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Sample records for human monocytes treated

  1. Interleukin-1 production by antibiotic-treated human monocytes.

    PubMed

    Roche, Y; Fay, M; Gougerot-Pocidalo, M A

    1988-05-01

    The effects of penicillin, macrolides (spiramycin and erythromycin), cephalosporins (cefaclor and cefadroxil), tetracycline (doxycycline) and quinolones (pefloxacin, ciprofloxacin and ofloxacin) on extracellular and cell-associated interleukin 1 (IL-1) activity from human adherent mononuclear leucocyte cells were investigated in vitro. When cells were treated with an antibiotic concentration of 10 mg/l, no apparent effect could be detected for penicillin, erythromycin, cephalosporins or quinolones, while a slight increase of extracellular IL-1 activity associated with a decrease of intracellular IL-1 activity was observed with spiramycin and doxycycline. When high antibiotic concentration were used, extracellular IL-1 activity was increased by macrolides and tetracycline, while both cell-associated and class II human monocyte antigen expression were decreased. A toxic effect may have been exerted by these antimicrobial agents, since cell viability was altered when they were used at high concentrations. In contrast, extracellular IL-1 activity was found to be decreased by quinolones and cephalosporins. Intracellular IL-1 activity was also decreased by cephalosporins, while quinolones did not modify either cell-associated IL-1 activity or class II human monocyte antigen expression. The effect induced by quinolones and cephalosporins occurred without modification of cell viability. IL-1 activity was shown to be affected by antibiotics over the same range of concentrations which are known to inhibit mononuclear leucocyte proliferation. Our data may help in defining the mechanism by which the mitogen-induced mononuclear proliferative response is suppressed by antimicrobial agents since this appears to involve the inhibition of IL-1 production or of its release.

  2. Absence of oxysterol-like side effects in human monocytic cells treated with phytosterols and oxyphytosterols.

    PubMed

    Vejux, Anne; Montange, Thomas; Martine, Lucy; Zarrouk, Amira; Riedinger, Jean-Marc; Lizard, Gérard

    2012-04-25

    Oxysterols, found in some commonly consumed foods, can induce a wide range of cytotoxic effects, which have been extensively studied. On the other hand, the side effects of phytosterols and oxyphytosterols are less well-known. Over the past few years, different types of foods have been enriched with phytosterols on the basis of the properties of these compounds that reduce circulating cholesterol levels in certain experimental conditions. It is therefore important to gain better knowledge of the risks and benefits of this type of diet. In this study, conducted in human monocytic U937 cells, the ability of phytosterols (sitosterol, campesterol) and oxyphytosterols (7β-hydroxysitosterol, 7-ketositosterol) to induce cell death, polar lipid accumulation, and pro-inflammatory cytokine (MCP-1; IL-8) secretion was determined and compared to that of oxysterols (7-ketocholesterol, 7β-hydroxycholesterol). Phytosterols and oxyphytosterols had no significant effects on the parameters studied; only 7β-hydroxysitosterol slightly increased cell death, whereas at the concentration used (20 μg/mL), strong cytotoxic effects were observed with the oxysterols. With sitosterol, campesterol, and 7-ketositosterol, IL-8 secretion was decreased, and with campesterol the intracellular polar lipid level was reduced. The data show that phytosterols and oxyphytosterols have no oxysterol-like side effects, and they rather argue in favor of phytosterols' beneficial effects.

  3. Monocyte heterogeneity in human cardiovascular disease.

    PubMed

    Zawada, Adam M; Rogacev, Kyrill S; Schirmer, Stephan H; Sester, Martina; Böhm, Michael; Fliser, Danilo; Heine, Gunnar H

    2012-12-01

    Atherosclerosis has been characterized as an inflammatory process, in which monocytes and monocyte-derived macrophages are of paramount importance. Contrasting with their established role in atherosclerosis, monocytes have not unanimously been found to predict cardiovascular events in large epidemiological studies. However, in these studies human monocyte heterogeneity has been largely overlooked so far. Three human monocyte subsets can be distinguished: classical CD14(++)CD16(-), intermediate CD14(++)CD16(+) and nonclassical CD14(+)CD16(++) monocytes. Of note, correct enumeration of subset counts requires appropriate staining and gating strategies that encompass a pan-monocytic marker (e.g. HLA-DR or CD86). In experimental studies on murine atherogenesis a monocyte subset-specific contribution to atherosclerosis has been established. However, major interspecies differences in atherogenesis itself, as well as in the immune system (including monocyte subset phenotype and distribution) preclude a direct extrapolation to human pathology. Experimental and pilot clinical studies point to a prominent involvement of intermediate CD14(++)CD16(+) monocytes in human atherosclerosis. Future clinical studies should analyze monocyte heterogeneity in cardiovascular disease. If a specific contribution of intermediate monocytes should be confirmed, immunomodulation of this monocyte subset could represent a future therapeutic target in atherosclerosis.

  4. Expression of leukocyte-endothelial cell adhesion molecules on monocyte adhesion to human endothelial cells on plasma treated PET and PTFE in vitro.

    PubMed

    Pu, F R; Williams, R L; Markkula, T K; Hunt, J A

    2002-12-01

    We used a coculture model to evaluate the inflammatory potential of ammonia gas plasma modified PET and PTFE by flow cytometry and immunohistochemistry. In these studies, human endothelial cells from umbilical cord (HUVEC) and promonocytic U937 cells were used. HUVECs grown on polystyrene tissue culture coverslips and HUVECs stimulated with tumour necrosis factor (TNF-alpha) were used as controls. U937 adhesion to endothelium on each surface was evaluated at day 1 and day 7. To further investigate the role of leukocyte-endothelial cell adhesion molecules (CAMs) in cell-to-cell interaction on material surfaces, the expression of the leukocyte-endothelial CAMs: ICAM-1, VCAM-1, PECAM-1, and E-selectin on HUVECs were evaluated after U937 cell adhesion. The results demonstrated that plasma treated PET (T-PET) and treated PTFE (T-PTFE) did not increase U937 cell adhesion compared to the negative control. Maximal adhesion of U937 cells to HUVEC was observed on TNF-alpha stimulated endothelium with significant differences between day 1 and day 7, which is consistent with our prior observation that T-PET and T-PTFE did not cause HUVECs to increase the expression of adhesion molecules. After U937 cell adhesion, the expression of ICAM-1 and VCAM-1 of HUVECs were not different on T-PET and T-PTFE compared with the negative control. However, the expression of E-selectin was reduced on day 1, but not on day 7. The effects of plasma treated PET and PTFE on HUVEC adhesion and proliferation were also studied. On day 1 there were slight increases in the growth of HUVECs on both of T-PET and T-PTFE but this was not statistically significant. On day 7, the cell number increased significantly on the surfaces compared to the negative control. The results demonstrate that the plasma treatment of PET and PTFE with ammonia improves the adhesion and growth of endothelial cells and these surfaces do not exhibit a direct inflammatory effect in terms of monocyte adhesion and expression of

  5. Successful Treatment of Human Monocytic Ehrlichiosis with Rifampin

    PubMed Central

    Ajmal, Saira; Hughes, Laura

    2016-01-01

    Currently recommended treatment regimens for human monocytic ehrlichiosis (HME) include doxycycline or tetracycline. Antibiotic susceptibility studies demonstrate that rifampin has in vitro bactericidal activity against Ehrlichia. Case reports have suggested clinical response with rifampin treatment of human granulocytic anaplasmosis (HGA). We report the first case of HME successfully treated with rifampin. PMID:26918212

  6. Lactic acid delays the inflammatory response of human monocytes

    SciTech Connect

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

    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.

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

    PubMed

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

    2015-04-01

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

  8. Initial Characterization of Monoclonal Antibodies against Human Monocytes

    NASA Astrophysics Data System (ADS)

    Ugolini, Valentina; Nunez, Gabriel; Smith, R. Graham; Stastny, Peter; Capra, J. Donald

    1980-11-01

    Three monoclonal antibodies against human monocytes have been produced by somatic cell fusion. Extensive specificity analysis suggests that these antibodies react with most if not all human peripheral blood monocytes and not with highly purified T or B cells. Initial chemical characterization of the monocyte antigen recognized by two of these antibodies is presented. The molecule is a single polypeptide chain with an apparent molecular weight of 200,000. These reagents should prove useful in the clinical definition of disorders of monocyte differentiation, in studies of monocyte function, and in the elucidation of the genetics and structure of monocyte cell surface antigens.

  9. Effect of the Monocyte Chemoattractant Protein-1/CC Chemokine Receptor 2 System on Nephrin Expression in Streptozotocin-Treated Mice and Human Cultured Podocytes

    PubMed Central

    Tarabra, Elena; Giunti, Sara; Barutta, Federica; Salvidio, Gennaro; Burt, Davina; Deferrari, Giacomo; Gambino, Roberto; Vergola, Daniela; Pinach, Silvia; Perin, Paolo Cavallo; Camussi, Giovanni; Gruden, Gabriella

    2009-01-01

    OBJECTIVE Monocyte chemoattractant protein-1 (MCP-1), a chemokine binding to the CC chemokine receptor 2 (CCR2) and promoting monocyte infiltration, has been implicated in the pathogenesis of diabetic nephropathy. To assess the potential relevance of the MCP-1/CCR2 system in the pathogenesis of diabetic proteinuria, we studied in vitro if MCP-1 binding to the CCR2 receptor modulates nephrin expression in cultured podocytes. Moreover, we investigated in vivo if glomerular CCR2 expression is altered in kidney biopsies from patients with diabetic nephropathy and whether lack of MCP-1 affects proteinuria and expression of nephrin in experimental diabetes. RESEARCH DESIGN AND METHODS Expression of nephrin was assessed in human podocytes exposed to rh-MCP-1 by immunofluorescence and real-time PCR. Glomerular CCR2 expression was studied in 10 kidney sections from patients with overt nephropathy and eight control subjects by immunohistochemistry. Both wild-type and MCP-1 knockout mice were made diabetic with streptozotocin. Ten weeks after the onset of diabetes, albuminuria and expression of nephrin, synaptopodin, and zonula occludens-1 were examined by immunofluorescence and immunoblotting. RESULTS In human podocytes, MCP-1 binding to the CCR2 receptor induced a significant reduction in nephrin both mRNA and protein expression via a Rho-dependent mechanism. The MCP-1 receptor, CCR2, was overexpressed in the glomerular podocytes of patients with overt nephropathy. In experimental diabetes, MCP-1 was overexpressed within the glomeruli and the absence of MCP-1 reduced both albuminuria and downregulation of nephrin and synaptopodin. CONCLUSIONS These findings suggest that the MCP-1/CCR2 system may be relevant in the pathogenesis of proteinuria in diabetes. PMID:19587356

  10. MicroRNA profiling of human intermediate monocytes.

    PubMed

    Zawada, Adam M; Zhang, Lu; Emrich, Insa E; Rogacev, Kyrill S; Krezdorn, Nicolas; Rotter, Björn; Fliser, Danilo; Devaux, Yvan; Ziegler-Heitbrock, Loems; Heine, Gunnar H

    2017-03-01

    Among the three human monocyte subsets, intermediate CD14++CD16+ monocytes have been characterized as particularly proinflammatory cells in experimental studies and as potential biomarkers of cardiovascular risk in clinical cohorts. To further substantiate the distinct role of intermediate monocytes within human monocyte heterogeneity, we assessed subset-specific expression of miRNAs as central epigenetic regulators of gene expression. We hypothesized that intermediate monocytes have a distinct miRNA profile compared to classical and non-classical monocytes. By using small RNA-seq we analyzed 662 miRNAs in the three monocyte subsets. We identified 38 miRNAs that are differentially expressed in intermediate monocytes compared to both classical and non-classical monocytes with a p value of <10(-10), of which two miRNAs - miR-6087 (upregulated) and miR-150-5p (downregulated) - differed in their expression more than ten-fold. Pathway analysis of the 38 differentially expressed miRNAs linked intermediate monocytes to distinct biological processes such as gene regulation, cell differentiation, toll-like receptor signaling as well as antigen processing and presentation. Moreover, differentially expressed miRNAs were connected to those genes that we previously identified as markers of intermediate monocytes. In aggregation, we provide first genome-wide miRNA data in the context of monocyte heterogeneity, which substantiate the concept of monocyte trichotomy in human immunity. The identification of miRNAs that are specific for intermediate monocytes may allow to develop strategies, which particularly target this cell population while sparing the other two subsets.

  11. Borrelia burgdorferi induces chemokines in human monocytes.

    PubMed Central

    Sprenger, H; Krause, A; Kaufmann, A; Priem, S; Fabian, D; Burmester, G R; Gemsa, D; Rittig, M G

    1997-01-01

    Lyme disease is clinically and histologically characterized by strong inflammatory reactions that contrast the paucity of spirochetes at lesional sites, indicating that borreliae induce mechanisms that amplify the inflammatory response. To reveal the underlying mechanisms of chemoattraction and activation of responding leukocytes, we investigated the induction of chemokines in human monocytes exposed to Borrelia burgdorferi by a dose-response and kinetic analysis. Lipopolysaccharide (LPS) derived from Escherichia coli was used as a positive control stimulus. The release of the CXC chemokines interleukin-8 (IL-8) and GRO-alpha and the CC chemokines MIP-1alpha, MCP-1, and RANTES was determined by specific enzyme-linked immunosorbent assays, and the corresponding gene expression patterns were determined by Northern blot analysis. The results showed a rapid and strong borrelia-inducible gene expression which was followed by the release of chemokines with peak levels after 12 to 16 h. Spirochetes and LPS were comparably effective in stimulating IL-8, GRO-alpha, MCP-1, and RANTES expression, whereas MIP-1alpha production preceded and exceeded chemokine levels induced by LPS. Unlike other bacteria, the spirochetes themselves did not bear or release factors with intrinsic chemotactic activity for monocytes or neutrophils. Thus, B. burgdorferi appears to be a strong inducer of chemokines which may, by the attraction and activation of phagocytic leukocytes, significantly contribute to inflammation and tissue damage observed in Lyme disease. PMID:9353009

  12. Effects of adherence, activation and distinct serum proteins on the in vitro human monocyte maturation process.

    PubMed

    Akiyama, Y; Griffith, R; Miller, P; Stevenson, G W; Lund, S; Kanapa, D J; Stevenson, H C

    1988-03-01

    Elutriator-purified human monocytes were cultured in a serum-free (SF) medium, and various serum proteins and functional activating agents were assessed for their effects on the in vitro maturation of human monocytes to macrophages. Following 3 days of suspension culture in Teflon labware, 60% of the monocytes were easily recovered. When varying concentrations of human AB serum (HuAB) were employed, human monocyte maturation progressed rapidly; the kinetics of this maturation process during cell suspension culture were very similar to the pattern observed following adherence culture. In contrast, when SF medium was employed, a marked retardation of the monocyte maturation process was observed; this could not be attributed to any changes in cell recovery and/or viability. Thus, cells could be maintained in their monocytoid form for 3 days when cultured in SF medium. When HuAB was added after 3 days of culture, human monocyte maturation into macrophages proceeded at a normal rate. We attempted to characterize certain of the serum protein(s) found in HuAB which promoted the monocyte maturation process. Human immunoglobulin G (IgG) was found to be the most potent serum protein in increasing 5'-N activity and decreasing peroxidase activity of suspension cultured monocytes. Immunoglobulin M (IgM) and albumin (Alb) were shown not to have significant monocyte maturation activity. Heat-treated human gamma globulin and IgG purified by high-performance liquid chromatography (HPLC) were shown to have patterns identical with that of untreated HGG and IgG with regard to promoting monocyte maturation; F(ab')2 was not an active maturation promoter, indicating the need for an intact Fc portion of the IgG molecule. Fibrinogen and fibronectin also had maturation promoting activity. Finally, addition of the potent monocyte functional activators, muramyl dipeptide (MDP), polyriboinosinic:polyribocytidilic acid (Poly I:C), and lipopolysaccharide (LPS) had no effect on the monocyte

  13. Interaction between human peripheral blood monocytes and tumor promoters: Effect on growth differentiation and function in vitro

    SciTech Connect

    Keisari, Y.; Bucana, C.; Markovich, S.; Campbell, D.E. )

    1990-08-01

    Studies on the differentiation and activation of human monocytes in tissue cultures have usually been limited by the deterioration of human monocytes and macrophages in long-term cultures. In this study, we attempted to establish long-term human monocyte/macrophage cultures using the phorbol ester 12-0 tetradecanoyl-phorbol-13-acetate (TPA), and we studied the morphology, function, and biochemical properties of such treated human blood monocytes. Enriched suspensions of monocytes were obtained using Ficoll-Hypaque gradient and cultured in the absence or presence of various concentrations of TPA. Samples were removed at different times and processed for scanning electron microscopy. Parallel samples were examined for numbers of adherent cells, phagocytosis, oxidative burst, beta-galactosidase assays, and lectin-mediated erythrolysis. TPA-treated monocytes survived in larger numbers in culture for up to 7 weeks and were more pleomorphic and exhibited higher beta-galactosidase activities after 14 days in culture than untreated monocytes. TPA-treated cells and untreated cells in long-term cultures showed a decrease in their oxidative burst activity while their phagocytic activity was not affected, and the TPA treatment augmented the lysis of wheat germ agglutinin-opsonized erythrocytes by the cultured monocytes. TPA treatment of adherent human monocytes resulted in cell cultures with increased numbers of viable and functionally adherent cells for extended periods of time and does not seem to interfere with the differentiation and maturation of the cells in culture.

  14. Activated platelets signal chemokine synthesis by human monocytes.

    PubMed Central

    Weyrich, A S; Elstad, M R; McEver, R P; McIntyre, T M; Moore, K L; Morrissey, J H; Prescott, S M; Zimmerman, G A

    1996-01-01

    Human blood monocytes adhere rapidly and for prolonged periods to activated platelets that display P-selectin, an adhesion protein that recognizes a specific ligand on leukocytes, P-selectin glycoprotein-1. We previously demonstrated that P-selectin regulates expression and secretion of cytokines by stimulated monocytes when it is presented in a purified, immobilized form or by transfected cells. Here we show that thrombin-activated platelets induce the expression and secretion of monocyte chemotactic protein-1 and IL-8 by monocytes. Enhanced monokine synthesis requires engagement of P-selectin glycoprotein-1 on the leukocyte by P-selectin on the platelet. Secretion of the chemokines is not, however, directly signaled by P-selectin; instead, tethering of the monocytes by P-selectin is required for their activation by RANTES (regulated upon activation normal T cell expressed presumed secreted), a platelet chemokine not previously known to induce immediate-early gene products in monocytes. Adhesion of monocytes to activated platelets results in nuclear translocation of p65 (RelA), a component of the NF-kappaB family of transcription factors that binds kappaB sequences in the regulatory regions of monocyte chemotactic protein-1, IL-8, and other immediate-early genes. However, expression of tissue factor, a coagulation protein that also has a kappaB sequence in the 5' regulatory region of its gene, is not induced in monocytes adherent to activated platelets. Thus, contact of monocytes with activated platelets differentially affects the expression of monocyte products. These experiments suggest that activated platelets regulate chemokine secretion by monocytes in inflammatory lesions in vivo and provide a model for the study of gene regulation in cell-cell interactions. PMID:8617886

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

    SciTech Connect

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

    1984-06-01

    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.

  16. Chromatin Immunoprecipitation for Human Monocyte Derived Macrophages

    PubMed Central

    Wooden, Jessica; Ciborowski, Pawel

    2014-01-01

    The importance of Chromatin Immunoprecipitation (ChIP) technology has grown exponentially along with an increased interest in epigenetic regulation. The correlation of transcription factors with histone marks is now well established as the center of epigenetic studies; therefore, precise knowledge about histone marks is critical to unravel their molecular function and to understand their role in biological systems. This knowledge constantly accumulates and is provided openly in the expanding hubs of information such as the USCS Genome Browser. Nevertheless, as we gain more knowledge, we realize that the DNA-protein interactions are not driven by a “one size fits all” rule. Also, the diversity of interactions between DNA, histones, and transcriptional regulators is much bigger than previously considered. Besides a detailed protocol of sample preparation for the ChIP assay from primary human monocyte-derived macrophages (MDM)a, we show that differences between various types of cells exist. Furthermore, we can postulate that such variations exist between transformed macrophage-like cell lines and primary macrophages obtained from healthy volunteers. We found that the most efficient fixation time for MDM is 10 minutes. Finally, to perform multiple analytical assays, we showed that even with thorough methodology, the yield of material obtained from primary cells is the major challenge. PMID:25220915

  17. Human monocyte differentiation stage affects response to arachidonic acid.

    PubMed

    Escobar-Alvarez, Elizabeth; Pelaez, Carlos A; García, Luis F; Rojas, Mauricio

    2010-01-01

    AA-induced cell death mechanisms acting on human monocytes and monocyte-derived macrophages (MDM), U937 promonocytes and PMA-differentiated U937 cells were studied. Arachidonic acid induced apoptosis and necrosis in monocytes and U937 cells but only apoptosis in MDM and U937D cells. AA increased both types of death in Mycobacterium tuberculosis-infected cells and increased the percentage of TNFalpha+ cells and reduced IL-10+ cells. Experiments blocking these cytokines indicated that AA-mediated death was TNFalpha- and IL-10-independent. The differences in AA-mediated cell death could be explained by high ROS, calpain and sPLA-2 production and activity in monocytes. Blocking sPLA-2 in monocytes and treatment with antioxidants favored M. tuberculosis control whereas AA enhanced M. tuberculosis growth in MDM. Such evidence suggested that AA-modulated effector mechanisms depend on mononuclear phagocytes' differentiation stage.

  18. Design of phosphorylated dendritic architectures to promote human monocyte activation.

    PubMed

    Poupot, Mary; Griffe, Laurent; Marchand, Patrice; Maraval, Alexandrine; Rolland, Olivier; Martinet, Ludovic; L'Faqihi-Olive, Fatima-Ezzahra; Turrin, Cédric-Olivier; Caminade, Anne-Marie; Fournié, Jean-Jacques; Majoral, Jean-Pierre; Poupot, Rémy

    2006-11-01

    As first defensive line, monocytes are a pivotal cell population of innate immunity. Monocyte activation can be relevant to a range of immune conditions and responses. Here we present new insights into the activation of monocytes by a series of phosphonic acid-terminated, phosphorus-containing dendrimers. Various dendritic or subdendritic structures were synthesized and tested, revealing the basic structural requirements for monocyte activation. We showed that multivalent character and phosphonic acid capping of dendrimers are crucial for monocyte targeting and activation. Confocal videomicroscopy showed that a fluorescein-tagged dendrimer binds to isolated monocytes and gets internalized within a few seconds. We also found that dendrimers follow the phagolysosomial route during internalization by monocytes. Finally, we performed fluorescence resonance energy transfer (FRET) experiments between a specifically designed fluorescent dendrimer and phycoerythrin-coupled antibodies. We showed that the typical innate Toll-like receptor (TLR)-2 is clearly involved, but not alone, in the sensing of dendrimers by monocytes. In conclusion, phosphorus-containing dendrimers appear as precisely tunable nanobiotools able to target and activate human innate immunity and thus prove to be good candidates to develop new drugs for immunotherapies.

  19. Monocyte chemoattractant protein-1 in human atheromatous plaques.

    PubMed Central

    Nelken, N A; Coughlin, S R; Gordon, D; Wilcox, J N

    1991-01-01

    Monocytes appear to be central to atherogenesis both as the progenitors of foam cells and as a potential source of growth factors mediating intimal hyperplasia, but the chemical messages which stimulate the influx of monocytes into human atheroma remain unknown. Monocyte chemoattractant protein-1 (MCP-1) is a recently described molecule with powerful monocyte chemotactic activity expressed by monocytes, vascular endothelial cells, and smooth muscle cells in culture. To begin to address the role of MCP-1 in vivo, we examined 10 normal arteries and 14 diseased human arteries for MCP-1 expression by in situ hybridization. MCP-1 mRNA was detected in 16% of 10,768 cells counted in human carotid endarterectomy specimens with highest expression seen in organizing thrombi (33%) and in macrophage rich areas bordering the necrotic lipid core (24%) as compared to the fibrous cap (8%) and the necrotic lipid core itself (5%). Based on immunohistochemical staining of serial sections and on cell morphology, MCP-1 mRNA appeared to be expressed by vascular smooth muscle cells (VSMC), mesenchymal appearing intimal cells (MICs), and macrophages. By contrast, few cells expressing MCP-1 mRNA were found in normal arteries (less than 0.1%). These data suggest a potential role for MCP-1 in mediating monocytic infiltration of the artery wall. Images PMID:1843454

  20. Tumoricidal activity of human monocytes activated in vitro by free and liposome-encapsulated human lymphokines.

    PubMed Central

    Kleinerman, E S; Schroit, A J; Fogler, W E; Fidler, I J

    1983-01-01

    Human peripheral blood mononuclear cells from normal donors obtained by separation on a Percoll gradient were incubated with free or liposome-entrapped lymphokines produced from concanavalin A-stimulated lymphocytes and then were tested for cytotoxic activity against tumor cells. The treated monocytes lysed tumorigenic melanoma and glioblastoma target cells, but had no effect on three types of nontumorigenic target cells. The activation of monocytes to become tumoricidal was caused by macrophage-activating factor (MAF) and not by contamination with endotoxins, concanavalin A, or interferon. The endocytosis of liposomes containing MAF, but not of those containing control supernatants, led to the activation of cytotoxic properties in the monocytes. Activation by liposome-encapsulated MAF was very efficient and required less than 1/800th of the amount of free MAF necessary to achieve the same levels of cytotoxicity. Thus, the encapsulation of mitogen-induced MAF in liposomes could provide an effective approach for the activation of blood monocytes in situ. Images FIGURE 2 PMID:6348087

  1. Derivation of multipotent progenitors from human circulating CD14+ monocytes.

    PubMed

    Seta, Noriyuki; Kuwana, Masataka

    2010-07-01

    Circulating CD14(+) monocytes are originated from hematopoietic stem cells in the bone marrow and believed to be committed precursors for phagocytes, such as macrophages. Recently, we have reported a primitive cell population termed monocyte-derived multipotential cells (MOMCs), which has a fibroblast-like morphology in culture and a unique phenotype positive for CD14, CD45, CD34, and type I collagen. MOMCs are derived from circulating CD14(+) monocytes, but circulating precursors for MOMCs still remain undetermined. Comparative analysis of gene expression profiles of MOMCs and other monocyte-derived cells has revealed that embryonic stem cell markers, Nanog and Oct-4, are specifically expressed by MOMCs. In vitro generation of MOMCs requires binding to fibronectin and exposure to soluble factors derived from activated platelets. MOMCs contain progenitors with capacity to differentiate into a variety of nonphagocytes, including bone, cartilage, fat, skeletal and cardiac muscle, neuron, and endothelium, indicating that circulating monocytes are more multipotent than previously thought. In addition, MOMCs are capable of promoting ex vivo expansion of human hematopoietic progenitor cells through direct cell-to-cell contact and secretion of a variety of hematopoietic growth factors. These findings obtained from the research on MOMCs indicate that CD14(+) monocytes in circulation are involved in a variety of physiologic functions other than innate and acquired immune responses, such as repair and regeneration of the damaged tissue.

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

    NASA Astrophysics Data System (ADS)

    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.

  3. Identification of Motile Sperm Domain–Containing Protein 2 as Regulator of Human Monocyte Migration

    PubMed Central

    Yacov, Niva; Salem, Yaniv; Propheta-Meiran, Oshrat; Ishai, Eti; Breitbart, Eyal

    2017-01-01

    Binding of chemokines to their cognate receptors on monocytes instigates a cascade of events that directs these cells to migrate to sites of inflammation and cancerous tissues. Although targeting of selected chemokine receptors on monocytes exhibited preclinical efficacy, attempts to translate these studies to the clinic have failed thus far, possibly due to redundancy of the target receptor. We reveal that motile sperm domain–containing protein 2 (MOSPD2), a protein with a previously unknown function, regulates monocyte migration in vitro. This protein was found to be expressed on the cytoplasmic membrane of human monocytes. Silencing or neutralizing MOSPD2 in monocytes restricted their migration when induced by different chemokines. Mechanistically, silencing MOSPD2 inhibited signaling events following chemokine receptor ligation. When tested for expression in other immune cell subsets, MOSPD2 was apparent also, though less abundantly, in neutrophils, but not in lymphocytes. Thus, in the presence of neutralizing Abs, neutrophil migration was inhibited to some extent whereas lymphocyte migration remained intact. In view of these results, we suggest MOSPD2 as a potential target protein for treating diseases in which monocyte and neutrophil accumulation is correlated with pathogenesis. PMID:28137892

  4. Identification of Motile Sperm Domain-Containing Protein 2 as Regulator of Human Monocyte Migration.

    PubMed

    Mendel, Itzhak; Yacov, Niva; Salem, Yaniv; Propheta-Meiran, Oshrat; Ishai, Eti; Breitbart, Eyal

    2017-03-01

    Binding of chemokines to their cognate receptors on monocytes instigates a cascade of events that directs these cells to migrate to sites of inflammation and cancerous tissues. Although targeting of selected chemokine receptors on monocytes exhibited preclinical efficacy, attempts to translate these studies to the clinic have failed thus far, possibly due to redundancy of the target receptor. We reveal that motile sperm domain-containing protein 2 (MOSPD2), a protein with a previously unknown function, regulates monocyte migration in vitro. This protein was found to be expressed on the cytoplasmic membrane of human monocytes. Silencing or neutralizing MOSPD2 in monocytes restricted their migration when induced by different chemokines. Mechanistically, silencing MOSPD2 inhibited signaling events following chemokine receptor ligation. When tested for expression in other immune cell subsets, MOSPD2 was apparent also, though less abundantly, in neutrophils, but not in lymphocytes. Thus, in the presence of neutralizing Abs, neutrophil migration was inhibited to some extent whereas lymphocyte migration remained intact. In view of these results, we suggest MOSPD2 as a potential target protein for treating diseases in which monocyte and neutrophil accumulation is correlated with pathogenesis.

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

    PubMed

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

    2014-01-01

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

  6. Soy isoflavones attenuate human monocyte adhesion to endothelial cell-specific CD54 by inhibiting monocyte CD11a.

    PubMed

    Nagarajan, Shanmugam; Stewart, Bradford W; Badger, Thomas M

    2006-09-01

    Soy-based diets have been shown to protect against the development of atherosclerosis; however, the underlying mechanism(s) remain unknown. Interaction between activated monocytes and inflamed endothelial cells is an early event in atherogenesis. Therefore, we examined whether treatment of monocytes with soy phytochemicals could inhibit their adhesion to the endothelial cell-specific protein, CD54, a key factor in monocyte adhesion. Female Sprague-Dawley rats were fed AIN-93G diets containing soy protein isolate or casein. Sera from soy-fed rats inhibited CD54-dependent monocyte adhesion, whereas sera from casein-fed rats did not. To determine whether isoflavones in the sera of soy-fed rats were involved in this inhibition, monocytes were preincubated with soy isoflavones. Isoflavone treatment inhibited monocyte adhesion to CD54 protein, as well as to endothelial cells expressing CD54. Monocyte expression of CD11a, the cognate receptor for CD54, was unaffected by isoflavones. However, binding of the activation epitope-specific antibody mAb24, which binds specifically to the active form of CD11a, was significantly lower in soy isoflavone-treated monocytes than in media-treated cells. These findings suggest that inhibition of CD54-dependent monocyte adhesion by soy isoflavones is mediated in part by affinity regulation of CD11a. Inhibition of monocyte adhesion to endothelial cells by isoflavones resulted in reduced expression of the inflammatory cytokines IL-6 and IL-8. Collectively, these data suggest that the athero-protective effect of soy diets may be mediated by blocking monocyte-endothelial cell interaction.

  7. Growth hormone activation of human monocytes for superoxide production but not tumor necrosis factor production, cell adherence, or action against Mycobacterium tuberculosis.

    PubMed Central

    Warwick-Davies, J; Lowrie, D B; Cole, P J

    1995-01-01

    We have previously demonstrated that growth hormone (GH) is a human macrophage-activating factor which primes monocytes for enhanced production of H2O2 in vitro. This report extends our observations to other monocyte functions relevant to infection. We find that GH also primes monocytes for O2- production, to a degree similar to the effect of gamma interferon. Neither macrophage-activating factor alone stimulates monocytes to release bioactive tumor necrosis factor. However, GH, unlike gamma interferon, does not synergize with endotoxin for enhanced tumor necrosis factor production. In further contrast, GH does not alter monocyte adherence or morphology, while phagocytosis and killing of Mycobacterium tuberculosis by GH-treated monocytes are also unaffected. Therefore, despite the multiplicity of the effects of GH on the immune system in vivo, its effects on human monocytes in vitro appear to be limited to priming for the release of reactive oxygen intermediates. PMID:7591064

  8. The monocyte binding domain(s) on human immunoglobulin G.

    PubMed

    Woof, J M; Nik Jaafar, M I; Jefferis, R; Burton, D R

    1984-06-01

    Monocyte binding has previously been assigned to the C gamma 3 domain of human immunoglobulin G (IgG) largely on the ability of the pFc' fragment to inhibit the monocyte-IgG interaction. This ability is markedly reduced compared to the intact parent IgG. We find this result with a conventional pFc' preparation but this preparation is found to contain trace contamination of parent IgG as demonstrated by reactivity with monoclonal antibodies directed against C gamma 2 domain and light-chain epitopes of human IgG. Extensive immunoaffinity purification of the pFc' preparation removes its inhibitory ability indicating that this originates in the trace contamination of parent IgG (or Fc). Neither of the human IgG1 paraproteins TIM, lacking the C gamma 2 domain, or SIZ, lacking the C gamma 3 domain, are found to inhibit the monocyte-IgG interaction. The hinge-deleted IgG1 Dob protein shows little or no inhibitory ability. Indirect evidence for the involvement of the C gamma 2 domain in monocyte binding is considered. We suggest finally that the site of interaction is found either on the C gamma 2 domain alone or between the C gamma 2 and C gamma 3 domains.

  9. The inability of human immunodeficiency virus to infect chimpanzee monocytes can be overcome by serial viral passage in vivo.

    PubMed Central

    Gendelman, H E; Ehrlich, G D; Baca, L M; Conley, S; Ribas, J; Kalter, D C; Meltzer, M S; Poiesz, B J; Nara, P

    1991-01-01

    Studies of lentivirus infection in ruminants, nonhuman primates, and humans suggest that virus infection of macrophages plays a central role in the disease process. To investigate whether human immunodeficiency virus type 1 (HIV-1) can infect chimpanzee macrophages, we recovered monocytes from peripheral blood mononuclear cells of HIV-1-negative animals and inoculated these and control human monocytes with a panel of four human-passaged monocytotropic virus strains and one chimpanzee-passaged isolate. HIV-1 infected human monocytes synthesized proviral DNA, viral mRNA, p24 antigen, and progeny virions. In contrast, except for the chimpanzee-passaged HIV-1 isolate, chimpanzee monocytes failed to support HIV-1 replication when cultured under both identical and a variety of other conditions. Proviral DNA was demonstrated only at background levels in these cell cultures by polymerase chain reaction for gag- and env-related sequences. Interestingly, the chimpanzee-passaged HIV-1 isolate did not replicate in human monocytes; viral p24 antigens and progeny virions were not detected. The same monocytotropic panel of HIV-1 strains replicated in both human and chimpanzee CD4+ T lymphoblasts treated with phytohemagglutinin and interleukin-2. The failure of HIV-1 to infect chimpanzee monocytes, which can be overcome by serial in vivo viral passage, occurs through a block early in the viral life cycle. Images PMID:1674968

  10. Effects of botulinum toxin type D on secretion of tumor necrosis factor from human monocytes

    SciTech Connect

    Imamura, K.; Spriggs, D.; Ohno, T.; Kufe, D.

    1989-05-01

    Botulinum toxins are potent neurotoxins which block the release of neurotransmitters. The effects of these toxins on hematopoietic cells, however, are unknown. Monocytes secrete a variety of polypeptide growth factors, including tumor necrosis factor (TNF). In the study reported here, the effects of botulinum toxin type D on the secretion of TNF from human monocytes were examined. The results demonstrate that biotulinum toxin type D inhibits the release of TNF from monocytes activated by lipopolysaccharide (LPS) but not by 12-O-tetradecanoylphorbol-13-acetate. Botulinum toxin type D had no detectable effect on intracellular TNF levels in LPS-treated monocytes, indicating that the effects of this toxin involve the secretory process. This inhibitory effect of botulinum toxin type D on TNF secretion from LPS-treated monocytes was partially reversed by treatment with 12-O-tetradecanoylphorbol-13-acetate or introduction of guanosine 5'-(/gamma/-thio)t-riphosphate into these cells. The results demonstrate that TNF secretion is regulated by at least two distinct guanine nucleotide-binding proteins, one responsible for the activation of phospholiphase C and another which acts as a substrate for botulinum toxin type D. ADP-ribosylation of monocyte membranes by botulinum toxin type D demonstrated the presence of three substrates with M/sub r/s of 45,000, 21,000, and 17,000. While the role of these substrates in exocytosis is unknown, the results suggest that the M/sub r/ 21,000 substrate is involved in a process other than TNF secretion.

  11. Glucocorticoids enhance the in vivo migratory response of human monocytes.

    PubMed

    Yeager, Mark P; Pioli, Patricia A; Collins, Jane; Barr, Fiona; Metzler, Sara; Sites, Brian D; Guyre, Paul M

    2016-05-01

    Glucocorticoids (GCs) are best known for their potent anti-inflammatory effects. However, an emerging model for glucocorticoid (GC) regulation of in vivo inflammation also includes a delayed, preparatory effect that manifests as enhanced inflammation following exposure to an inflammatory stimulus. When GCs are transiently elevated in vivo following exposure to a stressful event, this model proposes that a subsequent period of increased inflammatory responsiveness is adaptive because it enhances resistance to a subsequent stressor. In the present study, we examined the migratory response of human monocytes/macrophages following transient in vivo exposure to stress-associated concentrations of cortisol. Participants were administered cortisol for 6h to elevate in vivo cortisol levels to approximate those observed during major systemic stress. Monocytes in peripheral blood and macrophages in sterile inflammatory tissue (skin blisters) were studied before and after exposure to cortisol or placebo. We found that exposure to cortisol induced transient upregulation of monocyte mRNA for CCR2, the receptor for monocyte chemotactic protein-1 (MCP-1/CCL2) as well as for the chemokine receptor CX3CR1. At the same time, mRNA for the transcription factor IκBα was decreased. Monocyte surface expression of CCR2 but not CX3CR1 increased in the first 24h after cortisol exposure. Transient exposure to cortisol also led to an increased number of macrophages and neutrophils in fluid derived from a sterile inflammatory site in vivo. These findings suggest that the delayed, pro-inflammatory effects of cortisol on the human inflammatory responses may include enhanced localization of effector cells at sites of in vivo inflammation.

  12. Anticancer drug bortezomib increases interleukin-8 expression in human monocytes.

    PubMed

    Sanacora, Shannon; Urdinez, Joaquin; Chang, Tzu-Pei; Vancurova, Ivana

    2015-05-01

    Bortezomib (BZ) is the first clinically approved proteasome inhibitor that has shown remarkable anticancer activity in patients with hematological malignancies. However, many patients relapse and develop resistance; yet, the molecular mechanisms of BZ resistance are not fully understood. We have recently shown that in solid tumors, BZ unexpectedly increases expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8), while it inhibits expression of other NFκB-regulated genes. Since monocytes and macrophages are major producers of IL-8, the goal of this study was to test the hypothesis that BZ increases the IL-8 expression in human monocytes and macrophages. Here, we show that BZ dramatically increases the IL-8 expression in lipopolysaccharide (LPS)-stimulated U937 macrophages as well as in unstimulated U937 monocytes and peripheral blood mononuclear cells, while it inhibits expression of IL-6, IL-1 and tumor necrosis factor-α. In addition, our results show that the underlying mechanisms involve p38 mitogen-activated protein kinase, which is required for the BZ-induced IL-8 expression. Together, these data suggest that the BZ-increased IL-8 expression in monocytes and macrophages may represent one of the mechanisms responsible for the BZ resistance and indicate that targeting the p38-mediated IL-8 expression could enhance the BZ effectiveness in cancer treatment.

  13. A curated compendium of monocyte transcriptome datasets of relevance to human monocyte immunobiology research

    PubMed Central

    Rinchai, Darawan; Boughorbel, Sabri; Presnell, Scott; Quinn, Charlie; Chaussabel, Damien

    2016-01-01

    Systems-scale profiling approaches have become widely used in translational research settings. The resulting accumulation of large-scale datasets in public repositories represents a critical opportunity to promote insight and foster knowledge discovery. However, resources that can serve as an interface between biomedical researchers and such vast and heterogeneous dataset collections are needed in order to fulfill this potential. Recently, we have developed an interactive data browsing and visualization web application, the Gene Expression Browser (GXB). This tool can be used to overlay deep molecular phenotyping data with rich contextual information about analytes, samples and studies along with ancillary clinical or immunological profiling data. In this note, we describe a curated compendium of 93 public datasets generated in the context of human monocyte immunological studies, representing a total of 4,516 transcriptome profiles. Datasets were uploaded to an instance of GXB along with study description and sample annotations. Study samples were arranged in different groups. Ranked gene lists were generated based on relevant group comparisons. This resource is publicly available online at http://monocyte.gxbsidra.org/dm3/landing.gsp. PMID:27158452

  14. Priming of human monocytes for enhanced lipopolysaccharide responses: expression of alpha interferon, interferon regulatory factors, and tumor necrosis factor.

    PubMed Central

    Hayes, M P; Zoon, K C

    1993-01-01

    Culture of human monocytes with either granulocyte-macrophage colony-stimulating factor or gamma interferon (IFN-gamma) results in a primed state, during which these cells express heightened responses to bacterial lipopolysaccharide (LPS). The production of IFN-alpha in response to LPS by human monocytes has an absolute requirement for priming. Tumor necrosis factor (TNF) expression is also greatly enhanced in primed monocytes after LPS stimulation, but unlike IFN-alpha, TNF is readily expressed in unprimed monocytes as well. In an effort to determine the molecular events associated with IFN-alpha induction in this system, freshly isolated human monocytes were primed by culture with either IFN-gamma or granulocyte-macrophage colony-stimulating factor and then treated with LPS; expression of IFN-alpha subtype 2 (IFN-alpha 2), IFN regulatory factors (IRFs), and TNF was assessed by Northern (RNA blot) analysis. IRF-1 mRNA is expressed at high levels in monocytes and is regulated by both LPS and priming cytokines, but its expression alone does not correlate with the induction of IFN-alpha 2 expression. IRF-2 mRNA is expressed in a more gradual manner following LPS stimulation, implying a possible feedback mechanism for inhibiting IFN-alpha expression. However, nuclear run-on analysis indicates that IFN-alpha 2 is not transcriptionally modulated in this system, in striking contrast to TNF, which is clearly regulated at the transcriptional level. In addition, IFN-alpha 2 mRNA accumulation is superinduced when primed monocytes are treated with LPS plus cycloheximide, while TNF mRNA is relatively unaffected. The results demonstrate that priming can affect subsequent LPS-induced gene expression at different levels in human monocytes. Images PMID:8335353

  15. Bacillus anthracis’ lethal toxin induces broad transcriptional responses in human peripheral monocytes

    PubMed Central

    2012-01-01

    Background Anthrax lethal toxin (LT), produced by the Gram-positive bacterium Bacillus anthracis, is a highly effective zinc dependent metalloprotease that cleaves the N-terminus of mitogen-activated protein kinase kinases (MAPKK or MEKs) and is known to play a role in impairing the host immune system during an inhalation anthrax infection. Here, we present the transcriptional responses of LT treated human monocytes in order to further elucidate the mechanisms of LT inhibition on the host immune system. Results Western Blot analysis demonstrated cleavage of endogenous MEK1 and MEK3 when human monocytes were treated with 500 ng/mL LT for four hours, proving their susceptibility to anthrax lethal toxin. Furthermore, staining with annexin V and propidium iodide revealed that LT treatment did not induce human peripheral monocyte apoptosis or necrosis. Using Affymetrix Human Genome U133 Plus 2.0 Arrays, we identified over 820 probe sets differentially regulated after LT treatment at the p <0.001 significance level, interrupting the normal transduction of over 60 known pathways. As expected, the MAPKK signaling pathway was most drastically affected by LT, but numerous genes outside the well-recognized pathways were also influenced by LT including the IL-18 signaling pathway, Toll-like receptor pathway and the IFN alpha signaling pathway. Multiple genes involved in actin regulation, signal transduction, transcriptional regulation and cytokine signaling were identified after treatment with anthrax LT. Conclusion We conclude LT directly targets human peripheral monocytes and causes multiple aberrant gene responses that would be expected to be associated with defects in human monocyte’s normal signaling transduction pathways and function. This study provides further insights into the mechanisms associated with the host immune system collapse during an anthrax infection, and suggests that anthrax LT may have additional downstream targets outside the well-known MAPK

  16. Monophosphoryl lipid A stimulated up-regulation of nitric oxide synthase and nitric oxide release by human monocytes in vitro.

    PubMed

    Saha, D C; Astiz, M E; Lin, R Y; Rackow, E C; Eales, L J

    1997-10-01

    Monophosphoryl lipid A (MPL) is a derivative of lipopolysaccharide (LPS) with reduced toxicity which has been shown to modulate various immune functions in monocytes. We examined whether human monocytes can be stimulated to produce nitric oxide (NO) and its catalytic enzyme nitric oxide synthase (NOS). Monocytes were stimulated with LPS or MPL and both NOS and NO (as nitrite) production were measured. MPL at high doses (> 100 micrograms/ml) stimulated monocytes to release NO that was significantly greater than both the control and LPS-treated monocytes (p < 0.05). NO release by control cells and the LPS treated cells was not significantly different. Both arginase and N-monomethyl arginine (NMLA) inhibited the MPL stimulated release of NO (p < 0.01). MPL significantly increased inducible NOS (iNOS) expression as measured by both fluorescent microscopy and flow cytometry (p < 0.05). Similarly, both soluble NOS (sNOS) and particulate NOS (pNOS) activity were significantly up-regulated by MPL (p < 0.05). Significant correlations were found between pNOS expression and sNOS release (r = 0.72, p < 0.0001) and between 12 h NO release and sNOS production (r = 0.44, p < 0.005). These experiments confirm that human monocytes can be stimulated with MPL to produce NO in vitro and suggest that up-regulation of pNOS does not preclude NO release.

  17. Monocytes are Essential for the Neuroprotective Effect of Human Cord Blood Cells Following Middle Cerebral Artery Occlusion in Rat

    PubMed Central

    Womble, T. A.; Green, S.; Shahaduzzaman, M.; Grieco, J.; Sanberg, P. R.; Pennypacker, K. R.; Willing, A. E.

    2014-01-01

    Systemic administration of human umbilical cord blood (HUCB) mononuclear cells (MNC) following middle cerebral artery occlusion (MCAO) in the rat reduces infarct size and, more importantly, restores motor function. The HUCB cell preparation is composed of immature T-cells, B-cells, monocytes and stem cells. In this study we examined whether the beneficial effects of HUCB injection were attributable to one of these cell types. Male Sprague Dawley rats underwent permanent MCAO followed 48 hours later by intravenous administration of HUCB MNC preparations depleted of either CD14+ monocytes, CD133+ stem cells, CD2+ T-cells or CD19+ B cells. Motor function was measured prior to MCAO and 30 days post-stroke. When CD14+ monocytes were depleted from the HUCB MNC, activity and motor asymmetry were similar to the MCAO only treated animals. Monocyte depletion prevented HUCB cell treatment from reducing infarct size while monocyte enrichment was sufficient to reduce infarct size. Administration of monocyte-depleted HUCB cells did not suppress Iba1 labeling of microglia in the infarcted area relative to treatment with the whole HUCB preparation. These data demonstrate that the HUCB monocytes provide the majority of the efficacy in reducing infarct volume and promoting functional recovery. PMID:24472845

  18. Homocysteine stimulates the expression of monocyte chemoattractant protein-1 receptor (CCR2) in human monocytes: possible involvement of oxygen free radicals.

    PubMed Central

    Wang, G; O, K

    2001-01-01

    Homocysteinaemia is an independent risk factor for atherosclerosis. The development of atherosclerosis involves monocyte chemoattractant protein 1 (MCP-1)-mediated monocyte recruitment to the lesion site. The action of MCP-1 is mostly via its interaction with MCP-1 receptor (CCR2), which is the major receptor for MCP-1 on the surface of monocytes. The objective of the present study was to investigate the effect of homocysteine on CCR2 expression in human THP-1 monocytes. Cells were incubated with various concentrations of homocysteine for 6, 12, 24 and 48 h. The expression of CCR2 mRNA was determined by nuclease protection assay and the CCR2 protein was measured by Western immunoblotting analysis. The binding of MCP-1 to CCR2 as a functional receptor on the monocyte surface was determined by flow cytometry. Homocysteine (0.05-0.2 mM) significantly enhanced the expression of CCR2 mRNA (129-209% of the control) and CCR2 protein (up to 183% of control) in these cells after 24 h of incubation. Stimulation of CCR2 expression was associated with a parallel increase in the binding activity of CCR2 (129-191% of control) as well as an enhanced chemotactic response of homocysteine-treated monocytes. Further investigation revealed that the levels of superoxide were significantly elevated in cells incubated with homocysteine for 12-48 h. The addition of superoxide dismutase, a scavenger of superoxide, to the culture medium abolished the stimulatory effect of homocysteine on CCR2 expression as well as the binding activity of the receptor. The stimulatory effect of homocysteine on the expression of CCR2 mRNA and the levels of CCR2 protein was also observed in human peripheral blood monocytes. In conclusion, the present study has clearly demonstrated that homocysteine stimulates CCR2 expression in monocytes, leading to an enhanced binding activity and chemotatic response. Homocysteine-induced superoxide formation might serve as one of the underlying mechanisms for this effect

  19. Monocytic Differentiation Inhibits Infection and Granulocytic Differentiation Potentiates Infection by the Agent of Human Granulocytic Ehrlichiosis

    PubMed Central

    Klein, Marina B.; Hayes, Stanley F.; Goodman, Jesse L.

    1998-01-01

    Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne infection with a specific tropism for granulocytes. We previously isolated and cultivated the HGE agent in the promyelocytic leukemia cell line HL-60 and have also demonstrated the susceptibility of both granulocytic and monocytic human marrow progenitors. Circulating monocytes have not been observed to be infected, suggesting that cell susceptibility may be differentiation specific. To evaluate this hypothesis, HL-60 cells were differentiated towards granulocytes (with dimethyl sulfoxide or all-trans retinoic acid) or toward monocytes-macrophages (with 12-O-tetradecanoylphorbol-13-acetate [TPA], gamma interferon, or 1,25-dihydroxyvitamin D3) and then challenged with HGE. HGE binding, internalization, and proliferation were compared in differentiated and untreated control HL-60 cells by immunofluorescence, electron microscopy, and Giemsa staining. Granulocytic differentiation resulted in a doubling of HGE binding and enhanced infection consistent with the agent’s clinical tropism for neutrophils. Granulocytic cells were unable to kill internalized ehrlichiae even after activation induced by N-formyl-Met-Leu-Phe alone or together with tumor necrosis factor alpha. In contrast, monocyte-macrophage differentiation with TPA resulted in complete resistance to infection through at least two distinct mechanisms: (i) reduction in binding and uptake and (ii) killing of any internalized organisms. Diminished binding in TPA-treated cells correlated with their reduced expression of sialyl Lewis x (CD15s), a putative cellular receptor component for HGE. The degree of monocytic differentiation and activation induced (i.e., TPA > gamma interferon > vitamin D3) correlated with resistance to HGE. Thus, HL-60 cells exhibit a striking differentiation-specific susceptibility to HGE. Differentiation-induced changes in bacterial adhesion and killing capacity underlie the tropism of HGE for granulocytic HL-60 cells and

  20. Stimulation of tumor necrosis factor alpha production in human monocytes by inhibitors of protein phosphatase 1 and 2A

    PubMed Central

    1992-01-01

    The protein phosphatase 1 and 2A inhibitor, okadaic acid, has been shown to stimulate many cellular functions by increasing the phosphorylation state of phosphoproteins. In human monocytes, okadaic acid by itself stimulates tumor necrosis factor alpha (TNF-alpha) mRNA accumulation and TNF-alpha synthesis. Calyculin A, a more potent inhibitor of phosphatase 1, has similar effects. TNF-alpha mRNA accumulation in okadaic acid-treated monocytes is due to increased TNF- alpha mRNA stability and transcription rate. The increase in TNF-alpha mRNA stability is more remarkable in okadaic acid-treated monocytes than the mRNA stability of other cytokines, such as interleukin 1 alpha (IL-1 alpha), IL-1 beta, and IL-6. Gel retardation studies show the stimulation of AP-1, AP-2, and NF-kappa B binding activities in okadaic acid-stimulated monocytes. This increase may correlate with the increase in TNF-alpha mRNA transcription rate. In addition to the stimulation of TNF-alpha secretion by monocytes, okadaic acid appears to modulate TNF-alpha precursor processing, as indicated by a marked increase in the cell-associated 26-kD precursor. These results suggest that active basal phosphorylation/dephosphorylation occurs in monocytes, and that protein phosphatase 1 or 2A is important in regulating TNF-alpha gene transcription, translation, and posttranslational modification. PMID:1324971

  1. RNA-sequencing analysis of high glucose-treated monocytes reveals novel transcriptome signatures and associated epigenetic profiles

    PubMed Central

    Miao, Feng; Chen, Zhuo; Zhang, Lingxiao; Wang, Jinhui; Gao, Harry; Wu, Xiwei

    2013-01-01

    We performed high throughput transcriptomic profiling with RNA sequencing (RNA-Seq) to uncover network responses in human THP-1 monocytes treated with high glucose (HG). Our data analyses revealed that interferon (IFN) signaling, pattern recognition receptors, and activated interferon regulatory factors (IRFs) were enriched among the HG-upregulated genes. Motif analysis identified an HG-responsive IRF-mediated network in which interferon-stimulated genes (ISGs) were enriched. Notably, this network showed strong overlap with a recently discovered IRF7-driven network relevant to Type 1 diabetes. We next examined if the HG-regulated genes possessed any characteristic chromatin features in the basal state by profiling 15 active and repressive chromatin marks under normal glucose conditions using chromatin immunoprecipitation linked to promoter microarrays. Composite profiles revealed higher histone H3 lysine-9-acetylation levels around the promoters of HG-upregulated genes compared with all RefSeq promoters. Interestingly, within the HG-upregulated genes, active chromatin marks were enriched not only at high CpG content promoters, but surprisingly also at low CpG content promoters. Similar results were obtained with peripheral blood monocytes exposed to HG. These new results reveal a novel mechanism by which HG can exercise IFN-α-like effects in monocytes by upregulating a set of ISGs poised for activation with multiple chromatin marks. PMID:23386205

  2. Characterization of calcium oxalate crystal-induced changes in the secretome of U937 human monocytes.

    PubMed

    Sintiprungrat, Kitisak; Singhto, Nilubon; Thongboonkerd, Visith

    2016-03-01

    In kidney stone disease, migratory monocytes have been found to mediate progressive renal inflammation through the secretion of numerous inflammatory mediators. However, whether calcium oxalate monohydrate (COM), which is the major crystalline compound of kidney stones, has any effects on proteins secreted from monocytes remained largely unknown. The present study aimed to characterize changes in the secretome of U937 human monocytes induced by COM crystals. The viability of cells in serum/protein-free medium was serially evaluated and the data revealed that an exposure time of 16 h was optimal for this study, whereas prolonged incubation for 24 h resulted in declined cell viability. Using this optimal time-point, the secreted proteins recovered from serum/protein-free culture supernatants of controlled and COM-treated cells were resolved in 2-DE and stained with Deep Purple fluorescent dye. Quantitative intensity analysis revealed statistically significant changes in levels of 18 secreted proteins (14 increased and 4 decreased) from COM-treated cells. These significantly altered secreted proteins were then identified by Q-TOF MS and/or MS/MS analyses. Among these, the increased levels of secreted heat shock protein 90 (HSP90), HSP70 and β-actin were confirmed by Western blot analysis. The increased level of extracellular HSP90 was confirmed on the COM-treated cell surface by the immunofluorescence study, whereas the increased secretion of IFN-α was validated by ELISA. Global protein network analysis, literature search and bioinformatics revealed that these significantly altered secreted proteins were involved mainly in immune response and cell survival. Therefore, changes in the secretome of monocytes induced by COM crystals may be related, at least in part, to progressive renal inflammation found in kidney stone disease.

  3. Relationship of superoxide production to cytoplasmic free calcium in human monocytes.

    PubMed Central

    Scully, S P; Segel, G B; Lichtman, M A

    1986-01-01

    Calcium has been proposed as an intracellular second messenger for activation of secretion, phagocytosis, and the oxidative burst of neutrophils. We have examined the role of calcium in human monocyte activation. Concanavalin A (Con A)-stimulated monocytes displayed an increment in cytoplasmic ionized calcium at 31 +/- 6 s and the onset of superoxide production at 61 +/- 9 s. The increase in cytoplasmic calcium invariably preceded the onset of superoxide production. If the external calcium concentration was reduced to less than 28 nM by the addition of 10 mM EGTA, superoxide production was not diminished at 5 min; however, superoxide production decreased thereafter. The Con A-evoked increment in cytoplasmic ionized calcium was blunted upon the addition of EGTA and decreased further with time. Both the production of superoxide and the Con A-evoked increment in cytoplasmic ionized calcium displayed a 50% inhibition after 15 min of calcium depletion and were completely inhibited after 60 min. Total cell calcium fell from 0.7 to 0.5 fmol/cell, and the basal level of ionized calcium fell from 83 to 30 nM after 60 min. Histidine, a strong chelator of divalent cations other than calcium and magnesium, had no effect on monocyte superoxide production or on ionized calcium concentrations, indicating that EGTA inhibition was due to cell calcium depletion. In calcium-depleted cells, Con A did not evoke superoxide production until calcium was restored to the incubation medium. The restoration of calcium to Con A-treated, calcium-depleted monocytes permitted a rapid rise in the cytoplasmic ionized calcium, and the production of superoxide within 9 s. These data suggest that an increase in ionized cytoplasmic calcium is necessary for the activation of monocyte superoxide production by Con A. The rise in ionized calcium in response to Con A results, in part, from an internal redistribution of calcium, which is sufficient to permit superoxide generation. PMID:3007579

  4. C-reactive protein (CRP) induces chemokine secretion via CD11b/ICAM-1 interaction in human adherent monocytes.

    PubMed

    Montecucco, Fabrizio; Steffens, Sabine; Burger, Fabienne; Pelli, Graziano; Monaco, Claudia; Mach, François

    2008-10-01

    Several studies support C-reactive protein (CRP) as a systemic cardiovascular risk factor. The recent detection of CRP in arterial intima suggests a dual activity in atherosclerosis as a circulating and tissue mediator on vascular and immune cells. In the present paper, we focused on the inflammatory effects of CRP on human monocytes, which were isolated by Ficoll-Percoll gradients and cultured in adherence to polystyrene, endothelial cell monolayer, or in suspension. Chemokine levels, adhesion molecule, and chemokine receptor expression were detected by ELISA, flow cytometry, and real-time RT-PCR. Migration assays were performed in a Boyden chamber. Stimulation with CRP induced release of CCL2, CCL3, and CCL4 in adherent monocytes through the binding to CD32a, CD32b, and CD64, whereas no effect was observed in suspension culture. This was associated with CRP-induced up-regulation of adhesion molecules membrane-activated complex 1 (Mac-1) and ICAM-1 on adherent monocytes. Blockade of Mac-1/ICAM-1 interaction inhibited the CRP-induced chemokine secretion. In addition, CRP reduced mRNA and surface expression of corresponding chemokine receptors CCR1, CCR2, and CCR5 in adherent monocytes. This effect was a result of chemokine secretion, as coincubation with neutralizing anti-CCL2, anti-CCL3, and anti-CCL4 antibodies reversed the effect of CRP. Accordingly, a reduced migration of CRP-treated monocytes to CCL2 and CCL3 was observed. In conclusion, our data suggest an in vitro model to study CRP activities in adherent and suspension human monocytes. CRP-mediated induction of adhesion molecules and a decrease of chemokine receptors on adherent monocytes might contribute to the retention of monocytes within atherosclerotic lesions and recruitment of other circulating cells.

  5. Expression of ACAT-1 protein in human atherosclerotic lesions and cultured human monocytes-macrophages.

    PubMed

    Miyazaki, A; Sakashita, N; Lee, O; Takahashi, K; Horiuchi, S; Hakamata, H; Morganelli, P M; Chang, C C; Chang, T Y

    1998-10-01

    The acyl coenzyme A:cholesterol acyltransferase (ACAT) gene was first cloned in 1993 (Chang et al, J Biol Chem. 1993;268:20747-20755; designated ACAT-1). Using affinity-purified antibodies raised against the N-terminal portion of human ACAT-1 protein, we performed immunohistochemical localization studies and showed that the ACAT-1 protein was highly expressed in atherosclerotic lesions of the human aorta. We also performed cell-specific localization studies using double immunostaining and showed that ACAT-1 was predominantly expressed in macrophages but not in smooth muscle cells. We then used a cell culture system in vitro to monitor the ACAT-1 expression in differentiating monocytes-macrophages. The ACAT-1 protein content increased by up to 10-fold when monocytes spontaneously differentiated into macrophages. This increase occurred within the first 2 days of culturing the monocytes and reached a plateau level within 4 days of culturing, indicating that the increase in ACAT-1 protein content is an early event during the monocyte differentiation process. The ACAT-1 protein expressed in the differentiating monocytes-macrophages was shown to be active by enzyme assay in vitro. The high levels of ACAT-1 present in macrophages maintained in culture can explain the high ACAT-1 contents found in atherosclerotic lesions. Our results thus support the idea that ACAT-1 plays an important role in differentiating monocytes and in forming macrophage foam cells during the development of human atherosclerosis.

  6. Atypical Activin A and IL-10 Production Impairs Human CD16+ Monocyte Differentiation into Anti-Inflammatory Macrophages.

    PubMed

    González-Domínguez, Érika; Domínguez-Soto, Ángeles; Nieto, Concha; Flores-Sevilla, José Luis; Pacheco-Blanco, Mariana; Campos-Peña, Victoria; Meraz-Ríos, Marco A; Vega, Miguel A; Corbí, Ángel L; Sánchez-Torres, Carmen

    2016-02-01

    Human CD14(++)CD16(-) and CD14(+/lo)CD16(+) monocyte subsets comprise 85 and 15% of blood monocytes, respectively, and are thought to represent distinct stages in the monocyte differentiation pathway. However, the differentiation fates of both monocyte subsets along the macrophage (Mϕ) lineage have not yet been elucidated. We have now evaluated the potential of CD14(++) CD16(-) and CD16(+) monocytes to differentiate and to be primed toward pro- or anti-inflammatory Mϕs upon culture with GM-CSF or M-CSF, respectively (subsequently referred to as GM14, M14, GM16, or M16). Whereas GM16 and GM14 were phenotypic and functionally analogous, M16 displayed a more proinflammatory profile than did M14. Transcriptomic analyses evidenced that genes associated with M-CSF-driven Mϕ differentiation (including FOLR2, IL10, IGF1, and SERPINB2) are underrepresented in M16 with respect to M14. The preferential proinflammatory skewing of M16 relative to M14 was found to be mediated by the secretion of activin A and the low levels of IL-10 produced by M16. In fact, activin A receptor blockade during the M-CSF-driven differentiation of CD16(+) monocytes, or addition of IL-10-containing M14-conditioned medium, significantly enhanced their expression of anti-inflammatory-associated molecules while impairing their acquisition of proinflammatory-related markers. Thus, we propose that M-CSF drives CD14(++)CD16- monocyte differentiation into bona fide anti-inflammatory Mϕs in a self-autonomous manner, whereas M-CSF-treated CD16(+) monocytes generate Mϕs with a skewed proinflammatory profile by virtue of their high activin A expression unless additional anti-inflammatory stimuli such as IL-10 are provided.

  7. CTLA-4 is expressed by human monocyte-derived dendritic cells and regulates their functions.

    PubMed

    Laurent, Stefania; Carrega, Paolo; Saverino, Daniele; Piccioli, Patrizia; Camoriano, Marta; Morabito, Anna; Dozin, Beatrice; Fontana, Vincenzo; Simone, Rita; Mortara, Lorenzo; Mingari, Maria Cristina; Ferlazzo, Guido; Pistillo, Maria Pia

    2010-10-01

    Cytotoxic T lymphocyte antigen-4 (CTLA-4) is the major negative regulator of T-cell responses, although growing evidence supports its wider role as an immune attenuator that may also act in other cell lineages. Here, we have analyzed the expression of CTLA-4 in human monocytes and monocyte-derived dendritic cells (DCs), and the effect of its engagement on cytokine production and T-cell stimulatory activity by mature DCs. CTLA-4 was highly expressed on freshly isolated monocytes, then down-modulated upon differentiation toward immature DCs (iDCs) and it was markedly upregulated on mature DCs obtained with different stimulations (lipopolysaccharides [LPS], Poly:IC, cytokines). In line with the functional role of CTLA-4 in T cells, treatment of mDCs with an agonistic anti-CTLA-4 mAb significantly enhanced secretion of regulatory interleukin (IL)-10 but reduced secretion of IL-8/IL-12 pro-inflammatory cytokines, as well as autologous CD4+ T-cell proliferation in response to stimulation with recall antigen purified protein derivative (PPD) loaded-DCs. Neutralization of IL-10 with an anti-IL-10 antibody during the mDCs-CD4+ T-cell co-culture partially restored the ability of anti-CTLA-4-treated mDCs to stimulate T-cell proliferation in response to PPD. Taken together, our data provide the first evidence that CTLA-4 receptor is expressed by human monocyte-derived mDCs upon their full activation and that it exerts immune modulatory effects.

  8. Comparative uptake of grepafloxacin and ciprofloxacin by a human monocytic cell line, THP-1.

    PubMed

    Hara, T; Takemura, H; Kanemitsu, K; Yamamoto, H; Shimada, J

    2000-09-01

    The present study was designed to compare the uptake of grepafloxacin by a human monocytic cell line, THP-1, with that of ciprofloxacin. THP-1 cells were incubated with 20 microg/ml of either drug, and the entry of the drugs into the cells was determined using a velocity gradient centrifugation technique and HPLC assay. Antibiotic uptake by the cells was expressed as the ratio of the intracellular to the extracellular drug concentration (IC/EC). Grepafloxacin entered THP-1 cells readily within 5 min, and at steady-state (37 degrees C; 60 min), the IC/EC ratio of grepafloxacin (11.9 +/- 1.7; n = 13) was about 2.4-fold higher than that of ciprofloxacin (5.0 +/- 1.3; n = 13). The ratios decreased at low incubation temperature (4 degrees C), in paraformaldehyde-treated dead cells, and at low extracellular pH (pH 6.0), but were not influenced by high extracellular pH (pH, 9.0). Characterization of fluoroquinolone uptake suggests that these drugs penetrate the THP-1 membrane by passive diffusion, and also, in part, via an active transport system. We also examined the uptake of the two fluoroquinolones in phorbol 12 myristate 13-acetate (PMA)-stimulated adherent THP-1 cells (THP-1 macrophages). The IC/EC ratios for both fluoroquinolones in the THP-1 macrophages were significantly higher than those in the THP-1 monocytes. Further the uptake of three other fluoroquinolones, levofloxacin, tosufloxacin, and sparfloxacin, by THP-1 monocytes was examined in comparative studies. The IC/EC ratio of grepafloxacin was comparable to that of sparfloxacin and significantly higher than that of the other fluoroquinolones. Our results indicate that grepafloxacin exhibits better intracellular accumulation than ciprofloxacin and other fluoroquinolones in human monocytic and macrophage-like cells.

  9. Identification of Genes Responsive to Solar Simulated UV Radiation in Human Monocyte-Derived Dendritic Cells

    PubMed Central

    de la Fuente, Hortensia; Lamana, Amalia; Mittelbrunn, María; Perez-Gala, Silvia; Gonzalez, Salvador; García-Diez, Amaro; Vega, Miguel; Sanchez-Madrid, Francisco

    2009-01-01

    Ultraviolet (UV) irradiation has profound effects on the skin and the systemic immune system. Several effects of UV radiation on Dendritic cells (DCs) functions have been described. However, gene expression changes induced by UV radiation in DCs have not been addressed before. In this report, we irradiated human monocyte-derived DCs with solar-simulated UVA/UVB and analyzed regulated genes on human whole genome arrays. Results were validated by RT-PCR and further analyzed by Gene Set Enrichment Analysis (GSEA). Solar-simulated UV radiation up-regulated expression of genes involved in cellular stress and inflammation, and down-regulated genes involved in chemotaxis, vesicular transport and RNA processing. Twenty four genes were selected for comparison by RT-PCR with similarly treated human primary keratinocytes and human melanocytes. Several genes involved in the regulation of the immune response were differentially regulated in UVA/UVB irradiated human monocyte-derived DCs, such as protein tyrosine phosphatase, receptor type E (PTPRE), thrombospondin-1 (THBS1), inducible costimulator ligand (ICOSL), galectins, Src-like adapter protein (SLA), IL-10 and CCR7. These results indicate that UV-exposure triggers the regulation of a complex gene repertoire involved in human-DC–mediated immune responses. PMID:19707549

  10. Human T Cell Leukemia Virus Type 1 Infection of the Three Monocyte Subsets Contributes to Viral Burden in Humans

    PubMed Central

    de Castro-Amarante, Maria Fernanda; McKinnon, Katherine; Washington Parks, Robyn; Galli, Veronica; Omsland, Maria; Andresen, Vibeke; Massoud, Raya; Brunetto, Giovanna; Caruso, Breanna; Venzon, David; Jacobson, Steven

    2015-01-01

    ABSTRACT Because the viral DNA burden correlates with disease development, we investigated the contribution of monocyte subsets (classical, intermediate, and nonclassical monocytes) to the total viral burden in 22 human T cell leukemia virus type 1 (HTLV-1)-infected individuals by assessing their infectivity status, frequency, as well as chemotactic and phagocytic functions. All three monocyte subsets sorted from HTLV-1-infected individuals were positive for viral DNA, and the frequency of classical monocytes was lower in the blood of HTLV-1-infected individuals than in that of uninfected individuals, while the expression levels of the chemokine receptors CCR5, CXCR3, and CX3CR1 in classical monocytes were higher in HTLV-1-infected individuals than uninfected individuals; the percentage of intermediate monocytes and their levels of chemokine receptor expression did not differ between HTLV-1-infected and uninfected individuals. However, the capacity of intermediate monocytes to migrate to CCL5, the ligand for CCR5, was higher, and a higher proportion of nonclassical monocytes expressed CCR1, CXCR3, and CX3CR1. The level of viral DNA in the monocyte subsets correlated with the capacity to migrate to CCL2, CCL5, and CX3CL1 for classical monocytes, with lower levels of phagocytosis for intermediate monocytes, and with the level of viral DNA in CD8+ and CD4+ T cells for nonclassical monocytes. These data suggest a model whereby HTLV-1 infection augments the number of classical monocytes that migrate to tissues and become infected and the number of infected nonclassical monocytes that transmit virus to CD4+ and CD8+ T cells. These results, together with prior findings in a macaque model of HTLV-1 infection, support the notion that infection of monocytes by HTLV-1 is likely a requisite for viral persistence in humans. IMPORTANCE Monocytes have been implicated in immune regulation and disease progression in patients with HTLV-1-associated inflammatory diseases. We detected

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

    SciTech Connect

    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.

  12. CD13 mediates phagocytosis in human monocytic cells.

    PubMed

    Licona-Limón, Ileana; Garay-Canales, Claudia A; Muñoz-Paleta, Ofelia; Ortega, Enrique

    2015-07-01

    CD13 is a membrane-bound ectopeptidase, highly expressed on monocytes, macrophages, and dendritic cells. CD13 is involved in diverse functions, including degradation of peptide mediators, cellular adhesion, migration, viral endocytosis, signaling, and positive modulation of phagocytosis mediated by FcγRs and other phagocytic receptors. In this work, we explored whether besides acting as an accessory receptor, CD13 by itself is a primary phagocytic receptor. We found that hCD13 mediates efficient phagocytosis of large particles (erythrocytes) modified so as to interact with the cell only through CD13 in human macrophages and THP-1 monocytic cells. The extent of this phagocytosis is comparable with the phagocytosis mediated through the canonical phagocytic receptor FcγRI. Furthermore, we demonstrated that hCD13 expression in the nonphagocytic cell line HEK293 is sufficient to enable these cells to internalize particles bound through hCD13. CD13-mediated phagocytosis is independent of other phagocytic receptors, as it occurs in the absence of FcγRs, CR3, and most phagocytic receptors. Phagocytosis through CD13 is independent of its enzymatic activity but is dependent on actin rearrangement and activation of PI3K and is partially dependent on Syk activation. Moreover, the cross-linking of CD13 with antibodies rapidly induced pSyk in human macrophages. Finally, we observed that antibody-mediated cross-linking of hCD13, expressed in the murine macrophage-like J774 cell line, induces production of ROS. These results demonstrate that CD13 is a fully competent phagocytic receptor capable of mediating internalization of large particles.

  13. NFκB2/p100 is a key factor for endotoxin tolerance in human monocytes: a demonstration using primary human monocytes from patients with sepsis.

    PubMed

    Cubillos-Zapata, Carolina; Hernández-Jiménez, Enrique; Toledano, Víctor; Esteban-Burgos, Laura; Fernández-Ruíz, Irene; Gómez-Piña, Vanesa; Del Fresno, Carlos; Siliceo, María; Prieto-Chinchiña, Patricia; Pérez de Diego, Rebeca; Boscá, Lisardo; Fresno, Manuel; Arnalich, Francisco; López-Collazo, Eduardo

    2014-10-15

    Endotoxin tolerance (ET) is a state of reduced responsiveness to endotoxin stimulation after a primary bacterial insult. This phenomenon has been described in several pathologies, including sepsis, in which an endotoxin challenge results in reduced cytokine production. In this study, we show that the NFκ L chain enhancer of activated B cells 2 (NFκB2)/p100 was overexpressed and accumulated in a well-established in vitro human monocyte model of ET. The p100 accumulation in these cells inversely correlated with the inflammatory response after LPS stimulation. Knocking down NFκB2/p100 using small interfering RNA in human monocytes further indicated that p100 expression is a crucial factor in the progression of ET. The monocytes derived from patients with sepsis had high levels of p100, and a downregulation of NFκB2/p100 in these septic monocytes reversed their ET status.

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

    PubMed Central

    Hussain, Salik; Al-Nsour, Faris; Rice, Annette B; Marshburn, Jamie; Ji, Zhaoxia; Zink, Jeffery I; Yingling, Brenda; Walker, Nigel J; Garantziotis, Stavros

    2012-01-01

    Background Cerium dioxide (CeO2) nanoparticles have potential therapeutic applications and are widely used for industrial purposes. However, the effects of these nanoparticles on primary human cells are largely unknown. The ability of nanoparticles to exacerbate pre-existing inflammatory disorders is not well documented for engineered nanoparticles, and is certainly lacking for CeO2 nanoparticles. We investigated the inflammation-modulating effects of CeO2 nanoparticles at noncytotoxic concentrations in human peripheral blood monocytes. Methods CD14+ cells were isolated from peripheral blood samples of human volunteers. Cells were exposed to either 0.5 or 1 μg/mL of CeO2 nanoparticles over a period of 24 or 48 hours with or without lipopolysaccharide (10 ng/mL) prestimulation. Modulation of the inflammatory response was studied by measuring secreted tumor necrosis factor-alpha, interleukin-1beta, macrophage chemotactic protein-1, interferon-gamma, and interferon gamma-induced protein 10. Results CeO2 nanoparticle suspensions were thoroughly characterized using dynamic light scattering analysis (194 nm hydrodynamic diameter), zeta potential analysis (−14 mV), and transmission electron microscopy (irregular-shaped particles). Transmission electron microscopy of CD14+ cells exposed to CeO2 nanoparticles revealed that these nanoparticles were efficiently internalized by monocytes and were found either in vesicles or free in the cytoplasm. However, no significant differences in secreted cytokine profiles were observed between CeO2 nanoparticle-treated cells and control cells at noncytotoxic doses. No significant effects of CeO2 nanoparticle exposure subsequent to lipopolysaccharide priming was observed on cytokine secretion. Moreover, no significant difference in lipopolysaccharide-induced cytokine production was observed after exposure to CeO2 nanoparticles followed by lipopolysaccharide exposure. Conclusion CeO2 nanoparticles at noncytotoxic concentrations neither

  15. Cerium Dioxide Nanoparticles Induce Apoptosis and Autophagy in Human Peripheral Blood Monocytes

    PubMed Central

    Hussain, Salik; Al-Nsour, Faris; Rice, Annette B.; Marshburn, Jamie; Yingling, Brenda; Ji, Zhaoxia; Zink, Jeffrey I.; Walker, Nigel J.; Garantziotis, Stavros

    2015-01-01

    Cerium dioxide nanoparticles (CeO2 NPs) have diversified industrial uses and novel therapeutic applications are actively being pursued. There is lack of mechanistic data concerning the effects of CeO2 NPs on primary human cells. We aimed at characterizing the cytotoxic effects of CeO2 NPs in human peripheral blood monocytes. CeO2 NPs and their suspensions were thoroughly characterized, including using transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis. Blood from healthy human volunteers was drawn through phlebotomy and CD14+ cells were isolated. Cells were exposed to CeO2 NPs (0.5–10 μg/mL) for 20 or 40 hours and mechanisms of cell injury were studied. TEM revealed that CeO2 NPs are internalized by monocytes and are found either in vesicles or free in the cytoplasm. CeO2 NP exposure leads to decrease in cell viability and treated cells exhibit characteristic hallmarks of apoptosis (activation of Bax, loss of mitochondrial membrane potential, DNA fragmentation). CeO2 NP toxicity is caused by mitochondrial damage leading to apoptosis inducing factor (AIF) release, but not due to caspase activation or reactive oxygen species production. Moreover, CeO2 NP exposure leads to autophagy, which is further increased after pharmacological inhibition of tumour suppressor protein p53. Inhibition of autophagy partially reverses cell death by CeO2 NPs. It is concluded that CeO2 NPs are toxic to primary human monocytes at relatively low doses. PMID:22717232

  16. Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols.

    PubMed

    Figueroa, Gloria; Parira, Tiyash; Laverde, Alejandra; Casteleiro, Gianna; El-Mabhouh, Amal; Nair, Madhavan; Agudelo, Marisela

    2016-10-18

    Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded > 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

  17. Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS.

    PubMed

    Momen-Heravi, Fatemeh; Bala, Shashi; Kodys, Karen; Szabo, Gyongyi

    2015-05-14

    Hepatocyte damage and inflammation in monocytes/macrophages are central to the pathogenesis of alcoholic hepatitis (AH). MicroRNAs (miRNAs) regulate all of these processes. MiRNA-122 is abundantly expressed in hepatocytes while monocytes/macrophages have low levels. The role of exosomes in AH and possible cross talk between hepatocyte-derived exosomes and immune cells is not explored yet. Here, we show that the number of exosomes significantly increases in the sera of healthy individuals after alcohol binge drinking and in mice after binge or chronic alcohol consumption. Exosomes isolated from sera after alcohol consumption or from in vitro ethanol-treated hepatocytes contained miRNA-122. Exosomes derived from ethanol-treated Huh7.5 cells were taken up by the recipients THP1 monocytes and horizontally transferred a mature form of liver-specific miRNA-122. In vivo, liver mononuclear cells and Kupffer cells from alcohol-fed mice had increased miRNA-122 levels. In monocytes, miRNA-122 transferred via exosomes inhibited the HO-1 pathway and sensitized to LPS stimulation and increased levels of pro-inflammatory cytokines. Finally, inflammatory effects of exosomes from ethanol-treated hepatocytes were prevented by using RNA interference via exosome-mediated delivery of a miRNA-122 inhibitor. These results demonstrate that first, exosomes mediate communication between hepatocytes and monocytes/macrophages and second, hepatocyte-derived miRNA-122 can reprogram monocytes inducing sensitization to LPS.

  18. Monocyte subset dynamics in human atherosclerosis can be profiled with magnetic nano-sensors.

    PubMed

    Wildgruber, Moritz; Lee, Hakho; Chudnovskiy, Aleksey; Yoon, Tae-Jong; Etzrodt, Martin; Pittet, Mikael J; Nahrendorf, Matthias; Croce, Kevin; Libby, Peter; Weissleder, Ralph; Swirski, Filip K

    2009-05-22

    Monocytes are circulating macrophage and dendritic cell precursors that populate healthy and diseased tissue. In humans, monocytes consist of at least two subsets whose proportions in the blood fluctuate in response to coronary artery disease, sepsis, and viral infection. Animal studies have shown that specific shifts in the monocyte subset repertoire either exacerbate or attenuate disease, suggesting a role for monocyte subsets as biomarkers and therapeutic targets. Assays are therefore needed that can selectively and rapidly enumerate monocytes and their subsets. This study shows that two major human monocyte subsets express similar levels of the receptor for macrophage colony stimulating factor (MCSFR) but differ in their phagocytic capacity. We exploit these properties and custom-engineer magnetic nanoparticles for ex vivo sensing of monocytes and their subsets. We present a two-dimensional enumerative mathematical model that simultaneously reports number and proportion of monocyte subsets in a small volume of human blood. Using a recently described diagnostic magnetic resonance (DMR) chip with 1 microl sample size and high throughput capabilities, we then show that application of the model accurately quantifies subset fluctuations that occur in patients with atherosclerosis.

  19. Cannabidiol induced a contrasting pro-apoptotic effect between freshly isolated and precultured human monocytes

    SciTech Connect

    Wu, Hsin-Ying; Chang, An-Chi; Wang, Chia-Chi; Kuo, Fu-Hua; Lee, Chi-Ya; Liu, Der-Zen; Jan, Tong-Rong

    2010-08-01

    It has been documented that cannabidiol (CBD) induced apoptosis in a variety of transformed cells, including lymphocytic and monocytic leukemias. In contrast, a differential sensitivity between normal lymphocytes and monocytes to CBD-mediated apoptosis has been reported. The present study investigated the pro-apoptotic effect of CBD on human peripheral monocytes that were either freshly isolated or precultured for 72 h. CBD markedly enhanced apoptosis of freshly isolated monocytes in a time- and concentration-dependent manner, whereas precultured monocytes were insensitive. By comparison, both cells were sensitive to doxorubicin-induced apoptosis. CBD significantly diminished the cellular thiols and glutathione in freshly isolated monocytes. The apoptosis induced by CBD was abrogated in the presence of N-acetyl-{sub L}-cysteine, a precursor of glutathione. In addition, precultured monocytes contained a significantly greater level of glutathione and heme oxygenase-1 (HO-1) compared to the freshly isolated cells. The HO-1 competitive inhibitor zinc protoporphyrin partially but significantly restored the sensitivity of precultured monocytes to CBD-mediated apoptosis. Collectively, our results demonstrated a contrasting pro-apoptotic effect of CBD between precultured and freshly isolated monocytes, which was closely associated with the cellular level of glutathione and the antioxidative capability of the cells.

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

    PubMed

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

    2015-11-14

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

  1. Lipid profiling of polarized human monocyte-derived macrophages.

    PubMed

    Montenegro-Burke, J Rafael; Sutton, Jessica A; Rogers, Lisa M; Milne, Ginger L; McLean, John A; Aronoff, David M

    2016-12-01

    The highly orchestrated transcriptional and metabolic reprogramming during activation drastically transforms the main functions and physiology of human macrophages across the polarization spectrum. Lipids, for example, can modify protein function by acting remotely as signaling molecules but also locally by altering the physical properties of cellular membranes. These changes play key roles in the functions of highly plastic immune cells due to their involvement in inflammation, immune responses, phagocytosis and wound healing processes. We report an analysis of major membrane lipids of distinct phenotypes of resting (M0), classically activated (M1), alternatively activated (M2a) and deactivated (M2c) human monocyte derived macrophages from different donors. Samples were subjected to supercritical fluid chromatography-ion mobility-mass spectrometry analysis, which allowed separations based on lipid class, facilitating the profiling of their fatty acid composition. Different levels of arachidonic acid mobilization as well as other fatty acid changes were observed for different lipid classes in the distinct polarization phenotypes, suggesting the activation of highly orchestrated and specific enzymatic processes in the biosynthesis of lipid signaling molecules and cell membrane remodeling. Thromboxane A2 production appeared to be a specific marker of M1 polarization. These alterations to the global composition of lipid bi-layer membranes in the cell provide a potential methodology for the definition and determination of cellular and tissue activation states.

  2. Interleukin-6 production by human monocytes stimulated with Cryptococcus neoformans components.

    PubMed

    Delfino, D; Cianci, L; Lupis, E; Celeste, A; Petrelli, M L; Curró, F; Cusumano, V; Teti, G

    1997-06-01

    In order to ascertain if Cryptococcus neoformans components can induce interleukin-6 (IL-6) production, we stimulated human whole blood with purified capsular products. Their potencies in stimulating IL-6 release were mannoproteins > galactoxylomannan = glucuronoxylomannan > alpha(1-3)glucan. IL-6 production was tumor necrosis factor alpha independent and required the presence of monocytes and plasma. Since IL-6 can stimulate replication of the human immunodeficiency virus in monocytic cells, these findings may be clinically relevant.

  3. Interleukin-6 production by human monocytes stimulated with Cryptococcus neoformans components.

    PubMed Central

    Delfino, D; Cianci, L; Lupis, E; Celeste, A; Petrelli, M L; Curró, F; Cusumano, V; Teti, G

    1997-01-01

    In order to ascertain if Cryptococcus neoformans components can induce interleukin-6 (IL-6) production, we stimulated human whole blood with purified capsular products. Their potencies in stimulating IL-6 release were mannoproteins > galactoxylomannan = glucuronoxylomannan > alpha(1-3)glucan. IL-6 production was tumor necrosis factor alpha independent and required the presence of monocytes and plasma. Since IL-6 can stimulate replication of the human immunodeficiency virus in monocytic cells, these findings may be clinically relevant. PMID:9169790

  4. DNA Microarray Analysis of Human Monocytes Early Response Genes upon Infection with Rickettsia rickettsii

    DTIC Science & Technology

    2004-11-15

    DNA Microarray Analysis of Human Monocytes Early Response Genes upon Infection with Rickettsia rickettsii Chien-Chung Chao Rickettsiae Diseases...TITLE AND SUBTITLE DNA Microarray Analysis of Human Monocytes Early Response Genes upon Infection with Rickettsia rickettsii 5a. CONTRACT NUMBER 5b...ANSI Std Z39-18 Rickettsiae • Gram negative coccobacillary bacteria • Obligate intracellular organisms • Arthropod-borne • Cause febrile diseases (mild

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

    SciTech Connect

    Islam, Zahidul; Gray, Jennifer S.; Pestka, James J. . E-mail: pestka@msu.edu

    2006-06-15

    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-1{beta} 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{sup +} cells. DON-induced p38 activation occurred exclusively in the CD14{sup +} 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.

  6. Profiling of the three circulating monocyte subpopulations in human obesity.

    PubMed

    Devêvre, Estelle F; Renovato-Martins, Mariana; Clément, Karine; Sautès-Fridman, Catherine; Cremer, Isabelle; Poitou, Christine

    2015-04-15

    Three subpopulations of circulating monocytes have been described: CD14(2+)CD16(-) (classical monocytes [CM]), CD14(2+)CD16(+) (intermediate monocytes [IM]), and CD14(+)CD16(2+) (nonclassical monocytes [NCM]). We previously showed that obesity is associated with an increased proportion of IM and NCM. Our objective is to decipher the migratory and inflammatory functions of each monocyte subset in obesity-related low-grade inflammation. Twenty-six healthy, normal-weight and nondiabetic volunteers (C) and 40 obese nondiabetic (Ob) individuals were included in this study. We explored the gene expression profile of 18 inflammatory genes in each subset of C and Ob subjects and measured protein expression of the upregulated genes. We then tested their functional response to TLR signaling in both groups. We showed an increased expression of CX3CR1 in all monocyte subpopulations and of CCR2 and CCR5 in CM and IM in the Ob group. We found negative correlation between CCR2 and CX3CR1 expressions and high-density lipoprotein-cholesterol, whereas CCR5 expression was positively linked to obesity-related metabolic traits. Production of inflammatory proteins upon bacterial LPS and viral ssRNA stimulation was higher in CM and NCM of the Ob group compared with the C group. Our work highlights an enhanced inflammatory phenotype of monocytes with a higher response to TLR4 and TLR8 stimulations in obesity. Moreover, it suggests an increased migration capacity of CM and IM subpopulations.

  7. The “Intermediate” CD14++CD16+ monocyte subset increases in severe peripheral artery disease in humans

    PubMed Central

    Wildgruber, Moritz; Aschenbrenner, Teresa; Wendorff, Heiko; Czubba, Maria; Glinzer, Almut; Haller, Bernhard; Schiemann, Matthias; Zimmermann, Alexander; Berger, Hermann; Eckstein, Hans-Henning; Meier, Reinhard; Wohlgemuth, Walter A.; Libby, Peter; Zernecke, Alma

    2016-01-01

    Monocytes are key players in atherosclerotic. Human monocytes display a considerable heterogeneity and at least three subsets can be distinguished. While the role of monocyte subset heterogeneity has already been well investigated in coronary artery disease (CAD), the knowledge about monocytes and their heterogeneity in peripheral artery occlusive disease (PAOD) still is limited. Therefore, we aimed to investigate monocyte subset heterogeneity in patients with PAOD. Peripheral blood was obtained from 143 patients suffering from PAOD (Rutherford stage I to VI) and three monocyte subsets were identified by flow cytometry: CD14++CD16− classical monocytes, CD14+CD16++ non-classical monocytes and CD14++CD16+ intermediate monocytes. Additionally the expression of distinct surface markers (CD106, CD162 and myeloperoxidase MPO) was analyzed. Proportions of CD14++CD16+ intermediate monocyte levels were significantly increased in advanced stages of PAOD, while classical and non-classical monocytes displayed no such trend. Moreover, CD162 and MPO expression increased significantly in intermediate monocyte subsets in advanced disease stages. Likewise, increased CD162 and MPO expression was noted in CD14++CD16− classical monocytes. These data suggest substantial dynamics in monocyte subset distributions and phenotypes in different stages of PAOD, which can either serve as biomarkers or as potential therapeutic targets to decrease the inflammatory burden in advanced stages of atherosclerosis. PMID:27991581

  8. Altered Monocyte and Endothelial Cell Adhesion Molecule Expression Is Linked to Vascular Inflammation in Human Immunodeficiency Virus Infection

    PubMed Central

    Kulkarni, Manjusha; Bowman, Emily; Gabriel, Janelle; Amburgy, Taylor; Mayne, Elizabeth; Zidar, David A.; Maierhofer, Courtney; Turner, Abigail Norris; Bazan, Jose A.; Koletar, Susan L.; Lederman, Michael M.; Sieg, Scott F.

    2016-01-01

    Background. Human immunodeficiency virus (HIV)-infected individuals have increased risk for vascular thrombosis, potentially driven by interactions between activated leukocytes and the endothelium. Methods. Monocyte subsets (CD14+CD16−, CD14+CD16+, CD14DimCD16+) from HIV negative (HIV−) and antiretroviral therapy-treated HIV positive (HIV+) participants (N = 19 and 49) were analyzed by flow cytometry for adhesion molecule expression (lymphocyte function-associated antigen 1 [LFA-1], macrophage-1 antigen [Mac-1], CD11c/CD18, very late antigen [VLA]-4) and the fractalkine receptor (CX3CR1); these receptors recognize ligands (intercellular adhesion molecules [ICAMs], vascular cell adhesion molecule [VCAM]-1, fractalkine) on activated endothelial cells (ECs) and promote vascular migration. Plasma markers of monocyte (soluble [s]CD14, sCD163) and EC (VCAM-1, ICAM-1,2, fractalkine) activation and systemic (tumor necrosis factor receptor [TNFR-I], TNFR-II) and vascular (lipoprotein-associated phospholipase A2 [Lp-PLA2]) inflammation were measured by enzyme-linked immunosorbent assay. Results. Proportions of CD16+ monocyte subsets were increased in HIV+ participants. Among all monocyte subsets, levels of LFA-1 were increased and CX3CR1 levels were decreased in HIV+ participants (P < .01). Levels of sCD163, sCD14, fractalkine, ICAM-1, VCAM-1, TNFR-II, and Lp-PLA2 were also increased in HIV+ participants (P < .05), and levels of sCD14, TNFR-I, and TNFR-II were directly related to ICAM-1 and VCAM-1 levels in HIV+ participants. Expression of CX3CR1 on monocyte subsets was inversely related to plasma Lp-PLA2 (P < .05 for all). Conclusions. Increased proportions of CD16+ monocytes, cells with altered adhesion molecule expression, combined with elevated levels of their ligands, may promote vascular inflammation in HIV infection. PMID:28066794

  9. Δ(9)-Tetrahydrocannabinol treatment during human monocyte differentiation reduces macrophage susceptibility to HIV-1 infection.

    PubMed

    Williams, Julie C; Appelberg, Sofia; Goldberger, Bruce A; Klein, Thomas W; Sleasman, John W; Goodenow, Maureen M

    2014-06-01

    The major psychoactive component of marijuana, Δ(9)-tetrahydrocannabinol (THC), also acts to suppress inflammatory responses. Receptors for THC, CB1, CB2, and GPR55, are differentially expressed on multiple cell types including monocytes and macrophages, which are important modulators of inflammation in vivo and target cells for HIV-1 infection. Use of recreational and medicinal marijuana is increasing, but the consequences of marijuana exposure on HIV-1 infection are unclear. Ex vivo studies were designed to investigate effects on HIV-1 infection in macrophages exposed to THC during or following differentiation. THC treatment of primary human monocytes during differentiation reduced HIV-1 infection of subsequent macrophages by replication competent or single cycle CCR5 using viruses. In contrast, treatment of macrophages with THC immediately prior to or continuously following HIV-1 exposure failed to alter infection. Specific receptor agonists indicated that the THC effect during monocyte differentiation was mediated primarily through CB2. THC reduced the number of p24 positive cells with little to no effect on virus production per infected cell, while quantitation of intracellular viral gag pinpointed the THC effect to an early event in the viral life cycle. Cells treated during differentiation with THC displayed reduced expression of CD14, CD16, and CD163 and donor dependent increases in mRNA expression of selected viral restriction factors, suggesting a fundamental alteration in phenotype. Ultimately, the mechanism of THC suppression of HIV-1 infection was traced to a reduction in cell surface HIV receptor (CD4, CCR5 and CXCR4) expression that diminished entry efficiency.

  10. The subcellular particulate NADPH-dependent O2.(-)-generating oxidase from human blood monocytes: comparison to the neutrophil system.

    PubMed

    Chaudhry, A N; Santinga, J T; Gabig, T G

    1982-10-01

    Highly purified preparations of normal human monocytes obtained from peripheral blood were shown to contain a subcellular particulate O2.(-)-generating oxidase system. This O2.(-)-generating activity was present in particulate preparations from monocytes that had been previously stimulated with phorbol myristate acetate but was low or absent in control preparations from unstimulated monocytes or stimulated monocytes from a patient with chronic granulomatous disease. In the stimulated preparations from normal monocytes, O2.(-)-generation was linearly proportional to cell protein concentration, insensitive to inhibition by azide, and dependent on NADPH as substrate. These characteristics are similar to the O2.(-)-generating oxidase system from human neutrophils. A significant difference in the apparent Km for NADPH was shown between preparations from stimulated monocytes and neutrophils (monocyte 83 +/- 16 microM, neutrophil 31 +/- 5 microM, mean +/- SE). Additionally, affinity of the stimulated monocyte particulate preparation for NADH was unmeasurably low.

  11. Effects of tropism and virulence of Leishmania parasites on cytokine production by infected human monocytes

    PubMed Central

    Meddeb-Garnaoui, A; Zrelli, H; Dellagi, K

    2009-01-01

    The nature of early interactions between Leishmania and macrophages which determine the outcome of infection can be related directly to parasite biological properties. Here we compared the capacity of L. major (Lm) strains, reported to be high (LmHV) and low virulent and (LmLV) in the mouse model and L. infantum (Li) strains, dermotropic (LiD) and viscerotropic (LiV), to infect and modulate cytokine production in human peripheral blood derived monocytes. Monocytes were infected with metacyclic promastigotes for 24, 48 and 72 h. Parasite burden was significantly higher in Lm- than in Li-infected monocytes. LmHV and LiD induced a significantly higher parasite burden than LmLV and LiV respectively. Cytokine production was evaluated in monocytes infected for 24 h. Contrary to interleukin (IL)-12p70, monocyte chemotactic protein-1 and transforming growth factor-β production was increased significantly in infected monocytes with no differences between strains. Lm isolates induced significantly higher quantities of tumour necrosis factor (TNF)-α than Li isolates. Low levels of IL-10 were induced by all Leishmania strains and, interestingly, co-stimulation with lipopolysaccharide (LPS) was accompanied by a dramatic increase in IL-10 production by infected monocytes. In conclusion, Lm isolates displaying different levels of virulence in mice exhibited significant differences in parasite burden but similar abilities to modulate cytokine production in human monocytes. Li strains showed weaker infectivity and TNF-α inducing-capacity compared with Lm strains. The dramatic increase of IL-10 production in infected monocytes co-stimulated by LPS may play a role in disease progression considering the presence of LPS during bacterial superinfections observed during human leishmaniasis. PMID:19040614

  12. Activation of tumoricidal properties in human blood monocytes by muramyl dipeptide requires specific intracellular interaction

    SciTech Connect

    Fogler, W.E.; Fidler, I.J.

    1986-03-15

    The purpose of this study was to identify the mechanism by which muramyl dipeptide (MDP) activates antitumor cytotoxic properties in normal and interferon-..gamma.. (IFN-..gamma..)-primed human peripheral blood monocytes. The structurally and functionally active MDP analog, nor-muramyl dipeptide (nor-MDP), and (/sup 3/H)nor-MDP were used as reference glycopeptides. Direct activation of normal, noncytotoxic monocytes by nor-MDP was enhanced its encapsulation within multilamellar vesicles (MLV). Studies with (/sup 3/H)nor-MDP revealed that the activation of monocytes by nor-MDP was not attributable to its interaction with a specific cell surface receptor, nor did it result merely from the internalization by monocytes of glycopeptide. Subthreshold concentrations of nor-MDP could activate tumor cytotoxic properties in IFN-..gamma..-primed monocytes. The intracellular interaction of (/sup 3/H)nor-MDP with IFN-..gamma..-primed monocytes was specific in that intracellular levels of radiolabeled material could be displaced and recovered as intact molecules by unlabeled nor-MDP, but not by a biologically inactive MDP stereoisomer. Collectively, these results suggest that the activation of tumoricidal properties in human blood monocytes by MDP occurs subsequent to intracellular interaction with specific MDP receptors.

  13. Monocyte prostaglandins inhibit procollagen secretion by human vascular smooth muscle cells: implications for plaque stability.

    PubMed

    Fitzsimmons, C; Proudfoot, D; Bowyer, D E

    1999-02-01

    Extracellular matrix remodelling occurs during atherosclerosis dictating the structure of the plaque and thus the resistance to rupture. Monocytes and macrophages are believed to play a role in this remodelling. In the present study, filter-separated co-culture has been used to study the effect of monocytes on procollagen turnover by human vascular smooth muscle cells (VSMC). In this system, freshly isolated human peripheral blood monocytes inhibited procollagen secretion from VSMC without affecting either degradation of procollagen, or DNA synthesis by the VSMC. Insertion of a 12 kDa dialysis membrane between the two cell types and treatment with indomethacin showed that the inhibitory factor was of low molecular weight and was cyclooxygenase-dependent. Pre-incubation of each cell type with indomethacin demonstrated that monocyte, but not VSMC cyclooxygenase was required. Thus, the inhibitory effect on procollagen secretion was due, most likely, to monocyte prostaglandins. Neither inhibition of thromboxane synthetase, nor blocking IL-1 activity, reduced the inhibitory activity. Addition of prostaglandins PGE1, PGE2 and PGF2alpha to VSMC cultures caused a reduction in procollagen secretion which was equivalent to, but was not additive with, the maximal effect achieved by monocytes. Monocytes and macrophages are a major source of prostaglandins and these molecules are likely to play an important role in collagen turnover within lesions.

  14. Binding of human C-reactive protein to monocytes: analysis by flow cytometry.

    PubMed Central

    Ballou, S P; Cleveland, R P

    1991-01-01

    An opsonic role has been proposed as a major function of C-reactive protein (CRP) in humans. In support of this hypothesis, recent radiolabelled ligand binding studies have provided evidence for the presence of specific receptors for soluble human CRP on human phagocytic cells, including neutrophils and monocytes. In order to confirm specific binding of CRP to monocytes and to quantify the percentage of such cells capable of expressing binding sites, we employed a sensitive biotin-avidin fluorescence assay to study the CRP-monocyte interaction. It was observed that 67% of monocytes bound biotinylated CRP in a dose-dependent manner, that the binding was calcium dependent, and that it could be inhibited by 60% in the presence of a greater than 20-fold excess of competing native CRP. In other experiments, neither IgG nor heat-aggregated IgG inhibited the binding of CRP to monocytes; and no significant binding to lymphocyte population could be detected. These studies confirm the ability of human CRP to bind to a majority of human monocytes in a calcium-dependent and specific manner, and provide further support for a biologically important interaction of this acute-phase protein with phagocytic cells. PMID:2025959

  15. Bryostatins trigger human polymorphonuclear neutrophil and monocyte oxidative metabolism: association with in vitro antineoplastic activity.

    PubMed

    Esa, A H; Warren, J T; Hess, A D; May, W S

    1995-01-01

    Bryostatin-1-but not bryostatin-13-a macrocyclic lactone isolated from the marine bryozoan Bugula neritina, triggered human polymorphonuclear neutrophil (PMN) and monocyte release of reactive oxygen radicals, as measured by the generation of lucigenin chemiluminescence and by the ferricytochrome c reduction assay. The release of oxygen radicals by bryostatins was sensitive to inhibitors of protein kinases, but resistant to the inhibition of phospholipase A2 activity and arachidonic acid metabolism (prior treatment with mepacrine or indomethacin). Comparison of the effect of protein kinase (PK) inhibitors H-8, H-7 and staurosporine on bryostatin-1-induced neutrophil oxygen radical release further suggested a requirement for activation of phospholipid-dependent PKC, but not for cGMP- or cAMP-dependent PK. In cytostatic assays, PMNs treated with bryostatin-1 inhibited the growth of the erythroleukaemic cell line K562 in a concentration-dependent manner. These findings suggest that the reported antineoplastic effect of bryostatins may result at least in part from activation of PMNs and monocytes.

  16. Suppression of human monocyte interleukin-1beta production by ajulemic acid, a nonpsychoactive cannabinoid.

    PubMed

    Zurier, Robert B; Rossetti, Ronald G; Burstein, Sumner H; Bidinger, Bonnie

    2003-02-15

    Oral administration of ajulemic acid (AjA), a cannabinoid acid devoid of psychoactivity, reduces joint tissue damage in rats with adjuvant arthritis. Because interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha) are central to the progression of inflammation and joint tissue injury in patients with rheumatoid arthritis, we investigated human monocyte IL-1beta and TNFalpha responses after the addition of AjA to cells in vitro. Peripheral blood and synovial fluid monocytes (PBM and SFM) were isolated from healthy subjects and patients with inflammatory arthritis, respectively, treated with AjA (0-30 microM) in vitro, and then stimulated with lipopolysaccharide. Cells were harvested for mRNA, and supernatants were collected for cytokine assay. Addition of AjA to PBM and SFM in vitro reduced both steady-state levels of IL-1beta mRNA and secretion of IL-1beta in a concentration-dependent manner. Suppression was maximal (50.4%) at 10 microM AjA (P<0.05 vs untreated controls, N=7). AjA did not influence TNFalpha gene expression in or secretion from PBM. Reduction of IL-1beta by AjA may help explain the therapeutic effects of AjA in the animal model of arthritis. Development of nonpsychoactive therapeutically useful synthetic analogs of Cannabis constituents, such as AjA, may help resolve the ongoing debate about the use of marijuana as medicine.

  17. Two distinct receptors account for recognition of maleyl-albumin in human monocytes during differentiation in vitro.

    PubMed Central

    Haberland, M E; Rasmussen, R R; Olch, C L; Fogelman, A M

    1986-01-01

    A comparison of the receptor-mediated interaction of malondialdehyde-low density lipoprotein and maleyl-albumin has been examined in human monocytes during differentiation in vitro. The recognition of both ligands by the scavenger receptor of these cells has been confirmed. We now report that human monocytes express a second cellular surface receptor for maleyl-albumin that is distinct from the scavenger receptor. The activity of the maleyl-albumin receptor, determined by both binding and lysosomal hydrolytic assays, substantially exceeds that of the scavenger receptor in freshly isolated monocytes. A dramatic and rapid decline in the activity of the maleyl-albumin receptor occurs within 72 to 96 h during differentiation in vitro. At day 7, while only 5-10% of the original activity of the maleyl-albumin receptor remains, it is similar to that of the maximally expressed scavenger receptor. Both the binding and hydrolysis of ligand mediated by the maleyl-albumin receptor are specifically inhibited by alpha-casein and alkaline-treated albumin; neither of these proteins is recognized by the scavenger receptor. The occurrence of the exceptionally active maleyl-albumin receptor on freshly isolated human monocytes suggests that it participates in processes necessary to the function of the cells that diminish in importance after differentiation of the monocytes into macrophages in vitro. Furthermore, while maleyl-albumin is a useful adjunct to studies of cellular events mediated by the scavenger receptor, the presence of a second receptor for maleyl-albumin must be taken into account as a potential contributing and complicating event. Images PMID:3949974

  18. Endotoxin triggers tumor necrosis factor (TNF)-dependent cytotoxicity from interferon-. gamma. (IFN-. gamma. ) primed and unprimed human monocytes

    SciTech Connect

    Kornbluth, R.S.; Edgington, T.S.

    1986-03-05

    Under endotoxin-free conditions, the authors have found that human peripheral blood mononuclear cells (PBM) lack spontaneous monocyte-mediated cytotoxicity against actinomycin D-treated WEHI 164 target cells in a 6 hr /sup 51/Cr release assay. However, small amounts of endotoxin (e.g., LPS) rapidly induce monocyte-mediated cytotoxicity. IFN-..gamma.. alone is incapable of inducing monocyte cytotoxicity. Instead, pretreatment of PBM with IFN-..gamma.. for 36 hr or more primes them for triggering by amounts of endotoxin that are almost 100-fold less than that required for unprimed cells. These conditions are analogous to the two step activation sequence described for mice where IFN-..gamma.. primes and LPS triggers macrophage cytotoxic capacity. Additionally, the authors have observed that neutralizing anti-TNF monoclonal antibody abolishes the cytotoxicity measured here; and rTNF is directly cytotoxic to the target cells used in this assay. Thus, TNF is both necessary and sufficient for the monocyte mediated cytotoxicity. Since IFN-..gamma.. is thought to be produced during a variety of immunological reactions, these findings may help to explain the augmented capacity of immunologically stimulated animals for LPS-triggered TNF production and their enhanced sensitivity to the lethal effects of endotoxin.

  19. FSH-Receptor Isoforms and FSH-dependent Gene Transcription in Human Monocytes and Osteoclasts

    PubMed Central

    Robinson, Lisa J; Tourkova, Irina; Wang, Yujuan; Sharrow, Allison C; Landau, Michael S; Yaroslavskiy, Beatrice B; Li, Sun; Zaidi, Mone; Blair, Harry C

    2010-01-01

    Cells of the monocyte series respond to follicle stimulating hormone (FSH) by poorly characterized mechanisms. We studied FSH-receptors (FSH-R) and FSH response in nontransformed human monocytes and in osteoclasts differentiated from these cells. Western blot and PCR confirmed FSH-R expression on monocytes or osteoclasts, although at low levels relative to ovarian controls. Monocyte and osteoclast FSH-Rs differed from FSH-R from ovarian cells, reflecting variable splicing in exons 8–10. Monocytes produced no cAMP, the major signal in ovarian cells, in response to FSH. However, monocytes or osteoclasts transcribed TNFα in response to the FSH. No relation of expression of osteoclast FSH-R to the sex of cell donors or to exposure to sex hormones was apparent. Controls for FSH purity and endotoxin contamination were negative. Unamplified cRNA screening in adherent CD14 cells after 2 hours in 25 ng/ml FSH showed increased transcription of RANKL signalling proteins. Transcription of key proteins that stimulate bone turnover, TNFα and TSG-6, increased 2–3 fold after FSH treatment. Smaller but significant changes occurred in transcripts of selected signalling, adhesion, and cytoskeletal proteins. We conclude that monocyte and osteoclast FSH response diverges from that of ovarian cells, reflecting, at least in part, varying FSH-R isoforms. PMID:20171950

  20. Cellular requirements for the formation of EA rosettes by human monocytes.

    PubMed Central

    Passwell, J H; Schneeberger, E; Merler, E

    1978-01-01

    The binding of sensitized red cells to Fc receptors in human monocytes was studied by evaluating the effects of various pharmacological reagents and other treatments on EA rosette formation. Cytochalasin B and 2-deoxyglucose inhibited rosette formation in a dose-dependent manner. Sodium azide and incubation at 4 degrees also inhibited rosette formation, while at 37 degrees increased numbers of RBCs bound to the monocytes. The microtubular poisons, vinblastine and colchicine at high concentrations resulted in decreased adherence of monocytes and inhibition of rosette formation, while at low concentrations of colchicine, enhanced rosette formation was sometimes observed. Contrary to the effects on rosette formation, binding of [125I] IgG to monocyte monolayers was not altered by treatment of the monocytes with drugs. Magnesium ions were required to promote monocyte adherence, but both magnesium and calcium were needed for the best rosette formation. We conclude that the formation of EA rosettes is dependent not merely on binding of IgG to the Fc receptor but requires metabolically active monocytes, an intact cytostructure and suitable environmental conditions (temperature and cation concentration). Images Figure 3 PMID:738764

  1. ACTIVATED NEUTROPHILS INHIBIT PHAGOCYTOSIS BY HUMAN MONOCYTE CELLS IN VITRO

    EPA Science Inventory

    We have previously reported the correlation of decreased phagocytosis of opsonized zymosan by sputum monocytic cells with the increase in sputum neutrophils in volunteers 6h after inhalation of endotoxin (20,000 EU) (Alexis, et al. JACI, 2003;112:353). To define whether an intrin...

  2. Immunomodulatory Drugs Regulate HMGB1 Release from Activated Human Monocytes

    PubMed Central

    Schierbeck, Hanna; Wähämaa, Heidi; Andersson, Ulf; Harris, Helena Erlandsson

    2010-01-01

    Several HMGB1-specific antagonists have provided beneficial results in multiple models of inflammatory disease–preclinical trials including arthritis. Since no HMGB1-specific targeted therapy has yet reached the clinic, we have performed in vitro studies to investigate whether any of a selection of well-established antirheumatic drugs inhibit HMGB1 release as part of its mode of action. Freshly purified peripheral blood monocytes from healthy donors were stimulated in cultures with LPS and IFNγ to cause HMGB1 and TNF release detected in ELISPOT assays. Effects on the secretion were assessed in cultures supplemented with dexamethasone, cortisone, chloroquine, gold sodium thiomalate, methotrexate, colchicine, etanercept or anakinra. Pharmacologically relevant doses of dexamethasone, gold sodium thiomalate and chloroquine inhibited the extracellular release of HMGB1 in a dose-dependent mode. Immunostaining demonstrated that dexamethasone caused intracellular HMGB1 retention. No effects on HMGB1 secretion were observed in cultures with activated monocytes by any of the other studied agents. TNF production in LPS/IFNγ-activated monocytes was readily downregulated by dexamethasone and, to some extent, by chloroquine and etanercept. We conclude that dexamethasone, gold sodium thiomalate and chloroquine share a capacity to inhibit HMGB1 release from activated monocytes. PMID:20386869

  3. Expression of the cytochrome P450 epoxygenase CYP2J2 in human monocytic leukocytes.

    PubMed

    Nakayama, Kaeko; Nitto, Takeaki; Inoue, Teruo; Node, Koichi

    2008-08-29

    CYP2J2 is one of the cytochrome P450 epoxygenases involved in the metabolism of arachidonic acid. CYP2J2 has been identified in several tissues, especially cardiovascular tissues. CYP2J2 has cardiovascular effects, as epoxyeicosatrienoic acid, one of its metabolites, has anti-inflammatory and vasodilative activities. We investigated the expression of CYP2J2 in human leukocytes using reverse transcription-polymerase chain reaction, immunoblotting and immunostaining. Human monocytic cells, but not human neutrophils, exhibited constitutive expression of CYP2J2. Furthermore, the expression of CYP2J2 mRNA increased when the human monocytic cell line THP-1 cells and human monocytes were stimulated with phorbol 12-myristate 13-acetate and macrophage-colony stimulating factor in combination with granulocyte/macrophage-colony stimulating factor, respectively. These results suggest that expression of CYP2J2 was up-regulated when human monocytes differentiated into macrophages and that human monocytic cells and macrophages have a pathway to metabolize arachidonic acid using CYP epoxygenases.

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

  5. PARK2 Mediates Interleukin 6 and Monocyte Chemoattractant Protein 1 Production by Human Macrophages

    PubMed Central

    de Léséleuc, Louis; Girard, Manon; Huong, Nguyen Thu; Ba, Nguyen Ngoc; Van Thuc, Nguyen; Truman, Richard; Spencer, John S.; Adams, Linda; Thai, Vu Hong; Alcais, Alexandre; Schurr, Erwin

    2013-01-01

    Leprosy is a persistent infectious disease caused by Mycobacterium leprae that still affects over 200,000 new patients annually. The host genetic background is an important risk factor for leprosy susceptibility and the PARK2 gene is a replicated leprosy susceptibility candidate gene. The protein product of PARK2, Parkin, is an E3 ubiquitin ligase that is involved in the development of various forms of Parkinsonism. The human macrophage is both a natural host cell of M. leprae as well as a primary mediator of natural immune defenses, in part by secreting important pro-inflammatory cytokines and chemokines. Here, we report that down-regulation of Parkin in THP-1 macrophages, human monocyte-derived macrophages and human Schwann cells resulted in a consistent and specific decrease in interleukin-6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1/CCL2) production in response to mycobacteria or LPS. Interestingly, production of IL-6 at 6 hours by THP-1 cells stimulated with live M. leprae and M. bovis BCG was dependent on pretreatment with 1,25-dihydroxyvitamin D3 (VD). Parkin knockdown in VD-treated cells blocked IL-6 induction by mycobacteria. However, IκB-α phosphorylation and levels of IκB-ξ, a nuclear protein required for IL-6 expression, were not affected by Parkin silencing. Phosphorylation of MAPK ERK1/2 and p38 was unaffected by Parkin silencing while JNK activation was promoted but did not explain the altered cytokine production. In a final set of experiments we found that genetic risk factors of leprosy located in the PARK2 promoter region were significantly correlated with M. leprae sonicate triggered CCL2 and IL6 transcript levels in whole blood assays. These results associated genetically controlled changes in the production of MCP-1/CCL2 and IL-6 with known leprosy susceptibility factors. PMID:23350010

  6. Epstein-Barr virus infection-induced inflammasome activation in human monocytes

    PubMed Central

    Torii, Yuka; Murata, Takayuki; Yoshiyama, Hironori; Kimura, Hiroshi; Ito, Yoshinori

    2017-01-01

    Inflammasomes are cytoplasmic sensors that regulate the activity of caspase-1 and the secretion of interleukin-1β (IL-1β) or interleukin-18 (IL-18) in response to foreign molecules, including viral pathogens. They are considered to be an important link between the innate and adaptive immune responses. However, the mechanism by which inflammasome activation occurs during primary Epstein-Barr virus (EBV) infection remains unknown. Human B lymphocytes and epithelial cells are major targets of EBV, although it can also infect a variety of other cell types. In this study, we found that EBV could infect primary human monocytes and the monocyte cell line, THP-1, inducing inflammasome activation. We incubated cell-free EBV with THP-1 cells or primary human monocytes, then confirmed EBV infection using confocal microscopy and flow cytometry. Lytic and latent EBV genes were detected by real-time RT-PCR in EBV-infected monocytes. EBV infection of THP-1 cells and primary human monocytes induced caspase-dependent IL-1β production, while EBV infection of B-cell or T-cell lines did not induce IL-1β production. To identify the sensor molecule responsible for inflammasome activation during EBV infection, we examined the mRNA and the protein levels of NLR family pyrin domain-containing 3 (NLRP3), absent in melanoma 2 (AIM2), and interferon-inducible protein 16 (IFI16). Increased AIM2 levels were observed in EBV-infected THP-1 cells and primary human monocytes, whereas levels of IFI16 and NLRP3 did not show remarkable change. Furthermore, knockdown of AIM2 by small interfering RNA attenuated caspase-1 activation. Taken together, our results suggest that EBV infection of human monocytes induces caspase-1-dependent IL-1β production, and that AIM2, acting as an inflammasome, is involved in this response. PMID:28369146

  7. Viral infection triggers rapid differentiation of human blood monocytes into dendritic cells.

    PubMed

    Hou, Wanqiu; Gibbs, James S; Lu, Xiuju; Brooke, Christopher B; Roy, Devika; Modlin, Robert L; Bennink, Jack R; Yewdell, Jonathan W

    2012-03-29

    Surprisingly little is known about the interaction of human blood mononuclear cells with viruses. Here, we show that monocytes are the predominant cell type infected when peripheral blood mononuclear cells are exposed to viruses ex vivo. Remarkably, infection with vesicular stomatitis virus, vaccinia virus, and a variety of influenza A viruses (including circulating swine-origin virus) induces monocytes to differentiate within 18 hours into CD16(-)CD83(+) mature dendritic cells with enhanced capacity to activate T cells. Differentiation into dendritic cells does not require cell division and occurs despite the synthesis of viral proteins, which demonstrates that monocytes counteract the capacity of these highly lytic viruses to hijack host cell biosynthetic capacity. Indeed, differentiation requires infectious virus and viral protein synthesis. These findings demonstrate that monocytes are uniquely susceptible to viral infection among blood mononuclear cells, with the likely purpose of generating cells with enhanced capacity to activate innate and acquired antiviral immunity.

  8. In Vitro Brucella suis Infection Prevents the Programmed Cell Death of Human Monocytic Cells

    PubMed Central

    Gross, Antoine; Terraza, Annie; Ouahrani-Bettache, Safia; Liautard, Jean-Pierre; Dornand, Jacques

    2000-01-01

    During the complex interaction between an infectious agent and a host organism, the pathogen can interfere with the host cell's programmed death to its own benefit. Induction or prevention of host cell apoptosis appears to be a critical step for determining the infection outcome. Members of the gram-negative bacterial genus Brucella are intracellular pathogens which preferentially invade monocytic cells and develop within these cells. We investigated the effect of Brucella suis infection on apoptosis of human monocytic phagocytes. The present study provides evidence that Brucella infection inhibited spontaneously occurring apoptosis in human monocytes. Prevention of monocyte apoptosis was not mediated by Brucella lipopolysaccharide and required bacterial survival within infected cells. Both invaded and noninvaded cells were protected, indicating that soluble mediators released during infection were involved in the phenomenon. Analysis of Brucella-infected monocytes revealed specific overexpression of the A1 gene, a member of the bcl-2 family implicated in the survival of hematopoietic cells. Brucella infection also rendered macrophage-like cells resistant to Fas ligand- or gamma interferon-induced apoptosis, suggesting that Brucella infection protected host cells from several cytotoxic processes occurring at different steps of the immune response. The present data clearly show that Brucella suis modulated the monocyte/macrophage's apoptotic response to the advantage of the pathogen, thus preventing host cell elimination. This might represent a strategy for Brucella development in infected hosts. PMID:10603407

  9. CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes.

    PubMed

    Yeap, Wei Hseun; Wong, Kok Loon; Shimasaki, Noriko; Teo, Esmeralda Chi Yuan; Quek, Jeffrey Kim Siang; Yong, Hao Xiang; Diong, Colin Phipps; Bertoletti, Antonio; Linn, Yeh Ching; Wong, Siew Cheng

    2016-09-27

    Antibody-dependent cellular cytotoxicity (ADCC) is exerted by immune cells expressing surface Fcγ receptors (FcγRs) against cells coated with antibody, such as virus-infected or transformed cells. CD16, the FcγRIIIA, is essential for ADCC by NK cells, and is also expressed by a subset of human blood monocytes. We found that human CD16- expressing monocytes have a broad spectrum of ADCC capacities and can kill cancer cell lines, primary leukemic cells and hepatitis B virus-infected cells in the presence of specific antibodies. Engagement of CD16 on monocytes by antibody bound to target cells activated β2-integrins and induced TNFα secretion. In turn, this induced TNFR expression on the target cells, making them susceptible to TNFα-mediated cell death. Treatment with TLR agonists, DAMPs or cytokines, such as IFNγ, further enhanced ADCC. Monocytes lacking CD16 did not exert ADCC but acquired this property after CD16 expression was induced by either cytokine stimulation or transient transfection. Notably, CD16+ monocytes from patients with leukemia also exerted potent ADCC. Hence, CD16+ monocytes are important effectors of ADCC, suggesting further developments of this property in the context of cellular therapies for cancer and infectious diseases.

  10. CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes

    PubMed Central

    Yeap, Wei Hseun; Wong, Kok Loon; Shimasaki, Noriko; Teo, Esmeralda Chi Yuan; Quek, Jeffrey Kim Siang; Yong, Hao Xiang; Diong, Colin Phipps; Bertoletti, Antonio; Linn, Yeh Ching; Wong, Siew Cheng

    2016-01-01

    Antibody-dependent cellular cytotoxicity (ADCC) is exerted by immune cells expressing surface Fcγ receptors (FcγRs) against cells coated with antibody, such as virus-infected or transformed cells. CD16, the FcγRIIIA, is essential for ADCC by NK cells, and is also expressed by a subset of human blood monocytes. We found that human CD16− expressing monocytes have a broad spectrum of ADCC capacities and can kill cancer cell lines, primary leukemic cells and hepatitis B virus-infected cells in the presence of specific antibodies. Engagement of CD16 on monocytes by antibody bound to target cells activated β2-integrins and induced TNFα secretion. In turn, this induced TNFR expression on the target cells, making them susceptible to TNFα-mediated cell death. Treatment with TLR agonists, DAMPs or cytokines, such as IFNγ, further enhanced ADCC. Monocytes lacking CD16 did not exert ADCC but acquired this property after CD16 expression was induced by either cytokine stimulation or transient transfection. Notably, CD16+ monocytes from patients with leukemia also exerted potent ADCC. Hence, CD16+ monocytes are important effectors of ADCC, suggesting further developments of this property in the context of cellular therapies for cancer and infectious diseases. PMID:27670158

  11. Adenylyl cyclase-associated protein 1 is a receptor for human resistin and mediates inflammatory actions of human monocytes.

    PubMed

    Lee, Sahmin; Lee, Hyun-Chae; Kwon, Yoo-Wook; Lee, Sang Eun; Cho, Youngjin; Kim, Joonoh; Lee, Soobeom; Kim, Ju-Young; Lee, Jaewon; Yang, Han-Mo; Mook-Jung, Inhee; Nam, Ky-Youb; Chung, Junho; Lazar, Mitchell A; Kim, Hyo-Soo

    2014-03-04

    Human resistin is a cytokine that induces low-grade inflammation by stimulating monocytes. Resistin-mediated chronic inflammation can lead to obesity, atherosclerosis, and other cardiometabolic diseases. Nevertheless, the receptor for human resistin has not been clarified. Here, we identified adenylyl cyclase-associated protein 1 (CAP1) as a functional receptor for human resistin and clarified its intracellular signaling pathway to modulate inflammatory action of monocytes. We found that human resistin directly binds to CAP1 in monocytes and upregulates cyclic AMP (cAMP) concentration, protein kinase A (PKA) activity, and NF-κB-related transcription of inflammatory cytokines. Overexpression of CAP1 in monocytes enhanced the resistin-induced increased activity of the cAMP-dependent signaling. Moreover, CAP1-overexpressed monocytes aggravated adipose tissue inflammation in transgenic mice that express human resistin from their monocytes. In contrast, suppression of CAP1 expression abrogated the resistin-mediated inflammatory activity both in vitro and in vivo. Therefore, CAP1 is the bona fide receptor for resistin leading to inflammation in humans.

  12. A new role for monocytes in modulating myometrial inflammation during human labor.

    PubMed

    Srikhajon, Khetsopon; Shynlova, Oksana; Preechapornprasert, Anyarin; Chanrachakul, Boonsri; Lye, Stephen

    2014-07-01

    Here we fully characterize the cytokine profile of laboring human myometrium using Luminex analysis of 48 cytokine proteins, and stereologically quantified infiltration of monocytes and neutrophils into the myometrium. We hypothesized that monocytes can regulate their accumulation in the myometrium by disruption of proinflammatory cytokines to prevent an uncontrolled inflammatory response after labor onset. We isolated primary human myometrial cells (HMCs) from term, nonlaboring myometrial biopsies. Confluent HMCs were cocultured directly with human monocytic (THP-1) or lymphocytic (U937) cells, and with the same cells spatially separated by a membrane insert. After 72 h, HMCs and THP-1 were harvested separately, and RNA was extracted and analyzed by quantitative PCR. Coculture supernatants were collected and analyzed by Luminex assay and ELISA. We found that the laboring human myometrium produces significantly higher amounts of interleukin (IL) 6, IL9, IL18, IL1RA, CCL2, CCL7, CXCL8, CSF3, and tumor necrosis factor alpha, which coincides with the influx of immune cells. The direct contact or presence of THP-1 monocytes (but not U937 cells) significantly decreased CCL2 protein levels and increased IL1RA protein levels secreted by HMCs. This time-dependent decrease of CCL2 was greater with increasing number of monocytes being in direct contact with HMCs. We suggest a novel mechanism by which monocytes are first recruited to the myometrium by multiple cytokines and contribute to the physiologic inflammation of labor. After completing transmigration, activated monocytes disrupt locally established CCL2 gradients (possible by CCR2-mediated consumption) to limit their accumulation in the uterus. This mechanism may serve as a negative feedback loop to control the local inflammation and promote a return to homeostasis.

  13. Cardiomyogenic potential of mesenchymal progenitors derived from human circulating CD14+ monocytes.

    PubMed

    Kodama, Hiroaki; Inoue, Takafumi; Watanabe, Ryuichi; Yasuoka, Hidekata; Kawakami, Yutaka; Ogawa, Satoshi; Ikeda, Yasuo; Mikoshiba, Katsuhiko; Kuwana, Masataka

    2005-12-01

    Previously, we reported a unique CD14+CD45+CD34+type I collagen+ cell fraction derived from human circulating CD14+ monocytes, named monocyte-derived mesenchymal progenitors (MOMPs). These primitive cells differentiate along mesenchymal lineages, including bone, cartilage, fat, and skeletal muscle. Here, we demonstrate that CD14+ monocytes generate MOMPs that differentiate into cardiomyocytes. MOMPs labeled with a fluorescent marker and co-cultivated with rat cardiomyocytes for 4 weeks expressed the cardiomyocyte-specific transcription factors Nkx2.5, GATA-4, eHAND, and MEF2 and the hematopoietic/monocytic markers CD45 and CD14 within 10 days and retained their proliferative capacity for up to 16 days. A subpopulation of MOMPs subsequently expressed the cardiomyocyte-specific markers micro-sarcomeric actinin, troponin I, and atrial natriuretic peptide on day 21. Furthermore, fluorescence-labeled, spontaneously beating cells that formed gap junctions with adjacent rat cardiomyocytes appeared in these cultures and these cells exhibited electrophysiological properties typical of ventricular myocytes. The co-cultivation of human MOMPs with rat GFP-tagged cardiomyocytes resulted in the generation of human cardiomyocytes lacking green fluorescent protein (GFP) staining, suggesting that our observations could not solely be explained by cell fusion. Our results demonstrate for the first time that human circulating CD14+ monocytes include progenitors capable of proliferating and differentiating along the cardiomyogenic lineage via their differentiation into MOMPs.

  14. Caspase-8 inhibition represses initial human monocyte activation in septic shock model

    PubMed Central

    Oliva-Martin, Maria Jose; Sanchez-Abarca, Luis Ignacio; Rodhe, Johanna; Carrillo-Jimenez, Alejandro; Vlachos, Pinelopi; Herrera, Antonio Jose; Garcia-Quintanilla, Albert; Caballero-Velazquez, Teresa; Perez-Simon, Jose Antonio; Joseph, Bertrand; Venero, Jose Luis

    2016-01-01

    In septic patients, the onset of septic shock occurs due to the over-activation of monocytes. We tested the therapeutic potential of directly targeting innate immune cell activation to limit the cytokine storm and downstream phases. We initially investigated whether caspase-8 could be an appropriate target given it has recently been shown to be involved in microglial activation. We found that LPS caused a mild increase in caspase-8 activity and that the caspase-8 inhibitor IETD-fmk partially decreased monocyte activation. Furthermore, caspase-8 inhibition induced necroptotic cell death of activated monocytes. Despite inducing necroptosis, caspase-8 inhibition reduced LPS-induced expression and release of IL-1β and IL-10. Thus, blocking monocyte activation has positive effects on both the pro and anti-inflammatory phases of septic shock. We also found that in primary mouse monocytes, caspase-8 inhibition did not reduce LPS-induced activation or induce necroptosis. On the other hand, broad caspase inhibitors, which have already been shown to improve survival in mouse models of sepsis, achieved both. Thus, given that monocyte activation can be regulated in humans via the inhibition of a single caspase, we propose that the therapeutic use of caspase-8 inhibitors could represent a more selective alternative that blocks both phases of septic shock at the source. PMID:27250033

  15. Leptin regulates CD16 expression on human monocytes in a sex‐specific manner

    PubMed Central

    Cannon, Joseph G.; Sharma, Gyanendra; Sloan, Gloria; Dimitropoulou, Christiana; Baker, R. Randall; Mazzoli, Andrew; Kraj, Barbara; Mulloy, Anthony; Cortez‐Cooper, Miriam

    2014-01-01

    Abstract Fat mass is linked mechanistically to the cardiovascular system through leptin, a 16 kDa protein produced primarily by adipocytes. In addition to increasing blood pressure via hypothalamic‐sympathetic pathways, leptin stimulates monocyte migration, cytokine secretion, and other functions that contribute to atherosclerotic plaque development. These functions are also characteristics of CD16‐positive monocytes that have been implicated in the clinical progression of atherosclerosis. This investigation sought to determine if leptin promoted the development of such CD16‐positive monocytes. Cells from 45 healthy men and women with age ranging from 20 to 59 years were analyzed. Circulating numbers of CD14++16++ monocytes, which are primary producers of TNFα, were positively related to plasma leptin concentrations (P < 0.0001), with a stronger correlation in men (P < 0.05 for leptin × sex interaction). In vitro, recombinant human leptin induced CD16 expression in a dose‐related manner (P = 0.02), with a stronger influence on monocytes from men (P = 0.03 for leptin × sex interaction). There were no sex‐related differences in total leptin receptor expression on any monocyte subtypes, relative expression of long versus short isoforms of the receptor, or soluble leptin receptor concentrations in the plasma. The number of circulating CD14+16++ monocytes, which preferentially migrate into nascent plaques, was positively related to systolic blood pressure (R = 0.56, P = 0.0008) and intima‐media thickness (R = 0.37, P = 0.03), and negatively related to carotid compliance (R = −0.39, P = 0.02). These observations indicate that leptin promotes the development of CD16‐positive monocyte populations in a sex‐specific manner and that these subpopulations are associated with diminished vascular function. PMID:25303952

  16. Differential Activation of Human Monocytes and Lymphocytes by Distinct Strains of Trypanosoma cruzi

    PubMed Central

    Magalhães, Luísa M. D.; Viana, Agostinho; Chiari, Egler; Galvão, Lúcia M. C.; Gollob, Kenneth J.; Dutra, Walderez O.

    2015-01-01

    Background Trypanosoma cruzi strains are currently classified into six discrete typing units (DTUs) named TcI to VI. It is known that these DTUs have different geographical distribution, as well as biological features. TcI and TcII are major DTUs found in patients from northern and southern Latin America, respectively. Our hypothesis is that upon infection of human peripheral blood cells, Y strain (Tc II) and Col cl1.7 (Tc I), cause distinct immunological changes, which might influence the clinical course of Chagas disease. Methodology/Principal Findings We evaluated the infectivity of CFSE-stained trypomastigotes of Col cl1.7 and Y strain in human monocytes for 15 and 72 hours, and determined the immunological profile of lymphocytes and monocytes exposed to the different isolates using multiparameter flow cytometry. Our results showed a similar percentage and intensity of monocyte infection by Y and Col cl1.7. We also observed an increased expression of CD80 and CD86 by monocytes infected with Col cl1.7, but not Y strain. IL-10 was significantly higher in monocytes infected with Col cl1.7, as compared to Y strain. Moreover, infection with Col cl1.7, but not Y strain, led to an increased expression of IL-17 by CD8+ T cells. On the other hand, we observed a positive correlation between the expression of TNF-alpha and granzyme A only after infection with Y strain. Conclusion/Significance Our study shows that while Col cl1.7 induces higher monocyte activation and, at the same time, production of IL-10, infection with Y strain leads to a lower monocyte activation but higher inflammatory profile. These results show that TcI and TcII have a distinct immunological impact on human cells during early infection, which might influence disease progression. PMID:26147698

  17. Regulation of nutrition-associated receptors in blood monocytes of normal weight and obese humans.

    PubMed

    Pivovarova, Olga; Hornemann, Silke; Weimer, Sandra; Lu, Ye; Murahovschi, Veronica; Zhuk, Sergei; Seltmann, Anne-Cathrin; Malashicheva, Anna; Kostareva, Anna; Kruse, Michael; Busjahn, Andreas; Rudovich, Natalia; Pfeiffer, Andreas F H

    2015-03-01

    Obesity, type 2 diabetes and associated metabolic diseases are characterized by low-grade systemic inflammation which involves interplay of nutrition and monocyte/macrophage functions. We suggested that some factors such as nutrient components, neuropeptides involved in the control of gastrointestinal functions, and gastrointestinal hormones might influence immune cell functions and in this way contribute to the disease pathogenesis. The aim of this study was to investigate the mRNA expression of twelve nutrition-associated receptors in peripheral blood mononuclear cells (PBMC), isolated monocytes and monocyte-derived macrophages and their regulation under the switching from the high-carbohydrate low-fat diet to the low-carbohydrate high-fat (LC/HFD) isocaloric diet in healthy humans. The mRNA expression of receptors for short chain fatty acids (GPR41, GPR43), bile acids (TGR5), incretins (GIPR, GLP1R), cholecystokinin (CCKAR), neuropeptides VIP and PACAP (VIPR1, VIPR2), and neurotensin (NTSR1) was detected in PBMC and monocytes, while GPR41, GPR43, GIPR, TGR5, and VIPR1 were found in macrophages. Correlations of the receptor expression in monocytes with a range of metabolic and inflammatory markers were found. In non-obese subjects, the dietary switch to LC/HFD induced the increase of GPR43 and VIPR1 expression in monocytes. No significant differences of receptor expression between normal weight and moderately obese subjects were found. Our study characterized for the first time the expression pattern of nutrition-associated receptors in human blood monocytes and its dietary-induced changes linking metabolic responses to nutrition with immune functions in health and metabolic diseases.

  18. Modulation of the effector function of human monocytes for Mycobacterium avium by human immunodeficiency virus-1 envelope glycoprotein gp120.

    PubMed Central

    Shiratsuchi, H; Johnson, J L; Toossi, Z; Ellner, J J

    1994-01-01

    Disseminated Mycobacterium avium infection in AIDS is associated with high tissue burdens (10(9)-10(10) mycobacteria/g tissue) of organism. The basis for the extraordinary susceptibility of AIDS to M. avium infection is unclear. HIV or its constituents may alter mononuclear phagocyte functions resulting in enhanced intracellular M. avium growth. The effects of an envelope glycoprotein (gp120), a transmembrane protein (p121), and core proteins of HIV-1 on M. avium infection of human monocytes were examined. Preculturing monocytes with gp120 inhibited M. avium phagocytosis and consistently enhanced intracellular growth of six M. avium strains. Pretreatment with p121, gag5, or p24 did not inhibit phagocytosis nor enhance intracellular growth of M. avium. Incubation of gp120 with soluble CD4 before addition to monocyte cultures or pretreatment of monocytes with OKT4A abrogated gp120 effects on M. avium phagocytosis and intracellular growth. gp120 also augmented cytokine production by infected monocytes. These results suggest that gp120, but not p121 or core proteins, modulate monocyte phagocytosis and enhance intracellular growth of M. avium at least in part through monocyte CD4 receptors. Direct effects of HIV-1 products may, therefore, contribute to the diathesis of AIDS to develop disseminated M. avium infection and to the extensive replication of the organisms within tissue macrophages. Images PMID:8113420

  19. Monocyte-expressed urokinase regulates human vascular smooth muscle cell migration in a coculture model.

    PubMed

    Kusch, Angelika; Tkachuk, Sergey; Lutter, Steffen; Haller, Hermann; Dietz, Rainer; Lipp, Martin; Dumler, Inna

    2002-01-01

    Interactions of vascular smooth muscle cells (VSMC) with monocytes recruited to the arterial wall at a site of injury, with resultant modulation of VSMC growth and migration, are central to the development of vascular intimal thickening. Urokinase-type plasminogen activator (uPA) expressed by monocytes is a potent chemotactic factor for VSMC and might serve for the acceleration of vascular remodeling. In this report, we demonstrate that coculture of human VSMC with freshly isolated peripheral blood-derived human monocytes results in significant VSMC migration that increases during the coculture period. Accordingly, VSMC adhesion was inhibited with similar kinetics. VSMC proliferation, however, was not affected and remained at the same basal level during the whole period of coculture. The increase of VSMC migration in coculture was equivalent to the uPA-induced migration of monocultured VSMC and was blocked by addition into coculture of soluble uPAR (suPAR). Analysis of uPA and uPAR expression in cocultured cells demonstrated that monocytes are a major source of uPA, whose expression increases in coculture five-fold, whereas VSMC display an increased expression of cell surface-associated uPAR. These findings indicate that upregulated uPA production by monocytes following vascular injury acts most likely as an endogenous activator of VSMC migration contributing to the remodeling of vessel walls.

  20. Monocyte human leukocyte antigen-DR transcriptional downregulation by cortisol during septic shock.

    PubMed

    Le Tulzo, Yves; Pangault, Celine; Amiot, Laurence; Guilloux, Valérie; Tribut, Olivier; Arvieux, Cédric; Camus, Christophe; Fauchet, Renée; Thomas, Rémi; Drénou, Bernard

    2004-05-15

    Monocyte deactivation has been identified as a major factor of immunosuppression in sepsis and is associated with a loss of surface human leukocyte antigen-DR (HLA-DR) expression on circulating monocytes. Using flow cytometry, quantitative reverse transcription-polymerase chain reaction, we investigated this phenomenon in septic patients. We confirmed the early loss of monocyte HLA-DR expression in all infected patients and demonstrated that this persistent lowered expression at Day 6 correlated with severity scores, secondary infection, and death. This phenomenon occurred at a transcriptional level via a decrease in the class II transactivator A (CIITA) transcription. Furthermore, these abnormalities correlated with the high cortisol levels observed in sepsis and not with those of other putative factors such as catecholamines or interleukin-10. Finally, in vitro studies evidenced that glucocorticoids decrease HLA-DR expression at a transcriptional level via a decrease in CIITA mRNA levels, mainly by down modulating its isoforms I and III. We conclude that in human sepsis, the loss of HLA-DR expression on circulating monocytes is associated with a poor outcome. We suggest that the high endogenous cortisol level observed in septic shock may be a possible new factor involved in the loss of HLA-DR expression on monocytes via its effect on HLA-DR and CIITA transcription.

  1. Inhibition of human arterial smooth muscle cell growth by human monocyte/macrophages: a co-culture study.

    PubMed

    Proudfoot, D; Fitzsimmons, C; Torzewski, J; Bowyer, D E

    1999-07-01

    Monocyte/macrophages produce a variety of substances which may influence the function of smooth muscle cells (SMC). During atherogenesis, macrophages are thought to modulate SMC migration, proliferation and synthesis of extracellular matrix. Such modulation is the balance between stimulatory and inhibitory influences. Thus, for example, our earlier studies have shown that macrophages not only secrete mitogens, but also produce small molecular weight inhibitors of SMC proliferation. In the present study, we have used a co-culture system in which human monocyte/macrophages were separated from human arterial SMC (hSMC) by a filter with the optional addition of a 12 kDa cut-off dialysis membrane, in order to assess their effect on hSMC growth. We have found that human peripheral blood-derived monocytes produced a substance of < 12 kDa that inhibited hSMC growth in the co-culture system. The monocyte-derived factor causing this effect was completely blocked by indomethacin, indicating that growth-inhibitory factors produced by the monocytes were cyclooxygenase products. We have shown that PGE1 and PGE2 inhibit hSMC growth, making them likely candidates for the effector molecules released from monocytes in our co-culture system.

  2. Induction of ceruloplasmin synthesis by IFN-gamma in human monocytic cells

    NASA Technical Reports Server (NTRS)

    Mazumder, B.; Mukhopadhyay, C. K.; Prok, A.; Cathcart, M. K.; Fox, P. L.

    1997-01-01

    Ceruloplasmin is a 132-kDa glycoprotein abundant in human plasma. It has multiple in vitro activities, including copper transport, lipid pro- and antioxidant activity, and oxidation of ferrous ion and aromatic amines; however, its physiologic role is uncertain. Although ceruloplasmin is synthesized primarily by the liver in adult humans, production by cells of monocytic origin has been reported. We here show that IFN-gamma is a potent inducer of ceruloplasmin synthesis by monocytic cells. Activation of human monoblastic leukemia U937 cells with IFN-gamma increased the production of ceruloplasmin by at least 20-fold. The identity of the protein was confirmed by plasmin fingerprinting. IFN-gamma also increased ceruloplasmin mRNA. Induction followed a 2- to 4-h lag and was partially blocked by cycloheximide, indicating a requirement for newly synthesized factors. Ceruloplasmin induction in monocytic cells was agonist specific, as IL-1, IL-4, IL-6, IFN-alpha, IFN-beta, TNF-alpha, and LPS were completely ineffective. The induction was also cell type specific, as IFN-gamma did not induce ceruloplasmin synthesis in endothelial or smooth muscle cells. In contrast, IFN-gamma was stimulatory in other monocytic cells, including THP-1 cells and human peripheral blood monocytes, and also in HepG2 cells. Ceruloplasmin secreted by IFN-gamma-stimulated U937 cells had ferroxidase activity and was, in fact, the only secreted protein with this activity. Monocytic cell-derived ceruloplasmin may contribute to defense responses via its ferroxidase activity, which may drive iron homeostasis in a direction unfavorable to invasive organisms.

  3. Potent toxicity of 2-chlorodeoxyadenosine toward human monocytes in vitro and in vivo. A novel approach to immunosuppressive therapy.

    PubMed Central

    Carrera, C J; Terai, C; Lotz, M; Curd, J G; Piro, L D; Beutler, E; Carson, D A

    1990-01-01

    Lymphoid cells were thought to be uniquely susceptible to excess 2'-deoxyadenosine (dAdo), when exposed to inhibitors of adenosine deaminase (ADA). However, we now find that human monocytes are as sensitive as lymphocytes to dAdo or to the ADA-resistant congener 2-chloro-2'-deoxyadenosine (CldAdo). Monocytes exposed in vitro to CldAdo, or to dAdo plus deoxycoformycin rapidly developed DNA strand breaks. Both the DNA damage and the toxicity of CldAdo or dAdo toward monocytes were blocked by deoxycytidine, but not by inhibitors of poly(ADP-ribose) polymerase. A partial decrease in RNA synthesis and a gradual decline of cellular NAD were early biochemical events associated with monocyte DNA damage. Low CldAdo concentrations (5-20 nM) inhibited monocyte phagocytosis and reduced the release of interleukin 6. Higher CldAdo concentrations led to a dose- and time-dependent loss of monocyte viability. Circulating monocytes disappeared within 1 wk in patients with cutaneous T cell lymphoma or with rheumatoid arthritis during continuous CldAdo infusion. The marked sensitivity of human monocyte function and survival to CldAdo in vitro, together with the monocyte depletion in patients receiving CldAdo chemotherapy, suggests that CldAdo or other dAdo analogues offer a novel therapeutic strategy for chronic inflammatory and autoimmune diseases characterized by inappropriate monocyte deployment or function. PMID:1700795

  4. Interleukin-1 and interleukin-6 gene expression in human monocytes stimulated with Salmonella typhimurium porins.

    PubMed Central

    Galdiero, M; Cipollaro de L'ero, G; Donnarumma, G; Marcatili, A; Galdiero, F

    1995-01-01

    The aim of this study was to verify whether Salmonella typhimurium porins can affect the expression of interleukin-1 (IL-1) and interleukin-6 (IL-6) genes. Human monocytes were treated with porins, and total RNAs were analysed by Northern blotting to evaluate the expression of IL-1 alpha, IL-1 beta and IL-6 in both treated and untreated cell cultures. Porins induced a significant increase in IL-1 and IL-6 transcripts. This increase was related to the dose of porins, and it peaked 5 hr after treatment. The same results were obtained when polymyxin B was added to the porin preparation to eliminate eventual traces of lipopolysaccharide (LPS) associated with porins. The porins-mediated increase in interleukin transcripts did not require de novo protein synthesis, and it was because of the enhanced half-life of IL-1 and IL-6 mRNAs, rather an increased rate of gene transcription. These data suggest that porins may affect inflammatory and immunological responses by enhancing the expression of cytokine genes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8567029

  5. The human homolog of the JE gene encodes a monocyte secretory protein.

    PubMed Central

    Rollins, B J; Stier, P; Ernst, T; Wong, G G

    1989-01-01

    The mouse fibroblast gene, JE, was one of the first platelet-derived growth factor-inducible genes to be described as such. The protein encoded by JE (mJE) is the prototype of a large family of secreted, cytokinelike glycoproteins, all of whose members are induced by a mitogenic or activation signal in monocytes macrophages, and T lymphocytes; JE is the only member to have been identified in fibroblasts. We report the identification of a human homolog for murine JE, cloned from human fibroblasts. The protein predicted by the coding sequence of human JE (hJE) is 55 amino acids shorter than mJE, and its sequence is identical to that of a recently purified monocyte chemoattractant. When expressed in COS cells, the human JE cDNA directed the secretion of N-glycosylated proteins of Mr 16,000 to 18,000 as well as proteins of Mr 15,500, 15,000, and 13,000. Antibodies raised against mJE recognized these hJE species, all of which were secreted by human fibroblasts. hJE expression was stimulated in HL60 cells during phorbol myristate acetate-induced monocytoid differentiation. However, resting human monocytes constitutively secreted hJE; treatment with gamma interferon did not enhance hJE expression in monocytes, and treatment with phorbol myristate acetate or lipopolysaccharide inhibited its expression. Thus, human JE encodes yet another member of the large family of JE-related cytokinelike proteins, in this case a novel human monocyte and fibroblast secretory protein. Images PMID:2513477

  6. Intracellular Survival of Brucella spp. in Human Monocytes Involves Conventional Uptake but Special Phagosomes

    PubMed Central

    Rittig, Michael G.; Alvarez-Martinez, Maria-Teresa; Porte, Françoise; Liautard, Jean-Pierre; Rouot, Bruno

    2001-01-01

    Brucella spp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated how B. suis and B. melitensis enter human monocytes and in which compartment they survive. Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h. The presence of Brucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca2+- and Mg2+-free medium reduced the uptake. Engulfment of all Brucella organisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP). Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria. Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival of Brucella. These results indicate that (i) human monocytes readily internalize Brucella in a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of some Brucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall. PMID:11349069

  7. Liver X receptor is a key regulator of cytokine release in human monocytes.

    PubMed

    Myhre, Anders E; Agren, Joanna; Dahle, Maria K; Tamburstuen, Margareth V; Lyngstadaas, Ståle P; Collins, A Jon L; Foster, Simon J; Thiemermann, Christoph; Aasen, Ansgar O; Wang, Jacob E

    2008-04-01

    Aberrant regulation of innate immune responses and uncontrolled cytokine bursts are hallmarks of sepsis and endotoxemia. Activation of the nuclear liver X receptor (LXR) was recently demonstrated to suppress inflammatory genes. Our aim was to investigate the expression of LXR in human monocytes under normal and endotoxemic conditions and to study the influence of LXR activation on endotoxin-induced cytokine synthesis and release. Adherent human monocytes or whole blood were pretreated with a synthetic LXR agonist (3-{3-[(2-chloro-3-trifluoromethyl-benzyl)-(2,2-diphenyl-ethyl)-amino]-propoxy}-phenyl)-acetic acid) and subsequently challenged with LPS (from Escherichia coli) or peptidoglycan (from Staphylococcus aureus). Cytokine release was assessed by a Multiplex antibody bead kit, and cytokine mRNA levels were measured by real-time reverse-transcriptase-polymerase chain reaction. We found that LXRalpha mRNA was up-regulated in CD14+ monocytes in LPS-challenged blood, whereas LXRbeta mRNA was not altered. Addition of 3-{3-[(2-chloro-3-trifluoromethyl-benzyl)-(2,2-diphenyl-ethyl)-amino]-propoxy}-phenyl)-acetic acid to monocytes suppressed the LPS-induced release of IL-1beta, IL-6, IL-8, IL-10, IL-12p40, TMF-alpha, macrophage inflammatory protein 1alpha, macrophage inflammatory protein 1beta, and monocyte chemoattractant protein 1 in a concentration-dependent manner. Surprisingly, an accompanying decrease in cytokine mRNA accumulation was not observed. The suppressed cytokine release could not be explained by a diminished transport of mRNA out of the nucleus or a decreased secretion of cytokines. We propose that LXR is a key regulator of cytokine release in LPS-challenged human monocytes, possibly by interfering with translational events.

  8. Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes

    PubMed Central

    1987-01-01

    Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative bacterium and a facultative intracellular parasite that multiplies in human monocytes and alveolar macrophages. In this paper, mutants of L. pneumophila avirulent for human monocytes were obtained and extensively characterized. The mutants were obtained by serial passage of wild-type L. pneumophila on suboptimal artificial medium. None of 44 such mutant clones were capable of multiplying in monocytes or exerting a cytopathic effect on monocyte monolayers. Under the same conditions, wild-type L. pneumophila multiplied 2.5-4.5 logs, and destroyed the monocyte monolayers. The basis for the avirulent phenotype was an inability of the mutants to multiply intracellularly. Both mutant and wild-type bacteria bound to and were ingested by monocytes, and both entered by coiling phagocytosis. Thereafter, their intracellular destinies diverged. The wild-type formed a distinctive ribosome-lined replicative phagosome, inhibited phagosome-lysosome fusion, and multiplied intracellularly. The mutant did not form the distinctive phagosome nor inhibit phagosome-lysosome fusion. The mutant survived intracellularly but did not replicate in the phagolysosome. In all other respects studied, the mutant and wild-type bacteria were similar. They had similar ultrastructure and colony morphology; both formed colonies of compact and diffuse type. They had similar structural and secretory protein profiles and LPS profile by PAGE. Both the mutant and wild-type bacteria were completely resistant to human complement in the presence or absence of high titer anti-L. pneumophila antibody. The mutant L. pneumophila have tremendous potential for enhancing our understanding of the intracellular biology of L. pneumophila and other parasites that follow a similar pathway through the mononuclear phagocyte. Such mutants also show promise for enhancing our understanding of immunity to L. pneumophila, and they may serve

  9. Human monocyte spreading induced by factor Bb of the alternative pathway of complement activation. A possible role for C5 in monocyte spreading

    PubMed Central

    1981-01-01

    The central serine esterase of the alternative pathway of complement (APC) activation, activated factor B (Bb), has been shown recently to induce murine macrophages and human monocytes to become spread on a glass substrata. It has also been established that to induce the spreading reaction, the catalytic site of the Bb enzyme must be structurally intact since treatment of Bb with heat (56 degrees C for 30 min) or diisopropylfluorophosphate (10(-3) M) destroyed both enzymatic and spreading activities. In the C3b,Bb complex, Bb exhibits restricted substrate specificity for C3 and C5. With this in mind, the role of C3 and C5 in the monocyte spreading reaction was explored in the present study. Expression of C3 and C5 on the surface of human peripheral blood monocytes was investigated by the direct fluorescent antibody technique employing fluorescein isothiocyanate-conjugated anti- C3 or C5 F(ab')2 antibody fragments. It was found that C3 and C5 were present on 6 +/- 7% of freshly prepared monocytes and that expression of C5, but not C3, increased to 70 +/- 6% when monocytes were incubated for 3 d in serum-free medium. Biosynthesis of C5 was indicated when it was found that under serum-free conditions, monocytes incorporated [3H]leucine into immunoprecipitable C5 with an apparent mol wt of 180,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The role of C3 and C5 in the monocyte spreading reaction induced by factor Bb was explored by testing for the ability of anti-C3 and anti- C5 Fab' antibody fragments to block monocyte spreading. It was found that anti-C5 Fab' inhibited by up to 100% the 3-h human monocyte spreading reaction induced by Bb; in contrast, anti-C3 Fab' or anti-C4 Fab' inhibited by less than 10%. That the inhibitory effect of anti-C5 Fab' was exerted directly on the monocyte was established when it was found that the 3-h monocyte spreading reaction was significantly inhibited by pretreating monocytes with anti-C5 Fab' for 20 min and then

  10. A new method of preparing monocyte suspensions from human whole blood.

    PubMed

    Stoll, H P; Krämer, S; Oberhausen, E

    1986-01-01

    A method of isolating monocytes from human whole blood is described. The technique is primarily based on simple centrifugation steps that follow Tylose-sedimentation as well as on the use of the new density gradient medium Nycodens. Counterflow centrifugation is not involved. The final monocyte suspension is free of platelets. The contaminating cells are predominantly lymphocytes. As a whole, the method is a modification of the Nycodens technique published by Boyum in 1983, which leads to a total elimination of platelet contamination in the final cell suspension.

  11. A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

    PubMed

    Schwartz, B S; Reitnauer, P J; Hank, J A; Sondel, P M

    1985-09-01

    A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.

  12. Circulating classical monocytes are associated with CD11c+ macrophages in human visceral adipose tissue

    PubMed Central

    Wouters, Kristiaan; Gaens, Katrien; Bijnen, Mitchell; Verboven, Kenneth; Jocken, Johan; Wetzels, Suzan; Wijnands, Erwin; Hansen, Dominique; van Greevenbroek, Marleen; Duijvestijn, Adriaan; Biessen, Erik A. L.; Blaak, Ellen E.; Stehouwer, Coen D. A.; Schalkwijk, Casper G.

    2017-01-01

    Immune cell accumulation in adipose tissue (AT) is associated with the development of AT inflammation, resulting in metabolic dysfunction. Circulating immune cell patterns may reflect immune cell accumulation in expanding AT. However, data linking human leukocytes in blood and AT is lacking. We investigated whether blood immune cell populations are associated with their counterparts in subcutaneous (scAT) or visceral AT (vAT). Flow cytometry was performed on blood, scAT and vAT from 16 lean and 29 obese men. Circulating natural killer (NK)-cells, classical monocytes and nonclassical monocytes were higher in obese individuals. vAT, but not scAT, of obese individuals contained more inflammatory CD11c+ “M1” macrophages and NK cells compared to lean individuals. Blood classical monocytes were associated with CD11c+ macrophages in vAT but not scAT. This association was unrelated to expression of the adhesion molecules CD11b and CD11c or of the chemokine receptor CX3CR1 on these monocytes. Other AT immune cells were not associated with their respective counterparts in blood. Finally, CD11c+ macrophages and CD4+ T-cells in vAT were associated with their counterparts in scAT. In conclusion, blood classical monocytes reflect CD11c+ macrophages in vAT. PMID:28198418

  13. Cytokine and Eicosanoid Production by Cultured Human Monocytes Exposed to Titanium Particulate Debris

    NASA Astrophysics Data System (ADS)

    Robinson, Timothy M.; Manley, Paul A.; Sims, Paul A.; Albrecht, Ralph; Darien, Benjamin J.

    1999-10-01

    Phagocytosis of particulate wear debris from arthroplasties by macrophages induces an inflammatory response that has been linked to implant loosening and premature failure of artificial joints. Inflammatory mediators released by phagocytic macrophages such as tumor necrosis factor-a (TNF-[alpha]), interleukin-1[beta] (IL-1[beta]), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) are believed to play a central role in the pathogenesis of aseptic loosening. The objective of this study was to characterize titanium alloy particulates that closely match wear debris found around joint arthroplasties and to study their effects on the biosynthesis of inflammatory mediators by cultured monocytes. Peripheral blood monocytes were isolated from healthy human volunteers. Monocytes were cultured in 96-well plates for 24 h, washed, and exposed to three concentrations of titanium particulates and controls from 18Ð24 h. Supernatants were assayed for TNF-[alpha], IL-1[beta], IL-6, and PGE2 activity. Energy dispersive X-ray spectroscopy (EDX) verified the titanium alloy to be Ti6A14V. Scanning electron microscopy (SEM) analysis showed significant titanium particulate heterogeneity with approximately 95% of the particles <1 micrometer in diameter. SEM and EDX technology was useful in the characterization of the titanium particulates utilized for in vitro models of titanium-induced cytokine release by monocytes. Incubation of titanium particulates (in concentrations similar to those found around loosened prosthetic joints) with cultured monocytes significantly increased their production of TNF-[alpha], IL-1[beta], and PGE2.

  14. A 48-kilodalton Mycoplasma fermentans membrane protein induces cytokine secretion by human monocytes.

    PubMed Central

    Kostyal, D A; Butler, G H; Beezhold, D H

    1994-01-01

    Mycoplasma fermentans is one of several Mycoplasma species that have been reported to stimulate tumor necrosis factor (TNF) secretion from monocytes. This activity has been associated primarily with the mycoplasma membrane fraction. In this article, we have characterized a membrane protein that stimulates TNF and interleukin 1 beta secretion. The TNF-releasing activity partitioned into the Triton X-114 detergent phase, suggesting that the molecules is hydrophobic. The secretion of TNF is elevated in the presence of serum, which suggests that a serum component may play a role in the interaction between this mycoplasma protein and monocytes. Treatment of monocytes with monoclonal anti-CD14 antibody had no effect on the levels of TNF-releasing activity. By using the monocyte Western blot (immunoblot) technique, we have determined the molecular mass of the active molecule to be 48 kDa. This molecule appears to be distinct from the recently described family of variable lipoproteins of M. fermentans. Mycoplasma particulate material treated with proteinase K lost all inducing activity, whereas lipoprotein lipase-treated samples retained some level of activity. Images PMID:7520421

  15. Differentiation of human CD14+ monocytes: an experimental investigation of the optimal culture medium and evidence of a lack of differentiation along the endothelial line

    PubMed Central

    Safi, Wajima; Kuehnl, Andreas; Nüssler, Andreas; Eckstein, Hans-Henning; Pelisek, Jaroslav

    2016-01-01

    The aim of this study was to determine the optimal culturing media for human CD14+ monocytes and to evaluate whether these cells are capable of differentiating into vascular endothelial cells. Human monocytes isolated from peripheral blood were cultured for 1, 3, 7, 10 or 14 days in different media containing either 10% fetal bovine serum (FBS), 10% autologous donor serum (Auto), 10% FBS with interleukin-3 and macrophage colony stimulating factor (FBS-WF) or 10% Auto and the same growth factors (AU-WF). The cells were differentiated using endothelial cell conditioning medium (EC). Viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the cells were characterized by histology, immunohistochemistry and western blot analysis. Monocytes treated with Auto, FBS-WF or AU-WF medium generated a significant higher yield of vital cells after 7 days in culture compared with FBS-only medium (mean difference (MD)=0.318, P=0.01; MD=1.83, P=0.04; or MD=0.271, P=0.01 and MD=0.318, P=0.102). All tested media led to the differentiation of monocytes into macrophages, identified by CD68, especially in the FBS-WF medium (MD=+18.3% P=0.04). Differentiation into ECs caused a significant decrease in cell viability in all media. Endothelial cell markers, including CD31, CD144, VEGF, VEGF-R2 and CD34, could not be detected. Autologous serum significantly increases the yield of monocyte-derived cells with a higher effectiveness than commonly used FBS-only serum. There is no further benefit in culturing monocytes longer than 7 days. The cultivation of monocytes in the tested media leads preferentially to differentiation into macrophages. Differentiation into endothelial cells did not take place. PMID:27080367

  16. Impaired antigen presentation and potent phagocytic activity identifying tumor-tolerant human monocytes.

    PubMed

    Soares-Schanoski, Alessandra; Jurado, Teresa; Córdoba, Raúl; Siliceo, María; Fresno, Carlos Del; Gómez-Piña, Vanesa; Toledano, Victor; Vallejo-Cremades, Maria T; Alfonso-Iñiguez, Sergio; Carballo-Palos, Arkaitz; Fernández-Ruiz, Irene; Cubillas-Zapata, Carolina; Biswas, Subhra K; Arnalich, Francisco; García-Río, Francisco; López-Collazo, Eduardo

    2012-06-29

    Monocyte exposure to tumor cells induces a transient state in which these cells are refractory to further exposure to cancer. This phenomenon, termed "tumor tolerance", is characterized by a decreased production of proinflammatory cytokines in response to tumors. In the past, we found that this effect comprises IRAK-M up regulation and TLR4 and CD44 activation. Herein we have established a human model of tumor tolerance and have observed a marked down-regulation of MHCII molecules as well as the MHCII master regulator, CIITA, in monocytes/macrophages. These cells combine an impaired capability for antigen presentation with potent phagocytic activity and exhibit an M2-like phenotype. In addition circulating monocytes isolated from Chronic Lymphocytic Leukemia patients exhibited the same profile as tumor tolerant cells after tumor ex vivo exposition.

  17. Human immunodeficiency virus type 1 induces cellular polarization, intercellular adhesion molecule-1 redistribution, and multinucleated giant cell generation in human primary monocytes but not in monocyte-derived macrophages.

    PubMed

    Fais, S; Borghi, P; Gherardi, G; Logozzi, M; Belardelli, F; Gessani, S

    1996-12-01

    In this study, we evaluated the effects of human immunodeficiency virus type 1 (HIV-1) on some morphologic and functional changes in cultured human monocytes/macrophages at different stages of differentiation. Freshly isolated monocytes infected with HIV-1 24 hours after seeding exhibited marked morphologic changes such as uropod formation, polarization of intercellular adhesion molecule-1 (ICAM-1) on the cytoplasmic projection, the redistribution of alpha-actinin on cell-membrane dots, and an increased release of soluble ICAM-1. These changes preceded the increase in monocyte-monocyte fusion and the formation of multinucleated giant cells. In contrast, HIV-1 infection did not affect monocyte-derived macrophages in terms of either cellular polarization or multinucleated giant cell formation. Immunocytochemistry showed that HIV-1 matrix protein was present mostly in bi- and trinucleated cells, which suggests that multinucleated giant cells may represent a long-lived and highly productive cellular source of HIV. The treatment of the HIV-1-infected monocytes with azidodeoxythymidine virtually abolished all viral-induced morphofunctional changes. On the whole, these results indicate that blood monocytes and differentiated macrophages may be affected differently by HIV infection, as monocytes seem to be much more prone to polarize, undergo homotypic fusion, and form multinucleated giant cells. These changes may confer to HIV-infected monocytes an increased ability to transmigrate through endothelia into tissues, whereas differentiated macrophages may have a predominant role as a widespread reservoir of HIV.

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

    PubMed

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

    2014-01-01

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

  19. Localized adhesion of monocytes to human atherosclerotic plaques demonstrated in vitro: implications for atherogenesis.

    PubMed Central

    Poston, R. N.; Johnson-Tidey, R. R.

    1996-01-01

    Blood-derived macrophages in the arterial intima are a characteristic feature of active atherosclerotic plaques. Adherent monocytes on the luminal surface and increased adhesion molecules on the endothelium have suggested that specific molecular mechanisms are involved in monocyte/macrophage traffic into the arterial wall. Adhesion of human monocytes and related cell lines was therefore studied in vitro to histological sections of human plaques. At 37 degrees C, these cells bound selectively to the plaques. Binding to the endothelium occurred and was also present extensively in the diseased intima. Inhibition studies showed that the endothelial and general intimal binding had largely similar molecular properties. Strong inhibition was produced by antibodies to the monocyte-specific adhesion molecule CD14, to beta2 integrins, and to ICAM-1. Likewise, a peptide containing the Arg-Gly-Asp sequence was strongly inhibitory, suggesting that binding of leukocyte integrins to arterial extracellular matrix was synergistic with cell-cell interactions. A P-selectin antibody was exceptional in giving selective inhibition of endothelial adhesion, which correlates with the specific endothelial localization of this adhesion molecule. These results show that monocytes adhere to atherosclerotic plaques through the focal activation of multiple arterial wall adhesion molecules, confirming the adhesion hypothesis. A positive feedback theory for the pathogenesis of atherosclerosis can be suggested, based on the ability of macrophages in the wall to activate the endothelium, induce adhesion molecules, and facilitate additional monocyte entry. The adhesion assay provides a means for the identification of adhesion inhibitors with therapeutic potential. Images Figure 2 PMID:8686764

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

    PubMed Central

    Bian, Tianying; Li, Houxuan; Zhou, Qian; Ni, Can; Zhang, Yangheng

    2017-01-01

    The aim of this study was to investigate the role of human β-defensin 3 (hBD3) in the initiation stage of atherosclerosis with human umbilical vein endothelial cells (HUVECs) triggered by tumor necrosis factor- (TNF-) α. The effects of hBD3 on TNF-α-induced endothelial injury and inflammatory response were evaluated. Our data revealed that first, hBD3 reduced the production of interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1 (MCP-1), and macrophage migration inhibitory factor (MIF) in HUVECs in a dose-dependent manner. In addition, hBD3 significantly prevented intracellular reactive oxygen species (ROS) production by HUVECs. Second, western blot analysis demonstrated that hBD3 dose-dependently suppressed the protein levels of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in TNF-α-induced HUVECs. As a result, hBD3 inhibited monocyte adhesion to TNF-α-treated endothelial cells. Additionally, hBD3 suppressed TNF-α-induced F-actin reorganization in HUVECs. Third, hBD3 markedly inhibited NF-κB activation by decreasing the phosphorylation of IKK-α/β, IκB, and p65 subunit within 30 min. Moreover, the phosphorylation of p38 and c-Jun N-terminal protein kinase (JNK) in the mitogen-activated protein kinase (MAPK) pathway were also inhibited by hBD3 in HUVECs. In conclusion, hBD3 exerts anti-inflammatory and antioxidative effects in endothelial cells in response to TNF-α by inhibiting NF-κB and MAPK signaling. PMID:28348463

  1. Exaggerated human vascular cell prostaglandin biosynthesis mediated by monocytes: role of monokines and interleukin 1.

    PubMed

    Albrightson, C R; Baenziger, N L; Needleman, P

    1985-09-01

    Incubation of cultured human umbilical vein endothelial cells with factors derived from human peripheral blood mononuclear cells (MNCF) or adherent monocytes (AMF) resulted in concentration-and time-dependent increases in prostacyclin and prostaglandin E2 (PGE2) production. MNCF and AMF also stimulated prostacyclin and PGE2 biosynthesis in cultured human arterial smooth muscle cells and human dermal fibroblasts. The effect of these monokines on endothelial cells and fibroblasts was mimicked by treatment with purified human interleukin 1 (IL 1). Mononuclear cell-conditioned medium subjected to gel filtration yielded fractions (Mr 12,000 to 18,000 daltons) which simultaneously contained endothelial cell and fibroblast prostaglandin-stimulating activity and IL 1 activity. Therefore, monokines, specifically IL 1, appear to serve as chemical mediators of the interaction between monocytes and vascular cells as would occur in blood vessel injury, inflammation, and atherosclerosis.

  2. Pathogenicity of Mycobacterium avium for human monocytes: absence of macrophage-activating factor activity of gamma interferon.

    PubMed Central

    Toba, H; Crawford, J T; Ellner, J J

    1989-01-01

    Mycobacterium avium is a frequent opportunistic pathogen in the acquired immunodeficiency syndrome (AIDS). We compared 12 strains of M. avium in an in vitro model of pathogenicity. Peripheral blood-derived monocytes from healthy individuals were infected with M. avium in vitro. Bacterial uptake and intracellular replication were assessed by microscopic count of acid-fast bacilli and CFU of bacteria, respectively, in lysed monocytes. The CFU assay showed that among five AIDS-associated strains, only one replicated in monocytes. Two of seven non-AIDS-associated strains replicated intracellularly. In addition, we examined the effect of gamma interferon (IFN-gamma) on M. avium infection. IFN-gamma treatment of monocytes decreased phagocytosis and had no effect on the intracellular replication of M. avium. Thus, most strains of M. avium do not multiply within monocytes from healthy individuals and IFN-gamma does not have macrophage-activating factor activity for M. avium infection of human monocytes. PMID:2491838

  3. Induction of release of tumor necrosis factor from human monocytes by staphylococci and staphylococcal peptidoglycans.

    PubMed Central

    Timmerman, C P; Mattsson, E; Martinez-Martinez, L; De Graaf, L; Van Strijp, J A; Verbrugh, H A; Verhoef, J; Fleer, A

    1993-01-01

    The role of cytokines in gram-positive infections is still relatively poorly defined. The purpose of this study was to establish whether or not intact staphylococci and purified peptidoglycans and peptidoglycan components derived from staphylococci are capable of stimulating the release of tumor necrosis factor (TNF) by human monocytes. We show here that intact staphylococci and purified peptidoglycans, isolated from three Staphylococcus epidermidis and three S. aureus strains, were indeed able to induce secretion of TNF by human monocytes in a concentration-dependent fashion. TNF release was detected by both enzyme immunoassay and the L929 fibroblast bioassay. In the enzyme immunoassay, a minimal concentration of peptidoglycan of 1 micrograms/ml was required to detect TNF release by monocytes, whereas in the bioassay a peptidoglycan concentration of 10 micrograms/ml was needed to detect a similar amount of TNF release. Peptidoglycan components such as the stem peptide, tetra- and pentaglycine, and muramyl dipeptide were unable to induce TNF release from human monocytes. PMID:8406805

  4. Antithrombin III, but not C1 esterase inhibitor reduces inflammatory response in lipopolysaccharide-stimulated human monocytes in an ex-vivo whole blood setting.

    PubMed

    Kellner, Patrick; Nestler, Frank; Leimert, Anja; Bucher, Michael; Czeslick, Elke; Sablotzki, Armin; Raspè, Christoph

    2014-12-01

    In order to examine the immunomodulatory effects of antithrombin III (AT-III) and C1 esterase inhibitor (C1-INH) in human monocytes, we investigated the intracellular expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in an ex-vivo laboratory study in a whole blood setting. Heparinized whole blood samples from 23 healthy male and female volunteers (mean age: 27±7years) were pre-incubated with clinically relevant concentrations of AT-III (n=11) and C1-INH (n=12), then stimulated with 0.2 ng/mL lipopolysaccharide (LPS) for 3h. After phenotyping CD14⁺ monocytes, intracellular expression of IL-6, IL-8, and TNF-α was assessed using flow cytometry. In addition, 12 whole blood samples (AT-III and C1-INH, n=6 each) were examined using hirudin for anticoagulation; all samples were processed in the same way. To exclude cytotoxicity effects, 7-amino-actinomycin D and Nonidet P40 staining were used to investigate probes. This study is the first to demonstrate the influence of C1-INH and AT-III on the monocytic inflammatory response in a whole blood setting, which mimics the optimal physiological setting. Cells treated with AT-III exhibited significant downregulation of the proportion of gated CD14⁺ monocytes for IL-6 and IL-8, in a dose-dependent manner; downregulation for TNF-α did not reach statistical significance. There were no significant effects on mean fluorescence intensity (MFI). In contrast, C1-INH did not significantly reduce the proportion of gated CD14⁺ monocytes or the MFI regarding IL-6, TNF-α, and IL-8. When using hirudin for anticoagulation, no difference in the anti-inflammatory properties of AT-III and C1-INH in monocytes occurs. Taken together, in contrast to TNF-α, IL-6 and IL-8 were significantly downregulated in monocytes in an ex-vivo setting of human whole blood when treated with AT-III. This finding implicates monocytes as an important point of action regarding the anti-inflammatory properties of AT-III in sepsis. C1

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

    SciTech Connect

    Li Song; Zhang Junjie

    2009-01-09

    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 {beta} 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.

  6. In vitro production of tumor necrosis factor-alpha by human monocytes stimulated with lipopolysaccharide is positively correlated with increased blood monocytes after exposure to a swine barn.

    PubMed

    Willson, P J; Khozani, T Talaei; Juurlink, B H J; Senthilselvan, A; Rennie, D C; Gerdts, V; Gawaziuk, J; Schneberger, D; Burch, Lauranell H; Dosman, J A

    2008-01-01

    Recently there has been interest in the air quality in and around intensive livestock production facilities, such as modern swine production barns, where agricultural workers and surrounding residents may be exposed to elevated levels of organic dusts. The health effects of these exposures are not completely understood. The study that is reported here is a component of a larger investigation of the relationships among the acute effects of high-concentration endotoxin exposure (swine barn dust), polymorphisms in the TLR4 gene, and respiratory outcomes following exposure to swine confinement buildings. The relationships among a mediator of acute lung inflammation, tumor necrosis factor alpha (TNF-alpha), and clinical responses to acute swine barn exposure were characterized. Analysis of the results showed that in vitro stimulation of human monocytes with as little as 1 ng/ml of lipopolysaccharide (LPS) produced a significant increase in the monocytes that produced TNF-alpha. Although the proportion of TNF-alpha-positive monocytes after in vitro stimulation with 1 ng/ml of LPS was not associated with gender or TLR4 genotype, it was positively associated with the concentration of monocytes in blood after barn exposure. Thus, these two responses to different forms of LPS exposure are significantly correlated, and more responsive monocytes in vitro indicate a forthcoming relative monocytosis, post barn exposure, which may initiate a cascade of chronic inflammation.

  7. Monocyte/macrophage-derived microparticles up-regulate inflammatory mediator synthesis by human airway epithelial cells.

    PubMed

    Cerri, Chiara; Chimenti, Daniele; Conti, Ilaria; Neri, Tommaso; Paggiaro, Pierluigi; Celi, Alessandro

    2006-08-01

    Cell-derived microparticles (MP) are membrane fragments shed by virtually all eukaryotic cells upon activation or during apoptosis that play a significant role in physiologically relevant processes, including coagulation and inflammation. We investigated whether MP derived from monocytes/macrophages have the potential to modulate human airway epithelial cell activation. Monocytes/macrophages were isolated from the buffy coats of blood donors by Ficoll gradient centrifugation, followed by overnight culture of the mononuclear cell fraction. Adherent cells were washed and incubated with the calcium ionophore, A23187, or with histamine. The MP-containing supernatant was incubated with cells of the human bronchial epithelial line BEAS-2B and of the human alveolar line A549. IL-8, MCP-1, and ICAM-1 production was assessed by ELISA and by RT-PCR. In some experiments, monocytes/macrophages were stained with the fluorescent lipid intercalating dye PKH67, and the supernatant was analyzed by FACS. Stimulation of monocytes/macrophages with A23187 caused the release of particles that retain their fluorescent lipid intercalating label, indicating that they are derived from cell membranes. Incubation with A549 and BEAS-2B cells up-regulate IL-8 synthesis. Ultrafiltration and ultracentrifugation of the material abolished the effect, indicating that particulate matter, rather than soluble molecules, is responsible for it. Up-regulation of MCP-1 and ICAM-1 was also demonstrated in A549 cells. Similar results were obtained with histamine. Our data show that human monocytes/macrophages release MP that have the potential to sustain the innate immunity of the airway epithelium, as well as to contribute to the pathogenesis of inflammatory diseases of the lungs through up-regulation of proinflammatory mediators.

  8. Baseline Gene Expression Signatures in Monocytes from Multiple Sclerosis Patients Treated with Interferon-beta

    PubMed Central

    Bustamante, Marta F.; Nurtdinov, Ramil N.; Río, Jordi; Montalban, Xavier; Comabella, Manuel

    2013-01-01

    Background A relatively large proportion of relapsing-remitting multiple sclerosis (RRMS) patients do not respond to interferon-beta (IFNb) treatment. In previous studies with peripheral blood mononuclear cells (PBMC), we identified a subgroup of IFNb non-responders that was characterized by a baseline over-expression of type I IFN inducible genes. Additional mechanistic experiments carried out in IFNb non-responders suggested a selective alteration of the type I IFN signaling pathway in the population of blood monocytes. Here, we aimed (i) to investigate whether the type I IFN signaling pathway is up-regulated in isolated monocytes from IFNb non-responders at baseline; and (ii) to search for additional biological pathways in this cell population that may be implicated in the response to IFNb treatment. Methods Twenty RRMS patients classified according to their clinical response to IFNb treatment and 10 healthy controls were included in the study. Monocytes were purified from PBMC obtained before treatment by cell sorting and the gene expression profiling was determined with oligonucleotide microarrays. Results and discussion Purified monocytes from IFNb non-responders were characterized by an over-expression of type I IFN responsive genes, which confirms the type I IFN signature in monocytes suggested from previous studies. Other relevant signaling pathways that were up-regulated in IFNb non-responders were related with the mitochondrial function and processes such as protein synthesis and antigen presentation, and together with the type I IFN signaling pathway, may also be playing roles in the response to IFNb. PMID:23637780

  9. Human THP-1 monocyte uptake and cellular disposition of 14C-grepafloxacin.

    PubMed

    Hall, Iris H; Schwab, Ute E; Ward, E Stacy; Rublein, John C; Butts, John D; Ives, Timothy J

    2004-02-01

    Uptake of (14)C-grepafloxacin into human mononuclear (THP-1) cells was determined at pH 7.4, 6.8, or 5.0 over a 4-log antibiotic concentration. Grepafloxacin was taken up by THP-1 monocytes rapidly by both a passive and an active transport mechanism at pH 7.4. Its uptake was initially linear, with equilibrium being reached after approximately 1 h. Efflux followed first-order clearance and was complete within 1 h, suggesting no longterm sequestering of the antibiotic occurred. Neither cell number nor serum protein binding appeared to have any effect on antibiotic uptake. High intracellular concentrations were achieved and the ratios of cellular to extracellular antibiotic concentration (IC/EC) were between 529 and 644 at 0.04 micro g/ml at pH 7.4 and 6.8, suggesting that monocytes may contain sufficient levels of grepafloxacin for affecting bacteriostatic killing. Grepafloxacin disposition within the THP-1 monocytes showed large amounts present in the nucleus and cell sap in stimulated and unstimulated cells, and its presence was evenly distributed throughout the cytosol, nuclei, lysosomes, mitochondria, and ribosomes. After stimulation by zymogen A, Staphylococcus aureus, or Streptococcus pneumoniae, increased amounts of grepafloxacin were found within THP-1 monocytes and isolated phagosome vacuoles. No antibiotic sequestration occurred inside stimulated monocytes, although a sufficient intracellular grepafloxacin concentration was available to kill phagocytized bacteria. Metabolic inhibitors, suppressors of K(+)/Cl(-) and Cl(-) transporters, inhibitors of the phagocytic process, low temperature, and low pH inhibited grepafloxacin uptake by THP-1 monocytes.

  10. Monocytes increase human cardiac myofibroblast-mediated extracellular matrix remodeling through TGF-β1.

    PubMed

    Mewhort, Holly E M; Lipon, Brodie D; Svystonyuk, Daniyil A; Teng, Guoqi; Guzzardi, David G; Silva, Claudia; Yong, V Wee; Fedak, Paul W M

    2016-03-15

    Following myocardial infarction (MI), cardiac myofibroblasts remodel the extracellular matrix (ECM), preventing mechanical complications. However, prolonged myofibroblast activity leads to dysregulation of the ECM, maladaptive remodeling, fibrosis, and heart failure (HF). Chronic inflammation is believed to drive persistent myofibroblast activity; however, the mechanisms are unclear. We assessed the influence of peripheral blood monocytes on human cardiac myofibroblast activity in a three-dimensional (3D) ECM microenvironment. Human cardiac myofibroblasts isolated from surgical biopsies of the right atrium and left ventricle were seeded into 3D collagen matrices. Peripheral blood monocytes were isolated from healthy human donors and cocultured with myofibroblasts. Monocytes increased myofibroblast activity measured by collagen gel contraction (baseline: 57.6 ± 5.9% vs. coculture: 65.2 ± 7.1% contraction; P < 0.01) and increased local ECM remodeling quantified by confocal microscopy. Under coculture conditions that allow indirect cellular interaction via paracrine factors but prevent direct cell-cell contact, monocytes had minimal effects on myofibroblast activity (17.9 ± 11.1% vs. 6.4 ± 7.0% increase, respectively; P < 0.01). When cells were cultured under direct contact conditions, multiplex analysis of the coculture media revealed an increase in the paracrine factors TGF-β1 and matrix metalloproteinase 9 compared with baseline (122.9 ± 10.1 pg/ml and 3,496.0 ± 190.4 pg/ml, respectively, vs. 21.5 ± 16.3 pg/ml and 183.3 ± 43.9 pg/ml; P < 0.001). TGF-β blockade abolished the monocyte-induced increase in cardiac myofibroblast activity. These data suggest that direct cell-cell interaction between monocytes and cardiac myofibroblasts stimulates TGF-β-mediated myofibroblast activity and increases remodeling of local matrix. Peripheral blood monocyte interaction with human cardiac myofibroblasts stimulates myofibroblast activity through release of TGF-β1

  11. Neurogenic potential of progenitors derived from human circulating CD14+ monocytes.

    PubMed

    Kodama, Hiroaki; Inoue, Takafumi; Watanabe, Ryuichi; Yasutomi, Daisuke; Kawakami, Yutaka; Ogawa, Satoshi; Mikoshiba, Katsuhiko; Ikeda, Yasuo; Kuwana, Masataka

    2006-04-01

    We previously reported a primitive cell fraction derived from human circulating CD14+ monocytes, named monocyte-derived multipotential cells (MOMC), that can differentiate along mesenchymal lineages, including bone, cartilage, fat, skeletal muscle and cardiac muscle. In this study, we investigated whether MOMC can differentiate into the neuronal lineage. MOMC were fluorescently labelled and cocultivated with a primary culture of rat neurons for up to 4 weeks. The protein and gene expressions of neuron-specific markers in the human MOMC were evaluated over time using immunohistochemistry, in situ hybridization and reverse transcription followed by PCR. Shortly after cocultivation with rat neurons, nearly all the MOMC expressed early neuroectodermal markers, Mash1, Neurogenin2 and NeuroD, together with nestin, an intermediate filament expressed in neurogenesis. After 14 days of coculture, a subpopulation of MOMC displayed a multipolar morphology with elongated neurites and expressed mature neuron-specific markers, including neurofilament, microtubule-associated protein type 2, beta3-tubulin, NeuN and Hu. Transdifferentiation of monocytes into the neuroectodermal lineage was shown by the simultaneous expression of proneural markers and CD45/CD14 early in the differentiation process. The cocultivated MOMC retained their proliferative capacity for at least 16 days. Finally, the neuronal differentiation of MOMC was observed when they were cultured with neurons without cell-to-cell contact. The capacity of MOMC to differentiate into both mesodermal and neuroectodermal lineages suggests that circulating CD14+ monocytes are more multipotential than previously thought.

  12. Extracellular adenosine triphosphate activates calcium mobilization in human phagocytic leukocytes and neutrophil/monocyte progenitor cells.

    PubMed Central

    Cowen, D S; Lazarus, H M; Shurin, S B; Stoll, S E; Dubyak, G R

    1989-01-01

    We have examined the ability of extracellular ATP to elicit intracellular Ca2+ mobilization in a broad range of human leukocytes at particular stages of hematopoietic differentiation. The average cytosolic [Ca2+] in various leukocyte populations was measured in Fura 2-loaded cell suspensions while the cytosolic [Ca2+] in individual, Indo 1-loaded leukocytes was assayed by flow cytometric methods. Utilizing normal blood- and marrow-derived cells, human leukemic cell lines, and mononuclear cell fractions derived from the blood of patients with various leukemias, we have found that ATP-induced Ca2+ mobilization appears restricted to leukocytes of neutrophil/monocyte ontogeny. Significant ATP-induced increases in cytosolic [Ca2+] were observed in neutrophils, monocytes, and myeloid progenitor cells as immature as myeloblasts, but not in lymphocytes. Extensive characterization of the ATP-induced changes in [Ca2+] observed in the HL-60 promyelocytic cell line have indicated these Ca2+-mobilizing effects of ATP can be correlated with an activation of inositol phospholipid breakdown via the occupation of P2-purinergic receptors Significantly, of the various agonists (FMLP, platelet-activating factor, LTB4, and ATP) which elicit equivalent and maximal Ca2+ mobilization in mature neutrophils and monocytes, ATP was the most efficacious stimulant of Ca2+ mobilization in immature neutrophil/monocyte precursors. Thus, expression of putative P2-purinergic receptors for ATP appears to precede expression of other receptor types known to activate the inositol phospholipid signaling cascades in terminally differentiated phagocytes. PMID:2708526

  13. DNA methylation profiling reveals differences in the 3 human monocyte subsets and identifies uremia to induce DNA methylation changes during differentiation

    PubMed Central

    Zawada, Adam M.; Schneider, Jenny S.; Michel, Anne I.; Rogacev, Kyrill S.; Hummel, Björn; Krezdorn, Nicolas; Müller, Soeren; Rotter, Björn; Winter, Peter; Obeid, Rima; Geisel, Jürgen; Fliser, Danilo; Heine, Gunnar H.

    2016-01-01

    ABSTRACT Human monocytes are a heterogeneous cell population consisting of 3 subsets: classical CD14++CD16-, intermediate CD14++CD16+ and nonclassical CD14+CD16++ monocytes. Via poorly characterized mechanisms, intermediate monocyte counts rise in chronic inflammatory diseases, among which chronic kidney disease is of particular epidemiologic importance. DNA methylation is a central epigenetic feature that controls hematopoiesis. By applying next-generation Methyl-Sequencing we now tested how far the 3 monocyte subsets differ in their DNA methylome and whether uremia induces DNA methylation changes in differentiating monocytes. We found that each monocyte subset displays a unique phenotype with regards to DNA methylation. Genes with differentially methylated promoter regions in intermediate monocytes were linked to distinct immunological processes, which is in line with results from recent gene expression analyses. In vitro, uremia induced dysregulation of DNA methylation in differentiating monocytes, which affected several transcription regulators important for monocyte differentiation (e.g., FLT3, HDAC1, MNT) and led to enhanced generation of intermediate monocytes. As potential mediator, the uremic toxin and methylation inhibitor S-adenosylhomocysteine induced shifts in monocyte subsets in vitro, and associated with monocyte subset counts in vivo. Our data support the concept of monocyte trichotomy and the distinct role of intermediate monocytes in human immunity. The shift in monocyte subsets that occurs in chronic kidney disease, a proinflammatory condition of substantial epidemiological impact, may be induced by accumulation of uremic toxins that mediate epigenetic dysregulation. PMID:27018948

  14. DNA methylation profiling reveals differences in the 3 human monocyte subsets and identifies uremia to induce DNA methylation changes during differentiation.

    PubMed

    Zawada, Adam M; Schneider, Jenny S; Michel, Anne I; Rogacev, Kyrill S; Hummel, Björn; Krezdorn, Nicolas; Müller, Soeren; Rotter, Björn; Winter, Peter; Obeid, Rima; Geisel, Jürgen; Fliser, Danilo; Heine, Gunnar H

    2016-04-02

    Human monocytes are a heterogeneous cell population consisting of 3 subsets: classical CD14++CD16-, intermediate CD14++CD16+ and nonclassical CD14+CD16++ monocytes. Via poorly characterized mechanisms, intermediate monocyte counts rise in chronic inflammatory diseases, among which chronic kidney disease is of particular epidemiologic importance. DNA methylation is a central epigenetic feature that controls hematopoiesis. By applying next-generation Methyl-Sequencing we now tested how far the 3 monocyte subsets differ in their DNA methylome and whether uremia induces DNA methylation changes in differentiating monocytes. We found that each monocyte subset displays a unique phenotype with regards to DNA methylation. Genes with differentially methylated promoter regions in intermediate monocytes were linked to distinct immunological processes, which is in line with results from recent gene expression analyses. In vitro, uremia induced dysregulation of DNA methylation in differentiating monocytes, which affected several transcription regulators important for monocyte differentiation (e.g., FLT3, HDAC1, MNT) and led to enhanced generation of intermediate monocytes. As potential mediator, the uremic toxin and methylation inhibitor S-adenosylhomocysteine induced shifts in monocyte subsets in vitro, and associated with monocyte subset counts in vivo. Our data support the concept of monocyte trichotomy and the distinct role of intermediate monocytes in human immunity. The shift in monocyte subsets that occurs in chronic kidney disease, a proinflammatory condition of substantial epidemiological impact, may be induced by accumulation of uremic toxins that mediate epigenetic dysregulation.

  15. Biological effects of double-walled carbon nanotubes on the innate immune system: An in vitro study on THP-1 human monocytes.

    PubMed

    Dekali, Samir; Bachelet, Christine; Maunoir-Regimbal, Séverine; Flahaut, Emmanuel; Debouzy, Jean-Claude; Crouzier, David

    2016-07-15

    DWCNTs have numerous industrial and biomedical applications and several studies reported that they could act as immunomodulator systems. The immune system is the first line of defence of the human body when exposed to particulate matter. In order to investigate DWCNTs' role on innate immunity, we used THP-1 monocytic cells for the purpose of this study. We showed that DWCNTs were not cytotoxic until 6h, 24h, 48h and 72h of incubation with THP-1 monocytic cells (concentrations tested from 10 to 50μg/mL). From 6h to 72h of incubation of THP-1 cells with DWCNTs, we measured a significant increase of the baseline cell index using xCELLigence(®) technology showing cell adhesion. After 24h of exposure, DWCNTs agglomerates were localized in THP-1 monocyte cytoplasm and cell adhesion was observed simultaneously with a significant increase in the expression of CD11b and CD14 cell surface proteins. Pro-inflammatory cytokine secretion (IL-1β, IL-6, IL-8, TNF-α and IL-10) was also measured in supernatants after 6h or 24h of exposure to DWCNTs. This pro-inflammatory response was increased in THP-1 monocytic cells pre-treated with LPS. Altogether, our data indicate that DWCNTs induce an increased pro-inflammatory response of THP-1 monocytes and seem to modulate cell surface protein expression confirming that DWCNTs could act as stimulators of innate immunity.

  16. Aryl hydrocarbon receptor activation inhibits in vitro differentiation of human monocytes and Langerhans dendritic cells.

    PubMed

    Platzer, Barbara; Richter, Susanne; Kneidinger, Doris; Waltenberger, Darina; Woisetschläger, Maximilian; Strobl, Herbert

    2009-07-01

    The transcription factor aryl hydrocarbon receptor (AhR) represents a promising therapeutic target in allergy and autoimmunity. AhR signaling induced by the newly described ligand VAF347 inhibits allergic lung inflammation as well as suppresses pancreatic islet allograft rejection. These effects are likely mediated via alterations in dendritic cell (DC) function. Moreover, VAF347 induces tolerogenic DCs. Langerhans cells (LCs) are immediate targets of exogenous AhR ligands at epithelial surfaces; how they respond to AhR ligands remained undefined. We studied AhR expression and function in human LCs and myelopoietic cell subsets using a lineage differentiation and gene transduction model of human CD34(+) hematopoietic progenitors. We found that AhR is highly regulated during myeloid subset differentiation. LCs expressed highest AhR levels followed by monocytes. Conversely, neutrophil granulocytes lacked AhR expression. AhR ligands including VAF347 arrested the differentiation of monocytes and LCs at an early precursor cell stage, whereas progenitor cell expansion or granulopoiesis remained unimpaired. AhR expression was coregulated with the transcription factor PU.1 during myeloid subset differentiation. VAF347 inhibited PU.1 induction during initial monocytic differentiation, and ectopic PU.1 restored monocyte and LC generation in the presence of this compound. AhR ligands failed to interfere with cytokine receptor signaling during LC differentiation and failed to impair LC activation/maturation. VAF347-mediated antiproliferative effect on precursors undergoing LC lineage differentiation occurred in a clinically applicable serum-free culture model and was not accompanied by apoptosis induction. In conclusion, AhR agonist signaling interferes with transcriptional processes leading to monocyte/DC lineage commitment of human myeloid progenitor cells.

  17. Effect of Bacterial Lipopolysaccharide Contamination on Gutta Percha- versus Resilon-Induced Human Monocyte Cell Line Toxicity

    PubMed Central

    Hadjati, Jamshid; Assadian, Hadi; Ghorbanzadeh, Abdollah; Nourizadeh, Maryam; Fattah, Tahereh; Shokouhinejad, Noushin

    2015-01-01

    Objectives: Cytotoxic effects of obturation materials were tested in presence and absence of endotoxin on human monocytes in vitro. Materials and Methods: Human monocytes from THP-1 cell line were cultured. Three millimeters from the tip of each Resilon and gutta percha points were cut and directly placed at the bottom of the culture wells. Cultured cells were exposed to gutta percha (groups G1 and G2) and Resilon (R1 and R2). Ten μg/ml bacterial lipopolysaccharide (LPS) was added to the culture wells in groups G1 and R1. Positive control included the bacterial LPS without the root canal filling material and the negative control contained the cells in culture medium only. Viability of cells was tested in all groups after 24, 48, and 72 hours using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay for at least 3 times to obtain reproducible results. Optical density values were read and the data were analyzed using three-way ANOVA and post hoc statistical test. Results: The results showed that cells in G2 had the lowest rate of viability at 24 hours, but the lowest rate of viable cells was recorded in G1 at 48 and 72 hours. The effect of LPS treatment was not statistically significant. Resilon groups showed cell viability values higher than those of gutta percha groups, although statistically non-significant (P=0.105). Cell viability values were lower in gutta percha than Resilon groups when LPS-treated and LPS-untreated groups were compared independently at each time point. Conclusion: It could be concluded that none of the tested root canal filling materials had toxic effects on cultured human monocyte cells whether in presence or absence of LPS contamination. PMID:26056523

  18. Alcohol-Induced miR-27a Regulates Differentiation and M2 Macrophage Polarization of Normal Human Monocytes

    PubMed Central

    Saha, Banishree; Bruneau, Johanna C.; Kodys, Karen; Szabo, Gyongyi

    2015-01-01

    Alcohol abuse is a leading cause of liver disease characterized by liver inflammation, fatty liver, alcoholic hepatitis, or liver cirrhosis. Immunomodulatory effects of alcohol on monocytes and macrophages contribute to alcoholic liver disease. Alcohol use, an independent risk factor for progression of hepatitis C virus (HCV) infection–mediated liver disease, impairs host defense and alters cytokine production and monocyte/macrophage activation. We hypothesized that alcohol and HCV have synergistic effects on the phenotype and function of monocytes. Our data show that acute alcohol binge drinking in healthy volunteers results in increased frequency of CD16+ and CD68+ and M2-type (CD206+, dendritic cell [DC]-SIGN+–expressing and IL-10–secreting) circulating CD14+ monocytes. Expression of HCV-induced CD68 and M2 markers (CD206 and DC-SIGN) in normal monocytes was further enhanced in the presence of alcohol. The levels of microRNA (miR)-27a was significantly upregulated in monocytes cultured in the presence of alcohol or alcohol and HCV as compared with HCV alone. The functional role of miR-27a in macrophage polarization was demonstrated by transfecting monocytes with an miR-27a inhibitor that resulted in reduced alcohol- and HCV- mediated monocyte activation (CD14 and CD68 expression), polarization (CD206 and DC-SIGN expression), and IL-10 secretion. Over-expression of miR-27a in monocytes enhanced IL-10 secretion via activation of the ERK signaling pathway. We found that miR-27a promoted ERK phosphorylation by downregulating the expression of ERK inhibitor sprouty2 in monocytes. Thus, we identified that sprouty2 is a target of miR-27a in human monocytes. In summary, our study demonstrates the regulatory role of miR-27a in alcohol-induced monocyte activation and polarization. PMID:25716995

  19. Human CD64-targeted non-viral siRNA delivery system for blood monocyte gene modulation

    PubMed Central

    Yong, Seok-Beom; Kim, Hyung Jin; Kim, Jang Kyoung; Chung, Jee Young; Kim, Yong-Hee

    2017-01-01

    A subset of phagocytes including inflammatory monocytes in blood migrate and give rise to macrophages in inflammatory tissues which generated the idea that blood monocytes are the therapeutic targets for drug delivery. Fc gamma receptor I (CD64) is a membrane receptor for the Fc region of immunoglobulin G, primarily expressed on monocyte-lineage, and H22 a monoclonal antibody for human CD64 had shown rapid blood monocyte binding and occupation in clinical studies. Small interfering RNA-mediated gene silencing as a therapeutic has been proposed and is a promising strategy in terms of its “knock-down” ability on the target gene prior to translation. However, its instability and off-targeting effect must be overcome for success in clinical studies. In this study, we developed a non-viral delivery system composed of oligo-nona-arginine (9R) and anti-human CD64 single chain antibodies (H22) for human monocyte-specific siRNA delivery. A targeted and efficient siRNA delivery mediated by anti-CD64 scFv-9R was observed in CD64 positive human leukemia cells, THP-1. With primary human blood cells, anti-CD64 scFv-9R mediated gene silencing was quantitatively confirmed representing blood monocyte selective gene delivery. These results demonstrate the potential of anti-CD64 scFv-9R mediated siRNA delivery for the treatment of human inflammatory diseases via blood monocytes gene delivery. PMID:28169353

  20. Iron oxide nanoparticles modulate lipopolysaccharide-induced inflammatory responses in primary human monocytes

    PubMed Central

    Grosse, Susann; Stenvik, Jørgen; Nilsen, Asbjørn M

    2016-01-01

    Co-stimulation of the immune system to more than one agent concomitantly is very common in real life, and considering the increasing use of engineered nanoparticles and nanomaterials, it is highly relevant to assess the ability of these materials to modulate key innate immune responses, which has not yet been studied in detail. We investigated the immunomodulatory effects of 10 nm and 30 nm iron oxide nanoparticles (IONPs) on primary human monocytes in the presence and absence of Toll-like receptor 4 agonist lipopolysaccharide (LPS). Prior to the cell studies, we characterized the physicochemical properties of the nanoparticles in cell culture medium and ensured that the nanoparticles were free from biological contamination. Cellular uptake of the IONPs in monocytes was assessed using transmission electron microscopy. Using enzyme-linked immunosorbent assay, we found that the IONPs per se did not induce the production of proinflammatory cytokines tumor necrosis factor-α, interleukin-6, and interleukin-1β. However, the IONPs had the ability to suppress LPS-induced nuclear factor kappa B activation and production of proinflammatory cytokines in primary human monocytes in an LPS and a particle dose-dependent manner. Using confocal microscopy and fluorescently labeled LPS, we showed that the effects correlated with impaired LPS internalization by monocytes in the presence of IONPs, which could be partly explained by LPS adsorption onto the nanoparticle surface. Additionally, the results from particle pretreatment experiments indicate that other cellular mechanisms might also play a role in the observed effects, which warrants further studies to elucidate the additional mechanisms underlying the capacity of IONPs to alter the reactivity of monocytes to LPS and to mount an appropriate cellular response. PMID:27695322

  1. Human antiphospholipid antibodies induce TNFalpha in monocytes via Toll-like receptor 8.

    PubMed

    Döring, Yvonne; Hurst, Julia; Lorenz, Mareike; Prinz, Nadine; Clemens, Natascha; Drechsler, Maik D; Bauer, Stefan; Chapman, Joab; Shoenfeld, Yehuda; Blank, Miri; Lackner, Karl J; von Landenberg, Philipp

    2010-03-01

    The antiphospholipid syndrome (APS) is characterized by recurrent arterial and/or venous thromboses, pregnancy loss and the presence of antiphospholipid antibodies (aPL). One of the discussed mechanisms of this thrombotic activity in APS patients is attributed to TNFalpha secretion in monocytes after aPL stimulation. To investigate this mechanism in detail, we employed a monoclonal aPL and IgG fractions of APS patients for stimulation of human peripheral monocytes. Stimulation with this monoclonal aPL resulted in an increased expression and secretion of TNFalpha, caused by specific upregulation of TLR8 mRNA and protein expression levels. To confirm the specificity of this finding we could demonstrate that the TNFalpha enhancement could be neutralized by a TLR8-specific inhibitory DNA-oligonucleotide and could be further increased by adding the specific ligands for TLR8. Using APS patients IgG fractions for stimulation of peripheral monocytes revealed a similar TLR8 mRNA elevation and increase in TNFalpha-production. Furthermore the TLR8 expression level in PBMC's of APS patients was as well significantly elevated. It could be demonstrated that the TNFalpha release in monocytes resulting from aPL stimulation was exclusively induced by TLR8 engagement. This could be confirmed in PBMC's of APS patients, hinting that endogenous stimulation of TLR8 in APS patients and consecutive elevation of TNFalpha promotes a proinflammatory environment.

  2. Glycosyltransferase and sulfotransferase gene expression profiles in human monocytes, dendritic cells and macrophages

    PubMed Central

    Trottein, François; Schaffer, Lana; Ivanov, Stoyan; Paget, Christophe; Vendeville, Catherine; Groux-Degroote, Sophie; Lee, Suzanna; Krzewinski-Recchi, Marie-Ange; Head, Steven R; Gosset, Philippe; Delannoy, Philippe

    2010-01-01

    Using a focused glycan-gene microarray, we compared the glycosyltransferase (GT) and sulfotransferase gene expression profile of human monocytes relative to immature and mature dendritic cells (DCs) or macrophages (Mφs). Microarray analysis indicated that monocytes express transcripts for a full set of enzymes involved in the biosynthesis of N- and O-glycans potentially elongated by poly-LacNAc chains with type II terminal sequences. Monocytes also express genes encoding enzymes involved in glycosaminoglycan biosynthesis but have a limited capacity for glycolipid synthesis. Among genes significantly expressed in monocytes (90 out of 175), 39 are modulated in DCs and/or Mφ, a large proportion being increased in both cell types. This change in GT and sulfotransferase genes might potentially enforce the capacity of differentiated cells to synthesize branched N-glycans and mucin-type O-glycans, and to remodel of cell surface proteoglycans during the differentiation process. Stimulation of DCs and Mφs with lipopolysaccharide caused a decrease in gene expression mainly affecting genes found to be positively modulated during the differentiation steps. Validation of this analysis was provided by quantitative real-time PCR and flow cytometry of cell surface glycan epitopes. Collectively, this study implies an important modification of the pattern of glycosylation in DCs and Mφs undergoing differentiation and maturation with potential biological consequences. PMID:19533340

  3. Angiotensin II activates the proinflammatory transcription factor nuclear factor-kappaB in human monocytes.

    PubMed

    Kranzhöfer, R; Browatzki, M; Schmidt, J; Kübler, W

    1999-04-21

    The renin-angiotensin system may contribute to the pathogenesis of atherosclerosis. A common feature of all stages of atherosclerosis is inflammation of the vessel wall. The transcription factor nuclear factor-kappaB (NF-kappaB) participates in most signaling pathways involved in inflammation. This study therefore examined the effect of angiotensin (ANG) II on NF-kappaB activation in monocytic cells, a major cellular component of human atheroma, by electrophoretic mobility shift assay. ANG II, like TNFalpha, caused rapid activation of NF-kappaB in human mononuclear cells isolated from peripheral blood by Ficoll density gradient. This ANG II effect was blocked by the angiotensin AT1 receptor antagonist losartan. Specificity of ANG II-induced NF-kappaB activation was ascertained by supershift and competition experiments. Moreover, ANG II stimulated NF-kappaB activation in human monocytes, but not in lymphocytes from the same preparation. Together, the data demonstrate the ability of the vasoactive peptide ANG II to activate inflammatory pathways in human monocytes.

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

    PubMed

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

    2013-04-01

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

  5. Expression of extracellular calcium (Ca2+o)-sensing receptor in human peripheral blood monocytes

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Olozak, I.; Chattopadhyay, N.; Butters, R. R.; Kifor, O.; Scadden, D. T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor playing key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone turnover and may play a role in the "reversal" phase of skeletal remodeling that follows osteoclastic resorption and precedes osteoblastic bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for such mononuclear cells present locally within the bone marrow microenvironment. Indeed, previous studies by other investigators have shown that raising Ca2+o either in vivo or in vitro stimulated the release of interleukin-6 (IL-6) from human peripheral blood monocytes, suggesting that these cells express a Ca2+o-sensing mechanism. In these earlier studies, however, the use of reverse transcription-polymerase chain reaction (RT-PCR) failed to detect transcripts for the CaR previously cloned from parathyroid and kidney in peripheral blood monocytes. Since we recently found that non-specific esterase-positive, putative monocytes isolated from murine bone marrow express the CaR, we reevaluated the expression of this receptor in human peripheral blood monocytes. Immunocytochemistry, flow cytometry, and Western blot analysis, performed using a polyclonal antiserum specific for the CaR, detected CaR protein in human monocytes. In addition, the use of RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products, identified CaR transcripts in the cells. Therefore, taken together, our data show that human peripheral blood monocytes possess both CaR protein and mRNA very similar if not identical to those expressed in parathyroid and kidney that could mediate the previously described, direct effects of Ca2+o on these cells. Furthermore, since mononuclear cells isolated from bone marrow also express the CaR, the latter might play some role in

  6. Interaction of Salmonella typhi strains with cultured human monocyte-derived macrophages.

    PubMed Central

    Sizemore, D R; Elsinghorst, E A; Eck, L C; Branstrom, A A; Hoover, D L; Warren, R L; Rubin, F A

    1997-01-01

    Human monocyte-derived macrophages (MDM) provided this laboratory with a tool to develop a primary-cell assay for evaluating the relative virulence of newly constructed Salmonella typhi carrier strains. In this study, the interaction with and survival within MDM were compared for delta aroA143-attenuated strains, wild-type virulent strains, and the current oral-vaccine strain, Ty21a. PMID:8975929

  7. In vitro activity, postantibiotic effect and human monocyte activity of grepafloxacin against Legionella species.

    PubMed

    Dubois, Jacques; St-Pierre, Claude

    1999-04-01

    OBJECTIVE: To determine the in vitro antimicrobial activity, postantibiotic effect (PAE) and human monocyte activity of grepafloxacin compared with sparfloxacin, ciprofloxacin, clarithromycin, erythromycin and rifampicin against 181 strains of Legionella pneumophila, nine strains of L. micdadei, 10 strains of L. dumoffii, seven strains of L. longbeachae and seven other Legionella strains. METHODS: MICs were determined by standard agar dilution using buffered yeast extract (BYE) agar. PAE and human monocyte activity were determined by standard culture techniques. RESULTS: Grepafloxacin, sparfloxacin and rifampicin were the most active agents against L. pneumophila (MIC90 human monocytes. However, only grepafloxacin and ciprofloxacin prevented regrowth or killed L. pneumophila after removal of extracellular antibiotic. CONCLUSIONS: Grepafloxacin showed effective antibacterial activity against the Legionella spp. tested, and has a PAE and activity within human monocytes that suggest it may be useful in the treatment of lower respiratory tract infections caused by Legionella spp.

  8. Distinct Expression and Function of FcεRII in Human B Cells and Monocytes.

    PubMed

    Peng, Wenming; Grobe, William; Walgenbach-Brünagel, Gisela; Flicker, Sabine; Yu, Chunfeng; Sylvester, Marc; Allam, Jean-Pierre; Oldenburg, Johannes; Garbi, Natalio; Valenta, Rudolf; Novak, Natalija

    2017-04-15

    FcεRII is a multifunctional low-affinity IgER that is involved in the pathogenesis of allergic, inflammatory, and neoplastic diseases. Although discrepancies in FcεRII-mediated functions are being increasingly recognized, the consequences of FcεRII activation are not completely understood. In this study, we evaluated the expression of FcεRII on human blood cells and found that it was primarily expressed on monocytes and B cells. Although IL-4 promoted expression of the FcεRIIb isoform on B cells and monocytes, the expression of the FcεRIIa isoform was not dependent on IL-4. Furthermore, FcεRII predominantly bound allergen-IgE complexes on B cells but not on monocytes. FcεRII-mediated allergen-IgE complex uptake by B cells directed Ags to MHC class II-rich compartments. FcεRII-bearing monocytes and B cells expressed high levels of the FcεRII sheddase a disintegrin and metalloproteinase 10, which implies that they are important sources of soluble FcεRII. Moreover, we identified that IgE immune complex stimulation of FcεRII activated intracellular tyrosine phosphorylation via Syk in B cells but not in monocytes. Importantly, FcεRII-mediated signaling by allergen-IgE immune complexes increased IFN-γ production in B cells of allergic patients during the build-up phase of allergen-specific immunotherapy. Together, our results demonstrate that FcεRII mediates cell type-dependent function in allergic reactions. In addition, the results identify a novel allergen-IgE complex/FcεRII/Syk/IFN-γ pathway in allergic responses and suggest that FcεRII may play a role in regulating allergic reactions via modulating IFN-γ production in B cells.

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

    PubMed

    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.

  10. Blood monocytes and tumor-infiltrating macrophages in human breast cancer: differences in activation level as assessed by lysozyme content.

    PubMed

    Steele, R J; Eremin, O; Brown, M

    1983-11-01

    The lysozyme content of tumor-infiltrating macrophages (TIM) from human breast carcinomas has been compared with that of blood monocytes both from breast cancer patients and tumor-free controls. Cells were identified as macrophages or monocytes with the use of rosetting reactions to detect receptors for the Fc portion of IgG and differentiation antigens, and lysozyme was detected by an immunoperoxidase technique on cytocentrifuge preparations of rosetted cells. Significantly more monocytes from patients with breast cancer contained lysozyme than monocytes from comparable controls, suggesting the presence of activated circulating blood monocytes. Conversely, TIM were virtually devoid of lysozyme. This lack of enzyme was not due to methodologic factors and may represent defective antitumor activity.

  11. Natural haemozoin induces expression and release of human monocyte tissue inhibitor of metalloproteinase-1.

    PubMed

    Polimeni, Manuela; Valente, Elena; Ulliers, Daniela; Opdenakker, Ghislain; Van den Steen, Philippe E; Giribaldi, Giuliana; Prato, Mauro

    2013-01-01

    Recently matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1) have been implicated in complicated malaria. In vivo, mice with cerebral malaria (CM) display high levels of both MMP-9 and TIMP-1, and in human patients TIMP-1 serum levels directly correlate with disease severity. In vitro, natural haemozoin (nHZ, malarial pigment) enhances monocyte MMP-9 expression and release. The present study analyses the effects of nHZ on TIMP-1 regulation in human adherent monocytes. nHZ induced TIMP-1 mRNA expression and protein release, and promoted TNF-α, IL-1β, and MIP-1α/CCL3 production. Blocking antibodies or recombinant cytokines abrogated or mimicked nHZ effects on TIMP-1, respectively. p38 MAPK and NF-κB inhibitors blocked all nHZ effects on TIMP-1 and pro-inflammatory molecules. Still, total gelatinolytic activity was enhanced by nHZ despite TIMP-1 induction. Collectively, these data indicate that nHZ induces inflammation-mediated expression and release of human monocyte TIMP-1 through p38 MAPK- and NF-κB-dependent mechanisms. However, TIMP-1 induction is not sufficient to counterbalance nHZ-dependent MMP-9 enhancement. Future investigation on proteinase-independent functions of TIMP-1 (i.e. cell survival promotion and growth/differentiation inhibition) is needed to clarify the role of TIMP-1 in malaria pathogenesis.

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

    SciTech Connect

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

    2008-11-01

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

  13. Tissue factor activity in human monocytes is regulated by plasma: implications for the high and low responder phenomenon.

    PubMed

    Nijziel, M; van Oerle, R; van 't Veer, C; van Pampus, E; Lindhout, T; Hamulyák, K

    2001-01-01

    The 'high and low responder phenomenon' of monocyte tissue factor (MTF) activity has been attributed to effects on monocytes by granulocytes, platelets and lipopolysaccharide (LPS). To study the possible contribution of plasma to the high and low responder phenomenon, we measured the MTF activity in isolated cryopreserved human monocytes from two donors (monocytes A and monocytes B) after incubation in a plasma environment depleted of granulocytes, platelets and LPS. In buffer only, MTF activity was 643 and 679 fM (fM = final concentration of tissue factor), in normal pooled plasma, it was 1478 and 1615 fM (P = 0.001), respectively, in monocytes A and in monocytes B. Incubation with individual plasma samples from healthy controls (n = 43) gave a median MTF of 1355 fM (range 1044-1976 fM) and 1329 fM (range 858-1951 fM) respectively. A plasma consistently induced a higher or lower level of MTF activity in both monocytes: r = 0.82 (P < 0.00001). Coumarin use did not influence the high and low responder phenomenon. In the absence of granulocytes, platelets and LPS, plasma determines the high and low responder phenomenon. This phenomenon is not influenced by coumarin treatment.

  14. Cell-to-cell contact of human monocytes with infected arterial smooth-muscle cells enhances growth of Chlamydia pneumoniae.

    PubMed

    Puolakkainen, Mirja; Campbell, Lee Ann; Lin, Tsun-Mei; Richards, Theresa; Patton, Dorothy L; Kuo, Cho-Chou

    2003-02-01

    Chlamydia pneumoniae can infect arterial cells. It has been shown that coculture of human monocytes (U937) and endothelial cells promotes infection of C. pneumoniae in endothelial cells and that the enhancement was mediated by a soluble factor (insulin-like growth factor 2) secreted by monocytes. In this study, it is shown that coculture of monocytes with C. pneumoniae enhances infection of C. pneumoniae in arterial smooth-muscle cells 5.3-fold at a monocyte-to-smooth-muscle cell ratio of 5. However, unlike endothelial cells, no enhancement was observed if monocytes were placed in cell culture inserts or if conditioned medium from monocyte cultures was used, which suggests that cell-to-cell contact is critical. The addition of mannose 6-phosphate or octyl glucoside, a nonionic detergent containing a sugar group, to cocultures inhibited the enhancement. These findings suggest that the monocyte-smooth-muscle cell interaction may be mediated by mannose 6-phosphate receptors present on monocytes.

  15. Antibody-Dependent Enhancement of Dengue Virus Growth in Human Monocytes as a Risk Factor for Dengue Hemorrhagic Fever

    DTIC Science & Technology

    1989-01-01

    Clamfication) Antibody-Dependent Enhancement of Dengue Virus Growth in Human Monocytes as a Risk Factor for Dengue Hemorrhagic Fever 𔃼 PERSONAL AjTHOR(S...FELD GROUP SUBGROUP Antibody-Dependent Enhancement of Dengue Virus Growth in Human Monocytes as a Risk Factor for Dengue Hemorrhagic Fever. 19...ABSTRAC7 (Continue on reverse if necessary and identify by block number) Serum specimens~collected during a prospective study of dengue infections among

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

    PubMed Central

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

    2016-01-01

    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. PMID:26857012

  17. A Single-Cell Gene-Expression Profile Reveals Inter-Cellular Heterogeneity within Human Monocyte Subsets

    PubMed Central

    Gren, Susanne T.; Rasmussen, Thomas B.; Janciauskiene, Sabina; Håkansson, Katarina; Gerwien, Jens G.; Grip, Olof

    2015-01-01

    Human monocytes are a heterogeneous cell population classified into three different subsets: Classical CD14++CD16-, intermediate CD14++CD16+, and non-classical CD14+CD16++ monocytes. These subsets are distinguished by their differential expression of CD14 and CD16, and unique gene expression profile. So far, the variation in inter-cellular gene expression within the monocyte subsets is largely unknown. In this study, the cellular variation within each human monocyte subset from a single healthy donor was described by using a novel single-cell PCR gene-expression analysis tool. We investigated 86 different genes mainly encoding cell surface markers, and proteins involved in immune regulation. Within the three human monocyte subsets, our descriptive findings show multimodal expression of key immune response genes, such as CD40, NFⱪB1, RELA, TLR4, TLR8 and TLR9. Furthermore, we discovered one subgroup of cells within the classical monocytes, which showed alterations of 22 genes e.g. IRF8, CD40, CSF1R, NFⱪB1, RELA and TNF. Additionally one subgroup within the intermediate and non-classical monocytes also displayed distinct gene signatures by altered expression of 8 and 6 genes, respectively. Hence the three monocyte subsets can be further subdivided according to activation status and differentiation, independently of the traditional classification based on cell surface markers. Demonstrating the use and the ability to discover cell heterogeneity within defined populations of human monocytes is of great importance, and can be useful in unravelling inter-cellular variation in leukocyte populations, identifying subpopulations involved in disease pathogenesis and help tailor new therapies. PMID:26650546

  18. Factor H Binds to Extracellular DNA Traps Released from Human Blood Monocytes in Response to Candida albicans

    PubMed Central

    Halder, Luke D.; Abdelfatah, Mahmoud A.; Jo, Emeraldo A. H.; Jacobsen, Ilse D.; Westermann, Martin; Beyersdorf, Niklas; Lorkowski, Stefan; Zipfel, Peter F.; Skerka, Christine

    2017-01-01

    Upon systemic infection with human pathogenic yeast Candida albicans (C. albicans), human monocytes and polymorph nuclear neutrophilic granulocytes are the first immune cells to respond and come into contact with C. albicans. Monocytes exert immediate candidacidal activity and inhibit germination, mediate phagocytosis, and kill fungal cells. Here, we show that human monocytes spontaneously respond to C. albicans cells via phagocytosis, decondensation of nuclear DNA, and release of this decondensed DNA in the form of extracellular traps (called monocytic extracellular traps: MoETs). Both subtypes of monocytes (CD14++CD16−/CD14+CD16+) formed MoETs within the first hours upon contact with C. albicans. MoETs were characterized by the presence of citrullinated histone, myeloperoxidase, lactoferrin, and elastase. MoETs were also formed in response to Staphylococcus aureus and Escherichia coli, indicating a general reaction of monocytes to infectious microbes. MoET induction differs from extracellular trap formation in macrophages as MoETs are not triggered by simvastatin, an inhibitor of cholesterol synthesis and inducer of extracellular traps in macrophages. Extracellular traps from both monocytes and neutrophils activate complement and C3b is deposited. However, factor H (FH) binds via C3b to the extracellular DNA, mediates cofactor activity, and inhibits the induction of the inflammatory cytokine interleukin-1 beta in monocytes. Altogether, the results show that human monocytes release extracellular DNA traps in response to C. albicans and that these traps finally bind FH via C3b to presumably support clearance without further inflammation. PMID:28133459

  19. Creatine kinase expression and creatine phosphate accumulation are developmentally regulated during differentiation of mouse and human monocytes

    PubMed Central

    1984-01-01

    We have studied the expression of creatine kinase (CK) and the accumulation of creatine phosphate during the differentiation of human and mouse peripheral blood monocytes. Mouse monocytes cultured for 24 h do not contain detectable levels of CK and creatine phosphate. However, resident tissue macrophages and inflammatory elicited macrophages obtained from the peritoneal cavities of mice have 70 and 300 mU per mg protein of CK activity and contain 3 and 6 mol of creatine phosphate per mol of ATP, respectively. The major isozyme of CK in these cells has been identified as the brain form. These findings suggest that the differentiation of monocytes into macrophages is associated with the expression of CK and the accumulation of creatine phosphate. We have found a similar pattern in human monocytes. Human blood monocytes, maintained in culture for 24 or 48 h, do not contain detectable levels of CK or creatine phosphate. Monocyte-derived macrophages (monocytes maintained in tissue cultures for 1 to 2 wk) have up to 100 mU per mg protein of CK activity and contain 0.5 mol of creatine phosphate per mol of ATP. Human macrophages express multiple isozymes of CK including the brain (BB) and possibly the mitochondrial forms of this enzyme. Thus, the expression of CK and the accumulation of creatine phosphate in human monocytes is induced by their in vitro cultivation. The induction of CK during in vitro cultivation occurs independently of the concentration of creatine in the medium. However, the size of the creatine phosphate pool varies with respect to extracellular creatine concentration. Creatine phosphate and CK are not detectable in freshly isolated human lymphocytes, polymorphonuclear leukocytes or erythrocytes, but are found in freshly isolated human platelets. PMID:6699543

  20. In vitro immunotoxicity assessment of culture-derived extracellular vesicles in human monocytes

    PubMed Central

    Rosas, Lucia E.; Elgamal, Ola A.; Mo, Xiaokui; Phelps, Mitch A.; Schmittgen, Thomas D.; Papenfuss, Tracey L.

    2016-01-01

    The potential to engineer extracellular vesicles (EV) that target specific cells and deliver a therapeutic payload has propelled a growing interest in their development as promising therapeutics. These EV are often produced from cultured cells. Very little is known about the interaction of cell culture-derived EV with cells of the immune system and their potential immunomodulatory effects. The present study evaluated potential immunotoxic effects of HEK293T-derived EV on the human monocytic cell lines THP-1 and U937. Incubation of cells with different doses of EV for 16–24 h was followed by assessment of cytotoxicity and cell function by flow cytometry. Changes in cell functionality were evaluated by the capacity of cells to phagocytize fluorescent microspheres. In addition, the internalization of labeled EV in THP-1 and U937 cells was evaluated. Exposure to EV did not affect the viability of THP-1 or U937 cells. Although lower doses of the EV increased phagocytic capacity in both cell lines, phagocytic efficiency of individual cells was not affected by EV exposure at any of the doses evaluated. This study also demonstrated that THP-1 and U937 monocytic cells are highly permissive to EV entry in a dose-response manner. These results suggest that, although HEK293T-derived EV are efficiently internalized by human monocytic cells, they do not exert a cytotoxic effect or alter phagocytic efficiency on the cell lines evaluated. PMID:27075513

  1. ER stress induced impaired TLR signaling and macrophage differentiation of human monocytes.

    PubMed

    Komura, Takuya; Sakai, Yoshio; Honda, Masao; Takamura, Toshinari; Wada, Takashi; Kaneko, Shuichi

    2013-03-01

    Endoplasmic reticulum (ER) stress causes impairment of the intracellular protein synthesis machinery, affecting various organ functions and homeostasis systems, including immunity. We found that ER stress induced by the N-linked glycosylation inhibitor, tunicamycin, caused susceptibility to apoptosis in the human monocytic cell line, THP-1 cells. Importantly, prior to tunicamycin-induced apoptosis, the proinflammatory response to toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) stimulation was attenuated with respect to the expression of the proinflammatory cytokines. This impaired expression of proinflammatory cytokines was a consequence of the inhibition of NF-κB activation. Moreover, tunicamycin-induced ER stress disturbed the differentiation of THP-1 cells into macrophages induced by phorbol-12-myristate-13-acetate treatment. We also confirmed that ER stress affected the response of primary human monocytes to TLR ligand and their ability to differentiate into macrophages. These data suggest that ER stress imposes an important pathological insult to the immune system, affecting the crucial functions of monocytes.

  2. Interaction between human monocytes and vascular smooth muscle cells induces vascular endothelial growth factor expression.

    PubMed

    Hojo, Y; Ikeda, U; Maeda, Y; Takahashi, M; Takizawa, T; Okada, M; Funayama, H; Shimada, K

    2000-05-01

    The objective of this study was to investigate whether synthesis of vascular endothelial growth factor (VEGF), a major mitogen for vascular endothelial cells, was induced by a cell-to-cell interaction between monocytes and vascular smooth muscle cells (VSMCs). Human VSMCs and THP-1 cells (human monocytoid cell) were cocultured. VEGF levels in the coculture medium were determined by enzyme-linked immunosorbent assay. Northern blot analysis of VEGF mRNA was performed using a specific cDNA probe. Immunohistochemistry was performed to determine which types of cell produce VEGF. Adding THP-1 cells to VSMCs for 24 h increased VEGF levels of the culture media, 8- and 10-fold relative to those of THP-1 cells and VSMCs alone, respectively. Northern blot analysis showed that VEGF mRNA expression was induced in the cocultured cells and peaked after 12 h. Immunohistochemistry disclosed that both types of cell in the coculture produced VEGF. Separate coculture experiments revealed that both direct contact and a soluble factor(s) contributed to VEGF production. Neutralizing anti-interleukin (IL)-6 antibody inhibited VEGF production by the coculture of THP-1 cells and VSMCs. A cell-to-cell interaction between monocytes and VSMCs induced VEGF synthesis in both types of cell. An IL-6 mediated mechanism is at least partially involved in VEGF production by the cocultures. Local VEGF production induced by a monocyte-VSMC interaction may play an important role in atherosclerosis and vascular remodeling.

  3. Saturated fatty acids activate caspase-4/5 in human monocytes, triggering IL-1β and IL-18 release.

    PubMed

    Pillon, Nicolas J; Chan, Kenny L; Zhang, Shitian; Mejdani, Marios; Jacobson, Maya R; Ducos, Alexandre; Bilan, Philip J; Niu, Wenyan; Klip, Amira

    2016-11-01

    Obesity is associated with metabolic tissue infiltration by monocyte-derived macrophages. Saturated fatty acids contribute to proinflammatory gene induction in tissue-embedded immune cells. However, it is unknown how circulating monocytes, the macrophage precursors, react to high-fat environments. In macrophages, saturated fatty acids activate inflammatory pathways and, notably, prime caspase-associated inflammasomes. Inflammasome-activated IL-1β contributes to type 2 diabetes. We hypothesized that 1) human monocytes from obese patients show caspase activation, and 2) fatty acids trigger this response and consequent release of IL-1β/IL-18. Human peripheral blood monocytes were sorted by flow cytometry, and caspase activity was measured with a FLICA dye-based assay. Blood monocytes from obese individuals exhibited elevated caspase activity. To explore the nature and consequence of this activity, human THP1 monocytes were exposed to saturated or unsaturated fatty acids. Caspase activity was revealed by isoform-specific cleavage and enzymatic activity; cytokine expression/release was measured by qPCR and ELISA. Palmitate, but not palmitoleate, increased caspase activity in parallel to the release of IL-1β and IL-18. Palmitate induced eventual monocyte cell death with features of pyroptosis (an inflammation-linked cell death program involving caspase-4/5), scored through LDH release, vital dye influx, cell volume changes, and nuclear morphology. Notably, selective gene silencing or inhibition of caspase-4/5 reduced palmitate-induced release of IL-1β and IL-18. In summary, monocytes from obese individuals present elevated caspase activity. Mechanistically, palmitate activates a pyroptotic program in monocytes through caspase-4/5, causing inflammatory cytokine release, additional to inflammasomes. These caspases represent potential, novel, therapeutic targets to taper obesity-associated inflammation.

  4. Interferon-α disables dendritic cell precursors: dendritic cells derived from interferon-α-treated monocytes are defective in maturation and T-cell stimulation

    PubMed Central

    Dauer, Marc; Pohl, Katrin; Obermaier, Bianca; Meskendahl, Tobias; Röbe, Julian; Schnurr, Max; Endres, Stefan; Eigler, Andreas

    2003-01-01

    Dendritic cells (DC) can be derived from monocytes in vitro by culture with granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). It is unknown whether this regimen reflects DC differentiation from blood precursors under physiological conditions. Induction of DC development from monocytes by interferon-α (IFN-α) may occur in vivo during infection or inflammation and thus may represent a more physiological approach to DC differentiation in vitro. Here, we show that incubation of GM-CSF-cultured monocytes with IFN-α does not induce DC differentiation: cells maintain their original phenotype and cytokine secretion pattern. Even after stimulation with pro-inflammatory or T-cell-derived activation signals, IFN-α-treated monocytes do not develop DC characteristics. Addition of IL-4 during stimulation of IFN-α-treated monocytes results in the rapid development of DC-like cells expressing co-stimulatory molecules, CD83 and chemokine receptor CCR7, indicating that some degree of developmental plasticity is preserved. However, DC pre-activated with IFN-α are less effective in inducing allogeneic or antigen-specific autologous T-cell proliferation, produce less IL-12 and express lower levels of CCR7 compared to DC generated by culture with GM-CSF and IL-4. Incubating GM-CSF-cultured monocytes simultaneously with IFN-α and IL-4 does not affect phenotypic maturation of DC, but reduces IL-12 production upon pro-inflammatory activation. We conclude that: (1) IFN-α fails to induce DC differentiation and thus cannot replace IL-4 in generating DC from monocytes in vitro; and (2) the presence of IFN-α prior to or during differentiation of DC from monocyte precursors alters their response to maturation stimuli and may affect their capacity to stimulate T helper type 1 immune responses in vivo. PMID:12941139

  5. Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells

    SciTech Connect

    Cushing, S.D.; Berliner, J.A.; Valente, A.J.; Territo, M.C.; Navab, M.; Parhami, F.; Gerrity, R.; Schwartz, C.J.; Fogelman, A.M.

    1990-07-01

    After exposure to low density lipoprotein (LDL) that had been minimally modified by oxidation (MM-LDL), human endothelial cells (EC) and smooth muscle cells (SMC) cultured separately or together produced 2- to 3-fold more monocyte chemotactic activity than did control cells or cells exposed to freshly isolated LDL. This increase in monocyte chemotactic activity was paralleled by increases in mRNA levels for a monocyte chemotactic protein 1 (MCP-1) that is constitutively produced by the human glioma U-105MG cell line. Antibody that had been prepared against cultured baboon smooth muscle cell chemotactic factor (anti-SMCF) did not inhibit monocyte migration induced by the potent bacterial chemotactic factor f-Met-Leu-Phe. However, anti-SMCF completely inhibited the monocyte chemotactic activity found in the media of U-105MG cells, EC, and SMC before and after exposure to MM-LDL. Moreover, monocyte migration into the subendothelial space of a coculture of EC and SMC that had been exposed to MM-LDL was completely inhibited by anti-SMCF. Anti-SMCF specifically immunoprecipitated 10-kDa and 12.5-kDa proteins from EC. Incorporation of (35S)methionine into the immunoprecipitated proteins paralleled the monocyte chemotactic activity found in the medium of MM-LDL stimulated EC and the levels of MCP-1 mRNA found in the EC. We conclude that SMCF is in fact MCP-1 and MCP-1 is induced by MM-LDL.

  6. Effects of monascin on anti-inflammation mediated by Nrf2 activation in advanced glycation end product-treated THP-1 monocytes and methylglyoxal-treated wistar rats.

    PubMed

    Lee, Bao-Hong; Hsu, Wei-Hsuan; Huang, Tao; Chang, Yu-Ying; Hsu, Ya-Wen; Pan, Tzu-Ming

    2013-02-13

    Hyperglycemia is associated with advanced glycation end products (AGEs). This study was designed to evaluate the inhibitory effects of monascin on receptor for advanced glycation end product (RAGE) signal and THP-1 monocyte inflammation after treatment with S100b, a specific ligand of RAGE. Monascin inhibited cytokine production by S100b-treated THP-1 monocytes via up-regulation of nuclear factor-erythroid 2-related factor-2 (Nrf2) and alleviated p47phox translocation to the membrane. Methylglyoxal (MG, 600 mg/kg bw) was used to induce diabetes in Wistar rats. Inhibitions of RAGE and p47phox by monascin were confirmed by peripheral blood mononuclear cells (PBMCs) of MG-induced rats. Silymarin (SM) was used as a positive control group. It was found that monascin promoted heme oxygenase-1 (HO-1) expression mediated by Nrf2. Suppressions of AGEs, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-β) in serum of MG-induced rats were attenuated in the monascin administration group treated with retinoic acid (RA). RA treatment resulted in Nrf2 inactivation by increasing RA receptor-α (RARα) activity, suggesting that RA acts as an inhibitor of Nrf2. The results showed that monascin exerted anti-inflammatory and antioxidative effects mediated by Nrf2 to prevent the development of diseases such as type 2 diabetes caused by inflammation.

  7. Lactoferrin inhibits or promotes Legionella pneumophila intracellular multiplication in nonactivated and interferon gamma-activated human monocytes depending upon its degree of iron saturation. Iron-lactoferrin and nonphysiologic iron chelates reverse monocyte activation against Legionella pneumophila.

    PubMed Central

    Byrd, T F; Horwitz, M A

    1991-01-01

    We have been exploring the role of iron in the pathogenesis of the intracellular bacterial pathogen Legionella pneumophila. In previous studies, we have demonstrated that L. pneumophila intracellular multiplication in human monocytes is iron dependent and that IFN gamma-activated monocytes inhibit L. pneumophila intracellular multiplication by limiting the availability of iron. In this study, we have investigated the effect on L. pneumophila intracellular multiplication of lactoferrin, an iron-binding protein which is internalized via specific receptors on monocytes, and of nonphysiologic iron chelates which enter monocytes by a receptor-independent route. Apolactoferrin completely inhibited L. pneumophila multiplication in nonactivated monocytes, and enhanced the capacity of IFN gamma-activated monocytes to inhibit L. pneumophila intracellular multiplication. In contrast, iron-saturated lactoferrin had no effect on the already rapid rate of L. pneumophila multiplication in nonactivated monocytes. Moreover, it reversed the capacity of activated monocytes to inhibit L. pneumophila intracellular multiplication, demonstrating that L. pneumophila can utilize iron from the lactoferrin-lactoferrin receptor pathway. The capacity of iron-lactoferrin to reverse monocyte activation was dependent upon its percent iron saturation and not just its total iron content. Similarly, the nonphysiologic iron chelates ferric nitrilotriacetate and ferric ammonium citrate completely reverse and ferric pyrophosphate partially reversed the capacity of IFN gamma-activated monocytes to inhibit L. pneumophila intracellular multiplication, demonstrating that L. pneumophila can utilize iron derived from nonphysiologic iron chelates internalized by monocytes independently of the transferrin and lactoferrin endocytic pathways. This study suggests that at sites of inflammation, lactoferrin may inhibit or promote L. pneumophila intracellular multiplication in mononuclear phagocytes depending upon

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

    PubMed Central

    Paijo, Jennifer; Döring, Marius; Spanier, Julia; Grabski, Elena; Nooruzzaman, Mohammed; Schmidt, Tobias; Witte, Gregor; Messerle, Martin; Hornung, Veit; Kaever, Volkhard; Kalinke, Ulrich

    2016-01-01

    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. PMID:27058035

  9. Human adipose tissue-resident monocytes exhibit an endothelial-like phenotype and display angiogenic properties

    PubMed Central

    2014-01-01

    Introduction Adipose tissue has the unique property of expanding throughout adult life, and angiogenesis is required for its growth. However, endothelial progenitor cells contribute minimally to neovascularization. Because myeloid cells have proven to be angiogenic, and monocytes accumulate in expanding adipose tissue, they might contribute to vascularization. Methods The stromal vascular fraction (SVF) cells from human adipose tissue were magnetically separated according to CD45 or CD14 expression. Adipose-derived mesenchymal stromal cells (MSCs) were obtained from SVF CD45- cells. CD14+ monocytes were isolated from peripheral blood (PB) mononuclear cells and then cultured with SVF-derived MSCs. Freshly isolated or cultured cells were characterized with flow cytometry; the conditioned media were analyzed for the angiogenic growth factors, angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF) with Luminex Technology; their angiogenic capacity was determined in an in vivo gelatinous protein mixture (Matrigel) plug angiogenesis assay. Results CD45+ hematopoietic cells within the SVF contain CD14+ cells that co-express the CD34 progenitor marker and the endothelial cell antigens VEGF receptor 2 (VEGFR2/KDR), VEGFR1/Flt1, and Tie2. Co-culture experiments showed that SVF-derived MSCs promoted the acquisition of KDR and Tie-2 in PB monocytes. MSCs secreted significant amounts of Ang-2 and HGF, but minimal amounts of bFGF, G-CSF, or GM-CSF, whereas the opposite was observed for SVF CD14+ cells. Additionally, SVF CD14+ cells secreted significantly higher levels of VEGF and bFGF than did MSCs. Culture supernatants of PB monocytes cultured with MSCs contained significantly higher concentrations of VEGF, HGF, G-CSF, and GM-CSF than did the supernatants from cultures without MSCs

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

    SciTech Connect

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

    1990-02-01

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

  11. Human β defensin-3 induces chemokines from monocytes and macrophages: diminished activity in cells from HIV-infected persons.

    PubMed

    Petrov, Velizar; Funderburg, Nicholas; Weinberg, Aaron; Sieg, Scott

    2013-12-01

    Human β defensin-3 (hBD-3) is an antimicrobial peptide with diverse functionality. We investigated the capacity of hBD-3 and, for comparison, Pam3CSK4 and LL-37 to induce co-stimulatory molecules and chemokine expression in monocytes. These stimuli differentially induced CD80 and CD86 on the surface of monocytes and each stimulant induced a variety of chemokines including monocyte chemoattractant protein 1 (MCP-1), Gro-α, macrophage-derived chemokine (MDC) and macrophage inflammatory protein 1β (MIP1β), while only hBD-3 and Pam3CSK4 significantly induced the angiogenesis factor, vascular endothelial growth factor (VEGF). Human BD-3 induced similar chemokines in monocyte-derived macrophages and additionally induced expression of Regulated upon activation normal T-cell expressed and presumably secreted (RANTES) in these cells. Comparison of monocytes from HIV(+) and HIV(-) donors indicated that monocytes from HIV(+) donors were more likely to spontaneously express certain chemokines (MIP-1α, MIP-1β and MCP-1) and less able to increase expression of other molecules in response to hBD-3 (MDC, Gro-α and VEGF). Chemokine receptor expression (CCR5, CCR2 and CXCR2) was relatively normal in monocytes from HIV(+) donors compared with cells from HIV(-) donors with the exception of diminished expression of the receptor for MDC, CCR4, which was reduced in the patrolling monocyte subset (CD14(+)  CD16(++) ) of HIV(+) donors. These observations implicate chemokine induction by hBD-3 as a potentially important mechanism for orchestrating cell migration into inflamed tissues. Alterations in chemokine production or their receptors in monocytes of HIV-infected persons could influence cell migration and modify the effects of hBD-3 at sites of inflammation.

  12. Phospholipid metabolism in zymosan stimulated human monocytes: modulation by cyclic AMP (cAMP)

    SciTech Connect

    Godfrey R.W.; Manzi, R.M.; Hoffstein, S.T.

    1986-05-01

    Oxygenated products of arachidonic acid (AA) are critical components in the development of the inflammatory response. Monocytes exposed to inflammatory stimuli are capable of converting free AA into these bioactive molecules. However, the limiting step in the formation of these compounds is thought to be the mechanism responsible for the release of esterified AA from phospholipids. When (/sup 3/H) AA labeled monocytes were challenged with opsonized zymosan, 28 +/- 2% of the incorporated counts were released compared to 8 +/- 1% for the control. Upon pretreatment with isobutyl methyl xanthine (IBMX) or dibutyrl cyclic AMP (d-cAMP) zymosan stimulated AA release was markedly reduced. The IC/sub 50/'s were 4 x 10/sup -4/M and 7 x 10/sup -4/M respectively. Analysis of (/sup 3/H) AA incorporation into cellular phospholipids showed that phosphatidylcholine (PC) and phosphatidylinositol (PI) were the primary pools labeled. Loss of label from both of these pools was evident after exposure to zymosan, however, pretreatment of cells with IBMX or d-cAMP inhibited release of (/sup 3/H)AA from the PC pool but not from the PI pool. The results show that human monocytes challenged with opsonized zymosan release arachidonic acid via cAMP-dependent and independent pathways. Furthermore, they suggest that a phospholipase activity (possibly A/sub 2/) against PC is modulated by cAMP.

  13. Tailored HIV-1 Vectors for Genetic Modification of Primary Human Dendritic Cells and Monocytes

    PubMed Central

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

    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. PMID:23077304

  14. Thrombin selectively induces transcription of genes in human monocytes involved in inflammation and wound healing.

    PubMed

    López, Mercedes L; Bruges, Gustavo; Crespo, Gustavo; Salazar, Victor; Deglesne, Pierre-Antoine; Schneider, Heike; Cabrera-Fuentes, Hector; Schmitz, M Lienhard; Preissner, Klaus T

    2014-11-01

    Thrombin is essential for blood coagulation but functions also as a mediator of cellular signalling. Gene expression microarray experiments in human monocytes revealed thrombin-induced upregulation of a limited subset of genes, which are almost exclusively involved in inflammation and wound healing. Among these, the expression of F3 gene encoding for tissue factor (TF) was enhanced indicating that this physiological initiator of coagulation cascade may create a feed-forward loop to enhance blood coagulation. Activation of protease-activated receptor type 1 (PAR1) was shown to play a main role in promoting TF expression. Moreover, thrombin induced phosphorylation of ERK1/2, an event that is required for expression of thrombin-regulated genes. Thrombin also increased the expression of TF at the protein level in monocytes as evidenced by Western blot and immunostaining. Furthermore, FXa generation induced by thrombin-stimulated monocytes was abolished by a TF blocking antibody and therefore it is entirely attributable to the expression of tissue factor. This cellular activity of thrombin provides a new molecular link between coagulation, inflammation and wound healing.

  15. Gram-positive pathogenic bacteria induce a common early response in human monocytes

    PubMed Central

    2010-01-01

    Background We infected freshly isolated human peripheral monocytes with live bacteria of three clinically important gram-positive bacterial species, Staphylococcus aureus, Streptococcus pneumoniae and Listeria monocytogenes and studied the ensuing early transcriptional response using expression microarrays. Thus the observed response was unbiased by signals originating from other helper and effector cells of the host and was not limited to induction by solitary bacterial constituents. Results Activation of monocytes was demonstrated by the upregulation of chemokine rather than interleukin genes except for the prominent expression of interleukin 23, marking it as the early lead cytokine. This activation was accompanied by cytoskeleton rearrangement signals and a general anti-oxidative stress and anti-apoptotic reaction. Remarkably, the expression profiles also provide evidence that monocytes participate in the regulation of angiogenesis and endothelial function in response to these pathogens. Conclusion Regardless of the invasion properties and survival mechanisms of the pathogens used, we found that the early response comprised of a consistent and common response. The common response was hallmarked by the upregulation of interleukin 23, a rather unexpected finding regarding Listeria infection, as this cytokine has been linked primarily to the control of extracellular bacterial dissemination. PMID:21044323

  16. Modulation of the cytokine response in human monocytes by mycobacterium leprae phenolic glycolipid-1.

    PubMed

    Manca, Claudia; Peixoto, Blas; Malaga, Wladimir; Guilhot, Christophe; Kaplan, Gilla

    2012-01-01

    Leprosy is a chronic but treatable infectious disease caused by the intracellular pathogen Mycobacterium leprae. M. leprae cell wall is characterized by a unique phenolic glycolipid-1 (PGL-1) reported to have several immune functions. We have examined the role of PGL-1 in the modulation of monocyte cytokine/chemokine production in naive human monocytes. PGL-1 in its purified form or expressed in a recombinant Mycobacterium bovis Bacillus Colmette-Guérin (BCG) background (rBCG-PGL-1) was tested. We found that PGL-1 selectively modulated the induction of specific monocyte cytokines and chemokines and, when used as prestimulus, exerted priming and/or inhibitory effects on the induction of selected cytokines/chemokines in response to a second stimulus. Taken together, the results of this study support a modulatory role for PGL-1 in the innate immune response to M. leprae. Thus, PGL-1 may play an important role in the development of the anergic clinical forms of disease and in tissue damage seen in lepromatous patients and during the reactional states of leprosy.

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

    PubMed

    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.

  18. Sequence and molecular characterization of human monocyte/neutrophil elastase inhibitor.

    PubMed Central

    Remold-O'Donnell, E; Chin, J; Alberts, M

    1992-01-01

    cDNA encoding human monocyte/neutrophil elastase inhibitor (EI), a M(r) approximately 42,000 protein with serpin-like functional properties, has been sequenced. The 1316-base-pair sequence was obtained from overlapping clones and amplified DNA from libraries of monocyte-like and neutrophil-like cells. Hybridization with EI cDNA identified three EI mRNA species of 1.5, 1.9, and 2.6 kilobases in U937 monocyte-like cells and no hybridizing mRNA in lymphoblastoid cells lacking detectable EI. The cDNA open reading frame encodes a 379-amino acid protein, of which 167 residues were confirmed by tryptic peptides. Although EI may function extracellularly as well as intracellularly, its deduced sequence lacks a typical cleavable N-terminal signal sequence. Sequence analysis established that EI is a member of the serpin superfamily. EI has greatest homology (50.1% identity of amino acids) with plasminogen activator inhibitor 2, also a monocyte protein, and ovalbumin and gene Y, which were previously grouped as an ancient branch of the serpin superfamily. The extent of EI identity with the functionally related serpin alpha 1 antitrypsin is only 30.1%. Sequence alignment indicates that the reactive center P1 residue is Cys-344, consistent with abrogation of elastase inhibitory activity by iodoacetamide and making EI a naturally occurring Cys-serpin. The cleavable bond, Cys-Met, suggests an oxidation-sensitive molecule capable of inhibiting more than one serine protease. Oxidation sensitivity would limit the place of action of EI to the immediate vicinity of carrier cells. The molecular structure will help clarify the likely role of EI in regulating protease action and preventing tissue damage by phagocytic cells. Images PMID:1376927

  19. Daily quadratic trend in basal monocyte expressed HSP72 in healthy human subjects.

    PubMed

    Taylor, Lee; Midgley, Adrian W; Chrismas, Bryna; Madden, Leigh A; Vince, Rebecca V; McNaughton, Lars R

    2010-05-01

    The inducible human stress protein heat shock protein 72 (HSP72) performs vital roles within the body at rest and during periods of stress. Recently it was shown over a 24 h period that basal HSP72 followed a diurnal variation. However, these results and previous literature demonstrate noticeable inter-subject variation in basal HSP72 expression. The notion of intra/inter-day variation in basal HSP72 expression has not been explored in detail. Basal monocyte expressed HSP72 was determined every 3 h, over a 9 h period in 12 healthy male subjects (20.2 +/- 1.9 years, 178.7 +/- 5.6 cm, 75.1 +/- 6.0 kg) within a temperature controlled laboratory. A significant quadratic trend was observed for time (F = 26.0, P = 0.001, partial eta(2) = 0.74), where HSP72 decreased between 0800 and 1100 hours (P < 0.001) and then increased between 1100 and 1400 hours (P = 0.015). The main effect for day (F = 2.6, P = 0.14) and the day x time interaction effect (F = 3.9, P = 0.08) were not significant. There was no correlation between serum and monocyte expressed HSP72, with no significant effect for time (F = 2.0, P = 0.21) in serum HSP72 expression. The results support findings by others that basal monocyte expressed HSP72 follows a diurnal variation which incorporates a quadratic trend, which is not compromised by any significant daily variation and that serum HSP72 expression has no endogenous circadian rhythm. The significant quadratic trend in basal monocyte HSP72 expression shown here highlights the need to tightly control variables, such as timing of sample collection, as it is known basal values influence the magnitude of HSP72 expression post-stressor/intervention.

  20. An evaluation of the putative human mammary tumour retrovirus associated with peripheral blood monocytes.

    PubMed Central

    Kahl, L. P.; Carroll, A. R.; Rhodes, P.; Wood, J.; Read, N. G.

    1991-01-01

    The primary aims of this study were purification and molecular cloning of a putative retrovirus designated human mammary tumour virus (HMTV). However, our preliminary unpublished data of negative reverse transcriptase (RT) activity in ostensibly 'infected' cells led us to re-examine the evidence for this virus; namely multinucleate giant cell (MNGC) formation and RT activity in cultured blood monocytes from breast cancer patients versus benign breast tumour and normal control subjects. MNGCs from by fusion of monocytes and we estimated the total number of cell fusions which had occurred after 10 days of culture in vitro by counting cells with two, three, four and five or more nuclei (n) and by measuring the density of adherent mononuclear cells for each subject studied. We found no clear-cut difference in MNGC formation between the three subject groups. Moreover, a substantial number of cultures, encompassing the three groups, showed far more MNGCs per 10(5) monocytes than previously reported. Various parametric and nonparametric statistical analyses were performed on the multinucleate cell data and only one parametric test, which utilised the density of monolayers as a co-variate, showed a statistically significant difference at the 5% level between the breast cancer and the normal subject groups. We observed marked subject-to-subject variation in multinucleate cell formation and we suggest that the evidence for a difference between the breast cancer and the normal groups is marginal. Further, MNGC formation by breast cancer monocytes may not be attributed to the presence of a retrovirus since 5'-Azacytidine (AZA), an agent known to stimulate replication of latent retroviruses showed no effect on the MNGC formation. In addition, culture supernatants from the three groups were assayed for RT activity and no test sample gave a significant signal above background. Preliminary transmission electron microscopy analysis failed to identify viral particles in MNGCs

  1. Manumycin A downregulates release of proinflammatory cytokines from TNF alpha stimulated human monocytes.

    PubMed

    Cecrdlova, Eva; Petrickova, Katerina; Kolesar, Libor; Petricek, Miroslav; Sekerkova, Alena; Svachova, Veronika; Striz, Ilja

    2016-01-01

    Macrolide antibiotics such as azithromycin or clarithromycin are known to have potent anti-inflammatory and immunomodulatory effects but these properties cannot be widely used due to a risk of bacterial resistance. We studied another polyketide antibiotic, structurally related manumycin A known as a streptomycete derived farnesyltransferase inhibitor with limited antibacterial effects, with respect to its potential regulation of mRNA expression of several genes associated with proinflammatory responses. Downregulation of mRNA for IL-6, TLR-8, IL-1 beta and IL-10 was found in THP-1 cells after 4h stimulation with TNF alpha in the presence of manumycin A and downregulated TLR-8 and EGR-1 genes were observed after 8h. Among the genes upregulated in response to manumycin were HMOX-1, TNFRSF10A, IL-1R1, TICAM2, NLRP12 after 4h and only IL-1R1 after 8h. Furthermore, manumycin A was found to inhibit IL-1beta, IL-6, and IL-8 production in TNF alpha stimulated THP-1 cells and peripheral blood monocytes in a dose dependent manner (0.25-1 μM of manumycin A) without affecting cell viability. Cell viability of blood monocytes decreased by about 30% at manumycin A doses of 2-5 μM. Manumycin A also inhibited IL-18 release from THP-1 cells, while in cultures of blood monocytes, this cytokine was not detectable. That manumycin A mediated downregulation of proinflammatory genes in human monocytes confirmed by a measurement of cytokine levels in culture supernatants, together with a very limited effect on cell viability, might suggest potential anti-inflammatory properties of this polyketide antibiotic.

  2. Ultrafine carbon particles down-regulate CYP1B1 expression in human monocytes

    PubMed Central

    Eder, Christiane; Frankenberger, Marion; Stanzel, Franz; Seidel, Albrecht; Schramm, Karl-Werner; Ziegler-Heitbrock, Loems; Hofer, Thomas PJ

    2009-01-01

    Background Cytochrome P450 monoxygenases play an important role in the defence against inhaled toxic compounds and in metabolizing a wide range of xenobiotics and environmental contaminants. In ambient aerosol the ultrafine particle fraction which penetrates deeply into the lungs is considered to be a major factor for adverse health effects. The cells mainly affected by inhaled particles are lung epithelial cells and cells of the monocyte/macrophage lineage. Results In this study we have analyzed the effect of a mixture of fine TiO2 and ultrafine carbon black Printex 90 particles (P90) on the expression of cytochrome P450 1B1 (CYP1B1) in human monocytes, macrophages, bronchial epithelial cells and epithelial cell lines. CYP1B1 expression is strongly down-regulated by P90 in monocytes with a maximum after P90 treatment for 3 h while fine and ultrafine TiO2 had no effect. CYP1B1 was down-regulated up to 130-fold and in addition CYP1A1 mRNA was decreased 13-fold. In vitro generated monocyte-derived macrophages (MDM), epithelial cell lines, and primary bronchial epithelial cells also showed reduced CYP1B1 mRNA levels. Benzo[a]pyrene (BaP) is inducing CYB1B1 but ultrafine P90 can still down-regulate gene expression at 0.1 μM of BaP. The P90-induced reduction of CYP1B1 was also demonstrated at the protein level using Western blot analysis. Conclusion These data suggest that the P90-induced reduction of CYP gene expression may interfere with the activation and/or detoxification capabilities of inhaled toxic compounds. PMID:19835593

  3. Adjuvant-induced Human Monocyte Secretome Profiles Reveal Adjuvant- and Age-specific Protein Signatures*

    PubMed Central

    Oh, Djin-Ye; Dowling, David J.; Ahmed, Saima; Choi, Hyungwon; Brightman, Spencer; Bergelson, Ilana; Berger, Sebastian T.; Sauld, John F.; Pettengill, Matthew; Kho, Alvin T.; Pollack, Henry J.; Steen, Hanno; Levy, Ofer

    2016-01-01

    Adjuvants boost vaccine responses, enhancing protective immunity against infections that are most common among the very young. Many adjuvants activate innate immunity, some via Toll-Like Receptors (TLRs), whose activities varies with age. Accordingly, characterization of age-specific adjuvant-induced immune responses may inform rational adjuvant design targeting vulnerable populations. In this study, we employed proteomics to characterize the adjuvant-induced changes of secretomes from human newborn and adult monocytes in response to Alum, the most commonly used adjuvant in licensed vaccines; Monophosphoryl Lipid A (MPLA), a TLR4-activating adjuvant component of a licensed Human Papilloma Virus vaccine; and R848 an imidazoquinoline TLR7/8 agonist that is a candidate adjuvant for early life vaccines. Monocytes were incubated in vitro for 24 h with vehicle, Alum, MPLA, or R848 and supernatants collected for proteomic analysis employing liquid chromatography-mass spectrometry (LC-MS) (data available via ProteomeXchange, ID PXD003534). 1894 non-redundant proteins were identified, of which ∼30 - 40% were common to all treatment conditions and ∼5% were treatment-specific. Adjuvant-stimulated secretome profiles, as identified by cluster analyses of over-represented proteins, varied with age and adjuvant type. Adjuvants, especially Alum, activated multiple innate immune pathways as assessed by functional enrichment analyses. Release of lactoferrin, pentraxin 3, and matrix metalloproteinase-9 was confirmed in newborn and adult whole blood and blood monocytes stimulated with adjuvants alone or adjuvanted licensed vaccines with distinct clinical reactogenicity profiles. MPLA-induced adult monocyte secretome profiles correlated in silico with transcriptome profiles induced in adults immunized with the MPLA-adjuvanted RTS,S malaria vaccine (Mosquirix™). Overall, adjuvants such as Alum, MPLA and R848 give rise to distinct and age-specific monocyte secretome profiles

  4. Inflammatory gene expression in monocytes of patients with schizophrenia: overlap and difference with bipolar disorder. A study in naturalistically treated patients.

    PubMed

    Drexhage, Roosmarijn C; van der Heul-Nieuwenhuijsen, Leonie; Padmos, Roos C; van Beveren, Nico; Cohen, Dan; Versnel, Marjan A; Nolen, Willem A; Drexhage, Hemmo A

    2010-11-01

    Accumulating evidence indicates an activated inflammatory response system as a vulnerability factor for schizophrenia (SZ) and bipolar disorder (BD). We aimed to detect a specific inflammatory monocyte gene expression signature in SZ and compare such signature with our recently described inflammatory monocyte gene signature in BD. A quantitative-polymerase chain reaction (Q-PCR) case-control gene expression study was performed on monocytes of 27 SZ patients and compared to outcomes collected in 56 BD patients (all patients naturalistically treated). For Q-PCR we used nine 'SZ specific genes' (found in whole genome analysis), the 19 BD signature genes (previously found by us) and six recently described autoimmune diabetes inflammatory monocyte genes. Monocytes of SZ patients had (similar to those of BD patients) a high inflammatory set point composed of three subsets of strongly correlating genes characterized by different sets of transcription/MAPK regulating factors. Subset 1A, characterized by ATF3 and DUSP2, and subset 1B, characterized by EGR3 and MXD1, were shared between BD and SZ patients (up-regulated in 67% and 51%, and 34% and 41%, respectively). Subset 2, characterized by PTPN7 and NAB2 was up-regulated in the monocytes of 62% BD, but down-regulated in the monocytes of 48% of SZ patients. Our approach shows that monocytes of SZ and BD patients overlap, but also differ in inflammatory gene expression. Our approach opens new avenues for nosological classifications of psychoses based on the inflammatory state of patients, enabling selection of those patients who might benefit from an anti-inflammatory treatment.

  5. Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin.

    PubMed

    Yuk, Jae-Min; Shin, Dong-Min; Lee, Hye-Mi; Yang, Chul-Su; Jin, Hyo Sun; Kim, Kwang-Kyu; Lee, Zee-Won; Lee, Sang-Hee; Kim, Jin-Man; Jo, Eun-Kyeong

    2009-09-17

    Autophagy and vitamin D3-mediated innate immunity have been shown to confer protection against infection with intracellular Mycobacterium tuberculosis. Here, we show that these two antimycobacterial defenses are physiologically linked via a regulatory function of human cathelicidin (hCAP-18/LL-37), a member of the cathelicidin family of antimicrobial proteins. We show that 1,25-dihydroxyvitamin D3 (1,25D3), the active form of vitamin D, induced autophagy in human monocytes via cathelicidin, which activated transcription of the autophagy-related genes Beclin-1 and Atg5. 1,25D3 also induced the colocalization of mycobacterial phagosomes with autophagosomes in human macrophages in a cathelicidin-dependent manner. Furthermore, the antimycobacterial activity in human macrophages mediated by physiological levels of 1,25D3 required autophagy and cathelicidin. These results indicate that human cathelicidin, a protein that has direct antimicrobial activity, also serves as a mediator of vitamin D3-induced autophagy.

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

    PubMed

    Pollak, Cora N; Delpino, M Victoria; Fossati, Carlos A; Baldi, Pablo C

    2012-01-01

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

  7. Control of pro-inflammatory cytokine release from human monocytes with the use of an interleukin-10 monoclonal antibody.

    PubMed

    Patel, Hardik; Davidson, Dennis

    2014-01-01

    The monocytes (MONOs) can be considered as "double-edge swords"; they have both important pro-inflammatory and anti-inflammatory functions manifested in part by cytokine production and release. Although MONOs are circulating cells, they are the major precursors of a variety of tissue-specific immune cells such as the alveolar macrophage, dendritic cells, microglial cells, and Kupffer cells. Unlike the polymorphonuclear leukocyte, which produces no or very little interleukin-10 (IL-10), the monocyte can produce this potent anti-inflammatory cytokine to control inflammation. IL-10, on an equimolar basis, is a more potent inhibitor of pro-inflammatory cytokines produced by monocytes than many anti-inflammatory glucocorticoids which are used clinically. This chapter describes how to isolate monocytes from human blood and the use of IL-10 monoclonal antibody to determine the effect and timing of endogenous IL-10 release on the production and release of pro-inflammatory cytokines.

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

    PubMed

    Ghavampour, Sharang; Lange, Carsten; Bottino, Cristina; Gerke, Volker

    2013-01-01

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

  9. Oxidatively modified high density lipoprotein promotes inflammatory response in human monocytes-macrophages by enhanced production of ROS, TNF-α, MMP-9, and MMP-2.

    PubMed

    Soumyarani, V S; Jayakumari, N

    2012-07-01

    It has been proposed that high-density lipoprotein (HDL) loses its cardioprotective ability through oxidative modifications by reactive oxygen species (ROS) and promote atherogenesis. However, the pro-atherogenic pathways undergone by oxidized HDL remain poorly understood. Since monocytes play a crucial role in atherogenesis, this study was aimed to investigate the influence of both native and oxidized HDL (oxHDL) on monocytes-macrophages functions relevant to atherogenesis. HDL particles were isolated from human blood samples by ultracentrifugation and subjected to in vitro oxidation with CuSO(4). The extent of oxidation was quantitated by measurement of lipid peroxides. Human peripheral blood mononuclear cells were isolated and cultured under standard conditions. Cells were treated with native and oxHDL at varying concentrations for different time intervals and used for several analyses. Intracellular ROS production was assessed based on ROS-mediated DCFH fluorescence of the cells. The release of TNF-α and matrix metalloproteinases (MMPs) was quantitated using ELISA kit and gelatine zymography, respectively. Treatment of cells with oxidized HDL enhanced the production of ROS in a concentration-dependent way, while native HDL had no such effect. Further, the release of TNF-α, MMP-9, and MMP-2 was found to be remarkably higher in cells incubated with oxHDL than that of native HDL. Results demonstrate that oxidative modification of HDL induces pro-inflammatory response and oxidative stress in human monocytes-macrophages.

  10. Measurement of reactive oxygen metabolites produced by human monocyte-derived macrophages exposed to mineral dusts.

    PubMed Central

    Nyberg, P.; Klockars, M.

    1990-01-01

    The aim of the present work was to develop an in-vitro model for studying mineral dust-induced production of reactive oxygen metabolites by human macrophages. Monocytes isolated from human buffy coats were cultured in vitro for 1-6 days. Quartz particles induced both luminol- and lucigenin-dependent chemiluminescence (CL) by the adherent cells. However, the luminol response decreased form day to day, obviously due to a decrease in the myeloperoxidase (MPO) activity of the cells, whereas the lucigenin response showed no such MPO dependence. The luminol response was inhibited by superoxide dismutase (SOD), catalase, and the MPO-inhibitor azide, while the lucigenin response was inhibited by SOD and catalase but stimulated by azide. There was a positive correlation between the lucigenin responses and the results obtained with the established cytochrome c assay for superoxide, when opsonized zymosan was used as a stimulant. The effects of quartz, titanium dioxide, chrysotile asbestos, and wollastonite particles were investigated with the lucigenin assay. Quartz and chrysotile caused prominent light emission by 6-day-old macrophages, whereas titanium dioxide and wollastonite caused weak responses. We conclude that mineral dusts induce production of reactive oxygen metabolites by human monocyte-derived macrophages, and that the quantitative responses depend on both physical and physicochemical dust properties, the nature of which are still to be defined. PMID:2169299

  11. Epstein-Barr virus induces MCP-1 secretion by human monocytes via TLR2.

    PubMed

    Gaudreault, Eric; Fiola, Stéphanie; Olivier, Martin; Gosselin, Jean

    2007-08-01

    Epstein-Barr virus (EBV) is a gammaherpesvirus infecting the majority of the human adult population in the world. TLR2, a member of the Toll-like receptor (TLR) family, has been implicated in the immune responses to different viruses including members of the herpesvirus family, such as human cytomegalovirus, herpes simplex virus type 1, and varicella-zoster virus. In this report, we demonstrate that infectious and UV-inactivated EBV virions lead to the activation of NF-kappaB through TLR2 using HEK293 cells cotransfected with TLR2-expressing vector along with NF-kappaB-Luc reporter plasmid. NF-kappaB activation in HEK293-TLR2 cells (HEK293 cells transfected with TLR2) by EBV was not enhanced by the presence of CD14. The effect of EBV was abrogated by pretreating HEK293-TLR2 cells with blocking anti-TLR2 antibodies or by preincubating viral particles with neutralizing anti-EBV antibodies 72A1. In addition, EBV infection of primary human monocytes induced the release of MCP-1 (monocyte chemotactic protein 1), and the use of small interfering RNA targeting TLR2 significantly reduced such a chemokine response to EBV. Taken together, these results indicate that TLR2 may be an important pattern recognition receptor in the immune response directed against EBV infection.

  12. Human Cytomegalovirus Promotes Survival of Infected Monocytes via a Distinct Temporal Regulation of Cellular Bcl-2 Family Proteins

    PubMed Central

    Collins-McMillen, Donna; Kim, Jung Heon; Nogalski, Maciej T.; Stevenson, Emily V.; Caskey, Joshua R.; Cieply, Stephen J.

    2015-01-01

    ABSTRACT Monocytes play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to target organ systems. To infect monocytes and reprogram them to deliver infectious virus, HCMV must overcome biological obstacles, including the short life span of monocytes and their antiviral proapoptotic response to infection. We have shown that virally induced upregulation of cellular Mcl-1 promotes early survival of HCMV-infected monocytes, allowing cells to overcome an early apoptotic checkpoint at around 48 h postinfection (hpi). Here, we demonstrate an HCMV-dependent shift from Mcl-1 as the primary antiapoptotic player to the related protein, Bcl-2, later during infection. Bcl-2 was upregulated in HCMV-infected monocytes beginning at 48 hpi. Treatment with the Bcl-2 antagonist ABT-199 only reduced the prosurvival effects of HCMV in target monocytes beginning at 48 hpi, suggesting that Mcl-1 controls survival prior to 48 hpi, while Bcl-2 promotes survival after 48 hpi. Although Bcl-2 was upregulated following viral binding/signaling through cellular integrins (compared to Mcl-1, which is upregulated through binding/activation of epidermal growth factor receptor [EGFR]), it functioned similarly to Mcl-1, adopting the early role of Mcl-1 in preventing caspase-3 cleavage/activation. This distinct, HCMV-induced shift from Mcl-1 to Bcl-2 occurs in response to a cellular upregulation of proapoptotic Bax, as small interfering RNA (siRNA)-mediated knockdown of Bax reduced the upregulation of Bcl-2 in infected monocytes and rescued the cells from the apoptotic effects of Bcl-2 inhibition. Our data demonstrate a distinct survival strategy whereby HCMV induces a biphasic regulation of cellular Bcl-2 proteins to promote host cell survival, leading to viral dissemination and the establishment of persistent HCMV infection. IMPORTANCE Hematogenous dissemination of HCMV via infected monocytes is a crucial component of the viral survival strategy and is required for the

  13. Glycodelin-A stimulates interleukin-6 secretion by human monocytes and macrophages through L-selectin and the extracellular signal-regulated kinase pathway.

    PubMed

    Lee, Cheuk-Lun; Lam, Eve Y F; Lam, Kevin K W; Koistinen, Hannu; Seppälä, Markku; Ng, Ernest H Y; Yeung, William S B; Chiu, Philip C N

    2012-10-26

    Macrophages represent the second major type of decidual leukocytes at the fetomaternal interface. Changes in macrophage number and activity are associated with fetal loss and pregnancy complications. Glycodelin-A (GdA) is an abundant glycoprotein in the first-trimester decidua. It is involved in fetomaternal defense and early placental development through its regulatory activities in various immune cells. The N-glycosylation of GdA mediates the binding and therefore the activities of the molecule. In this study, we studied the biological activities of GdA in the functions of human monocytes/macrophages. GdA was purified from amniotic fluid by affinity chromatography. GdA treatment did not affect the viability, cell death, or phagocytic activity of the monocytes/macrophages. GdA, but not recombinant glycodelin without glycosylation, induced IL-6 production as demonstrated by cytokine array, intracellular staining, and ELISA. GdA also induced phosphorylation of ERK in monocytes/macrophages. The involvement of ERKs in IL-6 induction was confirmed using pharmacological inhibitors. Co-immunoprecipitation showed that L-selectin on the monocytes/macrophages was the binding protein of GdA. Treatment with anti-L-selectin antibody reduced GdA binding and GdA-induced IL-6 production. GdA-treated macrophages suppressed IFN-γ expression by co-cultured T-helper cells in an IL-6-dependent manner. These results show that GdA interacts with L-selectin to induce IL-6 production in monocytes/macrophages by activating the ERK signaling pathway. In turn, the increased IL-6 production suppresses IFN-γ expression in T-helper cells, which may play an important role in inducing a Th-2-polarized cytokine environment that flavors the immunotolerance of the fetoplacental unit.

  14. Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment.

    PubMed

    Clark, R A; Wikner, N E; Doherty, D E; Norris, D A

    1988-08-25

    Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell

  15. Synergistic enhancement of cytokine-induced human monocyte matrix metalloproteinase-1 by C-reactive protein and oxidized LDL through differential regulation of monocyte chemotactic protein-1 and prostaglandin E2.

    PubMed

    Zhang, Yahong; Wahl, Larry M

    2006-01-01

    C-reactive protein (CRP) and oxidized LDL (ox-LDL) are associated with inflammatory lesions, such as coronary artery disease, in which monocytes and matrix metalloproteinases (MMPs) may play a major role in the rupture of atherosclerotic plaques. Monocytes are recruited to inflammation sites by monocyte chemoattractant protein-1 (MCP-1), which may also participate in the activation of monocytes. The objective of this study was to compare the individual and combined effect of CRP and ox-LDL on human monocyte MMP-1 and the role of MCP-1 in this effect. Although CRP or ox-LDL failed to induce MMP-1 in control monocytes, these molecules enhanced MMP-1 production induced by tumor necrosis factor alpha (TNF-alpha) and granulocyte macrophage-colony stimulating factor (GM-CSF) with a synergistic increase in MMP-1 occurring in the presence of both mediators. Enhancement of MMP-1 by CRP and ox-LDL was attributable to a differential increase in MCP-1 and prostaglandin E2(PGE2). CRP, at physiological concentrations, induced high levels of MCP-1 and relatively low levels of PGE2, whereas ox-LDL caused a significant enhancement of PGE2 with little affect on MCP-1. Accordingly, CRP- and ox-LDL-induced MMP-1 production by monocytes was inhibited by anti-MCP-1 antibodies and indomethacin, respectively. Moreover, addition of exogenous MCP-1 or PGE2 enhanced MMP-1 production by TNF-alpha- and GM-CSF-stimulated monocytes. These results show that the combination of CRP and ox-LDL can cause a synergistic enhancement of the role of monocytes in inflammation, first, by increasing MCP-1, which attracts more monocytes and directly enhances MMP-1 production by activated monocytes, and second, by elevating PGE2 production, which also leads to higher levels of MMP-1.

  16. Human podoplanin-positive monocytes and platelets enhance lymphangiogenesis through the activation of the podoplanin/CLEC-2 axis.

    PubMed

    Hur, Jin; Jang, Jae Hee; Oh, Il-Young; Choi, Jae-Il; Yun, Ji-Yeon; Kim, Joonoh; Choi, Young-Eun; Ko, Seung-Bum; Kang, Jin-A; Kang, Jeehoon; Lee, Sang Eun; Lee, Hwan; Park, Young-Bae; Kim, Hyo-Soo

    2014-08-01

    Emerging studies suggested that murine podoplanin-positive monocytes (PPMs) are involved in lymphangiogenesis. The goal of this study was to demonstrate the therapeutic lymphangiogenesis of human PPMs by the interaction with platelets. Aggregation culture of human peripheral blood mononuclear cells (PBMCs) resulted in cellular aggregates termed hematospheres. During 5-day culture, PPMs expanded exponentially and expressed several lymphatic endothelial cell-specific markers including vascular endothelial growth factor receptor (VEGFR)-3 and well-established lymphangiogenic transcription factors. Next, we investigated the potential interaction of PPMs with platelets that had C-type lectin-like receptor-2 (CLEC-2), a receptor of podoplanin. In vitro coculture of PPMs and platelets stimulated PPMs to strongly express lymphatic endothelial markers and upregulate lymphangiogenic cytokines. Recombinant human CLEC-2 also stimulated PPMs through Akt and Erk signaling. Likewise, platelets in coculture with PPMs augmented secretion of a lymphangiogenic cytokine, interleukin (IL)-1-β, via podoplanin/CLEC-2 axis. The supernatant obtained from coculture was able to enhance the migration, viability, and proliferation of lymphatic endothelial cell. Local injection of hematospheres with platelets significantly increased lymphatic neovascularization and facilitated wound healing in the full-thickness skin wounds of nude mice. Cotreatment with PPMs and platelets augments lymphangiogenesis through podoplanin/CLEC-2 axis, which thus would be a promising novel strategy of cell therapy to treat human lymphatic vessel disease.

  17. Modulation of the development of human monocyte-derived dendritic cells by lithium chloride.

    PubMed

    Liu, Ko-Jiunn; Lee, Yueh-Lun; Yang, Yi-Yuan; Shih, Neng-Yao; Ho, Chia-Chen; Wu, Yu-Chen; Huang, Tze-Sing; Huang, Ming-Chyi; Liu, Hsing-Cheng; Shen, Winston W; Leu, Sy-Jye

    2011-02-01

    Lithium has been used or explored to treat psychiatric and neurodegenerative diseases that are frequently associated with an abnormal immune status. It is likely that lithium may work through modulation of immune responses in these patients. Because dendritic cells (DC) play a central role in regulating immune responses, this study investigated the influence of lithium chloride (LiCl) on the development and function of DC. Exposure to LiCl during the differentiation of human monocyte-derived immature DCs (iDC) enhances CD86 and CD83 expression and increases the production of IL-1β, IL-6, IL-8, IL-10, and TNF-α. However, the presence of LiCl during LPS-induced maturation of iDC has the opposite effect. During iDC differentiation, LiCl suppresses the activity of glycogen synthase kinase (GSK)-3β, and activates PI3K and MEK. In addition, LiCl activates peroxisome proliferator-activated receptor γ (PPARγ) during iDC differentiation, a pathway not described before. Each of these signaling pathways appears to have distinct impact on the differentiating iDC. The enhanced CD86 expression by LiCl involves the PI3K/AKT and GSK-3β pathway. LiCl modulates the expression of CD83 in iDC mainly through MEK/ERK, PI3K/AKT, and PPARγ pathways, while the increased production of IL-1β and TNF-α mainly involves the MEK/ERK pathway. The effect of LiCl on IL-6/IL-8/IL-10 secretion in iDC is mediated through inhibition of GSK-3β. We have also demonstrated that PPARγ is downstream of GSK-3β and is responsible for the LiCl-mediated modulation of CD86/83 and CD1 expression, but not IL-6/8/10 secretion. The combined influence of these molecular signaling pathways may account for certain clinical effect of lithium.

  18. Investigating the Human Immunodeficiency Virus Type One-Infected Monocyte-Derived Macrophage Secretome

    PubMed Central

    Ciborowski, Pawel; Kadiu, Irena; Rozek, Wojciech; Smith, Lynette; Bernhardt, Kristen; Fladseth, Melissa; Ricardo-Dukelow, Mary; Gendelman, Howard E.

    2007-01-01

    Mononuclear phagocytes (bone marrow monocyte-derived macrophages, alveolar macrophages, perivascular macrophages, and microglia) are reservoirs and vehicles of dissemination for the human immunodeficiency virus type-1 (HIV-1). How virus alters mononuclear phagocyte immunoregulatory activities to complete its life cycle and influence disease is incompletely understood. In attempts to better understanding the influence of virus on macrophage functions, we used one-dimensional electrophoresis, and liquid chromatography tandem mass spectrometry to analyze the secretome of HIV-1 infected human monocyte-derived macrophages. We identified 111 proteins in culture supernatants of control (uninfected) and virus-infected cells. Differentially expressed cytoskeletal, enzymes, redox, and immunoregulatory protein classes were discovered and validated by Western-blot tests. These included, but were not limited to, cystatin C, cystatin B, chitinase 3-like 1 protein, cofilin-1, L-plastin, superoxide dismutase, leukotriene A4 hydrolase, and α-enolase. This study, through the use of a unique proteomics platform, provides novel insights into virus-host cell interactions that affect the functional role of macrophages in HIV disease. PMID:17320137

  19. Dynamic Modulation of Innate Immune Response by Varying Dosages of Lipopolysaccharide (LPS) in Human Monocytic Cells*

    PubMed Central

    Morris, Matthew C.; Gilliam, Elizabeth A.; Button, Julia; Li, Liwu

    2014-01-01

    Innate monocytes and macrophages can be dynamically programmed into distinct states depending upon the strength of external stimuli. Innate programming may bear significant relevance to the pathogenesis and resolution of human inflammatory diseases. However, systems analyses with regard to the dynamic programming of innate leukocytes are lacking. In this study, we focused on the dynamic responses of human promonocytic THP-1 cells to lipopolysaccharide (LPS). We observed that varying dosages of LPS differentially modulate the expression of selected pro- and anti- inflammatory mediators such as IL-6 and IL-33. Super-low dosages of LPS preferentially induced the pro-inflammatory mediator IL-6, while higher dosages of LPS induced both IL-6 and IL-33. Mechanistically, we demonstrated that super-low and high doses of LPS cause differential activation of GSK3 and Akt, as well as the transcription factors FoxO1 and CREB. Inhibition of GSK3 enabled THP-1 cells to express IL-33 when challenged with super-low dose LPS. On the other hand, activation of CREB with adenosine suppressed IL-6 expression. Taken together, our study reveals a dynamic modulation of monocytic cells in response to varying dosages of endotoxin, and may shed light on our understanding of the dynamic balance that controls pathogenesis and resolution of inflammatory diseases. PMID:24970893

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

    SciTech Connect

    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.

  1. Phospholipase D from Loxosceles laeta Spider Venom Induces IL-6, IL-8, CXCL1/GRO-α, and CCL2/MCP-1 Production in Human Skin Fibroblasts and Stimulates Monocytes Migration.

    PubMed

    Rojas, José M; Arán-Sekul, Tomás; Cortés, Emmanuel; Jaldín, Romina; Ordenes, Kely; Orrego, Patricio R; González, Jorge; Araya, Jorge E; Catalán, Alejandro

    2017-04-05

    Cutaneous loxoscelism envenomation by Loxosceles spiders is characterized by the development of a dermonecrotic lesion, strong inflammatory response, the production of pro-inflammatory mediators, and leukocyte migration to the bite site. The role of phospholipase D (PLD) from Loxosceles in the recruitment and migration of monocytes to the envenomation site has not yet been described. This study reports on the expression and production profiles of cytokines and chemokines in human skin fibroblasts treated with catalytically active and inactive recombinant PLDs from Loxosceles laeta (rLlPLD) and lipid inflammatory mediators ceramide 1-phosphate (C1P) and lysophosphatidic acid (LPA), and the evaluation of their roles in monocyte migration. Recombinant rLlPLD1 (active) and rLlPLD2 (inactive) isoforms induce interleukin (IL)-6, IL-8, CXCL1/GRO-α, and CCL2/monocyte chemoattractant protein-1 (MCP-1) expression and secretion in fibroblasts. Meanwhile, C1P and LPA only exhibited a minor effect on the expression and secretion of these cytokines and chemokines. Moreover, neutralization of both enzymes with anti-rLlPLD1 antibodies completely inhibited the secretion of these cytokines and chemokines. Importantly, conditioned media from fibroblasts, treated with rLlPLDs, stimulated the transmigration of THP-1 monocytes. Our data demonstrate the direct role of PLDs in chemotactic mediator synthesis for monocytes in human skin fibroblasts and indicate that inflammatory processes play an important role during loxoscelism.

  2. Tacaribe Virus but Not Junin Virus Infection Induces Cytokine Release from Primary Human Monocytes and Macrophages

    PubMed Central

    Groseth, Allison; Hoenen, Thomas; Weber, Michaela; Wolff, Svenja; Herwig, Astrid; Kaufmann, Andreas; Becker, Stephan

    2011-01-01

    The mechanisms underlying the development of disease during arenavirus infection are poorly understood. However, common to all hemorrhagic fever diseases is the involvement of macrophages as primary target cells, suggesting that the immune response in these cells may be of paramount importance during infection. Thus, in order to identify features of the immune response that contribute to arenavirus pathogenesis, we have examined the growth kinetics and cytokine profiles of two closely related New World arenaviruses, the apathogenic Tacaribe virus (TCRV) and the hemorrhagic fever-causing Junin virus (JUNV), in primary human monocytes and macrophages. Both viruses grew robustly in VeroE6 cells; however, TCRV titres were decreased by approximately 10 fold compared to JUNV in both monocytes and macrophages. Infection of both monocytes and macrophages with TCRV also resulted in the release of high levels of IL-6, IL-10 and TNF-α, while levels of IFN-α, IFN-β and IL-12 were not affected. However, we could show that the presence of these cytokines had no direct effect on growth of either TCRV of JUNV in macrophages. Further analysis also showed that while the production of IL-6 and IL-10 are dependent on viral replication, production of TNF-α also occurs after exposure to UV-inactivated TCRV particles and is thus independent of productive virus infection. Surprisingly, JUNV infection did not have an effect on any of the cytokines examined indicating that, in contrast to other viral hemorrhagic fever viruses, macrophage-derived cytokine production is unlikely to play an active role in contributing to the cytokine dysregulation observed in JUNV infected patients. Rather, these results suggest that an early, controlled immune response by infected macrophages may be critical for the successful control of infection of apathogenic viruses and prevention of subsequent disease, including systemic cytokine dysregulation. PMID:21572983

  3. Systems toxicology-based assessment of the candidate modified risk tobacco product THS2.2 for the adhesion of monocytic cells to human coronary arterial endothelial cells.

    PubMed

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

    2016-01-02

    Alterations of endothelial adhesive properties by cigarette smoke (CS) can progressively favor the development of atherosclerosis which may cause cardiovascular disorders. Modified risk tobacco products (MRTPs) are tobacco products developed to reduce smoking-related risks. A systems biology/toxicology approach combined with a functional in vitro adhesion assay was used to assess the impact of a candidate heat-not-burn technology-based MRTP, Tobacco Heating System (THS) 2.2, on the adhesion of monocytic cells to human coronary arterial endothelial cells (HCAECs) compared with a reference cigarette (3R4F). HCAECs were treated for 4h with conditioned media of human monocytic Mono Mac 6 (MM6) cells preincubated with low or high concentrations of aqueous extracts from THS2.2 aerosol or 3R4F smoke for 2h (indirect treatment), unconditioned media (direct treatment), or fresh aqueous aerosol/smoke extracts (fresh direct treatment). Functional and molecular investigations revealed that aqueous 3R4F smoke extract promoted the adhesion of MM6 cells to HCAECs via distinct direct and indirect concentration-dependent mechanisms. Using the same approach, we identified significantly reduced effects of aqueous THS2.2 aerosol extract on MM6 cell-HCAEC adhesion, and reduced molecular changes in endothelial and monocytic cells. Ten- and 20-fold increased concentrations of aqueous THS2.2 aerosol extract were necessary to elicit similar effects to those measured with 3R4F in both fresh direct and indirect exposure modalities, respectively. Our systems toxicology study demonstrated reduced effects of an aqueous aerosol extract from the candidate MRTP, THS2.2, using the adhesion of monocytic cells to human coronary endothelial cells as a surrogate pathophysiologically relevant event in atherogenesis.

  4. In vitro Effects of Selected Saponins on the Production and Release of Lysozyme Activity of Human Monocytic and Epithelial Cell Lines

    PubMed Central

    Helal, Racha; Melzig, Matthias F.

    2011-01-01

    Lysozyme is one of the most important factors of innate immunity and a unique enzybiotic in that it exerts not only antibacterial activity, but also antiviral, anti-inflammatory, anticancer and immunomodulatory activities. The purpose of the present study was to investigate whether in vitro exposure to saponins can affect the release and production of lysozyme activity in human monocytic cells THP-1, and in human epithelial cells HT-29. Lysozyme activity levels in cell culture fluids were measured using highly sensitive fluorescence-based lysozyme activity assay. Majority of the examined saponins were demonstrated to stimulate significantly the release of lysozyme activity of monocytes and epithelial cells after one hour treatment at non-toxic concentrations. On the contrary, cells treated with saponins for longer periods up to 72 hours showed tendency to decrease in the secretion and production of lysozyme activity. However, these inhibitory effects of saponins observed with long-term treatment periods were mostly associated with toxic effects of saponins to cells. The results suggested positive contribution of some saponins to lysozyme release of monocytes and epithelial cells upon short exposure. Furthermore, demonstrated ability of these saponins to enhance the release of lysozyme activity can present a new mechanism contribute to explaining important biological characteristics of saponins, including the antibacterial, antiviral, anti-inflammatory or immune-stimulating properties. PMID:21773070

  5. Monocytic cells derived from human embryonic stem cells and fetal liver share common differentiation pathways and homeostatic functions.

    PubMed

    Klimchenko, Olena; Di Stefano, Antonio; Geoerger, Birgit; Hamidi, Sofiane; Opolon, Paule; Robert, Thomas; Routhier, Mélanie; El-Benna, Jamel; Delezoide, Anne-Lise; Boukour, Siham; Lescure, Bernadette; Solary, Eric; Vainchenker, William; Norol, Françoise

    2011-03-17

    The early emergence of macrophages and their large pattern of tissue distribution during development suggest that they may play a critical role in the initial steps of embryogenesis. In the present study, we show that monocytic cells derived from human embryonic stem cells (hESCs) and from fetal liver follow a differentiation pathway different to that of adult cells, leading to specific functions. Embryonic and fetal monocytic cells differentiated from a CD14(low)CD16(-) precursor to form CD14(high)CD16(+) cells without producing the CD14(high)CD16(-) cell population that predominates in adult peripheral blood. Both demonstrated an enhanced expression of genes encoding tissue-degrading enzymes, chemokines, and scavenger receptors, as was previously reported for M2 macrophages. Compared with adult blood monocytes, embryonic and fetal monocytic cells secreted high amounts of proteins acting on tissue remodeling and angiogenesis, and most of them expressed the Tie2 receptor. Furthermore, they promoted vascular remodeling in xenotransplanted human tumors. These findings suggest that the regulation of human fetal and embryonic monocytic cell differentiation leads to the generation of cells endowed mainly with anti-inflammatory and remodeling functions. Trophic and immunosuppressive functions of M2-polarized macrophages link fetus and tumor development, and hESCs offer a valuable experimental model for in vitro studies of mechanisms sustaining these processes.

  6. IgE and IL-33-mediated triggering of human basophils inhibits TLR4-induced monocyte activation.

    PubMed

    Rivellese, Felice; Suurmond, Jolien; de Paulis, Amato; Marone, Gianni; Huizinga, Tom W J; Toes, René E M

    2014-10-01

    Basophils are circulating granulocytes, best known as effector cells in allergic reactions. Recent studies in mice suggest that they might also participate in the suppression of chronic inflammation. The aim of this study was to assess the ability of purified human basophils to modulate monocyte responses upon IL-33 and IgE triggering. Activation of human basophils with IL-33 induced the production of IL-4 and the release of histamine, and enhanced their IgE-mediated activation. In addition, basophils triggered with IL-33 and anti-IgE significantly suppressed the LPS-induced production of the proinflammatory cytokine TNF-α and the upregulation of the costimulatory molecule CD80 by monocytes. These effects were mainly explained by the release of histamine, as they could be inhibited by the histamine receptor 2 antagonist ranitidine, with a smaller contribution of IL-4. In contrast, basophil-derived IL-4 and histamine had opposing effects on the expression of the inhibitory Fc γ receptor IIb and the production of IL-10 by monocytes. Our data show that basophils can influence monocyte activation and suggest a previously unrecognized role for human basophils in the modulation of monocyte-mediated immune responses, through the balanced secretion of histamine and IL-4.

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

    SciTech Connect

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

    1990-05-15

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

  8. Comparison of Ultrastructural Cytotoxic Effects of Carbon and Carbon/Iron Particulates on Human Monocyte-Derived Macrophages

    PubMed Central

    Long, John F.; Waldman, W. James; Kristovich, Robert; Williams, Marshall; Knight, Deborah; Dutta, Prabir K.

    2005-01-01

    In this study, we tested the hypothesis that the presence of iron in carbon particulates enhances ultrastructural perturbation in human monocyte-derived macrophages (MDMs) after phagocytosis. We used 1-μm synthetic carbon-based particulates, designed to simulate environmental particulates of mass median aerodynamic diameter ≤ 2.5 μm (PM2.5). Cultures of human MDMs or T-lymphocytes (as a nonphagocytic control) were exposed to carbon or carbon/iron particulates for various time periods and examined by transmission electron microscopy for ultrastructural changes. T-cells failed to internalize either of the particulates and showed no organelle or nuclear changes. Conversely, MDMs avidly phagocytized the particulates. MDMs treated with C particulates exhibited morphologic evidence of macrophage activation but no evidence of lysis of organelles. In contrast, MDMs treated with C/Fe particulates exhibited coalescence of particulate-containing lysosomes. This phenomenon was not observed in the case of C particulates. By 24 hr there was a tendency of the C/Fe particulates to agglomerate into loose or compact clusters. Surrounding the compact C/Fe agglomerates was a uniform zone of nearly total organelle lysis. The lytic changes diminished in proportion to the distance from the agglomerate. In such cells, the nucleus showed loss of chromatin. Although C particles induced no detectable oxidative burst on treated MDMs, C/Fe particles induced a nearly 5-fold increase in the extracellular oxidative burst by treated MDMs compared with untreated controls. Iron bound to C particles catalyzed the decomposition of hydrogen peroxide to generate hydroxyl radicals. Results of these studies suggest that, among particulates of similar size, biologic activity can vary profoundly as a function of particulate physicochemical properties. PMID:15687054

  9. Human cytomegalovirus interleukin-10 polarizes monocytes toward a deactivated M2c phenotype to repress host immune responses.

    PubMed

    Avdic, Selmir; Cao, John Z; McSharry, Brian P; Clancy, Leighton E; Brown, Rebecca; Steain, Megan; Gottlieb, David J; Abendroth, Allison; Slobedman, Barry

    2013-09-01

    Several human cytomegalovirus (HCMV) genes encode products that modulate cellular functions in a manner likely to enhance viral pathogenesis. This includes UL111A, which encodes homologs of human interleukin-10 (hIL-10). Depending upon signals received, monocytes and macrophages become polarized to either classically activated (M1 proinflammatory) or alternatively activated (M2 anti-inflammatory) subsets. Skewing of polarization toward an M2 subset may benefit the virus by limiting the proinflammatory responses to infection, and so we determined whether HCMV-encoded viral IL-10 influenced monocyte polarization. Recombinant viral IL-10 protein polarized CD14(+) monocytes toward an anti-inflammatory M2 subset with an M2c phenotype, as demonstrated by high expression of CD163 and CD14 and suppression of major histocompatibility complex (MHC) class II. Significantly, in the context of productive HCMV infection, viral IL-10 produced by infected cells polarized uninfected monocytes toward an M2c phenotype. We also assessed the impact of viral IL-10 on heme oxygenase 1 (HO-1), which is an enzyme linked with suppression of inflammatory responses. Polarization of monocytes by viral IL-10 resulted in upregulation of HO-1, and inhibition of HO-1 function resulted in a loss of capacity of viral IL-10 to suppress tumor necrosis factor alpha (TNF-α) and IL-1β, implicating HO-1 in viral IL-10-induced suppression of proinflammatory cytokines by M2c monocytes. In addition, a functional consequence of monocytes polarized with viral IL-10 was a decreased capacity to activate CD4(+) T cells. This study identifies a novel role for viral IL-10 in driving M2c polarization, which may limit virus clearance by restricting proinflammatory and CD4(+) T cell responses at sites of infection.

  10. Monocyte/macrophage trafficking in acquired immunodeficiency syndrome encephalitis: lessons from human and nonhuman primate studies.

    PubMed

    Fischer-Smith, Tracy; Bell, Christie; Croul, Sidney; Lewis, Mark; Rappaport, Jay

    2008-08-01

    Here the authors discuss evidence in human and animal models supporting two opposing views regarding the pathogenesis of human immunodeficiency virus (HIV) in the central nervous system (CNS): (1) HIV infection in the CNS is a compartmentalized infection, with the virus-infected macrophages entering the CNS early, infecting resident microglia and astrocytes, and achieving a state of latency with evolution toward a fulminant CNS infection late in the course of disease; or alternatively, (2) events in the periphery lead to altered monocyte/macrophage (MPhi) homeostasis, with increased CNS invasion of infected and/or uninfected MPhis. Here the authors have reevaluated evidence presented in the favor of the latter model, with a discussion of phenotypic characteristics distinguishing normal resident microglia with those accumulating in HIV encephalitis (HIVE). CD163 is normally expressed by perivascular MPhi s but not resident microglia in normal CNS of humans and rhesus macaques. In agreement with other studies, the authors demonstrate expression of CD163 by brain MPhi s in HIVE and simian immunodeficiency virus encephalitis (SIVE). CNS tissues from HIV-sero positive individuals with HIVE or HIV-associated progressive multifocal leukoencephalopathy (PML) were also examined. In HIVE, the authors further demonstrate colocalization of CD163 and CD16 (Fcgamma III recptor) gene expression, the latter marker associated with HIV infection of monocyte in vivo and permissivity of infection. Indeed, CD163(+) MPhis and microglia are often productively infected in HIVE CNS. In SIV infected rhesus macaques, CD163(+) cells accumulate perivascularly, within nodular lesions and the parenchyma in animals with encephalitis. Likewise, parenchymal microglia and perivascular MPhi s are CD163(+) in HIVE. In contrast to HIVE, CD163(+)perivascular and parenchymal MPhi s in HIV-associated PML were only associated with areas of demyelinating lesions. Interestingly, SIV-infected rhesus macaques

  11. Granzyme K synergistically potentiates LPS-induced cytokine responses in human monocytes

    PubMed Central

    Wensink, Annette C.; Kemp, Vera; Fermie, Job; García Laorden, M. Isabel; van der Poll, Tom; Hack, C. Erik; Bovenschen, Niels

    2014-01-01

    Granzymes are serine proteases released by cytotoxic lymphocytes to induce apoptosis in virus-infected cells and tumor cells. Evidence is emerging that granzymes also play a role in controlling inflammation. Granzyme serum levels are elevated in patients with autoimmune diseases and infections, including sepsis. However, the function of extracellular granzymes in inflammation largely remains unknown. Here, we show that granzyme K (GrK) binds to Gram-negative bacteria and their cell-wall component lipopolysaccharide (LPS). GrK synergistically enhances LPS-induced cytokine release in vitro from primary human monocytes and in vivo in a mouse model of LPS challenge. Intriguingly, these extracellular effects are independent of GrK catalytic activity. GrK disaggregates LPS from micelles and augments LPS–CD14 complex formation, thereby likely boosting monocyte activation by LPS. We conclude that extracellular GrK is an unexpected direct modulator of LPS–TLR4 signaling during the antimicrobial innate immune response. PMID:24711407

  12. Chemotactic activity of Helicobacter pylori sonicate for human polymorphonuclear leucocytes and monocytes.

    PubMed Central

    Nielsen, H; Andersen, L P

    1992-01-01

    The immunopathology of Helicobacter pylori associated active chronic gastritis, which is characterised by predominance of polymorphonuclear leucocyte infiltration, is largely unknown. To evaluate the role of bacterial components as inflammatory mediators ultracentrifuged sonicated preparations were made of clinical isolates of Helicobacter pylori. The crude sonicates were shown to exhibit chemotactic activity for human polymorphonuclear leucocytes and blood monocytes in a concentration dependent fashion. The potency was comparable with previously described bacterial derived cytotaxins. The cytotaxin(s) was non-dialysable and completely destroyed by proteinase. Heat treatment did not decrease the chemotactic activity, but in sonicate subjected to 100 degrees C for 15 minutes all activity disappeared after dialysis suggesting the breakdown of a larger protein to small fragments that are still biological active. By ammonium sulphate precipitation at increasing concentrations the cytotaxin(s) was selectively found in 10% ammonium sulphate saturation, and by further molecular gel separation the chemotactic activity was found in the molecular size range from 25 to 35 kDa. The demonstration of a polymorphonuclear leucocyte and monocyte cytotaxin from Helicobacter pylori sonicate may help in understanding the mucosal immune response in gastric inflammatory diseases. PMID:1624151

  13. Structure of human monocyte chemoattractant protein 4 (MCP-4/CCL13)

    SciTech Connect

    Barinka, Cyril; Prahl, Adam; Lubkowski, Jacek

    2008-04-02

    Monocyte chemoattractant proteins (MCPs) belong to the CC chemokine family and are involved in many (patho)physiological processes characterized by mononuclear cell infiltration, including tissue remodeling, atherosclerosis and cancer metastasis. Here, the crystal structure of human monocyte chemoattractant protein 4 (MCP-4) refined at 1.70 {angstrom} resolution is reported with crystallographic values R = 0.180 and R{sub free} = 0.212. The overall MCP-4 fold reveals the typical tertiary features of the CC chemokine family. A central three-stranded antiparallel {beta}-sheet is C-terminally flanked by an overlaying {alpha}-helix, while the N-terminal part of the molecule forms an extended loop that is anchored to the rest of the molecule via two disulfide bridges, Cys11-Cys35 and Cys12-Cys51. The crystal packing suggests the existence of MCP-4 dimers with a dimerization interface similar to those previously reported for the X-ray structures of MCP-1 and MCP-2.

  14. Isolation and characterization of auxotrophic mutants of Legionella pneumophila that fail to multiply in human monocytes.

    PubMed Central

    Mintz, C S; Chen, J X; Shuman, H A

    1988-01-01

    Attempts to isolate auxotrophic mutants of Legionella pneumophila have been hampered by the complex nutritional composition of the media used to cultivate this organism. We developed a semidefined medium, designated CAA, to facilitate the isolation and characterization of Legionella auxotrophs. Unlike previously described chemically defined media for this organism, L. pneumophila formed colonies on CAA agar. Using this medium, we isolated several independent tryptophan auxotrophs of strain Philadelphia-1 after ethyl methanesulfonate mutagenesis and penicillin enrichment. Trimethoprim selection was used to isolate several independent thymidine-requiring mutants of the same strain. The thymidine auxotrophs exhibited a marked decrease in viability when they were deprived of thymidine. The results of monocyte infection experiments with both the tryptophan and thymidine auxotrophs indicated that the thymidine auxotrophs were incapable of intracellular survival or multiplication. In contrast, the tryptophan auxotrophs grew well in monocyte cultures. The isolation of additional auxotrophic mutants will facilitate the study of the nutritional requirements of L. pneumophila for growth in human mononuclear phagocytes. PMID:3372016

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

    SciTech Connect

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

    1985-09-01

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

  16. Gliadin activates arginase pathway in RAW264.7 cells and in human monocytes.

    PubMed

    Barilli, Amelia; Rotoli, Bianca Maria; Visigalli, Rossana; Dall'Asta, Valeria

    2014-09-01

    Celiac disease (CD) is an autoimmune enteropathy triggered in susceptible individuals by the ingestion of gliadin-containing grains. Recent studies have demonstrated that macrophages play a key role in the pathogenesis of CD through the release of inflammatory mediators such as cytokines and nitric oxide (NO). Since arginine is the obliged substrate of iNOS (inducible nitric oxide synthase), the enzyme that produces large amount of NO, the aim of this work is to investigate arginine metabolic pathways in RAW264.7 murine macrophages after treatment with PT-gliadin (PTG) in the absence and in the presence of IFNγ. Our results demonstrate that, besides strengthening the IFNγ-dependent activation of iNOS, gliadin is also an inducer of arginase, the enzyme that transforms arginine into ornithine and urea. Gliadin treatment increases, indeed, the expression and the activity of arginase, leading to the production of polyamines through the subsequent induction of ornithine decarboxylase. This effect is strengthened by IFNγ. The activation of these pathways takes advantage of the increased availability of arginine due to a decreased system y(+)l-mediated efflux, likely ascribable to a reduced expression of Slc7a6 transporter. A significant induction of arginase expression is also observed in human monocytes from healthy subject upon treatment with gliadin, thus demonstrating that gluten components trigger changes in arginine metabolism in monocyte/macrophage cells.

  17. Signal transduction pathways leading to the production of IL-8 by human monocytes are differentially regulated by dexamethasone.

    PubMed Central

    Anttila, H S; Reitamo, S; Ceska, M; Hurme, M

    1992-01-01

    Previous studies have shown that IL-8 gene expression is enhanced by various stimuli, which induce different signal transduction pathways. A lipopolysaccharide (LPS)-induced pathway has been reported to be inhibited by glucocorticoids in monocytes. We have now examined the effect of dexamethasone on the LPS-induced and other signal transduction pathways leading to the production of IL-8 by human monocytes. Dexamethasone inhibited the production of IL-8 stimulated with a cyclic adenosine monophosphate analog or LPS. In contrast, dexamethasone had no significant effect on a phorbol ester (PMA)-stimulated IL-8 production. These results suggest that the signal transduction pathways leading to the production of IL-8 by human monocytes are differentially regulated by dexamethasone. PMID:1325308

  18. Oxygen-Loaded Nanodroplets Effectively Abrogate Hypoxia Dysregulating Effects on Secretion of MMP-9 and TIMP-1 by Human Monocytes

    PubMed Central

    Gulino, Giulia Rossana; Magnetto, Chiara; Khadjavi, Amina; Panariti, Alice; Rivolta, Ilaria; Soster, Marco; Argenziano, Monica; Cavalli, Roberta; Giribaldi, Giuliana; Guiot, Caterina; Prato, Mauro

    2015-01-01

    Monocytes play a key role in the inflammatory stage of the healing process. To allow monocyte migration to injured tissues, the balances between secreted matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) must be finely modulated. However, a reduction of blood supply and local oxygen tension can modify the phenotype of immune cells. Intriguingly, hypoxia might be targeted by new effective oxygenating devices such as 2H,3H-decafluoropentane- (DFP-) based oxygen-loaded nanodroplets (OLNs). Here, hypoxia effects on gelatinase/TIMP release from human peripheral monocytes were investigated, and the therapeutic potential of dextran-shelled OLNs was evaluated. Normoxic monocytes constitutively released ~500 ng/mL MMP-9, ~1.3 ng/mL TIMP-1, and ~0.6 ng/mL TIMP-2 proteins. MMP-2 was not detected. After 24 hours, hypoxia significantly altered MMP-9/TIMP-1 balance by reducing MMP-9 and increasing TIMP-1, without affecting TIMP-2 secretion. Interestingly OLNs, not displaying toxicity to human monocytes after cell internalization, effectively counteracted hypoxia, restoring a normoxia-like MMP-9/TIMP-1 ratio. The action of OLNs was specifically dependent on time-sustained oxygen diffusion up to 24 h from their DFP-based core. Therefore, OLNs appear as innovative, nonconventional, cost-effective, and nontoxic therapeutic tools, to be potentially employed to restore the physiological invasive phenotype of immune cells in hypoxia-associated inflammation. PMID:25878404

  19. Role of endogenous ceruloplasmin in low density lipoprotein oxidation by human U937 monocytic cells.

    PubMed Central

    Ehrenwald, E; Fox, P L

    1996-01-01

    Oxidation of lipids and lipoproteins by macrophages is an important event during atherogenesis. Activation of monocytic cells by zymosan and other agonists results in the release of multiple oxidant species and consequent oxidation of LDL. We now show evidence that ceruloplasmin, a copper-containing acute phase reactant, is secreted by zymosan-activated U937 monocytic cells, and that the protein has an important role in LDL oxidation by these cells. In one approach, ceruloplasmin has been shown to exhibit oxidant activity under the appropriate conditions. Exogenous addition of purified human ceruloplasmin stimulates U937 cell oxidation of LDL to nearly the same extent as activation by zymosan. In contrast to previous cell-free experiments (Ehrenwald, E., G.M. Chisom, and P.L. Fox. 1994. Intact human ceruloplasmin oxidatively modifies low density lipoprotein. J. Clin. Invest. 93:1493-1501.) in which ceruloplasmin by itself (in PBS) oxidizes LDL, under the conditions of the current experiments (in RPMI 1640 medium) ceruloplasmin only oxidizes LDL in the presence of cells; the mechanism by which cells overcome the inhibition by medium components has not been ascertained. As further evidence for a role of ceruloplasmin, activation of U937 cells with zymosan induces ceruloplasmin mRNA and ceruloplasmin protein synthesis after a 5-6 h lag that is consistent with that preceding LDL oxidation. Finally, neutralization by a highly specific polyclonal antibody to human ceruloplasmin inhibits LDL oxidation by at least 65%. Moreover, multiple antisense oligodeoxynucleotides targeted to different regions of the ceruloplasmin mRNA block LDL oxidation by up to 95%. The specific action of the antisense oligonucleotides has been verified by showing inhibition of ceruloplasmin synthesis and by the ability of exogenous ceruloplasmin to overcome the inhibition. In summary, these results are consistent with a mechanism in which cell-derived ceruloplasmin participates in oxidation of LDL

  20. The Prognostic Value of Baseline Lymphocyte, Neutrophil, and Monocyte Counts in Locally Advanced Cervical Carcinoma Treated with Radiation

    PubMed Central

    Himler, Justin; Nagel, Christa I.; Resnick, Kimberly

    2017-01-01

    Background. To determine the prognostic significance of pretreatment levels of circulating lymphocyte (CLC), neutrophil (CNC), and monocyte (CMC) counts in patients with locally advanced cervical carcinoma (CC) treated with definitive radiation. Methods. A retrospective, dual-institution review of patients with Stage IB2-IVA CC from 2005 to 2015. Progression-free (PFS) and Overall Survival (OS) were determined for high and low CLC, CNC, and CMC groups. Multivariate analysis was used to confirm prognostic value of baseline leukocyte counts. Results. 181 patients were included. Median follow-up time was 26 (3–89) months. CNC had no effect on PFS or OS. PFS was similar between CMC groups; however, OS was significantly improved for patients with low CMC (62.5 versus 45.3 months, p = 0.016). High CLC was associated with improved PFS (48.5 versus 27.8 months, p = 0.048) and OS (58.4 versus 34.9 months, p = 0.048). On multivariate analysis, high CNC was associated with increased relapse risk (HR 1.12, p = 0.006) and low CLC was associated with increased mortality risk (HR 0.67, p = 0.027). Conclusion. This study demonstrates that leukocyte values can provide prognostic information in CC. These hypothesis-generating findings warrant further prospective investigations. PMID:28239396

  1. Natural isoprenoids inhibit LPS-induced-production of cytokines and nitric oxide in aminobisphosphonate-treated monocytes.

    PubMed

    Marcuzzi, Annalisa; Tommasini, Alberto; Crovella, Sergio; Pontillo, Alessandra

    2010-06-01

    The inhibition of mevalonate pathway through genetic defects (mevalonate kinase deficiency, MKD) or pharmacologic drugs (aminobisphosphonates) causes a shortage of intermediate compounds and, in particular, of geranylgeranyl-pyrophosphate (GGPP) associated to the activation of caspase-1 and IL-1beta release. Geraniol (GOH), farnesol (FOH), geranylgeraniol (GGOH) and menthol (MOH), due to their isoprenoid structure, are supposed to enter the mevalonate pathway and to by-pass the biochemical block, reconstituting the pathway. Considering the already known side effects of aminobisphosphonates, and the lack of a specific treatment for MKD, we evaluated the impact of these natural isoprenoids compounds in a RAW cell lines chemically treated with the aminobisphosphonate alendronate, and in monocytes isolated from 2 patients affected by MKD. GOH, FOH, GGOH and MOH were all capable to diminish inflammatory marker levels induced by LPS. These natural isoprenoids could be proposed as novel therapeutic approach for the still orphan drug MKD, but also considered for the evaluation of possible inflammatory side effects of aminobisphosphonates.

  2. Global Effect of Interleukin-10 on the Transcriptional Profile Induced by Neisseria meningitidis in Human Monocytes

    PubMed Central

    Øvstebø, Reidun; Olstad, Ole Kristoffer; Brusletto, Berit; Dalsbotten Aass, Hans Christian; Kierulf, Peter; Brandtzaeg, Petter; Berg, Jens Petter

    2012-01-01

    In meningococcal septic shock, the dominant inducer of inflammation is lipopolysaccharide (LPS) in the outer membrane of Neisseria meningitidis, while interleukin-10 (IL-10) is the principal anti-inflammatory cytokine. We have used microarrays and Ingenuity Pathway Analysis to study the global effects of IL-10 on gene expression induced by N. meningitidis, after exposure of human monocytes (n = 5) for 3 h to N. meningitidis (106 cells/ml), recombinant human IL-10 (rhIL-10) (25 ng/ml), and N. meningitidis combined with rhIL-10. N. meningitidis and IL-10 differentially expressed 3,579 and 648 genes, respectively. IL-10 downregulated 125 genes which were upregulated by N. meningitidis, including NLRP3, the key molecule of the NLRP3 inflammasome. IL-10 also upregulated 270 genes which were downregulated by N. meningitidis, including members of the leukocyte immunuglobulin-like receptor (LIR) family. Fifty-three genes revealed a synergistically increased expression when N. meningitidis and IL-10 were combined. AIM2 (the principal molecule of the AIM2 inflammasome) was among these genes (fold change [FC], 18.3 versus 7.4 and 9.4 after stimulation by N. meningitidis and IL-10, respectively). We detected reduced concentrations (92% to 40%) of six cytokines (IL-1b, IL-6, IL-8, tumor necrosis factor alpha [TNF-α], macrophage inflammatory protein alpha [MIP-α], MIP-β) in the presence of IL-10, compared with concentrations with stimulation by N. meningitidis alone. Our data analysis of the effects of IL-10 on gene expression induced by N. meningitidis suggests that high plasma levels of IL-10 in meningococcal septic shock plasma may have a profound effect on a variety of functions and cellular processes in human monocytes, including cell-to-cell signaling, cellular movement, cellular development, antigen presentation, and cell death. PMID:22966040

  3. Human antibodies against dengue enhance dengue viral infectivity without suppressing type I interferon secretion in primary human monocytes.

    PubMed

    Kou, Zhihua; Lim, Joanne Y H; Beltramello, Martina; Quinn, Matthew; Chen, Huiyuan; Liu, Shengyong; Liu, Shengyo-ng; Martinez-Sobrido, Luis; Martnez-Sobrido, Luis; Diamond, Michael S; Schlesinger, Jacob J; de Silva, Aravinda; Sallusto, Federica; Jin, Xia

    2011-02-05

    It remains unclear whether antibody-dependent-enhancement (ADE) of dengue infection merely augments viral attachment and entry through Fcγ receptors or immune complex binding to Fcγ receptors triggers an intrinsic signaling cascade that changes the viral permissiveness of the cell. Using human dengue-immune sera and novel human monoclonal antibodies against dengue in combination with virologic and immunologic techniques, we found that ADE infection increased the proportion of infected primary human monocytes modestly from 0.2% ± 0.1% (no Ab) to 1.7% ± 1.6% (with Ab) but the total virus output markedly from 2 ± 2 (× 10(3)) FFU to 120 ± 153 (× 10(3))FFU. However, this increased virus production was not associated with a reduced secretion of type I interferon or an elevated secretion of anti-inflammatory cytokine, IL-10. These results demonstrate that the regulation of virus production in ADE infection of primary human monocytes is more complex than previously appreciated.

  4. Inhibitory and stimulatory effects of Pseudomonas aeruginosa pyocyanine on human T and B lymphocytes and human monocytes.

    PubMed Central

    Ulmer, A J; Pryjma, J; Tarnok, Z; Ernst, M; Flad, H D

    1990-01-01

    Pyocyanine, a pigment produced by Pseudomonas aeruginosa, has dual dose-dependent stimulatory as well as inhibitory effects on immune responses in vitro as measured by DNA synthesis of human T and B lymphocytes, interleukin-2 (IL-2) production by human T lymphocytes, immunoglobulin production by human B lymphocytes, and monokine production by human monocytes. In general, stimulatory activity was found at low concentrations of pyocyanine, whereas high concentrations of the pigment resulted in an inhibition of responses. At a pyocyanine concentration of 0.1 micrograms/ml or less the proliferation of T and B lymphocytes was enhanced, but at 0.5 micrograms/ml it was suppressed. IL-2 production by T lymphocytes was enhanced at concentrations up to 0.5 micrograms/ml but totally inhibited at 1.0 micrograms/ml. The differentiation of B lymphocytes to become immunoglobulin-producing cells was also enhanced in the presence of low doses of pyocyanine, whereas secretion of immunoglobulin by B lymphocytes was suppressed at all concentrations of pyocyanine. In contrast to the dual effects of pyocyanine on lymphocyte response, lipopolysaccharide-induced IL-1 and tumor necrosis factor release by monocytes was markedly enhanced by low as well as high concentrations of pyocyanine. From these results we conclude that this property of pyocyanine may lead to suppression of specific defense mechanisms and enhance harmful inflammatory reactions of the host during infection with Pseudomonas aeruginosa. PMID:2106495

  5. Both plasmacytoid dendritic cells and monocytes stimulate natural killer cells early during human herpes simplex virus type 1 infections.

    PubMed

    Vogel, Karin; Thomann, Sabrina; Vogel, Benjamin; Schuster, Philipp; Schmidt, Barbara

    2014-12-01

    Herpes simplex virus type 1 (HSV-1), a member of the herpes virus family, is characterized by a short replication cycle, high cytopathogenicity and distinct neurotropism. Primary infection and reactivation may cause severe diseases in immunocompetent and immunosuppressed individuals. This study investigated the role of human plasmacytoid dendritic cells (pDC) in the activation of natural killer (NK) cells for the control of herpesviral infections. Within peripheral blood mononuclear cells, UV-inactivated HSV-1 and CpG-A induced CD69 up-regulation on NK cells, whereas infectious HSV-1 was particularly active in inducing NK cell effector functions interferon-γ (IFN-γ) secretion and degranulation. The pDC-derived IFN-α significantly contributed to NK cell activation, as evident from neutralization and cell depletion experiments. In addition, monocyte-derived tumour necrosis factor-α (TNF-α) induced after exposure to infectious HSV-1 was found to stimulate IFN-γ secretion. A minority of monocytes was shown to be non-productively infected in experiments using fluorescently labelled viruses and quantitative PCR analyses. HSV-1-exposed monocytes up-regulated classical HLA-ABC and non-classical HLA-E molecules at the cell surface in an IFN-α-dependent manner, whereas stress molecules MICA/B were not induced. Notably, depletion of monocytes reduced NK cell effector functions induced by infectious HSV-1 (P < 0.05). Altogether, our data suggest a model in which HSV-1-stimulated pDC and monocytes activate NK cells via secretion of IFN-α and TNF-α. In addition, infection of monocytes induces NK cell effector functions via TNF-α-dependent and TNF-α-independent mechanisms. Hence, pDC and monocytes, which are among the first cells infiltrating herpetic lesions, appear to have important bystander functions for NK cells to control these viral infections.

  6. Sargaquinoic Acid Inhibits TNF-α-Induced NF-κB Signaling, Thereby Contributing to Decreased Monocyte Adhesion to Human Umbilical Vein Endothelial Cells (HUVECs).

    PubMed

    Gwon, Wi-Gyeong; Lee, Bonggi; Joung, Eun-Ji; Choi, Min-Woo; Yoon, Nayoung; Shin, Taisun; Oh, Chul-Woong; Kim, Hyeung-Rak

    2015-10-21

    Sargaquinoic acid (SQA) has been known for its antioxidant and anti-inflammatory properties. This study investigated the effects of SQA isolated from Sargassum serratifolium on the inhibition of tumor necrosis factor (TNF)-α-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). SQA decreased the expression of cell adhesion molecules such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as chemotactic cytokines such as interleukin-8 and monocyte chemoattractant protein-1 in TNF-α-treated HUVECs. As a result, SQA prevented monocyte adhesion to TNF-α-induced adhesion. SQA also inhibited TNF-α-induced nuclear factor kappa B (NF-κB) translocation into the nucleus by preventing proteolytic degradation of inhibitor κB-α. Overall, SQA protects against TNF-α-induced vascular inflammation through inhibition of the NF-κB pathway in HUVECs. These data suggest that SQA may be used as a therapeutic agent for vascular inflammatory diseases such as atherosclerosis.

  7. Membrane type 1-matrix metalloproteinase is involved in migration of human monocytes and is regulated through their interaction with fibronectin or endothelium.

    PubMed

    Matías-Román, Salomón; Gálvez, Beatriz G; Genís, Laura; Yáñez-Mó, María; de la Rosa, Gonzalo; Sánchez-Mateos, Paloma; Sánchez-Madrid, Francisco; Arroyo, Alicia G

    2005-05-15

    Membrane type 1-matrix metalloproteinase (MT1-MMP) is involved in endothelial and tumor-cell migration, but its putative role in leukocyte migration has not been characterized yet. Here, we demonstrate that anti-MT1-MMP monoclonal antibody (mAb) impaired monocyte chemotactic protein-1 (MCP-1)-stimulated monocyte migration on fibronectin (FN), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). In addition, monocyte transmigration through tumor necrosis factor-alpha (TNF-alpha)-activated endothelium is also inhibited by anti-MT1-MMP mAb. Therefore, regulation of MT1-MMP in human peripheral blood monocytes was investigated. First, MT1-MMP clustering was observed at motility-associated membrane protrusions of MCP-1-stimulated monocytes migrating on FN, VCAM-1, or ICAM-1 and at the leading edge, together with profilin, of monocytes transmigrating through activated endothelial cells. In addition, up-regulation of MT1-MMP expression was induced in human monocytes upon attachment to FN in a manner dependent on alpha4beta1 and alpha5beta1 integrins. Binding of monocytes to TNF-alpha-activated human endothelial cells as well as to VCAM-1 or ICAM-1 also resulted in an increase of MT1-MMP expression. These findings correlated with an enhancement of MT1-MMP fibrinolytic activity in monocytes bound to FN, VCAM-1, or ICAM-1. Our data show that MT1-MMP is required during human monocyte migration and endothelial transmigration and that MT1-MMP localization, expression, and activity are regulated in monocytes upon contact with FN or endothelial ligands, pointing to a key role of MT1-MMP in monocyte recruitment during inflammation.

  8. Minocycline ameliorates LPS-induced inflammation in human monocytes by novel mechanisms including LOX-1, Nur77 and LITAF inhibition

    PubMed Central

    Pang, Tao; Wang, Juan; Benicky, Julius; Saavedra, Juan M.

    2012-01-01

    Background Minocycline exhibits anti-inflammatory properties independent of its antibiotic activity, ameliorating inflammatory responses in monocytes and macrophages. However, the mechanisms of minocycline anti-inflammatory effects are only partially understood. Methods Human circulating monocytes were cultured in the presence of lipopolysaccharide (LPS), 50 ng/ml, and minocycline (10–40 µM). Gene expression was determined by RT-PCR, cytokine and prostaglandin E2 (PGE2) release by ELISA, protein expression, phosphorylation and nuclear translocation by Western blotting. Results Minocycline significantly reduced the inflammatory response in LPS-challenged monocytes, decreasing LPS-induced transcription of pro-inflammatory tumor-necrosis factor alpha (TNF-α), interleukin-1 beta, interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2), and the LPS-stimulated TNF-α, IL-6 and PGE2 release. Minocycline inhibited LPS-induced activation of the lectin-like oxidized low density lipoprotein receptor-1 (LOX-1), NF-κB, LPS-induced TNF-α factor (LITAF) and the Nur77 nuclear receptor. Mechanisms involved in the anti-inflammatory effects of minocycline include a reduction of LPS-stimulated p38 mitogen-activated protein kinase (p38 MAPK) activation and stimulation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Conclusions We provide novel evidence demonstrating that the anti-inflammatory effects of minocycline in human monocytes include, in addition to decreased NF-κB activation, abrogation of the LPS-stimulated LOX-1, LITAF, Nur77 pathways, p38 MAPK inhibition and PI3K/Akt activation. Our results reveal that minocycline inhibits points of convergence of distinct and interacting signaling pathways mediating multiple inflammatory signals which may influence monocyte activation, traffic and recruitment into the brain. General significance Our results in primary human monocytes contribute to explain the profound anti-inflammatory and protective effects of minocycline in

  9. Tumor necrosis factor alpha gene expression in human monocytic THP-1 cells exposed to beryllium.

    PubMed

    Galbraith, G M; Pandey, J P; Schmidt, M G; Arnaud, P; Goust, J M

    1996-01-01

    Chronic beryllium disease, which results from occupational exposure to particulate beryllium, is characterized by the development of lung granulomas and progressive pulmonary fibrosis. Increased production of proinflammatory cytokines (e.g., tumor necrosis factor alpha and interleukin-1 beta) by pulmonary alveolar macrophages occurs in many chronic fibrotic lung diseases and is thought to contribute to the disease process. The purpose of the present study was to investigate cytokine production by human monocytic cells exposed to beryllium in vitro. The results indicated that such cells respond to beryllium ions in the presence of fluoride by accumulation of messenger ribonucleic acid for both tumor necrosis factor alpha and interleukin-1 beta. These findings suggest that inhaled beryllium may directly stimulate the production of these cytokines by alveolar macrophages in vitro.

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

    PubMed

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

    2016-01-01

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

  11. A phased strategy to differentiate human CD14+monocytes into classically and alternatively activated macrophages and dendritic cells.

    PubMed

    Zarif, Jelani C; Hernandez, James R; Verdone, James E; Campbell, Scott P; Drake, Charles G; Pienta, Kenneth J

    2016-01-01

    There are currently several in vitro strategies to differentiate human CD14(+) monocytes isolated from peripheral blood mononuclear cells (PBMCs) into the M1 or M2 macrophage cell types. Each cell type is then verified using flow cytometric analysis of cell-surface markers. Human CD14(+) monocytes have the potential to differentiate into M1 and M2 macrophages, both of which demonstrate varying degrees of cell-surface antigen overlap. Using multiple surface markers with current macrophage polarization protocols, our data reveal several limitations of currently used methods, such as highly ambiguous cell types that possess cell-surface marker overlap and functional similarities. Utilizing interleukin-6 (IL-6) and two phases of cytokine exposure, we have developed a protocol to differentiate human monocytes into M1, M2, or dendritic cells (DCs) with greater efficiency and fidelity relative to macrophages and DCs that are produced by commonly used methods. This is achieved via alterations in cytokine composition, dosing, and incubation times, as well as improvements in verification methodology. Our method reliably reproduces human in vitro monocyte-derived DCs and macrophage models that will aid in better defining and understanding innate and adaptive immunity, as well as pathologic states.

  12. Leucine-Rich Repeat Kinase 2 Influences Fate Decision of Human Monocytes Differentiated from Induced Pluripotent Stem Cells.

    PubMed

    Speidel, Anna; Felk, Sandra; Reinhardt, Peter; Sterneckert, Jared; Gillardon, Frank

    2016-01-01

    Mutations in Leucine-rich repeat kinase 2 (LRRK2) are strongly associated with familial Parkinson's disease (PD). High expression levels in immune cells suggest a role of LRRK2 in regulating the immune system. In this study, we investigated the effect of the LRRK2 (G2019S) mutation in monocytes, using a human stem cell-derived model expressing LRRK2 at endogenous levels. We discovered alterations in the differentiation pattern of LRRK2 mutant, compared to non-mutant isogenic controls, leading to accelerated monocyte production and a reduction in the non-classical CD14+CD16+ monocyte subpopulation in the LRRK2 mutant cells. LPS-treatment of the iPSC-derived monocytes significantly increased the release of pro-inflammatory cytokines, demonstrating a functional response without revealing any significant differences between the genotypes. Assessment of the migrational capacity of the differentiated monocytes revealed moderate deficits in LRRK2 mutant cells, compared to their respective controls. Our findings indicate a pivotal role of LRRK2 in hematopoietic fate decision, endorsing the involvement of the immune system in the development of PD.

  13. Leucine-Rich Repeat Kinase 2 Influences Fate Decision of Human Monocytes Differentiated from Induced Pluripotent Stem Cells

    PubMed Central

    Felk, Sandra; Reinhardt, Peter; Sterneckert, Jared; Gillardon, Frank

    2016-01-01

    Mutations in Leucine-rich repeat kinase 2 (LRRK2) are strongly associated with familial Parkinson’s disease (PD). High expression levels in immune cells suggest a role of LRRK2 in regulating the immune system. In this study, we investigated the effect of the LRRK2 (G2019S) mutation in monocytes, using a human stem cell-derived model expressing LRRK2 at endogenous levels. We discovered alterations in the differentiation pattern of LRRK2 mutant, compared to non-mutant isogenic controls, leading to accelerated monocyte production and a reduction in the non-classical CD14+CD16+ monocyte subpopulation in the LRRK2 mutant cells. LPS-treatment of the iPSC-derived monocytes significantly increased the release of pro-inflammatory cytokines, demonstrating a functional response without revealing any significant differences between the genotypes. Assessment of the migrational capacity of the differentiated monocytes revealed moderate deficits in LRRK2 mutant cells, compared to their respective controls. Our findings indicate a pivotal role of LRRK2 in hematopoietic fate decision, endorsing the involvement of the immune system in the development of PD. PMID:27812199

  14. Cell Surface Expression and Function of the Macromolecular C1 Complex on the Surface of Human Monocytes

    PubMed Central

    Hosszu, Kinga K.; Valentino, Alisa; Ji, Yan; Matkovic, Mara; Pednekar, Lina; Rehage, Nina; Tumma, Nithin; Peerschke, Ellinor I. B.; Ghebrehiwet, Berhane

    2011-01-01

    The synthesis of the subunits of the C1 complex (C1q, C1s, C1r), and its regulator C1 inhibitor (C1-Inh) by human monocytes has been previously established. However, surface expression of these molecules by monocytes has not been shown. Using flow cytometry and antigen-capture enzyme-linked immunosorbent assay, we show here for the first time that, in addition to C1q, peripheral blood monocytes, and the monocyte-derived U937 cells express C1s and C1r, as well as Factor B and C1-Inh on their surface. C1s and C1r immunoprecipitated with C1q, suggesting that at least some of the C1q on these cells is part of the C1 complex. Furthermore, the C1 complex on U937 cells was able to trigger complement activation via the classical pathway. The presence of C1-Inh may ensure that an unwarranted autoactivation of the C1 complex does not take place. Since C1-Inh closely monitors the activation of the C1 complex in a sterile or infectious inflammatory environment, further elucidation of the role of C1 complex is crucial to dissect its function in monocyte, dendritic cell, and T cell activities, and its implications in host defense and tolerance. PMID:22566921

  15. Proteoglycans regulate the chemotaxis of dendritic cells derived from human peripheral blood monocytes.

    PubMed

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

    2010-01-01

    Dendritic cells (DCs) are a type of antigen-presenting cell which play an essential role in the immune system. The transition from immature DC (iDCs) to mature DCs (mDCs) requires appropriate maturation stimuli, such as pro-inflammatory cytokines or pathogen-derived components. Proteoglycans (PGs), which are composed of core proteins and the glycosaminoglycans that bind to them, are one of the main components of the extracellular matrix around pathogens such as bacteria. This study investigated the effects of PG extracted from the nasal septum cartilage of whale (W-PG) on the maturation of DCs derived from human peripheral blood monocytes. iDCs were prepared from human monocytes using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The iDCs were stimulated by W-PG alone. In another type of experiment, the iDCs were stimulated by MIX (tumor necrosis factor-alpha (TNF-alpha), IL-1beta, IL-6 and prostaglandin E(2) (PGE(2))) or a combination of MIX plus W-PG. The stimulation of W-PG alone did not induce the phenotypic maturation from iDCs. However, W-PG promoted the up-regulation of chemokine receptor CCR7-surface expression and the chemotactic responsiveness to CCR7 ligand macrophage inflammatory protein-3beta on MIX-stimulated mDCs although W-PG did not influence matrix metalloproteinase-9 activity which is an important factor in DC migration through the extracellular matrix. The findings that W-PG can selectively regulate the chemotactic activity of DCs in vitro under inflammatory conditions therefore indicate that the interaction of PGs with immune cells including DCs plays an important role in the immune response under the milieu of innate immunity.

  16. Inability of pyrogenic, purified Bordetella pertussis lipid A to induce interleukin-1 release by human monocytes.

    PubMed Central

    Caroff, M; Cavaillon, J M; Fitting, C; Haeffner-Cavaillon, N

    1986-01-01

    Free lipid A of Bordetella pertussis, Neisseria meningitidis, and Escherichia coli lipopolysaccharide (LPS) was prepared by hydrolysis in acetate buffer (pH 4.5); in addition, lipid A from B. pertussis and E. coli was prepared by hydrolysis in mineral acid (HCl). The precipitates obtained were purified by extraction methods in toluene-methanol and are referred to as crude lipid A. Purified lipid A from N. meningitidis and B. pertussis was obtained by extraction in a mixture of chloroform-methanol-water-triethylamine. The different preparations were tested for their pyrogenicity (endogenous pyrogen; EP) and their capacity to trigger the release of interleukin-1 (IL-1; previously known as lymphocyte-activating factor; LAF) by human monocytes. Crude lipid A from E. coli and N. meningitidis were both IL-1 inducers. Crude B. pertussis lipid A (acetate buffer; pH 4.5), which contains a beta-1-6-linked D-glucosamine disaccharide, two phosphoryl groups, and five fatty acids, was pyrogenic and an IL-1 inducer (EP+/LAF+); but crude B. pertussis lipid A (0.25 N HCl), which lacked the glycosidic phosphoryl group, was 1,000-fold less pyrogenic than the diphosphorylated lipid A, yet it retained its IL-1-inducing capacity (EP-/LAF+). Purified N. meningitidis lipid A was not an inducer of IL-1 release and purified B. pertussis lipid A exhibited identical pyrogenicity as the parent LPS but was devoid of any IL-1-release inducing capacity (EP+/LAF-). These results demonstrate that for some endotoxins, purified lipid A is unable to induce IL-1 release by human monocytes; however, it is pyrogenic, supporting the hypothesis that IL-1 and EP are induced by different determinants on the LPS molecule. Images PMID:2876960

  17. Flagella from Five Cronobacter Species Induce Pro-Inflammatory Cytokines in Macrophage Derivatives from Human Monocytes

    PubMed Central

    Cruz-Córdova, Ariadnna; Rocha-Ramírez, Luz M.; Ochoa, Sara A.; Gónzalez-Pedrajo, Bertha; Espinosa, Norma; Eslava, Carlos; Hernández-Chiñas, Ulises; Mendoza-Hernández, Guillermo; Rodríguez-Leviz, Alejandra; Valencia-Mayoral, Pedro; Sadowinski-Pine, Stanislaw; Hernández-Castro, Rigoberto; Estrada-García, Iris; Muñoz-Hernández, Onofre; Rosas, Irma; Xicohtencatl-Cortes, Juan

    2012-01-01

    Cronobacter spp. are opportunistic pathogens linked to lie-threatening infections in neonates and contaminated powdered infant formula that has been epidemiologically associated with these cases. Clinical symptoms of Cronobacter include necrotizing enterocolitis, bacteremia, and meningitis. Flagella from C. sakazakii are involved in biofilm formation and its adhesion to epithelial cells. We investigated the role of flagella from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis during the activation of cytokines (IL-8, TNF-α, and IL-10) in macrophage derivatives from human monocytes, which has not been extensively studied. The production and identity of flagella from the five Cronobacter species were visualized and recognized with anti-flagella antibodies by immunogold labeling through transmission electron microscopy. Purified flagella were dissociated into monomers in 12% SDS-PAGE Coomassie blue-stained gels showing a band of ∼28 kDa and, in addition, mass spectrometry revealed the presence of several peptides that correspond to flagellin. Flagella (100 ng) induced the release of IL-8 (3314–6025 pg/ml), TNF-α (39–359 pg/ml), and IL-10 (2–96 pg/ml), in macrophage isolates from human monocytes and similar results were obtained when flagella were dissociated into monomers. Inhibition assays using three dilutions of anti-flagella antibodies (1∶10, 1∶100, and 1∶200) suppressed the secretion of IL-8, TNF-α, and IL-10 between 95–100% using 100 ng of protein. A transfection assay using 293-hTLR5 cells showed IL-8 release of 197 pg/ml and suppression in the secretion of IL-8 when anti-hTLR5-IgA antibodies were used at different concentrations. These observations suggest that flagella and flagellin are involved in an inflammatory response dependent on TLR5 recognition, which could contribute to the pathogenesis of the bacteria. PMID:23284883

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

    PubMed

    Cruz-Córdova, Ariadnna; Rocha-Ramírez, Luz M; Ochoa, Sara A; González-Pedrajo, Bertha; Gónzalez-Pedrajo, Bertha; Espinosa, Norma; Eslava, Carlos; Hernández-Chiñas, Ulises; Mendoza-Hernández, Guillermo; Rodríguez-Leviz, Alejandra; Valencia-Mayoral, Pedro; Sadowinski-Pine, Stanislaw; Hernández-Castro, Rigoberto; Estrada-García, Iris; Muñoz-Hernández, Onofre; Rosas, Irma; Xicohtencatl-Cortes, Juan

    2012-01-01

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

  19. Surface expression of Mo3e antigen by activated human monocytes and U-937 cells

    SciTech Connect

    Todd R.F. III; Bury, M.J.; Liu, D.Y.

    1986-03-05

    The surface expression of a protease-sensitive antigen, Mo3e, by activated human monocytes and U-937 cells is a plasma membrane feature of the activated state. Mo3e, which is an 80 kD protein on Western blot analysis, may represent the surface receptor for migration inhibitory factor (MIF), as evidenced by inhibition of MIF responsiveness produced by anti-Mo3e monoclonal antibody. Mo3e is barely detectable (by surface immunofluorescence) on freshly isolated monocytes but becomes expressed in high antigen density during 18-24 hrs culture in medium containing E. coli lipopolysaccharide (> 1 ng/ml), 4..beta..-phorbol 12-myristate 13-acetate (PMA) (5-10 nM), or muramyl dipeptide (0.1-1 ..mu..M). In U-937 cells, Mo3e surface expression is detectable after 24 hrs exposure to PMA and other pharmacological activators of protein kinase C: 4..beta..-phorbol 12, 13 dibutyrate, 4..beta..-phorbol 12, 13 didecanoate, mezerein, or Sn-1,2-dioctanoylglycerol. The biologically-inactivate phorbol compounds, 4..cap alpha..-phorbol 12, 13 didecanoate and 4/sub ..beta../-phorbol do not stimulate Mo3e expression. The calcium ionophore, ionomycin, has a synergistic effect on Mo3e expression stimulated by PMA; conversely, calcium antagonists block PMA-induced Mo3e expression. These results suggest the involvement of protein kinase C activation and intracellular calcium mobilization in the stimulated expression of Mo3e by activated human mononuclear phagocytes.

  20. Salvianolic acid B suppresses maturation of human monocyte-derived dendritic cells by activating PPARγ

    PubMed Central

    Sun, Aijun; Liu, Hongying; Wang, Shijun; Shi, Dazhuo; Xu, Lei; Cheng, Yong; Wang, Keqiang; Chen, Keji; Zou, Yunzeng; Ge, Junbo

    2011-01-01

    BACKGROUND AND PURPOSE Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is known to be effective in the prevention of atherosclerosis. Here, we tested the hypothesis that the anti-atherosclerotic effect of Sal B might be mediated by suppressing maturation of human monocyte-derived dendritic cells (h-monDC). EXPERIMENTAL APPROACH h-monDC were derived by incubating purified human monocytes with GM-CSF and IL-4. h-monDC were pre-incubated with or without Sal B and stimulated by oxidized low-density lipoprotein (ox-LDL) in the presence or absence of PPARγ siRNA. Expression of h-monDC membrane molecules (CD40, CD86, CD1a, HLA-DR) were analysed by FACS, cytokines were measured by elisa and the TLR4-associated signalling pathway was determined by Western blotting. KEY RESULTS Ox-LDL promoted h-monDC maturation, stimulated CD40, CD86, CD1a, HLA-DR expression and IL-12, IL-10, TNF-α production; and up-regulated TLR4 signalling. These effects were inhibited by Sal B. Sal B also triggered PPARγ activation and promoted PPARγ nuclear translocation, attenuated ox-LDL-induced up-regulation of TLR4 and myeloid differentiation primary-response protein 88 and inhibited the downstream p38-MAPK signalling cascade. Knocking down PPARγ with the corresponding siRNA blocked these effects of Sal B. CONCLUSIONS AND IMPLICATIONS Our data suggested that Sal B effectively suppressed maturation of h-monDC induced by ox-LDL through PPARγ activation. PMID:21649636

  1. Geranylated flavanone tomentodiplacone B inhibits proliferation of human monocytic leukaemia (THP-1) cells

    PubMed Central

    Kollár, Peter; Bárta, Tomáš; Závalová, Veronika; Šmejkal, Karel; Hampl, Aleš

    2011-01-01

    BACKGROUND AND PURPOSE Paulownia tomentosa is a rich source of geranylated flavanones, some of which we have previously shown to have cytotoxic activity. To identify members of this class of compounds with cytostatic effects, we assessed the effects of the geranylated flavanone tomentodiplacone B (TOM B) on cell cycle progression and cell cycle regulatory pathways of THP-1 human monocytic leukaemia cells. EXPERIMENTAL APPROACH Cell viability was measured by dye exclusion and proliferation by WST-1 assays; cell cycle was monitored by flow cytometry. Regulatory proteins were assessed by immunoprecipitation and kinase assays, and Western blotting. KEY RESULTS Tomentodiplacone B had no effect during the first 24 h of cell growth at concentrations between 1 and 2.5 µM, but inhibited cell growth in a dose-dependent manner at concentrations of 5 µM or higher. Growth inhibition during the first 24 h of exposure to TOM B was not accompanied by cytotoxicity as cells were accumulated in G1 phase dose-dependently. This G1 phase accumulation was associated with down-regulation of cyclin-dependent kinase 2 activity and also protein levels of cyclins E1 and A2. However, key stress-related molecules (γ-H2AX, p53 and p21) were not induced, suggesting that TOM B acts by directly inhibiting the cyclin-dependent kinase 2 signalling pathway rather than initiating DNA damage or cellular stress. CONCLUSIONS AND IMPLICATIONS Our study provides the first evidence that TOM B directly inhibits proliferation of human monocytic leukaemia cells, and thus is a potential anticancer agent, preventing leukaemia cells from progressing from G1 phase into DNA synthesis. PMID:21175584

  2. The urokinase receptor is required for human monocyte chemotaxis in vitro.

    PubMed Central

    Gyetko, M R; Todd, R F; Wilkinson, C C; Sitrin, R G

    1994-01-01

    Mononuclear phagocytes (Mphi) produce urokinase-type plasminogen activator (uPA) and also express a specific cell-surface receptor for urokinase, uPAR. The concomitant expression of these proteins provides a mechanism by which Mphi can degrade extracellular matrix proteins during directed cell migration. In this study, we sought to determine if uPAR plays a role in Mphi chemotaxis that is distinct from its role in matrix proteolysis. Exposing adherent monocytes to a chemotactic gradient causes plasma membrane uPAR to localize strongly to the leading edge of cell migration. Adherence alone or exposure to FMLP had no effect on uPAR expression. Using Boyden chamber chemotaxis assays, we demonstrate that treating mononuclear cells with an anti-uPAR mAb (either as an intact mAb or F[ab']2) ablates chemotaxis induced by FMLP and monocyte chemotactic peptide-1 (P < 0.001). Inactivating the catalytic activity of uPAR-bound uPA had no effect on chemotaxis. Similarly, blocking uPAR expression with an antisense oligonucleotide to uPAR completely ablates chemotaxis, but blocking uPA expression with an antisense oligonucleotide to uPA has a minimal effect. We therefore demonstrate that expression and unimpeded function of uPAR plays an obligate role in M phi chemotaxis by mechanisms that are largely independent of its ligand, uPA. Combined with its known role in mediating pericellular proteolysis, these observations demonstrate that uPAR is essential for both locomotion and traversing tissue barriers during M phi migration. Images PMID:8163642

  3. The monocyte locomotion inhibitory factor produced by Entamoeba histolytica inhibits induced nitric oxide production in human leukocytes.

    PubMed

    Rico, G; Leandro, E; Rojas, S; Giménez, J A; Kretschmer, R R

    2003-07-01

    The monocyte locomotion inhibitory factor, an anti-inflammatory pentapeptide produced by Entamoeba histolytica, inhibits the in vitro production of nitric oxide induced by cytokines (INF-gamma, TNF-alpha) or PMA in human leukocytes. This can be added to the other previously reported functional effects of this factor, such as the inhibition of monocyte locomotion and the synthesis of reactive oxygen intermediates in both monocytes and neutrophils. The decreased nitric oxide production may interfere with the killing of amebas by neutrophils in the early invasive stages of amebiasis, when oxidative mechanisms are used [reactive oxygen and nitrogen intermediates either individually or synergistically via peroxynitrite (ONOO(-))], and in the advanced stages, when both non-oxidative and oxidative (including nitric oxide) mechanisms are employed by macrophages. Diminished nitric oxide production by leukocytes may also contribute to the paucity of late inflammatory components in amebic abscess of the liver and other amebic lesions.

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

    PubMed Central

    2013-01-01

    Background Bacterial invasion through the blood-cerebrospinal fluid barrier (BCSFB) during bacterial meningitis causes secretion of proinflammatory cytokines/chemokines followed by the recruitment of leukocytes into the CNS. In this study, we analyzed the cellular and molecular mechanisms of polymorphonuclear neutrophil (PMN) and monocyte transepithelial transmigration (TM) across the BCSFB after bacterial infection. Methods Using an inverted transwell filter system of human choroid plexus papilloma cells (HIBCPP), we studied leukocyte TM rates, the migration route by immunofluorescence, transmission electron microscopy and focused ion beam/scanning electron microscopy, the secretion of cytokines/chemokines by cytokine bead array and posttranslational modification of the signal regulatory protein (SIRP) α via western blot. Results PMNs showed a significantly increased TM across HIBCPP after infection with wild-type Neisseria meningitidis (MC58). In contrast, a significantly decreased monocyte transmigration rate after bacterial infection of HIBCPP could be observed. Interestingly, in co-culture experiments with PMNs and monocytes, TM of monocytes was significantly enhanced. Analysis of paracellular permeability and transepithelial electrical resistance confirmed an intact barrier function during leukocyte TM. With the help of the different imaging techniques we could provide evidence for para- as well as for transcellular migrating leukocytes. Further analysis of secreted cytokines/chemokines showed a distinct pattern after stimulation and transmigration of PMNs and monocytes. Moreover, the transmembrane glycoprotein SIRPα was deglycosylated in monocytes, but not in PMNs, after bacterial infection. Conclusions Our findings demonstrate that PMNs and monoctyes differentially migrate in a human BCSFB model after bacterial infection. Cytokines and chemokines as well as transmembrane proteins such as SIRPα may be involved in this process. PMID:23448224

  5. TGF-beta and vitamin D3 utilize distinct pathways to suppress IL-12 production and modulate rapid differentiation of human monocytes into CD83+ dendritic cells.

    PubMed

    Lyakh, Lyudmila A; Sanford, Michael; Chekol, Sebel; Young, Howard A; Roberts, Anita B

    2005-02-15

    We previously demonstrated that agents known to signal infection or inflammation can rapidly and directly drive differentiation of human CD14+ monocytes into CD83+ dendritic cells (DCs) when introduced to cells under serum-free conditions. In this study, we evaluated the effects of TGF-beta and vitamin D3 (VitD3) on the proportion and function of monocytes that adopt DC characteristics. TGF-beta significantly decreased the proportion of cells that rapidly adopted stable DC characteristics in response to LPS, but had little or no effect on calcium ionophore-induced differentiation. In contrast, VitD3 showed no such pathway specificity and dramatically suppressed differentiation of monocytes into DCs in response to these agents. Both TGF-beta and VitD3 altered cytokine and chemokine production in LPS-treated monocytes, inhibited IL-12 and IL-10 secretion, and decreased the functional capacity of DCs. Despite the similar effects of TGF-beta and VitD3, there are significant differences in the signaling pathways used by these agents, as evidenced by their distinct effects on LPS- and calcium ionophore-induced DC differentiation, on LPS-induced secretion of IL-10, and on two members of the NF-kappaB family of transcription factors, RelB and cRel. These studies identify TGF-beta and VitD3 as potent regulatory factors that use distinct pathways to suppress both the differentiation of DCs as well as their capacity to secrete the Th1-polarizing cytokine IL-12. Because these agents are present in serum and negatively affect DC differentiation at physiological concentrations, our findings are likely to have significance regarding the in vivo role of TGF-beta and VitD3 in determining the type of immune responses.

  6. Human monocyte-derived soluble product(s) has an accessory function in the generation of histamine- and concanavalin A-induced suppressor T cells.

    PubMed

    Beer, D J; Dinarello, C A; Rosenwasser, L J; Rocklin, R E

    1982-08-01

    We have analyzed the cellular interactions required for the generation of histamine- and concanavalin A (Con A)-induced suppressor T cells by employing a co-culture assay and techniques for fractionation of human blood mononuclear cells (PBMC). PBMC cultured in the presence of histamine (0.1 mM-1 mM) or Con A (20 micrograms/ml) for 24 h, mitomycin treated and subsequently combined with autologous mitogen-stimulated mononuclear cells, significantly suppressed a subsequent blastogenic response. PBMC fractionated over nylon wool columns and depleted of adherent cells and enriched for T cells (NWNA-T) were unable to generate suppressor activity. However, suppressor cell function by NWNA-T cells was reconstituted by the addition of autologous monocytes. In both the histamine and ConA suppressor systems, the requirement for monocytes in the activation process was enhanced by suspending the NWNA-T population in supernatants derived from allogeneic monocytes stimulated with heat-killed Staphylococcus albus. These crude supernatants contained leukocytic pyrogen (LP) and lymphocyte activating factor (LAF). Sequential purification and separation of the crude supernatants using gel-filtration, immunoadsorption, and isoelectric focusing demonstrated that only those fractions containing LP and LAF were capable to reconstituting NWNA-T cell histamine and Con A-induced suppressor activity. Thus, these studies suggest that the accessory role of supernatants derived from activated monocytes in the generation of suppressor cells may be mediated by LP/LAF. Further studies are in progress to explore the mechanism by which soluble factors stimulate suppressor T cells.

  7. Assessing the Immunosafety of Engineered Nanoparticles with a Novel in Vitro Model Based on Human Primary Monocytes.

    PubMed

    Li, Yang; Italiani, Paola; Casals, Eudald; Valkenborg, Dirk; Mertens, Inge; Baggerman, Geert; Nelissen, Inge; Puntes, Victor F; Boraschi, Diana

    2016-10-13

    The possibility that nanomaterials could perturb the normal course of an inflammatory response is a key issue when assessing nanoimmunosafety. The alteration of the normal progress of an inflammatory response may have pathological consequences, since inflammation is a major defensive mechanism and its efficiency maintains the body's health. The immunosafety of engineered nanoparticles at nontoxic concentrations was investigated with the use of a human primary monocyte-based in vitro system, which reproduces in a simplified fashion the full course of the physiological inflammatory response, from initiation and development to resolution. The kinetics of expression and production of inflammatory and anti-inflammatory cytokines and the proteomic profiles were used for describing the inflammatory defensive response. We assessed the ability of gold and silver nanoparticles to trigger inflammation and to interfere with the course of an ongoing defensive reaction. While neither nanoparticle type was able to directly activate monocytes, silver nanoparticles could exacerbate the inflammatory response of monocytes but did not interfere with the resolution of the inflammatory reaction. These findings support the use of human primary monocyte-based in vitro assays for realistically investigating the effects of engineered nanoparticles on human innate immune responses, in order to predict the immunological risk of nanomaterials and implement safe nanoparticle-based applications.

  8. Stable extracellular RNA fragments of Mycobacterium tuberculosis induce early apoptosis in human monocytes via a caspase-8 dependent mechanism.

    PubMed

    Obregón-Henao, Andrés; Duque-Correa, María A; Rojas, Mauricio; García, Luis F; Brennan, Patrick J; Ortiz, Blanca L; Belisle, John T

    2012-01-01

    The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms. Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis.

  9. Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism

    PubMed Central

    Obregón-Henao, Andrés; Duque-Correa, María A.; Rojas, Mauricio; García, Luis F.; Brennan, Patrick J.; Ortiz, Blanca L.; Belisle, John T.

    2012-01-01

    The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms. Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis. PMID:22253841

  10. Monocyte procoagulant activity and plasminogen activator. Role in human renal allograft rejection

    SciTech Connect

    Cole, E.H.; Cardella, C.J.; Schulman, J.; Levy, G.A.

    1985-10-01

    Currently the mechanism of renal allograft rejection is not well understood. This study was designed to determine whether induction of monocyte procoagulant activity (MCPA) is important in the pathogenesis of renal allograft rejection. The MPCA assay was performed utilizing a one stage clotting assay both in normal and in factor-VII-deficient plasma. There was no increase in spontaneous MPCA in 20 patients with endstage renal failure and in 10 patients following abdominal or orthopedic operation, as compared with 20 normal controls. MPCA was assessed daily in 18 patients who had received renal allografts. Rejection episodes (RE) were predicted on the basis of persistent elevation in MPCA as compared with pretransplant levels. Rejection was diagnosed clinically and treated on the basis of standard criteria. Treated RE were compared with those predicted by elevated MPCA, and 3 patients were assessed as having no RE by MPCA and by standard criteria. In 8 RE, MPCA correlated temporally with RE (same day) when compared with standard criteria. In 12 RE, MPCA was predictive of rejection preceding standard criteria by at least 24 hr. There were 7 false-positive predictions on the basis of MPCA; however, there was only 1 false negative. MPCA was shown to be a prothrombinase by its dependence only on prothrombin and fibrinogen for full activity. MPCA may be important in the pathogenesis of allograft rejection, and additionally it may be a useful adjunct in the clinical management of this disease.

  11. Microbial Pattern Recognition Causes Distinct Functional Micro-RNA Signatures in Primary Human Monocytes

    PubMed Central

    Häsler, Robert; Jacobs, Gunnar; Till, Andreas; Grabe, Nils; Cordes, Christian; Nikolaus, Susanna; Lao, Kaiqin

    2012-01-01

    Micro-RNAs (miRNAs) are short, non-coding RNAs that regulate gene expression post transcriptionally. Several studies have demonstrated the relevance of miRNAs for a wide range of cellular mechanisms, however, the current knowledge on how miRNAs respond to relevant external stimuli, e.g. in disease scenarios is very limited. To generate a descriptive picture of the miRNA network associated to inflammatory responses, we quantified the levels of 330 miRNAs upon stimulation with a panel of pro-inflammatory components such as microbial pattern molecules (flagellin, diacylated lipopeptide lipopolysaccharide, muramyl dipeptide), infection with Listeria monocytogenes and TNF-α as pro-inflammatory control in primary human monocytes using real time PCR. As a result, we found distinct miRNA response clusters for each stimulus used. Additionally, we identified potential target genes of three selected miRNAs miR-129-5p, miR-146a and miR-378 which were part of PAMP-specific response clusters by transfecting THP1 monocytes with the corresponding pre- or anti-miRNAs and microfluidic PCR arrays. The miRNAs induced distinct transcriptomal signatures, e.g. overexpression of miRNA129-5p, which was selectively upregulated by the NOD2-elicitor MDP, led to an upregulation of DEFB1, IRAK1, FBXW7 and IKK γ (Nemo). Our findings on highly co-regulated clusters of miRNAs support the hypothesis that miRNAs act in functional groups. This study indicates that miRNAs play an important role in fine-tuning inflammatory mechanisms. Further investigation in the field of miRNA responses will help to understand their effects on gene expression and may close the regulatory gap between mRNA and protein expression in inflammatory diseases. PMID:22363568

  12. RANK Expression and Osteoclastogenesis in Human Monocytes in Peripheral Blood from Rheumatoid Arthritis Patients

    PubMed Central

    Kobashigawa, Tsuyoshi

    2016-01-01

    Rheumatoid arthritis (RA) appears as inflammation of synovial tissue and joint destruction. Receptor activator of NF-κB (RANK) is a member of the TNF receptor superfamily and a receptor for the RANK ligand (RANKL). In this study, we examined the expression of RANKhigh and CCR6 on CD14+ monocytes from patients with RA and healthy volunteers. Peripheral blood samples were obtained from both the RA patients and the healthy volunteers. Osteoclastogenesis from monocytes was induced by RANKL and M-CSF in vitro. To study the expression of RANKhigh and CCR6 on CD14+ monocytes, two-color flow cytometry was performed. Levels of expression of RANK on monocytes were significantly correlated with the level of osteoclastogenesis in the healthy volunteers. The expression of RANKhigh on CD14+ monocyte in RA patients without treatment was elevated and that in those receiving treatment was decreased. In addition, the high-level expression of RANK on CD14+ monocytes was correlated with the high-level expression of CCR6 in healthy volunteers. Monocytes expressing both RANK and CCR6 differentiate into osteoclasts. The expression of CD14+RANKhigh in untreated RA patients was elevated. RANK and CCR6 expressed on monocytes may be novel targets for the regulation of bone resorption in RA and osteoporosis. PMID:27822475

  13. Lipooligosaccharide from Bordetella pertussis induces mature human monocyte-derived dendritic cells and drives a Th2 biased response.

    PubMed

    Fedele, Giorgio; Celestino, Ignacio; Spensieri, Fabiana; Frasca, Loredana; Nasso, Maria; Watanabe, Mineo; Remoli, Maria Elena; Coccia, Eliana Marina; Altieri, Fabio; Ausiello, Clara Maria

    2007-06-01

    Bordetella pertussis has a distinctive cell wall lipooligosaccharide (LOS) that is released from the bacterium during bacterial division and killing. LOS directly participates in host-bacterial interactions, in particular influencing the dendritic cells' (DC) immune regulatory ability. We analyze LOS mediated toll-like receptor (TLR) activation and dissect the role played by LOS on human monocyte-derived (MD)DC functions and polarization of the host T cell response. LOS activates TLR4-dependent signaling and induces mature MDDC able to secrete IL-10. LOS-matured MDDC enhance allogeneic presentation and skew T helper (Th) cell polarization towards a Th2 phenotype. LOS protects MDDC from undergoing apoptosis, prolonging their longevity and their functions. Compared to Escherichia coli lipopolysaccharide (LPS), the classical DC maturation stimulus, LOS was a less efficient inducer of TLR4 signaling, MDDC maturation, IL-10 secretion and allogeneic T cell proliferation and it was not able to induce IL-12p70 production in MDDC. However, the MDDC apoptosis protection exerted by LOS and LPS were comparable. In conclusion, LOS treated MDDC are able to perform antigen presentation in a context that promotes licensing of Th2 effectors. Considering these properties, the use of LOS in the formulation of acellular pertussis vaccines to potentiate protective and adjuvant capacity should be taken into consideration.

  14. Tumor-Associated Monocytes/Macrophages Impair NK-Cell Function via TGFβ1 in Human Gastric Cancer.

    PubMed

    Peng, Liu-Sheng; Zhang, Jin-Yu; Teng, Yong-Sheng; Zhao, Yong-Liang; Wang, Ting-Ting; Mao, Fang-Yuan; Lv, Yi-Pin; Cheng, Ping; Li, Wen-Hua; Chen, Na; Duan, Mubing; Chen, Weisan; Guo, Gang; Zou, Quan-Ming; Zhuang, Yuan

    2017-03-01

    Natural killer (NK) cells are a major component of the host antitumor immune response in human cancer. However, the nature, functional regulation, and clinical relevance of NK cells in gastric cancer remain largely unknown. In this study, we showed that the percentages of NK cells in tumors were significantly decreased, and low percentages of tumor-infiltrating NK cells were positively correlated with poor survival and disease progression. Although the expression of activating and inhibitory receptors on NK cells was shown to be not different between tumor and nontumor tissues, NK cells in tumors had impaired effector functions, characterized by decreased IFNγ, TNFα, and Ki-67 expression. We found that tumor-infiltrating monocytes/macrophages were physically close to NK cells, and their percentages negatively correlated with IFNγ(+) and TNFα(+) NK-cell percentages. Ex vivo study showed that isolated tumor-associated monocytes/macrophages could impair NK-cell expression of IFNγ, TNFα, and Ki-67. Blockade of TGFβ1 attenuated such monocytes/macrophages-mediated impairment of NK-cell function. Our data suggest that human NK-cell function was impaired by tumor-associated monocytes/macrophages, and that restoring NK-cell function may be an important therapeutic strategy to prevent tumor immune escape in gastric cancer. Cancer Immunol Res; 5(3); 248-56. ©2017 AACR.

  15. Soluble CD100 functions on human monocytes and immature dendritic cells require plexin C1 and plexin B1, respectively.

    PubMed

    Chabbert-de Ponnat, Isabelle; Marie-Cardine, Anne; Pasterkamp, R Jeroen; Schiavon, Valérie; Tamagnone, Luca; Thomasset, Nicole; Bensussan, Armand; Boumsell, Laurence

    2005-04-01

    CD100 represents the first semaphorin described in the immune system. It is expressed as a 300-kDa homodimer at the surface of most hematopoietic cells, but is also found in a soluble form following a proteolytic cleavage upon cell activation. We herein established that soluble CD100 (sCD100) impaired the migration of human monocytes and immature dendritic cells (DCs), but not of mature DCs. Performing competition assays, we identified plexin C1 (VESPR/CD232) as being involved in sCD100-mediated effects on human monocytes. Interestingly, we observed a complete down-regulation of plexin C1 expression during the in vitro differentiation process of monocytes to immature DCs, while concomitantly the surface expression of plexin B1 was induced. The latter receptor then binds sCD100 on immature DCs, mediating its inhibitory effect on cell migration. Finally, we showed that sCD100 modulated the cytokine production from monocytes and immature DCs. Together these results suggest that sCD100 plays a critical role in the regulation of antigen-presenting cell migration and functions via a tightly regulated process of receptor expression.

  16. Induction of heme oxygenase 1 by arsenite inhibits cytokine-induced monocyte adhesion to human endothelial cells

    SciTech Connect

    Sun Xi; Pi Jingbo; Liu Wenlan; Hudson, Laurie G.; Liu Kejian; Feng Changjian

    2009-04-15

    Heme oxygenase-1 (HO-1) is an oxidative stress responsive gene upregulated by various physiological and exogenous stimuli. Arsenite, as an oxidative stressor, is a potent inducer of HO-1 in human and rodent cells. In this study, we investigated the mechanistic role of arsenite-induced HO-1 in modulating tumor necrosis factor {alpha} (TNF-{alpha}) induced monocyte adhesion to human umbilical vein endothelial cells (HUVEC). Arsenite pretreatment, which upregulated HO-1 in a time- and concentration-dependent manner, inhibited TNF-{alpha}-induced monocyte adhesion to HUVEC and intercellular adhesion molecule 1 protein expression by 50% and 40%, respectively. Importantly, knockdown of HO-1 by small interfering RNA abolished the arsenite-induced inhibitory effects. These results indicate that induction of HO-1 by arsenite inhibits the cytokine-induced monocyte adhesion to HUVEC by suppressing adhesion molecule expression. These findings established an important mechanistic link between the functional monocyte adhesion properties of HUVEC and the induction of HO-1 by arsenite.

  17. Atorvastatin promotes human monocyte differentiation toward alternative M2 macrophages through p38 mitogen-activated protein kinase-dependent peroxisome proliferator-activated receptor γ activation.

    PubMed

    Zhang, Ou; Zhang, Jinying

    2015-05-01

    M1 and M2 macrophages are detectable in human atherosclerotic lesions, and M2 macrophages are present at locations distant from the lipid core in more stable zones of the plaque and appear to exert anti-inflammatory properties on M1 macrophages. Peroxisome proliferator-activated receptor (PPAR) γ promotes the differentiation of monocytes into anti-inflammatory M2 macrophages. Although both statins and PPARγ ligands have been reported to protect against the progression of atherosclerosis, no data are currently available regarding the implication of statins in the alternative differentiation of human monocytes. In the present study, we hypothesized that atorvastatin may exert novel effects to prime human monocytes toward an anti-inflammatory alternative M2 phenotype. To this aim, we first found that abundant M2 markers were expressed in human circulating monocytes after atorvastatin treatment. Moreover, atorvastatin was able to induce PPARγ expression and activation in human monocytes in vivo and in vitro, resulting in priming primary human monocytes differentiation into M2 macrophages with a more pronounced paracrine anti-inflammatory activity in M1 macrophages. Additional data with molecular approaches revealed that p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase (ERK) 1/2 activation was involved in atorvastatin-mediated PPARγ activation and enhanced alternative M2 macrophage phenotype. Collectively, our data demonstrated that atorvastatin promotes human monocyte differentiation toward alternative M2 macrophages via p38 MAPK-dependent PPARγ activation.

  18. Differential Effects of Vitamins A and D on the Transcriptional Landscape of Human Monocytes during Infection

    PubMed Central

    Klassert, Tilman E.; Bräuer, Julia; Hölzer, Martin; Stock, Magdalena; Riege, Konstantin; Zubiría-Barrera, Cristina; Müller, Mario M.; Rummler, Silke; Skerka, Christine; Marz, Manja; Slevogt, Hortense

    2017-01-01

    Vitamin A and vitamin D are essential nutrients with a wide range of pleiotropic effects in humans. Beyond their well-documented roles in cellular differentiation, embryogenesis, tissue maintenance and bone/calcium homeostasis, both vitamins have attracted considerable attention due to their association with-immunological traits. Nevertheless, our knowledge of their immunomodulatory potential during infection is restricted to single gene-centric studies, which do not reflect the complexity of immune processes. In the present study, we performed a comprehensive RNA-seq-based approach to define the whole immunomodulatory role of vitamins A and D during infection. Using human monocytes as host cells, we characterized the differential role of both vitamins upon infection with three different pathogens: Aspergillus fumigatus, Candida albicans and Escherichia coli. Both vitamins showed an unexpected ability to counteract the pathogen-induced transcriptional responses. Upon infection, we identified 346 and 176 immune-relevant genes that were regulated by atRA and vitD, respectively. This immunomodulatory activity was dependent on the inflammatory stimulus, allowing us to distinguish regulatory patterns which were specific for each stimulatory setting. Moreover, we explored possible direct and indirect mechanisms of vitamin-mediated regulation of the immune response. Our findings highlight the importance of vitamin-monitoring in critically ill patients. Moreover, our results underpin the potential of atRA and vitD as therapeutic options for anti-inflammatory treatment. PMID:28094291

  19. Differential Effects of Vitamins A and D on the Transcriptional Landscape of Human Monocytes during Infection.

    PubMed

    Klassert, Tilman E; Bräuer, Julia; Hölzer, Martin; Stock, Magdalena; Riege, Konstantin; Zubiría-Barrera, Cristina; Müller, Mario M; Rummler, Silke; Skerka, Christine; Marz, Manja; Slevogt, Hortense

    2017-01-17

    Vitamin A and vitamin D are essential nutrients with a wide range of pleiotropic effects in humans. Beyond their well-documented roles in cellular differentiation, embryogenesis, tissue maintenance and bone/calcium homeostasis, both vitamins have attracted considerable attention due to their association with-immunological traits. Nevertheless, our knowledge of their immunomodulatory potential during infection is restricted to single gene-centric studies, which do not reflect the complexity of immune processes. In the present study, we performed a comprehensive RNA-seq-based approach to define the whole immunomodulatory role of vitamins A and D during infection. Using human monocytes as host cells, we characterized the differential role of both vitamins upon infection with three different pathogens: Aspergillus fumigatus, Candida albicans and Escherichia coli. Both vitamins showed an unexpected ability to counteract the pathogen-induced transcriptional responses. Upon infection, we identified 346 and 176 immune-relevant genes that were regulated by atRA and vitD, respectively. This immunomodulatory activity was dependent on the inflammatory stimulus, allowing us to distinguish regulatory patterns which were specific for each stimulatory setting. Moreover, we explored possible direct and indirect mechanisms of vitamin-mediated regulation of the immune response. Our findings highlight the importance of vitamin-monitoring in critically ill patients. Moreover, our results underpin the potential of atRA and vitD as therapeutic options for anti-inflammatory treatment.

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

    PubMed

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

    2014-09-09

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

  1. Molecular characteristics of cytochrome b558 isolated from human granulocytes, monocytes and HL60 and U937 cells differentiated into monocyte/macrophages.

    PubMed

    Capeillere-Blandin, C; Masson, A; Descamps-Latscha, B

    1991-08-13

    Enriched cytochrome b558 preparations were obtained from human mature monocytes (MN) and retinoic acid plus interferon gamma induced human myeloid leukemia cell lines HL-60 and U937, using an adaptation of the procedure described by A.W. Segal (Nature (1987) 326, 88-91) for purification of cytochrome b558 from human polymorphonuclear leukocytes (PMN). Spectral characteristics of cytochrome b558 were determined and found to be independent of cell type and specific heme b content of the preparation. To increase the sensitivity of the spectral assay, analysis in the gamma band were used and delta epsilon 427-413 was determined to be equal to 158 mM-1 cm-1. An alpha beta type heterodimeric cytochrome b558 was found for PMN and MN by the concordant elution of heme b spectral activity from heparin agarose and the detection of two polypeptide chains by SDS-PAGE. The expression of the lighter polypeptide alpha chain in the various human monocyte-like cell lines was assessed and its identity, as a component of cytochrome b, was confirmed by immunodetection using a rabbit polyclonal antibody reacting with the alpha subunit of PMN cytochrome b558. Immunoblotting studies detected the alpha subunit in monocyto-macrophagic differentiated HL-60 and U 937 cells and mature MN at 22 kDa, but not in uninduced cells which did not express the respiratory burst. Whatever the specific content or the cell origin of the cytochrome b558-enriched preparations, the heme b binding site was shown to be associated with the alpha subunit defined by a constant molecular mass of 22 kDa, as evidenced by the finding of a constant ratio between the silver stained band intensity and the corresponding heme b amount. The heavy polypeptide beta chain from MN cytochrome b was found to have a significantly higher molecular weight than the beta subunit from PMN at 94 +/- 5 kDa instead of 90 +/- 4 kDa. In contrast, in cytochrome b preparations from induced monocyto-macrophagic cells, isolated with a low heme

  2. Phenotypic and functional characterization of macrophages with therapeutic potential generated from human cirrhotic monocytes in a cohort study

    PubMed Central

    Moore, Joanna K.; Mackinnon, Alison C.; Wojtacha, Dvina; Pope, Caroline; Fraser, Alasdair R.; Burgoyne, Paul; Bailey, Laura; Pass, Chloe; Atkinson, Anne; Mcgowan, Neil W.A.; Manson, Lynn; Turner, Mark L.; Campbell, John D.M.; Forbes, Stuart J.

    2015-01-01

    Background aims Macrophages have complex roles in the liver. The aim of this study was to compare profiles of human monocyte-derived macrophages between controls and cirrhotic patients, to determine whether chronic inflammation affects precursor number or the phenotype, with the eventual aim to develop a cell therapy for cirrhosis. Methods Infusion of human macrophages in a murine liver fibrosis model demonstrated a decrease in markers of liver injury (alanine transaminase, bilirubin, aspartate transaminase) and fibrosis (transforming growth factor-β, α-smooth muscle actin, phosphatidylserine receptor) and an increase in markers of liver regeneration (matrix metalloproteinases [MMP]-9, MMP-12 and TNF-related weak inducer of apoptosis). CD14+ monocytes were then isolated from controls. Monocytes were matured into macrophages for 7 days using a Good Manufacturing Practice–compatible technique. Results There was no significant difference between the mean number of CD14+ monocytes isolated from cirrhotic patients (n = 9) and controls (n = 10); 2.8 ± SEM 0.54 × 108 and 2.5 ± 0.56 × 108, respectively. The mean yield of mature macrophages cultured was also not significantly different between cirrhotic patients and controls (0.9 × 108 ± 0.38 × 108, with more than 90% viability and 0.65 × 108 ± 0.16 × 108, respectively. Maturation to macrophages resulted in up-regulation of a number of genes (MMP-9, CCL2, interleukin [IL]-10 and TNF-related weak inducer of apoptosis). A cytokine and chemokine polymerase chain reaction array, comparing the control and cirrhotic macrophages, revealed no statistically significant differences. Conclusions Macrophages can be differentiated from cirrhotic patients' apheresis-derived CD14 monocytes and develop the same pro-resolution phenotype as control macrophages, indicating their suitability for clinical therapy. PMID:26342993

  3. Role of the cytokine environment and cytokine receptor expression on the generation of functionally distinct dendritic cells from human monocytes.

    PubMed

    Conti, Lucia; Cardone, Marco; Varano, Barbara; Puddu, Patrizia; Belardelli, Filippo; Gessani, Sandra

    2008-03-01

    Myeloid dendritic cells (DC) and macrophages evolve from a common precursor. However, factors controlling monocyte differentiation toward DC or macrophages are poorly defined. We report that the surface density of the GM-CSF receptor (GM-CSFR) alpha subunit in human peripheral blood monocytes varies among donors. Although no correlation was found between the extent of GM-CSFR and monocyte differentiation into DC driven by GM-CSF and IL-4, GM-CSFR expression strongly influenced the generation of CD1a(+) dendritic-like cells in the absence of IL-4. CD1a(+) cells generated in the presence of GM-CSF express CD40, CD80, MHC class I and II, DC-SIGN, MR, CCR5, and partially retain CD14 expression. Interestingly, they spontaneously induce the expansion of CD4(+) and CD8(+) allogeneic T lymphocytes producing IFN-gamma, and migrate toward CCL4 and CCL19. Upon stimulation with TLR ligands, they acquire the phenotypic features of mature DC. In contrast, the allostimulatory capacity is not further increased upon LPS activation. However, by blocking LPS-induced IL-10, a higher T cell proliferative response and IL-12 production were observed. Interestingly, IL-23 secretion was not affected by endogenous IL-10. These results highlight the importance of GM-CSFR expression in monocytes for cytokine-induced DC generation and point to GM-CSF as a direct player in the generation of functionally distinct DC.

  4. LDL acts as an opsonin enhancing the phagocytosis of group A Streptococcus by monocyte and whole human blood.

    PubMed

    Zhou, Lulei; Liu, Ling; Yang, Jinli; Li, Yuxin; Bai, Wencheng; Liu, Na; Li, Wenlong; Gao, Yumin; Xu, Liping; Liu, Zhi; Han, Runlin

    2016-04-01

    Low-density lipoprotein (LDL) binds to group A Streptococcus (GAS) through Sc11 protein, and scavenger receptor CD36 of monocyte mediates the endocytosis of native or modified LDL. Therefore, we hypothesized that LDL might be an opsonin enhancing the phagocytosis of LDL-bound GAS by monocyte. The results showed that LDL could significantly promote U937 cell to phagocytose M28 (ATCC BAA1064) and M41 (ATCC 12373, AM41)-type GAS, and the phagocytosis rates were significantly increased, compared with LDL-free group. LDL, however, did not enhance the phagocytosis of M41 (CMCC 32198, CM41) or M6 (ATCC BAA946)-type GAS since these two strains did not bind to LDL. CD36 was the major scavenger receptor mediating the uptake of LDL-bound GAS by monocyte U937 cells since anti-CD36 antibody abolished the phagocytosis of LDL-opsonized GAS but anti-CD4 antibody did not. Most of AM41-type GAS cells were killed in human blood, whereas only a few CM41-type cells were phagocytosed. Moreover, recombinant Scl1 (rScl1) derived from M41-type GAS could significantly decrease the opsonophagocytosis of AM41 but not CM41-type GAS because the rScl1 competitively blocked the binding of AM41-type GAS to LDL. Therefore, our findings suggest that LDL may be an opsonin to enhance CD36-dependent opsonic phagocytosis of GAS by monocyte.

  5. T-lymphocyte induction of human monocyte angiotensin converting enzyme (ACE) is not dependent upon T-lymphocyte proliferation

    SciTech Connect

    Vuk-Pavlovic, Z.; Rohrbach, M.S.

    1986-03-05

    Human peripheral blood monocytes cultured in serum free media for seven days show a basal activity of the ectoenzyme ACE which is augmented 2-3 times by the presence of autologous peripheral blood T-lymphocytes. Since these two cell types are also involved in autologous mixed lymphocyte reaction if serum is present, the authors compared the ability of T-cells to stimulate ACE activity in the presence or absence of proliferation (measured by /sup 3/H-thymidine incorporation). By the seventh day, cultures with 5% AB/sup +/ serum showed significant increase in proliferation but no increase in ACE activity compared to the serum free cultures. Even higher proliferation rate achieved by co-culturing T-lymphocytes with allogeneic monocytes did not increase ACE production; on the contrary, ACE activity remained at the basal level. Monocyte-T-cell co-cultures stimulated with increasing concentrations of ConA or PHA showed dose dependent increases in proliferation but parallel decreases in ACE activity. Addition of soluble antigen (Candida albicans) also enhanced proliferation but not ACE synthesis. They conclude that T-lymphocyte induction of monocyte ACE is a result of cooperation between autologous cells which is not dependent upon T-cell proliferation.

  6. Amelioration of glucolipotoxicity-induced endoplasmic reticulum stress by a "chemical chaperone" in human THP-1 monocytes.

    PubMed

    Lenin, Raji; Maria, Mariawilliam Sneha; Agrawal, Madhur; Balasubramanyam, Jayashree; Mohan, Viswanathan; Balasubramanyam, Muthuswamy

    2012-01-01

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

  7. High glucose concentrations induce TNF-α production through the down-regulation of CD33 in primary human monocytes

    PubMed Central

    2012-01-01

    Background CD33 is a membrane receptor containing a lectin domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) that is able to inhibit cytokine production. CD33 is expressed by monocytes, and reduced expression of CD33 correlates with augmented production of inflammatory cytokines, such as IL-1β, TNF-α, and IL-8. However, the role of CD33 in the inflammation associated with hyperglycemia and diabetes is unknown. Therefore, we studied CD33 expression and inflammatory cytokine secretion in freshly isolated monocytes from patients with type 2 diabetes. To evaluate the effects of hyperglycemia, monocytes from healthy donors were cultured with different glucose concentrations (15-50 mmol/l D-glucose), and CD33 expression and inflammatory cytokine production were assessed. The expression of suppressor of cytokine signaling protein-3 (SOCS-3) and the generation of reactive oxygen species (ROS) were also evaluated to address the cellular mechanisms involved in the down-regulation of CD33. Results CD33 expression was significantly decreased in monocytes from patients with type 2 diabetes, and higher levels of TNF-α, IL-8 and IL-12p70 were detected in the plasma of patients compared to healthy donors. Under high glucose conditions, CD33 protein and mRNA expression was significantly decreased, whereas spontaneous TNF-α secretion and SOCS-3 mRNA expression were increased in monocytes from healthy donors. Furthermore, the down-regulation of CD33 and increase in TNF-α production were prevented when monocytes were treated with the antioxidant α-tocopherol and cultured under high glucose conditions. Conclusion Our results suggest that hyperglycemia down-regulates CD33 expression and triggers the spontaneous secretion of TNF-α by peripheral monocytes. This phenomenon involves the generation of ROS and the up-regulation of SOCS-3. These observations support the importance of blood glucose control for maintaining innate immune function and suggest

  8. Isolation of IL-12p70-competent human monocyte-derived dendritic cells.

    PubMed

    Søndergaard, Jonas N; Brix, Susanne

    2012-12-14

    Diverse methodologies ranging from experimental immunological studies to immunotherapy involve the application of human monocyte-derived dendritic cells (moDCs). Considerable donor-dependent variations in the moDC production of IL-12p70 affect the outcome of these methodologies. It has been shown that moDCs generated under standard conditions develop into two subsets based on CD1a-expression with the CD1a+ moDCs being the main IL-12p70 producers. This has however not been generally accepted, which we show here because the subset described as CD1a-negative does express CD1a, but at a lower level than the other subset. We further characterize the phenotype of these two subsets, showing that the CD1a-hi subset has a greater immunogenic phenotype, making this subset more suitable for immunotherapy. The two subsets have previously been separated by cell sorting, but as this technique is not available to many laboratories and has incompatibility with clinical settings, a more widely useable technique is warranted. Therefore we tested if magnetic-activated cell sorting is useful for the purpose, and show that it is possible to isolate IL-12p70-competent CD1a-hi moDCs to a <92% purity, irrespective of the starting purity.

  9. Metabolic profiling during HIV-1 and HIV-2 infection of primary human monocyte-derived macrophages

    PubMed Central

    Hollenbaugh, Joseph A.; Montero, Catherine; Schinazi, Raymond F.; Munger, Joshua; Kim, Baek

    2016-01-01

    We evaluated cellular metabolism profiles of HIV-1 and HIV-2 infected primary human monocyte-derived macrophages (MDMs). First, HIV-2 GL-AN displays faster production kinetics and greater amounts of virus as compared to HIV-1s: YU-2, 89.6 and JR-CSF. Second, quantitative LC–MS/MS metabolomics analysis demonstrates very similar metabolic profiles in glycolysis and TCA cycle metabolic intermediates between HIV-1 and HIV-2 infected macrophages, with a few notable exceptions. The most striking metabolic change in MDMs infected with HIV-2 relative to HIV-1-infected MDMs was the increased levels of quinolinate, a metabolite in the tryptophan catabolism pathway that has been linked to HIV/AIDS pathogenesis. Third, both HIV-1 and HIV-2 infected MDMs showed elevated levels of ribose-5-phosphate, a key metabolic component in nucleotide biosynthesis. Finally, HIV-2 infected MDMs display increased dNTP concentrations as predicted by Vpx-mediated SAMHD1 degradation. Collectively, these data show differential metabolic changes during HIV-1 and HIV-2 infection of macrophages. PMID:26895248

  10. Influence of Acanthamoeba castellanii on intracellular growth of different Legionella species in human monocytes.

    PubMed

    Neumeister, B; Reiff, G; Faigle, M; Dietz, K; Northoff, H; Lang, F

    2000-03-01

    Previous studies using a murine model of coinhalation of Legionella pneumophila and Hartmannella vermiformis have shown a significantly enhanced intrapulmonary growth of L. pneumophila in comparison to inhalation of legionellae alone (J. Brieland, M. McClain, L. Heath, C. Chrisp, G. Huffnagle, M. LeGendre, M. Hurley, J. Fantone, and C. Engleberg, Infect. Immun. 64:2449-2456, 1996). In this study, we introduce an in vitro coculture model of legionellae, Mono Mac 6 cells (MM6) and Acanthamoeba castellanii, using a cell culture chamber system which separates both cell types by a microporous polycarbonate membrane impervious to bacteria, amoebae, and human cells. Whereas L. pneumophila has shown a maximal 4-log-unit multiplication within MM6, which could not be further increased by coculture with Acanthamoeba castellanii, significantly enhanced replication of L. gormanii, L. micdadei, L. steigerwaltii, L. longbeachae, and L. dumoffii was seen after coculture with amoebae. This effect was seen only with uninfected amoebae, not with Legionella-infected amoebae. The supporting effect for intracellular multiplication in MM6 could be reproduced in part by addition of a cell-free coculture supernatant obtained from a coincubation experiment with uninfected A. castellanii and Legionella-infected MM6, suggesting that amoeba-derived effector molecules are involved in this phenomenon. This coculture model allows investigations of molecular and biochemical mechanisms which are responsible for the enhancement of intracellular multiplication of legionellae in monocytic cells after interaction with amoebae.

  11. Copper egress is induced by PMA in human THP-1 monocytic cell line.

    PubMed

    Afton, Scott E; Caruso, Joseph A; Britigan, Bradley E; Qin, Zhenyu

    2009-06-01

    Copper egress is an essential regulator of the kinetics of cellular copper and is primarily regulated by ATP7A, a copper-transporting P-type ATPase. However, little is known under which physiological condition copper egress is induced and its molecular consequence. In current manuscript, using THP-1 cells, a human monocytic cell line, we found that ATP7A expression was increased in cells exposed to phorbol-12-myristate-13-acetate (PMA), a potent inducer of neovascularization and cancer. Inductively coupled plasma mass spectrometry revealed that PMA also induced copper egress. Inhibition of ATP7A expression using small interfering RNA abrogated PMA induced copper egress. PMA treatment in THP-1 cells resulted in increased expression of matrix metalloproteinase (MMP) 9 and vascular endothelial growth factor receptor 1 (VEGFR1), whereas inhibition of ATP7A resulted in suppression of PMA-induced expression of VEGFR1, but not MMP9. Finally, addition of exogenous copper into the conditioned medium did not change VEGFR1 expression in THP-1 cells. Collectively, we demonstrate that PMA induces copper egress in THP-1 cells, which is regulated by ATP7A, and ATP7A regulates VEGFR1 expression. Considering the involvement of copper in neovascularization, our current finding provides the potential evidence to interpret the molecular mechanism.

  12. Human endotoxin tolerance is associated with enrichment of the CD14+ CD16+ monocyte subset.

    PubMed

    Domínguez-Nieto, Aimée; Zentella, Alejandro; Moreno, José; Ventura, José L; Pedraza, Sigifredo; Velázquez, Juan R

    2015-01-01

    Prior exposure to lipopolysaccharides (LPS) induces a state of cell resistance to subsequent LPS restimulation, known as endotoxin tolerance, mainly by repressing the expression of pro-inflammatory cytokines. We established an endotoxin tolerance model in human monocytes Endotoxin-tolerant cells showed a decrease in IκBα degradation and diminished expression of Tumor necrosis factor (TNF) (both messenger RNA [mRNA] and protein content). The myeloid differentiation factor 88 (MyD88)/MyD88 splice variant (MyD88s) ratio, an indirect way to test the Toll-like receptor 4 (TLR4) MyD88-dependent signaling cascade, did not change in endotoxin-tolerant cells when compared to LPS-stimulated or -unstimulated ones. Remarkably, cell population analysis indicated a significant increase of the CD14+ CD16+ subset only under the endotoxin-tolerant condition. Furthermore, endotoxin-tolerant cells produced higher amounts of C-X-C motif chemokine 10 (CXCL10), a typical MyD88-independent cytokine.

  13. Influence of sex and genetic variability on expression of X-linked genes in human monocytes.

    PubMed

    Castagné, Raphaële; Zeller, Tanja; Rotival, Maxime; Szymczak, Silke; Truong, Vinh; Schillert, Arne; Trégouët, David-Alexandre; Münzel, Thomas; Ziegler, Andreas; Cambien, François; Blankenberg, Stefan; Tiret, Laurence

    2011-11-01

    In humans, the fraction of X-linked genes with higher expression in females has been estimated to be 5% from microarray studies, a proportion lower than the 25% of genes thought to escape X inactivation. We analyzed 715 X-linked transcripts in circulating monocytes from 1,467 subjects and found an excess of female-biased transcripts on the X compared to autosomes (9.4% vs 5.5%, p<2×10(-5)). Among the genes not previously known to escape inactivation, the most significant one was EFHC2 whose 20% of variability was explained by sex. We also investigated cis expression quantitative trait loci (eQTLs) by analyzing 15,703 X-linked SNPs. The frequency and magnitude of X-linked cis eQTLs were quite similar in males and females. Few genes exhibited a stronger genetic effect in females than in males (ARSD, DCX, POLA1 and ITM2A). These genes would deserve further investigation since they may contribute to sex pathophysiological differences.

  14. Expression of ESE-3 Isoforms in Immunogenic and Tolerogenic Human Monocyte-Derived Dendritic Cells

    PubMed Central

    Sprater, Florian; Hovden, Arnt-Ove; Appel, Silke

    2012-01-01

    Dendritic cells (DC) are the only hematopoietic cells expressing the epithelial specific Ets transcription factor ESE-3. Here we analyzed presence and quantity of isoforms ESE-3a, ESE-3b and ESE-3j in various immunogenic and tolerogenic human monocyte-derived DC (moDC) and blood DC populations using quantitative real time PCR and immunoblot analyses. ESE-3a and ESE-3b were detectable in all moDC populations with ESE-3b being the main transcript. ESE-3b expression was upregulated in immunogenic moDC and downregulated in tolerogenic moDC compared to immature moDC. ESE-3a had similar transcript levels in immature and immunogenic moDC and had very low levels in tolerogenic moDC. In blood DC populations only splice variant ESE-3b was detectable. ESE-3j was not detectable in any of the DC populations. These findings suggest that ESE-3b is the functionally most important ESE-3 isoform in DC. PMID:23185370

  15. Expression of ESE-3 isoforms in immunogenic and tolerogenic human monocyte-derived dendritic cells.

    PubMed

    Sprater, Florian; Hovden, Arnt-Ove; Appel, Silke

    2012-01-01

    Dendritic cells (DC) are the only hematopoietic cells expressing the epithelial specific Ets transcription factor ESE-3. Here we analyzed presence and quantity of isoforms ESE-3a, ESE-3b and ESE-3j in various immunogenic and tolerogenic human monocyte-derived DC (moDC) and blood DC populations using quantitative real time PCR and immunoblot analyses. ESE-3a and ESE-3b were detectable in all moDC populations with ESE-3b being the main transcript. ESE-3b expression was upregulated in immunogenic moDC and downregulated in tolerogenic moDC compared to immature moDC. ESE-3a had similar transcript levels in immature and immunogenic moDC and had very low levels in tolerogenic moDC. In blood DC populations only splice variant ESE-3b was detectable. ESE-3j was not detectable in any of the DC populations. These findings suggest that ESE-3b is the functionally most important ESE-3 isoform in DC.

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

    PubMed

    Raghavan, Ram K; Neises, Daniel; Goodin, Douglas G; Andresen, Daniel A; Ganta, Roman R

    2014-01-01

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

  17. Monocyte transferrin-iron uptake in hereditary hemochromatosis

    SciTech Connect

    Sizemore, D.J.; Bassett, M.L.

    1984-05-01

    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.

  18. Sinomenine influences capacity for invasion and migration in activated human monocytic THP-1 cells by inhibiting the expression of MMP-2, MMP-9, and CD147

    PubMed Central

    Ou, Yang-qiong; Chen, Li-hua; Li, Xue-jun; Lin, Zhi-bin; Li, Wei-dong

    2009-01-01

    Aim: The aim of this study was to investigate the mechanism of the effects of Sinomenine (SIN) on the invasion and migration ability of activated human monocytic THP-1 cells (A-THP-1). Sinomenine is a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum. Methods: Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA). Cells were treated with different concentrations of SIN. The invasion and migration ability of cells was tested by in vitro transwell assays. The levels of CD147 and MMPs were evaluated by flow cytometric analysis and zymographic analysis, respectively. The mRNA expression of CD147, MMP-2, and MMP-9 was measured by RT-PCR. Results: The invasion and migration ability of A-THP-1 cells was significantly inhibited by SIN in a concentration-dependent fashion; at the same time, the levels of CD147, MMP-2, and MMP-9 were markedly down-regulated. This inhibitory effect was most notable at concentrations of 0.25 mmol/L and 1.00 mmol/L (P<0.01). Conclusion: A possible mechanism of the inhibitory effect of SIN on cell invasion and migration ability is repression of the expression of MMP-2 and MMP-9, which strongly correlates with the inhibition of CD147 activity. PMID:19305422

  19. Diverging biological roles among human monocyte subsets in the context of tuberculosis infection.

    PubMed

    Balboa, Luciana; Barrios-Payan, Jorge; González-Domínguez, Erika; Lastrucci, Claire; Lugo-Villarino, Geanncarlo; Mata-Espinoza, Dulce; Schierloh, Pablo; Kviatcovsky, Denise; Neyrolles, Olivier; Maridonneau-Parini, Isabelle; Sánchez-Torres, Carmen; Sasiain, María del Carmen; Hernández-Pando, Rogelio

    2015-08-01

    Circulating monocytes (Mo) play an essential role in the host immune response to chronic infections. We previously demonstrated that CD16(pos) Mo were expanded in TB (tuberculosis) patients, correlated with disease severity and were refractory to dendritic cell differentiation. In the present study, we investigated whether human Mo subsets (CD16(neg) and CD16(pos)) differed in their ability to influence the early inflammatory response against Mycobacterium tuberculosis. We first evaluated the capacity of the Mo subsets to migrate and engage a microbicidal response in vitro. Accordingly, CD16(neg) Mo were more prone to migrate in response to different mycobacteria-derived gradients, were more resistant to M. tuberculosis intracellular growth and produced higher reactive oxygen species than their CD16(pos) counterpart. To assess further the functional dichotomy among the human Mo subsets, we carried out an in vivo analysis by adapting a hybrid mouse model (SCID/Beige, where SCID is severe combined immunodeficient) to transfer each Mo subset, track their migratory fate during M. tuberculosis infection, and determine their impact on the host immune response. In M. tuberculosis-infected mice, the adoptively transferred CD16(neg) Mo displayed a higher lung migration index, induced a stronger pulmonary infiltration of murine leucocytes expressing pro- and anti-inflammatory cytokines, and significantly decreased the bacterial burden, in comparison with CD16(pos) Mo. Collectively, our results indicate that human Mo subsets display divergent biological roles in the context of M. tuberculosis infection, a scenario in which CD16(neg) Mo may contribute to the anti-mycobacterial immune response, whereas CD16(pos) Mo might promote microbial resilience, shedding light on a key aspect of the physiopathology of TB disease.

  20. Responsiveness of human monocyte-derived dendritic cells to thimerosal and mercury derivatives.

    PubMed

    Migdal, C; Tailhardat, M; Courtellemont, P; Haftek, M; Serres, M

    2010-07-01

    Several cases of skin sensitization have been reported following the application of thimerosal, which is composed of ethyl mercury and thiosalicylic acid (TSA). However, few in vitro studies have been carried out on human dendritic cells (DCs) which play an essential role in the initiation of allergic contact dermatitis. The aim of the present study was to identify the effect of thimerosal and other mercury compounds on human DCs. To address this purpose, DCs derived from monocytes (mono-DCs) were used. Data show that thimerosal and mercury derivatives induced DC activation, as monitored by CD86 and HLA-DR overexpression associated with the secretion of tumor necrosis factor alpha and interleukin 8, similarly to lipopolysaccharide and the sensitizers, 1-chloro-2,4-dinitrobenzene (DNCB) and nickel sulfate, which were used as positive controls. In contrast, TSA, the non-mercury part of thimerosal, as well as dichloronitrobenzene, a DNCB negative control, and the irritant, sodium dodecyl sulfate, had no effect. Moreover, oxidative stress, monitored by ROS induction and depolarization of the mitochondrial membrane potential, was induced by thimerosal and mercury compounds, as well as DNCB, in comparison with hydrogen peroxide, used as a positive control. The role of thiol oxidation in the initiation of mono-DC activation was confirmed by a pre-treatment with N-acetyl-l-cysteine which strongly decreased chemical-induced CD86 overexpression. These data are in agreement with several clinical observations of the high relevance of thimerosal in patch-test reactions and prove that human mono-DCs are useful in vitro tools for determining the allergenic potency of chemicals.

  1. Responsiveness of human monocyte-derived dendritic cells to thimerosal and mercury derivatives

    SciTech Connect

    Migdal, C.; Tailhardat, M.; Courtellemont, P.; Haftek, M.; Serres, M.

    2010-07-15

    Several cases of skin sensitization have been reported following the application of thimerosal, which is composed of ethyl mercury and thiosalicylic acid (TSA). However, few in vitro studies have been carried out on human dendritic cells (DCs) which play an essential role in the initiation of allergic contact dermatitis. The aim of the present study was to identify the effect of thimerosal and other mercury compounds on human DCs. To address this purpose, DCs derived from monocytes (mono-DCs) were used. Data show that thimerosal and mercury derivatives induced DC activation, as monitored by CD86 and HLA-DR overexpression associated with the secretion of tumor necrosis factor {alpha} and interleukin 8, similarly to lipopolysaccharide and the sensitizers, 1-chloro-2,4-dinitrobenzene (DNCB) and nickel sulfate, which were used as positive controls. In contrast, TSA, the non-mercury part of thimerosal, as well as dichloronitrobenzene, a DNCB negative control, and the irritant, sodium dodecyl sulfate, had no effect. Moreover, oxidative stress, monitored by ROS induction and depolarization of the mitochondrial membrane potential, was induced by thimerosal and mercury compounds, as well as DNCB, in comparison with hydrogen peroxide, used as a positive control. The role of thiol oxidation in the initiation of mono-DC activation was confirmed by a pre-treatment with N-acetyl-L-cysteine which strongly decreased chemical-induced CD86 overexpression. These data are in agreement with several clinical observations of the high relevance of thimerosal in patch-test reactions and prove that human mono-DCs are useful in vitro tools for determining the allergenic potency of chemicals.

  2. Two P2X1 receptor transcripts able to form functional channels are present in most human monocytes.

    PubMed

    López-López, Cintya; Jaramillo-Polanco, Josue; Portales-Pérez, Diana P; Gómez-Coronado, Karen S; Rodríguez-Meléndez, Jessica G; Cortés-García, Juan D; Espinosa-Luna, Rosa; Montaño, Luis M; Barajas-López, Carlos

    2016-12-15

    To characterize the presence and general properties of P2X1 receptors in single human monocytes we used RT-PCR, flow cytometry, and the patch-clamp and the two-electrode voltage-clamp techniques. Most human monocytes expressed the canonical P2X1 (90%) and its splicing variant P2X1del (88%) mRNAs. P2X1 receptor immunoreactivity was also observed in 70% of these cells. Currents mediated by P2X1 (EC50=1.9±0.8µm) and P2X1del (EC50 >1000µm) channels, expressed in Xenopus leavis oocytes, have different ATP sensitivity and kinetics. Both currents mediated by P2X1 and P2X1del channels kept increasing during the continuous presence of high ATP concentrations. Currents mediated by the native P2X1 receptors in human monocytes showed an EC50=6.3±0.2µm. Currents have kinetics that resemble those observed for P2X1 and P2X1del receptors in oocytes. Our study is the first to demonstrate the expression of P2X1 transcript and its splicing variant P2X1del in most human monocytes. We also, for the first time, described functional homomeric P2X1del channels and demonstrated that currents mediated by P2X1 or P2X1del receptors, during heterologous expression, increased in amplitude when activated with high ATP concentrations in a similar fashion to those channels that increase their conductance under similar conditions, such as P2X7, P2X2, and P2X4 channels.

  3. Differential role of pannexin-1/ATP/P2X7 axis in IL-1β release by human monocytes.

    PubMed

    Parzych, Katarzyna; Zetterqvist, Anna V; Wright, William R; Kirkby, Nicholas S; Mitchell, Jane A; Paul-Clark, Mark J

    2017-02-28

    IL-1β release is integral to the innate immune system. The release of mature IL-1β depends on 2 regulated events: the de novo induction of pro-IL-1β, generally via NF-κB-dependent transduction pathways; and the assembly and activation of the NLRP3 inflammasome. This latter step is reliant on active caspase-1, pannexin-1, and P2X7 receptor activation. Pathogen-associated molecular patterns in gram-positive and gram-negative bacteria activate IL-1β release from immune cells via TLR2 and TLR4 receptors, respectively. We found that pro-IL-1β and mature IL-1β release from human monocytes is stimulated by the TLR2 agonists Pam3CSK4 or FSL-1 as well as the TLR4 agonist LPS in the absence of additional ATP. TLR2 agonists required pannexin-1 and P2X7 receptor activation to stimulate IL-1β release. In contrast, IL-1β release stimulated by the TLR4 agonist LPS is independent of both pannexin-1 and P2X7 activation. In the absence of exogenous ATP, P2X7 activation requires endogenous ATP release, which occurs in some cells via pannexin-1. In line with this, we found that LPS-stimulated human monocytes released relatively low levels of ATP, whereas cells stimulated with TLR2 agonists released high levels of ATP. These findings suggest that in human monocytes, both TLR2 and TLR4 signaling induce pro-IL-1β expression, but the mechanism by which they activate caspase-1 diverges at the level of the pannexin-1/ATP/P2X7 axis.-Parzych, K., Zetterqvist, A. V., Wright, W. R., Kirkby, N. S., Mitchell, J. A., Paul-Clark, M. J. Differential role of pannexin-1/ATP/P2X7 axis in IL-1β release by human monocytes.

  4. Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions.

    PubMed Central

    Ylä-Herttuala, S; Lipton, B A; Rosenfeld, M E; Särkioja, T; Yoshimura, T; Leonard, E J; Witztum, J L; Steinberg, D

    1991-01-01

    The recruitment of monocyte-macrophages into the artery wall is one of the earliest events in the pathogenesis of atherosclerosis. Monocyte chemoattractant protein 1 (MCP-1) is a potent monocyte chemoattractant secreted by many cells in vitro, including vascular smooth muscle and endothelial cells. To test whether it is expressed in the artery in vivo, we used Northern blot analysis, in situ hybridization, and immunocytochemistry to study the expression of MCP-1 in normal and atherosclerotic human and rabbit arteries. Northern blot analysis showed that MCP-1 mRNA could be isolated from rabbit atherosclerotic lesions but not from the intima media of normal animals. Furthermore, MCP-1 mRNA was extracted from macrophage-derived foam cells isolated from arterial lesions of ballooned cholesterol-fed rabbits, whereas alveolar macrophages isolated simultaneously from the same rabbits did not express MCP-1 mRNA. MCP-1 mRNA was detected by in situ hybridization in macrophage-rich regions of both human and rabbit atherosclerotic lesions. No MCP-1 mRNA was found in sublesional medial smooth muscle cells or in normal arteries. By using immunocytochemistry, MCP-1 protein was demonstrated in human lesions, again only in macrophage-rich regions. Immunostaining of the serial sections with an antiserum against malondialdehyde-modified low density lipoprotein indicated the presence of oxidized low density lipoprotein indicated the presence of oxidized low density lipoprotein and/or other oxidation-specific lipid-protein adducts in the same areas that contained macrophages and MCP-1. We conclude that (i) MCP-1 is strongly expressed in a small subset of cells in macrophage-rich regions of human and rabbit atherosclerotic lesions and (ii) MCP-1 may, therefore, play an important role in the ongoing recruitment of monocyte-macrophages into developing lesions in vivo. Images PMID:2052604

  5. Effects of an interleukin-1 receptor antagonist on human sleep, sleep-associated memory consolidation, and blood monocytes.

    PubMed

    Schmidt, Eva-Maria; Linz, Barbara; Diekelmann, Susanne; Besedovsky, Luciana; Lange, Tanja; Born, Jan

    2015-07-01

    Pro-inflammatory cytokines like interleukin-1 beta (IL-1) are major players in the interaction between the immune system and the central nervous system. Various animal studies report a sleep-promoting effect of IL-1 leading to enhanced slow wave sleep (SWS). Moreover, this cytokine was shown to affect hippocampus-dependent memory. However, the role of IL-1 in human sleep and memory is not yet understood. We administered the synthetic IL-1 receptor antagonist anakinra (IL-1ra) in healthy humans (100mg, subcutaneously, before sleep; n=16) to investigate the role of IL-1 signaling in sleep regulation and sleep-dependent declarative memory consolidation. Inasmuch monocytes have been considered a model for central nervous microglia, we monitored cytokine production in classical and non-classical blood monocytes to gain clues about how central nervous effects of IL-1ra are conveyed. Contrary to our expectation, IL-1ra increased EEG slow wave activity during SWS and non-rapid eye movement (NonREM) sleep, indicating a deepening of sleep, while sleep-associated memory consolidation remained unchanged. Moreover, IL-1ra slightly increased prolactin and reduced cortisol levels during sleep. Production of IL-1 by classical monocytes was diminished after IL-1ra. The discrepancy to findings in animal studies might reflect species differences and underlines the importance of studying cytokine effects in humans.

  6. Interactions of TANGO and leukocyte integrin CD11c/CD18 regulate the migration of human monocytes.

    PubMed

    Arndt, Stephanie; Melle, Christian; Mondal, Krishna; Klein, Gerd; von Eggeling, Ferdinand; Bosserhoff, Anja-Katrin

    2007-12-01

    The TANGO gene was originally identified as a new member of the MIA gene family. It codes for a protein of yet unknown function. TANGO revealed a very broad expression pattern in contrast to the highly restricted expression pattern determined for the other family members. The only cells lacking TANGO expression are cells of the hematopoietic system. One of the major differences between mature hematopoietic cells and other tissue cells is the lack of adhesion until these cells leave the bloodstream. In this study, we observed that TANGO expression was induced after adhesion of human monocytic cells to substrate. To understand the mechanism of TANGO function during monocyte adhesion we isolated interacting proteins and found an interaction between TANGO and the leukocyte-specific integrin CD11c. In functional assays, we observed reduced attachment of human monocytic cells to fibrinogen, ICAM-1 and to human microvascular endothelial cells (HMECs) after stimulation with recombinant TANGO protein. Additionally, the migrating capacity of premonocytic cells through fibrinogen or HMECs was increased after stimulation of these cells with recombinant TANGO. Therefore, we suggest that TANGO reduced the attachment to fibrinogen or other cell adhesion molecules. As TANGO does not compete for CD11c ligand binding directly, we hypothesize TANGO function by modulation of integrin activity. Taken together, the results from this study present TANGO as a novel ligand for CD11c, regulating migratory processes of hematopoietic cells.

  7. Supra-therapeutic plasma concentrations of haloperidol induce moderate inhibition of lipopolysaccharide-induced interleukin-8 release in human monocytes

    PubMed Central

    2016-01-01

    Background The clinical use of antipsychotics and mood-stabilizing drugs with proven efficacy is largely determined by the occurrence of treatment-emergent adverse events and routine clinical chemistry and haematology data, which together define the safety and tolerability profile of these psychopharmaceuticals. Whereas the effects of mood-stabilizing drugs on functional properties of blood cells have been poorly investigated, the effects of antipsychotics have received more attention. Such studies have yielded conflicting results. This study examined the effects of the mood-stabilizing drugs carbamazepine and valproic acid and of the antipsychotic drugs olanzapine, risperidone and haloperidol on the production of the pro-inflammatory chemokine interleukin-8 (IL-8), which is released from human monocytes when activated by Gram-negative lipopolysaccharide (LPS). Methods Peripheral human whole blood was diluted with Roswell Park Memorial Institute (RPMI) cell culture medium and stimulated with LPS. Accumulating IL-8 was quantified in the supernatant with an adapted enzyme-linked immunosorbent assay (ELISA) and the results correlated to the number of monocytes at venipuncture. Results At supra-therapeutic concentrations of 100 µM, haloperidol inhibited the LPS-induced release of IL-8 in peripheral human monocytes moderately, whereas olanzapine, risperidone, carbamazepine and valproic acid showed no such effect. Conclusions The results suggest that these mood-stabilizing drugs and antipsychotics are endowed with clinically favorable inertness rather than pro-inflammatory properties. PMID:27867948

  8. The CD16+ (FcγRIII+) Subset of Human Monocytes Preferentially Becomes Migratory Dendritic Cells in a Model Tissue Setting

    PubMed Central

    Randolph, Gwendalyn J.; Sanchez-Schmitz, Guzman; Liebman, Ronald M.; Schäkel, Knut

    2002-01-01

    Much remains to be learned about the physiologic events that promote monocytes to become lymph-homing dendritic cells (DCs). In a model of transendothelial trafficking, some monocytes become DCs in response to endogenous signals. These DCs migrate across endothelium in the ablumenal-to-lumenal direction (reverse transmigration), reminiscent of the migration into lymphatic vessels. Here we show that the subpopulation of monocytes that expresses CD16 (Fcγ receptor III) is predisposed to become migratory DCs. The vast majority of cells derived from CD16+ monocytes reverse transmigrated, and their presence was associated with migratory cells expressing high levels of CD86 and human histocompatibility leukocyte antigen (HLA)-DR, and robust capacity to induce allogeneic T cell proliferation. A minority of CD16− monocytes reverse transmigrated, and these cells stimulated T cell proliferation less efficiently. CD16 was not functionally required for reverse transmigration, but promoted cell survival when yeast particles (zymosan) were present as a maturation stimulus in the subendothelial matrix. The cell surface phenotype and migratory characteristics of CD16+ monocytes were inducible in CD16− monocytes by preincubation with TGFβ1. We propose that CD16+ monocytes may contribute significantly to precursors for DCs that transiently survey tissues and migrate to lymph nodes via afferent lymphatic vessels. PMID:12186843

  9. Decreased expression of human class II antigens on monocytes from patients with acquired immune deficiency syndrome. Increased expression with interferon-gamma.

    PubMed Central

    Heagy, W; Kelley, V E; Strom, T B; Mayer, K; Shapiro, H M; Mandel, R; Finberg, R

    1984-01-01

    The expression of HLA-DR (a class II histocompatibility antigen) on monocytes isolated from the peripheral blood of normal individuals and patients with acquired immune deficiency syndrome (AIDS) was investigated by the use of dual fluorescent staining and cytofluorometry. In animal models the absence of class II positive monocytes is linked to a failure of T cells to respond to antigens. We now report that patients with AIDS have a paucity of HLA-DR+ monocytes. The percentage of HLA-DR+ monocytes among eight normal individuals ranged from 49.3 to 95.0%+, and only one individual had less than 50% HLA-DR+ monocytes. HLA-DR expression on monocytes from homosexual male patients with lymphadenopathy was similar to that of normal subjects (range, 58.0 to 97.4%+). In contrast, seven of nine patients with AIDS had less than 50% HLA-DR+ monocytes (range, 13.4 to 78.8%+). The in vitro incubation of monocytes from AIDS patients with cloned human interferon-gamma resulted in an increase of the expression of HLA-DR to near normal levels. PMID:6439741

  10. Human Bone Marrow-Derived Mesenchymal Stromal Cells Differentially Inhibit Cytokine Production by Peripheral Blood Monocytes Subpopulations and Myeloid Dendritic Cells

    PubMed Central

    Laranjeira, Paula; Gomes, Joana; Pedrosa, Monia; Martinho, Antonio; Antunes, Brigida; Ribeiro, Tania; Santos, Francisco; Domingues, Rosario; Abecasis, Manuel; Trindade, Helder; Paiva, Artur

    2015-01-01

    The immunosuppressive properties of mesenchymal stromal/stem cells (MSC) rendered them an attractive therapeutic approach for immune disorders and an increasing body of evidence demonstrated their clinical value. However, the influence of MSC on the function of specific immune cell populations, namely, monocyte subpopulations, is not well elucidated. Here, we investigated the influence of human bone marrow MSC on the cytokine and chemokine expression by peripheral blood classical, intermediate and nonclassical monocytes, and myeloid dendritic cells (mDC), stimulated with lipopolysaccharide plus interferon (IFN)γ. We found that MSC effectively inhibit tumor necrosis factor- (TNF-) α and macrophage inflammatory protein- (MIP-) 1β protein expression in monocytes and mDC, without suppressing CCR7 and CD83 protein expression. Interestingly, mDC exhibited the highest degree of inhibition, for both TNF-α and MIP-1β, whereas the reduction of TNF-α expression was less marked for nonclassical monocytes. Similarly, MSC decreased mRNA levels of interleukin- (IL-) 1β and IL-6 in classical monocytes, CCL3, CCL5, CXCL9, and CXCL10 in classical and nonclassical monocytes, and IL-1β and CXCL10 in mDC. MSC do not impair the expression of maturation markers in monocytes and mDC under our experimental conditions; nevertheless, they hamper the proinflammatory function of monocytes and mDC, which may impede the development of inflammatory immune responses. PMID:26060498

  11. Human Bone Marrow-Derived Mesenchymal Stromal Cells Differentially Inhibit Cytokine Production by Peripheral Blood Monocytes Subpopulations and Myeloid Dendritic Cells.

    PubMed

    Laranjeira, Paula; Gomes, Joana; Pedreiro, Susana; Pedrosa, Monia; Martinho, Antonio; Antunes, Brigida; Ribeiro, Tania; Santos, Francisco; Domingues, Rosario; Abecasis, Manuel; Trindade, Helder; Paiva, Artur

    2015-01-01

    The immunosuppressive properties of mesenchymal stromal/stem cells (MSC) rendered them an attractive therapeutic approach for immune disorders and an increasing body of evidence demonstrated their clinical value. However, the influence of MSC on the function of specific immune cell populations, namely, monocyte subpopulations, is not well elucidated. Here, we investigated the influence of human bone marrow MSC on the cytokine and chemokine expression by peripheral blood classical, intermediate and nonclassical monocytes, and myeloid dendritic cells (mDC), stimulated with lipopolysaccharide plus interferon (IFN)γ. We found that MSC effectively inhibit tumor necrosis factor- (TNF-) α and macrophage inflammatory protein- (MIP-) 1β protein expression in monocytes and mDC, without suppressing CCR7 and CD83 protein expression. Interestingly, mDC exhibited the highest degree of inhibition, for both TNF-α and MIP-1β, whereas the reduction of TNF-α expression was less marked for nonclassical monocytes. Similarly, MSC decreased mRNA levels of interleukin- (IL-) 1β and IL-6 in classical monocytes, CCL3, CCL5, CXCL9, and CXCL10 in classical and nonclassical monocytes, and IL-1β and CXCL10 in mDC. MSC do not impair the expression of maturation markers in monocytes and mDC under our experimental conditions; nevertheless, they hamper the proinflammatory function of monocytes and mDC, which may impede the development of inflammatory immune responses.

  12. 5-Hydroxytryptamine modulates cytokine and chemokine production in LPS-primed human monocytes via stimulation of different 5-HTR subtypes.

    PubMed

    Dürk, Thorsten; Panther, Elisabeth; Müller, Tobias; Sorichter, Stephan; Ferrari, Davide; Pizzirani, Cinzia; Di Virgilio, Francesco; Myrtek, Daniel; Norgauer, Johannes; Idzko, Marco

    2005-05-01

    The neurotransmitter 5-hydroxytryptamine (5-HT), commonly known as serotonin, is released at peripheral sites from activated enterochromaffin cells, mast cells and platelets. In this study we analyzed the biological activity and intracellular signaling of 5-HT in human monocytes. By reverse transcription (RT) and PCR, messenger RNA (mRNA) expression of 5-HT receptor 1E (5-HTR(1E)), 5-HTR(2A), 5-HTR(3), 5-HTR(4) and 5-HTR(7) could be revealed. Functional studies showed that 5-HT modulates the release of IL-1beta, IL-6, IL-8/CXCL8, IL-12p40 and tumor necrosis factor-alpha (TNF-alpha), while it has no effect on the production of IL-18 and IFN-gamma in LPS-stimulated human blood monocytes. Moreover, RT and PCR revealed that 5-HT modulated mRNA levels of IL-6 and IL-8/CXCL8, but did not influence mRNA levels of IL-1beta and TNF-alpha. Pharmacological studies with isotype-selective receptor agonists allowed us to show that 5-HTR(3) subtype up-regulates the LPS-induced production of IL-1beta, IL-6 and IL-8/CXCL8, while it was not involved in TNF-alpha and IL-12p40 secretion. Furthermore, activation of the G(s)-coupled 5-HTR(4) and 5-HTR(7) subtypes increased intracellular cyclic AMP (cAMP) and secretion of IL-1beta, IL-6, IL-12p40 and IL-8/CXCL8, while, on the contrary, it inhibited LPS-induced TNF-alpha release. Interestingly, 5-HTR(1) and 5-HTR(2) agonists did not modulate the LPS-induced cytokine production in human monocytes. Our results point to a new role for 5-HT in inflammation by modulating cytokine production in monocytes via activation of 5-HTR(3), 5-HTR(4) and 5-HTR(7) subtypes.

  13. DNA from Porphyromonas gingivalis and Tannerella forsythia induce cytokine production in human monocytic cell lines.

    PubMed

    Sahingur, S E; Xia, X-J; Alamgir, S; Honma, K; Sharma, A; Schenkein, H A

    2010-04-01

    Toll-like receptor 9 (TLR9) expression is increased in periodontally diseased tissues compared with healthy sites indicating a possible role of TLR9 and its ligand, bacterial DNA (bDNA), in periodontal disease pathology. Here, we determine the immunostimulatory effects of periodontal bDNA in human monocytic cells (THP-1). THP-1 cells were stimulated with DNA of two putative periodontal pathogens: Porphyromonas gingivalis and Tannerella forsythia. The role of TLR9 in periodontal bDNA-initiated cytokine production was determined either by blocking TLR9 signaling in THP-1 cells with chloroquine or by measuring IL-8 production and nuclear factor-kappaB (NF-kappaB) activation in HEK293 cells stably transfected with human TLR9. Cytokine production (IL-1beta, IL-6, and TNF-alpha) was increased significantly in bDNA-stimulated cells compared with controls. Chloroquine treatment of THP-1 cells decreased cytokine production, suggesting that TLR9-mediated signaling pathways are operant in the recognition of DNA from periodontal pathogens. Compared with native HEK293 cells, TLR9-transfected cells demonstrated significantly increased IL-8 production (P < 0.001) and NF-kappaB activation in response to bDNA, further confirming the role of TLR9 in periodontal bDNA recognition. The results of PCR arrays demonstrated upregulation of proinflammatory cytokine and NF-kappaB genes in response to periodontal bDNA in THP-1 cells, suggesting that cytokine induction is through NF-kappaB activation. Hence, immune responses triggered by periodontal bacterial nucleic acids may contribute to periodontal disease pathology by inducing proinflammatory cytokine production through the TLR9 signaling pathway.

  14. Inflammatory Transcriptome Profiling of Human Monocytes Exposed Acutely to Cigarette Smoke

    PubMed Central

    Wright, William R.; Parzych, Katarzyna; Crawford, Damian; Mein, Charles; Mitchell, Jane A.; Paul-Clark, Mark J.

    2012-01-01

    Background Cigarette smoking is responsible for 5 million deaths worldwide each year, and is a major risk factor for cardiovascular and lung diseases. Cigarette smoke contains a complex mixture of over 4000 chemicals containing 1015 free radicals. Studies show smoke is perceived by cells as an inflammatory and xenobiotic stimulus, which activates an immune response. The specific cellular mechanisms driving cigarette smoke-induced inflammation and disease are not fully understood, although the innate immune system is involved in the pathology of smoking related diseases. Methodology/Principle findings To address the impact of smoke as an inflammagen on the innate immune system, THP-1 cells and Human PBMCs were stimulated with 3 and 10% (v/v) cigarette smoke extract (CSE) for 8 and 24 hours. Total RNA was extracted and the transcriptome analysed using Illumina BeadChip arrays. In THP-1 cells, 10% CSE resulted in 80 genes being upregulated and 37 downregulated by ≥1.5 fold after 8 hours. In PBMCs stimulated with 10% CSE for 8 hours, 199 genes were upregulated and 206 genes downregulated by ≥1.5 fold. After 24 hours, the number of genes activated and repressed by ≥1.5 fold had risen to 311 and 306 respectively. The major pathways that were altered are associated with cell survival, such as inducible antioxidants, protein chaperone and folding proteins, and the ubiquitin/proteosome pathway. Conclusions Our results suggest that cigarette smoke causes inflammation and has detrimental effects on the metabolism and function of innate immune cells. In addition, THP-1 cells provide a genetically stable alternative to primary cells for the study of the effects of cigarette smoke on human monocytes. PMID:22363418

  15. Nucleolin, a Shuttle Protein Promoting Infection of Human Monocytes by Francisella tularensis

    PubMed Central

    Barel, Monique; Meibom, Karin; Charbit, Alain

    2010-01-01

    Background Francisella tularensis is a highly virulent facultative intracellular bacterium, disseminating in vivo mainly within host mononuclear phagocytes. After entry into macrophages, F. tularensis initially resides in a phagosomal compartment, whose maturation is then arrested. Bacteria escape rapidly into the cytoplasm, where they replicate freely. We recently demonstrated that nucleolin, an eukaryotic protein able to traffic from the nucleus to the cell surface, acted as a surface receptor for F. tularensis LVS on human monocyte-like THP-1 cells. Methodology/Principal Findings Here, we followed the fate of nucleolin once F. tularensis has been endocytosed. We first confirmed by siRNA silencing experiments that expression of nucleolin protein was essential for binding of LVS on human macrophage-type THP-1 cells. We then showed that nucleolin co-localized with intracellular bacteria in the phagosomal compartment. Strikingly, in that compartment, nucleolin also co-localized with LAMP-1, a late endosomal marker. Co-immunoprecipation assays further demonstrated an interaction of nucleolin with LAMP-1. Co-localization of nucleolin with LVS was no longer detectable at 24 h when bacteria were multiplying in the cytoplasm. In contrast, with an iglC mutant of LVS, which remains trapped into the phagosomal compartment, or with inert particles, nucleolin/bacteria co-localization remained almost constant. Conclusions/Significance We herein confirm the importance of nucleolin expression for LVS binding and its specificity as nucleolin is not involved in binding of another intracellular pathogen as L. monocytogenes or an inert particle. Association of nucleolin with F. tularensis during infection continues intracellularly after endocytosis of the bacteria. The present work therefore unravels for the first time the presence of nucleolin in the phagosomal compartment of macrophages. PMID:21152024

  16. Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells

    SciTech Connect

    Inoue, Kazushi; Akiyama, Tetsu; Toyoshima, Kumao ); Wongsasant, Budsaba )

    1991-12-01

    The mutant c-fgr protein (p58{sup c-fgr/F523}) containing Phe-523 instead of Tyr-523 exhibited transforming activity in NIH 3T3 cells like other protein-tyrosine kinases of the src family, but normal p58{sup c-fgr} (p58{sup c-fgr/wt}) did not. The mutant protein showed tyrosine kinase activity threefold higher than that of the normal protein in vitro. Surprisingly, transfection of the normal c-fgr gene into NIH 3T3 cells resulted in induction of sodium fluoride (NaF)-sensitive {alpha}-naphthyl butyrate esterase ({alpha}-NBE), marker enzyme of cells of monocytic origin, which was not induced in v-src-, v-fgr-, or lyn-transfected NIH 3T3 cells. The NaF-sensitive {alpha}-NBE induced in c-fgr transfectants was shown by isoelectric focusing to have a pI of 5.2 to 5.4, a range which was the same as those for thioglycolate-induced murine peritoneal macrophages and 1{alpha}, 25-dihydroxyvitamin D{sub 3}-treated WEHI-3B cells. Immunoblotting studies with antophosphotyrosine antibodies revealed that 58-, 62-, 75-, 120-, 200-, and 230-kDa proteins were commonly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in cells transfected with the mutated c-fgr. These findings suggest that tyrosine phosphorylation of specific cellular substrate proteins is important in induction of NaF-sensitive {alpha}-NBE and cell transformation by p58{sup c-fgr}.

  17. A calcium-redox feedback loop controls human monocyte immune responses: The role of ORAI Ca2+ channels.

    PubMed

    Saul, Stephanie; Gibhardt, Christine S; Schmidt, Barbara; Lis, Annette; Pasieka, Bastian; Conrad, David; Jung, Philipp; Gaupp, Rosmarie; Wonnenberg, Bodo; Diler, Ebru; Stanisz, Hedwig; Vogt, Thomas; Schwarz, Eva C; Bischoff, Markus; Herrmann, Mathias; Tschernig, Thomas; Kappl, Reinhard; Rieger, Heiko; Niemeyer, Barbara A; Bogeski, Ivan

    2016-03-08

    In phagocytes, pathogen recognition is followed by Ca(2+) mobilization and NADPH oxidase 2 (NOX2)-mediated "oxidative burst," which involves the rapid production of large amounts of reactive oxygen species (ROS). We showed that ORAI Ca(2+) channels control store-operated Ca(2+) entry, ROS production, and bacterial killing in primary human monocytes. ROS inactivate ORAI channels that lack an ORAI3 subunit. Staphylococcal infection of mice reduced the expression of the gene encoding the redox-sensitive Orai1 and increased the expression of the gene encoding the redox-insensitive Orai3 in the lungs or in bronchoalveolar lavages. A similar switch from ORAI1 to ORAI3 occurred in primary human monocytes exposed to bacterial peptides in culture. These alterations in ORAI1 and ORAI3 abundance shifted the channel assembly toward a more redox-insensitive configuration. Accordingly, silencing ORAI3 increased the redox sensitivity of the channel and enhanced oxidation-induced inhibition of NOX2. We generated a mathematical model that predicted additional features of the Ca(2+)-redox interplay. Our results identified the ORAI-NOX2 feedback loop as a determinant of monocyte immune responses.

  18. Reduction of transition metals by human (THP-1) monocytes is enhanced by activators of protein kinase C.

    PubMed

    Wood, J L; Graham, A

    1999-11-01

    Macrophages oxidize low density lipoprotein (LDL) by enzymatic and non-enzymatic mechanisms; however, it is evident that macrophage reduction of transition metals can accelerate LDL oxidation in vitro, and possibly in vivo. Distinct cellular pathways contribute to this process, including trans-plasma membrane electron transport (TPMET), and production of free thiols or superoxide. Here, we explore the role of protein kinase C (PKC) in regulating transition metal reduction by each of these redox-active pathways, in human (THP-1) monocytes. We demonstrate that PKC agonists and/or inhibitors modulate reduction of transition metals by monocytes: both thiol-independent (direct) and thiol-dependent (indirect) pathways for transition metal reduction are enhanced by PKC activation, suggesting a potential strategy for therapeutic intervention.

  19. Human monocyte-derived dendritic cells turn into foamy dendritic cells with IL-17A1[S

    PubMed Central

    Salvatore, Giulia; Bernoud-Hubac, Nathalie; Bissay, Nathalie; Debard, Cyrille; Daira, Patricia; Meugnier, Emmanuelle; Proamer, Fabienne; Hanau, Daniel; Vidal, Hubert; Aricò, Maurizio; Delprat, Christine; Mahtouk, Karène

    2015-01-01

    Interleukin 17A (IL-17A) is a proinflammatory cytokine involved in the pathogenesis of chronic inflammatory diseases. In the field of immunometabolism, we have studied the impact of IL-17A on the lipid metabolism of human in vitro-generated monocyte-derived dendritic cells (DCs). Microarrays and lipidomic analysis revealed an intense remodeling of lipid metabolism induced by IL-17A in DCs. IL-17A increased 2–12 times the amounts of phospholipids, cholesterol, triglycerides, and cholesteryl esters in DCs. Palmitic (16:0), stearic (18:0), and oleic (18:ln-9c) acid were the main fatty acid chains present in DCs. They were strongly increased in response to IL-17A while their relative proportion remained unchanged. Capture of extracellular lipids was the major mechanism of lipid droplet accumulation, visualized by electron microscopy and Oil Red O staining. Besides this foamy phenotype, IL-17A induced a mixed macrophage-DC phenotype and expression of the nuclear receptor NR1H3/liver X receptor-α, previously identified in the context of atherosclerosis as the master regulator of cholesterol homeostasis in macrophages. These IL-17A-treated DCs were as competent as untreated DCs to stimulate allogeneic naive T-cell proliferation. Following this first characterization of lipid-rich DCs, we propose to call these IL-17A-dependent cells “foamy DCs” and discuss the possible existence of foamy DCs in atherosclerosis, a metabolic and inflammatory disorder involving IL-17A. PMID:25833686

  20. Effects of 1 alpha,25-dihydroxyvitamin D3 and cytokines on the expression of MHC antigens, complement receptors and other antigens on human blood monocytes and U937 cells: role in cell differentiation, activation and phagocytosis.

    PubMed Central

    Spittler, A; Willheim, M; Leutmezer, F; Ohler, R; Krugluger, W; Reissner, C; Lucas, T; Brodowicz, T; Roth, E; Boltz-Nitulescu, G

    1997-01-01

    The effect of calcitriol/1 alpha,25-dihydroxyvitamin D3, alone and in combination with cytokines, on the expression of various antigens (Ag) on human peripheral blood monocytes and U937 cells was studied by flow cytometry. Both constitutive and interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6 and tumour necrosis factor-alpha (TNF-alpha)-induced human leucocyte antigen (HLA)-DR, HLA-DP and HLA-DQ Ag expression on monocytes was significantly down-regulated by calcitriol, IL-10 and transforming growth factor-beta (TGF-beta). The effects of calcitriol were concentration dependent and reached maximal inhibitory levels after 3-5 days. Modulation of HLA-DR by calcitriol and IFN-gamma at the protein level correlated with the amount of mRNA specific for the HLA-DR alpha-chain, as judged by Northern blot analysis. The basal as well as IL-4, IL-6, IFN-gamma, TNF-alpha and TGF-beta-driven levels of HLA-ABC Ag were significantly diminished by calcitriol. On U937 cells calcitriol markedly induced CD11a and CD11b expression and weakly up-regulated CD11c whereas on monocytes, constitutive CD11a, CD11b and CD11c expression was significantly down-regulated by calcitriol. The expression of CD14 Ag was strongly induced on U937 cells but only modestly on monocytes. Both the basal level of CD71 and IL-4, IFN-gamma or TNF-alpha-driven expression was diminished on calcitriol-treated U937 cells. In addition, calcitriol suppressed the expression of CD71 Ag on monocytes. The ability of monocytes to phagocytize opsonized Escherichia coli was diminished by calcitriol. Our results demonstrate that calcitriol, alone or in combination with cytokines, modulates expression of MHC, CD11b, CD11c, CD14 and CD71 Ag on both monocytes and U937 cells, and impairs the phagocytic property of monocytes. Images Figure 2 PMID:9135559

  1. A novel approach to induce human DCs from monocytes by triggering 4-1BBL reverse signaling.

    PubMed

    Ju, Songwen; Ju, Songguang; Ge, Yan; Qiu, Hongxia; Lu, Binfeng; Qiu, Yuhua; Fu, Jingxiang; Liu, Gaoqin; Wang, Qin; Hu, Yumin; Shu, Yongqian; Zhang, Xueguang

    2009-10-01

    Dendritic cells (DCs) are responsible for the initiation of immune responses. Our study demonstrates a new pathway for generating a large quantity of stimulatory monocyte-derived DCs (Mo-DCs) from human monocytes using anti-4-1BB ligand (4-1BBL) mAb to trigger reverse signaling. The anti-4-1BBL-driven Mo-DCs (DCs(alpha-4-1BBL)) not only express higher levels of CD86, CD83 and HLA-DR, when compared with the Mo-DCs matured by tumor necrosis factor alpha, but also exhibit a unique phenotype that expresses lower levels of PD-L1. High levels of GM-CSF, M-CSF and Flt3 ligand (FL) were found in the anti-4-1BBL-differentiation culture. Neutralizing M-CSF, GM-CSF and FL inhibited Mo-DC proliferation stimulated by anti-4-1BBL mAb, suggesting that M-CSF, GM-CSF and FL are involved in cell proliferation stimulated by anti-4-1BBL. Further analysis of the DCs(alpha-4-1BBL) showed increased secretion of T(h)1-type cytokines IL-12 and IFN-gamma and decreased secretion of IL-10. DCs(alpha-4-1BBL) induced much stronger proliferative responses in the mixed lymphocyte reaction assay when compared with DCs derived by GM-CSF. Moreover, DCs(alpha-4-1BBL) preferentially induced T(h)1 responses. We have further demonstrated that anti-4-1BBL antibody stimulated nuclear translocation of NF-kappaB from the cytoplasm in monocytes, suggesting that reverse signaling by 4-1BBL is likely responsible for mediating DC differentiation. Collectively, we have found that reverse signaling of 4-1BBL promotes the differentiation of potent T(h)1-inducing DCs from human monocytes.

  2. Simultaneous activation of p38 and JNK by arachidonic acid stimulates the cytosolic phospholipase A2-dependent synthesis of lipid droplets in human monocytes

    PubMed Central

    Guijas, Carlos; Pérez-Chacón, Gema; Astudillo, Alma M.; Rubio, Julio M.; Gil-de-Gómez, Luis; Balboa, María A.; Balsinde, Jesús

    2012-01-01

    Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. This effect appears specific for AA in that it is not mimicked by other fatty acids, whether saturated or unsaturated. LDs are formed by two different routes: (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling, leading to increased triacylglycerol and cholesteryl ester formation utilizing fatty acids coming from the de novo biosynthetic route. Both routes can be dissociated by the arachidonyl-CoA synthetase inhibitor triacsin C, which prevents the former but not the latter. LD formation by AA-induced signaling predominates, accounting for 60–70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A2α (cPLA2α), pointing out this enzyme as a key regulator of AA-induced signaling. LD formation in AA-treated monocytes can also be blocked by the combined inhibition of the mitogen-activated protein kinase family members p38 and JNK, which correlates with inhibition of cPLA2α activation by phosphorylation. Collectively, these results suggest that concomitant activation of p38 and JNK by AA cooperate to activate cPLA2α, which is in turn required for LD formation possibly by facilitating biogenesis of this organelle, not by regulating neutral lipid synthesis. PMID:22949356

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

    PubMed

    Ghosh, Somnath; Ghosh, Anu; Krishna, Malini

    2015-12-01

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

  4. Induction and processing of oxidative clustered DNA lesions in 56Fe-ion-irradiated human monocytes.

    PubMed

    Tsao, Doug; Kalogerinis, Peter; Tabrizi, Isla; Dingfelder, Michael; Stewart, Robert D; Georgakilas, Alexandros G

    2007-07-01

    Space and cosmic radiation is characterized by energetic heavy ions of high linear energy transfer (LET). Although both low- and high-LET radiations can create oxidative clustered DNA lesions and double-strand breaks (DSBs), the local complexity of oxidative clustered DNA lesions tends to increase with increasing LET. We irradiated 28SC human monocytes with doses from 0-10 Gy of (56)Fe ions (1.046 GeV/ nucleon, LET = 148 keV/microm) and determined the induction and processing of prompt DSBs and oxidative clustered DNA lesions using pulsed-field gel electrophoresis (PFGE) and Number Average Length Analysis (NALA). The (56)Fe ions produced decreased yields of DSBs (10.9 DSB Gy(-1) Gbp(-1)) and clusters (1 DSB: approximately 0.8 Fpg clusters: approximately 0.7 Endo III clusters: approximately 0.5 Endo IV clusters) compared to previous results with (137)Cs gamma rays. The difference in the relative biological effectiveness (RBE) of the measured and predicted DSB yields may be due to the formation of spatially correlated DSBs (regionally multiply damaged sites) which result in small DNA fragments that are difficult to detect with the PFGE assay. The processing data suggest enhanced difficulty compared with gamma rays in the processing of DSBs but not clusters. At the same time, apoptosis is increased compared to that seen with gamma rays. The enhanced levels of apoptosis observed after exposure to (56)Fe ions may be due to the elimination of cells carrying high levels of persistent DNA clusters that are removed only by cell death and/or "splitting" during DNA replication.

  5. Daily hypoxia increases basal monocyte HSP72 expression in healthy human subjects.

    PubMed

    Taylor, Lee; Midgley, Adrian W; Chrismas, Bryna; Hilman, Angela R; Madden, Leigh A; Vince, Rebecca V; McNaughton, Lars R

    2011-02-01

    Heat shock protein 72 (HSP72) performs vital roles within the body at rest and during periods of stress. In vitro, research demonstrates HSP72 induction in response to hypoxia. Recently, in vivo, an acute hypoxic exposure (75 min at 2,980 m) was sufficient to induce significant increases in monocyte expressed HSP72 (mHSP72) and a marker of oxidative stress in healthy human subjects. The purpose of the current study was to identify the impact of 10 consecutive days of hypoxic exposures (75 min at 2,980 m) on mHSP72 and erythropoietin (EPO) expression, markers of oxidative stress, and maximal oxygen consumption in graded incremental aerobic exercise. Eight male subjects were exposed to daily normobaric hypoxic exposures for 75 min at 2,980 m for 10 consecutive days, commencing and ceasing at 0930 and 1045, respectively. This stressor was sufficient to induce significant increases in mHSP72, which was significantly elevated from day 2 of the hypoxic exposures until 48 h post-final exposure. Notably, this increase had an initial rapid (30% day on day compared to baseline) and final slow phase (16% day on day compared to baseline) of expression. The authors postulate that 7-day hypoxic exposure in this manner would be sufficient to induce near maximum hypoxia-mediated basal mHSP72 expression. Elevated levels of mHSP72 are associated with acquired thermotolerance and provide cross tolerance to non-related stressors in vivo, the protocol used here may provide a useful tool for elevating mHSP72 in vivo. Aside from these major findings, significant transient daily elevations were seen in a marker of oxidative stress, alongside sustained increases in EPO expression. However, no physiologically significant changes were seen in maximal oxygen consumption or time to exhaustion.

  6. The effect of polyethylene particle chemistry on human monocyte-macrophage function in vitro.

    PubMed

    Boynton, E L; Waddell, J; Meek, E; Labow, R S; Edwards, V; Santerre, J P

    2000-11-01

    Osteolysis remains the most important problem in orthopedic implant failure. Wear debris from the implant contains polyethylene (PE) particulate which has been shown to activate monocyte-derived macrophages (MDM). Although the response of MDM has been shown to be influenced by the size, shape, and chemical type of PE, the effect of chemically altered PE on MDM has not been studied. In this study, human MDM were seeded onto glass coverslips coated with virgin high density (HD)PE and chemically modified HDPE (impregnated with ppm levels of CoCl(2) and oxidized by heat) mixed with type I collagen and cultured for 96 h. Light microscopic evaluation demonstrated consistent phagocytosis of the HDPE particulate that was confirmed by scanning electron and transmission electron microscopy with little evidence of cytotoxicity. Evaluation of pro-inflammatory mediator secretion by MDMs in response to the virgin and chemically modified HDPE revealed significant differences in interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and IL-6 secretion. A significant elevation of IL-1 secretion was observed after initial exposure to virgin HDPE particles compared with controls (p = 0.001). IL-1 secretion was also elevated in the low oxidized particle groups (p = 0.001), whereas the highly oxidized particles were not different than controls. Secretion of both IL-6 (p = 0.03) and TNF-alpha (p = 0.007) were significantly elevated by the low oxidized HDPE particles whereas the virgin and highly oxidized groups showed no difference. The different effects on MDM activation when HDPE surface chemistry was altered, highlight the importance of defining the particle properties when studying the role of MDM activation in in vitro systems and extrapolating these observations to the in vivo situation.

  7. PSP activates monocytes in resting human peripheral blood mononuclear cells: immunomodulatory implications for cancer treatment.

    PubMed

    Sekhon, Bhagwant Kaur; Sze, Daniel Man-Yuen; Chan, Wing Keung; Fan, Kei; Li, George Qian; Moore, Douglas Edwin; Roubin, Rebecca Heidi

    2013-06-15

    Polysaccharopeptide (PSP), from Coriolus versicolor, has been used as an adjuvant to chemotherapy, and has demonstrated anti-tumor and immunomodulating effects. However its mechanism remains unknown. To elucidate how PSP affects immune populations, we compared PSP treatments both with and without prior incubation in phytohaemagglutinin (PHA) - a process commonly used in immune population experimentation. We first standardised a capillary electrophoresis fingerprinting technique for PSP identification and characterisation. We then established the proliferative capability of PSP on various immune populations in peripheral blood mononuclear cells, using flow cytometry, without prior PHA treatment. It was found that PSP significantly increased the number of monocytes (CD14(+)/CD16(-)) compared to controls without PHA. This increase in monocytes was confirmed using another antibody panel of CD14 and MHCII. In contrast, proliferations of T-cells, NK, and B-cells were not significantly changed by PSP. Thus, stimulating monocyte/macrophage function with PSP could be an effective therapeutic intervention in targeting tumors.

  8. Native LDL promotes differentiation of human monocytes to macrophages with an inflammatory phenotype.

    PubMed

    Al-Sharea, Annas; Lee, Man Kit Sam; Moore, Xiao-Lei; Fang, Lu; Sviridov, Dmitri; Chin-Dusting, Jaye; Andrews, Karen L; Murphy, Andrew J

    2016-04-01

    Recruitment of monocytes in atherosclerosis is dependent upon increased levels of plasma lipoproteins which accumulate in the blood vessel wall. The extracellular milieu can influence the phenotype of monocyte subsets (classical: CD14++CD16-, intermediate: CD14+CD16+ and non-classical: CD14dimCD16++) and macrophages (M1 or M2) and consequently the initiation, progression and/or regression of atherosclerosis. However, it is not known what effect lipoproteins, in particular native low-density lipoproteins (nLDL), have on the polarisation of monocyte-derived macrophages. Monocytes were differentiated into macrophages in the presence of nLDL. nLDL increased gene expression of the inflammatory cytokines TNFα and IL-6 in macrophages polarised towards the M1 phenotype while decreasing the M2 surface markers, CD206 and CD200R and the anti-inflammatory cytokines TGFβ and IL-10. Compared to the classical and intermediate subsets, the non-classical subset-derived macrophages had a reduced ability to respond to M1 stimuli (LPS and IFNγ). nLDL enhanced the TNFα and IL-6 gene expression in macrophages from all monocyte subsets, indicating an inflammatory effect of nLDL. Further, the classical and intermediate subsets both responded to M2 stimuli (IL-4) with upregulation of TGFβ and SR-B1 mRNA; an effect, which was reduced by nLDL. In contrast, the non-classical subset failed to respond to IL-4 or nLDL, suggesting it may be unable to polarise into M2 macrophages. Our data suggests that monocyte interaction with nLDL significantly affects macrophage polarisation and that this interaction appears to be subset dependent.

  9. A sea urchin lectin, SUL-1, from the Toxopneustid sea urchin induces DC maturation from human monocyte and drives Th1 polarization in vitro

    SciTech Connect

    Takei, Masao . E-mail: mtakei@fz-borstel.de; Nakagawa, Hideyuki

    2006-05-15

    The sea urchin Toxopneustes pileolus belonging to the family Toxopneustidae, they have well-developed globiferous pedicellariae with pharmacologically active substances. We have purified a novel sea urchin lectin-1 (SUL-1) from the large globiferous pedicellariae of T. pileolus. Dendritic cells (DC) are professional APC and play a pivotal role in controlling immune responses. This study investigated whether SUL-1 can drive DC maturation from human immature monocyte-derived DC in vitro. Human monocytes were cultured with GM-CSF and IL-4 for 6 days followed by another 1 day in the presence of SUL-1 or LPS. DC harvested on day 7 were examined using functional assays. The expression levels of CD1a, CD80, CD83, CD86 and HLA-DR as expressed by mean fluorescence intensity (MFI) on DC differentiated from immature DC after culture with 1.0 {mu}g/ml of SUL-1 for 1 day were enhanced and decreased endocytic activity. SUL-1-treated DC also displayed enhanced T cell stimulatory capacity in an MLR, as measured by T cell proliferation. Cell surface expression of CD80, CD83 and CD86 on SUL-1-treated DC was inhibited by anti-DC-SIGN mAb, while anti-DC-SIGN mAb had no influence on allogeneic T cell proliferation by SUL-1-treated DC. DC differentiated with SUL-1 induced the differentiation of naive T cell towards a helper T cell type 1 (Th1) response at DC/T (1:5) cells ratio depending on IL-12 secretion. In CTL assay, the production of IFN-{gamma} and {sup 51}Cr release on SUL-1-treated DC were more augmented than of immature DC or LPS-treated DC. SUL-1-treated DC expressed CCR7 and had a high migration to MIP-3{beta}. Intracellular Ca{sup 2+} mobilization in SUL-1-treated DC was also induced by MIP-3{beta}. These results suggest that SUL-1 bindings to DC-SIGN on surface of immature DC may lead to differentiate DC from immature DC. Moreover, it suggests that SUL-1 may be used on DC-based vaccines for cancer immunotherapy.

  10. Human and mouse monocytes display distinct signalling and cytokine profiles upon stimulation with FFAR2/FFAR3 short-chain fatty acid receptor agonists

    PubMed Central

    Ang, Zhiwei; Er, Jun Zhi; Tan, Nguan Soon; Lu, Jinhua; Liou, Yih-Cherng; Grosse, Johannes; Ding, Jeak Ling

    2016-01-01

    Knockout mice studies implicate the mammalian short-chain fatty acid (SCFA) receptors, FFAR2 and FFAR3– in colitis, arthritis and asthma. However, the correlation with human biology is uncertain. Here, we detected FFAR2 and FFAR3 expression in human monocytes via immunohistochemistry. Upon treatment with acetate SCFA or FFAR2- and FFAR3-specific synthetic agonists, human monocytes displayed elevated p38 phosphorylation and attenuated C5, CCL1, CCL2, GM-CSF, IL-1α, IL-1β and ICAM-1 inflammatory cytokine expression. Acetate and FFAR2 agonist treatment also repressed Akt and ERK2 signalling. Surprisingly, mouse monocytes displayed a distinct response to acetate treatment, elevating GM-CSF, IL-1α, and IL-1β cytokine expression. This effect persisted in FFAR2/3-knockout mouse monocytes and was not reproduced by synthetic agonists, suggesting a FFAR2/3 independent mechanism in mice. Collectively, we show that SCFAs act via FFAR2/3 to modulate human monocyte inflammatory responses– a pathway that is absent in mouse monocytes. PMID:27667443

  11. Sinomenine promotes differentiation but impedes maturation and co-stimulatory molecule expression of human monocyte-derived dendritic cells.

    PubMed

    Chen, Yongwen; Yang, Chengying; Jin, Naishi; Xie, Zhunyi; Fei, Lie; Jia, Zhengcai; Wu, Yuzhang

    2007-08-01

    Dendritic cells (DC) excel at presenting antigen to T cells and thus make a key contribution to the induction of primary and secondary immune responses. Sinomenine has been used for centuries in the treatment of patients with autoimmune diseases as it possesses immunosuppressive and anti-inflammatory activities. However, the effect of sinomenine on the differentiation, maturation, and functionality of DC derived from monocytes has not been studied. We show here that DC differentiation is promoted when monocytes are treated with GM-CSF and IL-4 (IL-4) in the presence of sinomenine (200 microg/ml), as evidenced by the upregulation of CD1a while CD14 was decreased. In addition, incubation of immature DC with sinomenine significantly blunted lipopolysaccharide (LPS)-induced DC maturation, as shown by the reduction of expression of the maturation marker CD83 and co-stimulatory molecules, including CD86, B7-H1, and CD40. Moreover, sinomenine also prevented decreases in antigen (FITC-Dextran or Lucifer Yellow) uptake by LPS-treated DC. Mixed lymphocyte reactions (MLRs) revealed that sinomenine-treated DC impede the secretion of the cytokines IL-2 and IFN-gamma by co-cultured CD4(+) T cells. Therefore, modulation of DC differentiation, maturation, and functionality by sinomenine is of potential relevance to its immunomodulatory effects in controlling specific immune responses in autoimmune diseases, transplantation, and other immune-mediated conditions.

  12. New evidence for the therapeutic potential of curcumin to treat nonalcoholic fatty liver disease in humans

    PubMed Central

    Inzaugarat, María Eugenia; De Matteo, Elena; Baz, Placida; Lucero, Diego; García, Cecilia Claudia; Gonzalez Ballerga, Esteban; Daruich, Jorge; Sorda, Juan Antonio; Wald, Miriam Ruth

    2017-01-01

    Introduction The immune system acts on different metabolic tissues that are implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Leptin and linoleic acid have the ability to potentially affect immune cells, whereas curcumin is a known natural polyphenol with antioxidant and anti-inflammatory properties. Aims This study was designed to evaluate the pro-inflammatory and pro-oxidant effects of leptin and linoleic acid on immune cells from patients with NAFLD and to corroborate the modulatory effects of curcumin and its preventive properties against the progression of NAFLD using a high-fat diet (HFD)-induced NAFLD/nonalcoholic steatohepatitis mouse model. Results The ex vivo experiments showed that linoleic acid increased the production of reactive oxygen species in monocytes and liver macrophages, whereas leptin enhanced tumor necrosis factor-α (TNF-α) production in monocytes and interferon-γ production in circulating CD4+ cells. Conversely, oral administration of curcumin prevented HFD-induced liver injury, metabolic alterations, intrahepatic CD4+ cell accumulation and the linoleic acid- and leptin- induced pro-inflammatory and pro-oxidant effects on mouse liver macrophages. Conclusion Our findings provide new evidence for the therapeutic potential of curcumin to treat human NAFLD. However, the development of a preventive treatment targeting human circulating monocytes and liver macrophages as well as peripheral and hepatic CD4+ cells requires additional research. PMID:28257515

  13. Biochemical and morphological characterization of the killing of human monocytes by a leukotoxin derived from Actinobacillus actinomycetemcomitans.

    PubMed Central

    Taichman, N S; Dean, R T; Sanderson, C J

    1980-01-01

    A potent, heat-labile leukotoxic material was extracted from Actinobacillus actinomycetemcomitans (strain Y4), an anaerobic gram-negative microorganism originally isolated from subgingival plaque in a patient with juvenile periodontitis. The cytopathic effects of Y4 toxin on purified monocytes were studied by the extracellular release of radioactive cytoplasmic markers and cell enzymes and by time-lapse microcinematography. Y4 toxin rapidly bound to the cells, producing dose- and time-dependent alterations culminating in cell death and release of intracellular constituents into the culture medium. The evidence to be presented suggests that the cell membrane of the monocyte may be the primary target in the development of these phenomena. Previous studies have shown that Y4 toxin also kills human polymorphonuclear leukocytes but not other cell types. It is conceivable that disruption of polymorphonuclear leukocytes and monocytes by Y4 toxin in the gingival crevice area may be relevant in the pathogenesis of juvenile periodontitis. Images Fig. 1 PMID:6155347

  14. Rhein lysinate inhibits monocyte adhesion to human umbilical vein endothelial cells by blocking p38 signaling pathway.

    PubMed

    Lin, Yajun; Zhen, Yongzhan; Liu, Jiang; Wei, Jie; Tu, Ping; Hu, Gang

    2013-11-01

    The objective of this study was to investigate the effect of rhein lysinate (RHL) on monocyte adhesion and its mechanism. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the growth inhibition by drugs. The monocyte chemoattractant protein (MCP)-1 levels were assayed using MCP-1 ELISA. The expression of proteins was detected by Western blotting analysis. The results indicated that RHL inhibited monocyte adhesion in a dose- and time-dependent manner. RHL (<20 μmol/L) and lipopolysaccharide (LPS) had no effect on viability of human umbilical vein endothelial cells. Therefore, 20 μmol/L RHL was selected for this study. RHL inhibited secretion of MCP-1 induced by LPS and expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1. In the meantime, both RHL and p38 inhibitor (SB203580) inhibited phosphorylation of p38 and mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2) and transcription and expression of ICAM-1 and VCAM-1. In conclusion, RHL inhibits the transcription and expression of ICAM-1 and VCAM-1 by the p38/MAPKAPK-2 signaling pathway, and the effect of RHL on transcription and expression of ICAM-1 and VCAM-1 is similar to p38 inhibitor. RHL could be a prophylactic drug for atherosclerosis.

  15. Methamphetamine and HIV-1 gp120 effects on lipopolysaccharide stimulated matrix metalloproteinase-9 production by human monocyte-derived macrophages.

    PubMed

    Reynolds, Jessica L; Mahajan, Supriya D; Aalinkeel, Ravikumar; Nair, Bindukumar; Sykes, Donald E; Schwartz, Stanley A

    2011-01-01

    Monocytes/macrophages are a primary source of human immunodeficiency virus (HIV-1) in the central nervous system (CNS). Macrophages infected with HIV-1 produce a plethora of factors, including matrix metalloproteinase-9 (MMP-9) that may contribute to the development of HIV-1-associated neurocognitive disorders (HAND). MMP-9 plays a pivotal role in the turnover of the extracellular matrix (ECM) and functions to remodel cellular architecture. We have investigated the role of methamphetamine and HIV-1 gp120 in the regulation of lipopolysaccaride (LPS) induced-MMP-9 production in monocyte-derived macrophages (MDM). Here, we show that LPS-induced MMP-9 gene expression and protein secretion are potentiated by incubation with methamphetamine alone and gp120 alone. Further, concomitant incubation with gp120 and methamphetamine potentiated LPS-induced MMP-9 expression and biological activity in MDM. Collectively methamphetamine and gp120 effects on MMPs may modulate remodeling of the extracellular environment enhancing migration of monocytes/macrophages to the CNS.

  16. [Effects of crocetin on VCAM-1 expression in human umbilical vein endothelial cells and monocyte-endothelial cell adhesion].

    PubMed

    Zheng, Shu-guo; Zhao, Meng-qiu; Ren, You-nan; Yang, Jie-ren; Qian, Zhi-yu

    2015-01-01

    Crocetin, a naturally occurring carotenoid, possesses antioxidant and antiatherosclerotic properties, of which the underlying mechanism remains unclear. In the present study, we examined the effects of crocetin (0.1, 1, 10 μmol·L(-1)) on angiotensin II (Ang II, 0.1 μmol·L(-1)) induced expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) and monocyte-endothelial cell adhesion. The effects of crocetin on the activation of nuclear factor kappa B (NF-κB) and intracellular reactive oxygen species (ROS) were also observed. The results demonstrated that crocetin notably suppressed Ang II induced NF-κB activation (P<0.01) and VCAM-1 expression (P<0.05, P<0.01) in HUVECs, accompanied by a markedly reduced monocyte-endothelial cell adhesion (P<0.05, P<0.01). In addition, preincubation with crocetin resulted in a significant enhancement of cellular antioxidant capacity (P<0.05, P<0.01), while Ang II induced intracellular ROS decreased markedly (P<0.05, P<0.01). These results indicated that crocetin was capable of suppressing Ang II induced VCAM-1 expression and monocyte-endothelial cell adhesion by suppression of NF-κB activation, which might be derived from the enhancement of antioxidant capacity and subsequent reduction of intracellular ROS.

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

    PubMed

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

    2015-07-01

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

  18. Successful Isolation of Infectious and High Titer Human Monocyte-Derived HIV-1 from Two Subjects with Discontinued Therapy

    PubMed Central

    Zhu, Haiying; Andrus, Thomas; Ivanov, Sergei B.; Pan, Charlotte; Dolores, Jazel; Dann, Gregory C.; Zhou, Michael; Forte, Dominic; Yang, Zihuan; Holte, Sarah; Corey, Lawrence; Zhu, Tuofu

    2013-01-01

    Background HIV-1 DNA in blood monocytes is considered a viral source of various HIV-1 infected tissue macrophages, which is also known as “Trojan horse” hypothesis. However, whether these DNA can produce virions has been an open question for years, due to the inability of isolating high titer and infectious HIV-1 directly from monocytes. Results In this study, we demonstrated successful isolation of two strains of M-HIV-1 (1690 M and 1175 M) from two out of four study subjects, together with their in vivo controls, HIV-1 isolated from CD4+ T-cells (T-HIV-1), 1690 T and 1175 T. All M- and T- HIV-1 isolates were detected CCR5-tropic. Both M- HIV-1 exhibited higher levels of replication in monocyte-derived macrophages (MDM) than the two T- HIV-1. Consistent with our previous reports on the subject 1175 with late infection, compartmentalized env C2-V3-C3 sequences were identified between 1175 M and 1175 T. In contrast, 1690 M and 1690 T, which were isolated from subject 1690 with relatively earlier infection, showed homogenous env C2-V3-C3 sequences. However, multiple reverse transcriptase (RT) inhibitor resistance-associated variations were detected in the Gag-Pol region of 1690 M, but not of 1690 T. By further measuring HIV DNA intracellular copy numbers post-MDM infection, 1690 M was found to have significantly higher DNA synthesis efficiency than 1690 T in macrophages, indicating a higher RT activity, which was confirmed by AZT inhibitory assays. Conclusions These results suggested that the M- and T- HIV-1 are compartmentalized in the two study subjects, respectively. Therefore, we demonstrated that under in vitro conditions, HIV-1 infected human monocytes can productively release live viruses while differentiating into macrophages. PMID:23741458

  19. SARS-CoV regulates immune function-related gene expression in human monocytic cells.

    PubMed

    Hu, Wanchung; Yen, Yu-Ting; Singh, Sher; Kao, Chuan-Liang; Wu-Hsieh, Betty A

    2012-08-01

    Severe acute respiratory syndrome (SARS) is characterized by acute respiratory distress syndrome (ARDS) and pulmonary fibrosis, and monocytes/macrophages are the key players in the pathogenesis of SARS. In this study, we compared the transcriptional profiles of SARS coronavirus (SARS-CoV)-infected monocytic cells against that infected by coronavirus 229E (CoV-229E). Total RNA was extracted from infected DC-SIGN-transfected monocytes (THP-1-DC-SIGN) at 6 and 24 h after infection, and the gene expression was profiled in oligonucleotide-based microarrays. Analysis of immune-related gene expression profiles showed that at 24 h after SARS-CoV infection: (1) IFN-α/β-inducible and cathepsin/proteasome genes were downregulated; (2) hypoxia/hyperoxia-related genes were upregulated; and (3) TLR/TLR-signaling, cytokine/cytokine receptor-related, chemokine/chemokine receptor-related, lysosome-related, MHC/chaperon-related, and fibrosis-related genes were differentially regulated. These results elucidate that SARS-CoV infection regulates immune-related genes in monocytes/macrophages, which may be important to the pathogenesis of SARS.

  20. HIV-1 gp120 envelope glycoprotein determinants for cytokine burst in human monocytes

    PubMed Central

    Coutu, Mathieu; Prévost, Jérémie; Brassard, Nathalie; Peres, Adam; Stegen, Camille; Madrenas, Joaquín; Kaufmann, Daniel E.; Finzi, Andrés

    2017-01-01

    The first step of HIV infection involves the interaction of the gp120 envelope glycoprotein to its receptor CD4, mainly expressed on CD4+ T cells. Besides its role on HIV-1 entry, the gp120 has been shown to be involved in the production of IL-1, IL-6, CCL20 and other innate response cytokines by bystander, uninfected CD4+ T cells and monocytes. However, the gp120 determinants involved in these functions are not completely understood. Whether signalling leading to cytokine production is due to CD4 or other receptors is still unclear. Enhanced chemokine receptor binding and subsequent clustering receptors may lead to cytokine production. By using a comprehensive panel of gp120 mutants, here we show that CD4 binding is mandatory for cytokine outburst in monocytes. Our data suggest that targeting monocytes in HIV-infected patients might decrease systemic inflammation and the potential tissue injury associated with the production of inflammatory cytokines. Understanding how gp120 mediates a cytokine burst in monocytes might help develop new approaches to improve the chronic inflammation that persists in these patients despite effective suppression of viremia by antiretroviral therapy. PMID:28346521

  1. Enhanced production of the chemotactic cytokines interleukin-8 and monocyte chemoattractant protein-1 in human abdominal aortic aneurysms.

    PubMed Central

    Koch, A. E.; Kunkel, S. L.; Pearce, W. H.; Shah, M. R.; Parikh, D.; Evanoff, H. L.; Haines, G. K.; Burdick, M. D.; Strieter, R. M.

    1993-01-01

    Inflammatory leukocytes play a central role in the pathogenesis of human atherosclerotic disease, from early atherogenesis to the late stages of atherosclerosis, such as aneurysm formation. We have shown previously that human abdominal aortic aneurysms are characterized by the presence of numerous chronic inflammatory cells throughout the vessel wall (Am J Pathol 1990, 137: 1199-1213). The signals that attract lymphocytes and monocytes into the aortic wall in aneurysmal disease remain to be precisely defined. We have studied the production of the chemotactic cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by aortic tissues obtained from 47 subjects. We compared the antigenic production of these cytokines by explants of: 1) human abdominal aneurysmal tissue, 2) occlusive (atherosclerotic) aortas, and 3) normal aortas. IL-8, which is chemotactic for neutrophils, lymphocytes, and endothelial cells was liberated in greater quantities by abdominal aortic aneurysms than by occlusive or normal aortas. Using immunohistochemistry, macrophages, and to a lesser degree endothelial cells, were found to be positive for the expression of antigenic IL-8. Similarly, MCP-1, a potent chemotactic cytokine for monocytes/macrophages, was released by explants from abdominal aortic aneurysms in greater quantities than by explants from occlusive or normal aortas. Using immunohistochemistry, the predominant MCP-1 antigen-positive cells were macrophages and to a lesser extent smooth muscle cells. Our results indicate that human abdominal aortic aneurysms produce IL-8 and MCP-1, both of which may serve to recruit additional inflammatory cells into the abdominal aortic wall, hence perpetuating the inflammatory reaction that may result in the pathology of vessel wall destruction and aortic aneurysm formation. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:8494046

  2. Induction of synthesis and secretion of interleukin 1 beta in the human monocytic THP-1 cells by human serum albumins modified with methylglyoxal and advanced glycation endproducts.

    PubMed

    Westwood, M E; Thornalley, P J

    1996-04-01

    Human serum albumin modified with 1-2 methylglyoxal residues per molecule of protein (MGmin-HSA) stimulated the synthesis and secretion of interleukin 1 beta (IL-1 beta) from human monocytic THP-1 cells in vitro. It was a more potent inducer of IL-1 beta synthesis than human serum albumin highly-modified with glucose-derived advanced glycation endproducts (AGE-HSA). With 20 microM ligand. IL-1 beta synthesis was (pg/10(6) cells): MGmin-HSA 484.5 +/- 50.3; AGE-HSA 30.6 +/- 2.0 (n = 3). IL-1 beta synthesis increased markedly with MGmin-HSA concentrations > 5 microM. IL-1 beta synthesis and secretion from monocytes in response to methylglyoxal-modified proteins in vivo may contribute to the development of macro- and micro-angiopathy, particularly in diabetes mellitus.

  3. Activation and measurement of NLRP3 inflammasome activity using IL-1β in human monocyte-derived dendritic cells.

    PubMed

    Fernandez, Melissa V; Miller, Elizabeth A; Bhardwaj, Nina

    2014-05-22

    Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control(1) (,) (2) . Interleukin-1β is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection(1-5). Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1β secretion(6). Pro-IL-1β protein expression is limited in resting cells; therefore a priming signal is required for IL-1β transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1β secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells (moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin. Monocyte derived DCs are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

  4. Immune complexes (IC) down-regulate the basal and interferon-γ-induced expression of MHC Class II on human monocytes

    PubMed Central

    Barrionuevo, P; Beigier-Bompadre, M; De La Barrera, S; Alves-Rosa, M F; Fernandez, G; Palermo, M S; Isturiz, M A

    2001-01-01

    The interaction of Fc receptors for IgG (FcγRs) on monocytes/macrophages with immune complexes (IC) triggers regulatory and effector functions. Previous studies have shown that FcγR–IC interactions inhibit the IFN-γ-induced expression of MHC class II in murine macrophages. However, the mechanism(s) responsible for these effects have not been elucidated. In addition, whether this IC-dependent effect also occurs in human cells is not known. Taking into account the fact that IC and IFN-γ are frequently found in infections and autoimmune disorders, together with the crucial role MHC class II molecules play in the regulation of immune response, we explored the effect and mechanism of IC-induced MHC class II down-regulation in human peripheral blood mononuclear cells (PBMC). This effect was studied either in the presence or absence of IFN-γ. We demonstrate that IC exert a drastic inhibition of basal and IFN-γ-induced expression of MHC class II on human monocytes. This effect was mediated through the interaction of IC with both FcγRI and FcγRII. Moreover, similar results were obtained using supernatants from IC-treated PBMC. The IC-induced down-regulation of MHC class II is abrogated by pepstatin and phosphoramidon, supporting the role of aspartic protease(s) and metalloprotease(s) in this process. In parallel with MHC class II expression, antigen presentation was markedly inhibited in the presence of IC. PMID:11529917

  5. Energy Metabolism of Monocytic Ehrlichia

    DTIC Science & Technology

    1989-03-01

    Security Classification) Energy metabolism of monocytic Ehrlichia 12. PERSONAL AU1TOR(S) Weiss E, Williams JC, Dasch GA, Kang Y 13a. TYPE OF REPORT 13b...monocytic Ehrlichia (intracellular bacteria/animal pathogens/human pathogens) EMILIO WEISS*t, JIM C. WILLIAMSO§, GREGORY A. DASCH*, AND YUAN-HSU KANG...by Carl R. Woese, December 1, 1988 ABSTRACT We investigated if the monocytic Ehrlichia some ATP from the metabolism of glutamine. as is the case are

  6. Human mesenchymal stem cells shift CD8+ T cells towards a suppressive phenotype by inducing tolerogenic monocytes.

    PubMed

    Hof-Nahor, Irit; Leshansky, Lucy; Shivtiel, Shoham; Eldor, Liron; Aberdam, Daniel; Itskovitz-Eldor, Joseph; Berrih-Aknin, Sonia

    2012-10-01

    The mechanisms underlying the immunomodulatory effects of mesenchymal stem cells (MSCs) have been investigated under extreme conditions of strong T cell activation, which induces the rapid death of activated lymphocytes. The objective of this study was to investigate these mechanisms in the absence of additional polyclonal activation. In co-cultures of peripheral mononuclear blood cells with human MSCs (hereafter referred to as hMSCs), we observed a striking decrease in the level of CD8 expression on CD8+ cells, together with decreased expression of CD28 and CD44, and impaired production of IFN-gamma and Granzyme B. This effect was specific to hMSCs, because it was not observed with several other cell lines. Downregulation of CD8 expression required CD14+ monocytes to be in direct contact with the CD8+ cells, whereas the effects of hMSCs on the CD14+ cells were essentially mediated by soluble factors. The CD14+ monocytes exhibited a tolerogenic pattern when co-cultured with hMSCs, with a clear decrease in CD80 and CD86 co-stimulatory molecules, and an increase in the inhibitory receptors ILT-3 and ILT-4. CD8+ cells that were preconditioned by MSCs had similar effects on monocytes and were able to inhibit lymphocyte proliferation. Injection of hMSCs in humanized NSG mice showed similar trends, in particular decreased levels of CD44 and CD28 in human immune cells. Our study demonstrates a new immunomodulation mechanism of action of hMSCs through the modulation of CD8+ cells towards a non-cytotoxic and/or suppressive phenotype. This mechanism of action has to be taken into account in clinical trials, where it should be beneficial in grafts and autoimmune diseases, but potentially detrimental in malignant diseases.

  7. The Pelargonium sidoides Extract EPs 7630 Drives the Innate Immune Defense by Activating Selected MAP Kinase Pathways in Human Monocytes

    PubMed Central

    Witte, Katrin; Koch, Egon; Volk, Hans-Dieter; Wolk, Kerstin; Sabat, Robert

    2015-01-01

    Pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. However, the effects of Pelargonium sidoides and a special extract prepared from its roots (EPs 7630) on human immune cells are not fully understood. Here we demonstrate that EPs 7630 induced a rapid and dose-dependent production of TNF-α, IL-6, and IL-10 by human blood immune cells. This EPs 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. The search for EPs 7630 target cells revealed that T-cells did not respond to EPs 7630 stimulation by production of TNF-α, IL-6, or IL-10. Furthermore, pretreatment of T-cells with EPs 7630 did not modulate their TNF-α, IL-6, and IL-10 secretion during subsequent activation. In contrast to lymphocytes, monocytes showed clear intracellular TNF-α staining after EPs 7630 treatment. Accordingly, EPs 7630 predominantly provoked activation of MAP kinases and inhibition of p38 strongly reduced the monocyte TNF-α production. The pretreatment of blood immune cells with EPs 7630 lowered their secretion of TNF-α and IL-10 and caused an IL-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. In summary, we demonstrate that EPs 7630 activates human monocytes, induces MAP kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive Th17 and Th22 cells. PMID:26406906

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

    PubMed

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

    1985-08-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  10. Carboxylated single-walled carbon nanotubes induce an inflammatory response in human primary monocytes through oxidative stress and NF-κB activation

    NASA Astrophysics Data System (ADS)

    Ye, Shefang; Zhang, Honggang; Wang, Yifang; Jiao, Fei; Lin, Cuilin; Zhang, Qiqing

    2011-09-01

    A mechanistic understanding of interactions between carbon nanotubes (CNTs) and living systems has become imperative owing to the growing nanomedicine applications and the mounting societal concerns on nanosafety. The addition of different chemical groups leads to a significant change in the properties of CNTs, and the resulting functionalized CNTs are generating great interest in many biological applications, such as biosensors and transporters. This study aimed to assess the toxicity exhibited by carboxylic acid functionalized single-walled CNTs (SWCNTs) (with a diameter of 1-2 nm and mean length of 500 nm) and to elucidate possible molecular mechanisms underlying the biological effects of carboxylated SWCNTs in human primary monocytes. The results demonstrated that carboxylated SWCNTs were cytotoxic, triggering apoptosis and G2/M phase arrest in human primary monocytes. Flow cytometric and confocal microscopic analysis indicated that internalized carboxylated SWCNTs were mainly accumulated in the cytoplasm. Exposure of human primary monocytes to carboxylated SWCNTs led to interleukin-8 (IL-8) and interleukin-6 (IL-6) expression, reactive oxygen species (ROS) production, and nuclear factor-kappa B (NF-κB) activation in human primary monocytes. Pretreatment of human primary monocytes with antioxidants or NF-κB-specific inhibitor before exposure to carboxylated SWCNTs significantly abolished carboxylated SWCNTs-induced IL-8 and IL-6 expression. These results provide novel insights into the carboxylated SWCNTs-mediated chemokine induction and inflammatory responses in vitro.

  11. Mitochondrial DAMPs Induce Endotoxin Tolerance in Human Monocytes: An Observation in Patients with Myocardial Infarction

    PubMed Central

    Hernández-Jiménez, Enrique; Moreno-González, Raúl; Toledano, Víctor; Fernández-Velasco, María; Vallejo-Cremades, Maria T.; Esteban-Burgos, Laura; de Diego, Rebeca Pérez; Llamas-Matias, Miguel A.; García-Arumi, Elena; Martí, Ramón; Boscá, Lisardo; Andreu, Antoni L.; López-Sendón, José Luis; López-Collazo, Eduardo

    2014-01-01

    Monocyte exposure to mitochondrial Danger Associated Molecular Patterns (DAMPs), including mitochondrial DNA (mtDNA), induces a transient state in which these cells are refractory to further endotoxin stimulation. In this context, IRAK-M up-regulation and impaired p65 activity were observed. This phenomenon, termed endotoxin tolerance (ET), is characterized by decreased production of cytokines in response to the pro-inflammatory stimulus. We also show that monocytes isolated from patients with myocardial infarction (MI) exhibited high levels of circulating mtDNA, which correlated with ET status. Moreover, a significant incidence of infection was observed in those patients with a strong tolerant phenotype. The present data extend our current understanding of the implications of endotoxin tolerance. Furthermore, our data suggest that the levels of mitochondrial antigens in plasma, such as plasma mtDNA, should be useful as a marker of increased risk of susceptibility to nosocomial infections in MI and in other pathologies involving tissue damage. PMID:24797663

  12. Virulent Shigella flexneri causes damage to mitochondria and triggers necrosis in infected human monocyte-derived macrophages.

    PubMed

    Koterski, James F; Nahvi, Massoumeh; Venkatesan, Malabi M; Haimovich, Beatrice

    2005-01-01

    Shigella flexneri is a gram-negative bacterium that causes bacillary dysentery in humans that is characterized by an acute inflammatory response of the colon. The fate of phagocytes that are infected in vitro with virulent Shigella has been the subject of some investigation and debate. In this study we found that virulent Shigella caused a rapid increase in the cell membrane permeability of infected human monocyte-derived macrophages (HMDM) but not in the cell membrane permeability of monocytes, as demonstrated by the uptake of fluorescent vital dyes. Within 2 h of infection, 59% +/- 6% of the HMDM and monocytes were stained with propidium iodide. Treatment of the cells with the inhibitors of caspases YVAD and zVAD, the antioxidants N-acetyl-l-cysteine and butylated hydroxyanisole, or an inhibitor of NADPH oxidase, diphenyleniodonium, did not alter the infection outcome. Importantly, we found that virulent Shigella caused a rapid drop in the ATP level to about 50% in infected HMDM. Furthermore, using a combination of fluorescent vital dyes and mitochondrial membrane potential-sensitive dyes, we observed that cells that exhibited a permeable cell membrane were not stained by the mitochondrion-specific dyes, indicating that the mitochondrial membrane potential was lost in these cells. We also observed infected cells that were not stained with either type of dye, indicating that the loss of the mitochondrial membrane potential preceded the increase in cell membrane permeability. Taken together, our studies showed that virulent Shigella flexneri targets the host cell mitochondria for destruction. This activity may account for the necrotic cell death precipitated by these pathogens.

  13. Short-term in vitro responses of human peripheral blood monocytes to ferritic stainless steel fiber networks.

    PubMed

    Spear, Rose L; Brooks, Roger A; Markaki, Athina E

    2013-05-01

    Beneficial effects on bone-implant bonding may accrue from ferromagnetic fiber networks on implants which can deform in vivo inducing controlled levels of mechanical strain directly in growing bone. This approach requires ferromagnetic fibers that can be implanted in vivo without stimulating undue inflammatory cell responses or cytotoxicity. This study examines the short-term in vitro responses, including attachment, viability, and inflammatory stimulation, of human peripheral blood monocytes to 444 ferritic stainless steel fiber networks. Two types of 444 networks, differing in fiber cross section and thus surface area, were considered alongside austenitic stainless steel fiber networks, made of 316L, a widely established implant material. Similar high percent seeding efficiencies were measured by CyQuant® on all fiber networks after 48 h of cell culture. Extensive cell attachment was confirmed by fluorescence and scanning electron microscopy, which showed round monocytes attached at various depths into the fiber networks. Medium concentrations of lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-α) were determined as indicators of viability and inflammatory responses, respectively. Percent LDH concentrations were similar for both 444 fiber networks at all time points, whereas significantly lower than those of 316L control networks at 24 h. All networks elicited low-level secretions of TNF-α, which were significantly lower than that of the positive control wells containing zymosan. Collectively, the results indicate that 444 networks produce comparable responses to medical implant grade 316L networks and are able to support human peripheral blood monocytes in short-term in vitro cultures without inducing significant inflammatory or cytotoxic effects.

  14. Immunomodulatory properties of human serum immunoglobulin A: anti-inflammatory and pro-inflammatory activities in human monocytes and peripheral blood mononuclear cells

    PubMed Central

    Olas, K; Butterweck, H; Teschner, W; Schwarz, H P; Reipert, B M

    2005-01-01

    Our study investigated the immunomodulatory activities of human plasma-derived serum immunoglobulin (Ig)A. Previous findings seem contradictory indicating either pro- or anti-inflammatory activities. We used serum IgA purified from large plasma pools and studied the modulation of the release of cytokines and chemokines from resting and lipopolysaccharide (LPS, endotoxin)-stimulated human adherent monocytes and human peripheral blood mononuclear cells (PBMC). Our results indicate that IgA down-modulates the release of the pro-inflammatory chemokines monocyte chemoattractant protein (MCP) 1, macrophage inflammatory protein (MIP) 1α and MIP1β from LPS-stimulated PBMC and the release of MCP1, MIP1α and MIP1β from LPS-stimulated monocytes. Furthermore, we confirmed previous reports that plasma-derived serum IgA down-modulates the release of the pro-inflammatory cytokines, interleukin (IL)-6 and tumour necrosis factor (TNF)-α, from LPS-stimulated monocytes and PBMC, and up-regulates the release of IL-1 receptor antagonist (IL-1RA) from resting and LPS-stimulated monocytes and resting PBMC. This IgA-mediated up-regulation of IL-1RA is independent of the simultaneous up-regulation of IL-1β release, as shown by blocking the biological activity of IL-1β with a neutralizing antibody. On the other hand, we also found an IgA-induced pro-inflammatory activity, namely IgA-mediated up-regutation of the release of pro-inflammatory IL-1β as well as down-regulation of the anti-inflammatory cytokines IL-10 and IL-12p40 from LPS-stimulated monocytes and PBMC and a down-regulation of transforming growth factor (TGF)-β from resting and LPS-stimulated PBMC. We conclude that human serum IgA has both an anti-inflammatory and a pro-inflammatory capacity and this dual capacity might contribute to the feedback mechanisms maintaining a balance between pro-inflammatory and anti-inflammatory activities. PMID:15932509

  15. Defects in mitochondrial clearance predispose human monocytes to interleukin-1β hypersecretion.

    PubMed

    van der Burgh, Robert; Nijhuis, Lotte; Pervolaraki, Kalliopi; Compeer, Ewoud B; Jongeneel, Lieneke H; van Gijn, Marielle; Coffer, Paul J; Murphy, Michael P; Mastroberardino, Pier G; Frenkel, Joost; Boes, Marianne

    2014-02-21

    Most hereditary periodic fever syndromes are mediated by deregulated IL-1β secretion. The generation of mature IL-1β requires two signals: one that induces synthesis of inflammasome components and substrates and a second that activates inflammasomes. The mechanisms that mediate autoinflammation in mevalonate kinase deficiency, a periodic fever disease characterized by a block in isoprenoid biosynthesis, are poorly understood. In studying the effects of isoprenoid shortage on IL-1 β generation, we identified a new inflammasome activation signal that originates from defects in autophagy. We find that hypersecretion of IL-1β and IL-18 requires reactive oxygen species and is associated with an oxidized redox status of monocytes but not lymphocytes. IL-1β hypersecretion by monocytes involves decreased mitochondrial stability, release of mitochondrial content into the cytosol and attenuated autophagosomal degradation. Defective autophagy, as established by ATG7 knockdown, results in prolonged cytosolic retention of damaged mitochondria and increased IL-1β secretion. Finally, activation of autophagy in healthy but not mevalonate kinase deficiency patient cells reduces IL-1β secretion. Together, these results indicate that defective autophagy can prime monocytes for mitochondria-mediated NLRP3 inflammasome activation, thereby contributing to hypersecretion of IL-1β in mevalonate kinase deficiency.

  16. Inflammatory responses induced by lipopolysaccharide are amplified in primary human monocytes but suppressed in macrophages by complement protein C5a.

    PubMed

    Seow, Vernon; Lim, Junxian; Iyer, Abishek; Suen, Jacky Y; Ariffin, Juliana K; Hohenhaus, Daniel M; Sweet, Matthew J; Fairlie, David P

    2013-10-15

    Monocytes and macrophages are important innate immune cells equipped with danger-sensing receptors, including complement and Toll-like receptors. Complement protein C5a, acting via C5aR, is shown in this study to differentially modulate LPS-induced inflammatory responses in primary human monocytes versus macrophages. Whereas C5a enhanced secretion of LPS-induced IL-6 and TNF from primary human monocytes, C5a inhibited these responses while increasing IL-10 secretion in donor-matched human monocyte-derived macrophages differentiated by GM-CSF or M-CSF. Gαi/c-Raf/MEK/ERK signaling induced by C5a was amplified in macrophages but not in monocytes by LPS. Accordingly, the Gαi inhibitor pertussis toxin and MEK inhibitor U0126 blocked C5a inhibition of LPS-induced IL-6 and TNF production from macrophages. This synergy was independent of IL-10, PI3K, p38, JNK, and the differentiating agent. Furthermore, C5a did not inhibit IL-6 production from macrophages induced by other TLR agonists that are selective for Toll/IL-1R domain-containing adapter inducing IFN-β (polyinosinic-polycytidylic acid) or MyD88 (imiquimod), demonstrating selectivity for C5a regulation of LPS responses. Finally, suppression of proinflammatory cytokines IL-6 and TNF in macrophages did not compromise antimicrobial activity; instead, C5a enhanced clearance of the Gram-negative bacterial pathogen Salmonella enterica serovar Typhimurium from macrophages. C5aR is thus a regulatory switch that modulates TLR4 signaling via the Gαi/c-Raf/MEK/ERK signaling axis in human macrophages but not monocytes. The differential effects of C5a are consistent with amplifying monocyte proinflammatory responses to systemic danger signals, but attenuating macrophage cytokine responses (without compromising microbicidal activity), thereby restraining inflammatory responses to localized infections.

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

    PubMed Central

    Nishibayashi, Ryoichiro; Inoue, Ryo; Harada, Yuri; Watanabe, Takumi; Makioka, Yuko; Ushida, Kazunari

    2015-01-01

    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. PMID:26083838

  18. Phenotypic and functional changes in peripheral blood monocytes during progression of human immunodeficiency virus infection. Effects of soluble immune complexes, cytokines, subcellular particulates from apoptotic cells, and HIV-1-encoded proteins on monocytes phagocytic function, oxidative burst, transendothelial migration, and cell surface phenotype.

    PubMed Central

    Trial, J; Birdsall, H H; Hallum, J A; Crane, M L; Rodriguez-Barradas, M C; de Jong, A L; Krishnan, B; Lacke, C E; Figdor, C G; Rossen, R D

    1995-01-01

    We postulated that changes in the cell surface display of molecules that facilitate cell-cell and cell-matrix adhesions may reflect the changing immunosurveillance capacity of blood monocytes during progression of human immunodeficiency virus (HIV) infections. In Centers for Disease Control (CDC) stage A patients, whose monocytes' ability to phagocytose bacteria and generate reactive oxygen intermediates is often increased, the frequency of monocytes expressing CD49d, HLA-DP, HLA-DQ, and an activation epitope of CD11a/CD18 was increased and monocyte transendothelial migration was unimpaired. In CDC stage B/C patients, whose monocytes' ability to phagocytose bacteria and migrate across confluent endothelial monolayers was diminished, surface expression of CD49e and CD62L and the percentage of monocytes expressing CD18, CD11a, CD29, CD49e, CD54, CD58, CD31, and HLA-I were significantly decreased. Incubating normal donor monocytes with immune complexes in vitro reproduced the phenotypic and functional abnormalities seen in stage B/C patients. By contrast, in vitro stimulation with subcellular particulates released by apoptotic lymphocytes reproduced changes seen in stage A patients' monocytes. Although circulating monocytes appear to be activated at all stages, these data suggest that the high levels of circulating immune complexes, found predominantly in the later stages of HIV infection, may be particularly instrumental in reducing the monocyte's capacity to maintain surveillance against infection. Images PMID:7706478

  19. Human Monoclonal Antibodies against Clostridium difficile Toxins A and B Inhibit Inflammatory and Histologic Responses to the Toxins in Human Colon and Peripheral Blood Monocytes

    PubMed Central

    Koon, Hon Wai; Shih, David Q.; Hing, Tressia C.; Yoo, Jun Hwan; Ho, Samantha; Chen, Xinhua; Kelly, Ciarán P.; Targan, Stephan R.

    2013-01-01

    Clostridium difficile infection (CDI) is a common and debilitating nosocomial infection with high morbidity and mortality. C. difficile mediates diarrhea and colitis by releasing two toxins, toxin A and toxin B. Since both toxins stimulate proinflammatory signaling pathways in human colonocytes and both are involved in the pathophysiology of CDI, neutralization of toxin A and B activities may represent an important therapeutic approach against CDI. Recent studies indicated that human monoclonal antibodies (MAbs) against toxins A and B reduce their cytotoxic and secretory activities and prevent CDI in hamsters. Moreover, anti-toxin A and anti-toxin B MAbs together with antibiotics also effectively reduced recurrent CDI in humans. However, whether these MAbs neutralize toxin A- and toxin B-associated immune responses in human colonic mucosa or human peripheral blood monocyte cells (PBMCs) has never been examined. We used fresh human colonic biopsy specimens and peripheral blood monocytes to evaluate the effects of these antibodies against toxin A- and B-associated cytokine release, proinflammatory signaling, and histologic damage. Incubation of anti-toxin A (MK3415) or anti-toxin B (MK6072) MAbs with human PBMCs significantly inhibited toxin A- and toxin B-mediated tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) expression. MK3415 and MK6072 also diminished toxin A- and toxin B-mediated NF-κB p65 phosphorylation in human monocytes, respectively, and significantly reduced toxin A- and B-induced TNF-α and IL-1β expression as well as histologic damage in human colonic explants. Our results underline the effectiveness of MK3415 and MK6072 in blocking C. difficile toxin A- and toxin B-mediated inflammatory responses and histologic damage. PMID:23629713

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

    PubMed Central

    Yonno, L.; Heaslip, R. J.; Weichman, B. M.

    1992-01-01

    The effect of selective PDE-I (vinpocetine), PDE-III (milrinone, CI-930), PDE-IV (rolipram, nitroquazone), and PDE-V (zaprinast) isozyme inhibitors on TNF-α and IL-1β production from LPS stimulated human monocytes was investigated. The PDE-IV inhibitors caused a concentration dependent inhibition of TNF-α production, but only partially inhibited IL-1β at high concentrations. High concentrations of the PDE-III inhibitors weakly inhibited TNF-α, but had no effect on IL-1β production. PDE-V inhibition was associated with an augmentation of cytokine secretion. Studies with combinations of PDE isozyme inhibitors indicated that PDE-III and PDE-V inhibitors modulate rolipram's suppression of TNF production in an additive manner. These data confirm that TNF-α and IL-1β production from LPS stimulated human monocytes are differentially regulated, and suggest that PDE-IV inhibitors have the potential to suppress TNF levels in man. PMID:18475493

  1. Optimization of protein solubilization for the analysis of the CD14 human monocyte membrane proteome using LC-MS/MS.

    PubMed

    Ye, Xiaoying; Johann, Donald J; Hakami, Ramin M; Xiao, Zhen; Meng, Zhaojing; Ulrich, Robert G; Issaq, Haleem J; Veenstra, Timothy D; Blonder, Josip

    2009-11-02

    Proteomic profiling of membrane proteins is of vital importance in the search for disease biomarkers and drug development. However, the slow pace in this field has resulted mainly from the difficulty to analyze membrane proteins by mass spectrometry (MS). The objective of this investigation was to explore and optimize solubilization of membrane proteins for shotgun membrane proteomics of the CD14 human monocytes by examining different systems that rely on: i) an organic solvent (methanol) ii) an acid-labile detergent 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), iii) a combination of both agents (methanol+PPS). Solubilization efficiency of different buffers was first compared using bacteriorhodopsin as a model membrane protein. Selected approaches were then applied on a membrane subproteome isolated from a highly enriched human monocyte population that was approximately 98% positive for CD14 expression as determined by FACS analysis. A methanol-based buffer yielded 194 proteins of which 93 (48%) were mapped as integral membrane proteins. The combination of methanol and acid-cleavable detergent gave similar results; 203 identified proteins of which 93 (46%) were mapped integral membrane proteins. However, employing PPS 216 proteins were identified of which 75 (35%) were mapped as integral membrane proteins. These results indicate that methanol alone or in combination with PPS yielded significantly higher membrane protein identification/enrichment than the PPS alone.

  2. A Novel Marker of Impaired Aortic Elasticity in Never Treated Hypertensive Patients: Monocyte/High-Density Lipoprotein Cholesterol Ratio

    PubMed Central

    Yayla, Kadriye Gayretli; Canpolat, Uğur; Yayla, Çagri; Akboğa, Mehmet Kadri; Akyel, Ahmet; Akdi, Ahmet; Çiçek, Gökhan; Ozcan, Firat; Turak, Osman; Aydoğdu, Sinan

    2017-01-01

    Background Monocyte to high density lipoprotein cholesterol ratio (MHR) is generally understood to be a candidate marker of inflammation and oxidative stress. Therefore, we aimed to assess the association between MHR and aortic elastic properties in hypertensive patients. Methods A total of 114 newly-diagnosed untreated patients with hypertension and 71 healthy subjects were enrolled. Aortic stiffness index, aortic strain and aortic distensibility were measured by using echocardiography. Results Patients with hypertension had a significantly higher MHR compared to the control group (p < 0.001). Also, aortic stiffness index (p < 0.001) was significantly higher and aortic distensibility (p < 0.001) was lower in the hypertensive group. There was a positive correlation of MHR with aortic stiffness index (r = 0.294, p < 0.001) and negative correlation with aortic distensibility (r = -0.281, p < 0.001). In addition, MHR and high sensitivity C-reactive protein have a positive correlation (r = 0.30, p < 0.001). Furthermore, MHR was found to be an independent predictor of aortic distensibility and aortic stiffness index. Conclusions In patients with newly-diagnosed untreated essential hypertension, higher MHR was significantly associated with impaired aortic elastic properties. PMID:28115806

  3. The PRKAA1/AMPKα1 pathway triggers autophagy during CSF1-induced human monocyte differentiation and is a potential target in CMML.

    PubMed

    Obba, Sandrine; Hizir, Zoheir; Boyer, Laurent; Selimoglu-Buet, Dorothée; Pfeifer, Anja; Michel, Gregory; Hamouda, Mohamed-Amine; Gonçalvès, Diogo; Cerezo, Michael; Marchetti, Sandrine; Rocchi, Stephane; Droin, Nathalie; Cluzeau, Thomas; Robert, Guillaume; Luciano, Frederic; Robaye, Bernard; Foretz, Marc; Viollet, Benoit; Legros, Laurence; Solary, Eric; Auberger, Patrick; Jacquel, Arnaud

    2015-01-01

    Autophagy is induced during differentiation of human monocytes into macrophages that is mediated by CSF1/CSF-1/M-CSF (colony stimulating factor 1 [macrophage]). However, little is known about the molecular mechanisms that link CSF1 receptor engagement to the induction of autophagy. Here we show that the CAMKK2-PRKAA1-ULK1 pathway is required for CSF1-induced autophagy and human monocyte differentiation. We reveal that this pathway links P2RY6 to the induction of autophagy, and we decipher the signaling network that links the CSF1 receptor to P2RY6-mediated autophagy and monocyte differentiation. In addition, we show that the physiological P2RY6 ligand UDP and the specific P2RY6 agonist MRS2693 can restore normal monocyte differentiation through reinduction of autophagy in primary myeloid cells from some but not all chronic myelomonocytic leukemia (CMML) patients. Collectively, our findings highlight an essential role for PRKAA1-mediated autophagy during differentiation of human monocytes and pave the way for future therapeutic interventions for CMML.

  4. Low-affinity IgE receptor (FcepsilonRII)-mediated activation of human monocytes by both monomeric IgE and IgE/anti-IgE immune complex.

    PubMed

    Ezeamuzie, Charles I; Al-Attiyah, Raja'a; Shihab, Puthiyaveetil K; Al-Radwan, Reem

    2009-08-01

    Monocytes and macrophages of individuals with allergic diseases express increased levels of the low-affinity IgE receptors (FcepsilonRII or CD23) on their surfaces. The cross-linking of CD23-bound IgE antibody by allergen activates the cells to release inflammatory mediators. In mast cells, the binding of IgE to the high-affinity IgE receptors (FcepsilonRI) has recently been shown to activate these cells independent of allergen. It has not been determined if such is true of the binding of IgE to the low-affinity receptors. The purpose of this study was, therefore, to determine whether monomeric IgE alone can activate CD23-bearing human monocytes and how this may relate to the activation by IgE/anti-IgE immune complex. Purified monocytes, cultured for 48 h with IL-4 to up-regulate CD23 were sensitized with human myeloma IgE and further cultured for 24 h with or without anti-human IgE antibody. The release of cytokines TNF-alpha and MIP-1alpha (as an index of activation) was determined by enzyme immunoassay. Results showed that in IL-4-treated/CD23-bearing monocytes, sensitization with IgE alone caused a release of TNF-alpha and MIP-1alpha. The addition of anti-IgE antibody to cross-link the bound IgE resulted in the enhancement of the response. Such activation by monomeric IgE and IgE/anti-IgE immune complex was blocked with an anti-CD23 antibody, confirming the specific involvement of CD23 molecules. Neither of the activation modalities elevated intracellular cAMP, contrary to previous report. These results show for the first time, that in CD23-bearing monocytes, IgE sensitization alone can activate monocytes, and that ligation of such IgE by anti-IgE antibody only enhances the response. These observations have implications for the understanding of the pathophysiology of IgE-dependent inflammation accompanying many allergic diseases.

  5. Human monocytes and macrophages express NADPH oxidase 5; a potential source of reactive oxygen species in atherosclerosis.

    PubMed

    Manea, Adrian; Manea, Simona-Adriana; Gan, Ana Maria; Constantin, Alina; Fenyo, Ioana Madalina; Raicu, Monica; Muresian, Horia; Simionescu, Maya

    2015-05-22

    Monocytes (Mon) and Mon-derived macrophages (Mac) orchestrate important oxidative and inflammatory reactions in atherosclerosis by secreting reactive oxygen species (ROS) due, in large part, to the upregulated NADPH oxidases (Nox). The Nox enzymes have been extensively investigated in human Mon and Mac. However, the expression and functional significance of the Nox5 subtypes is not known. We aimed at elucidating whether Nox5 is expressed in human Mon and Mac, and examine its potential role in atherosclerosis. Human monocytic THP-1 cell line and CD14(+) Mon were employed to search for Nox5 expression. RT-PCR, Western blot, lucigenin-enhanced chemiluminescence and dihydroethidium assays were utilized to examine Nox5 in these cells. We found that Nox5 transcription variants and proteins are constitutively expressed in THP-1 cells and primary CD14(+) Mon. Silencing of Nox5 protein expression by siRNA reduced the Ca(2+)-dependent Nox activity and the formation of ROS in Mac induced by A23187, a selective Ca(2+) ionophore. Exposure of Mac to increasing concentrations of IFNγ (5-100 ng/ml) or oxidized LDL (5-100 μg/ml) resulted in a dose-dependent increase in Nox5 protein expression and elevation in intracellular Ca(2+) concentration. Immunohistochemical staining revealed that Nox5 is present in CD68(+) Mac-rich area within human carotid artery atherosclerotic plaques. To the best of our knowledge, this is the first evidence that Nox5 is constitutively expressed in human Mon. Induction of Nox5 expression in IFNγ- and oxidized LDL-exposed Mac and the presence of Nox5 in Mac-rich atheroma are indicative of the implication of Nox5 in atherogenesis.

  6. ApoE production in human monocytes and its regulation by inflammatory cytokines.

    PubMed

    Braesch-Andersen, Sten; Paulie, Staffan; Smedman, Christian; Mia, Sohel; Kumagai-Braesch, Makiko

    2013-01-01

    The apoE production by tissue macrophages is crucial for the prevention of atherosclerosis and the aim of this study was to further elucidate how this apolipoprotein is regulated by cytokines present during inflammation. Here we studied apoE production in peripheral blood mononuclear cells (PBMC) and analysis was made with a newly developed apoE ELISpot assay. In PBMC, apoE secretion was restricted to monocytes with classical (CD14(++)CD16(-)) and intermediate (CD14(+)CD16(+)) monocytes being the main producers. As earlier described for macrophages, production was strongly upregulated by TGF-β and downregulated by bacterial lipopolysaccharide (LPS) and the inflammatory cytokines IFN-γ, TNF-α and IL-1β. We could here show that a similar down-regulatory effect was also observed with the type I interferon, IFN-α, while IL-6, often regarded as one of the more prominent inflammatory cytokines, did not affect TGF-β-induced apoE production. The TNF-α inhibitor Enbrel could partly block the down-regulatory effect of IFN-γ, IFN-α and IL-1β, indicating that inhibition of apoE by these cytokines may be dependent on or synergize with TNF-α. Other cytokines tested, IL-2, IL-4, IL-12, IL-13, IL-17A and IL-23, had no inhibitory effect on apoE production. In contrast to the effect on monocytes, apoE production by primary hepatocytes and the hepatoma cell line HepG2 was more or less unaffected by treatment with cytokines or LPS.

  7. Infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3.

    PubMed

    Le Nouën, Cyril; Munir, Shirin; Losq, Stéphanie; Winter, Christine C; McCarty, Thomas; Stephany, David A; Holmes, Kevin L; Bukreyev, Alexander; Rabin, Ronald L; Collins, Peter L; Buchholz, Ursula J

    2009-03-01

    Human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), and human parainfluenza virus type 3 (HPIV3) are common, important respiratory pathogens, but HRSV has a substantially greater impact with regard to acute disease, long-term effects on airway function, and frequency of re-infection. It has been reported to strongly interfere with the functioning of dendritic cells (DC). We compared HRSV to HMPV and HPIV3 with regard to their effects on human monocyte-derived immature DC (IDC). Side-by-side analysis distinguished between common effects versus those specific to individual viruses. The use of GFP-expressing viruses yielded clear identification of robustly infected cells and provided the means to distinguish between direct effects of robust viral gene expression versus bystander effects. All three viruses infected inefficiently based on GFP expression, with considerable donor-to donor-variability. The GFP-negative cells exhibited low, abortive levels of viral RNA synthesis. The three viruses induced low-to-moderate levels of DC maturation and cytokine/chemokine responses, increasing slightly in the order HRSV, HMPV, and HPIV3. Infection at the individual cell level was relatively benign, such that in general GFP-positive cells were neither more nor less able to mature compared to GFP-negative bystanders, and cells were responsive to a secondary treatment with lipopolysaccharide, indicating that the ability to mature was not impaired. However, there was a single exception, namely that HPIV3 down-regulated CD38 expression at the RNA level. Maturation by these viruses was anti-apoptotic. Inefficient infection of IDC and sub-optimal maturation might result in reduced immune responses, but these effects would be common to all three viruses rather than specific to HRSV.

  8. Hyperketonemia increases monocyte adhesion to endothelial cells and is mediated by LFA-1 expression in monocytes and ICAM-1 expression in endothelial cells.

    PubMed

    Rains, Justin L; Jain, Sushil K

    2011-08-01

    Frequent episodes of hyperketonemia are associated with a higher incidence of vascular disease. The objective of this study was to examine the hypothesis that hyperketonemia increases monocyte-endothelial cell (EC) adhesion and the development of vascular disease in diabetes. Human U937 and THP-1 monocyte cell lines and human umbilical vein endothelial cells (HUVECs) were cultured with acetoacetate (AA) (0-10 mM) or β-hydroxybutyrate (BHB) (0-10 mM) for 24 h prior to evaluating adhesion and adhesion molecule expression. The results demonstrate a significant (P < 0.01) increase in both U937 and THP-1 adhesion to HUVEC monolayers treated with 4 mM AA compared with control. Equal concentrations of BHB resulted in similar increases in monocyte-EC adhesion. Similarly, treatments of AA or BHB to isolated monocytes from human blood also show increases in adhesion to endothelial cells. intercellular adhesion molecule-1 (ICAM-1) was significantly increased on the surface of HUVECs and an increase in total protein expression with AA treatment compared with control. The expression level of lymphocyte function-associated antigen-1 (LFA-1) was increased in monocytes treated with AA, and LFA-1 affinity was altered from low to high affinity following treatment with both AA and BHB. Monocyte adhesion could be blocked when cells were preincubated with an antibody to ICAM-1 or LFA-1. Results also show a significant increase in IL-8 and MCP-1 secretion in monocytes and HUVECs treated with 0-10 mM AA. These results suggest that hyperketonemia can induce monocyte adhesion to endothelial cells and that it is mediated via increased ICAM-1 expression in endothelial cells and increased expression and affinity of LFA-1 in monocytes.

  9. Hyperketonemia increases monocyte adhesion to endothelial cells and is mediated by LFA-1 expression in monocytes and ICAM-1 expression in endothelial cells

    PubMed Central

    Rains, Justin L.

    2011-01-01

    Frequent episodes of hyperketonemia are associated with a higher incidence of vascular disease. The objective of this study was to examine the hypothesis that hyperketonemia increases monocyte-endothelial cell (EC) adhesion and the development of vascular disease in diabetes. Human U937 and THP-1 monocyte cell lines and human umbilical vein endothelial cells (HUVECs) were cultured with acetoacetate (AA) (0–10 mM) or β-hydroxybutyrate (BHB) (0–10 mM) for 24 h prior to evaluating adhesion and adhesion molecule expression. The results demonstrate a significant (P < 0.01) increase in both U937 and THP-1 adhesion to HUVEC monolayers treated with 4 mM AA compared with control. Equal concentrations of BHB resulted in similar increases in monocyte-EC adhesion. Similarly, treatments of AA or BHB to isolated monocytes from human blood also show increases in adhesion to endothelial cells. intercellular adhesion molecule-1 (ICAM-1) was significantly increased on the surface of HUVECs and an increase in total protein expression with AA treatment compared with control. The expression level of lymphocyte function-associated antigen-1 (LFA-1) was increased in monocytes treated with AA, and LFA-1 affinity was altered from low to high affinity following treatment with both AA and BHB. Monocyte adhesion could be blocked when cells were preincubated with an antibody to ICAM-1 or LFA-1. Results also show a significant increase in IL-8 and MCP-1 secretion in monocytes and HUVECs treated with 0–10 mM AA. These results suggest that hyperketonemia can induce monocyte adhesion to endothelial cells and that it is mediated via increased ICAM-1 expression in endothelial cells and increased expression and affinity of LFA-1 in monocytes. PMID:21540444

  10. Human Cytomegalovirus-Encoded Human Interleukin-10 (IL-10) Homolog Amplifies Its Immunomodulatory Potential by Upregulating Human IL-10 in Monocytes

    PubMed Central

    Avdic, Selmir; McSharry, Brian P.; Steain, Megan; Poole, Emma; Sinclair, John; Abendroth, Allison

    2016-01-01

    ABSTRACT The human cytomegalovirus (HCMV) gene UL111A encodes cytomegalovirus-encoded human interleukin-10 (cmvIL-10), a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). This viral homolog exhibits a range of immunomodulatory functions, including suppression of proinflammatory cytokine production and dendritic cell (DC) maturation, as well as inhibition of major histocompatibility complex (MHC) class I and class II. Here, we present data showing that cmvIL-10 upregulates hIL-10, and we identify CD14+ monocytes and monocyte-derived macrophages and DCs as major sources of hIL-10 secretion in response to cmvIL-10. Monocyte activation was not a prerequisite for cmvIL-10-mediated upregulation of hIL-10, which was dose dependent and controlled at the transcriptional level. Furthermore, cmvIL-10 upregulated expression of tumor progression locus 2 (TPL2), which is a regulator of the positive hIL-10 feedback loop, whereas expression of a negative regulator of the hIL-10 feedback loop, dual-specificity phosphatase 1 (DUSP1), remained unchanged. Engagement of the hIL-10 receptor (hIL-10R) by cmvIL-10 led to upregulation of heme oxygenase 1 (HO-1), an enzyme linked with suppression of inflammatory responses, and this upregulation was required for cmvIL-10-mediated upregulation of hIL-10. We also demonstrate an important role for both phosphatidylinositol 3-kinase (PI3K) and STAT3 in the upregulation of HO-1 and hIL-10 by cmvIL-10. In addition to upregulating hIL-10, cmvIL-10 could exert a direct immunomodulatory function, as demonstrated by its capacity to upregulate expression of cell surface CD163 when hIL-10 was neutralized. This study identifies a mechanistic basis for cmvIL-10 function, including the capacity of this viral cytokine to potentially amplify its immunosuppressive impact by upregulating hIL-10 expression. IMPORTANCE Human cytomegalovirus (HCMV) is a large, double-stranded DNA virus that causes significant human disease

  11. Rifampin Increases Cytokine-Induced Expression of the CD1b Molecule in Human Peripheral Blood Monocytes

    PubMed Central

    Tentori, L.; Graziani, G.; Porcelli, S. A.; Sugita, M.; Brenner, M. B.; Madaio, R.; Bonmassar, E.; Giuliani, A.; Aquino, A.

    1998-01-01

    In recent years, it has been shown that a nonclassical, major histocompatibility complex-independent system (i.e., CD1-restricted T-cell responses) is involved in T-cell immunity against nonpeptide antigens. The CD1 system appears to function by presenting microbial lipid antigens to specific T cells, and the antigens so far identified include several known constituents of mycobacterial cell walls. Among the four known human CD1 isoforms, the CD1b protein is the best characterized with regard to its antigen-presenting function. Expression of CD1b is upregulated on human blood monocytes upon exposure to granulocyte/macrophage-colony stimulating factor, alone or in combination with interleukin-4 (IL-4) (S. A. Porcelli, Adv. Immunol. 59:1–98, 1995). Rifampin (RFP) and its derivatives are widely used for chemoprophylaxis or chemotherapy against Mycobacterium tuberculosis. However, this agent was found to reduce the mitogen responsiveness of human B and T lymphocytes, chemotaxis, and delayed-type hypersensitivity. The present study extends the immunopharmacological profile of RFP by examining its effects on CD1b expression by human peripheral blood monocytes exposed to GM-CSF plus IL-4. The results showed that clinically attainable concentrations (i.e., 2 or 10 μg/ml for 24 h) of the agent produced a marked increase in CD1b expression on the plasma membrane, as evaluated by fluorescence-activated cell sorter analysis, whereas it had no effect on cytosolic fractions, as indicated by Western blot analysis. This was found to be the result of increased CD1b gene expression, as shown by Northern blot analysis of CD1b mRNA. These results suggest that RFP could be of potential value in augmenting the CD1b-restricted antigen recognition system, thereby enhancing protective cellular immunity to M. tuberculosis. PMID:9517931

  12. Continuous enteral and parenteral feeding each reduces heart rate variability but differentially influences monocyte gene expression in humans.

    PubMed

    Gale, Stephen C; Shanker, Beth-Ann; Coyle, Susette M; Macor, Marie A; Choi, Chun W; Calvano, Steve E; Corbett, Siobhan A; Lowry, Stephen F

    2012-08-01

    Enteral (EN) or parenteral (PN) nutrition is used to support critically ill patients until oral feeding resumes. Enteral nutrition is assumed preferable to PN, but the differential influence on immune function is not well defined. Autonomic nervous activity is known to influence innate immune responses, and we hypothesized that EN and PN could influence both autonomic signaling and gene activation in peripheral blood monocytes (PBMs). Ten subjects (aged 18-36 years) received continuous EN or PN for 72 h. Peripheral blood monocytes were isolated from whole blood before and after continuous feeding and were analyzed for gene expression using a microarray platform. Gene expression after feeding was compared from baseline and between groups. To measure autonomic outflow, subjects also underwent heart rate variability (HRV) monitoring during feeding. Time and frequency domain HRV data were compared between groups and five orally fed subjects for changes from baseline and changes over time. During continuous EN and PN, subjects exhibited declines in both time and frequency domain HRV parameters compared with baseline and with PO subjects, indicating a loss of vagal/parasympathetic tone. However, PN feeding had a much greater influence on PBM gene expression compared with baseline than EN, including genes important to innate immunity. Continuous EN and PN are both associated with decreasing vagal tone over time, yet contribute differently to PBM gene expression, in humans. These preliminary findings support assumptions that PN imposes a systemic inflammatory risk but also imply that continuous feeding, independent of route, may impart additional risk through different mechanisms.

  13. Long noncoding RNA LINC00305 promotes inflammation by activating the AHRR-NF-κB pathway in human monocytes

    PubMed Central

    Zhang, Dan-Dan; Wang, Wen-Tian; Xiong, Jian; Xie, Xue-Min; Cui, Shen-Shen; Zhao, Zhi-Guo; Li, Mulin Jun; Zhang, Zhu-Qin; Hao, De-Long; Zhao, Xiang; Li, Yong-Jun; Wang, Junwen; Chen, Hou-Zao; Lv, Xiang; Liu, De-Pei

    2017-01-01

    Accumulating data from genome-wide association studies (GWAS) have provided a collection of novel candidate genes associated with complex diseases, such as atherosclerosis. We identified an atherosclerosis-associated single-nucleotide polymorphism (SNP) located in the intron of the long noncoding RNA (lncRNA) LINC00305 by searching the GWAS database. Although the function of LINC00305 is unknown, we found that LINC00305 expression is enriched in atherosclerotic plaques and monocytes. Overexpression of LINC00305 promoted the expression of inflammation-associated genes in THP-1 cells and reduced the expression of contractile markers in co-cultured human aortic smooth muscle cells (HASMCs). We showed that overexpression of LINC00305 activated nuclear factor-kappa beta (NF-κB) and that inhibition of NF-κB abolished LINC00305-mediated activation of cytokine expression. Mechanistically, LINC00305 interacted with lipocalin-1 interacting membrane receptor (LIMR), enhanced the interaction of LIMR and aryl-hydrocarbon receptor repressor (AHRR), and promoted protein expression as well as nuclear localization of AHRR. Moreover, LINC00305 activated NF-κB exclusively in the presence of LIMR and AHRR. In light of these findings, we propose that LINC00305 promotes monocyte inflammation by facilitating LIMR and AHRR cooperation and the AHRR activation, which eventually activates NF-κB, thereby inducing HASMC phenotype switching. PMID:28393844

  14. Hypoxia reduces constitutive and TNF-{alpha}-induced expression of monocyte chemoattractant protein-1 in human proximal renal tubular cells

    SciTech Connect

    Li Xuan; Kimura, Hideki . E-mail: hkimura@fmsrsa.fukui-med.ac.jp; Hirota, Kiichi; Sugimoto, Hidehiro; Yoshida, Haruyoshi

    2005-10-07

    Chronic hypoxia has been reported to be associated with macrophage infiltration in progressive forms of kidney disease. Here, we investigated the regulatory effects of hypoxia on constitutive and TNF-{alpha}-stimulated expression of monocyte chemoattractant protein-1 (MCP-1) in cultured human proximal renal tubular cells (HPTECs). Hypoxia reduced constitutive MCP-1 expression at the mRNA and protein levels in a time-dependent fashion for up to 48 h. Hypoxia also inhibited MCP-1 up-regulation by TNF-{alpha}. Treatment with actinomycin D showed that hypoxic down-regulation of MCP-1 expression resulted mainly from a decrease in the transcription but not the mRNA stability. Immunoblot and immunofluorescence analyses revealed that treatment with hypoxia or an iron chelator, desferrioxamine, induced nuclear accumulation of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in HPTECs. Desferrioxamine mimicked hypoxia in the reduction of MCP-1 expression. However, overexpression of a dominant negative form of HIF-1{alpha} did not abolish the hypoxia-induced reduction of MCP-1 expression in HPTECs. These results suggest that hypoxia is an important negative regulator of monocyte chemotaxis to the renal inflamed interstitium, by reducing MCP-1 expression partly via hypoxia-activated signals other than the HIF-1 pathway.

  15. Long non-coding RNA-DANCR in human circulating monocytes: a potential biomarker associated with postmenopausal osteoporosis.

    PubMed

    Tong, Xiang; Gu, Peng-cheng; Xu, San-zhong; Lin, Xiang-jin

    2015-01-01

    Osteoporosis is a common disease characterized by low bone mineral density (BMD) and low trauma fractures, mainly resulting from exceeding bone resorption by osteoclasts over bone formation by osteoblasts. Circulating monocytes are directly involved in osteoclastogenesis, and lncRNAs are believed to be involved in the osteoblast differentiation. However, no study has been conducted to identify the roles of lncRNA in circulating monocytes associated with human osteoporosis. In this study, we found significant upregulation of DANCR in the blood mononuclear cells (MNCs) from low-BMD patients with the qRT-PCR analyses. We further found that DANCR promoted the expression of IL6 and TNF-α at both mRNA level and protein level in MNCs. After deletion of DANCR with siRNAs, the levels of IL6 and TNF-α are decreased in the MNCs from low-BMD postmenopausal women. Moreover, DANCR level was correlated with IL6 and TNF-α in postmenopausal women with low BMD. Furthermore, we found that DANCR-induced IL6 and TNF-α in MNCs had bone-resorbing activity. These results indicate that DANCR is involved in the pathology of osteoporosis and may be as a biomarker for postmenopausal osteoporosis.

  16. Differential expression of the fractalkine chemokine receptor (CX3CR1) in human monocytes during differentiation

    PubMed Central

    Panek, Cecilia Analia; Ramos, Maria Victoria; Mejias, Maria Pilar; Abrey-Recalde, Maria Jimena; Fernandez-Brando, Romina Jimena; Gori, Maria Soledad; Salamone, Gabriela Verónica; Palermo, Marina Sandra

    2015-01-01

    Circulating monocytes (Mos) may continuously repopulate macrophage (MAC) or dendritic cell (DC) populations to maintain homeostasis. MACs and DCs are specialized cells that play different and complementary immunological functions. Accordingly, they present distinct migratory properties. Specifically, whereas MACs largely remain in tissues, DCs are capable of migrating from peripheral tissues to lymphoid organs. The aim of this work was to analyze the expression of the fractalkine receptor (CX3CR1) during the monocytic differentiation process. Freshly isolated Mos express high levels of both CX3CR1 mRNA and protein. During the Mo differentiation process, CX3CR1 is downregulated in both DCs and MACs. However, MACs showed significantly higher CX3CR1 expression levels than did DC. We also observed an antagonistic CX3CR1 regulation by interferon (IFN)-γ and interleukin (IL)-4 during MAC activation through the classical and alternative MAC pathways, respectively. IFN-γ inhibited the loss of CX3CR1, but IL-4 induced it. Additionally, we demonstrated an association between CX3CR1 expression and apoptosis prevention by soluble fractalkine (sCX3CL1) in Mos, DCs and MACs. This is the first report demonstrating sequential and differential CX3CR1 modulation during Mo differentiation. Most importantly, we demonstrated a functional link between CX3CR1 expression and cell survival in the presence of sCX3CL1. PMID:25502213

  17. Differential expression of the fractalkine chemokine receptor (CX3CR1) in human monocytes during differentiation.

    PubMed

    Panek, Cecilia Analia; Ramos, Maria Victoria; Mejias, Maria Pilar; Abrey-Recalde, Maria Jimena; Fernandez-Brando, Romina Jimena; Gori, Maria Soledad; Salamone, Gabriela Verónica; Palermo, Marina Sandra

    2015-11-01

    Circulating monocytes (Mos) may continuously repopulate macrophage (MAC) or dendritic cell (DC) populations to maintain homeostasis. MACs and DCs are specialized cells that play different and complementary immunological functions. Accordingly, they present distinct migratory properties. Specifically, whereas MACs largely remain in tissues, DCs are capable of migrating from peripheral tissues to lymphoid organs. The aim of this work was to analyze the expression of the fractalkine receptor (CX3CR1) during the monocytic differentiation process. Freshly isolated Mos express high levels of both CX3CR1 mRNA and protein. During the Mo differentiation process, CX3CR1 is downregulated in both DCs and MACs. However, MACs showed significantly higher CX3CR1 expression levels than did DC. We also observed an antagonistic CX3CR1 regulation by interferon (IFN)-γ and interleukin (IL)-4 during MAC activation through the classical and alternative MAC pathways, respectively. IFN-γ inhibited the loss of CX3CR1, but IL-4 induced it. Additionally, we demonstrated an association between CX3CR1 expression and apoptosis prevention by soluble fractalkine (sCX3CL1) in Mos, DCs and MACs. This is the first report demonstrating sequential and differential CX3CR1 modulation during Mo differentiation. Most importantly, we demonstrated a functional link between CX3CR1 expression and cell survival in the presence of sCX3CL1.

  18. Comparison of two different strategies for human monocyte subsets gating within the large-scale prospective CARE FOR HOMe Study.

    PubMed

    Zawada, Adam M; Fell, Lisa H; Untersteller, Kathrin; Seiler, Sarah; Rogacev, Kyrill S; Fliser, Danilo; Ziegler-Heitbrock, Loems; Heine, Gunnar H

    2015-08-01

    Monocytes are heterogeneous cells consisting of (at least) three subsets: classical, intermediate, and nonclassical monocytes. Correct enumeration of cell counts necessitates well-defined gating strategies, which are essentially based upon CD14 and CD16 expression. For the delineation of intermediate from nonclassical monocytes, a "rectangular gating (RG) strategy" and a "trapezoid gating (TG) strategy" have been proposed. We compared the two gating strategies in a well-defined clinical cohort of patients with chronic kidney disease (CKD). Within the ongoing CARE FOR HOMe study, monocyte subsets were reanalyzed in 416 CKD patients, who were followed 3.6 ± 1.6 years for the occurrence of a cardiovascular event. Gating was performed by either RG or TG. We analyzed the expression of surface markers, and compared the predictive role of cell counts of monocyte subsets, as defined by RG and TG, respectively. With both gating strategies, higher intermediate monocyte counts predicted the cardiovascular endpoint in Kaplan-Meier analyses (P < 0.001 with RG; P < 0.001 with TG). After correction for confounders, intermediate monocyte counts remained independent predictors in Cox-Regression analyses (HR = 1.013 [95% CI: 1.006-1.020; P < 0.001] with RG; HR = 1.015 [95% CI: 1.006-1.024; P = 0.001] with TG). NRI was 3.9% when reclassifying patients from quartiles of intermediate monocyte counts with RG strategy toward quartiles of intermediate monocytes counts with TG strategy. In expression analysis, those monocytes which are defined as intermediate monocytes by the RG strategy and as nonclassical monocytes by the TG strategy share characteristics of both subsets. In conclusion, intermediate monocytes were independent predictors of cardiovascular outcome irrespective of the applied gating strategy. Future studies should aim to identify markers that allow for an unequivocal definition of intermediate monocytes, which may further improve their power to

  19. Triggering of human monocyte activation through CD69, a member of the natural killer cell gene complex family of signal transducing receptors

    PubMed Central

    1994-01-01

    The expression and function of CD69, a member of the natural killer cell gene complex family of signal transducing receptors, was investigated on human monocytes. CD69 was found expressed on all peripheral blood monocytes, as a 28- and 32-kD disulfide-linked dimer. Molecular cross-linking of CD69 receptors induced extracellular Ca2+ influx, as revealed by flow cytometry. CD69 cross-linking resulted also in phospholipase A2 activation, as detected by in vivo arachidonic acid release measurement from intact cells and by direct in vitro measurement of enzymatic activity using radiolabeled phosphatidylcholine vesicles. Prostaglandin E 2 alpha, 6-keto- prostaglandin F 1 alpha, and leukotriene B4 were detected by radioimmunoassay in supernatants from CD69-stimulated monocytes, suggesting the activation of both cyclooxygenase and lipoxygenase pathways after CD69 stimulation. CD69 cross-linking, moreover, was able to induce strong nitric oxide (NO) production from monocytes, as detected by accumulation of NO oxydixed derivatives, and cyclic GMP. It is important to note that NO generation was responsible for CD69- mediated increase in spontaneous cytotoxicity against L929 murine transformed fibroblast cell line and induction of redirected cytotoxicity towards P815 FcRII+ murine mastocytoma cell line. These data indicate that CD69 can act as a potent stimulatory molecule on the surface of human peripheral blood monocytes. PMID:7964477

  20. ADP and Other Metabolites Released from Acanthamoeba castellanii Lead to Human Monocytic Cell Death through Apoptosis and Stimulate the Secretion of Proinflammatory Cytokines

    PubMed Central

    Mattana, A.; Cappai, V.; Alberti, L.; Serra, C.; Fiori, P. L.; Cappuccinelli, P.

    2002-01-01

    Monocytes/macrophages are thought to be involved in Acanthamoeba infections. The aim of this work was to study whether soluble metabolites (ADP and other compounds) released by Acanthamoeba castellanii trophozoites could induce morphological and biochemical changes in human monocytic cells in vitro. We demonstrate here that ADP constitutively released in the medium by A. castellanii, interacting with specific P2y2 purinoceptors expressed on the monocytic cell membrane, caused a biphasic rise in [Ca2+]i, morphological changes characteristics of cells undergoing apoptosis, caspase-3 activation, and secretion of tumor necrosis factor alpha (TNF-α). The same results were found in monocytes exposed to purified ADP. Cell damage and TNF-α release induced by amoebic ADP were blocked by the P2y2 inhibitor suramin. Other metabolites contained in amoebic cell-free supernatants, with molecular masses of, respectively, >30 kDa and between 30 and 10 kDa, also caused morphological modifications and activation of intracellular caspase-3, characteristics of programmed cell death. Nevertheless, mechanisms by which these molecules trigger cell damage appeared to differ from that of ADP. In addition, other amoebic thermolable metabolites with molecular masses of <10 kDa caused the secretion of interleukin-1β. These findings suggest that pathogenic free-living A. castellanii by release of ADP and other metabolites lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines. PMID:12117953

  1. Detection of calcium activity in human monocytes by the fura-2 fluorescence method: in vitro differentiation sensitizes cells to dihydropyridine calcium channel modulators

    NASA Astrophysics Data System (ADS)

    Oraevsky, Alexander A.; Cabello, Olga A.; Shan, Qin; Tittel, Frank K.; Henry, Philip D.

    1994-07-01

    Dihydropyridine (DHP) calcium channel blockers have been shown to suppress atherogenesis in various species and controlled angiographic trials suggest that these drugs may retard the progression of occlusive coronary disease in humans. Because mononuclear leukocytes play a key role in the formation of early and advanced atheromatous lesions, we determined effects of DHP calcium channel modulators on calcium uptake by cells of the monocytic lineage. Human peripheral blood monocytes were evaluated before and after undergoing in vitro differentiation induced by two days of culture with fetal calf serum and FMLP. Changes in intracellular calcium activity were estimated with fura-2, a fluorescent calcium indicator. Freshly isolated (unactivated) monocytes were insensitive to DHP drugs both in the presence and absence of high potassium membrane depolarization. In contrast, nisoldipine, a DHP calcium channel blocker, and BAY K 8644, a DHP calcium channel activator, decreased and increased calcium uptake by KC1-depolarized differentiated monocytes. Results suggest that differentiation of monocytes to macrophages may involve a change in the expression and/or regulation of DHP- sensitive calcium channels.

  2. Exenatide (a GLP-1 agonist) improves the antioxidative potential of in vitro cultured human monocytes/macrophages.

    PubMed

    Bułdak, Łukasz; Łabuzek, Krzysztof; Bułdak, Rafał Jakub; Machnik, Grzegorz; Bołdys, Aleksandra; Okopień, Bogusław

    2015-09-01

    Macrophages are dominant cells in the pathogenesis of atherosclerosis. They are also a major source of reactive oxygen species (ROS). Oxidative stress, which is particularly high in subjects with diabetes, is responsible for accelerated atherosclerosis. Novel antidiabetic drugs (e.g., glucagon-like peptide-1 (GLP-1) agonists) were shown to reduce ROS level. Therefore, we conceived a study to evaluate the influence of exenatide, a GLP-1 agonist, on redox status in human monocytes/macrophages cultured in vitro, which may explain the beneficial effects of incretin-based antidiabetic treatment. Human macrophages obtained from 10 healthy volunteers were in vitro subjected to the treatment with GLP-1 agonist (exenatide) in the presence of lipopolysaccharide (LPS), antagonist of GLP-1 receptors (exendin 9-39), or protein kinase A inhibitor (H89). Afterwards, reactive oxygen species, malondialdehyde level, NADPH oxidase, and antioxidative enzymes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase] expression was evaluated. Finally, we estimated the activity of the abovementioned enzymes in the presence of H89. According to our findings, exenatide reduced ROS and malondialdyhyde (MDA) level by decreasing the expression of ROS-generating NADPH oxidase and by increasing the expression and activities of SOD and GSH-Px. We also showed that this effect was significantly inhibited by exendin 9-39 (a GLP-1 antagonist) and blocked by H89. Exenatide improved the antioxidative potential and reduced oxidative stress in cultured human monocytes/macrophages, and this finding may be responsible for the pleiotropic effects of incretin-based therapies. This effect relied on the stimulation of GLP-1 receptor.

  3. Expression of GM1, a marker of lipid rafts, defines two subsets of human monocytes with differential endocytic capacity and lipopolysaccharide responsiveness

    PubMed Central

    Moreno-Altamirano, M Maximina Bertha; Aguilar-Carmona, Israel; Sánchez-García, F Javier

    2007-01-01

    Monocytes constitute 5–10% of total human peripheral blood leucocytes and remain in circulation for several days before replenishing the tissue macrophage populations. Monocytes display heterogeneity in size, granularity and nuclear morphology, and in the expression of cell membrane molecules, such as CD14, CD16, CD32, CD64, major histocompatibility complex class II, CCR2, CCR5, among others. This has led to the suggestion that individual monocyte/macrophage populations have specialized functions within their microenvironments. This study provides evidence for the occurrence of two peripheral blood monocyte subpopulations on the basis of their differential expression of GM1, a sphingolipid found mostly in lipid rafts, a CD14+ GM1low population and a CD14+ GM1high population comprising about 97·5% and 2·5% of total CD14+ cells, respectively. GM1 expression correlates with functional differences in terms of endocytic activity, susceptibility to mycobacterial infection, and response to lipopolysaccharide (LPS) (modulation of Toll-like receptor-4 expression). CD14+ GM1low cells proved to be less endocytic and more responsive to LPS, whereas CD14+ GM1high cells are more endocytic and less responsive to LPS. In addition, during monocyte to macrophage differentiation in vitro, the percentage of CD14+ GM1high cells increases from about 2·5% at day 1 to more than 50% at day 7 of culture. These results suggest that GM1low and GM1high monocytes in peripheral blood, represent either different stages of maturation or different subsets with specialized activities. The expression of CD16 on GM1high favours the first possibility and, on the other hand that up-regulation of GM1 expression and probably lipid rafts function is involved in the monocyte to macrophage differentiation process. PMID:17250589

  4. Monocyte-induced downregulation of nitric oxide synthase in cultured aortic endothelial cells.

    PubMed

    Marczin, N; Antonov, A; Papapetropoulos, A; Munn, D H; Virmani, R; Kolodgie, F D; Gerrity, R; Catravas, J D

    1996-09-01

    Since endothelium-dependent vasodilation is altered in atherosclerosis and enhanced monocyte/endothelial interactions are implicated in early atherosclerosis, we evaluated the effects of monocytes on the endothelial nitric oxide (NO) pathway by estimating release of biologically active NO from cultured endothelial cells and levels of constitutive NO synthase (ecNOS). NO release was estimated in a short-term bioassay using endothelial cell-induced cGMP accumulation in vascular smooth muscle (SM) cells. Exposure of SM cells to porcine aortic endothelial cells (PAECs) and human aortic endothelial cells (HAECs) produced large increases in SM cGMP content; this increase was prevented by NG-nitro-L-arginine methyl ester, the inhibitor of endothelial NOS. Confluent monolayers of PAECs and HAECs cocultured with monocytes also stimulated SM cGMP formation; however, NO release from these cultures was attenuated in a coculture time (2 to 48 hours)- and monocyte concentration (20 to 200 x 10(3) per well)-dependent manner. This effect of monocyte adhesion appeared to be selective for NO release since other biochemical pathways, such as atriopeptin-and isoproterenol-induced cyclic nucleotide accumulation within the endothelial cells, were not altered by monocytes. The effects of adherent monocytes on NO release were mimicked by monocyte-derived cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 alpha. Furthermore, the conditioned medium of monocytes contained significant quantities of these cytokines. Conditioned medium, as well as monocytes physically separated from the endothelial cells, attenuated NO release, suggesting that soluble factors may mediate the effects of monocytes. An IL-1 beta neutralizing antibody fully prevented the NO dysfunction in response to directly adherent monocytes. Superoxide dismutase, catalase, 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), and exogenous L-arginine failed to improve NO release, suggesting that oxidant stress

  5. Diabetic conditions promote binding of monocytes to vascular smooth muscle cells and their subsequent differentiation.

    PubMed

    Meng, Li; Park, Jehyun; Cai, Qiangjun; Lanting, Linda; Reddy, Marpadga A; Natarajan, Rama

    2010-03-01

    Diabetes is associated with significantly accelerated rates of atherosclerosis, key features of which include the presence of excessive macrophage-derived foam cells in the subendothelial space. We examined the hypothesis that enhanced monocyte-vascular smooth muscle cell (VSMC) interactions leading to subendothelial monocyte retention and differentiation to macrophages under diabetic conditions may be underlying mechanisms. Human aortic VSMCs (HVSMCs) treated with diabetic stimuli high glucose (HG) or S100B, a ligand of the receptor for advanced glycation end products, exhibited significantly increased binding of THP-1 monocytic cells. Diabetic stimuli increased the expression of the adhesive chemokine fractalkine (FKN) in HVSMCs. Pretreatment of HVSMCs with FKN or monocyte chemoattractant protein-1 (MCP-1) neutralizing antibodies significantly inhibited monocyte-VSMC binding, whereas monocytes treated with FKN showed enhanced binding to VSMC. Mouse aortic VSMCs (MVSMCs) derived from type 2 diabetic db/db mice exhibited significantly increased FKN levels and binding to mouse WEHI78/24 monocytic cells relative to nondiabetic control db/+ cells. The enhanced monocyte binding in db/db cells was abolished by both FKN and MCP-1 antibodies. Endothelium-denuded aortas from db/db mice and streptozotocin-induced diabetic mice also exhibited enhanced FKN expression and monocyte binding, relative to respective controls. Coculture with HVSMCs increased CD36 expression in THP-1 cells, and this was significantly augmented by treatment of HVSMCs with S100B or HG. CD36 mRNA and protein levels were also significantly increased in WEHI78/24 cells after coincubation with db/db MVSMCs relative to control MVSMCs. These results demonstrate that diabetic conditions may accelerate atherosclerosis by inducing key chemokines in the vasculature that promote VSMC-monocyte interactions, subendothelial monocyte retention, and differentiation.

  6. Pathogenic bacteria and TNF do not induce production of macrophage migration inhibitory factor (MIF) by human monocytes.

    PubMed

    Temple, Suzanna E L; Cheong, Karey Y; Price, Patricia; Waterer, Grant W

    2009-06-01

    Elevated serum macrophage migration inhibitory factor (MIF) is associated with severe sepsis, but it is not clear whether bacteria stimulate synthesis of MIF by blood leukocytes directly or via induction of TNF. Here we assess production of MIF mRNA and protein by blood leukocytes from healthy human subjects (n=28) following exposure to bacteria commonly associated with sepsis (Escherichia coli and Streptococcus pneumoniae). Bacteria did not increase levels of MIF mRNA or secreted protein. CD14(+) monocytes were the main cell type producing MIF before and after stimulation. Exposure of leukocytes to TNF did not induce MIF. Hence elevated levels of serum MIF observed in sepsis may not reflect MIF produced by blood leukocytes stimulated directly by bacteria or TNF.

  7. Production of the second component of complement by human monocytes: stimulation by antigen-activated lymphocytes or lymphokines

    PubMed Central

    1977-01-01

    Human peripheral blood mononuclear cells cultured in the presence of antigen produced hemolytically active second complement component earlier and in larger amounts than did control cultures of the same cells without antigen. The increased amount of C2 in culture supernates came primarily from the adherent cell population and was due to increased synthesis as demonstrated by inhibition with 10(-4) M cycloheximide. Purified adherent monocytes produced more C2 when exposed to lymphokine-rich supernates from antigen-stimulated lymphocytes than when exposed to control supernates from unstimulated lymphocyte cultures. The increased synthesis of C2, which appeared to be mediated by a lymphokine, was partially inhibited specifically by 0.025 M alpha-L(-) fucose, a sugar which has previously been shown in inhibit the response of macrophages to migration inhibitory factor. PMID:858999

  8. Lipid-cell interactions in human monocytes investigated by doubly-resonant coherent anti-Stokes Raman scattering microscopy

    PubMed Central

    Weeks, Tyler; Schie, Iwan; den Hartigh, Laura J.; Rutledge, John C.; Huser, Thomas

    2011-01-01

    We demonstrate that doubly-resonant coherent anti-Stokes Raman scattering can provide enhanced and highly specific contrast for molecules containing unique Raman-active small molecular groups. This combination provides contrast for molecules that can otherwise be difficult to discriminate by Raman spectroscopy. Here, human monocytes were incubated with either deuterated oleic acid or 17-octadecynoic acid (a fatty acid with an end terminal acetylene group). The carbon-deuterium stretching vibration of the deuterated fatty acid, as well as the unique alkyne stretching vibration of the alkyne-containing fatty acid, were used to provide contrast for these exogenous free fatty acids. The combination of these unique modes with the common aliphatic carbon-hydrogen stretching vibration inherent to all fatty acid allowed for doubly-resonant detection of these unique molecules and enabled us to detect the presence of these lipids in areas within a cell where each molecular resonance by itself did not generate sufficient signal. PMID:21361680

  9. Abrasive Endoprosthetic Wear Particles Inhibit IFN-γ Secretion in Human Monocytes Via Upregulating TNF-α-Induced miR-29b.

    PubMed

    Bu, Yan-Min; Zheng, De-Zhi; Wang, Lei; Liu, Jun

    2017-02-01

    The adverse biological responses to prostheses wear particles commonly led to the failure of total hip arthroplasty. Among the released cytokines, interferon-γ (IFN-γ) has been found to be a critical functional factor during osteoclast differentiation. However, the molecular mechanism underlying the regulation of IFN-γ in wear particles-induced cells still needs to be determined. Four kinds of abrasive endoprosthetic wear particle were used to treat THP-1 cells, including polymethylmethacrylate (PMMA), zirconiumoxide (ZrO2), commercially pure titanium (cpTi), and titanium alloy (Ti-6Al-7Nb), with a concentration of 0.01, 0.05, 0.1, or 0.2 mg/ml for 48 h. The expression of IFN-γ and miR-29b was detected by real-time RT-PCR or ELISA. Luciferase reporter assay was performed to determine the regulation of miR-29b on IFN-γ. The effect of miR-29b inhibitor on the expression of wear particle-induced IFN-γ was detected. The expression of miR-29b was examined in THP-1 cells treated with tumor necrosis factor-alpha (TNF-α). The expression of IFN-γ was downregulated and the level of miR-29b was increased in THP-1 cells pretreated with wear particles. IFN-γ was a target of miR-29b. Wear particles inhibited the expression of IFN-γ through miR-29b. The expression of miR-29b was significantly reduced in THP-1 cells treated with TNF-α neutralizing antibody and particles comparing to that in the cells treated with particles alone. Wear particles inhibit the IFN-γ secretion in human monocytes, which was associated with the upregulating TNF-α-induced miR-29b.

  10. Role of MMPs in orchestrating inflammatory response in human monocytes via a TREM-1-PI3K-NF-κB pathway.

    PubMed

    Gómez-Piña, Vanesa; Martínez, Eriel; Fernández-Ruíz, Irene; Del Fresno, Carlos; Soares-Schanoski, Alessandra; Jurado, Teresa; Siliceo, María; Toledano, Victor; Fernández-Palomares, Rosa; García-Rio, Francisco; Arnalich, Francisco; Biswas, Subhra K; López-Collazo, Eduardo

    2012-06-01

    The MMPs constitute a family of endopeptidases that can cleavage extracellular proteins. They are involved in a number of events; some of these include inflammatory processes. One of its targets is the TREM-1, which has emerged as an important modulator of innate immune responses in mammals. This transmembrane glycoprotein possesses an Ig-like ectodomain readily shed by MMPs to generate sTREM-1. Whereas membrane-anchored TREM-1 amplifies inflammatory responses, sTREM-1 exhibits anti-inflammatory properties. Here we show that sustained cell surface expression of TREM-1 in human monocytes, through metalloproteinase inhibition, counteracts the well-characterized down-regulation of several proinflammatory cytokines during the ET time-frame, also known as M2 or alternative activation. In addition to the cytokines profile, other features of the ET phenotype were underdeveloped when TREM-1 was stabilized at the cell surface. These events were mediated by the signal transducers PI3Ks and Syk. We also show that sTREM-1 counteracts the proinflammatory response obtained by membrane TREM-1 stabilization but failed to induce ET on naïve human monocytes. As the sustained TREM-1 expression at the cell surface suffices to block the progress of a refractory state in human monocytes, our data indicate that TREM-1 and MMPs orchestrate an "adaptive" form of innate immunity by modulating the human monocytes response to endotoxin.

  11. Leukaemia inhibitory factor enhances tissue factor expression in human monocyte-derived macrophages: a gp130-mediated mechanism.

    PubMed

    Meisel, S R; Shimon, I; Edgington, T S; Melmed, S; Cercek, B; Shah, P K

    1999-12-01

    Leukaemia inhibitory factor (LIF) and interleukin (IL)-6 are members of a cytokine group that share a common signal transducer gp130 and induce pleiotropic biological effects in cells of diverse lineage. In monocytes, LIF facilitates differentiation, which may stimulate the biosynthesis of tissue factor (TF) that initiates the coagulation cascade. We tested the hypothesis that LIF would enhance TF expression in human monocyte-derived macrophages (MDMs). Human peripheral blood mononuclear cells separated from whole blood by density centrifugation were allowed to differentiate into MDMs in primary culture, and were then exposed to LIF, IL-6 and oncostatin M (OSM) for 24 h. LIF and IL-6 receptors, and gp130 were demonstrated in MDMs by immunocytochemistry and RT-PCR. TF procoagulant activity (TF-PCA) was measured by recalcification clotting time and TF protein by Western blotting. The results show that both TF procoagulant activity and TF protein increased significantly in response to LIF over the concentration range of 1-100 nM (P < 0.03). Although OSM and IL-6 tended to enhance TF expression by MDMs, the increase did not reach statistical significance. Anti-LIF receptor and anti-gp130 antibodies attenuated the effect of LIF on TF expression as assayed by both bioassay and flow-cytometry. In conclusion, LIF increases TF-PCA and TF protein in MDMs, and specific anti-LIF receptor antibodies attenuate this effect. Thus, LIF may regulate by a gp130-dependent pathway macrophage-mediated procoagulant function in diverse pathological states involving inflammation and thrombosis and seems to serve as an important mediator at the interface between these processes.

  12. Epicubenol and Ferruginol induce DC from human monocytes and differentiate IL-10-producing regulatory T cells in vitro

    SciTech Connect

    Takei, Masao . E-mail: mtakei@fz-borstel.de; Umeyama, Akemi; Arihara, Shigenobu

    2005-11-18

    Epicubenol and 19-hydroxyferruginol (Ferruginol) are sesquiterpenes isolated from the black heartwood of Cryptomeria japonica. Dendritic cells (DC) are specialized antigen-presenting cells that monitor the antigenic environment and activate naive T cells. The role of DC is not only to sense danger but also to tolerize the immune system to antigens encountered in the absence of maturation/inflammatory stimuli. In this study, we attempted to investigate the effects of Epicubenol and Ferruginol on the phenotypic and functional maturation of human monocytes-derived DC in vitro. Human monocytes were cultured with GM-CSF and IL-4 for 6 days under standard conditions, followed by another 2 days with Epicubenol or Ferruginol. The expression levels of CD1a, CD83, and HLA-DR as expressed by mean fluorescence intensity (MFI) on Epicubenol-primed DC or Ferruginol-primed DC were enhanced. Allogeneic Epicubenol-primed DC or Ferruginol-primed DC co-cultured with naive T cells at 1:5 ratio, secreted IL-10 and TGF-{beta}, but little IL-4. Moreover, T cells that develop in co-culture of Epicubenol-primed DC or Ferruginol-primed DC and naive T cells at 1:5 ratio suppressed the proliferation of autologous T cells at Treg cells: Ttarget cells and this suppression of proliferation was inhibited by anti-IL-10 mAb. The expression of FoxP3 mRNA on T cells that develop in co-culture of Epicubenol-primed DC or Ferruginol-primed DC and naive T cells was lower. From these results, Epicubenol and Ferruginol may induce IL-10-producing Treg 1 cells from naive T cells by modulating DC function. It seems that Epicubenol and Ferruginol appear to be a target for tolerance after transplantation and in autoimmune diseases.

  13. Human plasma enhances the infectivity of primary human immunodeficiency virus type 1 isolates in peripheral blood mononuclear cells and monocyte-derived macrophages.

    PubMed Central

    Wu, S C; Spouge, J L; Conley, S R; Tsai, W P; Merges, M J; Nara, P L

    1995-01-01

    Physiological microenvironments such as blood, seminal plasma, mucosal secretions, or lymphatic fluids may influence the biology of the virus-host cell and immune interactions for human immunodeficiency virus type 1 (HIV-1). Relative to media, physiological levels of human plasma were found to enhance the infectivity of HIV-1 primary isolates in both phytohemagglutinin-stimulated peripheral blood mononuclear cells and monocyte-derived macrophages. Enhancement was observed only when plasma was present during the virus-cell incubation and resulted in a 3- to 30-fold increase in virus titers in all of the four primary isolates tested. Both infectivity and virion binding experiments demonstrated a slow, time-dependent process generally requiring between 1 and 10 h. Human plasma collected in anticoagulants CPDA-1 and heparin, but not EDTA, exhibited this effect at concentrations from 90 to 40%. Furthermore, heat-inactivated plasma resulted in a loss of enhancement in peripheral blood mononuclear cells but not in monocyte-derived macrophages. Physiological concentrations of human plasma appear to recruit additional infectivity, thus increasing the infectious potential of the virus inoculum. PMID:7666510

  14. Analysis of the human monocyte-derived macrophage transcriptome and response to lipopolysaccharide provides new insights into genetic aetiology of inflammatory bowel disease.

    PubMed

    Baillie, J Kenneth; Arner, Erik; Daub, Carsten; De Hoon, Michiel; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Carninci, Piero; Forrest, Alistair R R; Hayashizaki, Yoshihide; Faulkner, Geoffrey J; Wells, Christine A; Rehli, Michael; Pavli, Paul; Summers, Kim M; Hume, David A

    2017-03-01

    The FANTOM5 consortium utilised cap analysis of gene expression (CAGE) to provide an unprecedented insight into transcriptional regulation in human cells and tissues. In the current study, we have used CAGE-based transcriptional profiling on an extended dense time course of the response of human monocyte-derived macrophages grown in macrophage colony-stimulating factor (CSF1) to bacterial lipopolysaccharide (LPS). We propose that this system provides a model for the differentiation and adaptation of monocytes entering the intestinal lamina propria. The response to LPS is shown to be a cascade of successive waves of transient gene expression extending over at least 48 hours, with hundreds of positive and negative regulatory loops. Promoter analysis using motif activity response analysis (MARA) identified some of the transcription factors likely to be responsible for the temporal profile of transcriptional activation. Each LPS-inducible locus was associated with multiple inducible enhancers, and in each case, transient eRNA transcription at multiple sites detected by CAGE preceded the appearance of promoter-associated transcripts. LPS-inducible long non-coding RNAs were commonly associated with clusters of inducible enhancers. We used these data to re-examine the hundreds of loci associated with susceptibility to inflammatory bowel disease (IBD) in genome-wide association studies. Loci associated with IBD were strongly and specifically (relative to rheumatoid arthritis and unrelated traits) enriched for promoters that were regulated in monocyte differentiation or activation. Amongst previously-identified IBD susceptibility loci, the vast majority contained at least one promoter that was regulated in CSF1-dependent monocyte-macrophage transitions and/or in response to LPS. On this basis, we concluded that IBD loci are strongly-enriched for monocyte-specific genes, and identified at least 134 additional candidate genes associated with IBD susceptibility from reanalysis

  15. Epstein-Barr Virus Interferes with the Amplification of IFNα Secretion by Activating Suppressor of Cytokine Signaling 3 in Primary Human Monocytes

    PubMed Central

    Michaud, François; Coulombe, François; Gaudreault, Eric; Paquet-Bouchard, Carine; Rola-Pleszczynski, Marek; Gosselin, Jean

    2010-01-01

    Background Epstein-Barr virus is recognized to cause lymphoproliferative disorders and is also associated with cancer. Evidence suggests that monocytes are likely to be involved in EBV pathogenesis, especially due to a number of cellular functions altered in EBV-infected monocytes, a process that may affect efficient host defense. Because type I interferons (IFNs) are crucial mediators of host defense against viruses, we investigated the effect of EBV infection on the IFNα pathway in primary human monocytes. Methodology/Principal Findings Infection of monocytes with EBV induced IFNα secretion but inhibited the positive feedback loop for the amplification of IFNα. We showed that EBV infection induced the expression of suppressor of cytokine signaling 3 (SOCS3) and, to a lesser extent, SOCS1, two proteins known to interfere with the amplification of IFNα secretion mediated by the JAK/STAT signal transduction pathway. EBV infection correlated with a blockage in the activation of JAK/STAT pathway members and affected the level of phosphorylated IFN regulatory factor 7 (IRF7). Depletion of SOCS3, but not SOCS1, by small interfering RNA (siRNA) abrogated the inhibitory effect of EBV on JAK/STAT pathway activation and significantly restored IFNα secretion. Finally, transfection of monocytes with the viral protein Zta caused the upregulation of SOCS3, an event that could not be recapitulated with mutated Zta. Conclusions/Significance We propose that EBV protein Zta activates SOCS3 protein as an immune escape mechanism that both suppresses optimal IFNα secretion by human monocytes and favors a state of type I IFN irresponsiveness in these cells. This immunomodulatory effect is important to better understand the aspects of the immune response to EBV. PMID:20689596

  16. Human Cytomegalovirus Stimulates the Synthesis of Select Akt-Dependent Antiapoptotic Proteins during Viral Entry To Promote Survival of Infected Monocytes

    PubMed Central

    Peppenelli, Megan A.; Arend, Kyle C.; Cojohari, Olesea; Moorman, Nathaniel J.

    2016-01-01

    ABSTRACT Primary peripheral blood monocytes are responsible for the hematogenous dissemination of human cytomegalovirus (HCMV) following a primary infection. To facilitate viral spread, we have previously shown HCMV to extend the short 48-h life span of monocytes. Mechanistically, HCMV upregulated two specific cellular antiapoptotic proteins, myeloid leukemia sequence 1 (Mcl-1) and heat shock protein 27 (HSP27), to block the two proteolytic cleavages necessary for the formation of fully active caspase 3 and the subsequent initiation of apoptosis. We now show that HCMV more robustly upregulated Mcl-1 than normal myeloid growth factors and that Mcl-1 was the only myeloid survival factor to rapidly induce HSP27 prior to the 48-h cell fate checkpoint. We determined that HCMV glycoproteins gB and gH signal through the cellular epidermal growth factor receptor (EGFR) and αvβ3 integrin, respectively, during viral entry in order to drive the increase of Mcl-1 and HSP27 in an Akt-dependent manner. Although Akt is known to regulate protein stability and transcription, we found that gB- and gH-initiated signaling preferentially and cooperatively stimulated the synthesis of Mcl-1 and HSP27 through mTOR-mediated translation. Overall, these data suggest that the unique signaling network generated during the viral entry process stimulates the upregulation of select antiapoptotic proteins allowing for the differentiation of short-lived monocytes into long-lived macrophages, a key step in the viral dissemination strategy. IMPORTANCE Human cytomegalovirus (HCMV) infection is endemic within the human population. Although primary infection is generally asymptomatic in immunocompetent individuals, HCMV is a significant cause of morbidity and mortality in the immunocompromised. The multiorgan inflammatory diseases associated with symptomatic HCMV infection are a direct consequence of the monocyte-mediated systemic spread of the virus. In order for peripheral blood monocytes to

  17. SuperSAGE evidence for CD14++CD16+ monocytes as a third monocyte subset.

    PubMed

    Zawada, Adam M; Rogacev, Kyrill S; Rotter, Björn; Winter, Peter; Marell, Rolf-R; Fliser, Danilo; Heine, Gunnar H

    2011-09-22

    Monocytes are a heterogeneous cell population with subset-specific functions and phenotypes. The differential expression of CD14 and CD16 distinguishes classical CD14(++)CD16(-), intermediate CD14(++)CD16(+), and nonclassical CD14(+)CD16(++) monocytes. Current knowledge on human monocyte heterogeneity is still incomplete: while it is increasingly acknowledged that CD14(++)CD16(+) monocytes are of outstanding significance in 2 global health issues, namely HIV-1 infection and atherosclerosis, CD14(++)CD16(+) monocytes remain the most poorly characterized subset so far. We therefore developed a method to purify the 3 monocyte subsets from human blood and analyzed their transcriptomes using SuperSAGE in combination with high-throughput sequencing. Analysis of 5 487 603 tags revealed unique identifiers of CD14(++)CD16(+) monocytes, delineating these cells from the 2 other monocyte subsets. Gene Ontology (GO) enrichment analysis suggests diverse immunologic functions, linking CD14(++)CD16(+) monocytes to Ag processing and presentation (eg, CD74, HLA-DR, IFI30, CTSB), to inflammation and monocyte activation (eg, TGFB1, AIF1, PTPN6), and to angiogenesis (eg, TIE2, CD105). In conclusion, we provide genetic evidence for a distinct role of CD14(++)CD16(+) monocytes in human immunity. After CD14(++)CD16(+) monocytes have earlier been discussed as a potential therapeutic target in inflammatory diseases, we are hopeful that our data will spur further research in the field of monocyte heterogeneity.

  18. Monocyte-induced recovery of inflammation-associated hepatocellular dysfunction in a biochip-based human liver model

    PubMed Central

    Gröger, Marko; Rennert, Knut; Giszas, Benjamin; Weiß, Elisabeth; Dinger, Julia; Funke, Harald; Kiehntopf, Michael; Peters, Frank T.; Lupp, Amelie; Bauer, Michael; Claus, Ralf A.; Huber, Otmar; Mosig, Alexander S.

    2016-01-01

    Liver dysfunction is an early event in sepsis-related multi-organ failure. We here report the establishment and characterization of a microfluidically supported in vitro organoid model of the human liver sinusoid. The liver organoid is composed of vascular and hepatocyte cell layers integrating non-parenchymal cells closely reflecting tissue architecture and enables physiological cross-communication in a bio-inspired fashion. Inflammation-associated liver dysfunction was mimicked by stimulation with various agonists of toll-like receptors. TLR-stimulation induced the release of pro- and anti-inflammatory cytokines and diminished expression of endothelial VE-cadherin, hepatic MRP-2 transporter and apolipoprotein B (ApoB), resulting in an inflammation-related endothelial barrier disruption and hepatocellular dysfunction in the liver organoid. However, interaction of the liver organoid with human monocytes attenuated inflammation-related cell responses and restored MRP-2 transporter activity, ApoB expression and albumin/urea production. The cellular events observed in the liver organoid closely resembled pathophysiological responses in the well-established sepsis model of peritoneal contamination and infection (PCI) in mice and clinical observations in human sepsis. We therefore conclude that this human liver organoid model is a valuable tool to investigate sepsis-related liver dysfunction and subsequent immune cell-related tissue repair/remodeling processes. PMID:26902749

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

    PubMed

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

    2016-04-01

    Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is observed in various conditions, including depression and obesity, which are also often related. Glucocorticoid (GC) resistance and desensitization of peripheral GC receptors (GRs) are often the case in HPA dysregulation seen in depression, and GC plays a critical role in regulation of inflammation. Given the growing evidence that inflammation is a central feature of some depression cases and obesity, we aimed to investigate the immune-regulatory role of GC-GR in relation to depressive mood and obesity in 35 healthy men and women. Depressive mood and level of obesity were assessed, using Beck Depression Inventory (BDI-Ia) and body mass index (BMI), respectively. We measured plasma cortisol levels via enzyme-linked immunosorbent assay and lipopolysaccharide-stimulated intracellular tumor necrosis factor (TNF) production by monocytes, using flow cytometry. Cortisol sensitivity was determined by the difference in monocytic TNF production between the conditions of 1 and 0 μM cortisol incubation ("cortisol-mediated inflammation regulation, CoMIR"). GR vs. mineralocorticoid receptor (MR) antagonism for CoMIR was examined by using mifepristone and spironolactone. A series of multiple regression analyses were performed to investigate independent contribution of depressive mood vs. obesity after controlling for age, gender, systolic blood pressure (SBP), and plasma cortisol in predicting CoMIR. CoMIR was explained by somatic subcomponents of depressive mood (BDI-S: β=-0.499, p=0.001), or BMI (β=-0.466, p<0.01) in separate models. The effects of BMI disappeared when BDI-S was controlled for in the model, while BDI-S remained a significant independent predictor for CoMIR (β=-0.369, p<0.05). However, BMI remained the only independent predictor when BDI-T or BDI-C were controlled for in the model. Mediation analyses also revealed that the relationship between BMI and CoMIR was mediated by BDI-S. The

  20. Powerful Identification of Cis-regulatory SNPs in Human Primary Monocytes Using Allele-Specific Gene Expression

    PubMed Central

    Almlöf, Jonas Carlsson; Lundmark, Per; Lundmark, Anders; Ge, Bing; Maouche, Seraya; Göring, Harald H. H.; Liljedahl, Ulrika; Enström, Camilla; Brocheton, Jessy; Proust, Carole; Godefroy, Tiphaine; Sambrook, Jennifer G.; Jolley, Jennifer; Crisp-Hihn, Abigail; Foad, Nicola; Lloyd-Jones, Heather; Stephens, Jonathan; Gwilliam, Rhian; Rice, Catherine M.; Hengstenberg, Christian; Samani, Nilesh J.; Erdmann, Jeanette; Schunkert, Heribert; Pastinen, Tomi; Deloukas, Panos; Goodall, Alison H.; Ouwehand, Willem H.; Cambien, François; Syvänen, Ann-Christine

    2012-01-01

    A large number of genome-wide association studies have been performed during the past five years to identify associations between SNPs and human complex diseases and traits. The assignment of a functional role for the identified disease-associated SNP is not straight-forward. Genome-wide expression quantitative trait locus (eQTL) analysis is frequently used as the initial step to define a function while allele-specific gene expression (ASE) analysis has not yet gained a wide-spread use in disease mapping studies. We compared the power to identify cis-acting regulatory SNPs (cis-rSNPs) by genome-wide allele-specific gene expression (ASE) analysis with that of traditional expression quantitative trait locus (eQTL) mapping. Our study included 395 healthy blood donors for whom global gene expression profiles in circulating monocytes were determined by Illumina BeadArrays. ASE was assessed in a subset of these monocytes from 188 donors by quantitative genotyping of mRNA using a genome-wide panel of SNP markers. The performance of the two methods for detecting cis-rSNPs was evaluated by comparing associations between SNP genotypes and gene expression levels in sample sets of varying size. We found that up to 8-fold more samples are required for eQTL mapping to reach the same statistical power as that obtained by ASE analysis for the same rSNPs. The performance of ASE is insensitive to SNPs with low minor allele frequencies and detects a larger number of significantly associated rSNPs using the same sample size as eQTL mapping. An unequivocal conclusion from our comparison is that ASE analysis is more sensitive for detecting cis-rSNPs than standard eQTL mapping. Our study shows the potential of ASE mapping in tissue samples and primary cells which are difficult to obtain in large numbers. PMID:23300628

  1. Spleen Tyrosine Kinase (Syk) Mediates IL-1β Induction by Primary Human Monocytes during Antibody-enhanced Dengue Virus Infection.

    PubMed

    Callaway, Justin B; Smith, Scott A; McKinnon, Karen P; de Silva, Aravinda M; Crowe, James E; Ting, Jenny P-Y

    2015-07-10

    Approximately 500,000 people are hospitalized with severe dengue illness annually. Antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is believed to contribute to the pathogenic cytokine storm described in severe dengue patients, but the precise signaling pathways contributing to elevated cytokine production are not elucidated. IL-1β is a potent inflammatory cytokine that is frequently elevated during severe dengue, and the unique dual regulation of IL-1β provides an informative model to study ADE-induced cytokines. This work utilizes patient-derived anti-DENV mAbs and primary human monocytes to study ADE-induced IL-1β and other cytokines. ADE of DENV serotype 2 (DENV-2) elevates mature IL-1β secretion by monocytes independent of DENV replication by 4 h postinoculation (hpi). Prior to this, DENV immune complexes activate spleen tyrosine kinase (Syk) within 1 hpi. Syk induces elevated IL1B, TNF, and IL6 mRNA by 2 hpi. Syk mediates elevated IL-1β secretion by activating ERK1/2, and both Syk and ERK1/2 inhibitors ablated ADE-induced IL-1β secretion. Maturation of pro-IL-1β during ADE requires caspase-1 and NLRP3, but caspase-1 is suboptimally increased by ADE and can be significantly enhanced by a typical inflammasome agonist, ATP. Importantly, this inflammatory Syk-ERK signaling axis requires DENV immune complexes, because DENV-2 in the presence of serotype-matched anti-DENV-2 mAb, but not anti-DENV-1 mAb, activates Syk, ERK, and IL-1β secretion. This study provides evidence that DENV-2 immune complexes activate Syk to mediate elevated expression of inflammatory cytokines. Syk and ERK may serve as new therapeutic targets for interfering with ADE-induced cytokine expression during severe dengue.

  2. Complex evaluation of human monocyte-derived dendritic cells for cancer immunotherapy

    PubMed Central

    Vopenkova, Katerina; Mollova, Klara; Buresova, Ivana; Michalek, Jaroslav

    2012-01-01

    Dendritic cell (DC) immunotherapy is capable of generating tumour-specific immune responses. Different maturation strategies were previously tested to obtain DC capable of anti-cancer responses in vitro, usually with limited clinical benefit. Mutual comparison of currently used maturation strategies and subsequent complex evaluation of DC functions and their stimulatory capacity on T cells was performed in this study to optimize the DC vaccination strategy for further clinical application. DC were generated from monocytes using granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, pulsed with whole tumour cell lysate and then matured with one of five selected maturation strategies or cultured without additional maturation stimulus. DC were characterized with regard to their surface marker expression, cytokine profiles, migratory capacity, allogeneic and autologous T cell stimulatory capacity as well as their specific cytotoxicity against tumour antigens. We were able to demonstrate extensive variability among different maturation strategies currently used in DC immunotherapeutic protocols that may at least partially explain limited clinical benefit of some clinical trials with such DC. We identified DC matured with interferon-γ and lipopolysaccharide as the most attractive candidate for future clinical trials in cancer immunotherapy. PMID:22882679

  3. Dual stimulation with bacterial and viral components increases the expression of hepcidin in human monocytes.

    PubMed

    Ripley, Delia A; Morris, Roger H; Maddocks, Sarah E

    2014-10-01

    Hepcidin belongs to the antimicrobial peptide (AMP) family and is the key regulator of iron metabolism. It modulates iron homeostasis by binding to, and degrading the iron exporter molecule, ferroportin, thus inhibiting cellular iron efflux. Many antimicrobial peptides have a dual function; some are able to act directly as an antimicrobial agent as well as having an immunoregulatory role in the host. Toll-like receptors (TLRs) bind to components of microorganisms, activate cellular signal transduction pathways and stimulate innate immune responses. The effect of TLR3 (poly I:C) and TLR9 (CpG) co-stimulation of THP-1-derived monocytes using purified TLR ligands showed that 24 h after exposure poly I:C and CpG ligands in combination, hepcidin expression was significantly increased (10-fold) when compared to the untreated control. This combination of TLR ligands mimics simultaneous bacterial and viral infections, thus suggesting a potential key role for hepcidin in combined infections. Additionally, using a chequerboard assay, we have shown that hepcidin has an antagonistic effect in combination with the antibiotics rifampicin and tetracycline against Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes, evidenced by a fractional inhibitory concentration index (FICI) > 4. This finding has important implications for future treatment regimens especially in an era of increasing antimicrobial resistance.

  4. Importance of hydroxyapatite particles characteristics on cytokines production by human monocytes in vitro.

    PubMed

    Laquerriere, Patrice; Grandjean-Laquerriere, Alexia; Jallot, Edouard; Balossier, Gerard; Frayssinet, Patrick; Guenounou, Moncef

    2003-07-01

    Calcium phosphate bioceramics have been applied as bone substitutes for several decades. Aseptic loosening after total joint arthroplasty is a major problem in orthopaedic surgery. Hydroxyapatite particles from materials wear have been reported as the main cause of implant failure. For this reason, an investigation into possible wear particles from materials used in the implant may lead to longevity after arthroplasty. Monocytes are among the first cells to colonize the inflammatory site. In the present study, we have evaluated the inflammatory response after exposition to particles with different characteristics (size, sintering temperature and shape). Our data demonstrate that the most important characteristic was the shape and the size of the particles. The needle shaped particles induced the larger production of TNF-alpha, IL-6 and IL-10 by cells. To a less manner, the smallest particles induced an increase of the expression and production of the cytokines studied (TNF-alpha, IL-6 and IL-10). The sintering temperature appeared to be a less important characteristic even though it was involved in the dissolution/precipitation process.

  5. Autophagy Induction by Endothelial-Monocyte Activating Polypeptide II Contributes to the Inhibition of Malignant Biological Behaviors by the Combination of EMAP II with Rapamycin in Human Glioblastoma

    PubMed Central

    Ma, Jun; Meng, Fanjie; Li, Shuai; Liu, Libo; Zhao, Lini; Liu, Yunhui; Hu, Yi; Li, Zhen; Yao, Yilong; Xi, Zhuo; Teng, Hao; Xue, Yixue

    2015-01-01

    This study aims to investigate the effect of endothelial-monocyte activating polypeptide II (EMAP II) on human glioblastoma (GBM) cells and glioblastoma stem cells (GSCs) as well as its possible mechanisms. In this study, EMAP II inhibited the cell viability and decreased the mitochondrial membrane potential in human GBM cells and GSCs, and autophagy inhibitor 3-methyl adenine (3-MA) blocked these effects. Autophagic vacuoles were formed in these cells after EMAP II treatment and this phenomenon was blocked by 3-MA. In addition, the up-regulation of microtubule-associated protein-1 light chain-3 (LC3)-II and the down-regulation of autophagic degraded substrate p62/SQSTM1 caused by EMAP II were observed. Cells treated with EMAP-II inhibited the PI3K/Akt/mTOR signal pathway, and PI3K/Akt agonist insulin-like growth factor-1 (IGF-1) blocked the effect of EMAP II on the expression of LC3-II and p62/SQSTM1. Cells exposed to EMAP-II experienced mitophagy and ER stress. Furthermore, the inhibition of cell proliferation, migration and invasion of GBM cells and GSCs were more remarkable by the combination of EMAP II and rapamycin than either agent alone in vitro and in vivo. The current study demonstrated that the cytotoxicity of EMAP II in human GBM cells and GSCs was induced by autophagy, accompanied by the inhibition of PI3K/Akt/mTOR signal pathway, mitophagy and ER stress. The combination of EMAP II with rapamycin demonstrated the inhibitory effect on the malignant biological behaviors of human GBM cells and GSCs in vitro and in vivo. PMID:26648842

  6. In vitro Catecholamine Exposure Produces Variable Effects on the B7 Costimulatory Pathway in Human Monocytic Cells

    NASA Technical Reports Server (NTRS)

    Salicru, A. N.; Crucian, B.; Sams, Clarence; Actor, J. K.; Marshall, G. D., Jr.

    2006-01-01

    Catecholamines have been associated with immunomodulation of the adaptive immune system towards a Th2 response in vitro. We therefore examined the role of in vitro epinephrine (EPI) and norepinephrine (NE) exposure on the B7 costimulatory expression of antigen presenting cells (APC) from human monocytic cell lines and human peripheral blood mononuclear cells (PBMC). THP1 monocytic cells and CD14+ cells from normal human PBMC were stimulated with lipopolysaccharide (LPS) and incubated with physiologic stress levels (10(exp -6) - 10(exp -8)M) of EPI or NE for 24 hours. Cells were subsequently stained with CD80 FITC, CD86 PE, and CD14 PC5 antibodies and analyzed by flow cytometry for changes in fluorescence and mean fluorescence intensity (MFI). Exposure of THP1 to EPI in vitro at concentrations of 10(exp -6), 10(exp -7) and 10(exp -8)M significantly decreased mean CD80 from 42 plus or minus 0.7% to 11 plus or minus 0.44%, 19.1 plus or minus 2.0%, and 30.7 plus or minus 2.1% expression, respectively (p less than 0.01). In addition, CD86 expression increased with EPI at 10(exp -6), 10(exp -7) and 10(exp -8) M from 9.2 plus or minus 0.52% to 41 plus or minus 3.8%, 26.4 plus or minus 1.9%, and 15.74 plus or minus 1.8% expression, respectively (p less than 0.01). Similar results for mean CD80 and CD86 percent expression were observed for CD14+ cells from PBMC with a sample size of N = 6 and for NE when substituted for EPI. The data show that in vitro exposure to catecholamines significantly decreases %CD86 expression and significantly increases %CD86 expression in THP1 cells and human CD14+ APC. Previous studies have suggested an association between increased CD86 expression and TH2 activity. Thus, these data suggest that immunomodulation by catecholamines results in part by the variable effects of the B7 costimulatory pathway in APC.

  7. Salusin-β induces foam cell formation and monocyte adhesion in human vascular smooth muscle cells via miR155/NOX2/NFκB pathway

    PubMed Central

    Sun, Hai-Jian; Zhao, Ming-Xia; Liu, Tong-Yan; Ren, Xing-Sheng; Chen, Qi; Li, Yue-Hua; Kang, Yu-Ming; Zhu, Guo-Qing

    2016-01-01

    Vascular smooth muscle cells (VSMCs) are indispensible components in foam cell formation. Salusin-β is a stimulator in the progression of atherosclerosis. Here, we showed that salusin-β increased foam cell formation evidenced by accumulation of lipid droplets and intracellular cholesterol content, and promoted monocyte adhesion in human VSMCs. Salusin-β increased the expressions and activity of acyl coenzyme A:cholesterol acyltransferase-1 (ACAT-1) and vascular cell adhesion molecule-1 (VCAM-1) in VSMCs. Silencing of ACAT-1 abolished the salusin-β-induced lipid accumulation, and silencing of VCAM-1 prevented the salusin-β-induced monocyte adhesion in VSMCs. Salusin-β caused p65-NFκB nuclear translocation and increased p65 occupancy at the ACAT-1 and VCAM-1 promoter. Inhibition of NFκB with Bay 11-7082 prevented the salusin-β-induced ACAT-1 and VCAM-1 upregulation, foam cell formation and monocyte adhesion in VSMCs. Scavenging ROS, inhibiting NADPH oxidase or knockdown of NOX2 abolished the effects of salusin-β on ACAT-1 and VCAM-1 expressions, p65-NFκB nuclear translocation, lipid accumulation and monocyte adhesion in VSMCs. Salusin-β increased miR155 expression, and knockdown of miR155 prevented the effects of salusin-β on ACAT-1 and VCAM-1 expressions, p65-NFκB nuclear translocation, lipid accumulation, monocyte adhesion and ROS production in VSMCs. These results indicate that salusin-β induces foam formation and monocyte adhesion via miR155/NOX2/NFκB-mediated ACAT-1 and VCAM-1 expressions in VSMCs. PMID:27004848

  8. Salusin-β induces foam cell formation and monocyte adhesion in human vascular smooth muscle cells via miR155/NOX2/NFκB pathway.

    PubMed

    Sun, Hai-Jian; Zhao, Ming-Xia; Liu, Tong-Yan; Ren, Xing-Sheng; Chen, Qi; Li, Yue-Hua; Kang, Yu-Ming; Zhu, Guo-Qing

    2016-03-23

    Vascular smooth muscle cells (VSMCs) are indispensible components in foam cell formation. Salusin-β is a stimulator in the progression of atherosclerosis. Here, we showed that salusin-β increased foam cell formation evidenced by accumulation of lipid droplets and intracellular cholesterol content, and promoted monocyte adhesion in human VSMCs. Salusin-β increased the expressions and activity of acyl coenzyme A:cholesterol acyltransferase-1 (ACAT-1) and vascular cell adhesion molecule-1 (VCAM-1) in VSMCs. Silencing of ACAT-1 abolished the salusin-β-induced lipid accumulation, and silencing of VCAM-1 prevented the salusin-β-induced monocyte adhesion in VSMCs. Salusin-β caused p65-NFκB nuclear translocation and increased p65 occupancy at the ACAT-1 and VCAM-1 promoter. Inhibition of NFκB with Bay 11-7082 prevented the salusin-β-induced ACAT-1 and VCAM-1 upregulation, foam cell formation and monocyte adhesion in VSMCs. Scavenging ROS, inhibiting NADPH oxidase or knockdown of NOX2 abolished the effects of salusin-β on ACAT-1 and VCAM-1 expressions, p65-NFκB nuclear translocation, lipid accumulation and monocyte adhesion in VSMCs. Salusin-β increased miR155 expression, and knockdown of miR155 prevented the effects of salusin-β on ACAT-1 and VCAM-1 expressions, p65-NFκB nuclear translocation, lipid accumulation, monocyte adhesion and ROS production in VSMCs. These results indicate that salusin-β induces foam formation and monocyte adhesion via miR155/NOX2/NFκB-mediated ACAT-1 and VCAM-1 expressions in VSMCs.

  9. Effect of natural porcine surfactant in Staphylococcus aureus induced pro-inflammatory cytokines and reactive oxygen species generation in monocytes and neutrophils from human blood.

    PubMed

    de Guevara, Yuliannis Lugones Ladrón; Hidalgo, Odalys Blanco; Santos, Sidnéia Sousa; Brunialti, Milena Karina Colo; Faure, Roberto; Salomao, Reinaldo

    2014-08-01

    Surfacen® is a clinical surfactant preparation of porcine origin. In the present study, we have evaluated the effect of Surfacen® in the modulation of oxidative burst in monocytes and neutrophils in human blood and pro-inflammatory cytokine production in peripheral blood mononuclear cells (PBMC). Reactive oxygen species (ROS) level was measured in monocytes and neutrophils by flow cytometry using 2,7-dichlorofluorescein diacetate (DCFH-DA) as substrate, while, tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were estimated in PBMC supernatant by enzyme-linked immunosorbent assays (ELISA). Our results show that Staphylococcus aureus-induced ROS level was slightly affected by Surfacen® added to whole blood monocytes and neutrophils. The time course experiments of pre-incubation with Surfacen® showed no significant increase of ROS level at 2h; however, the ROS level decreased when pre incubated for 4h and 6h with Surfacen®. Pre-incubation of PBMC cells with Surfacen® at 0.125 and 0.5mg/mL showed a dose-dependent suppression of TNF-α levels measured after 4h of S. aureus stimulation, an effect less impressive when cells were stimulated for 24h. A similar behavior was observed in IL-6 release. In summary, the present study provides experimental evidence supporting an anti-inflammatory role of Surfacen® in human monocytes and neutrophils in vitro.

  10. TNF-alpha is secreted by monocytes in transit to become macrophages, but not by peripheral blood monocytes, following OK-432 (lyophilized S. pyogenes) stimulation.

    PubMed

    Olsnes, C; Stavang, H; Olofsson, J; Aarstad, H J

    2007-12-01

    OK-432, penicillin-killed Streptococcus pyogenes, is used in treating lymphangiomas and carcinomas. We have studied proinflammatory interleukin (IL) secretion following OK-432 stimulation of total blood, peripheral blood mononuclear cell (PBMC) and purified monocytes in vitro. OK-432 stimulation of purified monocytes gave IL-1beta, IL-1RA, IL-6, IL-12p40 and tumour necrosis factor (TNF)-alpha response. OK-432 stimulation of cells within blood did, however, not yield TNF-alpha secretion. When PBMC or monocytes were cultured in low-attachment wells a decreased IL secretion was observed compared to adherent cells. Inhibition of Syk kinase with piceatannol, only at high, non-specific doses, but not PI3 kinase inhibition with LY294002 or Wortmannin, decreased monocyte IL response to OK-432. This shows that beta(1-3)-integrin receptor function is not necessary for monocyte OK-432-stimulated TNF-alpha secretion. Direct blockage of the beta(2)-integrin (CD18) receptor by anti-CD18 antibody was also unable to prevent the stimulating effects of OK-432 in human monocytes. On the other hand, Syk phosphorylation is elevated upon adherence of monocytes and this is further increased by OK-432 stimulation, as shown by Western blot. The Fc-receptor was also ruled out as a main receptor of the OK-432 monocyte response. In conclusion, TNF-alpha secretion is only found in monocytes removed from blood. This TNF-alpha secretion is not mediated through the beta(1-3)-integrin receptors. OK-432 may act as a target-seeking substance whereby only monocytes adhered, e.g. to a tumour cell, become cytotoxic in part explaining why OK-432 is well suited as a cancer treatment drug.

  11. Cigarette smoke activates human monocytes by an oxidant-AP-1 signaling pathway: implications for steroid resistance.

    PubMed

    Walters, Matthew J; Paul-Clark, Mark J; McMaster, Shaun K; Ito, Kazuhiro; Adcock, Ian M; Mitchell, Jane A

    2005-11-01

    Smoking cigarettes is a major risk factor for the development of cardiovascular and respiratory disease. Moreover, smoking-induced pathophysiology is often resistant to the anti-inflammatory effects of glucocorticoids. The nature of cigarette smoke-induced inflammation is still not defined, although neutrophil recruitment and activation seem to be consistent features. In the current study, we have used a range of approaches to demonstrate that cigarette smoke activates human monocytes and macrophages to release the CXC chemokine CXCL8 [(interleukin-8 (IL-8)]. Furthermore, we show for the first time that cigarette smoke synergizes with proinflammatory cytokines IL-1beta and tumor necrosis factor-alpha, and it is this interaction that confers steroid resistance to smoke-induced CXCL8 release. We go on to show that smoke-induced activation of human cells is an oxidant-mediated phenomenon acting through activator protein-1, but not nuclear factor kappaB, pathway. These observations add significantly to our understanding of smoke as an inflammatory stimulus that has implications for potential the development of treatments of smoking or related disease.

  12. Quantitative proteomics of extracellular vesicles released from human monocyte-derived macrophages upon β-glucan stimulation.

    PubMed

    Cypryk, Wojciech; Ohman, Tiina; Eskelinen, Eeva-Liisa; Matikainen, Sampsa; Nyman, Tuula A

    2014-05-02

    Fungal infections (mycoses) are common diseases of varying severity that cause problems, especially to immunologically compromised people. Fungi express a variety of pathogen-associated molecular patterns on their surface including β-glucans, which are important immunostimulatory components of fungal cell walls. During stimulatory conditions of infection and colonization, besides intensive intracellular response, human cells actively communicate on the intercellular level by secreting proteins and other biomolecules with several mechanisms. Vesicular secretion remains one of the most important paths for the proteins to exit the cell. Here, we have used high-throughput quantitative proteomics combined with bioinformatics to characterize and quantify vesicle-mediated protein release from β-glucan-stimulated human macrophages differentiated in vitro from primary blood monocytes. We show that β-glucan stimulation induces vesicle-mediated protein secretion. Proteomic study identified 540 distinct proteins from the vesicles, and the identified proteins show a proteomic signature characteristic for their cellular origin. Importantly, we identified several receptors, including cation-dependent mannose-6-phosphate receptor, macrophage scavenger receptor, and P2X7 receptor, that have not been identified from vesicles before. Proteomic data together with detailed pathway and network analysis showed that integrins and their cytoplasmic cargo proteins are highly abundant in extracellular vesicles released upon β-glucan stimulation. In conclusion, the present data provides a solid basis for further studies on the functional role of vesicular protein secretion upon fungal infection.

  13. Differential Induction of Cytokines by Human Neonatal, Adult, and Elderly Monocyte/Macrophages Infected with Dengue Virus

    PubMed Central

    Valero, Nereida; Levy, Alegria; Añez, Germán; Marcucci, Rafael; Alvarez-Mon, Melchor

    2014-01-01

    Abstract Immunosuppressive status against infections in monocytes from neonates and elderly subjects has been reported. The interaction between dengue virus and monocytes/macrophages plays an important role during dengue disease. The aim of this study was to determine the cytokine response of monocytes from individuals with different ages after infection with dengue virus. Monocyte/macrophage cultures from neonatal, adult, and elderly subjects (n=10 each group) were incubated with all four dengue virus types (DENV-1 to -4). After 1 and 3 days of culture, cytokine concentrations (TNF-α, IL-6, and IL-1β) were determined in culture supernatants by enzyme-linked immunosorbant assay. Increased production of all studied cytokines was induced by the different viral types in monocyte/macrophage cultures regardless of their source. However, lower cytokine concentrations were found in neonatal and elderly monocytes. The relative monocyte/macrophage immunosuppressive status observed in neonates and the elderly could be relevant during dengue infection in those age groups and important in innate and adaptive immunity responses against this virus. PMID:24801946

  14. Adrenergic stimulation alters the expression of inflammasome components and interleukins in primary human monocytes

    PubMed Central

    HORSTMANN, JOHANN-PHILIPP; MARZI, INGO; RELJA, BORNA

    2016-01-01

    Prior to their release, interleukin (IL)-1β and IL-18 are cleaved to their bioactive forms by a multiprotein complex known as an inflammasome, which is comprised of a number of elements that are subject to nuclear factor-κB-dependent transcription. Catecholamines have been indicated to exert an enhancing effect on the IL-1β release. The aim of the present study was to determine whether alterations in inflammasome gene expression may be responsible for the modified IL-1β and IL-18 secretion following lipopolysaccharide (LPS) and catecholamine co-stimulation. Monocytes were isolated from the peripheral blood of 21 healthy volunteers using CD14+ microbeads. Following stimulation with LPS (2 µg/ml) and/or phenylephrine (PE; 10 µM) for 24 h, the supernatants were subjected to ELISA to evaluate the ex vivo protein expression levels of IL-1β and IL-18. In addition, the gene expression levels of inflammasome components associated with the cleavage of IL-1β and IL-18, including NLRP1, NLRP3, caspase-1 and PYCARD were determined using polymerase chain reaction. The results indicated that LPS significantly increased IL-1β expression compared with the unstimulated control samples. Co-stimulation with LPS + PE significantly enhanced IL-1β expression compared with LPS alone. Furthermore, IL-18 expression was significantly reduced by LPS and LPS + PE co-stimulation. The gene expression levels of IL-18, NLRP1, caspase-1 and PYCARD were comparable in the LPS- and LPS + PE-stimulated cells. LPS significantly induced the expression levels of IL-1β and NLRP3, and to a lesser degree, the expression of NLRP1, compared with the control. By contrast, PE markedly induced the expression levels of IL-18 and NLRP1, while LPS reduced the gene expression of IL-18. In conclusion, adrenergic stimulation suppressed NLRP3 expression and enhanced NLRP1 expression, indicating that NLRP3 may regulate IL-1β secretion and NLRP1 may regulate the release of IL-18. PMID:26889257

  15. CD11c/CD18 Dominates Adhesion of Human Monocytes, Macrophages and Dendritic Cells over CD11b/CD18

    PubMed Central

    Ungai-Salánki, Rita; Orgován, Norbert; Szabó, Bálint; Horváth, Róbert; Erdei, Anna; Bajtay, Zsuzsa

    2016-01-01

    Complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) belong to the family of beta2 integrins and are expressed mainly by myeloid cell types in humans. Previously, we proved that CR3 rather than CR4 plays a key role in phagocytosis. Here we analysed how CD11b and CD11c participate in cell adhesion to fibrinogen, a common ligand of CR3 and CR4, employing human monocytes, monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells (MDDCs) highly expressing CD11b as well as CD11c. We determined the exact numbers of CD11b and CD11c on these cell types by a bead-based technique, and found that the ratio of CD11b/CD11c is 1.2 for MDDCs, 1.7 for MDMs and 7.1 for monocytes, suggesting that the function of CD11c is preponderant in MDDCs and less pronounced in monocytes. Applying state-of-the-art biophysical techniques, we proved that cellular adherence to fibrinogen is dominated by CD11c. Furthermore, we found that blocking CD11b significantly enhances the attachment of MDDCs and MDMs to fibrinogen, demonstrating a competition between CD11b and CD11c for this ligand. On the basis of the cell surface receptor numbers and the measured adhesion strength we set up a model, which explains the different behavior of the three cell types. PMID:27658051

  16. CD11c/CD18 Dominates Adhesion of Human Monocytes, Macrophages and Dendritic Cells over CD11b/CD18.

    PubMed

    Sándor, Noémi; Lukácsi, Szilvia; Ungai-Salánki, Rita; Orgován, Norbert; Szabó, Bálint; Horváth, Róbert; Erdei, Anna; Bajtay, Zsuzsa

    Complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) belong to the family of beta2 integrins and are expressed mainly by myeloid cell types in humans. Previously, we proved that CR3 rather than CR4 plays a key role in phagocytosis. Here we analysed how CD11b and CD11c participate in cell adhesion to fibrinogen, a common ligand of CR3 and CR4, employing human monocytes, monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells (MDDCs) highly expressing CD11b as well as CD11c. We determined the exact numbers of CD11b and CD11c on these cell types by a bead-based technique, and found that the ratio of CD11b/CD11c is 1.2 for MDDCs, 1.7 for MDMs and 7.1 for monocytes, suggesting that the function of CD11c is preponderant in MDDCs and less pronounced in monocytes. Applying state-of-the-art biophysical techniques, we proved that cellular adherence to fibrinogen is dominated by CD11c. Furthermore, we found that blocking CD11b significantly enhances the attachment of MDDCs and MDMs to fibrinogen, demonstrating a competition between CD11b and CD11c for this ligand. On the basis of the cell surface receptor numbers and the measured adhesion strength we set up a model, which explains the different behavior of the three cell types.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2017-01-01

    GM-CSF and IL-3 are hematopoietic cytokines that also modulate the effector functions of several immune cell subsets. In particular, GM-CSF and IL-3 exert a significant control on monocyte and macrophage effector functions, as assessed in experimental models of inflammatory and autoimmune diseases and also in human studies. Here, we sought to investigate the mechanisms and the extent to which GM-CSF and IL-3 modulate the pro-inflammatory, LPS-mediated, activation of human CD14+ monocytes taking into account the new concept of trained immunity (i.e., the priming stimulus modulates the response to subsequent stimuli mainly by inducing chromatin remodeling and increased transcription at relevant genetic loci). We demonstrate that GM-CSF and IL-3 priming enhances TNF-α production upon subsequent LPS stimulation (short-term model of trained immunity) in a p38- and SIRT2-dependent manner without increasing TNF primary transcript levels (a more direct measure of transcription), thus supporting a posttranscriptional regulation of TNF-α in primed monocytes. GM-CSF and IL-3 priming followed by 6 days of resting also results in increased TNF-α production upon LPS stimulation (long-term model of trained immunity). In this case, however, GM-CSF and IL-3 priming induces a c-Myc-dependent monocyte renewal and increase in cell number that is in turn responsible for heightened TNF-α production. Overall, our results provide insights to understand the biology of monocytes in health and disease conditions in which the hematopoietic cytokines GM-CSF and IL-3 play a role and also extend our knowledge of the cellular and molecular mechanisms of trained immunity. PMID:28138327

  19. Subcellular proteomic analysis of host-pathogen interactions using human monocytes exposed to Yersinia pestis and Yersinia pseudotuberculosis

    SciTech Connect

    Zhang, C G; Gonzales, A D; Choi, M W; Chromy, B A; Fitch, J P; McCutchen-Maloney, S L

    2004-05-20

    Yersinia pestis, the etiological agent of plague, is of concern to human health both from an infectious disease and a civilian biodefense perspective. While Y. pestis and Y. pseudotuberculosis share more than 90% DNA homology, they have significantly different clinical manifestations. Plague is often fatal if untreated, yet Y. pseudotuberculosis causes severe intestinal distress and is rarely fatal. A better understanding of host response to these closely related pathogens may help explain the different mechanisms of virulence and pathogenesis that result in such different clinical outcomes. The aim of this study was to characterize host protein expression changes in human monocyte-like U937 cells after exposure to Y. pestis and Y. pseudotuberculosis. In order to gain global proteomic coverage of host response, proteins from cytoplasmic, nuclear and membrane fractions of host cells were studied by 2-dimensional differential gel electrophoresis (2-D DIGE) and relative protein expression differences were quantitated. Differentially expressed proteins, with at least 1.5 fold expression changes and p values of 0.01 or less, were identified by MALDI-MS or LC/MS/MS. With these criteria, differential expression was detected in 16 human proteins after Y. pestis exposure and 13 human proteins after Y. pseudotuberculosis exposure, of which only two of the differentially expressed proteins identified were shared between the two exposures. Proteins identified in this study are reported to be involved in a wide spectrum of cellular functions and host defense mechanisms including apoptosis, cytoskeletal rearrangement, protein synthesis and degradation, DNA replication and transcription, metabolism, protein folding, and cell signaling. Notably, the differential expression patterns observed can distinguish the two pathogen exposures from each other and from unexposed host cells. The functions of the differentially expressed proteins identified provide insight on the different

  20. Selective p38α MAP kinase/MAPK14 inhibition in enzymatically modified LDL-stimulated human monocytes: implications for atherosclerosis.

    PubMed

    Cheng, Fei; Twardowski, Laura; Fehr, Sarah; Aner, Christoph; Schaeffeler, Elke; Joos, Thomas; Knorpp, Thomas; Dorweiler, Bernhard; Laufer, Stefan; Schwab, Matthias; Torzewski, Michael

    2017-02-01

    The first ATP-competitive p38α MAPK/MAPK14 inhibitor with excellent in vivo efficacy and selectivity, skepinone-L, is now available. We investigated the impact of selective p38α MAPK/MAPK14 inhibition on enzymatically modified LDL (eLDL) stimulated human monocytes with its implications for atherosclerosis. Among the different p38 MAPK isoforms, p38α/MAPK14 was the predominantly expressed and activated isoform in isolated human peripheral blood monocytes. Moreover, eLDL colocalized with macrophages positive for p38α MAPK/MAPK14 in human carotid endarterectomy specimens. Using the human leukemia cell line THP-1 and/or primary monocyte-derived macrophages, skepinone-L inhibited eLDL-induced activation of the p38 MAPK pathway, inhibited eLDL induced expression of both cluster of differentiation 36 (CD36) and ATP-binding cassette, subfamily A, member 1 (ABCA1), without a net effect on foam cell formation, had a cell- and time-dependent effect on eLDL-triggered apoptosis, and inhibited eLDL-stimulated secretion of IL-8 and MIP-1β/CCL4 (macrophage inflammatory protein-1β/chemokine, CC motif, ligand 4). Inhibition of a key signaling molecule of the p38 MAPK pathway, p38α MAPK/MAPK14, by selective inhibitors like skepinone-L, conclusively facilitates elucidation of the impact of the complex network of p38 MAPK signaling on atherogenesis and might provide a promising therapeutic tool to prevent inflammatory cascades in atherosclerosis.-Cheng, F., Twardowski, L., Fehr, S., Aner, C., Schaeffeler, E., Joos, T., Knorpp, T., Dorweiler, B., Laufer, S., Schwab, M., Torzewski, M. Selective p38α MAP kinase/MAPK14 inhibition in enzymatically modified LDL-stimulated human monocytes: implications for atherosclerosis.

  1. Evidence that cyclic AMP phosphodiesterase inhibitors suppress TNF alpha generation from human monocytes by interacting with a 'low-affinity' phosphodiesterase 4 conformer.

    PubMed Central

    Souness, J. E.; Griffin, M.; Maslen, C.; Ebsworth, K.; Scott, L. C.; Pollock, K.; Palfreyman, M. N.; Karlsson, J. A.

    1996-01-01

    1. We have investigated the inhibitory effects of RP 73401 (piclamilast) and rolipram against human monocyte cyclic AMP-specific phosphodiesterase (PDE4) in relation to their effects on prostaglandin (PG)E2-induced cyclic AMP accumulation and lipopolysaccharide (LPS)-induced TNF alpha production and TNF alpha mRNA expression. 2. PDE4 was found to be the predominant PDE isoenzyme in the cytosolic fraction of human monocytes. Cyclic GMP-inhibited PDE (PDE3) was also detected in the cytosolic and particulate fractions. Reverse transcription polymerase chain reaction (RT-PCR) of human monocyte poly (A+) mRNA revealed amplified products corresponding to PDE4 subtypes A and B of which the former was most highly expressed. A faint band corresponding in size to PDE4D was also observed. 3. RP 73401 was a potent inhibitor of cytosolic PDE4 (IC50: 1.5 +/- 0.6 nM, n = 3). (+/-)-Rolipram (IC50: 313 +/- 6.7 nM, n = 3) was at least 200 fold less potent than RP 73401. R-(-)-rolipram was approximately 3 fold more potent than S-(+)-rolipram against cytosolic PDE4. 4. RP 73401 (IC50: 9.2 +/- 2.1 nM, n = 6) was over 50 fold more potent than (+/-)-rolipram (IC50: 503 +/- 134 nM, n = 6) ) in potentiating PGE2-induced cyclic AMP accumulation. R-(-)-rolipram (IC50: 289 +/- 121 nM, n = 5) was 4.7 fold more potent than its S-(+)-enantiomer (IC50: 1356 +/- 314 nM, n = 5). A strong and highly-significant, linear correlation (r = 0.95, P < 0.01, n = 13) was observed between the inhibitory potencies of a range of structurally distinct PDE4 inhibitors against monocyte PDE4 and their ED50 values in enhancing monocyte cyclic AMP accumulation. A poorer, though still significant, linear correlation (r = 0.67, P < 0.01, n = 13) was observed between the potencies of the same compounds in potentiating PGE2-induced monocyte cyclic AMP accumulation and their abilities to displace [3H]-rolipram binding to brain membranes. 5. RP 73401 (IC50: 6.9 +/- 3.3 nM, n = 5) was 71 fold more potent than

  2. Xanthohumol from hop (Humulus lupulus L.) is an efficient inhibitor of monocyte chemoattractant protein-1 and tumor necrosis factor-alpha release in LPS-stimulated RAW 264.7 mouse macrophages and U937 human monocytes.

    PubMed

    Lupinacci, Elvira; Meijerink, Jocelijn; Vincken, Jean-Paul; Gabriele, Bartolo; Gruppen, Harry; Witkamp, Renger F

    2009-08-26

    Activated macrophages in adipose tissue play a major role in the chronic inflammatory process that has been linked to the complications of overweight and obesity. The hop plant (Humulus lupulus L.) has been described to possess both anti-inflammatory and antidiabetic effects. In the present study, the chemical composition of a hop crude extract (HCE) was investigated by ultrahigh-performance liquid chromatography (UHPLC). Next, HCE and various fractions obtained by preparative HPLC were tested for their ability to inhibit production of two pro-inflammatory cytokines, monocyte chemoattractant protein-1 (MCP-1, CCL2) and tumor necrosis factor-alpha (TNF-alpha), which play crucial roles in the complications of obesity. The hop chalcone xanthohumol was found to be the most potent inhibitor of both cytokines in LPS-activated RAW 264.7 mouse macrophages and U937 human monocytes. Moreover, other constituents, namely, iso-alpha-acids, in combination with the beta-acid hulupone, showed a moderate but selective inhibitory activity only on MCP-1 release. These findings underscore the potential health effects of hop and support further optimization, selection, and use of this plant.

  3. Monocyte-conditioned medium, interleukin-1, and tumor necrosis factor stimulate the acute phase response in human hepatoma cells in vitro

    PubMed Central

    1986-01-01

    Human hepatoma cells mimic the acute phase response after treatment with monocyte-conditioned medium. Levels of secreted fibrinogen, alpha- 1 acid glycoprotein, C-reactive protein, haptoglobin, and the third component of complement were elevated compared with control levels after 48 h of incubation with conditioned supernatant medium from an enriched fraction of normal peripheral monocytes. Albumin levels declined and alpha-1 antitrypsin remained unchanged. Levels of specific mRNA were measured by hybridization to slot blots and Northern blots and changed in correspondence with protein alterations. Interleukin-1 and tumor necrosis factor stimulated the third component of complement, but did not elevate any other member of the acute phase group and were therefore only partially active in this system. The identification of an in vitro model of the human acute phase response will permit analysis of the molecular basis for coordinate regulation of this group of facultative genes. PMID:3017995

  4. Bromelain inhibits lipopolysaccharide-induced cytokine production in human THP-1 monocytes via the removal of CD14.

    PubMed

    Huang, Jing-Rong; Wu, Chia-Chuan; Hou, Rolis Chien-Wei; Jeng, Kee-Ching

    2008-01-01

    Bromelain has been reported to have anti-inflammatory and immunomodulatory effects. However, the anti-inflammatory mechanism of bromelain is unclear. Therefore, we investigated the effect of bromelain on cytokine production from lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC) and monocytic leukemia THP-1 cells. The result showed that bromelain (50-100 microg/ml) significantly and reversibly reduced tumor necrosis factor (TNF)-alpha interleukin- (IL)-1beta and IL-6 from LPS-induced PBMC and THP-1 cells. This effect was correlated with reduced LPS-induced TNF-alpha mRNA and NF-kappaB activity in THP-1 cells. In addition, bromelain dose-dependently inhibited LPS-induced prostaglandin E(2), thromboxane B(2) and COX-2 mRNA but not COX-1 mRNA. Importantly, bromelain degraded TNF-alpha and IL-1beta molecules, reduced the expression of surface marker CD14 but not Toll-like receptor 4 from THP-1 cells. Taken together, the results suggest that the suppression of signaling pathways by bromelain's proteolytic activity may contribute to the anti-inflammatory activity of bromelain.

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

    PubMed Central

    Chauhan, Vinita; Howland, Matthew

    2012-01-01

    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. PMID:23097634

  6. Human Monocyte Heat Shock Protein 72 Responses to Acute Hypoxic Exercise after 3 Days of Exercise Heat Acclimation

    PubMed Central

    Lee, Ben J.; Mackenzie, Richard W. A.; Cox, Valerie; James, Rob S.; Thake, Charles D.

    2015-01-01

    The aim of this study was to determine whether short-term heat acclimation (STHA) could confer increased cellular tolerance to acute hypoxic exercise in humans as determined via monocyte HSP72 (mHSP72) expression. Sixteen males were separated into two matched groups. The STHA group completed 3 days of exercise heat acclimation; 60 minutes cycling at 50% V˙O2peak in 40°C 20% relative humidity (RH). The control group (CON) completed 3 days of exercise training in 20°C, 40% RH. Each group completed a hypoxic stress test (HST) one week before and 48 hours following the final day of CON or STHA. Percentage changes in HSP72 concentrations were similar between STHA and CON following HST1 (P = 0.97). STHA induced an increase in basal HSP72 (P = 0.03) with no change observed in CON (P = 0.218). Basal mHSP72 remained elevated before HST2 for the STHA group (P < 0.05) and was unchanged from HST1 in CON (P > 0.05). Percent change in mHSP72 was lower after HST2 in STHA compared to CON (P = 0.02). The mHSP72 response to hypoxic exercise was attenuated following 3 days of heat acclimation. This is indicative of improved tolerance and ability to cope with the hypoxic insult, potentially mediated in part by increased basal reserves of HSP72. PMID:25874231

  7. Antiphospholipid antibodies induce translocation of TLR7 and TLR8 to the endosome in human monocytes and plasmacytoid dendritic cells.

    PubMed

    Prinz, Nadine; Clemens, Natascha; Strand, Dennis; Pütz, Inge; Lorenz, Mareike; Daiber, Andreas; Stein, Pamela; Degreif, Adriana; Radsak, Markus; Schild, Hansjörg; Bauer, Stefan; von Landenberg, Philipp; Lackner, Karl J

    2011-08-25

    The antiphospholipid syndrome (APS) is an autoimmune disease characterized by thromboembolic events and/or fetal loss in the presence of antiphospholipid antibodies (aPLs). The mechanisms underlying the pathogenicity of aPLs are still poorly understood. Here we show that 3 human monoclonal aPLs as well as IgG fractions from patients with the APS increase mRNA expression of the intracellular toll-like receptor (TLR) 7 in plasmacytoid dendritic cells and TLR8 in monocytes. Simultaneously they induce the translocation of TLR7 or TLR8 from the endoplasmic reticulum to the endosome. These effects depend on the uptake of aPLs into the endosome, subsequent activation of endosomal NADPH oxidase, and generation of superoxide. As a consequence cells are dramatically sensitized to ligands for TLR7 and TLR8. This observation delineates a novel signal transduction pathway in innate immunity originating from the endosome. Because the overexpression of TLR7 can also be detected in plasmacytoid dendritic cells from patients with the APS ex vivo, our results provide an explanation for proinflammatory and procoagulant effects of aPLs. Because inappropriate expression of TLR7 has been implicated in the development of systemic autoimmunity, these findings may also be relevant for the understanding of autoimmunity.

  8. Effect of size of man-made and natural mineral fibers on chemiluminescent response in human monocyte-derived macrophages.

    PubMed

    Ohyama, M; Otake, T; Morinaga, K

    2001-10-01

    Fiber size is an important factor in the tumorigenicity of various mineral fibers and asbestos fibers in animal experiments. We examined the time course of the ability to induce lucigenin-dependent chemiluminescence (CL) from human monocyte-derived macrophages exposed to Japan Fibrous Material standard reference samples (glass wool, rock wool, micro glass fiber, two types of refractory ceramic fiber, refractory mullite fiber, potassium titanium whisker, silicon carbide whisker, titanium oxide whisker, and wollastonite). We determined how fiber length or width might modify the response of cells. We found that the patterns of time-dependent increase of CL (sigmoid type) were similar for each sample except wollastonite. We observed a strong correlation between geometric-mean length and ability to induce CL in seven samples > 6 microm in length over the time course (largest r(2) = 0.9760). Although we also observed a close positive correlation between geometric-mean width and the ability to induce CL in eight samples < 1.8 microm in width at 15 min (r(2) = 0.8760), a sample of 2.4 microm in width had a low ability to induce CL. Moreover, the relationship between width and the rate of increase in ability to induce CL had a negative correlation at 30-60 min (largest r(2) = 0.7473). Our findings suggest that the release of superoxide from macrophages occurs nonspecifically for various types of mineral fibers depending on fiber length.

  9. Monocytic differentiation and synthesis of proteins associated with apoptosis in human leukemia U-937 cells acquiring resistance to vincristine.

    PubMed

    Pantazis, P; Chatterjee, D; Han, Z; Wyche, J; DeJesus, A; Giovanella, B

    1996-07-01

    Human leukemia U-937/WT cells were exposed to stepwise increased concentrations of Vincristine so that Vincristine-resistant cell sublines (termed U-937/RV) were developed. Established U-937/RV cell sublines have continuously propagated over a year, both in absence and presence of VCR, and have demonstrated similar features. In contrast to U-937/WT cells, U-937/RV cells have longer doubling time, and are more differentiated as determined by appearance of distinct morphological features and synthesis of mRNA that codes for the monocyte colony-stimulating factor-1 receptor (c-fms). Both apoptosis-suppressing Bcl-2 and Bcl-XL proteins were undectable in U-937/WT cells, whereas Bcl-2 was nearly detectable and Bcl-XL readily detectable in U-937/RV cells. The apoptosis-promoting Bax protein was also absent in U-937/WT cells and readily detected in U-937/RV cells. Vincristine-resistant cells with different levels of resistance synthesize similar levels of c-fms mRNA and Bax protein. Finally, unlike U-937/WT cells, U-937/RV cells have no ability to induce tumors when xenografted in immunodeficient mice. The findings collectively suggest that development of resistance to Vincristine in U-937/WT cells may correlate with cell differentiation and synthesis of proteins that regulate apoptosis.

  10. Human monocyte heat shock protein 72 responses to acute hypoxic exercise after 3 days of exercise heat acclimation.

    PubMed

    Lee, Ben J; Mackenzie, Richard W A; Cox, Valerie; James, Rob S; Thake, Charles D

    2015-01-01

    The aim of this study was to determine whether short-term heat acclimation (STHA) could confer increased cellular tolerance to acute hypoxic exercise in humans as determined via monocyte HSP72 (mHSP72) expression. Sixteen males were separated into two matched groups. The STHA group completed 3 days of exercise heat acclimation; 60 minutes cycling at 50% V̇O2peak in 40°C 20% relative humidity (RH). The control group (CON) completed 3 days of exercise training in 20°C, 40% RH. Each group completed a hypoxic stress test (HST) one week before and 48 hours following the final day of CON or STHA. Percentage changes in HSP72 concentrations were similar between STHA and CON following HST1 (P = 0.97). STHA induced an increase in basal HSP72 (P = 0.03) with no change observed in CON (P = 0.218). Basal mHSP72 remained elevated before HST2 for the STHA group (P < 0.05) and was unchanged from HST1 in CON (P > 0.05). Percent change in mHSP72 was lower after HST2 in STHA compared to CON (P = 0.02). The mHSP72 response to hypoxic exercise was attenuated following 3 days of heat acclimation. This is indicative of improved tolerance and ability to cope with the hypoxic insult, potentially mediated in part by increased basal reserves of HSP72.

  11. The Effect of Regular Intake of Dry-Cured Ham Rich in Bioactive Peptides on Inflammation, Platelet and Monocyte Activation Markers in Humans.

    PubMed

    Martínez-Sánchez, Sara María; Minguela, Alfredo; Prieto-Merino, David; Zafrilla-Rentero, María Pilar; Abellán-Alemán, José; Montoro-García, Silvia

    2017-03-23

    Background and aims: Dietary studies have shown that active biopeptides provide protective health benefits, although the mediating pathways are somewhat uncertain. To throw light on this situation, we studied the effects of consuming Spanish dry-cured ham on platelet function, monocyte activation markers and the inflammatory status of healthy humans with pre-hypertension. Methods: Thirty-eight healthy volunteers with systolic blood pressure of >125 mmHg were enrolled in a two-arm crossover randomized controlled trial. Participants received 80 g/day dry-cured pork ham of >11 months proteolysis or 100 g/day cooked ham (control product) for 4 weeks followed by a 2-week washout before "crossing over" to the other treatment for 4 more weeks. Soluble markers and cytokines were analyzed by ELISA. Platelet function was assessed by measuring P-selectin expression and PAC-1 binding after ADP (adenosine diphosphate) stimulation using whole blood flow cytometry. Monocyte markers of the pathological status (adhesion, inflammatory and scavenging receptors) were also measured by flow cytometry in the three monocyte subsets after the interventional period. Results: The mean differences between dry-cured ham and cooked ham followed by a time period adjustment for plasmatic P-selectin and interleukin 6 proteins slightly failed (p = 0.062 and p = 0.049, respectively), notably increased for MCP-1 levels (p = 0.023) while VCAM-1 was not affected. Platelet function also decreased after ADP stimulation. The expression of adhesion and scavenging markers (ICAM1R, CXCR4 and TLR4) in the three subsets of monocytes was significantly higher (all p < 0.05). Conclusions: The regular consumption of biopeptides contained in the dry-cured ham but absent in cooked ham impaired platelet and monocyte activation and the levels of plasmatic P-selectin, MCP-1 and interleukin 6 in healthy subjects. This study strongly suggests the existence of a mechanism that links dietary biopeptides and beneficial

  12. Endogenous Epoxygenases Are Modulators of Monocyte/Macrophage Activity

    PubMed Central

    Sugden, Mary C.; Holness, Mark J.; Swales, Karen E.; Warner, Timothy D.; Edin, Matthew L.; Zeldin, Darryl C.; Gilroy, Derek W.; Bishop-Bailey, David

    2011-01-01

    Background Arachidonic acid is metabolized through three major metabolic pathways, the cyclooxygenase, lipoxygenase and CYP450 enzyme systems. Unlike cyclooxygenase and lipoxygenases, the role of CYP450 epoxygenases in monocyte/macrophage-mediated responses is not known. Methodology/Principal Findings When transfected in vitro, CYP2J2 is an efficient activator of anti-inflammatory pathways through the nuclear receptor peroxisome proliferator-activated receptor (PPAR) α. Human monocytes and macrophages contain PPARα and here we show they express the epoxygenases CYP2J2 and CYP2C8. Inhibition of constitutive monocyte epoxygenases using the epoxygenase inhibitor SKF525A induces cyclooxygenase (COX)-2 expression and activity, and the release of TNFα, and can be reversed by either add back of the endogenous epoxygenase products and PPARα ligand 11,12- epoxyeicosatrienoic acid (EET) or the addition of the selective synthetic PPARα ligand GW7647. In alternatively activated (IL-4-treated) monocytes, in contrast to classically activated cells, epoxygenase inhibition decreased TNFα release. Epoxygenases can be pro-inflammatory via superoxide anion production. The suppression of TNFα by SKF525A in the presence of IL-4 was associated with a reduction in superoxide anion generation and reproduced by the superoxide dismutase MnCl2. Similar to these acute activation studies, in monocyte derived macrophages, epoxygenase inhibition elevates M1 macrophage TNFα mRNA and further decreases M2 macrophage TNFα. Conclusions/Significance In conclusion, epoxygenase activity represents an important endogenous pathway which limits monocyte activation. Moreover endogenous epoxygenases are immuno-modulators regulating monocyte/macrophage activation depending on the underlying activation state. PMID:22028915

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

    PubMed

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

    2017-02-01

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

  14. Acanthamoeba castellanii Genotype T4 Stimulates the Production of Interleukin-10 as Well as Proinflammatory Cytokines in THP-1 Cells, Human Peripheral Blood Mononuclear Cells, and Human Monocyte-Derived Macrophages.

    PubMed

    Mattana, Antonella; Sanna, Manuela; Cano, Antonella; Delogu, Giuseppe; Erre, Giuseppe; Roberts, Craig W; Henriquez, Fiona L; Fiori, Pier Luigi; Cappuccinelli, Piero

    2016-10-01

    Free-living amoebae of the genus Acanthamoeba can cause severe and chronic infections in humans, mainly localized in immune privileged sites, such as the brain and the eye. Monocytes/macrophages are thought to be involved in Acanthamoeba infections, but little is known about how these facultative parasites influence their functions. The aim of this work was to investigate the effects of Acanthamoeba on human monocytes/macrophages during the early phase of infection. Here, THP-1 cells, primary human monocytes isolated from peripheral blood, and human monocyte-derived macrophages were either coincubated with trophozoites of a clinical isolate of Acanthamoeba (genotype T4) or stimulated with amoeba-derived cell-free conditioned medium. Production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-12), anti-inflammatory cytokine (IL-10), and chemokine (IL-8) was evaluated at specific hours poststimulation (ranging from 1.5 h to 23 h). We showed that both Acanthamoeba trophozoites and soluble amoebic products induce an early anti-inflammatory monocyte-macrophage phenotype, characterized by significant production of IL-10; furthermore, challenge with either trophozoites or their soluble metabolites stimulate both proinflammatory cytokines and chemokine production, suggesting that this protozoan infection results from the early induction of coexisting, opposed immune responses. Results reported in this paper confirm that the production of proinflammatory cytokines and chemokines by monocytes and macrophages can play a role in the development of the inflammatory response during Acanthamoeba infections. Furthermore, we demonstrate for the first time that Acanthamoeba stimulates IL-10 production in human innate immune cells, which might both promote the immune evasion of Acanthamoeba and limit the induced inflammatory response.

  15. Acanthamoeba castellanii Genotype T4 Stimulates the Production of Interleukin-10 as Well as Proinflammatory Cytokines in THP-1 Cells, Human Peripheral Blood Mononuclear Cells, and Human Monocyte-Derived Macrophages

    PubMed Central

    Sanna, Manuela; Cano, Antonella; Delogu, Giuseppe; Erre, Giuseppe; Roberts, Craig W.; Henriquez, Fiona L.; Fiori, Pier Luigi; Cappuccinelli, Piero

    2016-01-01

    Free-living amoebae of the genus Acanthamoeba can cause severe and chronic infections in humans, mainly localized in immune privileged sites, such as the brain and the eye. Monocytes/macrophages are thought to be involved in Acanthamoeba infections, but little is known about how these facultative parasites influence their functions. The aim of this work was to investigate the effects of Acanthamoeba on human monocytes/macrophages during the early phase of infection. Here, THP-1 cells, primary human monocytes isolated from peripheral blood, and human monocyte-derived macrophages were either coincubated with trophozoites of a clinical isolate of Acanthamoeba (genotype T4) or stimulated with amoeba-derived cell-free conditioned medium. Production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-12), anti-inflammatory cytokine (IL-10), and chemokine (IL-8) was evaluated at specific hours poststimulation (ranging from 1.5 h to 23 h). We showed that both Acanthamoeba trophozoites and soluble amoebic products induce an early anti-inflammatory monocyte-macrophage phenotype, characterized by significant production of IL-10; furthermore, challenge with either trophozoites or their soluble metabolites stimulate both proinflammatory cytokines and chemokine production, suggesting that this protozoan infection results from the early induction of coexisting, opposed immune responses. Results reported in this paper confirm that the production of proinflammatory cytokines and chemokines by monocytes and macrophages can play a role in the development of the inflammatory response during Acanthamoeba infections. Furthermore, we demonstrate for the first time that Acanthamoeba stimulates IL-10 production in human innate immune cells, which might both promote the immune evasion of Acanthamoeba and limit the induced inflammatory response. PMID:27481240

  16. Regulation of LPS-induced tissue factor expression in human monocytic THP-1 cells by curcumin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tissue factor (TF) is a transmembrane receptor, which initiates thrombotic episodes associated with various diseases. In addition to membrane-bound TF, we have discovered an alternatively spliced form of human TF mRNA. It was later confirmed that this form of TF mRNA expresses a soluble protein circ...

  17. Prevention and reversal of tumor cell-induced monocyte deactivation by cytokines, purified protein derivative (PPD), and anti-IL-10 antibody.

    PubMed

    Baj-Krzyworzeka, Monika; Baran, Jaroslaw; Szatanek, Rafal; Stankiewicz, Danuta; Siedlar, Maciej; Zembala, Marek

    2004-08-25

    Upon contact with tumor cells when cocultured in vitro, human monocytes become unresponsive (deactivated) to restimulation and demonstrate decreased production of TNF-alpha and IL-12, and enhanced IL-10 secretion. The present study was undertaken to determine whether immunomodulatory agents (proinflammatory cytokines and PPD of tuberculin) could either prevent or reverse the deactivation of monocytes. Monocytes were treated with the agents either before or after being cocultured with tumor cells. Pretreatment of monocytes with IFN-gamma, either alone or in combination with TNF-alpha, GM-CSF, or PPD, significantly enhanced TNF-alpha and IL-12 production by deactivated monocytes. TNF-alpha, GM-CSF, and PPD alone were inactive. Treatment of monocytes following coculture with IFN-gamma, TNF-alpha, GM-CSF, PPD or IFN-gamma in combination with these agents reversed the depressed TNF-alpha release, whereas IL-12 production was enhanced by IFN-gamma alone. All the agents had no or only a limited effect on the enhanced IL-10 secretion by deactivated monocytes. However, treatment of cocultured monocytes with anti-IL-10 mAb significantly increased the production of TNF-alpha and IL-12 by deactivated monocytes. Moreover, coengraftment of deactivated monocytes with human pancreatic carcinoma cells into SCID mice caused an enhancement of the tumor growth that was alleviated by the treatment of monocytes in vitro with IFN-gamma alone or in combination with GM-CSF or PPD. These results suggest that activation of monocytes with certain proinflammatory cytokines and/or selective inhibition of IL-10 by a mAb may prevent or reverse monocyte deactivation caused by tumor cells.

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

    PubMed Central

    Mohebbi, Sara; Erfurth, Florian; Hennersdorf, Philipp; Brakhage, Axel A.; Saluz, Hans Peter

    2016-01-01

    Hyperspectral imaging (HSI) is a technique based on the combination of classical spectroscopy and conventional digital image processing. It is also well suited for the biological assays and quantitative real-time analysis since it provides spectral and spatial data of samples. The method grants detailed information about a sample by recording the entire spectrum in each pixel of the whole image. We applied HSI to quantify the constituent pH variation in a single infected apoptotic monocyte as a model system. Previously, we showed that the human-pathogenic fungus Aspergillus fumigatus conidia interfere with the acidification of phagolysosomes. Here, we extended this finding to monocytes and gained a more detailed analysis of this process. Our data indicate that melanised A. fumigatus conidia have the ability to interfere with apoptosis in human monocytes as they enable the apoptotic cell to recover from mitochondrial acidification and to continue with the cell cycle. We also showed that this ability of A. fumigatus is dependent on the presence of melanin, since a non-pigmented mutant did not stop the progression of apoptosis and consequently, the cell did not recover from the acidic pH. By conducting the current research based on the HSI, we could measure the intracellular pH in an apoptotic infected human monocyte and show the pattern of pH variation during 35 h of measurements. As a conclusion, we showed the importance of melanin for determining the fate of intracellular pH in a single apoptotic cell. PMID:27727286

  19. Peroxisome proliferator-activated receptor gamma activators affect the maturation of human monocyte-derived dendritic cells.

    PubMed

    Gosset, P; Charbonnier, A S; Delerive, P; Fontaine, J; Staels, B; Pestel, J; Tonnel, A B; Trottein, F

    2001-10-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma ), a member of the nuclear receptor superfamily, has recently been described as a modulator of macrophage functions and as an inhibitor of T cell proliferation. Here, we investigated the role of PPARgamma in dendritic cells (DC), the most potent antigen-presenting cells. We showed that PPARgamma is highly expressed in immature human monocyte-derived DC (MDDC) and that it may affect the immunostimulatory function of MDDC stimulated with lipopolysaccharide (LPS) or via CD40 ligand (CD40L). We found that the synthetic PPARgamma agonist rosiglitazone (as well as pioglitazone and troglitazone) significantly increases on LPS- and CD40L-activated MDDC, the surface expression of CD36 (by 184% and 104%, respectively) and CD86 (by 54% and 48%), whereas it reduces the synthesis of CD80 (by 42% and 42%). Moreover, activation of PPARgamma resulted in a dramatic decreased secretion of the Th1-promoting factor IL-12 in LPS- and CD40L-stimulated cells (by 47% and 62%), while the production of IL-1beta, TNF-alpha, IL-6 and IL-10 was unaffected. Finally, PPARgamma ligands down-modulate the synthesis of IFN-gamma -inducible protein-10 (recently termed as CXCL10) and RANTES (CCL5), both chemokines involved in the recruitment of Th1 lymphocytes (by 49% and 30%), but not the levels of the Th2 cell-attracting chemokines,macrophage-derived chemokine (CCL22) and thymus and activation regulated chemokine (CCL17), in mature MDDC. Taken together, our data suggest that activation of PPARgamma in human DC may have an impact in the orientation of primary and secondary immune responses by favoring type 2 responses.

  20. Differential Activation of Human Monocyte-Derived and Plasmacytoid Dendritic Cells by West Nile Virus Generated in Different Host Cells▿

    PubMed Central

    Silva, Maria Carlan; Guerrero-Plata, Antonieta; Gilfoy, Felicia D.; Garofalo, Roberto P.; Mason, Peter W.

    2007-01-01

    Dendritic cells (DCs) play a central role in innate immunity and antiviral responses. In this study, we investigated the production of alpha interferon (IFN-α) and inducible chemokines by human monocyte-derived dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) infected with West Nile virus (WNV), an emergent pathogen whose infection can lead to severe cases of encephalitis in the elderly, children, and immunocompromised individuals. Our experiments demonstrated that WNV grown in mammalian cells (WNVVero) was a potent inducer of IFN-α secretion in pDCs and, to a lesser degree, in mDCs. The ability of WNVVero to induce IFN-α in pDCs did not require viral replication and was prevented by the treatment of cells with bafilomycin A1 and chloroquine, suggesting that it was dependent on endosomal Toll-like receptor recognition. On the other hand, IFN-α production in mDCs required viral replication and was associated with the nuclear translocation of IRF3 and viral antigen expression. Strikingly, pDCs failed to produce IFN-α when stimulated with WNV grown in mosquito cells (WNVC7/10), while mDCs responded similarly to WNVVero or WNVC7/10. Moreover, the IFN-dependent chemokine IP-10 was produced in substantial amounts by pDCs in response to WNVVero but not WNVC7/10, while interleukin-8 was produced in greater amounts by mDCs infected with WNVC7/10 than in those infected with WNVVero. These findings suggest that cell-specific mechanisms of WNV recognition leading to the production of type I IFN and inflammatory chemokines by DCs may contribute to both the innate immune response and disease pathogenesis in human infections. PMID:17913823

  1. Characterization of a constitutive type III nitric oxide synthase in human U937 monocytic cells: stimulation by soluble CD23.

    PubMed Central

    Roman, V; Dugas, N; Abadie, A; Amirand, C; Zhao, H; Dugas, B; Kolb, J P

    1997-01-01

    The soluble cleavage fragment of the low-affinity immunoglobulin E (IgE) receptor/CD23 (sCD23 25000 MW) and antibodies directed against their receptors on monocytes, CD11b and CD11c, stimulate the production of nitric oxide (NO) by these cells and we have suggested that the enzyme involved could be related to the endothelial constitutive type III nitric oxide synthase (ecNOS). In the present work, we have analysed the characteristic properties of this NOS isoform in the model of the human promonocytic cells U937 By reverse-transcription polymerase chain reaction (RT-PCR), the presence of an mRNA coding for type III NOS was found in U937 cells and the corresponding protein was detected by immunofluorescence in permeabilized cells with a specific anti-ecNOS monoclonal antibody (mAb). Membrane extracts displayed a NOS activity dependent on the presence of calcium and calmodulin in the reaction medium and that was abrogated in the presence of EGTA. Recombinant soluble CD23 (25000 MW) was found to trigger an NO-dependent cGMP accumulation in these cells, which was abrogated by calcium chelators and inhibitors of the calcium/calmodulin complex. Moreover, sCD23 elicited a transient augmentation of intracytoplasmic free calcium concentration [Ca2+]i that was dependent on the presence of calcium in the external buffer and was prevented in the presence of EGTA, indicating that it was due to a calcium influx. In conclusion, human promonocytic cells such as U937 exhibit a functional type III NOS that can be stimulated by calcium-raising agents, such as sCD23. Images Figure 1 PMID:9378507

  2. Hypericum perforatum differentially affects corticosteroid receptor-mRNA expression in human monocytic U-937 cells.

    PubMed

    Enning, F; Murck, H; Krieg, J-C; Vedder, H

    2011-09-01

    A dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) axis represents a prominent finding in major depression, possibly related to a dysfunction of the corticosteroid receptor system. Antidepressa