RESUMO
Upon tumor antigen recognition, cytotoxic T lymphocytes (CTLs) and target cells form specialized supramolecular structures, called cytotoxic immunological synapses, which are required for polarized delivery of cytotoxic granules. In previous reports, we described the accumulation of connexin 43 (Cx43)-formed gap junctions (GJs) at natural killer (NK) cell-tumor cell cytotoxic immunological synapse. In this report, we demonstrate the functional role of Cx43-GJs at the cytotoxic immunological synapse established between CTLs and melanoma cells during cytotoxicity. Using confocal microscopy, we evaluated Cx43 polarization to the contact site between CTLs isolated from pMEL-1 mice and B16F10 melanoma cells. We knocked down Cx43 expression in B16F10 cells and evaluated its role in the formation of functional GJs and the cytotoxic activity of CTLs, by calcein transfer and granzyme B activity assays, respectively. We found that Cx43 localizes at CTL/B16F10 intercellular contact sites via an antigen-dependent process. We also found that pMEL-1 CTLs but not wild-type naïve CD8+ T cells established functional GJs with B16F10 cells. Interestingly, we observed that Cx43-GJs were required for an efficient granzyme B activity in target B16F10 cells. Using an HLA-A2-restricted/MART-1-specific CD8+ T-cell clone, we confirmed these observations in human cells. Our results suggest that Cx43-channels are relevant components of cytotoxic immunological synapses and potentiate CTL-mediated tumor cell killing.
Assuntos
Conexina 43/imunologia , Junções Comunicantes/imunologia , Sinapses Imunológicas/imunologia , Melanoma/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Junções Comunicantes/patologia , Humanos , Sinapses Imunológicas/patologia , Melanoma/patologia , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/patologiaRESUMO
Gap junction (GJ) mediates intercellular communication through linked hemichannels from each of two adjacent cells. Using human and mouse models, we show that connexin 43 (Cx43), the main GJ protein in the immune system, was recruited to the immunological synapse during T cell priming as both GJs and stand-alone hemichannels. Cx43 accumulation at the synapse was Ag specific and time dependent, and required an intact actin cytoskeleton. Fluorescence recovery after photobleaching and Cx43-specific inhibitors were used to prove that intercellular communication between T cells and dendritic cells is bidirectional and specifically mediated by Cx43. Moreover, this intercellular cross talk contributed to T cell activation as silencing of Cx43 with an antisense or inhibition of GJ docking impaired intracellular Ca(2+) responses and cytokine release by T cells. These findings identify Cx43 as an important functional component of the immunological synapse and reveal a crucial role for GJs and hemichannels as coordinators of the dendritic cell-T cell signaling machinery that regulates T cell activation.
Assuntos
Conexina 43/imunologia , Junções Comunicantes/imunologia , Sinapses Imunológicas/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Animais , Comunicação Celular/imunologia , Separação Celular , Conexina 43/metabolismo , Citometria de Fluxo , Imunofluorescência , Junções Comunicantes/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Sinapses Imunológicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Receptor Cross-Talk/imunologia , Transdução de Sinais/imunologia , Linfócitos T/metabolismoRESUMO
Several patients affected by a new variant of endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF) have experienced a sudden death syndrome, including persons below the age of 50. El Bagre-EPF patients share several autoantigens with paraneoplastic pemphigus patients, such as reactivity to plakins. Further, paraneoplastic pemphigus patients have autoantibodies to the heart. Therefore, we tested 15 El Bagre-EPF patients and 15 controls from the endemic area for autoreactivity to heart tissue using direct and indirect immunofluorescence, confocal microscopy, immunohistochemistry, immunoblotting, and immunoelectron microscopy utilizing heart extracts as antigens. We found that 7 of 15 El Bagre patients exhibited a polyclonal immune response to several cell junctions of the heart, often colocalizing with known markers. These colocalizing markers included those for the area composita of the heart, such as anti-desmoplakins I and II; markers for gap junctions, such as connexin 43; markers for tight junctions, such as ezrin and junctional adhesion molecule A; and adherens junctions, such pan-cadherin. We also detected colocalization of the patient antibodies within blood vessels, Purkinje fibers, and cardiac sarcomeres. We conclude that El Bagre-EPF patients display autoreactivity to multiple cardiac epitopes, that this disease may resemble what is found in patients with rheumatic carditis, and further, that the cardiac pathophysiology of this disorder warrants further evaluation.
Assuntos
Autoanticorpos/metabolismo , Doenças Endêmicas , Miocárdio/metabolismo , Pênfigo/epidemiologia , Pênfigo/imunologia , Adulto , Animais , Complexo Antígeno-Anticorpo/metabolismo , Autoanticorpos/imunologia , Autoantígenos/imunologia , Autoantígenos/metabolismo , Caderinas/imunologia , Caderinas/metabolismo , Bovinos , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Colômbia , Conexina 43/imunologia , Conexina 43/metabolismo , Proteínas do Citoesqueleto/imunologia , Proteínas do Citoesqueleto/metabolismo , Morte Súbita Cardíaca , Desmoplaquinas/imunologia , Desmoplaquinas/metabolismo , Coração/fisiologia , Coração/fisiopatologia , Humanos , Imuno-Histoquímica , Junções Intercelulares/ultraestrutura , Camundongos , Microscopia Eletrônica , Pessoa de Meia-Idade , Miocárdio/imunologia , Miocárdio/ultraestrutura , Pênfigo/mortalidade , Pênfigo/fisiopatologia , Ratos , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Adulto JovemRESUMO
Immunocytochemical expression of Connexin 43 (Cx 43) in the rat Supraoptic Nucleus was analyzed following dehydration, using sequence-specific anti-Cx 43 antibodies (designated 13-8300, 71-0700, and sc-9059) that exhibit differential recognition of Cx 43. Punctate and longitudinally arranged immunostaining patterns of Cx 43 labeling, as evidenced by antibody sc-9059, was detected overlaying the nucleus of magnocellular neuroendocrine cells. This novel form of longitudinally arranged Cx 43 immunoreactivity was modified by dehydration and halothane exposure, but not lactation.
Assuntos
Conexina 43/análise , Conexina 43/imunologia , Desidratação/metabolismo , Núcleo Supraóptico/química , Animais , Conexina 43/biossíntese , Desidratação/imunologia , Feminino , Imuno-Histoquímica , Lactação/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Núcleo Supraóptico/metabolismoRESUMO
The aim of the present study was to determine the presence of the connexins Cx43, Cx32 and Cx26 in Bufo arenarum ovarian follicles during the breeding season as well as to analyse the possible alterations in the meiotic process when connexins are blocked by specific antibodies. Western blot analysis revealed that the Cx43 and Cx32 proteins were present but not Cx26. We demonstrated that the anti-Cx43 and anti-Cx32 antibodies produced the uncoupling of the gap junctions. When these junctions are blocked the maturation process is triggered in the oocytes. We determined that dbcAMP exerts an inhibitory effect on the maturation induced by the uncoupling of the gap junctions when the oocytes are injected or pretreated with this metabolite. We propose the idea that cAMP is the regulatory molecule in meiotic arrest in this amphibian species.