RESUMO
In this study, we demonstrate that HIV-1 Tat protein is able to induce IL-10 and TNF-alpha in human macrophages. We show that N-terminal Tat 1-45 fragment initiates the PKC pathway by acting at the membrane. Inhibition of PKC pathway, by chemical inhibitors or after PMA treatment, abolishes both IL-10 and TNF-alpha production. Among the eight PKC isoforms present in macrophages, we show that only PKC-betaIotaIota and -delta are activated by Tat or Tat 1-45 in human macrophages. However, their selective inhibition affects only IL-10 production. Downstream of PKC, Tat activates the MAP kinases p38 and ERK1/2 and the transcription factor NF-kappaB. Using chemical inhibitors we show that (i) both ERK1/2 MAP kinase and NF-kappaB transcription factor play an important role in IL-10 and TNF-alpha production, in macrophages stimulated by Tat. However, p38 MAP kinase seems to be involved only in IL-10 and not TNF-alpha production.
Assuntos
Interleucina-10/biossíntese , Macrófagos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteína Quinase C/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Produtos do Gene tat do Vírus da Imunodeficiência Humana/imunologia , Western Blotting , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Humanos , Isoenzimas/imunologia , Isoenzimas/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Proteína Quinase C-delta/imunologia , Proteína Quinase C-delta/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We have characterized the humoral and cellular immune responses of BALB/c mice immunized with HIV-1 Nef regulatory protein encapsulated in poly(DL-lactide-co-glycolide) PLG particles. Three groups of mice were immunized with Nef PLG, Nef in the presence of complete Freund's adjuvant (CFA) or Nef alone in PBS. When titers were compared 7 months after the last injection, anti-Nef titers in mice immunized with Nef PLG were still close to the maximum, whereas a significant decrease was observed in mice immunized with Nef alone (five times lower) or with Nef in CFA (three times lower). These results indicate that Nef PLG is at least a similar or better vector/adjuvant than Nef in CFA concerning the duration of the humoral immune response. The analysis of cytokine profiles (IL-5 and IL-10) and the isotypic patterns of anti-Nef antibodies (predominantly IgG1), in the three groups of mice, indicated a predominant Th2 immune response. Using synthetic peptides covering the entire sequence of Nef, we identified at least three linear epitopes within sequences 32-64, 118-167 and 185-205 in the sera of mice immunized with Nef PLG or Nef CFA. In contrast, anti-Nef antibodies against Nef alone failed to recognize synthetic peptides, indicating that the majority of anti-Nef antibodies were primarily directed against conformational epitopes. We then examined the ability of Nef PLG to prime for the antigen-specific proliferative responses in vitro. The data obtained indicate the presence of both B-cell and T-cell epitopes in the C-terminal fragment of the protein after immunization of mice with Nef encapsulated in PLG particles.