Exosomes from human macrophages and dendritic cells contain enzymes for leukotriene biosynthesis and promote granulocyte migration

Background

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.

Objective

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.

Methods

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.

Results

Exosomes from macrophages and DCs contain functional enzymes for LT biosynthesis. After incubation of intact cells with the LT biosynthesis intermediate LTA₄, LTB₄ was the major product of macrophages, whereas DCs primarily formed LTC₄. However, in exosomes from both cell types, LTC₄ was the predominant LTA₄ metabolite. Exosomal LTC₄ formation (per milligram protein) exceeded that of cells. In macrophages and DCs, TGF-β1 upregulated LTA₄ hydrolase along with increased LTB₄ 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 Ca²⁺-ionophore and arachidonic acid stimulation, exosomes produced chemotactic eicosanoids and induced granulocyte migration. Interestingly, active LTA₄ hydrolase and LTC₄ synthase were present also in exosomes from human plasma.

Conclusion

Our findings indicate that exosomes can contribute to inflammation by participation in LT biosynthesis and granulocyte recruitment.

Key words: Antigen-presenting cells, eicosanoids, exosomes, inflammation, leukotrienes, TGF-β1

Abbreviations used: AA, Arachidonic acid, APC, Antigen-presenting cell, cysLT, Cysteinyl leukotriene, DC, Dendritic cell, FITC, Fluorescein isothiocyanate, FLAP, 5-Lipoxygenase–activating protein, HETE, Hydroxy eicosatetraenoic acid, KETE, Keto eicosatetraenoic acid, LC, Langerhans cell, 5-LO, 5-Lipoxygenase, LT, Leukotriene, LTA₄H, Leukotriene A₄ hydrolase, LTC₄S, Leukotriene C₄ synthase, MDDC, Monocyte-derived dendritic cell, MDM, Monocyte-derived macrophage, PMNL, Polymorphonuclear leukocyte