Background Neutrophil extracellular traps (NETs) were first described as web-like structures that trap and neutralize microbes at sites of infection. NETs are comprised of chromatin components and neutrophil cytoplasmic proteins. Because these components of NETs might provide an immunogenic substrate for autoimmune responses during regular encounters with commensal and pathogenic microbes, it has been reported that NETs are involved in autoimmunity such as SLE or ANCA associated vasculitis (AAV). It has also been reported that autoantibodies themselves for important cytoplasmic auto-antigens of neutrophils such as MPO or PR3 induce NETs production. However, the role of other neutrophil cytoplasmic antigens for the production of NETs is unclear.
Objectives Thus, we investigated how auto-antibodies for these antigens other than PR3 and MPO are involved in NETs productions by neutrophils.
Methods Human peripheral blood neutrophils were obtained from healthy donors. Neutrophils were seed onto Polystyrene chamber slide system and incubated with PMA (control) or PMA plus several kinds of anti-neutrophil cytoplasmic antigens for 3 hours. Cells were fixed and stained with Hoechst 33342 and Sytox Green for DNA and anti-MPO antibody. The percentage NETs producing cells and the quantitation of nuclear decondensation were analyzed using Image J software. We distinguished cells that released NETs fiber from cells that just died of NETosis.
Results Anti-MPO antibody strongly promoted both NETosis induction and release of NETs fiber. Although anti-PR3 antibody promoted NETosis induction, it did not increase the release of NETs fiber. Antibody for cathepsin G, found in the azunophil granule, also promoted NETosis induction only. There were some other antibodies, such as anti-lactoferrin and anti-neutrophil elastase, that promoted both NETosis and release of NETs fiber.
Conclusions It has been thought that ANCAs directed against granule proteins of neutrophils are implicated in the pathogenesis of AAV, partly because ANCAs promote NETs production by neutrophils. We showed that not only anti-MPO and anti-PR3 but also antibodies for other neutrophil cytoplasmic antigens promote NETs production. The mechanism these antibodies for cytoplasmic protein induce NETosis and how important they are for the pathogenesis of AAV are to be elucidated.
Disclosure of Interest None Declared
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