RESUMEN
B lymphocytes that recognize soluble self-Ags are routinely found in normal individuals in a functionally inactive or anergic state. Current models indicate that this tolerant state is maintained by interactions with self-Ags that uncouple the BCR from downstream signaling pathways and increase levels of free calcium. Contrary to this expectation, B cells that harbor anti-insulin Ig transgenes (125Tg) are maintained in a tolerant state even though free calcium levels remain normal and tyrosine kinase substrate phosphorylation is preserved following BCR stimulation. Under basal conditions, intracellular levels of inositol 1,4,5-trisphosphate are increased and NFATc1 levels are reduced in 125Tg B cells. The 125Tg B cells are markedly impaired in their ability to mobilize calcium upon stimulation with ionomycin, and BCR-induced calcium mobilization from internal stores is decreased. In contrast, poisoning intracellular calcium pumps with thapsigargin increases calcium mobilization in 125Tg B cells. Changes in calcium signaling are accompanied by a failure of 125Tg B cells to translocate NFATc1 into the nucleus following stimulation with either anti-IgM or ionomycin. Thus, disassociation of BCR from multiple signaling pathways is not essential for maintaining tolerance in anti-insulin 125Tg B cells. Rather, BCRs that are occupied by autologous insulin deliver signals that induce changes in intracellular calcium mobilization and maintain tolerance by preventing activation of key transcription factors such as NFAT.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Señalización del Calcio/inmunología , Calcio/metabolismo , Tolerancia Inmunológica , Insulina/inmunología , Factores de Transcripción NFATC/metabolismo , Animales , Subgrupos de Linfocitos B/metabolismo , Calcio/antagonistas & inhibidores , Señalización del Calcio/genética , Células Cultivadas , Tolerancia Inmunológica/genética , Inmunoglobulina M/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factores de Transcripción NFATC/antagonistas & inhibidoresRESUMEN
BACKGROUND: Members of the Src family of tyrosine kinases (SFKs) are requisite signaling molecules activated by multiple receptors during immune responses. Their expression and catalytic activity has not been characterized in allograft rejection in vivo. METHODS: We measured expression and catalytic activity of SFKs in MHC- mismatched murine cardiac allografts. We also examined the effects of a Src inhibitor (CGP77675) with or without anti-CD154 mAb on graft survival, histology, and expression and catalytic activity of SFKs within the grafts. RESULTS: In acutely rejecting allografts from untreated controls, total activity of Hck and Lyn increased 10-fold, predominantly reflecting increases in the amount of protein. Total activity of Lck increased only fourfold, reflecting small changes in both the amount of protein and specific activity. One dose of anti-CD154 plus CGP77675 markedly diminished cellular infiltration, but survival was only moderately prolonged despite inhibition of all SFKs in the rejected grafts. Two doses of anti-CD154 plus CGP77675 allowed permanent graft acceptance in 60% of recipients even after discontinuation of the inhibitor. Both rejected and long surviving grafts showed increased activity of all SFKs. Recipients that rejected their grafts showed serum alloantibody production, and grafts rejected during treatment demonstrated deposition of complement indicating the contribution of antibody to rejection. CONCLUSIONS: The myeloid and B cell Src family kinases, Hck and Lyn, rather than the T cell Src kinase Lck, show the greatest increase in expression and total activity in rejecting allografts. Both rejected and long-surviving grafts show significant increases in SFK expression and acitivity.
Asunto(s)
Ligando de CD40/efectos de los fármacos , Rechazo de Injerto/enzimología , Rechazo de Injerto/prevención & control , Supervivencia de Injerto , Trasplante de Corazón , Familia-src Quinasas/antagonistas & inhibidores , Animales , Anticuerpos/farmacología , Ligando de CD40/inmunología , Linfocitos T CD8-positivos/inmunología , Rechazo de Injerto/inmunología , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/inmunología , Corazón/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Trasplante HomólogoRESUMEN
Fibroblast growth factor receptors are expressed by some T cells, and provide costimulation for these cells. Such receptors allow T cells to respond to fibroblast growth factors expressed in response to injury and inflammation and may provide a mechanism for 'context-dependent' responses to antigens within the local microenvironment. The mechanisms by which fibroblast growth factor receptors might interact with the TCR signalling pathway are not defined. Here we show that the TCR and fibroblast growth factor receptors co-localize during combined stimulation. Signalling via fibroblast growth factor receptors alone results in phosphorylation of Lck and induces nuclear translocation of nuclear factors of activated T cells. Combined stimulation via fibroblast growth factor receptors and the TCR synergistically enhances the activation of nuclear factors of activated T cells. The results suggest that peptide growth factors produced at sites of injury and inflammation can contribute to the outcome of T-cell encounters with antigen.
Asunto(s)
Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Linfocitos T/inmunología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Anticuerpos Monoclonales/farmacología , Complejo CD3/inmunología , Membrana Celular/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Humanos , Células Jurkat , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/efectos de los fármacos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Factores de Transcripción NFATC/metabolismo , Fosforilación/efectos de los fármacos , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/análisis , Linfocitos T/citologíaRESUMEN
Activated fibroblast growth factor receptor 1 (FGFR1) propagates FGF signals through multiple intracellular pathways via intermediates FRS2, PLCgamma, and Ras. Conflicting reports exist concerning the interaction between FGFR1 and Src family kinases. To address the role of c-Src in FGFR1 signaling, we compared proliferative responses of murine embryonic fibroblasts (MEF) deficient in c-Src, Yes, and Fyn to MEF expressing either endogenous levels or overexpressing c-Src. MEF with endogenous c-Src had significantly greater FGF-induced DNA synthesis and proliferation than cells lacking or overexpressing c-Src. This was related directly to c-Src expression by analysis of c-Src-deficient cells transfected with and sorted for varying levels of a c-Src expression vector. This suggests an "optimal" quantity of c-Src expression for FGF-induced proliferation. To determine if this was a general phenomenon for growth factor signaling pathways utilizing c-Src, responses to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and lysophosphatidic acid (LPA) were examined. As for FGF, responses to EGF were clearly inhibited when c-Src was absent or overexpressed. In contrast, varying levels of c-Src had little effect on responses to PDGF or LPA. The data show that mitogenic pathways activated by FGF-1 and EGF are regulated by c-Src protein levels and appear to differ significantly from those activated by PDGF and LPA.