Proteinase 3 Microparticles in Vasculitis: implications for inflammation

KR Martin1, M Pederzoli-Ribeil5, M Yin3, F Angelot2, P Frachet4, P Saas2, C Boulanger3, V Witko-Sarsat1,5. 1Inserm U1016, Paris, France, 2Inserm UMR1098, Besançon, France, 3INSERM U970, Paris, France, 4Joseph-Fourier University Grenoble and Institute of Structural Biology Jean-Pierre-Ebel, Grenoble, France, 5Université Paris Descartes, Paris, France

Introduction

Microparticles (MP) are small membrane vesicles generated from the plasma membrane of cells during the exocytic budding process following cellular activation or apoptosis. Ranging from 50 to 1000 nm in size, the majority of MP contain high levels of phosphatidylserine (PS) on their outer membrane (1). In addition to PS, these vesicles possess a wide array of molecules from the parent cell including both membrane constituents and cytoplasmic content. Functionally, MP are involved in intercellular communication, with studies suggesting that they play a role in inflammation, coagulation and vascular function. Proteinase 3 (PR3), the autoantigen in Granulomatosis with polyangiitis, is found within azurophilic granules of neutrophils, is expressed at the plasma membrane during activation and apoptosis and can be released into the extracellular environment. PR3 is a multifunctional protein with a number of well characterized pro-inflammatory activities. PR3 is capable of activating chemokines, controlling cell survival and proliferation and when presented on the surface of cells, can control macrophage activation (2). Using both surface plasmon resonance and a protein-lipid overlay assay, we have demonstrated that PR3 is a PS-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage.

Objective

The aim of our study was to determine whether PR3 is expressed on MP and if PR3 on cells affects MP production.

Methods and Results

To determine if PR3 is located on MP, soluble PR3 was incubated with MP isolated from either TNFα stimulated endothelial cells (HUVEC, 50 ng/mL TNFα for 24h followed, ultracentrifugation of media, 15000g; 90 minutes; 4°C) or plasma of healthy donors (ultracentrifugation, 15000g; 90 minutes; 4°C). Following incubation for 1h, MP were stained with anti-PR3 antibody (anti-CLB12.8-FITC, 1.36mg/mL) and flow cytometry performed with the aid of calibration beads. In these experiments, PR3 was detected on both endothelial and plasma-derived MP. To determine if PR3 affected MP production, we utilized rat basophilic leukemia cells transfected with either a control plasmid (RBL/pcDNA) or PR3 (RBL/PR3). To induce MP production, RBL cells were treated with either calcium ionophore (2µM for 30 minutes) or Gliotoxin (2 µg/ml for 2h). Annexin V staining and flow cytometry (Invitrogen, according to the manufacturer’s instructions) was used to detect PS+ MP. Our results indicate that RBL cells expressing PR3 produced significantly less MP compared to controls RBL cells during activation with calcium ionophore (2.06x105±0.22x105 vs 3.68x105±0.34 x105, p=.0014 Student’s t-test) and apoptosis induced by gliotoxin (2.29x105± 0.22x105 vs 3.45x105±0.25 x105, p=.0041 Student’s t-test).

Conclusion

Our data indicated that PR3 is expressed on MP and that PR3 expression on cells reduces MP production during both cell activation and apoptosis. Further studies are required to determine if the presence of PR3 affects MP function.

Reference

(1) Thery C et al. (2002) Nature Reviews Immunology 2:569-579

(2) Witko-Sarsat V et al. (2010) Curr Opin Rheumatol 22:1-7