Analysis of early calcium signals related to the differntiation of macrophages from monocytes

与巨噬细胞与单核细胞分化相关的早期钙信号分析

• 批准号: 09670056
• 负责人: ODA Shoji
• 金额: $ 1.79万
• 依托单位: Tokyo Women's Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670056/
• 关键词: MONOCYTE; MACROPHAGE; INTRACELLUlAR CALCIUM; ATP; LIPOPOLYSACCHARIDE; CD14; M-CSF; 細胞内カルシウムイオン; 細胞内Ca^<2+>; プリン受容体; リポポリサッカライド

Monocytes/macrophages (MOs/Mφs) are the multi-functional cell system that responds to inflammatory stimuli via receptors and accomplishes migration, adhesion, phagocytosis, immune cell activation, and cytotoxicity. The stimulated MOs differentiate into Mφs as they leave the circulation to various tissues and organs. In a wide variety of cells, CaィイD12+ィエD1 is the intracellular signal that couples receptor stimulation to cell activation. The present study aimed to examine the early response of MOs/Mφs to stimuli via purinoceptors and CD14 receptors and to lipopolysaccharide (LPS ; endotoxin of gram-negative bacteria) and its change during differentiation into Mφs, in terms of an increase in intracellular CaィイD12+ィエD1 concentration ([CaィイD12+ィエD1]ィイD2iィエD2). Human peripheral blood MOs were isolated by adhesion to glass dishes and cultured in vitro. [CaィイD12+ィエD1]ィイD2iィエD2 was measured in single cells using a CaィイD12+ィエD1 imaging method with the CaィイD12+ィエD1 indicator dye fura-2. The foll … More owing results were obtained during the term of the project.1) MOs/Mφs exhibited a transient [CaィイD12+ィエD1]ィイD2iィエD2 rise in response to extracellular ATP via PィイD22UィエD2 and PィイD22YィエD2 purinoceptors. The similar CaィイD12+ィエD1 response was induced by application of supernatant from tumor cells lysed by hypoosmotic treatment. A [CaィイD12+ィエD1]ィイD2iィエD2 rise occurred in MOs/Mφs in the vicinity of a single tumor cell that was attacked and permeabilized by a natural killer cell in a dish. These results support the view that MOs/Mφs respond to signal messengers discharged from damaged or dying cells to be ingested, and ATP is at least one of the messengers and causes a [CaィイD12+ィエD1]ィイD2iィエD2 rise via PィイD22UィエD2 and PィイD22YィエD2 purinoceptors.2) CD14 is known as the recognition site for LPS leading to production of cytokines. Stimulation of CD14 receptors by anti-CD14 monoclonal antibody caused [CaィイD12+ィエD1]ィイD2iィエD2 rises in MOs/Mφs in the form of damping oscillations. The signal transduction to the [CaィイD12+ィエD1]ィイD2iィエD2 rise through CD14 is interesting for future studies, since CD14 molecules are anchored in the lipid bilayer of the plasma membarne without the intracellular domain.3) LPS on its own at concentrations between 1 ng/ml and 100 μg/ml did not cause any substantial [CaィイD12+ィエD1]ィイD2iィエD2 rise in MOs/Mφs. Involvement of LPS-binding protein has been proposed in interaction between LPS and CA14.4) Macrophage-colony stimulating factor (M-CSF) produced CaィイD12+ィエD1 responses in about 45% of MOs/Mφs. MOs/Mφs differentiated to large Mφs, when cultured in the presence of M-CSF. The percentage of responding cells to anti-CD14 antibody as well as the magnitude of CaィイD12+ィエD1 responses was augmented during culture. Interestingly, about 45% of Mφs showed [CaィイD12+ィエD1]ィイD2iィエD2 rises in responses to LPS. It is likely that expression of CD14 molecules and/or the binding capacity of CD14 is enhanced during differentiation of Mφs. Less

单核细胞/巨噬细胞（MOs/Mφs）是多功能细胞系统，通过受体对炎症刺激做出反应，完成迁移、粘附、吞噬、免疫细胞激活和细胞毒作用。受刺激的 MO 在离开循环系统进入各种组织和器官时分化为 Mφ。在多种细胞中，CaィイD12+ィD1 是将受体刺激与细胞激活结合起来的细胞内信号。本研究旨在检查 MOs/Mφs 对嘌呤受体和 CD14 受体刺激以及脂多糖（LPS；革兰氏阴性菌内毒素）的早期反应及其在分化为 Mφs 过程中的变化，即细胞内 CaiiD12+ィD1 浓度的增加([CaィイD12+ィエD1]ィイD2iィD2)。通过粘附到玻璃皿上分离人外周血MO并进行体外培养。 [CaィイD12+ィエD1]ィイD2iエD2 使用CaィイD12+ィエD1 成像方法和CaィイD12+ィエD1 指示剂染料fura-2 在单细胞中测量。在项目期间获得了以下结果。1) MOs/Mφs 通过 PiiD22UiiD2 和 PiiD22YiiD2 嘌呤受体响应细胞外 ATP，表现出短暂的 [CaィイD12+ィエD1]ィイD2iィD2 上升。通过使用低渗处理裂解的肿瘤细胞的上清液诱导了类似的CaィイD12+ィD1反应。 [CaィイD12+ィエD1]ィイD2iエD2 升高发生在培养皿中被自然杀伤细胞攻击和通透的单个肿瘤细胞附近的 MOs/Mφs 中。这些结果支持这样的观点，即 MOs/Mφs 对受损或死亡细胞释放的信号信使作出反应并被摄取，并且 ATP 至少是信使之一，并通过 PiiD22UiiD2 和 PiiD22YiiD2 引起 [CaiiD12+iiiD1]iiiD2iiD2 上升嘌呤受体。2) CD14 被认为是 LPS 产生的识别位点 细胞因子。抗CD14单克隆抗体对CD14受体的刺激导致[CaィイD12+ィエD1]ィイD2iエD2以阻尼振荡的形式在MOs/Mφs中上升。通过 CD14 向 [CaィイD12+ィエD1]ィイD2iエD2 上升的信号转导对于未来的研究来说是有趣的，因为 CD14 分子锚定在血浆膜的脂质双层中，没有细胞内结构域。 3) 浓度在 1 ng/ml 和 100 μg/ml 之间的 LPS 本身不会引起任何实质性的影响。 [CaィイD12+ィエD1]ィイD2iィエD2 MOs/Mφs 上升。已提出 LPS 与 CA14 之间的相互作用涉及 LPS 结合蛋白。4) 巨噬细胞集落刺激因子 (M-CSF) 在约 45% 的 MO/Mφ 中产生 CaィイD12+ィD1 反应。当在 M-CSF 存在下培养时，MOs/Mφs 分化为大 Mφs。在培养过程中，对抗 CD14 抗体有反应的细胞百分比以及 CaiiD12+iiD1 反应的强度均得到增强。有趣的是，大约 45% 的 Mφ 显示 [CaィイD12+ィエD1]ィイD2iエD2 对 LPS 的反应有所上升。 CD14分子的表达和/或CD14的结合能力可能在Mφ的分化过程中增强。较少的

项目成果

Oshimi,Y.,Oda,S.and Miyazaki,S.: "Human monocytes and macrophages show [Ca^<2+>];rises in response to ATP and antibody against the CD14 receptor." Japanese Journal of Physiology. 48 Supplement. S43 (1998)

Oshimi,Y.、Oda,S. 和 Miyazaki,S.：“人类单核细胞和巨噬细胞表现出 [Ca^2>]；对 ATP 和抗 CD14 受体抗体的反应而升高。”

Oshimi, Y., Oda, S. and Miyazaki, S.: "Human monocytes and macrophages show [CaィイD12+ィエD1]ィイD2iィエD2 rises in response to ATP and antibody against the CD14 receptors"Jpn. J Physiol.. 48, Suppl.. S43 (1998)

Oshimi, Y.、Oda, S. 和 Miyazaki, S.：“人类单核细胞和巨噬细胞显示 [CaliD12+IeD1]IiD2iED2 响应 ATP 和针对 CD14 受体的抗体而升高”Jpn. J Physiol.. 48，增刊。 S43 (1998)

Oshimi, Y. Oda, S. and Miyazaki, S.: "Human monocytes and macrophages show(Ca^<2+>)i rises in response to ATP and antibody against the CD14 receptor"Japanese Journal of physiology. 48(supplement). 543 (1998)

Oshimi, Y. Oda, S. 和 Miyazaki, S.：“人类单核细胞和巨噬细胞显示 (Ca^2>)i 响应 ATP 和针对 CD14 受体的抗体而升高”《日本生理学杂志》。