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. 2013 Aug;12(8):2205-19.
doi: 10.1074/mcp.M113.028589. Epub 2013 May 8.

Heterogeneity in Neutrophil Microparticles Reveals Distinct Proteome and Functional Properties

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Free PMC article

Heterogeneity in Neutrophil Microparticles Reveals Distinct Proteome and Functional Properties

Jesmond Dalli et al. Mol Cell Proteomics. .
Free PMC article

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.

Figures

Fig. 1.
Fig. 1.
Differential stimulation of neutrophils yields microparticles with a distinct proteome. A, The physical properties of microparticles - obtained from neutrophils after stimulation in fluid-phase (FlP; in suspension) or immobilized-phase (ImP; post adhesion to a HUVEC monolayer - were assessed employing the forward and side scatter parameters on the dot-plot generated by flow-cytometric analysis. The origin from the neutrophil was ascertained by staining the microparticles by anti-CD66b staining. B, Venn diagrams representing the proteomic content identified in each of the neutrophil microparticle subsets as determined using tandem LC-MS-MS. C, Ingenuity Pathway Analysis software was used to highlight the top 15 functions of the various proteins expressed in the distinct microparticle subsets as illustrated. In all cases results are representative of four distinct analyses.
Fig. 2.
Fig. 2.
Stimulus-dependent protein expression in neutrophil microparticles. A, Western blotting selecting proteins that were either predominantly (alpha-2-macroglobulin, A2MG; ceruloplasmin, CERU) or uniquely (heat shock 70kDa protein 1, HSP71) expressed in one of the microparticle subsets, or expressed in both sets (Annexin A1, ANXA1; Lactoferrin, TRLF and β-actin, ACTB). ACTB was used as a loading control for densitometry analysis. and (B) flow cytometric analyses of a select group of proteins identified in the proteomic screen (see Methods for details). Results are mean ± S.E. of n = 3–4 distinct microparticle preparations.
Fig. 3.
Fig. 3.
Microparticle heterogeneity in human samples. Expression levels of A2MG, CERU, HSP71, ANXA1 in CD66b+ microparticles in plasma obtained from healthy volunteers (HV) or septic patients (Sep), as well as in exudates obtained from cantharidin-elicited skin blisters (BL). Data are mean ± S.E. of 10–16 samples tested in duplicate. *p < 0.05, ** p < 0.01 versus CT group; ++ p < 0.01 versus respective BL group.
Fig. 4.
Fig. 4.
Neutrophil microparticles respond to external stimuli. A, The ability of FlP and ImP microparticles (∼6 × 104) to produce ROS as assessed with lucigenin, following stimulation with fMLF (1 μm). B, The ability of FlP and ImP microparticles to produce ROS in a receptor dependent fashion was assessed after loading the microparticles with DCFDA and stimulation with 1 μm fMLF, with or without pertussis toxin (PTX,1 μg/ml). C, LTB4 release from microparticles (∼6 × 104) after incubation with arachidonic acid (AA, 100 nm), or in the presence of AA, (100 nm) and the calcium ionophore A23187 (10 nm, 15 min, 37 °C. D, Microparticle chemotaxis toward fMLF (1 μm) after 1 h at 37 °C. Microparticle counts in the chemtoaxis chamber of a ChemoTx ® were determined by flow cytometry (calibration conducted with 1 μm beads) as a function of AnxAV positive events. Results are mean ± S.E. n = 4 distinct microparticle preparations. *p < 0.05, ** p < 0.01 versus CT group; #p < 0.05 versus FIP microparticles.
Fig. 5.
Fig. 5.
Neutrophil microparticles modify endothelial cell gene expression profile. HUVEC were incubated with vehicle, FlP or ImP microparticles (∼8 × 105) for 6 h at 37 °C. After cell harvest and RNA extraction, gene expression profile was assessed using Illumina HT12v4 microarrays. A, Venn diagram showing the number of significantly (p < 0.05) regulated genes in the ImP and FlP microparticles treated versus vehicle cells (PBS). Selected genes are shown. B, Heatmap generated with the individual replicates showing intersample similarity. C, Real time-PCR validation of 15 distinct genes identified in the microarray analysis, showing very high correlation between the two techniques. Results are cumulative from three separate experiments with distinct microparticle and HUVEC preparations. (D, E) Functional analysis showing the top 15 pathways associated with ImP and FlP microparticles treated HUVEC respectively.

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