Neutrophil-derived microparticles induce myeloperoxidase-mediated damage of vascular endothelial cells.

Thassila Nogueira Pitanga,Thayna Meirelles,Lain Carlos Pontes-De-Carvalho,Alberto Augusto Noronha-Dutra,Viviane Costa Junqueira Rocha,Valéria Matos Borges,Washington Luis Conrado Dos-Santos,Luciana De Aragão França,Marilda Souza Gonçalves

doi:10.1186/1471-2121-15-21

Thassila Nogueira Pitanga, Thayna Meirelles + Show 7 more

Open Access

https://doi.org/10.1186/1471-2121-15-21

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Abstract

BackgroundUpon activation neutrophil releases microparticles - small plasma membrane vesicles that contain cell surface proteins and cytoplasmic matter, with biological activities. In this study we investigated the potential role of myeloperoxidase in the endothelial cell injury caused by neutrophil-derived microparticles.ResultsMicroparticles were produced by activating human neutrophils with a calcium ionophore and characterized by flow cytometry and transmission and scanning electron microscopy. Myeloperoxidase activity was measured by luminol-dependent chemiluminescence. Neutrophil microparticles-induced injuries and morphological alterations in human umbilical vein endothelial cells (HUVECs) were evaluated by microscopy and flow cytometry. Neutrophil microparticles were characterized as structures bounded by lipid bilayers and were less than 1 μm in diameter. The microparticles also expressed CD66b, CD62L and myeloperoxidase, which are all commonly expressed on the surface of neutrophils, as well as exposition of phosphatidylserine. The activity of the myeloperoxidase present on the microparticles was confirmed by hypochlorous acid detection. This compound is only catalyzed by myeloperoxidase in the presence of hydrogen peroxide and chloride ion. The addition of sodium azide or taurine inhibited and reduced enzymatic activity, respectively. Exposure of HUVEC to neutrophil microparticles induced a loss of cell membrane integrity and morphological changes. The addition of sodium azide or myeloperoxidase-specific inhibitor-I consistently reduced the injury to the endothelial cells. Taurine addition reduced HUVEC morphological changes.ConclusionsWe have demonstrated the presence of active myeloperoxidase in neutrophil microparticles and that the microparticle-associated myeloperoxidase cause injury to endothelial cells. Hence, the microparticle-associated myeloperoxidase-hydrogen peroxide-chloride system may contribute to widespread endothelial cell damage in conditions of neutrophil activation as observed in vasculitis and sepsis.

Highlights

Upon activation neutrophil releases microparticles - small plasma membrane vesicles that contain cell surface proteins and cytoplasmic matter, with biological activities
Characteristics of calcium ionophore-induced neutrophil MPs MPs were isolated by differential centrifugation of neutrophils activated with 2 μM calcium ionophore and characterized by electron microscopy and flow cytometry (Figure 1)
Exposure of human umbilical vein endothelial cells (HUVEC) to the MPs/Hydrogen peroxide (H2O2)/Cl- system Once the activity of MPO of neutrophil MPs was detected, we investigated whether this enzyme could mediate the damage caused by MPs to endothelial cells

Summary

Introduction

Upon activation neutrophil releases microparticles - small plasma membrane vesicles that contain cell surface proteins and cytoplasmic matter, with biological activities. Neutrophil activation by anti-neutrophil cytoplasmic antibody or complement plays a central role in the genesis of a variety of small vessels vasculitis. These conditions are associated with significant morbidity and mortality due to lesions in central nervous system, lungs and kidneys. A few studies have provided evidence that human neutrophil MPs contain active myeloperoxidase (MPO), suggesting that they may activate endothelial cells [1,8] This condition may give raise to lesions present in some vasculitis [3]. Little is known about the pathways of potential endothelial injury involving neutrophil MPs and ROS

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