Role of microparticles derived from monocytes, endothelial cells and platelets in the exacerbation of COPD

Abstract

Background: Microparticles (MPs) are shedding membrane vesicles released from activated blood and endothelial cells under inflammatory conditions. The role of endothelial MPs (EMPs) in pathophysiology of COPD is relatively well known. However, the release and function of MPs of other cellular origins, eg, platelets, red blood cells and leukocytes, are not clearly evaluated in COPD.

Purpose: The aim of this study was to measure EMPs and other cell-derived circulating MPs in stable and exacerbated COPD patients.

Patients and methods: A total of 50 patients with COPD and 19 healthy volunteers were enrolled in the study. EMPs (CD31+, CD62E+) and platelet-derived (CD61+, CD41+, CD42a+, PAC1+), red blood cell-derived (GlyA+) and leukocyte-derived (CD45+, CD13+, CD14+, CD56+) MPs were measured. Flow cytometry (FC) was performed on Beckman Coulter FC500 analyzer. MP reference gate was set using 0.3-0.5-0.9 µm microbeads with MP size gates of 0.5-1.0 µm.

Results: All the measured MPs were significantly (P<0.001) higher in COPD patients than in the controls. Furthermore, CD62E+, CD41+, CD42a+ and CD14+ MP values were significantly (P<0.001) increased in exacerbated COPD compared to stable COPD. These MPs showed significant (P<0.001) inverse correlation with FEV₁/FVC, as well.

Conclusion: In this study, we describe a reliable flow cytometric assay for MP analysis that was successfully applied in COPD. Besides EMPs, COPD is accompanied by an increased concentration of various MPs in the systemic circulation; particularly, platelet- and monocyte-derived MPs seem to be important in exacerbation.

Keywords: COPD; biomarker; cell-derived microparticles; flow cytometry.

Figure 1

FC gating strategy used for MP measurement. Notes: (A–C) MP size gate was determined using 0.3 µm, 0.5 µm, and 0.9 µm FITC-labeled polystyrene MBs. The lower side of the gate was set below 0.5 µm bead as a threshold, because the 0.3 µm and 0.5 µm bead histogram displayed an overlap indicating that the cytometer would not be able to discriminate individual MPs between these size ranges. The upper side of the gate was set at the upper and right sides of the 0.9 µm bead cloud. In this MP gate, the buffer and the sample containing MPs can be clearly distinguished. (D–G) MPs were defined as Annexin V+ events in the size gate, with fluorescence intensity above isotype control and the sample-free buffer. Abbreviations: Cy5, CyChrome (PE-Cy5 conjugate); FC, flow cytometry; FITC, fluorescein isothiocyanate; FS, forward scatter; Iso, isotype; MB, microbeads; MP, microparticle; PE, phycoerythrin.; SS, side scatter.

Figure 2

Comparison of PAC1+ MPs in patients with GOLD stage I–II vs stage III–IV. Note: PAC1+ MP numbers are significantly increased in patients with GOLD stage III–IV compared to patients with GOLD stage I–II (data are presented as median and 25–75th percentiles, Mann–Whitney test, P=0.031). Abbreviation: MPs, microparticles.