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

吞噬细胞如中性粒细胞和单核细胞从分子氧中产生超氧阴离子（0_2^-）。超氧阴离子自发或酶促转化为其他活性氧，如H_2O_2， ^。哦,HClO。这些反应物是杀死细菌所必需的。位于细胞表面的细胞色素b558被认为在超氧化物生成中起重要作用。我们已在人中性粒细胞中建立了抗细胞色素b558的单克隆抗体（MoAb） 7D5。我们的免疫细胞化学研究显示细胞色素在一些人外周血淋巴细胞上表达。在这项研究中，我们用fitc结合的针对淋巴细胞亚群的特异性抗体和pe标记的MoAb， 7D5对外周血淋巴细胞进行双重染色，以确定含有抗原的种类。用流式细胞术（FCM）同时检测红、绿荧光强度。发现细胞色素b558在大多数正常外周血B淋巴细胞表面表达。细胞色素未在来自x -联性慢性肉芽肿病（CGD）患者的外周T淋巴细胞、自然杀伤细胞或外周淋巴细胞表面发现。（这些患者的中性粒细胞不含细胞色素b558。）为了确定细胞色素b558在b淋巴形成的哪个阶段在细胞表面表达，我们用各种细胞表面标记物（抗cd10、抗igm、PCA-1）和抗细胞色素b558抗体7D5对正常骨髓细胞进行染色，并进行流式细胞术检测。大部分sIgM^+淋巴细胞被7D5抗体染色，大部分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细胞表面，但在B细胞早期出现。在b细胞向浆细胞的终末分化过程中，它从细胞表面消失。（Blood 75,458,1990）细胞色素b558由大小亚基组成。我们构建了针对大亚基和小亚基c端区域的序列特异性抗体，分别命名为alphalc和alphasc。利用这些抗体对纯化的B淋巴细胞进行Western blot。结果表明B淋巴细胞中存在细胞色素b558的大小亚基。分子量（90K宽频带和23K）与中性粒细胞相同。经PMA刺激后，至少一半的外周血B淋巴细胞产生超氧阴离子，sds敏感的NBT试验检测到。而B淋巴细胞产生的超氧化物少于相应中性粒细胞的1%。少

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