Dexamethasone Suppresses Oxysterol-Induced Differentiation of Monocytic Cells

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Yonghae Son

2016-01-01

Full Text Available Oxysterol like 27-hydroxycholesterol (27OHChol has been reported to induce differentiation of monocytic cells into a mature dendritic cell phenotype. We examined whether dexamethasone (Dx affects 27OHChol-induced differentiation using THP-1 cells. Treatment of monocytic cells with Dx resulted in almost complete inhibition of transcription and surface expression of CD80, CD83, and CD88 induced by 27OHChol. Elevated surface levels of MHC class I and II molecules induced by 27OHChol were reduced to basal levels by treatment with Dx. A decreased endocytosis ability caused by 27OHChol was recovered by Dx. We also examined effects of Dx on expression of CD molecules involved in atherosclerosis. Increased levels of surface protein and transcription of CD105, CD137, and CD166 by treatment with 27OHChol were significantly inhibited by cotreatment with Dx. These results indicate that Dx inhibits 27OHChol-induced differentiation of monocytic cells into a mature dendritic cell phenotype and expression of CD molecules whose levels are associated with atherosclerosis. In addition, we examined phosphorylation of AKT induced by 27OHChol and effect of Dx, where cotreatment with Dx inhibited the phosphorylation of AKT. The current study reports that Dx regulates oxysterol-mediated dendritic cell differentiation of monocytic cells.

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

Regulatory NK cells mediated between immunosuppressive monocytes and dysfunctional T cells in chronic HBV infection.

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Li, Haijun; Zhai, Naicui; Wang, Zhongfeng; Song, Hongxiao; Yang, Yang; Cui, An; Li, Tianyang; Wang, Guangyi; Niu, Junqi; Crispe, Ian Nicholas; Su, Lishan; Tu, Zhengkun

2017-09-12

HBV infection represents a major health problem worldwide, but the immunological mechanisms by which HBV causes chronic persistent infection remain only partly understood. Recently, cell subsets with suppressive features have been recognised among monocytes and natural killer (NK) cells. Here we examine the effects of HBV on monocytes and NK cells. Monocytes and NK cells derived from chronic HBV-infected patients and healthy controls were purified and characterised for phenotype, gene expression and cytokines secretion by flow cytometry, quantitative real-time (qRT)-PCR, ELISA and western blotting. Culture and coculture of monocytes and NK cells were used to determine NK cell activation, using intracellular cytokines staining. In chronic HBV infection, monocytes express higher levels of PD-L1, HLA-E, interleukin (IL)-10 and TGF-β, and NK cells express higher levels of PD-1, CD94 and IL-10, compared with healthy individuals. HBV employs hepatitis B surface antigen (HBsAg) to induce suppressive monocytes with HLA-E, PD-L1, IL-10 and TGF-β expression via the MyD88/NFκB signalling pathway. HBV-treated monocytes induce NK cells to produce IL-10, via PD-L1 and HLA-E signals. Such NK cells inhibit autologous T cell activation. Our findings reveal an immunosuppressive cascade, in which HBV generates suppressive monocytes, which initiate regulatory NK cells differentiation resulting in T cell inhibition. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

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.

Monocyte enrichment from leukapheresis products by using the Elutra cell separator.

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Kim, Sinyoung; Kim, Hyun Ok; Baek, Eun-Jung; Choi, Youjeong; Kim, Han-Soo; Lee, Min-Geul

2007-12-01

Dendritic cells (DCs), used in clinical trials for cancer immunotherapy, require processing on an expanded scale to conform to current good manufacturing practice guidelines. This study evaluated a large-scale monocyte enrichment procedure with a commercially available cell separator (Elutra, Gambro BCT) and analyzed the capacity of enriched monocytes to differentiate into DCs. Mononuclear cells were collected in two patients with malignant melanoma and seven healthy donors by leukapheresis. Continuous-counterflow elutriation with the Elutra was performed to enrich and purify monocytes from leukapheresis products. Purity and recovery of enriched monocytes were analyzed by flow cytometry. DCs were generated from the elutriated monocytes and characterized by phenotypic surface marker and stimulatory capacity in an allogeneic mixed lymphocyte reaction. In the leukapheresis products, the total MNC count was 7.3 x 10(9) +/- 0.7 x 10(9) and the mean percentage of CD14+ monocytes was 16.5 +/- 3.8 percent, which increased to 68.9 +/- 7.4 percent after elutriation with the Elutra. The mean monocyte recovery was 94.3 percent. Elutriated monocytes were successfully cultured into phenotypically and functionally mature DCs. These results indicate that the Elutra cell separator allows for fast and easy enrichment of monocytes within a closed system. Furthermore, these monocytes can be differentiated into functionally mature DCs. Compared to plastic adherence and immunomagnetic selection methods, the elutriation procedure is inexpensive, efficient, and very effective.

Immunomodulating effects of food compounds : a study using the THP-1 cell line

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Chanput, W.

2012-01-01

THP-1 is a human leukaemia monocytic cell line from the peripheral blood of a 1 year old human male. After exposure to phorbol-12-myristate-13-acetate (PMA), THP-1 cells in monocyte state start to adhere to culture plates and alter their morphology with an indication for differentiation into

Speeding up pyrogenicity testing: Identification of suitable cell components and readout parameters for an accelerated monocyte activation test (MAT).

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Stoppelkamp, Sandra; Würschum, Noriana; Stang, Katharina; Löder, Jasmin; Avci-Adali, Meltem; Toliashvili, Leila; Schlensak, Christian; Wendel, Hans Peter; Fennrich, Stefan

2017-02-01

Pyrogen testing represents a crucial safety measure for parental drugs and medical devices, especially in direct contact with blood or liquor. The European Pharmacopoeia regulates these quality control measures for parenterals. Since 2010, the monocyte activation test (MAT) has been an accepted pyrogen test that can be performed with different human monocytic cell sources: whole blood, isolated monocytic cells or monocytic cell lines with IL1β, IL6, or TNFα as readout cytokines. In the present study, we examined the three different cell sources and cytokine readout parameters with the scope of accelerating the assay time. We could show that despite all cell types being able to detect pyrogens, primary cells were more sensitive than the monocytic cell line. Quantitative real-time PCR revealed IL6 mRNA transcripts having the largest change in Ct-values upon LPS-stimulation compared to IL1β and TNFα, but quantification was unreliable. IL6 protein secretion from whole blood or peripheral blood mononuclear cells (PBMC) was also best suited for an accelerated assay with a larger linear range and higher signal-to-noise ratios upon LPS-stimulation. The unique combination with propan-2-ol or a temperature increase could additionally increase the cytokine production for earlier detection in PBMC. The increased incubation temperature could finally increase not only responses to lipopolysaccharides (LPS) but also other pyrogens by up to 13-fold. Therefore, pyrogen detection can be accelerated considerably by using isolated primary blood cells with an increased incubation temperature and IL6 as readout. These results could expedite assay time and thus help to promote further acceptance of the MAT. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

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

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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...

Monocytic leukemias.

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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.

EMMPRIN (CD147/basigin) mediates platelet-monocyte interactions in vivo and augments monocyte recruitment to the vascular wall.

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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.

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

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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)

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

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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.

In vitro effects of monophthalates on cytokine expression in the monocytic cell line THP-1 and in peripheral blood mononuclear cells from allergic and non-allergic donors

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Glue, C; Millner, A; Bødtger, Uffe

2002-01-01

It has recently been shown that plasticizers are present in indoor air dust, which may lead to human exposure via the inhalation route. Moreover, studies have indicated that plasticizers may possess adjuvant effects increasing the health damaging potential of allergens. The aim of this study...... was to investigate the in vitro effect of metabolites of phthalate plastisizers, such as whether an adjuvant effect is paralleled by changes of the cytokine expression in the monocytic cell line THP-1 and in peripheral blood mononuclear cells (PBMCs) from allergics and non-allergics. The toxicity monitored by cell...... viability was determined by incubating THP-1 cells with a 10-fold dilution series of monophthalates for 24 h. At different points in time cytokine expression (IL-1beta, IL-6, IL-12alpha (p35)) in THP-1 cells incubated with non-toxic concentrations of monophthalate (2-20 microg/ml)+/-LPS (1 microg/ml) were...

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

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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.

Indole-3-carbinol induces G1 cell cycle arrest and apoptosis through aryl hydrocarbon receptor in THP-1 monocytic cell line.

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Mohammadi, Saeed; Seyedhosseini, Fakhri Sadat; Behnampour, Nasser; Yazdani, Yaghoub

2017-10-01

The role of aryl hydrocarbon receptor (AhR) in carcinogenesis has been studied recently. Indole-3-carbinol (I3C) is an AhR agonist and a potential anticancer agent. Here, we investigated the effects of I3C on cell cycle progression and apoptosis through activation of AhR on THP-1 acute myeloid leukemia (AML) cell line. MTT viability assay was used to measure the cytotoxic effects of I3C on THP-1 cells. Apoptosis and cell cycle assays were investigated using flow cytometry. Real time RT-PCR was conducted to measure the alterations in the expression of AhR gene, key genes associated with AhR activation (IL1β and CYP1A1) and major genes involved in cell cycle regulation and apoptosis including P27, P21, CDK2, P53, BCL2 and FasR. Our findings revealed that I3C inhibits the proliferation of THP-1 cells in a dose- and time-dependent manner with minimal toxicity over normal monocytes. The AhR target genes (CYP1A1, IL1β) were overexpressed upon I3C treatment (p cycle arrest was also observed using flow cytometry. G1-acting cell cycle genes (P21, P27 and P53) were overexpressed (p cycle arrest in a dose- and time-dependent manner. Therefore, AhR could be targeted as a novel treatment possibility in AML.

Effect and possible mechanism of monocyte-derived VEGF on monocyte-endothelial cellular adhesion after electrical burns.

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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

Monocyte-derived dendritic cells are essential for CD8+ T cell activation and anti-tumor responses after local immunotherapy

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Sabine eKuhn

2015-11-01

Full Text Available Tumors harbor several populations of dendritic cells with the ability to prime tumor-specific T cells. However, these T cells mostly fail to differentiate into armed effectors and are unable to control tumor growth. We have previously shown that treatment with immunostimulatory agents at the tumor site can activate anti-tumor immune responses, and is associated with the appearance of a population of monocyte-derived dendritic cells in the tumor and tumor-draining lymph node. Here we use dendritic cell or monocyte depletion and monocyte transfer to show that these monocyte-derived dendritic cells are critical to the activation of anti-tumor immune responses. Treatment with the immunostimulatory agents Monosodium Urate crystals and Mycobacterium smegmatis induced the accumulation of monocytes in the draining lymph node, their upregulation of CD11c and MHCII, and expression of iNOS, TNFα and IL12p40. Blocking monocyte entry into the lymph node and tumor through neutralization of the chemokine CCL2 or inhibition of Colony Stimulating Factor-1 receptor signaling prevented the generation of monocyte-derived dendritic cells, the infiltration of tumor-specific T cells into the tumor, and anti-tumor responses. In a reciprocal fashion, monocytes transferred into mice depleted of CD11c+ cells were sufficient to rescue CD8+ T cell priming in lymph node and delay tumor growth. Thus monocytes exposed to the appropriate conditions become powerful activators of tumor-specific CD8+ T cells and anti-tumor immunity.

Differential effects of malignant mesothelioma cells on THP-1 monocytes and macrophages.

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Izzi, Valerio; Chiurchiù, Valerio; D'Aquilio, Fabiola; Palumbo, Camilla; Tresoldi, Ilaria; Modesti, Andrea; Baldini, Patrizia M

2009-02-01

Malignant mesothelioma (MM) is a highly fatal tumor arising from inner body membranes, whose extensive growth is facilitated by its week immunogenicity and by its ability to blunt the immune response which should arise from the huge mass of leukocytes typically infiltrating this tumor. It has been reported that the inflammatory infiltrate found in MM tissues is characterized by a high prevalence of macrophages. Thus, in this work we evaluated the ability of human MM cells to modulate the inflammatory phenotype of human THP-1 monocytes and macrophages, a widely used in vitro model of monocyte/macrophage differentiation. Furthermore, we tested the hypothesis that the exposure to MM cells could alter the differentiation of THP-1 monocytes favoring the development of alternatively activated, tumor-supporting macrophages. Our data prove for the first time that MM cells can polarize monocytes towards an altered inflammatory phenotype and macrophages towards an immunosuppressive phenotype. Moreover, we demonstrate that monocytes cocultivated with MM cells 'keep a memory' of their encounter with the tumor which influences their differentiation to macrophages. On the whole, we provide evidence that MM cells exert distinct, cell-specific effects on monocytes and macrophages. The thorough characterization of such effects may be of a crucial importance for the rational design of new immunotherapeutic protocols.

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.

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

Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells.

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Rossana Domenis

Full Text Available A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression, proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs. Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression.

Platelet-derived stromal cell-derived factor-1 is required for the transformation of circulating monocytes into multipotential cells.

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Noriyuki Seta

Full Text Available BACKGROUND: We previously described a primitive cell population derived from human circulating CD14(+ monocytes, named monocyte-derived multipotential cells (MOMCs, which are capable of differentiating into mesenchymal and endothelial lineages. To generate MOMCs in vitro, monocytes are required to bind to fibronectin and be exposed to soluble factor(s derived from circulating CD14(- cells. The present study was conducted to identify factors that induce MOMC differentiation. METHODS: We cultured CD14(+ monocytes on fibronectin in the presence or absence of platelets, CD14(- peripheral blood mononuclear cells, platelet-conditioned medium, or candidate MOMC differentiation factors. The transformation of monocytes into MOMCs was assessed by the presence of spindle-shaped adherent cells, CD34 expression, and the potential to differentiate in vitro into mesenchymal and endothelial lineages. RESULTS: The presence of platelets or platelet-conditioned medium was required to generate MOMCs from monocytes. A screening of candidate platelet-derived soluble factors identified stromal cell-derived factor (SDF-1 as a requirement for generating MOMCs. Blocking an interaction between SDF-1 and its receptor CXCR4 inhibited MOMC generation, further confirming SDF-1's critical role in this process. Finally, circulating MOMC precursors were found to reside in the CD14(+CXCR4(high cell population. CONCLUSION: The interaction of SDF-1 with CXCR4 is essential for the transformation of circulating monocytes into MOMCs.

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

Effects of extra virgin olive oil phenols on HL60 cell lines sensitive and resistant to anthracyclines

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M. Crescimanno

2009-01-01

Full Text Available The aim of our study was to evaluate the capability of a crude extract of phenols from extra virgin olive oil of Moraiolo cultivar to induce apoptosis and/or differentiation in sensitive and resistant HL60 cell lines to anticancer drugs (Typical Multidrug Resistance. Our data highlight that the crude extract is able to induce apoptosis on both sensitive and resistant cells, whereas the exposure to a number of anticancer drugs does not induce apoptosis in resistant cells. In differentiation experiments we investigated the capability of crude extract of phenols to induce the expression of CD11 granulocytic or CD14 monocytic cell surface antigen in sensitive and resistant HL60 cell lines. At IC50 dose level (17 ug/ml and 32 ug/ml respectively for sensitive and resistant cell lines, the crude extract induced differentiation associated with the expression of CD14 monocytic cell lines but not that of CD11 granulocityc cell surface antigen. Further investigations are in progress to better clarify the mechanism by which olive oil phenols induce diffentiation on this cell line.

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

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

2018-03-13

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

Adropin Contributes to Anti-Atherosclerosis by Suppressing Monocyte-Endothelial Cell Adhesion and Smooth Muscle Cell Proliferation

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Kengo Sato

2018-04-01

Full Text Available Adropin, a peptide hormone expressed in liver and brain, is known to improve insulin resistance and endothelial dysfunction. Serum levels of adropin are negatively associated with the severity of coronary artery disease. However, it remains unknown whether adropin could modulate atherogenesis. We assessed the effects of adropin on inflammatory molecule expression and human THP1 monocyte adhesion in human umbilical vein endothelial cells (HUVECs, foam cell formation in THP1 monocyte-derived macrophages, and the migration and proliferation of human aortic smooth muscle cells (HASMCs in vitro and atherogenesis in Apoe−/− mice in vivo. Adropin was expressed in THP1 monocytes, their derived macrophages, HASMCs, and HUVECs. Adropin suppressed tumor necrosis factor α-induced THP1 monocyte adhesion to HUVECs, which was associated with vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 downregulation in HUVECs. Adropin shifted the phenotype to anti-inflammatory M2 rather than pro-inflammatory M1 via peroxisome proliferator-activated receptor γ upregulation during monocyte differentiation into macrophages. Adropin had no significant effects on oxidized low-density lipoprotein-induced foam cell formation in macrophages. In HASMCs, adropin suppressed the migration and proliferation without inducing apoptosis via ERK1/2 and Bax downregulation and phosphoinositide 3-kinase/Akt/Bcl2 upregulation. Chronic administration of adropin to Apoe−/− mice attenuated the development of atherosclerotic lesions in the aorta, with reduced the intra-plaque monocyte/macrophage infiltration and smooth muscle cell content. Thus, adropin could serve as a novel therapeutic target in atherosclerosis and related diseases.

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

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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

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

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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.

Anti-CD20 B-cell depletion enhances monocyte reactivity in neuroimmunological disorders

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Hohlfeld Reinhard

2011-10-01

Full Text Available Abstract Background Clinical trials evaluating anti-CD20-mediated B-cell depletion in multiple sclerosis (MS and neuromyelitis optica (NMO generated encouraging results. Our recent studies in the MS model experimental autoimmune encephalomyelitis (EAE attributed clinical benefit to extinction of activated B-cells, but cautioned that depletion of naïve B-cells may be undesirable. We elucidated the regulatory role of un-activated B-cells in EAE and investigated whether anti-CD20 may collaterally diminish regulatory B-cell properties in treatment of neuroimmunological disorders. Methods Myelin oligodendrocyte glycoprotein (MOG peptide-immunized C57Bl/6 mice were depleted of B-cells. Functional consequences for regulatory T-cells (Treg and cytokine production of CD11b+ antigen presenting cells (APC were assessed. Peripheral blood mononuclear cells from 22 patients receiving anti-CD20 and 23 untreated neuroimmunological patients were evaluated for frequencies of B-cells, T-cells and monocytes; monocytic reactivity was determined by TNF-production and expression of signalling lymphocytic activation molecule (SLAM. Results We observed that EAE-exacerbation upon depletion of un-activated B-cells closely correlated with an enhanced production of pro-inflammatory TNF by CD11b+ APC. Paralleling this pre-clinical finding, anti-CD20 treatment of human neuroimmunological disorders increased the relative frequency of monocytes and accentuated pro-inflammatory monocyte function; when reactivated ex vivo, a higher frequency of monocytes from B-cell depleted patients produced TNF and expressed the activation marker SLAM. Conclusions These data suggest that in neuroimmunological disorders, pro-inflammatory APC activity is controlled by a subset of B-cells which is eliminated concomitantly upon anti-CD20 treatment. While this observation does not conflict with the general concept of B-cell depletion in human autoimmunity, it implies that its safety and

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

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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

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

Effector CD4+ T cells recognize intravascular antigen presented by patrolling monocytes.

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Westhorpe, Clare L V; Norman, M Ursula; Hall, Pam; Snelgrove, Sarah L; Finsterbusch, Michaela; Li, Anqi; Lo, Camden; Tan, Zhe Hao; Li, Songhui; Nilsson, Susan K; Kitching, A Richard; Hickey, Michael J

2018-02-21

Although effector CD4 + T cells readily respond to antigen outside the vasculature, how they respond to intravascular antigens is unknown. Here we show the process of intravascular antigen recognition using intravital multiphoton microscopy of glomeruli. CD4 + T cells undergo intravascular migration within uninflamed glomeruli. Similarly, while MHCII is not expressed by intrinsic glomerular cells, intravascular MHCII-expressing immune cells patrol glomerular capillaries, interacting with CD4 + T cells. Following intravascular deposition of antigen in glomeruli, effector CD4 + T-cell responses, including NFAT1 nuclear translocation and decreased migration, are consistent with antigen recognition. Of the MHCII + immune cells adherent in glomerular capillaries, only monocytes are retained for prolonged durations. These cells can also induce T-cell proliferation in vitro. Moreover, monocyte depletion reduces CD4 + T-cell-dependent glomerular inflammation. These findings indicate that MHCII + monocytes patrolling the glomerular microvasculature can present intravascular antigen to CD4 + T cells within glomerular capillaries, leading to antigen-dependent inflammation.

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

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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.

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

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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.

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

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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

Protective role of klotho protein on epithelial cells upon co-culture with activated or senescent monocytes

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Mytych, Jennifer, E-mail: jennifermytych@gmail.com [Institute of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Wos, Izabela; Solek, Przemyslaw; Koziorowski, Marek [Institute of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland)

2017-01-15

Monocytes ensure proper functioning and maintenance of epithelial cells, while good condition of monocytes is a key factor of these interactions. Although, it was shown that in some circumstances, a population of altered monocytes may appear, there is no data regarding their effect on epithelial cells. In this study, using direct co-culture model with LPS-activated and Dox-induced senescent THP-1 monocytes, we reported for the first time ROS-induced DNA damage, reduced metabolic activity, proliferation inhibition and cell cycle arrest followed by p16-, p21- and p27-mediated DNA damage response pathways activation, premature senescence and apoptosis induction in HeLa cells. Also, we show that klotho protein possessing anti-aging and anti-inflammatory characteristics reduced cytotoxic and genotoxic events by inhibition of insulin/IGF-IR and downregulation of TRF1 and TRF2 proteins. Therefore, klotho protein could be considered as a protective factor against changes caused by altered monocytes in epithelial cells. - Highlights: • Activated and senescent THP-1 monocytes induced cyto- and genotoxicity in HeLa cells. • Altered monocytes provoked oxidative and nitrosative stress-induced DNA damage. • DNA damage activated DDR pathways and lead to premature senescence and apoptosis. • Klotho reduced ROS/RNS-mediated toxicity through insulin/IGF-IR pathway inhibition. • Klotho protects HeLa cells from cyto- and genotoxicity induced by altered monocytes.

Protective role of klotho protein on epithelial cells upon co-culture with activated or senescent monocytes

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Mytych, Jennifer; Wos, Izabela; Solek, Przemyslaw; Koziorowski, Marek

2017-01-01

Monocytes ensure proper functioning and maintenance of epithelial cells, while good condition of monocytes is a key factor of these interactions. Although, it was shown that in some circumstances, a population of altered monocytes may appear, there is no data regarding their effect on epithelial cells. In this study, using direct co-culture model with LPS-activated and Dox-induced senescent THP-1 monocytes, we reported for the first time ROS-induced DNA damage, reduced metabolic activity, proliferation inhibition and cell cycle arrest followed by p16-, p21- and p27-mediated DNA damage response pathways activation, premature senescence and apoptosis induction in HeLa cells. Also, we show that klotho protein possessing anti-aging and anti-inflammatory characteristics reduced cytotoxic and genotoxic events by inhibition of insulin/IGF-IR and downregulation of TRF1 and TRF2 proteins. Therefore, klotho protein could be considered as a protective factor against changes caused by altered monocytes in epithelial cells. - Highlights: • Activated and senescent THP-1 monocytes induced cyto- and genotoxicity in HeLa cells. • Altered monocytes provoked oxidative and nitrosative stress-induced DNA damage. • DNA damage activated DDR pathways and lead to premature senescence and apoptosis. • Klotho reduced ROS/RNS-mediated toxicity through insulin/IGF-IR pathway inhibition. • Klotho protects HeLa cells from cyto- and genotoxicity induced by altered monocytes.

Prevention of UV irradiation induced suppression of monocyte functions by retinoids and carotenoids in vitro

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Schoen, D.J.; Watson, R.R.

1988-01-01

The effects of stimulation of human peripheral blood monocytes in vitro with retinoids and carotenoids, and subsequent exposure to ultraviolet light of the B wavelength were measured. The compounds were applied to the monocytes in culture for 24 h, and the washed cells were then exposed to UVB light up to 220 J/m 2 . The compounds tested protected the monocyte from UVB induced damage to phagocytic activity. This protection may be due to the antioxidant or UVB energy-quenching properties of these compounds. Monocyte cytotoxicity against a melanoma cell line was stimulated by exposure to the retinoids or carotenoids, but a protective effect in vitro against UVB damage was not seen for this cell function. (author)

Mycobacterium leprae upregulates IRGM expression in monocytes and monocyte-derived macrophages.

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Yang, Degang; Chen, Jia; Zhang, Linglin; Cha, Zhanshan; Han, Song; Shi, Weiwei; Ding, Ru; Ma, Lan; Xiao, Hong; Shi, Chao; Jing, Zhichun; Song, Ningjing

2014-08-01

Leprosy is caused by the infection of Mycobacterium leprae, which evokes a strong inflammatory response and leads to nerve damage. Immunity-related GTPase family M protein (IRGM) plays critical roles in controlling inflammation. The objective of the study was to investigate whether IRGM is involved in the infection of M. leprae. Levels of IRGM were assessed in M. leprae-infected CD4(+) T cells, monocytes, and monocyte-derived macrophages. Data revealed that both protein and mRNA levels of IRGM were increased in monocytes after M. leprae infection. Interestingly, monocyte-derived macrophages showed more prominent IRGM expression with M. leprae infection, whereas the bacteria did not affect IRGM in CD4(+) T cells. Furthermore, we assessed levels of IRGM in CD4(+) T cells and monocytes from 78 leprosy patients and 40 healthy controls, and observed upregulated protein level of IRGM in the monocytes from leprosy patients. Also, IRGM expression was inversely correlated with the severity of the disease. These findings suggested a close involvement of IRGM in M. leprae infection and indicated a potential mechanism of defending M. leprae infection.

Monocytes infiltrate the pancreas via the MCP-1/CCR2 pathway and differentiate into stellate cells.

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Kazuko Ino

Full Text Available Recent studies have shown that monocytes possess pluripotent plasticity. We previously reported that monocytes could differentiate into hepatic stellate cells. Although stellate cells are also present in the pancreas, their origin remains unclear. An accumulation of enhanced green fluorescent protein (EGFP(+CD45(- cells was observed in the pancreases and livers of chimeric mice, which were transplanted with a single hematopoietic stem cell isolated from EGFP-transgenic mice and treated with carbon tetrachloride (CCl4. Because the vast majority of EGFP(+CD45(- cells in the pancreas expressed stellate cell-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin, they were characterized as pancreatic stellate cells (PaSCs. EGFP(+ PaSCs were also observed in CCl4-treated mice adoptively transferred with monocytes but not with other cell lineages isolated from EGFP-transgenic mice. The expression of monocyte chemoattractant protein-1 (MCP-1 and angiotensin II (Ang II increased in the pancreas of CCl4-treated mice and their respective receptors, C-C chemokine receptor 2 (CCR2 and Ang II type 1 receptor (AT1R, were expressed on Ly6C(high monocytes isolated from EGFP-transgenic mice. We examined the effect of an AT1R antagonist, irbesartan, which is also a CCR2 antagonist, on the migration of monocytes into the pancreas. Monocytes migrated toward MCP-1 but not Ang II in vitro. Irbesartan inhibited not only their in vitro chemotaxis but also in vivo migration of adoptively transferred monocytes from peripheral blood into the pancreas. Irbesartan treatment significantly reduced the numbers of EGFP(+F4/80(+CCR2(+ monocytic cells and EGFP(+ PaSCs in the pancreas of CCl4-treated chimeric mice receiving EGFP(+ bone marrow cells. A specific CCR2 antagonist RS504393 inhibited the occurrence of EGFP(+ PaSCs in injured mice. We propose that CCR2(+ monocytes migrate into the pancreas possibly via the

Zinc oxide nanoparticles induce migration and adhesion of monocytes to endothelial cells and accelerate foam cell formation

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Suzuki, Yuka; Tada-Oikawa, Saeko [Graduate School of Regional Innovation Studies, Mie University, Tsu (Japan); Ichihara, Gaku [Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya (Japan); Yabata, Masayuki; Izuoka, Kiyora [Graduate School of Regional Innovation Studies, Mie University, Tsu (Japan); Suzuki, Masako; Sakai, Kiyoshi [Nagoya City Public Health Research Institute, Nagoya (Japan); Ichihara, Sahoko, E-mail: saho@gene.mie-u.ac.jp [Graduate School of Regional Innovation Studies, Mie University, Tsu (Japan)

2014-07-01

Metal oxide nanoparticles are widely used in industry, cosmetics, and biomedicine. However, the effects of exposure to these nanoparticles on the cardiovascular system remain unknown. The present study investigated the effects of nanosized TiO{sub 2} and ZnO particles on the migration and adhesion of monocytes, which are essential processes in atherosclerogenesis, using an in vitro set-up of human umbilical vein endothelial cells (HUVECs) and human monocytic leukemia cells (THP-1). We also examined the effects of exposure to nanosized metal oxide particles on macrophage cholesterol uptake and foam cell formation. The 16-hour exposure to ZnO particles increased the level of monocyte chemotactic protein-1 (MCP-1) and induced the migration of THP-1 monocyte mediated by increased MCP-1. Exposure to ZnO particles also induced adhesion of THP-1 cells to HUVECs. Moreover, exposure to ZnO particles, but not TiO{sub 2} particles, upregulated the expression of membrane scavenger receptors of modified LDL and increased cholesterol uptake in THP-1 monocytes/macrophages. In the present study, we found that exposure to ZnO particles increased macrophage cholesterol uptake, which was mediated by an upregulation of membrane scavenger receptors of modified LDL. These results suggest that nanosized ZnO particles could potentially enhance atherosclerogenesis and accelerate foam cell formation. - Highlights: • Effects of metal oxide nanoparticles on foam cell formation were investigated. • Exposure to ZnO nanoparticles induced migration and adhesion of monocytes. • Exposure to ZnO nanoparticles increased macrophage cholesterol uptake. • Expression of membrane scavenger receptors of modified LDL was also increased. • These effects were not observed after exposure to TiO{sub 2} nanoparticles.

Enhancement of proinflammatory and procoagulant responses to silica particles by monocyte-endothelial cell interactions

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Liu Xin

2012-09-01

Full Text Available Abstract Background Inorganic particles, such as drug carriers or contrast agents, are often introduced into the vascular system. Many key components of the in vivo vascular environment include monocyte-endothelial cell interactions, which are important in the initiation of cardiovascular disease. To better understand the effect of particles on vascular function, the present study explored the direct biological effects of particles on human umbilical vein endothelial cells (HUVECs and monocytes (THP-1 cells. In addition, the integrated effects and possible mechanism of particle-mediated monocyte-endothelial cell interactions were investigated using a coculture model of HUVECs and THP-1 cells. Fe3O4 and SiO2 particles were chosen as the test materials in the present study. Results The cell viability data from an MTS assay showed that exposure to Fe3O4 or SiO2 particles at concentrations of 200 μg/mL and above significantly decreased the cell viability of HUVECs, but no significant loss in viability was observed in the THP-1 cells. TEM images indicated that with the accumulation of SiO2 particles in the cells, the size, structure and morphology of the lysosomes significantly changed in HUVECs, whereas the lysosomes of THP-1 cells were not altered. Our results showed that reactive oxygen species (ROS generation; the production of interleukin (IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1, tumor necrosis factor (TNF-α and IL-1β; and the expression of CD106, CD62E and tissue factor in HUVECs and monocytes were significantly enhanced to a greater degree in the SiO2-particle-activated cocultures compared with the individual cell types alone. In contrast, exposure to Fe3O4 particles had no impact on the activation of monocytes or endothelial cells in monoculture or coculture. Moreover, using treatment with the supernatants of SiO2-particle-stimulated monocytes or HUVECs, we found that the enhancement of proinflammatory response by SiO2

Transcript and protein analysis reveals better survival skills of monocyte-derived dendritic cells compared to monocytes during oxidative stress.

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Ilse Van Brussel

Full Text Available BACKGROUND: Dendritic cells (DCs, professional antigen-presenting cells with the unique ability to initiate primary T-cell responses, are present in atherosclerotic lesions where they are exposed to oxidative stress that generates cytotoxic reactive oxygen species (ROS. A large body of evidence indicates that cell death is a major modulating factor of atherogenesis. We examined antioxidant defence systems of human monocyte-derived (moDCs and monocytes in response to oxidative stress. METHODS: Oxidative stress was induced by addition of tertiary-butylhydroperoxide (tert-BHP, 30 min. Cellular responses were evaluated using flow cytometry and confocal live cell imaging (both using 5-(and-6-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, CM-H(2DCFDA. Viability was assessed by the neutral red assay. Total RNA was extracted for a PCR profiler array. Five genes were selected for confirmation by Taqman gene expression assays, and by immunoblotting or immunohistochemistry for protein levels. RESULTS: Tert-BHP increased CM-H(2DCFDA fluorescence and caused cell death. Interestingly, all processes occurred more slowly in moDCs than in monocytes. The mRNA profiler array showed more than 2-fold differential expression of 32 oxidative stress-related genes in unstimulated moDCs, including peroxiredoxin-2 (PRDX2, an enzyme reducing hydrogen peroxide and lipid peroxides. PRDX2 upregulation was confirmed by Taqman assays, immunoblotting and immunohistochemistry. Silencing PRDX2 in moDCs by means of siRNA significantly increased CM-DCF fluorescence and cell death upon tert-BHP-stimulation. CONCLUSIONS: Our results indicate that moDCs exhibit higher intracellular antioxidant capacities, making them better equipped to resist oxidative stress than monocytes. Upregulation of PRDX2 is involved in the neutralization of ROS in moDCs. Taken together, this points to better survival skills of DCs in oxidative stress environments, such as atherosclerotic plaques.

Monocyte galactose/N-acetylgalactosamine-specific C-type lectin receptor stimulant immunotherapy of an experimental glioma. Part 1: stimulatory effects on blood monocytes and monocyte-derived cells of the brain

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Kushchayev SV

2012-09-01

Full Text Available Sergiy V Kushchayev,1 Tejas Sankar,1 Laura L Eggink,4,5 Yevgeniya S Kushchayeva,5 Philip C Wiener,1,5 J Kenneth Hoober,5,6 Jennifer Eschbacher,3 Ruolan Liu,2 Fu-Dong Shi,2 Mohammed G Abdelwahab,4 Adrienne C Scheck,4 Mark C Preul11Neurosurgery Research Laboratory, 2Neuroimmunology Laboratory, 3Department of Pathology, 4Neurooncology Research, Barrow Neurological Institute, St Joseph's Hospital and Medical Center, Phoenix, 5School of Life Sciences, Arizona State University, Tempe, 6Susavion Biosciences, Inc, Tempe, AZ, USAObjectives: Immunotherapy with immunostimulants is an attractive therapy against gliomas. C-type lectin receptors specific for galactose/N-acetylgalactosamine (GCLR regulate cellular differentiation, recognition, and trafficking of monocyte-derived cells. A peptide mimetic of GCLR ligands (GCLRP was used to activate blood monocytes and populations of myeloid-derived cells against a murine glioblastoma.Methods: The ability of GCLRP to stimulate phagocytosis by human microglia and monocyte-derived cells of the brain (MDCB isolated from a human glioblastoma was initially assessed in vitro. Induction of activation markers on blood monocytes was assayed by flow cytometry after administration of GCLRP to naive mice. C57BL/6 mice underwent stereotactic intracranial implantation of GL261 glioma cells and were randomized for tumor size by magnetic resonance imaging, which was also used to assess increase in tumor size. Brain tumor tissues were analyzed using flow cytometry, histology, and enzyme-linked immunosorbent assay with respect to tumor, peritumoral area, and contralateral hemisphere regions.Results: GCLRP exhibited strong stimulatory effect on MDCBs and blood monocytes in vitro and in vivo. GCLRP was associated with an increased percentage of precursors of dendritic cells in the blood (P = 0.003, which differentiated into patrolling macrophages in tumoral (P = 0.001 and peritumoral areas (P = 0.04, rather than into dendritic cells

Soluble fibrin inhibits monocyte adherence and cytotoxicity against tumor cells: implications for cancer metastasis

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Patel Shonak

2006-08-01

Full Text Available Abstract Background Soluble fibrin (sFn is a marker for disseminated intravascular coagulation and may have prognostic significance, especially in metastasis. However, a role for sFn in the etiology of metastatic cancer growth has not been extensively studied. We have reported that sFn cross-linked platelet binding to tumor cells via the major platelet fibrin receptor αIIbβ3, and tumor cell CD54 (ICAM-1, which is the receptor for two of the leukocyte β2 integrins (αLβ2 and aMβ2. We hypothesized that sFn may also affect leukocyte adherence, recognition, and killing of tumor cells. Furthermore, in a rat experimental metastasis model sFn pre-treatment of tumor cells enhanced metastasis by over 60% compared to untreated cells. Other studies have shown that fibrin(ogen binds to the monocyte integrin αMβ2. This study therefore sought to investigate the effect of sFn on β2 integrin mediated monocyte adherence and killing of tumor cells. Methods The role of sFn in monocyte adherence and cytotoxicity against tumor cells was initially studied using static microplate adherence and cytotoxicity assays, and under physiologically relevant flow conditions in a microscope perfusion incubator system. Blocking studies were performed using monoclonal antibodies specific for β2 integrins and CD54, and specific peptides which inhibit sFn binding to these receptors. Results Enhancement of monocyte/tumor cell adherence was observed when only one cell type was bound to sFn, but profound inhibition was observed when sFn was bound to both monocytes and tumor cells. This effect was also reflected in the pattern of monocyte cytotoxicity. Studies using monoclonal blocking antibodies and specific blocking peptides (which did not affect normal coagulation showed that the predominant mechanism of fibrin inhibition is via its binding to αMβ2 on monocytes, and to CD54 on both leukocytes and tumor cells. Conclusion sFn inhibits monocyte adherence and cytotoxicity of

Abnormalities in iNKT cells are associated with impaired ability of monocytes to produce IL-10 and suppress T-cell proliferation in sarcoidosis.

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Crawshaw, Anjali; Kendrick, Yvonne R; McMichael, Andrew J; Ho, Ling-Pei

2014-07-01

Sarcoidosis is a multisystem granulomatous disorder characterized by marked T-cell expansion of T helper 1 (Th1) cells. The cause of T-cell overactivity is unknown. We hypothesized that interleukin-10 (IL-10) production by a yet undefined cell type might be defective, resulting in loss of regulation of T-cell activity. Focusing on IL-10-producing monocytes, we first showed that monocytes isolated from the peripheral blood of corticosteroid-naïve sarcoidosis patients (n = 51) produced less IL-10 compared to controls, and were less able to suppress T-cell proliferation. In addition, monocytic IL-10 production correlated negatively with disease activity score. As invariant natural killer T (iNKT) cells are known to both interact with monocytes and be reduced in sarcoidosis patients, we then asked whether iNKT-specific defects might be responsible for this reduced IL-10 production. We found that greater numbers of circulating iNKT cells was associated with higher IL-10 production. Moreover, iNKT cells enhanced monocytic IL-10 production in vitro. Defective IL-10 production and T-cell suppression by sarcoidosis monocytes could be restored following their coculture with iNKT cells, in a CD1d- and cell contact-dependent process. We suggest that reduced iNKT-cell numbers in sarcoidosis may lead to impaired monocytic IL-10 production and unchecked T-cell expansion in sarcoidosis. These findings provide fresh insight into the mechanism of sarcoidosis disease, and interaction between iNKT cells and monocytes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Raman spectroscopy of individual monocytes reveals that single-beam optical trapping of mononuclear cells occurs by their nucleus

International Nuclear Information System (INIS)

Fore, Samantha; Chan, James; Taylor, Douglas; Huser, Thomas

2011-01-01

We show that laser tweezers Raman spectroscopy of eukaryotic cells with a significantly larger diameter than the tight focus of a single-beam laser trap leads to optical trapping of the cell by its optically densest part, i.e. typically the cell's nucleus. Raman spectra of individual optically trapped monocytes are compared with location-specific Raman spectra of monocytes adhered to a substrate. When the cell's nucleus is stained with a fluorescent live cell stain, the Raman spectrum of the DNA-specific stain is observed only in the nucleus of individual monocytes. Optically trapped monocytes display the same behavior. We also show that the Raman spectra of individual monocytes exhibit the characteristic Raman signature of cells that have not yet fully differentiated and that individual primary monocytes can be distinguished from transformed monocytes based on their Raman spectra. This work provides further evidence that laser tweezers Raman spectroscopy of individual cells provides meaningful biochemical information in an entirely non-destructive fashion that permits discerning differences between cell types and cellular activity

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

DYSFUNCTION OF MONOCYTES AND DENDRITIC CELLS IN PATIENTS WITH PREMATURE OVARIAN FAILURE

NARCIS (Netherlands)

HOEK, A; VAN KASTEREN, Y; DE HAAN-MEULMAN, M; SCHOEMAKER, J; DREXHAGE, HA

1993-01-01

PROBLEM: Due to the presence of ovarian antibodies it has been suggested that premature ovarian failure (POF) belongs to the autoimmune endocrinopathies. Monocytes and the monocyte-derived dendritic cells play a prominent role in the initial stages of endocrine autoimmune reactions: the accumulation

Tolerance of monocytes and macrophages in response to bacterial endotoxin

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Ewelina Wiśnik

2017-03-01

Full Text Available Monocytes belong to myeloid effector cells, which constitute the first line of defense against pathogens, also called the nonspecific immune system and play an important role in the maintenance of tissue homeostasis. In response to stimulation, monocytes differentiate into macrophages capable of microorganism phagocytosis and secrete factors that play a key role in the regulation of immune responses. However excessive exposure of monocytes/macrophages to the lipopolysaccharide (LPS of Gram negative bacteria leads to the acquisition of immune tolerance by these cells. Such state results from disruption of different biological processes, for example intracellular signaling pathways and is accompanied by a number of disease states (immune, inflammatory or neoplastic conditions. Regulation of monocytes/macrophages activity is controlled by miRNAs, which are involved in the modulation of immune tolerance acquired by these cells. Moreover, the tolerance to endotoxin is conditioned by the posttranscriptional processes and posttranslational epigenetic modifications leading to the impairment of normal immune response for example by alterations in the expression of many genes encoding immune signaling mediators. The aim of this paper is to provide an overview existing knowledge on the modulation of activity of monocytes/macrophages in response to bacterial endotoxin and impaired immune responses.

Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

DEFF Research Database (Denmark)

Hansen, Morten; Hjortø, Gertrud Malene; Met, Özcan

2011-01-01

Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...... in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...... at similar concentrations to the passive adsorption process, as measured by enzyme-linked immunosorbent assays, and the bound IL-4 did not leak into solution to any measurable extent during cell culture. However, covalently bound IL-4 was incapable of inducing monocyte differentiation. This may be caused...

Efficient elutriation of monocytes within a closed system (Elutra) for clinical-scale generation of dendritic cells.

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Berger, Thomas G; Strasser, Erwin; Smith, Richard; Carste, Curt; Schuler-Thurner, Beatrice; Kaempgen, Eckhart; Schuler, Gerold

2005-03-01

Dendritic cells (DC) are promising tools for the immunotherapy of cancer. The induction of tumor-specific T cells and clinical regressions have already been observed in early phase I/II vaccination trials. As DC vaccination is now facing trials with larger patient collectives it becomes increasingly important to obtain large numbers of cells suitable for therapeutic applications under labor- and cost-effective conditions. We describe here a procedure that uses a novel cell separator (Elutra, Gambro BCT) to enrich monocytes from an entire apheresis product within one hour. Cells are separated on the basis of size and to a lesser extent density, by elutriation in a 40-ml conical chamber. The total monocyte recovery following elutriation (n = 6) was 98.53% (+/-8.07%), the recovery in the monocyte-rich fraction 75.45% (+/-11.31%), and the mean purity 82.95% (+/-6.01%). These monocytes can be cultured either in conventional culture dishes or in closed cell culture bags and differentiated, by using GM-CSF+IL-4 followed by a maturation cocktail composed of IL-1beta+IL-6+TNF-alpha+PGE2, into fully mature DC. The Elutra separator allows for fast and easy enrichment of monocytes within a closed system. Subsequently, elutriated monocytes can be successfully cultured into phenotypically and functionally mature DC for immunotherapeutic approaches. The method neither requires a density gradient step to enrich PBMC from leucapheresis products nor does it apply (xenogeneic) antibodies to target monocytes. Isolation of monocytes with Elutra may greatly facilitate future DC-based vaccination approaches.

Proangiogenic hematopoietic cells of monocytic origin: roles in vascular regeneration and pathogenic processes of systemic sclerosis.

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Yamaguchi, Yukie; Kuwana, Masataka

2013-02-01

New blood vessel formation is critical, not only for organ development and tissue regeneration, but also for various pathologic processes, such as tumor development and vasculopathy. The maintenance of the postnatal vascular system requires constant remodeling, which occurs through angiogenesis, vasculogenesis, and arteriogenesis. Vasculogenesis is mediated by the de novo differentiation of mature endothelial cells from endothelial progenitor cells (EPCs). Early studies provided evidence that bone marrow-derived CD14⁺ monocytes can serve as a subset of EPCs because of their expression of endothelial markers and ability to promote neovascularization in vitro and in vivo. However, the current consensus is that monocytic cells do not give rise to endothelial cells in vivo, but function as support cells, by promoting vascular formation and repair through their immediate recruitment to the site of vascular injury, secretion of proangiogenic factors, and differentiation into mural cells. These monocytes that function in a supporting role in vascular repair are now termed monocytic pro-angiogenic hematopoietic cells (PHCs). Systemic sclerosis (SSc) is a multisystem connective tissue disease characterized by excessive fibrosis and microvasculopathy, along with poor vascular formation and repair. We recently showed that in patients with SSc, circulating monocytic PHCs increase dramatically and have enhanced angiogenic potency. These effects may be induced in response to defective vascular repair machinery. Since CD14⁺ monocytes can also differentiate into fibroblast-like cells that produce extracellular matrix proteins, here we propose a new hypothesis that aberrant monocytic PHCs, once mobilized into circulation, may also contribute to the fibrotic process of SSc.

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

HIV/SIV infection primes monocytes and dendritic cells for apoptosis.

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Mireille Laforge

2011-06-01

Full Text Available Subversion or exacerbation of antigen-presenting cells (APC death modulates host/pathogen equilibrium. We demonstrated during in vitro differentiation of monocyte-derived macrophages and monocyte-derived dendritic cells (DCs that HIV sensitizes the cells to undergo apoptosis in response to TRAIL and FasL, respectively. In addition, we found that HIV-1 increased the levels of pro-apoptotic Bax and Bak molecules and decreased the levels of anti-apoptotic Mcl-1 and FLIP proteins. To assess the relevance of these observations in the context of an experimental model of HIV infection, we investigated the death of APC during pathogenic SIV-infection in rhesus macaques (RMs. We demonstrated increased apoptosis, during the acute phase, of both peripheral blood DCs and monocytes (CD14(+ from SIV(+RMs, associated with a dysregulation in the balance of pro- and anti-apoptotic molecules. Caspase-inhibitor and death receptors antagonists prevented apoptosis of APCs from SIV(+RMs. Furthermore, increased levels of FasL in the sera of pathogenic SIV(+RMs were detected, compared to non-pathogenic SIV infection of African green monkey. We suggest that inappropriate apoptosis of antigen-presenting cells may contribute to dysregulation of cellular immunity early in the process of HIV/SIV infection.

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

Histamine type I (H1) receptor radioligand binding studies on normal T cell subsets, B cells, and monocytes

International Nuclear Information System (INIS)

Cameron, W.; Doyle, K.; Rocklin, R.E.

1986-01-01

A single, specific binding site for [ 3 H]pyrilamine on normal human T helper, T suppressor, B cells, and monocytes was documented. The binding of the radioligand to its receptor is reversible with cold H 1 antagonist, saturates at 40 to 60 nM, and binding equilibrium is achieved in 2 to 4 min. Using a computer program (Ligand), the authors calculated the dissociation constants, binding capacities, and numbers of receptors per cell for each of the different cell types. Monocytes were found to have the highest affinity for [ 3 H]pyrilamine, followed by T helper cells, B cells and T suppressor cells (K/sub D/ = 44.6 +/- 49.4 nM). T suppressor cells were found to express the higher number of H 1 receptors per cell followed by B cells, T helper cells, and monocytes. The binding affinity for [ 3 H]pyrilamine increased over a 48-hr period, whereas the number of receptors per T cell was essentially unchanged. In contrast, T cells stimulated with Con A or PHA were shown to have a greater than fourfold increase in the number of receptors per cell, whereas the binding affinity for [ 3 H]pyrilamine decreased over the 48-hr period. Although the function of H 1 receptors on T cells, B cells, and monocytes has not been completely defined, this receptor has the potential of playing an important role in the modulating the immune response

GM-CSF Monocyte-Derived Cells and Langerhans Cells As Part of the Dendritic Cell Family

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Manfred B. Lutz

2017-10-01

Full Text Available Dendritic cells (DCs and macrophages (Mph share many characteristics as components of the innate immune system. The criteria to classify the multitude of subsets within the mononuclear phagocyte system are currently phenotype, ontogeny, transcription patterns, epigenetic adaptations, and function. More recently, ontogenetic, transcriptional, and proteomic research approaches uncovered major developmental differences between Flt3L-dependent conventional DCs as compared with Mphs and monocyte-derived DCs (MoDCs, the latter mainly generated in vitro from murine bone marrow-derived DCs (BM-DCs or human CD14+ peripheral blood monocytes. Conversely, in vitro GM-CSF-dependent monocyte-derived Mphs largely resemble MoDCs whereas tissue-resident Mphs show a common embryonic origin from yolk sac and fetal liver with Langerhans cells (LCs. The novel ontogenetic findings opened discussions on the terminology of DCs versus Mphs. Here, we bring forward arguments to facilitate definitions of BM-DCs, MoDCs, and LCs. We propose a group model of terminology for all DC subsets that attempts to encompass both ontogeny and function.

Transfecting Human Monocytes with RNA.

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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.

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

TLR4-mediated expression of Mac-1 in monocytes plays a pivotal role in monocyte adhesion to vascular endothelium.

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Seung Jin Lee

Full Text Available Toll-like receptor 4 (TLR4 is known to mediate monocyte adhesion to endothelial cells, however, its role on the expression of monocyte adhesion molecules is unclear. In the present study, we investigated the role of TLR4 on the expression of monocyte adhesion molecules, and determined the functional role of TLR4-induced adhesion molecules on monocyte adhesion to endothelial cells. When THP-1 monocytes were stimulated with Kdo2-Lipid A (KLA, a specific TLR4 agonist, Mac-1 expression was markedly increased in association with an increased adhesion of monocytes to endothelial cells. These were attenuated by anti-Mac-1 antibody, suggesting a functional role of TLR4-induced Mac-1 on monocyte adhesion to endothelial cells. In monocytes treated with MK886, a 5-lipoxygenase (LO inhibitor, both Mac-1 expression and monocyte adhesion to endothelial cells induced by KLA were markedly attenuated. Moreover, KLA increased the expression of mRNA and protein of 5-LO, suggesting a pivotal role of 5-LO on these processes. In in vivo studies, KLA increased monocyte adhesion to aortic endothelium of wild-type (WT mice, which was attenuated in WT mice treated with anti-Mac-1 antibody as well as in TLR4-deficient mice. Taken together, TLR4-mediated expression of Mac-1 in monocytes plays a pivotal role on monocyte adhesion to vascular endothelium, leading to increased foam cell formation in the development of atherosclerosis.

Comparative analysis of signature genes in PRRSV-infected porcine monocyte-derived cells at differential activation statuses

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Activation statuses of monocytic cells are critically important for antiviral immunity. Devastating viruses like porcine reproductive and respiratory syndrome virus (PRRSV) are capable of directly infecting these cells, subverting host immunity. Monocyte-derived DCs (mDCs) are major target cells in ...

Oral Wild-Type Salmonella Typhi Challenge Induces Activation of Circulating Monocytes and Dendritic Cells in Individuals Who Develop Typhoid Disease.

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Franklin R Toapanta

2015-06-01

Full Text Available A new human oral challenge model with wild-type Salmonella Typhi (S. Typhi was recently developed. In this model, ingestion of 104 CFU of Salmonella resulted in 65% of subjects developing typhoid fever (referred here as typhoid diagnosis -TD- 5-10 days post-challenge. TD criteria included meeting clinical (oral temperature ≥38°C for ≥12 h and/or microbiological (S. Typhi bacteremia endpoints. One of the first lines of defense against pathogens are the cells of the innate immune system (e.g., monocytes, dendritic cells -DCs-. Various changes in circulating monocytes and DCs have been described in the murine S. Typhimurium model; however, whether similar changes are present in humans remains to be explored. To address these questions, a subset of volunteers (5 TD and 3 who did not develop typhoid despite oral challenge -NoTD- were evaluated for changes in circulating monocytes and DCs. Expression of CD38 and CD40 were upregulated in monocytes and DCs in TD volunteers during the disease days (TD-0h to TD-96h. Moreover, integrin α4β7, a gut homing molecule, was upregulated on monocytes but not DCs. CD21 upregulation was only identified in DCs. These changes were not observed among NoTD volunteers despite the same oral challenge. Moreover, monocytes and DCs from NoTD volunteers showed increased binding to S. Typhi one day after challenge. These monocytes showed phosphorylation of p38MAPK, NFkB and Erk1/2 upon stimulation with S. Typhi-LPS-QDot micelles. In contrast, monocytes from TD volunteers showed only a moderate increase in S. Typhi binding 48 h and 96 h post-TD, and only Erk1/2 phosphorylation. This is the first study to describe different activation and migration profiles, as well as differential signaling patterns, in monocytes and DCs which relate directly to the clinical outcome following oral challenge with wild type S. Typhi.

HIV-1 Latency in Monocytes/Macrophages

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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.

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

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

2017-08-25

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

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

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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.

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

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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.

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

Radiation-Induced Thymidine Phosphorylase Upregulation in Rectal Cancer Is Mediated by Tumor-Associated Macrophages by Monocyte Chemoattractant Protein-1 From Cancer Cells

International Nuclear Information System (INIS)

Kim, Tae-Dong; Li Ge; Song, Kyoung-Sub; Kim, Jin-Man; Kim, Jun-Sang; Kim, Jong-Seok; Yun, Eun-Jin; Park, Jong-Il; Park, Hae-Duck; Hwang, Byung-Doo; Lim, Kyu; Yoon, Wan-Hee

2009-01-01

Purpose: The mechanisms of thymidine phosphorylase (TP) regulation induced by radiation therapy (XRT) in various tumors are poorly understood. We investigated the effect and mechanisms of preoperative XRT on TP expression in rectal cancer tissues. Methods and Materials: TP expression and CD68 and monocyte chemoattractant protein-1 (MCP-1) levels in rectal cancer tissues and cancer cell lines were evaluated before and after XRT in Western blotting, immunohistochemistry, enzyme-linked immunoassay, and reverse transcription-polymerase chain reaction studies. Isolated peripheral blood monocytes were used in the study of chemotaxis under the influence of MCP-1 released by irradiated colon cancer cells. Results: Expression of TP was significantly elevated by 9 Gy of XRT in most rectal cancer tissues but not by higher doses of XRT. In keeping with the close correlation of the increase in both TP expression and the number of tumor-associated macrophages (TAMs), anti-TP immunoreactivity was found in the CD68-positive TAMs and not the neoplastic cells. Expression of MCP-1 was increased in most cases after XRT, and this increase was strongly correlated with TP expression. However, this increase in MCP-1 expression occurred in tumor cells and not stromal cells. The XRT upregulated MCP-1 mRNA and also triggered the release of MCP-1 protein from cultured colon cancer cells. The supernatant of irradiated colon cancer cells showed strong chemotactic activity for monocyte migration, but this activity was completely abolished by neutralizing antibody. Conclusions: Use of XRT induces MCP-1 expression in cancer cells, which causes circulating monocytes to be recruited into TAMs, which then upregulate TP expression in rectal cancer tissues

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...

Phenotype and Function of CD209+ Bovine Blood Dendritic Cells, Monocyte-Derived-Dendritic Cells and Monocyte-Derived Macrophages.

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Kun Taek Park

Full Text Available Phylogenic comparisons of the mononuclear phagocyte system (MPS of humans and mice demonstrate phenotypic divergence of dendritic cell (DC subsets that play similar roles in innate and adaptive immunity. Although differing in phenotype, DC can be classified into four groups according to ontogeny and function: conventional DC (cDC1 and cDC2, plasmacytoid DC (pDC, and monocyte derived DC (MoDC. DC of Artiodactyla (pigs and ruminants can also be sub-classified using this system, allowing direct functional and phenotypic comparison of MoDC and other DC subsets trafficking in blood (bDC. Because of the high volume of blood collections required to study DC, cattle offer the best opportunity to further our understanding of bDC and MoDC function in an outbred large animal species. As reported here, phenotyping DC using a monoclonal antibody (mAb to CD209 revealed CD209 is expressed on the major myeloid population of DC present in blood and MoDC, providing a phenotypic link between these two subsets. Additionally, the present study demonstrates that CD209 is also expressed on monocyte derived macrophages (MoΦ. Functional analysis revealed each of these populations can take up and process antigens (Ags, present them to CD4 and CD8 T cells, and elicit a T-cell recall response. Thus, bDC, MoDC, and MoΦ pulsed with pathogens or candidate vaccine antigens can be used to study factors that modulate DC-driven T-cell priming and differentiation ex vivo.

Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype

International Nuclear Information System (INIS)

Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique; Licona-Limón, Ileana; Huerta, Leonor

2017-01-01

Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4"+ T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependent phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. - Highlights: • Jurkat T cells expressing the HIV-1 envelope fuse with THP-1 monocytes. • Heterokaryons display a dominant myeloid phenotype and monocyte function. • Heterokaryons exhibit activation features in the absence of activation agents. • Activation is not due to cell-cell interaction but requires cell-cell fusion. • The

Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype

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Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique; Licona-Limón, Ileana; Huerta, Leonor, E-mail: leonorhh@biomedicas.unam.mx

2017-03-01

Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4{sup +} T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependent phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. - Highlights: • Jurkat T cells expressing the HIV-1 envelope fuse with THP-1 monocytes. • Heterokaryons display a dominant myeloid phenotype and monocyte function. • Heterokaryons exhibit activation features in the absence of activation agents. • Activation is not due to cell-cell interaction but requires cell-cell fusion. • The

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.

Dendritic cell, monocyte and T cell activation and response to glatiramer acetate in multiple sclerosis

DEFF Research Database (Denmark)

Sellebjerg, F; Hesse, D; Limborg, S

2012-01-01

, monocytes and dendritic cells (DC) in relation to disease activity in MS patients treated with GA. Methods: Flow cytometry was used to study the activation of CD4+ T cells and T cell subsets (CD25high and CD26high cells), monocytes and DCs in a cross-sectional study of 39 untreated and 29 GA-treated MS......Background: Treatment with glatiramer acetate (GA) modestly decreases disease activity in multiple sclerosis (MS). The mechanism of action is incompletely understood and differences in the response to treatment between individuals may exist. Objective: To study the activation of CD4+ T cells...... (Bonferroni-corrected p=0.0005). The hazard ratio of relapse was 1.32 (95% confidence interval 1.05–1.64) per 1% increase in CD40+ DCs. Patients treated with GA had fewer CD4+ T cells expressing surface markers associated with T helper type 1 effector responses and more CD4+ T cells expressing surface markers...

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.

Oxidative stress induces monocyte necrosis with enrichment of cell-bound albumin and overexpression of endoplasmic reticulum and mitochondrial chaperones.

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Haiping Tang

Full Text Available In the present study, monocytes were treated with 5-azacytidine (azacytidine, gossypol or hydrogen peroxide to induce cell death through oxidative stress. A shift from apoptotic to necrotic cell death occurred when monocytes were treated with 100 µM azacytidine for more than 12 hours. Necrotic monocytes exhibited characteristics, including enrichment of cell-bound albumin and up-regulation of endoplasmic reticulum (ER- and mitochondrial-specific chaperones to protect mitochondrial integrity, which were not observed in other necrotic cells, including HUH-7, A2780, A549 and HOC1a. Our results show that the cell-bound albumin originates in the culture medium rather than from monocyte-derived hepatocytes, and that HSP60 is a potential binding partner of the cell-bound albumin. Proteomic analysis shows that HSP60 and protein disulfide isomerase are the most abundant up-regulated mitochondrial and ER-chaperones, and that both HSP60 and calreticulin are ubiquitinated in necrotic monocytes. In contrast, expression levels of the cytosolic chaperones HSP90 and HSP71 were down-regulated in the azacytidine-treated monocytes, concomitant with an increase in the levels of these chaperones in the cell culture medium. Collectively, our results demonstrates that chaperones from different organelles behave differently in necrotic monocytes, ER- and mitochondrial chaperones being retained and cytosolic and nuclear chaperones being released into the cell culture medium through the ruptured cell membrane. HSP60 may serve as a new target for development of myeloid leukemia treatment.

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.)

HIV-1 infection and first line ART induced differential responses in mitochondria from blood lymphocytes and monocytes: the ANRS EP45 "Aging" study.

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Sophie Perrin

Full Text Available The ANRS EP45 "Aging" study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The data reported focus on mitochondria, organelles known to be involved in cell senescence.49 HIV-1 infected patients untreated with antiretroviral therapy, together with 49 seronegative age- and sex-matched control subjects and 81 HIV-1 infected and treated patients, were recruited by 3 AIDS centres (Marseille, Montpellier, Nice; France; http://clinicaltrials.gov/, NCT01038999. In more than 88% of treated patients, the viral load was 500/mm(3. ROS (reactive oxygen species production and ΔΨm (inner membrane potential were measured by flow cytometry in blood lymphocytes and monocytes (functional parameters. Three mitochondrial network quantitative morphological parameters were computed using confocal microscopy and image analysis. Three PBMC mitochondrial proteins (porin and subunits 2 and 4 of cytochrome C oxidase encoded by mtDNA or nuclear DNA, respectively were analysed by western blotting.Quantitative changes in PBMC mitochondrial proteins were not induced by either HIV-1 infection or ART. Discriminant analysis integrating functional (ROS production and ΔΨm or morphological (network volume density, fragmentation and branching parameters revealed HIV-1 infection and ART differential effects according to cell type. First line ART tended to rescue lymphocyte mitochondrial parameters altered by viral infection, but induced slight changes in monocytes. No statistical difference was found between the effects of three ART regimens on mitochondrial parameters. Correlations between functional parameters and viral load confirmed the damaging effects of HIV-1 in lymphocyte mitochondria.In patients considered to be clinically stable, mitochondria exhibited functional and morphological modifications in PBMCs resulting from either direct or indirect effects of HIV-1 infection (lymphocytes, or from first line ART

Evaluation of Medicinal Plant Hepatotoxicity in Co-cultures of Hepatocytes and Monocytes

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Bashar Saad

2006-01-01

Full Text Available Non-parenchymal cells might play an important role in the modulation of xenobiotic metabolism in liver and its pharmacological and toxicological consequences. Therefore, the role of cell-to-cell interactions in herbal induced liver toxicity was investigated in monocultures of cells from the human hepatocyte cell line (HepG2 and in co-cultures of cells from the HepG2 cell line and cells from the human monocyte cell line (THP1. Cells were treated with various concentrations (1–500 µg ml−1 of extracts of Pistacia palaestina, Juglans regia and Quercus ithaburensis for 24 h. Extracts from Cleome droserifolia, a known toxic plant, were taken as positive control. In the co-culture system, toxic effects were observed after exposure to extracts of Pistacia palestina and C. droserifolia. These two extracts significantly reduced by cell viability as measured the MTT test and the LDH assay. Whereas in hepatocyte cultures, only extracts of C. droserifolia were found to affect the cell viability. The production levels of albumin from hepatocytes were not affected by treatment with plant extracts in both culture systems. It seems that the observed reduction in cell viability after exposure to extracts of P. palestina in co-cultures but not in monocultures is a result of monocyte-derived factors. The use of liver cell co-cultures is therefore a useful approach to investigate the influence of intercellular communication on xenobiotic metabolism in liver.

Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines

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Gulec, Cagri, E-mail: cagri.gulec@gmail.com; Coban, Neslihan, E-mail: neslic@istanbul.edu.tr; Ozsait-Selcuk, Bilge, E-mail: ozsaitb@istanbul.edu.tr; Sirma-Ekmekci, Sema, E-mail: semasirma@gmail.com; Yildirim, Ozlem, E-mail: ozlm-yildirim@hotmail.com; Erginel-Unaltuna, Nihan, E-mail: nihanerginel@yahoo.com

2017-04-01

ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses.

Mitochondrial functions of THP-1 monocytes following the exposure to selected natural compounds.

Science.gov (United States)

Schultze, Nadin; Wanka, Heike; Zwicker, Paula; Lindequist, Ulrike; Haertel, Beate

2017-02-15

The immune system is an important target of various xenobiotics, which may lead to severe adverse effects including immunosuppression or inappropriate immunostimulation. Mitochondrial toxicity is one possibility by which xenobiotics exert their toxic effects in cells or organs. In this study, we investigated the impact of three natural compounds, cyclosporine A (CsA), deoxynivalenol (DON) and cannabidiol (CBD) on mitochondrial functions in the THP-1 monocytic cell line. The cells were exposed for 24h to two different concentrations (IC 10 and IC 50 determined by MTT) of each compound. The cells showed concentration-dependent elevated intracellular reactive oxygen species (iROS) and induction of apoptosis (except DON) in response to the three test compounds. Mitochondrial functions were characterized by using bioenergetics profiling experiments. In THP-1 monocytes, the IC 50 of CsA decreased basal and maximal respiration as well as ATP production with an impact on spare capacity indicating a mitochondrial dysfunction. Similar reaction patterns were observed following CBD exposure. The basal respiration level and ATP-production decreased in the THP-1 cells exposed to the IC 50 of DON with no major impact on mitochondrial function. In conclusion, impaired mitochondrial function was accompanied by elevated iROS and apoptosis level in a monocytic cell line exposed to CsA and CBD. Mitochondrial dysfunction may be one explanation for the cytotoxicity of CBD and CsA also in other in immune cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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.

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

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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.

Generation of dendritic cells for immunotherapy is minimally impaired by granulocytes in the monocyte preparation.

Science.gov (United States)

ten Brinke, Anja; Karsten, Miriam L; Dieker, Miranda C; Zwaginga, Jaap Jan; Vrielink, Hans; Marieke van Ham, S

2006-01-01

The growing number of clinical studies, using monocyte-derived DC therapy, requires protocols where a sufficient number of dendritic cell (DCs) are produced according to current Good Manufacturing Practice guidelines. Therefore, a closed culture system for the generation of DCs is inevitable. One cost-effective way to isolate monocytes directly from leukapheresis material in a closed system is by elutriation with the Elutra cell separation system. In the Elutra, granulocytes co-purify with the monocytes. Therefore, we studied if and to what extent the presence of granulocytes in a monocyte product affects the generation of mature DCs. The presence of up to 16% granulocytes in the monocyte product had no significant effects on the quality of the DCs formed. The presence of higher granulocyte percentages, however, gradually altered DC quality. In this respect, the presence of higher number of granulocytes induced significant lower migratory capacity of the DCs and lower expression levels of CD80, CD40 and CD86. No effects were observed on the DC yield, cytokine production or the stimulatory capacity of the DCs in MLR. In conclusion, the presence of 20-30% granulocytes in a monocyte product has no major influence on the quality of the DCs generated from monocytes. Therefore, the Elutra is a suitable closed system apparatus to separate monocytes from other blood components for the generation of DCs, even from leukapheresis material which contains a high number of granulocytes.

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

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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.

ADMA induces monocyte adhesion via activation of chemokine receptors in cultured THP-1 cells.

Science.gov (United States)

Chen, Meifang; Li, Yuanjian; Yang, Tianlun; Wang, Yongjin; Bai, Yongping; Xie, Xiumei

2008-08-01

Asymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor, is also an important inflammatory factor contributing to the development of atherosclerosis (AS). The present study was to test the effect of ADMA on angiotensin (Ang) II-induced monocytic adhesion. Human monocytoid cells (THP-1) or isolated peripheral blood monocyte cells (PBMCs) were incubated with Ang II (10(-6)M) or exogenous ADMA (30 microM) for 4 or 24h in the absence or presence of losartan or antioxidant PDTC. In cultured THP-1 cells, Ang II (10(-6)M) for 24h elevated the level of ADMA in the medium, upregulated the protein expression of protein arginine methyltransferase (PRMT) and decreased the activity of dimethylarginine dimethylaminohydrolase (DDAH). Both of Ang II and ADMA increased monocytic adhesion to human umbilical vein endothelial cells (HUVECs), elevated the levels of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-8 and tumor necrosis factor (TNF)-alpha and upregulated CCR(2) and CXCR(2) mRNA expression, concomitantly with increase in reactive oxygen species (ROS) generation and activation of nuclear factor (NF)-kappaB. Pretreatment with losartan (10 microM) or PDTC (10 microM) abolished the effects mediated by Ang II or ADMA. In isolated PBMCs from healthy individuals, ADMA upregulated the expression of CXCR(2) mRNA, which was attenuated by losartan (10 microM), however, ADMA had no effect on surface protein expression of CCR(2). The present results suggest that ADMA may be involved in monocytic adhesion induced by Ang II via activation of chemokine receptors by ROS/NF-kappaB pathway.

'Fluorescent Cell Chip' for immunotoxicity testing: Development of the c-fos expression reporter cell lines

International Nuclear Information System (INIS)

Trzaska, Dominika; Zembek, Patrycja; Olszewski, Maciej; Adamczewska, Violetta; Ulleras, Erik; Dastych, JarosIaw

2005-01-01

The Fluorescent Cell Chip for in vitro immunotoxicity testing employs cell lines derived from lymphocytes, mast cells, and monocytes-macrophages transfected with various EGFP cytokine reporter gene constructs. While cytokine expression is a valid endpoint for in vitro immunotoxicity screening, additional marker for the immediate-early response gene expression level could be of interest for further development and refinement of the Fluorescent Cell Chip. We have used BW.5147.3 murine thymoma transfected with c-fos reporter constructs to obtain reporter cell lines expressing ECFP under the control of murine c-fos promoter. These cells upon serum withdrawal and readdition and incubation with heavy metal compounds showed paralleled induction of c-Fos expression as evidenced by Real-Time PCR and ECFP fluorescence as evidenced by computer-supported fluorescence microscopy. In conclusion, we developed fluorescent reporter cell lines that could be employed in a simple and time-efficient screening assay for possible action of chemicals on c-Fos expression in lymphocytes. The evaluation of usefulness of these cells for the Fluorescent Cell Chip-based detection of immunotoxicity will require additional testing with a larger number of chemicals

Curcumin modulates endothelial permeability and monocyte transendothelial migration by affecting endothelial cell dynamics.

Science.gov (United States)

Monfoulet, Laurent-Emmanuel; Mercier, Sylvie; Bayle, Dominique; Tamaian, Radu; Barber-Chamoux, Nicolas; Morand, Christine; Milenkovic, Dragan

2017-11-01

Curcumin is a phenolic compound that exhibits beneficial properties for cardiometabolic health. We previously showed that curcumin reduced the infiltration of immune cells into the vascular wall and prevented atherosclerosis development in mice. This study aimed to investigate the effect of curcumin on monocyte adhesion and transendothelial migration (TEM) and to decipher the underlying mechanisms of these actions. Human umbilical vein endothelial cells (HUVECs) were exposed to curcumin (0.5-1μM) for 3h prior to their activation by Tumor Necrosis Factor alpha (TNF-α). Endothelial permeability, monocyte adhesion and transendothelial migration assays were conducted under static condition and shear stress that mimics blood flow. We further investigated the impact of curcumin on signaling pathways and on the expression of genes using macroarrays. Pre-exposure of endothelial cells to curcumin reduced monocyte adhesion and their transendothelial migration in both static and shear stress conditions. Curcumin also prevented changes in both endothelial permeability and the area of HUVECs when induced by TNF-α. We showed that curcumin modulated the expression of 15 genes involved in the control of cytoskeleton and endothelial junction dynamic. Finally, we showed that curcumin inhibited NF-κB signaling likely through an antagonist interplay with several kinases as suggested by molecular docking analysis. Our findings demonstrate the ability of curcumin to reduce monocyte TEM through a multimodal regulation of the endothelial cell dynamics with a potential benefit on the vascular endothelial function barrier. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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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.

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.

Mycoplasma hyorhinis-Contaminated Cell Lines Activate Primary Innate Immune Cells via a Protease-Sensitive Factor.

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Simon Heidegger

Full Text Available Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.

Transcriptome Analysis of Circulating Immune Cell Subsets Highlight the Role of Monocytes in Zaire Ebola Virus Makona Pathogenesis

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Andrea R. Menicucci

2017-10-01

Full Text Available Existing models of Ebola virus disease (EVD suggest antigen-presenting cells are initial targets of Zaire ebolavirus (ZEBOV. In vitro studies have shown that ZEBOV infection of monocytes and macrophages results in the production of inflammatory mediators, which may cause lymphocyte apoptosis. However, these findings have not been corroborated by in vivo studies. In this study, we report the first longitudinal analysis of transcriptional changes in purified monocytes, T-cells, and B-cells isolated from cynomolgus macaques following infection with ZEBOV-Makona. Our data reveal monocytes as one of the major immune cell subsets that supports ZEBOV replication in vivo. In addition, we report a marked increase in the transcription of genes involved in inflammation, coagulation, and vascular disease within monocytes, suggesting that monocytes contribute to EVD manifestations. Further, genes important for antigen presentation and regulation of immunity were downregulated, potentially subverting development of adaptive immunity. In contrast, lymphocytes, which do not support ZEBOV replication, showed transcriptional changes limited to a small number of interferon-stimulated genes (ISGs and a failure to upregulate genes associated with an antiviral effector immune response. Collectively, these data suggest that ZEBOV-infected monocytes play a significant role in ZEBOV-Makona pathogenesis and strategies to suppress virus replication or modify innate responses to infection in these cells should be a priority for therapeutic intervention.

HLA class I antibodies trigger increased adherence of monocytes to endothelial cells by eliciting an increase in endothelial P-selectin and, depending on subclass, by engaging FcγRs.

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Valenzuela, Nicole M; Mulder, Arend; Reed, Elaine F

2013-06-15

Ab-mediated rejection (AMR) of solid organ transplants is characterized by intragraft macrophages. It is incompletely understood how donor-specific Ab binding to graft endothelium promotes monocyte adhesion, and what, if any, contribution is made by the Fc region of the Ab. We investigated the mechanisms underlying monocyte recruitment by HLA class I (HLA I) Ab-activated endothelium. We used a panel of murine mAbs of different subclasses to crosslink HLA I on human aortic, venous, and microvascular endothelial cells and measured the binding of human monocytic cell lines and peripheral blood monocytes. Both anti-HLA I murine (m)IgG1 and mIgG2a induced endothelial P-selectin, which was required for monocyte adhesion to endothelium irrespective of subclass. mIgG2a but not mIgG1 could bind human FcγRs. Accordingly, HLA I mIgG2a but not mIgG1 treatment of endothelial cells significantly augmented recruitment, predominantly through FcγRI, and, to a lesser extent, FcγRIIa. Moreover, HLA I mIgG2a promoted firm adhesion of monocytes to ICAM-1 through Mac-1, which may explain the prominence of monocytes during AMR. We confirmed these observations using human HLA allele-specific mAbs and IgG purified from transplant patient sera. HLA I Abs universally elicit endothelial exocytosis leading to monocyte adherence, implying that P-selectin is a putative therapeutic target to prevent macrophage infiltration during AMR. Importantly, the subclass of donor-specific Ab may influence its pathogenesis. These results imply that human IgG1 and human IgG3 should have a greater capacity to trigger monocyte infiltration into the graft than IgG2 or IgG4 due to enhancement by FcγR interactions.

Effects of HIV infection and ART on phenotype and function of circulating monocytes, natural killer, and innate lymphoid cells.

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Nabatanzi, Rose; Cose, Stephen; Joloba, Moses; Jones, Sarah Rowland; Nakanjako, Damalie

2018-03-15

HIV infection causes upregulation of markers of inflammation, immune activation and apoptosis of host adaptive, and innate immune cells particularly monocytes, natural killer (NK) and innate lymphoid cells (ILCs). Although antiretroviral therapy (ART) restores CD4 T-cell counts, the persistent aberrant activation of monocytes, NK and ILCs observed likely contributes to the incomplete recovery of T-cell effector functions. A better understanding of the effects of HIV infection and ART on the phenotype and function of circulating monocytes, NK, and ILCs is required to guide development of novel therapeutic interventions to optimize immune recovery.

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

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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.

HLA class I antibodies trigger increased adherence of monocytes to endothelial cells by eliciting an increase in endothelial P-selectin and, depending on subclass, by engaging FcγRs1

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Valenzuela, Nicole M; Mulder, Arend; Reed, Elaine F

2013-01-01

Antibody-mediated rejection of solid organ transplants is characterized by intragraft macrophages. It is incompletely understood how donor specific antibody binding to graft endothelium promotes monocyte adhesion, and what, if any, contribution is made by the Fc region of the antibody. We investigated the mechanisms underlying monocyte recruitment by HLA class I antibody-activated endothelium. We used a panel of murine monoclonal antibodies of different subclasses to crosslink HLA I on human aortic, venous and microvascular endothelial cells, and measured the binding of human monocytic cell lines and peripheral blood monocytes. Both anti-HLA I murine IgG1 and mIgG2a induced endothelial P-selectin, which was required for monocyte adhesion to endothelium irrespective of subclass. Mouse IgG2a but not mIgG1 could bind human FcγRs. Accordingly, HLA I mIgG2a but not mIgG1 treatment of endothelial cells significantly augmented recruitment, predominantly through FcγRI, and, to a lesser extent, FcγRIIa. Moreover, HLA I mIgG2a promoted firm adhesion of monocytes to ICAM-1 through Mac-1, which may explain the prominence of monocytes during antibody mediated rejection. We confirmed these observations using human HLA allele specific monoclonal antibodies and IgG purified from transplant patient sera. HLA I antibodies universally elicit endothelial exocytosis leading to monocyte adherence, implying that P-selectin is a putative therapeutic target to prevent macrophage infiltration during antibody-mediated rejection. Importantly, the subclass of donor specific antibody may influence its pathogenesis. These results imply that hIgG1 and hIgG3 should have a greater capacity to trigger monocyte infiltration into the graft than IgG2 or IgG4 due to enhancement by FcγR interactions. PMID:23690477

Endogenous pyrogen production by Hodgkin's disease and human histiocytic lymphoma cell lines in vitro.

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Bodel, P; Ralph, P; Wenc, K; Long, J C

1980-02-01

Fever not explained by infection may occur in patients with malignant lymphoma presumably caused by a release of endogenous pyrogen. Although pyrogen has been found in some tumors with a mixed cell population, production of endogenous pyrogen by the neoplastic cells has not been demonstrated. This report documents the apparently spontaneous synthesis and release of such pyrogen by two human tumor cell lines derived from patients with Hodgkin's disease and histiocytic lymphoma. The endogenous pyrogen from the two cell lines was similar and closely resembled that produced by normal human monocytes in antigenic properties as well as heat and pronase sensitivity. The Hodgkin's disease and histiocytic lymphoma cell lines do not require specific stimulation for the production of endogenous pyrogen suggesting that the mechanism of pyrogen release by neoplastic macrophage-related cells differs from that of normal phagocytic cells. The tumor-associated fever in some patients with malignant lymphoma may be caused by a release of endogenous pyrogen by proliferating neoplastic cells.

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

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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.

Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases*

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Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J.; Nie, Guangjun

2016-01-01

Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. PMID:26895960

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.

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

Characterization of monocyte-derived dendritic cells maturated with IFN-alpha

DEFF Research Database (Denmark)

Svane, I M; Nikolajsen, K; Walter, M R

2006-01-01

Dendritic cells (DC) are promising candidates for cancer immunotherapy. These cells can be generated from peripheral blood monocytes cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). In order to obtain full functional capacity, maturation is required......, maturation with IFN-alpha has only a small effect on induction of autologous T-cell stimulatory capacity of the DC. However, an increase in DC allogeneic T-cell stimulatory capacity was observed. These data suggest that IFN-alpha has a potential as a maturation agent used in DC-based cancer vaccine trials...

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

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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.

Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases.

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Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J; Nie, Guangjun

2016-04-15

Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Establishing porcine monocyte-derived macrophage and dendritic cell systems for studying the interaction with PRRSV-1

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Helen eSingleton

2016-06-01

Full Text Available Monocyte-derived macrophages (MoMØ and monocyte-derived dendritic cells (MoDC are two model systems well established in human and rodent systems that can be used to study the interaction of pathogens with host cells. Porcine reproductive and respiratory syndrome virus (PRRSV is known to infect myeloid cells, such as macrophages (MØ and dendritic cells (DC. Therefore, this study aimed to establish systems for the differentiation and characterization of MoMØ and MoDC for subsequent infection with PRRSV-1. M-CSF differentiated monocyte-derived macrophages (MoMØ were stimulated with activators for classical (M1 or alternative (M2 activation. GM-CSF and IL-4 generated monocyte-derived dendritic cells (MoDC were activated with the well established maturation cocktail containing PAMPs and cytokines. In addition, MoMØ and MoDC were treated with dexamethasone and IL-10, which are known immuno-suppressive reagents. Cells were characterized by morphology, phenotype and function and porcine MØ subsets highlighted some divergence from described human counterparts, while MoDC, appeared more similar to mouse and human DCs. The infection with PRRSV-1 strain Lena demonstrated different replication kinetics between MoMØ and MoDC and within subsets of each cell type. While MoMØ susceptibility was significantly increased by dexamethasone and IL-10 with an accompanying increase in CD163/CD169 expression, MoDC supported only a minimal replication of PRRSV These findings underline the high variability in the susceptibility of porcine myeloid cells towards PRRSV-1 infection.

Protective properties of artichoke (Cynara scolymus) against oxidative stress induced in cultured endothelial cells and monocytes.

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Zapolska-Downar, Danuta; Zapolski-Downar, Andrzej; Naruszewicz, Marek; Siennicka, Aldona; Krasnodebska, Barbara; Kołdziej, Blanka

2002-11-01

It is currently believed that oxidative stress and inflammation play a significant role in atherogenesis. Artichoke extract exhibits hypolipemic properties and contains numerous active substances with antioxidant properties in vitro. We have studied the influence of aqueous and ethanolic extracts from artichoke on intracellular oxidative stress stimulated by inflammatory mediators (TNFalpha and LPS) and ox-LDL in endothelial cells and monocytes. Oxidative stress which reflects the intracellular production of reactive oxygen species (ROS) was followed by measuring the oxidation of 2', 7'-dichlorofluorescin (DCFH) to 2', 7'-dichlorofluorescein (DCF). Agueous and ethanolic extracts from artichoke were found to inhibit basal and stimulated ROS production in endothelial cells and monocytes in dose dependent manner. In endothelial cells, the ethanolic extract (50 microg/ml) reduced ox-LDL-induced intracellular ROS production by 60% (partichoke extracts have marked protective properties against oxidative stress induced by inflammatory mediators and ox-LDL in cultured endothelial cells and monocytes.

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

Edaravone attenuates monocyte adhesion to endothelial cells induced by oxidized low-density lipoprotein

International Nuclear Information System (INIS)

Li, Zhijuan; Cheng, Jianxin; Wang, Liping

2015-01-01

Oxidized low-density lipoprotein (oxLDL) plays a vital role in recruitment of monocytes to endothelial cells, which is important during early stages of atherosclerosis development. Edaravone, a potent and novel scavenger of free radicals inhibiting hydroxyl radicals, has been clinically used to reduce the neuronal damage following ischemic stroke. In the present study, Edaravone was revealed to markedly reduce oxLDL-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). The inhibitory mechanism of Edaravone was associated with suppression of the chemokine MCP-1 and adhesion molecule VCAM-1 and ICAM-1 expression. In addition, luciferase reporter assay results revealed that administration of Edaravone attenuated the increase in NF-κB transcriptional activity induced by oxLDL. Notably, it's also shown that Edaravone treatment blocked oxLDL induced p65 nuclear translocation in HUVECs. Results indicate that Edaravone negatively regulates endothelial inflammation. - Highlights: • Edaravone reduces oxLDL-induced monocyte adhesion to HUVECs. • Edaravone attenuates oxLDL-induced expression of MCP-1, VCAM-1, and ICAM-1. • Edaravone reduces NF-κB transcriptional activity and p65 nuclear translocation.

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

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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.

Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

DEFF Research Database (Denmark)

Hansen, Morten; Hjortø, Gertrud Malene; Met, Ozcan

2011-01-01

in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...... at similar concentrations to the passive adsorption process, as measured by enzyme-linked immunosorbent assays, and the bound IL-4 did not leak into solution to any measurable extent during cell culture. However, covalently bound IL-4 was incapable of inducing monocyte differentiation. This may be caused...

Infection Rate and Tissue Localization of Murine IL-12p40-Producing Monocyte-Derived CD103+ Lung Dendritic Cells during Pulmonary Tuberculosis

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Leepiyasakulchai, Chaniya; Taher, Chato; Chuquimia, Olga D.; Mazurek, Jolanta; Söderberg-Naucler, Cecilia; Fernández, Carmen; Sköld, Markus

2013-01-01

Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103+ dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40+ cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype. PMID:23861965

Infection rate and tissue localization of murine IL-12p40-producing monocyte-derived CD103(+) lung dendritic cells during pulmonary tuberculosis.

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Leepiyasakulchai, Chaniya; Taher, Chato; Chuquimia, Olga D; Mazurek, Jolanta; Söderberg-Naucler, Cecilia; Fernández, Carmen; Sköld, Markus

2013-01-01

Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103(+) dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40(+) cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype.

Evaluating the Effects of Cytomegalovirus Glycoprotein B on the Maturation and Function of Monocyte-derived dendritic cells

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Afsson shariat

2015-11-01

Full Text Available Background & Objectives: Interaction of cytomegalovirus glycoprotein B with toll-like receptors of dendritic cells leads to early signaling and innate immune responses. The aim of this study is to evaluate the effects of cytomegalovirus glycoprotein B on the maturation and function of monocyte-derived dendritic cells in treated groups in comparison with control groups. Materials & Methods: Blood samples were taken from 5 healthy volunteers. Following the generation of monocyte-derived dendritic cells on the fifth day of cell culture, half of the immature dendritic cells were treated with cytomegalovirus glycoprotein B, and the rest of them were induced to mature dendritic untreated cells and were used as the control group. The maturation and function of dendritic cells were evaluated in these two groups. Results: The gene expression level of toll-like receptor-4 significantly increased in the group treated with glycoprotein B (p < 0.05, whereas there were no significant differences in the expression rates of CD83, CD86, CD1a, and HLA-DR and the secretion of IL-23 from monocyte-derived dendritic cells between the treated groups and the controls. Conclusion: The increase in the gene expression of toll-like receptor-4 in monocyte-derived dendritic cells treated with cytomegalovirus glycoprotein B showed that cell contact is required to elicit cellular antiviral response and toll-like receptor activation. Thus, it is critical to recognize the viral and cellular determinants of the immune system in order to develop new therapeutic strategies against cytomegalovirus.

Combination of Cobe AutoPBSC and Gambro Elutra as a platform for monocyte enrichment in dendritic cell (DC) therapy: clinical study.

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Chen, Ying; Hoecker, Paul; Zeng, Jia; Dettke, Markus

2008-01-01

Monocytes are a common source for generating dendritic cells (DCs). The aim of the present study was to evaluate the efficiency of a platform for monocyte collection and enrichment in a clinical setting. The platform was based on the combination of two semiautomated devices; the Cobe Spectra Auto PBSC for mononuclear cells (MNC) collection followed by counterflow elutriation for monocyte enrichment (Gambro BCT Elutra). Twenty-four patients with various types of epithelial cancer participated in the study. MNC collections were first performed as large volume leukapheresis (LVL). Subsequently, MNC products were processed with an elutriation system for monocyte isolation. LVL resulted in the collection of MNC at a median of 8.1 x 10(9) cells, containing of 31.4% monocytes. A similar efficacy was also shown in patients with lower peripheral blood counts. Elutriation of the MNC product with the Cobe Elutra device resulted in the enrichment of monocytes at a median of 2.7 x 10(9) cells, with a recovery of 80.2% and a purity of 90.7%. These monocytes were then successfully developed into DCs for clinical therapy after in vitro manipulation. These data suggest that the combination of the Cobe Spectra Auto PBSC and the Gambro BCT Elutra is an effective platform for monocyte enrichment in clinical practice according to GCP standards and GMP guidelines, and can be easily implemented in the clinical routine under current DC protocols. Copyright 2008 Wiley-Liss, Inc.

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

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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

iNKT Cell Emigration out of the Lung Vasculature Requires Neutrophils and Monocyte-Derived Dendritic Cells in Inflammation

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Ajitha Thanabalasuriar

2016-09-01

Full Text Available iNKT cells are a subset of innate T cells that recognize glycolipids presented on CD1d molecules and protect against bacterial infections, including S. pneumoniae. Using lung intravital imaging, we examined the behavior and mechanism of pulmonary iNKT cell activation in response to the specific iNKT cell ligand α-galactosylceramide or S. pneumoniae infection. In untreated mice, the major fraction of iNKT cells resided in the vasculature, but a small critical population resided in the extravascular space in proximity to monocyte-derived DCs. Administration of either α-GalCer or S. pneumoniae induced CD1d-dependent rapid recruitment of neutrophils out of the vasculature. The neutrophils guided iNKT cells from the lung vasculature via CCL17. Depletion of monocyte-derived DCs abrogated both the neutrophil and subsequent iNKT cell extravasation. Moreover, impairing iNKT cell recruitment by blocking CCL17 increased susceptibility to S. pneumoniae infection, suggesting a critical role for the influx of iNKT cells in host defense.

The role of monocytes and T cells in 1,25-dihydroxyvitamin D3 mediated inhibition of B cell function in vitro

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Müller, K; Heilmann, C; Poulsen, L K

1991-01-01

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) inhibits immunoglobulin production by human mononuclear cells (MNC) in vitro. The present study was undertaken to evaluate the role of T cells and monocytes in 1,25-(OH)2D3 induced suppression of B cell functions. The synthetic vitamin D3 analogue MC 903...... was examined in parallel. 1,25-(OH)2D3 and MC 903 showed a dose-related inhibition of IgM, IgG and IgA plaque-forming cells in poke-weed mitogen (PWM) activated cultures of MNC. This effect was most likely mediated through impairment of T cell and monocyte functions. First, the inhibitory effect was seen after...

Static high-gradient magnetic fields affect the functionality of monocytic cells

Czech Academy of Sciences Publication Activity Database

Syrovets, T.; Schmidt, Z.; Buechele, B.; Zablotskyy, Vitaliy A.; Dejneka, Alexandr; Dempsey, N.; Simmet, T.

2014-01-01

Roč. 28, č. 1 (2014), s. 1-2 ISSN 0892-6638 Institutional support: RVO:68378271 Keywords : static high-gradient * magnet ic fields * affect the functionality * monocytic cells Subject RIV: BM - Solid Matter Physics ; Magnet ism OBOR OECD: Condensed matter physics (including formerly solid state physics, supercond.)

Endotoxin-induced monocytic microparticles have contrasting effects on endothelial inflammatory responses.

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Beryl Wen

Full Text Available Septic shock is a severe disease state characterised by the body's life threatening response to infection. Complex interactions between endothelial cells and circulating monocytes are responsible for microvasculature dysfunction contributing to the pathogenesis of this syndrome. Here, we intended to determine whether microparticles derived from activated monocytes contribute towards inflammatory processes and notably vascular permeability. We found that endotoxin stimulation of human monocytes enhances the release of microparticles of varying phenotypes and mRNA contents. Elevated numbers of LPS-induced monocytic microparticles (mMP expressed CD54 and contained higher levels of transcripts for pro-inflammatory cytokines such as TNF, IL-6 and IL-8. Using a prothrombin time assay, a greater reduction in plasma coagulation time was observed with LPS-induced mMP than with non-stimulated mMP. Co-incubation of mMP with the human brain endothelial cell line hCMEC/D3 triggered their time-dependent uptake and significantly enhanced endothelial microparticle release. Unexpectedly, mMP also modified signalling pathways by diminishing pSrc (tyr416 expression and promoted endothelial monolayer tightness, as demonstrated by endothelial impedance and permeability assays. Altogether, these data strongly suggest that LPS-induced mMP have contrasting effects on the intercellular communication network and display a dual potential: enhanced pro-inflammatory and procoagulant properties, together with protective function of the endothelium.

Stimulated monocyte IL-6 secretion predicts survival of patients with head and neck squamous cell carcinoma

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Olofsson Jan

2008-01-01

Full Text Available Abstract Background This study was performed in order to determine whether monocyte in vitro function is associated with presence, stage and prognosis of head and neck squamous cell carcinoma (HNSCC disease. Methods Prospective study describing outcome, after at least five years observation, of patients treated for HNSCC disease in relation to their monocyte function. Sixty-five patients with newly diagnosed HNSCC and eighteen control patients were studied. Monocyte responsiveness was assessed by measuring levels of monocyte in vitro interleukin (IL-6 and monocyte chemotactic peptide (MCP-1 secretion after 24 hours of endotoxin stimulation in cultures supplied either with 20% autologous serum (AS or serum free medium (SFM. Survival, and if relevant, cause of death, was determined at least 5 years following primary diagnosis. Results All patients, as a group, had higher in vitro monocyte responsiveness in terms of IL-6 (AS (t = 2.03; p t = 2.49; p in vitro monocyte IL-6 endotoxin responsiveness under the SFM condition was associated with decreased survival rate (Hazard ratio (HR = 2.27; Confidence interval (CI = 1.05–4.88; p p p Conclusion In HNSCC patients, changed monocyte in vitro response to endotoxin, as measured by increased IL-6 (SFM and decreased MCP-1 (AS responsiveness, are negative prognostic factors.

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

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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.

Intermediate Monocytes but Not TIE2-Expressing Monocytes Are a Sensitive Diagnostic Indicator for Colorectal Cancer

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Schauer, Dominic; Starlinger, Patrick; Reiter, Christian; Jahn, Nikolaus; Zajc, Philipp; Buchberger, Elisabeth; Bachleitner-Hofmann, Thomas; Bergmann, Michael; Stift, Anton; Gruenberger, Thomas; Brostjan, Christine

2012-01-01

We have conducted the first study to determine the diagnostic potential of the CD14++CD16+ intermediate monocytes as compared to the pro-angiogenic subset of CD14++CD16+TIE2+ TIE2-expressing monocytes (TEMs) in cancer. These monocyte populations were investigated by flow cytometry in healthy volunteers (N = 32) and in colorectal carcinoma patients with localized (N = 24) or metastatic (N = 37) disease. We further determined blood levels of cytokines associated with monocyte regulation. The results revealed the intermediate monocyte subset to be significantly elevated in colorectal cancer patients and to show the highest frequencies in localized disease. Multivariate regression analysis identified intermediate monocytes as a significant independent variable in cancer prediction. With a cut-off value at 0.37% (intermediate monocytes of total leukocytes) the diagnostic sensitivity and specificity ranged at 69% and 81%, respectively. In contrast, TEM levels were elevated in localized cancer but did not differ significantly between groups and none of the cytokines correlated with monocyte subpopulations. Of interest, in vitro analyses supported the observation that intermediate monocytes were more potently induced by primary as opposed to metastatic cancer cells which may relate to the immunosuppressive milieu established in the advanced stage of metastatic disease. In conclusion, intermediate monocytes as compared to TIE2-expressing monocytes are a more sensitive diagnostic indicator of colorectal cancer. PMID:22973451

Intermediate monocytes but not TIE2-expressing monocytes are a sensitive diagnostic indicator for colorectal cancer.

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Dominic Schauer

Full Text Available We have conducted the first study to determine the diagnostic potential of the CD14++CD16+ intermediate monocytes as compared to the pro-angiogenic subset of CD14++CD16+TIE2+ TIE2-expressing monocytes (TEMs in cancer. These monocyte populations were investigated by flow cytometry in healthy volunteers (N = 32 and in colorectal carcinoma patients with localized (N = 24 or metastatic (N = 37 disease. We further determined blood levels of cytokines associated with monocyte regulation. The results revealed the intermediate monocyte subset to be significantly elevated in colorectal cancer patients and to show the highest frequencies in localized disease. Multivariate regression analysis identified intermediate monocytes as a significant independent variable in cancer prediction. With a cut-off value at 0.37% (intermediate monocytes of total leukocytes the diagnostic sensitivity and specificity ranged at 69% and 81%, respectively. In contrast, TEM levels were elevated in localized cancer but did not differ significantly between groups and none of the cytokines correlated with monocyte subpopulations. Of interest, in vitro analyses supported the observation that intermediate monocytes were more potently induced by primary as opposed to metastatic cancer cells which may relate to the immunosuppressive milieu established in the advanced stage of metastatic disease. In conclusion, intermediate monocytes as compared to TIE2-expressing monocytes are a more sensitive diagnostic indicator of colorectal cancer.

Monocytes/macrophages support mammary tumor invasivity by co-secreting lineage-specific EGFR ligands and a STAT3 activator

International Nuclear Information System (INIS)

Vlaicu, Philip; Mertins, Philipp; Mayr, Thomas; Widschwendter, Peter; Ataseven, Beyhan; Högel, Bernhard; Eiermann, Wolfgang; Knyazev, Pjotr; Ullrich, Axel

2013-01-01

Tumor-associated macrophages (TAM) promote malignant progression, yet the repertoire of oncogenic factors secreted by TAM has not been clearly defined. We sought to analyze which EGFR- and STAT3-activating factors are secreted by monocytes/macrophages exposed to tumor cell-secreted factors. Following exposure of primary human monocytes and macrophages to supernatants of a variety of tumor cell lines, we have analyzed transcript and secreted protein levels of EGFR family ligands and of STAT3 activators. To validate our findings, we have analyzed TAM infiltration levels, systemic and local protein levels as well as clinical data of primary breast cancer patients. Primary human monocytes and macrophages respond to tumor cell-derived factors by secreting EGFR- and STAT3-activating ligands, thus inducing two important oncogenic pathways in carcinoma cells. Tumor cell-secreted factors trigger two stereotype secretory profiles in peripheral blood monocytes and differentiated macrophages: monocytes secrete epiregulin (EREG) and oncostatin-M (OSM), while macrophages secrete heparin-binding EGF-like growth factor (HB-EGF) and OSM. HB-EGF and OSM cooperatively induce tumor cell chemotaxis. HB-EGF and OSM are co-expressed by TAM in breast carcinoma patients, and plasma levels of both ligands correlate strongly. Elevated HB-EGF levels accompany TAM infiltration, tumor growth and dissemination in patients with invasive disease. Our work identifies systemic markers for TAM involvement in cancer progression, with the potential to be developed into molecular targets in cancer therapy

Edaravone attenuates monocyte adhesion to endothelial cells induced by oxidized low-density lipoprotein

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Li, Zhijuan, E-mail: zjlee038@163.com; Cheng, Jianxin; Wang, Liping

2015-10-30

Oxidized low-density lipoprotein (oxLDL) plays a vital role in recruitment of monocytes to endothelial cells, which is important during early stages of atherosclerosis development. Edaravone, a potent and novel scavenger of free radicals inhibiting hydroxyl radicals, has been clinically used to reduce the neuronal damage following ischemic stroke. In the present study, Edaravone was revealed to markedly reduce oxLDL-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). The inhibitory mechanism of Edaravone was associated with suppression of the chemokine MCP-1 and adhesion molecule VCAM-1 and ICAM-1 expression. In addition, luciferase reporter assay results revealed that administration of Edaravone attenuated the increase in NF-κB transcriptional activity induced by oxLDL. Notably, it's also shown that Edaravone treatment blocked oxLDL induced p65 nuclear translocation in HUVECs. Results indicate that Edaravone negatively regulates endothelial inflammation. - Highlights: • Edaravone reduces oxLDL-induced monocyte adhesion to HUVECs. • Edaravone attenuates oxLDL-induced expression of MCP-1, VCAM-1, and ICAM-1. • Edaravone reduces NF-κB transcriptional activity and p65 nuclear translocation.

HCMV Displays a Unique Transcriptome of Immunomodulatory Genes in Primary Monocyte-Derived Cell Types.

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Ellen Van Damme

Full Text Available Human cytomegalovirus (HCMV is a betaherpesvirus which rarely presents problems in healthy individuals, yet may result in severe morbidity in immunocompromised patients and in immune-naïve neonates. HCMV has a large 235 kb genome with a coding capacity of at least 165 open reading frames (ORFs. This large genome allows complex gene regulation resulting in different sets of transcripts during lytic and latent infection. While latent virus mainly resides within monocytes and CD34+ progenitor cells, reactivation to lytic infection is driven by differentiation towards terminally differentiated myeloid dendritic cells and macrophages. Consequently, it has been suggested that macrophages and dendritic cells contribute to viral spread in vivo. Thus far only limited knowledge is available on the expression of HCMV genes in terminally differentiated myeloid primary cells and whether or not the virus exhibits a different set of lytic genes in primary cells compared with lytic infection in NHDF fibroblasts. To address these questions, we used Illumina next generation sequencing to determine the HCMV transcriptome in macrophages and dendritic cells during lytic infection and compared it to the transcriptome in NHDF fibroblasts. Here, we demonstrate unique expression profiles in macrophages and dendritic cells which significantly differ from the transcriptome in fibroblasts mainly by modulating the expression of viral transcripts involved in immune modulation, cell tropism and viral spread. In a head to head comparison between macrophages and dendritic cells, we observed that factors involved in viral spread and virion composition are differentially regulated suggesting that the plasticity of the virion facilitates the infection of surrounding cells. Taken together, this study provides the full transcript expression analysis of lytic HCMV genes in monocyte-derived type 1 and type 2 macrophages as well as in monocyte-derived dendritic cells. Thereby

Polarization of migrating monocytic cells is independent of PI 3-kinase activity.

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Silvia Volpe

Full Text Available BACKGROUND: Migration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization. METHODOLOGY/PRINCIPAL FINDINGS: We present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the alpha(2A-adrenoceptor (alpha(2AAR display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homologue UK 14'304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microscopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET of alpha(2AAR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinase activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes--in contrast to neutrophils--rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation. CONCLUSIONS/SIGNIFICANCE: Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the

Functional Defects in Type 3 Innate Lymphoid Cells and Classical Monocytes in a Patient with Hyper-IgE Syndrome.

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Chang, Yuna; Kang, Sung-Yoon; Kim, Jihyun; Kang, Hye-Ryun; Kim, Hye Young

2017-10-01

Hyper-IgE syndrome (HIES) is a very rare primary immune deficiency characterized by elevated serum IgE levels, recurrent bacterial infections, chronic dermatitis, and connective tissue abnormalities. Autosomal dominant (AD) HIES involves a mutation in signal transducer and activator of transcription 3 (STAT3) that leads to an impaired T H 17 response. STAT3 signaling is also involved in the function of RORγt + type 3 innate lymphoid cells (ILC3s) and RORγt + T H 17 cells. The aim of this study was to investigate the role of innate immune cells such as innate lymphoid cells (ILCs), granulocytes, and monocytes in a patient with HIES. Peripheral blood mononuclear cells (PBMCs) from a patient with HIES and three age-matched healthy controls were obtained for the analysis of the innate and adaptive immune cells. The frequencies of ILCs in PBMCs were lower in the patient with HIES than in the controls. Moreover, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-17A produced by ILC3s in PBMCs were lower in the patient with HIES than the controls. Compared with the controls, classical monocytes (CD14 + CD16 low ), which have a high antimicrobial capability, were also lower in the patient with HIES, while non-classical monocytes (CD14 low CD16 + ) as well as intermediate monocytes (CD14 + CD16 intermediate ) were higher. Taken together, these results indicate that the impaired immune defense against pathogenic microbes in the patient with HIES might be partially explained by functional defects in ILC3s and inflammatory monocytes.

The acute monocytic leukemias: multidisciplinary studies in 45 patients.

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Straus, D J; Mertelsmann, R; Koziner, B; McKenzie, S; de Harven, E; Arlin, Z A; Kempin, S; Broxmeyer, H; Moore, M A; Menendez-Botet, C J; Gee, T S; Clarkson, B D

1980-11-01

The clinical and laboratory features of 37 patients with variants of acute monocytic leukemia are described. Three of these 37 patients who had extensive extramedullary leukemic tissue infiltration are examples of true histiocytic "lymphomas." Three additional patients with undifferentiated leukemias, one patient with refractory anemia with excess of blasts, one patient with chronic myelomonocytic leukemia, one patient with B-lymphocyte diffuse "histiocytic" lymphoma and one patient with "null" cell, terminal deoxynucleotidyl transferase-positive lymphoblastic lymphoma had bone marrow cells with monocytic features. Another patient had dual populations of lymphoid and monocytoid leukemic cells. The true monocytic leukemias, acute monocytic leukemia (AMOL) and acute myelomonocytic leukemia (AMMOL), are closely related to acute myelocytic leukemia (AML) morphologically and by their response to chemotherapy. like AML, the leukemic cells from the AMMOL and AMOL patients form leukemic clusters in semisolid media. Cytochemical staining of leukemic cells for nonspecific esterases, presence of Fc receptor on the cell surface, phagocytic ability, low TdT activity, presence of surface "ruffles" and "ridges" on scanning EM, elevations of serum lysozyme, and clinical manifestations of leukemic tissue infiltration are features which accompanied monocytic differentiation in these cases.

Bone marrow chimeric mice reveal a role for CX₃CR1 in maintenance of the monocyte-derived cell population in the olfactory neuroepithelium.

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Vukovic, Jana; Blomster, Linda V; Chinnery, Holly R; Weninger, Wolfgang; Jung, Steffen; McMenamin, Paul G; Ruitenberg, Marc J

2010-10-01

Macrophages in the olfactory neuroepithelium are thought to play major roles in tissue homeostasis and repair. However, little information is available at present about possible heterogeneity of these monocyte-derived cells, their turnover rates, and the role of chemokine receptors in this process. To start addressing these issues, this study used Cx₃cr1(gfp) mice, in which the gene sequence for eGFP was knocked into the CX₃CR1 gene locus in the mutant allele. Using neuroepithelial whole-mounts from Cx₃cr1(gfp/+) mice, we show that eGFP(+) cells of monocytic origin are distributed in a loose network throughout this tissue and can be subdivided further into two immunophenotypically distinct subsets based on MHC-II glycoprotein expression. BM chimeric mice were created using Cx₃cr1(gfp/+) donors to investigate turnover of macrophages (and other monocyte-derived cells) in the olfactory neuroepithelium. Our data indicate that the monocyte-derived cell population in the olfactory neuroepithelium is actively replenished by circulating monocytes and under the experimental conditions, completely turned over within 6 months. Transplantation of Cx₃cr1(gfp/gfp) (i.e., CX₃CR1-deficient) BM partially impaired the replenishment process and resulted in an overall decline of the total monocyte-derived cell number in the olfactory epithelium. Interestingly, replenishment of the CD68(low)MHC-II(+) subset appeared minimally affected by CX₃CR1 deficiency. Taken together, the established baseline data about heterogeneity of monocyte-derived cells, their replenishment rates, and the role of CX₃CR1 provide a solid basis to further examine the importance of different monocyte subsets for neuroregeneration at this unique frontier with the external environment.

Comparative analysis of signature genes in porcine reproductive and respiratory syndrome virus (PRRSV)-infected porcine monocyte-derived dendritic cells at differential activation statuses

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Activation statuses of monocytic cells, e.g. monocytes, macrophages and dendritic cells (DCs), are critically important for antiviral immunity. In particular, some devastating viruses, including porcine reproductive and respiratory syndrome virus (PRRSV), are capable of directly infecting these cell...

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

Interleukin 17 receptor A modulates monocyte subsets and macrophage generation in vivo.

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Shuwang Ge

Full Text Available Interleukin (IL-17A signaling via Interleukin 17 receptor A (Il17ra contributes to the inflammatory host response by inducing recruitment of innate immune cells, but also plays a role in homeostatic neutrophilic granulocyte regulation. Monocytes, the other main innate immune cell, have a longer life span and can pursue multiple differentiation pathways towards tissue macrophages. Monocytes are divided into two subpopulations by expression of the Ly6C/Gr1 surface marker in mice. We here investigated the role of Il17ra in monocyte homeostasis and macrophage generation. In Il17ra(-/- and in mixed bone marrow chimeric wt/Il17ra(-/- mice, the concentrations of circulating Il17ra(-/- Gr1(low monocytes were significantly decreased compared to wt cells. Pulmonary, splenic and resident peritoneal Il17ra(-/- macrophages were significantly fewer than of wt origin. Bone marrow progenitor and monocyte numbers were equal, but the proportion of Il17ra(-/- Gr1(low monocytes was already decreased at bone marrow level. After monocyte depletion, initial Gr1(high and Gr1(low monocyte regeneration of Il17ra(-/- and wt cells was very similar. However, Il17ra(-/- Gr1(low counts were not sustained. After labeling with either fluorescent beads or BrdU, Il17ra(-/- Gr1(high monocyte transition to Gr1(low cells was not detectable unlike wt cells. Monocyte recruitment in acute peritonitis, which is known to be largely due to Gr1(high cell migration, was unaffected in an identical environment. Unilateral ureteral obstruction induces a less acute inflammatory and fibrotic kidney injury. Compared to wt cells in the same environment, Il17ra(-/- macrophage accumulation in the kidney was decreased. In the absence of Il17ra on all myeloid cells, renal fibrosis was significantly attenuated. Our data show that Il17ra modulates Gr1(low monocyte counts and suggest defective Gr1(high to Gr1(low monocyte transition as an underlying mechanism. Lack of Il17ra altered homeostatic tissue

Monocyte scintigraphy in rheumatoid arthritis: the dynamics of monocyte migration in immune-mediated inflammatory disease.

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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.

Induction of autophagy is essential for monocyte-macrophage differentiation

OpenAIRE

Zhang, Yan; Morgan, Michael J.; Chen, Kun; Choksi, Swati; Liu, Zheng-gang

2012-01-01

Monocytes are programmed to undergo apoptosis in the absence of stimulation. Stimuli that promote monocyte-macrophage differentiation not only cause cellular changes, but also prevent the default apoptosis of monocytes. In the present study, we demonstrate that autophagy is induced when monocytes are triggered to differentiate and that the induction of autophagy is pivotal for the survival and differentiation of monocytes. We also show that inhibition of autophagy results in apoptosis of cell...

Identification of a Monocyte Receptor on Herpesvirus-Infected Endothelial Cells

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Etingin, Orli R.; Silverstein, Roy L.; Hajjar, David P.

1991-08-01

The adhesion of circulating blood cells to vascular endothelium may be an initial step in atherosclerosis, inflammation, and wound healing. One mechanism for promoting cell-cell adhesion involves the expression of adhesion molecules on the surface of the target cell. Herpes simplex virus infection of endothelium induces arterial injury and has been implicated in the development of human atherosclerosis. We now demonstrate that HSV-infected endothelial cells express the adhesion molecule GMP140 and that this requires cell surface expression of HSV glycoprotein C and local thrombin generation. Monocyte adhesion to HSV-infected endothelial cells was completely inhibited by anti-GMP140 antibodies but not by antibodies to other adhesion molecules such as VCAM and ELAM-1. The induction of GMP140 expression on HSV-infected endothelium may be an important pathophysiological mechanism in virus-induced cell injury and inflammation.

Helicobacter pylori induces IL-1β and IL-18 production in human monocytic cell line through activation of NLRP3 inflammasome via ROS signaling pathway.

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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.

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

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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.

Heterogeneity of Bovine Peripheral Blood Monocytes

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Jamal Hussen

2017-12-01

human and bovine monocyte subsets are also reflected in their different clinical relevance for distinct diseases. In opposite to the human system, where higher blood cell number of non-classical monocytes was widely correlated with several human infectious and non-infectious diseases, higher counts of bovine intermediate monocytes are suggested as a potential biomarker for inflammatory responses postpartum.

Skin-resident CD4+ T cells protect against Leishmania major by recruiting and activating inflammatory monocytes

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Glennie, Nelson D.; Volk, Susan W.

2017-01-01

Tissue-resident memory T cells are required for establishing protective immunity against a variety of different pathogens, although the mechanisms mediating protection by CD4+ resident memory T cells are still being defined. In this study we addressed this issue with a population of protective skin-resident, IFNγ-producing CD4+ memory T cells generated following Leishmania major infection. We previously found that resident memory T cells recruit circulating effector T cells to enhance immunity. Here we show that resident memory CD4+ T cells mediate the delayed-hypersensitivity response observed in immune mice and provide protection without circulating T cells. This protection occurs rapidly after challenge, and requires the recruitment and activation of inflammatory monocytes, which limit parasites by production of both reactive oxygen species and nitric oxide. Overall, these data highlight a novel role for tissue-resident memory cells in recruiting and activating inflammatory monocytes, and underscore the central role that skin-resident T cells play in immunity to cutaneous leishmaniasis. PMID:28419151

Monocyte Trafficking, Engraftment, and Delivery of Nanoparticles and an Exogenous Gene into the Acutely Inflamed Brain Tissue - Evaluations on Monocyte-Based Delivery System for the Central Nervous System.

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Hsin-I Tong

Full Text Available The ability of monocytes and monocyte-derived macrophages (MDM to travel towards chemotactic gradient, traverse tissue barriers, and accumulate precisely at diseased sites makes them attractive candidates as drug carriers and therapeutic gene delivery vehicles targeting the brain, where treatments are often hampered by the blockade of the blood brain barrier (BBB. This study was designed to fully establish an optimized cell-based delivery system using monocytes and MDM, by evaluating their homing efficiency, engraftment potential, as well as carriage and delivery ability to transport nano-scaled particles and exogenous genes into the brain, following the non-invasive intravenous (IV cell adoptive transfer in an acute neuroinflammation mouse model induced by intracranial injection of Escherichia coli lipopolysaccharides. We demonstrated that freshly isolated monocytes had superior inflamed-brain homing ability over MDM cultured in the presence of macrophage colony stimulating factor. In addition, brain trafficking of IV infused monocytes was positively correlated with the number of adoptive transferred cells, and could be further enhanced by transient disruption of the BBB with IV administration of Mannitol, Bradykinin or Serotonin right before cell infusion. A small portion of transmigrated cells was detected to differentiate into IBA-1 positive cells with microglia morphology in the brain. Finally, with the use of superparamagnetic iron oxide nanoparticles SHP30, the ability of nanoscale agent-carriage monocytes to enter the inflamed brain region was validated. In addition, lentiviral vector DHIV-101 was used to introduce green fluorescent protein (GFP gene into monocytes, and the exogenous GFP gene was detected in the brain at 48 hours following IV infusion of the transduced monocytes. All together, our study has set up the optimized conditions for the more-in-depth tests and development of monocyte-mediated delivery, and our data supported

Osteopontin Prevents Monocyte Recirculation and Apoptosis

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Burdo, Tricia H.; Wood, Malcolm R.; Fox, Howard S.

2007-01-01

Cells of the monocyte/macrophage lineage have been shown to be the principal targets for productive HIV-1 replication within the central nervous system. In addition, HIV-1-associated dementia (HAD) has been shown to correlate with macrophage abundance in the brain. While increased entry of monocytes into the brain is thought to initiate this process, mechanisms that prevent macrophage egress from the brain and means that prevent macrophage death may also contribute to cell accumulation. We hy...

Stimulated monocyte IL-6 secretion predicts survival of patients with head and neck squamous cell carcinoma

International Nuclear Information System (INIS)

Heimdal, John-Helge; Kross, Kenneth; Klementsen, Beate; Olofsson, Jan; Aarstad, Hans Jørgen

2008-01-01

This study was performed in order to determine whether monocyte in vitro function is associated with presence, stage and prognosis of head and neck squamous cell carcinoma (HNSCC) disease. Prospective study describing outcome, after at least five years observation, of patients treated for HNSCC disease in relation to their monocyte function. Sixty-five patients with newly diagnosed HNSCC and eighteen control patients were studied. Monocyte responsiveness was assessed by measuring levels of monocyte in vitro interleukin (IL)-6 and monocyte chemotactic peptide (MCP)-1 secretion after 24 hours of endotoxin stimulation in cultures supplied either with 20% autologous serum (AS) or serum free medium (SFM). Survival, and if relevant, cause of death, was determined at least 5 years following primary diagnosis. All patients, as a group, had higher in vitro monocyte responsiveness in terms of IL-6 (AS) (t = 2.03; p < 0.05) and MCP-1 (SFM) (t = 2.49; p < 0.05) compared to controls. Increased in vitro monocyte IL-6 endotoxin responsiveness under the SFM condition was associated with decreased survival rate (Hazard ratio (HR) = 2.27; Confidence interval (CI) = 1.05–4.88; p < 0.05). The predictive value of monocyte responsiveness, as measured by IL-6, was also retained when adjusted for age, gender and disease stage of patients (HR = 2.67; CI = 1.03–6.92; p < 0.05). With respect to MCP-1, low endotoxin-stimulated responsiveness (AS), analysed by Kaplan-Meier method, predicted decreased survival (χ = 4.0; p < 0.05). In HNSCC patients, changed monocyte in vitro response to endotoxin, as measured by increased IL-6 (SFM) and decreased MCP-1 (AS) responsiveness, are negative prognostic factors

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

Peripherally administered nanoparticles target monocytic myeloid cells, secondary lymphoid organs and tumors in mice.

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Kourtis, Iraklis C; Hirosue, Sachiko; de Titta, Alexandre; Kontos, Stephan; Stegmann, Toon; Hubbell, Jeffrey A; Swartz, Melody A

2013-01-01

Nanoparticles have been extensively developed for therapeutic and diagnostic applications. While the focus of nanoparticle trafficking in vivo has traditionally been on drug delivery and organ-level biodistribution and clearance, recent work in cancer biology and infectious disease suggests that targeting different cells within a given organ can substantially affect the quality of the immunological response. Here, we examine the cell-level biodistribution kinetics after administering ultrasmall Pluronic-stabilized poly(propylene sulfide) nanoparticles in the mouse. These nanoparticles depend on lymphatic drainage to reach the lymph nodes and blood, and then enter the spleen rather than the liver, where they interact with monocytes, macrophages and myeloid dendritic cells. They were more readily taken up into lymphatics after intradermal (i.d.) compared to intramuscular administration, leading to ∼50% increased bioavailability in blood. When administered i.d., their distribution favored antigen-presenting cells, with especially strong targeting to myeloid cells. In tumor-bearing mice, the monocytic and the polymorphonuclear myeloid-derived suppressor cell compartments were efficiently and preferentially targeted, rendering this nanoparticulate formulation potentially useful for reversing the highly suppressive activity of these cells in the tumor stroma.

Peripherally administered nanoparticles target monocytic myeloid cells, secondary lymphoid organs and tumors in mice.

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Iraklis C Kourtis

Full Text Available Nanoparticles have been extensively developed for therapeutic and diagnostic applications. While the focus of nanoparticle trafficking in vivo has traditionally been on drug delivery and organ-level biodistribution and clearance, recent work in cancer biology and infectious disease suggests that targeting different cells within a given organ can substantially affect the quality of the immunological response. Here, we examine the cell-level biodistribution kinetics after administering ultrasmall Pluronic-stabilized poly(propylene sulfide nanoparticles in the mouse. These nanoparticles depend on lymphatic drainage to reach the lymph nodes and blood, and then enter the spleen rather than the liver, where they interact with monocytes, macrophages and myeloid dendritic cells. They were more readily taken up into lymphatics after intradermal (i.d. compared to intramuscular administration, leading to ∼50% increased bioavailability in blood. When administered i.d., their distribution favored antigen-presenting cells, with especially strong targeting to myeloid cells. In tumor-bearing mice, the monocytic and the polymorphonuclear myeloid-derived suppressor cell compartments were efficiently and preferentially targeted, rendering this nanoparticulate formulation potentially useful for reversing the highly suppressive activity of these cells in the tumor stroma.

Distinct Upstream Role of Type I IFN Signaling in Hematopoietic Stem Cell-Derived and Epithelial Resident Cells for Concerted Recruitment of Ly-6Chi Monocytes and NK Cells via CCL2-CCL3 Cascade.

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Erdenebileg Uyangaa

Full Text Available Type I interferon (IFN-I-dependent orchestrated mobilization of innate cells in inflamed tissues is believed to play a critical role in controlling replication and CNS-invasion of herpes simplex virus (HSV. However, the crucial regulators and cell populations that are affected by IFN-I to establish the early environment of innate cells in HSV-infected mucosal tissues are largely unknown. Here, we found that IFN-I signaling promoted the differentiation of CCL2-producing Ly-6Chi monocytes and IFN-γ/granzyme B-producing NK cells, whereas deficiency of IFN-I signaling induced Ly-6Clo monocytes producing CXCL1 and CXCL2. More interestingly, recruitment of Ly-6Chi monocytes preceded that of NK cells with the levels peaked at 24 h post-infection in IFN-I-dependent manner, which was kinetically associated with the CCL2-CCL3 cascade response. Early Ly-6Chi monocyte recruitment was governed by CCL2 produced from hematopoietic stem cell (HSC-derived leukocytes, whereas NK cell recruitment predominantly depended on CC chemokines produced by resident epithelial cells. Also, IFN-I signaling in HSC-derived leukocytes appeared to suppress Ly-6Ghi neutrophil recruitment to ameliorate immunopathology. Finally, tissue resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+ dendritic cells appeared to produce initial CCL2 for migration-based self-amplification of early infiltrated Ly-6Chi monocytes upon stimulation by IFN-I produced from infected epithelial cells. Ultimately, these results decipher a detailed IFN-I-dependent pathway that establishes orchestrated mobilization of Ly-6Chi monocytes and NK cells through CCL2-CCL3 cascade response of HSC-derived leukocytes and epithelium-resident cells. Therefore, this cascade response of resident-to-hematopoietic-to-resident cells that drives cytokine-to-chemokine-to-cytokine production to recruit orchestrated innate cells is critical for attenuation of HSV replication in inflamed tissues.

Fatty Acid Oxidation Compensates for Lipopolysaccharide-Induced Warburg Effect in Glucose-Deprived Monocytes

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Nora Raulien

2017-05-01

Full Text Available Monocytes enter sites of microbial or sterile inflammation as the first line of defense of the immune system and initiate pro-inflammatory effector mechanisms. We show that activation with bacterial lipopolysaccharide (LPS induces them to undergo a metabolic shift toward aerobic glycolysis, similar to the Warburg effect observed in cancer cells. At sites of inflammation, however, glucose concentrations are often drastically decreased, which prompted us to study monocyte function under conditions of glucose deprivation and abrogated Warburg effect. Experiments using the Seahorse Extracellular Flux Analyzer revealed that limited glucose supply shifts monocyte metabolism toward oxidative phosphorylation, fueled largely by fatty acid oxidation at the expense of lipid droplets. While this metabolic state appears to provide sufficient energy to sustain functional properties like cytokine secretion, migration, and phagocytosis, it cannot prevent a rise in the AMP/ATP ratio and a decreased respiratory burst. The molecular trigger mediating the metabolic shift and the functional consequences is activation of AMP-activated protein kinase (AMPK. Taken together, our results indicate that monocytes are sufficiently metabolically flexible to perform pro-inflammatory functions at sites of inflammation despite glucose deprivation and inhibition of the LPS-induced Warburg effect. AMPK seems to play a pivotal role in orchestrating these processes during glucose deprivation in monocytes.

Aminopeptidase N/CD13 is associated with raft membrane microdomains in monocytes

DEFF Research Database (Denmark)

Navarrete Santos, A; Roentsch, J; Danielsen, E M

2000-01-01

as in adhesion and cell-cell interactions. Here, we report for the first time that aminopeptidase N/CD13 in monocytes is partially localized in detergent-insoluble membrane microdomains enriched in cholesterol, glycolipids, and glycosylphosphoinositol-anchored proteins, referred to as "rafts." Raft fractions...... of monocytes were characterized by the presence of GM1 ganglioside as raft marker molecule and by the high level of tyrosine-phosphorylated proteins. Furthermore, similar to polarized cells, rafts in monocytic cells lack Na(+), K(+)-ATPase. Cholesterol depletion of monocytes by methyl-beta-cyclodextrin greatly...... reduces raft localization of aminopeptidase N/CD13 without affecting ala-p-nitroanilide cleaving activity of cells....

Edaravone attenuates monocyte adhesion to endothelial cells induced by oxidized low-density lipoprotein.

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Li, Zhijuan; Cheng, Jianxin; Wang, Liping

2015-10-30

Oxidized low-density lipoprotein (oxLDL) plays a vital role in recruitment of monocytes to endothelial cells, which is important during early stages of atherosclerosis development. Edaravone, a potent and novel scavenger of free radicals inhibiting hydroxyl radicals, has been clinically used to reduce the neuronal damage following ischemic stroke. In the present study, Edaravone was revealed to markedly reduce oxLDL-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). The inhibitory mechanism of Edaravone was associated with suppression of the chemokine MCP-1 and adhesion molecule VCAM-1 and ICAM-1 expression. In addition, luciferase reporter assay results revealed that administration of Edaravone attenuated the increase in NF-κB transcriptional activity induced by oxLDL. Notably, it's also shown that Edaravone treatment blocked oxLDL induced p65 nuclear translocation in HUVECs. Results indicate that Edaravone negatively regulates endothelial inflammation. Copyright © 2015. Published by Elsevier Inc.

Systems Biology Reveals Cigarette Smoke-Induced Concentration-Dependent Direct and Indirect Mechanisms That Promote Monocyte-Endothelial Cell Adhesion.

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Poussin, Carine; Laurent, Alexandra; Peitsch, Manuel C; Hoeng, Julia; De Leon, Hector

2015-10-01

Cigarette smoke (CS) affects the adhesion of monocytes to endothelial cells, a critical step in atherogenesis. Using an in vitro adhesion assay together with innovative computational systems biology approaches to analyze omics data, our study aimed at investigating CS-induced mechanisms by which monocyte-endothelial cell adhesion is promoted. Primary human coronary artery endothelial cells (HCAECs) were treated for 4 h with (1) conditioned media of human monocytic Mono Mac-6 (MM6) cells preincubated with low or high concentrations of aqueous CS extract (sbPBS) from reference cigarette 3R4F for 2 h (indirect treatment, I), (2) unconditioned media similarly prepared without MM6 cells (direct treatment, D), or (3) freshly generated sbPBS (fresh direct treatment, FD). sbPBS promoted MM6 cells-HCAECs adhesion following I and FD, but not D. In I, the effect was mediated at a low concentration through activation of vascular inflammation processes promoted in HCAECs by a paracrine effect of the soluble mediators secreted by sbPBS-treated MM6 cells. Tumor necrosis factor α (TNFα), a major inducer, was actually shed by unstable CS compound-activated TNFα-converting enzyme. In FD, the effect was triggered at a high concentration that also induced some toxicity. This effect was mediated through an yet unknown mechanism associated with a stress damage response promoted in HCAECs by unstable CS compounds present in freshly generated sbPBS, which had decayed in D unconditioned media. Aqueous CS extract directly and indirectly promotes monocytic cell-endothelial cell adhesion in vitro via distinct concentration-dependent mechanisms. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

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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.

The proliferative human monocyte subpopulation contains osteoclast precursors

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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

Sialoadhesin expressed on IFN-induced monocytes binds HIV-1 and enhances infectivity.

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Hans Rempel

2008-04-01

Full Text Available HIV-1 infection dysregulates the immune system and alters gene expression in circulating monocytes. Differential gene expression analysis of CD14(+ monocytes from subjects infected with HIV-1 revealed increased expression of sialoadhesin (Sn, CD169, Siglec 1, a cell adhesion molecule first described in a subset of macrophages activated in chronic inflammatory diseases.We analyzed sialoadhesin expression on CD14(+ monocytes by flow cytometry and found significantly higher expression in subjects with elevated viral loads compared to subjects with undetectable viral loads. In cultured CD14(+ monocytes isolated from healthy individuals, sialoadhesin expression was induced by interferon-alpha and interferon-gamma but not tumor necrosis factor-alpha. Using a stringent binding assay, sialoadhesin-expressing monocytes adsorbed HIV-1 through interaction with the sialic acid residues on the viral envelope glycoprotein gp120. Furthermore, monocytes expressing sialoadhesin facilitated HIV-1 trans infection of permissive cells, which occurred in the absence of monocyte self-infection.Increased sialoadhesin expression on CD14(+ monocytes occurred in response to HIV-1 infection with maximum expression associated with high viral load. We show that interferons induce sialoadhesin in primary CD14(+ monocytes, which is consistent with an antiviral response during viremia. Our findings suggest that circulating sialoadhesin-expressing monocytes are capable of binding HIV-1 and effectively delivering virus to target cells thereby enhancing the distribution of HIV-1. Sialoadhesin could disseminate HIV-1 to viral reservoirs during monocyte immunosurveillance or migration to sites of inflammation and then facilitate HIV-1 infection of permissive cells.

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.

Association of Canine Osteosarcoma and Monocyte Phenotype and Chemotactic Function.

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Tuohy, J L; Lascelles, B D X; Griffith, E H; Fogle, J E

2016-07-01

Monocytes/macrophages are likely key cells in immune modulation in dogs with osteosarcoma (OSA). Increased peripheral monocyte counts are negatively correlated with shorter disease-free intervals in dogs with OSA. Understanding the monocyte/macrophage's modulatory role in dogs with OSA can direct further studies in immunotherapy development for OSA. That OSA evades the immune response by down-regulating monocyte chemokine receptor expression and migratory function, and suppresses host immune responses. Eighteen dogs with OSA that have not received definitive treatment and 14 healthy age-matched controls Clinical study-expression of peripheral blood monocyte cell surface receptors, monocyte mRNA expression and cytokine secretion, monocyte chemotaxis, and survival were compared between clinical dogs with OSA and healthy control dogs. Cell surface expression of multiple chemokine receptors is significantly down-regulated in peripheral blood monocytes of dogs with OSA. The percentage expression of CCR2 (median 58%, range 2-94%) and CXCR2 expression (median 54%, range 2-92%) was higher in control dogs compared to dogs with OSA (CCR2 median 29%, range 3-45%, P = 0.0006; CXCR2 median 23%, range 0.2-52%, P = 0.0007). Prostaglandin E2 (PGE2 ) (OSA, median 347.36 pg/mL, range 103.4-1268.5; control, 136.23 pg/mL, range 69.93-542.6, P = .04) and tumor necrosis factor-alpha (TNF-α) (P = .02) levels are increased in OSA monocyte culture supernatants compared to controls. Peripheral blood monocytes of dogs with OSA exhibit decreased chemotactic function when compared to control dogs (OSA, median 1.2 directed to random migration, range 0.8-1.25; control, 1.6, range of 0.9-1.8, P = .018). Dogs with OSA have decreased monocyte chemokine receptor expression and monocyte chemotaxis, potential mechanisms by which OSA might evade the immune response. Reversal of monocyte dysfunction using immunotherapy could improve survival in dogs with OSA. Copyright © 2016 The Authors. Journal of

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

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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.

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.

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

Platelet density per monocyte predicts adverse events in patients after percutaneous coronary intervention.

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Rutten, Bert; Roest, Mark; McClellan, Elizabeth A; Sels, Jan W; Stubbs, Andrew; Jukema, J Wouter; Doevendans, Pieter A; Waltenberger, Johannes; van Zonneveld, Anton-Jan; Pasterkamp, Gerard; De Groot, Philip G; Hoefer, Imo E

2016-01-01

Monocyte recruitment to damaged endothelium is enhanced by platelet binding to monocytes and contributes to vascular repair. Therefore, we studied whether the number of platelets per monocyte affects the recurrence of adverse events in patients after percutaneous coronary intervention (PCI). Platelet-monocytes complexes with high and low median fluorescence intensities (MFI) of the platelet marker CD42b were isolated using cell sorting. Microscopic analysis revealed that a high platelet marker MFI on monocytes corresponded with a high platelet density per monocyte while a low platelet marker MFI corresponded with a low platelet density per monocyte (3.4 ± 0.7 vs 1.4 ± 0.1 platelets per monocyte, P=0.01). Using real-time video microscopy, we observed increased recruitment of high platelet density monocytes to endothelial cells as compared with low platelet density monocytes (P=0.01). Next, we classified PCI scheduled patients (N=263) into groups with high, medium and low platelet densities per monocyte and assessed the recurrence of adverse events. After multivariate adjustment for potential confounders, we observed a 2.5-fold reduction in the recurrence of adverse events in patients with a high platelet density per monocyte as compared with a low platelet density per monocyte [hazard ratio=0.4 (95% confidence interval, 0.2-0.8), P=0.01]. We show that a high platelet density per monocyte increases monocyte recruitment to endothelial cells and predicts a reduction in the recurrence of adverse events in patients after PCI. These findings may imply that a high platelet density per monocyte protects against recurrence of adverse events.

Fc receptors for mouse IgG1 on human monocytes: polymorphism and role in antibody-induced T cell proliferation.

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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

Effects of acute exercise on monocyte subpopulations in metabolic syndrome patients.

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Wonner, Ralph; Wallner, Stefan; Orsó, Evelyn; Schmitz, Gerd

2016-06-10

Acute exercise induces numerous changes in peripheral blood, e.g. counts of leukocytes. CD16 pos monocytes, which play a role in the pathogenesis of arteriosclerosis and the metabolic syndrome (MetS), are among the blood cells with the highest fold increase through exercise. So far no studies have investigated the effect of exercise on the blood cell composition of patients with MetS. Blood cell counts, a wide panel of laboratory tests, as well as lipid and protein content of monocytes and granulocytes were determined in healthy subjects, persons with metabolic risk and MetS patients before and after one minute of exercise at 400 W. Leukocyte counts increased significantly in all groups with CD14 pos CD16 pos monocytes showing the highest fold-change. In MetS patients the fold increase was smaller. They had a higher resting level of CD14 pos CD16 pos monocytes and a lower basal ratio of CD16 neg /CD16 pos monocytes. A similar ratio of these cells was induced in control and risk subjects after exercise. However, absolute counts of mobilized pro-inflammatory monocytes did not differ significantly. Furthermore, we detected a decrease in protein content of monocytes in controls, but not in MetS patients. As strenuous exercise is able to mobilize the same amount of pro-inflammatory monocytes in MetS patients as in healthy persons, the elevated basal level of these cells in MetS patients is likely to be caused by enhanced maturation rather than chronic mobilization. The removal of these monocytes from the endothelium might be part of the beneficial effect of exercise on vascular disease. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

M1 and M2 Monocytes in Rheumatoid Arthritis: A Contribution of Imbalance of M1/M2 Monocytes to Osteoclastogenesis

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Shoichi Fukui

2018-01-01

Full Text Available ObjectivesWe investigated the relationships among M1 monocytes, M2 monocytes, osteoclast (OC differentiation ability, and clinical characteristics in patients with rheumatoid arthritis (RA.MethodsPeripheral blood mononuclear cells (PBMCs were isolated from RA patients and healthy donors, and we then investigated the number of M1 monocytes or M2 monocytes by fluorescence-activated cell sorting. We also obtained and cultured CD14-positive cells from PBMCs from RA patients and healthy donors to investigate OC differentiation in vitro.ResultsForty RA patients and 20 healthy donors were included. Twenty-two patients (55% were anticitrullinated protein antibody (ACPA positive. The median M1/M2 ratio was 0.59 (0.31–1.11, interquartile range. There were no significant differences between the RA patients and healthy donors. There was a positive correlation between the M1/M2 ratio and the differentiated OC number in vitro in RA patients (ρ = 0.81, p < 0.001. The ACPA-positive patients had significantly higher M1/M2 ratios in vivo (p = 0.028 and significantly greater numbers of OCs in vitro (p = 0.005 than the ACPA-negative patients. Multivariable regression analysis revealed that the M1/M2 ratio was the sole significant contribution factor to in vitro osteoclastogenesis. RA patients with M1/M2 ratios >1 (having relatively more M1 monocytes had higher C-reactive protein and erythrocyte sedimentation rates than RA patients with M1/M2 ratios ≤1. M1-dominant monocytes in vitro produced higher concentrations of interleukin-6 upon stimulation with lipopolysaccharide than M2 monocytes.ConclusionM1/M2 monocytes imbalance strongly contributes to osteoclastogenesis of RA patients. Our findings cast M1 and M2 monocyte subsets in a new light as a new target of treatments for RA to prevent progression of osteoclastic bone destruction.

CD14+CD16+ monocytes are the main target of Zika virus infection in peripheral blood mononuclear cells in a paediatric study in Nicaragua.

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Michlmayr, Daniela; Andrade, Paulina; Gonzalez, Karla; Balmaseda, Angel; Harris, Eva

2017-11-01

The recent Zika pandemic in the Americas is linked to congenital birth defects and Guillain-Barré syndrome. White blood cells (WBCs) play an important role in host immune responses early in arboviral infection. Infected WBCs can also function as 'Trojan horses' and carry viruses into immune-sheltered spaces, including the placenta, testes and brain. Therefore, defining which WBCs are permissive to Zika virus (ZIKV) is critical. Here, we analyse ZIKV infectivity of peripheral blood mononuclear cells (PBMCs) in vitro and from Nicaraguan Zika patients and show CD14 + CD16 + monocytes are the main target of infection, with ZIKV replication detected in some dendritic cells. The frequency of CD14 + monocytes was significantly decreased, while the CD14 + CD16 + monocyte population was significantly expanded during ZIKV infection compared to uninfected controls. Viral RNA was detected in PBMCs from all patients, but in serum from only a subset, suggesting PBMCs may be a reservoir for ZIKV. In Zika patients, the frequency of infected cells was lower but the percentage of infected CD14 + CD16 + monocytes was significantly higher compared to dengue cases. The gene expression profile in monocytes isolated from ZIKV- and dengue virus-infected patients was comparable, except for significant differences in interferon-γ, CXCL12, XCL1, interleukin-6 and interleukin-10 levels. Thus, our study provides a detailed picture of the innate immune profile of ZIKV infection and highlights the important role of monocytes, and CD14 + CD16 + monocytes in particular.

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...

Stimulation of monocytes by placental microparticles involves Toll-like receptors and nuclear factor kappa-light-chain-enhancer of activated B cells

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Marianne Simone Joerger-Messerli

2014-04-01

Full Text Available Human pregnancy is accompanied by a mild systemic inflammatory response, which includes the activation of monocytes circulating in maternal blood. This response is exaggerated in preeclampsia, a placental-dependent disorder specific to human pregnancies. We and others showed that placental syncytiotrophoblast membrane microparticles (STBM generated in vitro from normal placentas stimulated peripheral blood monocytes, which suggests a contribution of STBM to the systemic maternal inflammation. Here, we analyzed the inflammatory potential of STBM prepared from preeclamptic placentas on primary monocytes and investigated the mode of action in vitro.STBM generated in vitro by placental villous explants of normal or preeclamptic placentas were co-incubated with human peripheral blood monocytes. In some cases, inhibitors of specific cellular functions or signaling pathways were used. The analysis of the monocytic response was performed by flow cytometry, enzyme-linked immunoassays, real-time PCR and fluorescence microscopy.STBM derived from preeclamptic placentas up-regulated the cell surface expression of CD54, and stimulated the secretion of the pro-inflammatory interleukin (IL-6 and IL-8 in a similar, dose-dependent manner as did STBM prepared from normal placentas. STBM bound to the cell surface of monocytes, but phagocytosis was not necessary for activation. STBM-induced cytokine secretion was impaired in the presence of inhibitors of toll-like receptor (TLR signaling or when nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB activation was blocked.Our results suggest that the inflammatory reaction in monocytes may be initiated by the interaction of STBM with TLRs, which in turn signal through NF-κB to mediate the transcription of genes coding for pro-inflammatory factors.

Periodontitis-activated monocytes/macrophages cause aortic inflammation

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Miyajima, Shin-ichi; Naruse, Keiko; Kobayashi, Yasuko; Nakamura, Nobuhisa; Nishikawa, Toru; Adachi, Kei; Suzuki, Yuki; Kikuchi, Takeshi; Mitani, Akio; Mizutani, Makoto; Ohno, Norikazu; Noguchi, Toshihide; Matsubara, Tatsuaki

2014-01-01

A relationship between periodontal disease and atherosclerosis has been suggested by epidemiological studies. Ligature-induced experimental periodontitis is an adequate model for clinical periodontitis, which starts from plaque accumulation, followed by inflammation in the periodontal tissue. Here we have demonstrated using a ligature-induced periodontitis model that periodontitis activates monocytes/macrophages, which subsequently circulate in the blood and adhere to vascular endothelial cells without altering the serum TNF-α concentration. Adherent monocytes/macrophages induced NF-κB activation and VCAM-1 expression in the endothelium and increased the expression of the TNF-α signaling cascade in the aorta. Peripheral blood-derived mononuclear cells from rats with experimental periodontitis showed enhanced adhesion and increased NF-κB/VCAM-1 in cultured vascular endothelial cells. Our results suggest that periodontitis triggers the initial pathogenesis of atherosclerosis, inflammation of the vasculature, through activating monocytes/macrophages. PMID:24893991

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

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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

Differential expression of the ufo/axl oncogene in human leukemia-lymphoma cell lines.

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Challier, C; Uphoff, C C; Janssen, J W; Drexler, H G

1996-05-01

The ufo protein (also termed axl) is a member of a new family of receptor tyrosine kinases and is encoded by a transforming gene that was initially isolated from primary human myeloid leukemia cells by DNA-mediated transformation of NIH/3T3 cells. The ligand, Gas6, a protein S-related molecule lacking any known function yet, has recently been identified. We report the expression pattern of ufo mRNA in a panel of 76 human continuous leukemia-lymphoma cell lines. The gene was not expressed in cell lines derived from lymphoid malignancies (n=28), but transcription was seen in 3/11 myeloid, 0/6 monocytic, 9/13 erythroid and 11/18 megakaryocytic cell lines. Several cell lines were treated with phorbol ester leading to significant upregulation of the ufo message in constitutively positive cells. An apparent ufo mRNA overexpression was not found in any of the positive leukemia cell lines, but was identified in the drug-resistant subclones of the cervix carcinoma cell line HeLa. Southern blot analysis of restriction enzyme-digested genomic DNA did not provide evidence for gene amplification, but the HeLa subclones showed banding patterns suggestive of gene rearrangement. Two main ufo mRNA bands of 3.2 and 5.0 kb were identified; no differences in the half-lives (t1/2 = 2.5 h) of these two mRNA species could be identified. In summary, ufo, representing a novel type of receptor tyrosine kinase, is expressed solely in myeloid and erythro-megakaryocytic leukemias but not in lymphoid malignancies. These and previous data suggest an involvement of the ufo receptor tyrosine kinase in normal and malignant myelopoiesis; however, its exact role, if any, and mode of operation in leukemogenesis remains to be determined.

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

A simple method for human peripheral blood monocyte Isolation

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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.

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.

Dengue Virus-Infected Dendritic Cells, but Not Monocytes, Activate Natural Killer Cells through a Contact-Dependent Mechanism Involving Adhesion Molecules.

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Costa, Vivian Vasconcelos; Ye, Weijian; Chen, Qingfeng; Teixeira, Mauro Martins; Preiser, Peter; Ooi, Eng Eong; Chen, Jianzhu

2017-08-01

Natural killer (NK) cells play a protective role against dengue virus (DENV) infection, but the cellular and molecular mechanisms are not fully understood. Using an optimized humanized mouse model, we show that human NK cells, through the secretion of gamma interferon (IFN-γ), are critical in the early defense against DENV infection. Depletion of NK cells or neutralization of IFN-γ leads to increased viremia and more severe thrombocytopenia and liver damage in humanized mice. In vitro studies using autologous human NK cells show that DENV-infected monocyte-derived dendritic cells (MDDCs), but not monocytes, activate NK cells in a contact-dependent manner, resulting in upregulation of CD69 and CD25 and secretion of IFN-γ. Blocking adhesion molecules (LFA-1, DNAM-1, CD2, and 2β4) on NK cells abolishes NK cell activation, IFN-γ secretion, and the control of DENV replication. NK cells activated by infected MDDCs also inhibit DENV infection in monocytes. These findings show the essential role of human NK cells in protection against acute DENV infection in vivo , identify adhesion molecules and dendritic cells required for NK cell activation, and delineate the sequence of events for NK cell activation and protection against DENV infection. IMPORTANCE Dengue is a mosquito-transmitted viral disease with a range of symptoms, from mild fever to life-threatening dengue hemorrhagic fever. The diverse disease manifestation is thought to result from a complex interplay between viral and host factors. Using mice engrafted with a human immune system, we show that human NK cells inhibit virus infection through secretion of the cytokine gamma interferon and reduce disease pathogenesis, including depletion of platelets and liver damage. During a natural infection, DENV initially infects dendritic cells in the skin. We find that NK cells interact with infected dendritic cells through physical contact mediated by adhesion molecules and become activated before they can control

High-dose vitamin D in Addison's disease regulates T-cells and monocytes: A pilot trial.

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Penna-Martinez, Marissa; Filmann, Natalie; Bogdanou, Dimitra; Shoghi, Firouzeh; Huenecke, Sabine; Schubert, Ralf; Herrmann, Eva; Koehl, Ulrike; Husebye, Eystein S; Badenhoop, Klaus

2018-05-01

On the basis of the immunomodulatory actions of vitamin D (VD), we investigated the effects of high-dose VD therapy over a 3 mo period on the immune response in patients with Addison's disease (AD). This randomized, controlled, crossover trial included 13 patients with AD who received either cholecalciferol (4000 IU/d) for 3 mo followed by 3 mo placebo oil or the sequential alternative placebo followed by verum. Glucocorticoid replacement doses remained stable. The primary outcome measures were changes in 25-hydroxyvitamin D3 (25(OH)D 3 ) levels and immune cells including T helper cells (Th; CD3 + CD4 + ), late-activated Th cells (CD3 + CD4 + HLA-DR + ), regulatory T cells (CD3 + CD4 + CD25 bright CD127 dim/neg ), cytotoxic T cells (Tc; CD3 + CD8 + ), late-activated Tc cells (CD3 + CD8 + HLA-DR + ), and monocytes. The explorative analysis included the correlation of changes with VD-related gene polymorphisms and 21-hydroxylase antibody titers. Ten of 13 patients (77%) were VD deficient. Median 25(OH)D 3 concentrations increased significantly to 41.5 ng/ml (median changes: 19.95 ng/ml; P = 0.0005) after 3 mo of cholecalciferol treatment. Within the T-cells, only the late-activated Th (median changes: 1.6%; P = 0.02) and late-activated Tc cells (median changes: 4.05%; P = 0.03) decreased, whereas monocytes (median changes: 1.05%; P = 0.008) increased after VD therapy. T-cell changes were associated with two polymorphisms (CYP27B1-rs108770012 and VDR-rs10735810), but no changes in the 21-hydroxylase antibody titers were observed. Three months of treatment with cholecalciferol achieved sufficient 25(OH)D 3 levels and can regulate late-activated T-cells and monocytes in patients with AD. Explorative analysis revealed potential genetic contributions. This pilot trial provides novel insights about immunomodulation in AD. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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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.

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

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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

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.

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

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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.

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.

Umbilical Cord-derived Mesenchymal Stem Cells Instruct Monocytes Towards an IL10-producing Phenotype by Secreting IL6 and HGF.

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Deng, Yinan; Zhang, Yingcai; Ye, Linsen; Zhang, Tong; Cheng, Jintao; Chen, Guihua; Zhang, Qi; Yang, Yang

2016-12-05

Human UC-MSCs are regarded as an attractive alternative to BM-MSCs for clinical applications due to their easy preparation, higher proliferation and lower immunogenicity. However, the mechanisms underlying immune suppression by UC-MSCs are still unclear. We studied the mechanism of inhibition by UC-MSCs during the differentiation of monocytes into DCs and focused on the specific source and the role of the involved cytokines. We found that UC-MSCs suppressed monocyte differentiation into DCs and instructed monocytes towards other cell types, with clear decreases in the expression of co-stimulatory molecules, in the secretion of inflammatory factors and in allostimulatory capacity. IL6, HGF and IL10 might be involved in this process because they were detected at higher levels in a coculture system. UC-MSCs produce IL-6 and HGF, and neutralization of IL-6 and HGF reversed the suppressive effect of UC-MSCs. IL10 was not produced by UC-MSCs but was exclusively produced by monocytes after exposure to UC-MSCs, IL-6 or HGF. In summary, we found that the UC-MSC-mediated inhibitory effect was dependent on IL6 and HGF secreted by UC-MSCs and that this effect induced monocyte-derived cells to produce IL10, which might indirectly strengthen the suppressive effect of UC-MSCs.

Distinct functional programming of human fetal and adult monocytes.

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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.

A distinguishing gene signature shared by tumor-infiltrating Tie2-expressing monocytes, blood "resident" monocytes, and embryonic macrophages suggests common functions and developmental relationships.

Science.gov (United States)

Pucci, Ferdinando; Venneri, Mary Anna; Biziato, Daniela; Nonis, Alessandro; Moi, Davide; Sica, Antonio; Di Serio, Clelia; Naldini, Luigi; De Palma, Michele

2009-07-23

We previously showed that Tie2-expressing monocytes (TEMs) have nonredundant proangiogenic activity in tumors. Here, we compared the gene expression profile of tumor-infiltrating TEMs with that of tumor-associated macrophages (TAMs), spleen-derived Gr1(+)Cd11b(+) neutrophils/myeloid-derived suppressor cells, circulating "inflammatory" and "resident" monocytes, and tumor-derived endothelial cells (ECs) by quantitative polymerase chain reaction-based gene arrays. TEMs sharply differed from ECs and Gr1(+)Cd11b(+) cells but were highly related to TAMs. Nevertheless, several genes were differentially expressed between TEMs and TAMs, highlighting a TEM signature consistent with enhanced proangiogenic/tissue-remodeling activity and lower proinflammatory activity. We validated these findings in models of oncogenesis and transgenic mice expressing a microRNA-regulated Tie2-GFP reporter. Remarkably, resident monocytes and TEMs on one hand, and inflammatory monocytes and TAMs on the other hand, expressed coordinated gene expression profiles, suggesting that the 2 blood monocyte subsets are committed to distinct extravascular fates in the tumor microenvironment. We further showed that a prominent proportion of embryonic/fetal macrophages, which participate in tissue morphogenesis, expressed distinguishing TEM genes. It is tempting to speculate that Tie2(+) embryonic/fetal macrophages, resident blood monocytes, and tumor-infiltrating TEMs represent distinct developmental stages of a TEM lineage committed to execute physiologic proangiogenic and tissue-remodeling programs, which can be co-opted by tumors.

REGULATION OF TLR/RLR GENE ACTIVITY AND SYNTHESIS OF CYTOKINES DURING PHORBOL MYRISTATE ACETATE (PMA-INDUCED DIFFERENTIATION OF THP-1 MONOCYTES INTO MACROPHAGE-LIKE CELLS

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T. M. Sokolova

2017-01-01

Full Text Available The levels of TLR/RLR gene expression and production of some cytokines were studied in monocytic THP-1 cell line during its differentiation to mature macrophage-like forms induced by phorbol 12-myristate 13-acetate (PMA treatment for 1 and 5 days in vitro. For the first time, we have shown high induction levels for the genes that encode signaling immune receptors and transcription factors in response to PMA, as well as inhibitory effects of TLR3, TLR7/TLR8, TLR9-agonists in mature macrophages. The PMAactivated THP-1 macrophage-like cells secreted large quantitities of inflammatory IL-1β and TNFα cytokines into culture medium.

Innate immune responses of equine monocytes cultured in equine platelet lysate.

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Naskou, Maria C; Norton, Natalie A; Copland, Ian B; Galipeau, Jacques; Peroni, John F

2018-01-01

Platelet lysate (PL) has been extensively used for the laboratory expansion of human mesenchymal stem cells (MSC) in order to avoid fetal bovine serum (FBS) which has been associated with immune-mediated host reactions and transmission of bovine-origin microbial contaminants. Before suggesting the routine use of PL for MSC culture, we wanted to further investigate whether PL alone might trigger inflammatory responses when exposed to reactive white blood cells such as monocytes. Our objectives were to evaluate the inflammatory profile of equine monocytes cultured with equine PL (ePL) and to determine if ePL can modulate the expression of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated monocytes. In a first experiment, equine monocytes were isolated and incubated with donor horse serum (DHS), FBS, six individual donors ePL or pooled ePL from all horses. In a second experiment, monocytes were stimulated with E. coli LPS in the presence of 1, 5 or 10% DHS and/or pooled ePL. After 6h of incubation, cell culture supernatants were assayed via ELISA for production of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and Interleukin 1β (IL-1β) as well as for the anti-inflammatory Interleukin 10 (IL-10). Equine monocytes incubated with pooled ePL produced significantly less TNF-α and significantly more IL-10 than monocytes incubated in FBS. A statistically significant difference was not identified for the production of IL-1β. The second experiment showed that pooled ePL added to LPS-stimulated equine monocytes resulted in a significant reduction in TNF-α and IL-1β production. IL-10 production was not significantly upregulated by the addition of ePL to LPS-stimulated monocytes. Finally, the addition of ePL to LPS-stimulated monocytes in the presence of various concentrations of DHS resulted to statistically significant decrease of TNF-α and IL-1β compared to the control groups. This is the first study to demonstrate that ePL suppresses

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.

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

HL60 human premyelocitic cell line as a model system for bystander response

International Nuclear Information System (INIS)

Dini, V.; Tabocchini, M.A.; Belli, M.; Simone, G.; Di Carlo, B.; Sapora, O.; Superiore di Sanita, Rome

2007-01-01

Complete text of publication follows. Objective: to evaluate HL60 human premyelocitic cell line as a model system to study bystander response. Methods: HL60 cell line, isolated from the blood of a patient affected by premyelocitic leukemia, has 45-46 chromosomes with abnormalities mainly on chromosomes 5, 8 and X and can undergo chemical-induced in vitro differentiation. Differentiation gives rise to granulocytes, monocytes or macrophages depending on the drug used. We define as proliferative (AP) cells those in log phase of growth with less than 10 passages from thawing and as differentiated (D) cells those treated with 10 nM TPA (phorbol ester) for 72 hours. Phorbol ester treatment induces differentiation to monocytes and macrophages. Differentiation has been evaluated through the expression of differentiation cluster membrane antigens (CD95, CD9 and CD14). Results: AP cells resulted positive for CD95 and negative for CD9 and CD14, while D cells resulted positive for CD9 and negative for CD95 and CD14. Our data on AP and D cells showed that: (i) the level of intracellular reactive oxygen and nitrogen species (ROS and RNS) is lower in D cells compared to AP cells; (ii) radiation induced DNA damage (single and double strand breaks, SSB and DSB, as measured with the comet assay technique) is lower in D cells than in AP cells. This different radiosensitivity can be related to the higher degree of compactness of nuclear structure in D cells. Radiation induced bystander effect (BE) was analyzed with the medium transfer technique. The medium from irradiated, with 0.5 Gy of γ-rays, AP cells was collected after 0, 2, 4 and 24 hours from irradiation and added to non irradiated log phase cells. The frequency of micronuclei formation in bystander cells was measured by using the cytokinesis block technique by adding cytochalasin B to the non irradiated culture together with the irradiated medium. Preliminary data indicate about 1.4-fold increase in micronuclei formation in

Generation of dendritic cells for immunotherapy is minimally impaired by granulocytes in the monocyte preparation

NARCIS (Netherlands)

ten Brinke, Anja; Karsten, Miriam L.; Dieker, Miranda C.; Zwaginga, Jaap Jan; Vrielink, Hans; van Ham, S. Marieke

2006-01-01

The growing number of clinical studies, using monocyte-derived DC therapy, requires protocols where a sufficient number of dendritic cell (DCs) are produced according to current Good Manufacturing Practice guidelines. Therefore, a closed culture system for the generation of DCs is inevitable. One

Delivery of TLR7 agonist to monocytes and dendritic cells by DCIR targeted liposomes induces robust production of anti-cancer cytokines

DEFF Research Database (Denmark)

Klauber, Thomas Christopher Bogh; Laursen, Janne Marie; Zucker, Daniel

2017-01-01

Tumor immune escape is today recognized as an important cancer hallmark and is therefore a major focus area in cancer therapy. Monocytes and dendritic cells (DCs), which are central to creating a robust anti-tumor immune response and establishing an anti-tumorigenic microenvironment, are directly...... targeted by the tumor escape mechanisms to develop immunosuppressive phenotypes. Providing activated monocytes and DCs to the tumor tissue is therefore an attractive way to break the tumor-derived immune suppression and reinstate cancer immune surveillance. To activate monocytes and DCs with high...... as their immune activating potential in blood-derived monocytes, myeloid DCs (mDCs), and plasmacytoid DCs (pDCs). Monocytes and mDCs were targeted with high specificity over lymphocytes, and exhibited potent TLR7-specific secretion of the anti-cancer cytokines IL-12p70, IFN-α 2a, and IFN-γ. This delivery system...

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...

EFFECTS OF SECRETABLE PLACENTAL FACTORS UPON SECRETION OF CYTOKINES BY THP-1 MONOCYTE-LIKE CELLS

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Ya. S. Onokhina

2013-01-01

Full Text Available Abstract. Мonocytes in feto-placental circulation are exposed to factors secreted by placental tissue. These factors influence monocyte functions in pregnancy. In present study, an in vitro model (monocyte-like THP-1 cells was used for assessing effects of soluble placental factors obtained from women with physiological pregnancies, or preeclampsia cases. The following effects of placental factors were revealed: increased secretion of VEGF by THP-1 cells along with decreased secretion of IL-6, IL-8 and MCP-1 under the influence of placental factors from the I. trimester of pregnancy in comparison with III. trimester. Secretion of IL-6 and MCP-1 by THP-1 cells was increased, and secretion of soluble TNFRII was decreased upon co-cultivation with soluble placental factors from the women with preeclampsia, as compared with placental products from physiological pregnancies.The work is supported by grants ГК № 02.740.11.0711 from Ministry of Education and Science, and НШ-3594.2010.7 grant from the President of Russian Federation.

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.

Monocytic myeloid-derived suppressor cells as prognostic factor in chronic myeloid leukaemia patients treated with dasatinib.

Science.gov (United States)

Giallongo, Cesarina; Parrinello, Nunziatina L; La Cava, Piera; Camiolo, Giuseppina; Romano, Alessandra; Scalia, Marina; Stagno, Fabio; Palumbo, Giuseppe A; Avola, Roberto; Li Volti, Giovanni; Tibullo, Daniele; Di Raimondo, Francesco

2018-02-01

Myeloid suppressor cells are a heterogeneous group of myeloid cells that are increased in patients with chronic myeloid leukaemia (CML) inducing T cell tolerance. In this study, we found that therapy with tyrosine kinase inhibitors (TKI) decreased the percentage of granulocytic MDSC, but only patients treated with dasatinib showed a significant reduction in the monocytic subset (M-MDSC). Moreover, a positive correlation was observed between number of persistent M-MDSC and the value of major molecular response in dasatinib-treated patients. Serum and exosomes from patients with CML induced conversion of monocytes from healthy volunteers into immunosuppressive M-MDSC, suggesting a bidirectional crosstalk between CML cells and MDSC. Overall, we identified M-MDSC as prognostic factors in patients treated with dasatinib. It might be of interest to understand whether MDSC may be a candidate predictive markers of relapse risk following TKI discontinuation, suggesting their potential significance as practice of precision medicine. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

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...

Investigating the Role of Surface Materials and Three Dimensional Architecture on In Vitro Differentiation of Porcine Monocyte-Derived Dendritic Cells.

Science.gov (United States)

Hartmann, Sofie Bruun; Mohanty, Soumyaranjan; Skovgaard, Kerstin; Brogaard, Louise; Flagstad, Frederikke Bjergvang; Emnéus, Jenny; Wolff, Anders; Summerfield, Artur; Jungersen, Gregers

2016-01-01

In vitro generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell culture techniques, engineers have developed fluidic micro-devises usually manufactured in materials other than polystyrene and applying three-dimensional structures more similar to the in vivo environment. Polydimethylsiloxane (PDMS) is an often used polymer for lab-on-a-chip devices but not much is known about the effect of changing the culture surface material from polystyrene to PDMS. In the present study the differentiation of porcine monocytes to monocyte-derived dendritic cells (moDCs) was investigated using CD172apos pig blood monocytes stimulated with GM-CSF and IL-4. Monocytes were cultured on surfaces made of two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS and carbonised three-dimensional PDMS. Cells cultured conventionally (on two-dimensional polystyrene) differentiated into moDCs as expected. Interestingly, gene expression of a wide range of cytokines, chemokines, and pattern recognition receptors was influenced by culture surface material and architecture. Distinct clustering of cells, based on similar expression patterns of 46 genes of interest, was seen for cells isolated from two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS. Changing the material from polystyrene to PDMS resulted in cells with expression patterns usually associated with macrophage expression (upregulation of CD163 and downregulation of CD1a, FLT3, LAMP3 and BATF3). However, this was purely based on gene expression level, and no functional assays were included in this study which would be necessary in order to classify the cells as being macrophages. When changing to three-dimensional culture the cells became increasingly activated in terms of IL6, IL8, IL10 and CCR5 gene expression. Further stimulation with LPS resulted

Investigating the Role of Surface Materials and Three Dimensional Architecture on In Vitro Differentiation of Porcine Monocyte-Derived Dendritic Cells.

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Sofie Bruun Hartmann

Full Text Available In vitro generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell culture techniques, engineers have developed fluidic micro-devises usually manufactured in materials other than polystyrene and applying three-dimensional structures more similar to the in vivo environment. Polydimethylsiloxane (PDMS is an often used polymer for lab-on-a-chip devices but not much is known about the effect of changing the culture surface material from polystyrene to PDMS. In the present study the differentiation of porcine monocytes to monocyte-derived dendritic cells (moDCs was investigated using CD172apos pig blood monocytes stimulated with GM-CSF and IL-4. Monocytes were cultured on surfaces made of two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS and carbonised three-dimensional PDMS. Cells cultured conventionally (on two-dimensional polystyrene differentiated into moDCs as expected. Interestingly, gene expression of a wide range of cytokines, chemokines, and pattern recognition receptors was influenced by culture surface material and architecture. Distinct clustering of cells, based on similar expression patterns of 46 genes of interest, was seen for cells isolated from two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS. Changing the material from polystyrene to PDMS resulted in cells with expression patterns usually associated with macrophage expression (upregulation of CD163 and downregulation of CD1a, FLT3, LAMP3 and BATF3. However, this was purely based on gene expression level, and no functional assays were included in this study which would be necessary in order to classify the cells as being macrophages. When changing to three-dimensional culture the cells became increasingly activated in terms of IL6, IL8, IL10 and CCR5 gene expression. Further stimulation

Umbilical Cord-derived Mesenchymal Stem Cells Instruct Monocytes Towards an IL10-producing Phenotype by Secreting IL6 and HGF

Science.gov (United States)

Deng, Yinan; Zhang, Yingcai; Ye, Linsen; Zhang, Tong; Cheng, Jintao; Chen, Guihua; Zhang, Qi; Yang, Yang

2016-01-01

Human UC-MSCs are regarded as an attractive alternative to BM-MSCs for clinical applications due to their easy preparation, higher proliferation and lower immunogenicity. However, the mechanisms underlying immune suppression by UC-MSCs are still unclear. We studied the mechanism of inhibition by UC-MSCs during the differentiation of monocytes into DCs and focused on the specific source and the role of the involved cytokines. We found that UC-MSCs suppressed monocyte differentiation into DCs and instructed monocytes towards other cell types, with clear decreases in the expression of co-stimulatory molecules, in the secretion of inflammatory factors and in allostimulatory capacity. IL6, HGF and IL10 might be involved in this process because they were detected at higher levels in a coculture system. UC-MSCs produce IL-6 and HGF, and neutralization of IL-6 and HGF reversed the suppressive effect of UC-MSCs. IL10 was not produced by UC-MSCs but was exclusively produced by monocytes after exposure to UC-MSCs, IL-6 or HGF. In summary, we found that the UC-MSC-mediated inhibitory effect was dependent on IL6 and HGF secreted by UC-MSCs and that this effect induced monocyte-derived cells to produce IL10, which might indirectly strengthen the suppressive effect of UC-MSCs. PMID:27917866

MONOCYTES AND MACROPHAGES IN PREGNANCY AND PREECLAMPSIA

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Marijke M Faas

2014-06-01

Full Text Available Preeclampsia is an important complication in pregnancy, characterized byhypertension and proteinuria in the second half of pregnancy. Generalizedactivation of the inflammatory response is thought to play a role in thepathogenesis of preeclampsia. Monocytes may play a central role in thisinflammatory response. Monocytes are short lived cells, that mature in thecirculation and invade into tissues upon an inflammatory stimulus anddevelop into macrophages. Macrophages are abundantly present in theendometrium and play a role in implantation and placentation in normalpregnancy. In preeclampsia, these macrophages appear to be present in largernumbers and are also activated. In the present review we focused on the roleof monocytes and macrophages in the pathophysiology of preeclampsia.

Measurement of the unfolded protein response (UPR) in monocytes.

LENUS (Irish Health Repository)

Carroll, Tomás P

2011-01-01

In mammalian cells, the primary function of the endoplasmic reticulum (ER) is to synthesize and assemble membrane and secreted proteins. As the main site of protein folding and posttranslational modification in the cell, the ER operates a highly conserved quality control system to ensure only correctly assembled proteins exit the ER and misfolded and unfolded proteins are retained for disposal. Any disruption in the equilibrium of the ER engages a multifaceted intracellular signaling pathway termed the unfolded protein response (UPR) to restore normal conditions in the cell. A variety of pathological conditions can induce activation of the UPR, including neurodegenerative disorders such as Parkinson\\'s disease, metabolic disorders such as atherosclerosis, and conformational disorders such as cystic fibrosis. Conformational disorders are characterized by mutations that modify the final structure of a protein and any cells that express abnormal protein risk functional impairment. The monocyte is an important and long-lived immune cell and acts as a key immunological orchestrator, dictating the intensity and duration of the host immune response. Monocytes expressing misfolded or unfolded protein may exhibit UPR activation and this can compromise the host immune system. Here, we describe in detail methods and protocols for the examination of UPR activation in peripheral blood monocytes. This guide should provide new investigators to the field with a broad understanding of the tools required to investigate the UPR in the monocyte.

Measurement of the unfolded protein response (UPR) in monocytes.

LENUS (Irish Health Repository)

Carroll, Tomas P

2012-02-01

In mammalian cells, the primary function of the endoplasmic reticulum (ER) is to synthesize and assemble membrane and secreted proteins. As the main site of protein folding and posttranslational modification in the cell, the ER operates a highly conserved quality control system to ensure only correctly assembled proteins exit the ER and misfolded and unfolded proteins are retained for disposal. Any disruption in the equilibrium of the ER engages a multifaceted intracellular signaling pathway termed the unfolded protein response (UPR) to restore normal conditions in the cell. A variety of pathological conditions can induce activation of the UPR, including neurodegenerative disorders such as Parkinson\\'s disease, metabolic disorders such as atherosclerosis, and conformational disorders such as cystic fibrosis. Conformational disorders are characterized by mutations that modify the final structure of a protein and any cells that express abnormal protein risk functional impairment. The monocyte is an important and long-lived immune cell and acts as a key immunological orchestrator, dictating the intensity and duration of the host immune response. Monocytes expressing misfolded or unfolded protein may exhibit UPR activation and this can compromise the host immune system. Here, we describe in detail methods and protocols for the examination of UPR activation in peripheral blood monocytes. This guide should provide new investigators to the field with a broad understanding of the tools required to investigate the UPR in the monocyte.

Outside-in HLA class I signaling regulates ICAM-1 clustering and endothelial cell-monocyte interactions via mTOR in transplant antibody-mediated rejection.

Science.gov (United States)

Salehi, Sahar; Sosa, Rebecca A; Jin, Yi-Ping; Kageyama, Shoichi; Fishbein, Michael C; Rozengurt, Enrique; Kupiec-Weglinski, Jerzy W; Reed, Elaine F

2018-05-01

Antibody-mediated rejection (AMR) resulting in transplant allograft vasculopathy (TAV) is the major obstacle for long-term survival of solid organ transplants. AMR is caused by donor-specific antibodies to HLA, which contribute to TAV by initiating outside-in signaling transduction pathways that elicit monocyte recruitment to activated endothelium. Mechanistic target of rapamycin (mTOR) inhibitors can attenuate TAV; therefore, we sought to understand the mechanistic underpinnings of mTOR signaling in HLA class I Ab-mediated endothelial cell activation and monocyte recruitment. We used an in vitro model to assess monocyte binding to HLA I Ab-activated endothelial cells and found mTOR inhibition reduced ezrin/radixin/moesin (ERM) phosphorylation, intercellular adhesion molecule 1 (ICAM-1) clustering, and monocyte firm adhesion to HLA I Ab-activated endothelium. Further, in a mouse model of AMR, in which C57BL/6. RAG1 -/- recipients of BALB/c cardiac allografts were passively transferred with donor-specific MHC I antibodies, mTOR inhibition significantly reduced vascular injury, ERM phosphorylation, and macrophage infiltration of the allograft. Taken together, these studies indicate mTOR inhibition suppresses ERM phosphorylation in endothelial cells, which impedes ICAM-1 clustering in response to HLA class I Ab and prevents macrophage infiltration into cardiac allografts. These findings indicate a novel therapeutic application for mTOR inhibitors to disrupt endothelial cell-monocyte interactions during AMR. © 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

Phenotypic heterogeneity of peripheral monocytes in healthy dogs.

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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.

The glial scar-monocyte interplay: a pivotal resolution phase in spinal cord repair.

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Ravid Shechter

Full Text Available The inflammatory response in the injured spinal cord, an immune privileged site, has been mainly associated with the poor prognosis. However, recent data demonstrated that, in fact, some leukocytes, namely monocytes, are pivotal for repair due to their alternative anti-inflammatory phenotype. Given the pro-inflammatory milieu within the traumatized spinal cord, known to skew monocytes towards a classical phenotype, a pertinent question is how parenchymal-invading monocytes acquire resolving properties essential for healing, under such unfavorable conditions. In light of the spatial association between resolving (interleukin (IL-10 producing monocytes and the glial scar matrix chondroitin sulfate proteoglycan (CSPG, in this study we examined the mutual relationship between these two components. By inhibiting the de novo production of CSPG following spinal cord injury, we demonstrated that this extracellular matrix, mainly known for its ability to inhibit axonal growth, serves as a critical template skewing the entering monocytes towards the resolving phenotype. In vitro cell culture studies demonstrated that this matrix alone is sufficient to induce such monocyte polarization. Reciprocal conditional ablation of the monocyte-derived macrophages concentrated at the lesion margins, using diphtheria toxin, revealed that these cells have scar matrix-resolving properties. Replenishment of monocytic cell populations to the ablated mice demonstrated that this extracellular remodeling ability of the infiltrating monocytes requires their expression of the matrix-degrading enzyme, matrix metalloproteinase 13 (MMP-13, a property that was found here to be crucial for functional recovery. Altogether, this study demonstrates that the glial scar-matrix, a known obstacle to regeneration, is a critical component skewing the encountering monocytes towards a resolving phenotype. In an apparent feedback loop, monocytes were found to regulate scar resolution. This

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.

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

Peripheral blood monocyte subsets predict antiviral response in chronic hepatitis C.

Science.gov (United States)

Rodríguez-Muñoz, Y; Martín-Vílchez, S; López-Rodríguez, R; Hernández-Bartolomé, A; Trapero-Marugán, M; Borque, M J; Moreno-Otero, R; Sanz-Cameno, P

2011-10-01

Hepatitis C virus infection evolves into chronic progressive liver disease in a significant percentage of patients. Monocytes constitute a diverse group of myeloid cells that mediate innate and adaptive immune response. In addition to proinflammatory CD16+ monocytes, a Tie-2+ subgroup - Tie-2 expressing monocytes (TEMs) - that has robust proangiogenic potential has been recently defined. To study the heterogeneity of peripheral blood monocytes in chronic hepatitis C (CHC) patients and to examine their proposed pathophysiological roles on disease progression and response to antiviral therapy. We studied CD16+ and Tie-2+ peripheral monocyte subpopulations in 21 healthy subjects and 39 CHC patients in various stages of disease and responses to antiviral treatment using flow cytometry. Expression profiles of proangiogenic and tissue remodelling factors in monocyte supernatants were measured using ELISA and protein arrays. Intrahepatic expression of CD14, CD31 and Tie-2 was analysed using immunofluorescence. Increases of certain peripheral monocyte subsets were observed in the blood of CHC patients, wherein those cells with proinflammatory (CD16+) or proangiogenic (TEMs) potential expanded (P TEMs were significantly increased in nonresponders, particularly those with lower CD16 expression. In addition, many angiogenic factors were differentially expressed by peripheral monocytes from control or CHC patients, such as angiopoietin-1 and angiogenin (P TEMs were distinguished within portal infiltrates of CHC patients. These findings suggest for the first time the relevance of peripheral monocytes phenotypes for the achievement of response to treatment. Hence, the study of monocyte subset regulation might effect improved CHC prognoses and adjuvant therapies. © 2011 Blackwell Publishing Ltd.

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.

THP-1 monocytes but not macrophages as a potential alternative for CD34+ dendritic cells to identify chemical skin sensitizers

International Nuclear Information System (INIS)

Lambrechts, Nathalie; Verstraelen, Sandra; Lodewyckx, Hanne; Felicio, Ana; Hooyberghs, Jef; Witters, Hilda; Tendeloo, Viggo van; Cauwenberge, Paul van; Nelissen, Inge; Heuvel, Rosette van den; Schoeters, Greet

2009-01-01

Early detection of the sensitizing potential of chemicals is an emerging issue for chemical, pharmaceutical and cosmetic industries. In our institute, an in vitro classification model for prediction of chemical-induced skin sensitization based on gene expression signatures in human CD34 + progenitor-derived dendritic cells (DC) has been developed. This primary cell model is able to closely mimic the induction phase of sensitization by Langerhans cells in the skin, but it has drawbacks, such as the availability of cord blood. The aim of this study was to investigate whether human in vitro cultured THP-1 monocytes or macrophages display a similar expression profile for 13 predictive gene markers previously identified in DC and whether they also possess a discriminating capacity towards skin sensitizers and non-sensitizers based on these marker genes. To this end, the cell models were exposed to 5 skin sensitizers (ammonium hexachloroplatinate IV, 1-chloro-2,4-dinitrobenzene, eugenol, para-phenylenediamine, and tetramethylthiuram disulfide) and 5 non-sensitizers (L-glutamic acid, methyl salicylate, sodium dodecyl sulfate, tributyltin chloride, and zinc sulfate) for 6, 10, and 24 h, and mRNA expression of the 13 genes was analyzed using real-time RT-PCR. The transcriptional response of 7 out of 13 genes in THP-1 monocytes was significantly correlated with DC, whereas only 2 out of 13 genes in THP-1 macrophages. After a cross-validation of a discriminant analysis of the gene expression profiles in the THP-1 monocytes, this cell model demonstrated to also have a capacity to distinguish skin sensitizers from non-sensitizers. However, the DC model was superior to the monocyte model for discrimination of (non-)sensitizing chemicals.

Allogeneic transplantation of programmable cells of monocytic origin (PCMO) improves angiogenesis and tissue recovery in critical limb ischemia (CLI): a translational approach.

Science.gov (United States)

Berndt, Rouven; Hummitzsch, Lars; Heß, Katharina; Albrecht, Martin; Zitta, Karina; Rusch, Rene; Sarras, Beke; Bayer, Andreas; Cremer, Jochen; Faendrich, Fred; Groß, Justus

2018-04-27

Employing growth factor-induced partial reprogramming in vitro, peripheral human blood monocytes can acquire a state of plasticity along with expression of various markers of pluripotency. These so-called programmable cells of monocytic origin (PCMO) hold great promise in regenerative therapies. The aim of this translational study was to explore and exploit the functional properties of PCMO for allogeneic cell transplantation therapy in critical limb ischemia (CLI). Using our previously described differentiation protocol, murine and human monocytes were differentiated into PCMO. We examined paracrine secretion of pro-angiogenic and tissue recovery-associated proteins under hypoxia and induction of angiogenesis by PCMO in vitro. Allogeneic cell transplantation of PCMO was performed in a hind limb ischemia mouse model in comparison to cell transplantation of native monocytes and a placebo group. Moreover, we analyzed retrospectively four healing attempts with PCMO in patients with peripheral artery disease (PAD; Rutherford classification, stage 5 and 6). Statistical analysis was performed by using one-way ANOVA, Tukey's test or the Student's t test, p < 0.05. Cell culture experiments revealed good resilience of PCMO under hypoxia, enhanced paracrine release of pro-angiogenic and tissue recovery-associated proteins and induction of angiogenesis in vitro by PCMO. Animal experiments demonstrated significantly enhanced SO 2 saturation, blood flow, neoangiogenesis and tissue recovery after treatment with PCMO compared to treatment with native monocytes and placebo. Finally, first therapeutic application of PCMO in humans demonstrated increased vascular collaterals and improved wound healing in patients with chronic CLI without exaggerated immune response, malignant processes or extended infection after 12 months. In all patients minor and/or major amputations of the lower extremity could be avoided. In summary, PCMO improve angiogenesis and tissue recovery in chronic

Enhanced lysis of herpes simplex virus type 1-infected mouse cell lines by NC and NK effectors

Energy Technology Data Exchange (ETDEWEB)

Colmenares, C.; Lopez, C.

1986-05-01

Spontaneously cytotoxic murine lymphocytes lysed certain cell types infected by herpes simplex virus type 1 (HSV-1) better than uninfected cells. Although HSV-1 adsorbed to the surface of all the target cells, those in which the virus replicated more efficiently were lysed to a greater extent. As targets, the authors used cell lines that, when uninfected, were spontaneously lysed by NK cells (YAC-1) or by NC cells (WEHI-164). They also used a fibroblastoid cell line (M50) and a monocytic tumor line (PU51R), which were not spontaneously killed. NK cells lysed HSV-1-infected YAC cells better than uninfected cells, and an NC-like activity selectively lysed HSV-1-infected WEHI cells. These findings were consistent with the results of experiments performed to define the role of interferon in induction of virus-augmented cytolysis. Increased lysis of YAC-HSV and PU51R-HSV was entirely due to interferon activation and was completely abolished by performing the /sup 51/Cr-release assay in the presence of anti-interferon serum. The data show that HSV-1 infection of NK/NC targets induces increased cytotoxity, but the effector cell responsible for lysis is determined by the uninfected target, or by an interaction between the virus and target cell, rather than by a viral determinant alone.

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

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

Directory of Open Access Journals (Sweden)

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.

CD16+ Monocyte Subsets Are Increased in Large Abdominal Aortic Aneurysms and Are Differentially Related with Circulating and Cell-Associated Biochemical and Inflammatory Biomarkers

Directory of Open Access Journals (Sweden)

Giorgio Ghigliotti

2013-01-01

Full Text Available Proinflammatory components are present in abdominal aortic aneurysm (AAA. Circulating monocytes display heterogeneity, and three subsets have been identified, based on the differential expression for CD14 and CD16 receptors: CD14+CD16-, classical, CD14+CD16+, intermediate and CD14dim CD16+, non-classical monocytes. Increased proinflammatory CD16+ monocytes with high expression of CD143 are present in CKD patients. D-dimer is increased in AAA patients, and might contribute to the pro-inflammatory response associated to circulating monocytes. We aimed to investigate the frequency of CD14+CD16+, CD14dim CD16+ monocytes and monocyte CD143 expression in AAA patients, and their relationship with D-dimer, eGFR and other inflammatory parameters. Blood from 74 AAA patients and 30 healthy controls was analyzed to determine the frequency of CD14+, CD16+, CD14dim CD16+ monocytes and the monocyte CD143 expression by means of flow-cytometry. AAA patients had expanded CD16+ SUPsets (CD14+CD16+: 7.66 ± 0.31% vs 5.42 ± 0.27%; CD14dim CD16+: 7.43 ± 0.48% vs 5.54 ± 0.38%, AAA vs controls, mean ± SE, both p<0.05. CD14+ CD16+ cells were associated to D-dimer and age, and to reduced eGFR. CD14dim CD16+ cells were associated to uric acid, surface CD143, and reduced count of total leukocytes and neutrophils. Within AAA patients, the two CD16+ supsets and the monocyte CD143 expression display different relationships with D-dimer, parameters of renal function and circulating biochemical and inflammatory biomarkers.

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.

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.

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.

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.

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

Enhanced interleukin-8 production in THP-1 human monocytic cells by lipopolysaccharide from oral microorganisms and granulocyte-macrophage colony-stimulating factor.

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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.

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...

Intriguing interplay between feline infectious peritonitis virus and its receptors during entry in primary feline monocytes.

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Van Hamme, Evelien; Desmarets, Lowiese; Dewerchin, Hannah L; Nauwynck, Hans J

2011-09-01

Two potential receptors have been described for the feline infectious peritonitis virus (FIPV): feline aminopeptidase N (fAPN) and feline dendritic cell-specific intercellular adhesion molecule grabbing non-integrin (fDC-SIGN). In cell lines, fAPN serves as a receptor for serotype II, but not for serotype I FIPV. The role of fAPN in infection of in vivo target cells, monocytes, is not yet confirmed. Both serotype I and II FIPVs use fDC-SIGN for infection of monocyte-derived cells but how is not known. In this study, the role of fAPN and fDC-SIGN was studied at different stages in FIPV infection of monocytes. First, the effects of blocking the potential receptor(s) were studied for the processes of attachment and infection. Secondly, the level of co-localization of FIPV and the receptors was determined. It was found that FIPV I binding and infection were not affected by blocking fAPN while blocking fDC-SIGN reduced FIPV I binding to 36% and practically completely inhibited infection. Accordingly, 66% of bound FIPV I particles co-localized with fDC-SIGN. Blocking fAPN reduced FIPV II binding by 53% and infection by 80%. Further, 60% of bound FIPV II co-localized with fAPN. fDC-SIGN was not involved in FIPV II binding but infection was reduced with 64% when fDC-SIGN was blocked. In conclusion, FIPV I infection of monocytes depends on fDC-SIGN. Most FIPV I particles already interact with fDC-SIGN at the plasma membrane. For FIPV II, both fAPN and fDC-SIGN are involved in infection with only fAPN playing a receptor role at the plasma membrane. Copyright © 2011 Elsevier B.V. All rights reserved.

Intravital live cell triggered imaging system reveals monocyte patrolling and macrophage migration in atherosclerotic arteries

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McArdle, Sara; Chodaczek, Grzegorz; Ray, Nilanjan; Ley, Klaus

2015-02-01

Intravital multiphoton imaging of arteries is technically challenging because the artery expands with every heartbeat, causing severe motion artifacts. To study leukocyte activity in atherosclerosis, we developed the intravital live cell triggered imaging system (ILTIS). This system implements cardiac triggered acquisition as well as frame selection and image registration algorithms to produce stable movies of myeloid cell movement in atherosclerotic arteries in live mice. To minimize tissue damage, no mechanical stabilization is used and the artery is allowed to expand freely. ILTIS performs multicolor high frame-rate two-dimensional imaging and full-thickness three-dimensional imaging of beating arteries in live mice. The external carotid artery and its branches (superior thyroid and ascending pharyngeal arteries) were developed as a surgically accessible and reliable model of atherosclerosis. We use ILTIS to demonstrate Cx3cr1GFP monocytes patrolling the lumen of atherosclerotic arteries. Additionally, we developed a new reporter mouse (Apoe-/-Cx3cr1GFP/+Cd11cYFP) to image GFP+ and GFP+YFP+ macrophages "dancing on the spot" and YFP+ macrophages migrating within intimal plaque. ILTIS will be helpful to answer pertinent open questions in the field, including monocyte recruitment and transmigration, macrophage and dendritic cell activity, and motion of other immune cells.

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

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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.

SAMHD1 controls cell cycle status, apoptosis and HIV-1 infection in monocytic THP-1 cells

International Nuclear Information System (INIS)

Bonifati, Serena; Daly, Michele B.; St Gelais, Corine; Kim, Sun Hee; Hollenbaugh, Joseph A.; Shepard, Caitlin; Kennedy, Edward M.; Kim, Dong-Hyun; Schinazi, Raymond F.; Kim, Baek; Wu, Li

2016-01-01

SAMHD1 limits HIV-1 infection in non-dividing myeloid cells by decreasing intracellular dNTP pools. HIV-1 restriction by SAMHD1 in these cells likely prevents activation of antiviral immune responses and modulates viral pathogenesis, thus highlighting a critical role of SAMHD1 in HIV-1 physiopathology. Here, we explored the function of SAMHD1 in regulating cell proliferation, cell cycle progression and apoptosis in monocytic THP-1 cells. Using the CRISPR/Cas9 technology, we generated THP-1 cells with stable SAMHD1 knockout. We found that silencing of SAMHD1 in cycling cells stimulates cell proliferation, redistributes cell cycle population in the G_1/G_0 phase and reduces apoptosis. These alterations correlated with increased dNTP levels and more efficient HIV-1 infection in dividing SAMHD1 knockout cells relative to control. Our results suggest that SAMHD1, through its dNTPase activity, affects cell proliferation, cell cycle distribution and apoptosis, and emphasize a key role of SAMHD1 in the interplay between cell cycle regulation and HIV-1 infection.

SAMHD1 controls cell cycle status, apoptosis and HIV-1 infection in monocytic THP-1 cells

Energy Technology Data Exchange (ETDEWEB)

Bonifati, Serena [Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH (United States); Daly, Michele B. [Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA (United States); St Gelais, Corine; Kim, Sun Hee [Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH (United States); Hollenbaugh, Joseph A.; Shepard, Caitlin [Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA (United States); Kennedy, Edward M. [Department of Molecular Genetics and Microbiology, Duke University, Durham, NC (United States); Kim, Dong-Hyun [Department of Pharmacy, School of Pharmacy, Kyung-Hee University, Seoul (Korea, Republic of); Schinazi, Raymond F. [Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA (United States); Kim, Baek, E-mail: baek.kim@emory.edu [Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA (United States); Department of Pharmacy, School of Pharmacy, Kyung-Hee University, Seoul (Korea, Republic of); Wu, Li, E-mail: wu.840@osu.edu [Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH (United States)

2016-08-15

SAMHD1 limits HIV-1 infection in non-dividing myeloid cells by decreasing intracellular dNTP pools. HIV-1 restriction by SAMHD1 in these cells likely prevents activation of antiviral immune responses and modulates viral pathogenesis, thus highlighting a critical role of SAMHD1 in HIV-1 physiopathology. Here, we explored the function of SAMHD1 in regulating cell proliferation, cell cycle progression and apoptosis in monocytic THP-1 cells. Using the CRISPR/Cas9 technology, we generated THP-1 cells with stable SAMHD1 knockout. We found that silencing of SAMHD1 in cycling cells stimulates cell proliferation, redistributes cell cycle population in the G{sub 1}/G{sub 0} phase and reduces apoptosis. These alterations correlated with increased dNTP levels and more efficient HIV-1 infection in dividing SAMHD1 knockout cells relative to control. Our results suggest that SAMHD1, through its dNTPase activity, affects cell proliferation, cell cycle distribution and apoptosis, and emphasize a key role of SAMHD1 in the interplay between cell cycle regulation and HIV-1 infection.

Monocytes of patients with familial hypercholesterolemia show alterations in cholesterol metabolism

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Soufi Muhidien

2008-11-01

Full Text Available Abstract Background Elevated plasma cholesterol promotes the formation of atherosclerotic lesions in which monocyte-derived lipid-laden macrophages are frequently found. To analyze, if circulating monocytes already show increased lipid content and differences in lipoprotein metabolism, we compared monocytes from patients with Familial Hypercholesterolemia (FH with those from healthy individuals. Methods Cholesterol and oxidized cholesterol metabolite serum levels of FH and of healthy, gender/age matched control subjects were measured by combined gas chromatography – mass spectroscopy. Monocytes from patients with FH and from healthy subjects were isolated by antibody-assisted density centrifugation. Gene expression profiles of isolated monocytes were measured using Affymetrix HG-U 133 Plus 2.0 microarrays. We compared monocyte gene expression profiles from FH patients with healthy controls using a Welch T-test with correction for multiple testing (p Results Using microarray analysis we found in FH patients a significant up-regulation of 1,617 genes and a down-regulation of 701 genes compared to monocytes from healthy individuals. These include genes of proteins that are involved in the uptake, biosynthesis, disposition, and cellular efflux of cholesterol. In addition, plasma from FH patients contains elevated amounts of sterols and oxysterols. An increased uptake of oxidized as well as of native LDL by FH monocytes combined with a down-regulation of NPC1 and ABCA1 explains the lipid accumulation observed in these cells. Conclusion Our data demonstrate that circulating FH monocytes show differences in cell physiology that may contribute to the early onset of atherosclerosis in this disease.

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

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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

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

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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.

Evaluating the Use of Monocytes with a Degradable Polyurethane for Vascular Tissue Regeneration

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Battiston, Kyle Giovanni

Monocytes are one of the first cell types present following the implantation of a biomaterial or tissue engineered construct. Depending on the monocyte activation state supported by the biomaterial, monocytes and their derived macrophages (MDMs) can act as positive contributors to tissue regeneration and wound healing, or conversely promote a chronic inflammatory response that leads to fibrous encapsulation and implant rejection. A degradable polar hydrophobic iconic polyurethane (D-PHI) has been shown to reduce pro-inflammatory monocyte/macrophage response compared to tissue culture polystyrene (TCPS), a substrate routinely used for in vitro culture of cells, as well as poly(lactide- co-glycolide) (PLGA), a standard synthetic biodegradable biomaterial in the tissue engineering field. D-PHI has also shown properties suitable for use in a vascular tissue engineering context. In order to understand the mechanism through which D-PHI attenuates pro-inflammatory monocyte response, this thesis investigated the ability of D-PHI to modulate interactions with adsorbed serum proteins and the properties of D-PHI that were important for this activity. D-PHI was shown to regulate protein adsorption in a manner that produced divergent monocyte responses compared to TCPS and PLGA when coated with the serum proteins alpha2-macroglobulin or immunoglobulin G (IgG). In the case of IgG, D-PHI was shown to reduce pro-inflammatory binding site exposure as a function of the material's polar, hydrophobic, and ionic character. Due to the favourable monocyte activation state supported by D-PHI, and the importance of monocytes/macrophages in regulating the response of tissue-specific cell types in vivo, the ability of a D-PHI-stimulated monocyte/macrophage activation state to contribute to modulating the response of vascular smooth muscle cells (VSMCs) in a vascular tissue engineering context was investigated. D-PHI- stimulated monocytes promoted VSMC growth and migration through biomolecule

Transcriptome analysis of monocyte-HIV interactions

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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

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

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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.

Development of pro-inflammatory phenotype in monocytes after engulfing Hb-activated platelets in hemolytic disorders.

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Singhal, Rashi; Chawla, Sheetal; Rathore, Deepak K; Bhasym, Angika; Annarapu, Gowtham K; Sharma, Vandana; Seth, Tulika; Guchhait, Prasenjit

2017-02-01

Monocytes and macrophage combat infections and maintain homeostatic balance by engulfing microbes and apoptotic cells, and releasing inflammatory cytokines. Studies have described that these cells develop anti-inflammatory properties upon recycling the free-hemoglobin (Hb) in hemolytic conditions. While investigating the phenotype of monocytes in two hemolytic disorders-paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease (SCD), we observed a high number of pro-inflammatory (CD14 + CD16 hi ) monocytes in these patients. We further investigated in vitro the phenotype of these monocytes and found an estimated 55% of CD14 + cells were transformed into the CD14 + CD16 hi subset after engulfing Hb-activated platelets. The CD14 + CD16 hi monocytes, which were positive for both intracellular Hb and CD42b (platelet marker), secreted significant amounts of TNF-α and IL-1β, unlike monocytes treated with only free Hb, which secreted more IL-10. We have shown recently the presence of a high number of Hb-bound hyperactive platelets in patients with both diseases, and further investigated if the monocytes engulfed these activated platelets in vivo. As expected, we found 95% of CD14 + CD16 hi monocytes with both intracellular Hb and CD42b in both diseases, and they expressed high TNF-α. Furthermore our data showed that these monocytes whether from patients or developed in vitro after treatment with Hb-activated platelets, secreted significant amounts of tissue factor. Besides, these CD14 + CD16 hi monocytes displayed significantly decreased phagocytosis of E. coli. Our study therefore suggests that this alteration of monocyte phenotype may play a role in the increased propensity to pro-inflammatory/coagulant complications observed in these hemolytic disorders-PNH and SCD. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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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.

[Changes of monocyte and monocyte-platelet aggregates in different subgroups of thrombotic events in patients with acute myocardial infarction during PCI].

Science.gov (United States)

Wang, Sheng; Sun, Cuifang; Liao, Wang; Wu, Zhongwei; Wang, Yudai; Huang, Xiuxian; Lu, Sijia; Dong, Xiaoli; Shuai, Fujie; Li, Bin

2017-07-01

Objective To investigate the impact of thrombotic events on the alterations of monocyte and monocyte-platelet aggregates (MPAs) in patients with acute myocardial infarction (AMI) during percutaneous coronary intervention (PCI). Methods Blood was collected before PCI for flow cytometry. Monocyte subsets and MPAs were detected by four-color platform (CDl4-APC, CDl6-PE-Cy7, CD86-PE and CD41-Alexa Fluor R 488). According to the expression of the platelet surface marker CD41, the number of monocyte subsets and MPAs was analyzed using the fluorescent microspheres of absolute counting tube. The Wilcoxon rank sum test and receiver operating characteristic (ROC) curve analysis were performed. Results CD14 + CD16 ++ monocytes in intraprocedural thrombotic events (IPTE) group were significantly fewer than those in non-IPTE group, and the percentage in total mononuclear cells decreased. Compared with non-IPTE group, MPA binding ratio and monocyte subset MPA binding ratio were significantly higher in IPTE group. ROC analysis showed that MPA binding ratio and subgroup MPA binding ratio had a better predictive value for IPTE in patients with AMI. Conclusion The CD14 + CD16 ++ monocytes in IPTE group were significantly fewer than those in the non-IPTE group. MPA binding ratio and MPA binding ratio of monocyte subsets were significantly higher in the IPTE group than in the non-IPTE group, so they have a good predictive value for IPTE in patients with AMI.

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

Infrared spectroscopic characterization of monocytic microvesicles (microparticles) released upon lipopolysaccharide stimulation.

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Lee, Joonsup; Wen, Beryl; Carter, Elizabeth A; Combes, Valery; Grau, Georges E R; Lay, Peter A

2017-07-01

Microvesicles (MVs) are involved in cell-cell interactions, including disease pathogenesis. Nondestructive Fourier-transform infrared (FTIR) spectra from MVs were assessed as a technique to provide new biochemical insights into a LPS-induced monocyte model of septic shock. FTIR spectroscopy provided a quick method to investigate relative differences in biomolecular content of different MV populations that was complementary to traditional semiquantitative omics approaches, with which it is difficult to provide information on relative changes between classes (proteins, lipids, nucleic acids, carbohydrates) or protein conformations. Time-dependent changes were detected in biomolecular contents of MVs and in the monocytes from which they were released. Differences in phosphatidylcholine and phosphatidylserine contents were observed in MVs released under stimulation, and higher relative concentrations of RNA and α-helical structured proteins were present in stimulated MVs compared with MVs from resting cells. FTIR spectra of stimulated monocytes displayed changes that were consistent with those observed in the corresponding MVs they released. LPS-stimulated monocytes had reduced concentrations of nucleic acids, α-helical structured proteins, and phosphatidylcholine compared with resting monocytes but had an increase in total lipids. FTIR spectra of MV biomolecular content will be important in shedding new light on the mechanisms of MVs and the different roles they play in physiology and disease pathogenesis.-Lee, J., Wen, B., Carter, E. A., Combes, V., Grau, G. E. R., Lay, P. A. Infrared spectroscopic characterization of monocytic microvesicles (microparticles) released upon lipopolysaccharide stimulation. © FASEB.

Difference in membrane repair capacity between cancer cell lines and a normal cell line

DEFF Research Database (Denmark)

Frandsen, Stine Krog; McNeil, Anna K.; Novak, Ivana

2016-01-01

repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique...... cancer cell lines (p immortalized cell line (p

Human bladder uroepithelial cells synergize with monocytes to promote IL-10 synthesis and other cytokine responses to uropathogenic Escherichia coli.

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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.

Investigating the Role of Surface Materials and Three Dimensional Architecture on In Vitro Differentiation of Porcine Monocyte-Derived Dendritic Cells

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Hartmann, Sofie Bruun; Mohanty, Soumyaranjan; Skovgaard, Kerstin

2016-01-01

In vitro generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell culture techniques, engineers have developed fluidic micro-devises usually manufactured in materi......In vitro generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell culture techniques, engineers have developed fluidic micro-devises usually manufactured......-dimensional PDMS and carbonised three-dimensional PDMS. Cells cultured conventionally (on two-dimensional polystyrene) differentiated into moDCs as expected. Interestingly, gene expression of a wide range of cytokines, chemokines, and pattern recognition receptors was influenced by culture surface material...... and IL23A) but the influence of the surfaces was unchanged. These findings highlights future challenges of combining and comparing data generated from microfluidic cell culture-devices made using alternative materials to data generated using conventional polystyrene plates used by most laboratories today....

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.

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.

CD14-dependent monocyte isolation enhances phagocytosis of listeria monocytogenes by proinflammatory, GM-CSF-derived macrophages.

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Caroline Neu

Full Text Available Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF or macrophage colony-stimulating factor (M-CSF into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ stained positive for CD206 and M-CSF-derived macrophages (M-Mφ for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most

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.

ALV-J infection induces chicken monocyte death accompanied with the production of IL-1β and IL-18.

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Dai, Manman; Feng, Min; Xie, Tingting; Li, Yuanfang; Ruan, Zhuohao; Shi, Meiqing; Liao, Ming; Zhang, Xiquan

2017-11-21

Immunosuppression induced by avian leukosis virus subgroup J (ALV-J) causes serious reproduction problems and secondary infections in chickens. Given that monocytes are important precursors of immune cells including macrophages and dendritic cells, we investigated the fate of chicken monocytes after ALV-J infection. Our results indicated that most monocytes infected with ALV-J including field or laboratory strains could not successfully differentiate into macrophages due to cells death. And cells death was dependent upon viral titer and accompanied with increased IL-1β and IL-18 mRNA levels. In addition, ALV-J infection up-regulated caspase-1 and caspase-3 activity in monocytes. Collectively, we found that ALV-J could cause cell death in chicken monocytes, especially pyroptosis, which may be a significant reason for ALV-J induced immunosuppression.

Glatiramer acetate (GA) prevents TNF-α-induced monocyte adhesion to primary endothelial cells through interfering with the NF-κB pathway

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Wei, Guoqian; Zhang, Xueyan; Su, Zhendong; Li, Xueqi, E-mail: xueqili075@yeah.net

2015-01-30

Highlights: • GA inhibited TNF-α-induced binding of monocytes to endothelial cells. • GA inhibited the induction of adhesion molecules MCP-1, VCAM-1 and E-selectin. • GA inhibits NF-κB p65 nuclear translocation and transcriptional activity. • GA inhibits TNF-α-induced IκBα degradation. - Abstract: Pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) is considered to be the major one contributing to the process of development of endothelial dysfunction. Exposure to TNF-α induces the expression of a number of proinflammatory chemokines, such as monocyte chemotactic protein-1 (MCP-1), and adhesion molecules, including vascular adhesion molecule-1 (VCAM-1) and E-selectin, which mediate the interaction of invading monocytes with vascular endothelial cells. Glatiramer acetate (GA) is a licensed clinical drug for treating patients suffering from multiple sclerosis (MS). The effects of GA in vascular disease have not shown before. In this study, we found that GA significantly inhibited TNF-α-induced binding of monocytes to endothelial cells. Mechanistically, we found that GA ameliorated the upregulation of MCP-1, VCAM-1, and E-selectin induced by TNF-α. Notably, this process is mediated by inhibiting the nuclear translocation and activation of NF-κB. Our results also indicate that GA pretreatment attenuates the up-regulation of COX-2 and iNOS. These data suggest that GA might have a potential benefit in therapeutic endothelial dysfunction related diseases.

Effects of peritoneal fluid from endometriosis patients on the release of monocyte-specific chemokines by leukocytes.

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Na, Yong-Jin; Lee, Dong-Hyung; Kim, Seung-Chul; Joo, Jong-Kil; Wang, Ji-Won; Jin, Jun-O; Kwak, Jong-Young; Lee, Kyu-Sup

2011-06-01

Chemokines have been implicated in the pathological process of endometriosis. We compared the effects of peritoneal fluid obtained from patients with endometriosis (ePF) and controls without endometriosis (cPF) on the release of monocyte-specific CC chemokines such as monocyte chemotactic protein-1 (MCP-1), regulated upon activation normal T cell expressed and secreted (RANTES), and macrophage inflammatory protein-1α (MIP-1α) by neutrophils, monocytes, and T cells. Moreover, we evaluated the correlation between the levels of chemokines in ePF and their release by these cells. Cells were obtained from healthy young volunteers and cultured with ePF (n = 12) or cPF (n = 8). The chemokine levels in the ePF and the supernatants of cultured cells with ePF were then measured by ELISA. There was a positive correlation between the levels of MCP-1 and MIP-1α in ePF. The addition of ePF to the cell cultures failed to increase the release of MCP-1, RANTES, and MIP-1α when compared to cPF, but the levels of RANTES in ePF were positively correlated with the release of RANTES by ePF-treated monocytes and T cells. Moreover, there was a positive correlation between the levels of RANTES and MIP-1α released by neutrophils and between the levels of MCP-1 and MIP-1α released by T cells. Finally, the levels of RANTES released by monocyte-derived macrophages and monocytes cultured with ePF were positively correlated. These findings suggest that monocytes, neutrophils, and T cells release differential levels of MCP-1, RANTES, and MIP-1α in response to stimulation with ePF.

Uptake of silver nanoparticles by monocytic THP-1 cells depends on particle size and presence of serum proteins

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Kettler, Katja, E-mail: K.Kettler@science.ru.nl [Radboud University Nijmegen, Department of Environmental Science (Netherlands); Giannakou, Christina; Jong, Wim H. de [National Institute for Public Health and the Environment (RIVM) (Netherlands); Hendriks, A. Jan [Radboud University Nijmegen, Department of Environmental Science (Netherlands); Krystek, Petra [Philips Innovation Services (Netherlands)

2016-09-15

Human health risks by silver nanoparticle (AgNP) exposure are likely to increase due to the increasing number of NP-containing products and demonstrated adverse effects in various cell lines. Unfortunately, results from (toxicity) studies are often based on exposure dose and are often measured only at a fixed time point. NP uptake kinetics and the time-dependent internal cellular concentration are often not considered. Macrophages are the first line of defense against invading foreign agents including NPs. How macrophages deal with the particles is essential for potential toxicity of the NPs. However, there is a considerable lack of uptake studies of particles in the nanometer range and macrophage-like cells. Therefore, uptake rates were determined over 24 h for three different AgNPs sizes (20, 50 and 75 nm) in medium with and without fetal calf serum. Non-toxic concentrations of 10 ng Ag/mL for monocytic THP-1 cells, representing realistic exposure concentration for short-term exposures, were chosen. The uptake of Ag was higher in medium without fetal calf serum and showed increasing uptake for decreasing NP sizes, both on NP mass and on number basis. Internal cellular concentrations reached roughly 32/10 %, 25/18 % and 21/15 % of the nominal concentration in the absence of fetal calf serum/with fetal calf serum for 20-, 50- and 75-nm NPs, respectively. Our research shows that uptake kinetics in macrophages differ for various NP sizes. To increase the understanding of the mechanism of NP toxicity in cells, the process of uptake (timing) should be considered.

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

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Mukae Hiroshi

2005-08-01

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

Dielectrophoretic Separation of Live and Dead Monocytes Using 3D Carbon-Electrodes

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Yagmur Yildizhan

2017-11-01

Full Text Available Blood has been the most reliable body fluid commonly used for the diagnosis of diseases. Although there have been promising investigations for the development of novel lab-on-a-chip devices to utilize other body fluids such as urine and sweat samples in diagnosis, their stability remains a problem that limits the reliability and accuracy of readouts. Hence, accurate and quantitative separation and characterization of blood cells are still crucial. The first step in achieving high-resolution characteristics for specific cell subpopulations from the whole blood is the isolation of pure cell populations from a mixture of cell suspensions. Second, live cells need to be purified from dead cells; otherwise, dead cells might introduce biases in the measurements. In addition, the separation and characterization methods being used must preserve the genetic and phenotypic properties of the cells. Among the characterization and separation approaches, dielectrophoresis (DEP is one of the oldest and most efficient label-free quantification methods, which directly purifies and characterizes cells using their intrinsic, physical properties. In this study, we present the dielectrophoretic separation and characterization of live and dead monocytes using 3D carbon-electrodes. Our approach successfully removed the dead monocytes while preserving the viability of the live monocytes. Therefore, when blood analyses and disease diagnosis are performed with enriched, live monocyte populations, this approach will reduce the dead-cell contamination risk and achieve more reliable and accurate test results.

The distribution of IL-13 receptor alpha1 expression on B cells, T cells and monocytes and its regulation by IL-13 and IL-4.

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Graber, P; Gretener, D; Herren, S; Aubry, J P; Elson, G; Poudrier, J; Lecoanet-Henchoz, S; Alouani, S; Losberger, C; Bonnefoy, J Y; Kosco-Vilbois, M H; Gauchat, J F

1998-12-01

To study the expression of IL-13 receptor alpha1 (IL-13Ralpha1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL-13Ralpha1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38- B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD-CD38- B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up-regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL-13Ralpha1 expression levels on monocytes. While IL-13Ralpha1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL-13Ralpha1 might have functions unrelated to the capacity to form a type II IL-4/IL-13R with IL-4Ralpha.

Monocyte Trafficking to the Brain with Stress and Inflammation: A Novel Axis of Immune-to-Brain Communication that Influences Mood and Behavior

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Eric S Wohleb

2015-01-01

Full Text Available Psychological stressors cause physiological, immunological, and behavioral alterations in humans and rodents that can be maladaptive and negatively affect quality of life. Several lines of evidence indicate that psychological stress disrupts key functional interactions between the immune system and brain that ultimately affects mood and behavior. For example, activation of microglia, the resident innate immune cells of the brain, has been implicated as a key regulator of mood and behavior in the context of prolonged exposure to psychological stress. Emerging evidence implicates a novel neuroimmune circuit involving microglia activation and sympathetic outflow to the peripheral immune system that further reinforces stress-related behaviors by facilitating the recruitment of inflammatory monocytes to the brain. Evidence from various rodent models, including repeated social defeat (RSD, revealed that trafficking of monocytes to the brain promoted the establishment of anxiety-like behaviors following prolonged stress exposure. In addition, new evidence implicates monocyte trafficking from the spleen to the brain as key regulator of recurring anxiety following exposure to prolonged stress. The purpose of this review is to discuss mechanisms that cause stress-induced monocyte re-distribution in the brain and how dynamic interactions between microglia, endothelial cells, and brain macrophages lead to maladaptive behavioral responses.

Comparison of alpha-Type-1 polarizing and standard dendritic cell cytokine cocktail for maturation of therapeutic monocyte-derived dendritic cell preparations from cancer patients

DEFF Research Database (Denmark)

Trepiakas, Redas; Pedersen, Anders Elm; Met, Ozcan

2008-01-01

The current "gold standard" for generation of dendritic cell (DC) used in DC-based cancer vaccine studies is maturation of monocyte-derived DCs with tumor necrosis factor-alpha (TNF-alpha)/IL-1beta/IL-6 and prostaglandin E(2) (PGE(2)). Recently, a protocol for producing so-called alpha-Type-1...... polarized dendritic cells (alphaDC1) in serum-free medium was published based on maturation of monocyte-derived DCs with TNF-alpha/IL-1-beta/polyinosinic:polycytidylic acid (poly-I:C)/interferon (IFN)-alpha and IFN-gamma. This DC maturation cocktail was described to fulfill the criteria for optimal DC......-regulation of inhibitory molecules such as PD-L1, ILT2, ILT3 as compared to sDC. Although alphaDC1 matured DCs secreted more IL-12p70 and IL-23 these DCs had lower or similar stimulatory capacity compared to sDCs when used as stimulating cells in mixed lymphocyte reaction (MLR) or for induction of autologous influenza...

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.

Phenotypic, functional, and quantitative characterization of canine peripheral blood monocyte-derived macrophages

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R Bueno

2005-08-01

Full Text Available The yield as well as phenotypic and functional parameters of canine peripheral blood monocyte-derived macrophages were analyzed. The cells that remained adherent to Teflon after 10 days of culture had high phagocytic activity when inoculated with Leishmania chagasi. Flow cytometric analysis demonstrated that more than 80% of cultured cells were positive for the monocyte/macrophage marker CD14.

CD11c(hi) Dendritic Cells Regulate Ly-6C(hi) Monocyte Differentiation to Preserve Immune-privileged CNS in Lethal Neuroinflammation.

Science.gov (United States)

Kim, Jin Hyoung; Choi, Jin Young; Kim, Seong Bum; Uyangaa, Erdenebelig; Patil, Ajit Mahadev; Han, Young Woo; Park, Sang-Youel; Lee, John Hwa; Kim, Koanhoi; Eo, Seong Kug

2015-12-02

Although the roles of dendritic cells (DCs) in adaptive defense have been defined well, the contribution of DCs to T cell-independent innate defense and subsequent neuroimmunopathology in immune-privileged CNS upon infection with neurotropic viruses has not been completely defined. Notably, DC roles in regulating innate CD11b(+)Ly-6C(hi) monocyte functions during neuroinflammation have not yet been addressed. Using selective ablation of CD11c(hi)PDCA-1(int/lo) DCs without alteration in CD11c(int)PDCA-1(hi) plasmacytoid DC number, we found that CD11c(hi) DCs are essential to control neuroinflammation caused by infection with neurotropic Japanese encephalitis virus, through early and increased infiltration of CD11b(+)Ly-6C(hi) monocytes and higher expression of CC chemokines. More interestingly, selective CD11c(hi) DC ablation provided altered differentiation and function of infiltrated CD11b(+)Ly-6C(hi) monocytes in the CNS through Flt3-L and GM-CSF, which was closely associated with severely enhanced neuroinflammation. Furthermore, CD11b(+)Ly-6C(hi) monocytes generated in CD11c(hi) DC-ablated environment had a deleterious rather than protective role during neuroinflammation, and were more quickly recruited into inflamed CNS, depending on CCR2, thereby exacerbating neuroinflammation via enhanced supply of virus from the periphery. Therefore, our data demonstrate that CD11c(hi) DCs provide a critical and unexpected role to preserve the immune-privileged CNS in lethal neuroinflammation via regulating the differentiation, function, and trafficking of CD11b(+)Ly-6C(hi) monocytes.

Minocycline Inhibition of Monocyte Activation Correlates with Neuronal Protection in SIV NeuroAIDS

Science.gov (United States)

Campbell, Jennifer H.; Burdo, Tricia H.; Autissier, Patrick; Bombardier, Jeffrey P.; Westmoreland, Susan V.; Soulas, Caroline; González, R. Gilberto; Ratai, Eva-Maria; Williams, Kenneth C.

2011-01-01

Background Minocycline is a tetracycline antibiotic that has been proposed as a potential conjunctive therapy for HIV-1 associated cognitive disorders. Precise mechanism(s) of minocycline's functions are not well defined. Methods Fourteen rhesus macaques were SIV infected and neuronal metabolites measured by proton magnetic resonance spectroscopy (1H MRS). Seven received minocycline (4 mg/kg) daily starting at day 28 post-infection (pi). Monocyte expansion and activation were assessed by flow cytometry, cell traffic to lymph nodes, CD16 regulation, viral replication, and cytokine production were studied. Results Minocycline treatment decreased plasma virus and pro-inflammatory CD14+CD16+ and CD14loCD16+ monocytes, and reduced their expression of CD11b, CD163, CD64, CCR2 and HLA-DR. There was reduced recruitment of monocyte/macrophages and productively infected cells in axillary lymph nodes. There was an inverse correlation between brain NAA/Cr (neuronal injury) and circulating CD14+CD16+ and CD14loCD16+ monocytes. Minocycline treatment in vitro reduced SIV replication CD16 expression on activated CD14+CD16+ monocytes, and IL-6 production by monocytes following LPS stimulation. Conclusion Neuroprotective effects of minocycline are due in part to reduction of activated monocytes, monocyte traffic. Mechanisms for these effects include CD16 regulation, reduced viral replication, and inhibited immune activation. PMID:21494695

Indirect induction of endothelial cell injury by PU- or PTFE-mediated activation of monocytes.

Science.gov (United States)

Liu, Xin; Xue, Yang; Sun, Jiao

2010-01-01

Polyurethanes (PUs) and polytetrafluoroethylene (PTFE) are widely used for making cardiovascular devices, but thrombus formation on the surfaces of these devices is inevitable. Since endothelial injury can lead to thrombosis, most of the studies on PUs or PTFE focused on their damage to endothelial cells. However, few studies have attempted to clarify whether the use of foreign objects as biomaterials can cause endothelial injury by activating the innate immune system. In this study, we aimed to investigate the roles of PU- or PTFE-stimulated immune cells in endothelial-cell injury. First, monocytes (THP-1 cells) were stimulated with PU or PTFE for 24 h and, subsequently, human umbilical vein endothelial cells (HUVECs) were treated with the supernatants of the stimulated cells for 24 h. We measured the generation of intracellular reactive oxygen species (ROS) from THP-1 cells treated with PU and PTFE for 24 h, meanwhile hydrogen dioxide (H(2)O(2)), tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the supernatants were also detected. Then, we assessed the apoptosis rate of the HUVECs and determined the expression of NO, inducible nitric oxide synthase (iNOS), and apoptosis-related proteins (p53, Bax, Bcl-2) in the HUVECs. The results showed that large amounts of ROS and low levels of pro-inflammatory cytokines (TNF-α and IL-1β) were produced by the stimulated THP-1 cells. After culturing with the supernatants of the PU- or PTFE-stimulated THP-1 cells, the apoptosis rate, NO production and expression of iNOS, p53 and Bax in the HUVECs were up-regulated, while Bcl-2 expression was down-regulated. In conclusion, the release of ROS by PU- or PTFE-treated THP-1 cells may induce iNOS expression and cause apoptosis in HUVECs via the p53, Bax and Bcl-2 proteins. These data provide the interesting finding that endothelial injury in the process of biomaterial-induced thrombosis can be initiated through the release of soluble mediators by monocytes.

Inability of newborns' or pregnant women's monocytes to suppress pokeweed mitogen-induced responses

International Nuclear Information System (INIS)

Durandy, A.; Fischer, A.; Griscelli, C.

1982-01-01

Although an excess of human adult blood adherent cells inhibits the pokeweed mitogen- (PWM) induced normal adult lymphocyte proliferation and B cell maturation into immunoglobulin-containing cells (ICC), adherent cells collected from newborn infants or pregnant women at time of delivery were unable to exert a similar suppressor activity. After activation by Concanavalin A (Con A), newborns' and pregnant women's adherent cells acquired a suppressor activity comparable to that of control adult adherent cells. The adherent suppressor cell was shown to be radioresistant (3000 rad), indicating its probable monocytic orgin. Both monocyte-suppressor activities (MSA) observed in adulthood (spontaneously) and in the neonatal period (after activation) were dependent on prostaglandin E 2 (PGE 2 ) secretion, because they were abolished by indomethacin or a specific anti-PGE 2 anti-serum. Expression of MSA appeared to be under a negative regulation exerted by naturally occurring T suppressor lymphocytes present in the blood of newborns or pregnant women, because incubation of adult monocytes or Con A-activated newborn monocytes with newborns' or pregnant women's T lymphocytes resulted in a dramatic decrease of their MSA. These results strongly suggest that the lack of MSA in the neonatal period and in late pregnancy is a consequence of activation of T suppressor lymphocytes

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.

Radiosensitivity of mesothelioma cell lines

International Nuclear Information System (INIS)

Haekkinen, A.M.; Laasonen, A.; Linnainmaa, K.; Mattson, K.; Pyrhoenen, S.

1996-01-01

The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters α and β of the linear quadratic model (LQ-model) and mean inactivation dose (D MID ) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean α value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The α/β ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.)

Radiosensitivity of mesothelioma cell lines

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Haekkinen, A.M. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland); Laasonen, A. [Dept. of Pathology, Central Hospital of Etelae-Pohjanmaa, Seinaejoki (Finland); Linnainmaa, K. [Dept. of Industrial Hygiene and Toxicology, Inst. of Occupational Health, Helsinki (Finland); Mattson, K. [Dept. Pulmonary Medicine, Univ. Central Hospital, Helsinki (Finland); Pyrhoenen, S. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland)

1996-10-01

The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters {alpha} and {beta} of the linear quadratic model (LQ-model) and mean inactivation dose (D{sub MID}) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean {alpha} value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The {alpha}/{beta} ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.).

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

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

2006-02-01

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

Shear Stress Enhances Chemokine Secretion from Chlamydia pneumoniae-infected Monocytes.

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Evani, Shankar J; Dallo, Shatha F; Murthy, Ashlesh K; Ramasubramanian, Anand K

2013-09-01

Chlamydia pneumoniae is a common respiratory pathogen that is considered a highly likely risk factor for atherosclerosis. C. pneumoniae is disseminated from the lung into systemic circulation via infected monocytes and lodges at the atherosclerotic sites. During transit, C. pneumoniae -infected monocytes in circulation are subjected to shear stress due to blood flow. The effect of mechanical stimuli on infected monocytes is largely understudied in the context of C. pneumoniae infection and inflammation. We hypothesized that fluid shear stress alters the inflammatory response of C. pneumoniae -infected monocytes and contributes to immune cell recruitment to the site of tissue damage. Using an in vitro model of blood flow, we determined that a physiological shear stress of 7.5 dyn/cm 2 for 1 h on C. pneumoniae -infected monocytes enhances the production of several chemokines, which in turn is correlated with the recruitment of significantly large number of monocytes. Taken together, these results suggest synergistic interaction between mechanical and chemical factors in C. pneumoniae infection and associated inflammation.

Zinc oxide nanoparticles and monocytes: Impact of size, charge and solubility on activation status

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Prach, Morag; Stone, Vicki; Proudfoot, Lorna

2013-01-01

Zinc oxide (ZnO) particle induced cytotoxicity was dependent on size, charge and solubility, factors which at sublethal concentrations may influence the activation of the human monocytic cell line THP1. ZnO nanoparticles (NP; average diameter 70 nm) were more toxic than the bulk form ( 2+ ion with protein. This association with protein may influence interaction of the ZnO particles and NP with THP1 cells. After 24 h exposure to the ZnO particles and NP at sublethal concentrations there was little effect on immunological markers of inflammation such as HLA DR and CD14, although they may induce a modest increase in the adhesion molecule CD11b. The cytokine TNFα is normally associated with proinflammatory immune responses but was not induced by the ZnO particles and NP. There was also no effect on LPS stimulated TNFα production. These results suggest that ZnO particles and NP do not have a classical proinflammatory effect on THP1 cells. -- Highlights: ► ZnO is cytotoxic to THP-1 monocytes. ► ZnO nanoparticles are more toxic than the bulk form. ► Positive charge enhances ZnO nanoparticle cytotoxicity. ► Sublethal doses of ZnO particles do not induce classical proinflammatory markers.

Extracellular vesicles secreted from cancer cell lines stimulate secretion of MMP-9, IL-6, TGF-β1 and EMMPRIN.

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Jasmina S Redzic

Full Text Available Extracellular vesicles (EVs are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-β1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles.

Inflammatory Ly6Chigh Monocytes Protect against Candidiasis through IL-15-Driven NK Cell/Neutrophil Activation.

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Domínguez-Andrés, Jorge; Feo-Lucas, Lidia; Minguito de la Escalera, María; González, Leticia; López-Bravo, María; Ardavín, Carlos

2017-06-20

Neutrophils play a crucial role in defense against systemic candidiasis, a disease associated with a high mortality rate in patients receiving immunosuppressive therapy, although the early immune mechanisms that boost the candidacidal activity of neutrophils remain to be defined in depth. Here, we used a murine model of systemic candidiasis to explore the role of inflammatory Ly6C high monocytes in NK cell-mediated neutrophil activation during the innate immune response against C. albicans. We found that efficient anti-Candida immunity required a collaborative response between the spleen and kidney, which relied on type I interferon-dependent IL-15 production by spleen inflammatory Ly6C high monocytes to drive efficient activation and GM-CSF release by spleen NK cells; this in turn was necessary to boost the Candida killing potential of kidney neutrophils. Our findings unveil a role for IL-15 as a critical mediator in defense against systemic candidiasis and hold promise for the design of IL-15-based antifungal immunotherapies. Copyright © 2017 Elsevier Inc. All rights reserved.

Functional role of monocytes and macrophages for the inflammatory response in acute liver injury

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Henning W Zimmermann

2012-10-01

Full Text Available Different etiologies such as drug toxicity, acute viral hepatitis B or acetaminophen poisoning can cause acute liver injury (ALI or even acute liver failure (ALF. Excessive cell death of hepatocytes in the liver is known to result in a strong hepatic inflammation. Experimental murine models of liver injury highlighted the importance of hepatic macrophages, so-called Kupffer cells, for initiating and driving this inflammatory response by releasing proinflammatory cytokines and chemokines including tumor necrosis factor (TNF, interleukin-6 (IL-6, IL-1-beta or monocyte chemoattractant protein 1 (MCP-1, CCL2 as well as activating other non-parenchymal liver cells, e.g. endothelial or hepatic stellate cells (HSC. Many of these proinflammatory mediators can trigger hepatocytic cell death pathways, e.g. via caspase activation, but also activate protective signaling pathways, e.g. via nuclear factor kappa B (NF-kB. Recent studies in mice demonstrated that these macrophage actions largely depend on the recruitment of monocytes into the liver, namely of the inflammatory Ly6c+ (Gr1+ monocyte subset as precursors of tissue macrophages. The chemokine receptor CCR2 and its ligand MCP-1/CCL2 promote monocyte subset infiltration upon liver injury. In contrast, the chemokine receptor CX3CR1 and its ligand fractalkine (CX3CL1 are important negative regulators of monocyte infiltration by controlling their survival and differentiation into functionally diverse macrophage subsets upon injury. The recently identified cellular and molecular pathways for monocyte subset recruitment, macrophage differentiation and interactions with other hepatic cell types in the injured liver may therefore represent interesting novel targets for future therapeutic approaches in ALF.

Involvement of JNK and NF-κB pathways in lipopolysaccharide (LPS)-induced BAG3 expression in human monocytic cells.

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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.

Increased C-C chemokine receptor 2 gene expression in monocytes of severe obstructive sleep apnea patients and under intermittent hypoxia.

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Chuang, Li-Pang; Chen, Ning-Hung; Lin, Shih-Wei; Chang, Ying-Ling; Liao, Hsiang-Ruei; Lin, Yu-Sheng; Chao, I-Ju; Lin, Yuling; Pang, Jong-Hwei S

2014-01-01

Obstructive sleep apnea (OSA) is known to be a risk factor of coronary artery disease. The chemotaxis and adhesion of monocytes to the endothelium in the early atherosclerosis is important. This study aimed to investigate the effect of intermittent hypoxia, the hallmark of OSA, on the chemotaxis and adhesion of monocytes. Peripheral blood was sampled from 54 adults enrolled for suspected OSA. RNA was prepared from the isolated monocytes for the analysis of C-C chemokine receptor 2 (CCR2). The effect of intermittent hypoxia on the regulation and function of CCR2 was investigated on THP-1 monocytic cells and monocytes. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. Transwell filter migration assay and cell adhesion assay were performed to study the chemotaxis and adhesion of monocytes. Monocytic CCR2 gene expression was found to be increased in severe OSA patients and higher levels were detected after sleep. Intermittent hypoxia increased the CCR2 expression in THP-1 monocytic cells even in the presence of TNF-α and CRP. Intermittent hypoxia also promoted the MCP-1-mediated chemotaxis and adhesion of monocytes to endothelial cells. Furthermore, inhibitor for p42/44 MAPK or p38 MAPK suppressed the activation of monocytic CCR2 expression by intermittent hypoxia. This is the first study to demonstrate the increase of CCR2 gene expression in monocytes of severe OSA patients. Monocytic CCR2 gene expression can be induced under intermittent hypoxia which contributes to the chemotaxis and adhesion of monocytes.

Comparison of cellular effects of starch-coated SPIONs and poly(lactic-co-glycolic acid) matrix nanoparticles on human monocytes.

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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

Drug induced increases in CNS dopamine alter monocyte, macrophage and T cell functions: implications for HAND

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Gaskill, Peter J.; Calderon, Tina M.; Coley, Jacqueline S.; Berman, Joan W.

2013-01-01

Central nervous system (CNS) complications resulting from HIV infection remain a major public health problem as individuals live longer due to the success of combined antiretroviral therapy (cART). As many as 70% of HIV infected people have HIV associated neurocognitive disorders (HAND). Many HIV infected individuals abuse drugs, such as cocaine, heroin or methamphetamine, that may be important cofactors in the development of HIV CNS disease. Despite different mechanisms of action, all drugs of abuse increase extracellular dopamine in the CNS. The effects of dopamine on HIV neuropathogenesis are not well understood, and drug induced increases in CNS dopamine may be a common mechanism by which different types of drugs of abuse impact the development of HAND. Monocytes and macrophages are central to HIV infection of the CNS and to HAND. While T cells have not been shown to be a major factor in HIV-associated neuropathogenesis, studies indicate that T cells may play a larger role in the development of HAND in HIV infected drug abusers. Drug induced increases in CNS dopamine may dysregulate functions of, or increase HIV infection in, monocytes, macrophages and T cells in the brain. Thus, characterizing the effects of dopamine on these cells is important for understanding the mechanisms that mediate the development of HAND in drug abusers. PMID:23456305

Mycobacterium leprae alters classical activation of human monocytes in vitro.

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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.

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

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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

Vaccine adjuvant MF59 promotes the intranodal differentiation of antigen-loaded and activated monocyte-derived dendritic cells.

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Rossella Cioncada

Full Text Available MF59 is an oil-in-water emulsion adjuvant approved for human influenza vaccination in European Union. The mode of action of MF59 is not fully elucidated yet, but results from several years of investigation indicate that MF59 establishes an immunocompetent environment at injection site which promotes recruitment of immune cells, including antigen presenting cells (APCs, that are facilitated to engulf antigen and transport it to draining lymph node (dLN where the antigen is accumulated. In vitro studies showed that MF59 promotes the differentiation of monocytes to dendritic cells (Mo-DCs. Since after immunization with MF59, monocytes are rapidly recruited both at the injection site and in dLN and appear to have a morphological change toward a DC-like phenotype, we asked whether MF59 could play a role in inducing differentiation of Mo-DC in vivo. To address this question we immunized mice with the auto-fluorescent protein Phycoerythrin (PE as model antigen, in presence or absence of MF59. We measured the APC phenotype and their antigen uptake within dLNs, the antigen distribution within the dLN compartments and the humoral response to PE. In addition, using Ovalbumin as model antigen, we measured the capacity of dLN APCs to induce antigen-specific CD4 T cell proliferation. Here, we show, for the first time, that MF59 promotes differentiation of Mo-DCs within dLNs from intranodal recruited monocytes and we suggest that this differentiation could take place in the medullary compartment of the LN. In addition we show that the Mo-DC subset represents the major source of antigen-loaded and activated APCs within the dLN when immunizing with MF59. Interestingly, this finding correlates with the enhanced triggering of antigen-specific CD4 T cell response induced by LN APCs. This study therefore demonstrates that MF59 is able to promote an immunocompetent environment also directly within the dLN, offering a novel insight on the mechanism of action of

Prostaglandin E2 blocks menadione-induced apoptosis through the Ras/Raf/Erk signaling pathway in promonocytic leukemia cell lines.

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Yeo, Hyun-Seok; Shehzad, Adeeb; Lee, Young Sup

2012-04-01

Altered oxidative stress has long been observed in cancer cells, and this biochemical property of cancer cells represents a specific vulnerability that can be exploited for therapeutic benefit. The major role of an elevated oxidative stress for the efficacy of molecular targeted drugs is under investigation. Menadione is considered an attractive model for the study of oxidative stress, which can induce apoptosis in human leukemia HL-60 cell lines. Prostaglandin E(2) (PGE(2)) via its receptors not only promotes cell survival but also reverses apoptosis and promotes cancer progression. Here, we present evidence for the biological role of PGE(2) as a protective agent of oxidative stress-induced apoptosis in monocytic cells. Pretreatment of HL-60 cells with PGE(2) markedly ameliorated the menadione-induced apoptosis and inhibited the degradation of PARP and lamin B. The EP(2) receptor antagonist AH6809 abrogated the inhibitory effect of PGE(2), suggesting the role of the EP(2)/cAMP system. The PKA inhibitor H89 also reversed apoptosis and decreased the PKA activity that was elevated 10-fold by PGE(2). The treatment of HL-60 cells with NAC or zinc chloride showed a similar protective effect as with PGE(2) on menadione-treated cells. Furthermore, PGE(2) activated the Ras/Raf/MEK pathway, which in turn initiated ERK activation, and ultimately protected menadione-induced apoptosis. These results imply that PGE(2) via cell survival pathways may protect oxidative stress-induced apoptosis in monocytic cells. This study warrants further pre-clinical investigation as well as application towards leukemia clinics.

Plasminogen activator inhibitor-2 in patients with monocytic leukemia.

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Scherrer, A; Kruithof, E K; Grob, J P

1991-06-01

Plasma and tumor cells from 103 patients with leukemia or lymphoma at initial presentation were investigated for the presence of plasminogen activator inhibitor-2 (PAI-2) antigen, a potent inhibitor of urokinase. PAI-2 was detected in plasma and leukemic cells of the 21 patients with leukemia having a monocytic component [acute myelomonocytic (M4), acute monoblastic (M5), and chronic myelomonocytic leukemias], and in the three patients with acute undifferentiated myeloblastic leukemia (M0). In contrast, this serine protease inhibitor was undetectable in 79 patients with other subtypes of acute myeloid leukemia or other hematological malignancies. Serial serum PAI-2 determinations in 16 patients with acute leukemia at presentation, during therapy, remission, and relapse revealed that in the five patients with M4-M5, elevated PAI-2 levels rapidly normalized under therapy and during remission, but increased again in the patients with a relapse associated with an M4-M5 phenotype. Thus, PAI-2 seems to be a marker highly specific for the active stages of monocytic leukemia, i.e. presentation and relapse. The presence of PAI-2 in the plasma and cells of patients with M0 may give a clue to a monocytic origin of these cells.

Specific Depletion of Ly6Chi Inflammatory Monocytes Prevents Immunopathology in Experimental Cerebral Malaria

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Kuepper, Janina M.; Biswas, Aindrila; Djie-Maletz, Andrea; Limmer, Andreas; van Rooijen, Nico; Mack, Matthias; Hoerauf, Achim; Dunay, Ildiko Rita

2015-01-01

Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice leads to experimental cerebral malaria (ECM) that is commonly associated with serious T cell mediated damage. In other parasitic infection models, inflammatory monocytes have been shown to regulate Th1 responses but their role in ECM remains poorly defined, whereas neutrophils are reported to contribute to ECM immune pathology. Making use of the recent development of specific monoclonal antibodies (mAb), we depleted in vivo Ly6Chi inflammatory monocytes (by anti-CCR2), Ly6G+ neutrophils (by anti-Ly6G) or both cell types (by anti-Gr1) during infection with Ovalbumin-transgenic PbA parasites (PbTg). Notably, the application of anti-Gr1 or anti-CCR2 but not anti-Ly6G antibodies into PbTg-infected mice prevented ECM development. In addition, depletion of Ly6Chi inflammatory monocytes but not neutrophils led to decreased IFNγ levels and IFNγ+CD8+ T effector cells in the brain. Importantly, anti-CCR2 mAb injection did not prevent the generation of PbTg-specific T cell responses in the periphery, whereas anti-Gr1 mAb injection strongly diminished T cell frequencies and CTL responses. In conclusion, the specific depletion of Ly6Chi inflammatory monocytes attenuated brain inflammation and immune cell recruitment to the CNS, which prevented ECM following Plasmodium infection, pointing out a substantial role of Ly6C+ monocytes in ECM inflammatory processes. PMID:25884830

Inhibitory effects of Kaempferia parviflora extract on monocyte adhesion and cellular reactive oxygen species production in human umbilical vein endothelial cells.

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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.

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.

Glucose transporter expression differs between bovine monocyte and macrophage subsets and is influenced by milk production.

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Eger, M; Hussen, J; Koy, M; Dänicke, S; Schuberth, H-J; Breves, G

2016-03-01

The peripartal period of dairy cows is characterized by negative energy balance and higher incidences of infectious diseases such as mastitis or metritis. With the onset of lactation, milk production is prioritized and large amounts of glucose are transported into the mammary gland. Decreased overall energy availability might impair the function of monocytes acting as key innate immune cells, which give rise to macrophages and dendritic cells and link innate and adaptive immunity. Information on glucose requirements of bovine immune cells is rare. Therefore, this study aims to evaluate glucose transporter expression of the 3 bovine monocyte subsets (classical, intermediate, and nonclassical monocytes) and monocyte-derived macrophages and to identify influences of the peripartal period. Blood samples were either collected from nonpregnant healthy cows or from 16 peripartal German Holstein cows at d -14, +7, and +21 relative to parturition. Quantitative real-time PCR was applied to determine mRNA expression of glucose transporters (GLUT) 1, GLUT3, and GLUT4 in monocyte subsets and monocyte-derived macrophages. The low GLUT1 and GLUT3 expression in nonclassical monocytes was unaltered during differentiation into macrophages, whereas in classical and intermediate monocytes GLUT expression was downregulated. Alternatively activated M2 macrophages consumed more glucose compared with classically activated M1 macrophages. The GLUT4 mRNA was only detectable in unstimulated macrophages. Neither monocytes nor macrophages were insulin responsive. In the peripartum period, monocyte GLUT1 and GLUT3 expression and the GLUT3/GLUT1 ratio were negatively correlated with lactose production. The high-affinity GLUT3 transporter appears to be the predominant glucose transporter on bovine monocytes and macrophages, especially in the peripartal period when blood glucose levels decline. Glucose transporter expression in monocytes is downregulated as a function of lactose production, which

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

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Valenzuela, Nicole M.; Thomas, Kimberly A.; Mulder, Arend; Parry, Graham C.; Panicker, Sandip; Reed, Elaine F.

2017-01-01

Background Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. Methods Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). Results Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. Conclusions Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR. PMID:28640789

Microparticles engineered to highly express peroxisome proliferator-activated receptor-γ decreased inflammatory mediator production and increased adhesion of recipient monocytes.

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Sahler, Julie; Woeller, Collynn F; Phipps, Richard P

2014-01-01

Circulating blood microparticles are submicron vesicles released primarily by megakaryocytes and platelets that act as transcellular communicators. Inflammatory conditions exhibit elevated blood microparticle numbers compared to healthy conditions. Direct functional consequences of microparticle composition, especially internal composition, on recipient cells are poorly understood. Our objective was to evaluate if microparticle composition could impact the function of recipient cells, particularly during inflammatory provocation. We therefore engineered the composition of megakaryocyte culture-derived microparticles to generate distinct microparticle populations that were given to human monocytes to assay for influences recipient cell function. Herein, we tested the responses of monocytes exposed to either control microparticles or microparticles that contain the anti-inflammatory transcription factor, peroxisome proliferator-activated receptor-γ (PPARγ). In order to normalize relative microparticle abundance from two microparticle populations, we implemented a novel approach that utilizes a Nanodrop Spectrophotometer to assay for microparticle density rather than concentration. We found that when given to peripheral blood mononuclear cells, microparticles were preferentially internalized by CD11b+ cells, and furthermore, microparticle composition had a profound functional impact on recipient monocytes. Specifically, microparticles containing PPARγ reduced activated monocyte production of the proinflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared to activated monocytes exposed to control microparticles. Additionally, treatment with PPARγ microparticles greatly increased monocyte cell adherence. This change in morphology occurred simultaneously with increased production of the key extracellular matrix protein, fibronectin and increased expression of the fibronectin-binding integrin, ITGA5. PPARγ microparticles also changed monocyte

Niacin results in reduced monocyte adhesion in patients with type 2 diabetes mellitus.

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Tavintharan, S; Woon, K; Pek, L T; Jauhar, N; Dong, X; Lim, S C; Sum, C F

2011-03-01

Patients with type 2 diabetes have increased expression of cell adhesion molecules (CAMs). CAMs and monocyte adhesion mediate essential processes in atherogenesis. It remains unclear if monocytes from patients on niacin have reduced adhesion function. We studied the variation of monocyte adhesion in patients with type 2 diabetes and low HDL-cholesterol, taking either extended release niacin (Niaspan®, Abbott Laboratories) or controls not on niacin. Biochemical parameters including adiponectin, CAMs and fresh monocytes from whole blood for adhesion assays, were studied at baseline and 12-weeks. Niacin 1500 mg daily raised HDL-cholesterol from 0.8 mmol/l (95% CI: 0.7-0.9) to 0.9 mmol/l (95% CI: 0.8-1.1), p=0.10, and significantly reduced PECAM-1 by 24.9% (95% CI: 10.9-39.0; p<0.05), increased adiponectin by 30.5% (95% CI: 14.1-47.0; p<0.05), with monocyte adhesion reduced by 9.2% (95%CI: 0.7-17.7; p<0.05) in endothelial cells treated in basal conditions, and 7.8% (95% CI: 3.1-12.5; p<0.05) after TNF-α stimulation. Monocytes isolated from patients on niacin had reduced adhesion to endothelial cells. Our findings suggest niacin has broad range of effects apart from lipid-modification, and these could be important in cardiovascular risk reduction. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

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.

Increased MCP-1 gene expression in monocytes of severe OSA patients and under intermittent hypoxia.

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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.

Orf virus IL-10 reduces monocyte, dendritic cell and mast cell recruitment to inflamed skin.

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Bennett, Jared R; Lateef, Zabeen; Fleming, Stephen B; Mercer, Andrew A; Wise, Lyn M

2016-02-02

Orf virus (ORFV) is a zoonotic parapoxvirus that causes pustular dermatitis of sheep, and occasionally humans. Despite causing sustained infections, ORFV induces only a transient increase in pro-inflammatory signalling and the trafficking of innate immune cells within the skin seems to be impaired. An explanation for this tempered response to ORFV infection may lie in its expression of a homolog of the anti-inflammatory cytokine, interleukin (IL)-10. Using a murine model in which inflammation was induced by bacterial lipopolysaccharide, we examined the effects of the ORFV-IL-10 protein on immune cell trafficking to and from the skin. ORFV-IL-10 limited the recruitment of blood-derived Gr-1(int)/CD11b(int) monocytes, CD11c(+ve)/MHC-II(+ve) dendritic cells and c-kit(+ve)/FcεR1(+ve) mature mast cells into inflamed skin. ORFV-IL-10 also suppressed the activation of CD11c(+ve)/MHC-II(+ve) dendritic cells within the skin, reducing their trafficking to the draining lymph node. These findings suggest that expression of IL-10 by ORFV may contribute to the impaired trafficking of innate immune cells within infected skin. Copyright © 2015 Elsevier B.V. All rights reserved.

Thioredoxin 80-Activated-Monocytes (TAMs) Inhibit the Replication of Intracellular Pathogens

DEFF Research Database (Denmark)

Cortes-Bratti, Ximena; Brasseres, Eugenie; Herrera-Rodriquez, Fabiola

2011-01-01

Background: Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced primarily by monocytes. Trx80 induces differentiation of human monocytes into a novel cell type, named Trx80-activated-monocytes (TAMs). Principal Findings: In this investigation we present evidence...... for a role of TAMs in the control of intracellular bacterial infections. As model pathogens we have chosen Listeria monocytogenes and Brucella abortus which replicate in the cytosol and the endoplasmic reticulum respectively. Our data indicate that TAMs efficiently inhibit intracellular growth of both L...... in TAMs compared to that observed in control cells 24 h post-infection, indicating that TAMs kill bacteria by preventing their escape from the endosomal compartments, which progress into a highly degradative phagolysosome. Significance: Our results show that Trx80 potentiates the bactericidal activities...

Purification of monocytes from cryopreserved mobilized apheresis products by elutriation with the Elutra device.

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Lemarie, Claude; Sugaye, Romina; Kaur, Indreshpaul; Taga, Tim; Chabannon, Christian; Schuyler, Robert; Mcmannis, John

2007-01-10

The Elutra biomedical device allows semi-automatic enrichment of monocytes by elutriation, using a single-use, closed and cGMP compliant tubing set, in a cost effective way. The procedure has been validated using fresh apheresis products from nonmobilized donors. We here evaluated the possibility of using Elutra to enrich monocytes from frozen/thawed apheresis products collected from mobilized healthy donors. Frozen apheresis products from 6 G CSF mobilized donors were thawed and used in 16 elutriation procedures. We compared the recovery and purity of enriched monocytes using different buffer compositions and elutriation profiles. Elutriated monocytes were cultured to generate mature dendritic cells (DCs). Depending in part of the initial granulocyte contamination in the apheresis product, the use of Desoxyribo Nuclease (DNAse) to avoid aggregation, was needed through only the initial steps or throughout the elutriation process. The average monocyte recovery was 85+/-31%. The average purity was 73+/-9%. The recovery of mature DC at d8 of culture was 20+/-6% of the input monocyte numbers. We conclude that Elutra allows the purification of monocytes from thawed mobilized apheresis. It requires no pre-processing of the cell product before elutriation, and allows the generation of phenotypically mature DC in quantities that are compatible with a clinical use.

Glycoengineering of therapeutic antibodies enhances monocyte/macrophage-mediated phagocytosis and cytotoxicity.

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Herter, Sylvia; Birk, Martina C; Klein, Christian; Gerdes, Christian; Umana, Pablo; Bacac, Marina

2014-03-01

Therapeutic Abs possess several clinically relevant mechanisms of action including perturbation of tumor cell signaling, activation of complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), and induction of adaptive immunity. In view of the important role of phagocytic lineage cells in the mechanism of action of therapeutic Abs, we analyzed FcγR receptor-dependent effector functions of monocytes and macrophages triggered by glycoengineered (GE) Abs (having enhanced FcγRIIIa [CD16a] binding affinity) versus their wild-type (WT) counterparts under different experimental conditions. We first defined the precise FcγR repertoire on classical and nonclassical intermediate monocytes--M1 and M2c macrophage populations. We further show that WT and GE Abs display comparable binding and induce similar effector functions (ADCC and ADCP) in the absence of nonspecific, endogenous IgGs. However, in the presence of these IgGs (i.e., in a situation that more closely mimics physiologic conditions), GE Abs display significantly superior binding and promote stronger monocyte and macrophage activity. These data show that in addition to enhancing CD16a-dependent NK cell cytotoxicity, glycoengineering also enhances monocyte and macrophage phagocytic and cytotoxic activities through enhanced binding to CD16a under conditions that more closely resemble the physiologic setting.

Effective clinical-scale production of dendritic cell vaccines by monocyte elutriation directly in medium, subsequent culture in bags and final antigen loading using peptides or RNA transfection.

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Erdmann, Michael; Dörrie, Jan; Schaft, Niels; Strasser, Erwin; Hendelmeier, Martin; Kämpgen, Eckhart; Schuler, Gerold; Schuler-Thurner, Beatrice

2007-09-01

Dendritic cell (DC) vaccination approaches are advancing fast into the clinic. The major obstacle for further improvement is the current lack of a simple functionally "closed" system to generate standardized monocyte-derived (mo) DC vaccines. Here, we significantly optimized the use of the Elutra counterflow elutriation system to enrich monocytic DC precursors by (1) developing an algorithm to avoid red blood cell debulking and associated monocyte loss before elutriation, and (2) by elutriation directly in culture medium rather than phosphate-buffered saline. Upon elutriation the bags containing the collected monocytes are simply transferred into the incubator to generate DC progeny as the final "open" washing step is no longer required. Elutriation resulted in significantly more (> or = 2-fold) and purer DC than the standard gradient centrifugation/adherence-based monocyte enrichment, whereas morphology, maturation markers, viability, migratory capacity, and T cell stimulatory capacity were identical. Subsequently, we compared RNA transfection, as this is an increasingly used approach to load DC with antigen. Elutra-derived and adherence-derived DC could be electroporated with similar, high efficiency (on average >85% green fluorescence protein positive), and appeared also equal in antigen expression kinetics. Both Elutra-derived and adherence-derived DC, when loaded with the MelanA peptide or electroporated with MelanA RNA, showed a high T cell stimulation capacity, that is, priming of MelanA-specific CD8+ T cells. Our optimized Elutra-based procedure is straightforward, clearly superior to the standard gradient centrifugation/plastic adherence protocol, and now allows the generation of large numbers of peptide-loaded or RNA-transfected DC in a functionally closed system.

Gas6 Promotes Inflammatory (CCR2hiCX3CR1lo) Monocyte Recruitment in Venous Thrombosis.

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Laurance, Sandrine; Bertin, François-René; Ebrahimian, Talin; Kassim, Yusra; Rys, Ryan N; Lehoux, Stéphanie; Lemarié, Catherine A; Blostein, Mark D

2017-07-01

Coagulation and inflammation are inter-related. Gas6 (growth arrest-specific 6) promotes venous thrombosis and participates to inflammation through endothelial-innate immune cell interactions. Innate immune cells can provide the initiating stimulus for venous thrombus development. We hypothesize that Gas6 promotes monocyte recruitment during venous thrombosis. Deep venous thrombosis was induced in wild-type and Gas6-deficient (-/-) mice using 5% FeCl 3 and flow reduction in the inferior vena cava. Total monocyte depletion was achieved by injection of clodronate before deep venous thrombosis. Inflammatory monocytes were depleted using an anti-C-C chemokine receptor type 2 (CCR2) antibody. Similarly, injection of an anti-chemokine ligand 2 (CCL2) antibody induced CCL2 depletion. Flow cytometry and immunofluorescence were used to characterize the monocytes recruited to the thrombus. In vivo, absence of Gas6 was associated with a reduction of monocyte recruitment in both deep venous thrombosis models. Global monocyte depletion by clodronate leads to smaller thrombi in wild-type mice. Compared with wild type, the thrombi from Gas6 -/- mice contain less inflammatory (CCR2 hi CX 3 CR1 lo ) monocytes, consistent with a Gas6-dependent recruitment of this monocyte subset. Correspondingly, selective depletion of CCR2 hi CX 3 CR1 lo monocytes reduced the formation of venous thrombi in wild-type mice demonstrating a predominant role of the inflammatory monocytes in thrombosis. In vitro, the expression of both CCR2 and CCL2 were Gas6 dependent in monocytes and endothelial cells, respectively, impacting monocyte migration. Moreover, Gas6-dependent CCL2 expression and monocyte migration were mediated via JNK (c-Jun N-terminal kinase). This study demonstrates that Gas6 specifically promotes the recruitment of inflammatory CCR2 hi CX 3 CR1 lo monocytes through the regulation of both CCR2 and CCL2 during deep venous thrombosis. © 2017 American Heart Association, Inc.

Baseline and Trend of Lymphocyte-to-Monocyte Ratio as Prognostic Factors in Epidermal Growth Factor Receptor Mutant Non-Small Cell Lung Cancer Patients Treated with First-Line Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors.

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Yu-Mu Chen

Full Text Available Patients with early-stage lung cancer who have a high baseline lymphocyte-to-monocyte ratio (LMR have a favorable prognosis. However, the prognostic significance of LMR in patients with advanced-stage EGFR-mutant non-small cell lung cancer (NSCLC receiving first-line epidermal growth factor receptor (EGFR-tyrosine kinase inhibitors (TKIs has not been established. We conducted a retrospective analysis to investigate the influence of LMR on clinical outcomes including progression-free survival (PFS and overall survival (OS in EGFR-mutant patients with NSCLC.Of 1310 lung cancer patients diagnosed between January 2011 and October 2013, 253 patients receiving first-line EGFR-TKIs for EGFR-mutant NSCLC were included. The cut-off values for baseline and the 1-month-to-baseline ratio of LMR (MBR, determined by using receiver operating characteristic curves, were 3.29 and 0.63, respectively. Patients were divided into 3 prognostic groups: high LMR and MBR, high LMR or MBR, and low LMR and MBR.The mean patient age was 65.2 years, and 41% were men. The median PFS and OS were 10.3 and 22.0 months, respectively. The PFS in patients with high LMR and MBR, high LMR or MBR, and low LMR and MBR were 15.4, 7.1, and 2.0 months, respectively (p < 0.001, whereas the OS were 32.6, 13.7, and 5.1 months, respectively (p < 0.001.A combination of baseline and trend of LMR can be used to identify patients with a high mortality risk in EGFR-mutant NSCLC patients receiving first-line EGFR-TKIs.

Olopatadine Suppresses the Migration of THP-1 Monocytes Induced by S100A12 Protein

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2006-01-01

Full Text Available Olopatadine hydrochloride (olopatadine is an antiallergic drug with histamine H 1 receptor antagonistic activity. Recently, olopatadine has been shown to bind to S100A12 which is a member of the S100 family of calcium-binding proteins, and exerts multiple proinflammatory activities including chemotaxis for monocytes and neutrophils. In this study, we examined the possibility that the interaction of olopatadine with S100A12 inhibits the proinflammatory effects of S100A12. Pretreatment of olopatadine with S100A12 reduced migration of THP-1, a monocyte cell line, induced by S100A12 alone, but did not affect recombinant human regulated upon activation, normal T cell expressed and secreted (RANTES-induced migration. Amlexanox, which also binds to S100A12, inhibited the THP-1 migration induced by S100A12. However, ketotifen, another histamine H 1 receptor antagonist, had little effect on the activity of S100A12. These results suggest that olopatadine has a new mechanism of action, that is, suppression of the function of S100A12, in addition to histamine H 1 receptor antagonistic activity.

Cell lines authentication and mycoplasma detection as minimun quality control of cell lines in biobanking.

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Corral-Vázquez, C; Aguilar-Quesada, R; Catalina, P; Lucena-Aguilar, G; Ligero, G; Miranda, B; Carrillo-Ávila, J A

2017-06-01

Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.

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

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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.

Monocyte and lymphocyte surface molecules in severe sepsis and non-septic critically ill Patients.

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Jämsä, Joel; Syrjälä, Hannu; Huotari, Virva; Savolainen, Eeva-Riitta; Ala-Kokko, Tero

2017-06-01

The aim of the present study was to investigate whether expression of monocyte and lymphocyte surface molecules differs between patients with severe sepsis and non-septic patients treated in the intensive care unit (ICU). The expression of monocyte CD14, CD40, CD80 and HLA-DR, and lymphocyte CD69 were analyzed using quantitative flow cytometry on three consecutive days in 27 patients with severe sepsis and in 15 non-septic patients. Receiver operating characteristic analyses were performed and each corresponding area under the curve (AUC) was determined. The results showed that the expression levels of CD40 on monocytes and CD69 on CD4+ T cells and on natural killer (NK) cells were highest in patients with severe sepsis (p sepsis and positive blood culture compared with those with negative blood culture (p sepsis detection were 0.836 for CD40, 0.872 for CD69 on NK cells, and 0.795 for CD69 on CD4+ T cells. These findings suggest that monocyte CD40 and CD69 on NK cells and CD4+ T cells could prove useful for new approaches in the identification of severe sepsis in the ICU. © 2017 APMIS. Published by John Wiley & Sons Ltd.

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

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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.

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

Expression profiling feline peripheral blood monocytes identifies a transcriptional signature associated with type two diabetes mellitus.

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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.

Specific depletion of Ly6C(hi) inflammatory monocytes prevents immunopathology in experimental cerebral malaria.

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Schumak, Beatrix; Klocke, Katrin; Kuepper, Janina M; Biswas, Aindrila; Djie-Maletz, Andrea; Limmer, Andreas; van Rooijen, Nico; Mack, Matthias; Hoerauf, Achim; Dunay, Ildiko Rita

2015-01-01

Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice leads to experimental cerebral malaria (ECM) that is commonly associated with serious T cell mediated damage. In other parasitic infection models, inflammatory monocytes have been shown to regulate Th1 responses but their role in ECM remains poorly defined, whereas neutrophils are reported to contribute to ECM immune pathology. Making use of the recent development of specific monoclonal antibodies (mAb), we depleted in vivo Ly6C(hi) inflammatory monocytes (by anti-CCR2), Ly6G+ neutrophils (by anti-Ly6G) or both cell types (by anti-Gr1) during infection with Ovalbumin-transgenic PbA parasites (PbTg). Notably, the application of anti-Gr1 or anti-CCR2 but not anti-Ly6G antibodies into PbTg-infected mice prevented ECM development. In addition, depletion of Ly6C(hi) inflammatory monocytes but not neutrophils led to decreased IFNγ levels and IFNγ+CD8+ T effector cells in the brain. Importantly, anti-CCR2 mAb injection did not prevent the generation of PbTg-specific T cell responses in the periphery, whereas anti-Gr1 mAb injection strongly diminished T cell frequencies and CTL responses. In conclusion, the specific depletion of Ly6C(hi) inflammatory monocytes attenuated brain inflammation and immune cell recruitment to the CNS, which prevented ECM following Plasmodium infection, pointing out a substantial role of Ly6C+ monocytes in ECM inflammatory processes.

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...

Complement 5a Enhances Hepatic Metastases of Colon Cancer via Monocyte Chemoattractant Protein-1-mediated Inflammatory Cell Infiltration*

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Piao, Chunmei; Cai, Lun; Qiu, Shulan; Jia, Lixin; Song, Wenchao; Du, Jie

2015-01-01

Complement 5a (C5a), a potent immune mediator generated by complement activation, promotes tumor growth; however, its role in tumor metastasis remains unclear. We demonstrate that C5a contributes to tumor metastases by modulating tumor inflammation in hepatic metastases of colon cancer. Colon cancer cell lines generate C5a under serum-free conditions, and C5a levels increase over time in a murine syngeneic colon cancer hepatic metastasis model. Furthermore, in the absence of C5a receptor or upon pharmacological inhibition of C5a production with an anti-C5 monoclonal antibody, tumor metastasis is severely impaired. A lack of C5a receptor in colon cancer metastatic foci reduces the infiltration of macrophages, neutrophils, and dendritic cells, and the role for C5a receptor on these cells were further verified by bone marrow transplantation experiments. Moreover, C5a signaling increases the expression of the chemokine monocyte chemoattractant protein-1 and the anti-inflammatory molecules arginase-1, interleukin 10, and transforming growth factor β, but is inversely correlated with the expression of pro-inflammatory molecules, which suggests a mechanism for the role of C5a in the inflammatory microenvironment required for tumor metastasis. Our results indicate a new and potentially promising therapeutic application of complement C5a inhibitor for the treatment of malignant tumors. PMID:25739439

Adhesion kinetics of human primary monocytes, dendritic cells, and macrophages: Dynamic cell adhesion measurements with a label-free optical biosensor and their comparison with end-point assays.

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Orgovan, Norbert; Ungai-Salánki, Rita; Lukácsi, Szilvia; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Szabó, Bálint; Horvath, Robert

2016-09-01

Monocytes, dendritic cells (DCs), and macrophages (MFs) are closely related immune cells that differ in their main functions. These specific functions are, to a considerable degree, determined by the differences in the adhesion behavior of the cells. To study the inherently and essentially dynamic aspects of the adhesion of monocytes, DCs, and MFs, dynamic cell adhesion assays were performed with a high-throughput label-free optical biosensor [Epic BenchTop (BT)] on surfaces coated with either fibrinogen (Fgn) or the biomimetic copolymer PLL-g-PEG-RGD. Cell adhesion profiles typically reached their maximum at ∼60 min after cell seeding, which was followed by a monotonic signal decrease, indicating gradually weakening cell adhesion. According to the biosensor response, cell types could be ordered by increasing adherence as monocytes, MFs, and DCs. Notably, all three cell types induced a larger biosensor signal on Fgn than on PLL-g-PEG-RGD. To interpret this result, the molecular layers were characterized by further exploiting the potentials of the biosensor: by measuring the adsorption signal induced during the surface coating procedure, the authors could estimate the surface density of adsorbed molecules and, thus, the number of binding sites potentially presented for the adhesion receptors. Surfaces coated with PLL-g-PEG-RGD presented less RGD sites, but was less efficient in promoting cell spreading than those coated with Fgn; hence, other binding sites in Fgn played a more decisive role in determining cell adherence. To support the cell adhesion data obtained with the biosensor, cell adherence on Fgn-coated surfaces 30-60 min after cell seeding was measured with three complementary techniques, i.e., with (1) a fluorescence-based classical adherence assay, (2) a shear flow chamber applying hydrodynamic shear stress to wash cells away, and (3) an automated micropipette using vacuum-generated fluid flow to lift cells up. These techniques confirmed the results

Monocytes/Macrophages Control Resolution of Transient Inflammatory Pain

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

2014-01-01

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

Radiation sensitivity of Merkel cell carcinoma cell lines

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Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

1995-07-30

Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

Radiation sensitivity of Merkell cell carcinoma cell lines

International Nuclear Information System (INIS)

Leonard, J. Helen; Ramsay, Jonathan R.; Kearsley, John H.; Birrell, Geoff W.

1995-01-01

Purpose: Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Methods and Materials: Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after γ irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. Results: We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to γ irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Conclusion: Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution

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

Microparticles engineered to highly express peroxisome proliferator-activated receptor-γ decreased inflammatory mediator production and increased adhesion of recipient monocytes.

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Julie Sahler

Full Text Available Circulating blood microparticles are submicron vesicles released primarily by megakaryocytes and platelets that act as transcellular communicators. Inflammatory conditions exhibit elevated blood microparticle numbers compared to healthy conditions. Direct functional consequences of microparticle composition, especially internal composition, on recipient cells are poorly understood. Our objective was to evaluate if microparticle composition could impact the function of recipient cells, particularly during inflammatory provocation. We therefore engineered the composition of megakaryocyte culture-derived microparticles to generate distinct microparticle populations that were given to human monocytes to assay for influences recipient cell function. Herein, we tested the responses of monocytes exposed to either control microparticles or microparticles that contain the anti-inflammatory transcription factor, peroxisome proliferator-activated receptor-γ (PPARγ. In order to normalize relative microparticle abundance from two microparticle populations, we implemented a novel approach that utilizes a Nanodrop Spectrophotometer to assay for microparticle density rather than concentration. We found that when given to peripheral blood mononuclear cells, microparticles were preferentially internalized by CD11b+ cells, and furthermore, microparticle composition had a profound functional impact on recipient monocytes. Specifically, microparticles containing PPARγ reduced activated monocyte production of the proinflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared to activated monocytes exposed to control microparticles. Additionally, treatment with PPARγ microparticles greatly increased monocyte cell adherence. This change in morphology occurred simultaneously with increased production of the key extracellular matrix protein, fibronectin and increased expression of the fibronectin-binding integrin, ITGA5. PPARγ microparticles

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.

Lactic acid delays the inflammatory response of human monocytes

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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.

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

All trans retinoic acid abrogates spontaneous monocytic growth in juvenile chronic myelomonocytic leukaemia.

Science.gov (United States)

Cambier, N; Menot, M L; Schlageter, M H; Balitrand, N; Leblanc, T; Bordigoni, P; Rohrlich, P; Lamagnère, J P; Donadieu, J; Herbelin, C; Puissant, C; Gourand, F; Baruchel, A; Chomienne, C

2001-01-01

All trans retinoic acid, the active metabolite of vitamin A, exerts profound effects on cell differentiation. On normal myeloid progenitors, retinoids switch the differentiation program of granulo-macrophagic progenitors towards the granulocytic lineage and consequently reduce CFU-M colony formation. Bone marrow and peripheral blood mononuclear cells from children with Juvenile Chronic Myelomonocytic Leukaemia show typical spontaneous monocytic growth. We questioned whether in this disease, retinoids could switch myelomonocytic growth and inhibit the abnormal CFU-M colony proliferation. Ten JCML samples were studied in the presence of ATRA in methyl cellulose colony assay, before (CFU-C) or after (pre-CFU) liquid suspension culture. In vitro characteristics of JCML such as spontaneous monocytic growth in the absence of growth factor was noted in all patients. In the presence of leucocyte-conditioned medium, nine samples showed only CFU-M growth and one sample CFU-GM growth. Incubation with ATRA inhibited CFU-M colony formation in nine cases. Enhancement of granulocytic differentiation (CFU-G) was noted in nine cases. ATRA also inhibited CD34+ JCML monocytic growth and GM-CSF hypersensitivity. These data suggest that, in JCML progenitors, retinoid pathways are functional and inhibition of immature monocytic progenitors cells may be achieved with retinoids, without impeding granulocytic cell growth.

Circulating microparticles in acute diabetic Charcot foot exhibit a high content of inflammatory cytokines, and support monocyte-to-osteoclast cell induction.

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Pasquier, Jennifer; Thomas, Binitha; Hoarau-Véchot, Jessica; Odeh, Tala; Robay, Amal; Chidiac, Omar; Dargham, Soha R; Turjoman, Rebal; Halama, Anna; Fakhro, Khalid; Menzies, Robert; Jayyousi, Amin; Zirie, Mahmoud; Al Suwaidi, Jassim; Rafii, Arash; Malik, Rayaz A; Talal, Talal; Abi Khalil, Charbel

2017-11-27

Circulating microparticles (MPs) are major mediators in cardiovascular complications of type 2 diabetes (T2D); however, their contribution to Charcot foot (CF) disease is not known. Here, we purified and assessed the origin, concentration and content of circulating MPs from 33 individuals: 11 with T2D and acute CF, 11 T2D patients with equivalent neuropathy and 11 non-diabetic controls. First, we demonstrated that there were no differences in the distribution of MPs of endothelial, platelet origin among the 3 groups. However, MPs from leukocytes and monocytes origin were increased in CF patients. Moreover, we demonstrated that monocytes-derived MPs originated more frequently from intermediate and non-classical monocytes in CF patients. Five cytokines (G-CSF, GM-CSF, IL-1-ra, IL-2 and IL-16) were significantly increased in MPs from acute CF patients. Applying ingenuity pathways analysis, we found that those cytokines interacted well and induced the activation of pathways that are involved in osteoclast formation. Further, we treated THP-1 monocytes and monocytes sorted from healthy patients with CF-derived MPs during their differentiation into osteoclasts, which increased their differentiation into multinucleated osteoclast-like cells. Altogether, our study suggests that circulating MPs in CF disease have a high content of inflammatory cytokines and could increase osteoclast differentiation in vitro.

[Role of phosphoinositide 3 kinase/protein kinase B signal pathway in monocyte-endothelial adhesion induced by serum of rats with electrical burn].

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