RESUMO
Eosinophils play an important role in the pathogenesis of asthma and can be activated by extracellular nucleotides released following cell damage or inflammation. For example, increased ATP concentrations were reported in bronchoalveolar lavage fluids of asthmatic patients. Although eosinophils are known to express several subtypes of P2 receptors for extracellular nucleotides, their function and contribution to asthma remain unclear. In this article, we show that transcripts for P2X1, P2X4, and P2X5 receptors were expressed in healthy and asthmatic eosinophils. The P2X receptor agonist α,ß-methylene ATP (α,ß-meATP; 10 µM) evoked rapidly activating and desensitizing inward currents (peak 18 ± 3 pA/pF at -60 mV) in healthy eosinophils, typical of P2X1 homomeric receptors, which were abolished by the selective P2X1 antagonist NF449 (1 µM) (3 ± 2 pA/pF). α,ß-meATP-evoked currents were smaller in eosinophils from asthmatic patients (8 ± 2 versus 27 ± 5 pA/pF for healthy) but were enhanced following treatment with a high concentration of the nucleotidase apyrase (17 ± 5 pA/pF for 10 IU/ml and 11 ± 3 pA/pF for 0.32 IU/ml), indicating that the channels are partially desensitized by extracellular nucleotides. α,ß-meATP (10 µM) increased the expression of CD11b activated form in eosinophils from healthy, but not asthmatic, donors (143 ± 21% and 108 ± 11% of control response, respectively). Furthermore, α,ß-meATP increased healthy (18 ± 2% compared with control 10 ± 1%) but not asthmatic (13 ± 1% versus 10 ± 0% for control) eosinophil adhesion. Healthy human eosinophils express functional P2X1 receptors whose activation leads to eosinophil αMß2 integrin-dependent adhesion. P2X1 responses are constitutively reduced in asthmatic compared with healthy eosinophils, probably as the result of an increase in extracellular nucleotide concentration.
Assuntos
Asma/imunologia , Adesão Celular , Eosinófilos/fisiologia , Receptores Purinérgicos P2X1/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Apirase/farmacologia , Asma/fisiopatologia , Benzenossulfonatos/farmacologia , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Voluntários Saudáveis , Humanos , Contagem de Leucócitos , Agonistas do Receptor Purinérgico P2X/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores Purinérgicos P2X1/genética , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X5/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The entry of neutrophils into tissue has been well characterised; however the fate of these cells once inside the tissue microenvironment is not fully understood. A variety of signal transduction pathways including those involving class I PI3 Kinases have been suggested to be involved in neutrophil migration. This study aims to determine the involvement of PI3 Kinases in chemokinetic and chemotactic neutrophil migration in response to CXCL8 and GM-CSF in a three-dimensional collagen gel, as a model of tissue. Using a three-dimensional collagen assay chemokinetic and chemotactic migration induced by CXCL8 was inhibited with the pan PI3 Kinase inhibitor wortmannin. Analysis of the specific Class I PI3 Kinase catalytic isoforms alpha, delta and gamma using the inhibitors PIK-75, PIK-294 and AS-605240 respectively indicated differential roles in CXCL8-induced neutrophil migration. PIK-294 inhibited both chemokinetic and chemotactic CXCL8-induced migration. AS-605240 markedly reduced CXCL8 induced chemokinetic migration but had no effect on CXCL8 induced chemotactic migration. In contrast PIK-75 inhibited chemotactic migration but not chemokinetic migration. At optimal concentrations of GM-CSF the inhibitors had no effect on the percentage of neutrophil migration in comparison to the control however at suboptimal concentrations wortmannin, AS-605240 and PIK-294 inhibited chemokinesis. This study suggests that PI3 Kinase is necessary for CXCL8 induced migration in a 3D tissue environment but that chemokinetic and chemotactic migration may be controlled by different isoforms with gamma shown to be important in chemokinesis and alpha important in chemotaxis. Neutrophil migration in response to suboptimal concentrations of GM-CSF is dependent on PI3 Kinase, particularly the gamma and delta catalytic isoforms.
Assuntos
Movimento Celular/fisiologia , Colágeno/química , Neutrófilos/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Movimento Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Géis/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-8/farmacologia , Masculino , Neutrófilos/citologia , Inibidores de Fosfoinositídeo-3 QuinaseRESUMO
Microglia are the resident immune cells of the brain, which are important therapeutic targets for regulating the inflammatory responses particularly neurodegeneration in the aging human brain. The activation, chemotaxis and migration of microglia are regulated through G-protein coupled receptors by chemokines such as stromal cell-derived factor (SDF)-1α and bioactive lysophospholipids such as lysophosphatidic acid (LPA). Potassium channels play important roles in microglial function and cell fate decisions; however, the regulation of microglial potassium channels has not been fully elucidated. Here we show reciprocal action of SDF-1α and LPA, on potassium currents through Kir2.1 channels in primary murine microglia. The potassium channel modulation is mediated by the same small GTPases, Rac and Rho that regulate the actin cytoskeleton. SDF-1α rapidly increased the Kir2.1 current amplitude and cell spreading. These effects were mimicked by dialysing the cells with constitutively active Rac1 protein, and they were blocked by inhibiting the phosphatidylinositol 3-kinase (PI3K) with wortmannin. In contrast, LPA and constitutively active RhoA decreased the Kir2.1 currents and stimulated cell contraction. Thus, SDF-1α and LPA regulate both the actin cytoskeleton and the Kir2.1 potassium channels through the same Rho GTPase signaling pathways. The inhibition of Kir2.1 with chloroethylclonidine produced cell contraction independently of chemokine action. This suggests that potassium channels are essential for the morphological phenotype and functioning of microglia. In conclusion, the small GTPases, Rac and Rho, modulate Kir2.1 channels and block of Kir2.1 channels causes changes in microglia morphology.
Assuntos
Quimiocina CXCL12/farmacologia , Lisofosfolipídeos/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Animais Recém-Nascidos , Antígenos de Diferenciação/metabolismo , Tamanho Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Clonidina/análogos & derivados , Clonidina/farmacologia , Camundongos , Microscopia Confocal , Técnicas de Patch-Clamp , Fosfatidilinositol 3-Quinases/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transfecção , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Asthma is a common disease that causes considerable morbidity. Increased numbers of airway eosinophils are a hallmark of asthma. Mechanisms controlling the entry of eosinophils into asthmatic lung have been intensively investigated, but factors regulating migration within the tissue microenvironment are less well understood. We modeled this by studying chemoattractant and growth factor-mediated human eosinophil migration within a three-dimensional collagen matrix. Stimulation with GM-CSF induced dose-dependent, random migration with a maximum of 77 +/- 4.7% of cells migrating. In contrast, CCL11 and C5a caused a more modest although significant degree of migration (19 +/- 1.8% and 20 +/- 2.6%, respectively). Migration to GM-CSF was partially dependent on Ca(2+) and alpha(M)beta(2) integrins. The Rho family of small GTPases regulates intracellular signaling of cell migration. GM-CSF-induced migration was only partially dependent on Rho kinase/Rho-associated kinase (ROCK) and was independent of RhoA activation. In contrast, CCL11-induced migration was fully dependent on both RhoA and ROCK. Activation of RhoA was therefore neither necessary nor sufficient to cause eosinophil migration in a three-dimensional collagen environment. This study suggests that eosinophil growth factors are likely to be required for eosinophil migration within the bronchial mucosa, and this involves signal transduction pathways distinct from those used by G protein-associated chemoattractants.
Assuntos
Quimiocina CCL11/fisiologia , Quimiotaxia de Leucócito/imunologia , Eosinófilos/enzimologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Transdução de Sinais/imunologia , Proteínas rho de Ligação ao GTP/fisiologia , Anticorpos Bloqueadores/farmacologia , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Cálcio/antagonistas & inibidores , Cálcio/fisiologia , Inibição de Migração Celular/imunologia , Quelantes/farmacologia , Colágeno Tipo I/fisiologia , Complemento C5a/fisiologia , Ácido Edético/farmacologia , Eosinófilos/citologia , Eosinófilos/metabolismo , Géis , Humanos , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
Many nuclear hormones have physiological effects that are too rapid to be explained by changes in gene expression and are often attributed to unidentified or novel G protein-coupled receptors. Thyroid hormone is essential for normal human brain development, but the molecular mechanisms responsible for its effects remain to be identified. Here, we present direct molecular evidence for potassium channel stimulation in a rat pituitary cell line (GH(4)C(1)) by a nuclear receptor for thyroid hormone, TRbeta, acting rapidly at the plasma membrane through phosphatidylinositol 3-kinase (PI3K) to slow the deactivation of KCNH2 channels already in the membrane. Signaling was disrupted by heterologous expression of TRbeta receptors with mutations in the ligand-binding domain that are associated with neurological disorders in humans, but not by mutations that disrupt DNA binding. More importantly, PI3K-dependent signaling was reconstituted in cell-free patches of membrane from CHO cells by heterologous expression of human KCNH2 channels and TRbeta, but not TRalpha, receptors. TRbeta signaling through PI3K provides a molecular explanation for the essential role of thyroid hormone in human brain development and adult lipid metabolism.
Assuntos
Membrana Celular/metabolismo , Transdução de Sinais , Receptores beta dos Hormônios Tireóideos/metabolismo , Tri-Iodotironina/metabolismo , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Cricetinae , Canal de Potássio ERG1 , Eletrofisiologia , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Ativação do Canal Iônico , Técnicas de Patch-Clamp , Fosfatidilinositol 3-Quinases/metabolismo , Transporte Proteico , Ratos , Transdução de Sinais/efeitos dos fármacos , Receptores beta dos Hormônios Tireóideos/genética , Fatores de Tempo , Tri-Iodotironina/farmacologiaRESUMO
The mechanisms regulating retrograde neuronal degeneration and subsequent death of thalamic neurons following cortical injury are not well understood. However, the delay in the onset of retrograde cell death and observed morphological changes are consistent with apoptosis. Our previous studies demonstrated that monocyte chemoattractant protein-1 (MCP-1), a beta-chemokine that attracts cells of monocytic origin to sites of injury, is rapidly and specifically expressed in the lateral geniculate nucleus following visual cortical lesions. To determine the potential role of MCP-1 in retrograde degeneration, the present study examined the effect of genetic deletion of MCP-1 (MCP-1 KO or -/-) or its high affinity receptor CCR2 (CCR2 KO or -/-) on thalamic microglial activation and neuronal cell death following aspiration lesions of the visual cortex in adult mice. Deletion of the MCP-1 gene delayed microglial activation and transiently improved the survival of thalamic neurons. Deletion of the CCR2 receptor resulted in a significant increase in apoptosis as measured by nucleosomal fragmentation after injury compared to wild-type mice, but did not alter neuron survival, suggesting that glial apoptosis is increased in the receptor knockout mice. Investigation of Bcl-2, Bax, Fas, Fas ligand (FasL) and activated caspase-3, key regulators of apoptosis that can be modulated by cytokines, revealed complex alterations of mRNA and protein levels in MCP-1(-/-) and CCR2(-/-) mice. As examples, Bcl-2 protein was detected in wild-type, but not in MCP-1(-/-) mice. Caspase-3 activity was higher in MCP-1(-/-) mice compared to wild-type and CCR2(-/-) mice at 5 days after injury. High levels of activated caspase-3 correlate with the beginning of a period of delayed, but rapid cell death in the thalami of MCP-1(-/-) mice. In summary, our data strongly suggest that MCP-1 is involved in early microglial response to axotomy and that modulation of this chemokine could provide a novel strategy for improved neuronal survival following injury to the central nervous system.