RESUMEN
Calcium ion (Ca(2+)) signaling is a typical phenomenon mediated through immune receptors, such as the B-cell antigen receptor (BCR), and it is important for their biological activities. To analyze the signaling of immune receptors together with their in vivo dynamics, we generated stable transgenic mice with the Föster/fluorescence resonance energy transfer (FRET)-based Ca(2+) indicator yellow cameleon 3.60 (YC3.60), based on the Cre/loxP system (YC3.60(flox)). We successfully obtained mice with specific YC3.60 expression in immune or nerve cells as well as mice with ubiquitous expression of this indicator. We established five-dimensional (5D) (x, y, z, time, and Ca(2+)) intravital imaging of lymphoid tissues, including the bone marrow. Furthermore, in autoimmune-prone models, the CD22(-/-) and C57BL/6- lymphoproliferation (lpr)/lpr mouse, Ca(2+) fluxes were augmented, although they did not induce autoimmune disease. Intravital imaging of Ca(2+) signals in lymphocytes may improve assessment of the risk of autoimmune diseases in model animals.
Asunto(s)
Técnicas Biosensibles/métodos , Señalización del Calcio , Calcio/metabolismo , Linfocitos/metabolismo , Imagen Molecular/métodos , Animales , Antígenos CD19/genética , Antígenos CD19/metabolismo , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/metabolismo , Autoinmunidad , Médula Ósea/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Genes Reporteros , Ratones , Ratones Transgénicos , Ganglios Linfáticos Agregados/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Bazo/metabolismoRESUMEN
Many chemical mediators regulate neutrophil recruitment to inflammatory sites. Although the actions of each chemical mediator have been demonstrated with neutrophils in vitro, how such chemical mediators act cooperatively or counteractively in vivo remains largely unknown. Here, by in vivo two-photon excitation microscopy with transgenic mice expressing biosensors based on Förster resonance energy transfer, we time-lapse-imaged the activities of extracellular signal-regulated kinase (ERK) and protein kinase A (PKA) in neutrophils in inflamed intestinal tissue. ERK activity in neutrophils rapidly increased during spreading on the endothelial cells and showed positive correlation with the migration velocity on endothelial cells or in interstitial tissue. Meanwhile, in the neutrophils migrating in the interstitial tissue, high PKA activity correlated negatively with migration velocity. In contradiction to previous in vitro studies that showed ERK activation by prostaglandin E2 (PGE2) engagement with prostaglandin receptor EP4, intravenous administration of EP4 agonist activated PKA, inhibited ERK, and suppressed migration of neutrophils. The opposite results were obtained using nonsteroidal antiinflammatory drugs (NSAIDs). Therefore, NSAID-induced enteritis may be caused at least partially by the inhibition of EP4 receptor signaling of neutrophils. Our results demonstrate that ERK positively regulates the neutrophil recruitment cascade by promoting adhesion and migration steps.