RESUMO
We have investigated the role of the mitogen-activated protein kinase (MAPK) pathway in the differentiation of CD4+ and CD8+ T cells by looking specifically at the effects of inhibitors of MAPK-activating enzyme, MAPK/extracellular signal-related kinase (ERK) kinase (MEK), during the positive selection step from double-positive to single-positive (SP) thymocytes. Using a variety of transgenic/knockout mouse strain combinations that fail to differentiate individual lineages of SP thymocytes together with genetically engineered F(ab')2 reagents that induce maturation preferentially to either the CD4 or CD8 subpopulations, we show that induction of CD4 differentiation cells is highly sensitive to levels of MEK inhibition that have no effect on CD8 maturation. In addition, the presence of MEK inhibitor is able to modify signals that normally induce CD4 differentiation to instead promote CD8 differentiation. Finally, we show that continuous culture in the presence of inhibitor interferes with TCR up-regulation in SP thymocytes, suggesting that MAPK signaling may be involved in final maturation steps for both lineages. These data indicate that there is discrimination in the biochemical pathways that are necessary to specify CD4 and CD8 lineage commitment and can reconcile previously conflicting reports on the influence of MAPK activation in commitment and maturation of thymocytes.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD8-Positivos/enzimologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Transdução de Sinais/imunologia , Animais , Complexo CD3/imunologia , Complexo CD3/fisiologia , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , Feminino , Flavonoides/farmacologia , Soros Imunes/farmacologia , Fragmentos de Imunoglobulinas/fisiologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Transdução de Sinais/efeitos dos fármacos , Timo/citologia , Timo/imunologiaRESUMO
The association of cyclosporin A (CsA) immunosuppression with inhibition of transcription factor-dependent lymphokine gene activation formed the basis of our decision to investigate nuclear-associated Cyp isoforms. Immunofluorescence microscopy of mouse macrophages cell line with a monoclonal antibody mAb7F1 raised against CypA shows a co-localisation of CypA in the nucleus and in the cytosol. Nuclear CypA binds to DNA in a zinc ion-dependent manner, in contrast to recombinant CypB. Peptidyl-prolyl cisltrans isomerase (PPIase) activity of nuclear CypA is inhibited by zinc ions. The zinc inhibited CypA does not bind cyclosporin A (CsA). We suggest that nuclear Cyp in complex with zinc ions recognizes DNA sequences and is involved in transcription modulating processes.
