Competitively disrupting the neutrophil-specific receptor-autoantigen CD177:proteinase 3 membrane complex reduces anti-PR3 antibody-induced neutrophil activation

CD177 is a neutrophil-specific receptor presenting the 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 CD177(pos)/mPR3(high) subset has implications for antineutrophil cytoplasmic autoantibody (ANCA)-associated autoimmune vasculitis, wherein patients harbor PR3-specific ANCAs that activate neutrophils for degranulation. Here, 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 nonactivating Fab fragments of a PR3 blocker and nonblocker that bound specifically to CD177(pos) neutrophils. We observed that Fab blocker clone 40, but not nonblocker 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 with either monoclonal antibodies to PR3 or PR3-ANCA immunoglobulin G from ANCA-associated autoimmune vasculitis patients. After separating the two CD177/mPR3 neutrophil subsets from individual donors by magnetic sorting, we found that PR3-ANCAs 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.

Automated summary

CD177 is a receptor specific to neutrophils, presenting the PR3 autoantigen on their surface. The CD177:PR3 membrane complex plays a crucial role in the severity of PR3-ANCA diseases. High-affinity anti-CD177 monoclonal antibodies have been generated to interfere with PR3 binding to CD177 and reduce neutrophil activation.