cytotoxicity, which were strongly inhibited by mono-sialoganglioside (G(M1)), di-sialoganglioside (G(D1a)) and tri-sialoganglioside (G(T1b)). In contrast, honeybee venom-derived phospholipase A(2) induced the net degranulation directly without cytotoxicity, which was not inhibited by G(M1), G(D1a) and G(T1b). For analysis of distribution of Gα(q) and Gα(i) protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of Gα(q) and Gα(i) at lipid raft, but gangliosides stabilized the rafts. As a result from this cell-based study, bee venom-induced anaphylactoid reaction can be explained with melittin cytotoxicity and phospholipase A(2)-induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A(2)-induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting. Copyright © 2011 Elsevier Inc. All rights reserved.

Bid-Induced Release of AIF/EndoG from Mitochondria Causes Apoptosis of Macrophages during Infection with Leptospira interrogans

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Wei-Lin Hu

2017-11-01

Full Text Available Leptospirosis is a global zoonotic infectious disease caused by pathogenic Leptospira species. Leptospire-induced macrophage apoptosis through the Fas/FasL-caspase-8/3 pathway plays an important role in the survival and proliferation of the pathogen in hosts. Although, the release of mitochondrial apoptosis-inducing factor (AIF and endonuclease G (EndoG in leptospire-infected macrophages has been described, the mechanisms linking caspase and mitochondrion-related host-cell apoptosis has not been determined. Here, we demonstrated that leptospire-infection induced apoptosis through mitochondrial damages in macrophages. Apoptosis was caused by the mitochondrial release and nuclear translocation of AIF and/or EndoG, leading to nuclear DNA fragmentation. However, the mitochondrion-related CytC-caspase-9/3 pathway was not activated. Next, we found that the release and translocation of AIF and/or EndoG was preceded by the activation of the BH3-interacting domain death agonist (Bid. Furthermore, our data demonstrated that caspase-8 was activated during the infection and caused the activation of Bid. Meanwhile, high reactive oxygen species (ROS trigged by the infection caused the dephosphorylation of Akt, which also activated Bid. In conclusion, Bid-mediated mitochondrial release of AIF and/or EndoG followed by nuclear translocation is a major mechanism of leptospire- induced apoptosis in macrophages, and this process is modulated by both caspase-8 and ROS-Akt signal pathways.

Hyperglycemia induces mixed M1/M2 cytokine profile in primary human monocyte-derived macrophages.

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Moganti, Kondaiah; Li, Feng; Schmuttermaier, Christina; Riemann, Sarah; Klüter, Harald; Gratchev, Alexei; Harmsen, Martin C; Kzhyshkowska, Julia

2017-10-01

Hyperglycaemia is a key factor in diabetic pathology. Macrophages are essential regulators of inflammation which can be classified into two major vectors of polarisation: classically activated macrophages (M1) and alternatively activated macrophages (M2). Both types of macrophages play a role in diabetes, where M1 and M2-produced cytokines can have detrimental effects in development of diabetes-associated inflammation and diabetic vascular complications. However, the effect of hyperglycaemia on differentiation and programming of primary human macrophages was not systematically studied. We established a unique model to assess the influence of hyperglycaemia on M1 and M2 differentiation based on primary human monocyte-derived macrophages. The effects of hyperglycaemia on the gene expression and secretion of prototype M1 cytokines TNF-alpha and IL-1beta, and prototype M2 cytokines IL-1Ra and CCL18 were quantified by RT-PCR and ELISA. Hyperglycaemia stimulated production of TNF-alpha, IL-1beta and IL-1Ra during macrophage differentiation. The effect of hyperglycaemia on TNF-alpha was acute, while the stimulating effect on IL-1beta and IL-1Ra was constitutive. Expression of CCL18 was supressed in M2 macrophages by hyperglycaemia. However the secreted levels remained to be biologically significant. Our data indicate that hyperglycaemia itself, without additional metabolic factors induces mixed M1/M2 cytokine profile that can support of diabetes-associated inflammation and development of vascular complications. Copyright © 2016 Elsevier GmbH. All rights reserved.

Orphan Nuclear Receptor ERRα Controls Macrophage Metabolic Signaling and A20 Expression to Negatively Regulate TLR-Induced Inflammation.

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Yuk, Jae-Min; Kim, Tae Sung; Kim, Soo Yeon; Lee, Hye-Mi; Han, Jeongsu; Dufour, Catherine Rosa; Kim, Jin Kyung; Jin, Hyo Sun; Yang, Chul-Su; Park, Ki-Sun; Lee, Chul-Ho; Kim, Jin-Man; Kweon, Gi Ryang; Choi, Hueng-Sik; Vanacker, Jean-Marc; Moore, David D; Giguère, Vincent; Jo, Eun-Kyeong

2015-07-21

The orphan nuclear receptor estrogen-related receptor α (ERRα; NR3B1) is a key metabolic regulator, but its function in regulating inflammation remains largely unknown. Here, we demonstrate that ERRα negatively regulates Toll-like receptor (TLR)-induced inflammation by promoting Tnfaip3 transcription and fine-tuning of metabolic reprogramming in macrophages. ERRα-deficient (Esrra(-/-)) mice showed increased susceptibility to endotoxin-induced septic shock, leading to more severe pro-inflammatory responses than control mice. ERRα regulated macrophage inflammatory responses by directly binding the promoter region of Tnfaip3, a deubiquitinating enzyme in TLR signaling. In addition, Esrra(-/-) macrophages showed an increased glycolysis, but impaired mitochondrial respiratory function and biogenesis. Further, ERRα was required for the regulation of NF-κB signaling by controlling p65 acetylation via maintenance of NAD(+) levels and sirtuin 1 activation. These findings unravel a previously unappreciated role for ERRα as a negative regulator of TLR-induced inflammatory responses through inducing Tnfaip3 transcription and controlling the metabolic reprogramming. Copyright © 2015 Elsevier Inc. All rights reserved.

Cisplatin Induces a Mitochondrial-ROS Response That Contributes to Cytotoxicity Depending on Mitochondrial Redox Status and Bioenergetic Functions

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Marullo, Rossella; Werner, Erica; Degtyareva, Natalya; Moore, Bryn; Altavilla, Giuseppe; Ramalingam, Suresh S.; Doetsch, Paul W.

2013-01-01

Cisplatin is one of the most effective and widely used anticancer agents for the treatment of several types of tumors. The cytotoxic effect of cisplatin is thought to be mediated primarily by the generation of nuclear DNA adducts, which, if not repaired, cause cell death as a consequence of DNA replication and transcription blockage. However, the ability of cisplatin to induce nuclear DNA (nDNA) damage per se is not sufficient to explain its high degree of effectiveness nor the toxic effects exerted on normal, post-mitotic tissues. Oxidative damage has been observed in vivo following exposure to cisplatin in several tissues, suggesting a role for oxidative stress in the pathogenesis of cisplatin-induced dose-limiting toxicities. However, the mechanism of cisplatin-induced generation of ROS and their contribution to cisplatin cytotoxicity in normal and cancer cells is still poorly understood. By employing a panel of normal and cancer cell lines and the budding yeast Saccharomyces cerevisiae as model system, we show that exposure to cisplatin induces a mitochondrial-dependent ROS response that significantly enhances the cytotoxic effect caused by nDNA damage. ROS generation is independent of the amount of cisplatin-induced nDNA damage and occurs in mitochondria as a consequence of protein synthesis impairment. The contribution of cisplatin-induced mitochondrial dysfunction in determining its cytotoxic effect varies among cells and depends on mitochondrial redox status, mitochondrial DNA integrity and bioenergetic function. Thus, by manipulating these cellular parameters, we were able to enhance cisplatin cytotoxicity in cancer cells. This study provides a new mechanistic insight into cisplatin-induced cell killing and may lead to the design of novel therapeutic strategies to improve anticancer drug efficacy. PMID:24260552

Antibacterial and cytotoxic activities of the sesquiterpene lactones cnicin and onopordopicrin.

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Bach, Sandra M; Fortuna, Mario A; Attarian, Rodgoun; de Trimarco, Juliana T; Catalán, César A N; Av-Gay, Yossef; Bach, Horacio

2011-02-01

The antimicrobial and cytotoxic activities of chloroform extracts from the weeds Centaurea tweediei and C. diffusa, and the main sesquiterpene lactones isolated from these species, onopordopicrin and cnicin, respectively, were assayed. Results show that the chloroform extracts from both Centaurea species possess antibacterial activities against a panel of Gram-positive and Gram-negative bacteria. Remarkable antibacterial activity against methicillin-resistant Staphylococcus aureus was also measured. Both the extracts and the purified sesquiterpene lactones show high cytotoxicity against human-derived macrophages. Despite this cytotoxicity, C. diffusa chloroform extract and cnicin are attractive candidates for evaluation as antibiotics in topical preparations against skin-associated pathogens.

Macrophage recruitment, but not interleukin 1 beta activation, enhances noise-induced hearing damage.

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Mizushima, Yu; Fujimoto, Chisato; Kashio, Akinori; Kondo, Kenji; Yamasoba, Tatsuya

2017-11-18

It has been suggested that macrophages or inflammatory monocytes participate in the pathology of noise-induced hearing loss (NIHL), but it is unclear how extensively these cells contribute to the development of temporary and/or permanent NIHL. To address this question, we used clodronate liposomes to deplete macrophages and monocytes. After clodronate liposome injection, mice were exposed to 4-kHz octave band noise at 121 dB for 4 h. Compared to vehicle-injected controls, clodronate-treated mice exhibited significantly reduced permanent threshold shifts at 4 and 8 kHz and significantly smaller outer hair cell losses in the lower-apical cochlear turn. Following noise exposure, the stria vascularis had significantly more cells expressing the macrophage-specific protein F4/80, and this effect was significantly suppressed by clodronate treatment. These F4/80-positive cells expressed interleukin 1 beta (IL-1β), which noise exposure activated. However, IL-1β deficient mice did not exhibit significant resistance to intense noise when compared to wild-type mice. These findings suggest that macrophages that enter the cochlea after noise exposure are involved in NIHL, whereas IL-1β inhibition does not reverse this cochlear damage. Therefore, macrophages may be a promising therapeutic target in human sensorineural hearing losses such as NIHL. Copyright © 2017 Elsevier Inc. All rights reserved.

Curcumin inhibits EMMPRIN and MMP-9 expression through AMPK-MAPK and PKC signaling in PMA induced macrophages.

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Cao, Jiatian; Han, Zhihua; Tian, Lei; Chen, Kan; Fan, Yuqi; Ye, Bozhi; Huang, Weijian; Wang, Changqian; Huang, Zhouqing

2014-09-21

In coronary arteries, plaque disruption, the major acute clinical manifestations of atherosclerosis, leads to a subsequent cardiac event, such as acute myocardial infarction (AMI) and unstable angina pectoris (UA). Numerous reports have shown that high expression of MMP-9 (matrix metalloproteinase-9), MMP-13 (matrix metalloproteinase-13) and EMMPRIN (extracellular matrix metalloproteinase induce) in monocyte/macrophage results in the plaque progression and destabilization. Curcumin exerts well-known anti-inflammatory and antioxidant effects and probably has a protective role in the atherosclerosis. The purpose of our study was to investigate the molecular mechanisms by which curcumin affects MMP-9, MMP13 and EMMPRIN in PMA (phorbol 12-myristate 13-acetate) induced macrophages. Human monocytic cells (THP-1 cells) were pretreated with curcumin or compound C for 1 h, and then induced by PMA for 48 h. Total RNA and proteins were collected for real-time PCR and Western blot analysis, respectively. In the present study, the exposure to curcumin resulted in attenuated JNK, p38, and ERK activation and decreased expression of MMP-9, MMP-13 and EMMPRIN in PMA induced macrophages. Moreover, we demonstrated that AMPK (AMP-activated protein kinase) and PKC (Protein Kinase C) was activated by PMA during monocyte/macrophage differentiation. Furthermore, curcumin reversed PMA stimulated PKC activation and suppressed the chronic activation of AMPK, which in turn reduced the expression of MMP-9, MMP-13 and EMMPRIN. Therefore, it is suggested that curcumin by inhibiting AMPK-MAPK (mitogen activated protein kinase) and PKC pathway may led to down-regulated EMMPRIN, MMP-9 and MMP-13 expression in PMA-induced THP-1 cells.

Immunoregulation of antitumor response; differential secretion of arachidonic acid metabolites by macrophages during stimulation ''in vitro'' with BCG and ''Corynebacterium parvum''

International Nuclear Information System (INIS)

Tomecki, Jaroslaw; Sukiennik, Jadwiga; Kordowiak, Anna

1993-01-01

The level of arachidonic acid (AA) metabolites in the supernatants of cultures peritoneal exudate cells (PEC) were studied under various conditions using BCG and ''Corynebacterium parvum'' as stimulators. The metabolite levels were analyzed by thin layer chromatography (TLC). The degree of macrophage cytotoxic/cytostatic activity was dependent on the dose and character of stimulators used and the source of macrophages. The application of micro cytotoxicity assay for the evaluation of tumor cell lysis (lung sarcoma SaL-1) ''in vitro'' revealed that peritoneal macrophages from healthy and tumor bearing BALB/c mice may affect the degree of antitumor response. In the supernatants of cultured PEC from tumor bearing mice AA level increased (by 10-fold) in comparison with PEC from healthy mice. Stimulation with BCG induced over a double level of AA in PEC isolated from tumor bearing mice non-stimulated or stimulated with ''C.parvum''. A lower level of prostaglandins (PGs) was found in the supernatants of cultured PEC isolated from healthy mice (stimulated and non-stimulated), but the highest level of PGs was observed in the supernatants of cultured PEC isolated from tumor bearing mice stimulated with BCG. The unique metabolite of AA was found only in the supernatants form non-stimulated PEC from tumor bearing mice. PEC from tumor bearing mice produced metabolites of AA which were not detected in control group. These results suggest that macrophages also play a regulatory role by secretion of AA. This process can be modified by bacterial antigens. (author). 21 refs, 7 figs

Curcumin protects against cytotoxic and inflammatory effects of quartz particles but causes oxidative DNA damage in a rat lung epithelial cell line

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Li Hui; Berlo, Damien van; Shi Tingming; Speit, Guenter; Knaapen, Ad M.; Borm, Paul J.A.; Albrecht, Catrin; Schins, Roel P.F.

2008-01-01

Chronic inhalation of high concentrations of respirable quartz particles has been implicated in various lung diseases including lung fibrosis and cancer. Generation of reactive oxygen species (ROS) and oxidative stress is considered a major mechanism of quartz toxicity. Curcumin, a yellow pigment from Curcuma longa, has been considered as nutraceutical because of its strong anti-inflammatory, antitumour and antioxidant properties. The aim of our present study was to investigate whether curcumin can protect lung epithelial cells from the cytotoxic, genotoxic and inflammatory effects associated with quartz (DQ12) exposure. Electron paramagnetic resonance (EPR) measurements using the spin-trap DMPO demonstrated that curcumin reduces hydrogen peroxide-dependent hydroxyl-radical formation by quartz. Curcumin was also found to reduce quartz-induced cytotoxicity and cyclooxygenase 2 (COX-2) mRNA expression in RLE-6TN rat lung epithelial cells (RLE). Curcumin also inhibited the release of macrophage inflammatory protein-2 (MIP-2) from RLE cells as observed upon treatment with interleukin-1 beta (IL-1β) and tumour necrosis factor-alpha (TNFα). However, curcumin failed to protect the RLE cells from oxidative DNA damage induced by quartz, as shown by formamidopyrimidine glycosylase (FPG)-modified comet assay and by immunocytochemistry for 8-hydroxydeoxyguanosine. In contrast, curcumin was found to be a strong inducer of oxidative DNA damage itself at non-cytotoxic and anti-inflammatory concentrations. In line with this, curcumin also enhanced the mRNA expression of the oxidative stress response gene heme oxygenase-1 (ho-1). Curcumin also caused oxidative DNA damage in NR8383 rat alveolar macrophages and A549 human lung epithelial cells. Taken together, these observations indicate that one should be cautious in considering the potential use of curcumin in the prevention or treatment of lung diseases associated with quartz exposure

Anti-inflammatory and cytotoxic activities of five Veronica species.

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Harput, U Sebnem; Saracoglu, Iclal; Inoue, Makoto; Ogihara, Yukio

2002-04-01

Biological activities of five Veronica species (Scrophulariaceae), V. cymbalaria, V. hederifolia, V. pectinata var. glandulosa, V. persica and V. polita were studied for their anti-inflammatory and cytotoxic activities. Their methanol extracts showed both the inhibitory activity of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophages and cytotoxic activity against KB epidermoid carcinoma and B16 melanoma. When the methanol extracts were fractionated between water and chloroform, water fractions significantly inhibited NO production without any cytotoxicity, while chloroform fractions showed cytotoxicity dose-dependently. When the radical scavenging activity was determined using 2,2-diphenyl-1-picryl-hydrazyl (DPPH), water fractions of the five Veronica species scavenged free radicals effectively, suggesting that the inhibitory effect of this species on NO production was due to their radical scavenging activity. On the other hand, chloroform fractions of Veronica species except for V. cymbalaria showed similar cytotoxic activity against KB and B16 melanoma cells.

Adipocyte-Macrophage Cross-Talk in Obesity.

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Engin, Ayse Basak

2017-01-01

Obesity is characterized by the chronic low-grade activation of the innate immune system. In this respect, macrophage-elicited metabolic inflammation and adipocyte-macrophage interaction has a primary importance in obesity. Large amounts of macrophages are accumulated by different mechanisms in obese adipose tissue. Hypertrophic adipocyte-derived chemotactic monocyte chemoattractant protein-1 (MCP-1)/C-C chemokine receptor 2 (CCR2) pathway also promotes more macrophage accumulation into the obese adipose tissue. However, increased local extracellular lipid concentrations is a final mechanism for adipose tissue macrophage accumulation. A paracrine loop involving free fatty acids and tumor necrosis factor-alpha (TNF-alpha) between adipocytes and macrophages establishes a vicious cycle that aggravates inflammatory changes in the adipose tissue. Adipocyte-specific caspase-1 and production of interleukin-1beta (IL-1beta) by macrophages; both adipocyte and macrophage induction by toll like receptor-4 (TLR4) through nuclear factor-kappaB (NF-kappaB) activation; free fatty acid-induced and TLR-mediated activation of c-Jun N-terminal kinase (JNK)-related pro-inflammatory pathways in CD11c+ immune cells; are effective in macrophage accumulation and in the development of adipose tissue inflammation. Old adipocytes are removed by macrophages through trogocytosis or sending an "eat me" signal. The obesity-induced changes in adipose tissue macrophage numbers are mainly due to increases in the triple-positive CD11b+ F4/80+ CD11c+ adipose tissue macrophage subpopulation. The ratio of M1-to-M2 macrophages is increased in obesity. Furthermore, hypoxia along with higher concentrations of free fatty acids exacerbates macrophage-mediated inflammation in obesity. The metabolic status of adipocytes is a major determinant of macrophage inflammatory output. Macrophage/adipocyte fatty-acid-binding proteins act at the interface of metabolic and inflammatory pathways. Both macrophages and

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

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Schwarze Per E

2006-06-01

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

Interactions between Streptomyces californicus and Stachybotrys chartarum can induce apoptosis and cell cycle arrest in mouse RAW264.7 macrophages

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Penttinen, Piia; Pelkonen, Jukka; Huttunen, Kati; Toivola, Mika; Hirvonen, Maija-Riitta

2005-01-01

Exposure to complex mixtures of bacteria and fungi in moisture-damaged buildings is a potential cause of inflammatory related symptoms among occupants. The present study assessed interactions between two characteristic moldy house microbes Streptomyces californicus and Stachybotrys chartarum. Differences in cytotoxic and inflammatory responses in mouse (RAW264.7) macrophages were studied after exposure to the spores of co-cultivated microbes, the mixture of separately cultivated spores, and the spores of either of these microbes cultivated alone. The RAW264.7 cells were exposed to six doses (1 x 10 4 to 3 x 10 6 spores/ml) for 24 h, and the time course of the induced responses was evaluated after 4, 8, 16, and 24 h of exposure (1 x 10 6 spores/ml). The cytotoxic potential of the spores was characterized by the MTT test, DNA content analysis, and enzyme assay for caspase-3 activity. The production of cytokines (IL-1β, IL-6, IL-10, TNFα, and MIP2) was measured immunochemically and nitric oxide by the Griess method. Co-cultivation increased the ability of the spores to cause apoptosis by more than 4-fold and the proportion of RAW264.7 cells at the G 2 /M stage increased nearly 2-fold when compared to the response induced by the mixture of spores. In contrast, co-cultivation decreased significantly the ability of the spores to trigger the production of NO and IL-6 in RAW264.7 cells. In conclusion, these data suggest that co-culture of S. californicus and S. chartarum can result in microbial interactions that significantly potentiate the ability of the spores to cause apoptosis and cell cycle arrest in mammalian cells

Minocycline Has Anti-inflammatory Effects and Reduces Cytotoxicity in an Ex Vivo Spinal Cord Slice Culture Model of West Nile Virus Infection.

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Quick, Eamon D; Seitz, Scott; Clarke, Penny; Tyler, Kenneth L

2017-11-15

West Nile virus (WNV) is a neurotropic flavivirus that can cause significant neurological disease. Mouse models of WNV infection demonstrate that a proinflammatory environment is induced within the central nervous system (CNS) after WNV infection, leading to entry of activated peripheral immune cells. We utilized ex vivo spinal cord slice cultures (SCSC) to demonstrate that anti-inflammatory mechanisms may also play a role in WNV-induced pathology and/or recovery. Microglia are a type of macrophage that function as resident CNS immune cells. Similar to mouse models, infection of SCSC with WNV induces the upregulation of proinflammatory genes and proteins that are associated with microglial activation, including the microglial activation marker Iba1 and CC motif chemokines CCL2, CCL3, and CCL5. This suggests that microglia assume a proinflammatory phenotype in response to WNV infection similar to the proinflammatory (M1) activation that can be displayed by other macrophages. We now show that the WNV-induced expression of these and other proinflammatory genes was significantly decreased in the presence of minocycline, which has antineuroinflammatory properties, including the ability to inhibit proinflammatory microglial responses. Minocycline also caused a significant increase in the expression of anti-inflammatory genes associated with alternative anti-inflammatory (M2) macrophage activation, including interleukin 4 (IL-4), IL-13, and FIZZ1. Minocycline-dependent alterations to M1/M2 gene expression were associated with a significant increase in survival of neurons, microglia, and astrocytes in WNV-infected slices and markedly decreased levels of inducible nitric oxide synthase (iNOS). These results demonstrate that an anti-inflammatory environment induced by minocycline reduces viral cytotoxicity during WNV infection in ex vivo CNS tissue. IMPORTANCE West Nile virus (WNV) causes substantial morbidity and mortality, with no specific therapeutic treatments available

The cytotoxic effect of oxybuprocaine on human corneal epithelial cells by inducing cell cycle arrest and mitochondria-dependent apoptosis.

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Fan, W-Y; Wang, D-P; Wen, Q; Fan, T-J

2017-08-01

Oxybuprocaine (OBPC) is a widely used topical anesthetic in eye clinic, and prolonged and repeated usage of OBPC might be cytotoxic to the cornea, especially to the outmost corneal epithelium. In this study, we characterized the cytotoxic effect of OBPC on human corneal epithelial (HCEP) cells and investigated its possible cellular and molecular mechanisms using an in vitro model of non-transfected HCEP cells. Our results showed that OBPC at concentrations ranging from 0.025% to 0.4% had a dose- and time-dependent cytotoxicity to HCEP cells. Moreover, OBPC arrested the cells at S phase and induced apoptosis of these cells by inducing plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation. Furthermore, OBPC could trigger the activation of caspase-2, -3, and -9, downregulate the expression of Bcl-xL, upregulate the expression of Bax along with the cytoplasmic amount of mitochondria-released apoptosis-inducing factor, and disrupt mitochondrial transmembrane potential. Our results suggest that OBPC has a dose- and time-dependent cytotoxicity to HCEP cells by inducing cell cycle arrest and cell apoptosis via a death receptor-mediated mitochondria-dependent proapoptotic pathway, and this novel finding provides new insights into the acute cytotoxicity and its toxic mechanisms of OBPC on HCEP cells.

Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

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Wang, Yi-Fen; Shyu, Huey-Wen [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chang, Yi-Chuang [Department of Nursing, Fooyin University, Kaohsiung, Taiwan (China); Tseng, Wei-Chang [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Huang, Yeou-Lih [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chen, Chang-Yu, E-mail: mt037@mail.fy.edu.tw [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China)

2012-03-01

Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

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Wang, Yi-Fen; Shyu, Huey-Wen; Chang, Yi-Chuang; Tseng, Wei-Chang; Huang, Yeou-Lih; Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling; Chen, Chang-Yu

2012-01-01

Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

Pharmacological Inhibition of Macrophage Toll-like Receptor 4/Nuclear Factor-kappa B Alleviates Rhabdomyolysis-induced Acute Kidney Injury.

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Huang, Rong-Shuang; Zhou, Jiao-Jiao; Feng, Yu-Ying; Shi, Min; Guo, Fan; Gou, Shen-Ju; Salerno, Stephen; Ma, Liang; Fu, Ping

2017-09-20

Acute kidney injury (AKI) is the most common and life-threatening systemic complication of rhabdomyolysis. Inflammation plays an important role in the development of rhabdomyolysis-induced AKI. This study aimed to investigate the kidney model of AKI caused by rhabdomyolysis to verify the role of macrophage Toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) signaling pathway. C57BL/6 mice were injected with a 50% glycerin solution at bilateral back limbs to induce rhabdomyolysis, and CLI-095 or pyrrolidine dithiocarbamate (PDTC) was intraperitoneally injected at 0.5 h before molding. Serum creatinine levels, creatine kinase, the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, and hematoxylin and eosin stainings of kidney tissues were tested. The infiltration of macrophage, mRNA levels, and protein expression of TLR4 and NF-κB were investigated by immunofluorescence double-staining techniques, reverse transcriptase-quantitative polymerase chain reaction, and Western blotting, respectively. In vitro, macrophage RAW264.7 was stimulated by ferrous myoglobin; the cytokines, TLR4 and NF-κB expressions were also detected. In an in vivo study, using CLI-095 or PDTC to block TLR4/NF-κB, functional and histologic results showed that the inhibition of TLR4 or NF-κB alleviated glycerol-induced renal damages (P rhabdomyolysis-induced AKI by the regulation of proinflammatory cytokine production and macrophage infiltration.

Identification of M2 macrophages in anterior pituitary glands of normal rats and rats with estrogen-induced prolactinoma.

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Fujiwara, Ken; Yatabe, Megumi; Tofrizal, Alimuddin; Jindatip, Depicha; Yashiro, Takashi; Nagai, Ryozo

2017-05-01

Macrophages are present throughout the anterior pituitary gland. However, the features and function of macrophages in the gland are poorly understood. Recent studies have indicated that there are two main macrophage classes: M1 (classically activated) and M2 (alternatively activated). In this study, we examine whether both M1 and M2 macrophages are present in the anterior pituitary gland of rats. Our findings indicate that macrophages that are positive for CD68 (a pan-macrophage marker) were localized near capillaries in rat anterior pituitary gland. These macrophages were positive for iNOS or mannose receptor (MR), which are markers of M1 and M2 macrophages, respectively. To determine the morphological characteristics of M2 macrophages under pathological conditions, diethylstilbestrol (DES)-treated rats were used as an animal model of prolactinoma. After 2 weeks of DES treatment, a number of MR-immunopositive cells were present in the gland. Immunoelectron microscopy revealed that MR-immunopositive M2 macrophages had many small vesicles and moderately large vacuoles in cytoplasm. Phagosomes were sometimes present in cytoplasm. Interestingly, M2 macrophages in prolactinoma tissues did not usually exhibit distinct changes or differences during the normal, hyperplasia and adenoma stages. This study is the first to confirm that both M1 and M2 macrophages are present in the anterior pituitary gland of rats. Moreover, the number of M2 macrophages was greatly increased in rats with DES-induced prolactinoma. Future studies should attempt to characterize the functional role of M2 macrophages in the gland.

Modulation of macrophage Ia expression by lipopolysaccharide: Stem cell requirements, accessory lymphocyte involvement, and IA-inducing factor production

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Wentworth, P.A.; Ziegler, H.K.

1989-01-01

The mechanism of induction of murine macrophage Ia expression by lipopolysaccharide (LPS) was studied. Intraperitoneal injection of 1 microgram of LPS resulted in a 3- to 10-fold increase in the number of IA-positive peritoneal macrophages (flow cytometry and immunofluorescence) and a 6-to 16-fold increase by radioimmunoassay. The isolated lipid A moiety of LPS was a potent inducer of macrophage Ia expression. Ia induction required a functional myelopoietic system as indicated by the finding that the response to LPS was eliminated in irradiated (900 rads) mice and reinstated by reconstitution with bone marrow cells. Comparison of LPS-induced Ia expression in normal and LPS-primed mice revealed a faster secondary response to LPS. The memory response could be adoptively transferred to normal mice with nonadherent spleen cells prepared 60 days after LPS injection. Spleen cells prepared 5 days after LPS injection caused Ia induction in LPS-nonresponder mice; such induction was not observed in irradiated (900 rads) recipients. The cell responsible for this phenomenon was identified as a Thy-1+, immunoglobulin-negative nonadherent cell. The biosynthesis and expression of Ia were not increased by direct exposure of macrophages to LPS in vitro. Small amounts of LPS inhibited Ia induction by gamma interferon. LPS showed positive regulatory effects on Ia expression by delaying the loss of Ia expression on cultured macrophages and by stimulating the production of Ia-inducing factors. Supernatants from cultured spleen cells stimulated with LPS in vitro contained antiviral and Ia-inducing activity that was acid labile, indicating that the active factor is gamma interferon. We conclude that induction of Ia expression by LPS in vivo is a bone-marrow-dependent, radiation-sensitive process which involves the stimulation of a gamma interferon-producing accessory lymphocyte and a delay in Ia turnover

Inhibitors of the 5-lipoxygenase arachidonic acid pathway induce ATP release and ATP-dependent organic cation transport in macrophages.

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da Silva-Souza, Hercules Antônio; Lira, Maria Nathalia de; Costa-Junior, Helio Miranda; da Cruz, Cristiane Monteiro; Vasconcellos, Jorge Silvio Silva; Mendes, Anderson Nogueira; Pimenta-Reis, Gabriela; Alvarez, Cora Lilia; Faccioli, Lucia Helena; Serezani, Carlos Henrique; Schachter, Julieta; Persechini, Pedro Muanis

2014-07-01

We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca(2+) concentration ([Ca(2+)]i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca(2+)]i. Chelating Ca(2+) ions in the extracellular medium suppressed the intracellular Ca(2+) signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca(2+)- and P2X7-independent transport mechanism in macrophages. Copyright © 2014 Elsevier B.V. All rights reserved.

Inflammatory Macrophages Promotes Development of Diabetic Encephalopathy.

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Wang, Beiyun; Miao, Ya; Zhao, Zhe; Zhong, Yuan

2015-01-01

Diabetes and Alzheimer's disease are often associated with each other, whereas the relationship between two diseases is ill-defined. Although hyperglycemia during diabetes is a major cause of encephalopathy, diabetes may also cause chronic inflammatory complications including peripheral neuropathy. Hence the role and the characteristics of inflammatory macrophages in the development of diabetic encephalopathy need to be clarified. Diabetes were induced in mice by i.p. injection of streptozotocin (STZ). Two weeks after STZ injection and confirmation of development of diabetes, inflammatory macrophages were eliminated by i.p. injection of 20µg saporin-conjugated antibody against a macrophage surface marker CD11b (saporin-CD11b) twice per week, while a STZ-treated group received injection of rat IgG of same frequency as a control. The effects of macrophage depletion on brain degradation markers, brain malondialdehyde (MDA), catalase, superoxidase anion-positive cells and nitric oxide (NO) were measured. Saporin-CD11b significantly reduced inflammatory macrophages in brain, without affecting mouse blood glucose, serum insulin, glucose responses and beta cell mass. However, reduced brain macrophages significantly inhibited the STZ-induced decreases in brain MDA, catalase and superoxidase anion-positive cells, and the STZ-induced decreases in brain NO. Inflammatory macrophages may promote development of diabetic encephalopathy. © 2015 S. Karger AG, Basel.

Immunogenic Eimeria tenella glycosylphosphatidylinositol-anchored surface antigens (SAGs induce inflammatory responses in avian macrophages.

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Yock-Ping Chow

Full Text Available At least 19 glycosylphosphatidylinositol (GPI-anchored surface antigens (SAGs are expressed specifically by second-generation merozoites of Eimeria tenella, but the ability of these proteins to stimulate immune responses in the chicken is unknown.Ten SAGs, belonging to two previously defined multigene families (A and B, were expressed as soluble recombinant (r fusion proteins in E. coli. Chicken macrophages were treated with purified rSAGs and changes in macrophage nitrite production, and in mRNA expression profiles of inducible nitric oxide synthase (iNOS and of a panel of cytokines were measured. Treatment with rSAGs 4, 5, and 12 induced high levels of macrophage nitric oxide production and IL-1β mRNA transcription that may contribute to the inflammatory response observed during E. tenella infection. Concomitantly, treatment with rSAGs 4, 5 and 12 suppressed the expression of IL-12 and IFN-γ and elevated that of IL-10, suggesting that during infection these molecules may specifically impair the development of cellular mediated immunity.In summary, some E. tenella SAGs appear to differentially modulate chicken innate and humoral immune responses and those derived from multigene family A (especially rSAG 12 may be more strongly linked with E. tenella pathogenicity associated with the endogenous second generation stages.

Toxicity and oxidative stress induced by semiconducting polymer dots in RAW264.7 mouse macrophages

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Ye, Fangmao; White, Collin C.; Jin, Yuhui; Hu, Xiaoge; Hayden, Sarah; Zhang, Xuanjun; Gao, Xiaohu; Kavanagh, Terrance J.; Chiu, Daniel T.

2015-05-01

The rapid development and acceptance of PDots for biological applications depends on an in depth understanding of their cytotoxicity. In this paper, we performed a comprehensive study of PDot cytotoxicity at both the gross cell effect level (such as cell viability, proliferation and necrosis) and more subtle effects (such as redox stress) on RAW264.7 cells, a murine macrophage cell line with high relevance to in vivo nanoparticle disposition. The redox stress measurements assessed were inner mitochondrial membrane lipid peroxidation (nonyl-acridine orange, NAO), total thiol level (monobromobimane, MBB), and pyridine nucleotide redox status (NAD(P)H autofluorescence). Because of the extensive work already performed with QDots on nanotoxicity and also because of their comparable size, QDots were chosen as a comparison/reference nanoparticle for this study. The results showed that PDots exhibit cytotoxic effects to a much lesser degree than their inorganic analogue (QDots) and are much brighter, allowing for much lower concentrations to be used in various biological applications. In addition, at lower dose levels (2.5 nM to 10 nM) PDot treatment resulted in higher total thiol level than those found with QDots. At higher dose levels (20 nM to 40 nM) QDots caused significantly higher thiol levels in RAW264.7 cells, than was seen with PDots, suggesting that QDots elicit compensation to oxidative stress by upregulating GSH synthesis. At the higher concentrations of QDots, NAD(P)H levels showed an initial depletion, then repletion to a level that was greater than vehicle controls. PDots showed a similar trend but this was not statistically significant. Because PDots elicit less oxidative stress and cytotoxicity at low concentrations than QDots, and because they exhibit superior fluorescence at these low concentrations, PDots are predicted to have enhanced utility in biomedical applications.

Effect of bleaching agent extracts on murine macrophages.

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Fernandes, Aletéia M M; Vilela, Polyana G F; Valera, Marcia C; Bolay, Carola; Hiller, Karl Anton; Schweikl, Helmut; Schmalz, Gottfried

2018-05-01

The aim of this study was to evaluate the cytotoxicity and the influence of bleaching agents on immunologically cell surface antigens of murine macrophages in vitro. RAW 264.7 cells were exposed to bleaching gel extracts (40% hydrogen peroxide or 20% carbamide peroxide) and different H 2 O 2 concentrations after 1 and 24-h exposure periods and 1-h exposure and 23-h recovery. Tests were performed with and without N-acetyl cysteine (NAC) and buthionine sulfoximine (BSO). Cell viability was determined by MTT assay. The expression of surface markers CD14, CD40, and CD54 with and without LPS stimulation was detected by flow cytometry, while the production of TNF-α was measured by ELISA. Statistical analysis was performed using the Mann-Whitney U test (α = 0.05). Extracts of bleaching agents were cytotoxic for cells after a 1-h exposure; cells could not recover after 24 h. This effect can be mitigated by the antioxidant NAC and increased by BSO, an inhibitor of glutathione (GSH) synthesis. LPS stimulated expression of all surface markers and TNF-α production. Exposure to bleaching agent extracts and H 2 O 2 leads to a reduction of TNF-α, CD14, and CD40 expression, while the expression of CD54 was upregulated at non-cytotoxic concentrations. Whereas NAC reduced this effect, it was increased in the presence of BSO. Extracts of bleaching agents were irreversibly cytotoxic to macrophages after a 1-h exposure. Only the expression of CD54 was upregulated. The reactions are mediated by the non-enzymatic antioxidant GSH. The addition of an antioxidant can downregulate unfavorable effects of dental bleaching.

Prevention of Paclitaxel-induced allodynia by Minocycline: Effect on loss of peripheral nerve fibers and infiltration of macrophages in rats

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Xin Wen-Jun

2010-11-01

Full Text Available Abstract Background Although paclitaxel is a frontline antineoplastic agent for treatment of solid tumors, the paclitaxel-evoked pain syndrome is a serious problem for patients. There is currently no valid drug to prevent or treat the paclitaxel-induced allodynia, partly due to lack of understanding regarding the cellular mechanism. Studies have shown that minocycline, an inhibitor of microglia/macrophage, prevented neuropathic pain and promoted neuronal survival in animal models of neurodegenerative disease. Recently, Cata et al also reported that minocycline inhibited allodynia induced by low-dose paclitaxel (2 mg/kg in rats, but the mechanism is still unclear. Results Here, we investigate by immunohistochemistry the change of intraepidermal nerve fiber (IENF in the hind paw glabrous skin, expression of macrophage and activating transcription factor 3 (ATF3 in DRG at different time points after moderate-dose paclitaxel treatment (cumulative dose 24 mg/kg; 3 × 8 mg/kg in rats. Moreover, we observe the effect of minocycline on the IENF, macrophages and ATF3. The results showed that moderate-dose paclitaxel induced a persisted, gradual mechanical allodynia, which was accompanied by the loss of IENF in the hind paw glabrous skin and up-regulation of macrophages and ATF3 in DRG in rats. The expressions of ATF3 mainly focus on the NF200-positive cells. More importantly, we observed that pretreatment of minocycline at dose of 30 mg/kg or 50 mg/kg, but not 5 mg/kg, prevented paclitaxel-evoked allodynia. The evidence from immunohistochemistry showed that 30 mg/kg minocycline rescued the degeneration of IENF, attenuated infiltration of macrophages and up-regulation of ATF3 induced by paclitaxel treatment in rats. Conclusions Minocycline prevents paclitaxel-evoked allodynia, likely due to its inhibition on loss of IENF, infiltration of macrophages and up-regulation of ATF3 in rats. The finding might provide potential target for preventing paclitaxel-induced

Sensitization of TNF-induced cytotoxicity in lung cancer cells by concurrent suppression of the NF-κB and Akt pathways

International Nuclear Information System (INIS)

Wang Xia; Chen Wenshu; Lin Yong

2007-01-01

Blockage of either nuclear factor-κB (NF-κB) or Akt sensitizes cancer cells to TNF-induced apoptosis. In this study, we investigated the undetermined effect of concurrent blockage of these two survival pathways on TNF-induced cytotoxicity in lung cancer cells. The results show that Akt contributes to TNF-induced NF-κB activation in lung cancer cells through regulating phosphorylation of the p65/RelA subunit of NF-κB. Although individually blocking IKK or Akt partially suppressed TNF-induced NF-κB activation, concurrent suppression of these pathways completely inhibited TNF-induced NF-κB activation and downstream anti-apoptotic gene expression, and synergistically potentiated TNF-induced cytotoxicity. Moreover, suppression of Akt inhibited the Akt-mediated anti-apoptotic pathway through dephosphorylation of BAD. These results indicate that concurrent suppression of NF-κB and Akt synergistically sensitizes TNF-induced cytotoxicity through blockage of distinct survival pathways downstream of NF-κB and Akt, which may be applied in lung cancer therapy

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

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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.

Prevention of phosphine-induced cytotoxicity by nutrients in HepG2 cells

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Marzieh Rashedinia

2016-01-01

Interpretation & conclusions: The results supported the hypothesis that phosphine-induced cytotoxicity was due to decrease of ATP levels. ATP suppliers could prevent its toxicity by generating ATP through glycolysis. α-keto compounds such as dihydroxyacetone and α-ketoglutarate may bind to phosphine and restore mitochondrial respiration.

Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

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Aoki, Manabu; Yamaguchi, Rui; Yamamoto, Takatoshi; Ishimaru, Yasuji; Ono, Tomomichi; Sakamoto, Arisa; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-04-01

Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

Cytotoxic activity of Justicia spicigera is inhibited by bcl-2 proto-oncogene and induces apoptosis in a cell cycle dependent fashion.

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Cáceres-Cortés, J R; Cantú-Garza, F A; Mendoza-Mata, M T; Chavez-González, M A; Ramos-Mandujano, G; Zambrano-Ramírez, I R

2001-12-01

Identification of organic compounds from plants is of clinical significance because of the effect that they might have in patients with haematopoietic disorders. We studied the effect of the plant extract Justicia spicigera (Acanthaceae) in different haematopoietic cells: human leukaemic cell lines, umbilical cord blood cells, and mouse bone marrow cells. By examining colony formation and performing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay it was shown that the plant extract of Justicia spicigera contains cytotoxic factors for leukaemic cells and has no proliferative activity on normal haematopoietic progenitor cells. Our results show that this plant extract induces apoptosis in the human leukaemia cell line TF-1, but not in the bcl-2 transfectant cell line TB-1. Similar results were obtained using a haemopoietic cell line 32D and 32DBcl2. The cultures of umbilical cord blood cells and mouse bone marrow that contain granulocyte-macrophage colony-stimulating factor (GM-CSF) do not proliferate or become terminally differentiated in the presence of the infusion of Justicia spicigera. GM-CSF that acts by abrogating programmed cell death is not sufficient to inhibit the apoptotic stimulus in TF-1 and 32D cells. Moreover mouse fibroblasts (3T3) and two cervical carcinoma cell lines CALO and INBL, undergo apoptosis in the presence of different concentrations of an infusion from the plant. Our data show that there is a strong correlation between the cytotoxic effect and cell proliferation. Together, these results indicate that the plant infusion of Justicia spicigera does not contain any haematopoietic activity, induces apoptosis inhibited by bcl-2 and is linked to cell proliferation. Copyright 2001 John Wiley & Sons, Ltd.

Nur77 inhibits oxLDL induced apoptosis of macrophages via the p38 MAPK signaling pathway

International Nuclear Information System (INIS)

Shao, Qin; Han, Fei; Peng, Shi; He, Ben

2016-01-01

The interaction between macrophages and oxLDL plays a crucial role in the initiation and progression of atherosclerosis. As a key initiator in a number of plaque promoting processes, oxLDL induces variable effects such as cell apoptosis or proliferation. Orphan nuclear receptor Nur77 is potently induced in macrophages by diverse stimuli, suggesting that it is of importance in vascular inflammation resulting in atherosclerosis, but whether Nur77 induction is detrimental or protective is unclear. In our study, we explore the role of Nur77 in the regulation of oxLDL-induced macrophage apoptosis and the signaling pathways that are involved. We found that oxLDL induced Nur77 expression in a dose and time dependent fashion, and cell viability was decreased in parallel. To determine whether Nur77 induction contributes to the loss of cell viability or is a protective mechanism, the effect of Nur77 overexpression was examined. Importantly, Nur77 overexpression inhibited the oxLDL-induced decrease of cell viability, inhibited the production of apoptotic bodies and restored DNA synthesis following oxLDL exposure. Furthermore, we found that Nur77 induction is mediated through the p38 MAPK signaling pathway. After pretreatment with SB203580, cell viability was decreased, the expression of CyclinA2 and PCNA was attenuated and the percentage of cell apoptosis was enhanced. Likewise, Nur77 overexpression increased the expression of the cell cycle genes PCNA and p21, and attenuated the increase in caspase-3. On the other hand, knockdown of Nur77 expression by specific siRNA resulted in the increased expression of caspase 3. The results demonstrate that Nur77 is induced by oxLDL via the p38 MAPK signaling pathway, which is involved in the regulation of cell survival. Nur77 enhanced cell survival via suppressing apoptosis, without affecting cell proliferation of activated macrophages, which may be beneficial in patients with atherosclerosis. - Highlights: • oxLDL could induce Nur77

Nur77 inhibits oxLDL induced apoptosis of macrophages via the p38 MAPK signaling pathway

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Shao, Qin; Han, Fei; Peng, Shi; He, Ben, E-mail: heben@medmail.com.cn

2016-03-18

The interaction between macrophages and oxLDL plays a crucial role in the initiation and progression of atherosclerosis. As a key initiator in a number of plaque promoting processes, oxLDL induces variable effects such as cell apoptosis or proliferation. Orphan nuclear receptor Nur77 is potently induced in macrophages by diverse stimuli, suggesting that it is of importance in vascular inflammation resulting in atherosclerosis, but whether Nur77 induction is detrimental or protective is unclear. In our study, we explore the role of Nur77 in the regulation of oxLDL-induced macrophage apoptosis and the signaling pathways that are involved. We found that oxLDL induced Nur77 expression in a dose and time dependent fashion, and cell viability was decreased in parallel. To determine whether Nur77 induction contributes to the loss of cell viability or is a protective mechanism, the effect of Nur77 overexpression was examined. Importantly, Nur77 overexpression inhibited the oxLDL-induced decrease of cell viability, inhibited the production of apoptotic bodies and restored DNA synthesis following oxLDL exposure. Furthermore, we found that Nur77 induction is mediated through the p38 MAPK signaling pathway. After pretreatment with SB203580, cell viability was decreased, the expression of CyclinA2 and PCNA was attenuated and the percentage of cell apoptosis was enhanced. Likewise, Nur77 overexpression increased the expression of the cell cycle genes PCNA and p21, and attenuated the increase in caspase-3. On the other hand, knockdown of Nur77 expression by specific siRNA resulted in the increased expression of caspase 3. The results demonstrate that Nur77 is induced by oxLDL via the p38 MAPK signaling pathway, which is involved in the regulation of cell survival. Nur77 enhanced cell survival via suppressing apoptosis, without affecting cell proliferation of activated macrophages, which may be beneficial in patients with atherosclerosis. - Highlights: • oxLDL could induce Nur77

Metabolomic study of corticosterone-induced cytotoxicity in PC12 cells by ultra performance liquid chromatography-quadrupole/time-of-flight mass spectrometry.

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Zhang, Hongye; Zheng, Hua; Zhao, Gan; Tang, Chaoling; Lu, Shiyin; Cheng, Bang; Wu, Fang; Wei, Jinbin; Liang, Yonghong; Ruan, Junxiang; Song, Hui; Su, Zhiheng

2016-03-01

Glucocorticoids (GCs) have been proved to be an important pathogenic factor of some neuropsychiatric disorders. Usually, a classical injury model based on corticosterone-induced cytotoxicity of differentiated rat pheochromocytoma (PC12) cells was used to stimulate the state of GC damage of hippocampal neurons and investigate its potential mechanisms involved. However, up to now, the mechanism of corticosterone-induced cytotoxicity in PC12 cells was still looking forward to further elucidation. In this work, the metabolomic study of the biochemical changes caused by corticosterone-induced cytotoxicity in differentiated PC12 cells with different corticosterone concentrations was performed for the first time, using the ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS). Partial least squares-discriminate analysis (PLS-DA) indicated that metabolic profiles of different corticosterone treatment groups deviated from the control group. A total of fifteen metabolites were characterized as potential biomarkers involved in corticosterone-induced cytotoxicity, which were corresponding to the dysfunctions of five pathways including glycerophospholipid metabolism, sphingolipid metabolism, oxidation of fatty acids, glycerolipid metabolism and sterol lipid metabolism. This study indicated that the rapid and holistic cell metabolomics approach might be a powerful tool to further study the pathogenesis mechanism of corticosterone-induced cytotoxicity in PC12 cells.

Extracts of Porphyra tenera (Nori Seaweed) Activate the Immune Response in Mouse RAW264.7 Macrophages via NF-κB Signaling.

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Song, Ji-Hye; Kang, Hee-Bum; Park, Seung-Ho; Jeong, Ji-Hoon; Park, Jeongjin; You, Yanghee; Lee, Yoo-Hyun; Lee, Jeongmin; Kim, Eungpil; Choi, Kyung-Chul; Jun, Woojin

2017-12-01

Porphyra tenera, also known as nori, is a red algal species of seaweed. It is cultivated in Asia for culinary purposes. We report that P. tenera extract (PTE) enhances the immune response in mouse macrophages. We found that P. tenera extract regulates the NF-κB IκB kinase (IKK) signaling pathway, and we assessed the expression and translocation of p65, a subunit of NF-κB, in RAW264.7 mouse macrophage cells after treatment with PTE. We also investigated the effects of 10% ethanol PTE (PTE10) in RAW264.7 cells. The production of IL-10, IL-6, TNF-α, and IFN-γ was induced by PTE treatment of the macrophages, and PTE also enhanced p-IκB and p-AKT. PTE10 showed no cytotoxicity at 10-20 μg/mL in RAW264.7 cells. PTE10, in fact, increased cell viability at 24 h, stimulated macrophage cells, and induced the phosphorylation of Akt. Akt stimulates IKK activity through the phosphorylation of IKKα and enhances immune activity through the activation of NF-κB. In this study, NF-κB activation was induced by increasing p-NF-κB and p-IKK. A subunit of NF-κB, p65, was located in the nucleus and increased the expression of various cytokines. PTE thus enhanced the immune response through IκB-α immunostimulation signaling in RAW264.7 cells. PTE10 has potential therefore for development of future treatments requiring immune system stimulation.

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

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C-D. Arreto

1997-01-01

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

The influence of N-acetyl-L-cysteine on oxidative stress and nitric oxide synthesis in stimulated macrophages treated with a mustard gas analogue

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Smith Milton

2008-06-01

Full Text Available Abstract Background Sulphur mustard gas, 2, 2'-dichlorodiethyl sulphide (HD, is a chemical warfare agent. Both mustard gas and its monofunctional analogue, 2-chloroethyl ethyl sulphide (CEES, are alkylating agents that react with and diminish cellular thiols and are highly toxic. Previously, we reported that lipopolysaccharide (LPS significantly enhances the cytotoxicity of CEES in murine RAW 264.7 macrophages and that CEES transiently inhibits nitric oxide (NO production via suppression of inducible NO synthase (iNOS protein expression. NO generation is an important factor in wound healing. In this paper, we explored the hypotheses that LPS increases CEES toxicity by increasing oxidative stress and that treatment with N-acetyl-L-cysteine (NAC would block LPS induced oxidative stress and protect against loss of NO production. NAC stimulates glutathione (GSH synthesis and also acts directly as a free radical scavenger. The potential therapeutic use of the antibiotic, polymyxin B, was also evaluated since it binds to LPS and could thereby block the enhancement of CEES toxicity by LPS and also inhibit the secondary infections characteristic of HD/CEES wounds. Results We found that 10 mM NAC, when administered simultaneously or prior to treatment with 500 μM CEES, increased the viability of LPS stimulated macrophages. Surprisingly, NAC failed to protect LPS stimulated macrophages from CEES induced loss of NO production. Macrophages treated with both LPS and CEES show increased oxidative stress parameters (cellular thiol depletion and increased protein carbonyl levels. NAC effectively protected RAW 264.7 cells simultaneously treated with CEES and LPS from GSH loss and oxidative stress. Polymyxin B was found to partially block nitric oxide production and diminish CEES toxicity in LPS-treated macrophages. Conclusion The present study shows that oxidative stress is an important mechanism contributing to CEES toxicity in LPS stimulated macrophages and

Evaluation of Trace Elements in Augmentation of Statin-Induced Cytotoxicity in Uremic Serum-Exposed Human Rhabdomyosarcoma Cells

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Hitoshi Uchiyama

2018-01-01

Full Text Available Patients with end-stage kidney disease (ESKD are at higher risk for rhabdomyolysis induced by statin than patients with normal kidney function. Previously, we showed that this increase in the severity of statin-induced rhabdomyolysis was partly due to uremic toxins. However, changes in the quantity of various trace elements in ESKD patients likely contribute as well. The purpose of this study is to determine the effect of trace elements on statin-induced toxicity in rhabdomyosarcoma cells exposed to uremic serum (US cells for a long time. Cell viability, apoptosis, mRNA expression, and intracellular trace elements were assessed by viability assays, flow cytometry, real-time RT-PCR, and ICP-MS, respectively. US cells exhibited greater simvastatin-induced cytotoxicity than cells long-time exposed with normal serum (NS cells (non-overlapping 95% confidence intervals. Intracellular levels of Mg, Mn, Cu, and Zn were significantly less in US cells compared to that in NS cells (p < 0.05 or 0.01. Pre-treatment with TPEN increased simvastatin-induced cytotoxicity and eliminated the distinction between both cells of simvastatin-induced cytotoxicity. These results suggest that Zn deficiencies may be involved in the increased risk for muscle complaints in ESKD patients. In conclusion, the increased severity of statin-induced rhabdomyolysis in ESKD patients may be partly due to trace elements deficiencies.

The Nuclear Orphan Receptor NR4A1 is Involved in the Apoptotic Pathway Induced by LPS and Simvastatin in RAW 264.7 Macrophages.

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Kim, Yong Chan; Song, Seok Bean; Lee, Sang Kyu; Park, Sang Min; Kim, Young Sang

2014-04-01

Macrophage death plays a role in several physiological and inflammatory pathologies such as sepsis and arthritis. In our previous work, we showed that simvastatin triggers cell death in LPS-activated RAW 264.7 mouse macrophage cells through both caspase-dependent and independent apoptotic pathways. Here, we show that the nuclear orphan receptor NR4A1 is involved in a caspase-independent apoptotic process induced by LPS and simvastatin. Simvastatin-induced NR4A1 expression in RAW 264.7 macrophages and ectopic expression of a dominant-negative mutant form of NR4A1 effectively suppressed both DNA fragmentation and the disruption of mitochondrial membrane potential (MMP) during LPS- and simvastatin-induced apoptosis. Furthermore, apoptosis was accompanied by Bcl-2-associated X protein (Bax) translocation to the mitochondria. Our findings suggest that NR4A1 expression and mitochondrial translocation of Bax are related to simvastatin-induced apoptosis in LPS-activated RAW 264.7 macrophages.

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

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

2015-04-01

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

Inhibition of EMMPRIN and MMP-9 Expression by Epigallocatechin-3-Gallate through 67-kDa Laminin Receptor in PMA-Induced Macrophages.

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Wang, Qi-Ming; Wang, Hao; Li, Ya-Fei; Xie, Zhi-Yong; Ma, Yao; Yan, Jian-Jun; Gao, Yi Fan Wei; Wang, Ze-Mu; Wang, Lian-Sheng

2016-01-01

It is well documented that overexpression of EMMPRIN (extracellular matrix metalloproteinase inducer) and MMPs (matrix metalloproteinases) by monocytes/macrophages plays an important role in atherosclerotic plaque rupture. Green tea polyphenol epigallocatechin-3-gallate (EGCG) has a variety of pharmacological properties and exerts cardiovascular protective effects. Recently, the 67-kD laminin receptor (67LR) has been identified as a cell surface receptor of EGCG. The aim of the present study was to evaluate the effects of EGCG on the expression of EMMPRIN and MMP-9 in PMA-induced macrophages, and the potential mechanisms underlying its effects. Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA). Protein expression and MMP-9 activity were assayed by Western blot and Gelatin zymography, respectively. Real-time PCR was used to examine EMMPRIN and MMP-9 mRNA expression. We showed that EGCG (10-50µmol/L) significantly inhibited the expression of EMMPRIN and MMP-9 and activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and c-Jun N-terminal kinase (JNK) in PMA-induced macrophages. Downregulation of EMMPRIN by gene silencing hindered PMA-induced MMP-9 secretion and expression, indicating an important role of EMMPRIN in the inhibition of MMP-9 by EGCG. Moreover, 67LR was involved in EGCG-mediated suppression of EMMPRIN and MMP-9 expression. Anti-67LR antibody treatment led to abrogation of the inhibitory action of EGCG on the expression of EMMPRIN and MMP-9 and activation of ERK1/2, p38, and JNK. Our results indicate that EGCG restrains EMMPRIN and MMP-9 expression via 67LR in PMA-induced macrophages, which also suggests that EGCG may be a possible therapeutic agent for stabilizing atherosclerotic plaque. © 2016 The Author(s) Published by S. Karger AG, Basel.

Inhibition of EMMPRIN and MMP-9 Expression by Epigallocatechin-3-Gallate through 67-kDa Laminin Receptor in PMA-Induced Macrophages

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Qi-Ming Wang

2016-11-01

Full Text Available Background/Aims: It is well documented that overexpression of EMMPRIN (extracellular matrix metalloproteinase inducer and MMPs (matrix metalloproteinases by monocytes/macrophages plays an important role in atherosclerotic plaque rupture. Green tea polyphenol epigallocatechin-3-gallate (EGCG has a variety of pharmacological properties and exerts cardiovascular protective effects. Recently, the 67-kD laminin receptor (67LR has been identified as a cell surface receptor of EGCG. The aim of the present study was to evaluate the effects of EGCG on the expression of EMMPRIN and MMP-9 in PMA-induced macrophages, and the potential mechanisms underlying its effects. Methods: Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA. Protein expression and MMP-9 activity were assayed by Western blot and Gelatin zymography, respectively. Real-time PCR was used to examine EMMPRIN and MMP-9 mRNA expression. Results: We showed that EGCG (10-50µmol/L significantly inhibited the expression of EMMPRIN and MMP-9 and activation of extracellular signal-regulated kinase 1/2 (ERK1/2, p38 and c-Jun N-terminal kinase (JNK in PMA-induced macrophages. Downregulation of EMMPRIN by gene silencing hindered PMA-induced MMP-9 secretion and expression, indicating an important role of EMMPRIN in the inhibition of MMP-9 by EGCG. Moreover, 67LR was involved in EGCG-mediated suppression of EMMPRIN and MMP-9 expression. Anti-67LR antibody treatment led to abrogation of the inhibitory action of EGCG on the expression of EMMPRIN and MMP-9 and activation of ERK1/2, p38, and JNK. Conclusion: Our results indicate that EGCG restrains EMMPRIN and MMP-9 expression via 67LR in PMA-induced macrophages, which also suggests that EGCG may be a possible therapeutic agent for stabilizing atherosclerotic plaque.

Exosomes derived from hypoxic epithelial ovarian cancer deliver microRNA-940 to induce macrophage M2 polarization.

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Chen, Xin; Ying, Xiang; Wang, Xinjing; Wu, Xiaoli; Zhu, Qinyi; Wang, Xipeng

2017-07-01

Hypoxia is a common feature of solid tumors. It is closely related to tumor progression. Exosomal microRNAs derived from cancers are considered to be mediators between cancer cells and the tumor microenvironment. In addition, the number of tumor-associated macrophages (TAMs) in the tumor microenvironment has also been demonstrated to correlate with tumor development. However, the relationship between tumor-secreted exosomes and TAM polarization under hypoxic conditions during tumor progression is not clear. Herein, we demonstrated that hypoxia induces the high expression of microRNA-940 (miR‑940) in exosomes derived from epithelial ovarian cancer (EOC). We also found that miR‑940 is highly expressed in exosomes isolated from ascites of EOC patients. Moreover, the overexpression of miR‑940 in macrophages delivered by exosomes stimulated M2 phenotype polarization, while the M2 subtype macrophages promoted EOC proliferation and migration. These results highlight the function of hypoxia in enhancing the high level of expression of miR‑940 in tumor exosomes taken up by macrophages. We also showed that the tumor-promoting function of miR‑940 is mediated by TAM polarization in EOC. These findings show that tumor-derived exosomal miR‑940 induced by hypoxia plays an important role in stimulating TAM polarization in the progression of EOC.

Effect of Shark Liver Oil on Peritoneal Murine Macrophages in Responses to Killed-Candida albicans

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Monire Hajimoradi

2009-09-01

Full Text Available Objective(sShark Liver Oil (SLO is an immunomodulator. Macrophages play a key role in host defense against pathogens like fungi. Candida albicans have mechanisms to escape immune system. We determined the effect of killed-Candida on the in vitro viability of macrophages and the effect of SLO on augmentation of this potency.Materials and MethodsPeritoneal macrophages were separated and cultured (3×105/well. At first, the effect of killed-Candida (200 cells/well on macrophage viability was evaluated, using MTT test. Then, MTT was performed on macrophages stimulated with killed-Candida in the presence of SLO. ResultsKilled-Candida suppressed the ability of MTT reduction and hence macrophages viability (P=0.026, but addition of SLO (100 mg/ml significantly enhanced cell viability (P=0.00. So, SLO could neutralize the inhibitory effect of Candida.ConclusionSimultaneous with cytotoxic effect of killed-Candida cells on macrophages viability, SLO augment macrophages viability. So, it can be applied in candidiasis as a complement.

Primary Tr1 cells from metastatic melanoma eliminate tumor-promoting macrophages through granzyme B- and perforin-dependent mechanisms.

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Yan, Hongxia; Zhang, Ping; Kong, Xue; Hou, Xianglian; Zhao, Li; Li, Tianhang; Yuan, Xiaozhou; Fu, Hongjun

2017-04-01

In malignant melanoma, tumor-associated macrophages play multiple roles in promoting tumor growth, such as inducing the transformation of melanocytes under ultraviolet irradiation, increasing angiogenesis in melanomas, and suppressing antitumor immunity. Because granzyme B- and perforin-expressing Tr1 cells could specifically eliminate antigen-presenting cells of myeloid origin, we examined whether Tr1 cells in melanoma could eliminate tumor-promoting macrophages and how the interaction between Tr1 cells and macrophages could affect the growth of melanoma cells. Tr1 cells were characterized by high interleukin 10 secretion and low Foxp3 expression and were enriched in the CD4 + CD49b + LAG-3 + T-cell fraction. Macrophages derived from peripheral blood monocytes in the presence of modified melanoma-conditioned media demonstrated tumor-promoting capacity, exemplified by improving the proliferation of cocultured A375 malignant melanoma cells. But when primary Tr1 cells were present in the macrophage-A375 coculture, the growth of A375 cells was abrogated. The conventional CD25 + Treg cells, however, were unable to inhibit macrophage-mediated increase in tumor cell growth. Further analyses showed that Tr1 cells did not directly eliminate A375 cells, but mediated the killing of tumor-promoting macrophages through the secretion of granzyme B and perforin. The tumor-infiltrating interleukin 10 + Foxp3 - CD4 + T cells expressed very low levels of granzyme B and perforin, possibly suggested the downregulation of Tr1 cytotoxic capacity in melanoma tumors. Together, these data demonstrated an antitumor function of Tr1 cells through the elimination of tumor-promoting macrophages, which was not shared by conventional Tregs.

Enhancement of CD147 on M1 macrophages induces differentiation of Th17 cells in the lung interstitial fibrosis.

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Geng, Jie-jie; Zhang, Kui; Chen, Li-na; Miao, Jin-lin; Yao, Meng; Ren, Ying; Fu, Zhi-guang; Chen, Zhi-nan; Zhu, Ping

2014-09-01

Lung interstitial fibrosis is a chronic lung disease, and few effective therapies are available to halt or reverse the progression of the disease. In murine and human lung fibrosis, the expression of CD147 is increased. However, the role of CD147 in lung fibrosis has not been identified, and it remains to be determined whether lung fibrosis would be improved by decreasing the expression of CD147. A murine bleomycin-induced lung interstitial fibrosis model was used in the experiments, and HAb18 mAbs and CsA were administered during the induction of lung fibrosis. In our study, we found that the HAb18 mAbs markedly reduced the collagen score and down-regulated M1 macrophages and Th17 cells. In vitro, flow cytometry analysis showed that M1 macrophages induced higher Th17 differentiation than M2 macrophages. After treatment with HAb18 mAbs or after reducing the expression of CD147 by lentivirus interference in M1 macrophages, the level of Th17 cells were significantly inhibited. In conclusion, HAb18 mAbs or CsA treatment ameliorates lung interstitial fibrosis. CD147 promoted M1 macrophage and induced the differentiation of Th17 cells in lung interstitial fibrosis, perhaps by regulating some cytokines such as IL-6, IL-1β, IL-12 and IL-23. These results indicated that CD147 may play an important role in the development of lung interstitial fibrosis. Copyright © 2014 Elsevier B.V. All rights reserved.

The Poly-γ-D-Glutamic Acid Capsule of Bacillus licheniformis, a Surrogate of Bacillus anthracis Capsule Induces Interferon-Gamma Production in NK Cells through Interactions with Macrophages.

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Lee, Hae-Ri; Jeon, Jun Ho; Rhie, Gi-Eun

2017-05-28

The poly-γ- D -glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis , provides protection of the bacterium from phagocytosis and allows its unimpeded growth in the host. We investigated crosstalk between murine natural killer (NK) cells and macrophages stimulated with the PGA capsule of Bacillus licheniformis , a surrogate of the B. anthracis capsule. PGA induced interferon-gamma production from NK cells cultured with macrophages. This effect was dependent on macrophage-derived IL-12 and cell-cell contact interaction with macrophages through NK cell receptor NKG2D and its ligand RAE-1. The results showed that PGA could enhance NK cell activation by inducing IL-12 production in macrophages and a contact-dependent crosstalk with macrophages.

Differential Macrophage Response to Slow- and Fast-Growing Pathogenic Mycobacteria

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A. Cecilia Helguera-Repetto

2014-01-01

Full Text Available Nontuberculous mycobacteria (NTM have recently been recognized as important species that cause disease even in immunocompetent individuals. The mechanisms that these species use to infect and persist inside macrophages are not well characterised. To gain insight concerning this process we used THP-1 macrophages infected with M. abscessus, M. fortuitum, M. celatum, and M. tuberculosis. Our results showed that slow-growing mycobacteria gained entrance into these cells with more efficiency than fast-growing mycobacteria. We have also demonstrated that viable slow-growing M. celatum persisted inside macrophages without causing cell damage and without inducing reactive oxygen species (ROS, as M. tuberculosis caused. In contrast, fast-growing mycobacteria destroyed the cells and induced high levels of ROS. Additionally, the macrophage cytokine pattern induced by M. celatum was different from the one induced by either M. tuberculosis or fast-growing mycobacteria. Our results also suggest that, in some cases, the intracellular survival of mycobacteria and the immune response that they induce in macrophages could be related to their growth rate. In addition, the modulation of macrophage cytokine production, caused by M. celatum, might be a novel immune-evasion strategy used to survive inside macrophages that is different from the one reported for M. tuberculosis.

Macrophage migration inhibitory factor triggers chemotaxis of CD74+CXCR2+ NKT cells in chemically induced IFN-γ-mediated skin inflammation.

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Hsieh, Chia-Yuan; Chen, Chia-Ling; Lin, Yee-Shin; Yeh, Trai-Ming; Tsai, Tsung-Ting; Hong, Ming-Yuan; Lin, Chiou-Feng

2014-10-01

IFN-γ mediates chemically induced skin inflammation; however, the mechanism by which IFN-γ-producing cells are recruited to the sites of inflammation remains undefined. Secretion of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, from damaged cells may promote immune cell recruitment. We hypothesized that MIF triggers an initial step in the chemotaxis of IFN-γ-producing cells in chemically induced skin inflammation. Using acute and chronic models of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mouse ears, MIF expression was examined, and its role in this process was investigated pharmacologically. The cell populations targeted by MIF, their receptor expression patterns, and the effects of MIF on cell migration were examined. TPA directly caused cytotoxicity accompanied by MIF release in mouse ear epidermal keratinocytes, as well as in human keratinocytic HaCaT cells. Treatment with the MIF antagonist (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester considerably attenuated TPA-induced ear swelling, leukocyte infiltration, epidermal cell proliferation, and dermal angiogenesis. Inhibition of MIF greatly diminished the dermal infiltration of IFN-γ(+) NKT cells, whereas the addition of exogenous TPA and MIF to NKT cells promoted their IFN-γ production and migration, respectively. MIF specifically triggered the chemotaxis of NKT cells via CD74 and CXCR2, and the resulting depletion of NKT cells abolished TPA-induced skin inflammation. In TPA-induced skin inflammation, MIF is released from damaged keratinocytes and then triggers the chemotaxis of CD74(+)CXCR2(+) NKT cells for IFN-γ production. Copyright © 2014 by The American Association of Immunologists, Inc.

miR-146a down-regulation alleviates H2O2-induced cytotoxicity of PC12 cells by regulating MCL1/JAK/STAT pathway : miR-146a down-regulation relieves H2O2-induced PC12 cells cytotoxicity by MCL1/JAK/STAT.

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Yang, Xuecheng; Mao, Xin; Ding, Xuemei; Guan, Fengju; Jia, Yuefeng; Luo, Lei; Li, Bin; Tan, Hailin; Cao, Caixia

2018-02-26

Oxidative stress and miRNAs have been confirmed to play an important role in neurological diseases. The study aimed to explore the underlying effect and mechanisms of miR-146a in H 2 O 2 -induced injury of PC12 cells. Here, PC12 cells were stimulated with 200 μM of H 2 O 2 to construct oxidative injury model. Cell injury was evaluated on the basis of the changes in cell viability, migration, invasion, apoptosis, and DNA damage. Results revealed that miR-146a expression was up-regulated in H 2 O 2 -induced PC12 cells. Functional analysis showed that down-regulation of miR-146a alleviated H 2 O 2 -induced cytotoxicity in PC12 cells. Dual-luciferase reporter and western blot assay verified that MCL1 was a direct target gene of miR-146a. Moreover, anti-miR-146a-mediated suppression on cell cytotoxicity was abated following MCL1 knockdown in H 2 O 2 -induced PC12 cells. Furthermore, MCL1 activated JAK/STAT signaling pathway and MCL1 overexpression attenuated H 2 O 2 -induced cytotoxicity in PC12 cells by JAK/STAT signaling pathway. In conclusion, this study suggested that suppression of miR-146a abated H 2 O 2 -induced cytotoxicity in PC12 cells via regulating MCL1/JAK/STAT pathway.

A macrophage activation switch (MAcS)-index for assessment of monocyte/macrophage activation

DEFF Research Database (Denmark)

Maniecki, Maciej Bogdan; Lauridsen, Mette; Knudsen, Troels Bygum

2008-01-01

, simplified by the M1-M2 dichotomy of classically activated (M1), pro-inflammatory cells and alternatively activated (M2), anti-inflammatory cells. Macrophages, however, display a large degree of flexibility and are able to switch between activation states (1). The hemoglobin scavenger receptor CD163...... is expressed exclusively on monocytes and macrophages, and its expression is strongly induced by anti-inflammatory stimuli like IL10 and glucocorticoid, making CD163 an ideal M2 macrophage marker (2). Furthermore a soluble variant of CD163 (sCD163) is shed from the cell surface to plasma by protease mediated.......058-5139) (panti-inflammatory state.   CONCLUSION: We present a CD163-derived macrophage activation switch (MAcS)-index, which seems able to differentiate between (predominantly) pro-inflammatory and anti-inflammatory macrophage activation. The index needs...

Corn silk induces nitric oxide synthase in murine macrophages.

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Kim, Kyung A; Choi, Sang Kyu; Choi, Hye Seon

2004-12-31

Corn silk has been purified as an anticoagulant previously and the active component is a polysaccharide with a molecular mass of 135 kDa. It activates murine macrophages to induce nitric oxide synthase (NOS) and generate substantial amounts of NO in time and dose-dependent manners. It was detectable first at 15 h after stimulation by corn silk, peaked at 24 h, and undetectable by 48 h. Induction of NOS is inhibited by pyrolidine dithiocarbamate (PDTC) and genistein, an inhibitor of nuclear factor kappa B (NF-kappaB) and tyrosine kinase, respectively, indicating that iNOS stimulated by corn silk is associated with tyrosine kinase and NF-kappaB signaling pathways. IkappaB-alpha degradation was detectible at 10 min, and the level was restored at 120 min after treatment of corn silk. Corn silk induced nuclear translocation of NF-kappaB by phosphorylation and degradation of IkappaB-alpha.

Reduced cadmium-induced cytotoxicity in cultured liver cells following 5-azacytidine pretreatment

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Waalkes, M.P.; Wilson, M.J.; Poirier, L.A.

1985-01-01

Recent work indicated that administration of the pyrimidine analog 5-azacytidine (AZA), either to cells in culture or to rats, results in an enhancement of expression of the metallothionein (MT) gene. Since MT is thought to play a central role in the detoxification of cadmium, the present study was designed to assess the effect of AZA pretreatment on cadmium cytotoxicity. Cultured rat liver cells in log phase of growth were first exposed to AZA (8 microM). Forty-eight hours later, cadmium was added. A modest increase in MT amounts over control was detected after AZA treatment alone. Cadmium alone resulted in a 10-fold increase in MT concentrations. The combination of AZA pretreatment followed by cadmium exposure caused a 23-fold increase in MT concentrations over control. Treatment with the DNA synthesis inhibitor hydroxyurea (HU) eliminated the enhancing effect of AZA pretreatment on cadmium induction of MT, indicating that cell division is required. AZA-pretreated cells were also harvested and incubated in suspension with cadmium for 0 to 90 min. AZA-pretreated cells showed marked reductions in cadmium-induced cytotoxicity as reflected by reduced intracellular potassium loss, glutamic-oxaloacetic transaminase loss, and lipid peroxidation following cadmium exposure. Results suggest that AZA pretreatment induces tolerance to cadmium cytotoxicity which appears to be due to an increased capacity to synthesize MT rather than high quantities of preexisting MT at the time of cadmium exposure

Recruitment of macrophages from the spleen contributes to myocardial fibrosis and hypertension induced by angiotensin II

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Ning-Ping Wang

2017-05-01

Full Text Available Introduction: The purpose of this study was to determine whether macrophages migrated from the spleen are associated with angiotensin II-induced cardiac fibrosis and hypertension. Methods: Sprague-Dawley rats were subjected to angiotensin II infusion in vehicle (500 ng/kg/min for up to four weeks. In splenectomy, the spleen was removed before angiotensin II infusion. In the angiotensin II AT1 receptor blockade, telmisartan was administered by gastric gavage (10 mg/kg/day during angiotensin II infusion. The heart and aorta were isolated for Western blot analysis and immunohistochemistry. Results: Angiotensin II infusion caused a significant reduction in the number of monocytes in the spleen through the AT1 receptor-activated monocyte chemoattractant protein-1. Comparison of angiotensin II infusion, splenectomy and telmisartan comparatively reduced the recruitment of macrophages into the heart. Associated with this change, transforming growth factor β1 expression and myofibroblast proliferation were inhibited, and Smad2/3 and collagen I/III were downregulated. Furthermore, interstitial/perivascular fibrosis was attenuated. These modifications occurred in coincidence with reduced blood pressure. At week 4, invasion of macrophages and myofibroblasts in the thoracic aorta was attenuated and expression of endothelial nitric oxide synthase was upregulated, along with a reduction in aortic fibrosis. Conclusions: These results suggest that macrophages when recruited into the heart and aorta from the spleen potentially contribute to angiotensin II-induced cardiac fibrosis and hypertension.

Commensal Bacteria-Induced Inflammasome Activation in Mouse and Human Macrophages Is Dependent on Potassium Efflux but Does Not Require Phagocytosis or Bacterial Viability.

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Kejie Chen

Full Text Available Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß. Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3 inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis.

The Current State of Nanoparticle-Induced Macrophage Polarization and Reprogramming Research

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Xiaoyuan Miao

2017-02-01

Full Text Available Macrophages are vital regulators of the host defense in organisms. In response to different local microenvironments, resting macrophages (M0 can be polarized into different phenotypes, pro-inflammatory (M1 or anti-inflammatory (M2, and perform different roles in different physiological or pathological conditions. Polarized macrophages can also be further reprogrammed by reversing their phenotype according to the changed milieu. Macrophage polarization and reprogramming play essential roles in maintaining the steady state of the immune system and are involved in the processes of many diseases. As foreign substances, nanoparticles (NPs mainly target macrophages after entering the body. NPs can perturb the polarization and reprogramming of macrophages, affect their immunological function and, therefore, affect the pathological process of disease. Optimally-designed NPs for the modulation of macrophage polarization and reprogramming might provide new solutions for treating diseases. Systematically investigating how NPs affect macrophage polarization is crucial for understanding the regulatory effects of NPs on immune cells in vivo. In this review, macrophage polarization by NPs is summarized and discussed.

Flavonoid glycosides from leaves and straw of Oryza sativa and their effects of cytotoxicity on a macrophage cell line and allelopathic on weed germination

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Ill-Min Chung

2018-03-01

Full Text Available Five new flavonoids namely, 5-hydroxy-6-isoprenyl-7,4′-dimethoxyflavonol-3-O-β-d-arabinofuranoside (1, 5,7-dihydroxy-4′-methoxyflavone-7-O-β-d-arabinopyranosyl-2′′-n-decan-1′′′-oate (2, 3-butanoyl-5,6,8-trihydroxy-7,4′-dimethoxyflavonol--5-O-β-d-glucopyranoside (3, 7, 4′-dimethoxy-5-hydroxyflavone-5-O-α-d-arabinopyranosyl-(2′′ → 1′′′-O-α-d-arabinopyranoside (4, and 5,6-dihydroxy-7, 4′-dimethoxyflavone-5-O-α-d-glucopyranoside (5, together with two known compounds, were isolated from the methanol extract of Oryza sativa leaves and straw. Their structures of new compounds were elucidated by 1D and 2D NMR spectral methods, viz: COSY, HMBC and HSQC aided by mass techniques and IR spectroscopy. The cytotoxicity of these compounds (1–7 were assessed by using (RAW 264.7 mouse macrophages cell line, and allelopathic effects of compounds (1–7 on the germination characteristics of barnyardgrass (Echinochloa oryzicola and pigweed (Chenopodium album L. were also evaluated. The compounds 1, 6 and 7 showed cytotoxicity and compounds 1–7 exhibited significant inhibitory activity on the seed germination of two weed species.

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�

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

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Gammelsrud, A.; Solhaug, A.; Dendelé, B.; Sandberg, W.J.; Ivanova, L.; Kocbach Bølling, A.; Lagadic-Gossmann, D.; Refsnes, M.; Becher, R.; Eriksen, G.; Holme, J.A.

2012-01-01

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�

Effects of habitual exercise on the eHsp72-induced release of inflammatory cytokines by macrophages from obese Zucker rats.

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Garcia, J J; Martin-Cordero, L; Hinchado, M D; Bote, M E; Ortega, E

2013-06-01

Regular exercise is a good non-pharmacological treatment of metabolic syndrome in that it improves obesity, diabetes, and inflammation. The 72 kDa extracellular heat shock protein (eHsp72) is released during exercise, thus stimulating the inflammatory responses. The aim of the present work was to evaluate the effect of regular exercise on the eHsp72-induced release of IL-1β, IL-6, and TNFα by macrophages from genetically obese Zucker rats (fa/fa) (ObZ), using lean Zucker (LZ) rats (Fa/fa) to provide reference values. ObZ presented a higher plasma concentration of eHsp72 than LZ, and exercise increased that concentration. In response to eHsp72, the macrophages from ObZ released less IL-1β and TNFα, but more IL-6, than macrophages from LZ. While eHsp72 stimulated the release of IL-1β, TNFα, and IL-6 in the macrophages from healthy LZ (with respect to the constitutive release), it inhibited the release of IL-1β and IL-6 in macrophages from ObZ. The habitual exercise improved the release of inflammatory cytokines by macrophages from ObZ in response to eHsp72 (it increased IL-1β and TNFα, and decreased IL-6), tending to values closer to those determined in healthy LZ. A deregulated macrophage inflammatory and stress response induced by eHsp72 underlies MS, and this is improved by habitual exercise. © Georg Thieme Verlag KG Stuttgart · New York.

Moderate restriction of macrophage-tropic human immunodeficiency virus type 1 by SAMHD1 in monocyte-derived macrophages.

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Taya, Kahoru; Nakayama, Emi E; Shioda, Tatsuo

2014-01-01

Macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains are able to grow to high titers in human monocyte-derived macrophages. However, it was recently reported that cellular protein SAMHD1 restricts HIV-1 replication in human cells of the myeloid lineage, including monocyte-derived macrophages. Here we show that degradation of SAMHD1 in monocyte-derived macrophages was associated with moderately enhanced growth of the macrophage-tropic HIV-1 strain. SAMHD1 degradation was induced by treating target macrophages with vesicular stomatitis virus glycoprotein-pseudotyped human immunodeficiency virus type 2 (HIV-2) particles containing viral protein X. For undifferentiated monocytes, HIV-2 particle treatment allowed undifferentiated monocytes to be fully permissive for productive infection by the macrophage-tropic HIV-1 strain. In contrast, untreated monocytes were totally resistant to HIV-1 replication. These results indicated that SAMHD1 moderately restricts even a macrophage-tropic HIV-1 strain in monocyte-derived macrophages, whereas the protein potently restricts HIV-1 replication in undifferentiated monocytes.

Cytokine overproduction and crosslinker hypersensitivity are unlinked in Fanconi anemia macrophages.

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Garbati, Michael R; Hays, Laura E; Rathbun, R Keaney; Jillette, Nathaniel; Chin, Kathy; Al-Dhalimy, Muhsen; Agarwal, Anupriya; Newell, Amy E Hanlon; Olson, Susan B; Bagby, Grover C

2016-03-01

The Fanconi anemia proteins participate in a canonical pathway that repairs cross-linking agent-induced DNA damage. Cells with inactivated Fanconi anemia genes are universally hypersensitive to such agents. Fanconi anemia-deficient hematopoietic stem cells are also hypersensitive to inflammatory cytokines, and, as importantly, Fanconi anemia macrophages overproduce such cytokines in response to TLR4 and TLR7/8 agonists. We questioned whether TLR-induced DNA damage is the primary cause of aberrantly regulated cytokine production in Fanconi anemia macrophages by quantifying TLR agonist-induced TNF-α production, DNA strand breaks, crosslinker-induced chromosomal breakage, and Fanconi anemia core complex function in Fanconi anemia complementation group C-deficient human and murine macrophages. Although both M1 and M2 polarized Fanconi anemia cells were predictably hypersensitive to mitomycin C, only M1 macrophages overproduced TNF-α in response to TLR-activating signals. DNA damaging agents alone did not induce TNF-α production in the absence of TLR agonists in wild-type or Fanconi anemia macrophages, and mitomycin C did not enhance TLR responses in either normal or Fanconi anemia cells. TLR4 and TLR7/8 activation induced cytokine overproduction in Fanconi anemia macrophages. Also, although TLR4 activation was associated with induced double strand breaks, TLR7/8 activation was not. That DNA strand breaks and chromosome breaks are neither necessary nor sufficient to account for the overproduction of inflammatory cytokines by Fanconi anemia cells suggests that noncanonical anti-inflammatory functions of Fanconi anemia complementation group C contribute to the aberrant macrophage phenotype and suggests that suppression of macrophage/TLR hyperreactivity might prevent cytokine-induced stem cell attrition in Fanconi anemia. © Society for Leukocyte Biology.

Anaplasma phagocytophilum-Related Defects in CD8, NKT, and NK Lymphocyte Cytotoxicity

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Diana G. Scorpio

2018-04-01

Full Text Available Human granulocytic anaplasmosis, caused by the tick-transmitted Anaplasma phagocytophilum, is not controlled by innate immunity, and induces a proinflammatory disease state with innate immune cell activation. In A. phagocytophilum murine infection models, hepatic injury occurs with production of IFNγ thought to be derived from NK, NKT cells, and CD8 T lymphocytes. Specific A. phagocytophilum ligands that drive inflammation and disease are not known, but suggest a clinical and pathophysiologic basis strikingly like macrophage activation syndrome (MAS and hemophagocytic syndrome (HPS. We studied in vivo responses of NK, NKT, and CD8 T lymphocytes from infected animals for correlates of lymphocyte-mediated cytotoxicity and examined in vitro interactions with A. phagocytophilum-loaded antigen-presenting cells (APCs. Murine splenocytes were examined and found deficient in cytotoxicity as determined by CD107a expression in vitro for specific CTL effector subsets as determined by flow cytometry. Moreover, A. phagocytophilum-loaded APCs did not lead to IFNγ production among CTLs in vitro. These findings support the concept of impaired cytotoxicity with A. phagocytophilum presentation by APCs that express MHC class I and that interact with innate and adaptive immune cells with or after infection. The findings strengthen the concept of an enhanced proinflammatory phenotype, such as MAS and HPS disease states as the basis of disease and severity with A. phagocytophilum infection, and perhaps by other obligate intracellular bacteria.

The influence of macrophage growth factors on Theiler's Murine Encephalomyelitis Virus (TMEV) infection and activation of macrophages.

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Schneider, Karin M; Watson, Neva B; Minchenberg, Scott B; Massa, Paul T

2018-02-01

Macrophages are common targets for infection and innate immune activation by many pathogenic viruses including the neurotropic Theiler's Murine Encephalomyelitis Virus (TMEV). As both infection and innate activation of macrophages are key determinants of viral pathogenesis especially in the central nervous system (CNS), an analysis of macrophage growth factors on these events was performed. C3H mouse bone-marrow cells were differentiated in culture using either recombinant macrophage colony stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), inoculated with TMEV (BeAn) and analyzed at various times thereafter. Cytokine RNA and protein analysis, virus titers, and flow cytometry were performed to characterize virological parameters under these culture conditions. GM-CSF-differentiated macrophages showed higher levels of TMEV viral RNA and proinflammatory molecules compared to infected M-CSF-differentiated cells. Thus, GM-CSF increases both TMEV infection and TMEV-induced activation of macrophages compared to that seen with M-CSF. Moreover, while infectious viral particles decreased from a peak at 12h to undetectable levels at 48h post infection, TMEV viral RNA remained higher in GM-CSF- compared to M-CSF-differentiated macrophages in concert with increased proinflammatory gene expression. Analysis of a possible basis for these differences determined that glycolytic rates contributed to heightened virus replication and proinflammatory cytokine secretion in GM-CSF compared to M-CSF-differentiated macrophages. In conclusion, we provide evidence implicating a role for GM-CSF in promoting virus replication and proinflammatory cytokine expression in macrophages, indicating that GM-CSF may be a key factor for TMEV infection and the induction of chronic TMEV-induced immunopathogenesis in the CNS. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dairy cows produce cytokine and cytotoxic T cell responses following vaccination with an antigenic fraction from Streptococcus uberis.

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Wedlock, D Neil; Buddle, Bryce M; Williamson, John; Lacy-Hulbert, S Jane; Turner, Sally-Anne; Subharat, Supatsak; Heiser, Axel

2014-07-15

Streptococcus uberis is a major cause of mastitis in dairy cows worldwide and currently, there is no vaccine commercially available against this form of mastitis. In the current study, cell-free extracts (CFE) were prepared from each of three different S. uberis strains, designated as #3, #24 and #363 representative of the three main sequence types of S. uberis that cause mastitis in New Zealand. These proteins were formulated into vaccines with Emulsigen-D and the immunogenicity of the vaccines was determined in both calves and dairy cows. Two groups of calves (n=5/group) were vaccinated subcutaneously with CFE from strain #24 or strains #3, #24 and #363 formulated with Emulsigen-D, respectively. A third group (n=5) was vaccinated with CFE from the three strains formulated with Emulsigen-D and also containing recombinant bovine granulocyte macrophage colony-stimulating factor while, a control group (n=5) was not vaccinated. Vaccinated animals produced strong antibody responses to the S. uberis antigens and an antigen-specific cytotoxic effect against blood monocytes/macrophages that had phagocytosed S. uberis, with no significant differences in responses observed between the three vaccinated groups. In a second trial, the safety and immunogenicity of the vaccine containing CFE from all three strains of S. uberis and Emulsigen-D was determined in dairy cows. A group of six cows were vaccinated subcutaneously at 3 and 1 week prior to dry off and revaccinated 2-3 weeks before calving. Immune responses in blood and mammary gland secretions (MGS) were monitored during the dry period and in the subsequent lactation. The vaccine was well tolerated with no adverse effect from vaccination observed in any of the cows. Vaccination induced an antigen-specific cytotoxic effect against blood monocytes/macrophages that had phagocytosed S. uberis, moderate antigen-specific IFN-γ responses in blood and strong antibody responses in both blood and MGS. In conclusion, the results

Vitamin K3 analogs induce selective tumor cytotoxicity in neuroblastoma.

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Kitano, Toru; Yoda, Hiroyuki; Tabata, Keiichi; Miura, Motofumi; Toriyama, Masaharu; Motohashi, Shigeyasu; Suzuki, Takashi

2012-01-01

We investigated the cytotoxicity of eight vitamin K3 (VK3) analogs against neuroblastoma cell lines (IMR-32, LA-N-1, NB-39, and SK-N-SH) and normal cell lines (human umbilical vein endothelial cells (HUVEC) and human dermal fibroblasts (HDF)) using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. 2-[(2-Methoxy)ethylthio]-3-methyl-1,4-naphthoquinone (VK3-OCH(3)) showed especially potent cytotoxic activities against neuroblastoma cells compared with normal cells. In a Hoechst 33342 staining experiment, apoptotic morphologies characterized by cell shrinkage, nuclear condensation, and nuclear fragmentation were observed in IMR-32 and LA-N-1 cells after 48 h of treatment with 10(-5) M of VK3-OCH(3). To clarify the molecular mechanisms of apoptosis induced by VK3-OCH(3), we examined the expression of apoptosis related proteins using a Proteome Profiler Array and western blotting. Heme oxygenase (HO)-1 was remarkably increased by VK3-OCH(3) compared with the control (173% in IMR-32 and 170% in LA-N-1 at 24 h). Moreover, caveolin-1 was induced by VK3-OCH(3) at 48 h. In addition, VK3-OCH(3) arrested the cell cycle at the G2/M phase in IMR-32 cells. These results suggest that VK3-OCH(3) exhibited a selective antitumor activity via HO-1-related mechanisms.

Nocardia brasiliensis induces formation of foamy macrophages and dendritic cells in vitro and in vivo.

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Irene Meester

Full Text Available Foamy cells have been described in various infectious diseases, for example in actinomycetoma induced by Nocardia brasiliensis. These cells are generally considered to be macrophages, although they present dendritic cell (DC-specific surface markers. In this study, we determined and confirmed the lineage of possible precursors of foamy cells in vitro and in vivo using an experimental actinomycetoma model in BALB/c mice. Bone marrow-derived macrophages (BMDM or DC (BMDC were infected in vitro with N. brasiliensis or labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE. Both, macrophages and DC, differentiated into foamy cells after in vitro infection. CFSE-labeled BMDM or BMDC were tested for phagocytosis and CD11c/CD11b receptors markers expression before being transferred into the actinomycetoma lesion site of infected mice. In vivo studies showed that BMDM and BMDC were traced at the site where foamy cells are present in the experimental actinomycetoma. Interestingly, many of the transferred BMDM and BMDC were stained with the lipid-droplet fluorophore Nile Red. In conclusion, macrophages and DC cells can be differentiated into foamy cells in vitro and in vivo during N. brasiliensis infection.

Nocardia brasiliensis induces formation of foamy macrophages and dendritic cells in vitro and in vivo.

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Meester, Irene; Rosas-Taraco, Adrian Geovanni; Salinas-Carmona, Mario Cesar

2014-01-01

Foamy cells have been described in various infectious diseases, for example in actinomycetoma induced by Nocardia brasiliensis. These cells are generally considered to be macrophages, although they present dendritic cell (DC)-specific surface markers. In this study, we determined and confirmed the lineage of possible precursors of foamy cells in vitro and in vivo using an experimental actinomycetoma model in BALB/c mice. Bone marrow-derived macrophages (BMDM) or DC (BMDC) were infected in vitro with N. brasiliensis or labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). Both, macrophages and DC, differentiated into foamy cells after in vitro infection. CFSE-labeled BMDM or BMDC were tested for phagocytosis and CD11c/CD11b receptors markers expression before being transferred into the actinomycetoma lesion site of infected mice. In vivo studies showed that BMDM and BMDC were traced at the site where foamy cells are present in the experimental actinomycetoma. Interestingly, many of the transferred BMDM and BMDC were stained with the lipid-droplet fluorophore Nile Red. In conclusion, macrophages and DC cells can be differentiated into foamy cells in vitro and in vivo during N. brasiliensis infection.

Comparative activation states of tumor-associated and peritoneal macrophages from mice bearing an induced fibrosarcoma.

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Valdez, J C; de Alderete, N; Meson, O E; Sirena, A; Perdigon, G

1990-11-01

Balb/c mice bearing a methylcholanthrene-induced fibrosarcoma were used to compare the activation levels of tumor-associated and peritoneal macrophages. Two stages of tumor growth were examined, namely "small" and "large" tumors, with average diameters of 10 and 30 mm, respectively. The activation state, determined by measurement of both phagocytic index and beta-glucuronidase content, was found to be markedly higher in tumor-associated macrophages than in their peritoneal counterparts and it was, in addition, independent of tumor progression. The percentage of tumor-associated macrophages, which were detected on the basis of Fc receptor expression, remained constant in the growing neoplasm, at approximately 23% of total cell population. None of these parameters were affected by inoculation with an immunopotentiating dose of heat-killed Candida albicans which, on the other hand, seemed not to alter the course of the tumor. These data suggest that within the tumor microenvironment macrophages would somehow be maintained at a constant proportion and at a highly activated state, while outside the tumor they would be at a lower activation level. Our results also suggest that TAM would not possess antitumor activity in vivo, although we have found this activity in vitro.

Role of macrophages and oxygen radicals in IgA induced lung injury in the rat

International Nuclear Information System (INIS)

Johnson, K.J.; Ward, P.A.; Kunkel, R.G.; Wilson, B.S.

1986-01-01

Acute lung injury in the rat has been induced by the instillation of affinity-purified mouse monoclonal IgA antibody with specific reactivity to dinitrophenol (DNP) coupled to albumin. This model of lung injury requires an intact complement system but not neutrophils, and evidence suggests that pulmonary macrophages are the critical effector cell. Macrophages retrievable from the lungs of the IgA immune complex treated rats are considerably increased in number as compared to control animals which received only the antibody. In addition these cells show evidence of activation in vivo with greater spontaneous generation of the superoxide anion (O 2 - ) as well as significantly enhanced O 2 - response in the presence of a second stimulus. Inhibition studies in vivo suggest that the lung injury is mediated by oxygen radical generation by the pulmonary macrophages. Pretreatment of rats with superoxide dismutase (SOD), catalase, the iron chelator deferoxamine or the hydroxyl radical scavenger dimethyl sulfoxide (DMSO) all markedly suppressed the development of the lung injury. In summary, these studies suggest that IgA immune complex injury in the rat lung is mediated by oxygen radical formation from pulmonary macrophages

Galectin-3 disruption impaired tumoral angiogenesis by reducing VEGF secretion from TGFβ1-induced macrophages

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Machado, Camila Maria Longo; Andrade, Luciana Nogueira Sousa; Teixeira, Verônica Rodrigues; Costa, Fabrício Falconi; Melo, Camila Morais; Santos, Sofia Nascimento dos; Nonogaki, Suely; Liu, Fu-Tong; Bernardes, Emerson Soares; Camargo, Anamaria Aranha; Chammas, Roger

2014-01-01

In order to study the role of galectin-3 in tumor angiogenesis associated with tumor-associated macrophages (TAM) and tumor parenchyma, the galectin-3 expression was reconstituted in Tm1 melanoma cell line that lacks this protein. Galectin-3-expressing cells (Tm1G3) and mock-vector transfected cells (Tm1N3) were injected into wild-type (WT) and galectin-3 knockout (KO) C57Bl/6 mice. Tumors originated from Tm1G3 were larger in tumor volume with enlarged functional vessels, decreased necrotic areas, and increased vascular endothelial growth factor (VEGF) protein levels. Galectin-3-nonexpressing-cells injected into WT and KO showed increased levels of transforming growth factor beta 1 (TGFβ1) and, in WT animals this feature was also accompanied by increased VEGFR2 expression and its phosphorylation. In KO animals, tumors derived from galectin-3-expressing cells were infiltrated by CD68 + -cells, whereas in tumors derived from galectin-3-nonexpressing-cells, CD68 + cells failed to infiltrate tumors and accumulated in the periphery of the tumor mass. In vitro studies showed that Tm1G3 secreted more VEGF than Tm1N3 cells. In the latter case, TGFβ1 induced VEGF production. Basal secretion of VEGF was higher in WT-bone marrow-derived macrophages (BMDM) than in KO-BMDM. TGFβ1 induced secretion of VEGF only in WT-BMDM. Tm1G3-induced tumors had the Arginase I mRNA increased, which upregulated alternative macrophage (M2)/TAM induction. M2 stimuli, such as interleukin-4 (IL4) and TGFβ1, increased Arginase I protein levels and galectin-3 expression in WT- BMDM, but not in cells from KO mice. Hence, we report that galectin-3 disruption in tumor stroma and parenchyma decreases angiogenesis through interfering with the responses of macrophages to the interdependent VEGF and TGFβ1 signaling pathways

Combination Immunotherapy of B16 Melanoma Using Anti–Cytotoxic T Lymphocyte–Associated Antigen 4 (Ctla-4) and Granulocyte/Macrophage Colony-Stimulating Factor (Gm-Csf)-Producing Vaccines Induces Rejection of Subcutaneous and Metastatic Tumors Accompanied by Autoimmune Depigmentation

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van Elsas, Andrea; Hurwitz, Arthur A.; Allison, James P.

1999-01-01

We examined the effectiveness of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) blockade, alone or in combination with a granulocyte/macrophage colony-stimulating factor (GM-CSF)–expressing tumor cell vaccine, on rejection of the highly tumorigenic, poorly immunogenic murine melanoma B16-BL6. Recently established tumors could be eradicated in 80% (68/85) of the cases using combination treatment, whereas each treatment by itself showed little or no effect. Tumor rejection was dependent on CD8+ and NK1.1+ cells but occurred irrespective of the presence of CD4+ T cells. Mice surviving a primary challenge rejected a secondary challenge with B16-BL6 or the parental B16-F0 line. The same treatment regimen was found to be therapeutically effective against outgrowth of preestablished B16-F10 lung metastases, inducing long-term survival. Of all mice surviving B16-BL6 or B16-F10 tumors after combination treatment, 56% (38/68) developed depigmentation, starting at the site of vaccination or challenge and in most cases progressing to distant locations. Depigmentation was found to occur in CD4-depleted mice, strongly suggesting that the effect was mediated by CTLs. This study shows that CTLA-4 blockade provides a powerful tool to enhance T cell activation and memory against a poorly immunogenic spontaneous murine tumor and that this may involve recruitment of autoreactive T cells. PMID:10430624

Sonodynamically-induced cytotoxicity by rose bengal derivative and microbubbles in isolated sarcoma 180 cells

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Sugita, Nami; Hosokawa, Mami; Sunaga, Naoki; Iwase, Yumiko; Yumita, Nagahiko; Ikeda, Toshihiko; Umemura, Shin-ichiro

2015-07-01

It is known that the combination of ultrasound and sonodynamic sensitizer (SDS) is effective in noninvasive tumor treatment, referred to as sonodynamic therapy (SDT). Microbubbles have been used in ultrasound therapy as well. The purpose of this paper is to clarify the effect of microbubbles on SDT. Sarcoma 180 cells were suspended in air-saturated phosphate-buffered saline and exposed to ultrasound with the SDS rose bengal derivative (RBD) in standing wave mode in the presence and absence of microbubbles [sonazoid (SZ)]. The ultrasonically induced cytotoxicity with RBD and SZ was about 20 times higher than without either, and about 80% of the SZ microbubbles were destructed by ultrasonic exposure in as short as five seconds. Since microbubbles induce significant cytotoxicity even with short duration, low intensity ultrasound, the application of microbubbles in SDT shows promise in anti-tumor treatment.

Brucella infection inhibits macrophages apoptosis via Nedd4-dependent degradation of calpain2.

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Cui, Guimei; Wei, Pan; Zhao, Yuxi; Guan, Zhenhong; Yang, Li; Sun, Wanchun; Wang, Shuangxi; Peng, Qisheng

2014-11-07

The calcium-dependent protease calpain2 is involved in macrophages apoptosis. Brucella infection-induced up-regulation of intracellular calcium level is an essential factor for the intracellular survival of Brucella within macrophages. Here, we hypothesize that calcium-dependent E3 ubiquitin ligase Nedd4 ubiquitinates calpain2 and inhibits Brucella infection-induced macrophage apoptosis via degradation of calpain2.Our results reveal that Brucella infection induces increases in Nedd4 activity in an intracellular calcium dependent manner. Furthermore, Brucella infection-induced degradation of calpain2 is mediated by Nedd4 ubiquitination of calpain2. Brucella infection-induced calpain2 degradation inhibited macrophages apoptosis. Treatment of Brucella infected macrophages with calcium chelator BAPTA or Nedd4 knock-down decreased Nedd4 activity, prevented calpain2 degradation, and resulted in macrophages apoptosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Lewis Lung Cancer Cells Promote SIGNR1(CD209b)-Mediated Macrophages Polarization Induced by IL-4 to Facilitate Immune Evasion.

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Yan, Xiaolong; Li, Wenhai; Pan, Lei; Fu, Enqing; Xie, Yonghong; Chen, Min; Mu, Deguang

2016-05-01

Tumor-associated macrophages are a prominent component of lung cancer and contribute to tumor progression by facilitating the immune evasion of cancer cells. DC-SIGN (CD209) assists in the immune evasion of a broad spectrum of pathogens and neoplasms by inhibiting the maturation of DCs and subsequent cytokines production. However, the expression of DC-SIGN in macrophages and its role in mediating immune evasion in lung cancer and the underlying mechanism remain unclear. Our study aimed to identify the immunosuppressive role of SIGNR1 in murine macrophage differentiation and lung cancer progression. We found that SIGNR1-positive RAW264.7 macrophages were enriched in mixed cultures with Lewis lung cancer cells (LLC) (ratio of RAW 264.7 to LLC being 1:1) after stimulation with IL-4. Moreover, LLC-educated macrophages exhibited significantly higher levels of IL-10 but lower IL-12 in response to IL-4 treatment as determined by RT-PCR and ELISA. However, inhibition of SIGNR1 markedly hampered the production of IL-10, indicating that SIGNR1 was indispensable for IL-4+LLC induced macrophage polarization towards the M2 subtype. Furthermore, polarized M2 cells immersed in a tumor microenvironment promoted the migration of LLCs, as measured by transwell assays, but migration was suppressed after blockade of SIGNR1 using CD209b antibody. In addition, IL-4+LLC-educated macrophages reduced the proliferation of the activated T cells and reduced IFN-γ-mediated Th1 response in T cells, while SIGNR1 inhibition rescued Th1 cell functions. In conclusion, murine SIGNR1 expressed in LLC-educated macrophages appears to mediate IL-4-induced RAW264.7 macrophage polarization and thus facilitate lung cancer evasion. © 2015 Wiley Periodicals, Inc.

Wld(S reduces paraquat-induced cytotoxicity via SIRT1 in non-neuronal cells by attenuating the depletion of NAD.

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Qiujing Yu

Full Text Available Wld(S is a fusion protein with NAD synthesis activity, and has been reported to protect axonal and synaptic compartments of neurons from various mechanical, genetic and chemical insults. However, whether Wld(S can protect non-neuronal cells against toxic chemicals is largely unknown. Here we found that Wld(S significantly reduced the cytotoxicity of bipyridylium herbicides paraquat and diquat in mouse embryonic fibroblasts, but had no effect on the cytotoxicity induced by chromium (VI, hydrogen peroxide, etoposide, tunicamycin or brefeldin A. Wld(S also slowed down the death of mice induced by intraperitoneal injection of paraquat. Further studies demonstrated that Wld(S markedly attenuated mitochondrial injury including disruption of mitochondrial membrane potential, structural damage and decline of ATP induced by paraquat. Disruption of the NAD synthesis activity of Wld(S by an H112A or F116S point mutation resulted in loss of its protective function against paraquat-induced cell death. Furthermore, Wld(S delayed the decrease of intracellular NAD levels induced by paraquat. Similarly, treatment with NAD or its precursor nicotinamide mononucleotide attenuated paraquat-induced cytotoxicity and decline of ATP and NAD levels. In addition, we showed that SIRT1 was required for both exogenous NAD and Wld(S-mediated cellular protection against paraquat. These findings suggest that NAD and SIRT1 mediate the protective function of Wld(S against the cytotoxicity induced by paraquat, which provides new clues for the mechanisms underlying the protective function of Wld(S in both neuronal and non-neuronal cells, and implies that attenuation of NAD depletion may be effective to alleviate paraquat poisoning.

Cytotoxicity and regenerative proliferation as the mode of action for diuron-induced urothelial carcinogenesis in the rat.

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da Rocha, Mitscheli S; Nascimento, Merielen G; Cardoso, Ana Paula F; de Lima, Patrícia L A; Zelandi, Edneia A; de Camargo, João Lauro V; de Oliveira, Maria Luiza C S

2010-01-01

Diuron, a substituted urea herbicide, is carcinogenic to the urinary bladder of rats at high dietary levels. Its proposed carcinogenic mode of action (MOA) includes urothelial cytotoxicity and necrosis followed by regenerative cell proliferation and sustained urothelial hyperplasia. Cytotoxicity could be induced either by urinary solids or by chemical toxicity by diuron and/or metabolites excreted in the urine. Diuron was not genotoxic in a previous single-cell gel (comet) assay, but possible cross-linking activity remained to be evaluated. The present study explored the MOA of diuron and the effect of urinary acidification on the development of urothelial lesions. Male Wistar rats were fed diuron (2500 ppm, about 130 mg/kg of body weight) either with or without NH(4)Cl 10,000 ppm to acidify the urine. Reversibility of urothelial changes was also examined. The animals were euthanized after 15, 25, or 30 weeks. Diuron-fed rats had urinary amorphous precipitate and magnesium ammonium phosphate crystals similar to control animals. Groups treated with diuron + NH(4)Cl showed decreased urinary pH and reduced amounts of urinary crystals and precipitate. Urothelial necrosis and simple hyperplasia were observed by light microscopy and scanning electron microscopy both in diuron- and in diuron + NH(4)Cl-treated groups. Cytotoxicity and proliferative changes were mostly reversible. A modified comet assay developed in vitro with Chinese hamster ovary cells showed that diuron did not induce DNA cross-links. These data suggest that cytotoxicity with consequent regenerative cell proliferation is the predominant MOA for diuron rat urothelial carcinogenesis, the cytotoxicity being chemically induced and not due to urinary solids.

Activation/proliferation and apoptosis of bystander goat lymphocytes induced by a macrophage-tropic chimeric caprine arthritis encephalitis virus expressing SIV Nef

International Nuclear Information System (INIS)

Bouzar, Baya Amel; Rea, Angela; Hoc-Villet, Stephanie; Garnier, Celine; Guiguen, Francois; Jin Yuhuai; Narayan, Opendra; Chebloune, Yahia

2007-01-01

Caprine arthritis encephalitis virus (CAEV) is the natural lentivirus of goats, well known for its tropism for macrophages and its inability to cause infection in lymphocytes. The viral genome lacks nef, tat, vpu and vpx coding sequences. To test the hypothesis that when nef is expressed by the viral genome, the virus became toxic for lymphocytes during replication in macrophages, we inserted the SIVsmm PBj14 nef coding sequences into the genome of CAEV thereby generating CAEV-nef. This recombinant virus is not infectious for lymphocytes but is fully replication competent in goat macrophages in which it constitutively expresses the SIV Nef. We found that goat lymphocytes cocultured with CAEV-nef-infected macrophages became activated, showing increased expression of the interleukin-2 receptor (IL-2R). Activation correlated with increased proliferation of the cells. Interestingly, a dual effect in terms of apoptosis regulation was observed in exposed goat lymphocytes. Nef was found first to induce a protection of lymphocytes from apoptosis during the first few days following exposure to infected macrophages, but later it induced increased apoptosis in the activated lymphocytes. This new recombinant virus provides a model to study the functions of Nef in the context of infection of macrophages, but in absence of infection of T lymphocytes and brings new insights into the biological effects of Nef on lymphocytes

TNF-alpha, produced by feline infectious peritonitis virus (FIPV)-infected macrophages, upregulates expression of type II FIPV receptor feline aminopeptidase N in feline macrophages.

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Takano, Tomomi; Hohdatsu, Tsutomu; Toda, Ayako; Tanabe, Maki; Koyama, Hiroyuki

2007-07-20

The pathogenicity of feline infectious peritonitis virus (FIPV) is known to depend on macrophage tropism, and this macrophage infection is enhanced by mediation via anti-S antibody (antibody-dependent enhancement, ADE). In this study, we found that TNF-alpha production was increased with viral replication in macrophages inoculated with a mixture of FIPV and anti-S antibody, and demonstrated that this culture supernatant had feline PBMC apoptosis-inducing activity. We also demonstrated that the expression level of the FIPV virus receptor, feline aminopeptidase N (fAPN), was increased in macrophages of FIP cats. For upregulation of TNF-alpha and fAPN in macrophages, viral replication in macrophages is necessary, and their expressions were increased by ADE of FIPV infection. It was demonstrated that a heat-resistant fAPN-inducing factor was present in the culture supernatant of FIPV-infected macrophages, and this factor was TNF-alpha: fAPN expression was upregulated in recombinant feline TNF-alpha-treated macrophages, and FIPV infectivity was increased in these macrophages. These findings suggested that FIPV replication in macrophages increases TNF-alpha production in macrophages, and the produced TNF-alpha acts and upregulates fAPN expression, increasing FIPV sensitivity.

The Evaluation of Melatonin Effect Against The Early Effect of Ionizing Radiation Induced Lung Injury

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raziyeh tahamtan

2014-06-01

Results: the results indicated that radiation increases the collapse and fibrosis and cause the abundance of macrophage compared to control group (p<0.05. Oral administration of melatonin before radiation therapy significantly increased the lymphocyte and macrophage frequency (p<0.001 and decreased the RBC (p<0.05 frequency compared to the radiation group. Conclusion: According to the results, melatonin can prevent early damages in irradiated lungs. Free radicals cause cytotoxicity and melatonin can directly decrease the radiation induced cell damages by converting the free radicals to non- toxic compounds and also through the activation of the major antioxidant enzymes.

Morphologic categorization of cell death induced by mild hyperthermia and comparison with death induced by ionizing radiation and cytotoxic drugs

International Nuclear Information System (INIS)

Allan, D.J.; Harmon, B.V.

1986-01-01

This paper presents a summary of the morphological categorization of cell death, results of two in vivo studies on the cell death induced by mild hyperthermia in rat small intestine and mouse mastocytoma, and a comparison of the cell death induced by hyperthermia, radiation and cytotoxic drugs. Two distinct forms of cell death, apoptosis and necrosis, can be recognized on morphologic grounds. Apoptosis appears to be a process of active cellular self-destruction to which a biologically meaningful role can usually be attributed, whereas necrosis is a passive degenerative phenomenon that results from irreversible cellular injury. Light and transmission electron microscopic studies showed that lower body hyperthermia (43 degrees C for 30 min) induced only apoptosis of intestinal epithelial cells, and of lymphocytes, plasma cells, and eosinophils. In the mastocytoma, hyperthermia (43 degrees C for 15 min) produced widespread tumor necrosis and also enhanced apoptosis of tumor cells. Ionizing radiation and cytotoxic drugs are also known to induce apoptosis in a variety of tissues. It is attractive to speculate that DNA damage by each agent is the common event which triggers the same process of active cellular self-destruction that characteristically effects selective cell deletion in normal tissue homeostasis

FC-99 ameliorates sepsis-induced liver dysfunction by modulating monocyte/macrophage differentiation via Let-7a related monocytes apoptosis.

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Zhao, Yarong; Zhu, Haiyan; Wang, Haining; Ding, Liang; Xu, Lizhi; Chen, Dai; Shen, Sunan; Hou, Yayi; Dou, Huan

2018-03-13

The liver is a vital target for sepsis-related injury, leading to inflammatory pathogenesis, multiple organ dysfunction and high mortality rates. Monocyte-derived macrophage transformations are key events in hepatic inflammation. N 1 -[(4-methoxy)methyl]-4-methyl-1,2-benzenediamine (FC-99) previously displayed therapeutic potential on experimental sepsis. However, the underlying mechanism of this protective effect is still not clear. FC-99 treatment attenuated the liver dysfunction in septic mice that was accompanied with reduced numbers of pro-inflammatory Ly6C hi monocytes in the peripheral blood and CD11b + F4/80 lo monocyte-derived macrophages in the liver. These effects were attributed to the FC-99-induced apoptosis of CD11b + cells. In PMA-differentiated THP-1 cells, FC-99 repressed the expression of CD11b, CD14 and caspase3 and resulted in a high proportion of Annexin V + cells. Moreover, let-7a-5p expression was abrogated upon CLP stimulation in vivo , whereas it was restored by FC-99 treatment. TargetScan analysis and luciferase assays indicated that the anti-apoptotic protein BCL-XL was targeted by let-7a-5p. BCL-XL was inhibited by FC-99 in order to induce monocyte apoptosis, leading to the impaired monocyte-to-macrophage differentiation. Murine acute liver failure was generated by caecal ligation puncture surgery after FC-99 administration; Blood samples and liver tissues were collected to determine the monocyte/macrophage subsets and the induction of apoptosis. Human acute monocytic leukemia cell line (THP-1) cells were pretreated with FC-99 followed by phorbol-12-myristate-13-acetate (PMA) stimulation, in order to induce monocyte-to-macrophage differentiation. The target of FC-99 and the mechanistic analyses were conducted by microarrays, qRT-PCR validation, TargetScan algorithms and a luciferase report assay. FC-99 exhibits potential therapeutic effects on CLP-induced liver dysfunction by restoring let-7a-5p levels.

Protective effect of kombucha mushroom (KM) tea on phenol-induced cytotoxicity in albino mice.

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Yapar, Kursad; Cavusoglu, Kultigin; Oruc, Ertan; Yalcin, Emine

2010-09-01

The present study was carried out to evaluate the protective role of kombucha mushroom (KM) tea on cytotoxicity induced by phenol (PHE) in mice. We used weight gain and micronucleus (MN) frequency as indicators of cytotoxicity and supported these parameters with pathological findings. The animals were randomly divided into seven groups: (Group I) only tap water (Group II) 1000 microl kg(-1) b. wt KM-tea, (Group III) 35 mg kg(-1) body wt. PHE (Group IV) 35 mg kg(-1) body wt. PHE + 250 microl kg(-1) b. wt KM-tea (Group V) 35 mg kg(-1) b. wt PHE + 500 microl kg(-1) b. wt KM-tea (Group VI) 35 mg kg(-1) b. wt PHE + 750 microl kg(-1) b. wt KM-tea, (Group VII) 35 mg kg(-1) b. wt PHE + 1000 microl kg(-1) b. wt KM-tea, for 20 consecutive days by oral gavage. The results indicated that all KM-tea supplemented mice showed a lower MN frequency than erythrocytes in only PHE-treated group. There was an observable regression on account of lesions in tissues of mice supplemented with different doses of KM-tea in histopathological observations. In conclusion, the KM-tea supplementation decreases cytotoxicity induced by PHE and its protective role is dose-dependent.

Monocytes/Macrophages Control Resolution of Transient Inflammatory Pain

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Willemen, Hanneke L. D. M.; Eijkelkamp, Niels; Carbajal, Anibal Garza; Wang, Huijing; Mack, Matthias; Zijlstra, Jitske; Heijnen, Cobi J.; Kavelaars, Annemieke

2014-01-01

Insights into mechanisms governing resolution of inflammatory pain are of great importance for many chronic pain–associated diseases. Here we investigate the role of macrophages/monocytes and the anti-inflammatory cytokine interleukin-10 (IL-10) in the resolution of transient inflammatory pain. Depletion of mice from peripheral monocytes/macrophages delayed resolution of intraplantar IL-1β- and carrageenan-induced inflammatory hyperalgesia from 1 to 3 days to >1 week. Intrathecal administration of a neutralizing IL-10 antibody also markedly delayed resolution of IL-1β- and carrageenan-induced inflammatory hyperalgesia. Recently, we showed that IL-1β- and carrageenan-induced hyperalgesia is significantly prolonged in LysM-GRK2+/− mice, which have reduced levels of G-protein-coupled receptor kinase 2 (GRK2) in LysM+ myeloid cells. Here we show that adoptive transfer of wild-type, but not of GRK2+/−, bone marrow-derived monocytes normalizes the resolution of IL-1β-induced hyperalgesia in LysM-GRK2+/− mice. Adoptive transfer of IL-10−/− bone marrow-derived monocytes failed to normalize the duration of IL-1β-induced hyperalgesia in LysM-GRK2+/− mice. Mechanistically, we show that GRK2+/− macrophages produce less IL-10 in vitro. In addition, intrathecal IL-10 administration attenuated IL-1β-induced hyperalgesia in LysM-GRK2+/− mice, whereas it had no effect in wild-type mice. Our data uncover a key role for monocytes/macrophages in promoting resolution of inflammatory hyperalgesia via a mechanism dependent on IL-10 signaling in dorsal root ganglia. Perspective We show that IL-10-producing monocytes/macrophages promote resolution of transient inflammatory hyperalgesia. Additionally, we show that reduced monocyte/macrophage GRK2 impairs resolution of hyperalgesia and reduces IL-10 production. We propose that low GRK2 expression and/or impaired IL-10 production by monocytes/macrophages represent peripheral biomarkers for the risk of developing

Exosomes from M1-Polarized Macrophages Potentiate the Cancer Vaccine by Creating a Pro-inflammatory Microenvironment in the Lymph Node.

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Cheng, Lifang; Wang, Yuhua; Huang, Leaf

2017-07-05

Exosomes are small membrane-bound vesicular particles generated by most cells for intercellular communication and regulation. During biogenesis, specific lipids, RNAs, proteins, and carbohydrates are enriched and packaged into the vesicles so that the exosomal contents reflect not only the source but also the physiological conditions of the parental cells. These exosomes transport materials or signals to the target cells for diverse physiological purposes. Our study focused on the exosomes derived from M1-polarized, proinflammatory macrophages for the possibility of using M1 exosomes as an immunopotentiator for a cancer vaccine. The M1 exosomes displayed a tropism toward lymph nodes after subcutaneous injection, primarily taken up by the local macrophages and dendritic cells, and they induced the release of a pool of Th1 cytokines. We found that M1, but not M2, exosomes enhanced activity of lipid calcium phosphate (LCP) nanoparticle-encapsulated Trp2 vaccine, and they induced a stronger antigen-specific cytotoxic T cell response. The M1 exosomes proved to be a more potent immunopotentiator than CpG oligonucleotide when used with LCP nanoparticle vaccine in a melanoma growth inhibition study. Thus, our study indicated that exosomes derived from M1-polarized macrophages could be used as a vaccine adjuvant. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Islet cytotoxicity of interleukin 1. Influence of culture conditions and islet donor characteristics

DEFF Research Database (Denmark)

Mandrup-Poulsen, T; Spinas, G A; Prowse, S J

1987-01-01

We recently demonstrated that the macrophage product interleukin 1 (IL-1) is cytotoxic to isolated pancreatic islets and hypothesized that IL-1 is responsible for beta-cell destruction in insulin-dependent diabetes mellitus (IDDM). We studied whether the variation in IDDM preponderance with age, ...

Interferon-β gene transfer induces a strong cytotoxic bystander effect on melanoma cells.

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Rossi, Úrsula A; Gil-Cardeza, María L; Villaverde, Marcela S; Finocchiaro, Liliana M E; Glikin, Gerardo C

2015-05-01

A local gene therapy scheme for the delivery of type I interferons could be an alternative for the treatment of melanoma. We evaluated the cytotoxic effects of interferon-β (IFNβ) gene lipofection on tumor cell lines derived from three human cutaneous and four canine mucosal melanomas. The cytotoxicity of human IFNβ gene lipofection resulted higher or equivalent to that of the corresponding addition of the recombinant protein (rhIFNβ) to human cells. IFNβ gene lipofection was not cytotoxic for only one canine melanoma cell line. When cultured as monolayers, three human and three canine IFNβ-lipofected melanoma cell lines displayed a remarkable bystander effect. As spheroids, the same six cell lines were sensitive to IFNβ gene transfer, two displaying a significant multicell resistance phenotype. The effects of conditioned IFNβ-lipofected canine melanoma cell culture media suggested the release of at least one soluble thermolabile cytotoxic factor that could not be detected in human melanoma cells. By using a secretion signal-free truncated human IFNβ, we showed that its intracellular expression was enough to induce cytotoxicity in two human melanoma cell lines. The lower cytoplasmatic levels of reactive oxygen species detected after intracellular IFNβ expression could be related to the resistance displayed by one human melanoma cell line. As IFNβ gene transfer was effective against most of the assayed melanomas in a way not limited by relatively low lipofection efficiencies, the clinical potential of this approach is strongly supported. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Size-dependent cytotoxicity of Fe3O4 nanoparticles induced by biphasic regulation of oxidative stress in different human hepatoma cells

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Xie Y

2016-07-01

Full Text Available Yuexia Xie,1,2,* Dejun Liu,3,* Chenlei Cai,1,* Xiaojing Chen,1 Yan Zhou,1 Liangliang Wu,1 Yongwei Sun,3 Huili Dai,1,2 Xianming Kong,1,2 Peifeng Liu1,2 1Central Laboratory, 2State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, 3Department of Biliary-Pancreatic Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: The application of Fe3O4 nanoparticles (NPs has made great progress in the diagnosis of disease and in the drug delivery system for cancer therapy, but the relative mecha­nisms of potential toxicity induced by Fe3O4 have not kept pace with its development in the application, which has hampered its further clinical application. In this article, we used two kinds of human hepatoma cell lines, SK-Hep-1 and Hep3B, to investigate the cytotoxic effects and the involved mechanisms of small Fe3O4 NPs with different diameters (6 nm, 9 nm, and 14 nm. Results showed that the size of NPs effectively influences the cytotoxicity of hepatoma cells: 6 nm Fe3O4 NPs exhibited negligible cytotoxicity and 9 nm Fe3O4 NPs affected cytotoxicity via cellular mitochondrial dysfunction and by inducing necrosis mediated through the mitochondria-dependent intracellular reactive oxygen species generation. Meanwhile, 14 nm Fe3O4 NPs induced cytotoxicity by impairing the integrity of plasma membrane and promoting massive lactate dehydrogenase leakage. These results explain the detailed mechanism of different diameters of small Fe3O4 NPs-induced cytotoxicity. We anticipate that this study will provide different insights into the cytotoxicity mechanism of Fe3O4 NPs, so as to make them safer to use in clinical application. Keywords: hepatoma cells, nanoparticles, cytotoxicity, mechanism, oxidative stress

Uranyl nitrate-exposed rat alveolar macrophages cell death: Influence of superoxide anion and TNF α mediators

International Nuclear Information System (INIS)

Orona, N.S.; Tasat, D.R.

2012-01-01

Uranium compounds are widely used in the nuclear fuel cycle, military and many other diverse industrial processes. Health risks associated with uranium exposure include nephrotoxicity, cancer, respiratory, and immune disorders. Macrophages present in body tissues are the main cell type involved in the internalization of uranium particles. To better understand the pathological effects associated with depleted uranium (DU) inhalation, we examined the metabolic activity, phagocytosis, genotoxicity and inflammation on DU-exposed rat alveolar macrophages (12.5–200 μM). Stability and dissolution of DU could differ depending on the dissolvent and in turn alter its biological action. We dissolved DU in sodium bicarbonate (NaHCO 3 100 mM) and in what we consider a more physiological vehicle resembling human internal media: sodium chloride (NaCl 0.9%). We demonstrate that uranyl nitrate in NaCl solubilizes, enters the cell, and elicits its cytotoxic effect similarly to when it is diluted in NaHCO 3 . We show that irrespective of the dissolvent employed, uranyl nitrate impairs cell metabolism, and at low doses induces both phagocytosis and generation of superoxide anion (O 2 − ). At high doses it provokes the secretion of TNFα and through all the range of doses tested, apoptosis. We herein suggest that at DU low doses O 2 − may act as the principal mediator of DNA damage while at higher doses the signaling pathway mediated by O 2 − may be blocked, prevailing damage to DNA by the TNFα route. The study of macrophage functions after uranyl nitrate treatment could provide insights into the pathophysiology of uranium‐related diseases. -- Highlights: ► Uranyl nitrate effect on cultured macrophages is linked to the doses and independent of its solubility. ► At low doses uranyl nitrate induces generation of superoxide anion. ► At high doses uranyl nitrate provokes secretion of TNFα. ► Uranyl nitrate induces apoptosis through all the range of doses tested.

Uranyl nitrate-exposed rat alveolar macrophages cell death: Influence of superoxide anion and TNF α mediators

Energy Technology Data Exchange (ETDEWEB)

Orona, N.S. [School of Science and Technology, National University of General Martín, Avda Gral Paz 5445 (1650) San Martín, Buenos Aires (Argentina); Tasat, D.R., E-mail: deborah.tasat@unsam.edu.ar [School of Science and Technology, National University of General Martín, Avda Gral Paz 5445 (1650) San Martín, Buenos Aires (Argentina); School of Dentistry, University of Buenos Aires, M. T. de Alvear 2142 (1122), Buenos Aires (Argentina)

2012-06-15

Uranium compounds are widely used in the nuclear fuel cycle, military and many other diverse industrial processes. Health risks associated with uranium exposure include nephrotoxicity, cancer, respiratory, and immune disorders. Macrophages present in body tissues are the main cell type involved in the internalization of uranium particles. To better understand the pathological effects associated with depleted uranium (DU) inhalation, we examined the metabolic activity, phagocytosis, genotoxicity and inflammation on DU-exposed rat alveolar macrophages (12.5–200 μM). Stability and dissolution of DU could differ depending on the dissolvent and in turn alter its biological action. We dissolved DU in sodium bicarbonate (NaHCO{sub 3} 100 mM) and in what we consider a more physiological vehicle resembling human internal media: sodium chloride (NaCl 0.9%). We demonstrate that uranyl nitrate in NaCl solubilizes, enters the cell, and elicits its cytotoxic effect similarly to when it is diluted in NaHCO{sub 3}. We show that irrespective of the dissolvent employed, uranyl nitrate impairs cell metabolism, and at low doses induces both phagocytosis and generation of superoxide anion (O{sub 2}{sup −}). At high doses it provokes the secretion of TNFα and through all the range of doses tested, apoptosis. We herein suggest that at DU low doses O{sub 2}{sup −} may act as the principal mediator of DNA damage while at higher doses the signaling pathway mediated by O{sub 2}{sup −} may be blocked, prevailing damage to DNA by the TNFα route. The study of macrophage functions after uranyl nitrate treatment could provide insights into the pathophysiology of uranium‐related diseases. -- Highlights: ► Uranyl nitrate effect on cultured macrophages is linked to the doses and independent of its solubility. ► At low doses uranyl nitrate induces generation of superoxide anion. ► At high doses uranyl nitrate provokes secretion of TNFα. ► Uranyl nitrate induces apoptosis through

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

Science.gov (United States)

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

2016-01-01

Metabolism and immune responses have been shown to be closely linked and as our understanding increases, so do the intricacies of the level of linkage. NAD+ has previously been shown to regulate tumour necrosis factor-α (TNF-α) synthesis and TNF-α has been shown to regulate NAD+ homoeostasis providing a link between a pro-inflammatory response and redox status. In the present study, we have used THP-1 differentiation into pro- (M1-like) and anti- (M2-like) inflammatory macrophage subset models to investigate this link further. Pro- and anti-inflammatory macrophages showed different resting NAD+ levels and expression levels of NAD+ homoeostasis enzymes. Challenge with bacterial lipopolysaccharide, a pro-inflammatory stimulus for macrophages, caused a large, biphasic and transient increase in NAD+ levels in pro- but not anti-inflammatory macrophages that were correlated with TNF-α release and inhibition of certain NAD+ synthesis pathways blocked TNF-α release. Lipopolysaccharide stimulation also caused changes in mRNA levels of some NAD+ homoeostasis enzymes in M1-like cells. Surprisingly, despite M2-like cells not releasing TNF-α or changing NAD+ levels in response to lipopolysaccharide, they showed similar mRNA changes compared with M1-like cells. These data further strengthen the link between pro-inflammatory responses in macrophages and NAD+. The agonist-induced rise in NAD+ shows striking parallels to well-known second messengers and raises the possibility that NAD+ is acting in a similar manner in this model. PMID:26764408

Extracts of human atherosclerotic lesions modify LDL inducing enhanced macrophage uptake

International Nuclear Information System (INIS)

Hoff, H.F.; O'Neill, J.

1986-01-01

Both an LDL-like fraction isolated from human aortic plaques and LDL incubated with cultured aortic endothelial or smooth muscle cells have been shown to be internalized by macrophages in vitro in an unregulated fashion leading to foam cell formation. Lipid peroxidation induced by free radicals released from cells was shown to be responsible for cell-modified LDL. The authors incubated LDL with a supernatant fraction of leached, i.e. non-homogenized, extracts of aortic plaques for one hour at 37 0 C, to determine whether extracellular components present in arteries were also capable of modifying LDL. Extract-treated LDL showed the following changes relative to untreated LDL: 1) increased electrophretic mobility, 2) altered pattern of B-100 on SDS-PAGE, i.e. presence of a doublet with higher M/sub r/ than B-100, and 3) enhanced uptake by cultured mouse peritoneal macrophages as measured by increased degradation of 125 I-LDL, and increased stimulation of cholesterol esterification using 14 C-oleate. Extracts from homogenized plaques and grossly normal intima induced similar changes. The modification was tissue specific in that extracts of arteries but not of liver, muscle or skin modified LDL. Protease degradation of LDL during incubation was probably not responsible since inhibitors did not prevent modification. It is possible that products of lipid peroxidation present in extracellular lipid of arteries may propagate free radicals or be incorporated into LDL, leading to modifications similar to those found in cell-modified LDL

Cervical Cancer Cell Supernatants Induce a Phenotypic Switch from U937-Derived Macrophage-Activated M1 State into M2-Like Suppressor Phenotype with Change in Toll-Like Receptor Profile

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Karina Sánchez-Reyes

2014-01-01

Full Text Available Cervical cancer (CC is the second most common cancer among women worldwide. Infection with human papillomavirus (HPV is the main risk factor for developing CC. Macrophages are important immune effector cells; they can be differentiated into two phenotypes, identified as M1 (classically activated and M2 (alternatively activated. Macrophage polarization exerts profound effects on the Toll-like receptor (TLR profile. In this study, we evaluated whether the supernatant of human CC cells HeLa, SiHa, and C-33A induces a shift of M1 macrophage toward M2 macrophage in U937-derived macrophages. Results. The results showed that soluble factors secreted by CC cells induce a change in the immunophenotype of macrophages from macrophage M1 into macrophage M2. U937-derived macrophages M1 released proinflammatory cytokines and nitric oxide; however, when these cells were treated with the supernatant of CC cell lines, we observed a turnover of M1 toward M2. These cells increased CD163 and IL-10 expression. The expression of TLR-3, -7, and -9 is increased when the macrophages were treated with the supernatant of CC cells. Conclusions. Our result strongly suggests that CC cells may, through the secretion of soluble factors, induce a change of immunophenotype M1 into M2 macrophages.

Role of macrophage migration inhibitory factor (MIF in allergic and endotoxin-induced airway inflammation in mice

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M. Korsgren

2000-01-01

Full Text Available Macrophage migration inhibitory factor (MIF has recently been forwarded as a critical regulator of inflammatory conditions, and it has been hypothesized that MIF may have a role in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD. Hence, we examined effects of MIF immunoneutralization on the development of allergen-induced eosinophilic inflammation as well as on lipopolysaccaride (LPS-induced neutrophilic inflammation in lungs of mice. Anti-MIF serum validated with respect to MIF neutralizing capacity or normal rabbit serum (NRS was administered i.p. repeatedly during allergen aerosol exposure of ovalbumin (OVA-immunized mice in an established model of allergic asthma, or once before instillation of a minimal dose of LPS into the airways of mice, a tentative model of COPD. Anti-MIF treatment did not affect the induced lung tissue eosinophilia or the cellular composition of bronchoalveolar lavage fluid (BALF in the asthma model. Likewise, anti-MIF treatment did not affect the LPS-induced neutrophilia in lung tissue, BALF, or blood, nor did it reduce BALF levels of tumor necrosis factor-α (TNF-α and macrophage inflammatory protein–1 α (MIP–1 α. The present data suggest that MIF is not critically important for allergen-induced eosinophilic, and LPS-induced neutrophilic responses in lungs of mice. These findings do not support a role of MIF inhibition in the treatment of inflammatory respiratory diseases.

Susceptibility of bone marrow-derived macrophages to influenza virus infection is dependent on macrophage phenotype.

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Campbell, Gillian M; Nicol, Marlynne Q; Dransfield, Ian; Shaw, Darren J; Nash, Anthony A; Dutia, Bernadette M

2015-10-01

The role of the macrophage in influenza virus infection is complex. Macrophages are critical for resolution of influenza virus infections but implicated in morbidity and mortality in severe infections. They can be infected with influenza virus and consequently macrophage infection is likely to have an impact on the host immune response. Macrophages display a range of functional phenotypes, from the prototypical pro-inflammatory classically activated cell to alternatively activated anti-inflammatory macrophages involved in immune regulation and wound healing. We were interested in how macrophages of different phenotype respond to influenza virus infection and therefore studied the infection of bone marrow-derived macrophages (BMDMs) of classical and alternative phenotype in vitro. Our results show that alternatively activated macrophages are more readily infected and killed by the virus than classically activated. Classically activated BMDMs express the pro-inflammatory markers inducible nitric oxide synthase (iNOS) and TNF-α, and TNF-α expression was further upregulated following infection. Alternatively activated macrophages express Arginase-1 and CD206; however, following infection, expression of these markers was downregulated whilst expression of iNOS and TNF-α was upregulated. Thus, infection can override the anti-inflammatory state of alternatively activated macrophages. Importantly, however, this results in lower levels of pro-inflammatory markers than those produced by classically activated cells. Our results showed that macrophage phenotype affects the inflammatory macrophage response following infection, and indicated that modulating the macrophage phenotype may provide a route to develop novel strategies to prevent and treat influenza virus infection.

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

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Russe, Otto Quintus; Möser, Christine V.; Kynast, Katharina L.; King, Tanya S.; Olbrich, Katrin; Grösch, Sabine; Geisslinger, Gerd; Niederberger, Ellen

2014-01-01

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

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

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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.

A fibroblast/macrophage co-culture model to evaluate the biocompatibility of an electrospun Dextran/PLGA scaffold and its potential to induce inflammatory responses

International Nuclear Information System (INIS)

Pan Hui; Kantharia, Sarah; Jiang Hongliang; Chen Weiliam

2011-01-01

Fibroblasts and macrophages are the two major types of cells responding to implanted biomaterials. They play crucial roles in inflammatory responses, host-material interactions and tissue remodeling. However, the synergistic interactions of these two cell types with biomaterials are not fully understood. In this investigation, an in vitro fibroblast/macrophage co-culture system was utilized to examine the biocompatibility and the potential to induce inflammatory responses of an electrospun Dextran/PLGA scaffold. The scaffold did not affect the morphologies, attachments, proliferations and viabilities of both the fibroblasts and macrophages, cultured separately or together. Moreover, it only activated a small subset of the macrophages implicating a low potential to induce either severe acute or chronic inflammatory response. Additionally, fibroblasts played a role in prolonging macrophage activation in the presence of the scaffolds. Using antibody arrays, IL-10, SDF-1, MIP-1 gamma and RANTES were found to be up-regulated when the cells were incubated with the scaffolds. The results of subdermal implantation of the Dextran/PLGA scaffolds confirmed its biocompatibility and low inflammatory potential.

A fibroblast/macrophage co-culture model to evaluate the biocompatibility of an electrospun Dextran/PLGA scaffold and its potential to induce inflammatory responses

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Pan Hui; Kantharia, Sarah [Department of Biomedical Engineering, State University of New York-Stony Brook, Stony Brook, NY 11794-2580 (United States); Jiang Hongliang [Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027 (China); Chen Weiliam, E-mail: weiliam.chen@nyumc.org [Division of Wound Healing and Regenerative Medicine, Department of Surgery, New York University School of Medicine, New York, NY 10016 (United States)

2011-12-15

Fibroblasts and macrophages are the two major types of cells responding to implanted biomaterials. They play crucial roles in inflammatory responses, host-material interactions and tissue remodeling. However, the synergistic interactions of these two cell types with biomaterials are not fully understood. In this investigation, an in vitro fibroblast/macrophage co-culture system was utilized to examine the biocompatibility and the potential to induce inflammatory responses of an electrospun Dextran/PLGA scaffold. The scaffold did not affect the morphologies, attachments, proliferations and viabilities of both the fibroblasts and macrophages, cultured separately or together. Moreover, it only activated a small subset of the macrophages implicating a low potential to induce either severe acute or chronic inflammatory response. Additionally, fibroblasts played a role in prolonging macrophage activation in the presence of the scaffolds. Using antibody arrays, IL-10, SDF-1, MIP-1 gamma and RANTES were found to be up-regulated when the cells were incubated with the scaffolds. The results of subdermal implantation of the Dextran/PLGA scaffolds confirmed its biocompatibility and low inflammatory potential.

Rare pneumoconiosis induced by long-term amorphous silica exposure: the histological characteristics and expression of cyclooxygenase-2 as an antifibrogenic mediator in macrophages.

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Kumasaka, Toshio; Akaike, Yasushi; Nakamura, Osamu; Yamazaki, Kazuma; Moriyama, Hiroshi; Takemura, Tamiko

2011-11-01

Pneumoconiosis induced by non-crystalline silica is considered rare, although silicosis resulting from contact with crystalline silica is a well-known hazard associated with progressive pulmonary fibrosis. Here we describe a patient with pneumoconiosis induced by diatomaceous earth composed of amorphous silica detected by two-dimensional imaging of chemical elements. The histology revealed that the disease was characterized by a granulomatous reaction in the lung. A large number of macrophages laden with yellow and black pigments accumulated in alveolar spaces and were incorporated into the interstitial sites. Bronchiolar walls were destroyed by palisade macrophages, suggesting airflow obstruction. Packed macrophages adhering to and covering the denuded interstitium indicated that macrophages might be incorporated into pulmonary interstitium in this fashion. Immunohistochemistry showed that cyclooxygenase-2, an antifibrogenic mediator, was intensely expressed in the macrophages compared with macrophages in control lungs. No birefringent material was found in the tissues. When two-dimensional analysis of chemical elements was performed using an electron probe microanalyzer with a wavelength-dispersive spectrometer, the resultant fine mapping of silicon and oxygen on the tissue indicated that the pigments phagocytosed by macrophages corresponded to amorphous silica. In conclusion, two-dimensional analysis of elements is very useful for pathologists in correlating the presence of chemical elements with histological changes. © 2011 The Authors. Pathology International © 2011 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.

Human native lipoprotein-induced de novo DNA methylation is associated with repression of inflammatory genes in THP-1 macrophages.

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Rangel-Salazar, Rubén; Wickström-Lindholm, Marie; Aguilar-Salinas, Carlos A; Alvarado-Caudillo, Yolanda; Døssing, Kristina B V; Esteller, Manel; Labourier, Emmanuel; Lund, Gertrud; Nielsen, Finn C; Rodríguez-Ríos, Dalia; Solís-Martínez, Martha O; Wrobel, Katarzyna; Wrobel, Kazimierz; Zaina, Silvio

2011-11-25

We previously showed that a VLDL- and LDL-rich mix of human native lipoproteins induces a set of repressive epigenetic marks, i.e. de novo DNA methylation, histone 4 hypoacetylation and histone 4 lysine 20 (H4K20) hypermethylation in THP-1 macrophages. Here, we: 1) ask what gene expression changes accompany these epigenetic responses; 2) test the involvement of candidate factors mediating the latter. We exploited genome expression arrays to identify target genes for lipoprotein-induced silencing, in addition to RNAi and expression studies to test the involvement of candidate mediating factors. The study was conducted in human THP-1 macrophages. Native lipoprotein-induced de novo DNA methylation was associated with a general repression of various critical genes for macrophage function, including pro-inflammatory genes. Lipoproteins showed differential effects on epigenetic marks, as de novo DNA methylation was induced by VLDL and to a lesser extent by LDL, but not by HDL, and VLDL induced H4K20 hypermethylation, while HDL caused H4 deacetylation. The analysis of candidate factors mediating VLDL-induced DNA hypermethylation revealed that this response was: 1) surprisingly, mediated exclusively by the canonical maintenance DNA methyltransferase DNMT1, and 2) independent of the Dicer/micro-RNA pathway. Our work provides novel insights into epigenetic gene regulation by native lipoproteins. Furthermore, we provide an example of DNMT1 acting as a de novo DNA methyltransferase independently of canonical de novo enzymes, and show proof of principle that de novo DNA methylation can occur independently of a functional Dicer/micro-RNA pathway in mammals.

Cell surface antigens of radiation leukemia virus-induced BALB/c leukemias defined by syngeneic cytotoxic T lymphocytes

International Nuclear Information System (INIS)

Kaneko, Yukio; Oettgen, H.F.; Obata, Yuichi; Nakayama, Eiichi.

1989-01-01

Two cell surface antigens of mouse leukemias were defined by BALB/c cytotoxic T lymphocytes (CTL) generated against syngeneic radiation leukemia virus (RadLV)-induced leukemia, BALBRV1 or BALBRVD. Hyperimmunization of BALB/c mice with irradiated leukemias followed by in vitro sensitization of primed spleen cells resulted in the generation of CTL with high killing activity. The specificity of CTL was examined by direct cytotoxicity assays and competitive inhibition assays. A shared cell surface antigen, designated as BALBRV1 antigen, was detected by BALB/c anti-BALBRV1 CTL. BALBRV1 antigen was expressed not only on RadLV-induced BALB/c leukemias except for BALBRVD, but also on spontaneous or X-ray-induced BALB/c leukemias, chemically-induced leukemias with the H-2 d haplotype and some chemically-induced BALB/c sarcomas. In contrast, a unique cell surface antigen, designated as BALBRVD antigen, was detected by BALB/c anti-BALBRVD CTL. BALBRVD antigen was expressed only on BALBRVD, but not on thirty-nine normal lymphoid or tumor cells. These two antigens could be distinguished from those previously defined on Friend, Moloney, Rauscher or Gross murine leukemia virus (MuLV) leukemias, or MuLV-related antigens. Both cytotoxic responses were blocked by antisera against H-2K d , but not H-2D d . The relationship of BALBRV1 antigen and BALBRVD antigen to endogenous MuLV is discussed with regard to the antigenic distribution on tumor cell lines. (author)

Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis.

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Shaw, Catherine A; Webb, David J; Rossi, Adriano G; Megson, Ian L

2009-05-07

Nitric oxide (NO) can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-). In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMvarphi), and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP) is able to limit apoptosis in this cell type. Characterisation of the NO-related species generated by (Z)-1- [2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-, chloride (GEA-3162) was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR) spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMvarphi. Resultant MDMvarphi were treated for 24 h with DETA/NO (100 - 1000 muM) or GEA-3162 (10 - 300 muM) in the presence or absence of BAY 41-2272 (1 muM), isobutylmethylxanthine (IBMX; 1 muM), 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 muM) or 8-bromo-cGMP (1 mM). Apoptosis in MDMvarphi was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO) had no effect on cell viability, but ONOO- (GEA-3162) caused a concentration-dependent induction of apoptosis in MDMvarphi. Preconditioning of MDMvarphi with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX), or the NO-independent stimulator of soluble guanylate cyclase, BAY 41-2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner. These results

Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis

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Rossi Adriano G

2009-05-01

Full Text Available Abstract Background Nitric oxide (NO can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-. In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMϕ, and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP is able to limit apoptosis in this cell type. Methods Characterisation of the NO-related species generated by (Z-1- [2-(2-aminoethyl-N-(2-ammonioethylamino]diazen-1-ium-1,2-diolate (DETA/NO and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl-, chloride (GEA-3162 was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMϕ. Resultant MDMϕ were treated for 24 h with DETA/NO (100 – 1000 μM or GEA-3162 (10 – 300 μM in the presence or absence of BAY 41–2272 (1 μM, isobutylmethylxanthine (IBMX; 1 μM, 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 μM or 8-bromo-cGMP (1 mM. Apoptosis in MDMϕ was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Results Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO had no effect on cell viability, but ONOO- (GEA-3162 caused a concentration-dependent induction of apoptosis in MDMϕ. Preconditioning of MDMϕ with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX, or the NO-independent stimulator of soluble guanylate cyclase, BAY 41–2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner

Purinergic signaling to terminate TLR responses in macrophages

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Kajal eHamidzadeh

2016-03-01

Full Text Available Macrophages undergo profound physiological alterations when they encounter pathogen associated molecular patterns (PAMPs. These alterations can result in the elaboration of cytokines and mediators that promote immune responses and contribute to the clearance of pathogens. These innate immune responses by myeloid cells are transient. The termination of these secretory responses is not due to the dilution of stimuli, but rather to the active down-regulation of innate responses induced by the very PAMPs that initiated them. Here we describe a purinergic autoregulatory program whereby TLR-stimulated macrophages control their activation state. In this program, TLR stimulated macrophages undergo metabolic alterations that result in the production of ATP and its release through membrane pannexin channels. This purine nucleotide is rapidly hydrolyzed to adenosine by ectoenzymes on the macrophage surface, CD39 and CD73. Adenosine then signals through the P1 class of seven transmembrane receptors to induce a regulatory state that is characterized by the down-regulation of inflammatory cytokines and the production of anti-inflammatory cytokines and growth factors. This purinergic autoregulatory system mitigates the collateral damage that would be caused by the prolonged activation of macrophages, and rather allows the macrophage to maintain homeostasis. The transient activation of macrophages can be prolonged by treating macrophages with IFN-γ. IFN-γ treated macrophages become less sensitive to the regulatory effects of adenosine, allowing them to sustain macrophage activation for the duration of an adaptive immune response.

Interaction of the antiemetics ondansetron and granisetron with the cytotoxicity induced by irradiation, epirubicin, bleomycin, estramustine, and cisplatin in vitro

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Behnam Motlagh, P. [Dept. of Oncology, Umeaa Univ. Hospital (Sweden); Henriksson, R. [Dept. of Oncology, Umeaa Univ. Hospital (Sweden); Grankvist, K. [Dept. of Clinical Chemistry, Umeaa Univ. Hospital (Sweden)

1995-12-31

At cancer treatment, the use of antiemetics are often needed due to induction of nausea and vomiting. Some antiemetics have been shown to interact with the direct cytotoxic effects. The newly developed antiemetics have, so far as we know, not been studied in this respect. In the present study, the effects of the 5-HT{sub 3} receptor antagonists ondansetron and granisetron were evaluated on the cytotoxicity, induced by irradiation, bleomycin, epirubicin, estramustine, and cisplatin using fibroblasts (V79) and lung cancer cells (P31) in vitro. Ondansetron or granisetron (10{sup -5} mol/l) had no effect on the survival of irradiated cells. Granisetron (10{sup -5} mol/l) significantly potentiated cytoxocity of 2.5 mg/l epirubicin on fibroblasts whereas the effect of granisetron (10{sup -7} mol/l) on the cytotoxic effect of 25 mg/l bleomycin, and estramustine (80 mg/l) seemed additive to lung cancer cells. Ondansetron was non-interactive with the cytotoxicity induced by any of the anti-cancer drugs. Although the encountered observation with an enhancing effect of granisetron on the epirubicin-induced cytotoxicity is seen in a specific experimental situation in vitro, the fact that 5-HT{sub 3} receptor antagonists are routinely used during cancer treatment indicate that attention should be given to a possible interaction with the antineoplastic action of cancer treatment. (orig.).

Interaction of the antiemetics ondansetron and granisetron with the cytotoxicity induced by irradiation, epirubicin, bleomycin, estramustine, and cisplatin in vitro

International Nuclear Information System (INIS)

Behnam Motlagh, P.; Henriksson, R.; Grankvist, K.

1995-01-01

At cancer treatment, the use of antiemetics are often needed due to induction of nausea and vomiting. Some antiemetics have been shown to interact with the direct cytotoxic effects. The newly developed antiemetics have, so far as we know, not been studied in this respect. In the present study, the effects of the 5-HT 3 receptor antagonists ondansetron and granisetron were evaluated on the cytotoxicity, induced by irradiation, bleomycin, epirubicin, estramustine, and cisplatin using fibroblasts (V79) and lung cancer cells (P31) in vitro. Ondansetron or granisetron (10 -5 mol/l) had no effect on the survival of irradiated cells. Granisetron (10 -5 mol/l) significantly potentiated cytoxocity of 2.5 mg/l epirubicin on fibroblasts whereas the effect of granisetron (10 -7 mol/l) on the cytotoxic effect of 25 mg/l bleomycin, and estramustine (80 mg/l) seemed additive to lung cancer cells. Ondansetron was non-interactive with the cytotoxicity induced by any of the anti-cancer drugs. Although the encountered observation with an enhancing effect of granisetron on the epirubicin-induced cytotoxicity is seen in a specific experimental situation in vitro, the fact that 5-HT 3 receptor antagonists are routinely used during cancer treatment indicate that attention should be given to a possible interaction with the antineoplastic action of cancer treatment. (orig.)

Suppressive effects of ketamine on macrophage functions

International Nuclear Information System (INIS)

Chang Yi; Chen, T.-L.; Sheu, J.-R.; Chen, R.-M.

2005-01-01

Ketamine is an intravenous anesthetic agent. Clinically, induction of anesthesia with ketamine can cause immunosuppression. Macrophages play important roles in host defense. In this study, we attempted to evaluate the effects of ketamine on macrophage functions and its possible mechanism using mouse macrophage-like Raw 264.7 cells as the experimental model. Exposure of macrophages to 10 and 100 μM ketamine, which correspond to 0.1 and 1 times the clinically relevant concentration, for 1, 6, and 24 h had no effect on cell viability or lactate dehydrogenase release. When the administered concentration reached 1000 μM, ketamine caused a release of lactate dehydrogenase and cell death. Ketamine, at 10 and 100 μM, did not affect the chemotactic activity of macrophages. Administration of 1000 μM ketamine in macrophages resulted in a decrease in cell migration. Treatment of macrophages with ketamine reduced phagocytic activities. The oxidative ability of macrophages was suppressed by ketamine. Treatment with lipopolysaccharide induced TNF-α, IL-1β, and IL-6 mRNA in macrophages. Administration of ketamine alone did not influence TNF-α, IL-1β, or IL-6 mRNA production. Meanwhile, cotreatment with ketamine and lipopolysaccharide significantly inhibited lipopolysaccharide-induced TNF-α, IL-1β, and IL-6 mRNA levels. Exposure to ketamine led to a decrease in the mitochondrial membrane potential. However, the activity of mitochondrial complex I NADH dehydrogenase was not affected by ketamine. This study shows that a clinically relevant concentration of ketamine (100 μM) can suppress macrophage function of phagocytosis, its oxidative ability, and inflammatory cytokine production possibly via reduction of the mitochondrial membrane potential instead of direct cellular toxicity

M2 macrophages induce ovarian cancer cell proliferation via a heparin binding epidermal growth factor/matrix metalloproteinase 9 intercellular feedback loop.

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Carroll, Molly J; Kapur, Arvinder; Felder, Mildred; Patankar, Manish S; Kreeger, Pamela K

2016-12-27

In ovarian cancer, a high ratio of anti-inflammatory M2 to pro-inflammatory M1 macrophages correlates with poor patient prognosis. The mechanisms driving poor tumor outcome as a result of the presence of M2 macrophages in the tumor microenvironment remain unclear and are challenging to study with current techniques. Therefore, in this study we utilized a micro-culture device previously developed by our lab to model concentrated paracrine signaling in order to address our hypothesis that interactions between M2 macrophages and ovarian cancer cells induce tumor cell proliferation. Using the micro-culture device, we determined that co-culture with M2-differentiated primary macrophages or THP-1 increased OVCA433 proliferation by 10-12%. This effect was eliminated with epidermal growth factor receptor (EGFR) or heparin-bound epidermal growth factor (HB-EGF) neutralizing antibodies and HBEGF expression in peripheral blood mononuclear cells from ovarian cancer patients was 9-fold higher than healthy individuals, suggesting a role for HB-EGF in tumor progression. However, addition of HB-EGF at levels secreted by macrophages or macrophage-conditioned media did not induce proliferation to the same extent, indicating a role for other factors in this process. Matrix metalloproteinase-9, MMP-9, which cleaves membrane-bound HB-EGF, was elevated in co-culture and its inhibition decreased proliferation. Utilizing inhibitors and siRNA against MMP9 in each population, we determined that macrophage-secreted MMP-9 released HB-EGF from macrophages, which increased MMP9 in OVCA433, resulting in a positive feedback loop to drive HB-EGF release and increase proliferation in co-culture. Identification of multi-cellular interactions such as this may provide insight into how to most effectively control ovarian cancer progression.

KRAS Mutation and Epithelial-Macrophage Interplay in Pancreatic Neoplastic Transformation.

Science.gov (United States)

Bishehsari, Faraz; Zhang, Lijuan; Barlass, Usman; Preite, Nailliw; Turturro, Sanja; Najor, Matthew S; Shetuni, Brandon B; Zayas, Janet P; Mahdavinia, Mahboobeh; Abukhdeir, Abde M; Keshavarzian, Ali

2018-05-14

Pancreatic ductal adenocarcinoma (PDA) is characterized by epithelial mutations in KRAS and prominent tumor-associated inflammation, including macrophage infiltration. But knowledge of early interactions between neoplastic epithelium and macrophages in PDA carcinogenesis is limited. Using a pancreatic organoid model, we found that the expression of mutant KRAS in organoids increased i) ductal to acinar gene expression ratios, ii) epithelial cells proliferation, and iii) colony formation capacity in vitro, and endowed pancreatic cells with the ability to generate neoplastic tumors in vivo. KRAS mutations induced a pro-tumorigenic phenotype in macrophages. Altered macrophages decreased epithelial Pigment Epithelial Derived Factor (PEDF) expression and induced a cancerous phenotype. We validated our findings using annotated patient samples from The Cancer Genome Atlas (TCGA) as well as in our human PDA specimens. Epithelium-macrophage cross talk occurs early in pancreatic carcinogenesis where KRAS directly induces cancer-related phenotypes in epithelium, and also promotes a pro-tumorigenic phenotype in macrophages, in turn augmenting neoplastic growth. This article is protected by copyright. All rights reserved. © 2018 UICC.

Macrophages Promote Axon Regeneration with Concurrent Neurotoxicity

NARCIS (Netherlands)

Gensel, J.C.; Nakamura, S.; Guan, Z.; Rooijen, van N.; Ankeny, D.P.; Popovich, P.G.

2009-01-01

Activated macrophages can promote regeneration of CNS axons. However, macrophages also release factors that kill neurons. These opposing functions are likely induced simultaneously but are rarely considered together in the same experimental preparation. A goal of this study was to unequivocally

Docosahexaenoic acid ester of phloridzin inhibit lipopolysaccharide-induced inflammation in THP-1 differentiated macrophages.

Science.gov (United States)

Sekhon-Loodu, Satvir; Ziaullah; Rupasinghe, H P Vasantha

2015-03-01

Phloridzin or phlorizin (PZ) is a predominant phenolic compound found in apple and also used in various natural health products. Phloridzin shows poor absorption and cellular uptake due to its hydrophilic nature. The aim was to investigate and compare the effect of docosahexaenoic acid (DHA) ester of PZ (PZ-DHA) and its parent compounds (phloridzin and DHA), phloretin (the aglycone of PZ) and cyclooxygenase inhibitory drugs (diclofenac and nimesulide) on production of pro-inflammatory biomarkers in inflammation-induced macrophages by lipopolysaccharide (LPS)-stimulation. Human THP-1 monocytes were seeded in 24-well plates (5×10(5)/well) and treated with phorbol 12-myristate 13-acetate (PMA, 0.1μg/mL) for 48h to induce macrophage differentiation. After 48h, the differentiated macrophages were washed with Hank's buffer and treated with various concentrations of test compounds for 4h, followed by the LPS-stimulation (18h). Pre-exposure of PZ-DHA ester was more effective in reducing tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) protein levels compared to DHA and nimesulide. However, diclofenac was the most effective in reducing prostaglandin (PGE2) level by depicting a dose-dependent response. However, PZ-DHA ester and DHA were the most effective in inhibiting the activation of nuclear factor-kappa B (NF-κB) among other test compounds. Our results suggest that PZ-DHA ester might possess potential therapeutic activity to treat inflammation related disorders such as type 2 diabetes, asthma, atherosclerosis and inflammatory bowel disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Macrophage Migration Inhibitory Factor Induces Inflammation and Predicts Spinal Progression in Ankylosing Spondylitis.

Science.gov (United States)

Ranganathan, Vidya; Ciccia, Francesco; Zeng, Fanxing; Sari, Ismail; Guggino, Guiliana; Muralitharan, Janogini; Gracey, Eric; Haroon, Nigil

2017-09-01

To investigate the role of macrophage migration inhibitory factor (MIF) in the pathogenesis of ankylosing spondylitis (AS). Patients who met the modified New York criteria for AS were recruited for the study. Healthy volunteers, rheumatoid arthritis patients, and osteoarthritis patients were included as controls. Based on the annual rate of increase in modified Stoke AS Spine Score (mSASSS), AS patients were classified as progressors or nonprogressors. MIF levels in serum and synovial fluid were quantitated by enzyme-linked immunosorbent assay. Predictors of AS progression were evaluated using logistic regression analysis. Immunohistochemical analysis of ileal tissue was performed to identify MIF-producing cells. Flow cytometry was used to identify MIF-producing subsets, expression patterns of the MIF receptor (CD74), and MIF-induced tumor necrosis factor (TNF) production in the peripheral blood. MIF-induced mineralization of osteoblast cells (SaOS-2) was analyzed by alizarin red S staining, and Western blotting was used to quantify active β-catenin levels. Baseline serum MIF levels were significantly elevated in AS patients compared to healthy controls and were found to independently predict AS progression. MIF levels were higher in the synovial fluid of AS patients, and MIF-producing macrophages and Paneth cells were enriched in their gut. MIF induced TNF production in monocytes, activated β-catenin in osteoblasts, and promoted the mineralization of osteoblasts. Our findings indicate an unexplored pathogenic role of MIF in AS and a link between inflammation and new bone formation. © 2017, American College of Rheumatology.

Protective effect of lavender oil on scopolamine induced cognitive deficits in mice and H2O2 induced cytotoxicity in PC12 cells.

Science.gov (United States)

Xu, Pan; Wang, Kezhu; Lu, Cong; Dong, Liming; Gao, Li; Yan, Ming; Aibai, Silafu; Liu, Xinmin

2016-12-04

Lavender essential oil (LO), an aromatic liquid extracted from Lavandula angustifolia Mill., has been traditionally used in the treatments of many nervous system diseases, and recently LO also reported to be effective for the Alzheimer's disease (AD). The improvement effect of lavender oil (LO) on the scopolamine-induced cognitive deficits in mice and H 2 O 2 induced cytotoxicity in PC12 cells have been evaluated. The relevant mechanism was also researched from the perspective of antioxidant effect and cholinergic system modulation. Cognitive deficits were induced in C57BL/6J mice treated with scopolamine (1mg/kg, i.p.) and were assessed by Morris water maze (MWM) and step-through passive avoidance tests. Then their hippocampus were removed for biochemical assays (acetylcholinesterase (AChE), superoxide dismutase (SOD), glutathione peroxidase (GPX) and malondialdehyde (MDA)). In vitro, the cytotoxicity were induced by 4h exposure to H 2 O 2 in PC12 and evaluated by cell viability (MTT), lactate dehydrogenase (LDH) level, nitric oxide (NO) release, reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP). The results demonstrated that LO (100mg/kg) could improve the cognitive performance of scopolamine induced mice in behavioral tests. Meanwhile, it significantly decreased the AChE activity, MDA level, and increase SOD and GPX activities of the model. Moreover, LO (12μg/mL) protected PC12 cells from H 2 O 2 induced cytotoxicity by reducing LDH, NO release, intracellular ROS accumulation and MMP loss. It was suggested that LO could show neuroprotective effect in AD model in vivo (scopolamine-treated mice) and in vitro (H 2 O 2 induced PC12 cells) via modulating oxidative stress and AChE activity. Copyright © 2016. Published by Elsevier Ireland Ltd.

Regulation and control of nitric oxide (NO) in macrophages

DEFF Research Database (Denmark)

Kovacevic, Zaklina; Sahni, Sumit; Lok, K.H.

2017-01-01

We recently demonstrated that a novel storage and transport mechanism for nitric oxide (NO) mediated by glutathione-S-transferase P1 (GSTP1) and multidrug resistance protein 1 (MRP1/ABCC1), protects M1-macrophage (M1-MØ) models from large quantities of endogenous NO. This system stores and transp......We recently demonstrated that a novel storage and transport mechanism for nitric oxide (NO) mediated by glutathione-S-transferase P1 (GSTP1) and multidrug resistance protein 1 (MRP1/ABCC1), protects M1-macrophage (M1-MØ) models from large quantities of endogenous NO. This system stores...... be responsible for delivering cytotoxic NO as DNICs via MRP1 from M1-MØs, to tumor cell targets....

Function of miR-146a-5p in Tumor Cells As a Regulatory Switch between Cell Death and Angiogenesis: Macrophage Therapy Revisited

Directory of Open Access Journals (Sweden)

Elina Simanovich

2018-01-01

Full Text Available Tumors survive and progress by evading killing mechanisms of the immune system, and by generating a tumor microenvironment (TME that reprograms macrophages in situ to produce factors that support tumor growth, angiogenesis, and metastasis. We have previously shown that by blocking the translation of the enzyme inducible nitric oxide synthase (iNOS, miR-146a-5p inhibits nitric oxide (NO production in a mouse renal carcinoma cell line (RENCA, thereby endowing RENCA cells with resistance to macrophage-induced cell death. Here, we expand these findings to the mouse colon carcinoma CT26 cell line and demonstrate that neutralizing miR-146a-5p’s activity by transfecting both RENCA and CT26 cells with its antagomir restored iNOS expression and NO production and enhanced susceptibility to macrophage-induced cell death (by 48 and 25%, respectively, p < 0.001. Moreover, miR-146a-5p suppression simultaneously inhibited the expression of the pro-angiogenic protein EMMPRIN (threefolds, p < 0.001, leading to reduced MMP-9 and vascular endothelial growth factor secretion (twofolds and threefolds, respectively, p < 0.05, and reduced angiogenesis, as estimated by in vitro tube formation and scratch assays. When we injected tumors with pro-inflammatory-stimulated RAW 264.7 macrophages together with i.v. injection of the miR-146a-5p antagomir, we found inhibited tumor growth (sixfolds, p < 0.001 and angiogenesis (twofolds, p < 0.01, and increased apoptosis (twofolds, p < 0.01. This combination therapy increased nitrites and reduced TGFβ concentrations in tumor lysates, alleviated immune suppression, and allowed enhanced infiltration of cytotoxic CD8+ T cells. Thus, miR-146a-5p functions as a control switch between angiogenesis and cell death, and its neutralization can manipulate the crosstalk between tumor cells and macrophages and profoundly change the TME. This strategy can be therapeutically utilized in combination with the macrophage

Macrophage specific MRI imaging for antigen induced arthritides. A potential new strategy for the diagnosis of rheumatoid arthritis

International Nuclear Information System (INIS)

Simon, G.H.; Rummeny, E.J.; Daldrup-Link, H.E.

2007-01-01

The present work describes the potential of iron oxides for the detection of macrophages in synovitis in experimental, antigen-induced arthritis. The pivotal role of macrophages in rheumatoid arthritis (RA) in humans and in antigen-induced arthritis (AIA) in animal models is discussed. The latter appear to be very similar in many aspects to the human RA. We show the potential for iron oxide-enhanced magnetic resonance imaging (MRI) to determine the macrophage content in the arthritic synovial membranes. The results of our own research, as well as those of other research groups, are presented and discussed. MRI after the intravenous (i.v.) administration of iron oxides enables the depiction of macrophage content in arthritic synovial membranes in AIA through the effects of the intracellular compartmentalisation of iron oxide particles. These effects can be demonstrated in 24-h delayed images after i.v. contrast application, on T2-weighted spin-echo or turbo-spin-echo sequences, and especially on T2 * -weighted gradient-echo sequences. The signal effects are not only apparent in high field strength (4.7 Tesla) but also on 1.5 Tesla clinical scanners. The use of iron oxides enables the determination of the macrophage content in synovitis in animals with AIA. This parameter represents a potential marker to determine disease activity, and possibly represents a marker to evaluate the effectiveness of specific therapies in human RA. Current knowledge of iron oxide-enhanced MRI is limited to animal models. The clinical evaluation of this new method in patients with RA has not yet been performed. However, based on the considerations presented here, significant progress in the diagnostic work-up of RA can be expected