Neuromodulation of the Antibody Response

抗体反应的神经调节

• 批准号: 6861694
• 负责人: VIRGINIA M SANDERS
• 金额: $ 36.88万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6861694
• 关键词: B lymphocyte; CD antigens; DNA binding protein; antibody formation; beta adrenergic receptor; biological signal transduction; enzyme inhibitors; enzyme linked immunosorbent assay; gel mobility shift assay; genetic enhancer element; genetic transcription; helper T lymphocyte; immunoglobulin G; immunoglobulin isotypes; immunoprecipitation; neuroimmunomodulation; norepinephrine; nuclear runoff assay; polymerase chain reaction; protein kinase A; protein localization; sympathetic nervous system; transfection; western blottings

DESCRIPTION (provided by applicant): The overall goal of this research project is to understand the mechanism by which norepinephrine (NE) and beta-2-adrenergic receptor (a2AR) stimulation regulate the Th2 cell-dependent antibody response. With the reality of bioterrorism, the need to elucidate the molecular and biochemical mechanisms by which the release of antigen- and stress-induced NE affects immune responsiveness is critical. Our laboratory has reported that NE stimulates the a2AR on a B cell to increase the level of IgG1 produced per cell, and that stimulation of CD86 (B7-2) increases this level further. Although the mechanisms by which CD40 and IL-4 receptor signaling affect IgG1 production are known, little is known about those induced by the a2AR and CD86. Preliminary data show that the level of mature IgG1 transcript, IgG1 protein, and nuclear protein binding to the 3'-IgH enhancer region of the IgH locus increase following a2AR and/or CD86 stimulation on a CD40L/IL-4-activated B cell, but that mature IgG1 transcript stability and IgG1 class switching are unaffected. Importantly, this finding dissociates the a2AR- and CD86-induced effect on the expression of the rearranged IgG1 gene from an effect on IgG1 class switching. Unlike the a2AR-activated signaling pathway, the CD86-activated signaling pathway remains unknown. We propose to test the hypothesis that a2AR and/or CD86 stimulation on a B cell increase the rate of mature IgG1 transcription through signaling intermediates that regulate 3'-IgH enhancer activity. The following specific aims are designed to test this hypothesis in vitro and in vivo by using B cells from wild type and a2AR- or CD86-deficient mice. We will determine if stimulating these receptors 1) Increases either IgG1 class switching and/or the rate of mature IgG1 transcription using real-time PCR and nuclear run-on analysis; 2) Increases the level of a DNA-binding protein at the 3'-IgH enhancer using EMSA, super shift, chromatin immunoprecipitation (CHIP), and a transient transfection gene reporter system; and 3) Increases the activation of a specific signaling pathway(s) using selective inhibitors for PKA, PKC, and MAPK. The significance of testing our hypothesis is that the findings will provide a molecular basis for the role of NE, a2AR, and CD86 stimulation in regulating the Th2 cell-dependent Ab response, thus providing a defined target for therapeutics and a potential mechanism for increasing the efficacy of vaccination protocols.

描述(申请人提供)：本研究项目的总体目标是了解去甲肾上腺素(NE)和β-2-肾上腺素能受体(A2AR)刺激调节Th2细胞依赖的抗体反应的机制。随着生物恐怖主义的现实，需要阐明抗原和应激诱导的NE释放影响免疫反应的分子和生化机制是至关重要的。我们的实验室已经报道，去甲肾上腺素刺激B细胞上的a2AR增加每个细胞产生的IgG1水平，而刺激CD86(B7-2)进一步增加这一水平。虽然CD40和IL-4受体信号影响IgG1产生的机制是已知的，但关于a2AR和CD86诱导的机制却知之甚少。初步数据显示，在CD40L/IL-4激活的B细胞上，经2AR和/或CD86刺激后，IgH基因3‘-IgH增强子区的成熟IgG1转录本、IgG1蛋白和核蛋白结合水平增加，但成熟IgG1转录本的稳定性和IgG1类转换不受影响。重要的是，这一发现将a2AR和CD86对重排的IgG1基因表达的影响与对IgG1类转换的影响分离开来。与a2AR激活的信号通路不同，CD86激活的信号通路仍然未知。我们建议检验这一假设，即对B细胞的a2AR和/或CD86刺激通过调节3‘-IgH增强子活性的信号中间产物增加成熟IgG1的转录速率。以下特定目的旨在通过使用野生型和a2AR或CD86缺陷小鼠的B细胞在体外和体内验证这一假说。我们将确定是否刺激这些受体1)增加免疫球蛋白G1类转换和/或成熟的免疫球蛋白1转录的速率通过实时定量聚合酶链式反应和核连续分析；2)增加DNA结合蛋白在3‘-免疫球蛋白增强子的水平使用EMSA，超级移位，染色质免疫沉淀(芯片)，和瞬时基因报告系统；以及3)增加特定的信号通路(S)的激活使用选择性的抑制蛋白激酶A，蛋白激酶C，和丝裂原活化蛋白激酶。验证我们假设的意义在于，这些发现将为NE、a2AR和CD86刺激在调节Th2细胞依赖的抗体反应中的作用提供一个分子基础，从而为治疗提供一个明确的靶点，并为提高疫苗接种方案的有效性提供一个潜在的机制。