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Abstract: TH-PO841

Targeting Neutrophil Serine Proteases in ANCA-Associated Vasculitis

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Jerke, Uwe, Experimental and Clinical Research Center (ECRC) Campus Buch, Berlin, Germany
  • Eulenberg-Gustavus, Claudia, Experimental and Clinical Research Center (ECRC) Campus Buch, Berlin, Germany
  • Rousselle, Anthony, Experimental and Clinical Research Center (ECRC) Campus Buch, Berlin, Germany
  • Schreiber, Adrian, Experimental and Clinical Research Center (ECRC) Campus Buch, Berlin, Germany
  • Korkmaz, Brice, INSERM U-1100, Tours, France
  • Kettritz, Ralph, Experimental and Clinical Research Center (ECRC) Campus Buch, Berlin, Germany
Background

ANCA-activated neutrophils and monocytes cause necrotizing vasculitis. Enzymatically active neutrophil serine proteases (NSPs) contribute to the injury. NSPs include neutrophil elastase (NE), cathepsin G (CatG), and proteinase 3 (PR3) that also provides a major ANCA antigen. NSPs are generated from inactive zymogens by cathepsin C (CatC) during neutrophil maturation in the bone marrow. CatC loss-of-function mutations prevent NSP maturation leading to zymogen degradation. We characterized NSPs in humans and mice and tested the hypothesis that pharmacological CatC inhibition eliminates NSPs in a neutrophil differentiation model and reduces PR3-ANCA induced respiratory burst as well as neutrophil-mediated endothelial injury.

Methods

We prepared highly purified human and murine neutrophils and monocytes. We assessed NSP proteins by immunoblotting and flow cytometry, and proteolytic activity using specific FRET substrates. We employed a reversible cyclopropyl nitrile CatC inhibitor (IcatC) to reduce NSPs in neutrophils differentiated from CD34+ HSC in vitro. We assessed superoxide production by ferricytochrome C reduction and endothelial injury by phalloidin staining.

Results

Human and murine neutrophils and monocytes expressed PR3, NE, and CG by immunoblotting and flow cytometry. All three NSPs were enzymatically active by FRET assays. Protein amounts and proteolytic activity were significantly higher in neutrophils compared to monocytes. In vitro differentiated human neutrophils progressively expressed active NSPs during maturation. IcatC significantly reduced NSP proteins and enzymatic activity. PR3 surface protein decreased by 79%±10% (p<0.01), cellular protein by 82%±4% (p<0.001) and PR3 enzymatic activity by 98%±1% (p<0.001). Anti-PR3 antibodies provoked less superoxide release (46%±6% of control, p<0.01) in neutrophils differentiated in the presence of IcatC compared to buffer control (n=4). In addition, supernatants from neutrophils that were differentiated in the presence of IcatC caused less cytoskeletal damage in HUVECs.

Conclusion

Pharmacological CatC inhibition down-regulates NSPs, abrogates PR3-ANCA induced respiratory burst in an in vitro neutrophil differentiation model, and reduced neutrophil-mediated endothelial damage. CatC inhibition may provide a future treatment strategy in ANCA vasculitis.

Funding

  • Government Support - Non-U.S.