Hampton M B, Kettle A J, Winterbourn C C
Department of Pathology, Christchurch School of Medicine, New Zealand.
Infect Immun. 1996 Sep;64(9):3512-7. doi: 10.1128/iai.64.9.3512-3517.1996.
We have used a quantitative assay that measures independent rate constants for phagocytosis and killing of Staphylococcus aureus to investigate the involvement of superoxide and myeloperoxidase in bacterial killing by human neutrophils. To inhibit superoxide-dependent processes, superoxide dismutase was cross-linked to immunoglobulin G and the conjugate was attached to the surface of S. aureus via protein A in its cell wall. Myeloperoxidase was inhibited with azide, and myeloperoxidase-deficient neutrophils were used. Adding the NADPH oxidase inhibitor diphenyleneiodonium, to prevent superoxide production, decreased the killing rate to 25%, indicating that oxidative killing mechanisms predominate in this system. The rate constant for killing of S. aureus with superoxide dismutase attached was 70% of that for control bacteria linked to inactivated enzyme. Superoxide dismutase had no effect in the presence of diphenyleneiodonium. The rate of killing was decreased to 33% in the presence of azide and to 40% with myeloperoxidase-deficient neutrophils. Superoxide dismutase had no effect in the presence of azide. On the assumption that the oxidative and nonoxidative components of killing can be considered separately, the oxidative rate was decreased by almost half by superoxide dismutase and was about six times lower when myeloperoxidase was inactive. We conclude that myeloperoxidase-dependent processes are strongly favored by human neutrophils as their prime mechanism of oxidative killing of S. aureus and that superoxide makes a direct contribution to killing. Our results also suggest that superoxide acts in conjunction with a myeloperoxidase-dependent pathway.
我们使用了一种定量测定方法，该方法可测量吞噬作用和杀灭金黄色葡萄球菌的独立速率常数，以研究超氧化物和髓过氧化物酶在人类中性粒细胞杀灭细菌过程中的作用。为抑制超氧化物依赖性过程，将超氧化物歧化酶与免疫球蛋白G交联，并通过其细胞壁中的蛋白A将偶联物附着于金黄色葡萄球菌表面。用叠氮化物抑制髓过氧化物酶，并使用髓过氧化物酶缺陷的中性粒细胞。添加NADPH氧化酶抑制剂二苯基碘鎓以防止超氧化物产生，使杀灭率降至25%，这表明氧化杀灭机制在该系统中占主导地位。附着有超氧化物歧化酶的金黄色葡萄球菌的杀灭速率常数是与失活酶连接的对照细菌的70%。在存在二苯基碘鎓的情况下，超氧化物歧化酶没有作用。在存在叠氮化物的情况下，杀灭率降至33%，而使用髓过氧化物酶缺陷的中性粒细胞时杀灭率降至40%。在存在叠氮化物的情况下，超氧化物歧化酶没有作用。假设杀灭的氧化和非氧化成分可以分开考虑，超氧化物歧化酶使氧化速率降低了近一半，而当髓过氧化物酶无活性时，氧化速率约低六倍。我们得出结论，人类中性粒细胞强烈倾向于依赖髓过氧化物酶的过程，将其作为氧化杀灭金黄色葡萄球菌的主要机制，并且超氧化物对杀灭有直接贡献。我们的结果还表明，超氧化物与依赖髓过氧化物酶的途径共同起作用。
