FLOW CYTOMETRIC EVALUATION OF SUPEROXIDE GENERATION IN GRANULOCYTES OF ASTHMATIC PATIENTS

• Published in: Japanese Journal of National Medical Services Vol. 49; no. 7; pp. 547 - 553
• Main Authors: AKIYAMA, Kazuo, IKEDA, Yasuko, SHIDA, Takao, MITA, Haruhisa
• Format: Journal Article
• Language: Japanese
• Published: Japanese Society of National Medical Services 1995; 一般社団法人 国立医療学会
• Subjects: bronchial asthma; flow cytometry; superoxide; フローサイトメリー; 気管支喘息; 活性酸素
• ISSN: 0021-1699; 1884-8729
• DOI: 10.11261/iryo1946.49.547

We have examined superoxide generation from peripheral leukocytes of asthmatic patients and control subjects by flow cytometry. After loading with DCFH-DA, a fluorescent probe for superoxide generation, the blood was stimulated with suboptimal concentration of phorbol myristate acetate (PMA, 20ng/ml) at 37°C for 5, 10, or 15 minutes and the fluorescence intensity of the cells was measured by flow cytometry. When assessed by two parameters, 1) fluorescence intensity of the cells in the absence of fluorescent probe was used as a cut-off value and the difference between the positive cells with PMA stimulation and those without stimulation was defined as Δ PCN, and 2) the difference in mean fluorescence intensity (Δ MFI) between the cells with PMA stimulation and those without stimulation, superoxide generation was significantly decreased in an asthmatic group with attack and in a group receiving steroid therapy. Those findings could be partially explained by the observations that these groups showed an increased fluorescence intensity of granulocytes in the absence of PMA stimulation. These results show that spontaneous superoxide generation without PMA stimulation was already increased in granulocytes from asthmatic patients with attack and steroid therapy, suggesting that the method presented in this paper may be valuable means to evaluate conditions of superoxide generation in asthmatic patients.