RESUMO
We previously identified a 65-kDa protein (p65) that was phosphorylated in activated macrophages. It has turned out to be a murine homologue of human L-plastin, which was identified as a novel protein in human cancer cells. p65/L-plastin is characterized by a series of Ca(2+)-, calmodulin-, and actin-binding domains, and is thought to play a crucial role in leukocytes and cancer cells. We have expressed a recombinant (r) p65/L-plastin in Escherichia coli that binds to beta-actin and prepared high-titer antibodies using large amounts of the protein as immunogen. Anti-rp65/L-plastin antibodies recognize native p65/L-plastin as well as rp65/L-plastin and have enabled us to detect the fine structures of intracellular p65/L-plastin, and it was found that its localization was extensively changed by stimulation with bacterial components. We further developed an enzyme-linked immunosorbent assay system and a flow cytometry method using these reagents, which made it possible to measure antibodies, including autoantibodies, against p65/L-plastin and to evaluate the maturation-dependent expression of the protein in leukocytes.
Assuntos
Anticorpos/imunologia , Clonagem Molecular , Fosfoproteínas/imunologia , Animais , Anticorpos/análise , Anticorpos/genética , Formação de Anticorpos , Proteínas do Citoesqueleto , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Citometria de Fluxo , Imuno-Histoquímica , Leucócitos/química , Camundongos , Proteínas dos Microfilamentos , Fosfoproteínas/análise , Fosfoproteínas/genética , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
IL-12 is a heterodimer composed of p40 and p35 and is a key cytokine that functions to protect the host from viral and microbial infections. IL-12 links the innate immune system with the acquired immune system during infection, and induces differentiation of type 1 T cells that play an important role in the eradication of microbes. The induction of the IL-12 p40 gene is regulated by NF-kappaB in the presence of IFN-gamma. IFN-gamma induces IFN regulatory factor-1 (IRF-1), which in turn induces the transcription of the IL-12 p40 gene. However, the IRF-1 binding site in the promoter region of the IL-12 p40 gene has not yet been formally determined. In the present study, we demonstrated that IRF-1 directly binds to the IL-12 p40 gene promoter and identified its binding site. The IRF-1 binding site in the promoter region of the IL-12 p40 gene is shown to be in the -72 to -58 area of the 5'-upstream region. The -63 to -61 position is the critical site within this region for the binding of IRF-1 to the IL-12 p40 gene promoter. While IFN-gamma must be present for IL-12 p40 gene induction, the p35 gene is strongly induced by LPS, even in the absence of IFN-gamma, and therefore the induction of the p35 gene is IRF-1 independent.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interferon gama/metabolismo , Interleucina-12/genética , Interleucina-12/metabolismo , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas/imunologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Regiões 5' não Traduzidas/fisiologia , Animais , Sequência de Bases , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Células Cultivadas , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/imunologia , Fator Regulador 1 de Interferon , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Interleucina-12/biossíntese , Subunidade p35 da Interleucina-12 , Subunidade p40 da Interleucina-12 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Fosfoproteínas/fisiologia , Ligação Proteica/genética , Ligação Proteica/imunologia , Subunidades Proteicas/biossíntese , RNA Mensageiro/biossíntese , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transcrição Gênica/imunologia , Ativação Transcricional , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Pathogenic infections lead to activation of innate immunity followed by induction of a type 1 T cell subset and, therefore, provide a good model to evaluate when T cells commit to type 1 T cells. Here we show a two-step mechanism of T cell subset commitment during pathogenic infection. The first step is mediated by the basal function of macrophage/dendritic cells and is antigen independent. This step modulates the committed precursor frequency of T cell subsets and influences the expression of T-box expressed in T cells (T-bet) and GATA-3 genes. IL-12 and NK cells are not required for this step. The second step requires antigenic stimulation of T cells together with IL-12 or IL-4, and influences on the expression of T-bet and GATA-3. We propose a two-step T cell subset commitment pathway based on these observations. Therefore, pathogenic infections influence functional T cell commitment before T cells encounter nominal antigen.
