Neuromodulation of the Antibody Response

描述（由申请人提供）：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（α 2 AR）刺激调节Th 2细胞依赖性抗体应答的机制。随着生物恐怖主义的现实，需要阐明的分子和生化机制，抗原和应力诱导NE的释放影响免疫反应是至关重要的。我们的实验室已经报道NE刺激B细胞上的α 2 AR以增加每个细胞产生的IgG 1的水平，并且刺激CD 86（B7-2）进一步增加该水平。虽然CD 40和IL-4受体信号传导影响IgG 1产生的机制是已知的，但对α 2 AR和CD 86诱导的机制知之甚少。初步数据显示，在CD 40 L/IL-4活化的B细胞上，α 2 AR和/或CD 86刺激后，成熟IgG 1转录物、IgG 1蛋白和与IgH基因座的3 ′-IgH增强子区域结合的核蛋白的水平增加，但成熟IgG 1转录物稳定性和IgG 1类别转换不受影响。重要的是，这一发现将α 2 AR和CD 86诱导的对重排IgG 1基因表达的影响与对IgG 1类别转换的影响分离。与a2 AR活化的信号传导途径不同，CD 86活化的信号传导途径仍然未知。我们建议测试这样的假设：对B细胞的a2 AR和/或CD 86刺激通过调节3 '-IgH增强子活性的信号中间体来增加成熟IgG 1转录的速率。设计以下具体目的以通过使用来自野生型和α 2 AR-或CD 86-缺陷小鼠的B细胞在体外和体内检验该假设。我们将确定刺激这些受体是否1）使用实时PCR和核连续分析增加IgG 1类别转换和/或成熟IgG 1转录的速率; 2）使用EMSA、超级移位、染色质免疫沉淀（CHIP）和瞬时转染基因报告系统增加3 '-IgH增强子处的DNA结合蛋白的水平;和3）使用PKA、PKC和MAPK的选择性抑制剂增加特异性信号传导途径的活化。检验我们的假设的意义在于，这些发现将为NE、α 2 AR和CD 86刺激在调节Th 2细胞依赖性Ab应答中的作用提供分子基础，从而为治疗提供确定的靶点和增加疫苗接种方案功效的潜在机制。

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.