Interaction Between Myeloperoxidase (MPO), Ceruloplasmin And Anti-MPO Antibodies From Patients With Microscopic Polyangiitis

[Background and Objective]Anti-neutrophil cytoplasmic antibodies (ANCA) associated systemic small vessel vasculitides are autoimmune disorder. ANCA are serological diagnostic markers for certain small vessel vasculitis. Using pre-cooled ethanol fixed neutrophils as substrate, ANCA can be divided into two staining patterns by indirect immunofluorescence technique: cytoplasmic (c-ANCA) and perinuclear (p-ANCA). Their major target antigens are Proteinase 3 (PR3) and myeloperoxidase (MPO) respectively. The C-ANCA/PR3-ANCA are mainly associated with Wegener's granulomatosis (WG); p-ANCA/MPO-ANCA are mainly associated with microscopic polyangiitis (MPA).MPA is the most common ANCA associated systemic vasculitis in Chinese. Anti-MPO antibodies are considered to be the serological marker for diagnosis of primary MPA. But the pathogenesis is not fully clear. MPO is the major oxidative enzyme of neutrophils and serum ceruloplasmin is the physiological inhibitor of MPO. The interaction between MPO, ceruloplasmin and anti-MPO antibodies may be involved in the pathogenesis of MPA.The current study aimed to investigate the inhibitory effects on MPO oxidation activity by affinity-purified anti-MPO antibodies from patients with primary MPA and to further investigate the interaction between MPO, ceruloplasmin and anti-MPO antibodies, which may be provide rational reasons of pathogenesis of primary MPA. [Methods]Sera were collected from hospitalized patients from October 1998 to April 2006 in Peking University First Hospital. IgG preparations were purified by protein G affinity chromatography from MPO-ANCA positive sera from eleven patients with primary MPA and 12 normal healthy blood donors as controls. Anti-MPO antibodies were further purified from anti-MPO antibody containing IgG fractions using MPO affinity chromatography. The enzyme activity of MPO was measured, in the presence of anti-MPO antibodies ceruloplasmin and normal IgG preparations, using a classical MPO oxidation assay. Interaction between ceruloplasmin, MPO and anti-MPO antibodies was further investigated using ELISA. The inhibition rate≥30% was considered as inhibitable type. [Results]1. Establish the reaction system that the final concentration of MPO was 40ng/ml. The Michaelis constant (Km) was 0.705mM.2. Anti-MPO antibodies from 7/11 patients with MPA could inhibit the MPO activity as non-competitive inhibitors in a dose-dependent manner. The average inhibitory rate was 32%. The average inhibitory constant (Ki) was 0.124mg/ml measured by the Dixon plot.3. Ceruloplasmin could competitively inhibit the oxidation activity of MPO in a dose dependent and time dependent manner.4. Anti-MPO antibodies could inhibit the binding between MPO and ceruloplasmin to a maximum of 75.4±11.6%.[Conclusions]1. Anti-MPO antibodies, from majority of patients with MPA, could inhibit the oxidation activity of MPO in a dose-dependent manner and in a non-competitive manner.2. Anti-MPO antibodies could interfere the binding between MPO and ceruloplasmin.3. The potential role of such inhibition effect may be involved in the pathogenesis of small vessel vasculitis.