Abstract
Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension or adherent to an endothelial monolayer, led to the production of microparticles containing >400 distinct proteins with only 223 being shared by the two subsets. For instance, postadherent microparticles were enriched in alpha-2 macroglobulin and ceruloplasmin, whereas microparticles produced by neutrophils in suspension were abundant in heat shock 70 kDa protein 1. Annexin A1 and lactotransferrin were expressed in both microparticle subsets. We next determined relative abundance of these proteins in three types of human microparticle samples: healthy volunteer plasma, plasma of septic patients and skin blister exudates finding that these proteins were differentially expressed on neutrophil microparticles from these samples reflecting in part the expression profiles we found in vitro. Functional assessment of the neutrophil microparticles subsets demonstrated that in response to direct stimulation neutrophil microparticles produced reactive oxygen species and leukotriene B4 as well as locomoted toward a chemotactic gradient. Finally, we investigated the actions of the two neutrophil microparticles subsets described herein on target cell responses. Microarray analysis with human primary endothelial cells incubated with either microparticle subset revealed a discrete modulation of endothelial cell gene expression profile. These findings demonstrate that neutrophil microparticles are heterogenous and can deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions.
Highlights
From the ‡The William Harvey Research Institute, Barts and The London School of Medical, Charterhouse Square, London EC1M 6BQ, United Kingdom; §Cardiovascular Division, The James Black Centre, King’s College, University of London,125 Coldharbour Lane, London SE5 9NU, United Kingdom; ¶Department of Intensive Care, Barts Health NHS Trust, London, United Kingdom; ʈDepartment of Rheumatology and Department of Haematology, Imperial College Healthcare NHS Trust, London, United Kingdom
We have recently identified the selective expression of the potent anti-inflammatory and proresolving protein Annexin A1 (ANXA1) on the surface of microparticles generated from neutrophils adherent to endothelial monolayers, when compared with those prepared from quiescent neutrophils [12]
We report that neutrophil stimulation can lead to the generation of heterogeneous microparticles characterized by distinct proteomes
Summary
Materials were obtained from SigmaAldrich Ltd (Poole, UK). Microparticle Generation and Characterization Generation of Different Samples of Microparticles—Human neutrophil microparticles were prepared from peripheral blood neutrophils obtained from healthy volunteers as previously described [12]. The second protocol involved pre-incubation of neutrophils, resuspended at 2 ϫ 107 cells/ml, over a HUVEC monolayer for 20 min at 37 °C before addition of fMLF (1 M; 20 min at 37 °C): this subset of microparticles will be referred to Immobilized Phase or ImP microparticles. HUVEC monolayers were washed with PBS before addition of the neutrophils In both cases, cell supernatants were collected and cells removed by two successive centrifugations at 3000 ϫ g for 10 min at 4 °C, before pelleting the microparticles by centrifugation at 100,000 ϫ g for 1 h at 4 °C, as described [12]. Tryptic peptides from the digests were separated on a nanoflow LC system (UltiMate3000, Thermo Fisher Scientific) and eluted with a 40 min gradient
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