Hoffman T, Lizzio E F, Suissa J, Rotrosen D, Sullivan J A, Mandell G L, Bonvini E
Division of Blood and Blood Products, United States Food and Drug Administration, Bethesda, MD 20892.
J Immunol. 1988 Jun 1;140(11):3912-8.
Human peripheral blood monocytes, prelabeled with [3H]arachidonic acid (AA), release labeled eicosanoids in response to soluble or particulate stimuli. Treatment with 12-O-tetradecanoate phorbol-13 acetate (20 nM), calcium ionophores, A23187 (2 microM) or ionomycin (1 microM), or serum-treated zymosan (300 micrograms) resulted in production of cyclooxygenase (CO) metabolites, 6-keto-PG-F1 alpha, thromboxane-B2, PGE2, PGF2 alpha, PGD2, PGB2, 12-L-hydroxy-5,8,10-heptadecatrienoic acid; 15-lipoxygenase products, including 15-hydroxyeicosatetraenoic acid (HETE); and unmetabolized AA. Labeled 5-lipoxygenase (LO) products, 5-HETE, and leukotriene-B4 were detected only after exposure to ionophore or serum-treated zymosan. The calcium dependence of 5-LO activation was confirmed in experiments where calcium was omitted from the incubation medium, and EGTA (0.5 mM) was added, as well as by direct measurement of increased intracellular calcium in phagocytosing monocytes. Combined or sequential treatment with two stimuli increased the release of unmetabolized AA without a commensurate augmentation of labeled metabolites, indicating that release of CO and LO metabolites does not necessarily reflect the extent of phospholipase activation. Quantitation of individual eicosanoids by RIA confirmed results by using radionuclides. These studies show the following. Activation of human monocyte phospholipase may be regulated by at least two pathways, one "12-O-tetradecanoate phorbol-13 acetate-like," which is largely independent of calcium, and another which is mediated by increased intracellular Ca2+ ("ionophore-like"). "Physiologic" stimulation of monocyte arachidonate release, such as that seen accompanying phagocytosis of opsonized particles, may occur via either a calcium-sensitive or calcium-insensitive pathway or both. Calcium may regulate eicosanoid formation at the level of phospholipase or 5-LO. Free AA, CO products, and 12- or 15-LO products are ordinarily released after phagocytosis, but leukotriene-B4, 5-HETE, or other 5-LO metabolites are produced only under conditions where calcium concentrations are optimal.
预先用[3H]花生四烯酸（AA）标记的人外周血单核细胞，会对可溶性或颗粒性刺激作出反应，释放出标记的类二十烷酸。用12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（20 nM）、钙离子载体A23187（2 microM）或离子霉素（1 microM）处理，或用经血清处理的酵母聚糖（300微克）处理，会导致环氧化酶（CO）代谢产物的产生，包括6 - 酮 - PG - F1α、血栓素 - B2、前列腺素E2、前列腺素F2α、前列腺素D2、前列腺素B2、12 - L - 羟基 - 5,8,10 - 十七碳三烯酸；15 - 脂氧合酶产物，包括15 - 羟基二十碳四烯酸（HETE）；以及未代谢的AA。仅在暴露于离子载体或经血清处理的酵母聚糖后，才检测到标记的5 - 脂氧合酶（LO）产物、5 - HETE和白三烯 - B4。在孵育培养基中省略钙并添加乙二醇双四乙酸（EGTA，0.5 mM）的实验，以及通过直接测量吞噬单核细胞中细胞内钙的增加，证实了5 - LO激活对钙的依赖性。用两种刺激联合或顺序处理会增加未代谢AA的释放，而标记代谢产物却没有相应增加，这表明CO和LO代谢产物的释放不一定反映磷脂酶激活的程度。通过放射免疫分析（RIA）对单个类二十烷酸进行定量，证实了使用放射性核素得到的结果。这些研究表明：人单核细胞磷脂酶的激活可能受至少两条途径调节，一条是“12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯样”途径，它在很大程度上不依赖于钙；另一条是由细胞内Ca2 +增加介导的途径（“离子载体样”途径）。单核细胞花生四烯酸释放的“生理性”刺激，如在吞噬调理过的颗粒时所见，可能通过钙敏感或钙不敏感途径或两者同时发生。钙可能在磷脂酶或5 - LO水平调节类二十烷酸的形成。通常在吞噬后会释放游离AA、CO产物以及12 - 或15 - LO产物，但仅在钙浓度最佳的条件下才会产生白三烯 - B4、5 - HETE或其他5 - LO代谢产物。
