RESUMO
Effector T cells are described to be primed in the lymph nodes draining the site of immunization and to recirculate to effector sites. Sublingual immunization generates effector T cells able to disseminate to the genital tract. Herein, we report an alternative mechanism that involves the recirculation of antigen-bearing dendritic cells (DCs) in remote lymphoid organs to prime T cells. Sublingual immunization with a muco-adhesive model antigen unable to diffuse through lymphatic or blood vessels induced genital CD8 T cells. The sublingual draining lymph nodes were not mandatory to generate these lymphocytes, and antigen-bearing DCs from distant lymph nodes and spleen were able to prime specific CD8 T cells in a time- and dose-dependent manner. This study demonstrates, for the first time, that antigen-bearing DCs originating from the site of immunization recirculate to distant lymphoid organs and provides insights into the mechanism of distant CD8 T-cell generation by sublingual immunization.
Assuntos
Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Tecido Linfoide/imunologia , Mucosa Bucal/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Comunicação Celular/imunologia , Movimento Celular/imunologia , Células Dendríticas/metabolismo , Linfonodos/imunologia , Camundongos , Soalho Bucal , Fatores de TempoRESUMO
Glial cell line-derived neurotrophic factor (GDNF) plays a crucial role in rescuing neural crest cells from apoptosis during their migration in the foregut. This survival factor binds to the heterodimer GDNF family receptor alpha1/Ret, inducing the Ret tyrosine kinase activity. ret loss-of-function mutations result in Hirschsprung's disease, a frequent developmental defect of the enteric nervous system. Although critical to enteric nervous system development, the intracellular signaling cascades activated by GDNF and their importance in neuroectodermic cell survival still remain elusive. Using the neuroectodermic SK-N-MC cell line, we found that the Ret tyrosine kinase activity is essential for GDNF to induce phosphatidylinositol 3-kinase (PI3K)/Akt and ERK pathways as well as cell rescue. We demonstrate that activation of PI3K is mandatory for GDNF-induced cell survival. In addition, evidence is provided for a critical up-regulation of the ERK pathway by PI3K at the level of Raf-1. Conversely, Akt inhibits the ERK pathway. Thus, both PI3K and Akt act in concert to finely regulate the level of ERK. We found that Akt activation is indispensable for counteracting the apoptotic signal on mitochondria, whereas ERK is partially involved in precluding procaspase-3 cleavage. Altogether, these findings underscore the importance of the Ret/PI3K/Akt pathway in GDNF-induced neuroectodermic cell survival.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Androstadienos/farmacologia , Apoptose , Western Blotting , Caspase 3 , Caspases/metabolismo , Linhagem Celular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Citometria de Fluxo , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Potenciais da Membrana , Mitocôndrias/metabolismo , Modelos Biológicos , Mutação , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas , WortmaninaRESUMO
Hirschsprung's disease (HSCR), a frequent developmental defect of the enteric nervous system is due to loss-of-function mutations of RET, a receptor tyrosine kinase essential for the mediation of glial cell-derived neurotrophic factor (GDNF)-induced cell survival. Instead, gain-of-function Cys mutations (e.g., Cys(609), Cys(620), and Cys(634)) of the same gene are responsible for thyroid carcinoma (MEN2A/familial medullary thyroid carcinoma) by causing a covalent Ret dimerization, leading to ligand-independent activation of its tyrosine kinase. In this context, the association of Cys(609)- or Cys(620)-activating mutations with HSCR is still an unresolved paradox. To address this issue, we have compared these two mutants with the Cys(634) Ret variant, which has never been associated with HSCR, for their ability to rescue neuroectodermic cells (SK-N-MC cells) from apoptosis. We show here that despite their constitutively activated kinase, the mere expression of these three mutants does not allow cell rescue. Instead, we demonstrate that like the wild-type Ret, the Cys(634) Ret variant can trigger antiapoptotic pathways only in response to GDNF. In contrast, Cys(609) or Cys(620) mutations, which impair the terminal Ret glycosylation required for its insertion at the plasma membrane, abrogate GDNF-induced cell rescue. Taken together, these data support the idea that sensitivity to GDNF is the mandatory condition, even for constitutively activated Ret mutants, to rescue neuroectodermic cells from apoptosis. These findings may help clarify how a gain-of-function mutation can be associated with a developmental defect.