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Proteinase 3 (PR3), the target antigen of antineutrophil cytoplasm autoantibodies, which are found in patients with Wegener granulomatosis, is a neutrophil serine protease localized within cytoplasmic granules. Recently, the human neutrophil antigen NB1 was identified as a specific neutrophil cell surface receptor of PR3. We hypothesized that the unique hydrophobic cluster of PR3 that is not present on human neutrophil elastase and cathepsin G and presumably is also missing in other human PR3 homologs accounts for its binding to the NB1 receptor expressed on the cellular surface of NB1 cells. Instead of generating and testing various artificial human PR3 mutants, we cloned and expressed the very closely related gibbon (Hylobates pileatus) PR3 homolog, which did not bind to the human NB1 receptor. Moreover, a human-gibbon hybrid constructed from the N- and C-terminal half of the human and gibbon PR3, respectively, also did not interact with human NB1. The C-terminal half of gibbon PR3 differs only by 9 residues from human PR3, among which four closely spaced hydrophobic residues are substituted in a nonconservative manner (F166L, W218R, G219A, and L223H). The NB1-bound PR3 was active and was cleared from the surface by a-1-protease inhibitor. Conformational distortion of the hydrophobic 217-225 loop by α-1-protease inhibitor most likely triggers rapid solubilization. [ABSTRACT FROM AUTHOR]

Proteinase 3 (PR3) is the main target antigen of antineutrophil cytoplasmic antibodies (ANCAs) in PR3-ANCA-associated vasculitis. A small fraction of PR3 is constitutively exposed on the surface of quiescent blood neutrophils in a proteolytically inactive form. When activated, neutrophils expose an induced form of membrane-bound PR3 (PR3mb) on their surface as well, which is enzymatically less active than unbound PR3 in solution due to its altered conformation. In this work, our objective was to understand the respective role of constitutive and induced PR3mb in the immune activation of neutrophils triggered by murine anti-PR3 mAbs and human PR3-ANCA. We quantified immune activation of neutrophils by the measurement of the production of superoxide anions and secreted protease activity in the cell supernatant before and after treatment of the cells by alpha-1 protease inhibitor that clears induced PR3mb from the cell surface. Incubation of TNFa-primed neutrophils with anti-PR3 antibodies resulted in a significant increase in superoxide anion production, membrane activation marker exposition, and secreted protease activity. When primed neutrophils were first treated with alpha-1 protease inhibitor, we observed a partial reduction in antibody-induced neutrophil activation, suggesting that constitutive PR3mb is sufficient to activate neutrophils. The pretreatment of primed neutrophils with purified antigenbinding fragments used as competitor significantly reduced cell activation by whole antibodies. This led us to the conclusion that PR3mb promoted immune activation of neutrophils. We propose that blocking and/or elimination of PR3mb offers a new therapeutic strategy to attenuate neutrophil activation in patients with PR3-ANCA-associated vasculitis. [ABSTRACT FROM AUTHOR]