RESUMO
Use of mice in which individual PI3K isoforms have been deleted or mutated by gene targeting, has determined that PI3Kgamma provides a key migratory signal for T lymphocyte migration. Since PI3Kgamma can be a dispensable signal for directional migration of human T cells, we have adopted a pharmacological and siRNA strategy to assess the contribution of individual PI3K isoforms to chemokine-stimulated migration of human T cells. The broad spectrum PI3K isoform inhibitor Ly294002 inhibits CXCL12-stimulated migration of freshly isolated T lymphocytes. Use of second generation inhibitors that can discriminate between individual PI3K isoforms, revealed that PI3Kgamma was the major contributor to CXCL12-induced migration and PI3K/Akt signaling (as assessed by S6 phosphorylation). Non-viral delivery of siRNA targeting class I (PI3Kgamma), class II (PI3KC2alpha and PI3KC2beta) and class III PI3Ks, followed by 3 days ex vivo culture, reduces the levels of isoform mRNA, but is insufficient to impact on cell migration responses. However, ex vivo maintenance of T cells alone, independently of siRNA treatment, resulted in the migratory response of T cells toward CXCL12 becoming insensitive to Ly294002. Remarkably, random migration remains sensitive to Ly294002. This study therefore, highlights that the migratory response of freshly isolated human T cells is dependent on PI3K signals that are provided predominantly by PI3Kgamma. However, the role of PI3K in cell migration is context-dependent and diminishes during ex vivo maintenance.
Assuntos
Técnicas de Cultura de Células , Quimiotaxia de Leucócito , Fosfatidilinositol 3-Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Linfócitos T/metabolismo , Transfecção/métodos , Células Cultivadas , Quimiocina CXCL12/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Classe Ib de Fosfatidilinositol 3-Quinase , Relação Dose-Resposta a Droga , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Fatores de TempoRESUMO
Cannabinoids have long been proposed to affect the immune system, especially as one of the cannabinoid receptors, the cannabinoid receptor-2 (CB(2)R) has been found almost exclusively on immune cells. Here, using human in vitro activated peripheral blood-derived T lymphocytes we investigated the long-term changes in cannabinoid receptor protein expression following cellular activation and the effects of cannabinoids on migration. We report that resting T lymphocytes do not detectably express either the cannabinoid receptor-1 (CB(1)R) or CB(2)R at the protein level. However, CB(2)R protein expression is upregulated in a biphasic manner in T lymphocytes following activation by superantigen. The cannabinoids 2-AG and JWH-133 were found to elicit activation of downstream biochemical effectors (as assessed by the phosphorylation of the ERK1/2 MAP kinases). Neither 2-AG nor JWH-133 induced chemotaxis in day 5 activated T lymphocytes, when receptor expression was at its highest. Interestingly, both 2-AG and JWH-133 inhibited CXCL12-induced chemotaxis, suggesting a modulatory role for cannabinoids in activated T lymphocytes.