Microvesicles derived from alveolar macrophages mediate epithelial activation via a TNF dependant mechanism

Background: Microvesicles (MVs), extracellular particles Aims/Objectives: We hypothesised that alveolar macrophages (AMs) release MVs which contain TNF and can activate other intra-alveolar cells during ALI. Methods: AMs, harvested from C57BL/6 mice, were treated with lipopolysaccharide and then stimulated to produce MVs with ATP/calcium ionophore. Cell-free supernatant underwent high-speed centrifugation to produce a MV pellet and MV-free supernatant. MVs were identified ( + , detergent sensitive) and examined for TNF expression by flow cytometry/ELISA. MVs were incubated with murine lung epithelial (MLE) cells and ICAM-1 was measured on MLE by flow cytometry (n=5-6 for all protocols). Results: AMs released CD11c + MVs (4250±814/µL mean±SD) and >87% were TNF positive. TNF content of MVs was confirmed by ELISA following sonication of samples (14±12 pg/ml in MV pellet vs. 0 pg/ml in MV-free supernatant). Co-culture of MLE cells with MVs led to an increase in MLE ICAM-1 expression compared to culture with MV-free supernatant (12.4±2.6 MFI vs. 7.8±1.8 MFI, p Conclusions: We demonstrate, for the first time, that AMs, an active source of pro-inflammatory cytokines during ALI, can release TNF-laden MVs, which activate MLE cells. These results suggest that MV-mediated inflammatory activation within the alveolar space may play a previously unappreciated, important role in the pathophysiology of ALI.