Interaction of human neutrophils with the mitochondria/sPLA₂-IIA complex promotes the release of proinflammatory mediators. (A) Human neutrophils associate with the mitochondria/sPLA₂-IIA complex in vitro as measured by flow cytometry analysis of human neutrophils incubated with fluorescently labeled mitochondria (MitoTracker Deep Red) in the (left) absence or (right) presence of Alexa488-conjugated sPLA₂-IIA. (B) Three-dimensional CSLM reconstruction of mitochondria (red) and sPLA₂-IIA (green) colocalizing within neutrophils (blue nuclei, Hoechst stain; gray cytoplasm, CellTracker CMTPX). (C) Mitochondria are internalized in human neutrophils via an endocytosis-dependent pathway. Graph bars representation of the relative localization (surface vs intracellular) of the mitochondria inside neutrophils following pretreatment with indicated inhibitors (nystatin for inhibition of caveolin-mediated endocytosis; chlorpromazine for inhibition of clathrin-mediated endocytosis; dynasore for inhibition of dynamin-mediated endocytosis; nocodazole for inhibition of polymerization of microtubule [endocytosis and phagocytosis]; cytochalasin B for inhibition of polymerization of actin [endocytosis and phagocytosis]). Data were obtained from 100 neutrophils per condition repeated 3 times (n = 3, *P < .01, **P < .001, and ***P < .0001, Mann-Whitney test compare with diluent). (D) Mitochondrial hydrolytic products derived from the action of the mitochondria/sPLA₂-IIA complex (Figure 4D-E) induce proinflammatory responses in human neutrophils. The total 5-lipoxygenase products (5-LO products) were quantified by high-performance liquid chromatography (n = 4; data are mean ± SEM, **P < .005 vs control, Student t test). (E) The freeMito fraction induces NET formation in vitro and is enhanced by sPLA₂-IIA. NET formation (left panel, DNA, blue, white dotted line) was confirmed by confocal imaging after treatment of mitochondria (red, right panel) with sPLA₂-IIA. sPLA₂-IIA significantly enhances NET formation by mitochondria (upper right panel, n ≥ 7; data are mean ± SEM, *P < .05 and **P < .005, Student t test). Hydrolysis products from mitochondria/sPLA2-IIA complex activity also induce significant NET formation (lower right panel, n ≥ 3; data are mean ± SEM, **P < .005 and ***P < .001, Student t test).