Takao S, Smith E H, Wang D, Chan C K, Bulkley G B, Klein A S
Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
Am J Physiol. 1996 Oct;271(4 Pt 1):C1278-84. doi: 10.1152/ajpcell.1996.271.4.C1278.
This study was designed to quantify the role of reactive oxygen metabolites (ROMs) in two distinct components of murine peritoneal macrophage activity, phagocytosis and killing, and to discriminate quantitatively the degree to which each component is dependent on NADPH oxidase and/or xanthine oxidase. A fluorochromatic vital staining technique was modified to simultaneously quantify phagocytosis and microbicidal activity of macrophages incubated with Candida parapsilosis targets. To determine the role of ROMs, macrophages were preincubated with free radical scavengers [superoxide dismutase (SOD) and/or catalase] or with selective inhibitors of xanthine oxidase (XO, e.g., allopurinol) or NADPH oxidase [diphenyleneiodonium (DPI)]. Phagocytosis was not affected by treatment of macrophages with SOD, catalase, allopurinol, or DPI. Candidacidal activity, however, was inhibited by SOD, allopurinol, or DPI. The inhibitory effects of DPI and allopurinol were additive. Histochemical and biochemical assays demonstrated substantial quantities of XO in murine peritoneal macrophages. The findings suggest that the generation of ROMs by XO- and NADPH oxidase-dependent pathways are each important for phagocytic killing by murine peritoneal macrophages.
本研究旨在量化活性氧代谢产物（ROMs）在小鼠腹腔巨噬细胞活性的两个不同组成部分（吞噬作用和杀伤作用）中的作用，并定量区分每个组成部分依赖于烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶和/或黄嘌呤氧化酶的程度。对一种荧光活体染色技术进行了改进，以同时量化与近平滑念珠菌靶标孵育的巨噬细胞的吞噬作用和杀菌活性。为了确定ROMs的作用，巨噬细胞先用自由基清除剂[超氧化物歧化酶（SOD）和/或过氧化氢酶]或黄嘌呤氧化酶（XO，例如别嘌呤醇）或NADPH氧化酶的选择性抑制剂[二苯基碘鎓（DPI）]进行预孵育。用SOD、过氧化氢酶、别嘌呤醇或DPI处理巨噬细胞对吞噬作用没有影响。然而，杀念珠菌活性受到SOD、别嘌呤醇或DPI的抑制。DPI和别嘌呤醇的抑制作用是相加的。组织化学和生化分析表明，小鼠腹腔巨噬细胞中存在大量XO。这些发现表明，XO和NADPH氧化酶依赖性途径产生的ROMs对小鼠腹腔巨噬细胞的吞噬杀伤作用均很重要。
