RESUMO
Lipopolysaccharide (LPS) enhances the production of nitric oxide (NO) in interferon (IFN)-gamma-stimulated vascular endothelial cells. We studied the mechanism by which LPS enhances IFN-gamma-induced NO production by using the murine vascular endothelial cell line, END-D. LPS enhanced IFN-gamma-induced NO production via augmented expression of inducible type NO synthase (iNOS) mRNA. LPS significantly augmented the activation of interferon regulatory factor (IRF)-1 in IFN-gamma-stimulated END-D cells, although it did not affect the activation of either MyD88-dependent nuclear factor (NF)-kappaB or MyD88-independent IRF-3. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), prevented the nuclear translocation of IRF-1 in LPS and IFN-gamma-stimulated END-D cells, and inhibited the iNOS expression and NO production in those cells. Therefore, it is proposed that LPS enhanced NO production in IFN-gamma-stimulated END-D cells via augmenting p38 MAPKmediated IRF-1 activation.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Fator Regulador 1 de Interferon/metabolismo , Lipopolissacarídeos/farmacologia , Óxido Nítrico/biossíntese , Animais , Aorta/citologia , Linhagem Celular , Células Endoteliais/citologia , Genes Reporter , Imuno-Histoquímica , Interferon gama/genética , Interferon gama/farmacologia , Luciferases/metabolismo , Camundongos , Óxido Nítrico/análise , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/análise , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
The effect of lipopolysaccharide on doxorubicin-induced cell death was studied by using mouse RAW 264.7 macrophage cells. Pretreatment with lipopolysaccharide at 10 ng/mL prevented doxorubicin-induced cell death and the inhibition was roughly dependent on the concentration of lipopolysaccharide. Posttreatment with lipopolysaccharide for 1 hour also prevented doxorubicin-induced cell death. Lipopolysaccharide inhibited DNA fragmentation and caspase-3 activation in doxorubicin-treated RAW 264.7 cells, suggesting the prevention of doxorubicin-induced apoptosis. Lipopolysaccharide did not significantly inhibit doxorubicin-induced DNA damage detected by single-cell gel electrophoresis (comet) assay. Lipopolysaccharide definitely inhibited the stabilization and nuclear translocation of p53 in doxorubicin-treated RAW 264.7 cells. Lipopolysaccharide, as well as being an inhibitor of p53, abolished doxorubicin-induced apoptosis. Therefore, p53 was suggested to play a pivotal role in the prevention of doxorubicin-induced apoptosis in RAW 264.7 cells by lipopolysaccharide.
Assuntos
Antibióticos Antineoplásicos/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Doxorrubicina/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Proteína Supressora de Tumor p53/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/efeitos adversos , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Doxorrubicina/efeitos adversos , Escherichia coli , Humanos , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína Supressora de Tumor p53/fisiologiaRESUMO
The effect of interferon (IFN)-alpha, beta and gamma on the growth of DHL-4 diffuse large B cell lymphoma cells was studied. IFN-beta significantly inhibited the cell growth, and the effect was stronger than that of IFN-alpha. IFN-gamma did not inhibit the cell growth because of lack of IFN-gamma receptors. IFN-beta caused apoptotic cell death which was accompanied by DNA fragmentation, caspase 3 activation and annexin V binding. IFN-beta lead to the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA. Anti-TRAIL antibody significantly prevented IFN-beta-induced apoptosis. It was suggested that IFN-beta might cause apoptosis in DHL-4 cells through TRAIL.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Interferon beta/farmacologia , Linfoma de Células B/patologia , Linfoma Difuso de Grandes Células B/patologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Reguladoras de Apoptose , Proliferação de Células , Dano ao DNA , Perfilação da Expressão Gênica , Humanos , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais CultivadasRESUMO
A new method, combining seminested polymerase chain reaction (PCR) with heteroduplex analysis, was utilized to detect follicular lymphoma (FL) cells in peripheral blood. The method, based on the detection of IgH rearrangements in DNA, detected the presence of monoclonal B cells in FL patients with a high frequency.
Assuntos
Rearranjo Gênico , Análise Heteroduplex/métodos , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma Folicular/genética , Ácidos Nucleicos Heteroduplexes/química , Reação em Cadeia da Polimerase/métodos , Linfócitos B/metabolismo , Eletroforese em Gel de Poliacrilamida , Genes de Imunoglobulinas , Humanos , Sensibilidade e EspecificidadeRESUMO
The effect of inhibition of mitogen and stress-activated protein kinases 1/2 (MSK1/2) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was investigated. Pretreatment with Ro 31-8220, an inhibitor of MSK1/2, induced cell death in LPS-stimulated RAW 264.7 cells. In contrast, calphostin C, another inhibitor of protein kinase C, did not cause cell death. Cell death was not mediated by the release of pro-inflammatory mediators from LPS-stimulated RAW 264.7 cells. Cell death was accompanied by DNA fragmentation and annexin V binding, suggesting apoptotic cell death. Further, several caspase inhibitors did not prevent LPS-induced cell death of Ro 31-8220-pretreated RAW 264.7 cells. Nuclear translocation of apoptosis-inducing factor (AIF) was detected in Ro 31-8220-pretreated cells after LPS stimulation. Cell death was due to mitochondrial damage. Ro 31-8220 exclusively inhibited the phosphorylation of cAMP-responsive element binding protein (CREB), a substrate of MSK1/2. RAW 264.7 cells transfected with the dominant-negative MSK1 clones underwent cell death in response to LPS. Hence, it was suggested that MSK1/2 might play a critical role in the survival of LPS-stimulated RAW 264.7 cells.
Assuntos
Apoptose , Lipopolissacarídeos/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Anexina A5/metabolismo , Fator de Indução de Apoptose , Inibidores de Caspase , Linhagem Celular , Núcleo Celular/química , Sobrevivência Celular , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fragmentação do DNA , Inibidores Enzimáticos/farmacologia , Flavoproteínas/metabolismo , Indóis/farmacologia , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Naftalenos/farmacologiaRESUMO
The present study was conducted to determine effects of U0126, a specific inhibitor of mitogen-activated kinase kinase 1/2, on production of nitric oxide (NO) in RAW264.7 macrophage cells. U0126 significantly enhanced NO production in lipopolysaccharide (LPS) but not CpG DNA or interferon-gamma-stimulated RAW264.7 cells. In contrast, U0124, a negative control for U0126, did not affect LPS-induced NO production. Further, a series of inhibitors of p38, phosphatidyl-inositol 3-kinase and Janus tyrosine kinase rather caused suppression in LPS-stimulated RAW264.7 cells. U0126 was found to definitely inhibit phosphorylation of extracellular signal-regulated kinase (Erk) 1/2 and augment the levels of inducible type of NO synthase. Antisense oligonucleotides of Erk1/2 also augmented LPS-induced NO production. Inactivation of Erk1/2 by U0126 furthermore inhibited LPS-induced activating protein-1 activation, but not nuclear factor-kappaB activation. The results suggest that Erk1/2 might negatively regulate NO production in LPS-stimulated RAW264.7 cells.
Assuntos
MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Macrófagos/metabolismo , Óxido Nítrico/biossíntese , Animais , Sequência de Bases , Butadienos/farmacologia , Linhagem Celular , Citocinas/biossíntese , DNA/genética , Inibidores Enzimáticos/farmacologia , Interferon beta/genética , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Nitrilas/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes , Fator de Necrose Tumoral alfa/genéticaRESUMO
The effect of C(2)-ceramide, a membrane-permeable ceramide analogue, on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was studied. The non-toxic concentration of C(2)-ceramide inhibited LPS-induced NO production. It was due to the attenuated expression of the inducible type of NO synthase (iNOS). C(2)-ceramide did not influence the phosphorylation of a series of mitogen-activated protein (MAP) kinases in response to LPS. On the other hand, C(2)-ceramide down-regulated the phosphorylation of Akt in LPS-stimulated RAW 264.7 cells, followed by the impairment of nuclear factor (NF)-kappaB activation. Moreover, the Akt dominant-negative mutant inhibited LPS-induced NO production. C(2)-ceramide was suggested to inhibit LPS-induced NO production through down-regulating the activation of Akt.
Assuntos
Inibidores Enzimáticos/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/biossíntese , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Animais , Linhagem Celular , Regulação para Baixo , Escherichia coli/imunologia , Macrófagos/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/biossíntese , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Fosforilação , Proteínas Proto-Oncogênicas c-akt , TransfecçãoRESUMO
The effect of butyrate, a natural bacterial product of colonic bacterial flora, on nitric oxide (NO) production in murine vascular endothelial cell line END-D in response to IFN-gamma and/or LPS was studied. Butyrate significantly augmented NO production in END-D cells in response to IFN-gamma or IFN-gamma + LPS, but not LPS alone. The NO production was augmented by the addition of butyrate until 6 h after the stimulation with IFN-gamma or IFN-gamma + LPS. The augmentation was abolished by the removal of butyrate from the cultures. Butyrate enhanced the expression of inducible type NO synthase (iNOS) in the stimulated END-D cells. Furthermore, butyrate-enhanced NO production in the presence of various signal inhibitors down-regulating the signal pathways using nuclear factor (NF)-kappaB, mitogen-activated protein (MAP) kinases and Janus tyrosine kinase. The putative mechanism of butyrate-induced augmentation of NO production in response to IFN-gamma or IFN-gamma + LPS is discussed.
Assuntos
Antineoplásicos/farmacologia , Butiratos/farmacologia , Células Endoteliais/fisiologia , Sequestradores de Radicais Livres/análise , Interferon gama/farmacologia , Óxido Nítrico/análise , Animais , Linhagem Celular , Regulação para Baixo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/farmacologia , NF-kappa B/farmacologia , Proteínas Tirosina Quinases/farmacologia , Transdução de SinaisRESUMO
A new, sensitive method combining seminested polymerase chain reaction (PCR) and heteroduplex analysis was used to detect follicular lymphoma (FL) cells in peripheral blood. Based on the detection of IgH rearrangement in DNA from peripheral blood leukocytes, the method demonstrated the presence of monoclonal B cells in FL patients with high frequency.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Antineoplásicos/genética , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Análise Heteroduplex , Linfoma Folicular/genética , Reação em Cadeia da Polimerase/métodos , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Feminino , Doença de Hodgkin/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Masculino , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Sensibilidade e EspecificidadeRESUMO
The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.
Assuntos
Galactosamina/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Óxido Nítrico/biossíntese , Animais , Linhagem Celular , Ativação de Macrófagos , Macrófagos/imunologia , Camundongos , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Espécies Reativas de Oxigênio/metabolismoRESUMO
The effect of piceatannol on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was examined. Piceatannol significantly inhibited NO production in LPS-stimulated RAW 264.7 cells. The inhibition was due to the reduced expression of an inducible isoform of NO synthase (iNOS). The inhibitory effect of piceatannol was mediated by down-regulation of LPS-induced nuclear factor (NF)-kappaB activation, but not by its cytotoxic action. Piceatannol inhibited IkappaB kinase (IKK)-alpha and beta phosphorylation, and subsequently IkappaB-alpha phosphorylation in LPS-stimulated RAW 264.7 cells. On the other hand, piceatannol did not affect activation of mitogen-activated protein (MAP) kinases including extracellular signal regulated kinase 1/2 (Erk1/2), p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Piceatannol inhibited the phosphorylation of Akt and Raf-1 molecules, which regulated the activation of IKK-alpha and beta phosphorylation. The detailed mechanism of the inhibition of LPS-induced NO production by piceatannol is discussed.
Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Estilbenos/farmacologia , Animais , Linhagem Celular , Quinase I-kappa B , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico/antagonistas & inibidores , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The role of p38 mitogen-activated protein kinase (MAPK) on vacuole formation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was examined. LPS definitely induced the formation of vacuoles in RAW 264.7 cells and SB202190 as a p38 specific inhibitor also induced slight vacuole formation. The simultaneous treatment with LPS and SB202190 induced many more vacuoles in RAW 264.7 cells than the treatment with LPS or SB202190 alone, and the vacuoles were extraordinarily large in size. On the other hand, an inactive inhibitor of p38 MAPK did not augment LPS-induced vacuole formation. Further, the inhibitors of other MAPKs and nuclear factor (NF)-kappaB pathways did not affect it. The extraordinarily large vacuoles in RAW 264.7 cells treated with LPS and SB202190 were possibly formed via fusion of small vacuoles. However, SB202190 did not augment vacuole formation in CpG DNA or interferon (IFN)-gamma-stimulated RAW 264.7 cells. The role of p38 MAPK in the vacuole formation in LPS-stimulated macrophages is discussed.
Assuntos
Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Vacúolos/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Escherichia coli/metabolismo , Macrófagos Peritoneais/imunologia , Camundongos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
The in vitro effects of gamma interferon (IFN-gamma) on the mouse CD5(+) B1-cell line, TH2.52, a hybridoma between mouse B lymphoma and mouse splenic B cells that expresses a series of B1 markers, were investigated. A significant number of macrophage-like cells appeared in the cultures of TH2.52 cells exposed to IFN-gamma, these adhering to plastic dishes and exhibiting phagocytic activity. Positive for esterase staining, the macrophage-like cells returned to the original TH2.52 morphology upon removal of IFN-gamma. The change was prevented by treatment with SB202190, an inhibitor of p38 mitogen-activated protein (MAP) kinase and by transfection of a p38 MAP kinase dominant-negative mutant. Further, interleukin-4 (IL-4) inhibited IFN-gamma-induced phosphorylation of p38 MAP kinase and the appearance of macrophage-like cells. IFN-gamma and IL-4 exhibited contradictory actions on morphological change of CD5(+) B1 cells into macrophage-like cells. Differential regulation of CD5(+) B1 cells by IFN-gamma, a Th1 cytokine, and IL-4, a Th2 cytokine, may have clear immunological significance.
Assuntos
Linfócitos B/efeitos dos fármacos , Antígenos CD5/análise , Interferon gama/farmacologia , Interleucina-4/farmacologia , Macrófagos/citologia , Animais , Linfócitos B/citologia , Linhagem Celular , Macrófagos/fisiologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The in vitro effect of gamma interferon (IFN-gamma) on nitric oxide (NO) production in a mouse CD5+ B1-like cell line, TH2.52, was studied. The TH2.52 cell line is the hybridoma line between mouse B lymphoma line and mouse splenic B cells and expresses a series of B1 markers. IFN-gamma induced a marked NO production in TH2.52 cells through the expression of an inducible type of NO synthase (iNOS). IFN-gamma-induced NO production was triggered by the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) pathway since it was inhibited by AG490, a JAK2 inhibitor. The growth of TH2.52 cells significantly was inhibited in the presence of IFN-gamma. A significant number of cells underwent apoptotic cell death, accompanied by the DNA fragmentation, annexin V binding, and caspase 3 activation. N(G)-monomethyl-L-arginine, an iNOS inhibitor, prevented IFN-gamma-induced cell death. Therefore, IFN-gamma-induced NO production was possible in causing cell death in TH2.52 cells. Further, IFN-gamma-induced NO production and cell death significantly were prevented by interleukin-4, a representative Th2 cytokine. The immunological significance of IFN-gamma-induced NO production in a mouse B1-like cell line is discussed.