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

Membrane-bound proteinase 3 (PR3) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis (GPA), a systemic small-vessel vasculitis. Binding of ANCA to PR3 triggers neutrophil activation with the secretion of enzymatically active PR3 and related neutrophil serine 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 PR3 and has therefore implications as a novel therapeutic approach in GPA. We first studied PR3 in neutrophils from 21 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 PR3 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 PR3 on PLS neutrophils, whereas the total amount of PR3 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 PR3, cellular PR3 protein and proteolytic PR3 activity whereas neutrophil differentiation was not compromised. Granulomatosis with polyangiitis (GPA) is a systemic small-vessel vasculitis most commonly affecting the upper and lower respiratory tract and kidneys (1,2). The main target autoantigen in GPA is the neutrophil serine protease (NSP) proteinase 3 (PR3) (EC 3.4.21.76) (3,4). GPA patients develop antineutrophil cytoplasmic autoantibodies to PR3 (PR3-ANCA) that bind to membrane-bound PR3 (PR3) on the neutrophil surface (5,6). The membrane expression of PR3 is mediated by a hydrophobic patch at the protease surface, which is not conserved in other related NSPs, such as human neutrophil elastase (HNE), cathepsin G (CG) and neutrophil serine protease 4 NSP-4 (7-9). Binding of PR3-ANCA to PR3 on cytokine-primed neutrophils induces cell activation resulting in neutrophil extracellular traps (NETs) production, and in granule protein and superoxide release (10,11). Secreted active proteases, including PR3 and related NSPs, exert proteolytic activity on endothelial cells thereby contributing to vascular necrosis (5,12). NETs are known to be directly implicated in ANCA induction as well as endothelial damage (13). There is no treatment for GPA that is based on disease-specific mechanisms and the current protocols involve combined administration of steroids with either cyclophosphamide or rituximab (14,15). These standard treatments are associated with toxicity highlighting the need to develop novel, more specific therapeutic strategies (16). Cathepsin C (CatC) (EC 3.4.14.1), also known as dipeptidyl peptidase I, is a lysosomal amino peptidase belonging to the papain superfamily of cysteine peptidases (17). CatC catalyzes the cleavage of two residues from the N-termini of peptides and proteins. CatC, which is ubiquitously expressed in mammals is considered to be a major intracellular processing enzyme. High concentrations of CatC are detected in immune defense cells including neutrophils, mast cells, lymphocytes and macrophages. CatC is the physiological activator of several immune cell-associated serine proteases such as NSPs (18,19). NSPs are synthetized as inactive zymogens containing a di-propeptide in the myeloblast/promyelocyte stage in the bone marrow (8,20). The proforms mature in this very early developmental stage, induced by CatC, through the cleavage of the Nterminal di-propeptide. The cleavage of the dipropeptide by CatC results in a re-orientation