Stimulation of Neutrophils in Whole Blood Enhances the Pro‐inflammatory Activity of Neutrophil‐Derived Microvesicles

Abstract

BackgroundAcute increases in circulating levels of neutrophil‐derived microvesicles (N‐MVs) during sepsis combined with evidence of enhanced uptake of MVs within the pulmonary microvasculature during endotoxemia in mice, suggests a role for N‐MVs in development of indirect acute lung injury (ALI). However, evidence for the pro‐inflammatory activity of N‐MVs is unclear, with various in vitro studies demonstrating anti‐inflammatory effects. Most in vitro investigations of N‐MV activity involve stimulation of purified neutrophils with single agonists such as formyl‐methionyl‐leucyl‐phenylalanine (fMLP). This reductionist approach excludes the complex in vivo interplay between different stimuli and other vascular cells contributing to neutrophil responses during sepsis. Here we investigated the proinflammatory activities of N‐MVs under more clinically relevant in vitro conditions, treating healthy volunteer whole blood with lipopolysaccharide (LPS) as a sepsis‐relevant inflammatory stimulus and fMLP as an acute N‐MV inducing stimulus. The activities of N‐MVs derived from whole blood as well as isolated neutrophils were evaluated in a monocyte‐neutrophil‐endothelial cell ‘tri‐culture’ model of pulmonary vascular inflammation.MethodsN‐MVs were generated from whole blood or neutrophils isolated by two‐step density gradient centrifugation, by stimulation with fMLP (1 µM, 30 min) alone or combined with LPS pre‐treatment (100 ng/ml, 1 h). N‐MVs were isolated from blood by anti‐CD66b immunomagnetic‐bead selection. For the tri‐culture assay, isolated neutrophils and immunomagnetic‐bead sorted monocytes were cultured with human lung microvascular endothelial cells. N‐MVs were incubated in the tri‐cultures for 3 h, and MV‐induced responses were determined by ELISA and flow cytometric quantification of cell activation markers on monocytes and neutrophils.ResultsN‐MVs derived from LPS‐fMLP‐stimulated (but not fMLP‐stimulated) whole blood induced significant responses in the tri‐culture assay, i.e. substantive tumour necrosis factor (TNF) release (375.2±251.4 vs untreated 2.2±1.4 pg/ml, p<0.01), increased tissue factor expression on monocytes (MFI: 231.1±90.0 vs untreated 9.2±2.5, p<0.001), and increased expression of CD45 (MFI: 478.3±131.2 vs untreated 281.4±37.5, p<0.05), CD11b (MFI: 208.0±33.0 vs untreated 112.7±20.1, p<0.05) and CD63 (MFI: 77.3±36.5 vs untreated 16.23±5.3, p<0.05) on neutrophils. In contrast, N‐MVs derived from isolated neutrophils showed no activity, with either the LPS‐fMLP or the fMLP stimulation protocols.ConclusionsWe demonstrated that LPS inflammatory ‘priming’ of neutrophils within a whole blood environment was necessary for generation of leukocyte‐activating N‐MVs in our in vitro model of pulmonary vascular inflammation. These findings support a role for circulating N‐MVs in the pathogenesis of indirect ALI, and also highlight the crucial importance of using physiologically relevant in vitro models to evaluate the function of circulating intravascular cell‐derived MVs.