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

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Title:Disrupting the CD177:proteinase 3 membrane complex reduces anti-PR3 antibody-induced neutrophil activation
Creators Name:Marino, S.F. and Jerke, U. and Rolle, S. and Daumke, O. and Kettritz, R.
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 for degranulation. 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, dosedependently 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.
Source:bioRxiv
Publisher:Cold Spring Harbor Laboratory Press
Article Number:2021.05.17.444335
Date:17 May 2021
Official Publication:https://doi.org/10.1101/2021.05.17.444335

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