RESUMO
The elimination of transformed and viral infected cells by natural killer (NK) cells requires a specialized junction between NK and target cells, denominated immunological synapse (IS). After initial recognition, the IS enables the directed secretion of lytic granules content into the susceptible target cell. The lymphocyte function-associated antigen (LFA)-1 regulates NK effector function by enabling NK-IS assembly and maturation. The pathways underlying LFA-1 accumulation at the IS in NK cells remained uncharacterized. A kinase anchoring protein 350 (AKAP350) is a centrosome/Golgi-associated protein, which, in T cells, participates in LFA-1 activation by mechanisms that have not been elucidated. We first evaluated AKAP350 participation in NK cytolytic activity. Our results showed that the decrease in AKAP350 levels by RNA interference (AKAP350KD) inhibited NK-YTS cytolytic activity, without affecting conjugate formation. The impairment of NK effector function in AKAP350KD cells correlated with decreased LFA-1 clustering and defective IS maturation. AKAP350KD cells that were exclusively activated via LFA-1 showed impaired LFA-1 organization and deficient lytic granule translocation as well. In NK AKAP350KD cells, activation signaling through Vav1 was preserved up to 10 min of interaction with target cells, but significantly decreased afterwards. Experiments in YTS and in ex vivo NK cells identified an intracellular pool of LFA-1, which partially associated with the Golgi apparatus and, upon NK activation, redistributed to the IS in an AKAP350-dependent manner. The analysis of Golgi organization indicated that the decrease in AKAP350 expression led to the disruption of the Golgi integrity in NK cells. Alteration of Golgi function by BFA treatment or AKAP350 delocalization from this organelle also led to impaired LFA-1 localization at the IS. Therefore, this study characterizes AKAP350 participation in the modulation of NK effector function, revealing the existence of a Golgi-dependent trafficking pathway for LFA-1, which is relevant for LFA-1 organization at NK-lytic IS.
Assuntos
Proteínas de Ancoragem à Quinase A , Sinapses Imunológicas , Células Matadoras Naturais , Antígeno-1 Associado à Função Linfocitária , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Centrossomo/metabolismo , Citotoxicidade Imunológica , Antígeno-1 Associado à Função Linfocitária/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Células Matadoras Naturais/metabolismoRESUMO
A severe re-emergingzoonosis, leptospirosis, is caused by pathogenic spirochetes of the genus Leptospira. Several studies have identified leptospiral surface proteins with the ability to bind ECM and plasma components, which could mediate adhesion and invasion through the hosts. It has been shown that Mce of pathogenic Leptospira spp. is an RGD (Arg-Gly-Asp)-motif-dependent virulence factor, responsible for infection of cells and animals. In the present article, we decided to further study the repertoire of the Mce activities in leptospiral biological properties. We report that the recombinant Mce is a broad-spectrum ECM-binding protein, capable of interacting with laminin, cellular and plasma fibronectin and collagen IV. Dose--r-esponse interaction was observed for all the components, fulfilling ligand--receptor requirements. Mce is a PLG binding protein capable to recruit this component from NHS, generating PLA in the presence of PLG activator. Binding of Mce was also observed with the leukocyte cell receptors αLß2 [(CD11a/CD18)-LFA-1] and αMß2 [(CD11b/CD18)-Mac-1], suggesting the involvement of this protein in the host immune response. Indeed, virulent Leptospira L1-130 was capable of binding both integrins, whereas culture-attenuated M-20 strain only bind to αMß2 [(CD11b/CD18)-Mac-1]. To the best of our knowledge, this is the first work to describe that Mce surface protein could mediate the attachment of Leptospira interrogans to human cell receptors αLß2(CD11a/CD18) and αMß2(CD11b/CD18).
Assuntos
Proteínas de Bactérias/metabolismo , Antígenos CD18/metabolismo , Matriz Extracelular/metabolismo , Leptospira interrogans/fisiologia , Leptospirose/metabolismo , Leptospirose/microbiologia , Plasminogênio/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Ativação Enzimática , Fibrinolisina/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Ligantes , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/metabolismo , Fases de Leitura Aberta , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Enflurane is a fluorinated volatile anesthetic, whose bioactive conformation is not known. Actually, a few studies have reported on the conformations of enflurane in nonpolar solution and gas phase. The present computational and spectroscopic (infrared and NMR) work shows that three pairs of isoenergetic conformers take place in the gas phase, neat liquid, polar, and nonpolar solutions. According to docking studies, a single conformation is largely preferred over its isoenergetic isomers to complex with the active site of Integrin LFA-1 enzyme (PDB code: 3F78 ), where the widely used anesthetic isoflurane (a constitutional isomer of enflurane) is known to bind. Weak hydrogen bonding from an electrostatic interaction between the CHF2 hydrogen and the central CF2 fluorines was not found to rule the conformational isomerism of enflurane. Moreover, intramolecular interactions based on steric, electrostatic, and hyperconjugative effects usually invoked to describe the anomeric effect are not responsible for the possible bioactive conformation of enflurane, which is rather governed by the enzyme induced fit.
Assuntos
Enflurano/química , Antígeno-1 Associado à Função Linfocitária/metabolismo , Domínio Catalítico , Antígeno-1 Associado à Função Linfocitária/química , Conformação Molecular , Simulação de Acoplamento Molecular , Teoria Quântica , Soluções , TermodinâmicaRESUMO
Although the exact etiology of Chagas' disease remains unknown, the inflammatory process and oxidative stress are believed to be the main contributors to the dysfunction and pathogenesis during chronic Trypanosoma cruzi infection. Our hypothesis is that melatonin administered for 2 months daily could modulate the oxidative stress and the inflammatory response during the chronic infection. Flow cytometric analysis of macrophages and antigen-presenting cells (APC), expression of RT1B as well as LFA-1 and MCP-1 in CD4(+) and CD8(+) T cells and levels of interleukin-17A were assessed. The oxidative stress was evaluated through lipid peroxidation (LPO) analysis on the plasma of thiobarbituric acid-reactive substances (TBARS) and nitric oxide production. Decreased concentrations of nitrite and TBARS were found in infected and melatonin-treated animals, as well as a rising trend in the production of IL-17A as compared to infected and untreated counterparts. A significant decrease was found in the percentages of CD4(+) and CD8(+) T lymphocytes MCP-1 producers for infected and melatonin-treated rats. Reduced percentage of CD8(+) T cells producing LFA-1 was observed in control and melatonin-treated animals as compared to untreated rats. The cellular response of peritoneal APC cells and macrophages significantly dropped in infected and treated animals. As an endpoint, the use of antioxidant compounds such as melatonin emerges as a new and promising approach to control the oxidative stress during the chronic Chagas' disease partially mediated through the abrogation of LPO and the prevention of the inflammatory response and can be used for further investigation on treatment trials for other infectious diseases.
Assuntos
Interleucina-17/metabolismo , Melatonina/farmacologia , Animais , Antioxidantes/farmacologia , Citometria de Fluxo , Inflamação/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Receptores CCR2/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismoRESUMO
Kinins are pro-inflammatory peptides that mimic the cardinal features of inflammation. We examined the concept that expression levels of endothelial intercellular adhesion molecule-1 (ICAM-1) and neutrophil integrins Mac-1 and LFA-1 are modulated by the kinin B1 receptor (B1R) agonist, Lys-des[Arg(9)]bradykinin (LDBK). Stimulation of endothelial cells with LDBK increased the levels of ICAM-1 mRNA transcripts/protein, and also of E-selectin and platelet endothelial adhesion molecule-1. ICAM-1 levels increased in a magnitude comparable with that produced by TNF-α. This stimulatory effect was reduced when endothelial cells, which had been previously transfected with a B1R small interfering RNA, were stimulated with LDBK, under comparable conditions. Similarly, LDBK produced a significant increase in protein levels of LFA-1 and Mac-1 integrins in human neutrophils, an effect that was reversed by pretreatment of cells with 10 µg/ml cycloheximide or a B1R antagonist. Functional experiments performed with post-confluent monolayers of endothelial cells stimulated with LDBK and neutrophils primed with TNF-α, and vice versa, resulted in enhanced adhesiveness between both cells. Neutralizing Abs to ICAM-1 and Mac-1 reduced the adhesion between them. Our results indicate that kinin B1R is a novel modulator that promotes adhesion of leukocytes to endothelial cells, critically enhancing the movement of neutrophils from the circulation to sites of inflammation.
Assuntos
Células Endoteliais/imunologia , Inflamação/imunologia , Calidina/análogos & derivados , Neutrófilos/efeitos dos fármacos , Receptor B1 da Bradicinina/metabolismo , Adesão Celular/efeitos dos fármacos , Comunicação Celular , Movimento Celular , Células Cultivadas , Cicloeximida/farmacologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Calidina/farmacologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/metabolismo , Neutrófilos/imunologia , RNA Interferente Pequeno/genética , Receptor B1 da Bradicinina/agonistas , Receptor B1 da Bradicinina/genéticaRESUMO
Galectin-8 belongs to a family of mammalian lectins that recognize glycoconjugates present on different cell surface components and modulate a variety of cellular processes. A role of Gal-8 in the immune system has been proposed based on its effects in immune cells, including T and B lymphocytes, as well as the presence of anti-Gal-8 autoantibodies in the prototypic autoimmune disease systemic lupus erythematosus (SLE). We have previously described that Gal-8 induces apoptosis in activated T cells interacting with certain ß1 integrins and this effect is counteracted by the anti-Gal-8 autoantibodies. Given that Gal-8 can potentially interact with several glycoproteins, here we analyzed the ß2 integrin Lymphocyte Function-Associated Antigen-1 (LFA-1), which is involved in leukocyte cell adhesion and immunological synapses. We show by GST-pull down assays that Gal-8 interacts with LFA-1 and this interaction is inhibited by anti-Gal-8 autoantibodies isolated from SLE patients. In cell adhesion assays, Gal-8 precluded the interaction of LFA-1 with its ligand Intracellular Adhesion Molecule-1 (ICAM-1). These results suggest that Gal-8 can exert immunosuppressive action not only by inducing apoptosis in activated T cells but also by negatively modulating the crucial function of LFA-1 in the immune system, while function-blocking autoantibodies counteract these effects.
Assuntos
Galectinas/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Adesão Celular , HumanosRESUMO
Galectin-8 belongs to a family of mammalian lectins that recognize glycoconjugates present on different cell surface components and modulate a variety of cellular processes. A role of Gal-8 in the immune system has been proposed based on its effects in immune cells, including T and B lymphocytes, as well as the presence of anti-Gal-8 autoantibodies in the prototypic autoimmune disease systemic lupus erythematosus (SLE). We have previously described that Gal-8 induces apoptosis in activated T cells interacting with certain β1 integrins and this effect is counteracted by the anti-Gal-8 autoantibodies. Given that Gal-8 can potentially interact with several glycoproteins, here we analyzed the β2 integrin Lymphocyte Function-Associated Antigen-1 (LFA-1), which is involved in leukocyte cell adhesion and immunological synapses. We show by GST-pull down assays that Gal-8 interacts with LFA-1 and this interaction is inhibited by anti-Gal-8 autoantibodies isolated from SLE patients. In cell adhesion assays, Gal-8 precluded the interaction of LFA-1 with its ligand Intracellular Adhesion Molecule-1 (ICAM-1). These results suggest that Gal-8 can exert immunosuppressive action not only by inducing apoptosis in activated T cells but also by negatively modulating the crucial function of LFA-1 in the immune system, while function-blocking autoantibodies counteract these effects.
Assuntos
Humanos , Galectinas/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Adesão CelularRESUMO
PURPOSE: To compare gene expression of the chemokines RANTES and eotaxin-2, its receptor, CCR-3, adhesion molecule ICAM-1 and its receptor LFA-1 in eosinophilic polyps and in control normal nasal mucosa. METHODS: Gene expression was quantified by Real Time PCR in polyps (n=35) and in healthy nasal mucosa (n=15). RESULTS: Eosinophilic polyps showed a higher expression of eotaxin-2 and RANTES, but not of CCR-3, ICAM-1 or LFA-1 compared to control nasal mucosa. CONCLUSION: Eosinophilic polyps present greater expression of eotaxin-2 and RANTES, but not of CCR-3, ICAM-1 or LFA-1 compared to control nasal mucosa.
Assuntos
Pólipos Nasais/metabolismo , Rinite/metabolismo , Sinusite/metabolismo , Estudos de Casos e Controles , Quimiocina CCL24/genética , Quimiocina CCL24/metabolismo , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Doença Crônica , Expressão Gênica , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Mucosa Nasal , Pólipos Nasais/complicações , Reação em Cadeia da Polimerase , Receptores CCR3/genética , Receptores CCR3/metabolismo , Rinite/complicações , Sinusite/complicaçõesRESUMO
PURPOSE: To compare gene expression of the chemokines RANTES and eotaxin-2, its receptor, CCR-3, adhesion molecule ICAM-1 and its receptor LFA-1 in eosinophilic polyps and in control normal nasal mucosa. METHODS: Gene expression was quantified by Real Time PCR in polyps (n=35) and in healthy nasal mucosa (n=15). RESULTS: Eosinophilic polyps showed a higher expression of eotaxin-2 and RANTES, but not of CCR-3, ICAM-1 or LFA-1 compared to control nasal mucosa. CONCLUSION: Eosinophilic polyps present greater expression of eotaxin-2 and RANTES, but not of CCR-3, ICAM-1 or LFA-1 compared to control nasal mucosa.
OBJETIVO: Comparar a expressão gênica das quimiocinas RANTES e eotaxina-2, do seu receptor CCR-3, da molécula de adesão ICAM-1 e do seu receptor LFA-1 entre pólipos nasais eosinofílicos (PE) (n=35) e mucosa nasal controle (n=15). MÉTODOS: Quantificou-se a expressão gênica dos mediadores citados pela técnica de PCR em tempo real em PEs e em mucosas de concha média de pacientes sem doenças nasais ou alteração endoscópica. RESULTADOS: Pólipos eosinofílicos apresentam maior expressão de eotaxina-2 e RANTES, mas não de CCR-3, ICAM-1 e LFA-1, quando comparados as mucosas nasais controles. CONCLUSÃO: Pólipos eosinofícios apresentaram maior expressão de eotaxin-2 and RANTES, mas não de CCR-3, ICAM-1 ou LFA-1,comparada à mucosa nasal controle.
Assuntos
Humanos , Pólipos Nasais/metabolismo , Rinite/metabolismo , Sinusite/metabolismo , Estudos de Casos e Controles , Doença Crônica , /genética , /metabolismo , /genética , /metabolismo , Expressão Gênica , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Mucosa Nasal , Pólipos Nasais/complicações , Reação em Cadeia da Polimerase , /genética , /metabolismo , Rinite/complicações , Sinusite/complicaçõesRESUMO
Hyperglycemia during diabetes is one of the causes of encephalopathy. However, diabetes causes chronic inflammatory complications and among them is peripheral neuropathy. Since, diabetes is one of the major risk factors for cerebrovascular disease, inflammatory process could take place in central nervous system (CNS). To test that hypothesis, experiments to determine inflammatory events in CNS during streptozotocin-induced diabetes were performed. Diabetes was induced by intravenous injection of streptozotocin (STZ). Brain angiotensin II (Ang II), monocyte/macrophage (ED-1 positive cells), CD8, the intercellular adhesion molecule-1 (ICAM-1), the lymphocyte function-associated antigen-1 (LFA-1) and superoxide anion were determined by hystochemical and immunohistochemical methods. Nitric oxide (NO), malondialdehyde (MDA) and catalase activity were measured in brain homogenates by enzymatic and biochemical methods. This research showed increased expressions of Ang II, ICAM-1, LFA-1 and CD8 positive cells in diverse zones of cerebrum and cerebellum of diabetic rats (week 8). Treatment of diabetic animals with losartan or enalapril reduced the expression of those molecules. Values of lipid peroxidation, nitrite content and superoxide anion expression remained similar to control rats. Only decreased activity of catalase was observed in diabetic animals, but losartan or enalapril failed to modify catalase activity. This study suggests the presence of Ang II-mediated brain inflammatory events in diabetes probably mediated by AT1 receptors.