Neutrophils from ANCA-associated vasculitis patients show an increased capacity to activate the complement system via the alternative pathway after ANCA stimulation.

Sophie Ohlsson,Christina Hansson,Lena Gunnarsson,Susanne M Ohlsson,Lisa Holm,Lillemor Skattum,Åsa Pettersson,Nades Palaniyar

doi:10.1371/journal.pone.0218272

Abstract

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), are autoimmune conditions associated with small vessel inflammation. Earlier studies indicate that complement activation via the alternative pathway plays a major role in the pathogenesis. In this study we have investigated if ANCA-activation of neutrophils from AAV patients leads to activation of the alternative complement pathway. C5a-primed neutrophils (PMN) from 10 AAV patients and 10 healthy controls (HC) were stimulated with PMA or IgG purified from PR3-ANCA positive patients (ANCA IgG). The supernatants were analyzed for release of complement proteins and markers of different granules by ELISA, and release of microparticles (MP) by flow cytometry. The ability of the supernatants to activate the alternative complement pathway was determined by incubation with normal serum and C3bBbP and C5a were measured by ELISA. MP were analyzed by flow cytometry and removed by centrifugation. The supernatants from the AAV patients’ neutrophils produced significantly more C3bBbP compared with HCs (p = 0.0001). C3bBbP levels correlated with the number of MP. After removal of MP from the supernatants, alternative pathway activation was significantly lower. This study shows that primed and ANCA-stimulated neutrophils from AAV patients have a greater ability to activate the alternative complement pathway compared to primed neutrophils from healthy controls. This finding emphasizes the role of complement in the pathogenesis of AAV - underlining the therapeutic potential of C5a and other complement blockade.

Highlights

Primary systemic vasculitis is characterized by relapsing-remitting inflammation and necrosis of blood vessel walls and sometimes granuloma formation
primed neutrophils (PMN) from associated vasculitides (AAV) patients and healthy controls (HC) degranulated to a similar extent upon anti-neutrophil cytoplasmic autoantibodies (ANCA)-stimulation after priming with tumor necrosis factor alpha (TNF-α), as it has been shown previously [2] and the same was seen after priming with complement factor 5a (C5a)
In order to obtain an extra effect, on top of the priming effect, with ANCA stimulation, the PMN had to be pre-incubated with cytochalasin B (CLB) and there was a variation in the effect of individual ANCA Immunoglobulin G fraction from PR3-ANCA positive patients (IgG)

Summary

Introduction

Primary systemic vasculitis is characterized by relapsing-remitting inflammation and necrosis of blood vessel walls and sometimes granuloma formation. Small-vessel vasculitis lesions with little or no immune complex deposition (pauci-immune) in conjunction with anti-neutrophil cytoplasmic autoantibodies (ANCA) characterize ANCA-associated vasculitides (AAV). In AAV, immune complexes and complement were previously considered not to be involved in the pathogenesis, since depositions detected by immunofluorescence are generally absent or scanty in the lesions, which differs from immune complex-mediated and anti-GBM glomerulonephritis [4]. Low levels of immune complexes and complement do exist at sites of vascular inflammation and necrosis. Haas and Eustace performed studies on 126 renal biopsies from patients with crescentic glomerulonephritis associated with positive ANCA serology and/or necrotizing small vessel arteritis and found immune complex deposits in 54% of these [5]. In the majority of these cases immunofluorescence was positive for Ig and/or C3

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