RESUMO
Interleukin-21 (IL-21) has broad actions on T and B cells, but its actions in innate immunity are poorly understood. Here we show that IL-21 induced apoptosis of conventional dendritic cells (cDCs) via STAT3 and Bim, and this was inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). ChIP-Seq analysis revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3. Expression of IL-21 in vivo decreased cDC numbers, and this was prevented by GM-CSF. Moreover, repetitive α-galactosylceramide injection of mice induced IL-21 but decreased GM-CSF production by natural killer T (NKT) cells, correlating with decreased cDC numbers. Furthermore, adoptive transfer of wild-type CD4+ T cells caused more severe colitis with increased DCs and interferon-γ (IFN-γ)-producing CD4+ T cells in Il21r(-/-)Rag2(-/-) mice (which lack T cells and have IL-21-unresponsive DCs) than in Rag2(-/-) mice. Thus, IL-21 and GM-CSF exhibit cross-regulatory actions on gene regulation and apoptosis, regulating cDC numbers and thereby the magnitude of the immune response.
Assuntos
Apoptose/imunologia , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Interleucinas/imunologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Western Blotting , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , DNA Intergênico/genética , DNA Intergênico/imunologia , DNA Intergênico/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Citometria de Fluxo , Galactosilceramidas/imunologia , Galactosilceramidas/farmacologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucinas/genética , Interleucinas/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica/imunologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Interleucina-21/deficiência , Receptores de Interleucina-21/genética , Receptores de Interleucina-21/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologiaRESUMO
IL-1ß is a proinflammatory cytokine important for local and systemic immunity. However, aberrant production of this cytokine is implicated in pathogenic mechanisms of a number of inflammatory diseases, including Behçet's disease and age-related macular degeneration. In this study, we report the increased secretion of IL-1ß in the retina by neutrophils, macrophages, and dendritic cells during ocular inflammation and show that loss of IL-1R signaling confers protection from experimental autoimmune uveitis. Moreover, the amelioration of experimental autoimmune uveitis in Il1r-deficient mice was associated with reduced infiltration of inflammatory cells into the retina and decreased numbers of uveitogenic Th17 cells that mediate uveitis. These findings indicate the possible utility of IL-1R-blocking agents for the treatment of ocular inflammatory diseases.
Assuntos
Doenças Autoimunes/imunologia , Interleucina-1beta/imunologia , Receptores de Interleucina-1/imunologia , Transdução de Sinais , Uveíte/imunologia , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/imunologiaRESUMO
IL-21 is a type I cytokine essential for immune cell differentiation and function. Although IL-21 can activate several STAT family transcription factors, previous studies focused mainly on the role of STAT3 in IL-21 signaling. Here, we investigated the role of STAT1 and show that STAT1 and STAT3 have at least partially opposing roles in IL-21 signaling in CD4(+) T cells. IL-21 induced STAT1 phosphorylation, and this was augmented in Stat3-deficient CD4(+) T cells. RNA-Seq analysis of CD4(+) T cells from Stat1- and Stat3-deficient mice revealed that both STAT1 and STAT3 are critical for IL-21-mediated gene regulation. Expression of some genes, including Tbx21 and Ifng, was differentially regulated by STAT1 and STAT3. Moreover, opposing actions of STAT1 and STAT3 on IFN-γ expression in CD4(+) T cells were demonstrated in vivo during chronic lymphocytic choriomeningitis infection. Finally, IL-21-mediated induction of STAT1 phosphorylation, as well as IFNG and TBX21 expression, were higher in CD4(+) T cells from patients with autosomal dominant hyper-IgE syndrome, which is caused by STAT3 deficiency, as well as in cells from STAT1 gain-of-function patients. These data indicate an interplay between STAT1 and STAT3 in fine-tuning IL-21 actions.
Assuntos
Linfócitos T CD4-Positivos/citologia , Interleucinas/imunologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Citocinas/imunologia , Citometria de Fluxo , Regulação da Expressão Gênica , Imunoglobulina E/imunologia , Interferon gama/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Fosforilação , Análise de Sequência de RNA , Transdução de Sinais , Proteínas com Domínio T/metabolismoRESUMO
BACKGROUND: Chemical restraint is often required to control agitation induced by methamphetamine. Dexmedetomidine is an α-2 adrenergic receptor agonist with sedative, analgesic, and sympatholytic properties. Its use in the emergency department (ED) to control methamphetamine-induced agitation has not been reported. OBJECTIVE: To report two cases of methamphetamine-induced agitation successfully sedated with dexmedetomidine in the ED. CASE REPORT: The first case was a 42-year-old man with unstable emotion and violent behaviours after smoking methamphetamine. His agitation did not respond to a large cumulative dose of benzodiazepines (10mg of diazepam and 332mg of midazolam) administered over 48h and sedation was achieved with dexmedetomidine. The second case was a 38-year-old methamphetamine user with unstable emotion and recurrent episodes of agitation despite repeated doses of benzodiazepines, whose agitation was controlled with dexmedetomidine infusion. DISCUSSION: In both cases, dexmedetomidine apparently reduced the dose of benzodiazepines needed to achieve adequate sedation. Transient falls in blood pressure and slowing of the heart rate were noted, which resolved either spontaneously or after reducing the infusion rate without requiring drug treatment. CONCLUSION: Dexmedetomidine can be considered as an adjunct for chemical restraint when standard treatment fails to control the agitation induced by methamphetamine, but patient's hemodynamic state should be monitored closely during administration. Its efficacy and safety in the ED warrant further evaluation with prospective controlled trials.
Assuntos
Inibidores da Captação Adrenérgica/efeitos adversos , Agonistas de Receptores Adrenérgicos alfa 2/uso terapêutico , Sintomas Afetivos/tratamento farmacológico , Agressão , Acatisia Induzida por Medicamentos/tratamento farmacológico , Confusão/tratamento farmacológico , Dexmedetomidina/uso terapêutico , Metanfetamina/efeitos adversos , Adulto , Sintomas Afetivos/induzido quimicamente , Acatisia Induzida por Medicamentos/etiologia , Confusão/induzido quimicamente , Serviço Hospitalar de Emergência , Humanos , MasculinoRESUMO
IL-21 is a cytokine with pleiotropic actions, promoting terminal differentiation of B cells, increased Ig production, and the development of Th17 and T follicular helper cells. IL-21 is also implicated in the development of autoimmune disease and has antitumor activity. In this study, we investigated the role of IL-21 in host defense to pneumonia virus of mice (PVM), which initiates an infection in mice resembling that of respiratory syncytial virus disease in humans. We found that PVM-infected mice expressed IL-21 in lung CD4(+) T cells. Following infection, Il21r(-/-) mice exhibited less lung infiltration by neutrophils than did wild-type (WT) mice and correspondingly had lower levels of the chemokine CXCL1 in bronchoalveolar lavage fluid and lung parenchyma. CD8(+), CD4(+), and γδ T cell numbers were also lower in the lungs of PVM-infected Il21r(-/-) mice than in infected WT mice, with normal Th17 cytokines but diminished IL-6 production in PVM-infected Il21r(-/-) mice. Strikingly, Il21r(-/-) mice had enhanced survival following PVM infection, and moreover, treatment of WT mice with soluble IL-21R-Fc fusion protein enhanced their survival. These data reveal that IL-21 promotes the pathogenic inflammatory effect of PVM and indicate that manipulating IL-21 signaling may represent an immunomodulatory strategy for controlling PVM and potentially other respiratory virus infections.
Assuntos
Interleucinas/imunologia , Vírus da Pneumonia Murina/imunologia , Infecções por Pneumovirus/imunologia , Infecções por Pneumovirus/patologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Quimiocina CXCL1/biossíntese , Quimiocina CXCL1/imunologia , Interleucina-6/biossíntese , Interleucina-6/deficiência , Interleucinas/biossíntese , Interleucinas/metabolismo , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Vírus da Pneumonia Murina/patogenicidade , Receptores de Interleucina-21/imunologia , Células Th17/imunologiaRESUMO
The engagement of TCR induces T-cell activation, which initiates multiple characteristic changes such as increase in cell size, cell division, and the production of cytokines and other effector molecules. The mammalian target of rapamycin (mTOR) regulates protein synthesis, transcription, cell survival, and autophagy. Critical roles of mTOR in T-cell activation and effector/memory differentiation have been revealed using chemical inhibitors or by genetic ablation of mTOR in T cells. However, the connection between mTOR signaling and other signaling cascades downstream of TCR is unclear. We demonstrate that diacylglycerol (DAG) and TCR engagement activate signaling in both mTOR complexes 1 and 2 through the activation of the Ras-mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (Mek1/2)-extracellular signal-regulated kinase 1/2 (Erk1/2)-activator protein 1 (AP-1), known collectively as the Ras-Mek1/2-Erk1/2-AP-1 pathway. Deficiency of RasGRP1 or inhibition of Mek1/2 activity drastically decreases TCR-induced mTOR activation, whereas constitutively active Ras or Mek1 promotes mTOR activation. Although constitutively active Akt promotes TCR-induced mTOR activation, such activation is attenuated by Mek1/2 inhibition. We demonstrated further that DAG kinases (DGKs) α and ζ, which terminate DAG-mediated signaling, synergistically inhibit TCR-induced mTOR activation by inhibiting the Ras-Mek1/2-Erk/12 pathway. These observations provide novel insights into the regulation of mTOR activation.
Assuntos
Diacilglicerol Quinase/metabolismo , Ativação Linfocitária/fisiologia , Sistema de Sinalização das MAP Quinases/imunologia , Linfócitos T/enzimologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Diacilglicerol Quinase/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Memória Imunológica/fisiologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Camundongos , Camundongos Mutantes , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Serina-Treonina Quinases TOR/genética , Timo/citologia , Timo/imunologia , Timo/metabolismo , Fator de Transcrição AP-1/metabolismo , Proteínas ras/metabolismoRESUMO
Both diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers involved in signal transduction from many immune cell receptors and can be generated and metabolized through multiple mechanisms. Recent studies indicate that diacylglycerol kinases (DGKs), the enzymes that catalyze phosphorylation of DAG to produce PA, play critical roles in regulating the functions of multiple immune cell lineages. In T cells, two DGK isoforms, alpha and zeta, inhibit DAG-mediated signaling following T-cell receptor engagement and prevent T-cell hyperactivation. DGK alpha and zeta synergistically promote T-cell anergy and are critical for T-cell tolerance. In mast cells, DGKzeta plays differential roles in their activation by promoting degranulation but attenuating cytokine production following engagement of the high affinity receptor for immunoglobulin E. In dendritic cells and macrophages, DGKzeta positively regulates Toll-like receptor-induced proinflammatory cytokine production through its product PA and is critical for host defense against Toxoplasma gondii infection. These studies demonstrate pivotal roles of DGKs in regulating immune cell function by acting both as signal terminator and initiator.
Assuntos
Diacilglicerol Quinase/imunologia , Ácidos Fosfatídicos/imunologia , Tolerância a Antígenos Próprios/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Degranulação Celular/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Diacilglicerol Quinase/metabolismo , Humanos , Imunidade Inata/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Ácidos Fosfatídicos/metabolismo , Receptores de IgE/imunologia , Receptores de IgE/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismoRESUMO
Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that convert DAG to phosphatidic acid (PA), the physiologic functions of which have been poorly defined. We report here that DGK alpha and zeta synergistically promote T cell maturation in the thymus. Absence of both DGKalpha and zeta (DGKalpha(-/-)zeta(-/-)) results in a severe decrease in the number of CD4(+)CD8(-) and CD4(-)CD8(+) single-positive thymocytes correlating with increased DAG-mediated signaling. Positive selection, but not negative selection, is impaired in DGKalpha(-/-)zeta(-/-) mice. The developmental blockage in DGKalpha(-/-)zeta(-/-) mice can be partially overcome by treatment with PA. Furthermore, decreased DGK activity also promotes thymic lymphomagenesis accompanying elevated Ras and Erk1/2 activation. Our data demonstrate a synergistic and critical role of DGK isoforms in T cell development and tumor suppression, and indicate that DGKs not only terminate DAG signaling but also initiate PA signaling in thymocytes to promote positive selection.
Assuntos
Diferenciação Celular/imunologia , Diacilglicerol Quinase/metabolismo , Linfoma/imunologia , Linfoma/patologia , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Diacilglicerol Quinase/deficiência , Diacilglicerol Quinase/genética , Ativação Enzimática , Feminino , Isoenzimas/metabolismo , Linfoma/enzimologia , Linfoma/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Timo/enzimologia , Técnicas de Cultura de Tecidos , Proteínas ras/metabolismoRESUMO
The mis-regulation of nuclear factor-kappa B (NF-kappaB) signal pathway is involved in a variety of inflammatory diseases that leds to the production of inflammatory mediators. Our studies using human U937 promonocytes cells suggested that magnolol, a low molecular weight lignan isolated from the medicinal plant Magnolia officinalis, differentially down-regulated the pharmacologically induced expression of NF-kappaB-regulated inflammatory gene products MMP-9, IL-8, MCP-1, MIP-1alpha, TNF-alpha. Pre-treatment of magnolol blocked TNF-alpha-induced NF-kappaB activation in different cell types as evidenced by EMSA. Magnolol did not directly affect the binding of p65/p50 heterodimer to DNA. Immunoblot analysis demonstrated that magnolol inhibited the TNF-alpha-stimulated phosphorylation and degradation of the cytosolic NF-kappaB inhibitor IkappaBalpha and the effects were dose-dependent. Mechanistically, a non-radioactive IkappaB kinases (IKK) assay using immunoprecipitated IKKs protein demonstrated that magnolol inhibited both intrinsic and TNF-alpha-stimulated IKK activity, thus suggesting a critical role of magnolol in abrogating the phosphorylation and degradation of IkappaBalpha. The involvement of IKK was further verified in a HeLa cell NF-kappaB-dependent luciferase reporter system. In this system magnolol suppressed luciferase expression stimulated by TNF-alpha and by the transient transfection and expression of NIK (NF-kappaB-inducing kinase), wild type IKKbeta, constitutively active IKKalpha and IKKbeta, or the p65 subunit. Magnolol was also found to inhibit the nuclear translocation and phosphorylation of p65 subunit of NF-kappaB. In line with the observation that NF-kappaB activation may up-regulate anti-apoptotic genes, it was shown in U937 cells that magnolol enhanced TNF-alpha-induced apoptotic cell death. Our results suggest that magnolol or its derivatives may have potential anti-inflammatory actions through IKK inactivation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Compostos de Bifenilo/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Quinase I-kappa B/antagonistas & inibidores , Lignanas/farmacologia , NF-kappa B/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Citocinas/genética , Dimerização , Regulação para Baixo , Humanos , Quinase I-kappa B/metabolismo , Lipopolissacarídeos/farmacologia , Metaloproteinase 9 da Matriz/genética , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/antagonistas & inibidores , Subunidade p50 de NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Triptolide, a bioactive component of the Chinese medicinal herb Tripterygium wilfordii Hook F., induces p53-mediated apoptosis in cancer cells. This study demonstrated that triptolide activated an alternative p53-independent apoptotic pathway in HL-60 cells. In the absence of an intact p53 and without changing Bax level, at nM range triptolide induced apoptosis with concomitant DNA fragmentation, S phase cell cycle arrest, mitochondrial cytochrome c release and the activation of caspases. Besides, both caspases 8 and 9 were activated and the simultaneous inhibition of both was required to completely block triptolide's apoptotic effect. Importantly, triptolide induced the appearance of a truncated 23kD Bcl-2 which was inhibited by the general caspase inhibitor Z-VAD-FMK. In the MCF-7 cells that possessed the wild type p53 but lacked caspases 3, triptolide induced cell death with an increase in p53 but Bcl-2 remained unaltered. On the other hand, transfected cells overexpressing the 28kD Bcl-2 became more resistant to triptolide and upon triptolide treatment accumulated in the G(1) instead of S phase. After 36h treatment, triptolide activated JNK pathways, at the same time inactivated the ERK and p38 pathways. However, SP600125, a specific JNK inhibitor, could not inhibit the triptolide-mediated cleavage of caspase 3, indicated that activation of JNK might not be related to the apoptotic effects of triptolide. Our data suggest that in the absence of an intact p53 and without altering Bax level triptolide induces apoptosis activates a positive amplification loop involving caspase-mediated Bcl-2 cleavage/activation, mitochondrial cytochrome c release and further activation of caspases.
Assuntos
Diterpenos/farmacologia , Fenantrenos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Caspases/metabolismo , Ciclo Celular , Citocromos c/metabolismo , Ativação Enzimática , Compostos de Epóxi , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HL-60 , Humanos , Hidrólise , Mitocôndrias/enzimologia , Proteína Supressora de Tumor p53/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Through an extensive herbal drug screening program, we found that gomisin A, a dibenzocyclooctadiene compound isolated from Schisandra chinensis, reversed multidrug resistance (MDR) in Pgp-overexpressing HepG2-DR cells. Gomisin A was relatively non-toxic but without altering Pgp expression, it restored the cytotoxic actions of anticancer drugs such as vinblastine and doxorubicin that are Pgp substrates but may act by different mechanisms. Several lines of evidence suggest that gomisin A alters Pgp-substrate interaction but itself is neither a Pgp substrate nor competitive inhibitor. (1) First unlike Pgp substrates gomisin A inhibited the basal Pgp-associated ATPase (Pgp-ATPase) activity. (2) The cytotoxicity of gomisin A was not affected by Pgp competitive inhibitors such as verapamil. (3) Gomisin A acted as an uncompetitive inhibitor for Pgp-ATPase activity stimulated by the transport substrates verapamil and progesterone. (4) On the inhibition of rhodamine-123 efflux the effects of gomisin A and the competitive inhibitor verapamil were additive, so were the effects of gomisin A and the ATPase inhibitor vanadate. (5) Binding of transport substrates with Pgp would result in a Pgp conformational change favoring UIC-2 antibody reactivity but gomisin A impeded UIC-2 binding. (6) Photocrosslinking of Pgp with its transport substrate [125I]iodoarylazidoprazosin was inhibited by gomisin A in a concentration-dependent manner. Taken together our results suggest that gomisin A may bind to Pgp simultaneously with substrates and alters Pgp-substrate interaction.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Ciclo-Octanos/farmacologia , Dioxóis/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Lignanas/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Anticarcinógenos/química , Anticarcinógenos/farmacologia , Azidas/farmacologia , Western Blotting/métodos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Octanos/química , Dioxóis/química , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Hidrólise/efeitos dos fármacos , Lignanas/química , Prazosina/análogos & derivados , Prazosina/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rodamina 123/metabolismo , Vanadatos/farmacologia , Verapamil/farmacologia , Vimblastina/farmacologiaRESUMO
IL-21 is a type I cytokine produced by T cells and natural killer T cells that has pleiotropic actions on a wide range of immune and non-immune cell types. Since its discovery in 2000, extensive studies on the biological actions of IL-21 have been performed in vitro and in vivo. Recent reports describing patients with primary immunodeficiency caused by mutations of IL21 or IL21R have further deepened our knowledge of the role of this cytokine in host defense. Elucidation of the molecular mechanisms that mediate IL-21's actions has provided the rationale for targeting IL-21 and IL-21 downstream mediators for therapeutic purposes. The use of next-generation sequencing technology has provided further insights into the complexity of IL-21 signaling and has identified transcription factors and co-factors involved in mediating the actions of this cytokine. In this review, we discuss recent advances in the biology and signaling of IL-21 and how this knowledge can be potentially translated into clinical settings.
RESUMO
Honokiol, a small molecular weight lignan originally isolated from Magnolia officinalis, shows anti-angiogenic, anti-invasive and anti-proliferative activities in a variety of cancers. In this study, we investigated whether honokiol affects the transcription factor nuclear factor-kappa B (NF-kappaB) which controls a large number of genes involved in angiogenesis, metastasis and cell survival. We observed that the tumor necrosis factor-alpha (TNF-alpha)-induced NF-kappaB activation was blocked by honokiol in four different cancer cell lines as evidenced by EMSA. Honokiol did not directly affect the NF-kappaB-DNA binding. Immunoblot experiments demonstrated that honokiol inhibited the TNF-alpha-stimulated phosphorylation and degradation of the cytosolic NF-kappaB inhibitor IkappaBalpha. Furthermore, honokiol suppressed the intrinsic and TNF-alpha-stimulated upstream IkappaB kinases (IKKs) activities measured by a non-radioactive kinase assay using immunoprecipitated IKKs, suggesting a critical role of honokiol in abrogating the phosphorylation and degradation of IkappaBalpha. In a HeLa cell NF-kappaB-dependent luciferase reporter system, honokiol suppressed luciferase expression stimulated by TNF-alpha and by the transient transfection and expression of NIK (NF-kappaB-inducing kinase), wild type IKKbeta, constitutively active IKKalpha and IKKbeta, or the p65 subunit. Honokiol was also found to inhibit the nuclear translocation and phosphorylation of p65 subunit of NF-kappaB. RT-PCR results showed that honokiol suppressed NF-kappaB-regulated inflammatory and carcinogenic gene products including MMP-9, TNF-alpha, IL-8, ICAM-1 and MCP-1. In line with the observation that NF-kappaB activation may up-regulate anti-apoptotic genes, it was shown that honokiol enhanced TNF-alpha-induced apoptotic cell death. In summary, our results demonstrate that honokiol suppresses NF-kappaB activation and NF-kappaB-regulated gene expression through the inhibition of IKKs, which provides a possible mechanism for its anti-tumor actions.
Assuntos
Compostos de Bifenilo/farmacologia , Quinase I-kappa B/metabolismo , Lignanas/farmacologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/química , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Expressão Gênica/efeitos dos fármacos , Genes Reporter/fisiologia , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/efeitos dos fármacos , Lignanas/química , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Luciferases/antagonistas & inibidores , Luciferases/efeitos dos fármacos , Luciferases/genética , Linfotoxina-alfa/metabolismo , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/efeitos dos fármacos , Subunidade p50 de NF-kappa B/metabolismo , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/efeitos dos fármacos , Fator de Transcrição RelA/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The canonical pathway for IL-1ß production requires TLR-mediated NF-κB-dependent Il1b gene induction, followed by caspase-containing inflammasome-mediated processing of pro-IL-1ß. Here we show that IL-21 unexpectedly induces IL-1ß production in conventional dendritic cells (cDCs) via a STAT3-dependent but NF-κB-independent pathway. IL-21 does not induce Il1b expression in CD4(+) T cells, with differential histone marks present in these cells versus cDCs. IL-21-induced IL-1ß processing in cDCs does not require caspase-1 or caspase-8 but depends on IL-21-mediated death and activation of serine protease(s). Moreover, STAT3-dependent IL-1ß expression in cDCs at least partially explains the IL-21-mediated pathologic response occurring during infection with pneumonia virus of mice. These results demonstrate lineage-restricted IL-21-induced IL-1ß via a non-canonical pathway and provide evidence for its importance in vivo.
Assuntos
Células Dendríticas/metabolismo , Regulação da Expressão Gênica/fisiologia , Interleucina-1beta/metabolismo , Interleucinas/metabolismo , Animais , Antígenos CD1/metabolismo , Células da Medula Óssea , Linfócitos T CD4-Positivos/metabolismo , Epigênese Genética , Glicoproteínas/metabolismo , Humanos , Interleucina-1beta/genética , Interleucinas/genética , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Baço/citologiaRESUMO
Herbal drugs were screened for their activity in reversing multidrug resistance (MDR) in P-glycoprotein (P-gp) over-expressing cancer cells. Through bio-assay guided fractionation an active compound was isolated from Rhizoma Alismatis, the underground part of Alisma orientale and the chemical structure of the isolate compound was confirmed by HPLC, LC-MS and NMR as Alisol B 23-acetate (ABA). ABA restored the sensitivity of MDR cell lines HepG2-DR and K562-DR to anti-tumor agents that have different modes of action but are all P-gp substrates. It restored the activity of vinblastine, a P-gp substrate, in causing G2/M arrest in MDR cells. In a dose-dependent manner, ABA increased doxorubicin accumulation and slowed down the efflux of rhodamin-123 from MDR cells. ABA inhibited the photoaffinity labeling of P-gp by [125I]iodoarylazidoprazosin and stimulated the ATPase activity of P-gp in a concentration-dependent manner, suggesting that it could be a transporter substrate for P-gp. In addition, ABA was also a partial non-competitive inhibitor of P-gp when verapamil was used as a substrate. Our results suggest that ABA may be a potential MDR reversal agent and could serve as a lead compound in the development of novel drugs.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Colestenonas/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Expressão Gênica/efeitos dos fármacos , Humanos , Radioisótopos do Iodo , Células K562 , Marcadores de Fotoafinidade/metabolismo , Ródio/metabolismo , Células Tumorais Cultivadas , Vimblastina/farmacologiaRESUMO
1alpha,25-Dihydroxyvitamin D(3) (VD(3)), the biologically active form of vitamin D, may have either pro- or anti-inflammatory activities because of its diverse actions on nuclear factor kappa B (NF-kappaB). Previous studies indicated that VD(3) can either activate or inhibit NF-kappaB via Akt-induced I kappaB alpha phosphorylation and increase in I kappaB alpha synthesis respectively. At present, the relevant contribution of each mechanism has not been fully explored. We observed a VD(3)-mediated NF-kappaB inhibitory effect in vitamin D receptor (VDR)-positive MCF-7 breast cancer cells. We showed that VD(3) induced VDR-dependent I kappaB alpha expression but still able to lead on transient NF-kappaB p65 nuclear translocation through Akt-induced I kappaB alpha phosphorylation. Upon TNFalpha stimulation, VD(3) was not capable to inhibit I kappaB alpha degradation, p65 nuclear translocation and p65/p50-DNA binding. Here, we found that VD(3) strongly repressed p65 transactivation in MCF-7 cells using Gal4-p65 chimeras system. VDR was required for the VD(3)-mediated transrepression and mutations in VDR affected its suppressive ability. We also demonstrated that neither inhibition of p65 phosphorylation nor acetylation was responsible for the transrepression. In fact, we found that treatment of MCF-7 cells with histone deacetylase inhibitors abrogated VD(3)-induced p65 transrepression. In addition, knockdown of two nuclear corepressors HDAC3 and SMRT relieved p65 transactivation and particular TNFalpha-triggered gene expression. In conclusion, the reduction of gene activation by VD(3) in breast cancer cells was caused by the interference of the transactivation potential of NF-kappaB p65 subunit. Our studies provide a scientific background for rational use of vitamin D in the prevention and treatment of inflammatory diseases.
Assuntos
Calcitriol/farmacologia , NF-kappa B/metabolismo , Ativação Transcricional/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Animais , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Regulação para Baixo/genética , Feminino , Células HeLa , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/genética , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Correpressor 2 de Receptor Nuclear/genética , Correpressor 2 de Receptor Nuclear/metabolismo , Fosforilação/efeitos dos fármacos , Interferência de RNA , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/farmacologia , Vitaminas/farmacologiaRESUMO
1,25-dihydroxyvitamin D(3) (VD(3)) induces differentiation in a number of leukemia cell lines and under various conditions is able to either stimulate or inhibit nuclear factor kappa B (NF-kappaB) activity. Here we report a time-dependent biphasic regulation of NF-kappaB in VD(3)-treated HL-60 leukemia cells. After VD(3) treatment there was an early approximately 4 h suppression and a late 8-72 h prolonged reactivation of NF-kappaB. The reactivation of NF-kappaB was concomitant with increased IKK activities, IKK-mediated IkappaBalpha phosphorylation, p65 phosphorylation at residues S276 and S536, p65 nuclear translocation and p65 recruitment to the NF-kappaB/vitamin D responsive element promoters. In parallel with NF-kappaB stimulation, there was an up-regulation of NF-kappaB controlled inflammatory and anti-apoptotic genes such as TNFalpha, IL-1beta and Bcl-xL. VD(3)-triggered reactivation of NF-kappaB was associated with PI3K/Akt phosphorylation. PI3K/Akt antagonists suppressed VD(3)-stimulated IkappaBalpha phosphorylation as well as NF-kappaB-controlled gene expression. The early approximately 4 h VD(3)-mediated NF-kappaB suppression coincided with a prolonged increase of IkappaBalpha protein which require de novo protein synthesis, lasted for as least 72 h and was insensitive to MAPK, IKK or PI3K/Akt inhibitors. Our data suggest a novel biphasic regulation of NF-kappaB in VD(3)-treated leukemia cells and our results may have provided the first molecular explanation for the contradictory observations reported on VD(3)-mediated immune-regulation.
Assuntos
Calcitriol/farmacologia , Diferenciação Celular , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Antígeno CD11b/metabolismo , Células HL-60 , Humanos , Proteínas I-kappa B/antagonistas & inibidores , Interleucina-1beta/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/biossíntese , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , Proteína bcl-X/metabolismoRESUMO
Recent studies have shown that dibenzocyclooctadiene lignans may reverse P-glycoprotein-mediated multidrug resistance (Pgp-MDR) in cancer cells; however, the mechanism of action remains unknown. Through screening of herbs, we found that schisandrol A (SCH) isolated from Fructus Schisandrae (the dried fruit of Schisandra chinensis (Turcz.) Baill.) sensitized Pgp-MDR HepG2-DR cells by interfering with the function of Pgp-substrate complexes. In Pgp-MDR cells, SCH enhanced the cytotoxicity of cancer drugs that are Pgp substrates and restored vinblastine-induced G2/M arrest without lowering Pgp expression. SCH increased cellular retention of Pgp substrates such as rhodamine 123. In Pgp-overexpressing membrane preparations, SCH stimulated basal Pgp-ATPase thus showing some substrate-like function. However, SCH was not a competitive inhibitor for verapamil or progesterone and decreased their Km. In the presence of substrates, SCH decreased the reactivity between Pgp and the monoclonal antibody UIC-2 which is normally increased with active substrate-Pgp complexes. The labeling of active Pgp transport sites by [125I]-iodoarylazidoprazosin was partially blocked by SCH. SCH did not affect the activity of the mutant Pgp F983A suggesting that SCH acted differently than the thioxanthene type of Pgp allosteric inhibitors. Our results suggest that SCH acts by affecting the normal formation and functioning of the Pgp-substrate complexes.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Ciclo-Octanos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Lignanas/farmacologia , Fitoterapia , Extratos Vegetais/farmacologia , Schisandra , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/uso terapêutico , Western Blotting , Linhagem Celular Tumoral/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Octanos/administração & dosagem , Ciclo-Octanos/uso terapêutico , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Citometria de Fluxo , Frutas , Humanos , Concentração Inibidora 50 , Lignanas/administração & dosagem , Lignanas/uso terapêutico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Extratos Vegetais/administração & dosagem , Extratos Vegetais/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Betulinic acid (BA) is a pentacyclic triterpene found in a number of medicinal plants and has been shown to cause apoptosis in a number of cell lines. We report here that BA may also have an effect on HL-60 cell differentiation. BA was cytotoxic to HL-60 cells with an IC50 of 5.7 microM after a 72-h treatment. Flow cytometry analysis showed that after exposure to 1-12 microM of BA for 72 h, approximately 10% of viable cells were in the sub-G1, presumably apoptotic, phase. At the same time differentiation was induced in approximately 10% (at 1 microM BA) to a maximum of 20% (at 6 microM BA) of cells as judged by the NBT-reduction test, and the expression of membrane markers CD11b and CD14. On the other hand, at 1 and 5 nM, 1alpha,25-dihydroxyvitamin D3 (DHD3) induced differentiation in approximately 10 and 70% of cells, respectively. At 1 nM DHD3, the addition of 1 microM BA increased differentiated cells from 10 to 43% and with 3 microM BA the increase was to 80%. BA also enhanced the effects of DHD3 in the expansion of the G1 cell population with a concomitant decrease of S phase cells. The effects of DHD3 and BA on CD11b and CD14 expression were inhibited by PD98059, a MEK inhibitor. Our results suggest that BA may enhance the effect of DHD3 in inducing mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase-mediated HL-60 cell differentiation.