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FRI0247 The Release of Nets from MPO-AAV Neutrophils was Increased by Anti-MPO Antibody and Correlated with Disease Activity
  1. K. Murakami,
  2. Y. Komagata,
  3. C. Mori,
  4. K. Fukuoka,
  5. M. Karube,
  6. S. Kaname,
  7. Y. Arimura
  1. 1st depertment of internal medicine, Kyorin university hospital, Tokyo, Japan

Abstract

Background NETosis, a unique form of cell death of neutrophils, is characterized by the active release of chromatin fibers called NETs, that trap and kill invading microbes extracellularly. Although NETosis plays a crucial role in host defense, excessive NETs formation becomes self-defeating by promoting tissue injury and organ damage. It has been known that NETs are implicated also in the pathogenesis of autoimmune vasculitis such as SLE, RA and ANCA –associated vasculitis (AAV).

Objectives We observed NETs formation of neutrophils from MPO-AAV patients which is a majority type of AAV in Asia, examined the effects of anti-MPO antibody and immunosuppressive therapy on the release of NETs in order to investigate the role of NETs and MPO-ANCA in MPO-AAV.We also observed the damage of human renal glomerular endothelial cells (HRGECs) by neutrophils in order to investigate the role of NETs in AAV.

Methods Isolated peripheral blood neutrophils from healthy donors, pre/post-treatment MPO-AAV patients were incubated with phorbol myristate acetate (PMA), which is known as a strong inducer of NETs, PMA plus anti-MPO antibody or PMA plus anti-PR3 antibody. Neutrophils were stained with Hoechst 33342, SYTOX Green and the percentage of NETs producing cells were calculated. Brd-U labeled HRGECs stimulated by PMA were co-cultured with neutrophils. A cellular DNA fragmentation ELISA was used to quantitatively determine HRGECs damage.

Results Neutrophils from pre-treatment MPO-AAV patients produced much more fiber-like NETs than neutrophils from controls and the shape of fiber-like NETs were different from controls. Anti-MPO antibody increased the release of fiber-like NETs both in controls and MPO-AAV patients. The release of fiber-like NETs decreased in tandem with the decrease of ANCA titer after initial treatment and increased with the rise of ANCA titer in some MPO-AAV patients. Although PMA-stimulated HRGEC were damaged in the presence of neutrophils, HRGEC without PMA stimulation were not damaged.

Conclusions Neutrophils from pre-treatment MPO-AAV patients released different type of fiber-like NETs and the amount of fiber-like NETs correlated with the activity of AAV. HRGECs stimulated with PMA were damaged in the presence of neutrophils. These data suggest the release of fiber-like NETs by neutrophils at vascular endothelium is important for the pathogenesis of AAV.

References

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  2. Arimura Y et al: Serum myeloperoxidase and serum cytokines in anti-myeloperoxidase antibody-associated glomerulonephritis, Clin Nephrol, 40: 256-264, 1993.

  3. Kessenbrock K et al: Netting neutrophils in autoimmune small-vessel vasculitis, Nat Med, 15: 623-625, 2009.

  4. Gupta AK et al: Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated cell death, FEBS Lett, 584: 3193-3197, 2010.

  5. Kambas K et al: Tissue factor expression in neutrophil extracellular traps and neutrophil derived microparticles in antineutrophil cytoplasmic antibody associated vasculitis may promote thromboinflammation and the thrombophilic state associated with the disease, Ann Rheum Dis, 73: 1854-1863, 2014.

Disclosure of Interest None declared

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