Disrupting the CD177:proteinase 3 membrane complex reduces anti-PR3 antibody-induced neutrophil activation

ABSTRACT CD177 is a neutrophil-specific receptor presenting proteinase 3 (PR3) autoantigen on the neutrophil surface. CD177 expression is restricted to a neutrophil subset resulting in CD177 pos /mPR3 high and CD177 neg /mPR3 low populations. The size of the CD177 pos /mPR3 high subset has implications for anti-neutrophil cytoplasmic autoantibody (ANCA)-associated autoimmune vasculitis (AAV) where patients harbor PR3-specific ANCA that activate neutrophils. We generated high affinity anti-CD177 monoclonal antibodies, some of which interfered with PR3 binding to CD177 (PR3 “blockers”) as determined by surface plasmon resonance spectroscopy, and used them to test the effect of competing PR3 from the surface of CD177 pos neutrophils. Because intact anti-CD177 antibodies also caused neutrophil activation, we prepared non-activating Fab fragments of a PR3 blocker and non-blocker that bound specifically to CD177 pos neutrophils by flow cytometry. We observed that Fab blocker clone 40, but not non-blocker clone 80, dose-dependently reduced anti-PR3 antibody binding to CD177 pos neutrophils. Importantly, preincubation with clone 40 significantly reduced respiratory burst in primed neutrophils challenged either with monoclonal antibodies to PR3 or PR3-ANCA IgG from AAV patients. After separating the two CD177/mPR3 neutrophil subsets from individual donors by magnetic sorting, we found that PR3-ANCA provoked significantly more superoxide production in CD177 pos /mPR3 high than in CD177 neg /mPR3 low neutrophils, and that anti-CD177 Fab clone 40 reduced the superoxide production of CD177 pos cells to the level of the CD177 neg cells. Our data demonstrate the importance of the CD177:PR3 membrane complex in maintaining a high ANCA epitope density and thereby underscore the contribution of CD177 to the severity of PR3-ANCA diseases.