Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
J Allergy Clin Immunol. 2010 Nov;126(5):1032-40, 1040.e1-4. doi: 10.1016/j.jaci.2010.06.039. Epub 2010 Aug 21.
Leukotrienes (LTs) are potent proinflammatory lipid mediators with key roles in the pathogenesis of asthma and inflammation. Recently, nanovesicles (exosomes), released from macrophages and dendritic cells (DCs), have become increasingly appreciated as messengers in immunity.
We investigated whether exosomes from human macrophages, DCs, and plasma contain enzymes for LT biosynthesis and studied potential roles for exosomes in transcellular LT metabolism and granulocyte chemotaxis.
The presence of LT pathway enzymes and LT biosynthesis in exosomes and cells was analyzed by Western blot, immunoelectron microscopy, and enzyme activity assays. Surface marker expression was evaluated by flow cytometry, and granulocyte migration was assessed in a multiwell chemotaxis system.
Exosomes from macrophages and DCs contain functional enzymes for LT biosynthesis. After incubation of intact cells with the LT biosynthesis intermediate LTA(4), LTB(4) was the major product of macrophages, whereas DCs primarily formed LTC(4). However, in exosomes from both cell types, LTC(4) was the predominant LTA(4) metabolite. Exosomal LTC(4) formation (per milligram protein) exceeded that of cells. In macrophages and DCs, TGF-β1 upregulated LTA(4) hydrolase along with increased LTB(4) formation also in the exosomes. Moreover, TGF-β1 modified the expression of surface marker proteins on cells and exosomes and reduced the exosome yield from macrophages. On Ca(2+)-ionophore and arachidonic acid stimulation, exosomes produced chemotactic eicosanoids and induced granulocyte migration. Interestingly, active LTA(4) hydrolase and LTC(4) synthase were present also in exosomes from human plasma.
Our findings indicate that exosomes can contribute to inflammation by participation in LT biosynthesis and granulocyte recruitment.
白三烯（LTs）是具有关键作用的炎症介质，在哮喘和炎症的发病机制中发挥作用。最近，巨噬细胞和树突状细胞（DCs）释放的纳米囊泡（外泌体）作为免疫中的信使，越来越受到关注。
我们研究了人类巨噬细胞、DC 和血浆来源的外泌体是否含有 LT 生物合成的酶，并研究了外泌体在细胞间 LT 代谢和嗜中性粒细胞趋化中的潜在作用。
通过 Western blot、免疫电子显微镜和酶活性测定分析外泌体和细胞中 LT 途径酶和 LT 生物合成的存在。通过流式细胞术评估表面标记物的表达，并用多孔趋化系统评估嗜中性粒细胞的迁移。
巨噬细胞和 DC 的外泌体含有功能性 LT 生物合成酶。在用 LT 生物合成中间体 LTA（4）孵育完整细胞后，巨噬细胞的主要产物是 LTB（4），而 DC 主要形成 LTC（4）。然而，在两种细胞类型的外泌体中，LTC（4）是主要的 LTA（4）代谢物。外泌体中 LTC（4）的形成（每毫克蛋白）超过细胞。在巨噬细胞和 DC 中，TGF-β1 上调 LTA（4）水解酶，同时也增加了外泌体中 LTB（4）的形成。此外，TGF-β1 修饰了细胞和外泌体表面标记蛋白的表达，并减少了巨噬细胞外泌体的产量。在 Ca（2+）离子载体和花生四烯酸刺激下，外泌体产生趋化性类花生酸和诱导嗜中性粒细胞迁移。有趣的是，人血浆来源的外泌体中也存在活性 LTA（4）水解酶和 LTC（4）合成酶。
我们的研究结果表明，外泌体可以通过参与 LT 生物合成和嗜中性粒细胞募集来促进炎症。
