ROLE OF SUPEROXIDE ANION GENERATION IN PHAGOCYTIC BACTERICIDAL ACTIVITY - STUDIES WITH NORMAL AND CHRONIC GRANULOMATOUS DISEASE LEUKOCYTES
ROLE OF SUPEROXIDE ANION GENERATION IN PHAGOCYTIC BACTERICIDAL ACTIVITY - STUDIES WITH NORMAL AND CHRONIC GRANULOMATOUS DISEASE LEUKOCYTES
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DOI：
10.1172/jci108055
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发表时间：
1975-01-01
影响因子：
15.9
通讯作者：
RAJAGOPALAN, KV
中科院分区：
文献类型：
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作者：
JOHNSTON, RB;KEELE, BB;RAJAGOPALAN, KV
利用正常人和慢性肉芽肿病（CGD）患者的白细胞，探讨了人吞噬细胞产生超氧阴离子（O2-）（O的自由基）的能力，以及这种自由基或其衍生物在杀死吞噬细菌中的可能作用。超氧化物歧化酶，清除O2-，一贯抑制吞噬相关的硝基四氮唑蓝（NBT）的减少，表明O2-在这个过程中的参与。超氧化物歧化酶抑制了吞噬作用产生的发光，这可能与O2-有关，可能是通过其自发歧化为单线态O。正常和CGD白细胞匀浆的亚细胞组分在NADH作为底物的存在下有效地产生O2。在9例CGD患者中，完整细胞在吞噬过程中产生的O2明显减少。从母亲的白细胞被确定为携带者的X-连锁隐性CGD的中间吞噬减少NBT阐述O2-到一个中间的程度，进一步证明NBT减少和O2-生成之间的相互关系，在吞噬细胞。超氧化物歧化酶的活性，负责保护细胞免受O2-的破坏作用的酶，在正常和CGD粒细胞的匀浆中大致相等。聚丙烯酰胺电泳分离这种活动成一个次要的频带，似乎是锰的含超氧化物歧化酶与线粒体和更集中，氰化物敏感，细胞质形式的酶与电泳迁移率，对应于红细胞铜锌超氧化物歧化酶。超氧化物歧化酶抑制大肠杆菌、金黄色葡萄球菌和草绿色链球菌的吞噬杀伤作用。用去除H2 O2的过氧化氢酶观察到类似的抑制作用。两种酶都不能抑制细菌的摄入。过氧化物和O2-被认为相互作用以产生有效的氧化剂，羟基自由基（·OH）。在吞噬杀菌事件中对OH的需要可以解释这种活性对O2-和H2 O2的明显需要。与这种可能性相一致，苯甲酸盐和甘露醇，OH的清除剂，抑制吞噬细胞的杀菌活性。从O2-和·OH产生单线态O也可以解释这些发现。粒细胞产生O2-作为伴随吞噬作用发生的呼吸爆发的伴随物。O2-中固有的能量在多大程度上通过O2-本身、H2 O2、OH、单线态O或某些其它试剂转化为微生物死亡仍有待明确定义。
The capacity of human phagocytes to generate superoxide anion (O2-), a free radical of O, and a possible role for this radical or its derivatives in the killing of phagocytized bacteria were explored using leukocytes from normal individuals and patients with chronic granulomatous disease (CGD). Superoxide dismutase, which removes O2-, consistently inhibited phagocytosis-associated nitroblue tetrazolium (NBT) reduction indicating the involvement of O2- in this process. Superoxide dismutase inhibited the luminescence that occurs with phagocytosis, implicating O2- in this phenomenon, perhaps through its spontaneous dismutation into singlet O. Subcellular fractions from homogenates of normal and CGD leukocytes generated O2- effectively in the presence of NADH as substrate. O2- generation by intact cells during phagocytosis was markedly diminished in 9 patients with CGD. Leukocytes from mothers determined to be carriers of X-linked recessive CGD by intermediate phagocytic reduction of NBT elaborated O2- to an intermediate extent, further demonstrating the interrelationship between NBT reduction and O2- generation in phagocytizing cells. Activity of superoxide dismutase, the enzyme responsible for protecting the cell from the damaging effects of O2-, was approximately equal in homogenates of normal and CGD granulocytes. Polyacrylamide electrophoresis separated this activity into a minor band that appeared to be the Mn-containing superoxide dismutase associated with mitochondria and a more concentrated, cyanide-sensitive, cytosol form of the enzyme with electrophoretic mobility that corresponded to that of erythrocyte cuprozinc superoxide dismutase. Superoxide dismutase inhibited the phagocytic killing of Escherichia coli, Staphylococcus aureus and Streptococcus viridans. A similar inhibitory effect was noted with catalse which removes H2O2. Neither enzyme inhibited the ingestion of bacteria. Peroxide and O2- are believed to interact to generate the potent oxidant, hydroxyl radical (.cntdot.OH). A requirement for .cntdot.OH in the phagocytic bactericidal event might explain the apparent requirement for O2- and H2O2 for such activity. In agreement with this possibility, benzoate and mannitol, scavengers of .cntdot.OH, inhibited phagocytic bactericidal activity. Generation of singlet O from O2- and .cntdot.OH also might explain these findings. The granulocyte elaborates O2- as concomitant of the respiratory burst that occurs with phagocytosis. To what extent the energy inherent in O2- is translated into microbial death through O2- itself, H2O2, .cntdot.OH, singlet O or some other agent remains to be clearly defined.