Consequences of cathepsin C inactivation on membrane exposure of proteinase 3, the target antigen in autoimmune vasculitis

Membrane-bound proteinase 3 (PR3m) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis (GPA). Binding of ANCA to PR3m triggers neutrophil activation with the secretion of enzymatically active proteases, thereby contributing to vascular damage. PR3 and related proteases are activated from proforms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce PR3m and has therefore implications as a novel therapeutic approach in GPA. We first studied neutrophilic PR3 from 24 patients with Papillon-Lefèvre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced, but still detectable (0.5-4%) PR3 activity when compared to healthy control cells. Despite extremely low levels of cellular PR3, the amount of constitutive PR3m expressed on the surface of quiescent neutrophils, and the typical bimodal membrane distribution pattern, was similar to what was observed in healthy neutrophils. However, following cell activation, there was no significant increase in the total amount of PR3m on PLS neutrophils, whereas the total amount of PR3m on healthy neutrophils was significantly increased. We then explored the effect of pharmacological CatC inhibition on PR3 expression in normal neutrophils using a potent cell permeable CatC inhibitor and a CD34+ hematopoietic stem cell model. Human CD34+ hematopoietic stem cells were treated with the inhibitor during neutrophil differentiation over 10 days. We observed strong reductions in PR3m, cellular PR3 protein and proteolytic PR3 activity whereas neutrophil differentiation was not compromised