| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1990: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
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
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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
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