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Sample records for u937 human monocytes

  1. [Linezolid-induced Apoptosis through Mitochondrial Damage and Role of Superoxide Dismutase-1 in Human Monocytic Cell Line U937].

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    Fujii, Satoshi; Muraoka, Sanae; Miyamoto, Atsushi; Sakurai, Koichi

    2018-01-01

     Cytopenia is a major adverse event associated with linezolid therapy. The objective of this study was to examine whether the cytotoxicity of linezolid to eukaryotic cells was associated with mitochondrial dysfunction and apoptosis-like cell death in human leukemic monocyte lymphoma cell line U937. Apoptosis-like cell death was clearly observed when cells were incubated with linezolid, depending on the duration and linezolid concentration. Mitochondrial membrane potential of cells treated with linezolid collapsed in a short period of time, but the number of mitochondria did not decrease. Cytotoxicity of linezolid was relieved by the knockdown of superoxide dismutase-1 in U937 cells. On the other hand, no autophagy was observed in cells treated with linezolid. These results suggest that mitochondrial damages would be linked to the induction of apoptosis in U937 cells treated with linezolid and that its mechanism does not involve autophagy.

  2. Synthesis of complement factor B by the human monocyte U-937 cell line. Augmentation by immunostimulatory agents

    International Nuclear Information System (INIS)

    Minta, J.O.

    1988-01-01

    The human pre-monocytic cell line U-937 was shown to synthesize and to secrete increasing amounts of factor B in short term cultures in serum-free medium containing BSA. The kinetics of factor B production were higher on day 2 than on days 1 and 3. The production of factor B was reversibly inhibited by cycloheximide, indicating de novo synthesis. Metabolic labeling with [ 35 S]-methionine and SDS-PAGE analysis revealed that both intracellular and secreted factor B were single-chain proteins with similar m.w. (90,000), which co-migrated with purified plasma factor B. Incubation of U-937 cells with the immunostimulants PMA, LPS, IFN-gamma, and IL-1 resulted in a dose-dependent augmentation of factor B production. A 24-h exposure to IL-1 was shown to be required for maximal stimulation. A combination of suboptimal doses of LPS and IFN-gamma was shown to exert a synergistic effect on factor B production. The U-937 cell line is thus a valuable model for the study of the regulation of the factor B gene expression

  3. NRF2 Signaling Negatively Regulates Phorbol-12-Myristate-13-Acetate (PMA-Induced Differentiation of Human Monocytic U937 Cells into Pro-Inflammatory Macrophages.

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    Min-Gu Song

    Full Text Available Blood monocytes are recruited to injured tissue sites and differentiate into macrophages, which protect against pathogens and repair damaged tissues. Reactive oxygen species (ROS are known to be an important contributor to monocytes' differentiation and macrophages' function. NF-E2-related factor 2 (NRF2, a transcription factor regulating cellular redox homeostasis, is known to be a critical modulator of inflammatory responses. We herein investigated the role of NRF2 in macrophage differentiation using the human monocytic U937 cell line and phorbol-12-myristate-13-acetate (PMA. In U937 cells with NRF2 silencing, PMA-stimulated cell adherence was significantly facilitated when compared to control U937 cells. Both transcript and protein levels for pro-inflammatory cytokines, including interleukine-1β (IL-1β, IL-6, and tumor necrosis factor-α (TNFα were highly elevated in PMA-stimulated NRF2-silenced U937 compared to the control. In addition, PMA-inducible secretion of monocyte chemotactic protein 1 (MCP-1 was significantly high in NRF2-silenced U937. As an underlying mechanism, we showed that NRF2-knockdown U937 retained high levels of cellular ROS and endoplasmic reticulum (ER stress markers expression; and subsequently, PMA-stimulated levels of Ca2+ and PKCα were greater in NRF2-knockdown U937 cells, which caused enhanced nuclear accumulation of nuclear factor-ҡB (NFҡB p50 and extracellular signal-regulated kinase (ERK-1/2 phosphorylation. Whereas the treatment of NRF2-silenced U937 cells with pharmacological inhibitors of NFҡB or ERK1/2 largely blocked PMA-induced IL-1β and IL-6 expression, indicating that these pathways are associated with cell differentiation. Taken together, our results suggest that the NRF2 system functions to suppress PMA-stimulated U937 cell differentiation into pro-inflammatory macrophages and provide evidence that the ROS-PKCα-ERK-NFҡB axis is involved in PMA-facilitated differentiation of NRF2-silenced U937

  4. shRNA-mediated EMMPRIN silencing inhibits human leukemic monocyte lymphoma U937 cell proliferation and increases chemosensitivity to adriamycin.

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    Gao, Hui; Jiang, Qixiao; Han, Yantao; Peng, Jianjun; Wang, Chunbo

    2015-03-01

    EMMPRIN is a widely distributed cell surface glycoprotein, which plays an important role in tumor progression and confers resistance to some chemotherapeutic drugs. Recent studies have shown that EMMPRIN overexpression indicates poor prognosis in acute myeloid leukemia (AML). However, little was known on the role of EMMPRIN in leukemia. Human leukemia cell line U937 was stably transfected with a EMMPRIN-targeted shRNA-containing vector to investigate the effect of EMMPRIN on cellular functions. EMMPRIN expression was monitored by qRT-PCR and Western blotting. Cell viability and proliferation were determined by trypan blue exclusion and BrdU labeling, respectively. Cell cycle and apoptosis were analyzed by flow cytometry. Cytotoxicity of chemotherapeutic agent adriamycin on cells was assessed by MTT assay. Knockdown of EMMPRIN gene significantly inhibited cell viability and decreased cell proliferation. Fluorescence-activated cell-sorting analysis revealed that the reduced EMMPRIN expression resulted in cell cycle arrest at G1 phase and induced apoptosis. Meanwhile, western blotting analysis showed that EMMPRIN knockdown was associated with downregulation of cell cycle- and apoptosis-related molecules including cyclin D1, cyclin E, as well as increase in cleavage of caspase-3 and PARP. This study also showed that silencing of EMMPRIN sensitized U937 cells to Adriamycin. EMMPRIN is involved in proliferation, growth, and chemosensitivity of human AML line U937, indicating that EMMPRIN may be a promising therapeutic target for AML.

  5. Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

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    Hatton, Jason P.; Gaubert, Francois; Cazenave, Jean-Pierre; Schmitt, Didier; Hashemi, B. B. (Principal Investigator); Hughes-Fulford, M. (Principal Investigator)

    2002-01-01

    Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.

  6. Time Dependent Production of Cytokines and Eicosanoids by Human Monocytic Leukaemia U937 Cells; Effects of Glucocorticosteroids

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    Ingrid M. Garrelds

    1999-01-01

    Full Text Available In the present study the human monoblast cell line U937 has been used as a model to study the function of human mononuclear phagocytes in asthma. The kinetics of the production of eicosanoids and cytokines, which are thought to play a role in the pathogenesis of asthma, were studied. In addition, the effects of glucocorticosteroids were investigated, as these drugs are of great importance for the treatment of asthmatic patients. After stimulation with phorbol-12 myristate acetate (PMA for 24h, U937 cells were cultured in the absence or presence of lipopolysaccharide (LPS: 1 and 5 μg ml-1 and glucocorticosteroids (budesonide, fluticasone propionate and prednisolone: 10-11, 10-9 and 10-7 M for 96h. The production of interleukin- 1β (IL-1β, interleukin-6 (IL-6, prostaglandin E2 (PGE2 and thromboxane B2 (TxB2 gradually increased in time after stimulation with LPS, whereas the transient production of tumor necrosis factor alpha (TNF-α reached its maximum between 6 and 12 h. Interferon-gamma (IFN-γ, interleukin-10 (IL-10 and leukotriene B4 (LTB4 were not detectable. All three glucocorticosteroids (budesonide, fluticasone propionate and prednisolone completely inhibited the production of both eicosanoids and cytokines. The production of eicosanoids was more sensitive to these glucocorticoids than the production of cytokines. The observed differences in the kinetics of the production of eicosanoids and cytokines stress the importance of time course experiments in studies on the effect of drugs on mononuclear cells.

  7. Polypeptide structure of a human dermal fibroblast-activating factor (FAF) derived from the U937 cultured line of human monocyte-like cells

    International Nuclear Information System (INIS)

    Cooke, M.P.; Allar, W.J.; Goetzl, E.J.; Dohlman, J.G.

    1986-01-01

    Six liter batches of 1 x 10 6 U937 cells/ml of serum-free RPMI medium were incubated with 100 ng/ml of phorbol myristate acetate for 48 hr at 37 0 C in 5% CO 2 in air to generate FAFs, as quantified by the stimulation of uptake of [ 3 H]thymidine by quiescent human dermal fibroblasts. Filtration of the supernatants on Sephadex G-75 resolved two FAFs of approximately 40,000 and 10-13,000 daltons. The latter principle was purified to homogeneity by sequential Sephadex G-50 filtration, revealing an apparent m.w. of 7-8000, Mono-Q FPLC anion-exchange chromatography with a linear gradient from 20 mM Tris-HCl (pH 8.3) to 0.5 M NaCl-20 mM Tris-HCl in 30 min, and two cycles of high-performance liquid chromatography (HPLC) on a 300 A pore 10 μm C4 column at 1 ml/min with 0.05% trifluoroacetic acid (TFA) in water to 30:70 (v:v) and then to 60:40 (v:v) acetonitrile: 0.05% TFA linearly in 15 min and 30 min, respectively, The FAF activity eluted from HPLC in a sharp peak of O.D. 215 nm at 45% acetonitrile. Analyses of amino acid composition of the highly purified 7-8000 dalton FAF-U937 revealed 37% hydrophobic, 14% basic, and 21% acidic or amide residues, as well as one tryrosine and one methionine. This U937 cell-derived FAF appears to be a unique acidic polypeptide growth factor

  8. Structure-activity relationships of dimethylsphingosine (DMS) derivatives and their effects on intracellular pH and Ca2+ in the U937 monocyte cell line.

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    Chang, Young-Ja; Lee, Yun-Kyung; Lee, Eun-Hee; Park, Jeong-Ju; Chung, Sung-Kee; Im, Dong-Soon

    2006-08-01

    We recently reported that dimethylsphingosine (DMS), a metabolite of sphingolipids, increased intracellular pH and Ca2+ concentration in U937 human monocytes. In the present study, we found that dimethylphytosphingosine (DMPH) induced the above responses more robustly than DMS. However, phytosphingosine, monomethylphytosphingosine or trimethylsphingosine showed little or no activity. Synthetic C3 deoxy analogues of sphingosine did show similar activities, with the C16 analogue more so than C18. The following structure-activity relationships were observed between DMS derivatives and the intracellular pH and Ca2+ concentrations in U937 monocytes; 1) dimethyl modification is important for the DMS-induced increase of intracellular pH and Ca2+, 2) the addition of an OH group on C4 enhances both activities, 3) the deletion of the OH group on C3 has a negligible effect on the activities, and 4) C16 appears to be more effective than C18. We also found that W-7, a calmodulin inhibitor, blocked the DMS-induced pH increase, whereas, KN-62, ML9, and MMPX, specific inhibitors for calmodulin-dependent kinase II, myosin light chain kinase, and Ca(2+)-calmodulin-dependent phosphodiesterase, respectively, did not affect DMS-induced increases of pH in the U937 monocytes.

  9. Effects of vinegar–egg on growth inhibition, differentiation human leukemic U937 cells and its immunomodulatory activity

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    Shiu-Yu Wang

    2018-04-01

    Full Text Available Vinegar and eggs have rich nutrients. In this study, the mixed form of both derived products, vinegar–egg solution and its products (vinegar–egg concentrate and vinegar–egg condensate were chosen for an assessment of their biological activity. To further our understanding regarding the anticancer and immunomodulatory effects of vinegar–egg, we investigated its effects on the proliferation and differentiation of U937 cells. Vinegar–egg was treated using spray drying, freeze drying and vacuum concentration and used to stimulate human mononuclear cells. The conditioned media obtained from these cultures by filtration were used to treat U937 cells. Three conditioned media inhibited U937 cell growth by 22.1–67.25% more effectively than PHA-treated control (22.53%. CD11b and CD14 expression on the treated U937 cells were 29.1–45.4% and 31.6–47.2%, respectively. High levels of cytokines IL-1β, IFN-γ and TNF-α were detected in the three conditioned media. Vinegar–egg stimulates human mononuclear cells to secrete cytokines, which inhibit the growth of U937 cells and induce their differentiation. Keywords: Cytokines, Differentiation, Immunomodulatory activity, Leukemic U937 cells, Vinegar–egg

  10. Cucurbitacin E as a new inhibitor of cofilin phosphorylation in human leukemia U937 cells.

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    Nakashima, Souichi; Matsuda, Hisashi; Kurume, Ai; Oda, Yoshimi; Nakamura, Seikou; Yamashita, Masayuki; Yoshikawa, Masayuki

    2010-05-01

    Cucurbitane-type triterpenes, cucurbitacins B and E, were reported to exhibit cytotoxic effects in several cell lines mediated by JAK/STAT3 signaling. However, neither compound inhibited phosphorylation of STAT3 in human leukemia (U937) cells at low concentrations. We therefore synthesized a biotin-linked cucurbitacin E to isolate target proteins based on affinity for the molecule. As a result, cofilin, which regulates the depolymerization of actin, was isolated and suggested to be a target. Cucurbitacins E and I inhibited the phosphorylation of cofilin in a concentration-dependent manner, and their effective concentrations having the same range as the concentrations at which they had cytotoxic effects in U937 cells. In addition, the fibrous-/globular-actin ratio was decreased after treatment with cucurbitacin E in HT1080 cells. These findings suggested that the inhibition of cofilin's phosphorylation increased the severing activity of cofilin, and then the depolymerization of actin was enhanced after treatment with cucurbitacin E at lower concentrations. 2010 Elsevier Ltd. All rights reserved.

  11. Isolation and identification of gene mediating radiation-induced apoptosis in human leukemia U937 cells

    International Nuclear Information System (INIS)

    Tong Xin; Luo Ying; Dong Yan; Sun Zhixian

    1998-01-01

    Objective: Increasing evidences suggest that Caspase family proteases play an important role in the effector mechanism of apoptotic cell death. Radiation (IR) can induce apoptosis in tumor cells, so it is very important to isolate and identify the member of the Caspase family proteases involved in IR-induced apoptosis, and this would contribute to the understanding of the mechanism responsible for apoptosis execution. Methods: A PCR approach to isolate genes for IR-induced apoptosis was developed. The approach used degenerated oligonucleotide encoding the highly conserved peptides that were present in all known Caspases. Results: Protease inhibitors special for Caspases could block the apoptotic cell death caused by IR, and Caspase-3 was isolated from irradiated human leukemia U937 cells. Conclusion: Caspases involve in IR-induced apoptosis, and Caspase-3 is the pivotal element of IR-induced apoptosis

  12. Oxidative damage of U937 human leukemic cells caused by hydroxyl radical results in singlet oxygen formation.

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    Marek Rác

    Full Text Available The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules.

  13. Apoptotic Effect of Nigella sativa on Human Lymphoma U937 Cells.

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    Arslan, Belkis Atasever; Isik, Fatma Busra; Gur, Hazal; Ozen, Fatih; Catal, Tunc

    2017-10-01

    Nigella sativa is from botanical Ranunculaceae family and commonly known as black seed. Apoptotic effect of N. sativa and its apoptotic signaling pathways on U937 lymphoma cells are unknown. In this study, we investigated selective cytotoxic and apoptotic effects of N. sativa extract and its apoptotic mechanisms on U937 cells. In addition, we also studied selective cytotoxic activity of thymoquinone that is the most active essential oil of N. sativa . Our results showed that N. sativa extract has selective cytotoxicity and apoptotic effects on U937 cells but not ECV304 control cells. However, thymoquinone had no significant cytotoxicity against on both cells. N. sativa extract increased significantly caspase-3, BAD, and p53 gene expressions in U937 cells. N. sativa may have anticancer drug potential and trigger p53-induced apoptosis in U937 lymphoma cells. This is the first study showing the apoptotic effect of Nigella sativa extract on U937 cells. Abbreviations used: CI: Cytotoxicity index, DMEM: Dulbecco's Modified Eagle Medium, HL: Hodgkin's lymphoma, MTT: 3-(4,5-dimethy lthiazol-2yl)-2,5-diphenyl tetrazolium bromide, RPMI: Roswell Park Memorial Institute medium.

  14. Naja nigricollis CMS-9 enhances the mitochondria-mediated death pathway in adaphostin-treated human leukaemia U937 cells.

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    Chen, Ying-Jung; Wang, Jeh-Jeng; Chang, Long-Sen

    2011-11-01

    1. The aim of the present study was to explore the effect of the Naja nigricollis phospholipase A(2) CMS-9 on adaphostin-induced death of human leukaemia U937 cells. 2. Leukaemia U937 cells (Bcr/Abl-negative cells) were treated with adaphostin (0-10 μmol/L) and CMS-9 (0-1 μmol/L). The effects of CMS-9, adaphostin and their combination on cell viability, the generation reactive oxygen species (ROS), [Ca(2+) ](i) , p38 mitogen-activated protein kinase (MAPK) activation, Akt and extracellular signal-regulated kinase (ERK) inactivation, mitochondrial membrane potential (ΔΨ(m) ) and Bcl-2 family proteins were analysed. 3. Both adaphostin and CMS-9 induced U937 cell apoptosis, characterized by dissipation of ΔΨ(m) and ROS generation. Combined treatment further increased ΔΨ(m) loss and reduced the viability of adaphostin-treated cells. Unlike in CMS-9-treated cells, in adaphostin-treated cells ROS-induced increases in [Ca(2+) ](i) were observed. CMS-9-induced ROS generation resulted in p38 MAPK activation, whereas adaphostin treatment elicited ROS/Ca(2+) -mediated inactivation of Akt and ERK. Moreover, Akt was found to be involved in ERK phosphorylation. Suppression of p38 MAPK activation blocked CMS-9-induced ΔΨ(m) loss and Bcl-xL downregulation. Overexpression of constitutively active Akt and mitogen-activated protein kinase kinase (MEK) 1 rescued adaphostin-induced ΔΨ(m) loss and Bcl-2 downregulation. Similarly, CMS-9 augmented adaphostin toxicity in human leukaemia K562 cells via increased mitochondrial alterations. 4. The results suggest that two distinct pathways mediate adaphostin- and CMS-9-induced mitochondrial damage (i.e. the ROS-Ca(2+) -Akt-ERK and ROS-p38 MAPK pathways, respectively). These distinct pathway explain the augmentation by CMS-9 of ΔΨ(m) loss and apoptosis in adaphostin-treated U937 cells. © 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.

  15. Intracellular fate of recombinant human interferon-gamma (rIFN) in U937 cells

    International Nuclear Information System (INIS)

    Finbloom, D.S.

    1986-01-01

    After IFN binds to specific receptors on macrophages, both modulation of surface molecules and induction of microbicidal and tumoricidal activity occurs 24-48 hr later. Since the intracellular events required to insure these responses are poorly defined, the fate of radiolabeled rIFN in U937 cells was examined. Endocytosis was determined by exposing cells to pH 2.5 to allow rIFN to dissociate leaving only intracellular ligand. Degradation was measured as trichloroacetic acid soluble radioactivity in the media. Of the 4-5000 molecules of rIFN that specifically and saturably (at 300 U/ml) bound at 4 0 C, 40% dissociated during 15-30 min after cells were warmed to 37 0 C. However, if cells were continuously exposed at 37 0 C to lower levels of rIFN (60-100 U/ml), 30-40% of those molecules capable of binding to the cell at that concentration were internalized. Furthermore, 60% of the molecules were degraded during 3-4 hr of additional culture. Since exposure of cells to chloroquine and monensin resulted in only partial inhibition of degradation (75% and 43%, respectively), there may also be degradation within endosomes or on the cell following binding to its receptor. Soon thereafter, degradation products are measurable. Since many biological responses require prolonged incubation with the molecule, intracellular processing of IFN may be important for expression of these effects

  16. Localization of urokinase-type plasminogen activator receptor on U937 cells

    DEFF Research Database (Denmark)

    Hansen, S H; Behrendt, N; Danø, K

    1990-01-01

    The binding of human urokinase-type plasminogen activator (u-PA) to the surface of the human monocytic cell line U937 was studied by immunological detection of bound u-PA or binding of biotinylated diisopropyl fluorophosphate-inactivated human u-PA visualized by light or electron microscopy...

  17. A novel assay system for macrophage-activating factor activity using a human U937 cell line.

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    Ishikawa, Mami; Inoue, Takahiro; Inui, Toshio; Kuchiike, Daisuke; Kubo, Kentaro; Uto, Yoshihiro; Nishikata, Takahito

    2014-08-01

    Macrophages play important roles in antitumor immunity, and immunotherapy with the group-specific component protein-derived macrophage-activating factor (GcMAF) has been reported to be effective in patients with various types of cancers. However, in macrophage research, it is important to properly evaluate macrophage activity. U937 macrophages were induced by 12-O-tetradecanoyl-13-phorbolacetate (TPA). The phagocytic activity of macrophages was evaluated as the internalized beads ratio. The MAF activity was assessed at 30 min after MAF addition as the activation ratio. We established a novel assay for phagocytic activities using differentiated U937 macrophages. The novel protocol was simple and rapid and was sensitive for GcMAF. This protocol should be useful not only for basic studies, such as those on molecular mechanisms underlying macrophage activation, but also for clinical studies, such as assessment of GcMAF activity prior to clinical use. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  18. Mechanism of Hericium erinaceus (Yamabushitake) Mushroom-Induced Apoptosis of U937 Human Monocytic Leukemia Cells

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    Phytochemicals in some foods are a potential source of bioactive safe compounds for cancer chemoprevention. In the present study, we evaluated hot water (HWE), microwaved 50% ethanol (MWE), acidic (ACE), and alkaline (AKE) extracts of the fruit body (sporocarp) of edible Hericium erinaceus (Yamabus...

  19. Combination of doxorubicin and low-intensity ultrasound causes a synergistic enhancement in cell killing and an additive enhancement in apoptosis induction in human lymphoma U937 cells.

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    Yoshida, Toru; Kondo, Takashi; Ogawa, Ryohei; Feril, Loreto B; Zhao, Qing-Li; Watanabe, Akihiko; Tsukada, Kazuhiro

    2008-04-01

    Potential clinical use of ultrasound (US) in enhancing the effects of anticancer drugs in the treatment of cancers has been highlighted in previous reports. Increased uptake of drugs by the cancer cells due to US has been suggested as a mechanism. However, the precise mechanism of the enhancement has not yet been elucidated. Here, the combined effects of low-intensity pulsed US and doxorubicin (DOX) on cell killing and apoptosis induction of U937 cells, and mechanisms involved were investigated. Human myelomonocytic lymphoma U937 cells were used for the experiments. Experiments were conducted in 4 groups: (1) non-treated, (2) DOX treated (DOX), (3) US treated (US), and (4) combined (DOX + US). In DOX +US, cells were exposed to 5 microM DOX for 30 min and sonicated by 1 MHz pulsed US (PRF 100 Hz, DF 10%) at intensities of 0.2-0.5 W/cm(2) for 60 s. The cells were washed and incubated for 6 h. The viability was evaluated by Trypan blue dye exclusion test and apoptosis and incorporation of DOX was assessed by flow cytometry. Involvement of sonoporation in molecular incorporation was evaluated using FITC-dextran, hydroxyl radical formation was measured by electron paramagnetic resonance-spin trapping, membrane alteration including lipid peroxidation and membrane fluidity by DOX was evaluated using cis-parinaric acid and perylene fluorescence polarization method, respectively. Synergistic enhancement in cell killing and additive enhancement in induction of apoptosis were observed at and above 0.3 W/cm(2). No enhancement was observed at 0.2 W/cm(2) in cell killing and induction of apoptosis. Hydroxyl radicals formation was detected at and above 0.3 W/cm(2). The radicals were produced more in the DOX + US than US alone. Incorporation of DOX was increased 13% in DOX + US (vs. DOX) at 0.5 W/cm(2). Involvement of sonoporation for increase of drug uptake was suggested by experiment using FITC-labeled dextran. We made the hypothesis that DOX treatment made the cells weaken

  20. Gamma interferon augments Fc gamma receptor-mediated dengue virus infection of human monocytic cells.

    OpenAIRE

    Kontny, U; Kurane, I; Ennis, F A

    1988-01-01

    It has been reported that anti-dengue antibodies at subneutralizing concentrations augment dengue virus infection of monocytic cells. This is due to the increased uptake of dengue virus in the form of virus-antibody complexes by cells via Fc gamma receptors. We analyzed the effects of recombinant human gamma interferon (rIFN-gamma) on dengue virus infection of human monocytic cells. U937 cells, a human monocytic cell line, were infected with dengue virus in the form of virus-antibody complexe...

  1. Identification of latent tuberculosis infection-related microRNAs in human U937 macrophages expressing Mycobacterium tuberculosis Hsp16.3.

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    Meng, Qing-Lin; Liu, Fei; Yang, Xing-Yuan; Liu, Xiao-Mei; Zhang, Xia; Zhang, Chun; Zhang, Zong-De

    2014-02-12

    Latent tuberculosis infection (LTBI) relies on a homeostasis of macrophages and Mycobacterium tuberculosis (Mtb). The small heat shock protein, Mtb Hsp16.3 (also known as latency-associated antigen), plays an important role in Mtb persistence within macrophages. However, the mechanism of LTBI remains elusive. The aim of this study was to delineate LTBI-related miRNA expression in U937 macrophages expressing Mtb Hsp16.3 protein. U937 macrophages were infected with an integrase-deficient Lentivirus vector to transiently express Mtb Hsp16.3, and green fluorescent protein (GFP) as a control. We used a microRNA (miRNA) microarray chip containing more than 1000 probes to identify the significant differentially expressed miRNAs in the infected U937 cells, and employed real-time quantitative polymerase chain reaction (qRT-PCR) for validation. Furthermore, we confirmed these candidate LTBI-related miRNAs in peripheral blood mononuclear cells from subjects with LTBI and in healthy control individuals. Functional annotation prediction of miRNA target genes and pathway enrichment analyses were used to explore the putative links between these miRNAs and LTBI. Analysis of the miRNA expression profile identified 149 miRNAs that were differentially expressed in U937 macrophages expressing Mtb Hsp16.3 compared with the control expressing GFP. The expression level of seven miRNAs (miR-424-5p, miR-493-5p, miR-296-5p, miR-27b-3p, miR-377-5p, miR-3680-5p, miR-191-5p) were validated by qRT-PCR. The expression level of four miRNAs (miR-424-5p, miR-27b-3p, miR-377-5p, miR-3680-5p) in the peripheral blood mononuclear cells samples from LTBI and healthy participants reflected the altered patterns observed in the microarray profile. The bioinformatic analyses suggest that the miRNAs may regulate Mtb latent infection by affecting the development of macrophage cells. The results suggest that miRNA expression may play a considerable role in the pathogenesis of LTBI, and this would increase our

  2. Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

    OpenAIRE

    Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

    1988-01-01

    Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding t...

  3. Cell shape and organelle modification in apoptotic U937 cells

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    MR Montinari

    2009-12-01

    Full Text Available U937 cells induced to apoptosis, progressively and dramatically modified their cell shape by intense blebbing formation, leading to the production of apoptotic bodies. The blebs evolved with time; milder forms of blebbing involving only a region or just the cortical part of the cytoplasm were observed within the first hour of incubation with puromycin; blebbing involving the whole cell body with very deep constrictions is the most frequent event observed during late times of incubation. The ultrastructural analysis of apoptotic cells revealed characteristic features of nuclear fragmentation (budding and cleavage mode and cytoplasmatic modifications. The cytoplasm of blebs does not contain organelles, such as ribosomes or mitochondria. Scarce presence of endoplasmic reticulum can be observed at the site of bleb detachment. However, blebbing is a dispensable event as evaluated by using inhibitor of actin polymerization. In the present study, the progressive modifications of the nucleus, mitochondria, nuclear fragmentation, cytoplasmic blebs formation and production of apoptotic bodies in U937 monocytic cells induced to apoptosis by puromycin (an inhibitor of protein synthesis were simultaneously analyzed.

  4. IL-4 induces cAMP and cGMP in human monocytic cells

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    B. Dugas

    1995-01-01

    Full Text Available Human monocytes, preincubated with IFN-γ respond to IL-4 by a cGMP increase through activation of an inducible NO synthase. Here, IL-4 was found to induce an accumulation of cGMP (1 – 3 min and cAMP (20 – 25 min in unstimulated monocytes. This was impaired with NOS inhibitors, but also with EGTA and calcium/calmodulin inhibitors. These results suggest that: (1 IL-4 may stimulate different NOS isoforms in resting and IFN-γ activated monocytes, and (2 cAMP accumulation may be partially dependent on the NO pathway. By RT-PCR, a type III constitutive NOS mRNA was detected in U937 monocytic cells. IL-4 also increased the [Ca2+]i in these cells. Different NOS may thus be expressed in monocytic cells depending on their differentiation and the signals they receive.

  5. CoCr wear particles generated from CoCr alloy metal-on-metal hip replacements, and cobalt ions stimulate apoptosis and expression of general toxicology-related genes in monocyte-like U937 cells

    Energy Technology Data Exchange (ETDEWEB)

    Posada, Olga M., E-mail: O.M.PosadaEstefan@leeds.ac.uk [Biomedical Engineering Department, University of Strathclyde, Wolfson Centre, Glasgow G4 0NW (United Kingdom); Gilmour, Denise [Pure and Applied Chemistry Department, University of Strathclyde, Thomas Graham Building, Glasgow G1 1XL (United Kingdom); Tate, Rothwelle J., E-mail: r.j.tate@strath.ac.uk [Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE (United Kingdom); Grant, M. Helen [Biomedical Engineering Department, University of Strathclyde, Wolfson Centre, Glasgow G4 0NW (United Kingdom)

    2014-11-15

    Cobalt-chromium (CoCr) particles in the nanometre size range and their concomitant release of Co and Cr ions into the patients' circulation are produced by wear at the articulating surfaces of metal-on-metal (MoM) implants. This process is associated with inflammation, bone loss and implant loosening and led to the withdrawal from the market of the DePuy ASR™ MoM hip replacements in 2010. Ions released from CoCr particles derived from a resurfacing implant in vitro and their subsequent cellular up-take were measured by ICP-MS. Moreover, the ability of such metal debris and Co ions to induce both apoptosis was evaluated with both FACS and immunoblotting. qRT-PCR was used to assess the effects on the expression of lymphotoxin alpha (LTA), BCL2-associated athanogene (BAG1), nitric oxide synthase 2 inducible (NOS2), FBJ murine osteosarcoma viral oncogene homolog (FOS), growth arrest and DNA-damage-inducible alpha (GADD45A). ICP-MS showed that the wear debris released significant (p < 0.05) amounts of Co and Cr ions into the culture medium, and significant (p < 0.05) cellular uptake of both ions. There was also an increase (p < 0.05) in apoptosis after a 48 h exposure to wear debris. Analysis of qRT-PCR results found significant up-regulation (p < 0.05) particularly of NOS2 and BAG1 in Co pre-treated cells which were subsequently exposed to Co ions + debris. Metal debris was more effective as an inducer of apoptosis and gene expression when cells had been pre-treated with Co ions. This suggests that if a patient receives sequential bilateral CoCr implants, the second implant may be more likely to produce adverse effects than the first one. - Highlights: • Effects of CoCr nanoparticles and Co ions on U937 cells were investigated. • Ions released from wear debris play an important role in cellular response, • Toxicity of Co ions could be related to NO metabolic processes and apoptosis. • CoCr particles were a more effective inducer of apoptosis after cell

  6. Basic study on apoptosis induction into cancer cells U-937 and EL-4 by ultrasound exposure.

    Science.gov (United States)

    Takeuchi, Shinichi; Udagawa, Yoshiko; Oku, Yumiko; Fujii, Takuma; Nishimura, Hiroyuki; Kawashima, Norimichi

    2006-12-22

    Recently, the low invasive cancer treatments with small aftereffects have been considered. We are studying on the suppression methods of cancer cell proliferation with ultrasound. Cancer cells of mouse T lymphoma (EL-4) have been used in our study. The human histitocytic lymphoma cells (U-937) was used in this time. The cancer cells were cultured in a culture medium of RPMI1640. The standing wave acoustic field was formed in a water tank of our ultrasound exposure system by a vibrating plate driven with a Langevine type transducer. The U-937 and EL-4 were exposed to ultrasound in the acoustic field with spatial average acoustic intensity of 350 mW/cm(2) at 150 kHz. The viable rate of EL-4 decreased with the lapse of culture time after ultrasound exposure. U-937 did not show the remarkable decrease tendency. The proliferation of U-937 which exposed to ultrasound with 700 mW/cm(2) was suppressed. It can be thought that apoptosis was induced in the cancer cells in this condition. We observed the morphological change on the U-937 exposed to ultrasound with this condition. The morphological changes by apoptosis like the shrink of cells, formation of apoptotic bodies etc. can be observed with an optical microscope and a phase contrast microscope.

  7. Characterization of NF-κB Reporter U937 Cells and Their Application for the Detection of Inflammatory Immune-Complexes.

    Directory of Open Access Journals (Sweden)

    Csilla Kecse-Nagy

    Full Text Available Our study tested the hypothesis that immunoglobulins differ in their ability to activate the nuclear factor-κB pathway mediated cellular responses. These responses are modulated by several properties of the immune complex, including the ratio of antibody isotypes binding to antigen. Immunoassays allow the measurement of antigen specific antibodies belonging to distinct immunoglobulin classes and subclasses but not the net biological effect of the combination of these antibodies. We set out to develop a biosensor that is suitable for the detection and characterization of antigen specific serum antibodies. We genetically modified the monocytoid U937 cell line carrying Fc receptors with a plasmid encoding NF-κB promoter-driven GFP. This clone, U937-NF-κB, was characterized with respect to FcR expression and response to solid-phase immunoglobulins. Human IgG3, IgG4 and IgG1 induced GFP production in a time- and dose-dependent manner, in this order of efficacy, while IgG2 triggered no activation at the concentrations tested. IgA elicited no response alone but showed significant synergism with IgG3 and IgG4. We confirmed the importance of activation via FcγRI by direct stimulation with monoclonal antibody and by competition assays. We used citrullinated peptides and serum from rheumatoid arthritis patients to generate immune complexes and to study the activation of U937-NF-κB, observing again a synergistic effect between IgG and IgA. Our results show that immunoglobulins have distinct pro-inflammatory potential, and that U937-NF-κB is suitable for the estimation of biological effects of immune-complexes, offering insight into monocyte activation and pathogenesis of antibody mediated diseases.

  8. Transfecting Human Monocytes with RNA.

    Science.gov (United States)

    Dannull, Jens; Nair, Smita K

    2016-01-01

    Targeting monocytes as a delivery system for drugs or nucleic acids, and thereby harnessing their natural tissue-infiltrating capacity, has become an area of intense investigation in both basic and clinical research. Herein we describe an efficient method to deliver mRNA (messenger RNA) or siRNA (small interfering RNA) into human monocytes by electroporation. This method can be applied in the laboratory to monocytes isolated via magnetic bead-based techniques, or in a clinical setting using monocytes that were collected via counterflow centrifugation elutriation using the Elutra(®) Cell Separation System. We further demonstrate that electroporation of monocytes with RNA represents a robust and highly relevant approach to modify monocytes for cell-based therapies. Last, the procedure described can readily be adapted to monocytes from different species, hence facilitating research in animal models.

  9. Dimethyl sulfoxide potentiates death receptor-mediated apoptosis in the human myeloid leukemia U937 cell line through enhancement of mitochondrial membrane depolarization

    Czech Academy of Sciences Publication Activity Database

    Vondráček, Jan; Souček, Karel; Sheard, M. A.; Chramostová, Kateřina; Andrysík, Zdeněk; Hofmanová, Jiřina; Kozubík, Alois

    2006-01-01

    Roč. 30, č. 1 (2006), s. 81-89 ISSN 0145-2126 R&D Projects: GA ČR(CZ) GA524/03/0766 Institutional research plan: CEZ:AV0Z50040507 Keywords : human myeloid leukemia * DMSO * apoptosis Subject RIV: BO - Biophysics Impact factor: 2.483, year: 2006

  10. Suppression of multiple bioactivities of interleukin-1 and interleukin-2 production by U937 conditioned medium

    International Nuclear Information System (INIS)

    Wiblin, R.T.; Edmonds, K.; Ellner, J.J.

    1986-01-01

    The human macrophage-like cell line U937 spontaneously produces a factor which blocks interleukin-1 (IL-1) activity for mouse thymocytes but not mitosis of T-lymphoblastoid cells. The authors examined the effects of U937 conditioned medium (CM) on other IL-1 activities and on interleukin-2 (IL-2) production. U937 was cultured at 5 x 10 6 /ml in RPMI-1640 at 37 0 C for 5 days. The resulting CM inhibited the mitogenic response of C3H/HeJ mouse thymocytes to an IL-1 standard, with an inhibitory of activity of 6.64 U/ml (1 U = reciprocal dilution producing 50% inhibition of maximal response). Similarly, CM inhibited (10.12 U/ml) the fibroblast stimulation promoter activity of IL-1. The effect of CM on IL-2 production was assessed in a direct assay in which IL-2 production by γ-irradiated (12,000 rads) MLA-144 lymphosarcoma cells was assayed as 3 H-thymidine incorporation in CTLL-20 cells. The suppressive activity of CM was 4.95 U/ml; CM did not interfere with the response of CTLL-20 to IL-2. These studies establish that U937 produces factors with multiple, related biological activities; U937 CM blocks IL-2 dependent (thymocyte mitogenesis) and IL-2 independent (fibroblast proliferation) IL-1 activities and interferes with production of, but not response to, IL-2. U937 is an excellent model to study growth inhibitory properties of mononuclear phagocytes

  11. Binding of recombinant HIV coat protein gp120 to human monocytes

    International Nuclear Information System (INIS)

    Finbloom, D.S.; Hoover, D.L.; Meltzer, M.S.

    1991-01-01

    Inasmuch as the exact level of CD4 Ag expression on macrophages is controversial and because HIV may interact with macrophages in a manner different from that on T cells, we analyzed the binding of gp120 to freshly isolated and cultured monocytes. rgp120 was iodinated using the lactoperoxidase method to a sp. act. of 600 Ci/mmol. Highly purified monocytes (greater than 90%) were isolated from the leukapheresed blood of normal volunteers by Ficoll-Hypaque sedimentation followed by countercurrent centrifugal elutriation and cultured 7 days in DMEM supplemented with 1000 U/ml macrophage CSF in 10% human serum. Whereas MOLT/4 cells consistently bound freshly prepared 125I-rgp120 at 80% specificity with 5100 +/- 700 mol/cell, MCSF cultured monocytes bound rgp120 at only 0 to 20% specificity and 420 +/- 200 mol/cell. Most of the radioactivity bound by these cells could not be blocked by the addition of unlabeled rgp120. In contrast, the U937 myeloid cell line bound rgp120 with 50% specificity and about 2500 mol/cell. Whereas the antibody OKT4a (anti-CD4) blocked 80% of the binding on MOLT/4 cells and 50% on U937 cells, binding was only inhibited on the average of 6% on cultured monocytes. When soluble rCD4 was used as an inhibitor, binding to MOLT/4 cells was blocked by 80%. In contrast, binding to cultured monocytes was inhibited by 28%. HIV infectivity was blocked by similar concentrations of OKT4a. These observations suggest that although most binding of gp120 to cultured monocytes is not to the CD4 determinant, several hundred molecules do bind to a CD4-like molecule which promotes virus entry and replication

  12. Stimulation of the Angiotensin II AT2 Receptor is Anti-inflammatory in Human Lipopolysaccharide-Activated Monocytic Cells

    DEFF Research Database (Denmark)

    Menk, Mario; Graw, Jan Adriaan; von Haefen, Clarissa

    2015-01-01

    and the translational level over course of time. Treatment with C21 attenuated the expression of TNFα, IL-6, and IL-10 after LPS challenge in both cell lines in a time- and dose-dependent manner. We conclude that selective AT2 receptor stimulation acts anti-inflammatory in human monocytes. Modulation of cytokine......Recently, AT2 receptors have been discovered on the surface of human immunocompetent cells such as monocytes. Data on regulative properties of this receptor on the cellular immune response are poor. We hypothesized that direct stimulation of the AT2 receptor mediates anti-inflammatory responses...... in these cells. Human monocytic THP-1 and U937 cells were stimulated with lipopolysaccharide (LPS) and the selective AT2 receptor agonist Compound 21 (C21). Expression of pro- and anti-inflammatory cytokines IL-6, IL-10, tumor necrosis factor-α (TNFα), and IL-1β were analyzed on both the transcriptional...

  13. Role of ATM in bystander signaling between human monocytes and lung adenocarcinoma cells.

    Science.gov (United States)

    Ghosh, Somnath; Ghosh, Anu; Krishna, Malini

    2015-12-01

    The response of a cell or tissue to ionizing radiation is mediated by direct damage to cellular components and indirect damage mediated by radiolysis of water. Radiation affects both irradiated cells and the surrounding cells and tissues. The radiation-induced bystander effect is defined by the presence of biological effects in cells that were not themselves in the field of irradiation. To establish the contribution of the bystander effect in the survival of the neighboring cells, lung carcinoma A549 cells were exposed to gamma-irradiation, 2Gy. The medium from the irradiated cells was transferred to non-irradiated A549 cells. Irradiated A549 cells as well as non-irradiated A549 cells cultured in the presence of medium from irradiated cells showed decrease in survival and increase in γ-H2AX and p-ATM foci, indicating a bystander effect. Bystander signaling was also observed between different cell types. Phorbol-12-myristate-13-acetate (PMA)-stimulated and gamma-irradiated U937 (human monocyte) cells induced a bystander response in non-irradiated A549 (lung carcinoma) cells as shown by decreased survival and increased γ-H2AX and p-ATM foci. Non-stimulated and/or irradiated U937 cells did not induce such effects in non-irradiated A549 cells. Since ATM protein was activated in irradiated cells as well as bystander cells, it was of interest to understand its role in bystander effect. Suppression of ATM with siRNA in A549 cells completely inhibited bystander effect in bystander A549 cells. On the other hand suppression of ATM with siRNA in PMA stimulated U937 cells caused only a partial inhibition of bystander effect in bystander A549 cells. These results indicate that apart from ATM, some additional factor may be involved in bystander effect between different cell types. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Autocrine secretion of tumor necrosis factor under the influence of interferon-γ amplifies HLA-DR gene induction in human monocytes

    International Nuclear Information System (INIS)

    Arenzana-Seisdedos, F.; Mogensen, S.C.; Vuillier, F.; Fiers, W.; Virelizier, J.L.

    1988-01-01

    Recombinant interferon-γ (IFN-γ) induced HLA-DR gene expression in both U937 and THP-1 human monocytic cell lines, although the former was only very weakly inducible. Combination of recombinant tumor necrosis factor (TNF) and IFN-γ resulted in a synergistic enhancement of DR mRNA and protein induction in both cell lines. TNF alone increased the constitutive expression of the DR gene in THP-1 cells. In the HLA class II-negative U937 cells, TNF used alone was not able to induce DR gene expression. Such a negative result was not due to a lack of TNF receptor expression in U937 cells, since TNF clearly induced HLA class I and TNF gene expression in this cell line. THP-1, but not U937, cells secreted TNF under the influence of IFN-γ. Neutralization of TNF by a specific antibody decreased IFN-γ-induced DR antigen expression in THP-1 cultures. These observations indicate that TNF is not able to directly induce DR gene expression, but rather amplifies ongoing expression of this gene, whether constitutive or induced by IFN-γ. In the two cell lines tested, the level of DR inducibility under the influence of IFN-γ used alone depended on a different inducibility of TNF secretion by IFN-γ. Altogether, the observations indicate that TNF, whether exogenous or endogenously produced under the influence of IFN-γ, amplifies DR gene expression in monocytes, a phenomenon that may provide to such antigen-presenting cells a selective sensitivity to the DR-inducing effects of IFN-γ

  15. hCLP46 regulates U937 cell proliferation via Notch signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Wenzhan; Du, Jie; Chu, Qiaoyun [College of Life Science, Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Wang, Youxin [School of Public Health and Family Medicine, Capital Medical University, Beijing 100069 (China); Liu, Lixin [College of Life Science, Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Song, Manshu [School of Public Health and Family Medicine, Capital Medical University, Beijing 100069 (China); Wang, Wei, E-mail: wei6014@yahoo.com [College of Life Science, Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); School of Public Health and Family Medicine, Capital Medical University, Beijing 100069 (China)

    2011-04-29

    Highlights: {yields} Knock down of hCLP46 by RNAi impairs mammalian Notch signaling. {yields} hCLP46 affects neither cell surface Notch1 expression nor ligand-receptor binding. {yields} Knock down of hCLP46 inhibits U937 cell-growth by up-regulation of CDKN1B. -- Abstract: Human CAP10-like protein 46 kDa (hCLP46) is the homolog of Rumi, which is the first identified protein O-glucosyltransferase that modifies Notch receptor in Drosophila. Dysregulation of hCLP46 occurs in many hematologic diseases, but the role of hCLP46 remains unclear. Knockdown of hCLP46 by RNA interference resulted in decreased protein levels of endogenous Notch1, Notch intracellular domain (NICD) and Notch target gene Hes-1, suggesting the impairment of the Notch signaling. However, neither cell surface Notch expression nor ligand binding activities were affected. In addition, down-regulated expression of hCLP46 inhibited the proliferation of U937 cells, which was correlated with increased cyclin-dependent kinase inhibitor (CDKI) CDKN1B (p27) and decreased phosphorylation of retinoblastoma (RB) protein. We showed that lack of hCLP46 results in impaired ligand induced Notch activation in mammalian cell, and hCLP46 regulates the proliferation of U937 cell through CDKI-RB signaling pathway, which may be important for the pathogenesis of leukemia.

  16. Assessment of the U937 cell line for the detection of contact allergens

    International Nuclear Information System (INIS)

    Python, Francois; Goebel, Carsten; Aeby, Pierre

    2007-01-01

    The human myeloid cell line U937 was evaluated as an in vitro test system to identify contact sensitizers in order to develop alternatives to animal tests for the cosmetic industry. Specific culture conditions (i.e., presence of interleukin-4, IL-4) were applied to obtain a dendritic cell-like phenotype. In the described test protocol, these cells were exposed to test chemicals and then analyzed by flow cytometry for CD86 expression and by quantitative real-time reverse transcriptase-polymerase chain reaction for IL-1β and IL-8 gene expressions. Eight sensitizers, three non-sensitizers and five oxidative hair dye precursors were examined after 24-, 48- and 72-h exposure times. Test item-specific modulations of the chosen activation markers (CD86, IL-1β and IL-8) suggest that this U937 activation test could discriminate test items classified as contact sensitizers or non-sensitizers in the local lymph node assay in mice (LLNA). More specifically, a test item can be considered as a potential sensitizer when it significantly induced the upregulation of the expression of at least two markers. Using this approach, we could correctly evaluate the dendritic cell (DC) activation potential for 15 out of 16 tested chemicals. We conclude that the U937 activation test may represent an useful tool in a future in vitro test battery for predicting sensitizing properties of chemicals

  17. Icariside II induces apoptosis in U937 acute myeloid leukemia cells: role of inactivation of STAT3-related signaling.

    Directory of Open Access Journals (Sweden)

    Sang-Hun Kang

    Full Text Available BACKGROUND: The aim of this study is to determine anti-cancer effect of Icariside II purified from the root of Epimedium koreanum Nakai on human acute myeloid leukemia (AML cell line U937. METHODOLOGY/PRINCIPAL FINDINGS: Icariside II blocked the growth U937 cells in a dose- and time-dependent manner. In this anti-proliferation process, this herb compound rendered the cells susceptible to apoptosis, manifested by enhanced accumulation of sub-G1 cell population and increased the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL-positive cells. Icariside II was able to activate caspase-3 and cleaved poly (ADP-ribose polymerase (PARP in a time-dependent manner. Concurrently, the anti-apoptotic proteins, such as bcl-x(L and survivin in U937 cells, were downregulated by Icariside II. In addition, Icariside II could inhibit STAT3 phosphorylation and function and subsequently suppress the activation of Janus activated kinase 2 (JAK2, the upstream activators of STAT3, in a dose- and time-dependent manner. Icariside II also enhanced the expression of protein tyrosine phosphatase (PTP SH2 domain-containing phosphatase (SHP-1, and the addition of sodium pervanadate (a PTP inhibitor prevented Icariside II-induced apoptosis as well as STAT3 inactivation in STAT3 positive U937 cells. Furthermore, silencing SHP-1 using its specific siRNA significantly blocked STAT3 inactivation and apoptosis induced by Icariside II in U937 cells. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated that via targeting STAT3-related signaling, Icariside II sensitizes U937 cells to apoptosis and perhaps serves as a potent chemotherapeutic agent for AML.

  18. Coordinate viral induction of tumor necrosis factor α and interferon β in human B cells and monocytes

    International Nuclear Information System (INIS)

    Goldfeld, A.E.; Maniatis, T.

    1989-01-01

    Human tumor necrosis factor α (TNF-α) gene expression can be induced primarily in cells of the monocyte/macrophage lineage by a variety of inducers, including lipopolysaccharide, phorbol esters such as phorbol 12-myristate 13-acetate, and virus or synthetic double-stranded RNA [poly(I)·poly(C)]. In this paper the authors show that the TNF-α gene also responds to virus and phorbol 12-myristate 13-acetate in B lymphocytes and that virus is the most potent inducer of TNF-α mRNA in both monocyte and B-cell lines. In addition, they show that viral infection coinduces the expression of TNF-α and interferon β mRNA and that viral induction of both genes is blocked by the kinase inhibitor 2-aminopurine. Inhibition of protein synthesis with cycloheximide had no effect on mRNA expression of the genes in one of three cell lines tested (U937) but blocked the viral induction of both genes in another (Namalwa). Thus, the regulatory factors required for mRNA induction of both genes are present prior to the addition of virus in U937 but not in Namalwa cells. However, in a third cell line (JY), cycloheximide blocked viral induction of the interferon β gene but not the TNF-α gene. Taken together, these observations suggest that viral induction of TNF-α and interferon β gene expression may involve overlapping pathways with both common and distinct regulatory factors

  19. CD1 molecule expression on human monocytes induced by granulocyte-macrophage colony-stimulating factor.

    Science.gov (United States)

    Kasinrerk, W; Baumruker, T; Majdic, O; Knapp, W; Stockinger, H

    1993-01-15

    In this paper we demonstrate that granulocyte-macrophage CSF (GM-CSF) specifically induces the expression of CD1 molecules, CD1a, CD1b and CD1c, upon human monocytes. CD1 molecules appeared upon monocytes on day 1 of stimulation with rGM-CSF, and expression was up-regulated until day 3. Monocytes cultured in the presence of LPS, FMLP, PMA, recombinant granulocyte-CSF, rIFN-gamma, rTNF-alpha, rIL-1 alpha, rIL-1 beta, and rIL-6 remained negative. The induction of CD1 molecules by rGM-CSF was restricted to monocytes, since no such effect was observed upon peripheral blood granulocytes, PBL, and the myeloid cell lines Monomac1, Monomac6, MV4/11, HL60, U937, THP1, KG1, and KG1A. CD1a mRNA was detectable in rGM-CSF-induced monocytes but not in those freshly isolated. SDS-PAGE and immunoblotting analyses of CD1a mAb VIT6 immunoprecipitate from lysate of rGM-CSF-activated monocytes revealed an appropriate CD1a polypeptide band of 49 kDa associated with beta 2-microglobulin. Expression of CD1 molecules on monocytes complements the distribution of these structures on accessory cells, and their specific induction by GM-CSF strengthens the suggestion that CD1 is a family of crucial structures required for interaction between accessory cells and T cells.

  20. Tumor Necrosis Factor-α Induced Apoptosis in U937 Cells Promotes Cathepsin D-Independent Stefin B Degradation.

    Science.gov (United States)

    Bidovec, Katja; Božič, Janja; Dolenc, Iztok; Turk, Boris; Turk, Vito; Stoka, Veronika

    2017-12-01

    Lysosomal cathepsins were previously found to be involved in tumor necrosis factor-α (TNFα)-induced apoptosis. However, there are opposing views regarding their role as either initiators or amplifiers of the signaling cascade as well as the order of molecular events during this process. In this study, we investigated the role of cathepsin D (catD) in TNFα/cycloheximide-induced apoptosis in U937 human monocytic cells. TNFα-induced apoptosis proceeds through caspase-8 activation, processing of the pro-apoptotic molecule Bid, mitochondrial membrane permeabilization, and caspase-3 activation. The translocation of lysosomal catD into the cytosol was a late event, suggesting that lysosomal membrane permeabilization and the release of cathepsins are not required for the induction of apoptosis, but rather amplifies the process through the generation of reactive oxygen species. For the first time, we show that apoptosis is accompanied by degradation of the cysteine cathepsin inhibitor stefin B (StfB). CatD did not exhibit a crucial role in this step. However, this degradation was partially prevented through pre-incubation with the antioxidant N-acetyl cysteine, although it did not prevent apoptosis and its progression. These results suggest that the degradation of StfB, as a response to TNFα, could induce a cell death amplification effect as a result of progressive damage to lysosomes during TNFα treatment. J. Cell. Biochem. 118: 4813-4820, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  1. Mammalian protein secretion without signal peptide removal. Biosynthesis of plasminogen activator inhibitor-2 in U-937 cells

    International Nuclear Information System (INIS)

    Ye, R.D.; Wun, T.C.; Sadler, J.E.

    1988-01-01

    Plasminogen activator inhibitor-2 (PAI-2) is a serine protease inhibitor that regulates plasmin generation by inhibiting urokinase and tissue plasminogen activator. The primary structure of PAI-2 suggests that it may be secreted without cleavage of a single peptide. To confirm this hypothesis we have studied the glycosylation and secretion of PAI-2 in human monocytic U-937 cells by metabolic labeling, immunoprecipitation, glycosidase digestion, and protein sequencing. PAI-2 is variably glycosylated on asparagine residues to yield intracellular intermediates with zero, one, two, or three high mannose-type oligosaccharide units. Secretion of the N-glycosylated species began by 1 h of chase and the secreted molecules contained both complex-type N-linked and O-linked oligosaccharides. Enzymatically deglycosylated PAI-2 had an electrophoretic mobility identical to that of the nonglycosylated precursor and also to that of PAI-2 synthesized in vitro in a rabbit reticulocyte lysate from synthetic mRNA derived from full length PAI-2 cDNA. The amino-terminal protein sequence of secreted PAI-2 began with the initiator methionine residue. These results indicate that PAI-2 is glycosylated and secreted efficiently without the cleavage of a signal peptide. PAI-2 shares this property with its nearest homologue in the serine protease inhibitor family, chicken ovalbumin, and appears to be the first well characterized example of this phenomenon among natural mammalian proteins

  2. A role for inflammatory mediators in heterologous desensitization of CysLT1 receptor in human monocytes

    Science.gov (United States)

    Capra, Valérie; Accomazzo, Maria Rosa; Gardoni, Fabrizio; Barbieri, Silvia; Rovati, G. Enrico

    2010-01-01

    Cysteinyl-leukotrienes (cysteinyl-LT) are rapidly generated at sites of inflammation and, in addition to their role in asthma, rhinitis, and other immune disorders, are increasingly regarded as significant inflammatory factors in cancer, gastrointestinal, cardiovascular diseases. We recently demonstrated that in monocyte/macrophage–like U937 cells, extracellular nucleotides heterologously desensitize CysLT1 receptor (CysLT1R)-induced Ca2+ transients. Given that monocytes express a number of other inflammatory and chemoattractant receptors, this study was aimed at characterizing transregulation between these different stimuli. We demonstrate that in U937 cells and in primary human monocytes, a series of inflammatory mediators activating Gi-coupled receptor (FPR1, BLT1) desensitize CysLT1R-induced Ca2+ response unidirectionally through activation of PKC. Conversely, PAF-R, exclusively coupled to Gq, cross-desensitizes CysLT1R without the apparent involvement of any kinase. Interestingly, Gs-coupled receptors (β2AR, H1/2R, EP2/4R) are also able to desensitize CysLT1R response through activation of PKA. Heterologous desensitization seems to affect mostly the Gi-mediated signaling of the CysLT1R. The hierarchy of desensitization among agonists may be important for leukocyte signal processing at the site of inflammation. Considering that monocytes/macrophages are likely to be the major source of cysteinyl-LT in many immunological and inflammatory processes, shedding light on how their receptors are regulated will certainly help to better understand the role of these cells in orchestrating this complex network of integrated signals. PMID:19965602

  3. Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor

    International Nuclear Information System (INIS)

    Antalis, T.M.; Clark, M.A.; Barnes, T.; Lehrbach, P.R.; Devine, P.L.; Schevzov, G.; Goss, N.H.; Stephens, R.W.; Tolstoshev, P.

    1988-01-01

    Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A) + RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the λ P/sub L/ promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated M/sub r/ of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators

  4. Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

    Science.gov (United States)

    Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

    1988-02-01

    Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.

  5. The crosstalk between α-irradiated Beas-2B cells and its bystander U937 cells through MAPK and NF-κB signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Jiamei; Yuan, Dexiao; Xiao, Linlin; Tu, Wenzhi; Dong, Chen; Liu, Weili; Shao, Chunlin, E-mail: clshao@shmu.edu.cn

    2016-01-15

    Highlights: • α-irradiated Beas-2B cells induced bystander effects in macrophage U937 cells. • The neighboring macrophages enhanced the damage of α-irradiated Beas-2B cells. • MAPK and NF-κB pathways were activated in U937 cells after cell co-culture. • NF-κB and MAPK pathways participated in the bilateral bystander responses. - Abstract: Although accumulated evidence suggests that α-particle irradiation induced bystander effect may relevant to lung injury and cancer risk assessment, the exact mechanisms are not yet elucidated. In the present study, a cell co-culture system was used to investigate the interaction between α-particle irradiated human bronchial epithelial cells (Beas-2B) and its bystander macrophage U937 cells. It was found that the cell co-culture amplified the detrimental effects of α-irradiation including cell viability decrease and apoptosis promotion on both irradiated cells and bystander cells in a feedback loop which was closely relevant to the activation of MAPK and NF-κB pathways in the bystander U937 cells. When these two pathways in U937 cells were disturbed by special pharmacological inhibitors before cell co-culture, it was found that a NF-κB inhibitor of BAY 11-7082 further enhanced the proliferation inhibition and apoptosis induction in bystander U937 cells, but MAPK inhibitors of SP600125 and SB203580 protected cells from viability loss and apoptosis and U0126 presented more beneficial effect on cell protection. For α-irradiated epithelial cells, the activation of NF-κB and MAPK pathways in U937 cells participated in detrimental cellular responses since the above inhibitors could largely attenuate cell viability loss and apoptosis of irradiated cells. Our results demonstrated that there are bilateral bystander responses between irradiated lung epithelial cells and macrophages through MAPK and NF-κB signaling pathways, which accounts for the enhancement of α-irradiation induced damage.

  6. The crosstalk between α-irradiated Beas-2B cells and its bystander U937 cells through MAPK and NF-κB signaling pathways

    International Nuclear Information System (INIS)

    Fu, Jiamei; Yuan, Dexiao; Xiao, Linlin; Tu, Wenzhi; Dong, Chen; Liu, Weili; Shao, Chunlin

    2016-01-01

    Highlights: • α-irradiated Beas-2B cells induced bystander effects in macrophage U937 cells. • The neighboring macrophages enhanced the damage of α-irradiated Beas-2B cells. • MAPK and NF-κB pathways were activated in U937 cells after cell co-culture. • NF-κB and MAPK pathways participated in the bilateral bystander responses. - Abstract: Although accumulated evidence suggests that α-particle irradiation induced bystander effect may relevant to lung injury and cancer risk assessment, the exact mechanisms are not yet elucidated. In the present study, a cell co-culture system was used to investigate the interaction between α-particle irradiated human bronchial epithelial cells (Beas-2B) and its bystander macrophage U937 cells. It was found that the cell co-culture amplified the detrimental effects of α-irradiation including cell viability decrease and apoptosis promotion on both irradiated cells and bystander cells in a feedback loop which was closely relevant to the activation of MAPK and NF-κB pathways in the bystander U937 cells. When these two pathways in U937 cells were disturbed by special pharmacological inhibitors before cell co-culture, it was found that a NF-κB inhibitor of BAY 11-7082 further enhanced the proliferation inhibition and apoptosis induction in bystander U937 cells, but MAPK inhibitors of SP600125 and SB203580 protected cells from viability loss and apoptosis and U0126 presented more beneficial effect on cell protection. For α-irradiated epithelial cells, the activation of NF-κB and MAPK pathways in U937 cells participated in detrimental cellular responses since the above inhibitors could largely attenuate cell viability loss and apoptosis of irradiated cells. Our results demonstrated that there are bilateral bystander responses between irradiated lung epithelial cells and macrophages through MAPK and NF-κB signaling pathways, which accounts for the enhancement of α-irradiation induced damage.

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

    NARCIS (Netherlands)

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

    2009-01-01

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

  8. Different mechanisms between premitotic apoptosis and postmitotic apoptosis in X-irradiated U937 cells

    International Nuclear Information System (INIS)

    Shinomiya, Nariyoshi; Kuno, Yukie; Yamamoto, Fuyumi; Fukasawa, Masashi; Okumura, Atsushi; Uefuji, Megumi; Rokutanda, Makoto

    2000-01-01

    Purpose: Apoptosis is currently being evaluated for its importance as a pathway of radiation-induced cell death. However, the difference in the mechanisms between premitotic and postmitotic apoptosis following X-irradiation remains not well understood. We show here that the human monoblastoid cell line U937 can be induced to undergo these two different types of apoptosis. Methods and Materials: U937 cells were irradiated at a dose of 5 or 20 Gy, and the DNA fragmentation rate was measured by both flow cytometric analysis and gel electrophoresis. Activation of caspase-3 was detected by Western blot analysis and fluorogenic assay using acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin (Ac-DEVD-AMC). Detection of mitochondrial transmembrane potential (no. DELTAno. no. PSIno. ) was performed by using Rho123. Chasing of S-phase fraction following X-irradiation was performed after labeling with 5-bromo-2'-deoxyuridine (BrdU). Thymidine was used for synchronization of the cells. Inhibition of caspase-3 activity was achieved by Acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). Results: Time courses of the apoptotic rates, caspase activation, and no. DELTAno. no. PSIno. indicated that two different types of cell death were induced by the different X-ray doses. High-dose X-ray (20 Gy) induced a rapid and strong apoptosis, whereas low-dose X-ray (5 Gy) induced a slow and mild apoptosis. Cell-cycle analyses revealed that there was cell death before cell division in the former apoptosis but the cells must be dying after cell division in the latter apoptosis. By means of cell-cycle synchronization, the S-phase cells proved to be the most sensitive fraction to premitotic apoptosis, but an obvious difference in the susceptibility to cell death among the cell-cycle phases was not observed in postmitotic apoptosis. Ac-DEVD-CHO treatment effectively blocked caspase activity and premitotic apoptosis, but it failed to block postmitotic apoptosis. Conclusions: Irradiation of U937 cells at

  9. p38 mitogen-activated protein kinase mediates IL-8 induction by the ribotoxin deoxynivalenol in human monocytes

    International Nuclear Information System (INIS)

    Islam, Zahidul; Gray, Jennifer S.; Pestka, James J.

    2006-01-01

    The effects of the ribotoxic trichothecene deoxynivalenol (DON) on mitogen-activated protein kinase (MAPK)-mediated IL-8 expression were investigated in cloned human monocytes and peripheral blood mononuclear cells (PBMC). DON (250 to 1000 ng/ml) induced both IL-8 mRNA and IL-8 heteronuclear RNA (hnRNA), an indicator of IL-8 transcription, in the human U937 monocytic cell line in a concentration-dependent manner. Expression of IL-8 hnRNA, mRNA and protein correlated with p38 phosphorylation and was completely abrogated by the p38 MAPK inhibitor SB203580. DON at 500 ng/ml similarly induced p38-dependent IL-8 protein and mRNA expression in PBMC cultures from healthy volunteers. Significantly increased IL-6 and IL-1β intracellular protein and mRNA expression was also observed in PBMC treated with DON (500 ng/ml) which were also partially p38-dependent. Flow cytometry of PBMC revealed that DON-induced p38 phosphorylation varied among individuals relative to both threshold toxin concentrations (25-100 ng/ml) and relative increases in percentages of phospho-p38 + cells. DON-induced p38 activation occurred exclusively in the CD14 + monocyte population. DON was devoid of agonist activity for human Toll-like receptors 2, 3, 4, 5, 7, 8 and 9. However, two other ribotoxins, emetine and anisomycin, induced p38 phosphorylation in PBMC similarly to DON. Taken together, these data suggest that (1) p38 activation was required for induction of IL-8 and proinflammatory gene expression in the monocyte and (2) DON induced p38 activation in human monocytes via the ribotoxic stress response

  10. Characterization of a receptor for human monocyte-derived neutrophil chemotactic factor/interleukin-8

    International Nuclear Information System (INIS)

    Grob, P.M.; David, E.; Warren, T.C.; DeLeon, R.P.; Farina, P.R.; Homon, C.A.

    1990-01-01

    Monocyte-derived neutrophil chemotactic factor/interleukin-8 (MDNCF/IL-8) is an 8,000-dalton protein produced by monocytes which exhibits activity as a chemoattractant for neutrophils with maximal activity achieved at a concentration of 50 ng/ml. This polypeptide has been iodinated by chloramine-T methodology (350 Ci/mM), and specific receptors for MDNCF/IL-8 have been detected on human neutrophils, U937 cells, THP-1 cells, and dimethyl sulfoxide-differentiated HL-60 cells. The binding of MDNCF/IL-8 to human neutrophils is not inhibited by interleukin-1 alpha, tumor necrosis factor-alpha, insulin, or epidermal growth factor. In addition, chemoattractants such as C5a, fMet-Leu-Phe, leukotriene B4, and platelet-activating factor fail to inhibit binding, suggesting that MDNCF/IL-8 utilizes a unique receptor. The receptor for MDNCF/IL-8 is apparently glycosylated since ligand binding is inhibited by the presence of wheat germ agglutinin, a lectin with a binding specificity for N-acetylglucosamine and neuraminic acid. Steady state binding experiments indicate Kd values of 4 and 0.5 nM and receptor numbers of 75,000 and 7,400 for human neutrophils and differentiated HL-60 cells, respectively. 125I-MDNCF/IL-8 bound to human neutrophils is rapidly internalized and subsequently released from cells as trichloroacetic acid-soluble radioactivity. Affinity labeling experiments suggest that the human neutrophil MDNCF/IL-8 receptor exhibits a mass of approximately 58,000 daltons

  11. Deoxynivalenol (DON) is toxic to human colonic, lung and monocytic cell lines, but does not increase the IgE response in a mouse model for allergy

    International Nuclear Information System (INIS)

    Instanes, Christine; Hetland, Geir

    2004-01-01

    We examined whether the common crop mycotoxin deoxynivalenol (DON) from Fusarium species is toxic to human colonic (Caco-2), lung (A549) and monocytic (U937) cell lines. Moreover, since DON reportedly induces increased levels of Th2 cytokines and total IgE, and we have observed that mould extracts adjuvated allergy development in mice, possible adjuvant effect of DON on allergy was studied in a mouse model. For all the cells, exposure to DON for 24 h reduced cellular protein synthesis, proliferation and survival rate dose-dependently. In addition, production of IL-8 in the U937 cell line increased up to eight-fold at levels of DON just lower than the most toxic one, suggesting that IL-8 can be used as an additional index for cytotoxicity in mononuclear phagocytes. However, DON did not increase levels of allergen-specific IgE or IgG1 in the mouse model for allergy. These results suggest that DON, when inhaled or ingested, may have toxic effect on human alveolar macrophages and epithelial cells in lungs and colon, but does not increase the allergic response to allergens

  12. The crosstalk between α-irradiated Beas-2B cells and its bystander U937 cells through MAPK and NF-κB signaling pathways.

    Science.gov (United States)

    Fu, Jiamei; Yuan, Dexiao; Xiao, Linlin; Tu, Wenzhi; Dong, Chen; Liu, Weili; Shao, Chunlin

    2016-01-01

    Although accumulated evidence suggests that α-particle irradiation induced bystander effect may relevant to lung injury and cancer risk assessment, the exact mechanisms are not yet elucidated. In the present study, a cell co-culture system was used to investigate the interaction between α-particle irradiated human bronchial epithelial cells (Beas-2B) and its bystander macrophage U937 cells. It was found that the cell co-culture amplified the detrimental effects of α-irradiation including cell viability decrease and apoptosis promotion on both irradiated cells and bystander cells in a feedback loop which was closely relevant to the activation of MAPK and NF-κB pathways in the bystander U937 cells. When these two pathways in U937 cells were disturbed by special pharmacological inhibitors before cell co-culture, it was found that a NF-κB inhibitor of BAY 11-7082 further enhanced the proliferation inhibition and apoptosis induction in bystander U937 cells, but MAPK inhibitors of SP600125 and SB203580 protected cells from viability loss and apoptosis and U0126 presented more beneficial effect on cell protection. For α-irradiated epithelial cells, the activation of NF-κB and MAPK pathways in U937 cells participated in detrimental cellular responses since the above inhibitors could largely attenuate cell viability loss and apoptosis of irradiated cells. Our results demonstrated that there are bilateral bystander responses between irradiated lung epithelial cells and macrophages through MAPK and NF-κB signaling pathways, which accounts for the enhancement of α-irradiation induced damage. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Signal transduction of p53-independent apoptotic pathway induced by hexavalent chromium in U937 cells

    International Nuclear Information System (INIS)

    Hayashi, Yoko; Kondo, Takashi; Zhao Qingli; Ogawa Ryohei; Cui Zhengguo; Feril, Loreto B.; Teranishi, Hidetoyo; Kasuya, Minoru

    2004-01-01

    It has been reported that the hexavalent chromium compound (Cr(VI)) can induce both p53-dependent and p53-independent apoptosis. While a considerable amount of information is available on the p53-dependent pathway, only little is known about the p53-independent pathway. To elucidate the p53-independent mechanism, the roles of the Ca 2+ -calpain- and mitochondria-caspase-dependent pathways in apoptosis induced by Cr(VI) were investigated. When human lymphoma U937 cells, p53 mutated cells, were treated with 20 μM Cr(VI) for 24 h, nuclear morphological changes and DNA fragmentation were observed. Production of hydroxyl radicals revealed by electron paramagnetic resonance (EPR)-spin trapping, and increase of intracellular calcium ion concentration monitored by digital imaging were also observed in Cr(VI)-treated cells. An intracellular Ca 2+ chelator, BAPTA-AM, and calpain inhibitors suppressed the Cr(VI)-induced DNA fragmentation. The number of cells showing low mitochondrial membrane potential (MMP), high level of superoxide anion radicals (O 2 - ), and high activity of caspase-3, which are indicators of mitochondria-caspase-dependent pathway, increased significantly in Cr(VI)-treated cells. An antioxidant, N-acetyl-L-cysteine (NAC), decreased DNA fragmentation and inhibited the changes in MMP, O 2 - formation, and activation of caspase-3 induced by Cr(VI). No increase of the expressions of Fas and phosphorylated JNK was observed after Cr(VI) treatment. Cell cycle analysis revealed that the fraction of G2/M phase tended to increase after 24 h of treatment, suggesting that Cr(VI)-induced apoptosis is related to the G2 block. These results indicate that Ca 2+ -calpain- and mitochondria-caspase-dependent pathways play significant roles in the Cr(VI)-induced apoptosis via the G2 block, which are independent of JNK and Fas activation. The inhibition of apoptosis and all its signal transductions by NAC suggests that intracellular reactive oxygen species (ROS) are

  14. The proliferative human monocyte subpopulation contains osteoclast precursors

    Science.gov (United States)

    Lari, Roya; Kitchener, Peter D; Hamilton, John A

    2009-01-01

    Introduction Immediate precursors of bone-resorbing osteoclasts are cells of the monocyte/macrophage lineage. Particularly during clinical conditions showing bone loss, it would appear that osteoclast precursors are mobilized from bone marrow into the circulation prior to entering tissues undergoing such loss. The observed heterogeneity of peripheral blood monocytes has led to the notion that different monocyte subpopulations may have special or restricted functions, including as osteoclast precursors. Methods Human peripheral blood monocytes were sorted based upon their degree of proliferation and cultured in macrophage colony-stimulating factor (M-CSF or CSF-1) and receptor activator of nuclear factor-kappa-B ligand (RANKL). Results The monocyte subpopulation that is capable of proliferation gave rise to significantly more multinucleated, bone-resorbing osteoclasts than the bulk of the monocytes. Conclusions Human peripheral blood osteoclast precursors reside in the proliferative monocyte subpopulation. PMID:19222861

  15. Macrophage Activation Mechanisms in Human Monocytic Cell Line-derived Macrophages.

    Science.gov (United States)

    Sumiya, Yu; Ishikawa, Mami; Inoue, Takahiro; Inui, Toshio; Kuchiike, Daisuke; Kubo, Kentaro; Uto, Yoshihiro; Nishikata, Takahito

    2015-08-01

    Although the mechanisms of macrophage activation are important for cancer immunotherapy, they are poorly understood. Recently, easy and robust assay systems for assessing the macrophage-activating factor (MAF) using monocytic cell line-derived macrophages were established. Gene-expression profiles of U937- and THP-1-derived macrophages were compared using gene expression microarray analysis and their responses against several MAFs were examined by in vitro experiments. Activated states of these macrophages could not be assigned to a specific sub-type but showed, however, different unique characteristics. The unique of monocytic cell line-derived macrophages could provide clues to understand the activation mechanism of macrophages and, therefore, help to develop effective cancer immunotherapy with MAFs. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  16. Induction of Programmed Cell Death by Parvovirus H-1 in U937 Cells: Connection with the Tumor Necrosis Factor Alpha Signalling Pathway

    Science.gov (United States)

    Rayet, Béatrice; Lopez-Guerrero, José-Antonio; Rommelaere, Jean; Dinsart, Christiane

    1998-01-01

    The human promonocytic cell line U937 undergoes apoptosis upon treatment with tumor necrosis factor alpha (TNF-α). This cell line has previously been shown to be very sensitive to the lytic effect of the autonomous parvovirus H-1. Parvovirus infection leads to the activation of the CPP32 ICE-like cysteine protease which cleaves the enzyme poly(ADP-ribose)polymerase and induces morphologic changes that are characteristic of apoptosis in a way that is similar to TNF-α treatment. This effect is also observed when the U937 cells are infected with a recombinant H-1 virus which expresses the nonstructural (NS) proteins but in which the capsid genes are replaced by a reporter gene, indicating that the induction of apoptosis can be assigned to the cytotoxic nonstructural proteins in this cell system. The c-Myc protein, which is overexpressed in U937 cells, is rapidly downregulated during infection, in keeping with a possible role of this product in mediating the apoptotic cell death induced by H-1 virus infection. Interestingly, four clones (designated RU) derived from the U937 cell line and selected for their resistance to H-1 virus (J. A. Lopez-Guerrero et al., Blood 89:1642–1653, 1997) failed to decrease c-Myc expression upon treatment with differentiation agents and also resisted the induction of cell death after TNF-α treatment. Our data suggest that the RU clones have developed defense strategies against apoptosis, either by their failure to downregulate c-Myc and/or by activating antiapoptotic factors. PMID:9765434

  17. Comparative DNA microarray analysis of human monocyte derived dendritic cells and MUTZ-3 cells exposed to the moderate skin sensitizer cinnamaldehyde

    International Nuclear Information System (INIS)

    Python, Francois; Goebel, Carsten; Aeby, Pierre

    2009-01-01

    The number of studies involved in the development of in vitro skin sensitization tests has increased since the adoption of the EU 7th amendment to the cosmetics directive proposing to ban animal testing for cosmetic ingredients by 2013. Several studies have recently demonstrated that sensitizers induce a relevant up-regulation of activation markers such as CD86, CD54, IL-8 or IL-1β in human myeloid cell lines (e.g., U937, MUTZ-3, THP-1) or in human peripheral blood monocyte-derived dendritic cells (PBMDCs). The present study aimed at the identification of new dendritic cell activation markers in order to further improve the in vitro evaluation of the sensitizing potential of chemicals. We have compared the gene expression profiles of PBMDCs and the human cell line MUTZ-3 after a 24-h exposure to the moderate sensitizer cinnamaldehyde. A list of 80 genes modulated in both cell types was obtained and a set of candidate marker genes was selected for further analysis. Cells were exposed to selected sensitizers and non-sensitizers for 24 h and gene expression was analyzed by quantitative real-time reverse transcriptase-polymerase chain reaction. Results indicated that PIR, TRIM16 and two Nrf2-regulated genes, CES1 and NQO1, are modulated by most sensitizers. Up-regulation of these genes could also be observed in our recently published DC-activation test with U937 cells. Due to their role in DC activation, these new genes may help to further refine the in vitro approaches for the screening of the sensitizing properties of a chemical.

  18. A simple method for human peripheral blood monocyte Isolation

    Directory of Open Access Journals (Sweden)

    Marcos C de Almeida

    2000-04-01

    Full Text Available We describe a simple method using percoll gradient for isolation of highly enriched human monocytes. High numbers of fully functional cells are obtained from whole blood or buffy coat cells. The use of simple laboratory equipment and a relatively cheap reagent makes the described method a convenient approach to obtaining human monocytes.

  19. Effects of alpha-mangostin on the expression of anti-inflammatory genes in U937 cells

    Directory of Open Access Journals (Sweden)

    Liu Szu-Hsiu

    2012-08-01

    Full Text Available Abstract Background α-Mangostin (α-MG is a main constituent of the fruit hull of the mangosteen. Previous studies have shown that α-MG has pharmacological activities such as antioxidant, antitumor, anti-inflammatory, antiallergic, antibacterial, antifungal and antiviral effects. This study aims to investigate the anti-inflammatory molecular action of α-MG on gene expression profiles. Methods U937 and EL4 cells were treated with different concentrations of α-MG in the presence of 0.1 ng/mL lipopolysaccharide (LPS for 4 h. The anti-inflammatory effects of α-MG were measured by the levels of tumor necrosis factor (TNF-α and interleukin (IL-4 in cell culture media, which were determined with enzyme-linked immunosorbent assay kits. The gene expression profiles of all samples were analyzed with a whole human genome microarray, Illumina BeadChip WG-6 version 3, containing 48804 probes. The protein levels were determined by Western blotting analyses. Results α-MG decreased the LPS induction of the inflammatory cytokines TNF-α (P = 0.038 and IL-4 (P = 0.04. α-MG decreased the gene expressions in oncostatin M signaling via mitogen-activated protein kinase (MAPK pathways, including extracellular signal-regulated kinases (P = 0.016, c-Jun N-terminal kinase (P = 0.01 , and p38 (P = 0.008. α-MG treatment of U937 cells reduced the phosphorylation of MAPK kinase 3 / MAPK kinase 6 (P = 0.0441, MAPK-activated protein kinase-2 (P = 0.0453, signal transducers and activators of transcription-1 (STAT1 (P = 0.0012, c-Fos (P = 0.04, c-Jun (P = 0.019 and Ets-like molecule 1 (Elk-1 (P = 0.038. Conclusion This study demonstrates that α-MG attenuates LPS-mediated activation of MAPK, STAT1, c-Fos, c-Jun and EIK-1, inhibiting TNF-α and IL-4 production in U937 cells.

  20. Plant extracts of spices and coffee synergistically dampen nuclear factor-κB in U937 cells.

    Science.gov (United States)

    Kolberg, Marit; Paur, Ingvild; Balstad, Trude R; Pedersen, Sigrid; Jacobs, David R; Blomhoff, Rune

    2013-10-01

    A large array of bioactive plant compounds (phytochemicals) has been identified and synergy among these compounds might contribute to the beneficial effects of plant foods. The transcription factor nuclear factor-κB (NF-κB) has been suggested as a target for many phytochemicals. Due to the complexity of mechanisms involved in NF-κB regulation, including numerous feedback loops, and the large number of phytochemicals which regulate NF-κB activity, we hypothesize that synergistic or antagonistic effects are involved. The objectives of our study were to develop a statistical methodology to evaluate the concept of synergy and antagonism and to use this methodology in a monocytic cell line (U937 expressing an NF-κB-luciferase reporter) treated with lipopolysaccharide and phytochemical-rich plant extracts. Both synergistic and antagonistic effects were clearly observed. Observed synergy was most pronounced for the combinations of oregano and coffee, and thyme and oregano. For oregano and coffee the synergistic effect was highest at 5 mg/mL with 13.9% (P oregano the highest synergistic effects was at 3 mg/mL with 13.7% (P phytochemical-rich plants may exert synergistic and antagonistic effects on NF-κB regulation. Such complex mechanistic interactions between phytochemicals are likely to underlie the protective effects of a plant-based diet on life-style related diseases. © 2013 Elsevier Inc. All rights reserved.

  1. Effects of Cigarette Smoke Condensate on Oxidative Stress, Apoptotic Cell Death, and HIV Replication in Human Monocytic Cells.

    Directory of Open Access Journals (Sweden)

    Pss Rao

    Full Text Available While cigarette smoking is prevalent amongst HIV-infected patients, the effects of cigarette smoke constituents in cells of myeloid lineage are poorly known. Recently, we have shown that nicotine induces oxidative stress through cytochrome P450 (CYP 2A6-mediated pathway in U937 monocytic cells. The present study was designed to examine the effect of cigarette smoke condensate (CSC, which contains majority of tobacco constituents, on oxidative stress, cytotoxicity, expression of CYP1A1, and/or HIV-1 replication in HIV-infected (U1 and uninfected U937 cells. The effects of CSC on induction of CYP1 enzymes in HIV-infected primary macrophages were also analyzed. The results showed that the CSC-mediated increase in production of reactive oxygen species (ROS in U937 cells is dose- and time-dependent. Moreover, CSC treatment was found to induce cytotoxicity in U937 cells through the apoptotic pathway via activation of caspase-3. Importantly, pretreatment with vitamin C blocked the CSC-mediated production of ROS and induction of caspase-3 activity. In U1 cells, acute treatment of CSC increased ROS production at 6H (>2-fold and both ROS (>2 fold and HIV-1 replication (>3-fold after chronic treatment. The CSC mediated effects were associated with robust induction in the expression of CYP1A1 mRNA upon acute CSC treatment of U937 and U1 cells (>20-fold, and upon chronic CSC treatment to U1 cells (>30-fold. In addition, the CYP1A1 induction in U937 cells was mediated through the aromatic hydrocarbon receptor pathway. Lastly, CSC, which is known to increase viral replication in primary macrophages, was also found to induce CYP1 enzymes in HIV-infected primary macrophages. While mRNA levels of both CYP1A1 and CYP1B1 were elevated following CSC treatment, only CYP1B1 protein levels were increased in HIV-infected primary macrophages. In conclusion, these results suggest a possible association between oxidative stress, CYP1 expression, and viral replication in

  2. A micro-Raman spectroscopic investigation of leukemic U-937 cells in aged cultures

    Science.gov (United States)

    Fazio, Enza; Trusso, Sebastiano; Franco, Domenico; Nicolò, Marco Sebastiano; Allegra, Alessandro; Neri, Fortunato; Musolino, Caterina; Guglielmino, Salvatore P. P.

    2016-04-01

    Recently it has been shown that micro-Raman spectroscopy combined with multivariate analysis is able to discriminate among different types of tissues and tumoral cells by the detection of significant alterations and/or reorganizations of complex biological molecules, such as nucleic acids, lipids and proteins. Moreover, its use, being in principle a non-invasive technique, appears an interesting clinical tool for the evaluation of the therapeutical effects and of the disease progression. In this work we analyzed molecular changes in aged cultures of leukemia model U937 cells with respect to fresh cultures of the same cell line. In fact, structural variations of individual neoplastic cells on aging may lead to a heterogeneous data set, therefore falsifying confidence intervals, increasing error levels of analysis and consequently limiting the use of Raman spectroscopy analysis. We found that the observed morphological changes of U937 cells corresponded to well defined modifications of the Raman contributions in selected spectral regions, where markers of specific functional groups, useful to characterize the cell state, are present. A detailed subcellular analysis showed a change in cellular organization as a function of time, and correlated to a significant increase of apoptosis levels. Besides the aforementioned study, Raman spectra were used as input for principal component analysis (PCA) in order to detect and classify spectral changes among U937 cells.

  3. Radiation effects on cultured human monocytes and on monocyte-derived macrophages

    International Nuclear Information System (INIS)

    Buescher, E.S.; Gallin, J.I.

    1984-01-01

    Prior to administration, leukocyte transfusions are commonly irradiated with up to 5,000 R to eliminate lymphocytes and thereby prevent graft-versus-host disease in the recipient. It has been widely believed that phagocytes are resistant to this irradiation. In a recent report, it was noted that phagocyte oxidative metabolism was compromised during preparation of white cells for transfusion. As part of the effort to examine the basis for this inhibition of phagocyte function during white cell preparation, an assessment was made of the effects of irradiation on the long-lived monocytes that have been shown to persist at inflammatory foci posttransfusion. Human monocytes were irradiated for up to 3 min, receiving 2,500-5,000 R. This irradiation damaged human monocytes, significantly decreasing their in vitro survival for the first 3 wk of culture, and growth as assessed by two-dimensional cell size measurements during the first 2 wk of culture. Despite smaller cell size, total cell protein was significantly increased over time in irradiated cultures. Extracellular release of lysozyme and beta-glucuronidase per cell was not affected by irradiation, but extracellular lactate dehydrogenase (LDH) release was significantly increased after irradiation. Irradiated monocytes killed Listeria monocytogenes at a slower rate than the nonirradiated controls. Thus, the data indicate that irradiation in doses used to prevent graft-versus-host disease in leukocyte transfusion recipients has a deleterious effect on in vitro human monocyte survival and function

  4. Reduction of Monocyte Chemoattractant Protein-1 and Interleukin-8 Levels by Ticlopidine in TNF-α Stimulated Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Chaur-Jong Hu

    2009-01-01

    Full Text Available Atherosclerosis and its associated complications represent major causes of morbidity and mortality in the industrialized or Western countries. Monocyte chemoattractant protein-1 (MCP-1 is critical for the initiating and developing of atherosclerotic lesions. Interleukin-8 (IL-8, a CXC chemokine, stimulates neutrophil chemotaxis. Ticlopidine is one of the antiplatelet drugs used to prevent thrombus formation relevant to the pathophysiology of atherothrombosis. In this study, we found that ticlopidine dose-dependently decreased the mRNA and protein levels of TNF-α-stimulated MCP-1, IL-8, and vascular cell adhesion molecule-1 (VCAM-1 in human umbilical vein endothelial cells (HUVECs. Ticlopidine declined U937 cells adhesion and chemotaxis as compared to TNF-α stimulated alone. Furthermore, the inhibitory effects were neither due to decreased HUVEC viability, nor through NF-kB inhibition. These results suggest that ticlopidine decreased TNF-α induced MCP-1, IL-8, and VCAM-1 levels in HUVECs, and monocyte adhesion. Therefore, the data provide additional therapeutic machinery of ticlopidine in treatment and prevention of atherosclerosis.

  5. Distinct functional programming of human fetal and adult monocytes.

    Science.gov (United States)

    Krow-Lucal, Elisabeth R; Kim, Charles C; Burt, Trevor D; McCune, Joseph M

    2014-03-20

    Preterm birth affects 1 out of 9 infants in the United States and is the leading cause of long-term neurologic handicap and infant mortality, accounting for 35% of all infant deaths in 2008. Although cytokines including interferon-γ (IFN-γ), interleukin-10 (IL-10), IL-6, and IL-1 are produced in response to in utero infection and are strongly associated with preterm labor, little is known about how human fetal immune cells respond to these cytokines. We demonstrate that fetal and adult CD14(+)CD16(-) classical monocytes are distinct in terms of basal transcriptional profiles and in phosphorylation of signal transducers and activators of transcription (STATs) in response to cytokines. Fetal monocytes phosphorylate canonical and noncanonical STATs and respond more strongly to IFN-γ, IL-6, and IL-4 than adult monocytes. We demonstrate a higher ratio of SOCS3 to IL-6 receptor in adult monocytes than in fetal monocytes, potentially explaining differences in STAT phosphorylation. Additionally, IFN-γ signaling results in upregulation of antigen presentation and costimulatory machinery in adult, but not fetal, monocytes. These findings represent the first evidence that primary human fetal and adult monocytes are functionally distinct, potentially explaining how these cells respond differentially to cytokines implicated in development, in utero infections, and the pathogenesis of preterm labor.

  6. Bioinformatic prediction and functional characterization of human KIAA0100 gene

    Directory of Open Access Journals (Sweden)

    He Cui

    2017-02-01

    Full Text Available Our previous study demonstrated that human KIAA0100 gene was a novel acute monocytic leukemia-associated antigen (MLAA gene. But the functional characterization of human KIAA0100 gene has remained unknown to date. Here, firstly, bioinformatic prediction of human KIAA0100 gene was carried out using online softwares; Secondly, Human KIAA0100 gene expression was downregulated by the clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated (Cas 9 system in U937 cells. Cell proliferation and apoptosis were next evaluated in KIAA0100-knockdown U937 cells. The bioinformatic prediction showed that human KIAA0100 gene was located on 17q11.2, and human KIAA0100 protein was located in the secretory pathway. Besides, human KIAA0100 protein contained a signalpeptide, a transmembrane region, three types of secondary structures (alpha helix, extended strand, and random coil , and four domains from mitochondrial protein 27 (FMP27. The observation on functional characterization of human KIAA0100 gene revealed that its downregulation inhibited cell proliferation, and promoted cell apoptosis in U937 cells. To summarize, these results suggest human KIAA0100 gene possibly comes within mitochondrial genome; moreover, it is a novel anti-apoptotic factor related to carcinogenesis or progression in acute monocytic leukemia, and may be a potential target for immunotherapy against acute monocytic leukemia.

  7. Lactic acid delays the inflammatory response of human monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Peter, Katrin, E-mail: katrin.peter@ukr.de [Department of Internal Medicine III, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg (Germany); Rehli, Michael, E-mail: michael.rehli@ukr.de [Department of Internal Medicine III, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg (Germany); RCI Regensburg Center for Interventional Immunology, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg (Germany); Singer, Katrin, E-mail: katrin.singer@ukr.de [Department of Internal Medicine III, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg (Germany); Renner-Sattler, Kathrin, E-mail: kathrin.renner-sattler@ukr.de [Department of Internal Medicine III, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg (Germany); Kreutz, Marina, E-mail: marina.kreutz@ukr.de [Department of Internal Medicine III, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg (Germany); RCI Regensburg Center for Interventional Immunology, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg (Germany)

    2015-02-13

    Lactic acid (LA) accumulates under inflammatory conditions, e.g. in wounds or tumors, and influences local immune cell functions. We previously noted inhibitory effects of LA on glycolysis and TNF secretion of human LPS-stimulated monocytes. Here, we globally analyze the influence of LA on gene expression during monocyte activation. To separate LA-specific from lactate- or pH-effects, monocytes were treated for one or four hours with LPS in the presence of physiological concentrations of LA, sodium lactate (NaL) or acidic pH. Analyses of global gene expression profiles revealed striking effects of LA during the early stimulation phase. Up-regulation of most LPS-induced genes was significantly delayed in the presence of LA, while this inhibitory effect was attenuated in acidified samples and not detected after incubation with NaL. LA targets included genes encoding for important monocyte effector proteins like cytokines (e.g. TNF and IL-23) or chemokines (e.g. CCL2 and CCL7). LA effects were validated for several targets by quantitative RT-PCR and/or ELISA. Further analysis of LPS-signaling pathways revealed that LA delayed the phosphorylation of protein kinase B (AKT) as well as the degradation of IκBα. Consistently, the LPS-induced nuclear accumulation of NFκB was also diminished in response to LA. These results indicate that the broad effect of LA on gene expression and function of human monocytes is at least partially caused by its interference with immediate signal transduction events after activation. This mechanism might contribute to monocyte suppression in the tumor environment. - Highlights: • Lactic acid broadly delays LPS-induced gene expression in human monocytes. • Expression of important monocyte effector molecules is affected by lactic acid. • Interference of lactic acid with TLR signaling causes the delayed gene expression. • The profound effect of lactic acid might contribute to immune suppression in tumors.

  8. Lactic acid delays the inflammatory response of human monocytes

    International Nuclear Information System (INIS)

    Peter, Katrin; Rehli, Michael; Singer, Katrin; Renner-Sattler, Kathrin; Kreutz, Marina

    2015-01-01

    Lactic acid (LA) accumulates under inflammatory conditions, e.g. in wounds or tumors, and influences local immune cell functions. We previously noted inhibitory effects of LA on glycolysis and TNF secretion of human LPS-stimulated monocytes. Here, we globally analyze the influence of LA on gene expression during monocyte activation. To separate LA-specific from lactate- or pH-effects, monocytes were treated for one or four hours with LPS in the presence of physiological concentrations of LA, sodium lactate (NaL) or acidic pH. Analyses of global gene expression profiles revealed striking effects of LA during the early stimulation phase. Up-regulation of most LPS-induced genes was significantly delayed in the presence of LA, while this inhibitory effect was attenuated in acidified samples and not detected after incubation with NaL. LA targets included genes encoding for important monocyte effector proteins like cytokines (e.g. TNF and IL-23) or chemokines (e.g. CCL2 and CCL7). LA effects were validated for several targets by quantitative RT-PCR and/or ELISA. Further analysis of LPS-signaling pathways revealed that LA delayed the phosphorylation of protein kinase B (AKT) as well as the degradation of IκBα. Consistently, the LPS-induced nuclear accumulation of NFκB was also diminished in response to LA. These results indicate that the broad effect of LA on gene expression and function of human monocytes is at least partially caused by its interference with immediate signal transduction events after activation. This mechanism might contribute to monocyte suppression in the tumor environment. - Highlights: • Lactic acid broadly delays LPS-induced gene expression in human monocytes. • Expression of important monocyte effector molecules is affected by lactic acid. • Interference of lactic acid with TLR signaling causes the delayed gene expression. • The profound effect of lactic acid might contribute to immune suppression in tumors

  9. Resveratrol strongly enhances the retinoic acid-induced superoxide generating activity via up-regulation of gp91-phox gene expression in U937 cells.

    Science.gov (United States)

    Kikuchi, Hidehiko; Mimuro, Hitomi; Kuribayashi, Futoshi

    2018-01-01

    The membrane bound cytochrome b 558 composed of gp91-phox and p22-phox proteins, and cytosolic proteins p40-, p47-and p67-phox are important components of superoxide (O 2 - )-generating system in phagocytes. Here, we describe that resveratrol, a pleiotropic phytochemical belonging to the stilbenoids, dramatically activates the O 2 - -generating system during retinoic acid (RA)-induced differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. When U937 cells were cultured in the presence of RA and resveratrol, the O 2 - -generating activity increased more than 5-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and resveratrol strongly enhanced transcription of the gp91-phox compared with those of the RA-treatment only. On the other hand, immunoblot analysis revealed that co-treatment with RA and resveratrol caused remarkable accumulation of protein levels of gp91-phox (to 4-fold), p22-phox (to 5-fold) and p47-phox (to 4-fold) compared with those of the RA-treatment alone. In addition, ChIP assay suggested that resveratrol participates in enhancing the gene expression of gp91-phox via promoting acetylation of Lys-9 residues and Lys-14 residues of histone H3 within chromatin around the promoter regions of the gene. These results suggested that resveratrol strongly enhances the RA-induced O 2 - -generating activity via up-regulation of gp91-phox gene expression in U937 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Suppression of NRF2–ARE activity sensitizes chemotherapeutic agent-induced cytotoxicity in human acute monocytic leukemia cells

    International Nuclear Information System (INIS)

    Peng, Hui; Wang, Huihui; Xue, Peng; Hou, Yongyong; Dong, Jian; Zhou, Tong; Qu, Weidong; Peng, Shuangqing; Li, Jin; Carmichael, Paul L.; Nelson, Bud; Clewell, Rebecca; Zhang, Qiang; Andersen, Melvin E.; Pi, Jingbo

    2016-01-01

    Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the antioxidant response element (ARE)-dependent transcription, plays a pivotal role in chemical detoxification in normal and tumor cells. Consistent with previous findings that NRF2–ARE contributes to chemotherapeutic resistance of cancer cells, we found that stable knockdown of NRF2 by lentiviral shRNA in human acute monocytic leukemia (AML) THP-1 cells enhanced the cytotoxicity of several chemotherapeutic agents, including arsenic trioxide (As 2 O 3 ), etoposide and doxorubicin. Using an ARE-luciferase reporter expressed in several human and mouse cells, we identified a set of compounds, including isonicotinic acid amides, isoniazid and ethionamide, that inhibited NRF2–ARE activity. Treatment of THP-1 cells with ethionamide, for instance, significantly reduced mRNA expression of multiple ARE-driven genes under either basal or As 2 O 3 -challenged conditions. As determined by cell viability and cell cycle, suppression of NRF2–ARE by ethionamide also significantly enhanced susceptibility of THP-1 and U937 cells to As 2 O 3 -induced cytotoxicity. In THP-1 cells, the sensitizing effect of ethionamide on As 2 O 3 -induced cytotoxicity was highly dependent on NRF2. To our knowledge, the present study is the first to demonstrate that ethionamide suppresses NRF2–ARE signaling and disrupts the transcriptional network of the antioxidant response in AML cells, leading to sensitization to chemotherapeutic agents. - Highlights: • Identification of novel inhibitors of ARE-dependent transcription • Suppression of NRF2–ARE sensitizes THP-1 cells to chemotherapy. • Ethionamide suppresses ARE-dependent transcriptional activity. • Ethionamide and isoniazid increase the cytotoxicity of As 2 O 3 in AML cells. • Sensitization of THP-1 cells to As 2 O 3 toxicity by ethionamide is NRF2-dependent.

  11. Maturation and demise of human primary monocytes by carbon nanotubes

    Science.gov (United States)

    De Nicola, Milena; Mirabile Gattia, Daniele; Traversa, Enrico; Ghibelli, Lina

    2013-06-01

    The possibility of exploiting carbon nanotubes (CNT) in biomedical practices requires thorough analysis of the chemical or bulk effects they may exert on the immune system, the complex network that recognizes and eliminates foreign particles. In particular, the phagocytosing ability of cells belonging to the monocyte/macrophage lineage may render these immune cells an ideal toxicological target of pristine CNT, which may form aggregates of size exceeding monocyte/macrophage phagocytosing plasticity. To shed light on this issue, we analyzed the effects that pristine multi-walled CNT (MWCNT) without metal or biological impurities exert on survival and activation of freshly explanted human peripheral blood monocytes, analyzing in parallel the non-phagocytosing lymphocytes, and using graphite as control carbon material. MWCNT (diameter 10-50 nm, length up to 10 μm) exert two different toxic effects on mononuclear leukocytes: a minor apoptogenic effect (on lymphocytes > monocytes), and a major, apoptosis-independent effect that exclusively and deeply affect monocyte homeostasis. Analysis of monocyte number, adhesion, redox equilibrium, and the differentiation markers CD14 and CD11b reveals that MWCNT cause the selective disappearance of phagocytosis-competent monocytes by mechanisms related to the presence of large nanoparticle aggregates, suggesting phenomena of bulk toxicity possibly consisting of frustrated phagocytosis. At the same time, MWCNT stimulate adhesion of the phagocytosis-incompetent monocytes, and their differentiation toward a peculiar maturation asset. These observations point out novel mechanisms of CNT toxicity, renewing concerns that they may impair the innate immune system deranging the inflammatory responses.

  12. Maturation and demise of human primary monocytes by carbon nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    De Nicola, Milena, E-mail: milena.de.nicola@uniroma2.it [University of Rome ' Tor Vergata' , Department of Biology (Italy); Mirabile Gattia, Daniele, E-mail: daniele.mirabile@enea.it [UTTMAT, ENEA-C.R. Casaccia (Italy); Traversa, Enrico, E-mail: Enrico.Traversa@kaust.edu.sa [King Abdullah University of Science and Technology (KAUST), Division of Physical Science and Engineering (Saudi Arabia); Ghibelli, Lina, E-mail: ghibelli@uniroma2.it [University of Rome ' Tor Vergata' , Department of Biology (Italy)

    2013-06-15

    The possibility of exploiting carbon nanotubes (CNT) in biomedical practices requires thorough analysis of the chemical or bulk effects they may exert on the immune system, the complex network that recognizes and eliminates foreign particles. In particular, the phagocytosing ability of cells belonging to the monocyte/macrophage lineage may render these immune cells an ideal toxicological target of pristine CNT, which may form aggregates of size exceeding monocyte/macrophage phagocytosing plasticity. To shed light on this issue, we analyzed the effects that pristine multi-walled CNT (MWCNT) without metal or biological impurities exert on survival and activation of freshly explanted human peripheral blood monocytes, analyzing in parallel the non-phagocytosing lymphocytes, and using graphite as control carbon material. MWCNT (diameter 10-50 nm, length up to 10 {mu}m) exert two different toxic effects on mononuclear leukocytes: a minor apoptogenic effect (on lymphocytes > monocytes), and a major, apoptosis-independent effect that exclusively and deeply affect monocyte homeostasis. Analysis of monocyte number, adhesion, redox equilibrium, and the differentiation markers CD14 and CD11b reveals that MWCNT cause the selective disappearance of phagocytosis-competent monocytes by mechanisms related to the presence of large nanoparticle aggregates, suggesting phenomena of bulk toxicity possibly consisting of frustrated phagocytosis. At the same time, MWCNT stimulate adhesion of the phagocytosis-incompetent monocytes, and their differentiation toward a peculiar maturation asset. These observations point out novel mechanisms of CNT toxicity, renewing concerns that they may impair the innate immune system deranging the inflammatory responses.

  13. Maturation and demise of human primary monocytes by carbon nanotubes

    International Nuclear Information System (INIS)

    De Nicola, Milena; Mirabile Gattia, Daniele; Traversa, Enrico; Ghibelli, Lina

    2013-01-01

    The possibility of exploiting carbon nanotubes (CNT) in biomedical practices requires thorough analysis of the chemical or bulk effects they may exert on the immune system, the complex network that recognizes and eliminates foreign particles. In particular, the phagocytosing ability of cells belonging to the monocyte/macrophage lineage may render these immune cells an ideal toxicological target of pristine CNT, which may form aggregates of size exceeding monocyte/macrophage phagocytosing plasticity. To shed light on this issue, we analyzed the effects that pristine multi-walled CNT (MWCNT) without metal or biological impurities exert on survival and activation of freshly explanted human peripheral blood monocytes, analyzing in parallel the non-phagocytosing lymphocytes, and using graphite as control carbon material. MWCNT (diameter 10–50 nm, length up to 10 μm) exert two different toxic effects on mononuclear leukocytes: a minor apoptogenic effect (on lymphocytes > monocytes), and a major, apoptosis-independent effect that exclusively and deeply affect monocyte homeostasis. Analysis of monocyte number, adhesion, redox equilibrium, and the differentiation markers CD14 and CD11b reveals that MWCNT cause the selective disappearance of phagocytosis-competent monocytes by mechanisms related to the presence of large nanoparticle aggregates, suggesting phenomena of bulk toxicity possibly consisting of frustrated phagocytosis. At the same time, MWCNT stimulate adhesion of the phagocytosis-incompetent monocytes, and their differentiation toward a peculiar maturation asset. These observations point out novel mechanisms of CNT toxicity, renewing concerns that they may impair the innate immune system deranging the inflammatory responses.

  14. Maturation and demise of human primary monocytes by carbon nanotubes

    KAUST Repository

    De Nicola, Milena D.

    2013-05-17

    The possibility of exploiting carbon nanotubes (CNT) in biomedical practices requires thorough analysis of the chemical or bulk effects they may exert on the immune system, the complex network that recognizes and eliminates foreign particles. In particular, the phagocytosing ability of cells belonging to the monocyte/macrophage lineage may render these immune cells an ideal toxicological target of pristine CNT, which may form aggregates of size exceeding monocyte/macrophage phagocytosing plasticity. To shed light on this issue, we analyzed the effects that pristine multi-walled CNT (MWCNT) without metal or biological impurities exert on survival and activation of freshly explanted human peripheral blood monocytes, analyzing in parallel the non-phagocytosing lymphocytes, and using graphite as control carbon material. MWCNT (diameter 10-50 nm, length up to 10 μm) exert two different toxic effects on mononuclear leukocytes: a minor apoptogenic effect (on lymphocytes > monocytes), and a major, apoptosis-independent effect that exclusively and deeply affect monocyte homeostasis. Analysis of monocyte number, adhesion, redox equilibrium, and the differentiation markers CD14 and CD11b reveals that MWCNT cause the selective disappearance of phagocytosis-competent monocytes by mechanisms related to the presence of large nanoparticle aggregates, suggesting phenomena of bulk toxicity possibly consisting of frustrated phagocytosis. At the same time, MWCNT stimulate adhesion of the phagocytosis-incompetent monocytes, and their differentiation toward a peculiar maturation asset. These observations point out novel mechanisms of CNT toxicity, renewing concerns that they may impair the innate immune system deranging the inflammatory responses. © 2013 Springer Science+Business Media Dordrecht.

  15. Oxalomalate, a competitive inhibitor of NADP+ -dependent isocitrate dehydrogenase, regulates lipid peroxidation-mediated apoptosis in U937 cells.

    Science.gov (United States)

    Yang, Eun Sun; Yang, Joon-Hyuck; Park, Ji Eun; Park, Jeen-Woo

    2005-01-01

    Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Recently, we demonstrated that the control of cytosolic redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic NADP+ -dependent isocitrate dehydrogenase (IDPc) through to supply NADPH for antioxidant systems. The protective role of IDPc against lipid peroxidation-mediated apoptosis in U937 cells was investigated in control and cells pre-treated with oxlalomalate, a competitive inhibitor of IDPc. Upon exposure to 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the susceptibility to apoptosis was higher in oxalomalate-treated cells as compared to control cells. The results suggest that IDPc plays an important protective role in apoptosis of U937 cells induced by lipid peroxidation-mediated oxidative stress.

  16. Regulation of ICAM-1 in Cells of the Monocyte/Macrophage System in Microgravity

    Directory of Open Access Journals (Sweden)

    Katrin Paulsen

    2015-01-01

    Full Text Available Cells of the immune system are highly sensitive to altered gravity, and the monocyte as well as the macrophage function is proven to be impaired under microgravity conditions. In our study, we investigated the surface expression of ICAM-1 protein and expression of ICAM-1 mRNA in cells of the monocyte/macrophage system in microgravity during clinostat, parabolic flight, sounding rocket, and orbital experiments. In murine BV-2 microglial cells, we detected a downregulation of ICAM-1 expression in clinorotation experiments and a rapid and reversible downregulation in the microgravity phase of parabolic flight experiments. In contrast, ICAM-1 expression increased in macrophage-like differentiated human U937 cells during the microgravity phase of parabolic flights and in long-term microgravity provided by a 2D clinostat or during the orbital SIMBOX/Shenzhou-8 mission. In nondifferentiated U937 cells, no effect of microgravity on ICAM-1 expression could be observed during parabolic flight experiments. We conclude that disturbed immune function in microgravity could be a consequence of ICAM-1 modulation in the monocyte/macrophage system, which in turn could have a strong impact on the interaction with T lymphocytes and cell migration. Thus, ICAM-1 can be considered as a rapid-reacting and sustained gravity-regulated molecule in mammalian cells.

  17. Aliphatic alcohols in spirits inhibit phagocytosis by human monocytes.

    Science.gov (United States)

    Pál, László; Árnyas, Ervin M; Bujdosó, Orsolya; Baranyi, Gergő; Rácz, Gábor; Ádány, Róza; McKee, Martin; Szűcs, Sándor

    2015-04-01

    A large volume of alcoholic beverages containing aliphatic alcohols is consumed worldwide. Previous studies have confirmed the presence of ethanol-induced immunosuppression in heavy drinkers, thereby increasing susceptibility to infectious diseases. However, the aliphatic alcohols contained in alcoholic beverages might also impair immune cell function, thereby contributing to a further decrease in microbicidal activity. Previous research has shown that aliphatic alcohols inhibit phagocytosis by granulocytes but their effect on human monocytes has not been studied. This is important as they play a crucial role in engulfment and killing of pathogenic microorganisms and a decrease in their phagocytic activity could lead to impaired antimicrobial defence in heavy drinkers. The aim of this study was to measure monocyte phagocytosis following their treatment with those aliphatic alcohols detected in alcoholic beverages. Monocytes were separated from human peripheral blood and phagocytosis of opsonized zymosan particles by monocytes treated with ethanol and aliphatic alcohols individually and in combination was determined. It was shown that these alcohols could suppress the phagocytic activity of monocytes in a concentration-dependent manner and when combined with ethanol, they caused a further decrease in phagocytosis. Due to their additive effects, it is possible that they may inhibit phagocytosis in a clinically meaningful way in alcoholics and episodic heavy drinkers thereby contribute to their increased susceptibility to infectious diseases. However, further research is needed to address this question.

  18. Mycobacterium leprae alters classical activation of human monocytes in vitro.

    Science.gov (United States)

    Fallows, Dorothy; Peixoto, Blas; Kaplan, Gilla; Manca, Claudia

    2016-01-01

    Macrophages play a central role in the pathogenesis of leprosy, caused by Mycobacterium leprae. The polarized clinical presentations in leprosy are associated with differential immune activation. In tuberculoid leprosy, macrophages show a classical activation phenotype (M1), while macrophages in lepromatous disease display characteristics of alternative activation (M2). Bacille Calmette-Guérin (BCG) vaccination, which protects against leprosy, can promote sustained changes in monocyte response to unrelated pathogens and may preferentially direct monocytes towards an M1 protective phenotype. We previously reported that M. leprae can dampen the response of naïve human monocytes to a strong inducer of pro-inflammatory cytokines, such as BCG. Here, we investigated the ability of the pathogen to alter the direction of macrophage polarization and the impact of BCG vaccination on the monocyte response to M. leprae. We show that in vitro exposure of monocytes from healthy donors to M. leprae interferes with subsequent M1 polarization, indicated by lower levels of M1-associated cytokine/chemokines released and reduced expression of M1 cell surface markers. Exposure to M. leprae phenolic glycolipid (PGL) 1, instead of whole bacteria, demonstrated a similar effect on M1 cytokine/chemokine release. In addition, we found that monocytes from 10-week old BCG-vaccinated infants released higher levels of the pro-inflammatory cytokines TNF-α and IL-1β in response to M. leprae compared to those from unvaccinated infants. Exposure to M. leprae has an inhibitory effect on M1 macrophage polarization, likely mediated through PGL-1. By directing monocyte/macrophages preferentially towards M1 activation, BCG vaccination may render the cells more refractory to the inhibitory effects of subsequent M. leprae infection.

  19. Anti-Cancerous Effect of Inonotus taiwanensis Polysaccharide Extract on Human Acute Monocytic Leukemia Cells through ROS-Independent Intrinsic Mitochondrial Pathway.

    Science.gov (United States)

    Chao, Tsai-Ling; Wang, Ting-Yin; Lee, Chin-Huei; Yiin, Shuenn-Jiun; Ho, Chun-Te; Wu, Sheng-Hua; You, Huey-Ling; Chern, Chi-Liang

    2018-01-29

    Acute leukemia is one of the commonly diagnosed neoplasms and causes human death. However, the treatment for acute leukemia is not yet satisfactory. Studies have shown that mushroom-derived polysaccharides display low toxicity and have been used clinically for cancer therapy. Therefore, we set out to evaluate the anti-cancerous efficacy of a water-soluble polysaccharide extract from Inonotus taiwanensis (WSPIS) on human acute monocytic leukemia THP-1 and U937 cell lines in vitro. Under our experimental conditions, WSPIS elicited dose-dependent growth retardation and induced apoptotic cell death. Further analysis showed that WSPIS-induced apoptosis was associated with a mitochondrial apoptotic pathway, such as the disruption of mitochondrial membrane potential (MMP), followed by the activation of caspase-9, caspase-3, and PARP (poly(ADP-ribose) polymerase) cleavage. However, a broad caspase inhibitor, Z-VAD.fmk, could not prevent WSPIS-induced apoptosis. These data imply that mechanism(s) other than caspase might be involved. Thus, the involvement of endonuclease G (endoG), a mediator arbitrating caspase-independent oligonucleosomal DNA fragmentation, was examined. Western blotting demonstrated that WSPIS could elicit nuclear translocation of endoG. MMP disruption after WSPIS treatment was accompanied by intracellular reactive oxygen species (ROS) generation. However, pretreatment with N -acetyl-l-cysteine (NAC) could not attenuate WSPIS-induced apoptosis. In addition, our data also show that WSPIS could inhibit autophagy. Activation of autophagy by rapamycin decreased WSPIS-induced apoptosis and cell death. Taken together, our findings suggest that cell cycle arrest, endonuclease G-mediated apoptosis, and autophagy inhibition contribute to the anti-cancerous effect of WSPIS on human acute monocytic leukemia cells.

  20. Anti-Cancerous Effect of Inonotus taiwanensis Polysaccharide Extract on Human Acute Monocytic Leukemia Cells through ROS-Independent Intrinsic Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Tsai-Ling Chao

    2018-01-01

    Full Text Available Acute leukemia is one of the commonly diagnosed neoplasms and causes human death. However, the treatment for acute leukemia is not yet satisfactory. Studies have shown that mushroom-derived polysaccharides display low toxicity and have been used clinically for cancer therapy. Therefore, we set out to evaluate the anti-cancerous efficacy of a water-soluble polysaccharide extract from Inonotus taiwanensis (WSPIS on human acute monocytic leukemia THP-1 and U937 cell lines in vitro. Under our experimental conditions, WSPIS elicited dose-dependent growth retardation and induced apoptotic cell death. Further analysis showed that WSPIS-induced apoptosis was associated with a mitochondrial apoptotic pathway, such as the disruption of mitochondrial membrane potential (MMP, followed by the activation of caspase-9, caspase-3, and PARP (poly(ADP-ribose polymerase cleavage. However, a broad caspase inhibitor, Z-VAD.fmk, could not prevent WSPIS-induced apoptosis. These data imply that mechanism(s other than caspase might be involved. Thus, the involvement of endonuclease G (endoG, a mediator arbitrating caspase-independent oligonucleosomal DNA fragmentation, was examined. Western blotting demonstrated that WSPIS could elicit nuclear translocation of endoG. MMP disruption after WSPIS treatment was accompanied by intracellular reactive oxygen species (ROS generation. However, pretreatment with N-acetyl-l-cysteine (NAC could not attenuate WSPIS-induced apoptosis. In addition, our data also show that WSPIS could inhibit autophagy. Activation of autophagy by rapamycin decreased WSPIS-induced apoptosis and cell death. Taken together, our findings suggest that cell cycle arrest, endonuclease G-mediated apoptosis, and autophagy inhibition contribute to the anti-cancerous effect of WSPIS on human acute monocytic leukemia cells.

  1. Alterations in calcium metabolism during human monocyte activation

    International Nuclear Information System (INIS)

    Scully, S.P.

    1984-01-01

    Human peripheral blood monocytes have been prepared from plateletpheresis residues by counterflow centrifugal elutriation in sufficient quantities to enable quantitative studies of cell calcium. Kinetic analysis of 45 Ca exchange data in resting monocytes was compatible with a model of cellular calcium containing three exchangeable calcium pools. These pools are thought to represent a putative ectocellular pool, a putative cytoplasmic chelated pool, and a putative organelle sequestered pool. Exposure of monocytes to the plant lectin Con A at a concentration that maximally simulated superoxide production caused an increase in the size and a doubling in the exchange rate of the putative cytoplasmic pool without a change in the other cellular pools. The cytoplasmic ionized calcium, [Ca]/sub i/, measured with the fluorescent probe, Quin 2 rose from a resting level of 83 nM to 165 mN within 30 sec of exposure to Con A. This increase in cytoplasmic calcium preceded the release of superoxide radicals. Calcium transport and calcium ATPase activities were identified and characterized in plasma membrane vesicles prepared from monocytes. Both activities were strictly dependent on ATP and Mg, had a Km/sub Ca/ in the submicromolar range and were stimulated by calmodulin. Thus, it seems that monocyte calcium is in a dynamic steady state that is a balance between efflux and influx rates, and that the activation of these cells results in the transition to a new steady state. The alteration in [Ca]/sub i/ that accompany the new steady state are essential for superoxide production by human monocytes

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

    Lifescience Database Archive (English)

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

  3. NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

    International Nuclear Information System (INIS)

    Wang, Yi; Wang, Xiang; Sun, Minghui; Zhang, Zhenyu; Cao, Heng; Chen, Xiaoqing

    2011-01-01

    Highlights: → Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. → Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. → P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. → Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kB (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the regulation of foam

  4. NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yi, E-mail: wangyi2004a@126.com [Department of Cardiology, Shanghai First People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080 (China); Wang, Xiang; Sun, Minghui; Zhang, Zhenyu; Cao, Heng; Chen, Xiaoqing [Department of Cardiology, Shanghai First People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080 (China)

    2011-08-05

    Highlights: {yields} Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. {yields} Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. {yields} P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. {yields} Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kB (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the

  5. Proteasome (Prosome Subunit Variations during the Differentiation of Myeloid U937 Cells

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    Laurent Henry

    1997-01-01

    Full Text Available 20S proteasomes (prosomes/multicatalytic proteinase are protein particles built of 28 subunits in variable composition. We studied the changes in proteasome subunit composition during the differentiation of U937 cells induced by phorbol‐myristate‐acetate or retinoic acid plus 1,25‐dihydroxy‐cholecalciferol by western blot, flow cytometry and immuno‐fluorescence. p25K (C3, p27K (IOTA and p30/33K (C2 subunits were detected in both the nucleus and cytoplasm of undifferentiated cells. Flow cytometry demonstrated a biphasic decrease in proteasome subunits detection during differentiation induced by RA+VD. PMA caused an early transient decrease in these subunits followed by a return to their control level, except for p30/33K, which remained low. Immuno‐fluorescence also showed differences in the cytolocalization of the subunits, with a particular decrease in antigen labeling in the nucleus of RA+VD‐induced cells, and a scattering in the cytoplasm and a reorganization in the nucleus of PMA‐induced cells. Small amounts of proteasomal proteins were seen on the outer membrane of non‐induced cells; these membrane proteins disappeared when treated with RA+VD, whereas some increased on PMA‐induced cells. The differential changes in the distribution and type of proteasomes in RA+VD and PMA‐induced cells indicate that, possibly, 20S proteasomes may play a role in relation to the mechanisms of differentiation and the inducer used.

  6. Differentiation of U937 cells induced by 5,8,11,14-eicosatetraynoic acid, a competitive inhibitor of arachidonic acid metabolism

    International Nuclear Information System (INIS)

    Ondrey, F.; Anderson, K.; Hoeltgen, D.; Harris, J.

    1988-01-01

    5,8,11,14-Eicosatetraynoic acid, a competitive inhibitor of arachidonic acid metabolism, rapidly and reversibly inhibited DNA synthesis in U937 cells. This inhibition was not due to cytotoxicity, as judged by studies with trypan blue, release of 51 Cr-labeled proteins, and its reversibility. When cells were cultured in the presence of ETYA for several days, morphologic, enzymatic, and functional changes consistent with differentiation occurred. The cells enlarged, the ratio of cytoplasm to nuclei increased, secretory granules and vacuoles developed, the apparent activity of nonspecific esterase rose, and ingestion of latex particles increased. A morphology consistent with that of an immature monocyte was evident by electron microscopy. When cells differentiated by ETYA were cultured in media free of the inhibitor, DNA synthesis reinitiated and the cell number increased; differentiation was phenotypic and not genotypic. To examine whether ETYA-induced differentiation was obligatorily related to its suppression of DNA synthesis, cells were incubated in 50 μM hydroxyurea and DNA synthesis was inhibited for 24 to 36 h without morphologic evidence of cellular differentiation. However, addition of ETYA to cells prevented from dividing by hydroxyurea and subsequent culture for 72 h induced morphologic evidence of differentiation. The effects of ETYA on cell division and cell differentiation are closely related but can be dissociated

  7. Staurosporine induces necroptotic cell death under caspase-compromised conditions in U937 cells.

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    Zsuzsanna A Dunai

    Full Text Available For a long time necrosis was thought to be an uncontrolled process but evidences recently have revealed that necrosis can also occur in a regulated manner. Necroptosis, a type of programmed necrosis is defined as a death receptor-initiated process under caspase-compromised conditions. The process requires the kinase activity of receptor-interacting protein kinase 1 and 3 (RIPK1 and RIPK3 and mixed lineage kinase domain-like protein (MLKL, as a substrate of RIPK3. The further downstream events remain elusive. We applied known inhibitors to characterize the contributing enzymes in necroptosis and their effect on cell viability and different cellular functions were detected mainly by flow cytometry. Here we report that staurosporine, the classical inducer of intrinsic apoptotic pathway can induce necroptosis under caspase-compromised conditions in U937 cell line. This process could be hampered at least partially by the RIPK1 inhibitor necrotstin-1 and by the heat shock protein 90 kDa inhibitor geldanamycin. Moreover both the staurosporine-triggered and the classical death ligand-induced necroptotic pathway can be effectively arrested by a lysosomal enzyme inhibitor CA-074-OMe and the recently discovered MLKL inhibitor necrosulfonamide. We also confirmed that the enzymatic role of poly(ADP-ribosepolymerase (PARP is dispensable in necroptosis but it contributes to membrane disruption in secondary necrosis. In conclusion, we identified a novel way of necroptosis induction that can facilitate our understanding of the molecular mechanisms of necroptosis. Our results shed light on alternative application of staurosporine, as a possible anticancer therapeutic agent. Furthermore, we showed that the CA-074-OMe has a target in the signaling pathway leading to necroptosis. Finally, we could differentiate necroptotic and secondary necrotic processes based on participation of PARP enzyme.

  8. Suppression of NRF2–ARE activity sensitizes chemotherapeutic agent-induced cytotoxicity in human acute monocytic leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Hui [The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC (United States); Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing (China); Wang, Huihui [School of Public Health, China Medical University, 77 Puhe Road, Shenyang North New Area, Shenyang (China); Xue, Peng [The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC (United States); Key Laboratory of the Public Health Safety, Ministry of Education, School of Public Health, Fudan University, Shanghai (China); Hou, Yongyong [School of Public Health, China Medical University, 77 Puhe Road, Shenyang North New Area, Shenyang (China); Dong, Jian [The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC (United States); Institute of Biology and Medicine, Wuhan University of Science and Technology, Wuhan (China); Zhou, Tong [The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC (United States); Qu, Weidong [Key Laboratory of the Public Health Safety, Ministry of Education, School of Public Health, Fudan University, Shanghai (China); Peng, Shuangqing [Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing (China); Li, Jin; Carmichael, Paul L. [Unilever, Safety & Environmental Assurance Centre, Colworth Science Park, Sharnbrook, Bedfordshire MK44 1LQ (United Kingdom); Nelson, Bud; Clewell, Rebecca; Zhang, Qiang; Andersen, Melvin E. [The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC (United States); Pi, Jingbo, E-mail: jpi@mail.cmu.edu.cn [School of Public Health, China Medical University, 77 Puhe Road, Shenyang North New Area, Shenyang (China); The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC (United States)

    2016-02-01

    Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the antioxidant response element (ARE)-dependent transcription, plays a pivotal role in chemical detoxification in normal and tumor cells. Consistent with previous findings that NRF2–ARE contributes to chemotherapeutic resistance of cancer cells, we found that stable knockdown of NRF2 by lentiviral shRNA in human acute monocytic leukemia (AML) THP-1 cells enhanced the cytotoxicity of several chemotherapeutic agents, including arsenic trioxide (As{sub 2}O{sub 3}), etoposide and doxorubicin. Using an ARE-luciferase reporter expressed in several human and mouse cells, we identified a set of compounds, including isonicotinic acid amides, isoniazid and ethionamide, that inhibited NRF2–ARE activity. Treatment of THP-1 cells with ethionamide, for instance, significantly reduced mRNA expression of multiple ARE-driven genes under either basal or As{sub 2}O{sub 3}-challenged conditions. As determined by cell viability and cell cycle, suppression of NRF2–ARE by ethionamide also significantly enhanced susceptibility of THP-1 and U937 cells to As{sub 2}O{sub 3}-induced cytotoxicity. In THP-1 cells, the sensitizing effect of ethionamide on As{sub 2}O{sub 3}-induced cytotoxicity was highly dependent on NRF2. To our knowledge, the present study is the first to demonstrate that ethionamide suppresses NRF2–ARE signaling and disrupts the transcriptional network of the antioxidant response in AML cells, leading to sensitization to chemotherapeutic agents. - Highlights: • Identification of novel inhibitors of ARE-dependent transcription • Suppression of NRF2–ARE sensitizes THP-1 cells to chemotherapy. • Ethionamide suppresses ARE-dependent transcriptional activity. • Ethionamide and isoniazid increase the cytotoxicity of As{sub 2}O{sub 3} in AML cells. • Sensitization of THP-1 cells to As{sub 2}O{sub 3} toxicity by ethionamide is NRF2-dependent.

  9. Transcription of human resistin gene involves an interaction of Sp1 with peroxisome proliferator-activating receptor gamma (PPARgamma.

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    Anil K Singh

    2010-03-01

    Full Text Available Resistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin.We show that the minimal promoter of human resistin lies within approximately 80 bp sequence upstream of the transcriptional start site (-240 whereas binding sites for cRel, CCAAT enhancer binding protein alpha (C/EBP-alpha, activating transcription factor 2 (ATF-2 and activator protein 1 (AP-1 transcription factors, important for induced expression, are present within sequences up to -619. Specificity Protein 1(Sp1 binding site (-276 to -295 is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPARgamma is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPARgamma in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPARgamma on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPARgamma interaction. Chromatin immunoprecipitation (ChIP assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPARgamma, chromatin modifier histone deacetylase 1 (HDAC1 and the acetylated form of histone H3 but not cRel, C/EBP-alpha and phospho c-Jun during resistin gene transcription.Our findings suggest a complex interplay of Sp1 and PPARgamma along with other transcription factors that drives the expression of resistin in human monocytic U937 cells.

  10. Susceptibility and response of human blood monocyte subsets to primary dengue virus infection.

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    Kok Loon Wong

    Full Text Available Human blood monocytes play a central role in dengue infections and form the majority of virus infected cells in the blood. Human blood monocytes are heterogeneous and divided into CD16(- and CD16(+ subsets. Monocyte subsets play distinct roles during disease, but it is not currently known if monocyte subsets differentially contribute to dengue protection and pathogenesis. Here, we compared the susceptibility and response of the human CD16(- and CD16(+ blood monocyte subsets to primary dengue virus in vitro. We found that both monocyte subsets were equally susceptible to dengue virus (DENV2 NGC, and capable of supporting the initial production of new infective virus particles. Both monocyte subsets produced anti-viral factors, including IFN-α, CXCL10 and TRAIL. However, CD16(+ monocytes were the major producers of inflammatory cytokines and chemokines in response to dengue virus, including IL-1β, TNF-α, IL-6, CCL2, 3 and 4. The susceptibility of both monocyte subsets to infection was increased after IL-4 treatment, but this increase was more profound for the CD16(+ monocyte subset, particularly at early time points after virus exposure. These findings reveal the differential role that monocyte subsets might play during dengue disease.

  11. Maturation and demise of human primary monocytes by carbon nanotubes

    KAUST Repository

    De Nicola, Milena D.; Mirabile Gattia, Daniele; Traversa, Enrico; Ghibelli, Lina

    2013-01-01

    -competent monocytes by mechanisms related to the presence of large nanoparticle aggregates, suggesting phenomena of bulk toxicity possibly consisting of frustrated phagocytosis. At the same time, MWCNT stimulate adhesion of the phagocytosis-incompetent monocytes

  12. Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation in vivo.

    Science.gov (United States)

    Iqbal, Asif J; McNeill, Eileen; Kapellos, Theodore S; Regan-Komito, Daniel; Norman, Sophie; Burd, Sarah; Smart, Nicola; Machemer, Daniel E W; Stylianou, Elena; McShane, Helen; Channon, Keith M; Chawla, Ajay; Greaves, David R

    2014-10-09

    The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo, we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115(+) monocytes of adult blood, spleen, and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX3CR1(GFP) monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1(GFP) monocytes, which downregulate GFP expression on differentiation into macrophages in this model, CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages, allowing continued cell tracking during resolution of inflammation. In summary, this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation. © 2014 by The American Society of Hematology.

  13. Differential induction from X-irradiated human peripheral blood monocytes to dendritic cells

    International Nuclear Information System (INIS)

    Yoshino, Hironori; Takahashi, Kenji; Monzen, Satoru; Kashiwakura, Ikuo

    2008-01-01

    Dendritic cells (DCs) are a type of antigen-presenting cell which plays an essential role in the immune system. To clarify the influences of ionizing radiation on the differentiation to DCs, we focused on human peripheral blood monocytes and investigated whether X-irradiated monocytes can differentiate into DCs. The non-irradiated monocytes and 5 Gy-irradiated monocytes were induced into immature DCs (iDCs) and mature DCs (mDCs) with appropriate cytokine stimulation, and the induced cells from each monocyte expressed each DC-expressing surface antigen such as CD40, CD86 and HLA-DR. However, the expression levels of CD40 and CD86 on the iDCs derived from the 5 Gy-irradiated monocytes were higher than those of iDCs derived from non-irradiated monocytes. Furthermore, the mDCs derived from 5 Gy-irradiated monocytes had significantly less ability to stimulate allogeneic T cells in comparison to the mDCs derived from non-irradiated monocytes. There were no significant differences in the phagocytotic activity of the iDCs and cytokines detected in the supernatants conditioned by the DCs from the non-irradiated and irradiated monocytes. These results suggest that human monocytes which are exposed to ionizing radiation can thus differentiate into DCs, but there is a tendency that X-irradiation leads to an impairment of the function of DCs. (author)

  14. Serologic Evidence of Human Monocytic and Granulocytic Ehrlichiosis in Israel

    Science.gov (United States)

    Keysary, Avi; Amram, Lili; Keren, Gershon; Sthoeger, Zev; Potasman, Israel; Jacob, Amir; Strenger, Carmella; Dawson, Jacqueline E.

    1999-01-01

    We conducted a retrospective serosurvey of 1,000 persons in Israel who had fever of undetermined cause to look for Ehrlichia chaffeensis antibodies. Four of five cases with antibodies reactive to E. chaffeensis were diagnosed in the summer, when ticks are more active. All patients had influenzalike symptoms with high fever. None of the cases was fatal. Three serum samples were also seroreactive for antibodies to E. canis, and one was also reactive to the human granulocytic ehrlichiosis (HGE) agent. The titer to the HGE agent in this patient was higher than the serum titer to E. chaffeensis, and the Western blot analysis also indicated that the HGE agent was the primary cause of infection. We present the first serologic evidence that the agents of human monocytic ehrlichiosis (HME) and HGE are present in Israel. Therefore, human ehrlichiosis should be included in the differential diagnoses for persons in Israel who have been exposed to ticks and have influenzalike symptoms. PMID:10603210

  15. A case of human monocytic ehrlichiosis in Serbia

    Directory of Open Access Journals (Sweden)

    Arsić Bogdan

    2014-01-01

    Full Text Available Introduction. Ehrlichiosis is a bacterial zoonosis transmitted by hematophagous arthropods - ticks. In humans, it occurs as monocytic, granulocytic, and ewingii ehrlichiosis. Pathological process is based on parasitic presence of Ehrlichia organisms within peripheral blood cells - monocytes and granulocytes. Case Outline. Fifty-two year old patient was admitted to hospital due to high fever of over 40°C that lasted two days, accompanied with chills, muscle aches, malaise, loss of appetite, headache, confusion, breathing difficulties, and mild dry cough. The history suggested tick bite that occurred seven days before the onset of disease. Doxycycline was introduced and administered for 14 days, causing the disease to subside. Indirect immunofluorescence assay was used to analyze three serum samples obtained from this patient for Ehrlichia chaffeensis antibodies, and peripheral blood smear was evaluated for the presence of Ehrlichia and Ehrlichia aggregation into morulae. Conclusion. Ehrlichiosis should be considered in each case where there is a history of tick bite together with the clinical picture (high fever, chills, muscle aches, headache, generalized weakness and malaise, and possible maculopapular rash. The presence of Ehrlichia chaffeensis antibodies was confirmed in a patient with the history of tick bite, appropriate clinical picture and indirect immunofluorescence assay. This confirmed the presence of human monocytotropic ehrlichiosis, a disease that is uncommonly identified in our country.

  16. In vitro studies of the toxic effects of silver nanoparticles on HeLa and U937 cells

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    Kaba SI

    2015-03-01

    Full Text Available Said I Kaba, Elena M Egorova Institute of General Pathology and Pathophysiology, Moscow, Russia Abstract: In the last decade, much attention has been paid to studies of the effect of silver nanoparticles (Ag NPs on tumor cells. Apart from elucidation of the mechanism of NPs’ interaction with mammalian cells, these studies are aimed at discovering new effective antitumor drugs. In this work, we report about the toxic effects of Ag NPs observed on two types of tumor cells: HeLa (adhesive cells and U937 (suspension cells. The Ag NPs were obtained by an original method of biochemical synthesis. Particle size was 13.2±4.72 nm, and zeta potential was -61.9±3.2 mV. The toxicity of Ag NPs in the concentration range 0.5–8.0 µg Ag/mL was determined by means of 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay and cytofluorometry after 4 and 24 hours' incubation. It was found that Ag NPs had high toxicity toward both cell types. The minimal concentrations where a toxicity effect was registered (toxicity thresholds lied in the range 0.5–2.0 µg Ag/mL. In parallel with the Ag NP solution, cells were incubated with water solutions of the NP stabilizer (aerosol-OT and Ag+ ions (as silver nitrate. It was shown that aerosol-OT had no effect on the viability on HeLa cells, but was moderately toxic toward U937, though less dangerous for these cells than Ag NPs. With Ag+ ions, for HeLa no toxic effect was observed, while for U937 they were as toxic as the Ag NPs. The data obtained indicate that Ag NPs as used in this study may prove to be useful for the creation of medicines for cancer therapy. Keywords: silver nanoparticles, cell viability, apoptosis, tumor cells

  17. Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer

    OpenAIRE

    Venneri, Mary Anna; De Palma, Michele; Ponzoni, Maurilio; Pucci, Ferdinando; Scielzo, Cristina; Zonari, Erika; Mazzieri, Roberta; Doglioni, Claudio; Naldini, Luigi

    2007-01-01

    Tumor-infiltrating myeloid cells, including tumor-associated macrophages (TAMs), have been implicated in tumor progression. We recently described a lineage of mouse monocytes characterized by expression of the Tie2 angiopoietin receptor and required for the vascularization and growth of several tumor models. Here, we report that TIE2 expression in human blood identifies a subset of monocytes distinct from classical inflammatory monocytes and comprised within the less abundant "resident" popul...

  18. Effect of Surface-Modified Paclitaxel Nanowires on U937 Cells In Vitro: A Novel Drug Delivery Vehicle

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    Mohamed H. Abumaree

    2012-01-01

    Full Text Available We have fabricated surface-modified paclitaxel nanowires (SM-PNs with a precise diameter and an average length of 50 μm. The surface of these nanowires is coated with a monolayer of octadecylsiloxane (ODS, which prevents aggregation and enhances dispersivity in aqueous media. This system constitutes a novel drug delivery vehicle based on one-dimensional (1D nanostructures with a large drug to vehicle ratio. We assayed the cytotoxicity of different diameter SM-PNs (200, 80, 35, and 18 nm with U937 cells and compared their activity to microcrystalline paclitaxel. SM-PNs reduced U937 cell proliferation in culture followed by cell death. For the same amount of paclitaxel, different diameter SM-PNs displayed different cytotoxic effect at the same incubation time period. SM-PNs with 35 nm diameters were the most efficient in completely halting cell proliferation following the first 24 hours of treatment, associated with 42% cell death. SM-PNs with 18 nm diameters were least effective. These SM-PNs can be tailored to fit a certain treatment protocol by simply choosing the appropriate diameter.

  19. High-Density Lipoprotein Reduction Differentially Modulates to Classical and Nonclassical Monocyte Subpopulations in Metabolic Syndrome Patients and in LPS-Stimulated Primary Human Monocytes In Vitro

    Science.gov (United States)

    Grün, Johanna L.; Manjarrez-Reyna, Aaron N.; Gómez-Arauz, Angélica Y.; Leon-Cabrera, Sonia; Bueno-Hernández, Nallely; Islas-Andrade, Sergio

    2018-01-01

    The effect of metabolic syndrome on human monocyte subpopulations has not yet been studied. Our main goal was to examine monocyte subpopulations in metabolic syndrome patients, while also identifying the risk factors that could directly influence these cells. Eighty-six subjects were divided into metabolic syndrome patients and controls. Monocyte subpopulations were quantified by flow cytometry, and interleukin- (IL-) 1β secretion levels were measured by ELISA. Primary human monocytes were cultured in low or elevated concentrations of high-density lipoprotein (HDL) and stimulated with lipopolysaccharide (LPS). The nonclassical monocyte (NCM) percentage was significantly increased in metabolic syndrome patients as compared to controls, whereas classical monocytes (CM) were reduced. Among all metabolic syndrome risk factors, HDL reduction exhibited the most important correlation with monocyte subpopulations and then was studied in vitro. Low HDL concentration reduced the CM percentage, whereas it increased the NCM percentage and IL-1β secretion in LPS-treated monocytes. The LPS effect was abolished when monocytes were cultured in elevated HDL concentrations. Concurring with in vitro results, IL-1β serum values significantly increased in metabolic syndrome patients with low HDL levels as compared to metabolic syndrome patients without HDL reduction. Our data demonstrate that HDL directly modulates monocyte subpopulations in metabolic syndrome. PMID:29850624

  20. High-Density Lipoprotein Reduction Differentially Modulates to Classical and Nonclassical Monocyte Subpopulations in Metabolic Syndrome Patients and in LPS-Stimulated Primary Human Monocytes In Vitro

    Directory of Open Access Journals (Sweden)

    Johanna L. Grün

    2018-01-01

    Full Text Available The effect of metabolic syndrome on human monocyte subpopulations has not yet been studied. Our main goal was to examine monocyte subpopulations in metabolic syndrome patients, while also identifying the risk factors that could directly influence these cells. Eighty-six subjects were divided into metabolic syndrome patients and controls. Monocyte subpopulations were quantified by flow cytometry, and interleukin- (IL- 1β secretion levels were measured by ELISA. Primary human monocytes were cultured in low or elevated concentrations of high-density lipoprotein (HDL and stimulated with lipopolysaccharide (LPS. The nonclassical monocyte (NCM percentage was significantly increased in metabolic syndrome patients as compared to controls, whereas classical monocytes (CM were reduced. Among all metabolic syndrome risk factors, HDL reduction exhibited the most important correlation with monocyte subpopulations and then was studied in vitro. Low HDL concentration reduced the CM percentage, whereas it increased the NCM percentage and IL-1β secretion in LPS-treated monocytes. The LPS effect was abolished when monocytes were cultured in elevated HDL concentrations. Concurring with in vitro results, IL-1β serum values significantly increased in metabolic syndrome patients with low HDL levels as compared to metabolic syndrome patients without HDL reduction. Our data demonstrate that HDL directly modulates monocyte subpopulations in metabolic syndrome.

  1. DMPD: Differential responses of human monocytes and macrophages to IL-4 and IL-13. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 10534111 Differential responses of human monocytes and macrophages to IL-4 and IL-1...):575-8. (.png) (.svg) (.html) (.csml) Show Differential responses of human monocytes and macrophages to IL-...4 and IL-13. PubmedID 10534111 Title Differential responses of human monocytes an

  2. 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.

  3. Matrix metalloproteinase-2 and -9 are induced differently by metal nanoparticles in human monocytes: The role of oxidative stress and protein tyrosine kinase activation

    International Nuclear Information System (INIS)

    Wan Rong; Mo Yiqun; Zhang Xing; Chien Sufan; Tollerud, David J.; Zhang Qunwei

    2008-01-01

    Recently, many studies have shown that nanoparticles can translocate from the lungs to the circulatory system. As a particulate foreign body, nanoparticles could induce host responses such as reactive oxygen species (ROS) generation, inflammatory cytokine and matrix metalloproteinase (MMP) release which play a major role in tissue destruction and remodeling. However, the direct effects of nanoparticles on leukocytes, especially monocytes, are still unclear. The objective of the present study was to compare the ability of Nano-Co and Nano-TiO 2 to cause alteration of transcription and activity of MMPs and to explore possible mechanisms. We hypothesized that non-toxic doses of some transition metal nanoparticles stimulate an imbalance of MMP/TIMP that cause MMP production that may contribute to their health effects. To test this hypothesis, U937 cells were treated with Nano-Co and Nano-TiO 2 and cytotoxic effects and ROS generation were measured. The alteration of MMP-2 and MMP-9 expression and activity of MMP-2 and MMP-9 after exposure to these metal nanoparticles were subsequently determined. To investigate the potential signaling pathways involved in the Nano-Co-induced MMP activation, the ROS scavengers or inhibitors, AP-1 inhibitor, and protein tyrosine kinase (PTK) inhibitors were also used to pre-treat U937 cells. Our results demonstrated that exposure of U937 cells to Nano-Co, but not to Nano-TiO 2 , at a dose that does not cause cytotoxicity, resulted in ROS generation and up-regulation of MMP-2 and MMP-9 mRNA expression .. Our results also showed dose- and time-related increases in pro-MMP-2 and pro-MMP-9 gelatinolytic activities in conditioned media after exposure of U937 cells to Nano-Co, but not to Nano-TiO 2 . Nano-Co-induced pro-MMP-2 and pro-MMP-9 activity increases were inhibited by pre-treatment with ROS scavengers or inhibitors. We also demonstrated dose- and time-related decreases in tissue inhibitors of metalloproteinases 2 (TIMP-2) in U937 cells

  4. Modulation of the expression of chondroitin sulfate proteoglycan in stimulated human monocytes

    International Nuclear Information System (INIS)

    Uhlin-Hansen, L.; Eskeland, T.; Kolset, S.O.

    1989-01-01

    Proteoglycan biosynthesis was studied in human monocytes and monocyte-derived macrophages (MDM) after exposure to typical activators of the monocyte/macrophage system: interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). By morphological examination, both monocytes and MDM were stimulated by these activators. Treatment with IFN-gamma resulted in a slight decrease in the expression of [35S]chondroitin sulfate proteoglycan (CSPG) in both monocytes and MDM, whereas LPS treatment increased the [35S]CSPG expression 1.8 and 2.2 times, respectively. PMA, in contrast, decreased the CSPG expression 0.4 times in monocytes, whereas MDM were stimulated to increase the biosynthesis 1.9 times. An increase in the sulfate density of the chondroitin sulfate chains was evident following differentiation of monocytes into MDM due to the expression of disulfated disaccharide units of the chondroitin sulfate E type (CS-E). However, monocytes exposed to PMA did also express disaccharides of the chondroitin sulfate E type. Furthermore, the expression of CS-E in MDM was increased 2 times following PMA treatment. An inactive phorbol ester, phorbol 12,13-diacetate, did not affect the expression of CS-E in either monocytes or MDM when compared with control cultures, suggesting that protein kinase C-dependent signal pathways may be involved in the regulation of sulfation of CSPG. Exposure to LPS or IFN-gamma did not lead to any changes in the sulfation of the chondroitin sulfate chains

  5. A comparative study of U937 cell size changes during apoptosis initiation by flow cytometry, light scattering, water assay and electronic sizing.

    Science.gov (United States)

    Yurinskaya, Valentina; Aksenov, Nikolay; Moshkov, Alexey; Model, Michael; Goryachaya, Tatyana; Vereninov, Alexey

    2017-10-01

    A decrease in flow cytometric forward light scatter (FSC) is commonly interpreted as a sign of apoptotic cell volume decrease (AVD). However, the intensity of light scattering depends not only on the cell size but also on its other characteristics, such as hydration, which may affect the scattering in the opposite way. That makes estimation of AVD by FSC problematic. Here, we aimed to clarify the relationship between light scattering, cell hydration (assayed by buoyant density) and cell size by the Coulter technique. We used human lymphoid cells U937 exposed to staurosporine, etoposide or hypertonic stress as an apoptotic model. An initial increase in FSC was found to occur in apoptotic cells treated with staurosporine and hypertonic solutions; it is accompanied by cell dehydration and is absent in apoptosis caused by etoposide that is consistent with the lack of dehydration in this case. Thus, the effect of dehydration on the scattering signal outweighs the effect of reduction in cell size. The subsequent FSC decrease, which occurred in parallel to accumulation of annexin-positive cells, was similar in apoptosis caused by all three types of inducers. We conclude that an increase, but not a decrease in light scattering, indicates the initial cell volume decrease associated with apoptotic cell dehydration.

  6. Interaction studies reveal specific recognition of an anti-inflammatory polyphosphorhydrazone dendrimer by human monocytes.

    Science.gov (United States)

    Ledall, Jérémy; Fruchon, Séverine; Garzoni, Matteo; Pavan, Giovanni M; Caminade, Anne-Marie; Turrin, Cédric-Olivier; Blanzat, Muriel; Poupot, Rémy

    2015-11-14

    Dendrimers are nano-materials with perfectly defined structure and size, and multivalency properties that confer substantial advantages for biomedical applications. Previous work has shown that phosphorus-based polyphosphorhydrazone (PPH) dendrimers capped with azabisphosphonate (ABP) end groups have immuno-modulatory and anti-inflammatory properties leading to efficient therapeutic control of inflammatory diseases in animal models. These properties are mainly prompted through activation of monocytes. Here, we disclose new insights into the molecular mechanisms underlying the anti-inflammatory activation of human monocytes by ABP-capped PPH dendrimers. Following an interdisciplinary approach, we have characterized the physicochemical and biological behavior of the lead ABP dendrimer with model and cell membranes, and compared this experimental set of data to predictive computational modelling studies. The behavior of the ABP dendrimer was compared to the one of an isosteric analog dendrimer capped with twelve azabiscarboxylate (ABC) end groups instead of twelve ABP end groups. The ABC dendrimer displayed no biological activity on human monocytes, therefore it was considered as a negative control. In detail, we show that the ABP dendrimer can bind both non-specifically and specifically to the membrane of human monocytes. The specific binding leads to the internalization of the ABP dendrimer by human monocytes. On the contrary, the ABC dendrimer only interacts non-specifically with human monocytes and is not internalized. These data indicate that the bioactive ABP dendrimer is recognized by specific receptor(s) at the surface of human monocytes.

  7. Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer.

    Science.gov (United States)

    Venneri, Mary Anna; De Palma, Michele; Ponzoni, Maurilio; Pucci, Ferdinando; Scielzo, Cristina; Zonari, Erika; Mazzieri, Roberta; Doglioni, Claudio; Naldini, Luigi

    2007-06-15

    Tumor-infiltrating myeloid cells, including tumor-associated macrophages (TAMs), have been implicated in tumor progression. We recently described a lineage of mouse monocytes characterized by expression of the Tie2 angiopoietin receptor and required for the vascularization and growth of several tumor models. Here, we report that TIE2 expression in human blood identifies a subset of monocytes distinct from classical inflammatory monocytes and comprised within the less abundant "resident" population. These TIE2-expressing monocytes (TEMs) accounted for 2% to 7% of blood mononuclear cells in healthy donors and were distinct from rare circulating endothelial cells and progenitors. In human cancer patients, TEMs were observed in the blood and, intriguingly, within the tumors, where they represented the main monocyte population distinct from TAMs. Conversely, TEMs were hardly detected in nonneoplastic tissues. In vitro, TEMs migrated toward angiopoietin-2, a TIE2 ligand released by activated endothelial cells and angiogenic vessels, suggesting a homing mechanism for TEMs to tumors. Purified human TEMs, but not TEM-depleted monocytes, markedly promoted angiogenesis in xenotransplanted human tumors, suggesting a potentially critical role of TEMs in human cancer progression. Human TEMs may provide a novel, biologically relevant marker of angiogenesis and represent a previously unrecognized target of cancer therapy.

  8. Inhibition of the differentiation of monocyte-derived dendritic cells by human gingival fibroblasts.

    Directory of Open Access Journals (Sweden)

    Sylvie Séguier

    Full Text Available We investigated whether gingival fibroblasts (GFs can modulate the differentiation and/or maturation of monocyte-derived dendritic cells (DCs and analyzed soluble factors that may be involved in this immune modulation. Experiments were performed using human monocytes in co-culture with human GFs in Transwell® chambers or using monocyte cultures treated with conditioned media (CM from GFs of four donors. The four CM and supernatants from cell culture were assayed by ELISA for cytokines involved in the differentiation of dendritic cells, such as IL-6, VEGF, TGFβ1, IL-13 and IL-10. The maturation of monocyte-derived DCs induced by LPS in presence of CM was also studied. Cell surface phenotype markers were analyzed by flow cytometry. In co-cultures, GFs inhibited the differentiation of monocyte-derived DCs and the strength of this blockade correlated with the GF/monocyte ratio. Conditioned media from GFs showed similar effects, suggesting the involvement of soluble factors produced by GFs. This inhibition was associated with a lower stimulatory activity in MLR of DCs generated with GFs or its CM. Neutralizing antibodies against IL-6 and VEGF significantly (P<0.05 inhibited the inhibitory effect of CM on the differentiation of monocytes-derived DCs and in a dose dependent manner. Our data suggest that IL-6 is the main factor responsible for the inhibition of DCs differentiation mediated by GFs but that VEGF is also involved and constitutes an additional mechanism.

  9. Endogenous pyrogen production by human blood monocytes stimulated by staphylococcal cell wall components.

    OpenAIRE

    Oken, M M; Peterson, P K; Wilkinson, B J

    1981-01-01

    To determine the properties of Staphylococcus aureus contributing to its pyrogenicity, we compared, in human monocytes, endogenous pyrogen production stimulated by heat-killed S. aureus with that stimulated by purified S. aureus cell walls or by particulate peptidoglycan prepared from the same strain. Peptidoglycan, but not the purified cell wall preparation, was found comparable to S. aureus as an endogenous pyrogen stimulus. This finding was associated with a more effective monocyte phagocy...

  10. Virulent Type A Francisella tularensis actively suppresses cytokine responses in human monocytes

    Science.gov (United States)

    Gillette, Devyn D.; Curry, Heather M.; Cremer, Thomas; Ravneberg, David; Fatehchand, Kavin; Shah, Prexy A.; Wewers, Mark D.; Schlesinger, Larry S.; Butchar, Jonathan P.; Tridandapani, Susheela; Gavrilin, Mikhail A.

    2014-01-01

    Background: Human monocyte inflammatory responses differ between virulent and attenuated Francisella infection. Results: A mixed infection model showed that the virulent F. tularensis Schu S4 can attenuate inflammatory cytokine responses to the less virulent F. novicida in human monocytes. Conclusion: F. tularensis dampens inflammatory response by an active process. Significance: This suppression may contribute to enhanced pathogenicity of F. tularensis. Francisella tularensis is a Gram-negative facultative bacterium that can cause the disease tularemia, even upon exposure to low numbers of bacteria. One critical characteristic of Francisella is its ability to dampen or subvert the host immune response. Previous work has shown that monocytes infected with highly virulent F. tularensis subsp. tularensis strain Schu S4 responded with a general pattern of quantitatively reduced pro-inflammatory signaling pathway genes and cytokine production in comparison to those infected with the less virulent related F. novicida. However, it has been unclear whether the virulent Schu S4 was merely evading or actively suppressing monocyte responses. By using mixed infection assays with F. tularensis and F. novicida, we show that F. tularensis actively suppresses monocyte pro-inflammatory responses. Additional experiments show that this suppression occurs in a dose-dependent manner and is dependent upon the viability of F. tularensis. Importantly, F. tularensis was able to suppress pro-inflammatory responses to earlier infections with F. novicida. These results lend support that F. tularensis actively dampens human monocyte responses and this likely contributes to its enhanced pathogenicity. PMID:24783062

  11. Toxicity of nanotitanium dioxide (TiO2-NP) on human monocytes and their mitochondria.

    Science.gov (United States)

    Ghanbary, Fatemeh; Seydi, Enaytollah; Naserzadeh, Parvaneh; Salimi, Ahmad

    2018-03-01

    The effect of nanotitanium dioxide (TiO 2 -NP) in human monocytes is still unknown. Therefore, an understanding of probable cytotoxicity of TiO 2 -NP on human monocytes and underlining the mechanisms involved is of significant interest. The aim of this study was to assess the cytotoxicity of TiO 2 -NP on human monocytes. Using biochemical and flow cytometry assessments, we demonstrated that addition of TiO 2 -NP at 10 μg/ml concentration to monocytes induced cytotoxicity following 12 h. The TiO 2 -NP-induced cytotoxicity on monocytes was associated with intracellular reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) collapse, lysosomal membrane injury, lipid peroxidation, and depletion of glutathione. According to our results, TiO 2 -NP triggers oxidative stress and organelles damages in monocytes which are important cells in defense against foreign agents. Finally, our findings suggest that use of antioxidants and mitochondrial/lysosomal protective agents could be of benefit for the people in the exposure with TiO 2 -NP.

  12. Decreased glucose uptake by hyperglycemia is regulated by different mechanisms in human cancer cells and monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chae Kyun; Chung, June Key; Lee, Yong Jin; Hong, Mee Kyoung; Jeong, Jae Min; Lee, Dong Soo; Lee, Myung Chul [College of Medicine, Seoul National Univ., Seoul (Korea, Republic of)

    2002-04-01

    To clarify the difference in glucose uptake between human cancer cells and monocytes, we studied ({sup 18}F) fluorodeoxyglucose (FDG) uptake in three human colon cancer cell lines (SNU-C2A, SNU-C4, SNU-C5), one human lung cancer cell line (NCI-H522), and human peripheral blood monocytes. The FDG uptake of both cancer cells and monocytes was increased in glucose-free medium, but decreased in the medium containing 16.7 mM glucose (hyperglycemic). The level of Glut1 mRNA decreased in human colon cancer cells and NCI-H522 under hyperglycemic condition. Glut1 protein expression was also decreased in the four human cancer cell lines under hyperglycemic condition, whereas it was consistently undetectable in monocytes. SNU-C2A, SNU-C4 and NCI-H522 showed a similar level of hexokinase activity (7.5-10.8 mU/mg), while SNU-C5 and moncytes showed lower range of hexokinase activity (4.3-6.5 mU/mg). These data suggest that glucose uptake is regulated by different mechanisms in human cancer cells and monocytes.

  13. Decreased glucose uptake by hyperglycemia is regulated by different mechanisms in human cancer cells and monocytes

    International Nuclear Information System (INIS)

    Kim, Chae Kyun; Chung, June Key; Lee, Yong Jin; Hong, Mee Kyoung; Jeong, Jae Min; Lee, Dong Soo; Lee, Myung Chul

    2002-01-01

    To clarify the difference in glucose uptake between human cancer cells and monocytes, we studied ( 18 F) fluorodeoxyglucose (FDG) uptake in three human colon cancer cell lines (SNU-C2A, SNU-C4, SNU-C5), one human lung cancer cell line (NCI-H522), and human peripheral blood monocytes. The FDG uptake of both cancer cells and monocytes was increased in glucose-free medium, but decreased in the medium containing 16.7 mM glucose (hyperglycemic). The level of Glut1 mRNA decreased in human colon cancer cells and NCI-H522 under hyperglycemic condition. Glut1 protein expression was also decreased in the four human cancer cell lines under hyperglycemic condition, whereas it was consistently undetectable in monocytes. SNU-C2A, SNU-C4 and NCI-H522 showed a similar level of hexokinase activity (7.5-10.8 mU/mg), while SNU-C5 and moncytes showed lower range of hexokinase activity (4.3-6.5 mU/mg). These data suggest that glucose uptake is regulated by different mechanisms in human cancer cells and monocytes

  14. Andrographolide Analogue Induces Apoptosis and Autophagy Mediated Cell Death in U937 Cells by Inhibition of PI3K/Akt/mTOR Pathway.

    Directory of Open Access Journals (Sweden)

    Deepak Kumar

    Full Text Available Current chemotherapeutic agents based on apoptosis induction are lacking in desired efficacy. Therefore, there is continuous effort to bring about new dimension in control and gradual eradication of cancer by means of ever evolving therapeutic strategies. Various forms of PCD are being increasingly implicated in anti-cancer therapy and the complex interplay among them is vital for the ultimate fate of proliferating cells. We elaborated and illustrated the underlying mechanism of the most potent Andrographolide analogue (AG-4 mediated action that involved the induction of dual modes of cell death-apoptosis and autophagy in human leukemic U937 cells.AG-4 induced cytotoxicity was associated with redox imbalance and apoptosis which involved mitochondrial depolarisation, altered apoptotic protein expressions, activation of the caspase cascade leading to cell cycle arrest. Incubation with caspase inhibitor Z-VAD-fmk or Bax siRNA decreased cytotoxic efficacy of AG-4 emphasising critical roles of caspase and Bax. In addition, AG-4 induced autophagy as evident from LC3-II accumulation, increased Atg protein expressions and autophagosome formation. Pre-treatment with 3-MA or Atg 5 siRNA suppressed the cytotoxic effect of AG-4 implying the pro-death role of autophagy. Furthermore, incubation with Z-VAD-fmk or Bax siRNA subdued AG-4 induced autophagy and pre-treatment with 3-MA or Atg 5 siRNA curbed AG-4 induced apoptosis-implying that apoptosis and autophagy acted as partners in the context of AG-4 mediated action. AG-4 also inhibited PI3K/Akt/mTOR pathway. Inhibition of mTOR or Akt augmented AG-4 induced apoptosis and autophagy signifying its crucial role in its mechanism of action.Thus, these findings prove the dual ability of AG-4 to induce apoptosis and autophagy which provide a new perspective to it as a potential molecule targeting PCD for future cancer therapeutics.

  15. A curated compendium of monocyte transcriptome datasets of relevance to human monocyte immunobiology research [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Darawan Rinchai

    2016-04-01

    Full Text Available Systems-scale profiling approaches have become widely used in translational research settings. The resulting accumulation of large-scale datasets in public repositories represents a critical opportunity to promote insight and foster knowledge discovery. However, resources that can serve as an interface between biomedical researchers and such vast and heterogeneous dataset collections are needed in order to fulfill this potential. Recently, we have developed an interactive data browsing and visualization web application, the Gene Expression Browser (GXB. This tool can be used to overlay deep molecular phenotyping data with rich contextual information about analytes, samples and studies along with ancillary clinical or immunological profiling data. In this note, we describe a curated compendium of 93 public datasets generated in the context of human monocyte immunological studies, representing a total of 4,516 transcriptome profiles. Datasets were uploaded to an instance of GXB along with study description and sample annotations. Study samples were arranged in different groups. Ranked gene lists were generated based on relevant group comparisons. This resource is publicly available online at http://monocyte.gxbsidra.org/dm3/landing.gsp.

  16. Effect of Robola and Cabernet Sauvignon extracts on platelet activating factor enzymes activity on U937 cells.

    Science.gov (United States)

    Xanthopoulou, M N; Asimakopoulos, D; Antonopoulou, S; Demopoulos, C A; Fragopoulou, E

    2014-12-15

    A number of studies support the anti-atherogenic effect of wine compounds. The scope of this study was to examine the effect of a red (Cabernet Sauvignon-CS) and a white (Robola-R) wine, as well as resveratrol and quercetin, on the platelet activating factor (PAF) biosynthetic enzymes, acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAF-AT) and DTT-insensitive CDP-choline 1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-CPT), and its main catabolic enzyme (PAF acetylhydrolase; PAF-AH), on U937 cells, in cell free and in intact cell experiments. In cell free experiments, phenolic compounds and wine extracts inhibited PAF biosynthetic enzymes, however in higher concentrations than intact cell experiments. In the latter cases, polar lipids of both wines inhibited in the same order of magnitude the action of lyso-PAF-AT and of PAF-CPT. The water fractions possessed a dual action, in lower concentrations they activated both enzymes, while in higher concentrations only inhibited PAF-CPT. All fractions either did not affect or slightly activated PAF-AH activity. In conclusion, wine compounds may exert their anti-inflammatory activity by reducing PAF levels through modulation of the PAF metabolic enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Evaluation of the Genetic Response of U937 and Jurkat Cells to 10-Nanosecond Electrical Pulses (nsEP.

    Directory of Open Access Journals (Sweden)

    Caleb C Roth

    Full Text Available Nanosecond electrical pulse (nsEP exposure activates signaling pathways, produces oxidative stress, stimulates hormone secretion, causes cell swelling and induces apoptotic and necrotic death. The underlying biophysical connection(s between these diverse cellular reactions and nsEP has yet to be elucidated. Using global genetic analysis, we evaluated how two commonly studied cell types, U937 and Jurkat, respond to nsEP exposure. We hypothesized that by studying the genetic response of the cells following exposure, we would gain direct insight into the stresses experienced by the cell and in turn better understand the biophysical interaction taking place during the exposure. Using Ingenuity Systems software, we found genes associated with cell growth, movement and development to be significantly up-regulated in both cell types 4 h post exposure to nsEP. In agreement with our hypothesis, we also found that both cell lines exhibit significant biological changes consistent with mechanical stress induction. These results advance nsEP research by providing strong evidence that the interaction of nsEPs with cells involves mechanical stress.

  18. Signaling factors and pathways of α-particle irradiation induced bilateral bystander responses between Beas-2B and U937 cells

    International Nuclear Information System (INIS)

    Fu, Jiamei; Wang, Juan; Wang, Xiangdong; Wang, Ping; Xu, Jinping; Zhou, Cuiping; Bai, Yang; Shao, Chunlin

    2016-01-01

    Highlights: • Radiation damage of Beas-2B cells was enhanced by macrophage-mediated bilateral bystander responses. • Expressions of TNF-α and IL-8 in the α-irradiated Beas-2B cells were dependent on ERK and p38 pathways. • The neighboring U937 cells further increased the generation of TNF-α and IL-8 in the α-irradiated Beas-2B cells. • NF-κB dependent upregulation of TNF-α and IL-8 was induced in the bystander U937 cells. - Abstract: Although radiation induced bystander effects (RIBE) have been investigated for decades for their potential health risk, the underlying gene regulation is still largely unclear, especially the roles of immune system and inflammatory response in RIBE. In the present study, macrophage U937 cells and epithelial Beas-2B cells were co-cultured to disclose the cascades of bystander signaling factors and intercellular communications. After α-particle irradiation, both ERK and p38 pathways were activated in Beas-2B cells and were associated with the autocrine and paracrine signaling of TNF-α and IL-8, resulting in direct damage to the irradiated cells. Similar upregulation of TNF-α and IL-8 was induced in the bystander U937 cells after co-culture with α-irradiated Beas-2B cells. This upregulation was dependent on the activation of NF-κB pathway and was responsible for the enhanced damage of α-irradiated Beas-2B cells. Interestingly, the increased expressions of TNF-α and IL-8 mRNAs in the bystander U937 cells were clearly relayed on the activated ERK and p38 pathways in the irradiated Beas-2B cells, and the upregulation of TNF-α and IL-8 mRNAs in co-cultured Beas-2B cells was also partly due to the activated NF-κB pathway in the bystander U937 cells. With the pretreatment of U0126 (MEK1/2 inhibitor), SB203580 (p38 inhibitor) or BAY 11-7082 (NF-κB inhibitor), the aggravated damage in the α-irradiated Beas-2B cells could be largely alleviated. Our results disclosed novel signaling cascades of macrophage-mediated bilateral

  19. Synergism between arsenite and proteasome inhibitor MG132 over cell death in myeloid leukaemic cells U937 and the induction of low levels of intracellular superoxide anion

    International Nuclear Information System (INIS)

    Lombardo, Tomás; Cavaliere, Victoria; Costantino, Susana N.; Kornblihtt, Laura; Alvarez, Elida M.; Blanco, Guillermo A.

    2012-01-01

    Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitt's lymphoma-derived Raji cells, by the Chou–Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O 2 − ) levels. Our results showed that combined arsenite + MG132 produced low levels of O 2 − at 6 h and 24 h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80 μM hydrogen peroxide together with arsenite + MG132 changed synergism on cell death to antagonism at all effect levels while increasing O 2 − levels. Arsenite + hydrogen peroxide also resulted in antagonism with increased O 2 − levels in U937 cells. In Raji cells, arsenite + MG132 also produced low levels of O 2 − at 6 h and 24 h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite + CAPE showed high levels of O 2 − production at 6 h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O 2 − levels at early time points after exposure. -- Highlights: ► Arsenic combined cytotoxic and anti-proliferative effects by Chou–Talalay method. ► Cytotoxic effect associated with superoxide levels as assessed by flow cytometry. ► Synergism

  20. Signaling factors and pathways of α-particle irradiation induced bilateral bystander responses between Beas-2B and U937 cells

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Jiamei; Wang, Juan; Wang, Xiangdong; Wang, Ping; Xu, Jinping; Zhou, Cuiping; Bai, Yang; Shao, Chunlin, E-mail: clshao@shmu.edu.cn

    2016-07-15

    Highlights: • Radiation damage of Beas-2B cells was enhanced by macrophage-mediated bilateral bystander responses. • Expressions of TNF-α and IL-8 in the α-irradiated Beas-2B cells were dependent on ERK and p38 pathways. • The neighboring U937 cells further increased the generation of TNF-α and IL-8 in the α-irradiated Beas-2B cells. • NF-κB dependent upregulation of TNF-α and IL-8 was induced in the bystander U937 cells. - Abstract: Although radiation induced bystander effects (RIBE) have been investigated for decades for their potential health risk, the underlying gene regulation is still largely unclear, especially the roles of immune system and inflammatory response in RIBE. In the present study, macrophage U937 cells and epithelial Beas-2B cells were co-cultured to disclose the cascades of bystander signaling factors and intercellular communications. After α-particle irradiation, both ERK and p38 pathways were activated in Beas-2B cells and were associated with the autocrine and paracrine signaling of TNF-α and IL-8, resulting in direct damage to the irradiated cells. Similar upregulation of TNF-α and IL-8 was induced in the bystander U937 cells after co-culture with α-irradiated Beas-2B cells. This upregulation was dependent on the activation of NF-κB pathway and was responsible for the enhanced damage of α-irradiated Beas-2B cells. Interestingly, the increased expressions of TNF-α and IL-8 mRNAs in the bystander U937 cells were clearly relayed on the activated ERK and p38 pathways in the irradiated Beas-2B cells, and the upregulation of TNF-α and IL-8 mRNAs in co-cultured Beas-2B cells was also partly due to the activated NF-κB pathway in the bystander U937 cells. With the pretreatment of U0126 (MEK1/2 inhibitor), SB203580 (p38 inhibitor) or BAY 11-7082 (NF-κB inhibitor), the aggravated damage in the α-irradiated Beas-2B cells could be largely alleviated. Our results disclosed novel signaling cascades of macrophage-mediated bilateral

  1. Synergism between arsenite and proteasome inhibitor MG132 over cell death in myeloid leukaemic cells U937 and the induction of low levels of intracellular superoxide anion

    Energy Technology Data Exchange (ETDEWEB)

    Lombardo, Tomás [Laboratorio de Immunotoxicologia (LaITO), IDEHU-CONICET, Hospital de Clínicas, José de San Martín, Universidad de Buenos Aires (UBA), Buenos Aires (Argentina); Cavaliere, Victoria; Costantino, Susana N. [Laboratorio de Inmunología Tumoral (LIT), IDEHU-CONICET, Facultad de Farmacia y Bioquímica, UBA, Buenos Aires (Argentina); Kornblihtt, Laura [Servicio de Hematología, Hospital de Clínicas, José de San Martín (UBA), Buenos Aires (Argentina); Alvarez, Elida M. [Laboratorio de Inmunología Tumoral (LIT), IDEHU-CONICET, Facultad de Farmacia y Bioquímica, UBA, Buenos Aires (Argentina); Blanco, Guillermo A., E-mail: gblanco@ffyb.uba.ar [Laboratorio de Immunotoxicologia (LaITO), IDEHU-CONICET, Hospital de Clínicas, José de San Martín, Universidad de Buenos Aires (UBA), Buenos Aires (Argentina)

    2012-02-01

    Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitt's lymphoma-derived Raji cells, by the Chou–Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O{sub 2}{sup −}) levels. Our results showed that combined arsenite + MG132 produced low levels of O{sub 2}{sup −} at 6 h and 24 h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80 μM hydrogen peroxide together with arsenite + MG132 changed synergism on cell death to antagonism at all effect levels while increasing O{sub 2}{sup −} levels. Arsenite + hydrogen peroxide also resulted in antagonism with increased O{sub 2}{sup −} levels in U937 cells. In Raji cells, arsenite + MG132 also produced low levels of O{sub 2}{sup −} at 6 h and 24 h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite + CAPE showed high levels of O{sub 2}{sup −} production at 6 h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O{sub 2}{sup −} levels at early time points after exposure. -- Highlights: ► Arsenic combined cytotoxic and anti-proliferative effects by Chou–Talalay method. ► Cytotoxic effect

  2. The effect of short-chain fatty acids on human monocyte-derived dendritic cells

    DEFF Research Database (Denmark)

    Nastasi, Claudia; Candela, Marco; Bonefeld, Charlotte Menné

    2015-01-01

    negligible effects, while both butyrate and propionate strongly modulated gene expression in both immature and mature human monocyte-derived DC. An Ingenuity pathway analysis based on the differentially expressed genes suggested that propionate and butyrate modulate leukocyte trafficking, as SCFA strongly......The gut microbiota is essential for human health and plays an important role in the pathogenesis of several diseases. Short-chain fatty acids (SCFA), such as acetate, butyrate and propionate, are end-products of microbial fermentation of macronutrients that distribute systemically via the blood....... The aim of this study was to investigate the transcriptional response of immature and LPS-matured human monocyte-derived DC to SCFA. Our data revealed distinct effects exerted by each individual SCFA on gene expression in human monocyte-derived DC, especially in the mature ones. Acetate only exerted...

  3. Characterization and molecular features of the cell surface receptor for human granulocyte-macrophage colony-stimulating factor

    International Nuclear Information System (INIS)

    Chiba, S.; Tojo, A.; Kitamura, T.; Urabe, A.; Miyazono, K.; Takaku, F.

    1990-01-01

    The receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF) on the surfaces of normal and leukemic myeloid cells were characterized using 125I-labeled bacterially synthesized GM-CSF. The binding was rapid, specific, time dependent, and saturable. Scatchard analysis of the 125I-GM-CSF binding to peripheral blood neutrophils indicated the presence of a single class of binding site (Kd = 99 +/- 21 pM; 2,304 +/- 953 sites/cell). However, for peripheral blood monocytes and two GM-CSF-responsive myeloid cell lines (U-937 and TF-1), the Scatchard plots were biphasic curvilinear, which were best fit by curves derived from two binding site model: one with high affinity (Kd1 = 10-40 pM) and the other with low affinity (Kd2 = 0.9-2.0 nM). For U-937 cells, the number of high-affinity receptors was 1,058 +/- 402 sites/cell and that of low-affinity receptors was estimated to be 10,834 +/- 2,396 sites/cell. Cross-linking studies yielded three major bands with molecular masses of 150 kDa, 115 kDa, and 95 kDa, which were displaced by an excess amount of unlabeled GM-CSF, suggesting 135-kDa, 100-kDa, and 80-kDa species for the individual components of the human GM-CSF receptor. These bands comigrated for different cell types including peripheral blood neutrophils, U-937 cells and TF-1 cells. In experiments using U-937 cells, only the latter two bands appeared to be labeled in a dose-dependent manner in a low-affinity state. These results suggest that the human GM-CSF receptor possibly forms a multichain complex

  4. L-Cysteine in vitro can restore cellular glutathione and inhibits the expression of cell adhesion molecules in G6PD-deficient monocytes.

    Science.gov (United States)

    Parsanathan, Rajesh; Jain, Sushil K

    2018-04-06

    L-Cysteine is a precursor of glutathione (GSH), a potent physiological antioxidant. Excess glucose-6-phosphate dehydrogenase (G6PD) deficiency in African Americans and low levels of L-cysteine diet in Hispanics can contributes to GSH deficiency and oxidative stress. Oxidative stress and monocyte adhesion was considered to be an initial event in the progression of vascular dysfunction and atherosclerosis. However, no previous study has investigated the contribution of GSH/G6PD deficiency to the expression of monocyte adhesion molecules. Using human U937 monocytes, this study examined the effect of GSH/G6PD deficiency and L-cysteine supplementation on monocyte adhesion molecules. G6PD/GSH deficiency induced by either siRNA or inhibitors (6AN/BSO, respectively) significantly (p adhesion molecules (ICAM-1, VCAM-1, SELL, ITGB1 and 2); NADPH oxidase (NOX), reactive oxygen species (ROS) and MCP-1 were upregulated, and decreases in levels of GSH, and nitric oxide were observed. The expression of ICAM-1 and VCAM-1 mRNA levels increased in high glucose, MCP-1 or TNF-α-treated G6PD-deficient compared to G6PD-normal cells. L-Cysteine treatment significantly (p adhesion molecules. Thus, GSH/G6PD deficiency increases susceptibility to monocyte adhesion processes, whereas L-cysteine supplementation can restore cellular GSH/G6PD and attenuates NOX activity and expression of cell adhesion molecules.

  5. A System Dynamics Model to Predict the Human Monocyte Response to Endotoxins

    Directory of Open Access Journals (Sweden)

    Enrique Álvarez

    2017-08-01

    Full Text Available System dynamics is a powerful tool that allows modeling of complex and highly networked systems such as those found in the human immune system. We have developed a model that reproduces how the exposure of human monocytes to lipopolysaccharides (LPSs induces an inflammatory state characterized by high production of tumor necrosis factor alpha (TNFα, which is rapidly modulated to enter into a tolerant state, known as endotoxin tolerance (ET. The model contains two subsystems with a total of six states, seven flows, two auxiliary variables, and 14 parameters that interact through six differential and nine algebraic equations. The parameters were estimated and optimized to obtain a model that fits the experimental data obtained from human monocytes treated with various LPS doses. In contrast to publications on other animal models, stimulation of human monocytes with super-low-dose LPSs did not alter the response to a second LPSs challenge, neither inducing ET, nor enhancing the inflammatory response. Moreover, the model confirms the low production of TNFα and increased levels of C–C motif ligand 2 when monocytes exhibit a tolerant state similar to that of patients with sepsis. At present, the model can help us better understand the ET response and might offer new insights on sepsis diagnostics and prognosis by examining the monocyte response to endotoxins in patients with sepsis.

  6. Differential oxidative stress induced by dengue virus in monocytes from human neonates, adult and elderly individuals.

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    Nereida Valero

    Full Text Available Changes in immune response during lifespan of man are well known. These changes involve decreased neonatal and elderly immune response. In addition, it has been shown a relationship between immune and oxidative mechanisms, suggesting that altered immune response could be associated to altered oxidative response. Increased expression of nitric oxide (NO has been documented in dengue and in monocyte cultures infected with different types of dengue virus. However, there is no information about the age-dependent NO oxidative response in humans infected by dengue virus. In this study, monocyte cultures from neonatal, elderly and adult individuals (n = 10 each group were infected with different dengue virus types (DENV- 1 to 4 and oxidative/antioxidative responses and apoptosis were measured at days 1 and 3 of culture. Increased production of NO, lipid peroxidation and enzymatic and nonenzymatic anti-oxidative responses in dengue infected monocyte cultures were observed. However, neonatal and elderly monocytes had lower values of studied parameters when compared to those in adult-derived cultures. Apoptosis was present in infected monocytes with higher values at day 3 of culture. This reduced oxidant/antioxidant response of neonatal and elderly monocytes could be relevant in the pathogenesis of dengue disease.

  7. Endogenous pyrogen production by human blood monocytes stimulated by staphylococcal cell wall components.

    Science.gov (United States)

    Oken, M M; Peterson, P K; Wilkinson, B J

    1981-01-01

    To determine the properties of Staphylococcus aureus contributing to its pyrogenicity, we compared, in human monocytes, endogenous pyrogen production stimulated by heat-killed S. aureus with that stimulated by purified S. aureus cell walls or by particulate peptidoglycan prepared from the same strain. Peptidoglycan, but not the purified cell wall preparation, was found comparable to S. aureus as an endogenous pyrogen stimulus. This finding was associated with a more effective monocyte phagocytosis of S. aureus and peptidoglycan as compared with that of purified cell walls. Lysostaphin digestion of peptidoglycan markedly reduced its pyrogenicity. To test whether the chemical composition of the ingested particles is important, latex particles were tested as possible stimuli for monocyte endogenous pyrogen release. Although 40 to 68% of monocytes ingested latex particles during the first hour, there was no evidence of endogenous pyrogen activity in the supernatant even when supernatants equivalent to 5.2 X 10(6) monocytes were tested. This study demonstrates that the pyrogenic moiety of the S. aureus cell wall resides in the peptidoglycan component. Phagocytosis is not in itself a pyrogenic stimulus, but rather serves as an effective mechanism to bring about contact between the chemical stimulus and the monocyte.

  8. Cinnamic Acid Is Partially Involved in Propolis Immunomodulatory Action on Human Monocytes

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    Bruno José Conti

    2013-01-01

    Full Text Available Propolis is a beehive product used in traditional medicine due to its biological properties. It shows a complex chemical composition including phenolics, such as cinnamic acid (Ci. The mechanisms of action of propolis have been the subject of research recently; however, the involvement of Ci on propolis activity was not investigated on immune cells. Ci effects were evaluated on human monocytes, assessing the expression of Toll-like receptors (TLRs, HLA-DR, and CD80. Cytokine production (TNF-α and IL-10 and the fungicidal activity of monocytes were evaluated as well. Data showed that Ci downregulated TLR-2, HLA-DR, and CD80 and upregulated TLR-4 expression by human monocytes. High concentrations of Ci inhibited both TNF-α and IL-10 production, whereas the same concentrations induced a higher fungicidal activity against Candida albicans. TNF-α and IL-10 production was decreased by blocking TLR-4, while the fungicidal activity of monocytes was not affected by blocking TLRs. These results suggest that Ci modulated antigen receptors, cytokine production, and the fungicidal activity of human monocytes depending on concentration, and TLR-4 may be involved in its mechanism of action. Ci seemed to be partially involved in propolis activities.

  9. The effects of exogenous fatty acids and niacin on human monocyte-macrophage plasticity.

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    Montserrat-de la Paz, Sergio; Rodriguez, Dolores; Cardelo, Magdalena P; Naranjo, Maria C; Bermudez, Beatriz; Abia, Rocio; Muriana, Francisco J G; Lopez, Sergio

    2017-08-01

    Macrophage plasticity allows adapting to different environments, having a dual activity in inflammatory-related diseases. Our hypothesis is that the type of dietary fatty acids into human postprandial triglyceride-rich lipoproteins (TRLs), alone or in combination with niacin (vitamin B3), could modulate the plasticity of monocytes-macrophages. We isolated TRLs at the postprandial peak from blood samples of healthy volunteers after the ingestion of a meal rich in saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) or MUFAs plus omega-3 long-chain polyunsaturated fatty acids (LCPUFAs). Autologous monocytes isolated at fasting were first induced to differentiate into naïve macrophages. We observed that postprandial TRL-MUFAs, particularly in combination with niacin, enhance competence to monocytes to differentiate and polarise into M2 macrophages. Postprandial TRL-SFAs made polarised macrophages prone to an M1 phenotype. In contrast to dietary SFAs, dietary MUFAs in the meals plus immediate-release niacin primed circulating monocytes for a reduced postprandial pro-inflammatory profile. Our study underlines a role of postprandial TRLs as a metabolic entity in regulating the plasticity of the monocyte-macrophage lineage and also brings an understanding of the mechanisms by which dietary fatty acids are environmental factors fostering the innate immune responsiveness in humans. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. miR-223 is upregulated in monocytes from patients with tuberculosis and regulates function of monocyte-derived macrophages.

    Science.gov (United States)

    Liu, Yanhua; Wang, Ruo; Jiang, Jing; Yang, Bingfen; Cao, Zhihong; Cheng, Xiaoxing

    2015-10-01

    Tuberculosis (TB) is a serious infectious disease that most commonly affects the lungs. Macrophages are among the first line defenders against establishment of Mycobacterium tuberculosis infection in the lungs. In this study, we found that activation and cytokine production in monocyte-derived macrophages (MDM) from patients with active TB was impaired. miR-223 expression was significantly elevated in monocytes and MDM from patients with TB compared with healthy controls. To determine the functional role of miR-223 in macrophages, stable miR-223-expressing and miR-223 antisense-expressing U937 cells were established. Compared with empty vector controls, expression of IL-1β, IL-6, TNF-α and IL-12p40 genes was significantly higher in miR-223 antisense-expressing U937 cells, but lower in miR-223-expressing U937 cells. miR-223 can negatively regulate activation of NF-κB by inhibition of p65 phosphorylation and nuclear translocation. It is concluded that miR-223 can regulate macrophage function by inhibition of cytokine production and NF-κB activation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Palmitate and insulin synergistically induce IL-6 expression in human monocytes

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    Lumpkin Charles K

    2010-11-01

    Full Text Available Abstract Background Insulin resistance is associated with a proinflammatory state that promotes the development of complications such as type 2 diabetes mellitus (T2DM and atherosclerosis. The metabolic stimuli that initiate and propagate proinflammatory cytokine production and the cellular origin of proinflammatory cytokines in insulin resistance have not been fully elucidated. Circulating proinflammatory monocytes show signs of enhanced inflammation in obese, insulin resistant subjects and are thus a potential source of proinflammatory cytokine production. The specific, circulating metabolic factors that might stimulate monocyte inflammation in insulin resistant subjects are poorly characterized. We have examined whether saturated nonesterified fatty acids (NEFA and insulin, which increase in concentration with developing insulin resistance, can trigger the production of interleukin (IL-6 and tumor necrosis factor (TNF-α in human monocytes. Methods Messenger RNA and protein levels of the proinflammatory cytokines IL-6 and TNF-α were measured by quantitative real-time PCR (qRT-PCR and Luminex bioassays. Student's t-test was used with a significance level of p Results Esterification of palmitate with coenzyme A (CoA was necessary, while β-oxidation and ceramide biosynthesis were not required, for the induction of IL-6 and TNF-α in THP-1 monocytes. Monocytes incubated with insulin and palmitate together produced more IL-6 mRNA and protein, and more TNF-α protein, compared to monocytes incubated with palmitate alone. Incubation of monocytes with insulin alone did not affect the production of IL-6 or TNF-α. Both PI3K-Akt and MEK/ERK signalling pathways are important for cytokine induction by palmitate. MEK/ERK signalling is necessary for synergistic induction of IL-6 by palmitate and insulin. Conclusions High levels of saturated NEFA, such as palmitate, when combined with hyperinsulinemia, may activate human monocytes to produce

  12. CD14+ monocytes promote the immunosuppressive effect of human umbilical cord matrix stem cells

    International Nuclear Information System (INIS)

    Wang, Ding; Chen, Ke; Du, Wei Ting; Han, Zhi-Bo; Ren, He; Chi, Ying

    2010-01-01

    Here, the effect of CD14 + monocytes on human umbilical cord matrix stem cell (hUC-MSC)-mediated immunosuppression was studied in vitro. hUC-MSCs exerted a potent inhibitory effect on the proliferation and interferon-γ (IFN-γ) secretion capacities of CD4 + and CD8 + T cells in response to anti-CD3/CD28 stimulation. Transwell co-culture system revealed that the suppressive effect was primarily mediated by soluble factors. Addition of prostaglandin synthesis inhibitors (indomethacin or NS-398) almost completely abrogated the immunosuppression activity of hUC-MSCs, identifying prostaglandin E 2 (PGE 2 ) as an important soluble mediator. CD14 + monocytes were found to be able to enhance significantly the immunosuppressive effect of hUC-MSCs in a dose-dependent fashion. Moreover, the inflammatory cytokine IL-1β, either exogenously added or produced by CD14 + monocytes in culture, could trigger expression of high levels of PGE 2 by hUC-MSCs, whereas inclusion of the IL-1 receptor antagonist (IL-1RA) in the culture down-regulated not only PGE 2 expression, but also reversed the promotional effect of CD14 + monocytes and partially restored CD4 + and CD8 + T cell proliferation and IFN-γ secretion. Our data demonstrate an important role of monocytes in the hUC-MSC-induced immunomodulation, which may have important implications in future efforts to explore the clinical potentials of hUC-MSCs.

  13. Studies on the mechanism of endogenous pyrogen production. III. Human blood monocytes.

    Science.gov (United States)

    Bodel, P

    1974-10-01

    The characteristics of pyrogen production and release by human blood monocytes were investigated. A dose-response assay of monocyte pyrogen in rabbits indicated a linear relationship of temperature elevation to dose of pyrogen at lower doses. Monocytes did not contain pyrogen when first obtained, nor did they release it spontaneously even after 5 days of incubation in vitro. Pyrogen production was apparent 4 h after stimulation by endotoxin or phagocytosis, and continued for 24 h or more. Puromycin, an inhibitor of protein synthesis, prevented both initiation and continuation of pyrogen production and release. Pyrogen-containing supernates retained most pyrogenic activity during overnight incubation even in the presence of activated cells. Lymphocytes appeared to play no role in either initiation or continuation of pyrogen production in these studies.

  14. Novel ex vivo culture method for human monocytes uses shear flow to prevent total loss of transendothelial diapedesis function.

    Science.gov (United States)

    Tsubota, Yoshiaki; Frey, Jeremy M; Raines, Elaine W

    2014-01-01

    Monocyte recruitment to inflammatory sites and their transendothelial migration into tissues are critical to homeostasis and pathogenesis of chronic inflammatory diseases. However, even short-term suspension culture of primary human monocytes leads to phenotypic changes. In this study, we characterize the functional effects of ex vivo monocyte culture on the steps involved in monocyte transendothelial migration. Our data demonstrate that monocyte diapedesis is impaired by as little as 4 h culture, and the locomotion step is subsequently compromised. After 16 h in culture, monocyte diapedesis is irreversibly reduced by ∼90%. However, maintenance of monocytes under conditions mimicking physiological flow (5-7.5 dyn/cm²) is sufficient to reduce diapedesis impairment significantly. Thus, through the application of shear during ex vivo culture of monocytes, our study establishes a novel protocol, allowing functional analyses of monocytes not currently possible under static culture conditions. These data further suggest that monocyte-based therapeutic applications may be measurably improved by alteration of ex vivo conditions before their use in patients.

  15. Lipopolysaccharide induces autotaxin expression in human monocytic THP-1 cells

    International Nuclear Information System (INIS)

    Li Song; Zhang Junjie

    2009-01-01

    Autotaxin (ATX) is a secreted enzyme with lysophospholipase D (lysoPLD) activity, which converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a bioactive phospholipid involved in numerous biological activities, including cell proliferation, differentiation, and migration. In the present study, we found that bacterial lipopolysaccharide (LPS), a well-known initiator of the inflammatory response, induced ATX expression in monocytic THP-1 cells. The activation of PKR, JNK, and p38 MAPK was required for the ATX induction. The LPS-induced ATX in THP-1 cells was characterized as the β isoform. In the presence of LPC, ATX could promote the migrations of THP-1 and Jurkat cells, which was inhibited by pertussis toxin (PTX), an inhibitor of Gi-mediated LPA receptor signaling. In summary, LPS induces ATX expression in THP-1 cells via a PKR, JNK and p38 MAPK-mediated mechanism, and the ATX induction is likely to enhance immune cell migration in proinflammatory response by regulating LPA levels in the microenvironment.

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

    Science.gov (United States)

    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.

  17. Elastolytic activity of human blood monocytes characterized by a new monoclonal antibody against human leucocyte elastase. Relationship to rheumatoid arthritis

    DEFF Research Database (Denmark)

    Jensen, H S; Christensen, L D

    1990-01-01

    The leucocyte elastase of human blood monocytes was investigated by applying a new monoclonal antibody which did not block the enzyme activity against elastin. In a fixed population of mononuclear cells (MNC) and using fluorescence activated cell sorting (FACS), the human leucocyte elastase (HLE...

  18. Presence of estrogen receptors in human myeloid monocytic cells (THP-1 cell line).

    Science.gov (United States)

    Cutolo, M; Villaggio, B; Bisso, A; Sulli, A; Coviello, D; Dayer, J M

    2001-01-01

    To test THP-1 cells for the presence of estrogen receptors (ER) since studies have demonstrated in vivo and in vitro, the influence of estrogens on cells involved in immune response (i.e. macrophages), and since it has been demonstrated that human myeloid monocytic THP-1 cells acquire phenotypic and functional macrophage-like features after incubation with several cytokines or pharmacological agents. Stimulation of THP-1 cells with phorbol myristate acetate (PMA) to prompt their differentiation into macrophage-like cells and evaluation of the possible induction of ER. The expression of ER was analyzed by immunocytochemical assay, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. After stimulation by PMA, the human myeloid monocytic THP-1 cells showed the presence of ER, together with markers of monocytic cell differentiation such as CD68, CD54 and HLA-DR. Estrogen effects may be exerted directly through ER on monocytes/macrophages. PMA-treated THP-1 cells may constitute a useful in vitro model to determine the effects of estrogens on macrophage-like cells and their implications in the inflammatory and immune processes.

  19. Virulent Type A Francisella tularensis actively suppresses cytokine responses in human monocytes

    Directory of Open Access Journals (Sweden)

    Devyn D Gilette

    2014-04-01

    Full Text Available Francisella tularensis is a Gram-negative facultative bacterium that can cause the disease tularemia, even upon exposure to low numbers of bacteria. One critical characteristic of Francisella is its ability to dampen or subvert the host immune response. Previous work has shown that monocytes infected with highly virulent F. tularensis subsp. tularensis strain Schu S4 responded with a general pattern of quantitatively reduced pro-inflammatory signaling pathway genes and cytokine production in comparison to those infected with the less virulent related F. novicida. However, it has been unclear whether the virulent Schu S4 was merely evading or actively suppressing monocyte responses. By using mixed infection assays with F. tularensis and F. novicida, we show that F. tularensis actively suppresses monocyte pro-inflammatory responses. Additional experiments show that this suppression occurs in a dose-dependent manner and is dependent upon the viability of F. tularensis. Importantly, F. tularensis was able to suppress pro-inflammatory responses to earlier infections with F. novicida. These results lend support that F. tularensis actively dampens human monocyte responses and this likely contributes to its enhanced pathogenicity.

  20. Monocyte-Derived Signals Activate Human Natural Killer Cells in Response to Leishmania Parasites

    Science.gov (United States)

    Messlinger, Helena; Sebald, Heidi; Heger, Lukas; Dudziak, Diana; Bogdan, Christian; Schleicher, Ulrike

    2018-01-01

    Activated natural killer (NK) cells release interferon (IFN)-γ, which is crucial for the control of intracellular pathogens such as Leishmania. In contrast to experimental murine leishmaniasis, the human NK cell response to Leishmania is still poorly characterized. Here, we investigated the interaction of human blood NK cells with promastigotes of different Leishmania species (Leishmania major, Leishmania mexicana, Leishmania infantum, and Leishmania donovani). When peripheral blood mononuclear cells or purified NK cells and monocytes (all derived from healthy blood donors from Germany without a history of leishmaniasis) were exposed to promastigotes, NK cells showed increased surface expression of the activation marker CD69. The extent of this effect varied depending on the Leishmania species; differences between dermotropic and viscerotropic L. infantum strains were not observed. Upregulation of CD69 required direct contact between monocytes and Leishmania and was partly inhibitable by anti-interleukin (IL)-18. Unexpectedly, IL-18 was undetectable in most of the supernatants (SNs) of monocyte/parasite cocultures. Confocal fluorescence microscopy of non-permeabilized cells revealed that Leishmania-infected monocytes trans-presented IL-18 to NK cells. Native, but not heat-treated SNs of monocyte/Leishmania cocultures also induced CD69 on NK cells, indicating the involvement of a soluble heat-labile factor other than IL-18. A role for the NK cell-activating cytokines IL-1β, IL-2, IL-12, IL-15, IL-21, and IFN-α/β was excluded. The increase of CD69 was not paralleled by NK cell IFN-γ production or enhanced cytotoxicity. However, prior exposure of NK cells to Leishmania parasites synergistically increased their IFN-γ release in response to IL-12, which was dependent on endogenous IL-18. CD1c+ dendritic cells were identified as possible source of Leishmania-induced IL-12. Finally, we observed that direct contact between Leishmania and NK cells reduced the

  1. Monocyte-Derived Signals Activate Human Natural Killer Cells in Response to Leishmania Parasites

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    Helena Messlinger

    2018-01-01

    Full Text Available Activated natural killer (NK cells release interferon (IFN-γ, which is crucial for the control of intracellular pathogens such as Leishmania. In contrast to experimental murine leishmaniasis, the human NK cell response to Leishmania is still poorly characterized. Here, we investigated the interaction of human blood NK cells with promastigotes of different Leishmania species (Leishmania major, Leishmania mexicana, Leishmania infantum, and Leishmania donovani. When peripheral blood mononuclear cells or purified NK cells and monocytes (all derived from healthy blood donors from Germany without a history of leishmaniasis were exposed to promastigotes, NK cells showed increased surface expression of the activation marker CD69. The extent of this effect varied depending on the Leishmania species; differences between dermotropic and viscerotropic L. infantum strains were not observed. Upregulation of CD69 required direct contact between monocytes and Leishmania and was partly inhibitable by anti-interleukin (IL-18. Unexpectedly, IL-18 was undetectable in most of the supernatants (SNs of monocyte/parasite cocultures. Confocal fluorescence microscopy of non-permeabilized cells revealed that Leishmania-infected monocytes trans-presented IL-18 to NK cells. Native, but not heat-treated SNs of monocyte/Leishmania cocultures also induced CD69 on NK cells, indicating the involvement of a soluble heat-labile factor other than IL-18. A role for the NK cell-activating cytokines IL-1β, IL-2, IL-12, IL-15, IL-21, and IFN-α/β was excluded. The increase of CD69 was not paralleled by NK cell IFN-γ production or enhanced cytotoxicity. However, prior exposure of NK cells to Leishmania parasites synergistically increased their IFN-γ release in response to IL-12, which was dependent on endogenous IL-18. CD1c+ dendritic cells were identified as possible source of Leishmania-induced IL-12. Finally, we observed that direct contact between Leishmania and NK cells

  2. Imatinib and Nilotinib Off-Target Effects on Human NK Cells, Monocytes, and M2 Macrophages.

    Science.gov (United States)

    Bellora, Francesca; Dondero, Alessandra; Corrias, Maria Valeria; Casu, Beatrice; Regis, Stefano; Caliendo, Fabio; Moretta, Alessandro; Cazzola, Mario; Elena, Chiara; Vinti, Luciana; Locatelli, Franco; Bottino, Cristina; Castriconi, Roberta

    2017-08-15

    Tyrosine kinase inhibitors (TKIs) are used in the clinical management of hematological neoplasms. Moreover, in solid tumors such as stage 4 neuroblastomas (NB), imatinib showed benefits that might depend on both on-target and immunological off-target effects. We investigated the effects of imatinib and nilotinib on human NK cells, monocytes, and macrophages. High numbers of monocytes died upon exposure to TKI concentrations similar to those achieved in patients. Conversely, NK cells were highly resistant to the TKI cytotoxic effect, were properly activated by immunostimulatory cytokines, and degranulated in the presence of NB cells. In NB, neither drug reduced the expression of ligands for activating NK receptors or upregulated that of HLA class I, B7-H3, PD-L1, and PD-L2, molecules that might limit NK cell function. Interestingly, TKIs modulated the chemokine receptor repertoire of immune cells. Acting at the transcriptional level, they increased the surface expression of CXCR4, an effect observed also in NK cells and monocytes of patients receiving imatinib for chronic myeloid leukemia. Moreover, TKIs reduced the expression of CXCR3 (in NK cells) and CCR1 (in monocytes). Monocytes also decreased the expression of M-CSFR, and low numbers of cells underwent differentiation toward macrophages. M0 and M2 macrophages were highly resistant to TKIs and maintained their phenotypic and functional characteristics. Importantly, also in the presence of TKIs, the M2 immunosuppressive polarization was reverted by TLR engagement, and M1-oriented macrophages fully activated autologous NK cells. Our results contribute to better interpreting the off-target efficacy of TKIs in tumors and to envisaging strategies aimed at facilitating antitumor immune responses. Copyright © 2017 by The American Association of Immunologists, Inc.

  3. CD14{sup +} monocytes promote the immunosuppressive effect of human umbilical cord matrix stem cells

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    Wang, Ding, E-mail: qqhewd@gmail.com [The State Key Laboratory of Experimental Hematology, Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of Medical Sciences and Peking Union of Medical College, 288 Nanjing Road, Tianjin 300020 (China); TEDA Life and Technology Research Center, Institute of Hematology, Chinese Academy of Medical Sciences, TEDA, Tianjin (China); Chen, Ke, E-mail: chenke_59@hotmail.com [The State Key Laboratory of Experimental Hematology, Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of Medical Sciences and Peking Union of Medical College, 288 Nanjing Road, Tianjin 300020 (China); TEDA Life and Technology Research Center, Institute of Hematology, Chinese Academy of Medical Sciences, TEDA, Tianjin (China); Du, Wei Ting, E-mail: duwtpumc@yahoo.com.cn [The State Key Laboratory of Experimental Hematology, Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of Medical Sciences and Peking Union of Medical College, 288 Nanjing Road, Tianjin 300020 (China); Han, Zhi-Bo, E-mail: zhibohan@hotmail.com [The State Key Laboratory of Experimental Hematology, Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of Medical Sciences and Peking Union of Medical College, 288 Nanjing Road, Tianjin 300020 (China); TEDA Life and Technology Research Center, Institute of Hematology, Chinese Academy of Medical Sciences, TEDA, Tianjin (China); Ren, He, E-mail: knifesharp2000@hotmail.com [National Engineering Research Center of Cell Products, AmCellGene Co. Ltd, TEDA, Tianjin (China); Chi, Ying, E-mail: caizhuying@hotmail.com [The State Key Laboratory of Experimental Hematology, Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of Medical Sciences and Peking Union of Medical College, 288 Nanjing Road, Tianjin 300020 (China); TEDA Life and Technology Research Center, Institute of Hematology, Chinese Academy of Medical Sciences, TEDA, Tianjin (China); and others

    2010-09-10

    Here, the effect of CD14{sup +} monocytes on human umbilical cord matrix stem cell (hUC-MSC)-mediated immunosuppression was studied in vitro. hUC-MSCs exerted a potent inhibitory effect on the proliferation and interferon-{gamma} (IFN-{gamma}) secretion capacities of CD4{sup +} and CD8{sup +} T cells in response to anti-CD3/CD28 stimulation. Transwell co-culture system revealed that the suppressive effect was primarily mediated by soluble factors. Addition of prostaglandin synthesis inhibitors (indomethacin or NS-398) almost completely abrogated the immunosuppression activity of hUC-MSCs, identifying prostaglandin E{sub 2} (PGE{sub 2}) as an important soluble mediator. CD14{sup +} monocytes were found to be able to enhance significantly the immunosuppressive effect of hUC-MSCs in a dose-dependent fashion. Moreover, the inflammatory cytokine IL-1{beta}, either exogenously added or produced by CD14{sup +} monocytes in culture, could trigger expression of high levels of PGE{sub 2} by hUC-MSCs, whereas inclusion of the IL-1 receptor antagonist (IL-1RA) in the culture down-regulated not only PGE{sub 2} expression, but also reversed the promotional effect of CD14{sup +} monocytes and partially restored CD4{sup +} and CD8{sup +} T cell proliferation and IFN-{gamma} secretion. Our data demonstrate an important role of monocytes in the hUC-MSC-induced immunomodulation, which may have important implications in future efforts to explore the clinical potentials of hUC-MSCs.

  4. New Approach in Translational Medicine: Effects of Electrolyzed Reduced Water (ERW on NF-κB/iNOS Pathway in U937 Cell Line under Altered Redox State

    Directory of Open Access Journals (Sweden)

    Sara Franceschelli

    2016-09-01

    Full Text Available It is known that increased levels of reactive oxygen species (ROS and reactive nitrogen species (RNS can exert harmful effects, altering the cellular redox state. Electrolyzed Reduced Water (ERW produced near the cathode during water electrolysis exhibits high pH, high concentration of dissolved hydrogen and an extremely negative redox potential. Several findings indicate that ERW had the ability of a scavenger free radical, which results from hydrogen molecules with a high reducing ability and may participate in the redox regulation of cellular function. We investigated the effect of ERW on H2O2-induced U937 damage by evaluating the modulation of redox cellular state. Western blotting and spectrophotometrical analysis showed that ERW inhibited oxidative stress by restoring the antioxidant capacity of superoxide dismutase, catalase and glutathione peroxidase. Consequently, ERW restores the ability of the glutathione reductase to supply the cell of an important endogenous antioxidant, such as GSH, reversing the inhibitory effect of H2O2 on redox balance of U937 cells. Therefore, this means a reduction of cytotoxicity induced by peroxynitrite via a downregulation of the NF-κB/iNOS pathway and could be used as an antioxidant for preventive and therapeutic application. In conclusion, ERW can protect the cellular redox balance, reducing the risk of several diseases with altered cellular homeostasis such as inflammation.

  5. CD147 induces up-regulation of vascular endothelial growth factor in U937-derived foam cells through PI3K/AKT pathway.

    Science.gov (United States)

    Zong, JiaXin; Li, YunTian; Du, DaYong; Liu, Yang; Yin, YongJun

    2016-11-01

    Intraplaque angiogenesis has been recognized as an important risk factor for the rupture of advanced atherosclerotic plaques in recent years. CD147, also called Extracellular Matrix Metalloproteinase Inducer, has been found the ability to promote angiogenesis in many pathological conditions such as cancer diseases and rheumatoid arthritis via the up-regulation of vascular endothelial growth factor (VEGF), a critical mediator of angiogenesis. We investigated whether CD147 would also induce the up-regulation of VEGF in the foam cells formation process and explored the probable signaling pathway. The results showed the expression of CD147 and VEGF was significantly higher in U937-derived foam cells. After CD147 stealth siRNA transfection treatment, the production of VEGF was reduced depended on the inhibition efficiency of CD147 siRNAs.The special signaling pathway inhibitors LY294002, SP600125, SB203580 and U0126 were added to cultures respectively and the results showed LY294002 dose-dependently inhibited the expression of VEGF. The reduction of phospho-Akt was observed in both LY294002 and siRNA groups, suggested that the phosphatidylinositol 3-kinase/Akt pathway may be the probable signaling pathway underlying CD147 induced up-regulation of VEGF in U937-derived foam cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Synthesis of pro-inflammatory cytokines and adhesion molecules expression by the irradiated human monocyte/macrophage

    International Nuclear Information System (INIS)

    Pons, I.

    1997-09-01

    As lesions induced by ionizing radiations are essentially noticed in organs the functional and structural organisation of which depend on the highly proliferative stem cell pool, the author reports an in-vivo investigation of the effect of a gamma irradiation on the expression and secretion of pro-inflammatory cytokines par human monocytes/macrophages. In order to study the role of the cell environment in the radiation-induced inflammation, the author studied whether a co-stimulation of monocytes/macrophages by gamma irradiation, or the exposure of co-cultures of monocytes/macrophages and lymphocytes, could modulate the regulation of inflammatory cytokines. The author also studied the modulation of the expression of adhesion molecules mainly expressed by the monocyte/macrophage, and the membrane density of the CD14 receptor after irradiation of monocytes/macrophages during 24 hours, and of totally differentiated macrophages after seven days of culture

  7. Leishmania major surface protease Gp63 interferes with the function of human monocytes and neutrophils in vitro

    DEFF Research Database (Denmark)

    Sørensen, A L; Hey, A S; Kharazmi, A

    1994-01-01

    In the present study the effect of Leishmania major surface protease Gp63 on the chemotaxis and oxidative burst response of human peripheral blood monocytes and neutrophils was investigated. It was shown that prior incubation of cells with Gp63 inhibited chemotaxis of neutrophils but not monocytes...... towards the chemotactic peptide f-met-leu-phe. On the other hand, chemotaxis of both neutrophils and monocytes towards zymosan-activated serum containing C5a was inhibited by Gp63. Monocyte and neutrophil chemiluminescence response to opsonized zymosan was reduced by preincubation of the cells with Gp63...... in a concentration-dependent manner. Notably, monocytes were inhibited to a much greater degree than neutrophils by a given concentration of Gp63, and they were also inhibited at much lower concentrations of the protease. The inhibitory effect of Gp63 on chemotaxis and chemiluminescence was completely abolished...

  8. Human monocytes undergo functional re-programming during differentiation to dendritic cell mediated by human extravillous trophoblasts.

    Science.gov (United States)

    Zhao, Lei; Shao, Qianqian; Zhang, Yun; Zhang, Lin; He, Ying; Wang, Lijie; Kong, Beihua; Qu, Xun

    2016-02-09

    Maternal immune adaptation is required for a successful pregnancy to avoid rejection of the fetal-placental unit. Dendritic cells within the decidual microenvironment lock in a tolerogenic profile. However, how these tolerogenic DCs are induced and the underlying mechanisms are largely unknown. In this study, we show that human extravillous trophoblasts redirect the monocyte-to-DC transition and induce regulatory dendritic cells. DCs differentiated from blood monocytes in the presence of human extravillous trophoblast cell line HTR-8/SVneo displayed a DC-SIGN(+)CD14(+)CD1a(-) phenotype, similar with decidual DCs. HTR8-conditioned DCs were unable to develop a fully mature phenotype in response to LPS, and altered the cytokine secretory profile significantly. Functionally, conditioned DCs poorly induced the proliferation and activation of allogeneic T cells, whereas promoted CD4(+)CD25(+)Foxp3(+) Treg cells generation. Furthermore, the supernatant from DC and HTR-8/SVneo coculture system contained significant high amount of M-CSF and MCP-1. Using neutralizing antibodies, we discussed the role of M-CSF and MCP-1 during monocyte-to-DCs differentiation mediated by extravillous trophoblasts. Our data indicate that human extravillous trophoblasts play an important role in modulating the monocyte-to-DC differentiation through M-CSF and MCP-1, which facilitate the establishment of a tolerogenic microenvironment at the maternal-fetal interface.

  9. The CD157-integrin partnership controls transendothelial migration and adhesion of human monocytes.

    Science.gov (United States)

    Lo Buono, Nicola; Parrotta, Rossella; Morone, Simona; Bovino, Paola; Nacci, Giulia; Ortolan, Erika; Horenstein, Alberto L; Inzhutova, Alona; Ferrero, Enza; Funaro, Ada

    2011-05-27

    CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of β(1) and β(2) integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes.

  10. The CD157-Integrin Partnership Controls Transendothelial Migration and Adhesion of Human Monocytes*

    Science.gov (United States)

    Lo Buono, Nicola; Parrotta, Rossella; Morone, Simona; Bovino, Paola; Nacci, Giulia; Ortolan, Erika; Horenstein, Alberto L.; Inzhutova, Alona; Ferrero, Enza; Funaro, Ada

    2011-01-01

    CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of β1 and β2 integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes. PMID:21478153

  11. Deciphering the transcriptional circuitry of microRNA genes expressed during human monocytic differentiation

    KAUST Repository

    Schmeier, Sebastian; MacPherson, Cameron R; Essack, Magbubah; Kaur, Mandeep; Schaefer, Ulf; Suzuki, Harukazu; Hayashizaki, Yoshihide; Bajic, Vladimir B.

    2009-01-01

    Background: Macrophages are immune cells involved in various biological processes including host defence, homeostasis, differentiation, and organogenesis. Disruption of macrophage biology has been linked to increased pathogen infection, inflammation and malignant diseases. Differential gene expression observed in monocytic differentiation is primarily regulated by interacting transcription factors (TFs). Current research suggests that microRNAs (miRNAs) degrade and repress translation of mRNA, but also may target genes involved in differentiation. We focus on getting insights into the transcriptional circuitry regulating miRNA genes expressed during monocytic differentiation. Results: We computationally analysed the transcriptional circuitry of miRNA genes during monocytic differentiation using in vitro time-course expression data for TFs and miRNAs. A set of TF?miRNA associations was derived from predicted TF binding sites in promoter regions of miRNA genes. Time-lagged expression correlation analysis was utilised to evaluate the TF?miRNA associations. Our analysis identified 12 TFs that potentially play a central role in regulating miRNAs throughout the differentiation process. Six of these 12 TFs (ATF2, E2F3, HOXA4, NFE2L1, SP3, and YY1) have not previously been described to be important for monocytic differentiation. The remaining six TFs are CEBPB, CREB1, ELK1, NFE2L2, RUNX1, and USF2. For several miRNAs (miR-21, miR-155, miR-424, and miR-17-92), we show how their inferred transcriptional regulation impacts monocytic differentiation. Conclusions: The study demonstrates that miRNAs and their transcriptional regulatory control are integral molecular mechanisms during differentiation. Furthermore, it is the first study to decipher on a large-scale, how miRNAs are controlled by TFs during human monocytic differentiation. Subsequently, we have identified 12 candidate key controllers of miRNAs during this differentiation process. 2009 Schmeier et al; licensee Bio

  12. Deciphering the transcriptional circuitry of microRNA genes expressed during human monocytic differentiation

    KAUST Repository

    Schmeier, Sebastian

    2009-12-10

    Background: Macrophages are immune cells involved in various biological processes including host defence, homeostasis, differentiation, and organogenesis. Disruption of macrophage biology has been linked to increased pathogen infection, inflammation and malignant diseases. Differential gene expression observed in monocytic differentiation is primarily regulated by interacting transcription factors (TFs). Current research suggests that microRNAs (miRNAs) degrade and repress translation of mRNA, but also may target genes involved in differentiation. We focus on getting insights into the transcriptional circuitry regulating miRNA genes expressed during monocytic differentiation. Results: We computationally analysed the transcriptional circuitry of miRNA genes during monocytic differentiation using in vitro time-course expression data for TFs and miRNAs. A set of TF?miRNA associations was derived from predicted TF binding sites in promoter regions of miRNA genes. Time-lagged expression correlation analysis was utilised to evaluate the TF?miRNA associations. Our analysis identified 12 TFs that potentially play a central role in regulating miRNAs throughout the differentiation process. Six of these 12 TFs (ATF2, E2F3, HOXA4, NFE2L1, SP3, and YY1) have not previously been described to be important for monocytic differentiation. The remaining six TFs are CEBPB, CREB1, ELK1, NFE2L2, RUNX1, and USF2. For several miRNAs (miR-21, miR-155, miR-424, and miR-17-92), we show how their inferred transcriptional regulation impacts monocytic differentiation. Conclusions: The study demonstrates that miRNAs and their transcriptional regulatory control are integral molecular mechanisms during differentiation. Furthermore, it is the first study to decipher on a large-scale, how miRNAs are controlled by TFs during human monocytic differentiation. Subsequently, we have identified 12 candidate key controllers of miRNAs during this differentiation process. 2009 Schmeier et al; licensee Bio

  13. Effects of transforming growth factor-beta on long-term human cord blood monocyte cultures

    International Nuclear Information System (INIS)

    Orcel, P.; Bielakoff, J.; De Vernejoul, M.C.

    1990-01-01

    Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes

  14. Extracellular lipase of Pseudomonas aeruginosa: biochemical characterization and effect on human neutrophil and monocyte function in vitro

    DEFF Research Database (Denmark)

    Jaeger, K E; Kharazmi, A; Høiby, N

    1991-01-01

    concentrations of this lipase preparation were preincubated with human peripheral blood neutrophils and monocytes. The chemotaxis and chemiluminescence of these cells were then determined. It was shown that lipase inhibited the monocyte chemotaxis and chemiluminescence, whereas it had no or very little effect...... on neutrophils. The inhibitory effect was concentration dependent and was abolished by heat treatment of the enzyme at 100 degrees C. Since monocytes are one of the important cells of the host defence system the inhibition of the function of these cells may contribute to the pathogenesis of infections caused...

  15. cGAS Senses Human Cytomegalovirus and Induces Type I Interferon Responses in Human Monocyte-Derived Cells.

    Directory of Open Access Journals (Sweden)

    Jennifer Paijo

    2016-04-01

    Full Text Available Human cytomegalovirus (HCMV infections of healthy individuals are mostly unnoticed and result in viral latency. However, HCMV can also cause devastating disease, e.g., upon reactivation in immunocompromised patients. Yet, little is known about human immune cell sensing of DNA-encoded HCMV. Recent studies indicated that during viral infection the cyclic GMP/AMP synthase (cGAS senses cytosolic DNA and catalyzes formation of the cyclic di-nucleotide cGAMP, which triggers stimulator of interferon genes (STING and thus induces antiviral type I interferon (IFN-I responses. We found that plasmacytoid dendritic cells (pDC as well as monocyte-derived DC and macrophages constitutively expressed cGAS and STING. HCMV infection further induced cGAS, whereas STING expression was only moderately affected. Although pDC expressed particularly high levels of cGAS, and the cGAS/STING axis was functional down-stream of STING, as indicated by IFN-I induction upon synthetic cGAMP treatment, pDC were not susceptible to HCMV infection and mounted IFN-I responses in a TLR9-dependent manner. Conversely, HCMV infected monocyte-derived cells synthesized abundant cGAMP levels that preceded IFN-I production and that correlated with the extent of infection. CRISPR/Cas9- or siRNA-mediated cGAS ablation in monocytic THP-1 cells and primary monocyte-derived cells, respectively, impeded induction of IFN-I responses following HCMV infection. Thus, cGAS is a key sensor of HCMV for IFN-I induction in primary human monocyte-derived DC and macrophages.

  16. Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kleinhenz, M.E.; Ellner, J.J.

    1985-07-01

    In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the (/sup 3/H)thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced (/sup 3/H)thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% (SD)) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition.

  17. Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

    International Nuclear Information System (INIS)

    Kleinhenz, M.E.; Ellner, J.J.

    1985-01-01

    In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the [ 3 H]thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced [ 3 H]thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% [SD]) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition

  18. Tailored HIV-1 vectors for genetic modification of primary human dendritic cells and monocytes.

    Science.gov (United States)

    Durand, Stéphanie; Nguyen, Xuan-Nhi; Turpin, Jocelyn; Cordeil, Stephanie; Nazaret, Nicolas; Croze, Séverine; Mahieux, Renaud; Lachuer, Joël; Legras-Lachuer, Catherine; Cimarelli, Andrea

    2013-01-01

    Monocyte-derived dendritic cells (MDDCs) play a key role in the regulation of the immune system and are the target of numerous gene therapy applications. The genetic modification of MDDCs is possible with human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LVs) but requires high viral doses to bypass their natural resistance to viral infection, and this in turn affects their physiological properties. To date, a single viral protein is able to counter this restrictive phenotype, Vpx, a protein derived from members of the HIV-2/simian immunodeficiency virus SM lineage that counters at least two restriction factors present in myeloid cells. By tagging Vpx with a short heterologous membrane-targeting domain, we have obtained HIV-1 LVs incorporating high levels of this protein (HIV-1-Src-Vpx). These vectors efficiently transduce differentiated MDDCs and monocytes either as previously purified populations or as populations within unsorted peripheral blood mononuclear cells (PBMCs). In addition, these vectors can be efficiently pseudotyped with receptor-specific envelopes, further restricting their cellular tropism almost uniquely to MDDCs. Compared to conventional HIV-1 LVs, these novel vectors allow for an efficient genetic modification of MDDCs and, more importantly, do not cause their maturation or affect their survival, which are unwanted side effects of the transduction process. This study describes HIV-1-Src-Vpx LVs as a novel potent tool for the genetic modification of differentiated MDDCs and of circulating monocyte precursors with strong potential for a wide range of gene therapy applications.

  19. Binding of α2-macroglobulin-thrombin complexes and methylamine-treated α2-macroglobulin to human blood monocytes

    International Nuclear Information System (INIS)

    Straight, D.L.; Jakoi, L.; McKee, P.A.; Snyderman, R.

    1988-01-01

    The binding of α 2 -macroglobulin (α 2 M) to human peripheral blood monocytes was investigated. Monocytes, the precursors of tissue macrophages, were isolated from fresh blood by centrifugal elutriation or density gradient centrifugation. Binding studies were performed using 125 I-labeled α 2 M. Cells and bound ligand were separated from free ligand by rapid vacuum filtration. Nonlinear least-squares analysis of data obtained in direct binding studies at 0 0 C showed that monocytes bound the α 2 M-thrombin complex with a K/sub d/ 3.0 +- .09 nM and the monocyte had 1545 +- 153 sitescell. Thrombin alone did not compete for the site. Binding was divalent cation dependent. Direct binding studies also demonstrated that monocytes bound methylamine-treated α 2 M in a manner similar to α 2 M-thrombin. Competitive binding studies showed that α 2 M-thrombin and methylamine-treated α 2 M bound to the same sites on the monocyte. In contrast, native α 2 M did not compete with α 2 M-thrombin for the site. Studies done at 37 0 C suggested that after binding, the monocyte internalized and degraded α 2 M-thrombin and excreted the degradation products. Receptor turnover and degradation of α 2 M-thrombin complexes were blocked in monocytes treated with chloroquine, an inhibitor of lysosomal function. The results indicate that human monocytes have a divalent cation dependent, high-affinity binding site for α 2 M-thrombin and methylamine-treated α 2 M which may function to clear α 2 M-proteinase complexes from the circulation

  20. Prostaglandin E2 and thromboxane B2 release from human monocytes treated with bacterial lipopolysaccharide

    International Nuclear Information System (INIS)

    Nichols, F.C.; Garrison, S.W.; Davis, H.W.

    1988-01-01

    We investigated the capacity of counterflow-isolated human monocytes to independently synthesize thromboxane B2 (TxB2) and prostaglandin E2 (PGE2) when stimulated with bacterial lipopolysaccharide (LPS). Independent metabolism was confirmed by establishing different specific activities (dpm/ng) of TxB2 and PGE2 released from LPS-treated cells. For metabolites released during the initial 2-hr treatment period, the specific activity of PGE2 was approximately threefold higher than that of TxB2 regardless of labeling with [3H]arachidonic acid (AA) or [14C]AA. Cells that were pulse-labeled for 2 hr with [3H]AA demonstrated a decreasing PGE2 specific activity over 24 hr, whereas the TxB2 specific activity remained unchanged. In contrast, cells continuously exposed to [14C]AA demonstrated an increasing TxB2 specific activity that approached the level of PGE2 by 24 hr. These results suggest the presence of at least 2 cyclooxygenase metabolic compartments in counterflow-isolated monocytes. Although freshly isolated monocytes have been reported to contain variable numbers of adherent platelets, additional experiments demonstrated that counterflow-isolated platelets are not capable of releasing elevated levels of TxB2 or PGE2 when treated with LPS. It is proposed from these findings that at least two subsets of monocytes exist in peripheral blood that can be distinguished on the basis of independent conversion of AA to TxB2 and PGE2

  1. Cerium dioxide nanoparticles do not modulate the lipopolysaccharide-induced inflammatory response in human monocytes

    Directory of Open Access Journals (Sweden)

    Hussain S

    2012-03-01

    Full Text Available Salik Hussain1,*, Faris Al-Nsour1,*, Annette B Rice1, Jamie Marshburn1, Zhaoxia Ji2, Jeffery I Zink2, Brenda Yingling1, Nigel J Walker3, Stavros Garantziotis11Clinical Research Unit, National Institute of Environmental Health Sciences/National Institute of Health, Research Triangle Park, NC, 2UC Center for Environmental Implications of Nanotechnology University of California, Los Angeles, CA, 3Division of National Toxicology Program, National Institute of Environmental Health Sciences/National Institute of Health, Research Triangle Park, NC, USA*Both are principal authorsBackground: Cerium dioxide (CeO2 nanoparticles have potential therapeutic applications and are widely used for industrial purposes. However, the effects of these nanoparticles on primary human cells are largely unknown. The ability of nanoparticles to exacerbate pre-existing inflammatory disorders is not well documented for engineered nanoparticles, and is certainly lacking for CeO2 nanoparticles. We investigated the inflammation-modulating effects of CeO2 nanoparticles at noncytotoxic concentrations in human peripheral blood monocytes.Methods: CD14+ cells were isolated from peripheral blood samples of human volunteers. Cells were exposed to either 0.5 or 1 µg/mL of CeO2 nanoparticles over a period of 24 or 48 hours with or without lipopolysaccharide (10 ng/mL prestimulation. Modulation of the inflammatory response was studied by measuring secreted tumor necrosis factor-alpha, interleukin-1beta, macrophage chemotactic protein-1, interferon-gamma, and interferon gamma-induced protein 10.Results: CeO2 nanoparticle suspensions were thoroughly characterized using dynamic light scattering analysis (194 nm hydrodynamic diameter, zeta potential analysis (-14 mV, and transmission electron microscopy (irregular-shaped particles. Transmission electron microscopy of CD14+ cells exposed to CeO2 nanoparticles revealed that these nanoparticles were efficiently internalized by monocytes and

  2. Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

    Directory of Open Access Journals (Sweden)

    N.E. Gomes

    2010-09-01

    Full Text Available Lipopolysaccharide (LPS activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R and smooth (S forms signal through Toll-like receptor 4 (TLR4, but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS and nitric oxide (NO generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

  3. TREM2 expression in the human brain: a marker of monocyte recruitment?

    Science.gov (United States)

    Fahrenhold, Marie; Rakic, Sonja; Classey, John; Brayne, Carol; Ince, Paul G; Nicoll, James A R; Boche, Delphine

    2017-10-07

    Mutation in the triggering receptor expressed on myeloid cells (TREM) 2 gene has been identified as a risk factor for several neurodegenerative diseases including Alzheimer's disease (AD). Experimental studies using animal models of AD have highlighted a number of functions associated with TREM2 and its expression by microglial cells. It has therefore been assumed that this is also the case in humans. However, there is very limited information concerning the cellular expression of TREM2 in the human brain. As part of investigations of microglia using post-mortem resources provided by the Medical Research Council Cognitive Function and Ageing Studies (MRC-CFAS), we immunostained the cerebral cortex of 299 participants for TREM2 using the Sigma antibody HPA010917 and compared with the macrophage/microglial markers Iba1 and CD68. As expected, Iba1 and CD68 labeled microglia and perivascular macrophages. However, in most cases (284/299), the TREM2 antibody labelled monocytes within vascular lumens, but not microglia or perivascular macrophages. In contrast, in 5 out of 6 cases with acute infarcts, TREM2 immunoreaction identified cells within the brain parenchyma interpreted as recruited monocytes. Six cases with old infarcts contained phagocytic foamy macrophages which were CD68-positive but TREM2 negative. Our observations, using the HPA010917 anti-TREM2 antibody, suggest that TREM2 is not expressed by microglia but instead seems to be a marker of recruited monocytes in the human brain. This finding has implications with regards to the role of TREM2 as a risk factor, emphasizing the importance of systemic immune responses in the development and progression of Alzheimer's disease. © 2017 International Society of Neuropathology.

  4. Fibroblast growth factor 23 inhibits extrarenal synthesis of 1,25-dihydroxyvitamin D in human monocytes.

    Science.gov (United States)

    Bacchetta, Justine; Sea, Jessica L; Chun, Rene F; Lisse, Thomas S; Wesseling-Perry, Katherine; Gales, Barbara; Adams, John S; Salusky, Isidro B; Hewison, Martin

    2013-01-01

    Vitamin D is a potent stimulator of monocyte innate immunity, and this effect is mediated via intracrine conversion of 25-hydroxyvitamin D (25OHD) to 1,25-dihydroxyvitamin D (1,25(OH)(2) D). In the kidney, synthesis of 1,25(OH)(2) D is suppressed by fibroblast growth factor 23 (FGF23), via transcriptional suppression of the vitamin D-activating enzyme 1α-hydroxylase (CYP27B1). We hypothesized that FGF23 also suppresses CYP27B1 in monocytes, with concomitant effects on intracrine responses to 1,25(OH)(2) D. Healthy donor peripheral blood mononuclear cell monocytes (PBMCm) and peritoneal dialysate monocyte (PDm) effluent from kidney disease patients were assessed at baseline to confirm the presence of mRNA for FGF23 receptors (FGFRs), with Klotho and FGFR1 being more strongly expressed than FGFR2/3/4 in both cell types. Immunohistochemistry showed coexpression of Klotho and FGFR1 in PBMCm and PDm, with this effect being enhanced following treatment with FGF23 in PBMCm but not PDm. Treatment with FGF23 activated mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) pathways in PBMCm, demonstrating functional FGFR signaling in these cells. FGF23 treatment of PBMCm and PDm decreased expression of mRNA for CYP27B1. In PBMCm this was associated with downregulation of 25OHD to 1,25(OH)(2) D metabolism, and concomitant suppression of intracrine induced 24-hydroxylase (CYP24A1) and antibacterial cathelicidin (LL37). FGF23 suppression of CYP27B1 was particularly pronounced in PBMCm treated with interleukin-15 to stimulate synthesis of 1,25(OH)(2) D. These data indicate that FGF23 can inhibit extra-renal expression of CYP27B1 and subsequent intracrine responses to 1,25(OH)(2) D in two different human monocyte models. Elevated expression of FGF23 may therefore play a crucial role in defining immune responses to vitamin D and this, in turn, may be a key determinant of infection in patients with chronic kidney disease (CKD). Copyright © 2013 American Society for

  5. An antigen shared by human granulocytes, monocytes, marrow granulocyte precursors and leukemic blasts.

    Science.gov (United States)

    Shumak, K H; Rachkewich, R A

    1983-01-01

    An antibody to human granulocytes was raised in rabbits by immunization with granulocytes pretreated with rabbit antibody to contaminating antigens. The antibody reacted not only with granulocytes but also with monocytes and bone marrow granulocyte precursors including colony-forming units in culture (CFU-C). In tests with leukemic cells, the antibody reacted with blasts from most (8 of 9) patients with acute myelomonoblastic leukemia and from some patients with acute myeloblastic leukemia, morphologically undifferentiated acute leukemia and chronic myelogenous leukemia in blast crisis. The antibody did not react with blasts from patients with acute lymphoblastic leukemia nor with leukemic cells from patients with chronic lymphocytic leukemia.

  6. Transcriptional profiling of human monocytes identifies the inhibitory receptor CD300a as regulator of transendothelial migration.

    Directory of Open Access Journals (Sweden)

    Sharang Ghavampour

    Full Text Available Local inflammatory responses are characterized by the recruitment of circulating leukocytes from the blood to sites of inflammation, a process requiring the directed migration of leukocytes across the vessel wall and hence a penetration of the endothelial lining. To identify underlying signalling events and novel factors involved in these processes we screened for genes differentially expressed in human monocytes following their adhesion to and passage through an endothelial monolayer. Functional annotation clustering of the genes identified revealed an overrepresentation of those associated with inflammation/immune response, in particular early monocyte to macrophage differentiation. Among the gene products so far not implicated in monocyte transendothelial migration was the inhibitory immune receptor CD300a. CD300a mRNA and protein levels were upregulated following transmigration and engagement of the receptor by anti-CD300a antibodies markedly reduced monocyte transendothelial migration. In contrast, siRNA mediated downregulation of CD300a in human monocytes increased their rate of migration. CD300a colocalized and cosedimented with actin filaments and, when activated, caused F-actin cytoskeleton alterations. Thus, monocyte transendothelial migration is accompanied by an elevation of CD300a which serves an inhibitory function possibly required for termination of the actual transmigration.

  7. Integrin αMβ2 is differently expressed by subsets of human osteoclast precursors and mediates adhesion of classical monocytes to bone

    Energy Technology Data Exchange (ETDEWEB)

    Sprangers, Sara, E-mail: s.l.sprangers@acta.nl [Department of Oral Cell Biology and Functional Anatomy, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam The Netherlands (Netherlands); Schoenmaker, Ton, E-mail: t.schoenmaker@acta.nl [Department of Oral Cell Biology and Functional Anatomy, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam The Netherlands (Netherlands); Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam The Netherlands (Netherlands); Cao, Yixuan, E-mail: y.cao@acta.nl [Department of Oral Cell Biology and Functional Anatomy, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam The Netherlands (Netherlands); Everts, Vincent, E-mail: v.everts@acta.nl [Department of Oral Cell Biology and Functional Anatomy, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam The Netherlands (Netherlands); Vries, Teun J. de, E-mail: teun.devries@acta.nl [Department of Oral Cell Biology and Functional Anatomy, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam The Netherlands (Netherlands); Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam The Netherlands (Netherlands)

    2017-01-01

    Bone-degrading osteoclasts are formed through fusion of their monocytic precursors. In the population of human peripheral blood monocytes, three distinct subsets have been identified: classical, intermediate and non-classical monocytes. We have previously shown that when the monocyte subsets are cultured on bone, significantly more osteoclasts are formed from classical monocytes than from intermediate or non-classical monocytes. Considering that this difference does not exist when monocyte subsets are cultured on plastic, we hypothesized that classical monocytes adhere better to the bone surface compared to intermediate and non-classical monocytes. To investigate this, the different monocyte subsets were isolated from human peripheral blood and cultured on slices of human bone in the presence of the cytokine M-CSF. We found that classical monocytes adhere better to bone due to a higher expression of the integrin αMβ2 and that their ability to attach to bone is significantly decreased when the integrin is blocked. This suggests that integrin αMβ2 mediates attachment of osteoclast precursors to bone and thereby enables the formation of osteoclasts.

  8. p38 MAPK protects human monocytes from postprandial triglyceride-rich lipoprotein-induced toxicity.

    Science.gov (United States)

    Lopez, Sergio; Jaramillo, Sara; Varela, Lourdes M; Ortega, Almudena; Bermudez, Beatriz; Abia, Rocio; Muriana, Francisco J G

    2013-05-01

    Postprandial triglyceride (TG)-rich lipoproteins (TRLs) transport dietary fatty acids through the circulatory system to satisfy the energy and structural needs of the tissues. However, fatty acids are also able to modulate gene expression and/or induce cell death. We investigated the underlying mechanism by which postprandial TRLs of different fatty acid compositions can induce cell death in human monocytes. Three types of dietary fat [refined olive oil (ROO), high-palmitic sunflower oil (HPSO), and butter] with progressively increasing SFA:MUFA ratios (0.18, 0.41, and 2.08, respectively) were used as a source of postprandial TRLs (TRL-ROO, TRL-HPSO, and TRL-BUTTER) from healthy men. The monocytic cell line THP-1 was used as a model for this study. We demonstrated that postprandial TRLs increased intracellular lipid accumulation (31-106%), reactive oxygen species production (268-349%), DNA damage (133-1467%), poly(ADP-ribose) polymerase 1 (800-1710%) and caspase-3 (696-1244%) activities, and phosphorylation of c-Jun NH2-terminal kinase (JNK) (54 kDa, 141-288%) and p38 (24-92%). These effects were significantly greater with TRL-BUTTER, and TRL-ROO did not induce DNA damage, DNA fragmentation, or p38 phosphorylation. In addition, blockade of p38, but not of JNK, significantly decreased intracellular lipid accumulation and increased cell death in postprandial TRL-treated cells. These results suggest that in human monocytes, p38 is involved in survival signaling pathways that protect against the lipid-mediated cytotoxicity induced by postprandial TRLs that are abundant in saturated fatty acids.

  9. A human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes.

    Science.gov (United States)

    Mesel-Lemoine, Mariana; Millet, Jean; Vidalain, Pierre-Olivier; Law, Helen; Vabret, Astrid; Lorin, Valérie; Escriou, Nicolas; Albert, Matthew L; Nal, Béatrice; Tangy, Frédéric

    2012-07-01

    Human coronaviruses are associated with upper respiratory tract infections that occasionally spread to the lungs and other organs. Although airway epithelial cells represent an important target for infection, the respiratory epithelium is also composed of an elaborate network of dendritic cells (DCs) that are essential sentinels of the immune system, sensing pathogens and presenting foreign antigens to T lymphocytes. In this report, we show that in vitro infection by human coronavirus 229E (HCoV-229E) induces massive cytopathic effects in DCs, including the formation of large syncytia and cell death within only few hours. In contrast, monocytes are much more resistant to infection and cytopathic effects despite similar expression levels of CD13, the membrane receptor for HCoV-229E. While the differentiation of monocytes into DCs in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 requires 5 days, only 24 h are sufficient for these cytokines to sensitize monocytes to cell death and cytopathic effects when infected by HCoV-229E. Cell death induced by HCoV-229E is independent of TRAIL, FasL, tumor necrosis factor alpha, and caspase activity, indicating that viral replication is directly responsible for the observed cytopathic effects. The consequence of DC death at the early stage of HCoV-229E infection may have an impact on the early control of viral dissemination and on the establishment of long-lasting immune memory, since people can be reinfected multiple times by HCoV-229E.

  10. Properties of human blood monocytes. I. CD91 expression and log orthogonal light scatter provide a robust method to identify monocytes that is more accurate than CD14 expression.

    Science.gov (United States)

    Hudig, Dorothy; Hunter, Kenneth W; Diamond, W John; Redelman, Doug

    2014-03-01

    This study was designed to improve identification of human blood monocytes by using antibodies to molecules that occur consistently on all stages of monocyte development and differentiation. We examined blood samples from 200 healthy adults without clinically diagnosed immunological abnormalities by flow cytometry (FCM) with multiple combinations of antibodies and with a hematology analyzer (Beckman LH750). CD91 (α2 -macroglobulin receptor) was expressed only by monocytes and to a consistent level among subjects [mean median fluorescence intensity (MFI) = 16.2 ± 3.2]. Notably, only 85.7 ± 5.82% of the CD91(+) monocytes expressed high levels of the classical monocyte marker CD14, with some CD91(+) CD16(+) cells having negligible CD14, indicating that substantial FCM under-counts will occur when monocytes are identified by high CD14. CD33 (receptor for sialyl conjugates) was co-expressed with CD91 on monocytes but CD33 expression varied by nearly ten-fold among subjects (mean MFI = 17.4 ± 7.7). In comparison to FCM analyses, the hematology analyzer systematically over-counted monocytes and eosinophils while lymphocyte and neutrophil differential values generally agreed with FCM methods. CD91 is a better marker to identify monocytes than CD14 or CD33. Furthermore, FCM (with anti-CD91) identifies monocytes better than a currently used clinical CBC instrument. Use of anti-CD91 together with anti-CD14 and anti-CD16 supports the identification of the diagnostically significant monocyte populations with variable expression of CD14 and CD16. Copyright © 2013 Clinical Cytometry Society.

  11. Age-Related Gene Expression Differences in Monocytes from Human Neonates, Young Adults, and Older Adults.

    Science.gov (United States)

    Lissner, Michelle M; Thomas, Brandon J; Wee, Kathleen; Tong, Ann-Jay; Kollmann, Tobias R; Smale, Stephen T

    2015-01-01

    A variety of age-related differences in the innate and adaptive immune systems have been proposed to contribute to the increased susceptibility to infection of human neonates and older adults. The emergence of RNA sequencing (RNA-seq) provides an opportunity to obtain an unbiased, comprehensive, and quantitative view of gene expression differences in defined cell types from different age groups. An examination of ex vivo human monocyte responses to lipopolysaccharide stimulation or Listeria monocytogenes infection by RNA-seq revealed extensive similarities between neonates, young adults, and older adults, with an unexpectedly small number of genes exhibiting statistically significant age-dependent differences. By examining the differentially induced genes in the context of transcription factor binding motifs and RNA-seq data sets from mutant mouse strains, a previously described deficiency in interferon response factor-3 activity could be implicated in most of the differences between newborns and young adults. Contrary to these observations, older adults exhibited elevated expression of inflammatory genes at baseline, yet the responses following stimulation correlated more closely with those observed in younger adults. Notably, major differences in the expression of constitutively expressed genes were not observed, suggesting that the age-related differences are driven by environmental influences rather than cell-autonomous differences in monocyte development.

  12. Human glioblastoma-associated microglia/monocytes express a distinct RNA profile compared to human control and murine samples.

    Science.gov (United States)

    Szulzewsky, Frank; Arora, Sonali; de Witte, Lot; Ulas, Thomas; Markovic, Darko; Schultze, Joachim L; Holland, Eric C; Synowitz, Michael; Wolf, Susanne A; Kettenmann, Helmut

    2016-08-01

    Glioblastoma (GBM) is the most aggressive brain tumor in adults. It is strongly infiltrated by microglia and peripheral monocytes that support tumor growth. In the present study we used RNA sequencing to compare the expression profile of CD11b(+) human glioblastoma-associated microglia/monocytes (hGAMs) to CD11b(+) microglia isolated from non-tumor samples. Hierarchical clustering and principal component analysis showed a clear separation of the two sample groups and we identified 334 significantly regulated genes in hGAMs. In comparison to human control microglia hGAMs upregulated genes associated with mitotic cell cycle, cell migration, cell adhesion, and extracellular matrix organization. We validated the expression of several genes associated with extracellular matrix organization in samples of human control microglia, hGAMs, and the hGAMs-depleted fraction via qPCR. The comparison to murine GAMs (mGAMs) showed that both cell populations share a significant fraction of upregulated transcripts compared with their respective controls. These genes were mostly related to mitotic cell cycle. However, in contrast to murine cells, human GAMs did not upregulate genes associated to immune activation. Comparison of human and murine GAMs expression data to several data sets of in vitro-activated human macrophages and murine microglia showed that, in contrast to mGAMs, hGAMs share a smaller overlap to these data sets in general and in particular to cells activated by proinflammatory stimulation with LPS + INFγ or TNFα. Our findings provide new insights into the biology of human glioblastoma-associated microglia/monocytes and give detailed information about the validity of murine experimental models. GLIA 2016 GLIA 2016;64:1416-1436. © 2016 Wiley Periodicals, Inc.

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

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    Allison Groseth

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

  14. [Peptide fragments of chemokine domain of fractalkine: effect on human monocyte migration].

    Science.gov (United States)

    Kukhtina, N B; Aref'eva, T I; Ruleva, N Iu; Sidorova, M V; Az'muko, A A; Bespalova, Zh D; Krasnikova, T L

    2012-01-01

    Leukocyte chemotaxis to the area of tissue damage is mediated by chemokines. According to the primary structure, chemokines are divided into four families, fractalkine (CX3CL1) is the only one member of CX3C family and the only membrane-bound chemokine. Fractalkine molecule includes the extracellular N-terminal chemokine domain, mucin-like rod, the transmembrane and the intracellular domains. In membrane-bound state fractalkine has the properties of an adhesion molecule. Chemokine domain of fractalkine (CDF) is released from cell membrane by proteolysis, and this soluble form acts as a chemoattractant for leukocytes expressing fractalkine receptor CX3CR1. Fractalkine is involved in development of a number of pathological processes caused by inflammation, and therefore a search for fractalkine inhibitors is very important. For this purpose we identified several antigenic determinants--the fragments of CDF, and the following peptides were synthesized--P41-52 H-Leu-Glu-Thr-Arg-Gln-His-Arg-Leu-Phe-Cys-Ala-Asp-NH2, P53-60 H-Pro-Lys-Glu-Gln-Trp-Val-Lys-Asp-NH2 and P60-71 H-Asp-Ala-Met-Gln-His-Leu-Asp-Arg-Gln-Ala-Ala-Ala-NH2. The peptide effects on adhesion and migration of human peripheral blood monocytes expressing fractalkine receptors were investigated. In the presence of CDF and P41-52 we observed the increased adhesion and migration of monocytes compared with spontaneous values. Peptides P53-60 and P60-71 significantly inhibited monocyte adhesion and migration stimulated by CDF. Since the chemotactic activity of chemokines was shown to be dependent on their binding to glycosaminoglycans of the cell surface and extracellular matrix, the effect ofpeptides on the interaction of CDF with heparin was analyzed by ELISA. Peptide P41-52 competed with CDF for heparin binding, while peptides P53-60 and P60-71 had no significant activity.

  15. Monoclonal antibody to a subset of human monocytes found only in the peripheral blood and inflammatory tissues

    Energy Technology Data Exchange (ETDEWEB)

    Zwadlo, G.; Schlegel, R.; Sorg, C.

    1986-07-15

    A monoclonal antibody is described that was generated by immunizing mice with cultured human blood monocytes. The antibody (27E10) belongs to the IgG1 subclass and detects a surface antigen at M/sub r/ 17,000 that is found on 20% of peripheral blood monocytes. The antigen is increasingly expressed upon culture of monocytes, reaching a maximum between days 2 and 3. Stimulation of monocytes with interferon-..gamma.. (IFN-..gamma..), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and lipopolysaccharide (LPS) Ylalanine (fMLP) increased the 27E10 antigen density. The amount of 27E10-positive cells is not or is only weakly affected. The antigen is absent from platelets, lymphotyces, and all tested human cell lines, yet it cross-reacts with 15% of freshly isolated granulocytes. By using the indirect immunoperoxidase technique, the antibody is found to be negative on cryostat sections of normal human tissue (skin, lung, and colon) and positive on only a few monocyte-like cells in liver and on part of the cells of the splenic red pulp. In inflammatory tissue, however, the antibody is positive on monocytes/macrophages and sometimes on endothelial cells and epidermal cells, depending on the stage and type of inflammation, e.g., BCG ranulomas are negative, whereas psoriasis vulgaris, atopic dermatitis, erythrodermia, pressure urticaria, and periodontitis contain positively staining cells. In contact eczemas at different times after elicitation (6 hr, 24 hr, and 72 hr), the 27E10 antigen is seen first after 24 hr on a few infiltrating monocytes/macrophages, which increase in numbers after 72 hr.

  16. Human monocytes undergo excessive apoptosis following temozolomide activating the ATM/ATR pathway while dendritic cells and macrophages are resistant.

    Directory of Open Access Journals (Sweden)

    Martina Bauer

    Full Text Available Immunodeficiency is a severe therapy-limiting side effect of anticancer chemotherapy resulting from sensitivity of immunocompetent cells to DNA damaging agents. A central role in the immune system is played by monocytes that differentiate into macrophages and dendritic cells (DCs. In this study we compared human monocytes isolated from peripheral blood and cytokine matured macrophages and DCs derived from them and assessed the mechanism of toxicity of the DNA methylating anticancer drug temozolomide (TMZ in these cell populations. We observed that monocytes, but not DCs and macrophages, were highly sensitive to the killing effect of TMZ. Studies on DNA damage and repair revealed that the initial DNA incision was efficient in monocytes while the re-ligation step of base excision repair (BER can not be accomplished, resulting in an accumulation of DNA single-strand breaks (SSBs. Furthermore, monocytes accumulated DNA double-strand breaks (DSBs following TMZ treatment, while DCs and macrophages were able to repair DSBs. Monocytes lack the DNA repair proteins XRCC1, ligase IIIα and PARP-1 whose expression is restored during differentiation into macrophages and DCs following treatment with GM-CSF and GM-CSF plus IL-4, respectively. These proteins play a key role both in BER and DSB repair by B-NHEJ, which explains the accumulation of DNA breaks in monocytes following TMZ treatment. Although TMZ provoked an upregulation of XRCC1 and ligase IIIα, BER was not enhanced likely because PARP-1 was not upregulated. Accordingly, inhibition of PARP-1 did not sensitize monocytes, but monocyte-derived DCs in which strong PARP activation was observed. TMZ induced in monocytes the DNA damage response pathways ATM-Chk2 and ATR-Chk1 resulting in p53 activation. Finally, upon activation of the Fas-receptor and the mitochondrial pathway apoptosis was executed in a caspase-dependent manner. The downregulation of DNA repair in monocytes, resulting in their selective

  17. Unsaponifiable fraction isolated from grape (Vitis vinifera L.) seed oil attenuates oxidative and inflammatory responses in human primary monocytes.

    Science.gov (United States)

    Millan-Linares, Maria C; Bermudez, Beatriz; Martin, Maria E; Muñoz, Ernesto; Abia, Rocio; Millan, Francisco; Muriana, Francisco J G; Montserrat-de la Paz, Sergio

    2018-04-25

    Grape (Vitis vinifera L.) seed has well-known potential for production of oil as a byproduct of winemaking and is a rich source of bioactive compounds. Herein, we report that the unsaponifiable fraction (UF) isolated from grape seed oil (GSO) possesses anti-oxidative and anti-inflammatory properties towards human primary monocytes. The UF isolated from GSO was phytochemically characterized by GC-MS and HPLC. Freshly obtained human monocytes were used to analyse the effects of GSOUF (10-100 μg mL-1) on oxidative and inflammatory responses using FACS analysis, RT-qPCR, and ELISA procedures. GSOUF skewed the monocyte plasticity towards the anti-inflammatory non-classical CD14+CD16++ monocytes and reduced the inflammatory competence of LPS-treated human primary monocytes diminishing TNF-α, IL-1β, and IL-6 gene expression and secretion. In addition, GSOUF showed a strong reactive oxygen species (ROS)-scavenging activity, reducing significantly nitrite levels with a significant decrease in Nos2 gene expression. Our results suggest that the UF isolated from GSO has significant potential for the management of inflammatory and oxidative conditions and offer novel benefits derived from the consumption of GSO in the prevention of inflammation-related diseases.

  18. FGF23 inhibits extra-renal synthesis of 1,25-dihydroxyvitamin D in human monocytes

    Science.gov (United States)

    Bacchetta, Justine; Sea, Jessica L; Chun, Rene F; Lisse, Thomas S; Wesseling-Perry, Katherine; Gales, Barbara; Adams, John S.; Salusky, Isidro B; Hewison, Martin

    2012-01-01

    Vitamin D is a potent stimulator of monocyte innate immunity, with this effect being mediated via intracrine conversion of 25-hydroxyvitamin D (25OHD) to 1,25-dihydroxyvitamin D (1,25(OH)2D). In the kidney synthesis of 1,25(OH)2D is suppressed by fibroblast growth factor 23 (FGF23), via transcriptional suppression of the vitamin D-activating enzyme 1α-hydroxylase (CYP27B1). We hypothesized that FGF23 also suppresses CYP27B1 in monocytes, with concomitant effects on intracrine responses to 1,25(OH)2D. Monocytes from healthy donor peripheral blood mononuclear cells (PBMCm) and from peritoneal dialysate effluent from kidney disease patients (PDm) were assessed at baseline to confirm the presence of mRNA for FGF23 receptors (FGFRs), with Klotho and FGFR1 being more strongly expressed than FGFR2/3/4 in both cell types. Immunohistochemistry showed co-expression of Klotho and FGFR1 in PBMCm and PDm, with this effect being enhanced following treatment with FGF23 in PBMCm but not PDm. Treatment with FGF23 activated MAP kinase (MAPK) and Akt pathways in PBMCm, demonstrating functional FGFR signaling in these cells. FGF23 treatment of PBMCm and PDm decreased expression of mRNA for CYP27B1. In PBMCm this was associated with downregulation of 25OHD to 1,25(OH)2D metabolism, and concomitant suppression of intracrine induced 24-hydroxylase (CYP24A1) and antibacterial cathelicidin (LL37). FGF23 suppression of CYP27B1 was particularly pronounced in PBMCm treated with interleukin-15 to stimulate synthesis of 1,25(OH)2D. These data indicate that FGF23 can inhibit extra-renal expression of CYP27B1 and subsequent intracrine responses to 1,25(OH)2D in two different human monocyte models. Elevated expression of FGF23 may therefore play a crucial role in defining immune responses to vitamin D and this, in turn, may be a key determinant of infection in patients with CKD. PMID:22886720

  19. Effects of Platelets on Platelet Concentrate Product on the Activation of Human Peripheral Blood Monocyte Cells

    Directory of Open Access Journals (Sweden)

    N Sadat Razavi Hoseini

    2016-02-01

    Full Text Available Introduction: Monocytes can interact with platelets due to their surface molecules such as P-selectin glycoprotein ligand-1 (PSGL-1, and form monocyte-platelet complex. In the present study, the effects of platelets interaction of platelet concentrates (PCs and peripheral blood monocytes were investigated in vitro as a model to predict the probable interactions of these cells and consequently activation of monocytes. Methods: In this experimental study, units of whole blood and PCs were prepared from Tehran Blood Transfusion Center. After isolation of monocytes from the whole blood, these cells were treated with PC- derived platelets. The activation of monocytes was assessed before and after treatment by the analysis of the respiratory burst of monocytes using dihydrorhodamine 123 (DHR-123. The study data were analyzed using the non-parametric test of Wilcoxon. Results: The purity of monocytes was determined as 86.1±2 using NycoPrep method. The respiratory burst of monocytes was increased after exposure with platelets. In fact, the difference was significant when platelets were used on the 5th day of storage (P=0.001. Conclusions: The study findings revealed that platelets have an efficient capacity to stimulate and activate monocytes. The possible involvement of molecules in the interaction of platelet-monocyte demand to be further studied in future.

  20. Nanoparticles as Antituberculosis Drugs Carriers: Effect on Activity Against Mycobacterium tuberculosis in Human Monocyte-Derived Macrophages

    International Nuclear Information System (INIS)

    Anisimova, Y.V.; Gelperina, S.I.; Peloquin, C.A.; Heifets, L.B.

    2000-01-01

    This is the first report evaluating the nanoparticle delivery system for three antituberculosis drugs: isoniazid, rifampin, and streptomycin. The typical particle size is 250 nm. We studied accumulation of these drugs in human monocytes as well as their antimicrobial activity against Mycobacterium tuberculosis residing in human monocyte-derived macrophages. Nanoparticle encapsulation increased the intracellular accumulation (cell-association) of all three tested drugs, but it enhanced the antimicrobial activity of isoniazid and streptomycin only. On the other hand, the activity of encapsulated rifampin against intracellular bacteria was not higher than that of the free drug

  1. Triglyceride-rich lipoprotein regulates APOB48 receptor gene expression in human THP-1 monocytes and macrophages.

    Science.gov (United States)

    Bermudez, Beatriz; Lopez, Sergio; Varela, Lourdes M; Ortega, Almudena; Pacheco, Yolanda M; Moreda, Wenceslao; Moreno-Luna, Rafael; Abia, Rocio; Muriana, Francisco J G

    2012-02-01

    The postprandial metabolism of dietary fats implies that the production of TG-rich lipoproteins (TRL) contributes to the progression of plaque development. TRL and their remnants cause rapid receptor-mediated monocyte/macrophage lipid engorgement via the cell surface apoB48 receptor (apoB48R). However, the mechanistic basis for apoB48 receptor (APOB48R) regulation by postprandial TRL in monocytes and macrophages is not well established. In this study, we investigated the effects of postprandial TRL from healthy volunteers on the expression of APOB48R mRNA and lipid uptake in human THP-1 monocytes and THP-1-derived macrophages. The expression of APOB48R mRNA was upregulated in THP-1 monocytes, but downregulated in THP-1-derived macrophages when treated with postprandial TRL (P < 0.05), in a dose- and time-dependent manner. TG and free cholesterol were dramatically increased in THP-1-derived macrophages (140 and 50%, respectively; P < 0.05) and in THP-1 monocytes (160 and 95%, respectively; P < 0.05). This lipid accumulation was severely decreased (~50%; P < 0.05) in THP-1-derived macrophages by small interfering RNA (siRNA) targeting of APOB48R. Using PPAR and retinoid X receptor (RXR) agonists, antagonists, and siRNA, our data indicate that PPARα, PPARγ, and RXRα are involved in postprandial TRL-induced APOB48R transcriptional regulation. Co-incubation with acyl-CoA synthetase or acyl-CoA:cholesterol acyltransferase inhibitors potentiated the effects of postprandial TRL on the expression of APOB48R mRNA in THP-1 monocytes and THP-1-derived macrophages. Our findings collectively suggest that APOB48R represents a molecular target of postprandial TRL via PPAR-dependent pathways in human THP-1 monocytes and macrophages and advance a potentially important link between postprandial metabolism of dietary fats and atherogenesis.

  2. Synergistic in-vitro effects of combining an antiglycolytic, 3-bromopyruvate, and a bromodomain-4 inhibitor on U937 myeloid leukemia cells.

    Science.gov (United States)

    Kapp, Nicolette; Stander, Xiao X; Stander, Barend A

    2018-06-01

    This project investigated the in-vitro effects of a glycolytic inhibitor, 3-bromopyruvate (3-BrP), in combination with and a new in silico-designed inhibitor of the bromodomain-4 (BRD-4) protein, ITH-47, on the U937 acute myeloid leukemia cell line. 3-BrP is an agent that targets the altered metabolism of cancer cells by interfering with glucose metabolism in the glycolytic pathway. ITH-47 is an acetyl-lysine inhibitor that displaces bromdomain 4 proteins from chromatin by competitively binding to the acetyl-lysine recognition pocket of this bromodomain and extraterminal (BET) BRD protein, thereby preventing transcription of cancer-associated genes and further cell growth. Cell growth studies determined the IC50 after 48 h exposure for 3-BrP and ITH-47 to be 6 and 2 μmol/l, respectively. When combined, 2.4 and 1 μmol/l of 3-BrP and ITH-47, respectively, inhibited 50% of the cell population, yielding a synergistic combination index of 0.9. Subsequent mechanistic studies showed that the IC50 concentrations of ITH-47 and 3-BrP and the combination increased observable apoptotic bodies and cell shrinkage in U937 cells treated for 48 h. Cell cycle analysis showed an increase in the sub-G1 fraction in all treated cells, suggesting that cell death was increased in the treated samples. Annexin-V-FITC apoptosis analysis showed a statistically significant increase in the number of cells in early and late apoptosis, indicating that cell death occurred through apoptosis and not necrosis. Only U937 cells exposed to ITH-47 showed a decrease in mitochondrial membrane potential compared with the vehicle control. Reactive oxygen species production was decreased in all treated samples. ITH-47-exposed cells showed a decrease in c-Myc, Bcl-2, and p53 gene expressions. 3-BrP-treated cells showed an increase in c-myc and p53 gene expressions. The combination of ITH-47 and 3-BrP lead to downregulation of c-myc and Bcl-2 genes. ITH-47 exposure conditions yielded a marked decrease

  3. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    International Nuclear Information System (INIS)

    Holers, V.M.; Kotzin, B.L.

    1985-01-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases

  4. Suppressive oligodeoxynucleotides containing TTAGGG motifs inhibit cGAS activation in human monocytes.

    Science.gov (United States)

    Steinhagen, Folkert; Zillinger, Thomas; Peukert, Konrad; Fox, Mario; Thudium, Marcus; Barchet, Winfried; Putensen, Christian; Klinman, Dennis; Latz, Eicke; Bode, Christian

    2018-04-01

    Type I interferon (IFN) is a critical mediator of autoimmune diseases such as systemic lupus erythematosus (SLE) and Aicardi-Goutières Syndrome (AGS). The recently discovered cyclic-GMP-AMP (cGAMP) synthase (cGAS) induces the production of type I IFN in response to cytosolic DNA and is potentially linked to SLE and AGS. Suppressive oligodeoxynucleotides (ODN) containing repetitive TTAGGG motifs present in mammalian telomeres have proven useful in the treatment of autoimmune diseases including SLE. In this study, we demonstrate that the suppressive ODN A151 effectively inhibits activation of cGAS in response to cytosolic DNA, thereby inhibiting type I IFN production by human monocytes. In addition, A151 abrogated cGAS activation in response to endogenous accumulation of DNA using TREX1-deficient monocytes. We demonstrate that A151 prevents cGAS activation in a manner that is competitive with DNA. This suppressive activity of A151 was dependent on both telomeric sequence and phosphorothioate backbone. To our knowledge this report presents the first cGAS inhibitor capable of blocking self-DNA. Collectively, these findings might lead to the development of new therapeutics against IFN-driven pathologies due to cGAS activation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT.

    Science.gov (United States)

    Sakaguchi, H; Ashikaga, T; Miyazawa, M; Yoshida, Y; Ito, Y; Yoneyama, K; Hirota, M; Itagaki, H; Toyoda, H; Suzuki, H

    2006-08-01

    Recent regulatory changes have placed a major emphasis on in vitro safety testing and alternative models. In regard to skin sensitization tests, dendritic cells (DCs) derived from human peripheral blood have been considered in the development of new in vitro alternatives. Human cell lines have been also reported recently. In our previous study, we suggested that measuring CD86 and/or CD54 expression on THP-1 cells (human monocytic leukemia cell line) could be used as an in vitro skin sensitization method. An inter-laboratory study among two laboratories was undertaken in Japan in order to further develop an in vitro skin sensitization model. In the present study, we used two human cell lines: THP-1 and U-937 (human histiocytic lymphoma cell line). First we optimized our test protocol (refer to the related paper entitled "optimization of the h-CLAT protocol" within this journal) and then we did an inter-laboratory validation with nine chemicals using the optimized protocol. We measured the expression of CD86 and CD54 on the above cells using flow cytometry after a 24h and 48h exposure to six known allergens (e.g., DNCB, pPD, NiSO(4)) and three non-allergens (e.g., SLS, tween 80). For the sample test concentration, four doses (0.1x, 0.5x, 1x, and 2x of the 50% inhibitory concentration (IC(50))) were evaluated. IC(50) was calculated using MTT assay. We found that allergens/non-allergens were better predicted using THP-1 cells compared to U-937 cells following a 24 h and a 48 h exposure. We also found that the 24h treatment time tended to have a better accuracy than the 48 h treatment time for THP-1 cells. Expression of CD86 and CD54 were good predictive markers for THP-1 cells, but for U-937 cells, expression of CD86 was a better predictor than CD54, at the 24h and the 48 h treatment time. The accuracy also improved when both markers (CD86 and CD54) were used as compared with a single marker for THP-1 cells. Both laboratories gave a good prediction of allergen

  6. Application of fluorescent monocytes for probing immune complexes on antigen microarrays.

    Directory of Open Access Journals (Sweden)

    Zoltán Szittner

    Full Text Available Microarrayed antigens are used for identifying serum antibodies with given specificities and for generating binding profiles. Antibodies bind to these arrayed antigens forming immune complexes and are conventionally identified by secondary labelled antibodies.In the body immune complexes are identified by bone marrow derived phagocytic cells, such as monocytes. In our work we were looking into the possibility of replacing secondary antibodies with monocytoid cells for the generation of antibody profiles. Using the human monocytoid cell line U937, which expresses cell surface receptors for immune complex components, we show that cell adhesion is completely dependent on the interaction of IgG heavy chains and Fcγ receptors, and this recognition is susceptible to differences between heavy chain structures and their glycosylation. We also report data on a possible application of this system in autoimmune diagnostics.Compared to secondary antibodies, fluorescent monocytesas biosensors are superior in reflecting biological functions of microarray-bound antibodies and represent an easy and robust alternative for profiling interactions between serum proteins and antigens.

  7. 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.

  8. Lipopolysaccharide-Elicited TSLPR Expression Enriches a Functionally Discrete Subset of Human CD14+ CD1c+ Monocytes.

    Science.gov (United States)

    Borriello, Francesco; Iannone, Raffaella; Di Somma, Sarah; Vastolo, Viviana; Petrosino, Giuseppe; Visconte, Feliciano; Raia, Maddalena; Scalia, Giulia; Loffredo, Stefania; Varricchi, Gilda; Galdiero, Maria Rosaria; Granata, Francescopaolo; Del Vecchio, Luigi; Portella, Giuseppe; Marone, Gianni

    2017-05-01

    Thymic stromal lymphopoietin (TSLP) is a cytokine produced mainly by epithelial cells in response to inflammatory or microbial stimuli and binds to the TSLP receptor (TSLPR) complex, a heterodimer composed of TSLPR and IL-7 receptor α (CD127). TSLP activates multiple immune cell subsets expressing the TSLPR complex and plays a role in several models of disease. Although human monocytes express TSLPR and CD127 mRNAs in response to the TLR4 agonist LPS, their responsiveness to TSLP is poorly defined. We demonstrate that TSLP enhances human CD14 + monocyte CCL17 production in response to LPS and IL-4. Surprisingly, only a subset of CD14 + CD16 - monocytes, TSLPR + monocytes (TSLPR + mono), expresses TSLPR complex upon LPS stimulation in an NF-κB- and p38-dependent manner. Phenotypic, functional, and transcriptomic analysis revealed specific features of TSLPR + mono, including higher CCL17 and IL-10 production and increased expression of genes with important immune functions (i.e., GAS6 , ALOX15B , FCGR2B , LAIR1 ). Strikingly, TSLPR + mono express higher levels of the dendritic cell marker CD1c. This evidence led us to identify a subset of peripheral blood CD14 + CD1c + cells that expresses the highest levels of TSLPR upon LPS stimulation. The translational relevance of these findings is highlighted by the higher expression of TSLPR and CD127 mRNAs in monocytes isolated from patients with Gram-negative sepsis compared with healthy control subjects. Our results emphasize a phenotypic and functional heterogeneity in an apparently homogeneous population of human CD14 + CD16 - monocytes and prompt further ontogenetic and functional analysis of CD14 + CD1c + and LPS-activated CD14 + CD1c + TSLPR + mono. Copyright © 2017 by The American Association of Immunologists, Inc.

  9. Captopril increases the intensity of monocyte infection by Trypanosoma cruzi and induces human T helper type 17 cells.

    Science.gov (United States)

    Coelho dos Santos, J S; Menezes, C A S; Villani, F N A; Magalhães, L M D; Scharfstein, J; Gollob, K J; Dutra, W O

    2010-12-01

    The anti-hypertensive drug captopril is used commonly to reduce blood pressure of patients with severe forms of Chagas disease, a cardiomyopathy caused by chronic infection with the intracellular protozoan Trypanosoma cruzi. Captopril acts by inhibiting angiotensin-converting enzyme (ACE), the vasopressor metallopeptidase that generates angiotensin II and promotes the degradation of bradykinin (BK). Recent studies in mice models of Chagas disease indicated that captopril can potentiate the T helper type 1 (Th1)-directing natural adjuvant property of BK. Equipped with kinin-releasing cysteine proteases, T. cruzi trypomastigotes were shown previously to invade non-professional phagocytic cells, such as human endothelial cells and murine cardiomyocytes, through the signalling of G protein-coupled bradykinin receptors (B(2) KR). Monocytes are also parasitized by T. cruzi and these cells are known to be important for the host immune response during infection. Here we showed that captopril increases the intensity of T. cruzi infection of human monocytes in vitro. The increased parasitism was accompanied by up-regulated expression of ACE in human monocytes. While T. cruzi infection increased the expression of interleukin (IL)-10 by monocytes significantly, compared to uninfected cells, T. cruzi infection in association with captopril down-modulated IL-10 expression by the monocytes. Surprisingly, studies with peripheral blood mononuclear cells revealed that addition of the ACE inhibitor in association with T. cruzi increased expression of IL-17 by CD4(+) T cells in a B(2) KR-dependent manner. Collectively, our results suggest that captopril might interfere with host-parasite equilibrium by enhancing infection of monocytes, decreasing the expression of the modulatory cytokine IL-10, while guiding development of the proinflammatory Th17 subset. © 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.

  10. A micro-Raman spectroscopic investigation of leukemic U-937 cells treated with Crotalaria agatiflora Schweinf and the isolated compound madurensine

    Science.gov (United States)

    le Roux, Karlien; Prinsloo, Linda C.; Hussein, Ahmed A.; Lall, Namrita

    In South Africa traditional medicine plays an important role in primary health care and therefore it is very important that the medicinal use of plants is scientifically tested for toxicity and effectiveness. It was established that the ethanolic extract of the leaves of Crotalaria agatiflora, as well as the isolated compound madurensine, is moderately toxic against leukemic U-937 cells. Light microscopic investigations indicated that symptoms of cell death are induced during treatments, but flow cytometry analysis of treated cells, using annexin-V and propidium iodide, showed that apoptosis and necrosis are insignificantly induced. The Raman results suggested that protein extraction and DNA melting occur in the cells during treatment with the ethanolic extracts (IC50 value 73.9 μg/mL), drastically changing the molecular content of the cells. In contrast, treatment with madurensine (IC50 value 136.5 μg/mL), an isolated pyrrolizidine alkaloid from the ethanolic extract of the leaves, did not have the same effect. The results are also compared to that of cells treated with actinomycin D, a compound known to induce apoptosis. The investigation showed that micro-Raman spectroscopy has great promise to be used for initial screening of samples to determine the effects of different treatments on cancerous cell lines together with conventional methods. The results highlight the fact that for many natural products used for medicinal purposes, the therapeutic effect of the crude plant extract tends to be significantly more effective than the particular action of its individual constituents.

  11. Flagella from five Cronobacter species induce pro-inflammatory cytokines in macrophage derivatives from human monocytes.

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    Ariadnna Cruz-Córdova

    Full Text Available Cronobacter spp. are opportunistic pathogens linked to lie-threatening infections in neonates and contaminated powdered infant formula that has been epidemiologically associated with these cases. Clinical symptoms of Cronobacter include necrotizing enterocolitis, bacteremia, and meningitis. Flagella from C. sakazakii are involved in biofilm formation and its adhesion to epithelial cells. We investigated the role of flagella from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis during the activation of cytokines (IL-8, TNF-α, and IL-10 in macrophage derivatives from human monocytes, which has not been extensively studied. The production and identity of flagella from the five Cronobacter species were visualized and recognized with anti-flagella antibodies by immunogold labeling through transmission electron microscopy. Purified flagella were dissociated into monomers in 12% SDS-PAGE Coomassie blue-stained gels showing a band of ∼28 kDa and, in addition, mass spectrometry revealed the presence of several peptides that correspond to flagellin. Flagella (100 ng induced the release of IL-8 (3314-6025 pg/ml, TNF-α (39-359 pg/ml, and IL-10 (2-96 pg/ml, in macrophage isolates from human monocytes and similar results were obtained when flagella were dissociated into monomers. Inhibition assays using three dilutions of anti-flagella antibodies (1∶10, 1∶100, and 1∶200 suppressed the secretion of IL-8, TNF-α, and IL-10 between 95-100% using 100 ng of protein. A transfection assay using 293-hTLR5 cells showed IL-8 release of 197 pg/ml and suppression in the secretion of IL-8 when anti-hTLR5-IgA antibodies were used at different concentrations. These observations suggest that flagella and flagellin are involved in an inflammatory response dependent on TLR5 recognition, which could contribute to the pathogenesis of the bacteria.

  12. Impact of psychostimulants and atomoxetine on the expression of 8-hydroxyguanine glycosylase 1 in human cells.

    Science.gov (United States)

    Schmidt, Andreas Johannes; Clement, Hans-Willi; Gebhardt, Stefan; Hemmeter, Ulrich Michael; Schulz, Eberhard; Krieg, Jürgen-Christian; Kircher, Tilo; Heiser, Philip

    2010-06-01

    Oxidative DNA damage as one sign of reactive oxygen species induced oxidative stress is an important factor in the pathogenesis of various psychiatric disorders. Altered levels of DNA base damage products as well as the expression of the main repair enzyme 8-hydroxyguanine glycosylase 1 have been described. The aim of the present study was to examine the effects of drugs (amphetamine, methylphenidate and atomoxetine) used in the treatment of attention deficit-hyperactivity disorder on the expression of this enzyme via reverse transcriptase-polymerase chain reaction in human neuroblastoma SH-SY5Y and human monocytic U-937 cells at concentrations of 50, 500 and 5,000 ng/ml. We observed decreased expression of this enzyme for all applied substances. In U-937 cells, the significance level was reached after treatment with 5,000 ng/ml amphetamine as well as after treatment with 50, 500 and 5,000 ng/ml atomoxetine. Incubation of SH-SY5Y cells with 50 and 5,000 ng/ml amphetamine and 5,000 ng/ml methylphenidate led to significant decreases of 8-hydroxyguanine glycosylase 1. As a positive correlation between the expression of 8-hydroxyguanine glycosylase 1 and the level of oxidative DNA damage products has been described, we accordingly consider these substances (amphetamine, methylphenidate and atomoxetine) to possibly play a protective role in this process.

  13. [EVALUATION OF THE HUMAN SENSITIVITY TO SMALLPOX VIRUS BY THE PRIMARY CULTURES OF THE MONOCYTE-MACROPHAGES].

    Science.gov (United States)

    Zamedyanskaya, A S; Titova, K A; Sergeev, Al A; Kabanov, A S; Bulychev, L E; Sergeev, Ar A; Galakhova, D O; Nesterov, A E; Nosareva, O V; Shishkina, L N; Taranov, O S; Omigov, V V; Agafonov, A P; Sergeev, A N

    2016-01-01

    Studies of the primary cultures of granulocytes, mononuclear, and monocyte-macrophage cells derived from human blood were performed using variola virus (VARV) in the doses of 0.001-0.021 PFU/cell (plaques-forming units per cell). Positive dynamics of the virus accumulation was observed only in the monocyte-macrophages with maximum values of virus concentration (5.0-5.5 Ig PFU/ml) mainly within six days after the infection. The fact of VARV replication in the monocyte-macrophages was confirmed by the data of electron microscopy. At the same time, virus vaccines when tested in doses 3.3 and 4.2 Ig PFU/ml did not show the ability to reproduce in these human cells. The people sensitivity to VARV as assessed from the data obtained on human monocyte-macrophages corresponded to -1 PFU (taking into account the smooth interaction of the virus in the body to the cells of this type), which is consistent to previously found theoretical data on the virus sensitivity. The human susceptibility to VARV assessed experimentally can be used to predict the adequacy of developed smallpox models (in vivo) based on susceptible animals. This is necessary for reliable assessment of the efficiency of development of drugs for treatment and prophylaxis of the smallpox.

  14. 17 beta-estradiol and progesterone do not influence the production of cytokines from lipopolysaccharide-stimulated monocytes in humans

    NARCIS (Netherlands)

    Schipper, M; Heineman, MJ; Faas, M; Bouman, A.

    2004-01-01

    Objective: To test whether 17beta-estradiol or progesterone influence the cytokine productive capacity of lipopolysaccharide (LPS)-stimulated monocytes in humans. Design: Prospective study. Setting: Academic research institution. Patient(s): Seven women in the luteal phase of a normal ovarian cycle,

  15. OSCAR Is a Receptor for Surfactant Protein D That Activates TNF-α Release from Human CCR2+ Inflammatory Monocytes

    DEFF Research Database (Denmark)

    Barrow, Alexander D; Palarasah, Yaseelan; Bugatti, Mattia

    2015-01-01

    of recombinant SP-D and captured native SP-D from human bronchoalveolar lavage. OSCAR localized in an intracellular compartment of alveolar macrophages together with SP-D. Moreover, we found OSCAR on the surface of interstitial lung and blood CCR2(+) inflammatory monocytes, which secreted TNF-α when exposed...

  16. 17beta-estradiol and progesterone do not influence the production of cytokines from lipopolysaccharide-stimulated monocytes in humans

    NARCIS (Netherlands)

    Bouman, Annechien; Schipper, Martin; Heineman, Maas Jan; Faas, Marijke

    2004-01-01

    OBJECTIVE: To test whether 17beta-estradiol or progesterone influence the cytokine productive capacity of lipopolysaccharide (LPS)-stimulated monocytes in humans. DESIGN: Prospective study. SETTING: Academic research institution. PATIENT(S): Seven women in the luteal phase of a normal ovarian cycle,

  17. Human monocytes undergo functional re-programming during sepsis mediated by hypoxia-inducible factor-1α.

    Science.gov (United States)

    Shalova, Irina N; Lim, Jyue Yuan; Chittezhath, Manesh; Zinkernagel, Annelies S; Beasley, Federico; Hernández-Jiménez, Enrique; Toledano, Victor; Cubillos-Zapata, Carolina; Rapisarda, Annamaria; Chen, Jinmiao; Duan, Kaibo; Yang, Henry; Poidinger, Michael; Melillo, Giovanni; Nizet, Victor; Arnalich, Francisco; López-Collazo, Eduardo; Biswas, Subhra K

    2015-03-17

    Sepsis is characterized by a dysregulated inflammatory response to infection. Despite studies in mice, the cellular and molecular basis of human sepsis remains unclear and effective therapies are lacking. Blood monocytes serve as the first line of host defense and are equipped to recognize and respond to infection by triggering an immune-inflammatory response. However, the response of these cells in human sepsis and their contribution to sepsis pathogenesis is poorly understood. To investigate this, we performed a transcriptomic, functional, and mechanistic analysis of blood monocytes from patients during sepsis and after recovery. Our results revealed the functional plasticity of monocytes during human sepsis, wherein they transited from a pro-inflammatory to an immunosuppressive phenotype, while enhancing protective functions like phagocytosis, anti-microbial activity, and tissue remodeling. Mechanistically, hypoxia inducible factor-1α (HIF1α) mediated this functional re-programming of monocytes, revealing a potential mechanism for their therapeutic targeting to regulate human sepsis. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. SIMULTANEOUS EXPRESSION AND REGULATION OF G-CSF AND IL-6 MESSENGER-RNA IN ADHERENT HUMAN MONOCYTES AND FIBROBLASTS

    NARCIS (Netherlands)

    VELLENGA, E; VANDERVINNE, B; DEWOLF, JTM; HALIE, MR

    The regulation of granulocyte-colony stimulating factor (G-CSF) and interleukin-6 (IL-6) mRNA was studied in human adherent monocytes in response to the protein kinase C activator, oleolyl-acetylglycerol (OAG), the calcium-ionophore A23187 and the cyclic AMP elevating agents, dibutyryl c-AMP

  19. Transmigration of polymorphnuclear neutrophils and monocytes through the human blood-cerebrospinal fluid barrier after bacterial infection in vitro.

    Science.gov (United States)

    Steinmann, Ulrike; Borkowski, Julia; Wolburg, Hartwig; Schröppel, Birgit; Findeisen, Peter; Weiss, Christel; Ishikawa, Hiroshi; Schwerk, Christian; Schroten, Horst; Tenenbaum, Tobias

    2013-02-28

    Bacterial invasion through the blood-cerebrospinal fluid barrier (BCSFB) during bacterial meningitis causes secretion of proinflammatory cytokines/chemokines followed by the recruitment of leukocytes into the CNS. In this study, we analyzed the cellular and molecular mechanisms of polymorphonuclear neutrophil (PMN) and monocyte transepithelial transmigration (TM) across the BCSFB after bacterial infection. Using an inverted transwell filter system of human choroid plexus papilloma cells (HIBCPP), we studied leukocyte TM rates, the migration route by immunofluorescence, transmission electron microscopy and focused ion beam/scanning electron microscopy, the secretion of cytokines/chemokines by cytokine bead array and posttranslational modification of the signal regulatory protein (SIRP) α via western blot. PMNs showed a significantly increased TM across HIBCPP after infection with wild-type Neisseria meningitidis (MC58). In contrast, a significantly decreased monocyte transmigration rate after bacterial infection of HIBCPP could be observed. Interestingly, in co-culture experiments with PMNs and monocytes, TM of monocytes was significantly enhanced. Analysis of paracellular permeability and transepithelial electrical resistance confirmed an intact barrier function during leukocyte TM. With the help of the different imaging techniques we could provide evidence for para- as well as for transcellular migrating leukocytes. Further analysis of secreted cytokines/chemokines showed a distinct pattern after stimulation and transmigration of PMNs and monocytes. Moreover, the transmembrane glycoprotein SIRPα was deglycosylated in monocytes, but not in PMNs, after bacterial infection. Our findings demonstrate that PMNs and monoctyes differentially migrate in a human BCSFB model after bacterial infection. Cytokines and chemokines as well as transmembrane proteins such as SIRPα may be involved in this process.

  20. Dyslipidemic Diet-Induced Monocyte “Priming” and Dysfunction in Non-Human Primates Is Triggered by Elevated Plasma Cholesterol and Accompanied by Altered Histone Acetylation

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    John D. Short

    2017-08-01

    Full Text Available Monocytes and the recruitment of monocyte-derived macrophages into sites of inflammation play a key role in atherogenesis and other chronic inflammatory diseases linked to cardiometabolic syndrome and obesity. Previous studies from our group have shown that metabolic stress promotes monocyte priming, i.e., enhanced adhesion and accelerated chemotaxis of monocytes in response to chemokines, both in vitro and in dyslipidemic LDLR−/− mice. We also showed that metabolic stress-induced monocyte dysfunction is, at least to a large extent caused by the S-glutathionylation, inactivation, and subsequent degradation of mitogen-activated protein kinase phosphatase 1. Here, we analyzed the effects of a Western-style, dyslipidemic diet (DD, which was composed of high levels of saturated fat, cholesterol, and simple sugars, on monocyte (dysfunction in non-human primates (NHPs. We found that similar to mice, a DD enhances monocyte chemotaxis in NHP within 4 weeks, occurring concordantly with the onset of hypercholesterolemia but prior to changes in triglycerides, blood glucose, monocytosis, or changes in monocyte subset composition. In addition, we identified transitory decreases in the acetylation of histone H3 at the lysine residues 18 and 23 in metabolically primed monocytes, and we found that monocyte priming was correlated with the acetylation of histone H3 at lysine 27 after an 8-week DD regimen. Our data show that metabolic stress promotes monocyte priming and hyper-chemotactic responses in NHP. The histone modifications accompanying monocyte priming in primates suggest a reprogramming of the epigenetic landscape, which may lead to dysregulated responses and functionalities in macrophages derived from primed monocytes that are recruited to sites of inflammation.

  1. Methylglyoxal-bis-guanylhydrazone inhibits osteopontin expression and differentiation in cultured human monocytes.

    Science.gov (United States)

    Jin, Xia; Xu, Hua; McGrath, Michael S

    2018-01-01

    Monocyte activation and polarization play essential roles in many chronic inflammatory diseases. An imbalance of M1 and M2 macrophage activation (pro-inflammatory and alternatively activated, respectively) is believed to be a key aspect in the etiology of these diseases, thus a therapeutic approach that regulates macrophage activation could be of broad clinical relevance. Methylglyoxal-bis-guanylhydrazone (MGBG), a regulator of polyamine metabolism, has recently been shown to be concentrated in monocytes and macrophages, and interfere with HIV integration into the DNA of these cells in vitro. RNA expression analysis of monocytes from HIV+ and control donors with or without MGBG treatment revealed the only gene to be consistently down regulated by MGBG to be osteopontin (OPN). The elevated expression of this pro-inflammatory cytokine and monocyte chemoattractant is associated with various chronic inflammatory diseases. We demonstrate that MGBG is a potent inhibitor of secreted OPN (sOPN) in cultured monocytes with 50% inhibition achieved at 0.1 μM of the drug. Furthermore, inhibition of OPN RNA transcription in monocyte cultures occurs at similar concentrations of the drug. During differentiation of monocytes into macrophages in vitro, monocytes express cell surface CD16 and the cells undergo limited DNA synthesis as measured by uptake of BrdU. MGBG inhibited both activities at similar doses to those regulating OPN expression. In addition, monocyte treatment with MGBG inhibited differentiation into both M1 and M2 classes of macrophages at non-toxic doses. The inhibition of differentiation and anti-OPN effects of MGBG were specific for monocytes in that differentiated macrophages were nearly resistant to MGBG activities. Thus MGBG may have potential therapeutic utility in reducing or normalizing OPN levels and regulating monocyte activation in diseases that involve chronic inflammation.

  2. Uric acid priming in human monocytes is driven by the AKT–PRAS40 autophagy pathway

    Science.gov (United States)

    Crişan, Tania O.; Cleophas, Maartje C. P.; Novakovic, Boris; Erler, Kathrin; van de Veerdonk, Frank L.; Stunnenberg, Hendrik G.; Netea, Mihai G.; Dinarello, Charles A.; Joosten, Leo A. B.

    2017-01-01

    Metabolic triggers are important inducers of the inflammatory processes in gout. Whereas the high serum urate levels observed in patients with gout predispose them to the formation of monosodium urate (MSU) crystals, soluble urate also primes for inflammatory signals in cells responding to gout-related stimuli, but also in other common metabolic diseases. In this study, we investigated the mechanisms through which uric acid selectively lowers human blood monocyte production of the natural inhibitor IL-1 receptor antagonist (IL-1Ra) and shifts production toward the highly inflammatory IL-1β. Monocytes from healthy volunteers were first primed with uric acid for 24 h and then subjected to stimulation with lipopolysaccharide (LPS) in the presence or absence of MSU. Transcriptomic analysis revealed broad inflammatory pathways associated with uric acid priming, with NF-κB and mammalian target of rapamycin (mTOR) signaling strongly increased. Functional validation did not identify NF-κB or AMP-activated protein kinase phosphorylation, but uric acid priming induced phosphorylation of AKT and proline-rich AKT substrate 40 kDa (PRAS 40), which in turn activated mTOR. Subsequently, Western blot for the autophagic structure LC3-I and LC3-II (microtubule-associated protein 1A/1B-light chain 3) fractions, as well as fluorescence microscopy of LC3-GFP–overexpressing HeLa cells, revealed lower autophagic activity in cells exposed to uric acid compared with control conditions. Interestingly, reactive oxygen species production was diminished by uric acid priming. Thus, the Akt–PRAS40 pathway is activated by uric acid, which inhibits autophagy and recapitulates the uric acid-induced proinflammatory cytokine phenotype. PMID:28484006

  3. Uric acid priming in human monocytes is driven by the AKT-PRAS40 autophagy pathway.

    Science.gov (United States)

    Crişan, Tania O; Cleophas, Maartje C P; Novakovic, Boris; Erler, Kathrin; van de Veerdonk, Frank L; Stunnenberg, Hendrik G; Netea, Mihai G; Dinarello, Charles A; Joosten, Leo A B

    2017-05-23

    Metabolic triggers are important inducers of the inflammatory processes in gout. Whereas the high serum urate levels observed in patients with gout predispose them to the formation of monosodium urate (MSU) crystals, soluble urate also primes for inflammatory signals in cells responding to gout-related stimuli, but also in other common metabolic diseases. In this study, we investigated the mechanisms through which uric acid selectively lowers human blood monocyte production of the natural inhibitor IL-1 receptor antagonist (IL-1Ra) and shifts production toward the highly inflammatory IL-1β. Monocytes from healthy volunteers were first primed with uric acid for 24 h and then subjected to stimulation with lipopolysaccharide (LPS) in the presence or absence of MSU. Transcriptomic analysis revealed broad inflammatory pathways associated with uric acid priming, with NF-κB and mammalian target of rapamycin (mTOR) signaling strongly increased. Functional validation did not identify NF-κB or AMP-activated protein kinase phosphorylation, but uric acid priming induced phosphorylation of AKT and proline-rich AKT substrate 40 kDa (PRAS 40), which in turn activated mTOR. Subsequently, Western blot for the autophagic structure LC3-I and LC3-II (microtubule-associated protein 1A/1B-light chain 3) fractions, as well as fluorescence microscopy of LC3-GFP-overexpressing HeLa cells, revealed lower autophagic activity in cells exposed to uric acid compared with control conditions. Interestingly, reactive oxygen species production was diminished by uric acid priming. Thus, the Akt-PRAS40 pathway is activated by uric acid, which inhibits autophagy and recapitulates the uric acid-induced proinflammatory cytokine phenotype.

  4. Free hemoglobin enhances tumor necrosis factor-alpha production in isolated human monocytes.

    Science.gov (United States)

    Carrillo, Eddy H; Gordon, Laura E; Richardson, J David; Polk, Hiram C

    2002-03-01

    A systemic inflammatory response (SIR) is seen in approximately 75% of patients with complex blunt liver injuries treated nonoperatively. Many feel this response is caused by blood, bile, and necrotic tissue accumulation in the peritoneal cavity. Our current treatment for these patients is a delayed laparoscopic washout of the peritoneal cavity, resulting in a dramatic resolution of the SIR. Spectrophotometric analysis of the intraperitoneal fluid has confirmed the presence of high concentrations of free hemoglobin (Hb). We hypothesize that free Hb enhances the local peritoneal response by increasing tumor necrosis factor-alpha (TNF-alpha) production by monocytes, contributing to the local inflammatory response and SIR. Monocytes from five healthy volunteers were isolated and cultured in RPMI-1640 for 24 hours. Treatment groups included saline controls, lipopolysaccharide ([LPS], 10 ng/mL, from Escherichia coli), human Hb (25 microg/mL), and Hb + LPS. Supernatants were analyzed by enzyme-linked immunosorbent assay. Student's t test with Mann-Whitney posttest was used for statistical analysis with p < or = 0.05 considered significant. Free Hb significantly increased TNF-alpha production 915 +/- 223 pg/mL versus saline (p = 0.02). LPS and Hb + LPS further increased TNF-alpha production (2294 pg/mL and 2501 pg/mL, respectively, p < 0.001) compared with saline controls. These data confirm that free Hb is a proinflammatory mediator resulting in the production of significant amounts of TNF-alpha. These in vitro findings support our clinical data in which timely removal of intraperitoneal free hemoglobin helps prevent its deleterious local and systemic inflammatory effects in patients with complex liver injuries managed nonoperatively.

  5. Induction of Chemokine Secretion and Monocyte Migration by Human Choroidal Melanocytes in Response to Proinflammatory Cytokines

    DEFF Research Database (Denmark)

    Jehs, Tina; Faber, Carsten; Udsen, Maja S.

    2016-01-01

    of 10 HCM donors induced a high initial level of monocyte migration, which decreased upon stimulation with either TCM or IFN-γ and TNF-α. The supernatants from three HCM donors initially showed a low level of monocyte attraction, which increased after exposure to proinflammatory cytokines. Direct...

  6. In Vitro experimental model of trained innate immunity in human primary monocytes

    DEFF Research Database (Denmark)

    Bekkering, S.; Blok, B. A.; Joosten, Leo A B

    2016-01-01

    experimental protocol of monocyte training using three of the most commonly used training stimuli from the literature: β-glucan, the bacillus Calmette-Guérin (BCG) vaccine, and oxidized low-density lipoprotein (ox-LDL). We investigated and optimized a protocol of monocyte trained immunity induced by an initial....... All Rights Reserved....

  7. IκK-16 decreases miRNA-155 expression and attenuates the human monocyte inflammatory response.

    Directory of Open Access Journals (Sweden)

    Norman James Galbraith

    Full Text Available Excessive inflammatory responses in the surgical patient may result in cellular hypo-responsiveness, which is associated with an increased risk of secondary infection and death. microRNAs (miRNAs, such as miR-155, are powerful regulators of inflammatory signalling pathways including nuclear factor κB (NFκB. Our objective was to determine the effect of IκK-16, a selective blocker of inhibitor of kappa-B kinase (IκK, on miRNA expression and the monocyte inflammatory response. In a model of endotoxin tolerance using primary human monocytes, impaired monocytes had decreased p65 expression with suppressed TNF-α and IL-10 production (P < 0.05. miR-155 and miR-138 levels were significantly upregulated at 17 h in the impaired monocyte (P < 0.05. Notably, IκK-16 decreased miR-155 expression with a corresponding dose-dependent decrease in TNF-α and IL-10 production (P < 0.05, and impaired monocyte function was associated with increased miR-155 and miR-138 expression. In the context of IκK-16 inhibition, miR-155 mimics increased TNF-α production, while miR-155 antagomirs decreased both TNF-α and IL-10 production. These data demonstrate that IκK-16 treatment attenuates the monocyte inflammatory response, which may occur through a miR-155-mediated mechanism, and that IκK-16 is a promising approach to limit the magnitude of an excessive innate inflammatory response to LPS.

  8. High Cellular Monocyte Activation in People Living With Human Immunodeficiency Virus on Combination Antiretroviral Therapy and Lifestyle-Matched Controls Is Associated With Greater Inflammation in Cerebrospinal Fluid

    NARCIS (Netherlands)

    Booiman, Thijs; Wit, Ferdinand W.; Maurer, Irma; de Francesco, Davide; Sabin, Caroline A.; Harskamp, Agnes M.; Prins, Maria; Garagnani, Paolo; Pirazzini, Chiara; Franceschi, Claudio; Fuchs, Dietmar; Gisslén, Magnus; Winston, Alan; Reiss, Peter; Kootstra, Neeltje A.; Schouten, J.; Kooij, K. W.; van Zoest, R. A.; Elsenga, B. C.; Janssen, F. R.; Heidenrijk, M.; Zikkenheiner, W.; van der Valk, M.; Booiman, T.; Harskamp-Holwerda, A. M.; Boeser-Nunnink, B.; Maurer, I.; Mangas Ruiz, M. M.; Girigorie, A. F.; Villaudy, J.; Frankin, E.; Pasternak, A.; Berkhout, B.; van der Kuyl, T.; Portegies, P.; Schmand, B. A.; Geurtsen, G. J.; ter Stege, J. A.; Klein Twennaar, M.; Majoie, C. B. L. M.; Caan, M. W. A.; Su, T.; Weijer, K.; Bisschop, P. H. L. T.; Kalsbeek, A.; Wezel, M.; Visser, I.; Ruhé, H. G.; Franceschi, C.; Garagnani, P.

    2017-01-01

    Increased monocyte activation and intestinal damage have been shown to be predictive for the increased morbidity and mortality observed in treated people living with human immunodeficiency virus (PLHIV). A cross-sectional analysis of cellular and soluble markers of monocyte activation, coagulation,

  9. High Cellular Monocyte Activation in People Living With Human Immunodeficiency Virus on Combination Antiretroviral Therapy and Lifestyle-Matched Controls Is Associated With Greater Inflammation in Cerebrospinal Fluid

    NARCIS (Netherlands)

    Booiman, Thijs; Wit, Ferdinand W N M; Maurer, Irma; De Francesco, Davide; Sabin, Caroline A; Harskamp, Agnes M; Prins, Maria; Garagnani, Paolo; Pirazzini, Chiara; Franceschi, Claudio; Fuchs, Dietmar; Gisslén, Magnus; Winston, Alan; Reiss, Peter; Kootstra, Neeltje A; Kalsbeek, A.

    2017-01-01

    BACKGROUND: Increased monocyte activation and intestinal damage have been shown to be predictive for the increased morbidity and mortality observed in treated people living with human immunodeficiency virus (PLHIV). METHODS: A cross-sectional analysis of cellular and soluble markers of monocyte

  10. Increased platelet reactivity is associated with circulating platelet-monocyte complexes and macrophages in human atherosclerotic plaques.

    Directory of Open Access Journals (Sweden)

    Bert Rutten

    Full Text Available Platelet reactivity, platelet binding to monocytes and monocyte infiltration play a detrimental role in atherosclerotic plaque progression. We investigated whether platelet reactivity was associated with levels of circulating platelet-monocyte complexes (PMCs and macrophages in human atherosclerotic carotid plaques.Platelet reactivity was determined by measuring platelet P-selectin expression after platelet stimulation with increasing concentrations of adenosine diphosphate (ADP, in two independent cohorts: the Circulating Cells cohort (n = 244 and the Athero-Express cohort (n = 91. Levels of PMCs were assessed by flow cytometry in blood samples of patients who were scheduled for percutaneous coronary intervention (Circulating Cells cohort. Monocyte infiltration was semi-quantitatively determined by histological examination of atherosclerotic carotid plaques collected during carotid endarterectomy (Athero-Express cohort.We found increased platelet reactivity in patients with high PMCs as compared to patients with low PMCs (median (interquartile range: 4153 (1585-11267 area under the curve (AUC vs. 9633 (3580-21565 AUC, P<0.001. Also, we observed increased platelet reactivity in patients with high macrophage levels in atherosclerotic plaques as compared to patients with low macrophage levels in atherosclerotic plaques (mean ± SD; 8969 ± 3485 AUC vs. 7020 ± 3442 AUC, P = 0.02. All associations remained significant after adjustment for age, sex and use of drugs against platelet activation.Platelet reactivity towards ADP is associated with levels of PMCs and macrophages in human atherosclerotic carotid plaques.

  11. Estradiol coupling to human monocyte nitric oxide release is dependent on intracellular calcium transients: evidence for an estrogen surface receptor.

    Science.gov (United States)

    Stefano, G B; Prevot, V; Beauvillain, J C; Fimiani, C; Welters, I; Cadet, P; Breton, C; Pestel, J; Salzet, M; Bilfinger, T V

    1999-10-01

    We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase (cNOS) activity in human peripheral monocytes by acting on an estrogen surface receptor. NO release was measured in real time with an amperometric probe. 17beta-estradiol exposure to monocytes stimulated NO release within seconds in a concentration-dependent manner, whereas 17alpha-estradiol had no effect. 17beta-estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17beta-estradiol and E2-BSA, whereas ICI 182,780, a selective inhibitor of the nuclear estrogen receptor, had no effect. We further showed, using a dual emission microfluorometry in a calcium-free medium, that the 17beta-estradiol-stimulated release of monocyte NO was dependent on the initial stimulation of intracellular calcium transients in a tamoxifen-sensitive process. Leeching out the intracellular calcium stores abolished the effect of 17beta-estradiol on NO release. RT-PCR analysis of RNA obtained from the cells revealed a strong estrogen receptor-alpha amplification signal and a weak beta signal. Taken together, a physiological dose of estrogen acutely stimulates NO release from human monocytes via the activation of an estrogen surface receptor that is coupled to increases in intracellular calcium.

  12. MIR144* inhibits antimicrobial responses against Mycobacterium tuberculosis in human monocytes and macrophages by targeting the autophagy protein DRAM2.

    Science.gov (United States)

    Kim, Jin Kyung; Lee, Hye-Mi; Park, Ki-Sun; Shin, Dong-Min; Kim, Tae Sung; Kim, Yi Sak; Suh, Hyun-Woo; Kim, Soo Yeon; Kim, In Soo; Kim, Jin-Man; Son, Ji-Woong; Sohn, Kyung Mok; Jung, Sung Soo; Chung, Chaeuk; Han, Sang-Bae; Yang, Chul-Su; Jo, Eun-Kyeong

    2017-02-01

    Autophagy is an important antimicrobial effector process that defends against Mycobacterium tuberculosis (Mtb), the human pathogen causing tuberculosis (TB). MicroRNAs (miRNAs), endogenous noncoding RNAs, are involved in various biological functions and act as post-transcriptional regulators to target mRNAs. The process by which miRNAs affect antibacterial autophagy and host defense mechanisms against Mtb infections in human monocytes and macrophages is largely uncharacterized. In this study, we show that Mtb significantly induces the expression of MIR144*/hsa-miR-144-5p, which targets the 3'-untranslated region of DRAM2 (DNA damage regulated autophagy modulator 2) in human monocytes and macrophages. Mtb infection downregulated, whereas the autophagy activators upregulated, DRAM2 expression in human monocytes and macrophages by activating AMP-activated protein kinase. In addition, overexpression of MIR144* decreased DRAM2 expression and formation of autophagosomes in human monocytes, whereas inhibition of MIR144* had the opposite effect. Moreover, the levels of MIR144* were elevated, whereas DRAM2 levels were reduced, in human peripheral blood cells and tissues in TB patients, indicating the clinical significance of MIR144* and DRAM2 in human TB. Notably, DRAM2 interacted with BECN1 and UVRAG, essential components of the autophagic machinery, leading to displacement of RUBCN from the BECN1 complex and enhancement of Ptdlns3K activity. Furthermore, MIR144* and DRAM2 were critically involved in phagosomal maturation and enhanced antimicrobial effects against Mtb. Our findings identify a previously unrecognized role of human MIR144* in the inhibition of antibacterial autophagy and the innate host immune response to Mtb. Additionally, these data reveal that DRAM2 is a key coordinator of autophagy activation that enhances antimicrobial activity against Mtb.

  13. Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression in U937 cells.

    Science.gov (United States)

    Chihara, Kazuyasu; Kato, Yuji; Yoshiki, Hatsumi; Takeuchi, Kenji; Fujieda, Shigeharu; Sada, Kiyonao

    2017-09-13

    The adaptor protein c-Abl SH3 domain binding protein-2 (3BP2) is tyrosine phosphorylated by Syk in response to cross-linking of antigen receptors, which in turn activates various immune responses. Recently, a study using the mouse model of cherubism, a dominant inherited disorder caused by mutations in the gene encoding 3BP2, showed that 3BP2 is involved in the regulation of phagocytosis mediated by Fc receptor for IgG (FcγR) in macrophages. However, the molecular mechanisms underlying 3BP2-mediated regulation of phagocytosis and the physiological relevance of 3BP2 tyrosine phosphorylation remains elusive. In this study, we established various gene knockout U937 cell lines using the CRISPR/Cas9 system and found that 3BP2 is rapidly tyrosine phosphorylated by Syk in response to cross-linking of FcγRI. Depletion of 3BP2 caused significant reduction in the Fc receptor γ chain (FcRγ)-mediated phagocytosis in addition to the FcγRI-mediated induction of chemokine mRNA for IL-8, CCL3L3 and CCL4L2. Syk-dependent tyrosine phosphorylation of 3BP2 was required for overcoming these defects. Finally, we found that the PH and SH2 domains play important roles on FcγRI-mediated tyrosine phosphorylation of 3BP2 in HL-60 cells. Taken together, these results indicate that Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression.

  14. Extracellular Histones Increase Tissue Factor Activity and Enhance Thrombin Generation by Human Blood Monocytes.

    Science.gov (United States)

    Gould, Travis J; Lysov, Zakhar; Swystun, Laura L; Dwivedi, Dhruva J; Zarychanski, Ryan; Fox-Robichaud, Alison E; Liaw, Patricia C

    2016-12-01

    Sepsis is characterized by systemic activation of inflammatory and coagulation pathways in response to infection. Recently, it was demonstrated that histones released into the circulation by dying/activated cells may contribute to sepsis pathology. Although the ability of extracellular histones to modulate the procoagulant activities of several cell types has been investigated, the influence of histones on the hemostatic functions of circulating monocytes is unknown. To address this, we investigated the ability of histones to modulate the procoagulant potential of THP-1 cells and peripheral blood monocytes, and examined the effects of plasmas obtained from septic patients to induce a procoagulant phenotype on monocytic cells. Tissue factor (TF) activity assays were performed on histone-treated THP-1 cells and blood monocytes. Exposure of monocytic cells to histones resulted in increases in TF activity, TF antigen, and phosphatidylserine exposure. Histones modulate the procoagulant activity via engagement of Toll-like receptors 2 and 4, and this effect was abrogated with inhibitory antibodies. Increased TF activity of histone-treated cells corresponded to enhanced thrombin generation in plasma determined by calibrated automated thrombography. Finally, TF activity was increased on monocytes exposed to plasma from septic patients, an effect that was attenuated in plasma from patients receiving unfractionated heparin (UFH). Our studies suggest that increased levels of extracellular histones found in sepsis contribute to dysregulated coagulation by increasing TF activity of monocytes. These procoagulant effects can be partially ameliorated in sepsis patients receiving UFH, thereby identifying extracellular histones as a potential therapeutic target for sepsis treatment.

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

    Science.gov (United States)

    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.

  16. The transcriptome of Legionella pneumophila-infected human monocyte-derived macrophages.

    Directory of Open Access Journals (Sweden)

    Christopher T D Price

    Full Text Available Legionella pneumophila is an intracellular bacterial pathogen that invades and replicates within alveolar macrophages through injection of ∼ 300 effector proteins by its Dot/Icm type IV translocation apparatus. The bona fide F-box protein, AnkB, is a nutritional virulence effector that triggers macrophages to generate a surplus of amino acids, which is essential for intravacuolar proliferation. Therefore, the ankB mutant represents a novel genetic tool to determine the transcriptional response of human monocyte-derived macrophages (hMDMs to actively replicating L. pneumophila.Here, we utilized total human gene microarrays to determine the global transcriptional response of hMDMs to infection by wild type or the ankB mutant of L. pneumophila. The transcriptomes of hMDMs infected with either actively proliferating wild type or non-replicative ankB mutant bacteria were remarkably similar. The transcriptome of infected hMDMs was predominated by up-regulation of inflammatory pathways (IL-10 anti-inflammatory, interferon signaling and amphoterin signaling, anti-apoptosis, and down-regulation of protein synthesis pathways. In addition, L. pneumophila modulated diverse metabolic pathways, particularly those associated with bio-active lipid metabolism, and SLC amino acid transporters expression.Taken together, the hMDM transcriptional response to L. pneumophila is independent of intra-vacuolar replication of the bacteria and primarily involves modulation of the immune response and metabolic as well as nutritional pathways.

  17. Recognition and uptake of free and nanoparticle‐bound betalactoglobulin – a food allergen – by human monocytes

    DEFF Research Database (Denmark)

    Marengo, Mauro; Bonomi, Francesco; Iametti, Stefania

    2011-01-01

    Scope: To improve our understanding of the interaction of food allergens with cells of the immune system, the endocytosis by human monocytes of bovine β‐lactoglobulin (BLG) and ovomucoid (OM) – two major food allergens – and human serum albumin (HSA) was studied. Methods and results: BLG......, and HSA were conjugated to MNPs also labeled with a fluorescent probe. The uptake of these materials by human monocytes was monitored through flow cytometry, and compared with fluorescent MNPs and the free fluorescently labeled proteins, confirming higher uptake of the BLG‐conjugated MNPs versus non......‐conjugated MNPs. OM but not HSA conjugation to particles enhanced uptake of the MNPs. Confocal microscopy provided direct evidence of the actual internalization of BLG–MNP conjugates into the cytoplasm. Conclusions: These results contribute to the current understanding of the interaction between food allergens...

  18. Monocytic leukemias.

    Science.gov (United States)

    Shaw, M T

    1980-05-01

    The monocytic leukemias may be subdivided into acute monocytic leukemia, acute myelomonocytic leukemia, and subacute and chronic myelomonocytic leukemia. The clinical features of acute monocytic and acute myelomonocytic leukemias are similar and are manifestations of bone marrow failure. Gingival hypertrophy and skin infiltration are more frequent in acute monocytic leukemia. Cytomorphologically the blast cells in acute monocytic leukemia may be undifferentiated or differentiated, whereas in the acute myelomonocytic variety there are mixed populations of monocytic and myeloblastic cells. Cytochemical characteristics include strongly positive reactions for nonspecific esterase, inhibited by fluoride. The functional characteristics of acute monocytic and acute myelomonocytic cells resemble those of monocytes and include glass adherence and phagocytoses, the presence of Fc receptors for IgG and C'3, and the production of colony stimulating activity. Subacute and chronic myelomonocytic leukemias are insidious and slowly progressive diseases characterized by anemia and peripheral blood monocytosis. Atypical monocytes called paramyeloid cells are characteristic. The drugs used in the treatment of acute monocytic and acute myelomonocytic leukemias include cytosine arabinoside, the anthracyclines, and VP 16-213. Drug therapy in subacute and chronic myelomonocytic leukemias is not usually indicated, although VP 16-213 has been claimed to be effective.

  19. Isolation of human monocytes by double gradient centrifugation and their differentiation to macrophages in teflon-coated cell culture bags.

    Science.gov (United States)

    Menck, Kerstin; Behme, Daniel; Pantke, Mathias; Reiling, Norbert; Binder, Claudia; Pukrop, Tobias; Klemm, Florian

    2014-09-09

    Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

  20. Amelioration of Glucolipotoxicity-Induced Endoplasmic Reticulum Stress by a “Chemical Chaperone” in Human THP-1 Monocytes

    Directory of Open Access Journals (Sweden)

    Raji Lenin

    2012-01-01

    Full Text Available Chronic ER stress is emerging as a trigger that imbalances a number of systemic and arterial-wall factors and promote atherosclerosis. Macrophage apoptosis within advanced atherosclerotic lesions is also known to increase the risk of atherothrombotic disease. We hypothesize that glucolipotoxicity might mediate monocyte activation and apoptosis through ER stress. Therefore, the aims of this study are (a to investigate whether glucolipotoxicity could impose ER stress and apoptosis in THP-1 human monocytes and (b to investigate whether 4-Phenyl butyric acid (PBA, a chemical chaperone could resist the glucolipotoxicity-induced ER stress and apoptosis. Cells subjected to either glucolipotoxicity or tunicamycin exhibited increased ROS generation, gene and protein (PERK, GRP-78, IRE1α, and CHOP expression of ER stress markers. In addition, these cells showed increased TRPC-6 channel expression and apoptosis as revealed by DNA damage and increased caspase-3 activity. While glucolipotoxicity/tunicamycin increased oxidative stress, ER stress, mRNA expression of TRPC-6, and programmed the THP-1 monocytes towards apoptosis, all these molecular perturbations were resisted by PBA. Since ER stress is one of the underlying causes of monocyte dysfunction in diabetes and atherosclerosis, our study emphasize that chemical chaperones such as PBA could alleviate ER stress and have potential to become novel therapeutics.

  1. Functional contribution of elevated circulating and hepatic non-classical CD14CD16 monocytes to inflammation and human liver fibrosis.

    Directory of Open Access Journals (Sweden)

    Henning W Zimmermann

    Full Text Available BACKGROUND: Monocyte-derived macrophages critically perpetuate inflammatory responses after liver injury as a prerequisite for organ fibrosis. Experimental murine models identified an essential role for the CCR2-dependent infiltration of classical Gr1/Ly6C(+ monocytes in hepatic fibrosis. Moreover, the monocyte-related chemokine receptors CCR1 and CCR5 were recently recognized as important fibrosis modulators in mice. In humans, monocytes consist of classical CD14(+CD16(- and non-classical CD14(+CD16(+ cells. We aimed at investigating the relevance of monocyte subpopulations for human liver fibrosis, and hypothesized that 'non-classical' monocytes critically exert inflammatory as well as profibrogenic functions in patients during liver disease progression. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed circulating monocyte subsets from freshly drawn blood samples of 226 patients with chronic liver disease (CLD and 184 healthy controls by FACS analysis. Circulating monocytes were significantly expanded in CLD-patients compared to controls with a marked increase of the non-classical CD14(+CD16(+ subset that showed an activated phenotype in patients and correlated with proinflammatory cytokines and clinical progression. Correspondingly, CD14(+CD16(+ macrophages massively accumulated in fibrotic/cirrhotic livers, as evidenced by immunofluorescence and FACS. Ligands of monocyte-related chemokine receptors CCR2, CCR1 and CCR5 were expressed at higher levels in fibrotic and cirrhotic livers, while CCL3 and CCL4 were also systemically elevated in CLD-patients. Isolated monocyte/macrophage subpopulations were functionally characterized regarding cytokine/chemokine expression and interactions with primary human hepatic stellate cells (HSC in vitro. CD14(+CD16(+ monocytes released abundant proinflammatory cytokines. Furthermore, CD14(+CD16(+, but not CD14(+CD16(- monocytes could directly activate collagen-producing HSC. CONCLUSIONS/SIGNIFICANCE: Our data

  2. Bayesian spatio-temporal analysis and geospatial risk factors of human monocytic ehrlichiosis.

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    Ram K Raghavan

    Full Text Available Variations in spatio-temporal patterns of Human Monocytic Ehrlichiosis (HME infection in the state of Kansas, USA were examined and the relationship between HME relative risk and various environmental, climatic and socio-economic variables were evaluated. HME data used in the study was reported to the Kansas Department of Health and Environment between years 2005-2012, and geospatial variables representing the physical environment [National Land cover/Land use, NASA Moderate Resolution Imaging Spectroradiometer (MODIS], climate [NASA MODIS, Prediction of Worldwide Renewable Energy (POWER], and socio-economic conditions (US Census Bureau were derived from publicly available sources. Following univariate screening of candidate variables using logistic regressions, two Bayesian hierarchical models were fit; a partial spatio-temporal model with random effects and a spatio-temporal interaction term, and a second model that included additional covariate terms. The best fitting model revealed that spatio-temporal autocorrelation in Kansas increased steadily from 2005-2012, and identified poverty status, relative humidity, and an interactive factor, 'diurnal temperature range x mixed forest area' as significant county-level risk factors for HME. The identification of significant spatio-temporal pattern and new risk factors are important in the context of HME prevention, for future research in the areas of ecology and evolution of HME, and as well as climate change impacts on tick-borne diseases.

  3. Activation of the TREM-1 pathway in human monocytes by periodontal pathogens and oral commensal bacteria.

    Science.gov (United States)

    Varanat, M; Haase, E M; Kay, J G; Scannapieco, F A

    2017-08-01

    Periodontitis is a highly prevalent disease caused in part by an aberrant host response to the oral multi-species biofilm. A balance between the oral bacteria and host immunity is essential for oral health. Imbalances in the oral microbiome lead to an uncontrolled host inflammatory response and subsequent periodontal disease (i.e. gingivitis and periodontitis). TREM-1 is a signaling receptor present on myeloid cells capable of acting synergistically with other pattern recognition receptors leading to amplification of inflammatory responses. The aim of this study was to investigate the activation of the TREM-1 pathway in the human monocyte-like cell line THP-1 exposed to both oral pathogens and commensals. The relative expression of the genes encoding TREM-1 and its adapter protein DAP12 were determined by quantitative real-time polymerase chain reaction. The surface expression of TREM-1 was determined by flow cytometry. Soluble TREM-1 and cytokines were measured by enzyme-linked immunosorbent assay. The results demonstrate that both commensal and pathogenic oral bacteria activate the TREM-1 pathway, resulting in a proinflammatory TREM-1 activity-dependent increase in proinflammatory cytokine production. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. TLR2 ligands induce NF-κB activation from endosomal compartments of human monocytes.

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    Karim J Brandt

    Full Text Available Localization of Toll-like receptors (TLR in subcellular organelles is a major strategy to regulate innate immune responses. While TLR4, a cell-surface receptor, signals from both the plasma membrane and endosomal compartments, less is known about the functional role of endosomal trafficking upon TLR2 signaling. Here we show that the bacterial TLR2 ligands Pam3CSK4 and LTA activate NF-κB-dependent signaling from endosomal compartments in human monocytes and in a NF-κB sensitive reporter cell line, despite the expression of TLR2 at the cell surface. Further analyses indicate that TLR2-induced NF-κB activation is controlled by a clathrin/dynamin-dependent endocytosis mechanism, in which CD14 serves as an important upstream regulator. These findings establish that internalization of cell-surface TLR2 into endosomal compartments is required for NF-κB activation. These observations further demonstrate the need of endocytosis in the activation and regulation of TLR2-dependent signaling pathways.

  5. Pathway Interaction Network Analysis Identifies Dysregulated Pathways in Human Monocytes Infected by Listeria monocytogenes

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    Wufeng Fan

    2017-01-01

    Full Text Available In our study, we aimed to extract dysregulated pathways in human monocytes infected by Listeria monocytogenes (LM based on pathway interaction network (PIN which presented the functional dependency between pathways. After genes were aligned to the pathways, principal component analysis (PCA was used to calculate the pathway activity for each pathway, followed by detecting seed pathway. A PIN was constructed based on gene expression profile, protein-protein interactions (PPIs, and cellular pathways. Identifying dysregulated pathways from the PIN was performed relying on seed pathway and classification accuracy. To evaluate whether the PIN method was feasible or not, we compared the introduced method with standard network centrality measures. The pathway of RNA polymerase II pretranscription events was selected as the seed pathway. Taking this seed pathway as start, one pathway set (9 dysregulated pathways with AUC score of 1.00 was identified. Among the 5 hub pathways obtained using standard network centrality measures, 4 pathways were the common ones between the two methods. RNA polymerase II transcription and DNA replication owned a higher number of pathway genes and DEGs. These dysregulated pathways work together to influence the progression of LM infection, and they will be available as biomarkers to diagnose LM infection.

  6. Human β-Defensin 3 Reduces TNF-α-Induced Inflammation and Monocyte Adhesion in Human Umbilical Vein Endothelial Cells

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    Tianying Bian

    2017-01-01

    Full Text Available The aim of this study was to investigate the role of human β-defensin 3 (hBD3 in the initiation stage of atherosclerosis with human umbilical vein endothelial cells (HUVECs triggered by tumor necrosis factor- (TNF- α. The effects of hBD3 on TNF-α-induced endothelial injury and inflammatory response were evaluated. Our data revealed that first, hBD3 reduced the production of interleukin-6 (IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1, and macrophage migration inhibitory factor (MIF in HUVECs in a dose-dependent manner. In addition, hBD3 significantly prevented intracellular reactive oxygen species (ROS production by HUVECs. Second, western blot analysis demonstrated that hBD3 dose-dependently suppressed the protein levels of intracellular adhesion molecule-1 (ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1 in TNF-α-induced HUVECs. As a result, hBD3 inhibited monocyte adhesion to TNF-α-treated endothelial cells. Additionally, hBD3 suppressed TNF-α-induced F-actin reorganization in HUVECs. Third, hBD3 markedly inhibited NF-κB activation by decreasing the phosphorylation of IKK-α/β, IκB, and p65 subunit within 30 min. Moreover, the phosphorylation of p38 and c-Jun N-terminal protein kinase (JNK in the mitogen-activated protein kinase (MAPK pathway were also inhibited by hBD3 in HUVECs. In conclusion, hBD3 exerts anti-inflammatory and antioxidative effects in endothelial cells in response to TNF-α by inhibiting NF-κB and MAPK signaling.

  7. Augmented TLR2 expression on monocytes in both human Kawasaki disease and a mouse model of coronary arteritis.

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    I-Chun Lin

    Full Text Available BACKGROUND: Kawasaki disease (KD of unknown immunopathogenesis is an acute febrile systemic vasculitis and the leading cause of acquired heart diseases in childhood. To search for a better strategy for the prevention and treatment of KD, this study compared and validated human KD immunopathogenesis in a mouse model of Lactobacillus casei cell wall extract (LCWE-induced coronary arteritis. METHODS: Recruited subjects fulfilled the criteria of KD and were admitted for intravenous gamma globulin (IVIG treatment at the Kaohsiung Chang Gung Memorial Hospital from 2001 to 2009. Blood samples from KD patients were collected before and after IVIG treatment, and cardiovascular abnormalities were examined by transthoracic echocardiography. Wild-type male BALB/c mice (4-week-old were intraperitoneally injected with LCWE (1 mg/mL to induce coronary arteritis. The induced immune response in mice was examined on days 1, 3, 7, and 14 post injections, and histopathology studies were performed on days 7 and 14. RESULTS: Both human KD patients and LCWE-treated mice developed coronary arteritis, myocarditis, valvulitis, and pericarditis, as well as elevated plasma levels of interleukin (IL-2, IL-6, IL-10, monocyte chemoattractant protein (MCP-1, and tumor necrosis factor (TNF-α in acute phase. Most of these proinflammatory cytokines declined to normal levels in mice, whereas normal levels were achieved in patients only after IVIG treatment, with a few exceptions. Toll-like receptor (TLR-2, but not TLR4 surface enhancement on circulating CD14+ monocytes, was augmented in KD patients before IVIG treatment and in LCWE-treated mice, which declined in patients after IVIG treatment. CONCLUSION: This result suggests that that not only TLR2 augmentation on CD14+ monocytes might be an inflammatory marker for both human KD patients and LCWE-induced CAL mouse model but also this model is feasible for studying therapeutic strategies of coronary arteritis in human KD by

  8. Augmented TLR2 expression on monocytes in both human Kawasaki disease and a mouse model of coronary arteritis.

    Science.gov (United States)

    Lin, I-Chun; Kuo, Ho-Chang; Lin, Ying-Jui; Wang, Feng-Shen; Wang, Lin; Huang, Shun-Chen; Chien, Shao-Ju; Huang, Chien-Fu; Wang, Chih-Lu; Yu, Hong-Ren; Chen, Rong-Fu; Yang, Kuender D

    2012-01-01

    Kawasaki disease (KD) of unknown immunopathogenesis is an acute febrile systemic vasculitis and the leading cause of acquired heart diseases in childhood. To search for a better strategy for the prevention and treatment of KD, this study compared and validated human KD immunopathogenesis in a mouse model of Lactobacillus casei cell wall extract (LCWE)-induced coronary arteritis. Recruited subjects fulfilled the criteria of KD and were admitted for intravenous gamma globulin (IVIG) treatment at the Kaohsiung Chang Gung Memorial Hospital from 2001 to 2009. Blood samples from KD patients were collected before and after IVIG treatment, and cardiovascular abnormalities were examined by transthoracic echocardiography. Wild-type male BALB/c mice (4-week-old) were intraperitoneally injected with LCWE (1 mg/mL) to induce coronary arteritis. The induced immune response in mice was examined on days 1, 3, 7, and 14 post injections, and histopathology studies were performed on days 7 and 14. Both human KD patients and LCWE-treated mice developed coronary arteritis, myocarditis, valvulitis, and pericarditis, as well as elevated plasma levels of interleukin (IL)-2, IL-6, IL-10, monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF)-α in acute phase. Most of these proinflammatory cytokines declined to normal levels in mice, whereas normal levels were achieved in patients only after IVIG treatment, with a few exceptions. Toll-like receptor (TLR)-2, but not TLR4 surface enhancement on circulating CD14+ monocytes, was augmented in KD patients before IVIG treatment and in LCWE-treated mice, which declined in patients after IVIG treatment. This result suggests that that not only TLR2 augmentation on CD14+ monocytes might be an inflammatory marker for both human KD patients and LCWE-induced CAL mouse model but also this model is feasible for studying therapeutic strategies of coronary arteritis in human KD by modulating TLR2-mediated immune activation on CD14

  9. Importance of large conductance calcium-activated potassium channels (BKCa) in interleukin-1b-induced adhesion of monocytes to endothelial cells.

    Science.gov (United States)

    Burgazli, K M; Venker, C J; Mericliler, M; Atmaca, N; Parahuleva, M; Erdogan, A

    2014-01-01

    The present study investigated the role of the large conductance calcium-activated potassium channels (BKCa) in interleukin-1b (IL-1b) induced inflammation. Human umbilical vein endothelial cells (HUVECs) were isolated and cultured. Endothelial cell membrane potential measurements were accomplished using the fluorescent dye DiBAC4(3). The role of BKCa was assessed using iberiotoxin, a highly selective BKCa inhibitor. Changes in the calcium intracellular calcium were investigated using Fura-2-AM imaging. Fluorescent dyes DCF-AM and DAF-AM were further used in order to measure the formation of reactive oxygen species (ROS) and nitric oxide (NO) synthesis, respectively. Endothelial cell adhesion tests were conducted with BCECF-AM adhesion assay and tritium thymidine uptake using human monocytic cells (U937). Expression of cellular adhesion molecules (ICAM-1, VCAM-1) was determined by flow cytometer. Interleukin-1b induced a BKCa dependent hyperpolarization of HUVECs. This was followed by an increase in the intracellular calcium concentration. Furthermore, IL-1b significantly increased the synthesis of NO and ROS. The increase of intracellular calcium, radicals and NO resulted in a BKCa dependent adhesion of monocytes to HUVECs. Endothelial cells treated with IL-1b expressed both ICAM-1 and VCAM-1 in significantly higher amounts as when compared to controls. It was further shown that the cellular adhesion molecules ICAM-1 and VCAM-1 were responsible for the BKCa-dependent increase in cellular adhesion. Additionally, inhibition of the NADPH oxidase with DPI led to a significant downregulation of IL-1b-induced expression of ICAM and VCAM, as well as inhibition of eNOS by L-NMMA, and intracellular calcium by BAPTA. Activation of the endothelial BKCa plays an important role in the IL-1b-induced monocyte adhesion to endothelial cells.

  10. Elimination of clonogenic tumor cells from HL-60, Daudi, and U-937 cell lines by laser photoradiation therapy: implications for autologous bone marrow purging

    International Nuclear Information System (INIS)

    Gulliya, K.S.; Pervaiz, S.

    1989-01-01

    Laser photoradiation therapy was tested in an in vitro model for its efficacy in the elimination of non-Hodgkin's lymphoma cells. Results show that at 31.2 J/cm2 of laser light in the presence of 20 micrograms/mL of merocyanine 540 (MC540) there was greater than 5 log reduction in Burkitt's lymphoma (Daudi) cells. Similar tumor cell kill was obtained for leukemia (HL-60) cells at a laser light dose of 93.6 J/cm2. However, to obtain the same efficiency of killing for histiocytic lymphoma (U-937) cells, a higher dose of MC540 (25 micrograms/mL) was required. Clonogenic tumor stem cell colony formation was reduced by greater than 5 logs after laser photoradiation therapy. Under identical conditions for each cell line the percent survival for granulocyte-macrophage colony-forming units (CFU-GM, 45.9%, 40%, 17.5%), granulocyte/erythroid/macrophage/megakaryocyte (GEMM, 40.1%, 20.1%, 11.5%), colony-forming units (CFU-C, 16.2%, 9.1%, 1.8%), and erythroid burst-forming units (BFU-E, 33.4%, 17.8%, 3.9%) was significantly higher than the tumor cells. Mixing of gamma ray-irradiated normal marrow cells with tumor cells (1:1 and 10:1 ratio) did not interfere with the elimination of tumor cells. The effect of highly purified recombinant interferon alpha (rIFN) on laser photoradiation therapy of tumor cells was also investigated. In the presence of rIFN (30 to 3,000 U/mL), the viability of leukemic cells was observed to increase from 0% to 1.5% with a concurrent decrease in membrane polarization, suggesting an increase in fluidity of cell membrane in response to rIFN. However, at higher doses of rIFN (6,000 to 12,000 U/mL) this phenomenon was not observed. The viability of lymphoma cells remained unaffected at all doses of rIFN tested

  11. Uptake of [18F]fluorodeoxyglucose in human monocyte-macrophages in vitro

    International Nuclear Information System (INIS)

    Deichen, Jan Thiess; Prante, Olaf; Gack, Michaela; Schmiedehausen, Kristin; Kuwert, Torsten

    2003-01-01

    The fact that fluorine-18 fluorodeoxyglucose ([ 18 F]FDG) accumulates in inflammatory lesions as well as in tumours reduces the diagnostic specificity of positron emission tomography (PET) in oncology. The aim of this study was to characterise the uptake of [ 18 F]FDG in isolated human monocyte-macrophages (HMMs) in vitro in comparison with that in human glioblastoma (GLI) and pancreatic carcinoma cells (PAN). The purity of HMM preparations was determined by immunohistochemical staining and their functional integrity was assessed by long-term incubation with iodine-131 acetylated bovine serum albumin. [ 18 F]FDG uptake in HMMs was quantified as percent of whole [ 18 F]FDG activity per well (% ID) or as % ID in relation to total protein mass. [ 18 F]FDG uptake in HMMs significantly increased with culture duration, yielding 7.5%±0.9% (% ID/100 μg) at day 14. Stimulation by lipopolysaccharide further enhanced [ 18 F]FDG uptake in HMMs by a factor of 2. [ 18 F]FDG uptake significantly decreased with increasing glucose concentration in the medium. Radio-thin layer chromatography of intracellular metabolites revealed that [ 18 F]FDG was trapped by HMMs mainly as [ 18 F]FDG-6-phosphate and [ 18 F]FDG-1,6-diphosphate. [ 18 F]FDG uptake was in the range of uptake values measured in GLI and PAN. By accumulating [ 18 F]FDG in a manner analogous to uptake by tumour cells, activated HMMs may contribute to the [ 18 F]FDG uptake values measured by PET in neoplasms. (orig.)

  12. Effect of β-agonist on the dexamethasone-induced expression of aromatase by the human monocyte cells

    Directory of Open Access Journals (Sweden)

    Masatada Watanabe

    2017-02-01

    Full Text Available Emerging evidence suggests that sex steroids are important for human skin health. In particular, estrogen improves skin thickness, elasticity and moisture of older women. The major source of circulating estrogen is the ovary; however, local estrogen synthesis and secretion have important roles in, for example, bone metabolism and breast cancer development. We hypothesized that infiltrated peripheral monocytes are one of the sources of estrogen in skin tissues. We also hypothesized that, during atopic dermatitis under stress, a decline in the hypothalamus–pituitary–adrenal axis (HPA and facilitation of the (hypothalamus–sympathetic–adrenomedullary system (SAM attenuates estrogen secretion from monocytes. Based on this hypothesis, we tested aromatase expression in the human peripheral monocyte-derived cell line THP-1 in response to the synthetic glucocorticoid dexamethasone (Dex, the synthetic β-agonist isoproterenol (Iso and the β-antagonist propranolol (Pro. Dex mimics glucocorticoid secreted during excitation of the HPA, and Iso mimics catecholamine secreted during excitation of the SAM. We found that aromatase activity and the CYP19A1 gene transcript were both upregulated in THP-1 cells in the presence of Dex. Addition of Iso induced their downregulation and further addition of Pro rescued aromatase expression. These results may suggest that attenuation of estrogen secretion from peripheral monocytes could be a part of the pathology of stress-caused deterioration of atopic dermatitis. Further examination using an in vitro human skin model including THP-1 cells might be a valuable tool for investigating the therapeutic efficacy and mechanism of estrogen treatment for skin health.

  13. Suppression of blood monocyte and neutrophil chemotaxis in acute human malaria

    DEFF Research Database (Denmark)

    Nielsen, H; Kharazmi, A; Theander, T G

    1986-01-01

    tested monocyte chemotactic responsiveness in 19 patients with acute primary attack malaria. In addition, the neutrophil chemotaxis was measured in 12 patients. Before the initiation of antimalarial treatment a significant depression of monocyte chemotaxis was observed in approximately half...... of the patients when compared with healthy control subjects. The depression was found in Plasmodium falciparum malaria as well as in P. vivax or P. ovale malaria patients. The defective responsiveness was not receptor specific, since the responses towards casein and zymosan activated serum proved to be equally...... of treatment, and nearly normalized after 7 days (87% of controls). Furthermore, monocyte phagocytic and candidacidal activities were assessed in the same patients on admission and during the follow-up. In contrast to chemotaxis, these functions were normal in all of the patients whenever measured...

  14. Maturation of human dendritic cells by monocyte-conditioned medium is dependent upon trace amounts of lipopolysaccharide inducing tumour necrosis factor alpha

    DEFF Research Database (Denmark)

    Nersting, Jacob; Svenson, Morten; Andersen, Vagn

    2003-01-01

    We investigated the ability of monocyte-conditioned medium (MCM), generated by monocytes cultured on plastic-immobilised immunoglobulin, to stimulate maturation of human monocyte-derived dendritic cells (DC). Earlier reports suggest that MCM is a strong inducer of irreversible DC maturation......, whereas we find, that adding a small amount of lipopolysaccharide (LPS) to the MCM-generating cultures is required for the production of a DC-stimulatory MCM. Moreover, compared with addition of LPS directly to the DC cultures, stimulation via MCM cultures increases by several fold the DC...

  15. Induction of oxygen free radical generation in human monocytes by lipoprotein(a)

    DEFF Research Database (Denmark)

    Riis Hansen, P; Kharazmi, A; Jauhiainen, M

    1994-01-01

    The mechanism behind the association of elevated plasma lipoprotein(a) [Lp(a)] levels with atherosclerotic disease is unknown. In the present study, Lp(a) induced generation of oxygen free radicals by monocytes from selected healthy individuals in vitro. This observation may provide a link between...

  16. Cationic liposomal drug delivery system for specific targeting of human cd14+ monocytes in whole blood

    DEFF Research Database (Denmark)

    2013-01-01

    blood when compared to adherence to granulocytes, T-lymphocytes, B- lymphocytes and/or NK cells in freshly drawn blood, to a lipid-based pharmaceutical composition comprising said liposomes and their use in monocytic associated prophylaxis, treatment or amelioration of a condition such as cancer...

  17. Palmitate-induced inflammatory pathways in human adipose microvascular endothelial cells promote monocyte adhesion and impair insulin transcytosis.

    Science.gov (United States)

    Pillon, Nicolas J; Azizi, Paymon M; Li, Yujin E; Liu, Jun; Wang, Changsen; Chan, Kenny L; Hopperton, Kathryn E; Bazinet, Richard P; Heit, Bryan; Bilan, Philip J; Lee, Warren L; Klip, Amira

    2015-07-01

    Obesity is associated with inflammation and immune cell recruitment to adipose tissue, muscle and intima of atherosclerotic blood vessels. Obesity and hyperlipidemia are also associated with tissue insulin resistance and can compromise insulin delivery to muscle. The muscle/fat microvascular endothelium mediates insulin delivery and facilitates monocyte transmigration, yet its contribution to the consequences of hyperlipidemia is poorly understood. Using primary endothelial cells from human adipose tissue microvasculature (HAMEC), we investigated the effects of physiological levels of fatty acids on endothelial inflammation and function. Expression of cytokines and adhesion molecules was measured by RT-qPCR. Signaling pathways were evaluated by pharmacological manipulation and immunoblotting. Surface expression of adhesion molecules was determined by immunohistochemistry. THP1 monocyte interaction with HAMEC was measured by cell adhesion and migration across transwells. Insulin transcytosis was measured by total internal reflection fluorescence microscopy. Palmitate, but not palmitoleate, elevated the expression of IL-6, IL-8, TLR2 (Toll-like receptor 2), and intercellular adhesion molecule 1 (ICAM-1). HAMEC had markedly low fatty acid uptake and oxidation, and CD36 inhibition did not reverse the palmitate-induced expression of adhesion molecules, suggesting that inflammation did not arise from palmitate uptake/metabolism. Instead, inhibition of TLR4 to NF-κB signaling blunted palmitate-induced ICAM-1 expression. Importantly, palmitate-induced surface expression of ICAM-1 promoted monocyte binding and transmigration. Conversely, palmitate reduced insulin transcytosis, an effect reversed by TLR4 inhibition. In summary, palmitate activates inflammatory pathways in primary microvascular endothelial cells, impairing insulin transport and increasing monocyte transmigration. This behavior may contribute in vivo to reduced tissue insulin action and enhanced tissue

  18. Human platelet lysate is a successful alternative serum supplement for propagation of monocyte-derived dendritic cells.

    Science.gov (United States)

    Švajger, Urban

    2017-04-01

    Clinical protocols for dendritic cell (DC) generation from monocytes require the use of animal serum-free supplements. Serum-free media can also require up to 1% of serum supplementation. In addition, recommendations based on the 3Rs (Refinement, Reduction, Replacement) principle also recommend the use of non-animal sera in in vitro studies. The aim of this study was to explore the potential use of platelet lysate (PL) for generation of optimally differentiated DCs from monocytes. Cells were isolated from buffy coats from healthy volunteers using immunomagnetic selection. DCs were differentiated in RPMI1640 supplemented with either 10% fetal bovine serum (FBS), 10% AB serum or 10% PL with the addition of granulocyte monocyte colony stimulating factor and interleukin-4. Generated DCs were assessed for their morphology, viability, endocytotic capacity, surface phenotype (immature, mature and tolerogenic DCs) and activation of important signaling pathways. DC function was evaluated on the basis of their allostimulatory capacity, cytokine profile and ability to induce different T-helper subsets. DCs generated with PL displayed normal viability, morphology and endocytotic capacity. Their differentiation and maturation phenotype was comparable to FBS-cultured DCs. They showed functional plasticity and up-regulated tolerogenic markers in response to their environment. PL-cultured mature DCs displayed unhindered allostimulatory potential and the capacity to induce Th1 responses. The use of PL allowed for activation of crucial signaling proteins associated with DC differentiation and maturation. This study demonstrates for the first time that human PL represents a successful alternative to FBS in differentiation of DCs from monocytes. DCs display the major phenotypic and functional characteristics compared with existing culture protocols. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  19. Matrix metalloproteinase-12 gene regulation by a PPAR alpha agonist in human monocyte-derived macrophages

    International Nuclear Information System (INIS)

    Souissi, Imen Jguirim; Billiet, Ludivine; Cuaz-Perolin, Clarisse; Slimane, Mohamed-Naceur; Rouis, Mustapha

    2008-01-01

    MMP-12, a macrophage-specific matrix metalloproteinase with large substrate specificity, has been reported to be highly expressed in mice, rabbits and human atherosclerotic lesions. Increased MMP-12 from inflammatory macrophages is associated with several degenerative diseases such as atherosclerosis. In this manuscript, we show that IL-1β, a proinflammatory cytokine found in atherosclerotic plaques, increases both mRNA and protein levels of MMP-12 in human monocyte-derived macrophages (HMDM). Since peroxisome proliferator-activated receptors (PPARs), such as PPARα and PPARγ, are expressed in macrophages and because PPAR activation exerts an anti-inflammatory effect on vascular cells, we have investigated the effect of PPARα and γ isoforms on MMP-12 regulation in HMDM. Our results show that MMP-12 expression (mRNA and protein) is down regulated in IL-1β-treated macrophages only in the presence of a specific PPARα agonist, GW647, in a dose-dependent manner. In contrast, this inhibitory effect was abolished in IL-1β-stimulated peritoneal macrophages isolated from PPARα -/- mice and treated with the PPARα agonist, GW647. Moreover, reporter gene transfection experiments using different MMP-12 promoter constructs showed a reduction of the promoter activities by ∼ 50% in IL-1β-stimulated PPARα-pre-treated cells. However, MMP-12 promoter analysis did not reveal the presence of a PPRE response element. The IL-1β effect is known to be mediated through the AP-1 binding site. Mutation of the AP-1 site, located at - 81 in the MMP-12 promoter region relative to the transcription start site, followed by transfection analysis, gel shift and ChIP experiments revealed that the inhibitory effect was the consequence of the protein-protein interaction between GW 647-activated PPARα and c-Fos or c-Jun transcription factors, leading to inhibition of their binding to the AP-1 motif. These studies suggest that PPARα agonists may be used therapeutically, not only for lipid

  20. Robust and highly-efficient differentiation of functional monocytic cells from human pluripotent stem cells under serum- and feeder cell-free conditions.

    Directory of Open Access Journals (Sweden)

    Masakatsu D Yanagimachi

    Full Text Available Monocytic lineage cells (monocytes, macrophages and dendritic cells play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3 × 10(6 ± 0.3 × 10(6 floating monocytes from approximately 30 clusters of ESCs/iPSCs 5-6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.

  1. Lipopolysaccharide (LPS) stimulates fresh human monocytes to lyse actinomycin D-treated WEHI-164 target cells via increased secretion of a monokine similar to tumor necrosis factor

    International Nuclear Information System (INIS)

    Chen, A.R.; McKinnon, K.P.; Koren, H.S.

    1985-01-01

    The effects of lipopolysaccharide (LPS) on tumoricidal activity of human monocytes freshly isolated from peripheral blood were studied. Actinomycin D-treated WEHI-164 cells were used as targets because they are NK insensitive and are lysed rapidly by monocytes in 6-hr 51 Cr-release assays. Monocytes exhibited significant spontaneous activity without endotoxin. Monocytes either pretreated for 1 hr with LPS or assayed in the presence of LPS exhibited 100- to 1000-fold increased cytolytic activity. Cytolytic activity was heat labile and trypsin sensitive, and was recovered from Sepharose S-200 columns in a single peak with an apparent m.w. between 25,000 and 40,000. Actinomycin D or cycloheximide treatment of monocytes before the addition of LPS inhibited cytolytic monokine production. Cytolytic monokine activity was practically neutralized by specific rabbit antisera to human tumor necrosis factor (TNF). It was concluded that, although fresh human monocytes exhibit spontaneous tumoricidal activity, LPS is a potent activating agent. Its stimulatory effects depend on new transcription and translation and are mediated by enhanced secretion of a cytolytic monokine similar to TNF

  2. Unlike PPARγ, PPARα or PPARβ/δ activation does not promote human monocyte differentiation toward alternative macrophages

    International Nuclear Information System (INIS)

    Bouhlel, Mohamed Amine; Brozek, John; Derudas, Bruno; Zawadzki, Christophe; Jude, Brigitte; Staels, Bart; Chinetti-Gbaguidi, Giulia

    2009-01-01

    Macrophages adapt their response to micro-environmental signals. While Th1 cytokines promote pro-inflammatory M1 macrophages, Th2 cytokines promote an 'alternative' anti-inflammatory M2 macrophage phenotype. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors expressed in macrophages where they control the inflammatory response. It has been shown that PPARγ promotes the differentiation of monocytes into anti-inflammatory M2 macrophages in humans and mice, while a role for PPARβ/δ in this process has been reported only in mice and no data are available for PPARα. Here, we show that in contrast to PPARγ, expression of PPARα and PPARβ/δ overall does not correlate with the expression of M2 markers in human atherosclerotic lesions, whereas a positive correlation with genes of lipid metabolism exists. Moreover, unlike PPARγ, PPARα or PPARβ/δ activation does not influence human monocyte differentiation into M2 macrophages in vitro. Thus, PPARα and PPARβ/δ do not appear to modulate the alternative differentiation of human macrophages.

  3. [The effect of isoflurane on the secretion of TNF-alpha and IL-1 beta from LPS-stimulated human peripheral blood monocytes].

    Science.gov (United States)

    Sato, W; Enzan, K; Masaki, Y; Kayaba, M; Suzuki, M

    1995-07-01

    The cytokines such as tumor necrosis factor and interleukin-1 secreted from macrophages/monocytes proved to play important roles in the pathogenesis of endotoxemia, severe pancreatitis and other surgical injuries. However, it is still unclear how inhalational anesthetic agents influence the secretion of these cytokines from macrophages/monocytes. We investigated the effects of isoflurane on TNF-alpha and IL-1 beta secretions from human peripheral blood monocytes stimulated by lipopolysaccharide. TNF-alpha and IL-1 beta secretions increased after LPS stimulation and this increase was inhibited by isoflurane in dose-dependent fashion. The inhibitory action of isoflurane disappeared between 1 and 3 hours after stopping isoflurane inhalation. We concluded that isoflurane could inhibit TNF-alpha and IL-1 beta secretions from peripheral blood monocytes stimulated by LPS in a dose-dependent fashion and that the inhibitory action of isoflurane was reversible.

  4. Human circulating monocytes internalize 125I-insulin in a similar fashion to rat hepatocytes: relevance to receptor regulation in target and nontarget tissues

    International Nuclear Information System (INIS)

    Grunberger, G.; Robert, A.; Carpentier, J.L.; Dayer, J.M.; Roth, A.; Stevenson, H.C.; Orci, L.; Gorden, P.

    1985-01-01

    Circulating monocytes bind 125 I-insulin in a specific fashion and have been used to analyze the ambient receptor status in humans. When freshly isolated circulating monocytes are incubated with 125 I-insulin and examined by electron microscopic autoradiography, approximately 18% of the labeled material is internalized after 15 minutes at 37 degrees C. By 2 hours at 37 degrees C, approximately one half of the 125 I-insulin is internalized. Internalization occurs also at 15 degrees C but at a slower rate. Furthermore, the monocytes bind and internalize 125 I-insulin in a manner that mirrors that of major target tissues, such as rat hepatocytes. These data suggest that the insulin receptor of the circulating monocyte might be regulated by adsorptive endocytosis in a manner analogous to that of target tissue, such as the liver

  5. The Effect of Aggregatibacter actinomycetemcomitans Leukotoxin on Cell-to-Cell Communication in Human Monocytes

    OpenAIRE

    Hussein, Bejyan; Tamali, Ali

    2014-01-01

    Periodontitis is a complex and multifactorial dental disease and without proper treatment it eventually leads to the loss of teeth. Individuals with a higher number of Aggregatibacter actinomycetemcomitans in their micro-flora have a greater risk of developing periodontitis and the bacterium is associated with local aggressive periodontitis that affects younger populations. Aa produces a toxic virulent factor named Leukotoxin-A that can activate monocytes by a cellular chain reaction resultin...

  6. Immature dendritic cells generated from cryopreserved human monocytes show impaired ability to respond to LPS and to induce allogeneic lymphocyte proliferation.

    Directory of Open Access Journals (Sweden)

    Guilherme Ferreira Silveira

    Full Text Available Dendritic cells play a key role in the immune system, in the sensing of foreign antigens and triggering of an adaptive immune response. Cryopreservation of human monocytes was investigated to understand its effect on differentiation into immature monocyte-derived dendritic cells (imdDCs, the response to inflammatory stimuli and the ability to induce allogeneic lymphocyte proliferation. Cryopreserved (crp-monocytes were able to differentiate into imdDCs, albeit to a lesser extent than freshly (frh-obtained monocytes. Furthermore, crp-imdDCs had lower rates of maturation and cytokine/chemokine secretion in response to LPS than frh-imdDCs. Lower expression of Toll-like receptor 4 (at 24 and 48 h and higher susceptibility to apoptosis in crp-imdDCs than in fresh cells would account for the impaired maturation and cytokine/chemokine secretion observed. A mixed leukocyte reaction showed that lymphocyte proliferation was lower with crp-imdDCs than with frh-imdDCs. These findings suggested that the source of monocytes used to generate human imdDCs could influence the accuracy of results observed in studies of the immune response to pathogens, lymphocyte activation, vaccination and antigen sensing. It is not always possible to work with freshly isolated monocytes but the possible effects of freezing/thawing on the biology and responsiveness of imdDCs should be taken into account.

  7. Filarial excretory-secretory products induce human monocytes to produce lymphangiogenic mediators.

    Directory of Open Access Journals (Sweden)

    Tiffany Weinkopff

    2014-07-01

    Full Text Available The nematodes Wuchereria bancrofti and Brugia spp. infect over 120 million people worldwide, causing lymphedema, elephantiasis and hydrocele, collectively known as lymphatic filariasis. Most infected individuals appear to be asymptomatic, but many exhibit sub-clinical manifestations including the lymphangiectasia that likely contributes to the development of lymphedema and elephantiasis. As adult worm excretory-secretory products (ES do not directly activate lymphatic endothelial cells (LEC, we investigated the role of monocyte/macrophage-derived soluble factors in the development of filarial lymphatic pathology. We analyzed the production of IL-8, IL-6 and VEGF-A by peripheral blood mononuclear cells (PBMC from naïve donors following stimulation with filarial ES products. ES-stimulated PBMCs produced significantly more IL-8, IL-6 and VEGF-A compared to cells cultured in medium alone; CD14(+ monocytes appear to be the primary producers of IL-8 and VEGF-A, but not IL-6. Furthermore, IL-8, IL-6 and VEGF-A induced in vitro tubule formation in LEC Matrigel cultures. Matrigel plugs supplemented with IL-8, IL-6, VEGF-A, or with supernatants from ES-stimulated PBMCs and implanted in vivo stimulated lymphangiogenesis. Collectively, these data support the hypothesis that monocytes/macrophages exposed to filarial ES products may modulate lymphatic function through the secretion of soluble factors that stimulate the vessel growth associated with the pathogenesis of filarial disease.

  8. Terbinafine stimulates the pro-inflammatory responses in human monocytic THP-1 cells through an ERK signaling pathway.

    Science.gov (United States)

    Mizuno, Katsuhiko; Fukami, Tatsuki; Toyoda, Yasuyuki; Nakajima, Miki; Yokoi, Tsuyoshi

    2010-10-23

    Oral antifungal terbinafine has been reported to cause liver injury with inflammatory responses in a small percentage of patients. However the underlying mechanism remains unknown. To examine the inflammatory reactions, we investigated whether terbinafine and other antifungal drugs increase the release of pro-inflammatory cytokines using human monocytic cells. Dose- and time-dependent changes in the mRNA expression levels and the release of interleukin (IL)-8 and tumor necrosis factor (TNF)α from human monocytic THP-1 and HL-60 cells with antifungal drugs were measured. Effects of terbinafine on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK)1/2 were investigated. The release of IL-8 and TNFα from THP-1 and HL-60 cells was significantly increased by treatment with terbinafine but not by fluconazole, suggesting that terbinafine can stimulate monocytes and increase the pro-inflammatory cytokine release. Terbinafine also significantly increased the phosphorylation of ERK1/2 and p38 MAP kinase in THP-1 cells. Pretreatment with a MAP kinase/ERK kinase (MEK)1/2 inhibitor U0126 significantly suppressed the increase of IL-8 and TNFα levels by terbinafine treatment in THP-1 cells, but p38 MAPK inhibitor SB203580 did not. These results suggested that an ERK1/2 pathway plays an important role in the release of IL-8 and TNFα in THP-1 cells treated with terbinafine. The release of inflammatory mediators by terbinafine might be one of the mechanisms underlying immune-mediated liver injury. This in vitro method may be useful to predict adverse inflammatory reactions that lead to drug-induced liver injury. Copyright © 2010 Elsevier Inc. All rights reserved.

  9. From human monocytes to genome-wide binding sites--a protocol for small amounts of blood: monocyte isolation/ChIP-protocol/library amplification/genome wide computational data analysis.

    Directory of Open Access Journals (Sweden)

    Sebastian Weiterer

    Full Text Available Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next generation sequencing. We developed a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyse the distribution of histone modifications in a genome-wide manner.The protocol describes the whole work flow from isolating monocytes from human blood samples followed by a high-sensitivity and small-scale chromatin immunoprecipitation assay with guidance for generating libraries compatible with next generation sequencing from small amounts of immunoprecipitated DNA.

  10. RNA of Enterococcus faecalis Strain EC-12 Is a Major Component Inducing Interleukin-12 Production from Human Monocytic Cells.

    Directory of Open Access Journals (Sweden)

    Ryoichiro Nishibayashi

    Full Text Available Interleukin-12 (IL-12 is an important cytokine for the immunomodulatory effects of lactic acid bacteria (LAB. Using murine immune cells, we previously reported that the RNA of Enterococcus faecalis EC-12, a LAB strain exerting probiotic-like beneficial effects, is the major IL-12-inducing immunogenic component. However, it was recently revealed that bacterial RNA can be a ligand for Toll-like receptor (TLR 13, which is only expressed in mice. Because TLR13 is not expressed in humans, the immuno-stimulatory and -modulatory effects of LAB RNA in human cells should be augmented excluding TLR13 contribution. In experiment 1 of this study, the role of LAB RNA in IL-12 induction in human immune cells was studied using three LAB strains, E.faecalis EC-12, Lactobacillus gasseri JCM5344, and Bifidobacterium breve JCM1192. RNase A treatment of heat-killed LAB significantly decreased the IL-12 production of human peripheral blood mononuclear cells on stimulation, while RNase III treatment revealed virtually no effects. Further, IL-12 production against heat-killed E. faecalis EC-12 was abolished by depleting monocytes. These results demonstrated that single stranded RNA (ssRNA of LAB is a strong inducer of IL-12 production from human monocytes. In experiment 2, major receptor for ssRNA of E. faecalis EC-12 was identified using THP-1 cells, a human monocytic cell line. The type of RNA molecules of E. faecalis EC-12 responsible for IL-12 induction was also identified. IL-12 production induced by the total RNA of E. faecalis EC-12 was significantly reduced by the treatment of siRNA for TLR8 but not for TLR7. Furthermore, both 23S and 16S rRNA, but not mRNA, of E. faecalis EC-12 markedly induced IL-12 production from THP-1 cells. These results suggested that the recognition of ssRNA of E. faecalis EC-12 was mediated by TLR8 and that rRNA was the RNA molecule that exhibited IL-12-inducing ability in human cells.

  11. Ubiquinol decreases monocytic expression and DNA methylation of the pro-inflammatory chemokine ligand 2 gene in humans

    Directory of Open Access Journals (Sweden)

    Fischer Alexandra

    2012-10-01

    Full Text Available Abstract Background Coenzyme Q10 is an essential cofactor in the respiratory chain and serves in its reduced form, ubiquinol, as a potent antioxidant. Studies in vitro and in vivo provide evidence that ubiquinol reduces inflammatory processes via gene expression. Here we investigate the putative link between expression and DNA methylation of ubiquinol sensitive genes in monocytes obtained from human volunteers supplemented with 150 mg/ day ubiquinol for 14 days. Findings Ubiquinol decreases the expression of the pro-inflammatory chemokine (C-X-C motif ligand 2 gene (CXCL2 more than 10-fold. Bisulfite-/ MALDI-TOF-based analysis of regulatory regions of the CXCL2 gene identified six adjacent CpG islands which showed a 3.4-fold decrease of methylation status after ubiquinol supplementation. This effect seems to be rather gene specific, because ubiquinol reduced the expression of two other pro-inflammatory genes (PMAIP1, MMD without changing the methylation pattern of the respective gene. Conclusion In conclusion, ubiquinol decreases monocytic expression and DNA methylation of the pro-inflammatory CXCL2 gene in humans. Current Controlled Trials ISRCTN26780329.

  12. Differential regulation of TNF-α and IL-1β production from endotoxin stimulated human monocytes by phosphodiesterase inhibitors

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    K. L. Molnar-Kimber

    1992-01-01

    Full Text Available The effect of selective PDE-I (vinpocetine, PDE-III (milrinone, CI-930, PDE-IV (rolipram, nitroquazone, and PDE-V (zaprinast isozyme inhibitors on TNF-α and IL-1β production from LPS stimulated human monocytes was investigated. The PDE-IV inhibitors caused a concentration dependent inhibition of TNF-α production, but only partially inhibited IL-1β at high concentrations. High concentrations of the PDE-III inhibitors weakly inhibited TNF-α, but had no effect on IL-1β production. PDE-V inhibition was associated with an augmentation of cytokine secretion. Studies with combinations of PDE isozyme inhibitors indicated that PDE-III and PDE-V inhibitors modulate rolipram's suppression of TNF production in an additive manner. These data confirm that TNF-α and IL-1β production from LPS stimulated human monocytes are differentially regulated, and suggest that PDE-IV inhibitors have the potential to suppress TNF levels in man.

  13. Gefitinib and pyrrolidine dithiocarbamate decrease viral replication and cytokine production in dengue virus infected human monocyte cultures.

    Science.gov (United States)

    Duran, Anyelo; Valero, Nereida; Mosquera, Jesús; Fuenmayor, Edgard; Alvarez-Mon, Melchor

    2017-12-15

    The epidermal growth factor receptor (EGFR) and nucleotide-binding and oligomerization-domain containing 2 (NOD2) are important in cancer and in microbial recognition, respectively. These molecules trigger intracellular signaling pathways inducing the expression of inflammatory genes by NF-kB translocation. Gefitinib (GBTC) and pyrrolidine dithiocarbamate (PDTC) are capable of inhibiting EGFR/NOD2 and NF-kB, respectively. In earlier stages of dengue virus (DENV) infection, monocytes are capable of sustaining viral replication and increasing cytokine production, suggesting that monocyte/macrophages play an important role in early DENV replication. GBTC and PDTC have not been used to modify the pathogenesis of DENV in infected cells. This study was aimed to determine the effect of GBTC and PDTC on viral replication and cytokine production in DENV serotype 2 (DENV2)-infected human monocyte cultures. GBTC and PDTC were used to inhibit EGFR/NOD2 and NF-kB, respectively. Cytokine production was measured by ELISA and viral replication by plaque forming unit assay. Increased DENV2 replication and anti-viral cytokine production (IFN-α/β, TNF-α, IL-12 and IL-18) in infected cultures were found. These parameters were decreased after EGFR/NOD2 or NF-kB inhibitions. The inhibitory effects of GBTC and PDTC on viral replication and cytokine production can be beneficial in the treatment of patients infected by dengue and suggest a possible role of EGFR/NOD2 receptors and NF-kB in dengue pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. A rapid crosstalk of human gammadelta T cells and monocytes drives the acute inflammation in bacterial infections.

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    Matthias Eberl

    2009-02-01

    Full Text Available Vgamma9/Vdelta2 T cells are a minor subset of T cells in human blood and differ from other T cells by their immediate responsiveness to microbes. We previously demonstrated that the primary target for Vgamma9/Vdelta2 T cells is (E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP, an essential metabolite produced by a large range of pathogens. Here we wished to study the consequence of this unique responsiveness in microbial infection. The majority of peripheral Vgamma9/Vdelta2 T cells shares migration properties with circulating monocytes, which explains the presence of these two distinct blood cell types in the inflammatory infiltrate at sites of infection and suggests that they synergize in anti-microbial immune responses. Our present findings demonstrate a rapid and HMB-PP-dependent crosstalk between Vgamma9/Vdelta2 T cells and autologous monocytes that results in the immediate production of inflammatory mediators including the cytokines interleukin (IL-6, interferon (IFN-gamma, tumor necrosis factor (TNF-alpha, and oncostatin M (OSM; the chemokines CCL2, CXCL8, and CXCL10; and TNF-related apoptosis-inducing ligand (TRAIL. Moreover, under these co-culture conditions monocytes differentiate within 18 hours into inflammatory dendritic cells (DCs with antigen-presenting functions. Addition of further microbial stimuli (lipopolysaccharide, peptidoglycan induces CCR7 and enables these inflammatory DCs to trigger the generation of CD4(+ effector alphabeta T cells expressing IFN-gamma and/or IL-17. Importantly, our in vitro model replicates the responsiveness to microbes of effluent cells from peritoneal dialysis (PD patients and translates directly to episodes of acute PD-associated bacterial peritonitis, where Vgamma9/Vdelta2 T cell numbers and soluble inflammatory mediators are elevated in patients infected with HMB-PP-producing pathogens. Collectively, these findings suggest a direct link between invading pathogens, microbe

  15. Mitochondrial DAMPs induce endotoxin tolerance in human monocytes: an observation in patients with myocardial infarction.

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    Irene Fernández-Ruiz

    Full Text Available Monocyte exposure to mitochondrial Danger Associated Molecular Patterns (DAMPs, including mitochondrial DNA (mtDNA, induces a transient state in which these cells are refractory to further endotoxin stimulation. In this context, IRAK-M up-regulation and impaired p65 activity were observed. This phenomenon, termed endotoxin tolerance (ET, is characterized by decreased production of cytokines in response to the pro-inflammatory stimulus. We also show that monocytes isolated from patients with myocardial infarction (MI exhibited high levels of circulating mtDNA, which correlated with ET status. Moreover, a significant incidence of infection was observed in those patients with a strong tolerant phenotype. The present data extend our current understanding of the implications of endotoxin tolerance. Furthermore, our data suggest that the levels of mitochondrial antigens in plasma, such as plasma mtDNA, should be useful as a marker of increased risk of susceptibility to nosocomial infections in MI and in other pathologies involving tissue damage.

  16. Gallic Acid Decreases Inflammatory Cytokine Secretion Through Histone Acetyltransferase/Histone Deacetylase Regulation in High Glucose-Induced Human Monocytes.

    Science.gov (United States)

    Lee, Wooje; Lee, Sang Yeol; Son, Young-Jin; Yun, Jung-Mi

    2015-07-01

    Hyperglycemia contributes to diabetes and several diabetes-related complications. Gallic acid is a polyhydroxy phenolic compound found in various natural products. In this study, we investigated the effects and mechanism of gallic acid on proinflammatory cytokine secretion in high glucose-induced human monocytes (THP-1 cells). THP-1 cells were cultured under normoglycemic or hyperglycemic conditions, in the absence or presence of gallic acid. Hyperglycemic conditions significantly induced histone acetylation, nuclear factor-κB (NF-κB) activation, and proinflammatory cytokine release from THP-1 cells, whereas gallic acid suppressed NF-κB activity and cytokine release. It also significantly reduced CREB-binding protein/p300 (CBP/p300, a NF-κB coactivator) gene expression, acetylation levels, and CBP/p300 histone acetyltransferase (HAT) activity. In addition, histone deacetylase 2 (HDAC2) expression was significantly induced. These results suggest that gallic acid inhibits hyperglycemic-induced cytokine production in monocytes through epigenetic changes involving NF-κB. Therefore, gallic acid may have potential for the treatment and prevention of diabetes and its complications.

  17. An in vitro model for dengue virus infection that exhibits human monocyte infection, multiple cytokine production and dexamethasone immunomodulation

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    Sônia Regina Nogueira Ignácio Reis

    2007-12-01

    Full Text Available An important cytokine role in dengue fever pathogenesis has been described. These molecules can be associated with haemorrhagic manifestations, coagulation disorders, hypotension and shock, all symptoms implicated in vascular permeability and disease worsening conditions. Several immunological diseases have been treated by cytokine modulation and dexamethasone is utilized clinically to treat pathologies with inflammatory and autoimmune ethiologies. We established an in vitro model with human monocytes infected by dengue virus-2 for evaluating immunomodulatory and antiviral activities of potential pharmaceutical products. Flow cytometry analysis demonstrated significant dengue antigen detection in target cells two days after infection. TNF-alpha, IFN-alpha, IL-6 and IL-10 are produced by in vitro infected monocytes and are significantly detected in cell culture supernatants by multiplex microbead immunoassay. Dexamethasone action was tested for the first time for its modulation in dengue infection, presenting optimistic results in both decreasing cell infection rates and inhibiting TNF-alpha, IFN-alpha and IL-10 production. This model is proposed for novel drug trials yet to be applyed for dengue fever.

  18. Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cytokine release from monocytes.

    Science.gov (United States)

    Rasmussen, P B; Bjørn, S; Hastrup, S; Nielsen, P F; Norris, K; Thim, L; Wiberg, F C; Flodgaard, H

    1996-07-15

    Neutrophil-derived heparin-binding protein (HBP) is a strong chemoattractant for monocytes. We report here for the first time the expression of recombinant HBP. A baculovirus containing the human HBP cDNA mediated in insect cells the secretion of a 7-residue N-terminally extended HBP form (pro-HBP). Deletion of the pro-peptide-encoding cDNA sequence resulted in correctly processed HBP at the N-terminus. Electrospray mass spectrum analysis of recombinant HBP yielded a molecular weight of 27.237 +/- 3 amu. Consistent with this mass is a HBP form of 225 amino acids (mature part +3 amino acid C-terminal extension). The biological activity of recombinant HBP was confirmed by its chemotactic action towards monocytes. Furthermore, we have shown that recombinant HBP stimulates in a dose-dependent manner the lipopolysaccharide (LPS)-induced cytokine release from human monocytes.

  19. Outer membrane vesicles from Brucella abortus promote bacterial internalization by human monocytes and modulate their innate immune response.

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    Cora N Pollak

    Full Text Available Outer membrane vesicles (OMVs released by some gram-negative bacteria have been shown to exert immunomodulatory effects that favor the establishment of the infection. The aim of the present study was to assess the interaction of OMVs from Brucella abortus with human epithelial cells (HeLa and monocytes (THP-1, and the potential immunomodulatory effects they may exert. Using confocal microscopy and flow cytometry, FITC-labeled OMVs were shown to be internalized by both cell types. Internalization was shown to be partially mediated by clathrin-mediated endocytosis. Pretreatment of THP-1 cells with Brucella OMVs inhibited some cytokine responses (TNF-α and IL-8 to E. coli LPS, Pam3Cys or flagellin (TLR4, TLR2 and TLR5 agonists, respectively. Similarly, pretreatment with Brucella OMVs inhibited the cytokine response of THP-1 cells to B. abortus infection. Treatment of THP-1 cells with OMVs during IFN-γ stimulation reduced significantly the inducing effect of this cytokine on MHC-II expression. OMVs induced a dose-dependent increase of ICAM-1 expression on THP-1 cells and an increased adhesion of these cells to human endothelial cells. The addition of OMVs to THP-1 cultures before the incubation with live B. abortus resulted in increased numbers of adhered and internalized bacteria as compared to cells not treated with OMVs. Overall, these results suggest that OMVs from B. abortus exert cellular effects that promote the internalization of these bacteria by human monocytes, but also downregulate the innate immune response of these cells to Brucella infection. These effects may favor the persistence of Brucella within host cells.

  20. Glucocorticoid mediated regulation of inflammation in human monocytes is associated with depressive mood and obesity.

    Science.gov (United States)

    Cheng, Tiefu; Dimitrov, Stoyan; Pruitt, Christopher; Hong, Suzi

    2016-04-01

    Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is observed in various conditions, including depression and obesity, which are also often related. Glucocorticoid (GC) resistance and desensitization of peripheral GC receptors (GRs) are often the case in HPA dysregulation seen in depression, and GC plays a critical role in regulation of inflammation. Given the growing evidence that inflammation is a central feature of some depression cases and obesity, we aimed to investigate the immune-regulatory role of GC-GR in relation to depressive mood and obesity in 35 healthy men and women. Depressive mood and level of obesity were assessed, using Beck Depression Inventory (BDI-Ia) and body mass index (BMI), respectively. We measured plasma cortisol levels via enzyme-linked immunosorbent assay and lipopolysaccharide-stimulated intracellular tumor necrosis factor (TNF) production by monocytes, using flow cytometry. Cortisol sensitivity was determined by the difference in monocytic TNF production between the conditions of 1 and 0 μM cortisol incubation ("cortisol-mediated inflammation regulation, CoMIR"). GR vs. mineralocorticoid receptor (MR) antagonism for CoMIR was examined by using mifepristone and spironolactone. A series of multiple regression analyses were performed to investigate independent contribution of depressive mood vs. obesity after controlling for age, gender, systolic blood pressure (SBP), and plasma cortisol in predicting CoMIR. CoMIR was explained by somatic subcomponents of depressive mood (BDI-S: β=-0.499, p=0.001), or BMI (β=-0.466, pcortisol dose (1 μM). There was initial indication that greater obesity and somatic depressive symptoms were associated with smaller efficacy of the blockers, which warrants further investigation. Our findings, although in a preclinical sample, signify the shared pathophysiology of immune dysregulation in depression and obesity and warrant further mechanistic investigation. Published by Elsevier Ltd.

  1. Similarities and differences between helminth parasites and cancer cell lines in shaping human monocytes: Insights into parallel mechanisms of immune evasion.

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    Prakash Babu Narasimhan

    2018-04-01

    Full Text Available A number of features at the host-parasite interface are reminiscent of those that are also observed at the host-tumor interface. Both cancer cells and parasites establish a tissue microenvironment that allows for immune evasion and may reflect functional alterations of various innate cells. Here, we investigated how the phenotype and function of human monocytes is altered by exposure to cancer cell lines and if these functional and phenotypic alterations parallel those induced by exposure to helminth parasites. Thus, human monocytes were exposed to three different cancer cell lines (breast, ovarian, or glioblastoma or to live microfilariae (mf of Brugia malayi-a causative agent of lymphatic filariasis. After 2 days of co-culture, monocytes exposed to cancer cell lines showed markedly upregulated expression of M1-associated (TNF-α, IL-1β, M2-associated (CCL13, CD206, Mreg-associated (IL-10, TGF-β, and angiogenesis associated (MMP9, VEGF genes. Similar to cancer cell lines, but less dramatically, mf altered the mRNA expression of IL-1β, CCL13, TGM2 and MMP9. When surface expression of the inhibitory ligands PDL1 and PDL2 was assessed, monocytes exposed to both cancer cell lines and to live mf significantly upregulated PDL1 and PDL2 expression. In contrast to exposure to mf, exposure to cancer cell lines increased the phagocytic ability of monocytes and reduced their ability to induce T cell proliferation and to expand Granzyme A+ CD8+ T cells. Our data suggest that despite the fact that helminth parasites and cancer cell lines are extraordinarily disparate, they share the ability to alter the phenotype of human monocytes.

  2. Similarities and differences between helminth parasites and cancer cell lines in shaping human monocytes: Insights into parallel mechanisms of immune evasion.

    Science.gov (United States)

    Narasimhan, Prakash Babu; Akabas, Leor; Tariq, Sameha; Huda, Naureen; Bennuru, Sasisekhar; Sabzevari, Helen; Hofmeister, Robert; Nutman, Thomas B; Tolouei Semnani, Roshanak

    2018-04-01

    A number of features at the host-parasite interface are reminiscent of those that are also observed at the host-tumor interface. Both cancer cells and parasites establish a tissue microenvironment that allows for immune evasion and may reflect functional alterations of various innate cells. Here, we investigated how the phenotype and function of human monocytes is altered by exposure to cancer cell lines and if these functional and phenotypic alterations parallel those induced by exposure to helminth parasites. Thus, human monocytes were exposed to three different cancer cell lines (breast, ovarian, or glioblastoma) or to live microfilariae (mf) of Brugia malayi-a causative agent of lymphatic filariasis. After 2 days of co-culture, monocytes exposed to cancer cell lines showed markedly upregulated expression of M1-associated (TNF-α, IL-1β), M2-associated (CCL13, CD206), Mreg-associated (IL-10, TGF-β), and angiogenesis associated (MMP9, VEGF) genes. Similar to cancer cell lines, but less dramatically, mf altered the mRNA expression of IL-1β, CCL13, TGM2 and MMP9. When surface expression of the inhibitory ligands PDL1 and PDL2 was assessed, monocytes exposed to both cancer cell lines and to live mf significantly upregulated PDL1 and PDL2 expression. In contrast to exposure to mf, exposure to cancer cell lines increased the phagocytic ability of monocytes and reduced their ability to induce T cell proliferation and to expand Granzyme A+ CD8+ T cells. Our data suggest that despite the fact that helminth parasites and cancer cell lines are extraordinarily disparate, they share the ability to alter the phenotype of human monocytes.

  3. The alpha hemolisina of Escherichia Coli induces increases in the calcium citoplasmico of neutrofilos and monocytes human beings

    International Nuclear Information System (INIS)

    Garcia, J.

    2000-01-01

    Escherichia coli alpha hemolysin (AH) and the calcium ionophores ionomycin and 4 Br A23187 caused increases in cell fluorescence, indicative of elevations in cytoplasmic calcium, in fura 2-loaded human polymorphonuclear leukocytes(PMN) and monocytes (MN). The increase in fluorescence caused by AH was dose dependent. Quelation of extracellular calcium with EGTA prevented fluorescence increases in PMN exposed to 2 HU50/ml AH, but did not prevent a small increase in 4 μM, ionomycin-treated PMN, indicating that ionomycin treatment under conditions of calcium quelation can mobilize calcium from internal stores, and that entry of external calcium accounts for most of the increases in cell fluorescence in cells treated with both AH and calcium ionophores. AH, as well as calcium ionophores and the chemotactic peptide FMLP caused rease of myeloperoxidase (MPO) from PMM suggesting that increments in intracellular calcium cause degramulation with release of granule contents (Author) [es

  4. Gram-negative, but not Gram-positive, bacteria elicit strong PGE2 production in human monocytes.

    Science.gov (United States)

    Hessle, Christina C; Andersson, Bengt; Wold, Agnes E

    2003-12-01

    Gram-positive and Gram-negative bacteria induce different cytokine patterns in human mononuclear cells. We have seen that Gram-positives preferentially induce IL-12 and TNF-alpha, whereas Gram-negatives induce more IL-10, IL-6, and IL-8. In this study, we compared the capacity of these two groups of bacteria to induce PGE2. Monocytes stimulated with Gram-negative bacterial species induced much more PGE2 than did Gram-positive bacteria (5600 +/- 330 vs. 1700 +/- 670 pg/mL, p Gram-positive and Gram-negative bacteria. We suggest that Gram-positive and Gram-negative bacteria may stimulate different innate effector functions; Gram-positive bacteria promoting cell-mediated effector functions whereas Gram-negative bacteria inducing mediators inhibiting the same.

  5. Efficient, long term production of monocyte-derived macrophages from human pluripotent stem cells under partly-defined and fully-defined conditions.

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    Bonnie van Wilgenburg

    Full Text Available Human macrophages are specialised hosts for HIV-1, dengue virus, Leishmania and Mycobacterium tuberculosis. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens, because available myeloid cell lines are, by definition, not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined, feeder- and serum-free culture conditions and produces very consistent, pure, high yields across both human Embryonic Stem Cell (hESC and multiple human induced Pluripotent Stem Cell (hiPSC lines over time periods of up to one year. Cumulatively, up to ∼3×10(7 monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14(+, CD16(low, CD163(+. Differentiation with M-CSF produces macrophages that are highly phagocytic, HIV-1-infectable, and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate, as they recognise foreign nucleic acids; the lentivector system described here overcomes this, as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells, surmounting issues of transgene silencing. Overall, the method we describe here is an efficient, effective, scalable system for the reproducible production and genetic modification of human macrophages, facilitating the interrogation of human macrophage biology.

  6. Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines

    Science.gov (United States)

    Rojnik, Matija; Jevnikar, Zala; Mirkovic, Bojana; Janes, Damjan; Zidar, Nace; Kikelj, Danijel; Kos, Janko

    2011-01-01

    Background Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines. Materials and methods BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing. Results In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity. Conclusions Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression. PMID:22933963

  7. Soluble vascular endothelial growth factor (VEGF) receptor-1 inhibits migration of human monocytic THP-1 cells in response to VEGF.

    Science.gov (United States)

    Zhu, Cansheng; Xiong, Zhaojun; Chen, Xiaohong; Lu, Zhengqi; Zhou, Guoyu; Wang, Dunjing; Bao, Jian; Hu, Xueqiang

    2011-08-01

    We aimed to investigate the regulation and contribution of vascular endothelial growth factor (VEGF) and sFlt-1(1-3) to human monocytic THP-1 migration. Ad-sFlt-1/FLAG, a recombinant adenovirus carrying the human sFlt-1(1-3) (the first three extracellular domains of FLT-1, the hVEGF receptor-1) gene, was constructed. L929 cells were infected with Ad-sFlt-1/FLAG and the expression of sFlt-1 was detected by immunofluorescent assay and ELISA. Corning(®) Transwell(®) Filter Inserts containing polyethylene terephthalate (PET) membranes with pore sizes of 3 μm were used as an experimental model to simulate THP-1 migration. Five VEGF concentrations (0, 0.1, 1, 10 and 100 ng/ml), four concentrations of sFlt-1(1-3)/FLAG expression supernatants (0.1, 1, 10 and 100 ng/ml), and monocyte chemoattractant protein-1 (MCP-1, 10 ng/ml) were used to test the ability of THP-1 cells to migrate through PET membranes. The sFlt-1(1-3) gene was successfully recombined into Ad-sFlt-1/FLAG. sFlt-1(1-3) was expressed in L929 cells transfected with Ad-sFlt-1/FLAG. THP-1 cell migration increased with increasing concentrations of VEGF, while cell migration decreased with increasing concentrations of sFlt1(1-3)/FLAG. sFlt1(1-3)/FLAG had no effect on MCP-1-induced cell migration. This study demonstrated that VEGF is able to elicit a migratory response in THP-1 cells, and that sFlt-1(1-3) is an effective inhibitor of THP-1 migration towards VEGF.

  8. Expression of adhesion molecules, monocyte interactions and oxidative stress in human endothelial cells exposed to wood smoke and diesel exhaust particulate matter

    DEFF Research Database (Denmark)

    Forchhammer, Lykke; Loft, Steffen; Roursgaard, Martin

    2012-01-01

    Toxicological effects of wood smoke particles are less investigated than traffic-related combustion particles. We investigated the effect of wood smoke particles, generated by smouldering combustion conditions, on human umbilical endothelial cells (HUVECs) co-cultured with or without monocytic TH...

  9. Glutamine and alanine-induced differential expression of intracellular IL-6, IL-8, and TNF-α in LPS-stimulated monocytes in human whole-blood.

    Science.gov (United States)

    Raspé, C; Czeslick, E; Weimann, A; Schinke, C; Leimert, A; Kellner, P; Simm, A; Bucher, M; Sablotzki, A

    2013-04-01

    To investigate the effects of the commonly-used immunomodulators l-glutamine, l-alanine, and the combination of both l-alanyl-l-glutamine (Dipeptamin(®)) on intracellular expression of IL-6, IL-8, and TNF-α during endotoxemia, lipopolysaccharide (LPS)-stimulated human monocytes in a whole blood system were investigated by flow cytometry. Whole blood of twenty-seven healthy volunteers was stimulated with LPS and incubated with three different amino acid solutions (1. l-glutamine, 2. l-alanine, 3. l-alanyl-l-glutamine, each concentration 2 mM, 5 mM, incubation time 3 h). CD14(+) monocytes were phenotyped in whole-blood and intracellular expression of cytokines was assessed by flow cytometry. Our investigations showed for the first time in whole blood probes, imitating best physiologically present cellular interactions, that l-glutamine caused a dose-independent inhibitory effect on IL-6 and TNF-α production in human monocytes stimulated with LPS. However, l-alanine had contrary effects on IL-6 expression, significantly upregulating expression of IL-6 in LPS-treated monocytes. The impact of l-alanine on the expression of TNF-α was comparable with glutamine. Neither amino acid was able to affect IL-8 production in LPS-stimulated monocytes. The combination of both did not influence significantly IL-6 and IL-8 expression in monocytes during endotoxemia, however strongly reduced TNF-α production. For the regulation of TNF-α, l-glutamine, l-alanine and the combination of both show a congruent and exponentiated downregulating effect during endotoxemia, for the modulation of IL-6, l-glutamine and l-alanine featured opposite regulation leading to a canceling impact of each other when recombining both amino acids. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Ureaplasma Species Differentially Modulate Pro- and Anti-Inflammatory Cytokine Responses in Newborn and Adult Human Monocytes Pushing the State Toward Pro-Inflammation

    Science.gov (United States)

    Glaser, Kirsten; Silwedel, Christine; Fehrholz, Markus; Waaga-Gasser, Ana M.; Henrich, Birgit; Claus, Heike; Speer, Christian P.

    2017-01-01

    Background: Ureaplasma species have been associated with chorioamnionitis and preterm birth and have been implicated in the pathogenesis of neonatal short and long-term morbidity. However, being mostly commensal bacteria, controversy remains on the pro-inflammatory capacity of Ureaplasma. Discussions are ongoing on the incidence and impact of prenatal, perinatal, and postnatal infection. The present study addressed the impact of Ureaplasma isolates on monocyte-driven inflammation. Methods: Cord blood monocytes of term neonates and adult monocytes, either native or LPS-primed, were cultured with Ureaplasma urealyticum (U. urealyticum) serovar 8 (Uu8) and Ureaplasma parvum serovar 3 (Up3). Using qRT-PCR, cytokine flow cytometry, and multi-analyte immunoassay, we assessed mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8, IL-12p40, IL-10, and IL-1 receptor antagonist (IL-1ra) as well as Toll-like receptor (TLR) 2 and TLR4. Results: Uu8 and Up3 induced mRNA expression and protein release of TNF-α, IL-1β and IL-8 in term neonatal and adult monocytes (p Ureaplasma-stimulated cells paralleled those results. Ureaplasma-induced cytokine levels did not significantly differ from LPS-mediated levels except for lower intracellular IL-1β in adult monocytes (Uu8: p ureaplasmas did not induce IL-12p40 response and promoted lower amounts of anti-inflammatory IL-10 and IL-1ra than LPS, provoking a cytokine imbalance more in favor of pro-inflammation (IL-1β/IL-10, IL-8/IL-10 and IL-8/IL-1ra: p Ureaplasma isolates in human monocytes. Stimulating pro-inflammatory cytokine responses while hardly inducing immunomodulatory and anti-inflammatory cytokines, ureaplasmas might push monocyte immune responses toward pro-inflammation. Inhibition of LPS-induced cytokines in adult monocytes in contrast to sustained inflammation in term neonatal monocytes indicates a differential modulation of host immune responses to a second stimulus. Modification of

  11. Redox Stimulation of Human THP-1 Monocytes in Response to Cold Physical Plasma

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    Sander Bekeschus

    2016-01-01

    Full Text Available In plasma medicine, cold physical plasma delivers a delicate mixture of reactive components to cells and tissues. Recent studies suggested a beneficial role of cold plasma in wound healing. Yet, the biological processes related to the redox modulation via plasma are not fully understood. We here used the monocytic cell line THP-1 as a model to test their response to cold plasma in vitro. Intriguingly, short term plasma treatment stimulated cell growth. Longer exposure only modestly compromised cell viability but apparently supported the growth of cells that were enlarged in size and that showed enhanced metabolic activity. A significantly increased mitochondrial content in plasma treated cells supported this notion. On THP-1 cell proteome level, we identified an increase of protein translation with key regulatory proteins being involved in redox regulation (hypoxia inducible factor 2α, differentiation (retinoic acid signaling and interferon inducible factors, and cell growth (Yin Yang 1. Regulation of inflammation is a key element in many chronic diseases, and we found a significantly increased expression of the anti-inflammatory heme oxygenase 1 (HMOX1 and of the neutrophil attractant chemokine interleukin-8 (IL-8. Together, these results foster the view that cold physical plasma modulates the redox balance and inflammatory processes in wound related cells.

  12. Redox Stimulation of Human THP-1 Monocytes in Response to Cold Physical Plasma.

    Science.gov (United States)

    Bekeschus, Sander; Schmidt, Anke; Bethge, Lydia; Masur, Kai; von Woedtke, Thomas; Hasse, Sybille; Wende, Kristian

    2016-01-01

    In plasma medicine, cold physical plasma delivers a delicate mixture of reactive components to cells and tissues. Recent studies suggested a beneficial role of cold plasma in wound healing. Yet, the biological processes related to the redox modulation via plasma are not fully understood. We here used the monocytic cell line THP-1 as a model to test their response to cold plasma in vitro. Intriguingly, short term plasma treatment stimulated cell growth. Longer exposure only modestly compromised cell viability but apparently supported the growth of cells that were enlarged in size and that showed enhanced metabolic activity. A significantly increased mitochondrial content in plasma treated cells supported this notion. On THP-1 cell proteome level, we identified an increase of protein translation with key regulatory proteins being involved in redox regulation (hypoxia inducible factor 2α), differentiation (retinoic acid signaling and interferon inducible factors), and cell growth (Yin Yang 1). Regulation of inflammation is a key element in many chronic diseases, and we found a significantly increased expression of the anti-inflammatory heme oxygenase 1 (HMOX1) and of the neutrophil attractant chemokine interleukin-8 (IL-8). Together, these results foster the view that cold physical plasma modulates the redox balance and inflammatory processes in wound related cells.

  13. Characterization of two types of osteoclasts from human peripheral blood monocytes

    International Nuclear Information System (INIS)

    Yuasa, Kimitaka; Mori, Kouki; Ishikawa, Hitoshi; Sudo, Akihiro; Uchida, Atsumasa; Ito, Yasuhiko

    2007-01-01

    The two osteoclastogenesis pathways, receptor activator nuclear factor (NF)-κB ligand (RANKL)-mediated and fusion regulatory protein-1 (FRP-1)-mediated osteoclastogenesis, have recently been reported. There were significant differences in differentiation and activation mechanisms between the two pathways. When monocytes were cultured with FRP-1 without adding M-CSF, essential for the RANKL system, TRAP-positive polykaryocyte formation occurred. FRP-1-mediated osteoclasts formed larger pits on mineralized calcium phosphate plates than RANKL+M-CSF-mediated osteoclasts did. Lacunae on dentin surfaces induced by FRP-1-mediated osteoclasts were inclined to be single and isolated. However, osteoclasts induced by RANKL+M-CSF made many connected pits on dentin surfaces as if they crawled on there. Interestingly, FRP-1 osteoclastogenesis was enhanced by M-CSF/IL-1α, while chemotactic behavior to the dentin slices was not effected. There were differences in pH and concentration of HCO3- at culture endpoint and in adherent feature to dentin surfaces. Our findings indicate there are two types of osteoclasts with distinct properties

  14. Alcohol and cannabinoids differentially affect HIV infection and function of human monocyte-derived dendritic cells (MDDC

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    Marisela eAgudelo

    2015-12-01

    Full Text Available During human immunodeficiency virus (HIV infection, alcohol has been known to induce inflammation while cannabinoids have been shown to have an anti-inflammatory role. For instance cannabinoids have been shown to reduce susceptibility to HIV-1 infection and attenuate HIV replication in macrophages. Recently, we demonstrated that alcohol induces cannabinoid receptors and regulates cytokine production by monocyte-derived dendritic cells (MDDC. However, the ability of alcohol and cannabinoids to alter MDDC function during HIV infection has not been clearly elucidated yet. In order to study the potential impact of alcohol and cannabinoids on differentiated MDDC infected with HIV, monocytes were cultured for 7 days with GM-CSF and IL-4, differentiated MDDC were infected with HIV-1Ba-L and treated with EtOH (0.1 and 0.2%, THC (5 and 10 uM, or JWH-015 (5 and 10 uM for 4-7 days. HIV infection of MDDC was confirmed by p24 and Long Terminal Repeats (LTR estimation. MDDC endocytosis assay and cytokine array profiles were measured to investigate the effects of HIV and substances of abuse on MDDC function. Our results show the HIV+EtOH treated MDDC had the highest levels of p24 production and expression when compared with the HIV positive controls and the cannabinoid treated cells. Although both cannabinoids, THC and JWH-015 had lower levels of p24 production and expression, the HIV+JWH-015 treated MDDC had the lowest levels of p24 when compared to the HIV+THC treated cells. In addition, MDDC endocytic function and cytokine production were also differentially altered after alcohol and cannabinoid treatments. Our results show a differential effect of alcohol and cannabinoids, which may provide insights into the divergent inflammatory role of alcohol and cannabinoids to modulate MDDC function in the context of HIV infection.

  15. A human monocytic NF-κB fluorescent reporter cell line for detection of microbial contaminants in biological samples.

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    Claire Battin

    Full Text Available Sensing of pathogens by innate immune cells is essential for the initiation of appropriate immune responses. Toll-like receptors (TLRs, which are highly sensitive for various structurally and evolutionary conserved molecules derived from microbes have a prominent role in this process. TLR engagement results in the activation of the transcription factor NF-κB, which induces the expression of cytokines and other inflammatory mediators. The exquisite sensitivity of TLR signalling can be exploited for the detection of bacteria and microbial contaminants in tissue cultures and in protein preparations. Here we describe a cellular reporter system for the detection of TLR ligands in biological samples. The well-characterized human monocytic THP-1 cell line was chosen as host for an NF-ᴋB-inducible enhanced green fluorescent protein reporter gene. We studied the sensitivity of the resultant reporter cells for a variety of microbial components and observed a strong reactivity towards TLR1/2 and TLR2/6 ligands. Mycoplasma lipoproteins are potent TLR2/6 agonists and we demonstrate that our reporter cells can be used as reliable and robust detection system for mycoplasma contaminations in cell cultures. In addition, a TLR4-sensitive subline of our reporters was engineered, and probed with recombinant proteins expressed in different host systems. Bacterially expressed but not mammalian expressed proteins induced strong reporter activity. We also tested proteins expressed in an E. coli strain engineered to lack TLR4 agonists. Such preparations also induced reporter activation in THP-1 cells highlighting the importance of testing recombinant protein preparations for microbial contaminations beyond endotoxins. Our results demonstrate the usefulness of monocytic reporter cells for high-throughput screening for microbial contaminations in diverse biological samples, including tissue culture supernatants and recombinant protein preparations. Fluorescent reporter

  16. Mortality in Severe Human Immunodeficiency Virus-Tuberculosis Associates With Innate Immune Activation and Dysfunction of Monocytes.

    Science.gov (United States)

    Janssen, Saskia; Schutz, Charlotte; Ward, Amy; Nemes, Elisa; Wilkinson, Katalin A; Scriven, James; Huson, Mischa A; Aben, Nanne; Maartens, Gary; Burton, Rosie; Wilkinson, Robert J; Grobusch, Martin P; Van der Poll, Tom; Meintjes, Graeme

    2017-07-01

    Case fatality rates among hospitalized patients diagnosed with human immunodeficiency virus (HIV)-associated tuberculosis remain high, and tuberculosis mycobacteremia is common. Our aim was to define the nature of innate immune responses associated with 12-week mortality in this population. This prospective cohort study was conducted at Khayelitsha Hospital, Cape Town, South Africa. Hospitalized HIV-infected tuberculosis patients with CD4 counts tuberculosis blood cultures were performed in all. Ambulatory HIV-infected patients without active tuberculosis were recruited as controls. Whole blood was stimulated with Escherichia coli derived lipopolysaccharide, heat-killed Streptococcus pneumoniae, and Mycobacterium tuberculosis. Biomarkers of inflammation and sepsis, intracellular (flow cytometry) and secreted cytokines (Luminex), were assessed for associations with 12-week mortality using Cox proportional hazard models. Second, we investigated associations of these immune markers with tuberculosis mycobacteremia. Sixty patients were included (median CD4 count 53 cells/µL (interquartile range [IQR], 22-132); 16 (27%) died after a median of 12 (IQR, 0-24) days. Thirty-one (52%) grew M. tuberculosis on blood culture. Mortality was associated with higher concentrations of procalcitonin, activation of the innate immune system (% CD16+CD14+ monocytes, interleukin-6, tumour necrosis factor-ɑ and colony-stimulating factor 3), and antiinflammatory markers (increased interleukin-1 receptor antagonist and lower monocyte and neutrophil responses to bacterial stimuli). Tuberculosis mycobacteremia was not associated with mortality, nor with biomarkers of sepsis. Twelve-week mortality was associated with greater pro- and antiinflammatory alterations of the innate immune system, similar to those reported in severe bacterial sepsis. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

  17. The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines.

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    Rojnik, Matija; Jevnikar, Zala R; Doljak, Bojan; Turk, Samo; Zidar, Nace; Kos, Janko

    2012-10-01

    Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type. Copyright © 2012 Elsevier GmbH. All rights reserved.

  18. Effects of bee venom against Propionibacterium acnes-induced inflammation in human keratinocytes and monocytes.

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    Kim, Jung-Yeon; Lee, Woo-Ram; Kim, Kyung-Hyun; An, Hyun-Jin; Chang, Young-Chae; Han, Sang-Mi; Park, Yoon-Yub; Pak, Sok Cheon; Park, Kwan-Kyu

    2015-06-01

    Propionibacterium acnes (P. acnes) cause inflammatory acne and play an important role in the pathogenesis of acne by inducing inflammatory mediators. P. acnes contributes to the inflammatory responses of acne by activating inflammatory cells, keratinocytes and sebocytes to secrete pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-8. Bee venom has traditionally been used in the treatment of certain immune-related diseases. However, there has not yet been a robust trial to prove the therapeutic effect of bee venom in skin inflammation. The aim of the present study was to investigate anti-inflammatory properties of bee venom in skin inflammation induced by P. acnes using keratinocytes (HaCaT) and monocytes (THP-1). P. acnes is known to stimulate the production of pro-inflammatory cytokines such as IL-1, IL-8, IL-12 and TNF-α. In the present study, the production of interferon-γ (IFN-γ), IL-1β, IL-8 and TNF-α was increased by P. acnes treatment in HaCaT and THP-1 cells. By contrast, bee venom effectively inhibited the secretion of IFN-γ, IL-1β, IL-8 and TNF-α. Furthermore, P. acnes treatment activated the expression of IL-8 and toll-like receptor 2 (TLR2) in HaCaT cells. However, bee venom inhibited the expression of IL-8 and TLR2 in heat-killed P. acnes. Based on these results, it is concluded that bee venom has an effective anti-inflammatory activity against P. acnes in HaCaT and THP-1 cells. Therefore, we suggest that bee venom is an alternative treatment to antibiotic therapy of acne.

  19. The MAPK ERK5, but not ERK1/2, inhibits the progression of monocytic phenotype to the functioning macrophage

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xuening [Department of Pathology and Laboratory Medicine, Rutgers, NJ Medical School, 185 South Orange Ave, Newark, NJ 07103 (United States); Pesakhov, Stella [Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, PO Box 653, 84105 Beer-Sheva (Israel); Harrison, Jonathan S [Department of Medicine, Rutgers, Robert Wood Johnson Medical School, New Brunswick, NJ 08903 (United States); Kafka, Michael; Danilenko, Michael [Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, PO Box 653, 84105 Beer-Sheva (Israel); Studzinski, George P, E-mail: studzins@njms.rutgers.edu [Department of Pathology and Laboratory Medicine, Rutgers, NJ Medical School, 185 South Orange Ave, Newark, NJ 07103 (United States)

    2015-01-01

    Intracellular signaling pathways present targets for pharmacological agents with potential for treatment of neoplastic diseases, with some disease remissions already recorded. However, cellular compensatory mechanisms usually negate the initial success. For instance, attempts to interrupt aberrant signaling downstream of the frequently mutated ras by inhibiting ERK1/2 has shown only limited usefulness for cancer therapy. Here, we examined how ERK5, that overlaps the functions of ERK1/2 in cell proliferation and survival, functions in a manner distinct from ERK1/2 in human AML cells induced to differentiate by 1,25D-dihydroxyvitamin D{sub 3} (1,25D). Using inhibitors of ERK1/2 and of MEK5/ERK5 at concentrations specific for each kinase in HL60 and U937 cells, we observed that selective inhibition of the kinase activity of ERK5, but not of ERK1/2, in the presence of 1,25D resulted in macrophage-like cell morphology and enhancement of phagocytic activity. Importantly, this was associated with increased expression of the macrophage colony stimulating factor receptor (M-CSFR), but was not seen when M-CSFR expression was knocked down. Interestingly, inhibition of ERK1/2 led to activation of ERK5 in these cells. Our results support the hypothesis that ERK5 negatively regulates the expression of M-CSFR, and thus has a restraining function on macrophage differentiation. The addition of pharmacological inhibitors of ERK5 may influence trials of differentiation therapy of AML. - Highlights: • ERK5 has at least some functions in AML cells which are distinct from those of ERK1/2. • ERK5 activity negatively controls the expression of M-CSFR. • ERK5 retards the progression of differentiation from monocyte to functional macrophage.

  20. Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses.

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    Valeria de Turris

    Full Text Available Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.

  1. Interaction of Coxiella burnetii Strains of Different Sources and Genotypes with Bovine and Human Monocyte-Derived Macrophages

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    Katharina Sobotta

    2018-01-01

    Full Text Available Most human Q fever infections originate from small ruminants. By contrast, highly prevalent shedding of Coxiella (C. burnetii by bovine milk rarely results in human disease. We hypothesized that primary bovine and human monocyte-derived macrophages (MDM represent a suitable in vitro model for the identification of strain-specific virulence properties at the cellular level. Twelve different C. burnetii strains were selected to represent different host species and multiple loci variable number of tandem repeat analysis (MLVA genotypes. Infection efficiency and replication of C. burnetii were monitored by cell culture re-titration and qPCR. Expression of immunoregulatory factors after MDM infection was measured by qRT-PCR and flow cytometry. Invasion, replication and MDM response differed between C. burnetii strains but not between MDMs of the two hosts. Strains isolated from ruminants were less well internalized than isolates from humans and rodents. Internalization of MLVA group I strains was lower compared to other genogroups. Replication efficacy of C. burnetii in MDM ranged from low (MLVA group III to high (MLVA group IV. Infected human and bovine MDM responded with a principal up-regulation of pro-inflammatory cytokines such as IL-1β, IL-12, and TNF-α. However, MLVA group IV strains induced a pronounced host response whereas infection with group I strains resulted in a milder response. C. burnetii infection marginally affected polarization of MDM. Only one C. burnetii strain of MLVA group IV caused a substantial up-regulation of activation markers (CD40, CD80 on the surface of bovine and human MDM. The study showed that replication of C. burnetii in MDM and the subsequent host cell response is genotype-specific rather than being determined by the host species pointing to a clear distinction in C. burnetii virulence between the genetic groups.

  2. Phagocytosis of haemozoin (malarial pigment enhances metalloproteinase-9 activity in human adherent monocytes: Role of IL-1beta and 15-HETE

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    Giribaldi Giuliana

    2008-08-01

    Full Text Available Abstract Background It has been shown previously that human monocytes fed with haemozoin (HZ or trophozoite-parasitized RBCs displayed increased matrix metalloproteinase-9 (MMP-9 enzyme activity and protein/mRNA expression and increased TNF production, and showed higher matrix invasion ability. The present study utilized the same experimental model to analyse the effect of phagocytosis of: HZ, delipidized HZ, beta-haematin (lipid-free synthetic HZ and trophozoites on production of IL-1beta and MMP-9 activity and expression. The second aim was to find out which component of HZ was responsible for the effects. Methods Native HZ freshly isolated from Plasmodium falciparum (Palo Alto strain, Mycoplasma-free, delipidized HZ, beta-haematin (lipid-free synthetic HZ, trophozoites and control meals such as opsonized non-parasitized RBCs and inert latex particles, were fed to human monocytes. The production of IL-1beta by differently fed monocytes, in presence or absence of specific MMP-9 inhibitor or anti-hIL-1beta antibodies, was quantified in supernatants by ELISA. Expression of IL-1beta was analysed by quantitative real-time RT-PCR. MMP-9 activity and protein expression were quantified by gelatin zymography and Western blotting. Results Monocytes fed with HZ or trophozoite-parasitized RBCs generated increased amounts of IL-1beta and enhanced enzyme activity (in cell supernatants and protein/mRNA expression (in cell lysates of monocyte MMP-9. The latter appears to be causally related to enhanced IL-1beta production, as enhancement of both expression and enzyme activity were abrogated by anti-hIL-1beta Abs. Upregulation of IL-1beta and MMP-9 were absent in monocytes fed with beta-haematin or delipidized HZ, indicating a role for HZ-attached or HZ-generated lipid components. 15-HETE (15(S,R-hydroxy-6,8,11,13-eicosatetraenoic acid a potent lipoperoxidation derivative generated by HZ from arachidonic acid via haem-catalysis was identified as one mediator

  3. Chemoresistance of human monocyte-derived dendritic cells is regulated by IL-17A.

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    Selma Olsson Åkefeldt

    Full Text Available Dendritic cells initiate adaptive immune responses, leading either to control cancer by effector T cells or to exacerbate cancer by regulatory T cells that inhibit IFN-γ-mediated Th1-type response. Dendritic cells can also induce Th17-type immunity, mediated by IL-17A. However, the controversial role of this cytokine in cancer requires further investigations. We generated dendritic cells from peripheral blood monocytes to investigate lifespan, phenotype and chemoresistance of dendritic cells, treated with IL-17A with or without IFN-γ. Studying the expression of Bcl-2 family members, we demonstrated that dendritic cells constitutively express one pro-survival Bcl-2 member: MCL1. Immature dendritic cells were CD40(lowHLADR(low CD1a(+ MCL1(+, did not express CD14, CD68 or BCL2A1, and displayed a short 2-day lifespan. IL-17A-treated DC exhibited a semi-mature (CD40(high HLADR(low pre-M2 (CCL22(+ CD206(+ CD163(+ IL1RN(+ IL-10(- CXCL10(- IL-12(- mixed (CD1a(+ CD14+ CD68(+ macrophage-dendritic cell phenotype. They efficiently exerted mannose receptor-mediated endocytosis and did not produce superoxide anions, in the absence of TLR engagement. Interestingly, IL-17A promoted a long-term survival of dendritic cells, beyond 12 days, that correlated to BCL2A1 induction, a pro-survival Bcl-2 family member. BCL2A1 transcription was activated by NF-κB, downstream of IL-17A transduction. Thus, immature dendritic cells only express MCL1, whereas IL-17A-treated dendritic cells concomitantly expressed two pro-survival Bcl-2 family members: MCL1 and BCL2A1. These latter developed chemoresistance to 11 of the 17 chemotherapy agents tested. However, high doses of either vinblastine or cytarabine decreased MCL1 expression and induced dendritic cell death. When IL-17A is produced in vivo, administration of anti-IL-17A biotherapy may impair dendritic cell survival by targeting BCL2A1 expression. Consequently, depending on the effector or regulatory role of dendritic

  4. alpha-MSH and its receptors in regulation of tumor necrosis factor-alpha production by human monocyte/macrophages.

    Science.gov (United States)

    Taherzadeh, S; Sharma, S; Chhajlani, V; Gantz, I; Rajora, N; Demitri, M T; Kelly, L; Zhao, H; Ichiyama, T; Catania, A; Lipton, J M

    1999-05-01

    The hypothesis that macrophages contain an autocrine circuit based on melanocortin [ACTH and alpha-melanocyte-stimulating hormone (alpha-MSH)] peptides has major implications for neuroimmunomodulation research and inflammation therapy. To test this hypothesis, cells of the THP-1 human monocyte/macrophage line were stimulated with lipopolysaccharide (LPS) in the presence and absence of alpha-MSH. The inflammatory cytokine tumor necrosis factor (TNF)-alpha was inhibited in relation to alpha-MSH concentration. Similar inhibitory effects on TNF-alpha were observed with ACTH peptides that contain the alpha-MSH amino acid sequence and act on melanocortin receptors. Nuclease protection assays indicated that expression of the human melanocortin-1 receptor subtype (hMC-1R) occurs in THP-1 cells; Southern blots of RT-PCR product revealed that additional subtypes, hMC-3R and hMC-5R, also occur. Incubation of resting macrophages with antibody to hMC-1R increased TNF-alpha concentration; the antibody also markedly reduced the inhibitory influence of alpha-MSH on TNF-alpha in macrophages treated with LPS. These results in cells known to produce alpha-MSH at rest and to increase secretion of the peptide when challenged are consistent with an endogenous regulatory circuit based on melanocortin peptides and their receptors. Targeting of this neuroimmunomodulatory circuit in inflammatory diseases in which myelomonocytic cells are prominent should be beneficial.

  5. Regulation of tumor necrosis factor gene expression by ionizing radiation in human myeloid leukemia cells and peripheral blood monocytes

    International Nuclear Information System (INIS)

    Sherman, M.L.; Datta, R.; Hallahan, D.E.; Weichselbaum, R.R.; Kufe, D.W.

    1991-01-01

    Previous studies have demonstrated that ionizing radiation induces the expression of certain cytokines, such as TNF alpha/cachectin. However, there is presently no available information regarding the molecular mechanisms responsible for the regulation of cytokine gene expression by ionizing radiation. In this report, we describe the regulation of the TNF gene by ionizing radiation in human myeloid leukemia cells. The increase in TNF transcripts by x rays was both time- and dose-dependent as determined by Northern blot analysis. Similar findings were obtained in human peripheral blood monocytes. Transcriptional run-on analyses have demonstrated that ionizing radiation stimulates the rate of TNF gene transcription. Furthermore, induction of TNF mRNA was increased in the absence of protein synthesis. In contrast, ionizing radiation had little effect on the half-life of TNF transcripts. These findings indicate that the increase in TNF mRNA observed after irradiation is regulated by transcriptional mechanisms and suggest that production of this cytokine by myeloid cells may play a role in the pathophysiologic effects of ionizing radiation

  6. Heterogeneity of Bovine Peripheral Blood Monocytes

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    Jamal Hussen

    2017-12-01

    Full Text Available Peripheral blood monocytes of several species can be divided into different subpopulations with distinct phenotypic and functional properties. Herein, we aim at reviewing published work regarding the heterogeneity of the recently characterized bovine monocyte subsets. As the heterogeneity of human blood monocytes was widely studied and reviewed, this work focuses on comparing bovine monocyte subsets with their human counterparts regarding their phenotype, adhesion and migration properties, inflammatory and antimicrobial functions, and their ability to interact with neutrophilic granulocytes. In addition, the differentiation of monocyte subsets into functionally polarized macrophages is discussed. Regarding phenotype and distribution in blood, bovine monocyte subsets share similarities with their human counterparts. However, many functional differences exist between monocyte subsets from the two species. In contrast to their pro-inflammatory functions in human, bovine non-classical monocytes show the lowest phagocytosis and reactive oxygen species generation capacity, an absent ability to produce the pro-inflammatory cytokine IL-1β after inflammasome activation, and do not have a role in the early recruitment of neutrophils into inflamed tissues. Classical and intermediate monocytes of both species also differ in their response toward major monocyte-attracting chemokines (CCL2 and CCL5 and neutrophil degranulation products (DGP in vitro. Such differences between homologous monocyte subsets also extend to the development of monocyte-derived macrophages under the influence of chemokines like CCL5 and neutrophil DGP. Whereas the latter induce the differentiation of M1-polarized macrophages in human, bovine monocyte-derived macrophages develop a mixed M1/M2 macrophage phenotype. Although only a few bovine clinical trials analyzed the correlation between changes in monocyte composition and disease, they suggest that functional differences between

  7. Supernatants from oral epithelial cells and gingival fibroblasts modulate human immunodeficiency virus type 1 promoter activation induced by periodontopathogens in monocytes/macrophages.

    Science.gov (United States)

    González, O A; Ebersole, J L; Huang, C B

    2010-04-01

    Bacterial and host cell products during coinfections of Human Immunodeficiency Virus type 1-positive (HIV-1(+)) patients regulate HIV-1 recrudescence in latently infected cells (e.g. T cells, monocytes/macrophages), impacting highly active antiretroviral therapy (HAART) failure and progression of acquired immunodeficiency syndrome. A high frequency of oral opportunistic infections (e.g. periodontitis) in HIV-1(+) patients has been demonstrated; however, their potential to impact HIV-1 exacerbation is unclear. We sought to determine the ability of supernatants derived from oral epithelial cells (OKF4) and human gingival fibroblasts (Gin-4) challenged with periodontal pathogens, to modulate the HIV-1 promoter activation in monocytes/macrophages. BF24 monocytes/macrophages transfected with the HIV-1 promoter driving the expression of chloramphenicol acetyltransferase (CAT) were stimulated with Porphyromonas gingivalis, Fusobacterium nucleatum, or Treponema denticola in the presence of supernatants from OKF4 or Gin4 cells either unstimulated or previously pulsed with bacteria. CAT levels were determined by enzyme-linked immunosorbent assay and cytokine production was evaluated by Luminex beadlyte assays. OKF4 and Gin4 supernatants enhanced HIV-1 promoter activation particularly related to F. nucleatum challenge. An additive effect was observed in HIV-1 promoter activation when monocytes/macrophages were simultaneously stimulated with gingival cell supernatants and bacterial extracts. OKF4 cells produced higher levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukins -6 and -8 in response to F. nucleatum and P. gingivalis. Preincubation of OKF4 supernatants with anti-GM-CSF reduced the additive effect in periodontopathogen-induced HIV-1 promoter activation. These results suggest that soluble mediators produced by gingival resident cells in response to periodontopathogens could contribute to HIV-1 promoter activation in monocytes

  8. GM-CSF and IL-3 Modulate Human Monocyte TNF-α Production and Renewal in In Vitro Models of Trained Immunity.

    Science.gov (United States)

    Borriello, Francesco; Iannone, Raffaella; Di Somma, Sarah; Loffredo, Stefania; Scamardella, Eloise; Galdiero, Maria Rosaria; Varricchi, Gilda; Granata, Francescopaolo; Portella, Giuseppe; Marone, Gianni

    2016-01-01

    GM-CSF and IL-3 are hematopoietic cytokines that also modulate the effector functions of several immune cell subsets. In particular, GM-CSF and IL-3 exert a significant control on monocyte and macrophage effector functions, as assessed in experimental models of inflammatory and autoimmune diseases and also in human studies. Here, we sought to investigate the mechanisms and the extent to which GM-CSF and IL-3 modulate the pro-inflammatory, LPS-mediated, activation of human CD14 + monocytes taking into account the new concept of trained immunity (i.e., the priming stimulus modulates the response to subsequent stimuli mainly by inducing chromatin remodeling and increased transcription at relevant genetic loci). We demonstrate that GM-CSF and IL-3 priming enhances TNF-α production upon subsequent LPS stimulation (short-term model of trained immunity) in a p38- and SIRT2-dependent manner without increasing TNF primary transcript levels (a more direct measure of transcription), thus supporting a posttranscriptional regulation of TNF-α in primed monocytes. GM-CSF and IL-3 priming followed by 6 days of resting also results in increased TNF-α production upon LPS stimulation (long-term model of trained immunity). In this case, however, GM-CSF and IL-3 priming induces a c-Myc-dependent monocyte renewal and increase in cell number that is in turn responsible for heightened TNF-α production. Overall, our results provide insights to understand the biology of monocytes in health and disease conditions in which the hematopoietic cytokines GM-CSF and IL-3 play a role and also extend our knowledge of the cellular and molecular mechanisms of trained immunity.

  9. Mesenchymal Stem Cells Induce Expression of CD73 in Human Monocytes In Vitro and in a Swine Model of Myocardial Infarction In Vivo

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    Marta Monguió-Tortajada

    2017-11-01

    Full Text Available The ectoenzymes CD39 and CD73 regulate the purinergic signaling through the hydrolysis of adenosine triphosphate (ATP/ADP to AMP and to adenosine (Ado, respectively. This shifts the pro-inflammatory milieu induced by extracellular ATP to the anti-inflammatory regulation by Ado. Mesenchymal stem cells (MSCs have potent immunomodulatory capabilities, including monocyte modulation toward an anti-inflammatory phenotype aiding tissue repair. In vitro, we observed that human cardiac adipose tissue-derived MSCs (cATMSCs and umbilical cord MSCs similarly polarize monocytes toward a regulatory M2 phenotype, which maintained the expression of CD39 and induced expression of CD73 in a cell contact dependent fashion, correlating with increased functional activity. In addition, the local treatment with porcine cATMSCs using an engineered bioactive graft promoted the in vivo CD73 expression on host monocytes in a swine model of myocardial infarction. Our results suggest the upregulation of ectonucleotidases on MSC-conditioned monocytes as an effective mechanism to amplify the long-lasting immunomodulatory and healing effects of MSCs delivery.

  10. Age-dependent alterations of monocyte subsets and monocyte-related chemokine pathways in healthy adults

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    Trautwein Christian

    2010-06-01

    Full Text Available Abstract Background Recent experimental approaches have unraveled essential migratory and functional differences of monocyte subpopulations in mice. In order to possibly translate these findings into human physiology and pathophysiology, human monocyte subsets need to be carefully revisited in health and disease. In analogy to murine studies, we hypothesized that human monocyte subsets dynamically change during ageing, potentially influencing their functionality and contributing to immunosenescence. Results Circulating monocyte subsets, surface marker and chemokine receptor expression were analyzed in 181 healthy volunteers (median age 42, range 18-88. Unlike the unaffected total leukocyte or total monocyte counts, non-classical CD14+CD16+ monocytes significantly increased with age, but displayed reduced HLA-DR and CX3CR1 surface expression in the elderly. Classical CD14++CD16- monocyte counts did not vary dependent on age. Serum MCP-1 (CCL2, but not MIP1α (CCL3, MIP1β (CCL4 or fractalkine (CX3CL1 concentrations increased with age. Monocyte-derived macrophages from old or young individuals did not differ with respect to cytokine release in vitro at steady state or upon LPS stimulation. Conclusions Our study demonstrates dynamic changes of circulating monocytes during ageing in humans. The expansion of the non-classical CD14+CD16+ subtype, alterations of surface protein and chemokine receptor expression as well as circulating monocyte-related chemokines possibly contribute to the preserved functionality of the monocyte pool throughout adulthood.

  11. Attenuation of LPS-induced inflammation by ICT, a derivate of icariin, via inhibition of the CD14/TLR4 signaling pathway in human monocytes.

    Science.gov (United States)

    Wu, Jinfeng; Zhou, Junmin; Chen, Xianghong; Fortenbery, Nicole; Eksioglu, Erika A; Wei, Sheng; Dong, Jingcheng

    2012-01-01

    To evaluate the anti-inflammatory potential of ICT in LPS stimulated human innate immune cells. 3, 5, 7-Trihydroxy-4'-methoxy-8-(3-hydroxy-3- methylbutyl)-flavone (ICT) is a novel derivative of icariin, the major active ingredient of Herba Epimedii, an herb used in traditional Chinese medicine. We previously demonstrated its anti-inflammatory potential in a murine macrophage cell line as well as in mouse models. We measured TNF-α production by ELISA, TLR4/CD14 expression by flow cytometry, and NF-κB and MAPK activation by western blot all in LPS-stimulated PBMC, human monocytes, or THP-1 cells after treatment with ICT. ICT inhibited LPS-induced TNF-α production in THP-1 cells, PBMCs and human monocytes in a dose-dependent manner. ICT treatment resulted in down-regulation of the expression of CD14/TLR4 and attenuated NF-κB and MAPK activation induced by LPS. We illustrate the anti-inflammatory property of ICT in human immune cells, especially in monocytes. These effects were mediated, at least partially, via inhibition of the CD14/TLR4 signaling pathway. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Integrating genome-wide genetic variations and monocyte expression data reveals trans-regulated gene modules in humans.

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    Maxime Rotival

    2011-12-01

    Full Text Available One major expectation from the transcriptome in humans is to characterize the biological basis of associations identified by genome-wide association studies. So far, few cis expression quantitative trait loci (eQTLs have been reliably related to disease susceptibility. Trans-regulating mechanisms may play a more prominent role in disease susceptibility. We analyzed 12,808 genes detected in at least 5% of circulating monocyte samples from a population-based sample of 1,490 European unrelated subjects. We applied a method of extraction of expression patterns-independent component analysis-to identify sets of co-regulated genes. These patterns were then related to 675,350 SNPs to identify major trans-acting regulators. We detected three genomic regions significantly associated with co-regulated gene modules. Association of these loci with multiple expression traits was replicated in Cardiogenics, an independent study in which expression profiles of monocytes were available in 758 subjects. The locus 12q13 (lead SNP rs11171739, previously identified as a type 1 diabetes locus, was associated with a pattern including two cis eQTLs, RPS26 and SUOX, and 5 trans eQTLs, one of which (MADCAM1 is a potential candidate for mediating T1D susceptibility. The locus 12q24 (lead SNP rs653178, which has demonstrated extensive disease pleiotropy, including type 1 diabetes, hypertension, and celiac disease, was associated to a pattern strongly correlating to blood pressure level. The strongest trans eQTL in this pattern was CRIP1, a known marker of cellular proliferation in cancer. The locus 12q15 (lead SNP rs11177644 was associated with a pattern driven by two cis eQTLs, LYZ and YEATS4, and including 34 trans eQTLs, several of them tumor-related genes. This study shows that a method exploiting the structure of co-expressions among genes can help identify genomic regions involved in trans regulation of sets of genes and can provide clues for understanding the

  13. Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte macrophages

    International Nuclear Information System (INIS)

    Haberland, M.E.; Fogelman, A.M.

    1985-01-01

    Maleyl bovine plasma albumin competed on an equimolar basis with malondialdehyde low density lipoprotein (LDL) in suppressing the lysosomal hydrolysis of 125 I-labeled malondialdehyde LDL mediated by the scavenger receptor of human monocyte macrophages. Maleyl bovine plasma albumin, in which 94% of the amino groups were modified, exhibited an anodic mobility in agarose electrophoresis 1.7 times that of the native protein. Incubation of maleyl bovine plasma albumin at pH 3.5 regenerated the free amino groups and restored the protein to the same electrophoretic mobility as native albumin. Although ligands recognized by the scavenger receptor typically are anionic, the authors propose that addition of new negative charge achieved by maleylation, rather than directly forming the receptor binding site(s), induces conformational changes in albumin as a prerequisite to expression of the recognition domain(s). They conclude that the primary sequence of albumin, rather than addition of new negative charge, provides the recognition determinant(s) essential for interaction of maleyl bovine plasma albumin with the scavenger receptor

  14. Lymph node hemophagocytosis in rickettsial diseases: a pathogenetic role for CD8 T lymphocytes in human monocytic ehrlichiosis (HME?

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    Dumler J Stephen

    2006-07-01

    Full Text Available Abstract Background Human monocytic ehrlichiosis (HME and Rocky Mountain spotted fever (RMSF are caused by Ehrlichia chaffeensis and Rickettsia rickettsii, respectively. The pathogenesis of RMSF relates to rickettsia-mediated vascular injury, but it is unclear in HME. Methods To study histopathologic responses in the lymphatic system for correlates of immune injury, lymph nodes from patients with HME (n = 6 and RMSF (n = 5 were examined. H&E-stained lymph node tissues were examined for five histopathologic features, including hemophagocytosis, cellularity, necrosis, and vascular congestion and edema. The relative proportions of CD68 macrophages, CD8 and CD4 T lymphocytes, and CD20 B lymphocytes were evaluated by immunohistochemical staining. Results Hemophagocytosis was similar in HME and RMSF, and was greater than in control cases (p = .015. Cellularity in HME was not different from controls, whereas RMSF lymph nodes were markedly less cellular (p E. chaffeensis-infected mononuclear phagocytes were infrequent compared to R. rickettsii-infected endothelial cells. More CD8 cells in lymph nodes were observed with HME (p Conclusion Hemophagocytosis, CD8 T cell expansion, and the paucity of infected cells in HME, suggest that E. chaffeensis infection leads to macrophage activation and immune-mediated injury.

  15. Genomic Profiling of a Human Leukemic Monocytic Cell-Line (THP-1 Exposed to Alpha Particle Radiation

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    Vinita Chauhan

    2012-01-01

    Full Text Available This study examined alpha (α- particle radiation effects on global changes in gene expression in human leukemic monocytic cells (THP-1 for the purposes of mining for candidate biomarkers that could be used for the development of a biological assessment tool. THP-1 cells were exposed to α-particle radiation at a dose range of 0 to 1.5 Gy. Twenty-four hours and three days after exposure gene expression was monitored using microarray technology. A total of 16 genes were dose responsive and classified as early onset due to their expression 24 h after exposure. Forty-eight transcripts were dose responsive and classified as late-onset as they were expressed 72 h after exposure. Among these genes, 6 genes were time and dose responsive and validated further using alternate technology. These transcripts were upregulated and associated with biological processes related to immune function, organelle stability and cell signalling/communication. This panel of genes merits further validation to determine if they are strong candidate biomarkers indicative of α-particle exposure.

  16. Circulating CD14brightCD16+ 'intermediate' monocytes exhibit enhanced parasite pattern recognition in human helminth infection.

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    Joseph D Turner

    2014-04-01

    Full Text Available Circulating monocyte sub-sets have recently emerged as mediators of divergent immune functions during infectious disease but their role in helminth infection has not been investigated. In this study we evaluated whether 'classical' (CD14brightCD16-, 'intermediate' (CD14brightCD16+, and 'non-classical' (CD14dimCD16+ monocyte sub-sets from peripheral blood mononuclear cells varied in both abundance and ability to bind antigenic material amongst individuals living in a region of Northern Senegal which is co-endemic for Schistosoma mansoni and S. haematobium. Monocyte recognition of excretory/secretory (E/S products released by skin-invasive cercariae, or eggs, of S. mansoni was assessed by flow cytometry and compared between S. mansoni mono-infected, S. mansoni and S. haematobium co-infected, and uninfected participants. Each of the three monocyte sub-sets in the different infection groups bound schistosome E/S material. However, 'intermediate' CD14brightCD16+ monocytes had a significantly enhanced ability to bind cercarial and egg E/S. Moreover, this elevation of ligand binding was particularly evident in co-infected participants. This is the first demonstration of modulated parasite pattern recognition in CD14brightCD16+ intermediate monocytes during helminth infection, which may have functional consequences for the ability of infected individuals to respond immunologically to infection.

  17. Effect of 60Co γ-ray irradiation on cytoskeleton of human peripheral blood monocytes with whole mount cell electron microscopy in vitro

    International Nuclear Information System (INIS)

    Chen Xiaomei; Guo Yuhua; Yin Zhiwei

    1992-01-01

    Whole mount cell electron microscopy was used in combination with selective extraction to prepare cytoskeletal framework. Cytoskeleton prepared by Triton X-100 treatment of human peripheral blood monocytes appeared on electron microscopy as a highly organized and interconnected three-dimensional matrix of different fibrous elements. By three-dimensional visualization of Triton X-100 resistant cytoskeletons it was demonstrated that different doses of 60 Co γ-rays caused distinctive and reproducible alterations of the cytoskeleton of intact human peripheral blood monocytes in vitro. The alterations were similar to those caused by cytochalasin B and by colchicine. From these observations and other workers'studies, it is presumed that 60 Co γ-ray irradiation may inhibit cytoplasmic microtubule and microfilament assembling

  18. Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

    Science.gov (United States)

    Posner, M R; Santos, D J; Elboim, H S; Tumber, M B; Frackelton, A R

    1989-04-01

    Peripheral blood mononuclear cells from a patient with chronic myelogenous leukemia (CML), in remission, were depleted of CD8-positive T-cells and cultured with Epstein-Barr virus. Four of 20 cultures (20%) secreted human IgG antibodies selectively reactive with the cell surfaces of certain human leukemia cell lines. Three polyclonal, Epstein-Barr virus-transformed, B-cell lines were expanded and fused with the human-mouse myeloma analogue HMMA2.11TG/O. Antibody from secreting clones HL 1.2 (IgG1), HL 2.1 (IgG3), and HL 3.1 (IgG1) have been characterized. All three react with HL-60 (promyelocytic), RWLeu4 (CML promyelocytic), and U937 (monocytic), but not with KG-1 (myeloblastic) or K562 (CML erythroid). There is no reactivity with T-cell lines, Burkitt's cell lines, pre-B-leukemia cell lines, or an undifferentiated CML cell line, BV173. Leukemic cells from two of seven patients with acute myelogenous leukemia and one of five with acute lymphocytic leukemia react with all three antibodies. Normal lymphocytes, monocytes, polymorphonuclear cells, red blood cells, bone marrow cells, and platelets do not react. Samples from patients with other diverse hematopoietic malignancies showed no reactivity. Immunoprecipitations suggest that the reactive antigen(s) is a lactoperoxidase iodinatable series of cell surface proteins with molecular weights of 42,000-54,000 and a noniodinatable protein with a molecular weight of 82,000. Based on these data these human monoclonal antibodies appear to react with myelomonocytic leukemic cells and may detect a leukemia-specific antigen or a highly restricted differentiation antigen.

  19. CD16(+) monocytes with smooth muscle cell characteristics are reduced in human renal chronic transplant dysfunction

    NARCIS (Netherlands)

    Boersema, M.; van den Born, Joost; van Ark, J.; Harms, Geertruida; Seelen, M. A.; van Dijk, M. C. R. F.; van Goor, H.; Navis, G. J.; Popa, E. R.; Hillebrands, J. L.

    In chronic transplant dysfunction (CTD), persistent (allo)immune-mediated inflammation eventually leads to tissue remodeling including neointima formation in intragraft arteries. We previously showed that recipient-derived neointimal alpha-SMA(+) smooth muscle-like cells are present in human renal

  20. Ursodeoxycholic acid inhibits TNFα-induced IL-8 release from monocytes.

    Science.gov (United States)

    O'Dwyer, Aoife M; Lajczak, Natalia K; Keyes, Jennifer A; Ward, Joseph B; Greene, Catherine M; Keely, Stephen J

    2016-08-01

    Monocytes are critical to the pathogenesis of inflammatory bowel disease (IBD) as they infiltrate the mucosa and release cytokines that drive the inflammatory response. Ursodeoxycholic acid (UDCA), a naturally occurring bile acid with anti-inflammatory actions, has been proposed as a potential new therapy for IBD. However, its effects on monocyte function are not yet known. Primary monocytes from healthy volunteers or cultured U937 monocytes were treated with either the proinflammatory cytokine, TNFα (5 ng/ml) or the bacterial endotoxin, lipopolysaccharide (LPS; 1 μg/ml) for 24 h, in the absence or presence of UDCA (25-100 μM). IL-8 release into the supernatant was measured by ELISA. mRNA levels were quantified by qPCR and changes in cell signaling proteins were determined by Western blotting. Toxicity was assessed by measuring lactate dehydrogenase (LDH) release. UDCA treatment significantly attenuated TNFα-, but not LPS-driven, release of IL-8 from both primary and cultured monocytes. UDCA inhibition of TNFα-driven responses was associated with reduced IL-8 mRNA expression. Both TNFα and LPS stimulated NFκB activation in monocytes, while IL-8 release in response to both cytokines was attenuated by an NFκB inhibitor, BMS-345541. Interestingly, UDCA inhibited TNFα-, but not LPS-stimulated, NFκB activation. Finally, TNFα, but not LPS, induced phosphorylation of TNF receptor associated factor (TRAF2), while UDCA cotreatment attenuated this response. We conclude that UDCA specifically inhibits TNFα-induced IL-8 release from monocytes by inhibiting TRAF2 activation. Since such actions would serve to dampen mucosal immune responses in vivo, our data support the therapeutic potential of UDCA for IBD. Copyright © 2016 the American Physiological Society.

  1. Inhibition of osteoclast differentiation by overexpression of NDRG2 in monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Kyeongah; Nam, Sorim; Kim, Bomi; Lim, Ji Hyun; Yang, Young; Lee, Myeong-Sok; Lim, Jong-Seok, E-mail: jslim@sookmyung.ac.kr

    2015-12-25

    N-Myc downstream-regulated gene 2 (NDRG2), a member of the NDRG family of differentiation-related genes, has been characterized as a regulator of dendritic cell differentiation from monocytes, CD34{sup +} progenitor cells, and myelomonocytic leukemic cells. In this study, we show that NDRG2 overexpression inhibits the differentiation of U937 cells into osteoclasts in response to stimulation with a combination of macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of NF-κB ligand (RANKL). U937 cells stably expressing NDRG2 are unable to differentiate into multinucleated osteoclast-like cells and display reduced tartrate-resistant acid phosphatase (TRAP) activity and resorption pit formation. Furthermore, NDRG2 expression significantly suppresses the expression of genes that are crucial for the proliferation, survival, differentiation, and function of osteoclasts, including c-Fos, Atp6v0d2, RANK, and OSCAR. The activation of ERK1/2 and p38 is also inhibited by NDRG2 expression during osteoclastogenesis, and the inhibition of osteoclastogenesis by NDRG2 correlates with the down-regulation of the expression of the transcription factor PU.1. Taken together, our results suggest that the expression of NDRG2 potentially inhibits osteoclast differentiation and plays a role in modulating the signal transduction pathway responsible for osteoclastogenesis. - Highlights: • The expression of NDRG2 significantly impairs osteoclast differentiation. • PU.1 and p38 MAPK inhibitions by NDRG2 are critical for the inhibition of osteoclastogenesis. • Knockdown of NDRG2 rescues the ability of monocytes to differentiate into osteoclasts. • NDRG2 expression in BM and primary macrophages also impairs osteoclast differentiation. • This study implies the potential of NDRG2 expression in the inhibition of osteoclastogenesis.

  2. IL-17A influences essential functions of the monocyte/macrophage lineage and is involved in advanced murine and human atherosclerosis.

    Science.gov (United States)

    Erbel, Christian; Akhavanpoor, Mohammadreza; Okuyucu, Deniz; Wangler, Susanne; Dietz, Alex; Zhao, Li; Stellos, Konstantinos; Little, Kristina M; Lasitschka, Felix; Doesch, Andreas; Hakimi, Maani; Dengler, Thomas J; Giese, Thomas; Blessing, Erwin; Katus, Hugo A; Gleissner, Christian A

    2014-11-01

    Atherosclerosis is a chronic inflammatory disease. Lesion progression is primarily mediated by cells of the monocyte/macrophage lineage. IL-17A is a proinflammatory cytokine, which modulates immune cell trafficking and is involved inflammation in (auto)immune and infectious diseases. But the role of IL-17A still remains controversial. In the current study, we investigated effects of IL-17A on advanced murine and human atherosclerosis, the common disease phenotype in clinical care. The 26-wk-old apolipoprotein E-deficient mice were fed a standard chow diet and treated either with IL-17A mAb (n = 15) or irrelevant Ig (n = 10) for 16 wk. Furthermore, essential mechanisms of IL-17A in atherogenesis were studied in vitro. Inhibition of IL-17A markedly prevented atherosclerotic lesion progression (p = 0.001) by reducing inflammatory burden and cellular infiltration (p = 0.01) and improved lesion stability (p = 0.01). In vitro experiments showed that IL-17A plays a role in chemoattractance, monocyte adhesion, and sensitization of APCs toward pathogen-derived TLR4 ligands. Also, IL-17A induced a unique transcriptome pattern in monocyte-derived macrophages distinct from known macrophage types. Stimulation of human carotid plaque tissue ex vivo with IL-17A induced a proinflammatory milieu and upregulation of molecules expressed by the IL-17A-induced macrophage subtype. In this study, we show that functional blockade of IL-17A prevents atherosclerotic lesion progression and induces plaque stabilization in advanced lesions in apolipoprotein E-deficient mice. The underlying mechanisms involve reduced inflammation and distinct effects of IL-17A on monocyte/macrophage lineage. In addition, translational experiments underline the relevance for the human system. Copyright © 2014 by The American Association of Immunologists, Inc.

  3. Hyperspectral Imaging Using Intracellular Spies: Quantitative Real-Time Measurement of Intracellular Parameters In Vivo during Interaction of the Pathogenic Fungus Aspergillus fumigatus with Human Monocytes.

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    Sara Mohebbi

    Full Text Available Hyperspectral imaging (HSI is a technique based on the combination of classical spectroscopy and conventional digital image processing. It is also well suited for the biological assays and quantitative real-time analysis since it provides spectral and spatial data of samples. The method grants detailed information about a sample by recording the entire spectrum in each pixel of the whole image. We applied HSI to quantify the constituent pH variation in a single infected apoptotic monocyte as a model system. Previously, we showed that the human-pathogenic fungus Aspergillus fumigatus conidia interfere with the acidification of phagolysosomes. Here, we extended this finding to monocytes and gained a more detailed analysis of this process. Our data indicate that melanised A. fumigatus conidia have the ability to interfere with apoptosis in human monocytes as they enable the apoptotic cell to recover from mitochondrial acidification and to continue with the cell cycle. We also showed that this ability of A. fumigatus is dependent on the presence of melanin, since a non-pigmented mutant did not stop the progression of apoptosis and consequently, the cell did not recover from the acidic pH. By conducting the current research based on the HSI, we could measure the intracellular pH in an apoptotic infected human monocyte and show the pattern of pH variation during 35 h of measurements. As a conclusion, we showed the importance of melanin for determining the fate of intracellular pH in a single apoptotic cell.

  4. Unsaturated long-chain fatty acids induce the respiratory burst of human neutrophils and monocytes in whole blood

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    Osthaus Wilhelm A

    2008-07-01

    Full Text Available Abstract Background It is increasingly recognized that infectious complications in patients treated with total parenteral nutrition (TPN may be caused by altered immune responses. Neutrophils and monocytes are the first line of defence against bacterial and fungal infection through superoxide anion production during the respiratory burst. To characterize the impact of three different types of lipid solutions that are applied as part of TPN formulations, we investigated the unstimulated respiratory burst activation of neutrophils and monocytes in whole blood. Methods Whole blood samples were incubated with LCT (Intralipid®, LCT/MCT (Lipofundin® and LCT-MUFA (ClinOleic® in three concentrations (0.06, 0.3 and 0.6 mg ml-1 for time periods up to one hour. Hydrogen peroxide production during the respiratory burst of neutrophils and monocytes was measured by flow cytometry. Results LCT and LCT-MUFA induced a hydrogen peroxide production in neutrophils and monocytes without presence of a physiological stimulus in contrast to LCT/MCT. Conclusion We concluded that parenteral nutrition containing unsaturated oleic (C18:1 and linoleic (C18:2 acid can induce respiratory burst of neutrophils and monocytes, resulting in an elevated risk of tissue damage by the uncontrolled production of reactive oxygen species. Contradictory observations reported in previous studies may in part be the result of different methods used to determine hydrogen peroxide production.

  5. Involvement of mitogen-activated protein kinases and NFκB in LPS-induced CD40 expression on human monocytic cells

    International Nuclear Information System (INIS)

    Wu Weidong; Alexis, Neil E.; Chen Xian; Bromberg, Philip A.; Peden, David B.

    2008-01-01

    CD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanisms regulating lipopolysaccharide (LPS)-induced CD40 expression have not been adequately characterized in human monocytic cells. In this study we used a human monocytic cell line, THP-1, to investigate the possible mechanisms of CD40 expression following LPS exposure. Exposure to LPS resulted in a dose- and time-dependent increase in CD40 expression. Further studies using immunoblotting and pharmacological inhibitors revealed that mitogen-activated protein kinases (MAPKs) and NFκB were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NFκB activation. TLR4 was expressed on THP-1 cells and pretreatment of cells with a Toll-like receptor 4 (TLR4) neutralizing antibody (HTA125) significantly blunted LPS-induced MAPK and NFκB activation and ensuing CD40 expression. Additional studies with murine macrophages expressing wild type and mutated TLR4 showed that TLR4 was implicated in LPS-induced ERK and NFκB activation, and CD40 expression. Moreover, blockage of MAPK and NFκB activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NFκB

  6. Mycobacterium tuberculosis-infected human monocytes down-regulate microglial MMP-2 secretion in CNS tuberculosis via TNFα, NFκB, p38 and caspase 8 dependent pathways

    Directory of Open Access Journals (Sweden)

    Elkington Paul T

    2011-05-01

    Full Text Available Abstract Tuberculosis (TB of the central nervous system (CNS is a deadly disease characterized by extensive tissue destruction, driven by molecules such as Matrix Metalloproteinase-2 (MMP-2 which targets CNS-specific substrates. In a simplified cellular model of CNS TB, we demonstrated that conditioned medium from Mycobacterium tuberculosis-infected primary human monocytes (CoMTb, but not direct infection, unexpectedly down-regulates constitutive microglial MMP-2 gene expression and secretion by 72.8% at 24 hours, sustained up to 96 hours (P M.tb-infected monocyte-dependent networks paradoxically involves the pro-inflammatory mediators TNF-α, p38 MAP kinase and NFκB in addition to a novel caspase 8-dependent pathway.

  7. A hot water extract of Curcuma longa inhibits adhesion molecule protein expression and monocyte adhesion to TNF-α-stimulated human endothelial cells.

    Science.gov (United States)

    Kawasaki, Kengo; Muroyama, Koutarou; Yamamoto, Norio; Murosaki, Shinji

    2015-01-01

    The recruitment of arterial leukocytes to endothelial cells is an important step in the progression of various inflammatory diseases. Therefore, its modulation is thought to be a prospective target for the prevention or treatment of such diseases. Adhesion molecules on endothelial cells are induced by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), and contribute to the recruitment of leukocytes. In the present study, we investigated the effect of hot water extract of Curcuma longa (WEC) on the protein expression of adhesion molecules, monocyte adhesion induced by TNF-α in human umbilical vascular endothelial cells (HUVECs). Treatment of HUVECs with WEC significantly suppressed both TNF-α-induced protein expression of adhesion molecules and monocyte adhesion. WEC also suppressed phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) induced by TNF-α in HUVECs, suggesting that WEC inhibits the NF-κB signaling pathway.

  8. In vitro differentiation of human monocytes to macrophages: change of PDE profile and its relationship to suppression of tumour necrosis factor-α release by PDE inhibitors

    Science.gov (United States)

    Gantner, Florian; Kupferschmidt, Rochus; Schudt, Christian; Wendel, Albrecht; Hatzelmann, Armin

    1997-01-01

    During in vitro culture in 10% human AB serum, human peripheral blood monocytes acquire a macrophage-like phenotype. The underlying differentiation was characterized by increased activities of the macrophage marker enzymes unspecific esterase (NaF-insensitive form) and acid phosphatase, as well as by a down-regulation in surface CD14 expression. In parallel, a dramatic change in the phosphodiesterase (PDE) profile became evident within a few days that strongly resembled that previously described for human alveolar macrophages. Whereas PDE1 and PDE3 activities were augmented, PDE4 activity, which represented the major cyclic AMP-hydrolysing activity of peripheral blood monocytes, rapidly declined. Monocytes and monocyte-derived macrophages responded to lipopolysaccharide (LPS) with the release of tumour necrosis factor-α (TNF). In line with the change in CD14 expression, the EC50 value of LPS for induction of TNF release increased from approximately 0.1 ng ml−1 in peripheral blood monocytes to about 2 ng ml−1 in macrophages. Both populations of cells were equally susceptible towards inhibition of TNF release by cyclic AMP elevating agents such as dibutyryl cyclic AMP, prostaglandin E2 (PGE2) or forskolin, which all led to a complete abrogation of TNF production in a concentration-dependent manner and which were more efficient than the glucocorticoid dexamethasone. In monocytes, PDE4 selective inhibitors (rolipram, RP73401) suppressed TNF formation by 80%, whereas motapizone, a PDE3 selective compound, exerted a comparatively weak effect (10–15% inhibition). Combined use of PDE3 plus PDE4 inhibitors resulted in an additive effect and fully abrogated LPS-induced TNF release as did the mixed PDE3/4 inhibitor tolafentrine. In monocyte-derived macrophages, neither PDE3- nor PDE4-selective drugs markedly affected TNF generation when used alone (<15% inhibition), whereas in combination, they led to a maximal inhibition of TNF formation by about 40–50

  9. Screening of Compounds Toxicity against Human Monocytic cell line-THP-1 by Flow Cytometry

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    Pick Neora

    2004-01-01

    Full Text Available The worldwide rapid increase in bacterial resistance to numerous antibiotics requires on-going development of new drugs to enter the market. As the development of new antibiotics is lengthy and costly, early monitoring of compound's toxicity is essential in the development of novel agents. Our interest is in a rapid, simple, high throughput screening method to assess cytotoxicity induced by potential agents. Some intracellular pathogens, such as Mycobacterium tuberculosis primary site of infection is human alveolar macrophages. Thus, evaluation of candidate drugs for macrophage toxicity is crucial. Protocols for high throughput drug toxicity screening of macrophages using flow cytometry are lacking in the literature. For this application we modified a preexisting technique, propidium iodide (PI exclusion staining and utilized it for rapid toxicity tests. Samples were prepared in 96 well plates and analyzed by flow cytometry, which allowed for rapid, inexpensive and precise assessment of compound's toxicity associated with cell death.

  10. Acute stress reduces wound-induced activation of microbicidal potential of ex vivo isolated human monocyte-derived macrophages.

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    Ulrike Kuebler

    Full Text Available BACKGROUND: Psychological stress delays wound healing but the precise underlying mechanisms are unclear. Macrophages play an important role in wound healing, in particular by killing microbes. We hypothesized that (a acute psychological stress reduces wound-induced activation of microbicidal potential of human monocyte-derived macrophages (HMDM, and (b that these reductions are modulated by stress hormone release. METHODS: Fourty-one healthy men (mean age 35 ± 13 years were randomly assigned to either a stress or stress-control group. While the stress group underwent a standardized short-term psychological stress task after catheter-induced wound infliction, stress-controls did not. Catheter insertion was controlled. Assessing the microbicidal potential, we investigated PMA-activated superoxide anion production by HMDM immediately before and 1, 10 and 60 min after stress/rest. Moreover, plasma norepinephrine and epinephrine and salivary cortisol were repeatedly measured. In subsequent in vitro studies, whole blood was incubated with norepinephrine in the presence or absence of phentolamine (norepinephrine blocker before assessing HMDM microbicidal potential. RESULTS: Compared with stress-controls, HMDM of the stressed subjects displayed decreased superoxide anion-responses after stress (p's <.05. Higher plasma norepinephrine levels statistically mediated lower amounts of superoxide anion-responses (indirect effect 95% CI: 4.14-44.72. Norepinephrine-treated HMDM showed reduced superoxide anion-production (p<.001. This effect was blocked by prior incubation with phentolamine. CONCLUSIONS: Our results suggest that acute psychological stress reduces wound-induced activation of microbicidal potential of HMDM and that this reduction is mediated by norepinephrine. This might have implications for stress-induced impairment in wound healing.

  11. TIE-2 and VEGFR kinase activities drive immunosuppressive function of TIE-2-expressing monocytes in human breast tumors.

    Science.gov (United States)

    Ibberson, Mark; Bron, Sylvian; Guex, Nicolas; Faes-van't Hull, Eveline; Ifticene-Treboux, Assia; Henry, Luc; Lehr, Hans-Anton; Delaloye, Jean-François; Coukos, George; Xenarios, Ioannis; Doucey, Marie-Agnès

    2013-07-01

    Tumor-associated TIE-2-expressing monocytes (TEM) are highly proangiogenic cells critical for tumor vascularization. We previously showed that, in human breast cancer, TIE-2 and VEGFR pathways control proangiogenic activity of TEMs. Here, we examine the contribution of these pathways to immunosuppressive activity of TEMs. We investigated the changes in immunosuppressive activity of TEMs and gene expression in response to specific kinase inhibitors of TIE-2 and VEGFR. The ability of tumor TEMs to suppress tumor-specific T-cell response mediated by tumor dendritic cells (DC) was measured in vitro. Characterization of TEM and DC phenotype in addition to their interaction with T cells was done using confocal microscopic images analysis of breast carcinomas. TEMs from breast tumors are able to suppress tumor-specific immune responses. Importantly, proangiogenic and suppressive functions of TEMs are similarly driven by TIE-2 and VEGFR kinase activity. Furthermore, we show that tumor TEMs can function as antigen-presenting cells and elicit a weak proliferation of T cells. Blocking TIE-2 and VEGFR kinase activity induced TEMs to change their phenotype into cells with features of myeloid dendritic cells. We show that immunosuppressive activity of TEMs is associated with high CD86 surface expression and extensive engagement of T regulatory cells in breast tumors. TIE-2 and VEGFR kinase activity was also necessary to maintain high CD86 surface expression levels and to convert T cells into regulatory cells. These results suggest that TEMs are plastic cells that can be reverted from suppressive, proangiogenic cells into cells that are able to mediate an antitumoral immune response. ©2013 AACR.

  12. Activation of the aryl hydrocarbon receptor affects activation and function of human monocyte-derived dendritic cells.

    Science.gov (United States)

    Wang, C; Ye, Z; Kijlstra, A; Zhou, Y; Yang, P

    2014-08-01

    Aryl hydrocarbon receptor (AhR) is well known for mediating the toxic effects of dioxin-containing pollutants, but has also been shown to be involved in the natural regulation of the immune response. In this study, we investigated the effect of AhR activation by its endogenous ligands 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) on the differentiation, maturation and function of monocyte-derived DCs in Behçet's disease (BD) patients. In this study, we showed that AhR activation by FICZ and ITE down-regulated the expression of co-stimulatory molecules including human leucocyte antigen D-related (HLA-DR), CD80 and CD86, while it had no effect on the expression of CD83 and CD40 on DCs derived from BD patients and normal controls. Lipopolysaccharide (LPS)-treated dendritic cells (DCs) from active BD patients showed a higher level of interleukin (IL)-1β, IL-6, IL-23 and tumour necrosis factor (TNF)-α production. FICZ or ITE significantly inhibited the production of IL-1β, IL-6, IL-23 and TNF-α, but induced IL-10 production by DCs derived from active BD patients and normal controls. FICZ or ITE-treated DCs significantly inhibited the T helper type 17 (Th17) and Th1 cell response. Activation of AhR either by FICZ or ITE inhibits DC differentiation, maturation and function. Further studies are needed to investigate whether manipulation of the AhR pathway may be used to treat BD or other autoimmune diseases. © 2014 British Society for Immunology.

  13. The role and mechanism of KCa3.1 channels in human monocyte migration induced by palmitic acid.

    Science.gov (United States)

    Ma, Xiao-Zhen; Pang, Zheng-Da; Wang, Jun-Hong; Song, Zheng; Zhao, Li-Mei; Du, Xiao-Jun; Deng, Xiu-Ling

    2018-05-21

    Monocyte migration into diseased tissues contributes to the pathogenesis of diseases. Intermediate-conductance Ca 2+ -activated K + (K Ca 3.1) channels play an important role in cell migration. However, the role of K Ca 3.1 channels in mediating monocyte migration induced by palmitic acid (PA) is still unclear. Using cultured THP-1 cells and peripheral blood mononuclear cells from healthy subjects, we investigated the role and signaling mechanisms of K Ca 3.1 channels in mediating the migration induced by PA. Using methods of Western blotting analysis, RNA interference, cell migration assay and ELISA, we found that PA-treated monocytes exhibited increment of the protein levels of K Ca 3.1 channel and monocyte chemoattractant protein-1 (MCP-1), and the effects were reversed by co-incubation of PA with anti-TLR2/4 antibodies or by specific inhibitors of p38-MAPK, or NF-κB. In addition, PA increased monocyte migration, which was abolished by a specific K Ca 3.1 channel blocker, TRAM-34, or K Ca 3.1 small interfering RNA (siRNA). The expression and secretion of MCP-1 induced by PA was also similarly prevented by TRAM-34 and K Ca 3.1 siRNA. These results demonstrate for the first time that PA upregulates K Ca 3.1 channels through TLR2/4, p38-MAPK and NF-κB pathway to promote the expression of MCP-1, and then induce the trans-endothelial migration of monocytes. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Ganoderma lucidum polysaccharides in human monocytic leukemia cells: from gene expression to network construction

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    Ou Chern-Han

    2007-11-01

    Full Text Available Abstract Background Ganoderma lucidum has been widely used as a herbal medicine for promoting health and longevity in China and other Asian countries. Polysaccharide extracts from Ganoderma lucidum have been reported to exhibit immuno-modulating and anti-tumor activities. In previous studies, F3, the active component of the polysaccharide extract, was found to activate various cytokines such as IL-1, IL-6, IL-12, and TNF-α. This gave rise to our investigation on how F3 stimulates immuno-modulating or anti-tumor effects in human leukemia THP-1 cells. Results Here, we integrated time-course DNA microarray analysis, quantitative PCR assays, and bioinformatics methods to study the F3-induced effects in THP-1 cells. Significantly disturbed pathways induced by F3 were identified with statistical analysis on microarray data. The apoptosis induction through the DR3 and DR4/5 death receptors was found to be one of the most significant pathways and play a key role in THP-1 cells after F3 treatment. Based on time-course gene expression measurements of the identified pathway, we reconstructed a plausible regulatory network of the involved genes using reverse-engineering computational approach. Conclusion Our results showed that F3 may induce death receptor ligands to initiate signaling via receptor oligomerization, recruitment of specialized adaptor proteins and activation of caspase cascades.

  15. Ganoderma lucidum polysaccharides in human monocytic leukemia cells: from gene expression to network construction.

    Science.gov (United States)

    Cheng, Kun-Chieh; Huang, Hsuan-Cheng; Chen, Jenn-Han; Hsu, Jia-Wei; Cheng, Hsu-Chieh; Ou, Chern-Han; Yang, Wen-Bin; Chen, Shui-Tein; Wong, Chi-Huey; Juan, Hsueh-Fen

    2007-11-09

    Ganoderma lucidum has been widely used as a herbal medicine for promoting health and longevity in China and other Asian countries. Polysaccharide extracts from Ganoderma lucidum have been reported to exhibit immuno-modulating and anti-tumor activities. In previous studies, F3, the active component of the polysaccharide extract, was found to activate various cytokines such as IL-1, IL-6, IL-12, and TNF-alpha. This gave rise to our investigation on how F3 stimulates immuno-modulating or anti-tumor effects in human leukemia THP-1 cells. Here, we integrated time-course DNA microarray analysis, quantitative PCR assays, and bioinformatics methods to study the F3-induced effects in THP-1 cells. Significantly disturbed pathways induced by F3 were identified with statistical analysis on microarray data. The apoptosis induction through the DR3 and DR4/5 death receptors was found to be one of the most significant pathways and play a key role in THP-1 cells after F3 treatment. Based on time-course gene expression measurements of the identified pathway, we reconstructed a plausible regulatory network of the involved genes using reverse-engineering computational approach. Our results showed that F3 may induce death receptor ligands to initiate signaling via receptor oligomerization, recruitment of specialized adaptor proteins and activation of caspase cascades.

  16. Suppression of human monocyte tissue factor induction by red wine phenolics and synthetic derivatives of resveratrol.

    Science.gov (United States)

    Kaur, Gurjeet; Roberti, Marinella; Raul, Francis; Pendurthi, Usha R

    2007-01-01

    Prevention of cardiovascular disease through nutritional supplements is growing in popularity throughout the world. Multiple epidemiologic studies found that moderate consumption of alcohol, particularly red wine, lowers mortality rates from coronary heart diseases (CHD). Chronic inflammation and atherosclerosis associated with CHD culminate in aberrant intravascular expression of tissue factor (TF), which triggers blood coagulation leading to thrombosis, a major cause for heart attack. We showed earlier that two red wine phenolics, resveratrol and quercetin, suppressed TF induction in endothelial cells. In the present study, we investigated efficacy of seven resveratrol derivatives, which were shown to be effective in regulating cancer cell growth in vitro at much lower concentrations than the parent compound resveratrol, in inhibiting TF induction in peripheral blood mononuclear cells (PBMCs). We also tested possible synergistic effects of resveratrol and quercetin with the other major red wine phenolics in suppression of lipopolysaccharide-induced TF expression in human PBMCs. We found that several resveratrol derivatives were 2- to 10-fold more efficient than resveratrol in inhibiting TF induction. Our study found no evidence for synergism among red wine polyphenolics. These data suggest that structural alterations of resveratrol can be effective in producing potent antithrombotic agents that will have therapeutic potential in the improvement of cardiovascular health and prevention of CHD. Among major red wine phenolics, quercetin appears to be the predominant suppressor of TF induction.

  17. Suppression of human monocyte tissue factor induction by red wine phenolics and synthetic derivatives of resveratrol

    Science.gov (United States)

    Kaur, Gurjeet; Roberti, Marinella; Raul, Francis; Pendurthi, Usha R.

    2010-01-01

    Prevention of cardiovascular disease through nutritional supplements is growing in popularity throughout the world. Multiple epidemiologic studies found that moderate consumption of alcohol, particularly red wine, lowers mortality rates from coronary heart diseases (CHD). Chronic inflammation and atherosclerosis associated with CHD culminate in aberrant intravascular expression of tissue factor (TF), which triggers blood coagulation leading to thrombosis, a major cause for heart attack. We showed earlier that two red wine phenolics, resveratrol and quercetin, suppressed TF induction in endothelial cells. In the present study, we investigated efficacy of seven resveratrol derivatives, which were shown to be effective in regulating cancer cell growth in vitro at much lower concentrations than the parent compound resveratrol, in inhibiting TF induction in peripheral blood mononuclear cells (PBMCs). We also tested possible synergistic effects of resveratrol and quercetin with the other major red wine phenolics in suppression of lipopolysaccharide-induced TF expression in human PBMCs. We found that several resveratrol derivatives were 2- to 10-fold more efficient than resveratrol in inhibiting TF induction. Our study found no evidence for synergism among red wine polyphenolics. These data suggest that structural alterations of resveratrol can be effective in producing potent antithrombotic agents that will have therapeutic potential in the improvement of cardiovascular health and prevention of CHD. Among major red wine phenolics, quercetin appears to be the predominant suppressor of TF induction. PMID:16507316

  18. Structure of gene and pseudogenes of human apoferritin H

    Energy Technology Data Exchange (ETDEWEB)

    Costanzo, F; Colombo, M; Staempfli, S; Santoro, C; Marone, M; Frank, K; Delius, H; Cortese, R

    1986-01-24

    Ferritin is composed of two subunits, H and L. cDNA's coding for these proteins from human liver, lymphocytes and from the monocyte-like cell line U937 have been cloned and sequenced. Southern blot analysis on total human DNA reveals that there are many DNA segments hybridizing to the apoferritin H and L cDNA probes. In view of the tissue heterogeneity of ferritin molecules, it appeared possible that apoferritin molecules could be coded by a family of genes differentially expressed in various tissues. In this paper, the authors describe the cloning and sequencing of the gene coding for human apoferritin H. This gene has three introns; the exon sequence is identical to that of cDNAs isolated from human liver, lymphocytes, HeLa cells and endothelial cells. In addition they show that at least 15 intronless pseudogenes exist, with features suggesting that there were originated by reverse transcription and insertion. On the basis of these results they conclude that only one gene is responsible for the synthesis of the majority of apoferritin H mRNA in various tissues examined, and that probably all the other DNA segments hybridizing with apoferritin cDNA are pseudogenes.

  19. Fibrin(ogen) is internalized and degraded by activated human monocytoid cells via Mac-1 (CD11b/CD18): a nonplasmin fibrinolytic pathway.

    Science.gov (United States)

    Simon, D I; Ezratty, A M; Francis, S A; Rennke, H; Loscalzo, J

    1993-10-15

    Fibrin(ogen) (FGN) is important for hemostasis and wound healing and is cleared from sites of injury primarily by the plasminogen activator system. However, there is emerging evidence in plasminogen activator-deficient transgenic mice that nonplasmin pathways may be important in fibrin(ogen)olysis, as well. Given the proximity of FGN and monocytes within the occlusive thrombus at sites of vascular injury, we considered the possibility that monocytes may play an ancillary role in the degradation and clearance of fibrin. We found that monocytes possess an alternative fibrinolytic pathway that uses the integrin Mac-1, which directly binds and internalizes FGN, resulting in its lysosomal degradation. At 4 degrees C, FGN binds to U937 monocytoid cells in a specific and saturable manner with a kd of 1.8 mumol/L. Binding requires adenosine diphosphate stimulation and is calcium-dependent. At 37 degrees C, FGN and fibrin monomer (FM) are internalized and degraded at rates of 0.37 +/- 0.13 and 0.55 +/- 0.03 microgram/10(6) cells/h by U937 cells, 1.38 +/- 0.02 and 1.20 +/- 0.30 microgram/10(6) cells/h by THP-1 cells, and 2.10 +/- 0.20 and 2.52 +/- 0.18 micrograms/10(6) cells/h by human peripheral blood mononuclear cells, respectively. The serine protease inhibitors, PPACK and aprotinin, and the specific elastase inhibitor, AAPVCK, do not significantly inhibit degradation. However, degradation is inhibited by chloroquine, suggesting that a lysosomal pathway is involved. Factor X, a competitive ligand with FGN for the Mac-1 receptor, also blocks degradation, as does a monoclonal antibody to the alpha-subunit of Mac-1. Autoradiography of radioiodinated, internalized FGN shows that FGN proteolysis by the pathway produces a unique degradation pattern distinct from that observed with plasmin. In a fibrin clot lysis assay, Mac-1-mediated fibrinolysis contributed significantly to total fibrinolysis. In summary, FGN is internalized and degraded by activated human monocytoid cells via

  20. The effects of dietary fatty acids on the postprandial triglyceride-rich lipoprotein/apoB48 receptor axis in human monocyte/macrophage cells.

    Science.gov (United States)

    Varela, Lourdes M; Ortega-Gomez, Almudena; Lopez, Sergio; Abia, Rocio; Muriana, Francisco J G; Bermudez, Beatriz

    2013-12-01

    Intestinally produced triglyceride-rich lipoproteins (TRL) play an important role in the progression of atherosclerosis. In this study, we investigated the relevance of monounsaturated fatty acid (MUFA) and saturated fatty acid (SFA) in postprandial TRL in affecting the transcriptional activity of the apolipoprotein-B48 receptor (ApoB48R) and its functionality in human monocyte/macrophage cells. Healthy male volunteers were administered four standardized high-fat meals containing butter, high-palmitic sunflower oil, olive oil (ROO) or a mixture of vegetable and fish oils (50 g/m(2) body surface area) to obtain a panel of postprandial TRL with gradual MUFA oleic acid-to-SFA palmitic acid ratios. The increase in this ratio was linearly associated with a decrease of ApoB48R up-regulation and lipid accumulation in THP-1 and primary monocytes. ApoB48R mRNA levels and intracellular triglycerides were also lower in the monocytes from volunteers after the ingestion of the ROO meal when compared to the ingestion of the butter meal. In THP-1 macrophages, the increase in the MUFA oleic acid-to-SFA palmitic acid ratio in the postprandial TRL was linearly correlated with an increase in ApoB48R down-regulation and a decrease in lipid accumulation. We also revealed that the nuclear receptor transcription factors PPARα, PPARβ/δ, and PPARγ and the PPAR-RXR transcriptional complex were involved in sensing the proportion of MUFA oleic acid and SFA palmitic acid, and these were also involved in adjusting the transcriptional activity of ApoB48R. The results of this study support the notion that MUFA-rich dietary fats may prevent excessive lipid accumulation in monocyte/macrophage cells by targeting the postprandial TRL/ApoB48R axis. © 2013.

  1. Fc receptors for mouse IgG1 on human monocytes: polymorphism and role in antibody-induced T cell proliferation.

    Science.gov (United States)

    Tax, W J; Hermes, F F; Willems, R W; Capel, P J; Koene, R A

    1984-09-01

    In previous studies, it was shown that there is polymorphism in the mitogenic effect of mouse IgG1 monoclonal antibodies against the T3 antigen of human T cells. This polymorphism implies that IgG1 anti-T3 antibodies are not mitogenic for T cells from 30% of healthy individuals. The present results demonstrate that this polymorphism is caused by polymorphism of an Fc receptor for mouse IgG1, present on human monocytes. The Fc receptor for murine IgG1 could be detected by a newly developed rosetting assay on monocytes from all individuals responsive to the mitogenic effect of IgG1 anti-T3 antibodies. This Fc receptor was not detectable on monocytes from those individuals exhibiting no mitogenic responses to IgG1 anti-T3 monoclonal antibodies. Cross-linking of T3 antigens appears to be essential for antibody-induced mitosis of T cells, because mononuclear cells that did not proliferate in response to WT 31 (an IgG1 antibody against T3 antigen) showed a proliferative response to Sepharose beads coated with WT 31. The Fc receptor--if functionally present--may be involved in the cross-linking of T3 antigens through anti-T3 antibodies. Further evidence for the involvement of this Fc receptor in antibody-induced T cell proliferation was provided by inhibition studies. Immune complexes containing IgG1 antibodies were able to inhibit the proliferative response to IgG1 anti-T3 antibodies. This inhibition by immune complexes appears to be mediated through the monocyte Fc receptor for mouse IgG1. These findings are important for the interpretation of previously described inhibitory effects of anti-T cell monoclonal antibodies on T cell proliferation, and show that such inhibitory effects may be monocyte-mediated (via immune complexes) rather than caused by a direct involvement of the respective T cell antigens in T cell mitosis. The Fc receptor for mouse IgG1 plays a role in antibody-induced T cell proliferation. Its polymorphism may have important implications for the

  2. The Toll-like receptor 1/2 agonists Pam(3) CSK(4) and human β-defensin-3 differentially induce interleukin-10 and nuclear factor-κB signalling patterns in human monocytes.

    Science.gov (United States)

    Funderburg, Nicholas T; Jadlowsky, Julie K; Lederman, Michael M; Feng, Zhimin; Weinberg, Aaron; Sieg, Scott F

    2011-10-01

    Human β-defensin 3 (hBD-3) activates antigen-presenting cells through Toll-like receptors (TLRs) 1/2. Several TLR1/2 agonists have been identified but little is known about how they might differentially affect cellular activation. We compared the effects of hBD-3 with those of another TLR1/2 agonist, Pam(3) CSK(4) , in human monocytes. Monocytes incubated with hBD-3 or Pam(3) CSK(4) produced interleukin-6 (IL-6), IL-8 and IL-1β, but only Pam(3) CSK(4) induced IL-10. The IL-10 induction by Pam(3) CSK(4) caused down-modulation of the co-stimulatory molecule, CD86, whereas CD86 expression was increased in monocytes exposed to hBD-3. Assessment of signalling pathways linked to IL-10 induction indicated that mitogen-activated protein kinases were activated similarly by hBD-3 or Pam(3) CSK(4) , whereas the non-canonical nuclear factor-κB pathway was only induced by Pam(3) CSK(4) . Our data suggest that the lack of non-canonical nuclear factor-κB signalling by hBD-3 could contribute to the failure of this TLR agonist to induce production of the anti-inflammatory cytokine, IL-10, in human monocytes. © 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.

  3. Functional role of CD11c+ monocytes in atherogenesis associated with hypercholesterolemia

    Science.gov (United States)

    Monocyte activation and migration into the arterial wall are key events in atherogenesis associated with hypercholesterolemia. CD11c/CD18, a beta2 integrin expressed on human monocytes and a subset of mouse monocytes, has been shown to play a distinct role in human monocyte adhesion on endothelial c...

  4. Platelet-derived growth factor (PDGF-BB-mediated induction of monocyte chemoattractant protein 1 in human astrocytes: implications for HIV-associated neuroinflammation

    Directory of Open Access Journals (Sweden)

    Bethel-Brown Crystal

    2012-12-01

    Full Text Available Abstract Chemokine (C-C motif ligand 2, also known as monocyte chemoattractant protein 1 (MCP-1 is an important factor for the pathogenesis of HIV-associated neurocognitive disorders (HAND. The mechanisms of MCP-1-mediated neuropathogenesis, in part, revolve around its neuroinflammatory role and the recruitment of monocytes into the central nervous system (CNS via the disrupted blood-brain barrier (BBB. We have previously demonstrated that HIV-1/HIV-1 Tat upregulate platelet-derived growth factor (PDGF-BB, a known cerebrovascular permeant; subsequently, the present study was aimed at exploring the regulation of MCP-1 by PDGF-BB in astrocytes with implications in HAND. Specifically, the data herein demonstrate that exposure of human astrocytes to HIV-1 LAI elevated PDGF-B and MCP-1 levels. Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels. MCP-1 induction was regulated by activation of extracellular-signal-regulated kinase (ERK1/2, c-Jun N-terminal kinase (JNK and p38 mitogen-activated protein (MAP kinases and phosphatidylinositol 3-kinase (PI3K/Akt pathways and the downstream transcription factor, nuclear factor κB (NFκB. Chromatin immunoprecipitation (ChIP assays demonstrated increased binding of NFκB to the human MCP-1 promoter following PDGF-BB exposure. Conditioned media from PDGF-BB-treated astrocytes increased monocyte transmigration through human brain microvascular endothelial cells (HBMECs, an effect that was blocked by STI-571, a tyrosine kinase inhibitor (PDGF receptor (PDGF-R blocker. PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays. Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND. These results suggest that astrocyte

  5. Human bladder uroepithelial cells synergize with monocytes to promote IL-10 synthesis and other cytokine responses to uropathogenic Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Benjamin L Duell

    Full Text Available Urinary tract infections are a major source of morbidity for women and the elderly, with Uropathogenic Escherichia coli (UPEC being the most prevalent causative pathogen. Studies in recent years have defined a key anti-inflammatory role for Interleukin-10 (IL-10 in urinary tract infection mediated by UPEC and other uropathogens. We investigated the nature of the IL-10-producing interactions between UPEC and host cells by utilising a novel co-culture model that incorporated lymphocytes, mononuclear and uroepithelial cells in histotypic proportions. This co-culture model demonstrated synergistic IL-10 production effects between monocytes and uroepithelial cells following infection with UPEC. Membrane inserts were used to separate the monocyte and uroepithelial cell types during infection and revealed two synergistic IL-10 production effects based on contact-dependent and soluble interactions. Analysis of a comprehensive set of immunologically relevant biomarkers in monocyte-uroepithelial cell co-cultures highlighted that multiple cytokine, chemokine and signalling factors were also produced in a synergistic or antagonistic fashion. These results demonstrate that IL-10 responses to UPEC occur via multiple interactions between several cells types, implying a complex role for infection-related IL-10 during UTI. Development and application of the co-culture model described in this study is thus useful to define the degree of contact dependency of biomarker production to UPEC, and highlights the relevance of histotypic co-cultures in studying complex host-pathogen interactions.

  6. Inhibitory effects of Kaempferia parviflora extract on monocyte adhesion and cellular reactive oxygen species production in human umbilical vein endothelial cells.

    Science.gov (United States)

    Horigome, Satoru; Yoshida, Izumi; Ito, Shihomi; Inohana, Shuichi; Fushimi, Kei; Nagai, Takeshi; Yamaguchi, Akihiro; Fujita, Kazuhiro; Satoyama, Toshiya; Katsuda, Shin-Ichi; Suzuki, Shinobu; Watai, Masatoshi; Hirose, Naoto; Mitsue, Takahiro; Shirakawa, Hitoshi; Komai, Michio

    2017-04-01

    The rhizome of Kaempferia parviflora (KP) is used in traditional Thai medicine. In this study, we investigated the effects of an ethanol KP extract and two of its components [5,7-dimethoxyflavone (DMF) and 5-hydroxy-3,7,3',4'-tetramethoxyflavone (TMF)] on monocyte adhesion and cellular reactive oxygen species (ROS) production in human umbilical vein endothelial cells (HUVECs), which provide an in vitro model of events relevant to the development and progression of atherosclerosis. RAW264.7 mouse macrophage-like cells were incubated with various concentrations of KP extract or polymethoxyflavonoids and stimulated with lipopolysaccharide prior to measuring nitrite levels in the culture media. Monocyte adhesion was evaluated by measuring the fluorescently labeled human monocytic leukemia THP-1 cells that is attached to tumor necrosis factor-α (TNF-α)-stimulated HUVECs. Cellular ROS production was assessed by measuring cellular antioxidant activity using pyocyanin-stimulated HUVECs. KP extract and DMF reduced nitrite levels (as indicator of nitric oxide production) in LPS-stimulated RAW264.7 cells and also inhibited THP-1 cell adhesion to HUVECs. These treatments induced mRNA expression of endothelial nitric oxide synthase in TNF-α-stimulated HUVECs and downregulated that of various cell adhesion molecules, inflammatory mediators, and endothelial function-related genes. Angiotensin-converting enzyme activity was inhibited by KP extract in vitro. Furthermore, KP extract, DMF, and TMF inhibited the production of cellular ROS in pyocyanin-stimulated HUVECs. KP extract, DMF, and TMF showed potential anti-inflammatory and antioxidant effects in these in vitro models, properties that would inhibit the development and progression of atherosclerosis.

  7. Age Increases Monocyte Adhesion on Collagen

    Science.gov (United States)

    Khalaji, Samira; Zondler, Lisa; Kleinjan, Fenneke; Nolte, Ulla; Mulaw, Medhanie A.; Danzer, Karin M.; Weishaupt, Jochen H.; Gottschalk, Kay-E.

    2017-05-01

    Adhesion of monocytes to micro-injuries on arterial walls is an important early step in the occurrence and development of degenerative atherosclerotic lesions. At these injuries, collagen is exposed to the blood stream. We are interested whether age influences monocyte adhesion to collagen under flow, and hence influences the susceptibility to arteriosclerotic lesions. Therefore, we studied adhesion and rolling of human peripheral blood monocytes from old and young individuals on collagen type I coated surface under shear flow. We find that firm adhesion of monocytes to collagen type I is elevated in old individuals. Pre-stimulation by lipopolysaccharide increases the firm adhesion of monocytes homogeneously in older individuals, but heterogeneously in young individuals. Blocking integrin αx showed that adhesion of monocytes to collagen type I is specific to the main collagen binding integrin αxβ2. Surprisingly, we find no significant age-dependent difference in gene expression of integrin αx or integrin β2. However, if all integrins are activated from the outside, no differences exist between the age groups. Altered integrin activation therefore causes the increased adhesion. Our results show that the basal increase in integrin activation in monocytes from old individuals increases monocyte adhesion to collagen and therefore the risk for arteriosclerotic plaques.

  8. The effects of 60Co γ-ray irradiation on the cytoskeleton of mouse peritoneal macrophages and human peripheral blood monocytes in vitro

    International Nuclear Information System (INIS)

    Chen Xiaomei; Guo Yuhua; Yin Zhiwei; Mao Zijun

    1990-03-01

    The whole mount cell electron microscopy in combination with selective extraction method for preparing cytoskeletal framework was applied. Cy toskeleton prepared by Triton X-100 treatment of mouse peritoneal macrophages and human peripheral blood monocytes appeared in electron microscopy as a highly organized and interconnected three-dimensional matrix of different fibrous elements. Since such cytoskeletons are open membrane-free system, individual fibrous organizations can be identified by specific antibodies. An indirect immunogold procedure using monoclonal anti-tubulin or anti-actin antibodies was applied to visualize tubulin-or actin-containing structures. The three-dimensional visualization of Triton X-100 resistant cytoskeletons had been used to demonstrate that different doses of 60 Co γ-ray caused a distinctive and reproducible alterations of the cytoskeletons of intact mouse peritoneal macrophages and human peripheral blood monocytes in vitro. The results showed that there were some similar alterations with those caused by cytochalasin B and by colchicine. From these observations and other workers' studies, it's likely that 60 Co γ-ray irradiation may inhibit cytoplasmic microtubule and microfilament assembling

  9. Involvement of JNK and NF-κB pathways in lipopolysaccharide (LPS)-induced BAG3 expression in human monocytic cells.

    Science.gov (United States)

    Wang, Hua-Qin; Meng, Xin; Liu, Bao-Qin; Li, Chao; Gao, Yan-Yan; Niu, Xiao-Fang; Li, Ning; Guan, Yifu; Du, Zhen-Xian

    2012-01-01

    Lipopolysaccharide (LPS) is an outer-membrane glycolipid component of Gram-negative bacteria known for its fervent ability to activate monocytic cells and for its potent proinflammatory capabilities. Bcl-2-associated athanogene 3 (BAG3) is a survival protein that has been shown to be stimulated during cell response to stressful conditions, such as exposure to high temperature, heavy metals, proteasome inhibition, and human immunodeficiency virus 1 (HIV-1) infection. In addition, BAG3 regulates replication of Varicella-Zoster Virus (VZV) and Herpes Simplex Virus (HSV) replication, suggesting that BAG3 could participate in the host response to infection. In the current study, we found that LPS increased the expression of BAG3 in a dose- and time-dependent manner. Actinomycin D completely blocked the LPS-induced BAG3 accumulation, as well as LPS activated the proximal promoter of BAG3 gene, supported that the induction by LPS occurred at the level of gene transcription. LPS-induced BAG3 expression was blocked by JNK or NF-κB inhibition, suggesting that JNK and NF-κB pathways participated in BAG3 induction by LPS. In addition, we also found that induction of BAG3 was implicated in monocytic cell adhesion to extracellular matrix induced by LPS. Overall, the data support that BAG3 is induced by LPS via JNK and NF-κB-dependent signals, and involved in monocytic cell-extracellular matrix interaction, suggesting that BAG3 may have a role in the host response to LPS stimulation. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Performance comparison of two microarray platforms to assess differential gene expression in human monocyte and macrophage cells

    Directory of Open Access Journals (Sweden)

    Montalescot Gilles

    2008-06-01

    Full Text Available Abstract Background In this study we assessed the respective ability of Affymetrix and Illumina microarray methodologies to answer a relevant biological question, namely the change in gene expression between resting monocytes and macrophages derived from these monocytes. Five RNA samples for each type of cell were hybridized to the two platforms in parallel. In addition, a reference list of differentially expressed genes (DEG was generated from a larger number of hybridizations (mRNA from 86 individuals using the RNG/MRC two-color platform. Results Our results show an important overlap of the Illumina and Affymetrix DEG lists. In addition, more than 70% of the genes in these lists were also present in the reference list. Overall the two platforms had very similar performance in terms of biological significance, evaluated by the presence in the DEG lists of an excess of genes belonging to Gene Ontology (GO categories relevant for the biology of monocytes and macrophages. Our results support the conclusion of the MicroArray Quality Control (MAQC project that the criteria used to constitute the DEG lists strongly influence the degree of concordance among platforms. However the importance of prioritizing genes by magnitude of effect (fold change rather than statistical significance (p-value to enhance cross-platform reproducibility recommended by the MAQC authors was not supported by our data. Conclusion Functional analysis based on GO enrichment demonstrates that the 2 compared technologies delivered very similar results and identified most of the relevant GO categories enriched in the reference list.

  11. Modulation of neutrophil and monocyte function by recombinant human granulocyte macrophage colony-stimulating factor in patients with lymphoma

    DEFF Research Database (Denmark)

    Kharazmi, A; Nielsen, H; Hovgaard, D

    1991-01-01

    by up to 43-fold. rhGM-CSF treatment did not affect degranulation of the neutrophils as measured by release of vitamin B12 binding protein. Degree of modulation of neutrophil and monocyte function by rhGM-CSF was independent of rhGM-CSF dosages administered. These data suggest that phagocytic defence...... and chemiluminescence responses to f-Met-Leu-Phe, zymosan activated serum (ZAS) and opsonized zymosan (OZ) were determined. It was observed that chemotactic response of neutrophils to f-Met-Leu-Phe and ZAS was reduced, whereas the chemiluminescence response of both cell types to f-Met-Leu-Phe and zymosan was enhanced...

  12. Monocyte scintigraphy in rheumatoid arthritis: the dynamics of monocyte migration in immune-mediated inflammatory disease.

    Directory of Open Access Journals (Sweden)

    Rogier M Thurlings

    2009-11-01

    Full Text Available Macrophages are principal drivers of synovial inflammation in rheumatoid arthritis (RA, a prototype immune-mediated inflammatory disease. Conceivably, synovial macrophages are continuously replaced by circulating monocytes in RA. Animal studies from the 1960s suggested that macrophage replacement by monocytes is a slow process in chronic inflammatory lesions. Translation of these data into the human condition has been hampered by the lack of available techniques to analyze monocyte migration in man.We developed a technique that enabled us to analyze the migration of labelled autologous monocytes in RA patients using single photon emission computer tomography (SPECT. We isolated CD14+ monocytes by CliniMACS in 8 patients and labeled these with technetium-99m (99mTc-HMPAO. Monocytes were re-infused into the same patient. Using SPECT we calculated that a very small but specific fraction of 3.4 x 10(-3 (0.95-5.1 x 10(-3 % of re-infused monocytes migrated to the inflamed joints, being detectable within one hour after re-infusion.The results indicate monocytes migrate continuously into the inflamed synovial tissue of RA patients, but at a slow macrophage-replacement rate. This suggests that the rapid decrease in synovial macrophages that occurs after antirheumatic treatment might rather be explained by an alteration in macrophage retention than in monocyte influx and that RA might be particularly sensitive to treatments targeting inflammatory cell retention.

  13. Human tissue factor: cDNA sequence and chromosome localization of the gene

    International Nuclear Information System (INIS)

    Scarpati, E.M.; Wen, D.; Broze, G.J. Jr.; Miletich, J.P.; Flandermeyer, R.R.; Siegel, N.R.; Sadler, J.E.

    1987-01-01

    A human placenta cDNA library in λgt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, λHTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of λHTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of ∼ 3.2 kilobases in poly(A) + RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased several fold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of λHTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(a) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser. The 3'-noncoding region contains an inverted Alu family repetitive sequence. The tissue factor gene was localized to chromosome 1 by hybridization of the cDNA insert of λHTF8 to flow-sorted human chromosomes

  14. Generation of dendritic cells from human bone marrow mononuclear cells: advantages for clinical application in comparison to peripheral blood monocyte derived cells.

    Science.gov (United States)

    Bai, L; Feuerer, M; Beckhove, P; Umansky, V; Schirrmacher, V

    2002-02-01

    Dendritic cells (DCs) currently used for vaccination in clinical studies to induce immunity against malignant cells are normally generated from peripheral blood-derived monocytes. Here we studied conditions for the generation of DCs from unseparated human bone marrow (BM) mononuclear cells and compared them functionally with DCs from blood. The two types of DCs, from bone marrow (BM-DC) and peripheral blood (BL-DC), were generated in parallel from the same normal healthy donors by culturing in serum-free X-VIVO 20 medium containing GM-CSF and IL-4, and then the phenotypes and functions were compared. BM-DC generation occurred in 14 days and involved proliferative expansion from CD34 stem cells and differentiation while BL-DC generation occurred in 7 days from CD14 monocytes and involved only differentiation. A 7- to 25-fold higher number of DCs could be obtained from BM than from blood. BM-DC had similar phenotypes as BL-DC. The capacity to stimulate MLR reactivity in allogeneic T lymphocytes was higher with BM-DC than that with BL-DC. Also, the capacity to stimulate autologous memory T cell responses to tetanus toxoid (TT) or tuberculin (PPD) was higher with BM-DC than with BL-DC. These results suggest that BM-DC as produced here may be a very economic and useful source of professional antigen-presenting cells for anti-tumor immunotherapeutic protocols.

  15. Interferon-α-conditioned human monocytes combine a Th1-orienting attitude with the induction of autologous Th17 responses: role of IL-23 and IL-12.

    Directory of Open Access Journals (Sweden)

    Stefano M Santini

    Full Text Available IFN-α exerts multiple effects leading to immune protection against pathogens and cancer as well to autoimmune reactions by acting on monocytes and dendritic cells. We analyzed the versatility of human monocytes conditioned by IFN-α towards dendritic cell differentiation (IFN-DC in shaping the autologous T-helper response. Priming of naïve CD4 T cells with autologous IFN-DC in the presence of either SEA or anti-CD3, resulted, in addition to a prominent expansion of CXCR3+ IFN-γ-producing CD4 Th1 cells, in the emergence of two distinct subsets of IL-17-producing CD4 T cells: i a predominant Th17 population selectively producing IL-17 and expressing CCR6; ii a minor Th1/Th17 population, producing both IL-17 and IFN-γ. After phagocytosis of apoptotic cells, IFN-DC induced Th17 cell expansion and IL-17 release. Notably, the use of neutralizing antibodies revealed that IL-23 was an essential cytokine in mediating Th17 cell development by IFN-DC. The demonstration of the IFN-DC-induced expansion of both Th1 and Th17 cell populations reveals the intrinsic plasticity of these DC in orienting the immune response and provides a mechanistic link between IFN-α and the onset of autoimmune phenomena, which have been correlated with both IL-17 production and exposure to IFN-α.

  16. Functional characterization and phenotypic monitoring of human hematopoietic stem cell expansion and differentiation of monocytes and macrophages by whole-cell mass spectrometry

    Directory of Open Access Journals (Sweden)

    Guido Vogel

    2018-01-01

    Full Text Available The different facets of macrophages allow them to play distinct roles in tissue homeostasis, tissue repair and in response to infections. Individuals displaying dysregulated macrophage functions are proposed to be prone to inflammatory disorders or infections. However, this being a cause or a consequence of the pathology remains often unclear. In this context, we isolated and expanded CD34+ HSCs from healthy blood donors and derived them into CD14+ myeloid progenitors which were further enriched and differentiated into macrophages. Aiming for a comprehensive phenotypic profiling, we generated whole-cell mass spectrometry (WCMS fingerprints of cell samples collected along the different stages of the differentiation process to build a predictive model using a linear discriminant analysis based on principal components. Through the capacity of the model to accurately predict sample's identity of a validation set, we demonstrate that WCMS profiles obtained from bona fide blood monocytes and respectively derived macrophages mirror profiles obtained from equivalent HSC derivatives. Finally, HSC-derived macrophage functionalities were assessed by quantifying cytokine and chemokine responses to a TLR agonist in a 34-plex luminex assay and by measuring their capacity to phagocytise mycobacteria. These functional read-outs could not discriminate blood monocytes-derived from HSC-derived macrophages. To conclude, we propose that this method opens new avenues to distinguish the impact of human genetics on the dysregulated biological properties of macrophages in pathological conditions.

  17. Consequences of gamma-irradiation on inflammatory cytokine regulation in human monocytes/macrophages; Consequences de l`irradiation gamma sur la regulation des cytokines de l`inflammation dans les monocytes/macrophages humains

    Energy Technology Data Exchange (ETDEWEB)

    Pons, I.; Gras, G.; Dormont, D.

    1995-12-31

    Inflammation is a frequent radiation-induced damage, especially after therapeutic irradiation. In this study, we have investigated, the inflammatory cytokine regulation after ionizing irradiation of monocytes/macrophages from four donors. Semi-quantitative RT-PCR revealed, after in vitro 24 h-differentiated monocytes irradiation between 5 to 40 Gy, no induction of interleukin-I{beta} (IL I{beta}), interleukin-6 (IL-6) and tumor necrosis factor-{alpha} (TNF-{alpha} mRNA) expression. Moreover, protein quantitation shows no significant increase of post-irradiation secretion. (author). 6 refs.

  18. Introducing directly induced microglia-like (iMG cells from fresh human monocytes: A novel translational research tool for psychiatric disorders.

    Directory of Open Access Journals (Sweden)

    Masahiro eOhgidani

    2015-05-01

    Full Text Available Microglia, glial cells with immunological functions, have been implicated in various neurological diseases and psychiatric disorders in rodent studies, and human postmortem and PET studies. However, the deeper molecular implications of living human microglia have not been clarified.Here, we introduce a novel translational research approach focusing on human microglia. We have recently developed a new technique for creating induced microglia-like (iMG cells from human peripheral blood. Two cytokines, GM-CSF and IL-34, converted human monocytes into the iMG cells within 14 days, which show various microglial characterizations; expressing markers, forming a ramified morphology, and phagocytic activity with various cytokine releases. We have already confirmed the applicability of this technique by analyzing iMG cells from a patient of Nasu-Hakola disease (Ohgidani et al., Sci Rep 2014. We herein show possible applications of the iMG cells in translational research.We believe that this iMG technique will open the door to explore various unknown dynamic aspects of human microglia in psychiatric disorders. This also opens new routes for psychopharmacological approach such as drug efficacy screening and personalized medicine.

  19. Tumour-cytolytic human monocyte-derived macrophages: a simple and efficient method for the generation and long-term cultivation as non-adherent cells in a serum-free medium.

    Science.gov (United States)

    Streck, R J; Hurley, E L; Epstein, D A; Pauly, J L

    1992-01-01

    We report a simple and efficient culture procedure for the generation of tumour-cytolytic human monocyte-derived macrophages (MAC). In this method, normal human peripheral blood mononuclear cells, isolated using a conventional Ficoll-Hypaque density gradient procedure, are cultured as a heterogenous leukocyte population in Teflon or other hydrophobic cultureware, in a commercially available serum-free culture medium (M-SFM) that has been formulated specifically for the cultivation and ex vivo stimulation of human monocytes and MAC, and in the absence of exogenous mitogens, antigens, cytokines or other stimulants. This procedure features a negative-selection technique that takes advantage of the differential survival of blood leukocytes. Using the prescribed in vitro conditions, lymphocytes survived relatively poorly, whereas monocytes differentiated in the absence of exogenous stimulants into mature tumour-cytolytic MAC. The MAC were present as non-adherent, single cells that expressed good viability (greater than 95%) for a prolonged period (greater than 60 days). When compared to conventional procedures for generating MAC, the prescribed technique is thought to offer several important advantages in that it: (a) eliminates the tedious and cumbersome monocyte isolation procedures, thus providing a significant savings not only in time and money but also in eliminating repetitive cell manipulations that have often been associated with damage to monocyte morphology and/or function; (b) reduces the loss of monocyte subsets that are not recovered during specific isolation procedures; (c) facilitates harvesting a single cell, non-adherent suspension of immunocompetent MAC suitable for various examinations including analyses defining MAC morphology, cytochemistry, phenotype and function; and (d) eliminates variability and artifacts associated with different sera that are utilised frequently as medium supplements. The utility of the prescribed method is illustrated by the

  20. Maturation of the viral core enhances the fusion of HIV-1 particles with primary human T cells and monocyte-derived macrophages

    International Nuclear Information System (INIS)

    Jiang Jiyang; Aiken, Christopher

    2006-01-01

    HIV-1 infection requires fusion of viral and cellular membranes in a reaction catalyzed by the viral envelope proteins gp120 and gp41. We recently reported that efficient HIV-1 particle fusion with target cells is linked to maturation of the viral core by an activity of the gp41 cytoplasmic domain. Here, we show that maturation enhances the fusion of a variety of recombinant viruses bearing primary and laboratory-adapted Env proteins with primary human CD4 + T cells. Overall, HIV-1 fusion was more dependent on maturation for viruses bearing X4-tropic envelope proteins than for R5-tropic viruses. Fusion of HIV-1 with monocyte-derived macrophages was also dependent on particle maturation. We conclude that the ability to couple fusion to particle maturation is a common feature of HIV-1 Env proteins and may play an important role during HIV-1 replication in vivo

  1. The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

    DEFF Research Database (Denmark)

    Behrendt, N; Rønne, E; Ploug, M

    1990-01-01

    The receptor for human urokinase-type plasminogen activator (u-PA) was purified from phorbol 12-myristate 13-acetate-stimulated U937 cells by temperature-induced phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fluorophosphate-treated u...

  2. A matrix of cholesterol crystals, but not cholesterol alone, primes human monocytes/macrophages for excessive endotoxin-induced production of tumor necrosis factor-alpha. Role in atherosclerotic inflammation?

    DEFF Research Database (Denmark)

    Bendtzen, Klaus; Christensen, Ole; Nielsen, Claus Henrik

    2014-01-01

    When exposed to small amounts of bacterial endotoxin, matrices of cholesterol crystals, but not cholesterol itself, primed human monocytes/macrophages to a highly augmented (>10-fold) production of inflammatory tumor necrosis factor-α. Priming also sensitized the cells, as 10- to 100-fold lower...

  3. Ectopic overexpression of LAPTM5 results in lysosomal targeting and induces Mcl-1 down-regulation, Bak activation, and mitochondria-dependent apoptosis in human HeLa cells.

    Directory of Open Access Journals (Sweden)

    Do Youn Jun

    Full Text Available Human lysosomal-associated protein multispanning membrane 5 (LAPTM5 was identified by an ordered differential display-polymerase chain reaction (ODD-PCR as an up-regulated cDNA fragment during 12-O-tetradecanoylphorbol 13-acetate (TPA-induced differentiation of U937 cells into monocytes/macrophages. After TPA-treatment, the levels of LAPTM5 mRNA and protein increased and reached a maximum at 18-36 h. In healthy human tissues, LAPTM5 mRNA was expressed at high levels in hematopoietic cells and tissues, at low levels in the lung and fetal liver, and was not detected in other non-hematopoietic tissues. LAPTM5 mRNA was detected in immature malignant cells of myeloid lineage, such as K562, HL-60, U937, and THP-1 cells, and in unstimulated peripheral T cells, but was absent or barely detectable in lymphoid malignant or non-hematopoietic malignant cells. The LAPTM5 level in HL-60 cells increased more significantly during TPA-induced monocyte/macrophage differentiation than during DMSO-induced granulocyte differentiation. Ectopic expression of GFP-LAPTM5 or LAPTM5 in HeLa cells exhibited the localization of LAPTM5 to the lysosome. In HeLa cells overexpressing LAPTM5, the Mcl-1 and Bid levels declined markedly and apoptosis was induced via Bak activation, Δψm loss, activation of caspase-9, -8 and -3, and PARP degradation without accompanying necrosis. However, these LAPTM5-induced apoptotic events except for the decline of Bid level were completely abrogated by concomitant overexpression of Mcl-1. The pan-caspase inhibitor (z-VAD-fmk could suppress the LAPTM5-induced apoptotic sub-G1 peak by ~40% but failed to block the induced Δψm loss, whereas the broad-range inhibitor of cathepsins (Cathepsin Inhibitor I could suppress the LAPTM5-induced apoptotic sub-G1 peak and Δψm loss, by ~22% and ~23%, respectively, suggesting that the LAPTM5-mediated Δψm loss was exerted at least in part in a cathepsin-dependent manner. Together, these results

  4. Ectopic overexpression of LAPTM5 results in lysosomal targeting and induces Mcl-1 down-regulation, Bak activation, and mitochondria-dependent apoptosis in human HeLa cells

    Science.gov (United States)

    Jun, Do Youn; Kim, Hyejin; Jang, Won Young; Lee, Ji Young; Fukui, Kiyoshi; Kim, Young Ho

    2017-01-01

    Human lysosomal-associated protein multispanning membrane 5 (LAPTM5) was identified by an ordered differential display-polymerase chain reaction (ODD-PCR) as an up-regulated cDNA fragment during 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced differentiation of U937 cells into monocytes/macrophages. After TPA-treatment, the levels of LAPTM5 mRNA and protein increased and reached a maximum at 18–36 h. In healthy human tissues, LAPTM5 mRNA was expressed at high levels in hematopoietic cells and tissues, at low levels in the lung and fetal liver, and was not detected in other non-hematopoietic tissues. LAPTM5 mRNA was detected in immature malignant cells of myeloid lineage, such as K562, HL-60, U937, and THP-1 cells, and in unstimulated peripheral T cells, but was absent or barely detectable in lymphoid malignant or non-hematopoietic malignant cells. The LAPTM5 level in HL-60 cells increased more significantly during TPA-induced monocyte/macrophage differentiation than during DMSO-induced granulocyte differentiation. Ectopic expression of GFP-LAPTM5 or LAPTM5 in HeLa cells exhibited the localization of LAPTM5 to the lysosome. In HeLa cells overexpressing LAPTM5, the Mcl-1 and Bid levels declined markedly and apoptosis was induced via Bak activation, Δψm loss, activation of caspase-9, -8 and -3, and PARP degradation without accompanying necrosis. However, these LAPTM5-induced apoptotic events except for the decline of Bid level were completely abrogated by concomitant overexpression of Mcl-1. The pan-caspase inhibitor (z-VAD-fmk) could suppress the LAPTM5-induced apoptotic sub-G1 peak by ~40% but failed to block the induced Δψm loss, whereas the broad-range inhibitor of cathepsins (Cathepsin Inhibitor I) could suppress the LAPTM5-induced apoptotic sub-G1 peak and Δψm loss, by ~22% and ~23%, respectively, suggesting that the LAPTM5-mediated Δψm loss was exerted at least in part in a cathepsin-dependent manner. Together, these results demonstrate that

  5. Prediction of the contact sensitizing potential of chemicals using analysis of gene expression changes in human THP-1 monocytes.

    Science.gov (United States)

    Arkusz, Joanna; Stępnik, Maciej; Sobala, Wojciech; Dastych, Jarosław

    2010-11-10

    The aim of this study was to find differentially regulated genes in THP-1 monocytic cells exposed to sensitizers and nonsensitizers and to investigate if such genes could be reliable markers for an in vitro predictive method for the identification of skin sensitizing chemicals. Changes in expression of 35 genes in the THP-1 cell line following treatment with chemicals of different sensitizing potential (from nonsensitizers to extreme sensitizers) were assessed using real-time PCR. Verification of 13 candidate genes by testing a large number of chemicals (an additional 22 sensitizers and 8 nonsensitizers) revealed that prediction of contact sensitization potential was possible based on evaluation of changes in three genes: IL8, HMOX1 and PAIMP1. In total, changes in expression of these genes allowed correct detection of sensitization potential of 21 out of 27 (78%) test sensitizers. The gene expression levels inside potency groups varied and did not allow estimation of sensitization potency of test chemicals. Results of this study indicate that evaluation of changes in expression of proposed biomarkers in THP-1 cells could be a valuable model for preliminary screening of chemicals to discriminate an appreciable majority of sensitizers from nonsensitizers. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  6. Electroporated Antigen-Encoding mRNA Is Not a Danger Signal to Human Mature Monocyte-Derived Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Stefanie Hoyer

    2015-01-01

    Full Text Available For therapeutic cancer vaccination, the adoptive transfer of mRNA-electroporated dendritic cells (DCs is frequently performed, usually with monocyte-derived, cytokine-matured DCs (moDCs. However, DCs are rich in danger-sensing receptors which could recognize the exogenously delivered mRNA and induce DC activation, hence influencing the DCs’ immunogenicity. Therefore, we examined whether electroporation of mRNA with a proper cap and a poly-A tail of at least 64 adenosines had any influence on cocktail-matured moDCs. We used 16 different RNAs, encoding tumor antigens (MelanA, NRAS, BRAF, GNAQ, GNA11, and WT1, and variants thereof. None of those RNAs induced changes in the expression of CD25, CD40, CD83, CD86, and CD70 or the secretion of the cytokines IL-8, IL-6, and TNFα of more than 1.5-fold compared to the control condition, while an mRNA encoding an NF-κB-activation protein as positive control induced massive secretion of the cytokines. To determine whether mRNA electroporation had any effect on the whole transcriptome of the DCs, we performed microarray analyses of DCs of 6 different donors. None of 60,000 probes was significantly different between mock-electroporated DCs and MelanA-transfected DCs. Hence, we conclude that no transcriptional programs were induced within cocktail-matured DCs by electroporation of single tumor-antigen-encoding mRNAs.

  7. Haloperidol Abrogates Matrix Metalloproteinase-9 Expression by Inhibition of NF-κB Activation in Stimulated Human Monocytic Cells

    Directory of Open Access Journals (Sweden)

    Yueh-Lun Lee

    2018-01-01

    Full Text Available Much evidence has indicated that matrix metalloproteinases (MMPs participate in the progression of neuroinflammatory disorders. The present study was undertaken to investigate the inhibitory effect and mechanism of the antipsychotic haloperidol on MMP activation in the stimulated THP-1 monocytic cells. Haloperidol exerted a strong inhibition on tumor necrosis factor- (TNF- α-induced MMP-9 gelatinolysis of THP-1 cells. A concentration-dependent inhibitory effect of haloperidol was observed in TNF-α-induced protein and mRNA expression of MMP-9. On the other hand, haloperidol slightly affected cell viability and tissue inhibition of metalloproteinase-1 levels. It significantly inhibited the degradation of inhibitor-κB-α (IκBα in activated cells. Moreover, it suppressed activated nuclear factor-κB (NF-κB detected by a mobility shift assay, NF-κB reporter gene, and chromatin immunoprecipitation analyses. Consistent with NF-κB inhibition, haloperidol exerted a strong inhibition of lipopolysaccharide- (LPS- induced MMP-9 gelatinolysis but not of transforming growth factor-β1-induced MMP-2. In in vivo studies, administration of haloperidol significantly attenuated LPS-induced intracerebral MMP-9 activation of the brain homogenate and the in situ in C57BL/6 mice. In conclusion, the selective anti-MMP-9 activation of haloperidol could possibly involve the inhibition of the NF-κB signal pathway. Hence, it was found that haloperidol treatment may represent a bystander of anti-MMP actions for its conventional psychotherapy.

  8. Surface modification of biomaterials based on high-molecular polylactic acid and their effect on inflammatory reactions of primary human monocyte-derived macrophages: perspective for personalized therapy.

    Science.gov (United States)

    Stankevich, Ksenia S; Gudima, Alexandru; Filimonov, Victor D; Klüter, Harald; Mamontova, Evgeniya M; Tverdokhlebov, Sergei I; Kzhyshkowska, Julia

    2015-06-01

    Polylactic acid (PLA) based implants can cause inflammatory complications. Macrophages are key innate immune cells that control inflammation. To provide higher biocompatibility of PLA-based implants with local innate immune cells their surface properties have to be improved. In our study surface modification technique for high-molecular PLA (MW=1,646,600g/mol) based biomaterials was originally developed and successfully applied. Optimal modification conditions were determined. Treatment of PLA films with toluene/ethanol=3/7 mixture for 10min with subsequent exposure in 0.001M brilliant green dye (BGD) solution allows to entrap approximately 10(-9)mol/cm(2) model biomolecules. The modified PLA film surface was characterized by optical microscopy, SERS, FT-IR, UV and TG/DTA/DSC analysis. Tensile strain of modified films was determined as well. The effect of PLA films modified with BGD on the inflammatory reactions of primary human monocyte-derived macrophages was investigated. We developed in vitro test-system by differentiating primary monocyte-derived macrophages on a coating material. Type 1 and type 2 inflammatory cytokines (TNFα, CCL18) secretion and histological biomarkers (CD206, stabilin-1) expression were analyzed by ELISA and confocal microscopy respectively. BGD-modified materials have improved thermal stability and good mechanical properties. However, BGD modifications induced additional donor-specific inflammatory reactions and suppressed tolerogenic phenotype of macrophages. Therefore, our test-system successfully demonstrated specific immunomodulatory effects of original and modified PLA-based biomaterials, and can be further applied for the examination of improved coatings for implants and identification of patient-specific reactions to implants. Copyright © 2015. Published by Elsevier B.V.

  9. Cis and trans acting factors involved in human cytomegalovirus experimental and natural latent infection of CD14 (+ monocytes and CD34 (+ cells.

    Directory of Open Access Journals (Sweden)

    Cyprian C Rossetto

    Full Text Available The parameters involved in human cytomegalovirus (HCMV latent infection in CD14 (+ and CD34 (+ cells remain poorly identified. Using next generation sequencing we deduced the transcriptome of HCMV latently infected CD14 (+ and CD34 (+ cells in experimental as well as natural latency settings. The gene expression profile from natural infection in HCMV seropositive donors closely matched experimental latency models, and included two long non-coding RNAs (lncRNAs, RNA4.9 and RNA2.7 as well as the mRNAs encoding replication factors UL84 and UL44. Chromatin immunoprecipitation assays on experimentally infected CD14 (+ monocytes followed by next generation sequencing (ChIP-Seq were employed to demonstrate both UL84 and UL44 proteins interacted with the latent viral genome and overlapped at 5 of the 8 loci identified. RNA4.9 interacts with components of the polycomb repression complex (PRC as well as with the MIE promoter region where the enrichment of the repressive H3K27me3 mark suggests that this lncRNA represses transcription. Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE, which identifies nucleosome-depleted viral DNA, was used to confirm that latent mRNAs were associated with actively transcribed, FAIRE analysis also showed that the terminal repeat (TR region of the latent viral genome is depleted of nucleosomes suggesting that this region may contain an element mediating viral genome maintenance. ChIP assays show that the viral TR region interacts with factors associated with the pre replication complex and a plasmid subclone containing the HCMV TR element persisted in latently infected CD14 (+ monocytes, strongly suggesting that the TR region mediates viral chromosome maintenance.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. 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

  12. Specific interaction of aurintricarboxylic acid with the human immunodeficiency virus/CD4 cell receptor

    International Nuclear Information System (INIS)

    Schols, D.; Baba, M.; Pauwels, R.; Desmyter, J.; De Clercq, E.

    1989-01-01

    The triphenylmethane derivative aurintricarboxylic acid (ATA), but not aurin, selectively prevented the binding of OKT4A/Leu-3a monoclonal antibody (mAb) and, to a lesser extent, OKT4 mAb to the CD4 cell receptor for human immunodeficiency virus type 1 (HIV-1). The effect was seen within 1 min at an ATA concentration of 10 μM in various T4 + cells (MT-4, U-937, peripheral blood lymphocytes, and monocytes). It was dose-dependent and reversible. ATA prevented the attachment of radiolabeled HIV-1 particles to MT-4 cells, which could be expected as the result of its specific binding to the HIV/CD4 receptor. Other HIV inhibitors such as suramin, fuchsin acid, azidothymidine, dextran sulfate, heparin, and pentosan polysulfate did not affect OKT4A/Leu-3a mAb binding to the CD4 receptor, although the sulfated polysaccharides suppressed HIV-1 adsorption to the cells at concentrations required for complete protection against HIV-1 cytopathogenicity. Thus, ATA is a selective marker molecule for the CD4 receptor. ATA also interfered with the staining of membrane-associated HIV-1 glycoprotein gp120 by a mAb against it. These unusual properties of a small molecule of nonimmunological origin may have important implications for the study of CD4/HIV/AIDS pathogenesis and possibly treatment

  13. Anti-Inflammatory effect of Buddleja officinalis on vascular inflammation in human umbilical vein endothelial cells.

    Science.gov (United States)

    Lee, Yun Jung; Moon, Mi Kyoung; Hwang, Sun Mi; Yoon, Jung Joo; Lee, So Min; Seo, Kwan Soo; Kim, Jin Sook; Kang, Dae Gill; Lee, Ho Sub

    2010-01-01

    Vascular inflammation process has been suggested to be an important risk factor in the initiation and development of atherosclerosis. In this study, we investigated whether and by what mechanisms an aqueous extract of Buddleja officinalis (ABO) inhibited the expressions of cellular adhesion molecules, which are relevant to inflammation and atherosclerosis. Pretreatment of human umbilical vein endothelial cells (HUVEC) with ABO (1-10 microg/ml) for 18 hours dose-dependently inhibited TNF-alpha-induced adhesion U937 monocytic cells, as well as mRNA and protein expressions of vascular cell adhesion molecule-1 (VCAM-1), and intercellular cell adhesion molecule-1 (ICAM-1). Pretreatment with ABO also blocked TNF-alpha-induced ROS formation. Nuclear factor-kappa B (NF-kappaB) is required in the transcription of these adhesion molecule genes. Western blot analysis revealed that ABO inhibits the translocation of the p65 subunit of NF-kappaB to the nucleus. ABO inhibited the TNF-alpha-induced degradation of IkappaB-alpha, an inhibitor of NF-kappaB, by inhibiting the phosphorylation of IkappaB-alpha in HUVEC. Taken together, ABO could reduce cytokine-induced endothelial adhesiveness throughout down-regulating intracellular ROS production, NF-kappaB, and adhesion molecule expression in HUVEC, suggesting that the natural herb Buddleja officinalis may have potential implications in atherosclerosis.

  14. The Fc-receptor III of cultured human monocytes. Structural similarity with FcRIII of natural killer cells and role in the extracellular lysis of sensitized erythrocytes

    NARCIS (Netherlands)

    Klaassen, R. J.; Ouwehand, W. H.; Huizinga, T. W.; Engelfriet, C. P.; von dem Borne, A. E.

    1990-01-01

    FcRIII is not present on peripheral blood monocytes, but becomes expressed upon culturing and can be demonstrated on tissue macrophages. We studied the expression of FcRIII of cultured monocytes in detail and compared its structure with FcRIII of neutrophils and NK cells. The cell density of FcRIII

  15. Structure and function of the IFNγ receptor on human mononuclear phagocytes

    International Nuclear Information System (INIS)

    Schreiber, R.D.; Celada, A.

    1986-01-01

    Human mononuclear phagocytes bear a receptor that binds 125 I-IFNγ in a saturable, reversible and specific manner. The receptor consists minimally of a 70 kD polypeptide chain and its expression (5000/cell) and binding affinity (Ka=10 9 M -1 ) are unaffected by cellular activation or differentiation. The receptor's biological relevance was validated by correlating receptor occupancy with induction of a cellular response. 50% maximal induction of Fc receptors on U937 was effected by 0.8 nM IFNγ; the same concentration needed to half saturate U937 IFNγ receptors. Ligand-receptor interaction displayed species specificity but not cellular specificity. The receptors on U937 and human fibroblasts displayed identical ligand binding affinities (1.5-1.8 x 10 9 M -1 ). At 37 0 C, IFNγ bound to U937 in a biphasic manner. The high affinity binding component was due to ligand internalization since purified cell membranes and paraformaldehyde fixed cells displayed only the lower Ka and ligand internalization could be directly demonstrated. Using lysosomotropic amines, the internalized IFNγ-IFNγ receptor complex was tracked into an acid compartment where dissociation occurred. Free intracellular IFNγ was then degraded while free receptor entered an intracellular pool and eventually recycled back to the cell surface

  16. HIV-1 Latency in Monocytes/Macrophages

    Directory of Open Access Journals (Sweden)

    Amit Kumar

    2014-04-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 targets CD4+ T cells and cells of the monocyte/macrophage lineage. HIV pathogenesis is characterized by the depletion of T lymphocytes and by the presence of a population of cells in which latency has been established called the HIV-1 reservoir. Highly active antiretroviral therapy (HAART has significantly improved the life of HIV-1 infected patients. However, complete eradication of HIV-1 from infected individuals is not possible without targeting latent sources of infection. HIV-1 establishes latent infection in resting CD4+ T cells and findings indicate that latency can also be established in the cells of monocyte/macrophage lineage. Monocyte/macrophage lineage includes among others, monocytes, macrophages and brain resident macrophages. These cells are relatively more resistant to apoptosis induced by HIV-1, thus are important stable hideouts of the virus. Much effort has been made in the direction of eliminating HIV-1 resting CD4+ T-cell reservoirs. However, it is impossible to achieve a cure for HIV-1 without considering these neglected latent reservoirs, the cells of monocyte/macrophage lineage. In this review we will describe our current understanding of the mechanism of latency in monocyte/macrophage lineage and how such cells can be specifically eliminated from the infected host.

  17. Homotypic aggregation of human cell lines by HLA class II-, class Ia- and HLA-G-specific monoclonal antibodies

    DEFF Research Database (Denmark)

    Odum, Niels; Ledbetter, J A; Martin, P

    1991-01-01

    Major histocompatibility complex (MHC) class II molecules have been implicated in cell adhesion in two ways. In addition to the well-established role of class II antigens in low-affinity adhesion provided by interactions between class II and CD4, recent data indicated that class II may also induce...... adhesion between T and B cells by activating the CD18/CD11a (LFA-1) adhesion pathway. Here we report that monoclonal antibodies (mAb) against HLA-DR (L243, p4.1, HB10a, VI15) and certain broad class II reacting mAb (TU35, TU39), but not anti-DQ (TU22, Leu-10) mAb, induced homotypic aggregation of human...... class II-positive monocytic (I937) and T leukemic (HUT78) tumor cell lines and Epstein-Barr virus (EBV) transformed B-lymphoid cell lines (EBV-LCL). Class II-negative cell lines (U-937 and the EBV-LCL mutant line 616) were not induced to aggregate. An HLA-G-transfected EBV-LCL, 221-AGN...

  18. Anti-inflammatory properties of clovamide and Theobroma cacao phenolic extracts in human monocytes: evaluation of respiratory burst, cytokine release, NF-κB activation, and PPARγ modulation.

    Science.gov (United States)

    Zeng, Huawu; Locatelli, Monica; Bardelli, Claudio; Amoruso, Angela; Coisson, Jean Daniel; Travaglia, Fabiano; Arlorio, Marco; Brunelleschi, Sandra

    2011-05-25

    There is a great interest in the potential health benefits of biologically active phenolic compounds in cocoa (Theobroma cacao) and dark chocolate. We investigated the anti-inflammatory potential of clovamide (a N-phenylpropenoyl-L-amino acid amide present in cocoa beans) and two phenolic extracts from unroasted and roasted cocoa beans, by evaluating superoxide anion (O(2)(-)) production, cytokine release, and NF-κB activation in human monocytes stimulated by phorbol 12-myristate 13-acetate (PMA). The effects of rosmarinic acid are shown for comparison. Clovamide and rosmarinic acid inhibited PMA-induced O(2)(-) production and cytokine release (with a bell-shaped curve and maximal inhibition at 10-100 nM), as well as PMA-induced NF-κB activation; the two cocoa extracts were less effective. In all tests, clovamide was the most potent compound and also enhanced peroxisome proliferator-activated receptor-γ (PPARγ) activity, which may exert anti-inflammatory effects. These findings indicate clovamide as a possible bioactive compound with anti-inflammatory activity in human cells.

  19. Malarial pigment haemozoin, IFN-gamma, TNF-alpha, IL-1beta and LPS do not stimulate expression of inducible nitric oxide synthase and production of nitric oxide in immuno-purified human monocytes

    Directory of Open Access Journals (Sweden)

    Ceretto Monica

    2007-06-01

    Full Text Available Abstract Background Enhanced production of nitric oxide (NO following upmodulation of the inducible isoform of NO synthase (iNOS by haemozoin (HZ, inflammatory cytokines and LPS may provide protection against Plasmodium falciparum malaria by killing hepatic and blood forms of parasites and inhibiting the cytoadherence of parasitized erythrocytes (RBC to endothelial cells. Monocytes and macrophages are considered to contribute importantly to protective upregulation of iNOS and production of NO. Data obtained with murine phagocytes fed with human HZ and synthetic HZ (sHZ indicate that supplemental treatment of those cells with IFN-gamma elicited significant increases in protein and mRNA expression of iNOS and NO production, providing a potential mechanism linking HZ phagocytosis and increased production of NO. Purpose of this study was to analyse the effect of P. falciparum HZ and sHZ supplemental to treatment with IFN-gamma and/or a stimulatory cytokine-LPS mix on iNOS protein and mRNA expression in immuno-purified human monocytes. Methods Adherent immunopurified human monocytes (purity >85%, and murine phagocytic cell lines RAW 264.7, N11 and ANA1 were fed or not with P. falciparum HZ or sHZ and treated or not with IFN-gamma or a stimulatory cytokine-LPS mix. Production of NO was quantified in supernatants, iNOS protein and mRNA expression were measured after immunoprecipitation and Western blotting and quantitative RT-PCT, respectively. Results Phagocytosis of HZ/sHZ by human monocytes did not increase iNOS protein and mRNA expression and NO production either after stimulation by IFN-gamma or the cytokine-LPS mix. By contrast, in HZ/sHZ-laden murine macrophages, identical treatment with IFN-gamma and the cytokine-LPS mix elicited significant increases in protein and mRNA expression of iNOS and NOS metabolites production, in agreement with literature data. Conclusion Results indicate that human monocytes fed or not with HZ/sHZ were constantly

  20. A sea urchin lectin, SUL-1, from the Toxopneustid sea urchin induces DC maturation from human monocyte and drives Th1 polarization in vitro

    International Nuclear Information System (INIS)

    Takei, Masao; Nakagawa, Hideyuki

    2006-01-01

    The sea urchin Toxopneustes pileolus belonging to the family Toxopneustidae, they have well-developed globiferous pedicellariae with pharmacologically active substances. We have purified a novel sea urchin lectin-1 (SUL-1) from the large globiferous pedicellariae of T. pileolus. Dendritic cells (DC) are professional APC and play a pivotal role in controlling immune responses. This study investigated whether SUL-1 can drive DC maturation from human immature monocyte-derived DC in vitro. Human monocytes were cultured with GM-CSF and IL-4 for 6 days followed by another 1 day in the presence of SUL-1 or LPS. DC harvested on day 7 were examined using functional assays. The expression levels of CD1a, CD80, CD83, CD86 and HLA-DR as expressed by mean fluorescence intensity (MFI) on DC differentiated from immature DC after culture with 1.0 μg/ml of SUL-1 for 1 day were enhanced and decreased endocytic activity. SUL-1-treated DC also displayed enhanced T cell stimulatory capacity in an MLR, as measured by T cell proliferation. Cell surface expression of CD80, CD83 and CD86 on SUL-1-treated DC was inhibited by anti-DC-SIGN mAb, while anti-DC-SIGN mAb had no influence on allogeneic T cell proliferation by SUL-1-treated DC. DC differentiated with SUL-1 induced the differentiation of naive T cell towards a helper T cell type 1 (Th1) response at DC/T (1:5) cells ratio depending on IL-12 secretion. In CTL assay, the production of IFN-γ and 51 Cr release on SUL-1-treated DC were more augmented than of immature DC or LPS-treated DC. SUL-1-treated DC expressed CCR7 and had a high migration to MIP-3β. Intracellular Ca 2+ mobilization in SUL-1-treated DC was also induced by MIP-3β. These results suggest that SUL-1 bindings to DC-SIGN on surface of immature DC may lead to differentiate DC from immature DC. Moreover, it suggests that SUL-1 may be used on DC-based vaccines for cancer immunotherapy

  1. B. abortus RNA is the component involved in the down-modulation of MHC-I expression on human monocytes via TLR8 and the EGFR pathway

    Science.gov (United States)

    Milillo, M. Ayelén; Velásquez, Lis N.; Trotta, Aldana; Delpino, M. Victoria; Balboa, Luciana; Vermeulen, Mónica; Espindola, Sonia L.; Rodriguez-Rodrigues, Nahuel; Fernández, Gabriela C.; Oliveira, Sergio Costa; Giambartolomei, Guillermo H.

    2017-01-01

    Despite eliciting a potent CD8+ T cell response, Brucella abortus is able to persist and establish a chronic infection inside its host. We have previously reported that the infection of human monocytes/macrophages with B. abortus inhibits the IFN-γ-induced MHC-I cell surface expression down-modulating cytotoxic CD8+ T cell responses. MHC-I down-modulation depends on bacterial viability and results from the capacity of B. abortus to retain the MHC-I molecules within the Golgi apparatus. Furthermore, we recently demonstrated that epidermal growth factor receptor (EGFR) pathway is involved in this phenomenon and that this is an early event during infection. However, the components and mechanisms whereby B. abortus is able to down-modulate MHC-I remained to be elucidated. In this study we demonstrated that the down-modulation of MHC-I expression is not mediated by well-known Brucella virulence factors but instead by B. abortus RNA, a PAMP associated to viability (vita-PAMP). Surprisingly, completely degraded RNA was also able to inhibit MHC-I expression to the same extent as intact RNA. Accordingly, B. abortus RNA and its degradation products were able to mimic the MHC-I intracellular retention within the Golgi apparatus observed upon infection. We further demonstrated that TLR8, a single-stranded RNA and RNA degradation products sensor, was involved in MHC-I inhibition. On the other hand, neutralization of the EGFR reversed the MHC-I inhibition, suggesting a connection between the TLR8 and EGFR pathways. Finally, B. abortus RNA-treated macrophages display diminished capacity of antigen presentation to CD8+ T cells. Overall, our results indicate that the vita-PAMP RNA as well as its degradation products constitute novel virulence factors whereby B. abortus, by a TLR8-dependent mechanism and through the EGFR pathway, inhibits the IFN-γ-induced MHC-I surface expression on human monocytes/macrophages. Thus, bacteria can hide within infected cells and avoid the

  2. Enhanced interleukin-8 production in THP-1 human monocytic cells by lipopolysaccharide from oral microorganisms and granulocyte-macrophage colony-stimulating factor.

    Science.gov (United States)

    Baqui, A A; Meiller, T F; Falkler, W A

    1999-10-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-8 (IL-8) plays an important role in macrophage mediated inflammatory processes including exacerbation of periodontal diseases, one of the most common complications in GM-CSF receiving cancer patients. The effect of GM-CSF supplementation on IL-8 production was investigated in a human monocyte cell line THP-1, stimulated with lipopolysaccharide extracted from two oral microorganisms, Porphyromonas gingivalis and Fusobacterium nucleatum. Resting THP-1 cells were treated with lipopolysaccharide (1 microgram/ml) of P. gingivalis or F. nucleatum and/or GM-CSF (50 IU/ml) for varying time periods. The production of IL-8 in THP-1 cells was measured by a solid-phase enzyme-linked immunosorbent assay (ELISA). A very low level of the cytokine IL-8 was produced constitutive in THP-1 cells. Starting from 8 h of treatment and afterwards GM-CSF alone significantly increased IL-8 production in THP-1 cells. Lipopolysaccharide (1 microgram/ml) extracts from either F. nucleatum or P. gingivalis amplified IL-8 production 500-800 times in comparison to resting THP-1 cells. When lipopolysaccharide of F. nucleatum or P. gingivalis was supplemented with 50 IU/ml of GM-CSF, there was a statistically significant enhanced production of IL-8 by THP-1 cells after 1 day to 7 days of treatment as compared with lipopolysaccharide treatment alone. GM-CSF (50 IU/ml) also significantly increased IL-8 production from 2-7 days of treatment of THP-1 cells when supplemented with a positive control, phorbol-12-myristate-13 acetate (PMA), as compared to PMA treatment alone. These investigations using the in vitro THP-1 human monocyte cell model indicate that there may be an increase in the response on a cellular level to oral endotoxin following GM-CSF therapy as evidenced by enhanced production of the tissue-reactive inflammatory cytokine, IL-8.

  3. B. abortus RNA is the component involved in the down-modulation of MHC-I expression on human monocytes via TLR8 and the EGFR pathway.

    Directory of Open Access Journals (Sweden)

    M Ayelén Milillo

    2017-08-01

    Full Text Available Despite eliciting a potent CD8+ T cell response, Brucella abortus is able to persist and establish a chronic infection inside its host. We have previously reported that the infection of human monocytes/macrophages with B. abortus inhibits the IFN-γ-induced MHC-I cell surface expression down-modulating cytotoxic CD8+ T cell responses. MHC-I down-modulation depends on bacterial viability and results from the capacity of B. abortus to retain the MHC-I molecules within the Golgi apparatus. Furthermore, we recently demonstrated that epidermal growth factor receptor (EGFR pathway is involved in this phenomenon and that this is an early event during infection. However, the components and mechanisms whereby B. abortus is able to down-modulate MHC-I remained to be elucidated. In this study we demonstrated that the down-modulation of MHC-I expression is not mediated by well-known Brucella virulence factors but instead by B. abortus RNA, a PAMP associated to viability (vita-PAMP. Surprisingly, completely degraded RNA was also able to inhibit MHC-I expression to the same extent as intact RNA. Accordingly, B. abortus RNA and its degradation products were able to mimic the MHC-I intracellular retention within the Golgi apparatus observed upon infection. We further demonstrated that TLR8, a single-stranded RNA and RNA degradation products sensor, was involved in MHC-I inhibition. On the other hand, neutralization of the EGFR reversed the MHC-I inhibition, suggesting a connection between the TLR8 and EGFR pathways. Finally, B. abortus RNA-treated macrophages display diminished capacity of antigen presentation to CD8+ T cells. Overall, our results indicate that the vita-PAMP RNA as well as its degradation products constitute novel virulence factors whereby B. abortus, by a TLR8-dependent mechanism and through the EGFR pathway, inhibits the IFN-γ-induced MHC-I surface expression on human monocytes/macrophages. Thus, bacteria can hide within infected cells and

  4. EMMPRIN (CD147/basigin) mediates platelet-monocyte interactions in vivo and augments monocyte recruitment to the vascular wall.

    Science.gov (United States)

    Schulz, C; von Brühl, M-L; Barocke, V; Cullen, P; Mayer, K; Okrojek, R; Steinhart, A; Ahmad, Z; Kremmer, E; Nieswandt, B; Frampton, J; Massberg, S; Schmidt, R

    2011-05-01

    Platelets play a central role in hemostasis, in inflammatory diseases such as atherosclerosis, and during thrombus formation following vascular injury. Thereby, platelets interact intensively with monocytes and enhance their recruitment to the vascular wall. To investigate the role of the extracellular matrix metalloproteinase inducer (EMMPRIN) in platelet-monocyte interactions. Isolated human monocytes were perfused in vitro over firmly adherent platelets to allow investigation of the role of EMMPRIN in platelet-monocyte interactions under flow conditions. Monocytes readily bound to surface-adherent platelets. Both antibody blockade and gene silencing of monocyte EMMPRIN substantially attenuated firm adhesion of monocytes to platelets at arterial and venous shear rates. In vivo, platelet interactions with the murine monocyte cell line ANA-1 were significantly decreased when ANA-1 cells were pretreated with EMMPRIN-silencing small interfering RNA prior to injection into wild-type mice. Using intravital microscopy, we showed that recruitment of EMMPRIN-silenced ANA-1 to the injured carotid artery was significantly reduced as compared with control cells. Further silencing of EMMPRIN resulted in significantly fewer ANA-1-platelet aggregates in the mouse circulation as determined by flow cytometry. Finally, we identified glycoprotein (GP)VI as a critical corresponding receptor on platelets that mediates interaction with monocyte EMMPRIN. Thus, blocking of GPVI inhibited the effect of EMMPRIN on firm monocyte adhesion to platelets under arterial flow conditions in vitro, and abrogated EMMPRIN-mediated platelet-monocyte aggregate formation in vivo. EMMPRIN supports platelet-monocyte interactions and promotes monocyte recruitment to the arterial wall. Therefore, EMMPRIN might represent a novel target to reduce vascular inflammation and atherosclerotic lesion development. © 2011 International Society on Thrombosis and Haemostasis.

  5. Technical advance: Generation of human pDC equivalents from primary monocytes using Flt3-L and their functional validation under hypoxia.

    Science.gov (United States)

    Sekar, Divya; Brüne, Bernhard; Weigert, Andreas

    2010-08-01

    The division of labor between DC subsets is evolutionarily well-defined. mDC are efficient in antigen presentation, whereas pDC act as rheostats of the immune system. They activate NK cells, cause bystander activation of mDC, and interact with T cells to induce tolerance. This ambiguity positions pDC at the center of inflammatory diseases, such as cancer, arthritis, and autoimmune diseases. The ability to generate human mDC ex vivo made it possible to engineer them to suit therapy needs. Unfortunately, a similar, easily accessible system to generate human pDC is not available. We describe a method to generate human pDC equivalents ex vivo, termed mo-pDC from peripheral blood monocytes using Flt3-L. mo-pDC showed a characteristic pDC profile, such as high CD123 and BDCA4, but low CD86 and TLR4 surface expression and a low capacity to induce autologous lymphocyte proliferation and to phagocytose apoptotic debris in comparison with mDC. Interestingly, mo-pDC up-regulated the pDC lineage-determining transcription factor E2-2 as well as expression of BDCA2, which is under the transcriptional control of E2-2 but not its inhibitor ID2, during differentiation. mo-pDC produced high levels of IFN-alpha when pretreated overnight with TNF-alpha. Under hypoxia, E2-2 was down-regulated, and ID2 was induced in mo-pDC, whereas surface expression of MHCI, CD86, and BDCA2 was decreased. Furthermore, mo-pDC produced high levels of inflammatory cytokines when differentiated under hypoxia compared with normoxia. Hence, mo-pDC can be used to study differentiation and functions of human pDC under microenvironmental stimuli.

  6. The cachectic mediator proteolysis inducing factor activates NF-kappaB and STAT3 in human Kupffer cells and monocytes

    NARCIS (Netherlands)

    Watchorn, T.M.; Dowidar, N.; Dejong, C.H.; Waddell, I.D.; Garden, O.J.; Ross, J.A.

    2005-01-01

    A novel proteoglycan, proteolysis inducing factor (PIF), is capable of inducing muscle proteolysis during the process of cancer cachexia, and of inducing an acute phase response in human hepatocytes. We investigated whether PIF is able to activate pro-inflammatory pathways in human Kupffer cells,

  7. Src Family Kinases Regulate Interferon Regulatory Factor 1 K63 Ubiquitination following Activation by TLR7/8 Vaccine Adjuvant in Human Monocytes and B Cells

    Directory of Open Access Journals (Sweden)

    Lorenza Tulli

    2018-03-01

    Full Text Available Toll-like receptors (TLRs play a key role in the activation of innate immune cells, in which their engagement leads to production of cytokines and co-stimulatory molecules. TLRs signaling requires recruitment of toll/IL-1R (TIR domain-containing adaptors, such as MyD88 and/or TRIF, and leads to activation of several transcription factors, such as NF-κB, the AP1 complex, and various members of the interferon regulatory factor (IRF family, which in turn results in triggering of several cellular functions associated with these receptors. A role for Src family kinases (SFKs in this signaling pathway has also been established. Our work and that of others have shown that this type of kinases is activated following engagement of several TLRs, and that this event is essential for the initiation of specific downstream cellular response. In particular, we have previously demonstrated that activation of SFKs is required for balanced production of pro-inflammatory cytokines by monocyte-derived dendritic cells after stimulation with R848, an agonist of human TLRs 7/8. We also showed that TLR7/8 triggering leads to an increase in interferon regulatory factor 1 (IRF-1 protein levels and that this effect is abolished by inhibition of SFKs, suggesting a critical role of these kinases in IRF-1 regulation. In this study, we first confirmed the key role of SFKs in TLR7/8 signaling for cytokine production and accumulation of IRF-1 protein in monocytes and in B lymphocytes, two other type of antigen-presenting cells. Then, we demonstrate that TLR7 triggering leads to an increase of K63-linked ubiquitination of IRF-1, which is prevented by SFKs inhibition, suggesting a key role of these kinases in posttranslational regulation of IRF-1 in the immune cells. In order to understand the mechanism that links SFKs activation to IRF-1 K63-linked ubiquitination, we examined SFKs and IRF-1 possible interactors and proved that activation of SFKs is necessary for their

  8. Different Transcriptional Profiles of Human Monocyte-Derived Dendritic Cells Infected with Distinct Strains of Mycobacterium tuberculosis and Mycobacterium bovis Bacillus Calmette-Guérin

    Directory of Open Access Journals (Sweden)

    Nunzia Sanarico

    2011-01-01

    Full Text Available In order to analyze dendritic cells (DCs activation following infection with different mycobacterial strains, we studied the expression profiles of 165 genes of human monocyte-derived DCs infected with H37Rv, a virulent Mycobacterium tuberculosis (MTB laboratory strain, CMT97, a clinical MTB isolate, Mycobacterium bovis bacillus Calmette-Guérin (BCG, Aventis Pasteur, and BCG Japan, both employed as vaccine against tuberculosis. The analysis of the gene expression reveals that, despite a set of genes similarly modulated, DCs response resulted strain dependent. In particular, H37Rv significantly upregulated EBI3 expression compared with BCG Japan, while it was the only strain that failed to release a significant IL-10 amount. Of note, BCG Japan showed a marked increase in CCR7 and TNF-α expression regarding both MTB strains and it resulted the only strain failing in exponential intracellular growth. Our results suggest that DCs display the ability to elicit a tailored strain-specific immune response.

  9. Serum amyloid A stimulates matrix-metalloproteinase-9 upregulation via formyl peptide receptor like-1-mediated signaling in human monocytic cells

    International Nuclear Information System (INIS)

    Lee, Ha Young; Kim, Mi-Kyoung; Park, Kyoung Sun; Bae, Yun Hee; Yun, Jeanho; Park, Joo-In; Kwak, Jong-Young; Bae, Yoe-Sik

    2005-01-01

    In the present study, we found that serum amyloid A (SAA) stimulated matrix-metalloproteinase-9 (MMP-9) upregulation at the transcription and translational levels in THP-1 cells. SAA stimulated the activation of nuclear factor κB (NF-κB), which was required for the MMP-9 upregulation by SAA. The signaling events induced by SAA included the activation of ERK and intracellular calcium rise, which were found to be required for MMP-9 upregulation. Formyl peptide receptor like 1 (FPRL1) was found to be involved in the upregulation of MMP-9 by SAA. Among several FPRL1 agonists, including Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), SAA selectively stimulated MMP-9 upregulation. With respect to the molecular mechanisms involved in the differential action of SAA and WKYMVm, we found that SAA could not competitively inhibit the binding of 125 I-labeled WKYMVm to FPRL1. Taken together, we suggest that SAA plays a role in the modulation of inflammatory and immune responses via FPRL1, by inducing MMP-9 upregulation in human monocytic cells

  10. Induction of cytosine arabinoside-resistant human myeloid leukemia cell death through autophagy regulation by hydroxychloroquine.

    Science.gov (United States)

    Kim, Yundeok; Eom, Ju-In; Jeung, Hoi-Kyung; Jang, Ji Eun; Kim, Jin Seok; Cheong, June-Won; Kim, Young Sam; Min, Yoo Hong

    2015-07-01

    We investigated the effects of the autophagy inhibitor hydroxychloroquine (HCQ) on cell death of cytosine arabinoside (Ara-C)-resistant human acute myeloid leukemia (AML) cells. Ara-C-sensitive (U937, AML-2) and Ara-C-resistant (U937/AR, AML-2/AR) human AML cell lines were used to evaluate HCQ-regulated cytotoxicity, autophagy, and apoptosis as well as effects on cell death-related signaling pathways. We found that HCQ-induced dose- and time-dependent cell death in Ara-C-resistant cells compared to Ara-C-sensitive cell lines. The extent of cell death and features of HCQ-induced autophagic markers including increase in microtubule-associated protein light chain 3 (LC3) I conversion to LC3-II, beclin-1, ATG5, as well as green fluorescent protein-LC3 positive puncta and autophagosome were remarkably greater in U937/AR cells. Also, p62/SQSTM1 was increased in response to HCQ. p62/SQSTM1 protein interacts with both LC3-II and ubiquitin protein and is degraded in autophagosomes. Therefore, a reduction of p62/SQSTM1 indicates increased autophagic degradation, whereas an increase of p62/SQSTM1 by HCQ indicates inhibited autophagic degradation. Knock down of p62/SQSTM1 using siRNA were prevented the HCQ-induced LC3-II protein level as well as significantly reduced the HCQ-induced cell death in U937/AR cells. Also, apoptotic cell death and caspase activation in U937/AR cells were increased by HCQ, provided evidence that HCQ-induced autophagy blockade. Taken together, our data show that HCQ-induced apoptotic cell death in Ara-C-resistant AML cells through autophagy regulation. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  11. Human metapneumovirus M2-2 protein inhibits innate immune response in monocyte-derived dendritic cells.

    Directory of Open Access Journals (Sweden)

    Junping Ren

    Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in young children, the elderly and immunocompromised patients. Repeated hMPV infections occur throughout life. However, immune evasion mechanisms of hMPV infection are largely unknown. Recently, our group has demonstrated that hMPV M2-2 protein, an important virulence factor, contributes to immune evasion in airway epithelial cells by targeting the mitochondrial antiviral-signaling protein (MAVS. Whether M2-2 regulates the innate immunity in human dendritic cells (DC, an important family of immune cells controlling antigen presenting, is currently unknown. We found that human DC infected with a virus lacking M2-2 protein expression (rhMPV-ΔM2-2 produced higher levels of cytokines, chemokines and IFNs, compared to cells infected with wild-type virus (rhMPV-WT, suggesting that M2-2 protein inhibits innate immunity in human DC. In parallel, we found that myeloid differentiation primary response gene 88 (MyD88, an essential adaptor for Toll-like receptors (TLRs, plays a critical role in inducing immune response of human DC, as downregulation of MyD88 by siRNA blocked the induction of immune regulatory molecules by hMPV. Since M2-2 is a cytoplasmic protein, we investigated whether M2-2 interferes with MyD88-mediated antiviral signaling. We found that indeed M2-2 protein associated with MyD88 and inhibited MyD88-dependent gene transcription. In this study, we also identified the domains of M2-2 responsible for its immune inhibitory function in human DC. In summary, our results demonstrate that M2-2 contributes to hMPV immune evasion by inhibiting MyD88-dependent cellular responses in human DC.

  12. The continuum of monocyte phenotypes: Experimental evidence and prognostic utility in assessing cardiovascular risk.

    Science.gov (United States)

    Cignarella, Andrea; Tedesco, Serena; Cappellari, Roberta; Fadini, Gian Paolo

    2018-03-30

    The monocyte-macrophage cell lineage represents a major player in innate immunity, and is involved in many physiologic and pathologic conditions. Particularly, monocyte-macrophages play a very important role in atherosclerosis and cardiovascular disease. Monocyte heterogeneity is well recognized but the biologic and clinical meaning of the various monocyte subtypes is not entirely understood. Traditionally, monocytes can be divided in classical, intermediate, and nonclassical based on expression of the surface antigens CD14 and CD16. While macrophage diversity is now well recognized to organize as a continuum, monocyte subsets have long been considered as separated entities. However, mounting evidence obtained by tracking the ontology of human monocytes help clarifying that monocytes mature from classical to nonclassical ones, through an intermediate phenotype. This concept is therefore best depicted as a continuum, whereas the subdivision into discrete CD14/CD16 subsets appears an oversimplification. In this review, we discuss the evidence supporting the existence of a monocyte continuum along with the technical challenges of monocyte characterization. In particular, we describe the advantage of considering monocytes along a continuous distribution for the evaluation of cardiovascular risk. We make the point that small transition along the monocyte continuum better reflects cardiovascular risk than a simplified analysis of discrete monocyte subsets. Recognizing the monocyte continuum can be helpful to model other pathophysiologic conditions where these cells are involved. ©2018 Society for Leukocyte Biology.

  13. Leonurine protects against tumor necrosis factor-α-mediated inflammation in human umbilical vein endothelial cells.

    Science.gov (United States)

    Liu, Xinhua; Pan, Lilong; Wang, Xianli; Gong, Qihai; Zhu, Yi Zhun

    2012-05-01

    Leonurine, a bioactive alkaloid compound in Herba leonuri, has various pharmacological activities, including antioxidant and anti-apoptotic capacities. This study was conducted to test the hypothesis that leonurine was able to attenuate tumor necrosis factor (TNF)-α-induced human umbilical vein endothelial cells (HUVEC) activation and the underlying molecular mechanisms. Mitogen-activated protein kinases (MAPK) activation, nuclear factor-κB (NF-κB) activation, and inflammatory mediators expression were detected by Western blot or enzyme-liked immunosorbent assay, intracellular reactive oxygen species (ROS) and NF-κB p65 translocation were measured by immunofluorescence, endothelial cell-monocyte interaction was detected by microscope. Leonurine inhibited U937 cells adhesion to TNF-α-activated HUVEC in a concentration dependent manner. Treatment with leonurine blocked TNF-α-induced mRNA and protein expression of adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1), cyclooxygenase-2, and monocyte chemoattractant protein-1 in endothelial cells. In addition, leonurine attenuated TNF-α-induced intracellular ROS production in HUVEC. Furthermore, leonurine also suppressed the TNF-α-activated p38 phosphorylation and IκBα degradation. Subsequently, reduced NF-κB p65 phosphorylation, nuclear translocation, and DNA-binding activity were also observed. Our results demonstrated for the first time that the anti-inflammatory properties of leonurine in endothelial cells, at least in part, through suppression of NF-κB activation, which may have a potential therapeutic use for inflammatory vascular diseases. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  14. In vivo imaging of monocyte trafficking with 18F-fluorodeoxyglucose labeled monocytes

    International Nuclear Information System (INIS)

    Paik, Jin Young; Lee, Kyung Han; Han, Yu Mi; Choe, Yearn Seong; Kim, Byung Tae

    2000-01-01

    Since the ability to monitor in vivo monocyte trafficking would contribute to our understanding of the pathophysiology of various inflammatory disorders, we investigated the feasibility of labeling human monocytes with 18 F-FDG. Human monocytes were separated by Ficoll/Hypaque gradient and purity was assessed by flow cytometry. The influence of insulin and/or glucose on labeling efficiency was evaluated. Cell viability and activation was measured with trypan blue exclusion and hydrogen peroxide assays, respectively. Label stability was measured for up to 18 hr, and the effect of insulin pre-incubation on FDG washout was investigated. PET images were acquired in SD rats at various time points after injection of FDG labeled monocytes. Monocytes were >85% pure, and labeling efficiency was 35% for 1x106 cells after 40 min incubation with 2 mCi 18 F-FDG without insulin. Pre-incubation with 10∼100 nM insulin significantly increased FDG uptake which reached 400% of baseline levels, whereas presence of glucose or serum decreased FDG uptake. Labeled cells were >90% viable for up to 22 hr, and the labeling process did appear to significantly activate cells, Washout studies however, demonstrated gradual washout of the FDG from monocytes after initial uptake PET images of FDG labeled monocytes in SD rats showed consistent findings. Utilizing insulin effects on cellular glucose metabolism may be a feasible way of labeling monocytes with 18 F-FDG for PET imaging. However, gradual washout of FDG after initial uptake poses as a potential problem which needs to be addressed before practical application

  15. HSV-1-induced chemokine expression via IFI16-dependent and IFI16-independent pathways in human monocyte-derived macrophages

    DEFF Research Database (Denmark)

    Søby, Stine; Laursen, Rune R; Østergaard, Lars Jørgen

    2012-01-01

    ABSTRACT: BACKGROUND: Innate recognition is essential in the antiviral response against infection by herpes simplex virus (HSV). Chemokines are important for control of HSV via recruitment of natural killer cells, T lymphocytes, and antigen-presenting cells. We previously found that early HSV-1......-mediated chemokine responses are not dependent on TLR2 and TLR9 in human macrophages. Here, we investigated the role of the recently identified innate IFN-inducible DNA receptor IFI16 during HSV-1 infection in human macrophages. METHODS: Peripheral blood mononuclear cells were purified from buffy coats...

  16. Helicobacter pylori induces IL-1β and IL-18 production in human monocytic cell line through activation of NLRP3 inflammasome via ROS signaling pathway.

    Science.gov (United States)

    Li, Xiang; Liu, Sheng; Luo, Jingjing; Liu, Anyuan; Tang, Shuangyang; Liu, Shuo; Yu, Minjun; Zhang, Yan

    2015-06-01

    This study investigated whether Helicobacter pylori could activate the nucleotide-binding oligomerization domain-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome in human macrophages and the involvement of reactive oxygen species (ROS) in inflammasome activation. Phorbol-12-myristate-13-acetate (PMA)-differentiated human acute monocytic leukemia cell line THP-1 was infected with H. pylori. The levels of pro-inflammatory cytokines interleukin (IL)-1β and IL-18 in supernatant were measured by ELISA. Intracellular ROS level was analyzed by flow cytometry. Quantitative real-time PCR and western blot analysis were employed to determine the mRNA and protein expression levels of NLRP3 and caspase-1 in THP-1 cells, respectively. Our results showed that H. pylori infection could induce IL-1β and IL-18 production in PMA-differentiated THP-1 cells in a dose- and time-dependent manner. Moreover, secretion of IL-1β and IL-18 in THP-1 cells following H. pylori infection was remarkably reduced by NLRP3-specific small interfering RNA treatment. In addition, the intracellular ROS level was elevated by H. pylori infection, which could be eliminated by the ROS scavenger N-acetylcysteine (NAC). Furthermore, NAC treatment could inhibit NLRP3 inflammasome formation and caspase-1 activation and suppress the release of IL-1β and IL-18 from H. pylori-infected THP-1 cells. These findings provide novel insights into the innate immune response against H. pylori infection, which could potentially be used for the prevention and treatment of H. pylori-related diseases. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Phenotypic heterogeneity of peripheral monocytes in healthy dogs.

    Science.gov (United States)

    Gibbons, Natalie; Goulart, Michelle R; Chang, Yu-Mei; Efstathiou, Konstantinos; Purcell, Robert; Wu, Ying; Peters, Laureen M; Turmaine, Mark; Szladovits, Balazs; Garden, Oliver A

    2017-08-01

    Monocytes are key cells of the innate immune system. Their phenotypic and functional roles have been investigated in humans, mice and other animals, such as the rat, pig and cow. To date, detailed phenotypic analysis of monocytes has not been undertaken in dogs. Two important surface markers in human monocytes are CD14 and MHC class II (MHC II). By flow cytometry, we demonstrated that canine monocytes can be subdivided into three separate populations: CD14 pos MHC II neg , CD14 pos MHC II pos and CD14 neg MHC II pos . Both light and transmission electron microscopy confirmed the monocytic identity of all three populations. The CD14 pos MHC II neg population could be distinguished on an ultrastructural level by their smaller size, the presence of more numerous, larger granules, and more pseudopodia than both of the other populations. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Oxidative Mechanisms of Monocyte-Mediated Cytotoxicity

    Science.gov (United States)

    Weiss, Stephen J.; Lobuglio, Albert F.; Kessler, Howard B.

    1980-01-01

    Human monocytes stimulated with phorbol myristate acetate were able to rapidly destroy autologous erythrocyte targets. Monocyte-mediated cytotoxicity was related to phorbol myristate acetate concentration and monocyte number. Purified preparations of lymphocytes were incapable of mediating erythrocyte lysis in this system. The ability of phorbol myristate acetate-stimulated monocytes to lyse erythrocyte targets was markedly impaired by catalase or superoxide dismutase but not by heat-inactivated enzymes or albumin. Despite a simultaneous requirement for superoxide anion and hydrogen peroxide in the cytotoxic event, a variety of hydroxyl radical and singlet oxygen scavengers did not effect cytolysis. However, tryptophan significantly inhibited cytotoxicity. The myeloperoxidase inhibitor cyanide enhanced erythrocyte destruction, whereas azide reduced it modestly. The inability of cyanide to reduce cytotoxicity coupled with the protective effect of superoxide dismutase suggests that cytotoxicity is independent of the classic myeloperoxidase system. We conclude that monocytes, stimulated with phorbol myristate acetate, generate superoxide anion and hydrogen peroxide, which together play an integral role in this cytotoxic mechanism.

  19. Monocyte functions in diabetes mellitus

    DEFF Research Database (Denmark)

    Geisler, C; Almdal, T; Bennedsen, J

    1982-01-01

    The aim of this study was to investigate the functions of monocytes obtained from 14 patients with diabetes mellitus (DM) compared with those of monocytes from healthy individuals. It was found that the total number of circulating monocytes in the 14 diabetic patients was lower than that from...... for the elucidation of concomitant infections in diabetic patients are discussed....

  20. Augmented TLR2 Expression on Monocytes in both Human Kawasaki Disease and a Mouse Model of Coronary Arteritis

    OpenAIRE

    Lin, I-Chun; Kuo, Ho-Chang; Lin, Ying-Jui; Wang, Feng-Shen; Wang, Lin; Huang, Shun-Chen; Chien, Shao-Ju; Huang, Chien-Fu; Wang, Chih-Lu; Yu, Hong-Ren; Chen, Rong-Fu; Yang, Kuender D.

    2012-01-01

    BACKGROUND: Kawasaki disease (KD) of unknown immunopathogenesis is an acute febrile systemic vasculitis and the leading cause of acquired heart diseases in childhood. To search for a better strategy for the prevention and treatment of KD, this study compared and validated human KD immunopathogenesis in a mouse model of Lactobacillus casei cell wall extract (LCWE)-induced coronary arteritis. METHODS: Recruited subjects fulfilled the criteria of KD and were admitted for intravenous gamma globul...

  1. Human Monocytes Accelerate Proliferation and Blunt Differentiation of Preadipocytes in Association With Suppression of C/Ebpα mRNA

    Science.gov (United States)

    Couturier, Jacob; Patel, Sanjeet G.; Iyer, Dinakar; Balasubramanyam, Ashok; Lewis, Dorothy E.

    2015-01-01

    Obesity, type 2 diabetes, and HIV-associated lipodystrophy are associated with abnormalities in adipocyte growth and differentiation. In persons with these conditions, adipose depots contain increased numbers of macrophages, but the origins of these cells and their specific effects are uncertain. Peripheral blood mononuclear cells (PBMC)-derived monocytes, but not T cells, cocultured via transwells with primary subcutaneous preadipocytes, increased proliferation (approximately twofold) and reduced differentiation (~50%) of preadipocytes. Gene expression analyses in proliferating preadipocytes (i.e., prior to hormonal induction of terminal differentiation) revealed that monocytes down-regulated mRNA levels of CCAAT/enhancer binding protein, alpha (C/EBPα) and up-regulated mRNA levels of G0/G1 switch 2 (G0S2) message, genes important for the regulation of adipogenesis and the cell cycle. These data indicate that circulating peripheral blood monocytes can disrupt adipogenesis by interfering with a critical step in C/EBPα and G0S2 transcription required for preadipocytes to make the transition from proliferation to differentiation. Interactions between preadipocytes and monocytes also increased the inflammatory cytokines IL-6 and IL-8, as well as a novel chemotactic cytokine, CXCL1. Additionally, the levels of both IL-6 and CXCL1 were highest when preadipocytes and monocytes were cultured together, compared to each cell in culture alone. Such cross-talk amplifies the production of mediators of tissue inflammation. PMID:21869759

  2. The aqueous extract of Hibiscus sabdariffa calices modulates the production of monocyte chemoattractant protein-1 in humans.

    Science.gov (United States)

    Beltrán-Debón, R; Alonso-Villaverde, C; Aragonès, G; Rodríguez-Medina, I; Rull, A; Micol, V; Segura-Carretero, A; Fernández-Gutiérrez, A; Camps, J; Joven, J

    2010-03-01

    Diet supplementation and/or modulation is an important strategy to significantly improve human health. The search of plants as additional sources of bioactive phenolic compounds is relevant in this context. The aqueous extract of Hibiscus sabdariffa is rich in anthocyanins and other phenolic compounds including hydroxycitric and chlorogenic acids. Using this extract we have shown an effective protection of cultured peripheral blood mononuclear cells from the cellular death induced by H(2)O(2) and a significant role in the production of inflammatory cytokines. In vitro, the extract promotes the production of IL-6 and IL-8 and decreases the concentration of MCP-1 in supernatants in a dose-dependent manner. In humans, the ingestion of an acute dose of the extract (10g) was well tolerated and decreased plasma MCP-1 concentrations significantly without further effects on other cytokines. This effect was not due to a concomitant increase in the antioxidant capacity of plasma. Instead, its mechanisms probably involve a direct inhibition of inflammatory and/or metabolic pathways responsible for MCP-1 production, and may be relevant in inflammatory and chronic conditions in which the role of MCP-1 is well established. If beneficial effects are confirmed in patients, Hibiscus sabdariffa could be considered a valuable traditional herbal medicine for the treatment of chronic inflammatory diseases with the advantage of being devoid of caloric value or potential alcohol toxicity. Copyright 2009 Elsevier GmbH. All rights reserved.

  3. In vitro impact of bisphenols BPA, BPF, BPAF and 17β-estradiol (E2) on human monocyte-derived dendritic cell generation, maturation and function.

    Science.gov (United States)

    Švajger, Urban; Dolenc, Marija Sollner; Jeras, Matjaž

    2016-05-01

    Bisphenols (BPs) are widely spread pollutants that act as estrogen-like endocrine disruptors and are potentially affecting human health on a long run. We explored the effects of BPA, BPF and BPAF, on in vitro differentiation and maturation of MDDCs. Monocytes were treated with 17β-estradiol (E2) and each BP at the beginning of their differentiation into iMDDCs. We found that 10 and 50 μM of BPA and BPF, 10 and 30μM of BPAF and 10 and 50 nM of E2 did not affect cell viability. However, 50 μM of BPA and BPF, as well as 10 and 30 μM of BPAF, significantly decreased the endocytotic capacity of iMDDCs. Both, BPA (50 μM) and BPAF (30 μM) decreased the expression of CD1a and increased the amount of DC-SIGN molecules on iMDDCs. The E2 pre-treatment moderately decreased expression of CD80, CD86 and CD83 co-stimulatory molecules while increasing the numbers of HLA-DR on mMDDCs. Only BPAF significantly influenced the expression of CD80 and CD86 (both decreased), as well as CD83 and HLA-DR molecules (both increased) on mMDDCs. In addition, BPAF modulated DC maturation signaling pathways by lowering the phosphorylation of p65 NF-κB (nuclear factor-kappaB) and ERK (extracellular signal regulated kinase) 1/2 proteins. Consequently, the in vitro proliferation of allogeneic T cells, stimulated with differently pre-treated iMDDCs and mMDDCs, was significantly reduced only in case of BPAF. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Interleukin-6 production by human monocytes treated with granulocyte-macrophage colony-stimulating factor in the presence of lipopolysaccharide of oral microorganisms.

    Science.gov (United States)

    Baqui, A A; Meiller, T F; Chon, J J; Turng, B F; Falkler, W A

    1998-06-01

    This study focused on the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide of the putative periodontal pathogens Porphyromonas gingivalis or Fusobacterium nucleatum on IL-6 production by THP-1 cells (a human monocytic cell line). Resting THP-1 cells were alternatively treated with GM-CSF (50 IU/ml) and lipopolysaccharide of P. gingivalis or F. nucleatum, in varying concentrations for varying time periods. IL-6 production in supernatant fluids of treated cells was evaluated by an enzyme-linked immunosorbent assay (ELISA) and a reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate gene expression. Untreated THP-1 cells did not produce IL-6 as determined by ELISA. RT-PCR also failed to detect IL-6 mRNA in untreated THP-1 cells, indicating that IL-6 was not constitutively produced. After stimulation of THP-1 cells with lipopolysaccharide of F. nucleatum or P. gingivalis, IL-6 was produced, peaking at 4 h (200-300 pg/ml) and thereafter sharply declining by 8 h. When GM-CSF was added together with lipopolysaccharide of P. gingivalis or F. nucleatum, there was a synergistic quantitative increase in production of IL-6 as measured by ELISA as compared with lipopolysaccharide alone. IL-6 mRNA was detected by RT-PCR, 15 min after stimulation with lipopolysaccharide of either P. gingivalis or F. nucleatum. GM-CSF supplementation with lipopolysaccharide of P. gingivalis shortened the transcription of IL-6 mRNA to 5 min, a shift which was not observed with lipopolysaccharide of F. nucleatum, possibly indicating a different mechanism of initiation of transcription. Production of IL-6 by GM-CSF-treated THP-1 cells in the presence of lipopolysaccharide of oral microorganisms may provide a model for studying the role of macrophages in acute and chronic periodontal diseases, including the clinical periodontal exacerbation as observed in chemotherapy patients receiving GM-CSF for bone marrow recovery.

  5. cGAMP Quantification in Virus-Infected Human Monocyte-Derived Cells by HPLC-Coupled Tandem Mass Spectrometry.

    Science.gov (United States)

    Paijo, Jennifer; Kaever, Volkhard; Kalinke, Ulrich

    2017-01-01

    Upon virus infection, cells of the innate immune system such as dendritic cells and macrophages can mount type I interferon (IFN-I) responses that restrict viral dissemination. To inform host cells of virus infection, detection of cytosolic DNA is one important mechanism. Inappropriate sensing of endogenous DNA and subsequent induction of IFN-I responses can also cause autoimmunity, highlighting the need to tightly regulate DNA sensing. The cyclic GMP-AMP synthase (cGAS) was recently identified to be the major sensor of cytosolic DNA that triggers IFN-I expression. Upon DNA binding, cGAS synthesizes the second messenger cyclic guanosine-adenosine monophosphate (cGAMP) that induces IFN-I expression by the activation of the stimulator of interferon genes (STING). Notably, cGAMP does not only act in infected cells, but can also be relocated to noninfected bystander cells to there trigger IFN-I expression. Thus, direct quantification of cGAMP in cells of the innate immune system is an important approach to study where, when, and how DNA is sensed and IFN-I responses are induced. Here, we describe a method that allows specific quantification of cGAMP from extracts of virus-infected human myeloid cells by HPLC-coupled tandem mass spectrometry.

  6. Postprandial Monocyte Activation in Individuals With Metabolic Syndrome

    Science.gov (United States)

    Khan, Ilvira M.; Pokharel, Yashashwi; Dadu, Razvan T.; Lewis, Dorothy E.; Hoogeveen, Ron C.; Wu, Huaizhu

    2016-01-01

    Context: Postprandial hyperlipidemia has been suggested to contribute to atherogenesis by inducing proinflammatory changes in monocytes. Individuals with metabolic syndrome (MS), shown to have higher blood triglyceride concentration and delayed triglyceride clearance, may thus have increased risk for development of atherosclerosis. Objective: Our objective was to examine fasting levels and effects of a high-fat meal on phenotypes of monocyte subsets in individuals with obesity and MS and in healthy controls. Design, Setting, Participants, Intervention: Individuals with obesity and MS and gender- and age-matched healthy controls were recruited. Blood was collected from participants after an overnight fast (baseline) and at 3 and 5 hours after ingestion of a high-fat meal. At each time point, monocyte phenotypes were examined by multiparameter flow cytometry. Main Outcome Measures: Baseline levels of activation markers and postprandial inflammatory response in each of the three monocyte subsets were measured. Results: At baseline, individuals with obesity and MS had higher proportions of circulating lipid-laden foamy monocytes than controls, which were positively correlated with fasting triglyceride levels. Additionally, the MS group had increased counts of nonclassical monocytes, higher CD11c, CX3CR1, and human leukocyte antigen-DR levels on intermediate monocytes, and higher CCR5 and tumor necrosis factor-α levels on classical monocytes in the circulation. Postprandial triglyceride increases in both groups were paralleled by upregulation of lipid-laden foamy monocytes. MS, but not control, subjects had significant postprandial increases of CD11c and percentages of IL-1β+ and tumor necrosis factor-α+ cells in nonclassical monocytes. Conclusions: Compared to controls, individuals with obesity and MS had increased fasting and postprandial monocyte lipid accumulation and activation. PMID:27575945

  7. Monocyte function is severely impaired by the fluorochrome calcein acetomethylester

    International Nuclear Information System (INIS)

    Czepluch, Frauke S.; Olieslagers, Serve J.F.; Waltenberger, Johannes

    2007-01-01

    For rapid chemotaxis quantification, cell prelabelling is often performed with the fluorochrome calcein acetomethylester (calcein AM). We investigated whether calcein AM-prelabelling is reliable for monocyte migration analysis. Human monocytes were either preexposed to calcein AM or unlabelled. Monocyte migration towards the potent chemoattractants transforming growth factor-β1 (TGF-β1) and N-formyl-Methionin-Leucin-Phenylalanin (fMLP) was assessed using a 48-well micro-chemotaxis chamber. For quantification, cells were visualized by light microscopy and counted. Surprisingly, random migration of calcein AM-prelabelled cells was significantly impaired compared to the unlabelled control. Accordingly, monocyte chemotaxis towards either TGF-β1 or fMLP dramatically declined. Adherence of calcein AM-labelled monocytes on plastic was also significantly decreased compared to control cells. As adhesion is regarded as an essential component of monocyte migration, the reduced migration observed in calcein AM-labelled monocytes might be explained by a fluorochrome-induced adhesion defect. Therefore, use of the fluorochrome calcein AM cannot be recommended for functional testing of monocytes

  8. Anti-inflammatory effects of the new generation synthetic surfactant CHF5633 on Ureaplasma-induced cytokine responses in human monocytes.

    Science.gov (United States)

    Glaser, Kirsten; Fehrholz, Markus; Henrich, Birgit; Claus, Heike; Papsdorf, Michael; Speer, Christian P

    2017-02-01

    Synthetic surfactants represent a promising alternative to animal-derived preparations in the treatment of neonatal respiratory distress syndrome. The synthetic surfactant CHF5633 has proven biophysical effectiveness and, moreover, demonstrated anti-inflammatory effects in LPS-stimulated monocytes. With ureaplasmas being relevant pathogens in preterm lung inflammation, the present study addressed immunomodulatory features on Ureaplasma-induced monocyte cytokine responses. Ureaplasma parvum-stimulated monocytes were exposed to CHF5633. TNF-α, IL-1β, IL-8, IL-10, TLR2 and TLR4 expression were analyzed using qPCR and flow cytometry. CHF5633 did not induce pro-inflammation, and did not aggravate Ureaplasma-induced pro-inflammatory cytokine responses. It suppressed U. parvum-induced intracellular TNF-α (p Ureaplasma-induced TNF-α mRNA (p Ureaplasma-modulated IL-8, IL-10, TLR2 and TLR4 were unaffected. CHF5633 does neither act pro-apoptotic nor pro-inflammatory in native and Ureaplasma-infected monocytes. Suppression of Ureaplasma-induced TNF-α and IL-1β underlines anti-inflammatory features of CHF5633.

  9. Respective roles and interactions of T-lymphocyte and PGE2-mediated monocyte suppressive activities in human newborns and mothers at the time of delivery

    International Nuclear Information System (INIS)

    Durandy, A.; Fischer, A.; Mamas, S.; Dray, F.; Griscelli, C.

    1982-01-01

    Recently the concept of a poorly functional humoral immune response in the newborn was proposed. Data have been presented indicating that the impaired newborn B cell maturation, as shown in vitro in a pokeweed mitogen-induced B cell maturation system, is due both to an immaturity of lymphocyte subsets and to an increased suppressive T activity. In the present work, we present evidence that there exists a predominance of a naturally occurring T lymphocyte suppressive activity in the cord blood in that the removal of the suppressive activity by irradiation allows a normal maturation of newborn B cells. Such normal maturation of newborn B cells can also be obtained using mixed cultures of adult T cells and newborn B cells. Newborn suppressor T cells belong to both EA gamma (+) and EA gamma (-) fractions, and it is not known whether these two groups do or do not belong to different subsets. The PGE2-dependent monocyte suppressive activity does not play any role in the suppression observed in newborns since newborn monocytes are poorly suppressive and since they produce a smaller amount of PGE2 than adult monocytes. Some observations suggest, on the contrary, that the suppressive T lymphocytes can regulate the level of the PGE2-dependent monocyte suppressive activity. It should be noticed that similar observations about T lymphocyte and PGE2-dependent monocyte suppressive activities have been made at the same time using mothers' cells. These observations suggest the possibility that such changes in B cell immune regulation may result from an interaction between maternal and fetal lymphoid cells

  10. High Uric Acid Activates the ROS-AMPK Pathway, Impairs CD68 Expression and Inhibits OxLDL-Induced Foam-Cell Formation in a Human Monocytic Cell Line, THP-1

    Directory of Open Access Journals (Sweden)

    Chaohuan Luo

    2016-11-01

    Full Text Available Background/Aims: Hyperuricemia is part of the metabolic-syndrome cluster of abdominal obesity, impaired glucose tolerance, insulin resistance, dyslipidemia, and hypertension. Monocytes/macrophages are critical in the development of metabolic syndrome, including gout, obesity and atherosclerosis. However, how high uric acid (HUA exposure affects monocyte/macrophage function remains unclear. In this study, we investigated the molecular mechanism of HUA exposure in monocytes/macrophages and its impact on oxidized low-density lipoprotein (oxLDL-induced foam-cell formation in a human monocytic cell line, THP-1. Methods: We primed THP-1 cells with phorbol-12-myristate-13-acetate (PMA for differentiation, then exposed cells to HUA and detected the production of reactive oxygen species (ROS and analyzed the level of phospho-AMPKα. THP-1 cells were pre-incubated with Compound C, an AMPK inhibitor, or N-acetyl-L-cysteine (NAC, a ROS scavenger, or HUA before PMA, to assess CD68 expression and phospho-AMPKα level. PMA-primed THP-1 cells were pre-treated with oxLDL before Compound C and HUA treatment. Western blot analysis was used to examine the levels of phospho-AMPKα, CD68, ABCG1, ABCA1, cyclooxygenase-2 (COX-2 and NF-κB (p65. Flow cytometry was used to assess ROS production and CD68 expression in live cells. Oil-red O staining was used to observe oxLDL uptake in cells. Results: HUA treatment increased ROS production in PMA-primed THP-1 cells; NAC blocked HUA-induced oxidative stress. HUA treatment time-dependently increased phospho-AMPKα level in PMA-primed THP-1 cells. The HUA-induced oxidative stress increased phospho-AMPKα levels, which was blocked by NAC. HUA treatment impaired CD68 expression during cell differentiation by activating the AMPK pathway, which was reversed by Compound C treatment. Finally, HUA treatment inhibited oxLDL uptake in the formation of foam cells in THP-1 cells, which was blocked by Compound C treatment. HUA treatment

  11. Granulocyte-Macrophage Colony-Stimulating Factor Amplification of Interleukin-1β and Tumor Necrosis Factor Alpha Production in THP-1 Human Monocytic Cells Stimulated with Lipopolysaccharide of Oral Microorganisms

    OpenAIRE

    Baqui, A. A. M. A.; Meiller, Timothy F.; Chon, Jennifer J.; Turng, Been-Foo; Falkler, William A.

    1998-01-01

    Cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), are used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) play important roles in inflammatory processes, including exacerbation of periodontal diseases, one of the most common complications in patients who undergo this therapy. A human monocyte cell line (THP-1) was utilized to investigate IL-1β and TNF-α production following GM-CSF suppl...

  12. Comparison of cellular effects of starch-coated SPIONs and poly(lactic-co-glycolic acid) matrix nanoparticles on human monocytes.

    Science.gov (United States)

    Gonnissen, Dominik; Qu, Ying; Langer, Klaus; Öztürk, Cengiz; Zhao, Yuliang; Chen, Chunying; Seebohm, Guiscard; Düfer, Martina; Fuchs, Harald; Galla, Hans-Joachim; Riehemann, Kristina

    Within the last years, progress has been made in the knowledge of the properties of medically used nanoparticles and their toxic effects, but still, little is known about their influence on cellular processes of immune cells. The aim of our comparative study was to present the influence of two different nanoparticle types on subcellular processes of primary monocytes and the leukemic monocyte cell line MM6. We used core-shell starch-coated superparamagnetic iron oxide nanoparticles (SPIONs) and matrix poly(lactic-co-glycolic acid) (PLGA) nanoparticles for our experiments. In addition to typical biocompatibility testing like the detection of necrosis or secretion of interleukins (ILs), we investigated the impact of these nanoparticles on the actin cytoskeleton and the two voltage-gated potassium channels Kv1.3 and Kv7.1. Induction of necrosis was not seen for PLGA nanoparticles and SPIONs in primary monocytes and MM6 cells. Likewise, no alteration in secretion of IL-1β and IL-10 was detected under the same experimental conditions. In contrast, IL-6 secretion was exclusively downregulated in primary monocytes after contact with both nanoparticles. Two-electrode voltage clamp experiments revealed that both nanoparticles reduce currents of the aforementioned potassium channels. The two nanoparticles differed significantly in their impact on the actin cytoskeleton, demonstrated via atomic force microscopy elasticity measurement and phalloidin staining. While SPIONs led to the disruption of the respective cytoskeleton, PLGA did not show any influence in both experimental setups. The difference in the effects on ion channels and the actin cytoskeleton suggests that nanoparticles affect these subcellular components via different pathways. Our data indicate that the alteration of the cytoskeleton and the effect on ion channels are new parameters that describe the influence of nanoparticles on cells. The results are highly relevant for medical application and further

  13. Monocyte transferrin-iron uptake in hereditary hemochromatosis

    International Nuclear Information System (INIS)

    Sizemore, D.J.; Bassett, M.L.

    1984-01-01

    Transferrin-iron uptake by peripheral blood monocytes was studied in vitro to test the hypothesis that the relative paucity of mononuclear phagocyte iron loading in hereditary hemochromatosis results from a defect in uptake of iron from transferrin. Monocytes from nine control subjects and 17 patients with hemochromatosis were cultured in the presence of 59Fe-labelled human transferrin. There was no difference in 59Fe uptake between monocytes from control subjects and monocytes from patients with hemochromatosis who had been treated by phlebotomy and who had normal body iron stores. However, 59Fe uptake by monocytes from iron-loaded patients with hemochromatosis was significantly reduced compared with either control subjects or treated hemochromatosis patients. It is likely that this was a secondary effect of iron loading since iron uptake by monocytes from treated hemochromatosis patients was normal. Assuming that monocytes in culture reflect mononuclear phagocyte iron metabolism in vivo, this study suggests that the relative paucity of mononuclear phagocyte iron loading in hemochromatosis is not related to an abnormality in transferrin-iron uptake by these cells

  14. Requirement of the coiled-coil domain of PML-RARα oncoprotein for localization, sumoylation, and inhibition of monocyte differentiation

    International Nuclear Information System (INIS)

    Kim, Young-Eui; Kim, Dong-Yeon; Lee, Jang-Mi; Kim, Seong-Tae; Han, Tae-Hee; Ahn, Jin-Hyun

    2005-01-01

    Homo-oligomerization via a coiled-coil (C-C) domain has been shown to be necessary for the promyelocytic leukemia (PML)-retinoic acid receptor-α (RARα) fusion protein to acquire oncogenic potential in acute promyelocytic leukemia. We show here that PML(ΔC-C)-RARα, which contains a deletion in its C-C domain, is neither localized as characteristic microspeckles nor modified by small ubiquitin-like modifiers (SUMO). The absence of sumoylation of the ΔC-C mutant was due to the lack of binding to Ubc9, a SUMO conjugation enzyme. The integrity of RING finger domain was also needed for both sumoylation and microspeckle formation. In GAL4-DNA tethering assays, the ΔC-C mutant completely lost the inhibitory effect on retinoic acid (RA)-mediated transactivation. Furthermore, the expression of CD14 in U937 cells expressing the ΔC-C mutant in response to vitamin D3 was markedly higher than in cells expressing PML-RARα. However, the RA-mediated induction of C/EBPβ in cells expressing the ΔC-C mutant was comparable to that of control cells. Thus, our results suggest that the C-C domain-associated functions of sumoylation, localization as microspeckles, and the inhibition of monocyte differentiation all contribute to the oncogenic activity of PML-RARα

  15. Statins attenuate polymethylmethacrylate-mediated monocyte activation.

    LENUS (Irish Health Repository)

    Laing, Alan J

    2012-02-03

    BACKGROUND: Periprosthetic osteolysis precipitates aseptic loosening of components, increases the risk of periprosthetic fracture and, through massive bone loss, complicates revision surgery and ultimately is the primary cause for failure of joint arthroplasty. The anti-inflammatory properties of HMG-CoA reductase inhibitors belonging to the statin family are well recognized. We investigated a possible role for status in initiating the first stage of the osteolytic cycle, namely monocytic activation. METHODS: We used an in vitro model of the human monocyte\\/macrophage inflammatory response to poly-methylmethacrylate (PMMA) particles after pretreat-ing cells with cerivastatin, a potent member of the statin family. Cell activation based upon production of TNF-alpha and MCP-1 cytokines was analyzed and the intracellular Raf-MEK-ERK signal transduction pathway was evaluated using western blot analysis, to identify its role in cell activation and in any cerivastatin effects observed. RESULTS: We found that pretreatment with cerivastatin significantly abrogates the production of inflammatory cytokines TNF-alpha and MCP-1 by human monocytes in response to polymethylmethacrylate particle activation. This inflammatory activation and attenuation appear to be mediated through the intracellular Raf-MEK-ERK pathway. INTERPRETATION: We propose that by intervening at the upstream activation stage, subsequent osteoclast activation and osteolysis can be suppressed. We believe that the anti-inflammatory properties of statins may potentially play a prophylactic role in the setting of aseptic loosening, and in so doing increase implant longevity.

  16. Stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes by human monocytes Estágios da fagocitose in vitro por monócitos humanos de eritrócitos infectados por Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Maria Imaculada Muniz-Junqueira

    2009-04-01

    Full Text Available Monocytes/macrophages play a critical role in the defense mechanisms against malaria parasites, and are the main cells responsible for the elimination of malaria parasites from the blood circulation. We carried out a microscope-aided evaluation of the stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes, by human monocytes. These cells were obtained from healthy adult individuals by means of centrifugation through a cushion of Percoll density medium and were incubated with erythrocytes infected with Plasmodium falciparum that had previously been incubated with a pool of anti-plasmodial immune serum. We described the stages of phagocytosis, starting from adherence of infected erythrocytes to the phagocyte membrane and ending with their destruction within the phagolisosomes of the monocytes. We observed that the different erythrocytic forms of the parasite were ingested by monocytes, and that the process of phagocytosis may be completed in around 30 minutes. Furthermore, we showed that phagocytosis may occur continuously, such that different phases of the process were observed in the same phagocyte.Monócitos/macrófagos desempenham uma função crítica nos mecanismos de defesa antiplasmódio e constituem as principais células responsáveis pela eliminação das formas eritrocitárias do plasmódio da circulação sangüínea. Realizamos uma avaliação microscópica dos estágios da fagocitose in vitro de eritrócitos infectados por Plasmodium falciparum por monócitos humanos. Essas células foram obtidas de indivíduos adultos sadios por centrifugação em Percoll e incubadas com eritrócitos infectados por Plasmodium falciparum previamente incubados com um pool de soro imune contra plasmódio. Descrevemos os estágios da fagocitose, desde a aderência dos eritrócitos infectados até sua destruição nos fagolisossomas dos monócitos. Observou-se que eritrócitos infectados por todos os diferentes est

  17. Gallic Acid Is the Major Active Component of Cortex Moutan in Inhibiting Immune Maturation of Human Monocyte-Derived Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Ben Chung Lap Chan

    2015-09-01

    Full Text Available Atopic dermatitis (AD is a widely prevalent and chronically relapsing inflammatory skin disease. Penta Herbs Formula (PHF is efficacious in improving the quality of life and reducing topical corticosteroid used in children with AD and one of the active herbs it contains is Cortex Moutan. Recent studies showed that altered functions of dendritic cells (DC were observed in atopic individuals, suggesting that DC might play a major role in the generation and maintenance of inflammation by their production of pro-inflammatory cytokines. Hence, the aims of the present study were to identify the major active component(s of Cortex Moutan, which might inhibit DC functions and to investigate their possible interactions with conventional corticosteroid on inhibiting the development of DC from monocytes. Monocyte-derived dendritic cells (moDC culture model coupled with the high-speed counter-current chromatography (HSCCC, high pressure liquid chromatography (HPLC and Liquid Chromatography-Mass Spectrometry (LCMS analyses were used. Gallic acid was the major active component from Cortex Moutan which could dose dependently inhibit interleukin (IL-12 p40 and the functional cluster of differentiation (CD surface markers CD40, CD80, CD83 and CD86 expression from cytokine cocktail-activated moDC. Gallic acid could also lower the concentration of hydrocortisone required to inhibit the activation of DC.

  18. Bioactivity screening and mass spectrometric confirmation for the detection of PPAR-delta agonists that increase type 1 muscle fibres

    NARCIS (Netherlands)

    Bovee, T.F.H.; Blokland, M.H.; Kersten, A.H.; Hamers, A.R.M.; Heskamp, H.H.; Essers, M.L.; Nielen, M.W.F.; Ginkel, van L.A.

    2014-01-01

    Sensitive and robust bioassays able to detect nuclear receptor activation are very useful for veterinary and doping control, pharmaceutical industry and environmental scientists. Here, we used bioassays based on human leukemic monocyte lymphoma U937 and human liver hepatocellular carcinoma HepG2

  19. Transcriptome analysis of monocyte-HIV interactions

    Directory of Open Access Journals (Sweden)

    Tran Huyen

    2010-06-01

    Full Text Available Abstract Background During HIV infection and/or antiretroviral therapy (ART, monocytes and macrophages exhibit a wide range of dysfunctions which contribute significantly to HIV pathogenesis and therapy-associated complications. Nevertheless, the molecular components which contribute to these dysfunctions remain elusive. We therefore applied a parallel approach of genome-wide microarray analysis and focused gene expression profiling on monocytes from patients in different stages of HIV infection and/or ART to further characterise these dysfunctions. Results Processes involved in apoptosis, cell cycle, lipid metabolism, proteasome function, protein trafficking and transcriptional regulation were identified as areas of monocyte dysfunction during HIV infection. Individual genes potentially contributing to these monocyte dysfunctions included several novel factors. One of these is the adipocytokine NAMPT/visfatin, which we show to be capable of inhibiting HIV at an early step in its life cycle. Roughly half of all genes identified were restored to control levels under ART, while the others represented a persistent dysregulation. Additionally, several candidate biomarkers (in particular CCL1 and CYP2C19 for the development of the abacavir hypersensitivity reaction were suggested. Conclusions Previously described areas of monocyte dysfunction during HIV infection were confirmed, and novel themes were identified. Furthermore, individual genes associated with these dysfunctions and with ART-associated disorders were pinpointed. These genes form a useful basis for further functional studies concerning the contribution of monocytes/macrophages to HIV pathogenesis. One such gene, NAMPT/visfatin, represents a possible novel restriction factor for HIV. Background Both macrophages and T lymphocyte subsets express the CD4 receptor and either the CXCR4 and/or the CCR5 coreceptor which confer susceptibility to infection with the Human Immunodeficiency Virus

  20. Activation of the canonical Wnt/β-catenin pathway enhances monocyte adhesion to endothelial cells

    International Nuclear Information System (INIS)

    Lee, Dong Kun; Nathan Grantham, R.; Trachte, Aaron L.; Mannion, John D.; Wilson, Colleen L.

    2006-01-01

    Monocyte adhesion to vascular endothelium has been reported to be one of the early processes in the development of atherosclerosis. In an attempt to develop strategies to prevent or delay atherosclerosis progression, we analyzed effects of the Wnt/β-catenin signaling pathway on monocyte adhesion to various human endothelial cells. Adhesion of fluorescein-labeled monocytes to various human endothelial cells was analyzed under a fluorescent microscope. Unlike sodium chloride, lithium chloride enhanced monocyte adhesion to endothelial cells in a dose-dependent manner. We further demonstrated that inhibitors for glycogen synthase kinase (GSK)-3β or proteosome enhanced monocyte-endothelial cell adhesion. Results of semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) indicated that activation of Wnt/β-catenin pathway did not change expression levels of mRNA for adhesion molecules. In conclusion, the canonical Wnt/β-catenin pathway enhanced monocyte-endothelial cell adhesion without changing expression levels of adhesion molecules

  1. Regulation of CD43-induced U937 homotypic aggregation

    Czech Academy of Sciences Publication Activity Database

    Cho, J. Y.; Chain, B.; M. Vives, J.; Hořejší, Václav; Katz, D. R.

    2003-01-01

    Roč. 290, č. 1 (2003), s. 155-167 ISSN 0014-4827 R&D Projects: GA MŠk LN00A026 Institutional research plan: CEZ:AV0Z5052915 Keywords : CD43 * cell adhesion Subject RIV: EC - Immunology Impact factor: 3.949, year: 2003

  2. Prion protein induced signaling cascades in monocytes

    International Nuclear Information System (INIS)

    Krebs, Bjarne; Dorner-Ciossek, Cornelia; Schmalzbauer, Ruediger; Vassallo, Neville; Herms, Jochen; Kretzschmar, Hans A.

    2006-01-01

    Prion proteins play a central role in transmission and pathogenesis of transmissible spongiform encephalopathies. The cellular prion protein (PrP C ), whose physiological function remains elusive, is anchored to the surface of a variety of cell types including neurons and cells of the lymphoreticular system. In this study, we investigated the response of a mouse monocyte/macrophage cell line to exposure with PrP C fusion proteins synthesized with a human Fc-tag. PrP C fusion proteins showed an attachment to the surface of monocyte/macrophages in nanomolar concentrations. This was accompanied by an increase of cellular tyrosine phosphorylation as a result of activated signaling pathways. Detailed investigations exhibited activation of downstream pathways through a stimulation with PrP fusion proteins, which include phosphorylation of ERK 1,2 and Akt kinase. Macrophages opsonize and present antigenic structures, contact lymphocytes, and deliver cytokines. The findings reported here may become the basis of understanding the molecular function of PrP C in monocytes and macrophages

  3. Pertussis Toxin Exploits Host Cell Signaling Pathways Induced by Meningitis-Causing E. coli K1-RS218 and Enhances Adherence of Monocytic THP-1 Cells to Human Cerebral Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Laura Julia Starost

    2016-10-01

    Full Text Available Pertussis toxin (PTx, the major virulence factor of the whooping cough-causing bacterial pathogen Bordetella pertussis, permeabilizes the blood–brain barrier (BBB in vitro and in vivo. Breaking barriers might promote translocation of meningitis-causing bacteria across the BBB, thereby facilitating infection. PTx activates several host cell signaling pathways exploited by the neonatal meningitis-causing Escherichia coli K1-RS218 for invasion and translocation across the BBB. Here, we investigated whether PTx and E. coli K1-RS218 exert similar effects on MAPK p38, NF-κB activation and transcription of downstream targets in human cerebral endothelial TY10 cells using qRT-PCR, Western blotting, and ELISA in combination with specific inhibitors. PTx and E. coli K1-RS218 activate MAPK p38, but only E. coli K1-RS218 activates the NF-κB pathway. mRNA and protein levels of p38 and NF-κB downstream targets including IL-6, IL-8, CxCL-1, CxCL-2 and ICAM-1 were increased. The p38 specific inhibitor SB203590 blocked PTx-enhanced activity, whereas E. coli K1-RS218’s effects were inhibited by the NF-κB inhibitor Bay 11-7082. Further, we found that PTx enhances the adherence of human monocytic THP-1 cells to human cerebral endothelial TY10 cells, thereby contributing to enhanced translocation. These modulations of host cell signaling pathways by PTx and meningitis-causing E. coli support their contributions to pathogen and monocytic THP-1 cells translocation across the BBB.

  4. Comparison of cellular effects of starch-coated SPIONs and poly(lactic-co-glycolic acid matrix nanoparticles on human monocytes

    Directory of Open Access Journals (Sweden)

    Gonnissen D

    2016-10-01

    Full Text Available Dominik Gonnissen,1 Ying Qu,1,2 Klaus Langer,3 Cengiz Öztürk,4 Yuliang Zhao,2 Chunying Chen,2 Guiscard Seebohm,5 Martina Düfer,6 Harald Fuchs,1 Hans-Joachim Galla,7 Kristina Riehemann11Center for Nanotechnology, Institute of Physics, University of Münster, Münster, Germany; 2National Center for Nanoscience and Technology, Chinese Academy of Sciences, Beijing, People’s Republic of China; 3Institute of Pharmaceutical Technology and Biopharmacy, University of Münster, Münster, 4chemicell GmbH, Berlin, 5Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases, University Hospital Münster, 6Department of Pharmacology, Institute of Pharmaceutical and Medicinal Chemistry, 7Department of Cell Biology/Biophysics, Institute of Biochemistry, University of Münster, Münster, GermanyAbstract: Within the last years, progress has been made in the knowledge of the properties of medically used nanoparticles and their toxic effects, but still, little is known about their influence on cellular processes of immune cells. The aim of our comparative study was to present the influence of two different nanoparticle types on subcellular processes of primary monocytes and the leukemic monocyte cell line MM6. We used core-shell starch-coated superparamagnetic iron oxide nanoparticles (SPIONs and matrix poly(lactic-co-glycolic acid (PLGA nanoparticles for our experiments. In addition to typical biocompatibility testing like the detection of necrosis or secretion of interleukins (ILs, we investigated the impact of these nanoparticles on the actin cytoskeleton and the two voltage-gated potassium channels Kv1.3 and Kv7.1. Induction of necrosis was not seen for PLGA nanoparticles and SPIONs in primary monocytes and MM6 cells. Likewise, no alteration in secretion of IL-1β and IL-10 was detected under the same experimental conditions. In contrast, IL-6 secretion was exclusively downregulated in primary monocytes after contact with both

  5. Lipopolysaccharide regulated protein expression is only partly impaired in monocytes from patients with type I diabetes

    Directory of Open Access Journals (Sweden)

    Abke Sabine

    2006-03-01

    Full Text Available Abstract Background Monocytes play an important role in innate immunity and atherosclerosis. A disturbed secretion of cytokines in lipopolysaccharide (LPS activated monocytes from type 1 diabetes (T1D patients has been described and may contribute to the impaired inflammatory response in these individuals. In the present study the influence of LPS on five different proteins with a function in immunity and atherosclerosis was analyzed in monocytes from controls and T1D patients. Methods Monocytes were isolated from controls and T1D patients and the LPS-stimulated increase of IL-6, CXCL8, monocyte chemotactic protein 1 (CCL2, MCP-1 and superoxide dismutase (SOD 2, as well as the LPS-mediated decrease of apolipoprotein E (Apo E in primary human monocytes from controls and T1D patients was determined. Results CCL2 and IL-6 secretion in response to LPS was found significantly reduced in monocytes from T1D patients when compared to controls whereas basal CCL2 release was similar in control and T1D cells. In contrast, CXCL8 and apolipoprotein E secretion and SOD 2 expression upon LPS stimulation is similar from T1D and control monocytes. Conclusion These data indicate that LPS-mediated protein expression is only partly disturbed in monocytes from T1D patients. Reduced secretion of IL-6 and CCL2 in activated monocytes of these patients may contribute to an impaired inflammatory response and vascular disease.

  6. Novel leads from Heliotropium ovalifolium, 4,7,8-trimethoxy-naphthalene-2-carboxylic acid and 6-hydroxy-5,7-dimethoxy-naphthalene-2-carbaldehyde show specific IL-6 inhibitory activity in THP-1 cells and primary human monocytes.

    Science.gov (United States)

    Kulkarni-Almeida, Asha; Suthar, Ashish; Goswami, Hitesh; Vishwakarma, Ram; Chauhan, Vijay Singh; Balakrishnan, Arun; Sharma, Somesh

    2008-12-01

    From our screening program, we identified the anti-inflammatory effects of the extracts of Heliotropium ovalifolium in its ability to inhibit specific cytokines. The H. ovalifolium extract was found to be moderately active with an IC(50) equaling 10 microg/ml for inhibition of interleukin-6 (IL-6) in a human monocytic cell line. Interleukin-6 is a pleiotropic cytokine with implications in the regulation of the immune response, inflammation and hematopoiesis. This prompted us to examine and identify the active molecules that are responsible for the bioactivity in THP-1 cells. Bioassay guided fractionation identified two compounds 4,7,8-trimethoxy-naphthalene-2-carboxylic acid and 6-hydroxy-5,7-dimethoxy-naphthalene-2-carbaldehyde with an IC(50) of 2.4 and 2.0 microM for IL-6 inhibition and an IC(50) of 15.6 and 7.0 microM for tumor necrosis factor-alpha (TNF-alpha) inhibition in THP-1 cells. The protein expression data were supported by the inhibitory effect on mRNA gene expression. The compounds isolated from H. ovalifolium were also non-toxic in human peripheral blood monocytes from normal donors and the activity profile was similar to that obtained on THP-1 cells. Thus, we believe that these scaffolds may be of interest to develop leads for treating rheumatoid arthritis, psoriasis, ulcerative colitis, Crohn's disease and other inflammatory disorders. However, more detailed investigations need to be carried out to explain the efficacy of these compounds as drugs.

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

    NARCIS (Netherlands)

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

    1999-01-01

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

  8. Carica Papaya Seed Extract Enhances Cellular Response to Stress ...

    African Journals Online (AJOL)

    Therefore, the present study was carried out to investigate the role of Carica papaya seed (CPS) extract that contains, Benzyl Isothiocyanates, one of the inducers of phase II enzymes in the regulation of cellular stress. The cellular responses were observed in U937 cells (human monocyte/macrophage cell line) at the ...

  9. The differential role of human macrophage in triggering secondary bystander effects after either gamma-ray or carbon beam irradiation.

    Science.gov (United States)

    Dong, Chen; He, Mingyuan; Tu, Wenzhi; Konishi, Teruaki; Liu, Weili; Xie, Yuexia; Dang, Bingrong; Li, Wenjian; Uchihori, Yukio; Hei, Tom K; Shao, Chunlin

    2015-07-10

    The abscopal effect could be an underlying factor in evaluating prognosis of radiotherapy. This study established an in vitro system to examine whether tumor-generated bystander signals could be transmitted by macrophages to further trigger secondary cellular responses after different irradiations, where human lung cancer NCI-H446 cells were irradiated with either γ-rays or carbon ions and co-cultured with human macrophage U937 cells, then these U937 cells were used as a bystander signal transmitter and co-cultured with human bronchial epithelial cells BEAS-2B. Results showed that U937 cells were only activated by γ-irradiated NCI-H446 cells so that the secondary injuries in BEAS-2B cells under carbon ion irradiation were weaker than γ-rays. Both TNF-α and IL-1α were involved in the γ-irradiation induced secondary bystander effect but only TNF-α contributed to the carbon ion induced response. Further assay disclosed that IL-1α but not TNF-α was largely responsible for the activation of macrophages and the formation of micronucleus in BEAS-2B cells. These data suggest that macrophages could transfer secondary bystander signals and play a key role in the secondary bystander effect of photon irradiation, while carbon ion irradiation has conspicuous advantage due to its reduced secondary injury. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. A novel peptide derived from human pancreatitis-associated protein inhibits inflammation in vivo and in vitro and blocks NF-kappa B signaling pathway.

    Directory of Open Access Journals (Sweden)

    Xiaolu Yang

    Full Text Available BACKGROUND: Pancreatitis-associated protein (PAP is a pancreatic secretory protein belongs to the group VII of C-type lectin family. Emerging evidence suggests that PAP plays a protective effect in inflammatory diseases. In the present study, we newly identified a 16-amino-acid peptide (named PAPep derived from C-type lectin-like domain (CTLD of human PAP with potent anti-inflammatory activity using both in vivo and in vitro assays. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the anti-inflammatory effect of PAPep on endotoxin-induced uveitis (EIU in rats and demonstrated that intravitreal pretreatment of PAPep concentration-dependently attenuated clinical manifestation of EIU rats, reduced protein leakage and cell infiltration into the aqueous humor (AqH, suppressed tumor necrosis factor (TNF-α, interleukin (IL-6, intercellular adhesion molecule-1 (ICAM-1 and monocyte chemoattractant protein (MCP-1 production in ocular tissues, and improved histopathologic manifestation of EIU. Furthermore, PAPep suppressed the LPS-induced mRNA expression of TNF-α and IL-6 in RAW 264.7 cells, inhibited protein expression of ICAM-1 in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs as well as U937 cells adhesion to HUVECs. Western blot analysis in ocular tissues and different cell lines revealed that the possible mechanism for this anti-inflammatory effect of PAPep may depend on its ability to inhibit the activation of NF-kB signaling pathway. CONCLUSIONS/SIGNIFICANCE: Our studies provide the first evidence that the sequence of PAPep is within the critically active region for the anti-inflammatory function of PAP and the peptide may be a promising candidate for the management of ocular inflammatory diseases.

  11. STAT3 activation in monocytes accelerates liver cancer progression

    International Nuclear Information System (INIS)

    Wu, Wen-Yong; Li, Jun; Wu, Zheng-Sheng; Zhang, Chang-Le; Meng, Xiang-Ling

    2011-01-01

    Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor ubiquitously expressed in different cell types. STAT3 plays an essential role in cell survival, proliferation, and differentiation. Aberrantly hyper-activated STAT3 signaling in cancer cells and in the tumor microenvironment has been detected in a wide variety of human cancers and is considered an important factor for cancer initiation, development, and progression. However, the role of STAT3 activation in monocytes in the development of HCC has not been well understood. Immunohistochemical analysis of phosphorylated STAT3 was performed on tissue microarray from HCC patients. Using a co-culture system in vivo, HCC cell growth was determined by the MTT assay. In vivo experiments were conducted with mice given diethylinitrosamine (DEN), which induces HCC was used to investigate the role of STAT3 expression in monocytes on tumor growth. Real-time PCR was used to determine the expression of cell proliferation and cell arrest associated genes in the tumor and nontumor tissue from liver. Phosphorylated STAT3 was found in human hepatocellular carcinoma tissue samples and was expressed in tumor cells and also in monocytes. Phosphorylated STAT3 expression in monocyte was significantly correlated to advanced clinical stage of HCC and a poor prognosis. Using a co-culture system in vivo, monocytes promoted HCC cell growth via the IL-6/STAT3 signaling pathway. The STAT3 inhibitor, NSC 74859, significantly suppressed tumor growth in vivo in mice with diethylinitrosamine (DEN)-induced HCC. In this animal model, blockade of STAT3 with NSC 74859 induced tumor cell apoptosis, while inhibiting both tumor cells and monocytes proliferation. Furthermore, NSC 74859 treatment suppressed cancer associated inflammation in DEN-induce HCC. Our data suggest constitutively activated STAT3 monocytes promote liver tumorigenesis in clinical patients and animal experiments. Thus, STAT3 in tumor

  12. Ex vivo expanded human regulatory T cells delay islet allograft rejection via inhibiting islet-derived monocyte chemoattractant protein-1 production in CD34+ stem cells-reconstituted NOD-scid IL2rγnull mice.

    Science.gov (United States)

    Xiao, Fang; Ma, Liang; Zhao, Min; Huang, Guocai; Mirenda, Vincenzo; Dorling, Anthony; Lechler, Robert; Lombardi, Giovanna

    2014-01-01

    Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo.

  13. Ex Vivo Expanded Human Regulatory T Cells Delay Islet Allograft Rejection via Inhibiting Islet-Derived Monocyte Chemoattractant Protein-1 Production in CD34+ Stem Cells-Reconstituted NOD-scid IL2rγnull Mice

    Science.gov (United States)

    Xiao, Fang; Ma, Liang; Zhao, Min; Huang, Guocai; Mirenda, Vincenzo; Dorling, Anthony

    2014-01-01

    Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo. PMID:24594640

  14. Ex vivo expanded human regulatory T cells delay islet allograft rejection via inhibiting islet-derived monocyte chemoattractant protein-1 production in CD34+ stem cells-reconstituted NOD-scid IL2rγnull mice.

    Directory of Open Access Journals (Sweden)

    Fang Xiao

    Full Text Available Type 1 diabetes mellitus (T1DM is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo.

  15. Induction of IL-12 Production in Human Peripheral Monocytes by Trypanosoma cruzi Is Mediated by Glycosylphosphatidylinositol-Anchored Mucin-Like Glycoproteins and Potentiated by IFN-γ and CD40-CD40L Interactions

    Directory of Open Access Journals (Sweden)

    Lúcia Cristina Jamli Abel

    2014-01-01

    Full Text Available Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi, is characterized by immunopathology driven by IFN-γ secreting Th1-like T cells. T. cruzi has a thick coat of mucin-like glycoproteins covering its surface, which plays an important role in parasite invasion and host immunomodulation. It has been extensively described that T. cruzi or its products—like GPI anchors isolated from GPI-anchored mucins from the trypomastigote life cycle stage (tGPI-mucins—are potent inducers of proinflammatory responses (i.e., cytokines and NO production by IFN-γ primed murine macrophages. However, little is known about whether T. cruzi or GPI-mucins exert a similar action in human cells. We therefore decided to further investigate the in vitro cytokine production profile from human mononuclear cells from uninfected donors exposed to T. cruzi as well as tGPI-mucins. We observed that both living T. cruzi trypomastigotes and tGPI-mucins are potent inducers of IL-12 by human peripheral blood monocytes and this effect depends on CD40-CD40L interaction and IFN-γ. Our findings suggest that the polarized T1-type cytokine profile seen in T. cruzi infected patients might be a long-term effect of IL-12 production induced by lifelong exposure to T. cruzi tGPI-mucins.

  16. Distinct Properties of Human M-CSF and GM-CSF Monocyte-Derived Macrophages to Simulate Pathological Lung Conditions In Vitro: Application to Systemic and Inflammatory Disorders with Pulmonary Involvement.

    Science.gov (United States)

    Lescoat, Alain; Ballerie, Alice; Augagneur, Yu; Morzadec, Claudie; Vernhet, Laurent; Fardel, Olivier; Jégo, Patrick; Jouneau, Stéphane; Lecureur, Valérie

    2018-03-17

    Macrophages play a central role in the pathogenesis of inflammatory and fibrotic lung diseases. However, alveolar macrophages (AM) are poorly available in humans to perform in vitro studies due to a limited access to broncho-alveolar lavage (BAL). In this study, to identify the best alternative in vitro model for human AM, we compared the phenotype of AM obtained from BAL of patients suffering from three lung diseases (lung cancers, sarcoidosis and Systemic Sclerosis (SSc)-associated interstitial lung disease) to human blood monocyte-derived macrophages (MDMs) differentiated with M-CSF or GM-CSF. The expression of eight membrane markers was evaluated by flow cytometry. Globally, AM phenotype was closer to GM-CSF MDMs. However, the expression levels of CD163, CD169, CD204, CD64 and CD36 were significantly higher in SSc-ILD than in lung cancers. Considering the expression of CD204 and CD36, the phenotype of SSc-AM was closer to MDMs, from healthy donors or SSc patients, differentiated by M-CSF rather than GM-CSF. The comparative secretion of IL-6 by SSc-MDMs and SSc-AM is concordant with these phenotypic considerations. Altogether, these results support the M-CSF MDM model as a relevant in vitro alternative to simulate AM in fibrotic disorders such as SSc.

  17. Flavonoid metabolites reduce tumor necrosis factor-α secretion to a greater extent than their precursor compounds in human THP-1 monocytes.

    Science.gov (United States)

    di Gesso, Jessica L; Kerr, Jason S; Zhang, Qingzhi; Raheem, Saki; Yalamanchili, Sai Krishna; O'Hagan, David; Kay, Colin D; O'Connell, Maria A

    2015-06-01

    Flavonoids are generally studied in vitro, in isolation, and as unmetabolized precursor structures. However, in the habitual diet, multiple flavonoids are consumed together and found present in the circulation as complex mixtures of metabolites. Using a unique study design, we investigated the potential for singular or additive anti-inflammatory effects of flavonoid metabolites relative to their precursor structures. Six flavonoids, 14 flavonoid metabolites, and 29 combinations of flavonoids and their metabolites (0.1-10 μM) were screened for their ability to reduce LPS-induced tumor necrosis factor-α (TNF-α) secretion in THP-1 monocytes. One micromolar peonidin-3-glucoside, cyanidin-3-glucoside, and the metabolites isovanillic acid (IVA), IVA-glucuronide, vanillic acid-glucuronide, protocatechuic acid-3-sulfate, and benzoic acid-sulfate significantly reduced TNF-α secretion when in isolation, while there was no effect on TNF-α mRNA expression. Four combinations of metabolites that included 4-hydroxybenzoic acid (4HBA) and/or protocatechuic acid also significantly reduced TNF-α secretion to a greater extent than the precursors or metabolites alone. The effects on LPS-induced IL-1β and IL-10 secretion and mRNA expression were also examined. 4HBA significantly reduced IL-1β secretion but none of the flavonoids or metabolites significantly modified IL-10 secretion. This study provides novel evidence suggesting flavonoid bioactivity results from cumulative or additive effects of circulating metabolites. © 2015 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Flavonoid metabolites reduce tumor necrosis factor‐α secretion to a greater extent than their precursor compounds in human THP‐1 monocytes

    Science.gov (United States)

    di Gesso, Jessica L.; Kerr, Jason S.; Zhang, Qingzhi; Raheem, Saki; Yalamanchili, Sai Krishna; O'Hagan, David; Kay, Colin D.; O'Connell, Maria A.

    2015-01-01

    1 Scope Flavonoids are generally studied in vitro, in isolation, and as unmetabolized precursor structures. However, in the habitual diet, multiple flavonoids are consumed together and found present in the circulation as complex mixtures of metabolites. Using a unique study design, we investigated the potential for singular or additive anti‐inflammatory effects of flavonoid metabolites relative to their precursor structures. 2 Methods and results Six flavonoids, 14 flavonoid metabolites, and 29 combinations of flavonoids and their metabolites (0.1–10 μM) were screened for their ability to reduce LPS‐induced tumor necrosis factor‐α (TNF‐α) secretion in THP‐1 monocytes. One micromolar peonidin‐3‐glucoside, cyanidin‐3‐glucoside, and the metabolites isovanillic acid (IVA), IVA‐glucuronide, vanillic acid‐glucuronide, protocatechuic acid‐3‐sulfate, and benzoic acid‐sulfate significantly reduced TNF‐α secretion when in isolation, while there was no effect on TNF‐α mRNA expression. Four combinations of metabolites that included 4‐hydroxybenzoic acid (4HBA) and/or protocatechuic acid also significantly reduced TNF‐α secretion to a greater extent than the precursors or metabolites alone. The effects on LPS‐induced IL‐1β and IL‐10 secretion and mRNA expression were also examined. 4HBA significantly reduced IL‐1β secretion but none of the flavonoids or metabolites significantly modified IL‐10 secretion. 3 Conclusion This study provides novel evidence suggesting flavonoid bioactivity results from cumulative or additive effects of circulating metabolites. PMID:25801720

  19. The radioactive labeling of monocytes

    International Nuclear Information System (INIS)

    Ensing, G.J.

    1985-01-01

    With the aim of studying a possible relationship between circulating monocytes and Sternberg-Reed cells investigations were started on the specific labeling of monocytes. In this thesis the literature on the pertinent data has been reviewed and a series of experiments on the monocyte labeling procedure has been described. The principles of cell labeling with radioactive compounds were discussed. 1. Total separation of the particular cell population to be labeled and subsequent labeling with a non-specific radiopharmaceutical. 2. Specific cell labeling in a mixture of cell types based on a well defined affinity of the cell under study for the radiopharmaceutical used. Next the radionuclides that can be used for cell labeling purposes were discussed with special attention for 111 In and its chelates. The principles of radiodosimetry were also discussed shortly. This section was focussed on the radiation dose the labeled cells receive because of the intracellular localized radioactivity. The radiation burden is high in comparison to amounts of radiation known to affect cell viability. A newly developed method for labeling monocytes specifically by phagocytosis of 111 In-Fe-colloid without apparent loss of cells was described in detail. (Auth.)

  20. Generation of Adducts of 4-Hydroxy-2-nonenal with Heat Shock 60 kDa Protein 1 in Human Promyelocytic HL-60 and Monocytic THP-1 Cell Lines

    Directory of Open Access Journals (Sweden)

    Alessia Arcaro

    2015-01-01

    Full Text Available Heat shock 60 kDa protein 1 (HSP60 is a chaperone and stress response protein responsible for protein folding and delivery of endogenous peptides to antigen-presenting cells and also a target of autoimmunity implicated in the pathogenesis of atherosclerosis. By two-dimensional electrophoresis and mass spectrometry, we found that exposure of human promyelocytic HL-60 cells to a nontoxic concentration (10 μM of 4-hydroxy-2-nonenal (HNE yielded a HSP60 modified with HNE. We also detected adducts of HNE with putative uncharacterized protein CXorf49, the product of an open reading frame identified in various cell and tissue proteomes. Moreover, exposure of human monocytic THP-1 cells differentiated with phorbol 12-myristate 13-acetate to 10 μM HNE, and to light density lipoprotein modified with HNE (HNE-LDL or by copper-catalyzed oxidation (oxLDL, but not to native LDL, stimulated the formation of HNE adducts with HSP60, as detected by immunoprecipitation and western blot, well over basal levels. The identification of HNE-HSP60 adducts outlines a framework of mutually reinforcing interactions between endothelial cell stressors, like oxLDL and HSP60, whose possible outcomes, such as the amplification of endothelial dysfunction, the spreading of lipoxidative damage to other proteins, such as CXorf49, the activation of antigen-presenting cells, and the breaking of tolerance to HSP60 are discussed.

  1. Increased MCP-1 gene expression in monocytes of severe OSA patients and under intermittent hypoxia.

    Science.gov (United States)

    Chuang, Li-Pang; Chen, Ning-Hung; Lin, Yuling; Ko, Wen-Shan; Pang, Jong-Hwei S

    2016-03-01

    Obstructive sleep apnea (OSA) is known to be a risk factor of coronary artery disease. Monocyte chemoattractant protein-1 (MCP-1), as a critical factor for monocyte infiltration, is known to play a role in the development of atherosclerosis. This study aimed to investigate the effect of intermittent hypoxia, the hallmark of OSA, on the MCP-1 expression of monocytes. Peripheral blood was sampled from 61 adults enrolled for suspected OSA. RNA was prepared from the isolated monocytes for the analysis of MCP-1. The effect of in vitro intermittent hypoxia on the regulation and function of MCP-1 was investigated on THP-1 monocytic cells and human monocytes. The mRNA and secreted protein levels were investigated by RT/real-time PCR and enzyme-linked immunosorbent assay, respectively. Monocytic MCP-1 gene expression was found to be increased significantly in severe OSA patients. In vitro intermittent hypoxia was demonstrated to increase the mRNA and protein expression levels of MCP-1 dose- and time-dependently in THP-1 monocytic cells. The MCP-1 mRNA expression in monocytes isolated from OSA patient was induced to a much higher level compared to that from normal control. Pre-treatment with inhibitor for p42/44 MAPK or p38 MAPK suppressed the activation of MCP-1 expression by intermittent hypoxia. This is the first study to demonstrate the increase of MCP-1 gene expression in monocytes of severe OSA patients. In addition, monocytic MCP-1 gene expression can be induced under intermittent hypoxia.

  2. Expression profiling feline peripheral blood monocytes identifies a transcriptional signature associated with type two diabetes mellitus.

    Science.gov (United States)

    O'Leary, Caroline A; Sedhom, Mamdouh; Reeve-Johnson, Mia; Mallyon, John; Irvine, Katharine M

    2017-04-01

    Diabetes mellitus is a common disease of cats and is similar to type 2 diabetes (T2D) in humans, especially with respect to the role of obesity-induced insulin resistance, glucose toxicity, decreased number of pancreatic β-cells and pancreatic amyloid deposition. Cats have thus been proposed as a valuable translational model of T2D. In humans, inflammation associated with adipose tissue is believed to be central to T2D development, and peripheral blood monocytes (PBM) are important in the inflammatory cascade which leads to insulin resistance and β-cell failure. PBM may thus provide a useful window to study the pathogenesis of diabetes mellitus in cats, however feline monocytes are poorly characterised. In this study, we used the Affymetrix Feline 1.0ST array to profile peripheral blood monocytes from 3 domestic cats with T2D and 3 cats with normal glucose tolerance. Feline monocytes were enriched for genes expressed in human monocytes, and, despite heterogeneous gene expression, we identified a T2D-associated expression signature associated with cell cycle perturbations, DNA repair and the unfolded protein response, oxidative phosphorylation and inflammatory responses. Our data provide novel insights into the feline monocyte transcriptome, and support the hypothesis that inflammatory monocytes contribute to T2D pathogenesis in cats as well as in humans. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. HCMV Reprogramming of Infected Monocyte Survival and Differentiation: A Goldilocks Phenomenon

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    Emily V. Stevenson

    2014-02-01

    Full Text Available The wide range of disease pathologies seen in multiple organ sites associated with human cytomegalovirus (HCMV infection results from the systemic hematogenous dissemination of the virus, which is mediated predominately by infected monocytes. In addition to their role in viral spread, infected monocytes are also known to play a key role in viral latency and life-long persistence. However, in order to utilize infected monocytes for viral spread and persistence, HCMV must overcome a number of monocyte biological hurdles, including their naturally short lifespan and their inability to support viral gene expression and replication. Our laboratory has shown that HCMV is able to manipulate the biology of infected monocytes in order to overcome these biological hurdles by inducing the survival and differentiation of infected monocytes into long-lived macrophages capable of supporting viral gene expression and replication. In this current review, we describe the unique aspects of how HCMV promotes monocyte survival and differentiation by inducing a “finely-tuned” macrophage cell type following infection. Specifically, we describe the induction of a uniquely polarized macrophage subset from infected monocytes, which we argue is the ideal cellular environment for the initiation of viral gene expression and replication and, ultimately, viral spread and persistence within the infected host.

  4. Low-frequency electromagnetic fields do not alter responses of inflammatory genes and proteins in human monocytes and immune cell lines

    NARCIS (Netherlands)

    Bouwens, M.; Kleijn, de S.; Cuppen, J.J.M.; Savelkoul, H.F.J.; Verburg-van Kemenade, B.M.L.

    2012-01-01

    The effects of low frequency electromagnetic fields (LF EMF) on human health are the subject of on-going research and serious public concern. These fields potentially elicit small effects that have been proposed to have consequences, either positive or negative, for biological systems. To reveal

  5. Effect and possible mechanism of monocyte-derived VEGF on monocyte-endothelial cellular adhesion after electrical burns.

    Science.gov (United States)

    Ruan, Qiongfang; Zhao, Chaoli; Ye, Ziqing; Ruan, Jingjing; Xie, Qionghui; Xie, Weiguo

    2015-06-01

    One of the major obstacles in the treatment of severe electrical burns is properly handling the resulting uncontrolled inflammation. Such inflammation often causes secondary injury and necrosis, thus complicating patient outcomes. Vascular endothelial grow factor (VEGF) has emerged as an important mediator for the recruitment of monocytes to the site inflammation. This study was designed to explore the effects and possible mechanism of VEGF on monocyte-endothelial cellular adhesion. To do so, we used a cultured human monocytic cell line (THP-1) that was stimulated with serum derived from rats that had received electrical burns. Serum was obtained from rats that had received electrical burns. Both the VEGF and soluble flt-1 (sflt-1) concentrations of the serum were determined by double-antibody sandwich ELISA. The concentrations of VEGF, sflt-1, and TNF-α obtained from the cell-free cultured supernatant of THP-1 cells that had been exposed to the serum were then determined by double-antibody sandwich ELISA. Serum-stimulated THP-1 cells were added to wells with a monolayer of endothelial cells to detect the level of monocyte-endothelial cells adhesion. Finally, the state of phosphorylation of AKT was determined by Western blotting. Both in vivo and in vitro studies showed that compared to controls, the levels of VEGF were significantly increased after electrical burns. This increased was accompanied by a reduction of sflt-1 levels. Furthermore, the serum of rats that had received electrical burns was able to both activate monocytes to secrete TNF-α and enhance monocyte-endothelial cell adhesion. Treatment with the serum also resulted in an up-regulation of the phosphorylation of AKT, but had no effect on the total levels of AKT. Phosphatidylinositide 3-kinases (PI3K) inhibition decreased the number of THP-1 cells that were adhered to endothelial cells. Finally, sequestering VEGF with sflt-1 was able to reduce the effect on monocyte-endothelial cells adhesion by

  6. Psychedelic N,N-dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine modulate innate and adaptive inflammatory responses through the sigma-1 receptor of human monocyte-derived dendritic cells.

    Directory of Open Access Journals (Sweden)

    Attila Szabo

    Full Text Available The orphan receptor sigma-1 (sigmar-1 is a transmembrane chaperone protein expressed in both the central nervous system and in immune cells. It has been shown to regulate neuronal differentiation and cell survival, and mediates anti-inflammatory responses and immunosuppression in murine in vivo models. Since the details of these findings have not been elucidated so far, we studied the effects of the endogenous sigmar-1 ligands N,N-dimethyltryptamine (NN-DMT, its derivative 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT and the synthetic high affinity sigmar-1 agonist PRE-084 hydrochloride on human primary monocyte-derived dendritic cell (moDCs activation provoked by LPS, polyI:C or pathogen-derived stimuli to induce inflammatory responses. Co-treatment of moDC with these activators and sigma-1 receptor ligands inhibited the production of pro-inflammatory cytokines IL-1β, IL-6, TNFα and the chemokine IL-8, while increased the secretion of the anti-inflammatory cytokine IL-10. The T-cell activating capacity of moDCs was also inhibited, and dimethyltryptamines used in combination with E. coli or influenza virus as stimulators decreased the differentiation of moDC-induced Th1 and Th17 inflammatory effector T-cells in a sigmar-1 specific manner as confirmed by gene silencing. Here we demonstrate for the first time the immunomodulatory potential of NN-DMT and 5-MeO-DMT on human moDC functions via sigmar-1 that could be harnessed for the pharmacological treatment of autoimmune diseases and chronic inflammatory conditions of the CNS or peripheral tissues. Our findings also point out a new biological role for dimethyltryptamines, which may act as systemic endogenous regulators of inflammation and immune homeostasis through the sigma-1 receptor.

  7. Estrogen inhibits RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of estrogen receptor-α with BCAR1 and Traf6

    International Nuclear Information System (INIS)

    Robinson, Lisa J.; Yaroslavskiy, Beatrice B.; Griswold, Reed D.; Zadorozny, Eva V.; Guo, Lida; Tourkova, Irina L.; Blair, Harry C.

    2009-01-01

    The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at ∼ 1 nM reduced RANKL-dependent osteoclast differentiation by 40-50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-β-estradiol. Estrogen receptor-α (ERα) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ERα. However, ERα was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ERα in the presence of estrogen, was abundant. Immunoprecipitation showed rapid (∼ 5 min) estrogen-dependent formation of ERα-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-κB activity, precipitated with this complex. Reduction of NF-κB nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of IκB in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ERα.

  8. Estrogen inhibits RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of estrogen receptor-{alpha} with BCAR1 and Traf6

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, Lisa J., E-mail: robinsonlj@msx.upmc.edu [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Yaroslavskiy, Beatrice B.; Griswold, Reed D.; Zadorozny, Eva V.; Guo, Lida; Tourkova, Irina L. [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Blair, Harry C. [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Veteran' s Affairs Medical Center, Pittsburgh, PA 15243 (United States)

    2009-04-15

    The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at {approx} 1 nM reduced RANKL-dependent osteoclast differentiation by 40-50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-{beta}-estradiol. Estrogen receptor-{alpha} (ER{alpha}) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ER{alpha}. However, ER{alpha} was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ER{alpha} in the presence of estrogen, was abundant. Immunoprecipitation showed rapid ({approx} 5 min) estrogen-dependent formation of ER{alpha}-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-{kappa}B activity, precipitated with this complex. Reduction of NF-{kappa}B nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of I{kappa}B in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ER{alpha}.

  9. Human T-lymphotropic virus type I tax regulates the expression of the human lymphotoxin gene.

    Science.gov (United States)

    Tschachler, E; Böhnlein, E; Felzmann, S; Re