Novel mechanisms of Ca2+ signaling in B lymphocytes

B 淋巴细胞中 Ca2 信号传导的新机制

• 批准号: 6923251
• 负责人: BRUCE D FREEDMAN
• 金额: $ 39.63万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6923251
• 关键词: B cell receptor; B lymphocyte; antigen presentation; biological signal transduction; calcium ion; cell biology; gel mobility shift assay; genetically modified animals; immunity; laboratory mouse; leukocyte activation /transformation; voltage /patch clamp; western blottings

DESCRIPTION (provided by applicant): B lymphocytes play a critical role in host defenses; however critical mechanisms of their activation and differentiation are poorly understood. For example, intracellular Ca2+ is recognized as being central, yet relatively little is known about how it is regulated. Much of what we know about Ca2+ in lymphocytes has been learned from studies of T cells. For example, Ca2+-Release Activated Calcium (CRAC) channels, which are believed to be their major, and possibly only, antigen receptor-operated Ca2+ channel in T cells, has been studied in great detail, but it has not yet been described in primary B cells. In fact, limited previous studies and our preliminary data demonstrate that the mechanisms of Ca2+ signaling in B cells are more complex than described for T cells, and whether CRAC channels play a similar indispensable role in B cell immunological functions now seems unlikely. We show that B cells utilize CRAC channels, but also additional Ca2+-permeant Non-Selective Cation Channels (NSCC). We further demonstrate that these NSCCs are activated by stimuli associated with innate immune responses including viral and bacterial agonists of Toll like receptors (TLR) called Pathogen-Associated Molecular Patterns (PAMPs), and by mechanical forces. These responses are not observed in T cells. We speculate that these dissimilarities between B and T cells reflect the fundamental difference in the way that these two lymphocyte populations recognize antigens. B cells recognize antigens directly through contact with immunoglobulin molecules at their surface. In contrast, antigen recognition by T cells is indirect and depends upon permissive interactions with antigen presenting cells (APC's). As a safeguard against autoimmunity, recognition of antigen alone is not sufficient to trigger activation; a second signal indicating the presence or absence of danger - TLR ligands such as double-stranded RNA, LPS, LTA, unmethylated CpG DNA that are unique to pathogens - is required. Much of the decision making for a T cell is made by the APC, while the B cell is on its own in terms of integrating costimulatory information and this will require the more complex signaling that we have uncovered. One of the central underlying ideas of this application, therefore, is that appropriate responses of B cells will reflect the coordinated activation of distinct Ca2+ permeant (NSC and CRAC) channels by the overlapping pathways activated by the BCR and innate stimuli. We will use single cell patch clamp recording and calcium imaging methods to biophysically define these channels activated by BCR and innate stimuli, but also a novel modification of this approach to unravel the complex intracellular signaling pathways that coordinate their activation and functions. Our results will have fundamental importance for understanding the basis of immune competence, immune deficiency, and autoimmunity.

描述（由申请人提供）：B淋巴细胞在宿主防御中起关键作用;然而，对其活化和分化的关键机制知之甚少。例如，细胞内Ca 2+被认为是中心的，但对它是如何调节的知之甚少。我们对淋巴细胞中Ca 2+的了解大部分来自对T细胞的研究。例如，Ca 2+释放激活钙（CRAC）通道被认为是T细胞中主要且可能是唯一的抗原受体操纵的Ca 2+通道，已被非常详细地研究，但尚未在原代B细胞中描述。事实上，有限的先前研究和我们的初步数据表明，B细胞中的Ca 2+信号传导机制比T细胞中描述的更为复杂，CRAC通道是否在B细胞免疫功能中发挥类似的不可或缺的作用现在似乎不太可能。我们表明，B细胞利用CRAC通道，但也有额外的Ca 2+渗透性非选择性阳离子通道（NSCC）。我们进一步证明，这些神经干细胞被激活的刺激与先天性免疫反应，包括病毒和细菌激动剂的Toll样受体（TLR）称为病原体相关的分子模式（PAMP），并通过机械力。这些反应在T细胞中没有观察到。我们推测，B和T细胞之间的这些差异反映了这两种淋巴细胞群体识别抗原的方式的根本差异。B细胞通过与其表面的免疫球蛋白分子接触直接识别抗原。相反，T细胞的抗原识别是间接的，并且依赖于与抗原呈递细胞（APC）的容许相互作用。作为对自身免疫的保护措施，单独识别抗原不足以触发激活;需要指示危险存在或不存在的第二个信号- TLR配体，如双链RNA，LPS，LTA，病原体特有的未甲基化CpG DNA。T细胞的大部分决策是由APC做出的，而B细胞在整合共刺激信息方面是独立的，这将需要我们发现的更复杂的信号传导。因此，本申请的中心基本思想之一是，B细胞的适当反应将反映不同的Ca 2+渗透（NSC和CRAC）通道通过由BCR和先天刺激激活的重叠途径的协调激活。我们将使用单细胞膜片钳记录和钙成像方法来生物病理学地定义这些由BCR和先天刺激激活的通道，而且还对这种方法进行了新的修改，以解开协调其激活和功能的复杂细胞内信号传导通路。我们的研究结果对于理解免疫能力、免疫缺陷和自身免疫的基础具有根本的重要性。