The Expression of Cytochrome b558 in Human Peripheral Lymphocytes and its role in Lymphocytes

细胞色素b558在人外周淋巴细胞中的表达及其在淋巴细胞中的作用

• 批准号: 01570670
• 负责人: KOBAYASHI Sonoko
• 金额: $ 1.28万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01570670/
• 关键词: Cytochrome b558; B lymphocytes; Active oxygen (Superoxide); Flow cytometry; CGD; NBT test; B系株化細胞; surface IgM; ケミルミネッセンス; b型チトクロ-ム; リンパ球分化; ス-パ-オキサイド

Phagocytes such as neutrophils and monocytes produce superoxide anion (0_2^-) from molecular oxygen. Superoxide anion is converted spontaneously and/or enzymatically to other active oxygen species such as H_2O_2, ^.OH, HClO. These reactants are essential for the killing of bacteria. Cytochrome b558, located on the cell surface, is considered to play an important role in superoxide generation.Previously we have established the monoclonal antibody (MoAb) 7D5 against the cytochrome b558 in human neutrophils. Our immunocytochemical studies revealed that the cytochrome was expressed on some human peripheral lymphocytes.In this study we double-stained the peripheral lymphocytes with FITC-conjugated specific antibodies against lymphocyte subsets and a PE-labelled MoAb, 7D5 to determine the species which contained the antigen. The red and green fluorescence intensities was measured simultaneously by flow cytometry (FCM). It was found that cytochrome b558 was expressed on the cell surface of mo … More st normal periperal B lymphocytes. The cytochrome was not found on the surface of peripheral T lymphocytes, natural killer cells, or peripheral lymphocytes derived from patients with X-linked Chronic Granulomatous disease (CGD). (The neutrophils from these patients do not contain cytochrome b558.)To determine at what stages in B-lymphopoiesis cytochrome b558 is expressed on the cell surface, we stained normal bone marrow cells with various cell surface markers (anti-CD10, anti-IgM, PCA-1) and the anti-cytochrome b558 antibody 7D5, and examined them flow-cytometrically. Most sIgM^+ lymphocytes were stained with the antibody 7D5, and most sIgM^- lymphocytes were not stained. In other words cytochrome b558 was not present on the surface of pre-pre B cells or pre-B cells, but did appear at the early B-cell stage. It disappeared from the B-cell surface during terminal differentiation to plasma cells. (Blood 75, 458, 1990)Cytochrome b558 is composed of large and small subunits. We raised sequence specific antibodies against C-terminal regions of large and small subunits, which were designated asalphaLc andalphaSc. Using these antibodies Western blot of purified B lymphocyte fraction was performed. The results indicated B lymphocytes have both large and small subunits of cytochrome b558. The molecular weights (90K broad band and 23K) were the same as those in neutrophils.Upon stimulation with PMA, at least half of peripheral B lymphocytes was found to generate superoxide anion which was detected by SDS-sensitive NBT test. The amount of superoxide generated by B lymphocytes was, however, less than 1% of that of corresponding neutrophils. Less

嗜中性粒细胞和单核细胞等吞噬细胞从分子氧中产生超氧阴离子（0_2^-）。超氧阴离子可自发和/或酶促转化为其它活性氧物种，如H_2O_2、H_2OH、HClO等。这些反应物对于杀死细菌是必不可少的。细胞色素b558位于细胞表面，在超氧阴离子的产生中起重要作用，我们已经建立了抗人中性粒细胞细胞色素b558的单克隆抗体7 D5。免疫细胞化学研究表明，细胞色素在某些人外周血淋巴细胞上有表达，本研究用FITC标记的淋巴细胞亚群特异性抗体和PE标记的单克隆抗体7 D5对人外周血淋巴细胞进行双重染色，以确定含有该抗原的种类。用流式细胞仪（FCM）同时测定红、绿色荧光强度。细胞色素b558表达于细胞膜表面。 ...更多信息 正常人外周B淋巴细胞。在外周T淋巴细胞、自然杀伤细胞或来自X连锁慢性肉芽肿病（CGD）患者的外周淋巴细胞表面未发现细胞色素。(The来自这些患者的嗜中性粒细胞不含细胞色素b558。为了确定细胞色素b558在B淋巴细胞生成的哪个阶段在细胞表面上表达，我们用各种细胞表面标记物（抗CD 10、抗IgM、PCA-1）和抗细胞色素b558抗体7 D5对正常骨髓细胞进行染色，并对其进行流式细胞术检查。大多数sIgM^+淋巴细胞被抗体7 D5染色，大多数sIgM^-淋巴细胞未被染色。换句话说，细胞色素b558不存在于前-前B细胞或前-B细胞的表面上，但确实出现在早期B细胞阶段。在终末分化为浆细胞的过程中，它从B细胞表面消失。（Blood 75，458，1990）细胞色素b558由大小亚基组成。我们针对大小亚基的C-末端区域提出了序列特异性抗体，它们被命名为α Lc和α Sc。使用这些抗体进行纯化的B淋巴细胞级分的Western印迹。结果表明，B淋巴细胞具有细胞色素b558的大小亚基。经PMA刺激后，至少有一半的外周B淋巴细胞产生超氧阴离子，SDS敏感的NBT试验检测到超氧阴离子。然而，由B淋巴细胞产生的超氧化物的量小于相应的中性粒细胞的1%。少

项目成果

Sonoko Kobayashi: "Occurrence of cytochrome b558 in B-cell Lineage of Human Lymphocytes" Blood. 75. 458-461 (1990)

Sonoko Kobayashi：“细胞色素 b558 在人类淋巴细胞 B 细胞谱系中的出现”血液。

Kobayashi S.: "Occurrencc of cytochrome b558 in human B cell linecye of human lymphocytes" Blood. 75. 458-461 (1990)

Kobayashi S.：“细胞色素 b558 在人淋巴细胞的人 B 细胞系中的出现”血液。

Muhlbach TJ.: "Analysis of vorious types of chronic granulomatous disease with the monoclonal antibody 705 directeu the small subunit of cytochrome b558." British J.Haemotol.

Muhlbach TJ.：“使用细胞色素 b558 小亚基的单克隆抗体 705 分析各种类型的慢性肉芽肿性疾病。”

Muhlbach TJ.: "Analysis of various types of chronic granulomatous disease with the monoclonal antibody 705 directeu the small subunit of cytochrome b558." British J.Haemotol.

Muhlbach TJ.：“使用细胞色素 b558 小亚基的单克隆抗体 705 分析各种类型的慢性肉芽肿性疾病。”

Friedrich-Ernst Maly: "Superoxide-dependent Nitroblue Tetrazolium Reduction and Expression of Cytochrome b-245 Components by Human Tonsillar B Lymphocytes and B Cell Lines" J.Immunol.142. 1260-1267 (1989)

Friedrich-Ernst Maly：“人扁桃体 B 淋巴细胞和 B 细胞系的超氧化物依赖性硝基蓝四唑还原和细胞色素 b-245 成分的表达”J.Immunol.142。