RESUMO
Myeloperoxidase (MPO) is an important enzyme in the front-line protection against microorganisms. In peripheral blood, it is accepted that MPO is only produced by myeloid-lineage cells. Thus, MPO presence is unexpected in lymphocytes. We showed recently that B1-lymphocytes from mice have MPO. Here, we showed that subsets of human peripheral B, CD4(+) and CD8(+) T lymphocytes express MPO. The content of MPO in lymphocytes was very low compared to neutrophils/monocytes with a preferential distribution in the nucleus and perinuclear region. Also, we performed a MPO mRNA expression analysis from human blood cells derived from microarray raw data publicly available, showing that MPO is modulated in infectious disease. MPO was increased in CD4(+) T lymphocytes from HIV chronic infection and in CD8(+) T lymphocytes from HCV-positive patients. Our study points out MPO as a multifunctional protein due to its subcellular localization and expression modulation in lymphocytes indicating alternative unknown functions for MPO in lymphocytes.
Assuntos
Linfócitos B/enzimologia , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD8-Positivos/enzimologia , Peroxidase/biossíntese , Linfócitos B/imunologia , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Separação Celular , Citometria de Fluxo , Infecções por HIV/enzimologia , Infecções por HIV/imunologia , Hepatite C/enzimologia , Hepatite C/imunologia , Humanos , Imunofenotipagem , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Peroxidase/imunologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Indoleamine 2,3-dioxygenase (IDO) is an interferon-γ (IFN-γ)-induced tryptophan-degrading enzyme, producing kynurenine (KYN) that participates in the mechanism of tumor immune tolerance. Thus, IDO inhibition has been considered a strategy for anticancer therapy. The aim of this study was to identify whether the metabolites originated from the competitive routes of tryptophan metabolism, such as the serotonergic or N, N-dimethyltryptamine (DMT) pathways, have inhibitory effects on recombinant human IDO (rhIDO) activity. Serotonin and melatonin had no effect; on the other hand, tryptamine (TRY) and DMT modulated the activity of rhIDO as classical non-competitive inhibitors, with Ki values of 156 and 506 µM, respectively. This inhibitory effect was also observed on constitutively expressed or IFN-γ-induced IDO in the A172 human glioma cell line. TRY and DMT increased the cytotoxic activity of peripheral blood mononuclear cells (PBMCs) in co-culture assays. We conclude that the IDO inhibition by TRY and DMT contributed to a more effective tumor-reactive response by the PBMCs.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Leucócitos Mononucleares/efeitos dos fármacos , N,N-Dimetiltriptamina/farmacologia , Triptaminas/farmacologia , Ligação Competitiva , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Ensaios Enzimáticos , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Cinética , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/enzimologia , Leucócitos Mononucleares/imunologia , Ligação Proteica , Proteínas Recombinantes/metabolismo , Triptofano/metabolismoRESUMO
Generation of hypochlorous acid (HOCl), an important microbicidal agent, is considered to be the main function of myeloperoxidase (MPO), an enzyme present in phagocytes. High amounts of MPO are present in neutrophil azurophilic granules, which are mobilized into the phagolysosome vacuole during phagocytosis. MPO is also present in monocytes and macrophages, although to a lesser degree than in neutrophils. In the present study, we investigated the distribution of MPO in murine peritoneal cells using flow cytometry, confocal microscopy (CM) and transmission electron microscopy (TEM). MPO was observed in macrophages, and surprisingly, we detected MPO in B lymphocytes, specifically in B1-a. MPO was present in cytoplasmic granules, vesicles, mitochondria and the nucleus of murine peritoneal cells. Together, these findings suggest that, in addition to its known microbicidal activity, MPO has a myriad of other unanticipated cellular functions.
Assuntos
Líquido Ascítico/citologia , Linfócitos B , Macrófagos , Cavidade Peritoneal/citologia , Peroxidase/metabolismo , Animais , Linfócitos B/enzimologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Núcleo Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Vesículas Citoplasmáticas/metabolismo , Citometria de Fluxo , Ácido Hipocloroso/metabolismo , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Microscopia Confocal , Mitocôndrias/metabolismo , Neutrófilos/enzimologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Peroxidase/imunologiaRESUMO
Our data suggest that impaired activity of myeloperoxidase (MPO) may play an important role in the dysfunction of neutrophils from hyperglycemic rats. Neutrophil biochemical pathways include the NADPH oxidase system and the MPO enzyme. They both play important role in the killing function of neutrophils. The effect of hyperglycemia on the activity of these enzymes and the consequences with regard to Candida albicans phagocytosis and the microbicidal property of rat peritoneal neutrophils is evaluated here. The NADPH oxidase system activity was measured using chemiluminescence and cytochrome C reduction assays. MPO activity was measured by monitoring HOCl production, and MPO protein expression was analysed using Western blot and immunofluorescence. C. albicans phagocytosis and death were evaluated by optical microscopy using the May-Grunwald-Giemsa staining method. ROS generation kinetic was slightly delayed in the diabetic group. MPO expression levels were higher in diabetic neutrophils; however, MPO activity was decreased in these same neutrophils compared with the controls. C. albicans phagocytosis and killing were lower in the diabetic neutrophils. Based on our experimental model, the phagocytic and killing functions of neutrophil phagocytosis are impaired in diabetic rats because of the decreased production of HOCl, highlighting the importance of MPO in the microbicidal function of neutrophils.
Assuntos
Hiperglicemia/fisiopatologia , Neutrófilos/enzimologia , Peroxidase/metabolismo , Animais , Candida albicans/patogenicidade , Hiperglicemia/metabolismo , Ácido Hipocloroso/metabolismo , Medições Luminescentes , Masculino , NADPH Oxidases/metabolismo , Neutrófilos/fisiologia , Fagocitose , Ratos , Ratos WistarRESUMO
Induction of apoptotic cell death in response to chemotherapy and other external stimuli has proved extremely difficult in melanoma, leading to tumor progression, metastasis formation and resistance to therapy. A promising approach for cancer chemotherapy is the inhibition of proteasomal activity, as the half-life of the majority of cellular proteins is under proteasomal control and inhibitors have been shown to induce cell death programs in a wide variety of tumor cell types. 4-Nerolidylcatechol (4-NC) is a potent antioxidant whose cytotoxic potential has already been demonstrated in melanoma tumor cell lines. Furthermore, 4-NC was able to induce the accumulation of ubiquitinated proteins, including classic targets of this process such as Mcl-1. As shown for other proteasomal inhibitors in melanoma, the cytotoxic action of 4-NC is time-dependent upon the pro-apoptotic protein Noxa, which is able to bind and neutralize Mcl-1. We demonstrate the role of 4-NC as a potent inducer of ROS and p53. The use of an artificial skin model containing melanoma also provided evidence that 4-NC prevented melanoma proliferation in a 3D model that more closely resembles normal human skin.
Assuntos
Catecóis/farmacologia , Melanoma/patologia , Inibidores de Proteassoma , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/patologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Avaliação Pré-Clínica de Medicamentos , Sequestradores de Radicais Livres/farmacologia , Humanos , Modelos Biológicos , Inibidores de Proteases/farmacologia , Células Tumorais CultivadasRESUMO
Triptofano (TRP) é metabolizado por duas vias, a via serotonérgica e a via das quinureninas. Na via serotonérgica, TRP é metabolizado a serotonina (5-HT) e, em algumas células, à melatonina (MLT) que pode ser oxidada à N1-acetil-N2-formil-5- metoxiquinuramina (AFMK) e N1-acetil-5-metoxiquinuramina (AMK) por ação de peroxidases. Na via das quinureninas o TRP é diretamente metabolizado à N formilquinurenina (NFK) e em seguida a quinurenina (QUIN). A enzima indolamina 2, 3 dioxigenase (IDO) é uma das responsáveis por esta reação. Dada a importância da IDO na tolerância imunológica e pelo fato desta enzima ser induzível nos propusemos a avaliar a existência de uma regulação cruzada entre esta enzima e a via serotonérgica. Avaliando a interferência de AMK sobre a ação de IDO e a interferência de QUIN sobre a formação de AFMK por peroxidases, observamos uma possível interação entre as vias. AMK é um inibidor competitivo clássico de IDO e o Ki encontrado foi de 0,98 mM. QUIN é um inibidor acompetitivo linear simples da formação de AFMK e o Ki encontrado foi de 0,1 mM. A inibição da formação de AFMK também ocorre para a peroxidase humana (mieloperoxidase, MPO). Além de representarem uma regulação cruzada utilizada in vivo, as inibições encontradas podem ser relevantes para a proposta de novos inibidores de IDO e MPO na terapia imunomodulatória. Dado o nosso interesse pelas enzimas IDO e MPO, avaliamos ainda a localização intracelular destas enzimas em células de peritônio de camundongo, tanto residente como ativada com concanavalina A (Con A). O estímulo com Con A representa uma ativação de linfócitos T mediado por interferon gama (IFN-γ) e foi usado como modelo experimental para avaliar condições de localização em células ativadas. Por imunocitoquímica verificamos que IDO e MPO localizam-se próxima à membrana plasmática sendo que uma leve dispersão apenas de MPO foi observada em células ativadas com Con A. A localização intracelular das duas enzimas é no...
Tryptophan (TRP) is metabolized by two mains pathways, the serotoninergic pathway and the kynurenine pathway. In the serotoninergic pathway, TRP is metabolized to serotonin (5-HT) and, in some cells, to melatonin (MLT). The later can even be oxidized to acetyl-N1-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5 -methoxykynuramine (AMK) by peroxidases. In the kynurenine pathway, TRP is metabolized to N-formylkynurenine (NFK) and to kynurenine (KYN). Indoleamine 2, 3 dioxygenase (IDO) is one of those responsible for this reaction. Since IDO is importat in immune tolerance and the fact that this enzyme is inducible by cytokines we proposed whether there is a cross regulation between this enzyme and the serotoninergic pathway. A possible interaction between MLT and TRP oxidation pathways was shown by the AMK influence on IDO activity and QUIN interference on AFMK formation by peroxidases. AMK was shown to be an IDO classical competitive inhibitor with a Ki of 0.98 mM. QUIN was a peroxidase (horseradish peroxidase, HRP) classical uncompetitive inhibitor and Ki was found to be 0,1 mM. AFMK formation inhibition was also found in human peroxidase (myeloperoxidase, MPO). Beyond the in vivo crosstalk, new IDO and MPO inhibitors in immunomodulatory therapy would be proposed by the compounds shown in this study. Given our interest in IDO and MPO, we also evaluated their intracellular localization in both resident and concanavalin A (Con A) activated mice peritoneum cells. Con A stimulation is a IFN-γ mediated T lymphocytes activation and was our experimental model to evaluate activated cells. In light microscopy we observed IDO and MPO localization near the membrane and MPO only had a dispersed localization in Con A activated cells. Cytoplasm, nucleus and vesicles were the intracellular localization of both enzymes. Interestingly, we found MPO in isolated cells and in cell clusters of two or more cells. MPO was founded on macrophages, B1 cells and cell clusters by...
Assuntos
Dioxigenases/análise , Peroxidase/análise , Triptofano/metabolismo , Quinurenina 3-Mono-Oxigenase , Linfócitos/fisiologia , MacrófagosRESUMO
Activated phagocytes oxidize the hormone melatonin to N1-acethyl-N2-formyl-5-methoxykynuramine (AFMK) in a superoxide anion- and myeloperoxidase-dependent reaction. We examined the effect of melatonin, AFMK and its deformylated-product N-acetyl-5-methoxykynuramine (AMK) on the phagocytosis, the microbicidal activity and the production of hypochlorous acid by neutrophils. Neither neutrophil and bacteria viability nor phagocytosis were affected by melatonin, AFMK or AMK. However these compounds affected the killing of Staphylococcus aureus. After 60 min of incubation, the percentage of viable bacteria inside the neutrophil increased to 76% in the presence of 1 mM of melatonin, 34% in the presence of AFMK and 73% in the presence of AMK. The sole inhibition of HOCl formation, expected in the presence of myeloperoxidase substrates, was not sufficient to explain the inhibition of the killing activity. Melatonin caused an almost complete inhibition of HOCl formation at concentrations of up to 0.05 mM. Although less effective, AMK also inhibited the formation of HOCl. However, AFMK had no effect on the production of HOCl. These findings corroborate the present view that the killing activity of neutrophils is a complex phenomenon, which involves more than just the production of reactive oxygen species. Furthermore, the action of melatonin and its oxidation products include additional activities beyond their antioxidant property. The impairment of the neutrophils' microbicidal activity caused by melatonin and its oxidation products may have important clinical implications, especially in those cases in which melatonin is pharmacologically administered in patients with infections.
Assuntos
Cinuramina/análogos & derivados , Cinuramina/farmacologia , Melatonina/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Células Cultivadas , Humanos , Ácido Hipocloroso/metabolismo , Cinuramina/química , Cinuramina/metabolismo , Ativação de Neutrófilo/imunologia , Neutrófilos/microbiologia , Neutrófilos/fisiologia , Oxirredução , Fagocitose , Staphylococcus aureus/imunologiaRESUMO
O objetivo deste estudo foi avaliar a presença de tolerância à vancomicina em cepas de Staphylococus aureus resistentes à oxacilina (ORSA) isoladas de quatro hospitais da cidade de São Paulo. Foram estudadas 395 cepas ORSA isoladas de pacientes hospitalizados entre outubro de 1998 e maio de 2000. A determinação da concentração inibitória mínima (CIM) e da concentração bactericida mínima (CBM) para vancomicina foi realizada conforme padronizado pelo National Committee for Clinical Laboratory Standards (NCCLS). A tolerância à vancomicina foi definida como a razão CBM/CIM maior ou igual 32. Do total de cepas estudadas, 10,4 por cento apresentaram CIM de 0,5µg/ml para vancomicina; 41,3 por cento, CIM de 1µg/ml; 42,2 por cento, CIM de 2µg/ml; e 6,1 por cento, CIM de 4µg/ml. Em média, 49,1 por cento dos Orsa apresentaram tolerância à vancomicina entre as cepas Orsa foi considerada elevada. Conseqüentemente, aumentam as chances de falhas no tratamento com vancomicina, além de aumentar o risco da emergência de Staphylococcus aureus vancomicina-intermediário