RESUMO
We previously reported that cucurbitacin D isolated from Trichosanthes kirilowii has anti-tumor roles to leukemia cells. However, the effect of cucurbitacin D on immune cells is not fully understood although there is no toxic activity to normal cells. In this study, immunomodulating activities of cucurbitacin D were investigated in macrophages. Cucurbitacin D could increase LPS-induced interleukin (IL)-1ß production in culture supernatant of THP-1 cells, peritoneal exudate cells (PECs), bone marrow derived macrophages (BMDMs), and RAW264 cells. At the transcriptional level, cucurbitacin D enhanced LPS-induced IL-1ß mRNA expression through activation of ERK1/2 mitogen-activated protein kinases (MAPKs). At the posttranscriptional level, the activation of caspase-1 induced by cucurbitacin D has also been demonstrated following treatment with a caspase-1 inhibitor and siRNA. Importantly, cucurbitacin D has further been shown to induce inflammasome activation independent of ERK1/2 activation. Western blotting showed interaction of NOD-like receptor family, pyrin domain containing 3 (NALP3) and apoptosis-associated speck-like protein containing a caspase-activating and recruitment domain (ASC), suggesting activation of the inflammasome and a possible reason for activation of caspase-1. Taken together, these results suggest that cucurbitacin D could initiate immunomodulating activity in macrophages to lead to inflammasome activation as well as enhancement of LPS signaling.
Assuntos
Fatores Imunológicos/farmacologia , Interleucina-1beta/imunologia , Macrófagos/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Células da Medula Óssea/citologia , Linhagem Celular , Células Cultivadas , Inflamassomos/efeitos dos fármacos , Inflamassomos/imunologia , Interleucina-1beta/genética , Lipopolissacarídeos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/imunologiaRESUMO
BACKGROUND: Human T-cell leukemia is an aggressive malignancy of T lymphocytes. T-cell leukemia has a very poor prognosis, even with intensive chemotherapy, indicating the need for development of new drugs to treat the disease. Triterpenoid cucurbitacins have been shown to have antitumor activity, but the mechanism of this activity is not fully understood. METHODS: The effects of cucurbitacin D on the proliferation and apoptotic induction of T-cell leukemia cells using the Cell viability assay and Annexin V staining were evaluated. To investigate the mechanisms of apoptosis, antiapoptotic protein, NF-κB, and the proteasome activity of leukemia cells treated with cucurbitacin D were evaluated by Western blotting both in vitro and in vivo. RESULTS: In this study, cucurbitacin D was found to inhibit proliferation and to induce apoptosis of T-cell leukemia cells. Constitutively activated NF-κB was inhibited by cucurbitacin D in the nucleus, which resulted in accumulation of NF-κB in the cytoplasm, leading to down-regulation of the expression of antiapoptotic proteins Bcl-xL and Bcl-2. Furthermore, cucurbitacin D induced the accumulation of inhibitor of NF-κB (IκB)α by inhibition of proteasome activity. Low doses of cucurbitacin D synergistically potentiated the antiproliferative effects of the histone deacetylase inhibitor VPA. Finally, the proapoptotic and proteasome inhibitory activities of cucurbitacin D also were demonstrated using SCID mice in an in vivo study. CONCLUSIONS: Cucurbitacin D induced apoptosis through suppression of proteasome activity both in vitro and in vivo, making cucurbitacin D a promising candidate for clinical applications in the treatment of T-cell leukemia.
Assuntos
Apoptose/efeitos dos fármacos , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Inibidores de Proteassoma , Triterpenos/farmacologia , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Feminino , Inibidores de Histona Desacetilases/farmacologia , Humanos , Leucemia-Linfoma de Células T do Adulto/patologia , Camundongos , Camundongos SCID , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/análise , Triterpenos/uso terapêutico , UbiquitinaçãoRESUMO
Toluene has been extensively examined for effects on the central nervous system. To investigate the influence of low-level inhalation of toluene on the naive immune cells, male C3H/HeN mice were exposed to filtered air (control) and 50 ppm of toluene for 3 weeks. Low-level exposure resulted in (1) increased proliferation of thymocytes, (2) IL-2 production induced in thymocytes and (3) activation of the transcription factors NF-κB, STAT5 and NF-AT in thymocytes. These results suggest that thymocytes are sensitive cells and T cell activators are candidates for biomarkers for low-level exposure to toluene on naive immune cells.
Assuntos
Ativação Linfocitária/imunologia , NF-kappa B/fisiologia , Fatores de Transcrição NFATC/fisiologia , Fator de Transcrição STAT5/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Timo/citologia , Tolueno/farmacologia , Administração por Inalação , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Interleucina-2/biossíntese , Interleucina-2/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Fatores de Tempo , Tolueno/administração & dosagemRESUMO
To determine the potent immunomodulating activities of different types of Japanese rice bran, we analyzed the effects of extracts of red, brown and black rice brans on the cell proliferation and cytokine production of mouse immunocompetent cells by cell culture experiments. Methanol extracts of these rice brans showed suppressive activities against the proliferative response (3H-thymidine incorporation) of mouse spleen cells induced by concanavalin A (Con A) or lipopolysaccharide (LPS) in the cell culture experiments. Although the black and brown rice bran extracts showed suppressive effects on the production of interferon gamma (IFN gamma) or interleukin 6 (IL-6) in mouse spleen cells induced by Con A or LPS, the red rice bran extract exhibited stimulatory effects on the same cytokine-producing systems. Furthermore, when the effects of these extracts on the production of macropahage-derived inflammatory cytokines such as interleukin-1alpha (IL-1alpha) and tumor necrosis factor alpha (TNF-alpha) were assayed, the red rice bran extract caused a stimulatory effect on the IL-1alpha production from mouse macrophages induced by LPS, but did not show a significant effect on TNF-alpha production. However, the brown and black rice bran extracts exhibited significant inhibitory effects on the production of IL-1alpha and TNF-alpha in the same macrophage culture experiment. A possible mechanism of the immunomodulating activities of the rice bran extracts and the immunopharmacological significance of these findings are discussed.
Assuntos
Fatores Imunológicos/análise , Oryza/química , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/biossíntese , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Organismos Livres de Patógenos Específicos , Baço/citologiaRESUMO
Although the NF-kappaB transcription factors participate in both innate and adaptive immune responses, little is known about the role of the RelA subunit because mice lacking the rela gene die at embryonic day 14. To elucidate the role of RelA in Leishmania major infection, we prepared fetal liver chimeric mice by adoptively transferring embryonic day 13.5 rela(-/-) or rela(+/+) fetal liver into lethally irradiated host mice. About 90% of the peripheral lymphocytes of the chimeric mice had differentiated from rela fetal liver cells. The rela(-/-) fetal liver chimeric mice were highly sensitive to infection with L. major and died within 11 wk after infection. Despite the severity of the disease, parasite Ag-reactive Th1 cells developed normally. The rela(-/-) macrophages were less able to control intracellular parasite replication than rela(+/+) macrophages, despite showing equally efficient phagocytosis. Both in vitro NO production of macrophages and in vivo expression of NO synthase 2 in the lesions and draining lymph nodes was reduced in rela(-/-) fetal liver chimeric mice. Moreover, up-regulation of Fas in rela(-/-) macrophages was impaired both after in vitro stimulation with LPS and after in vivo infection with L. major, implying a defect in their ability to eliminate infected cells. Thus, RelA is necessary for macrophages to be resistant to intracellular parasite infection.
Assuntos
Leishmania major/imunologia , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Transcrição RelA/metabolismo , Regulação para Cima , Receptor fas/metabolismo , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Indução Enzimática , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Células Th1/citologia , Células Th1/imunologia , Fator de Transcrição RelA/deficiência , Fator de Transcrição RelA/genética , Receptor fas/imunologiaRESUMO
1-Bromopropane (1-BP) has been widely used as an alternative to ozone-depleting chlorofluorocarbons in various industries. Although the neurotoxicity of 1-BP has been recently reported, there is little information about the effect of 1-BP on the cells in brain by experimental approach. Here we studied the effect of 1-BP on brain-derived neurotrophic factor (BDNF) expression in astrocytes in vitro. The BDNF mRNA level was remarkably decreased by 1-BP in a human astrocytoma cell line, U251, and in mouse primary astrocytes. The DNA-binding and specific reporter activity of cAMP response element-binding transcription factor (CREB), which is one of the key molecules regulating BDNF expression, were reduced by 1-BP in U251 and/or mouse primary astrocytes. Additionally, protein kinase A (PKA) activity was suppressed by 1-BP in U251. These results suggest that BDNF expression was affected by 1-BP through at least PKA.
Assuntos
Astrócitos/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Animais , Astrócitos/metabolismo , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Linhagem Celular Tumoral , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Humanos , Hidrocarbonetos Bromados/farmacologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The aim of the present study is to examine the effects of the anti-tumor component isolated from Trichosanthes kirilowii on human hepatocellular carcinoma cells. Using Sephadex G-25 column chromatography, Sep-Pak Plus C18 cartridge and high-performance liquid chromatography (HPLC), we isolated the active component from trichosanthes extract. By fast atom bombardment mass spectrometric analysis, the molecular mass of the active fraction was determined, the active components identified, and their mechanisms of action were analyzed by cell growth assay, cell cycle analysis, TUNEL staining and Western blot analysis. We found that the anti-tumor components isolated from the extract of trichosanthes (EOT) are cucurbitacin D and dihydrocucurbitacin D, and suggest that cucurbitacin D induces apoptosis through caspase-3 and phosphorylation of JNK in hepatocellular carcinoma cells. These results suggest that cucurbitacin D isolated from Trichosanthes kirilowii could be a valuable candidate for anti-tumor drug.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Carcinoma Hepatocelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Triterpenos/farmacologia , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Humanos , MAP Quinase Quinase 4/efeitos dos fármacos , MAP Quinase Quinase 4/metabolismo , Medicina Tradicional , Raízes de Plantas/química , Trichosanthes/química , Triterpenos/isolamento & purificaçãoRESUMO
It has been reported that the numbers of people suffering from occupational asthma and skin rashes triggered by various chemicals in indoor air have increased markedly. Two-ethyl-hexanol (2-EH) is known to be an indoor air pollutant and its influence on health is of great concern. However, there are only a few reports regarding its effect on immune cells. Thus, we investigated the effects of 2-EH on immune responses in vitro with respect to effects on regulation of transcription factors as well as on 2-EH induced proliferation of spleen cells in vitro. The production of interleukin (IL)-6 and immunoglobulin were not induced by 2-EH. To characterize the effector cells of 2-EH, we prepared CD4-positive, CD8-positive, and peritoneal exudate cells (PEC). IL-2 was induced by 2-EH in CD4 cells, but not in CD8 cells. CD3-induced IL-2 expression was enhanced by 2-EH in CD4 cells, but not in CD8 cells. Moreover, IL-6 production was not induced by 2-EH in PEC. Nuclear factor-kappa B, nuclear factor of activated T, and signal transducer and activator of transcription-5 were activated by 2-EH in CD4 cells. Taken together, 2-EH activated CD4 cells, where this was accompanied by the activation of transcription factors. This suggested that the indoor pollutant 2-EH could function as a modulator of immune response.
Assuntos
Poluentes Ocupacionais do Ar/toxicidade , Linfócitos T CD4-Positivos/efeitos dos fármacos , Hexanóis/toxicidade , Ativação Linfocitária/efeitos dos fármacos , Plastificantes/toxicidade , Baço/efeitos dos fármacos , Poluição do Ar em Ambientes Fechados , Animais , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Interleucina-2/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Baço/metabolismoRESUMO
N-Acetylcysteine (NAC) has been widely used as an antioxidant in research, however, it has also been found to reduce the binding of TNF to its receptor independent of its antioxidative role. In this study, we investigated the effect of NAC on NF-kappaB activation. In HeLa cells, Hep3B cells, and A549 cells, DNA-binding activity of NF-kappaB was induced by NAC without any other stimulation but not by tetramethylthiourea (TMTU) or vitamin C, suggesting that ROS is not involved in the effect of NAC. The degradation of IkappaBalpha and nuclear translocation of NF-kappaB were not induced by NAC. The phosphorylation of p65 at serine 536 was induced by NAC, which is known to contribute to the enhancement of DNA-binding activity of NF-kappaB, however, NAC did not directly phosphorylate p65. The NAC-induced DNA-binding activity of NF-kappaB and phosphorylation of p65 were sensitive to a phosphatidylinositol (PI) 3-kinase inhibitor, partially sensitive to an IkappaB kinase (IKK) inhibitor, but not sensitive to a Bruton's tyrosine kinase (Btk) inhibitor. Moreover, both the DNA-binding activity and phosphorylation induced by NAC were reduced by the overexpression of a dominant negative Akt in HeLa cells. These results suggest that NAC activates mainly PI3K to phosphorylate p65 and subsequently induces DNA-binding activity of NF-kappaB, independent of its antioxidative function.
Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , DNA/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Transporte Ativo do Núcleo Celular , Tirosina Quinase da Agamaglobulinemia , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacologia , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , NF-kappa B/antagonistas & inibidores , Fosforilação , Ligação Proteica , Proteínas Tirosina Quinases/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Tioureia/análogos & derivados , Tioureia/farmacologia , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismoRESUMO
There are immediate, late-phase, and delayed-type reactions to exogenous agents. In IFN-gamma-knockout (IFN-gamma(-/-)) and wild-type B6 mice, we examined the response to picryl chloride (PCl) for assessing delayed-type reactions, and the responses to repeatedly challenged FITC for immediate and late-phase reactions. The delayed-type hypersensitivity was depressed in IFN-gamma(-/-) mice, and the immediate and late-phase reactions were enhanced in IFN-gamma(-/-) mice. As skin-infiltrating lymphocytes were scarce at the PCl-challenged site of IFN-gamma(-/-) mice, we investigated chemokine production by keratinocytes and Langerhans cells (LCs). A real-time PCR analysis demonstrated that Th1 chemokines (CXCL9 and CXCL10) and Th2 chemokines (CCL17 and CCL22) were derived mainly from keratinocytes and LCs, respectively. Challenge with PCl or FITC augmented keratinocyte expression of Th1 chemokines in wild-type but not in IFN-gamma(-/-) mice, and Th2 chemokine production by LCs was induced by repeated FITC in IFN-gamma(-/-) mice. Finally, transfer of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled draining lymph node cells from hapten-sensitized B6 mice or lymph node cells from sensitized green fluorescent protein (GFP) mice to naive IFN-gamma(-/-) mice revealed less infiltration of CFSE(+) or GFP(+) lymphocytes at the challenged site. Our study suggests that one of the crucial actions of IFN-gamma is upregulation of keratinocyte production of Th1 chemokines and downregulation of LC production of Th2 chemokines.
Assuntos
Quimiocinas/genética , Dermatite de Contato/etiologia , Haptenos/imunologia , Interferon gama/fisiologia , Queratinócitos/imunologia , Células de Langerhans/imunologia , Células Th1/imunologia , Células Th2/imunologia , Transferência Adotiva , Animais , Feminino , Imunoglobulina E/biossíntese , Interleucina-4/genética , Camundongos , Camundongos Endogâmicos C57BL , Cloreto de Picrila/toxicidade , Receptores CXCR3/genética , Triazóis/toxicidadeRESUMO
OBJECT: Microglia are one of the members of monocyte/macrophage lineage in the central nervous system (CNS) and exist as ramified microglia in a normal resting state, but they are activated by various stimuli, such as tumors. Activated microglia induce immune responses in the CNS, but the precise functions of microglia in glioma microenvironments are not clear. It has been reported that glioma cells produce prostaglandin (PG)E2, which promotes the growth of tumor cells and possesses immunosuppressive activity. The authors previously reported that PGE2 production by peritoneal macrophages was enhanced by glioma-derived soluble factors, which induce an immunosuppressive state. In this study, they investigated PGE2 production by microglia treated with glioma cells and assessed the role of microglia in glioma microenvironments in the mouse. METHODS: Microglia and peritoneal macrophages were cultured in vitro with or without lipopolysaccharide, and tumor necrosis factor (TNF) and PGE2 in the culture supernatant were measured using L929 bioassay and enzyme immunoassay. The expression of mRNA was measured using reverse transcriptase polymerase chain reaction, and the protein expression was assayed with Western blotting. In some experiments glioma cells and conditioned glioma medium were added to the microglia cultures. RESULTS: Glioma cells studied in this report did not produce a significant amount of PGE2. However, the coculture of microglia with glioma cells or conditioned glioma medium led to the production of a large amount of PGE2. The enhancement of PGE2 production by microglia was more significant than that by peritoneal macrophages. The expression of cyclooxygenase (COX)-2 and particularly the expression of microsomal PGE synthase (mPGES)-1 (a terminal enzyme of the arachidonate cascade) in microglia were enhanced by conditioned glioma medium. The enhancement of mPGES-1 expression in microglia was more significant than that in peritoneal macrophages. The production of TNF was suppressed when culturing microglia with conditioned glioma medium, but this suppression was abrogated by the addition of a COX inhibitor (NS-398) and a PGE2 receptor (EP4) antagonist. Furthermore, TNF production was not suppressed in microglia from mPGES-1-deficient mice. CONCLUSIONS: These results indicate that PGE2 production by microglia is enhanced by conditioned glioma medium, which induces an immunosuppressive state in the CNS. Therefore, the manipulation of microglia, from the standpoint of PGE2, provides investigators with an important strategy to induce an effective antiglioma immune response.
Assuntos
Dinoprostona/análise , Glioma/metabolismo , Oxirredutases Intramoleculares/análise , Microglia/metabolismo , Microssomos/enzimologia , Animais , Western Blotting , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados , Ciclo-Oxigenase 2/análise , Inibidores de Ciclo-Oxigenase 2/farmacologia , Feminino , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Nitrobenzenos/farmacologia , Prostaglandina-E Sintases , RNA Mensageiro/análise , Receptores de Prostaglandina E/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologia , Fator de Necrose Tumoral alfa/análiseRESUMO
IL-23 is a heterodimeric cytokine composed of a unique p19 subunit and a common p40 subunit is shared with IL-12. IL-23 promotes the inflammatory response by inducing the expansion of CD4(+) cells producing IL-17. The regulation of p19 gene expression has been less studied than that of p40 subunit expression, which in macrophages is well known to be dependent on NF-kappaB. To clarify the role of NF-kappaB in expression of the p19 gene, we analyzed mRNA levels in NF-kappaB-deficient macrophages. As reported to occur in dendritic cells, p19 expression was dramatically reduced in c-rel-deficient macrophages. Moreover, we found that p19 expression was halved in rela-deficient macrophages, but it was enhanced in p52-deficient macrophages. The p19 promoter contains three putative kappaB sites, located at nt -642 to -632 (kappaB-642), nt -513 to -503 (kappaB-513), and nt -105 to -96 (kappaB-105), between the transcription start site and -937 bp upstream in the p19 promoter region. Although EMSA analysis indicated that both kappaB-105 and kappaB-642, but not kappaB-513, bound to NF-kappaB in vitro, luciferase-based reporter assays showed that the most proximal kappaB site, kappaB-105, was uniquely indispensable to the induction of p19 transcription. Chromatin immunoprecipitation demonstrated in vivo association of RelA, c-Rel, and p50 with kappaB-105 of the p19 promoter. These results provide the evidence that the association of RelA and c-Rel with the proximal kappaB site in the p19 promoter is required to induce of p19 expression.
Assuntos
Regulação da Expressão Gênica/fisiologia , Subunidade p19 da Interleucina-23/biossíntese , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Elementos de Resposta/fisiologia , Fator de Transcrição RelA/metabolismo , Transcrição Gênica/fisiologia , Animais , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Subunidade p40 da Interleucina-12/biossíntese , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/imunologia , Interleucina-17/biossíntese , Interleucina-17/genética , Interleucina-17/imunologia , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , NF-kappa B/genética , NF-kappa B/imunologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-rel/genética , Proteínas Proto-Oncogênicas c-rel/imunologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologiaRESUMO
Reactive oxygen species (ROS) produced by the innate immune system work as effectors to destroy pathogens and to control cellular responses. However, their role in the adaptive immune response remains unclear. Here we studied the effect of exogenous ROS on CD40-induced B cell activation. H2O2 treatment inhibited CD40-induced immunoglobulin production of B cells, DNA binding of NF-kappaB, IkappaBalpha degradation and IKK phosphorylation. On the other hand, H2O2 treatment did not induce obvious B cell death after 30 min of stimulation. Although the ligation of anti-CD40 antibody was not disturbed by H2O2, TRAF2 recruitment to CD40 was inhibited. These results suggest that exogenous ROS play a negative role in CD40 signaling during B cell activation.
Assuntos
Linfócitos B/imunologia , Antígenos CD40/imunologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Anticorpos/farmacologia , Linfócitos B/efeitos dos fármacos , Antígenos CD40/antagonistas & inibidores , DNA/metabolismo , Feminino , Peróxido de Hidrogênio/farmacologia , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Imunoglobulinas/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/farmacologiaRESUMO
We have previously reported that peritoneal and splenic macrophages from Th2-dominant BALB/c mice produced higher amounts of prostaglandin (PG) E2 than cells from C57BL/6 mice. In this study, we investigated how macrophages from BALB/c mice acquire the ability of enhanced PGE2 production, using bone marrow-derived macrophages differentiated by M-CSF, GM-CSF or IL-3. There is no strain difference in PGE2 production by GM-CSF- and M-CSF-differentiated macrophages; however, IL-3-differentiated macrophages from BALB/c mice produced higher amounts of PGE2 and lower amounts of type I cytokines than cells from C57BL/6 mice. IL-3-differentiated macrophages from BALB/c mice expressed larger amounts of mRNA of membrane-bound (microsomal) PGE synthase-1 (mPGES-1). The amounts of PGE2 produced by macrophages were significantly reduced in mPGES-1-deficient mice, and these mice displayed enhanced Th1 responses after Propionibacterium acnes treatment compared with wild-type mice. Microarray analysis revealed 63 genes that are differentially expressed more than fivefold in macrophages between C57BL/6 and BALB/c mice. These results indicate that mPGES-1-mediated PGE2 produced by macrophages regulates immune responses, and IL-3 is an important factor for the differentiation of macrophages that produce higher amounts of PGE2 through mPGES-1 activity in BALB/c mice.
Assuntos
Dinoprostona/biossíntese , Interleucina-3/imunologia , Macrófagos/citologia , Camundongos Endogâmicos BALB C/imunologia , Camundongos Endogâmicos C57BL/imunologia , Animais , Western Blotting , Células da Medula Óssea/imunologia , Diferenciação Celular/imunologia , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Regulação da Expressão Gênica/imunologia , Oxirredutases Intramoleculares/imunologia , Oxirredutases Intramoleculares/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C/genética , Camundongos Endogâmicos C57BL/genética , Análise de Sequência com Séries de Oligonucleotídeos , Prostaglandina-E Sintases , RNA Mensageiro , Especificidade da EspécieRESUMO
BACKGROUND: The invasion of first trimester extravillous trophoblast (EVT) to decidua is an important event in placentation. Leukemia inhibitory factor (LIF) is an essential factor for mouse implantation, and it is reported that LIF may be involved in human first trimester EVT invasion. Prostaglandin E2 (PGE2) is also known as a critical factor for first trimester EVT invasion. In this study, we investigated the role of LIF in PGE2 production and EVT invasion using a human first trimester EVT cell line, HTR-8/SVneo. METHODS AND RESULTS: Co-stimulation with LIF and IL-1beta induced higher amounts of PGE2 production and further migration of HTR-8/SVneo cells compared with that by stimulation with LIF or IL-1beta alone. Enhanced PGE2 production was most probably due to the enhanced expression of cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1). PGE2 produced by HTR-8/SVneo cells promoted the migration of HTR-8/SVneo cells. A COX-2 inhibitor suppressed PGE2 production and the migration of HTR-8/SVneo cells. Agonists to PGE2 receptors, EP1, EP2 and EP4, promoted the migration of HTR-8/SVneo cells. Moreover, stimulation with LIF up-regulated EP1, EP2 and EP4 expression in HTR-8/SVneo cells. CONCLUSIONS: It is suggested that LIF participates in placentation through EVT invasion by up-regulating PGE2 production and PGE2 receptor expression in first trimester EVT.
Assuntos
Dinoprostona/biossíntese , Regulação da Expressão Gênica , Fator Inibidor de Leucemia/metabolismo , Trofoblastos/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Contactinas , Ciclo-Oxigenase 2/metabolismo , Feminino , Humanos , Oxirredutases Intramoleculares/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Gravidez , Primeiro Trimestre da Gravidez , Prostaglandina-E Sintases , Receptores de OSM-LIF/metabolismoRESUMO
Recent studies have shown that cytokines and cyclooxygenase (COX)-2 are up-regulated in the brain of human epilepsy patients and animal models of epilepsy. We investigated the effect of inflammatory responses induced by intramuscular injection of turpentine on the epileptic phenomenon in genetically epileptic El mice. As parameters of epileptic seizure, seizure threshold (number of toss-ups to induce convulsion), duration of actual convulsion and duration of post actual convulsive period (period from the offset of convulsion to full recovery) were evaluated. The post actual convulsive period was prolonged without any change of seizure threshold or duration of actual convulsion 24 h after turpentine injection. Although pretreatment with indomethacin for one week did not change the seizure parameters, indomethacin suppressed the prolongation of the post actual convulsive period induced by turpentine. The mRNA expression of IL-1beta, IL-6 and COX-2 in the cerebral cortex was detected by RT-PCR. There was no difference in the mRNA expression in the cerebral cortex before and 24 h after seizure. The mRNA levels of IL-1beta, IL-6 and COX-2 in the cerebral cortex were up-regulated 24 h after turpentine injection. On the other hand, the up-regulated mRNA levels of IL-1beta, IL-6 and COX-2 in the cerebral cortex after turpentine treatment were not suppressed by indomethacin. These results suggest that prostaglandins induced with COX-2 in the cerebral cortex seem to play an important role in the maintenance of the post convulsive period, but not in induction and maintenance of the actual convulsive state.
Assuntos
Ciclo-Oxigenase 2/fisiologia , Epilepsia/fisiopatologia , Prostaglandina-Endoperóxido Sintases/fisiologia , Animais , Córtex Cerebral/química , Ciclo-Oxigenase 2/análise , Modelos Animais de Doenças , Epilepsia/congênito , Epilepsia/enzimologia , Indometacina , Injeções Intramusculares , Interleucina-1/fisiologia , Interleucina-6/análise , Interleucina-6/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos , RNA Mensageiro/análise , Convulsões/fisiopatologia , Terebintina/administração & dosagemRESUMO
Pioglitazone, a peroxisome proliferator-activated receptor-gamma agonist, delayed the development of seizure responses and mildly shortened the duration of convulsion of genetically epileptic EL mice. mRNA levels of IL-1beta, IL-6 and TNF-alpha before seizure and mRNA levels of IL-6 and TNF-alpha after seizure were decreased in the brains of the mice with pioglitazone. These results suggest that pioglitazone may have ameliorative effects on epileptic seizure responses partly through the reduction of inflammatory responses in the brain.
Assuntos
Anticonvulsivantes/uso terapêutico , Epilepsia , Convulsões/tratamento farmacológico , Tiazolidinedionas/uso terapêutico , Animais , Encéfalo/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Citocinas/genética , Citocinas/metabolismo , Epilepsia/tratamento farmacológico , Epilepsia/genética , Epilepsia/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Pioglitazona , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
OBJECT: It has been reported that glioma cells produce prostaglandin (PG)E2, which promotes the growth of tumor cells and possesses immunosuppressive activity, and that cyclooxygenase (COX) inhibitors impede tumor growth and infiltration. Macrophages in tumor-bearing hosts are activated to produce PGE2, which induces an immunosuppressive state. Note, however, that the precise mechanism by which PGE2 induces an immunosuppressive state is still unclear. In this study, the authors investigated the mechanism of PGE2 production in glioma-bearing hosts. METHODS: The human and murine glioma cells that were studied did not produce a significant amount of PGE2. However, the coculture of human peripheral blood mononuclear cells or murine peritoneal macrophages with glioma cells or conditioned glioma medium led to the production of a large amount of PGE2. In contrast, production of tumor necrosis factor and interleukin (IL)-12p70 by macrophages and cytotoxic T lymphocyte induction were suppressed by culturing with conditioned glioma medium; this suppression was abrogated by the addition of the COX inhibitor indomethacin. The macrophagic expression of COX-2, and particularly the expression of microsomal PGE synthase (mPGES)-1, a terminal enzyme of the arachidonate cascade, was enhanced by the glioma-derived soluble factors. Furthermore, IL-12p70 production was not clearly suppressed in macrophages from mPGES-1-deficient mice. The glioma-derived soluble factors were sensitive to treatment with heat and papain. CONCLUSIONS: These results indicated that PGE2 production by macrophages is enhanced by glioma-derived soluble factors, which induce an immunosuppressive state in glioma-bearing hosts. Therefore, the inhibition of PGE2 synthesis, targeting COX-2 and mPGES-1, is an effective treatment for the induction of antiglioma immune responses.
Assuntos
Neoplasias Encefálicas/imunologia , Dinoprostona/metabolismo , Tolerância Imunológica/imunologia , Ativação de Macrófagos/fisiologia , Macrófagos/imunologia , Células Tumorais Cultivadas/imunologia , Animais , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/imunologia , Evasão Tumoral/imunologiaRESUMO
Mast cells play an important role for the induction and the expression of allergic responses. In this report, we studied the strain difference of bone marrow-derived murine mast cell (BMMC) functions in vitro. BMMC were induced by in vitro culture of bone marrow cells from BALB/c and C57BL/6 mice with interleukin (IL)-3 for 4 weeks, stimulated with immunoglobulin E antibody and antigen, and mediators and cytokines released in the culture supernatant were assayed. BMMC from C57BL/6 mice released a higher amount of granule-associated mediators, beta-hexosaminidase, and histamine than that from BALB/c mice. The expression of mRNA of histidine decarboxylase was higher in C57BL/6 mice. Conversely, the productions of newly synthesized mediators, prostaglandin D2 (PGD2), IL-6, and monocyte chemoattractant protein-1, and the mRNA expression of IL-5 were higher in BALB/c BMMC than C57BL/6 BMMC. Although mRNA and protein expression levels of cyclooxygenase-2 were equal in two strains, both expression levels of hematopoietic PGD synthase (hPGDS) were higher in BALB/c BMMC. Mast cells, freshly obtained from mice, also showed the same strain difference concerning the mediator release. These results indicate that the strain difference exists in mast cell functions in mice, and this difference can be considered to induce the susceptibility difference to allergic reactions in mouse strains.
Assuntos
Células da Medula Óssea/imunologia , Mastócitos/imunologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Técnicas de Cultura de Células , Quimiocinas/biossíntese , Citocinas/biossíntese , Feminino , Histamina/biossíntese , Histidina Descarboxilase/biossíntese , Imunoglobulina E/imunologia , Imunoglobulina E/farmacologia , Interleucina-3/imunologia , Interleucina-3/farmacologia , Oxirredutases Intramoleculares/biossíntese , Lipocalinas , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Prostaglandina D2/biossíntese , Prostaglandina-Endoperóxido Sintases/biossíntese , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/imunologia , Especificidade da Espécie , beta-N-Acetil-Hexosaminidases/biossínteseRESUMO
Sodium-dependent glutamate transporters of astrocytes have been reported to maintain extracellular concentration of glutamate below toxic level in the central nervous system and to be concerned with neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). In this study, the effects of inflammatory mediators including prostaglandin (PG) E2, interleukin (IL)-1beta and IL-6 on Na(+)-dependent L-glutamate transport of astrocytes were analyzed using primary murine astrocytes. The exposure of astrocytes to PGE2 for 24 h elicited a dose-dependent increase of L-glutamate uptake. Neither IL-beta nor IL-6 alone had any effect on L-glutamate uptake. However, IL-1beta enhanced the PGE2-induced increase of L-glutamate uptake. IL-6 suppressed the increase of L-glutamate uptake induced by PGE2. Kinetic analysis of L-glutamate uptake showed that PGE2 and PGE2 with IL-1beta increased V(max) value with no significant effect on Km value for Na(+)-dependent L-glutamate uptake. IL-6 suppressed the PGE2-induced V(max) value. These results suggest that IL-1beta, IL-6 and PGE2 modulate glutamate transport of astrocytes and play a role in the pathogenesis of neurodegenerative disorders such as ALS and AD.