NDT Advance Access originally published online on October 24, 2008
Nephrology Dialysis Transplantation 2009 24(4):1150-1157; doi:10.1093/ndt/gfn580
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Published by Oxford University Press on behalf of ERA-EDTA [2008].
Anti-proteinase 3 antibodies both stimulate and prime human neutrophils
1 Department of Medicine 2 Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine 3 Veterans Affairs Medical Center, Louisville, KY, USA
Correspondence and offprint requests to: Richard A. Ward, Kidney Disease Program, University of Louisville, 615 S. Preston Street, Louisville, KY 40202-1718, USA. Tel: +1-502-852-5757; Fax: +1-502-852-7643; E-mail: richard.ward{at}louisville.edu
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Abstract |
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Background. Anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) are postulated to injure vascular endothelium by inducing cytokine-primed neutrophils to release proteolytic enzymes and generate reactive oxygen species. Anti-PR3 induce exocytosis, and since priming is associated with upregulation of plasma membrane proteins by exocytosis of intracellular granules, we tested the hypothesis that anti-PR3 prime neutrophils in the absence of cytokines.
Methods. Isolated human neutrophils were incubated with or without anti-PR3. Superoxide release was determined by measuring the reduction of ferricytochrome C. Exocytosis of secretory vesicles and specific granules was determined by measuring the expression of CD35 and CD66b, respectively, using flow cytometry.
Results. Anti-PR3 (15 µg/mL) directly stimulated superoxide production and enhanced FMLP-stimulated superoxide production. Anti-PR3 (0.5 µg/mL) did not stimulate superoxide production but did enhance FMLP-stimulated superoxide production. Incubation of neutrophils with anti-PR3 resulted in time-dependent exocytosis of secretory vesicles and specific granules. Anti-PR3-induced exocytosis, but not superoxide production, was dependent on p38 mitogen-activated protein kinase.
Conclusions. These data demonstrate that anti-PR3 can directly stimulate production of reactive oxygen species by neutrophils without cytokine priming, and that anti-PR3 prime neutrophils for increased FMLP-stimulated reactive oxygen species production. Anti-PR3 also induce exocytosis via a mechanism separate from their effect on reactive oxygen species production. These findings suggest that anti-PR3 ANCA may activate neutrophils and cause endothelial cell injury by multiple pathways, including some that are independent of priming by a second agent.
Keywords: ANCA; exocytosis; neutrophil; priming; reactive oxygen species
Received for publication: 25. 2.08
Accepted in revised form: 24. 9.08