Williams J D, Lee T H, Lewis R A, Austen F
J Immunol. 1985 Apr;134(4):2624-30.
The cellular and extracellular distribution of leukotriene B4 (LTB4) generated in human neutrophilic polymorphonuclear leukocytes (PMN) stimulated with unopsonized zymosan has been compared with that generated in PMN activated by the calcium ionophore. The amounts of extracellular and intracellular LTB4 were quantitated by radioimmunoassay. The authenticity of the immunoreactive LTB4 was confirmed by the elution of a single immunoreactive peak after reverse phase-high performance liquid chromatography (RP-HPLC) at the retention time of synthetic LTB4, by the identical elution time of a peak of radiolabeled product derived from [3H]arachidonic acid-labeled PMN with the immunoreactive product, and by the comparable chemotactic activity on a weight basis of immunoreactive LTB4 and synthetic LTB4 standard. Under optimal conditions of stimulation by unopsonized zymosan, more than 78% of the generated immunoreactive LTB4 remained intracellular, whereas with optimal activation by the ionophore, less than 8.6% of immunoreactive LTB4 was retained. Resolution by RP-HPLC of the products from the supernatants and cell extracts of [3H]arachidonic acid-labeled PMN stimulated with unopsonized zymosan and those stimulated with calcium ionophore allowed identification and measurement of 5-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-LTB4, LTB4, and omega oxidation products of LTB4 by radioactivity. With zymosan stimulation of PMN, 5-HETE and the 6-trans-LTB4 diastereoisomers were not released, LTB4 was partially released, and the omega oxidation products of LTB4 were preferentially extracellular in distribution. In contrast, with ionophore stimulation, only 5-HETE had any duration of intracellular residence being equally distributed intra- and extracellularly throughout the 30-min period of observation; 6-trans-LTB4, LTB4, and the omega oxidation products of LTB4 were retained at less than 19%. The respective distributions of 5-HETE after zymosan and ionophore stimulation were not altered by the introduction of albumin to the reaction mixtures to prevent reacylation, or by hydrolysis of the cell extract to uncover any product that had been reacylated. The finding that stimulation of PMN with unopsonized zymosan results in the cellular retention of 5-lipoxygenase products suggests that release of these metabolites may be an event that is regulated separately from their generation.
将未调理酵母聚糖刺激的人嗜中性多形核白细胞（PMN）中产生的白三烯B4（LTB4）的细胞内和细胞外分布，与钙离子载体激活的PMN中产生的LTB4分布进行了比较。通过放射免疫测定法定量细胞外和细胞内LTB4的量。通过在合成LTB4的保留时间进行反相高效液相色谱（RP-HPLC）后洗脱单个免疫反应峰、[3H]花生四烯酸标记的PMN衍生的放射性标记产物峰与免疫反应产物的洗脱时间相同、以及免疫反应性LTB4和合成LTB4标准品在重量基础上具有可比的趋化活性，证实了免疫反应性LTB4的真实性。在未调理酵母聚糖刺激的最佳条件下，产生的免疫反应性LTB4中超过78%保留在细胞内，而在离子载体的最佳激活下，免疫反应性LTB4的保留率低于8.6%。通过RP-HPLC对[3H]花生四烯酸标记的PMN用未调理酵母聚糖刺激的上清液和细胞提取物产物以及用钙离子载体刺激的产物进行分离，通过放射性鉴定和测量5-羟基二十碳四烯酸（5-HETE）、6-反式-LTB4、LTB4以及LTB4的ω氧化产物。在用酵母聚糖刺激PMN时，5-HETE和6-反式-LTB4非对映异构体未释放，LTB4部分释放，LTB4的ω氧化产物在分布上优先位于细胞外。相比之下，在用离子载体刺激时，只有5-HETE在细胞内有任何停留时间，在整个30分钟观察期内细胞内和细胞外分布均匀；6-反式-LTB4、LTB4以及LTB4的ω氧化产物的保留率低于19%。在反应混合物中引入白蛋白以防止再酰化，或对细胞提取物进行水解以揭示任何已被再酰化的产物，均未改变酵母聚糖和离子载体刺激后5-HETE的各自分布。未调理酵母聚糖刺激PMN导致5-脂氧合酶产物细胞内保留这一发现表明，这些代谢产物的释放可能是一个与其生成分开调节的事件。
