The Pathogenicity of BPI-ANCA in a Patient With Systemic Vasculitis

Abstract

Objective: ANCA associated vasculitis (AAV) is characterized by systemic necrotizing vasculitis with the presence of ANCA. Although BPI-ANCA is one of the atypical ANCAs and is occasionally seen in patients with vasculitis, the pathogenicity of BPI-ANCA remains unclear. This study was performed to examine the pathogenic role of BPI-ANCA against neutrophils. Methods: A 76-year-old Japanese man showed BPI-ANCA positive systemic vasculitis with a medical history of Pseudomonas aeruginosa infection. BPI-ANCA IgGs were eluted from the patient serum using an immunoadsorbent column. In vitro experiment, healthy donor neutrophils were treated with BPI-AAV IgGs, MPO-AAV IgGs, healthy control IgGs under TNFα stimulation. After 3 h incubation, neutrophil extracellular trap (NET) was assessed by immunofluorescent imaging. To determine the pathogenicity of BPI-ANCA, TNFα-primed neutrophils were incubated with monoclonal BPI-ANCA in the presence or absence of recombinant BPI. Results: BPI-AAV IgGs-treated neutrophils showed NET formation with histone citrullination. Interestingly, the monoclonal BPI-ANCA did not induce NET, but the immune complexes (ICs) of recombinant BPI and BPI-ANCA induced TNFα-dependent NET formation with hypercitrullination. Furthermore, TNFα increased the expression of BPIs in neutrophils and the BPIs were translocated to cell surface. Conclusion: BPI-ANCA could affect neutrophils leading to NET formation and may play a role in the development of systemic vasculitis as pathogenic autoantibody.

Keywords: ANCA-associated vasculitis; BPI-ANCA; Bactericidal/permeability-increasing protein (BPI); immunity; neutrophil extracellular traps (NETs).

Figure 1

Clinical course of a patient.

Figure 2

BPI-ANCAs induce NETs with hyper citrullinated histones. (A) Representative NETs staining of healthy or BPI-AAV patient IgGs-stimulated neutrophils using CitH3 (red) and DAPI (BLUE). (B) Representative Sytox-positive neutrophils live image. From the left, healthy IgGs (n = 3), MPO-AAV patient IgGs (n = 5), BPI-AAV patient IgGs. Right figure shows Sytox-positive areas quantified by Image J. (C) Representative CitH3-positive NETs and Sytox-positive neutrophils treated with TNFα, TNFα+mBPI-Ab, TNFα+mBPI-Ab+rBPI. Right figures show CitH3-positive area (upper) and Sytox-positive area (lower) under each situation. Data represent the mean±SEM of three independent experiments and were analyzed using unpaired t-test (PRISM software, GraphPad). ^*p < 0.05. (D) BPI expression with or without TNFα stimulation. Green; BPI, BLUE; DAPI. (E) Sytox positivity of neutrophils treated with 3% serum, or 3% IgG depleted serum of patients with BPI-AAV, MPO-AAV (n = 3) and healthy control (n = 3). Scale bar in all figures, 50 mm. CitH3, citrullinated histone3; DAPI, 49,6-diamidin-2-phenylindol; BPI, bactericidal/permeability increasing protein; ANCA, anti neutrophil cytoplasmic antibody; AAV, ANCA associated vasculitis; IgG, immunoglobulin G; TNFα, tumor necrosis factor alpha; mBPI-Ab, monoclonal BPI antibody; rBPI, recombinant BPI.