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Updated: Apr 28, 2026
Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies
Published on: March 2, 2018
Neutrophil-derived microparticles induce myeloperoxidase-mediated damage of vascular endothelial cells
Thassila Nogueira Pitanga, Luciana de Aragão França, Viviane Costa Junqueira Rocha
1Fundação Oswaldo Cruz-BA, Gonçalo Moniz Research Center, Salvador, BA, Brazil. wluis@bahia.fiocruz.br.
Background:
Upon 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.
Results:
Microparticles 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.
Conclusions:
We 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.
Insights
Neutrophil microparticles contain active myeloperoxidase (MPO) that damages endothelial cells. Inhibiting MPO reduces this injury, suggesting a role in inflammatory diseases like vasculitis and sepsis.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Neutrophils release microparticles upon activation.
- These microparticles carry proteins and cytoplasmic matter with biological activities.
- The role of myeloperoxidase in microparticle-induced endothelial cell injury was investigated.
Purpose of the Study:
- To investigate the potential role of myeloperoxidase (MPO) in endothelial cell injury caused by neutrophil-derived microparticles.
- To characterize neutrophil microparticles and confirm the activity of MPO associated with them.
- To evaluate the impact of MPO-containing microparticles on human umbilical vein endothelial cells (HUVECs).
Main Methods:
- Neutrophil activation using a calcium ionophore.
- Characterization of microparticles via flow cytometry and electron microscopy.
- Measurement of MPO activity using luminol-dependent chemiluminescence and hypochlorous acid detection.
- Assessment of HUVEC injury and morphological changes using microscopy and flow cytometry.
Main Results:
- Neutrophil microparticles, <1 μm in diameter, were characterized by lipid bilayers and expressed CD66b, CD62L, MPO, and phosphatidylserine.
- Active MPO on microparticles was confirmed by hypochlorous acid production, inhibited by sodium azide and reduced by taurine.
- Exposure to neutrophil microparticles caused HUVEC membrane damage and morphological alterations.
- Sodium azide and an MPO-specific inhibitor significantly reduced HUVEC injury, while taurine mitigated morphological changes.
Conclusions:
- Active myeloperoxidase is present in neutrophil microparticles.
- Microparticle-associated MPO directly causes injury to endothelial cells.
- The MPO-H2O2-Cl system in microparticles may contribute to endothelial damage in inflammatory conditions like vasculitis and sepsis.
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