Exosome secretion as an effective mechanism of LTB4 mediated signal relay in migrating neutrophils

Wednesday, September 24, 2014 — Poster Session IV
10:00 a.m. –12:00 p.m.	FAES Academic Center	NCI	CELLBIO-9

Authors

• R Majumdar
• AT Tameh
• PW Kriebel
• CA Parent

Abstract

Leukotriene B4 (LTB4) is a secondary chemoattractant, involved in signal relay during neutrophil chemotaxis. Its mechanism of secretion and its role in neutrophil chemotaxis is, however, not well understood. We hypothesize that LTB4 secreted by neutrophils are packaged into exosomes that are released periodically to form a stable gradient. We show that upon stimulation with the fMLP, neutrophils secrete vesicles that are enriched with the exosomal markers CD63 and HSP70 and contain LTB4. Importantly, we also discovered that LTB4 synthesizing enzymes, 5-lipoxygenase (5-LO) and LTA4hydrolase, are present in purified exosomes, suggesting active synthesis of LTB4 in secreted exosomes. Furthermore, 5-LO-mCherry, which primarily localizes to the nucleus in resting neutrophils, co-localize in CD63-positive vesicles in chemotaxing neutrophils and were found to dock at the trailing edge of a migrating cell. Exogenous addition of exosomes to resting primary neutrophils lead to rapid polarization and activation of neutrophils, a response inhibited by LTB4 receptor-1 inhibitor LY22392. Finally, decrease of exosome secretion by inhibiting n-Sphingomylienase2 activity led to reduction of neutrophil recruitment and LTB4 relay, an effect that recapitulates inhibition of LTB4 synthesis using the FLAP inhibitor MK886. Together, these findings strongly suggest that LTB4 secretion is mediated by exosome release during neutrophil chemotaxis.

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