Synthesis and release of leukotriene C4 by human eosinophils.
Synthesis and release of leukotriene C4 by human eosinophils.
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DOI：
10.4049/jimmunol.138.2.532
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发表时间：
1987-01
影响因子：
4.4
通讯作者：
W. Owen;R. Soberman;T. Yoshimoto;A. Sheffer;R. Lewis;K. Austen
中科院分区：
文献类型：
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作者：
W. Owen;R. Soberman;T. Yoshimoto;A. Sheffer;R. Lewis;K. Austen
用钙离子载体A23187或N-甲酰基-L-甲硫基-L-亮氨酰-L-苯丙氨酸刺激分离出纯度为92.5%+/-6.9的人外周血嗜酸粒细胞时，免疫反应性白三烯C4首先定位于细胞内，随后以动力学可分辨的步骤释放到外部介质中。在含有20 mM L丝氨酸的情况下，用2.5微米的A23187刺激嗜酸性粒细胞，后者是一种次氯酸清除剂，可防止硫多肽白三烯的氧化代谢。细胞内和胞外LTC4的合成在5~10分钟内即可完成。在5、10和30min时，分别有65.9%+/-15.2、42.3%+/-24.3和5.5%+/-3.9的LTC4留在细胞内。用5微克/毫升细胞松弛素B和0.5微米FMLP刺激嗜酸性粒细胞，细胞内合成和细胞外释放免疫活性LTC4的时间进程与用离子载体刺激的相似，尽管LTC4的总产量仅为10%左右。细胞内LTC4的鉴定通过反相高压液相色谱洗脱、扫描紫外光谱、放射免疫测定和生物测定来确认。在没有L丝氨酸的情况下，用A23187刺激嗜酸性粒细胞将新合成的LTC4代谢成6-反式LTB4非对映异构体和亚类特异性的非对映异构体亚硫醚，这些异构体仅在细胞外介质中被鉴定。因此，纯化的嗜酸性粒细胞对两种不同刺激的反应表明，生物活性LTC4在细胞内短暂积累，细胞外释放明显，氧化代谢明显限制在细胞外位置。
When human peripheral blood eosinophils isolated to 92.5% +/- 6.9 purity were stimulated with either the calcium ionophore A23187 or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), immunoreactive leukotriene C4 (LTC4) was initially localized intracellularly and was subsequently released to the external medium in kinetically distinguishable steps. Eosinophils were stimulated with 2.5 microM A23187 in the presence of 20 mM L-serine, a hypochlorous acid scavenger that prevents the oxidative metabolism of sulfidopeptide leukotrienes. Total production of immunoreactive LTC4, the sum of intra- and extracellular LTC4, was complete within 5 to 10 min. At 5, 10, and 30 min, 65.9% +/- 15.2, 42.3% +/- 24.3, and 5.5% +/- 3.9, respectively, of the total amount of LTC4 measured remained intracellular as detected after the media and cells were separated and the latter was extracted with methanol. The time course for the intracellular synthesis and extracellular release of immunoreactive LTC4 from eosinophils pretreated with 5 micrograms/ml cytochalasin B and stimulated with 0.5 microM FMLP was like that obtained with ionophore, although the total LTC4 production was only approximately 10%. The identity of the intracellular LTC4 was confirmed by elution with reverse-phase high pressure liquid chromatography followed by scanning UV spectroscopy, radioimmunoassay, and bioassay. Eosinophils that were stimulated with A23187 in the absence of L-serine metabolized newly synthesized LTC4 to 6-trans-LTB4 diastereoisomers and subclass-specific diastereoisomeric sulfoxides that were identified only in the extracellular medium. Thus the response of purified eosinophils to two different stimuli demonstrates a transient intracellular accumulation of biologically active LTC4, the distinct extracellular release, and the apparent limitation of oxidative metabolism to the extracellular location.