RESUMO
The serine-threonine mitogen-activated protein kinase (MAPK) family includes extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 kinases. In NK cells, spontaneous or Ab-mediated recognition of target cells leads to activation of an ERK-2 MAPK-dependent biochemical pathway(s) involved in the regulation of NK cell effector functions. Here we assessed the roles of p38 and JNK MAPK in NK cell-mediated cytotoxicity. Our data indicate that p38 is activated in primary human NK cells upon stimulation with immune complexes and interaction with NK-sensitive target cells. FcgammaRIIIA-induced granule exocytosis and both spontaneous and Ab-dependent cytotoxicity were reduced in a dose-dependent manner in cells pretreated with either of two specific inhibitors of this kinase. Target cell-induced IFN-gamma and FcgammaRIIIA-induced TNF-alpha mRNA accumulation was similarly affected under the same conditions. Lack of inhibition of NK cell cytotoxicity in cells overexpressing an inactive form of JNK1 indicates that this kinase, activated only upon FcgammaRIIIA ligation, does not play a significant role in cytotoxicity. These data underscore the involvement of p38, but not JNK1, in the molecular mechanisms regulating NK cell cytotoxicity.
Assuntos
Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/imunologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Actinas/metabolismo , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linhagem Celular , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Citocinas/metabolismo , Grânulos Citoplasmáticos/imunologia , Testes Imunológicos de Citotoxicidade , Ativação Enzimática/imunologia , Exocitose/imunologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Células K562 , Células Matadoras Naturais/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Receptores de IgG/fisiologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The role of epitope expression levels in CD8+ T cell priming has been controversial. Yet this parameter is of great importance in the design of rational approaches to optimize CTL responses to a variety of pathogens. In this paper we examine the influence of epitope production on CD8+ T cell priming by exploiting a system that allows a 200-fold range of cell surface epitope expression in vitro with a fixed dose of vaccinia virus. Our results demonstrate that, with the exception of a notable decline at the highest level of epitope, the magnitude of the responding CTL population generated in vivo following equivalent viral infections is essentially proportional to epitope density.
Assuntos
Antígenos Virais/imunologia , Epitopos de Linfócito T/biossíntese , Proteínas de Ligação a RNA , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Animais , Antígenos de Superfície/metabolismo , Antígenos Virais/biossíntese , Antígenos Virais/genética , Citotoxicidade Imunológica , Relação Dose-Resposta Imunológica , Proteínas do Ovo/genética , Proteínas do Ovo/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/metabolismo , Feminino , Hibridomas , Vírus da Influenza A/imunologia , Células L , Ativação Linfocitária , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteínas do Nucleocapsídeo , Nucleoproteínas/biossíntese , Nucleoproteínas/genética , Nucleoproteínas/imunologia , Ovalbumina/genética , Ovalbumina/imunologia , Fragmentos de Peptídeos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/virologia , Vaccinia virus/genética , Vaccinia virus/imunologia , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
Extracellular signal-regulated kinases (ERK, also known as mitogen-activated protein kinases) are serine-threonine kinases transducing signals elicited upon ligand binding to several tyrosine kinase-associated receptors. We have reported that ERK2 phosphorylation and activation follows engagement of the low affinity receptor for the Fc portion of IgG (CD16) on NK cells, and is necessary for CD16-induced TNF-alpha mRNA expression. Here, we analyzed the involvement of ERK in NK cell-mediated cytotoxicity and IFN-gamma expression induced upon stimulation with targets cells, coated or not with Abs. Our data indicate that, as with immune complexes, ERK2 phosphorylation occurs in human primary NK cells upon interaction with target cells sensitive to granule exocytosis-mediated spontaneous cytotoxicity, and that this regulates both target cell- and immune complex-induced cytotoxicity and IFN-gamma mRNA expression. A specific inhibitor of mitogen-activated protein kinase kinase reduced both spontaneous and Ab-dependent cytotoxicity in a dose-dependent manner involving, at least in part, inhibition of granule exocytosis without affecting effector/target cell interaction and rearrangement of the cytoskeleton proteins actin and tubulin. Involvement of ERK in the regulation of Ca2+-dependent cell-mediated cytotoxicity was confirmed, using a genetic approach, in primary NK cells infected with a recombinant vaccinia virus encoding an ERK inactive mutant. These data indicate that the biochemical pathways elicited in NK cells upon engagement of receptors responsible for either spontaneous or Ab-dependent recognition of target cells, although distinct, utilize ERK as one of their downstream molecules to regulate effector functions.