RESUMO
Resistance to Leishmania major in mice is associated with the appearance of distinct T helper type 1 (Th1) and Th2 subsets. T cells from lymph nodes draining cutaneous lesions of resistant mice are primarily interferon gamma (IFN-gamma)-producing Th1 cells. In contrast, T cells from susceptible mice are principally Th2 cells that generate interleukin 4 (IL-4). Although existing evidence is supportive of a role for IFN-gamma in the generation of Th1 cells, additional factors may be required for a protective response to be maintained. A potential candidate is IL-12, a heterodimeric cytokine produced by monocytes and B cells that has multiple effects on T and natural killer cell function, including inducing IFN-gamma production. Using an experimental leishmanial model we have observed that daily intraperitoneal administration at the time of parasite challenge of either 0.33 micrograms IL-12 (a consecutive 5 d/wk for 5 wk) or 1.0 micrograms IL-12 per mouse (only a consecutive 5 d) caused a > 75% reduction in parasite burden at the site of infection, in highly susceptible BALB/c mice. Delay of treatment by 1 wk had less of a protective effect. Concomitant with these protective effects was an increase in IFN-gamma and a decrease in IL-4 production, as measured by enzyme-linked immunosorbent assay of supernatants generated from popliteal lymph node cells stimulated with leishmanial antigen in vitro. The reduction in parasite numbers induced by IL-12 therapy was still apparent at 10 wk postinfection. In addition, we observed that the administration of a rabbit anti-recombinant murine IL-12 polyclonal antibody (200 micrograms i.p. every other day for 25 d) at the time of infection to resistant C57Bl/6 mice exacerbated disease. These effects were accompanied by a shift in IFN-gamma production in vitro by antigen-stimulated lymph node cells indicative of a Th2-like response. These findings suggest that IL-12 has an important role in initiating a Th1 response and protective immunity.
Assuntos
Interleucinas/farmacologia , Leishmaniose Cutânea/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Feminino , Imunidade Inata/efeitos dos fármacos , Interferon gama/biossíntese , Interleucina-12 , Leishmania tropica , Leishmaniose Cutânea/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/farmacologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacosRESUMO
We describe the identification and characterization of a novel gene, sur-8, that positively regulates Ras-mediated signal transduction during C. elegans vulval development. Reduction of sur-8 function suppresses an activated ras mutation and dramatically enhances phenotypes of mpk-1 MAP kinase and ksr-1 mutations, while increase of sur-8 dosage enhances an activated ras mutation. sur-8 appears to act downstream of or in parallel to ras but upstream of raf. sur-8 encodes a conserved protein that is composed predominantly of leucine-rich repeats. The SUR-8 protein interacts directly with Ras but not with the Ras(P34G) mutant protein, suggesting that SUR-8 may mediate its effects through Ras binding. A structural and functional SUR-8 homolog in humans specifically binds K-Ras and N-Ras but not H-Ras in vitro.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Genes de Helmintos , Proteínas ras/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/embriologia , Sequência Conservada , Indução Embrionária , Feminino , Dosagem de Genes , Leucina , Mamíferos/genética , Proteína Quinase 1 Ativada por Mitógeno , Dados de Sequência Molecular , Mutação , Fenótipo , Ligação Proteica , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Supressão Genética , Vulva/embriologiaRESUMO
We describe evidence that a regulatory B subunit of protein phosphatase 2A (PP2A) positively regulates an RTK-Ras-MAP kinase signaling cascade during Caenorhabditis elegans vulval induction. Although reduction of sur-6 PP2A-B function causes few vulval induction defects in an otherwise wild-type background, sur-6 PP2A-B mutations suppress the Multivulva phenotype of an activated ras mutation and enhance the Vulvaless phenotype of mutations in lin-45 raf, sur-8, or mpk-1. Double mutant analysis suggests that sur-6 PP2A-B acts downstream or in parallel to ras, but likely upstream of raf, and functions with ksr-1 in a common pathway to positively regulate Ras signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Caenorhabditis elegans , Proteínas de Helminto/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Transdução de Sinais , Vulva/embriologia , Proteínas ras/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/embriologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA de Helmintos , Indução Embrionária , Feminino , Proteínas de Helminto/genética , Humanos , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Fosfatase 2 , Homologia de Sequência de AminoácidosRESUMO
Mycobacterium tuberculosis and its antigens are potent inducers of cytokine expression by mononuclear phagocytes. In this study, the ability of live M. tuberculosis to stimulate interleukin-12 (IL-12) expression by human monocytes was examined. Monocytes were purified from peripheral blood mononuclear cells by adherence and either infected with M. tuberculosis or exposed to soluble protein antigens of M. tuberculosis (purified protein derivative [PPD]). Live M. tuberculosis (10(6) to 10(7) CFU/ml) was a potent stimulus for interleukin-12 (IL-12). By using reverse transcription-PCR, p40 mRNA was detected at 3 h, peaked at 6 to 12 h, and decayed to baseline levels at 18 to 24 h following infection. Bioactive IL-12 (p70) was measured by the phytohemagglutinin blast proliferation assay and confirmed the p40 mRNA results. In contrast, soluble PPD at concentrations known to readily induce IL-1 and tumor necrosis factor alpha expression by monocytes (10 to 100 microg/ml) was a poor stimulus for IL-12 p40 mRNA expression. The different efficiencies of M. tuberculosis bacilli and PPD for IL-12 expression by monocytes was in part due to a requirement for phagocytosis. Induction of IL-12 in response to M. tuberculosis was reduced by cytochalasin D. Furthermore, phagocytosis of dead M. tuberculosis or inert 2-micron-diameter polystyrene beads by monocytes induced IL-12 p40 mRNA. In contrast, 0.5-micron-diameter beads, which can enter cells through pinocytosis, did not stimulate IL-12 expression. Functionally, IL-12 readily enhanced PPD-stimulated IFN-gamma production and CD4+ T-cell-mediated cytotoxicity by peripheral blood mononuclear cells from healthy tuberculin-positive donors but induced less enhancement when live M. tuberculosis was the antigen. These results suggest that IL-12 is upregulated as part of the early cytokine response of mononuclear phagocytes to M. tuberculosis and that the cellular events associated with phagocytosis are themselves a potent signal for IL-12 production. IL-12 released by infected macrophages in turn can further upregulate M. tuberculosis-specific CD4+ T-cell effector function.