Background/Purpose:

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 diseases such as SLE, RA and ANCA –associated vasculitis (AAV). We observed NETs formation of neutrophils from MPO-AAV patients which is a majority type of AAV in Asia, and examined the effects of immunosuppressive therapy on the release of NETs in order to investigate the role of NETs 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. When HRGEC and neutrophils were separated by transwell chamber, damage of HRGEC by neutrophils were substantially suppressed.

Conclusion:

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. It is suggested that both some soluble factors and fiber-NETs released from neutrophils are involved in the damage of HRGECs. These data suggest the release of fiber-like NETs by neutrophils at vascular endothelium is important for the pathogenesis of AAV.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/stimulated-renal-glomerular-endothelial-cells-were-damaged-by-fiber-like-nets-released-by-neutrophils-in-anca-associated-vasculitis/