Guthrie L A, McPhail L C, Henson P M, Johnston R B
J Exp Med. 1984 Dec 1;160(6):1656-71. doi: 10.1084/jem.160.6.1656.
We investigated the capacity of bacterial endotoxin (lipopolysaccharide, LPS) to modify the oxidative metabolic response to membrane stimulation of human neutrophils. Neutrophils were pretreated for 60 min with LPS, 10 ng/ml, then stimulated by exposure to fixed immune complexes, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), or phorbol myristate acetate. Release of superoxide anion (O-2) was up to 7-times greater in cells preincubated with LPS, depending upon the stimulus used. Consumption of oxygen and release of hydrogen peroxide (H2O2) were similarly increased, using FMLP as stimulus. The enhancement was accompanied by a reduction in lag time and an increase in the rate of the response, but the duration of the oxidative events was not changed. The molecular basis for the augmented oxidative response of LPS-pretreated cells was investigated. Preincubation with LPS at 0 degrees C prevented priming, but preincubation in the presence of cycloheximide or chelation of extracellular calcium ion did not. Neutrophils preincubated with LPS had slightly decreased numbers of binding sites and equivalent binding affinity for radiolabeled FMLP. Possible changes in the enzyme responsible for the oxidative burst were analyzed by studying NADPH-dependent generation of O-2 by particulate fractions from cells preincubated with LPS or buffer, then stimulated before cell disruption. The fraction prepared from LPS-pretreated neutrophils exhibited greater release of O-2 over a wide range of concentrations of NADPH. The calculated apparent Km for NADPH was equivalent in the two fractions, but the Vmax was increased 2.5-fold in the subcellular fraction from LPS-pretreated cells. These results suggest that LPS could increase neutrophil-mediated host defense or the tissue damage associated with endotoxemia by enhancing the generation of oxygen metabolites by neutrophils. These results also support the concept that the neutrophil is not an end-stage cell in regard to function or metabolic activity.
我们研究了细菌内毒素（脂多糖，LPS）改变人中性粒细胞对膜刺激的氧化代谢反应的能力。中性粒细胞用10 ng/ml LPS预处理60分钟，然后通过暴露于固定免疫复合物、趋化肽甲酰甲硫氨酰亮氨酰苯丙氨酸（FMLP）或佛波酯肉豆蔻酸乙酸酯进行刺激。根据所使用的刺激物，用LPS预孵育的细胞中超氧阴离子（O₂⁻）的释放量增加高达7倍。以FMLP作为刺激物时，氧气消耗和过氧化氢（H₂O₂）释放同样增加。这种增强伴随着延迟时间的缩短和反应速率的增加，但氧化事件的持续时间没有改变。我们研究了LPS预处理细胞氧化反应增强的分子基础。在0℃下用LPS预孵育可防止启动，但在环己酰亚胺存在下预孵育或细胞外钙离子螯合则不能。用LPS预孵育的中性粒细胞对放射性标记FMLP的结合位点数量略有减少，但结合亲和力相当。通过研究用LPS或缓冲液预孵育，然后在细胞裂解前进行刺激的细胞颗粒部分中NADPH依赖性O₂⁻的生成，分析了负责氧化爆发的酶可能发生的变化。从用LPS预处理的中性粒细胞制备的部分在广泛的NADPH浓度范围内表现出更大的O₂⁻释放。计算得出的NADPH的表观Km在两个部分中相当，但来自LPS预处理细胞的亚细胞部分的Vmax增加了2.5倍。这些结果表明，LPS可通过增强中性粒细胞产生氧代谢产物来增加中性粒细胞介导的宿主防御或与内毒素血症相关的组织损伤。这些结果也支持了中性粒细胞在功能或代谢活性方面不是终末细胞的概念。
