Mediation of endothelial cell damage by serine proteases, but not superoxide, released from antineutrophil cytoplasmic antibody-stimulated neutrophils.
OBJECTIVE
To evaluate potential mediators of endothelial cell injury in systemic vasculitis associated with antineutrophil cytoplasmic antibodies (ANCAs), we investigated the factors controlling the neutrophil respiratory burst and endothelial release of von Willebrand factor (vWF) during neutrophil-endothelial cell interactions.


METHODS
Superoxide release from neutrophils binding to purified P-selectin or to tumor necrosis factor-activated endothelial cells was measured under flow or static conditions using the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c. Neutrophils were activated with fMLP, normal IgG, or ANCA IgG. Enzyme-linked immunosorbent assay was used to measure vWF. Serine protease activity was measured enzymatically.


RESULTS
ANCA IgG or fMLP induced superoxide release when perfused over neutrophils that were rolling over P-selectin, but not those that were binding to endothelial cells. In static assays, endothelial cells inhibited superoxide production by neutrophils. Adenosine inhibited the respiratory burst, and, in cocultures, adenosine deaminase overcame the inhibitory effects of endothelial cells. Serine proteases were released during activated neutrophil-endothelial cell coculture. There was enhanced release of vWF during activated neutrophil-endothelial cell coculture; this was not inhibited by diphenyleneiodonium or by SOD plus catalase, but was inhibited by diisopropylfluorophosphate.


CONCLUSIONS
Endothelial cells inhibit superoxide generation by fMLP and ANCA-activated neutrophils. The release of vWF occurs during coculture and is sensitive to serine protease, but not NADPH oxidase inhibition. Serine proteases may play a more important role than reactive oxygen species as mediators of endothelial injury during ANCA-associated systemic vasculitis.
Lu
X
X
University of Birmingham, Birmingham, UK.
Garfield
A
A
Rainger
G E
GE
Savage
C O S
CO
Nash
G B
GB
D019268:Antibodies, Antineutrophil Cytoplasmic / Q000502:physiology*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000473:pathology*; D006801:Humans; D009504:Neutrophils / Q000378:metabolism* / Q000502:physiology; D012697:Serine Endopeptidases / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism*
D019268:Antibodies, Antineutrophil Cytoplasmic; D013481:Superoxides; D012697:Serine Endopeptidases