Anti-Neutrophil Cytoplasmic Antibodies Stimulate Release of Neutrophil Microparticles

Ying Hong

Despina Eleftheriou

Abdullah Hussain

Fiona Price-Kuehne

^{Department of Paediatric Rheumatology, and}

^{Department of Infectious Diseases and Microbiology, UCL Institute of Child Health, London, United Kingdom;}

^{Department of Renal Immunobiology, Medical School, University of Birmingham, Birmingham, United Kingdom;}

^{Vasculitis and Lupus Clinic, Addenbrookes Hospital, Cambridge, United Kingdom; and}

^{UCL Centre for Nephrology, Royal Free Hospital, London, United Kingdom}

^{Corresponding author.}

Correspondence: Dr. Ying Hong, Infectious Diseases and Microbiology Unit, 30 Guilford Street, London WC1N 1EH, United Kingdom. Email: ku.ca.lcu.hci@gnoh.y

Received 2011 Mar 24; Accepted 2011 Aug 26.

Abstract

The mechanisms by which anti-neutrophil cytoplasmic antibodies (ANCAs) may contribute to the pathogenesis of ANCA-associated vasculitis are not well understood. In this study, both polyclonal ANCAs isolated from patients and chimeric proteinase 3–ANCA induced the release of neutrophil microparticles from primed neutrophils. These microparticles expressed a variety of markers, including the ANCA autoantigens proteinase 3 and myeloperoxidase. They bound endothelial cells via a CD18-mediated mechanism and induced an increase in endothelial intercellular adhesion molecule-1 expression, production of endothelial reactive oxygen species, and release of endothelial IL-6 and IL-8. Removal of the neutrophil microparticles by filtration or inhibition of reactive oxygen species production with antioxidants abolished microparticle-mediated endothelial activation. In addition, these microparticles promoted the generation of thrombin. In vivo, we detected more neutrophil microparticles in the plasma of children with ANCA-associated vasculitis compared with that in healthy controls or those with inactive vasculitis. Taken together, these results support a role for neutrophil microparticles in the pathogenesis of ANCA-associated vasculitis, potentially providing a target for future therapeutics.

Abstract

The anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) include Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), and Churg–Strauss syndrome.¹2 These rare diseases are associated with significant mortality and morbidity due to small vessel vasculitis resulting in pauci-immune GN,¹ respiratory tract vasculitis resulting in alveolar hemorrhage, and thrombosis.³4 ANCAs directed against neutrophil antigens, typically proteinase 3 (PR3-ANCA) in WG or myeloperoxidase (MPO-ANCA) in MPA, are found in the sera of most affected patients.⁵ Data from in vitro studies,⁶7 animal models,⁸9 and clinical observations in humans¹⁰12 indicate that ANCAs are directly involved in the pathogenesis of vasculitis. One proposed paradigm of ANCA pathogenesis is that neutrophils, after cytokine priming, are fully activated by ANCAs either in the blood or within lesional tissue and firmly adhere to the vascular endothelium.⁵7 These neutrophils degranulate and release numerous cytotoxic mediators provoking endothelial injury and vasculitis.¹³ There are additional mechanisms postulated, including complement activation.¹⁴

Although there is supportive evidence for all of these mechanisms, important unanswered questions concerning the pathogenesis of AAVs remain. These include how ANCAs bind to endothelium independently of ANCA antigens to cause endothelial activation¹⁵ even though endothelial cells have not been conclusively demonstrated to produce MPO or PR3,¹⁶17 and why patients with AAV have evidence of increased hypercoagulability.³18 Finally, ANCA levels do not always correlate with disease activity,¹⁹ and it is unknown how therapeutic plasma exchange mediates its beneficial effects,²⁰21 as this does not appear to be the result of ANCA removal from the circulation alone. Further understanding of the interaction between ANCAs, leukocytes, endothelium, and coagulation pathways could address some of these important but as yet unexplained observations.

Increased cellular microparticles (MPs) have been described in AAVs,²²24 although their pathologic significance in this context is currently unknown. MPs are membrane vesicles released upon activation or apoptosis from various cell types including neutrophils, platelets, and endothelial cells.²⁵26 Loss of phospholipid asymmetry and increased surface expression of phosphatidylserine are crucial events in this process.²²25 In children with active vasculitis, we previously demonstrated elevated platelet and endothelial MPs that correlated with disease activity,²²27 an observation subsequently confirmed in adults with AAVs.²⁴ In adults with AAVs, Daniel et al.²³ observed increased plasma neutrophil microparticles (NMPs) expressing CD66b, although the pathogenic potential of these were not investigated. As they convey various bioactive effectors originating from the parent cells, MPs may exhibit a wide spectrum of biological activities relevant to the pathogenesis of acute vasculitis including participation in inflammation and involvement in hemostatic/thrombotic pathways.²⁸

Because neutrophil activation is a central event in the initiation of vasculitis caused by ANCAs, we hypothesized that stimulation of cytokine-primed neutrophils with ANCAs would result in the release of NMPs, and that these NMPs could be potent mediators of vasculitis and thrombin generation. In this study, we demonstrated for the first time that NMPs carrying adhesion molecules and the ANCA autoantigens MPO and PR3 are increased in the plasma of children with active AAV and vary with disease activity. We show that polyclonal PR3-ANCA and MPO-ANCA from patients and chimeric mouse/human PR3-ANCA directly stimulate cytokine-primed neutrophils to release NMPs. In addition, these NMPs can bind to and activate endothelium via induction of reactive oxygen species (ROS) and generate thrombin.

Classification of the vasculitic syndromes was based on the recent EULAR/PRINTO/PRES classification criteria for pediatric vasculitis. KD was identified based on five of six of the American Heart Criteria. Immunomagnetic bead extraction was used for enumeration of circulating endothelial cells. ENT, ear, nose, and throat; EULAR/PRINTO/PRES, European League Against Rheumatism/Pediatric Rheumatology International trials Organisation/Pediatric Rheumatology European Society.

Acknowledgments

This study and Y.H. were supported in part by a grant from Arthritis Research UK, by a National Institute for Health Research Biomedical Research Centre grant (to Y.H.), and by an Action Medical Research grant (to D.E.).

Acknowledgments

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

Footnotes

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