Objectives Inappropriate activation of neutrophils plays a pathological role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The aim of this study was to investigate the functions of semaphorin 4D (SEMA4D) in regulation of neutrophil activation, and its involvement in AAV pathogenesis.
Methods Serum levels of soluble SEMA4D were evaluated by ELISA. Blood cell-surface expression of membrane SEMA4D was evaluated by flow cytometry. To determine the functional interactions between neutrophil membrane SEMA4D and endothelial plexin B2, wild-type and SEMA4D −/− mice neutrophils were cultured with an endothelial cell line (MS1) stained with SYTOX green, and subjected to neutrophil extracellular trap (NET) formation assays. The efficacy of treating human neutrophils with recombinant plexin B2 was assessed by measuring the kinetic oxidative burst and NET formation assays.
Results Serum levels of soluble SEMA4D were elevated in patients with AAV and correlated with disease activity scores. Cell-surface expression of SEMA4D was downregulated in neutrophils from patients with AAV, a consequence of proteolytic cleavage of membrane SEMA4D. Soluble SEMA4D exerted pro-inflammatory effects on endothelial cells. Membranous SEMA4D on neutrophils bound to plexin B2 on endothelial cells, and this interaction decreased NET formation. Recombinant plexin B2 suppressed neutrophil Rac1 activation through SEMA4D’s intracellular domain, and inhibited pathogen-induced or ANCA-induced oxidative burst and NET formation.
Conclusions Neutrophil surface SEMA4D functions as a negative regulator of neutrophil activation. Proteolytic cleavage of SEMA4D as observed in patients with AAV may amplify neutrophil-mediated inflammatory responses. SEMA4D is a promising biomarker and potential therapeutic target for AAV.
- Autoimmune Diseases
- Granulomatosis with polyangiitis
- Systemic vasculitis
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Contributors AK designed and supervised the project. MN carried out most of experiments. SN, DI, TT and YN conducted some of experiments. HT, SK, SK, TK, KM, EM and AK contributed to preparation of materials and provided advice on project planning and data interpretation. MN, TH, YH, YK, YK, TN, TH, YS and MN recruited and clinically characterised patients. MN, SN, JP and AK wrote the manuscript. SN and AK provided funding for the study. All authors contributed to the discussion of the results and edited and approved the final version.
Competing interests None declared.
Patient consent All human samples were obtained after informed consent was provided by the subjects, in accordance with the Declaration of Helsinki and with approval from the ethical review board of the Graduate School of Medicine, Osaka University, Japan (no. 10237).
Ethics approval The ethical review board of the Graduate School of Medicine, Osaka University, Japan (no. 10237). This study was registered in the University Hospital Medical Information Network Clinical Trials Registry (UMIN000013076).
Provenance and peer review Not commissioned; externally peer reviewed.
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