RESUMO
INTRODUCTION: The formation of neutrophil extracellular traps (NETs) is a process in which several kinds of enzymes participate generating posttranslational modifications of proteins. NETs have been associated with infectious, autoimmune, and inflammatory diseases. Inhibition of several proteases reduces the formation of NETs. In the present work, we analyzed the role of several broad-acting and specific inhibitors of proteases in the formation of NETs. METHODS: Neutrophils were isolated from peripheral blood of healthy individuals by density gradient. The neutrophils were quantified and seeded into cell culture plates. Phorbol myristate acetate and A23187 were used as NETs inducers, and several specific inhibitors of proteases were used. The cells were stained for cytoskeleton or DNA. The cell-free supernatants were used to assess DNA release. Statistical analysis was carried out by a Kruskal-Wallis or ANOVA test. RESULTS: We observed marked changes in actin organization after the induction of NETs, suggesting that the cytoskeleton is being actively regulated. When we used protease inhibitors, the release of DNA was reduced, suggesting the participation of actin remodeling in the process. Further characterization of the specific proteases revealed that calpain modulates the reorganization of actin cytoskeleton and DNA release. Preservation of part of the actin cytoskeleton suggests that DNA release is not only a mechanic process associated to the chromatin decondensation; rather the process is highly regulated by active proteases that promote cytoskeleton reorganization and chromatin decondensation that culminates in DNA release. CONCLUSION: Calpain mediates the DNA release in the NET formation process by the modification of cortical actin cytoskeleton in a calcium-dependent manner.
Assuntos
Calpaína/metabolismo , Citoesqueleto/metabolismo , DNA/metabolismo , Armadilhas Extracelulares/imunologia , Neutrófilos/metabolismo , Actinas/metabolismo , Cálcio/metabolismo , Células Cultivadas , Humanos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Inibidores de Proteases/farmacologiaRESUMO
The first degree relatives of rheumatoid arthritis (RA) patients have a higher risk of developing RA, which is related to the expression of autoantibodies against citrullinated proteins (ACPA). Remarkably, prior to the onset of RA, cartilage damage is already initiated, whereas ACPA autoantibodies are already expressed. Here we show that both TNF-α and IL-6 are also increased prior to the onset of RA. Furthermore, when the levels of DKK1 and Sclerostin were evaluated in first degree relatives of RA patients, we found that the serum levels of TNF- α correlate with the expression levels of both DKK1 and Sclerostin. Interestingly, when the disease is already established, the correlation of TNF- α with DKK1 is lost in RA patients, whereas the correlation of Sclerostin with both TNF- α and IL-6 is further increased. Our data suggest a subclinical inflammation in patients at high risk of developing RA, which might lead to an increase in the levels of both DKK1 and Sclerostin, contributing to joint damage in the preclinical phase of the disease linked to the expression of ACPA autoantibodies.
Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Doenças Assintomáticas , Cartilagem Articular/imunologia , Cartilagem Articular/patologia , Família , Proteínas Adaptadoras de Transdução de Sinal/sangue , Adulto , Anticorpos Antiproteína Citrulinada/sangue , Artrite Reumatoide/sangue , Artrite Reumatoide/diagnóstico , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/sangueRESUMO
Dopaminergic neurons have the ability to release Dopamine from their axons as well as from their soma and dendrites. This somatodendritically-released Dopamine induces an autoinhibition of Dopaminergic neurons mediated by D2 autoreceptors, and the stimulation of neighbor GABAergic neurons mediated by D1 receptors (D1r). Here, our results suggest that the somatodendritic release of Dopamine in the substantia nigra (SN) may stimulate GABAergic neurons that project their axons into the hippocampus. Using semiquantitative multiplex RT-PCR we show that chronic blockade of the Dopaminergic neurotransmission with both AMPT and reserpine specifically decreases the expression levels of D1r, remarkably this may be the result of an antagonistic effect between AMPT and reserpine, as they induced the expression of a different set of genes when treated by separate. Furthermore, using anterograde and retrograde tracing techniques, we found that the GABAergic neurons that express D1r also project their axons in to the CA1 region of the hippocampus. Finally, we also found that the same treatment that decreases the expression levels of D1r in SN, also induces an impairment in the performance in an appetitive learning task that requires the coding of reward as well as navigational skills. Overall, our findings show the presence of a GABAergic interconnection between the SNr and the hippocampus mediated by D1r.
Assuntos
Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Receptores de Dopamina D1/biossíntese , Reserpina/farmacologia , Substância Negra/metabolismo , alfa-Metiltirosina/farmacologia , Inibidores da Captação Adrenérgica/farmacologia , Animais , Antagonistas dos Receptores de Dopamina D2/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Dopamina/biossíntese , Neurônios Dopaminérgicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D1/genética , Receptores de Dopamina D2/biossíntese , Receptores de Dopamina D2/genética , Substância Negra/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid glutamate receptors have been shown to modulate the morphology of the lamelar processes of Bergmann glia cells in the molecular layer of the cerebellum. Here we suggest that reorganization of F-actin may underlay the changes in the morphology of the lamelar processes. Using the fluorescent staining of F-actin with Phalloidin and the quantification of RhoA activation through immunoprecipitation or pull-down assays, we show that RhoA is activated after stimulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors and leads to the reorganization of the actin cytoskeleton of Bergmann fibers. This reorganization of the actin cytoskeleton is reflected in the form of an increase in the intensity of the F-actin staining as well as in the loss of the number of Bergmann fibers stained with Phalloidin. Moreover, using a pharmacological approach, we show that activation of RhoA and the change in the intensity of the F-actin staining depends on the activation of PI3-K, focal adhesion kinase, and protein kinase C, whereas changes in the number of Bergmann fibers depend on external calcium in a RhoA independent manner. Our findings show that glutamate may induce a form of structural plasticity in Bergmann glia cells through the reorganization of the actin cytoskeleton. This may have implications in the way the synaptic transmission is processed in the cerebellum.
Assuntos
Actinas/metabolismo , Ácido Glutâmico/metabolismo , Neuroglia/metabolismo , Receptores de AMPA/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Cerebelo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais/fisiologiaRESUMO
Glycolytic enzymes have, in addition to their role in energy production, other functions in the regulation of cellular processes. Aldolase A has been reported to be present in sperm, playing a key role in glycolysis; however, despite its reported interactions with actin and WAS, little is known about a non-glycolytic role of aldolase A in sperm. Here, we show that in guinea pig spermatozoa, aldolase A is tightly associated to cytoskeletal structures where it interacts with actin, WAS, and Arp2/3. We show that aldolase A spermatozoa treatment increases their polymerized actin levels. In addition, we show that there is a direct correlation between the levels of polymerized actin and the levels of aldolase A-actin interaction. Our results suggest that aldolase A functions as a bridge between filaments of actin and the actin-polymerizing machinery.