THP-1 cell line: an in vitro cell model for immune modulation approach.

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Chanput, Wasaporn; Mes, Jurriaan J; Wichers, Harry J

2014-11-01

THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions. Copyright © 2013. Published by Elsevier B.V.

PLACENTAL SECRETORY FACTORS INFLUENCE TO THP-1 CELLS PHENOTYPE AND THP-1 CELLS TRANSENDOTHELIAL MIGRATION

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O. I. Stepanova

2013-01-01

Full Text Available Decidual and placental macrophage pools are renewed due to its transendothelial monocyte migration from peripheral blood. Tissue macrophages control placental development and provide fetomaternal immunological tolerance. Preeclamptic pregnancy is accompanied by increased monocyte migration to decidual tissue and local inflammatory events. Regulatory mechanisms of monocyte recruitment to placental and decidual tissues is still unclear. Therefore we investigated the influence soluble placental factors (SPFs during the first- and third-trimester normal pregnancy, as compared to effects of these factors in preeclamptic pregnancy. We studied biological actions of SPF upon transendothelial migration of monocyte-like THP-1 cells and their phenotypic pattern. Transendothelial migration of THP-1 cells was more intensive with firsttrimester SPFs from normal pregnancy, when compared with third-trimester samples, and it was accompanied by decreased CD11a expression. SPFs from pre-eclamptic pregnancy caused an increase in transendothelial migration of THP-1 cells, as compared to SPFs from normal pregnancies, being accompanied by increased CD11b expression. The present study was supported by grants ГК №  02.740.11.0711, НШ-3594.2010.7, МД-150.2011.7 and a grant from St.-Petersburg Goverment for young scientists.

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

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Sander Bekeschus

2016-01-01

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

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

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

2016-01-01

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

Granulocyte-Macrophage Colony-Stimulating Factor Amplification of Interleukin-1β and Tumor Necrosis Factor Alpha Production in THP-1 Human Monocytic Cells Stimulated with Lipopolysaccharide of Oral Microorganisms

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Baqui, A. A. M. A.; Meiller, Timothy F.; Chon, Jennifer J.; Turng, Been-Foo; Falkler, William A.

1998-01-01

Cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), are used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) play important roles in inflammatory processes, including exacerbation of periodontal diseases, one of the most common complications in patients who undergo this therapy. A human monocyte cell line (THP-1) was utilized to investigate IL-1β and TNF-α production following GM-CSF suppl...

M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide

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Genin, Marie; Clement, Francois; Fattaccioli, Antoine; Raes, Martine; Michiels, Carine

2015-01-01

Tumor associated macrophages (TAMs) are present in high density in solid tumors. TAMs share many characteristics with alternatively activated macrophages, also called M2. They have been shown to favor tumor development and a role in chemoresistance has also been suggested. Here, we investigated the effects of M2 in comparison to M1 macrophages on cancer cell sensitivity to etoposide. We set up a model of macrophage polarization, starting from THP-1 monocytes differentiated into macrophages using PMA (Phorbol 12-myristate 13-acetate). Once differentiated (M0 macrophages), they were incubated with IL-4 and IL-13 in order to obtain M2 polarized macrophages or with IFN-gamma and LPS for classical macrophage activation (M1). To mimic the communication between cancer cells and TAMs, M0, M1 or M2 macrophages and HepG2 or A549 cancer cells were co-cultured during respectively 16 (HepG2) or 24 (A549) hours, before etoposide exposure for 24 (HepG2) or 16 (A549) hours. After the incubation, the impact of etoposide on macrophage polarization was studied and cancer cell apoptosis was assessed by western-blot for cleaved caspase-3 and cleaved PARP-1 protein, caspase activity assay and FACS analysis of Annexin V and PI staining. mRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1-derived macrophages, which provide a new, easy and well-characterized model of polarized human macrophages. Etoposide-induced cancer cell apoptosis was markedly reduced in the presence of THP-1 M2 macrophages, while apoptosis was increased in cells co-cultured with M1 macrophages. On the other hand, etoposide did not influence M1 or M2 polarization. These results evidence for the first time a clear protective effect of M2 on the contrary to M1 macrophages on etoposide-induced cancer cell apoptosis

ST2 suppresses IL-6 production via the inhibition of IκB degradation induced by the LPS signal in THP-1 cells

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Takezako, Naoki; Hayakawa, Morisada; Hayakawa, Hiroko; Aoki, Shinsuke; Yanagisawa, Ken; Endo, Hitoshi; Tominaga, Shin-ichi

2006-01-01

LPS induces the production of inflammatory cytokines via the stimulation of Toll-like receptors. In this study, we demonstrated that a soluble secreted form of the ST2 gene product (ST2), a member of the interleukin-1 receptor family, suppressed the production of IL-6 in an LPS-stimulated human monocytic leukemia cell line, THP-1. Immunofluorescence confocal microscopy revealed the binding of ST2 to the surface of the THP-1 cells, in which ST2 led to decreased binding of nuclear factor-κB to the IL-6 promoter. Furthermore, the degradation of IκB in the cytoplasm after LPS stimulation was reduced by pretreatment with ST2. These results demonstrated that ST2 negatively regulates LPS-induced IL-6 production via the inhibition of IκB degradation in THP-1 cells

Genome-wide immunity studies in the rabbit: transcriptome variations in peripheral blood mononuclear cells after in vitro stimulation by LPS or PMA-Ionomycin.

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Jacquier, Vincent; Estellé, Jordi; Schmaltz-Panneau, Barbara; Lecardonnel, Jérôme; Moroldo, Marco; Lemonnier, Gaëtan; Turner-Maier, Jason; Duranthon, Véronique; Oswald, Isabelle P; Gidenne, Thierry; Rogel-Gaillard, Claire

2015-01-23

Our purpose was to obtain genome-wide expression data for the rabbit species on the responses of peripheral blood mononuclear cells (PBMCs) after in vitro stimulation by lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) and ionomycin. This transcriptome profiling was carried out using microarrays enriched with immunity-related genes, and annotated with the most recent data available for the rabbit genome. The LPS affected 15 to 20 times fewer genes than PMA-Ionomycin after both 4 hours (T4) and 24 hours (T24), of in vitro stimulation, in comparison with mock-stimulated PBMCs. LPS induced an inflammatory response as shown by a significant up-regulation of IL12A and CXCL11 at T4, followed by an increased transcription of IL6, IL1B, IL1A, IL36, IL37, TNF, and CCL4 at T24. Surprisingly, we could not find an up-regulation of IL8 either at T4 or at T24, and detected a down-regulation of DEFB1 and BPI at T24. A concerted up-regulation of SAA1, S100A12 and F3 was found upon stimulation by LPS. PMA-Ionomycin induced a very early expression of Th1, Th2, Treg, and Th17 responses by PBMCs at T4. The Th1 response increased at T24 as shown by the increase of the transcription of IFNG and by contrast to other cytokines which significantly decreased from T4 to T24 (IL2, IL4, IL10, IL13, IL17A, CD69) by comparison to mock-stimulation. The granulocyte-macrophage colony-stimulating factor (CSF2) was by far the most over-expressed gene at both T4 and T24 by comparison to mock-stimulated cells, confirming a major impact of PMA-Ionomycin on cell growth and proliferation. A significant down-regulation of IL16 was observed at T4 and T24, in agreement with a role of IL16 in PBMC apoptosis. We report new data on the responses of PBMCs to LPS and PMA-Ionomycin in the rabbit species, thus enlarging the set of mammalian species for which such reports exist. The availability of the rabbit genome assembly together with high throughput genomic tools should pave the way for more

Anti-Inflammatory Effects of Pomegranate Peel Extract in THP-1 Cells Exposed to Particulate Matter PM10

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Soojin Park

2016-01-01

Full Text Available Epidemiological and experimental evidence support health risks associated with the exposure to airborne particulate matter with a diameter of <10 μM (PM10. PM10 stimulates the production of reactive oxygen species (ROS and inflammatory mediators. Thus, we assumed that natural antioxidants might provide health benefits attenuating hazardous effects of PM10. In the present study, we examined the effects of pomegranate peel extract (PPE on THP-1 monocytic cells exposed to PM10. PM10 induced cytotoxicity and the production of ROS. It also increased the expression and secretion of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α, interleukin-1β (IL-1β, and monocyte chemoattractant protein-1 (MCP-1, and cell adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1. PPE at 10–100 μg mL−1 attenuated the production of ROS and the expression of TNF-α, IL-1β, MCP-1, and ICAM-1, but not VCAM-1, in THP-1 cells stimulated by PM10 (100 μg mL−1. PPE also attenuated the adhesion of PM10-stimulated THP-1 cells to EA.hy926 endothelial cells. PPE constituents, punicalagin and ellagic acid, attenuated PM10-induced monocyte adhesion to endothelial cells, and punicalagin was less cytotoxic compared to ellagic acid. The present study suggests that PPE and punicalagin may be useful in alleviating inflammatory reactions due to particulate matter.

Lipopolysaccharide induces autotaxin expression in human monocytic THP-1 cells

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Li Song; Zhang Junjie

2009-01-01

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

Influence of the protein kinase C activator phorbol myristate acetate on the intracellular activity of antibiotics against hemin- and menadione-auxotrophic small-colony variant mutants of Staphylococcus aureus and their wild-type parental strain in human THP-1 cells.

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Garcia, Laetitia G; Lemaire, Sandrine; Kahl, Barbara C; Becker, Karsten; Proctor, Richard A; Tulkens, Paul M; Van Bambeke, Françoise

2012-12-01

In a previous study (L. G. Garcia et al., Antimicrob. Agents Chemother. 56:3700-3711, 2012), we evaluated the intracellular fate of menD and hemB mutants (corresponding to menadione- and hemin-dependent small-colony variants, respectively) of the parental COL methicillin-resistant Staphylococcus aureus strain and the pharmacodynamic profile of the intracellular activity of a series of antibiotics in human THP-1 monocytes. We have now examined the phagocytosis and intracellular persistence of the same strains in THP-1 cells activated by phorbol 12-myristate 13-acetate (PMA) and measured the intracellular activity of gentamicin, moxifloxacin, and oritavancin in these cells. Postphagocytosis intracellular counts and intracellular survival were lower in PMA-activated cells, probably due to their higher killing capacities. Gentamicin and moxifloxacin showed a 5- to 7-fold higher potency (lower static concentrations) against the parental strain, its hemB mutant, and the genetically complemented strain in PMA-activated cells and against the menD strain in both activated and nonactivated cells. This effect was inhibited when cells were incubated with N-acetylcysteine (a scavenger of oxidant species). In parallel, we observed that the MICs of these drugs were markedly reduced if bacteria had been preexposed to H(2)O(2). In contrast, the intracellular potency of oritavancin was not different in activated and nonactivated cells and was not decreased by the addition of N-acetylcysteine, regardless of the phenotype of the strains. The oritavancin MIC was also unaffected by preincubation of the bacteria with H(2)O(2). Thus, activation of THP-1 cells by PMA may increase the intracellular potency of certain antibiotics (probably due to synergy with reactive oxygen species), but this effect cannot be generalized to all antibiotics.

[Use of THP-1 for allergens identification method validation].

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Zhao, Xuezheng; Jia, Qiang; Zhang, Jun; Li, Xue; Zhang, Yanshu; Dai, Yufei

2014-05-01

Look for an in vitro test method to evaluate sensitization using THP-1 cells by the changes of the expression of cytokines to provide more reliable markers of the identification of sensitization. The monocyte-like THP-1 cells were induced and differentiated into THP-1-macrophages with PMA (0.1 microg/ml). The changes of expression of cytokines at different time points after the cells being treated with five known allergens, 2,4-dinitrochlorobenzene (DNCB), nickel sulfate (NiSO4), phenylene diamine (PPDA) potassium dichromate (K2Cr2O7) and toluene diisocyanate (TDI) and two non-allergens sodium dodecyl sulfate (SDS) and isopropanol (IPA) at various concentrations were evaluated. The IL-6 and TNF-alpha production was measured by ELISA. The secretion of IL-1beta and IL-8 was analyzed by Cytometric Bead Array (CBA). The section of the IL-6, TNF-alpha, IL-1beta and IL-8 were the highest when THP-1 cells were exposed to NiSO4, DNCB and K2Cr2O7 for 6h, PPDA and TDI for 12h. The production of IL-6 were approximately 40, 25, 20, 50 and 50 times for five kinds chemical allergens NiSO4, DNCB, K2Cr2O7, PPDA and TDI respectively at the optimum time points and the optimal concentration compared to the control group. The expression of TNF-alpha were 20, 12, 20, 8 and 5 times more than the control group respectively. IL-1beta secretion were 30, 60, 25, 30 and 45 times respectively compared to the control group. The production of IL-8 were approximately 15, 12, 15, 12 and 7 times respectively compared to the control group. Both non-allergens SDS and IPA significantly induced IL-6 secretion in a dose-dependent manner however SDS cause a higher production levels, approximately 20 times of the control. Therefore IL-6 may not be a reliable marker for identification of allergens. TNF-alpha, IL-1beta and IL-8 expressions did not change significantly after exposed to the two non-allergens. The test method using THP-1 cells by detecting the productions of cytokines (TNF-alpha, IL-1beta and

Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naive THP-1 cell line.

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Yoshida, Y; Sakaguchi, H; Ito, Y; Okuda, M; Suzuki, H

2003-04-01

It has been known that dendritic cells (DCs) including Langerhans cells (LCs) play a critical role in the skin sensitization process. Many attempts have been made to develop in vitro sensitization tests that employ DCs derived from peripheral blood mononuclear cells (PBMC-DC) or CD34+ hematopoietic progenitor cells (CD34+ HPC) purified from cord blood or bone marrow. However, the use of the DCs in in vitro methods has been difficult due to the nature of these cells such as low levels in the source and/or donor-to-donor variability. In our studies, we employed the human monocytic leukemia cell line, THP-1, in order to avoid some of these difficulties. At the start, we examined whether treatment of the cells with various cytokines could produce DCs from THP-1. Treatment of THP-1 cells with cytokines such as GM-CSF, IL-4, TNF-alpha, and/or PMA did induce some phenotypic changes in THP-1 cells that were characteristic of DCs. Subsequently, responses to a known sensitizer, dinitrochlorobenzene (DNCB), and a non-sensitizer, dimethyl sulfoxide (DMSO) or sodium lauryl sulfate (SLS), on the expression of co-stimulatory molecules, CD54 and CD86, were examined between the naive cells and the cytokine-treated cells. Interestingly, the naive THP-1 cells responded only to DNCB and the response to the sensitizer was more distinct than cytokine-treated THP-1 cells. Similar phenomena were also observed in the human myeloid leukemia cell line, KG-1. Furthermore, with treatment of DNCB, naive THP-1 cells showed augmented expression of HLA, CD80 and secretion of IL-1 beta. The response of THP-1 cells to a sensitizer was similar to that of LCs/DCs. Upon demonstrating the differentiation of monocyte cells in our system, we then evaluated a series of chemicals, including known sensitizers and non-sensitizers, for their potential to augment CD54 and CD86 expression on naive THP-1 cells. Indeed, known sensitizers such as PPD and 2-MBT significantly augmented CD54 and CD86 expression in a

[Effect of DOT1L gene silence on proliferation of acute monocytic leukemia cell line THP-1].

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Zhang, Yu-Juan; Li, Hua-Wen; Chang, Guo-Qiang; Zhang, Hong-Ju; Wang, Jian; Lin, Ya-Ni; Zhou, Jia-Xi; Li, Qing-Hua; Pang, Tian-Xiang

2013-08-01

This study was aimed to investigate the influence of short hairpin RNA (shRNA) on proliferation of human leukemia cell line THP-1. The shRNA targeting the site 732-752 of DOT1L mRNA was designed and chemically synthesized, then a single-vector lentiviral, tet-inducible shRNA-DOT1L system (Plko-Tet-On) was generated. Thereafter, the THP-1 cells with lentivirus were infected to create stable cell line with regulatable shRNA expression. The expression of DOT1L in the THP-1 cell line was assayed by RT-PCR. Effect of shRNA-DOT1L on the proliferation of THP-1 cells was detected with MTT method,and the change of colony forming potential of THP-1 cells was analyzed by colony forming unit test. Cell cycle distribution was tested by flow cytometry. The results indicated that the expression of DOT1L was statistically lower than that in the control groups. The proliferation and colony forming capacity of THP-1 cells were significantly inhibited. The percentage of cells at G0/G1 phase increased in THP-1/shRNA cells treated with Dox while the percentage of cells at S phase significantly decreased as compared with that in the control group. It is concluded that the shRNA targeting DOT1L can effectively inhibit the proliferation of acute monocytic leukemia cell line THP-1.

Myelomonocytic THP-1 cells for in vitro testing of immunomodulatory properties of nanoparticles.

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Schroecksnadel, Sebastian; Jenny, Marcel; Fuchs, Dietmar

2011-02-01

The use of nanoparticles for new therapeutic and diagnostics options represents a new risk for individuals exposed to such compounds. The myelomonocytic cell line THP-1 could be a useful alternative to human peripheral blood mononuclear cells (PBMC) to test for effects of drugs and compounds. Stimulation degree of cells can be monitored by measurement of neopterin and/or the kynurenine to tryptophan ratio. The method is robust and reproducible in the range of 0.1-1.0 microg/ml of LPS. However, compared to the PBMC assay it will not reveal any effect on the T-cell interaction.

Establishment of an in vitro photoassay using THP-1 cells and IL-8 to discriminate photoirritants from photoallergens.

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Martínez, V; Galbiati, V; Corsini, E; Martín-Venegas, R; Vinardell, M P; Mitjans, M

2013-09-01

At present, there are no in vivo or in vitro methods developed which has been adopted by regulatory authorities to assess photosensitization induced by chemicals. Recently, we have proposed the use of THP-1 cells and IL-8 release to identify the potential of chemicals to induce skin sensitization. Based on the assumption that sensitization and photosensitization share common mechanisms, the aim of this work was to explore the THP-1 model as an in vitro model to identify photoallergenic chemicals. THP-1 cells were exposed to 7 photoallergens and 3 photoirritants and irradiated with UVA light or kept in dark. Non phototoxic allergens or irritants were also included as negative compounds. Following 24h of incubation, cytotoxicity and IL-8 release were measured. At subtoxic concentrations, photoallergens produced a dose-related increase in IL-8 release after irradiation. Some photoirritants also produced a slight increase in IL-8 release. However, when the overall stimulation indexes of IL-8 were calculated for each chemical, 6 out of 7 photoallergens tested reached a stimulation index above 2, while the entire set of negative compounds had stimulation indexes below 2. Our data suggest that this assay may become a useful cell-based in vitro test for evaluating the photosensitizing potential of chemicals. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Effect of LPXN Overexpression on the Proliferation, Adhesion and Invasion of THP-1 Cells and Its Mechamisms].

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Dai, Hai-Ping; Zhu, Guo-Hua; Wu, Li-Li; Wang, Qian; Yao, Hong; Wang, Qin-Rong; Wen, Li-Jun; Qiu, Hui-Ying; Shen, Qun; Chen, Su-Ning; Wu, De-Pei

2017-06-01

To explore the effect of LPXN overexpression on the proliferation, adhesion and invasion of THP-1 cells and its possible mechanism. A THP-1 cell line with stable overexpression of LPXN was constucted by using a lentivirus method, CCK-8 was used to detect the proliferation of cells, adhesion test was used to evaluate adhesion ablity of cells to Fn. Transwell assay was used to detect the change of invasion capability. Western blot was used to detect expression of LPXN, ERK, pERK and integrin α4, α5, β1, the Gelatin zymography was applied to detect activity of MMP2/MMP9 secreted by the THP-1 cells. Successful establishment of THP-1 cells with LPXN overexpression (THP-1 LPXN) was confirmed with Western blot. THP-1 LPXN cells were shown to proliferate faster than the control THP-1 vector cells. Adhesion to Fn and expression of ERK, integrin α4, α5 and β1 in the THP-1 LPXN cells were higher than that in the control cells. Invasion across matrigel and enhanced activity of MMP2 could be detected both in the THP-1 LPXN cells as compared with the control cells. Ectopically ovexpression of LPXN may promote proliferation of THP-1 cells through up-regulation of ERK; promote adhesion of THP-1 cells through up-regulating the integrin α4/β1 as well as integrin α5/β1 complex; promote invasion of THP-1 cells through activating MMP2.

Thrombospondin-1 production is enhanced by Porphyromonas gingivalis lipopolysaccharide in THP-1 cells.

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Misa Gokyu

Full Text Available Periodontitis is a chronic inflammatory disease caused by gram-negative anaerobic bacteria. Monocytes and macrophages stimulated by periodontopathic bacteria induce inflammatory mediators that cause tooth-supporting structure destruction and alveolar bone resorption. In this study, using a DNA microarray, we identified the enhanced gene expression of thrombospondin-1 (TSP-1 in human monocytic cells stimulated by Porphyromonas gingivalis lipopolysaccharide (LPS. TSP-1 is a multifunctional extracellular matrix protein that is upregulated during the inflammatory process. Recent studies have suggested that TSP-1 is associated with rheumatoid arthritis, diabetes mellitus, and osteoclastogenesis. TSP-1 is secreted from neutrophils, monocytes, and macrophages, which mediate immune responses at inflammatory regions. However, TSP-1 expression in periodontitis and the mechanisms underlying TSP-1 expression in human monocytic cells remain unknown. Here using real-time RT-PCR, we demonstrated that TSP-1 mRNA expression level was significantly upregulated in inflamed periodontitis gingival tissues and in P. gingivalis LPS-stimulated human monocytic cell line THP-1 cells. TSP-1 was expressed via Toll-like receptor (TLR 2 and TLR4 pathways. In P. gingivalis LPS stimulation, TSP-1 expression was dependent upon TLR2 through the activation of NF-κB signaling. Furthermore, IL-17F synergistically enhanced P. gingivalis LPS-induced TSP-1 production. These results suggest that modulation of TSP-1 expression by P. gingivalis plays an important role in the progression and chronicity of periodontitis. It may also contribute a new target molecule for periodontal therapy.

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

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Min-Gu Song

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

Fenoterol inhibits LPS-induced AMPK activation and inflammatory cytokine production through β-arrestin-2 in THP-1 cell line

International Nuclear Information System (INIS)

Wang, Wei; Zhang, Yuan; Xu, Ming; Zhang, You-Yi; He, Bei

2015-01-01

The AMP-activated protein kinase (AMPK) pathway is involved in regulating inflammation in several cell lines. We reported that fenoterol, a β 2 -adrenergic receptor (β 2 -AR) agonist, had anti-inflammatory effects in THP-1 cells, a monocytic cell line. Whether the fenoterol anti-inflammatory effect involves the AMPK pathway is unknown. In this study, we explored the mechanism of β 2 -AR stimulation with fenoterol in a lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in THP-1 cells. We studied whether fenoterol and β-arrestin-2 or AMPKα1 subunit knockdown could affect LPS-induced AMPK activation, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion. LPS-induced AMPK activation and interleukin 1β (IL-1β) release were reduced with fenoterol pretreatment of THP-1 cells. SiRNA knockdown of β-arrestin-2 abolished the fenoterol inhibition of LPS-induced AMPK activation and interleukin 1β (IL-1β) release, thus β-arrestin-2 mediated the anti-inflammatory effects of fenoterol on LPS-treated THP-1 cells. In addition, siRNA knockdown of AMPKα1 significantly attenuated the LPS-induced NF-κB activation and IL-1β release, so AMPKα1 was a key signaling molecule involved in LPS-induced inflammatory cytokine production. These results suggested the β 2 -AR agonist fenoterol inhibited LPS-induced AMPK activation and IL-1β release via β-arrestin-2 in THP-1 cells. The exploration of these mechanisms may help optimize therapeutic agents targeting these pathways in inflammatory diseases. - Highlights: • β 2 -AR agonist fenoterol exerts its protective effect on LPS-treated THP-1 cells. • Fenoterol inhibits LPS-induced AMPK activation and IL-1β production. • β-arrestin2 mediates fenoterol-inhibited AMPK activation and IL-1β release. • AMPKα1 is involved in LPS-induced NF-κB activation and IL-1β production

Fenoterol inhibits LPS-induced AMPK activation and inflammatory cytokine production through β-arrestin-2 in THP-1 cell line

Energy Technology Data Exchange (ETDEWEB)

Wang, Wei [Department of Respiratory Medicine, Peking University Third Hospital, Beijing (China); Department of Infectious Diseases, Peking University Third Hospital, Beijing (China); Zhang, Yuan [Department of Respiratory Medicine, Peking University Third Hospital, Beijing (China); Xu, Ming; Zhang, You-Yi [Department of Institute of Vascular Medicine and Beijing Key Laboratory of Cardiovascular Receptors Research, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides, Peking University Third Hospital, Beijing (China); He, Bei, E-mail: puh3_hb@bjmu.edu.cn [Department of Respiratory Medicine, Peking University Third Hospital, Beijing (China)

2015-06-26

The AMP-activated protein kinase (AMPK) pathway is involved in regulating inflammation in several cell lines. We reported that fenoterol, a β{sub 2}-adrenergic receptor (β{sub 2}-AR) agonist, had anti-inflammatory effects in THP-1 cells, a monocytic cell line. Whether the fenoterol anti-inflammatory effect involves the AMPK pathway is unknown. In this study, we explored the mechanism of β{sub 2}-AR stimulation with fenoterol in a lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in THP-1 cells. We studied whether fenoterol and β-arrestin-2 or AMPKα1 subunit knockdown could affect LPS-induced AMPK activation, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion. LPS-induced AMPK activation and interleukin 1β (IL-1β) release were reduced with fenoterol pretreatment of THP-1 cells. SiRNA knockdown of β-arrestin-2 abolished the fenoterol inhibition of LPS-induced AMPK activation and interleukin 1β (IL-1β) release, thus β-arrestin-2 mediated the anti-inflammatory effects of fenoterol on LPS-treated THP-1 cells. In addition, siRNA knockdown of AMPKα1 significantly attenuated the LPS-induced NF-κB activation and IL-1β release, so AMPKα1 was a key signaling molecule involved in LPS-induced inflammatory cytokine production. These results suggested the β{sub 2}-AR agonist fenoterol inhibited LPS-induced AMPK activation and IL-1β release via β-arrestin-2 in THP-1 cells. The exploration of these mechanisms may help optimize therapeutic agents targeting these pathways in inflammatory diseases. - Highlights: • β{sub 2}-AR agonist fenoterol exerts its protective effect on LPS-treated THP-1 cells. • Fenoterol inhibits LPS-induced AMPK activation and IL-1β production. • β-arrestin2 mediates fenoterol-inhibited AMPK activation and IL-1β release. • AMPKα1 is involved in LPS-induced NF-κB activation and IL-1β production.

Evaluation of amentoflavone isolated from Cnestis ferruginea Vahl ex DC (Connaraceae) on production of inflammatory mediators in LPS stimulated rat astrocytoma cell line (C6) and THP-1 cells.

Science.gov (United States)

Ishola, I O; Chaturvedi, J P; Rai, S; Rajasekar, N; Adeyemi, O O; Shukla, R; Narender, T

2013-03-27

Cnestisferruginea (CF) Vahl ex DC (Connaraceae) is a shrub widely used in traditional African medicine for the treatment of various psychiatric illness and inflammatory conditions. This study was carried out to investigate the effect of amentoflavone isolated from methanolic root extract of CF on lipopolysaccharide (LPS)-induced neuroinflammatory cascade of events associated to the oxidative and nitrative stress, and TNF-α production in rat astrocytoma cell line (C6) and human monocytic leukemia cell line (THP-1), respectively. Rat astrocytoma cells (C6) were stimulated with LPS (10μg/ml) alone and in the presence of different concentrations of amentoflavone (0.1-3μg/ml) for 24h incubation period. Nitrite release, reactive oxygen species (ROS), malondialdehyde (MDA) and reduced-glutathione (GSH) in C6 cells were estimated; while the TNF-α level was estimated in THP-1 cell lysate. In vivo analgesic activity was evaluated using mouse writhing and hot plate tests while the anti-inflammatory effect was investigated using carrageenan-induced oedema test. LPS (10μg/ml) significantly (PTHP-1 cells. Amentoflavone (6.25-50mg/kg) significantly (Ptest. It produced time course significant (P<0.05) decrease in oedema formation in rodents. Findings in this study demonstrate the anti-neuroinflammatory and antinoceptive effects of amentoflavone which may suggest its beneficial roles in neuroinflammation associated disorders. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Effects of HSP27 chaperone on THP-1 tumor cell apoptosis.

Science.gov (United States)

Kaigorodova, E V; Ryazantseva, N V; Novitskii, V V; Maroshkina, A N; Belkina, M V

2012-11-01

The role of Hsp27 (heat shock protein 27) chaperone in regulation of THP-1 tumor cell apoptosis was studied. Realization of tumor cell apoptosis under conditions of in vitro culturing with Hsp27 specific inhibitor (KRIBB3) was evaluated by fluorescent microscopy with FITC-labeled annexin V and propidium iodide. Measurements of Bcl-2 family proteins (Bcl-2, Bax, Bad) in tumor cells incubated with Hsp27 inhibitor were carried out by Western blotting. Chaperone Hsp27 acted as apoptosis inhibitor in THP-1 tumor cells modulating the proportion of antiapoptotic (Bcl-2) and proapoptotic (Bax and Bad) proteins.

Evaluation of the sensitizing potential of antibiotics in vitro using the human cell lines THP-1 and MUTZ-LC and primary monocyte‐derived dendritic cells

International Nuclear Information System (INIS)

Sebastian, Katrin; Ott, Hagen; Zwadlo-Klarwasser, Gabriele; Skazik-Voogt, Claudia; Marquardt, Yvonne; Czaja, Katharina; Merk, Hans F.; Baron, Jens Malte

2012-01-01

Since the 7th amendment to the EU cosmetics directive foresees a complete ban on animal testing, alternative in vitro methods have been established to evaluate the sensitizing potential of small molecular weight compounds. To find out whether these novel in vitro assays are also capable to predict the sensitizing potential of small molecular weight drugs, model compounds such as beta-lactams and sulfonamides – which are the most frequent cause of adverse drug reactions – were co-incubated with THP-1, MUTZ-LC, or primary monocyte‐derived dendritic cells for 48 h and subsequent expression of selected marker genes (IL-8, IL-1β, CES1, NQO1, GCLM, PIR and TRIM16) was studied by real time PCR. Benzylpenicillin and phenoxymethylpenicillin were recognized as sensitizing compounds because they are capable to induce the mRNA expression of these genes in moDCs and, except for IL-8, in THP-1 cells but not in MUTZ-LC. Ampicillin stimulated the expression of some marker genes in moDCs and THP-1 cells. SMX did not affect the expression of these genes in THP-1, however, in moDCs, at least PIR was enhanced and there was an increase of the release of IL-8. These data reveal that novel in vitro DC based assays might play a role in the evaluation of the allergenic potential of novel drug compounds, but these systems seem to lack the ability to detect the sensitizing potential of prohaptens that require metabolic activation prior to sensitization and moDCs seem to be superior with regard to the sensitivity compared with THP-1 and MUTZ-3 cell lines. -- Highlights: ► We tested the sensitizing potential of small molecular weight drugs in vitro. ► In vitro assays were performed with moDCs and THP-1 cells. ► Beta-lactam antibiotics can be recognized as sensitizing compounds. ► They affect the expression of metabolic enzymes, cytokines and transcription factors. ► Sulfamethoxazole has no measurable effect on THP-1 cells and moDCs.

Evaluation of the sensitizing potential of antibiotics in vitro using the human cell lines THP-1 and MUTZ-LC and primary monocyte‐derived dendritic cells

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Sebastian, Katrin, E-mail: ksebastian@ukaachen.de [Department of Dermatology and Allergology, RWTH Aachen University Hospital, D-52074 Aachen (Germany); Ott, Hagen [Department of Dermatology and Allergology, RWTH Aachen University Hospital, D-52074 Aachen (Germany); Zwadlo-Klarwasser, Gabriele [IZKF (BIOMAT), RWTH Aachen University Hospital, D-52074 Aachen (Germany); Skazik-Voogt, Claudia; Marquardt, Yvonne; Czaja, Katharina; Merk, Hans F.; Baron, Jens Malte [Department of Dermatology and Allergology, RWTH Aachen University Hospital, D-52074 Aachen (Germany)

2012-08-01

Since the 7th amendment to the EU cosmetics directive foresees a complete ban on animal testing, alternative in vitro methods have been established to evaluate the sensitizing potential of small molecular weight compounds. To find out whether these novel in vitro assays are also capable to predict the sensitizing potential of small molecular weight drugs, model compounds such as beta-lactams and sulfonamides – which are the most frequent cause of adverse drug reactions – were co-incubated with THP-1, MUTZ-LC, or primary monocyte‐derived dendritic cells for 48 h and subsequent expression of selected marker genes (IL-8, IL-1β, CES1, NQO1, GCLM, PIR and TRIM16) was studied by real time PCR. Benzylpenicillin and phenoxymethylpenicillin were recognized as sensitizing compounds because they are capable to induce the mRNA expression of these genes in moDCs and, except for IL-8, in THP-1 cells but not in MUTZ-LC. Ampicillin stimulated the expression of some marker genes in moDCs and THP-1 cells. SMX did not affect the expression of these genes in THP-1, however, in moDCs, at least PIR was enhanced and there was an increase of the release of IL-8. These data reveal that novel in vitro DC based assays might play a role in the evaluation of the allergenic potential of novel drug compounds, but these systems seem to lack the ability to detect the sensitizing potential of prohaptens that require metabolic activation prior to sensitization and moDCs seem to be superior with regard to the sensitivity compared with THP-1 and MUTZ-3 cell lines. -- Highlights: ► We tested the sensitizing potential of small molecular weight drugs in vitro. ► In vitro assays were performed with moDCs and THP-1 cells. ► Beta-lactam antibiotics can be recognized as sensitizing compounds. ► They affect the expression of metabolic enzymes, cytokines and transcription factors. ► Sulfamethoxazole has no measurable effect on THP-1 cells and moDCs.

Effect of bisphosphonates on macrophagic THP-1 cell survival in bisphosphonate-related osteonecrosis of the jaw (BRONJ).

Science.gov (United States)

Hoefert, Sebastian; Sade Hoefert, Claudia; Munz, Adelheid; Schmitz, Inge; Grimm, Martin; Yuan, Anna; Northoff, Hinnak; Reinert, Siegmar; Alexander, Dorothea

2016-03-01

Immune deficiency and bacterial infection have been suggested to play a role in the pathophysiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). Zoledronate was previously found to promote THP-1 cell death. To examine this hypothesis with all commonly prescribed bisphosphonates, we tested the effect of (nitrogen-containing) ibandronate, risedronate, alendronate, pamidronate, and (non-nitrogen-containing) clodronate on macrophagic THP-1 cells. Activated THP-1 cells were exposed to .5 to 50 μM of nitrogen-containing bisphosphonates and .5 to 500 μM of clodronate. Cell adherence and survival were assessed in vitro using the xCELLigence real-time monitoring system. Results were confirmed histologically and verified with Live/Dead staining. All bisphosphonates inhibited THP-1 cell adherence and survival dose and time dependently, significant for zoledronate, alendronate, pamidronate, and clodronate in high concentrations (50 μM and 500 μM; P THP-1 cell survival compared with controls (P THP-1 cells exhibited no cytomorphologic changes at all concentrations. Commonly prescribed bisphosphonates inhibit the survival of macrophagic THP-1 cells dose-dependently without altering morphology. This may suggest a local immune dysfunction reflective of individual bisphosphonate potency leading to the pathogenesis of BRONJ. Copyright © 2016 Elsevier Inc. All rights reserved.

An in vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THP-G8.

Science.gov (United States)

Takahashi, Toshiya; Kimura, Yutaka; Saito, Rumiko; Nakajima, Yoshihiro; Ohmiya, Yoshihiro; Yamasaki, Kenshi; Aiba, Setsuya

2011-12-01

Several studies have suggested that interleukin (IL)-8 can serve as a biomarker for discrimination of skin sensitizers from nonsensitizers. We established a stable THP-1-derived IL-8 reporter cell line, THP-G8, which harbors SLO and SLR luciferase genes under the control of IL-8 and glyceraldehyde 3-phosphate dehydrogenase promoters, respectively. After 6 h treatment with chemicals, normalized SLO luciferase activity (nSLO-LA) was calculated by dividing SLO-LA by SLR-LA, and the fold induction of nSLO-LA (FInSLO-LA) was calculated by dividing nSLO-LA of chemically treated cells by that of nontreated cells. The nSLO-LA of THP-G8 cells increased in response to lipopolysaccharide (LPS) and several sensitizers. The FInSLO-LA in THP-G8 cells induced by LPS or sensitizers positively correlated with their induction of IL-8 messenger RNA in THP-1 cells. The nSLO-LA value of THP-G8 cells was significantly increased (FInSLO-LA ≥ 1.4) by 13 of the 15 sensitizers as well as by 5 of the 7 nonsensitizers. Interestingly, pretreatment with N-acetylcysteine suppressed the increase in FInSLO-LA induced by all sensitizers (inhibition index (II) ≤ 0.8) but did not suppress that induced by most of the nonsensitizers. We then evaluated the performance of this assay using values of FInSLO-LA ≥ 1.4 and II ≤ 0.8 in at least two of three independent experiments as the criteria of a sensitizer, which resulted in test accuracies of 82% for the 22 chemicals used and of 88% for the chemicals proposed by European Center for the Validation of Alternative Methods. This newly developed assay is a candidate replacement for animal tests of skin sensitization because of its accuracy, convenience, and high throughput performance.

Modulation of the human equilibrative nucleoside transporter1 (hENT1) activity by IL-4 and PMA in B cells from chronic lymphocytic leukemia.

Science.gov (United States)

Fernández Calotti, Paula; Galmarini, Carlos María; Cañones, Cristian; Gamberale, Romina; Saénz, Daniel; Avalos, Julio Sánchez; Chianelli, Mónica; Rosenstein, Ruth; Giordano, Mirta

2008-02-15

Nucleoside transporters (NTs) are essential for the uptake of therapeutic nucleoside analogs, broadly used in cancer treatment. The mechanisms responsible for NT regulation are largely unknown. IL-4 is a pro-survival signal for chronic lymphocytic leukemia (CLL) cells and has been shown to confer resistance to nucleoside analogs. The aim of this study was to investigate whether IL-4 is able to modulate the expression and function of the human equilibrative NT1 (hENT1) in primary cultures of CLL cells and, consequently, to affect cytotoxicity induced by therapeutic nucleosides analogs. We found that treatment with IL-4 (20 ng/ml for 24 h) increased mRNA hENT1 expression in CLL cells without affecting that of normal B cells. Given that the enhanced mRNA levels of hENT1 in CLL cells did not result in increased transport activity, we examined the possibility that hENT1 induced by IL-4 may require post-translational modifications to become active. We found that the acute stimulation of PKC in IL-4-treated CLL cells by short-term incubation with PMA significantly increased hENT1 transport activity and favoured fludarabine-induced apoptosis. By contrast, and in line with previous reports, IL-4 plus PMA protected CLL cells from a variety of cytotoxic agents. Our findings indicate that the combined treatment with IL-4 and PMA enhances hENT1 activity and specifically sensitizes CLL cells to undergo apoptosis induced by fludarabine.

Pma1 is an alkali/alkaline earth metal cation ATPase that preferentially transports Na(+) and K(+) across the Mycobacterium smegmatis plasma membrane.

Science.gov (United States)

Ayala-Torres, Carlos; Novoa-Aponte, Lorena; Soto, Carlos Y

2015-07-01

Mycobacterium smegmatis Pma1 is the orthologue of M. tuberculosis P-type ATPase cation transporter CtpF, which is activated under stress conditions, such as hypoxia, starvation and response to antituberculous and toxic substances. The function of Pma1 in the mycobacterial processes across the plasma membrane has not been characterised. In this work, bioinformatic analyses revealed that Pma1 likely contains potential sites for, Na(+), K(+) and Ca(2+) binding and transport. Accordingly, RT-qPCR experiments showed that M. smegmatis pma1 transcription is stimulated by sub-lethal doses of Na(+), K(+) and Ca(2+); in addition, the ATPase activity of plasma membrane vesicles in recombinant Pma1-expressing M. smegmatis cells is stimulated by treatment with these cations. In contrast, M. smegmatis cells homologously expressing Pma1 displayed tolerance to high doses of Na(+) and K(+) but not to Ca(2+) ions. Consistently, the recombinant protein Km embedded in plasma membrane demonstrated that Ca(2+) has more affinity for Pma1 than Na(+) and K(+) ions; furthermore, the estimation of Vmax/Km suggests that Na(+) and K(+) ions are more efficiently translocated than Ca(2+). Thus, these results strongly suggest that Pma1 is a promiscuous alkali/alkaline earth cation ATPase that preferentially transports Na(+) and/or K(+) across the mycobacterial plasma membrane. Copyright © 2015 Elsevier GmbH. All rights reserved.

[Acetyl-11-keto-beta-boswellic acid and arsenic trioxide regulate the productions and activities of matrix metalloproteinases in human skin fibroblasts and human leukemia cell line THP-1].

Science.gov (United States)

Liang, Ya-hui; Li, Ping; Zhao, Jing-xia; Liu, Xin; Huang, Qi-fu

2010-11-01

In order to reveal the treatment mechanism of Chinese medicine with the effect of activating blood and resolving putridity, we selected acetyl-11-keto-beta-boswellic acid (AKBA) and arsenic trioxide (ATO), the main monomeric components of frankincense and arsenolite which are two most commonly used Chinese medicine with effect of activating blood and resolving putridity. We combined AKBA and ATO as a compound, and explored its regulatory role in productions and activities of matrix metalloproteinase (MMP)-1, MMP-2 and MMP-9 in human skin fibroblasts (HSFbs) and human acute monocytic leukemia cell line THP-1 in inflammatory state. In order to simulate the inflammatory micro-environment of chronic wounds, we established 3 cell models: HSFb model activated by tumor necrosis factor-alpha (TNF-α), THP-1 cell model activated by phorbol-12-myristate-13-acetate (PMA) and HSFb-THP-1 cell coculture system. AKBA and ATO were cocultured with these cell models. Enzyme-linked immunosorbent assay (ELISA), gelatin zymography assay and reverse transcription-polymerase chain reaction (RT-PCR) were used to test the secretions, activities and mRNA expressions of MMP-1, MMP-2 and MMP-9. In the study of the regulatory mechanism of AKBA and ATO on MMPs, AKBA and ATO were cocultured with the cell models. ELISA was used to test the secretions of TNF-α and interleukin-1beta (IL-β) and Western blot was used to test the phosphorylation levels of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated proteinkinase (p38MAPK). Compound of AKBA and ATO inhibited MMP-1, MMP-2 and MMP-9 mRNA expressions, secretions and activities respectively in HSFbs and THP-1 cells in inflammatory state (PTHP-1 cells and cell coculture system (PTHP-1 cells (P<0.05, P<0.01). The combined use of AKBA and ATO which in line with the rule of activating blood and resolving putridity inhibits fibroblasts and inflammatory cells in producing MMPs in inflammatory state through inhibiting the

The opposing effects of calmodulin, adenosine 5 prime -triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells

Energy Technology Data Exchange (ETDEWEB)

Sekiya, M.; Frohlich, E.D.; Cole, F.E. (Alton Ochsner Medical Foundation, New Orleans, LA (USA))

1991-01-01

In the present study, we investigated the effects of calmodulin, adenosine 5{prime}-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation. In conclusion, these results demonstrated that PMA inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.

Stimulation of prostaglandin E2 production by phorbol esters and epidermal growth factor in porcine thyroid cells

International Nuclear Information System (INIS)

Kasai, K.; Hiraiwa, M.; Emoto, T.; Akimoto, K.; Takaoka, T.; Shimoda, S.I.

1987-01-01

Effects of phorbol esters and epidermal growth factor (EGF) on prostaglandin E 2 production by cultured porcine thyroid cells were examined. Both phorbol 12-myristate 13-acetate (PMA) and EGF stimulated prostaglandin E 2 production by the cells in dose related fashion. PMA stimulated prostaglandin E 2 production over fifty-fold with the dose of 10 -7 M compared with control. EGF (10 -7 M) also stimulated it about ten-fold. The ED 50 values of PMA and EGF were respectively around 1 x 10 -9 M and 5 x 10 -10 M. Thyroid stimulating hormone (TSH), however, did not stimulate prostaglandin E 2 production from 1 to 24-h incubation. The release of radioactivity from [ 3 H]-arachidonic acid prelabeled cells was also stimulated by PMA and EGF, but not by TSH. These results indicate that both PMA and EGF are potent stimulators of prostaglandin E 2 production, associated with the activity to stimulate arachidonic acid release in porcine thyroid cells. 36 references, 2 figures, 1 table

The Anti-Inflammatory Effect of Algae-Derived Lipid Extracts on Lipopolysaccharide (LPS)-Stimulated Human THP-1 Macrophages.

Science.gov (United States)

Robertson, Ruairi C; Guihéneuf, Freddy; Bahar, Bojlul; Schmid, Matthias; Stengel, Dagmar B; Fitzgerald, Gerald F; Ross, R Paul; Stanton, Catherine

2015-08-20

Algae contain a number of anti-inflammatory bioactive compounds such as omega-3 polyunsaturated fatty acids (n-3 PUFA) and chlorophyll a, hence as dietary ingredients, their extracts may be effective in chronic inflammation-linked metabolic diseases such as cardiovascular disease. In this study, anti-inflammatory potential of lipid extracts from three red seaweeds (Porphyra dioica, Palmaria palmata and Chondrus crispus) and one microalga (Pavlova lutheri) were assessed in lipopolysaccharide (LPS)-stimulated human THP-1 macrophages. Extracts contained 34%-42% total fatty acids as n-3 PUFA and 5%-7% crude extract as pigments, including chlorophyll a, β-carotene and fucoxanthin. Pretreatment of the THP-1 cells with lipid extract from P. palmata inhibited production of the pro-inflammatory cytokines interleukin (IL)-6 (p lipid extracts. The lipid extracts effectively inhibited the LPS-induced pro-inflammatory signaling pathways mediated via toll-like receptors, chemokines and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling molecules. These results suggest that lipid extracts from P. lutheri, P. palmata, P. dioica and C. crispus can inhibit LPS-induced inflammatory pathways in human macrophages. Therefore, algal lipid extracts should be further explored as anti-inflammatory ingredients for chronic inflammation-linked metabolic diseases.

Anti-inflammatory effects of polyunsaturated fatty acids in THP-1 cells

International Nuclear Information System (INIS)

Zhao Guixiang; Etherton, Terry D.; Martin, Keith R.; Vanden Heuvel, John P.; Gillies, Peter J.; West, Sheila G.; Kris-Etherton, Penny M.

2005-01-01

The effects of linoleic acid (LA), α-linolenic acid (ALA), and docosahexaenoic acid (DHA) were compared to that of palmitic acid (PA), on inflammatory responses in human monocytic THP-1 cells. When cells were pre-incubated with fatty acids for 2-h and then stimulated with lipopolysaccharide for 24-h in the presence of fatty acids, secretion of interleukin (IL)-6, IL-1β, and tumor necrosis factor-α (TNFα) was significantly decreased after treatment with LA, ALA, and DHA versus PA (P 12,14 -prostaglandin J2 (15d-PGJ2) and were dose-dependent. In addition, LA, ALA, and DHA decreased IL-6, IL-1β, and TNFα gene expression (P < 0.05 for all) and nuclear factor (NF)-κB DNA-binding activity, whereas peroxisome proliferator-activated receptor-γ (PPARγ) DNA-binding activity was increased. The results indicate that the anti-inflammatory effects of polyunsaturated fatty acids may be, in part, due to the inhibition of NF-κB activation via activation of PPARγ

Association of RANTES with the replication of severe acute respiratory syndrome coronavirus in THP-1 cells.

Science.gov (United States)

Li, D; Wu, N; Yao, H; Bader, A; Brockmeyer, Norbert H; Altmeyer, P

2005-03-29

Severe acute respiratory syndrome (SARS) is a novel infectious disease which is characterized by an overaggressive immune response. Chemokines are important inflammatory mediators and regulate disease due to viral infection. In previous study, we found that SARS-CoV has the ability to replicate in mononuclear cells. In present work, we sought to characterize the replication of SARS-CoV at the presence of RANTES in THP-1 cells. To determine whether RANTES play an role in the process of SARS, THP-1 cells were incubated with heat-inactivated SARS-CoV and ELISA was used to test RANTES levels in the supernatants; Then the effect of dexamethasone on the induced secretion was evaluated. Real-time PCR was used to investigate the effort of RANTES on the replication of SARS-CoV in vitro. Macrophages, induced by THP-1 cells, were used as cell model. Inactive SARS-CoV could induce THP-1 cells secret RANTES and this increase effect could not be suppressed by DXM. RANTES itself could inhibit the replication of SARS-CoV in THP-1 cells when it was added into the culture before or at the same time with the virus; No inhibition effect was shown when RANTES were added into the culture after SARS-CoV infected the cells.

Hemocompatibility and biocompatibility of antibacterial biomimetic hybrid films

Energy Technology Data Exchange (ETDEWEB)

Coll Ferrer, M. Carme [Department of Anesthesiology and Critical Care, University of Pennsylvania, Philadelphia, PA 19104 (United States); Department of Materials Science and Engineering, University of Pennsylvania, Philadelphia, PA 19104 (United States); Eckmann, Uriel N. [Department of Anesthesiology and Critical Care, University of Pennsylvania, Philadelphia, PA 19104 (United States); Composto, Russell J. [Department of Materials Science and Engineering, University of Pennsylvania, Philadelphia, PA 19104 (United States); Eckmann, David M., E-mail: eckmanndm@uphs.upenn.edu [Department of Anesthesiology and Critical Care, University of Pennsylvania, Philadelphia, PA 19104 (United States)

2013-11-01

In previous work, we developed novel antibacterial hybrid coatings based on dextran containing dispersed Ag NPs (∼ 5 nm, DEX-Ag) aimed to offer dual protection against two of the most common complications associated with implant surgery, infections and rejection of the implant. However, their blood-material interactions are unknown. In this study, we assess the hemocompatibility and biocompatibility of DEX-Ag using fresh blood and two cell lines of the immune system, monocytes (THP-1 cells) and macrophages (PMA-stimulated THP-1 cells). Glass, polyurethane (PU) and bare dextran (DEX) were used as reference surfaces. PU, DEX and DEX-Ag exhibited non-hemolytic properties. Relative to glass (100%), platelet attachment on PU, DEX and DEX-Ag was 15%, 10% and 34%, respectively. Further, we assessed cell morphology and viability, pro-inflammatory cytokines expression (TNF-α and IL-1β), pro-inflammatory eicosanoid expression (Prostaglandin E{sub 2}, PGE{sub 2}) and release of reactive oxygen species (ROS, superoxide and H{sub 2}O{sub 2}) following incubation of the cells with the surfaces. The morphology and cell viability of THP-1 cells were not affected by DEX-Ag whereas DEX-Ag minimized spreading of PMA-stimulated THP-1 cells and caused a reduction in cell viability (16% relative to other surfaces). Although DEX-Ag slightly enhanced release of ROS, the expression of pro-inflammatory cytokines remained minimal with similar levels of PGE{sub 2}, as compared to the other surfaces studied. These results highlight low toxicity of DEX-Ag and hold promise for future applications in vivo. - Highlights: • We examined specific blood-contact reactions of dextran doped with Ag NPs coatings. • Biocompatibility was assessed with THP-1 cells and PMA-stimulated THP-1 cells. • Glass, polyurethane and dextran were used as reference surfaces. • Hybrid coatings exhibited non-hemolytic properties. • Low toxicity, inflammatory response and ROS suggest potential for in vivo use.

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

Science.gov (United States)

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

2011-08-01

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

Nanomechanical measurement of adhesion and migration of leukemia cells with phorbol 12-myristate 13-acetate treatment.

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Zhou, Zhuo Long; Ma, Jing; Tong, Ming-Hui; Chan, Barbara Pui; Wong, Alice Sze Tsai; Ngan, Alfonso Hing Wan

The adhesion and traction behavior of leukemia cells in their microenvironment is directly linked to their migration, which is a prime issue affecting the release of cancer cells from the bone marrow and hence metastasis. In assessing the effectiveness of phorbol 12-myristate 13-acetate (PMA) treatment, the conventional batch-cell transwell-migration assay may not indicate the intrinsic effect of the treatment on migration, since the treatment may also affect other cellular behavior, such as proliferation or death. In this study, the pN-level adhesion and traction forces between single leukemia cells and their microenvironment were directly measured using optical tweezers and traction-force microscopy. The effects of PMA on K562 and THP1 leukemia cells were studied, and the results showed that PMA treatment significantly increased cell adhesion with extracellular matrix proteins, bone marrow stromal cells, and human fibroblasts. PMA treatment also significantly increased the traction of THP1 cells on bovine serum albumin proteins, although the effect on K562 cells was insignificant. Western blots showed an increased expression of E-cadherin and vimentin proteins after the leukemia cells were treated with PMA. The study suggests that PMA upregulates adhesion and thus suppresses the migration of both K562 and THP1 cells in their microenvironment. The ability of optical tweezers and traction-force microscopy to measure directly pN-level cell-protein or cell-cell contact was also demonstrated.

A20 is critical for the induction of Pam3CSK4-tolerance in monocytic THP-1 cells.

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Jinyue Hu

Full Text Available A20 functions to terminate Toll-like receptor (TLR-induced immune response, and play important roles in the induction of lipopolysacchride (LPS-tolerance. However, the molecular mechanism for Pam3CSK4-tolerance is uncertain. Here we report that TLR1/2 ligand Pam3CSK4 induced tolerance in monocytic THP-1 cells. The pre-treatment of THP-1 cells with Pam3CSK4 down-regulated the induction of pro-inflammatory cytokines induced by Pam3CSK4 re-stimulation. Pam3CSK4 pre-treatment also down-regulated the signaling transduction of JNK, p38 and NF-κB induced by Pam3CSK4 re-stimulation. The activation of TLR1/2 induced a rapid and robust up-regulation of A20, suggesting that A20 may contribute to the induction of Pam3CSK4-tolerance. This hypothesis was proved by the observation that the over-expression of A20 by gene transfer down-regulated Pam3CSK4-induced inflammatory responses, and the down-regulation of A20 by RNA interference inhibited the induction of tolerance. Moreover, LPS induced a significant up-regulation of A20, which contributed to the induction of cross-tolerance between LPS and Pam3CSK4. A20 was also induced by the treatment of THP-1 cells with TNF-α and IL-1β. The pre-treatment with TNF-α and IL-1β partly down-regulated Pam3CSK4-induced activation of MAPKs. Furthermore, pharmacologic inhibition of GSK3 signaling down-regulated Pam3CSK4-induced A20 expression, up-regulated Pam3CSK4-induced inflammatory responses, and partly reversed Pam3CSK4 pre-treatment-induced tolerance, suggesting that GSK3 is involved in TLR1/2-induced tolerance by up-regulation of A20 expression. Taken together, these results indicated that A20 is a critical regulator for TLR1/2-induced pro-inflammatory responses.

Nanotubo de carbono-chitosan en células HOS y THP-1 Carbon nanotubes-chitosan in HOS and THP-1 cells

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John Jairo Castillo León

2011-04-01

Full Text Available Introducción: Los nanotubos de carbono (NTC son estructuras nanométricas utilizadas en el tratamiento de enfermedades, principalmente en la entrega de fármacos para terapias en cáncer. Objetivos: Estudiar la internalización de NTC acoplado a quitosan (NTC-Q en células de osteosarcoma humano (HOS y monocitos humanos de leucemia aguda (THP-1. Materiales y métodos: Los NTC solubilizados con quitosan 30% fueron caracterizados espectroscópicamente por UV-Vis, fluorescencia y Raman. Las células HOS y THP-1 fueron tratadas con NTC-Q y se evaluó la internalización por tinción de Giemsa en microscopio de luz y la citotóxicidad utilizando la prueba fluorométrica de Azul de Alamar. Resultados: Los espectros Raman y de fluorescencia mostraron la funcionalización de los NTC con quitosan. Los NTC fueron internalizados por las líneas celulares después de 24 h mostrando una ubicación citoplasmática sin presentar citotóxicidad en ninguna de las células evaluadas. Discusión: Las características presentadas por los NTC-Q les brinda la posibilidad de ser utilizados como transportadores de fármacos. Salud UIS 2011; 43(1: 21-26Introduction: Carbon nanotubes (CNT are nanometer-sized structures used in medicine in the treatment of diseases, mainly in drug delivery in therapies against cancer. Objectives: To Study the internalization of carbon nanotubes modified with chitosan (CNT-CH in human osteosarcom cells (HOS and human monocytes of acute leukemia (THP1. Materials and methods: The CNTs solubilized in chitosan 30% were characterized spectroscopically by UV-Vis, fluorescence and Raman. HOS cells and THP-1 were treated with CNT-CH, the internalization was evaluated by Giemsa staining with light microscopy, and cytotoxicity was determined using Alamar Blue assay. Results: Raman and fluorescence spectra showed the functionalization of the CNT with chitosan. After 24 h the NTC were internalized in the cell lines showing a cytoplasmic location and

Immunomodulatory/inflammatory effects of geopropolis produced by Melipona fasciculata Smith in combination with doxorubicin on THP-1 cells.

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Oliveira, Lucas Pires Garcia; Conte, Fernanda Lopes; Cardoso, Eliza de Oliveira; Conti, Bruno José; Santiago, Karina Basso; Golim, Marjorie de Assis; Cruz, Maria Teresa; Sforcin, José Maurício

2016-12-01

Geopropolis (GEO) in combination with doxorubicin (DOX) reduced HEp-2 cells viability compared to GEO and DOX alone. A possible effect of this combination on the innate immunity could take place, and its effects were analysed on THP-1 cell - a human leukaemia monocytic cell line used as a model to study monocyte activity and macrophage activity, assessing cell viability, expression of cell markers and cytokine production. THP-1 cells were incubated with GEO, DOX and their combination. Cell viability was assessed by MTT assay, cell markers expression by flow cytometry and cytokine production by ELISA. GEO + DOX did not affect cell viability. GEO alone or in combination increased TLR-4 and CD80 but not HLA-DR and TLR-2 expression. GEO stimulated TNF-α production while DOX alone or in combination did not affect it. GEO alone or in combination inhibited IL-6 production. GEO exerted a pro-inflammatory profile by increasing TLR-4 and CD80 expression and TNF-α production, favouring the activation of the immune/inflammatory response. GEO + DOX did not affect cell viability and presented an immunomodulatory action. Lower concentrations of DOX combined to GEO could be used in cancer patients, avoiding side effects and benefiting from the biological properties of GEO. © 2016 Royal Pharmaceutical Society.

A Study of the Differential Effects of Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) on Gene Expression Profiles of Stimulated Thp-1 Macrophages.

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Allam-Ndoul, Bénédicte; Guénard, Frédéric; Barbier, Olivier; Vohl, Marie-Claude

2017-04-25

Background: An appropriate intake of omega-3 ( n -3) fatty acids (FAs) such as eicosapentaenoic and docosahexaenoic acid (EPA/DHA) from marine sources is known to have anti-inflammatory effects. However, molecular mechanisms underlying their beneficial effects on health are not fully understood. The aim of the present study was to characterize gene expression profiles of THP-1 macrophages, incubated in either EPA or DHA and stimulated with lipopolysaccharide (LPS), a pro-inflammatory agent. Methods: THP-1 macrophages were incubated into 10, 50 and 75 µM of EPA or DHA for 24 h, and 100 nM of LPS was added to the culture media for 18 h. Total mRNA was extracted and gene expression examined by microarray analysis using Illumina Human HT-12 expression beadchips (Illumina). Results: Pathway analysis revealed that EPA and DHA regulate genes involved in cell cycle regulation, apoptosis, immune response and inflammation, oxidative stress and cancer pathways in a differential and dose-dependent manner. Conclusions: EPA and DHA appear to exert differential effects on gene expression in THP-1 macrophages. Specific effects of n -3 FAs on gene expression levels are also dose-dependent.

Activation of CD147 with Cyclophilin A Induces the Expression of IFITM1 through ERK and PI3K in THP-1 Cells

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Ju-Young Kim

2010-01-01

Full Text Available CD147, as a receptor for Cyclophilins, is a multifunctional transmembrane glycoprotein. In order to identify genes that are induced by activation of CD147, THP-1 cells were stimulated with Cyclophilin A and differentially expressed genes were detected using PCR-based analysis. Interferon-induced transmembrane 1 (IFITM1 was detected to be induced and it was confirmed by RT-PCR and Western blot analysis. CD147-induced expression of IFITM1 was blocked by inhibitors of ERK, PI3K, or NF-κB, but not by inhibitors of p38, JNK, or PKC. IFITM1 appears to mediate inflammatory activation of THP-1 cells since cross-linking of IFITM1 with specific monoclonal antibody against it induced the expression of proinflammatory mediators such as IL-8 and MMP-9. These data indicate that IFITM1 is one of the pro-inflammatory mediators that are induced by signaling initiated by the activation of CD147 in macrophages and activation of ERK, PI3K, and NF-κB is required for the expression of IFITM1.

Cu/Zn superoxide dismutase and the proton ATPase Pma1p of Saccharomyces cerevisiae

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Baron, J. Allen; Chen, Janice S.; Culotta, Valeria C., E-mail: vculott1@jhu.edu

2015-07-03

In eukaryotes, the Cu/Zn containing superoxide dismutase (SOD1) plays a critical role in oxidative stress protection as well as in signaling. We recently demonstrated a function for Saccharomyces cerevisiae Sod1p in signaling through CK1γ casein kinases and identified the essential proton ATPase Pma1p as one likely target. The connection between Sod1p and Pma1p was explored further by testing the impact of sod1Δ mutations on cells expressing mutant alleles of Pma1p that alter activity and/or post-translational regulation of this ATPase. We report here that sod1Δ mutations are lethal when combined with the T912D allele of Pma1p in the C-terminal regulatory domain. This “synthetic lethality” was reversed by intragenic suppressor mutations in Pma1p, including an A906G substitution that lies within the C-terminal regulatory domain and hyper-activates Pma1p. Surprisingly the effect of sod1Δ mutations on Pma1-T912D is not mediated through the Sod1p signaling pathway involving the CK1γ casein kinases. Rather, Sod1p sustains life of cells expressing Pma1-T912D through oxidative stress protection. The synthetic lethality of sod1Δ Pma1-T912D cells is suppressed by growing cells under low oxygen conditions or by treatments with manganese-based antioxidants. We now propose a model in which Sod1p maximizes Pma1p activity in two ways: one involving signaling through CK1γ casein kinases and an independent role for Sod1p in oxidative stress protection. - Highlights: • In yeast, the anti-oxidant enzyme SOD1 promotes activity of the proton ATPase Pma1p. • Cells expressing a T912D variant of Pma1p are not viable without SOD1. • SOD1 is needed to protect Pma1-T912D expressing cells from severe oxidative damage. • SOD1 activates Pma1p through casein kinase signaling and oxidative stress protection.

Cu/Zn superoxide dismutase and the proton ATPase Pma1p of Saccharomyces cerevisiae

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Baron, J. Allen; Chen, Janice S.; Culotta, Valeria C.

2015-01-01

In eukaryotes, the Cu/Zn containing superoxide dismutase (SOD1) plays a critical role in oxidative stress protection as well as in signaling. We recently demonstrated a function for Saccharomyces cerevisiae Sod1p in signaling through CK1γ casein kinases and identified the essential proton ATPase Pma1p as one likely target. The connection between Sod1p and Pma1p was explored further by testing the impact of sod1Δ mutations on cells expressing mutant alleles of Pma1p that alter activity and/or post-translational regulation of this ATPase. We report here that sod1Δ mutations are lethal when combined with the T912D allele of Pma1p in the C-terminal regulatory domain. This “synthetic lethality” was reversed by intragenic suppressor mutations in Pma1p, including an A906G substitution that lies within the C-terminal regulatory domain and hyper-activates Pma1p. Surprisingly the effect of sod1Δ mutations on Pma1-T912D is not mediated through the Sod1p signaling pathway involving the CK1γ casein kinases. Rather, Sod1p sustains life of cells expressing Pma1-T912D through oxidative stress protection. The synthetic lethality of sod1Δ Pma1-T912D cells is suppressed by growing cells under low oxygen conditions or by treatments with manganese-based antioxidants. We now propose a model in which Sod1p maximizes Pma1p activity in two ways: one involving signaling through CK1γ casein kinases and an independent role for Sod1p in oxidative stress protection. - Highlights: • In yeast, the anti-oxidant enzyme SOD1 promotes activity of the proton ATPase Pma1p. • Cells expressing a T912D variant of Pma1p are not viable without SOD1. • SOD1 is needed to protect Pma1-T912D expressing cells from severe oxidative damage. • SOD1 activates Pma1p through casein kinase signaling and oxidative stress protection

Neopterin negatively regulates expression of ABCA1 and ABCG1 by the LXRα signaling pathway in THP-1 macrophage-derived foam cells.

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Yan, Jin-quan; Tan, Chun-zhi; Wu, Jin-hua; Zhang, Dong-cui; Chen, Ji-ling; Zeng, Bin-yuan; Jiang, Yu-ping; Nie, Jin; Liu, Wei; Liu, Qin; Dai, Hao

2013-07-01

To investigate the effects of neopterin on ABCA1 expression and cholesterol efflux in human THP-1 macrophage-derived foam cells, and to explore the role of the liver X receptor alpha (LXRα) involved. In the present study, THP-1 cells were pre-incubated with ox-LDL to become foam cells. The protein and mRNA expression were examined by Western blot assays and real-time quantitative PCR, respectively. Liquid scintillation counting and high performance liquid chromatography assays were used to test cellular cholesterol efflux and cholesterol content. Neopterin decreased ABCA1 expression and cholesterol efflux in a time- and concentration-dependent manner in THP-1 macrophage-derived foam cells, and the LXRα siRNA can reverse the inhibitory effects induced by neopterin. Neoterin has a negative regulation on ABCA1 expression via the LXRα signaling pathway, which suggests the aggravated effects of neopterin on atherosclerosis.

Mixed metal oxide nanoparticles inhibit growth of Mycobacterium tuberculosis into THP-1 cells.

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Jafari, A R; Mosavi, T; Mosavari, N; Majid, A; Movahedzade, F; Tebyaniyan, M; Kamalzadeh, M; Dehgan, M; Jafari, S; Arastoo, S

2016-12-01

Humans have been in a constant battle with tuberculosis (TB). Currently, overuse of antibiotics has resulted in the spread of multidrug-resistant Mycobacterium tuberculosis (MDR), leading to antibiotic ineffectiveness at controlling the spread of TB infection in host cells and especially macrophages. Additionally, the Mycobacterium tuberculosis (Mtb) has developed methods to evade the immune system and survive. With the discovery of nanoparticle (NP)-based drugs, it is necessary to research their anti-mycobacterial properties and bactericidal mechanisms. In this study, we synthesized mixed metal oxide NPs and tested their ability to inhibit Mtb growth into macrophages and investigated the cytotoxic effects of NPs in THP-1 cells. Silver (Ag) NPs and zinc oxide (ZnO) NPs were synthesized by chemical reduction and chemical deposition in aqueous solution, and the diffraction light scattering, scanning electron microscopy, transmission electron microscopy, and ultraviolet-visible light-absorption spectra were used to identify NP properties. Ag and ZnO NPs were mixed together at a ratio of 8 ZnO /2 Ag and diluted into Löwenstein-Jensen medium followed by the addition of bacteria and incubation for 28days at 37°C. The toxicity of NPs to THP-1 cells was assessed by MTT test, and macrophages were infected with Mtb for 4h at 37°C under 5% CO 2 . Nano-sized particles were estimated at ∼30-80nm, and the initial concentration of Ag NPs and ZnO NPs were estimated at ∼20ppm and ∼60ppm. The minimal inhibitory concentration ratio of 8 ZnO /2 Ag NPs against Mtb was detected at ∼1/32 of the initial concentration. Ag NPs in the range of concentrations exhibited no anti-Mtb effects, whereas ZnO NPs showed potent antibacterial activity at ∼1/128 of the initial concentration. ZnO NPs at all concentrations showed cytotoxic activity, whereas 100% of THP-1 cells remained viable in the presence of Ag NPs at ∼1/32 and ∼1/64 of the initial concentrations. However, at ratios of

Serum lipoproteins attenuate macrophage activation and Toll-Like Receptor stimulation by bacterial lipoproteins

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James Richard W

2010-09-01

Full Text Available Abstract Background Chlamydia trachomatis was previously shown to express a lipoprotein, the macrophage infectivity potentiator (Mip, exposed at the bacterial surface, and able to stimulate human primary monocytes/macrophages through Toll Like Receptor (TLR2/TLR1/TLR6, and CD14. In PMA-differentiated THP-1 cells the proinflammatory activity of Mip was significantly higher in the absence than in the presence of serum. The present study aims to investigate the ability of different serum factors to attenuate Mip proinflammatory activity in PMA-differentiated THP-1 cells and in primary human differentiated macrophages. The study was also extend to another lipoprotein, the Borrelia burgdorferi outer surface protein (OspA. The proinflammatory activity was studied through Tumor Necrosis Factor alpha (TNF-α and Interleukin (IL-8 release. Finally, TLR1/2 human embryonic kidney-293 (HEK-293 transfected cells were used to test the ability of the serum factors to inhibit Mip and OspA proinflammatory activity. Results In the absence of any serum and in the presence of 10% delipidated FBS, production of Mip-induced TNF-α and IL-8 in PMA-differentiated THP-1 cells were similar whereas they were significantly decreased in the presence of 10% FBS suggesting an inhibiting role of lipids present in FBS. In the presence of 10% human serum, the concentrations of TNF-α and IL-8 were 2 to 5 times lower than in the presence of 10% FBS suggesting the presence of more potent inhibitor(s in human serum than in FBS. Similar results were obtained in primary human differentiated macrophages. Different lipid components of human serum were then tested (total lipoproteins, HDL, LDL, VLDL, triglyceride emulsion, apolipoprotein (apoA-I, B, E2, and E3. The most efficient inhibitors were LDL, VLDL, and apoB that reduced the mean concentration of TNF-α release in Mip-induced macrophages to 24, 20, and 2%, respectively (p Conclusions These results demonstrated the ability of

Hydroxyoctadecadienoic acids regulate apoptosis in human THP-1 cells in a PPARγ-dependent manner.

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Vangaveti, Venkat N; Shashidhar, Venkatesh M; Rush, Catherine; Malabu, Usman H; Rasalam, Roy R; Collier, Fiona; Baune, Bernhard T; Kennedy, Richard L

2014-12-01

Macrophage apoptosis, a key process in atherogenesis, is regulated by oxidation products, including hydroxyoctadecadienoic acids (HODEs). These stable oxidation products of linoleic acid (LA) are abundant in atherosclerotic plaque and activate PPARγ and GPR132. We investigated the mechanisms through which HODEs regulate apoptosis. The effect of HODEs on THP-1 monocytes and adherent THP-1 cells were compared with other C18 fatty acids, LA and α-linolenic acid (ALA). The number of cells was reduced within 24 hours following treatment with 9-HODE (p labelling of cells (p blocked by the caspase inhibitor DEVD-CHO. The PPARγ antagonist T0070907 further increased apoptosis, suggestive of the PPARγ-regulated apoptotic effects induced by 9-HODE. The use of siRNA for GPR132 showed no evidence that the effect of HODEs was mediated through this receptor. 9-HODE and 13-HODE are potent--and specific--regulators of apoptosis in THP-1 cells. Their action is PPARγ-dependent and independent of GPR132. Further studies to identify the signalling pathways through which HODEs increase apoptosis in macrophages may reveal novel therapeutic targets for atherosclerosis.

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

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

2017-10-01

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

RasGRP1 confers the phorbol ester-sensitive phenotype to EL4 lymphoma cells.

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Han, Shujie; Knoepp, Stewart M; Hallman, Mark A; Meier, Kathryn E

2007-01-01

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to the tumor promoter phorbol 12-myristate 13-acetate (PMA). In sensitive EL4 cells, PMA causes robust Erk mitogen-activated protein kinase activation that results in growth arrest. In resistant cells, PMA induces minimal Erk activation, without growth arrest. PMA stimulates IL-2 production in sensitive, but not resistant, cells. The role of RasGRP1, a PMA-activated guanine nucleotide exchange factor for Ras, in EL4 phenotype was examined. Endogenous RasGRP1 protein is expressed at much higher levels in sensitive than in resistant cells. PMA-induced Ras activation is observed in sensitive cells but not in resistant cells lacking Ras-GRP1. PMA induces down-regulation of RasGRP1 protein in sensitive cells but increases RasGRP1 in resistant cells. Transfection of RasGRP1 into resistant cells enhances PMA-induced Erk activation. In the reverse experiment, introduction of small interfering RNA (siRNA) for RasGRP1 suppresses PMA-induced Ras and Erk activations in sensitive cells. Sensitive cells incubated with siRNA for RasGRP1 exhibit the PMA-resistant phenotype, in that they are able to proliferate in the presence of PMA and do not secrete IL-2 when stimulated with PMA. These studies indicate that the PMA-sensitive phenotype, as previously defined for the EL4 cell line, is conferred by endogenous expression of RasGRP1 protein.

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

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Ya. S. Onokhina

2013-01-01

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

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

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Li, Xiang; Liu, Sheng; Luo, Jingjing; Liu, Anyuan; Tang, Shuangyang; Liu, Shuo; Yu, Minjun; Zhang, Yan

2015-06-01

This study investigated whether Helicobacter pylori could activate the nucleotide-binding oligomerization domain-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome in human macrophages and the involvement of reactive oxygen species (ROS) in inflammasome activation. Phorbol-12-myristate-13-acetate (PMA)-differentiated human acute monocytic leukemia cell line THP-1 was infected with H. pylori. The levels of pro-inflammatory cytokines interleukin (IL)-1β and IL-18 in supernatant were measured by ELISA. Intracellular ROS level was analyzed by flow cytometry. Quantitative real-time PCR and western blot analysis were employed to determine the mRNA and protein expression levels of NLRP3 and caspase-1 in THP-1 cells, respectively. Our results showed that H. pylori infection could induce IL-1β and IL-18 production in PMA-differentiated THP-1 cells in a dose- and time-dependent manner. Moreover, secretion of IL-1β and IL-18 in THP-1 cells following H. pylori infection was remarkably reduced by NLRP3-specific small interfering RNA treatment. In addition, the intracellular ROS level was elevated by H. pylori infection, which could be eliminated by the ROS scavenger N-acetylcysteine (NAC). Furthermore, NAC treatment could inhibit NLRP3 inflammasome formation and caspase-1 activation and suppress the release of IL-1β and IL-18 from H. pylori-infected THP-1 cells. These findings provide novel insights into the innate immune response against H. pylori infection, which could potentially be used for the prevention and treatment of H. pylori-related diseases. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The yeast H+-ATPase Pma1 promotes Rag/Gtr-dependent TORC1 activation in response to H+-coupled nutrient uptake.

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Saliba, Elie; Evangelinos, Minoas; Gournas, Christos; Corrillon, Florent; Georis, Isabelle; André, Bruno

2018-03-23

The yeast Target of Rapamycin Complex 1 (TORC1) plays a central role in controlling growth. How amino acids and other nutrients stimulate its activity via the Rag/Gtr GTPases remains poorly understood. We here report that the signal triggering Rag/Gtr-dependent TORC1 activation upon amino-acid uptake is the coupled H + influx catalyzed by amino-acid/H + symporters. H + -dependent uptake of other nutrients, ionophore-mediated H + diffusion, and inhibition of the vacuolar V-ATPase also activate TORC1. As the increase in cytosolic H + elicited by these processes stimulates the compensating H + -export activity of the plasma membrane H + -ATPase (Pma1), we have examined whether this major ATP-consuming enzyme might be involved in TORC1 control. We find that when the endogenous Pma1 is replaced with a plant H + -ATPase, H + influx or increase fails to activate TORC1. Our results show that H + influx coupled to nutrient uptake stimulates TORC1 activity and that Pma1 is a key actor in this mechanism. © 2018, Saliba et al.

The stimulation of EL-4 cells to produce interleukin-2 and its potential use in immunocytotoxicity testing

International Nuclear Information System (INIS)

Lasek, W.; Steer, S.; Clothier, R.; Balls, M.

1989-01-01

The ability of EL-4 thymoma cells to produce interleukin-2 (IL-2) following exposure to phorbol-12-myristate 13-acetate (PMA) and Concanavalin A (Con A) has been studied in vitro using medium containing either 10% or 1% fetal calf serum (FCS). The potent stimulatory effect of PMA on IL-2 production by EL-4 cells has been confirmed by measuring 3H-thymidine incorporation by the IL-2-dependent T cell line, CTLL-2, in the presence of conditioned medium (CM) from stimulated cultures. EL-4 cells produced several times more IL-2 when cultured in medium containing 10% FCS than when only 1% FCS was present. Added together, PMA and Con A acted synergistically in some EL-4 cell cultures. The ability of E:-4 cells to produce IL-2 was maintained after further incubation without stimulants. CM with IL-2 activity from stimulated EL-4 cells could prove useful in immunotoxicity testing

Involvement of Resveratrol and ω-3 Polyunsaturated Fatty Acids on Sirtuin 1 Gene Expression in THP1 Cells.

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Tsuchiya, Takafumi; Endo, Ayano; Tsujikado, Kyoko; Inukai, Toshihiko

2017-10-01

Resveratrol, a kind of polyphenol, has the potential to activate the longevity gene in several cells, in the same manner as calorie restriction. We investigated the effect of resveratrol and ω-3-line polyunsaturated fatty acid on surtuin 1 (SIRT1) gene expression in human monocytes (THP1) cells. We examined the gene expression of THP1 cells using real-time polymerase chain reaction and Western blotting analysis. Resveratol, eicosapentaenoic acid (EPA) and docosahexaeanoic acid (DHA) as n-3 polyunsaturated fatty acid were added on THP1 cells. We observed the changes in the SIRT1 gene expression in those cells, under various doses of agents and in time courses. Then, we examined the interaction of glucose and mannitol on those agents׳ effect of the gene expression. The concentration range of glucose and mannitol was from 5-20mM, respectively. The SIRT1 gene expression could be defined in 24 and 48 hours both in real-time polymerase chain reaction analysis and in Western blotting. Resveratrol showed SIRT1 gene expression in a dose-dependent manner in the range of 0-20μM in both analyses. Although EPA at 10μM showed marked increase in SIRT1 gene expression compared to control condition in Western blotting, this phenomenon was not in dose-dependent manner. DHA did not exhibit any augmentation of SIRT1 gene expression in a dose-dependent manner in the range of 0-20μM in both analyses. We refined the dose-dependent inhibition of the SIRT1 gene expression within 20mM glucose medium. Although 20mM did not exhibit any inhibition, 10μM resveratrol induced the gene expression compared to control medium. Both 5 and 15mM mannitol medium did not significantly alter basic gene expression and 10μM resveratrol-induced gene expression. The present results suggest that resveratrol and EPA, but not DHA, markedly activated the SIRT1 gene expression in THP1 cells, and that high glucose medium could inhibit the basic gene expression, but not powerful resveratrol-induced gene

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

Science.gov (United States)

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

2009-02-01

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

SIRT1 inactivation induces inflammation through the dysregulation of autophagy in human THP-1 cells

International Nuclear Information System (INIS)

Takeda-Watanabe, Ai; Kitada, Munehiro; Kanasaki, Keizo; Koya, Daisuke

2012-01-01

Highlights: ► SIRT1 inactivation decreases autophagy in THP-1 cell. ► Inhibition of autophagy induces inflammation. ► SIRT1 inactivation induces inflammation through NF-κB activation. ► The p62/Sqstm1 accumulation by impairment of autophagy is related to NF-κB activation. ► SIRT1 inactivation is involved in the activation of mTOR and decreased AMPK activation. -- Abstract: Inflammation plays a crucial role in atherosclerosis. Monocytes/macrophages are some of the cells involved in the inflammatory process in atherogenesis. Autophagy exerts a protective effect against cellular stresses like inflammation, and it is regulated by nutrient-sensing pathways. The nutrient-sensing pathway includes SIRT1, a NAD + -dependent histone deacetylase, which is implicated in the regulation of a variety of cellular processes including inflammation and autophagy. The mechanism through which the dysfunction of SIRT1 contributes to the regulation of inflammation in relation to autophagy in monocytes/macrophages is unclear. In the present study, we demonstrate that treatment with 2-[(2-Hydroxynaphthalen-1-ylmethylene)amino]-N-(1-phenethyl)benzamide (Sirtinol), a chemical inhibitor of SIRT1, induces the overexpression of inflammation-related genes such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 through nuclear factor (NF)-κB signaling activation, which is associated with autophagy dysfunction, as shown through p62/Sqstm1 accumulation and decreased expression of light chain (LC) 3 II in THP-1 cells. The autophagy inhibitor, 3-methyladenine, also induces inflammation-related NF-κB activation. In p62/Sqstm1 knockdown cells, Sirtinol-induced inflammation through NF-κB activation is blocked. In addition, inhibition of SIRT1 is involved in the activation of the mammalian target of rapamycin (mTOR) pathway and is implicated in decreased 5′-AMP activated kinase (AMPK) activation, leading to the impairment of autophagy. The mTOR inhibitor, rapamycin, abolishes

SIRT1 inactivation induces inflammation through the dysregulation of autophagy in human THP-1 cells

Energy Technology Data Exchange (ETDEWEB)

Takeda-Watanabe, Ai; Kitada, Munehiro; Kanasaki, Keizo [Diabetology and Endocrinology, Kanazawa Medical University, Kahoku-Gun, Ishikawa (Japan); Koya, Daisuke, E-mail: koya0516@kanazawa-med.ac.jp [Diabetology and Endocrinology, Kanazawa Medical University, Kahoku-Gun, Ishikawa (Japan)

2012-10-12

Highlights: Black-Right-Pointing-Pointer SIRT1 inactivation decreases autophagy in THP-1 cell. Black-Right-Pointing-Pointer Inhibition of autophagy induces inflammation. Black-Right-Pointing-Pointer SIRT1 inactivation induces inflammation through NF-{kappa}B activation. Black-Right-Pointing-Pointer The p62/Sqstm1 accumulation by impairment of autophagy is related to NF-{kappa}B activation. Black-Right-Pointing-Pointer SIRT1 inactivation is involved in the activation of mTOR and decreased AMPK activation. -- Abstract: Inflammation plays a crucial role in atherosclerosis. Monocytes/macrophages are some of the cells involved in the inflammatory process in atherogenesis. Autophagy exerts a protective effect against cellular stresses like inflammation, and it is regulated by nutrient-sensing pathways. The nutrient-sensing pathway includes SIRT1, a NAD{sup +}-dependent histone deacetylase, which is implicated in the regulation of a variety of cellular processes including inflammation and autophagy. The mechanism through which the dysfunction of SIRT1 contributes to the regulation of inflammation in relation to autophagy in monocytes/macrophages is unclear. In the present study, we demonstrate that treatment with 2-[(2-Hydroxynaphthalen-1-ylmethylene)amino]-N-(1-phenethyl)benzamide (Sirtinol), a chemical inhibitor of SIRT1, induces the overexpression of inflammation-related genes such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-6 through nuclear factor (NF)-{kappa}B signaling activation, which is associated with autophagy dysfunction, as shown through p62/Sqstm1 accumulation and decreased expression of light chain (LC) 3 II in THP-1 cells. The autophagy inhibitor, 3-methyladenine, also induces inflammation-related NF-{kappa}B activation. In p62/Sqstm1 knockdown cells, Sirtinol-induced inflammation through NF-{kappa}B activation is blocked. In addition, inhibition of SIRT1 is involved in the activation of the mammalian target of rapamycin (mTOR) pathway and

Toll-like receptor 4 is involved in the cell cycle modulation and required for effective human cytomegalovirus infection in THP-1 macrophages

Energy Technology Data Exchange (ETDEWEB)

Arcangeletti, Maria-Cristina, E-mail: mariacristina.arcangeletti@unipr.it [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy); Germini, Diego; Rodighiero, Isabella [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy); Mirandola, Prisco [Department of Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma (Italy); De Conto, Flora; Medici, Maria-Cristina [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy); Gatti, Rita [Department of Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma (Italy); Chezzi, Carlo; Calderaro, Adriana [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy)

2013-05-25

Suitable host cell metabolic conditions are fundamental for the effective development of the human cytomegalovirus (HCMV) lytic cycle. Indeed, several studies have demonstrated the ability of this virus to interfere with cell cycle regulation, mainly by blocking proliferating cells in G1 or G1/S. In the present study, we demonstrate that HCMV deregulates the cell cycle of THP-1 macrophages (a cell line irreversibly arrested in G0) by pushing them into S and G2 phases. Moreover, we show that HCMV infection of THP-1 macrophages leads to Toll-like receptor 4 (TLR4) activation. Since various studies have indicated TLR4 to be involved in promoting cell proliferation, here we investigate the possible role of TLR4 in the observed HCMV-induced cell cycle perturbation. Our data strongly support TLR4 as a mediator of HCMV-triggered cell cycle activation in THP-1 macrophages favouring, in turn, the development of an efficient viral lytic cycle. - Highlights: ► We studied HCMV infection impact on THP-1 macrophage cell cycle. ► We analysed the role played by Toll-like receptor (TLR) 4 upon HCMV infection. ► HCMV pushes THP-1 macrophages (i.e. resting cells) to re-enter the cell cycle. ► TLR4 pathway inhibition strongly affects the effectiveness of HCMV replication. ► TLR4 pathway inhibition significantly decreases HCMV-induced cell cycle re-entry.

Development of an in vitro skin sensitization test based on ROS production in THP-1 cells.

Science.gov (United States)

Saito, Kazutoshi; Miyazawa, Masaaki; Nukada, Yuko; Sakaguchi, Hitoshi; Nishiyama, Naohiro

2013-03-01

Recently, it has been reported that reactive oxygen species (ROS) produced by contact allergens can affect dendritic cell migration and contact hypersensitivity. The aim of the present study was to develop a new in vitro assay that could predict the skin sensitizing potential of chemicals by measuring ROS production in THP-1 (human monocytic leukemia cell line) cells. THP-1 cells were pre-loaded with a ROS sensitive fluorescent dye, 5-(and 6-)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), for 15min, then incubated with test chemicals for 30min. The fluorescence intensity was measured by flow cytometry. For the skin sensitizers, 25 out of 30 induced over a 2-fold ROS production at more than 90% of cell viability. In contrast, increases were only seen in 4 out of 20 non-sensitizers. The overall accuracy for the local lymph node assay (LLNA) was 82% for 50 chemicals tested. A correlation was found between the estimated concentration showing 2-fold ROS production in the ROS assay and the EC3 values (estimated concentration required to induce positive response) of the LLNA. These results indicated that the THP-1 cell-based ROS assay was a rapid and highly sensitive detection system able to predict skin sensitizing potentials and potency of chemicals. Copyright © 2012 Elsevier Ltd. All rights reserved.

Nuclear translocation of Nrf2 and expression of antioxidant defence genes in THP-1 cells exposed to carbon nanotubes.

Science.gov (United States)

Brown, David M; Donaldson, Kenneth; Stone, Vicki

2010-06-01

Carbon nanotubes have a wide range of applications in various industries and their use is likely to rise in the future. Currently, a major concern is that with the increasing use and production of these materials, there may be increased health risks to exposed workers. Long (> 15 microm) straight nanotubes may undergo frustrated phagocytosis which is likely to result in reduced clearance. We examine here the effects of multiwalled carbon nanotubes of different sizes on monocytic THP-1 cells, with regard to their ability to stimulate increased expression of the HO-1 and GST genes and their ability to produce nuclear translocation of the transcription factor, Nrf2, as well as the release of several pro-inflammatory cytokines and mediators of inflammation. Our results suggest that long (50 microm) carbon nanotubes (62.5 microg/ml for 4 hours) produce increased nuclear translocation of Nrf2 and increased HO-1 gene expression compared with shorter entangled nanotubes. There was no increased gene expression for GST. The long nanotubes (NT1) caused increased release of the proinflammatory cytokine IL-1beta, an effect which was diminished by the antioxidant trolox, suggesting a role of oxidative stress in the upregulation of this cytokine. Tentatively, our study suggests that long carbon nanotubes may exert their effect in THP-1 cells in part via an oxidative stress mechanism.

Secretion of Galectin-9 as a DAMP during Dengue Virus Infection in THP-1 Cells.

Science.gov (United States)

Dapat, Isolde C; Pascapurnama, Dyshelly Nurkartika; Iwasaki, Hiroko; Labayo, Hannah Karen; Chagan-Yasutan, Haorile; Egawa, Shinichi; Hattori, Toshio

2017-07-28

Damage-associated molecular patterns (DAMPs) are endogenous cellular molecules released to the extracellular environment in response to stress conditions such as virus infection. Galectins are β-galactoside-binding proteins that are widely expressed in cells and tissues of the immune system, are localized in the cell cytoplasm, and have roles in inflammatory responses and immune responses against infection. Elevated levels of galectin-9 (Gal-9) in natural human infections have been documented in numerous reports. To investigate the effect of dengue virus (DENV) infection on expression of endogenous Gal-9, monocytic THP-1 cells were infected with varying doses of DENV-3 (multiplicity of infection (MOI) 0.01, 0.03 and 0.1) and incubated at varying time points (Day 1, Day 2, Day 3). Results showed augmentation of Gal-9 levels in the supernatant, reduction of Gal-9 levels in the cells and decreased expression of LGALS9 mRNA, while DENV-3 mRNA copies for all three doses remained stable through time. Dengue virus induced the secretion of Gal-9 as a danger response; in turn, Gal-9 and other inflammatory factors, and stimulated effector responses may have limited further viral replication. The results in this pilot experiment add to the evidence of Gal-9 as a potential DAMP.

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

Energy Technology Data Exchange (ETDEWEB)

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

2017-04-01

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

Oleoylethanolamide exerts anti-inflammatory effects on LPS-induced THP-1 cells by enhancing PPARα signaling and inhibiting the NF-κB and ERK1/2/AP-1/STAT3 pathways.

Science.gov (United States)

Yang, Lichao; Guo, Han; Li, Ying; Meng, Xianglan; Yan, Lu; Dan Zhang; Wu, Sangang; Zhou, Hao; Peng, Lu; Xie, Qiang; Jin, Xin

2016-10-10

The present study aimed to examine the anti-inflammatory actions of oleoylethanolamide (OEA) in lipopolysaccharide (LPS)-induced THP-1 cells. The cells were stimulated with LPS (1 μg/ml) in the presence or absence of OEA (10, 20 and 40 μM). The pro-inflammatory cytokines were evaluated by qRT-PCR and ELISA. The THP-1 cells were transiently transfected with PPARα small-interfering RNA, and TLR4 activity was determined with a blocking test using anti-TLR4 antibody. Additionally, a special inhibitor was used to analyse the intracellular signaling pathway. OEA exerted a potent anti-inflammatory effect by reducing the production of pro-inflammatory cytokines and TLR4 expression, and by enhancing PPARα expression. The modulatory effects of OEA on LPS-induced inflammation depended on PPARα and TLR4. Importantly, OEA inhibited LPS-induced NF-κB activation, IκBα degradation, expression of AP-1, and the phosphorylation of ERK1/2 and STAT3. In summary, our results demonstrated that OEA exerts anti-inflammatory effects by enhancing PPARα signaling, inhibiting the TLR4-mediated NF-κB signaling pathway, and interfering with the ERK1/2-dependent signaling cascade (TLR4/ERK1/2/AP-1/STAT3), which suggests that OEA may be a therapeutic agent for inflammatory diseases.

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

Science.gov (United States)

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

1993-01-15

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

The Role of FAK in the Secretion of MMP9 after CD147 Stimulation in Macrophages.

Science.gov (United States)

Yu, Chen; Lixia, Yang; Ruiwei, Guo; Yankun, Shi; Jinshan, Ye

2018-03-30

To investigate whether focal adhesion kinase (FAK) can participate in the secretion of matrix metalloproteinase 9 (MMP9) after CD147 stimulation in THP-1 induced macrophages; thus, to explore the potential treatment perspectives for acute coronary syndrome (ACS).Phorbol-12-myristate-13-acetate (PMA) was used to induce THP-1 cells to differentiate into macrophages. To confirm the peak mRNA and protein expression of FAK and MMP9 after the stimulation of CD147, the macrophages were divided into 5 groups (0, 3, 6, 9, and 12 hours), with 0 hours group as control group. To investigate the role of FAK in the secretion of MMP9, with stimulation of CD147 for 9 hours, FAK inhibitor 14 was used to inhibit FAK Y397 phosphorylation. The mRNA and protein expressions were quantified by qRT-PCR and western blotting, respectively. (1) Relative mRNA expression of FAK and MMP9 were both significantly up-regulated (all P CD147, FAK peaked at 9 hours (3.908 ± 0.106 versus 1, P CD147 stimulation (all P CD147 up-regulates FAK, pFAK, and MMP9 mRNA and protein expressions in a dose-dependent manner. (4) FAK inhibitor 14 significantly reduced the relative protein expression level of pFAK (0.077 ± 0.012 versus 1, P CD147 stimulation.The results demonstrated that FAK Y397 phosphorylation was involved in the secretion of MMP9 after CD147 stimulation in macrophages and may play a role in the regulation of ACS.

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

DEFF Research Database (Denmark)

Glue, C; Millner, A; Bødtger, Uffe

2002-01-01

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

PMA Induces SnoN Proteolysis and CD61 Expression through an Autocrine Mechanism

Science.gov (United States)

Li, Chonghua; Peart, Natoya; Xuan, Zhenyu; Lewis, Dorothy E; Xia, Yang; Jin, Jianping

2014-01-01

Phorbol-12-myristate-13-acetate, also called PMA, is a small molecule that activates protein kinase C and functions to differentiate hematologic lineage cells. However, the mechanism of PMA-induced cellular differentiation is not fully understood. We found that PMA triggers global enhancement of protein ubiquitination in K562, a myelogenous leukemia cell line and one of the enhanced-ubiquitination targets is SnoN, an inhibitor of the Smad signaling pathway. Our data indicated that PMA stimulated the production of Activin A, a cytokine of the TGF-β family. Activin A then activated the phosphorylation of both Smad2 and Smad3. In consequence, SnoN is ubiquitinated by the APCCdh1 ubiquitin ligase with the help of phosphorylated Smad2. Furthermore, we found that SnoN proteolysis is important for the expression of CD61, a marker of megakaryocyte. These results indicate that protein ubiquitination promotes megakaryopoiesis via degrading SnoN, an inhibitor of CD61 expression, strengths the roles of ubiquitination in cellular differentiation. PMID:24637302

Docosahexaenoic acid ester of phloridzin inhibit lipopolysaccharide-induced inflammation in THP-1 differentiated macrophages.

Science.gov (United States)

Sekhon-Loodu, Satvir; Ziaullah; Rupasinghe, H P Vasantha

2015-03-01

Phloridzin or phlorizin (PZ) is a predominant phenolic compound found in apple and also used in various natural health products. Phloridzin shows poor absorption and cellular uptake due to its hydrophilic nature. The aim was to investigate and compare the effect of docosahexaenoic acid (DHA) ester of PZ (PZ-DHA) and its parent compounds (phloridzin and DHA), phloretin (the aglycone of PZ) and cyclooxygenase inhibitory drugs (diclofenac and nimesulide) on production of pro-inflammatory biomarkers in inflammation-induced macrophages by lipopolysaccharide (LPS)-stimulation. Human THP-1 monocytes were seeded in 24-well plates (5×10(5)/well) and treated with phorbol 12-myristate 13-acetate (PMA, 0.1μg/mL) for 48h to induce macrophage differentiation. After 48h, the differentiated macrophages were washed with Hank's buffer and treated with various concentrations of test compounds for 4h, followed by the LPS-stimulation (18h). Pre-exposure of PZ-DHA ester was more effective in reducing tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) protein levels compared to DHA and nimesulide. However, diclofenac was the most effective in reducing prostaglandin (PGE2) level by depicting a dose-dependent response. However, PZ-DHA ester and DHA were the most effective in inhibiting the activation of nuclear factor-kappa B (NF-κB) among other test compounds. Our results suggest that PZ-DHA ester might possess potential therapeutic activity to treat inflammation related disorders such as type 2 diabetes, asthma, atherosclerosis and inflammatory bowel disease. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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

2016-01-01

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

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

Directory of Open Access Journals (Sweden)

Qi-Ming Wang

2016-11-01

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

Flow cytometric evaluation of the effects of 3-bromopyruvate (3BP) and dichloracetate (DCA) on THP-1 cells: a multiparameter analysis

NARCIS (Netherlands)

Verhoeven, H.A.; Griensven, van L.J.L.D.

2012-01-01

Two human leukemia cells K562 and THP-1, the breast cancer lines MCF-7 and ZR-75-1, and the melanoma line MDA-MB-435S were compared by flowcytometry for their behaviour at increasing levels of 3BP. K562 and THP-1 responded to 3BP by membrane depolarization and increased ROS; MCF-7 and ZR-75-1 showed

Mixed metal oxide nanoparticles inhibit growth of Mycobacterium tuberculosis into THP-1 cells

Directory of Open Access Journals (Sweden)

A R Jafari

2016-01-01

Conclusion: Although Ag NPs exhibited low cytotoxicity, they were unable to inhibit Mtb growth in vitro. ZnO NPs exhibited strong anti-Mtb activity and inhibited bacterial growth, but exhibited high cytotoxicity to human macrophage cells. By mixing Ag and ZnO NPs at a ratio of 8ZnO/2Ag, we acquired a mixture that exhibited potent antibacterial activity against Mtb and no cytotoxic effects on THP-1 cells, resulting in inhibition of both in vitro and ex vivo Mtb growth [Figure 1],[Figure 2],[Figure 3], [Table 1],[Table 2],[Table 3].{Figure 1}{Figure 2}{Figure 3} {Table 1}{Table 2}{Table 3}

HMOX1 and NQO1 genes are upregulated in response to contact sensitizers in dendritic cells and THP-1 cell line: role of the Keap1/Nrf2 pathway.

Science.gov (United States)

Ade, Nadège; Leon, Fanny; Pallardy, Marc; Peiffer, Jean-Luc; Kerdine-Romer, Saadia; Tissier, Marie-Hélène; Bonnet, Pierre-Antoine; Fabre, Isabelle; Ourlin, Jean-Claude

2009-02-01

Electrophilicity is one of the most common features of skin contact sensitizers and is necessary for protein haptenation. The Keap1 (Kelch-like ECH-associated protein 1)/Nrf2 -signaling pathway is dedicated to the detection of electrophilic stress in cells leading to the upregulation of genes involved in protection or neutralization of chemical reactive species. Signals provided by chemical stress could play an important role in dendritic cell activation and the aim of this work was to test whether contact sensitizers were specific activators of the Keap1/Nrf2 pathway. CD34-derived dendritic cells (CD34-DC) and the THP-1 myeloid cell line were treated by a panel of sensitizers (Ni, 1-chloro 2,4-dinitrobenzene, cinnamaldehyde, 7-hydroxycitronellal, 1,4-dihydroquinone, alpha-methyl-trans-cinnamaldehyde, 2-4-tert-(butylbenzyl)propionaldehyde or Lilial, and 1,4-phenylenediamine), irritants (sodium dodecyl sulfate, benzalkonium chloride), and a nonsensitizer molecule (chlorobenzene). Three well-known Nrf2 activators (tert-butylhydroquinone, lipoic acid, sulforaphane) were also tested. Expression of hmox1 and nqo1 was measured using real-time PCR and cellular accumulation of Nrf2 was assessed by Western blot. Our results showed an increased expression at early time points of hmox1 and nqo1 mRNAs in response to sensitizers but not to irritants. Accumulation of the Nrf2 protein was also observed only with chemical sensitizers. A significant inhibition of the expression of hmox1 and nqo1 mRNAs and CD86 expression was found in 1-chloro 2,4-dinitrobenzene-treated THP-1 cells preincubated with N-acetyl cysteine, a glutathione precursor. Altogether, these data suggested that the Keap1/Nrf2-signaling pathway was activated by electrophilic molecules including sensitizers in dendritic cells and in the THP-1 cell line. Monitoring of this pathway may provide new biomarkers (e.g., Nrf2, hmox1) for the detection of the sensitization potential of chemicals.

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

International Nuclear Information System (INIS)

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

2014-01-01

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

Aggregatibacter actinomycetemcomitans-Induced AIM2 Inflammasome Activation Is Suppressed by Xylitol in Differentiated THP-1 Macrophages.

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Kim, Seyeon; Park, Mi Hee; Song, Yu Ri; Na, Hee Sam; Chung, Jin

2016-06-01

Aggressive periodontitis is characterized by rapid destruction of periodontal tissue caused by Aggregatibacter actinomycetemcomitans. Interleukin (IL)-1β is a proinflammatory cytokine, and its production is tightly regulated by inflammasome activation. Xylitol, an anticaries agent, is anti-inflammatory, but its effect on inflammasome activation has not been researched. This study investigates the effect of xylitol on inflammasome activation induced by A. actinomycetemcomitans. The differentiated THP-1 macrophages were stimulated by A. actinomycetemcomitans with or without xylitol and the expressions of IL-1β and inflammasome components were detected by real time PCR, ELISA, confocal microscopy and Immunoblot analysis. The effects of xylitol on the adhesion and invasion of A. actinomycetemcomitans to cells were measured by viable cell count. A. actinomycetemcomitans increased pro IL-1β synthesis and IL-1β secretion in a multiplicity of infection- and time-dependent manner. A. actinomycetemcomitans also stimulated caspase-1 activation. Among inflammasome components, apoptosis-associated speck-like protein containing a CARD (ASC) and absent in melanoma 2 (AIM2) proteins were upregulated by A. actinomycetemcomitans infection. When cells were pretreated with xylitol, proIL-1β and IL-1β production by A. actinomycetemcomitans infection was significantly decreased. Xylitol also inhibited ASC and AIM2 proteins and formation of ASC puncta. Furthermore, xylitol suppressed internalization of A. actinomycetemcomitans into differentiated THP-1 macrophages without affecting viability of A. actinomycetemcomitans within cells. A. actinomycetemcomitans induced IL-1β production and AIM2 inflammasome activation. Xylitol inhibited these effects, possibly by suppressing internalization of A. actinomycetemcomitans into cells. Thus, this study proposes a mechanism for IL-1β production via inflammasome activation and discusses a possible use for xylitol in periodontal inflammation

Centella asiatica modulates cancer cachexia associated inflammatory cytokines and cell death in leukaemic THP-1 cells and peripheral blood mononuclear cells (PBMC's).

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Naidoo, Dhaneshree Bestinee; Chuturgoon, Anil Amichund; Phulukdaree, Alisa; Guruprasad, Kanive Parashiva; Satyamoorthy, Kapaettu; Sewram, Vikash

2017-08-01

Cancer cachexia is associated with increased pro-inflammatory cytokine levels. Centella asiatica (C. asiatica) possesses antioxidant, anti-inflammatory and anti-tumour potential. We investigated the modulation of antioxidants, cytokines and cell death by C. asiatica ethanolic leaf extract (C LE ) in leukaemic THP-1 cells and normal peripheral blood mononuclear cells (PBMC's). Cytotoxcity of C LE was determined at 24 and 72 h (h). Oxidant scavenging activity of C LE was evaluated using the 2, 2-diphenyl-1 picrylhydrazyl (DPPH) assay. Glutathione (GSH) levels, caspase (-8, -9, -3/7) activities and adenosine triphosphate (ATP) levels (Luminometry) were then assayed. The levels of tumour necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β and IL-10 were also assessed using enzyme-linked immunosorbant assay. C LE decreased PBMC viability between 33.25-74.55% (24 h: 0.2-0.8 mg/ml C LE and 72 h: 0.4-0.8 mg/ml C LE ) and THP-1 viability by 28.404% (72 h: 0.8 mg/ml C LE ) (p cachexia.

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

International Nuclear Information System (INIS)

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

2009-01-01

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

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

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Vinita Chauhan

2012-01-01

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

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

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

2017-02-01

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

Stimulation of granulocytic cell iodination by pine cone antitumor substances

International Nuclear Information System (INIS)

Unten, S.; Sakagami, H.; Konno, K.

1989-01-01

Antitumor substances (Fractions VI and VII) prepared from the NaOH extract of pine cone significantly stimulated the iodination (incorporation of radioactive iodine into an acid-insoluble fraction) of human peripheral blood adherent mononuclear cells, polymorphonuclear cells (PMN), and human promyelocytic leukemic HL-60 cells. In contrast, these fractions did not significantly increase the iodination of nonadherent mononuclear cells, red blood cells, other human leukemic cell lines (U-937, THP-1, K-562), human diploid fibroblast (UT20Lu), or mouse cell lines (L-929, J774.1). Iodination of HL-60 cells, which were induced to differentiate by treatment with either retinoic acid or tumor necrosis factor, were stimulated less than untreated cells. The stimulation of iodination of both PMN and HL-60 cells required the continuous presence of these fractions and was almost completely abolished by the presence of myeloperoxidase inhibitors. The stimulation activity of these fractions was generally higher than that of various other immunopotentiators. Possible mechanisms of extract stimulation of myeloperoxidase-containing cell iodination are discussed

Establishment of a common acute lymphoblastic leukemia cell line (LC4-1) and effects of phorbol myristate acetate (PMA) on the surface antigen expression of the cell line.

Science.gov (United States)

Yoshimura, T; Mayumi, M; Yorifuji, T; Kim, K M; Heike, T; Miyanomae, T; Shinomiya, K; Mikawa, H

1987-09-01

A common acute lymphoblastic leukemia (ALL) cell line, designated LC4-1, was established from peripheral blood mononuclear cells of a patient with acute non-T-cell ALL. LC4-1 cells were characteristically positive for Ia, B4, and common ALL antigens (CALLA), but negative for B2, Tac, T3, T4, T8, T11, and M1 antigens and E-rosette formation. Approximately 30% of LC4-1 cells expressed detectable amounts of B1 antigens. LC4-1 cells expressed neither Epstein-Barr-virus-associated nuclear antigen (EBNA), cytoplasmic immunoglobulins (cIg) nor surface immunoglobulins (sIg). Gene rearrangements had already occurred in LC4-1 in the D-J region of immunoglobulin heavy chain genes, but not in T-cell receptor (beta-chain) genes, suggesting that LC4-1 is a progenitor cell line of B-cell lineage earlier than pre-B-cells. The expression of cell surface antigens of LC4-1 was changed by treatment with 4-phorbol 12-myristate 13-acetate (PMA) (0.1 ng/ml) for 2 days. Before treatment with PMA, about 98% of LC4-1 cells were positive for B4, CALLA, and Ia. However, following treatment they lost CALLA expression without any change in expression of Ia and B4. There was no change in B1-positive population. The change in surface antigens on LC4-1 cells seems to be due to differentiation induced in the cells by PMA. These results support the hypothesis that CALLA is a differentiation antigen and suggest one possible differentiation pathway for pre-B-cells.

CD4+ T cell-derived novel peptide Thp5 induces interleukin-4 production in CD4+ T cells to direct T helper 2 cell differentiation.

Science.gov (United States)

Khan, Mohd Moin; Chatterjee, Samit; Dwivedi, Ved Prakash; Pandey, Nishant Kumar; Singh, Yogesh; Tousif, Sultan; Bhavesh, Neel Sarovar; Van Kaer, Luc; Das, Jyoti; Das, Gobardhan

2012-01-20

The differentiation of naïve CD4(+) T cells into T helper 2 (Th2) cells requires production of the cytokine IL-4 in the local microenvironment. It is evident that naïve/quiescently activated CD4(+) T cells produce the IL-4 that drives Th2 cell differentiation. Because early production of IL-4 in naïve T cells leads to preferential Th2 cell differentiation, this process needs to be tightly regulated so as to avoid catastrophic and misdirected Th2 cell differentiation. Here, we show that Thp5, a novel peptide with structural similarity to vasoactive intestinal peptide, regulates production of early IL-4 in newly activated CD4(+) T cells. Induction of IL-4 in CD4(+) T cells by Thp5 is independent of the transcription factor STAT6 but dependent on ERK1/2 signaling. Furthermore, cytokines (IL-12 and TGF-β) that promote the differentiation of Th1 or Th17 cells inhibit Thp5 induction, thus suppressing Th2 cell differentiation. We further showed that Thp5 enhances Th2 responses and exacerbates allergic airway inflammation in mice. Taken together, our findings reveal that early activated CD4(+) T cells produce Thp5, which plays a critical role as a molecular switch in the differentiation of Th cells, biasing the response toward the Th2 cell phenotype.

The effects of exogenous lipid on THP-1 cells: an in vitro model of airway aspiration?

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Yvette A. Hayman

2017-03-01

Full Text Available Chronic inflammatory diseases of the airways are associated with gastro-oesophageal reflux (GOR and aspiration events. The observation of lipid-laden macrophages (LLMs within the airway may indicate aspiration secondary to GOR. The proposed mechanism, that lipid droplets from undigested or partially digested food are aspirated leading to accumulation in scavenging macrophages, led us to hypothesise that an activated population of LLMs could interact with other immune cells to induce bronchial inflammation. To test this, we generated an in vitro model using differentiated THP-1 cells, which were treated with a high-fat liquid feed. Here, we show that THP-1 cells can take up lipid from the high-fat feed independent of actin polymerisation or CD36-dependent phagocytosis. These cells did not exhibit M1 or M2 polarisation. Gene array analysis confirmed over 8000 genes were upregulated by at least twofold following high fat exposure, and IL-8 was the most upregulated gene. Pathway analysis revealed upregulation of genes known to be involved in chronic obstructive pulmonary disease (COPD pathophysiology. We suggest that aspiration and macrophage phagocytosis may be important mechanisms in the aetiology of diseases such as COPD and cystic fibrosis that are characterised by high levels of IL-8 within the airways.

The effects of exogenous lipid on THP-1 cells: an in vitro model of airway aspiration?

Science.gov (United States)

Hayman, Yvette A; Sadofsky, Laura R; Williamson, James D; Hart, Simon P; Morice, Alyn H

2017-01-01

Chronic inflammatory diseases of the airways are associated with gastro-oesophageal reflux (GOR) and aspiration events. The observation of lipid-laden macrophages (LLMs) within the airway may indicate aspiration secondary to GOR. The proposed mechanism, that lipid droplets from undigested or partially digested food are aspirated leading to accumulation in scavenging macrophages, led us to hypothesise that an activated population of LLMs could interact with other immune cells to induce bronchial inflammation. To test this, we generated an in vitro model using differentiated THP-1 cells, which were treated with a high-fat liquid feed. Here, we show that THP-1 cells can take up lipid from the high-fat feed independent of actin polymerisation or CD36-dependent phagocytosis. These cells did not exhibit M1 or M2 polarisation. Gene array analysis confirmed over 8000 genes were upregulated by at least twofold following high fat exposure, and IL-8 was the most upregulated gene. Pathway analysis revealed upregulation of genes known to be involved in chronic obstructive pulmonary disease (COPD) pathophysiology. We suggest that aspiration and macrophage phagocytosis may be important mechanisms in the aetiology of diseases such as COPD and cystic fibrosis that are characterised by high levels of IL-8 within the airways.

P2X receptor-dependent erythrocyte damage by α-hemolysin from Escherichia coli triggers phagocytosis by THP-1 cells

DEFF Research Database (Denmark)

Fagerberg, Steen Kåre; Skals, Marianne Gerberg; Leipziger, Jens Georg

2013-01-01

, which is known to be a keen trigger for phagocytosis. We hypothesize that exposure to HlyA elicits removal of the damaged erythrocytes by phagocytic cells. Cultured THP-1 cells as a model for erythrocytal phagocytosis was verified by a variety of methods, including live cell imaging. We consistently...

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

Science.gov (United States)

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

2008-08-01

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

[Effect of P38MAPK signal transduction pathway on apoptosis of THP-1 induced by allicin].

Science.gov (United States)

Liao, Yang; Chen, Jianbin; Tang, Weixue; Ge, Qunfang; Lu, Qianwei; Yang, Zesong

2009-06-01

The objective of this paper was to study the change of P38MAPK and Fas in the apoptosis of THP-1 cells induced by allicin. The proliferation inhibition rates of THP-1 cells after various treatments were examined by MTT assay. Apoptosis rate was determined with Annexin V- FITC/PI double staining by flow cytometry. The expression and distribution change of the phosphorylation p38MAPK (P-p38MAPK) were detected by immunohistochemical staining. The changes of P-p38 MAPK and Fas proteins were detected by Western blot. The proliferations of leukemia cell line THP-1 are inhibited by allicin. MTT assay showed that allicin can inhibit the proliferation of the THP-1 cell, and the inhibition was dependent on both dose and time. The IC50 of 72 hours was 12.8 mg x L(-1). Apoptosis rate detected by Annexin V-FITC/PI was proportional to the concentration of the allicin. After the immunohistochemical staining test, the P-p38MAPK was located in the cell nucleus and plasma, showing deep brown, when adding allicin to THP-1 cell. Western blot test showed that the P-p38MAPK proteins expression was proportional to the concentration of Allicin and was also dose dependent. The levels of P-p38MAPK in negative control group, 1/2 IC50 of 72 hours group and IC50 of 72 hours group were 0.259 8 +/- 0.013 2, 0.61 2 +/- 0.008 3 and 0.505 6 +/- 0.005 5 respectively, and the levels of Fas proteins were 0.287 4 +/- 0.008 9, 0.426 8 +/- 0.007 9 and 0.597 1 +/- 0.010 9 respectively. The difference was statistically significant when compared with the negative control group (P THP-1 cells apoptosis, and its mechanism may be related to the activation of P38MAPK/Fas.

Saikosaponin-a Attenuates Oxidized LDL Uptake and Prompts Cholesterol Efflux in THP-1 Cells.

Science.gov (United States)

He, Dan; Wang, Hongyan; Xu, Ling; Wang, Xiaoqing; Peng, Kuang; Wang, Lili; Liu, Pixu; Qu, Peng

2016-06-01

Saikosaponins-a (Ssa) is a major bioactive extract of Radix Bupleuri which is a traditional Chinese medicine. The roles of inflammatory response and lipid transportation in the process of atherosclerosis have drawn increasing attention. We explored the regulation of lipid transportation and immune-inflammatory role of Ssa in early atherosclerosis. The antiatherogenic actions and possible molecular mechanisms of Ssa were texted in THP-1 cells. We examined the effect of Ssa on oxidized low-density lipoprotein (ox-LDL)-induced lipid uptake, cholesterol efflux, immune-inflammatory response. THP-1 macrophages were treated with Ssa followed by ox-LDL for 24 hours. Results from western blot showed that Ssa obviously reduced lipoprotein uptake to block foam cell formation and the expression of Density Lipoprotein Receptor-1 and CD36. Ssa also significantly boosted cholesterol efflux and the expression of ATP binding cassettetransporter A1 and peroxisome proliferator-activated receptor γ. The results also indicated that Ssa inhibited ox-LDL-induced activation of AKT and nuclear factor-κB, assembly of NLRP3 inflammasome and production of proinflammatory cytokines. It is suggested that the ability against immune inflammatory response of Ssa is due to modulation of the PI3K/AKT/NF-κB/NLRP3 pathway. In conclusion, this study provides new insight into Ssa's molecular mechanism and its therapeutic potential in the treatment of atherosclerosis.

NK cells of the oldest seniors represent constant and resistant to stimulation high expression of cellular protective proteins SIRT1 and HSP70.

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Kaszubowska, Lucyna; Foerster, Jerzy; Kaczor, Jan Jacek; Schetz, Daria; Ślebioda, Tomasz Jerzy; Kmieć, Zbigniew

2018-01-01

Natural killer cells (NK cells) are cytotoxic lymphocytes of innate immunity that reveal some immunoregulatory properties, however, their role in the process of ageing is not completely understood. The study aimed to analyze the expression of proteins involved in cellular stress response: sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in human NK cells with reference to the process of ageing. Non-stimulated and stimulated with IL-2, LPS or PMA with ionomycin cells originated from peripheral blood samples of: seniors aged over 85 ('the oldest'; n  = 25; 88.5 ± 0.5 years, mean ± SEM), seniors aged under 85 ('the old'; n  = 30; 75.6 ± 0.9 years) and the young ( n  = 31; 20.9 ± 0.3 years). The relationships between the levels of expression of cellular protective proteins in the studied population were also analyzed. The concentrations of carbonyl groups and 8-isoprostanes, markers of oxidative stress, in both stimulated and non-stimulated cultured NK cells were measured to assess the level of the oxidative stress in the cells. The oldest seniors varied from the other age groups by significantly higher expression of SIRT1 and HSP70 both in non-stimulated and stimulated NK cells. These cells also appeared to be resistant to further stimulations with IL-2, LPS or PMA with ionomycin. Highly positive correlations between SIRT1 and intracellular HSP70 in both stimulated and non-stimulated NK cells were observed. SOD2 presented low expression in non-stimulated cells, whereas its sensitivity to stimulation increased with age of donors. High positive correlations between SOD2 and surface HSP70 were observed. We found that the markers of oxidative stress in NK cells did not change with ageing. The oldest seniors revealed well developed adaptive stress response in NK cells with increased, constant levels of SIRT1 and intracellular HSP70. They presented also very high positive correlations between

Urotensin II increases foam cell formation by repressing ABCA1 expression through the ERK/NF-κB pathway in THP-1 macrophages

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Wang, Yan [Department of Anesthesiology, The Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan (China); Wu, Jian-Feng [Department of Cardiovascular Medicine, The Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan (China); Tang, Yan-Yan; Zhang, Min; Li, Yuan [Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, University of South China, Hengyang 421001, Hunan (China); Chen, Kong; Zeng, Meng-Ya [Department of Cardiovascular Medicine, The Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan (China); Yao, Feng; Xie, Wei [Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, University of South China, Hengyang 421001, Hunan (China); Zheng, Xi-Long [Department of Biochemistry and Molecular Biology, Libin Cardiovascular Institute of Alberta, University of Calgary, Health Sciences Center, 3330 Hospital Dr NW, Calgary, Alberta T2N 4N1 (Canada); Zeng, Gao-Feng, E-mail: qichingnudou@tom.com [Department of Cardiovascular Medicine, The Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan (China); Tang, Chao-Ke, E-mail: tangchaoke@qq.com [Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, University of South China, Hengyang 421001, Hunan (China)

2014-10-03

Highlights: • U II reduces cholesterol efflux in THP-1 macrophages. • U II decreases the expression of ABCA1. • Inhibition of the ERK/NF-κB pathway reduces U II effects on ABCA1 expression and cholesterol efflux. - Abstract: Objective: Foam cell formation in the arterial wall plays a key role in the development of atherosclerosis. Recent studies showed that Urotensin II (U II) is involved in the pathogenesis of atherosclerosis. Here we examined the effects of human U II on ATP-binding cassette transporter A1 (ABCA1) expression and the underlying mechanism in THP-1 macrophages. Methods and results: Cultured THP-1 macrophages were treated with U II, followed by measuring the intracellular lipid contents, cholesterol efflux and ABCA1 levels. The results showed that U II dramatically decreased ABCA1 levels and impaired cholesterol efflux. However, the effects of U II on ABCA1 protein expression and cellular cholesterol efflux were partially reversed by inhibition of extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa B (NF-κB) activity, suggesting the potential roles of ERK1/2 and NF-κB in ABCA1 expression, respectively. Conclusion: Our current data indicate that U II may have promoting effects on the progression of atherosclerosis, likely through suppressing ABCA1 expression via activation of the ERK/NF-κB pathway and reducing cholesterol efflux to promote macrophage foam cell formation.

Lipopolysaccharide (LPS) of Porphyromonas gingivalis induces IL-1beta, TNF-alpha and IL-6 production by THP-1 cells in a way different from that of Escherichia coli LPS.

Science.gov (United States)

Diya Zhang; Lili Chen; Shenglai Li; Zhiyuan Gu; Jie Yan

2008-04-01

Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been shown to differ from enterobacterial LPS in structure and function; therefore, the Toll-like receptors (TLRs) and the intracellular inflammatory signaling pathways are accordingly different. To elucidate the signal transduction pathway of P. gingivalis, LPS-induced pro-inflammatory cytokine production in the human monocytic cell line THP-1 was measured by ELISA, and the TLRs were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors as well as Phospho-ELISA kits were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. In this study, P. gingivalis LPS showed the ability to induce cytokine production in THP-1 cells and its induction was significantly (P THP-1 cells, and that the TLR2-JNK pathway might play a significant role in P. gingivalis LPS-induced chronic inflammatory periodontal disease.

Effects of miR-33a-5P on ABCA1/G1-mediated cholesterol efflux under inflammatory stress in THP-1 macrophages.

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Min Mao

Full Text Available The present study is to investigate whether inflammatory cytokines inhibit ABCA1/ABCG1-mediated cholesterol efflux by regulating miR-33a-5P in THP-1 macrophages. We used interleukin-6 and tumor necrosis factor-alpha in the presence or absence of native low density lipoprotein (LDL to stimulate THP-1 macrophages. THP-1 macrophages were infected by either control lentivirus vectors or lentivirus encoding miR-33a-5P or antisense miR-33a-5P. The effects of inflammatory cytokines, miR-33a-5P and antisense miR-33a-5P on intracellular lipids accumulation and intracellular cholesterol contents were assessed by oil red O staining and quantitative intracellular cholesterol assay. ApoA-I-mediated cholesterol efflux was examined using the fluorescent sterol (BODIPY-cholesterol. The gene and protein expressions of the molecules involved in cholesterol trafficking were examined using quantitative real-time polymerase chain reaction and Western blotting. Inflammatory cytokines or miR-33a-5P increased intracellular lipid accumulation and decreased apoA-I-mediated cholesterol efflux via decreasing the expression of ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. However, antisense miR-33a-5P reversed the effects of inflammatory cytokines on intracellular lipid accumulation, cholesterol efflux, and the expression of miR-33a-5P, ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. This study indicated that inflammatory cytokines inhibited ABCA1/ABCG1-mediated cholesterol efflux by up-regulating miR-33a-5P in THP-1 macrophages.

Role of p38 MAPK in the selective release of IL-8 induced by chemical allergen in naive THp-1 cells.

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Mitjans, Montserrat; Viviani, Barbara; Lucchi, Laura; Galli, Corrado L; Marinovich, Marina; Corsini, Emanuela

2008-03-01

At present, the assessment of the allergenic potential of chemicals is carried out using animal models. Over the last decade, several in vitro methods mainly using primary dendritic cells have been proposed to identify the potential of chemicals to induce skin sensitization to meet current animal welfare and public opinions. The major limitations of such tests are the donor-to-donor variability, the low levels in the source, and a possible shortage of human sources. The aim of the present investigation was to establish an in vitro test to identify chemical allergens using the human promyelocytic cell line THP-1 in order to avoid some of these difficulties. We investigated whether the chemokine interleukin-8 or CXCL8 (IL-8) production could provide a methodology for the detection of both respiratory and contact allergens. THP-1 cells were exposed to contact allergens (cinnamaldehyde, dinitrochlorobenzene, nickel sulfate, penicillin G, p-phenylenediamine, tetramethylthiuram disulfide), to respiratory allergens (ammonium hexachloroplatinate, diphenylmethane diisocyanate, trimellitic anhydride) and to irritants (salicylic acid, phenol, sodium lauryl sulphate). Following 48 h of incubation, the release of IL-8 was evaluated by sandwich ELISA. IL-8 production was significantly increased after stimulation with all allergens tested, with the exception of trimellitic anhydride, whereas irritants exposure failed to induce IL-8 release. The lack of IL-8 production by trimellitic anhydride can be explained by the rapid hydrolysis of this chemical in water to trimellitic acid, which is not an allergen. In contrast to IL-8 release, CD54 and CD86 expression did not provide a sensitive method failing to correctly identify approximately 30% of the tested compounds. Although CD86 appears to be a more sensitive marker than CD54 when discriminating allergens from irritants neither of these markers provided robust methodology. We also investigated if a common activation pathway in

Resolution of PMA-Induced Skin Inflammation Involves Interaction of IFN-γ and ALOX15

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Guojun Zhang

2013-01-01

Full Text Available Background. Acute inflammation and its timely resolution play important roles in the body’s responses to the environmental stimulation. Although IFN-γ is well known for the induction of inflammation, its role in the inflammation resolution is still poorly understood. Methodology and Principal Findings. In this study, we investigated the function of interferon gamma (IFN-γ during the resolution of PMA-induced skin inflammation in vivo. The results revealed that the expression levels of IL-6, TNF-α, and monocyte chemoattractant protein 1 (MCP-1 in skin decreased during the resolution stage of PMA-induced inflammation, while IFN-γ is still maintained at a relatively high level. Neutralization of endogenous IFN-γ led to accelerated reduction of epidermal thickness and decreased epithelial cell proliferation. Similarly, decreased infiltration of inflammatory cells (Gr1+ or CD11b+ cells and a significant reduction of proinflammatory cytokines were also observed upon the blockade of IFN-γ. Furthermore, neutralization of IFN-γ boosted ALOX15 expression of the skin during inflammation resolution. In accordance, application of lipoxin A4 (LXA4, a product of ALOX15 obtained a proresolution effect similar to neutralization of IFN-γ. These results demonstrated that through upregulating ALOX15-LXA4 pathway, blockage of IFN-γ can promote the resolution of PMA-induced skin inflammation.

Inhibition of the NLRP3 inflammasome attenuates foam cell formation of THP-1 macrophages by suppressing ox-LDL uptake and promoting cholesterol efflux.

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Chen, Liang; Yao, Qiying; Xu, Siwei; Wang, Hongyan; Qu, Peng

2018-01-01

The NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) inflammasome plays an important role in the development of atherosclerosis. The activated NLRP3 inflammasome has been reported to promote macrophage foam cell formation, but not all studies have obtained the same result, and how NLRP3 inflammasome is involved in the formation of foam cells remains elusive. We used selective NLRP3 inflammasome inhibitors and NLRP3-deficient THP-1 cells to assess the effect of NLRP3 inflammasome inhibition on macrophage foam cell formation, oxidized low-density lipoprotein (ox-LDL) uptake, esterification, and cholesterol efflux, as well as the expression of associated proteins. Inhibition of the NLRP3 inflammasome attenuated foam cell formation, diminished ox-LDL uptake, and promoted cholesterol efflux from THP-1 macrophages. Moreover, it downregulated CD36, acyl coenzyme A: cholesterol acyltransferase-1 and neutral cholesterol ester hydrolase expression; upregulated ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression; but had no effect on the expression of scavenger receptor class A and ATP-binding cassette transporter G1. Collectively, our findings show that inhibition of the NLRP3 inflammasome decreases foam cell formation of THP-1 macrophages via suppression of ox-LDL uptake and enhancement of cholesterol efflux, which may be due to downregulation of CD36 expression and upregulation of ABCA1 and SR-BI expression, respectively. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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

2012-02-01

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

Differential regulation by agonist and phorbol ester of cloned m1 and m2 muscarinic acetylcholine receptors in mouse Y1 adrenal cells and in Y1 cells deficient in cAMP-dependent protein kinase

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Scherer, N.M.; Nathanson, N.M.

1990-01-01

Cloned muscarinic acetylcholine m1 and m2 receptors were expressed in stably transfected mouse Y1 adrenal cells and in a variant Y1 line, Kin-8, which is deficient in cAMP-dependent protein kinase activity (PKA - ). m1 and m2 receptors were rapidly internalized following exposure of transfected PKA + or PKA - cells to the muscarinic agonist carbachol. Thus, agonist-dependent internalization of m1 and m2 did not require PKA activity. A differential effect of PKA on regulation by agonist of the m2 receptor, but not the m1 receptor, was unmasked in PKA - cells. These data indicate that the basal activity of PKA may modulate the agonist-dependent internalization of the m2 receptor, but not the m1 receptor. The internalization of the m1 and m2 receptors in both PKA + and PKA - cells was accompanied by desensitization of functional responses. Exposure of PKA + cells to 10 -7 M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, resulted in a 30 ± 9% decrease in the number of m1 receptors on the cell surface. The m2 receptor was not internalized following treatment of either PKA + or PKA - cells with PMA. Thus, the m1 and m2 receptors show differential sensitivity to internalization by PMA. Agonist-dependent internalization of the m1 receptor appeared to be independent of activation of PKC because (1) agonist-dependent internalization of m1 was not attenuated in PKA - cells, (2) the rate and extent of internalization of m1 in cells exposed to PMA were less than those in cells exposed to agonist, and (3) treatment of cells with concanavalin A selectivity blocked internalization of m1 in cells exposed to PMA, but not to agonist. The effects of agonist and PMA on receptor internalization were not additive. Exposure of PKA + or PKA - cells to PMA reduced the magnitude of pilocarpine-stimulated PI hydrolysis by about 25%

[6]-Gingerol induces caspase-dependent apoptosis and prevents PMA-induced proliferation in colon cancer cells by inhibiting MAPK/AP-1 signaling.

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E K Radhakrishnan

Full Text Available We report mechanism-based evidence for the anticancer and chemopreventive efficacy of [6]-gingerol, the major active principle of the medicinal plant, Ginger (Zingiber officinale, in colon cancer cells. The compound was evaluated in two human colon cancer cell lines for its cytotoxic effect and the most sensitive cell line, SW-480, was selected for the mechanistic evaluation of its anticancer and chemopreventive efficacy. The non-toxic nature of [6]-gingerol was confirmed by viability assays on rapidly dividing normal mouse colon cells. [6]-gingerol inhibited cell proliferation and induced apoptosis as evidenced by externalization of phosphatidyl serine in SW-480, while the normal colon cells were unaffected. Sensitivity to [6]-gingerol in SW-480 cells was associated with activation of caspases 8, 9, 3 &7 and cleavage of PARP, which attests induction of apoptotic cell death. Mechanistically, [6]-gingerol down-regulated Phorbol Myristate Acetate (PMA induced phosphorylation of ERK1/2 and JNK MAP kinases and activation of AP-1 transcription factor, but had only little effects on phosphorylation of p38 MAP kinase and activation of NF-kappa B. Additionally, it complemented the inhibitors of either ERK1/2 or JNK MAP kinase in bringing down the PMA-induced cell proliferation in SW-480 cells. We report the inhibition of ERK1/2/JNK/AP-1 pathway as a possible mechanism behind the anticancer as well as chemopreventive efficacy of [6]-gingerol against colon cancer.

Suitability of macrophage inflammatory protein-1beta production by THP-1 cells in differentiating skin sensitizers from irritant chemicals.

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Lim, Yeon-Mi; Moon, Seong-Joon; An, Su-Sun; Lee, Soo-Jin; Kim, Seo-Young; Chang, Ih-Seop; Park, Kui-Lea; Kim, Hyoung-Ah; Heo, Yong

2008-04-01

Worldwide restrictions in animal use for research have driven efforts to develop alternative methods. The study aimed to test the efficacy of the macrophage inflammatory protein-1beta (MIP-1beta) assay for testing chemicals' skin-sensitizing capacity. The assay was performed using 9 chemicals judged to be sensitizing and 7 non-sensitizing by the standard in vivo assays. THP-1 cells were cultured in the presence or absence of 4 doses, 0.01x, 0.1x, 0.5x, or 1x IC(50) (50% inhibitory concentration for THP-1 cell proliferation) of these chemicals for 24 hr, and the MIP-1beta level in the supernatants was determined. Skin sensitization by the test chemicals was determined by MIP-1beta production rates. The MIP-1beta production rate was expressed as the relative increase in MIP-1beta production in response to chemical treatment compared with vehicle treatment. When the threshold MIP-1beta production rate used was 100% or 105% of dimethyl sulfoxide, all the sensitizing chemicals tested (dinitrochlorobenzene, hexyl cinnamic aldehyde, eugenol, hydroquinone, dinitrofluorobenzene, benzocaine, nickel, chromium, and 5-chloro-2-methyl-4-isothiazolin-3-one) were positive, and all the non-sensitizing chemicals (methyl salicylate, benzalkonium chloride, lactic acid, isopropanol, and salicylic acid), with the exception of sodium lauryl sulfate, were negative for MIP-1beta production. These results indicate that MIP-1beta could be a biomarker for classification of chemicals as sensitizers or non-sensitizers.

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

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

2017-02-15

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

Stimulation of phosphatidylcholine breakdown and diacylglycerol production by growth factors in Swiss-3T3 cells.

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Price, B D; Morris, J D; Hall, A

1989-01-01

The effect of a number of growth factors on phosphatidylcholine (PtdCho) turnover in Swiss-3T3 cells was studied. Phorbol 12-myristate 13-acetate (PMA), bombesin, platelet-derived growth factor (PDGF) and vasopressin rapidly stimulated PtdCho hydrolysis, diacylglycerol (DAG) production, and PtdCho synthesis. Insulin and prostaglandin F2 alpha (PGF2 alpha) stimulated PtdCho synthesis, but not its breakdown, whereas epidermal growth factor (EGF) and bradykinin were without effect. Stimulation of PtdCho hydrolysis by the above ligands resulted in increased production of phosphocholine and DAG (due to phospholipase C activity) and significant amounts of choline, suggesting activation of a phospholipase D as well. CDP-choline and glycerophosphocholine levels were unchanged. Down-regulation of protein kinase C with PMA (400 nM, 40 h) abolished the stimulation of PtdCho hydrolysis and PtdCho synthesis by PMA, bombesin, PDGF and vasopressin, but not the stimulation of PtdCho synthesis by insulin and PGF2 alpha. PtdCho hydrolysis therefore occurs predominantly by activation of protein kinase C (either by PMA or PtdIns hydrolysis) leading to elevation of DAG levels derived from non-PtdIns(4,5)P2 sources. PtdCho synthesis occurs by both a protein kinase C-dependent pathway (stimulated by PMA, PDGF, bombesin and vasopressin) and a protein kinase C-independent pathway (stimulated by insulin and PGF2 alpha). DAG production from PtdCho hydrolysis is not the primary signal to activate protein kinase C, but may contribute to long-term activation of this kinase. PMID:2690829

Salvianolic acid B accelerated ABCA1-dependent cholesterol efflux by targeting PPAR-γ and LXRα

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Yue, Jianmei [Department of Endocrinology, Shandong Province Hospital Affiliated to Shandong University, 324# Jing 5 Road, Jinan 255021 (China); Department of Endocrinology, The First Hospital of Zibo, 4# E Mei Shan Dong Road, Zibo 255200 (China); Li, Bo, E-mail: libosubmit@163.com [Department of Cardiology, Central Hospital of Zibo, 54# Gong Qing Tuan Xi Road, Zibo, Shandong Province (China); Jing, Qingping [Department of Endocrinology, The First Hospital of Zibo, 4# E Mei Shan Dong Road, Zibo 255200 (China); Guan, Qingbo, E-mail: guanqingbosubmit@163.com [Department of Endocrinology, Shandong Province Hospital Affiliated to Shandong University, 324# Jing 5 Road, Jinan 255021 (China)

2015-07-03

Objectives: Cholesterol efflux has been thought to be the main and basic mechanism by which free cholesterol is transferred from extra hepatic cells to the liver or intestine for excretion. Salvianolic acid B (Sal B) has been widely used for the prevention and treatment of atherosclerotic diseases. Here, we sought to investigate the effects of Sal B on the cholesterol efflux in THP-1 macrophages. Methods: After PMA-stimulated THP-1 cells were exposed to 50 mg/L of oxLDL and [{sup 3}H] cholesterol (1.0 μCi/mL) for another 24 h, the effect of Sal B on cholesterol efflux was evaluated in the presence of apoA-1, HDL{sub 2} or HDL{sub 3}. The expression of ATP binding cassette transporter A1 (ABCA1), peroxisome proliferator-activated receptor-gamma (PPAR-γ), and liver X receptor-alpha (LXRα) was detected both at protein and mRNA levels in THP-1 cells after the stimulation of Sal B. Meanwhile, specific inhibition of PPAR-γ and LXRα were performed to investigate the mechanism. Results: The results showed that Sal B significantly accelerated apoA-I- and HDL-mediated cholesterol efflux in both dose- and time-dependent manners. Meanwhile, Sal B treatment also enhanced the expression of ABCA1 at both mRNA and protein levels. Then the data demonstrated that Sal B increased the expression of PPAR-γ and LXRα. And the application of specific agonists and inhibitors of further confirmed that Sal exert the function through PPAR-γ and LXRα. Conclusion: These results demonstrate that Sal B promotes cholesterol efflux in THP-1 macrophages through ABCA1/PPAR-γ/LXRα pathway. - Highlights: • Sal B promotes the expression of ABCA1. • Sal B promotes cholesterol efflux in macrophages. • Sal B promotes the expression of ABCA1 and cholesterol efflux through PPAR-γ/LXRα signaling pathway.

Bactericidal impact of Ag, ZnO and mixed AgZnO colloidal nanoparticles on H37Rv Mycobacterium tuberculosis phagocytized by THP-1 cell lines.

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Jafari, Alireza; Mosavari, Nader; Movahedzadeh, Farahnaz; Nodooshan, Saeedeh Jafari; Safarkar, Roya; Moro, Rossella; Kamalzadeh, Morteza; Majidpour, Ali; Boustanshenas, Mina; Mosavi, Tahereh

2017-09-01

The purpose of this research project was to infection of human macrophages (THP-1) cell lines by H 37 Rv strain of Mycobacterium tuberculosis (H 37 RvMTB) and find out the ratio/dilution of mixture silver (Ag NPs) and zinc oxide nanoparticles (ZnO NPs) whose ability to eliminate phagocytized bacteria compared to rifampicin. The colloidal Ag NPs and ZnO NPs were synthesized and their characteristics were evaluated. The THP-1 cell lines were infected with different concentration of H 37 RvMTB. Next, the infected cells were treated with different ratios/dilutions of Ag NPs, ZnO NPs and rifampicin. The THP-1 were lysed and were cultured in Lowenstein-Jensen agar medium, for eight weeks. The TEM and AFM images of NPs and H 37 RvMTB were supplied. It is observed that Ag NPs, 2 Ag :8 ZnO and 8 Ag :2 ZnO did not have any anti-tubercular effects on phagocytized H 37 RvMTB. Conversely, ZnO NPs somehow eliminated 18.7 × 10 4  CFU ml -1 of H 37 RvMTB in concentration of ∼ 0.468 ppm. To compare with 40 ppm of rifampicin, ∼ 0.663 ppm of 5 Ag :5 ZnO had the ability to kill of H 37 RvMTB, too. Based on previous research, ZnO NPs had strong anti-tubercular impact against H 37 RvMTB to in-vitro condition, but it was toxic in concentration of ∼ 0.468 ppm to both of THP-1 and normal lung (MRC-5) cell lines. It also seems that 5 Ag :5 ZnO is justified because in concentration of ∼ 0.663 ppm of 5 Ag :5 ZnO , phagocytized H 37 RvMTB into the THP-1 had died without any toxicity effects against THP-1 and also MRC-5 cell lines. It is obvious that the mixture of colloidal silver and zinc oxide NPs with ratio of 5 Ag :5 ZnO would be trustworthy options as anti-tubercular nano-drugs in future researches. Copyright © 2017. Published by Elsevier Ltd.

Biosynthesis of promatrix metalloproteinase-9/chondroitin sulphate proteoglycan heteromer involves a Rottlerin-sensitive pathway.

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Nabin Malla

Full Text Available BACKGROUND: Previously we have shown that a fraction of the matrix metalloproteinase-9 (MMP-9 synthesized by the macrophage cell line THP-1 was bound to a chondroitin sulphate proteoglycan (CSPG core protein as a reduction sensitive heteromer. Several biochemical properties of the enzyme were changed when it was bound to the CSPG. METHODOLOGY/PRINCIPAL FINDINGS: By use of affinity chromatography, zymography, and radioactive labelling, various macrophage stimulators were tested for their effect on the synthesis of the proMMP-9/CSPG heteromer and its components by THP-1 cells. Of the stimulators, only PMA largely increased the biosynthesis of the heteromer. As PMA is an activator of PKC, we determined which PKC isoenzymes were expressed by performing RT-PCR and Western Blotting. Subsequently specific inhibitors were used to investigate their involvement in the biosynthesis of the heteromer. Of the inhibitors, only Rottlerin repressed the biosynthesis of proMMP-9/CSPG and its two components. Much lower concentrations of Rottlerin were needed to reduce the amount of CSPG than what was needed to repress the synthesis of the heteromer and MMP-9. Furthermore, Rottlerin caused a minor reduction in the activation of the PKC isoenzymes δ, ε, θ and υ (PKD3 in both control and PMA exposed cells. CONCLUSIONS/SIGNIFICANCE: The biosynthesis of the proMMP-9/CSPG heteromer and proMMP-9 in THP-1 cells involves a Rottlerin-sensitive pathway that is different from the Rottlerin sensitive pathway involved in the CSPG biosynthesis. MMP-9 and CSPGs are known to be involved in various physiological and pathological processes. Formation of complexes may influence both the specificity and localization of the enzyme. Therefore, knowledge about biosynthetic pathways and factors involved in the formation of the MMP-9/CSPG heteromer may contribute to insight in the heteromers biological function as well as pointing to future targets for therapeutic agents.

Probing cell internalisation mechanics with polymer capsules.

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Chen, Xi; Cui, Jiwei; Ping, Yuan; Suma, Tomoya; Cavalieri, Francesca; Besford, Quinn A; Chen, George; Braunger, Julia A; Caruso, Frank

2016-10-06

We report polymer capsule-based probes for quantifying the pressure exerted by cells during capsule internalisation (P in ). Poly(methacrylic acid) (PMA) capsules with tuneable mechanical properties were fabricated through layer-by-layer assembly. The P in was quantified by correlating the cell-induced deformation with the ex situ osmotically induced deformation of the polymer capsules. Ultimately, we found that human monocyte-derived macrophage THP-1 cells exerted up to approximately 360 kPa on the capsules during internalisation.

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

International Nuclear Information System (INIS)

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

2017-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2017-03-01

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

Prediction of preservative sensitization potential using surface marker CD86 and/or CD54 expression on human cell line, THP-1.

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Sakaguchi, Hitoshi; Miyazawa, Masaaki; Yoshida, Yukiko; Ito, Yuichi; Suzuki, Hiroyuki

2007-02-01

Preservatives are important components in many products, but have a history of purported allergy. Several assays [e.g., guinea pig maximization test (GPMT), local lymph node assay (LLNA)] are used to evaluate allergy potential of preservatives. We recently developed the human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test using human THP-1 cells. This test evaluates the augmentation of CD86 and CD54 expression, which are key events in the sensitization process, as an indicator of allergy following treatment with test chemical. Earlier, we found that a sub-toxic concentration was needed for the up-regulation of surface marker expression. In this study, we further evaluate the capability of h-CLAT to predict allergy potential using eight preservatives. Cytotoxicity was determined using propidium iodide with flow cytometry analysis and five doses that produce a 95, 85, 75, 65, and 50% cell viability were selected. If a material did not have any cytotoxicity at the highest technical dose (HTD), five doses are set using serial 1.3 dilutions of the HTD. The test materials used were six known allergic preservatives (e.g., methylchloroisothiazolinone/methylisothiazolinone, formaldehyde), and two non-allergic preservatives (methylparaben and 4-hydroxybenzoic acid). All allergic preservatives augmented CD86 and/or CD54 expression, indicating h-CLAT correctly identified the allergens. No augmentation was observed with the non-allergic preservatives; also correctly identified by h-CLAT. In addition, we report two threshold concentrations that may be used to categorize skin sensitization potency like the LLNA estimated concentration that yield a three-fold stimulation (EC3) value. These corresponding values are the estimated concentration which gives a relative fluorescence intensity (RFI) = 150 for CD86 and an RFI = 200 for CD54. These data suggest that h-CLAT, using THP-1 cells, may be able to predict the allergy potential of preservatives and

Xylitol, an anticaries agent, exhibits potent inhibition of inflammatory responses in human THP-1-derived macrophages infected with Porphyromonas gingivalis.

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Park, Eunjoo; Na, Hee Sam; Kim, Sheon Min; Wallet, Shannon; Cha, Seunghee; Chung, Jin

2014-06-01

Xylitol is a well-known anticaries agent and has been used for the prevention and treatment of dental caries. In this study, the anti-inflammatory effects of xylitol are evaluated for possible use in the prevention and treatment of periodontal infections. Cytokine expression was stimulated in THP-1 (human monocyte cell line)-derived macrophages by live Porphyromonas gingivalis, and enzyme-linked immunosorbent assay and a commercial multiplex assay kit were used to determine the effects of xylitol on live P. gingivalis-induced production of cytokine. The effects of xylitol on phagocytosis and the production of nitric oxide were determined using phagocytosis assay, viable cell count, and Griess reagent. The effects of xylitol on P. gingivalis adhesion were determined by immunostaining, and costimulatory molecule expression was examined by flow cytometry. Live P. gingivalis infection increased the production of representative proinflammatory cytokines, such as tumor necrosis factor-α and interleukin (IL)-1β, in a multiplicity of infection- and time-dependent manner. Live P. gingivalis also enhanced the release of cytokines and chemokines, such as IL-12 p40, eotaxin, interferon γ-induced protein 10, monocyte chemotactic protein-1, and macrophage inflammatory protein-1. The pretreatment of xylitol significantly inhibited the P. gingivalis-induced cytokines production and nitric oxide production. In addition, xylitol inhibited the attachment of live P. gingivalis on THP-1-derived macrophages. Furthermore, xylitol exerted antiphagocytic activity against both Escherichia coli and P. gingivalis. These findings suggest that xylitol acts as an anti-inflammatory agent in THP-1-derived macrophages infected with live P. gingivalis, which supports its use in periodontitis.

The toxicity of rifampicin polylactic acid nanoparticles against Mycobacterium bovis BCG and human macrophage THP-1 cell line

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Erokhina, M; Rybalkina, E; Lepekha, L; Barsegyan, G; Onishchenko, G

2015-01-01

Tuberculosis is rapidly becoming a major health problem. The rise in tuberculosis incidence stimulates efforts to develop more effective delivery systems for the existing antituberculous drugs while decreasing the side effects. The nanotechnology may provide novel drug delivery tools allowing controlled drug release. Rifampicin is one of the main antituberculous drugs, characterized by high toxicity, and Poly (L-lactic acid) (PLLA) is a biodegradable polymer used for the preparation of encapsulated drugs. The aim of our work was to evaluate the toxicity of rifampicin-PLLA nanoparticles against Mycobacterium bovis BCG using human macrophage THP-1 cell line. Our data demonstrate that rifampicin-PLLA is effective against M. bovis BCG in the infected macrophages. The drug is inducing the dysfunction of mitochondria and apoptosis in the macrophages and is acting as a potential substrate of Pgp thereby modulating cell chemosensitivity. The severity of the toxic effects of the rifampicin-PLLA nanoparticles is increasing in a dose-dependent manner. We suggest that free rifampicin induces death of M. bovis BCG after PLLA degradation and diffusion from phago-lysosomes to cytoplasm causing mitochondria dysfunction and affecting the Pgp activity. (paper)

Involvement of PKCα in PMA-induced facilitation of exocytosis and vesicle fusion in PC12 cells

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Xue Renhao; Zhao Yanying; Chen Peng

2009-01-01

Phorbol-12-myristate-13-acetate, a stable analog of the important signaling membrane lipid diacylglycerol (DAG), is known to potentiate exocytosis and modulate vesicle fusion kinetics in neurons and endocrine cells. The exact mechanisms underlying the actions of PMA, however, is often not clear, largely because of the diversity of the DAG/PMA receptors involved in the exocytotic process, which include, most notably, various isoforms of protein kinase C (PKC). In this study, the roles of PKCα in PMA-mediated regulation of exocytosis were investigated by over-expressing wild-type PKCα (wt-PKCα) or dominant negative PKCα (dn-PKCα). Amperometric measurements based on carbon fiber microelectrodes demonstrated that PKCα has a key role in the PMA-mediated facilitation of exocytosis and vesicle fusion in neuroendocrine PC12 cells.

Pertussis Toxin Exploits Host Cell Signaling Pathways Induced by Meningitis-Causing E. coli K1-RS218 and Enhances Adherence of Monocytic THP-1 Cells to Human Cerebral Endothelial Cells

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Laura Julia Starost

2016-10-01

Full Text Available Pertussis toxin (PTx, the major virulence factor of the whooping cough-causing bacterial pathogen Bordetella pertussis, permeabilizes the blood–brain barrier (BBB in vitro and in vivo. Breaking barriers might promote translocation of meningitis-causing bacteria across the BBB, thereby facilitating infection. PTx activates several host cell signaling pathways exploited by the neonatal meningitis-causing Escherichia coli K1-RS218 for invasion and translocation across the BBB. Here, we investigated whether PTx and E. coli K1-RS218 exert similar effects on MAPK p38, NF-κB activation and transcription of downstream targets in human cerebral endothelial TY10 cells using qRT-PCR, Western blotting, and ELISA in combination with specific inhibitors. PTx and E. coli K1-RS218 activate MAPK p38, but only E. coli K1-RS218 activates the NF-κB pathway. mRNA and protein levels of p38 and NF-κB downstream targets including IL-6, IL-8, CxCL-1, CxCL-2 and ICAM-1 were increased. The p38 specific inhibitor SB203590 blocked PTx-enhanced activity, whereas E. coli K1-RS218’s effects were inhibited by the NF-κB inhibitor Bay 11-7082. Further, we found that PTx enhances the adherence of human monocytic THP-1 cells to human cerebral endothelial TY10 cells, thereby contributing to enhanced translocation. These modulations of host cell signaling pathways by PTx and meningitis-causing E. coli support their contributions to pathogen and monocytic THP-1 cells translocation across the BBB.

KCl stimulation increases norepinephrine transporter function in PC12 cells.

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Mandela, Prashant; Ordway, Gregory A

2006-09-01

The norepinephrine transporter (NET) plays a pivotal role in terminating noradrenergic signaling and conserving norepinephrine (NE) through the process of re-uptake. Recent evidence suggests a close association between NE release and regulation of NET function. The present study evaluated the relationship between release and uptake, and the cellular mechanisms that govern these processes. KCl stimulation of PC12 cells robustly increased [3H]NE uptake via the NET and simultaneously increased [3H]NE release. KCl-stimulated increases in uptake and release were dependent on Ca2+. Treatment of cells with phorbol-12-myristate-13-acetate (PMA) or okadaic acid decreased [3H]NE uptake but did not block KCl-stimulated increases in [3H]NE uptake. In contrast, PMA increased [3H]NE release and augmented KCl-stimulated release, while okadaic acid had no effects on release. Inhibition of Ca2+-activated signaling cascades with KN93 (a Ca2+ calmodulin-dependent kinase inhibitor), or ML7 and ML9 (myosin light chain kinase inhibitors), reduced [3H]NE uptake and blocked KCl-stimulated increases in uptake. In contrast, KN93, ML7 and ML9 had no effect on KCl-stimulated [3H]NE release. KCl-stimulated increases in [3H]NE uptake were independent of transporter trafficking to the plasma membrane. While increases in both NE release and uptake mediated by KCl stimulation require Ca2+, different intracellular mechanisms mediate these two events.

Synergistic effect of hydrogen peroxide on polyploidization during the megakaryocytic differentiation of K562 leukemia cells by PMA.

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Ojima, Yoshihiro; Duncan, Mark Thompson; Nurhayati, Retno Wahyu; Taya, Masahito; Miller, William Martin

2013-08-15

The human myelogenous cell line, K562 has been extensively used as a model for the study of megakaryocytic (MK) differentiation, which could be achieved by exposure to phorbol 12-myristate 13-acetate (PMA). In this study, real-time PCR analysis revealed that the expression of catalase (cat) was significantly repressed during MK differentiation of K562 cells induced by PMA. In addition, PMA increased the intracellular reactive oxygen species (ROS) concentration, suggesting that ROS was a key factor for PMA-induced differentiation. PMA-differentiated K562 cells were exposed to hydrogen peroxide (H2O2) to clarify the function of ROS during MK differentiation. Interestingly, the percentage of high-ploidy (DNA content >4N) cells with H2O2 was 34.8±2.3% at day 9, and was 70% larger than that without H2O2 (21.5±0.8%). Further, H2O2 addition during the first 3 days of PMA-induced MK differentiation had the greatest effect on polyploidization. In an effort to elucidate the mechanisms of enhanced polyploidization by H2O2, the BrdU assay clearly indicated that H2O2 suppressed the division of 4N cells into 2N cells, followed by the increased polyploidization of K562 cells. These findings suggest that the enhancement in polyploidization mediated by H2O2 is due to synergistic inhibition of cytokinesis with PMA. Although H2O2 did not increase ploidy during the MK differentiation of primary cells, we clearly observed that cat expression was repressed in both immature and mature primary MK cells, and that treatment with the antioxidant N-acetylcysteine effectively blocked and/or delayed the polyploidization of immature MK cells. Together, these findings suggest that MK cells are more sensitive to ROS levels during earlier stages of maturation. Copyright © 2013 Elsevier Inc. All rights reserved.

Serum amyloid A stimulates matrix-metalloproteinase-9 upregulation via formyl peptide receptor like-1-mediated signaling in human monocytic cells

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Lee, Ha Young; Kim, Mi-Kyoung; Park, Kyoung Sun; Bae, Yun Hee; Yun, Jeanho; Park, Joo-In; Kwak, Jong-Young; Bae, Yoe-Sik

2005-01-01

In the present study, we found that serum amyloid A (SAA) stimulated matrix-metalloproteinase-9 (MMP-9) upregulation at the transcription and translational levels in THP-1 cells. SAA stimulated the activation of nuclear factor κB (NF-κB), which was required for the MMP-9 upregulation by SAA. The signaling events induced by SAA included the activation of ERK and intracellular calcium rise, which were found to be required for MMP-9 upregulation. Formyl peptide receptor like 1 (FPRL1) was found to be involved in the upregulation of MMP-9 by SAA. Among several FPRL1 agonists, including Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), SAA selectively stimulated MMP-9 upregulation. With respect to the molecular mechanisms involved in the differential action of SAA and WKYMVm, we found that SAA could not competitively inhibit the binding of 125 I-labeled WKYMVm to FPRL1. Taken together, we suggest that SAA plays a role in the modulation of inflammatory and immune responses via FPRL1, by inducing MMP-9 upregulation in human monocytic cells

Stable Toll-Like Receptor 10 Knockdown in THP-1 Cells Reduces TLR-Ligand-Induced Proinflammatory Cytokine Expression

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Hai Van Le

2016-06-01

Full Text Available Toll-like receptor 10 (TLR10 is the only orphan receptor whose natural ligand and function are unknown among the 10 human TLRs. In this study, to test whether TLR10 recognizes some known TLR ligands, we established a stable TLR10 knockdown human monocytic cell line THP-1 using TLR10 short hairpin RNA lentiviral particle and puromycin selection. Among 60 TLR10 knockdown clones that were derived from each single transduced cell, six clones were randomly selected, and then one of those clones, named E7, was chosen for the functional study. E7 exhibited approximately 50% inhibition of TLR10 mRNA and protein expression. Of all the TLRs, only the expression of TLR10 changed significantly in this cell line. Additionally, phorbol 12-myristate 13-acetate-induced macrophage differentiation of TLR10 knockdown cells was not affected in the knockdown cells. When exposed to TLR ligands, such as synthetic diacylated lipoprotein (FSL-1, lipopolysaccharide (LPS, and flagellin, significant induction of proinflammatory cytokine gene expression including Interleukin-8 (IL-8, Interleukin-1 beta (IL-1β, Tumor necrosis factor-alpha (TNF-α and Chemokine (C–C Motif Ligand 20 (CCL20 expression, was found in the control THP-1 cells, whereas the TLR10 knockdown cells exhibited a significant reduction in the expression of IL-8, IL-1β, and CCL20. TNF-α was the only cytokine for which the expression did not decrease in the TLR10 knockdown cells from that measured in the control cells. Analysis of putative binding sites for transcription factors using a binding-site-prediction program revealed that the TNF-α promoter does not have putative binding sites for AP-1 or c-Jun, comprising a major transcription factor along with NF-κB for TLR signaling. Our results suggest that TLR10 is involved in the recognition of FSL-1, LPS, and flagellin and TLR-ligand-induced expression of TNF-α does not depend on TLR10.

Stable Toll-Like Receptor 10 Knockdown in THP-1 Cells Reduces TLR-Ligand-Induced Proinflammatory Cytokine Expression.

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Le, Hai Van; Kim, Jae Young

2016-06-01

Toll-like receptor 10 (TLR10) is the only orphan receptor whose natural ligand and function are unknown among the 10 human TLRs. In this study, to test whether TLR10 recognizes some known TLR ligands, we established a stable TLR10 knockdown human monocytic cell line THP-1 using TLR10 short hairpin RNA lentiviral particle and puromycin selection. Among 60 TLR10 knockdown clones that were derived from each single transduced cell, six clones were randomly selected, and then one of those clones, named E7, was chosen for the functional study. E7 exhibited approximately 50% inhibition of TLR10 mRNA and protein expression. Of all the TLRs, only the expression of TLR10 changed significantly in this cell line. Additionally, phorbol 12-myristate 13-acetate-induced macrophage differentiation of TLR10 knockdown cells was not affected in the knockdown cells. When exposed to TLR ligands, such as synthetic diacylated lipoprotein (FSL-1), lipopolysaccharide (LPS), and flagellin, significant induction of proinflammatory cytokine gene expression including Interleukin-8 (IL-8), Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha (TNF-α) and Chemokine (C-C Motif) Ligand 20 (CCL20) expression, was found in the control THP-1 cells, whereas the TLR10 knockdown cells exhibited a significant reduction in the expression of IL-8, IL-1β, and CCL20. TNF-α was the only cytokine for which the expression did not decrease in the TLR10 knockdown cells from that measured in the control cells. Analysis of putative binding sites for transcription factors using a binding-site-prediction program revealed that the TNF-α promoter does not have putative binding sites for AP-1 or c-Jun, comprising a major transcription factor along with NF-κB for TLR signaling. Our results suggest that TLR10 is involved in the recognition of FSL-1, LPS, and flagellin and TLR-ligand-induced expression of TNF-α does not depend on TLR10.

PMA Induces Vaccine Adjuvant Activity by the Modulation of TLR Signaling Pathway

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Dool-Ri Oh

2014-01-01

Full Text Available Toll-like receptor (TLR ligands are being developed for use as vaccine adjuvants and as immunomodulators because of their ability to stimulate innate and adaptive immune responses. Flagellin, a TLR5 ligand, was reported to show potent mucosal vaccine adjuvant activity. To identify ligands that potentiate the adjuvant activity of flagellin, we screened a plant library using HEK293T cells transiently cotransfected with phTLR5 and pNF-κB-SEAP plasmids. The 90% EtOH extract from Croton tiglium showed significant NF-κB transactivation in a TLR5-independent manner along with the increase of a flagellin activity. We have studied to characterize an active component from Croton tiglium and to elucidate the action mechanisms. Phorbol 12-myristate 13-acetate (PMA was isolated as an active component of Croton tiglium by activity-guided fractionation, column chromatography, HPLC, NMR, and MS. PMA at a range of nM induced PKC-dependent NF-κB activation and IL-8 production in both TLR5− and TLR5+ assay systems. In in vivo mouse vaccination model, PMA induced antigen-specific IgG and IgA antibody responses and increased IL-12 production corresponding to T cell responses in spleen lymphocytes. These results suggest that PMA would serve as an efficacious mucosal vaccine adjuvant.

Protein kinase C isoforms in bovine aortic endothelial cells: role in regulation of P2Y- and P2U-purinoceptor-stimulated prostacyclin release.

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Patel, V; Brown, C; Boarder, M R

1996-05-01

1. Enhanced synthesis of prostacyclin (PGI2) and inositol polyphosphates in bovine aortic endothelial cells in response to ATP and ADP is mediated by co-existing P2Y- and P2U-purinoceptors. Here we examine the regulation of these responses by isoforms of protein kinase C (PKC). 2. Immunoblots with antisera specific for 8 different PKC isoforms revealed the presence of alpha, epsilon and zeta, while no immunoreactivity was found for beta, gamma, delta, eta and theta isoforms. PKC-alpha was largely cytosolic in unstimulated cells and almost all translocated to the membrane (Triton X-100 soluble) after a 1 min treatment with the PKC activating phorbol myristate acetate (PMA); PKC-epsilon was always in a Triton X-100 insoluble membrane fraction, while PKC-zeta was found in both soluble and membrane bound (Triton X-100 soluble) forms in the unstimulated cells and was unaffected by PMA. 3. Treatment with PMA for 6 h led to a 90% downregulation of PKC-alpha, while the immunoreactivity to the epsilon and zeta isoforms remained largely unchanged. 4. After either 10 min or 6 h exposure to PMA the PGI2 response to activation of both receptors was enhanced, while the inositol 1,4,5-trisphosphate response to P2Y-purinoceptor activation was substantially attenuated and the P2U-purinoceptor response was unchanged. Thus the PGI2 response to PMA under conditions when 90% of the PKC-alpha was lost resembles that seen on acute stimulation of PKC by PMA, and the PGI2 response does not correlate with phospholipase C response. 5. Inhibition of PKC with the isoform non-selective inhibitors, Ro 31-8220 and Go 6850 abolished the PGI2 response to both P2U- and P2Y-purinoceptor stimulation. However, Go 6976, which preferentially inhibits Ca2+ sensitive isoforms (such as PKC-alpha) and not Ca2+ insensitive isoforms (such as PKC-epsilon), had no effect on the PGI2 response. 6. The results show that there is a requirement for PKC in the stimulation of PGI2 production by endothelial P2Y- and P2U

Role of Protein Kinase C in Endothelin Converting Enzyme-1 trafficking and shedding from endothelial cells

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Kuruppu, Sanjaya, E-mail: Sanjaya.Kuruppu@med.monash.edu.au [Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Vic. 3800 (Australia); Tochon-Danguy, Natalie; Ian Smith, A. [Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Vic. 3800 (Australia)

2010-07-23

Research highlights: {yields} PKC activation increases the trafficking of ECE-1 to the cell surface. {yields} This in turn leads to an increase in the amount of ECE-1 shed. {yields} Only the catalytically active C-terminal region is shed from the cell surface. -- Abstract: This study aimed to determine the consequences of Protein Kinase C (PKC) mediated Endothelin Converting Enzyme-1 (ECE-1) phosphorylation and its relationship to ECE-1 expression and shedding. The proteins on the surface of EA.hy926 cells were labelled with EZ-Link NHS-SS-Biotin both prior to (control) and following stimulation by 2 {mu}M phorbol 12-myristate 13-acetate (PMA) which activates PKC. The biotinylated proteins were isolated using neutravidin beads, resolved by gel electrophoresis and analysed by western blotting using anti-ECE-1 antibodies. Significant increase in ECE-1 expression at the cell surface was observed following stimulation by PMA, compared to unstimulated control cells (170 {+-} 32.3% of control, n = 5). The ECE-1 activity (expressed as {mu}M substrate cleaved/min) was determined by monitoring the cleavage of a quenched fluorescent substrate. The specificity of cleavage was confirmed using the ECE-1 inhibitor (CGS35066). The stimulation of cells by PMA (1 {mu}M, 6 h) significantly increased the ECE-1 activity (0.28 {+-} 0.02; n = 3) compared to the control (0.07 {+-} 0.02; n = 3). This increase was prevented by prior incubation with the PKC inhibitor bisindolymaleimide (BIM; 2 {mu}M for 1 h; 0.10 {+-} 0.01; n = 3). Treatment with PMA also increased the activity of ECE-1 in the media (0.18 {+-} 0.01; n = 3) compared to control (0.08 {+-} 0.01; n = 3). In addition, this study confirmed by western immunoblotting that only the extracellular region of ECE-1 is released from the cell surface. These data indicate for the first time that PKC activation induces the trafficking and shedding of ECE to and from the cell surface, respectively.

Hydrolysis products generated by lipoprotein lipase and endothelial lipase differentially impact THP-1 macrophage cell signalling pathways.

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Essaji, Yasmin; Yang, Yanbo; Albert, Carolyn J; Ford, David A; Brown, Robert J

2013-08-01

Macrophages express lipoprotein lipase (LPL) and endothelial lipase (EL) within atherosclerotic plaques; however, little is known about how lipoprotein hydrolysis products generated by these lipases might affect macrophage cell signalling pathways. We hypothesized that hydrolysis products affect macrophage cell signalling pathways associated with atherosclerosis. To test our hypothesis, we incubated differentiated THP-1 macrophages with products from total lipoprotein hydrolysis by recombinant LPL or EL. Using antibody arrays, we found that the phosphorylation of six receptor tyrosine kinases and three signalling nodes--most associated with atherosclerotic processes--was increased by LPL derived hydrolysis products. EL derived hydrolysis products only increased the phosphorylation of tropomyosin-related kinase A, which is also implicated in playing a role in atherosclerosis. Using electrospray ionization-mass spectrometry, we identified the species of triacylglycerols and phosphatidylcholines that were hydrolyzed by LPL and EL, and we identified the fatty acids liberated by gas chromatography-mass spectrometry. To determine if the total liberated fatty acids influenced signalling pathways, we incubated differentiated THP-1 macrophages with a mixture of the fatty acids that matched the concentrations of liberated fatty acids from total lipoproteins by LPL, and we subjected cell lysates to antibody array analyses. The analyses showed that only the phosphorylation of Akt was significantly increased in response to fatty acid treatment. Overall, our study shows that macrophages display potentially pro-atherogenic signalling responses following acute treatments with LPL and EL lipoprotein hydrolysis products.

Ubiquitous hazardous metal lead induces TNF-{alpha} in human phagocytic THP-1 cells: Primary role of ERK 1/2

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Khan, Mohd Imran [Fiber Toxicology Division, Indian Institute of Toxicology Research, Council of Scientific and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O Box 80, Lucknow 226001, U.P. (India); Islam, Najmul [Department of Biochemistry, J.N Medical College, Aligarh Muslim University, Aligarh (India); Sahasrabuddhe, Amogh A. [Molecular and Structural Biology Division, Central Drug Research Institute, Lucknow (India); Mahdi, Abbas Ali [Department of Biochemistry, C.S.M. Medical University, Lucknow (India); Siddiqui, Huma; Ashquin, Mohd [Fiber Toxicology Division, Indian Institute of Toxicology Research, Council of Scientific and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O Box 80, Lucknow 226001, U.P. (India); Ahmad, Iqbal, E-mail: ahmadi@sify.com [Fiber Toxicology Division, Indian Institute of Toxicology Research, Council of Scientific and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O Box 80, Lucknow 226001, U.P. (India)

2011-05-15

Induction of tumor necrosis factor-{alpha} (TNF-{alpha}) in response to lead (Pb) exposure has been implicated in its immunotoxicity. However, the molecular mechanism by which Pb upregulates the level of TNF-{alpha} is wagely known. An attempt was therefore made to elucidate the mechanistic aspect of TNF-{alpha} induction, mainly focusing transcriptional and post transcriptional regulation via mitogen activated protein kinases (MAPKs) activation. We observed that exposure of Pb to human monocytic THP-1 cells resulted in significant enhanced production of TNF-{alpha} m-RNA and protein secretion. Moreover, the stability of TNF-{alpha} m-RNA was also increased as indicated by its half life. Notably, activation of ERK 1/2, p38 and JNK in Pb exposed THP-1 was also evident. Specific inhibitor of ERK1/2, PD 98059 caused significant inhibition in production and stability of TNF-{alpha} m-RNA. However, SB 203580 partially inhibited production and stability of TNF-{alpha} m-RNA. Interestingly, a combined exposure of these two inhibitors completely blocked modulation of TNF-{alpha} m-RNA. Data tends to suggest that expression and stability of TNF-{alpha} induction due to Pb exposure is mainly regulated through ERK. Briefly, these observations are useful in understanding some mechanistic aspects of proinflammatory and immunotoxicity of Pb, a globally acknowledged key environmental contaminant.

Ubiquitous hazardous metal lead induces TNF-α in human phagocytic THP-1 cells: Primary role of ERK 1/2

International Nuclear Information System (INIS)

Khan, Mohd Imran; Islam, Najmul; Sahasrabuddhe, Amogh A.; Mahdi, Abbas Ali; Siddiqui, Huma; Ashquin, Mohd; Ahmad, Iqbal

2011-01-01

Induction of tumor necrosis factor-α (TNF-α) in response to lead (Pb) exposure has been implicated in its immunotoxicity. However, the molecular mechanism by which Pb upregulates the level of TNF-α is wagely known. An attempt was therefore made to elucidate the mechanistic aspect of TNF-α induction, mainly focusing transcriptional and post transcriptional regulation via mitogen activated protein kinases (MAPKs) activation. We observed that exposure of Pb to human monocytic THP-1 cells resulted in significant enhanced production of TNF-α m-RNA and protein secretion. Moreover, the stability of TNF-α m-RNA was also increased as indicated by its half life. Notably, activation of ERK 1/2, p38 and JNK in Pb exposed THP-1 was also evident. Specific inhibitor of ERK1/2, PD 98059 caused significant inhibition in production and stability of TNF-α m-RNA. However, SB 203580 partially inhibited production and stability of TNF-α m-RNA. Interestingly, a combined exposure of these two inhibitors completely blocked modulation of TNF-α m-RNA. Data tends to suggest that expression and stability of TNF-α induction due to Pb exposure is mainly regulated through ERK. Briefly, these observations are useful in understanding some mechanistic aspects of proinflammatory and immunotoxicity of Pb, a globally acknowledged key environmental contaminant.

Differentiated THP-1 Cells Exposed to Pathogenic and Nonpathogenic Borrelia Species Demonstrate Minimal Differences in Production of Four Inflammatory Cytokines.

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Stokes, John V; Moraru, Gail M; McIntosh, Chelsea; Kummari, Evangel; Rausch, Keiko; Varela-Stokes, Andrea S

2016-11-01

Tick-borne borreliae include Lyme disease and relapsing fever agents, and they are transmitted primarily by ixodid (hard) and argasid (soft) tick vectors, respectively. Tick-host interactions during feeding are complex, with host immune responses influenced by biological differences in tick feeding and individual differences within and between host species. One of the first encounters for spirochetes entering vertebrate host skin is with local antigen-presenting cells, regardless of whether the tick-associated Borrelia sp. is pathogenic. In this study, we performed a basic comparison of cytokine responses in THP-1-derived macrophages after exposure to selected borreliae, including a nonpathogen. By using THP-1 cells, differentiated to macrophages, we eliminated variations in host response and reduced the system to an in vitro model to evaluate the extent to which the Borrelia spp. influence cytokine production. Differentiated THP-1 cells were exposed to four Borrelia spp., Borrelia hermsii (DAH), Borrelia burgdorferi (B31), B. burgdorferi (NC-2), or Borrelia lonestari (LS-1), or lipopolysaccharides (LPS) (activated) or media (no treatment) controls. Intracellular and secreted interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured using flow cytometric and Luminex-based assays, respectively, at 6, 24, and 48 h postexposure time points. Using a general linear model ANOVA for each cytokine, treatment (all Borrelia spp. and LPS compared to no treatment) had a significant effect on secreted TNF-α only. Time point had a significant effect on intracellular IFN-γ, TNF-α and IL-6. However, we did not see significant differences in selected cytokines among Borrelia spp. Thus, in this model, we were unable to distinguish pathogenic from nonpathogenic borreliae using the limited array of selected cytokines. While unique immune profiles may be detectable in an in vitro model and may reveal predictors for pathogenicity in borreliae

Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA)

International Nuclear Information System (INIS)

Vesper, Stephen; McKinstry, Craig A.; Hartmann, Chris; Neace, Michelle; Yoder, Stephanie; Vesper, Alex

2007-01-01

A method is described to discriminate between live and dead cells of the infectious fungi Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Mucor racemosus, Rhizopus stolonifer and Paecilomyces variotii. To test the method, conidial suspensions were heat inactivated at 85 C or held at 5 C (controls) for 1 h. Polycarbonate filters (25 mm diameter, 0.8 (micro)m pore size) were placed on 'welled' slides (14 mm diameter) and the filters treated with either PBS or PMA. Propidium monoazide (PMA), which enters dead cells but not live cells, was incubated with cell suspensions, exposed to blue wavelength light-emitting diodes (LED) to inactivate remaining PMA and secure intercalation of PMA with DNA of dead cells. Treated cells were extracted and the live and dead cells evaluated with quantitative PCR (QPCR). After heat treatment and DNA modification with PMA, all fungal species tested showed an approximate 100- to 1000-fold difference in cell viability estimated by QPCR analysis which was consistent with estimates of viability based on culturing.

Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed

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Makoveichuk, Elena; Sukonina, Valentina; Kroupa, Olessia; Thulin, Petra; Ehrenborg, Ewa; Olivecrona, Thomas; Olivecrona, Gunilla

2012-01-01

Highlights: ► Lipoprotein lipase (LPL) activity is controlled by ANGPTL4 in THP-1 macrophages. ► Both LPL and ANGPTL4 bind to THP-1 macrophages in a heparin-releasable fashion. ► Only monomers of ANGPTL4 are present within THP-1 macrophages. ► Covalent oligomers of ANGPTL4 appear on cell surface and in medium. ► Inactivation of LPL coincide with ANGPTL4 oligomer formation on cell surfaces. -- Abstract: Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPARδ agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.

Evaluation of bioactivityoffucoidanfrom laminariawith in vitrohuman cell culture(THP-1

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Magdalena M. Stefaniak–Vidarsson

2017-09-01

Full Text Available Background: Seaweeds represent one of the few remaining food sources available globally which are not being fully utilized or even over utilized. Kelps (Laminaria spp. are one of the numerous species of brown seaweeds, a popular marine vegetable, which has been used as a source of iodine and minerals for centuries. Kelps contain anionic polysaccharides called fucoidans heteroglycans with L – fucose units. Their monosaccharide composition, physicochemical and bioactive properties vary between seaweed species. The objective of this work was to evaluate the bioactive properties of laminaria fucoidan (L. digitata and L. hyperborea toward THP–1 macrophages, a human macrophage like cell line, and investigate its potential antioxidant and immunomodulatory characteristics. Methods: THP-1 macrophages were incubated with five fucoidan concentrations. The Oxygen Radical Absorbance Capacity (ORAC assay was determined for cell lysates and for the fucoidan extract, in addition to Total Polyphenol Content (TPC. Cytotoxicity of fucoidan was assessed by light microscopy, followed by XTT proliferation assay. Enzyme–linked immunosorbant assays (ELISA were performed to determine concentrations of the secreted tumor necrosis factor α (TNF-α, interleukin 6 (IL–6, and interleukin 10 (IL–10. Results: Fucoidan did not affect macrophage ability to scavenge oxygen radicals (ORAC confirming its antioxidant properties toward activated macrophages. The laminaria fucoidan extract at 100 µg/ml concentration lowered macrophage viability. Lower concentrations of laminaria fucoidan did not have impact on cell viability. Very low concentration of fucoidan at 0.1 µg/ml triggered secretion of TNF-α. However, IL–6 and interleukin IL–10 were expressed when concentration of applied fucoidan was 10 µg/ml indicating bioactivity of laminaria fucoidan through immunomodulatory actions. Conclusions: The study demonstrated how laminaria fucoidan may have bioactive

Expression of surface markers on the human monocytic leukaemia cell line, THP-1, as indicators for the sensitizing potential of chemicals.

Science.gov (United States)

An, Susun; Kim, Seoyoung; Huh, Yong; Lee, Tae Ryong; Kim, Han-Kon; Park, Kui-Lea; Eun, Hee Chul

2009-04-01

Evaluation of skin sensitization potential is an important part of the safety assessment of cosmetic ingredients and topical drugs. Recently, evaluation of changes in surface marker expression induced in dendritic cells (DC) or DC surrogate cell lines following exposure to chemicals represents one approach for in vitro test methods. The study aimed to test the change of expression patterns of surface markers on THP-1 cells by chemicals as a predictive in vitro method for contact sensitization. We investigated the expression of CD54, CD86, CD83, CD80, and CD40 after a 1-day exposure to sensitizers (1-chloro-2,4-dinitrobenzene; 2,4-dinitrofluorobenzene; benzocaine; 5-chloro-2-methyl-4-isothiazolin-3-one; hexyl cinnamic aldehyde; eugenol; nickel sulfate hexahydrate; potassium dichromate; cobalt sulfate; 2-mercaptobenzothiazole; and ammonium tetrachloroplatinate) and non-sensitizers (sodium lauryl sulfate, benzalkonium chloride, lactic acid, salicylic acid, isopropanol, and dimethyl sulphoxide). The test concentrations were 0.1x, 0.5x, and 1x of the 50% inhibitory concentration, and the relative fluorescence intensity was used as an expression indicator. By evaluating the expression patterns of CD54, CD86, and CD40, we could classify the chemicals as sensitizers or non-sensitizers, but CD80 and CD83 showed non-specific patterns of expression. These data suggest that the THP-1 cells are good model for screening contact sensitizers and CD40 could be a useful marker complementary to CD54 and CD86.

Interleukin-6 production by human monocytes treated with granulocyte-macrophage colony-stimulating factor in the presence of lipopolysaccharide of oral microorganisms.

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Baqui, A A; Meiller, T F; Chon, J J; Turng, B F; Falkler, W A

1998-06-01

This study focused on the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide of the putative periodontal pathogens Porphyromonas gingivalis or Fusobacterium nucleatum on IL-6 production by THP-1 cells (a human monocytic cell line). Resting THP-1 cells were alternatively treated with GM-CSF (50 IU/ml) and lipopolysaccharide of P. gingivalis or F. nucleatum, in varying concentrations for varying time periods. IL-6 production in supernatant fluids of treated cells was evaluated by an enzyme-linked immunosorbent assay (ELISA) and a reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate gene expression. Untreated THP-1 cells did not produce IL-6 as determined by ELISA. RT-PCR also failed to detect IL-6 mRNA in untreated THP-1 cells, indicating that IL-6 was not constitutively produced. After stimulation of THP-1 cells with lipopolysaccharide of F. nucleatum or P. gingivalis, IL-6 was produced, peaking at 4 h (200-300 pg/ml) and thereafter sharply declining by 8 h. When GM-CSF was added together with lipopolysaccharide of P. gingivalis or F. nucleatum, there was a synergistic quantitative increase in production of IL-6 as measured by ELISA as compared with lipopolysaccharide alone. IL-6 mRNA was detected by RT-PCR, 15 min after stimulation with lipopolysaccharide of either P. gingivalis or F. nucleatum. GM-CSF supplementation with lipopolysaccharide of P. gingivalis shortened the transcription of IL-6 mRNA to 5 min, a shift which was not observed with lipopolysaccharide of F. nucleatum, possibly indicating a different mechanism of initiation of transcription. Production of IL-6 by GM-CSF-treated THP-1 cells in the presence of lipopolysaccharide of oral microorganisms may provide a model for studying the role of macrophages in acute and chronic periodontal diseases, including the clinical periodontal exacerbation as observed in chemotherapy patients receiving GM-CSF for bone marrow recovery.

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

DEFF Research Database (Denmark)

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

2015-01-01

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

iTRAQ quantitative proteomics-based identification of cell adhesion as a dominant phenotypic modulation in thrombin-stimulated human aortic endothelial cells.

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Wang, Huang-Joe; Chen, Sung-Fang; Lo, Wan-Yu

2015-05-01

The phenotypic changes in thrombin-stimulated endothelial cells include alterations in permeability, cell shape, vasomotor tone, leukocyte trafficking, migration, proliferation, and angiogenesis. Previous studies regarding the pleotropic effects of thrombin on the endothelium used human umbilical vein endothelial cells (HUVECs)-cells derived from fetal tissue that does not exist in adults. Only a few groups have used screening approaches such as microarrays to profile the global effects of thrombin on endothelial cells. Moreover, the proteomic changes of thrombin-stimulated human aortic endothelial cells (HAECs) have not been elucidated. HAECs were stimulated with 2 units/mL thrombin for 5h and their proteome was investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and the MetaCore(TM) software. A total of 627 (experiment A) and 622 proteins (experiment B) were quantified in the duplicated iTRAQ analyses. MetaCore(TM) pathway analysis identified cell adhesion as a dominant phenotype in thrombin-stimulated HAECs. Replicated iTRAQ data revealed that "Cell adhesion_Chemokines and adhesion," "Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity," and "Cell adhesion_Integrin-mediated cell adhesion and migration" were among the top 10 statistically significant pathways. The cell adhesion phenotype was verified by increased THP-1 adhesion to thrombin-stimulated HAECs. In addition, the expression of ICAM-1, VCAM-1, and SELE was significantly upregulated in thrombin-stimulated HAECs. Several regulatory pathways are altered in thrombin-stimulated HAECs, with cell adhesion being the dominant altered phenotype. Our findings show the feasibility of the iTRAQ technique for evaluating cellular responses to acute stimulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Nyctanthes arbortristis mediated synthesis of silver nanoparticles: Cytotoxicity assay against THP-1 human leukemia cell lines

Science.gov (United States)

Kumari, Priti; Kumari, Niraj; Jha, Anal K.; Singh, K. P.; Prasad, K.

2018-05-01

Green synthesis, characterizations and applications of nanoparticles have become an important branch of nanotechnology now a day. In this paper, green synthesis of silver nanoparticles (AgNPs) using the aqueous extract of Nyctanthes arbortristis as a reducing and stabilizing agent, has been discussed. Present synthetic method is very handy, cost-effective and reproducible. Formation of AgNPs was characterized by X-ray diffraction, dynamic light scattering, scanning electron microscopy and UV-visible spectroscopy techniques. The phytochemicals responsible for nano-transformation were principally flavonoids, phenols and glycosides present in the leaves. Further, the dose dependent cytotoxicity assay of biosynthesized AgNPs against THP-1 human leukemia cell lines showed the encouraging results.

21 CFR 814.37 - PMA amendments and resubmitted PMA's.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false PMA amendments and resubmitted PMA's. 814.37 Section 814.37 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... heading and paragraph (b), effective Aug. 16, 2010. For the convenience of the user, the revised text is...

Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed

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Makoveichuk, Elena; Sukonina, Valentina; Kroupa, Olessia [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden); Thulin, Petra; Ehrenborg, Ewa [Atherosclerosis Research Unit, Department of Medicine, Karolinska Institutet, SE-171 76 Stockholm (Sweden); Olivecrona, Thomas [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden); Olivecrona, Gunilla, E-mail: Gunilla.Olivecrona@medbio.umu.se [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden)

2012-08-24

Highlights: Black-Right-Pointing-Pointer Lipoprotein lipase (LPL) activity is controlled by ANGPTL4 in THP-1 macrophages. Black-Right-Pointing-Pointer Both LPL and ANGPTL4 bind to THP-1 macrophages in a heparin-releasable fashion. Black-Right-Pointing-Pointer Only monomers of ANGPTL4 are present within THP-1 macrophages. Black-Right-Pointing-Pointer Covalent oligomers of ANGPTL4 appear on cell surface and in medium. Black-Right-Pointing-Pointer Inactivation of LPL coincide with ANGPTL4 oligomer formation on cell surfaces. -- Abstract: Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPAR{delta} agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.

Generation of Adducts of 4-Hydroxy-2-nonenal with Heat Shock 60 kDa Protein 1 in Human Promyelocytic HL-60 and Monocytic THP-1 Cell Lines

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Alessia Arcaro

2015-01-01

Full Text Available Heat shock 60 kDa protein 1 (HSP60 is a chaperone and stress response protein responsible for protein folding and delivery of endogenous peptides to antigen-presenting cells and also a target of autoimmunity implicated in the pathogenesis of atherosclerosis. By two-dimensional electrophoresis and mass spectrometry, we found that exposure of human promyelocytic HL-60 cells to a nontoxic concentration (10 μM of 4-hydroxy-2-nonenal (HNE yielded a HSP60 modified with HNE. We also detected adducts of HNE with putative uncharacterized protein CXorf49, the product of an open reading frame identified in various cell and tissue proteomes. Moreover, exposure of human monocytic THP-1 cells differentiated with phorbol 12-myristate 13-acetate to 10 μM HNE, and to light density lipoprotein modified with HNE (HNE-LDL or by copper-catalyzed oxidation (oxLDL, but not to native LDL, stimulated the formation of HNE adducts with HSP60, as detected by immunoprecipitation and western blot, well over basal levels. The identification of HNE-HSP60 adducts outlines a framework of mutually reinforcing interactions between endothelial cell stressors, like oxLDL and HSP60, whose possible outcomes, such as the amplification of endothelial dysfunction, the spreading of lipoxidative damage to other proteins, such as CXorf49, the activation of antigen-presenting cells, and the breaking of tolerance to HSP60 are discussed.

Prediction of the contact sensitizing potential of chemicals using analysis of gene expression changes in human THP-1 monocytes.

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Arkusz, Joanna; Stępnik, Maciej; Sobala, Wojciech; Dastych, Jarosław

2010-11-10

The aim of this study was to find differentially regulated genes in THP-1 monocytic cells exposed to sensitizers and nonsensitizers and to investigate if such genes could be reliable markers for an in vitro predictive method for the identification of skin sensitizing chemicals. Changes in expression of 35 genes in the THP-1 cell line following treatment with chemicals of different sensitizing potential (from nonsensitizers to extreme sensitizers) were assessed using real-time PCR. Verification of 13 candidate genes by testing a large number of chemicals (an additional 22 sensitizers and 8 nonsensitizers) revealed that prediction of contact sensitization potential was possible based on evaluation of changes in three genes: IL8, HMOX1 and PAIMP1. In total, changes in expression of these genes allowed correct detection of sensitization potential of 21 out of 27 (78%) test sensitizers. The gene expression levels inside potency groups varied and did not allow estimation of sensitization potency of test chemicals. Results of this study indicate that evaluation of changes in expression of proposed biomarkers in THP-1 cells could be a valuable model for preliminary screening of chemicals to discriminate an appreciable majority of sensitizers from nonsensitizers. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

DEFF Research Database (Denmark)

Kolkova, K; Novitskaya, V; Pedersen, N

2000-01-01

, inhibitors of the nonreceptor tyrosine kinase p59(fyn), PLC, PKC and MEK and an activator of PKC, phorbol-12-myristate-13-acetate (PMA). MEK2 transfection rescued cells treated with all inhibitors. The same was found for PMA treatment, except when cells concomitantly were treated with the MEK inhibitor....... Arachidonic acid rescued cells treated with antibodies to the FGF receptor or the PLC inhibitor, but not cells in which the activity of PKC, p59(fyn), FAK, Ras, or MEK was inhibited. Interaction of NCAM with a synthetic NCAM peptide ligand, known to induce neurite outgrowth, was shown to stimulate...... phosphorylation of the MAP kinases extracellular signal-regulated kinases ERK1 and ERK2. The MAP kinase activation was sustained, because ERK1 and ERK2 were phosphorylated in PC12-E2 cells and primary hippocampal neurons even after 24 hr of cultivation on NCAM-expressing fibroblasts. Based on these results, we...

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

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Liu, Xin; Xue, Yang; Sun, Jiao

2010-01-01

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

Inflammation response and cytotoxic effects in human THP-1 cells of size-fractionated PM10 extracts in a polluted urban site.

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Schilirò, T; Alessandria, L; Bonetta, S; Carraro, E; Gilli, G

2016-02-01

To contribute to a greater characterization of the airborne particulate matter's toxicity, size-fractionated PM10 was sampled during different seasons in a polluted urban site in Torino, a northern Italian city. Three main size fractions (PM10 - 3 μm; PM3 - 0.95 μm; PM THP-1 cells to evaluate their effects on cell proliferation, LDH activity, TNFα, IL-8 and CYP1A1 expression. The mean PM10 concentrations were statistically different in summer and in winter and the finest fraction PMtest) that could be used in the context of the different monitoring programs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cadmium Alters the Concentration of Fatty Acids in THP-1 Macrophages.

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Olszowski, Tomasz; Gutowska, Izabela; Baranowska-Bosiacka, Irena; Łukomska, Agnieszka; Drozd, Arleta; Chlubek, Dariusz

2018-03-01

Fatty acid composition of human immune cells influences their function. The aim of this study was to evaluate the effects of known toxicant and immunomodulator, cadmium, at low concentrations on levels of selected fatty acids (FAs) in THP-1 macrophages. The differentiation of THP-1 monocytes into macrophages was achieved by administration of phorbol myristate acetate. Macrophages were incubated with various cadmium chloride (CdCl 2 ) solutions for 48 h at final concentrations of 5 nM, 20 nM, 200 nM, and 2 μM CdCl 2 . Fatty acids were extracted from samples according to the Folch method. The fatty acid levels were determined using gas chromatography. The following fatty acids were analyzed: long-chain saturated fatty acids (SFAs) palmitic acid and stearic acid, very long-chain saturated fatty acid (VLSFA) arachidic acid, monounsaturated fatty acids (MUFAs) palmitoleic acid, oleic acid and vaccenic acid, and n-6 polyunsaturated fatty acids (PUFAs) linoleic acid and arachidonic acid. Treatment of macrophages with very low concentrations of cadmium (5-200 nM) resulted in significant reduction in the levels of arachidic, palmitoleic, oleic, vaccenic, and linoleic acids and significant increase in arachidonic acid levels (following exposure to 5 nM Cd), without significant reduction of palmitic and stearic acid levels. Treatment of macrophages with the highest tested cadmium concentration (2 μM) produced significant reduction in the levels of all examined FAs: SFAs, VLSFA, MUFAs, and PUFAs. In conclusion, cadmium at tested concentrations caused significant alterations in THP-1 macrophage fatty acid levels, disrupting their composition, which might dysregulate fatty acid/lipid metabolism thus affecting macrophage behavior and inflammatory state.

The effect of agglomeration state of silver and titanium dioxide nanoparticles on cellular response of HepG2, A549 and THP-1 cells.

Science.gov (United States)

Lankoff, Anna; Sandberg, Wiggo J; Wegierek-Ciuk, Aneta; Lisowska, Halina; Refsnes, Magne; Sartowska, Bożena; Schwarze, Per E; Meczynska-Wielgosz, Sylwia; Wojewodzka, Maria; Kruszewski, Marcin

2012-02-05

Nanoparticles (NPs) occurring in the environment rapidly agglomerate and form particles of larger diameters. The extent to which this abates the effects of NPs has not been clarified. The motivation of this study was to examine how the agglomeration/aggregation state of silver (20nm and 200nm) and titanium dioxide (21nm) nanoparticles may affect the kinetics of cellular binding/uptake and ability to induce cytotoxic responses in THP1, HepG2 and A549 cells. Cellular binding/uptake, metabolic activation and cell death were assessed by the SSC flow cytometry measurements, the MTT-test and the propidium iodide assay. The three types of particles were efficiently taken up by the cells, decreasing metabolic activation and increasing cell death in all the cell lines. The magnitude of the studied endpoints depended on the agglomeration/aggregation state of particles, their size, time-point and cell type. Among the three cell lines tested, A549 cells were the most sensitive to these particles in relation to cellular binding/uptake. HepG2 cells showed a tendency to be more sensitive in relation to metabolic activation. THP-1 cells were the most resistant to all three types of particles in relation to all endpoints tested. Our findings suggest that particle features such as size and agglomeration status as well as the type of cells may contribute to nanoparticles biological impact. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA)

Energy Technology Data Exchange (ETDEWEB)

Vesper, Stephen; McKinstry, Craig A.; Hartmann, Chris; Neace, Michelle; Yoder, Stephanie; Vesper, Alex

2007-11-28

A method is described to discriminate between live and dead cells of the infectious fungi Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Mucor racemosus, Rhizopus stolonifer and Paecilomyces variotii. To test the method, conidial suspensions were heat inactivated at 85 °C or held at 5 °C (controls) for 1 h. Polycarbonate filters (25 mm diameter, 0.8 μm pore size) were placed on "welled" slides (14 mm diameter) and the filters treated with either PBS or PMA. Propidium monoazide (PMA), which enters dead cells but not live cells, was incubated with cell suspensions, exposed to blue wavelength light-emitting diodes (LED) to inactivate remaining PMA and secure intercalation of PMAwith DNA of dead cells. Treated cells were extracted and the live and dead cells evaluated with quantitative PCR (QPCR). After heat treatment and DNA modification with PMA, all fungal species tested showed an approximate 100- to 1000-fold difference in cell viability estimated by QPCR analysis which was consistent with estimates of viability based on culturing.

Evaluation of the Effects of Some Brazilian Medicinal Plants on the Production of TNF-α and CCL2 by THP-1 Cells

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Grasielle S. Gusman

2015-01-01

Full Text Available Several plant species are traditionally used in Brazil to treat various inflammatory diseases. Tumor necrosis factor- (TNF- α and chemokine (C-C motif ligand 2 (CCL2 are key inflammatory mediators in diseases like rheumatoid arthritis and atherosclerosis, respectively; nevertheless, only a few extracts have been assayed against these targets. We herein report the effect of 19 plant extracts on TNF-α and CCL2 release by lipopolysaccharide- (LPS- stimulated THP-1 cells, a human monocytic leukemia cell line, along with their radical scavenging activity on DPPH. The extracts of Caryocar brasiliense, Casearia sylvestris, Coccoloba cereifera, and Terminalia glabrescens inhibited TNF-α production in a concentration-dependent manner. Fractionation of these extracts potentiated the anti-TNF-α effect, which was shown to concentrate in polar fractions, mainly composed by polyphenols. Significant CCL2 inhibition was elicited by Lippia sidoides and Terminalia glabrescens extracts, whose fractionation resulted in highly active low polar fractions. All assayed extracts showed strong radical scavenging activity, but antioxidant activity did not correlate with inhibition of TNF-α or CCL2 production. Our results allowed identifying extracts with selective capacity to block cytokine production; therefore, further purification of these extracts may yield molecules that could be useful in the treatment of chronic inflammatory diseases.

Brucella melitensis and Mycobacterium tuberculosis depict overlapping gene expression patterns induced in infected THP-1 macrophages.

Science.gov (United States)

Masoudian, M; Derakhshandeh, A; Ghahramani Seno, M M

2015-01-01

Pathogens infecting mammalian cells have developed various strategies to suppress and evade their hosts' defensive mechanisms. In this line, the intracellular bacteria that are able to survive and propagate within their host cells must have developed strategies to avert their host's killing attitude. Studying the interface of host-pathogen confrontation can provide valuable information for defining therapeutic approaches. Brucellosis, caused by the Brucella strains, is a zoonotic bacterial disease that affects thousands of humans and animals around the world inflicting discomfort and huge economic losses. Similar to many other intracellular dwelling bacteria, infections caused by Brucella are difficult to treat, and hence any attempt at identifying new and common therapeutic targets would prove beneficial for the purpose of curing infections caused by the intracellular bacteria. In THP-1 macrophage infected with Brucella melitensis we studied the expression levels of four host's genes, i.e. EMP2, ST8SIA4, HCP5 and FRMD5 known to be involved in pathogenesis of Mycobacterium tuberculosis. Our data showed that at this molecular level, except for FRMD5 that was downregulated, the other three genes were upregulated by B. melitensis. Brucella melitensis and M. tuberculosis go through similar intracellular processes and interestingly two of the investigated genes, i.e. EMP2 and ST4SIA8 were upregulated in THP-1 cell infected with B. melitensis similar to that reported for THP-1 cells infected with M. tuberculosis. At the host-pathogen interaction interface, this study depicts overlapping changes for different bacteria with common survival strategies; a fact that implies designing therapeutic approaches based on common targets may be possible.

[The influence of stinging nettle (Urtica dioica L.) extracts on the activity of catalase in THP1 monocytes/macrophages].

Science.gov (United States)

Wolska, Jolanta; Janda, Katarzyna; Szkyrpan, Sylwia; Gutowska, Izabela

2015-01-01

Stinging nettle (Urtica dioicd L.) is one of the most valuable plants used in phytotherapy. The herbal raw material is a herb (Urticae herba), leaves (Urticae folium), roots (Urticae radix) and seeds (Urticae semina). This plant is a good source of vitamins, minerals, fibre, protein and biologically active compounds with antioxidant properties. The literature provides limited information about the chemical composition and properties of the seed heads. No papers are available on the effect of extracts of this plant on catalase activity in human cells. The aim of this study was to investigate the impact of stinging nettle (Urtica dioica L.) extracts on the antioxidant activity of catalase in THP1 macrophages. Two types of extracts: water and alcohol, at two different concentrations, were used in experiments. Nettle was collected in September and October in 2012 in the area of Szczecin. The collected plant material was frozen and lyophilized. After those procedures water and alcohol extracts of nettle were prepared and then added to THP1 cells. The antioxidant activity of catalase was established with the spectrophotometric method. The study showed that both extracts (water and alcohol) significantly increased the antioxidant activity of catalase in THP1 cells. The increase in catalase was directly proportional to the concentration of the added alcohol extract.

Evaluation of selected biomarkers for the detection of chemical sensitization in human skin: a comparative study applying THP-1, MUTZ-3 and primary dendritic cells in culture.

Science.gov (United States)

Hitzler, Manuel; Bergert, Antje; Luch, Andreas; Peiser, Matthias

2013-09-01

Dendritic cells (DCs) exhibit the unique capacity to induce T cell differentiation and proliferation, two processes that are crucially involved in allergic reactions. By combining the exclusive potential of DCs as the only professional antigen-presenting cells of the human body with the well known handling advantages of cell lines, cell-based alternative methods aimed at detecting chemical sensitization in vitro commonly apply DC-like cells derived from myeloid cell lines. Here, we present the new biomarkers programmed death-ligand 1 (PD-L1), DC immunoreceptor (DCIR), IL-16, and neutrophil-activating protein-2 (NAP-2), all of which have been detectable in primary human DCs upon exposure to chemical contact allergens. To evaluate the applicability of DC-like cells in the prediction of a chemical's sensitization potential, the expression of cell surface PD-L1 and DCIR was analyzed. In contrast to primary DCs, only minor subpopulations of MUTZ-3 and THP-1 cells presented PD-L1 or DCIR at their surface. After exposure to increasing concentrations of nickel and cinnamic aldehyde, the expression level of PD-L1 and DCIR revealed much stronger affected on monocyte-derived DCs (MoDCs) or Langerhans cells (MoLCs) when compared to THP-1 and MUTZ-3 cells. Applying protein profiler arrays we further identified the soluble factors NAP-2, IL-16, IL-8 and MIP-1α as sensitive biomarkers showing the capacity to discriminate sensitizing from non-sensitizing chemicals or irritants. An allergen-specific release of IL-8 and MIP-1α could be detected in the supernatants of MoDCs and MoLCs and also in MUTZ-3 and THP-1 cells, though at much lower levels. On the protein and transcriptional level, NAP-2 and IL-16 indicated sensitizers most sensitively and specifically in MoDCs. Altogether, we have proven the reciprocal regulated surface molecules PD-L1 and DCIR and the soluble factors MIP-1α, NAP-2 and IL-16 as reliable biomarkers for chemical sensitization. We further show that primary

Effect of gamma-ray irradiated natural herbal extracts on NF-kB activation in HMC-1 cells

International Nuclear Information System (INIS)

Kim, Yong Soo; Lim, Youn Mook; Gwon, Hui Jeong; Choi, Bo Ram; Nho, Young Chang

2009-01-01

Recently, studies have documented various health benefits of some natural herbal extracts (NHE) such as Houttuynia cordata (H), Centella asiatica (C), Plantago asiatica (P), Morus alba L. (M), and Ulmus davidiana (U). The aim of the present study was to demonstrate the radiation effect on NF-kB activation of the NHE in the human mast cell line (HMC-1). The HMC-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) plus A23187. Both non-and irradiated NHE also significantly inhibited the PMA plus A23187-induced nuclear factor NF-kB activation and also suppressed the expression of activation of NF-kB. These results indicated that the NHE exerted a regulatory effect on inflammatory reactions mediated by mast cells

Effect of gamma-ray irradiated natural herbal extracts on NF-kB activation in HMC-1 cells

Energy Technology Data Exchange (ETDEWEB)

Kim, Yong Soo; Lim, Youn Mook; Gwon, Hui Jeong; Choi, Bo Ram; Nho, Young Chang [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

2009-12-15

Recently, studies have documented various health benefits of some natural herbal extracts (NHE) such as Houttuynia cordata (H), Centella asiatica (C), Plantago asiatica (P), Morus alba L. (M), and Ulmus davidiana (U). The aim of the present study was to demonstrate the radiation effect on NF-kB activation of the NHE in the human mast cell line (HMC-1). The HMC-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) plus A23187. Both non-and irradiated NHE also significantly inhibited the PMA plus A23187-induced nuclear factor NF-kB activation and also suppressed the expression of activation of NF-kB. These results indicated that the NHE exerted a regulatory effect on inflammatory reactions mediated by mast cells.

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

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2006-01-01

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

Serum Levels of IL-1β, IL-6, TGF-β, and MMP-9 in Patients Undergoing Carotid Artery Stenting and Regulation of MMP-9 in a New In Vitro Model of THP-1 Cells Activated by Stenting

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Rongrong Zhang

2015-01-01

Full Text Available Inflammation plays an important role in the pathophysiological process after carotid artery stenting (CAS. Monocyte is a significant source of inflammatory cytokines in vascular remodeling. Telmisartan could reduce inflammation. In our study, we first found that, after CAS, the serum IL-1β, IL-6, TGF-β, and MMP-9 levels were significantly increased, but only MMP-9 level was elevated no less than 3 months. Second, we established a new in vitro model, where THP-1 monocytes were treated with the supernatants of human umbilical vein endothelial cells (HUVECs that were scratched by pipette tips, which mimics monocytes activated by mechanical injury of stenting. The treatment enhanced THP-1 cell adhesion, migration and invasion ability, and the phosphorylation of ERK1/2 and Elk-1 and MMP-9 expression were significantly increased. THP-1 cells pretreated with PD98095 (ERK1/2 inhibitor attenuated the phosphorylation of ERK1/2 and Elk-1 and upregulation of MMP-9, while pretreatment with telmisartan merely decreased the phosphorylation of Elk-1 and MMP-9 expression. These results suggested that IL-1β, IL-6, TGF-β, and MMP-9 participate in the pathophysiological process after CAS. Our new in vitro model mimics monocytes activated by stenting. MMP-9 expression could be regulated through ERK1/2/Elk-1 pathway, and the protective effects of telmisartan after stenting are partly attributed to its MMP-9 inhibition effects via suppression of Elk-1.

Chronic Iron Overload Results in Impaired Bacterial Killing of THP-1 Derived Macrophage through the Inhibition of Lysosomal Acidification

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Kao, Jun-Kai; Wang, Shih-Chung; Ho, Li-Wei; Huang, Shi-Wei; Chang, Shu-Hao; Yang, Rei-Cheng; Ke, Yu-Yuan; Wu, Chun-Ying; Wang, Jiu-Yao; Shieh, Jeng-Jer

2016-01-01

Iron is essential for living organisms and the disturbance of iron homeostasis is associated with altered immune function. Additionally, bacterial infections can cause major complications in instances of chronic iron overload, such as patients with transfusion-dependent thalassemia. Monocytes and macrophages play important roles in maintaining systemic iron homoeostasis and in defense against invading pathogens. However, the effect of iron overload on the function of monocytes and macrophages is unclear. We elucidated the effects of chronic iron overload on human monocytic cell line (THP-1) and THP-1 derived macrophages (TDM) by continuously exposing them to high levels of iron (100 μM) to create I-THP-1 and I-TDM, respectively. Our results show that iron overload did not affect morphology or granularity of I-THP-1, but increased the granularity of I-TDM. Bactericidal assays for non-pathogenic E. coli DH5α, JM109 and pathogenic P. aeruginosa all revealed decreased efficiency with increasing iron concentration in I-TDM. The impaired P. aeruginosa killing ability of human primary monocyte derived macrophages (hMDM) was also found when cells are cultured in iron contained medium. Further studies on the bactericidal activity of I-TDM revealed lysosomal dysfunction associated with the inhibition of lysosomal acidification resulting in increasing lysosomal pH, the impairment of post-translational processing of cathepsins (especially cathepsin D), and decreased autophagic flux. These findings may explain the impaired innate immunity of thalassemic patients with chronic iron overload, suggesting the manipulation of lysosomal function as a novel therapeutic approach. PMID:27244448

PMA-PhyloChip DNA Microarray to Elucidate Viable Microbial Community Structure

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Venkateswaran, Kasthuri J.; Stam, Christina N.; Andersen, Gary L.; DeSantis, Todd

2011-01-01

Since the Viking missions in the mid-1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a "true" estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells. The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method. DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated

PVP-coated silver nanoparticles and silver ions induce reactive oxygen species, apoptosis and necrosis in THP-1 monocytes

DEFF Research Database (Denmark)

Foldbjerg, Rasmus; Olesen, Ping Liu; Hougaard, Mads

2009-01-01

, both Ag NPs and Ag+ were shown to induce apoptosis and necrosis in THP-1 cells depending on dose and exposure time. Furthermore, the presence of apoptosis could be confirmed by the TUNEL method. A number of studies have implicated the production of reactive oxygen species (ROS) in cytotoxicity mediated...... the effect of well characterized, PVP-coated Ag NPs (69 nm ± 3 nm) and Ag+ in a human monocytic cell line (THP-1). Characterization of the Ag NPs was conducted in both stock suspension and cell media with or without serum and antibiotics. By using the flowcytometric annexin V/propidium iodide (PI) assay...... by NPs. We used the fluorogenic probe, 2′,7′-dichlorofluorescein to assess the levels of intracellular ROS during exposure to Ag NPs and Ag+. A drastic increase in ROS levels could be detected after 6–24 h suggesting that oxidative stress is an important mediator of cytotoxicity caused by Ag NPs and Ag+....

PMA-Linked Fluorescence for Rapid Detection of Viable Bacterial Endospores

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LaDuc, Myron T.; Venkateswaran, Kasthuri; Mohapatra, Bidyut

2012-01-01

The most common approach for assessing the abundance of viable bacterial endospores is the culture-based plating method. However, culture-based approaches are heavily biased and oftentimes incompatible with upstream sample processing strategies, which make viable cells/spores uncultivable. This shortcoming highlights the need for rapid molecular diagnostic tools to assess more accurately the abundance of viable spacecraft-associated microbiota, perhaps most importantly bacterial endospores. Propidium monoazide (PMA) has received a great deal of attention due to its ability to differentiate live, viable bacterial cells from dead ones. PMA gains access to the DNA of dead cells through compromised membranes. Once inside the cell, it intercalates and eventually covalently bonds with the double-helix structures upon photoactivation with visible light. The covalently bound DNA is significantly altered, and unavailable to downstream molecular-based manipulations and analyses. Microbiological samples can be treated with appropriate concentrations of PMA and exposed to visible light prior to undergoing total genomic DNA extraction, resulting in an extract comprised solely of DNA arising from viable cells. This ability to extract DNA selectively from living cells is extremely powerful, and bears great relevance to many microbiological arenas.

Immunomodulatory role for membrane vesicles released by THP-1 macrophages and respiratory pathogens during macrophage infection.

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Volgers, Charlotte; Benedikter, Birke J; Grauls, Gert E; Savelkoul, Paul H M; Stassen, Frank R M

2017-11-13

During infection, inflammation is partially driven by the release of mediators which facilitate intercellular communication. Amongst these mediators are small membrane vesicles (MVs) that can be released by both host cells and Gram-negative and -positive bacteria. Bacterial membrane vesicles are known to exert immuno-modulatory and -stimulatory actions. Moreover, it has been proposed that host cell-derived vesicles, released during infection, also have immunostimulatory properties. In this study, we assessed the release and activity of host cell-derived and bacterial MVs during the first hours following infection of THP-1 macrophages with the common respiratory pathogens non-typeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa. Using a combination of flow cytometry, tunable resistive pulse sensing (TRPS)-based analysis and electron microscopy, we demonstrated that the release of MVs occurs by both host cells and bacteria during infection. MVs released during infection and bacterial culture were found to induce a strong pro-inflammatory response by naive THP-1 macrophages. Yet, these MVs were also found to induce tolerance of host cells to secondary immunogenic stimuli and to enhance bacterial adherence and the number of intracellular bacteria. Bacterial MVs may play a dual role during infection, as they can both trigger and dampen immune responses thereby contributing to immune defence and bacterial survival.

Molecular pathway profiling of T lymphocyte signal transduction pathways; Th1 and Th2 genomic fingerprints are defined by TCR and CD28-mediated signaling

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Smeets Ruben L

2012-03-01

Full Text Available Abstract Background T lymphocytes are orchestrators of adaptive immunity. Naïve T cells may differentiate into Th1, Th2, Th17 or iTreg phenotypes, depending on environmental co-stimulatory signals. To identify genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli and pathway inhibitors. Results from these experiments were validated in a human experimental setting using whole blood and purified CD4+ Tcells. Results Calcium-dependent activation of T cells using CD3/CD28 and PMA/CD3 stimulation induced a Th1 expression profile reflected by increased expression of T-bet, RUNX3, IL-2, and IFNγ, whereas calcium-independent activation via PMA/CD28 induced a Th2 expression profile which included GATA3, RXRA, CCL1 and Itk. Knock down with siRNA and gene expression profiling in the presence of selective kinase inhibitors showed that proximal kinases Lck and PKCθ are crucial signaling hubs during T helper cell activation, revealing a clear role for Lck in Th1 development and for PKCθ in both Th1 and Th2 development. Medial signaling via MAPkinases appeared to be less important in these pathways, since specific inhibitors of these kinases displayed a minor effect on gene expression. Translation towards a primary, whole blood setting and purified human CD4+ T cells revealed that PMA/CD3 stimulation induced a more pronounced Th1 specific, Lck and PKCθ dependent IFNγ production, whereas PMA/CD28 induced Th2 specific IL-5 and IL-13 production, independent of Lck activation. PMA/CD3-mediated skewing towards a Th1 phenotype was also reflected in mRNA expression of the master transcription factor Tbet, whereas PMA/CD28-mediated stimulation enhanced GATA3 mRNA expression in primary human CD4+ Tcells. Conclusions This study identifies stimulatory pathways and gene expression profiles for in vitro skewing of T helper cell

The relationship between CD86/CD54 expression and THP-1 cell viability in an in vitro skin sensitization test--human cell line activation test (h-CLAT).

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Sakaguchi, Hitoshi; Ashikaga, Takao; Miyazawa, Masaaki; Kosaka, Nanae; Ito, Yuichi; Yoneyama, Katsurako; Sono, Sakiko; Itagaki, Hiroshi; Toyoda, Hidekazu; Suzuki, Hiroyuki

2009-04-01

Recent regulations for cosmetics in Europe prohibit animal testing for evaluating the sensitization potential of chemicals to improve animal welfare. Yet, there is not an acceptable Organization for Economic Co-operation and Development non-animal skin sensitization test method. Several in vitro skin sensitization methods that focus on the activation of Langerhans cells, including human cell lines, are being evaluated as possible alternatives. In our previous study, we optimized our human cell line activation test (h-CLAT) using THP-1 cells (monocytic leukemia cell line) and conducted an inter-laboratory study. We found that measuring CD86/CD54 expression may be useful for predicting skin sensitization. The aim of this study was to confirm the relationship between CD86/CD54 expression and THP-1 cell viability in the h-CLAT. In this study, 21 allergens (e.g., dinitrochlorobenzene, p-phenylenediamine, Ni) and 8 non-allergens (e.g., SLS, lactic acid) were evaluated. For each chemical, more than 10 concentrations that gave a predicted cell viability range of 20-95% were used. The data showed that expression patterns of CD86/CD54 differed depending on chemical. For most allergens, cytotoxicity (65-90% cell viability) was needed for enhancement of CD86/CD54 expression. The criteria of "CD86 > or = 150 or CD54 > or = 200" resulted in an accuracy of 93%, which confirms appropriate cut-off criteria for h-CLAT. Furthermore, a good correlation was observed between EC3 of local lymph node assay and EC150(CD86) or EC200(CD54) of h-CLAT (12 or 16 chemicals, respectively), which would provide a useful estimate of allergic potency. These findings suggest that h-CLAT would be a good robust in vitro skin sensitization test.

Differences in Intracellular Fate of Two Spotted Fever Group Rickettsia in Macrophage-Like Cells.

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Curto, Pedro; Simões, Isaura; Riley, Sean P; Martinez, Juan J

2016-01-01

Spotted fever group (SFG) rickettsiae are recognized as important agents of human tick-borne diseases worldwide, such as Mediterranean spotted fever (Rickettsia conorii) and Rocky Mountain spotted fever (Rickettsia rickettsii). Recent studies in several animal models have provided evidence of non-endothelial parasitism by pathogenic SFG Rickettsia species, suggesting that the interaction of rickettsiae with cells other than the endothelium may play an important role in pathogenesis of rickettsial diseases. These studies raise the hypothesis that the role of macrophages in rickettsial pathogenesis may have been underappreciated. Herein, we evaluated the ability of two SFG rickettsial species, R. conorii (a recognized human pathogen) and Rickettsia montanensis (a non-virulent member of SFG) to proliferate in THP-1 macrophage-like cells, or within non-phagocytic cell lines. Our results demonstrate that R. conorii was able to survive and proliferate in both phagocytic and epithelial cells in vitro. In contrast, R. montanensis was able to grow in non-phagocytic cells, but was drastically compromised in the ability to proliferate within both undifferentiated and PMA-differentiated THP-1 cells. Interestingly, association assays revealed that R. montanensis was defective in binding to THP-1-derived macrophages; however, the invasion of the bacteria that are able to adhere did not appear to be affected. We have also demonstrated that R. montanensis which entered into THP-1-derived macrophages were rapidly destroyed and partially co-localized with LAMP-2 and cathepsin D, two markers of lysosomal compartments. In contrast, R. conorii was present as intact bacteria and free in the cytoplasm in both cell types. These findings suggest that a phenotypic difference between a non-pathogenic and a pathogenic SFG member lies in their respective ability to proliferate in macrophage-like cells, and may provide an explanation as to why certain SFG rickettsial species are not associated

Differences in intracellular fate of two spotted fever group Rickettsia in macrophage-like cells

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Pedro Curto

2016-07-01

Full Text Available Spotted fever group (SFG rickettsiae are recognized as important agents of human tick-borne diseases worldwide, such as Mediterranean spotted fever (R. conorii and Rocky Mountain spotted fever (R. rickettsii. Recent studies in several animal models have provided evidence of non-endothelial parasitism by pathogenic SFG Rickettsia species, suggesting that the interaction of rickettsiae with cells other than the endothelium may play an important role in pathogenesis of rickettsial diseases. These studies raise the hypothesis that the role of macrophages in rickettsial pathogenesis may have been underappreciated. Herein, we evaluated the ability of two SFG rickettsial species, R. conorii (a recognized human pathogen and R. montanensis (a non-virulent member of SFG to proliferate in THP-1 macrophage-like cells, or within non-phagocytic cell lines. Our results demonstrate that R. conorii was able to survive and proliferate in both phagocytic and epithelial cells in vitro. In contrast, R. montanensis was able to grow in non-phagocytic cells, but was drastically compromised in the ability to proliferate within both undifferentiated and PMA-differentiated THP-1 cells. Interestingly, association assays revealed that R. montanensis was defective in binding to THP-1-derived macrophages; however, the invasion of the bacteria that are able to adhere did not appear to be affected. We have also demonstrated that R. montanensis which entered into THP-1-derived macrophages were rapidly destroyed and partially co-localized with LAMP-2 and cathepsin D, two markers of lysosomal compartments. In contrast, R. conorii was present as intact bacteria and free in the cytoplasm in both cell types. These findings suggest that a phenotypic difference between a non-pathogenic and a pathogenic SFG member lies in their respective ability to proliferate in macrophage-like cells, and may provide an explanation as to why certain SFG rickettsial species are not associated with

Acidic conditions induce the suppression of CD86 and CD54 expression in THP-1 cells.

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Mitachi, Takafumi; Mezaki, Minori; Yamashita, Kunihiko; Itagaki, Hiroshi

2018-01-01

To evaluate the sensitization potential of chemicals in cosmetics, using non-animal methods, a number of in vitro safety tests have been designed. Current assays are based on the expression of cell surface markers, such as CD86 and CD54, which are associated with the activation of dendritic cells, in skin sensitization tests. However, these markers are influenced by culture conditions through activating danger signals. In this study, we investigated the relationship between extracellular pH and the expression of the skin sensitization test human cell line activation test (h-CLAT) markers CD86 and CD54. We measured expression levels after THP-1 cells were exposed to representative contact allergens, i.e., 2,4-dinitrochlorobenzene and imidazolidinyl urea, under acidic conditions. These conditions were set by exposure to hydrochloric acid, lactic acid, and citric acid. An acidic extracellular pH (6-7) suppressed the augmentation of CD86 and CD54 levels by the sensitizer. Additionally, when the CD86/CD54 expression levels were suppressed, a reduction in the intracellular pH was confirmed. Furthermore, we observed that Na + /H + exchanger 1 (NHE-1), a protein that contributes to the regulation of extracellular/intracellular pH, is involved in CD86 and CD54 expression. These findings suggest that the extracellular/intracellular pH has substantial effects on in vitro skin sensitization markers and should be considered in evaluations of the safety of mixtures and commercial products in the future.

Skin sensitizer identification by IL-8 secretion and CD86 expression on THP-1 cells.

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Parise, Carolina Bellini; Sá-Rocha, Vanessa Moura; Moraes, Jane Zveiter

2015-12-25

Substantial progress has been made in the development of alternative methods for skin sensitization in the last decade in several countries around the world. Brazil is experiencing an increasing concern about using animals for product development, since the publication of the Law 9605/1998, which prohibits the use of animals when an alternative method is available. In this way, an in vitro test to evaluate allergenic potential is a pressing need.This preliminary study started setting the use of myelomonocytic THP-1 cell line, according to the human cell line activation test (h-CLAT), already under validation process. We found that 48-h chemical exposure was necessary to identify 22 out of 23 sensitizers by the analyses of CD86 expression. In addition, the CD54 expression analyses presented a poor efficiency to discriminate sensitizers from non-sensitizers in our conditions. In view of these results, we looked for changes of pro-inflammatory interleukin profile. The IL-8 secretion analyses after 24-h chemical incubation seemed to be an alternative for CD54 expression assessing.Altogether, our findings showed that the combination of the analyses of CD86 expression and IL-8 secretion allowed predicting allergenicity.

Combination Treatments with Luteolin and Fisetin Enhance Anti-Inflammatory Effects in High Glucose-Treated THP-1 Cells Through Histone Acetyltransferase/Histone Deacetylase Regulation.

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Kim, Arang; Yun, Jung-Mi

2017-08-01

Hyperglycemia leads to diabetes and its diabetic complications. In this study, we investigated the synergistic effects of luteolin and fisetin on proinflammatory cytokine secretion and its underlying epigenetic regulation in human monocytes exposed to hyperglycemic (HG) concentrations. Human monocytic cells (THP-1) were cultured under controlled (14.5 mM mannitol), normoglycemic (5.5 mM glucose), or HG (20 mM glucose) conditions in the absence or presence of the two phytochemicals for 48 h. Whereas HG conditions significantly induced histone acetylation, nuclear factor-kappa B (NF-κB) activation, interleukin 6, and tumor necrosis factor-α release from THP-1 cells; combination treatments with the two phytochemicals (500 nM fisetin, and l μM and 500 nM luteolin) suppressed NF-κB activity and inflammatory cytokine release. Fisetin, luteolin, and their combination treatments also significantly decreased the activity of histone acetyltransferase, a known NF-κB coactivator; inhibited reactive oxygen species production; and activated sirtuin (SIRT)1 and forkhead box O3a (FOXO3a) expressions (P < .05). Thus, combination treatments with the two phytochemicals inhibited HG condition-induced cytokine production in monocytes, through epigenetic changes involving NF-κB activation. We, therefore, suggest that combination treatments with luteolin and fisetin may be a potential candidate for the treatment and prevention of diabetes and its complications.

Uptake of Eudragit Retard L (Eudragit® RL Nanoparticles by Human THP-1 Cell Line and Its Effects on Hematology and Erythrocyte Damage in Rats

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Mosaad A. Abdel-Wahhab

2014-02-01

Full Text Available The aim of this study was to prepare Eudragit Retard L (Eudragit RL nanoparticles (ENPs and to determine their properties, their uptake by the human THP-1 cell line in vitro and their effect on the hematological parameters and erythrocyte damage in rats. ENPs showed an average size of 329.0 ± 18.5 nm, a positive zeta potential value of +57.5 ± 5.47 mV and nearly spherical shape with a smooth surface. THP-1 cell lines could phagocyte ENPs after 2 h of incubation. In the in vivo study, male Sprague-Dawley rats were exposed orally or intraperitoneally (IP with a single dose of ENP (50 mg/kg body weight. Blood samples were collected after 4 h, 48 h, one week and three weeks for hematological and erythrocytes analysis. ENPs induced significant hematological disturbances in platelets, red blood cell (RBC total and differential counts of white blood cells (WBCs after 4 h, 48 h and one week. ENP increased met-Hb and Co-Hb derivatives and decreased met-Hb reductase activity. These parameters were comparable to the control after three weeks when administrated orally. It could be concluded that the route of administration has a major effect on the induction of hematological disturbances and should be considered when ENPs are applied for drug delivery systems.

Role of protein haptenation in triggering maturation events in the dendritic cell surrogate cell line THP-1

International Nuclear Information System (INIS)

Megherbi, Rym; Kiorpelidou, Evanthia; Foster, Brian; Rowe, Cliff; Naisbitt, Dean J.; Goldring, Christopher E.; Park, B. Kevin

2009-01-01

Dendritic cell (DC) maturation in response to contact sensitizers is a crucial step in the induction of sensitization reactions; however the underlying mechanism of activation remains unknown. To test whether the extent of protein haptenation is a determinant in DC maturation, we tested the effect of five dinitrophenyl (DNP) analogues of different reactivity, on maturation markers in the cell line, THP-1. The potencies of the test compounds in upregulating CD54 levels, inducing IL-8 release and triggering p38 MAPK phosphorylation did not correlate with their ability to deplete intracellular glutathione (GSH) levels or cause cell toxicity. However, the compounds' potency at inducing p38 phosphorylation was significantly associated with the amount of intracellular protein adducts formed (p < 0.05). Inhibition experiments show that, at least for DNFB, p38 MAP kinase signalling controls compound-specific changes in CD54 expression and IL-8 release. 2D-PAGE analysis revealed that all the DNP analogues appeared to bind similar proteins. The analogues failed to activate NFkB, however, they activated Nrf2, which was used as a marker of oxidative stress. Neither GSH depletion, by use of buthionine sulfoximine, nor treatment with the strongly lysine-reactive hapten penicillin elicited maturation. We conclude that protein haptenation, probably through reactive cysteine residues may be a trigger for maturation events in this in vitro model and that p38 activation may be a discriminatory marker for the classification of potency of chemical sensitizers.

Role of protein haptenation in triggering maturation events in the dendritic cell surrogate cell line THP-1.

Science.gov (United States)

Megherbi, Rym; Kiorpelidou, Evanthia; Foster, Brian; Rowe, Cliff; Naisbitt, Dean J; Goldring, Christopher E; Park, B Kevin

2009-07-15

Dendritic cell (DC) maturation in response to contact sensitizers is a crucial step in the induction of sensitization reactions; however the underlying mechanism of activation remains unknown. To test whether the extent of protein haptenation is a determinant in DC maturation, we tested the effect of five dinitrophenyl (DNP) analogues of different reactivity, on maturation markers in the cell line, THP-1. The potencies of the test compounds in upregulating CD54 levels, inducing IL-8 release and triggering p38 MAPK phosphorylation did not correlate with their ability to deplete intracellular glutathione (GSH) levels or cause cell toxicity. However, the compounds' potency at inducing p38 phosphorylation was significantly associated with the amount of intracellular protein adducts formed (p<0.05). Inhibition experiments show that, at least for DNFB, p38 MAP kinase signalling controls compound-specific changes in CD54 expression and IL-8 release. 2D-PAGE analysis revealed that all the DNP analogues appeared to bind similar proteins. The analogues failed to activate NFkB, however, they activated Nrf2, which was used as a marker of oxidative stress. Neither GSH depletion, by use of buthionine sulfoximine, nor treatment with the strongly lysine-reactive hapten penicillin elicited maturation. We conclude that protein haptenation, probably through reactive cysteine residues may be a trigger for maturation events in this in vitro model and that p38 activation may be a discriminatory marker for the classification of potency of chemical sensitizers.

Postprandial phase time influences the uptake of TAG from postprandial TAG-rich lipoproteins by THP-1 macrophages.

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Cabello-Moruno, Rosana; Sinausia, Laura; Botham, Kathleen M; Montero, Emilio; Avella, Michael; Perona, Javier S

2014-11-14

Postprandial TAG-rich lipoproteins (TRL) can be taken up by macrophages, leading to the formation of foam cells, probably via receptor-mediated pathways. The present study was conducted to investigate whether the postprandial time point at which TRL are collected modulates this process. A meal containing refined olive oil was given to nine healthy young men and TRL were isolated from their serum at 2, 4 and 6 h postprandially. The lipid class and apoB compositions of TRL were determined by HPLC and SDS-PAGE, respectively. The accumulation of lipids in macrophages was determined after the incubation of THP-1 macrophages with TRL. The gene expression of candidate receptors was measured by real-time PCR. The highest concentrations of TAG, apoB48 and apoB100 in TRL were observed at 2 h after the consumption of the test meal. However, excessive intracellular TAG accumulation in THP-1 macrophages was observed in response to incubation with TRL isolated at 4 h, when their particle size (estimated as the TAG:apoB ratio) was intermediate. The abundance of mRNA transcripts in macrophages in response to incubation with TRL was down-regulated for LDL receptor (LDLR), slightly up-regulated for VLDL receptor and remained unaltered for LDLR-related protein, but no effect of the postprandial time point was observed. In contrast, the mRNA expression of scavenger receptors SRB1, SRA2 and CD36 was higher when cells were incubated with TRL isolated at 4 h after the consumption of the test meal. In conclusion, TRL led to excessive intracellular TAG accumulation in THP-1 macrophages, which was greater when cells were incubated with intermediate-sized postprandial TRL isolated at 4 h and was associated with a significant increase in the mRNA expression of scavenger receptors.

Arctigenin promotes cholesterol efflux from THP-1 macrophages through PPAR-γ/LXR-α signaling pathway

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Xu, Xiaolin [Department of Cardiothoracic Surgery, Huashan Hospital, Fudan University, Shanghai 200032 (China); Li, Qian [Department of Integrative Medicine and Neurobiology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai (China); Pang, Liewen; Huang, Guoqian; Huang, Jiechun; Shi, Meng; Sun, Xiaotian [Department of Cardiothoracic Surgery, Huashan Hospital, Fudan University, Shanghai 200032 (China); Wang, Yiqing [Department of Cardiothoracic Surgery, Huashan Hospital, Fudan University, Shanghai 200032 (China)

2013-11-15

Highlights: •Arctigenin enhanced cholesterol efflux in oxLDL-loaded THP-1 macrophages. •The expression of ABCA1, ABCG1 and apoE was upregulated in arctigenin-treated cells. •Arctigenin promoted the expression of PPAR-γ and LXR-α. •Inhibition of PPAR-γ or LXR-α reversed arctigenin-mediated biological effects. •Arctigenin promotes cholesterol efflux via activation of PPAR-γ/LXR-α/ABCA1 pathway. -- Abstract: Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α.

Arctigenin promotes cholesterol efflux from THP-1 macrophages through PPAR-γ/LXR-α signaling pathway

International Nuclear Information System (INIS)

Xu, Xiaolin; Li, Qian; Pang, Liewen; Huang, Guoqian; Huang, Jiechun; Shi, Meng; Sun, Xiaotian; Wang, Yiqing

2013-01-01

Highlights: •Arctigenin enhanced cholesterol efflux in oxLDL-loaded THP-1 macrophages. •The expression of ABCA1, ABCG1 and apoE was upregulated in arctigenin-treated cells. •Arctigenin promoted the expression of PPAR-γ and LXR-α. •Inhibition of PPAR-γ or LXR-α reversed arctigenin-mediated biological effects. •Arctigenin promotes cholesterol efflux via activation of PPAR-γ/LXR-α/ABCA1 pathway. -- Abstract: Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α

Virus-producing cells determine the host protein profiles of HIV-1 virion cores

Science.gov (United States)

2012-01-01

Background Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized. Results We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of

Mitogen-activated protein kinase phosphatase-1 expression in macrophages is controlled by lymphocytes during macrophage activation.

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Luo, Chong; Yang, Xiqiang; Yao, Lan; Jiang, Liping; Liu, Wei; Li, Xin; Wang, Lijia

2012-01-01

The viewpoints on the control of innate immune cells by the adaptive immune system during sepsis remain controversial. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is essential to the negative control of innate immunity and suppresses the activation of macrophages by inhibiting activated mitogen-activated protein kinase (MAPK). The purpose of the current study was to observe inflammatory response and macrophage activation in mice with severe combined immunodeficiency (SCID) with endotoxemia and to determine the role of MKP-1 in the control of macrophage activation by the adaptive immune system. Endotoxemia was induced in wild-type and SCID mice by an intraperitoneal injection of lipopolysaccharide (LPS), and all of the SCID mice died. SCID mice produced more inflammatory cytokines than BALB/c mice systemically and locally. TNF-α mRNA expression was higher and MKP-1 mRNA expression was lower in peritoneal macrophages (PMa) from SCID mice compared to PMa from wild-type mice after and even before LPS injection. Thioglycollate-stimulated PMa from wild-type mice were stimulated with LPS in vitro in the presence or absence of pan-T cells. The levels of TNF-α and IL-6 were higher in the supernatants from PMa cultured alone compared to PMa co-cultured with pan-T cells, and PMa MKP-1 mRNA and protein expression were higher when PMa were co-cultured with pan-T cells. Therefore, pan-T cells can up-regulate MKP-1 expression in macrophages and inhibit the secretion of inflammatory cytokines secretion by macrophages. In SCID mice, lymphocyte deficiency, especially T cell deficiency, causes insufficient MKP-1 expression in macrophages, which can be responsible for the severe inflammation and bad prognosis of septic SCID mice. MKP-1 plays an important role in the control of macrophage activation by the adaptive immune system.

Hsp27 promotes ABCA1 expression and cholesterol efflux through the PI3K/PKCζ/Sp1 pathway in THP-1 macrophages.

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Kuang, Hai-Jun; Zhao, Guo-Jun; Chen, Wu-Jun; Zhang, Min; Zeng, Gao-Feng; Zheng, Xi-Long; Tang, Chao-Ke

2017-09-05

Heat shock protein 27 (Hsp27) is a putative biomarker and therapeutic target in atherosclerosis. This study was to explore the potential mechanisms underlying Hsp27 effects on ATP-binding cassette transporter A1 (ABCA1) expression and cellular cholesterol efflux. THP-1 macrophage-derived foam cells were infected with adenovirus to express wild-type Hsp27, hyper-phosphorylated Hsp27 mimic (3D Hsp27), antisense Hsp27 or hypo-phosphorylated Hsp27 mimic (3A Hsp27). Wild-type and 3D Hsp27 were found to up-regulate ABCA1 mRNA and protein expression and increase cholesterol efflux from cells. Expression of antisense or 3A Hsp27 suppressed the expression of ABCA1 and cholesterol efflux. Furthermore, over-expression of wild-type and 3D Hsp27 significantly increased the levels of phosphorylated specificity protein 1 (Sp1), protein kinase C ζ (PKCζ) and phosphatidylinositol 3-kinase (PI3K). In addition, the up-regulation of ABCA1 expression and cholesterol efflux induced by 3D Hsp27 was suppressed by inhibition of Sp1, PKCζ and PI3K with specific kinase inhibitors. Taken together, our results revealed that Hsp27 may up-regulate the expression of ABCA1 and promotes cholesterol efflux through activation of the PI3K/PKCζ/Sp1 signal pathway in THP-1 macrophage-derived foam cells. Our findings may partly explain the mechanisms underlying the anti-atherogenic effect of Hsp27. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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

2018-03-13

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

Inhibition of VDAC1 prevents Ca²⁺-mediated oxidative stress and apoptosis induced by 5-aminolevulinic acid mediated sonodynamic therapy in THP-1 macrophages.

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Chen, Haibo; Gao, Weiwei; Yang, Yang; Guo, Shuyuan; Wang, Huan; Wang, Wei; Zhang, Shuisheng; Zhou, Qi; Xu, Haobo; Yao, Jianting; Tian, Zhen; Li, Bicheng; Cao, Wenwu; Zhang, Zhiguo; Tian, Ye

2014-12-01

Ultrasound combined with endogenous protoporphyrin IX derived from 5-aminolevulinic acid (ALA-SDT) is known to induce apoptosis in multiple cancer cells and macrophages. Persistent retention of macrophages in the plaque has been implicated in the pathophysiology and progression of atherosclerosis. Here we investigated the effects of inhibition of voltage-dependent anion channel 1 (VDAC1) on ALA-SDT-induced THP-1 macrophages apoptosis. Cells were pre-treated with VDAC1 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) disodium salt for 1 h or downregulated VDAC1 expression by small interfering RNA and exposed to ultrasound. Cell viability was assessed by MTT assay, and cell apoptosis along with necrosis was evaluated by Hoechst 33342/propidium iodide staining and flow cytometry. Levels of cytochrome c release was assessed by confocal microscope and Western blot. The levels of full length caspases, caspase activation, and VDAC isoforms were analyzed by Western blot. Intracellular reactive oxygen species generation, mitochondrial membrane permeability, and intracellular Ca(2+) [Ca(2+)]i levels were measured with fluorescent probes. We confirmed that the pharmacological inhibition of VDAC1 by DIDS notably prevented ALA-SDT-induced cell apoptosis in THP-1 macrophages. Additionally, DIDS significantly inhibited intracellular ROS generation and apoptotic biochemical changes such as inner mitochondrial membrane permeabilization, loss of mitochondrial membrane potential, cytochrome c release and activation of caspase-3 and caspase-9. Moreover, ALA-SDT elevated the [Ca(2+)]i levels and it was also notably reduced by DIDS. Furthermore, both of intracellular ROS generation and cell apoptosis were predominately inhibited by Ca(2+) chelating reagent BAPTA-AM. Intriguingly, ALA-treatment markedly augmented VDAC1 protein levels exclusively, and the downregulation of VDAC1 expression by specific siRNA also significantly abolished cell apoptosis. Altogether, these

Enhanced uptake of multiple sclerosis-derived myelin by THP-1 macrophages and primary human microglia.

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Hendrickx, Debbie A E; Schuurman, Karianne G; van Draanen, Michael; Hamann, Jörg; Huitinga, Inge

2014-03-31

The pathological hallmark of multiple sclerosis (MS) is myelin phagocytosis. It remains unclear why microglia and macrophages demyelinate axons in MS, but previously found or yet-unknown changes in the myelin of MS patients could contribute to this process. We therefore studied whether myelin from normal-appearing white matter (NAWM) of MS donors is phagocytosed more efficiently than myelin from control donors. Myelin was isolated from 11 MS and 12 control brain donors and labeled with the pH-sensitive fluorescent dye pHrodo to quantify uptake in lysosomes. Phagocytosis by differentiated THP-1 macrophages and by primary human microglia was quantified with flow cytometry. Whereas myelin uptake by THP-1 macrophages reached a plateau after approximately 24 hours, uptake by primary human microglia showed an almost linear increase over a 72-hour period. Data were statistically analyzed with the Mann-Whitney U test. MS-derived myelin was phagocytosed more efficiently by THP-1 macrophages after 6-hour incubation (P = 0.001 for the percentage of myelin-phagocytosing cells and P = 0.0005 for total myelin uptake) and after 24-hour incubation (P = 0.0006 and P = 0.0001, respectively), and by microglia after 24-hour incubation (P = 0.0106 for total myelin uptake). This enhanced uptake was not due to differences in the oxidation status of the myelin. Interestingly, myelin phagocytosis correlated negatively with the age of myelin donors, whereas the age of microglia donors showed a positive trend with myelin phagocytosis. Myelin isolated from normal-appearing white matter of MS donors was phagocytosed more efficiently than was myelin isolated from control brain donors by both THP-1 macrophages and primary human microglia. These data indicate that changes in MS myelin might precede phagocyte activation and subsequent demyelination in MS. Identifying these myelin changes responsible for enhancing phagocytic ability could be an interesting therapeutic target to

Assessment of tobacco heating product THP1.0. Part 4: Characterisation of indoor air quality and odour.

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Forster, Mark; McAughey, John; Prasad, Krishna; Mavropoulou, Eleni; Proctor, Christopher

2018-03-01

The tobacco heating product THP1.0, which heats but does not burn tobacco, was tested as part of a modified-risk tobacco product assessment framework for its impacts on indoor air quality and residual tobacco smoke odour. THP1.0 heats the tobacco to less than 240 °C ± 5 °C during puffs. An environmentally controlled room was used to simulate ventilation conditions corresponding to residential, office and hospitality environments. An analysis of known tobacco smoke constituents, included CO, CO 2 , NO, NO 2 , nicotine, glycerol, 3-ethenyl pyridine, sixteen polycyclic aromatic hydrocarbons, eight volatile organic compounds, four carbonyls, four tobacco-specific nitrosamines and total aerosol particulate matter. Significant emissions reductions in comparison to conventional cigarettes were measured for THP1.0. Levels of nicotine, acetaldehyde, formaldehyde and particulate matter emitted from THP1.0 exceeded ambient air measurements, but were more than 90% reduced relative to cigarette smoke emissions within the laboratory conditions defined Residual tobacco smoke odour was assessed by trained sensory panels after exposure of cloth, hair and skin to both mainstream and environmental emissions from the test products. Residual tobacco smoke odour was significantly lower from THP1.0 than from a conventional cigarette. These data show that using THP1.0 has the potential to result in considerably reduced environmental emissions that affect indoor air quality relative to conventional cigarettes. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Structure-related clustering of gene expression fingerprints of thp-1 cells exposed to smaller polycyclic aromatic hydrocarbons.

Science.gov (United States)

Wan, B; Yarbrough, J W; Schultz, T W

2008-01-01

This study was undertaken to test the hypothesis that structurally similar PAHs induce similar gene expression profiles. THP-1 cells were exposed to a series of 12 selected PAHs at 50 microM for 24 hours and gene expressions profiles were analyzed using both unsupervised and supervised methods. Clustering analysis of gene expression profiles revealed that the 12 tested chemicals were grouped into five clusters. Within each cluster, the gene expression profiles are more similar to each other than to the ones outside the cluster. One-methylanthracene and 1-methylfluorene were found to have the most similar profiles; dibenzothiophene and dibenzofuran were found to share common profiles with fluorine. As expression pattern comparisons were expanded, similarity in genomic fingerprint dropped off dramatically. Prediction analysis of microarrays (PAM) based on the clustering pattern generated 49 predictor genes that can be used for sample discrimination. Moreover, a significant analysis of Microarrays (SAM) identified 598 genes being modulated by tested chemicals with a variety of biological processes, such as cell cycle, metabolism, and protein binding and KEGG pathways being significantly (p < 0.05) affected. It is feasible to distinguish structurally different PAHs based on their genomic fingerprints, which are mechanism based.

IMB2026791, a Xanthone, Stimulates Cholesterol Efflux by Increasing the Binding of Apolipoprotein A-I to ATP-Binding Cassette Transporter A1

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Zijian Xie

2012-03-01

Full Text Available It is known that the ATP-binding cassette transporter A1 (ABCA1 plays a major role in cholesterol homeostasis and high density lipoprotein (HDL metabolism. Several laboratories have demonstrated that ABCA1 binding to lipid-poor apolipoprotein A-I (apoA-I will mediate the assembly of nascent HDL and cellular cholesterol efflux, which suggests a possible receptor-ligand interaction between ABCA1 and apoA-I. In this study, a cell-based-ELISA-like high-throughput screening (HTS method was developed to identify the synthetic and natural compounds that can regulate binding activity of ABCA1 to apoA-I. The cell-based-ELISA-like high-throughput screen was conducted in a 96-well format using Chinese hamster ovary (CHO cells stably transfected with ABCA1 pIRE2-EGFP (Enhanced Green Fluorecence Protein expression vector and the known ABCA1 inhibitor glibenclamide as the antagonist control. From 2,600 compounds, a xanthone compound (IMB 2026791 was selected using this HTS assay, and it was proved as an apoA-I binding agonist to ABCA1 by a flow cytometry assay and western blot analysis. The [3H] cholesterol efflux assay of IMB2026791 treated ABCA1-CHO cells and PMA induced THP-1 macrophages (human acute monocytic leukemia cell further confirmed the compound as an accelerator of cholesterol efflux in a dose-dependent manner with an EC50 of 25.23 μM.

Enhancement of alpha particles-induced cell transformation by oxygen free radicals and tumor necrosis factor released from phagocytes

International Nuclear Information System (INIS)

Gong Yifen; Guo Renfeng; Zhu Maoxiang; Shou Jiang; Ge Guixiu; Yang Zhihua; Hieber, L.; Peters, K.; Schippel, C.

1997-01-01

To illustrate the role of several endogenous factors released from phagocytes under chronic inflammation in radiation-induced cancer. C 3 T 10 T 1/2 and SHE cells were used as targets, and 238 Pu alpha source was used in alpha irradiation. The enhancement of TF in alpha particles-induced cell transformation by PMA-stimulated human blood and zymosan-stimulated U-937 cells was studied using formation of transformed foci. Transformation frequency (TF) of C 3 H 10 T 1/2 cells exposed to alpha particles of 0.5 Gy increased 2.1 and 2.8 fold by PMA-and PMA-stimulated neutrophils, respectively. TF of irradiated SHE cells at a dose of 0.5 Gy increased 12 fold by the addition of the supernatant of macrophage-like U-937 cell line. It was shown that TF of irradiated SHE cells at above dose increased 8 fold by the supernatant treated with anti-TNF-α could be subcultured continuously in vitro. The cells at 40 th passage and two lines of monoclone cells have the ability to develop malignant tumors in nude mice. The overdose of free radicals and TNF-α released from neutrophils and macrophages have played an important role in low dose radiation-induced cancer

Eficiencia de cultivo in vitro de Toxoplasma gondii en las líneas celulares THP1 y Vero

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Jorge Andrés Cuellar

2012-03-01

Full Text Available Introducción. El cultivo in vitro es un método importante para la obtención de Toxoplasma gondii confines de diagnóstico clínico o biotecnológico. Objetivo. Determinar el porcentaje de invasión y producción de T. gondii en las líneas celulares THP1y Vero. Materiales y métodos. Se determinó la curva de crecimiento para las células Vero y THP1 por conteoen hemocitómetro. Posteriormente, se identificó el porcentaje de invasión de T. gondii en células THP1y Vero por citometría de flujo, en diferentes proporciones célula/taquizoíto de 1/5, 1/20, 1/50. Por otrolado, se calculó el índice de rendimiento de T. gondii, cepa RH, y del aislamiento CIBM1 en célulasTHP1. Resultados. Las células Vero crecen más rápidamente que las células THP1, con un crecimientoexponencial en un periodo de siete días. El aislamiento CIBM1 infecta las células THP1 en las tresproporciones diferentes de 1/5,1/20 y 1/50 con porcentajes de invasión de 57,1 %, 15,5 % y 12,2 %, yen células Vero, de 25,3 %, 17,8 % y 8,8 %. La cepa RH de T. gondii mostró porcentajes de invasiónmás bajos, de 32,6 %, 14,8 % y 8,1 % en células THP1 y de 22,3 %, 14,1 % y 3,4 % en células Vero. Conclusiones. El aislamiento CIBM1 presentó mayor rendimiento con respecto a la cepa RH de T.gondii en células THP1, siendo estas células una buena línea para estudiar el proceso de invasión yprobar candidatos farmacológicos para reducir la infección por T. gondii.   doi: http://dx.doi.org/10.7705/biomedica.v32i3.485

Identification of transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) as a novel factor for TNF-α expression upon lipopolysaccharide stimulation in human monocytes.

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Murata, H; Hattori, T; Maeda, H; Takashiba, S; Takigawa, M; Kido, J; Nagata, T

2015-08-01

Tumor necrosis factor alpha (TNF-α) is a major cytokine implicated in various inflammatory diseases. The nature of the nuclear factors associated with human TNF-α gene regulation is not well elucidated. We previously identified a novel region located from -550 to -487 in human TNF-α promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-κB) but showed lipopolysaccharide (LPS)-induced transcriptional activity. The purpose of this study is to identify novel factors that bind to the promoter region and regulate TNF-α expression. To identify DNA-binding proteins that bound to the target region of TNF-α promoter, a cDNA library from LPS-stimulated human monocytic cell line THP-1 was screened using a yeast one-hybrid system. Cellular localizations of the DNA-binding protein in the cells were examined by subcellular immunocytochemistry. Nuclear amounts of the protein in LPS-stimulated THP-1 cells were identified by western blot analysis. Expression of mRNA of the protein in the cells was quantified by real-time polymerase chain reaction. Electrophoretic mobility shift assays were performed to confirm the DNA-binding profile. Overexpression of the protein and knockdown of the gene were also performed to investigate the role for TNF-α expression. Several candidates were identified from the cDNA library and transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) was focused on. Western blot analysis revealed that nuclear TDP-43 protein was increased in the LPS-stimulated THP-1 cells. Expression of TDP-43 mRNA was already enhanced before TNF-α induction by LPS. Electrophoretic mobility shift assay analysis showed that nuclear extracts obtained by overexpressing FLAG-tagged TDP-43 bound to the -550 to -487 TNF-α promoter fragments. Overexpression of TDP-43 in THP-1 cells resulted in an increase of TNF-α expression. Knockdown of TDP-43 in THP-1 cells downregulated TNF-α expression. We identified TDP-43 as one of the novel

Cyr61 promotes CD204 expression and the migration of macrophages via MEK/ERK pathway in esophageal squamous cell carcinoma

International Nuclear Information System (INIS)

Shigeoka, Manabu; Urakawa, Naoki; Nishio, Mari; Takase, Nobuhisa; Utsunomiya, Soken; Akiyama, Hiroaki; Kakeji, Yoshihiro; Komori, Takahide; Koma, Yu-ichiro; Yokozaki, Hiroshi

2015-01-01

Tumor-associated macrophages (TAMs) are known to be involved in the progression of various human malignancies. We previously demonstrated that CD204 was a useful marker for TAMs contributing to the angiogenesis, progression, and prognosis of human esophageal squamous cell carcinoma (ESCC). We also showed that conditioned media of ESCC cell lines induced CD204 expression in THP-1 human monocytic leukemia cells. Here, we performed a cDNA microarray analysis between THP-1 cells stimulated with TPA (macrophage [MΦ]-like THP-1 cells) treated with and without conditioned medium of ESCC cell line to clarify the molecular characteristics of TAMs in ESCC. From the microarray data, we discovered that Cyr61 was induced in CD204-positive-differentiated THP-1 cells (TAM-like THP-1 cells). In the ESCC microenvironment, not only cancer cells but also TAMs expressed Cyr61. Interestingly, the expression levels of Cyr61 showed a significant positive correlation with the number of CD204-positive macrophages in ESCCs by immunohistochemistry. Recombinant human Cyr61 (rhCyr61) promoted cell migration and induced the expression of CD204 along with the activation of the MEK/ERK pathway in MΦ-like THP-1 cells. Pretreatment with a MEK1/2 inhibitor significantly inhibited not only the Cyr61-mediated migration but also the CD204 expression in the MΦ-like THP-1 cells. These results suggest that Cyr61 may contribute to the expression of CD204 and the promotion of cell migration via the MEK/ERK pathway in TAMs in the ESCC microenvironment

Haloperidol Abrogates Matrix Metalloproteinase-9 Expression by Inhibition of NF-κB Activation in Stimulated Human Monocytic Cells

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Yueh-Lun Lee

2018-01-01

Full Text Available Much evidence has indicated that matrix metalloproteinases (MMPs participate in the progression of neuroinflammatory disorders. The present study was undertaken to investigate the inhibitory effect and mechanism of the antipsychotic haloperidol on MMP activation in the stimulated THP-1 monocytic cells. Haloperidol exerted a strong inhibition on tumor necrosis factor- (TNF- α-induced MMP-9 gelatinolysis of THP-1 cells. A concentration-dependent inhibitory effect of haloperidol was observed in TNF-α-induced protein and mRNA expression of MMP-9. On the other hand, haloperidol slightly affected cell viability and tissue inhibition of metalloproteinase-1 levels. It significantly inhibited the degradation of inhibitor-κB-α (IκBα in activated cells. Moreover, it suppressed activated nuclear factor-κB (NF-κB detected by a mobility shift assay, NF-κB reporter gene, and chromatin immunoprecipitation analyses. Consistent with NF-κB inhibition, haloperidol exerted a strong inhibition of lipopolysaccharide- (LPS- induced MMP-9 gelatinolysis but not of transforming growth factor-β1-induced MMP-2. In in vivo studies, administration of haloperidol significantly attenuated LPS-induced intracerebral MMP-9 activation of the brain homogenate and the in situ in C57BL/6 mice. In conclusion, the selective anti-MMP-9 activation of haloperidol could possibly involve the inhibition of the NF-κB signal pathway. Hence, it was found that haloperidol treatment may represent a bystander of anti-MMP actions for its conventional psychotherapy.

68Ga-THP-PSMA: A PET Imaging Agent for Prostate Cancer Offering Rapid, Room-Temperature, 1-Step Kit-Based Radiolabeling.

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Young, Jennifer D; Abbate, Vincenzo; Imberti, Cinzia; Meszaros, Levente K; Ma, Michelle T; Terry, Samantha Y A; Hider, Robert C; Mullen, Greg E; Blower, Philip J

2017-08-01

The clinical impact and accessibility of 68 Ga tracers for the prostate-specific membrane antigen (PSMA) and other targets would be greatly enhanced by the availability of a simple, 1-step kit-based labeling process. Radiopharmacy staff are accustomed to such procedures in the daily preparation of 99m Tc radiopharmaceuticals. Currently, chelating agents used in 68 Ga radiopharmaceuticals do not meet this ideal. The aim of this study was to develop and evaluate preclinically a 68 Ga radiotracer for imaging PSMA expression that could be radiolabeled simply by addition of 68 Ga generator eluate to a cold kit. Methods: A conjugate of a tris(hydroxypyridinone) (THP) chelator with the established urea-based PSMA inhibitor was synthesized and radiolabeled with 68 Ga by adding generator eluate directly to a vial containing the cold precursors THP-PSMA and sodium bicarbonate, with no further manipulation. It was analyzed after 5 min by instant thin-layer chromatography and high-performance liquid chromatography. The product was subjected to in vitro studies to determine PSMA affinity using PSMA-expressing DU145-PSMA cells, with their nonexpressing analog DU145 as a control. In vivo PET imaging and ex vivo biodistribution studies were performed in mice bearing xenografts of the same cell lines, comparing 68 Ga-THP-PSMA with 68 Ga-HBED-CC-PSMA. Results: Radiolabeling was complete (>95%) within 5 min at room temperature, showing a single radioactive species by high-performance liquid chromatography that was stable in human serum for more than 6 h and showed specific binding to PSMA-expressing cells (concentration giving 50% inhibition of 361 ± 60 nM). In vivo PET imaging showed specific uptake in PSMA-expressing tumors, reaching 5.6 ± 1.2 percentage injected dose per cubic centimeter at 40-60 min and rapid clearance from blood to kidney and bladder. The tumor uptake, biodistribution, and pharmacokinetics were not significantly different from those of 68 Ga

Oxidative Stress, DNA Damage, and Inflammation Induced by Ambient Air and Wood Smoke Particulate Matter in Human A549 and THP-1 Cell Lines

DEFF Research Database (Denmark)

Danielsen, Pernille Høgh; Møller, Peter; Jensen, Keld Alstrup

2011-01-01

PM (WSPM) is poorly assessed. We assessed a wide spectrum of toxicity end points in human A549 lung epithelial and THP-1 monocytic cell lines comparingWSPM from high or low oxygen combustion and ambient PM collected in a village with many operating wood stoves and from a rural background area...... from the wood stove area. Expression of oxoguanine glycosylase 1, lymphocyte function-associated antigen-1, and interleukin-6 did not change. We conclude that WSPM has small particle size, high level of PAH, low level of water-soluble metals, and produces high levels of free radicals, DNA damage...

Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions.

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Corti, Daniele; Galbiati, Valentina; Gatti, Nicolò; Marinovich, Marina; Galli, Corrado L; Corsini, Emanuela

2015-10-01

Despite important impacts of systemic hypersensitivity induced by pharmaceuticals, for such endpoint no reliable preclinical approaches are available. We previously established an in vitro test to identify contact and respiratory allergens based on interleukin-8 (IL-8) production in THP-1 cells. Here, we challenged it for identification of pharmaceuticals associated with systemic hypersensitivity reactions, with the idea that drug sensitizers share common mechanisms of cell activation. Cells were exposed to drugs associated with systemic hypersensitivity reactions (streptozotocin, sulfamethoxazole, neomycin, probenecid, clonidine, procainamide, ofloxacin, methyl salicylate), while metformin was used as negative drug. Differently to chemicals, drugs tested were well tolerated, except clonidine and probenecid, with no signs of cytotoxicity up to 1-2mg/ml. THP-1 activation assay was adjusted, and conditions, that allow identification of all sensitizing drugs tested, were established. Next, using streptozotocin and selective inhibitors of PKC-β and p38 MAPK, two pathways involved in chemical allergen-induced cell activation, we tested the hypothesis that similar pathways were also involved in drug-induced IL-8 production and CD86 upregulation. Results indicated that drugs and chemical allergens share similar activation pathways. Finally, we made a structure-activity hypothesis related to hypersensitivity reactions, trying to individuate structural requisite that can be involved in immune mediated adverse reactions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of two second-site mutations preventing wild type protein aggregation caused by a dominant negative PMA1 mutant.

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Pilar Eraso

Full Text Available The correct biogenesis and localization of Pma1 at the plasma membrane is essential for yeast growth. A subset of PMA1 mutations behave as dominant negative because they produce aberrantly folded proteins that form protein aggregates, which in turn provoke the aggregation of the wild type protein. One approach to understand this dominant negative effect is to identify second-site mutations able to suppress the dominant lethal phenotype caused by those mutant alleles. We isolated and characterized two intragenic second-site suppressors of the PMA1-D378T dominant negative mutation. We present here the analysis of these new mutations that are located along the amino-terminal half of the protein and include a missense mutation, L151F, and an in-frame 12bp deletion that eliminates four residues from Cys409 to Ala412. The results show that the suppressor mutations disrupt the interaction between the mutant and wild type enzymes, and this enables the wild type Pma1 to reach the plasma membrane.

TOM1L Is Involved in a Novel Signaling Pathway Important for the IL-2 Production in Jurkat T Cells Stimulated by CD3/CD28 CoLigation

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Ahmed Elmarghani

2009-01-01

Full Text Available TOM1L (target of Myb-1 Like was identified as a binding partner for the full length and catalytically-active Lck in a yeast 2-hybrid screening assay. Here we show that in Jurkat T cells stimulated by CD3/CD28 coligation where the expression of TOM1L is reduced by lenti virus mediated-siRNA results in a dramatically lower IL-2 production. The production of IL-2 in siRNA treated cells stimulated with PMA/ionomycin was not affected indicating an involvement of TOM1L in a pathway proximal of TCR and CD28. The coexpression of Fyn with TOM1L increased the level of the phosphorylated form of Fyn indicating that TOM1L has the ability to activate Fyn. The ability of TOM1L to activate Fyn was further shown in a kinase assay using angiotensin II as a substrate. By confocal microscopy, we show that the expression of TOM1L in non-treated HeLa and SK-N-SH cells colocalizes with the mitochondrial membrane but not with lysosomal compartments or the trans-Golgi network. Furthermore, we show that the over-expression of TOM1L in Jurkat cells causes an increase of the STAT3 expression . Based on our results, we here propose that TOM1L is involved in a novel signaling pathway that is important for the IL-2 production in T cells.

E-selectin ligand-1 (ESL-1) is a novel adiponectin binding protein on cell adhesion

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Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi; Kochi, Ikoi; Matsumoto, Akane; Niinaga, Ryu [Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka (Japan); Funahashi, Tohru; Shimomura, Iichiro [Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka (Japan); Kihara, Shinji, E-mail: skihara@sahs.med.osaka-u.ac.jp [Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka (Japan)

2016-02-05

Background: Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. Methods and Results: In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partially abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Conclusion: Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. - Highlights: • E-selectin ligand (ESL)-1 was identified as an adiponectin (APN)-binding protein. • ESL-1 bound to APN at its N-terminal 6th-10th amino acids. • shESL-1 reduced the suppressive effect of APN on adhesion of THP-1 cells to HUVECs. • Interaction with ESL may be involved in the anti-atherogenic effects of APN.

E-selectin ligand-1 (ESL-1) is a novel adiponectin binding protein on cell adhesion

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Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi; Kochi, Ikoi; Matsumoto, Akane; Niinaga, Ryu; Funahashi, Tohru; Shimomura, Iichiro; Kihara, Shinji

2016-01-01

Background: Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. Methods and Results: In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partially abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Conclusion: Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. - Highlights: • E-selectin ligand (ESL)-1 was identified as an adiponectin (APN)-binding protein. • ESL-1 bound to APN at its N-terminal 6th-10th amino acids. • shESL-1 reduced the suppressive effect of APN on adhesion of THP-1 cells to HUVECs. • Interaction with ESL may be involved in the anti-atherogenic effects of APN.

Apoptosis of THP-1 derived macrophages induced by sonodynamic therapy using a new sonosensitizer hydroxyl acetylated curcumin.

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Longbin Zheng

Full Text Available Curcumin is extracted from the rhizomes of the traditional Chinese herb Curcuma longa. Our previous study indicated curcumin was able to function as a sonosensitizer. Hydroxyl acylated curcumin was synthesized from curcumin to eliminate the unstable hydroxy perssad in our group. The potential use of Hydroxyl acylated curcumin as a sonosensitizer for sonodynamic therapy (SDT requires further exploration. This study investigated the sonodynamic effect of Hydroxyl acylated curcumin on THP-1 macrophage. THP-1 macrophages were cultured with Hydroxyl acylated curcumin at a concentration of 5.0 μg/mL for 4 hours and then exposed to pulse ultrasound irradiation (0.5 W/cm2 with 1.0 MHz for 5 min, 10 min and 15 min. Six hours later, cell viability decreased significantly by CCK-8 assay. After ultrasound irradiation, the ratio of apoptosis and necrosis in SDT group was higher than that in control, Hydroxyl acylated curcumin alone and ultrasound alone. Moreover, the apoptotic rate was higher than necrotic rate with the flow cytometry analysis. Furthermore, Hydroxyl acylated curcumin-SDT induced reactive oxygen species (ROS generation in THP-1 macrophages immediately after the ultrasound treatment while ROS generation was reduced significantly with the scavenger of singlet oxygen Sodium azide (NaN3. Hydroxyl acylated curcumin-SDT led to a conspicuous loss of mitochondrial membrane potential (MMP compared with other groups, while MMP was increased significantly with the scavenger of singlet oxygen Sodium azide (NaN3, ROS inhibitor N-acetyl cysteine (NAC and Mitochondrial Permeability Transition Pore (MPTP inhibitor Cyclosporin A (CsA. The cytochrome C, cleaved-Caspase-9, cleaved-Caspase-3 and cleaved-PARP upregulated after SDT through Western blotting. These findings suggested that Hydroxyl acylated curcumin under low-intensity ultrasound had sonodynamic effect on THP-1 macrophages via generation of intracellular singlet oxygen and mitochondria

Quantification of viable bacteria in wastewater treatment plants by using propidium monoazide combined with quantitative PCR (PMA-qPCR).

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Li, Dan; Tong, Tiezheng; Zeng, Siyu; Lin, Yiwen; Wu, Shuxu; He, Miao

2014-02-01

The detection of viable bacteria in wastewater treatment plants (WWTPs) is very important for public health, as WWTPs are a medium with a high potential for waterborne disease transmission. The aim of this study was to use propidium monoazide (PMA) combined with the quantitative polymerase chain reaction (PMA-qPCR) to selectively detect and quantify viable bacteria cells in full-scale WWTPs in China. PMA was added to the concentrated WWTP samples at a final concentration of 100 micromol/L and the samples were incubated in the dark for 5 min, and then lighted for 4 min prior to DNA extraction and qPCR with specific primers for Escherichia coli and Enterococci, respectively. The results showed that PMA treatment removed more than 99% of DNA from non-viable cells in all the WWTP samples, while matrices in sludge samples markedly reduced the effectiveness of PMA treatment. Compared to qPCR, PMA-qPCR results were similar and highly linearly correlated to those obtained by culture assay, indicating that DNA from non-viable cells present in WWTP samples can be eliminated by PMA treatment, and that PMA-qPCR is a reliable method for detection of viable bacteria in environmental samples. This study demonstrated that PMA-qPCR is a rapid and selective detection method for viable bacteria in WWTP samples, and that WWTPs have an obvious function in removing both viable and non-viable bacteria. The results proved that PMA-qPCR is a promising detection method that has a high potential for application as a complementary method to the standard culture-based method in the future.

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

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Raji Lenin

2012-01-01

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

Modulation of transglutaminase 2 activity in H9c2 cells by PKC and PKA signalling: a role for transglutaminase 2 in cytoprotection

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Almami, Ibtesam; Dickenson, John M; Hargreaves, Alan J; Bonner, Philip L R

2014-01-01

BACKGROUND AND PURPOSE Tissue transglutaminase (TG2) has been shown to mediate cell survival in many cell types. In this study, we investigated whether the role of TG2 in cytoprotection was mediated by the activation of PKA and PKC in cardiomyocyte-like H9c2 cells. EXPERIMENTAL APPROACH H9c2 cells were extracted following stimulation with phorbol-12-myristate-13-acetate (PMA) and forskolin. Transglutaminase activity was determined using an amine incorporating and a protein crosslinking assay. The presence of TG isoforms (TG1, 2, 3) was determined using Western blot analysis. The role of TG2 in PMA- and forskolin-induced cytoprotection was investigated by monitoring H2O2-induced oxidative stress in H9c2 cells. KEY RESULTS Western blotting showed TG2 >> TG1 protein expression but no detectable TG3. The amine incorporating activity of TG2 in H9c2 cells increased in a time and concentration-dependent manner following stimulation with PMA and forskolin. PMA and forskolin-induced TG2 activity was blocked by PKC (Ro 31-8220) and PKA (KT 5720 and Rp-8-Cl-cAMPS) inhibitors respectively. The PMA- and forskolin-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Immunocytochemistry revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (β-tubulin) and novel (α-actinin) protein substrates for TG2. Pretreatment with PMA and forskolin reversed H2O2-induced decrease in MTT reduction and release of LDH. TG2 inhibitors R283 and Z-DON blocked PMA- and forskolin-induced cytoprotection. CONCLUSIONS AND IMPLICATIONS TG2 activity was stimulated via PKA- and PKC-dependent signalling pathways in H9c2 cells These results suggest a role for TG2 in cytoprotection induced by these kinases. PMID:24821315

Novel leads from Heliotropium ovalifolium, 4,7,8-trimethoxy-naphthalene-2-carboxylic acid and 6-hydroxy-5,7-dimethoxy-naphthalene-2-carbaldehyde show specific IL-6 inhibitory activity in THP-1 cells and primary human monocytes.

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Kulkarni-Almeida, Asha; Suthar, Ashish; Goswami, Hitesh; Vishwakarma, Ram; Chauhan, Vijay Singh; Balakrishnan, Arun; Sharma, Somesh

2008-12-01

From our screening program, we identified the anti-inflammatory effects of the extracts of Heliotropium ovalifolium in its ability to inhibit specific cytokines. The H. ovalifolium extract was found to be moderately active with an IC(50) equaling 10 microg/ml for inhibition of interleukin-6 (IL-6) in a human monocytic cell line. Interleukin-6 is a pleiotropic cytokine with implications in the regulation of the immune response, inflammation and hematopoiesis. This prompted us to examine and identify the active molecules that are responsible for the bioactivity in THP-1 cells. Bioassay guided fractionation identified two compounds 4,7,8-trimethoxy-naphthalene-2-carboxylic acid and 6-hydroxy-5,7-dimethoxy-naphthalene-2-carbaldehyde with an IC(50) of 2.4 and 2.0 microM for IL-6 inhibition and an IC(50) of 15.6 and 7.0 microM for tumor necrosis factor-alpha (TNF-alpha) inhibition in THP-1 cells. The protein expression data were supported by the inhibitory effect on mRNA gene expression. The compounds isolated from H. ovalifolium were also non-toxic in human peripheral blood monocytes from normal donors and the activity profile was similar to that obtained on THP-1 cells. Thus, we believe that these scaffolds may be of interest to develop leads for treating rheumatoid arthritis, psoriasis, ulcerative colitis, Crohn's disease and other inflammatory disorders. However, more detailed investigations need to be carried out to explain the efficacy of these compounds as drugs.

GEN-27, a Newly Synthetic Isoflavonoid, Inhibits the Proliferation of Colon Cancer Cells in Inflammation Microenvironment by Suppressing NF-κB Pathway

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Yajing Wang

2016-01-01

Full Text Available Nonresolving inflammation is one of the consistent features of the tumor microenvironment in the intestine and plays a critical role in the initiation and development of colon cancer. Here we reported the inhibitory effects of GEN-27, a new derivative of genistein, on the inflammation-related colon cancer cell proliferation and delineated the mechanism of its action. The results indicated that GEN-27 inhibited the proliferation of human colon tumor HCT116 cells stimulated by culture supernatants of LPS-induced human monocytes THP-1 cells and significantly decreased LPS-induced secretion of proinflammatory cytokines interleukin-6 and interleukin-1β in THP-1 cells. The HCT116 cell proliferation elicited by THP-1-conditioned medium could be blocked by the interleukin-1 receptor antagonist (IL-1RA. Further mechanistic study revealed that GEN-27 remarkably inhibited the nuclear translocation of NF-κB and phosphorylation of IκB and IKKα/β in both HCT116 and THP-1 cells. In addition, GEN-27 markedly suppressed the HCT116 cell proliferation stimulated by IL-1β treatment, which was dependent on the inhibition of NF-κB/p65 nuclear localization, as verified by p65 overexpression and BAY 11-7082, an NF-κB inhibitor. Taken together, our findings established that GEN-27 modulated NF-κB signaling pathway involved in inflammation-induced cancer cells proliferation and therefore could be a potential chemopreventive agent against inflammation-associated colon cancer.

GEN-27, a Newly Synthetic Isoflavonoid, Inhibits the Proliferation of Colon Cancer Cells in Inflammation Microenvironment by Suppressing NF-κB Pathway.

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Wang, Yajing; Lu, Ping; Zhang, Weifeng; Du, Qianming; Tang, Jingjing; Wang, Hong; Lu, Jinrong; Hu, Rong

2016-01-01

Nonresolving inflammation is one of the consistent features of the tumor microenvironment in the intestine and plays a critical role in the initiation and development of colon cancer. Here we reported the inhibitory effects of GEN-27, a new derivative of genistein, on the inflammation-related colon cancer cell proliferation and delineated the mechanism of its action. The results indicated that GEN-27 inhibited the proliferation of human colon tumor HCT116 cells stimulated by culture supernatants of LPS-induced human monocytes THP-1 cells and significantly decreased LPS-induced secretion of proinflammatory cytokines interleukin-6 and interleukin-1β in THP-1 cells. The HCT116 cell proliferation elicited by THP-1-conditioned medium could be blocked by the interleukin-1 receptor antagonist (IL-1RA). Further mechanistic study revealed that GEN-27 remarkably inhibited the nuclear translocation of NF-κB and phosphorylation of IκB and IKKα/β in both HCT116 and THP-1 cells. In addition, GEN-27 markedly suppressed the HCT116 cell proliferation stimulated by IL-1β treatment, which was dependent on the inhibition of NF-κB/p65 nuclear localization, as verified by p65 overexpression and BAY 11-7082, an NF-κB inhibitor. Taken together, our findings established that GEN-27 modulated NF-κB signaling pathway involved in inflammation-induced cancer cells proliferation and therefore could be a potential chemopreventive agent against inflammation-associated colon cancer.

Differential regulation of histamine- and bradykinin-stimulated phospholipase C in adrenal chromaffin cells: evidence for involvement of different protein kinase C isoforms.

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Sena, C M; Rosário, L M; Parker, P J; Patel, V; Boarder, M R

1996-03-01

In this report we investigate the isoforms of protein kinase C (PKC) present in cultured adrenal chromaffin cells with respect to their modulation by treatment with phorbol ester and their possible differential involvement in the regulation of responses to histamine and bradykinin. The presence of individual isoforms of PKC was investigated by using eight isoform specific antisera, as a result of which PKC-alpha, epsilon, and zeta were identified. To characterize down-regulation of these enzymes, cells were incubated for 6-48 h with 1 microM phorbol myristate acetate (PMA). PKC-epsilon down-regulated more rapidly than PKC-alpha. At 12 h, PMA pretreatment, for example, PKC-epsilon was maximally down-regulated (23 +/- 4% of controls), whereas PKC-alpha was unchanged. PKC-alpha showed partial down-regulation by 24 h of PMA pretreatment. PKC-zeta did not down-regulate at any of the times tested. Translocation from cytosol to membrane in response to PMA was also more rapid for PKC-epsilon than for PKC-alpha. The accumulation of total 3H-inositol (poly) phosphates in response to bradykinin or histamine was essentially abolished by prior treatment with 10-min PMA treatment (1 microM). However, with 12-h exposure to PMA, the bradykinin response was restored to the level seen with no prior PMA exposure. The histamine response showed no recovery by 12 h of PMA, but showed partial recovery by 24 h of PMA pretreatment. These observations showed that the restoration of the response to bradykinin corresponds to the loss of PKC-epsilon, whereas the restoration of the histamine response corresponds to the loss of PKC-alpha. This picture was confirmed with further studies on cytosolic Ca2+. The results show that chromaffin cells exhibit an unusual pattern of down-regulation of PKC isoforms on prolonged exposure to PMA, and that there is a differential effect of exposure to PMA on the histamine and bradykinin responses, suggesting that different PLC-linked receptors in chromafin

L-plastin nanobodies perturb matrix degradation, podosome formation, stability and lifetime in THP-1 macrophages.

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Sarah De Clercq

Full Text Available Podosomes are cellular structures acting as degradation 'hot-spots' in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein. The presence of the capping protein CapG in podosomes has not yet been investigated. We used an innovative approach to investigate the role of these proteins in macrophage podosomes by means of nanobodies or Camelid single domain antibodies. Nanobodies directed against distinct domains of gelsolin, L-plastin or CapG were stably expressed in macrophage-like THP-1 cells. CapG was not enriched in podosomes. Gelsolin nanobodies had no effect on podosome formation or function but proved very effective in tracing distinct gelsolin populations. One gelsolin nanobody specifically targets actin-bound gelsolin and was effectively enriched in podosomes. A gelsolin nanobody that blocks gelsolin-G-actin interaction was not enriched in podosomes demonstrating that the calcium-activated and actin-bound conformation of gelsolin is a constituent of podosomes. THP-1 cells expressing inhibitory L-plastin nanobodies were hampered in their ability to form stable podosomes. Nanobodies did not perturb Ser5 phosphorylation of L-plastin although phosphorylated L-plastin was highly enriched in podosomes. Furthermore, nanobody-induced inhibition of L-plastin function gave rise to an irregular and unstable actin turnover of podosomes, resulting in diminished degradation of the underlying matrix. Altogether these results indicate that L-plastin is indispensable for podosome formation and function in macrophages.

E-selectin ligand-1 (ESL-1) is a novel adiponectin binding protein on cell adhesion.

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Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi; Kochi, Ikoi; Matsumoto, Akane; Niinaga, Ryu; Funahashi, Tohru; Shimomura, Iichiro; Kihara, Shinji

2016-02-05

Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partially abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. Copyright © 2016 Elsevier Inc. All rights reserved.

Protein Kinase Cα and P-Type Ca2+ Channel CaV2.1 in Red Blood Cell Calcium Signalling

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Lisa Wagner-Britz

2013-06-01

Full Text Available Background/Aims: Protein kinase Cα (PKCα is activated by an increase in cytosolic Ca2+ in red blood cells (RBCs. Previous work has suggested that PKCα directly stimulates the CaV2.1 channel, whereas other studies revealed that CaV2.1 is insensitive to activation by PKC. The aim of this study was to resolve this discrepancy. Methods: We performed experiments based on a single cell read-out of the intracellular Ca2+ concentration in terms of Fluo-4 fluorescence intensity and phosphatidylserine exposure to the external membrane leaflet. Measurement modalities included flow cytometry and live cell imaging. Results: Treatment of RBCs with phorbol 12-myristate 13-acetate (PMA led to two distinct populations of cells with an increase in intracellular Ca2+: a weak-responding and a strong-responding population. The EC50 of PMA for the number of cells with Ca2+ elevation was 2.7±1.2 µM; for phosphatidylserine exposure to the external membrane surface, it was 2.8±0.5 µM; and for RBC haemolysis, it was 2.9±0.5 µM. Using pharmacological manipulation with the CaV2.1 inhibitor ω-agatoxin TK and the broad protein kinase C inhibitor Gö6983, we are able to show that there are two independent PMA-activated Ca2+ entry processes: the first is independent of CaV2.1 and directly PKCα-activated, while the second is associated with a likely indirect activation of CaV2.1. Further studies using lysophosphatidic acid (LPA as a stimulation agent have provided additional evidence that PKCα and CaV2.1 are not directly interconnected in a signalling chain. Conclusion: Although we provide evidence for a lack of interaction between PKCα and CaV2.1 in RBCs, further studies are required to decipher the signalling relationship between LPA, PKCα and CaV2.1.

Macrophage migration inhibitory factor stimulated by Helicobacter pylori increases proliferation of gastric epithelial cells

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Xia, Harry Hua-Xiang; Lam, Shiu Kum; Chan, Annie O.O.; Lin, Marie Chia Mi; Kung, Hsiang Fu; Ogura, Keiji; Berg, Douglas E.; Wong, Benjamin C. Y.

2005-01-01

AIM: Helicobacter pylori (H pylori) is associated with increased gastric inflammatory and epithelial expression of macrophage migration inhibitory factor (MIF) and gastric epithelial cell proliferation. This study aimed at determining whether H pylori directly stimulates release of MIF in monocytes, whether the cag pathogenicity island (PAI) is involved for this function, and whether MIF stimulated by H pylori increases gastric epithelial cell proliferation in vitro. METHODS: A cytotoxic wild-type H pylori strain (TN2)and its three isogenic mutants (TN2△cag, TN2△cagA and TN2△cagE) were co-cultured with cells of a human monocyte cell line, THP-1, for 24 h at different organism/cell ratios. MIF in the supernatants was measured by an ELISA. Cells of a human gastric cancer cell line, MKN45, were then co-cultured with the supernatants, with and without monoclonal anti-MIF antibody for 24 h. The cells were further incubated for 12 h after addition of 3H-thymidine, and the levels of incorporation of 3H-thymidine were measured with a liquid scintillation counter. RESULTS: The wild-type strain and the isogenic mutants, TN2△cagA and TN2△cagE, increased MIF release at organism/cell ratios of 200/1 and 400/1, but not at the ratios of 50/1 and 100/1. However, the mutant TN2△cag did not increase the release of MIF at any of the four ratios. 3H-thymidine readings for MKN-45 cells were significantly increased with supernatants derived from the wild-type strain and the mutants TN2△cagA and TN2△cagE, but not from the mutant TN2△cag. Moreover, in the presence of monoclonal anti-MIF antibody, the stimulatory effects of the wild-type strain on cell proliferation disappeared. CONCLUSION: H pylori stimulates MIF release in monocytes, likely through its cag PAI, but not related to cagA or cagE. H pylori-stimulated monocyte culture supernatant increases gastric cell proliferation, which is blocked by anti-MIF antibody, suggesting that MIF plays an important role in H

Prenylated Flavonoids from Morus alba L. Cause Inhibition of G1/S Transition in THP-1 Human Leukemia Cells and Prevent the Lipopolysaccharide-Induced Inflammatory Response

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Peter Kollar

2013-01-01

Full Text Available Morus alba L. (MA is a natural source of many compounds with different biological effects. It has been described to possess anti-inflammatory, antioxidant, and hepatoprotective activities. The aim of this study was to evaluate cytotoxicity of three flavonoids isolated from MA (kuwanon E, cudraflavone B, and 4′-O-methylkuwanon E and to determine their effects on proliferation of THP-1 cells, and on cell cycle progression of cancer cells. Anti-inflammatory effects were also determined for all three given flavonoids. Methods used in the study included quantification of cells by hemocytometer and WST-1 assays, flow cytometry, western blotting, ELISA, and zymography. From the three compounds tested, cudraflavone B showed the strongest effects on cell cycle progression and viability of tumor and/or immortalized cells and also on inflammatory response of macrophage-like cells. Kuwanon E and 4′-O-methylkuwanon E exerted more sophisticated rather than direct toxic effect on used cell types. Our data indicate that mechanisms different from stress-related or apoptotic signaling pathways are involved in the action of these compounds. Although further studies are required to precisely define the mechanisms of MA flavonoid action in human cancer and macrophage-like cells, here we demonstrate their effects combining antiproliferative and anti-inflammatory activities, respectively.

Inflammasome Inhibition Suppresses Alveolar Cell Permeability Through Retention of Neuregulin-1 (NRG-1

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Rajanbabu Venugopal

2015-07-01

Full Text Available Background: Neuregulin (NRG-1-human epidermal receptor (HER-2 signaling pathway is a key regulator of IL-1β-mediated pulmonary inflammation and epithelial permeability. The inflammasome is a newly discovered molecular platform required for caspase-1 activation and maturation of IL-1β. However, the role of the inflammasome in NRG-1-HER2 signaling-mediated alveolar cell permeability is unknown. Methods: The inflammasome was activated or inhibited in THP-1 cells; supernatants from these cells were added to A549 cells and human small airway epithelial cells (HSAEC. The protein expression of NRG-1 and phospho-HER2 (pHER2 were measured by Western blot analysis and epithelial permeability was measured using Lucifer yellow dye. Results: Results reveal that alveolar permeability in A549 cells and HSAEC is increased when treated with supernatants of inflammasome-activated THP-1 cells. Alveolar permeability is significantly suppressed when treated with supernatant of inflammasome-inhibited THP-1 cells. Inflammasome-mediated permeability is decreased when A549 cells and HSAEC are pretreated with IL-1β receptor antagonist (IL-1βRA. In addition, HER2 kinase inhibitor AG825 or NRG-1 inhibitor TAPI inhibits inflammasome-mediated permeability in A549 cells and HSAEC demonstrating critical roles of IL-1β, NRG-1, and HER2 in inflammasome-mediated alveolar permeability. Conclusion: These findings suggest that inflammasome-induced alveolar cell permeability is mediated by NRG-1/HER2 signaling through IL-1β regulation.

Anthocyanins and phenolic acids from a wild blueberry (Vaccinium angustifolium) powder counteract lipid accumulation in THP-1-derived macrophages

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Del Bo', Cristian; Cao, Yi; Roursgaard, Martin

2016-01-01

PURPOSE: Blueberries are a rich source of anthocyanins (ACNs) and phenolic acids (PA), which are hypothesized to protect against development of atherosclerosis. The present study examined the effect of an ACN- and PA-rich fractions, obtained from a wild blueberry powder, on the capacity...... to counteract lipid accumulation in macrophages derived from monocytic THP-1 cells. In addition, we tested the capacity of pure ACNs and their metabolites to alter lipid accumulation. METHODS: THP-1-derived macrophages were incubated with fatty acids (500 μM oleic/palmitic acid, 2:1 ratio) and different...... concentrations (from 0.05 to 10 μg mL(-1)) of ACN- and PA-rich fractions, pure ACN standards (malvidin, delphinidin and cyanidin 3-glucoside), and metabolites (syringic, gallic and protocatechuic acids). Lipid accumulation was quantified with the fluorescent dye Nile red. RESULTS: Lipid accumulation was reduced...

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

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

2013-01-01

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

Cytotoxic Capacity of IL-15-Stimulated Cytokine-Induced Killer Cells Against Human Acute Myeloid Leukemia and Rhabdomyosarcoma in Humanized Preclinical Mouse Models

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Rettinger, Eva; Meyer, Vida; Kreyenberg, Hermann [Department of Pediatric Hematology, Oncology and Hemostaseology, University Children’s Hospital of Frankfurt/Main, Goethe-University Frankfurt/Main, Frankfurt/Main (Germany); Volk, Andreas [Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Haus, Frankfurt/Main (Germany); Kuçi, Selim; Willasch, Andre [Department of Pediatric Hematology, Oncology and Hemostaseology, University Children’s Hospital of Frankfurt/Main, Goethe-University Frankfurt/Main, Frankfurt/Main (Germany); Koscielniak, Ewa [Department of Pediatric Oncology and Hematology, Olgahospital Stuttgart, Stuttgart (Germany); Fulda, Simone [Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt/Main, Frankfurt/Main (Germany); Wels, Winfried S. [Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Haus, Frankfurt/Main (Germany); Boenig, Halvard [Institute for Transfusion Medicine and Immunohematology, Goethe-University Frankfurt/Main, Division for Cell Processing, German Red Cross Blood Donor Service Baden-Württemberg-Hessen, Frankfurt/Main (Germany); Klingebiel, Thomas; Bader, Peter, E-mail: eva.rettinger@kgu.de, E-mail: peter.bader@kgu.de [Department of Pediatric Hematology, Oncology and Hemostaseology, University Children’s Hospital of Frankfurt/Main, Goethe-University Frankfurt/Main, Frankfurt/Main (Germany)

2012-04-09

Allogeneic stem cell transplantation (allo-SCT) has become an important treatment modality for patients with high-risk acute myeloid leukemia (AML) and is also under investigation for soft tissue sarcomas. The therapeutic success is still limited by minimal residual disease (MRD) status ultimately leading to patients’ relapse. Adoptive donor lymphocyte infusions based on MRD status using IL-15-expanded cytokine-induced killer (CIK) cells may prevent relapse without causing graft-versus-host-disease (GvHD). To generate preclinical data we developed mouse models to study anti-leukemic- and anti-tumor-potential of CIK cells in vivo. Immunodeficient mice (NOD/SCID/IL-2Rγc{sup −}, NSG) were injected intravenously with human leukemic cell lines THP-1, SH-2 and with human rhabdomyosarcoma (RMS) cell lines RH41 and RH30 at minimal doses required for leukemia or tumor engraftment. Mice transplanted with THP-1 or RH41 cells were randomly assigned for analysis of CIK cell treatment. Organs of mice were analyzed by flow cytometry as well as quantitative polymerase chain reaction for engraftment of malignant cells and CIK cells. Potential of CIK cells to induce GvHD was determined by histological analysis. Tissues of the highest degree of THP-1 cell expansion included bone marrow followed by liver, lung, spleen, peripheral blood (PB), and brain. RH30 and RH41 engraftment mainly took place in liver and lung, but was also detectable in spleen and PB. In spite of delayed CIK cell expansion compared with malignant cells, CIK cells injected at equal amounts were sufficient for significant reduction of RH41 cells, whereas against fast-expanding THP-1 cells 250 times more CIK than THP-1 cells were needed to achieve comparable results. Our preclinical in vivo mouse models showed a reliable 100% engraftment of malignant cells which is essential for analysis of anti-cancer therapy. Furthermore our data demonstrated that IL-15-activated CIK cells have potent cytotoxic capacity

Pneumatic muscle actuator (PMA) task-specific resistance for potential use in microgravity exercise.

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Hall, Kara L; Phillips, Chandler A; Reynolds, David B; Mohler, Stanley R; Neidhard-Doll, Amy T

2012-07-01

A pneumatic muscle actuator (PMA) is a device that mimics the behavior of skeletal muscle by contracting and generating force when activated. This type of actuator has a high power to weight ratio and unique characteristics which make it ideal for human interaction. PMAs, however, are difficult to control due to nonlinear dynamics. Our objective was to control a PMA as a source of task-specific resistance in simulated isokinetic strength training. Task-specific resistance will benefit those in need of strength training through a joint's range of motion, including astronauts who need to counteract muscle atrophy during prolonged spaceflight. The lightweight, clean, and compact PMA driven by pressurized air is able to produce resistance in microgravity. An open-loop control method based on a three-element phenomenological inverse model was developed to control the PMA. A motor was simultaneously controlled to act as simulated human quadriceps working against the PMA-produced resistance. For ankle weight replacement resistance profiles, the PMA control method produced resistance and PMA displacement tracking errors (RMSE) of 0.36-1.61 Nm and 0.55-1.59 mm, respectively. Motor position (simulated joint angle) tracking errors ranged from 0.47 to 2.82 degrees. Results indicate that the inverse model based control system produces task-specific PMA resistance and displacement. Closed-loop motor control was able to simulate isokinetic movement successfully. More complicated resistance profiles reveal the need for closed-loop control. Future work focuses on advancing both the PMA control strategies and the capabilities of the human simulator so that actual human operator applications can be realized.

Differential effects of phorbol 12-myristate 13-acetate and diacylglycerols on thromboxane A2-independent phospholipase A2 activation in collage-stimulated human platelets.

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Reddy, S; Rao, G H; Murthy, M

1994-04-01

We investigated the priming effects of protein kinase C (PKC) activators such as phorbol 12-myristate 13-acetate (PMA), 1,2-DiC8 and OAG, and 1,3-DiC8 (a poor activator of PKC) on thromboxane A2 (TxA2)-independent phospholipase A2 (PLA2) activation in human platelets using collagen and A23187 as agonists. We measured PLA2 activation in collagen-stimulated platelets in the presence of BW755C, which abolished TxA2 synthesis, rise in cytosolic Ca2+, and aggregation. In the presence of PMA (50 nM), the amount of arachidonic acid (AA) released in platelets stimulated with collagen and A23187 represented 300% (13.85 nmol versus 4.5 nmol) and 400% (28 nmol versus 7 nmol) of controls (without PMA), respectively, while 1,2-DiC8, OAG, and 1,3-DiC8 increased TxA2-independent AA release by 50% in A23187-stimulated platelets and had no effect on the release of AA in collagen-stimulated platelets. Interestingly, 1,3-DiC8, which is a poor activator of PKC, was as effective as the other two DAGs (OAG and 1,2-DiC8) in priming TxA2-independent PLA2 activation, but was less effective than PMA in platelets stimulated with A23187. These results suggest that the TXA2-dependent IP3-mediated rise in cytosolic Ca2+ may not be obligatory for priming PLA2 activation in the presence of PMA in collagen-stimulated platelets. In contrast, 1,2-DiC8, OAG, and 1,3-DiC8 likely enhanced PLA2 activation via intracellular Ca2+ as they selectively affect this enzyme only in A23187-stimulated platelets. We also observed a significant increase in both saturated (palmitic and stearic acids) and unsaturated fatty acids (oleic and linoleic acids) in platelets stimulated by collagen or A23187 in the presence of PMA (50 nM), but not in the presence of DAGs. These findings imply that PMA may also affect the activation of DAG/MAG lipases, PLA1, or nonspecific PLA2. Since both 1,2-DiC8 and OAG exert no significant effect on the release of these fatty acids, the effects observed with PMA on DAG lipase/PLA1 may not

Loss of BMI-1 dampens migration and EMT of colorectal cancer in inflammatory microenvironment through TLR4/MD-2/MyD88-mediated NF-κB signaling.

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Ye, Kai; Chen, Qi-Wei; Sun, Ya-Feng; Lin, Jian-An; Xu, Jian-Hua

2018-02-01

Increasing evidence from various clinical and experimental studies has demonstrated that the inflammatory microenvironment created by immune cells facilitates tumor migration. Epithelial-mesenchymal transition (EMT) is involved in the progression of cancer invasion and metastasis in an inflammatory microenvironment. B-lymphoma Moloney murine leukemia virus insertion region 1 (BMI-1) acts as an oncogene in various tumors. Ectopic expression of Bmi-1 have an effect on EMT and invasiveness. The purpose of this study was to investigate the efficacy of BMI-1 on inflammation-induced tumor migration and EMT and the underlying mechanism. We observed that the expression of BMI-1, TNF-α, and IL-1β was significantly increased in HT29 and HCT116 cells after THP-1 Conditioned-Medium (THP-1-CM) stimulation. Additionally, inhibition of BMI-1 impeded cell invasion induced by THP-1-CM-stimulation in both HT29 and HCT116 cells. BMI-1 knockdown remarkably repressed THP-1-CM-induced EMT by regulating the expression of EMT biomarkers with an increase in E-cadherin accompanied by decrease in N-cadherin and vimentin. Furthermore, downregulation of BMI-1 dramatically impeded THP-1-CM-triggered Toll-like receptor 4(TLR4)/myeloid differentiation protein 2(MD-2)/myeloid differentiation factor 88(MyD88) activity by repressing the expression of the TLR4/MD-2 complex and MyD88. Further data demonstrated that knockout of BMI-1 also dampened NF-κB THP-1-CM-triggered activity. Taken all data together, our findings established that BMI-1 modulated TLR4/MD-2/MyD88 complex-mediated NF-κB signaling involved in inflammation-induced cancer cells invasion and EMT, and therefore, could be a potential chemopreventive agent against inflammation-associated colorectal cancer. Establishment of an inflammatory microenvironment. Suppression of BMI-1 reverses THP-1-CM-induced inflammatory cytokine production in CRC. Loss of BMI-1 suppressed TLR4/MD-2/MyD88 complex-mediated NF-κB signaling. © 2017 Wiley

Development of an in vitro photosafety evaluation method utilizing intracellular ROS production in THP-1 cells.

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Toyoda, Akemi; Itagaki, Hiroshi

2018-01-01

Photoreactive compounds that may experience exposure to ultraviolet (UV) radiation can lead to the intracellular production of reactive oxygen species (ROS), which may cause phototoxic and photoallergenic responses. Here, we developed a novel in vitro photosafety assay and investigated whether it could be used to predict phototoxicity and photosensitivity by measuring changes in intracellular ROS production. THP-1 cells that had previously taken up 5-(and-6)-carboxy-2',7'-difluorodihydrofluorescein diacetate (carboxy-H 2 DFFDA), a ROS-sensitive fluorescent reagent, were exposed to photoreactive substances such as phototoxic and photoallergenic materials and then subjected to with UV-A irradiation (5 J/cm 2 ). The fluorescence intensity was subsequently measured using a flow cytometer, and the intracellular ROS production was calculated. A statistically significant increase in ROS following treatment with photoreactive substances was observed in cells irradiated with UV-A. In contrast, no significant increase was observed for non-photoreactive substances in comparison to the control solution. Next, to confirm the impact of intracellular ROS on the photosensitive response, changes in CD86 and CD54 expression were measured following quencher addition during the photo human cell line activation test (photo h-CLAT). The results confirmed the reduction of CD86 and CD54 expression in response to photoallergenic substances following quencher addition. Together, these findings suggest that intracellular ROS production is involved in photosensitizing reactions. Therefore, we suggest that the developed method utilizing intracellular ROS production as an index may be useful as a novel in vitro evaluation tool for photoreactive substances.

Effects of Porphyromonas gingivalis LipopolysaccharideTolerized Monocytes on Inflammatory Responses in Neutrophils.

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Xiang-Qing Zhu

Full Text Available Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, which is termed endotoxin tolerance. The role and mechanism of lipopolysaccharide (LPS-tolerized monocytes in inflammatory responses in neutrophils are currently unclear. Here, conditioned supernatants were collected from THP-1 cells treated with or without repeated 1 μg/ml Porphyromonas gingivalis (P.gingivalis LPS. The chemotactic response of freshly isolated neutrophils recruited by supernatants was determined by a transwell migration assay, which demonstrated a reduced migration of neutrophils stimulated with supernatants from tolerized THP-1 cells in comparison to non-tolerized THP-1 cells. In addition, there was a marked increase in reactive oxygen species (ROS generation and a significant decrease in Caspase 3 activities in neutrophils treated with supernatants from THP-1 cells that were treated repeatedly with P.gingivalis LPS in comparison to single treatment. A cytokine antibody array was then used to assess cytokine expression patterns in THP-1 cells. In tolerized THP-1 cells, 43 cytokine (43/170 expression levels were decreased, including chemokine ligand 23 (CCL23 and IFN-γ, while 11 cytokine (11/170 expression levels were increased, such as death receptor 6 (DR6. Furthermore, there was decreased production of IFN-γ and epithelial neutrophil activating peptide-78 (ENA-78 in THP-1 cells after stimulation with repeated P. gingivalis LPS in comparison to single challenge, which was confirmed by ELISA. Therefore, P.gingivalis LPS- tolerized THP-1 cells were able to depress neutrophil chemotaxis and apoptosis, and contribute to respiratory burst, which might be related to the changes in cytokine expression patterns in THP-1 cells.

Effects of an anti-oxidative ACAT inhibitor on apoptosis/necrosis and cholesterol accumulation under oxidative stress in THP-1 cell-derived foam cells.

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Miike, Tomohiro; Shirahase, Hiroaki; Jino, Hiroshi; Kunishiro, Kazuyoshi; Kanda, Mamoru; Kurahashi, Kazuyoshi

2008-01-02

THP-1 cell-derived foam cells were exposed to oxidative stress through combined treatment with acetylated LDL (acLDL) and copper ions (Cu2+). The foam cells showed caspase-dependent apoptotic changes on exposure to oxidative stress for 6 h, and necrotic changes with the leakage of LDH after 24 h. KY-455, an anti-oxidative ACAT inhibitor, and ascorbic acid (VC) but not YM-750, an ACAT inhibitor, prevented apoptotic and necrotic changes. These preventive effects of KY-455 and VC were accompanied by the inhibition of lipid peroxidation in culture medium containing acLDL and Cu2+, suggesting the involvement of oxidized acLDL in apoptosis and necrosis. Foam cells accumulated esterified cholesterol (EC) for 24 h in the presence of acLDL without Cu2+, which was suppressed by KY-455 and YM-750. Foam cells showed necrotic changes and died in the presence of acLDL and Cu2+. KY-455 but not YM-750 prevented cell death and reduced the amount of EC accumulated. The foam cells treated with VC further accumulated EC without necrotic changes for 24 h even in the presence of acLDL and Cu2+. YM-750 as well as KY-455 inhibited lipid accumulation when co-incubated with VC in foam cells exposed to oxidative stress. It is concluded that an anti-oxidative ACAT inhibitor or the combination of an antioxidant and an ACAT inhibitor protects foam cells from oxidative stress and effectively reduces cholesterol levels, which would be a promising approach in anti-atherosclerotic therapy.

Effects on proliferation and cell cycle of irradiated KG-1 cells stimulated by CM-CSF

International Nuclear Information System (INIS)

Guo Dehuang; Dong Bo; Wen Gengyun; Luo Qingliang; Mao Bingzhi

2000-01-01

In order to explore the variety of cell proliferation and cell cycle after exposure to ionizing radiation, the responses of irradiated KG-1 cells of the human myeloid leukemia stimulated by GM-CSF, the most common used cytokine in clinic, were investigated. The results showed that GM-CSF enhance KG-1 cells proliferation, reduce G0/G1 block, increase S phase and G2/M phase. The stimulation effects of the GM-CSF are more effective in irradiated group than in control group

Design, synthesis and biological evaluation of novel desmuramyldipeptide analogs.

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Jakopin, Žiga; Corsini, Emanuela; Gobec, Martina; Mlinarič-Raščan, Irena; Dolenc, Marija Sollner

2011-09-01

A series of novel desmuramyldipeptides have been designed and synthesized as part of our search for therapeutically useful muramyldipeptide (MDP) analogs. Their immunomodulatory properties were initially assessed in vitro, evaluating their effect on lipopolysaccharide (LPS)-induced cytokine release in THP-1 cells. Following the initial screening, selected compounds were further investigated for immunomodulatory properties using LPS and phorbol 12-myristate 13-acetate (PMA)/ionomycin-stimulated human peripheral blood mononuclear cells. The results confirmed the immunomodulatory properties of some of the synthesized desmuramyldipeptide analogs. Taken together, presented data confirmed the immunostimulatory effect of compound 44, MDP derivative incorporating a pyrido-fused [1,2]-benzisothiazole moiety, while for compounds 32 and 39, indole scaffold-based derivatives of MDP, an immunosuppressive effect was observed. Further studies will be necessary to address their potential therapeutic use as immunomodulatory drugs, both as immunostimulants or anti-inflammatory agents. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Activated factor X signaling via protease-activated receptor 2 suppresses pro-inflammatory cytokine production from LPS-stimulated myeloid cells.

LENUS (Irish Health Repository)

Gleeson, Eimear M

2013-07-19

Vitamin K-dependent proteases generated in response to vascular injury and infection enable fibrin clot formation, but also trigger distinct immuno-regulatory signaling pathways on myeloid cells. Factor Xa, a protease crucial for blood coagulation, also induces protease-activated receptor-dependent cell signaling. Factor Xa can bind both monocytes and macrophages, but whether factor Xa-dependent signaling stimulates or suppresses myeloid cell cytokine production in response to Toll-like receptor activation is not known. In this study, exposure to factor Xa significantly impaired pro-inflammatory cytokine production from lipopolysaccharide-treated peripheral blood mononuclear cells, THP-1 monocytic cells and murine macrophages. Furthermore, factor Xa inhibited nuclear factor-kappa B activation in THP-1 reporter cells, requiring phosphatidylinositide 3-kinase activity for its anti-inflammatory effect. Active-site blockade, γ-carboxyglutamic acid domain truncation and a peptide mimic of the factor Xa inter-epidermal growth factor-like region prevented factor Xa inhibition of lipopolysaccharide-induced tumour necrosis factor-α release. In addition, factor Xa anti-inflammatory activity was markedly attenuated by the presence of an antagonist of protease-activated receptor 2, but not protease-activated receptor 1. The key role of protease-activated receptor 2 in eliciting factor Xa-dependent anti-inflammatory signaling on macrophages was further underscored by the inability of factor Xa to mediate inhibition of tumour necrosis factor-α and interleukin-6 release from murine bone marrow-derived protease-activated receptor 2-deficient macrophages. We also show for the first time that, in addition to protease-activated receptor 2, factor Xa requires a receptor-associated protein-sensitive low-density lipoprotein receptor to inhibit lipopolysaccharide-induced cytokine production. Collectively, this study supports a novel function for factor Xa as an endogenous, receptor

Activity of novel oxazolidinones against Nocardia brasiliensis growing within THP-1 macrophages.

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Vera-Cabrera, Lucio; Espinoza-González, Nelly A; Welsh, Oliverio; Ocampo-Candiani, Jorge; Castro-Garza, Jorge

2009-11-01

Nocardia are organisms that can escape the effects of both immune response and antimicrobial agents, due to their potential capacity to grow intracellularly. In previous studies, we found that experimental oxazolidinones, DA-7157 and DA-7218, are active both in vitro and in vivo. In this study, we compare the ability of linezolid, DA-7157 and DA-7218 to inhibit intracellular growth of Nocardia brasiliensis within the human monocyte cell line THP-1. The addition of oxazolidinones to the infected macrophage monolayer at concentrations 0.25x, 1x, 4x and 16x the MIC for N. brasiliensis resulted in an inhibitory effect on bacterial growth as follows DA-7157 > or = DA-7218 > linezolid. The excellent intracellular antimicrobial activity detected suggests that these compounds could be effective in the treatment of actinomycetoma. However, more studies are needed both in vitro and in vivo, including clinical trials, to confirm this issue.

Anti-Inflammatory Activity of Haskap Cultivars is Polyphenols-Dependent

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H. P. Vasantha Rupasinghe

2015-06-01

Full Text Available Haskap (Lonicera caerulea L. berries have long been used for their health promoting properties against chronic conditions. The current study investigated the effect of Canadian haskap berry extracts on pro-inflammatory cytokines using a human monocytic cell line THP-1 derived macrophages stimulated by lipopolysaccharide. Methanol extracts of haskap from different growing locations in Canada were prepared and characterized for their total phenolic profile using colorimetric assays and liquid chromatography—Mass spectrometry (UPLC-MS/MS. Human THP-1 monocytes were seeded in 24-well plates (5 × 105/well and treated with phorbol 12-myristate 13-acetate (PMA, 0.1 μg/mL for 48 h to induce macrophage differentiation. After 48 h, the differentiated macrophages were washed with Hank’s buffer and treated with various concentrations of test compounds for 4 h, followed by the lipopolysaccharide (LPS-stimulation (18 h. Borealis cultivar showed the highest phenolic content, flavonoid content and anthocyanin content (p < 0.05. A negative correlation existed between the polyphenol concentration of the extracts and pro-inflammatory cytokines: Interleukin-6 (IL-6, tumour necrosis factor-alpha (TNF-α, prostaglandin (PGE2, and cyclooxygenase-2 (COX-2 enzyme. Borealis exhibited comparable anti-inflammatory effects to COX inhibitory drug, diclofenac. The results showed that haskap berry polyphenols has the potential to act as an effective inflammation inhibitor.

Optimal Method to Stimulate Cytokine Production and Its Use in Immunotoxicity Assessment

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Huiming Chen

2013-08-01

Full Text Available Activation of lymphocytes can effectively produce a large amount of cytokines. The types of cytokines produced may depend on stimulating reagents and treatments. To find an optimal method to stimulate cytokine production and evaluate its effect on immunotoxicity assessments, the authors analyzed production of IL-2, IL-4, IL-6, IL-10, IL-13, IFN-γ, TNF-α, GM-CSF, RANTES and TGF-β in undiluted rat whole blood culture (incubation for 0, 2, 4, 6, 8 or 10 h with different concentrations of PMA/ionomycin, PHA, Con A, LPS and PWM. We also evaluated the effects of cyclosporin A and azathioprine on cytokine production. The results revealed a rapid increase of IL-2, IFN-γ, TNF-α, RANTES and TGF-β secretion within 6 h after stimulation with 25 ng/mL PMA and 1 μg/mL ionomycin. The inhibition of these cytokine profiles reflected the effects of immunosuppressants on the immune system. Therefore, the results of this is study recommend the detection of cytokine profiles in undiluted whole blood stimulated 6 h with 25 ng/mL PMA and 1 μg/mL ionomycin as a powerful immunotoxicity assessment method.

Supercritical fluid extraction of oregano (Origanum vulgare) essentials oils: anti-inflammatory properties based on cytokine response on THP-1 macrophages.

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Ocaña-Fuentes, A; Arranz-Gutiérrez, E; Señorans, F J; Reglero, G

2010-06-01

Two fractions (S1 and S2) of an oregano (Origanum vulgare) extract obtained by supercritical fluid extraction have been used to test anti-inflammatory effects on activated human THP-1 cells. The main compounds present in the supercritical extract fractions of oregano were trans-sabinene hydrate, thymol and carvacrol. Fractions toxicity was assessed using the mitochondrial-respiration-dependent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction method for several concentrations during 24 and 48 h of incubation. Concentrations higher than 30 microg/mL of both supercritical S1 and S2 oregano fractions caused a reduction in cell viability in a dose-dependent manner. Oxidized-LDLs (oxLDLs) activated THP-1 macrophages were used as cellular model of atherogenesis and the release/secretion of cytokines (TNT-alpha, IL-1beta, IL-6 and IL-10) and their respective mRNA expressions were quantified both in presence or absence of supercritical oregano extracts. The results showed a decrease in pro-inflammatory TNF-alpha, IL-1beta and IL-6 cytokines synthesis, as well as an increase in the production of anti-inflammatory cytokine IL-10. These results may suggest an anti-inflammatory effect of oregano extracts and their compounds in a cellular model of atherosclerosis. Copyright 2010 Elsevier Ltd. All rights reserved.

Pomegranate inhibits neuroinflammation and amyloidogenesis in IL-1β-stimulated SK-N-SH cells.

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Velagapudi, Ravikanth; Baco, Gina; Khela, Sunjeet; Okorji, Uchechukwu; Olajide, Olumayokun

2016-06-01

Pomegranate fruit, Punica granatum L. (Punicaceae), and its constituents have been shown to inhibit inflammation. In this study, we aimed to assess the effects of freeze-dried pomegranate (PWE) on PGE2 production in IL-1β-stimulated SK-N-SH cells. An enzyme immunoassay (EIA) was used to measure prostaglandin E2 (PGE2) production from supernatants of IL-1β-stimulated SK-N-SH cells. Expression of COX-2, phospho-IκB, and phospho-IKK proteins was evaluated, while NF-κB reporter gene assay was carried out in TNFα-stimulated HEK293 cells to determine the effect of PWE on NF-κB transactivation. Levels of BACE-1 and Aβ in SK-N-SH cells stimulated with IL-1β were measured with an in cell ELISA. PWE (25-200 μg/ml) dose dependently reduced COX-2-dependent PGE2 production in SK-N-SH cells stimulated with IL-1β. Phosphorylation of IκB and IKK was significantly (p pomegranate inhibits inflammation, as well as amyloidogenesis in IL-1β-stimulated SK-N-SH cells. We propose that pomegranate is a potential nutritional strategy in slowing the progression of neurodegenerative disorders such as Alzheimer's disease.

SUMOylation of sPRDM16 promotes the progression of acute myeloid leukemia

International Nuclear Information System (INIS)

Dong, Song; Chen, Jieping

2015-01-01

In addition to genetic and epigenetic alteration, post-translational modification of proteins plays a critical role in the initiation, progression and maturation of acute myeloid leukemia (AML). The SUMOylation site of sPRDM16 at K568 was mutated to arginine by site-directed mutagenesis. THP-1 acute myeloid leukemia cells were transduced with a lentivirus containing wild type or K568 mutant sPRDM16. Proliferation, self-renewal and differentiation of transduced THP-1 cells were analyzed both in vitro cell culture and in mouse xenografts. Gene expression profiles were analyzed by RNA-seq. Overexpression of sPRDM16 promoted proliferation, enhanced self-renewal capacity, but inhibited differentiation of THP-1 acute myeloid leukemia cells. We further confirmed that K568 is a bona fide SUMOylation site on sPRDM16. Mutation of the sPRDM16 SUMOylation site at K568 partially abolished the capacity of sPRDM16 to promote proliferation and inhibit differentiation of acute myeloid leukemia cells both in vitro and in mouse xenografts. Furthermore, THP-1 cells overexpressing sPRDM16-K568R mutant exhibited a distinct gene expression profile from wild type sPRDM16 following incubation with PMA. Our results suggest that K568 SUMOylation of sPRDM16 plays an important role in the progression of acute myeloid leukemia

The efficacy and mechanism of apoptosis induction by hypericin-mediated sonodynamic therapy in THP-1 macrophages

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Li XS

2015-01-01

Full Text Available Xuesong Li,1,* Lei Gao,2,* Longbin Zheng,1 Jiayuan Kou,1 Xing Zhu,1 Yueqing Jiang,1 Zhaoyu Zhong,1 Juhua Dan,1 Haobo Xu,3 Yang Yang,3 Hong Li,1 Sa Shi,1 Wenwu Cao,4,5 Yajun Zhao,1 Ye Tian,1,3 Liming Yang1 1Department of Pathophysiology, Harbin Medical University, Harbin, People’s Republic of China; 2Electron Microscopy Centre, Harbin Medical University, Harbin, People’s Republic of China; 3Division of Cardiology, The First Affiliated Hospital, Harbin Medical University, Harbin, People’s Republic of China; 4Laboratory of Sono- and Photo-theranostic Technologies, Harbin Institute of Technology, Harbin, People’s Republic of China; 5Materials Research Institute, The Pennsylvania State University, University Park, PA, USA *These authors contributed equally to this work Purpose: To investigate the sonoactivity of hypericin (HY, together with its sonodynamic effect on THP-1 macrophages and the underlying mechanism.Materials and methods: CCK-8 was used to examine cell viability. Confocal laser scanning microscopy was performed to assess the localization of HY in cells, reactive oxygen species (ROS generation, and opening of the mitochondrial permeability transition pore (mPTP after different treatments. Apoptosis was analyzed using Hoechst–propidium iodide and transmission electron microscopy. Mitochondrial membrane potential (ΔΨm collapse was detected via fluorescence microscopy. Lipoprotein oxidation was determined in malondialdehyde (MDA assays. Western blotting was conducted to determine the translocation of BAX and cytochrome C and the expression of apoptosis-related proteins.Results: HY was sublocalized among the nuclei and the mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosome in the cytosol of THP-1 macrophages. Under low-intensity ultrasound irradiation, HY significantly decreased cell viability and induced apoptosis. Furthermore, greater ROS generation, higher MDA levels, and greater ΔΨm loss were observed in the

Rapid kit-based (68)Ga-labelling and PET imaging with THP-Tyr(3)-octreotate: a preliminary comparison with DOTA-Tyr(3)-octreotate.

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Ma, Michelle T; Cullinane, Carleen; Waldeck, Kelly; Roselt, Peter; Hicks, Rodney J; Blower, Philip J

2015-12-01

Ge/(68)Ga generators provide an inexpensive source of a PET isotope to hospitals without cyclotron facilities. The development of new (68)Ga-based molecular imaging agents and subsequent clinical translation would be greatly facilitated by simplification of radiochemical syntheses. We report the properties of a tris(hydroxypyridinone) conjugate of the SSTR2-targeted peptide, Tyr(3)-octreotate (TATE), and compare the (68)Ga-labelling and biodistribution of [(68)Ga(THP-TATE)] with the clinical radiopharmaceutical [(68)Ga(DOTATATE)]. A tris(hydroxypyridinone) with a pendant isothiocyanate group was conjugated to the primary amine terminus of H2N-PEG2-Lys(iv-Dde)(5)-TATE, and the resulting conjugate was deprotected to provide THP-TATE. THP-TATE was radiolabelled with (68)Ga(3+) from a (68)Ge/(68)Ga generator. In vitro uptake was assessed in SSTR2-positive 427-7 cells and SSTR2-negative 427 (parental) cells. Biodistribution of [(68)Ga(THP-TATE)] was compared with that of [(68)Ga(DOTATATE)] in Balb/c nude mice bearing SSTR2-positive AR42J tumours. PET scans were obtained 1 h post-injection, after which animals were euthanised and tissues/organs harvested and counted. [(68)Ga(THP-TATE)] was radiolabelled and formulated rapidly in negative cells, and receptor binding and internalisation were specific. Animals administered [(68)Ga(THP-TATE)] demonstrated comparable SSTR2-positive tumour activity (11.5 ± 0.6 %ID g(-1)) compared to animals administered [(68)Ga(DOTATATE)] (14.4 ± 0.8 %ID g(-1)). Co-administration of unconjugated Tyr(3)-octreotate effectively blocked tumour accumulation of [(68)Ga(THP-TATE)] (2.7 ± 0.6 %ID g(-1)). Blood clearance of [(68)Ga(THP-TATE)] was rapid and excretion was predominantly renal, although compared to [(68)Ga(DOTATATE)], [(68)Ga(THP-TATE)] exhibited comparatively longer kidney retention. Radiochemical synthesis of [(68)Ga(THP-TATE)] is significantly faster, proceeds under milder conditions, and requires less manipulation

Effects of Thyme Extract Oils (from Thymus vulgaris, Thymus zygis, and Thymus hyemalis on Cytokine Production and Gene Expression of oxLDL-Stimulated THP-1-Macrophages

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A. Ocaña

2012-01-01

Full Text Available Properties of thyme extracts from three different species (Thymus vulgaris, Thymus zygis, and Thymus hyemalis were examined. Two oil fractions from each species were obtained by CO2 supercritical fluid extraction. Main compounds presented in the supercritical extracts of the three thyme varieties were 1,8 cineole, thymol, camphor, borneol, and carvacrol. As a cellular model of inflammation/atherogenesis, we use human macrophages derived from THP-1 monocytes and activated by oxidized LDLs. These cells were incubated with the thyme fraction oils, and the productions and gene expressions of the inflammatory mediators TNF-α, IL-1B, IL-6, and IL-10 were determined. Thyme extracts significantly reduced production and gene expression of the proinflammatory mediators TNF-α, IL-1B, and IL-6 and highly increased these parameters on the anti-inflammatory IL-10 cytokine. Changes on production and gene expressions were dose dependent and according to the thyme content of each species. Taken together, these results may suggest that thyme extracts could have anti-inflammatory effects.

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) mediates repression of TNF-α by decreasing levels of acetylated histone H3 and H4 at its promoter

International Nuclear Information System (INIS)

Engdahl, Ryan; Monroy, M. Alexandra; Daly, John M.

2007-01-01

Prostaglandin metabolite 15-Deoxy-Δ 12,14 -prostaglandin J2 (15d-PGJ2) is known to inhibit a number of pro-inflammatory cytokines as well as being a ligand for nuclear receptor PPARγ. We investigated the ability of 15d-PGJ2 to inhibit TNF-α gene expression through mechanisms that involve histone modification. Pretreatment with 15d-PGJ2 (10 μM) inhibited LPS-stimulated TNF-α mRNA in THP-1 monocytes or PMA-differentiated cells to nearly basal levels. A specific PPARγ ligand, GW1929, failed to inhibit LPS-induced TNF-α mRNA expression nor did a PPARγ antagonist, GW9662, alter the repression of TNF-α mRNA in LPS-stimulated cells pretreated with 15d-PGJ2 suggesting a PPARγ-independent inhibition of TNF-α mRNA in THP-1 cells. Transfection studies with a reporter construct and subsequent treatment with 15d-PGJ2 demonstrated a dose-dependent inhibition of the TNF-α promoter. Additional studies demonstrated that inhibition of histone deacetylases with trichostatin A (TSA) or overexpression of histone acetyltransferase CBP could overcome 15d-PGJ2-mediated repression of the TNF-α promoter, suggesting that an important mechanism whereby 15d-PGJ2 suppresses a cytokine is through factors that regulate histone modifications. To examine the endogenous TNF-α promoter, chromatin immunoprecipitations (ChIP) were performed. ChIP assays demonstrated that LPS stimulation induced an increase in histone H3 and H4 acetylation at the TNF-α promoter, which was reduced in cells pretreated with 15d-PGJ2. These results highlight the ability of acetylation and deacetylation factors to affect the TNF-α promoter and demonstrate that an additional important mechanism whereby 15d-PGJ2 mediates TNF-α transcriptional repression by altering levels of acetylated histone H3 and H4 at its promoter

Aluminium based adjuvants and their effects on mitochondria and lysosomes of phagocytosing cells.

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Ohlsson, Lars; Exley, Christopher; Darabi, Anna; Sandén, Emma; Siesjö, Peter; Eriksson, Håkan

2013-11-01

Aluminium oxyhydroxide, Al(OH)3 is one of few compounds approved as an adjuvant in human vaccines. However, the mechanism behind its immune stimulating properties is still poorly understood. In vitro co-culture of an aluminium adjuvant and the human monocytic cell line THP-1 resulted in reduced cell proliferation. Inhibition occurred at concentrations of adjuvant several times lower than would be found at the injection site using a vaccine formulation containing an aluminium adjuvant. Based on evaluation of the mitochondrial membrane potential, THP-1 cells showed no mitochondrial rupture after co-culture with the aluminium adjuvant, instead an increase in mitochondrial activity was seen. The THP-1 cells are phagocytosing cells and after co-culture with the aluminium adjuvant the phagosomal pathway was obstructed. Primary or early phagosomes mature into phagolysosomes with an internal pH of 4.5 - 5 and carry a wide variety of hydrolysing enzymes. Co-culture with the aluminium adjuvant yielded a reduced level of acidic vesicles and cathepsin L activity, a proteolytic enzyme of the phagolysosomes, was almost completely inhibited. THP-1 cells are an appropriate in vitro model in order to investigate the mechanism behind the induction of a phagocytosing antigen presenting cell into an inflammatory cell by aluminium adjuvants. Much information will be gained by investigating the phagosomal pathway and what occurs inside the phagosomes and to elucidate the ultimate fate of phagocytosed aluminium particles. © 2013.

Cytotoxic capacity of IL-15-stimulated cytokine-induced killer cells against human acute myeloid leukemia and rhabdomyosarcoma in humanized preclinical mouse models

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Eva eRettinger

2012-04-01

Full Text Available Allogeneic stem cell transplantation (allo-SCT has become an important treatment modality for patients with high risk acute myeloid leukemia (AML and is also under investigation for soft tissue sarcomas. The therapeutic success is still limited by minimal residual disease (MRD status ultimately leading to patients’ relapse. Adoptive donor lymphocyte infusions (DLI based on MRD status using IL-15-expanded cytokine-induced killer (CIK cells may prevent relapse without causing graft-versus-host-disease (GvHD. To generate preclinical data we developed mouse models to study anti-leukemic- and anti-tumor-potential of CIK cells in vivo. Immunodeficient mice (NOD/SCID/IL2Rγc-, NSG were injected intravenously with human leukemic cell lines THP-1, SH-2 and with human rhabdomyosarcoma (RMS cell lines RH41 and RH30 at minimal doses required for leukemia or tumor engraftment. Mice transplanted with THP-1 or RH41 cells were randomly assigned for analysis of CIK cell treatment. Organs of mice were analyzed by flow cytometry as well as quantitative polymerase chain reaction (qPCR for engraftment of malignant cells and CIK cells. Potential of CIK cells to induce GvHD was determined by histological analysis. Tissues of the highest degree of THP-1 cell expansion included bone marrow (BM followed by liver, lung, spleen, peripheral blood (PB, and brain. RH30 and RH41 engraftment mainly took place in liver and lung, but was also detectable in spleen and PB. In spite of delayed CIK cell expansion compared with malignant cells, CIK cells injected at an effector to target cell (E:T ratio of 1:1 were sufficient for significant reduction of RH41 cells, whereas against fast-expanding THP-1 cells an E:T ratio of 250:1 was needed to achieve comparable results. Our preclinical in vivo mouse models showed a reliably 100% engraftment of malignant cells which is essential for analysis of anti-cancer therapy. Furthermore our data demonstrated that IL-15-activated CIK cells

T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers.

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Wai-Ping Fung-Leung

Full Text Available Kv1.3 is a voltage-gated potassium channel expressed on T cells that plays an important role in T cell activation. Previous studies have shown that blocking Kv1.3 channels in human T cells during activation results in reduced calcium entry, cytokine production, and proliferation. The aim of the present study was to further explore the effects of Kv1.3 blockers on the response of different human T cell subsets under various stimulation conditions. Our studies show that, unlike the immune suppressor cyclosporine A, the inhibitory effect of Kv1.3 blockers was partial and stimulation strength dependent, with reduced inhibitory efficacy on T cells under strengthened anti-CD3/CD28 stimulations. T cell responses to allergens including house dust mites and ragweed were partially reduced by Kv1.3 blockers. The effect of Kv1.3 inhibition was dependent on T cell subsets, with stronger effects on CCR7- effector memory compared to CCR7+ central memory CD4 T cells. Calcium entry studies also revealed a population of CD4 T cells resistant to Kv1.3 blockade. Activation of CD4 T cells was accompanied with an increase in Kv1.3 currents but Kv1.3 transcripts were found to be reduced, suggesting a posttranscriptional mechanism in the regulation of Kv1.3 activities. In summary, Kv1.3 blockers inhibit T cell activation in a manner that is highly dependent on the T cell identity and stimulation strength, These findings suggest that Kv1.3 blockers inhibit T cells in a unique, conditional manner, further refining our understanding of the therapeutic potential of Kv1.3 blockers.

Differential acute and chronic response of protein kinase C in cultured neonatal rat heart myocytes to alpha 1-adrenergic and phorbol ester stimulation.

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Henrich, C J; Simpson, P C

1988-12-01

Both alpha 1-adrenergic agonists (e.g. norepinephrine, NE*) and tumor-promoting phorbol esters (e.g. phorbol myristate acetate, PMA) are known to activate protein kinase C (PKC) (Abdel-Latif, 1986, Niedel and Blackshear, 1986). However, alpha 1 agonists and PMA produce very different effects on cardiac function (see Simpson, 1985; Benfey, 1987; Meidell et al., 1986; Leatherman et al., 1987; Yuan et al., 1987; for examples). PKC activation in heart cells has been studied only for PMA treated perfused heart (Yuan et al., 1987). Therefore, acute activation and chronic regulation of PKC by NE and PMA were compared in cultured neonatal rat heart myocytes. NE acutely and transiently activated PKC, as measured by translocation of PKC activity to the cell particulate fraction (Niedel and Blackshear, 1986). Particulate PKC activity peaked at 23% of total after NE for 30 s, as compared with 8% for control (P less than 0.001). By contrast, acute PKC activation by PMA was more pronounced and persistent, with particulate PKC activity 62% of total at 5 min (P less than 0.001). Calcium/lipid-independent kinase activity increased acutely with PMA, but not with NE. Chronic treatment with NE (24 to 48 h) increased total per cell PKC activity and 3H-phorbol dibutyrate (PDB) binding sites, an index of the number of PKC molecules (Niedel and Blackshear, 1986), by 30 to 60% over control (all P less than 0.05 to 0.01). In contrast with NE, chronic treatment with PMA down-regulated PKC, reducing total per cell PKC activity and 3H-PDB binding sites to 3% and 12% of control, respectively (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Virulent Mycobacterium bovis Beijing Strain Activates the NLRP7 Inflammasome in THP-1 Macrophages.

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Yang Zhou

Full Text Available Mycobacterium bovis is the causative agent of tuberculosis in a wide range of mammals, including humans. Macrophages are the first line of host defense. They secrete proinflammatory cytokines, such as interleukin-1 beta (IL-1β, in response to mycobacterial infection, but the underlying mechanisms by which human macrophages are activated and release IL-1β following M. bovis infection are poorly understood. Here we show that the 'nucleotide binding and oligomerization of domain-like receptor (NLR family pyrin domain containing 7 protein' (NLRP7 inflammasome is involved in IL-1β secretion and caspase-1 activation induced by M. bovis infection in THP-1 macrophages. NLRP7 inflammasome activation promotes the induction of pyroptosis as well as the expression of tumor necrosis factor alpha (TNF-α, Chemokine (C-C motif ligand 3 (CCL3 and IL-1β mRNAs. Thus, the NLRP7 inflammasome contributes to IL-1β secretion and induction of pyroptosis in response to M. bovis infection in THP-1 macrophages.

Inhibition of TNF-α production in LPS-activated THP-1 monocytic cells by the crude extracts of seven Bhutanese medicinal plants.

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Wangchuk, Phurpa; Keller, Paul A; Pyne, Stephen G; Taweechotipatr, Malai

2013-07-30

Seven studied medicinal plants; Aconitum laciniatum, Ajania nubigena, Codonopsis bhutanica, Corydalis crispa, Corydalis dubia, Meconopsis simplicifolia and Pleurospermum amabile, are currently used in the Bhutanese Traditional Medicine (BTM) for the management of different types of disorders including the diseases that bore relevance to various inflammatory conditions. This study aimed to evaluate the inhibition of TNF-α production in LPS-activated THP-1 monocytic cells by the crude extracts of seven selected Bhutanese medicinal plants. It is expected to; (a) generate a scientific basis for their use in the BTM and (b) form a basis for prioritization of the seven plants for further phytochemical and anti-inflammatory studies. Seven plants were selected using an ethno-directed bio-rational approach and their crude extracts were prepared using four different solvents (methanol, hexane, dichloromethane and chloroform). The TNF-α inhibitory activity of these extracts was determined by cytokine-specific sandwich quantitative enzyme-linked immunosorbent assays (ELISAs). The results were quantified statistically and the statistical significance were evaluated by GraphPad Prism version 5.01 using Student's t-test with one-tailed distribution. A p-value ≤0.05 was considered statistically significant. Of the seven plants studied, the crude extracts of six of them inhibited the production of pro-inflammatory cytokine, TNF-α in LPS-activated THP-1 monocytic cells. Amongst the six plants, Corydalis crispa gave the best inhibitory activity followed by Pleurospermum amabile, Ajania nubigena, Corydalis dubia, Meconopsis simplicifolia and Codonopsis bhutanica. Of the 13 extracts that exhibited statistically significant TNF-α inhibitory activity (p<0.05; p<0.01), five of them showed very strong inhibition when compared to the DMSO control and RPMI media. Six medicinal plants studied here showed promising TNF-α inhibitory activity. These findings rationalize the traditional

Radioimmunoassay of Alb, β2-M and THP

International Nuclear Information System (INIS)

Xie Zhiyuan; Li Shengqi; Ren Shusheng; Chen Yuying

1993-01-01

The combined determination of β 2 -M, THP, Alb, IgG may be used as the sensitive indication of the early degeneration of kidney function. The authors observed 107 cases, and found that the anomalous examine-out ratios of hemorrhage β 2 -M and UTHP are 50.75% and 65.67% respectively, 1.5-2.3 times higher than the C cr animals examine-out ratio. The radio-immunoassay of β 2 -M and THP is a better measure for discovering light kidney function injury in early period than C cr method. The clinical value of the radio-immunoassay of β 2 -M, THP is obviously better than the traditional and classical method. The diseases of anybody system may influence the kidney function through various mechanisms. The retrogression of kidney tissue of the patients in old-and mid-age leads to decrease of kidney function. The combination of β 2 -M and THP has the highest anomalous examine-out ratios of kidney function; the next is urine Alb

Effects of stem cell factor on hypoxia-inducible factor 1 alpha accumulation in human acute myeloid leukaemia and LAD2 mast cells.

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Bernhard F Gibbs

Full Text Available Stem cell factor (SCF is a hematopoietic growth factor that exerts its activity by signalling through the tyrosine kinase receptor known as Kit or CD117. SCF-Kit signalling is crucial for the survival, proliferation and differentiation of hematopoietic cells of myeloid lineage. Furthermore, since myeloid leukaemia cells express the Kit receptor, SCF may play an important role in myeloid leukaemia progression too. However, the mechanisms of this pathophysiological effect remain unclear. Recent evidence shows that SCF triggers accumulation of the inducible alpha subunit of hypoxia-inducible factor 1 (HIF-1 in hematopoietic cells--a transcription complex that plays a pivotal role in cellular adaptation to low oxygen availability. However, it is unknown how SCF impacts on HIF-1α accumulation in human myeloid leukaemia and mast cells. Here we show that SCF induces HIF-1α accumulation in THP-1 human myeloid leukaemia cells but not in LAD2 mast cells. We demonstrated that LAD2 cells have a more robust glutathione (GSH-dependent antioxidative system compared to THP-1 cells and are therefore protected against the actions of ROS generated in an SCF-dependent manner. BSO-induced GSH depletion led to a significant decrease in HIF-1α prolyl hydroxylase (PHD activity in THP-1 cells and to near attenuation of it in LAD2 cells. In THP-1 cells, SCF-induced HIF-1α accumulation is controlled via ERK, PI3 kinase/PKC-δ/mTOR-dependent and to a certain extent by redox-dependent mechanisms. These results demonstrate for the first time an important cross-talk of signalling pathways associated with HIF-1 activation--an important stage of the myeloid leukaemia cell life cycle.

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

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

2016-09-15

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

Induction of bacterial lipoprotein tolerance is associated with suppression of toll-like receptor 2 expression.

LENUS (Irish Health Repository)

Wang, Jiang Huai

2012-02-03

Tolerance to bacterial cell wall components including lipopolysaccharide (LPS) may represent an essential regulatory mechanism during bacterial infection. Two members of the Toll-like receptor (TLR) family, TLR2 and TLR4, recognize the specific pattern of bacterial cell wall components. TLR4 has been found to be responsible for LPS tolerance. However, the role of TLR2 in bacterial lipoprotein (BLP) tolerance and LPS tolerance is unclear. Pretreatment of human THP-1 monocytic cells with a synthetic bacterial lipopeptide induced tolerance to a second BLP challenge with diminished tumor necrosis factor-alpha and interleukin-6 production, termed BLP tolerance. Furthermore, BLP-tolerized THP-1 cells no longer responded to LPS stimulation, indicating a cross-tolerance to LPS. Induction of BLP tolerance was CD14-independent, as THP-1 cells that lack membrane-bound CD14 developed tolerance both in serum-free conditions and in the presence of a specific CD14 blocking monoclonal antibody (MEM-18). Pre-exposure of THP-1 cells to BLP suppressed mitogen-activated protein kinase phosphorylation and nuclear factor-kappaB activation in response to subsequent BLP and LPS stimulation, which is comparable with that found in LPS-tolerized cells, indicating that BLP tolerance and LPS tolerance may share similar intracellular pathways. However, BLP strongly enhanced TLR2 expression in non-tolerized THP-1 cells, whereas LPS stimulation had no effect. Furthermore, a specific TLR2 blocking monoclonal antibody (2392) attenuated BLP-induced, but not LPS-induced, tumor necrosis factor-alpha and interleukin-6 production, indicating BLP rather than LPS as a ligand for TLR2 engagement and activation. More importantly, pretreatment of THP-1 cells with BLP strongly inhibited TLR2 activation in response to subsequent BLP stimulation. In contrast, LPS tolerance did not prevent BLP-induced TLR2 overexpression. These results demonstrate that BLP tolerance develops through down-regulation of TLR2

In vitro methods of assessing ocular biocompatibility using THP-1-derived macrophages.

Science.gov (United States)

McCanna, David Joseph; Barthod-Malat, Aurore V; Gorbet, Maud B

2015-01-01

Macrophages play an important role in the elimination of infections, the removal of debris and in tissue repair after infection and trauma. In vitro models that assess ocular biomaterials for toxicity typically focus on the effects of these materials on epithelial or fibroblast cells. This investigation evaluated known ocular toxins deposited on model materials for their effects on the viability and activation of macrophages. THP-1-derived macrophages were cultured onto silicone films (used as a base biomaterial) deposited with chemical toxins (benzalkonium chloride (BAK), zinc diethyldithiocarbamate (ZDEC) and lipopolysaccharide (LPS)). Utilizing three fluorescent dyes calcein, ethidium homodimer-1 (EthD-1) and annexin V, the viability of macrophages attached to the biomaterial was determined using confocal microscopy. Propidium iodide (PI) staining and alamarBlue® (resazurin) reduction were used to assess cell death and metabolic activity. CD14, CD16, CD33, CD45, and CD54 expression of adherent macrophages, were also evaluated to detect LPS activation of macrophages using flow cytometry. The sensitivity of this test battery was demonstrated as significant toxicity from treated surfaces with ZDEC (0.001-0.01%), and BAK (0.001%-0.1%) was detected. Also, macrophage activation could be detected by measuring CD54 expression after exposure to adsorbed LPS. These in vitro methods will be helpful in determining the toxicity potential of new ocular biomaterials.

Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis.

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Karen K Y Lam

Full Text Available Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis.

Science.gov (United States)

Lam, Karen K Y; Zheng, Xingji; Forestieri, Roberto; Balgi, Aruna D; Nodwell, Matt; Vollett, Sarah; Anderson, Hilary J; Andersen, Raymond J; Av-Gay, Yossef; Roberge, Michel

2012-01-01

Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

Anti-inflammatory activity of olive seed polyphenolic extract in the THP1-XBLUE-CD14 human monocytes cell line

OpenAIRE

Cortés Castell, Ernesto; Veciana Galindo, C.; Torro Montell, L.; Sirvent Segura, E.; Rizo Baeza, M. M.; Gil Guillén, V.

2014-01-01

El objetivo de este trabajo es evaluar la actividad antiinflamatoria de un extracto de naturaleza polifenólica de huesos de oliva. Material y métodos: Se incubó la línea celular THP1-XBlue-CD14 (invivogen), 80.000 células/pocillo, provocando inflamación (activación de NF-kb) mediante 0.1 μg/ml LPS (lipopolisacárido de E. coli) durante 24 horas. Se evaluó la presencia del extracto (10 y 50 mg/l, concentraciones bioseguras) durante 2 horas a 37 ºC, previa (efecto preventivo) y posterior a la ac...

Aloin Inhibits Interleukin (IL)-1β-Stimulated IL-8 Production in KB Cells.

Science.gov (United States)

Na, Hee Sam; Song, Yu Ri; Kim, Seyeon; Heo, Jun-Young; Chung, Hae-Young; Chung, Jin

2016-06-01

Interleukin (IL)-1β, which is elevated in oral diseases including gingivitis, stimulates epithelial cells to produce IL-8 and perpetuate inflammatory responses. This study investigates stimulatory effects of salivary IL-1β in IL-8 production and determines if aloin inhibits IL-1β-stimulated IL-8 production in epithelial cells. Saliva was collected from volunteers to determine IL-1β and IL-8 levels. Samples from volunteers were divided into two groups: those with low and those with high IL-1β levels. KB cells were stimulated with IL-1β or saliva with or without IL-1 receptor agonist or specific mitogen-activated protein kinase (MAPK) inhibitors. IL-8 production was measured by enzyme-linked immunosorbent assay (ELISA). MAPK protein expression involved in IL-1β-induced IL-8 secretion was detected by Western blot. KB cells were pretreated with aloin, and its effect on IL-1β-induced IL-8 production was examined by ELISA and Western blot analysis. Saliva with high IL-1β strongly stimulated IL-8 production in KB cells, and IL-1 receptor agonist significantly inhibited IL-8 production. Low IL-1β-containing saliva did not increase IL-8 production. IL-1β treatment of KB cells induced activation of MAPK signaling molecules as well as nuclear factor-kappa B. IL-1β-induced IL-8 production was decreased by p38 and extracellular signal-regulated kinase (ERK) inhibitor treatment. Aloin pretreatment inhibited IL-1β-induced IL-8 production in a dose-dependent manner and inhibited activation of the p38 and ERK signaling pathway. Finally, aloin pretreatment also inhibited saliva-induced IL-8 production. Results indicated that IL-1β in saliva stimulates epithelial cells to produce IL-8 and that aloin effectively inhibits salivary IL-1β-induced IL-8 production by mitigating the p38 and ERK pathway. Therefore, aloin may be a good candidate for modulating oral inflammatory diseases.

Reduced response of splenocytes after mitogen-stimulation in the prion protein (PrP) gene-deficient mouse: PrPLP/Doppel production and cerebral degeneration

International Nuclear Information System (INIS)

Kim, Chi-Kyeong; Hirose, Yuko; Sakudo, Akikazu; Takeyama, Natsumi; Kang, Chung-Boo; Taniuchi, Yojiro; Matsumoto, Yoshitsugu; Itohara, Shigeyoshi; Sakaguchi, Suehiro; Onodera, Takashi

2007-01-01

Splenocytes of wild-type (Prnp +/+ ) and prion protein gene-deficient (Prnp -/- ) mice were treated with various activation stimuli such as T cell mitogen concanavalin A (ConA), phorbol 12-myristate 13-acetate (PMA) + ionomycin (Io), or B cell mitogen lipopolysaccharide (LPS). Cellular prion protein (PrP C ) expression was enhanced following ConA stimulation, but not PMA + Io or LPS in Prnp +/+ splenocytes. Rikn Prnp -/- splenocytes elicited lower cell proliferations than Prnp +/+ or Zrch I Prnp -/- splenocytes after LPS stimulation and showed sporadic nerve cells in the cerebral cortex and deeper structure. Around the degenerated nerve cells, mild vacuolation in the neuropil was observed. This neural alteration correlated well to the suppressed response of B cells in the spleen. The finding that discrete lesions within the central nervous systems induced marked modulation of immune function probably indicates the existence of a delicately balanced neural-endocrine network by PrP C and PrPLP/Doppel

Differential stimulation of luminol-enhanced chemiluminescence (CL) and arachidonic acid metabolism in rat peritoneal neutrophils

Energy Technology Data Exchange (ETDEWEB)

Sturm, R.J.; Adams, L.M.; Cullinan, C.A.; Berkenkopf, J.W.; Weichman, B.M.

1986-03-05

Phorbol 12-myristate, 13-acetate (PMA) induced the production of radical oxygen species (ROS) from rat peritoneal neutrophils as assessed by CL. ROS generation occurred in a time- (maximum at 13.5 min) and dose- (concentration range of 1.7-498 nM) related fashion. However, 166 nM PMA did not induce either cyclooxygenase (CO) or lipoxygenase (LPO) product formation by 20 min post-stimulation. Conversely, A23187, at concentrations between 0.1 and 10 ..mu..M, stimulated both pathways of arachidonic acid metabolism, but had little or no effect upon ROS production. When suboptimal concentrations of PMA (5.5 nM) and A23187 (0.1-1 ..mu..M) were coincubated with the neutrophils, a synergistic ROS response was elicited. However, arachidonic acid metabolism in the presence of PMA was unchanged relative to A12187 alone. Nordihydroguaiaretic acid (NDGA) inhibited both PMA-induced CL (IC/sub 50/ = 0.9 ..mu..M) and A23187-induced arachidonic acid metabolism (IC/sub 50/ = 1.7 ..mu..M and 6.0 ..mu..M for LPO and CO, respectively). The mixed LPO-CO inhibitor, BW755C, behaved in a qualitatively similar manner to NDGA, whereas the CO inhibitors, indomethacin, piroxicam and naproxen had no inhibitory effect on ROS generation at concentrations as high as 100 ..mu..M. These results suggest that NDGA and BW755C may inhibit CL and arachidonic acid metabolism by distinct mechanisms in rat neutrophils.

DNA Damage and DNA Damage Responses in THP-1 Monocytes after Exposure to Spores of either Stachybotrys chartarum or Aspergillus versicolor or to T-2 toxin

OpenAIRE

Rakkestad, Kirsten E.; Skaar, Ida; Ansteinsson, Vibeke E.; Solhaug, Anita; Holme, Jørn A.; Pestka, James J.; Samuelsen, Jan T.; Dahlman, Hans J.; Hongslo, Jan K.; Becher, Rune

2010-01-01

We have characterized cell death in THP-1 cells after exposure to heat-treated spores from satratoxin G–producing Stachybotrys chartarum isolate IBT 9631, atranone-producing S. chartarum isolate IBT 9634, and sterigmatocystin-producing Aspergillus versicolor isolate IBT 3781, as well as the trichothecenes T-2 and satratoxin G. Spores induced cell death within 3–6 h, with Stachybotrys appearing most potent. IBT 9631 induced both apoptosis and necrosis, while IBT 9634 and IBT 3781 induced mostl...