Date Published: June 19, 2019
Publisher: Public Library of Science
Author(s): Sophie Ohlsson, Lisa Holm, Christina Hansson, Susanne M. Ohlsson, Lena Gunnarsson, Åsa Pettersson, Lillemor Skattum, Nades Palaniyar.
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.
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). AAV affect small vessels in various organs, such as the kidneys, and include granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). ANCA are in most cases directed against proteinase 3 (PR3) or myeloperoxidase (MPO), two important enzymes in the host defense against bacteria which are located in the granules of neutrophils and monocytes .
The alternative complement pathway has received increasing attention in the context of AAV during the last 10 years. Both animal studies and a clinical trial on C5aR blockade have given us evidence of its importance in these traditionally pauci immune diseases. [9, 11–13] Previous studies have predominantly focused on MPO-ANCA-associated vasculitis and stimulation with MPO-ANCA, probably since the existing animal models are in MPO-ANCA associated disease. In this study we studied patients with PR3-ANCA-associated vasculitis and PR3-ANCA IgG preparations. Due to results from other studies, we focused on the potential role of the alternative pathway although our findings do not exclude involvement of other complement activation pathways. We could demonstrate increased C3bBbP generation in supernatants from AAV PMN compared with healthy controls. This effect correlated with intracellular ROS production and microparticle release, whereas no correlation could be seen neither with simply the degree of degranulation, nor with release of properdin or PMN contents of properdin. These findings imply a loss of inhibition in AAV PMN. Deficient function of factor H has recently been described in AAV by Chen et al, leading to decreased alternative pathway regulation [20–22]. Decreased regulation of the alternative pathway in the fluid phase would be expected to lead to increased production of anaphylatoxins, creating a vicious circle of increased complement activation and increased PMN activation, already suggested in the pathogenesis of AAV. In our degranulation assay, priming with C5a instead of TNF-α resulted in a 10-fold increased release of the ANCA antigens PR3 and MPO, implying an increased exposure and thereby possibly an increased risk of autoimmunity in C5a-driven inflammation.
This study shows that supernatants from stimulated AAV PMN have a greater ability to activate the alternative complement pathway compared to HC PMN. This increased ability could be caused by increased release of microparticles. Our findings emphasize the role of complement in the pathogenesis in AAV – underlining the therapeutic potential of C5a and other complement blockade.