Biological defense mechanisms. The effect of bacteria and serum on superoxide production by granulocytes.
Biological defense mechanisms. The effect of bacteria and serum on superoxide production by granulocytes.
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生物防御机制。
DOI：
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发表时间：
1974
影响因子：
15.9
通讯作者：
B. Babior
中科院分区：
文献类型：
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作者：
J. Curnutte;B. Babior
我们以前报道过，粒细胞能够产生超氧化物（O（2）（-）），一种由氧的单电子还原形成的高活性化合物。O（2）（-）产生的证明是基于观察到超氧化物歧化酶（一种催化O（2）（-）转化为过氧化氢和氧气的酶）极大地减少了粒细胞对细胞外细胞色素c的还原。在本报告中，研究了细菌和血清对O（2）（-）-依赖的粒细胞细胞色素c还原的影响。在最佳测定条件下，粒细胞对细胞外细胞色素c的O（2）（-）依赖性还原达到9.2+/-2.8 SD nmol/3 × 10（6）个细胞/20 min。（100个微生物/细胞）存在时，在其他类似条件下O（2）（-）依赖的细胞色素c还原增加了近4倍（34.5+/-9.4）。白蛋白或过氧化氢酶对细胞色素c还原没有影响，煮沸的歧化酶只有很小的影响。忽略粒细胞或用热杀死的细胞取代活细胞，可消除O（2）（-）依赖的细胞色素c还原。高压灭菌法杀死的细菌在刺激粒细胞O（2）（-）产生方面几乎与活菌一样有效。颗粒摄取和O（2）摄取的粒细胞的测量表明，超氧化物歧化酶并不影响非特异性粒细胞代谢，支持的结论是，在歧化酶的存在下，细胞色素c还原的减少是由于这种酶的O（2）（-）破坏。细菌对O（2）（-）产生的刺激强烈依赖于孵育混合物中血清的存在。血清加热到56摄氏度45分钟是一样有效的刺激O（2）（-）生产在细菌的存在下，未加热的血清，但煮沸的血清没有效果。其他实验表明，将细菌与血清一起孵育，会导致一种非颗粒状的热不稳定物质的释放，这种物质能够在没有细菌的情况下刺激O（2）（-）的产生。研究了O（2）（-）-依赖的粒细胞还原细胞色素c的某些特性，包括该过程对粒细胞、细胞色素c和细菌浓度的依赖性。此外，O（2）（-）依赖的细胞色素c还原作为时间的函数。静止粒细胞的速率恒定。对于细菌，时间过程更为复杂。一个明确的滞后之后是一个相当短暂的时期，非常激烈的细胞色素c还原。在此期间，细胞色素c还原的最大速率超过了在没有细菌的情况下观察到的速率的12倍。然后速率降低，直到40分钟，其已减慢至在不存在细菌的情况下观察到的速率。从上述结果可以得出结论，粒细胞暴露于细菌加血清启动了一个过程，在该过程中，在持续20-30分钟的快速爆发中形成规定量的O（2）（-）。可以想象，该过程产生的O（2）（-）可能参与粒细胞对细菌的杀灭。
We previously reported that granulocytes are able to produce superoxide (O(2) (-)), a highly reactive compound formed by the one-electron reduction of oxygen. The demonstration of O(2) (-) production was based on the observation that the reduction of extra-cellular cytochrome c by granulocytes was greatly diminished by superoxide dismutase, an enzyme catalyzing the conversion of O(2) (-) to hydrogen peroxide and oxygen. In the present report, studies concerning the effect of bacteria and serum on O(2) (-)-dependent cytochrome c reduction by granulocytes are described.In the absence of bacteria, the O(2) (-)-dependent reduction of extracellular cytochrome c by granulocytes under optimal assay conditions amounted to 9.2+/-2.8 SD nmol/3 x 10(6) cells/20 min. When bacteria (100 organisms/cell) were present, the O(2) (-)-dependent cytochrome c reduction under otherwise similar conditions increased by a factor of nearly four (34.5+/-9.4). There was no effect of albumin or catalase on cytochrome c reduction, and boiled dismutase had only a small effect. Omission of granulocytes or substitution of live cells by cells by cells killed by heat abolished O(2) (-)-dependent cytochrome c reduction. Bacteria killed by autoclaving were almost as effective as live bacteria in stimulating granulocyte O(2) (-) production. Measurements of particle uptake and O(2) uptake by granulocytes indicated that superoxide dismutase did not affect granulocyte metabolism nonspecifically, supporting the conclusion that the diminution of cytochrome c reduction in the presence of dismutase was due to the destruction of O(2) (-) by this enzyme. Stimulation of O(2) (-) production by bacteria was strongly dependent on the presence of serum in the incubation mixture. Serum heated to 56 degrees C for 45 min was as effective as unheated serum in stimulating O(2) (-) production in the presence of bacteria, but boiled serum had no effect. Other experiments suggested that incubation of bacteria with serum resulted in the release of a nonparticulate heat-labile substance capable of stimulating O(2) (-) production in the absence of bacteria. Certain characteristics of the O(2) (-)-dependent cytochrome c reduction by granulocytes were studied, including the dependence of this process on granulocyte, cytochrome c, and bacterial concentrations. In addition, O(2) (-)-dependent cytochrome c reduction was followed as a function of time. A constant rate was found with resting granulocytes. With bacteria the time course was more complex. A well-defined lag was followed by a fairly brief period of extremely vigorous cytochrome c reduction. During this period, the maximum rate of cytochrome c reduction exceeded the rate observed in the absence of bacteria by a factor of 12. The rate then decreased until by 40 min, it had slowed to the rate observed in the absence of bacteria. From the above results, it was concluded that the exposure of the granulocyte to bacteria plus serum initiates a process in which a defined quantity of O(2) (-) is formed in a rapid burst lasting 20-30 min. It is conceivable that the O(2) (-) generated by this process may be involved in the killing of bacteria by the granulocytes.